HANDBOOK OF

Media for
Environmental
Microbiology
SECOND EDITION

HANDBOOK OF

Media for
Environmental
Microbiology
SECOND EDITION

By

R O N A L D M . AT L A S

Published in 2005 by
CRC Press
Taylor & Francis Group
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Boca Raton, FL 33487-2742
© 2005 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group
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International Standard Book Number-10: 0-8493-3560-4 (Hardcover)
International Standard Book Number-13: 978-0-8493-3560-0 (Hardcover)
Library of Congress Card Number 2004065561
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Library of Congress Cataloging-in-Publication Data
Atlas, Ronald M., 1946Handbook of media for environmental microbiology / Ronald M. Atlas.--2nd ed.
p. cm.
Includes index.
ISBN 0-8493-3560-4 (alk. paper)
1. Microbiology--Cultures and culture media--Handbooks, manuals, etc. 2. Sanitary microbiology
--Handbooks, manuals, etc. 3. Water--Microbiology--Handbooks, manuals, etc. 4. Microbial ecology
--Handbooks, manuals, etc. I. Title.
QR66.3.A848 2005
579'.028--dc22

2004065561

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About the Author:
Ronald M. Atlas, Ph.D., is dean of the Graduate
School, professor of Biology, professor of Public
Health, and codirector of the Center for the Deterrence of Biowarfare and Bioterrorism at the University of Louisville. He received his B.S. from the
State University of New York at Stony Brook in
1968 and his M.S. and Ph.D. from Rutgers in 1970
and 1972, respectively. After spending a year at the
Jet Propulsion Laboratory in Pasadena, California,
he joined the faculty at the University of Louisville.
He also has served as an Adjunct Professor at the
University of Puerto Rico, External Examiner at the
National University of Singapore, External Examiner at the Chinese University in Hong Kong, and
Extraordinary Professor of Microbiology at the University of Pretoria. Dr. Atlas has received a number
of honors including: The University of Louisville
Excellence in Research Award, Johnson and
Johnson Fellowship for Biology, the American Society for Microbiology (ASM) Award in Applied and
Environmental Microbiology, the ASM Founders
Award, and the Edmund Youde Lectureship Award
in Hong Kong.
He has taught a variety of courses in microbiology at
the University of Louisville and has authored several
textbooks in general microbiology and microbial
ecology. He has written nearly 300 research papers
and authored or edited more than 20 books. He has
conducted studies on the fate of oil in the sea. As
part of these studies, he has extensively character-

ized marine bacterial populations and examined the
diversity of microorganisms. He pioneered the field
of bioremediation for marine oil spills. He also has
conducted studies on the application of molecular
techniques to environmental problems. His studies
have included the development of “suicide vectors”
for the containment of genetically engineered microorganisms and the use of gene probes and the polymerase chain reaction for environmental monitoring,
including the detection of pathogens and indicator
bacteria for water quality monitoring.
He served as president of the American Society for
Microbiology. Additionally, he has served on the
National Institutes of Health (NIH) Recombinant
Advisory Committee (RAC), as well as on various
advisory boards for the Environmental Protection
Agency (EPA), Federal Bureau of Investigation, and
Department of Homeland Security. He has been chairperson of the Environmental Committee and the Task
Force on Biological Weapons of the Public and Scientific Affairs Board of the American Society for
Microbiology. He has been a national lecturer for
Sigma Xi, an American Society for Microbiology
Foundation lecturer, and an Australian Society for
Microbiology national lecturer. He has served on the
editorial boards of Applied and Environmental Microbiology, Advances in Microbial Ecology, BioScience,
Biotechniques, Journal of Environmental Science,
Environmental Microbiology, Journal of Industrial
Microbiology, and Biosecurity and Bioterrorism. He
is editor of Critical Reviews in Microbiology.

Contents
INTRODUCTION .................................................................................................................................................................1
Overview ..........................................................................................................................................................................1
Organization .....................................................................................................................................................................1
Names of Media................................................................................................................................................................1
Trademarks .......................................................................................................................................................................1
Composition of Media ......................................................................................................................................................1
Agars..........................................................................................................................................................................2
Peptones.....................................................................................................................................................................2
Meat and Plant Extracts.............................................................................................................................................3
Growth Factors ..........................................................................................................................................................4
Selective Components ...............................................................................................................................................4
Differential Components ...........................................................................................................................................4
pH Buffers .................................................................................................................................................................4
Preparation of Media ........................................................................................................................................................4
Tyndallization ............................................................................................................................................................5
Inspissation ................................................................................................................................................................5
Autoclaving .............................................................................................................................................................. 5
Filtration ....................................................................................................................................................................5
Caution about Hazardous Components .....................................................................................................................5
Uses of Media ...................................................................................................................................................................6
References ........................................................................................................................................................................6
Web Resources..................................................................................................................................................................6
MEDIA, ALPHABETICAL..................................................................................................................................................7
A .......................................................................................................................................................................................7
B......................................................................................................................................................................................49
C......................................................................................................................................................................................78
D ...................................................................................................................................................................................123
E....................................................................................................................................................................................195
F ....................................................................................................................................................................................210
G ...................................................................................................................................................................................231
H ...................................................................................................................................................................................240
I .....................................................................................................................................................................................273
J.....................................................................................................................................................................................278
K ...................................................................................................................................................................................278
L....................................................................................................................................................................................282
M...................................................................................................................................................................................291
N ...................................................................................................................................................................................417
O ...................................................................................................................................................................................431
P ....................................................................................................................................................................................434
Q ...................................................................................................................................................................................473
R....................................................................................................................................................................................473
S ....................................................................................................................................................................................494
T....................................................................................................................................................................................552
U ...................................................................................................................................................................................612
V ...................................................................................................................................................................................614
W ..................................................................................................................................................................................620
X ...................................................................................................................................................................................623
Y ...................................................................................................................................................................................626
Z....................................................................................................................................................................................634
INDEX.................................................................................................................................................................................637

Introduction
Overview

Composition of Media

The second edition of the Handbook of Media for
Environmental Microbiology includes descriptions
of nearly 2000 media that are used for environmental microbial analyses, e.g., of water quality; for the
isolation of microorganisms from soils, waters, and
other environmental samples; and for the cultivation
and maintenance of environmentally relevant microorganisms, including plant pathogens. It provides
the formulations for the wide range of media needed
to cultivate the ever expanding diversity of microorganisms that are culurable.

Media for the cultivation of microorganisms contain
the substances necessary to support the growth of
microorganisms. Due to the diversity of microorganisms and their diverse metabolic pathways, there are
numerous media. Even slight differences in the composition of a medium can result in dramatically different growth characteristics of microorganisms.

Organization
The media described in the second edition of the
Handbook of Media for Environmental Microbiology are organized alphabetically. Synonyms for
media are listed. The description of each medium
includes its name(s), composition, instructions for
preparation, commercial sources, safety cautions
where needed, and uses.

Names of Media
Media often have numerous names. For the most
part the second edition of the Handbook of Media
for Environmental Microbiology retains the original
names assigned in the literature. In some cases
media with identical compositions produced by different companies have different names. For example
Trypticase™ Soy Agar produced as a BBL product
of BD Diagnostic Systems, Tryptone Soy Agar produced by Oxoid Unipath, and Tryptic Soy Agar produced as a Difco product of BD Diagnostic Systems
have identical compositions. Many media also are
known by acronyms. TSA, for example, is the common acronym for Trypticase Soy Agar.

Trademarks
The names of some media, components of media,
and other terms are registered trademarks. The
trademarked items referred to in the second edition
of the Handbook of Media for Environmental Microbiology are listed below.
American Type Culture Collection® and ATCC® are
trademarks of the American Type Culture Collection.
Bacto®, BiTek®, and Difco® are trademarks of Difco
Laboratories (registered trademarks owned by Becton
Dickinson and Company).
Oxoid® and Lab–Lemco® are trademarks of Unipath Ltd.
Acidase®, BBL®, Biosate®, CTA Medium®, DTA
Medium®, DCLS Agar®, Desoxycholate®, Desoxycholate Agar®, Desoxycholate Citrate Agar®, Enterococcosel®, Eugonagar®, Eugonbroth®, GC-Lect®, Gelysate®, IsoVitaleX®, Mycobactosel®, Mycophil®,
Mycosel®, Myosate®, Phytone®, Polypeptone®, Selenite-F Enrichment®, Thiotone®, Trichosel®, Trypticase®,
TSA II®, and TSI Agar® are trademarks of Becton Dickinson and Co.

When methods for culturing microorganisms were
first developed in the 19th century, largely by Robert
Koch and his colleagues, animal and plant tissues
were principally used as sources of nutrients used to
support microbial growth. One of the major discoveries of Fanny Hesse in Koch’s laboratory was that
agar could be used to form solidified culture media
on which microorganisms would grow. Extracts of
plants and animal tissues were prepared as broths or
mixed with agar to form a variety of culture media.
Virtually any plant, animal, or animal organ was
considered for use in preparing media. Infusions
were prepared from beef heart, calf brains, and beef
liver, as a few examples. These classic infusions still
form the primary components of many media that
are widely used.
The composition section of each medium describes
the ingredients that make up the medium, their
amounts, and the pH. It lists those ingredients in
order of decreasing amount. Solids are listed first
showing the weights to be added, followed by liquids showing the volumes to be included in the
medium.
The composition uses generic terms where these are
applicable. For example, pancreatic digest of casein
is marketed by various manufacturers as trypticase,
tryptone, and other commercial product names.
While there may well be differences between these
products, such differences are undefined. Variations
also occur between batches of products produced as
digests of animal tissues.
Media for the cultivation of microorganisms have a
source of carbon for incorporation into biomass. For
autotrophs, the carbon source most often is carbon
dioxide, which may be supplied as bicarbonate
within the medium. Carbohydrates, such as glucose,
or other organic compounds, such as acetate, various
lipids, proteins, hydrocarbons, and other organic
compounds, are included in media as sources of carbon for heterotrophs. These carbon sources may also
serve as the supply of energy. Other compounds,
such as ammonium ions, nitrite ions, elemental sulfur, and reduced iron, may be used as the sources of
energy for the cultivation of autotrophs. Nitrogen
also is required for microbial growth. It may be supplied as inorganic nitrogen compounds for the cultivation of some microorganisms but more commonly
is supplied as proteins, peptones, or amino acids.
Phosphates and metals, such as magnesium and iron,

2

Composition of Media

are also necessary components of microbiological
media. Phosphates may also serve as buffers to
maintain the pH of the medium within the growth
tolerance limits of the microorganism being cultivated. Various additional growth factors may also be
included in the media.
Agars
Agar is the most common solidifying agent used in
microbiological media. Agar is a polysaccharide
extract from marine algae. It melts at 84°C and
solidifies at 38°C. Agar concentrations of 15.0g/L
typically are used to form solid media. Lower concentrations of 7.5–10.0g/L are used to produce soft
agars or semisolid media. Below are some agars
used as solidifying agents in various media.
Agar Bacteriological (Agar No. 1)
An agar with low calcium and magnesium. Available from Oxoid Unipath.
Agar, Bacto
A purified agar with reduced pigmented compounds, salts, and extraneous matter. Available
from BD Diagnostic Systems.
Agar, BiTek™
Agar prepared as a special technical grade. Available from BD Diagnostic Systems.
Agar, Flake
A technical-grade agar. Available from BD Diagnostic Systems.
Agar, Grade A
A select-grade agar containing minerals. Available from BD Diagnostic Systems.
Agar, Granulated
A high-grade granulated agar that has been filtered, decolorized, and purified. Available from
BD Diagnostic Systems.
Agar, Purified
A very high-grade agar that has been filtered,
decolorized, and purified by washing and extraction of refined agars. It has reduced mineral content. Available from BD Diagnostic Systems.
Agar, Technical (Agar No. 3)
A technical-grade agar. Available from BD Diagnostic Systems and Oxoid Unipath.
Agarose
A low-sulfate neutral gelling fraction of agar that
is a complex galactose polysaccharide of near
neutral charge.
Ionagar
A purified agar. Available from Oxoid Unipath.
Noble Agar
An agar that has been extensively washed and is
essentially free of impurities. Available from BD
Diagnostic Systems.
Purified Agar
An agar that has been extensively washed and
extracted with water and organic solvent. Available from BD Diagnostic Systems and Oxoid Unipath.

Peptones
Many complex media, that is, media in which not all
the specific chemical components are known, contain peptones as the source of nitrogen. Peptones are
hydrolyzed proteins formed by enzymatic or acidic

digestion. Casein most often is used as the protein
substrate for forming peptones, but other substances,
such as soybean meal, also are commonly employed.
Below is a list of some of the peptones that are used
as ingredients in various media.
Acidase™ Peptone
A hydrochloric acid hydrolysate of casein. It has a
nitrogen content of 8% and is deficient in cystine
and tryptophan. Available from BD Diagnostic
Systems.
Bacto Casitone
A pancreatic digest of casein. Available from BD
Diagnostic Systems.
Bacto Peptamin
A peptic digest of animal tissues. Available from
BD Diagnostic Systems.
Bacto Peptone
An enzymatic digest of animal tissues. It has a
high concentration of low molecular weight peptones and amino acids. Available from BD Diagnostic Systems.
Bacto Proteose Peptone
An enzymatic digest of animal tissues. It has a
high concentration of high molecular weight peptones. Available from BD Diagnostic Systems.
Bacto Soytone
A enzymatic hydrolysate of soybean meal. Available from BD Diagnostic Systems.
Bacto Triplets
An enzymatic hydrolysate containing numerous
peptides, including those of higher molecular
weights. Available from BD Diagnostic Systems.
Bacto Tryptone
A pancreatic digest of casein. Available from BD
Diagnostic Systems.
Biosate™ Peptone
A hydrolysate of plant and animal proteins. Available from BD Diagnostic Systems.
Casein Hydrolysate
A hydrolysate of casein prepared with hydrochloric acid digestion under pressure and neutralized
with sodium hydroxide. It contains total nitrogen
of 7.6% and NaCl of 28.3%. Available from
Oxoid Unipath.
Gelatone
A pancreatic digest of gelatin. Available from BD
Diagnostic Systems.
Gelysate™ Peptone
A pancreatic digest of gelatin deficient in cystine
and tryptophan and which has a low carbohydrate
content. Available from Oxoid Unipath.
Lactoalbumin Hydrolysate
A pancreatic digest of lactoalbumin, a milk whey
protein. It has high levels of amino acids. It contains total nitrogen of 11.9% and NaCl of 1.4%.
Available from BD Diagnostic Systems and
Oxoid Unipath.
Liver Digest Neutralized
A papaic digest of liver that contains total nitrogen of 11.0% and NaCl of 1.6%. Available from
Oxoid Unipath.
Mycological Peptone
A peptone that contains total nitrogen of 9.5% and
NaCl of 1.1%. Available from Oxoid Unipath.

Composition of Media
Myosate™ Peptone
A pancreatic digest of heart muscle. Available
from BD Diagnostic Systems.
Neopeptone
An enzymatic digest of protein. Available from
from BD Diagnostic Systems.
Peptone Bacteriological Neutralized
A mixed pancreatic and papaic digest of animal
tissues. It contains total nitrogen of 14.0% and
NaCl of 1.6%. Available from BD Diagnostic
Systems and Oxoid Unipath.
Peptone P
A peptic digest of fresh meat that has a high sulfur
content and contains total nitrogen of 11.12% and
NaCl of 9.3%. Available from BD Diagnostic
Systems and Oxoid Unipath.
Peptonized Milk
A pancreatic digest of high-grade skim milk powder. It has a high carbohydrate and calcium concentration. It contains total nitrogen of 5.3% and
NaCl of 1.6%. Available from Oxoid Unipath.
Phytone™ Peptone
A papaic digest of soybean meal. It has a high
vitamin and a high carbohydrate content. Available from BD Diagnostic Systems.
Polypeptone™ Peptone
A mixture of peptones composed of equal parts of
pancreatic digest of casein and peptic digest of
animal tissue. Available from BD Diagnostic Systems.
Proteose Peptone
A specialized peptone prepared from a mixture of
peptones that contains a wide variety of high
molecular weight peptides. It contains total nitrogen of 12.7% and NaCl of 8.0%. Available from
BD Diagnostic Systems and Oxoid Unipath.
Proteose Peptone No. 2
An enzymatic digest of animal tissues with a high
concentration of high molecular weight peptones.
Available from BD Diagnostic Systems.
Proteose Peptone No. 3
An enzymatic digest of animal tissues. It has a
high concentration of high molecular weight peptones. Available from BD Diagnostic Systems.
Soya Peptone
A papaic digest of soybean meal with a high carbohydrate concentration. It contains total nitrogen
of 8.7% and NaCl of 0.4%. Available from Oxoid
Unipath.
Soytone
A papaic digest of soybean meal. Available from
BD Diagnostic Systems.
Special Peptone
A mixture of peptones, including meat, plant, and
yeast digests. It contains a wide variety of peptides, nucleotides, and minerals. It contains total
nitrogen of 11.7% and NaCl of 3.5%. Available
from Oxoid Unipath.
Thiotone™ E Peptone
An enzymatic digest of animal tissue. Available
from BD Diagnostic Systems.
Trypticase™ Peptone
A pancreatic digest of casein. It has a very low
carbohydrate content and a relatively high tryptophan content. Available from BD Diagnostic
Systems.

3

Tryptone
A pancreatic digest of casein. It contains total
nitrogen of 12.7% and NaCl of 0.4%. Available
from Oxoid Unipath.
Tryptone T
A pancreatic digest of casein with lower levels of
calcium, magnesium, and iron than tryptone. It
contains total nitrogen of 11.7% and NaCl of
4.9%. Available from BD Diagnostic Systems and
Oxoid Unipath.
Tryptose
An enzymatic hydrolysate containing high molecular weight peptides. It contains total nitrogen of
12.2% and NaCl of 5.7%. Available from BD
Diagnostic Systems and Oxoid Unipath.

Meat and Plant Extracts
Meat and plant infusions are aqueous extracts that
are commonly used as sources of nutrients for the
cultivation of microorganisms. Such infusions contain amino acids and low molecular weight peptides,
carbohydrates, vitamins, minerals, and trace metals.
Extracts of animal tissues contain relatively high
concentrations of water-soluble protein components
and glycogen. Extracts of plant tissues contain relatively high concentrations of carbohydrates.
With regard to infusions, many media list as an
ingredient infusion from beef heart or another animal tissue. This ingredient is prepared by boiling a
given amount of the animal tissue (e.g., 500.0g), and
then using the liquid or, more commonly, drying the
broth and using the solids from the infusion. The
actual weight of the dry solids extracted from the hot
water used to create the infusion varies, and so the
ingredient typically is simply listed as 500.0g beef
heart infusion, although the actual weight of solids
recovered from the infusion and used in the medium
is far less.
Below is a list of some of the meat and plant extracts
that are used as ingredients in various media.
Bacto Beef
A desiccated powder of lean beef. Available from
BD Diagnostic Systems.
Bacto Beef Extract
An extract of beef (paste). Available from BD
Diagnostic Systems.
Bacto Beef Extract Desiccated
An extract of desiccated beef. Available from BD
Diagnostic Systems.
Bacto Liver
A desiccated powder of beef liver. Available from
BD Diagnostic Systems.
Lab-Lemco
A meat extract powder. Available from Oxoid
Unipath.
Liver Desiccated
Dehydrated ox livers. Available from Oxoid Unipath.
Malt Extract
A water-soluble extract from germinated grain
dried by low-temperature evaporation. It has a
high carbohydrate content. It contains total nitrogen of 1.1% and NaCl of 0.1%.

4

Preparation of Media

Growth Factors
Many microorganisms have specific growth factor
requirements that must be included in media for
their successful cultivation. Vitamins, amino acids,
fatty acids, trace metals, and blood components
often must be added to media. In some cases, specific defined components are used to meet the
growth factor requirements. Incorporation of growth
factors are used to enrich, that is, to increase the
numbers of particular species of microorganisms.
Most often, mixtures of growth factors are used in
microbiological media. Acid hydrolysates of casein
commonly are used as sources of amino acids.
Extracts of yeast cells also are employed as sources
of amino acids and vitamins for the cultivation of
microorganisms. Many media, particularly those
employed in the clinical laboratory, contain blood or
blood components that serve as essential nutrients
for fastidious microorganisms. X factor (heme) and
V factor (nicotinamide adenine dinucleotide) often
are supplied by adding hemoglobin, IsoVitaleX, and/
or Supplement VX.
Selective Components
Many media contain selective components that
inhibit the growth of nontarget microorganisms.
Selective media are especially useful in the isolation
of specific microorganisms from mixed populations.
In many media for the study of microorganisms in
nature, compounds are included in the media as sole
sources of carbon or nitrogen so that only a few
types of microorganisms can grow. Selective toxic
compounds are also frequently used to select for the
cultivation of particular microbial species. The isolation of a pathogen from a stool specimen, for
example, where there is a high abundance of nonpathogenic normal microbiota, requires selective
media. Often, antimicrobics or other selectively
toxic compounds are incorporated into media to suppress the growth of the background microbiota while
permitting the cultivation of the target organism of
interest. Bile salts, selenite, tetrathionate, tellurite,
azide, phenylethanol, sodium lauryl sulfate, high
sodium chloride concentrations, and various dyes—
such as eosin, Crystal Violet, and Methylene Blue—
are used as selective toxic chemicals. Antimicrobial
agents used to suppress specific types of microorganisms include ampicillin, chloramphenicol, colistin, cycloheximide, gentamicin, kanamycin, nalidixic acid, sulfadiazine, and vancomycin. Various
combinations of antimicrobics are effective in suppressing classes of microorganisms, such as enteric
bacteria.
Differential Components
The differentiation of many microorganisms is
based upon the production of acid from various carbohydrates and other carbon sources or the decarboxylation of amino acids. Some media include
indicators, particularly of pH, that permit the visual
detection of changes in pH resulting from such metabolic reactions.

Below is a list of some commonly used pH indicators.
pH Indicator

pH Range

m-Cresol Purple

0.5 – 2.5

Acid
Color
Red

Thymol Blue

1.2 – 2.8

Red

Yellow

Yellow

Blue

Bromphenol Blue 3.0 – 4.6

Alkaline
Color
Yellow

Bromcresol Green 3.8 – 5.4

Yellow

Blue

Chlorcresol Green 4.0 – 5.6

Yellow

Blue

Methyl Red

4.2 – 6.3

Red

Yellow

Chlorphenol Red

5.0 – 6.6

Yellow

Red

Bromcresol Purple 5.2 – 6.8

Yellow

Purple

Bromothymol Blue 6.0 – 7.6

Yellow

Blue

Phenol Red

6.8 – 8.4

Yellow

Red

Cresol Red

7.2 – 8.8

Yellow

Red

m-Cresol Purple

7.4 – 9.0

Yellow

Purple

Thymol Blue

8.0 – 9.6

Yellow

Blue

Cresolphthalein

8.2 – 9.8

Colorless Red

Phenolphthalein

8.3 – 10.0

Colorless Red

pH Buffers
Maintaining the pH of media usually is accomplished by the inclusion of suitable buffers. Because
microorganisms grow optimally only within certain
limits of a pH range, the pH generally is maintained
within a few tenths of a pH unit. For the phosphage
buffers, the pH is established by using varying volumes of equimolar concentrations of Na2HPO4 and
NaH2PO4.
pH.
5.4
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0

Na2HPO4 (mL)
3.0
5.0
7.8
12.0
18.5
26.5
37.5
50.0
61.1
71.5
80.4
86.8
91.4
94.5

Preparation of Media

NaH2PO4 (mL)
97.0
95.0
92.2
88.0
81.5
73.5
62.5
50.0
38.9
28.5
19.6
13.2
8.6
5.5

The ingredients in a medium are usually dissolved,
and the medium is then sterilized. When agar is used
as a solidifying agent, the medium must be heated
gently, usually to boiling, to dissolve the agar. In
some cases where interactions of components, such
as metals, would cause precipitates, solutions must
be prepared and occasionally sterilized separately
before mixing the various solutions to prepare the

Inspissation using an autoclave employs exposure to 85°–90°C for 10 min achieved by having a mixture of air and steam in the chamber. or skin contact. A membrane with a pore size of 0. the temperature then is slowly lowered to less than 60°C. and cyanide are among the toxic components found in some media. sodium azide. inhalation. Media with human blood or human blood components must be handled with great caution to avoid exposure to human immunodeficiency virus and other pathogens that contaminate some blood supplies. Such exposure can be achieved using flowing steam in an Arnold sterilizer.6 105. and vitamin solutions are filter sterilized and added to media that have been cooled to temperatures below 50°C. Many media are sterilized by exposure to elevated temperatures. Exposure for 15 min to steam at 15 psi—121°C is most commonly used.2 116. John Tyndall.9 115.0 126. Appropriate safety precautions must be taken when using media with such components.2mm will trap bacterial cells and. the medium can be sterilized because all the endospores will have germinated and the heat exposure will have killed all the vegetative cells. Caution about Hazardous Components Some media contain components that are toxic or carcinogenic. generally under pressure. Basic fuchsin and acid fuchsin are carcinogens.7 129. Using an Arnold sterilizer or a specialized inspissator.0 126. These compounds are poisonous.6 118. Inspissation Inspissation is a heat exposure method that is employed with high-protein materials. after its discoverer.6 130. followed by a 15 min exposure during which the temperature is raised to 121°C using only steam under pressure in the chamber. the medium is exposed to 75°–80°C for 2 hr on each of 3 successive days. Media with sulfur-containing compounds may result in the formation of hydrogen sulfide. filtration renders fluids free of bacteria and eukaryotic microorganisms.6 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 5 113. and caution must be used in handling media with these compounds to avoid dangerous exposure that could lead to the development of malignancies.4 Filtration Filtration is commonly used to sterilize media containing heat-labile compounds. with small pore sizes. to kill microorganisms. and hence sterile. antibiotic solutions. that is.0 101. to form explosive metal azides. and steps must be taken to avoid ingestion. Tyndallization Exposure to steam at 100°C for 30 min will kill vegetative bacterial cells but not endospores.8 111. especially copper. Thallium salts. Care must be used to ensure proper ventilation. sodium biselenite. Below is a list of pressure– temperature relationships.8 128.4 109.0 122. The most common method is to autoclave the medium. free of living organisms. Pressure—psi 0 1 2 3 4 5 6 7 8 Temperature—°C 100. By allowing the medium to cool and incubate under conditions where endospore germination will occur and by repeating the 100°C–30 min exposure on 3 successive days. Autoclaving Autoclaving uses exposure to steam.9 103. .0 123. which is a toxic gas.9 121. Many carbohydrate solutions. Media containing carbohydrates often are sterilized at 116°–118°C in order to prevent the decomposition of the carbohydrate and the formation of toxic compounds that would inhibit microbial growth. The pH often is adjusted prior to sterilization.9 127. sometimes is called a bacteriological filter. The disposal of azides must avoid contact with copper or achieve sufficient dilution to avoid the formation of such hazardous explosive compounds. This process causes coagulation of the protein without greatly altering its chemical properties.9 108.8 119.3 106. therefore.0 124. that cannot withstand the high temperatures used in autoclaving. but in some cases sterile acid or base is used to adjust the pH of the medium following sterilization. Liquid media are passed through sintered glass or membranes.0 125.Preparation of Media complete medium. such as eggcontaining media. Such exposure kills vegetative bacterial cells and bacterial endospores.3 112. Different sterilization procedures are employed when heat-labile compounds are included in the formulation of the medium. Azides also react with many metals. typically made of cellulose acetate or nitrocellulose.4 117. By preventing the passage of microorganisms. This process of repetitive exposure to 100°C is called tyndallization.

6 Uses of Media Uses of Media The Handbook of Media for Environmental Microbiology.go.T.ecology. S. and H.cect. Russian Specialized Collection of Alkanotrophs. New York. Cold Spring Harbor. wfcc.riken. http://www.ac. Cold Spring Harbor Laboratory Press.jp Netherlands Centraalbureau voor Schimmelcultures (CBS).bbsrc. American Public Health Association Publications. Standard Methods for the Examination of Water and Wastewater. E. DasSarma. Unipath Ltd. L. a global biological resource in the United States with many services. http://www..ru/iegmcol Spanish Collection of Microorganisms (Colección Española de Cultivos Tipo Catalogo de Cepas).R. American Type Culture Collection (ATCC). Archaea: A Laboratory Manual: Methanogens.info .. England. http://www. C.inf. Archaea: A Laboratory Manual.ukncc.know. and E. DasSarma.M. and A. K.J. Robb. and A. Fleischmann. Cold Spring Harbor. Cold Spring Harbor Laboratory Press.belspo. cultivation. K. http://www. Sowers. Sparks. New York. Second Edition contains the media used for the testing of waters and wastewaters recommended by the USEPA for the standard methods examination of water and food. http://vm.atcc. Hampshire. It also includes the formulations of media used to cultivate the microorganisms of environmental significance that are held in the major culture collections of the world.org/catalogs/catalogs. Becton. Clesceri. Cold Spring Harbor. and differentiation of microorganisms. http://www. Difco & BBL Manual: Dehydrated Culture Media and Reagents for Microbiology. Bridson. New York. Eaton.htm United Kingdom National Collection of Yeast Cultures. Schreier. Valtion Teknillinen Tutkimuskeskus (VTT).fda. New York. http://www. F. 1990. 1998.C. psu. Robb. Greenberg. http://www.sci. 1995. and E. Place. http://www. Basingstoke. Dworkin.de Japanese Collection of Microorganisms and Microbial Cultures. Archaea: A Laboratory Manual: Thermophiles. http://www. Web Resources Below is a list of Web sites that provide information about media and microbial cultures. http://www. Dickinson and Co. F.html Belgian Coordinated Collections of Microorganisms of the Laboratorium Voor Microbiologie at the University of Gent (LMG).R. 1995. Cold Spring Harbor Laboratory Press.htm Czechoslovokian Collection of Microorganisms.E.know.nl/databases. Fleischmann.uk United States Food and Drug Administration Bacteriological Analytical Manual.immuni.be/bccm/ db/media.co. Place. A.J. A. Springer-Verlag Inc.ifr. Cold Spring Harbor Laboratory Press.cbs. 1999. 2003. H. The Oxoid Manual. M. Washington D.jcm.R. World Federation of Culture Collections. The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community.gbf-braunschweig.D. References Below is a list of references that can be consulted for further information about media used for the isolation. Maryland. New York.html. 1995. http://www. Schreier.cfsan.Y. Archaea: A Laboratory Manual: Halophiles. Cold Spring Harbor.S.R.org/english/index.uk/ncyc United Kingdom National Culture Collection Microbiological Resources.T. ed.cz/ccm Finish Culture Collection. http://www.fi German Collection of Microorganisms (DSMZ). 1995. Sowers.vtt. (CCM).gov/~embam/bam-toc.M.

...0g Salicin ..2g Bromothymol Blue ...............0g Lactose .......73g MgSO4·7H2O..5g Antibiotic solution .................. Mix thoroughly..........1....08g pH 6...................................................1.....................................................................2 at 25°C Salt Solution: Composition per 100....... Gently heat and bring to boiling...........................................9 ± 0.........0.. 15......0... Multiple dilutions of samples (3............................. Acetamide Agar Composition per liter: Use: For the detection of fecal coliforms in treated waste- Agar ............ 12.............0g MgSO4·7H2O...................1 at 25°C 7 Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10..... 20..................................................... Use: For the differentiation of nonfermentative Gram-negative bacteria. Mix thoroughly.. Cool tubes in a slanted position to produce a long slant........... 1................. 1.................... Can be used as a confirmatory test for water analysis.... 0................................................................15......................................................46g CaCl2·2H2O..................0g NaCl .5. 5..................0...............0g Agar ..................... Filter sterilize.9 ± 0............5g Triton™ X-100 . 0. 0....................................................................5g K2SO4 . Acetamide Broth Composition per liter: Acetamide...........................10.... except antibiotic solution..2mg Vitamin B12 ..0g Glucose .............................0g NaCl .... and seawater by a most-probable-number (MPN) method........... 1............0mL pH 5......... Filter through Whatman filter paper to remove particles...... 0.............. Autoclave for 15 min at 15 psi pressure– 121°C......0mL...........0mL pH 6...................0g K2HPO4 ................... 0.................................................................................. Autoclave for 15 min at 15 psi pressure–121°C............................................................................ Distribute into tubes or flasks.................... ACC Medium Composition per liter: Proteose peptone ............................. test tubes with gas accumulation in the Durham tubes are scored positive and those with no gas as negative......................................................... cent Pseudomonas species....0L...............10...................... 1.............. 20................................................................................................................10..0g Acetamide..............................5g Phenol Red ............................................. Distribute into tubes or flasks.......................0L........... 5... to distilled/deionized water and bring volume to 990......... Autoclave for 15 min at 15 psi pressure– 121°C.......................... 5..............................................1............................10...0............................................................. Source: This medium is available as a premixed powder from BD Diagnostic Systems............ Cool to 45°–50°C... Preparation of Medium: Add components. Mix thoroughly....0mL............................ Can be used as a confirmatory test for water analysis......0mL: Cycloheximide ...........0mL: MgSO4·7H2O ........................... especially Pseudomonas aeruginosa.................. Gently heat and bring to boiling....................................... After incubation................... Adjust pH..............5......................9 ± 0........0mL.... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....... 2.0g K2HPO4 ..........................3g L-Methionine..............5................ Antibiotic Solution: Composition per 10...........5 ± 0............. Mix thoroughly............. Mix thoroughly...................................................5.............................. Bacteria that deamidate acetamide turn the medium blue.............................73g ......................1..... 0..........1..... Acetamide Agar Composition per liter: Agar .................................................................................................................02g Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 100............ Autoclave for 15 min at 15 psi pressure–121°C..........0g NaCl ........................ Distribute into screw-capped tubes or flasks....................... Mix thoroughly..........................................................0mg Biotin .......9mg Thiamine ..........012g pH 6...0g Glycerol ...............................0125g deionized water and bring volume to 1....................................0L................ Cool tubes in a slanted position to produce a long slant............ Mix thoroughly............... Autoclave for 10 min at 15 psi pressure–121°C... 0............. Distribute into test tubes containing an inverted Durham tube............39g KH2PO4 ..... Adjust pH to 5...............Media..................2 ± 0.........0mL pH 7......................................... Gently heat and bring to boiling.........................2 at 25°C...05g Chloramphenicol..... especially Pseudomonas aeruginosa...... Adjust pH............0g NaCl ...... Pour into sterile Petri dishes or distribute into sterile tubes........... 15......................................0g NH4H2PO4 ...... Aseptically add sterile antibiotic solution..39g KH2PO4 ......... Alphabetical A 1 Broth Composition per liter: Pancreatic digest of casein .........2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems........ Proteose peptone ..................... 14.....0g K2HPO4 ...... Use: For the differentiation of nonfermentative Gram-negative bacteria...5g MgSO4·7H2O .......0µg Salt solution ..... or 10 replicates per dilution) are added to tubes containing A 1 broth.............0............ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............................0g Acetamide...........075g Ampicillin .........1................ A MPN table is consulted to determine the most probable number of fecal coliforms............. Gently heat and bring to boiling..... Use: For the cultivation of Acanthamoeba species.................15g FeCl3 .........................0g KH2PO4 .....2 at 25°C Acanthamoeba Medium Composition per liter: Preparation of Medium: Add components to distilled/ water.........................0L... Mix thoroughly.0........ 0...........1.......................... Bacteria that deamidate acetamide turn the medium blue.....15............................... Preparation of Medium: Add components to distilled/ Use: For the selective isolation and cultivation of fluores- deionized water and bring volume to 1............

....02g CuSO4·5H2O ............................. Add distilled/ deionized water to 1............02g Resazurin .. Kleenex tissue paper...0.... Vitamin Solution: Composition per liter: Pyridoxine·HCl........................ 1.................................................................................................................... Bacteria that can utilize acetate as the sole carbon source turn the medium blue...............................................................0g MnSO4·2H2O ...............18g ZnSO4·7H2O .. Mineral Solution 2: Composition per liter: (NH4)2SO4 ...... 2.............................0....................... or HCl-treated cotton)...................2 at 25°C Vitamin solution .................. Autoclave for 15 min at 15 psi pressure–121°C.....8 Acetate Agar MgSO4·7H2O . Autoclave for 15 min at 15 psi pressure–121°C..0g MgSO4·7H2O ............... 1.................. 0..... 5..0g K2HPO4 ...................... Distribute into tubes or flasks.............................0g pH 7..................................72g NaCl ........................................1g NiCl2·6H2O ....0...... Cellobiose Solution: Composition per 50............................................0 with 1N KOH.............. 1.9 ± 0...2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems.........012g pH 6................. Mix thoroughly..5g Phenol Red ............... 0. 0...........0L.....................................................0L........................................................5........ Mix thoroughly..................9g Preparation of Mineral Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 1. 0.....0mg Nicotinic acid .....0......0mg Thiamine·HCl .0mL K2HPO4 ...3......10................................... especially Pseudomonas aeruginosa............ Autoclave for 15 min at 15 psi pressure–121°C........................................5g CaCl2·2H2O.4g MgSO4·7H2O......0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50......... Mix thoroughly. Mix thoroughly..............................................2g CaCl2·2H2O ...........................................01g Na2SeO3·5H2O ................................................................................... Bacteria that deamidate acetamide turn the broth purplish red..... Adjust pH to 7......... Gently heat and bring to boiling............. Cool tubes in a slanted position...................................................0mL of distilled/deionized water............ Use: For the differentiation of nonfermentative Gram-neg- ative bacteria.......0........................................ 0...........0mL: D-Cellobiose .... Sparge under 100% N2 gas for 3 min........0mg p-Aminobenzoic acid ........................ 10.....0L.... Preparation of Medium: Add components to cold dis- tilled/deionized water and bring volume to 1...........0L............. Pour into sterile Petri dishes or leave in tubes............................................................................0mg Calcium DL-pantothenate ..5..................................0mL pH 7..........................2...........5...59g Preparation of Mineral Solution 2: Add components to distilled/deionized water and bring volume to 1...... Acetate Differential Agar (Sodium Acetate Agar) (Simmons’ Citrate Agar...............08g pH 6.........................................................0L.................. 15. Filter sterilize....................... Mix thoroughly.................0g (NH4)H2PO4 ................................025g KAI(SO4)2·12H2O ......75.......5.............. Adjust pH............................................0g NaCl .....10..... Store under N2 gas...........................50...........................0mg Lipoic acid...........0g Sodium acetate .......... Mix thoroughly.....0L...........0g K2HPO4 ........0.....................................................................1.....0mL Trace elements solution .....2..5g CoSO4·7H2O ........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500..................18g FeSO4·7H2O.........25g Na2S·9H2O....................................................................0L..... ...........25g FeSO4·7H2O.......... 0....0...................................................................................................0mL Mineral solution 2 ...01g H3BO3 .................0mL: Cellobiose or cellulose (MN 300.......................................001g Mineral solution 1 ......................................3.....................2 at 25°C Preparation of Medium: Add components to distilled/ Mineral Solution 1: Composition per liter: deionized water and bring volume to 1...... 0..................................0g Nitrilotriacetic acid........................................................0g Yeast extract.................0mL Cellobiose solution . Acetivibrio cellulolyticus Medium Composition per 1170........................................ 20.................. Mix thoroughly..................................................................0........0g L-Cysteine·HCl..0mg Biotin ................................................... Mix thoroughly........6.........................2 ± 0... Can be used as a confirmatory test for water analysis........... Gently heat and bring to boiling...........................5.................0mg Riboflavin.........0..............................0g NaCl ...............................................................8 ± 0................................................ Whatman CF II................................................................................ Modified) Composition per liter: Agar .....5 with KOH.......0g Pancreatic digest of casein ..........2.... Add remaining components......... Trace Elements Solution: Composition per liter: MgSO4·7H2O ................... 10........................................0mg Folic acid ................. Use: For the differentiation of Shigella species from Escherichia coli and also for the differentiation of nonfermenting Gram-negative bacteria........75..............5........ Dissolve by adjusting pH to 6.. 1............... Use: For the cultivation and maintenance of Caryophanon latum.....................1.............. 3.......0........0g Sodium acetate ...0. 2...........................0mL Reducing agent solution ..............2g Bromothymol Blue ........................................1........................... 2............ 0..........................................................0mL............... Acetate Agar Composition per liter: Agar ..............................................01g Na2MoO4·2H2O ..........1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.........................................................................0g NaHCO3 .......0........................0mg Vitamin B12.....................................................4 ± 0.1...................5..................................0................................ Distribute into tubes to produce a 1 cm butt and 30 cm slant...............................

..........0mL..... except cel- lobiose solution and reducing agent solution..... 0............0mL Solution E .......... Mix thoroughly..........................Acetobacter diazotrophicus Agar Reducing Agent Solution: Composition per 110.... Cool to room temperature........... Autoclave for 15 min at 15 psi pressure–121°C.. a nitrogen fixing endophyte.......... an anaerobic.......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................1............................ 0.0L..... Solution B: Composition per liter: FeCl2·4H2O ......04g NiCl2·6H2O ............. 0................................7......................5.0.......... Preparation of Medium: Add components................... Distribute anaerobically under 80% N2 + 20% CO2........................ Mix thoroughly.0mL Solution C ...0g Yeast extract .................................. Cool to room temperature... After autoclaving................ Boil under N2 gas for 1 min.. Mix thoroughly............. Cool to room temperature. Mix thoroughly.......................0g CaCO3.................. 40.0............ 2....................................... 2...... 0............ Gently heat and bring to boiling...............................5g CoCl2·6H2O .. 0........... Cool to room temperature..0mL...........02g CuCl2·2H2O .............. Add distilled/deionized water and bring volume to 1............7 ± 0............. Distribute under N2 gas in 10......................................... Cool rapidly to 50°–55°C...........0mL Solution B ...........................................0mL Solution D ..................................0g KCl........0mL...19g MnCl2·4H2O................ Add L-cysteine·HCl·H2O and dissolve... Bring pH to 5........................ 0..........10........869.. solution E............3g KH2PO4 . Solution D: Composition per 10........................................... Solution E: Composition per 10.............0mL..........0g Preparation of Solution C: Add the NaHCO3 to distilled/deionized water and bring volume to 100............. Cool to room temperature...................................0mL: 9 clave under 100% N2 for 15 min at 15 psi pressure–121°C.....5g Na2S·9H2O.............10.......15g Preparation of Medium: To 869.....................................0mL: Yeast extract ........ 0.... ...................... Pour into sterile Petri dishes or leave in tubes............................. Gas with 80% N2 + 20% CO2 to remove residual O2.................. Adjust pH to 7.... the pH of the medium will be 7...0mL..............................................1mL of sterile reducing agent solution and 0............ 100....5mL of sterile cellobiose solution to each tube containing 9. 10...............................0L....... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.............................5g MgCl2·6H2O.. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.............0µg Preparation of Solution E: Add components to distilled/ deionized water and bring volume to 10.................02g HCl...0g Thiamine·HCl ............ Auto- Desulfovibrio sapovorans which reduces sulfate.....0µg p-Aminobenzoic acid ...... Solution F: Composition per 10. Cool to room temperature under 80% N2 + 20% CO2................1g ZnCl2 ......... and solution F...................... 0...........07g H3BO3 ....0mL Preparation of Solution B: Add the FeCl2·4H2O to the HCl.50..............0mL flask.........0..0mL: Sodium butyrate ... Use: For the cultivation and maintenance of Acetobacter diazotrophicus.................. Adjust pH to 7..............0.................................... Prior to inoculation of cultures............................................. 1..............0mL.............................................. Na2SO4 ....0mL pH 7.....5............ Acetobacter diazotrophicus Agar Composition per liter: Glucose....................30................. 0........... Add washed Na2S·9H2O and dissolve............................................7g Sodium caproate ...0mL: L-Cysteine·HCl·H2O ... Prepare and autoclave part A under 80% N2 + 20% CO2... Distribute into tubes or flasks.......................... 3........................0mL: Na2S·9H2O .............100................... Autoclave for 15 min at 15 psi pressure–121°C......... aseptically add the remaining sterile solutions in the following order: solution B..10........ aseptically and anaerobically add 0.........0mL volumes into tubes.. Preparation of Solution A: Add components to dis- Use: For the cultivation of Acetivibrio ethanolgignens and tilled/deionized water and bring volume to 869....0g pH 5.25.......................... Adjust to pH 9 with 5N NaOH.................. 0....................... Filter sterilize....0mL of sterile cooled Part A.0g NaCl ................... Anaerobically distribute under 90% N2 + 10% CO2 into sterile tubes or flasks...... Mix thoroughly.........................................................10............. Mix thoroughly to evenly distribute CaCO3... Autoclave for 10 min at 15 psi pressure–121°C....... 0................ 25% .................10........ Acetivibrio Desulfovibrio Medium (LMG Medium 105) Composition per liter: Solution A ....4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.......... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C..............1..........2g CaCl2·2H2O..................06g Na2MoO4·2H2O ........... cellulolytic bacterium.... Mix thoroughly....5g Preparation of Reducing Agent Solution: Add 110.0µg D(+)-Biotin....................0g Agar ............0mL: NaHCO3 ......................................................2..... Autoclave for 15 min at 15 psi pressure–121°C........................................ Cool to room temperature........ 0......................................................... Add remaining components..................4g NH4Cl..4mL of sterile basal medium........................ Mix thoroughly..... Solution C: Composition per 100.....................................................................................0mL Solution F..............6 by gassing.................................. Gently heat and bring to boiling............ Use: For the cultivation and maintenance of Acetivibrio cellulolyticus....................15g Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10............... to distilled/ deionized water and bring volume to 940..... solution C..0mL of distilled/deionized water to a 250..3g Sodium octanoate .... Continue boiling for 3 min.............................. 1........................................2 at 25°C Solution A: Composition per 869............................ solution D.......................5 ± 0..

.....................1g NiCl2·6H2O ...................................0...............52g KCl .......................................... Mix thoroughly................0g K2HPO4 ...............5............. 0...0mL NaHCO3 solution...........0mg Riboflavin...0mL NaHCO3 solution..... 0.......................1g FeSO4·7H2O.0g Nitrilotriacetic acid .............................. 10......................................................20.................. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere. and 0..............5g NaCl ...................0..................1g Resazurin ................. Cool while sparging with 80% N2 + 20% CO2.................................................................. vitamin solution......................... Filter sterilize.........................................0mL...............0mL: Na-syringate................. Add distilled/deionized water to 1.........01g Na2MoO4·4H2O ........... to distilled/deionized water and bring volume to 900...................20mL sterile Na2CO3 solution to each 9...........10........ 0..................................0 ± 0.................................................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500...............0mL Trace mineral solution SL-10............................................ Cool to 25°C. For every 9.....0mL...........................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.. Add remaining components.........................0mg Folic acid ....... Sparge with 80% N2 + 20% CO2..... 1................................................0mg p-Aminobenzoic acid ..........10 Acetobacterium dehalogenans Medium Acetobacterium dehalogenans Medium (DSMZ Medium 787) Composition per liter: NaHCO3 ..............17mL cysteine solution........0..... Cysteine Solution: Composition per 10....................45g KH2PO4 .................................................5.......2 at 25°C L-Cysteine Solution: Composition per 10.............................33g CaCl2·2H2O ..... Boil for 5 min.......... 2............. 0.......33g MgSO4·7H2O ........ 10.... 0..... Sparge with 80% N2 + 20% CO2........0g Yeast extract............................................. and cysteine solution.............. 0...................... 1.....................................................5..........2.........................0.... 0........20.......... except NaHCO3 solution...15mL Na-syringate solution.................. 0..................... Trace Elements Solution: Composition per liter: MgSO4·7H2O ................... Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C..0mL............... 0.....5..... which dehalogenates methyl chloride......4......... 0...................... 0.........0mg Lipoic acid...........................................................18g CaCl2·2H2O....... 10.. Mix thoroughly.......................0mg Thiamine-HCl·2H2O ............................................................................ 10.......... 30............................5.......... Use: For the cultivation of Acetobacterium dehalogenans.......................0mL Cysteine solution..1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1............. 0.......................... Mix thoroughly..........................................2..............0mL........ 0...................0mg Vitamin B12......................................................... Autoclave for 15 min at 15 psi pressure–121°C.....0mL aliquots into serum bottles............ 1................................ Add components...............0...0mL: L-Cysteine·HCl·H2O ...................................2mL vitamin solution......025g KAl(SO4)2·12H2O.......... Mix thoroughly........................3g Preparation of Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10.18g ZnSO4·7H2O ...........01g CuSO4·5H2O .............0. Filter sterilize...........................0mL.........................................................................3g Preparation of L-Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10.2 at 25°C NaHCO3 Solution: Composition per 100.....................0mL Seven vitamin solution ................. Filter sterilize....20...0mL Na2CO3 solution.......0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20................. Mix thoroughly............... 0...........30....5 with KOH...... 10..................................................................... NaHCO3 Solution: Composition per 20... Aseptically and anaerobically add approximately 0............................................................................0L....................0.....0mg Nicotinic acid ....5g CoSO4·7H2O .................0mL pH 7............................. 1................................ Na2CO3 solution.......0mg NaHCO3 solution ....................0g MnSO4·2H2O ...10...........3g ...0mg D-Ca-pantothenate ...................................... Na-syringate solution................... 0........... Distribute 9...........................0mL Vitamin solution..................... Vitamin Solution: Composition per liter: Pyridoxine-HCl ....................................................................................0mL: L-Cysteine·HCl·H2O..................... 1.............................0mg Biotin ........5..... Dissolve by adjusting pH to 6............. Acetobacterium tundrae Medium (DSMZ Medium 900) Composition per liter: Yeast extract ........0g NH4Cl................. 5.............................0mL Na2S·9H2O solution ......................... Sparge with 100% N2..... Gently heat and bring to boiling......0mL of medium inject 1.................... 20..... Sparge with 100% N2.................................................................. 1............................. Sparge with 80% N2 + 20% CO2....... Filter sterilize...........0g MgCl2·6H2O ..... 0..................02g H3BO3 ....0mL of distilled/deionized water..................2g Preparation of Na-syringate Solution: Add Na-syringate to distilled/deionized water and bring volume to 20..... Na-syringate Solution: Composition per 20...........................................................................4 ± 0..........33g KH2PO4 ..............01g Na2SeO3·5H2O ........0mL: NaHCO3 ......................................0.... Sparge with 100% N2............0mL......0mL Trace elements solution ..........5mg Fructose solution .........0mL of medium so that pH is adjusted to 7....................0..............................................................1.............................33g NH4Cl ............. Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O ................0L..........0mL L-Cysteine solution............. Mix thoroughly.............................. 1.........0mL: NaHCO3 .. Mix thoroughly..........................................................................3g Resazurin ..........0mL Na-syringate solution ............... 3................ Mix thoroughly. 0............0mL pH 7........................... Autoclave for 15 min at 15 psi pressure–121°C..........

.........01g Preparation of Trace Minerals: Add nitrilotriacetic acid to 500...............................................................................................0mg p-Aminobenzoic acid ........0g MnSO4·H2O..1g FeCl2·4H2O ......5g CoCl2·6H2O . Mix thoroughly........ 4..... 0.........1g CoCl2·6H2O..............0L.......0mL............................. L-Cysteine solution.................0mL Clarified rumen fluid ....................................................................... 20..................................... Autoclave for 15 min at 15 psi pressure–121°C....0mg NiCl2·6H2O .....4g NH4Cl........................................................ Sparge with 100% N2.20....1.......................0mL Na2S·9H2O solution......20............................ and Na2S·9H2O solution..........0mg Niacinamide .................................. Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.................................... Vitamin Solution: Composition per liter: Pyridoxine·HCl...........0...............................6g NaCl ............0mL of distilled/deionized water and KH2PO4 in 230...............................................0mg Na2MoO4·2H2O .0mg i-Inositol ...........01g CuSO4·5H2O........................0mg Pyridoxal·HCl...... Aseptically and anaerobically add per liter of medium...............0mL nents to distilled/deionized water and bring volume to 1..............72g solve K2HPO4 in 600.............0L with distilled/deionized water........................................... Stock Salts Solution #2: Composition per liter: Trace Elements Solution SL-10: Composition per liter: CaCl2·2H2O ..........................................0mg p-Aminobenzoic acid .............0mL of distilled/deionized water.........0mL seven vitamin solution..... Mix thoroughly........................................................0.......0mg Biotin ................0............ Mix thoroughly. 80........................................ Add remaining components..............................5g NaCl .... 200........1mg ......................0L.....................5................................................................................... fructose solution............0mg Ascorbic acid............5........................................................0mg Calcium pantothenate ..0mg Thiamine·HCl ............................5....0mL fructose solution..........................................10.............................................................0mg Nicotinic acid ..........2.........................................0..............0mL Stock salts solution #2 ............................................ 36........... Bring volume to 1...........0mg D(+)-Biotin ............................ 100.01g H3BO3 ...................0................ The final pH should be 7....................0g NiCl2·6H2O ....................................4mL Resazurin (0.........................................................................0......... Mix thoroughly...................... and 10..................................0mg MnCl2·4H2O............................15.......... Mix thoroughly......0mL of distilled/deionized water...........1....0............................................0.............................................................0mg CuCl2·2H2O .. to distilled/deionized water and bring volume to 929.................................... 100.........................4.....................0.. 200.....0mg Thiamine-HCl·2H2O ........ Mix thoroughly......5......2...................... acetigenic bacterium from tundra soils... Stock Salts Solution #1: Composition per liter: KCl ..........0mL Preparation of Stock Salts Solution #2: Add compoTrace Minerals: Composition per liter: Nitrilotriacetic acid...................Acetogen Medium 11 Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10........... Distribute into anaerobe tubes or bottles. Use: For the cultivation of Acetobacterium tundrae.... 6.......... 10.....01g Na2MoO4·2H2O.................. Filter sterilize........ Add components..4..............0L.......................5g MgSO4·7H2O.0mg Vitamin B12 ..................................1...............8mL: NaHCO3 .................................. 20.1% solution) ..................0g Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...0mL NaHCO3 30............... 2............ a psychrophilic...............0mg Riboflavin......3.....0mL: Preparation of Fructose Solution: Add glucose to distilled/deionized water and bring volume to 30.................2g Yeast extract............4g MgSO4·7H2O.......40................................ Mix thoroughly................................................ 0.................................0mg Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1....... 5...............5......................0mL Potassium phosphate buffer .........1g FeSO4·7H2O ............ 190..............0mL Tungstate solution ..................4.... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere.........0mg ZnCl2 ...........................0mg Calcium pantothenate .................................................. 70........................0mL Reducing agent ................................0mg Lipoic acid. Filter sterilize..............................................68g KH2PO4 ......................................................................................... Dissolve by adjusting pH to 6................0mg Folic acid ................................ Preparation of Potassium Phosphate Buffer: Dis- Glucose ......................1g Na2SeO3·5H2O ................................................................................................................ Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.............4mL Fructose Solution: Composition per 30........... Add remaining components..............1.............0........10....0L..........5...0mg Nicotinic acid . 24............................ 100............................................ 300..5............0................... 2..............0................0mg HCl (25% solution) ...........0mL of HCl solution.............0mL: Potassium Phosphate Buffer: Composition per 830...... Sparge with 80% N2 + 20% CO2..............................1g CaCl2 ......... Mix thoroughly........ 0. 1............... Autoclave for 15 min at 15 psi pressure–121°C.. Cool to room temperature.. Mix thoroughly.................................................................... Sparge with 80% N2 + 20% CO2..0mg H3BO3 ..0mL L-Cysteine-HCl·H2O solution................. 1............................................1g ZnSO4·7H2O................................... Sparge with 100% N2..............0mL...........................................................................5...............0....0mL..........................0..... 4....... Acetogen Medium Composition per 421..............................................................................................5 with KOH.................................................... Add distilled/deionized water and bring volume to 1........... NaHCO3 solution........................................................2g Preparation of Stock Salts Solution #1: Add components to distilled/deionized water and bring volume to 1....................................................... K2HPO4 ...... Mix the two solutions together and use..............0mg Choline chloride .0......... Vitamin solution ....01g AlK(SO4)2·12H2O ............... 0..............0mg Vitamin B12......................... Mix thoroughly................5................5.........0mL Trace minerals......5......... except seven vitamin solution.......2g Stock salts solution #1 .

...... 1.......... aseptically and anaerobically add vitamin solution.................. Dissolve by adjusting pH to 6... Yeast Extract Solution: Composition per 5.............................. Autoclave for 10 min at 15 psi pressure–121°C.........0L....... Autoclave for 15 min at 15 psi pressure–121°C..........0mL: Yeast extract .......2–7.............10.......0mL Yeast extract solution ...0mL....................5.0g NaCl ...............2.........0...................0 with KOH...............0mg Thiamine·HCl ..... 150................................................. Gently heat and bring to boiling.....0mL of dis- tilled/deionized water to a 250.......6 ± 0.......................................................... yeast extract solution............... Mix thoroughly.01g Na2SeO3·5H2O.................... Adjust pH to 7.0g MnSO4·2H2O.. 0.........5....... Mix thoroughly................0mL: Na2S·9H2O ................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......................................................................................................................................................... 0................................. Cool to room temperature.........................................0mg Folic acid .0mL: NaHCO3 ..........................33g Resazurin .........0mg Preparation of Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.... Mix thoroughly..................................................... Store under N2 gas....5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O solution to distilled/deionized water and bring volume to 5.......................0mL: L-Cysteine·HCl·H2O ................5g of glycine betaine in place of trimethylamine·HCl.................... One may use 4.. 0..... Allow to cool to room temperature under 100% N2....5........................................ Autoclave for 15 min at 15 psi pressure–121°C.................... Adjust pH to 7.............................. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C......0mL pH 7............................................. Store frozen......................... Mix thoroughly............... Distribute into tubes or flasks under 100% N2...............................4g Preparation of Trimethylamine·HCl Solution: Add trimethylamine·HCl to distilled/deionized water and bring volume to 10.......... 0...................................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500...........0mg Vitamin B12... Add remaining components......................... 0................................... 2....5g CoSO4·7H2O.. 4...1g NiCl2·6H2O....... trimethylamine·HCl solution......... Mix thoroughly........18g FeSO4·7H2O ..0g MgCl2·6H2O.....0mL round-bottomed flask.0.................... Cool to 45°– 50°C................ 0........ Cool to room temperature.......................... Add L-cysteine·HCl and dissolve... Mix thoroughly.......... NaHCO3 Solution: Composition per 5........................ 1. to distilled/deionized water and bring volume to 1.................................................................. Adjust to pH 9 with 5N NaOH............................ 0.. Distribute into tubes or flasks under 80% N2 + 20% CO2.........4 with NaOH.........0mL............ 2...0... Sparge under 100% N2 gas for 3 min............... Sparge under 100% N2 gas for 3 min...............33g KCl................0mL of distilled/deionized water...... Autoclave for 15 min at 15 psi pressure–121°C. except NaHCO3 and reducing agent.. Boil for a few minutes.....................................2.............. ..............0mg Riboflavin......................0mL: NaCl .................................... 99............... 1........................................10....................... Store under N2 gas............. Filter sterilize.10.2.... Mix thoroughly................0mg p-Aminobenzoic acid .. Tungstate Solution: Composition per liter: Na2WO4·2H2O .... except vitamin solution............0mg Trace elements solution ......01g Na2MoO4·2H2O..................0mg Calcium DL-pantothenate ........33g NH4Cl...0mg Nicotinic acid ................2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O. 0............................. Store under N2 gas...025g KAI(SO4)2·12H2O ............................... Vitamin Solution: Composition per liter: Pyridoxine·HCl........ to distilled/deionized water and bring volume to 417.......18g ZnSO4·7H2O ......... trimethylamine·HCl solution..................5................5g Preparation of Reducing Agent: Add 110........5....................0g CaCl2·2H2O............................ NaHCO3 solu- .0mL NaHCO3 solution .......33g KH2PO4 ..................................... Store under N2 gas................ Na2S·9H2O Solution: Composition per 5..0mg Lipoic acid..................... Autoclave for 15 min at 15 psi pressure–121°C........5.................... exchange headspace with 80% H2 + 20% CO2... Sparge under 100% N2 gas for 3 min.................0g Nitrilotriacetic acid ...............................0...............0mL Trimethylamine·HCl solution ...................... Mix thoroughly...0mg Biotin .........5.................... and Na2S·9H2O solution.. 0................................0mL Vitamin solution.............................10............................ 0.............................. Mix thoroughly.8mL.................................. Boil under N2 gas for 1 min.....................0mL..............0L......... Use: For the cultivation and maintenance of acetogenic anaerobes such as some Clostridium species...0L..................... yeast extract solution... Add distilled/ deionized water to 1............ Add washed Na2S·9H2O and dissolve............... Distribute in amounts needed into tubes or flasks........ Autoclave for 15 min at 15 psi pressure– 121°C................. NaHCO3 solution................01g H3BO3 .........5..5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 5...............5...................... Preparation of Medium: Add components......... 0.........0L.... 0....02g CuSO4·5H2O......................... Add NaHCO3 and reducing agent..5 g CaCl2·2H2O ...0L.05g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 5.... Before inoculation.......................0mL: Trimethylamine·HCl........... Reducing Agent: Composition per 110.. 0... Gently heat and bring to boiling under 80% N2 + 20% CO2.........0mL Na2S·9H2O solution................................... Acetohalobium Medium Composition per 1025................1..... 3...5 with KOH..............12 Acetohalobium Medium Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.................. 0.......................................... After inoculation................ Trimethylamine·HCl Solution: Composition per 10....................... Preparation of Medium: Add components..............5g Na2S·9H2O... Sparge under 100% N2 gas for 3 min........0mL......................

.0 ± 0................... 0.................................................................. Preparation of Medium: Add components.0mL............4g NH4Cl...... Mix thoroughly................................... Adjust pH to about 7........................... including Rhodomicrobium vannielii and Rhodopseudomonas acidophila.................. 0..0mL Vitamin B12 solution ........ lactate.......25g Yeast extract..................................................0..............................02g CuCl2·2H2O.. Store under N2 gas.......................................... Autoclave for 15 min at 15 psi pressure–121°C.............. Sparge under 100% N2 gas for 3 min.Acid Tomato Broth 13 tion.............. aseptically and anaerobically add 0...5g (NH4)2SO4 ..1g .....0g K2HPO4 .... Distribute into tubes or flasks..............2g CaCl2·2H2O........................... Use: For the cultivation of Thiobacillus organoparus....................... Autoclave for 15 min at 15 psi pressure–121°C........................................................0................. Cool to room temperature.7 ± 0.0mL L-Cysteine solution........................ to distilled/deionized water and bring volume to 1050........................ Mix thoroughly. Acid Tomato Broth Composition per liter: Ferric citrate ...2g ZnSO4·7H2O........................7H2O ...................................................2................................. use a sterile syringe.......................... 0............4g Disodium succinate.......... Adjust pH to 6......................................0mL........................... 0..........0mL Trace elements solution SL-6 ................. Mix thoroughly................. 2....... Cool to room temperature.......4 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................0L.... Acid Rhodospirillaceae Medium Composition per 1050........................................ except neutralized sulfide solution.........................15g KH2PO4 ............... 5.... Boil for 3–4 min under a stream of 100% N2....................................05g Ferric citrate solution . Autoclave for 15 min at 15 psi pressure–121°C..................................................................03g NiCl2·6H2O............. 50......0... which is capable of chemolithotrophic growth on H2 + CO2 or CO................. 0.. Neutralized Sulfide Solution: Composition per 100............0L........................0mL of the prepared medium into 50......5mL Preparation of Ferric Citrate Solution: Add ferric L-Cysteine Ferric Citrate Solution: Composition per 10.........1.............. If necessary....... Stir the solution continuously to avoid precipitation of elemental sulfur................ 0...................... Distribute into tubes or flasks....... 2...... min................ Mix thoroughly........................... 1...............0mL.....1g KCl............ 0.3g CoCl2·6H2O...........................0mg Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 100.............................. 10....1g Na2MoO4·2H2O............05g Tomato juice ....................... Sparge under 100% N2 gas for 3 min............0mL: citrate to distilled/deionized water and bring volume to 10..... Before inoculation..................................... and of organotrophic growth on betaine.........1.......................................................0g Yeast extract...... 10.4g NaCl .............. etc...................2g MnSO4·4H2O ......1.... 20......................................25–0....01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0mL screw-capped bottle fitted with a butyl rubber septum and bring volume to 100........... 10............. Distribute 45..........................0L.......................10...................................0mg Ca(NO3)2 .............0...........................0g MgSO4·7H2O ............. Add a magnetic stir bar. Do not open the bottle to add H2SO4...................................................... Mix thoroughly.................. 0. 0..................................................................0g Triammonium citrate...0mL...............4mL Neutralized sulfide solution ................ Use: For the cultivation of bacteria that can utilize Nacetylglucosamine...................... Mix thoroughly...............10........... and Na2S·9H2O solution............0g Yeast extract .............0.....................................5g KH2PO4 .............50mL of neutralized sulfide solution.......... Mix thoroughly.......... adjust pH 7.......................0.0mL: Na2S·9H2O ........0mL: L-Cysteine. Use: For the cultivation and maintenance of Acetohalobi- Trace Elements Solution SL-6: Composition per liter: um arabaticum..... of methylotrophic growth on trimethylamine.............. Acetylglucosamine Medium (N-Acetylglucosamine Medium) Composition per liter: N-Acetylglucosamine........... 0...................................................... 0.0g Beef extract .........6 to with sterile anaerobic Na2CO3 solution..... 10...2 ± 0......2g MnSO4·4H2O ..10.... 250...........................................................................0mg pH 3........................... Use: For the cultivation and maintenance of members of the family Rhodospillaceae...0..............................0 mL: Ammonium acetate ...5g Preparation of Neutralized Sulfide Solution: Add Na2S·9H2O to distilled/deionized water in a 250..2 at 25°C MnCl2·4H2O ................... Sparge under 100% N2 gas for 3 Solution: Composition per 10....... The final solution should be clear and yellow in color..........................................0g Peptone.....0g Peptone ..................................05g Tween™ 80 ......5g H3BO3 ...... 0..........................5... Store under N2 gas......................................... Vitamin B12 Solution: Composition per 100......0..........................0mL screw-capped tubes that have been flushed with 100% N2...3 with sterile 2M H2SO4................... Autoclave for 15 min at 15 psi pressure–121°C..................................................... Acid Glucose Salts Medium Composition per liter: Glucose .0mg Glucose..................5...... 5.0g MgSO4·7H2O .......................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...............................................................0....... 0....................................... Autoclave for 15 min at 15 psi pressure–121°C.................................. variable pH 5............ 0..... Gently heat and bring to boiling........ Autoclave for 15 min at 15 psi pressure–121°C...............0mL pH 6..........0mL: Vitamin B12.......................5g MgSO4....................................................... 0.0g MgSO4·7H2O .........

...............0mL.............20..........2..............1....................................0mg MnCl2·4H2O...........................................................0........... 0..........................................................................1g Tween™ 80 ......................0g Arginine...... Mix thoroughly...............................................................................4 ± 0..............................5g DL-Tryptophan............................... 5.....0mg Calcium pantothenate ............................0L.......... to distilled/deionized water and bring volume to 999.......................................................................... NaHCO3 solution..................1............... 5.0mg Calcium DL-pantothenate..... 24.......... 1.................0..............................0g Pancreatic digest of casein ...........................................5..2g CaCl2·2H2O............. Acidaminobacter Medium Composition per liter: NaHCO3 ............. 5...................................... 0..................................2g Na2SO4 .0g Glycine .........................0.................................................0mg CuCl2·2H2O ....... 0........................5..................................... except vitamin solution...........3g KH2PO4 .4g MgCl2·6H2O....................... 2..1g of Lcysteine to distilled/deionized water and bring volume to 10..............5.....1g Guanine .....1........................0mL Na2S·9H2O solution..0L.............0.2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................. Aseptically distribute into sterile tubes or flasks.... Store under N2 gas............ 2............... 5................................................................................................................0mL NaHCO3 solution ....... 50. Mix thoroughly.... a fermentative bacterium from estuarine mud...........5..................... Add remaining components.... Autoclave for 15 min at 15 psi pressure–121°C.............0mg Nicotinic acid ............. Before inoculation.......................0g Sodium glutamate............. Mix thoroughly..............0mg Vitamin B12 ..................................................................... Sparge under 100% N2 gas for 3 min..............................................0g Yeast extract ..... 36............................................................. to distilled/deionized water and bring volume to 1............... Add distilled/deionized water and bring volume to 1..............................................................0.....................0mL pH 7......................................................0µg Vitamin solution...... 0. Mix thoroughly..........0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10........0....................................................................... Mix thoroughly...... Autoclave for 15 min at 15 psi pressure–121°C............. Filter sterilize................... Mix thoroughly...............................................................14 Acidaminobacter Medium Preparation of L-Cysteine Solution: Add 0.......................... 70........0g L-Cysteine·HCl ......... 5................................4.................... Na2S·9H2O Solution: Composition per 5. Acidaminococcus fermentans Medium Composition per liter: Casamino acids..................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O solution to distilled/deionized water and bring volume to 5..0mg Riboflavin ......1. Mix thoroughly... Mix thoroughly.......................0g Glucose......................................... 5.....0mg Na2MoO4·2H2O ......................... 0....0mg Thiamine.......2g KCl....................................0mg Folic acid.0L............0mg NiCl2·6H2O ........... Use: For the cultivation and maintenance of Acidaminobacter hydrogenoformans.......5........ 50............................ Autoclave for 15 min at 15 psi pressure–121°C....... 0.............2g Yeast extract............................................0µg p-Aminobenzoic acid ........................................... Mix thoroughly...... Sparge under 100% N2 gas for 3 min............... Distribute into tubes or flasks......0mL: Na2S·9H2O .......... 0....... 50..................0mg Biotin .5mL pH 7.... 1..........5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 5...15g Resazurin .............................................5mL of sterile L-cysteine solution..0g L-Cysteine·HCl·H2O.........0mg Lipoic acid ......................5...........................0mg Thiamine·HCl ............................. Allow to cool to room temperature under 100% N2......... Cool to room temperature........0g Glycine................35g DL-Tryptophan..................................8............0mg HCl (25% solution) ........................30................4.... Mix thoroughly.........................0mg H3BO3 ........... Mix thoroughly......................................... Autoclave for 15 min at 15 psi pressure–121°C............................................ 190..2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .................... Distribute into tubes or flasks under 100% N2.... aseptically and anaerobically add vitamin solution................ Adjust pH to 7....... 10.....................................................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.. 0........0µg Niacin .... 10......................... 1........... Adjust pH to 4...........10..0mL................ NaHCO3 Solution: Composition per 5.......................................... 2........................ 2........................................................ Use: For the cultivation of a variety of fungi..0 ± 0................0mg Na2SeO3·5H2O ......................................... Check that final pH is 7................................................0L..... Vitamin Solution: Composition per liter: Pyridoxine·HCl ......................................0g Glucose.................................... 100........................ 2................. and Na2S·9H2O solution.................................................... NaHCO3 solution.....5g CoCl2·6H2O ................... Gently heat and bring to boiling............. Aseptically add 0..............0µg Biotin .............0mL Trace elements solution SL-10 ..................0mL....... and Na2S·9H2O solution....................01g Hypoxanthine .......1......................................................................................................10..............01g Uracil .............01g Pyridoxal .....5mL................0g KH2PO4 .............................. Store under N2 gas.0.......... 6...................................................0mg p-Aminobenzoic acid .... Autoclave for 15 min at 15 psi pressure–121°C......... Preparation of Medium: Add components......................................0mg ZnCl2 ..........0mL: NaHCO3 ..................0......................................0µg ......................... Preparation of Medium: Add components....................................................................... Acidaminococcus Medium VR Composition per liter: Acid-hydrolyzed casein (vitamin and salt free) .... Boil for a few minutes... except Lcysteine solution.............. Use: For the cultivation and maintenance of Acidaminococcus fermentans from rumen.................... 0..... Filter sterilize.... 0....10..................0............................ 1.........5g NaCl ......4g Na2S·9H2O....................................0µg Riboflavin...................................0mL of HCl solution.

.. Preparation of Medium: Add components..........0mg Ferric citrate solution ....................................................................................10..5g Preparation of VR Salts A: Add components to dis- tilled/deionized water and bring volume to 500............. 0......................... a facultatively anaerobic thermoacidophilic archaean..........Acidic Rhodospirillaceae Medium Folic acid............................... 1...........2 at 25°C VR Salts A: Composition per 500.................................5g KCl....... 0.......02g Na2B4O7·10H2O .............................0L.......................................................................22mg CuCl2·2H2O.......0g Sulfur flowers .......................0mL.....0.............................................. Aseptically ..................... Autoclave for 15 min at 15 psi pressure–121°C...............0..............0.......... 3............0mL VR salts B ...........25g CoCl2·6H2O .............................. 37.............................125g Preparation of VR Salts B: Add components to dis- tilled/deionized water and bring volume to 700.....4g NaCl .........0µg Vitamin B12 ................................... except sul- fur flowers and yeast extract solution...... 12.1....................25g MnSO4·H2O .....................................................1...0g K2HPO4·3H2O.25g CuSO4·5H2O . Acidianus brierleyi.......... Adjust pH to 1.....0µg VR salts A .................. 0.................................. Adjust pH to 2.8mg ZnSO4·7H2O. Filter through paper..4g NH4Cl ..............01mg pH 2................... to distilled/deionized water and bring volume to 990. 5.................. Acidianus brierleyi Medium Composition per liter: Sulfur flowers ..............................4...................................0........0g of nitrilotriacetic acid to 300..............0....................... VR Salts B: Composition per liter: MgSO4·7H2O ................. Use: For the cultivation and maintenance of Acidianus brierleyi................... Add distilled/deionized water and bring volume to 1.................1........................................................... Mix thoroughly........................0mL: Yeast extract.....5g KH2PO4 ...................... Mix thoroughly....................5 with 10N H2SO4... Add 5..................................... 24.............................................................0................ Gently heat and bring to boiling.....................................................................5g MgSO4·7H2O....3g CoCl2·6H2O.................5 at 25°C Yeast Extract Solution: Composition per 10............................................0mL of concentrated HCl and heat until dissolved........................................ Acidianus infernus Medium Composition per liter: (NH4)2SO4 .4..........................0 ± 0...0....07g FeCl3·6H2O.....................................03g NiCl2·6H2O....0mL.. Slowly add the nitrilotriacetic acid solution to the larger volume of salt solutions while stirring until dissolved......................................... sterile sulfur flowers................ Mix thoroughly........ 0...............5 ± 0................ Distribute into tubes or flasks....................................0................. Preparation of Medium: Add components.......3g Yeast extract ................... except ferric citrate solution and trace elements solution...........0........................ Use: For the cultivation and maintenance of Acidaminococcus fermentans from rumen..................................................0........................ Mix thoroughly..0....0mL: Ferric citrate ............... 0..................05mg Na2MoO4·2H2O.......0mL pH 1......2g CaCl2·H2O . Filter sterilize............................................. Autoclave for 15 min at 15 psi pressure–121°C............. Mix thoroughly........ Preparation of Medium: Filter sterilize vitamins as separate solution...............0g KH2PO4 .......................................................... 0....................1......................................................................................................................................1g MnCl2·4H2O ......... If necessary......................5g FeSO4·7H2O.....0L..... Acidic Rhodospirillaceae Medium Composition per 1006.......1..........03mg CoSO4 ........................25g Na2MoO4·2H2O ...............0g KH2PO4 ...........0mL distilled/deionized water...............5mg MnCl2·4H2O ................................................5–2......... Mix thoroughly...........................0g (NH4)2SO4 ...1..................0mL....0.............1g Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 100........... Stir vigorously............03mg VOSO4·2H2O .......0L.4g Yeast extract .....0......................25g VSO4·7H2O.....0g CaCl2·2H2O.....5 with 6N H2SO4.................. Prepare and distribute medium anaerobically using Hungate techniques with 100% N2 gas....... Filter sterilize.......................................02g CuCl2·2H2O...... 0.....................30..... Trace Elements Solution: Composition per liter: H3BO3 .... 10............................................. 0....................0.......................................... Acidianus infernus...........................0mL............................50.................0..........................1g Ca(NO3)2 .............0....................................01g Yeast extract solution ......................................................... Mix thoroughly......5g MgSO4·7H2O ............................0.28g MgSO4·7H2O..........................0.. adjust pH with solid K2CO3 to 7.0mL Ferric Citrate Solution: Composition per 100..... Aseptically com- 15 bine the basal solution.........0.... 0................. and sterile yeast extract solution................................................ Autoclave for 15 min at 15 psi pressure–121°C.................. Add 2.... to distilled/ deionized water and bring volume to 1.................0.................................................................... and Desulfurolobus ambivalens............03g Na2MoO4·2H2O..................................................2g ZnSO4·7H2O......................................0.....0mL pH 7......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.25g CaCl2·2H2O ............................................................................. Store in a cool place........................0mL: Disodium succinate ................................................. 0.........1.....0............... Sulfur flowers are sterilized separately by steaming for 3 hr on 3 consecutive days.........0............0mL. Adjust pH with 10N NaOH to 7.5g ZnSO4·7H2O ..........................2g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.......0L.....0mL Trace elements solution...... Add aseptically to sterile basal medium.... 0........................ Use: For the aerobic cultivation and maintenance of Acidianus infernus...... 0.................................. Autoclave for 15 min at 15 psi pressure–121°C..........................................0mL: Na2HPO4 ..........0..................................01g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1....... 0........5–2........

.......... Yeast Extract Solution: Composition per 10......................1g pH 3.9g MgSO4·7H2O ..................... Preparation of Medium: Add components................... Distribute into tubes or flasks................... 0.......................................5-3...0...... 15........ Before inoculation....0..33g CaCl2·2H2O ..................0g Peptone..............5g (NH4)2SO4 . Adjust pH to 3...........................1g Yeast extract .. Mix thoroughly...................................... 0..................... aseptically add glucose solution and yeast extract solution....................... Mix well and warm gently until dissolved..........5g MgSO4·7H2O......................................0 ± 0....... Aseptically add 20.2g KCl............ Use: For the autotrophic cultivation of Sulfobacillus acidophilus..........0 using 1N H2SO4........................... Mix thoroughly....250...............8 ± 0..........................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10............................. Autoclave for 15 min at 15 psi pressure–121°C.................................................1g pH 1..........0g MgSO4·7H2O ...........0mL pH 3...... 15. Acidobacterium Medium Composition per liter: (NH4)2SO4 .................... 5.5g KCl ....................0..........7 with H2SO4.............. Mix thoroughly.0 with H2SO4............. Autoclave for 15 min at 15 psi pressure–121°C..0g Glucose ..................4g K2HPO4 ..........................................0mL of distilled/deionized water......................................................... except yeast extract solution.............33g KH2PO4 .. 10.........2g MnSO4·4H2O ......................................................... 0............. Use: For the cultivation of Rhodopseudomonas acidophila.................................................................................... 1.0mL.....................................................................................................0L.........................0mL..................... to distilled/deionized water and bring volume to 980. 0...............0..............10..........................................5g (NH4)2SO4 .0.....................2...0mL: Yeast extract............................... Aseptically distribute into sterile tubes or flasks.......................... Use: For the heterotrophic cultivation of Sulfobacillus aci- Acidiphilium Medium Composition per liter: (NH4)2SO4 ...............1....0mL of sterile trace elements solution........ Adjust pH to 3......5g KCl ...... 0.... Autoclave for 15 min at 15 psi pressure–121°C.......0 ± 0...........7 ± 0................ 10. Preparation of Medium: Add components...........0mL: Yeast extract .................................0g K2HPO4 ................... 10.... Mix thoroughly.....0mL: D-Glucose ............................. Distribute into tubes or flasks..........0...... Acidimicrobium Medium Composition per liter: Acidolobus aceticus Medium (DSMZ Medium 901) Composition per 1055mL: FeSO4·7H2O...0.............. Adjust pH to 2.. Filter sterilize............................ Cool to room temperature.......0mL Vitamin solution ...................................4g K2HPO4 ............1g FeSO4·7H2O..............0mL Na2S·9H2O solution .......................... Autoclave for 15 min at 15 psi pressure–121°C......2 at 25°C Preparation of Medium: Add solid components to 750........... Mix thoroughly...... Acidimicrobium Medium Composition per liter: MgSO4·7H2O ............... 13......................................................................1.............................................. Autoclave for 15 min at 15 psi pressure–121°C.......0.....................0mL. 10.....0g Yeast extract....8 at 25°C ......3g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10............ Add tomato juice........................................................... Pour into sterile Petri dishes............ 0.. 10....2g KCl............................................................................ 0............................. Mix thoroughly.......5 ± 0................................ Gently heat and bring to boiling............ Mix thoroughly..................................05g Tomato juice..0.....33g MgCl2·6H2O ................... Sulfur......................................... 0........... Autoclave for 15 min at 15 psi pressure–121°C...................0mL Yeast extract solution ........ 10..................2.. Mix thoroughly....................... Use: For the cultivation and maintenance of Leuconostoc oenos and other Leuconostoc species...............0L........ powdered .......16 Acidic Tomato Medium for Leuconostoc add 5. Autoclave for 15 min at 15 psi pressure–121°C..0mL pH 2....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................................................................0g NH4Cl ..... Use: For the cultivation of Acidobacterium capsulatum..33g KCl .33g Resazurin ..5g MgSO4·7H2O..................................................0mL of sterile yeast extract solution.... 0...0mL.... except glucose solution and yeast extract solution.........0................ Mix thoroughly........................0mL pH 3............................. Adjust pH to 1...................................................0.........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................... Aseptically distribute into sterile tubes or flasks........5 with H2SO4....1g Glucose solution ...............0.... Mix thoroughly..................................................5mg Yeast extract solution . 10.................0.........0mg Yeast extract solution ................0mL.0...................................... Acidic Tomato Medium for Leuconostoc Composition per liter: Agar ....................................20..........2 at 25°C Yeast Extract Solution: Composition per 20.0g K2HPO4 ........................... Use: For the cultivation and maintenance of Acidiphilium cryptum.............................0mL of sterile ferric citrate solution and 1......................... 0....................................0g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 20...................2 at 25°C Glucose Solution: Composition per 10..............0mL pH 4.............................................. dophilus......0mL Trace mineral solution SL-10................... 30.0g Glucose................................ to distilled/deionized water and bring volume to 1080.............

.............. Solution A .................. to distilled/deionized water and bring volume to 1.....8........................................ 24....0mL of solution A with 500...0........................... Adjust pH to 3............1mg Preparation of Medium: Aseptically mix 500.......................................... Sparge with 100% N2............. Mix thoroughly.... Heat to 90°C for 1 h on each of 3 successive days.... ... Na2S·9H2O solution............0mL..........20... Pour into sterile Petri dishes or distribute into sterile tubes... Mix thoroughly............... Mix thoroughly... 3..............................................5-3............... Mix thoroughly... Preparation of Medium: Prepare and dispense medium under 100% CO2.0mL: Agar ........................................5 with H2SO4........................ an acidophilic facultatively methylotrophic bacterium....0L.0g Yeast extract . Mix thoroughly................... Add distilled/deionized water and bring volume to 1........ 2..... Mix thoroughly.. Autoclave for 15 min at 15 psi pressure–121°C..........5g CoCl2·6H2O ................ 6. 500.............0mL: Yeast extract...........................0mg Folic acid................. Solution B: Composition per 500...............................0mL...............5.........0g Malt extract ...................... 0... Cool to 50°–55°C....0L.......0g MgSO4·7H2O ..............0mL: Glucose..........7g NaCl .... Mix thoroughly...........6g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20.....................2 at 25°C Solution A: Composition per 500......0g KH2PO4 .....0mg Na2MoO4·2H2O ......... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.................................... Na2S·9H2O Solution: Composition per 20................... 500................0............... 5......0mL of solution B...........0mL: Glucose..............3................3........................................................................... Sparge with 80% H2 + 20% CO2...0mL Solution B..............................................0mL........16g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500.......20. 1..................0g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1..............................................0g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 30... Adjust pH to 4......... 100...........0mL pH 4.........4g K2HPO4·3H2O ....0................................................................... 190.............................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.....0mL Solution B..... Distribute to anaerobe tubes or bottles.....0 ± 0........... Filter sterilize.. 36........10.................0L......... Neturalize to pH 7... 10..............0g (NH4)2SO4.....0mg Lipoic acid ................... The final pH should be 3.....................................0mL Solution A: Composition per 500................................ Use: For the cultivation and maintenance of Acidomonas methanolica.0mL..... Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly.......................................................... 2.................... Vitamin Solution: Composition per liter: 17 Acidomonas Agar Composition per liter: Solution A ..... Use: For the cultivation and maintenance of Acidomonas methanolica..............................10............ 2..................0mg ZnCl2 ...... Mix thoroughly..................... 70......0mg MnCl2·4H2O.....0mg NiCl2·6H2O .. Cool to 50°–55°C.........0mL of solution A and 500..................0mg Vitamin B12 ... 5........0................................0L...............................0mg Biotin .............0mg Thiamine-HCl·2H2O ............................... 5......... Sparge with 80% N2 + 20% CO2... Preparation of Medium: Aseptically mix 500....1................... Mix thoroughly........................0................ Preparation of Vitamin Solution: Add components to Acidomonas methanolica Agar Composition per liter: distilled/deionized water and bring volume to 1........ tilled/deionized water and bring volume to 500..................................0mL sterile yeast extract solution....... 10............... except vitamin solution........... Pour into sterile Petri dishes or leave in tubes............. Filter sterilize.......0mg D-Ca-pantothenate.... Solution B: Composition per 500.........0g Peptone ..... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.... Add components...0g Yeast extract ................ and yeast extract solution............ Mix thoroughly... 5...........0mL: Agar ..................0mg Nicotinic acid .......................... Cool to room temperature...... Use: For the cultivation of Acidilobus aceticus.............................................................. Autoclave for 15 min at 15 psi pressure–121°C.......0mL of HCl solution.. Cool to room temperature.............. Cool to 50°–55°C..... 500....... an acidophilic facultatively methylotrophic bacterium.. Add remaining components............. a novel anaerobic thermoacidophilic archaeon from continental hot vents in Kamchatka.0......0mg Riboflavin ...5g Ca(NO3)2·4H2O ...............................0g Preparation of Solution B: Add 20............. 15....... Gently heat and bring to boiling...20...............0mL sterile vitamin solution. Aseptically and anaerobically add per liter of medium.......................0mg HCl (25% solution) ........ Gently heat and bring to boiling.....................0mL of solution B............... Mix thoroughly.............................0mg H3BO3 ...5....... Autoclave for 15 min at 15 psi pressure–121°C............Acidomonas methanolica Agar Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ..0 with HCl............................................................0mL...........................0mL: Na2S·9H2O................................. 5..0mg CuCl2·2H2O ...0mL of sterile Na2S·9H2O solution and 30..................................................... Aseptically adjust pH to 4................0g of agar to dis- Pyridoxine-HCl .....................3........................... Yeast Extract Solution: Composition per 30. Cool to 50°–55°C.......................................................0mg p-Aminobenzoic acid ..............................................................0g Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 500.......... 0................. 500........... 5...................

......... Cool to 50°C........................................................... Adjust pH to 7................. 5....... Adjust pH to 4.................0mg ZnSO4·7H2O..............................................0g Pancreatic digest of casein .1................0L of distilled/deionized water.........................0mg CoCl2·6H2O..............0mL pH 5.......................... Pour into Petri dishes or leave in tubes..........200.............................................................900......................0mL of solution B................................................................................................ 0......................................................................................... Mix thoroughly.. Autoclave for 15 min at 15 psi pressure–121°C......................3.....0L.................1g KCl ..........0g Na2HPO4·12H2O ............................0mL Part A ..... Autoclave for 15 min at 15 psi pressure–121°C............................0g Na2SO3 .................. Mix thoroughly.1 at 25°C Trace Elements Solution TS2: Composition per liter: Na2MoO4·4H2O.....10................... Solution B: Composition per 500.....................0g KH2PO4 ...........0mL..............0mL: Agar .............0mL............... 0.............. 12....0g Na2HPO4 ....................................10.......................... to distilled/deionized water and bring volume to 900.. 50.......... Bring pH to 3..............................0mL: Soluble starch...........15...................................0g KH2PO4 .......................0mL pH 7.... Activated Carbon Medium (DSMZ Medium 811) Composition per liter: Agar ....5g MgSO4·7H2O..........................................................0g KH2PO4 ....50.....5g NH4Cl ..0mL: Mannitol........ 1..................................... 1...... Part B: Composition per 600........0..........400.. 5........2 at 25°C Preparation of Medium: Dissolve all components in 1.5 ± 0.0mg CuCl2·2H2O................... Solution A .. Dextrin.....5........... Gently heat and bring to boiling..5.........................2 at 25°C ...0L with distilled/deionized water............ Use: For the cultivation and maintenance of Bacillus stearothermophilus and other acidophilic Bacillus species.................... 5.................5g cus.18 Acidophilic Bacillus stearothermophilus Agar Acidophilic Bacillus stearothermophilus Agar Composition per liter: Part B .......................1...7................ Pour into sterile Petri dishes...........5g MnCl2·4H2O....0mL of solution A with 500................................. Gently heat and bring to boiling................................................................. 1.20............20........................0g Pancreatic digest of casein ................................................................................ 10...........................0mL Solution B ...............................0g CaCl2·2H2O...........................0mL...0............... Cool to 50°C.....................................0mg H3BO3 ......... Cool to 50°C...............1......................0g CaCl2·2H2O..................9............................0.............................5............. Mix thoroughly.15......................0g Preparation of Part B: Add agar to distilled/deionized water and bring volume to 600....................... 0....... Autoclave for 15 min at 15 psi pressure–121°C............. Autoclave for 15 min at 15 psi pressure–121°C..................................... except acti- vated carbon.............0mg Preparation of Solution B: Add components to dis- tilled/deionized water and bring volume to 1...0mg MnCl2·4H2O ............................13.....................2g CaCl2·2H2O ........5 Autoclave for 15 min at 15 psi pressure–121°C...................500. Mix thoroughly.....0............................0mg Ca(NO3)2 .............................. 10......75g NaCl ..........0mL.......................................0mg KH2PO4 .........0g Activated carbon .......... 20.......................0 ± 0...................... After all components are dissolved add activated carbon............. Distribute into tubes or flasks..............0mL: Aeromonas Differential Agar (Dextrin Fuchsin Sulfite Agar) Composition per liter: Agar ..600.........5g MnCl2·4H2O.................. Mix thoroughly........... 5.............................6g Acid Fuchsin solution......... Autoclave for 15 min at 15 psi pressure– 121°C..............0g Agar .................. Preparation of Medium: Aseptically combine 500....0mg NiCl2·6H2O...................5g pH 5.................7......................................5g Preparation of Part A: Add components to distilled/ deionized water and bring volume to 400..............................................0mg Na2SeO3............................................................10..................50...................................................................... Bring volume to 1................0mL pH 3.............. Use: For the cultivation and maintenance of Bacillus stearothermophilus and other acidophilic Bacillus species.......................................... Gently heat and bring to boiling. Use: For the cultivation and maintenance of Acidiphilium cryptum and other Acidiphilium species..............................30...........1...........0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 1..........0g Pancreatic digest of casein . 0..... 0...........................................0g Beef extract ..........................5 ± 0................ Mix thoroughly..0g Yeast extract.0 ± 0..2 at 25°C Part A: Composition per 400.............................................. Acidophilium Agar Composition per liter: (NH4)2SO4 ................0g Yeast extract.................. Cool to 50°C........................................................... Pour into sterile Petri dishes or aseptically distribute into sterile tubes.......... Adjust to pH 5.......................300.............500....................2mg Trace elements solution TS2 . Adjust pH to 5............0mg NH4-Fe-III-Citrate...................1g Tryptone soya broth............... Preparation of Medium: Aseptically combine solution A and solution B.........2 at 25°C Use: For the cultivation of Streptomyces thermoautotrophi- Solution A: Composition per 500...........................................0L.5 ± 0........................... Acidophilic Bacillus stearothermophilus Broth Composition per liter: Soluble starch......0g Preparation of Solution A: Add agar to distilled/deion- ized water and bring volume to 500.......................... Mix thoroughly...................0g MgSO4·7H2O .................................... Preparation of Medium: Add components................................ Mix thoroughly......... 1......20........................................................................0mL pH 7............100....0................

...........02g pH 6........... Aeromonas Medium (Ryan’s Aeromonas Medium) Composition per liter: Agar ................ Specimens with low numbers of Aeromonas may first be enriched by growth in starch broth for 4–9 days........8.......1 at 25°C Source: This medium is available as a dehydrated powder from Oxoid Unipath........... Autoclave for 12 min at 15 psi pressure–121°C....15...................................4g Bromcresol Purple ................................ 3..5g Biotin solution .... 3...... Gently heat while stirring and bring to boiling................................... 5% .............. 2....................0g Mannitol...............0mL: Biotin ................5g Na2S2O3.........N´. Distribute into tubes or flasks......6 ± 0.....................25g Aquesou dioxan........ phila.. Use: For the cultivation and maintenance of Agrobacterium rhizogenes and Agrobacterium tumefaciens...................................................................................5mm)........50.....25g Preparation of Medium: Add components to distilled/ Preparation of Medium: Add components to tap water and bring volume to 1...................................0............................ Aeromonas species appear as small (0.......... A positive Nadi reaction (dextrin degradation) is indicated by a purple color at the periphery of the colony.... Mix thoroughly........0g Inositol ............... 5.............. 3......... 10.1mL pH 6.......................... Mix thoroughly............................... Agrobacterium rhizogenes................................. Gently heat and bring to boiling..............................................................2...................... 10..............................7........................................ 0..0g (NH4)2SO4 ...................................6 ± 0.0g NaCl ...67g Proteose peptone ......................0........................................... 12......0g NaCl ............................ 2..0...........0mL: Acid Fuchsin ............... 3............ Mix thoroughly.. Gently heat and bring to boiling... Use: For the isolation and cultivation of Aeromonas hydro- Use: For the cultivation and maintenance of Agrobacterium azotophilum......Agrobacterium Agar with Biotin Acid Fuchsin Solution: Composition per 50................ Caution: Acid Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contamination of the skin................................................................................ 3.. 3.........2 at 25°C Biotin Solution: Composition per 10...........10...................................................... Mix thoroughly............. 10.............75g L-Lysine·HCl......................................................................................5...........0g Ferric ammonium citrate.... 0.. Agrobacterium tumefaciens.................................... Autoclave for 15 min at 15 psi pressure–121°C.0g KH2PO4 .........................................0g Sorbitol........0g Pancreatic digest of casein .........0g (NH4)2SO4.................... Adjust pH to 6. deionized water and bring volume to 1...........................0g NaCl ...8.................................. Distribute into tubes or flasks.....0g Yeast extract..0mg of ampicillin......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....................................2 at 25°C Agar . Autoclave for 15 min at 15 psi pressure–121°C.....2mg ...........................................04g pH 8........................0g Glucose.................... Gently heat until dissolved...................... Dextrin fermentation is also indicated by red colonies...............0g Yeast extract ........................................... Mix well to dissolve...... Pour into sterile Petri dishes or leave in tubes... Agrobacterium radiobacter.........................0g Yeast extract .....................0L................0g Yeast extract .......................................5–1... dark red colonies with a purple periphery... 1........5..........15........................................ dark green colonies with darker centers...................................15........................5g Ferric ammonium citrate....... 3............. Pour into sterile Petri dishes or leave in tubes...........2..........................10..............0g Xylose ................................... Do not autoclave... Aeromonas species appear as large............... 5....................................1........................0mL volumes................. Distribute into tubes or flasks.5g pH 6.......N´-tetramethyl-p-phenylene-diammonium dichloride).............. Distribute into tubes in 5..... 3.. Pour into sterile Petri dishes.....0g Mannitol ......... Agrobacterium Agar Composition per liter: Agar ..............8g Agrobacterium Agar with Biotin Composition per liter: Agar . 5............ 19 Bromothymol Blue ...............................N..............6.................................. Preparation of Medium: Add components to distilled/ Use: For the isolation and selective differentiation of Aeromonas hydrophila and other Aeromonas species..... species from other Gram-negative rods such as Pseudomonas and Enterobacteriaceae................................0........................ 0... Autoclave for 15 min at 15 psi pressure–121°C.....0...0g Proteose peptone ................. colonies are sprayed with Nadi reagent (1% solution of N............................. Gently heat and bring to boiling.......0mL Preparation of Acid Fuchsin Solution: Add Acid Fuchsin to 50.................................. Pour into sterile Petri dishes or leave in tubes...................0g Casamino acids.................. Mix thoroughly.....0g L-Ornithine·HCl ...5...5...................................................... 0.............................................................................................................. After 24 hrs of growth on this agar.0g Lactose ............. Cool to 50°C and aseptically add 5..........0g (NH4)2SO4..............................0g Casamino acids.............................0L... convex...............................5g L-Arginine·HCl . Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........................................................................................0 ± 0..........0L.................5g Na2S2O3·5H2O ......0mL of 5% aqueous dioxan........................................ 0...................0L.0g Agar ......... and Agrobacterium vitis...... 0....0g Bile salts No.................................................. 1.......................................................0............. Use: For the isolation and differentiation of Aeromonas Agrobacterium Agar Composition per liter: deionized water and bring volume to 1......04g Thymol Blue.........0L.... 5.... Adjust pH to 6..........................5g Yeast extract..................0g Mannitol ..........7 ± 0........................................ Aeromonas hydrophila Medium Composition per liter: Inositol ...... Agrobacterium rubi................

...................0...........14g Cycloheximide solution......................9g KH2PO4 ........................................................................................................ 20................................................. Filter sterilize...0mL Cycloheximide Solution: Composition per 10...........................................025g Preparation of Cycloheximide Solution: Add cyclo- heximide to distilled/deionized water and bring volume to 10...5g Yeast extract... Mix thoroughly............6.....0L...............0g Mannitol.................................................................................. 2...... 10.....0g Calcium propionate....................................2 at 25°C Supplement: Composition per 100..............................................................0mL.......075g Supplement ..............2g Yeast extract ................................... Mix thoroughly..........................075g NaHCO3 .....................0g Adoniitol.....................1g Na2SeO3 .....................................................0........... Mix thoroughly.................. Filter sterilize............. Mix thoroughly.......................0..... 0................. 0.. 0............0g L-Glutamate.....................................2g NaCl ... Pour into sterile Petri dishes or distribute into sterile tubes....... 0... 1.. 2. 0....0g MgSO4·7H2O................... 0... 0.............. Adjust pH to 7..............................03g Tyrothricin.......0mg Preparation of Supplement: Add components to distilled/deionized water and bring volume to 100...........................................9mL..........0g LiCl..............0g K2HPO4 .......0mL D-Cycloserine solution ............................................ 0..0mL of sterile supplement......................... except sup- plement................................ Filter sterilize........................ Cool to 50°–55°C......... Use: For the cultivation and maintenance of slow-growing Agrobacterium rhizogenes and Agrobacterium tumefaciens........ 0........... 0...................................................................0g Mannitol ................ Agrobacterium Medium Composition per liter: Agar ..02g pH 7................................................ Adjust pH to 6..................... Agrobacterium Medium D1 Composition per liter: Agar ..........................0g KH2PO4 .....................................0................................................................. 1...........................2g Bacitracin ............ Mix thoroughly...........0.... 10...... Cool to 45°–50°C......................1mL of sterile biotin solution.......1g MgCO3 ............................................................................100......2g Preparation of D-Cycloserine Solution: Add D-cycloserine to distilled/deionized water and bring volume to 10. except biotin solution....................... 0...............275g Cycloheximide ................................... Distribute into tubes or flasks.5...2g MgSO4 ...2g Mg3(PO4)2 .2g pH 7................0mL...............2g Bromothymol Blue ...................................0mL Triphenyl tetrazolium chloride solution ........... Filter sterilize.........20 Agrobacterium Mannitol Medium Preparation of Biotin Solution: Add biotin to distilled/deionized water and bring volume to 10......0mL..0...... ......0mL pH 7....................................... Gently heat and bring to boiling.........0mL: Trimethoprim...............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................... 4.....................2 ± 0............ Agrobacterium Mannitol Medium Composition per liter: Mannitol............. Filter sterilize.... Use: For the selective isolation and cultivation of Agrobacterium species........ Mix thoroughly...........0mL... Mix thoroughly........ to distilled/deionized water and bring volume to 900..........................1................. 1.................................................. Triphenyl Tetrazolium Chloride Solution: Composition per 10... to distilled/deionized water and bring volume to 999......... 0.......0.................................... Gently heat and bring to boiling........... Preparation of Medium: Add components....... Add a drop of dilute HCl..............0mL...0mL: Triphenyl tetrazolium chloride ....... Cool to 45°–50°C.......0g MgCl2 .........0L...........1g Ca(NO3)2·4H2O . Use: For the selective isolation and cultivation of Agrobacterium species.............................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................1g Penicillin G .................................. Filter sterilize............................. Mix thoroughly. Gently heat while mixing until dissolved.......... Trimethoprim Solution: Composition per 10............ 0........................0....0.....................................1 ± 0.... 10........................................................................................... Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly..............15..... Autoclave for 15 min at 15 psi pressure–121°C............................................................. Agrobacterium tumefaciens Modified Roy and Sasser Medium for Grapevine Strains Composition per 1020mL: Agar ........15................... Autoclave for 15 min at 15 psi pressure– 121°C......0..............0.................... D-Cycloserine Solution: Composition per 10......................................................0mL......3g MgSO4·7H2O .........0... Gently heat and bring to boiling............ Aseptically add 100.....................0mL..06g Streptomycin sulfate ...... Aseptically add 0...7g NaCl ................. Use: For the cultivation of Agrobacterium rhizogenes..... Preparation of Medium: Add components.................................... Pour into sterile Petri dishes or distribute into sterile tubes...................0.. Adjust pH to 7..................................................15.........................2........... 0.......8g Preparation of Triphenyl Tetrazolium Chloride Solution: Add triphenyl tetrazolium chloride to distilled/ deionized water and bring volume to 10........................ Mix thoroughly............ Autoclave for 15 min at 15 psi pressure–121°C..........0g NaNO3 ........................ Mix thoroughly...............2......0g H3BO3 ................0g K2HPO4 ..............2g MgSO4·7H2O......0g NaNO3 ............. 1. Mix thoroughly....0mL: Berberine.................6........... 1..............................0mL: D-Cycloserine .......... Pour into sterile Petri dishes or leave in tubes................0mL: Cycloheximide .................0 ± 0..........................................................4.........0mL Trimethoprin solution...025g Preparation of Trimethoprim Solution: Add trimethoprim to distilled/deionized water and bring volume to 10........

........0mL............ Cool to 50°C. 0.0mL: NaOH ................................10..................................0mL into flasks..0g Erythritol ................. Filter sterilize.0......... Mix thoroughly. Gently heat and bring to boiling...............0g heximide to distilled/deionized water and bring volume to 10...........0mL sterile selenite solution and 1............1mL sterile D-cycloserine solution......16g Cycloheximide solution . 0................................................... 0..................... 0....5................0.............. Aseptically pour into sterile Petri dishes..........................2.0......................................................................................... Gently heat and bring to boiling........... Aseptically pour into sterile Petri dishes......25g NH4NO3 ................... Gently heat and bring to boiling.0mL of medium.................... 0.....0mL: NaOH ..0mL Selenite Solution: Composition per 10............................0mL of medium..0L................... Aseptically add per 100..0mL: Preparation of Cycloheximide Solution: Add cyclo- NaHCO3 .......... 1...0g Yeast extract ..............25g NH4NO3 .............. dium bicarbonate solution...................... 1...........29g MgSO4·7H2O ..........0mL cycloheximide solution............................................... Prepare medium freshly.................2g CaCl2· 2H2O .. except so- species....... cloheximide solution and selenite solution........ Aseptically add per 100.............0....................... Aseptically distribute into sterile tubes or flasks....................................0mL..... to distilled/ deionized water and bring volume to 1...... 1. Mix thoroughly.02g Preparation of Cycloheximide Solution: Add cyclo- heximide to distilled/deionized water and bring volume to 10................1........0mL: 21 Malachite Green (0........................................................0mL Preparation of Medium: Add components. except cycloheximide solution and tetrazolium chloride solution............... Aseptically add sterile sodium bicarbonate solution........2...............................................1 mL sterile tetrazolium chloride solution......... Distribute 100........ Autoclave for 15 min at 15 psi pressure–121°C.............................. AKI Medium Composition per liter: Peptone .........0g KH2PO4 ..........................................1g Preparation of Selenite Solution: Add components to distilled/deionized water and bring volume to 1........0L...............................5g Na2SeO3·5H2O .........................0......0L............0mL pH 7........................0g L(−)Arabitol ................. 0...04g KH2PO4 .... Autoclave for 15 min at 15 psi pressure–121°C.......................................... Filter sterilize.....................2 at 25°C Cycloheximide .......16g Cycloheximide solution ..0mL of medium. Aseptically add per 100................0......0mL Yeast extract (1% solution) ............................. 5............. Mix thoroughly............................ 0.................................. 3....... Dcycloserine solution...... Adjust pH to 7........ Distribute 100......................... 0............... Mix thoroughly... Mix thoroughly...0mg FeCl3 ..... 0...5g Na2SeO3·5H2O ............................. except cycloheximide solution and selenite solution..........54g Sodium taurocholate ......0g NaCl .......... Use: For the selective cultivation of Agrobacterium tumefaciens biovar 2.................................0mL: Cycloheximide ..4mg Na2MoO4·2H2O...02g Sodium Bicarbonate Solution: Composition per 100.... 0............ except cy- Preparation of Sodium Bicarbonate Solution: Add sodium bicarbonate to distilled/deionized water and bring volume to 100... 15.................... Use freshly prepared solution........................................0mL into flasks.. 0.. Filter sterilize... Preparation of Medium: Add components.. Mix thoroughly.0mL into flasks.............................................................. 0...............................10........... Cool to 45°–50°C.....2 ± 0........ Cool to 50°C.................10................5.............0.........................0mg ... Alcaligenes N5 Medium Composition per liter: Sodium succinate·2H2O .........15.......................................0mL Selenite solution.............................. and 1............................. Autoclave for 15 min at 15 psi pressure–121°C........ Preparation of Medium: Add components....................... Distribute 100. 15...... Agrobacterium tumefaciens Selective Medium Composition per 1020mL: Agar .................... Mix thoroughly. Cycloheximide Solution: Composition per 10...............10....................................2..................... Mix thoroughly..... Agrobacterium tumefaciens Selective Medium Composition per 1020mL: Agar .0mL cycloheximide solution............04g KH2PO4 ................... Mix thoroughly.......................3........ Mix thoroughly. Mix thoroughly............... Aseptically pour into sterile Petri dishes.................... Filter sterilize.................................................................... 0.........04g K2HPO4 ................. Filter sterilize....4......0g Sodium bicarbonate solution ........1% solution) . Cycloheximide Solution: Composition per 10..29g MgSO4·7H2O ............. Mix thoroughly......Alcaligenes N5 Medium Preparation of Medium: Add components.....0mL sterile selenite solution and 1........................0mL cycloheximide solution...... Autoclave for 15 min at 15 psi pressure–121°C...................................................0mL Selenite solution.. to distilled/deionized water and bring volume to 970........ to distilled/ deionized water and bring volume to 1.1% solution) .0mL.......... Mix thoroughly.................................75g NH4Cl . 1...0mL Selenite Solution: Composition per 10............... 1. 3..0L..............67g K2HPO4 .....1mL sterile trimethoprin solution.........................10.............................. 30................... Use: For the selective cultivation of Agrobacterium tumefaciens biovar 3............0............05g K2HPO4 ....03g MnCl2·4H2O ..............1g Preparation of Selenite Solution: Add components to distilled/deionized water and bring volume to 1.61g MgSO4·7H2O.............................0L..0mL Crystal Violet (0...54g Sodium taurocholate ............ Cool to 50°C................................................ 0... Use: For the selective cultivation of Agrobacterium tume- Use: For the cultivation of Vibrio cholerae and other Vibrio faciens biovar 1............... and trimethoprin solution to distilled/ deionized water and bring volume to 1..........0mL..............

. Use: For the cultivation and maintenance of Alcaligenes faecalis...................0.......... Mix thoroughly................................. Alcaligenes NA YE Medium (Alcaligenes Nutrient Agar Yeast Extract Medium) Composition per liter: Agar ..... 3.............2g MgCl2 ..... Gently heat and bring to boiling....................... 0.......... Mix thoroughly........5-10......................... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.....5....... Cool to room temperature.....0..............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...... Cool to room temperature....................................5............ Sparge with 100% N2..3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.............................10............4g KH2PO4 ..... and vitamin solution..................0L..................................................................1. Mix thoroughly.....0L........01g Na2MoO4·4H2O........... 50...1..... Preparation of Medium: Prepare and dispense medium under 100% N2 gas atmosphere..................0mL........2g Resazurin ...........7g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.......... Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................................ Filter sterilize...........01g Na2SeO3·5H2O ..........10...5.............10...................................0..... Autoclave for 15 min at 15 psi pressure–121°C......................................0mL Trace elements .....................02g H3BO3 ...................................0mL Na2S·9H2O solu- .................0 ± 0....0 at 25°C Sucrose Solution: Composition per 50.....0........0mL Yeast extract solution .........................0mg p-Aminobenzoic acid ..... Mix thoroughly.............................0mL yeast extract solution..... Sparge with 80% H2 + 20% CO2............................. Mix thoroughly... Cool to room temperature while sparging with 100% N2.......0...... Cool to room temperature..0g Pancreatic digest of gelatin ..........................18g CaCl2·2H2O ........................................................... to distilled/deionized water and bring volume to 920...................0mL sucrose solution....................... 0..........0mg D-Ca-pantothenate ........................0..........10..........................0mL: Yeast extract ................................... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C............................... Pour into sterile Petri dishes or leave in tubes....................................0............. Na2S·9H2O solution... Mix thoroughly......................0......1g NH4Cl........6g NaHCO3 ...............................0mL.............................0mg Biotin ...................... Mix thoroughly......................5 with KOH...... Mix thoroughly................................. Mix thoroughly.............. Gently heat while stirring and bring to boiling..........0mg Nicotinic acid .....5g NaCl ..................... Add solid NaHCO3.............................................. Autoclave for 15 min at 15 psi pressure–121°C...0................................. 10......................................................... 63......................01g Sucrose solution ................. Pour into sterile Petri dishes or leave in tubes.............................0...........................1g FeSO4·7H2O ... 5.................................................0g Beef extract .....0L.... Distribute into tubes or flasks............... 0........................................ Add distilled/deionized water to 1.................................................................................................0mL Na2S·9H2O solution.. Distribute into tubes or flasks................ 10..........0mL: Sucrose........5g CoSO4·7H2O..........0mg Riboflavin............... 0............ Alcaliphilic Amphibacillus Strains Medium (DSMZ Medium 931) Composition per liter: Na2CO3 ...... Use: For the cultivation and maintenance of Alcaligenes species......0mg Thiamine-HCl·2H2O ........0g Beef extract .. Alcaligenes NB YE Agar (Alcaligenes Nutrient Broth Yeast Extract Agar) Composition per liter: Agar ... Na2CO3................................0L..............5.......1................ Use: For the cultivation and maintenance of Alcaligenes faecalis.....0L...................0g pH 7............. Trace Elements Solution: Composition per liter: MgSO4·7H2O. NH4Cl........ 0.....5................... 15................... 3.......... and Na2CO3......0g MnSO4·2H2O............ 5.................0.....0mL........................0mL of distilled/deionized water....... Aseptically and anaerobically add per liter of medium 50.......... Autoclave for 15 min at 15 psi pressure–121°C............................01g CuSO4·5H2O.............................2.........................................5g KCl............0mL pH 9. yeast extract solution. Mix thoroughly........... Add components............ Distribute into anaerobe tubes or bottles.......... Autoclave for 15 min at 15 psi pressure–121°C.................................2..0g Pancreatic digest of gelatin ..... Yeast Extract Solution: Composition per 10................................ sucrose solution..0.......... Autoclave for 15 min at 15 psi pressure–121°C.......0mL Vitamin solution....3..............2g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.................................0mL...............0............................................. 5.........0mg Lipoic acid.......... Gently heat while stirring and bring to boiling.......... Gently heat while stirring and bring to boiling.. Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O ........................................ Dissolve by adjusting pH to 6..............0g Nitrilotriacetic acid................................0g Preparation of Sucrose Solution: Add sucrose to distilled/deionized water and bring volume to 50....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....0g Yeast extract.....0g Yeast extract.22 Alcaligenes NA YE Medium Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....5............................0mg Folic acid ...... 5.............. Sparge with 100% N2..50............................ 5.................... Boil for 5 min...025g KAl(SO4)2·12H2O ........................................... NH4Cl........................................... except NaHCO3.............................0.................0mg Vitamin B12..........................................1g NiCl2·6H2O................................18g ZnSO4·7H2O................................................................. Add remaining components.................. 15................................................................ Distribute into tubes or flasks..............................

.0mL: Solution A ................ except trace elements solution SL-6 and agar solution...........0................. Use: For the cultivation of microorganisms that can utilize alginate as a carbon source. Mix thoroughly...........0g Preparation of Agar Solution: Add agar to distilled/ deionized water and bring volume to 500.........................................................0 ± 0.............. Mix thoroughly.............. 0.........2g ZnSO4·7H2O............. Preparation of Solution B: Add components to dis- Preparation of Medium: Aseptically combine 500.......2.......................................................0g tilled/deionized water and bring volume to 400. Cool to 45°C.................................... Autoclave for 15 min at 15 psi pressure–121°C................ Preparation of Medium: Aseptically combine solutions A..........500...........3g CoCl2·6H2O...................0mL of ................................0mL Solution B ...................................5g H3BO3 ..................... Alginate Utilization Medium Composition per liter: Solution B ..0mL Solution C ......................0................ Autoclave for 15 min at 15 psi pressure–121°C.............5g H3BO3 ........... 0...0mL.................02g CuCl2·2H2O........0.. and 500................................ 0....................... Mix thoroughly.........0mL Solution A ....0mL of solution C.................0mL: Tris·HCl buffer.. Autoclave for 15 min at 15 psi pressure–121°C.................0mL.... 20..................... 500........................... Filter sterilize..................................0........................... Agar ...................... Mix thoroughly.............................................................................2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ...................100...................................0mL Preparation of Solution B: Add components to dis- Solution A: Composition per 400......15......... Mix thoroughly...... Agar ..............0mL pH 4........ Autoclave for 15 min at 15 psi pressure–121°C...................................3µg Biotin .........................0L....... Preparation of Medium: Add components......0............................. 0................0mL of solution A................................................................................ 0...............................02g CuCl2·2H2O......0.0g MgSO4·7H2O..3........ 0....... Use: For the cultivation and maintenance of Alicyclobacillus acidoterrestris..........Alicyclobacillus Agar 23 tion..................0mL: ized water and bring volume to 500.. and C.............................................................01g Amphibacillus tropicus.......................... Solution B: Composition per liter: Use: For the cultivation of Amphibacillus fermentum and MnCl2·4H2O ................................ Distribute into tubes or flasks....................400.....0mL Trace Elements Solution SL-6..................0.................5mg Sodium glycerophosphate................67mg Thiamine·HCl .................................... Mix thoroughly.....1g Na2MoO4·2H2O...... Cool to 50°–55°C.................. 3...067g NaNO3 ............................................................. Cool to 50°–55°C...............2 at 25°C Solution A: Composition per 500..0g tilled/deionized water and bring volume to 500.......0mL..............0L.........1..0g Preparation of Solution C: Add agar to distilled/deion- Solution B: Composition per 500......................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1..................0mL Solution C ......5g CaCl2·7H2O.5-10................... Filter sterilize....0......................047g Ferric EDTA..................................... The final pH should be 9...............25g (NH4)2 SO4.... Adjust pH to 4....................0.............................5g CaCl2·2H2O .............. Mix thoroughly.........0.................. Warm to 50°–55°C............................ Autoclave for 15 min at 15 psi pressure–121°C....... Cool to 45°C.1...........................500............. Mix thoroughly......0 ± 0............. Mix thoroughly................... For liquid medium................................0L.....0......................0........ and 10....... 38...............................................................25g (NH4)2SO4 .0............................................... 66....... 6.......03g NiCl2·6H2O.................... Cool to room temperature............................... Preparation of Solution A: Add marine salts to dis- Solution C: Composition per 500........ B.. 1.............................0mL pH 4........... Growth on alginate (production of alginase) is a diagnostic test used in the differentiation of Vibrio species................ Solution C: Composition per 100..............................0...........500.0L..... Pour into sterile Petri dishes or distribute into sterile tubes......0mL of solution B.........................2g Agar solution ................................................................... to distilled/ deionized water and bring volume to 499............................1g Na2MoO4·2H2O.... Alicyclobacillus acidoterrestris Agar Composition per 1001...0........................ 10..........0............. Adjust pH to 4...0µg Vitamin B12 ..........0g Yeast extract ........... Alicyclobacillus Agar (DSMZ Medium 402) Composition per liter: Glucose.......................... omit agar from solution B.........0........ Autoclave for 15 min at 15 psi pressure–121°C.....................................0g Sodium alginate ......... 0.......................2g ZnSO4·7H2O...........................................................................2g Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 500..... Aseptically add 1...................67µg Preparation of Solution C: Add components to distilled/deionized water and bring volume to 100.......0g MgSO4·7H2O .......0mL vitamin solution..........................................03g NiCl2·6H2O...................... Gently heat and bring to boiling.........0mL: Marine salts............. 0..0mL: Glucose .. 0................0g Yeast extract...... Mix thoroughly..3g CoCl2·6H2O..... Gently heat and bring to boiling...............................................................0mL.0g KH2PO4 .................... Autoclave for 15 min at 15 psi pressure–121°C......... 2. 1................ Agar Solution: Composition per 500mL: Agar ...0mL.....................................................0mL: tilled/deionized water and bring volume to 1.........................0g KH2PO4 ..5...........................15..................................................................67. 5......

..................02g CuCl2·2H2O..03g NiCl2·6H2O ......0mL: Agar Solution: Composition per 500........0g Peptone .............0 ± 0....0mL: Yeast extract.. Autoclave for 15 min at 15 psi pressure–121°C.......... Mix thoroughly............... 0...........3...............0mL: Solution A . Cool to 45°–50°C............................................................................................... Autoclave for 15 min at 15 psi pressure– 121°C.........................................................................0mL Agar solution..0.0 with sterile 9% Na2CO3 solution........................................... Autoclave for 15 min at 15 psi pressure–121°C... 1... 10......................0mL.......0g Glucose ....0g Yeast extract ................................4 with 6% NaHCO3 solution............................................ 0. Pour into sterile Petri dishes or aseptically distribute into sterile tubes or flasks.............................................. and other Bacillus species..0g Agar ............... Aseptically add sterile Na2CO3 solution.......................................0g Glucose ................... Mix thoroughly............0 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..... 5........0g K2HPO4 ................ 0......................... Adjust to pH 4........0mL of solution B............... Mix thoroughly........... Gently heat and bring to boiling...........0mL sterile trace elements solution SL-6.............0L.......................0mL pH 4.0g Cellulose powder MN 300 ........................4.............0.................0g Peptone ..0mL............... 3............................................................4g Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 900....... Gently heat while stirring and bring to boiling..........5........................ Autoclave for 15 min at 15 psi pressure–121°C...................................... 15............ Autoclave for 15 min at 15 psi pressure–121°C.....0mL agar solution....... and Bacillus naganoensis............................2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500.....0mL.........03g Na2MoO4·H2O ...1.......................................5g CaCl2 ........1g MnCl2·4H2O.....0g MgSO4·7H2O ..........5g CaCl2·2H2O... Mix thoroughly.. Cool to 45°-50°C........0mL: Agar ....... Aseptically pour into sterile Petri dishes or distribute into sterile tubes............ Autoclave for 15 min at 10 psi pressure–115°C.... Mix thoroughly............................. 0... 900...................................... Mix thoroughly.. Cool to 50°–55°C.........0mL.............................................................0mL of solution A with 100................................. 0.................0g Na2CO3 solution.0g pH 9....... Use: For the cultivation and maintenance of cellulose-utilizing bacteria.... Filter sterilize......... Agar ...............................................0mL: Preparation of Solution B: Add 0.... Mix thoroughly.......... 500..........5g of Na2CO3 to distilled/deionized water and bring volume to 100...0...................................... Mix thoroughly..........................0mL sterile agar solution and 1...... 0. Use: For the cultivation and maintenance of alkalophilic microorganisms such as Bacillus alcalophilus.............................0 at 25°C Na2CO3 Solution: Composition per 100..............1....0g Beef extract ........ Na2CO3 solution....................... Distribute into tubes or flasks. to distilled/deionized water and bring volume to 900........24 Alicyclobacillus cycloheptanicus Agar sterile trace elements solution SL-6 and 500............ Na2CO3 ...........5–11.............. Adjust pH to 9..... 5........ 30.. Alkaline Cellulose Agar Composition per liter: Solution A ...................... Gently heat and bring to boiling.....4g MgSO4·7H2O............3g CoCl2·6H2O .....0g K2HPO4 ....0L........................................0g Preparation of Agar Solution: Add agar to distilled/ deionized water and bring volume to 500................................................2 at 25°C Solution A: Composition per 500.................... 100...............0mL solution A................0mL: Na2CO3 ...... Mix thoroughly......................................15.... 10.............. except Alicyclobacillus cycloheptanicus Agar (LMG Medium 174) Composition per 1001.......................... Alkaline Bacillus Medium Composition per liter: Agar .............................. Filter sterilize...... Aseptically adjust to pH 9........................25g (NH4)2SO4 ......... Preparation of Medium: Aseptically combine 500..........0.......................... Cool to 50°–55°C.... 10....................................1.................. ..................... Preparation of Na2CO3 Solution: Add Na2CO3 to dis- Use: For the cultivation and maintenance of Alicyclobacillus spp........0 with 1N HCl........5-10........................ Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1............................ Bacillus circulans.....................500.................................................. 5..............0g K2HPO4 .............0g Yeast extract..0mL Trace elements solution SL-6 ......... Pour into sterile Petri dishes or distribute into sterile tubes..5g Preparation of Medium: Aseptically combine 900......0mL................................ Use: For the cultivation of Alicyclobacillus cycloheptani- Alkaline Nutrient Agar Composition per liter: cus............500..0mL Solution B... 0.....................01g tilled/deionized water and bring volume to 100.... 0............. Mix thoroughly...................0mL Solution A: Composition per 900........0mL.......... Gently heat and bring to boiling.........0g NH4NO3...............15. Preparation of Medium: Add components....0mL pH 8..........5–10............................................ Pour into sterile Petri dishes or leave in tubes... 0..................... Solution B: Composition per 100............100.............................. Trace Elements Solution SL-6: Composition per liter: H3BO3 ..............2g ZnSO4·7H2O ....15...............2............... Cool to 45°-50°C... Mix thoroughly............0.... Pour into sterile Petri dishes or leave in tubes.......... Use: For the cultivation and maintenance of Bacillus alcalophilus and Bacillus species........0g Peptone.................................. Adjust pH to 3.......... Adjust pH to 7.............. 0.............................. Bacillus sp...............

................................. Use: For the cultivation of pectinolytic bacteria..........4 ± 0...................... Warm to 50°–55°C...0........................................0L....................... includ- 25 MgSO4·7H2O........... 10....0.0mL.......................... Autoclave for 15 min at 15 psi pressure–121°C...................0g Yeast extract ... Cool to 50°–55°C...................0mL.............2g MnSO4 .... Mix thoroughly................................................................................ 6..5g CoCl2·6H2O..................0g bacteria......................... Add remaining components............10......... Alkaline Starch Agar Composition per liter: Starch.......... ing Bacillus alcalophilus...........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..1g ZnSO4·7H2O..........................5.0g Agar ............................................................................ 16.............................................. Autoclave for 15 min at 15 psi pressure–121°C....7..................... Gently heat and bring to boiling....................0................0........... Autoclave for 20 min at 15 psi pressure–121°C..................0L.......0L....5mg Wolfe’s mineral solution ..................................................... 5....0g K2HPO4 ............... 1..0g Beef extract ..... 15..0g NaCl .... Add distilled/deionized water to 1..0g Peptone.02g CuSO4·5H2O..........................8.................10........0g NaCl .......................................................................................................................025g KAl(SO4)2·12H2O .........0..........0g Sodium polypectate........................................... Mix thoroughly...... Aseptically add sterile sodium sesquicarbonate solution........01g Na2MoO4·2H2O............................................ 1............. Preparation of Medium: Add components................................0g Methanol...1............................................................16.................Alkaliphilic Methanogen Medium Alkaline Nutrient Agar Composition per liter: Agar ..............0..................................01g Na2SeO3·5H2O ..............................................................0g Agar ............ Gently heat and bring to boiling..............0mL Selenite-tungstate solution ........ Pour into sterile Petri dishes or distribute into sterile tubes....0g Sodium sesquicarbonate solution...............................0mg pH 9................ Adjust pH to 8...........................................0g Yeast extract.............. 2................................................................. Adjust pH to 8........................5...........................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................. Adjust pH to 6........ Mix thoroughly........................................ Mix thoroughly...........3..........................15.. to distilled/deionized water and bring volume to 900........... Bring pH to 10.4............7.......................... 5.............................................................. Allow tubes to cool in a slanted position..........................0....0g Na2CO3 ..0g MgSO4·7H2O..... 1.................................0g Peptone............................0g Na2CO3 ........ 10.....................0g Na2S·9H2O ...........0..0g pH 8.......... Adjust pH to 6....................................................0g Peptone ...............0..... Use: For the cultivation of Vibrio cholerae and other Vibrio species.................3g NiCl2·6H2O......... Gently heat and bring to boiling..................0g Yeast extract.1g CaCl2·2H2O ..............7 ± 0.............2g MnSO4 ............................................6...................................4...................................................42g Preparation of Sodium Sesquicarbonate Solution: Add components to distilled/deionized water and bring volume to 100.................................1...............................................0g K2HPO4 .. Filter sterilize........................1g NiCl2·6H2O..1...................0.5 with KOH....................1g FeSO4·7H2O ..........0......................... Distribute into tubes........................ Mix thoroughly... Gently heat and bring to boiling..40..................0g Nitrilotriacetic acid... and other Bacillus species........0mL pH 9......... 15........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............................0mg Resazurin .................6g NaHCO3 ..................................................................................................... Distribute into tubes or flasks.............0mL of distilled/deionized water............. Mix thoroughly.........5g KH2PO4 .................................... Distribute into tubes or flasks. 3. ...0mL pH 9.2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.............2–9.................1.......................40.................4 at 25°C Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O...............3. Alkaline Polypectate Agar Composition per liter: Agar ................................................... 10.0g Peptone.0g Na2CO3 ......................0.. 5.................... Autoclave for 15 min at 15 psi pressure–121°C.........................................................0.......... 8........................2.......2 at 25°C Sodium Sesquicarbonate Solution: Composition per 100..............................0g pH 8..................0 ± 0.......0...............0mg pH 10...........................................5 ± 0..... 20......................... Bring pH to 9.. 5....................... Pour into sterile Petri dishes or leave in tubes.....5g Yeast extract .........7 ± 0............................................................................................. 10.....01g H3BO3 ..... Bacillus cohnii............. anhydrous............. Mix thoroughly.......... Distribute into tubes or flasks................................... Use: For the cultivation of alkiliniphilic starch-utilizing Alkaline Peptone Agar Composition per liter: Alkaliphilic Methanogen Medium Composition per liter: NaCl ............................ Pour into sterile Petri dishes or leave in tubes......................................................................................3mg Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500............20..........5g NaCl ..0L.....0L..........0............ Use: For the cultivation of a variety of alkalophilic microorganisms..... Use: For the cultivation of alkaliniphilic bacteria........................... Adjust pH to 9........... except so- dium sesquicarbonate solution...0mL: Na2CO3.................................. NaCl ........................................................................ 10....................0g NH4Cl .....0...........................0g NaHCO3 .......................... Autoclave for 15 min at 15 psi pressure– 121°C.................0............................ Alkaline Peptone Water Composition per liter: Peptone......100...................................0.......0g MnSO4·2H2O.......................................

................ Filter sterilize... The pH should be about 10... Mix thoroughly... 10...0mL pH 10................. 4........... Sparge with 100% N2.............................0g NaCl .....0mL Wolfe’s vitamin solution ....................0mL: Na2CO3 ................................................1......0mg Riboflavin .......................................10....0mL KSCN solution ...........0mL Sucrose solution ... 0.......20... 1..................................... Adjust pH to 9............. Cool to room temperature while sparging with 100% N2..............................................7 ± 0............. Sparge with 100% N2.......4.....0............... Na2S·9H2O solution....................... Autoclave for 15 min at 15 psi pressure–121°C........ to distilled/deionized water and bring volume to 910......................0L................................. Mix thoroughly....................... 0.....0mL: NaHCO3 .... Aseptically and anaerobically add 50.........................................30............................0.............0mL pH 9.... Alkaliphilic Sulfur Respiring Strains Medium (DSMZ Medium 925) Composition per liter: Mineral base .. Continue boiling for 5 min... Sparge with 100% N2................10......0mL Magnesium chloride solution ...............100................................ 20........... 2.................................................................................. Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl .....0.........................................0mL Trace elements solution SL-6 ................ Autoclave for 20 min at 110°C..0mg Lipoic acid ...................0 ± 0..................................2g ZnSO4·7H2O......... 0..................................300..........................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 50......................... Before use...................0 with sterile HCl..... Spirochaeta alkalica.......0L.......... 10.............................................................5....... Mix thoroughly.... 997.............. and Wolfe’s vitamin solution.........0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1... Calcium DL-pantothenate ................0mg Na2SeO3·5H2O ..2g Yeast extract...............2................0mL Na2S·9H2O solution................................... 0............0mL.... Mix thoroughly........................ 5......5g H3BO3 .......... Sparge with 100% N2.........................0g NH4Cl...... 5........................... Use: For the cultivation of Spirochaeta africana..................10.0mL: Na2S·9H2O.5g NaHCO3 solution ....................... Preparation of Medium: Prepare and dispense medium under 100% N2....... sucrose solution.....................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.....................0mL: Sucrose....0mL of sterile Wolfe’s vitamin solution.......................... except NaHCO3 and Na2S·9H2O.....0mg p-Aminobenzoic acid ............................................................................... Sucrose Solution: Composition per 20......................2.......................7 with 6N NaOH (about 15....10.............. Mix thoroughly..........0mg Nicotinic acid ..................0g Preparation of Sucrose Solution: Add sucrose to distilled/deionized water and bring volume to 20...............5.....30........................26 Alkaliphilic Spirochete Medium Selenite-Tungstate Solution: Composition per liter: NaOH ....................................... Autoclave for 15 min at 15 psi pressure–121°C..... Cool to room temperature while sparging with 100% N2.....................0L....... Autoclave for 15 min at 15 psi pressure–121°C.....0................... Gently heat and bring to boiling.......... 5.02g CuCl2·2H2O................ Add components...................................................................0mL............... Mix thoroughly...20..........0mg tilled/deionized water and bring volume to 1.............0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O Preparation of Mineral Base: Add components to dis- to distilled/deionized water and bring volume to 10. Mix thoroughly................. 0.......................2 at 25°C Mineral Base: Composition per liter: Na2S·9H2O Solution: Composition per 10..................... Add components............... Preparation of Medium: Prepare and dispense medium under 100% N2........ Autoclave for 15 min at 15 psi pressure–121°C....0mg Thiamine·HCl .................. Mix thoroughly...........1g Na2MoO4·2H2O.............3g CoCl2·6H2O............................................................... Mix thoroughly.................. to distilled/deionized water and bring volume to 1........0mL of sterile sucrose solution.........0mL of sterile NaHCO3 solution..............................................................0mL)........0g K2HPO4 ......................20...........................................0mg Na2MoO4·4H2O........... Gently heat and bring to boiling................. Sparge with 100% N2....0mL Trace elements solution......................................................0.............5g Na2WO4·2H2O .. 5.........2–9....0mg MnCl2·4H2O ...................1............. Sparge with 100% N2.......................0mL.. Anaerobically distribute into tubes................ except NaHCO3 solution.........0g K2HPO4 .............................................0g NaCl ..............0mg Biotin . 1......... neutralize to pH 7....0mL.................................................0mg CoCl2·6H2O.....0mL of sterile Na2S·9H2O solution..0mg Folic acid ............50.................2 at 25°C NaHCO3 Solution: Composition per 50..............03g NiCl2·6H2O..............................0mg NiCl2·6H2O....0mg Vitamin B12.......................0g NaHCO3 .......... Mix thoroughly............................ and 10.......... Autoclave for 15 min at 15 psi pressure–121°C.............................................. Adjust pH to 9......................................... Filter sterilize......................... 10........................... Alkaliphilic Spirochete Medium Composition per 1011............. Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ...................0mg ..... 15....................... 10...0mg CuCl2·2H2O........0mg ZnSO4·7H2O....... 1............................................................. Add NaHCO3 and Na2S·9H2O................. Aseptically distribute into sterile tubes or flasks.......0.............0L......................... and Spirochaeta asiatica............ Mix thoroughly....0L......................... 5...... 5.......................................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1............. 0.......... Use: For the cultivation of Methanohalophilus zhilinae..... 10.............. 3......2g KCl.....200..............0........ Trace Elements Solution: Composition per liter: H3BO3 ..........0g Na2CO3 ......

.......................................................... 20............... Add remaining components.............................................0mL: Na2S·9H2O .. 0...........5 with KOH............ Polysulfide Solution: Composition per 100...........................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500....................2........0mL sterile trace elements solution..........0g Preparation of Magnesium Chloride Solution: Add MgCl2·6H2O to distilled/deionized water and bring volume to 10....................0mL Glycine solution ...........18g 27 ZnSO4·7H2O.0.......... Dissolve by adjusting pH to 6.................... The solution will be dark yellow.. Mix thoroughly...0L of dis- tilled/deionized water with 100% N2 to remove O2.......................... Aseptically and anaerobically distribute to sterile tubes or bottles.........5g CoSO4·7H2O .... Adjust pH to 3..................................0mL....... Mix thoroughly...0.....0L.............. 27...........5g NaCl .........................0mL distilled/deionized water with 100% N2........ Autoclave for 15 min at 15 psi pressure–121°C...................................0...................... Sparge with 100% N2................................... Autoclave for 15 min at 15 psi pressure–121°C...............0mL sterile polysulfide solution to 1.............0..........0g Preparation of Glycine Solution: Add glycine to distilled/deionized water and bring volume to 100................. Mix thoroughly...........0mL sterile KSCN solution......................... Sparge with 100% N2...16g Preparation of Polysulfide Solution: Sparge 100.......... Add Na2S·9H2O....................... Mix thoroughly......................................... Cool to room temperature...........50... Preparation of Medium: Aseptically add 10........0mL........................ Yeast Extract Solution: Composition per 100............. Trace Elements Solution: Composition per liter: MgSO4·7H2O ... 15.0g MnSO4·2H2O ...0g Peptone ..............................5g Preparation of KSCN Solution: Add KSCN to dis- tilled/deionized water and bring volume to 10................................. 3...10......5......1000........ Add sulfur..... Mix thoroughly.02g H3BO3 ..... Do not adjust the pH......... Do not adjust pH............ 1. 1..............0mL: MgCl2·6H2O...................................20.0mL Preparation of Base Solution: Sparge 2...... 0.2g Sulfur ..7g MgSO4·7H2O ... 5............9.....0.......... Cool to room temperature......................05mg Trace elements solution .. Alkaliphilic Thermococcus Medium (DSMZ Medium 926) Composition per 1082................1g H3BO3 .................. 0................ Autoclave for 15 min at 15 psi pressure–121°C................ 1.......0g Beef extract .............................................................................. Autoclave for 15 min at 15 psi pressure–121°C.............. Mix thoroughly..01g CuSO4·5H2O.......... 2........... 7.........0mL Yeast extract solution .... Cool to room temperature..025g KAl(SO4)2·12H2O .................... 0.. Mix thoroughly............................. ........................................................................................... 0................. 1...................12............0.......................... flasks.............................. Cool to room temperature................0L sterile base solution.....0mg Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 1.............................................. or bottles.................0mL of distilled/deionized water.............. Autoclave for 15 min at 15 psi pressure–121°C.....................................................................0mL Base Solution: Composition per 2000................0mL: Yeast extract .................. Autoclave for 15 min at 15 psi pressure–121°C.................................5......................0g Yeast extract .............. Alkalophile Medium Composition per liter: Agar .......................0.................0 with HCl....0mL: Base solution..... 2.. Glycine Solution: Composition per 100............18g CaCl2·2H2O ................................................ 2..........................0mg FeSO4·7H2O.. Autoclave for 15 min at 15 psi pressure– 121°C..............65g NaHCO3 ... Sparge with 100% N2.1..0mL.......................... and 1...1g NiCl2·6H2O..........................03g KI ............1.............0mL sterile magnesium chloride solution to 987............... Mix thoroughly.................................................0........................0g Nitrilotriacetic acid ........0g Sodium sesquicarbonate solution .. A white colloid will dissolve after mixing............... Sparge with 100% N2.....0mL Polysulfide solution ......................0mL: Glycine ...........1g FeSO4·7H2O .......01g Na2MoO4·4H2O............0g Preparation of Yeast Extract Solution: Add yeast ex- tract to distilled/deionized water and bring volume to 100..01g Na2SeO3·5H2O ............20... Use: For the cultivation of Thermococcus alcaliphilus.................0L.....................0mL..... Preparation of Medium: Aseptically and anaeriobically add 50................................................ 0.........................................................................0mL: Sodium sesquicarbonate ..............0g KCl............................. KSCN Solution: Composition per 10mL: KSCN..............................0mL sterile mineral base...5g K2HPO4 ......0.................... Cool to room temperature............................0........0mL of O2-free distilled/deionized water.........2 at 25°C Sodium Sesquicarbonate Solution: Composition per 100......................32g NaBr....................0mg CaCl2·2H2O...........................................................................0g MgCl2·6H2O...... and 12............................ Mix thoroughly...................... Magnesium Chloride Solution: Composition per 10...............0mL sterile glycine solution..............................0g NaCl ................................ Use: For the cultivation of Thialkalivibrio paradoxus and Thialkalivibrio thiocyanoxidans.. Filter sterilize............ Add distilled/deionized water to 1...........0mL: NaCl ...........5 ± 0.......... 15........................................................................................0L............... 5........0g Preparation of Sodium Sesquicarbonate Solution: Add sodium sesquicarbonate to distilled/deionized water and bring volume to 100...............15..... 0...15...............0mL pH 9.......... Mix thoroughly.............................Alkalophile Medium EDTA ... Aseptically distribute to sterile tubes.0mL sterile yeast extract solution... Add components to 2000.......................................................... Sparge with 100% N2.........

Alkalophilic Halophile Agar Composition per liter: Solution A .........0L: MgSO4·7H2O (4% solution) ....0L.....0 at 25°C Solution A: Composition per 500...5g K2HPO4 .... Natronobacterium vacuolata...0mg NH4VO3..0mL: H3BO3 ... Add 13..................... AMB Agar Composition per liter: Agar ........ 50.............0mL: tilled/deionized water and bring volume to 500..........................................................................0mL Solution B ............20...... Sparge solution with filtered air until color changes....13...5.... K2HPO4 ...36mg Preparation of Solution B: Add components to distilled/deionized water and bring volume to 500............................25mL of solution B aseptically after sterilization.........................0mL of filter-sterilized sodium sesquicarbonate solution to adjust pH to 9.............................360.........0mg MoO3 ......0g Pancreatic digest of casein ..............................28...0mL: NaCl ..0mL: Disodium EDTA·2H2O.8% solution) .0mg MnCl2·4H2O... Bring pH to 9.... and other Bacillus species............................0mL pH 9..........................15................0mL......................................0g Microelements Stock Solution: Composition per 1090.0...... 200............................................212g Solution A ......................572.... Distribute into tubes or flasks..0g Na2CO3·10H2O ..............................0mL of solution A with 500...........................0mL Microelements stock solution ..................................................... Autoclave for 15 min at 15 psi pressure–121°C......0.....0mL Solution B ............................................ 10...0L............0mL of distilled/deionized water............ Add 25...... Use: For the cultivation and maintenance of Natronobacte- rium gregoryi........ Mix thoroughly........ 0.....0mL Preparation of Solution A: Prepare individual solutions and combine........0............... 3............. Autoclave for 15 min at 15 psi pressure–121°C..........5 ± 1..................0g Preparation of Solution B: Add K2HPO4 to distilled/ deionized water and bring volume to 500..0g FeSO4·7H2O............0g KCl................................ Bacillus submarinus...0g Casamino acids ....500...............0L.. 20..............0mg CuSO4·5H2O......................................................... 0........ 0................................... Mix well..... 2............ Bacillus circulans.7g KOH ...25................ Gently heat and bring to boiling........................................2g Preparation of Solution A: Add components to dis- Preparation of A & A FeEDTA Solution: Dissolve 5........0mg MnCl2·4H2O ......................0g Pancreatic digest of casein ....... .........................0mL NaCl (3.....................0g KNO3 .........0g Yeast extract..................... Pour into sterile Petri dishes or distribute into sterile tubes..28 Alkalophilic Halophile Agar Preparation of Medium: Add components......500.................................................. Add 6.....8mg CoCl2·6H2O............ Preparation of Medium: Add agar......2g of KOH in 186...................................4g of disodium EDTA·2H2O........................ Autoclave for 15 min at 15 psi pressure–121°C.................................. Gently heat and bring to boiling......................2.......... to distilled/deionized water and bring volume to 985.......... Pour into sterile Petri dishes or distribute into sterile tubes...................0g Starch..................................................... Use: For the cultivation of myxobacteria....................................................0mL Preparation of Microelements Stock Solution: Add components to distilled/deionized water and bring volume to 1090..........7g of FeSO4·7H2O to 364.....................................5. Allen and Arnon Medium with Nitrate Composition per 1000.......................................... Agar .........................................8.....25mL Solution A: Composition per 2.. 7....... 10..................25mL: Noble agar...............5g Trisodium citrate ................5..... Distribute into tubes or flasks...25g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....................0L....................... 50.253g NaNO3 .............................. Combine the EDTA solution with the FeSO4 solution...................2% solution)......0L...... soluble .......5g MgSO4·7H2O............0mL of solution B..................................................25g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................. 1..................... Mix thoroughly....................36.............5..44................0mL.................. soluble .500. Preparation of Medium: Aseptically combine 500................................. and NaNO3 to distilled/deionized water and bring volume to 969..0.................. Natronobacterium magadii.....500............ KNO3.........0mg ZnSO4·7H2O............... Cool to 50°–55°C............... Mix thoroughly. Mix thoroughly.. AMB Broth Composition per liter: Starch....5..................................................0g MgSO4·7H2O ............... Add 20.. Use: For the cultivation of myxobacteria................................ pH about 3.......6........................ Gently heat and bring to boiling..................................500.... Mix thoroughly....................5g MgSO4·7H2O........ Cool to 50°–55°C............................. Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C...... and Natronococcus occultus......................... A & A FeEDTA Solution: Composition per 550............6mg A & A FeEDTA solution.500...0mL of solution A.........4......5g K2HPO4 .................. Pour into sterile Petri dishes or leave in tubes...................0mL CaCl2·2H2O (1......................... Mix thoroughly..... Use: For the cultivation of Bacillus alcalophilus. except so- dium sesquicarbonate solution.0mL of distilled/deionized water......................................... Use: For the cultivation and maintenance of Anabaena species and Nostoc species............. Solution B: Composition per liter: Solution B: Composition per 500... Aseptically add 15.... Mix thoroughly........... 160......................... Autoclave for 15 min at 15 psi pressure–121°C........................2............0mL..... Cool to 50°–55°C...15........ Natronobacterium pharaonis...............4g FeSO4·7H2O ........................... Autoclave for 15 min at 15 psi pressure–121°C......5......................................................

.....................................0g KH2PO4 .............................................. Sparge with 100% N2 for 30 min...................................01mg pH 6...... aseptically and anaerobically add 0...............0mL aliquots....... Autoclave for 15 min at 15 psi pressure–121°C........5g NaCl ...................................... 2.......0g DL-Amino-butyric acid .0L.............4...........0mL of distilled/deionized water...... 1...............0....................0g MgSO4·7H2O...................... Mix thoroughly..................... Add sterile methanol to a concentration of 0..............0g MgSO4·7H2O.................0................... Adjust pH to 6.........4........... 0..10........1mL of sterile KNO3 to each tube.....5g CaCl2·2H2O .....................................3.................................................................................................................1mg Na2MoO4·2H2O....... to distilled/deionized water and bring volume to 1............................0g Preparation of KNO3 Solution: Add KNO3 to distilled/deionized water and bring volume to 100........................................................... Preparation of Medium: Prepare and dispense medium under 100% N2..................... Add components.................... Use: For the cultivation and maintenance of bacteria that can utilize methanol as a carbon source...............................1g FeSO4·7H2O...................................8 ± 0.............0 ± 0.. 0......................... 5.............................. AMS Agar without Methanol (Ammonium Mineral Salts Agar without Methanol) Composition per liter: Agar ..................................... Mix thoroughly..........0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.06mg MnCl2·4H2O .......0................................................................7g KH2PO4 ...1g CaCl2·2H2O...0L................. 0...........................0g Na2S·9H2O........................................1g ZnSO4·7H2O ..............5g CaCl2·2H2O ........... Autoclave for 15 min at 15 psi pressure–121°C...........................0mL pH 5........... 0..0......... Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ....... 0.......................................0g MgSO4·7H2O ..........................................7g KH2PO4 ............................................................06g FeSO4·7H2O...........22g (NH4)2SO4 ............. 0.............10.........0g Glucose ....................................................................... 0..3mg CoCl2·6H2O........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....................................2g MgSO4·7H2O .........2g FeSO4·7H2O .......................... AMS Agar (Ammonium Mineral Salts Agar) Composition per liter: Agar .........5g pH 7..............................................5 with 20% HCl.......................... Mix thoroughly........ Use: For the cultivation and maintenance of Serratia marcescens and other microorganisms that can utilize amino-butyric acid as a carbon source. Add distilled/deionized water to 1................... 0.......................... Autoclave for 15 min at 15 psi pressure–121°C......0mL Wolfe’s mineral solution ........................................ 1........................ and Methylophilus species.................. Mix thoroughly............0L.......06mg MnCl2·4H2O ............................................ 3.......... 0................. Autoclave for 15 min at 15 psi pressure–121°C.............. Use: For the cultivation of Ammonifex species that form ammonium from nitrate during chemolithoautotrophic growth of this extremely thermophilic bacterium............ 0................. 0.............. Dispense anaerobically into tubes in 10...................................................................54g NH4Cl ...........................................................0mg Na2SeO3·5H2O .............................2 at 25°C Selenite-Tungstate Solution: Composition per liter: NaOH ......................................... 4.........1..... Gently heat and bring to boiling...............................................................1.................. Sparge with 100% N2..............................0........2mg Resazurin ..................0..................................15............................025g KAl(SO4)2·12H2O.......................... Gently heat and bring to boiling.............0.....................5mg KNO3 solution.................02g CuSO4·5H2O ......... 0...................................................................0g MnSO4·2H2O .... Adjust pH to 6.................................... 0.. Methylomonas species........... Prior to use...0................0g K2HPO4 ............................................. 0........... 15...........................3mg CoCl2·6H2O...................................................... 0...... 0......................5 with KOH....0.......................5g CoCl2·6H2O ..................... except Na2S·9H2O and KNO3 solution............... 10......................................................3g KH2PO4 ........5% aseptically to cooled basal medium...................................................................0mL: NaCl ...........0.........0mL Selenite-tungstate solution ............ Mix thoroughly........................................AMS Agar without Methanol Amino-butyric Acid Medium Composition per liter: Agar ........... 7.................................. such as Methylobacterium species.....1g NiCl2·6H2O ... 1................ Adjust pH to 5......................22g NaHCO3 ... 0............. Autoclave for 15 min at 15 psi pressure–121°C............................01g Na2SeO3·5H2O ......0mg H3BO4 ..............0mL.............0L. 1..................................................09g CaCl2·2H2O........................................ Ammonifex Medium Composition per 1010......................1mg Na2MoO4·2H2O........................0mg H3BO4 ........4 ± 0....................................................................2g FeSO4·7H2O .................................03mg NiCl2·6H2O....5g K2HPO4·3H2O.. 0...................1...................................................... Add Na2S·9H2O...............0L....01g H3BO3 .....................................................................01g Na2MoO4·2H2O ..............0..................... Pressurize tubes to 200 kPa with 80% H2 + 20% CO2 gas mixture.......... Pour into sterile Petri dishes................................................0.............0.................................................. Mix thoroughly.................................................................... 29 KNO3 Solution: Composition per 100.............0.03mg .......... 3............................................................................0g Nitrilotriacetic acid ........................................................0...........2mg ZnSO4·7H2O...................... 0........................0g K2HPO4 ..............................................3mg Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500......................................... 3... 0...................0mg NiCl2·6H2O ................... 0. Sparge with 100% N2...............2mg ZnSO4·7H2O.0mL: KNO3 ..0............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............ Adjust pH to 6..........................................0g K2HPO4 ................... Evacuate headspace under vacuum.8.......15.....0g (NH4)2SO4 ...........................0............ Add remaining components.................................................................5g Na2WO4·2H2O ............02mg CuCl2·2H2O......... 0.....4.................0...0....................................................54g NH4Cl .................................0.........

............. Na2S·9H2O Solution: Composition per 100..........10........ Store anaerobically..8.......................0g NaCl ............................... Allow to cool under 100% N2...........................................................................0g Preparation of Disodium Thioglycolate Solution: Add disodium thioglycolate to distilled/deionized water and bring volume to 10................0g L-Cysteine·HCl ..... Mix thoroughly............ 0........5g Sodium acetate .. Pour into sterile Petri dishes or leave in tubes....... Mix thoroughly.............................. ...0g KH2PO4 .........0g K2HPO4 .............0g Yeast extract ...................................... Distribute into sterile tubes or flasks under anaerobic N2............5g Disodium thioglycolate solution ...................................0mL pH 7.............0mL Salt solution.....8 ± 0........ Adjust pH to 7.5g K2HPO4 ................ except Lcysteine.................... Autoclave for 15 min at 15 psi pressure–121°C............... 0...... Aseptically and anaerobically combine the basal solution................................ Cultures are grown under an atmosphere of 50% methane....................2g pH 7.0mL sterile Na2S·9H2O solution and 10.....1.25g Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 1.......................................... Powdered sulfur is sterilized separately by steaming for 3 hr on 3 consecutive days..........10.....0g ............................................................................................................................2 at 25°C Salt Solution: Composition per liter: NaHCO3 .0L..... 0................................................... Gently heat and bring to boiling................................ 50..... Mix thoroughly....................................................................... Enriched Composition per liter: Agar ....................... Use: For the cultivation and maintenance of Flexibacter columnaris.....0mL....0g Resazurin ..............1 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........... trichosporium and other methane-oxidizing bacteria...................... Mix thoroughly.0± 0.......0g Agar ......5........0mL............0L. Mix thoroughly..........................................0mg Na2S·9H2O solution .... Autoclave for 15 min at 15 psi pressure–121°C.... and Na2S·9H2 solution..........................................2 at 25°C Disodium Thioglycolate Solution: Composition per 10..0..... Mix thoroughly..........3 ± 0.............................. Gently heat and bring to boiling.............. 5.......... Preparation of Medium: Add components............................ Use: For the cultivation of Thermococcus species.............. 0...................... 10...................... Aseptically add 50............ Anaerobic Agar Composition per liter: Pancreatic digest of casein ......... sterile powdered sulfur........................ Mix thoroughly...........0g Beef extract .... Anacker and Ordal Medium Composition per liter: Agar ...................... 8. 2...0 with 8N NaOH.................... Sparge with N2.................................... Anacker and Ordal Medium......................................... 1................................5g CaCl2·2H2O ..02mg CuCl2·2H2O ...0mL Glucose solution .1.......................................2g Beef extract ... Use: For the cultivation and maintenance of Anaerobaculum thermoterrenum....0g K2HPO4 .........................2g pH 7................................ Cool to 25°C.....0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100..... 0...................................0g Glucose.................................................. 0...................................................................0g Pancreatic digest of casein ................0mg pH 7.................2 at 25°C Gently heat and bring to boiling................................. 0..............15..............0L................ powdered...........1 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... Preparation of Medium: Add components to distilled/ Use: For the cultivation and maintenance of Flexibacter Use: For the cultivation and maintenance of Methylosinus Anaerobaculum thermoterrenum Medium (DSMZ Medium 104a) Composition per liter: deionized water and bring volume to 1......0mL: Na2S·9H2O .. Distribute into tubes or flasks.... 40.......... AN1 Medium Composition per liter: Pancreatic digest of casein . 0... 10...............0g Pancreatic digest of casein ....0mL: D-Glucose ....... Autoclave for 15 min at 15 psi pressure–121°C.............................. Autoclave for 15 min at 15 psi pressure–121°C................................. Add L-cysteine..................... Gently heat and bring to boiling....0mL sterile glucose solution....................... glucose solution.............0g Yeast extract.................. Autoclave for 15 min at 15 psi pressure–121°C..... Autoclave for 15 min at 15 psi pressure–121°C....................................... 10...............3 ± 0......2.0g Trypticase peptone.................... Distribute into tubes or flasks..0mL.... Preparation of Medium: Add components....10......0....... to distilled/deionized water and bring volume to 990...........0g Sulfur.............. and sterile disodium thioglycolate solution under 100% N2.........................5. 1..........5g Sodium acetate .... Glucose Solution: Composition per 100............ to distilled/deionized water and bring volume to 940......... Mix thoroughly.. Sparge with N2..0mL....... Autoclave for 15 min at 15 psi pressure– 121°C................... Sparge with CO2............................ Gently heat and bring to boiling........... Pour into sterile Petri dishes or leave in tubes....... Mix thoroughly... Store anaerobically........2g Beef extract .................... Cool to 25°C. Filter sterilize.....................................0g MgSO4·7H2O..................................0g NaCl ..............................0g Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100.......15............. psychrophilus..............0g NaCl ........ 1..................0mL: Disodium thioglycolate........... 10..0g Peptone ....................0mL........................5....5g Resazurin .......................................5.............................................2....................0L.....................................1....... 0...1... except powdered sulfur and disodium thioglycolate solution..20...............3 ± 0..01mg pH 6.......0.......30 AN1 Medium NiCl2·6H2O .5g Yeast extract..... Yeast extract .................................................5............ Cool to 25°C..........................

...................0g Cellobiose ...........................................................0L with remaining water and add remaining compounds one at a time....................................0g Nitrilotriacetic acid ...............................................5g CaCl2 ............ Filter sterilize............................. 1........ 3..........5........0g Nitrilotriacetic acid...................... Mix thoroughly......................................01g H3BO3 ................ Boil medium under 80% N2 + 10% CO2 + 10% H2 until medium is colorless...... Add other compounds........ 0.......... 0......................... 0..................................................................01g AlK(SO4)2·12H2O..............................................................3.............. Cool to room temperature while sparging with 80% N2 + 20% CO2......1g ZnSO4·7H2O............ Anaerobic Citrate Medium Composition per liter: Ferric citrate .......1mL of sterile Na2S·9H2O solution to each tube.......2.....................0mL Na2S·9H2O solution............. 10...................0mg Riboflavin.......... 1................................................. 5..................................................01g Na2MoO4·2H2O.....0........................ Mix thoroughly..9 ± 0.................6g KCl ........1g Trace elements solution..... 1......................0g Sodium formaldehyde sulfoxylate .......0....0g Sodium thioglycolate ..............0mL...............0mg Folic acid .................................................................................................5g NH4Cl ...............................................0mg Wolfe’s mineral solution .............. Mix thoroughly...............01g Na2MoO4·2H2O ... Adjust pH to 7................... 2...........0mL of distilled/ deionized water and adjust pH to 6............0L with distilled/deionized water.....................................5g NaCl ............. 1.............. Na2CO3 Solution: Composition per 100................................................................0mL Na2CO3 solution.......... 0..........................1.........1g CaCl2 .......... 0................. and Bacillus lentimorbus..................5 with KOH to dissolve the compound............................10.............2 at 25°C Stopper the tubes anaerobically.......................... 0.5g K2HPO4 ......... Na2S·9H2O Solution: Composition per 100....1 at 25°C Wolfe’s Mineral Solution: Composition per liter MgSO4·7H2O ......................... .. Bring volume to 1....... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2..... Anaerobic Cellulolytic Medium Composition per liter: NH4Cl...................5g NaH2PO4·H2O .......................................... 1........0g Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100...... 10...............0mL...............0mL of boiling distilled/deionized water..............................................5....5g L-Cysteine·HCl·H2O ......................................... 0...2........................................................0g MnSO4·H2O.0mL pH 6...........0...........................................................................4g Resazurin ..........................................................0mL.......................8g NaHCO3 ............... 0............. es- pecially Bacillus larvae.................. Use: For the anaerobic cultivation of Bacillus species..........2................ Mix thoroughly.................................................................................. 0.......1g CoCl2·6H2O..................................10...................................................1g CoCl2·6H2O ........5...............0mg Calcium DL-pantothenate ..........0. 0........... to distilled/ deionized water and bring volume to 980.................0mL pH 7......2 ± 0..........1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..1...... Preparation of Medium: Add components to distilled/ Use: For the cultivation and maintenance of microorgan- deionized water and bring volume to 1..........................0mL of water and adjust to pH 6....0mg Nicotinic acid ...............0........................... 1.............................0.... Cool to room temperature.................................................0mL: Na2S·9H2O....................... 1..........1g FeSO4·7H2O ...............................................0.....................17............................ Autoclave for 15 min at 15 psi pressure–121°C.......01g H3BO3 ........................................5................................... Preparation of Trace Elements Solution: Combine components.....1g ZnSO4·7H2O ...................... Gently heat and bring to boiling....................................5g NaCl .....................................................0................................................6..... Bring volume to 1....................0 ± 0.. 0................. Add ferric citrate to 500..................0mg Biotin .........01g CuSO4·5H2O..............0mg p-Aminobenzoic acid .01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to approximately 500.................1mL of sterile Na2CO3 solution and 0.. except Na2CO3 solution and Na2S·9H2O solution.................................. Mix thoroughly.................................0L...................................................................................................2 at 25°C Trace Elements Solution: Composition per liter: Na2WO4 ...................................0g pH 7............................................Anaerobic Citrate Medium 31 NaCl ............................0mL Wolfe’s vitamin solution ..............1g AlK(SO4)2·12H2O ........0L..... such as Clostridium cellulovorans..................................0g Sodium acetate .........5g KCl.................0................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100....5........................0mL: Na2CO3 . 0......... Distribute into tubes until medium is 3 inches deep....................2.......10.....................................0mg Lipoic acid........ Mix thoroughly..........................................................................01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to approximately 500.................... Preparation of Medium: Add components........................25........................................... Filter sterilize...................... Mix thoroughly...0......... Mix thoroughly.......0............................................... 0...... 10..5 with KOH to dissolve............................ Bacillus popilliae.......1g CuSO4·5H2O ...........0g MnSO4·H2O ........... isms that can utilize cellobiose as sole carbon source.0g Yeast extract.....5g FeSO4·7H2O............................................0mg Thiamine·HCl ............................................ Autoclave for 20 min at 15 psi pressure–121°C............0mg Wolfe’s mineral solution .......0g MgSO4 ......0mg Vitamin B12..................... Aseptically add 0...........................................................5............................................. 15... Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl....... 1....................................0..............1............0mg NiCl2·6H2O.. Cool and distribute anaerobically into test tubes in 10.......20...........................24...................................0mL volumes using 80% N2 + 10% CO2 + 10% H2............................0L Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O............................

...................1................................. 0...................0.. 0.... Cool to 25°C while sparging with 100% N2.......................................................... and 50.......125g Na2S·9H2O .........18g CaCl2·2H2O........................................18g ZnSO4·7H2O ... Autoclave for 15 min at 15 psi pressure–121°C.................................................... Anaerobically distribute into tubes.....5........................ Autoclave for 15 min at 15 psi pressure–121°C....... 0................0g Na2HPO4·2H2O .....................................................................5................5....... Adjust pH to 7...................0mg Resazurin ........ Vitamin Solution: Composition per 100mL: Pyridoxine-HCl ...........05g FeSO4·7H2O.................0mL: Na2CO3 ........ 2.................................................................................................. Continue sparging with 80% N2 + 20% CO2...........................................0................................................... 0................. except starch solution...................................10.................... a novel thermoalkaliphilic bacterium that grows anaerobically at high pH and temperature................0mL of distilled/deionized water.................. 0...............0mg p-Aminobenzoic acid .........................................................5.......................................0mg Thiamine·HCl ........ Distribute to anaerobe tubes or bottles......................... 0............................0mg Calcium DL-pantothenate .0mg p-Aminobenzoic acid .................. Mix thoroughly...................1g NiCl2·6H2O ........................1g MgSO4·7H2O .................................0mg Nicotinic acid .......5g KCl ...... Add distilled/deionized water to 1............................................. 5...... 5..........0L..........0mg D-Ca-pantothenate........... Preparation of Medium: Add components....5............ Mix thoroughly....................................5.........................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500....................................... Sparge with 100% N2..2...... 10..............0mL sterile starch solution..........1............................................................0.........0mg Biotin .... Mix thoroughly...................5..................5g L-Cysteine·HCl·H2O.......5mg Na2CO3solution......0mg Biotin ....................1..................................................................................0mL Vitamin solution..........0mg Nicotinic acid ........................0g Preparation of Starch Solution: Add starch to distilled/deionized water and bring volume to 50....25g Tryptone ..... 0. Filter sterilize...........5g CoSO4·7H2O ................................ 5................. Sparge with 100% N2......................................1...0..........5g L-cysteine..................................................................... Use: For the cultivation of Anaerobranca gottschalkii........2................................ Mix thoroughly................................................. 0.........0mL: Starch.........................................................................................025g KAl(SO4)2·12H2O................. 2. 10..................... 10 fold conc....20..5...........0mL pH 9....0mL.................................................................. Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O.....1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.. 5........... Cool to 25°C.....0mL: Yeast extract ..............0mL Wolfe’s mineral solution .......................................... Aseptically and anaerobically add....5... 0.......... 20................. 0..... 5.............. and L-cysteine..............................0mg Lipoic acid .......... Dissolve by adjusting pH to 6.............0g MnSO4·2H2O............ 0...............2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O ....................................................................................01g Na2MoO4·4H2O ............................................................................................................. 0.................0mL Trace elements solution ....................0mL pH 8.....................................9g Sodium fumarate ................................... soluble ...............................................0mg Thiamine-HCl·2H2O ............................5g NH4Cl...........................................0mL...........01g CuSO4·5H2O ..........1g CaCl2·2H2O........................................................................................... 0.5 ± 0.......5g NaCl ....................... 1........ Na2CO3 solution....................................................................0....5 with KOH.............125g Wolfe’s vitamin solution ....5g CoCl2·6H2O..............................................0mg Vitamin B12 . 5...............................................................0..... Use: For the cultivation of Geobacter metallireducens.....3..........................3....................5g NaCl ..................0 with NaOH....50.........4g Yeast extract......0mL sterile Na2CO3 solution.......1g .....0g MnSO4·2H2O .......... Starch Solution: Composition per 50.... to distilled/deionized water and bring volume to 970.........0mg Riboflavin ....................... Anaerobranca gottschalkii Medium (DSMZ Medium 895) Composition per 1020mL: NaCl .................. 0.............................................0............. per liter of medium........................................... Mix thoroughly............................ 0.. Gently heat and bring to boiling.......................................................... Mix thoroughly.......0g Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100.....................01g Na2SeO3·5H2O ...................... Autoclave for 15 min at 15 psi pressure–121°C................. Sparge with 80% H2 + 20% CO2......... Mix thoroughly. Na2CO3 Solution: Composition per 100..............1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.......................................1g FeSO4·7H2O..........................0mg Vitamin B12........0g Nitrilotriacetic acid........................0. 5........ 0....... 0..10.........1g CaCl2·2H2O ..................................................................................0mg Folic acid .............0g Nitrilotriacetic acid ....................32 Anaerobranca gottschalkii Medium Add remaining components...................................0mg Riboflavin.............................................................................. 3........... 0................................... Filter sterilize......... 1..........1g ZnSO4·7H2O...........................0mL.............................02g H3BO3 ............ Anaerobranca Medium Composition per 1015......................... 10...........2 at 25°C Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl............................0mg Folic acid........................ Add remaining components............5g KH2PO4 ...........0L.............................0......... 0..............25g Na2S2O3·5H2O .............0L............... Add 0............................ Add distilled/deionized water and bring volume to 1........ Mix thoroughly........................................ 0....3-9.......... 2....5.......5g L-Cysteine ....................... 0..0mg Lipoic acid....0g K2HPO4 .............................. 1.................0mL Soluble starch solution......................0g (NH4)2SO4 ...................... Autoclave for 15 min at 15 psi pressure–121°C... Final pH is 9......................4 ± 0.......0mL.

..............0mg Biotin ..................... Mix thoroughly....Anaerocellum Medium FeSO4·7H2O..............................................0mL of HCl solution....2.......................33g MgCl2·6H2O... 5......................0mL......8.. 5..................5.....0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100...............3 at 25°C NaHCO3 Solution: Composition per 100................................ Anaerocellum Medium Composition per liter: NaHCO3 ..................50............................ Gently heat and bring to boiling..............01g Na2SeO3·5H2O .............5g Na2S·9H2O ...... 0.......3mg Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500......................... Cellobiose or Starch Solution: Composition per 50..................... Autoclave for 15 min at 15 psi pressure–121°C... Sparge with 100% N2....................01g Na2MoO4·2H2O .................... Mix thoroughly. 100......... Adjust pH to 6..........5...... Aseptically and anaerobically add sterile NaHCO3 solution....0mg Na2MoO4·2H2O...........02g CuSO4·5H2O ......01g H3BO3 . Add remaining components.................................. 0........................ ........33g KCl ......................100................................190.........0mg p-Aminobenzoic acid ..............33g KH2PO4 ..............................................33g NH4Cl.....0mg MnCl2·4H2O .................... to distilled/deionized water and bring volume to 990....................0.....................................0mg 33 Calcium DL-pantothenate ......................................................5 with KOH..............5mg NaHCO3 solution...................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10...............0.33g Yeast extract .......................5g Na2S·9H2O........... Na2S·9H2O Solution: Composition per 10.............. Use: For the cultivation of Anaerobranca horikoshii.......................................... Gas under 100% N2........ 0......1....................................................... Filter sterilize....0mg Lipoic acid............................. 0..... 0.................................................. Gas under 80% N2 + 20% CO2..... and sterile Na2S·9H2O solution.. Add distilled/deionized water and bring volume to 1......................................................................... Mix thoroughly.......36.......................................... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O..............5g CaCl2·2H2O................................0L.......................33g KH2PO4 ... 1...........................0mL Vitamin solution ..........................................0L....................0mg ZnCl2 ............................................................2......... Autoclave for 15 min at 15 psi pressure–121°C.........6.. 0...................... Anaerocellum Medium Composition per liter: Cellobiose or starch .................... Add components............025g KAl(SO4)2·12H2O...............................1g NiCl2·6H2O .................0mL Vitamin solution................................. Gas under 100% N2.......0mg Folic acid .................100....10........33g NH4Cl .................................................0.......0mg NiCl2·6H2O...........0mL Na2S·9H2O solution.........................................0mL: Na2S·9H2O.........0........ Continue boiling for 3–4 min.0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100............... Mix thoroughly.0mL pH 7.................. Mix thoroughly... 0.........................1–7........ 0..................... Mix thoroughly......0mg Nicotinic acid ............................................................... Mix thoroughly................ 0. Filter sterilize......... Mix thoroughly...................0mg Thiamine·HCl ..0mg CuCl2·2H2O................5g CaCl2·2H2O .....................0mL of sterile Wolfe’s vitamin solution................................. Gas under 100% N2....... Use: For the cultivation and maintenance of Anaerocellum thermophilum and Dictyoglomus turgidus....................... to distilled/deionized water and bring volume to 830..2.............................................................0mg HCl (25% solution) .................................... Filter sterilize..24.........70..5mg NaHCO3 solution ...........1................................................................................. Aseptically and anaerobically add 10.0mL............................ and Na2S·9H2O solution.................................0....................................... Mix thoroughly....0mL Na2S·9H2O solution ............................. 10.........0mL Trace elements solution SL-10 .................. 10.................................0mL: NaHCO3 ..........5..........33g Resazurin ......................................0.............................. Adjust pH to 8....................0mL: NaHCO3 ......5...............................0mL: Cellobiose or starch ...10.........0mL....0........0mg H3BO3 .................0mL.. Gas under 80% N2 + 20% CO2..................................... Mix thoroughly..3 at 25°C NaHCO3 Solution: Composition per 100.............................................................................................................................................0mL Cellobiose or starch solution ........................................................ Mix thoroughly..............0mL Trace elements solution SL-10...........................0mL.. Allow to cool to room temperature under 80% N2 + 20% CO2.................1.................................5............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......0g NaHCO3 . 5..................................... Add distilled/deionized water to 1. Adjust pH to 6......33g KCl................................ 10....................... 0....................1–7........ 0..... Preparation of Medium: Prepare and dispense medium under 100% N2..5g Yeast extract..............33g MgCl2·6H2O .... 0................. Preparation of Medium: Add components.....................................................0mL..... Add remaining components............0.0mL of distilled/deionized water.............. 1................................... Vitamin Solution: Composition per liter: Pyridoxine·HCl .............. Aseptically and anaerobically distribute into sterile tubes or bottles............. Distribute into bottles under 80% N2 + 20% CO2..................................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............................................. cellobiose or starch solution...................... 50...........................5g CoCl2·6H2O...........0L.. sterile cellobiose or starch solution.......................... except NaHCO3 solution.............5................. 0............................0g Preparation of Cellobiose or Starch Solution: Add cellobiose or starch to distilled/deionized water and bring volume to 50.............................0mL pH 7........... 0.........................0mg Riboflavin...... 0...... 10...................... Autoclave for 15 min at 15 psi pressure–121°C...0...... except Wolfe’s vitamin solution.................................5.......................... 0.0mL Cellobiose or starch solution.............5...0mg Vitamin B12...............2g Resazurin ............................. Autoclave for 15 min at 15 psi pressure–121°C..................................

.....................................4g NH4Cl .............8–7..................... Filter sterilize................................................................................................................................................... Mix thoroughly...... Gas under 80% N2 + 20% CO2.34 Anaerovibrio burkinabensis Medium Cellobiose or Starch Solution: Composition per 50....870.....................100.....0mg Riboflavin ..................................................0mL: Cellobiose or starch ..........................................0mg Lipoic acid ...................................0....0g KCl .................................5g CoCl2·6H2O .............0mg Na2MoO4·2H2O ............. Add remaining components.. cellobiose or starch solution.......................................................................0......... Gas under 100% N2......................0g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100.....70...................................... 10........... Allow to cool to room temperature under 80% N2 + 20% CO2.....................5....... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ......................................3g KH2PO4 ......... Solution B (Trace Elements Solution SL-10 ): Composition per liter: FeCl2·4H2O..........0mg Vitamin B12......................0L................................... 1..0...0mg Thiamine·HCl .. Solution C: Composition per 100............................................................................ 0..............0mL pH 6..... Mix thoroughly..........5.0mg Folic acid........................................... and Na2S·9H2O solution.....................................................................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10...................... Na2S·9H2O Solution: Composition per 10......0mg p-Aminobenzoic acid ............0mL...............0mL: Na2S·9H2O.............5.....0mL...................................... 1......................................................................0mL.........................................0mg Nicotinic acid ...0mg Riboflavin.............. Seal the flask under 80% N2 + 20% CO2..........0mg CuCl2·2H2O.......... 6............ 5................................................2.......0....0mL...............................5.....................................................0mg Biotin ......... Autoclave for 15 min at 15 psi pressure–121°C..............0mL Solution B (Trace elements solution SL-10) ....5.....100......................................................... sterile cellobiose or starch solution................................................................................................. Allow to cool to room temperature while gassing under 80% N2 + 20% CO2.0mg ZnCl2 ........... Continue boiling for 3–4 min.........0mL................................... Mix thoroughly.........0mL......................................15g Resazurin .................. Use: For the cultivation and maintenance of Anaerocellum thermophilum and Dictyoglomus turgidus.... 10........ 36.....10.. 190......10..........................................................................2 at 25°C Solution A: Composition per 870............0mL of HCl solution...... Distribute into bottles under 80% N2 + 20% CO2...... Autoclave for 15 min at 15 psi pressure–121°C...........................0mL of HCl solution... Autoclave for 15 min at 15 psi pressure–121°C............................................................. Mix thoroughly......0mg H3BO3 ......... 100..........0.......................0mg MnCl2·4H2O ...................................... Vitamin Solution: Composition per liter: Pyridoxine·HCl .......0.................................. Preparation of Medium: Add components...0mg H3BO3 ...............0mL Solution D .. Mix thoroughly...........2g CaCl2·2H2O ...........................1...0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10...... 5.......... Add distilled/deionized water and bring volume to 1..... Gas under 100% N2.......................................... Mix thoroughly.................. 70... Continue gassing until pH reaches below 6......... Gently heat and bring to boiling.................... 10...... to distilled/deionized water and bring volume to 830................... Gas under 100% N2.......... 2... Mix thoroughly............................10....5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10..................... Add distilled/deionized water and bring volume to 1.....0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870..........................................................................0L.....0mg HCl (25% solution) ........0mg Biotin ................0mg Lipoic acid....... Mix thoroughly.............0.......5..0mg NiCl2·6H2O .0L....... 24........................................0mL Solution C ..................................0mg p-Aminobenzoic acid ..............10...................5............0mL: NaHCO3 .........................1mg ...5g Preparation of Solution D: Add sodium lactate· to distilled/deionized water and bring volume to 10.......0mL Solution E (Vitamin solution) .................................................................... Mix thoroughly..................................................................0mg MnCl2·4H2O.0mg CuCl2·2H2O ........... except NaHCO3 solution..................... 5.................0mg Thiamine·HCl .........5g MgCl2·6H2O .......0mL: Na2SO4 .................... Mix thoroughly............ 5...............................................190.........1....................5g CoCl2·6H2O................................................ Mix thoroughly...24...................................... Gas under 100% N2........ Gas under 100% N2.......................3............. 0....2........................ 5.......2.... 5................. 5..........0mg HCl (25% solution) .............................................................0mg Folic acid ........ 2.................0mg Calcium DL-pantothenate.......................................................................0mL: Sodium lactate .. Add remaining components.1....0mL: Solution A ........ and sterile Na2S·9H2O solution..................................0g Preparation of Cellobiose or Starch Solution: Add cellobiose or starch to distilled/deionized water and bring volume to 50....0mL Solution F ........36..............6........... Autoclave for 15 min at 15 psi pressure–121°C.....0mg Vitamin B12 ..................2........ Aseptically and anaerobically add sterile NaHCO3 solution.......... Autoclave for 15 min at 15 psi pressure–121°C.0mg ZnCl2 ....1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.........................................0mg NiCl2·6H2O... 2.......................0mg Calcium DL-pantothenate ..................... Filter sterilize...................... 10................. Mix thoroughly.................................................... Anaerovibrio burkinabensis Medium Composition per 1001..........0g NaCl ................................. Autoclave for 15 min at 15 psi pressure–121°C..0mg Nicotinic acid ......0mg Na2MoO4·2H2O.....0mL Solution G ...... Solution E (Vitamin Solution): Composition per liter: Pyridoxine·HCl.......... Gently heat and bring to boiling................... Continue boiling for 3-4 min......... Solution D: Composition per 10.

.................. Ancylobacter-Spirosoma Agar Composition per liter: Agar ......... aged seawater ............................0g Andrade’s indicator ...... Pour into sterile Petri dishes or leave in tubes............. to distilled/deionized water and bring volume to 250.... Preparation of Medium: Add components..8 ± 0.... Solution G: Composition per 10. Bring pH to 7.......0g Yeast extract............................... trehalose.....5g Yeast extract ....4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............0g pH 6....3...................... Use: For the cultivation of Vibrio species............... Shewanella putrefaciens............................... Flectobacillus major..... mannitol.... fructose.............5g FePO4 .....5.... sorbitol............ Preparation of Medium: Aseptically and anaerobically combine solution A with solution B...2......... in that order....... Distribute into tubes or flasks... 20................... 1...0g Glucose .............. glucose..........................5g Yeast extract ......4–7..........1g Filtered............................................0... Use: For the cultivation and maintenance of Spirosoma lin- coccus species..... and solution G. Mix thoroughly.....0mL..................... 15.... 1...............10................ Use: For the cultivation and maintenance of Anaerovibrio burkinabensis..........4 ± 0.....5g FePO4 ......0mL.... Mix thoroughly.....................0L........0g NaCl ............0...4–7........ solution D..........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.. Mix thoroughly.......... Distribute into tubes or flasks... 750... Solution F: Composition per 10.0g Beef extract ........0L...........Andrade’s Broth Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1..... Autoclave for 15 min at 15 psi pressure–121°C... cellobiose.... inositol..........5g FePO4 ..... sucrose.... in tubes containing adonitol..... and Vibrio species.............. Mix thoroughly. Gas under 100% N2........ Gently heat and bring to boiling...... raffinose......................2 at 25°C Source: This medium is available as a prepared medium from BD Diagnostic Systems............ NaOH (1N solution) .............................. a lactate-fermenting bacterium isolated from rice field soils............................ or xylose....... Autoclave for 15 min at 15 psi pressure–121°C....... galactose................ 16...........1g Filtered...6... Distribute into tubes or flasks.............4....................................... Pour into sterile Petri dishes or leave in tubes.....0mL: Yeast extract... Autoclave for 15 min at 15 psi pressure–121°C...2....................................4–7.0.................0mL: Na2S·9H2O............0g ............................. maltose.................................................... aged seawater .....................0g Yeast extract. Anderson’s Marine Broth Composition per liter: Peptone ....... 1..........................0mL pH 7. solution E... Andrade’s Broth Composition per liter: Pancreatic digest of gelatin .......... Gently heat and bring to boiling........................0mL pH 7............4–7..................... Aseptically add 750.............................0mL of sterile aged seawater........ 750..2............1g Anderson’s Marine Agar Composition per liter: Preparation of Andrade’s Indicator: Add Acid Fuchsin to NaOH solution and bring volume to 100...0mL pH 7... 0......2..... 1.......... Methylobacterium mesophilicum.. Aseptically distribute into sterile tubes or flasks.1g Filtered........ except seawater........................0mL Acid Fuchsin ............ Use: For the cultivation of Ancylobacter aquaticus.....................2 at 25°C 35 Peptone .. Pseudomonas fluorescens..........0g Peptone............. 10. 15.....................................................................4 ± 0............ Mix thoroughly.... Runella slithyformis............ 50............ 1........ Agar . solution C........... arabinose..0L................... solution F..........0mL: guale.... Autoclave for 15 min at 15 psi pressure–121°C............ Gently heat and bring to boiling.........6 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............. Micro- Preparation of Medium: Add components to distilled/ lobacter species.......6.....0mL pH 7... Ancy- Use: For the cultivation of Flavobacterium species.............................. 1................. Ancylobacter-Spirosoma Medium (DSMZ Medium 7) Composition per liter: Agar ......0L. 1............2.............0g Peptone... lactose...................... salicin.. rhamnose.........................0g Glucose ............. Autoclave for 15 min at 15 psi pressure–121°C........ Mix thoroughly....0mL with distilled/deionized water............0g Preparation of Solution F: Add yeast extract to distilled/deionized water and bring volume to 10........... Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2................................ Adjust pH to 7................ Aquaspirillum serpens............. aged seawater .... Mix thoroughly. Anderson’s Marine Medium Composition per liter: Peptone . Gas under 100% N2............ Andrade’s Indicator Composition per 100. Distribute into tubes or flasks. Aquaspirillum metamorphum.....0L...1 ± 0.... Planococcus species.................. Autoclave for 15 min at 15 psi pressure–121°C.......0g pH 7.................... Mix thoroughly....... and Spirosoma linguale..................5g Yeast extract ............... Adjust pH to 7.................................................................. Autoclave for 15 min at 15 psi pressure–121°C....2 at 25°C deionized water and bring volume to 1.........................6 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................... dulcitol....0mL..................8...... Mix thoroughly................. 750......0..... Adjust pH to 6..0mL Carbohydrate solution .... Gas under 100% N2.. Use: For the cultivation and maintenance of Vibrio species..... Pour into sterile Petri dishes or leave in tubes................ Mix thoroughly... Autoclave for 15 min at 15 psi pressure–121°C.........................2.......

........ Autoclave for 15 min at 15 psi pressure–121°C........154g CuSO4·5H2O.......... phosphate......................................0mL Preparation of Metals Solution: Solubilize nitrilotriacetic acid with KOH...............0..0mg Cyanocobalamin....20.................................2.................................36 AO Agar Carbohydrate Solution: Composition per 100..1g KNO3 ......025g Na2B4O7·10H2O ..... Pour into sterile Petri dishes or leave in tubes.............................................. 15................................... 0............7g Nitrilotriacetic acid ......................................... Saprospira species..............0g CaCl2·2H2O.......2 ± 0..... 5.... 0....................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................5g Yeast extract.................20...................... mannitol..... Add aseptically to sterile basal medium..............018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100....5g Beef extract .................................. 0..2g pH 7... Use: For the isolation and cultivation of Cytophaga spe- cies...... raffinose........................................ Adjust pH to 7.......5.........0g Yeast extract....................................... Gently heat and bring to boiling..........................0mL pH 7.......................... Phosphate Solution: Composition per liter: K2HPO4 .. Cool to 25°C...................................0g Glucose........ A Durham tube is used to collect gas produced during the fermentation reaction............. Distribute into tubes or flasks........5g KH2PO4 . Vitamin Solution: Composition per liter: Pyridoxine·HCl...... 100...........................................................................................5g EDTA....... except carbohydrate solution..0g Peptone . Mix thoroughly.................................... Add 0...................... Filter sterilize and add aseptically to sterile basal medium..............................25g MnSO4·7H2O..2 with KOH or H2SO4...................... 5......................................... Add aseptically to sterile basal medium....................0g MgSO4·7H2O . Herpetosiphon species........ 0..................... Aolpha Medium Composition per 1041.......0g Preparation of Carbohydrate Solution: Add carbo- hydrate to distilled/deionized water and bring volume to 100................................... glucose................. Aplanobacterium Medium Composition per liter: Agar .........................0g Peptone............. Mix thoroughly............................ 5...... maltose.......0......0L........................ Dissolve remaining ingredients.................................... dulcitol.. AO Agar Composition per liter: Agar .............................0g KCl............................ Use: For the determination of carbohydrate fermentation reactions of microorganisms...................................0L.... sucrose..................0g Yeast extract ..................0mg Thiamine·HCl .................... phosphate solution....0mg Riboflavin.. cellobiose......... 29... 0. 10.........0mL........................... Mix thoroughly.....................0mg Na2MoO4·2H2O....................................... and vitamin solutions....0mg Calcium pantothenate .................. Mix thoroughly.2.............1g FeSO4·7H2O ............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................0L...... arabinose...7mg “Metals 44” ...............1. Autoclave for 15 min at 15 psi pressure–121°C...................... Autoclave for 15 min at 15 psi pressure–121°C........... Autoclave for 15 min at 15 psi pressure–121°C.......5g MgCl2·6H2O......................5. xylose........ Mix thoroughly............. or other carbohydrates may be used.............0.......... 1... Mix thoroughly....................0mL: Carbohydrate.................................... Preparation of Medium: Add components..........04g Co(NO3)2·6H2O. Autoclave for 15 min at 15 ..........................................................................................................................................................20.................... Gently heat and bring to boiling..................0mL: ZnSO4·7H2O... 0............12......0mL volumes into test tubes containing inverted Durham tubes......0L..................0.......... Filter sterilize.. 0.......... “Metals 44”: Composition per 100.........................................1g Metals solution...................... fructose.. particularly members of the Enterobacteriaceae...........0g Sodium acetate ......0L.. Mix thoroughly.5...................................5................2 ± 0.......... 3..................................................................................0g Agar . Mix thoroughly....................... salicin....................... 99. 9...........0mL Phosphate solution ............................... Distribute in 10......................0...............................5g Pancreatic digest of casein ....... lactose................................ Adjust pH of basal medium to 7................. Add aseptically to sterile basal medium.......1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1. Autoclave for 15 min at 15 psi pressure–121°C............... and vitamin solution—to distilled/deionized water and bring volume to 1................. inositol.0g CaCl2·2H2O................................2.......... sorbitol....5.............0mL...... 11........0mL: NaCl ..................... Acid production is indicated by a pink color...................................... Adonitol................2g (NH4)2SO4 ....... galactose.................0.0...............10......5................................... and Flexithrix species...........................................................0mg Biotin .5g Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 1....... to distilled/deionized water and bring volume to 1........... Use: For the cultivation and maintenance of Halomonas meridiana and other Halomonas species.10.... Gently heat and bring to boiling...................... rhamnose.............. Preparation of Medium: Add components—except “Metals 44”...0mL Vitamin solution...........2 at 25°C Metals Solution: Composition per liter: MgSO4·7H2O ........................................ Cool to 50°C and aseptically add the “Metals 44”.......1........... 10..0L....3g FeSO4·7H2O........................................................................ trehalose.....5mL of sterile carbohydrate solution to each tube.......................................................0mg Nicotinamide ......0mg Folic acid .... Caution: Acid Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin......2.0g pH 7...........................0 ± 0............. 50...0.................. Gently heat and bring to boiling................... Autoclave for 15 min at 15 psi pressure– 121°C..........

.......... Aquaspirillum Heterotrophic Agar Composition per liter: Noble agar .................. Mix thoroughly........1............... Pour into sterile Petri dishes or distribute into sterile tubes..........................3...............................5g NH4Cl ..................0g MgSO4·7H2O...................... 0.....................................2g CaCl2·2H2O ................... 0..........9........ Mix thoroughly... Adjust pH to 3..........................................0L............................... Autoclave for 15 min at 15 psi pressure–121°C...................... 5......1.0mg ZnSO4·7H2O.................................................... 30.....0L.....15....0.........................30...... 10............0g MgSO4·7H2O........1...................... 3............................................................. 0.........0g Peptone.....................03g Na2MoO4·H2O .........5..2g NaCl ..................................... Gently heat and bring to boiling...0mg NiCl2·6H2O....................... Use: For the cultivation of Zymomonas species...0mL Trace elements solution.Aquaspirillum Heterotrophic Agar 37 psi pressure–121°C.............................................................. Preparation of Medium: Add components...............................10...................................... add agar to 200......................15........ Trace Elements Solution: Composition per liter: H3BO3 ..................8 ± 0......... Autoclave for 15 min at 15 psi pressure–121°C..................................9............... For autotrophic growth...............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.............................0mg CoCl2·6H2O.. 0..........0........................ 1.......................................................... 0................................. 0.....................................5..................0mg MnCl2·4H2O ..03g NiCl2·6H2O .................................................. Mix thoroughly.............................0mg MnCl2·4H2O .................. Mix thoroughly............................... Aquabacter spiritensis Medium (LMG Medium 225) Sodium succinate ........2g CaCl2·2H2O..0mg ........ Aseptically add 20...........0L........................................ Pour into sterile Petri dishes or leave in tubes.............. Autoclave for 15 min at 15 psi pressure–121°C................. Aseptically add sterile NaHCO3 solution.......... except NaHCO3 solution...................0mg Ferric citrate ..................................0L of apple juice................................................................1 ± 0..............0mL: Agar .........................01g Ferric ammonium citrate ....... species................20......................10.........1........................... Mix thoroughly.....1 ± 0................. Use: For the autotrophic cultivation and maintenance of Aquaspirillum autotrophicum..1................. Adjust pH to 4..............................................2g MgSO4·7H2O .........4.................. 2.........................................................................................0L................................ 1.......... Filter sterilize...... 0......... except vita- min solution................5g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 10.....................0....................0mL SL-6 Trace elements solution .... Mix thoroughly.............................................................................0g Apple juice .............. Cool to 45°–50°C......................8.............................. Cool to 45°–50°C...02g CuCl2·2H2O..........................................0mg Biotin .. Autoclave for 10 min at 9 psi pressure–114°C........................ to 980.................................. 5...........................3.. 0...........3...... to double-distilled water and bring volume to 990..01g Preparation of SL-6 Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1........................................................................... 0..................... Aseptically combine the sterile apple juice solution with the sterile agar solution................................................. 0. Gently heat and bring to boiling.................................0mg CoCl2·6H2O........................2 at 25°C SL-6 Trace Elements Solution: Composition per liter: H3BO3 ........... 5....................................... 3..0g Na2HPO4·12H2O .. incubate under 85% H2 + 10% CO2 + 5% O2..... Mix thoroughly................................1.......................... In a separate flask..........................................................0 ± 0...................0mL pH 7........................................................................................................................ Preparation of Medium: Add components..... Mix thoroughly....................0mg ZnSO4·7H2O...............................................0mL distilled/deionized water...0.......... 0.................3... Vitamin Solution: Composition per liter: Calcium DL-pantothenate... Mix thoroughly..........1g MnCl2·4H2O............1...........5g NH4Cl ....... Pour into sterile Petri dishes................... Use: For the cultivation and maintenance of Xanthomonas Use: For the cultivation of Aquabacter spiritensis.0mL sterile vitamin solu- Aquaspirillum Autotrophic Agar Composition per liter: Noble agar ............0mg Vitamin solution...........20...................0.... 10...................... Cool to 45°–50°C..........0g Sodium succinate................................30.0mg Na2MoO4·2H2O.......................0mg Folic acid....0g Na2HPO4·12H2O ............... 2...................0mL pH 7.........0mL of distilled/deionized water and bring volume to 1................................0mg Trace elements solution...0mg Vitamin B12 ...................0mg CuCl2·2H2O......... 2......................0g Yeast extract..................0mg Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 1.............. 10.................................................... Aseptically distribute to sterile tubes or flasks.................................................................................................................... 0..................0L pH 4.............. Cool to 25°C............................................2 at 25°C Trace Elements Solution: Composition per liter: H3BO3 .0mg Thiamine·HCl ...01g Ferric ammonium citrate ...........0mL....0mL: NaHCO3 ..........2.1........0g KH2PO4 .............................................2 at 25°C NaHCO3 Solution: Composition per 10........................................0mL pH 7.2g ZnSO4·7H2O ...... 5.. Apple Juice Yeast Extract Medium (AJYE Medium) Composition per 1200...........0mg NaHCO3 solution..................2g CaCl2·2H2O ....0mg Na2MoO4·2H2O..20.................3g CoCl2·6H2O .........0g KH2PO4 ..0g KH2PO4 ...... tion............... Filter sterilize............................. Mix thoroughly...................2 at 25°C Preparation of Medium: Add yeast extract to 1.............................................................0mL......... Mix thoroughly...........................4g NH4Cl.....................0g Yeast extract.......0mg Riboflavin ...

Add Na2S·9H2O to distilled/deionized water and bring volume to 50....................0g Preparation of Na2S·9H2O Solution: Prepare and dispense solution anaerobically under with 80% N2 + 20% CO2.............................0L.... 2....................0........................................0...... 50.......0mL of HCl solution..........5. to distilled/deionized water and bring volume to 1................................ 36..........................................................0mL Trace elements solution.......0mL pH 6... 1...............................45g Sodium L-lactate...........................0L.........................0mg Riboflavin .....5g (NH4)2SO4 ...0...........................................5g H3BO3 ....................... 1.......... 30.............................. 100...0g Nitrilotriacetic acid........ 24.............................................. Autoclave for 15 min at 15 psi pressure–121°C...........0g MgCl2·6H2O ........0. 1........................0mg Sodium succinate solution ........0............ 50..................... Autoclave for 15 min at 15 psi pressure–121°C................ Sodium Succinate Solution: Composition per 10mL: Sodium succinate........................ Preparation of Medium: Add components.. 50................................... 10........ Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ......... Autoclave for 15 min at 15 psi pressure–121°C........................... Cool to room temperature...........................0g Na2S2O3·5H2O ........................... Mix thoroughly.......5g CaCl2·6H2O....4...1............ Use: For the heterotrophic cultivation and maintenance of Aquaspirillum autotrophicum....... Cool to room temperature...................................0....... Autoclave for 15 min at 15 psi pressure–121°C............... Mix thoroughly...............0mg ZnCl2 ........................................5........0g MgSO4·7H2O ................................... Add remaining components.....01g H3BO3 ...0...............0mL................9 ± 0...........................5g NaCl ........... 2.0.....................................0L with Thiosulfate Solution: Composition per 10mL: ............0mg Ca-pantothenate ......................... 10..5g Yeast extract ...............................0mL sterile thiosulfate solution.... Autoclave for 15 min at 15 psi pressure–121°C..0L.................................... Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500................................ 1...................... Use: For the cultivation of Aquaspirillum spp...........0............. and trace elements solution SL-10................0mg CoCl2·6H2O ...0g MgSO4·7H2O..............................14g Fe(NH4)2(SO4)2·7H2O ............................. 300........ 190...................5g CoSO4·7H2O......................... 25.......................0mL pH 7.......1g CaCl2·2H2O ..........................................1......... Mix thoroughly.... 1..................................0g NaHCO3 .............................34g NH4Cl ................0mg Na2MoO4·2H2O ...... 0.......... and 1............................0...........1.......................................2...... Sparge with 100% N2............01g Na2MoO4·2H2O.........................................5g KCl ...........................0g MnSO4·2H2O................................................................................................... Mix thoroughly........................... Aquaspirillum Medium (DSMZ Medium 888) Composition per 1026mL: Casamino acids ............................. 5.........................0mg CuCl2·2H2O ............... Gently heat and bring to boiling..................... 0......................................................0.5.........3...................0mg Folic acid.............................. Adjust pH to 7............................................0mg Na2H2PO4..1mg Preparation of Standard Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.............1....0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1..........................10. Archaeoglobus Medium Composition per liter: NaCl .................. standard vitamin solution........................... 0.0L..0mL Thiosulfate solution .............................................01g Na2SeO3·5H2O ...................18.......2 at 25°C Standard Vitamin Solution: Composition per 100mL: Thiamine-HCl·2H2O .........1...0........0mg CuCl2·2H2O .1..........................................0mg Pyridoxine-HCl ..14g K2HPO4 ... 1.......0mL sterile standard vitamin solution...........1. Filter sterilize.................................0mg NiCl2·6H2O . 1.................. Cool to room temperature....................... Add remaining components...................... Mix thoroughly.........................................02g CuSO4·5H2O...5........2mg Biotin .................. except so- dium succinate solution...............5 ± 0...................................... Autoclave for 15 min at 15 psi pressure–121°C....... Mix thoroughly....0mL sterile trace elements solution SL-10.....................................................0mg Vitamin B12 ..............0mg Resazurin ....................................................0mg Na2S·9H2O solution .........38 Aquaspirillum Medium NiCl2·6H2O ........... Mix thoroughly............................. Mix thoroughly...................3...............................................0.............7...................0g Preparation of Sodium Succinate Solution: Add sodium succinate to distilled/deionized water and bring volume to 10.................. Bring volume to 1.... Preparation of Medium: Add components to doubledistilled water and bring volume to 1.......0mL sterile sodium succinate solution...3mg Na2S·9H2O Solution: Composition per 50....................................... Mix thoroughly.. Pour into sterile Petri dishes or distribute into sterile tubes..........................................1g NiCl2·6H2O....... Add distilled/deionized water and bring volume to 1.................. Aseptically distribute into sterile tubes or flasks...........................................18g FeSO4·7H2O ...0g Agar .......0mL.....................0mg HCl (25% solution) .....0mL Trace elements solution SL-10 ....................................................................0mL Standard vitamin solution ....0..........................7mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.. Dissolve by adding KOH and adjust pH to 6...............0.......................................................... Mix thoroughly....................0mL: Na2S·9H2O ... 10............................18g ZnSO4·7H2O..................0mL............ 70................................................................025g KAl(SO4)2·l2H2O ...... Preparation of Thiosulfate Solution: Add Na2S2O3·5H2O to distilled/deionized water and bring volume to 10............0.........................10........................................0mL..........0mL of distilled/ deionized water...........0mg Nicotinic acid ...........................0......................................2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O.............................................................. Adjust pH to 7.. Aseptically add 10.................................... Cool to room temperature.................................................... 1................. thiosulfate solution......................................................25g CaCl2·2H2O ...................................................................0mg MnCl2·4H2O................. 50....

.5 ± 0.....................Archaeoglobus veneficus Medium additional distilled/deionized water. except NaHCO3 and Na2S·9H2O solution... Prior to inoculation of cultures....10...............18g CaCl2·2H2O ..................5g CuSO4·5H2O..........0g MnCl2·4H2O .............25...................................0g FeSO4·7H2O .......................05g Na2MO4·4H2O...................................................................................... Archaeoglobus veneficus Medium (DSMZ Medium 796) Composition per liter: NaCl ...0...................................... 1..........................5g KCl............. 0....0........... 4............................................. Filter sterilize..... Adjust pH to 7..........34g NH4Cl...................................14g Fe(NH4)2(SO4)2·6H2O ..........18...... Archaeoglobus profundus Medium Composition per liter: NaCl .......... 3...... 0.............. Sparge with N2..............0g KCl ... 0.....0................................................................ 39 Preparation of Medium: Add components.........0........... 0......0 with KOH.......................... Sparge with 100% N2....................................0mg Resazurin .............. 0.01g Na2SeO3·5H2O ....... Use: For the cultivation and maintenance of Archaeoglobus profundus.. 0............................................................. Filter sterilize........ 1.........0..............................................0mL: Na2SO3 ....................................... Autoclave for 15 min at 15 psi pressure–121°C.................................. 1. Autoclave for 15 min at 15 psi pressure–121°C............................... except NaHCO3 and Na2S·9H2O solution............ Distribute anaerobically under 80% N2 + 20% CO2 and pressurize sealed containers up to 2 bar pressure........ 18.....................................0.0g NaHCO3 ..0mL Trace elements solution.....................................15g NaHCO3 ................ 0...........25g K2HPO4·3H2O .33g NH4Cl ......0mL Na-acetate solution ....... Cool to 25°C............................................... Add NaHCO3 and adjust pH to 6..........................0mg Na2S·9H2O solution.. 0...................0mL of distilled/ deionized water.....0mL........01g H3BO3 ...................... Sparge with 100% N2......................................... 0...............1g CaCl2·2H2O............0mg Resazurin ........5g Preparation of Na-acetate Solution: Add Na-acetate to distilled/deionized water and bring volume to 10.......... Mix thoroughly...........14g Fe(NH4)2(SO4)2·7H2O .......................................... Add Na2S·9H2O to distilled/deionized water and bring volume to 50................25mL of sterile Na2S·9H2O solution to each tube containing 9..........................................................05g ...........0................. Mix well and heat to boiling for a few min...................................9...................................................................0mL Na2S·9H2O solution ................... Na2SO3 Solution: Composition per 10............. Cool rapidly to room temperature while gassing with 80% N2 + 20% CO2...............................0g MgCl2·6H2O .................................5g NaCl .45g Na2SO4 .............................................0mL...0g MgSO4·7H2O ...................7.5g CoSO4·7H2O ..........................0g Preparation of Na2S·9H2O Solution: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2..... Mix thoroughly... Bring volume to 1..................... Add NaHCO3 and adjust pH to 6...............0mL: Na2S·9H2O............................. Preparation of Medium: Add components...................0.....................................0g Nitrilotriacetic acid ............................................0...................025g KAl(SO4)2·l2H2O............................................................... 0....................... Distribute anaerobically under 80% N2 + 20% CO2 and pressurize sealed containers up to 2 bar pressure... Use: For the cultivation and maintenance of Archaeoglobus fulgidus.....................................................3mg Na2S·9H2O Solution: Composition per 50.......................................................... add 0......75mL of sterile basal medium........0L...........................................................................0......2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O .............................................. Adjust pH to 7....................25mL of sterile Na2S·9H2O solution to each tube containing 9...................... Mix well and heat to boiling for a few min.0L.......0.................0mL pH 6............01g Na2MoO4·2H2O ....5. Prior to inoculation of cultures....................... to distilled/deionized water and bring volume to 1.....................................................................18g FeSO4·7H2O............ Trace Element Solution: Composition per liter: NaCl ......................0g MgCl2·6H2O......................................1g NiCl2·6H2O .................... 1............9..1..................... Store anaerobically......................................0mL Trace elements solution ....75mL of sterile basal medium................................. 0.......5g CaCl2·2H2O ..................................... 20....................02g CuSO4·5H2O ......... 0.......................0................ 2......0g MnSO4·2H2O .................................. 1.... Na-acetate Solution: Composition per 10...0L with additional distilled/deionized water....14g K2HPO4 .......... 1........................... to distilled/deionized water and bring volume to 1......0 ± 0................................................5g CoCl2·6H2O............0g Yeast extract.............5g ZnSO4·7H2 .... 0............0mL.....................0mL.............................................................................. 0............................0mL: Na-acetate................................................9g (NH4)2Ni(SO4)2 ...5....... 2.... 10.. 4.........5mg Na2SO3solution .............................................................................. Preparation of Trace Elements Solution: Add ni- trilotriacetic acid to approximately 500....0..0 with KOH...........2...........................0.....2...........................2 at 25°C Na2S·9H2O Solution: Composition per 20..0mL pH 1........... Adjust pH to 7...... Autoclave for 15 min at 15 psi pressure– 121°C........ 1..........18g ZnSO4·7H2O ......25g CaCl2·2H2O................................................ Cool rapidly to room temperature while gassing with 80% N2 + 20% CO2.5..............0......0mL: Na2S·9H2O .................................................5g Preparation of Na2SO3 Solution: Add Na2SO3 to distilled/deionized water and bring volume to 10.......... add 0.......05g KAl(SO4)2·12H2O ..........................................................05g H3BO3 ............................................ Autoclave for 15 min at 15 psi pressure–121°C.............................................................................. Add remaining components.... Mix thoroughly................ Dissolve by adding KOH and adjust pH to 6....... 0. 3................................2....0...................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20................................7g Sodium acetate ........................ 0..........................................................

..........5g KCl ........................... to distilled/deionized water and bring volume to 990........ 50. Equilibrate with the 80% N2 + 20% CO2 gas... 0.......................0.... Autoclave for 15 min at 15 psi pressure–121°C..0mL Artificial organic lake vitamin solution.............1g L-Valine ....... Archangium violaceum Medium Composition per liter: Monosodium glutamate ......9............0g Yeast extract......154g Co(NO3)2·6H2O .................... Phosphate Supplement: Composition per liter: K2HPO4 ... 0......0mL.............. and NaHCO3.............................4g CaCl2·2H2O....................................... 0................ 0.............1g L-Asparagine .............. Cool to 25°C................................................ Boil for 5 min........2 at 25°C Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O ......... Adjust pH to 1.....................0.............1..................... Pour into sterile Petri dishes or leave in tubes...... Mix thoroughly.3g L-Proline............................. Add 10...................25g MnSO4·7H2O .....7g Nitrilotriacetic acid...............34g FeSO4·7H2O............................. Mix thoroughly............................ Mix thoroughly................. 0................................................................. 25........04g EDTA..................................0g Noble agar......1mL sterile Na-acetate solution and 0....... After inoculation pressurize to 1 bar atmosphere with 80% H2 + 20% CO2 gas.......... Store at 5°C..0... Mix thoroughly............4............................0g L-Leucine ....................................................1g FeSO4·7H2O........................... 0.................................................................5.5g KH2PO4 ................... 0.... Mix thoroughly............0g CaCl2·2H2O.......................5g Preparation of Phosphate Supplement: Add components to distilled/deionized water and bring volume to .....0mL medium in 100mL serum bottles................. Use: For the cultivation of Archaeoglobus veneficus...........10.0L pH 7......................................... to 986.............................. Preparation of Medium: Add components.......................................................................... 1....05g Na2SeO3·5H2O ............10....................................0..................................................... 20....02g KCl................................0 with HCl........0.................. pH 7... Artificial Organic Lake Peptone Medium Composition per 1001..........................................29...................0g Peptone ...............................3 ± 0.................... Artificial Deep Lake Medium Composition per liter: NaCl .................0..........................25mg “Metals 44” .......... 10.............3...............0mL: NaCl .........1g L-Threonine.. There may be a slight precipitate................99....................... Use: For the cultivation and maintenance of Halobacterium lacusprofundi....... Na2S·9H2O solution............... 20............................................................................ 1............................0mL pH 7.. Filter sterilize and add aseptically to sterile basal medium.0.... 30...................... 0...0.........................0mL Phosphate supplement ...................1................................................... 1.............. .......................... Add components..............10. Cool to 50°C...............1............................ Adjust medium to pH 7........ Cool to 25°C while sparging with 80% N2 + 20% CO2........5g CuSO4·5H2O ......................................02g Tris(hydroxymethyl)aminomethane buffer (0...... 75....................................1g L-Serine .................. 0.......5g L-Isoleucine..................... 0..05g L-Histidine...................... except vitamin solution........... .............5.....1g L-Alanine..................................0mg Preparation of Trace Element Solution: Add components to distilled/deionized water and bring volume to 1...............................................40 Archangium violaceum Medium Na2WO4·2H2O ................. 1..2 at 25°C Vitamin Solution: Composition per liter: Biotin ................ 0..0g MgCl2·6H2O. Autoclave for 15 min at 15 psi pressure–121°C..018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100........................................................................................0mL medium..0mg Thiamine·HCl ...........................2g KNO3 .... Gently heat and bring to boiling........................... Add other components and adjust the pH to 7.......... 0........0g CaCl2·2H2O .......0g MgSO4·7H2O.........................0L..............9...................................0mg distilled/deionized water and bring volume to 1... 15..... Aseptically add 10..................................................20........................... Aseptically distribute into tubes or flasks................. 7...................................1g Modified Hutner’s basal salts........0g MgSO4·7H2O .......0g Sodium succinate ...............0mL of vitamin solution............5) ......... Mix thoroughly...............0mL Na2S·9H2O solution...........2g L-Lysine.25g MgSO4·7H2O ...... Add the solid NaHCO3............................................... 7............... 0.............................................................05g Cyanocobalamin................................................05g Ca3(PO4)2 ..0g Yeast extract ..............02m solution......................................................................................................... Mix thoroughly...0mg Ammonium molybdate.....................................................0.................................0g Vitamin solution..5g L-Tyrosine ........................................05g L-Methionine. Sparge with 100% N2.................................0.....................................................2....................................0L... Adjust pH to 7...15g L-Arginine ...4g KCl.................................................... 0...................................2 at 25°C Preparation of Medium: Add solid components to 1......... Add aseptically to sterile modified Hutner’s basal salts solution...................4 ± 0.........................0L of Tris buffer.................................. Autoclave for 15 min at 15 psi pressure–121°C................................. 180..................................5 ± 0................... “Metals 44”: Composition per liter: ZnSO4·7H2O ....................................1g (NH4)2SO4 .... 0...5mL Na2SO3 solution per 25.................2 with KOH or H2SO4..... 0.................................................................................................................. Filter sterilize..... Gently heat and bring to boiling....0..............0mL pH 7... Aseptically and anaerobically inject 0........ Distribute into serum bottles under 80% N2 + 20% CO2 gas.....0..0mL Preparation of Modified Hutner’s Basal Salts: Dissolve the nitrilotracetic acid first and neutralize the solution with KOH.......................05g L-Glycine.............. except Na-acetate solution............ Preparation of Vitamin Solution: Add components to Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture.................................. Use: For the cultivation of Archangium violaceum...2....025g Na2B4O7·10H2O....30.................................................0mL distilled/deionized water................. 0.......................................0mL..........

........30.75g CaCl2·2H2O.... 0......................10.........0g ........ Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C....... 0.............................. Asticcacaulis Medium Composition per liter: Agar .........................................100................. Use: For the cultivation of Halomonas meridiana.... Gently heat and bring to boiling.......... Mix well until dissolved......5g KCl...0mL of sterile phosphate supplement solution............................................ Cool to 45°–50°C.............................ASTM Nutrient Salts Agar 20.......... A-5 Trace Metals: Composition per liter: H3BO3 .... Use: For the isolation and cultivation of Asticcacaulis species.81g ZnSO4·7H2O................................................. and 1.. 0...... 15............ 0...................................0mg Thiamine·HCl ......................... 15.............................0.................... that can utilize atmospheric N2 as sole nitrogen source.........0mg Folic acid..................0mL...... Ashbey’s Nitrogen-Free Agar Composition per liter: Agar .......0g K2HPO4 ..................................0mL of sterile modified Hutner’s basal salts solution...... Preparation of Medium: Add components.....................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....86g MnCl2·4H2O ......................0...............................039g Preparation of A-5 Trace Metals: Add components to distilled/deionized water and bring volume to 1............... Pour into sterile Petri dishes or distribute into sterile tubes.....0mL pH 7............................. Mix thoroughly........... Aseptically add 20.......................2g MgSO4·7H2O ........0mL A-5 trace metals ........................ 5...................... 10. Cool to 50°C...0mg Nicotinamide............................................ Artificial Organic Lake Vitamin Solution: Composition per liter: Pyridoxine·HCl ..................... Asparagine Broth Composition per liter: DL-Asparagine..... 3.....0g Preparation of Agar Solution: Add agar to glass distilled water and bring volume to 100.................... For the isolation of cyanobacteria from marine habitats......0mg Magnesium EDTA ....... Use: For the isolation and cultivation of bacteria..0mg Biotin .......... 25.......................1................... 2............................................................ phosphate supplement solution........ 3....................... to glass distilled water and bring volume to 900........ Use: For a presumptive test medium in the differentiation of nonfermentative Gram-negative bacteria..9 and 7..... such as Azotobacter species and cyanobacteria................5g Yeast extract .5g Sodium acetate ........... ASN-III Agar Composition per liter: NaCl ..........1..... and vitamin solution................................................................. Adjust pH to between 6. Distribute into tubes or flasks......1...................................................2... 2....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............................0g CaCl2·2H2O.....2..............................................1mg Preparation of Artificial Organic Lake Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..................0mL..... Filter sterilize. Pour into sterile Petri dishes or leave in tubes............0g Pancreatic digest of casein .......... 20.............0........... 2...............................................10...............2g pH 6.........0L........ to distilled/deionized water and bring volume to 959...........0mg Ferric ammonium citrate...............................0L......15..0g NaNO3 ....0mL: Noble agar .0................ For use in the multiple-tube technique in the microbiological analysis of recreational waters..........................................9–7.................5mg Vitamin B12 ................ 0.....0mL..................... 0................0.......... Autoclave for 15 min at 15 psi pressure–121°C..5g MgCl2·6H2O.........................0..0L......5g Na2CO3 ..................................... except agar solution..............05mL pH 7...........0mL of sterile artificial organic lake vitamin solution........ Store at 5°C..... 0...................3....................................................................... Preparation of Medium: Add components................. Use: For the cultivation of Xenococcus species.......... Autoclave for 15 min at 15 psi pressure–121°C................................................... Pour into sterile Petri dishes or leave in tubes.......0mg Calcium DL-pantothenate...3 ± 0....... 5..........0µg Agar solution....02g Citric acid....................... Gently heat and bring to boiling..........................0...... Mix thoroughly.................................. Aseptically add agar solution..........................2g MoO3 (10% solution).... Mix thoroughly......... Mix thoroughly............ 3....... especially Pseudomonas aeruginosa.15.......................................... Distribute into tubes or flasks............................0............ 5...........................................75g K2HPO4·3H2O...0L....................0...................... Mix thoroughly...........0g MgSO4·7H2O .. ASTM Nutrient Salts Agar (American Society for Testing and Materials Nutrient Salts Agar) Composition per liter: Agar ......... Mix well and heat gently until dissolved............ Autoclave for 15 min at 15 psi pressure– 121°C.............079g Co(NO3)2·6H2O.............................. 0.....................2 at 25°C 41 Agar Solution: Composition per 100.............................1mL FeCl3 (10% solution.................222g CuSO4·5H2O......... Mix thoroughly.................2g K2HPO4 ..... 0........... Aseptically distribute into sterile tubes or flasks.................................8 ± 0..................................0mg Cyanocobalamin ....... Warm to 45°–50°C... except modified Hutner’s basal salts solution......................................0mL............. Distribute into tubes or flasks.......................0...................................5g pH 6....................................................................... Autoclave for 15 min at 15 psi pressure–121°C............................0L...... Bring pH to 7........ Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly..................0g MgSO4·7H2O...............2 ± 0.............................0mg Riboflavin ................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... Gently heat and bring to boiling.............. 0...0g Mannitol.049g Na2MoO4·2H2O........... 5........................................... Filter sterilize....................... Mix thoroughly........

........................................0g KH2PO4 ........................1........ especially those from wastewater.................... 1................0mL Chelated iron solution ..................................................0g KH2PO4 .................0mg ZnSO4.................5mg Preparation of Trace Metal Solution: Add components to distilled/deionized water and bring volume to 100..........6g CaCl2·2H2O..............0mL: Use: For the cultivation and enumeration of fungi isolated Use: For the cultivation of Porphyridium purpureum.......0g K2HPO4 ............................... Chelated Iron Solution: Composition per 100.............................................. 0....0mL: H3BO3 .10.......0mL of sterile oil to 990.....0M.......................... Atlas Oil Agar Composition per liter: Preparation of Medium: Add components............. Filter sterilize.................. Mix thoroughly............. .....................................0........................ Distribute into tubes or flasks............0L.......................0mL of sterile aureomycin solution..................0mg KHCO3 .......0....0L..0mg CuCl2·2H2O ..... Autoclave for 15 min at 15 psi pressure–121°C....................................2g FeCl3 ......................................................... 1................................................................................ Mix thoroughly................................. 0..2 at 25°C from sewage and polluted waters..................0mg MnSO4·H2O .................. 2..............0mL................ Mix thoroughly......... 5...............10.........................7g NH4NO3 .............7g K2HPO4 .....0L....... AT5N Medium Composition per liter: CaCO3..................... Use: For the cultivation of bacteria that oxidize ammonia...15.................................................. Aseptically add 200.....5 ± 0............. Gently heat and bring to boiling..0mg Preparation of Medium: Add components to tap water and bring volume to 1....0g NH4NO3.......................07g NaHCO3 ............................... Gently heat and bring to boiling..........5g MgSO4 ..............0mg pH 6..................................... 2............ Aseptically add 10....04g Tris-HCl buffer (1...........4 ± 0.......................................................................0mg CaCl2·2H2O ............................................................................0g MgSO4·7H2O ...............0g Peptone . Cool to 60°C.....................42 ASW Medium KH2PO4 ..20..............6....................................0mg ZnCl2 .................. 40......5g KNO3 ...................................0mL....................0mg (NH4)6Mo7O24·4H2O . 1........... Autoclave for 15 min at 15 psi pressure–121°C....1......0g KH2PO4 .............................. fungal degradation...............0mL............... Use: For the cultivation and enumeration of hydrocarbonutilizing bacteria by direct plating of water and sediment samples...... Autoclave for 15 min at 15 psi pressure–121°C................................... 0................. Use: For determination of the susceptibility of plastics to Preparation of Medium: Filter sterilize oil...................................................................... Mix thoroughly.. Pour into sterile Petri dishes or leave in tubes.................... 37......................5g K2HPO4 ............. 200.. Autoclave for 15 min at 15 psi pressure–121°C........................................ Mix thoroughly....................................... Gently heat and bring to boiling......................20......................................1....5g Rose Bengal.... Gently heat and bring to boiling........... Gently heat and bring to boiling..................... Mix thoroughly.....0.........................0mL: FeCl3·4H2O ......6g Preparation of Chelated Iron Solution: Add EDTA to distilled/deionized water and bring volume to 100..0mL Oil .0mL pH 7................ sterile Bushnell-Haas agar..........0.................. 15...............0mL Trace Metal Solution: Composition per 100..................... 0.................................................0mL pH 5............................................................................ 1.................7g MgSO4·7H2O ..................................5................ Bushnell-Haas agar ......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......0mL: Aureomycin·HCl ............0g NaCl ...............................................0............... ASW Medium (Artificial Seawater Medium) Composition per liter: NaCl ........0g Agar ...........0mg CoCl2·6H2O ............. Pour into sterile Petri dishes or distribute into sterile tubes.............0g Glucose.....2 at 25°C Agar ............................ Put mixture into a sterile blender container................ to distilled/deionized water and bring volume to 800.............0mL......... Cool to 45°–50°C................... Autoclave for 15 min at 15 psi pressure–121°C.................................................................................... 240.............2 at 25°C Aureomycin Solution: Composition per 200...............................................1.. 5.0g MgSO4·7H2O...............................0mL of cooled...........20................... pH 7.............................................. Distribute into tubes or flasks....0g MgSO4·7H2O...........................1........... except aureomycin solution........ Mix thoroughly............................10..................... Adjust pH to 7.02g Preparation of Medium: Add components to tap water Preparation of Bushnell-Haas Agar: Add components to distilled/deionized water and bring volume to 990....6g MgCl2·6H2O.................................... 4........0mL. 6...... Mix thoroughly....... 4.............................990...... Aureomycin® Rose Bengal Glucose Peptone Agar Composition per liter: Agar ...................07g Preparation of Aureomycin Solution: Add aureomycin·HCl to distilled/deionized water and bring volume to 200........... Pour into sterile Petri dishes...0g (NH4)2SO4 .............05g CaCl2·2H2O ............................. 1........ 27..................................................... Pour into sterile Petri dishes or leave in tubes..... Bushnell-Haas Agar: Composition per 990.......................... 60......... Distribute into tubes or flasks........50................... 0............. Mix thoroughly..... Add FeCl3·4H2O..........1............................................30.6)...................................................0mg MnCl2·4H2O...................................................0..................... and bring volume to 1.............................................................................. Mix thoroughly..............035g Aureomycin solution .0.............................0mL Trace metal solution....0mg FeSO4·7H2O.............................................0mg EDTA . Blend on low speed to minimize the incorporation of air into the medium................. 14..............1..0 ± 0...

..............0g Stock solution.....15..... 97...........0g NaCl .........0L.............................................................................................. Azides also react with metals and disposal must be highly diluted...................0mL Trace elements solution .............0g Pancreatic digest of casein ................ 0...........0mL.7.......... 1.................................................0L...........................................0mL pH 7..................................0mL pH 7...........................3........5............ 50...... Mix thoroughly................ Azide Blood Agar Composition per liter: distilled/deionized water and bring volume to 1.................. Adjust pH to 8... 0....................... 49........15....0g Beef extract ......0g Beef extract ................. Aseptically add 50.. Distribute into tubes or flasks....0mg Caution: Sodium azide is toxic....... Azides also react with NaCl ........................................... Mix thoroughly.................0g NaN3 .......................5....1.......................10mg H3BO3 ......... Trace Elements Solution: Composition per liter: (NH4)Mo7O2 . Use: For the isolation and enumeration of fecal streptococci from nonclinical specimens such as water............................. FeSO4·7H2O.................0g Glucose.. Autoclave for 15 min at 15 psi pressure–121°C.......................2 at 25°C Stock Solution: Composition per liter: 43 Adjust pH to 8... Mix thoroughly..................... Allow tubes to cool in a slanted position........................................ Azide Broth (Azide Glucose Broth) (Azide Dextrose Broth) Composition per liter: Pancreatic digest of casein .................. Preparation of Medium: Add components...5..................... 97...........................................15..... Autoclave for 15 min at 15 psi pressure–121°C.............0g KH2PO4 .....................................100................. Cool to 45–50°C........ Use: For the cultivation of Nitrobacter winogradskyi.........................5g MgSO4·7H2O .............5.....80mg CuSO2·5H2O ..............0L............2 ± 0.....................7..........................07g Preparation of Stock Solution: Add components to distilled/deionized water and bring volume to 1.. Mix thoroughly.2 at 25°C Caution: Sodium azide is toxic. 437........................................1.5g CaCO3 .......... to distilled/deionized water and bring volume to 950.....2 at 25°C Trace Elements Solution: Composition per liter: Source: This medium is available as a premixed powder from BD Diagnostic Systems....... 50.......0.............. Also used for the isolation of Streptococcus pneumoniae and Erysipelothrix rhusiopathiae...........0mL pH 7.....5 ± 0.100........ except ponents to distilled/deionized water and bring volume to 1........................................................ except sheep blood......................... Agar ........................0mL......... Mix thoroughly.....0L.5g MgSO4·7H2O .... 33............................. 5.. Aseptically add 50.3......8mg CuSO4·5H2O ................ 2.....0g Peptic digest of animal tissue ................ defibrinated..............................................................5.....0g KH2PO4 ............... Azide Blood Agar with Crystal Violet (Packer’s Agar) Composition per liter: Agar ................................ to distilled/deionized water and bring volume to 950.............................................................................................. Allow to stand for 23 days so that pH adjusts itself to 7................ defibrinated. 25..............................................1mg (NH4)6Mo7O24·4H2O ....................5g NaCl ........................... Mix thoroughly.. 5.......5 ± 0............................5g CaCO3 .2.......40mg MnSO4·H2O ...30mg ZnSO4·7H2O .....0L................................. 25........................ 2.0............... 43.............................................................................1mg MnSO4·2H2O . Use: For the cultivation of autotrophic Nitrobacter spp....................................0g Stock solution.........................5..........0mL of sterile defibrinated sheep blood................. 1........6. 39............................... Autoclave for 15 min at 15 psi pressure–121°C.......0mL of sterile defibrinated sheep blood......................0g Peptic digest of animal tissue ... sheep blood.. Preparation of Medium: Add components.........................00mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.......... and Oxoid Unipath............................ 43....2 at 25°C Stock Solution: Composition per liter: NaCl ....0g NaCl ................07g Preparation of Stock Solution: Add components to metals and disposal must be highly diluted...............................................5g .......... and staphylococci from specimens containing mixed flora and from nonclinical specimens such as water and sewage...........2g Sheep blood.................................................... Pour into sterile Petri dishes or distribute into sterile tubes......................0mg Sheep blood...................0g Pancreatic digest of casein ...... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........ 37....0L.....................................4mg ZnSO4·7H2O ....4-7.. Distribute into tubes or flasks........ Pour into sterile Petri dishes or distribute into sterile tubes.....3mg H3BO3 ......6.....Azide Broth Autotrophic Nitrobacter Medium (DSMZ Medium 756c) Composition per liter: NaNO2 ... Gently heat and bring to boiling.................0mL Trace elements solution ................... 0.0mL pH 7.........................................................2 ± 0................. Preparation of Medium: Add components to distilled/ Use: For the isolation and differentiation of streptococci Preparation of Trace Elements Solution: Add com- deionized water and bring volume to 1..0g NaN3 .......................... Allow the medium to stand for 2-3 days so that the pH can adjust itself to pH 7.......9g Crystal Violet.........10mg FeSO4·7H2O................. Mix thoroughly..............4-7................... Allow tubes to cool in a slanted position..................................... Autotrophic Nitrobacter Medium (LMG Medium 247) Composition per liter: NaNO2 ................................................................... 0........ Cool to 45°–50°C............. Gently heat and bring to boiling.........................................6 with NaOH.......... 33............................................................ Mix thoroughly..... Autoclave for 15 min at 15 psi pressure–121°C..................6...........................................

......................2g NaCl ..... Azide Broth..0.........1mg NH4Cl ..........0g KOH ................0g K2HPO4 ........... 5....................0L.................................... 5....... Mix thoroughly.............. 1........ 0...0g MgSO4·7H2O.................................................................. Rothe (Azide Glucose Broth.......2..............1... Preparation of Medium: Aseptically combine 750..0g NaCl ....10.... 10...0 ± 0................66......................0mL... Adjust pH to 7..................0mg Biotin .....5g NaN3.............0L...0mL........... 5..................8 ± 0..........2......... 2...... 4..7g NaN3..................................................................................................1..........................2g pH 6.....4.................................0mg Na2MoO4·2H2O.......... Use: For the detection and enrichment of fecal streptococci in water and sewage.............................................................................1................... Gently heat and bring to boiling.............................................5...........................0g NaCl ..0mL distilled/deionized water.................... Mix thoroughly.................. metals and disposal must be highly diluted.1g K2HPO4 ..................... Distribute into tubes or flasks.......................25g pH 7..........0mg MnSO4·H2O...............................2g pH 7......................................066g Preparation of Ferric EDTA Solution: Add ferric EDTA to distilled/deionized water and bring volume to 10..........20.......................... 4................. Autoclave for 15 min at 15 psi pressure–121°C........ Mix thoroughly............ Autoclave for 15 min at 15 psi pressure–121°C.............. Autoclave for 15 min at 15 psi pressure–121°C................................. Adjust pH to 6...........0L..................0g Glucose ................... Rothe Beef extract ...0mL: Malic acid ........................... Adjust pH to 6..44 Azide Broth............ Bring volume to 750..........5.........................................................0mL sterile phosphate buffer solution......... 20...........8........................ Use: For the detection and enrichment of fecal streptococci in water and sewage..... 5........ Agar ...............................4........................ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......2g CaCl2 .................. Prepare double-strength broth for samples larger than 1................... 20.......................................7g KH2PO4 ........... Preparation of Medium: Add components to distilled/ Phosphate Buffer Solution: Composition per liter: deionized water and bring volume to 1............................................... Aseptically distribute to sterile tubes or flasks................................0L.......................0mL........................................ Distrib- .......8........... Cool to 25°C........................................................................................................................ Azides also react with metals and disposal must be highly diluted.....................0g Sodium citrate ............................0g KH2PO4 .......................0L...................................................................5g KOH .............. Autoclave for 15 min at 15 psi pressure–121°C.........0g Yeast extract.................... Rothe) Composition per liter: Azoarcus Medium (LMG Medium 202) Composition per liter: Solution A ................. Distribute into tubes or flasks...0g Yeast extract ......................................... Azide Citrate Broth Composition per liter: Azoarcus VM Medium (LMG Medium 252) Composition per liter: Pancreatic digest of casein .........5g NaN3................................................. Mix thoroughly....3................ Mix thoroughly..............8....................... Peptone..........0g Beef extract .........0.................. Caution: Sodium azide is toxic.........8 ± 0. Prepare double-strength broth for samples larger than 1............................ Source: This medium is available as a premixed powder from Oxoid Unipath.......8g KH2PO4 ..................0g malic acid to 500.5mg pH 6..... Also used in the multiple-tube technique as a presumptive test for the presence of fecal streptococci.............. Gently heat and bring to boiling. 2................................................................................................. Use: For the detection of enterococci in water and sewage..............0...0...............2g Fe EDTA..............0mL..................................... Rothe) (Azide Dextrose Broth............................................................ Prepare double-strength broth for samples larger than 1......5g NaCl .............. Autoclave for 15 min at 15 psi pressure–118°C...................................0......0mL Phosphate buffer solution....... Azides also react with Preparation of Phosphate Buffer Solution: Add components to distilled/deionized water and bring volume to 1...........................................................2 at 25°C Preparation of Solution A: Add 5.........................0......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........................0mg Na2MoO4·2H2O...................0mg MnSO4·H2O...........2 at 25°C Caution: Sodium azide is toxic.. 0................ Cool to 25°C.................0........................................8 ± 0....... 750.......0mg Ferric EDTA solution ...5g KH2PO4 ... Gently heat and bring to boiling.......0mL. 5........ Adjust pH to 6...1g CaCl2 ...................... Gently heat and bring to boiling....................................... Mix thoroughly.5g MgSO4·7H2O................0g DL-malic acid............0 with KOH (approximate amount of 4.......... Distribute into tubes or flasks............................2 ± 0................................. Na2HPO4·2H2O ................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......... Azides also react with metals and disposal must be highly diluted...... Use: For the cultivation of Azoarcus indigens.....0mL pH 6....0g Glucose .......................0mL sterile solution A with 250.2 at 25°C Solution A: Composition per 750.. 10.....................0g K2HPO4 ................................................15...2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems........................2..............2....5g)...............................0mL Ferric EDTA Solution: Composition per liter: Ferric EDTA .... 0...1..... 250... Mix thoroughly.......... Add other components.....10.....5g Caution: Sodium azide is toxic................

... Azospirillum lipoferum Medium Composition per liter: Calcium malate...........0L........................ 20..........................5g Ethanol ........................................................................ Mix thoroughly...............................................0................................................................................0mL of ethanol............... Distribute into tubes or flasks..........15g K2HPO4 ..................0.. Use: For the cultivation and maintenance of Azorhizophilus paspali......... Pour into sterile Petri dishes or leave in tubes.................01g Na2MoO4·2H2O ....0.......... Use: For the cultivation of Azospirillum lipoferum......................................... Autoclave for 15 min at 15 psi pressure–121°C.... Autoclave for 15 min at 15 psi pressure–121°C............0...................................................................0mg Bromothymol Blue solution.2..........................................0....................... 10.........0mg Bromothymol Blue solution ...............4g MgSO4·7H2O........... Mix thoroughly............1.........................5..........0g Beef extract ..................................01g pH 6............. Mix thoroughly.......0g MgSO4·7H2O...0mL pH 6................0g MgSO4 ................ Autoclave for 15 min at 15 psi pressure–121°C........05g FeCl3 ....0L.........................02g FeCl3 .....................................................0g Sucrose.....24g Glucose........ Gently heat and bring to boiling.................................... 5.................15............................ 20.......................... Pour into sterile Petri dishes or leave in tubes.................... 1......... Autoclave for 15 min at 15 psi pressure–121°C..........Azotobacter Agar 45 ute into tubes or flasks..................................................................................................... Use: For the isolation and cultivation of Azospirillum lipoferum................................. Bromothymol Blue to 100......................................................................................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......0...........1g Yeast extract ........ 0.................5.. 15...........0mL pH 6............... 0...0g Agar ........................................................... Autoclave for 15 min at 15 psi pressure–121°C.......................... 0. Distribute into tubes or flasks........0g Agar .. 0.. Azospirillum Medium Composition per liter: Sodium malate................................. amazonense.................................................. 1.......................1g CaCl2·2H2O ....02g pH 6..........2g K2HPO4 ......75g KH2PO4 ..........0.......................5g MgSO4·7H2O..2 at 25°C Bromothymol Blue Solution: Composition per 10..............................75g KH2PO4 .........0mL of 0..................... Mix thoroughly..2g KH2PO4 .......0mL: Bromothymol Blue ................ deionized water and bring volume to 1.................10.... Gently heat and bring to boiling..........................2N KOH.........0g ............0..............................................8 ± 0.......... Autoclave for 15 min at 15 psi pressure–121°C............................2 at 25°C Bromothymol Blue Solution: Composition per 100......5g deionized water and bring volume to 1...........2g CaCl2·2H2O.......................................0....0g Na2MoO4·2H2O...................................... Pour into sterile Petri dishes or leave in tubes........................5g KH2PO4 ..0g Agar ...0..............0...................................................................................... 0....... 2.........0L.......... Mix thoroughly........ 0............................................................................. 5....... Preparation of Medium: Add components to distilled/ Use: For the cultivation of Azospirillum species isolated from roots..... Mix thoroughly......... 0.. Autoclave for 15 min at 15 psi pressure–121°C......... Preparation of Medium: Add components to distilled/ Preparation of Bromothymol Blue Solution: Add deionized water and bring volume to 1.............5 ± 0............................................ Agar .1g CaCl2·2H2O ..............................1g NaCl ..................0g Yeast extract....... 0..................0g Sucrose..........................................................01g Na2MoO4·2H2O...... 5......... Use: For the cultivation and maintenance of Azospirillum Use: For the cultivation and maintenance of Azospira oryz- Azospirillum lipoferum Agar Medium Composition per liter: ae and Azonexus fungiphilus............ 5....................... Mix thoroughly.........0L..................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... Mix thoroughly..02g FeCl3 ..............................0L...............................20..............................2g FeCl3·6H2O. Azospirillum amazonense Medium (LGI Medium) Composition per liter: Sucrose................................ 5... 0...... 0........ Distribute into tubes or flasks..............................................0........................9 ± 0.................................................................. Distribute into tubes or flasks............0g K2HPO4 ...................... Azotobacter Agar Composition per liter: Agar ..................................02g Na2MoO4·2H2O..................................5g CaCl2·2H2O ............................................. Distribute into tubes or flasks..................0...... Gently heat and bring to boiling..........................0L....................................................................... 0.......................02g pH 6............. Azorhizophilus paspali Agar Composition per liter: Agar ...............................................8g MgSO4·7H2O................... Pour into sterile Petri dishes or leave in tubes............0mL: Bromothymol Blue .........................................0..0g Peptone...............................................0g K2HPO4 .................................1......2g MgSO4·7H2O .................6g K2HPO4 ....................................0...0..........0mL Preparation of Bromothymol Blue Solution: Add Bromothymol Blue to 10..............9 ± 0..........................0 ± 0..15......................... 0...... Distribute into tubes or flasks........ Pour into sterile Petri dishes or leave in tubes....................... Use: For the cultivation and maintenance of Azorhizobium caulinodans..2 at 25°C Preparation of Medium: Add components to distilled/ Preparation of Medium: Add components to distilled/ Azorhizobium caulinodans Agar (LMG 119) Composition per liter: deionized water and bring volume to 1............... Gently heat and bring to boiling.....................................

..02g Na2MoO4................. Use: For the cultivation and maintenance of Azorhizophilus paspali............0mg Na2MoO4·2H2O..........................2g KH2PO4 .............0g K2HPO4 .......................2.......................... Pour into sterile Petri dishes or leave in tubes.........................................2.......... 0..0mL.....02g Na2MoO4 ....0..0.....................................0L...... Azotobacter vinelandii.....................05g CaCl2 .................. Autoclave for 15 min at 15 psi pressure–121°C....................................... 0................................ 100................................. Azotobacter Basal Agar Composition per liter: Use: For the cultivation of Azotobacter species....................05g CaCl2 ..... Adjust pH to 7. Distribute into tubes or flasks.... Mix thoroughly...... Autoclave for 15 min at 15 psi pressure–121°C.................5g Na2CO3 ....... 1........ Gently heat and bring to boiling...................................................................05g CaCl2 ............... 1.......................................................... Mix thoroughly.............0g KH2PO4 .............................. Gently heat and bring to boiling............................0mg FeCl3 ..................... 2.. FeSO4·7H2O ....................................... 0.. Autoclave for 15 min at 15 psi pressure–121°C...15g ....................0µg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......... Modified II Composition per liter: Use: For the cultivation and maintenance of Azotobacter beijerinckii...........0.....................15g MgSO4·7H2O .................................................................................02g Na2MoO4 ............... Adjust pH to 6....................................10....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.............. 10... 1....................0mg pH 7... 0...............................5.....2 ± 0.......................................9g CaCl2·2H2O .......0g MgSO4·7H2O...0g K2HPO4 ..0........................... Autoclave for 15 min at 15 psi pressure–121°C........................... to tap water and bring volume to 1. Gently heat and bring to boiling....... 10.....................................10............ Pour into sterile Petri dishes or leave in tubes............ 0...........................15g CaSO4·2H2O....................05g CaCl2 . Distribute into tubes or flasks.....................0L.....15g K2HPO4 .. Adjust pH to 7............................................. Azotobacter Broth........ 20.......2.................................................1g MgSO4·7H2O... Autoclave for 15 min at 15 psi pressure–121°C.0..................................0g Sucrose..................... Gently heat and bring to boiling.......... Modified I Composition per liter: Agar ..0....................................................................................002g FeCl3 ........................... 0..1g K2HPO4 ......... Autoclave for 60 min at 15 psi pressure–121°C....................0g Glucose..........0mg Na2MoO4·2H2O...............................2.........................0g KH2PO4 .... species and Beijerinckia derxii...........................................0mg pH 7.... 15............ Pour into sterile Petri dishes or leave in tubes.. Autoclave for 15 min at 15 psi pressure–121°C......................................0mL pH 7...................2 ± 0...............0g MgSO4·7H2O......... 15..................3 ± 0............................................. 0.............2 at 25°C Soil Extract: Composition per 200. Modified II Composition per liter: Sucrose ...........................................0g MgSO4·7H2O ......02g Na2MoO4...0g Agar ....0mg Soil extract........1g KH2PO4 ................................................. 0..................0mL: African Violet soil ........................................ Pour into sterile Petri dishes or leave in tubes.... 0............. Azotobacter Agar.......................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........................................2 ± 0..........................5......................0..............................................................................10......................... 1.................0L....................................20.................0..0................................................................. 0...................0.................. Distribute into tubes or flasks.............. species.....2 at 25°C Preparation of Medium: Add components to distilled/ Use: For the cultivation and maintenance of Azotobacter deionized water and bring volume to 1..... Modified I Composition per liter: Sucrose .....1.1g FeSO4·7H2O .................46 Azotobacter Agar..........................5g Preparation of Soil Extract: Add components to tap water and bring volume to 200................................. Mix thoroughly.................. Distribute into tubes or flasks................................0L......... Gently heat and bring to boiling.......................0g Glucose .......................................... 15.....0mg pH 7........... and others when a carbon source is added..... Filter through Whatman filter paper...................0mg pH 6.....................0............ 1...15g CaSO4·2H2O .............2g KH2PO4 ..... including Azomonas species................................................... Azotobacter Agar..............................2g Azotobacter Broth.......................................0mg FeCl3 ...........................0...................10.0mg FeCl3 ...................................2 ± 0........................................................2g KH2PO4 ........... including filtered soil extract.....1...................5............. Gently heat and bring to boiling......................................0g CaCO3........0mg Na2MoO4·2H2O ..........2g NaCl ......................... 0..................................................................................... Azotobacter species......................1............... 0........................................................................ Mix thoroughly......... 0............................ Modified I Sucrose...... and Derxia gummosa......0mg Na2MoO4·2H2O ........................................... 2............................................. Pour into sterile Petri dishes or leave in tubes.................. Preparation of Medium: Add components....0g MgSO4·7H2O .............. Sucrose........... 0...1g K2HPO4 ........... Use: For the cultivation of a variety of bacteria..................................................................... Azotobacter Broth Composition per liter: Glucose................................ Distribute into tubes or flasks........................... Distribute into tubes or flasks........................ 0............ Agar ..................... Mix thoroughly............................2 at 25°C Preparation of Medium: Add components to distilled/ Use: For the cultivation and maintenance of Azotobacter deionized water and bring volume to 1......................2g K2HPO4 .........................................0L.... Mix thoroughly.............. 0......................0L................................................. Azotobacter chroococcum................... 10.................................

Warm to 50°–55°C........ 1.5g KH2PO4 .....................0g K2HPO4 ................1g MgSO4·7H2O ................... 0.....................................1...0mg FeCl3 .................. Pour into sterile Petri dishes or leave in tubes... 0.............................................0L.... Use: For the cultivation and maintenance of a variety of bacteria...1g FeCl3 ..0... and Rhodococcus erythropolis....................1.......................................... Distribute into tubes or flasks........ 0..0mg Na2MoO4·2H2O............... Use: For the cultivation and maintenance of Azotobacter chroococcum............................................................................... 5................................. 0..........15g K2HPO4 ......0mg pH 7.................................02g Na2MoO4...............0....................................................................2g MgSO4·7H2O....... 0..................... Autoclave for 15 min at 15 psi pressure–121°C...........05g pH 7........... 5........................... including Azotobacter species........ Filter sterilize.......................................................0mg Na2MoO4·2H2O .0mL: Mannitol........................................... Filter sterilize....................................0...... 0.............0g K2HPO4 ...... 5.......................................................... including Azomonas species............. Mix thoroughly.........................................8g Yeast extract ....... Mix thoroughly................. 0.........................0....................................... to distilled/deionized water and bring volume to 950................ except glucose solution and mannitol solution...................0mg Na2MoO4·2H2O............... Mannitol Solution: Composition per 25....... Aseptically add 25...........05g CaCl2 ..0g K2HPO4 ............1....................0L...........................15.... Distribute into tubes or flasks.......2mg pH 7............ Gently heat and bring to boiling...........2 ± 0..........0mg FeCl3 .......... Autoclave for 15 min at 15 psi pressure–121°C.......0g Glucose.......................... Mix thoroughly............................ 0... Azotobacter Medium Composition per liter: Agar ..0g MgSO4·7H2O...........................10................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 25.0mL: D-Glucose.......... 0..........0g Glucose ............. Azotobacter Medium (ATCC Medium 240) Composition per liter: Agar ..2. Distribute into tubes or flasks.............. Pseudomonas azotocolligans.. Mix thoroughly....2 ± 0................. Distribute into tubes or flasks.. 1.0..................Azotobacter Medium MgSO4·7H2O ..................................................0g CaCO3 ........................................01g Na2MoO4·2H2O ......................................2..................... Autoclave for 15 min at 15 psi pressure–121°C..... Gently heat and bring to boiling............................................................................1g FeSO4·7H2O.............0...........05g CaCl2 .........1g MgSO4·7H2O...... ..............2 ± 0....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........0mg pH 6..................... Warm to 50°–55°C.................................0L.............................................................. Azotobacter Medium (ATCC Medium 1771) Composition per liter: Agar ................ 5......... Use: For the cultivation of a variety of bacteria..........................................0mL.......................2 ± 0.... Mix thoroughly.... Autoclave for 15 min at 15 psi pressure–121°C.................1g Na2MoO4·2H2O ................................... Use: For the cultivation and maintenance of Azotobacter species......0mL Mannitol solution .. Pour agar medium into sterile Petri dishes or leave in tubes.........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................... 0..................................................2g KH2PO4 ..1g KH2PO4 ............................................. Gently heat and bring to boiling... Beijerinckia derxii........................3 ± 0....0mL...................... 20. Mix thoroughly...........0. 0........................................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................ Adjust pH to 6...5g KH2PO4 .. Autoclave for 15 min at 15 psi pressure–121°C....0mL pH 7............. Mix thoroughly.............................. Pour into sterile Petri dishes or distribute into sterile tubes.........................................................................................25.058g K2HPO4 ..........0g Preparation of Mannitol Solution: Add mannitol to distilled/deionized water and bring volume to 25..................0mL.0mL of sterile glucose solution 47 and 25....0....0...0mg Glucose solution ....................................................... Azotobacter chroococcum Agar Composition per liter: Agar .....................0............. 2.0g KH2PO4 ......................................... 20...................................0.........................0..0L....................15.......................................................... Azotobacter species.......... Pour into sterile Petri dishes or leave in tubes........................2g FeCl3·6H2O ..................... Autoclave for 15 min at 15 psi pressure–121°C......................... Cool to 50°– 55°C. Preparation of Medium: Add components..098g NaCl ...02g Na2MoO4·2H2O...........................20....................8g MgSO4·7H2O ........................................................................................................................................................................................................... Mix thoroughly............................... 20........................ Distribute into tubes or flasks..... Gently heat and bring to boiling......25...................2 at 25°C Glucose Solution: Composition per 25.0g CaCO3 .. Pour into sterile Petri dishes or leave in tubes..................2g K2HPO4 ............6 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......0mg pH 7............................... Gently heat and bring to boiling..0mL of sterile mannitol solution...................................0g Agar ............15.... 0............ 15.............................0L................................................. 0.........................................4–7..........................................................................2g CaSO4·2H2O.0.....................................................5..0.....................22g CaSO4·2H2O...........................0.................. Mix thoroughly.......... Azotobacter Medium (ATCC Medium 14) Composition per liter: Sucrose .........058g FeSO4·7H2O ..2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................ Use: For the cultivation of Azotobacter species and Beijerinckia derxii.........................................9g CaCl2·2H2O.................

........1................................................................ Mix thoroughly...........0mL................................. Filter sterilize...................02g Na2MoO4·2H20 ..5...5g Preparation of Soil Extract: Add components to tap water and bring volume to 200.........0g Sucrose...............15............0........................ to tap water and bring volume to 900..... Autoclave for 15 min at 15 psi pressure–121°C...........................2g FeSO4·7H2O .0mL pH 7...... 100..............0g Preparation of Mannitol Solution: Add mannitol to distilled/deionized water and bring volume to 100....................0mL Glucose solution .............0mL.........................................6 ± 0.................. Filter sterilize....0mL of sterile mannitol solution..........................0mL...................................0L...................... Gently heat and bring to boiling......... Autoclave for 60 min at 15 psi pressure–121°C...0g CaCO3 ..................................2g NaCl .100........15.............0mg Soil extract....................................2g FeSO4·7H2O ................................................................. Filter through Whatman filter paper...........0mL: African Violet soil ............5..............0g MgSO4·7H2O ..................................... Autoclave for 15 min at 15 psi pressure–121°C..................0................. Filter through Whatman filter paper...................................................................05g CaCl2 .15g K2HPO4 ......................... 2.... 0...........5g Ethanol ....... Mix thoroughly....... 0........ Mannitol ..0mL Mannitol solution ...5g Na2CO3 .............................................. Autoclave for 60 min at 15 psi pressure–121°C........2 at 25°C Soil Extract: Composition per 200.0mL................................0mL: Preparation of Soil Extract: Add components to distilled/deionized water and bring volume to 200.......2g NaCl ..... Glucose Solution: Composition per 100............ Pour into sterile Petri dishes or leave in tubes................................0mg Soil extract ........................ Azotobacter paspali Medium Composition per liter: Agar .........................0..... 0.. to tap water and bring volume to 900.......0g MgSO4·7H2O........0g K2HPO4 ....... Preparation of Medium: Add components. 1................ 5....................................... 20......... Adjust pH to7...6.......... including Azotobacter species.......................... Aseptically add 100........................... Mix thoroughly................ 20..............0mL pH 6....................0mL: Glucose.... except mannitol solution........... 0......0mL........ Mix thoroughly..............................0mL pH 7....... Filter through Whatman filter paper..5g Preparation of Soil Extract: Add components to tap water and bring volume to 200..............0mL of sterile glucose solution........... Adjust pH to 6...... Distribute into tubes or flasks.. Mix thoroughly...........10.....0mL FeCl3 (10% solution) ........... Pour into sterile Petri dishes or leave in tubes................... Mix thoroughly.....................................................0.............................0mL..........................................0......0mL Bromothymol Blue to 10................ Preparation of Medium: Add components........0g K2HPO4 .... Azotobacter Supplement (ATCC Medium 11) Composition per liter: Agar .......................................... Aseptically add 100.. Aseptically add ...........................20.......2g KH2PO4 ................... 0..................... Cool to 50°–55°C........0mL Glucose solution ......0mg Soil extract.............................. 100............................................ Use: For the cultivation and maintenance of Azotobacter paspali..........100.2g FeSO4·7H2O... 0......2 at 25°C Soil Extract: Composition per 200........................... 0. Autoclave for 15 min at 15 psi pressure–121°C........................................2 at 25°C Soil Extract: Composition per 200..........0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100..................0mL: African Violet soil ..20............. 0...........2g NaCl .0.............0.............. Mix thoroughly....... Use: For the cultivation of Azotobacter species and Azomonas species........................0mL of ethanol....................................... 15...........0............ Autoclave for 15 min at 15 psi pressure–121°C............................5g Na2CO3 .........................................................0mL.. Filter sterilize.................. 100............................1........0mL....0g MgSO4·7H2O.... Autoclave for 60 min at 15 psi pressure–121°C...... Preparation of Medium: Add components........ Preparation of Bromothymol Blue Solution: Add Mannitol Solution: Composition per 100........................1mL pH 7........ Mix thoroughly.........48 Azotobacter paspali Medium Use: For the cultivation and maintenance of a variety of bacteria.0......0 ± 0..... Glucose Solution: Composition per 100...........2 at 25°C Azotobacter Supplement (ATCC Medium 12) Composition per liter: Agar ....................................... except glucose solution..........0mL: Bromothymol Blue .........6 ± 0....................10.............0mL: African Violet soil...........6............ except glu- cose solution............................... 20....0g MgSO4·7H2O ................................................ Mix thoroughly..............0g K2HPO4 ..................0 ± 0.......0mg Bromothymol Blue solution............... 100...................5g Bromothymol Blue Solution: Composition per 10.... Use: For the cultivation of Azomonas agilis and Azotobacter chroococcum.......................................5g Na2CO3 .......... Cool to 50°–55°C.......... to tap water and bring volume to 900....... 0..................... Azotobacter Supplement (ATCC Medium 13) Composition per liter: Agar ......................................................................... Adjust pH to 7... Cool to 50°–55°C...................... 1.............0....................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................................................................... Pour into sterile Petri dishes or leave in tubes...0mL: Glucose ..... Mix thoroughly..0mL.........................................................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100................................

...............................0mL of sterile mannitol solution...0mL Wolfe’s mineral solution ...0........................ Adjust pH to 6............. 0...5............... 2......0g MgSO4·7H2O ........0mL of distilled/deionized water............. Before use........5.......0g Yeast extract.............0mL.............. Autoclave for 15 min at 15 psi pressure–121°C............0 with sterile HCl...........0mL......................0mg Soil extract ..................................... Autoclave for 15 min at 15 psi pressure–121°C........5.........0..... Mix thoroughly.........2g FeSO4·7H2O.........05g Resazurin .5g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.01g Na2MoO4·2H2O...............10.................... Mannitol Solution: Composition per 100.. Aseptically and anaerobically distribute into sterile tubes or bottles......... 0........ Mix thoroughly.................... 49 Cellobiose Solution: Composition per 50.........01g H3BO3 ........................0 ± 0................................ Filter sterilize.... 0.......5..............0mL.......... Azotobacter vinelandii Medium Composition per liter: Sodium benzoate...... Use: For the cultivation of Azotobacter vinelandii from water samples........... Adjust pH to 6.. Filter through Whatman filter paper...........0mg Lipoic acid............................. Distribute into tubes or flasks...................0mL: Soil Extract: Composition per 200...........................................0mL: Mannitol....................... Pour into sterile Petri dishes or leave in tubes.............................. Add remaining components......... 0............................1...............0mL: Wolfe’s Mineral Solution: Composition per liter: African Violet soil...............0mL of sterile Na2S·9H2O solution............................... Aseptically and anaerobically add 50.................0g K2HPO4 .........02g CuSO4·5H2O.........................0.4g MgCl2·6H2O....................... Mix thoroughly..0mL Na2S·9H2O solution.............................................................. 20....................................... to tap water and bring volume to 900..............................3mg Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500................50..100..................................................5 with KOH...... Mix thoroughly............5g CoCl2·6H2O............ MgSO4·7H2O.....5.............5g Mannitol....10....... 0............................ Mix thoroughly................ 0.............. 0....................... Mix thoroughly......... Autoclave for 15 min at 15 psi pressure–121°C............0L........................... Cellobiose........5g NaCl ........0......................................1g CaCl2·2H2O..5g Preparation of Soil Extract: Add components to distilled/deionized water and bring volume to 200.................................................... and Wolfe’s vitamin solution................................................... 0.......... Sparge with 100% N2..... Aseptically add 100................................ Add distilled/deionized water to 1.0..........01g Na2SeO3·5H2O ........................................0mg Thiamine·HCl ..............0mg Nicotinic acid ........... Mix thoroughly.....1g ZnSO4·7H2O...........0g Nitrilotriacetic acid......................... 0...............0mL..................................... Use: For the cultivation of Caldicellulosiruptor lactoaceticus.............. 0.025g KAl(SO4)2·12H2O ..............2g NaCl ...... Autoclave for 60 min at 15 psi pressure–121°C. Sparge with 80% N2 + 20% CO2 gas mixture.................. Na2S·9H2O solution..0......................2................................... Filter sterilize................ Preparation of Cellobiose Solution: Add cellobiose Agar ...........................0.........................0mL of sterile Wolfe’s mineral solution.........................0mL......25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10..............3..................................0mL of sterile cellobiose solution..................................0mL.......... Mix thoroughly...........................................................................0g Azotobacter Supplement (ATCC Medium 15) Composition per liter: to distilled/deionized water and bring volume to 50............................................................5g Na2CO3 ........... Preparation of Medium: Add components.9–7....1g CaCl2·2H2O ...................................................................... 0........................0......... Add components......................1.5..0mL: Use: For the cultivation of Beijerinckia species...........5mg Cellobiose solution .. .............................. Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl...................................................10....0...1g FeSO4·7H2O ...........0mg Folic acid ......................................................... 1.................................0................................................... Filter sterilize....................... BA Medium with Cellobiose Composition per liter: NaHCO3 ........................................................ Mix thoroughly........... Use: For the cultivation of Azomonas macrocytogenes............0g MnSO4·2H2O.........0mL pH 6..0g K2HPO4 .1g NaCl ...............0mL Wolfe’s vitamin solution .........0................................................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.. neutralize to pH 7.................................0mg Riboflavin. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture..................... Pour into sterile Petri dishes or leave in tubes....0mL of sterile Wolfe’s vitamin solution...................................... 15......................................................BA Medium with Cellobiose sterile glucose solution.......... 1......... except cellobiose solution...... except mannitol solution.....0mL Mannitol solution .......................6g NH4Cl................ 10......................................0... and 10.........................2....... Wolfe’s mineral solution..............................0g Preparation of Mannitol Solution: Add mannitol to distilled/deionized water and bring volume to 100......0mg Biotin .... 5................. Mix thoroughly..............................75g K2HPO4·3H2O.................................................................1g NiCl2·6H2O.............. Autoclave for 15 min at 15 psi pressure–121°C.. Cool to 50°–55°C......0mg Vitamin B12............ 10.............................. Adjust pH to 6....100....................................0............... to distilled/ deionized water and bring volume to 920........ 1.........0.8....................................2 at 25°C Na2S·9H2O Solution: Composition per 10...0L...10............0mL pH 6.............................0L.......................... 0............................................0mg Calcium DL-pantothenate .0mg p-Aminobenzoic acid ...................0 at 25°C Na2S·9H2O ...............................4...............

...... and component III.....5g Yeast extract ... Modified with 2................................. Autoclave for 15 min at 15 psi pressure–121°C................................ Mix thoroughly...5. and Thermodesulfobacterium species...... Mix thoroughly..... 1..0g Ferrous Ammonium Sulfate Solution: Composition per 20....................400.....2 at 25°C Component I: Composition per 400....0g Ferrous Ammonium Sulfate Solution: Composition per 20..... Adjust pH to 7...........1.......................................... Autoclave for 15 min at 15 psi pressure–121°C.... Mix thoroughly...1...................................0mL: NaCl ..1....400.......................0mL: Component I ...................................................... Use: For the cultivation and maintenance of Desulfotomac- ulum nigrificans............ Autoclave for 15 min at 15 psi pressure–121°C.. Preparation of Medium: Add components..................... Mix thoroughly..............0mL.10........................................3...............0mL......................2 at 25°C Component I: Composition per 400.. Adjust pH to 7............................0mL.....................................................................0mL volumes into tubes under 97% N2 + 3% H2....... Aseptically add 10.............................................10............. Modified Composition per 1020.........1mL of sterile ferrous ammonium sulfate solution to 5.............5g MgSO4·7H2O ........ 0...................... Mix thoroughly... Mix thoroughly............5g Preparation of Ferrous Ammonium Sulfate Solution: Add Fe(NH4)2(SO4)2 to distilled/deionized water and bring volume to 10.........0g MgSO4 ...5.5................2 at 25°C Preparation of Component III: Add components to distilled/deionized water and bring volume to 400.............. Mix thoroughly....5.....0mL Component II..0mL: Component I ....... Autoclave for 15 min at 15 psi pressure–121°C...........0g K2HPO4 ............................................................... 0.5g Preparation of Component II: Add K2HPO4 to dis- tilled/deionized water and bring volume to 200.................... Adjust pH to 7....1.5................0mL: Yeast extract......................0mL Yeast extract solution .............................. Mix thoroughly.....0..............................0mL.................... Preparation of Component I: Add components to distilled/deionized water and bring volume to 400...0g . Use: For the cultivation and maintenance of Desulfovibrio.......... Baar’s Medium for Sulfate Reducers.... K2HPO4 ...0mL........................0g Preparation of Ferrous Ammonium Sulfate Solution: Add Fe(NH4)2(SO4)2 to distilled/deionized water and bring volume to 20.....2..........................................5 ± 0...5.... Desulfobulbus....... Autoclave for 15 min at 15 psi pressure–121°C..0mL............ Mix thoroughly.....0mL: K2HPO4 ..... to tap water and bring volume to 980.. Component II: Composition per 200. Gently heat and bring to boiling....................5g Yeast extract .............................................0mL Ferrous ammonium sulfate solution .....0g Sodium lactate ...25............0g NH4Cl .................. 1.......................0mL pH 7.............. Sodium lactate .......0g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10. Mix thoroughly..........0mL Component III................0mL pH 7........ Aseptically add 0.......0mL Ferrous ammonium sulfate solution ..........................................................................0mL: Sodium citrate ................0mL: Fe(NH4)2(SO4)2 .............. Cool to 45°–50°C..... Autoclave for 15 min at 15 psi pressure–121°C................................ Baar’s Medium for Sulfate Reducers.............. Component III: Composition per 400.0g NH4Cl...............0g Preparation of Component I: Add components to distilled/deionized water and bring volume to 400........5 ± 0..............0mL of medium immediately prior to inoculation.......................... 1...0mL: Fe(NH4)2(SO4)2 .......0mL...............5g Preparation of Component II: Add K2HPO4 to distilled/deionized water and bring volume to 200........ 200.......... Autoclave for 15 min at 15 psi pressure–121°C.......... Adjust pH to 7.... Filter sterilize......1......................... Ferrous Ammonium Sulfate Solution: Composition per 10...........3...0mL: Sodium lactate.. 1... Mix thoroughly.................20....................................................0mL of sterile ferrous ammonium sulfate solution and sterile yeast extract solution........5% NaCl Composition per 1020.......................................................0mL: Fe(NH4)2(SO4)2 .. Adjust pH to 7......0mL: Preparation of Component III: Add components to distilled/deionized water and bring volume to 400..................................... 2... 5. Distribute 5....200...0g Sodium citrate ........................... 1........... 3......... 2......... Desulfotomaculum. Aseptically distribute into tubes or flasks......0g CaSO4...........................0mL pH 7............. 20.......0mL: Component II: Composition per 200.0g CaSO4........................50 Baar’s Medium for Sulfate Reducers Baar’s Medium for Sulfate Reducers Composition per liter: Component III: Composition per 400...1......................0g CaSO4 ...5 ± 0.......5..................... Yeast Extract Solution: Composition per 10. Preparation of Medium: Aseptically combine component I.... Autoclave for 15 min at 15 psi pressure–121°C................... 0..........0mL...0mL..... Autoclave for 15 min at 15 psi pressure–121°C............................. Add medium to tubes while still warm to exclude as much O2 as possible.0g NH4Cl.......................0g MgSO4 ................................. except fer- rous ammonium sulfate solution and yeast extract solution.................0mL Component II ......... 400............. component II........... Adjust pH to 7. Mix thoroughly...5g Ferrous ammonium sulfate solution .... 400..........0mL Component III .................0mL.............................................................

.............................................0–4........0mL of sterile solution A.................0–4............... Pour into sterile Petri dishes or distribute into sterile tubes.............3g CoCl2·6H2O.....................................0g MgSO4·7H2O ........0 ± 0..................................0–4....... Gently heat and bring to boil- ...0g KH2PO4 ..02g CuCl2·2H2O..... Alicyclobacillus acidoterrestris........................................... bring volume to 20............... Mix thoroughly................ Mix thoroughly.........1....... and 1.... Use: For the cultivation of Desulfovibrio africanus and other Desulfovibrio species that prefer 2....... Cool to 50°–55°C.....0g Yeast extract.0mL....................................... 0.....0mL..................... Autoclave for 10 min at 15 psi pressure–121°C........0g MgSO4·7H2O..................................................................... component II......0.................................. 0.........0g Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 500............0mL..5g CaCl2·2H2O........................0–4........... Bacillus acidoterrestris Agar Composition per 1001......0. Autoclave solutions separately for 15 min at 15 psi pressure–121°C.................... Use: For the cultivation and maintenance of Bacillus acidoterrestris...0mL Solution B (Trace elements solution SL-6).........0.............0L.......................0g (NH4)2SO4 ........... Autoclave for 15 min at 15 psi pressure–121°C.............1... Aseptically add 0..............0mL volumes into tubes under 97% N2 + 3% H2.................................................................... Bacillus Agar........ Use: For the cultivation and maintenance of Bacillus (Alicyclobacillus) acidocaldarius.................. and component III..... Use: For the cultivation of acidophilic Bacillus species such as Bacillus acidocaldarius........................0mL pH 4........... 0..0g Yeast extract . Mix thoroughly..............0..... 5...................................................0..500.......................................................0 ± 0........................................................................ Mix thoroughly....0............................07g FeCl3 ........2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500..................0mL of sterile solution C.0...................0 at 25°C Preparation of Medium: Add components........ nent I...... and Alicyclobacillus cycloheptanicus.......1.................. Mix thoroughly........1mL of sterile ferrous ammonium sulfate solution to 5..0mL: Agar .................0............6g MgSO4·7H2O............. Prepare a separate agar solution by adding 20...1..... 0...... This procedure avoids acid hydrolysis of the agar..5........0mL of distilled/deionized water...... Mix thoroughly.0g Solution B (Trace Elements Solution SL-6): Composition per liter: MnCl2·4H2O ... 1.........0mL: Glucose ...................... Preparation of Medium: Aseptically mix 500...0mL of solution B..... Adjust pH to 3...............0mL of solution A and 500...............0..5g H3BO3 .....25g (NH4)2SO4 ............... Adjust pH to 3... 0.......01g Preparation of Solution B (Trace Elements Solution SL-6): Add components to distilled/deionized water and bring volume to 1................................0mL.. Mix thoroughly.................0g Yeast extract .. Agar ...........5% NaCl......................0 at 25°C Solution A: Composition per 500.......................0mL.................................. Preparation of Medium: Aseptically combine 500......... 30................ to distilled/deionized water and bring volume to 500..........0.....5g CaCl2·2H2O ......................................................15............................... Gently heat and bring to boiling.............. except agar and glucose. 500........... 2..1g Na2MoO4·2H2O......... Pour into sterile Petri dishes or leave in tubes..........................2 at 25°C Solution A: Composition per 500............................................................................... Adjust pH to 4..........................20................................................................. Bacillus Agar Composition per liter: Agar .......... Autoclave for 15 min at 15 psi pressure–121°C.....................................0...................... 0.25g (NH4)2SO4 .................. Distribute 5...... 3.........25g (NH4)2SO4 .......0g KH2PO4 .............................................. Gently heat and bring to boiling.......................500...... Gently heat and bring to boiling..............0. 0......... Autoclave for 15 min at 15 psi pressure–121°C........ Autoclave for 15 min at 15 psi pressure– 121°C........0............... Pour into sterile Petri dishes or distribute into sterile tubes......................Bacillus Agar........ Filter sterilize.. Modified 51 Preparation of Ferrous Ammonium Sulfate Solution: Add Fe(NH4)2(SO4)2 to distilled/deionized water and Solution C: Composition per 500........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 500..........0g/500.0...........3g Glucose...................0mL: Solution A ................. Aseptically adjust pH to 3...............................................................................0........................................................2g pH 3....... Mix thoroughly....................................................20.......0mL: KH2PO4 .2g ZnSO4·7H2O...................................................0mL..................................0g Yeast extract........02g pH 4.........1............0mL Solution B ............ Mix thoroughly............................. Solution B: Composition per 500................................. Mix thoroughly......................500............0g Glucose ..........5g CaCl2·2H2O ...........0mL of sterile solution B........ Cool to 50°–55°C... Cool to 50°–55°C... Bacillus acidocaldarius Agar Composition per liter: Solution A .............0mL: Preparation of Medium: Aseptically combine compo- Preparation of Solution C: Add agar to distilled/deionized water and bring volume to 500..03g NiCl2·6H2O.............0mL pH 3. 3....................500............................0...0g KH2PO4 .1...0mL...........................2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500..... Cool to 50°–55°C.....................0mL of medium immediately prior to inoculation. Add medium to tubes while still warm to exclude as much O2 as possible........0mL Solution C ................25g CaCl2·2H2O ........................................................ 1....... 0.....................0g Glucose..... Modified Composition per liter: Agar ..............37g MgSO4·7H2O........... Aseptically combine both solutions..

............................. except magnesium nitrate solution.0............ Adjust pH to 3....... Cool to 50°–55°C...0mL Solution B (Trace Elements solution SL-6)..2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................2 at 25°C Magnesium Nitrate Solution: Composition per 100... Gently heat and bring to boiling...5....................0mL Solution C ..............0g KH2PO4 ........ Adjust pH to 4. Adjust pH to 7.................... 0..10..................03g Trace elements solution .................................................0.....5....0g Preparation of Solution C: Add agar to distilled/deionized water and bring volume to 500....2 at 25°C of sterile solution A................................................1..0L........0.. 0...0 with 10N H2SO4....................................2mL pH 7................0mL of distilled/deionized water............................... Distribute into tubes or flasks...0mL: Mg(NO3)2·6H2O ...........5g CaCl2·2H2O ......... 0......... Mix thoroughly.................................0g Yeast extract .....0g KH2PO4 .................3g CoCl2·6H2O.............................0g Mg(NO3)2·6H2O .........................................0........ Pour into sterile Petri dishes or leave in tubes...0mL.................1........................ 0.......... Prepare a separate agar and glucose solution by adding 20.................................. Autoclave for 15 min at 15 psi pressure– 121°C....................0L...................0g Na2HPO4·12H2O .........................25g CaCl2·2H2O................ 2...0g Peptone.......0mL pH 7................................................................................ Mix thoroughly.....0g Yeast extract.... 0..... 0........................................................0.... Autoclave for 15 min at 15 psi pressure–121°C...... 0......15............0L....................................................................... Bacillus cycloheptanicus Agar Composition per 1001............ 1...0mL of sterile solution B....................... Preparation of Medium: Add components to distilled/ Solution A: Composition per liter: Yeast extract ... and 1...... Preparation of Medium: Aseptically combine 500............................1......... Alicyclobacillus acidoterrestris............................................3...................2 ± 0.1g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100..................25g (NH4)2SO4 ...0g Yeast extract. Gently heat and bring to boiling...07g FeCl3 ....... 6....................................................1g Na2MoO4·2H2O....0mL... Mix thoroughly.. 0..... Use: For the cultivation of acidophilic Bacillus species such as Bacillus acidocaldarius.....0... 0....................................................... Preparation of Medium: Add components........................................................ Bacillus halodenitrificans Agar (LMG Medium 142) Composition per liter: NaCl .....5g H3BO3 ............................................................................................................. Use: For the cultivation of acidophilic Bacillus species such as Bacillus acidocaldarius................................... 15...500..........0mL: Agar .. and Alicyclobacillus cycloheptanicus.................. 1...... zoevorans...............0g Preparation of Magnesium Nitrate Solution: Add Mg(NO3)2·6H2O to distilled/deionized water and bring volume to 100.................... Mix thoroughly...................................... Aseptically combine both solutions.... Mix thoroughly......2 with KOH....................... Adjust pH to 4.......... Pour into sterile Petri dishes or distribute into sterile tubes........................ 500................................................. Cool to 50°–55°C.................................... 1....0g Na2HPO4 ........................6g Sodium benzoate......................0mL Use: For the cultivation and maintenance of Bacillus ben- Use: For the cultivation and maintenance of Bacillus cycloheptanicus...............0g of glucose to 500......................................................... Autoclave for 15 min at 15 psi pressure–121°C............................5.................3g (NH4)2SO4 .0L...0g of agar and 1.........................0 ± 0............................. Autoclave for 15 min at 15 psi pressure–121°C.1...................... 0.............................. 0..... 0......1.. Solution C: Composition per 500.....0mL..................0g Magnesium nitrate solution ............................ Bacillus benzoevorans Agar Composition per liter: Agar ...............37g MgSO4·7H2O .............0g KH2PO4 ............. Filter sterilize................3g Glucose ....2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.100......... Autoclave solutions separately for 15 min at 15 psi pressure–121°C............. Autoclave for 15 min at 15 psi pressure–121°C.............................................0....................0.........................0 ± 0.. Distribute into tubes or flasks. 1.... .................. deionized water and bring volume to 1.........................0......................................................................1...1g ZnSO4·7H2O ......0mL pH 4.................... Bacillus Broth Composition per liter: (NH4)2SO4 ..... Autoclave for 15 min at 15 psi pressure–121°C..........52 Bacillus benzoevorans Agar ing...................................................... Mix thoroughly..01g Preparation of Solution B (Trace Elements Solution SL-6): Add components to distilled/deionized water and bring volume to 1..0g MgSO4·7H2O................................15..........................................................................................................................................03g NiCl2·6H2O............2g ZnSO4·7H2O.5g MgSO4·7H2O.................0g Sodium acetate·3H2O ..............................500..........................0mL: Solution A .. 5..........0g Beef extract ....0mL............0...0g NaCl ... This procedure avoids acid hydrolysis of the agar.....................02g pH 4.................................................................. Mix thoroughly........0mL: FeSO4·7H2O ....................2 at 25°C Trace Elements Solution: Composition per 100.........02g CuCl2·2H2O..................................8g KH2PO4 ............................................0–7.................. to distilled/deionized water and bring volume to 900...... Pour into sterile Petri dishes or leave in tubes..98g NH4Cl.....0g Agar ............ Gently heat and bring to boiling.3............................................... 100.......................................... 3............ Mix thoroughly........... Solution B (Trace Elements Solution SL-6): Composition per liter: MnCl2·4H2O ............0g Glucose.......... 5.....................0mL of sterile solution C...............................................................1g MnCl2·4H2O ....................

...........................................10................................................2 at 25°C Yeast extract ................................. Autoclave for 15 min at 15 psi pressure–121°C............0mg Biotin ................0g Citric acid......... Aseptically pour into sterile Petri dishes or distribute into sterile tubes.......................................20.0................... 20...0L............0mg NaMoO4·2H2O ....... flasks.......0g Agar ...................... Mix thoroughly..........................0mg Calcium pantothenate ....0g Peptone...................................0.. Pullulan................... 20............4mg KI......................... 5........................................0g Beef extract ........5.0...............................0g Yeast extract... Aseptically distribute into sterile tubes or flasks............... Gently heat and bring to boiling.........0g KH2PO4 ................................................... Bacillus polymyxa Agar Composition per liter: Agar .................. Swirl medium to resuspend starch...0g Yeast extract.......................1.........0L.......................0g Meat extract .............. Mix thoroughly................0g L-Glutamic acid...................... Pour into sterile Petri dishes or leave in tubes.........................................0g Agar ....................................0g Starch........ 5................................................4 ± 0................................5.................... 5............................................................... Mix thoroughly........0L........0mg Riboflavin...................................................................0..........0L............................................................ erans................... 2.................... 4.....0 ± 0..........0mg Cyanocobalamin....................0mg Nicotinic acid .......................................................................5.......................................... Preparation of Medium: Add components to distilled/ Use: For the cultivation of Bacillus licheniformis......... 5. Vitamin Solution: Composition per liter: Pyridoxine·HCl.. Filter sterilize........................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................0g K2HPO4 ............................ Gently heat and bring to boiling..... Bacillus Medium (ATCC Medium 21) Composition per liter: Use: For the cultivation and maintenance of Bacillus mac- Bacillus popilliae Medium Composition per liter: Glycerol ..0mg Thiamine·HCl ................................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....0mL Vitamin solution ........0mg Na2SeO3.......0g Polypeptone™.2 ± 0.............0............... Distribute into tubes or flasks.........................0mg Na2WO4·2H2O.......... 10................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................ Autoclave for 15 min at 15 psi pressure–121°C....................................... Mix thoroughly...........0g Starch.............................................. Use: For the cultivation and maintenance of Bacillus subtilis.................................................0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................... 10..........................................0...............2 at 25°C Trace Mineral Solution: Composition per liter: CoCl2·6H2O...... 0..................................................0g Acid hydrolysate of casein .........3.5g NaCl ......... 5.. Bacillus Medium (ATCC Medium 455) Composition per liter: Bacillus Pullulan Salts Composition per liter: Soluble starch......................................... 10.95g K2HPO4 .0g NH4Cl ........................ Autoclave for 15 min at 15 psi pressure–121°C..............................5......... 3..................................1................................. 0.................36g Trehalose .....................................1............1g MnCl2·4H2O ................0........................ Bacillus polymyxa..................................................1....... and Bacillus thermoglucosidasius..... soluble...........................................................................................................................2g FeSO4·7H2O .....1g CaCl2·2H2O ...............2..............20............................................................ 15....Bacillus Pullulan Salts 53 Cool to 45°-50°C........................0g pH 7...........0mg Folic acid .0L.. Autoclave for 15 min at 15 psi pressure–121°C........5............... Pour into sterile Petri dishes or distribute into sterile tubes... 30.......0mg H3BO3 ................... 0............................................1..... Bacillus pasteurii Agar Composition per liter: Urea............................................................................................................................... Use: For the cultivation of Bacillus halodenitrificans................................. Distribute into tubes or flasks................ Also used to detect amylase-producing microorganisms...05g Trace mineral solution.................... Mix thoroughly............... Use: For the cultivation and maintenance of Bacillus pasteurii and Sporosarcina ureae..................................................................................................................3 ± 0................................... Distribute into tubes or deionized water and bring to 1...10..13g ZnCl2·2H2O ....... Do not overheat............ Mix thoroughly.............0.......................................... Gently heat and bring to boiling................5g MgSO4 ...2........................ ................................5g Ferric ammonium citrate.........5.5g pH 7........................... Pour into sterile Petri dishes or leave in tubes.......................................................... 20.....7...........2...........68g pH 7.......20....... 20.0........0L.....................0mL pH 6..................................................... Filter sterilize. Use: For the cultivation of Bacillus popilliae......0.1mg Preparation of Trace Mineral Solution: Add components to distilled/deionized water and bring volume to 1.........5mg CuSO4·5H2O.............................0mL sterile magnesium nitrate solution............ Gently heat until dissolved.... Gently heat and bring to boiling.0mg p-Aminobenzoic acid ..........................0........... Aseptically add 100............0.............1 at 25°C Preparation of Medium: Add components to tap water and bring volume to 1. Mix thoroughly...............................0g Peptone...................................................................0L...1g CaCl2·2H2O .................... Adjust pH to 7....0mg Thioctic acid ...................................................0g pH 7......0 ± 0..................................................................0...5g Yeast extract ..............0............5g MgSO4·7H2O.

....................0.....................................................................70g NaNO3 ...................................2 at 25°C Trace Elements Solution: Composition per liter: H3BO3 ...............0mg MnCl2·4H2O.................................................0mL Trace elements solution SL-10......... Pour into sterile Petri dishes or leave in tubes............. Mix thoroughly................................ Adjust pH to 7................................25g Yeast extract .....1g Na2MoO4·2H2O.........................................0L.......................... Aseptically add sterile vitamin solution.......08g MgSO4 ......................... 10.......................................... 0.......... 10..........................1 ± 0................ Mix thoroughly.................................3g CoCl2·6H2O . 9...........8 ± 0... 1...........0....................................1.........5g NH4Cl....... Incubation is at 65°C under an atmosphere of 5% O2.......................... Adjust pH to 6.....0.....0.....................0.........0mL Na2S·9H2O solution . Distribute into tubes or flasks........01g Ferric ammonium citrate.............. 45% H2..............0.....................0mL pH 7................... 4... Autoclave for 15 min at 15 psi pressure–121°C.........0mL pH 7........2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O..... Use: For the chemolithotrophic cultivation of Bacillus schlegelii.. 0...............02g CuCl2·2H2O.............................................................. 1........................ 5..............2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O .............................1g Na2MoO4·2H2O ...........................03g NiCl2·6H2O.....0g Na2CO3 ...2g ZnSO4·7H2O .......4............0mg CuCl2·2H2O ... 1.............. 20...................... 10...................................0L.............1 ± 0............... Use: For the cultivation of Bacillus schlegelii..........6g NaHCO3 ..................0g MgSO4·7H2O ......0L...............1g Na2MoO4·2H2O .............. 1.................................................................................................0............................................................ 0....................................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.............................. Bacillus selenitireducens Medium (DSMZ Medium 968) Composition per liter: NaCl ........0g KH2PO4 .......................................... 5.. 0...................3g CoCl2·6H2O.............................. Use: For the heterotrophic cultivation of Bacillus schlegelii.....................1 ± 0.. 0....................................... 0................................... Distribute into tubes or flasks....................0L..............01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1......01g CaCl2·2H2O ...................2g MnSO4·2H2O.................................. Autoclave for 15 min at 15 psi pressure–121°C......0mg CaCl2·2H2O........................... 0.........0mg Resazurin .....................................................................................................5g KH2PO4 ........................................................................ 1....0mL Selenite-tungstate solution ................0mg Ferric ammonium citrate.............................................................................2 at 25°C ........................................... 1...... except vitamin solution............................................. 0......0mL..................... Bacillus schlegelii Chemolithotrophic Growth Medium (DSMZ Medium 261) Composition per liter: Na2HPO4·2H2O.................................................5g H3BO3 .........................................0mg Trace elements solution SL-6........ Mix thoroughly.............3.......................................... Mix thoroughly.90................................... 10.........0..............................2g K2HPO4 ............... 0.........................................5g NH4Cl...15g (NH4)SO4.......................... 10.....2g MnSO4·2H2O ........................ 0.........5g H3BO3 .0g MgSO4·7H2O..... 0..01g Ferric ammonium citrate ....2g MnSO4·H2O .....................................0L............................... Distribute into tubes or flasks..........0..... Bacillus schlegelii Agar (LMG Medium 85) Composition per liter: Agar .....0mg Trace elements solution ......0g Na2HPO4·12 H2O..........5g NH4Cl ... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................0.. 30..................... 0............................................................1............................................................1.......01g CaCl2·2H2O...................................... 0............................ Incubation is at 65°C........................... Bacillus schlegelii Heterotrophic Growth Medium (DSMZ Medium 260) Composition per liter: Na2HPO4·2H2O ............ 1......................3g CoCl2·6H2O ..... Use: For the cultivation and maintenance of Bacillus species that can degrade pullulan........1........................54 Bacillus schlegelii Agar Preparation of Medium: Add components...2g Na-lactate ...................... 30......0mL pH 9...............................................................5g KH2PO4 ................................................. Distribute into tubes or flasks..................0mg NiCl2·6H2O ............. Mix thoroughly.. 0...................1............... Autoclave for 15 min at 15 psi pressure–121°C................................0....................25.......2g ZnSO4·7H2O.02g CuCl2·2H2O......0mg Trace elements solution SL-6 .........................0L.......................... 30..........................0......................................................... to distilled/deionized water and bring volume to 990................................................. Gently heat and bring to boiling..............................................5g Na-Pyruvate................5.......................................0.................................... 10% CO2............................ Mix thoroughly.... Autoclave for 15 min at 15 psi pressure–121°C.................................................................1.0mL pH 7.........10......................0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1........ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........................................0............0.......2g ZnSO4·7H2O ........03g NiCl2·6H2O ................1g KH2PO4 ........5g Sodium pyruvate ........................................................... 3.............3........................................................1............. Aseptically distribute into sterile tubes or flasks.................0g MgSO4·7H2O ....................................5mg Cysteine solution .................... Gently heat and bring to boiling.. Mix thoroughly... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................4.......... Mix thoroughly........................ Cool to 25°C.......... 0.................................................... Adjust pH to 7....................................................................

..........................................................0mg HCl (25% solution) ..........................0mL L-Isoleucine (1% solution) ...............0mL Potassium phosphate buffer... 1............... Sparge with 100% N2...................... Mix thoroughly.................... L-Cysteine·HCl·H2O ...........0g MgSO4 ................................. Use: For the cultivation of Bacillus selenitireducens and Bacillus arseniciselenatis..... 1....... Sparge with N2.......................... 0............................................. Selenite-Tungstate Solution: Composition per liter: NaOH ... Gently heat and bring to boiling....................................5mL L-Glutamine (1% solution)...... Cool to room temperature while sparging with 80% N2 gas.......2 at 25°C Mineral Salts Solution: Composition per liter: NaCl .............4mL L-Serine (1% solution)......... Sparge with 100% N2...............................10......................0mg MnCl2·4H2O.............30........3.............................. 0..... cysteine solution........4g ..... Bacillus stearothermophilus Defined Broth Composition per 100............................................................................. Aseptically and anaerobically add per liter 10................. 190.................. 0................................. 0.............................0L.......2 ± 0.......0mL Nicotinic acid (0....0mg H3BO3 ..0mg Na2SeO3·5H2O ... and Na2S·9H2O solution... 0..................................5mL L-Cystine (1% solution)......................................0L.....5g Na2WO4·2H2O .10.0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1....... Autoclave for 15 min at 15 psi pressure–121°C........................................ 3.......................................64mL L-Lysine·HCl (1% solution) ....... 0................. 2.............3mL CaCl2 (5% solution)................... Mix thoroughly.......................................................... 1..... 100................... Na2S·9H2O Solution: Composition per 10....... 5.............. 0...... Mix thoroughly........05% solution).......... 0.................... Bacillus thermoalcalophilus Medium Composition per liter: Yeast extract .......................125.......................................0mL sterile cysteine solution and 10.......10.52mL Glycine (1% solution) ......................................0mg ZnCl2 .......0mg NiCl2·6H2O ....................... 0..... 0. Use: For the cultivation of Bacillus stearothermophilus in a chemically defined medium..... 0........... Mix thoroughly..........5mL L-Asparagine·H2O (1% solution) .......... 10.........0mg CuCl2·2H2O .............. 5..... Use: For the cultivation of Bacillus stearothermophilus.......................... 1....5mL L-Histidine·HCl·H2O (1% solution) .........01mL ZnSO4·7H2O (5% solution) ......... 24....................1............................................0mL: Mineral salts solution .........25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10................................................... Mix thoroughly....3 ± 0..................... Preparation of Medium: Add components...0g KCl ....84mL L-Threonine (1% solution) .................... Cool to 25°C...........01% solution) ............... 6...................... Adjust pH to 9................. 1.....................8g Na2SO4 ........0mL........................... Autoclave for 15 min at 15 psi pressure–121°C.....................................................................0mL Glucose (20% solution) ..........0g Sodium acetate ................. Mix thoroughly.............56mL L-Methionine (1% solution) ..2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........0g KH2PO4 ................................ 0.................................. Filter sterilize.......... 0.........25g Preparation of Cysteine Solution: Add 55 L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10..0mg Na2MoO4·2H2O ....8........................................5g CoCl2·6H2O ..... Add remaining components.................................... Distribute to anaerobe tubes or bottles.......... 4.......0mL Thiamine·HCl (0................ 36.......... 0... 1.... Potassium Phosphate Buffer: Composition per 500........................ 0....01% solution) ..................................... Mix thoroughly....................... Add distilled/deionized water and bring volume to 1.................. Autoclave for 15 min at 15 psi pressure–121°C.. Mix thoroughly............ to distilled/deionized water and bring volume to 980...................... Filter sterilize...... 2.............. 4.. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ..0mL L-Glutamate·HCl (1% solution) .......42mL L-Tryptophan (1% solution) ................... Autoclave for 15 min at 15 psi pressure–121°C.................................... 1............ Mix thoroughly.... 1........01mL MnCl2 (10mm solution) ...................10......Bacillus thermoalcalophilus Medium Cysteine Solution: Composition per 10......... Mix thoroughly.................................... Add solid NaHCO3 and Na2CO3.0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10................................................0mL: K2HPO4 ....01mL pH 7.................4..............0g Yeast extract.... Sparge with 80% N2 + 20% CO2..........0mL: Distribute into tubes or flasks.............. 0..........01% solution) ....0g NH4Cl .....................................0g K2HPO4 .0mL.................0mL sterile Na2S·9H2O solution...........0g Preparation of Potassium Phosphate Buffer: Add components to distilled/deionized water and bring volume to 500....................................... 0........................................ Autoclave for 15 min at 15 psi pressure–121°C................................. 1......0g pH 7.......... 70............0mL of HCl solution.0mL L-Phenylalanine (1% solution) ..... 1...................... except NaHCO3..........64mL L-Tyrosine (1% solution)......0mL L-Proline (1% solution) ... 1...................0mL: Na2S·9H2O.....0........86mL L-Alanine (1% solution) ........0L.......... Na2CO3.........................................................0L................................... Boil for 3 min..........................3mL L-Valine (1% solution).............0mL. 1.............................0g Preparation of Mineral Salts Solution: Add components to distilled/deionized water and bring volume to 1.... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 100...... Bacillus stearothermophilus Broth Composition per liter: Pancreatic digest of casein ..............0mL...4mL L-Aspartate (1% solution) ........ 0...... 10..01mL FeCl3·6H2O (0.............84mL L-Arginine·HCl (1% solution) .................................... Cool to room temperature....0mL L-Leucine (1% solution)...........26mL Biotin (0....... 0..........................0mL......

............ 20........................0g MgSO4·7H2O................................... 0.......0mL of sterile ferric ammonium citrate solution and 5........0 ± 0................. Use: For the cultivation of Bacillus thuringiensis. Use: For the cultivation of Bacillus thermoalcalophilus.................. 0......0L.....0.......... 0...3........5g MgSO4·7H2O................................500..............05g CaCl2·2H2O ...3g MgSO4 ............. For chemolithotropic growth..................................... Autoclave for 15 min at 15 psi pressure–121°C.......... Aseptically distribute into sterile tubes or flasks........................... Cool to 50°–55°C............. Distribute into tubes or flasks....................05g pH 7.............. Cool to room temperature............................................................ Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O .......................................................0................2........6–5........0mL........8.....................0mL pH 4.....................0...0g NaCl .......5g NaHCO3 ................ Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C....... Gently heat and bring to boiling....................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1........................................................................ 6.............................................2g ..... Allow residue to settle...... Autoclave for 15 min at 15 psi pressure–121°C...........0...5g H3BO3 .0................. Autoclave for 15 min at 15 psi pressure–121°C... incubate the culture under 2% O2 + 10% CO2 + 60% H2 + 28% N2.........................................................02g CuCl2·2H2O....01g MnSO4·H2O.........................................0...........0L..............................................2g ZnSO4·7H2O...... 3.....0g (NH4)2SO4 ............................. Gently heat and bring to boiling.........................3g NH4Cl .56 Bacillus thermoantarcticus Medium K2HPO4 ................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................................................................ 1............6–5............ Autoclave for 15 min at 15 psi pressure–121°C.....0mL: Ferric ammonium citrate ............. Bacillus thermoleovorans Medium Composition per liter: Bacillus thuringiensis Medium Composition per liter: Glucose.................0L......................... Pour into sterile Petri dishes or distribute into sterile tubes......... Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly............................................... 5................... n-Heptadecane .......... ric ammonium citrate solution and trace elements solution SL-6............ Adjust pH to 4.................9g KH2PO4 ........ air dried ....... 0.......................0............0.....2... Autoclave for 60 min at 15 psi pressure–121°C...........0...2.............. Mix thoroughly.................. 0...........2............. Add soil to 960......... 1........................ 0.................0g Peptone..........................03g NiCl2·6H2O..................................3..0g Soil extract .3g KH2PO4 .................. Store at room temperature until clear................ 3.................................... Bacillus tusciae Medium Composition per liter: Na2HPO4·2H2O .....1.. Use: For the cultivation and maintenance of Bacillus thermoglucosidasius.........................0......................... Mix thoroughly..................8 at 25°C Soil Extract: Composition per liter: Garden soil.............................................0......0g Yeast extract ...........05g Preparation of Ferric Ammonium Citrate Solution: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20..........0g Preparation of Soil Extract: Pass 400............0.................. Adjust pH to 8...........................................0mL of tap water.................................... Mix thoroughly........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................0g K2HPO4·3H2O .. Preparation of Medium: Add components...0g Yeast extract...0L.0......0g Beef extract ......... Adjust pH to 7..................................... Mix thoroughly.............0L................................. 5....... Filter through Whatman filter paper................ Distribute into tubes or flasks........................ 0. 0............................................ Mix thoroughly...............3g CoCl2·6H2O............... Autoclave for 15 min at 15 psi pressure–121°C...................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......... Bacillus thermoantarcticus Medium Composition per liter: Yeast extract.............0mL Trace elements solution SL-6................... except fer- Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............................1g Na2MoO4·2H2O..................... 3................................................................2................. to distilled/deionized water and bring volume to 975..................................... Mix thoroughly..0L......... 3...........0...........................0g KCl.......0mL.......2g CaCl2·2H2O ......0mL pH 5............ Aseptically add 20.............. Decant supernatant solution..08g MnSO4·4H2O.............. Autoclave for 15 min at 15 psi pressure–121°C.....0g pH 7............................ Use: For the cultivation of Bacillus thermoantarcticus.....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...2 at 25°C Ferric Ammonium Citrate Solution: Composition per 20.................................................... Use: For the cultivation of Bacillus thermoleovorans......... Distribute into bottles in 200.....................0g (NH4)2HPO4 ............0g KH2PO4 .......... 1...0.................0mL of sterile trace elements solution SL-6........... Distribute into tubes or flasks...0g Soluble starch..................0 ± 0............................. 30............................................ Adjust pH to 7.............. Adjust pH to 5.......2g MgSO4·7H2O ..................0g of air-dried garden soil through a coarse sieve.........0........................01g pH 8..................3 ± 0.....0mL volumes.............2 ± 0.......... Autoclave for 15 min at 15 psi pressure– 121°C..... Bacillus thermoglucosidasius Agar Composition per liter: Agar .2g FeSO4 .. Use: For the chemolithotrophic growth of Bacillus tusciae.01g Ferric ammonium citrate solution ..0g Yeast extract.............5g Fe(NH4) citrate ....... 400.. Mix thoroughly.. 10............................................................. Distribute into tubes or flasks................0................................

..0........................3g L-Cysteine·HCl·H2O.....0 with KOH................0mL: Ferric ammonium citrate...................01g Na2SeO3·5H2O ................025 g KAI(SO4)2·12H2O ...............0L of distilled/deionized water.......0mL of sterile carbon source........................0.25g Na2S·9H2O ..........................................................................85% saline....... Cool to 45°– 50°C............ 0.............. Wash the cells four times in sterile 0...1....0mL distilled/ deionized water....................................................................0.............03g NiCl2·6H2O ...................... Autoclave for 15 min at 15 psi pressure–121°C.......05g CaCl2·2H2O.1g NiCl2·6H2O ........01g H3BO3 ... 0................ 0.......................................0g MnSO4·2H2O .. 17............................................................. Mix thoroughly.................0............. In a separate flask....................0L....... 2............0mg Lipoic acid....................................................... 1................0g NaCl ..0mg Riboflavin...............................................................0...................68g Beef extract .................................. trace elements solution SL6.......................0mL of sterile trace elements solution SL-6.................0g NaCl ................. Mix thoroughly.......10.. Carbon Source: Composition per 10....0.......... Resuspend the cell pellet in 25.... add 15....................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1................0.......... Autoclave for 15 min at 15 psi pressure–121°C......5....................5.......................................................................0...............0g NaHCO3 ........................................................2 at 25°C Ferric Ammonium Citrate Solution: Composition per 20..................... Mix thoroughly.............................................0.................................. and carbon source....0mg Trace elements solution................ Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O......02g CuSO4·5H2O .....0mg Calcium DL-pantothenate ..................................... to distilled/deionized water and bring volume to 965......................... 10.0g NH4Cl .................... 5. 0..............0g Na2HPO4·2H2O........................ Autoclave for 15 min at 15 psi pressure– 121°C..........................0mL of distilled/deionized water.......02g Resazurin ...........2............... Cool to 30°C...0mL Vitamin solution ............5...........06g FeSO4·7H2O .............................. 10......... Aseptically distribute into sterile tubes or flasks.... Add remaining components........0g Preparation of Carbon Source: Add carbohydrate or organic acid to distilled/deionized water and bring volume to 10..............................................................................5g Fe(NH4) citrate.............9g KH2PO4 .0.0..0g Nitrilotriacetic acid.....0mL of cooled...........................................................0.. 1...................................0mg Thiamine·HCl .........................................2g ZnSO4·7H2O .........................0mL...................... Autoclave for 15 min at 15 psi pressure– 121°C...0mL Carbon source ....0g or organic acid ....................... 10.............01g Ferric ammonium citrate solution...........0g of agar to 1..................... Autoclave for 15 min at 15 psi pressure–121°C...... Bacteroides cellulosolvens Medium Composition per liter: Cellobiose or cellulose .......... Pour into sterile Petri dishes......5 g CoSO4·7H2O ...................1g Na2MoO4·2H2O ..3..0mg p-Aminobenzoic acid .......................................................................... Autoclave for 15 min at 15 psi pressure–121°C...................................18 g FeSO4·7H2O.............0L with additional distilled/deionized water...............................................2g Yeast extract........... 0........5.............................................. Use: For the cultivation of freshwater Myxobacterium spe- cies....................................0mL Trace elements solution SL-6 ...2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O ...........0...01g MnSO4·H2O .....0mg ........... 0..........................18g (NH4)2SO4 ..................................................... Aseptically add 20................25g KH2PO4 .. to distilled/deionized water and bring volume to 1...................................... sterile agar solution....20....................0g MgSO4·7H2O ..........................................0mL pH 7........................ 0....36g Agar ... Mix thoroughly......................................... Mix thoroughly......................... except agar............................................................................ 0................. 2............0mL of washed cells and 250................................ Dissolve by adding KOH and adjust pH to 6...............0mg Nicotinic acid ............................0L................................................................ Vitamin Solution: Composition per liter: Pyridoxine·HCl..................................................... 1.....................................0........... Adjust pH to 4.............................0............................0 ± 0...................... 0............................. Inoculate with a culture of Aeromonas hydrophila..0.....................................................................7g pH 7......................5g NaHCO3 ................. Mix thoroughly..............2 at 25°C 57 Preparation of Medium: Add components. 0.5......... Cool to 45°–50°C..0mL pH 4...0mL.......................05g Preparation of Ferric Ammonium Citrate Solution: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20................................. Aseptically combine 25............10... 0.............................1..............000 × g for 10 min.....0................. Preparation of Medium: Add components......... 8.............0mL.........1.....................................................................3 mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500................15g MgSO4·7H2O.................. except fer- ric ammonium citrate solution.. Bacterial Cell Agar (BCA) Composition per liter: Tryptose ....... Mix thoroughly......0 ± 0.....5g H3BO3 ..0........................... 0....... Filter sterilize....................................3g NH4Cl................................. Gently heat and bring to boiling......................68g K2HPO4 ............................ 15... Bring volume to 1...........5............01g Na2MoO4·2H2O .......0mL: Carbohydrate...............3g CoCl2·6H2O ......1.............................5........................5................................................3 ± 0................... Use: For the heterotrophic growth of Bacillus tusciae........0mL of sterile ferric ammonium citrate solution.............. Adjust pH to 7............ 0................................... and 5......... Cool to 50°–55°C..................... Centrifuge culture in 40.....................................0mL volumes at 10....0....... Incubate with shaking at 30°C for 72 hr......12g CaCl2·2H2O ..................................................................... 15.........Bacteroides cellulosolvens Medium Bacillus tusciae Medium Composition per liter: Agar ................... Mix thoroughly.......5 g CaCl2·2H2O...18 g ZnSO4·7H2O ...........................02g CuCl2·2H2O ........... 2..5..............................................0............... Gently heat and bring to boiling...................

.......... Gently heat and bring to boiling.2 at 25°C Trace Elements: Composition per liter: CaCl2·2H2O ....................................0............0mg K2TeO3 ...........0.............................................................. BAM SM Agar (ATCC Medium 1656) Composition per liter: Agar ...........0g LiCl ..............................0mL: Bovine fibrinogen ....20.......................................................... 1..................................... Preparation of Cellobiose Solution: Add cellobiose Use: For the selective isolation and enumeration of coagu- Cellobiose Solution: Composition per 50..................................... Mix the two solutions together........................................................1g Preparation of Trace Elements: Add components to 1........................ to distilled/deionized water and bring volume to 800.................... Pour into sterile Petri dishes........ Gently heat and bring to boiling.0g Sodium pyruvate ...........25..........0........ Aseptically distribute into sterile tubes or flasks........................ Mix thoroughly.. Mix thoroughly............0mL.... 1............... BAM Agar (ATCC Medium 1655) Composition per liter: Agar ...... Mix thoroughly................. 10.0mL pH 7.......................... 5....................... Mix thoroughly..................25g (NH4)2SO4 ..5..............................0......... Gently heat and bring to boiling..................16g MnSO4·4H2O.............................................2 at 25°C RPF Supplement: Composition per 100............0..........................................2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath and BD Diagnostic Systems.. Filter sterilize.......................................................................................................0g Glucose....................... 5................0L of distilled/deionized water... 3............0L......................0g LiCl .................................18g CuSO4·5H2O........................ Preparation of Medium: Add components................................0...0.............66g Na2MoO4·2H2O............................5........................... 12............ 5......... Use: Used as a base for the preparation of egg-tellurite-glycine-pyruvate agar for the selective isolation and enumeration of coagulase-positive staphylococci from soil.............. 25...........0g KH2PO4 ......58 Baird-Parker Agar Biotin .... 1.......................0mL............. 17........................................ air............ Baird-Parker Agar Composition per liter: Agar .. 2..... 10.....0g Yeast extract .............................0g Yeast extract.0 ± 0... Colonies surrounded by an opaque zone of coagulated plasma are diagnostic for Staphylococcus aureus..0 with H2SO4...............................30.....................0g Preparation of Medium: Add components...0g Yeast extract ..0g RPF supplement........0mL. Cool to 45°–50°C.........0g Glycine.............................................75g Trypsin inhibitor ...0g pH 7................... Adjust medium to pH 4.......... 0...............................3............................... 5............1mg Preparation of RPF Supplement: Add components to Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.................. D-Cellobiose (or cellulose) .........0g Pancreatic digest of casein ......3...........0g Glucose............................. Mix thoroughly.............................. Preparation of Medium: Add components........................................................................................................... 1......................................0mg Vitamin B12 ......................................................... 2...................0g Sodium pyruvate ...15g H3BO3 . Add agar to 200.... For the differentiation and identification of staphylococci on the basis of their ability to coagulate plasma...............................0mL.......................................................5g CaCl2·2H2O ..........................................0mL: (or cellulose) to distilled/deionized water and bring volume to 50.......... Mix thoroughly...3g ZnSO4·7H2O.......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........100................0 ± 0..................... Filter sterilize.....0g Beef extract ................... 10.... Aseptically add 100.......... 25............................ and other materials.0mL............... except RPF supplement.............. Supplemented Composition per liter: Agar ................................................................................... Autoclave for 15 min at 15 psi pressure–121°C....1..................0mg Folic acid............ Use: For the cultivation and maintenance of Bacillus acidoterrestris...........0g Glycine................0....5g CaCl2·2H2O ................................0g Yeast extract..0mL of distilled/deionized water... Use: For the cultivation and maintenance of Bacteroides cellulosolvens.......................... Autoclave for 15 min at 15 psi pressure–121°C.................. Mix thoroughly... Mix thoroughly but gently...........0mg Rabbit plasma .... and other materials.............. to distilled/deionized water and bring volume to 900.........0mL pH 4...................................... Baird-Parker Agar..........0...................................... Mix thoroughly.................................................................................... Cool to 45°–50°C..... 17..........0mL pH 4................0g MgSO4·7H2O....... Mix thoroughly............2g Trace elements.....2 at 25°C .......... except agar........................0g MgSO4·7H2O.....................................18g CoCl2·6H2O..0................. Autoclave for 15 min at 15 psi pressure–121°C.................2g Trace elements.................. to distilled/deionized water and bring volume to 950.............................0L.0............... Aseptically add 50...................0g KH2PO4 ................. except cellobiose solution..................0 ± 0........... lase-positive staphylococci from soil..........0........................0g Beef extract ...... air.........0mL Caution: Potassium tellurite is toxic.....6..... Autoclave for 15 min at 15 psi pressure–121°C.................... 5..... 12..0 ± 0.0...... distilled/deionized water and bring volume to 100..............0g Pancreatic digest of casein ......................................................0mL of sterile cellobiose (or cellulose) solution.................. Autoclave agar separately to avoid acid hydrolysis........ Pour into sterile Petri dishes or distribute into sterile tubes............0mL of filter-sterilized RPF supplement......................... Pour into sterile Petri dishes..........................25g (NH4)2SO4 ............ 10........

........................1mg 59 MnSO4·4H2O............................................ Solution 3: Composition per 50............................. Trace Elements Solution: Composition per liter: Nitrilotriacetic acid......................... 0.................................................................................................... 0... Pour into sterile Petri dishes or distribute into sterile tubes....2g Trace elements ................... Add remaining components and bring volume to 1.............0mL Solution 1: Composition per 850........................0L.........................................................................................................15g H3BO3 ......... Mix thoroughly.......................................Basal Thermophile Medium Trace Elements: Composition per liter: CaCl2·2H2O..........................0..........0mL: Pancreatic digest of casein ...........................2% solution) ........0mL..0g Preparation of Solution 3: Add glucose to distilled/ deionized water and bring volume to 50...................0 ± 0.2 at 25°C Trace Elements: Composition per liter: CaCl2·2H2O...............18g CuSO4·5H2O ...........1......................... 9.......................0...................... 0. Use: For the cultivation and maintenance of Bacillus cycloheptanicus............. 0.....0..........9g KH2PO4 .............01g Disodium EDTA ..................................8g K2HPO4 ......................0... 0......1g Preparation of Trace Elements: Add components to 1..0.. Add agar to 200.........0L............... Preparation of Medium: Add components..........0mL Vitamin solution ..................... Autoclave for 15 min at 15 psi pressure–121°C............................ 0. except agar.......................................................0........ Cool to 45°–50°C.0mg H3BO3 .66g Na2MoO4·2H2O ...............0 with H2SO4.......................................................15g H3BO3 ..0mL: Yeast extract ............................................. Mix thoroughly...................5g CaCl2·2H2O...3g ZnSO4·7H2O .................... 0................. Adjust medium to pH 4...................................................................................03mL Preparation of Solution 1: Add components to dis- tilled/deionized water and bring volume to 850...............................................................................1g CuCl2 ........... Mix thoroughly......3......... 5....................................................................0L of distilled/deionized water....................02g CoCl2·6H2O..0mL Solution 3 .......0mg CaSO4·2H2O...01g Na2MoO4·2H2O............ 0.....................................................66g Na2MoO4·2H2O ...................5g NaCl .....0mg ........... Cool to 45°–50°C................... Use: For the cultivation and maintenance of Bacillus cycloheptanicus....................................5g NH4Cl ..... Gently heat and bring to boiling.................. Mix the two solutions together.......................................0......................1mg ZnSO4·7H2O ............... Adjust medium to pH 4..0mL: Glucose....................................7g MgSO4·7H2O ..02g Na2SeO3...............10...................... Autoclave for 45 min at 15 psi pressure–121°C...................10g Preparation of Trace Elements: Add components to 1............ 0.........017g H3BO3 ................0mL.......... Autoclave for 45 min at 15 psi pressure–121°C..........................................1g CaCl2·2H2O . Autoclave agar separately to avoid acid hydrolysis....25g (NH4)2SO4 ..................................................................... Mix thoroughly..... 850. 0..............0...... Pour into sterile Petri dishes or distribute into sterile tubes................0...................................0mL of distilled/deionized water..............................................0mL Resazurin (0.....................0mL of distilled/deionized water.2g MnCl2·4H2O . 0...............................................16g MnSO4·4H2O . Autoclave for 15 min at 15 psi pressure–121°C........ 0...........................01mg CuSO4·5H2O.................... Mix thoroughly.0g Yeast extract.................................................................................................................. Mix thoroughly....................................... 0...... 0.......................................... Autoclave agar separately to avoid acid hydrolysis........................0mL............1....02mg Co(NO3)2 ...... Mix thoroughly........... Add agar to 200............0..............0mL pH 4.... 1...... Cool to 45°–50°C................................................... 50........10..................5 with KOH.............. Mix thoroughly...12.....0mL........................................................... except agar..................... 0.............................. 100.......2g Trace elements solution..18g CoCl2·6H2O ....01mg NaMoO4·2H2O ... to distilled/deionized water and bring volume to 800...........................................0g MgSO4·7H2O ....................................................................................... 1...........2mg FeSO4·7H2O...............0g KH2PO4 ............................................. Adjust pH to 6..........0g Preparation of Solution 2: Add yeast extract to distilled/deionized water and bring volume to 100................................................................0mL FeSO4·7H2O (10% solution) .........30g ZnSO4·7H2O .................... Gently heat and bring to boiling....0mL...... 9.............. 0..0........................... 0....................................18g CuSO4·5H2O .................................. 2........................ 5............................. Basal Thermophile Medium Composition per liter: Solution 1 ............. Use: For the cultivation of Beggiatoa species................................. Preparation of Medium: Add components.................. 0......0............ 7......18g CoCl2·6H2O .... 0........... BAM SM Agar....... Mix thoroughly.............................................................. Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl........ Solution 2: Composition per 100..0g K2HPO4 ....................................................0........ Autoclave for 45 min at 15 psi pressure–121°C.......1.........................5µg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................................... to distilled/deionized water and bring volume to 800..0g Glucose ................ 0.................................................5.16g MnSO4·4H2O ....................... 3.............................................. Mix the two solutions together.............................................................. 30............................75g MgCl2·6H2O ... Filter sterilize.0g FeCl3·4H2O........................................................0mL of distilled/deionized water...........................................................0mL Solution 2 .................... Basal Mineral Medium Composition per liter: NH4Cl...0 with H2SO4..................................................0L of distilled/deionized water............ 0................................ 0....... 0.......... 0...... Modified Composition per liter: Agar ...0................0................01g Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 100.......................1g ZnCl2 ................

.............................0L with distilled/deionized water......0g to distilled/deionized water and bring volume to 100..... Add remaining components one at a time..........................12..........................2......................... Mix thoroughly...0.........................0mL: Preparation of Vitamin Mixture: Add components to distilled/deionized water and bring volume to 1......... 2.........8g Preparation of Mineral Solution 2: Add components to distilled/deionized water and bring volume to 1.................................................5g MnSO4·H2O.................................4g MgSO4·7H2O ........75.......................1g CoCl2·6H2O..............................5g Na2S·9H2O ...........................2.. Preparation of FeSO4·7H2O Solution: Dissolve FeSO4·7H2O in 100....... 12............ 2................ Add three drops of concentrated HCl......0............................... Distribute into tubes in 10............. Fervidobacterium nodosum................................................ and Thermoanaerobium brockii..0g Yeast extract ....................0....0mg Cyanocobalamin .01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500...........0mL Wolfe’s mineral solution ......................................................0mL of cysteine-sulfide reducing solution.............................................. Mineral Solution 2: Composition per liter: NH4Cl......... Mix thoroughly.........0.... 5....0mL Cysteine-sulfide reducing solution ....... 5....................0............................................................... Prepare in a chemical fume hood.............. The pH should be 7.............................................................................75......................0g Na2SO4 .....0g NaHCO3 .......2g to distilled/deionized water and bring volume to 800. Mix thoroughly.....0g FeSO4 ·7H2O ..... Mix thoroughly.............................................. Cysteine-Sulfide Reducing Solution: Composition per 200...... 3............................................. Use: For the cultivation and maintenance of Acetivibrio cellulolyticus....................................... 2.........10.....................1..........................0mg Thiamine·HCl .......... Bring volume to 1............1% solution)..........1g CaCl2 ........................................0L..............................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..5g Preparation of Cysteine-Sulfide Reducing Solution: Add L-cysteine·HCl·H2O to 50........... Preparation of Na2S·9H2O Solution: Add Na2S·9H2O Wolfe’s Mineral Solution: Composition per liter Na2S·9H2O...........0mL..................... Add 4........ FeSO4·7H2O Solution: Composition per 100....................... 5. Add 8................................5 with KOH to dissolve.................0mg Cyanocobalamin..............0mg Thioctic acid ......0..................0mL pH 7........0mL Resazurin (0...........6 ± 0............................01g Na2MoO4·2H2O.........8mL FeSO4·7H2O solution ...........0mg p-Aminobenzoic acid . and other cellulose-degrading microorganisms.....................0mg Folic acid........................0mL of distilled/deionized water...... Heat and boil under 90% N2 + 10% CO2...........10.......................................0....................................................9g Preparation of Medium: Add cellulose and NaHCO3 Caution: This medium contains sodium sulfide and may produce toxic H2S gas.......0mg Riboflavin ................................... Preparation of Medium: Aseptically combine solution 1....0mL: FeSO4·7H2O.....................................5...............0mL Mineral solution 2 .......10.............. 0......................8mL more of cysteine-sulfide reducing solution..................01g AlK(SO4)2·12H2O ..... 0.....5mg Na2S·9H2O Solution: Composition per 100........0mL volumes and cap.....1..........................................................................0mg Lipoic acid (thioctic acid) ......................0mg Nicotinic acid ..........6 at room temperature........ Immediately prior to inoculation................ 3................1mL of sterile Na2S·9H2O solution to each tube...............10.................. Mix thoroughly......... Transfer anaerobically to tubes or flasks and stopper.... Add Na2S·9H2O......5.... Cool and continue flushing under 90% N2 + 10% CO2..... and solution 3 under 100% N2..................... Preparation of Mineral Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 1..... 5.............................0mg p-Aminobenzoic acid ....... 5........................................10...................................................0mg Biotin ....................0mg Biotin .............................................................0mL with distilled/deionized water.............. Bacteroides cellulosolvens.......0L.........3.................5g KH2PO4 ........ 1............................2 at 25°C MgSO4·7H2O.......................... Store below −20°C.............................................1g CuSO4·5H2O....................... Add all other components except cysteine-sulfide reducing solution................... Autoclave for 15 min at 15 psi pressure–121°C....5g NaCl ......... do not adjust.. 2....0mL: L-Cysteine·HCl·H2O.................... BCP Azide Broth (Bromcresol Purple Azide Broth) Composition per liter: Casein peptone ........ BC Medium (Medium for Acetivibrio cellulolyticus) Composition per liter: Cellulose powder ........................................ Boil under 100% N2............5................................2.....0g ...........1g ZnSO4·7H2O............................... 10............. 5......0............ Acetivibrio cellulosolvens.................. Use: For the cultivation and maintenance of Clostridium species..................................0mL Vitamin mixture ...........2g CaCl2·2H2O....................................0L..............0mL volumes under 100% N2..........0mL of distilled/deionized water and adjust to pH 6............................ Bring volume to 200.............................. 0...... Quickly adjust pH to 10 with fresh 3N NaOH and flush under 100% N2.0g Nitriloacetic acid ........................................ Vitamin Mixture: Composition per liter: Pyridoxine·HCl........................0mL of distilled/ Mineral Solution 1: Composition per liter: deionized water.. K2HPO4 ........................................................0g Mineral solution 1 .....01g H3BO3 ...........0.10. Distribute anaerobically into tubes in 7...........0L................................0.......... aseptically add 0.................. solution 2.60 BC Medium Thiamine·HCl ......................0mg Calcium pantothenate ...1... 2............... Autoclave for 15 min at 15 psi pressure– 121°C....................................0mL.....

BCYE Selective Agar with CCVC D-Glucose.....1...........9 ± 0. Preparation of Medium: Add components........................0g Charcoal...0g Yeast extract........................ activated................. 2.................... 1..... cultivation.......0............... 2....................25g Antibiotic solution............... Filter sterilize............................ and maintenance of Le- gionella pneumophila and other Legionella species from environmental water samples.....0mL of a 10% solution of L-cysteine·HCl·H2O that has been filter sterilized........ Filter sterilize.... Adjust medium to pH 6............................. BCYE Differential Agar (Buffered Charcoal Yeast Extract Differential Agar) Composition per liter: Agar .15............................................ Azides also react with metals and disposal must be highly diluted............... Heat gently and bring to boil for 1 min....0. Gently heat to boiling.................. activated ............. Cool to 50°– 55°C.. and Cycloheximide) Composition per 1014..0g Fe4(P2O7)3·9H2O ................ Colistin........ Preparation of Medium: Add components distilled/ deionized water to 1......0g K2HPO4 .......4g Fe4(P2O7)3·9H2O ....... 10......0g α-Ketoglutarate.....5g Bromcresol Purple ......................9 with 1N KOH.......2 at 25°C Caution: Sodium azide is toxic............ Autoclave for 15 min at 15 psi pressure–121°C......... For the presumptive differential identification of Legionella species based on colony color and morphology.......0g Charcoal..............7g KH2PO4 ........032g pH 6.................................. Autoclave for 15 min at 15 psi pressure–121°C........................... to distilled/deionized water and bring volume to 1................................................................. 5..................0L.................. Use: For the isolation.......................................................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid).......................01g Bromothymol Blue ............... Mix thoroughly. 0...........9 61 with 1N KOH....0g Yeast extract. activated...............0mL pH 6........................................................................................0g NaCl ..................10........... 5...... Cool to 50°–55°C............ Mix thoroughly....................... 15................... 10................ BCYE Agar (BCYE Alpha Base) (Buffered Charcoal Yeast Extract Agar) Composition per liter: Agar ..16........0mg Cephalothin .....9 with 1N KOH.........0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) .........................................0mL.................................... 10.................. except Lcysteine and antibiotic solutions....... L-Cysteine·HCl·H2O Solution: Composition per 10........ except cys- teine.......25g Bromcresol Purple ......2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems........9 ± 0............................ to distilled/deionized water and bring volume to 1.....................9 ± 0.................................................... Mix thoroughly............ Autoclave for 15 min at 15 psi pressure–121°C..0g α-Ketoglutarate.................. except Lcysteine·HCl·H2O............................... 15.. Preparation of Medium: Add components.......................... Heat gently and bring to boil for 1 min.......9 ± 0.............................7g NaN3........................ 0................................... Adjust medium to pH 6.................... and maintenance of Legionella pneumophila and other Legionella species from environmental water samples...0mL...................................................4g Fe4(P2O7)3·9H2O .... Mix thoroughly............0g L-Cysteine·HCl·H2O ..........0mL: Agar ........ cultivation............ Adjust medium to pH 6.............. . Mix thoroughly.... Add 4............... Preparation of Medium: Add components.....2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension...............................25g pH 6.. Legionella micdadei appears as blue/gray or dark blue colonies........................................... 10.....5mg Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10.0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10..... Legionella pneumophila appears as light blue/green colonies.......... 0............................. 4.0g α-Ketoglutarate . Use: For the isolation................0mL: L-Cysteine·HCl·H2O....... 0........... Cool to 50°–55°C.......... 0.................... For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from environmental water samples.. 1.............................. and maintenance of Legionella pneumophila and other Legionella species from environmental samples....... 0.... 2..0mL of sterile antibiotic solution..........................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) .... to distilled/deionized water and bring volume to 1....80.... Add 4..... cultivation.......... Mix thoroughly............................0L.............0L............ Heat gently and bring to boil for 1 min............... Mix thoroughly... 10..........0mL of L-cysteine·HCl·H2O solution and 10.0mg Colistin .................. Vancomycin....... 2... Use: For use in the confirmation test for the presence of fecal streptococci in water and wastewater..........0mL: Cycloheximide ....1.............4......... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.........0g Yeast extract .......... Add 4.10.................2..... Mix thoroughly...... Mix thoroughly..... 0.......2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems...0g L-Cysteine·HCl·H2O ..... Use: For the isolation...........0mg Vancomycin . 0.....0mL Cysteine·HCl·H2O solution ......0L................ Antibiotic Solution: Composition per 10...............0mL of a 10% solution of L-cysteine·HCl·H2O that has been filter sterilized....01g pH 6.. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.................................. BCYE Selective Agar with CCVC (Buffered Charcoal Yeast Extract Selective Agar with Cephalothin... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.....................................0g Charcoal.....

..... except Lcysteine·HCl·H2O solution and antibiotic solution........0mL L-Cysteine·HCl·H2O solution ....... Polymyxin B.....................................................08g Vancomycin ................ 4....... Mix thor- ..........0........000 units Anisomycin .........0mL pH 6............................... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension..0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10..........0mL.........0L......25g Antibiotic solution ..........................0g Fe4(P2O7)3·9H2O ....0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) .0g α-Ketoglutarate...0g Cycloheximide ....................0mL: Agar ................ cultivation.... Mix thoroughly........................... Antibiotic Solution: Composition per 10..........0g Yeast extract.....0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid)...........................0mL of sterile antibiotic solution...........15. Preparation of Medium: Add components..0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10....................... 5.4..........0g Fe4(P2O7)3·9H2O ..... Filter sterilize.......... Filter sterilize......................................2...0mg Cefamandole.. Heat gently and bring to boil for 1 min. 1............10.. BCYE Selective Agar with GVPC (Buffered Charcoal Yeast Extract Selective Agar with Glycine..4.................... For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples......... Mix thoroughly.. Antibiotic Solution: Composition per 10........................... 1.. and maintenance of Le- gionella pneumophila from environmental samples.. 0............. 10.......... Polymyxin B......0mL......0mL... Adjust medium to pH 6..............0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10..........................0L.................. Mix thoroughly.......0g α-Ketoglutarate................ and Cycloheximide) Composition per 1014.......................... 10............0L............. Antibiotic Solution: Composition per 10........................... L-Cysteine·HCl·H2O Solution: Composition per 10........... Heat gently and bring to boil for 1 min...............0mL: Glycine ......................0mg Polymyxin B ......................... 15......................... Mix thoroughly.....................0mg Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10..................200U Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10............................ 15........................................................0mL of Lcysteine·HCl·H2O solution and 10.....0mL of sterile antibiotic solution.......... Anisomycin............................................0mL: Agar .......9 ± 0.....................................08g Vancomycin . Mix thoroughly..... activated.79........ Use: For the isolation....... L-Cysteine·HCl·H2O Solution: Composition per 10................ cultivation.. Add 4........................2 at 25°C L-Cysteine·HCl·H2O Solution: Composition per 10.........................0mg Polymyxin B........ 3.........................0mL: Polymyxin B............................... Adjust medium to pH 6...............0g Yeast extract.....62 BCYE Selective Agar with GPVA BCYE Selective Agar with GPVA (Buffered Charcoal Yeast Extract Selective Agar with Glycine........ 0...2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems....................0g Charcoal. Mix thoroughly...0g Yeast extract ........ Mix thoroughly...........0mL: L-Cysteine·HCl·H2O..9 ± 0...... 0........... Vancomycin..9 ± 0........................ Use: For the isolation.......2..................2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath. to distilled/deionized water and bring volume to 1................ 100.....................0mL: Glycine.....9 with 1N KOH..........0mL: L-Cysteine·HCl·H2O ..0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ..............0mL: Agar ....................... Mix thoroughly.0mL........ except Lcysteine·HCl·H2O solution and antibiotic solution.............0mL L-Cysteine·HCl·H2O solution ......1........000U Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10....1......... except Lcysteine·HCl·H2O solution and antibiotic solution........ Autoclave for 15 min at 15 psi pressure–121°C...........................................................0g Anisomycin .......0mL..........................................0mL pH 6............................0mL pH 6...............0mL: L-Cysteine·HCl·H2O... Mix thoroughly.10...25g Antibiotic solution .... Add 4.......................... Vancomycin...... Filter sterilize...........................3..........10..0.................................... activated... Filter sterilize............. 10..................................... 10..0g Fe4(P2O7)3·9H2O ....................... 2.................................. 2...........0mL L-Cysteine·HCl·H2O solution ............................................. to distilled/deionized water and bring volume to 1.0mL of L-cysteine·HCl·H2O solution and 10.......................0g Charcoal.............. Filter sterilize........ 10...10.................................0g Charcoal. Autoclave for 15 min at 15 psi pressure–121°C.......1........ Cool to 50°–55°C...................9 with 1N KOH. Preparation of Medium: Add components..25g Antibiotic solution............. Cool to 50°–55°C..... 1........80................ For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples..................0g α-Ketoglutarate ............ Mix thoroughly....0mL.. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension..................... Filter sterilize....................... Preparation of Medium: Add components..................... and maintenance of Legionella pneumophila and other Legionella species from environmental samples........................ and Anisomycin) Composition per 1014....................1... to distilled/deionized water and bring volume to 1.................. and Cefamandole) Composition per 1014.80........................... BCYE Selective Agar with PAC (Buffered Charcoal Yeast Extract Selective Agar with Polymyxin B............... activated .

....25g Preparation of Fe4(P2O7)3·9H2O Solution: Add Fe4(P2O7)3·9H2O to distilled/deionized water and bring volume to 10. cultivation.....1........0......... Mix thoroughly.........0......................................... cultivation.......................... 80.0g Bovine serum albumin solution.. and Vancomycin) (Wadowsky–Yee Medium) Composition per 1014.0mL.. BCYEα without L-Cysteine (Buffered Charcoal Yeast Extract Agar without L-Cysteine) Composition per liter: Agar .......... 10...000 units Anisomycin ........ Heat gently and bring to boil for 1 min.......0mL........0mL............ Fe4(P2O7)3·9H2O solution................................. 10.... Adjust medium to pH 6..................0g α-Ketoglutarate ..... Preparation of Medium: Add components—except Lcysteine·HCl·H2O solution.. Mix thoroughly. For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples.............. Filter sterilize. to distilled/deionized water and bring volume to 1......0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ............. Mix thoroughly..... and maintenance of Legionella pneumophila and other Legionella species from environmental samples. Autoclave for 15 min at 15 psi pressure– 121°C... 0..............0g Charcoal............9 ± 0....................0mL: Fe4(P2O7)3·9H2O . Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension........... Mix thoroughly.............4g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.....................0mL L-Cysteine·HCl·H2O solution .....0mL pH 6.............0mL: Fe4(P2O7)3·9H2O ...0mL: Polymyxin B ..........0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid).. Mix thoroughly... Filter sterilize.. Filter sterilize......2 at 25°C Bovine Serum Albumin Solution: Composition per 10......9 with 1N KOH.. BCYE Selective Agar with PAV (Buffered Charcoal Yeast Extract Selective Agar with Polymyxin B.. Adjust medium to pH 6..0mL..0mL pH 6..... Add 4........9 with 1N KOH......................10.0mL......10.... Add 4... activated......... Mix thoroughly........................ Mix thoroughly....0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10..0g Charcoal.................................. 40............. and maintenance of Legionella pneumophila and other Legionella species from environmental samples.... Mix thoroughly......0mL: Bovine serum albumin ....................0mL pH 6.. Use: For the isolation.........................0mL Fe4(P2O7)3·9H2O solution ...........0mL of L-cysteine·HCl·H2O solution and 10............................ Use: For the isolation...... Use: For the isolation....................................................... except Lcysteine and antibiotic solution.... For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples............2.........................0mL of sterile antibiotic solution..................... Preparation of Medium: Add components........0g Yeast extract ................0mL. 10.. 2......... 15..........1...... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension. Heat gently and bring to boil for 1 min...............25g Preparation of Fe4(P2O7)3·9H2O Solution: Add Fe4(P2O7)3·9H2O to distilled/deionized water and bring volume to 10...............................................0.............0mL: Agar ...........................25g Antibiotic solution ........ Antibiotic Solution: Composition per 10..0mL of sterile antibiotic solution..... Autoclave for 15 min at 15 psi pressure–121°C...4............................9 ± 0................ L-Cysteine·HCl·H2O Solution: Composition per 10................................... Filter sterilize............... BCYEα with Alb (Buffered Charcoal Yeast Extract Agar with Albumin) Composition per liter: Agar ........10. 1. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension. Mix thoroughly......... Adjust medium to pH 6......0mL..2 at 25°C Fe4(P2O7)3·9H2O Solution: Composition per 10. Mix thoroughly.. to distilled/deionized water and bring volume to 990.............................0mL..15.. Adjust medium ........ Cool to 50°–55°C.2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems.......................0mL of sterile bovine serum albumin solution.BCYEα without L-Cysteine oughly..........0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid).................... Fe4(P2O7)3·9H2O solution................0g α-Ketoglutarate....................10........................................ Preparation of Medium: Add components..... L-Cysteine·HCl·H2O Solution: Composition per 10.0g Yeast extract....... Mix thoroughly.... and bovine serum albumin solution—to distilled/deionized water and bring volume to 970...... activated ..........0mL of L-cysteine·HCl·H2O solution and 10........ Autoclave for 15 min at 15 psi pressure–121°C... Cool to 50°–55°C...... Heat gently and bring to boil for 1 min...0g α-Ketoglutarate ............ Aseptically add the L-cysteine·HCl·H2O solution............................. Anisomycin.....0mL: L-Cysteine·HCl·H2O ......................... 10....... Fe4(P2O7)3·9H2O Solution: Composition per 10..5mg Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10..................................... 10...........1g Preparation of Bovine Serum Albumin Solution: Add bovine serum albumin to distilled/deionized water and bring volume to 10...0mL: L-Cysteine·HCl·H2O.2................ activated ... and maintenance of Legionella pneumophila and other Legionella species from environmental samples........... except Fe4(P2O7)3·9H2O solution...0..0g Fe4(P2O7)3·9H2O solution ............10....................... Mix thoroughly.................. 0......... Cool to 50°–55°C... Filter sterilize.......0mg Vancomycin ...0g Fe4(P2O7)3·9H2O ...... 1............9 ± 0...........................................0mL L-Cysteine·HCl·H2O solution ...........................0g Charcoal..................................................0g 63 Yeast extract ........ and 10... cultivation. 15............. Filter sterilize...... 10...............................0L.9 with 1N KOH.

..........1% solution)...................0g Sodium acetate ................ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........4 ± 0.......... Autoclave for 15 min at 15 psi pressure–121°C............................2... 3................. Mix thoroughly............................... 3.........2 at 25°C ......... Use: For the cultivation of Beggiatoa species and myxotrophic Thiothrix species........ Adjust pH to 7. Pour the base layer agar into a sterile Petri dish..... Autoclave for 15 min at 15 psi pressure–121°C................0mL: FeSO4 ......... or Pseudomonas putida............ Preparation of Medium: Inoculate appropriate bacterial host into 10..........1............. Distribute into tubes or flasks...0mL............ Gently heat and bring to boiling..... 1....................... Mix thoroughly... Cool the semisolid agar to 40°–45°C..... 0...............2 ± 0........................0mL of the previously grown host culture.........0g Beef extract ..... 20...................6g Preparation of Base Layer Agar: Add components to distilled/deionized water and bring volume to 1.2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................01% solution) ................... 6.........0g Peptone.............4 ± 0.......................................... Pour over the solidified base layer agar.............. Mix thoroughly................1g Catalase ....................................... Mix thoroughly........ Mix thoroughly....................0mL Trace elements............. Escherichia coli. Use: For the cultivation and maintenance of a wide variety of microorganisms. Allow base layer agar to solidify........... Hosts include Erwinia amylovora................ 10..........9 with 1N KOH........... Add 1...... Heat gently and bring to boil for 1 min........... Base Layer Agar: Composition per liter: Agar .2. Melt the base layer agar and semisolid agar...... Pour into Petri dishes or leave in tubes........... 1............0L... Mix thoroughly............0mL Host Medium: Composition per liter: Yeast extract.......0mL K2HPO4 (1% solution) .....................0....0mL of sterile Fe4(P2O7)3·9H2O solution.........................0mL CuSO4·5H2O (0............. Gently heat and bring to boiling................ Adjust pH to 7...........................10.....................0L...................................6g Preparation of Semisolid Agar: Add components to distilled/deionized water and bring volume to 1.................................. 10........................0mL NH4Cl (4% solution) ............................ 10.................. Adjust pH to 7... Incubate host culture for 24–48 hr at 30°C.................................0mL volumes...0L..0mL Trace Elements: Composition per liter: EDTA solution.................0g Peptone..... Recommended for the culture of microorganisms from water.......0mL of host medium.......... Mix thoroughly....0g pH 7..................... Distribute into tubes or flasks.......................5................... Autoclave for 15 min at 15 psi pressure–121°C...0mL Host medium.............. 19........0mL Semisolid agar........ Heat gently and bring to boiling.......................... Autoclave for 15 min at 15 psi pressure–121°C...............02% solution)....2 Distribute into tubes in 10...0mL volumes..00005% solution). 3.......... Mix thoroughly.................................6g Preparation of Host Medium: Add components to distilled/deionized water and bring volume to 1......... 3.... Autoclave for 15 min at 15 psi pressure–121°C..... Semisolid Agar: Composition per liter: Agar ............ 10........... EDTA Solution: Composition per 100.........................0g Agar ... Use: For the isolation....... concentrated..............0mL Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 1.......0g Yeast extract..... Beggiatoa and Thiothrix Medium Composition per liter: CaSO4·2H2O (saturated solution).............................. Serratia marcescens................0mL Na2MoO4·2H2O (0......0mL ZnSO4·7H2O (0........0g Beef extract .7... Autoclave for 15 min at 15 psi pressure–121°C...............1% solution) ............ 5..................................5g CaCl2 .......................................................................0...... 1.............. 5....... Recommended for the culture of microorganisms from water........0mL MnSO4·4H2O (0............. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension...................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..0L........ Beggiatoa Medium (ATCC Medium 138) Composition per liter: Yeast extract .2 Distribute into tubes in 10.....10..........64 Bdellovibrio Medium to pH 6............0mL volumes.......2......... Bdellovibrio Medium Composition per Petri dish: Base layer agar.........2 Distribute into tubes in 10......................000U pH 7...............................0g pH 7......... 10....... 0...................0L.............0L.................................. Beef Extract Broth Composition per liter: Peptone ..........................0mL MgSO4·7H2O (1% solution)....... Heat gently and bring to boiling.... Mix thoroughly........... Autoclave for 15 min at 15 psi pressure–121°C......0g Peptone. cultivation.............................. Aseptically add 10..........3..............0mL Co(NO3)2 (0..........0mL H3BO3 (0................................................................. Use: For the cultivation of Bdellovibrio bacteriovorus and Bdellovibrio starrii.............0mL Preparation of EDTA Solution: Add EDTA and FeSO4 to concentrated HCl...................... Distribute into tubes or flasks. Use: For the cultivation and maintenance of a wide variety of microorganisms................ 15................ Beef Extract Agar Composition per liter: Agar ..... 20...............0L.......................... 10...0g Yeast extract..........................................01% solution) ... Cool to 50°–55°C.................... 0........0g Peptone......................................0g EDTA.......... 5........... and maintenance of Legionella pneumophila and other Legionella species from environmental samples...... Mix thoroughly....0g HCl......................10........... Carefully add to distilled/deionized water and bring volume to 100...

and Clostridium barkeri.................................0................................... Aseptically add 100.........................................................0g K2HPO4 .................. 2........ 5...... Mix thoroughly..............0g Sodium benzoate . deionized water and bring volume to 1........... Preparation of Medium: Add components... Autoclave for 15 min at 15 psi pressure–121°C..... and Beijerinckia mobilis...... Mix thoroughly...............................0g Preparation of Na2S2O3 Solution: Add Na2S2O3 to distilled/deionized water and bring volume to 100........... Autoclave for 15 min at 15 psi pressure–121°C.... except cat- alase........................................................... Autoclave for 15 min at 15 psi pressure–121°C..0g K2HPO4 ..............................................0mL pH 6................1...........................0mg ZnSO4·6H2O ..0L... 0........ 25......5g Sodium acetate ................ 0........................................ Mix thoroughly..........................5 ± 0..............1g NH4Cl ...............................................0..... Beijerinckia mobilis......................... 0... 10............ to tap water and bring volume to 1..........0mL................... NaHCO3 Solution: Composition per 10..................................0mL: D-Glucose............0mL of sterile NaHCO3 solution........................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 50... Beijerinckia fluminensis....................5g MgSO4·7H2O............... 5.............0........................................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10........................... to distilled/deionized water and bring volume to 950.... Beijerinckia species.....2 at 25°C Glucose Solution: Composition per 50............ Cool to 45°–50°C..............0mL............................ Beggiatoa Medium (ATCC Medium 1193) Composition per liter: Sodium sulfide .......... 20......5........... Autoclave for 15 min at 15 psi pressure–121°C...................................0mL of sterile glucose solution.50..2g MgCl2 .....1g FeSO4·7H2O...................................................0................ Mix thoroughly.......... Beijerinckia indica........................... Aseptically add 10..1.................... except glu- cose solution....................................2g Benzoate solution .....10..........3mg ZnSO4·7H2O............................................1.1g FeSO4·7H2O ........................................0mg Na2MoO4·2H2O .................3..............................0............................5g pH 5.20............ Beijerinckia fluminensis.........5 ± 0........2g MgSO4·7H2O............................................0mL: Na2S2O3 ............................... Pour into sterile Petri dishes or distribute into sterile tubes Use: For the cultivation and maintenance of Beijerinckia derxii.................. Mix thoroughly....... 0......... Preparation of Medium: Add components..................................... Autoclave for 15 min at 15 psi pressure–121°C.... to distilled/deionized water and bring volume to 890..................0mL: NaHCO3 ......................... Distribute into tubes or flasks...............0g Glucose.... 15............................................0mg Na2MoO4·2H2O............0mL of sterile Na2S2O3 solution and 10..0L........... Mix thoroughly...................................0mg pH 6........................... Use: For the isolation and cultivation of Beijerinckia derxii......... 0.......................5...............0g NaCl ................. Gently heat and bring to boiling..................0mL . Mix thoroughly............................ Beijerinckia Agar Composition per liter: Agar ......................................................................................0mg Glucose solution ..................0 ± 0.0mL pH 7.......................... Mix thoroughly.2g Yeast extract .... Benzoate Medium Composition per liter: Noble agar .5...........01g pH 7..0L.......8g KH2PO4 ...01g Yeast extract.................... Mix thoroughly....... Pour into sterile Petri dishes or leave in tubes................. 100..Benzoate Medium Preparation of Medium: Add components....................... Use: For the cultivation of Beggiatoa alba........ Mix thoroughly.............2g MgSO4·7H2O .........01g Nutrient broth.................... Modified Composition per liter: Agar ............ Use: For the cultivation and maintenance of Thiobacillus thermophilica........................................0L..........2 at 25°C Preparation of Medium: Add components to distilled/ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........2 at 25°C Na2S2O3 Solution: Composition per 100...........20...............................2 at 25°C 65 Beijerinckia Medium..........1g Na2S2O3 solution ..0mL...0..000 units of sterile catalase..... Beijerinckia Medium Composition per liter: Glucose .............4.. Beijerinck’s Thiobacillus Medium Composition per liter: Noble agar ....0mL NaHCO3 solution............................................................8g KH2PO4 . Autoclave for 15 min at 15 psi pressure–121°C..... Autoclave for 15 min at 15 psi pressure–121°C.......................................................0mg MnSO4·6H2O ......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....... Use: For the cultivation and maintenance of Beggiatoa alba and Vitreoscilla species................20...............................0g (NH4)2HPO4 ............................... 20.0mL..................... Mix thoroughly...............................0........0g KH2PO4 ..................................................................... 4.............................................. 0........3.....0mg CuSO4·6H2O .. Distribute into tubes or flasks..0g MgSO4·7H2O ... Gently heat and bring to boiling.........................................................................5. Filter sterilize...... Use: For the cultivation of Beijerinckia species.0g KH2PO4 .................0mg MnSO4·H2O.. Beijerinckia indica..... Pour into sterile Petri dishes or leave in tubes.............. 0............................. Filter sterilize......0mg CuSO4·5H2O......................0.............0–7............... 10.... 0................0mL....... except Na2S2O3 solution and NaHCO3 solution.. 1...... Distribute into tubes or flasks................................................ Aseptically add 10..0g Na2HPO4 ........................................................5 ± 0...........15...........................................................

......... 2.......0................. Mix thoroughly....................... Autoclave for 15 min at 15 psi pressure–121°C..............0mL Benzoate Solution: Composition per 25................... anhydrous . Sparge with air overnight to oxidize the Fe2+ to Fe3+.2g Yeast extract.... Aseptically add 25.....................10............. Mix thoroughly.0mL: Sodium benzoate........2g FeSO4·7H2O........50......................0mg Na2B4O7·10H2O ..... Filter sterilize..........0mL: EDTA..............0g NaNH4HPO4·4H2O ............2....... Benzoate Minimal Salts Medium Composition per liter: K2HPO4 ... Autoclave for 15 min at 15 psi pressure–121°C........... 700............. In a separate flask........2g CaCl2 . Autoclave for 15 min at 15 psi pressure–121°C........0mL...................................................0mL pH 7..0mL Preparation of Trace Metals Solution: Add components to distilled/deionized water and bring volume to 1.................... Mix thoroughly...................................5g Preparation of Phosphate Buffer: Add components to distilled/deionized water and bring volume to 200............................ Benzoate Nitrate Salts Medium (BNS) Composition per liter: Solution A .... Use: For the cultivation of microorganisms that can utilize benzoate as a carbon source..2 at 25°C Benzoate Solution: Composition per 25... Aseptically combine the two autoclavesterilized solutions........2 at 25°C Solution A: Composition per 700.......................0mL..0mL... 0....1.....12g KH2PO4 .. Mix thoroughly........ Preparation of Medium: Add components....0mg Trace metals solution............ Gently heat and bring to boiling.....................0.......................................................................... 3.... Mix thoroughly........ Cool to 45°– 50°C...0mg ZnSO4·7H2O..............2mg Ferric EDTA solution ....... .............0g NaCl ........ 1. Aseptically distribute into sterile tubes or flasks........................0g Sodium benzoate ....7g KOH . to distilled/deionized water and bring volume to 600............................................ Autoclave for 15 min at 15 psi pressure–121°C....... 200.......................... Cool to 45°–50°C................67g KH2PO4 .............................. add agar to distilled/deionized water and bring volume to 375. Mix thor- oughly................................. Cool to room temperature... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 950........................................... Filter sterilize......2g Benzoate solution.......1....................... Pour into sterile Petri dishes or leave in tubes................. Aseptically add the sterile benzoate solution...............13... 1............................... Mix thoroughly...............................0mL: Sodium benzoate..................0mL.. Cool to 45°C.............. 10.................... Use: For the cultivation of Pseudomonas putida and other microorganisms that can utilize benzoate as a carbon source........................................................23g Preparation of Ferric EDTA Solution: Add EDTA and KOH to distilled/deionized water and bring volume to 186.0g (NH4)2HPO4 .......... Heat gently to boiling.. Cool to room temperature.......... Filter sterilize. Solution B: Composition per 300......0mL..... Ferric EDTA Solution: Composition per 550..0mL of sterile benzoate solution.....5g MgSO4·7H2O ................ add FeSO4·7H2O to distilled/deionized water and bring volume to 364......0......... Autoclave for 15 min at 15 psi pressure–121°C........................................ Adjust pH to 9.10.........0mL Solution B.......2 ± 0... Cool to 45°–50°C...............3..........0mL......................... Mix thoroughly......... Aseptically add 25....... 0.3g Phosphate buffer solution.................................0mL Phosphate Buffer Solution: Composition per 200. Mix thoroughly....... Store in the dark..0 with KOH....5L: Noble agar..................... Mix thoroughly........0mL: Sodium benzoate................................................................................................................. except agar and sodium benzoate..............5g MgSO4·7H2O ...............0mL............. Mix thoroughly..0mL: K2HPO4 ......................................0mL................ Mix thoroughly.............0mL Preparation of Solution B: Add components to distilled/deionized water and bring volume to 300.0mL............2g Citric acid.. 300..0mL sterile benzoate solution......0mL..................0L........................ Preparation of Medium: Add components except ben- zoate solution to distilled/deionized water and bring volume to 975..........25.....................................0mg Na2MoO4·2H2O.......... 10.........................0mg CuSO4 .......0mg Co(NO3)2·6H2O........................ Trace Metals Solution: Composition per 300... Combine the two solutions............... In a separate flask.....1g Benzoate solution....66 Benzoate Medium II Benzoate Solution: Composition per 25........0g Preparation of Benzoate Solution: Add sodium ben- zoate to distilled/deionized water and bring volume to 25.................... Use: For the cultivation of Pseudomonas putida and other microorganisms which can utilize benzoate as a carbon source... Mix thoroughly...0................0g Preparation of Benzoate Solution: Add sodium ben- zoate to distilled/deionized water and bring volume to 25.....10.......0mL: KNO3 ............. 3....................................... 0.. 30....... 3.......................................................0mL.......0mL: MgSO4·7H2O..........0mL pH 8..........................................................................0mL: MnSO4·H2O....................................... Autoclave for 15 min at 15 psi pressure–121°C....................................................................... Mix thoroughly........................... Adjust pH to 7......... 0.0 ± 0.....0mL.......50.................................... Mix thoroughly......... Mix thoroughly.............................................5.......17........... 1............9g FeSO4·7H2O .5g Preparation of Benzoate Solution: Add sodium ben- zoate to distilled/deionized water and bring volume to 25.............................. 0.10............ Preparation of Solution A: Add components to distilled/deionized water and bring volume to 700.......................0g NH4Cl ....... Benzoate Medium II Composition per 1. Mix thoroughly and pour into sterile Petri dishes or leave in tubes............. 1.25.................

.. For solid medium..............................................1..........................................................................0mL. 2............................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................................................................................0mg EDTA disodium salt .................... 6................................... Vitamin B12 Solution: Composition per 100............... Filter sterilize.....0L.......................................... 1...... 6.......0.........1.....02g Citric acid ......... 0........................86g MnCl2·4H2O ................1............. BG 11 Uracil Agar Composition per liter: Agar .................86g MnCl2·4H2O...81g Na2MoO4·2H2O....... Heat gently to boiling.............................1 ± 0......................0mg Vitamin B12 solution............0mL of sterile Vitamin B12 solution...............................0L...................................0mg Ferric ammonium citrate................. 10..................0g NaNO3 ............................0L.............. 0........ 10....................................5g MgSO4·7H2O .......................... Vitamin B12 Solution: Composition per 100.......39g ZnSO4·7H2O .........0mg Disodium EDTA ..........02g Citric acid...... For the isolation of cyanobacteria from freshwater habitats.... Mix thoroughly...........................................10...................... 0............ Fill tubes completely............... 1............ Mix thoroughly.............0g ... Autoclave for 15 min at 15 psi pressure–121°C.................................81g Na2MoO4·2H2O ..................................... 0...............................0mg EDTA disodium salt................ BG 11 Marine Broth (Medium BG 11 for Marine Cyanobacteria) Composition per liter: NaCl .......... Heat gently to boiling. Adjust pH to 8...86g MnCl2·4H2O.........................................5g MgSO4·7H2O..049g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1................................ 100........................................................................................................................................ 0............................04g CaCl2·2H2O..1 ± 0.................................222g CuSO4·5H2O ............................................................................ Mix thoroughly....... 1..... Aseptically add 100....................... Autoclave for 15 min at 15 psi pressure–121°C............... 0....................0mg Ferric ammonium citrate.........0. 0...................... 1........ Use: For the cultivation and maintenance of a wide variety of cyanobacteria..................2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 .........................0mL: BG 11 Agar (Medium BG 11 for Cyanobacteria) Composition per liter: Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 100............................. 1.. Use: For the cultivation and maintenance of Synechococcus species.100......... Mix thoroughly....... Pour into sterile Petri dishes or leave in tubes..............0mL Trace metal mix A5 ................................. 0.....036g Na2CO3 ............075g K2HPO4 ...........................1.........................0mg Vitamin B12 solution ..................0g NaNO3 ................. Preparation of Medium: Add components............. Autoclave for 15 min at 15 psi pressure–121°C........0L...................................... Mix thoroughly..0.......................0mg Ferric ammonium citrate .............................. 1..........................0mL: Vitamin B12.....075g K2HPO4 ..5g MgSO4·7H2O .........04g CaCl2·2H2O................................................. Filter sterilize.................0mL......... 0............036g Na2CO3 ... to distilled/deionized water and bring volume to 900..........02g Citric acid..... 0.0mL........................................ 1.0g NaCl ......................................................... 0................ 1......................................... 6.........................049g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1..10......................0mL of sterile Vitamin B12 solution.........................BG 11 Uracil Agar Preparation of Medium: Aseptically combine 700.0................222g CuSO4·5H2O .............6......................................036g Na2CO3 ... BG 11 Marine Agar (Medium BG 11 for Marine Cyanobacteria) Composition per liter: Agar ...................0mL pH 7....................2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 .....................................................................................................0mL pH 7...................... 2........................... pour into sterile Petri dishes or leave in tubes...0..........049g Vitamin B12.......................0..1........................................................ Heat gently to boiling.................2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 ...................... Use: For the cultivation of Alcaligenes xylosoxydans.....0...0mL.... Distribute into sterile tubes or flasks..... Mix thoroughly.................................................6.................... 1...... For the isolation of cyanobacteria from freshwater habitats.... 67 Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1....0mL pH 7...........81g Na2MoO4·2H2O .............................079g Co(NO3)2·6H2O................079g Co(NO3)2·6H2O ........... 0........ 0.................... 10..0g NaNO3 ............... Mix thoroughly.....0µg Preparation of Medium: Add components...........................................................................................2..............................0µg Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 100... Mix thoroughly......39g ZnSO4·7H2O..... Agar ..................................................................................................................222g CuSO4·5H2O.................... 0. Mix thoroughly.......... Use: For the cultivation and maintenance of Synechococ- cus species. 0..075g K2HPO4 ...............079g Co(NO3)2·6H2O ........ Aseptically distribute into sterile screwcapped tubes... except Vi- tamin B12 solution............ 6.........................................39g ZnSO4·7H2O ...................................... except Vi- tamin B12 solution.................... to distilled/deionized water and bring volume to 900........................0mL Trace metal mix A5....... Distribute into tubes or flasks....... 0...............................................................0mL of sterile solution B....2...........1 ± 0...0mg Trace metal mix A5............ Mix thoroughly.............................................0.....04g CaCl2·2H2O .................................................0mL of sterile solution A with 300................ Aseptically add 100..

............................222g CuSO4·5H2O ................................ Mix thoroughly......................... 10....2g Glucose solution .... Pour into sterile Petri dishes..........16.................... 0..................................2 at 25°C ..........1g MnCl2·4H2O ..0L.............................................0g Pancreatic digest of casein ............................................................0....................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........1 ± 0......2mL of medium for each membrane filter.......................0g Agar .... Biphenyl Agar (DSMZ Medium 457d) Composition per liter: Agar ............ 0.......................................................0mg Trace metal mix A5............... Heat gently to boiling... Blastobacter denitrificans Agar (LMG Medium 157) Composition per liter: Agar ........8....................5g MgSO4·7H2O .................................................. 0...036g Na2CO3 ... Use 2........................0g Lab Lemco beef extract....04g CaCl2·2H2O........... Use: For the selective isolation of Salmonella typhi and other enteric bacilli and for the detection of Salmonella by the membrane filter method....... 0.....0g NaCl ..................... 0......5 ± 0.............................................................................. 0........................................ 0...................0g FeSO4·7H2O ...................... 6.............68 Bile Salts Gelatin Agar Uracil ..........................10.................0.....................25g MgSO4·7H2O ............................................. Distribute into tubes or flasks................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................. 1................................................................................ Preparation of Medium: Add components to distilled/ Preparation of Medium: Add components............................................0g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1.... 0......................................................................................10............0g Peptic digest of animal tissue .81g Na2MoO4·2H2O ........................ 2............................ Distribute into tubes or flasks............. Gently heat and bring to boiling.. 1.............................44g KH2PO4.2g Trace elements solution SL-6 ................. 1....... Do not autoclave...........03g NiCl2·6H2O........................................................3 ± 0................................ Mix thoroughly...................4.........................39g ZnSO4·7H2O ................... Bismuth Sulfite Broth (m-Bismuth Sulfite Broth) Composition per liter: Bi2(SO3)3 ................ Heat and gently bring to boiling... Use: For the cultivation and maintenance of Anabaena variabilis.25g/ L biphenyl..... Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1........05g Trace elements solution SL-4 ......................................0mL pH 8......... 1.................. Preparation of Biphenyl Solution: Add biphenyl to 1..................5g Na2CO3 .... 0.......0...........0g Beef extract ......0g Sodium taurocholate ..............0mL: Gelatin.... Mix thoroughly............................................................................... 2................ 0..6g pH 7. 0.................... Pour into sterile Petri dishes or leave in tubes..... Adjust pH to 6.0mL pH 6...............01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1............................. Add an aliquot of the biphenyl solution to the lid of a sterile Petri dish so that the final concentration will be approximately 0...........................5g Pancreatic digest of casein ......................................................................... Use: For the cultivation of biphenyl-utilizing bacteria...................................15............0L distilled/deionized water................................................................................................ 1.......................................10..079g Co(NO3)2·6H2O .............100...........................................................86g MnCl2·4H2O................ 6.....0......0L.................... Mix thoroughly and heat with frequent agitation until boiling..............075g K2HPO4 .....................52g (NH4)2SO4 ...........2g CaCl2·2H2O...................... 0................. Use: For the cultivation of Vibrio cholerae.................................................2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 ............ and let the ethanol evaporate so that the crystals of biphenyl coat the lid of the Petri dish...........0g Na2HPO4 ........0mg EDTA disodium salt............................................................................0.................................0......0mL phenyl solution..........................................................5g Yeast extract .....0L............................................ Mix to disperse the precipitate and aseptically distribute into sterile tubes or flasks..........0.......................0L............................2 at 25°C Trace Elements Solution SL-4: Composition per liter: EDTA ....0..0mL pH 7....10...02g Citric acid.... to 1..5g Sodium acetate .....................10...2g ZnSO4·7H2O..................2..................8g NaNO3 ........049g Trace Elements Solution SL-6: Composition per liter: H3BO3 ........5g FeSO4·7H2O...............1.0....................................................... 0.....................0g Na2HPO4 ................... 2. 1.............. 3...... Autoclave for 15 min at 15 psi pressure–121°C..........................0g Tryptone ................9 ± 0..................................................................... 0.....................................................10.....0mL pH 7..........03g Na2MoO4·H2O....... Adjust pH to 3..5g Biphenyl... 0.....7 ± 0.... 1...............................100................................................ except bi- deionized water and bring volume to 1........................... Aseptically add sterile agar medium to the crystal-layered Petri dish............0......................... Bile Salts Gelatin Agar Composition per 100................ Mix thoroughly.. Filter sterilize using a cellulose filter membrane...........................0L ethanol..0mg Ferric ammonium citrate................................................. 0.............................. Biphenyl Solution: Composition per liter: Biphenyl ................... Autoclave for 15 min at 15 psi pressure–121°C........... 15................9....... Autoclave for 15 min at 15 psi pressure–121°C......................................................3g CoCl2·6H2O......................................0L......... Cool to 45°–50°C............... Boil for 1 min.....02g CuCl2··2H2O ..................................0L.......................................0–2..0. Cool to 50°C..........0g Glucose.... Mix thoroughly.... Mix thoroughly.............1g Water ............................................

....................0........0....0mL................ Cool the basal medium to 45°–50°C..............................0g tamin B12 solution.......0g MgSO4·7H2O..............079g Co(NO3)2·6H2O................. Aseptically pour into sterile Petri dishes or distribute into sterile tubes............1.8 ± 0........0mL pH 6....... Gently heat while stirring and bring to boiling... Filter sterilize....5g Trace Metal Mix A5: Composition per liter: Preparation of Glucose Cystine Solution: Add com- H3BO3 ............................................................................................................... Mix thoroughly.0........ Use: For the cultivation of Francisella tularensis.........0g NaNO3 .....0.............0L..................0mL: Vitamin B12.............5g MgSO4·7H2O.0mg Ferric ammonium citrate ...........0g pH 6.......................................0mL: Noble agar . Preparation of Medium: Add compents.................... infusion from ....1................2..................0....079g Co(NO3)2·6H2O.....5% NaCl Composition per liter: Beef heart...........6...............5... Use: For the cultivation and maintenance of Synechococcus species.......0........1.......... 5..... 500.................................................. Cool to 45°–50°C...................1 ± 0.............. Preparation of Nutrient Agar: Add components to distilled/deionized water and bring volume to 1....036g Na2CO3 ................................ aseptically add 10......0mL Trace metal mix A5 .....................0..........39g ZnSO4·7H2O....... Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 50..........86g MnCl2·4H2O ... 1......0....... Use: For the cultivation of Blastobacter denitrificans.................. Preparation of Medium: To 85... Autoclave for 15 min at 15 psi pressure–121°C........ .................... Add Vitamin B12 solution.........................................049g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1..................................... Mix thoroughly............... Pour into sterile Petri dishes or distribute into sterile tubes....... 69 Blue-Green Agar Composition per liter: Agar .................................. Autoclave for 15 min at 15 psi pressure–121°C..... Filter sterilize.......................................... 50.....2 at 25°C Nutrient Agar: Composition per liter: Agar .........................................2...............10................ Pour into sterile Petri dishes or distribute into sterile tubes............................ Blood Glucose Cystine Agar Composition per 100....0..................0mg Ferric ammonium citrate ............................................049g Source: Nutrient agar is available as a premixed powder from BD Diagnostic Systems................................. Mix thoroughly..............................036g Na2CO3 .0g Beef extract ............... Mix thoroughly.....................................0mL Glucose cystine solution ................02g Citric acid .......................... Filter sterilize...8 ± 0.........02g Citric acid .... Distribute into tubes or flasks..........................0mL...........01g Use: For the cultivation of Paracoccus halodenitrificans.0g NaCl ...............0mL of cooled................ 1.......0g Agar .................222g CuSO4·5H2O............04g CaCl2·2H2O ..................0mL glucose solution..........6.......................0mg Vitamin B12 solution.............................................................................0L........................................................075g K2HPO4 .......0........................1.................................. Mix thoroughly and pour into sterile Petri dishes.......................... Mix thoroughly...222g CuSO4·5H2O............... except glucose solution..................10.......0L...............................0.. Autoclave for 15 min at 15 psi pressure–121°C......... to distilled/deionized water and bring volume to 990. Autoclave for 15 min at 15 psi pressure–121°C. except Vi- Nutrient agar .2 at 25°C Glucose ..................... 10...............39g ZnSO4·7H2O.. Aseptically add 10...6..................................................0..0mL: Glucose ........... Cool the basal medium to 45°–50°C............................................................................5g Preparation of Glucose Solution: Add glucose to 10....0mL pH 7..............0mL of distilled/deionized water.... 3.......85..6..10. 15. Vitamin B12 Solution: Composition per 50..... 15................. sterile agar solution...............0mL: Preparation of Medium: Add components.............. 0.0......... Blue-Green Nitrogen-Fixing Agar Composition per liter: Glucose Cystine Solution: Composition per 50..........0mL pH 7......... defibrinated blood to a final concentration of 5%.............................1 ± 0...... Cool to 45°-50°C.............................................. Mix thoroughly........0mg EDTA disodium salt ........................................0mL Human blood.............................0...............................0L...... Blood Agar Base with 2....................81g Na2MoO4·2H2O..............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..0g Pancreatic digest of gelatin .... 12......... to glass-distilled water and bring volume to 950...............................0mL of human blood......... 30. Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1.............................0mL.................0g Tryptose ........... Mix thoroughly.... 2.........1..................0mL of sterile glucose cystine solution and 5................................ Heat gently and bring to boiling....... Aseptically add sterile... ponents to distilled/deionized water and bring volume to 50......................0mL...................2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 ........................0mg EDTA disodium salt .......................... fresh.075g K2HPO4 .0mg Trace metal mix A5 ........................ Mix thoroughly....................................0.......... Mix thoroughly.....................86g MnCl2·4H2O ...........................Blue-Green Nitrogen-Fixing Agar Glucose Solution: Composition per 10..............................................................0............04g CaCl2·2H2O .................................................................... Heat with frequent agitation and boil for 1 min to completely dissolve...................................................................................................81g Na2MoO4·2H2O......................................0...... Mix thoroughly.........................5g L-Cystine·HCl .. Mix thoroughly......... Mix thoroughly..........

....................0g KH2PO4..08g Cefamandole ... 2..................0mL Vitamin solution...................... Autoclave for 15 min at 15 psi pressure–121°C..... Mix thoroughly......1....... Autoclave for 15 min at 15 psi pressure–121°C................................................. Mix thoroughly.... Filter through cotton............ Cool to 50°–55°C........ peel........0mL Albumin fatty acid supplement .........0mL: Bosea thiooxidans Medium (DSMZ Medium 763) Composition per liter: L-Cysteine·HCl·H2O .............................0mL ..... Leptospira Medium) Composition per liter: Basal medium .....0mL................................... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.......0mL..........0mL of potato extract solution.....................0......5....20...................... Fis- Bromothymol Blue Solution: Composition per 100...............0mL...5g KOH...............................0mL of the sterile antibiotic solution. Aseptically add 1.0mL Basal Medium: Composition per liter: Na2HPO4.............. 2.............. Filter sterilize........................... 20.................... Heat gently and bring to boiling for 1 min..........5g Ethanol.................................................... Bovine Albumin Tween 80 Medium....................5g Yeast extract . Modified (Albumin Fatty Acid Broth..................... 900................. Cool to 50°–55°C..................................10............... Mix thoroughly........000U Preparation of Antibiotic Inhibitor: Add compo- and slice several large potatoes.. Antibiotic Inhibitor: Composition per 10....0.......0g lipoferum......................................................0mL Bromothymol Blue ....0g L-Malic acid .......... Add 950. 1....0g KH2PO4 ...0g α-Ketoglutarate......0mL of distilled/deionized water..0mL: Anisomycin ............ Mix thoroughly..........0mg Polymyxin B ............5......... Autoclave for 15 min at 15 psi pressure– 121°C...0mL of 95% ethanol....9 with 1N KOH................ Add 20.................5g MgCl2 ............. Adjust medium to pH 6......... Vitamin Solution: Composition per 100............ 1............................1....... anhydrous............................... 0................................. BMS Agar Composition per liter: Agar . 2.............................. Add 1.. Mix thoroughly......................4.....................0 by adding KOH so that the solution is green......................................................... 2.......0mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 100........... 80..............................0mL of sterile vitamin solution....................... except cys- teine and antibiotic inhibitor... Place the bag with the potatoes in 1.............................1..................... Pour into sterile Petri dishes or distribute into sterile tubes..9 ± 0...0L of distilled/deionized water......0......0g Potato extract solution .1g Na-glutamate ..... Preparation of Medium: Dissolve 2.. Heat gently and bring to boiling.......................0g Na2HPO4 . Use: For the cultivation and maintenance of Azospirillum L-Cysteine·HCl·H2O Solution: Composition per 10.....0g Pyridoxine .............................. 0.....0 ± 0.................................... 0.................................................... 95% .............. 200........0mL Glycerol (10% solution) ... Pour into sterile Petri dishes or distribute into sterile tubes...........................0g of agar and 2...........0mL of Bromothymol Blue solution............................................0. 0................. Filter sterilize................05g Antibiotic inhibitor ..........................0L..... Distribute into tubes or flasks................ Filter sterilize..2g Fe4(P2O7)3·9H2O ....... Boil for 30 min... Gently heat and bring to boiling.................5g of sucrose....1g pH 7........ 100....... Na2S2O3·5H2O..............0mL.............................................0g Yeast extract............5-8....................0mL of the sterile L-cysteine·HCl·H2O solution and 10.....0mL: Biotin .............................. activated... Use: For the cultivation of Bosea thiooxidans.. Mix thoroughly....70 BMPA-α Medium Preparation of Medium: Add components to glass dis- Preparation of Potato Extract Solution: Wash.............. Adjust pH to 7............. Mix thoroughly.... to distilled/deionized water and bring volume to 980....0mL L-Cysteine·HCl·H2O solution ... and Nostoc species....1... 10.... Autoclave for 15 min at 15 psi pressure–121°C........10........................ 4............. Preparation of Medium: Add components.0mL Preparation of Bromothymol Blue Solution: Add Bromothymol Blue to 100.1......0g NaCl ...08g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.................. cherella.. Mix thoroughly............... Mix thoroughly.............2 at 25°C Source: This medium is available as premixed vials from Oxoid Unipath....... Use: For the cultivation and maintenance of Calothrix................................. and sliced .. nents to distilled/deionized water and bring volume to 10.................. Add 10.... Mix thorougly....................................... 13.................................................... Place the poltato slices in a gauze bag......5g of L-malic acid in 50............................ 2.0mL: tilled water and bring volume to 1...........................950.................................0mL pH 7...... anhydrous..............0g Charcoal...0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ...................0.. Bromothymol Blue . Ellinghausen and McCullough.. 100. Use: For the selective isolation and cultivation of Legionella pneumophila and other Legionella species....5g Sucrose.5 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........... BMPA-α Medium (Edelstein BMPA-α Medium) Composition per liter: Agar ...........................................0mL pH 6..0g Na-succinate .........2 at 25°C Potato Extract Solution: Composition per liter: Potatoes.... washed.....................................0L...... Mix thoroughly................ peeled............3g NH4Cl (25% solution) ...... Check pH after autoclaving and readjust if necessary............10...............

...0mL Thiamine (0.................................0mL Cyanocobalamin (0................20...............2.............. 20.........0mL Thiamine·HCl (0..4% solution) .........5mL FeSO4 (0...........20..............0mL of sterile basal medium.. Mix thoroughly......0mL: Bovine albumin fraction V ..............1..0mL with distilled/deionized water......2% solution) .............0mL: Bovine albumin ........... 1............. Mix thoroughly......................................0.... Aseptically distribute into sterile tubes or flasks........5% solution).. Ellinghausen and McCullough.........................................2mL 71 Preparation of Albumin Fatty Acid Supplement: Add bovine albumin to 100............0mL: Bovine albumin fraction V .....05g Artificial seawater ................0g Tween 80 (10% solution) ..... 20... Mix thoroughly..... 1................. Adjust pH to 7............1.........0mL Albumin supplement ....900..2 at 25°C Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 1............0mL CuSO4·5H2O (0.. 100.......5g Preparation of MgCl2-CaCl2 Solution: Add components to distilled/deionized water and bring volume to 100.... 1.2% solution) .........4% solution) ....................................2. 1.............5% solution) .................... 1...5% solution). Mix thoroughly... Preparation of Medium: Aseptically combine 100............................2.0mL Preparation of Albumin Supplement: Add compo- nents to distilled/deionized water and bring volume to 100..........10.....0mL NH4Cl (25% solution) .... Adjust pH to 7.......4 ± 0.......... Use: For the cultivation and maintenance of Leptospira species..................0mL with distilled/deionized water..............0mL FeSO4·7H2O (0.................5g MgCl2·6H2O ................0mL: CaCl2·2H2O ....0mL of cooled................. Mix thoroughly...............0....5% solution)..0mL Basal Medium: Composition per liter: Agar ...................... Mix thoroughly......0g NaCl .0L....0g NaCl . 1.............................................................0g Na2HPO4 ....... Mix thoroughly..........................0mL Vitamin B12 (0.0g KH2PO4 ...5% solution).....................................5% solution) ... Albumin Fatty Acid Supplement: Composition per 200........................................0mL pH 7....Brackish Acetate Sodium pyruvate (10% solution) ..........................................25........................2 at 25°C Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 1..........1...3g Glycerol (10% solution) .................. 1............................2.................0mL ZnSO4 (0.. Filter sterilize.......................................................................5% solution).... Bovine Albumin Tween 80 Semisolid Medium............... Autoclave for 15 min at 15 psi pressure–121°C....................... Mix thoroughly.....2..0mL ZnSO4·7H2O (0.......0mL of sterile albumin supplement................... Autoclave for 15 min at 15 psi pressure–121°C..........1.3% solution) ............ 0.. Bring volume to 200... Bring volume to 200............... MgCl2-CaCl2 Solution: Composition per 100.................................... Filter sterilize. Adjust pH to 7......... Add remaining components while stirring........4.......0mL....0mL ......0g KH2PO4...........................0g Polysorbate (Tween) 80 (10% solution) .. Mix thoroughly.....................................0g KNO3 .........1............ Modified) Composition per liter: Basal medium ..0mL MgCl2-CaCl2 solution ............ Use: For the cultivation of Leptospira species.....................0mL Vitamin B12 (0....... Mix thoroughly............................. Adjust pH to 7...25....5% solution)................ Add remaining components while stirring...................... Cool to 25°C........2....................0mL of distilled/deionized water. Gently heat and bring to boiling..0mL CaCl2·2H2O (1..................................................0mL CuSO4·5H2O (0...................4...............0L......0mL FeSO4·7H2O (0. 1.... Aseptically distribute into sterile tubes or flasks....0mL of sterile basal medium....... Brackish Acetate Composition per liter: Sodium acetate ............0mL of sterile albumin fatty acid supplement and 900...................... Use: For the cultivation of Leptospira species... 10......................................4..1... Bovine Serum Albumin Tween 80 Agar (BSA Tween 80 Agar) Composition per liter: Basal medium .........0mL of sterile albumin fatty acid supplement and 900.... Cool to 25°C........ anhydrous ..............0........... Albumin Fatty Acid Supplement: Composition per 200.................................... Preparation of Medium: To 900.....0mL Basal Medium: Composition per liter: Agar ..0L...............5% solution)..............4. Filter sterilize....100.2...3% solution) ............................ Albumin Supplement: Composition per 100.............5% solution ...........2mL Preparation of Albumin Fatty Acid Supplement: Add bovine albumin to 100.....................0mL Albumin fatty acid supplement. sterile basal medium..........0mL CaCl2·2H2O (1.........................................................0mL ZnSO4·7H2O (0.....0mL Sodium pyruvate (10% solution) ............1.....................3g NH4Cl (25% solution).... anhydrous ....2..... Adjust pH to 7........4% solution) .............4............................ Preparation of Medium: Aseptically combine 100......02% solution) ....................0mL Thiamine·HCl (0................0mL of distilled/deionized water........... 250..... Autoclave for 15 min at 15 psi pressure–121°C........................11..................1........ 900............0g Polysorbate (Tween) 80 (10% solution) .... Cool to 25°C.....1... 2............. Adjust pH to 7..1........0mL Glycerol (10% solution).....0g NaH2PO4·2H2O ..................0mL pH 7..0mL Sodium pyruvate (10% solution)................ 12............1.0mL MgCl2·2H2O (1........ Modified (Albumin Fatty Acid Semisolid Medium.... aseptically add 100.................0....................................4 ± 0...........................2g Na2HPO4..............1.....................0mL Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 1..... Aseptically distribute into sterile tubes or flasks.... Store at −20°C.......................0mL MgCl2·2H2O (1...... Gently heat and bring to boiling. 1.................. Store at −20°C....0mL......................4........

..............026g SrCl2 ..0............. 3...................................0mg Folic acid .....0mg Vitamin B12 .............. “Metals 44”: Composition per 100.....................................................................................................................10............ Distribute into tubes or flasks...........................................................................................66g NaHCO3 ..................................................................................2........................................0............................................................ 5........917g CaCl2 .......................................................................... Add other components and adjust the pH to 7...................7g Nitrilotriacetic acid ......................0mg Riboflavin.......................................19g KBr...........0g CaCl2·2H2O .................................................. 0............................................2 ± 0...........................................................................0mg D-Calcium pantothenate.1g (NH4)2MoO4 .......................... There may be a slight precipitate.....................0.......... Autoclave for 15 min at 15 psi pressure–121°C..................................2...................154g CuSO4·5H2O........... 50..............0mL of artificial seawater and 720.......................... Autoclave for 15 min at 15 psi pressure–121°C............. Mix thoroughly.................................................1g KCl............................................154g CuSO4·5H2O ................... “Metals 44”: Composition per 100... 2...........................0L..0mg Biotin ......025g Na2B4O7·10H2O..................... Mix thoroughly.......................................... Add components........ Mix thoroughly............1....................... 0.34g FeSO4·7H2O......... Mix thoroughly............5g EDTA...................5.. Use: For the cultivation of Filomicrobium fusiforme............ Aseptically add “Metals 44” and vitamin solutions............................... 0....50...................................................................... 3....................... 23........................................................0........................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...........................................................2 ± 0.............026g SrCl2 ..............25g Yeast extract .....................................0g Peptone ..............018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100...............................................................................................0.........25g MnSO4·7H2O..........1..............664g NaHCO3 .72 Brackish Prosthecomicrobium Medium Modified Hutner’s basal salts ............................................................................ Store at 5°C..................0.........0...................................................................... Add aseptically to sterile basal medium. Aseptically distribute into sterile tubes or flasks.................1g FeSO4·7H2O.......................9g CaCl2 ......................................................5g EDTA .... Store at 5°C........................................0mg Biotin . 0.........................25mg “Metals 44” .....04g Co(NO3)2·6H2O ............ 3......................................................................................... 0.......................................................................................................9........................................................0mg Riboflavin ...............................................0.................096g H3BO3 ....... 20....20................0................0mg D-Calcium pantothenate ....018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100...2 with KOH or H2SO4................ except for “Metals 44” and vitamin solutions..... Preparation of Medium: Add a few drops of H2SO4 to the distilled water to retard precipitation of the metal salts...............................................................0......................1mg .......................... Vitamin Solution: Composition per liter: Thiamine·HCl ..............3................................................... Add aseptically to sterile basal medium.... 10.......0mL Preparation of Modified Hutner’s Basal Salts: Dissolve the nitrilotriacetic acid first and neutralize the solution with KOH...................................0mL of distilled/deionized water.........................................................0g Na2SO4 ...0mL: ZnSO4·7H2O ... 2....5..................... 0..............................................0mL Modified Hutner’s basal salts...... Brackish Prosthecomicrobium Medium Composition per liter: Agar .......192g KBr .......................... There may be a slight precipitate..0mL pH 7...............0.024g NaF ................................ 5................... Mix thoroughly..........25g MnSO4·7H2O .................. 250................................. 10...........102g KCl ....... 0.... Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O.................0..5.............................. 0.2 at 25°C Artificial Seawater: Composition per liter: NaCl .............................0mL.......0mL: ZnSO4·7H2O.....................2...................................................................4............................ 5....23..........0mL Vitamins ............................................ 0... Mix thoroughly............ 0.... Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O ....................0mg Vitamin B12.....................5g MgCl2 .. Adjust pH to 7..........15........0mL.......25mg “Metals 44” .....0........0............................................................................10...................................0mL pH 7......................2....25g Artificial seawater .. 1.....0mg Preparation of Artificial Seawater: Add components to distilled/deionized water and bring volume to 100..................................... Filter sterilize and add aseptically to sterile basal medium.....................................981g Na2SO4 ..........................................0mg Preparation of Artificial Seawater: Add components to distilled/deionized water and bring volume to 100....1g (NH4)2MoO4 ......................................................................................................... 0...........29.......................0mL...............................................1g H3BO3 .34g FeSO4·7H2O ..............024g NaF........................................................025g Na2B4O7·10H2O .............3............................................25g Glucose......0mL Preparation of Modified Hutner’s Basal Salts: Dissolve the nitrilotriacetic acid first and neutralize the solution with KOH.1g FeSO4·7H2O ............................................ Vitamin Solution: Composition per liter: Thiamine·HCl ...3............................. to 250....................0..............................04g Co(NO3)2·6H2O........................... Sterilize by autoclaving for 15 min at 15 lbs pressure–121°C.......... 1............ 0.......................................2 at 25°C Artificial Seawater: Composition per liter: NaCl ......... 5.............0g CaCl2·2H2O.. Add other ingredients and readjust the pH with KOH and/or H2SO4 to 7....0mL Vitamin solution........................... 29.............0mL..................................................................................................0...0mg Folic acid......................7g Nitrilotriacetic acid............. 9.... 0.......................................477g MgCl2 ..........

..................0g KH2PO4 .................0mL Riboflavin Solution: Composition per 10.... Brevundimonas Agar (LMG Medium 221) Composition per liter: Agar ................... Autoclave for 15 min at 15 psi pressure–121°C............ Mix thoroughly.. 2................... 6.......................0g Na2HPO4 .................. from soils...... except ri- boflavin................... Filter sterilize............................ Autoclave for 15 min at 15 psi pressure–121°C.. and Enterobacter cloacae..........2.. Filter sterilize and add aseptically to cooled.................... Brevibacterium Medium (ATCC Medium 677) Composition per liter: Agar ..10........................... 1.....................0g Preparation of Soil Extract: Autoclave for 60 min at 15 psi pressure–121°C.........................2........................0...... except “Metals 44” and vitamin solutions....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........ including Coccidioides immitis......2......................0mL: Vitamin B12 .......0mg Preparation of Riboflavin Solution: Add riboflavin to 10.......... Distribute into tubes or flasks......0...................... Use: For the cultivation and maintenance of Brevibacterium spp.................... Autoclave for 15 min at 15 psi pressure–121°C............ Blend for 30 min in a blender to disperse the n-hexadecane..............................5g pH 7...0µg Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 1...2g CaCl2 .......................5g Glucose ........... Mix thoroughly....0g pH 5.........0L.................................................................. Mix thoroughly........ Sterilize by autoclaving for 15 min at 15 lbs pressure–121°C..0mL of Vitamin B12 solution. Brain Heart Infusion with 3% Sodium Chloride Composition per liter: NaCl ......... Mix thoroughly................................5g Brain heart....................................................0g Na2HPO4 ..............................0g Pancreatic digest of casein ..........250........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..0g Brain heart.. 8..............0mL Vitamin B12 solution .....1g MnSO4 .. Autoclave for 20 min at 15 psi pressure–121°C.......... Use: For the cultivation of Prosthecomicrobium litoralum............. Preparation of Medium: Add components.............0mL: African Violet soil.........................0g Yeast extract .........0g Tween™ 60............................ Add components...............0g Yeast extract ...................................... 2..............................0L...............15................ especially fastidious species from soil..........4 ± 0.. yeasts........ Distribute into tubes or flasks....... Autoclave for 15 min at 15 psi pressure–121°C.......0mL.......5...........0g Peptic digest of animal tissue......................0g Pancreatic digest of gelatin .......... Mix thoroughly... and Vitamin B12 solution... solids from infusion ......................... Filter sterilize..01g n-Hexadecane .....2......................................................0....0mL of distilled/deionized water...... 3....0g Na2CO3 ... 2.. For the isolation of Histoplasma capsulatum and other pathogenic fungi....0mL of distilled/deionized water....... sterile basal medium................2.......0mL of tap water............. Preparation of Medium: Add components....................................30....2 at 25°C Soil Extract: Composition per 400.....0mL of artificial seawater and 720... Adjust pH to 7..............................................5..2.0.0g Glucose ............ Preparation of Medium: Add a few drops of H2SO4 to the distilled water to retard precipitation of the metal salts............ yeast extract........0–6............. 5.2g FeSO4·7H2O.................................... Filter sterilize and add aseptically to sterile basal medium................... Gently heat and bring to boiling. 5....................................0mL: Riboflavin............. 50... Aseptically distribute into sterile tubes or flasks...0mL.....0g MgSO4·7H2O..................................1..............................................................................................................0g Peptone ..................... Add yeast extract and glucose to 200............ Aseptically distribute into sterile tubes or flasks.. 30.........................0mL pH 7.................. Mix thoroughly........1g Riboflavin solution ....2 ± 0............ to tap water and bring volume to 799..................... Use: For the cultivation of a wide variety of microorgan- isms........................0g NaCl .................... 5................................ Distribute into tubes or flasks.............. 14............................... and molds.0g MgSO4·7H2O.............0 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.............................0mL pH 7.............. Brevibacterium Medium (ATCC Medium 681) Composition per liter: Glucose.........................0L...... Use: For the cultivation of Vibrio parahaemolyticus.... Use: For the cultivation and maintenance of Brevibacterium alkanophilum and other microorganisms that can utilize hexadecane as a carbon source.......... solids from infusion .................0g Peptic digest of animal tissue. 16.................2.........0mL............. 1.............................................2...................... Aseptically add 73 1..0g Soil extract ........... Mix thoroughly.. Distribute into tubes or flasks...0g Yeast extract ......................0g Na2HPO4 ..............0g (NH4)2SO4 ................. Filter through paper before using in medium. Cool to .... 20..................................................... Brain Heart Infusion Soil Extract Medium Composition per liter: Yeast extract............................................................................... to 250...0......0g Peptone .............. Mix thoroughly..10...............................0L..........Brevundimonas Agar Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..................................... Mix thoroughly...............2.......... 6............ Mix thoroughly..................................... Vitamin B12 Solution: Composition per 1......0 ± 0.... including bacteria.... Aseptically add “Metals 44” and vitamin solutions...... to distilled/deionized water and bring volume to 995..................... except glu- cose.............

....... 20.... cool the broth rapidly.10.. 0................013g pH 7........ Gently heat with frequent mixing........0mL or less of sample............0g Yeast extract ..... Gently heat and bring to boiling...... The presence of Eschrichia coli and other coliforms is determined by the presence of fluorescence in the tube........................... dehydrated............................................................................ Escherichia coli appears as dark red colonies with a pink halo........... 2... as well as in other materials of sanitary importance................05g Brilliant Green.....................6......0. 10.......10........0295g KH2PO4 ....... After sterilization..... in 10..5........ dehydrated... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................................... Mix thoroughly............2 ± 0. 10.... Bacteria that ferment the specific carbohydrate turn the medium yellow..................................... cool the broth rapidly. When bacteria produce gas......... Distribute into test tubes that contain an inverted Durham tube...............................025g pH 6...........0L.................0g Lactose .20...........................0..... Distribute into tubes containing inverted Durham tubes.......0L..........013g pH 7................. Distribute into tubes containing inverted Durham tubes.................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems............................... dehydrated....................9g Na2SO3 ................015g Oxgall......078g Erioglaucine ............0g Beef extract .............................. deionized water and bring volume to 1.. 1.................. MUG (4-Methyl umbelliferylβ-D-glucuronide) ....16g Brilliant Green......................... Mix thoroughly.. Use: For the detection of coliform microorganisms in water Preparation of Medium: Add components to distilled/ and wastewater as well as in other materials of sanitary importance....2 at 25°C Carbohydrate Solution: Composition per 10..................................... Pour into sterile Petri dishes. Mix thoroughly......................... Use: For the selective isolation and differentiation of Sal- monella from polluted water by the membrane filter method....0g Phenol Red ..................... Mix thoroughly.0g Bromcresol Purple.....................................0L........2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems and Oxoid Unipath............................... Use: For the detection and enumeration of coliform bacteria in materials of sanitary importance such as water and sewage.................................. 0.......0L........................... 0.........0g Lactose ........................0...............................................9 ± 0..... Bromcresol Purple Broth Composition per liter: Peptone ............... Brilliant Green Bile Broth (Brilliant Green Lactose Bile Broth) Composition per liter: Oxgall.....0g Brilliant Green ..................... Gently heat and bring to boiling.... 0... Brilliant Green Bile Agar Composition per liter: Noble agar....... Autoclave for 12 min (not longer than 15 min) at 15 psi pressure– 121°C........... the gas is trapped in the Durham tube...........................20..............065g FeCl3 ......95mg Brilliant Green .....................0mL: Carbohydrate ......... 20.........................2 ± 0...0 ± 0...................5..................... Cool to 25°C.........................0mL amounts for testing 1...........0mL amounts for testing 1..................................................... Aseptically add 5.......... After sterilization..0g NaCl ........0mL............. Filter sterilize............. Brilliant Green Bile Broth with MUG Composition per liter: Use: For the differentiation of a variety of microorganisms Oxgall.......2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems....... Enterobacter species appear as pink colonies......0g Pancreatic digest of gelatin ... Use: For the cultivation of Brevundimonas spp............ Care should be taken to avoid exposure of the prepared medium to light.. Mix thoroughly.............. 10..15g Pancreatic digest of gelatin .. Use: For the detection of coliform microorganisms in water and wastewater......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......... prepare the medium double strength...........0g NaCl ..0mL samples..........0mL pH 7..................0mL or less of sample.............................. For plating 10.......... 0.................................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems...............205g Basic Fuchsin ..25g Lactose ......04g Carbohydrate solution ....... 8...........03mg pH 6........ 0.............................. Mix thoroughly........................................................ in 10... Autoclave for 12 min (not longer than 15 min) at 15 psi pressure– 121°C.. Caution: Basic Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contamination of the skin.................. Turbidity in the broth and gas in the Durham tube are positive indications of Escherichia coli..................................... 3......0g Sucrose ...............0g Pancreatic digest of gelatin .... Pour into sterile Petri dishes or distribute into sterile tubes............... Preparation of Medium: Add components to distilled/ deionized water and bring volume to1....0L.... 10........ ....3.............................................. 0........0mL to each sterile absorbent filter used. Medium is sensitive to light...... and Caulobacter henricii........ Mix thoroughly........0.............0...........74 Brilliant Green Bile Agar 45°–50°C.......... 10........... 10................ Autoclave for 10 min at 15 psi pressure–121°C. Preparation of Medium: Add components to distilled/ deionized water and bring volume to1..........................................................0g Preparation of Carbohydrate Solution: Add carbo- hydrate to distilled/deionized water and bring volume to 10... Autoclave for 15 min at 15 psi pressure– 121°C........ Brilliant Green Broth (m-Brilliant Green Broth) Composition per liter: Proteose peptone No..........................................0g Lactose .. Distribute into tubes or flasks....... Add 2.....9 ± 0.0mL sterile riboflavin.......... Boil for 1 min....20....0g based on their fermentation of specific carbohydrates......

.. 10.........0g Bromcresol Purple solution .. Use: For the cultivation and differentiation of bacteria based on their ability to ferment glucose................. 500........ L-Cysteine·HCl·H2O Solution: Composition per 10....................... Pour into sterile Petri dishes or distribute into sterile tubes................ Filter sterilize separately the lactose solution......................... Mix thoroughly..0mL.....1...................0mL pH 7.......... Cool to 45°–50°C..... Mix thoroughly......... except Lcysteine·HCl·H2O solution and antibiotic solution. Cool to 50°–55°C.0mL of sterile sodium thiosulfate solution...1.. To the cooled.17. Cool to 45°–50°C........................ Mix thoroughly..........17........8 at 25°C Preparation of Medium: Add agar....................3 ± 0..... sodium thiosulfate solution.................. Mix thoroughly....................5g Bovine serum........................01g Antibiotic solution.................................................1.................0mL of sterile L-cysteine·HCl·H2O solution and 10......................................3........... Distribute into tubes in 12–15mL volumes...... Add 4..........................2 at 25°C Preparation of Medium: Add components...................................0mL of sterile lactose solution....... Mix thoroughly.................. 70........0................. 1........... Bromothymol Blue Agar Composition per liter: Agar ..................... activated ............2 at 25°C Antibiotic Solution: Composition per 10..............0mL of horse serum......................0mL Sodium thiosulfate (50% solution) .... Bacteria that ferment glucose turn the medium yellow..0mL of sterile antibiotic solution.................................0mL.........0mL of sterile Bromothymol Blue solution..........5...........02g Horse serum ... Mix thoroughly......50.........0mL of ethanol.........................................0mL.............10...............2.....2............................... 2...............0mL: Bromcresol Purple ..................0mL: Vancomycin .......................................10........... . 0.................................. Mix thoroughly............................... 10......... Adjust pH to 7......7–7.................2mL of sterile glucose solution and1.... and maranil solution........0L... Autoclave for 15 min at 15 psi pressure–121°C.................................. 0.........0g Peptone........ 5..15.............0g Yeast extract ......... 1....... 1........2...................................0mL................0mL Bromothymol Blue (1% solution)...... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.. Mix thoroughly......2 at 25°C Bromcresol Purple Solution: Composition per 10.....0mL of sterile maranil solution....... 10.......0g Charcoal.0mL Preparation of Bromcresol Purple Solution: Add Bromcresol Purple to 10.. 10......... Mix thoroughly. 5...........0g K2HPO4 ..............0mL: Agar .........0mL Glucose (33% solution) ...............0mL pH 7........................ to distilled/deionized water and bring volume to 900....... 10........................ Filter sterilize.0 ± 0........0mL: L-Cysteine·HCl·H2O. Filter sterilize.......................0.....10.0g Peptone.. NaCl. Adjust pH to 8....................................... Use: For the isolation and cultivation of Simonsiella species and Alysiella species....0g NaCl ................... glucose solution.... Autoclave for 15 min at 15 psi pressure–121°C..........................0L......0mL pH 7................4................................... sterile agar solution aseptically add 27..............8.........0g Papaic digest of soybean meal ............. 3............................................................................ except horse serum......... Aseptically distribute into sterile tubes or flasks.........................0g Yeast extract . Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension....1...0L...........0g Sorbitol..0mg Polymyxin B...........0mL pH 7................................................ Use: For the cultivation of Spiroplasma citri........ Autoclave for 15 min at 15 psi 75 pressure–121°C.................................... Adjust medium to pH 6.27........................0g NaCl .... Use: For the selective isolation and cultivation of members of the Enterobacteriaceae........... Cool to 45°–50°C.... 4....... Autoclave for 20 min at 15 psi pressure–121°C.............25g Bromcresol Purple............ Bromothymol Blue solution......................................................0g Lactose (33% solution) ............... 10.......................0g Agar .........................0g Tryptose ......1................................................................ BSR Medium Composition per liter: Beef heart........ Mix thoroughly.... 100................0g NaCl ............10.....000U Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10....................................... Mix thoroughly............................................5g Glucose.................................. Autoclave for 15 min at 15 psi pressure–121°C.............01g Bromothymol Blue . Aseptically add sterile bovine serum............................ Buffered Charcoal Yeast Extract Differential Agar (DIFF/BCYE) Composition per 1014............... to distilled/deionized water and bring volume to 900........0mL pH 6.......................16g Ethanol (95% solution) ... Pour into sterile Petri dishes or distribute into sterile tubes..............................................0g Yeast extract........7–7.......9 ± 0....100.......... Gently heat and bring to boiling.......................... 5............2mL Maranil solution (5% solution) ..................................................Buffered Charcoal Yeast Extract Differential Agar Bromcresol Purple Dextrose Broth (BCP Broth) Composition per liter: Glucose ........... BSTSY Agar Composition per liter: Pancreatic digest of casein .....................6 ± 0..0g Beef extract .......0g Phenol Red ...........................0...............................0..... solids from infusion.... 5............... 11........ Aseptically add 100.......2 at 25°C Preparation of Medium: Add components..............0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10............2..................... except bo- vine serum.................0g Sucrose..0g Fructose....0mL L-Cysteine·HCl·H2O solution .......5g Fe4(P2O7)3·9H2O ........9 with 1N KOH..........0g ACES (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) buffer ........... and yeast extract to distilled/deionized water and bring volume to 1.... to distilled/deionized water and bring volume to 1........... Preparation of Medium: Add components.........0g Glucose ........ Heat gently and bring to boil for 1 min...... peptone....... Mix thoroughly.......

............. 0.........0.................................0L.................. 3...............................5g ...........50....6................................ For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from environmental water samples................ Autoclave for 15 min at 15 psi pressure–121°C.........0g MgSO4·7H2O....................20.......................................... Mix thoroughly...............0... 0................0....................... Butanediol Medium Composition per liter: NaH2PO4·H2O ................. There may be a slight precipitate... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.............................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................................... 0......................... Mix thoroughly......................0g Agar ....0... 9...72g MgSO4·7H2O.................02g KH2PO4 ..............0L..........................01g Adenine ...................................................................... 0............0mL pH 6...........05g CaSO4·2H2O ...........................01g NaCl ......... 29...............15........5mg pH 7........................................0g NH4NO3...1. Autoclave for 15 min at 15 psi pressure–121°C.......19g NaHCO3 .................1......................................................................01g FeSO4·7H2O.................0L.................................................................................0.... 85.......................... 24..........................................04g Co(NO3)2·6H2O ..189g NaHCO3 .......................................................011g SrCl2·6H2O ..............1-1...........................................................0... Adjust pH to 6...................... cultivation.....5mg pH 7.......5g MgSO4·7H2O.......................................5g EDTA ................................ 5..................................20.............................. 0....................................4g NaH2PO4·H2O...............02g pH 7....................0.. For use in cultivating hydrocarbon-utilizing bacteria......................................2 at 25°C 1M Phosphate Buffer.............. Gently heat and bring to boiling......76 Buffered Marine Yeast Medium Use: For the isolation............... Dissolve the nitrilotriacetic acid first and neutralize the solution with KOH....................................................0 ± 0................................. 0.......01g (NH4)2MoO4 .... 0............................ layer 0............. 1.....7g Nitrilotriacetic acid ..........................0..... Add aseptically to sterile basal medium...........0.......................................7..8....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...............................................................................0g K2HPO4 ................................01g NaCl .........................................................0g NH4Cl ...... 20............ pH 6...................... Also for the cultivation of Nocardia species..................1g FeSO4·7H2O.....................4-Butanediol ......... Distribute into tubes or flasks...............1...........................1g 1..........................................................2g Na2HPO4 .............................................. Buffered Marine Yeast Medium Composition per liter: NaCl ...............8: Composition per liter: K2H2PO4 ...............0..... Mix thoroughly................ Hutner’s Mineral Base: Composition per liter: MgSO4·7H2O............................................0L............................018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100...................8 ± 0...... Mix thoroughly...................................2.. Store at 5°C.......... Burke’s Modified Nitrogen-Free Medium Composition per liter: MgSO4·7H2O ................0mL Hutner’s mineral base ......................................0g Yeast extract.................05g CaSO4·2H2O..........2g FeCl3 ................011g SrCl2·6H2O ............................ Burke’s Modified Nitrogen-Free Medium with Benzoate Composition per liter: Sodium benzoate ............. Use: For the cultivation and maintenance of Pseudomonas species.... 0...0...............................0mg Na2MoO3 .....0..........4g Preparation of 1M Phosphate Buffer....................................................................................... 10...........01g Adenine ..........154g CuSO4·5H2O ................................................... Mix thoroughly......... Distribute into tubes or flasks........8 ± 0........ Bushnell-Haas Agar Composition per liter: Agar ......... 0........ Use: For examining fuels for microbial contamination and for studying hydrocarbon utilization by microorganisms................0..........................................................................................0g 1M Phosphate buffer................ 70........0....02g KH2PO4 ........................................................................01g FeSO4·7H2O.......0% hydrocarbon on agar surface or aseptically add sterile hydrocarbon to cooled agar prior to pouring plates....1............................... Mix thoroughly.............0..................... Distribute into tubes or flasks..........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....0..................0mg Na2MoO3 .... Pour into sterile Petri dishes or leave in tubes......8 ± 0.... Add other ingredients and adjust the pH to 7..25g MnSO4·7H2O ................2 with KOH and/or H2SO4.............8: Add components to distilled/deionized water and bring volume to 1.......0.......0g CaCl2·2H2O .. Autoclave for 15 min at 15 psi pressure–121°C..........8 ......................0..0mL: ZnSO4·7H2O ..........025g Na2B4O7·10H2O.........................0g KH2PO4 ........................ pH 6...................... Use: For the cultivation of Pseudomonas species and other microorganisms which can utilize benzoate as sole carbon source....... “Metals 44”: Composition per 100... Autoclave for 15 min at 15 psi pressure–121°C...............................1................34g FeSO4·7H2O.................................. 0.0g NaCl .................... and maintenance of Le- gionella pneumophila and other Legionella species from environmental samples...........0mL Preparation of Hutner’s Mineral Base: Initially add a few drops of H2SO4 to the distilled water to retard precipitation.25mg “Metals 44” .............6................................. 0................................0mL KOH (1N ) .......2g Na2HPO4 .................0g CaCl2·2H2O..... pH 6.......................... Use: For the cultivation of Azotobacter vinelandii............0mL.............................................................. 0..............1.......................................................0........................... Distribute into tubes or flasks................................................................... Autoclave for 15 min at 15 psi pressure–121°C.....................................................................05g CaCl2·2H2O .......0L.........................................................................

..................... using a syringe......0mL of sterile L-cysteine·HCl·H2O solution.....0g Beef extract .................0g Starch.......0g KH2PO4 .............................................0mL of distilled/ deionized water..............0g Cellobiose.... 1.......................................01g Na2SeO3 ................ ...........................0g Preparation of Carbohydrate Solution: Add components to distilled/deionized water and bring volume to 20.....0mL Minerals solution ................. and 10........... Autoclave for 15 min at 15 psi pressure–121°C..........2 at 25°C Modified Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ...................5g NaCl ..........BY Agar Medium K2HPO4 .......... 0............. Add remaining components one at a time.......................................0.................... Adjust pH to 6................20............1mL pH 6...................................................0g Maltose .....1g MgSO4·7H2O......................0g MgSO4·7H2O ....................25mL Preparation of Volatile Fatty Acid Mixture: Com- bine components.................... sterile Na2S·9H2O solution................................................... Autoclave for 15 min at 15 psi pressure–121°C................ 0.............. Aseptically and anaerobically add 20.05g pH 7................. 3....1. L-Cysteine·HCl·H2O Solution: Composition per 10...... Autoclave for 15 min at 15 psi pressure–121°C........1g AlK(SO4)2·12H2O.....01g NiC12·6H2O ............................5 with KOH...........0mL....... Adjust pH to 7.................................................. 0..0mL pH 7........................ 0..... Gently heat and bring to boiling........................6g Preparation of Minerals Solution: Add components to distilled/deionized water and bring volume to 1.......0 ± 0....................................5g CaCl2 ...........................0g Yeast extract .................. 1......................... Continue boiling for 5 min..1............................................ Sparge under 100% CO2............ Autoclave for 15 min at 15 psi pressure–121°C......0g MnSO4·H2O ...............01g Na2MoO4·2H2O .............. Continue sparging with 100% CO2 until pH reaches 6.....0mL DL-2-Methyl butyric acid .........................0g Glycerol ....0mL: Na2S·9H2O .... 1.25mL iso-Valeric acid ...... 0..3.....10...............1g ZnSO4·7H2O ................0g Nitrilotriacetic acid ......... 0..25g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10...........1..........................01g CuSO4·5H2O . 0.........0L.0g Pancreatic digest of casein ............................0.................................. 0................... Add distilled/deionized water to 1.....................................0mL..................... and sterile L-cysteine·HCl·H2O solution into individual tubes containing medium........................................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.. 0. 1.................0g (NH4)2SO4 ......... clarified ................1...... Adjust pH to 6.0g Pancreatic digest of casein ... 12.... 77 Na2S·9H2O Solution: Composition per 10........10........... 1................................................................................................ 0................................0L............ Mix thoroughly........................... Add components...... Mix thoroughly....................................0...........................0L...........25mL iso-Butyric acid ............................ 0....8..5.........................5........ 0................0L..........................................................................................................2 ± 0.... Preparation of Medium: Prepare and dispense medium under 100% CO2..0mL: Na2CO3 .........................3g Hemin......................................................................................................................... L-cysteine·HCl·H2O solution............0mL L-Cysteine·HCl·H2O solution ..0g K2HPO4 .................................................................. Volatile Fatty Acid Mixture: Composition per 7...................... 6... Sparge with 100% CO2...................................................................................................0mL of sterile Na2S·9H2O solution or....................................................... 0....................0mL Na2S·9H2O solution.75...5g CaCl2·2H2O...............1g CoCl2·6H2O ....150.....0mL Volatile fatty acid mixture.................. Sparge with 100% CO2...............................................1g FeSO4·7H2O.......................................................................... 1...................................8............ 4....1.................. 2....10.........................25mL n-Valeric acid...............01g Preparation of Modified Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.......... Mix thoroughly............ Autoclave for 15 min at 15 psi pressure–121°C.................... except carbohydrate solution........ 0............................... 1....................................... 2....50mL Butyric acid ..................0mg Rumen fluid..... Butyrivibrio species Medium Composition per 1001. Use: For the cultivation of Pseudomonas putida. Gently heat and bring to boiling. Distribute into tubes or flasks...........7 ± 0.............................. Mix thoroughly..0.... Distribute into tubes or ................................5...... to distilled/deionized water and bring volume to 960..............................................5g K2HPO4 .........0mL Mix thoroughly....0mg Resazurin ................................................................... 0................................... 4.......................25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.....75mL: Acetic acid...3g Yeast extract........... 0.0g NaCl ............0mL: Glucose............0.........2................ soluble ............. Cool to room temperature while sparging with 100% CO2........................................... 0.......................... Add Na2CO3................................................................ 10......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............ inject the appropriate amount of sterile carbohydrate solution....................................0g Yeast extract............0mL......................................... 0.......2g Modified Wolfe’s mineral solution .........2 at 25°C Minerals Solution: Composition per liter: NaCl ............ Mix thoroughly. Carbohydrate Solution: Composition per 20.....................................................15.. Autoclave for 15 min at 15 psi pressure–121°C.....25mL Propionic acid...... Mix thoroughly............................01g H3BO3 .................. 2................................ 0..........0mL Carbohydrate solution..............................0mL: L-Cysteine·HCl·H2O ... Na2CO3....01g NaWO4·2H2O................................. BY Agar Medium (ATCC Medium 2038) Composition per liter: Agar ...0mL of sterile carbohydrate solution... and Na2S·9H2O solution..................... Mix thoroughly..... Use: For the cultivation of Butyrivibrio species............................... 6............ Distribute into rubber-stoppered tubes under 100% CO2.................

....................................0................................................................................................................ 2........................... C/10 Agar Composition per liter: Agar .... Remove supernatant solution.............02g Phenol Red ...................2g MgSO4·7H2O...................................0............................03g L-Alanine ..0...................0....................025mg Niacinamide ............. 1..................................................0g Phenol Red .............025mg Pyridoxine .................. Filter sterilize...................01mg Folic acid .........................4..02g L-Histidine·HCl·H2O ......................0................................ 25....................................5mg Cocarboxylase ........... Use: For the cultivation of Legionella pneumophila................2g D-Glucose ....0.....05mg Niacin ............................................... and Myxococcus xanthus........................... Autoclave for 15 min at 15 psi pressure–121°C..............................1mg Inositol....... Myxococcus fulvus......................0...... pressed......................0......................075g L-Arginine·HCl..............................0..........03g L-Threonine ........................025g L-Cystine .07g L-Lysine·HCl .................78 BYEB flasks........................025mg Pyridoxal·HCl................0... 10......... BYEB (Buffered Yeast Extract Broth) Composition per liter: ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ........5 ± 0.........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............6.....................................................4g L-Cysteine·HCl·H2O........................................................ C 3G Spiroplasma Medium Composition per liter: Sucrose............................................... ................ 1..........0....0.. Gently heat and bring to boiling............................7..0mL pH 7...................2......................8g NaHCO3 ...........0mL Fresh yeast extract solution..........................8................................................52g Peptone...............................................................................................................................................0......... infusion from ..............................................01g Deoxyguanosine .....26g CaCl2......................0mg Nicotinamide adenine dinucleotide phosphate.................................................0.....32g NaCl .................0mL of distilled/deionized water.......................0g Yeast extract..........25g pH 6...................................................... Mix thoroughly.................025g L-Serine ......................05g L-Proline ........ Mix thoroughly........ Pour into sterile Petri dishes or leave in tubes............... Pour into sterile Petri dishes or leave in tubes........2.................0.......................................5mg Cholesterol ..... 15..0..............02g L-Methionine ..0..............9.1...2 ± 0................0mg Choline chloride ............................0............0g CaCl2·2H2O.......................0.0..01g L-Tryptophan .............0mg PPLO broth without Crystal Violet....................0..............................................0.................................... Adjust pH to 6.....................................04g L-Tyrosine....01mg Source: CMRL-1066 medium is available as a premixed powder from BD Diagnostics.....................6– 6...............................................0.....500.5.02g L-Isoleucine ..............................01mg Thiamine·HCl .........36g pH 7.......0.................01mg D-Calcium pantothenate ............2mg 5-Methyldeoxycytidine ............................................................0mL: Baker’s yeast..........0mL CMRL-1066 medium.....0L.............0L...............................07g L-Leucine...............................................................0. 0......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................2mg Coenzyme A ..................0....0....................0.....................................................0.......05mg p-Aminobenzoic acid ...... Distribute into tubes or flasks...............................025g L-Valine ...................0...... Preparation of CMRL-1066 Medium: Add components to distilled/deionized water and bring volume to 1........................................................2g NaH2PO4·H2O ................ Flexi- bacter filiformis.....0mg Uridine triphosphate .0mL: Beef heart..................................15g Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 500.......................150..............................0..........................0..............0........................................4g Fe4(P2O7)3·9H2O .........................................04g L-Aspartic acid .................06g Glycine ................0.. Adjust pH to 7.............................. Use: For the cultivation and maintenance of Paracoccus thiocyanatus....................1......0mg Tween 80 ..................0...0.....0................................................................ Allow to stand..0g Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100....... Filter sterilize..... Mix thoroughly...................................1.....0................................................................................ 3.............................................. Adjust pH to 7............ live.1.0g L-Cysteine·HCl·H2O ....0...01g Nicotinamide adenine dinucleotide ......50.083g L-Glutamic acid .....................01mg Riboflavin................................ 100...........................01g Thymidine ...........................................................2........ Autoclave for 90 min at 15 psi pressure–121°C........................................0....0.............0.................. reduced ................ PPLO Broth without Crystal Violet: Composition per 500.......................................................... CMRL-1066 Medium: Composition per liter: NaCl ....0g Pancreatic digest of casein .........0g KCl ....... Adjust pH to 6.................. anhydrous.5........0..............................015g Deoxyadenosine ..........................................................14g L-Glutamine...................0mg Sodium glucoronate·H2O ......................................................................................... Autoclave for 15 min at 15 psi pressure–121°C.....................1g Sodium acetate·3H2O ...........01g Glutathione..................0................................................0mL Horse serum .......0g α-Ketoglutarate................ Use: For the cultivation of Cytophaga flevensis............................................0...................9 ± 0...............................1.......2........0L.........................025mg Biotin ............................... Fresh Yeast Extract Solution: Composition per 100..0.......025g L-Phenylalanine ................................. 10............................................................................0mL.. 10................. 0..................... starch-free...........................................................2 at 25°C Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems............................. Aseptically distribute into sterile tubes or flasks.......... 11......................................... Mix thoroughly....................................... Filter sterilize.....0....................................................01g Deoxycytidine ............0mg Flavin adenine dinucleotide ..............................01g Hydroxy-L-proline.................................... 1.........05g Ascorbic acid................................

............0mL........... and CMRL medium—to distilled/deionized water and bring volume to 75........................................ Adjust pH to 7.......................0........................................05g L-Proline....................................... Filter sterilize.....1g Sodium acetate·3H2O.................................... Distribute into sterile tubes or flasks................20.................................................. Preparation of Medium: Add components—except horse serum...........................................................0g Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100....................0mg Tween 80 ......... 0.............1......... fresh yeast extract.......01g Deoxycytidine ...........................................5mg Cocarboxylase ..................................52g Peptone......................2g NaH2PO4·H2O... 5............04g L-Aspartic acid .............................2mg Coenzyme A ...........2g MgSO4·7H2O .........01mg D-Calcium pantothenate ....5g Yeast extract . 2........CAL Agar Preparation of Medium: Add components—except horse serum............0....................05mg p-Aminobenzoic acid ...........0g L–Arginine·HCl .......... Adjust pH to 7..................5........................................................02g L-Isoleucine .............. and 0.....01g Glutathione................0......6– 6...........0........ 12............. Adjust pH to 6......083g L-Glutamic acid.075g L-Arginine·HCl ..02g L-Methionine ....0..........................................................................03g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................4g L-Cysteine·HCl·H2O ..............................03g L-Threonine.........0............................................02g Phenol Red ................................................................................................................................ 0.........................................................01g Thymidine ............................ Autoclave for 15 min at 15 psi pressure– 121°C..................26g CaCl2......01mg Thiamine·HCl .............................0......... 0.............0................................... C 3N Spiroplasma Medium Composition per 100....................................... 0...............0......2g D-Glucose... 0.............................................0mL: Sucrose.......................0mL of sterile fresh yeast extract solution..................................50........................... Allow to stand....05mg Niacin .....................................................5....................................................................... Aseptically add 20.................2 at 25°C PPLO Broth without Crystal Violet: Composition per 500................................................................................................................0L...................................................................................0mL Fresh yeast extract solution...............0mg Nicotinamide adenine dinucleotide phosphate...6......0....................4.... 0...0mg PPLO broth without Crystal Violet.............................01g L-Tryptophan ..............5mg Cholesterol ...........025g L-Valine ...0L.........................................5mL of sterile CMRL medium.0....................1.............................5......................5................5g NaCl ......06g Glycine.......................... 0................... 0...........025g L-Phenylalanine......... reduced ................................0mg Sodium glucoronate·H2O ...................................................................... pressed............0g Cellobiose............. Remove supernatant solution.............3. Mix thoroughly........... CMRL-1066 Medium: Composition per liter: NaCl ...............5mL pH 7...........................0........... anhydrous .........02g 79 L-Histidine·HCl·H2O ......... Pour into sterile Petri dishes........0.................................................0mL CMRL-1066 medium..................................................0g Sodium deoxycholate ...................... infusion from ......................................0mL of sterile yeast extract. and 5......1mg Inositol...............0.................................. Distribute into sterile tubes or flasks.................................... 0.....................................8g NaHCO3 ..................0..............................................................................2.............. starch-free................0....0mL............... Fresh Yeast Extract Solution: Composition per 100....2mg 5-Methyldeoxycytidine ................................................................... live..................0.... 0............... Use: For the cultivation and maintenance of Spiroplasma species...07g L-Lysine·HCl................................................................. CAL Agar (Cellobiose Arginine Lysine Agar) (Yersinia Isolation Agar) Composition per liter: Agar .... 0................0mL Horse serum ....... 0.... 1... 2........... Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 500..................0...........................14g L-Glutamine .1.........0... fresh yeast extract..................................................................0mL: Baker’s yeast.......................................20..........................15g Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems...........1.8....................... 10..................04g L-Tyrosine ..................025g L-Serine .......................0mL of distilled/deionized water.....................0.01mg Source: CMRL-1066 medium is available as a premixed powder from BD Diagnostics....................... 0..01mg Riboflavin..01g Nicotinamide adenine dinucleotide ..........................0mL of sterile horse serum.. 6...............6.................................................... Preparation of CMRL-1066 Medium: Add components to distilled/deionized water and bring volume to 1.................... Aseptically add 150. and CMRL medium—to distilled/deionized water and bring volume to 795......................................... 0............................................................. 0................................................................................01mg Folic acid ...025g L-Cystine .............0mL of sterile CMRL medium.......0.. 0...............1..........5g Neutral Red .................. 0......................0........... Heat to boiling..................................................0mg Flavin adenine dinucleotide ... Filter sterilize..... Autoclave for 15 min at 15 psi pressure– 121°C......0mL: Beef heart.............. 0.........0........................07g L-Leucine ..... 11................................................................................................ 0.......... Autoclave for 90 min at 15 psi pressure–121°C..............5g L–Lysine·HCl.. 50............ Do not autoclave.................0mg Choline chloride ............025mg Pyridoxine ..............025mg Pyridoxal·HCl.............3...................... 0....................................7......0....025mg Niacinamide ....5..............................................................0. Adjust pH to 7........0mL of sterile horse serum........................................ ............... Mix thoroughly......................015g Deoxyadenosine .................................03g L-Alanine...................................0mL.........5 ± 0..05g Ascorbic acid ...................... 25..................................025mg Biotin ..........32g NaCl .0g KCl........................................... 0.....................0mg Uridine triphosphate .............................0............... 0................01g Hydroxy-L-proline...........0g Phenol Red ..0............................................2.........................................................................01g Deoxyguanosine .......................................... 1.... Use: For the cultivation and maintenance of Spiroplasma kunkelii.. Mix thoroughly..

......................... 1..................2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .................................... Spread 0.. Use: For the cultivation and maintenance of Pseudomonas putida...............................................0L................................80 Caldicellulosiruptor Medium Use: For the isolation and characterization of Yersinia enterocolitica and enumeration of Yersinia enterocolitica from water and other liquid specimens......................5....0mg HCl (25% solution) .........0mL of HCl solution......... Autoclave for 15 min at 15 psi pressure– 121°C................................................... Mix thoroughly.......................................... 10...................................0mL 100× Salt Solution: Composition per liter: MgSO4 ........2............................... Mix thoroughly.................................................................0mL.................................................................0mg MnCl2·4H2O...................................... Autoclave for 15 min at 15 psi pressure–121°C..0g K2HPO4 .........1 g NaOH (1N solution) ........................................5mg Resazurin ................. Carbohydrate Solution: Composition per 100........0mL pH 7.......................................................................... 70..............................3g Preparation of 100× Salt Solution: Add components to distilled/deionized water and bring volume to 1..... Pour into sterile Petri dishes........................................0L.................. 190..........................0g Carbohydrate solution .......20.... 2.......................................0g CaCl2·2H2O ......................................0g K2HPO4 ................................0g Ascorbic acid... Preparation of Medium: Prepare and dispense medium under 100% N2......0g Preparation of Carbohydrate Solution: Add carbo- hydrate to distilled/deionized water and bring volume to ..............2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .................4g FeCl3·6H2O .. 36...........0mg CuCl2·2H2O...... 1........... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... 2....10.............................................................. Mix thoroughly.......................................................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.4.0mL: Acid Fuchsin ......................0g K2HPO4 ............................... Preparation of Medium: Prepare and dispense medium under 100% N2.............................. 16........ Gently heat and bring to boiling....... 10................9g KH2PO4 ....................9g NH4Cl............. Sparge with 100% N2............................................................................................75g Resazurin ..........0mg ZnCl2 ................0mg ZnCl2 ...................... 1.......... 0............................ 36............ 0... 1......75g MgCl2·6H2O.....4g FeCl3·6H2O ... Add distilled/deionized water and bring volume to 1.............5g CoCl2·6H2O ....... 2.............. Mix thoroughly........................................................................................0g Yeast extract.....0mg H3BO3 .................................... 6. Mix thoroughly................ Camphor Minimal Medium Composition per liter: Agar .2 ± 0.. 190........... H3BO3 ...............................0L.0mL pH 7..............................................10.................. Autoclave for 15 min at 15 psi pressure–121°C......................................0mL Andrade’s indicator ............................... Use: For the cultivation of Caldicellulosiruptor saccharolyticus.......0g NaCl ...............................................0L.... Anaerobically distribute into tubes or flasks.....................................................................................5mg L-Cysteine·HCl.............................0L....................... Carbohydrate Fermentation Broth Composition per liter: Peptone ......0mg NiCl2·6H2O .......................0g Cellobiose .............0mL: Pancreatic digest of casein ............. Mix thoroughly........1 ± 0.........................................................0mg NiCl2·6H2O ............................. Sparge with 100% N2...............0mL Caldicellulosiruptor Medium Composition per liter: Preparation of Trace Elements Solution SL-10: Pancreatic digest of casein .....................10.........0.......................... 0..9g NH4Cl.........19...........................5mg pH 7............ 0.. 50...............................................................0g NaCl ...... Anaerobically distribute into tubes or flasks.5..............................................5mg Trace elements solution SL-10 ............................ Add components to distilled/deionized water and bring volume to 1..9g KH2PO4 ..1..............6...........0g Yeast extract.............................. 100.............. Add remaining components.................................................. 1........... 0............ 1........5g CoCl2·6H2O ...... Use: For the cultivation of Caldicellulosiruptor saccharolyticus....2 at 25°C Andrade’s Indicator: Composition per 100.........................................................................0mg Na2MoO4·2H2O . Add components to distilled/deionized water and bring volume to 1..............0mg Na2MoO4·2H2O ............0g Meat extract.............0mg CuCl2·2H2O .2mL of 2m D-(+) camphor solution in methylene chloride (CH2Cl2) on the inside cover of each plate.....................................0mL Preparation of Andrade’s Indicator: Add compo- nents to distilled/deionized water and bring volume to 100...................................... 0.......................................................................................................................................0mg Add FeCl2·4H2O to 10.1g KH2PO4 .....0g MnSO4·H2O....75g MgCl2·6H2O.............................................................................. 0.....5g Cellobiose ...................................... Sparge with 100% N2.........2.75g L-Cysteine·HCl................. 2...................................................2 ± 0........... 0............ 70.........0L.......................3.......................0mg MnCl2·4H2O.. 24..... 1..........0g NaCl ....................................... Sparge with 100% N2..... 10......... Add remaining components..................................................................... Invert Petri dishes.......................................................1................ Mix thoroughly.................. 0. Autoclave for 15 min at 15 psi pressure–121°C....................... Caldicellulosiruptor Medium Composition per 1001.....................4g NH4Cl .......................................... Mix thoroughly.........5..........................0............... 2...... Allow to cool to room temperature....................................... 100. Autoclave for 15 min at 15 psi pressure–121°C..............................0mL of HCl solution...................................... 0......................................... Add distilled/deionized water and bring volume to 1............................................................................................................................................5g Cellulose ... 0. 0...5g FeSO4·7H2O ....0mg HCl (25% solution) .. 1....................0mL: Carbohydrate ............. 1........................7g 100× salt solution .................. 24.......1..........

............. Filter sterilize..................2g Resazurin ........ Modified Composition per 1001.......0g MgSO4·7H2O....2g CaCl2·2H2O.......................... Carbon Monoxide Oxidizers Agar... 0...... maltose...........1g AlK(SO4)2·12H2O ............................................ 20. Aseptically add sterile carbon source....0mL of sterile NaHCO3 solution and 10.. 10...................0mL pH 6............................. Preparation of Medium: Add components.........0mg Na2SeO3 ......................... Mix thoroughly...0.. cellobiose................ sucrose.0µg Preparation of Thiamine·HCl Solution: Add thiamine·HCl to distilled/deionized water and bring volume to 10............ 3... 10.....0mL volumes into test tubes containing inverted Durham tubes............................................................................................................. rhamnose.........0...........................5mL of sterile carbohydrate solution to each tube............................................... Mix thoroughly......0g MgCl2·6H2O ...0....... Distribute in 10.5mg Sodium potassium phosphate buffer (0.......... Add 0.......0mL: Carbon source...... Caution: Acid Fuchsin is a potential carcinogen and care Use: For the cultivation of Carbophilus carboxidus and Zavarzinia compransoris..................................0g NH4Cl ....................... 1.................................................................................................................1.....1g ZnSO4·7H2O................0...9g H3BO3 ...................... except car- Carbon Utilization Test Composition per liter: must be taken to avoid inhalation of the powdered dye and contact with the skin................................................... 10.................... Carbonate-Buffered Medium CMB4 with Glucose Composition per 1002................. Gently heat and bring to boiling....0mL of sterile thiamine·HCl solution...................0mg NiCl2·6H2O .......75g MgSO4·7H2O ............. galactose........4........................0....................... Use: For the determination of carbohydrate fermentation reactions of microorganisms.....0....................................................................................01g Na2MoO4·2H2O........ 4.............. salicin... Preparation of Medium: Add components................................ except NaHCO3 solution and thiamine·HCl solution........................ Gently heat and bring to boiling...............0mL...... Mix thoroughly..0g Preparation of Carbon Source: Add carbon source to distilled/deionized water and bring volume to 10....................... NaHCO3 Solution: Composition per 10.1g MnCl2·4H2O......................0mL: Agar ................0mL.............0...33M solution........................................... Autoclave for 15 min at 15 psi pressure–121°C................... Filter sterilize... Cool to 45°–50°C............... oughly...... 0............ 12........1....................................... Preparation of Medium: Add components............ 0..........0..0....0mg Preparation of Trace Elements Solution: Add components to distilled/deinonized water and bring volume to 1...............3g KH2PO4 ....5g Sodium acetate ....................................1g CoCl2·6H2O.........................0mL Trace Elements Solution: Composition per liter: Na2MoO4·2H2O ....................................................... arabinose........................... raffinose........................0g NaHCO3 ............0g Na2HPO4·12H2O.............0L Carbon source............... Acid production is indicated by a pink reaction...................0mL.....................0............................. Filter sterilize........................0g NH4Cl.....................5g NH4Cl ..................0..................... sorbitol.. Mix thoroughly..01g CuSO4·5H2O.................0g MnSO4·H2O..............01g . inositol........1.......... 0......... bohydrate solution...........0mL: NaHCO3 .................20.................................... Autoclave for 15 min at 15 psi pressure–121°C..... Mix thor- Ionagar.....................................1........10....................... Mix thoroughly......0mg Trace elements solution .................2 at 25°C Modified Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O...................0................................. lactose..0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10................ xylose..5g CaCl2 ............................. trehalose............0mL Sulfide-calcium solution ............................ 2........0L..................... 0. Use: For the cultivation and differentiation of Pseudomonas species based on their ability to utilize a specific carbon source......... pH 6.....8g KCl ... Pour into sterile Petri dishes or distribute into sterile tubes............................................0mL Wolfe’s vitamin solution ...................................................................................................................9 ± 0..0mL. dulcitol......................... Mix thoroughly..............10....2 at 25°C Carbon Source: Composition per 10........0mL NaHCO3 solution .......................0mL: Thiamine·HCl ........ 18..........5g NaCl ...............................2...........................0mL pH 6.............................0mL.0mL: NaCl ................ A Durham tube is used to collect gas produced during the fermentation reaction.......5g Ferric ammonium citrate .........0.................0mg CuCl2·2H2O ...1............0mg Ferric ammonium citrate.............................10..05g CaCl2 .0.....................5g KH2PO4 ...... to distilled/deionized water and bring volume to 1............................... 0................... Cool to 50°–55°C...... Thiamine·HCl Solution: Composition per 10....................2g ZnSO4·7H2O ....... 30....1......................... Autoclave for 15 min at 15 psi pressure–121°C.............. 30.......8) ..0................................... except car- bon source.......................1g FeSO4·7H2O ......................................... 20.. Cool to 25°C.....................................................................................3........... 10... 1.............. Mix thoroughly. Filter sterilize............. mannitol............ to distilled/ deionized water and bring volume to 980...................... fructose....0mL Thiamine·HCl solution...3g CoCl2·6H2O ........................................ Adonitol....... 1...... glucose............................ to distilled/deionized water and bring volume to 990..................................01g H3BO3 ................0mg Modified Wolfe’s mineral solution....8 ± 0......................... Aseptically add 10....0................................0L................ or other carbohydrates may be used........ particularly members of the Enterobacteriaceae...........................................5g Glucose..............................................Carbonate-Buffered Medium CMB4 with Glucose 81 100....................0g Nitrilotriacetic acid..................................................0mL..................1... Gently heat and bring to boiling........ Pour into sterile Petri dishes or distribute into sterile tubes......

............ Use: For the autotrophic cultivation of Oligotropha carboxidovorans.....0g Sodium acetate .............5g FeSO4·7H2O .........................0mg CuCl2·2H2O .............0................ After inoculation.................... 0...................5g MgSO4·7H2O ................. Add NaHCO3........ Distribute into tubes or flasks...........0mg Na2SeO3................... Carboxydothermus Medium Composition per 1030........ 5......................................................................... incubate in an atmosphere of 50% CO + 50% air...........................................0L....................................0mg Lipoic acid ... Adjust pH to 7...................... 10.................3g CoCl2·6H2O....33g KH2PO4 ....82 Carboxydobacterium Medium Na2SeO3 ..............5g NH4Cl .............................20....... Adjust pH to 6..................5mg Trace elements solution SL-4..................................0mL Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1... 20...........................................1.................... 0.......................... Mix thoroughly..............................................................0mL of distilled/ Caution: Carbon monoxide (CO) is a toxic gas..............5 with KOH........ Gently heat and bring to boiling..................................0.. 0.................0mg Vitamin B12 .0mL: MgCl2·6H2O .... 100...................... 10......0g CaCl2·2H2O............. 0.....................0mg Biotin ......................3...................................2g ZnSO4·7H2O.9 ± 0...... 1.......................................... 5...........0L......... 2......2g CaCl2·2H2O..... Aseptically and anaerobically add 0......... Mix thoroughly.......8................ 1........... Add remaining components one at a time.......... Add components.. Distribute into tubes or flasks..................................................................3g CoCl2·6H2O .............0mL Na2S·9H2O solution ..0................. Add distilled/deionized water to 1..........................................1g MnCl2·4H2O......................... 5..0mg Folic acid...................... Mix thoroughly.......2mg TS2 trace elements solution .....0.. Autoclave for 15 min at 15 psi pressure–121°C......................................0mg Preparation of TS2 Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.......... deionized water and bring volume to 1.....2..................................... 20..............0L.................0mL pH 6. 10..... Anaerobically distribute into tubes.......................... 30. 15..............0.. Preparation of Medium: Add components to distilled/ deionized water..0mg components to distilled/deionized water and bring volume to 1....1.......................................................................... 2...............................................................0g KH2PO4 ............................0L... 10.......0L.. Carboxydobacterium Medium Composition per liter: Na2HPO4·12H2O..................................................0................................... 0........ to distilled/deionized water and bring volume to 1...0mg Nicotinic acid .............. 9.............................................................................................. 0........................... Sparge with 100% N2........... 36.......................................................9g H3BO3 ........ Autoclave for 15 min at 15 psi pressure–121°C..................... Carboxydobacterium Medium Composition per liter: Na2HPO4·12H2O ............. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2.......0L................0mg p-Aminobenzoic acid ......... Mix thoroughly........................0mg Na2SeO3 ............. ......................... Cool to room temperature while sparging with 80% N2 + 20% CO2..........01g Preparation of TS2 Trace Elements Solution: Add Preparation of Modified Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.. except NaHCO3 and sulfide-calcium solution................................0g Preparation of Sulfide-Calcium Solution: Add components to distilled/deionized water and bring volume to 1..................... Mix thoroughly.0mg CuCl2·2H2O.....1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........ Mix thoroughly........0..2g ZnSO4·7H2O ........................................... 1....................................0mg Ferric ammonium citrate...... Adjust pH to 6........................................................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........0....0mg Ferric ammonium citrate ................................ 0.........01g NiC12·6H2O ...................................0mg NiCl2·6H2O.........2g Trace elements solution SL-6........................ 0.. Sulfide-Calcium Solution: Composition per liter: Na2S·9H2O................................................................................................................0......2g CaCl2·2H2O ......... Use: For the organotrophic cultivation of Oligotropha carboxidovorans...................... Autoclave for 15 min at 15 psi pressure–121°C.........20........................................0mg NiCl2·6H2O ....9g H3BO3 ............................................... Mix thoroughly............... After inoculation................................0mg Riboflavin ............0g KH2PO4 ..................... 5...................... 10............... 5............33g NH4Cl ............. 10..............2 at 25°C Trace Elements Solution SL-4: Composition per liter: EDTA.0mL TS2 Trace Elements Solution: Composition per liter: Na2MoO4·2H2O................02mL of sterile sulfide-calcium solution to each tube............................52g KCl ............................................. 1............. incubate in air................. Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ................ Mix thoroughly.............29g Resazurin ............................. 0..........................1g MnCl2·4H2O ...................................................................5g NH4Cl..... 20..30................................01g NaWO4·2H2O........................ Continue boiling for 3 min................0mL volumes into anaerobic tubes......................5g MgSO4·7H2O............ Use: For the cultivation of Spirochaeta thermophila.0mL Vitamin solution .0mL TS2 Trace Elements Solution: Composition per liter: Na2MoO4·2H2O ... Adjust pH to 6......0mg Calcium DL-pantothenate.. Anaerobically distribute 10................. Autoclave at 121°C for 15 min................................................................2mg TS2 trace elements solution ........................0L........10........................................................20....0.........0mg Thiamine·HCl ............. Mix thoroughly...........................1......................................0......0........0L..33g CaCl2·2H2O ......................................... Mix thoroughly............... 0...............1................0L.................................................................................................................9.................................... 5......9.........................................................0mL Yeast extract solution ..........................................................

........0..........................................02g CuCl2·2H2O ........................................................................ Add Na2S·9H2O to distilled/deionized water and bring volume to 10......................................... 0.70. 0.......................0mg Vitamin B12 ...... Add distilled/deionized water and bring volume to 1..............................6.............................3g Na2HPO4 .45g Preparation of L-Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10..0mL Pyruvate solution..5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10....100.................................................................................. Mix thoroughly....... Autoclave for 15 min at 15 psi–121°C....................5........24...... Sparge under 100% N2 for 10 min.................0mL................ Mix thoroughly...........................................................5g CoCl2·6H2O.... 20................. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.................................. except vitamin solution. Adjust pH to 7............. Autoclave for 15 min at 15 psi pressure–121°C...0...............0mL........................................................0mg Nicotinic acid ....................0L...............0mL pH 7................................ 0......15g Yeast extract ...0L.....0mg H3BO3 . 0....... Mix thoroughly................. Mix thoroughly........ Mix thoroughly..... hermus hydrogenoformans..................03g Resazurin .............. 0.......................................... 0.....................10....................0L........190... 0..................... Cool to 25°C............. 0.. 0...................................................................5mg Vitamin solution ...........2......0............... 0.............0....0mg NiCl2·6H2O...........................0mL Na2S·9H2O solution ................ Must be prepared freshly.....0mL of sterile yeast extract solution......... Mix thoroughly............01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1................................ 0....5.. Filter sterilize..........0............................5..... and Na2S·9H2O solution..0..... Mix thoroughly........ Vitamin Solution: Composition per liter: Pyridoxine·HCl ....0mg Na2MoO4·2H2O...0mL.....................................3g CoCl2·6H2O ...............0mg .........0mL...........0mg D-Ca-pantothenate ............... Mix thoroughly...........................0mg Folic acid....0 ± 0........................ 5............................... Add remaining components..................... FeCl2·4H2O............................0mL: Na-pyruvate .................. Sparge with 100% N2......................................... Autoclave for 15 min at 15 psi pressure–121°C...............................0mL: NaHCO3 ...........0g MgSO4·7H2O .......0mg Thiamine·HCl .0mL: Na2S·9H2O.........0mL NaHCO3 solution..............................0mL Trace elements solution SL-10........................................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................. Sparge with 100% N2............... 10........................................................ Na2S·9H2O Solution: Composition per 10.......................................7g Preparation of Na2S·9H2O Solution: Prepare and dis- pense solution anaerobically under 100% N2....05g Na2SiO3 .. Mix thoroughly.....................0mg p-Aminobenzoic acid ...................45g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....................... 5.......................................0mg Biotin .......... Trace Elements Solution SL-10: Composition per liter: Preparation of Medium: Add components.................................Carcboxydobrachium pacificum Medium Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O.......0mg Nicotinic acid ..0mg Riboflavin .....9g KCl.......0..............................1.... 10.............................................................................................. Na2S·9H2O Solution: Composition per 10. L-Cysteine Solution: Composition per 10.... Autoclave for 15 min at 15 psi pressure–121°C........36.........5g H3BO3 ................. Pressurize inoculation flask with CO (carbon monoxide) gas at 2 bar pressure........................0mg HCl (25% solution) ........ 10... Mix thoroughly..... Aseptically and anaerobically combine with 10............................................................0mg Thiamine-HCl·2H2O ......... Sparge with 100% N2............ Use: For the cultivation and maintenance of Carboxydot- Add FeCl2·4H2O to 10....................................................0mg CuCl2·2H2O.................... 5...................0mL of sterile Na2S·9H2O solution.............0mg MnCl2·4H2O ..5g Preparation of Pyruvate Solution: Add pyruvate to distilled/deionized water and bring volume to 10.0L.........................0mL of sterile vitamin solution..................0mL: Na2S·9H2O .0mg Lipoic acid ......... Sparge with 100% N2...2g ZnSO4·7H2O ........0mg Riboflavin.............................2 at 25°C Pyruvate Solution: Composition per 10............................4g Preparation of Trace Elements Solution SL-10: Vitamin Solution: Composition per liter: Pyridoxine-HCl ............ and 10............................................................... 2. Sparge with 100% N2...............0mL of HCl solution.......................................... 5............... Autoclave for 15 min at 15 psi pressure–121°C................. 10. Mix thoroughly. 5.......... to distilled/deionized water and bring volume to 1.......................................... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.......5....... 1............................... Carcboxydobrachium pacificum Medium (DSMZ Medium 902) Composition per 1050mL: NaCl ........... NaHCO3 Solution: Composition per 10........................................0mL Caution: Carbon monoxide is a toxic gas...................................................................2............................ Autoclave for 15 min at 15 psi pressure–121°C.... 10..0mL................... 83 NH4Cl .............. 5.......................... Yeast Extract Solution: Composition per 10.......... yeast extract solution.......0mL L-Cysteine solution........0mg ZnCl2 .......0mL: L-Cysteine·HCl·H2O.........................0mL.......................................0mL: Yeast extract.................. 10.........1g Na2MoO4·2H2O .... Mix thoroughly.........................03g NiCl2·6H2O ...............................0mg Calcium DL-pantothenate.0mg Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10............0............................ 10.......................... 10...................0...............................................7g CaCl2·2H2O........................... 3............................ 10........................................................... 2.....................

.....0L with distilled/deionized water............................5...............................................10.............. Distribute into anaerobe tubes or bottles....................2....................... Mix thoroughly......................0............3..... Adjust pH to 7.......... Mix thoroughly......1000... Gently heat and bring to boiling.....0g MgCl2·6H2O ....0mL pH 7........................ Vitamin Solution: Composition per liter: Pyridoxine·HCL ..............0mg Biotin ............ Pour into sterile Petri dishes or leave in tubes................... The final pH should be 7............................. Autoclave for 15 min at 15 psi pressure–121°C..... 2. Mix thoroughly.......0mL Vitamin solution.............. Adjust pH to 7....................................... 5........................1mg Preparation of Medium: Combine components... Pour into sterile Petri dishes or leave in tubes...... Mix thoroughly.................. Solution B (Metallic Salts): Composition per 100.................................................... Add remaining components............ 1............0mL L-Cysteine-HCl·H2O solution................................... 2..0mL.....................................................4...... 10.... 2...............0mL Na2S·9H2O.................................2 ± 0................................0g Agar ..........10.......0L. Autoclave for 15 min at 15 psi pressure–121°C..................................................2....1g FeSO4·7H20 .. Sparge with 80% H2 + 20% CO2....... to distilled/deionized water and bring volume to 1...................... NaHCO3 solution..............................34g FeSO4·7H2O ........01g Calcium pantothenate ......... Use: For the cultivation of myxobacteria.......0L........0mg Lipoic acid ..............................5.20............ Gently heat to boiling..5. and 10...............................................1......0mL.................................................................................. Distribute into tubes or flasks......3g MnSO4·H2O......0L.........20......... Caryophanon latum Medium Composition per liter: Papaic digest of soybean meal .........................................2g pH 7.......................0.5............ Distribute into tubes or flasks......... Add components to acidified distilled/deionized water and bring volume to 100.0mg Vitamin B12 .0mg Nicotinamide .................... Mix thoroughly.1g Thiamine·HCl .............0........... Dissolve by adjusting pH to 6.......0mg Riboflavin.......0.......... Use: For the cultivation of Carboxydibrachium pacificum (Carboxydobrachium pacificum)................... pH 7.........8 ± 0........ except vitamin solution...27g Sodium glutamate ............0mL Preparation of Solution A (Mineral Salts): Add nitrilotriacetic acid to 500............ Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..................25g pH 6.......0g K2HPO4 ............0g Vitamin-free casamino acids.5......................................................67g Nitrilotriacetic acid.20.............. 10...........................0.............................0.0g Sodium acetate ................................. Preparation of Medium: Prepare and dispense medium under 100% N2.......5................................................................................. Autoclave for 15 min at 15 psi pressure–121°C.....................12....................0L....................... Autoclave for 15 min at 15 psi pressure– 121°C......05mg Tris/HCl-buffer 10mM...........3g CuSO4·5H2O......................0g Solution A (Mineral Salts) .........0............... 1.........0... CAS Medium Composition per liter: Pancreatic digest of casein ...........0g MgSO4·7H2O .......................................0mL of distilled/deionized water............. 10.0mg Folic acid ............7g NaMoO4·2H2O .......... 50..0g CaCl2·2H20 ......................1g Solution B (Metallic salts)...... 0...............2 with H2SO4 or KOH........................................................................0g Pancreatic digest of casein .......... Aseptically and anaerobically add per liter.........................0L...................................................... Add components........2......0mg Thiamine·HCl ...... 10..... Autoclave for 15 min at 15 psi pressure–121°C.................... Mix thoroughly...........................4 ± 0....................... Mix thoroughly...................................................04g CoCL2·6H20 ......... 2.................. L-Cysteine-HCl·H2O solution.. Mix thoroughly...250................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 950................................ Bring volume to 1........................... 1.....5 with KOH....................................0L.29...................0g Yeast extract .......02g Na2B4O7·10H20 .5g Ethylenediaminetetraacetic acid...0mL pyruvate solution.....0g Yeast extract............................................0g Casein hydrolysate ......... Casamino Acids Glucose Medium (CAGV Medium) Composition per liter: Agar ......2 at 25°C Solution A (Mineral Salts): Composition per liter: MgSO4·7H2O...0mg Folic acid.84 Caryophanon latum Medium p-Aminobenzoic acid ..1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......0g K2HPO4 .............................2mg Biotin .............................0....0mL NaHCO3 solution....................................................... Add distilled/deionized water to 1........................ 2...........0mL vitamin solution.......... 10.......... pyruvate solution...............0mL: ZnSO4·7H2O.................................. 0.8 .......... Distribute into tubes or flasks...0g MgSO4·7H2O ....... ................... Mix thoroughly..................0g KCl ....... Distribute into tubes or flasks.......... Use: For the cultivation and maintenance of Caryophanon Preparation of Medium: Add components to distilled/ latum and Vitreoscilla stercoraria...... 0.... 0.... Sparge with 100% N2 of at least 30 min...............0mL pH 7....................... 20.............................. Casein Medium Composition per liter: NaCl ..........0g Glucose ............................. Readjust pH to 7............................02g Preparation of Solution B (Metallic Salts): Add a few drops of H2SO4 to distilled/deionized water to inhibit precipitate formation.................0mg Vitamin B12. and Na2S·9H2O solution......................................................0g CaCl2·2H2O .............................................0mg Biotin ..... Filter sterilize..........0............. 5............................................................................0...................... 1. 1...0... 0....... 0.2 at 25°C deionized water and bring volume to 1..................................................... Filter sterilize..... Use: For the cultivation and maintenance of Microcyclus aquaticus...2 ± 0..........

....... Mix thoroughly................................ For the cultivation of Chloroflexus species and Fischerella species......................3mg Trace metal solution..... 0.......1...................0..........................0L with distilled/deionized water........................................................ of iron and sulfur bacteria from the Sphaerotilus group............................................................ 1...... Adjust pH to 7.........2 ± 0..................0L.........................................0g Potassium phosphate buffer (0...........................5........025g Na2MoO4·2H2O .... Castenholz DG Medium (Medium DG) Composition per liter: Glycyl-glycine buffer .............................................0.. Autoclave for 15 min at 15 psi pressure–121°C.................................................................................................... Casitone Agar Composition per liter: Pancreatic digest of casein . 2.........5g Co(NO3)2·6H2O..........5mL pH 7............... 0.......2..............................5 ± 0...................................0...................................69g Na2HPO4 ......... 1.... Use: For the isolation of cyanobacteria........ Mix thoroughly............................0L....... 1..28g H3BO3 ...... Bring volume to 1.025g CuSO4·5H2O ...........0.................. Autoclave for 15 min at 15 psi pressure–121°C...........................10..5g ZnSO4·7H2O ...........06g FeCl3 ...................... 0..............0..........01M solution..................0............2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O ............0.... Mix thoroughly... and enumeration..... cultivation...................0... Mix thoroughly................................. Modified (Medium D.......0............. Mix thoroughly.............28g Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1.......... 15.......................2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O....... Bring volume to 1.........5g CoCl2·6H2O ...........0g Agar ...............................0g Yeast autolysate......1g Nitrilotriacetic acid....................... 8.............. 0..............5 with KOH......0mg FeCl3 solution...............0L pH 7..............28g H3BO3 ..................... Pour into sterile Petri dishes or leave in tubes....................... 0...... Use: For the cultivation and maintenance of Myxococcus species...............................................0L with distilled/deionized water.............................................. 20........2 at 25°C Preparation of Medium: Combine components................................................................... 0...........0g NaNO3 ...........................................................................................2 at 25°C Trace Metal Solution..................025g H2SO4 ......111g KNO3 ......... 0.. Dissolve by adjusting pH to 6............. Autoclave for 15 min at 15 psi pressure–121°C..........0..0...0...................................0mg FeCl3 solution ..0.............................0............ .2..... 0........2) ........... Distribute into tubes or flasks..........................................................1g Nitrilotriacetic acid......5 ± 0........................ 85 Preparation of Medium: Add nitrilotriacetic acid to 500..................................................8.........0mL Micronutrient solution ................... 0.... 0......0...... including thermophilic species.........28g Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1.........8g NaNO3 ...... Mix thoroughly..........11g KNO3 .................................................103g MgSO4·7H2O........... Dissolve by adjusting pH to 6............. Autoclave for 15 min at 15 psi pressure– 121°C..........5g ZnSO4·7H2O.................. Castenholz: Add components to distilled/deionized water and bring volume to 1....................... Mix thoroughly........................ Mix thoroughly................. Gently heat to boiling......0.............. Castenholz D Medium (Medium D) Composition per liter: NaNO3 ... Preparation of Medium: Add nitrilotriacetic acid to 500.160........................... Readjust pH to 7...........................................................1g CaSO4·2H2O........................................ 5. 0.. 2...... Add remaining components.............................................0L........................11g KNO3 ................................................ Castenholz D Medium........5mL Preparation of Trace Metal Solution.........7g Na2HPO4 ...............2.. Modified) Composition per liter: NaCl .0mL Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................................0............................................. Readjust pH to 7.............................. Mix thoroughly..0g MgSO4·7H2O ...........................................025g Na2MoO4·2H2O...........06g NaCl ... Castenholz .........................0........................1g CaSO4·2H2O ...... Use: For the isolation of halophilic cyanobacteria... Distribute into tubes or flasks............ Distribute into screw-capped tubes or flasks..................... Castenholz: Composition per liter: MnSO4·H2O.06g NaCl .5mL Preparation of Micronutrient Solution: Add com- ponents to distilled/deionized water and bring volume to 1................................................... Add remaining components......... 0..........................................5.................................................... Mix thoroughly.......................1g MgSO4·7H2O...0L............. Micronutrient Solution: Composition per liter: MnSO4·H2O ...........................................1g Nitrilotriacetic acid .....0.......................... Distribute into tubes or flasks..5mL pH 7......5 with KOH.....................................0mL of distilled/deionized water............. 0......0mL pH 7..............................7g Na2HPO4 ..5 ± 0......................................................0mL Micronutrient solution.................0L..........1g MgSO4·7H2O ......................................................................... 0......Castenholz DG Medium Use: For the cultivation and maintenance of Halobacterium species and other halophilic bacteria................0g Glycerol ......................................................................0mL of distilled/deionized water..... Casitone Glycerol Yeast Autolysate Broth (CGY Autolysate Broth) Composition per liter: Pancreatic digest of casein .1............... 1........1g CaSO4·2H2O.....025g CuSO4·5H2O.................................0....................................................................................025g H2SO4 ....... Use: For the isolation... pH 7...............

...............0mg KNO3 . 0....1g CaSO4·2H2O ............1g Nitrilotriacetic acid......8g NaNO3 ..5 with KOH... Mix thoroughly............................................... 25....................................... Dissolve by adjusting pH to 6............................................... 0.........2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O ...................... Distribute into tubes or flasks.......... Mix thoroughly.....11g KNO3 ... .... 0..................................................... Bring volume to 1.............0mg Na2HPO4·2H2O .....06g NaCl .......5 ± 0....................025g CuSO4·5H2O .0................5 ± 0.5g ZnSO4·7H2O.......0mg Nitrilotriacetic acid.....2 with NaOH.... Autoclave for 15 min at 15 psi pressure– 121°C................... 0................................... Mix thoroughly............. 0...........2mg H3BO3 ....... Readjust pH to 8... 0.................................. including thermophilic species..........0mg MnSO4·H2O...................... Autoclave for 15 min at 15 psi pressure– 121°C........................................ Use: For the isolation of cyanobacteria........2 ± 0........ 46...................................................0.................................................025g Na2MoO4·2H2O... 0..................................... including thermophilic species........ Mix thoroughly............. 1................. 0..........................0mg NaCl ....................................... Castenholz DGN Medium (Medium DGN) Composition per liter: Glycyl-glycine buffer.......2....28g H3BO3 ........ 8..........................8....................... Preparation of Medium: Add nitrilotriacetic acid to 500..................................0mL of distilled/deionized water.......................06g NaCl ...............................................025g Na2MoO4·2H2O ......................025g CuSO4·5H2O .................................................................................5g ZnSO4·7H2O ....100..................................... Add remaining components......................025g CuSO4·5H2O........... Mix thoroughly.................5g CoCl2·6H2O..................28g Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1.0g Yeast extract ....0mg CaSO4·2H2O..... Distribute into tubes or flasks.....................0mg FeCl3 solution ......... 0.689................................ Dissolve by adjusting pH to 6..................................2.................. Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1..............0...........1.025g H2SO4 ................. Mix thoroughly...................0......................................................0L with distilled/deionized water............ 0...................................100................ Mix thoroughly.......................60........28g H3BO3 ..... Bring volume to 1......................................11g MgSO4·7H2O.............. Mix thoroughly.0L with distilled/deionized water..........................................................0...0µg CuSO4·5H2O........................5mL Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1.7g NH4Cl......04µg Na2MoO4·2H2O...............................0..................... Distribute into tubes or flasks... Bring volume to 1......... Readjust pH to 8............................................025g H2SO4 .......................0........................1g MgSO4·7H2O ............................................ Mix thoroughly.................................................................................. Micronutrient Solution: Composition per liter: Micronutrient Solution: Composition per liter: MnSO4·H2O ..................1...............................................2......................0............... Use: For the isolation of cyanobacteria.........................................................5g CoCl2·6H2O ... Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Thermus aquaticus....5mg ZnSO4·7H2O.......28g Castenholz Medium Composition per liter: Tryptone .................... 2..... 0.......... 0.... 0.......5g ZnSO4·7H2O ...................................0.................0L with distilled/deionized water.................2 at 25°C Preparation of Medium: Combine components........... Dissolve by adjusting pH to 6.......................... Add remaining components........................................5mg FeCl3·6H2O................................................................................ 0.............2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O................. Adjust pH to 8.........................................................5g CoCl2·6H2O .................. 0..............1...........2g Na2HPO4 ............. 0....................1....5mL Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1.................................. Distribute into tubes or flasks...2.....0mg MgSO4·7H2O............. Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1...........0L..................... Preparation of Medium: Add nitrilotriacetic acid to Preparation of Medium: Add nitrilotriacetic acid to 500.......................... 2...................0.. Mix thoroughly..........................................................................0mL of distilled/deionized water................................. that require reduced nitrogen concentrations....................1g Nitrilotriacetic acid ............025g H2SO4 .....0mL of distilled/deionized water...................................025g Na2MoO4·2H2O .47mg CoCl2·6H2O......................................0L..0L.......... Mix thoroughly........5mL MnSO4·H2O.....................0L............................ 0....5mL pH 7.......................................5 with KOH....................0g NaNO3 ...........0. 0....0............... 25...............0................................86 Castenholz DGN Medium Micronutrient Solution: Composition per liter: MnSO4·H2O ....8.... including thermophilic species.........................103.......................0µg pH 8...2........... 0...........5mL pH 7.. Autoclave for 15 min at 15 psi pressure– 121°C............... Castenholz ND Medium (Medium ND) Composition per liter: Na2HPO4 .. Readjust pH to 8................................................................................0......0mL Micronutrient solution................................................................... Add remaining components.................................................................0L........... Mix thoroughly...............................................1g CaSO4·2H2O......................................... Use: For the isolation of cyanobacteria.......... 2...................................................0mL Micronutrient solution .............................0.. 500......0mg FeCl3 solution.....5 with KOH............................................ 0........140.....................................................28g H3BO3 ................................

......... Preparation of Medium: Aseptically combine 500..............0g Yeast extract........................................... 2... Use: For the cultivation and maintenance of Thermonema lapsum and Thermus species................... 0..................................0mL of sterile 2% TYE................................. Nitsch’s Trace Elements: Composition per liter: MnSO4 ............... 30..... 0... 0.2g H3BO3 ............................5mL Preparation of Nitsch’s Trace Elements: Add components to distilled/deionized water and bring volume to 1...............................0..............................0L...........03% solution) .......0mL pH 7........2 at 25°C Castenholz Salts.....................0................. 2×: Composition per liter: Agar .......0....................4g Na2HPO4 . Autoclave for 15 min at 15 psi pressure– 121°C.............. 0......2g MgSO4·7H2O .................1....................0L... 10..........0mL of sterile Castenholz salts.... 2× .....2.............................................................................. and 400. Caulobacter Medium Composition per liter: Agar ....2 at 25°C Preparation of Medium: Add components to tap water and bring volume to 1................Caulobacter Medium Castenholz TYE Medium (Castenholz Trypticase Yeast Extract Medium) Composition per liter: Castenholz salts....... Mix thoroughly.......................... 2×...12g NaCl .............................. Mix thoroughly......................... Adjust pH to 7................. 2× ..........4g Na2HPO4 ................0L.... 0............0mL of sterile Castenholz salts....... 0......... Distribute into tubes or flasks...............................................................0mL Nitsch’s trace elements .................................0.........................................2..................03% solution) ........2g Riboflavin.................... 0.........................................5g ZnSO4..... 0............. Mix thoroughly....................................................0L............................................... 0....6 ± 0................ .. Gently heat and bring to boiling...0L....... 1.....................................2g Nitrilotriacetic acid .................. Gently heat and bring to boiling............ 100.........................0...................2.................................. Adjust pH to 8..........................................0g Peptone ................. 1% TYE: Composition per liter: Pancreatic digest of casein .......... Gently heat and bring to boiling................0mL Nitsch’s trace elements...........................................................................0mL pH 7...0 ± 0.............................................025g CuSO4 .................0mL 1% TYE ............ 0.................2 at 25°C Castenholz Salts.....500.0g Peptone .......20.....12g 87 NaCl ...... 2....................................... 30.. 2×...........................016g H2SO4 ....0mL 2% TYE ............................................ 0..0g NaNO3 ....................................0g MgSO4·7H2O...0mg pH 7........ 0.............................. 10.............................. Pour into sterile Petri dishes or leave in tubes... Gently heat and bring to boiling...........................0g Preparation of 1% TYE: Add components to distilled/ deionized water and bring volume to 1.. Pour into sterile Petri dishes or leave in tubes..............................10............................................ 1..........................................5g CoCl2·6H2O ...................... 0....................... Castenholz TYE Medium with 2% Trypticase Yeast Extract Composition per liter: Castenholz salts....................6........0.......................................... Adjust pH to 7...........................20.....21g Nitrilotriacetic acid ........................... Autoclave for 15 min at 15 psi pressure–121°C.............0 ± 0............................................100.......0L........................................................................................................... Mix thoroughly.......................016g H2SO4 ........................................................5g ZnSO4 . Adjust pH to 8...........................................................2g CaSO4·2H2O ....................................................10.... Autoclave for 15 min at 15 psi pressure–121°C......025g CuSO4 .............. 0.......... Use: For the cultivation and maintenance of Thermus species............................. Nitsch’s Trace Elements: Composition per liter: MnSO4 .5mL Preparation of Nitsch’s Trace Elements: Add components to distilled/deionized water and bring volume to 1.....0........ 2×: Composition per liter: Agar ............................0g Yeast extract ...1.................0g NaNO3 ............... 0........2..............046g Na2MoO4 ............................................................................... Distribute into tubes or flasks................... 2×: Add components to distilled/deionized water and bring volume to 1...................... Autoclave for 15 min at 15 psi pressure–121°C...0mL of sterile 1% TYE......016g FeCl3 solution (0.............................016g FeCl3 (0....0mL Preparation of Castenholz Salts....0L pH 7..... 100..0mL of sterile distilled/deionized water................................... Autoclave for 15 min at 15 psi pressure–121°C.................. Use: For the cultivation of Caulobacter species from marine isolates................... Mix thoroughly........2g H3BO3 ....... 0.....0..........................2...0L............500................5g CoCl2·6H2O.................................................. Autoclave for 15 min at 15 psi pressure–121°C............................22g KNO3 ...046g Na2MoO4..................... 0.................... Use: For the cultivation of Caulobacter species from fresh water...... and 400............. 2% TYE: Composition per liter: Pancreatic digest of casein ......... 2×: Add components to distilled/deionized water and bring volume to 1.............. Caulobacter Medium Composition per liter: Agar ..............0......100.........2............... Mix thoroughly... 1.............0g Preparation of 2% TYE: Add components to distilled/ deionized water and bring volume to 1............................................ filtered.....21g MgSO4·7H2O ............. Mix thoroughly.6 ± 0......................................22g KNO3 ..6.5g Seawater........................... 2...............................................0g Yeast extract ....0mL of sterile distilled/deionized water......... Mix thoroughly............. Preparation of Medium: Aseptically combine 500............................2 at 25°C Preparation of Medium: Combine components....0mL Preparation of Castenholz Salts................2g CaSO4·2H2O .........................

............................... 0.. 0............. 9.............0mL. Adjust pH to 6..02mg Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 100.................2mg (NH4)6Mo7O24·7H2O .... Distribute into tubes or flasks... Distribute into tubes or flasks..................0mL.............. 1..0g Peptone......................... Use: For the selective isolation and cultivation of Legionella species from environmental samples.....20........................................................................................................... Preparation of BCYE-Alpha Base: Add components.... Mix thoroughly.........................0mL...9 ± 0..................................................0mL: L-Cysteine·HCl·H2O.............................0g Yeast extract................. Distribute into tubes or flasks................................... 0... 2............................. Adjust pH to 7..... Pour into sterile Petri dishes or leave in tubes.................................................. Filter sterilize........................ Pedomicrobium species... Mix thoroughly...................10............... CCVC Medium (Cephalothin Cycloheximide Vancomycin Colistin Medium Composition per liter: BCYE-alpha base ...................................................0L........................................................................... Adjust medium to pH 6........0mL Salt Solution: Composition per 100...................... Heat gently and bring to boiling for 1 min..0g Yeast extract.............................066g MgSO4 ..............3mg NaBO3·4H2O...............0mL: EDTA .................. Add 4.. Use: For the enrichment of Stella species from polluted waters...........5mg Preparation of Antibiotic Supplement Solution: Add components to 10...... Mix thoroughly...... Pseudomonas carboxydohydrogena...... 990.................. Preparation of Medium: To cooled BCYE-alpha base...........................0g α-Ketoglutarate ........ Antibiotic Supplement Solution: Composition per 10................0mL of L-cysteine·HCl·H2O solution................................. Adjust pH to 7.4g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.......................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................... BCYE-Alpha Base: Composition per liter: Agar ...2.............4.63mg MgCl2·4H2O............................. Autoclave for 15 min at 15 psi pressure–121°C............0g Peptone.....1......... 0.......... Autoclave for 15 min at 15 psi pressure–121°C.....................0g Yeast extract ................0mL: Cycloheximide ...22mg CuSO4·5H2O .......4 at 25°C Preparation of Medium: Add components to filtered aged seawater and bring volume to 1...... Cool to 50°–55°C....... 0. Autoclave for 15 min at 15 psi pressure–121°C..........15.. 1.16. Filter sterilize....... 0........... Mix thoroughly..............65g ............................ 1.....................................3..................... Caulobacter species...1.....0g Salt solution ..........033g FeSO4·7H2O........ Caulobacter Medium with Riboflavin Composition per liter: Peptone............0mg Cephalothin .........0mL pH 6..................................................1...................................0g Riboflavin. 10...0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid).............................81mg CaCl2 . 1........0L........... 1....0L...............0L pH 7............... activated .......................... Gently heat to boiling.................................. Use: For the cultivation of Caulobacter vibrioides....... to distilled/deionized water and bring volume to 990.......0... 0.................. Distribute into tubes or flasks.0g K2HPO4 .....................88 Caulobacter Medium Caulobacter Medium Composition per liter: Glucose ....... Use: For the cultivation and maintenance of Asticcacaulis excentricus........................9 with sterile 1N KOH................................100....0L pH 7.......................... 1......0mg Colistin . Caulobacter Medium II Composition per liter: Peptone..............4 at 25°C Preparation of Medium: Add components to filtered aged seawater and bring volume to 1..........80.......0g NH4Cl ..0g Yeast extract... Mix thoroughly...0mg Vancomycin ................................................2–7.....................0mL L-Cysteine·HCl·H2O Solution: Composition per 10...1g KNO3 ..2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems............ Mix thoroughly...0.......................... 10.............0g Fe4(P2O7)3·9H2O ....................... 10........... add 10.......0............................... 1.................25g L-Cysteine·HCl·H2O solution ..... Caulobacter Medium Composition per liter: Agar ....................... and Stella species.......... Autoclave for 15 min at 15 psi pressure–121°C...................................................... Mix thoroughly....0mL Antibiotic supplement ......................0g Yeast extract...................0g MgSO4·7H2O ....................................0mg Seawater . Mix thoroughly............................... 2................................... Use: For the cultivation of Caulobacter halobacteroides and Caulobacter maris.....................0g Charcoal.........................0mL of distilled/deionized water............................... 10......2–7......................... 3...................................... Autoclave for 15 min at 15 psi pressure–121°C.........0mg ZnSO4·7H2O ...........................4...........10..................................2................................................................................... 10................... Pirellula staleyi............9 with 1N KOH.......0g Seawater.... 3... Labrys monachus............. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension........................................ 0..............079mg Co(NO3)2·H2O ..................1.................................2–7...............................................0mL sterile antibiotic supplement..........................2g Preparation of Medium: Add components to tap water and bring volume to 1................. Mix thoroughly.......................... Cellulolytic Agar with Sea Salts Composition per liter: Agar ............................. 1...............................0L........................1g K2HPO4 ....................................... except L-cysteine·HCl·H2O solution...........................................

.......................6g L-Cysteine·HCl·H2O ........................................................................... Adjust pH to 7.......................................................0mg Thiamine·HCl ...........18g (NH4)2SO4 .................................................0L................ 0......0mL Mineral solution ...........0mL pH 7............................... Mix thoroughly....................0...........................................0cm) of Whatman No.... Add components to distilled/deionized water and bring volume to 1.......................1% solution) .5g CaCl2 ......0mL pH 7..5................................................Cellulolytic Medium Yeast extract.........0mg CoCl2·H2O. 1 filter paper..... Mineral Solution: Composition per liter: NaCl ............... Distribute into tubes or flasks..........................................................................................................................................2 ± 0. Adjust pH to 7......................1................................................................ Whatman CF11 ............................0g Preparation of Cellulose Suspension: Add cellulose powder to 200...........65g NH4SO4 ...........................................0mg ZnSO4·7H2O..........................0..................6..................................................................................................................................... 0......0g CaCl2 ..........................................................0g CaCl2 ....5g Cellulose suspension.............................................................0mL: Cellulose powder.......................................................5g 89 Filtered seawater ..............................0mL Mineral solution ..................2 with 5m NaOH..................0............ 100..........0g (NH4)2SO4 ..............0g NH4Cl ...........................................2.0mg CuSO4·5H2O..... 8...........................................1mg KAl(SO4)2·12H2O .................... Add components to distilled/ deionized water and bring volume to 1....0L...................0mg Biotin ....................06g Cellulose.............................2 at 25°C Cellulose Suspension: Composition per 200........0g Yeast extract........6g Preparation of Mineral Solution: Add components to distilled/deionized water and bring volume to 1..................................0mg FeSO4·7H2O .............................1.296g KH2PO4 ................ 30....0mg Resazurin ...........................0......................................0mL Resazurin (0.....12g CaCl2·2H2O .......... 1.............5..................................................................1........................................96g L-Cysteine·HCl·H2O ..1% solution)....0.... Autoclave for 15 min at 15 psi pressure– 121°C....... 1................5............................................... 0............................................. 0...............0mg Thioctic acid .........5.......2 with 5M NaOH.............. 0.........................................21......... Cellulolytic Agar for Thermophiles Composition per liter: Agar ................ 6......0g K2HPO4 ...0g Preparation of Cellulose Suspension: Add cellulose powder to 200...........68g K2HPO4 ...................................................2 at 25°C Mineral Solution: Composition per liter: NaCl ...................1mg Wolfe’s vitamin solution .....0mg Riboflavin............. 1............................. Preparation of Medium: Prepare and dispense medium anaerobically in 100% N2..........................................0mg Folic acid ...........................................150.2.......0............................0mL pH 7...... Preparation of Medium: Prepare and dispense medium anaerobically under 100% N2. Adjust pH to 7...................... 0.....................1mg Na2MoO4·2H2O..... 0.......................0L..............5..... 0...................0mg p-Aminobenzoic acid ................................................0g (NH4)2SO4 ...................61............6g Preparation of Mineral Solution: Add components to distilled/deionized water and bring volume to 1..1mg H3BO3 ........... . Cellulolytic Medium Composition per liter: NaHCO3 ..................................................................6g MgSO4 ...... 200...................1% solution). Add components to distilled/ deionized water and bring volume to 1...........0mL pH 7........0...........10.....0L........................ 150.......... Whatman CF11 ...... Mix thoroughly...................................0mL of distilled/deionized water and mix thoroughly.....................................6........ Use: For the cultivation and maintenance of Clostridium papyrosolvens and other marine bacteria that can utilize cellulose as a carbon source.........2 with 5m NaOH.. Autoclave for 15 min at 15 psi pressure–121°C....0mg Cyanocobalamin.................0mL Resazurin (0.............................0......................................200..................................2 at 25°C Cellulose Suspension: Composition per 200............ Autoclave for 15 min at 15 psi pressure–121°C.....................0................................ Filter sterilize.....................................................................0mL L-Cysteine·HCl·H2O solution ....................15g MgSO4·7H2O...............10...............0............................... 1. 1...... 5...................................................................... Distribute into tubes or flasks that contain cellulose as a strip (4.......................................0g NaCl ................................ Preparation of Medium: Prepare and dispense medium anaerobically in 100% N2 atmosphere..5cm × 1................. 1.......6g MgSO4 ..............0mL Resazurin (0...................................0mg Calcium DL-pantothenate ............... Distribute into tubes or flasks.................................................................................................................................................65g NH4Cl............................5........... 0...15..........096g MgSO4 ........................................................................... Cellulolytic Broth with Sea Salts Composition per liter: K2HPO4 .............0µg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...................................0L.......................200...............5 ± 0................ Mix thoroughly...........0mg NaCl ...0mL: Cellulose powder..................................0................................6g Yeast extract.....5.......... 1.............. Mix thoroughly....0mg MnSO4·H2O ....1...2 ± 0....0mL Filtered seawater .........................................0mg Nicotinic acid .......................0mg Nitrilotriacetic acid....................2 ± 0..................................................1. Use: For cultivation of Clostridium stercorarium and other bacteria that can utilize cellulose as a carbon source.......200...2.....2 at 25°C Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl.......... 6.... Mix thoroughly. Use: For the cultivation and maintenance of Clostridium papyrosolvens and other marine bacteria that can utilize cellulose as a carbon source...... 8.0L.........................0mL of distilled/deionized water and mix thoroughly...........096g Cellulose suspension..2g L-Cysteine·HCl·H2O ...............................2................................................. 10.................................

........................................................................................................0mL.................9g (NH4)2SO4 .................................................5...... Aseptically add 10. NaHCO3 solution.2......... Use: For the cultivation of cellulolytic bacteria........................ 0.........0mL........25g Na2S·9H2O................................ Cool slightly.............. 5..........0mL........................................ Aseptically and anaerobically add 50......... 10..................................8....................... 1...........25.............8g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10........25g Preparation of Alkaline Solution: Add components to 25.............. and L-cysteine·HCl solution............ except Wolfe’s vitamin solution and L-cysteine·HCl·H2O solution........................................................0mL NaHCO3 solution.................... Mix thoroughly...0mL pH 6................................1g Cellulose...... Preparation of Medium: Add components................5g (NH4)2SO4 ................ 5.45g KH2PO4 ................................0g NaCl ....................... 1... 0........... 0........................... Heat to boiling and cool to 25°C.......... NaHCO3 Solution: Composition per 10.......0mL: NaHCO3 ........................... Preparation of Medium: Add components.........90 Cellulolytic Medium with Rumen Fluid and Soluble Starch L-Cysteine·HCl·H2O Solution: Composition per 20.............0mL...0......................... 15.... 1........................0mL of freshly prepared alkaline solution......0mL of sterile cellobiose solution....... Cellulomonas fermentans Medium Composition per liter: Yeast extract.................... Aseptically distribute into sterile tubes or flasks................... 5.........................5.0mL: D-Cellobiose (or cellulose)............ Use: For the cultivation and maintenance of Cellulomonas species...................... 0...........................................................0mL: Agar ......... Alkaline Solution: Composition per 25........... .......................................... 6............. 0................25mg Resazurin ......... L-Cysteine·HCl Solution: Composition per 10........................37g Pancreatic digest of casein ............................0mL of sterile Wolfe’s vitamin solution and 5......5....... Distribute into tubes using anaerobic techniques under a gas phase of 98% CO2 + 2% H2................................. 5...................02g FeSO4·7H2O ... Cellulolytic Medium with Rumen Fluid and Soluble Starch Composition per liter: Basal medium ............... Gently heat and bring to boiling under a gas phase of 98% CO2 + 2% H2...................18g CaCl2 ...8 ± 0..........0mL of basal medium....0.......0g Yeast extract ...975....0mL of sterile L-cysteine·HCl solution......................... Filter sterilize..0mg Cellobiose solution ........ Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.................... Preparation of Medium: Prepare 975........ Mix thoroughly....0g Glucose.....1g CaCl2·2H2O..............................2 at 25°C Basal Medium: Composition per 975....................5..................0g NH4Cl ..... Pour into sterile Petri dishes or leave in tubes.........................................................................................0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 20...... Distribute into tubes or flasks.......5g Preparation of L-Cysteine·HCl Solution: Add Lcysteine·HCl to distilled/deionized water and bring volume to 10..........0mL Alkaline solution.........................0g Preparation of Cellobiose Solution: Add cellobiose (or cellulose) to distilled/deionized water and bring volume to 50.......................... Cellulose Anaerobe Medium (LMG Medium 94) Composition per liter: NaHCO3 .........0g Soluble starch.................... Mix thoroughly.... Adjust pH to 6...............................3g MgCl2·6H2O......................... 0....................................... Mix thoroughly.0mg Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 975. Autoclave for 15 min at 15 psi pressure–121°C...18g ....... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.. 10......... to distilled/deionized water and bring volume to 930.0.......1.. Prepare freshly........ Mix thoroughly............9g K2HPO4 ................0mL of sterile L-cysteine·HCl·H2O solution.............................. Mix thoroughly...............................0mL..2 at 25°C Cellobiose Solution: Composition per 50..... 10....................................................................... Mix thoroughly................2..........................................5...........0mL of distilled/deionized water...... Mix thoroughly....0mL: L-Cysteine·HCl ...........1...................0.......0mL.......................0g Peptone ....0mL L-Cysteine·HCl solution ........................15..................... 0.................0L.. Autoclave for 15 min at 15 psi pressure–121°C..0g K2HPO4 ...............2....... Use: For the cultivation of Selenomonas ruminantium and Succinimonas amylolytica...... and 10.................... Sparge under 100% N2 for 10 min.. Add 25.............0g Cellobiose .0g NaHCO3 ..... Use: For the cultivation and maintenance of Cellulomonas fermentans......0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............0mL: L-Cysteine·HCl·H2O ............ Autoclave for 15 min at 15 psi pressure– 121°C.......................4 ± 0....21g KH2PO4 .......................................... except cel- lobiose solution......68g KH2PO4 ........................................0mL.. 0.......................... Mix thoroughly............0mL of sterile NaHCO3 solution.... Mix thoroughly........................................................................ Gently heat and bring to boiling.............45g MgSO4·7H2O .............................0g Pancreatic digest of casein .................................................... to distilled/deionized water and bring volume to 985............0mL: L-Cysteine·HCl·H2O ................... 0............... Mix thoroughly.........0mL pH 7................... Cellulomonas PTYG Medium (Cellulomonas Peptone Tryptone Yeast Extract Glucose Medium) Composition per liter: Agar ................ Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.........09g Resazurin ..... 50.......... 0.............. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.............. 1............

.... 21............................................................ 650............. Cool to 45°–50°C.....................................0mg p-Aminobenzoic acid ................................... powdered.....0...................0mL....0mg Resazurin ................. 10.................... 61....................02g KBr ......0mL Solution A: Composition per 350.....................0mL: CaCl2·2H2O ...................................................... 5...0mg Riboflavin ...............................................................01g NaMoO4·2H2O .......0mg Biotin ..........5......... Preparation of Stan 5 Agar: Aseptically combine 350...........1........................................................1........ Cool to 45°–50°C.....................................5.......................................Cellulose Overlay Agar (NH4)2SO4 ........................... 0...................0mg SnCl2·2H2O ............ Cool to 50°C..........5 at 25°C Preparation of Medium: Add cellulose to 100...... Mix thoroughly..................0mL Solution A: Composition per 350........ 2............ 0... 0......0mL of distilled/deionized water. L-Cysteine Solution: Composition per 10.......................................................................................................2g CaCl2·2H2O.........................0g K2HPO4 .......................0mg LiCl.............. and Flexithrix species.......... 2.0mL sterile cysteine hydrochloride solution.. Mix thoroughly....................................................................................0mL of cooled........................0..................................... Autoclave for 15 min at 15 psi pressure–121°C..................... 5...................................................8...........................................0mL of cooled............ Autoclave for 15 min at 15 psi pressure–121°C.. 5........0mL..................................... 0................................ 0.............................. In a separate flask......... Adjust pH to 6..............................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.................. 350................................................... 5... Cool to 45°–50°C.....0mg CaCl2·2H2O................. Solution B: Composition per 650.. 0......................................................0mL Cysteine hydrochloride solution .......... 1..............0mg Thiamine·HCl ....... Mix thoroughly............................................... 0......... Autoclave for 15 min at 15 psi pressure–121°C.... Aseptically add 10.................. 0..................5....0mL: L-Cysteine·HCl·H2O ................................ Adjust pH to 7....... Cellulose Overlay Agar: Composition per liter: Solution A .......................... 1...............0g (NH4)2SO4 ...................0mL: CaCl2·2H2O ....................... Preparation of Medium: Add nitrilotriacetic acid to 500................................ Mix thoroughly............................0mL Cellulose overlay agar ......................... Aseptically distribute into sterile tubes or flasks.......... Mix thoroughly.....................................................................................0mg ZnSO4·7H2O ............. 1.......................................02g CuSO4 .....................................................................................0g Preparation of Solution B: Add components to distilled/deionized water and bring volume to 650...................................01g BaCl2 ........1 ± 0....... Mix thoroughly..0mg CoCl2·6H2O ............1mg Vitamin solution...........2 at 25°C Vitamin Solution: Composition per liter: Pyridoxine·HCl ..... 1.............................. 5.......................................... Gently heat and bring to boiling..0mL ...................1. 1.................................... Mix thoroughly...... Aseptically combine the two sterile solutions.12g K2HPO4 ........................ 1.........................................1.0... 350...........0mg MnSO4·H2O ........................5 with KOH...0g MnCl2·4H2O .....0mg Folic acid.......................................0g MgSO4·7H2O..................0g MgSO4·7H2O ......................................................0L...............0..........................................................................1.................................5g Preparation of L-Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10....... Gently heat and bring to boiling...................................0..... sterile solution B......................1........0mg NaCl ................................................ Mix thoroughly.0.............................. 15.1mg KAl(SO4)2·12H2O......................0mL distilled/deionized water..... 1.....................10.............0mL Solution B............................0mg FeSO4·7H2O................................. Saprospira species.......................................... Autoclave for 15 min at 15 psi pressure–121°C...................... 296.. add remaining components to distilled/deionized water and bring volume to 900............... 0..0mL.................................... Mix thoroughly.......................0g Trace elements solution. 5........0mg Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 1......0mg Lipoic acid ........ 15...............0mL: Agar ............0mL Solution A ............ Trace Elements Solution: Composition per liter: EDTA....................0mg CuSO4·5H2O .................................... 5.....02g pH 7....................0mL pH 7............. Auto- 91 clave both solutions separately for 15 min at 15 psi pressure–121°C.0L.............1mg Na2MoO4·2H2O ..........................................................1g CoCl2 ............0mg Nitrilotriacetic acid ................1.............1g FeCl3 .......0mL Preparation of Solution A: Add components to distilled/deionized water and bring volume to 350...........................0g (NH4)2SO4 ................... Use: For the cultivation of cellulose-utilizing anerobic bacteria.......01g H3BO3 ...........02g ZnCl2 ............1mg H3BO3 ........................................ Add 485.5....................................0g K2HPO4 ......................... 0...................... 650...... 5..............................1.............. Cellulose Broth Composition per liter: Cellulose....0................... sterile solution A and 650...........................0mL of distilled/deionized water...............................0g Trace elements solution............................0mL of sterile vitamin solution and 5.........................0g (NH4)2SO4 ............................. Add remaining components except vitamin solution and cysteine hydrochloride solution. Sparge with 100% N2..........15g MgSO4·7H2O ...... Cellulose Overlay Agar Composition per plate: Stan 5 agar ... 10.....0mg Nicotinic acid ..............................................0mL............. Herpetosiphon species................................................. Use: For the isolation and cultivation of Cytophaga spe- cies.. 0.......... Filter sterilize.......................................................................0mg Calcium DL-pantothenate....... 1............0mL Stan 5 Agar: Composition per liter: Solution B......0g MgSO4·7H2O.......0mg Vitamin B12 ...............0–7.............. Mix thoroughly.5........ Aeptically distribute to tubes or flasks............................................

.............. 10............ Use: For the cultivation of Rhodocista centenaria=Rhodospirillum centenum.....................................0mg NaVO3·H2O .................... Cytophaga hutchinsonii.................................. 0.....92 Cellvibrio Medium Preparation of Solution A: Add components to distilled/deionized water and bring volume to 350..................0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.......01g BaCl2 ...............1g Na2MoO4·4H2O..............................0g Yeast extract ...... Mix thoroughly........... Mix thoroughly.....................02g CuSO4 . Cool to 45°–50°C.............................. 0...............0g HCl .......... Cool to 25°C.......................................2g K2HPO4 ...........................0...................0mL Preparation of Chelated Iron Solution: Add compo- nents to distilled/deionized water and bring volume to 900......9g KH2PO4 . 1........0mg LiCl .................0g K2HPO4 .0g MgSO4·7H2O ..................... 5.................... 1.........................................01g H3BO3 ....................................................................0mL with additional distilled/deionized water............2......1......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.... 5................0L....................0g NaNO3 ..............5g MgSO4·7H2O ...................... 10....................................................................................... Trace Elements Solution: Composition per liter: EDTA ................ 0........................................................................2......................0–7....0mL Chelated iron solution .....5..0................ Preparation of Medium: Add components...............................................0.................................. Sparge with 100% N2.............. 20....................6g Na2S2O3·5H2O ....50..0g Preparation of Solution B: Add components to distilled/deionized water and bring volume to 650...0mL of cooled......10......... 5.0mg CoCl2·2H2O.......................................0g MgSO4 .........................0............................ Pour into sterile Petri dishes or leave in tubes.............75.................... Autoclave for 15 min at 15 psi pressure–121°C.............. Distribute into tubes or flasks.....................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................... Mix thoroughly............ Mix thoroughly.............. 4...50............................ Chelated Iron Solution: Composition per 900mL: EDTA..........0mL sterile Na-pyruvate solution............................ Filter sterilize.................................. 0...............................2....................0g MnCl2·4H2O....................... sterile solution A and 650................................... Autoclave for 15 min at 15 psi pressure–121°C....... Mix thoroughly............................................................. 0..................................................................................0mL of cooled...........2 at 25°C Na-pyruvate Solution: Composition per 10........................................... and Sporocytophaga myxococcoides..............................0 ± 0................................................................................0L... 0....10....... Distribute into tubes or flasks.............................................................................................. 0..............0mL................. 0.....................................................0mL distilled/deionized water...............................1g ZnCl2 ........... Autoclave for 15 min at 15 psi pressure–121°C..............................2 ± 0...0mg CuCl2·2H2O...............................20............................................... Aseptically add 10.. 0............ except Na- pyruvate solution...........................0mg Na2SeO3....................................................0mg pH 7....................2g Preparation of Na-pyruvate Solution: Add Na-pyruvate to distilled/deionized water and bring volume to 10............................ 10..0g K2HPO4 ...................0µg Na-pyruvate solution .............................................. Cool to 45°–50°C.............................1...................5.... 2............. Overlay each plate with 5.... True Blue Trace Elements: Composition per 250mL: EDTA......................... Mix thoroughly.... Preparation of Cellulose Overlay Agar: Aseptically combine 350.................................... Mix thoroughly... 3.........1g CoCl2 ..................... 5...........................02g ZnCl2 ........ sterile solution B..............20.... 0...........0mL volumes............... to distilled/deionized water and bring volume to 990........... Cytophaga aurantiaca.........0mg SnCl2·2H2O........................5.................. Mix thoroughly.... CENS Medium (DSMZ Medium 748) Composition per liter: Soytone ................................... Centenum Medium Composition per liter of tap water: Agar .0µg Biotin .......................................... Gently heat and bring to boiling.................. Gently heat and bring to boiling. sterile Stan 5 agar into sterile Petri dishes in 15................................................ 1....... 0.. Allow agar to solidify. .................................................. Preparation of Medium: Pour cooled.........................................02mg pH 7................ 15.................5g Vitamin B12.......................0g FeCl2·4H2O.................................................... Autoclave for 15 min at 15 psi pressure–121°C.0mL....0g Sodium pyruvate ...........0mg Vitamin B12.....................0L.................................................0mL... Bring final volume to 250.................................................. Gently heat and bring to boiling.........0mL True Blue Trace elements.....................0mL.... Cellvibrio Medium Composition per liter: Agar ............................... Aseptically distribute into sterile tubes or flasks.......2g CaCl2·2H2O ...............0mg Preparation of True Blue Trace Elements: Add components one at a time to approximately 200....2g H3BO3 ........0mg NiCl2·6H2O............ 15.........01g NaMoO4·2H2O.. Use: For the cultivation of myxobacteria.....5g KCl......0mL of cellulose overlay agar................................................................................................................0mg EDTA............................ Pour into sterile Petri dishes or leave in tubes..............5g MnCl2·4H2O ..............................5g FeSO4·7H2O.................... 1........0..............0mL pH 7................................................. Use: For the cultivation and maintenance of Cellvibrio mixtus...0mL: Na-pyruvate ........0g NH4Cl.............0g K2HPO4 ... 8..... 0....2.............0L..02g KBr................... 0................... Solution B: Composition per 650.............. Autoclave for 15 min at 15 psi pressure–121°C.........................0mL: Agar .. Mix thoroughly................. Gently heat and bring to boiling............

.0mL of cold concentrated HCl..0L................ Pour into sterile Petri dishes or leave in tubes............................................................. Use: For the isolation and cultivation of Cytophaga spe- cies. nonfermentative bacteria..0mL: (NH4)2SO4 .....................01g MnCl2·4H2O . and identifica- tion of Pseudomonas aeruginosa and other Gram-negative................................................1.......... 0...0g Tryptose ................................................ concentrated.................................... 1. Filter the solution through Whatman #1 filter paper..... Cetrimide Agar..01g Na2MoO2·2H2O ..................0mg Chitin.......................................... Gently heat and bring to boiling..............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....0g Agar .......0g MgSO4·7H2O ...........0L of distilled/deionized water at 5°C................... Mix thoroughly..... Pour into sterile Petri dishes or leave in tubes.................................................................. Mix thoroughly........ Mix thoroughly...0.......................................................................2 ± 0.................01g ZnSO4·7H2O .............................0g Chitin.9g pH 7......................... 0..................... Non-USP Composition per liter: Beef heart...0g (NH4)2SO4 .............................. 20.................................................. Add this solution to 2.................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...... Use: For the selective isolation and cultivation of streptomycetes............ Pour into sterile Petri dishes or leave in tubes...............5g Cyclohexane carboxylic acid ...............................5g ........ 0........7g MgSO4·7H2O..........001g pH 8..... Distribute into tubes or flasks......................... Autoclave for 15 min at 15 psi pressure–121°C.....3............. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....6g K2SO4 .. precipitated .. Pour into sterile Petri dishes or leave in tubes...................... 5....1g Yeast extract.....................0g Na2HPO4 .......... 13..........................3g Glycerol .......0..............................0L.............................. Pour into sterile Petri dishes or leave in tubes......................................................4g Cetrimide ................................................ 2....0g NH4NO3 ......... anhydrous................................10...... Mix thoroughly.0................. For the cultivation and maintenance of Arthrobacter globiformis......5g KH2PO4 ............................ cultivation.....0.................................0.....................0.................0g Sodium acetate........ Autoclave for 20 min at 15 psi pressure–121°C................0L.............. Mix thoroughly...5L: Chitin .... Saprospira species................ Autoclave for 15 min at 13 psi pressure–118°C...2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................2 ± 0..5g FeSO4·7H2O. Gently heat and bring to boiling..0.. Use: For the cultivation of Chlamydomonas reinhardtii...............................0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................................... Precipitated: Composition per 2................................................... cultivation.................................................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems.0 ± 0......................15..................... 10............ and Flexithrix species....... 1.....0g Chitin .......................... CHCA Salts Medium (Cyclohexane Carboxylic Acid Salts Medium) Composition per liter: K2HPO4 ...................................... 500......0g K2HPO4 ............... solids from infusion................................ Autoclave for 15 min at 15 psi pressure–121°C... Mix thoroughly................ Distribute into tubes or flasks.......... Autoclave for 15 min at 15 psi pressure–121°C.....1g CaCl2·2H2O.............. Dialyze the precipitated chitin against tap water for 12 hr...........0mL pH 7.....................................40................... and identifica- tion of Pseudomonas aeruginosa and other Gram-negative..2g FeSO4 ..................................................001g ZnSO4·7H2O................................... 0............ 10...0mg MnSO4 ................. 1........... 15.. 0............................................................................... Gently heat and bring to boiling...................................... Distribute into tubes or flasks.0............................ Adjust pH to 7........................... Gently heat and bring to boiling....0....1...........0 with KOH............................................. 0....2........ Herpetosiphon species...... 2.......................0g NaCl ..................4............................. nonfermentative bacteria..... 93 Chitin Agar Composition per liter: Agar ... Gently heat and bring to boiling.............1g KH2PO4 ...........0g Tryptose.......... USP (Pseudosel® Agar) Composition per liter: Pancreatic digest of gelatin ..........................0g HCl..... Use: For the selective isolation.....20...0mL Preparation of Chitin......................01g pH 7........0L..............................................................................................................0 ± 0..................7g MgSO4·7H2O......0L.......................Chlorinated Fatty Acid Medium Use: For the cultivation and maintenance of Rhodospirillum species.........................5g KH2PO4 ......................... Autoclave for 15 min at 13 psi pressure–118°C............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.. Chlorinated Fatty Acid Medium Composition per 1001..........0L.......3g FeSO4·7H2O ....... 400.. Cetrimide Agar............................................1..................................0g Yeast extract ............................. Distribute into tubes or flasks.............. Chitin Agar Composition per liter: Agar ......... 3....... Pour into sterile Petri dishes or leave in tubes...0g Cetrimide .......0g MgCl2 ...... Gently heat and bring to boiling. Adjust the pH to 7..........................................2........................0g Agar .............. 1...................... Distribute into tubes or flasks......... 0........................15....... Use: For the cultivation and maintenance of bacteria that can utilize cyclohexane carboxylic acid as a carbon source..........0. Distribute into tubes or flasks................................... Use: For the selective isolation....... Precipitated: Add chitin to 400......... Chlamydomonas Enriched Medium Composition per liter: Agar ............ 0.......................

...07g H3BO3 .........................................0......................................................... Autoclave for 15 min at 15 psi pressure–121°C.................................0g Co(NO3)2·6H2O ..................0mL H2O ...................................... 60.................... Tightly close cap... Distribute into tubes or flasks....19g MnCl2·4H2O ...............................1................................................................................................................... 0.......5g CoCl2·6H2O................................ Chlorobiaceae Medium 1 Composition per 4990...0L of sterile.....0mg Preparation of Medium: To 4.......................8 ± 0...1g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water.............................. Mix thoroughly.. Saturate with CO2 by stirring under 100% CO2 for 30 min.......... 1............ Aseptically distribute into sterile 100.......................................................... 2....7g CaCl2·2H2O................. Preparation of Medium: Combine components....................................7g NH4Cl.......................... CO2-satu- rated solution 1.. 0.............. 6.........................0g MnSO4·H2O ......................0L O2-free water...0..0mL Trace elements solution .....................................05g Trace elements solution SL-8.............................5..0mL NaHCO3 solution ............1g Ca(NO3)2 ....................................0L........................................ Autoclave for 15 min at 15 psi pressure–121°C......................................0g Disodium EDTA......2 ± 0................0mL NaHCO3 solution...............2 at 25°C Trace Elements Solution: Composition per liter: FeSO4·7H2O...... 7...8............................................. 1... Use: For the isolation and cultivation of members of the Chlorobiaceae....04g CuCl2·2H2O.......................... Autoclave for 15 min at 15 psi pressure–121°C.................................................................. Autoclave for 45 min at 15 psi pressure–121°C...1...........0mL pH 6...........................................................................1g ZnCl2 .......... Filter sterilize with positive CO2 pressure..............0................0mL Na2S·9H2O solution........0.............................5mL Preparation of Trace Elements Solution: Add com- Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 1............1g ZnCl2 ......0. Vitamin B12.....0mL: Solution 1 ........................................1g NiSO4·6H2O..............................................................06g NaMoO4·2H2O ....0....0mL........... 40..... Cool to 25°C under 100% N2.......................04g CuCl2·2H2O............ 0.. 1.............. Cool to 45°–50°C. Na2S·9H2O Solution: Composition per 100...............860............... 0....................0.. 1............0mL: KH2PO4 ... Mix thoroughly........0mL Preparation of Solution 1: Add components to dis- Preparation of O2-Free Water: Autoclave H2O for 15 min at 15 psi pressure–121°C.....0g NH4Cl ...0mL.........................02g HCl (25% solution) .............1.................... 950.....075g 2-Chloropropionate .................... Chlorobiaceae Medium 2 Composition per 1051..................................4g CaCl2·2H2O .........8 ± 0....................... Mix thoroughly..94 Chlorobiaceae Medium 1 MgSO4·7H2O ............. 10.............................1....0mL: Na2S·9H2O........ tilled/deionized water and bring volume to 950.......................................... 0...860..............................................7g KH2PO4 .. 0.......5g KCl... Trace Elements Solution: Composition per liter: FeCl2·4H2O.........0..............................02g NiCl2·6H20 ........0....................25g Preparation of Solution 1: Add components to dis- tilled/deionized water and bring volume to 4...........................................2................................. Mix thoroughly..07g H3BO3 .......0mL pH 6.................................. Filter sterilize............. aseptically add the remaining components................. 1.................................25g H3BO3 ................. 1....................2 at 25°C Solution 1: Composition per 4...................................................0mL Vitamin B12 solution ... ponents to distilled/deionized water and bring volume to 1....................................................0mL: Solution 1 ................... Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 100.............................5....................................0mL bottles using positive pressure of 95% N2 + 5% CO2...............................2g FeCl2·4H2O............ Mix thoroughly...0..............0L: MgSO4·7H2O .......................................25g Na2MoO4·2H2O ..... 0.........0mL pH 7...............0....................... NaHCO3 Solution: Composition per 100...............0L Trace elements solution ............ Vitamin B12 Solution: Composition per 100...4..........................................................................5g MgSO4·7H2O.......0... pH 7..........0mL: Use: For the cultivation of Alcaligenes species. 0............................................................ Completely fill bottles with medium except for a pea-sized air bubble..................................................0.............................................06g NaMoO4·2H2O ..... 0...................0mL: Trace Elements Solution SL-8: Composition per liter: NaHCO3 ................................ Mix thoroughly.......................1...........0.......................................................................................100......... Autoclave for 15 min at 15 psi pressure–121°C.......................................................0L....25g ZnCl2 .54g Phosphate buffer (20mM solution.......................................02g NiCl2·6H20 ........2) ............25g CuCl2·2H2O ........... Gas with 100% CO2 for 20 min............0..................................5g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 100. Cool to 25°C...................0L... Mix thoroughly................................................1g NH4VO3 ........... Cool to 25°C under 100% N2.....02g ...................................... Cool to 25°C.......................... Mix thoroughly........0mL Na2S·9H2O solution ..........0....................0mL... Gas with 100% N2 for 15 min in a screw-capped bottle............0mL Vitamin B12 solution.......5g CoCl2·6H2O.....................................................19g MnCl2·4H2O ....................20...................... Mix thoroughly...................... Adjust pH to 6.............................................. 1..........................................0......5.................. 1....... 0........................................0mL: O2-Free Water: Composition per 860.........2 at 25°C Solution 1: Composition per 950......................................

.......................... aseptically add 60............................0mL of medium...........................0mL: NaHCO3 .....................................0.........0mL of sterile Na2S·9H2O solution.........0.................. and 0......................0.............................0mL of cooled................. Adjust pH to 7....4......... solution B...................0mL.................................... Vitamin B12 Solution: Composition per 100............29g Preparation of FeCl3 Solution: Add FeCl3 to distilled/ deionized water and bring volume to 1......................................0mL....0................................. Mix thoroughly.....................................2–8.......................................0L........ 2..1g NaNO3 .. Mix thoroughly.............................1g Co(NO3)2·6H2O.0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100..............1.............. Distribute into tubes or flasks.2.0–7............... Autoclave for 15 min at 15 psi pressure–121°C....................................0L..................0g Preparation of Solution B: Add Na2S·9H2O to distilled/deionized water and bring volume to 100...............8..........0g MgCl2·6H2O......0mL Trace elements solution .............................. B12 to distilled/deionized water and bring volume to 100.................0mL Solution B ...0mL: Na2S·9H2O.......................0mg 1-Chlorobutane..............0mL Solution C ..............................................................................0mg Yeast extract ...0... 5................ Completely fill bottles with medium except for a pea-sized air bubble.............5.....................................................0 ± 0................ Mix thoroughly......................0L Mix thoroughly....0mL Micronutrient solution........ Aseptically add 0........ Adjust pH to 6..............5......... 1....... Na2S·9H2O Solution: Composition per 100................ 5........ Filter sterilize................ Use: For the cultivation and maintenance of Chlorobium limnicola.........0.................................................................................................... Chloroflexus Agar Composition per liter: Agar ...........0.......2g MgSO4·7H2O..20.... Mix thoroughly.0mg MgSO4·7H2O........ Aseptically distribute into sterile 50........ Mix thoroughly........0mL pH 7... 40....1..........689g Na2HPO4 ..10..................................................................1......0mL: Na2S2O3·9H2O ........0g NH4Cl............. Chlorobutane Medium Composition per 1002......5g Na2S. Mix thoroughly............................ Filter sterilize............... 10.................5g Solution A ......... 2..0.... Mix thoroughly....10..................... sterile solution 1.......... Preparation of Medium: Add components............. Mix thoroughly........ Aseptically distribute into sterile tubes or flasks... to distilled/deionized water and bring volume to 1..............0mg CuSO4·5H2O.....................5.................................................. to distilled/deionized water and bring volume to 1......5g CaSO4·2H2O..0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.........2 at 25°C Use: For the isolation and cultivation of freshwater and soil members of the Chlorobiaceae.... Cool to 45°–50°C.............................. Use: For the cultivation of Corynebacterium species.................................................................................................... Bring pH to 7..0mL of sterile NaHCO3 solution......0mL............................ Mix thoroughly...0mg MnCl2·6H2O ......... Aseptically add 2......................................... 0...........0mg FeSO4·7H2O ..............0mL of sterile Vitamin B12 solution............ Preparation of Medium: Add components...2mL of sterile solution A........................................................0g KH2PO4 ...................... Autoclave for 15 min at 15 psi pressure–121°C..........................................0......................2 with H3PO4... Mix thoroughly...............0.. 1......................................1g ZnSO4·7H2O.................. 10.....................0mg Preparation of Vitamin B12 Solution: Add Vitamin 95 Trace Elements Solution: Composition per liter: FeCl3·6H2O............................0mL: NaHCO3 .. except so- lution A....... Chlorobium thiosulfatophilum Medium Composition per 1050.................0mg FeCl3 solution......20...............................0mL: Na2S·9H2O...10................... 1.............0mL of sterile 1-chlorobutane..........0mL: Preparation of Medium: To 950....0mL: KH2PO4 .......... and 1.......15....0 ± 0........ 0....... Solution C: Composition per 100........................ and solution C.8 with sterile H2SO4 or Na2CO3..Chloroflexus Agar Preparation of Trace Elements Solution SL-8: Add components to distilled/deionized water and bring volume to 1......................06g NaCl .................0mL pH 8................7g H3BO3 .............4 at 25°C FeCl3 Solution: Composition per liter: FeCl3 ................................. 1.............. 0............. NH4NO3.1g Nitrilotriacetic acid..........5.. Autoclave for 15 min at 15 psi pressure–121°C..................2mL of sterile solution B......0mL or 100.....0....0.....0L..............5g Yeast extract ....... Use immediately..0g Preparation of Solution A: Add NaHCO3 to distilled/ deionized water and bring volume to 100......0g Glycyl-glycine ..0mL.......................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100.................................. except 1- chlorobutane...........5g NH4Cl .................................... 10........ Autoclave for 15 min at 15 psi pressure–121°C....... Autoclave for 15 min at 15 psi pressure–121°C....................................................0mL: Vitamin B12 ............1mL of sterile solution C for each 10........................0mL bottles with metal screwcaps and rubber seals....... NaHCO3 Solution: Composition per 100................ Mix thoroughly...................103g CaSO4·2H2O........... .................................................................. Preparation of 1-Chlorobutane: Filter sterilize..............0.. Autoclave for 15 min at 15 psi pressure–121°C.......0mL..2 at 25°C Solution A: Composition per 100.......0mL pH 7.................................... Solution B: Composition per 100..........0mL..50...0L....................... Mix thoroughly......5g Na2HPO4·12H2O .........................0g Preparation of Solution C: Add Na2S2O3·9H2O to distilled/deionized water and bring volume to 100..111g KNO3 ............

................................................. Adjust pH to 8....0........................................0L.............1mg Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 100................. Chloroflexus aggregans Medium (DSMZ Medium 87a) Composition per 1061........5g ZnSO4·7H2O ..... 0..................02g CuCl2·2H2O................ 1......... Do not open the bottle to add H2SO4....1...5mL Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1.......0......................................... Adjust pH to about 7........................................... Autoclave for 15 min at 15 psi pressure–121°C..... 0.............................045g CuSO4·2H2O ......... 0....................................................... 0...............................................................0mL Ferric citrate solution ............0mL: Na2S·9H2O ...............01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1........0mL Trace elements solution SL-6 .4...................0g NaNO3 .....3 with sterile 2M H2SO4...... Pour into sterile Petri dishes or leave in tubes... For heavy cell suspension supplement periodically with sterile yeast extract solution to yield a final concentration of 0......................0mL pH 8.......... 0............................ to distilled/deionized water and bring volume to 1..0g Glycyl-glycine ..0g Yeast extract .............................................................3g CoCl2·6H2O................................... The final solution should be clear and yellow in color.....................................................2–8..............05g Neutralized sulfide solution ...................05g Neutralized sulfide solution ....0L.................................................. to distilled/deionized water and bring volume to 1050...............5g Preparation of Neutralized Sulfide Solution: Add Na2S·9H2O to distilled/deionized water in a 250mL screwcapped bottle fitted with a butyl rubber septum and bring volume to 100...................0................1..... Do not open the bottle to add H2SO4..........................................................................................0mL...... 0.............2 ± 0................................ 0... 1....... Add a magnetic stir bar................ Incubate the culture at 50°C at a light intensity of 300–500 lux.............0mL of medium into 100mL screw-capped bottles with rubber septa under 100% N2......... Readjust pH to 8..............1g MgSO4·7H2O....03g NiCl2·6H2O.............................. Using a sterile syringe... 0....................................0mL: Na2S·9H2O.......025g Na2MoO4·2H2O .........................................3g CoCl2·6H2O ............................................................... Sparge under 100% N2 gas for 3 min..............................................03g NiCl2·6H2O ...............0mL of neutralized sulfide solution into each bottle..............................0.......... except neu- tralized sulfide solution.................02g CuCl2·2H2O ..........0mL Mix thoroughly..........................1g NaCl .1g Na2MoO4·2H2O ..5g CoCl2·6H2O ........1%........0mL: Yeast extract. Adjust pH to about 7......................025g H2SO4..............5.....0................................................. Mix thoroughly..0 mL: Glycyl-glycine ....... Mix thoroughly........ Gently heat and bring to boiling................ Sparge under 100% N2 gas for 3 min..1g Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 100....... Distribute 90.......2–8.............................................2g ZnSO4·7H2O ... 0...................0..... inject 1.................................96 Chloroflexus aggregans Medium Micronutrient Solution: Composition per liter: MnSO4·7H2O ....1g KNO3 ............. Cool to room temperature.. 0...................................... 0........ 2................ 0...................................... Autoclave for 15 min at 15 psi pressure–121°C............................................. ... Autoclave for 15 min at 15 psi pressure–121°C.... Continue boiling for 3–4 min under 100% N2........................................................................................2 at 25°C Neutralized Sulfide Solution: Composition per 100..... Neutralized Sulfide Solution: Composition per 100................2 at 25°C Ferric Citrate Solution: Composition per 100......0L.................................... Sparge under 100% N2 gas for 3 min........................................2.......... Ferric Citrate Solution: Composition per 100.... 1....... 0..............0mL: Ferric citrate ..5g Preparation of Neutralized Sulfide Solution: Add Na2S·9H2O to distilled/deionized water in a 250mL screwcapped bottle fitted with a butyl rubber septum and bring volume to 100... 0............... 11.....................1............... Use: For the cultivation of Chloroflexus aurantiacus..........1g Na2MoO4·2H2O.......2 ± 0. Adjust pH to 8..........0L...............5g KNO3 ............. Autoclave for 15 min at 15 psi pressure–121°C.. Mix thoroughly........................0mL pH 8.1g CaCl2·2H2O.......0.............. Chloroflexus Medium.........1g MgSO4·7H2O ... Preparation of Medium: Add components........0mL.........................0..........................1g CaCl2·2H2O . Mix thoroughly............... Cool to room temperature............................. 1......................... use a sterile syringe.......... 0..4........0mL: Ferric citrate ............................. 0....0mL...1............................................... 0.............................................. Preparation of Medium: Add components.....................0................. Gently heat and bring to boiling.........................0mL Trace elements solution SL-6.... 0... Mix thoroughly.......01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1...... Mix thoroughly...................... use a sterile syringe.11.... Distribute into tubes or flasks............................... Add a magnetic stir bar.........1g Na2HPO4·2H2O ..................0g NaNO3 .... 1............. except Na2S........... Mix thoroughly.... The final solution should be clear and yellow in color.....5g H3BO3 ................................... Stir the solution continuously to avoid precipitation of elemental sulfur.................28g H3BO3 ..1g NaCl .0mL....................................... Stir the solution continuously to avoid precipitation of elemental sulfur................... concentrated ...................... Modified Composition per 1001......................... Use: For the growth and maintenance of Chloroflexus aggregans and Roseiflexus castenholzii............................ Add Na2S.. 0................5g H3BO3 .........................5g Na2HPO4·7H2O......... 0............ Mix thoroughly..0.........0..... 0....... Cool to room temperature........3 with sterile 2M H2SO4.. Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ...... Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O... 0.........0mL Ferric citrate solution ....................... 0..........2g ZnSO4·7H2O....................................

..................................... Distribute 90......................................................0..................................... 25..0mL of neutralized sulfide solution into each bottle........................... Grind................. 1.................. Gently heat and bring to boiling while stirring..................................... 2...................................... Add meat and NaOH to distilled/deionized water and bring volume to 1..............0L...0L...............................1g FeSO4·7H2O....................0 ± 0.................0mL pH 7........ 4......................0mL Rumen fluid...............................1.............. Preparation of Medium: To 1.0mL Mix thoroughly.................................0g Ox bile...... 200................ to distilled/deionized water and bring volume to 990....................................... Remove fat from surface.......................0g NaCl ...0g Yeast extract...... Use: For the cultivation of bacteria that can utilize 5-chloro-hydroxybenzoic acid...................0g Ferric citrate ........................................ Using a sterile syringe....... Gently heat and bring to boiling while stirring.............Chopped Meat Carbohydrate Medium with Rumen Fluid Preparation of Medium: Add components..... Cholera Medium TCBS Composition per liter: Sucrose...................500....0g Sodium citrate ...........................0g NaOH (1N solution) .0mL Resazurin (0................... 10.........0........ Cool to room temperature.............0 ± 0............. Cool to room temperature................0mg CoSO4 ............................................. add the remaining components............... Filter... Cap with rubber stoppers and place tubes in a press.0mL of distilled/deionized water...... Add the L-cysteine·HCl·H2O.... Adjust pH to 7...........................0g NaOH (1N solution) .......................... 3............ Eubacterium species.............................2g Nitrilotriacetic acid .......0mg ZnSO4·7H2O ....0L Chopped meat extract solids .0g L-Cysteine·HCl·H2O.......... Add meat and NaOH to distilled/deionized water and bring volume to 1............... except L-cysteine·HCl·H2O and chopped meat solids................. 0..0g Cellobiose ............ Remove fat from surface..................... Add the L-cysteine·HCl·H2O................. 25.....5g MgSO4·7H2O ......0g K2HPO4 ..............0L of chopped meat extract filtrate..... Use: For the growth of Vibrio cholerae.............. For the cultivation of ATCC strain 35944................. Autoclave for 15 min at 15 psi pressure–121°C............................ Filter................... inject 1. Vibrio parahaemolyticus......2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath............................ 8............................ 0........................ 3.................... Add distilled/deionized water to filtrate and bring volume to 1................................... Gently heat and bring to boiling.......... 1.. Continue boiling for 3–4 min under 100% N2..... Dry agar plates before using...................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....................0mL Preparation of Chopped Meat Extract: Use lean beef or horse meat...... Gently heat to boiling.0L of chopped meat extract filtrate...............0-7............... Mix thoroughly................................... 0....................0g NaH2PO4·H2O....................0g Yeast extract................................................. 0..........0g Agar .... 20............................0g Yeast extract ...2 at 25°C Chopped Meat Extract: Composition per liter: Beef or horse meat..0L......... 4....0mL Resazurin (0..............5. Pour into sterile Petri dishes............... 1...012g MnSO4·H2O ............................0g L-Cysteine·HCl·H2O........................................ 1..................................................... Cool to room temperature.................................. Autoclave for 15 min at 15 psi pressure–121°C.............. add the remaining components.. Adjust pH to 7.......... 150.......... 4................... 0.0...............0 with NaOH...........30.... except neu- tralized sulfide solution............ Adjust pH to 7................. 14...........1..........0mL: Peptone.. Use: For the cultivation of anaerobic bacteria.. 0................... Mix thoroughly......... 10...........0L....... and Prevotella ruminicola....................0mL Rumen fluid.......................................... Use: For the growth and maintenance of Chloroflexus au- rantiacus......25g NH4Cl.........................................................................................................1......................0g Glucose ............. .......... 1. Reserve ground meat particles and filtrate.... Gently heat and bring to boiling. 200............... Chopped Meat Carbohydrate Medium with Rumen Fluid (ATCC Medium 1016) Composition per 1390...........................0g Na2S2O3·5H2O .............................. Add distilled/deionized water to filtrate and bring volume to 1..4 at 25°C Preparation of Medium: Add 5-chloro-2-hydroxybenzoic acid to 800...5..0g K2HPO4 .0mL Preparation of Chopped Meat Extract: Use lean beef or horse meat........1...... Remove fat and connective tissue........................ except L-cysteine·HCl·H2O and chopped meat solids................ Distribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2............. and other Vibrio species................................................... Mix thoroughly.... 1... Preparation of Medium: To 1..............0g Starch.................0......... Do not autoclave... Gently heat to boiling....0g Peptone..................0mL of medium into 100mL screwcapped bottles under 100% N2......................0L........................ Cool to 25°C........04g pH 8......................... Add remaining components and bring volume to 1......2 at 25°C Chopped Meat Extract: Composition per liter: Beef or horse meat.......0g Bromothymol Blue ...................0mL pH 7.....025% solution) ..............................................0g Maltose .................. 4.. 10.............. Reserve ground meat particles and filtrate..5......................................5g Chopped meat extract filtrate ............ including Fusobacterium prausnitzii..................1................ 150..................................................0L.................................................0g Cellobiose..0g 97 Maltose .......0mL: Peptone .500..5g Chopped meat extract filtrate .. 5........................ Chlorohydroxybenzoic Acid Medium Composition per liter: K2HPO4·3H2O..0mg pH 7........0g 5-Chloro-2-hydroxybenzoic acid ............... 5.............. Chopped Meat Carbohydrate Medium with Rumen Fluid Composition per 1390.................................................. Distribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by ... Distribute into tubes or flasks............. Grind.............. 10.......... 30....6 ± 0..... Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust....... Cool to 25°C.0g Starch...........0L Chopped meat extract solids . Remove fat and connective tissue............................025% solution) ..................04g Thymol Blue ..

...........5g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 100...............7g CaCl2·2H2O ..................7.......................0mL: NaHCO3 .... Pour into sterile Petri dishes.................02g HCl (25% solution) ....... Do not overheat. Use: For the differentiation and presumptive identification Solution 1 . Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water....... 860................ Do not overheat.........0mL Na2S·9H2O solution ...... Mix thoroughly..... 20...........................0L.. Cool to 25°C. Mix thoroughly.......... Mix thoroughly..0L..07g H3BO3 .................0mL Vitamin B12 solution.. Gently heat in a boiling water bath or steam bath............ CHROMagar ECC Composition per liter: Proprietary.......... Cap with rubber stoppers and place tubes in a press..............0g deionized water and bring volume to 1.......1... Shake periodically during heating to dissolve components.......................................... Vibrio cholerae form turquoise blue colonies and Vibrio alginolyitcus colonies are colorless.................. Heat long enough with shaking every 5 min to ensure complete dissolution.... Shake periodically during heating to dissolve components....25g Preparation of Solution 1: Add components to dis- tilled/deionized water and bring volume to 4.... O2-Free Water: Composition per 860................................................. Adding tellurite can increase specificity.................... 860..... Gently heat in a boiling water bath or steam bath............................... Mix thoroughly...............0L: MgSO4·7H2O....0.. Cool to 45-50°C............ Gas with 100% N2 for 15 min in a screw-capped bottle............... Cool to 25°C under 100% N2....... Pour into sterile Petri dishes...................................0mL: Na2S·9H2O .............. Source: CHROMagar Vibrio is available from CHROM- Use: For the cultivation of anaerobic bacteria............. Cool to 45-50°C........................................ Do not overheat....0L............. and Ruminococcus species...................... Heat long enough with shaking every 5 min to ensure complete dissolution..........3 ± 0........0mL: H2O........ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................ Tightly close cap. 6.. Gas with 100% CO2 for 20 min.... Pour into sterile Petri dishes. Heat long enough with shaking every 5 min to ensure complete dissolution..... Use: For the differentiation and presumptive identification of Escherichia coli which forms blue colonies......................... Adding tellurite can increase specificity.....................1.................................0mL..... Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.0L O2-free water ...... of Vibrio parahaemolyiticus which form mauve colonies...0.... Source: CHROMagar O157 is available from CHROMagar Microbiology.........04g CuCl2·2H2O............. Adding tellurite can increase specificity............................ coli volume) into tubes under O2-free 97% N2 + 3% H2.7g NH4Cl ..1... coli Composition per liter: Chromatiaceae Medium 1 Composition per 4990..................2 at 25°C Solution 1: Composition per 4. Cool to 45-50°C....................................... 4......... 100.........1.......0mL: Proprietary........................ Shake periodically during heating to dissolve components..... Na2S·9H2O Solution: Composition per 100....................................... Saturate with CO2 by stirring under 100% CO2 for 30 min... CHROMagar O157 Composition per liter: Proprietary...............................5mL Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to ...... 5... CHROMagar E.5g CoCl2·6H2O.........06g NaMoO4·2H2O .... Eubacterium species. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......................1...02g NiCl2·6H20 .. CHROMagar Vibrio Composition per liter: Proprietary.....0...........2.... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........................10................... Mix thoroughly......................... Gently heat in a boiling water bath or steam bath..........0mL NaHCO3 solution................... Gently heat in a boiling water bath or steam bath.................0mL Preparation of O2-Free Water: Autoclave H2O for 15 min at 15 psi pressure–121°C...0.................. Filter sterilize with positive CO2 pressure............. FeCl2·4H2O.. Source: CHROMagar E..............7g KH2PO4 ...... coli is available from CHROM- agar Microbiology... Pour into sterile Petri dishes.5g KCl .......... Use: For the differentiation and presumptive identification Trace Elements Solution: Composition per liter: of Escherichia coli O157... Use: For the differentiation and presumptive identification of Escherichia coli and other coliform bacteria which form red colonies................ Autoclave for 45 min at 15 psi pressure–121°C............. Source: CHROMagar EEC is available from CHROMagar Microbiology...... Cool to 25°C under 100% N2.......0..... Do not overheat. NaHCO3 Solution: Composition per 100........0mL pH 7...... including Butyrivibrio crossotus...0............ Adding tellurite can increase specificity......1g ZnCl2 .....................0mL Trace elements solution................................................................19g MnCl2·4H2O ........................................................ Shake periodically during heating to dissolve components...... Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust..........................0L...98 CHROMagar E... Cool to 45-50°C..0........... Mix thoroughly........0L. Heat long enough with shaking every 5 min to ensure complete dissolution............ 5....... Preparation of Medium: Add components to distilled/ agar Microbiology..........

......... Mix thoroughly.............................0................... Filter sterilize........................5g MgSO4·7H2O .........0mL Na2S·9H2O solution ................0mg B12 to distilled/deionized water and bring volume to 100......................04g CuCl2·2H2O ....................................................................... NaHCO3 .......................0................................................ 2.............. Filter sterilize...07g VOSO4·2H2O ...............01g Trace elements.......................................... Adjust pH to 7... 1......0L....... Mix thoroughly.......06g NaMoO4·2H2O.................1g ZnCl2 .....0... 0...................................... Preparation of Medium: To 950.....0mL of sterile Vitamin B12 solution................0mL or 100..................0g NH4Cl.... NaHCO3 Solution: Composition per 100........... 0............................................ 5...............................................................0mL: Vitamin B12 Solution: Composition per 100.................. Completely fill bottles with medium except for a pea-sized air bubble.........0.1......6..... Cool to 45°–50°C......5g CoCl2·6H2O......... 1........ Autoclave for 15 min at 15 psi pressure–121°C...............................................5g NH4Cl .... Filter sterilize........... Aseptically distribute into sterile 100...............0mL.... 40..5g CoCl2·6H2O .07g H3BO3 ......0mL Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.............2......0mL of sterile Na2S·9H2O solution..Chromatium Medium 99 1..............0 ± 0...0...0mL bottles with metal screwcaps and rubber seals............................................................................................ Mix thoroughly......... Preparation of Medium: To 4...........0mL..1.............. 50.........................0............40.............. Mix thoroughly........................................................................3 ± 0....................... Filter sterilize........................2......... 5............0mL of cooled................ Preparation of Vitamin B12 Solution: Add compo- Preparation of Vitamin B12 Solution: Add Vitamin rated solution 1. Aseptically distribute into sterile 50.......0mL NaHCO3 solution........................................3.............................0mL bottles using positive pressure of 95% N2 + 5% CO2....................... Mix thoroughly...................0mL: Vitamin B12...................................5g .3 with sterile H2SO4 or Na2CO3...........................1.........................................................60.............................. Trace Elements Solution SL-8: Composition per liter: Disodium EDTA ..02g NiCl2·6H20 .........0mL pH 7.......02g KH2PO4 .................. Vitamin B12 Solution: Composition per 100......................................19g MnCl2·4H2O.. Use freshly prepared solution...0..................0mL: Na2S·9H2O...................4g MgSO4·7H2O....................................................... 0.......0mg Na2SeO3.................................................0mL....................0L..................0mL.............................. Autoclave for 15 min at 15 psi pressure–121°C.........0mL............. 5................ Cool to 45°–50°C................0g distilled/deionized water and bring volume to 50.................0....0mL: KH2PO4 .......................188g MnCl2·4H2O .................2g CaCl2·2H2O ......................................... Completely fill bottles with medium except for a pea-sized air bubble.....025g H3BO3 .........0g to distilled/deionized water and bring volume to 100..........2g FeCl2·4H2O........... aseptically add 60.......................................................................................0mL: Na2S·9H2O Solution: Composition per 100......0L of sterile..... Adjust pH to 7.1................................0................................... Sodium Pyruvate Solution: Composition per 50...... Cool to 25°C............0mL: Chromatium Medium (ATCC Medium 1449) Composition per liter: nents to distilled/deionized water and bring volume to 100........... 50..................0mL Sodium pyruvate solution........................0mL pH 7....... Chromatiaceae Medium 2 Composition per 1051...........................................................2g FeCl2·4H2O ..........05g Disodium EDTA............................. Na2S·9H2O .... Use: For the isolation and cultivation of members of the Use: For the isolation and cultivation of freshwater and soil Chlorobiaceae..........................................................................2.................0mL NaHCO3 solution ....0. Mix thoroughly.. 0... members of the Chromatiaceae........................0mL: Sodium pyruvate ..........0mg CuCl2·2H2O........ 0.............. Use freshly prepared solution....... 0...................0mL................. NaHCO3 Solution: Composition per 50. 0.............................. and 1.................5.... sterile solution 1..... 0.....0L......0mL of sterile NaHCO3 solution.............. 1.......0mL: NaHCO3 ................................................ Autoclave for 15 min at 15 psi pressure–121°C..........................950.................... 50......0mL Na2S·9H2O solution..........2..................................4g CaCl2·2H2O...... CO2-satu- Solution 1 ......0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.....................05g Trace elements solution SL-8 .......................0mL: Preparation of NaHCO3 Solution: Add NaHCO3 to Preparation of Na2S·9H2O Solution: Add Na2S·9H2O Na2S·9H2O Solution: Composition per 50... 0..........................2 at 25°C Trace Elements: Composition per liter: Disodium EDTA................................ Mix thoroughly.... Mix thoroughly..... aseptically add the remaining components................................1g ZnCl2 .. Mix thoroughly.................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 50........03g NiCl2·6H2O..... 0.......0mL Vitamin B12 solution .......0..............................0mg Vitamin B12 .....................0mL............0................... Preparation of Trace Elements Solution SL-8: Add components to distilled/deionized water and bring volume to 1.............. Autoclave for 15 min at 15 psi pressure–121°C.2 at 25°C Solution 1: Composition per 950...19g Na2MoO4·2H2O.....0mg Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 1.................

...... Completely fill bottles with medium except for a pea-sized air bubble........... 1.............. Preparation of Solution C (Vitamin B12 Solution): Add Vitamin B12 to distilled/deionized water and bring volume to 5........... Mix thoroughly............... 40......................................................... ...............2............25g Sodium acetate .............0L...............5g KH2PO4 ........ Mix thoroughly..0L................ 0....1......................... 30............. Cool to room temperature under 100% N2 in an anaerobic jar....... Add a teflon-coated magnetic stir bar to the flask..............................0mL Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.1......... 860.......................................10g ZnCl2 ........... in that order.............................................0mL bottles with metal screwcaps and rubber seals...................19g MnCl2·4H2O.. 100.............................................. and the sterile sodium pyruvate solution...............................0..... Use: For the cultivation and maintenance of Chromatium species.....................0mg Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100..... Use: For the cultivation of Ectothiorhodospira marismortui and Ectothiorhodospira mobilis..........0.................1..0mL: Solution 1 ........0mL of sterile NaHCO3 solution........ Preparation of Medium: Add components............. Mix thoroughly........ 860....................................0mL... Preparation of Solution B: Add 860.....................0mL. sterile solution 1...................0mL: Vitamin B12..........0............0mL: NaCl ....................0g MgCl2·6H2O .................... 0.............................. Dispense into a 5-liter flask with four openings at the top (two openings are in a central silicon rubber stopper and two openings are gas-tight screw caps).............0g NH4Cl.............02g Preparation of Trace Elements Solution SL-8: Add components to distilled/deionized water and bring volume to 1..............3 ± 0.......0g Vitamin B12............... 5............ aseptically add 60.0mL Na2S·9H2O solution..............0mL Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100................ NaHCO3 Solution: Composition per 100...........................................................................................100................... 5.................................... Sodium acetate may be substituted for the sodium pyruvate...............0mL of sterile Na2S·9H2O solution......... 5... Autoclave for 15 min at 15 psi pressure–121°C.................0mL Solution F ............0mL Vitamin B12 solution .................. 4000...............02g NiCl2·6H20 .......................................... Filter sterilize........................ 0................... Cool to room temperature. Filter sterilize...............0mL or 100.0mL.............0mL Solution D (Trace elements solution SL-12B) ......................3 with sterile H2SO4 or Na2CO3...............60....0mL Solution C (Vitamin B12 solution)......................... 0... Na2S·9H2O Solution: Composition per 100.....1.0mL Solution B............ Fill to capacity..... Adjust pH to 6....................0mg Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 100.....................06g NaMoO4·2H2O....... 0.. Filter sterilize...0mL of sterile Vitamin B12 solution............0mL NaHCO3 solution ............................ Solution B: Composition per 860.............. 0......7g CaCl2·2H2O ................ to distilled deionized water and bring volume to 850.................05–0.......... 2.... Add the sterile NaHCO3 solution.............................................4g CaCl2·2H2O.................................................0mL: Distilled/deionized water.....................................0mL: NaHCO3 .......... Cool to room temperature under 100% N2 at 0.......0mL......... 0.... and sodium pyruvate solution....0..........5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 4......1.................5g Na2S2O3 .................1.................0mL of cooled. Cool to 45°–50°C.....0mL of distilled/ deionized water to a cotton-stoppered flask.0g MgSO4 ........................................1 atm pressure (use a manometer to measure low pressure)...................................... Preparation of Medium: To 950...............05g Trace elements solution SL-8 .................. 5........0mL pH 3-4 at 25°C Solution 1: Composition per 950.................1. Chromatium Medium with NaCl Composition per 1051.... Filter sterilize.100 Chromatium Medium with NaCl Preparation of Sodium Pyruvate Solution: Add NaHCO3 to distilled/deionized water and bring volume to 50...... Distribute into screw-capped tubes or flasks...0mL: Solution A ........ Mix thoroughly.........................04g CuCl2·2H2O ...........................2g FeCl2·4H2O ... and 1.............40........................................ 0.....7g KCl ...... Use freshly prepared solution.................7g NH4Cl ...............07g H3BO3 ................... Mix thoroughly.. except NaHCO3 solution.. Adjust the pH to 7................................... Na2S·9H2O solution................................. the sterile Na2S·9H2O solution.................. Chromatium salexigens Medium Composition per 4990...... 1............950............0g Vitamin B12 Solution: Composition per 100.........0mL: Solution C (Vitamin B12 Solution): Composition per 5.................0mL.............. Adjust pH to 7..................0mL: Na2S·9H2O...........5g MgSO4·7H2O ........ Trace Elements Solution SL-8: Composition per liter: Disodium EDTA .......0mL....0g KH2PO4 .....................0mL Solution E ............0mL................ Autoclave for 15 min at 15 psi pressure–121°C......................................3..................... Mix thoroughly....................0mL: NaCl .... Autoclave for 20 min at 15 psi–121°C........... 0.......................... 20.......... Autoclave for 15 min at 15 psi pressure– 121°C..5g CoCl2·6H2O ....0mL pH 7.................. Cool to 45°–50°C................................................... Aseptically distribute into sterile 50........... 0.............................. Mix thoroughly......... 5.........................2 at 25°C Solution A: Composition per 4000....... Autoclave for 45 min at 15 psi pressure–121°C..........

...........10..............0mg NaCl . Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100............................ 50..... 0......................... Mix thoroughly....... 18......... Autoclave for 15 min at 15 psi pressure–121°C.......... 20......1..... Sparge with 100% N2 for 3–4 min..0mg Preparation of Trace Elements Solutions: Add components to distilled/deionized water and bring volume to 1........... Mix thoroughly......0mg NiCl2·6H2O ...........................0mL of solution B.................................0mg CuCl2·2H2O ....................2 at 25°C Solution A: Composition per 100...... Preparation of Medium: Add components...............0mL... 20............0mL of solution C........... Autoclave for 15 min at 15 psi pressure–121°C.......1......188....................0mL: NaHCO3 .............. 5..........0mg Na2MoO4·2H2O..........0g FeSO4·7H2O....... Aseptically add 20..0mg H3BO3 ... 400............0mL: Na2S·9H2O ...................... Chromatium/Thiocapsa Medium Composition per 1060..........0mL: NH4Cl....................................0mL............................0g MgCl2·6H2O .............................................0mL bottles under 95% N2 and 5% CO2 at 0...................................... Adjust pH to 7......................................................................... Adjust pH to 7..........0mg MnCl2·4H2O .0mL pH 7.....................................2g FeCl2·4H2O.................... 10..0mL Na2S·9H2O solution..................................19g MnCl2·2H2O......... Chromatium tepidum Medium Composition per 1001.............0mL: NaHCO3 ........................................0mL of sterile NaHCO3 solution and 20........... 2......................0L............................ Mix thoroughly........................................ Mix thoroughly.......5g Preparation of Solution E: Add NaHCO3 to distilled/ deionized water and bring volume to 100............... and 20.... 0......... to distilled/ deionized water and bring volume to 960...........0mL: Na2S·9H2O ...... 0..........2.................. Autoclave for 15 min at 15 psi pressure–121°C........................20........05–0..........................0g NH4Cl ..........0mL Solution D .................. 5................0L....0mL screw-capped bottle.................6.........................1 atm pressure..........0mg Na2WO4·2H2O...1.................0mg MgSO4·7H2O ....................0mg Ammonium acetate (or sodium pyruvate) ............10...............1....2...............................0mL: KH2PO4 ......................... 42................0mL..25.............. Solution B: Composition per 100...... Distribute into sterile screw-capped tubes or bottles so that medium completely fills container.............0mL of solution E... 0.0g Preparation of Solution F: Add Na2S·9H2O to dis- tilled/deionized water and bring volume to 100..........................0mL...... 10.......0mL Solution B....................................0g ..0mg Disodium ethylendiamine-tetraacetate (Disodium EDTA) ........5g CoCl2·6H2O.................................................Chromatium/Thiocapsa Medium Solution D (Trace Elements Solution SL-12B): Composition per liter: Disodium ethylendiamine-tetraacetate (Disodium EDTA) ........................0mL Trace elements solution ......... After 24 hr.0mg CuCl2·2H2O....................................05–0....................... 7.......... Sparge with 100% CO2 until saturated................................................3 with sterile 2M HCl or sterile 2M Na2CO3 solution...................30............................ 50.....0mL........... the iron in the medium will precipitate out of solution as black flocs..........0mL of solution D............5........... Filter sterilize under 100% CO2 into a sterile..0mg Na2MoO4·2H2O .... Mix thoroughly................................1g H3BO3 ............. 10....0mL pH 7..........................2 at 25°C 101 NaHCO3 Solution: Composition per 20. 200.......0mg ZnCl2 ....5mg NaHCO3 solution .............0mg NiCl2·6H2O............................ Mix thoroughly.........................2..........................0mL: NaHCO3 ...3g CoCl2·6H2O .......0mL Solution C.0mg CaCl2·2H2O........... Leave a small gas bubble in each bottle to accommodate pressure changes.1 atm pressure for 30 min with magnetic stirring... Na2S·9H2O Solution: Composition per 20....... 100.................... Use: For the growth and maintenance of Chromatium tepi- dum................................................0mL: Na2S·9H2O.................... Autoclave for 15 min at 15 psi–121°C.................0mg ZnCl2 ..0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20..............................0mL of solution F through one of the screw-capped openings under 95% N2 and 5% CO2 with magnetic stirring..... 24....................................................................................0mL of sterile Na2S·9H2O solution.......................... Solution F: Composition per 100................... Autoclave for 15 min at 15 psi pressure–121°C............ 400.....0 ± 0........0 ± 0................................................ 1............ Preparation of Medium: Saturate cooled solution A under 100% CO2 at 0.......................................................................................................0mg Preparation of Solution D (Trace Elements Solution SL-12B): Add components to distilled/deionized wa- ter and bring volume to 1... Use: For the growth and maintenance of Chromatium salexigens...........................................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20.................... Mix thoroughly.................1...5g Solution A ................0mg VOSO4·2H2O .............0....................................... Mix thoroughly.....190.............. 10.....................0g Preparation of Solution A: Add NaHCO3 to distilled/ deionized water and bring volume to 100............... gas-tight 100..... Autoclave for 15 min at 15 psi pressure– 121°C..............20................... except NaHCO3 solution and Na2S·9H2O solution.......................0mL..0mL Trace elements solution........ Dispense into a screw-capped bottle............................................ Mix thoroughly...................100................................................. Trace Elements Solutions: Composition per liter: Disodium ethylendiamine-tetraacetate (Disodium EDTA) ...........70........... Solution E: Composition per 100..... 1. 3. Add 860..............................0............5mg KH2PO4 ............................................

... Autoclave for 15 min at 15 psi pressure–121°C...... Use: For the cultivation and maintenance of Chromobacterium species and Alteromonas luteoviolacea....................................049g Preparation of Trace Metal Solution A5 with Cobalt: Add components to distilled/deionized water and bring volume to 1.............................................. Mix thoroughly........... Mix thoroughly...................04g Na2CO3 .044g MgSO4·7H2O...................... 1................................................... 15............0L Mix thoroughly.....................5mg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1. Distribute into tubes or flasks... Autoclave for 15 min at 15 psi pressure–121°C.......................................................3......0......................4g Peptone.........................................3........ Chromobacterium Medium Composition per liter: NaCl .........39g ZnSO4·7H2O......... Autoclave for 15 min at 15 psi pressure–121°C.....................0..................15............0 ± 0................................2mL of sterile solution B............................. Chu’s No................. Mix thoroughly..................2.............8g MgSO4 ............................... Aseptically add 0..... Mix thoroughly...............0mL..... and 0..........................................................1.0mL: Na2S2O3·9H2O .........02g CuCl2·2H2O..0................... 5...................0g CaCl2 ..................................0L........................ Trace Elements Solution: Composition per liter: FeCl3·6H2O ................. 10.........102 Chromobacterium Medium Preparation of Solution B: Add Na2S·9H2O to distilled/deionized water and bring volume to 100.......................................0........................................... 1....................0.................. 0...............0................. 5........1mL of sterile solution C.............. .................................... 0................ Autoclave for 15 min at 15 psi pressure–121°C. 1.... 10................. Use: For the cultivation and maintenance of Anabaena species and Plectomena boryanum....... 0..................................................................0mL................................................. Bring pH to 7......0L.......................... 5........................................ Solution D: Composition per 100....................................................... solution B.........06g CaCl2·2H2O ....................................7g pH 7.......................025g Na2CO3 ........... Mix thoroughly.......................................... Gently heat to boiling...........079g Co(NO3)2·6H2O.........................................0mg CuSO4·5H2O ....................................0L................................1g Co(NO3)2·6H2O ......... Mix thoroughly....0...................................................86g MnCl2·4H2O .................... Autoclave for 15 min at 15 psi pressure–121°C...............................0mg Ferric ammonium citrate ................ Use: For the cultivation of Chlorobium limnicola and Chromatium species....... 30....................................0–7.......0mg Seawater ...0.. 0.............. 50..................................................................... 0............................................ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...............0L...........0g MgCl2 ...............2 with H3PO4..................4g MnCl2·4H2O ...........6...............232g Na2SiO3·5H2O .............. 10 Medium Composition per liter: Agar ....025g Na2CO3 .......0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.................................................................... Pour into sterile Petri dishes or leave in tubes.. 10 Medium...0.......................0..................0. 0.........0mg MnCl2·6H2O......02g Citric acid ......3.1.......... Preparation of Medium: Add components...................................0g KCl................................0mL Metal Solution: Composition per liter: H3BO3 .................... 999.... solution C....................... Distribute into tubes or flasks...... Solution C: Composition per 100.........044g MgSO4·7H2O .0mg EDTA....0mL...........7g H3BO3 .. Modified Composition per liter: NaNO3 .......................................................... Mix thoroughly.222g CuSO4·H2O...0g Citric acid ...................0L............................. to distilled/ deionized water and bring volume to 1..........5 ± 0.....4g ZnCl2 ............................................................................................. Autoclave for 15 min at 15 psi pressure–121°C..............0.. Pour into sterile Petri dishes or leave in tubes.................................... Gently heat to boiling............................. 0.0.............................................. 0..................0mL pH 7............3................................ Distribute into tubes or flasks........ 2..... Mix thoroughly...........0mL Trace metal solution A5 with cobalt .0mL of medium..................... Use immediately................. Mix thoroughly.......5g MgSO4·7H2O...................4g CoCl2·6H2O................................ 5.....................6.............................................. Distribute into tubes or flasks....................1g ZnSO4·7H2O ......................0.................................01g Citric acid .....................................................................0g Ca(NO3)2·4H2O.........0.........0L............. Autoclave for 15 min at 15 psi pressure–121°C......08g Na2SiO3·9H2O .1mg Preparation of Metal Solution: Add components to distilled/deionized water and bring volume to 1.................1................................................... 0........................ except so- lution A............. and solution D.1..............................2.........0g Preparation of Solution D: Add sodium malate to distilled/deionized water and bring volume to 100........02g K2HPO4 ............................ Mix thoroughly...................... Chu’s No.2mL of sterile solution A......... Use: For the cultivation and maintenance of Anabaena species and Plectomena boryanum.................... Preparation of Solution C: Add Na2S2O3·9H2O to distilled/deionized water and bring volume to 100............... 0........0........04g K2HPO4·3H2O ............0g Ca(NO3)2·4H2O .......................5mg Ferric citrate .......... 10...............81g Na2MoO4·2H2O.....02g K2HPO4 .....................0.......5mg Metal solution........................................ 11 Medium............5mg Ferric citrate ............ Modified Composition per liter: Sodium malate .....................................................................................01g Agar ............232g Na2SiO3·5H2O...........0mL: Chu’s No........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........2 at 25°C Trace Metal Solution A5 with Cobalt: Composition per liter: H3BO3 .......1mL of sterile solution D for each 10............

...........................................................................................5................5g MgSO4·7H2O............... 2.............................0mL Solution B ..........0L........................ ............. 0................1...................0g Peptic digest of animal tissue..... until boiling................................................ Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly.............................0g CaCl2 ............................... Use: For the cultivation and differentiation of bacteria based on their ability to utilize citrate as a carbon source.......... Mix thoroughly.......................... Use: For the selective isolation and differentiation of Yersinia enterocolitica from a variety of nonclinical specimens based on mannitol fermentation.. Adjust pH to 6.................................... Distribute into tubes in 5......................0g Sodium deoxycholate................. anhydrous ....... CK1 Medium Composition per liter: MgSO4·7H2O........ Pour into sterile Petri dishes or distribute into sterile tubes.......................... Koser’s Modified Composition per liter: NaCl ... 1.........................0g KNO3 ....................................0g Yeast extract...................................... Mix thoroughly.....0mL: Agarose (electrophoresis grade).......................... Immediately distribute into sterile tubes or flasks... 1........................................... Combine solutions A.....................0mL volumes..................................................................0.....................0g Yeast extract .0g 103 Citric acid.........0mL: D-Glucose ............. except cef- sulodin and novobiocin.... 0.......... 10.................................................................10...... 5............................... CIN Agar (Yersinia Selective Agar) (Cefsulodin Irgasan® Novobiocin Agar) Composition per liter: Mannitol.....................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems...................... Do not autoclave..........................1...........015g Irgasan (triclosan) ...................... Preparation of Medium: Prepare solutions A........................0g (NH4)H2PO4 ...200..... to distilled/deionized water and bring volume to 1...1.....0.. 2.. Use: For the isolation and cultivation of cyanobacteria from marine habitats........5 ± 0........0mL.................02g Glucose solution ............................................................... and C.........0...................................0L..... Cool to 45°–50°C............ Citrate Medium....... Mix thoroughly.............................................. 1................... 20...............................................0g Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100.....5g Neutral Red .........10...25g Ferric citrate ....................................................................................................2g pH 6............ Autoclave for 15 min at 15 psi pressure–121°C..........6..0g KNO3 . 0........... Citrate Phosphate Buffered Glucose Medium Composition per liter: Solution A ..........0....... 2......0g NaCl ................4 ± 0....0g CaCl2 ....... Autoclave solutions separately for 15 min at 15 psi pressure–121°C........0mL......................8.0mL: Glucose................................ Yersinia enterocolitica appears as “bull’s eye” colonies with deep red centers surrounded by a transparent periphery... Autoclave for 15 min at 15 psi pressure–121°C...................0..............................................0g MgSO4·7H2O ..............................0mg pH 7......... Mix thoroughly.................................................5g KCl ............ Distribute into tubes or flasks......................23g MgSO4·7H2O........................................................5g K2HPO4 .. Mix thoroughly....................15............2......2.................. Mix thoroughly........................... Preparation of Medium: Add components................... CK Agar Composition per liter: Agar ................................0g Preparation of Solution B: Add components to distilled/deionized water and bring volume to 50........... Solution C: Composition per 200....................0mL K2HPO4 solution .........01g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 750......02g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........................2 at 25°C Solution A: Composition per 750...........1g Ca(NO3)2·4H2O ...................0g K2HPO4 .................750... 0.............................. B.......................1..................... Gently heat and bring to boiling............................................03g Cefsulodin ...0...... 5..................... 2....................... 12.................................................................................. Heat.....................0mL pH 3.................................50... Mix thoroughly..0g Agar ....4g Ferric citrate ................................. Gently heat and bring to boiling.0g Preparation of Solution C: Add agarose to distilled/ deionized water and bring volume to 200........................ 10........0mL: (NH4)2SO4 ..0mL Glucose Solution: Composition per 100... and C..1... Autoclave for 15 min at 15 psi pressure– 121°C.............CK1 Medium Preparation of Medium: Add components to seawater and bring volume to 1................................................................................................... Pour into sterile Petri dishes or leave in tubes............... 1......0g Pancreatic digest of gelatin ............................................................ Solution B: Composition per 50................0L...0g Glucose.......... mixing continuously....................0g Citric acid.1............................... Cool to 50°–55°C.0mg Novobiocin......... 5..........0g Beef extract .......................... Use: For the cultivation and maintenance of Acidiphilium organovorum..92g Na2HPO4 .0mL Solution C ...02g FeSO4·7H2O ..............0L.0mL...... 3..... 100................0mL..2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............................0g Sodium pyruvate ....................................0.........5mg Crystal Violet ..... Cool to 25°C....... Aseptically add cefsulodin and novobiocin.......3.................................... B.......................... Distribute into tubes or flasks. Use: For the cultivation of myxobacteria.............................8 ± 0.............. Mix thoroughly............. 4................

..... Aseptically add sterile glucose solution and K2HPO4 solution.. 5....................................................0g Pancreatic digest of casein ..... Sodium caproate .. Mix thoroughly............................... Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly............................0mg Na2MoO4·2H2O...................... 40. Sparge with 80% N2 + 20% CO2...................... 5.......70.0mL of sterile sodium caproate solution............................4.......... and 1.................. Mix thoroughly..... Autoclave for 15 min at 15 psi pressure–121°C.................0mL: Na2S·9H2O Solution: Composition per 20.......................0mL: Thiamine·HCl ......... Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20..0g MgCl2·6H2O........ Mix thoroughly..................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20........ Mix thoroughly.5g NH4Cl.0mL: NaHCO3 ..0mL Vitamin solution.........20...................................................... Cool to 25°C..0g Cellobiose................................. 10....20.0g Sodium thioglycolate ........ to distilled/deionized water and bring volume to 890............0mL.....5.....2 ± 0...........0g Maltose .... Pour into sterile Petri dishes or leave in tubes.... 100......0g Polygalacturonic acid solution............................. 0....................................................... Clostridium bryantii Medium Composition per liter: NaCl .......... 4............................... Mix thoroughly.............................0mL..............0g Glucose................... 2...............0g Agar ................0mL.............5.50...............6................15g Resazurin .....................0mL Na2S·9H2O solution..............6g Preparation of Polygalacturonic Acid Solution: Dissolve polygalacturonic acid or pectin in small amounts of distilled/deionized water neutralized with 10% NaOH to pH 7............0mL pH 7.......2g CaCl2·2H2O..0mL of sterile trace elements solution SL10....1.................... 5......5g CoCl2·6H2O............ except NaHCO3 solution................190.....................100.... fat free ... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1. 5......5 ± 0...... Clostridium beijerinckii Agar Composition per liter: Agar ...............0mg NaHCO3 solution .............................500............................20.......................20............ Aseptically and anaerobically distribute into sterile screw-capped bottles................... except glu- Sodium Caproate Solution: Composition per 20.................1............ Bring volume to 50................0g K2HPO4 ...30......................0mL: Ground meat.......... Adjust pH to 7.......................... sodium caproate solution.................. Filter sterilize........................................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10..0g Proteose peptone No.. 0... Mix thoroughly...... 10.. Autoclave for 15 min at 15 psi pressure–121°C......0mL.... 3............................2..0µg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 20..................0L...... Aseptically distribute into sterile tubes or flasks..........2 at 25°C Polygalacturonic Acid Solution: Composition per liter: Polygalacturonic acid (or pectin) ............0L.....0g Yeast extract .................................36..........................2 at 25°C NaHCO3 Solution: Composition per 20.........0mL....................................................0mL: K2HPO4 .................0mL.1g Na2SO4 ...................................................0mL Sodium caproate solution...........1...................... 1........ Preparation of Medium: Add components.................................. Autoclave for 15 min at 15 psi pressure–121°C....0mL of HCl solution....................... Sparge with 80% N2 + 20% CO2...........0mg CuCl2·2H2O................................................................. Mix thoroughly.0mL: cose solution and K2HPO4 solution.......... Autoclave for 15 min at 15 psi pressure–121°C....................0mg HCl (25% solution) ........ 0... Add remaining components...................................................................... 20........ Sparge with 80% N2 + 20% CO2..................... Na2S·9H2O solution...................................5mg Na2S·9H2O .................. Sparge with 80% N2 + 20% CO2.....................................0mg H3BO3 .......................................... Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.............................104 Clostridium beijerinckii Agar K2HPO4 Solution: Composition per 10........ Sparge with 80% N2 + 20% CO2...........0g K2HPO4 ............. Add distilled/deionized water and bring volume to 1.....5.......0mg MnCl2·4H2O ...........0mL pH 7.....0mg ZnCl2 .. Vitamin Solution: Composition per 20................................................................ Use: For the cultivation of myxobacteria......1. to distilled/ deionized water and bring volume to 919............. Autoclave for 15 min at 15 psi pressure–121°C.......................2.... Distribute into tubes or flasks... and trace elements solution SL-10........15................. 20.......................0g Yeast extract..............4g Preparation of K2HPO4 Solution: Add K2HPO4 to distilled/deionized water and bring volume to 10...............0mL of sterile Na2S·9H2O solution.3g KH2PO4 .. 20.......................0mg NiCl2·6H2O.. Mix thoroughly.................... Mix thoroughly........... Preparation of Medium: Add components................................ Aseptically and anaerobically add 20....... Sparge with 80% N2 + 20% CO2.........0.........0g ............................0mL with distilled/deionized water........... 3 ................................ Autoclave for 15 min at 15 psi pressure–121°C...........................0µg p-Aminobenzoic acid ..... 3..... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O...................0mL of sterile NaHCO3 solution.... Mix thoroughly...... 0.......24... 21.....................................................................................................0µg D(+)-Biotin . Gently heat and bring to boiling................................................. Clostridium cellobioparum Agar Composition per 1025................... Use: For the cultivation and maintenance of Syntrophospora bryantii....................0mL Trace elements solution SL-10 .1..............................0g KCl...................................................... Mix intensively.......4g Preparation of Sodium Caproate Solution: Add sodium caproate to distilled/deionized water and bring volume to 20...... Mix thoroughly......................... 5. Use: For the cultivation and maintenance of Clostridium beijerinckii............................0mL.........................

.0g MgCl2·6H2O ................................ NaHCO3 solution......... Aseptically and anaerobically distribute into sterile screw-capped bottles.....20......1...2 at 25°C Cellobiose Solution: Composition per 50...........3g FeSO4 ..........50...2.................. Add ground meat to 25......0mL: Na2S·9H2O......... Heat to boiling... 0.....................................................................0mL pH 7.......0 ± 0...........25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20..........15g Resazurin ...0.................................0mL: NaCl ........0g Na2CO3·10H2O....................... Cool to room temperature...............................0g Polypeptone™ ......................0g Avicel) ....................... NaHCO3 Solution: Composition per 30.........8 ± 0.0.......................0mL of sterile L-cysteine·HCl solution.....................................6.......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........0g Cellulose.............5g CaCl2 ..... Mix thoroughly.............0g K2HPO4 ........ Autoclave for 15 min at 15 psi pressure– 121°C................................................0g KH2PO4 .......... L-Cysteine·HCl Solution: Composition per 10............................. Mix thoroughly........................................................ Distribute 7..... Sparge with 100% N2...... Preparation of Medium: Add components..0mg NaOH (1N solution).... Prereduce under 100% N2...300........ Skim fat off surface..... Sparge under 100% N2 gas for 3 min.... except L-cysteine·HCl·H2O.................. except Lcysteine·HCl·H2O.......... Bring volume of filtrate to 1...............5g (NH4)2SO4 ..5g K2HPO4·3H2O ....1.............. 1...............5g (NH4)2SO4 ............ Autoclave for 15 min at 15 psi pressure–121°C...................1..................... Use: For the cultivation and maintenance of Clostridium cellulolyticum and other bacteria that can degrade cellulose...................9g Yeast extract ......... Cool to 50°–55°C..............................................0g CaCO3...........0mL Cellobiose solution ........................................................0mg pH 7...................0mL pH 6............................... 1.0..........15g FeSO4·7H2O ... 10.......................25..... to distilled/deionized water and bring volume to 1..................... 0.. Adjust pH to 7............0L................................ 0.5g CaCl2·2H2O ...........2g Yeast extract ..............................0g CaCl2·2H2O . Aseptically and anaerobically add 50... 0..... 30............0mL...... and 10.................................. 1. Use: For the cultivation of Clostridium cellobioparum.. Sparge with 100% CO2.................................0mL.0mL...... 0............ Add remaining components......... Distribute into tubes under 100% N2.0........10............. Clostridium Cellulolytic Medium Composition per liter: Agar ..................................................................0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50....................0mL of sterile NaHCO3 solution............... Store under N2 gas...... Clostridium cellobioparum Medium Composition per 1010.........20................................................0g L-Cysteine·HCl·H2O ..........1g MgSO4·7H2O......3g MgCl2·6H2O .............0L with distilled/deionized water.. Mix thoroughly... Na2S·9H2O solution...........................0L..5........5g KH2PO4 ......... 0................................. Add L-cysteine·HCl·H2O..1..................................0mL........ Add 1..........0mL............................ Autoclave for 15 min at 15 psi pressure–121°C.0mL: NaHCO3 .................................... Mix thoroughly.........................................10................................... Adjust pH to 7...... Heat to boiling.............20..... Continue boiling for 15 min while stirring..........Clostridium cellulovorans Medium Soluble starch....30.. Pour into sterile Petri dishes or distribute into sterile tubes..1....................0mL of agar into tubes containing meat particles (use 1 part meat particles to 4–5 parts fluid)........................... Gently heat and bring to boiling....0 ± 0..... Mix thoroughly......0mg Rumen fluid........ Mix thoroughly............................... Sparge with 100% N2...................................................2.........2 at 25°C Preparation of Medium: Add components....5 ± 0..... Clostridium Cellulose Medium Composition per liter: Agar ..........................25g L-Cysteine·HCl·H2O......................................4................0mL NaHCO3 solution ...................0g KH2PO4 .........1..............................0mL L-Cysteine·HCl solution.......................................................................................1...0mL of sterile Na2S·9H2O solution..........1..................... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C................. Use: For the cultivation and maintenance of Clostridium cellobioparum and Clostridium polysaccharolyticum......2...................5g (NH4)2SO4 . 20.............. Autoclave for 15 min at 15 psi pressure–121°C.............. Add L-cysteine·HCl·H2O..... Clostridium cellulovorans Medium Composition per liter: K2HPO4·3H2O .........7.. except cel- lobiose solution....... Cap tubes with rubber stoppers................. Mix thoroughly........... Autoclave for 15 min at 15 psi pressure–121°C.. 0.0g K2HPO4 ...................2 at 25°C Preparation of Medium: Remove fat and connective tissue from lean beef or horse meat..........0mg pH 7....2.........0g Filter paper (or 5...................... Use: For the cultivation and maintenance of Clostridium cellulolyticum and other bacteria that can degrade cellulose............... Mix thoroughly.......5....................... to distilled/deionized water and bring volume to 900.............25g Preparation of L-Cysteine·HCl Solution: Add L- cysteine·HCl to distilled/deionized water and bring volume 105 to 10............... Autoclave for 15 min at 15 psi pressure–121°C.............1.................... and L-cysteine·HCl solution......................................................5g Resazurin .....0mL Na2S·9H2O solution....... Filter sterilize.0mL of 1N NaOH.................2................................... 1........0L of distilled/ deionized water.......................................................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 30......... Na2S·9H2O Solution: Composition per 20..................................0g L-Cysteine·HCl·H2O....................0mL of sterile cellobiose solution.................................. Mix thoroughly...................... Grind meat finely.......0mL: D-Cellobiose.....................1g Resazurin ....................................... Filter suspension and retain the filtrate and the meat particles....... Gently heat and bring to boiling....... 5...................... 0.......5...........................0g .....................0mL: L-Cysteine·HCl........................................... clarified .............

.............0mL: Na2S·9H2O..... Filter sterilize...............................0g Sodium thioglycolate.............0...............20..............0mg MnCl2·4H2O............................0...........................0mL............... and Na2S·9H2O solution.0mg H3BO3 ............2g ZnSO4·7H2O......................0mL.......................0L...........................3g CoCl2·6H2O..............................10.0g Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 30..................................................... MN 300 or cellobiose solution. 1........... Clostridium halophilum Medium Composition per liter: Solution A ................................................... 5...........................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10......................75g Pyridoxine·HCl............... Mix thoroughly....... 50....02g CuCl2·2H2O................. Autoclave for 15 min at 15 psi pressure–121°C..................................0mL Na2CO3 solution..............0... 0............................................1......... Mix thoroughly.. Mix thoroughly.0mL pH 7..............0mL Na2S·9H2O....... Filter sterilize....................... 70............2 at 25°C Trace Elements Solution SL-4: Composition per liter: EDTA............0mL NaHCO3 solution.0.0L................................................................... 900.. Mix thoroughly.......0mg Cellulose...............................0mL of sterile Na2CO3 solution.........0mL of sterile cellobiose solution...... Na2CO3 solution......................................0mL of sterile fructose solution and 30...........................30..0mL of sterile NaHCO3 solution....... Sparge with 100% N2.....10..1................... Mix thoroughly...............0 ± 0.0mL Trace Elements Solutions SL-6: Composition per liter: MnCl2·4H2O ....................0mg HCl (25% solution) ........5g CoCl2·6H2O ................................. Mix thoroughly............................20.. 1...............0mL........................5g L-Cysteine·HCl·H2O ...0g Yeast extract .................... Cool to 50°C while sparging with 100% CO2.... 100........................5g FeSO4·7H2O . Mix thoroughly. Mix thoroughly........ Aseptically and anaerobically distribute into sterile screw-capped bottles.....................................................................................................5.0 ± 0.............. Mix thoroughly.......0mg ZnCl2 .... Na2CO3 Solution: Composition per 30.................................. 1.............................. Autoclave for 15 min at 15 psi pressure–121°C............. Mix thoroughly...0mL .. MN 300 or D-cellobiose......... Autoclave for 15 min at 15 psi pressure–121°C......................................................0mL Trace elements solution SL-4...0. 0................ Adjust pH to 8.....................1g Na2MoO4·2H2O.........0mL: Na2CO3 .2 at 25°C Cellobiose Solution: Composition per 50............ Sparge under 100% N2 gas for 3 min...... Na2S·9H2O Solution: Composition per 20......... Use: For the cultivation and maintenance of Clostridium cellulovorans.............. Aseptically and anaerobically pour into sterile Petri dishes or distribute into sterile screw-capped bottles under 100% CO2.....................0.................................................................................... 80.................................... to distilled/deionized water and bring volume to 920........0mg Resazurin .........5g Pancreatic digest of casein ........... 1..0mL: NaHCO3 ................................. Aseptically and anaerobically add 50...................... Store under N2 gas.................2g Trace elements solution SL-6......0L......0mg NiCl2·6H2O . Autoclave for 15 min at 15 psi pressure–121°C................ 0.................. 0.................. except cel- lobiose solution.............0mL Rumen fluid... 36. 0.....01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1......... 190... Sparge with 100% N2.......... NaHCO3 Solution: Composition per 30......5g H3BO3 .........................................0mL of HCl solution......... 24....................... Gently heat and bring to boiling.........................0mL: Cellulose.....................03g NiCl2·6H2O........................................... Autoclave for 15 min at 15 psi pressure–121°C.........................1.......0g KCl.... Store under N2 gas.0g Preparation of Cellobiose Solution: Add cellulose or cellobiose to distilled/deionized water and bring volume to 50.................. Mix thoroughly.........................0mL of sterile Na2S·9H2O solution. Preparation of Medium: Add components......................0.......................... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ..........................15....................... Sparge with 100% N2................0mg Fructose solution .......... Aseptically and anaerobically add 50....................................................... Sparge under 100% N2 gas for 3 min....................................15g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20...... 10............................................ 0....... Add distilled/deionized water and bring volume to 1.................................................. Use: For the cultivation and maintenance of Clostridium formicoaceticum..................... Mix thoroughly.............................................. 2...0g K2HPO4 .. Add remaining components...... 0..................... Mix thoroughly.........10...15g Resazurin ........................0mL Solution B..............5g MgSO4·7H2O ....................0mL....................................0................ Clostridium formicoaceticum Agar Composition per liter: Agar .........0mg CuCl2·2H2O ...................0mL: Fructose .....................................0mL.5........0mg Na2MoO4·2H2O .106 Clostridium formicoaceticum Agar NH4Cl... 0.0mL Trace elements solution SL-10 .0mL... Sparge with 100% CO2.............50............ Fructose Solution: Composition per 50.. 6..................................... Preparation of Medium: Add components................................. clarified ...............................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 30.......................... 0... 100....... except fructose solution and NaHCO3 solution.............. 30.............................................. and 20.................... Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1..5g Yeast extract....... to distilled/deionized water and bring volume to 900.................................0mL..0g Preparation of Fructose Solution: Add fructose to distilled/deionized water and bring volume to 50........0mL pH 8...... 30...

..................................................8mL volumes into anaerobe tubes....1g ...................................0mg Lipoic acid.........0mg Nicotinic acid ........................................... 60....... 2........................................0mL of HCl solution.................0mg ZnCl2 .......................0mg Biotin .0L Wolfe’s vitamin solution ........ Add components to distilled/ deionized water and bring volume to 900.................................................................0µg Wolfe’s vitamin solution .......................0mg NiCl2·6H2O ..............................................................................................................................1. Autoclave for 15 min at 15 psi pressure–121°C....... 5............................1....................0mg Riboflavin ..........0........................0.........................................................................0.......................10............................... 24.................................1g CaCl2·2H2O .....0g Preparation of Solution B: Add NaHCO3 to distilled/ deionized water and bring volume to 80..... 5...... Cool to room temperature while sparging with 80% N2 + 20% CO2.............0........................................ 5..................... 10...1g FeSO4·7H2O .............................0......10......................................................1...........................................................................................................................0mL Trace elements solution SL-10 ............................................. 5................. Use: For the cultivation of Clostridium halophilum...5................. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .............0mg Na2WO4·2H2O................... 2.................................................................................................................................... Adjust pH to 8........................... pH 7... 1...0mL Solution D ........0mg Folic acid ....86g MgSO4·7H2O ................0L........................................................................................... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2........................... Autoclave for 15 min at 15 psi pressure–121°C........3g Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10.........................................................0mg Calcium DL-pantothenate..........2..0mg CuCl2·2H2O ....................................0mg Folic acid.... Aseptically and anaerobically add 0.1g L-Lysine·HCl .......................................................0mg Biotin ...2................0g CaC12·2H2O............... Mix thoroughly...........0mg Nicotinic acid ..... Add remaining components.......0mL Wolfe’s mineral solution .............. Sparge with 100% N2...025g Resazurin ...............1.............0mL Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ..0mL..0mL......10.........0g L-Alanine....223g 107 Preparation of Solution C: Add components to distilled/deionized water and bring volume to 10.....................2g NH4Cl..............................0mL: NaCl ........... 1............................................0mg H3BO3 .................................... Mix thoroughly.....................0...........................05mg Sodium/potassium phosphate buffer (0.....0.........0mg p-Aminobenzoic acid ......................................0) ....................5g CoCl2·6H2O................................................................. 5.. Solution B: Composition per 80......................................0....... 5....Clostridium hydroxybenzoicum Medium Solution C ..................5g NaCl ......... 5................... 36.... Mix thoroughly.................0............................ Gently heat and bring to boiling..........................5g CoCl2·6H2O ..............................................................................................0mg Thiamine·HCl ..........................................................................................1......02M solution...........................................1g CaCl2·2H2O ....................................................2.........0mg HCl (25% solution) ..........................................................0mL. Anaerobically distribute 9.. 0....10.......0mg p-Aminobenzoic acid .10.......... Mix thoroughly........ Mix thoroughly........0mg Calcium DL-pantothenate ........................... Clostridium hydroxybenzoicum Medium Composition per 1055......................... Mix thoroughly...0mg Na2MoO4·2H2O .....4g Na2S·9H2O ......0mL pH 7...............................15.......................0g L-Arginine·HCl...........................0....0mL................1mL of sterile solution D to each tube.5...0L................ 25......................0mg Lipoic acid .................................................... 2...2 at 25°C Solution A: Composition per 900.....0 ± 0..........................................5g L-Cysteine·HCl ..........................................................................5.......................................................................... Add distilled/deionized water and bring volume to 1..................................................0g MnSO4·2H2O........... Sparge with 100% N2.....0.....0mg Thiamine·HCl . 1..........................................................................358g KH2PO4 ............0mL: Na2S·9H2O ......0mL: NaHCO3 ............................. 0..........................................................75g NH4Cl ......0mL of cooled solution A with 80.......................................0.3............... Mix thoroughly................0mg MnCl2·4H2O... 1.............................................3.......... Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O..............0...... Mix thoroughly.................................0mg Vitamin B12..... 5...0g Yeast extract .............................5........................................ Preparation of Solution A: Prepare and dispense medium under 80% N2 + 20% CO2..0mL: NaCl ..............2 at 25°C Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl.....8g Glycine .... Mix thoroughly....................1g ZnSO4·7H2O.............. Anaerobically combine 900........................0mg Riboflavin...... Mix thoroughly.....10...........0g Yeast extract..0mL: K2HPO4 ........................... 6................... Continue boiling for 3 min........0mg Vitamin B12 .... 0.......... 100...5..............................................................3 ± 0........................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10........................... 50............... 70.................... Solution D: Composition per 10..............................................................10.......... Autoclave for 15 min at 15 psi pressure–121°C................................................................. 5...........................................0mL of sparged solution B..... Solution C: Composition per 10... 190........................0g Betaine ...1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...................... 2.................................................................... 1...............4g MgCl2·7H2O .....................0mL pH 8..................................0mg Resazurin ............ 5...........................0L. Sparge with 100% N2 for 20 min.................1mL of sterile solution C and 0..........0g Nitrilotriacetic acid........1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..........5..........................0..................................0mg Na2SeO3·5H2O ..........

....................25...............................0mg MgSO4 ........................................................1...........02g CuSO4·5H2O .................................. and CaCO3. Use: For the cultivation of Clostridium lentocellum and other Clostridium species...................96g L-Cysteine·HCl................2............. Clostridium lentocellum Agar Composition per 1201..2............ 23............................6......0L........................2..................................................... Adjust pH to 7................................................0mL pH 8........................................50............0mg FeSO4·7H2O................................................0mg NH4·Cl.........0mg MgSO4·7H2O .......0g Preparation of Cellulose Suspension: Add components to distilled/deionized water and bring volume to 100........1% solution)....5.....0mg MnSO4·H2O ......... 4............................1...5 with KOH..................... Use: For the cultivation and maintenance of Clostridium kluyveri...... Mix thoroughly.........................10..... 10................. 80..0mg Thiamine·HCl ........... Add components to distilled/ deionized water and bring volume to 900...........01g Na2MoO4·2H2O ............................... 0........ Mix thoroughly....................5...........................0mg Resazurin .5g CoCl2·6H2O..............................5g CaC12·2H2O .0g K2HPO4 ........ Anaerobically distribute into tubes or flasks...............................1............ Use: For the cultivation of Clostridium hydroxybenzoicum.......................6g Yeast extract.............................................................................. Gently heat and bring to boiling.......... 0...0mL Solution C.........................................................0mg Na2SeO3·5H2O ........................... 50... Adjust pH to 6. 2........ 0...200................ 15.......................2 at 25°C Cellulose Suspension: Composition per 100.............................................................................0mg K2HPO4 ........................1...............0mg Biotin ...................0.......................................0mg Na2MoO4·2H2O......................0mg Calcium DL-pantothenate ........................................................................0µg Resazurin ........................... ethanol........................................ Mix thoroughly.0mg Vitamin B12..........................................0mg H3BO3 .......................1............................................ 10.......01g H3BO3 .......................................................0µg Wolfe’s vitamin solution ..................................0mg Lipoic acid............................... 1...........................................................108 Clostridium kluyveri Agar NiCl2·6H2O ...........................0mL.. Mix thoroughly..........................0mg MnCl2·4H2O .0mg KH2PO4 ............................................. 10......................................... 96...... except so- dium thioglycolate....0g Yeast extract ...... Add remaining components...........................................0mg Cellulose suspension............................................................................... Mix thoroughly..............0mL: Potassium acetate ............ Preparation of Solution A: Prepare and dispense medi- um under 80% N2 + 20% CO2.................................2g NH4Cl .................0L.... 0................................5.......... 0............36.......... 0................... 25............ 10..........50.. Clostridium kluyveri Agar Composition per 100......................0µg Biotin ....0g MgSO4·7H2O....01g Na2SeO3·5H2O ...................................................0....0mL Resazurin (0.........0mg Riboflavin. Sparge with 100% N2..........0mL: Whatman CF cellulose powder..................... Adjust pH to 6........0mL Solution D ...........70.0mg HCl (25% solution) .........24............... Clostridium litorale Medium Composition per liter: Solution A .................0L........ Gently heat and bring to boiling....................86g L-Alanine ...............0µg p-Aminobenzoic acid ................0mL of HCl solution.0mg CuCl2·2H2O...................................................0mg Na2MoO4·2H2O ................................................2 at 25°C Preparation of Medium: Add components................................... Add distilled/deionized water and bring volume to 1..........0mg CaCl2·2H2O............. Mix thoroughly.0mg Folic acid ... to distilled/deionized water and bring volume to 100................... Add remaining components.....................0mL....................................................... Continue boiling for 5 min........................0mL pH 7...............100............ 96.........3mg Preparation of Wolfe’s Mineral Solution: Add ni- trilotriacetic acid to 500......................... Autoclave for 15 min at 15 psi pressure–121°C............. Cool to room temperature while sparging with 80% N2 + 20% CO2..... 1. Mix thoroughly..................0µg CaCO3 .......0 ± 0. Gently heat and bring to boiling............ Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O......................................... variable Ethanol ................................0mg NiCl2·6H2O................................8...................5g CaCl2 ...... 1.................... 30.....................5.......................................................... Store anaerobically....... Pour into sterile Petri dishes or leave in tubes............. 0.... Autoclave for 15 min at 15 psi pressure–121°C..........0mg Nicotinic acid ............................ 900.................... Distribute into tubes containing a small amount of CaCO3........................2 at 25°C Solution A: Composition per 900.................0mg ZnCl2 .............................................10..................................................................3 ± 0..........0mg p-Aminobenzoic acid ... Add sodium thioglycolate and ethanol.................. Add distilled/deionized water to 1................................2 ± 0.....................0mL.... 0......... 31......... 20......................................................0mL pH 7...............................0mL Trace elements solution SL-10.............5...................................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10....................................................... 0......................................025g KAl(SO4)2·12H2O........................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...0mL: NaCl .... Preparation of Medium: Prepare and dispense medium under 100% N2..................5........... Autoclave for 15 min at 15 psi pressure–121°C................................................................................ 1... 1................................................................................................ 2..0g NaCl .....................10........................................................................2.....................................0mL of distilled/deionized water.................... Continue boiling for 3 min.................................2.. Cool rapidly to 50°C..............................0g Sodium thioglycolate . Distribute into tubes or flasks.............2mg Agar ......0mL: Agar .... Mix thoroughly......20.............................................................0mL Solution B... 1.............. ..190........0L...........1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1............................................................ Combine components.........0mL Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl..............2.65g NH4SO4 .............................0g Betaine.....5......................

..........0mL of cooled solution A with 80..........0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10........... Add remaining components.0mg Riboflavin......... 10.................................. Boil for 10 min.0mL trace elements solu- ...........................0................0mL..5............................. Mix thoroughly..0mg D-Ca-pantothenate .......01g CuSO4·5H2O......10........ Sparge with 80% H2 + 20% CO2.... Mix thoroughly.................1g CaCl2·2H2O............ Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere................................ Use: For the cultivation of Clostridium litorale.........................................3g Preparation of Solution B: Add NaHCO3 to distilled/ Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10........................................ Cool to 25°C.................0..........................0mL Na2S·9H2O solution. Filter sterilize..........................025g KAl(SO4)2·12H2O ............................ MgSO4·7H2O....... Autoclave for 15 min at 15 psi pressure–121°C........ Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2........................8g MgSO4·7H2O .......0mL Cysteine solution.....0............................0........ 10. Adjust pH to 8...........................................0mL NaHCO3 solution................ to distilled/deionized water and bring volume to 900..............0.......1mL of sterile solution C and 0.5g NaCl ............0mL vitamin solution........................223g L-Cysteine·HCl·H2O.....................0..Clostridium ljungdahlii Medium Solution B: Composition per 80............................0mg Nicotinic acid . Mix thoroughly................. cysteine solution.0mL..... 1.............0mL.....................0mL Trace elements solution ..................... Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly...........0...... Sparge with N2.................0mL pH 5.0mL: Fructose........................ 0..........0..............5.....8mL volumes into anaerobe tubes........................... Sparge with 100% N2.. Mix thoroughly................ 1............. Store anaerobically....................................... Mix thoroughly........0mg p-Aminobenzoic acid .............0mg Thiamine-HCl·2H2O ..........3...........0mL NaHCO3 solution ...........................01g Na2MoO4·4H2O.. Sparge with 100% N2. Mix thoroughly............................................................................ Sparge with 100% N2........ 0.....................5... 0...........0mL of sparged solution B................................................0g NaCl ..........................................0mL: NaHCO3 ...............5..........20mg Fructose solution... Dissolve by adjusting pH to 6..........0g Nitrilotriacetic acid...0mL: Trace Elements Solution: Composition per liter: Na2S·9H2O................................................... Vitamin Solution: Composition per liter: Pyridoxine-HCl ................................................1.....0mL fructose solution................................................0mL cysteine solution....................................1g NiCl2·6H2O.........0mL........................... Anaerobically distribute 9................. teine·HCl·H2O to distilled/deionized water and bring volume to 10........................ 10................................. 10.. 5................................... Solution C: Composition per 10.............0mL: L-Cysteine Solution: Composition per 10................. Gently heat and bring to boiling......................................50.........2.....................................1g KH2PO4 .0mL............................ deionized water and bring volume to 80....................10..................................................0g Yeast extract...............................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.......0g Preparation of Fructose Solution: Add fructose to distilled/deionized water and bring volume to 50...................... 5....10... Autoclave for 15 min at 15 psi pressure–121°C.............10.....0...... Filter sterilize...1g FeSO4·7H2O ....2g KCl..3g Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10..........02g Na2WO4·2H2O ..................................1mL of sterile solution D to each tube...........0mL................18g CaCl2·2H2O ......0. Fructose Solution: Composition per 50.......5........... Solution D: Composition per 10.......... vitamin solution... Na2S·9H2O solution..................................... 0...........................0mg Folic acid .......0mL: 109 Na2S·9H2O Solution: Composition per 10.................................2 at 25°C NaHCO3 Solution: Composition per 10..... Autoclave for 15 min at 15 psi pressure–121°C...0mg Lipoic acid.............. 0...... Add distilled/deionized water to 1.................. Anaerobically combine 900...........................................................................................0mg Biotin .... Autoclave for 15 min at 15 psi pressure– 121°C...........................0L.............01g Na2SeO3·5H2O ................ 0.. 0........ Mix thoroughly........................3....... except NaHCO3 solution...................9 ± 0.........0mL of distilled/deionized water........................... 1.................................................. 0.. Filter sterilize.........18g ZnSO4·7H2O.....0.....................1..0mL: K2HPO4 ......................................5 with KOH...5g CoSO4·7H2O......0mL Na2S·9H2O solution................ Sparge with 80% N2 + 20% CO2.. Aseptically and anaerobically add 50........ 0.............................................0mg Vitamin B12....................2.................. and trace elements solution SL-10............................... Aseptically and anaerobically add 0..........................0g Na2S·9H2O .......... Add components...............................0mL...............................0.0mL... Cool to room temperature while sparging with 80% N2 + 20% CO2.10.......02g H3BO3 ................0L................10...... Sparge with 100% N2.. Mix thoroughly........................... fructose solution...........................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.... Autoclave for 15 min at 15 psi pressure–121°C...0mL Vitamin solution..3g Preparation of Solution C: Add components to dis- Preparation of L-Cysteine Solution: Add L-cys- tilled/deionized water and bring volume to 10...........5... Clostridium ljungdahlii Medium (DSMZ Medium 879) Composition per liter: NH4Cl. Mix thoroughly............0. Mix thoroughly......... Sparge with 100% N2 for 20 min..............0mL: NaHCO3 ..0...... and 10....0g MnSO4·2H2O...................................................................................358g KH2PO4 ..

.................................................0mg Riboflavin ................0mL......................0mL: Solution A ................2 at 25°C Phosphate Solution: Composition per 100...................2 at 25°C Solution A: Composition per 890........................................0mg Thiamine·HCl ........................ Autoclave for 15 min at 15 psi pressure–121°C.................. 2...0mg Na2SeO3·5H2O ..........................10.... Autoclave for 15 min at 15 psi pressure–121°C..........................................0mL pH 7..... Mix thoroughly...................0mL.....0mL pH 7.............2........0mg p-Aminobenzoic acid . Adjust pH to 7................................................ except phosphate solution and Na2S·9H2O solution.............0mg Seawater........................................................... 50........ 100...... 5........................... 0........ Filter sterilize...........9..................................... Mix thoroughly......3 ± 0...............0L... ly........................................................0mg HCl (25% solution) ..............0mg Calcium DL-pantothenate...... 5........0mL Solution D .............2...... 70.................0............ Sparge with 100% N2.... Final pH is 5...............0g Resazurin .............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........ Cellobiose Solution: Composition per 50....... 5.............. 5.. 100............0mg H3BO3 ........0mL...0mg NiCl2·6H2O .....0mg MnCl2·4H2O...........................110 Clostridium M1 Medium tion SL-10...... 2.............................0mL Vitamin solution................ 36.............0g Yeast extract ......0mg Na2MoO4·2H2O ............................................... Mix thoroughly............................................... Sparge with 100% N2.....................0g NaHCO3 .............0g MgSO4·7H2O .............................................. Mix thoroughly............ filtered.........................................0mL Na2S·9H2O solution.... 10..... Mix thoroughly..................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.................1............................ Aseptically and anaerobically distribute into sterile screwcapped bottles under 100% N2..5.....2g CaCl2·2H2O ...............6g L-Cysteine·HCl ................................................................ 1..................................................100.0................................ 2.................... to distilled/deionized water and bring volume to 950......0mL: K2HPO4 .0mg Lipoic acid .......................... Use: For the cultivation of Clostridium ljungdahlii..... 10................................ Add distilled/deionized water and bring volume to 1...................... Mix thoroughly..........0mg Resazurin ..................................... 10................. Mix thoroughly...........................0mL pH 7.....................0mg CuCl2·2H2O .......0mg ZnCl2 ..... Mix thoroughly........ 890............10.... 6... Use: For the cultivation and maintenance of Clostridium papyrosolvens............................... Aseptically and anaerobically add 100........................................................................0mL......0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50.................0mL of HCl solution... 0................................0mg Folic acid................................1......................................0.. Mix thoroughly................................................ Mix thoroughly............................................................ Vitamin Solution: Composition per liter: Pyridoxine·HCl ........0mL of sterile cellobiose solution.....10......... 25.......... Add remaining components.................................................0mL of sterile phosphate solution and 10........................ 0......... Sparge under 100% N2 gas for 3 min............0mL Cellobiose solution .0g Yeast extract .................................................................................................. Mix thorough- Use: For the cultivation and maintenance of Clostridium halophilum and Clostridium litorale....15..1..............0L......... Clostridium pfennigii Medium Composition per 1001.................................8g Preparation of Mineral Salt Solution: Add components to distilled/deionized water and bring volume to 1........................................................................ 1.....................0mL: Na2S·9H2O.......... to distilled/ deionized water and bring volume to 890.................... Clostridium papyrosolvens Medium Composition per liter: K2HPO4 ...................... 1...............0µg Phosphate solution ........ Preparation of Medium: Add components....................... Na2S·9H2O Solution: Composition per 10..........................0mL Solution C...............................5g CaCl2·2H2O................0mL: Sodium vanillate........................0mg Nicotinic acid ................ Sparge with 100% N2.... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ......................................0mg Vitamin B12 ........0mL Trace elements solution SL-10 . Clostridium M1 Medium Composition per liter: NaCl .......................2g NH4Cl.......6............1... 190................................... Aseptically and anaerobically distribute into sterile screw-capped bottles under 80% N2 + 20% CO2.........2 ± 0..5g Resazurin .........358g KH2PO4 .................................. Aseptically and anaerobically distribute into sterile tubes or bottles........................... 24......................... 1........................... 150......0–7......0g NaCl .............................. 5........... Mix thoroughly.. 0......... Filter sterilize..........3g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....0mg ...........2 at 25°C Mineral Salt Solution: Composition per liter: (NH4)2SO4 .....................0g MgSO4·7H2O............. Aseptically and anaerobically add 50........................0mL...............0g Yeast extract........................ Autoclave for 15 min at 15 psi pressure–121°C..........................223g Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 100................................... 5.....0g L-Alanine..0L............................................................6.............................0mL Solution B..0mL Mineral salt solution......5g CoCl2·6H2O .............................................................................................................. except cellobiose solution......1....2 with 5N NaOH.......0mL: D-Cellobiose .. 200.............................65g NH4Cl ......................................................... 0.....................0g Betaine·H2O .... 2.... 5.... 1......................................................0mg Biotin .................. Autoclave for 15 min at 15 psi pressure–121°C.1.......... 6......... Sparge with 80% N2 + 20% CO2........................................................................ Preparation of Medium: Add components.....................0mL of sterile Na2S·9H2O solution...................... Sparge with 100% N2........................

.................. Add remaining components.. Mix thoroughly...................1.........0mg CuCl2·2H2O ... 8.....0g L-Arginine·HCl......... 10...................................5g CoCl2·6H2O ............... Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10..... Mix thoroughly..............................3g MgSO4·7H2O..0L......0................ Filter sterilize......... Mix thoroughly.. 70...... Adjust pH to 7....... Adjust pH to 6................. 5....................0mL: Preparation of Trace Elements Solution SL-10: NaHCO3 .................. ...0... Clostridium propionicum Medium Composition per 1007................. 2....0g Preparation of Mineral Solution: Add components to distilled/deionized water and bring volume to 1...0mL CaSO4...... Autoclave for 15 min at 15 psi pressure– 121°C..............0 ± 0...................................................1.........9mL into anaerobic tubes under 80% N2 + 20% CO2.........................................25mg Pantothenic acid ......... 0.......... 8.....0mg NiCl2·6H2O ......................................4....0mg ZnCl2 .........1 .............. Use: For the cultivation and maintenance of Clostridium propionicum............0.........................................0mL: Preparation of Medium: Add components.................................... Distribute into tubes or flasks..........................0mL..... Sparge with 80% N2 + 20% CO2 for 20 min...0g NaCl .........................0mL Trace elements solution SL-10 ............. to distilled/deionized water and bring volume to 1..... Na2S·H2O solution...................24g Preparation of Solution C: Add L-cysteine to distilled/ deionized water and bring volume to 10...5mg p-Aminobenzoic acid .............. 10...0mg H3BO3 .....0mg FeSO4·7H2O ....0L.0g L-Alanine ............................0g NH4Cl... Mix thoroughly...................25mg Thiamine·HCl .... and 0................................... 0..........0L..............267... Mix thoroughly.............................0mL Mineral solution ..2...3g Preparation of Solution B: Add NaHCO3 to distilled/ deionized water and bring volume to 100......................................3..... Mix thoroughly........... Solution C: Composition per 10....0g Sodium formate ....... Use: For the cultivation of Clostridium sticklandii.0mg HCl (25% solution) ..................0g K2HPO4 .... Mix thoroughly................................................................................1mL of sterile solution C........ Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .....................0g Na2S·9H2O .1mL of sterile Na2S·H2O solution to each 10.......... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C..... Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C........0mL............................................... 100.....9.3............................................. Clostridium sticklandii Medium Composition per liter: Add FeCl2·4H2O to 10...................................................0mL Vitamin solution...................................2...........................2mg Nicotinic acid ...........................5................................ Mix thoroughly......... Distribute 8.......6g CaCl2·2H2O............................................... 6...0mL: Na2S·9H2O Solution: Composition per 10...............................................0mg Na2MoO4·2H2O .....................................................0L..0.....0mg Na2S·H2O solution................. 1............. Distribute into tubes or bottles under 100% N2...........0mL of medium........................................0.........................................................................50.............. Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C..............0..................................................5mL Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........5mL: Yeast extract ...............0mL.....................75g MgSO4·7H2O.................... Yeast extract .............................0g L-Lysine·HCl .....01mL of sterile solution D.. Mix thoroughly................... 6.......................... Mix thoroughly........10.........0mg MnCl2·4H2O...... 0............0mL 111 Solution D: Composition per 1............ add (using a syringe) 1.......................... clarified ........................0.... Mix thoroughly.... Sparge with 80% N2 + 20% CO2 for 20 min................... Autoclave for 15 min at 15 psi pressure–121°C......................................0g MgCl2·6H2O...................................Clostridium sticklandii Medium Rumen fluid..................................................................0L....... Adjust pH to 7.................................. 36.. Mineral Solution: Composition per liter: KH2PO4 ..........62mg Biotin ....5............................................................0mL of HCl solution............ 190.. 1.......2......................... 24.....................018g Resazurin ......... 0............................2 at 25°C Solution B: Composition per 100.............0mg Potassium phosphate buffer solution.................. Bring pH to 7.....................................0g L-Cysteine·HCl ....2g CaCl2·2H2O ............. Vitamin Solution: Composition per liter: Pyridoxine·HCl .......................... Add distilled/deionized water and bring volume to 1.. Sparge with 100% N2 for 20 min................................10.......0mL: Na2S·9H2O ..........0mL of sterile solution B.... saturated solution................................ Aseptically add 0...................10..................... 1........................... 2.1................ pH 7......................... Preparation of Medium: To each tube containing 8.........1g FeSO4·7H2O ...........1................. 6......................................... 2....2...................................... Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C.......0mg Preparation of Solution D: Add Na2S·9H2O to distilled/deionized water and bring volume to 1.................. except L-Cysteine .................0mL Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 890............................. 5..0g Peptone ..........25mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1. Use: For the cultivation and maintenance of Clostridium pfennigii............................ 1..................................................9mL of sterile solution A............0mL............................................. 1M..........0g NH4Cl ...0mL............78......0mL pH 7....

........................0............... 4..................................0mg CuCl2·2H2O ..0mg NiCl2·6H2O .............8g NH4Cl . Autoclave for 15 min at 15 psi pressure–121°C......0g KCl..1...............................................................................112 Clostridium termitidis Medium Clostridium termitidis Medium Composition per liter: NaCl .. Aseptically and anaerobically add 50.... Sparge with 80% N2 + 20% CO2 until pH reaches below 6............................................. 0......... Sparge with 100% CO2..0mg Riboflavin..........0......18g CaCl2·2H2O ............................ 24............................................. 0...........................0mL..................................... Sparge under 100% N2 gas for 3 min...........3g KH2PO4 ...0g MnSO4·2H2O.................... Autoclave for 15 min at 15 psi pressure–121°C..................................................... Cool to 25°C while sparging with 100% ................................5g CoCl2·6H2O ........ to distilled/deionized water and bring volume to 985................................. 5.......................... Autoclave for 15 min at 15 psi pressure–121°C.......... 10...............01g Na2MoO4·4H2O............................................................................0mg Folic acid ........................... 100...... NaHCO3 solution..0................................ Na2S·9H2O Solution: Composition per 10.......................................................................5..0.......... 5. 1...3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500...................... Mix thoroughly.. 0......... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C........5g MgCl2·6H2O........................0mg HCl (25% solution) ...........2.......0...0mg MnCl2·4H2O.......................................................................................... Mix thoroughly......... Add remaining components..........0......4g NH4Cl..........50............1.... 1..0................0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50.......... NaHCO3 Solution: Composition per 20.....0mg Vitamin B12................................0mL pH 7............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...............................0mg Trace elements solution..... Preparation of Medium: Add components.........0mg Resazurin ........................................................0....... Cool to room temperature. Filter sterilize.....................0mg Trace elements solution SL-10 ...........................1......................... 0................................. Mix thoroughly...0mL pH 7................. Mix thoroughly.......................0mg Biotin .....0. Sparge with 80% H2 + 20% CO2............................0..................1...0mL of distilled/deionized water.... 190........................................................................... Use: For the cultivation and maintenance of Clostridium termitidis.......................2g CaCl2·2H2O......5......................................0L........ Mix thoroughly......0g Nitrilotriacetic acid......0mL Na2S·9H2O solution..........0mL: Na2S·9H2O.............................. Sparge with 100% N2............... Add remaining components.............0mg Nicotinic acid ....................0mg Lipoic acid...........................................0mL: D-Cellobiose..........0L.....................0g Na2HPO4·12H2O ............................... Clostridium thermoaceticum Medium (TYE-CO) (DSMZ Medium 316) Composition per liter: Trypticase ..........5 with KOH.0mL.............2............ Mix thoroughly..0L................ Add distilled/deionized water to 1............. and Na2S·9H2O solution..................10.5g NaCl ..........15g Resazurin ........................................6g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............0mg D-Ca-pantothenate .......0mL of sterile Na2S·9H2O solution...... Mix thoroughly......................................5g Yeast extract............................ 70....0mL of sterile NaHCO3 solution......................0............10...0mg Na2MoO4·2H2O ............................... Mix thoroughly............................................................................... except cellobiose solution....................0................0..................................025g KAl(SO4)2·12H2O ... Trace Elements Solution: Composition per liter: MgSO4·7H2O............ Filter sterilize.......................0.................0 ± 0.........................0g KH2PO4 ...... Preparation of Medium: Add components.......5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20.....................................01g CuSO4·5H2O.................... 0........ Sparge with 100% N2...........0mL..........0mg Thiamine-HCl·2H2O .......................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10............1g NiCl2·6H2O......................02g H3BO3 ........................... and Na2S·9H2O solution.........2g FeSO4·7H2O ......0mL Na2S·9H2O solution ..........................10.......... 20.....................2 at 25°C Vitamin Solution: Composition per liter: Pyridoxine-HCl ............................................20....................... Mix thoroughly......0..... Add distilled/deionized water and bring volume to 1......2......................................18g ZnSO4·7H2O.............0mg ZnCl2 .................3g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.....................0..........0mL Cellobiose solution ............................................ 0...................0mL Vitamin solution ........... 6.......1..................................2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ........... Dissolve by adjusting pH to 6..01g Na2SeO3·5H2O ............................................. 0......0mL of HCl solution...........................................0mL.......................1g FeSO4·7H2O ........................0g Yeast extract ....... Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O .................5........... 2. 1...............5............1..................5g CoSO4·7H2O..................................... 10.................................................................................................................................. except vitamin solution... 36...................0mL.................. Cellobiose Solution: Composition per 50..................................0mg H3BO3 .................................. and bring volume to 920.................................................3.....................................0mL......................................0mL NaHCO3 solution ..................5..................................................0 ± 0.......................... Autoclave for 15 min at 15 psi pressure–121°C.... Mix thoroughly..........................................................................3g MgCl2·6H2O .0mL of sterile cellobiose solution...........................................................................................................3.....................5............................0mg p-Aminobenzoic acid ..................10. Aseptically and anaerobically distribute into sterile screw-capped bottles under 80% N2 + 20% CO2................... Mix thoroughly.... and 10..0mL: NaHCO3 .........................

.........5g Yeast extract............................................................5.............0mL Na2S·9H2O solution .................. 0..5... Autoclave for 15 min at 15 psi pressure–121°C...............................0L.......... Use: For the cultivation of Moorella thermoacetica=Clostridium thermaceticum.....................................................................15g Na2SO4 ................................................0mL: NaHCO3 ... NaHCO3 Solution: Composition per 10.................. 0.......................03 g Preparation of Casamino Acids Solution: Add casamino acids to distilled/deionized water and bring volume to 10.. Mix thoroughly and sparge with 95% N2+ 5% CO2 gas..........................1................... 10..............0mL: Na2HPO4 .............0g Cellulose ............................... Mix thoroughly.....5.......2g Na2S2O3·5H2O .........0mL NaHCO3 solution.. Sparge with 100% N2 for 15 min..... Yeast Extract Solution: Composition per 10.....0mg Riboflavin................................................... 0...... 0............ 6................... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C......................................5....................................................... Na2S·9H2O Solution: Composition per 10....... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C..............................................................0.........0... Mix thoroughly.... Casamino Acids Solution: Composition per 10...............0mg ..........15mg Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10... Mix thoroughly.............. Distribute into tubes or flasks........................................................................ Sparge with 80% N2 + 20% CO2............................................... Pour into sterile Petri dishes or leave in tubes............0mg Folic acid ...........1............................... 0............................................21g CaCl2·2H2O....................................... 0....................................................................27g KH2PO4 ........................0mL: Casamino acids..........2..... and 10...................... 1..............................0mL.0mg pH 7.......5g CoCl2·6H2O................0.1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......... Mix thoroughly...................... 1.......................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........0mg Thiamine·HCl .0 ± 0...........................................0mL Casamino acids solution...0mg Vitamin B12........... 10......................................................5g MgCl2·6H2O....................................................Clostridium thermosuccinogenes Medium CO2........................................................... Use: For the cultivation and maintenance of Clostridium thermocellum...........25g FeSO4·7H2O...8 ± 0..2g Na2SO3 ....... Sparge with 80% N2 + 20% CO2......... 1............................................................ 10......................................................................................................... Sparge with 100% N2................. Use: For the cultivation and maintenance of Clostridium thermohydrosulfuricum.........0g Pancreatic digest of casein ......... Clostridium thermosuccinogenes Medium Composition per 1011..................0mL...0mL: Inulin .... and Thermoanaerobacter thermohydrosulfuricus..........5........4g KCl................ Autoclave for 15 min at 15 psi pressure–121°C............. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O........... 0........................03 g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.................................. Aseptically and anaerobically add 10........................0L under 95% N2+ 5% CO2 gas atmosphere................6g KH2PO4 .............0..........................................0mL Yeast extract solution .....................0mL vitamin solution...0mg pH 6.................0g NaCl ..................0g Sodium-beta-glycerophophate ..................................................................................................08g Resazurin .....13g Glutathione.0mL Trace elements solution SL-10..0mg Lipoic acid. Autoclave for 30 min at 15 psi pressure–121°C.2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........0mg Nicotinic acid ..................... 30...............................................................................3g CaCl2·2H2O........... 10......................................... 2................................................66g Preparation of Na2HPO4 Solution: Add Na2HPO4 to distilled/deionized water and bring volume to 20.0g FeSO4·7H2O.......... 0................................................. Sparge with 100% N2.......2................ 0............... 10........................................ 1...2 at 25°C Na2HPO4 Solution: Composition per 20......................................... Mix thoroughly...........................1g Resazurin .........3g NH4Cl.... 1..0mg Biotin ...0g K2HPO4 ............0mg Na2HPO4 solution ................. Autoclave for 15 min at 15 psi pressure–121°C.............0L................. 0.....0mL: Na2S·9H2O ....... Adjust pH to 7................................................. Vitamin Solution: Composition per liter: Pyridoxine·HCl........10................................................... Mix thoroughly. 1..0g Yeast extract...... Aseptically and anaerobically distribute into sterile tubes or flasks..2......0mL pH 7. Clostridium thermohydrosulfuricum Medium Composition per liter: Sucrose........................................................... 10..........................................0mL of sterile Na2S·9H2O solution..... 1........190....................................... Clostridium thermocellum Medium (LMG Medium 42) Composition per liter: Agar ............................5...... Thermoanaerobacter ethanolicus...........20......................... 2......43g (NH4)2SO4 .0mg Calcium DL-pantothenate ............................. 5.............0................................................0mL. 5.....................0mL................1mg Resazurin ......................... 10.....0mg p-Aminobenzoic acid .........1 ± 0.... 4........ Autoclave for 15 min at 15 psi pressure–121°C.........0mL................ Clostridium thermosaccharolyticum........... Gently heat and bring to boil- ing..............0mL 113 Vitamin solution ..................................0mg Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10....................................... 0.................... 1..................................... Autoclave for 15 min at 15 psi pressure–121°C...........0mL: Yeast extract ....... Mix thoroughly................... Mix thoroughly.......2g MgCl2·6H2O........... 10.........

......0mg p-Aminobenzoic acid ...02g CoCl2·6H2O .......0................ 1.......3g NaCl ........................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 50.......... and 1...... Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C..........................0mL Vitamin solution......5g (NH4)2SO4 .................0mL of sterile NaHCO3 solution...6..... 10........0L.............................. 10........... 0..........5..........1g CaCl2·2H2O .. 10....10................ 0...0mL of distilled/deionized water.....0mg Thiamine·HCl ........0mg Folic acid ............... Mix thoroughly................10......................1....... 1............................ Adjust pH to 7......0......017g H3BO3 ..........................0mL.................... 0..............0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.................................... 0..................5........................................0g FeCl3·4H2O.................1.................................. Use: For the cultivation and maintenance of Thermoanaerobacterium thermosulfurogenes..................75g Sodium thioglycolate..............0mg Riboflavin..0 ± 0..............0 with 1N HCl before use....................... Aseptically and anaerobically distribute into tubes or bottles............0L............................. Mix thoroughly.... 70............... Add remaining components. Aseptically and anaerobically add 50.... Add distilled/deionized water and bring volume to 1.......... Add remaining components.................................0132g Resazurin (1.............0.... casamino acids solution.............. CM4 Medium Composition per liter: Cellobiose...0mL............................................0L. 0........................... Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C..0mg Biotin ............. Mix thoroughly.............. Aseptically and anaerobically distribute into tubes or bottles.......................0.....................0mg Vitamin B12...................................................................01g Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500........5............ Autoclave for 15 min at 15 psi pressure–121°C.............................0mg Glucose solution ....................0mg NiCl2·6H2O ...................................... 24...............1g ZnCl2 .. Clostridium thermosulfurogenes Medium Composition per 1015...0mL of sterile vitamin solution........................................0mg Calcium DL-pantothenate ...........................................0mg Nicotinic acid ............................ Adjusting pH to 6.....2................ Na2S·9H2O Solution: Composition per 10........................................0mL of sterile Na2S·9H2O solution.................................0mL........0g MgCl2 .......... 0......... and bring volume to 940.................................0mg Na2MoO4·2H2O ...0mL: D-Glucose............5... Add distilled/deionized water to 1.0mL pH 6............................................ Gently heat until boiling.........................................0mL Trace elements solution .... Vitamin Solution: Composition per liter: Pyridoxine·HCl..... 0.......... Aseptically and anaerobically add 20............................................. NaHCO3 solution............0mL: Na2S·9H2O .... 0.......................................01g Na2MoO4·2H2O....................0mL of HCl solution.................0mL Na2S·9H2O solution........................5g NaCl .........0mL of sterile casamino acids solution.......5........................................... Use: For the cultivation and maintenance of Clostridium thermosuccinogenes................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1............... 10......................... indicating a reduced state..3g MgCl2·6H2O................ 2...........10......0mL.......0g KH2PO4 ........................... Cool......2 at 25°C Glucose Solution: Composition per 50...........25% solution).......................9g KH2PO4 .... Mix thoroughly.......0mL of sterile trace elements solution SL-10. ...............2g MnCl2·4H2O ............. Autoclave for 15 min at 15 psi pressure–121°C..................0g K2HPO4 ...... Pour into sterile Petri dishes or leave in tubes........... 0...... 5...10........................................... 0.................................. 1....................... Boil until color changes from red to colorless............................................ vitamin solution............. Preparation of Medium: Add components..........02g Na2SeO3 .........0mg HCl (25% solution) .........................02g CuCl2 ....................0g Yeast extract.........114 Clostridium thermosulfurogenes Medium MnCl2·4H2O... 5. Sparge with 100% N2.........0mL: Na2HPO4·12H2O.0% solution) ....................................0mL of sterile Na2S·9H2O solution..................................................... Adjust pH to 7....... 1................ Sparge with 100% N2....0mg H3BO3 ............................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10..........................................1mL Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................................................................................................50..0mL of sterile glucose solution and 10........ 1............................... Cap with rubber stoppers............... Na2S·9H2O solution......5............................ 100............0g Yeast extract ........................0mL of sterile yeast extract solution..............5mg Resazurin ..5........ 36................. 10.............. Mix thoroughly.2g FeSO4·7H2O...................... Autoclave for 15 min at 15 psi pressure–121°C............. except glucose solution and Na2S·9H2O solution...............................................2mL FeSO4 (1...........................0mg CuCl2·2H2O .................................0mg ZnCl2 ....................... Distribute into tubes or flasks under 97% N2 + 3% H2........................................................ Sparge with 80% N2 + 100% CO2........ Mix thoroughly.........5...........................................................0mg Lipoic acid.. 0.2........3g NH4Cl.............................. 0...... Autoclave for 15 min at 15 psi pressure–121°C.... to distilled/ deionized water and bring volume to 930........................0mL of sterile Na2HPO4 solution...............................................................................................2.....5 with KOH.. Sparge with 100% N2......................... Trace Elements Solution: Composition per liter: Nitrilotriacetic acid ... 0......... Mix thoroughly...... Sparge with 80% N2 + 100% CO2.................1.....5g CaCl2 ........................................ 6.. except Na2HPO4 solution... Use: For the cultivation and maintenance of Clostridium species and other bacteria that can utilize cellobiose as a carbon source........................................................................ and trace elements solution SL-10..0................... Sparge with 100% N2............ yeast extract solution......................................................0L....0.................... Preparation of Medium: Add components....... 12......

.........................................................................0g Na2SO4·10H2O .......................0g Chitin......................0g Ammonium tartrate .............. Cool to 50°C..0mL..........5.........0mg pH 7................................................7g MgSO4·5H2O ............... 0...............10.............. Pour into sterile Petri dishes or leave in tubes..... Colloidal Chitin Agar Composition per liter: Agar ......01g MnCl2 ............................. Distribute into tubes or flasks..........0 ± 0... Lysobacter brunescens..................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....0mL of sterile trimethylamine solution......................................................................2 at 25°C Salt Solution: Composition per 500mL: Na2HPO4 ..................... colloidal .2.2g NaCl ....... Hyphomicrobium species............... 1. Hyphomicrobium facilis...........................75g Yeast extract ........................0mg FeCl2·4H2O.10.................... 1.......................0.0.. Mix thoroughly...............0g Preparation of Trimethylamine Solution: Add trim- ethylamine to distilled/deionized water and bring volume to 10.. and Methylophilus methylotrophus...........0mL Thiamine solution....0mL of sterile salt solution and 1..............0........ Gently heat and bring to boiling..0g KH2PO4 ........ noble.... Aseptically add 10....... 0................... Mix thoroughly............................ 8.............. Use: For the cultivation of Aminobacter aminovorans...........................0g KH2PO4 ...... 0............ Mix thoroughly.75g Malt extract ...........0mL of sterile thiamine solution......... 1.................................. and other Lysobacter species................... Mix thoroughly...........2 at 25°C Trimethylamine Solution Composition per 10.............. 1... Adjust pH to 7...... Lysobacter gummosus.... Mix thoroughly.................. Hyphomicrobium zavarzinii......0µg H3BO3 .. Pour into sterile Petri dishes or leave in tubes...............................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...... Mix thoroughly...2. Methylobacterium extorquens.... Use: For the cultivation of Lysobacter antibioticus.......0L....... Use: For the cultivation of Aminobacter aminovorans...0 ± 0..0g pH 7................8 ± 0..................... Aseptically add 25. 25. and Methylophilus methylotrophus....2............................ Cool to 45°–50°C.....................................................3 ± 0.......................0mg Preparation of Thiamine Solution: Add thiamine to distilled/deionized water and bring volume to 100.62g (NH4)2SO4 ........ 0.......................... Hyphomicrobium aestuarii.........................0g K2HPO4 ....0mL pH 6.........0µg CoCl2·6H2O ......2g KH2PO4 ..... Gently heat and bring to boiling.........10... Hyphomicrobium species........................................... except salt solution and thiamine solution....5g KH2PO4 ............. Preparation of Medium: Add components........................ 0..... Mix thoroughly......................................4g 115 CaCl2·6H2O .........0g Asparagine...... 8.............................. For the germination of spores of Micromonospora species... Distribute into tubes or flasks....0µg Na2MoO4·2H2O .................................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......................... Ba- cillus species........................ Gently heat and bring to boiling...0µg MnSO4·5H2O ..1g CaCl2·6H2O...... Hyphomicrobium facilis................................ Thiamine Solution: Composition per 100mL: Thiamine.................................... Mix thoroughly..........................60g Salt solution.......5g MgSO4·7H2O ...................................................10..................10........0mL pH 7..........0g Pancreatic digest of casein .... Autoclave for 15 min at 15 psi pressure– 121°C............... 0............ 1...5....................... ............................................................................................5...........................................................0g Glucose........... Gently heat and bring to boiling... soil...... Preparation of Medium: Add components...... 20............ Hyphomicrobium variabile.0 ± 0.6g Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 500mL..............3g FeSO4·7H2O...........0µg FeCl3·6H2O ...............................0mg ZnSO4.....0L..0g Pancreatic digest of casein ............... Lysobacter enzymogenes.....0L......................................... Distribute into tubes or flasks...10....................... Hyphomicrobium variabile.................0mg pH 7..........0µg CuSO4·5H2O ......... 4......0L.................. or sediment............ 0.20....... Methylobacterium species......................... Hyphomicrobium zavarzinii...................... 29.................................................45........................ Autoclave for 15 min at 15 psi pressure–121°C..... 20.................... 0.................................. Mix thoroughly.......................................0mL: Trimethylamine................................... Pour into sterile Petri dishes or distribute into sterile tubes.... Use: For the isolation and cultivation of Micromonospora species from water........... Filter sterilize.....................0.......... Cook’s Cytophaga Agar Composition per liter: Agar .............0g Pancreatic digest of casein ......................0mL...........0g K2HPO4 . 0..20.......................................... Coprinus Medium Composition per 1026mL: Agar ....... Mix thoroughly........... Pour into sterile Petri dishes or distribute into sterile tubes.....05g ZnSO4·7H2O ........... to distilled/deionized water and bring volume to 990... Methylobacterium extorquens................. Autoclave for 15 min at 15 psi pressure–121°C...................................... Autoclave for 15 min at 15 psi pressure–121°C............................................... 1.................Coprinus Medium Colby and Zatman Agar Composition per liter: Agar...0g MgCl2·6H2O..................................................... Filter sterilize........................ except tri- methylamine solution..33..................................10......... Autoclave for 15 min at 15 psi pressure–121°C.......................................................0mg Trimethylamine solution ............... Filter sterilize.............................................. Methylobacterium species................. to distilled/deionized water and bring volume to 1...........70.......... 20.........0mL...... Colwella psychroerythrus Medium Composition per liter: NaCl ............. 5..................... Ba- cillus species............................... Hyphomicrobium aestuarii..........................

.....0L....0mL: Na2S . 0............................................................ 40..0mL: African Violet soil ..... 100. Pour into sterile Petri dishes or leave in tubes................................. 200..................0g Preparation of Solution B: Add K2HPO4·3H2O to distilled/deionized water and bring volume to 1.. Filter through Whatman #1 filter paper..............................0mL Resazurin solution.......................... Add 10........ 0.0mL of mineral salts solution with pipets which have been flushed for a few times with 100% N2.......................... 100.............. Mix thoroughly..0g Preparation of NaOH Solution: Add NaOH to distilled/deionized water and bring volume to 1. Heat to 60°C for 1 hr...... Add the Na2S.................. 2...... Add agar to filtrate......2...........0g pH 7......... 10mg NaMoO4·2H2O...................... Add 50................... One hour prior to inoculation.................................... Autoclave for 15 min at 15 psi pressure–121°C... 20mg H2SeO3 ..............................................................2g Preparation of Resazurin Solution: Add resazurin to distilled/deionized water and bring volume to 100.............0 ± 0.....2g Preparation of Soil Extract: Add components to 200. Sparge with 100% N2 for 10 min. Mix thoroughly................ Mix thoroughly.................50....5g CoCl2·H2O .......0L.......................................0L.............0mL: 100.............0g of Trypticase.......................0g tion with 100% CO2 for 30 min..............................................15....................... Preparation of Gelatin Solution: Gently heat Use: For the cultivation of Coprothermobacter proteolyti- NaOH Solution: Composition per liter: NaOH ....................................77.............0L Gelatin solution....... Mix thoroughly..................... Gently heat and bring to boiling....7............................. 10mg H3BO4 ... 40mg Na2WO4·2H2O ................................116 Coprothermobacter proteolyticus Medium Use: For the cultivation and maintenance of Coprinus cinereus.................... Na2S Solution: Composition per 100...............0L of NaOH solu- Gelatin...... Autoclave for 10 min at 15 psi pressure–121°C............................. Leave overnight in refrigerator.................. Pour into sterile Petri dishes or distribute into sterile tubes....... Mix thoroughly. cus................. Boil for 5 min.........0g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1..2 at 25°C Preparation of Medium: Add cornmeal polenta to distilled/deionized water and bring volume to 1.............................6mL Solution A ............0mL: Resazurin . Autoclave for 15 min at 15 psi pressure–121°C..... 0................. 100 ml Preparation of Na2S Solution: Gently heat 100............... Mix thoroughly............... Sparge with 100% N2 for 15 min..0mL Solution B ............0mL of medium.................. Preparation of Medium: Sparge 1............. Mix thoroughly.. Autoclave for 15 min at 15 psi pressure–121°C................5mL Solution B: Composition per liter: K2HPO4·3H2O ..................... Adjust pH to 4 with HCl......... Add the gelatin................................. 0...... 2......... Gently heat and bring to boiling...... Gently heat and bring to boiling.....15g MnCl2·4H2O...................113.. Sparge with 100% N2 for 10 min............... 30mg CuCl2·2H2O ....................0mL............................ 3...... .....1mL: Yeast extract..................................0g NaOH solution ... Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C. Coprothermobacter proteolyticus Medium Composition per 1168..............0g of yeast extract and 2..............................................1g AlCl3·H2O .................................................... and Trichophyton violaceum.............0mL Na2S solution .................... Anaerobically distribute 9.0mL of solution A....................0mL.................0g CaCl2·2H2O...........22...0g Cornmeal polenta ......................................................................0mL of soil extract.......... 2.............................5g Distilled water.......... Continue boiling for 30 min.....0g Na2CO3 ......... Add agar...0mL of distilled/deionized water to 100°C.......... Use: For the cultivation and maintenance of many filamentous fungi....1g ZnCl2 ................ 2.......0g Trypticase.. 20mg NiSO4·H2O........................ Cornmeal Agar with Soil Extract Composition per liter: Cornmeal .........0mL of solution B.................................................... Add 2...... 0...... Adjust pH to 3 with HCl..........0.........................0......0mL volumes into anaerobic tubes fitted with butyl rubber stoppers.................................... 4............. Filter through paper and reserve filtrate.............. Distribute into tubes or flasks..................... Autoclave for 60 min at 15 psi pressure–121°C............................ add 1..0L.. Solution A: Composition per liter: NH4Cl.................................1g FeSO4·7H2O...........................................0L with distilled/deionized water.....0L.................................... 50..................................................................................5mL of resazurin solution...................................... Mix thoroughly...................2mL of sterile Na2S solution to each 9...............0mL Mineral salts solution.........................10.........10.......................0g MgCl2·H2O...............0g Agar ........ Gelatin Solution: Composition per 100...........0mL Soil Extract: Composition per 200.................. Sparge with 100% N2 for 15 min.............. Bring volume to 1..0.... Resazurin Solution: Composition per 100..................0mL of distilled/deionized water to 80°C......................... 0........... and 10.... 10mg Preparation of Mineral Salts Solution: Add components to distilled/deionized water and bring volume to 1.............................5g Soil extract..............0mL of sterile gelatin solution and 0. Mineral Salts Solution: Composition per liter: EDTA·2H2O .................. Preparation of Medium: Add cornmeal to distilled/ deionized water and bring volume to 800......................1.. Dendrophoma obscurans.................................... Mix thoroughly..0mL of distilled/deionized water.......... Mix thoroughly.20... Cornmeal Agar (CMA) Composition per liter: Agar ............

..........0 at 25°C Preparation of Medium: Add components. Gently heat and bring to boiling......... Mortierella humilis....0...... Mix thoroughly...................................................... Gently heat until boiling...................... 15...............65. Autoclave for 15 min at 15 psi pressure–121°C........2 ± 0.................0L.........0mL Na2S·9H2O solution ...1....................................... except pimaricin and vancomycin......0mL pH 7.............. Cornmeal Yeast Extract Seawater Agar (ATCC Medium 2422) Composition per liter: Instant Ocean ....................10... except pimaricin and vancomycin...... tryptic digest .. Aseptically add pimaricin and vancomycin.........0L.5... Gently heat until boiling........... Mortierella minutissima.. Rhodococcus fascians. solids from infusion .. Filter through cheesecloth......... Pour into sterile Petri dishes or leave in tubes.... 15..400...0mL pH 7.... Mycobacterium vaccae......1g Pimaricin ................0g CaCl2·2H2O ...... Bring volume to 1........................0g pH 7.................0mg pH 5.... Add agar......... to distilled/deionized water and bring volume to 1....0L............... Sporolactobacillus inulinus........... Autoclave for 15 min at 15 psi pressure–121°C..... Microsporum distortum....0g Preparation of Medium: Add cow manure to tap water and bring volume to 1........3 ± 0...... Gently heat and bring to boiling....... Corynebacterium glutamicum................................................ Pour into sterile Petri dishes or leave in tubes.......2.4g Resazurin ..........................................................0g Glucose........................... Use: For the cultivation of Streptomyces species...............0g NH4Cl ...... Brevibacterium linens.....5................. Corynebacterium ammoniagenes................. 17..0L with tap water......0g Vancomycin .. Rhodococcus equi............ Use: For the cultivation and maintenance of fungi. Use: For the cultivation and maintenance of Nesterenkonia halobia=Micrococcus halobius..0g Agar ..........................0L.......0g Cornmeal infusion............ CP Medium for Coprothermobacter proteolyticus Composition per 1010... and Nigrospora sphaerica.................0g deionized water and bring volume to 1.............................................. Mix thoroughly.....6–6....................... 1. Distribute into tubes or flasks.......................... Filter through Whatman filter paper..0g Yeast extract ....2 at 25°C Cornmeal Phytophthora Isolation Medium No............. Cow Manure Agar Composition per liter: Cow manure .. 15......0g NaCl .....................................4g K2HPO4·3H2O ..... 0...........0L.0L with distilled/deionized water..... Distribute into tubes or flasks.0L...... Mix thoroughly...0g MgCl2·6H2O ...................................6–6..10.... 0. Gently heat and bring to boiling.........................................10......................... Mix thoroughly......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.............. 2. Gently heat and bring to boiling....................................................................................................................0...... 0...2. Return volume to 1. 2.... Corynebacterium Agar Composition per liter: Agar ..................................0 at 25°C Preparation of Medium: Add components................. Gently heat and bring to boiling..........................01g pH 5.0g Yeast extract......15..... 5...................0g Peptone .......................... Microbacterium laevaniformans......0g Cornmeal....5mg Gelatin solution .................... and yeast extract to 400............ Use: For the cultivation of Phytophthora species............. Autoclave for 15 min at 15 psi pressure–121°C............................. Mix thoroughly..... 100..........................2 at 25°C .......................................CP Medium for Coprothermobacter proteolyticus 117 Use: For the cultivation and maintenance of Helicodendron tubulosum.......0mL: NaHCO3 .. Use: For the cultivation and maintenance of Brevibacteri- um helvolum. Deinococcus radiodurans..................... Cellulomonas cellasea.. Distribute into tubes or flasks..................0 liter........... Adjust pH to 7........5 at 25°C Cornmeal Infusion: Composition per liter: Yellow cornmeal ......0mL Wolfe’s mineral solution ....................................... 0........... Mortierella hygrophila......................... Preparation of Medium: Add instant ocean... 0......0......... to distilled/deionized water and bring volume to 1.0 ± 0.... solids from infusion .. Corynebacterium Medium with Salt (DSMZ Medium 229) Composition per liter: NaCl ................................. 2 Composition per liter: Agar .......0mL cornmeal infusion and bring volume to 1.. 50....3g Pentachloronitrobenzene (PCNB)....................... Beef extract .........5g Agar ................. Simmer for 10 minutes... Pour into sterile Petri dishes............................................................. Boil for 1 hr.... Autoclave for 15 min at 15 psi pressure– 121°C........... Pour into sterile Petri dishes or distribute into sterile tubes........ other Corynebacterium species........... and Streptococcus mutans.............. Mix thoroughly.... 10..........4g Pancreatic digest of casein ...........025g Pimaricin ....................................................................... Use: For the cultivation of Phytophthora species....8...........15..... Autoclave for 15 min at 15 psi pressure–121°C...........0g Agar .......2g Pentachloronitrobenzene (PCNB).... Cornmeal Phytophthora Isolation Medium No.......................0g Vancomycin ........................0g Casein peptone.............50.................................2–7..1..0g Preparation of Cornmeal Infusion: Add cornmeal to distilled/deionized water and bring volume to 1............................. Filter through cheese cloth...........................0g Cornmeal.................0g pH 7....................... 10...........5....... 15........................ Autoclave for 15 min at 15 psi pressure–121°C.................................. Mix thoroughly......2.... Curtobacterium flaccumfaciens................. Aseptically add pimaricin and vancomycin............................. Corynebacterium callunae.... Brochothrix thermosphacta.............. Pour into sterile Petri dishes or leave in tubes........................................ 1 Composition per liter: Preparation of Medium: Add components to distilled/ Agar .............. agan.. Gently heat and bring to boiling............0g Yeast extract . Pour into sterile Petri dishes.............................

..............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.2 at 25°C Use: For the cultivation of myxobacteria..... 0...........3.............5mg pH 7........... 0.......15........... Mix thoroughly...............3............. Agar .......................0 with sterile HCl......................... 0..........0mL of sterile Wolfe’s mineral solution to each tube...0g Yeast extract ...................................2...... Distribute into tubes or flasks........................................ Adjust pH to 7....8............0g Yeast extract ........................2................0mL of distilled/deionized water................ Aseptically combine the two solu- Sucrose ..... Use: For the cultivation of Coprothermobacter proteolyti- cus.......... Mix thoroughly............................... neutralize to pH 7..................................... Preparation of Medium: Add agar................ CT Medium Composition per liter: Agar ...................0mL pH 7........................................... Mix thoroughly.. 0..........01g H3BO3 ............. 10...5g KCl .1.......0g NaNO3 ...6 ± 0. Sparge with 100% CO2 for 20 min........................................ Na2S·9H2O Solution: Composition per 10..........0mL of sterile gelatin solution....0g CaCl2·2H2O .............................0mL..........0g Pancreatic digest of casein .3........ Autoclave for 15 min at 15 psi pressure–121°C........ 3...........6) ..0.118 CT Agar Gelatin Solution: Composition per 100........... except gelatin solution................... Distribute into tubes or flasks................... Preparation of Medium: Combine components................15.. Mix thoroughly.............0g Cyanocobalamin................................... Aseptically and anaerobically add 100...........0. Mix thoroughly...... 0. Na2S·9H2O solution... Gently heat and bring to boiling...................................500..........0mL pH 7.......1g CaCl2·2H2O.5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............... pH 7...................6...5g CYC Medium Composition per liter: .........0g K2HPO4 ........ Pour into sterile Petri dishes or distribute into sterile tubes..... Sparge with 100% N2............... Cool to room temperature while sparging with 100% CO2........................................... Autoclave for 15 min at 15 psi pressure–121°C.....21g Potassium phosphate buffer (1 mM.. Add remaining components................................... Pour into sterile Petri dishes or leave in tubes.........2 ± 0......................6 ± 0. Preparation of Medium: Prepare medium anaerobically under 100% CO2........0g Pancreatic digest of casein ...................... Adjust pH to 7... vitamin free ...........0g Preparation of Gelatin Solution: Add gelatin to distilled/deionized water and bring volume to 100.......................................... Cool to 25°C.................01g Na2MoO4·2H2O .............................0g Casamino acids......0g Yeast extract .... Mix thoroughly.. Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ............ CY Agar Composition per liter: Agar ............................1g NiCl2·6H2O ..............................0g Preparation of Magnesium Sulfate Solution: Add MgSO4·7H2O to 10......... 0.................6..01g Na2SeO3·5H2O ...............5g CoCl2·6H2O ............... 20........0mL: Na2S·9H2O..............0mL of sterile Na2S·9H2O solution................................0mL of distilled/deionized water... 0.......0L...... Aseptically add sterile components.. Gently heat and bring to boiling...0g Tris(hydroxymethyl)aminomethane buffer ........0mL................ Mix thoroughly.......15...5 with KOH........ Mix thoroughly.......... 0...............0g Pancreatic digest of casein .. Autoclave agar–pancreatic digest of casein-MgSO4·7H2O solution and potassium phosphate buffer solution separately for 15 min at 15 psi pressure– 121°C...0L Magnesium sulfate solution .............0mL......................................10................. and Wolfe’s mineral solution..................................10................................................................. to distilled/deionized water and bring volume to 880........................5g NaCl ............................................... Gently heat until boiling.. Pour into sterile Petri dishes or leave in tubes.......................0mL............................... 2... 0.... 0.......................0mL: MgSO4·7H2O....................................................... Use: For the cultivation of myxobacteria... CTT Medium Composition per liter: Agar .............. Mix thoroughly................... Add distilled/deionized water to 1.......................... pH 7............. 10....0g Potassium phosphate buffer (0... Mix thoroughly. Adjust pH to 6........0mL: Gelatin............................................................... Pour into sterile Petri dishes or leave in tubes................... CT Agar Composition per liter: tions. pancreatic digest of casein.............0g Nitrilotriacetic acid .0g MgSO4·7H2O.................. Use: For the cultivation of myxobacteria............0.................... Autoclave for 15 min at 15 psi pressure–121°C...... Autoclave for 15 min at 15 psi pressure–121°C.....................0g MgSO4·7H2O ....................................... Autoclave for 15 min at 15 psi pressure–121°C. 1..................................................... 3................3mg Preparation of Wolfe’s Mineral Solution: Add ni- trilotriacetic acid to 500..1g ZnSO4·7H2O .......... Distribute into tubes or flasks..................................1g FeSO4·7H2O............. 0. and MgSO4·7H2O to distilled/deionized water and bring volume to 500...0L....... Gently heat and bring to boiling.................. Add components..96g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....1......................................................... and 10............................. Gently heat and bring to boiling..30...............................6).. Adjust pH to 6..........02g CuSO4·5H2O ..........................0g MnSO4·2H2O ....................1................... 20............... Sparge with 100% N2..... Before use......1................025g KAl(SO4)2·12H2O......2 at 25°C Magnesium Sulfate Solution: Composition per 10.......0................ 1...................................02M solution................ 0..........................5g MgSO4 ...0L.................... Autoclave for 15 min at 15 psi pressure–121°C....0....................... 1.......... Use: For the cultivation and maintenance of Stigmatella aurantiaca...........0g Pancreatic digest of casein ...

.. to distilled/deionized water and bring volume to 999................................................................0g .............5g KCl.....CYE-ACES Agar FeSO4·7H2O............... Mix thoroughly......0mL pH 6....... 2.. Use: For the cultivation and maintenance of Thermomonospora curvata........................................... Cyclohexanone Medium Composition per liter: NH4NO3..................... Pour into sterile Petri dishes or leave in tubes.........................0.....................................15..0L..0mg ZnSO4·7H2O ...........0g K2HPO4 .............................................0g KH2PO4 .... Preparation of Medium: Add components........................ Gently heat and bring to boiling..................................... to distilled/deionized water and bring volume to 990.......................... Mix thoroughly........................... Cool to 50°C. 10..... Cyclohexanecarboxylic Acid Medium Composition per liter: K2HPO4 ..................................0g KH2PO4 .. 2............ Mix thoroughly..................2..................................................5g Yeast extract ..................0g MgSO4·7H2O ..........01g pH 7..........1 at 25°C Solution A: Composition per 490mL: Yeast extract ...................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................. Distribute into tubes or flasks..................................................................................... Autoclave for 15 min at 15 psi pressure–121°C.................................. 0................. 1............. 0.0mL Solution D ........................0L........................................0.... Cyclohexanecarboxylic Acid Agar Composition per liter: Agar..........................0g MgSO4·7H2O...................................01g FeCl3·6H2O............................................................................................... Gently heat and bring to boiling.....................0g Yeast extract...2g Yeast extract................................. 10...................... Mix thoroughly......................................................... and Thermobifida alba..............01g NaMoO4·7H2O .......................................................................2................................ Autoclave for 15 min at 15 psi pressure–121°C.......................1g FeSO4·7H2O................................................................................ 1........... 5.....1..................................................... Autoclave for 20 min at 15 psi pressure–121°C.....................................5g Tryptophan ....................... Autoclave for 15 min at 15 psi pressure–121°C.......... Mix thoroughly......... 1............... Mix thoroughly........0mg CaCl2·2H2O.............. 10.... Filter sterilize............ 0. Mix thoroughly.......... Distribute into tubes or flasks............ 490........ Filter sterilize cyclohexanone..................................2 at 25°C Antibiotic Solution: Composition per 10.. Saccharopolyspora thermophila.. CYC Medium....................0L....0mg MnSO4·4H2O .3.........5g NH4NO3........ Aseptically distribute into sterile tubes...... Thermoactinomyces vulgaris........................0............. Mix thoroughly.............................025g Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10.. Aseptically add 1................ Adjust pH to 7........................... 1.........................1.........1g NaNO3 ................. except an- tibiotic solution.....................0..................... 15... CYE-ACES Agar (DSMZ Medium 585) Composition per liter: Solution A ...........01g FeCl3·6H2O.................................................. 0.............. Aseptically add sterile antibiotic solution..... 2............................ 6...... 2............0g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 490.................. Mix thoroughly...... Distribute into tubes or flasks............. Thermobifida fusca.... 0.....................................02g FeSO4·7H2O... Cross and Attwell Modification (DSMZ Medium 550) Composition per liter: Sucrose..2 ± 0..........2g CaCl2·2H2O ... Use: For the isolation and cultivation of Thermoactinomy- ces species.........0mg Cyclohexanone .. 30........................................................ Distribute into tubes or flasks.......................... Autoclave for 15 min at 15 psi pressure–121°C....... Gently heat and bring to boiling......10... 15.................................................0mL: Cycloheximide .............. 0.5g Cyclohexanecarboxylic acid ............... Cool to 45°–50°C.3.........0mL...........0 ± 0..............0..............0g Cyclohexanecarboxylic acid ...0mL...0mL Solution C...10......0g MgSO4·7H2O .............0mL........1..0...........................................0..........................................................0g Activated charcoal...........................................................................2 ± 0.........1g CaCl2·2H2O .....................................0mL....................0mg pH7........................................... Autoclave for 15 min at 15 psi pressure–121°C.............................. 0............. Pour into sterile Petri dishes or leave in tubes...01g ZnSO4·7H2O... 1.........01g Antibiotic solution ......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....................................................25g MgSO4·7H2O..............................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.... Use: For the cultivation and maintenance of Nocardia species and other bacteria that can utilize cyclohexanone as a carbon source.................................2 ± 0.0mL of cyclohexanone.......................0mL pH 7.........0g (NH4)2SO4 ..............................................0................... except cyclohexanone.......9 ± 0....10............0g ACES (N-2-acetamido-2-aminoethanesulfonic acid).... 0........ Use: For the cultivation and maintenance of Alcaligenes faecalis and other bacteria that can utilize cyclohexanecarboxylic acid as a carbon source.......05g Novobiocin.............. 490........0mL Preparation of Medium: Add components..........................0g Agar ......................................0g K2HPO4 ........ noble........0......01g pH 7....0g K2HPO4 ........................................................................ 3..... 0........ 119 Use: For the cultivation and maintenance of Corynebacterium cyclohexanicum and Saccharomyces cerevisiae.2.......................0g Casamino acids ........0mL Solution B..................................................... Solution B: Composition per 490mL: Agar ...........

Fe4(P2O7)3 Solution: Composition per liter: Fe4(P2O7)3.. The mixture dissolves readily in a 50°C water bath........ This is a critical step............................0mL: L-Cysteine·HCl·H2O.. Swirl medium in flask during dispensing to petri dishes or tubes to keep charcoal suspended.0g Pancreatic digest of casein ......0mL solution A.....5mg Glucose solution . 0.................... 10......................... and Xylella fastidiosa...0. 0.. Mix thoroughly.......... Autoclave for 15 min at 15 psi pressure–121°C... Filter sterilize............ Autoclave for 15 min at 15 psi pressure–121°C.......0mL: L-Cysteine·HCl·H2O ....0 N KOH........0–4........ Autoclave for 15 min at 15 psi pressure– 121°C................ Preparation of Medium: Add components......0g Yeast extract ..... 10....... L- cysteine·HCl·H2O solution and Fe4(P2O7)3 solution. acid-washed............... L-Cysteine·HCl·H2O ..........1... Gently heat to boiling. Mix thor- CYG Agar Composition per liter: Agar ...... 17....0g Yeast extract..... Legionella longbeachae............. Mix thoroughly..0mL... Filter sterilize.05 at 50°C by adding 4...............0mL of sterile Fe4(P2O7)3 solution.....0 to 4... acid-washed ...............0mL..... CYE Agar (Charcoal Yeast Extract Agar) Composition per liter: Agar ..........0mL: D-Glucose ..................... Pour in 20...... Mix thoroughly............................0g CaCl2·2H2O .............................2...................0mL...0mL.. Solution D: Composition per 10mL: Fe4(PO4)2........................10.........0mL solution C and then 10.....2 ± 0.............0g ACES buffer (N-2-acetamido2-aminoethane sulfonic acid) .. Buffered) Composition per liter: Agar ........ Adjust the pH 6................ to distilled/deionized water and bring volume to 980.... Preparation of Medium: Add 10...........10............................................0mL........3........0mL Fe4(P2O7)3 solution..... Finally... The mixture dissolves readily in a 50°C water bath............. Cool to 50°C.0mL volumes into sterile Petri dishes...... add 490......... Mix thoroughly.......0mL solution D to 490................... to distilled/deionized water and bring volume to 980.....0 N KOH...1......... Adjust pH to 6... Mix thoroughly............. Pour into Petri dishes or aseptically distribute into sterile tubes.......9 ± 0.0mL.. Mix thoroughly........0mL pH 7......... Filter sterilize........ 100...... Add 10........9 ± ...5mL of 1... 0....................0mL solution B...... Preparation of Medium: Add components.... CYE Agar. Filter sterilize..0g L-Cysteine·HCl·H2O solution .... Gently heat and bring to boiling............................. 2........ The soluble Fe4(P2O7)3 must be kept dry and in the dark..0mL volumes into sterile Petri dishes..0g Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100............ Mix thoroughly... Autoclave for 15 min at 15 psi pressure–121°C.....................9 ± ..................5 ml of sterile 1...0mL pH 6....... 10....5mL of 1........... Mix thoroughly..120 CYE Agar Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 490. Store in the dark.0g L-Cysteine·HCl·H2O solution . Pour in 20.....25g Preparation of Solution D: Add Fe4(PO4)2 to distilled/ deionized water and bring volume to 10.......10.. Heat to 5055°C to dissolve Fe4(PO4)2..............................0mL.....0........... Mix thoroughly............ The pH of the medium is critical. Do not use if brown or yellow..... This is a critical step...0mL Fe4(P2O7)3 solution .....0N KOH...................................0mL. Use: For the cultivation and maintenance of Legionella species and Xylella fastidiosa..... The soluble Fe4(P2O7)3 must be kept dry and in the dark..... Cool to 25°C..............4g Preparation of L-Cysteine·HCl·H2O solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0...0mL of sterile Fe4(P2O7)3 solution............ Prepare solutions freshly..... Swirl medium while pouring to keep charcoal in suspension.............0mL..... Do not use if chemical loses its green color and becomes brown or yellow..10.................15........... Legionella pneumophila................05 at 50°C L-Cysteine·HCl·H2O Solution: Composition per 10..............9 at 50°C by adding 4....9 at 50°C by adding 4. activated..... Cool to 50°C.....0mL of sterile L-cysteine·HCl·H2O solution and 10...0mL...................... Filter sterilize................. Do not heat over 60°C to dissolve............... Gently heat to boiling......................0g Yeast extract . Adjust pH to 6.... ...... activated............. Mix thoroughly................... Use: For the cultivation of Afipia clevelandensis.. Cool to 50°C........0g Cyanocobalamin....17....... Do not use if brown or yellow........ Prepare solutions freshly..........................5.2 at 25°C Glucose Solution: Composition per 100....................0g Charcoal..25g Preparation of Fe4(P2O7)3 Solution: Add soluble Fe4(P2O7)3 to distilled/deionized water and bring volume to 10........0mL of sterile L-cysteine·HCl·H2O solution and 10........ Buffered (Charcoal Yeast Extract Agar..05 at 50°C L-Cysteine·HCl·H2O Solution: Composition per 10..... Filter sterilize..................... except L- cysteine·HCl·H2O solution and Fe4(P2O7)3 solution..... Add 10......... Swirl medium while pouring to keep charcoal in suspension...... Mix thoroughly......0–4........................ Afipia broomeae.0mL pH 6...... Do not heat over 60°C to dissolve.... Mix thoroughly........25g Preparation of Fe4(P2O7)3 Solution: Add soluble Fe4(P2O7)3 to distilled/deionized water and bring volume to 10....... Fe4(P2O7)3 Solution: Composition per liter: Fe4(P2O7)3 .....4g Preparation of Solution C: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.....4g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10....... Solution C: Composition per 10mL: Use: For the cultivation and maintenance of Legionella species and Tatlockia micdadei...... 0........ except oughly.... Afipia felis.....0g Charcoal.

........................................... 30.........57g (NH4)6Mo7O24·4H2O .... and Na2S·9H2O solution..............................................5.....2mL pH 7..5g Yeast extract ............................1..........0 with KOH................. Autoclave for 15 min at 15 psi pressure–121°C.............................28g MnSO4·6H2O........0.........................2 at 25°C ..........2 ± 0.................15........ Cool to 45°–50°C..................1g CaCl2 ........ Mix thoroughly..0mL.... Autoclave for 15 min at 15 psi pressure–121°C.....5......... Aseptically add 10................................................ 1....... Herpetosiphon species....0..................30......5g NH4Cl....................0g Yeast extract....0............................................. Cool to 50°– 55°C................ Filter sterilize.............1g Preparation of Vishniac and Santer Trace Element Mixture: Add components to distilled/deionized water and bring volume to 1..... except glucose solution.....................................................0g Agar .............. and 1.......... Autoclave for 15 min at 15 psi pressure–121°C.......................................................... Gently heat and bring to boiling....................0g MgSO4·7H2O ............................................................0mL: NaHCO3 .......................0.................................5....... to distilled/deionized water and bring volume to 900...2................................3g Na2S·9H2O ................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.......................................... Mix thoroughly.............0L....................0g CaCl2·2H2O .............0..........1............................0............ 1.......02g Cu(NO3)2·3H2O.......................... 15....................................................... 50............................5g CaCl2·H2O. Pour into sterile Petri dishes or distribute into sterile tubes............................0.......................................2 at 25°C Vishniac and Santer Trace Element Mixture: Composition per liter: Ethylenediamine tetraacetic acid (EDTA) ..... Distribute into tubes or flasks.. Mix thoroughly.......... Use: For the cultivation of Cytophaga agarovorans..............................1.... Mix thoroughly........................ Gently heat and bring to boiling........................1....0mL...0mL: Na2S·9H2O ..... Use: For the cultivation of agar-digesting Cytophaga fermentans... Mix thoroughly..... Mix thoroughly...........0mL Na2S·9H2O solution....Cytophaga hutchinsonii Agar Preparation of Medium: Add components..........0g MgCl2·6H2O ........ 1... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................................0mg FeCl3·H2O .......0mL..........0g Pancreatic digest of casein ............ Saprospira species..........0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100................................... 1........................................................................................ Autoclave for 15 min at 15 psi pressure–121°C............. Cytophaga hutchinsonii Agar Composition per liter: Agar ..02g Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 100...............25mg Glucose solution ............ Filter sterilize................. Pour into sterile Petri dishes or leave in tubes...... NaHCO3 solution..................................................................10.....................0...................................1................. 1........2 at 25°C Trace Elements Solution: Composition per 100.......... Cytophaga Agarase Agar (ATCC Medium 793) Composition per liter: Agar . 1.........0 ± 0.............................. Adjust pH to 6.0g Cellobiose solution .........04g Ferric citrate (4mM solution) .................06g FeSO4·7H2O................................. 5.................................0g Agar ................................................ 50...... 0.....................................................................................025g CoCl2·6H2O..0mL: NaCl ....0L...36g Yeast extract .. 50......................0..... Mix thoroughly......................0g KH2PO4 ....0g KH2PO4 ........ 10.............. 1..............................0mL: D-Glucose..0mL NaHCO3 solution ..1..........54g MnCl2·4H2O........................................................... Aseptically add sterile glucose solution.....................10.......................0......2 ± 0...........0mL: H3BO3 ...............................................0g NaHCO3 . except glucose solution............................................. and Flexithrix species..... 0..................................................0mL of sterile NaHCO3 solution............. Adjust pH to 7.........0mL Trace elements solution......................0mL pH 7................................... Filter sterilize.........................................0mL pH 7............ to distilled/deionized water and bring volume to 980..... 2.............. Mix thoroughly........................................ Mix thoroughly....................0mL.........0g MgSO4·7H2O ............ 1......0mL of sterile glucose solution...................... 121 NaHCO3 Solution: Composition per 100.................0mL..0g KH2PO4 ..... 15....0mL of sterile Na2S·9H2O solution............................................ 22...................................61g CuSO4·5H2O . 1..0g ZnSO4·7H2O ...........21g Na2MoO4·2H2O................ Mix thoroughly................. Preparation of Medium: Add components..............0g CaCl2·2H2O................................... 1.....................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10..................... Use: For the cultivation and maintenance of Cytophaga flevensis....... Use: For the isolation and cultivation of Cytophaga spe- cies....... 5.....0g NH4Cl .....................0....................... 5................ Distribute into tubes or flasks.......................0g NH4Cl............075g Zn(NO3)2·6H2O .....0g CaCl2 .............. Cytophaga agarovorans Agar (LMG Medium 99) Composition per 1001....................... Na2S·9H2O Solution: Composition per 10.....................................0mL........ 0.....................................................99g CoCl2·6H2O . 4..............0mL Glucose Solution: Composition per 10.......... Pour into sterile Petri dishes or distribute into sterile tubes.... Cytophaga fermentans Medium Composition per liter: NaCl . Gently heat and bring to boiling..0L...........02g Vishniac and Santer trace element mixture........................................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....3..................................

...................... except su- crose.....0mL........ Preparation of Medium: Add components.............................. Autoclave for 15 min at 15 psi pressure–121°C..................... Saprospira grandis................... Autoclave for 15 min at 15 psi pressure–121°C.........................................0mL............................................... Filter sterilize... and sterile Na2S·9H2O solution......0mL.......0g MgSO4·7H2O .........10... except so- dium bicarbonate and calcium chloride solution to distilled/ deionized water and bring volume to 900............................ to distilled/deionized water and bring volume to 889............ Autoclave for 15 min at 15 psi pressure– 121°C.2g Yeast extract.................0mL......... Czapek Dox Agar.........................................................10....0g Yeast extract . Autoclave for 15 min at 15 psi pressure–121°C.............3 ± 0........................................ Mix thoroughly. Marinilabilia salmonicolor.... Cool to 50°–55°C.... 0..0mL sterile calcium chloride solution...........15..........0...............0mL Glucose solution ......................... Microscilla sp......................0mL: NaHCO3 .......0g KH2PO4 ...................5g CaCl2 ...... Aseptically add sterile NaHCO3 solution.......1............................. 24.......0mL: D-Cellobiose............................................................. Mix thoroughly...................1....................... Preparation of Medium: Add components..25mg NaHCO3 solution......................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100......0g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 100............0mL.................... Pour into sterile Petri dishes or distribute into sterile tubes... 1..........0mL: NaHCO3 ... Mix thoroughly....0....... Flexibacter tractuosus..0mL pH 7........ 1.......................................2 at 25°C Sodium Bicarbonate Solution: Composition per 10..................................0mL... Persicobacter diffluens......... Cool to 50°C..1..10......30............... add sucrose to distilled/deionized water and bring volume to 100........5g FeSO4·7H2O ....5.............. except NaHCO3 solution........... Mix thoroughly.... Mix thoroughly.3. 1..........0mL..................... Mix thoroughly.. Use: For the cultivation and maintenance of Cytophaga aurantiaca and Cytophaga hutchinsonii..... 100..... except cellobiose solution........ sterile glucose solution..0mL............ Cytophaga marinoflava....... to distilled/deionized water and bring volume to 950.............. Aseptically add 10..................................................... Use: For the cultivation and maintenance of Streptomyces species.... Pour into sterile Petri dishes or distribute into sterile tubes............... and Cellulophaga lytica...................10.................... Mix thoroughly..........0g NaNO3 ......................122 Cytophaga Marine Medium Cellobiose Solution: Composition per 50...............0....0mL: Na2S·9H2O ................................... 10.................0g KCl ............0mL Na2S·9H2O solution .......0g NH4Cl . Czapek Agar (ATCC Medium 312) Composition per liter: Sucrose .............................. 15....... Filter sterilize.......................6g CaCl2·2H2O.. Calcium Chloride Solution: Composition per 10......... Cool to 45-50°C.............................. Cytophaga Marine Medium (DSMZ Medium 172) Composition per liter: NaCl .......... Mix thoroughly..0mL Glucose Solution: Composition per 100....0g K2HPO4 .......... Adjust pH to 7.........................1........0g MgSO4 ......... 1.....2....2 at 25°C Preparation of Medium: Add components...0mL Calcium chloride solution ........................................................... For the cultivation of Actinoplanaceae..0mL: Glucose................0 ± 0. NaHCO3 Solution: Composition per 100.......................................0g Agar .... Modified Composition per liter: Sucrose .. Combine the sterile solutions.....................0............0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50.............. Na2S·9H2O Solution: Composition per 100.................... Autoclave for 15 min at 15 psi pressure–121°C......................................... Mix thoroughly.....................3g MgCl2·6H2O.... Marinilabilia salmonicolor.......................................... Aseptically add 50..... Pour into sterile Petri dishes or distribute into sterile tubes........ 0....................................... Autoclave both solutions separately for 15 min at 15 psi pressure– 121°C....................................... Mix thoroughly. Mix thoroughly.......... Use: For the cultivation and maintenance of Cellulophaga lytica.. In a separate flask. 6...........30.. 30...0g Tryptone .0mL........... Flammeovirga aprica............0mL: CaCl2·2H2O............... Mix thoroughly....................0g KCl..................... Flexithrix dorotheae........ 15......0mL................. Cytophaga Medium Composition per liter: NaCl .... Autoclave for 15 min at 15 psi pressure–121°C......... 6..........0g Agar ..................04g FeCl3·6H2O..............7g Sodium bicarbonate solution......0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100................01g pH 7....0mL of sterile bicarbonate solution and 10...................0......................2g Preparation of Calcium Chloride Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10................. glucose solution.............................................................................0mL.... Distribute into tubes or flasks............2g Preparation of Sodium Bicarbonate Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10............ 1...............................0mL of sterile cellobiose solution....0g .................. Pour into sterile Petri dishes or distribute into sterile tubes.. Gently heat and bring to boiling.................... Cool to 50°C.............1...........5g MgSO4·7H2O...................................7g Agar ......................... Preparation of Medium: Add components....................... Flexibacter litoralis.... Autoclave for 15 min at 15 psi pressure–121°C....................... Mix thoroughly... to distilled/deionized water and bring volume to 900..... 4...................... Mix thoroughly..... Cytophaga latercula...... Use: For the cultivation of Cytophaga agarovorans............... Gently heat and bring to boiling............................... and Na2S·9H2O solution................

..................... Autoclave medium and mineral oil separately for 15 min at 15 psi pressure–121°C....... 200....0g K2HPO4 .................. 10....0g Meat peptone ............5g K2SO4 ..............................................0g Agar ........... 5..................................2g Ethanol .0g MgSO4·7H2O .......... Gently heat and bring to boiling.......... 0.............0g Peptone ......... Preparation of Medium: Add components. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................ Autoclave medium and mineral oil separately for 15 min at 15 psi pressure–121°C.... Amino acid .....................Decarboxylase Medium..................................3 ± 0... After inoculation..... L-lysine............5g KCl.. deionized water and bring volume to 1...5g KCl............ For amino acid...0mg Pyridoxal .............0g NaNO3 .. 10...2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems................. Falkow Composition per liter: gal species...... Pour into sterile Petri dishes or leave in tubes....................8 ± 0......................................................... except min- Use: For the cultivation and maintenance of a variety of eral oil....................................01g pH 6..............5........ lysine.... Distribute into screw-capped tubes in 5............................01g pH 7..0L............................ Preparation of Medium: Add components to distilled/ 123 Source: This medium is available as a premixed powder from BD Diagnostic Systems..................5 ± 0.0L......0g NaNO3 ................................ 1..... except mineral oil.................................0g KCl......... Distribute into tubes or flasks............. Mix thoroughly..........5g MgSO4·7H2O .................. use L-arginine.... Use: For the cultivation and differentiation of bacteria based on their ability to decarboxylate the amino acid.......... to distilled/deionized water and bring volume to 1....... Pour into sterile Petri dishes or leave in tubes............................. or ornithine turn the medium turbid purple......................5..01g Cresol Red.................. Preparation of Medium: Add components.0L.0mL volumes...................0g NaNO3 ........... fungal and bacterial species that can use nitrate as sole nitrogen source.... 0..... 30..............0g Bromcresol Purple.. Bring volume to 100.......0g Peptone.35g FeSO4 .....................0mL pH 6.........0mL with distilled/deionized water........... 0....0mL volumes........................... Mix thoroughly.................... or L-ornithine..0mL pH 5.............0mL Preparation of Bromcresol Purple Solution: Add Bromcresol Purple to ethanol..... Autoclave for 15 min at 15 psi pressure–121°C... Distribute into screw-capped tubes in 5. or L-ornithine...............5g FeSO4·7H2O............................. lysine....................................0g Yeast extract ............................0mL of sterile mineral oil per tube............200. After inoculation............. 1.......... 3.....3............0.... Mix thoroughly............................. overlay medium with 1................ Adjust pH to 5........... 12.......................... Distribute into screw-capped tubes............................................ Bacteria that decarboxylate arginine.............2 at 25°C Bromcresol Purple Solution: Composition per 100.............02g Mineral oil ...............2 at 25°C Decarboxylase Medium......................................... Use: For the cultivation and maintenance of osmophilic fungi. Decarboxylase Base.............................................. overlay medium with 1....................................0L.......... 0........... Autoclave for 15 min at 15 psi pressure–121°C.......................... 0.... Ornithine Modified Agar ....................................................5 with HCl or NaOH............. 2.............0mL pH 6................ to distilled/deionized water and bring volume to 1.0mg Mineral oil............0mg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............3.5g FeSO4·7H2O............... L-lysine................ Gently heat until dissolved..............0g Beef extract ......................................... . Bacteria that decarboxylate arginine........ 20................................1.................... or ornithine turn the medium turbid purple....................................... Ornithine Modified Composition per liter: L-Ornithine .......... Sucrose...................... 0........ Mix thoroughly................ Distribute into tubes or flasks.....................5.................0g Glucose .............. Use: For the cultivation and differentiation of bacteria based on their ability to decarboxylate ornithine......0g Yeast extract .. 50..........0mL: Bromcresol Purple.......... 0.............0g Magnesium glycerophosphate ......0g Bromcresol Purple solution .8 ± 0.............................. 5.................................................................................................................... 5........ For amino acid.......0g Glucose........ 200...........................................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems... Use: For the cultivation and maintenance of numerous fun- Decarboxylase Medium Base................0L................2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath......... Mix thoroughly............ Bacteria that decarboxylate ornithine turn the medium turbid purple................. Czapek Dox Broth Composition per liter: Sucrose.................... Autoclave for 15 min at 15 psi pressure–121°C................ Distribute into tubes or flasks............... 3... 5........................................10............ 0.................5g Bromcresol Purple .................. Autoclave for 15 min at 15 psi pressure–121°C....................................... 0..........................0L.................................. Czapek Solution Agar with Sucrose Composition per liter: Use: For the cultivation and differentiation of bacteria based on their ability to decarboxylate the amino acid........0............................................ Møller Composition per liter: Amino acid.... 0....................... 5........ Mix thoroughly........................ Mix thoroughly......................0g K2HPO4 ........ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................ Mix thoroughly.............................. use L-arginine........0mL of sterile mineral oil per tube.......................................................... Filter sterilize.....0 ± 0......... 0..................

... 3...................2 at 25°C NaHCO3 Solution: Composition per 10...........7g Citric acid ...................................................................................... 25.04g NaCl ........0mL: NaHCO3 .............................................19g Na2MoO4·2H2O........................ Preparation of Medium: Add components.................................... 0................ Vitamin Solution: Composition per liter: Pyridoxine-HCl ..... TK6-3 Solution: Composition per liter: ZnSO4·7H2O.................. 0..........................0mL: Glucose.......... Adjust pH to 7........................0mL....0........................... 0...............................................18g CaCl2·2H2O........................0mg Folic acid.......0mL............................0g Na-acetate ......... 10......................... Defined Glucose Medium EMSY-1 Composition per liter: Na2HPO4 .0mL sterile vitamin solution and 10.................................0mg Riboflavin ........0mg Nicotinic acid .......... Mix thoroughly...............................................0............. Mix thoroughly.......................33g MgCl2·6H2O..........0mL TK6-3 solution ...04g H2SO4 (1N solution)..33g KCl.......................................0mL of sterile yeast extract solution....................0mg Biotin ....... 5........... 10.......0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.............................................................................................................. to distilled/deionized water and bring volume to 890. Defined Medium with Povidone Iodine Composition per 1025.................0....................... 5.......................... 5..............................................................0g Sulfur.. 0.. 0........4g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10............................. 5...........0mL distilled/deionized water.0mg Thiamine-HCl·2H2O .... Autoclave for 15 min at 15 psi pressure–121°C........... Sparge with 80% H2 + 20% CO2............ and sulfur to 980....................................15g NaHCO3 solution .............................................................................................................84mg Glucose solution ......................................... 0...................................... Mix thoroughly.. Aseptically distribute into sterile tubes or flasks... Autoclave for 15 min at 15 psi pressure–121°C............................... Mix thoroughly............ Anaerobically under 100% N2 distribute into tubes of bottles containing the sulfur (0......... 0..................................0mL.............................................. 0............... Aseptically add 100..........10......................0mg D-Ca-pantothenate.................5g CoSO4·7H2O ........0mL sterile NaHCO3 solution..... Filter sterilize...............................0mL Preparation of TK6-3 Solution: Add components to distilled/deionized water and bring volume to 1......... 1.........0mL ..............................0.... Gently heat and bring to boiling....1g FeSO4·7H2O...................................... Use: For the cultivation and maintenance of Xanthomonas campestris......0g MnSO4·2H2O ....0mg Lipoic acid ..................0.....................0mL of sterile glucose solution and 10............................................... Yeast Extract Solution: Composition per 10..................0g KNO3 ...............1g NiCl2·6H2O .0................ Mix thoroughly............ Filter sterilize..........2 ± 0........0mL pH 7........ Cool to 25°C........................... Cool to room temperature while sparging with 100% N2..........................................1g sulfur per 10mL medium)...............................................45g CuSO4·5H2O.2 at 25°C Glucose Solution: Composition per 100............................................. Dissolve by adjusting pH to 6..... Add distilled/deionized water to 1.......................1.....0mg p-Aminobenzoic acid .......................0g Nitrilotriacetic acid .......... except glucose solution and yeast extract solution.................18g ZnSO4·7H2O .............5g NH4Cl........ 10........01g Na2MoO4·4H2O . Gently heat and bring to boiling.....41g CaCl2·2H2O ............... 1......................... The final pH should be 7. 100............ Autoclave for 20 min at 110°C..................................... except vitamin solution....................0L........ Mix thoroughly..................................0L.................. Mix thoroughly.........................1.............. Add remaining components.79g KH2PO4 .. Filter sterilize......................................0.....................................0mg Vitamin B12 ................... 0..........................4.........................................................................01g Na2SeO3·5H2O ....... 10.............................. 0............5g NH4Cl ...........33g Yeast extract.....................................0 ± 0.. Mix thoroughly............3g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.....76g MnSO4·H2O...............33g CaCl2·2H2O..1.......................................0.......0mL pH 7.......0L.......................................025g KAl(SO4)2·12H2O....124 Deferribacter Medium Deferribacter Medium (DSMZ Medium 935) Composition per liter: NaCl .......10....0mL: Yeast extract .............................. 0..................0...... Must be prepared freshly....... 0. Trace Elements Solution: Composition per liter: MgSO4·7H2O ...............5g NaCl ....0...... 1......................................... 0.........................................0mL Solution A ..................0mL Vitamin solution......................................... 2.............................. Cool rapidly to 25°C............... 1................. 1............................................................0mL: Basal solution ......... 5...............................................0mL Yeast extract solution .........................................02g H3BO3 ............... 10.......................................33g KH2PO4 ...........5 with KOH................................ 0..03g FeCl3·6H2O............ Mix thoroughly....10..............0............................................................................................... 0.01g CuSO4·5H2O ................ 5.......................................0mL Trace elements solution ....................................................................................43g MgSO4·7H2O..0mL.. NaHCO3 solution......0mL of distilled/deionized water.... 0............................................................... 2..................0...................0L Solution B..........10.................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.......................... Aseptically and anaerobically add 10..............17g KI........................0mL Solution C.............................................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500........................................ powdered............0. Preparation of Medium: Add components...................... 0............... 0......................... Use: For the cultivation of Deferribacter desulfuricans and Deferribacter thermophilus...................... Boil for 3 min.....31g H3BO3 .................................................. 2.............................. 0....................................................... 0......................................... 5...

..................4g NH4Cl......... Defined Medium for Rhodopseudomonas Composition per liter: Malic acid ........................190........................................................................................................................ 0.....11g Preparation of Solution C: Add components to dis- tilled/deionized water and bring volume to 10................. 4............... 125 Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............................................................ 1................. Autoclave for 15 min at 15 psi pressure–121°C................................................................................................... Solution C: Composition per 100..... 100......................... 0.....9g KH2PO4 ............ Filter sterilize....0.......................0................................ 0.....................0mL Solution E .......... Cool to room temperature under 80% H2 + 20% CO2 gas............................. Close bottles............ Distribute 9 ml volumes into 50 mL serum bottles under 80% H2 + 20% CO2 gas....................................1mg Trace elements solution SL-10................19g ZnSO4·7H2O ................. Solution D: Composition per 10mL: Na2-EDTA ............... 0....................... Filter sterilize.................05g Cu(NO3)2·3H2O ...........................8 ± 0. Autoclave for 15 min at 15 psi pressure– 121°C... Mix thoroughly........................0mL Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O..............06g CoCl2·6H2O ..........................................................460g NaH2PO4·H2O ........36................................................................ 10.................................................................8g KH2PO4 .............7g MnSO4·H2O .........1........................................................ sterile bas- al solution..173g Peptone ..0 mL in bottles..................................1g Resazurin .............. Cool to 45°–50°C................................................. 0...... Preparation of Medium: To 1..... Gently heat and bring to boiling............................0mg ZnCl2 ........................................... Solution A: Composition per 100......................................................... 900........0...0mL...........0mL Solution F .................................................................5 bar overpressure...... 0.................................. 1....... Mix thoroughly.....1g Preparation of Solution C: Add povidone-iodine to distilled/deionized water and bring volume to 100..................................................0mL: D-Glucose......................0mg FeCl2·4H2O.....................................8............................................2 at 25°C Trace Elements: Composition per 250..........12g CaCl2·2H2O ... 10..............075g EDTA ........0mL... Adjust pH to 6........ Pour into sterile Petri dishes or distribute into sterile tubes.................0L.................653g Na-acetate............0..................................................................02g FeSO4·7H2O.........5g CoCl2·6H2O.................... aseptically add 5..0mg .............................0g Preparation of Solution B: Add glucose to distilled/ deionized water and bring volume to 100........... Flush solution with 80% N2 + 20% CO2 gas........................... Use: For the cultivation and maintenance of Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia....................................... Mix thoroughly....................................................... 0..... 4.1.....................................0mL: H3BO3 ...1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 100........0mL: Povidone-iodine .... Close bottles....Dehalobacter restrictus Medium Basal Solution: Composition per liter: Agar ..............6g MgSO4·7H2O ...............0.........................................0...................100............................0g K2HPO4 ...... 10...........0.........0mL......5.................0mL of sterile solution C.............................. Autoclave for 15 min at 15 psi pressure–121°C...........0mL pH 6..... 1..............0mL......................... Distribute into tubes or flasks......0mL Solution C..... 0...............0mL of sterile solution A......................... 4.. Mix thoroughly.....................................0g CaCl2·2H2O.................730g NH4HCO3 .... Mix thoroughly............................... 0........................................... and 10.....2 at 25°C Solution A: Composition per liter: K2HPO4 .... Solution B: Composition per 100........ Flush solution with 100% N2 for 20 min............ 0................4g Na2MoO4·2H2O .......................................................................0mL: Ferric ammonium citrate............. Autoclave for 15 min at 15 psi pressure– 121°C............................. 10............0mL Solution B............... Dehalobacter restrictus Medium (DSMZ Medium 732) Composition per 1046mL: Solution A ........ Mix thoroughly.......................015mg Trace elements ...0......................................... 0...............0L of cooled... 1........................ Autoclave for 15 min at 15 psi pressure–121°C.........................0 L Mix thoroughly........................ 0...................... Solution C: Composition per 10mL: MgCl2·6H2O .................0g MgSO4·7H2O ...................0g Na2HPO4 ...5..........................2g CaCl2·2H2O.0............................................5mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 1............................... Mix thoroughly... Gently heat and bring to boiling........ Pressurize closed bottles with H2 + CO2 gas to 0......3........................................0g (NH4)2SO4 ........... Solution B: Composition per 100mL: NaHCO3 .....0mL pH 7......443g Preparation of Solution B: Add components to distilled/deionized water and bring volume to 100...........0mg Na2MoO4·2H2O.0mg AlCl3.................. 0......01g Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 250........................................ 10......70..... Mix thoroughly........0L. 15................................ Mix thoroughly.......................................................... Filter sterilize.............5g Preparation of Basal Solution: Add components to distilled/deionized water and bring volume to 1.............................. 20.........0mL Solution D ........... 10.........0mg MnCl2·4H2O ............... 0............ 0..............................................012g Thiamine ..................2 ± 0... 0.....0mg Biotin ................................................... 1.0mL Solution G .0mL of sterile solution B.................... Use: For the cultivation and maintenance of Rhodobacter capsulatus...0 mL in bottles...........

.1................................................. Autoclave for 15 min at 15 psi pressure–121°C...0mL pH 7.2 at 25°C Source: This medium is available as a premixed powder and supplement from BD Diagnostic Systems..................0mL sterile tetrachloroethene to the 45............ 5.........................................0g Glucose......................026g NaHCO3 solution.......................... 6............................. Sparge with 100% N2.................100......... Mix thoroughly....0mL Preparation of Hexadecane: Add hexadecane to a 100mL serum bottle. Mix thoroughly.. Autoclave for 15 min at 15 psi pressure–121°C.......................................................0mg Thiamine-HCl·2H2O ............ 200......................10..........5 ± 0....4.0mg Calcium pantothenate ..... Sparge with 100% N2..0g Acriflavin·HCl.... 2........ 5............................................................................................0mg Vitamin B12 ... to distilled/deionized water and bring volume to 990... Mix thoroughly................... Preparation of Medium: Add 9mL sterile solution A to a 50 mL sterile bottle........................................... Mix thoroughly.......................10............. Sparge with 100% N2.........0mL of sterile NaHCO3 solution.0...................................... 10.............................................0mL: Na2S·9H2O..................0mL: NaHCO3 .... Solution E: Composition per liter: Vitamin solution.0mg Vitamin B12 ......................................0mg Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......................................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10............. Then add by injection 1............0g Proteose peptone No... 100........... FeCl2·4H2O.......... Deleya halophila Medium Composition per liter: NaCl ................2 ± 0.................................... 45....................... 5......................................................................... Autoclave for 15 min at 15 psi pressure–121°C................ 100.................... 80...................................................................... 0........................................0mg p-Aminobenzoic acid .................. Tetrachloroethene: Tetrachloroethene ..........0mL Vitamin Solution: Composition per liter: Pyridoxine-HCl ........................................3....................... Mix thoroughly..............2....................126 Deleya halophila Medium NiCl2·6H2O .................... Aseptically distribute into sterile tubes or flasks.....................0mL Tetrachloroethene........3g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....................... 10....3................................... Preparation of Solution D: Add Na2-EDTA.............................. Mix thoroughly............................5............0mL sterile hexadecane in the 100mL serum bottle.........35g Esculin ................................................0g KH2PO4 . Adjust pH to 7........1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..45.................................... Add remaining components.............. 0...6g Yeast extract .......................... Cool to 50°C. ........2 at 25°C NaHCO3 Solution Composition per 10............... 2.....0mg Ferric ammonium citrate supplement. Demi-Fraser Broth Composition per liter: NaCl ........0L...........................0mg Lipoic acid ............................................... Filter sterilize.................. 200........................................................0mg Thiamine-HCl·2H2O . 0.... except NaHCO3 solution...................10....... Preparation of Solution E: Combine 900...... Add distilled/deionized water and bring volume to 1..12..... Use: For the cultivation of Deleya halophila......... 5......... 2................ Autoclave for 15 min at 15 psi pressure–121°C........... Mix thoroughly............................................... Solution G: Hexadecane ....0g KCl ..0mL of HCl solution......................0mL Preparation of Tetrachloroethene: Add tetrachloro- ethene to a 10mL serum bottle........................ Solution F: Composition per 10..................0mL pH 7....1mL sterile solution E..........0mg H3BO3 ...........0mg D(+)-Biotin ...0g MnCl2 ......................... Aseptically add 10................... Autoclave for 15 min at 15 psi pressure–121°C...........................................................0mL seven vitamin solution..........................0mL Seven vitamin solution....1mL sterile solution C....0mg HCl (25% solution) ..0mL vitamin solution and 100................. 5............................0g LiCl............19.......... Close bottles............................................ 10...06g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.........15mL sterile solution G.....47g NaBr ..........0mL sterile solution B..0L.......... Preparation of Medium: Add components.5...................1.....10..... 10............. 300....... Inoculate the culture into the medium.................................................0mg Nicotinic acid . Sparge with 100% N2..................................0L................ Sparge with 100% N2...........0mg CuCl2·2H2O ..... Use: For the cultivation of Dehalobacter restrictus and Sulfurospirillum halorespiran................................................... aseptically inject 5...... Hexadecane: Hexadecane ....0 mL trace solution SL-10 in a Hungate bottle.........0mL.......1mL sterile solution F....0mg Folic acid.........5..........................900...................................... and 0....................0.5mg Nalidixic acid ..............0mL...............0g Na2HPO4 ..... Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...................................................................0mg p-Aminobenzoic acid .......... Flush solution with 100% N2 for 20 min..............20........0g MgSO4 . 24.......................01mL sterile solution D..........................................0mg D-Ca-pantothenate...................................0g Yeast extract ....... 20.. Sparge with 100% N2............................ Then add by injection 0...........0mg Biotin ... Mix thoroughly........................ and AlCl3 to 10......................... Autoclave for 15 min at 15 psi pressure–121°C........0mg Nicotinic acid ......... Sparge with 100% N2........... 0................ Filter sterilize.0mL Preparation of Solution G: Using a syringe..................5.0g Tryptose...........5..... 5........ Mix thoroughly.................................................0........0g CaCl2 ..................... 0.....................81........... Autoclave for 15 min at 15 psi pressure–121°C......9.......6g Beef extract ...0mL........ Mix thoroughly.......................1................................0mg Riboflavin ....................

0..................0g Ferric citrate .................0mL of HCl solution............................................................................6.................................... 0.....0g Lactose ..................... to distilled/deionized water and bring volume to 949........ Preparation of Medium: Prepare and dispense medium under 100% N2 gas atmosphere.....................0g Pancreatic digest of casein ....................................................................Deoxycholate Agar 127 Ferric Ammonium Citrate Supplement: Composition per 10........36............ Aseptically add 10.......033g pH 7.................15g Na2SO4 ................... Aseptically and anaerobically add 10.0mg ZnCl2 ..................................................... Autoclave for 15 min at 15 psi pressure–121°C.5g CoCl2·6H2O.10.. 1... Deoxycholate Agar Composition per liter: of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.......................... 100.................. Mix thoroughly........25g CaCl2·2H2O..................... Do not autoclave. Cool to room temperature........................ Sparge with 80% N2 + 20% CO2.16.................................................0L....................................0mg NiCl2·6H2O.....................100...0mL.................. Denitrovibrio Medium (DSMZ Medium 881) Composition per 1032mL: NaCl ...2 at 25°C NaHCO3 Solution: Composition per 10...........1....................................................................5............0mg Thiamine-HCl·2H2O ........ Autoclave for 15 min at 15 psi pressure–121°C............ 10... Mix thoroughly.........1........64g Preparation of Na-Acetate Solution: Add Na-acetate p-Aminobenzoic acid . Use: For the cultivation of Listeria species from environmental samples..................0g Neutral Red . Adjust pH to 6.......0g KH2PO4 ... Escherichia coli appears as large............................ Use: For the selective isolation.. Sparge with 100% N2... Gently heat and bring to boiling....................5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10......................... Sparge with 80% N2 + 20% CO2......... Pour into sterile Petri dishes.................0mL NaHCO3 solution......................0mg HCl (25% solution) .....................0L.. Cool to 45°–50°C..24... Filter sterilize................ 200..................0mg Nicotinic acid ......0mL................. Add remaining components............... 1.0mL: Na-acetate .................. pale colonies with a pink center.......................0... Add components......5.....0mL...............0mL Trace elements solution SL-10 ......................... Preparation of Medium: Add components.................................................................0mL Na-acetate solution...............5g NH4Cl.................. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C....0mL Na2S·9H2O solution... 200............. Na-acetate solution................ Mix thoroughly...20.........0mL.......... and trace elements solution SL-10........... Enterobacter and Klebsiella species appear as large.............................. Sparge with 80% N2 + 20% CO2.........................0g NaNO3 ................................................................................10.............................................0mg Vitamin B12 ....0mL.............0mL: D(+)-Biotin ....... to distilled/deionized water and bring volume to 990. Filter sterilize.......0mL of sterile ferric ammonium citrate supplement........ 0. Mix thoroughly................0mL NaHCO3 solution ... Mix thoroughly....5g water and bring volume to 10. Mix thoroughly.1......0mg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.7g KCl........................8-7.............. Mix thoroughly. 1.... ................. cultivation..... colorless to tan colonies....... Mix thoroughly............02g Resazurin ....................... 20...........................................2 at 25°C Na-Acetate Solution: Composition per 10.................. Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O..2.0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10............ enumeration.......... Add distilled/deionized water and bring volume to 1.0mL Na2S·9H2O solution... rose-red colonies.. Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride .................................................... 0............ Mix thoroughly...0mL seven vitamin solution..................10........0mg Calcium pantothenate .....8-7...............................5...................... Filter sterilize............... except NaHCO3 solution...............0g MgCl2·6H2O......... 0...... to distilled/deionized water and bring volume to 10..................................................................0mg MnCl2·4H2O ...................... 3.... flat...0g K2HPO4 . Agar ........ 2...0g NaCl ......................................................0mg Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O......0g Sodium deoxycholate ....................10..0L.0mL trace elements solution SL-10............. Ferric ammonium citrate.............. and differentiation of Gram-negative enteric microorganisms from a variety of clinical and nonclinical specimens.............0mg CuCl2·2H2O... Shigella species appear as colorless to pink colonies.......... 300.... 0.....................................................................0mL pH 6.1........... Use: For the cultivation of Denitrovibrio acetiphilus. Filter sterilize....................0mL.. 10..........................................3 ± 0... Mix thoroughly.... 0............ 10.................... mucoid.....70..............................0mL Seven vitamin solution... except fer- ric ammonium citrate supplement................0mg Preparation of Ferric Ammonium Citrate Supplement: Add ferric ammonium citrate to distilled/deionized Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................................. Proteus and Salmonella species appear as large. Mix thoroughly.....5g Preparation NaHCO3 ........... seven vitamin solution............80. 0.........................0g Peptic digest of animal tissue .................... Autoclave for 15 min at 15 psi pressure–121°C..... Mix thoroughly............2.......1... and 1.............................0mL: Source: This medium is available as a premixed powder from BD Diagnostic Systems.. 100..... Aseptically distribute into sterile tubes or flasks.................................................... Na2S·9H2O solution.....0mg Na2MoO4·2H2O.............................................. Sparge with 100% N2................2...........................................0g Sodium citrate ...... Aseptically and anaerobically distribute into sterile tubes or bottles..0mg H3BO3 ..........................190...............1........5mg Na-acetate solution .............................

.....0mL... rose-red colonies..... 15............ 0............200................................................................................................................ Proteus and Salmonella species appear as large................................... 5........... .0g Agar .........0g Beef extract ..... Deoxycholate Citrate Agar Composition per liter: Sodium citrate ...... Autoclave for 15 min at 15 psi pressure–121°C.0mL pH 7.................... Autoclave for 15 min at 15 psi pressure–121°C...........5......................................2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath.........................................0mg Thiamine-HCl·2H2O .......................................24..1g KH2PO4 .0g Lactose ......................................... cultivation.0g Sodium deoxycholate...................... 10........... especially Salmonella and Shigella species.............................190... Do not autoclave..................... 10............................... Autoclave for 15 min at 15 psi pressure–121°C..................... flat..................................0g NaCl .....36.....................0mg NiCl2·6H2O........................ Shigella species appear as colorless to pink colonies.................... Mix thoroughly........ Do not autoclave...................0g Sodium deoxycholate........................... 1...... 0........................................0g Yeast extract..................... colorless to tan colonies.................0L.............5g Ferric citrate ...................................0g Glucose . 5.................... 10.70........2.................... pale colonies with a pink center................... 5...............................0g Preparation of Solution C (NaHCO3 Solution): Add NaHCO3 to distilled/deionized water and bring volume to 100...0g Ferric citrate ........................ 1.............................3 ± 0... Mix thoroughly.. enumeration.... Gently heat and bring to boiling.......0mg H3BO3 ....................5mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 920......0mg Nicotinic acid .................. 50....... Autoclave for 15 min at 15 psi pressure–121°C................................................... 5...............200.0g Peptone........... Pour into sterile Petri dishes.............0g Sodium citrate .....0g Na2S2O3·5H2O .........................0mL of HCl solution............................300.....0mg Vitamin B12.............56g KCl ..........100....................................................0mg D(+)-Biotin .5g CoCl2·6H2O..3 ± 0.. and differentiation of Gram-negative enteric microorganisms from a variety of clinical and nonclinical specimens............................ Mix thoroughly.. Solution D (Seven Vitamin Solution): Composition per liter: Dermabacter Medium Composition per liter: Pyridoxine hydrochloride ..1............ Deoxycholate Agar (Desoxycholate Agar) Composition per liter: Agar ........... 1............0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10...............08g Resazurin ....0g Lactose ........................................100.... Cool to 45°–50°C.....0mg CuCl2·2H2O..................................................................................0.... Mix thoroughly..... 10................ Distribute into tubes or flasks..................................... Add distilled/deionized water and bring volume to 1........................................0mL Solution F ......025g pH 7..........80............................................................0........................................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.............................................................0mL Solution B (Trace elements solution SL-10) ...0mg Preparation of Medium: Add components to distilled/ Preparation of Solution D (Seven Vitamin Solution): Add components to distilled/deionized water and Pancreatic digest of casein ................................................. Escherichia coli appears as large...................................................................4g MgSO4·7H2O............0mg ZnCl2 ..... 1...4 ± 0........ 1.....2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath and BD Diagnostic Systems.........................24g CaCl2·2H2O ..................... 2...................0L............................................ Add remaining components..........0mg p-Aminobenzoic acid .. Dry the agar surface before use.... Use: For the cultivation and maintenance of Dermabacter hominus..........0mg Na2MoO4·2H2O........... 13...........................................100................................. 5....................... Mix thoroughly............... Solution C (NaHCO3 Solution): Composition per 100...........128 Deoxycholate Agar Pseudomonas species appear as irregular colorless to brown colonies........................... mucoid... Pseudomonas species appear as irregular colorless to brown colonies...............0mg Calcium pantothenate ......................................... Mix thoroughly..................... 50... Desulfacinum hydrothermale Medium (DSMZ Medium 875) Composition per 1004mL: Solution A ..................0mL Solution D (Seven vitamin solution) ...................................0mL Solution E ......................................... 5..........0g K2HPO4 ....0g Neutral Red .............................. 10.................. Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O...10..........................2.......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........................................................................2 at 25°C deionized water and bring volume to 1.................................... Pour into sterile Petri dishes.........0L.......................... Mix thoroughly............................ 5.......................29g NH4Cl ...............0mg MnCl2·4H2O .......6........................ 10......... Sparge with 100% N2................................................. Sparge with 80% N2 + 20% CO2.............0mg HCl (25% solution) ............... 2....................................0mL: NaHCO3 ....0g NaCl .......... Use: For the selective isolation.............. Enterobacter and Klebsiella species appear as large......72g MgCl2·6H2O ......................................2................0L........ Gently heat and bring to boiling..............0g Neutral Red ....... 10....................................................0........................... Use: For the selective isolation and cultivation of enteric pathogens........ Cool to 50°C........ 920........3 at 25°C Solution A: Composition per 920mL: NaCl .............................. 15..............................................0g Peptone................ Sparge with 100% N2............0...............0mL Solution C (NaHCO3 solution).......0-7......0g pH 7....................................................0mL................................0.....................20........................03g pH 7....................

0mL: Na2S·9H2O . 10........2g Yeast extract...... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere.....................0mL..............0.............................5 ± 0........0-7....0mL..................................2g CaCl2 ..................................0mL of sterile solution A with 25........................... 1..................................... 0.............. Sparge with 100% N2..............................1g CoCl2·6H2O ...........0mL........ Mix thoroughly... 0....................................... 0.........................1g CaCl2 ................ 5...0g tilled/deionized water and bring volume to 100.............0L..................................5g FeSO4·7H2O ............................... Mix thoroughly.... Autoclave for 15 min at 15 psi pressure–121°C...........................................................3g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............... Sodium Pyruvate Solution: Composition per 10............... Mix thoroughly..............................................6g NH4Cl .......2g Preparation of Solution B: Add sodium pyruvate to distilled/deionized water and bring volume to 25.................................................................. Mix thoroughly....0....... Adjust pH to 6....................1...... Filter sterilize......................................0mL Sodium pyruvate solution.....10..Desulfitobacterium hafniense Medium bring volume to 1...................... Use: For the cultivation of Desulfitobacterium dehalogen- ans...... 1.......................0mL sterile solution D........................... 1.................................................................. Solution E: Composition per 10.. Sparge with 100% CO2.......0mL: NaHCO3 . Adjust pH to 7....0mg Lipoic acid................................................................0..0mL of sterile solution B and 20...0mL Na2S2O3 solution .................1g CuSO4·5H2O.............. 3................ Add remaining components one at a time........ Autoclave for 15 min at 15 psi pressure– 121°C...0g MnSO4·H2O.................................... 10.0mL sterile solution C.. Solution F: Composition per 100...............0mL sterile solution E...............4g MgCl2·6H2O ..................5g L-Cysteine·HCl·H2O ........ 0.......................................5g Preparation of Solution E: Add Na-lactate to distilled/ deionized water and bring volume to 10............................0.... 1............. except Lcysteine·HCl·H2O................................5 with KOH..........05g Resazurin ................ 2....0mg Thiamine·HCl ................3.................................0mL: NaHCO3 ... 0.....3........................................................ 10........... 2..........................1g CaCl2·2H2O .............. Cool to room temperature while sparging with 90% N2 + 10% CO2..................................955.......20..... Autoclave for 15 min at 15 psi pressure–121°C.................01g H3BO3 ......................... Preparation of Solution A: Add components........... Sparge with 100% N2.....0mL Solution C ................................0mg p-Aminobenzoic acid ................................ Add distilled/deionized water to 1............... 13........... Add L-cysteine·HCl·H2O.........0mL Solution B ................................7g NH4Cl..................0mL...............0mL Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O... Autoclave for 15 min at 15 psi pressure–121°C...................0mL sterile solution F................................0...................0mL: Na2S·9H2O................... Filter sterilize.......... 0........... Solution C: Composition per 20................................0mL sterile solution B.........0mL...................................... 0...............2 at 25°C Na2S·9H2O Solution: Composition per 10...........0g 3-Chloro-4-hydroxy-phenylacetic acid........................2....0mg Riboflavin........... 1.......... Mix thoroughly....1....................... 3.........1g ZnSO4·7H2O ......... Aseptically and anaerobically distribute into sterile tubes or flasks..............44g MgCl2·6H2O ...5..........0mg Wolfe’s mineral solution ...............5...... The pH of the completed medium should be 7................................ Desulfitobacterium hafniense Medium Composition per 1005... Autoclave for 15 min at 15 psi pressure–121°C......................5.. Filter sterilize........... to distilled/deionized water and bring volume to 955.......0L......0mL sterile solution A.0g Yeast extract ................... Add 50..........01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.............0mL: Sodium pyruvate ..................................1.................... 0.......................... Solution B: Composition per 25........................ 0................................................ Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 20...................................... Mix thoroughly...5mg Na2S·9H2O solution ......0mL Selenite-tungstate solution ............................ Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl.................................. 0......0g K2HPO4·3H2O ............. 0...................... Mix thoroughly.......................................... Mix thoroughly.......01g A1K(SO4)2·12H2O ......................0mL: Na-lactate .......................... Mix thoroughly........2.......0mL of sterile solution C............0g Nitrilotriacetic acid ....................................................................0...0g Preparation of Solution F: Add Na2S·9H2O to dis- 129 to boiling...............2.....01g Na2MoO4·2H2O.........0mL: Na2HPO4 .... 0........... Sparge with 100% N2. 0....................... 2...............5g NaCl . 10......... Sparge with 100% N2..5g Preparation of Sodium Pyruvate Solution: Add sodium pyruvate to distilled/deionized water and bring volume to 10.....5..................0mL Solution A: Composition per 955.......... to 920.. Sparge with 100% N2..........................0mL: Sodium pyruvate ..............0mL............... Gently heat and bring combine 955............................... Aseptically and anaerobically distribute into sterile tubes or bottles.. 10......0mL Trace elements solution SL-10 with EDTA .10. Mix thoroughly........ Sparge with 100% N2................ Desulfitobacterium dehalogenans Medium Composition per liter: Solution A ....... Preparation of Medium: Aseptically and anaerobically Use: For the cultivation of Desulfacinum hydrothermale..............................................................1g NaCl .............0mg ...0mL Wolfe’s vitamin solution ..................................0mg Nicotinic acid .. and 1...... 25....5g KH2PO4 ..............................................5..........................0mL pH 7.........................25.....0mL of distilled/deionized water.................................0mL.

.........0L..1... Cysteine Solution: Composition per 10................ Mix thoroughly...........10.................5g Preparation of Na-pyruvate Solution: Add Napyruvate to distilled/deionized water and bring volume to 10..................... 190.................................................................... Trace Elements Solution SL-10 with EDTA: Composition per liter: Na2S·9H2O Solution: Composition per 10mL: FeCl2·4H2O ............ 10............................1mL of sterile vitamin solution.............................................. Add remaining components...........0mL Cysteine solution ....................... Mix thoroughly.............. Na-thiosulfate Solution: Composition per 10........ Selenite-Tungstate Solution: Composition per liter: NaOH ....................... Cool to room temperature........... Na2S2O3 Solution: Composition per 10...........................2.......................................................................... 2.........................................................0mL: Na2S2O3·5H2O .... and 0.... Adjust pH to 7..0mg Riboflavin................................. Dissolve by adjusting pH to 6.....................0mL Preparation of Trace Elements Solution SL-10 with EDTA: Add FeCl2·4H2O to 10.......... 6........ 0............ 20............... 0...0mL Trace elements solution. 5. Filter sterilize............ 10...........................0............ Mix thoroughly.......................................1g CaCl2·2H2O .....................................................5..........................35g NaCl .......................................... 0......026g CuCl2·2H2O....0mg Folic acid..........................................0mL Na-pyruvate solution ...............................................................................1.................... Mix thoroughly..................................0............5 with KOH..5g CoCl2·6H2O ............... Mix thoroughly....0mg D-Ca-pantothenate ......2........ and Na2S2O3·5H2O solution................. Mix thoroughly.....5g Disodium EDTA ...0mL. Mix thoroughly...........0mg CuCl2·2H2O ................... Na2S2O3·5H2O to distilled/deionized water and bring volume to 10......0mg ZnCl2 .................................. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2...................0L.............. Add remaining components................0mg ............4g Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10..................................................... Sparge with 100% N2.......0....... Mix thoroughly.0mL.................... 0......0mL Na-thiosulfate solution ..............................................0mL of distilled/deionized water................ 2.......................0g NH4Cl...........032g Na2S·9H2O ........... 0.......... 0.........................................0mg Resazurin ....................... Filter sterilize................................................. Gently heat and bring to boiling... except NaHCO3..............0mg NiCl2·6H2O ...........................................130 Desulfitobacterium Medium Calcium DL-pantothenate.......... Filter sterilize...0mg MnCl2·4H2O.............. 0................0mL.......... Desulfitobacterium Medium (DSMZ Medium 663) Composition per liter: KH2PO4 ........................ Add distilled/deionized water and bring volume to 1........................... Trace Elements Solution: Composition per liter: Nitrilotriacetic acid..............0mL pH 7................................ 5............... vitamin solution........0..................024g H3BO3 ...........................0L.......................... Autoclave for 15 min at 15 psi pressure–121°C...........0mg Nicotinic acid ............5g Preparation of Na-thiosulfate Solution: Add 2....0mL: L-Cysteine·HCl·H2O............................... Sparge with 100% N2.......0............2 at 25°C Na-pyruvate Solution: Composition per 10.........0mg Na2MoO4·2H2O ..... Anaerobically distribute 9.24g NiCl2·6H2O..................0....05mL of sterile Na2S2O3·5H2O solution to each tube..........18g CaCl2·2H2O.... Vitamin Solution: Composition per liter: Pyridoxine-HCl .....10...............5........ Aseptically and anaerobically add 0.. 10............. 36.....................5g Na2WO4·2H2O ..... Use: For the cultivation of Desulfitobacterium hafniense...................................................... 70.................................0.0...... to distilled/ deionized water and bring volume to 970......... Add components..........5...0mL.......0mg Thiamine-HCl·2H2O .......................0mg H3BO3 .01g Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500......1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...............................8.......................................................... Autoclave for 15 min at 15 psi pressure–121°C................................5 ± 0................ Mix thoroughly........................................................5g Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1........ 5.... 1....0..................... Mix thoroughly.1mL of sterile sodium pyruvate solution.................................0mL............ Sparge with 100% N2.....................5mg Vitamin solution ................... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.. Add NaHCO3............ Mix thoroughly..........................44g Yeast extract................0............. Add distilled/deionized water to 1........ Mix thoroughly..............0mL................0L... 3... 2.......................... 10................... Continue boiling for 3 min.....................................................0mL: Na2S2O3·5H2O................... Na2S·9H2O solution............5g MgCl2·2H2O............................0g CoCl2·4H2O...................................8g FeCl3·6H2O...........0....................0mg Biotin ......12g MnCl2·4H2O ...............................................0mL Na2S·9H2O solution ....0mL: Na-pyruvate .........5.........................1g ZnCl2 .......... 4.......................................... 0.......................4g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10. sodium pyruvate solution..7mL volumes into anaerobic tubes...................0mg HCl (25% solution) ........ Adjust pH to 6.. Autoclave for 15 min at 15 psi pressure– 121°C..............0mg Vitamin B12 ................. Sparge with 100% N2........ 100...........................................................0mL of HCl solution........ Mix thoroughly.........025g Na2MoO4·4H2O.5g Preparation of Na2S2O3 Solution: Add Na2S2O3·5H2O to distilled/deionized water and bring volume to 10.....0mg Na2SeO3·5H2O ...........1g Na2SeO3·5H2O ......12..... Sparge with 100% N2. Cool to room temperature while sparging with 80% N2 + 20% CO2............. 1............ 2............0.............1mL of sterile Na2S·9H2O solution................... 24..............0.........................................

..................................0mL: KOH ..................................0mL Na-lactate .....................0mg Vitamin B12.. Autoclave for 15 min at 15 psi pressure–121°C....................................................0mL Na2S·9H2O solution............0mg Vitamin B12........0L.... 0.............................................0mg NiCl2·6H2O....................... Mix thoroughly..... 5........................... 4.................... 5.. Mix thoroughly......................0mg Nicotinic acid ....... NaHCO3 Solution: Composition per 100....................20......... Aseptically and anaerobically distribute into sterile tubes or flasks...............................................0........1g Yeast extract............. Mix thoroughly................. Sparge with 100% N2.......................200............ 0. 3.......0mL: NaHCO3 ................................ Mix thoroughly.......................................................................5. KOH Solution: Composition per 100....................... Cool to 25°C...10...................5g to distilled/deionized water and bring volume to 10..........................................................0mg ZnCl2 ......0mg Na2SeO3·5H2O .......... Sparge with 80% H2 + 20% CO2..0mL..........2...........................2..............0mL............0mg D-Ca-pantothenate .88g MgSO4·7H2O ...........25...................................................0mg HCl (25% solution) .........1.........0mg H3BO3 ............. cysteine solution.................... Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride .....................................................................................................0mg p-Aminobenzoic acid .....10.....................................................................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........0mL..... Sparge with 100% N2..................3.....0mL Selenite-tungstate solution ..................0L................................1...1....... to distilled/deionized water and bring volume to 940...........0mL...............0mg Na2S·9H2O Solution: Composition per 10mL: Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.................200.....0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1........................................... Autoclave for 15 min at 15 psi pressure–121°C................ ............... 0.....................0mg Biotin ....0mL of sterile Na2S·9H2O solution....................... Mix thoroughly........0mg Na2MoO4·2H2O..........................................0mL sterile Na-thiosulfate solution............ Na-pyruvate solution..Desulfitobacterium PCE Medium p-Aminobenzoic acid ..0mg Vitamin B12 ..................190.................... Desulfitobacterium PCE Medium (DSMZ Medium 717) Composition per liter: (NH4)H2PO4 ...0mL KOH solution .. Mix thoroughly...............20.. Sparge with 100% N2............... 2........5g Na2WO4·2H2O ........................... Use: For the cultivation of Desulfitobacterium dehalogen- ans...............5.6.. Filter sterilize... Add remaining components..... 0.0mg CuCl2·2H2O.................. Mix thoroughly..................................... 10..0L......................... and Na2S·9H2O solution.......0mL.............. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O....0mL Na-fumarate ........................5.................. Preparation of Medium: Prepare and dispense medium under an oxygen-free 100% N2....05g Resazurin .............1mg NaHCO3 solution ...................... Filter sterilize... Add components. 10...0mg Lipoic acid..........................0L... 0.......................................................0mL sterile vitamin solution.............0g Na-lactate ..............................................10......................0mL Vitamin solution.................... Filter sterilize.......20.............0mL: Na2S·9H2O...........0mg Thiamine-HCl·2H2O ... Filter sterilize.......0mL of HCl solution........ Mix thoroughly....................0mg Riboflavin........................... 0......1....................100..................... Sparge with 100% N2.........................................0mg Biotin .....................................0g Preparation of KOH Solution: Add KOH to distilled/ deionized water and bring volume to 100.....0mL of sterile cysteine solution 10................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......2..36............0mL sterile....................50..1g Ca(NO3)2·4H2O....1 ± 0......................... 5....................... 131 Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0L.......................... 2..300..............................................................0mL Trace elements solution ..0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10....... 10............................ Autoclave for 15 min at 15 psi pressure–121°C................................. 0.5....0mL pH 7........70...............................5..... Mix thoroughly................................ Sparge with 80% H2 + 20% CO2...100.......2 at 25°C Selenite-Tungstate Solution Composition per liter: NaOH .............. Preparation of Na2S·9H2O Solution: Add Na2S·9H2O Na-lactate Solution: Composition per 100........... Na-thiosulfate solution...............20.100............ Sparge with 80% N2 + 20% CO2.........3mL Seven vitamin solution........ Mix thoroughly.................................. Cool to room temperature.......................... Na-pyruvate solution........... Cool to room temperature.................. Vitamin Solution: Composition per liter: Pyridoxine-HCl ................................................0mg Thiamine-HCl·2H2O ...........0mg Calcium pantothenate .....0mg D(+)-Biotin ............................................. Add distilled/deionized water and bring volume to 1.......................0mg p-Aminobenzoic acid ......... Adjust pH to 7........ Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C. Mix thoroughly....0mg Lipoic acid .................. and 10............................0mg Folic acid..........0mg Nicotinic acid .....................................0mg Folic acid .........5.............................. except vitamin solution................0g Preparation of Na-lactate Solution: Add Na-lactate to distilled/deionized water and bring volume to 100........................................... Sparge with 80% N2 + 20% CO2...5g CoCl2·6H2O.....0mg MnCl2·4H2O ... Aseptically and anaerobically add 20............ 2.................................................. Filter sterilize....................................................... Filter sterilize............................ Sparge with 100% N2..................... Mix thoroughly............24................................80...........5.....

. 0..........6.4 at 25°C Solution A: Composition per 870........................0mL Seven vitamin solution................................. Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O .... Sparge with 80% N2 + 20% CO2.....5g Na2WO4·2H2O............ 70...................0-7.....0L........................0mL selenite-tungstate solution.... seven vitamin solution........ Mix thoroughly..2.... Adjust pH to 7... 10...................... Cool to room temperature.. 200......... 1..........0mL NaHCO3 solution.................... Sparge with 80% N2 + 20% CO2............. and 1............. 10........ 200............................... Adjust pH to 7.............0mL pH 7.. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.....0g Na-acetate ....... Aseptically and anaerobically add 50...... 100............ Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere........................................... Mix thoroughly..............0g MgCl2·6H2O .... 1....0mL: Na-fumarate ..................0mL trace elements solution SL-10..... Mix thoroughly.................3.......... 100.....................0mL: CaCl2·2H2O .....0g Na2SO4 ............................................1......................................................0mg ZnCl2 ..0g .......... Cool to room temperature.....0mL Na2S·9H2O solution..................0mL Solution F ...........0mL: Solution A ............................... Autoclave for 15 min at 15 psi pressure–121°C............... Mix thoroughly...0mg Na2MoO4·2H2O ... to distilled/deionized water and bring volume to 977........3.......0L. Mix thoroughly........................5mg Na2S·9H2O solution..........................................7mL..................... Filter sterilize........... 3..............................................2mL seven vitamin solution.............0mg Calcium pantothenate ............. 0........... 20.................................... Mix thoroughly........ 20...................... 3.....0mg D(+)-Biotin ..................... Desulfobacca Medium (DSMZ Medium 728) Composition per liter: NaHCO3 ..................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10............................... 100.. 10....................... CaCl2 Solution: Composition per 10.........1g Resazurin .....2mL pH 7....... 10.......................... 100...... Na-fumarate solution................. and trace elements solution SL-10... Sparge with 100% N2.0mg Vitamin B12 . Autoclave for 15 min at 15 psi pressure–121°C.0mL vitamin solution............ Sparge with 100% N2.....................0g Preparation of Na-fumarate Solution: Add Na-fumarate to distilled/deionized water and bring volume to 100....0mL CaCl2 solution ............0mL Nalactate solution.... Mix thoroughly......0mL Na-fumarate solution............ Autoclave for 15 min at 15 psi pressure–121°C.......................... 20......... Mix thoroughly..... 870.. 20.... Add distilled/deionized water and bring volume to 1..... 300.........53g KH2PO4 .....................................0mL Selenite-tungstate solution ..... Add components.........0mg HCl (25% solution) ...............0mg Thiamine-HCl·2H2O .........................................1 ± 0...... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere.......................64g Na2HPO4·2H2O.... vitamin solution.................................. Use: For the cultivation of Desulfobacca acetoxidans........0.... except Na2S·9H2O solution... and 1.......................2 at 25°C Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ................... 4.. 0.0mL..3..............................0mg MnCl2·4H2O.. Na2S·9H2O solution...3g NaCl ... 1.......2..0mL Solution C.....10...................0mg NiCl2·6H2O............5g CoCl2·6H2O .... Sparge with 80% N2 + 20% CO2......... Aseptically and anaerobically distribute into sterile tubes or bottles..............0mg Na2SeO3·5H2O ...............................1–7....................0mL trace elements solution SL-10...........0mg H3BO3 . 10. 1.................... Filter sterilize..............2.................... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .................0mL.......................................................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10......................................4...........3mL Na2S·9H2O solution..........132 Desulfobacca Medium Na-fumarate Solution: Composition per 100................. Filter sterilize................................ Mix thoroughly....... Add components.............................................. 10...........................................0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.................. Aseptically and anaerobically add 10....................................0mg Nicotinic acid .......0mg p-Aminobenzoic acid .....21.....0mL Solution E (Vitamin solution) ...............................0mL KOH solution.............................. Sparge with 80% N2 + 20% CO2......0.......... 0................................ CaCl2 solution. 1...0mL selenite-tungstate solution.................. to distilled/deionized water and bring volume to 873............ Na-lactate solution.................0mg Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.........11g MgCl2·6H2O....................0mL of HCl solution..... selenite-tungstate solution.......... 0......... 0.........8mL...0mL: NaCl .........24.10.............................. and Desulfitobacterium hafniense.....0................................................................................. 190.... 16.................... Desulfobacter Medium Composition per 1001....0mL...............0mL Solution D ........................0mL Trace elements solution SL-10 ..................... Add remaining components...0mg CuCl2·2H2O...................... 36.0mL Solution B (Trace elements solution SL-10) .... 1..0............ Use: For the cultivation of Desulfitobacterium sp.................. selenite-tungstate solution................. 80.... Selenite-Tungstate Solution Composition per liter: NaOH ....1.. Mix thoroughly..... 0....................................0mL CaCl2 solution............................................. Mix thoroughly..1g Na2SO4 .......................3g CaCl2·2H2O..................................................0L.......... KOH solution....11g Preparation of CaCl2 Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10..........0-7....... 0...........0mL seven vitamin solution...... seven vitamin solution......... except NaHCO3 solution....... and trace elements solution SL-10............... Sparge with 100% N2........................41g NH4Cl...... Autoclave for 15 min at 15 psi pressure–121°C.................................................. Aseptically and anaerobically distribute into sterile tubes or bottles.....

.....................................................................................0mL Solution B (Trace elements solution SL-10) ................................................0mg MnCl2·4H2O ... 5.....190........... Preparation of Medium: Aseptically and anaerobically combine solution A with solution B..........4g Na-acetate·3H2O.0.0mL: Sodium acetate·3H20.................... Mix thoroughly................................................ 10.............0...............................1.0mg Nicotinic acid . Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O.... Seal the flask under 80% N2 + 20% CO2. Gently heat and bring to boiling..................................................................... 6........................0mL Mix thoroughly.................................... 2.............. Mix ............................. Solution F: Composition per 10..0mL............. Mix thoroughly....................0L...2g CaCl2·2H2O ..................... 5......................0mL of HCl solution................................................... Filter sterilize..................70..................................................................... Gas under 80% N2 + 20% CO2..... solution E.................. Autoclave for 15 min at 15 psi pressure–121°C...... 1......................0mg NiCl2·6H2O .....0mg CuCl2·2H2O ................................ 36..................... Mix thoroughly.0mL...0mg Riboflavin ............. Autoclave for 15 min at 15 psi pressure–121°C........15g Resazurin ....................... 2....0mg Lipoic acid ...............0mL: Solution A ...........0mg Calcium DL-pantothenate...............36..........................0mg NiCl2·6H2O. Continue gassing until pH reaches below 6.. Solution C: Composition per 100......................................................................... Mix thoroughly.......0g distilled/deionized water and bring volume to 10. 5.......................................0mL Solution F ...................................................5g NH4Cl ............................................ 5...............................0mL Pyridoxine·HCl ...5g NH4Cl.......................................0mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870........ Mix thoroughly... Autoclave for 15 min at 15 psi pressure–121°C.....................3g KH2PO4 ...... 0............. 0...........................5g CoCl2·6H2O........... Gas under 100% N2........0mL.....................0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10.............................. and solution F................................... 2........... 100............... Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1.................... Add distilled/deionized water and bring volume to 1................. Gas under 100% N2..0mL: NaCl ..4 at 25°C Solution A: Composition per 870....0.. Autoclave for 15 min at 15 psi pressure–121°C........... 5........1mg Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10..................2g CaCl2·2H2O. 5................... 0.................... 0..............0mL: Solution D: Composition per 10.............. solution D..................... Solution E (Vitamin Solution): Composition per liter: 133 thoroughly..................0L..........................................0g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100...... Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2................................................5........ Filter sterilize.......................... Mix Mix thoroughly............ 24...6.............................................................. Autoclave for 15 min at 15 psi pressure–121°C...0mg ZnCl2 ....2.......................... Mix thoroughly.....0L..........5g Preparation of Solution F: Add Na2S·9H2O to dis- Preparation of Solution D: Add Na-acetate·3H2O to tilled/deionized water and bring volume to 10........0mg HCl (25% solution) ....................0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870..0mg Na2MoO4·2H2O..................................0mL Mix thoroughly................................. Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O . 190...................5g Preparation of Solution D: Add sodium acetate to distilled/deionized water and bring volume to 10................. Desulfobacter postgatei Medium (DSMZ Medium 193) Composition per 1001........2............................ 1......................................0mL pH 7....................................3.......0mL: NaHCO3 .. Flush with 80% N2 + 20% CO2 to remove dissolved oxygen.....................................................3g KCl .. 10........0mg H3BO3 ...........1..........0mg MnCl2·4H2O...... Mix thoroughly............................................0mg Biotin ............ Add distilled/deionized water and bring volume to 1.................. 0....................................................................................................................................................................................................................................10.. 70............................... 2............................0mL. 0..........0mL: Na2S·9H2O.......................... Mix thoroughly...............0.................0g Na2SO4 .......................0mg ZnCl2 ..0g MgCl2·6H2O .0mg H3BO3 ........0mg Thiamine·HCl ...............................................0mg Vitamin B12 ...3g KH2PO4 ..Desulfobacter postgatei Medium KCl.......100........... Continue boiling for 3-4 min.....................0mg Na2MoO4·2H2O ................0mL: NaHCO3 ...............................................0mL of HCl solution.......................................................1-7....0mg Folic acid.......0mg CuCl2·2H2O......1....................... Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100...................... 1......... Autoclave for 15 min at 15 psi pressure–121°C.......0mL Solution C............ in that order. Gas under 100% N2............. 10....................... 10.............................0mL Solution E (Vitamin solution) .... solution C.......................................... Solution C: Composition per 100........7............0mL Solution D ...0mL. Add remaining components..................................... Gas under 100% N2.......5g CoCl2·6H2O .........................................................15g Resazurin ..................... Solution D: Composition per 10............. Use: For the cultivation and maintenance of Desulfobacter species and Malonomonas rubra.................. 5.. Allow to cool to room temperature while gassing under 80% N2 + 20% CO2...... Add remaining components........................................ Sparge with 100% N2........ 870......0mg HCl (25% solution) ....................................................24.... 100.......................0.....0mg p-Aminobenzoic acid ............... 10...........

....................0mg MnCl2·4H2O ....0mg p-Aminobenzoic acid .......0mg Folic acid................10.............. Mix thoroughly............ Autoclave for 15 min at 15 psi pressure–121°C..................................................2...... may stimulate growth..................... Medium thoroughly....................2.............. Sparge with 100% N2...6.0mg Riboflavin .........0mL Solution E (Vitamin solution) .................... 3...........134 Desulfobacter sp.........0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10........................... Sparge with 100% N2...0mg Vitamin B12.............................24............................................10mg Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1.................0mg D-Ca-pantothenate................... Boil solution A for a few minutes.... 10.......................... Preparation of Solution F: Add Na2S·9H2O to dis- Solution D: Composition per 10..........................0mL solution C.. .. Mix thoroughly............................ Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly..0... 3..2......................... Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels... 1..... may stimulate growth. 0............................ Paracoccus solventivorans..........1-7............5................... Use: For the cultivation of Desulfobacter species.. 10...............0g MgCl2·6H2O......4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....... freshly prepared under N2 and filter-sterilized.... Sparge with 100% N2..........................5g NH4Cl.......... 100...........5...... Autoclave for 15 min at 15 psi pressure–121°C.... Autoclave for 15 min at 15 psi pressure–121°C...................0mL solution D............................................0mL Mix thoroughly........0g KCl.... Filter sterilize......2......0mg Nicotinic acid .............. 10......70........................ Medium (DSMZ Medium 195) Composition per 991......................... Sequentially add 1............................................0mL: NaHCO3 ........... Autoclave for 15 min at 15 psi pressure–121°C...........0....................................................................0mg Thiamine-HCl·2H2O .............0mg HCl (25% solution) .............................................0mL....................... Solution E (Vitamin Solution): Composition per liter: Pyridoxine-HCl .............0mg Biotin ........ 0..... 0................36...................... Cool to room temperature............... and Desulfotomaculum sp.................................15g Resazurin ....... Sparge with 100% N2... Add distilled/deionized water and bring volume to 1...........................0mL Mix thoroughly.... Cool to room temperature.....4g tilled/deionized water and bring volume to 10.............. 2................................... 5................... 5..0mL Solution D .....5......................................... Sparge with 100% N2.........0mg D-Ca-pantothenate ............0mg CuCl2·2H2O......... Preparation of Medium: Gently heat solution A and bring to boiling...0mL solution F..... Boil solution A for a few minutes.. 0.............0mL: Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100......................... 5.......... Mix thoroughly...0mg Folic acid ......................... Solution E (Vitamin Solution): Composition per liter: Pyridoxine-HCl .0mg Nicotinic acid .0mg Biotin .....0mg Na2MoO4·2H2O.....................0mL.............0mg ZnCl2 ................. Sparge with 100% N2............0mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870............. Use: For the cultivation of Desulfobacter postgatei..............5..........5.. 21.............0mL Solution C .10................. Autoclave for 15 min at 15 psi pressure–121°C........................... Cool to room temperature.......................... 10...........................................0mg Thiamine-HCl·2H2O ............................................10.......4 at 25°C Solution A: Composition per 870...................... Mix thoroughly.......... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O ........................................................0mg Riboflavin.............. 5...............5............................. Sparge with 100% N2............. Autoclave for 15 min at 15 psi pressure–121°C.................... 5. Solution F: Composition per 10... Sequentially add 1......0mL solution D............0L. Mix thoroughly.............................100.....1g Na2SO4 ................190....0mL solution B...........0mL: Na2S·9H2O .......................0mL of HCl solution......5g Na-acetate·3H2O........0mg Lipoic acid.................. Gas with 80% N2 + 20% CO2 gas mixture to reach a pH below 6..... Autoclave for 15 min at 15 psi pressure–121°C..............0mg H3BO3 .............0mL: NaCl ...............0mL.......5g Preparation of Solution D: Add Na-acetate·3H2O to distilled/deionized water and bring volume to 10................... 0....5............ 10...................... Flush with 80% N2 + 20% CO2 to remove dissolved oxygen........ 5.... Desulfobacter sp.. Mix thoroughly........................ Cool to room temperature.........0mg Vitamin B12 ..............0mL solution E.............0mL solution C..... 100..... and 10................................... Addition of 10–20mg sodium dithionite per liter from a 5% (w/v) solution..... and 10. 2........................ Solution C: Composition per 100... CoCl2·6H2O......0mg Lipoic acid ..........................................................870.......0mg NiCl2·6H2O........... Gas with 80% N2 + 20% CO2 gas mixture to reach a pH below 6. 0...........................0mL pH 7........0L.........................10mg Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1.......... Addition of 10–20mg sodium dithionite per liter from a 5% (w/v) solution......................... Add remaining components................................0g Solution F: Composition per 10...0mL solution F............................................................ 1............................................................................100.....................3g KH2PO4 .........0mL: Solution A ...............0L......... 10................................0mL: Na2S·9H2O.... Preparation of Medium: Gently heat solution A and bring to boiling................................................................................................................................. Autoclave for 15 min at 15 psi pressure–121°C....2g CaCl2·2H2O.....0mL Solution B (Trace elements solution SL-10)......1................................0mL solution E.........0mg p-Aminobenzoic acid .................................................... Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels...0mL solution B............ freshly prepared under N2 and filter-sterilized..

..................... 1............................ 5........... Allow to cool to room temperature while gassing under 80% N2 + 20% CO2......................5g Preparation of Solution D: Add sodium acetate to dis- tilled/deionized water and bring volume to 10....................0mL Solution D .............. Autoclave for 15 min at 15 psi pressure–121°C.. Solution C: Composition per 100........................0............10.......0mL..............3g KCl....................0mL pH 7........................................................ 5........... Gas under 80% N2 + 20% CO2.............0mg Nicotinic acid ...0mL Solution G ........4 at 25°C Solution A: Composition per 870......2............5g NH4Cl...5g NH4Cl ..........0mL.....................0mg ZnCl2 ..........1............................ Gently heat and bring to boiling.. 1..........0mL Solution D ......... 3......... Continue boiling for 3-4 min...................0mL Solution G .......5g CoCl2·6H2O . Mix thoroughly.................................... Mix thoroughly..... solution C................................................................. solution D................................................190.......................15g Resazurin ...........................................870............................ Solution G: Composition per 10...................... 24................................................0mL: NaHCO3 ..............4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.............. 10............. Solution E (Vitamin Solution): Composition per liter: Pyridoxine·HCl ...0.........................0mg CuCl2·2H2O .. 1.......... 0....0mg 135 Riboflavin............ 5...........................................................................0................................1–7.....................0mg ZnCl2 ........... 0.........2.................0mL........2g CaCl2·2H2O................... Mix thoroughly.................................0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870.......0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10.... Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2............................0mL pH 7.. solution F........................................... Gas under 80% N2 + 20% CO2................................................10...................... 6................ Prepare solution freshly.0L...................... 7............... 5............0mL of HCl solution........0mg Calcium DL-pantothenate..... 10. 2........0mL Solution C........ 36......4 at 25°C Solution A: Composition per 870................... in that order.3........................0mL Solution F ..............0mg Thiamine·HCl ............... Add distilled/deionized water and bring volume to 1..0mg HCl (25% solution) ..................3g KH2PO4 ................................. Gently heat and bring to boiling...............10...... Autoclave for 15 min at 15 psi pressure–121°C.... 190................1.0mL..1....................... Filter sterilize...........................................................0mg Folic acid ............ Seal the flask under 80% N2 + 20% CO2......0mg Biotin ................... Gas under 100% N2.............. Autoclave for 15 min at 15 psi pressure–121°C...............Desulfobacterium anilini Medium Desulfobacterium anilini Medium Composition per 1011..0mL: Solution A .............2g CaCl2·2H2O .0mg .....0... and solution G....................... Autoclave for 15 min at 15 psi pressure–121°C.............100..............................................0mL Solution B (Trace elements solution SL-10).......... Mix thoroughly........5...... Autoclave for 15 min at 15 psi pressure–121°C.......................0L..................10......0mg MnCl2·4H2O............... Allow to cool to room temperature while gassing under 80% N2 + 20% CO2.............................................. Mix thoroughly............................. solution E...0mL: Sodium acetate·3H20..... Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C............ Preparation of Medium: Aseptically and anaerobically combine solution A with solution B.. 2......0mg Preparation of Solution G: Add phenol to distilled/ deionized water and bring volume to 10........0mg NiCl2·6H2O .................................0mL..........................................0mL: NaCl ........ Desulfobacterium anilini Medium Composition per 1002....0mg Vitamin B12...... 70.................................. Continue gassing until pH reaches below 6...........................................................0g Na2SO4 .................................... Mix thoroughly............................0mL Solution F............ Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O .......5g CoCl2·6H2O..0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870..... Gas under 100% N2......0mL Solution B (Trace elements solution SL-10) ........... Gas under 100% N2.1mg Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1........................0mg Na2MoO4·2H2O .....1–7..............................0mg Lipoic acid ...................7...................................... Mix thoroughly. Gas under 100% N2................ 10.... Filter sterilize.................................................0mg MnCl2·4H2O .............................3g KH2PO4 ...............................................................................0mL: Phenol....................... 0................................. 870.........................................15g Resazurin .................................1.0mL: Solution A ..............0.......................................................................0mL: Na2S·9H2O ........................ 1.........................3g KCl ............. 10..................0g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100........................0g MgCl2·6H2O ................................................100................................................... Solution F: Composition per 10...................................................................................0g Na2SO4 ..................................................0mg p-Aminobenzoic acid .............0mL Solution E (Vitamin solution) ... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O..... Continue boiling for 3-4 min....................0g MgCl2·6H2O..........5.......0mL Solution E (Vitamin solution) .................. 5... Mix thoroughly................ 0......................0mL Solution C .......... Continue gassing until pH reaches below 6................................................ Seal the flask under 80% N2 + 20% CO2.........................................................0................ Add remaining components............................. Solution D: Composition per 10..............................................................0mL....................0mL: NaCl ........... 1.......0mg H3BO3 . 100.........................94.................. Use: For the cultivation of Desulfobacterium anilini.................................................................10....................................70.......................0.......................0........... Mix thoroughly........................... 100.......

...................................0mg Calcuim pantothenate .........0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870.. Gas under 100% N2......................... Solution D: Composition per 10.... Mix thoroughly............................................ Gas under 100% N2............0..............24.........5....... Solution E (Vitamin Solution): Composition per 100................. Mix thoroughly....................0mL Solution E (Vitamin solution) ..70..................................... Autoclave for 15 min at 15 psi pressure–121°C..........0mg NiCl2·6H2O .....0mL...................0.................0mg Biotin .0mg HCl (25% solution) ............... Autoclave for 15 min at 15 psi pressure–121°C.......... in that order........0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10.................. Autoclave for 15 min at 15 psi pressure–121°C......................0mg Thiamine dichloride ...............36.....4g NH4Cl ..... Continue boiling for 3-4 min.6...... 10......... 1.......................................................... 10.................... 10............................ Add remaining components.0mg MnCl2·4H2O . Gently heat and bring to boiling..................... 10............................0mg NiCl2·6H2O.............. Desulfobacterium catecholicum Medium Composition per 1001................ 10......... Mix thoroughly........................... Mix thoroughly....0mg Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 100................................ 20..............................1....................0mg CuCl2·2H2O.........................0mg p-Aminobenzoic acid ...0mg CuCl2·2H2O .................................0...........4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............ Autoclave for 15 min at 15 psi pressure–121°C.................0...0mg Nicotinic acid .. 10.0g KCl .....0mL: NaHCO3 ............................ Mix thoroughly............................................................................0mL Solution C.....0mg ...................0mg Na2MoO4·2H2O.... Mix thoroughly..... 10...2g CaCl2·2H2O ....0mL of HCl solution........0mg H3BO3 .... Autoclave for 15 min at 15 psi pressure–121°C......0mL Solution F ...............................100............................0mg ZnCl2 ...............................................0..... 2....... Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2 Use: For the cultivation and maintenance of Desulfobacterium anilini....5g Na2WO4·2H2O ..........055g Preparation of Solution D: Add sodium benzoate and catechol to distilled/deionized water and bring volume to 10.......... Seal the flask under 80% N2 + 20% CO2....5...................................0mL: Phenol .................................0mL...................................0mg Vitamin B12 ................... 0... Filter sterilize..........5g MgCl2·6H2O ........1. Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O.........................................................0mL: Na2SO4 . 6......... Gas under 100% N2........0mL: Solution A ...................................... Solution E (Vitamin Solution): Composition per liter: Pyridoxine·HCl................................ Mix thoroughly.........4 at 25°C Solution A: Composition per 870.........0mL: Sodium benzoate ..... Mix thoroughly..5................... Gas under 100% N2.... Add distilled/deionized water and bring volume to 1...............0mg H3BO3 ..................136 Desulfobacterium catecholicum Medium Na2MoO4·2H2O ....... Solution D: Composition per 10.....0mL pH 7...................... Gas under 80% N2 + 20% CO2............................ 4..0.............................................0........ 5...... Mix thoroughly....2....................1–7...............................................0L....................................0mL Solution B (Trace elements solution SL-10) ...................0mL of HCl solution.........0...... Solution F: Composition per 10........... Gas under 100% N2..........0L.....0mL........0mg Na2SeO3·5H2O....................................................0mL.............. Gas under 100% N2...................................................... solution E..0mg HCl (25% solution) . Mix thoroughly.... 100......................... Mix thoroughly...........0mL.................. Preparation of Medium: Aseptically and anaerobically combine solution A with solution B........... Solution G: Composition per liter: NaOH .............................. Continue gassing until pH reaches below 6.......... Filter sterilize.............0g NaCl .............0mL Nicotinic acid amide .....................15g Resazurin ............................10.. Filter sterilize....... solution D..0mg Calcium DL-pantothenate ..10.........0mL Solution D .............................0mg p-Aminobenzoic acid ........................................................... Gas under 80% N2 + 20% CO2................ 0....190...0mg Pyridoxal·HCl .........................0mL............0mL..................................0g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100.................. Add distilled/deionized water and bring volume to 1........................5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100...................... 0.................................................. 36................................................................................... 5.................... solution F............................... Filter sterilize....... 30.........................0L............5.......0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10.. Mix thoroughly..................... 870....0mg Preparation of Solution G: Add components to distilled/deionized water and bring volume to 1........................ Allow to cool to room temperature while gassing under 80% N2 + 20% CO2............................................................ solution C................. Add remaining components.........0mL: NaHCO3 .............. 35................................................... Solution C: Composition per 100.......2.............. Autoclave for 15 min at 15 psi pressure–121°C........................37g Catechol........................1................... 24......................3.......... Gas under 100% N2......... Solution C: Composition per 100............... and solution G............................5g CoCl2·6H2O.094g Preparation of Solution D: Add phenol to distilled/ deionized water and bring volume to 10............ Mix thoroughly.3g KH2PO4 .......0mL: Na2S·9H2O................................................................................ 3.............................0mg Lipoic acid......................

............0mg NiCl2·6H2O ..................0mL.0mg H3BO3 . Use: For the cultivation of Desulfobacterium cetonicum......0mg Biotin ..............................................0.....................................0mg Vitamin B12 . Sparge with 100% N2. 36...........................................................0mg Preparation of Solution A: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2..... Autoclave for 15 min at 15 psi pressure–121°C.............................. 0.....................0mL of sterile solution B.Desulfobacterium indolicum Medium Riboflavin ...........................1g ZnCl2 ...............0mL Solution E (Wolfe’s vitamin solution).......................................2g CaCl2·2H2O ............................ and 10........................... 24............................... and solution F.....0mL Solution C ...............................0mL Solution E .....0mg MnCl2·4H2O............................... 5....................0mL..................... Continue boiling until resazurin turns colorless............. 0..........0mL: NaHCO3 ........................................................................2–7.......... Mix thoroughly....................... 6.................. 0.... 10.....40........0.................. Desulfobacterium indolicum Medium Composition per 1002.................................................... Mix thoroughly.........0mL: Solution A .....0mL of sterile solution C...........................0mL of sterile solution A with 1...0mL............2g Preparation of Solution C: Add sodium butyrate to distilled/deionized water and bring volume to 10......... 100...........0mg Thiamine·HCl . Solution D: Composition per 40........0mL: NaCl ......... Mix thoroughly...... Sparge with 100% N2..........5g NH4Cl ......................... 10.............. Preparation of Medium: Aseptically and anaerobically 137 Solution C: Composition per 10............... 190.........2 at 25°C Solution A: Composition per 900.............................. solution D........................ Autoclave for 15 min at 15 psi pressure–121°C...............0mL Solution F ...............10....................0................0mL Solution D ... in that order....4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.......... Mix thoroughly.......................2...........................................................................................................0g Preparation of Solution D: Add NaHCO3 to distilled/ deionized water and bring volume to 40......................0mL Solution G ...........0g MgCl2·6H2O ..................................3g combine solution A with solution B... 2.............................0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10.... Cool to 45°–50°C.................................................. Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O ....................3.......4......... 0.........0mL...............10........07g ... Autoclave for 15 min at 15 psi pressure–121°C.........15g Resazurin ......... Solution F: Composition per 10................. Mix thoroughly..........................................0mL Solution H ... Add components to distilled/deionized water and bring volume to 900.0mL Solution B (Trace elements solution SL-10) ..10............0mL Solution C...............0mL Solution D ........................... Autoclave for 15 min at 15 psi pressure–121°C...... 30.... Gas under 100% N2............1.. 0......................... Preparation of Solution E: Add Na2S·9H2O to dis- Use: For the cultivation and maintenance of Desulfobacterium catecholicum...... 5...1mg Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1......................................................4mL: Solution A .................................................................. 1........... Mix thoroughly.........................................................................3g NH4Cl......................0mL: Na2S·9H2O ................................. tilled/deionized water and bring volume to 10............19g MnCl2·4H2O .................. 0.0L......................4 at 25°C Solution A: Composition per 950........0mL..... Autoclave for 15 min at 15 psi pressure–121°C........... 900..................... Add remaining components............ Aseptically and anaerobically combine 950........0g Na2SO4 ....................................1.......0mL.................... 70.....................................0mL of sterile solution D..........0mg CuCl2·2H2O ..................6 ± 0.......... 2.0......................................................1............... Preparation of Medium: Prepare and dispense medium Desulfobacterium cetonicum Medium Composition per 1011....0mg ZnCl2 .................950.......... 50...............................05g Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 950.............................0......................................3g CaCl2·2H2O....................... 2...............21....................... solution E.... Cap with rubber stoppers..........1....................................... Check that final pH is 7....7g MgCl2·6H2O................... Sparge with 80% N2 + 20% CO2..........................0mL of sterile solution E. Mix thoroughly... indicating reduction.......5g CoCl2·6H2O .............0..................... Autoclave for 15 min at 15 psi pressure–121°C............................................ 1.............................................................. solution C... Gas under 100% N2.....................0L........ Autoclave for 15 min at 15 psi pressure–121°C...0mL: Na2S·9H2O.................................. 10.... Mix thoroughly............4mL pH 7................................ Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2...............0mg HCl (25% solution) ....................0g KCl ............................. under 80% N2 + 20% CO2..0mL pH 7...3............5g CoCl2·6H2O..... Mix thoroughly............................................... Autoclave for 15 min at 15 psi pressure–121°C..............................0mL of HCl solution........................................ 0........ 1.....0mL: NaCl ...........0mL Solution B (Trace elements solution SL-10)....... Gently heat and bring to boiling..... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O.....8g KH2PO4 .............. Sparge with 100% N2..............2–7.......................0mL: Sodium butyrate ............. Solution E: Composition per 10..........0.............. 2.... 4.............................. Sparge with 100% N2................................ Add distilled/deionized water and bring volume to 1.....0...... Mix thoroughly.............. 10...................0mg Na2MoO4·2H2O ... Mix thoroughly.....................3g KH2PO4 .......................0mg Folic acid......0g Na2SO4 ........

................. Add components to distilled/deionized water and bring volume to 930...............................0mL................................0.......................... Mix thoroughly....... 5......................5g CoCl2·6H2O..................0.......................1mg Preparation of Solution E (Wolfe’s Vitamin Solution): Add components to distilled/deionized water and Autoclave for 15 min at 15 psi pressure–121°C....0mL with distilled/deionized water......... Preparation of Medium: To 900........... 2.........0mL: NaHCO3 ...................... Solution E (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl ...................0mL of sterile solution F....... 10...0mL of cooled.....0mL of sterile solution E..................21....... Cap with a rubber stopper........0mg Calcium pantothenate ..... Continue boiling until resazurin turns colorless..................................... Aseptically and anaerobically distribute into sterile tubes or flasks................................................................0mL: Na2S·9H2O...............0...0.................................. aseptically and anaerobically add 7...............036g NiCl2·6H2O.......... Solution B (Trace Elements Solution SL-10): Composition per liter: Solution F: Composition per liter: FeCl2·4H2O.... Mix thoroughly.036g NiCl2·6H2O ... Mix thoroughly...................... Gently heat and bring to boiling..1.................0 with NaOH. Mix thoroughly.............0mL MgCl2·6H2O (40% solution)... Aseptically gas under 100% N2 for 20 min... Gently heat while stirring until dissolved............... 6..........5g Preparation of Solution H: Add Na2S2O4 to distilled/ deionized water and bring volume to 10......... Filter sterilize.........................................0..................... Autoclave for 15 min at 15 psi pressure–121°C..4mL: Solution A ... 1........ Adjust pH to 6............................ 50...............1.....0mg HCl (25% solution) ................................................0mL of HCl solu- tion..........2 at 25°C Solution A: Composition per 930.........0mg CuCl2·2H2O. 2................................... Bring volume to 1... 5....................138 Desulfobacterium Medium Na2MoO4·2H2O ... Cool to 45°–50°C........... 10............................................024g H3BO3 ........................................... 5..... To 100...................0mg Riboflavin ...............10.. 5.......0mg Nicotinic acid ........ 2..... Mix thoroughly......... 1... Bring volume to approximately 900..0mL Solution B (Trace elements solution SL-10) .............................. Add indole to distilled/deionized water and bring volume to 100.........................0mL Solution G ........0 ± 0...0g MgCl2·6H2O ...............................0L with ............................................................... 3..0mL Na2SeO3·5H2O ...... Solution H: Composition per 10.......................................... Bring volume to 1.0mL of sterile NaCl solution and 0.. aseptically and anaerobically add in the following order: 1.................................... Desulfobacterium Medium Composition per 1002.................4mL pH 7.5g NH4Cl .1..... Mix thoroughly..0 with NaOH............ Cool to 45°–50°C..... Mix thoroughly.... 0.... Mix thoroughly....7mL Preparation of Solution D: Prepare and dispense all solutions anaerobically under 100% N2....................................................0mL of HCl solu- tion.......0mg CuCl2·2H2O ........... Aseptically gas under 100% N2 for 20 min..............07g Na2MoO4·2H2O............................0............................... Prepare solution freshly.............. 0..0mg Cyanocobalamin ..................0mg Preparation of Solution A: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2. 2..................................... Mix thoroughly...3g NaCl (30% solution) ............................ Solution D: Composition per 107....................................0mL of sterile solution G............................. Cap with rubber stoppers...................0mg HCl (25% solution) ..0L............ 10..... Mix thoroughly..... 5........... Solution C: Composition per 50.....5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50.............................01g Thiamine·HCl ..............................................0mL of sterile solution C.............. 930..... Filter sterilize.......0mL of sterile solution D and 0................................................. sterile solution A.....3g KH2PO4 ................. Filter sterilize.....0...... Bring volume to approximately 900................................. bring volume to 1............1g ZnCl2 .. Gas under 100% N2 for 20 min.......................7................. Cool to 25°C...0mg Thioctic acid ................... indicating reduction............3.............................................. Mix thoroughly....0mL of sterile indole solution..024g H3BO3 .2................7mL of sterile MgCl2·6H2O solution...................... 0.....4g Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10...................................................0mL with distilled/deionized water.......................... 0....... Mix thoroughly...........0.....................0mL. 5....01M solution) ............ Adjust pH to 6................0mL: NaCl ........0mL Solution E ............................0....3..0g Na2SO4 ............................. Aseptically gas under 100% N2 for 20 min......0mg p-Aminobenzoic acid .......................0.......... 0.................. and 10.......................0mg NaOH (0.0mL Solution F ...................15g Resazurin ... Preparation of Solution B (Trace Elements Solution SL-10): Add the FeCl2·4H2O to 10..0mL Solution C..... Autoclave the three solutions separately for 15 min at 15 psi pressure–121°C............................... Prepare the NaCl solution and the MgCl2·6H2O solution separately.......6........0.......................0mL Preparation of Solution B (Trace Elements Solution SL-10): Add the FeCl2·4H2O to 10................................................................2g CaCl2·2H2O ......................... Filter sterilize................ aseptically and anaerobically add 30....0mg Biotin ..............0L with distilled/deionized water.................... 50.............................. Mix thoroughly.. Immediately prior to inoculation......0L of NaOH solution........0mL... Aseptically gas under 100% N2 for 20 min..................................................................0g KCl ........................ Aseptically gas under 80% N2 + 20% CO2 for 20 min.. 0....0mL Solution D (Wolfe’s vitamin solution) ....................0mL...19g MnCl2·4H2O .....................0mL of sterile solution B...........................................7mL: Indole ................................. 10..............4mL of sterile solution H.................. 0..................................................0L Preparation of Solution F: Add Na2SeO3·5H2O to 1.. 1.......... Use: For the cultivation and maintenance of Desulfobacterium indolicum......................... Filter sterilize..0mg Folic acid.................0mL................. Solution G: Composition per 10.0mL: Na2S2O4 ......

........0mg p-Aminobenzoic acid ............ Mix thoroughly............................ Aseptically gas under 100% N2 for 20 min..........2........................................................0mg p-Aminobenzoic acid ..........................0 with NaOH........0mL of HCl solu- Na2SeO3·5H2O ............. Immediately prior to inoculation......... Solution G: Composition per 10......930...........5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50........................................................................................................................... Prepare solution freshly.............. Cap with rubber stoppers...... Add components to distilled/deionized water and bring volume to 930.... Solution D (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl........................................................2 at 25°C Solution A: Composition per 930............... Filter sterilize........ Cap with a rubber stopper.................5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50. 10..........0........ Mix thoroughly.. Mix thoroughly. Mix thoroughly.............. 5.5g CoCl2·6H2O.........................01g Thiamine·HCl .....1mg Preparation of Solution D (Wolfe’s Vitamin Solution): Add components to distilled/deionized water and bring volume to 1............036g NiCl2·6H2O...5...........0mL of sterile solution D...................... Bring volume to approximately 900......4mL: Solution A ................ Solution D (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl .......0mg Thioctic acid . 10..............0....................................................................... Filter sterilize....... Mix thoroughly...............................0mL: NaHCO3 .................................. Solution C: Composition per 50... 0.............................. Solution C: Composition per 50....................5............... 1....................0mL Solution G .........0mg Calcium pantothenate ....... 10..0g MgCl2·6H2O ..19g MnCl2·4H2O .........................................0mL of sterile solution E........0mL.............................. Mix thoroughly........0L with distilled/deionized water............................................................................ Use: For the cultivation and maintenance of Desulfobacterium autotrophicum..................................0L of NaOH solution...... Solution E: Composition per liter: 139 Solution C...................... Aseptically gas under 100% N2 for 20 min.......01g Thiamine·HCl ..........2...0mL...........0.............1g KCl .............. Mix thoroughly.. Preparation of Medium: To 900..........0....................0mL: NaHCO3 ..............................0mg Calcium pantothenate .......................................0g Na2SO4 .......................................5g NH4Cl ...................................................................0......0.....0mg Folic acid........... 2.............. 50.............................. Filter sterilize...................................................................... 5.0mg Nicotinic acid ......................................0mg Biotin ..... 3...........3. 5........0mg Biotin ........ 5........... Bring volume to 1..............................0mg Thioctic acid .............0mL: Na2S2O4............................2................0.......... Aseptically gas under 100% N2 for 20 min.....1mg Preparation of Solution D (Wolfe’s Vitamin Solution): Add components to distilled/deionized water and ...........01M solution) ...................................................................0....024g H3BO3 .......0mL of sterile solution C..........................4mL of sterile solution G.Desulfobacterium Medium with Lactate distilled/deionized water.......3g KH2PO4 ............0mL Solution D (Wolfe’s vitamin solution) ............ Na2S·9H2O. Filter sterilize.............0mL of sterile solution B.................. aseptically and anaerobically add in the following order: 1............................0mL.........................070g Na2MoO4·2H2O..................................4g tilled/deionized water and bring volume to 10............ Aseptically gas under 80% N2 + 20% CO2 for 20 min......... 5...............................0.............................................. Aseptically gas under 100% N2 for 20 min..... 50.....2g CaCl2·2H2O .......... Solution B (Trace Elements Solution SL-10): Composition per liter: Solution F: Composition per 10..... sterile solution A.....................................0mL Solution E ........ Mix thoroughly..........................0............. Autoclave for 15 min at 15 psi pressure–121°C...........0mg Riboflavin....21..........................0mL..................... 0.............5.......0mL tion........ Mix thoroughly.....0mL of sterile solution F........................................1...... Gas under 100% N2 for 20 min...... Continue boiling until resazurin turns colorless......... 5................. Autoclave for 15 min at 15 psi pressure–121°C......................................................................... 0......................0.......................................5.......0mL of cooled......1.....................10g ZnCl2 ......2......0mL Preparation of Solution F: Add Na2S·9H2O to dis- Preparation of Solution B (Trace Elements Solution SL-10): Add the FeCl2·4H2O to 10.. Gently heat and bring to boiling............................. Mix thoroughly.... 0..........5...... 0................. indicating reduction....................................0mg Folic acid ......4mL pH 7.............................................5g Preparation of Solution G: Add Na2S2O4 to distilled/ deionized water and bring volume to 10.............................. Aseptically gas under 80% N2 + 20% CO2 for 20 min..................... 2..............0mL: FeCl2·4H2O................... 10.0mg NaOH (0... Desulfobacterium Medium with Lactate Composition per 1002...... and 10.............0mg Riboflavin ........................... Filter sterilize............5..................0L Preparation of Solution E: Add Na2SeO3·5H2O to 1......... Filter sterilize..................0mg Nicotinic acid ..........1.................................. 1. Cool to 25°C..................................................................... sodium salt .........................0L..............................0mg Cyanocobalamin . 2.........0mg CuCl2·2H2O......0mL with distilled/deionized water..........0mL Solution B (Trace elements solution SL-10) ............................ Aseptically gas under 100% N2 for 20 min............0g Lactic acid...... aseptically and anaerobically add 0..........0mg HCl (25% solution) ..................... Filter sterilize.. 1....................................... Adjust pH to 6...............0 ± 0..0mg Preparation of Solution A: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2...........................................15g Resazurin ..........0mL: NaCl .........0mg Cyanocobalamin..................0mL Solution F ..3.....6............. Aseptically and anaerobically distribute into sterile tubes or flasks.0mL.... Cool to 45°–50°C........................1..................

..0........... Aseptically gas under 100% N2 for 20 min............... Solution E: Composition per liter: Solution B (Trace Elements Solution SL-10): Composition per liter: Na2SeO3·5H2O ....0......................................... Solution G: Composition per 10............................0L............10........0 with NaOH..................................1......01g Thiamine·HCl ..............0L with distilled/deionized water....024g H3BO3 .............................. Mix thoroughly.....................................0mL: NaCl ... Continue boiling until resazurin turns colorless...................0mg NaOH (0. Aseptically and anaerobically distribute into sterile tubes or flasks....0L of NaOH solution....................... Add components to distilled/deionized water and bring volume to 920..............2.5.............................0mL Solution D ............... 1............................................................................ 1....... aseptically and anaerobically add 0.......3g KH2PO4 . in the following order.................... Use: For the cultivation of Desulfobacterium autotrophi- cum....................0L..0mL of sterile solution F........... aseptically and anaerobically add............. 0................140 Desulfobacterium Medium......2 at 25°C Solution A: Composition per 920.0mL..............................................0mL Solution H ......................................................5................................. Adjust pH to 6......0........... Filter sterilize..........0g KCl.......................0L Preparation of Solution E: Add Na2SeO3·5H2O to 1......0mL Solution B (Trace elements solution SL-10)...................................01M solution) . Aseptically gas under 100% N2 for 20 min.... Solution F: Composition per 10......0.....1. Mix thoroughly...................036g NiCl2·6H2O...... Modified Composition per 1002.....................................................10................. Bring volume to 1............0L Preparation of Solution F: Add Na2SeO3·5H2O to 1....15g Resazurin ...................................................0mL: Na2S·9H2O ........................................................................................................ Autoclave for 15 min at 15 psi pressure–121°C..................................................3... 10..0mL......... Cap with rubber stoppers........ 0.07g Na2MoO4·2H2O..19g MnCl2·4H2O ..............0mL.......................0mL of cooled..................2.......... Mix thoroughly............................... Gently heat and bring to boiling.......................... Solution D: Composition per 10......1mg Preparation of Solution E (Wolfe’s Vitamin Solution): Add components to distilled/deionized water and bring volume to 1... Solution G: Composition per 10............................................................... Cool to 45°–50°C.... Gas under 100% N2 for 20 min................................................ Mix thoroughly.........................4g ..............0mL: Sodium acetate·3H2O ...............0mL Solution E (Wolfe’s vitamin solution) ....... Mix thoroughly............ sterile solution A......................... Mix thoroughly........... Filter sterilize.0mg Nicotinic acid ...........0mg HCl (25% solution) ........................................................0mL: Na2S·9H2O.. 0.........0L of NaOH solution.4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10..0..........2........................5g NH4Cl.... Autoclave for 15 min at 15 psi pressure–121°C. and 10..................5g CoCl2·6H2O................................5........................1g ZnCl2 .................920.1.......... 0.....0mg Cyanocobalamin.....................................6.......0mL of sterile solution B......................... Immediately prior to inoculation............. Filter sterilize....................... Cap with a rubber stopper...0g MgCl2·6H2O........ Solution E (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl.......4mL of sterile solution G.............. reduction........ Aseptically gas under 100% N2 for 20 min........... Cool to 45°– 50°C.. 0... Modified bring volume to 1... Desulfobacterium Medium.............0mg CuCl2·2H2O........10. Filter sterilize.........................50................................... Add sodium acetate to distilled/deionized water and bring volume to 10... 21...... Mix thoroughly...... Prepare solution freshly...................0mg p-Aminobenzoic acid .......................................................... Aseptically gas under 100% N2 for 20 min.......... 3..............0 ± 0......................................... 0............2.... Filter sterilize..... Autoclave for 15 min at 15 psi pressure–121°C........0mL: NaHCO3 .......... Bring volume to approximately 900........ Mix thoroughly. Aseptically gas under 80% N2 + 20% CO2 for 20 min............................0mL Solution G ............................... Cool to 25°C. Cap with rubber stopper............ Mix thoroughly...2g CaCl2·2H2O...0.................0mL of sterile solution E... 1.. Solution F: Composition per liter: Na2SeO3·5H2O . Mix thoroughly......... Preparation of Medium: To 900..2.5.....0mL with distilled/deionized water..........0. 50......................0mL: Na2S2O4....0mg NaOH (0.................... Filter sterilize...1. 10........5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50......0mL Solution C ....................... Aseptically gas under 100% N2 for 20 min..... Mix thoroughly......................0mg Riboflavin............ Mix thoroughly.............................01M solution).0mg Biotin ........0......0mL of HCl solu- tion............5....................................................4mL pH 7................... Aseptically gas under 100% N2 for 20 min. Filter sterilize....4mL: Solution A .......................5..............0mL Solution F....5g Preparation of Solution G: Add Na2S2O4 to distilled/ deionized water and bring volume to 10................................................................. 3.......0mg Calcium pantothenate . 1..............................0mg Thioctic acid ......0mL Preparation of Solution B (Trace Elements Solution SL-10): Add the FeCl2·4H2O to 10...0...................................... indicating FeCl2·4H2O.........................5g Preparation of Solution D: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2....0mL...0mg Preparation of Solution A: Prepare and dispense solu- tion anaerobically under 80% N2 + 20% CO2.............0g Na2SO4 ............0mL.................................0mL of sterile solution C..0mg Folic acid .................. 3.....0mL of sterile solution D..................... Solution C: Composition per 50.................

Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O .................0mL Solution G ...................................0mL.......................................... 0........ Mix thoroughly.........5mg Preparation of Solution F: Add Vitamin B12 to distilled/deionized water and bring volume to 10................... Solution D: Composition per 50.....................................3................................... solution will solidify and should be remelted before use.................. Cool to 25°C..........0..... Mix thoroughly....................................................... Solution H: Composition per 10..0mL of sterile solution B.............10..................0.........................0mL of sterile solution F..50..............0mL of sterile solution E.................0mL....................2 ± 0..................Desulfobacterium oleovorans Medium Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10..0mL: NaHCO3 .............. 0.........0mL Solution B (Trace elements solution SL-10)...13....0mg CuCl2·2H2O .......0mg Folic acid ........... Autoclave for 15 min at 15 psi pressure–121°C......... Immediately prior to inoculation...... ........0mg Preparation of Solution C (Selenite-Tungstate Solution): Add components to distilled/deionized water and bring volume to 1........ Filter sterilize.0mL Solution D .............0mg MnCl2·4H2O....... 2..4g MgCl2·6H2O ............... Filter sterilize......0mL pH 7.........0mL of sterile solution G..........5g Na2WO4·2H2O ...286..............2.....................1........................................... Mix thoroughly..0mg Lipoic acid.....0mL: Vitamin B12................................5.......5g Preparation of Solution H: Add Na2S2O4 to distilled/ deionized water and bring volume to 10............ sterile solution A.........................0mg Vitamin B12........................0L..............................................0mL of sterile solution D..................0mL................... In a closed bottle..0mg NiCl2·6H2O ....................................................0g NaCl ... 24......................... Aseptically and anaerobically distribute into sterile tubes or flasks..0g MgCl2·6H2O.....................0mL: Na2S2O4.....0mL of HCl solution.................................................... Gas under 100% N2................0mg Na2MoO4·2H2O ..........................0mg H3BO3 ...................................... Prepare solution freshly....0mL: Solution A ....85g NaOH (4..........................................0L..... Allow to cool to room temperature while gassing under 80% N2 + 20% CO2.2........... Mix thoroughly................................ 1........................ Preparation of Medium: To 920... 0.5............................5mL Preparation of Solution G: Add components to distilled/deionized water and bring volume to 80....................................................................... Solution C (Selenite-Tungstate Solution): Composition per liter: NaOH ............0mg ZnCl2 .................0mg Thiamine·HCl ...0mL Soluti