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

Many carbohydrate solutions.0 124. Liquid media are passed through sintered glass or membranes.9 103. and vitamin solutions are filter sterilized and added to media that have been cooled to temperatures below 50°C.3 112. A membrane with a pore size of 0. Thallium salts. Basic fuchsin and acid fuchsin are carcinogens. but in some cases sterile acid or base is used to adjust the pH of the medium following sterilization.2 116. This process of repetitive exposure to 100°C is called tyndallization. to form explosive metal azides.4 Filtration Filtration is commonly used to sterilize media containing heat-labile compounds.8 128. Pressure—psi 0 1 2 3 4 5 6 7 8 Temperature—°C 100. 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. Such exposure kills vegetative bacterial cells and bacterial endospores.6 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 5 113. Such exposure can be achieved using flowing steam in an Arnold sterilizer.9 127. Different sterilization procedures are employed when heat-labile compounds are included in the formulation of the medium.6 130. antibiotic solutions. Azides also react with many metals.8 111. sometimes is called a bacteriological filter.8 119. The most common method is to autoclave the medium.Preparation of Media complete medium. Caution about Hazardous Components Some media contain components that are toxic or carcinogenic. . 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. 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. and hence sterile. inhalation. that is.6 118. the medium can be sterilized because all the endospores will have germinated and the heat exposure will have killed all the vegetative cells. Tyndallization Exposure to steam at 100°C for 30 min will kill vegetative bacterial cells but not endospores. free of living organisms.0 122. after its discoverer. This process causes coagulation of the protein without greatly altering its chemical properties.3 106.0 125. sodium biselenite.0 123. Many media are sterilized by exposure to elevated temperatures.9 121.2mm will trap bacterial cells and. generally under pressure. which is a toxic gas.0 101. typically made of cellulose acetate or nitrocellulose. By preventing the passage of microorganisms. The pH often is adjusted prior to sterilization.4 117. with small pore sizes. and caution must be used in handling media with these compounds to avoid dangerous exposure that could lead to the development of malignancies. Using an Arnold sterilizer or a specialized inspissator. therefore. to kill microorganisms. Below is a list of pressure– temperature relationships. such as eggcontaining media.0 126. Appropriate safety precautions must be taken when using media with such components. followed by a 15 min exposure during which the temperature is raised to 121°C using only steam under pressure in the chamber. sodium azide. Inspissation Inspissation is a heat exposure method that is employed with high-protein materials. especially copper.4 109. Exposure for 15 min to steam at 15 psi—121°C is most commonly used.6 105. and cyanide are among the toxic components found in some media.9 115. that cannot withstand the high temperatures used in autoclaving. John Tyndall.9 108.7 129. The disposal of azides must avoid contact with copper or achieve sufficient dilution to avoid the formation of such hazardous explosive compounds. 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. Care must be used to ensure proper ventilation. Autoclaving Autoclaving uses exposure to steam. Media with sulfur-containing compounds may result in the formation of hydrogen sulfide. the temperature then is slowly lowered to less than 60°C. These compounds are poisonous.0 126. and steps must be taken to avoid ingestion. or skin contact. the medium is exposed to 75°–80°C for 2 hr on each of 3 successive days. filtration renders fluids free of bacteria and eukaryotic microorganisms.

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

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

....0g NaHCO3 ..0L....0mg Biotin ................ Kleenex tissue paper...................75................5g Phenol Red ..............0mL Reducing agent solution .................................................. Adjust pH.....0mL Trace elements solution . Pour into sterile Petri dishes or leave in tubes.....................................0mL...........012g pH 6...........................................0mL: D-Cellobiose ...............2.................................. Can be used as a confirmatory test for water analysis.....0g NaCl ............................................................0mg Folic acid .....5............. Mix thoroughly............0mg Riboflavin.................08g pH 6..................................................................6...... Modified) Composition per liter: Agar ..................................0g L-Cysteine·HCl... or HCl-treated cotton).......0mL of distilled/deionized water...............0L.. 2..............0g MnSO4·2H2O ............................................25g FeSO4·7H2O............................................. 1...............................0L......... Mineral Solution 2: Composition per liter: (NH4)2SO4 ............................................. Cellobiose Solution: Composition per 50.................0L.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......... 0............................................... 0............ Store under N2 gas.............. Trace Elements Solution: Composition per liter: MgSO4·7H2O ...0g Nitrilotriacetic acid.0g NaCl ........................................................................ Adjust pH to 7..................5g CaCl2·2H2O.0... Mix thoroughly.............. Acetate Differential Agar (Sodium Acetate Agar) (Simmons’ Citrate Agar.................3........................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.............. Gently heat and bring to boiling............................................................0mL: Cellobiose or cellulose (MN 300........72g NaCl ................................ Gently heat and bring to boiling....... Cool tubes in a slanted position..... .02g Resazurin ......... Use: For the differentiation of Shigella species from Escherichia coli and also for the differentiation of nonfermenting Gram-negative bacteria................ Add remaining components...0..0g MgSO4·7H2O ............................ 2........................................0L.............18g ZnSO4·7H2O ..........................0L.....5........... Whatman CF II.........0g Yeast extract......0. Add distilled/ deionized water to 1... 15.....5 with KOH.......................................................................................................................... Filter sterilize... 0. Acetate Agar Composition per liter: Agar ............................8 ± 0.........................................5.........5....................................4g MgSO4·7H2O.............................................................75.............................. Bacteria that can utilize acetate as the sole carbon source turn the medium blue.................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..........0mg Lipoic acid......................0g K2HPO4 ..............0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50......................... 0................. Preparation of Medium: Add components to cold dis- tilled/deionized water and bring volume to 1................... Use: For the differentiation of nonfermentative Gram-neg- ative bacteria.............59g Preparation of Mineral Solution 2: Add components to distilled/deionized water and bring volume to 1..................................1...........1g NiCl2·6H2O ................. Mix thoroughly......... Mix thoroughly..8 Acetate Agar MgSO4·7H2O .....0............ 2................................. Use: For the cultivation and maintenance of Caryophanon latum...............................................01g Na2SeO3·5H2O .. Autoclave for 15 min at 15 psi pressure–121°C......................................................................0mL Cellobiose solution .................................................3................50.......0mg p-Aminobenzoic acid ... 0............0mg Vitamin B12......................25g Na2S·9H2O......................025g KAI(SO4)2·12H2O ....0................. 1................2 at 25°C Preparation of Medium: Add components to distilled/ Mineral Solution 1: Composition per liter: deionized water and bring volume to 1.................................................................................. Sparge under 100% N2 gas for 3 min.......................................................................1............ 0...0g Sodium acetate ........ 0........................ Mix thoroughly............... Autoclave for 15 min at 15 psi pressure–121°C... Bacteria that deamidate acetamide turn the broth purplish red..................... 1....0........... 20............. especially Pseudomonas aeruginosa................0mL Mineral solution 2 ......0mg Thiamine·HCl ...............................................5...............................02g CuSO4·5H2O .10.........2g CaCl2·2H2O .....................4 ± 0.........0mL pH 7.........................0........................0........0....0g Pancreatic digest of casein .......0mL K2HPO4 ...........................5....................... 10.......... Acetivibrio cellulolyticus Medium Composition per 1170..........1......0 with 1N KOH.............10.0...................................2.....................................0g pH 7.......................2 ± 0..5g CoSO4·7H2O ...........2.9g Preparation of Mineral Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 1.18g FeSO4·7H2O..............0L................................................0g Sodium acetate ............................................... Mix thoroughly..... Mix thoroughly.................... 3............................................................................ Dissolve by adjusting pH to 6............................................................................001g Mineral solution 1 ......................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems............0mg Nicotinic acid .....................2 at 25°C Vitamin solution ..............0.... Vitamin Solution: Composition per liter: Pyridoxine·HCl..1....................0g K2HPO4 ......................9 ± 0..........................................................0mg Calcium DL-pantothenate .......................... Distribute into tubes or flasks....... 0....................................0..........................................01g Na2MoO4·2H2O ........ 5............................. Mix thoroughly................ 10................0g (NH4)H2PO4 .....0................5.................................. 1.......... Distribute into tubes to produce a 1 cm butt and 30 cm slant.......01g H3BO3 ................................2g Bromothymol Blue ..................

.....1g ZnCl2 .............................1mL of sterile reducing agent solution and 0.......0mL Solution D ........................7g Sodium caproate ..................0mL: Sodium butyrate ...................0mL flask...... Autoclave for 15 min at 15 psi pressure–121°C......................0mL...... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C........................ 0....................0mL Solution E .0g Preparation of Solution C: Add the NaHCO3 to distilled/deionized water and bring volume to 100.. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.................10..............5.......... Solution D: Composition per 10.......... Adjust pH to 7.......7....................0mL: 9 clave under 100% N2 for 15 min at 15 psi pressure–121°C.................... 2.................. solution D.........0mL: L-Cysteine·HCl·H2O ..5..............4mL of sterile basal medium..0mL Solution B ........................................................................ Acetobacter diazotrophicus Agar Composition per liter: Glucose... a nitrogen fixing endophyte........ Cool to room temperature. 0............................. Anaerobically distribute under 90% N2 + 10% CO2 into sterile tubes or flasks... Use: For the cultivation and maintenance of Acetobacter diazotrophicus.................0.............10......0g Yeast extract ..........0g pH 5. cellulolytic bacterium..... Use: For the cultivation and maintenance of Acetivibrio cellulolyticus.........0.... 0.0L.......... Mix thoroughly..... to distilled/ deionized water and bring volume to 940................................. Pour into sterile Petri dishes or leave in tubes.0mL of distilled/deionized water to a 250.................................0mL volumes into tubes............ except cel- lobiose solution and reducing agent solution............ Adjust to pH 9 with 5N NaOH......... Autoclave for 15 min at 15 psi pressure–121°C.. Gently heat and bring to boiling.......... Mix thoroughly...........0g NaCl ...........30.... Mix thoroughly..................................10........ Continue boiling for 3 min..0L......................... Solution B: Composition per liter: FeCl2·4H2O ..... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.....19g MnCl2·4H2O.. 1................ solution C.. Prepare and autoclave part A under 80% N2 + 20% CO2... Mix thoroughly to evenly distribute CaCO3................0........................................................................ Na2SO4 .5g MgCl2·6H2O.......0µg Preparation of Solution E: Add components to distilled/ deionized water and bring volume to 10...................................... Cool to room temperature..0g Agar ........... Add L-cysteine·HCl·H2O and dissolve.....2 at 25°C Solution A: Composition per 869. 0..........100............ 10..........5g Preparation of Reducing Agent Solution: Add 110...... 40...... Add distilled/deionized water and bring volume to 1................... Cool to room temperature..........................................04g NiCl2·6H2O ............0g Thiamine·HCl ....... 0....................... 25% ................ aseptically add the remaining sterile solutions in the following order: solution B...........0µg D(+)-Biotin.....06g Na2MoO4·2H2O ...............................2................................ Distribute anaerobically under 80% N2 + 20% CO2......25.............................................................................. Preparation of Medium: Add components..............................................................................0g CaCO3. Adjust pH to 7...................... Preparation of Solution A: Add components to dis- Use: For the cultivation of Acetivibrio ethanolgignens and tilled/deionized water and bring volume to 869..................................... solution E.. Gently heat and bring to boiling.. Cool rapidly to 50°–55°C.......................0......................................5mL of sterile cellobiose solution to each tube containing 9. Auto- Desulfovibrio sapovorans which reduces sulfate...07g H3BO3 .............................................. Autoclave for 15 min at 15 psi pressure–121°C...................0mL Solution F.........0mL Solution C ....................................... Prior to inoculation of cultures... Solution F: Composition per 10.............. Distribute into tubes or flasks......0mL: Na2S·9H2O ....... 2......................................................4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....... 0........... Cool to room temperature.............. Mix thoroughly..........................0mL................3g Sodium octanoate ............................7 ± 0.......0mL Preparation of Solution B: Add the FeCl2·4H2O to the HCl...3g KH2PO4 ..5g Na2S·9H2O.1......... Cool to room temperature..................0mL pH 7. ............... 3........ Solution E: Composition per 10... 0......Acetobacter diazotrophicus Agar Reducing Agent Solution: Composition per 110...... an anaerobic.....0mL: NaHCO3 ............0mL of sterile cooled Part A....................50........................... Mix thoroughly..................0mL... Gas with 80% N2 + 20% CO2 to remove residual O2.......4g NH4Cl...10.....15g Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10........6 by gassing...................0mL........ Add remaining components..... Mix thoroughly............. Solution C: Composition per 100... Distribute under N2 gas in 10.........................02g CuCl2·2H2O ..10............ 0........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...... 0............................ aseptically and anaerobically add 0....... Autoclave for 10 min at 15 psi pressure–121°C..........1.......5g CoCl2·6H2O ......................................................................... and solution F...............0mL....0mL. 0................... Acetivibrio Desulfovibrio Medium (LMG Medium 105) Composition per liter: Solution A .......... After autoclaving..................15g Preparation of Medium: To 869.........0mL: Yeast extract ...... 100....... Cool to room temperature.02g HCl.5 ± 0........................................... Mix thoroughly.............. 0.... 0..... Cool to room temperature under 80% N2 + 20% CO2............ Boil under N2 gas for 1 min........................................................2g CaCl2·2H2O............................................... Add washed Na2S·9H2O and dissolve............ Filter sterilize.0µg p-Aminobenzoic acid ................................................... Bring pH to 5......................0g KCl...... the pH of the medium will be 7.....................................................869.... 1......

...... For every 9..3g Preparation of L-Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10......5................0mL Na2S·9H2O solution ...........0mg Folic acid ....0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100......0mL....................... NaHCO3 Solution: Composition per 20.............. and cysteine solution.... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere.....................0mL pH 7....................15mL Na-syringate solution............................................................................... Cysteine Solution: Composition per 10.....45g KH2PO4 ....... Sparge with 80% N2 + 20% CO2.1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........0mL NaHCO3 solution.......5mg Fructose solution . 0..........0g MnSO4·2H2O ....................0mL Seven vitamin solution ............................0mg Lipoic acid...5................ 1.................. 0... 0........................ 0.................................... 1................33g CaCl2·2H2O ............0mL..................................................33g NH4Cl .................... 0.... 0........................................................0mg D-Ca-pantothenate .... Mix thoroughly............................................................0mL of medium so that pH is adjusted to 7...................................... except NaHCO3 solution................................ Aseptically and anaerobically add approximately 0.......................................................... Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C.......3g Preparation of Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10..... Sparge with 100% N2.3g Resazurin ......... Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O ............................................ Autoclave for 15 min at 15 psi pressure–121°C. Sparge with 100% N2...................0 ± 0.........................................................................0mg Vitamin B12....................1.. 0........10.................0g K2HPO4 ..................... Filter sterilize............... Mix thoroughly.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500...............................0mg Thiamine-HCl·2H2O ......20.......0mL pH 7........... 2...........................0mL...................................................................................................... 3... Cool to 25°C.............................0mL: NaHCO3 .........0mL L-Cysteine solution.. Sparge with 80% N2 + 20% CO2.............................4 ± 0............................................. Mix thoroughly...................................................33g KH2PO4 .....................5..............0.......................................... Filter sterilize.......................0............................................................. 0.................... Add distilled/deionized water to 1...0......................... 0.............................0mL of medium inject 1....... 0. 0.10......... 0..0...................................................................................01g Na2MoO4·4H2O ......................................0g Nitrilotriacetic acid .................0mg Riboflavin..............................0.......... 20........ 1...0mg p-Aminobenzoic acid ....... and 0..... Filter sterilize....................0L..........0mL aliquots into serum bottles........... 10............................................. 1.. Sparge with 80% N2 + 20% CO2..................0mL: Na-syringate....0..................................0mg NaHCO3 solution ............0mL........................................ Vitamin Solution: Composition per liter: Pyridoxine-HCl ...5 with KOH............ 1..................................10 Acetobacterium dehalogenans Medium Acetobacterium dehalogenans Medium (DSMZ Medium 787) Composition per liter: NaHCO3 ..... Add components..................................33g MgSO4·7H2O .. Mix thoroughly....................................... Use: For the cultivation of Acetobacterium dehalogenans................ 10....0................. Boil for 5 min.............0mL Trace mineral solution SL-10...................................0mL Cysteine solution........................18g ZnSO4·7H2O ...........02g H3BO3 .0............ Sparge with 100% N2........... Na-syringate Solution: Composition per 20..........1g Resazurin .......30.....................0g Yeast extract..........................................2 at 25°C L-Cysteine Solution: Composition per 10................ Dissolve by adjusting pH to 6. Mix thoroughly...............2.................................20............................................................ Filter sterilize.0mL: NaHCO3 ....0g NH4Cl.................0mL: L-Cysteine·HCl·H2O ...........0mg Nicotinic acid ............ Mix thoroughly.... to distilled/deionized water and bring volume to 900.......................................... 5........ vitamin solution..........................0mL of distilled/deionized water..........................2mL vitamin solution..0L....................5.... Trace Elements Solution: Composition per liter: MgSO4·7H2O ........0g MgCl2·6H2O .... Cool while sparging with 80% N2 + 20% CO2..............5g CoSO4·7H2O ..........0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20... 0..............1g NiCl2·6H2O ........... 1............................... 30.....2.............................0mL Na-syringate solution .......025g KAl(SO4)2·12H2O...............................3g ................................................ 0................................5................................. 10... Acetobacterium tundrae Medium (DSMZ Medium 900) Composition per liter: Yeast extract ...............17mL cysteine solution............20mL sterile Na2CO3 solution to each 9.....................................................52g KCl ............................. 10................ 10.... Na2CO3 solution...............................................................5g NaCl ...........................................2 at 25°C NaHCO3 Solution: Composition per 100...............0mL........................................ which dehalogenates methyl chloride..... 0..................... Distribute 9............... Add remaining components.................... Gently heat and bring to boiling...........................01g Na2SeO3·5H2O ....4......... Na-syringate solution.......... Mix thoroughly...............................5................0mL Trace elements solution .. 0..2g Preparation of Na-syringate Solution: Add Na-syringate to distilled/deionized water and bring volume to 20..............0mL...........0.............................. 0........0mL Vitamin solution...........................0mL NaHCO3 solution.....01g CuSO4·5H2O ...0mL: L-Cysteine·HCl·H2O... Mix thoroughly..0mL Na2CO3 solution.........20....... Autoclave for 15 min at 15 psi pressure–121°C.............0mg Biotin ......................18g CaCl2·2H2O... 1..................1g FeSO4·7H2O......................

............................................................................... Cool to room temperature....................5g CoCl2·6H2O ....5...2...................... Add components....................0mL fructose solution...........................................0mL Na2S·9H2O solution..............................................0...2.. Mix thoroughly.... Filter sterilize.1% solution) ......................................0mg Niacinamide ..............0....0mL Tungstate solution .................................. Aseptically and anaerobically add per liter of medium..................................1.................... 4....................0mg Na2MoO4·2H2O .......0mL: Potassium Phosphate Buffer: Composition per 830......................................................0mg Calcium pantothenate ..0mg Lipoic acid........ a psychrophilic...0.....................0g NiCl2·6H2O .0mL Preparation of Stock Salts Solution #2: Add compoTrace Minerals: Composition per liter: Nitrilotriacetic acid.......2g Preparation of Stock Salts Solution #1: Add components to distilled/deionized water and bring volume to 1.... Distribute into anaerobe tubes or bottles.............. Preparation of Potassium Phosphate Buffer: Dis- Glucose ...0g MnSO4·H2O..............5 with KOH.............................................................................................. L-Cysteine solution.........01g CuSO4·5H2O......................................5.......2g Stock salts solution #1 ..................... 5.......................... 80............................... 100........15............5.. Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10................................0............0mg H3BO3 . 100......0.................................................................................5... and Na2S·9H2O solution........................0mL...........0mL of distilled/deionized water and KH2PO4 in 230............................5g NaCl ........ 0....0......01g Preparation of Trace Minerals: Add nitrilotriacetic acid to 500...............0...................................................................4mL Resazurin (0..................Acetogen Medium 11 Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10................................... Vitamin Solution: Composition per liter: Pyridoxine·HCl................0L with distilled/deionized water..... to distilled/deionized water and bring volume to 929......................................................0mg Nicotinic acid ...........................................................0mg Riboflavin.0mg CuCl2·2H2O . Add remaining components....0mL: Preparation of Fructose Solution: Add glucose to distilled/deionized water and bring volume to 30................. Sparge with 100% N2................0mg Vitamin B12 ............................................................................................................................................................... Stock Salts Solution #1: Composition per liter: KCl ..0.0mg Pyridoxal·HCl............5........................................ 70............................20...........0mg Biotin .......................... except seven vitamin solution.....0mg Thiamine·HCl .......................0... NaHCO3 solution.... Add remaining components........................................1g FeSO4·7H2O ...........4...1g FeCl2·4H2O ..................0... Add distilled/deionized water and bring volume to 1....................40................................ 1.....0. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C... Mix thoroughly.................. 1.....0mL Trace minerals................................ Mix thoroughly...............5................. Acetogen Medium Composition per 421................ Stock Salts Solution #2: Composition per liter: Trace Elements Solution SL-10: Composition per liter: CaCl2·2H2O .................................. Sparge with 80% N2 + 20% CO2. 0...................................0mL Potassium phosphate buffer ................. 2...... Mix the two solutions together and use..0mL...0mg Nicotinic acid ........5.........4g MgSO4·7H2O...0mg Ascorbic acid............................................0mL nents to distilled/deionized water and bring volume to 1......1g ZnSO4·7H2O..........1g CaCl2 .................0mg HCl (25% solution) .........................................0mg ZnCl2 .... Autoclave for 15 min at 15 psi pressure–121°C.............. Mix thoroughly............. fructose solution......... Sparge with 100% N2............10.............68g KH2PO4 ......................0mL NaHCO3 30.......................... 0... Filter sterilize........................................................0mL seven vitamin solution.. Bring volume to 1.........................0L.4mL Fructose Solution: Composition per 30.......0mg Vitamin B12.............1g Na2SeO3·5H2O ........................................... Mix thoroughly.......... 100..............................0mL of distilled/deionized water..................................0mg D(+)-Biotin .......... Mix thoroughly................................................................. Dissolve by adjusting pH to 6....... Sparge with 80% N2 + 20% CO2.......................................5g MgSO4·7H2O.................1.......0mg i-Inositol ................2g Yeast extract.............0mL L-Cysteine-HCl·H2O solution....................................................................................... Mix thoroughly.................01g H3BO3 ...............................1................................................0mg p-Aminobenzoic acid ............................... 24...........................................0mg Calcium pantothenate ..............5............. K2HPO4 ...... Autoclave for 15 min at 15 psi pressure–121°C................0g Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ...........................1mg ...........0mL of HCl solution...1................................ Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere.......4........ 0.............. 190.................0L...............................................72g solve K2HPO4 in 600............01g AlK(SO4)2·12H2O ............0........... 36...........0mL Reducing agent ..........................................0.........................................5................01g Na2MoO4·2H2O................................................. 200.... 200........................................ 4..20...............0mg Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.. Mix thoroughly................. 10....................0mg NiCl2·6H2O .................................. acetigenic bacterium from tundra soils...............4........0L....0mL of distilled/deionized water........... 20...1g CoCl2·6H2O.....................0mg Thiamine-HCl·2H2O ....................................10........ 2...................................... The final pH should be 7...........0mL Stock salts solution #2 ................. 300................0.....5.....................0mL Clarified rumen fluid ..0mg p-Aminobenzoic acid ...................................... Mix thoroughly..........0mg MnCl2·4H2O..............................0L........................................ Vitamin solution ..................................................................0....................................... Use: For the cultivation of Acetobacterium tundrae.......................... 6....3......................................................0mg Folic acid ..4g NH4Cl........................................ 20.....0mg Choline chloride ..6g NaCl ........ and 10..............8mL: NaHCO3 ....0mL.............................. Mix thoroughly....5............

............. 0......... Mix thoroughly......................5 g CaCl2·2H2O ..................... exchange headspace with 80% H2 + 20% CO2...................... 0................0mL: NaHCO3 ..... Gently heat and bring to boiling...............................12 Acetohalobium Medium Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.. Dissolve by adjusting pH to 6........................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.........01g Na2SeO3·5H2O....... 150....................33g Resazurin .....0mg Preparation of Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.................................. Store frozen......................5........... yeast extract solution.................................5....... Cool to room temperature....2........... 4.....................................................0mg Thiamine·HCl .......................... NaHCO3 solution.......2.0g Nitrilotriacetic acid ............. Autoclave for 15 min at 15 psi pressure–121°C.0mL of dis- tilled/deionized water to a 250.. One may use 4.0L........ 1............................ Store under N2 gas..............0mL........ Autoclave for 15 min at 15 psi pressure–121°C.........................0mg Nicotinic acid .........2.......... 1.......................0mL pH 7......... Add distilled/ deionized water to 1......................................... 0.............. to distilled/deionized water and bring volume to 1......0mL of distilled/deionized water...... Store under N2 gas.....................................................0L...........0mL: Trimethylamine·HCl.........0mL round-bottomed flask..... Add L-cysteine·HCl and dissolve...........................0mg Riboflavin..... Yeast Extract Solution: Composition per 5.......... Adjust pH to 7.............................01g H3BO3 .................5g Preparation of Reducing Agent: Add 110........... 0....5........6 ± 0... Sparge under 100% N2 gas for 3 min............................................ Cool to 45°– 50°C.......0mL: Yeast extract ................................................5.. After inoculation......3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500..................... Sparge under 100% N2 gas for 3 min..............10....18g ZnSO4·7H2O .......0mg Calcium DL-pantothenate ........5g of glycine betaine in place of trimethylamine·HCl........... Acetohalobium Medium Composition per 1025......0mL....................0L..................... 99......5...........................................1............. Allow to cool to room temperature under 100% N2.................. Tungstate Solution: Composition per liter: Na2WO4·2H2O ......... Na2S·9H2O Solution: Composition per 5.................. aseptically and anaerobically add vitamin solution............................5g Na2S·9H2O............ Distribute into tubes or flasks under 100% N2. Mix thoroughly......................... yeast extract solution.. Mix thoroughly.................................................................. trimethylamine·HCl solution...0mg Trace elements solution ..........0mg Lipoic acid.......... Mix thoroughly.. Mix thoroughly....... Vitamin Solution: Composition per liter: Pyridoxine·HCl....... Sparge under 100% N2 gas for 3 min..................0mg Biotin ...................... Add washed Na2S·9H2O and dissolve............ 1............. Store under N2 gas... Store under N2 gas.................0......0mL Trimethylamine·HCl solution ............5.......... 0...........05g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 5......4g Preparation of Trimethylamine·HCl Solution: Add trimethylamine·HCl to distilled/deionized water and bring volume to 10...............................0g MnSO4·2H2O............0g MgCl2·6H2O. Filter sterilize. Mix thoroughly............ Preparation of Medium: Add components............ 0.... Autoclave for 15 min at 15 psi pressure–121°C.........0........ Mix thoroughly.0mL: NaCl ....8mL................01g Na2MoO4·2H2O....................................................10.....5g CoSO4·7H2O.................. NaHCO3 Solution: Composition per 5.........0mL: Na2S·9H2O .............5......0g NaCl .........33g KCl................ Boil for a few minutes..................................................................... 0....02g CuSO4·5H2O........................ Use: For the cultivation and maintenance of acetogenic anaerobes such as some Clostridium species..5..................0... Trimethylamine·HCl Solution: Composition per 10............33g NH4Cl.............................. Before inoculation..... 0......................................0mL..............................5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 5..................... ...0mL: L-Cysteine·HCl·H2O ............ Autoclave for 15 min at 15 psi pressure–121°C........ Mix thoroughly.............. Autoclave for 10 min at 15 psi pressure–121°C.................... Autoclave for 15 min at 15 psi pressure–121°C...0L............... 0................................................................0mL NaHCO3 solution ...0.....0L..0mL Vitamin solution..... Gently heat and bring to boiling under 80% N2 + 20% CO2........ Distribute into tubes or flasks under 80% N2 + 20% CO2.. 2.................................025g KAI(SO4)2·12H2O ... Cool to room temperature......................... except vitamin solution............................ Add NaHCO3 and reducing agent.........10..... 0......... 2..........0mL Yeast extract solution .... NaHCO3 solu- .. 3..0g CaCl2·2H2O...........1g NiCl2·6H2O.... 0.... 0..... Boil under N2 gas for 1 min...... Add remaining components.................................................... Mix thoroughly....5..................... and Na2S·9H2O solution............................... trimethylamine·HCl solution.... Autoclave for 15 min at 15 psi pressure– 121°C...................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O solution to distilled/deionized water and bring volume to 5...................................0 with KOH.............. Sparge under 100% N2 gas for 3 min...................................18g FeSO4·7H2O ......0mL................... Reducing Agent: Composition per 110.. Mix thoroughly.......... Preparation of Medium: Add components...33g KH2PO4 ..................................................... Adjust pH to 7........0mg Folic acid ........ 0............................ except NaHCO3 and reducing agent.......................2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O................................. 0.........5 with KOH... Adjust to pH 9 with 5N NaOH.....0mg Vitamin B12.................0mg p-Aminobenzoic acid ............... Distribute in amounts needed into tubes or flasks.....4 with NaOH...........2–7...............................................10. to distilled/deionized water and bring volume to 417.......................0mL Na2S·9H2O solution............................

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

..............0..........................................................1g Guanine ...................................................0.......................... NaHCO3 solution.....0L............................................................ except Lcysteine solution.....0...........................................................................0g Pancreatic digest of casein ......0mg p-Aminobenzoic acid .........................................0......... 0............................ 190..4 ± 0....................................... 0.....5mL.......... a fermentative bacterium from estuarine mud...............................0mL pH 7..................................... 2... Filter sterilize...............0..........................0L.................. Autoclave for 15 min at 15 psi pressure–121°C.... 50....0g Glucose...................................1....0mg Calcium DL-pantothenate.................1... 1.....................0g Yeast extract .................................0.......... Mix thoroughly...........3g KH2PO4 ............................. Distribute into tubes or flasks under 100% N2.5................ Vitamin Solution: Composition per liter: Pyridoxine·HCl ....... 1........................... and Na2S·9H2O solution..................... NaHCO3 Solution: Composition per 5.................................0.............................................. Store under N2 gas................ Preparation of Medium: Add components................................14 Acidaminobacter Medium Preparation of L-Cysteine Solution: Add 0..............................................0µg Biotin ...1g Tween™ 80 .........4.............................................................0g Glycine .... 0...0............................................................................................... Autoclave for 15 min at 15 psi pressure–121°C........................................................ 0......... Acidaminococcus fermentans Medium Composition per liter: Casamino acids....0mg Na2MoO4·2H2O ... 0.................................................5mL pH 7............................0mL...........................0mg Thiamine..01g Uracil .......... Sparge under 100% N2 gas for 3 min....................0mL.....5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 5... 0.................................. Na2S·9H2O Solution: Composition per 5........2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ..............0................................. 100............................... Mix thoroughly..................4g Na2S·9H2O................. 50............................. Before inoculation....................................15g Resazurin ............... 0......................... Aseptically distribute into sterile tubes or flasks..0mg NiCl2·6H2O ....... Acidaminobacter Medium Composition per liter: NaHCO3 ................0mL: Na2S·9H2O .......0mg Calcium pantothenate ...................................5.. Add distilled/deionized water and bring volume to 1.30........................................................................................20........................... 5...............0g Arginine..0µg Riboflavin........ 0....................................0mL of HCl solution..............................0mg Vitamin B12 .................... Filter sterilize..............0µg .........................1g of Lcysteine to distilled/deionized water and bring volume to 10....................................01g Hypoxanthine ..........................0µg p-Aminobenzoic acid .0g L-Cysteine·HCl·H2O.1..0mg Folic acid............. Mix thoroughly.................... to distilled/deionized water and bring volume to 999...............................2g Na2SO4 .......................... Check that final pH is 7..............5g NaCl ......4g MgCl2·6H2O..................................................................0L.....0mL.............. and Na2S·9H2O solution.0mg CuCl2·2H2O ...........8........................................ Mix thoroughly....................................................0 ± 0.................................... 2.....................................0mL NaHCO3 solution ...... 2....2g KCl............................................................0g L-Cysteine·HCl .............................0mg Nicotinic acid ....... Mix thoroughly. Boil for a few minutes. Adjust pH to 4.......................................0mg MnCl2·4H2O.......... Mix thoroughly........... 5.................................... 5............. Mix thoroughly....................................0g Sodium glutamate................................10.......01g Pyridoxal .................................. Autoclave for 15 min at 15 psi pressure–121°C....2............................. 50...0mg Na2SeO3·5H2O ................................................... Mix thoroughly............................. 0..... 2............. Add remaining components......................2g Yeast extract.....................................0L......................................0mg Thiamine·HCl .......... 10................................................... Distribute into tubes or flasks..........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........0g Glucose.0mL Trace elements solution SL-10 .........5........... Allow to cool to room temperature under 100% N2...... Mix thoroughly.10.....1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......... aseptically and anaerobically add vitamin solution.......................5g DL-Tryptophan...............0mL: NaHCO3 ......10.......................... Sparge under 100% N2 gas for 3 min....................................... Gently heat and bring to boiling.......... to distilled/deionized water and bring volume to 1......0mg ZnCl2 ................5. Preparation of Medium: Add components.. 70............ 6.............................. 5. Cool to room temperature............................ NaHCO3 solution..................4..................... 5...........0g Glycine.. 1.................1...................................... 1.....35g DL-Tryptophan... 10..................................................................................................... Store under N2 gas................ 0...................... 5........................... Acidaminococcus Medium VR Composition per liter: Acid-hydrolyzed casein (vitamin and salt free) .................. Mix thoroughly........ Aseptically add 0..... Use: For the cultivation and maintenance of Acidaminococcus fermentans from rumen..........0mg Riboflavin ............................0µg Niacin . Use: For the cultivation and maintenance of Acidaminobacter hydrogenoformans..5...............5............ Adjust pH to 7.5mL of sterile L-cysteine solution...................0mL Na2S·9H2O solution.......................0mg H3BO3 ......... 36.2g CaCl2·2H2O...............................................................................0g KH2PO4 ...................................................0mg Biotin ....... Autoclave for 15 min at 15 psi pressure–121°C. 2............... Autoclave for 15 min at 15 psi pressure–121°C. except vitamin solution.......... Use: For the cultivation of a variety of fungi.............................0mg HCl (25% solution) .........0µg Vitamin solution......................0mg Lipoic acid .............1...........0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.................................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O solution to distilled/deionized water and bring volume to 5... Mix thoroughly.5g CoCl2·6H2O ................................. 24......................

......................................... Aseptically com- 15 bine the basal solution.... sterile sulfur flowers.................................................................................................................0g (NH4)2SO4 .............0.......................5–2....0...........0.............................. VR Salts B: Composition per liter: MgSO4·7H2O ..................... adjust pH with solid K2CO3 to 7.......25g CaCl2·2H2O ......................................................................0g CaCl2·2H2O......3g CoCl2·6H2O.........0..................02g Na2B4O7·10H2O .........0g of nitrilotriacetic acid to 300........................5mg MnCl2·4H2O ..................... 0.......................07g FeCl3·6H2O..................22mg CuCl2·2H2O..................... 0............................................................. Use: For the cultivation and maintenance of Acidianus brierleyi...................................4g Yeast extract .......................................1............ 0.........................03mg CoSO4 ...... Stir vigorously......01g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1........................0.....2g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.................0g KH2PO4 ..................0.. Autoclave for 15 min at 15 psi pressure–121°C..................0g Sulfur flowers ........................ Acidianus infernus Medium Composition per liter: (NH4)2SO4 .... 24......................... 12..............................0...............5 with 6N H2SO4.............4.............................0............0..............30.................0................................................ Filter sterilize....................................5g MgSO4·7H2O .. Mix thoroughly..................................... 10.....................8mg ZnSO4·7H2O......................5 with 10N H2SO4.....0mL Ferric Citrate Solution: Composition per 100...........................................................10.........01mg pH 2.................................................... 0......... Acidianus brierleyi Medium Composition per liter: Sulfur flowers .02g CuCl2·2H2O.........01g Yeast extract solution .... 0.......0mL: Disodium succinate ............................0......... Mix thoroughly..................... 0.......0L.... Autoclave for 15 min at 15 psi pressure–121°C.......... and sterile yeast extract solution........................25g CoCl2·6H2O ...0µg VR salts A ... Mix thoroughly.......5g FeSO4·7H2O......0..............0......0.....................................................................................................1......................................................................1.0g KH2PO4 ................................................................................... Add distilled/deionized water and bring volume to 1................ Filter sterilize..2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............5 ± 0.............................................................................................................0.....5–2.......0mL.........................0.............03g Na2MoO4·2H2O.......... Add 2...........25g VSO4·7H2O.............................................................. 37.....0..........................................Acidic Rhodospirillaceae Medium Folic acid........ Preparation of Medium: Add components................................................................. Filter through paper..................03mg VOSO4·2H2O ......0mL pH 7.0mL....................1g Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 100. Preparation of Medium: Filter sterilize vitamins as separate solution...............................50.1g Ca(NO3)2 ..........5g KH2PO4 ............................0mL of concentrated HCl and heat until dissolved...................................2g ZnSO4·7H2O...........0.....0mL Trace elements solution....4...............0µg Vitamin B12 .............0mL: Ferric citrate .................................... Mix thoroughly............................. except ferric citrate solution and trace elements solution.. 0.........................0 ± 0............. Aseptically .0........... Preparation of Medium: Add components............. Adjust pH with 10N NaOH to 7.................125g Preparation of VR Salts B: Add components to dis- tilled/deionized water and bring volume to 700...................0mL.............1....... Slowly add the nitrilotriacetic acid solution to the larger volume of salt solutions while stirring until dissolved.............25g Na2MoO4·2H2O .................. to distilled/ deionized water and bring volume to 1.....5g Preparation of VR Salts A: Add components to dis- tilled/deionized water and bring volume to 500...........0mL distilled/deionized water................................... Add aseptically to sterile basal medium................................................. 5..................................................0mL: Yeast extract..... 1....................................... Adjust pH to 2........... 0............. Store in a cool place. Gently heat and bring to boiling..............0.................5 at 25°C Yeast Extract Solution: Composition per 10....0g K2HPO4·3H2O......................0mL...........................................5g KCl......................03g NiCl2·6H2O.............0L............. Acidic Rhodospirillaceae Medium Composition per 1006.................................. Mix thoroughly......4g NH4Cl ........... 3.... and Desulfurolobus ambivalens... Prepare and distribute medium anaerobically using Hungate techniques with 100% N2 gas..........1............................25g MnSO4·H2O ... Use: For the cultivation and maintenance of Acidaminococcus fermentans from rumen...................................................................................... 0....... Acidianus brierleyi................. 0........................0mL: Na2HPO4 ....0......0mL VR salts B ........1..0.........0mg Ferric citrate solution .........................5g MgSO4·7H2O....................... Add 5..............................0L..........3g Yeast extract ..........................2g CaCl2·H2O ..... Adjust pH to 1........05mg Na2MoO4·2H2O...0.........25g CuSO4·5H2O .......... 0....................................... Autoclave for 15 min at 15 psi pressure–121°C................................................... Distribute into tubes or flasks.............................. to distilled/deionized water and bring volume to 990..........................28g MgSO4·7H2O............ Use: For the aerobic cultivation and maintenance of Acidianus infernus................. Mix thoroughly..... 0........2 at 25°C VR Salts A: Composition per 500.......................... Acidianus infernus................ Trace Elements Solution: Composition per liter: H3BO3 . Mix thoroughly.....................5g ZnSO4·7H2O ...................1.........0L............... Sulfur flowers are sterilized separately by steaming for 3 hr on 3 consecutive days......................... If necessary.....0mL pH 1... a facultatively anaerobic thermoacidophilic archaean............0mL...1g MnCl2·4H2O . except sul- fur flowers and yeast extract solution........ 0.......... Autoclave for 15 min at 15 psi pressure–121°C...........0.4g NaCl ..

. Autoclave for 15 min at 15 psi pressure–121°C..0mL......................................... Use: For the cultivation and maintenance of Acidiphilium cryptum..............1g Glucose solution ................ Yeast Extract Solution: Composition per 10...2g KCl......... Filter sterilize...................................... Sulfur...2.....0...... Mix thoroughly.................................0mL of sterile yeast extract solution....................... Autoclave for 15 min at 15 psi pressure–121°C..............0mL pH 3......................................................................1g pH 3.............................0.......................... Preparation of Medium: Add components..................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 10............................. to distilled/deionized water and bring volume to 1080................... aseptically add glucose solution and yeast extract solution............... Gently heat and bring to boiling.............5g (NH4)2SO4 ......5g MgSO4·7H2O.....0mL..... Mix thoroughly.. Autoclave for 15 min at 15 psi pressure–121°C........7 ± 0.................................................0mL of sterile trace elements solution.. Mix thoroughly... 0... Autoclave for 15 min at 15 psi pressure–121°C...... Distribute into tubes or flasks..............................0mL pH 3................ 0......... 10..0........ Autoclave for 15 min at 15 psi pressure–121°C...... Use: For the cultivation and maintenance of Leuconostoc oenos and other Leuconostoc species...........3g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10................ 0........................ Pour into sterile Petri dishes...... Use: For the heterotrophic cultivation of Sulfobacillus aci- Acidiphilium Medium Composition per liter: (NH4)2SO4 ......... Mix well and warm gently until dissolved........................0mL: D-Glucose .......0g K2HPO4 ........... 0.............2 at 25°C Preparation of Medium: Add solid components to 750................... Aseptically distribute into sterile tubes or flasks. 0............... 15..... dophilus..... Use: For the cultivation of Acidobacterium capsulatum..0mL.......... 15..................0mL Na2S·9H2O solution ...................................... Mix thoroughly......... to distilled/deionized water and bring volume to 980..33g Resazurin ............................... Acidimicrobium Medium Composition per liter: Acidolobus aceticus Medium (DSMZ Medium 901) Composition per 1055mL: FeSO4·7H2O........................................................................... Add tomato juice..5g (NH4)2SO4 .............. except yeast extract solution................1g Yeast extract .............................9g MgSO4·7H2O ..............0g Peptone........................0 ± 0................................ Acidobacterium Medium Composition per liter: (NH4)2SO4 . Aseptically add 20....0..... 1.............4g K2HPO4 ... Adjust pH to 1...0g Glucose............0..............10....0g K2HPO4 ........ powdered ...............................4g K2HPO4 .... Adjust pH to 2. Mix thoroughly....... 5....2g KCl...................... 10.......................33g KH2PO4 ...............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..16 Acidic Tomato Medium for Leuconostoc add 5............................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...2g MnSO4·4H2O .................8 at 25°C ............. Mix thoroughly.......................1... except glucose solution and yeast extract solution............................ Acidic Tomato Medium for Leuconostoc Composition per liter: Agar .......................0mL of sterile ferric citrate solution and 1.......................................0 ± 0...................0.........0 with H2SO4.......0mL Trace mineral solution SL-10..................20............ Mix thoroughly....... 0...................0mL of distilled/deionized water....... 0......................0............................ Autoclave for 15 min at 15 psi pressure–121°C..5 ± 0...................................................................8 ± 0.................33g KCl .......................................................0 using 1N H2SO4.................0g MgSO4·7H2O ............................. Autoclave for 15 min at 15 psi pressure–121°C............. 30..............................................0g Glucose ..................05g Tomato juice............7 with H2SO4...............250.................................................................................5 with H2SO4...................0................................................5g MgSO4·7H2O.. 0.........................0mL Yeast extract solution ........................... Before inoculation......................................5g KCl .. Adjust pH to 3....0mL.................1g pH 1................................ Cool to room temperature...........................................0.......... 10...0.................0....... 10......0mL.................................................0......0..0g NH4Cl . 10..................................................... 10.................. Mix thoroughly..............................2. 13..................................... Aseptically distribute into sterile tubes or flasks......33g CaCl2·2H2O .............33g MgCl2·6H2O ...... Distribute into tubes or flasks..........................2 at 25°C Glucose Solution: Composition per 10....................1g FeSO4·7H2O...1....................... Mix thoroughly..........................................2 at 25°C Yeast Extract Solution: Composition per 20.......5g KCl ....0L.............5-3.................. 10..................... Acidimicrobium Medium Composition per liter: MgSO4·7H2O ............................0g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 20...............................5mg Yeast extract solution ..............................0mL: Yeast extract.......0g Yeast extract... Mix thoroughly.... Preparation of Medium: Add components...... Use: For the cultivation of Rhodopseudomonas acidophila...............0.......................0mg Yeast extract solution ............................................0mL pH 2....... 0................................ Adjust pH to 3.........................0mL Vitamin solution ...............0mL: Yeast extract ........... Use: For the autotrophic cultivation of Sulfobacillus acidophilus........................................... 0................................0L....................0mL pH 4.

.4g K2HPO4·3H2O ....................................... Mix thoroughly........................................0g of agar to dis- Pyridoxine-HCl ................. Mix thoroughly.................................... Vitamin Solution: Composition per liter: 17 Acidomonas Agar Composition per liter: Solution A .. 1.0g Peptone .........0mL: Glucose.............................. to distilled/deionized water and bring volume to 1.16g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500............... Aseptically and anaerobically add per liter of medium................5............1mg Preparation of Medium: Aseptically mix 500... Adjust pH to 3..... 190..........0g Preparation of Solution B: Add 20............................... Autoclave for 15 min at 15 psi pressure–121°C....................5. Cool to 50°–55°C..... Use: For the cultivation and maintenance of Acidomonas methanolica............................... Mix thoroughly.................. 500..................0mg NiCl2·6H2O ..... Neturalize to pH 7.............0mL of solution B.......................... 0....0g Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 500......................... 3.......0mg Vitamin B12 ....................... Mix thoroughly....................0..............0g Malt extract ..............................20................ Autoclave for 15 min at 15 psi pressure–121°C.0mg Riboflavin ......................0g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 30............. ...........................0mg ZnCl2 ............... Filter sterilize............................... Cool to 50°–55°C.........................2 at 25°C Solution A: Composition per 500............... 5......................................0mg HCl (25% solution) .............0mg Biotin ....0...................... 15... Gently heat and bring to boiling.............0mg p-Aminobenzoic acid ...............5-3.........20...........3.. Pour into sterile Petri dishes or distribute into sterile tubes.. Filter sterilize. Use: For the cultivation of Acidilobus aceticus............0mL..........0g Yeast extract ............... a novel anaerobic thermoacidophilic archaeon from continental hot vents in Kamchatka..0mg H3BO3 ............0........... Add components................................................................................20...0mL of HCl solution.... 70........................................................ 5...0mL pH 4.......0mg MnCl2·4H2O...........0g KH2PO4 ................................ an acidophilic facultatively methylotrophic bacterium.......0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10....... Add distilled/deionized water and bring volume to 1......................... 10......0mL sterile vitamin solution..... 2....7g NaCl ......0L..................... Autoclave for 15 min at 15 psi pressure–121°C.0..... Aseptically adjust pH to 4.......... Mix thoroughly....... Yeast Extract Solution: Composition per 30.............. Na2S·9H2O Solution: Composition per 20... except vitamin solution..............0mL of solution B............0...........1.... 10...............0 ± 0.................. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C..... Mix thoroughly................ Use: For the cultivation and maintenance of Acidomonas methanolica....................... Sparge with 80% H2 + 20% CO2..........................................10..................... 5................................................................ Sparge with 100% N2........ Cool to 50°–55°C...0mL of solution A and 500........0mg D-Ca-pantothenate.......0mg Na2MoO4·2H2O ....6g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20.0 with HCl......................0mg Thiamine-HCl·2H2O ........... Solution B: Composition per 500......10....... Cool to room temperature....................... 500...... 500..............0mL: Na2S·9H2O.Acidomonas methanolica Agar Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .......................... 24..........0mg Folic acid..... Mix thoroughly....0mL.... 5............................ Cool to 50°–55°C.................... Solution A ....... tilled/deionized water and bring volume to 500...............................0mL sterile yeast extract solution.... Solution B: Composition per 500.........3........................................... Mix thoroughly.......................... Mix thoroughly. 0.................... Preparation of Medium: Aseptically mix 500............... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C......... Distribute to anaerobe tubes or bottles.......0mL of solution A with 500. 2..................0mL Solution A: Composition per 500........................... The final pH should be 3......0L..... Preparation of Vitamin Solution: Add components to Acidomonas methanolica Agar Composition per liter: distilled/deionized water and bring volume to 1............0mg CuCl2·2H2O ... 36.............................. 2..........0mL Solution B.....0mL: Agar ............................................................3...........0mL......0mg Nicotinic acid ....0g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1....0mL: Agar ......... 5............. and yeast extract solution.........................................0L......... Autoclave for 15 min at 15 psi pressure–121°C...................................0mL...............0........................... Adjust pH to 4..............0mL: Glucose.......................... Cool to room temperature...................0mL: Yeast extract.....0mL............... Na2S·9H2O solution.................. 5...............0g MgSO4·7H2O ......0mL of sterile Na2S·9H2O solution and 30....................5g Ca(NO3)2·4H2O ...0mg Lipoic acid .... Gently heat and bring to boiling.. Add remaining components........ an acidophilic facultatively methylotrophic bacterium....................................5g CoCl2·6H2O ......................0L..........0mL Solution B......... 100.............................................8... Mix thoroughly.........0g Yeast extract ....... 6.......................... Heat to 90°C for 1 h on each of 3 successive days......... Preparation of Medium: Prepare and dispense medium under 100% CO2...................... Sparge with 80% N2 + 20% CO2...................................0g (NH4)2SO4.......... Mix thoroughly....5 with H2SO4.... Mix thoroughly...... 500..................... Pour into sterile Petri dishes or leave in tubes...................................

1g Tryptone soya broth..............18 Acidophilic Bacillus stearothermophilus Agar Acidophilic Bacillus stearothermophilus Agar Composition per liter: Part B .............................................. Use: For the cultivation and maintenance of Bacillus stearothermophilus and other acidophilic Bacillus species..........0g Na2HPO4 .100............................... Part B: Composition per 600.........................................................5....................0mg KH2PO4 ..........................7.............2mg Trace elements solution TS2 ........................0g Na2HPO4·12H2O ....................0L with distilled/deionized water.......................... Gently heat and bring to boiling...... Cool to 50°C.................................... Autoclave for 15 min at 15 psi pressure–121°C..5............. 1...0..........................................................................15....0mg H3BO3 .0mL of solution B..................... Solution A .................5g MgSO4·7H2O...0......... Mix thoroughly............. 0....... Adjust pH to 7......0...............0mg NH4-Fe-III-Citrate............................................................7.............1.................................................0mL: Soluble starch................ 1.........................5. Cool to 50°C.........................0mL pH 7. 5........................0g Na2SO3 ..................1.............0g Pancreatic digest of casein ........................................... Adjust pH to 5.................. Adjust pH to 4..................... 0....................... Use: For the cultivation and maintenance of Acidiphilium cryptum and other Acidiphilium species............. Autoclave for 15 min at 15 psi pressure–121°C...0g KH2PO4 ...........................0mg Na2SeO3.....................0g Preparation of Solution A: Add agar to distilled/deion- ized water and bring volume to 500................0g Activated carbon ...........0mL pH 5.............0 ± 0.. Mix thoroughly............1............................................................0L.....................................................................................................................................................0 ± 0......................15......................................................... Distribute into tubes or flasks..........................................................................................10............. 50... Activated Carbon Medium (DSMZ Medium 811) Composition per liter: Agar . 0........................ to distilled/deionized water and bring volume to 900..........0mL: Agar .............0g Pancreatic digest of casein ...900................... Dextrin.0g CaCl2·2H2O......................................0g CaCl2·2H2O.......2g CaCl2·2H2O .......... 20..........................................5 Autoclave for 15 min at 15 psi pressure–121°C......50....... Mix thoroughly.....................................0mL: Mannitol.20......................1..... Adjust to pH 5........................................................................................................0g KH2PO4 ........ Preparation of Medium: Aseptically combine solution A and solution B..... Mix thoroughly................... Preparation of Medium: Aseptically combine 500.......0mL Solution B .0mL.200..............5 ± 0....0g Agar .....20............... 5..................................0mg NiCl2·6H2O.......................................10...............0g Beef extract .............5g MnCl2·4H2O....................2 at 25°C . Autoclave for 15 min at 15 psi pressure– 121°C............................0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 1............................................. Bring pH to 3.0mL Part A .5g cus.....1g KCl ...........................400...3.......0g Yeast extract..................................0mL pH 3........................................................5g Preparation of Part A: Add components to distilled/ deionized water and bring volume to 400........... Preparation of Medium: Add components.................................................... Cool to 50°C........ Solution B: Composition per 500....0mg Preparation of Solution B: Add components to dis- tilled/deionized water and bring volume to 1.....0g KH2PO4 ..... Autoclave for 15 min at 15 psi pressure–121°C...... After all components are dissolved add activated carbon.... 10.....0mL of solution A with 500............0mg MnCl2·4H2O ........ Pour into Petri dishes or leave in tubes............................. Acidophilium Agar Composition per liter: (NH4)2SO4 .....................5g pH 5..................................300...... Mix thoroughly.. 0...... Mix thoroughly.0mg ZnSO4·7H2O.. except acti- vated carbon................9............................................................... 0............................0mL....0mg CoCl2·6H2O.......13... 5........ Bring volume to 1.......0L of distilled/deionized water.................................0g Pancreatic digest of casein .......1 at 25°C Trace Elements Solution TS2: Composition per liter: Na2MoO4·4H2O... Mix thoroughly..0mL............30........................................10..................0g Yeast extract...................... Pour into sterile Petri dishes or aseptically distribute into sterile tubes........................0...................0g MgSO4·7H2O .........50............................................... 5........2 at 25°C Preparation of Medium: Dissolve all components in 1. Pour into sterile Petri dishes.......................2 at 25°C Use: For the cultivation of Streptomyces thermoautotrophi- Solution A: Composition per 500.............. Autoclave for 15 min at 15 psi pressure–121°C...................6g Acid Fuchsin solution...............................................0mg CuCl2·2H2O........................75g NaCl ... 1................5 ± 0........ 10...... Gently heat and bring to boiling....................................................................0g Preparation of Part B: Add agar to distilled/deionized water and bring volume to 600.. Cool to 50°C..............500...................................5g NH4Cl ...... 1....................0...0mL: Aeromonas Differential Agar (Dextrin Fuchsin Sulfite Agar) Composition per liter: Agar ................................. Mix thoroughly...2 at 25°C Part A: Composition per 400.........20.600.......500........ Acidophilic Bacillus stearothermophilus Broth Composition per liter: Soluble starch..................... Gently heat and bring to boiling...............................0mL pH 7...........................0L................ 12............5g MnCl2·4H2O..............5 ± 0.0mL........................................................................... Use: For the cultivation and maintenance of Bacillus stearothermophilus and other acidophilic Bacillus species.......0mg Ca(NO3)2 ....................................

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

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

Mix thoroughly........................................ 1....... 0......... Aseptically add per 100.......0g heximide to distilled/deionized water and bring volume to 10...........67g K2HPO4 ............................04g KH2PO4 ... Mix thoroughly. Gently heat and bring to boiling. Aseptically pour into sterile Petri dishes...................................03g MnCl2·4H2O ...0................ Mix thoroughly............... Autoclave for 15 min at 15 psi pressure–121°C.............. 0...2 at 25°C Cycloheximide ...10.....................5.......................................................... 0..0................... 15.. Preparation of Medium: Add components... Distribute 100............. Filter sterilize............ Use: For the selective cultivation of Agrobacterium tume- Use: For the cultivation of Vibrio cholerae and other Vibrio faciens biovar 1..... Alcaligenes N5 Medium Composition per liter: Sodium succinate·2H2O ............... 3.0mL of medium............. 1..........4mg Na2MoO4·2H2O..................... Autoclave for 15 min at 15 psi pressure–121°C. 0... Distribute 100........0mL into flasks........1g Preparation of Selenite Solution: Add components to distilled/deionized water and bring volume to 1.................1mL sterile D-cycloserine solution.............. Mix thoroughly......... Agrobacterium tumefaciens Selective Medium Composition per 1020mL: Agar .................................... Dcycloserine solution... Cool to 50°C...0mL Selenite solution........0mL..............0g KH2PO4 . dium bicarbonate solution................25g NH4NO3 ...................................5......... Aseptically pour into sterile Petri dishes....... Mix thoroughly......... Aseptically add per 100..................................................0mL sterile selenite solution and 1.....10...................................................................................... Cycloheximide Solution: Composition per 10. Mix thoroughly...29g MgSO4·7H2O ........... 1........... 0............................0mL.............0mL Crystal Violet (0...... cloheximide solution and selenite solution.....0L... 0..............54g Sodium taurocholate ................1mL sterile trimethoprin solution.... Gently heat and bring to boiling........0g Sodium bicarbonate solution ................................... to distilled/ deionized water and bring volume to 1.....................25g NH4NO3 .... 0.............................. Mix thoroughly......0....................................... except cycloheximide solution and tetrazolium chloride solution..........................5g Na2SeO3·5H2O ................. Aseptically pour into sterile Petri dishes....... 1............0.0mL Selenite Solution: Composition per 10...54g Sodium taurocholate .. 15........0mL Selenite Solution: Composition per 10............1.......................................04g K2HPO4 ............................ Aseptically add per 100... 0... to distilled/ deionized water and bring volume to 1.0...........4..........0.................2 ± 0.0mL into flasks....................0mL Selenite solution........0mg ....16g Cycloheximide solution .........................1 mL sterile tetrazolium chloride solution........5g Na2SeO3·5H2O ....0........0mL Yeast extract (1% solution) .75g NH4Cl ...0mL of medium...................1% solution) ......... 1...................0g Erythritol ..... Filter sterilize........... Filter sterilize......................0mL Preparation of Medium: Add components....... Cycloheximide Solution: Composition per 10...............................1g Preparation of Selenite Solution: Add components to distilled/deionized water and bring volume to 1........0mL of medium... Cool to 50°C.... Preparation of Medium: Add components............ and 1...............29g MgSO4·7H2O ...........0mL................................................ Mix thoroughly........ Use: For the selective cultivation of Agrobacterium tumefaciens biovar 2............61g MgSO4·7H2O....... except cy- Preparation of Sodium Bicarbonate Solution: Add sodium bicarbonate to distilled/deionized water and bring volume to 100.0mL sterile selenite solution and 1......1% solution) ...............0mL: NaOH .............. Gently heat and bring to boiling......0mL cycloheximide solution............................ 0.........0L.... 0...... Filter sterilize..................0L.... except cycloheximide solution and selenite solution.... Autoclave for 15 min at 15 psi pressure–121°C............ Prepare medium freshly.......0g NaCl .... and trimethoprin solution to distilled/ deionized water and bring volume to 1.......0g Yeast extract ......05g K2HPO4 . 3........0mL: 21 Malachite Green (0.........................................16g Cycloheximide solution .................................02g Preparation of Cycloheximide Solution: Add cyclo- heximide to distilled/deionized water and bring volume to 10..............................................................0mL cycloheximide solution.. 0......... Adjust pH to 7... Distribute 100. 0............................... Mix thoroughly......................................................0g L(−)Arabitol ......... Aseptically add sterile sodium bicarbonate solution.0mL pH 7..................................................... 0................ Use: For the selective cultivation of Agrobacterium tumefaciens biovar 3...................... AKI Medium Composition per liter: Peptone ..2......0mL: Cycloheximide .......... Autoclave for 15 min at 15 psi pressure–121°C................................................... Mix thoroughly..........0mL.....Alcaligenes N5 Medium Preparation of Medium: Add components.................... Mix thoroughly...............................................10.0mL into flasks...............0............15...2g CaCl2· 2H2O ....0mL: NaOH .......................................0mL cycloheximide solution............... Aseptically distribute into sterile tubes or flasks................................................. Mix thoroughly.............0L...............2........10..................0L...0mg FeCl3 .. 5.............. Agrobacterium tumefaciens Selective Medium Composition per 1020mL: Agar ........................... Cool to 45°–50°C......... Filter sterilize........................................... except so- species......... to distilled/deionized water and bring volume to 970.. Mix thoroughly....... Cool to 50°C..............................2...10.........02g Sodium Bicarbonate Solution: Composition per 100.0mL: Preparation of Cycloheximide Solution: Add cyclo- NaHCO3 .............................. 30.......... 0......................3....... Use freshly prepared solution...................................................04g KH2PO4 ............................................

........................... Dissolve by adjusting pH to 6. Mix thoroughly......... 15..... Use: For the cultivation and maintenance of Alcaligenes faecalis.............0...1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1... except NaHCO3................ Mix thoroughly................................ 0................0mg Nicotinic acid ...... Distribute into tubes or flasks........0L.............0mg Riboflavin...................................................................0............0mL Na2S·9H2O solution...........................................................5...........0...0g Preparation of Sucrose Solution: Add sucrose to distilled/deionized water and bring volume to 50..............................................0mL sucrose solution..0.....................................................................5.................................... 5.................................0L......................18g CaCl2·2H2O ............ Trace Elements Solution: Composition per liter: MgSO4·7H2O...........0g Beef extract ..................................... Add solid NaHCO3.................025g KAl(SO4)2·12H2O ....2g MgCl2 ..............0mL............4g KH2PO4 ..........0mL of distilled/deionized water.............. Preparation of Medium: Prepare and dispense medium under 100% N2 gas atmosphere....5g NaCl .........5 with KOH............. Mix thoroughly.................................... Distribute into tubes or flasks......0............ Pour into sterile Petri dishes or leave in tubes.0......... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.................................... Distribute into anaerobe tubes or bottles....0g pH 7.0 at 25°C Sucrose Solution: Composition per 50. Cool to room temperature while sparging with 100% N2.............10..........2g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10....................18g ZnSO4·7H2O......................... Mix thoroughly....... Mix thoroughly............0....0g Pancreatic digest of gelatin ..........................................0................................... 50..............................................0mL yeast extract solution.......0mg Folic acid .....2.0L.............1g FeSO4·7H2O ...................................... Autoclave for 15 min at 15 psi pressure–121°C.2......1.0mL Na2S·9H2O solu- ..0g Beef extract ...... Gently heat while stirring and bring to boiling.............. NH4Cl..........01g Sucrose solution ......... Na2S·9H2O solution... Yeast Extract Solution: Composition per 10. Mix thoroughly.............................0mL: Yeast extract .............. 5............... 5..................... Alcaligenes NB YE Agar (Alcaligenes Nutrient Broth Yeast Extract Agar) Composition per liter: Agar ............0mg D-Ca-pantothenate ..6g NaHCO3 .1g NiCl2·6H2O.........2g Resazurin ....... Gently heat while stirring and bring to boiling..................................................5g CoSO4·7H2O..........................7g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.............50.......0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............0mL pH 9.........0mg Biotin ... Use: For the cultivation and maintenance of Alcaligenes faecalis.........................0mg Lipoic acid............3........ Mix thoroughly......... 5..... Mix thoroughly.01g Na2SeO3·5H2O ...........................0g Yeast extract........5g KCl..1g NH4Cl.... and Na2CO3................................................ Mix thoroughly................0g Nitrilotriacetic acid..........0mg p-Aminobenzoic acid ............ NH4Cl.................... 0.... Na2CO3...............................0mL Yeast extract solution .......0. Pour into sterile Petri dishes or leave in tubes.............0g Yeast extract.........................................0L.............................................. Boil for 5 min................... Distribute into tubes or flasks..... Mix thoroughly.................................. 0............. Add distilled/deionized water to 1........................................ Sparge with 100% N2.........0.........01g Na2MoO4·4H2O..............0mL Vitamin solution......02g H3BO3 ...................................................... 0......5......................0...........0mL Trace elements ..0mL........0.............................................................................10. Filter sterilize........................ 10....................................... Sparge with 80% H2 + 20% CO2.............. 5..... Autoclave for 15 min at 15 psi pressure–121°C.............1......................0mg Thiamine-HCl·2H2O ..... Add components..................................... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C...... yeast extract solution.... 0............................ and vitamin solution..... Alcaliphilic Amphibacillus Strains Medium (DSMZ Medium 931) Composition per liter: Na2CO3 ..... Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O . Gently heat while stirring and bring to boiling............ Aseptically and anaerobically add per liter of medium 50.........0........5..............0mL: Sucrose.................................0...........1........................0mL.............................................5-10........ 63............................ Cool to room temperature.....0L................................... Autoclave for 15 min at 15 psi pressure–121°C................. Cool to room temperature......... Vitamin Solution: Composition per liter: Pyridoxine-HCl .............................................. 15.................................................................................. Use: For the cultivation and maintenance of Alcaligenes species................22 Alcaligenes NA YE Medium Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................................5............3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500... Cool to room temperature........10........................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................................. Alcaligenes NA YE Medium (Alcaligenes Nutrient Agar Yeast Extract Medium) Composition per liter: Agar .0mg Vitamin B12......................5.... 10... sucrose solution...................................... Autoclave for 15 min at 15 psi pressure–121°C....................................0mL..........................................0g MnSO4·2H2O. Add remaining components..........................................................01g CuSO4·5H2O......0 ± 0.. Sparge with 100% N2....................10........ to distilled/deionized water and bring volume to 920..... 3...........0g Pancreatic digest of gelatin ............................. Autoclave for 15 min at 15 psi pressure–121°C... Gently heat and bring to boiling..................................................................... 3...

0g Yeast extract ...........................0............... Distribute into tubes or flasks..25g (NH4)2SO4 .1.............................0mL pH 4.... Alicyclobacillus Agar (DSMZ Medium 402) Composition per liter: Glucose...............0mL Solution C .................................0............................... Agar ........................................................................ to distilled/ deionized water and bring volume to 499.............................................0g tilled/deionized water and bring volume to 500...........5g CaCl2·2H2O .............0mL Solution A ...............0L....................................0......................0.......0mL: Solution A ...........5g H3BO3 ......................02g CuCl2·2H2O..................................................0mL pH 4....................2 at 25°C Solution A: Composition per 500...........................2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ................................................... Filter sterilize.............0µg Vitamin B12 ...................... B........................................................... Growth on alginate (production of alginase) is a diagnostic test used in the differentiation of Vibrio species..3...67µg Preparation of Solution C: Add components to distilled/deionized water and bring volume to 100.........Alicyclobacillus Agar 23 tion..............0g KH2PO4 ........0.0mL of solution C......... Warm to 50°–55°C................0................ Mix thoroughly.................. 0...... Mix thoroughly........0mL: Tris·HCl buffer.......... Mix thoroughly...................25g (NH4)2 SO4.. 66................... Use: For the cultivation and maintenance of Alicyclobacillus acidoterrestris..........100.....................0mL: Marine salts.2g Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 500.........................0 ± 0...............................5.. Preparation of Solution A: Add marine salts to dis- Solution C: Composition per 500...........................................0mL of ............................................0.. and C....................... Gently heat and bring to boiling.... 3.................... Autoclave for 15 min at 15 psi pressure–121°C...01g Amphibacillus tropicus..0...............3g CoCl2·6H2O.....0.............500........................................0mL vitamin solution....0mL Preparation of Solution B: Add components to dis- Solution A: Composition per 400....... except trace elements solution SL-6 and agar solution............0mL of solution B............0g MgSO4·7H2O.067g NaNO3 .. Cool to 45°C.2g ZnSO4·7H2O.............0L.................................... Cool to 45°C.................0mL Trace Elements Solution SL-6..................3µg Biotin .................500........ For liquid medium.. Preparation of Medium: Add components......0mL Solution C .............................0.......................047g Ferric EDTA...... Cool to room temperature....... and 10............. 0................................. 1............67mg Thiamine·HCl ......0g Preparation of Agar Solution: Add agar to distilled/ deionized water and bring volume to 500...................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1................................................. 0........................................... 0.......................... Adjust pH to 4.........03g NiCl2·6H2O........ Autoclave for 15 min at 15 psi pressure–121°C........ Autoclave for 15 min at 15 psi pressure–121°C.....................0................ 0..0L.................... 20...... Pour into sterile Petri dishes or distribute into sterile tubes.... 6...1.......... Mix thoroughly....0mL....15............................ 2.5g H3BO3 .........0g MgSO4·7H2O ............................0mL....................0g Preparation of Solution C: Add agar to distilled/deion- Solution B: Composition per 500.....500...0mL: ized water and bring volume to 500............ Aseptically add 1............................... Cool to 50°–55°C........................0mL....0 ± 0.............................. Cool to 50°–55°C....... Mix thoroughly... Autoclave for 15 min at 15 psi pressure–121°C....................... Adjust pH to 4..................1g Na2MoO4·2H2O....... Filter sterilize......................0g tilled/deionized water and bring volume to 400............................................. and 500................0mL: Glucose ............0mL Solution B ........... Agar Solution: Composition per 500mL: Agar ............2g ZnSO4·7H2O......................... Preparation of Solution B: Add components to dis- Preparation of Medium: Aseptically combine 500......................... Mix thoroughly.......................................... 500............... Mix thoroughly............... Solution C: Composition per 100....................................... Mix thoroughly....................0g KH2PO4 .. Autoclave for 15 min at 15 psi pressure–121°C...400.....................0g Yeast extract...................... Solution B: Composition per liter: Use: For the cultivation of Amphibacillus fermentum and MnCl2·4H2O ......................2. Gently heat and bring to boiling..0................0................................... 5.............................................................. Agar ....5g CaCl2·7H2O.......0L.......................................15.....0.............................. Autoclave for 15 min at 15 psi pressure–121°C...........0g Sodium alginate ...... Alicyclobacillus acidoterrestris Agar Composition per 1001......... 1.............................0....................................................0mL...............................2g Agar solution ..............0mL of solution A............................................................................................ 38........0mL: tilled/deionized water and bring volume to 1...... Use: For the cultivation of microorganisms that can utilize alginate as a carbon source...0........... The final pH should be 9.................3g CoCl2·6H2O......................................... Mix thoroughly.................... Alginate Utilization Medium Composition per liter: Solution B ..1g Na2MoO4·2H2O............ 0................. Preparation of Medium: Aseptically combine solutions A..........5mg Sodium glycerophosphate. 0....................................02g CuCl2·2H2O..............67.........03g NiCl2·6H2O................... 10......0............... 0..................................................................................................0mL.. Autoclave for 15 min at 15 psi pressure–121°C........................ omit agar from solution B..5-10....... Mix thoroughly.............. 0...........

.0g K2HPO4 ........ Mix thoroughly. Mix thoroughly............. except Alicyclobacillus cycloheptanicus Agar (LMG Medium 174) Composition per 1001.......... Bacillus sp.............0mL pH 8...................0 ± 0....... 15.......0mL...0mL: Yeast extract........................500...........................0L...................... Autoclave for 15 min at 15 psi pressure–121°C...........0mL. Gently heat and bring to boiling...0mL pH 4.............. Filter sterilize................. Use: For the cultivation of Alicyclobacillus cycloheptani- Alkaline Nutrient Agar Composition per liter: cus................................................5–10...................... 1............................0g Peptone .. to distilled/deionized water and bring volume to 900....................................... 0.....5g Preparation of Medium: Aseptically combine 900...... Adjust to pH 4..03g Na2MoO4·H2O ................. ...................0g Peptone........................................1.....15.......3g CoCl2·6H2O ................... Pour into sterile Petri dishes or leave in tubes......... Use: For the cultivation and maintenance of cellulose-utilizing bacteria..01g tilled/deionized water and bring volume to 100..............................2........... Mix thoroughly.....................................0...........................0g Beef extract ............0g pH 9....1g MnCl2·4H2O.......... 10......................................500..0.....5-10....... Autoclave for 15 min at 15 psi pressure–121°C......0g Peptone .. 10...........0g Cellulose powder MN 300 .................... 0.........................1.................................................... Distribute into tubes or flasks... Solution B: Composition per 100............0g Preparation of Agar Solution: Add agar to distilled/ deionized water and bring volume to 500......4g Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 900......0mL....0mL: Agar Solution: Composition per 500.........0......................... 5..............................2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500........0mL sterile agar solution and 1............................................4 with 6% NaHCO3 solution............................................................. Adjust pH to 7.... 0................ Cool to 50°–55°C...5g of Na2CO3 to distilled/deionized water and bring volume to 100......... Mix thoroughly.............. Agar .. Trace Elements Solution SL-6: Composition per liter: H3BO3 .....0mL solution A............................ Preparation of Na2CO3 Solution: Add Na2CO3 to dis- Use: For the cultivation and maintenance of Alicyclobacillus spp...... Preparation of Medium: Add components. Cool to 45°-50°C.0mL agar solution... Mix thoroughly..0mL sterile trace elements solution SL-6............... Pour into sterile Petri dishes or distribute into sterile tubes.........................0g NH4NO3..0 with sterile 9% Na2CO3 solution.....................0g MgSO4·7H2O .................. 0...0g Na2CO3 solution.. 0.............. 0....2g ZnSO4·7H2O .................................0....... Mix thoroughly......2 at 25°C Solution A: Composition per 500.. 900...... and other Bacillus species.......... Mix thoroughly................................................. 30......0mL: Na2CO3 ............................. Aseptically pour into sterile Petri dishes or distribute into sterile tubes.........0g K2HPO4 .............................................0g Yeast extract... Alkaline Cellulose Agar Composition per liter: Solution A ................................................0mL Solution B..............................1......................................... Aseptically add sterile Na2CO3 solution.............. Autoclave for 15 min at 15 psi pressure– 121°C. Gently heat while stirring and bring to boiling............... Autoclave for 15 min at 15 psi pressure–121°C.0g K2HPO4 .. Pour into sterile Petri dishes or leave in tubes................................. Mix thoroughly.......................................................0mL: Agar ......0 at 25°C Na2CO3 Solution: Composition per 100........... 0............... 0..................................................5.. 500. 10..........................0g Yeast extract ........... Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.....0g Glucose ........ and Bacillus naganoensis.... Pour into sterile Petri dishes or aseptically distribute into sterile tubes or flasks..........5g CaCl2 ...100.. Na2CO3 ........................................... Use: For the cultivation and maintenance of Bacillus alcalophilus and Bacillus species...... Alkaline Bacillus Medium Composition per liter: Agar ......................................... Autoclave for 15 min at 15 psi pressure–121°C........0L......... Autoclave for 15 min at 10 psi pressure–115°C................................. Gently heat and bring to boiling....................02g CuCl2·2H2O........... Adjust pH to 3..... 5...0mL....0mL..........................4g MgSO4·7H2O.......25g (NH4)2SO4 ..............................0mL Agar solution.......24 Alicyclobacillus cycloheptanicus Agar sterile trace elements solution SL-6 and 500................................................................. Filter sterilize... Gently heat and bring to boiling....................................................................................... 5..03g NiCl2·6H2O .. Mix thoroughly.............0mL.3.0mL Trace elements solution SL-6 ................................. Preparation of Medium: Aseptically combine 500............. Mix thoroughly....5–11......0 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....... 0...15............. 100........... Adjust pH to 9............................0g Glucose .... Use: For the cultivation and maintenance of alkalophilic microorganisms such as Bacillus alcalophilus.............. Cool to 50°–55°C.0 with 1N HCl..............0mL: Solution A .... Na2CO3 solution.0....................................... 3............................................................4.0g Agar .................... Aseptically adjust to pH 9......0mL: Preparation of Solution B: Add 0...............................0mL of solution B........................... Cool to 45°–50°C............................................. Mix thoroughly..............15................ 0....5g CaCl2·2H2O...................0mL of solution A with 100......... Cool to 45°-50°C...0mL Solution A: Composition per 900.................. Bacillus circulans.............

.0g Yeast extract ......0g Methanol.......................................0.............................0g MgSO4·7H2O............................................................2............................40............................ 16.................................................................. Gently heat and bring to boiling. 10......................................................0g Nitrilotriacetic acid............................................... 2...3.... Distribute into tubes or flasks..............0g Peptone.....0g NH4Cl .......1.....4...................... Pour into sterile Petri dishes or leave in tubes.......5g KH2PO4 ..... Mix thoroughly.....01g Na2SeO3·5H2O ................................0mL Selenite-tungstate solution ......0g NaCl ................................1g ZnSO4·7H2O......... except so- dium sesquicarbonate solution....... Autoclave for 15 min at 15 psi pressure–121°C................................................................................. Bacillus cohnii....0................................................... 10............0L..2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................................... 10.........0g Sodium polypectate...... Use: For the cultivation of a variety of alkalophilic microorganisms................................................................................................ Warm to 50°–55°C.................. 8....02g CuSO4·5H2O......................................... Alkaline Starch Agar Composition per liter: Starch....................6g NaHCO3 .................................. to distilled/deionized water and bring volume to 900.. Autoclave for 15 min at 15 psi pressure– 121°C......................... Adjust pH to 9...............................5g Yeast extract .............................0g Agar ......15.......... Add remaining components.................................................................. Mix thoroughly......... Mix thoroughly....................................................................1g CaCl2·2H2O .................................................... Aseptically add sterile sodium sesquicarbonate solution............................................. Adjust pH to 8...............0g pH 8........................... Alkaline Polypectate Agar Composition per liter: Agar ............................................... includ- 25 MgSO4·7H2O..................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........0.. 20...01g Na2MoO4·2H2O.............16......................... Autoclave for 15 min at 15 psi pressure–121°C..........................................0..........................................7 ± 0...5 ± 0.................................................8.................................. Mix thoroughly........0g Na2CO3 ...................0............................3mg Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.........0......0g Peptone .............................. 5.0mL...............0.... Alkaline Peptone Water Composition per liter: Peptone....42g Preparation of Sodium Sesquicarbonate Solution: Add components to distilled/deionized water and bring volume to 100..............0L........................................0g Peptone..........0L......0mL.............................................................0.0mg pH 9............ 3.....1...............................................0mL pH 9....... 1....0g K2HPO4 ................. 15.............................0...........................................0 ± 0.01g H3BO3 ......0g Peptone.......................................0................ Filter sterilize........................................................................................2 at 25°C Sodium Sesquicarbonate Solution: Composition per 100.................0mg pH 10..................Alkaliphilic Methanogen Medium Alkaline Nutrient Agar Composition per liter: Agar ..... Adjust pH to 6........................................ NaCl ...... Use: For the cultivation of alkiliniphilic starch-utilizing Alkaline Peptone Agar Composition per liter: Alkaliphilic Methanogen Medium Composition per liter: NaCl .5 with KOH....................... 5.................................................... Gently heat and bring to boiling.................0g pH 8..................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....... ing Bacillus alcalophilus.............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...............1g FeSO4·7H2O ........................ Use: For the cultivation of Vibrio cholerae and other Vibrio species....... Gently heat and bring to boiling...............................................0...0g NaHCO3 ............20.... 10..........10.................................... Allow tubes to cool in a slanted position....7...............................0mL: Na2CO3..0g NaCl ....................5.......... Pour into sterile Petri dishes or distribute into sterile tubes............... Bring pH to 10............................. Autoclave for 20 min at 15 psi pressure–121°C..4......................10.................................... Distribute into tubes or flasks.......................................................5...................2g MnSO4 .................... Distribute into tubes......6.............................0g Sodium sesquicarbonate solution...............3...........0g MnSO4·2H2O.0g Yeast extract.............. Preparation of Medium: Add components............. Cool to 50°–55°C.. Mix thoroughly......................................................................0g Na2S·9H2O ............................................0g Beef extract ............ .........0........................0g Agar ........0............................. Distribute into tubes or flasks.........0g K2HPO4 .........................4 ± 0...............................................025g KAl(SO4)2·12H2O .......5g NaCl . 10... Autoclave for 15 min at 15 psi pressure–121°C..0g Na2CO3 ...... Pour into sterile Petri dishes or leave in tubes............ and other Bacillus species......... Use: For the cultivation of pectinolytic bacteria..1....... 1.............1g NiCl2·6H2O..........3g NiCl2·6H2O..................4 at 25°C Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O..........0.... anhydrous.....0mL pH 9.......0L........ Gently heat and bring to boiling.0g Yeast extract..........1..........0L...............................................................0.............. Use: For the cultivation of alkaliniphilic bacteria........... Adjust pH to 6..2–9.................5g CoCl2·6H2O............. Mix thoroughly........ 5... 1................................. 6..40.....7................. Bring pH to 9.................2g MnSO4 ....................... 5................................0mg Resazurin ................ 15..............................................0g Na2CO3 ...................... Mix thoroughly...........7 ± 0..... Add distilled/deionized water to 1......................................................0mL of distilled/deionized water............................0......100....0.....................................0g bacteria..........0. Adjust pH to 8...5mg Wolfe’s mineral solution ....

.................................0mL Trace elements solution SL-6 ....0mL Wolfe’s vitamin solution ........0mg ZnSO4·7H2O............... 5............................ and 10.............1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1... The pH should be about 10...............................0mg Nicotinic acid ................ Anaerobically distribute into tubes... Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ....0mg tilled/deionized water and bring volume to 1............ Adjust pH to 9................ 5..20...0L....0mg Folic acid ...............................................................50..... Cool to room temperature while sparging with 100% N2....... Sparge with 100% N2...... Mix thoroughly....... 10................................................5.......................................................................... 20........................0mg Vitamin B12...........0....0mg CoCl2·6H2O. Aseptically and anaerobically add 50...............2.....................0mL............ 4.............................. 0...........0mL of sterile sucrose solution.. Mix thoroughly....... Mix thoroughly... 10.............................................. Add NaHCO3 and Na2S·9H2O........................0mL........5g NaHCO3 solution .. Gently heat and bring to boiling..........................................................5g H3BO3 ..... Trace Elements Solution: Composition per liter: H3BO3 ..2g Yeast extract........................0mg Biotin ..................................................... Aseptically distribute into sterile tubes or flasks.......................................... Mix thoroughly.................. and Spirochaeta asiatica...0g K2HPO4 ..............................20... 0..0mL of sterile NaHCO3 solution. 0...........................0mL of sterile Na2S·9H2O solution..............0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 50........ Before use.............0L.................................0mg Riboflavin ........... Autoclave for 15 min at 15 psi pressure–121°C...................... 0.............. Autoclave for 15 min at 15 psi pressure–121°C. Sparge with 100% N2.2g KCl.. Filter sterilize........................0mg .............0g NH4Cl........ Sucrose Solution: Composition per 20................................... Spirochaeta alkalica...0................3g CoCl2·6H2O.......................................................................0g NaCl ..........................2g ZnSO4·7H2O...............................................................0g K2HPO4 ........20.....0mg Na2SeO3·5H2O ............. Alkaliphilic Spirochete Medium Composition per 1011............................................................ 1.. Add components....................................0mL: NaHCO3 .... Sparge with 100% N2... Preparation of Medium: Prepare and dispense medium under 100% N2...................................... Preparation of Medium: Prepare and dispense medium under 100% N2..0mg NiCl2·6H2O........................................100.0mL pH 10.1g Na2MoO4·2H2O.......... Na2S·9H2O solution...0 ± 0.....0g Na2CO3 .............. 5.........................0mg MnCl2·4H2O ...............................0mL: Na2CO3 .....2–9..............................2......30........0............0L..............0L........................................................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.......... Sparge with 100% N2.......................... 997........0g NaCl .........0mg Thiamine·HCl ...03g NiCl2·6H2O....0g Preparation of Sucrose Solution: Add sucrose to distilled/deionized water and bring volume to 20.............................................0g NaHCO3 ...................................................................................................... sucrose solution........................0mg p-Aminobenzoic acid . 1. Mix thoroughly..0mL Na2S·9H2O solution...200.......... 5... Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl .. Sparge with 100% N2...................................0mL pH 9........ Mix thoroughly. 0...........0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.........0mL Sucrose solution ..................7 with 6N NaOH (about 15.........................10............0L.10............................................... Autoclave for 20 min at 110°C............................................................0mg Na2MoO4·4H2O..........................0mg Lipoic acid ..................0mL)....... Use: For the cultivation of Spirochaeta africana.. Alkaliphilic Sulfur Respiring Strains Medium (DSMZ Medium 925) Composition per liter: Mineral base ............................... neutralize to pH 7................ and Wolfe’s vitamin solution...... Autoclave for 15 min at 15 psi pressure–121°C....1.30...1..........0mL: Sucrose.. Mix thoroughly......0............ Filter sterilize............. 10... 2.............................................. Adjust pH to 9..300.............10.0mL Trace elements solution...................................... Mix thoroughly.............................0mL: Na2S·9H2O....0mL......................0..... 10.....02g CuCl2·2H2O. 1. Cool to room temperature while sparging with 100% N2......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..................... 5.................5g Na2WO4·2H2O ....0...................26 Alkaliphilic Spirochete Medium Selenite-Tungstate Solution: Composition per liter: NaOH ..0mL Magnesium chloride solution .............................. Autoclave for 15 min at 15 psi pressure–121°C.0mL KSCN solution ......................................... Sparge with 100% N2....................10... to distilled/deionized water and bring volume to 910....................................... 10..........7 ± 0..0mg CuCl2·2H2O....................... 15...............0mL...... 5.....................5............. Gently heat and bring to boiling........................ except NaHCO3 and Na2S·9H2O............... except NaHCO3 solution.....2 at 25°C NaHCO3 Solution: Composition per 50..................................4..... Calcium DL-pantothenate ..................0 with sterile HCl................................... Mix thoroughly............... to distilled/deionized water and bring volume to 1............. Use: For the cultivation of Methanohalophilus zhilinae........... Mix thoroughly................................................ Autoclave for 15 min at 15 psi pressure–121°C..........................0mL of sterile Wolfe’s vitamin solution.... Add components.................. 0.............................0............2 at 25°C Mineral Base: Composition per liter: Na2S·9H2O Solution: Composition per 10.. Mix thoroughly.......... Continue boiling for 5 min............................ 3........................................................

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

..5...2g of KOH in 186...0mL: H3BO3 ..500.... Autoclave for 15 min at 15 psi pressure–121°C................ Cool to 50°–55°C................... Add 25..0g Preparation of Solution B: Add K2HPO4 to distilled/ deionized water and bring volume to 500.........5g MgSO4·7H2O. Preparation of Medium: Aseptically combine 500.............25mL Solution A: Composition per 2......0mL of solution A with 500........................................................................... Autoclave for 15 min at 15 psi pressure–121°C........0.......... Natronobacterium vacuolata....36.... Mix well. 10.....................................0g Casamino acids ......................0mL..................... and Natronococcus occultus... Mix thoroughly.........6...4.0g FeSO4·7H2O............................500................... Mix thoroughly..................................................6mg A & A FeEDTA solution. Add 20..........................................8mg CoCl2·6H2O..... 2.....36mg Preparation of Solution B: Add components to distilled/deionized water and bring volume to 500.......... Bacillus submarinus.....................0mL CaCl2·2H2O (1. Use: For the cultivation of myxobacteria............15............. Alkalophilic Halophile Agar Composition per liter: Solution A ..........0g KNO3 ................25..............................4g of disodium EDTA·2H2O..0g Microelements Stock Solution: Composition per 1090....0mL pH 9.................... Gently heat and bring to boiling................ 0.............. Mix thoroughly...............212g Solution A .................................0mL of filter-sterilized sodium sesquicarbonate solution to adjust pH to 9...... AMB Broth Composition per liter: Starch.....................0mL....0g Pancreatic digest of casein ...............5... Mix thoroughly... Preparation of Medium: Add agar........5g Trisodium citrate .................................................................... Mix thoroughly........................................... 1................0mg CuSO4·5H2O.. Aseptically add 15... Pour into sterile Petri dishes or distribute into sterile tubes...5g MgSO4·7H2O....0mL: tilled/deionized water and bring volume to 500......................................0.........................................2......0mg ZnSO4·7H2O.5 ± 1.................................25g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........0mg MoO3 .................. Solution B: Composition per liter: Solution B: Composition per 500............0mg MnCl2·4H2O........... Natronobacterium magadii................................................................5g K2HPO4 ........... Cool to 50°–55°C..360.....0mL of solution B............................... Pour into sterile Petri dishes or leave in tubes......0mL Microelements stock solution ....... Pour into sterile Petri dishes or distribute into sterile tubes.......0g Starch...........0L.....0mL..5..........25mL: Noble agar..........................................................................................0mg NH4VO3..............0mL: Disodium EDTA·2H2O......................... Bacillus circulans............ except so- dium sesquicarbonate solution.. 0......8..........0L..................0mL Solution B ........ Mix thoroughly.........................5...... Add 6.......................................0g Pancreatic digest of casein .................. 0..................................0mL Preparation of Solution A: Prepare individual solutions and combine............ to distilled/deionized water and bring volume to 985...0mL of distilled/deionized water....25mL of solution B aseptically after sterilization...7g of FeSO4·7H2O to 364....................13................... soluble .... Agar .....0L......... KNO3...... soluble .. 10...5.............0 at 25°C Solution A: Composition per 500........................................ Use: For the cultivation and maintenance of Natronobacte- rium gregoryi..... Cool to 50°–55°C............ Gently heat and bring to boiling....... Autoclave for 15 min at 15 psi pressure–121°C.............0mL: NaCl .. 50................. ........0L.......4g FeSO4·7H2O ......0L........................ Gently heat and bring to boiling................................. Autoclave for 15 min at 15 psi pressure–121°C... 200........0L: MgSO4·7H2O (4% solution) ...... Mix thoroughly... 3..............2% solution)........0mL of solution A...................................... Distribute into tubes or flasks...................................................... pH about 3............................................... Bring pH to 9....................... Add 13....... Allen and Arnon Medium with Nitrate Composition per 1000.......................... 7.................................... and NaNO3 to distilled/deionized water and bring volume to 969.....0.....................2..............2g Preparation of Solution A: Add components to dis- Preparation of A & A FeEDTA Solution: Dissolve 5...............7g KOH ....0mL of distilled/deionized water..28 Alkalophilic Halophile Agar Preparation of Medium: Add components........... Use: For the cultivation of myxobacteria............. 160.................... and other Bacillus species................... Autoclave for 15 min at 15 psi pressure–121°C.0g Na2CO3·10H2O .0mg MnCl2·4H2O ........ Distribute into tubes or flasks....................500........0mL Preparation of Microelements Stock Solution: Add components to distilled/deionized water and bring volume to 1090.........................0........................................... AMB Agar Composition per liter: Agar ............................................................ K2HPO4 ...................0g KCl.............................0mL NaCl (3........ Mix thoroughly.........25g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........ Combine the EDTA solution with the FeSO4 solution............500....................................................500... Autoclave for 15 min at 15 psi pressure–121°C....... Use: For the cultivation and maintenance of Anabaena species and Nostoc species............... Use: For the cultivation of Bacillus alcalophilus..............0mL Solution B ..............5..... 20....572.15.................... Sparge solution with filtered air until color changes.0g MgSO4·7H2O .............. A & A FeEDTA Solution: Composition per 550........................... 50..253g NaNO3 ......500.....................28...8% solution) .....44...............20................... Natronobacterium pharaonis....................................................0g Yeast extract..............5g K2HPO4 .

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

2g Beef extract ...........5g Resazurin ....5g CaCl2·2H2O ............ except Lcysteine............... 0..................0mL pH 7......... Enriched Composition per liter: Agar ..........5.....................0g K2HPO4 .............................5.............. and sterile disodium thioglycolate solution under 100% N2.................. Autoclave for 15 min at 15 psi pressure–121°C......10..........................2g pH 7.......................... Autoclave for 15 min at 15 psi pressure–121°C....... Gently heat and bring to boiling...... Store anaerobically..........1 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......... Gently heat and bring to boiling....................................................... Gently heat and bring to boiling.5g Yeast extract..0mL......................................0mL sterile Na2S·9H2O solution and 10......0mL......... Filter sterilize................0............................. to distilled/deionized water and bring volume to 940.0g Pancreatic digest of casein ........... Cool to 25°C..........0g Trypticase peptone....................0g Glucose.............................. 0...............................1...............1....... Autoclave for 15 min at 15 psi pressure–121°C.5...................0g Beef extract ..........0.. Pour into sterile Petri dishes or leave in tubes......0g Pancreatic digest of casein ................................. Adjust pH to 7........................0mL: Na2S·9H2O .......... 0.......................................................5.0mg pH 7...............0mL: Disodium thioglycolate.................0g MgSO4·7H2O........... Mix thoroughly.................0g NaCl ......................... Anacker and Ordal Medium.................................0mL Salt solution......5g Sodium acetate ............................................................................. Distribute into tubes or flasks. 1..................................................... trichosporium and other methane-oxidizing bacteria............................. Use: For the cultivation and maintenance of Anaerobaculum thermoterrenum................. 0........................................... Powdered sulfur is sterilized separately by steaming for 3 hr on 3 consecutive days...0g Sulfur...............0g Yeast extract............................................... except powdered sulfur and disodium thioglycolate solution................3 ± 0........5g K2HPO4 ......................... Mix thoroughly.................0mg Na2S·9H2O solution ...0L............... to distilled/deionized water and bring volume to 990....... 2............................... Mix thoroughly............ 0.......... Use: For the cultivation and maintenance of Flexibacter columnaris...................................................... Mix thoroughly.................................0g Agar ......................... Distribute into sterile tubes or flasks under anaerobic N2.............. Gently heat and bring to boiling..0mL.................. Allow to cool under 100% N2.. AN1 Medium Composition per liter: Pancreatic digest of casein .......0L........................ Mix thoroughly.........................................................................10............ Mix thoroughly. 10.........................0L.........0mL: D-Glucose ....2g Beef extract ........01mg pH 6............... Yeast extract ................................1 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1. Store anaerobically..3 ± 0....0mL.................................................... 0...........................0g KH2PO4 ......0mL sterile glucose solution.....2.......................0mL..................0g Preparation of Disodium Thioglycolate Solution: Add disodium thioglycolate to distilled/deionized water and bring volume to 10............8..0g ..5............................................. glucose solution........0g Peptone ... Na2S·9H2O Solution: Composition per 100...... Cultures are grown under an atmosphere of 50% methane. Add L-cysteine...................8 ± 0........25g Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 1............................0 with 8N NaOH...............0g K2HPO4 ............. 1...............30 AN1 Medium NiCl2·6H2O .... Autoclave for 15 min at 15 psi pressure–121°C........ 0............................... Glucose Solution: Composition per 100............... Use: For the cultivation of Thermococcus species..... 10....................... sterile powdered sulfur............... 0.......... Mix thoroughly....0± 0.......................... Cool to 25°C.02mg CuCl2·2H2O ........15.2 at 25°C Disodium Thioglycolate Solution: Composition per 10..10.........5g Disodium thioglycolate solution . psychrophilus............0g Yeast extract .................. 50.5g Sodium acetate ........ powdered.. ....... Sparge with CO2........0g NaCl .......... 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....0........0L... Sparge with N2.................... Autoclave for 15 min at 15 psi pressure–121°C....... Preparation of Medium: Add components...........1.... 8................ Mix thoroughly............................................ Anacker and Ordal Medium Composition per liter: Agar ............... 40.......................0g NaCl ...... Autoclave for 15 min at 15 psi pressure– 121°C............................ 5.................2g pH 7................2 at 25°C Gently heat and bring to boiling.......... Preparation of Medium: Add components.............0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100........ Cool to 25°C.................................................................... 1.................... Aseptically add 50.........0g L-Cysteine·HCl .........................15.......0g Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100....... 10.....................3 ± 0..... Sparge with N2.. 10..................0g Resazurin ............20................1............................ and Na2S·9H2 solution.... Aseptically and anaerobically combine the basal solution.. Anaerobic Agar Composition per liter: Pancreatic digest of casein ............................0mL Glucose solution ............. 0............. Pour into sterile Petri dishes or leave in tubes....2 at 25°C Salt Solution: Composition per liter: NaHCO3 .2.................. Distribute into tubes or flasks.. Autoclave for 15 min at 15 psi pressure–121°C......................... Mix thoroughly................................................................

................... Autoclave for 20 min at 15 psi pressure–121°C............ 1....... 1...... 10............. 0...0mg Calcium DL-pantothenate ................1g FeSO4·7H2O ................0mg Lipoic acid......................... 0......0mL Na2S·9H2O solution................................... 0..................5g CaCl2 .........5g FeSO4·7H2O..................................................... Mix thoroughly...........10.............................0g MnSO4·H2O .... 0............................6.........................................0.........01g Na2MoO4·2H2O..............0L Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O. 10..................................1g CaCl2 ..........1mL of sterile Na2CO3 solution and 0....... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2...................................................................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100............................. Anaerobic Cellulolytic Medium Composition per liter: NH4Cl.........0............. Na2S·9H2O Solution: Composition per 100.....1g CoCl2·6H2O .....................................01g H3BO3 ............... 1........ 0..........0..............0g Sodium acetate .............................................5.........0.......... Preparation of Medium: Add components....8g NaHCO3 ...........01g AlK(SO4)2·12H2O..1.............................. 0......... 0....................... 1.............................................. Bacillus popilliae.......................0mg Riboflavin...................0g pH 7......................................................................................5. 2.........................0........... 0.........0mg p-Aminobenzoic acid ................... 0...................................0g Nitrilotriacetic acid ........................... Mix thoroughly....5...0g Cellobiose ..............0............0L................................... 1.................. Use: For the anaerobic cultivation of Bacillus species.............. Bring volume to 1........... Cool and distribute anaerobically into test tubes in 10.....01g CuSO4·5H2O..0L with distilled/deionized water...................1g AlK(SO4)2·12H2O .............. Preparation of Trace Elements Solution: Combine components.............. 10........................2 ± 0........... such as Clostridium cellulovorans.......5g NaCl ........................ Boil medium under 80% N2 + 10% CO2 + 10% H2 until medium is colorless.................................................1mL of sterile Na2S·9H2O solution to each tube........ isms that can utilize cellobiose as sole carbon source........ Na2CO3 Solution: Composition per 100............... 1... Filter sterilize...01g Na2MoO4·2H2O ... 3........................... Mix thoroughly......0.. Mix thoroughly.............. Adjust pH to 7........................5g NaH2PO4·H2O ............0.......................................................................................5...................... 5............................................................................................................................ and Bacillus lentimorbus..... Mix thoroughly......................................0mL Na2CO3 solution...........................................................0mL............................. 1...............0g Yeast extract....................................... to distilled/ deionized water and bring volume to 980..0g Sodium thioglycolate .5g L-Cysteine·HCl·H2O .......... 1........5g K2HPO4 ................0mg Folic acid .. 0.0mL of distilled/ deionized water and adjust pH to 6........................25..0mL..........................................4g Resazurin ..................0g MnSO4·H2O...............01g H3BO3 .........3........................................ 0.....................................................0mL of water and adjust to pH 6.....................0g MgSO4 ...............1............................ Mix thoroughly...............Anaerobic Citrate Medium 31 NaCl ...........................................................................................0 ± 0...........0........0............ 0...........................................................1g CuSO4·5H2O ..... Gently heat and bring to boiling............................................... Bring volume to 1.........................1g ZnSO4·7H2O............................. except Na2CO3 solution and Na2S·9H2O solution.................... Add other compounds...10.............1g ZnSO4·7H2O ...........0mg Wolfe’s mineral solution ..............1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................5g NH4Cl ............0L.6g KCl ........................................................0mL: Na2CO3 ...............................................................0mL: Na2S·9H2O..................................................................................................0mL volumes using 80% N2 + 10% CO2 + 10% H2................. Mix thoroughly.............0mg NiCl2·6H2O..................5 with KOH to dissolve the compound.17....................................1 at 25°C Wolfe’s Mineral Solution: Composition per liter MgSO4·7H2O ................................ Aseptically add 0...1g CoCl2·6H2O............... es- pecially Bacillus larvae............ Mix thoroughly...............0mg Thiamine·HCl ............................1..1g Trace elements solution......................... Cool to room temperature...................... 0..... Distribute into tubes until medium is 3 inches deep...........2......................................5g NaCl .........................0L with remaining water and add remaining compounds one at a time..............24........................9 ± 0..............5.2...........0g Nitrilotriacetic acid....................0.................0g Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100....................................................5.....01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to approximately 500.. Anaerobic Citrate Medium Composition per liter: Ferric citrate ......01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to approximately 500.......................................................0mL..........2 at 25°C Trace Elements Solution: Composition per liter: Na2WO4 ..............................................................0mL Wolfe’s vitamin solution ....5 with KOH to dissolve.......................... Filter sterilize..20......... 15...............................2.................................................................. Add ferric citrate to 500..................................0mL pH 6................2...........2 at 25°C Stopper the tubes anaerobically.......................... Cool to room temperature while sparging with 80% N2 + 20% CO2.......... .............0mg Vitamin B12................ Autoclave for 15 min at 15 psi pressure–121°C.......................................................................................0g Sodium formaldehyde sulfoxylate .......................................0mg Nicotinic acid .......................0mL of boiling distilled/deionized water....... Preparation of Medium: Add components to distilled/ Use: For the cultivation and maintenance of microorgan- deionized water and bring volume to 1.....0mL pH 7.................................0mg Biotin ..............................................................10..................0mg Wolfe’s mineral solution .....0.....................5g KCl..... Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl....

Sparge with 100% N2...........0g MnSO4·2H2O......................................................32 Anaerobranca gottschalkii Medium Add remaining components.....01g Na2SeO3·5H2O .0g Na2HPO4·2H2O ........ Cool to 25°C while sparging with 100% N2.......................................................0...........................................................................18g ZnSO4·7H2O ............. Sparge with 100% N2............. Na2CO3 Solution: Composition per 100..1g CaCl2·2H2O.......................0L.........0mL sterile Na2CO3 solution............. 10 fold conc...........................................5...125g Na2S·9H2O . 0............................................. a novel thermoalkaliphilic bacterium that grows anaerobically at high pH and temperature.................................. Mix thoroughly............................ 0................................ Autoclave for 15 min at 15 psi pressure–121°C.......3-9.... Mix thoroughly.......50....0mL pH 9..........5g L-Cysteine·HCl·H2O... Final pH is 9.............................................................10...................... 5......5g NH4Cl....................................................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.............................0mg Folic acid.........................................................................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.......5mg Na2CO3solution.................................... Mix thoroughly.................. Mix thoroughly............. Use: For the cultivation of Anaerobranca gottschalkii..............5g KH2PO4 ..................0mg Lipoic acid.. and L-cysteine...................0mg Vitamin B12 ........................................ to distilled/deionized water and bring volume to 970............ Add 0.......... 0..........1g ZnSO4·7H2O.................................................................................... Adjust pH to 7.............5g L-Cysteine ...................................................................0L...........................................5g KCl .................2 at 25°C Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl...................................................... Add distilled/deionized water to 1......... 0....... Vitamin Solution: Composition per 100mL: Pyridoxine-HCl .1g MgSO4·7H2O ........................ Gently heat and bring to boiling...................................................................................................1....................... 1.... 0......0mg Folic acid ...0.............................................5g L-cysteine..........3................1..5....................0mg Resazurin .......... Na2CO3 solution.................. 2................ Preparation of Medium: Add components.......... Anaerobranca gottschalkii Medium (DSMZ Medium 895) Composition per 1020mL: NaCl ....10........................ 0.. 0...0......... Filter sterilize....... 1.... 0......1...................0mL pH 8..........20........0mg Calcium DL-pantothenate ........................................ 0...................0mL: Na2CO3 ....0g Preparation of Starch Solution: Add starch to distilled/deionized water and bring volume to 50.......... Sparge with 80% H2 + 20% CO2.........................................3.. 5.....0mL.........01g Na2MoO4·4H2O ...................................0g K2HPO4 .............................................5............0......0mL sterile starch solution..............9g Sodium fumarate ..............0mL: Starch........................................................................... Add remaining components................................................................................0mg Biotin ................... 0................. Add distilled/deionized water and bring volume to 1............ 5. 2..........................1g .......... 5...... Cool to 25°C..................5g CoCl2·6H2O....0.......................................2..................... 0.......................0mg Lipoic acid ................................0mL Vitamin solution................................................................0 with NaOH....0L................ 0......................5g NaCl ..............................................5g NaCl .... 0.............. Autoclave for 15 min at 15 psi pressure–121°C.5g CoSO4·7H2O ..................... Autoclave for 15 min at 15 psi pressure–121°C..................2.................1g FeSO4·7H2O. 5.. Mix thoroughly...5................................. Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O.0mg Vitamin B12........1g NiCl2·6H2O ...............................................................0mL............................................. Mix thoroughly..................4 ± 0......0mL of distilled/deionized water.......18g CaCl2·2H2O..........................................0g MnSO4·2H2O .....................................................................0mg Thiamine·HCl ..................... 10.......................... Mix thoroughly.................... Anaerobically distribute into tubes.. soluble ...... 1. Autoclave for 15 min at 15 psi pressure–121°C.................0mL Wolfe’s mineral solution .................... 0..............................0mg Riboflavin......5................................................... 5...0.....5.5 with KOH.....0mL............0mL.......0mg Riboflavin . Distribute to anaerobe tubes or bottles.................0mL Soluble starch solution.............................................. 0............................5............................................... 0...........................................0mL Trace elements solution ...0............................................0g Nitrilotriacetic acid.............................25g Na2S2O3·5H2O ....0mg p-Aminobenzoic acid ................0mg Thiamine-HCl·2H2O .......0g Nitrilotriacetic acid ................................................. Starch Solution: Composition per 50.. except starch solution............. Mix thoroughly...................................................................................2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O .............. Filter sterilize.............025g KAl(SO4)2·12H2O......... 20............. 5...................5......... 0.............................................................. Use: For the cultivation of Geobacter metallireducens.....5 ± 0............... 10.02g H3BO3 ............................................. 0....1g CaCl2·2H2O ..... per liter of medium..... and 50..........................................0g (NH4)2SO4 .............0mg p-Aminobenzoic acid .......1................. Continue sparging with 80% N2 + 20% CO2.............0mg Nicotinic acid ...................................................125g Wolfe’s vitamin solution .................................0mg Nicotinic acid ................................ 2............................5.............................. 0.......... 10.........0mg Biotin .............................. Aseptically and anaerobically add.....0........................................................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................................ 0......5.......0....25g Tryptone ............................................................0g Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 100..... Dissolve by adjusting pH to 6....................0mg D-Ca-pantothenate.........................0mL: Yeast extract .4g Yeast extract........... Anaerobranca Medium Composition per 1015........................ 3......................................... 0.01g CuSO4·5H2O ...................05g FeSO4·7H2O..............................................................

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

....................................... Autoclave for 15 min at 15 psi pressure–121°C............ Anaerovibrio burkinabensis Medium Composition per 1001....0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10....................................0mg Biotin ..................0g NaCl .........................................0mg NiCl2·6H2O............. 24......5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10...................... Seal the flask under 80% N2 + 20% CO2......................................5.......................0mL: NaHCO3 .0mL Solution C . Preparation of Medium: Add components.....0mg Vitamin B12 .................1.......5............. Add remaining components.............. 1.........0g Preparation of Cellobiose or Starch Solution: Add cellobiose or starch to distilled/deionized water and bring volume to 50.0mL........... and Na2S·9H2O solution.. Mix thoroughly.... 2.... Gas under 80% N2 + 20% CO2................................................................................................... 100........ Continue boiling for 3–4 min..................................................... Mix thoroughly.....................................................................10....... Use: For the cultivation and maintenance of Anaerocellum thermophilum and Dictyoglomus turgidus......... Allow to cool to room temperature while gassing under 80% N2 + 20% CO2...............5...................... Continue gassing until pH reaches below 6..........0.0mg Folic acid.........0mg Calcium DL-pantothenate ................ 5..............0mL pH 6.......0mg ZnCl2 ......... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ..........................0mg Riboflavin......................... 70..........0........................6.....1mg ..............................................0g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100.... 10.. and sterile Na2S·9H2O solution......... Filter sterilize......................0mL: Solution A ... 5...... Solution D: Composition per 10.....0mL Solution E (Vitamin solution) ....... Autoclave for 15 min at 15 psi pressure–121°C............................... 5................0.....................................................10..............................................0mg Folic acid . sterile cellobiose or starch solution....... 0..........1....0mg Nicotinic acid .............. Gently heat and bring to boiling.........0mg ZnCl2 ...............0mg Lipoic acid ...........2 at 25°C Solution A: Composition per 870.......0mL of HCl solution..............0mL....................................... Mix thoroughly......0mL....2..............................................................0L....0mL Solution G ....................................................................... 10.. Mix thoroughly................ Gas under 100% N2...................................... 10..... 5.. Mix thoroughly...........................................100......... 6....................0mg MnCl2·4H2O .............................................................................5g CoCl2·6H2O...........34 Anaerovibrio burkinabensis Medium Cellobiose or Starch Solution: Composition per 50..... 1.............. Autoclave for 15 min at 15 psi pressure–121°C.....0mg p-Aminobenzoic acid ........................ 190...0mg Na2MoO4·2H2O.................................................. Add distilled/deionized water and bring volume to 1........ 10......................... to distilled/deionized water and bring volume to 830...........0....... Autoclave for 15 min at 15 psi pressure–121°C.............. Solution B (Trace Elements Solution SL-10 ): Composition per liter: FeCl2·4H2O.................................................. 5................................................................... Gently heat and bring to boiling.................................0mg p-Aminobenzoic acid .0mg H3BO3 .......... Distribute into bottles under 80% N2 + 20% CO2....0mL Solution F ........0mg HCl (25% solution) ....0mg Vitamin B12...............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........................... Add remaining components.............. Na2S·9H2O Solution: Composition per 10....5..5g MgCl2·6H2O ...............5g Preparation of Solution D: Add sodium lactate· to distilled/deionized water and bring volume to 10............... Gas under 100% N2......................................................................5.............0mL Solution D ....................................70.......................5............................. 0...........0mg Biotin ......2.................... Continue boiling for 3-4 min..0L.................0mL Solution B (Trace elements solution SL-10) ...0mg Thiamine·HCl ......................................... 36................................................................................................... Mix thoroughly....0mg HCl (25% solution) ......24...................................15g Resazurin .....................190..... Allow to cool to room temperature under 80% N2 + 20% CO2...0mg Riboflavin ...............0mg Na2MoO4·2H2O .......10......................0mL.........0mg Thiamine·HCl ..........0mg MnCl2·4H2O.................. Add distilled/deionized water and bring volume to 1.................. Solution C: Composition per 100.......100................................................................... 5...1......................... 2......................0mL: Cellobiose or starch ...............................3g KH2PO4 ...........................0mg H3BO3 ..................... Mix thoroughly..................................................................... Aseptically and anaerobically add sterile NaHCO3 solution....................... Mix thoroughly....................................0. 2.................0mL.........0mg CuCl2·2H2O.........0L.................. Vitamin Solution: Composition per liter: Pyridoxine·HCl .870............0mL: Na2SO4 ..............................0g KCl .............................0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870............ Mix thoroughly........................0mg CuCl2·2H2O .....0.............................0. Gas under 100% N2........................................0mg Calcium DL-pantothenate.........5......... 5...........................................3..... Autoclave for 15 min at 15 psi pressure–121°C...........36............2.. Filter sterilize.0mL of HCl solution........0mL...................................... Mix thoroughly...............5g CoCl2·6H2O ...4g NH4Cl ............................... Autoclave for 15 min at 15 psi pressure–121°C.......... Mix thoroughly.................................2g CaCl2·2H2O ....................................................... Gas under 100% N2..2..................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10. Gas under 100% N2..........................10............0mg NiCl2·6H2O ...........................0mL: Na2S·9H2O... except NaHCO3 solution................................................0mg Lipoic acid............................................... cellobiose or starch solution..................... Solution E (Vitamin Solution): Composition per liter: Pyridoxine·HCl......8–7...... Mix thoroughly.............................................0mL: Sodium lactate ..............0mg Nicotinic acid ...................................

........................................4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10................. Andrade’s Indicator Composition per 100....... salicin..... and Spirosoma linguale. Aquaspirillum serpens.... dulcitol.....1g Filtered.... Use: For the cultivation of Ancylobacter aquaticus.2 at 25°C 35 Peptone ...4–7............2.. Mix thoroughly.. Bring pH to 7.....................8.0mL.....0g Preparation of Solution F: Add yeast extract to distilled/deionized water and bring volume to 10....... 15.0mL Carbohydrate solution .......... 1............ Gas under 100% N2.....0......... Mix thoroughly......................... Autoclave for 15 min at 15 psi pressure–121°C............... Adjust pH to 7.............3............. 1... Autoclave for 15 min at 15 psi pressure–121°C...0mL: guale....... and solution G..0g NaCl ..... 1..... Autoclave for 15 min at 15 psi pressure–121°C. 1........... 750.......... Distribute into tubes or flasks...........0g Andrade’s indicator ........2 at 25°C deionized water and bring volume to 1... 15.......2 at 25°C Source: This medium is available as a prepared medium from BD Diagnostic Systems......................0.................. Gently heat and bring to boiling......0g Glucose ............... solution D........................ Ancylobacter-Spirosoma Medium (DSMZ Medium 7) Composition per liter: Agar ......................... raffinose...................................... solution E............. 750.... aged seawater ... in tubes containing adonitol.....Andrade’s Broth Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1................................. and Vibrio species. Micro- Preparation of Medium: Add components to distilled/ lobacter species..... 50.......................... inositol....... Agar ..............0L... Mix thoroughly.............0mL........................ Adjust pH to 6.... or xylose..... Use: For the cultivation and maintenance of Anaerovibrio burkinabensis...........................0mL: Na2S·9H2O....... Gently heat and bring to boiling........................................... mannitol.....6.............. Use: For the cultivation of Vibrio species......4 ± 0..4.......6 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............4–7..5................ Anderson’s Marine Medium Composition per liter: Peptone ..................0.. solution F.2.. Use: For the cultivation and maintenance of Spirosoma lin- coccus species.....0mL...............0L.................. solution C..........0g Beef extract . 1.......... glucose................10........8 ± 0.............0g Peptone..... Distribute into tubes or flasks........................... Aseptically distribute into sterile tubes or flasks....................... aged seawater ................. Autoclave for 15 min at 15 psi pressure–121°C............... Mix thoroughly.............4–7...........5g Yeast extract .....2........0g Yeast extract................................2. fructose......... except seawater.............0L.. Anderson’s Marine Broth Composition per liter: Peptone ............................ sorbitol.....0mL pH 7.. Gas under 100% N2................................. 16........... 20...... Mix thoroughly.0mL with distilled/deionized water.......... to distilled/deionized water and bring volume to 250............ Mix thoroughly.. Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2... Planococcus species... trehalose.... galactose...................1g Filtered......... Aquaspirillum metamorphum...........0... Ancylobacter-Spirosoma Agar Composition per liter: Agar ..... 1................... 1..........6.0g Peptone...... Pseudomonas fluorescens...... Solution F: Composition per 10.... in that order.................................. Pour into sterile Petri dishes or leave in tubes...4–7.6 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.. Preparation of Medium: Add components.................0mL of sterile aged seawater....... Mix thoroughly.... Distribute into tubes or flasks....0mL pH 7... Gas under 100% N2.. lactose.......................... 750............ Autoclave for 15 min at 15 psi pressure–121°C........................ Gently heat and bring to boiling. Solution G: Composition per 10........ 10........... Shewanella putrefaciens.0g pH 6................. Autoclave for 15 min at 15 psi pressure–121°C..... 0................ Autoclave for 15 min at 15 psi pressure–121°C.... Runella slithyformis......................1g Filtered............ arabinose...0g pH 7......................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...0L.................. Aseptically add 750...0L.. Mix thoroughly...1g Anderson’s Marine Agar Composition per liter: Preparation of Andrade’s Indicator: Add Acid Fuchsin to NaOH solution and bring volume to 100................. Andrade’s Broth Composition per liter: Pancreatic digest of gelatin . Use: For the cultivation and maintenance of Vibrio species............. Mix thoroughly........................................................... a lactate-fermenting bacterium isolated from rice field soils........0mL pH 7............... Ancy- Use: For the cultivation of Flavobacterium species.............. cellobiose.....................2........ Adjust pH to 7.0g Yeast extract........... Mix thoroughly............... aged seawater ............ Autoclave for 15 min at 15 psi pressure–121°C......0g Glucose ..4 ± 0. Preparation of Medium: Aseptically and anaerobically combine solution A with solution B...................0mL Acid Fuchsin ...............5g Yeast extract ............. maltose............................5g FePO4 ................................5g Yeast extract ......... Pour into sterile Petri dishes or leave in tubes........... Flectobacillus major......0g ...............1 ± 0... Distribute into tubes or flasks...... sucrose....................5g FePO4 .0mL: Yeast extract.... Methylobacterium mesophilicum............. Pour into sterile Petri dishes or leave in tubes...........................2...... rhamnose..............................0mL pH 7...... NaOH (1N solution) ...........................5g FePO4 ...................................

....0mL volumes into test tubes containing inverted Durham tubes................... 50......................... 0............ galactose........................... Cool to 25°C... cellobiose......... Preparation of Medium: Add components—except “Metals 44”.............2..............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................. Gently heat and bring to boiling...................................025g Na2B4O7·10H2O ......10............... glucose.........................0............................................... 0.0g Agar ..... to distilled/deionized water and bring volume to 1................ Aplanobacterium Medium Composition per liter: Agar .. Add 0.0mL.................5g EDTA........ Mix thoroughly. inositol....... Distribute into tubes or flasks.. Autoclave for 15 min at 15 ......................... except carbohydrate solution....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............................... AO Agar Composition per liter: Agar .................0mL: ZnSO4·7H2O..............0mL Vitamin solution........ Dissolve remaining ingredients... phosphate. 9.......... lactose...............2. “Metals 44”: Composition per 100.................0L........................................0 ± 0........................................ phosphate solution......... Acid production is indicated by a pink color.. Filter sterilize.......0L. Add aseptically to sterile basal medium.........................3g FeSO4·7H2O.... 11.......1g KNO3 ................................................................2.........................................0mg Thiamine·HCl .........5g MgCl2·6H2O.................................................................................0.................. Autoclave for 15 min at 15 psi pressure–121°C.......... sucrose.....0g pH 7........................ Filter sterilize and add aseptically to sterile basal medium.............................. Phosphate Solution: Composition per liter: K2HPO4 ............................ 1................................................... 5............... rhamnose........0mg Na2MoO4·2H2O......................................... Adonitol............. and Flexithrix species.........1g Metals solution...........5.........0g Yeast extract ................0g Glucose............. Mix thoroughly....................................0.......10...... 3.............................2 ± 0..............2 ± 0.................................0. Use: For the determination of carbohydrate fermentation reactions of microorganisms..................... sorbitol.......0mL Phosphate solution ...........0mg Cyanocobalamin..........0L.0mL pH 7....................... Autoclave for 15 min at 15 psi pressure–121°C............ Adjust pH of basal medium to 7.....0g Sodium acetate ....................5......................................................... 0.....0mL: Carbohydrate.......... dulcitol...... Autoclave for 15 min at 15 psi pressure–121°C.. Cool to 50°C and aseptically add the “Metals 44”.2g (NH4)2SO4 .......................0mL Preparation of Metals Solution: Solubilize nitrilotriacetic acid with KOH. trehalose..........................5g Beef extract ..........0g Peptone..........................1............. 5. salicin........ Gently heat and bring to boiling.....................................0mL.0............... Saprospira species.................. fructose.7g Nitrilotriacetic acid ...................................................0...5g KH2PO4 ......5...... 100...........154g CuSO4·5H2O...................5g Yeast extract.......20...........0g Peptone ...0g KCl.................................2 with KOH or H2SO4....... Use: For the isolation and cultivation of Cytophaga spe- cies....7mg “Metals 44” .............................................0L....................... arabinose........0........................... 10........................0L.............0g Preparation of Carbohydrate Solution: Add carbo- hydrate to distilled/deionized water and bring volume to 100............1g FeSO4·7H2O .... Use: For the cultivation and maintenance of Halomonas meridiana and other Halomonas species.............................36 AO Agar Carbohydrate Solution: Composition per 100.................. 0........................04g Co(NO3)2·6H2O......................... Autoclave for 15 min at 15 psi pressure–121°C.....0mg Biotin .......5mL of sterile carbohydrate solution to each tube.................. Adjust pH to 7............. 5.................. Mix thoroughly.............................. or other carbohydrates may be used.............0g CaCl2·2H2O....... Add aseptically to sterile basal medium....................................20................0...............................5g Preparation of Phosphate 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......... Mix thoroughly............ Mix thoroughly......2... Herpetosiphon species..................................... xylose.......... 0........... 0.............. 15............................ Pour into sterile Petri dishes or leave in tubes............................................................20........................................................... mannitol..0g Yeast extract. and vitamin solutions...2 at 25°C Metals Solution: Composition per liter: MgSO4·7H2O ....................................................12................... Mix thoroughly... Autoclave for 15 min at 15 psi pressure– 121°C...5.5.........................0mg Calcium pantothenate ..2g pH 7.......0L...0g CaCl2·2H2O...............0mg Riboflavin........... Preparation of Medium: Add components............................................ 99..........................25g MnSO4·7H2O........... and vitamin solution—to distilled/deionized water and bring volume to 1................5................................... Vitamin Solution: Composition per liter: Pyridoxine·HCl. 0..................5g Pancreatic digest of casein . Caution: Acid Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contact with the skin........0mL: NaCl ........ Add aseptically to sterile basal medium...0g MgSO4·7H2O ............... Distribute in 10............ maltose............ raffinose..... particularly members of the Enterobacteriaceae...... A Durham tube is used to collect gas produced during the fermentation reaction......... Gently heat and bring to boiling............................0mg Nicotinamide .................................................... 10... Mix thoroughly. Aolpha Medium Composition per 1041.............................................. 29.......1.........................018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100.............0mg Folic acid ..................................................... Autoclave for 15 min at 15 psi pressure–121°C.......................... Mix thoroughly................. Gently heat and bring to boiling...........

..0mg Na2MoO4·2H2O............................................ Autoclave for 15 min at 15 psi pressure–121°C................ Mix thoroughly........... Gently heat and bring to boiling......................................... to 980. 5..................... species............... Gently heat and bring to boiling..0mg ZnSO4·7H2O.............0g MgSO4·7H2O..................... 2..... 10......0mL pH 7.............2g NaCl ................................................................................................................. Cool to 45°–50°C......9......................... Aseptically add sterile NaHCO3 solution......0L pH 4.............0mL Trace elements solution.......................... Aseptically combine the sterile apple juice solution with the sterile agar solution.. 0............. Vitamin Solution: Composition per liter: Calcium DL-pantothenate..........0mg ....30.............. except NaHCO3 solution.......0g MgSO4·7H2O.................. 0...... Pour into sterile Petri dishes or distribute into sterile tubes................................................1....................................0L of apple juice...0mg CoCl2·6H2O................1g MnCl2·4H2O............... Trace Elements Solution: Composition per liter: H3BO3 ......30.......Aquaspirillum Heterotrophic Agar 37 psi pressure–121°C................. 3..........................03g Na2MoO4·H2O ..........2 at 25°C NaHCO3 Solution: Composition per 10............15.............................0mL: NaHCO3 ......20.............. Mix thoroughly................... Adjust pH to 4....2g ZnSO4·7H2O ................................ In a separate flask..........................0mL pH 7........ Mix thoroughly........... Mix thoroughly.........1. Preparation of Medium: Add components... 2...................................... Mix thoroughly.........0mg ZnSO4·7H2O...........0mg MnCl2·4H2O .........................5g NH4Cl ......................................................... Cool to 45°–50°C.....0L.........3... 30............................... Autoclave for 15 min at 15 psi pressure–121°C................................................ Mix thoroughly..............0mg NaHCO3 solution....... add agar to 200..............................15...............1 ± 0......3g CoCl2·6H2O ..........20.........0mg Biotin ................... 0.........10........0mg CoCl2·6H2O..............2g CaCl2·2H2O ............. 0.............................................................................0mg Riboflavin ...............0g Yeast extract..................................................................................0mL of distilled/deionized water and bring volume to 1...0mg Vitamin B12 ..................... 0............1......................................... 2... Filter sterilize.....................................0g Peptone...........4g NH4Cl............................2g MgSO4·7H2O .............................. 10......................1.............................................................................. 5........... 5.. 3.................................... 0............................................. 1..................................................................................0mL pH 7...............1....0mg Folic acid........0mL SL-6 Trace elements solution .....................0g Sodium succinate...0mL distilled/deionized water. 0........................................10...........0.....................0mg Trace elements solution.... 0......................5....................2 at 25°C SL-6 Trace Elements Solution: Composition per liter: H3BO3 ................................................................................... 10.........0mL: Agar .............................................1...... 0... For autotrophic growth.2...................0g Yeast extract..... Adjust pH to 3............0mL............................... 0............................................................... Pour into sterile Petri dishes................. 1......................0.............................0mg Thiamine·HCl ................................ tion............. Pour into sterile Petri dishes or leave in tubes....5...03g NiCl2·6H2O ... incubate under 85% H2 + 10% CO2 + 5% O2............................................. Mix thoroughly.......................................0L....0g Apple juice ................8................................ Mix thoroughly............ Mix thoroughly................. Aseptically distribute to sterile tubes or flasks........................................2 at 25°C Preparation of Medium: Add yeast extract to 1..01g Ferric ammonium citrate ..................................................................... Use: For the autotrophic cultivation and maintenance of Aquaspirillum autotrophicum....0L.....0g Na2HPO4·12H2O ..........0mg Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 1............................ 0................... Use: For the cultivation and maintenance of Xanthomonas Use: For the cultivation of Aquabacter spiritensis......8 ± 0.....................................................0mg CuCl2·2H2O...................0mg Vitamin solution.......0g KH2PO4 .................2 at 25°C Trace Elements Solution: Composition per liter: H3BO3 .................... Mix thoroughly.....1................01g Ferric ammonium citrate ..2g CaCl2·2H2O ..................0 ± 0...........0g KH2PO4 ..........3........0g KH2PO4 ... to double-distilled water and bring volume to 990...5g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 10.................. Aquabacter spiritensis Medium (LMG Medium 225) Sodium succinate ......... Preparation of Medium: Add components...................................4.0mg Na2MoO4·2H2O........3..... 5.................... Apple Juice Yeast Extract Medium (AJYE Medium) Composition per 1200......................0mg MnCl2·4H2O ......................................3..................................................... Autoclave for 10 min at 9 psi pressure–114°C.........0............................... 0.......................... Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.................... Cool to 25°C...............1................. except vita- min solution............................................................01g Preparation of SL-6 Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0mg Ferric citrate ...........................20............................0mL..0L........................................... Aseptically add 20.................................................................................................................0g Na2HPO4·12H2O ...........9... Cool to 45°–50°C........................... Aquaspirillum Heterotrophic Agar Composition per liter: Noble agar .................................................0mg NiCl2·6H2O.... Filter sterilize..............02g CuCl2·2H2O..........1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...................5g NH4Cl ...................0...................2g CaCl2·2H2O.. Use: For the cultivation of Zymomonas species........1 ± 0....................0............................0mL sterile vitamin solu- Aquaspirillum Autotrophic Agar Composition per liter: Noble agar ........

............5g CaCl2·6H2O.........18g ZnSO4·7H2O...................................0L... Gently heat and bring to boiling..0mL sterile standard vitamin solution.........4................................................................................ Mix thoroughly..................0mg Resazurin ..... 1............................................... Add distilled/deionized water and bring volume to 1..5 ± 0...........................................................................................0...................... Add remaining components........0mg Na2S·9H2O solution ... 300.. Filter sterilize..... Cool to room temperature.........0mg NiCl2·6H2O ..................................0..........0mL of HCl solution... 1............................5g KCl .. except so- dium succinate solution.....0g NaHCO3 ................... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ....................... 50.......9 ± 0.............................10........................... 0..............................0..................................................... Aquaspirillum Medium (DSMZ Medium 888) Composition per 1026mL: Casamino acids ............ 10.................0..............................0........34g NH4Cl ............ 1.... 25.........0mL Thiosulfate solution ........ 50..............5.............0mL Trace elements solution...5..................................... and trace elements solution SL-10......................0mg Vitamin B12 ....... to distilled/deionized water and bring volume to 1................2 at 25°C Standard Vitamin Solution: Composition per 100mL: Thiamine-HCl·2H2O ....0mg HCl (25% solution) .........................0........ Add Na2S·9H2O to distilled/deionized water and bring volume to 50.......18................................ Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500....2...........................................5g CoSO4·7H2O.........2mg Biotin ...................................0mg CoCl2·6H2O ....... 70.................................01g H3BO3 ..........0mL: Na2S·9H2O ................................ Cool to room temperature...0mg Na2MoO4·2H2O ......................................5g H3BO3 .............. 10.....0mL...5g Yeast extract ................................... 0...................18g FeSO4·7H2O .........3mg Na2S·9H2O Solution: Composition per 50.................0mg Ca-pantothenate .........1............ and 1............................ Archaeoglobus Medium Composition per liter: NaCl ......... Cool to room temperature....0mL Standard vitamin solution ..... Preparation of Medium: Add components to doubledistilled water and bring volume to 1.0g Preparation of Na2S·9H2O Solution: Prepare and dispense solution anaerobically under with 80% N2 + 20% CO2...........0L with Thiosulfate Solution: Composition per 10mL: ...................1............. Autoclave for 15 min at 15 psi pressure–121°C. 1..............38 Aquaspirillum Medium NiCl2·6H2O ..............................0mL....................1mg Preparation of Standard Vitamin Solution: Add components to distilled/deionized water and bring volume to 100........0L.. 1....1g NiCl2·6H2O......0mg Nicotinic acid ................ Mix thoroughly....................................................... Aseptically add 10..........................................................................3...0g Na2S2O3·5H2O .................... Use: For the heterotrophic cultivation and maintenance of Aquaspirillum autotrophicum.......0mg Pyridoxine-HCl ........0mL of distilled/ deionized water.......................5...................0........0mg Folic acid.....0mL sterile thiosulfate solution........025g KAl(SO4)2·l2H2O .........................................................................0L.................................... 2.............................................. Mix thoroughly......... 1........................0mL Trace elements solution SL-10 ..........0......... 30................ Autoclave for 15 min at 15 psi pressure–121°C.............................. Autoclave for 15 min at 15 psi pressure–121°C................0........................................0mL sterile trace elements solution SL-10.......3...............0...................................................0mg CuCl2·2H2O ........................0............ Cool to room temperature... Bring volume to 1.......................0mL pH 7......... Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly............................................... thiosulfate solution.......................5g NaCl ...0............. Sparge with 100% N2........1...............7.................1....................................0....................... 1............................................. Mix thoroughly.0g MgSO4·7H2O................ 50.....0mg ZnCl2 ..... 10.......................................................... Pour into sterile Petri dishes or distribute into sterile tubes.................0...........02g CuSO4·5H2O.................................. Mix thoroughly..................... 24.......01g Na2SeO3·5H2O ..................................................................................10.0L.......... 2.....................................0mL pH 6................... 190..........0...................5g (NH4)2SO4 ........ Dissolve by adding KOH and adjust pH to 6...........................0mL sterile sodium succinate solution....................................................................... 100...... Sodium Succinate Solution: Composition per 10mL: Sodium succinate...1........ 0.................................14g Fe(NH4)2(SO4)2·7H2O .............. Preparation of Medium: Add components......................... Mix thoroughly......................................... 36.................................................7mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10................................................... Add remaining components.......0g MnSO4·2H2O..............0g Preparation of Sodium Succinate Solution: Add sodium succinate to distilled/deionized water and bring volume to 10.............................................. 50..............................................0............... Aseptically distribute into sterile tubes or flasks....0mg Sodium succinate solution .........................................0mL.............25g CaCl2·2H2O ......... Adjust pH to 7.............. 5.. Adjust pH to 7.....0mg MnCl2·4H2O. 1......................................................0g Agar ......1g CaCl2·2H2O ................................................................ Mix thoroughly...................14g K2HPO4 ............0g MgSO4·7H2O ................. Autoclave for 15 min at 15 psi pressure–121°C...............0g Nitrilotriacetic acid....5...........0mg Riboflavin ........0mL.......................... standard vitamin solution............1........ Mix thoroughly.....01g Na2MoO4·2H2O.............. Mix thoroughly............... Preparation of Thiosulfate Solution: Add Na2S2O3·5H2O to distilled/deionized water and bring volume to 10........................................0........................2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O.0mg Na2H2PO4.................0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1...................................................45g Sodium L-lactate.........................0mg CuCl2·2H2O ...................0g MgCl2·6H2O ....... Autoclave for 15 min at 15 psi pressure–121°C.............................................................1......... Use: For the cultivation of Aquaspirillum spp.....................................

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

...... 7.....3g L-Proline.....................0 with HCl..................................1g L-Alanine..........5mL Na2SO3 solution per 25.............................................................0g CaCl2·2H2O ............5................... Preparation of Medium: Add components.... 30... Gently heat and bring to boiling....................2 with KOH or H2SO4.......................................................................... 0............... Mix thoroughly..........................................0mL Na2S·9H2O solution........ 75............................................................... 0......................................................0....05g L-Histidine...............25g MgSO4·7H2O ..0g Sodium succinate .1g L-Asparagine .2g KNO3 ....25g MnSO4·7H2O ........... Adjust pH to 7.................................................40 Archangium violaceum Medium Na2WO4·2H2O ......................2 at 25°C Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O ...............0...................... Mix thoroughly............. 25................................................0mL distilled/deionized water............. and NaHCO3......................30............................5 ± 0..5) .......34g FeSO4·7H2O..................4g KCl.......................... Add components....................0mL Artificial organic lake vitamin solution.............................................0mg Preparation of Trace Element Solution: Add components to distilled/deionized water and bring volume to 1......... Use: For the cultivation of Archangium violaceum.............05g Cyanocobalamin................................... Distribute into serum bottles under 80% N2 + 20% CO2 gas............................... Phosphate Supplement: Composition per liter: K2HPO4 ....................1.. There may be a slight precipitate..............................................................1g L-Valine ...........................................................................0mL medium..............................................................3 ± 0............................. 0...............................25mg “Metals 44” ...0g MgSO4·7H2O .............................. Archangium violaceum Medium Composition per liter: Monosodium glutamate ....................................0...........0....... Mix thoroughly...................9.......................... Mix thoroughly.................................... Add other components and adjust the pH to 7.....................................................04g EDTA.......1g Modified Hutner’s basal salts................5g CuSO4·5H2O .......................................0mL pH 7......... Add 10....... After inoculation pressurize to 1 bar atmosphere with 80% H2 + 20% CO2 gas.......... Sparge with 100% N2.................................................... Aseptically distribute into tubes or flasks..........0g MgSO4·7H2O.............................. Add the solid NaHCO3........ “Metals 44”: Composition per liter: ZnSO4·7H2O .................0mg Ammonium molybdate..... 0.................................................... 0.............. to 986............ Cool to 25°C................. Use: For the cultivation and maintenance of Halobacterium lacusprofundi............ Na2S·9H2O solution.............. Autoclave for 15 min at 15 psi pressure–121°C....................................1g (NH4)2SO4 ..............05g Na2SeO3·5H2O ..........0L............................... Use: For the cultivation of Archaeoglobus veneficus...... 0....... 10.....................154g Co(NO3)2·6H2O .....0mL medium in 100mL serum bottles.......2g L-Lysine...0mL..................4................... 20..................................05g Ca3(PO4)2 .................0mg Thiamine·HCl ............0............018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100........ 50.................................... 0......7g Nitrilotriacetic acid......... 1.....10.......... 7....5.........................0...........2....... pH 7.........2 at 25°C Vitamin Solution: Composition per liter: Biotin ...................................2.............................5g Preparation of Phosphate Supplement: Add components to distilled/deionized water and bring volume to ................. ...9....................99...............0g MgCl2·6H2O.............. Filter sterilize and add aseptically to sterile basal medium..............................................1............. Cool to 25°C while sparging with 80% N2 + 20% CO2........... Gently heat and bring to boiling.............02g Tris(hydroxymethyl)aminomethane buffer (0....................0mL: NaCl .......................................... 0.. Pour into sterile Petri dishes or leave in tubes.0....4g CaCl2·2H2O....... Boil for 5 min.............1..0.........5g L-Isoleucine.................. 0.0....... Filter sterilize................................. Adjust medium to pH 7..................3...0g CaCl2·2H2O..... to distilled/deionized water and bring volume to 990.........................................................0g Yeast extract ... Mix thoroughly......... 15.......... 1.............0L pH 7........02g KCl.....0......... 0....0mg distilled/deionized water and bring volume to 1.......................05g L-Methionine.10.... Cool to 50°C..........................................................0mL Preparation of Modified Hutner’s Basal Salts: Dissolve the nitrilotracetic acid first and neutralize the solution with KOH........ Aseptically add 10....... Autoclave for 15 min at 15 psi pressure–121°C.........................0g L-Leucine ...5g KCl ..0mL of vitamin solution........ Artificial Deep Lake Medium Composition per liter: NaCl ......... 180................... 0..............0.......0mL Phosphate supplement .................................... 20..............................................05g L-Glycine... Mix thoroughly....... Mix thoroughly.................0mL pH 7............. except vitamin solution..........5g L-Tyrosine ..................................................... Artificial Organic Lake Peptone Medium Composition per 1001........1g L-Threonine...........5g KH2PO4 ...0L............................................0........................................ 0......................................... 1.............. except Na-acetate solution.............................................................................0L of Tris buffer................ Store at 5°C.. Adjust pH to 1...............................025g Na2B4O7·10H2O...................................................................0g Noble agar.0g Yeast extract............................................................................... 0. 0........................................ Aseptically and anaerobically inject 0..........29..............................1g L-Serine ..0mL.4 ± 0.......20....... 1....................... ............ 0.......... Add aseptically to sterile modified Hutner’s basal salts solution...........02m solution.........0g Vitamin solution........... 0.................... Preparation of Vitamin Solution: Add components to Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture............1g FeSO4·7H2O....0g Peptone ................1mL sterile Na-acetate solution and 0........................................ Equilibrate with the 80% N2 + 20% CO2 gas...........................................10..................................15g L-Arginine ............................................ 0.2 at 25°C Preparation of Medium: Add solid components to 1........... Autoclave for 15 min at 15 psi pressure–121°C...........................

................................................................ Mix thoroughly............... Filter sterilize.......... Use: For the cultivation of Xenococcus species.............................. except agar solution......0............................ 0........................ 3.................0...0mL........................................ ASTM Nutrient Salts Agar (American Society for Testing and Materials Nutrient Salts Agar) Composition per liter: Agar ....................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..... 15.......0mg Ferric ammonium citrate...0mL of sterile modified Hutner’s basal salts solution............. except modified Hutner’s basal salts solution... 0...10................................ Mix thoroughly.............. Cool to 50°C.......................5g Na2CO3 ...... such as Azotobacter species and cyanobacteria.................................. Autoclave for 15 min at 15 psi pressure–121°C......0g NaNO3 ....5g Yeast extract ...................................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........0mL of sterile artificial organic lake vitamin solution....0.. 3......................... 5................ Aseptically add 20......................0L............0..............9 and 7... 25.................................0L..0.................... phosphate supplement solution....... 20........................... Preparation of Medium: Add components.0g Preparation of Agar Solution: Add agar to glass distilled water and bring volume to 100...... that can utilize atmospheric N2 as sole nitrogen source...... Pour into sterile Petri dishes or distribute into sterile tubes. Autoclave for 15 min at 15 psi pressure–121°C..............................0mL.....0g CaCl2·2H2O.......................81g ZnSO4·7H2O......... 0.......................... Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.0L.......0................................ Autoclave for 15 min at 15 psi pressure– 121°C......... Autoclave for 15 min at 15 psi pressure–121°C..................................................... 0.... Gently heat and bring to boiling........................0mL. Aseptically distribute into sterile tubes or flasks.............................1mL FeCl3 (10% solution.......... Preparation of Medium: Add components......................................................................... Gently heat and bring to boiling.......2g pH 6.5g Sodium acetate .........................8 ± 0..............ASTM Nutrient Salts Agar 20.........................................0... Mix well and heat gently until dissolved...75g K2HPO4·3H2O............................ 0..2g K2HPO4 ............ 5........1mg Preparation of Artificial Organic Lake Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..........2........0g MgSO4·7H2O..0mL pH 7.................................................. For the isolation of cyanobacteria from marine habitats....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.............. Distribute into tubes or flasks..... 0... Artificial Organic Lake Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................... 10..........0L...................................5mg Vitamin B12 ............................... Mix well until dissolved..........................2.................. Autoclave for 15 min at 15 psi pressure–121°C..0mg Folic acid..............0mg Magnesium EDTA ..........................0......... Filter sterilize... Cool to 45°–50°C........... 5... Mix thoroughly........................................................... 0. Mix thoroughly.....0mg Nicotinamide............02g Citric acid....................................... ASN-III Agar Composition per liter: NaCl . Pour into sterile Petri dishes or leave in tubes..............0mg Biotin .......15................. to glass distilled water and bring volume to 900.................................0mg Cyanocobalamin .......................049g Na2MoO4·2H2O....................05mL pH 7...0g ............ Use: For the cultivation of Halomonas meridiana.... Pour into sterile Petri dishes or leave in tubes.............................0mg Riboflavin ....................... Store at 5°C...................... Gently heat and bring to boiling... Asticcacaulis Medium Composition per liter: Agar .. Bring pH to 7.............. and 1............... 2..................................................2 at 25°C 41 Agar Solution: Composition per 100..........0mg Calcium DL-pantothenate.. Warm to 45°–50°C..........75g CaCl2·2H2O................... Use: For a presumptive test medium in the differentiation of nonfermentative Gram-negative bacteria..........................1...0mg Thiamine·HCl ... Mix thoroughly....0mL....039g Preparation of A-5 Trace Metals: Add components to distilled/deionized water and bring volume to 1............5g pH 6.......... 0.0..............................0L...................0..... and vitamin solution....0µg Agar solution.................................................................0mL A-5 trace metals .. Use: For the isolation and cultivation of bacteria....... Asparagine Broth Composition per liter: DL-Asparagine....... Mix thoroughly.................................... Adjust pH to between 6........................... For use in the multiple-tube technique in the microbiological analysis of recreational waters.....................................2 ± 0.....................................................9–7..............1........ especially Pseudomonas aeruginosa..................................100......... 3.......... 2..............................0mL of sterile phosphate supplement solution................................................................... A-5 Trace Metals: Composition per liter: H3BO3 .......................0mL: Noble agar ... 0.................... to distilled/deionized water and bring volume to 959..........................0g Pancreatic digest of casein ..5g MgCl2·6H2O..........10............................................... Mix thoroughly......... 5......................... 15............3..............86g MnCl2·4H2O ....0g K2HPO4 ...3 ± 0..........2g MgSO4·7H2O .............................. 2........0g MgSO4·7H2O .... Distribute into tubes or flasks....222g CuSO4·5H2O...........................30.5g KCl....... Distribute into tubes or flasks......1.... Mix thoroughly........ Aseptically add agar solution............................15............079g Co(NO3)2·6H2O................0g Mannitol....................2g MoO3 (10% solution)............................................. Use: For the isolation and cultivation of Asticcacaulis species........................ Ashbey’s Nitrogen-Free Agar Composition per liter: Agar ....... 0.................

.......0 ± 0.....0g Glucose.................................................10..............................50................ 6..........................................................0mL of sterile aureomycin solution........ Mix thoroughly............5mg Preparation of Trace Metal Solution: Add components to distilled/deionized water and bring volume to 100.................................................. Put mixture into a sterile blender container........................ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................................................................... Adjust pH to 7............ Autoclave for 15 min at 15 psi pressure–121°C........................... 5..................................... 1............ Gently heat and bring to boiling........5g Rose Bengal........ Aseptically add 200...................................................................2 at 25°C Agar .............0mg ZnCl2 ............... Atlas Oil Agar Composition per liter: Preparation of Medium: Add components................ Mix thoroughly........................................7g K2HPO4 ........... Autoclave for 15 min at 15 psi pressure–121°C.............................. Cool to 45°–50°C...........................0mL: H3BO3 .....0mg pH 6.........0M...................................................................................... Bushnell-Haas Agar: Composition per 990.........0mL Chelated iron solution .......5..................1. Mix thoroughly.............0mL Trace Metal Solution: Composition per 100...0mg CaCl2·2H2O .....................4 ± 0.....................................................1........... 0......................................0g MgSO4·7H2O..................6g Preparation of Chelated Iron Solution: Add EDTA to distilled/deionized water and bring volume to 100...........................1................................. 2.... 40..............................................................................0mL Trace metal solution...................................... 5....................................................................................................0mL of cooled.. Cool to 60°C....... 4........42 ASW Medium KH2PO4 .......... Gently heat and bring to boiling........ 1..........................0...........0mL Oil .......0.........................0mL............1......................... Mix thoroughly.............0g MgSO4·7H2O...................0mg CoCl2·6H2O ..... except aureomycin solution.......0mL..... Mix thoroughly......990... Pour into sterile Petri dishes or leave in tubes..............10...... and bring volume to 1..0g KH2PO4 ....0mL: Use: For the cultivation and enumeration of fungi isolated Use: For the cultivation of Porphyridium purpureum........... Gently heat and bring to boiling......................... 14................ 27.......07g Preparation of Aureomycin Solution: Add aureomycin·HCl to distilled/deionized water and bring volume to 200..................0mL.............................. Use: For determination of the susceptibility of plastics to Preparation of Medium: Filter sterilize oil...........5g KNO3 .........05g CaCl2·2H2O .................. to distilled/deionized water and bring volume to 800......... Pour into sterile Petri dishes............0g NH4NO3.................................... 0.0..0g MgSO4·7H2O ............20..................................... 15............................................................1........ Mix thoroughly.... Pour into sterile Petri dishes or distribute into sterile tubes..........0................0L............0mL: FeCl3·4H2O .....................20... Mix thoroughly.1........................... Use: For the cultivation of bacteria that oxidize ammonia.5 ± 0.................... Autoclave for 15 min at 15 psi pressure–121°C...... Distribute into tubes or flasks..........2 at 25°C Aureomycin Solution: Composition per 200... 37.......................................6......04g Tris-HCl buffer (1............................................... 200..........0mg EDTA .................................................. Mix thoroughly........................... 0...........0mL.............. Bushnell-Haas agar ........................... 1........1............... Distribute into tubes or flasks.....................6g MgCl2·6H2O......................... sterile Bushnell-Haas agar.....0mg (NH4)6Mo7O24·4H2O ................. 1.......... Gently heat and bring to boiling..................0..... Filter sterilize........................... 0......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........................ pH 7.... 240............0mL pH 7... AT5N Medium Composition per liter: CaCO3...............................30..............................................2 at 25°C from sewage and polluted waters.6)................................................................... ASW Medium (Artificial Seawater Medium) Composition per liter: NaCl ................................... 1..........0g (NH4)2SO4 ...0g Agar . Distribute into tubes or flasks......2g FeCl3 ..07g NaHCO3 .........0..........................................15.... fungal degradation.......035g Aureomycin solution .0g KH2PO4 ..... Aseptically add 10....................0g NaCl .0g K2HPO4 .............5g MgSO4 ...................0g KH2PO4 ......... Autoclave for 15 min at 15 psi pressure–121°C... 4..........0mL........0mL: Aureomycin·HCl .......................... Pour into sterile Petri dishes or leave in tubes.............................7g NH4NO3 ................. 0........................0mg MnSO4·H2O ...0mg MnCl2·4H2O............0mg FeSO4·7H2O.....................................................5g K2HPO4 .............................................10........ Add FeCl3·4H2O...................... Mix thoroughly............... Aureomycin® Rose Bengal Glucose Peptone Agar Composition per liter: Agar .................. ............. 2.....................0mL of sterile oil to 990.....0mg CuCl2·2H2O .......................6g CaCl2·2H2O........................... Mix thoroughly........0L...........................................................0L................................................... Blend on low speed to minimize the incorporation of air into the medium...... Gently heat and bring to boiling.......0..... especially those from wastewater.................................. Autoclave for 15 min at 15 psi pressure–121°C..............0g Peptone ...................20..............0mL pH 5.....................7g MgSO4·7H2O ........................................................................0mg ZnSO4.................................................... 60..... Use: For the cultivation and enumeration of hydrocarbonutilizing bacteria by direct plating of water and sediment samples... Chelated Iron Solution: Composition per 100....0mg Preparation of Medium: Add components to tap water and bring volume to 1.........................0mg KHCO3 ...............

................... Agar ........................ 33....07g Preparation of Stock Solution: Add components to metals and disposal must be highly diluted.....................................80mg CuSO2·5H2O ................................... Allow to stand for 23 days so that pH adjusts itself to 7............... FeSO4·7H2O...0.5g MgSO4·7H2O ..........5......... Azide Broth (Azide Glucose Broth) (Azide Dextrose Broth) Composition per liter: Pancreatic digest of casein ...................2 at 25°C Stock Solution: Composition per liter: 43 Adjust pH to 8........0mg Sheep blood.5....0mL pH 7.......................................5............... to distilled/deionized water and bring volume to 950.................................................. Use: For the cultivation of autotrophic Nitrobacter spp.......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....0g Stock solution...........................0g KH2PO4 ........................0........ Aseptically add 50........................... 43.. 43............................8mg CuSO4·5H2O ..............5g CaCO3 ............ Trace Elements Solution: Composition per liter: (NH4)Mo7O2 ........................ Mix thoroughly....................................................5.........................................1mg MnSO4·2H2O .. 33................... Mix thoroughly...6.......................................0g Peptic digest of animal tissue ..............................................0mL Trace elements solution .................................0mL of sterile defibrinated sheep blood..........5 ± 0..............10mg FeSO4·7H2O....0mL................ 97............................0g Glucose.. Pour into sterile Petri dishes or distribute into sterile tubes......1.......... and Oxoid Unipath............ 50.........5g ............ Pour into sterile Petri dishes or distribute into sterile tubes......................................................07g Preparation of Stock Solution: Add components to distilled/deionized water and bring volume to 1......................2....0g KH2PO4 .......... Autotrophic Nitrobacter Medium (LMG Medium 247) Composition per liter: NaNO2 .............4mg ZnSO4·7H2O .....0mL pH 7.................5g MgSO4·7H2O ............. 2......... 1............7.......................... Mix thoroughly..0L.....5.............. Autoclave for 15 min at 15 psi pressure–121°C.............2 at 25°C Trace Elements Solution: Composition per liter: Source: This medium is available as a premixed powder from BD Diagnostic Systems........................................ sheep blood.........................0mL pH 7............................ Adjust pH to 8.............................. Cool to 45°–50°C.. Autoclave for 15 min at 15 psi pressure–121°C......6 with NaOH...10mg H3BO3 ............................ Azide Blood Agar Composition per liter: distilled/deionized water and bring volume to 1..............5..6. and staphylococci from specimens containing mixed flora and from nonclinical specimens such as water and sewage.................................... 97... Preparation of Medium: Add components..... Mix thoroughly.........0L.......... Mix thoroughly.....................0g Pancreatic digest of casein ....... Azides also react with metals and disposal must be highly diluted.....................................3mg H3BO3 ................................... Also used for the isolation of Streptococcus pneumoniae and Erysipelothrix rhusiopathiae........................................0mL of sterile defibrinated sheep blood...2 at 25°C Stock Solution: Composition per liter: NaCl ....6................................. 0.............................................. defibrinated....................................................................15.3...............0mL pH 7. 5......0mL Trace elements solution ................................................................... Cool to 45–50°C.........0mL............ 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...0L....0g Stock solution......2 ± 0.......0g NaCl ............ Allow the medium to stand for 2-3 days so that the pH can adjust itself to pH 7......... 437.........0g NaN3 ......................................... 0..2g Sheep blood..........5g NaCl ................30mg ZnSO4·7H2O ... Mix thoroughly......7....0L. 1........... Aseptically add 50............ Allow tubes to cool in a slanted position................... 5........................................ Mix thoroughly..................0g Pancreatic digest of casein ........... 2......... 25.....................4-7............................................................15............................... Autoclave for 15 min at 15 psi pressure–121°C......................0L..... Mix thoroughly........ Azide Blood Agar with Crystal Violet (Packer’s Agar) Composition per liter: Agar ...............00mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1............ Azides also react with NaCl ...........1mg (NH4)6Mo7O24·4H2O .................... defibrinated......3.................................... Distribute into tubes or flasks....... 0................................... Use: For the isolation and enumeration of fecal streptococci from nonclinical specimens such as water......0mg Caution: Sodium azide is toxic.5 ± 0..................................................4-7.........0g NaCl . 39............2 at 25°C Caution: Sodium azide is toxic..................9g Crystal Violet...................0g Peptic digest of animal tissue ........ Preparation of Medium: Add components..100.... 49........................... 50.................................100... Distribute into tubes or flasks.........0g NaN3 .....................................................................40mg MnSO4·H2O ....... 25. 37......... Allow tubes to cool in a slanted position............................... 0......................... Gently heat and bring to boiling......................................0g Beef extract .. to distilled/deionized water and bring volume to 950....0g Beef extract ......... except ponents to distilled/deionized water and bring volume to 1.. except sheep blood.........1..................... Gently heat and bring to boiling................. Autoclave for 15 min at 15 psi pressure–121°C.....................5g CaCO3 ........Azide Broth Autotrophic Nitrobacter Medium (DSMZ Medium 756c) Composition per liter: NaNO2 ....................15....... Use: For the cultivation of Nitrobacter winogradskyi.............................................................0L..........2 ± 0..........................................................

...................................................................0g DL-malic acid........0................................................................................. Adjust pH to 6.......... Azides also react with metals and disposal must be highly diluted..........................0mg Na2MoO4·2H2O........... Gently heat and bring to boiling............5mg pH 6.............................0...... 2.......................5g).066g Preparation of Ferric EDTA Solution: Add ferric EDTA to distilled/deionized water and bring volume to 10.......5g NaN3......................8 ± 0................. Use: For the detection and enrichment of fecal streptococci in water and sewage...0...................... 5............... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............. Add other components.........................0g malic acid to 500...............1......0L...0mL Phosphate buffer solution...0mL...........................................0mL..........................1.....................1g CaCl2 ......15....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........ Rothe) (Azide Dextrose Broth..................0mL Ferric EDTA Solution: Composition per liter: Ferric EDTA ......................4...............5g Caution: Sodium azide is toxic... Autoclave for 15 min at 15 psi pressure–118°C.................. 4........ 250........................8 ± 0..8.......0 with KOH (approximate amount of 4.2.....2g Fe EDTA..........8 ± 0...................................0mL..............0mL pH 6..................................2g pH 7.......5g MgSO4·7H2O.0mL.........................................................0g Glucose ..........................2......................... Mix thoroughly..................0g MgSO4·7H2O......................................7g KH2PO4 .................0mL distilled/deionized water......... Preparation of Medium: Aseptically combine 750............................... Source: This medium is available as a premixed powder from Oxoid Unipath................................................ 750.............. Na2HPO4·2H2O ............... Distribute into tubes or flasks.............0mL sterile phosphate buffer solution..................... Preparation of Medium: Add components to distilled/ Phosphate Buffer Solution: Composition per liter: deionized water and bring volume to 1............................................. Use: For the detection of enterococci in water and sewage............................. Rothe (Azide Glucose Broth.............. 0....................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............20........................................ Rothe Beef extract ..0L. Adjust pH to 6...................... Use: For the detection and enrichment of fecal streptococci in water and sewage.....2 at 25°C Solution A: Composition per 750..................... Mix thoroughly................................0mg Na2MoO4·2H2O.........................0g Sodium citrate .0g KH2PO4 ....0L.......................5g NaCl .... Cool to 25°C...........................................................................................................2..............44 Azide Broth......................................0.................. 5................... 20....2g CaCl2 ......0g KOH ..........................................................8...0L.................................................................... Azides also react with metals and disposal must be highly diluted... 2. 5................................................................... Gently heat and bring to boiling.. 10........................... Mix thoroughly.0L........ 10.............1mg NH4Cl .........5g NaN3..............0 ± 0.......... Distribute into tubes or flasks............2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems......................................... 1......5......................... Rothe) Composition per liter: Azoarcus Medium (LMG Medium 202) Composition per liter: Solution A ...66....... Agar ...... Autoclave for 15 min at 15 psi pressure–121°C....... Also used in the multiple-tube technique as a presumptive test for the presence of fecal streptococci.0mg MnSO4·H2O.................0g Beef extract ........................0...0mL.....2.......0g NaCl ....................... Prepare double-strength broth for samples larger than 1.....8g KH2PO4 .......................... 5...................25g pH 7......................0......... Prepare double-strength broth for samples larger than 1......................................7g NaN3................. 0..... Mix thoroughly....10..............8...... Mix thoroughly..0g Yeast extract ........................................ 0...2 ± 0.................0g K2HPO4 ......... Autoclave for 15 min at 15 psi pressure–121°C...1g K2HPO4 ........................................... Adjust pH to 7.0mg Biotin ............................ Gently heat and bring to boiling........4..... 4............................................0mL sterile solution A with 250.................10...... Peptone............... Prepare double-strength broth for samples larger than 1....5g KOH ....................1.....................................................0g K2HPO4 ................. Distribute into tubes or flasks.........2 at 25°C Caution: Sodium azide is toxic....0g Yeast extract............2g pH 6.....................0mg MnSO4·H2O................................. 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... Cool to 25°C........ Autoclave for 15 min at 15 psi pressure–121°C...........0mg Ferric EDTA solution ................... Caution: Sodium azide is toxic..........5g KH2PO4 ...................1.....0g Glucose ................ Gently heat and bring to boiling.............. metals and disposal must be highly diluted.....................2g NaCl ................................................................... Use: For the cultivation of Azoarcus indigens........ 20........... Adjust pH to 6......... Aseptically distribute to sterile tubes or flasks..................................3..... Azide Broth...................... Mix thoroughly. Mix thoroughly............................... Distrib- ............................................. 5..5....... Autoclave for 15 min at 15 psi pressure–121°C....0g NaCl .................0.................... Azide Citrate Broth Composition per liter: Azoarcus VM Medium (LMG Medium 252) Composition per liter: Pancreatic digest of casein .....................................0mL: Malic acid ............................ Bring volume to 750.....

....... 0........... Azospirillum lipoferum Medium Composition per liter: Calcium malate.................. Distribute into tubes or flasks...........02g FeCl3 ................... Use: For the isolation and cultivation of Azospirillum lipoferum................................5g deionized water and bring volume to 1.... Azospirillum Medium Composition per liter: Sodium malate.. 20...............6g K2HPO4 ....................................................0g Peptone..............0.............. Gently heat and bring to boiling..........01g Na2MoO4·2H2O....................5g KH2PO4 ............02g pH 6...................................................... Pour into sterile Petri dishes or leave in tubes.........................2 at 25°C Bromothymol Blue Solution: Composition per 100......... Mix thoroughly............................... 0.........0mL: Bromothymol Blue ................................................ Mix thoroughly... Distribute into tubes or flasks...1........................................................2g MgSO4·7H2O ..... Mix thoroughly........................ Bromothymol Blue to 100............................. Distribute into tubes or flasks....................... Autoclave for 15 min at 15 psi pressure–121°C..................9 ± 0.......... 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.. Gently heat and bring to boiling.............................................................................................................................0g Beef extract .............................................................2g KH2PO4 ............................................ 5......................9 ± 0.... Mix thoroughly....... Autoclave for 15 min at 15 psi pressure–121°C...0........................................................................75g KH2PO4 ............. 5.... 1.....................................0....................... 0....... Distribute into tubes or flasks.............0g K2HPO4 ........................................................................15g K2HPO4 ............. Distribute into tubes or flasks.... Azotobacter Agar Composition per liter: Agar ... Autoclave for 15 min at 15 psi pressure–121°C..........0mL Preparation of Bromothymol Blue Solution: Add Bromothymol Blue to 10....... Pour into sterile Petri dishes or leave in tubes...0mg Bromothymol Blue solution...................................0................................................................................02g pH 6..............0...........................................0L..................................2 at 25°C Bromothymol Blue Solution: Composition per 10............. amazonense....5g MgSO4·7H2O....2N KOH.... 10...................................10...0................ 0............. Autoclave for 15 min at 15 psi pressure–121°C......5g Ethanol ........................0...........02g FeCl3 ........15.....2g CaCl2·2H2O................................................................ 0................0g Agar ........................0.......8 ± 0............... Use: For the cultivation and maintenance of Azorhizophilus paspali......................................... Mix thoroughly.................. Use: For the cultivation of Azospirillum lipoferum.................................. Mix thoroughly...... 0...............2............0mg Bromothymol Blue solution ...................0g Agar ...... 20.. 0.................................................20...................0g MgSO4·7H2O............................................ 5.. Pour into sterile Petri dishes or leave in tubes............... Mix thoroughly..................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...........0L................................... Pour into sterile Petri dishes or leave in tubes.................. Azospirillum amazonense Medium (LGI Medium) Composition per liter: Sucrose............... 0...........0mL: Bromothymol Blue ...............................................1................................ Mix thoroughly.............................................................................8g MgSO4·7H2O.. 0..1g CaCl2·2H2O ....0.......1g CaCl2·2H2O .............................................. 0.......Azotobacter Agar 45 ute into tubes or flasks....0g MgSO4 ...................2g K2HPO4 ............................................... Preparation of Medium: Add components to distilled/ Preparation of Bromothymol Blue Solution: Add deionized water and bring volume to 1....... 5......................................... deionized water and bring volume to 1.0.. 15....4g MgSO4·7H2O...................... 1.........................0L.............5 ± 0....0................................................................................................1g NaCl ....... Agar ................................................ 2.................................................0mL of ethanol..............................0g Na2MoO4·2H2O......0g Sucrose................75g KH2PO4 ...............15................24g Glucose..... 0.. 5......................... Gently heat and bring to boiling........1g Yeast extract ................................5g CaCl2·2H2O ...................2g FeCl3·6H2O.................0L........ Autoclave for 15 min at 15 psi pressure–121°C...........0L.. Use: For the cultivation and maintenance of Azorhizobium caulinodans....................... Azorhizophilus paspali Agar Composition per liter: Agar ...0...........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......0.05g FeCl3 ................................. Gently heat and bring to boiling...5..............................................0g Agar .............. Autoclave for 15 min at 15 psi pressure–121°C.......0mL of 0..0mL pH 6..02g Na2MoO4·2H2O. Distribute into tubes or flasks.........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........................0g K2HPO4 ....0 ± 0.......0g Yeast extract.0............0..........01g Na2MoO4·2H2O ....................................................... Pour into sterile Petri dishes or leave in tubes................0mL pH 6...............................................0g Sucrose.........................................................................................................01g pH 6..............0L............................................ Autoclave for 15 min at 15 psi pressure–121°C..........0............................ Preparation of Medium: Add components to distilled/ Use: For the cultivation of Azospirillum species isolated from roots......0g .... 0................................5....

.............. Azotobacter Agar...... Modified II Composition per liter: Use: For the cultivation and maintenance of Azotobacter beijerinckii. 0.....................0mL..... Filter through Whatman filter paper..........02g Na2MoO4........................ 0........................0.................................................46 Azotobacter Agar............................. 15.......................5........................................ 0..............10............. 20...................................0..........................................0mL pH 7....................10......0mg FeCl3 ...0mg FeCl3 ............1............0mg pH 6.. 0...0g K2HPO4 ........ 10...................2g NaCl ............ Adjust pH to 6..................... 0.............. 0........2g KH2PO4 .................0.......0mg Na2MoO4·2H2O ..................0g Sucrose.. 0...1g K2HPO4 ....... Mix thoroughly.............................2....................................3 ± 0.................. 0....................................0µg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........0.0L..................0mg Na2MoO4·2H2O ................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... Modified I Composition per liter: Agar ....... species and Beijerinckia derxii......................................................15g CaSO4·2H2O ........................... 0.................................. and Derxia gummosa.........0...........1g MgSO4·7H2O...............................0mg FeCl3 ........9g CaCl2·2H2O .................0g MgSO4·7H2O.......................... to tap water and bring volume to 1........................................................................... 0.0g MgSO4·7H2O .................................................... 15................ Modified II Composition per liter: Sucrose .. Adjust pH to 7...0L..................2....................................................... Azotobacter chroococcum... 1........ including Azomonas species..................................... 15.........1g K2HPO4 ..0mL: African Violet soil ............ 0........................................ Azotobacter species....... Sucrose..........5g Na2CO3 ...............0.......... Use: For the cultivation and maintenance of Azorhizophilus paspali.....................0mg Na2MoO4·2H2O.................0L.............................................. Use: For the cultivation of a variety of bacteria.................... Azotobacter Broth....................................................................................................... 0............................ Pour into sterile Petri dishes or leave in tubes........................ species............. Autoclave for 60 min at 15 psi pressure–121°C......... Autoclave for 15 min at 15 psi pressure–121°C......... Preparation of Medium: Add components.................................1g KH2PO4 .......................................................... Distribute into tubes or flasks....... Azotobacter vinelandii............................................................... FeSO4·7H2O ...................10... Modified I Sucrose......0mg pH 7...............................0L...... Mix thoroughly...0........................0. Azotobacter Basal Agar Composition per liter: Use: For the cultivation of Azotobacter species..... Pour into sterile Petri dishes or leave in tubes..................................................15g MgSO4·7H2O .... including filtered soil extract..0........................................................0L.............. Mix thoroughly.... Distribute into tubes or flasks.. Mix thoroughly..............................................................................0g Agar .. 10.....0g MgSO4·7H2O .................................... 0............05g CaCl2 ...................................................5.................... 1..................................... Autoclave for 15 min at 15 psi pressure–121°C....................................0g CaCO3......................... Pour into sterile Petri dishes or leave in tubes..2g KH2PO4 ............................ 0.2 ± 0...1.............................................................................................................05g CaCl2 ................ Distribute into tubes or flasks..........0g Glucose..2...............0g KH2PO4 .............................................. Distribute into tubes or flasks...... Gently heat and bring to boiling.............. Autoclave for 15 min at 15 psi pressure–121°C............0g MgSO4·7H2O...............2g KH2PO4 ........................................................................ Mix thoroughly............. 100..................................... Distribute into tubes or flasks...... 2......... 0.... Adjust pH to 7...................... Distribute into tubes or flasks............0mg Soil extract....................0mg pH 7............................. and others when a carbon source is added...........................................2 at 25°C Soil Extract: Composition per 200............... Gently heat and bring to boiling......................002g FeCl3 ....... Modified I Composition per liter: Sucrose ...........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 ...... Autoclave for 15 min at 15 psi pressure–121°C...................15g CaSO4·2H2O.............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.............................................................................................20......... Autoclave for 15 min at 15 psi pressure–121°C.........0mg Na2MoO4·2H2O.................................0mg pH 7........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................... 0...............02g Na2MoO4 ...........................1...0..................... 1.... Gently heat and bring to boiling................................. Pour into sterile Petri dishes or leave in tubes............................ Azotobacter Broth Composition per liter: Glucose...................02g Na2MoO4................... Azotobacter Agar......05g CaCl2 .........................................................0..........0g KH2PO4 .. Agar ....5g Preparation of Soil Extract: Add components to tap water and bring volume to 200..................... Gently heat and bring to boiling......2 ± 0.....0L......................................................................................0......................2g Azotobacter Broth........................................................1g FeSO4·7H2O . 2.................05g CaCl2 ...............2 ± 0....02g Na2MoO4 ...10................................. Pour into sterile Petri dishes or leave in tubes................ Autoclave for 15 min at 15 psi pressure–121°C.. Gently heat and bring to boiling........5........................2 ± 0.... 1.........0g Glucose .0g K2HPO4 ............ Mix thoroughly....................15g ..........15g K2HPO4 ....................................2....................................... Gently heat and bring to boiling............................................................ 1.................... 10...........

..25..................................10..................................0......0mL of sterile glucose solution 47 and 25.............. Mix thoroughly....................... Azotobacter Medium (ATCC Medium 1771) Composition per liter: Agar ................................................... Pseudomonas azotocolligans................. Filter sterilize...............2g K2HPO4 .......0mg Na2MoO4·2H2O................................................................................... Distribute into tubes or flasks.................................2 at 25°C 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................................................0mg pH 7..................0.... Mix thoroughly... Beijerinckia derxii............. 0...0mg Na2MoO4·2H2O ............... Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C.........................4–7......... Preparation of Medium: Add components.......2g CaSO4·2H2O................................................2mg pH 7........................................... Pour into sterile Petri dishes or distribute into sterile tubes.....................0g K2HPO4 .......................................................................................... Gently heat and bring to boiling................... Distribute into tubes or flasks..0..0mg FeCl3 ........ Adjust pH to 6......0L.. Warm to 50°–55°C............................................. 0.. Mix thoroughly.......................................................0............................................................................................1..0............................. Mannitol Solution: Composition per 25......................... 1......05g CaCl2 ............................. Gently heat and bring to boiling.....................0mL of sterile mannitol solution... Filter sterilize....1g KH2PO4 ................................................0.... Use: For the cultivation and maintenance of Azotobacter chroococcum................................... including Azotobacter species....2.. Pour into sterile Petri dishes or leave in tubes................................................................ 15......................................0g KH2PO4 .............2 ± 0.............. Use: For the cultivation and maintenance of a variety of bacteria................2 ± 0............................ Mix thoroughly..........................................0g K2HPO4 ....................3 ± 0.......0.......................5...8g Yeast extract ...............0g Agar ................................... 0.......... Cool to 50°– 55°C... 5..... 0.........25..................098g NaCl .....................2.. 0....0mL: Mannitol........22g CaSO4·2H2O......................... Autoclave for 15 min at 15 psi pressure–121°C................. except glucose solution and mannitol solution......0L............... 0......0L......................................................................... Mix thoroughly......... Distribute into tubes or flasks.................................................. 0.... 0..........0mg Glucose solution ..........2 at 25°C Glucose Solution: Composition per 25....... 0......................0mg FeCl3 .............. Autoclave for 15 min at 15 psi pressure–121°C.......................2 ± 0.........1g FeSO4·7H2O.. 0...........0g CaCO3 ..2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............................... including Azomonas species...... Pour agar medium into sterile Petri dishes or leave in tubes................... Mix thoroughly...............................2g MgSO4·7H2O........ Distribute into tubes or flasks.................................................8g MgSO4·7H2O ............... Azotobacter chroococcum Agar Composition per liter: Agar ............0g CaCO3 . Azotobacter species.......................................................... 20.0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 25............................................................ Autoclave for 15 min at 15 psi pressure–121°C............................................................05g pH 7.. and Rhodococcus erythropolis............ Pour into sterile Petri dishes or leave in tubes.......... 0.....2 ± 0.. Gently heat and bring to boiling.2g FeCl3·6H2O ............... Gently heat and bring to boiling...........................0g Glucose .............. 2.... 5...............1g MgSO4·7H2O .........1.....0g K2HPO4 ... Use: For the cultivation and maintenance of Azotobacter species...................................................... ......0mg pH 6.....................................01g Na2MoO4·2H2O ....0g Preparation of Mannitol Solution: Add mannitol to distilled/deionized water and bring volume to 25....................................0......5g KH2PO4 .............0.. to distilled/deionized water and bring volume to 950........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....058g FeSO4·7H2O .................0mL.........................................0L......................15.......................................0L................................ Gently heat and bring to boiling...... 20...................................15.....20.....0mL: D-Glucose...... Azotobacter Medium Composition per liter: Agar ....................1g MgSO4·7H2O... 5.6 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................................0........................................... Azotobacter Medium (ATCC Medium 14) Composition per liter: Sucrose .............................................................Azotobacter Medium MgSO4·7H2O ............... Autoclave for 15 min at 15 psi pressure–121°C.............................. 5.............................. Mix thoroughly... Pour into sterile Petri dishes or leave in tubes.................0g MgSO4·7H2O.... Azotobacter Medium (ATCC Medium 240) Composition per liter: Agar .......05g CaCl2 ... 0...............1g FeCl3 ................................... Mix thoroughly........................1g Na2MoO4·2H2O ........0........0g Glucose...0mL Mannitol solution ...................................0................................0mL pH 7........................... Use: For the cultivation of a variety of bacteria.................................0........9g CaCl2·2H2O.................0mg Na2MoO4·2H2O.....15g K2HPO4 .0mL.....0mg pH 7..... Aseptically add 25........5g KH2PO4 ....................... 20.....058g K2HPO4 ...............02g Na2MoO4....... 0.......................................15.............................................2g KH2PO4 ........ Warm to 50°–55°C............................................... Autoclave for 15 min at 15 psi pressure–121°C............................ 1.............................0................... Use: For the cultivation of Azotobacter species and Beijerinckia derxii.1.................0..0mL.............02g Na2MoO4·2H2O....... Distribute into tubes or flasks..................................

........2g FeSO4·7H2O ......0.0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100..... Azotobacter Supplement (ATCC Medium 11) Composition per liter: Agar ............... Aseptically add ......................................................... Glucose Solution: Composition per 100.................10................................15........................................ Mix thoroughly...0mL: African Violet soil................ to tap water and bring volume to 900....................................................................................................... 0............. Mix thoroughly..............................0.0mL............. 5.......0mL pH 7... Preparation of Bromothymol Blue Solution: Add Mannitol Solution: Composition per 100..............0mL of ethanol................. Glucose Solution: Composition per 100.................. Cool to 50°–55°C...........................................................................................................................0mg Bromothymol Blue solution...05g CaCl2 ...... 0.2g FeSO4·7H2O.........................5g Ethanol ........... Preparation of Medium: Add components.......... Cool to 50°–55°C.....................0mL Glucose solution ......................................0.............................................. Use: For the cultivation and maintenance of Azotobacter paspali.. 0.0........ Autoclave for 15 min at 15 psi pressure–121°C...... to tap water and bring volume to 900...2g KH2PO4 . Autoclave for 15 min at 15 psi pressure–121°C..........................................................................0mL: Preparation of Soil Extract: Add components to distilled/deionized water and bring volume to 200...0g MgSO4·7H2O................. Mix thoroughly......... 0..................................................0...........2g NaCl .................. Filter through Whatman filter paper.......... 20...................... 0. Gently heat and bring to boiling...... Adjust pH to 7......15g K2HPO4 .. Adjust pH to7.0mg Soil extract............ Azotobacter paspali Medium Composition per liter: Agar ..............48 Azotobacter paspali Medium Use: For the cultivation and maintenance of a variety of bacteria.. 0..... Autoclave for 60 min at 15 psi pressure–121°C......... Autoclave for 15 min at 15 psi pressure–121°C...........5g Na2CO3 .........2 at 25°C Azotobacter Supplement (ATCC Medium 12) Composition per liter: Agar .........0mL pH 6................................................... 100..... Aseptically add 100.............. except mannitol solution........2 at 25°C Soil Extract: Composition per 200..................................0mL...................0g K2HPO4 .0.....................0 ± 0........100...........................5..5.... 0.................................. Mix thoroughly.........................0mL: Glucose......................................0mL: African Violet soil ....5g Na2CO3 ..............0mL pH 7...............6 ± 0..... Mix thoroughly... Pour into sterile Petri dishes or leave in tubes................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1. Filter sterilize........02g Na2MoO4·2H20 ........................................................1.................20.............................0mL Mannitol solution ...0g K2HPO4 ..........15.........2g NaCl .............................20.........0g MgSO4·7H2O............ Azotobacter Supplement (ATCC Medium 13) Composition per liter: Agar ...........5g Bromothymol Blue Solution: Composition per 10.............................................................0mL Glucose solution .............2 at 25°C Soil Extract: Composition per 200..........................6 ± 0........ Mix thoroughly..0 ± 0................................................... 100....2g FeSO4·7H2O ................................ Autoclave for 15 min at 15 psi pressure–121°C........... Pour into sterile Petri dishes or leave in tubes.................................................2g NaCl ...................0mL FeCl3 (10% solution) ................0...... Cool to 50°–55°C.... Filter sterilize............... Preparation of Medium: Add components...100...... except glu- cose solution......0mL Bromothymol Blue to 10....0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100..........0.......... 0.......... Mix thoroughly..0L.0mL of sterile mannitol solution...............1................................... Mannitol . Filter through Whatman filter paper................0mL...............5g Na2CO3 .0mL........................ Autoclave for 60 min at 15 psi pressure–121°C...... Use: For the cultivation of Azotobacter species and Azomonas species...... Use: For the cultivation of Azomonas agilis and Azotobacter chroococcum.....................0g MgSO4·7H2O ...... 1.. 20.... Filter sterilize... Mix thoroughly.......................0mg Soil extract .0g MgSO4·7H2O ....................................... 100....... 20........................................................................................ including Azotobacter species...0mL of sterile glucose solution.......................... except glucose solution..............10.......6........ Distribute into tubes or flasks..........1mL pH 7..........0mL: Glucose .................6.......5g Preparation of Soil Extract: Add components to tap water and bring volume to 200....0mL: African Violet soil ....2 at 25°C Soil Extract: Composition per 200...0g Sucrose............................. 0...0mL........................................0mL.0g Preparation of Mannitol Solution: Add mannitol to distilled/deionized water and bring volume to 100.......................... Adjust pH to 6...............0mL................................................................0mL......... 1.....................0g CaCO3 ..0...............0g K2HPO4 .................................... 2............0mL: Bromothymol Blue .... Mix thoroughly.........5g Preparation of Soil Extract: Add components to tap water and bring volume to 200...........0..............0mL......................................................... Mix thoroughly..... Pour into sterile Petri dishes or leave in tubes. Filter through Whatman filter paper.......... 15.................... Preparation of Medium: Add components.. to tap water and bring volume to 900.................................................................................... Aseptically add 100............. 100... Autoclave for 60 min at 15 psi pressure–121°C........................................0mg Soil extract...

0.................................................. Autoclave for 60 min at 15 psi pressure–121°C............1g NiCl2·6H2O..... 49 Cellobiose Solution: Composition per 50......0mL..9–7....................0mL Wolfe’s mineral solution ....0mL of sterile cellobiose solution.. Cool to 50°–55°C....................................................... Adjust pH to 6............10.....0g MnSO4·2H2O........................................... Filter through Whatman filter paper.................0mL of distilled/deionized water...........................025g KAl(SO4)2·12H2O ..........0.............. Azotobacter vinelandii Medium Composition per liter: Sodium benzoate............... Autoclave for 15 min at 15 psi pressure–121°C..................100................ Autoclave for 15 min at 15 psi pressure–121°C......... Preparation of Medium: Add components.......................5g Preparation of Soil Extract: Add components to distilled/deionized water and bring volume to 200........................................ Use: For the cultivation of Azomonas macrocytogenes..................... 20......................... Aseptically add 100................................. 1...50......... Mix thoroughly.. Mix thoroughly.0g Nitrilotriacetic acid.......0mL...... Use: For the cultivation of Azotobacter vinelandii from water samples..............................5 with KOH.....................................................................................................................2 at 25°C Na2S·9H2O Solution: Composition per 10.............01g Na2MoO4·2H2O............. Mix thoroughly.. Mix thoroughly.0L.........................5................. 5............................. Autoclave for 15 min at 15 psi pressure–121°C..........................5g Mannitol.......0g K2HPO4 .5.............................0mL pH 6............................05g Resazurin .......................5................................................ Filter sterilize.......................0mL: Soil Extract: Composition per 200...0L.......0mg Nicotinic acid ....................0 with sterile HCl. except mannitol solution............................. Add distilled/deionized water to 1.................. and Wolfe’s vitamin solution.....0mg Riboflavin............................ 0....................................75g K2HPO4·3H2O........................................................................0mL Wolfe’s vitamin solution ........0.............0mg Folic acid .... Mix thoroughly.................................0mL......................................................................... 0................3mg Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500............ Aseptically and anaerobically distribute into sterile tubes or bottles...0mg Soil extract .......... Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl..0mL Na2S·9H2O solution.............0mL: Use: For the cultivation of Beijerinckia species...............2g FeSO4·7H2O.. Mannitol Solution: Composition per 100.........................BA Medium with Cellobiose sterile glucose solution................ Adjust pH to 6.......................................................0mL....... neutralize to pH 7............. 0.................0......2................5...... Cellobiose...........................................................................................................................3..................................................................0mg Calcium DL-pantothenate ..................5g NaCl ...............................1g CaCl2·2H2O.........5mg Cellobiose solution ..02g CuSO4·5H2O......................0g Yeast extract........ Mix thoroughly.......0.......................................0mg Vitamin B12.....0mL.............1g NaCl ... 0...........................................6g NH4Cl................ Mix thoroughly.......................................... MgSO4·7H2O......01g H3BO3 ...0........0............ Adjust pH to 6..5...................5g CoCl2·6H2O.....0mL of sterile mannitol solution........0mL of sterile Wolfe’s vitamin solution. to distilled/ deionized water and bring volume to 920...................1..............0mL pH 6........................... Wolfe’s mineral solution.0.... Distribute into tubes or flasks.........................................0mL: Mannitol......... 0... 0....................4g MgCl2·6H2O........ Add components.....0mL........ Preparation of Cellobiose Solution: Add cellobiose Agar ........................ Sparge with 80% N2 + 20% CO2 gas mixture. 10....2................................... Aseptically and anaerobically add 50......... ............................ Pour into sterile Petri dishes or leave in tubes.... 1........ 15.................... Mix thoroughly....... Mix thoroughly...........................0.............. BA Medium with Cellobiose Composition per liter: NaHCO3 ....... and 10.0mg Thiamine·HCl ................... Na2S·9H2O solution..................25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....................4.0L................. Add remaining components.....0mL of sterile Na2S·9H2O solution.0mL: Wolfe’s Mineral Solution: Composition per liter: African Violet soil.... Filter sterilize...................................... 0........ 2.......................................0.........01g Na2SeO3·5H2O ..................................................0mg p-Aminobenzoic acid .......0........................ except cellobiose solution..0......................................0g K2HPO4 ..........................0 at 25°C Na2S·9H2O ...... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas mixture...................0 ± 0..... Filter sterilize........... Use: For the cultivation of Caldicellulosiruptor lactoaceticus..............................0mL Mannitol solution ......2g NaCl ...0mL of sterile Wolfe’s mineral solution...........................0.................10.......... Pour into sterile Petri dishes or leave in tubes..8.....1g CaCl2·2H2O .......................................1g FeSO4·7H2O ..................................... 0............... 0.........0g Preparation of Mannitol Solution: Add mannitol to distilled/deionized water and bring volume to 100....... 1..... Autoclave for 15 min at 15 psi pressure–121°C....10...... 0........100.............................0mg Lipoic acid...................0g Azotobacter Supplement (ATCC Medium 15) Composition per liter: to distilled/deionized water and bring volume to 50.. Mix thoroughly..........0g MgSO4·7H2O .... Before use........5g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................0.................................................0.........................................1g ZnSO4·7H2O............. to tap water and bring volume to 900.........................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.5...............10.................. Sparge with 100% N2............................................. 10........ 0..............................1...........5g Na2CO3 ..................................................0mg Biotin ..............

............ Component III: Composition per 400.......0mL pH 7...........0mL: Component I ... Modified with 2.............0g Sodium citrate ...... Modified Composition per 1020.......................... Adjust pH to 7.. Use: For the cultivation and maintenance of Desulfotomac- ulum nigrificans...........................................................................................0g Preparation of Component I: Add components to distilled/deionized water and bring volume to 400............400..............................0g CaSO4 .......5g Ferrous ammonium sulfate solution ..... Adjust pH to 7..... 400..............0mL Component III... 2.....0...5...................................5.0mL: Component I ...................................................................... Adjust pH to 7..0g MgSO4 .............10......................... Filter sterilize............5.................5 ± 0..........0mL of sterile ferrous ammonium sulfate solution and sterile yeast extract solution....................................0g K2HPO4 ................ Mix thoroughly................................................0g CaSO4.................. Autoclave for 15 min at 15 psi pressure–121°C...................0mL: Preparation of Component III: Add components to distilled/deionized water and bring volume to 400......................5 ± 0............................ Autoclave for 15 min at 15 psi pressure–121°C.......0g CaSO4...1................. Mix thoroughly............1.. Aseptically distribute into tubes or flasks.. component II......................................5% NaCl Composition per 1020.............0mL...5g Preparation of Component II: Add K2HPO4 to dis- tilled/deionized water and bring volume to 200....................0mL...................0mL: Sodium citrate ............0g NH4Cl ..... Mix thoroughly.5.........5g Preparation of Component II: Add K2HPO4 to distilled/deionized water and bring volume to 200........................................... Mix thoroughly.................................................................................... Aseptically add 0............................................ 20............2 at 25°C Preparation of Component III: Add components to distilled/deionized water and bring volume to 400.......5g Yeast extract .............0g ......1.........5 ± 0...5g Preparation of Ferrous Ammonium Sulfate Solution: Add Fe(NH4)2(SO4)2 to distilled/deionized water and bring volume to 10..0mL pH 7............. Mix thoroughly...0mL: Yeast extract. Baar’s Medium for Sulfate Reducers.... Adjust pH to 7............................1........0mL: NaCl . Mix thoroughly..........................................400..............................5g MgSO4·7H2O ..........0mL...................0g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10. Baar’s Medium for Sulfate Reducers................................. Autoclave for 15 min at 15 psi pressure–121°C.....1mL of sterile ferrous ammonium sulfate solution to 5................0mL: Fe(NH4)2(SO4)2 .....1.... Autoclave for 15 min at 15 psi pressure–121°C.......................... Desulfobulbus....0mL: Sodium lactate..................... Mix thoroughly. Mix thoroughly......0mL of medium immediately prior to inoculation..... Preparation of Medium: Aseptically combine component I.......0mL Ferrous ammonium sulfate solution . 0........0mL: Fe(NH4)2(SO4)2 .. Mix thoroughly............ 3.................0mL Component II...... Adjust pH to 7.............3...... Autoclave for 15 min at 15 psi pressure–121°C...10..............0g NH4Cl......................... 1..0g Sodium lactate ......0g NH4Cl...0mL.................................... Component II: Composition per 200...... Aseptically add 10......... Preparation of Component I: Add components to distilled/deionized water and bring volume to 400...... 1. Gently heat and bring to boiling............. 0...........................................0mL: K2HPO4 .................0mL....................................2 at 25°C Component I: Composition per 400........... and component III. 200.........3............. 400...... Ferrous Ammonium Sulfate Solution: Composition per 10..............0g MgSO4 ...0g Preparation of Ferrous Ammonium Sulfate Solution: Add Fe(NH4)2(SO4)2 to distilled/deionized water and bring volume to 20. Autoclave for 15 min at 15 psi pressure–121°C..25....... and Thermodesulfobacterium species.2.... Yeast Extract Solution: Composition per 10...0mL volumes into tubes under 97% N2 + 3% H2.. 1.....................0g Ferrous Ammonium Sulfate Solution: Composition per 20....0mL.............0mL pH 7...................5.. Mix thoroughly.50 Baar’s Medium for Sulfate Reducers Baar’s Medium for Sulfate Reducers Composition per liter: Component III: Composition per 400...................... K2HPO4 .............0mL Ferrous ammonium sulfate solution ... Desulfotomaculum.................0mL......200.. Mix thoroughly............... to tap water and bring volume to 980.............. Sodium lactate ................. Autoclave for 15 min at 15 psi pressure–121°C.......................... 1............5. Cool to 45°–50°C... Adjust pH to 7..... 5..1..... Use: For the cultivation and maintenance of Desulfovibrio.......0mL.......... except fer- rous ammonium sulfate solution and yeast extract solution.........0mL: Component II: Composition per 200......2 at 25°C Component I: Composition per 400........0mL Yeast extract solution ........ Autoclave for 15 min at 15 psi pressure–121°C........................... 1.........5....5g Yeast extract ... Distribute 5........................................ Autoclave for 15 min at 15 psi pressure–121°C...............0mL Component III ...0mL...........0g Ferrous Ammonium Sulfate Solution: Composition per 20........................ Preparation of Medium: Add components...............................................0mL...........0mL Component II .. Add medium to tubes while still warm to exclude as much O2 as possible.... 2... 0. Mix thoroughly...............................................0mL: Fe(NH4)2(SO4)2 ....................................................20.........

..0g MgSO4·7H2O.....................20..........03g NiCl2·6H2O.................... Pour into sterile Petri dishes or leave in tubes.......1..............01g Preparation of Solution B (Trace Elements Solution SL-6): Add components to distilled/deionized water and bring volume to 1...............................0mL of sterile solution C......................0mL....... bring volume to 20.. Gently heat and bring to boiling. Gently heat and bring to boiling....................0.......................... Prepare a separate agar solution by adding 20..................... Preparation of Medium: Aseptically combine 500.... Adjust pH to 3........ 1.................. Use: For the cultivation of Desulfovibrio africanus and other Desulfovibrio species that prefer 2...0 at 25°C Preparation of Medium: Add components...1...........1g Na2MoO4·2H2O.......0 ± 0...5g CaCl2·2H2O .........................................0–4............... Aseptically adjust pH to 3.........0g Yeast extract.02g CuCl2·2H2O...........0mL..... Distribute 5........ Preparation of Medium: Aseptically mix 500.0mL of medium immediately prior to inoculation................................... 0...0g MgSO4·7H2O ....... Agar ...........5% NaCl...0mL of distilled/deionized water.........2g ZnSO4·7H2O.......... component II......0g Yeast extract ....0g Yeast extract.........0L.......2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500........500............ Mix thoroughly........ 5....0 at 25°C Solution A: Composition per 500.. Use: For the cultivation and maintenance of Bacillus (Alicyclobacillus) acidocaldarius.5g CaCl2·2H2O............................ 0................0mL. Mix thoroughly......... Autoclave for 15 min at 15 psi pressure–121°C..... Pour into sterile Petri dishes or distribute into sterile tubes........ Autoclave for 15 min at 15 psi pressure–121°C.......0.... Bacillus acidoterrestris Agar Composition per 1001..........1mL of sterile ferrous ammonium sulfate solution to 5.....0........................0g Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 500......................... Cool to 50°–55°C.......0..............................25g CaCl2·2H2O ....................0mL.......... Bacillus Agar Composition per liter: Agar ............................ Gently heat and bring to boiling......... Add medium to tubes while still warm to exclude as much O2 as possible......................07g FeCl3 .........0mL volumes into tubes under 97% N2 + 3% H2......................0mL: Solution A ...........................0mL............... except agar and glucose..............3g Glucose..........25g (NH4)2SO4 ...0........... Aseptically combine both solutions..............................0g (NH4)2SO4 ........................... Modified 51 Preparation of Ferrous Ammonium Sulfate Solution: Add Fe(NH4)2(SO4)2 to distilled/deionized water and Solution C: Composition per 500...........................................0g Yeast extract .................................................0g Glucose....... Pour into sterile Petri dishes or distribute into sterile tubes........... Alicyclobacillus acidoterrestris....................3g CoCl2·6H2O............................0........................................5.... 30..........37g MgSO4·7H2O..0mL of solution A and 500..0–4.......................................0mL of sterile solution B.............0mL Solution B ...............0g KH2PO4 ..... 0..............0g KH2PO4 ........................0mL Solution B (Trace elements solution SL-6)....................1..........................0mL: Agar ..0.. 0..20........................................0.. Mix thoroughly............................. to distilled/deionized water and bring volume to 500... Mix thoroughly.......... 0....... Cool to 50°–55°C....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 500...0...............02g pH 4.......................................... 3............................0....2 at 25°C Solution A: Composition per 500............................................................... Use: For the cultivation and maintenance of Bacillus acidoterrestris....................................0–4............................ Adjust pH to 4............................. 0...................................................... Cool to 50°–55°C.................. Use: For the cultivation of acidophilic Bacillus species such as Bacillus acidocaldarius...5g CaCl2·2H2O ...................... Autoclave solutions separately for 15 min at 15 psi pressure–121°C.....0mL pH 4............................................. Adjust pH to 3...................................... Mix thoroughly........................................ Gently heat and bring to boil- ....................................................... Mix thoroughly..... 500........0mL: Preparation of Medium: Aseptically combine compo- Preparation of Solution C: Add agar to distilled/deionized water and bring volume to 500................0mL: KH2PO4 ............................................. Aseptically add 0....................0–4... Bacillus acidocaldarius Agar Composition per liter: Solution A ..Bacillus Agar..................1........0mL pH 3...........2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500.. Solution B: Composition per 500... Mix thoroughly..............0......... and 1.1...............2g pH 3.....0mL: Glucose ......................................... Mix thoroughly............0mL of solution B......... This procedure avoids acid hydrolysis of the agar.................... Bacillus Agar............. Autoclave for 10 min at 15 psi pressure–121°C................................0..........0..... 0..................... 1............0mL Solution C ....25g (NH4)2SO4 ......................................0 ± 0................0g Solution B (Trace Elements Solution SL-6): Composition per liter: MnCl2·4H2O ....0g Glucose ............. 3....... Mix thoroughly.................................0g KH2PO4 .....0mL of sterile solution A....................................0...........5g H3BO3 ......................... nent I..1.............. Cool to 50°–55°C............................0mL.....0......................................................... Modified Composition per liter: Agar ........................................6g MgSO4·7H2O...................0mL....... and component III............ Autoclave for 15 min at 15 psi pressure– 121°C...500.......................500................................25g (NH4)2SO4 ..0g/500. Filter sterilize................15. Mix thoroughly............................0.. 2.................................. and Alicyclobacillus cycloheptanicus....................500...... 0...................................... Autoclave for 15 min at 15 psi pressure–121°C...................

....... Adjust pH to 7............................................ and 1.............................................. 0........................0g Yeast extract...................... 100...................1..5g MgSO4·7H2O...0g Mg(NO3)2·6H2O ..0g Yeast extract ....................................0 ± 0................. except magnesium nitrate solution............98g NH4Cl........... Gently heat and bring to boiling... This procedure avoids acid hydrolysis of the agar...........0L.........0g of glucose to 500. Pour into sterile Petri dishes or distribute into sterile tubes............... 0.....0 ± 0..........................0mL pH 7.... Cool to 50°–55°C.........0g KH2PO4 .....0g Preparation of Magnesium Nitrate Solution: Add Mg(NO3)2·6H2O to distilled/deionized water and bring volume to 100...........0 with 10N H2SO4........... 5... Adjust pH to 3............ Bacillus cycloheptanicus Agar Composition per 1001..... 0......... 500........................ Distribute into tubes or flasks................... Mix thoroughly............................................................................................................. Autoclave for 15 min at 15 psi pressure–121°C....................................................... Filter sterilize.................2mL pH 7...... 0..................................03g NiCl2·6H2O......................... 0......0g Agar .........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....... Aseptically combine both solutions...................................500.............................2 ± 0.................................1....................................................2g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1....2g ZnSO4·7H2O................................................... Autoclave for 15 min at 15 psi pressure–121°C...0mL Solution C ...................... Gently heat and bring to boiling..........1g MnCl2·4H2O ................. Adjust pH to 4..100.. Bacillus halodenitrificans Agar (LMG Medium 142) Composition per liter: NaCl ................ Preparation of Medium: Add components..................................................0mL of distilled/deionized water.6g Sodium benzoate................... 5......................1g ZnSO4·7H2O .................................................0g Beef extract ...... Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.......................0....................0g Magnesium nitrate solution .................. 6.03g Trace elements solution ..01g Preparation of Solution B (Trace Elements Solution SL-6): Add components to distilled/deionized water and bring volume to 1..... Pour into sterile Petri dishes or leave in tubes.......... Autoclave for 15 min at 15 psi pressure– 121°C....... and Alicyclobacillus cycloheptanicus.............. Alicyclobacillus acidoterrestris......0..................................... Mix thoroughly.....0..0mL.................5..........0...25g (NH4)2SO4 ....................3g CoCl2·6H2O........................0.......15..............0g Yeast extract............................................................0.2 at 25°C of sterile solution A...........................25g CaCl2·2H2O................ 1..........................8g KH2PO4 ....................0L........... 0.......0g of agar and 1............0............ 0............................... 3..............................1...................................... zoevorans..................................52 Bacillus benzoevorans Agar ing............1.. 0.....3g (NH4)2SO4 ................0mL: Mg(NO3)2·6H2O .0g NaCl ....................1g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100................................................. Autoclave solutions separately for 15 min at 15 psi pressure–121°C..... Mix thoroughly.....................0g Na2HPO4 ..0..0mL.....................0g MgSO4·7H2O... 15...... 2...... Gently heat and bring to boiling..0mL: Agar .15..500... Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C............................ ..2 with KOH........................02g CuCl2·2H2O.0mL of sterile solution B......3.............. 1................. Use: For the cultivation of acidophilic Bacillus species such as Bacillus acidocaldarius............................0g Preparation of Solution C: Add agar to distilled/deionized water and bring volume to 500.................. Adjust pH to 4........................ Mix thoroughly...........0g Glucose.......0mL pH 4.......................37g MgSO4·7H2O .............10......................................................................... Preparation of Medium: Aseptically combine 500.................0mL: Solution A ............0g Na2HPO4·12H2O .0mL..........0L..................... 0.....................0mL Use: For the cultivation and maintenance of Bacillus ben- Use: For the cultivation and maintenance of Bacillus cycloheptanicus........................1..................... 1.........3............................. Cool to 50°–55°C.................................................... to distilled/deionized water and bring volume to 900..0g Sodium acetate·3H2O .............................................................0........................... 1.............................07g FeCl3 ...............5g H3BO3 ............. Solution B (Trace Elements Solution SL-6): Composition per liter: MnCl2·4H2O .............................................................................. Mix thoroughly..... Bacillus benzoevorans Agar Composition per liter: Agar ...........................................................5g CaCl2·2H2O ...... Mix thoroughly..............2 at 25°C Magnesium Nitrate Solution: Composition per 100......0mL.....5............... Use: For the cultivation of acidophilic Bacillus species such as Bacillus acidocaldarius........0–7................................1g Na2MoO4·2H2O...............02g pH 4.. Autoclave for 15 min at 15 psi pressure–121°C......1...................................... 0.....................................................0g KH2PO4 ............. Bacillus Broth Composition per liter: (NH4)2SO4 ....................................................3g Glucose ..0g KH2PO4 ....... Mix thoroughly.......................................................0mL: FeSO4·7H2O ...... Preparation of Medium: Add components to distilled/ Solution A: Composition per liter: Yeast extract .... 0................5...................0mL of sterile solution C. 0................................... Solution C: Composition per 500............................................... Prepare a separate agar and glucose solution by adding 20.......0L..........................................................................0g Peptone..............0.2 at 25°C Trace Elements Solution: Composition per 100....... Distribute into tubes or flasks................ deionized water and bring volume to 1......

0............................................... soluble....................0..............................0L.............. Swirl medium to resuspend starch.....0g Peptone................................................................................................1g MnCl2·4H2O ............ Distribute into tubes or deionized water and bring to 1.... Bacillus pasteurii Agar Composition per liter: Urea.............................................1...................5.......2g FeSO4·7H2O .......0g KH2PO4 ....................20............................................................................... Mix thoroughly.....0...4 ± 0.... 10... Gently heat and bring to boiling.............1mg Preparation of Trace Mineral Solution: Add components to distilled/deionized water and bring volume to 1......... Use: For the cultivation of Bacillus halodenitrificans........................ 5.....................................................................5g pH 7............. Autoclave for 15 min at 15 psi pressure–121°C..................................................... Gently heat and bring to boiling......................................................2...................5...............5.......... Mix thoroughly.................1g CaCl2·2H2O .....0g Beef extract .5g MgSO4·7H2O.....05g Trace mineral solution.....95g K2HPO4 .............. ..........1g CaCl2·2H2O .............0g Meat extract ..... 5.....0............0mg Na2SeO3...............0mL pH 6........................................................................ Mix thoroughly................... Do not overheat......Bacillus Pullulan Salts 53 Cool to 45°-50°C.....................5..0mg Thioctic acid .....................0mg NaMoO4·2H2O ...... 5.. Aseptically distribute into sterile tubes or flasks.... 0..........20......0mg H3BO3 .................................................0mg Riboflavin...........................0mg Na2WO4·2H2O................... 0..................0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................................ Use: For the cultivation and maintenance of Bacillus subtilis..3 ± 0.................................. 5........ Aseptically pour into sterile Petri dishes or distribute into sterile tubes.... Gently heat and bring to boiling...........................7................................................. Distribute into tubes or flasks......... Bacillus Medium (ATCC Medium 21) Composition per liter: Use: For the cultivation and maintenance of Bacillus mac- Bacillus popilliae Medium Composition per liter: Glycerol ..................0g L-Glutamic acid......................................1..........................................0L......................................................................0g K2HPO4 .......5g Yeast extract ........................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..........1........2.......... Filter sterilize..........0mg p-Aminobenzoic acid ...0L......... 15.....0................................... 2............................................... Preparation of Medium: Add components to distilled/ Use: For the cultivation of Bacillus licheniformis..............................................1 at 25°C Preparation of Medium: Add components to tap water and bring volume to 1...................0...1............................................. 20.........................................................................5....2 at 25°C Yeast extract ..............0g Yeast extract..0... Also used to detect amylase-producing microorganisms....... Aseptically add 100............................ Bacillus polymyxa.......................5g NaCl ........................... Mix thoroughly.....0g Agar ........................... Mix thoroughly........................ 20.0L.............................................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........0 ± 0...............0mg Nicotinic acid ........................................0L..... Gently heat until dissolved............ Autoclave for 15 min at 15 psi pressure–121°C...............0g pH 7..1................0g Polypeptone™............................................ 10.........0.................................... Vitamin Solution: Composition per liter: Pyridoxine·HCl........0L...........................................2 ± 0.......................................... 20..2................. and Bacillus thermoglucosidasius......................... Distribute into tubes or flasks...........0g Citric acid.......0..0g Agar .0mL Vitamin solution ..............0. Use: For the cultivation and maintenance of Bacillus pasteurii and Sporosarcina ureae....36g Trehalose ........................ erans............ Gently heat and bring to boiling................0mg Cyanocobalamin.......... Autoclave for 15 min at 15 psi pressure–121°C.............5mg CuSO4·5H2O........... 30........ Bacillus polymyxa Agar Composition per liter: Agar ......................5...... flasks............2 at 25°C Trace Mineral Solution: Composition per liter: CoCl2·6H2O......................0L..................................20.................................................................... 20................................0 ± 0. Bacillus Medium (ATCC Medium 455) Composition per liter: Bacillus Pullulan Salts Composition per liter: Soluble starch.................................0mg Folic acid .............. Pullulan.......68g pH 7....0mL sterile magnesium nitrate solution............................5g MgSO4 .................................................................................. 5..................................4mg KI.......... Adjust pH to 7........5g Ferric ammonium citrate......... 0.....................10................ Pour into sterile Petri dishes or leave in tubes...............................................10............. Pour into sterile Petri dishes or leave in tubes............. Filter sterilize. 10......0g Acid hydrolysate of casein .........0..............................................................0...0.....................................13g ZnCl2·2H2O ........................... 3.....................................................................0g Yeast extract................................0mg Thiamine·HCl ..........0g Peptone........................ Use: For the cultivation of Bacillus popilliae....... Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly..................... 4...........................................................................................................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........0g Starch............................0mg Calcium pantothenate ................... Pour into sterile Petri dishes or distribute into sterile tubes......0.........0g NH4Cl ................................................................................3.....0mg Biotin ........... Mix thoroughly.........0g Starch..........0g pH 7..

.......0g MgSO4·7H2O.....1 ± 0............................2g MnSO4·H2O ...............2 at 25°C Trace Elements Solution: Composition per liter: H3BO3 .............................0L. 0....3g CoCl2·6H2O ......1g Na2MoO4·2H2O .............................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...25g Yeast extract ... 0................... 45% H2..................0.5g NH4Cl .........1.0L............................................................................................. Distribute into tubes or flasks..........................1g KH2PO4 .................... Distribute into tubes or flasks.0................................................ Mix thoroughly.......................................................................................................... 0...................................5g H3BO3 .................70g NaNO3 ..................................................0g KH2PO4 .5g KH2PO4 ......................................................................0L..............................2g MnSO4·2H2O.................... Bacillus schlegelii Agar (LMG Medium 85) Composition per liter: Agar ......................................5mg Cysteine solution ...................5g H3BO3 ................................15g (NH4)SO4............................... Aseptically add sterile vitamin solution......... 0.......... 0................0mL pH 7....................... Use: For the chemolithotrophic cultivation of Bacillus schlegelii.................... Use: For the cultivation of Bacillus schlegelii.....................................................0mL Na2S·9H2O solution ................................................................ 9..................................................................... 0...............................................................................5g Na-Pyruvate................. 0................0....................................................................1g Na2MoO4·2H2O....................0mL Selenite-tungstate solution .0..................................................0mg CuCl2·2H2O ........... 10............5g KH2PO4 ... Incubation is at 65°C under an atmosphere of 5% O2........................2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O .....................3g CoCl2·6H2O .........................1 ± 0............................... 1...... to distilled/deionized water and bring volume to 990................ Adjust pH to 7.................0g Na2HPO4·12 H2O............................................................4..........5g Sodium pyruvate ..........5g NH4Cl........ Mix thoroughly.... Mix thoroughly......0....... 30........................................................ Gently heat and bring to boiling..............0mg CaCl2·2H2O................. Distribute into tubes or flasks......1 ± 0..........2g K2HPO4 ....................................0g MgSO4·7H2O ................2g ZnSO4·7H2O .........................0mg Trace elements solution ...........08g MgSO4 ................0................................................0mL pH 7.................................................................................. 30......................... 1............................................................ 10% CO2..................................... 0.......................................2 at 25°C ... 5................................... 1.......... 30.......... Mix thoroughly....... Use: For the cultivation and maintenance of Bacillus species that can degrade pullulan..... Bacillus schlegelii Chemolithotrophic Growth Medium (DSMZ Medium 261) Composition per liter: Na2HPO4·2H2O...............5g NH4Cl.......................0g Na2CO3 ...........3........................2g MnSO4·2H2O ....0L..........3g CoCl2·6H2O...................................................................6g NaHCO3 ............................ 1....................0...0mL Trace elements solution SL-10... 5..........................................01g Ferric ammonium citrate..........................................................................0mg MnCl2·4H2O....................................0L..... Aseptically distribute into sterile tubes or flasks......................................................................................02g CuCl2·2H2O........................... 0............................03g NiCl2·6H2O........................... 0.... 0......2g ZnSO4·7H2O .......... Adjust pH to 6.................0..... 4..................5............................01g Ferric ammonium citrate ................................ Autoclave for 15 min at 15 psi pressure–121°C.........1.......................0...........4...........0. Mix thoroughly.........03g NiCl2·6H2O ................................................ 10...............0mL...............................................1................ Autoclave for 15 min at 15 psi pressure–121°C...... 0..0.....0.........................0mL pH 7......... Gently heat and bring to boiling..........................................0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.................. 10.... 0...............54 Bacillus schlegelii Agar Preparation of Medium: Add components........... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....................................1.2g Na-lactate .0mg Trace elements solution SL-6 .............................0.....................................................0mg Trace elements solution SL-6.90.......1........... 3........................... Bacillus schlegelii Heterotrophic Growth Medium (DSMZ Medium 260) Composition per liter: Na2HPO4·2H2O ..................3.....0mg Resazurin ................0L.8 ± 0................................................................................. 10.........1........................................... Mix thoroughly.................................. Incubation is at 65°C.. 10.10......................... Autoclave for 15 min at 15 psi pressure–121°C..01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1....................2g ZnSO4·7H2O.... 1....................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1...............0mg NiCl2·6H2O .............. 0........................................ 20.......... Distribute into tubes or flasks.... Mix thoroughly......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........ Mix thoroughly..........0............ 1.................................0mL pH 9......... Cool to 25°C.... Bacillus selenitireducens Medium (DSMZ Medium 968) Composition per liter: NaCl ............1.............0..... except vitamin solution............................ Pour into sterile Petri dishes or leave in tubes..........01g CaCl2·2H2O ............. Use: For the heterotrophic cultivation of Bacillus schlegelii...............02g CuCl2·2H2O.......................25..0mg Ferric ammonium citrate.......................... Autoclave for 15 min at 15 psi pressure–121°C................0g MgSO4·7H2O ...............................................................................................2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O.01g CaCl2·2H2O.................1g Na2MoO4·2H2O ............ 0.. Adjust pH to 7.......................... 1...........

................... Selenite-Tungstate Solution: Composition per liter: NaOH .............................................................. 0..................................................................................................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10....01mL pH 7...................... Mix thoroughly.............. 0.................................................2 at 25°C Mineral Salts Solution: Composition per liter: NaCl .......................... 1.....................0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1............. 0........... cysteine solution...............................64mL L-Lysine·HCl (1% solution) ....10.......0g pH 7......... Sparge with 100% N2............0L.0g KCl .................... 0........................0mL............. 1.......0mg NiCl2·6H2O ...... Cool to room temperature......... Distribute to anaerobe tubes or bottles........ Na2CO3..................... Bacillus stearothermophilus Defined Broth Composition per 100.0mL...84mL L-Threonine (1% solution) .......................0mL: Distribute into tubes or flasks........................................... Autoclave for 15 min at 15 psi pressure–121°C.............................0mL Potassium phosphate buffer...0mg CuCl2·2H2O .0mL: K2HPO4 ..................................0mL L-Glutamate·HCl (1% solution) ........0mg Na2MoO4·2H2O ............ 3......................................... Na2S·9H2O Solution: Composition per 10.......52mL Glycine (1% solution) ....................01mL FeCl3·6H2O (0.......10........ 4...0mg Na2SeO3·5H2O .....................0mL L-Isoleucine (1% solution) ................ and Na2S·9H2O solution.........5mL L-Glutamine (1% solution).............................................. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ....... except NaHCO3.............................. Add solid NaHCO3 and Na2CO3..........3mL CaCl2 (5% solution)...................................0mg HCl (25% solution) ........................ 0.....................5g CoCl2·6H2O .... Add distilled/deionized water and bring volume to 1.. 1......................................0mL............... 5......4.............. Aseptically and anaerobically add per liter 10...... 0.......... Sparge with 80% N2 + 20% CO2..........................0mL L-Phenylalanine (1% solution) ..................................... Use: For the cultivation of Bacillus stearothermophilus............5mL L-Cystine (1% solution).............................................0mg ZnCl2 ............................................ 0..........0mL Thiamine·HCl (0..................30............................84mL L-Arginine·HCl (1% solution) .....10.01% solution) ................ Preparation of Medium: Add components..........0mg MnCl2·4H2O.. Bacillus thermoalcalophilus Medium Composition per liter: Yeast extract .......... 1....... 0.................... 1...................... Boil for 3 min..01mL MnCl2 (10mm solution) ..............0g KH2PO4 ......26mL Biotin (0...125................01% solution) ...................4g .................... Filter sterilize........... Cool to 25°C..... 0.....................................0mL Nicotinic acid (0........0mL: Mineral salts solution ... Mix thoroughly...........................01% solution) ....... Autoclave for 15 min at 15 psi pressure–121°C..............25g Preparation of Cysteine Solution: Add 55 L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10................. 5.......0mL of HCl solution......... 190...........................3mL L-Valine (1% solution)......8g Na2SO4 ...... 6....................................0mL...................................................................................... 2.2 ± 0.............. 1...0mL: Na2S·9H2O.............................................. 0.. 2................... 1.............................64mL L-Tyrosine (1% solution)..........0L....0mL Glucose (20% solution) ........... 36......................01mL ZnSO4·7H2O (5% solution) ..............................0g NH4Cl ............... Mix thoroughly.............. Filter sterilize.......................0mL L-Leucine (1% solution)........................0........................0g MgSO4 ................ Autoclave for 15 min at 15 psi pressure–121°C....................1....0mL................ 0... 24......3................ 1... Mix thoroughly..4mL L-Serine (1% solution)..... Mix thoroughly.....0mL sterile Na2S·9H2O solution...8...........0g Yeast extract..0L......0mL sterile cysteine solution and 10..... 0............ 10...... 10............ Adjust pH to 9...................................... 1.......................... 0...5mL L-Asparagine·H2O (1% solution) ............................0g Preparation of Mineral Salts Solution: Add components to distilled/deionized water and bring volume to 1..5mL L-Histidine·HCl·H2O (1% solution) ...................................... Potassium Phosphate Buffer: Composition per 500......................... Sparge with 100% N2......42mL L-Tryptophan (1% solution) ........................ Autoclave for 15 min at 15 psi pressure–121°C.................. 0.........0mL L-Proline (1% solution) ........... Bacillus stearothermophilus Broth Composition per liter: Pancreatic digest of casein ...... 0..................... 4.....0L......Bacillus thermoalcalophilus Medium Cysteine Solution: Composition per 10.. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 100................... 1................................. Use: For the cultivation of Bacillus selenitireducens and Bacillus arseniciselenatis... Sparge with N2.......... Autoclave for 15 min at 15 psi pressure–121°C...................25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10........... 1.....86mL L-Alanine (1% solution) ........ Gently heat and bring to boiling.............. to distilled/deionized water and bring volume to 980........................................... 1. Add remaining components......0g Sodium acetate ............ L-Cysteine·HCl·H2O ................................................................................................. Mix thoroughly.0g Preparation of Potassium Phosphate Buffer: Add components to distilled/deionized water and bring volume to 500...... Use: For the cultivation of Bacillus stearothermophilus in a chemically defined medium.........05% solution).5g Na2WO4·2H2O ..3 ± 0................. 0............4mL L-Aspartate (1% solution) ... Cool to room temperature while sparging with 80% N2 gas.... 0..... Mix thoroughly...........10............. Mix thoroughly................................ 0................................. 70...0g K2HPO4 ............... Mix thoroughly...........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....0mg H3BO3 ................ 0................ Mix thoroughly................. 100.....56mL L-Methionine (1% solution) ........

.......0g of air-dried garden soil through a coarse sieve....03g NiCl2·6H2O........................ Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ......................56 Bacillus thermoantarcticus Medium K2HPO4 .......................0g KCl...................0.. Bacillus tusciae Medium Composition per liter: Na2HPO4·2H2O ..................................... Distribute into tubes or flasks........................................... Mix thoroughly...... 3.....................0g Yeast extract...... Distribute into tubes or flasks................500... 0..................................................0g Soluble starch........01g MnSO4·H2O.............3........ Autoclave for 60 min at 15 psi pressure–121°C........................................ Autoclave for 15 min at 15 psi pressure– 121°C.... Use: For the cultivation and maintenance of Bacillus thermoglucosidasius...........2g CaCl2·2H2O .... 3........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...............................8 at 25°C Soil Extract: Composition per liter: Garden soil...................................................... Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C...................................2g ...............................0 ± 0...................0g (NH4)2SO4 .. Cool to 50°–55°C.............. Distribute into tubes or flasks.............. Autoclave for 15 min at 15 psi pressure–121°C....01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1..... Bacillus thermoglucosidasius Agar Composition per liter: Agar ........................0mL: Ferric ammonium citrate ...................0..................................0mL..................................... 0............................01g Ferric ammonium citrate solution ... Preparation of Medium: Add components.............. Adjust pH to 7..........2........................................8..... Bacillus thermoantarcticus Medium Composition per liter: Yeast extract. Bacillus thermoleovorans Medium Composition per liter: Bacillus thuringiensis Medium Composition per liter: Glucose..................... air dried ............................. Autoclave for 15 min at 15 psi pressure–121°C..........................3g MgSO4 .......................... Mix thoroughly..................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............0g Soil extract .... Aseptically add 20.....2g ZnSO4·7H2O.............. to distilled/deionized water and bring volume to 975............05g pH 7........................5g NaHCO3 ....................................................... For chemolithotropic growth.............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. 3.........................0L.... Use: For the cultivation of Bacillus thermoalcalophilus.. except fer- Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..0g (NH4)2HPO4 ............... Store at room temperature until clear....................2.................................0L........................ Adjust pH to 8................................................................ Distribute into bottles in 200..6–5.......................................3g KH2PO4 .........................................................1......................... 3.............6–5... Use: For the cultivation of Bacillus thuringiensis.......2 at 25°C Ferric Ammonium Citrate Solution: Composition per 20...............................................................5g H3BO3 ........................................ Allow residue to settle......1g Na2MoO4·2H2O...0.......................... Gently heat and bring to boiling....... Autoclave for 15 min at 15 psi pressure–121°C..............2.....08g MnSO4·4H2O.....0g Peptone....... Cool to room temperature.. Autoclave for 15 min at 15 psi pressure–121°C................. 20...05g Preparation of Ferric Ammonium Citrate Solution: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20.........0...........................0mL......................................0g pH 7...........................0... Decant supernatant solution.....0...... 0.. 400.... Mix thoroughly.........................0.................. Adjust pH to 5.... Mix thoroughly...............5g MgSO4·7H2O............................................... Adjust pH to 7........................0g K2HPO4·3H2O .... n-Heptadecane .................................... Gently heat and bring to boiling..........................0 ± 0............. 0........0g MgSO4·7H2O. Autoclave for 15 min at 15 psi pressure–121°C...............0............ Autoclave for 15 min at 15 psi pressure–121°C.........3g CoCl2·6H2O................ Use: For the chemolithotrophic growth of Bacillus tusciae.. 5.. Mix thoroughly...........3 ± 0......... Distribute into tubes or flasks....... 1................5g Fe(NH4) citrate .........................2g FeSO4 ..................................0g Preparation of Soil Extract: Pass 400... Use: For the cultivation of Bacillus thermoleovorans....................................... 30........... Adjust pH to 4.......................0........... 0...0L.....0mL volumes................................. incubate the culture under 2% O2 + 10% CO2 + 60% H2 + 28% N2. Mix thoroughly..0................................. 1..........9g KH2PO4 ........................ 10...........................2....... Filter through Whatman filter paper....................................3...................0.....................0g Yeast extract..................05g CaCl2·2H2O .....0g Yeast extract ...0g Beef extract ....0............. ric ammonium citrate solution and trace elements solution SL-6........................................................2 ± 0.0mL of tap water..0g NaCl ...........................................2g MgSO4·7H2O ....................0L... Pour into sterile Petri dishes or distribute into sterile tubes.............................0......................... 1...................... Mix thoroughly....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....0mL pH 5.... Use: For the cultivation of Bacillus thermoantarcticus................0.........0mL of sterile trace elements solution SL-6... Add soil to 960.. Mix thoroughly...................... 6. 0..............01g pH 8.............0mL of sterile ferric ammonium citrate solution and 5....... Aseptically distribute into sterile tubes or flasks... 0.....02g CuCl2·2H2O....0L............0mL Trace elements solution SL-6.............0mL pH 4.............0L..............................................0................................... 5.0............................0.......... 0........2............................0g KH2PO4 .3g NH4Cl ......................

......0g Preparation of Carbon Source: Add carbohydrate or organic acid to distilled/deionized water and bring volume to 10......................0..................06g FeSO4·7H2O .................................................000 × g for 10 min...02g CuSO4·5H2O .......................................0mL of sterile ferric ammonium citrate solution...........................................2g Yeast extract........0mg Riboflavin................0......................0.....0.......2 at 25°C Trace Elements Solution: Composition per liter: MgSO4·7H2O ........05g CaCl2·2H2O.................2g ZnSO4·7H2O ............0..5....0mL of sterile carbon source................................................................................................ 10....................................25g KH2PO4 ........... Add remaining components....................................................................0........0............................ 15........................................1.....0mL pH 4.............0mL Trace elements solution SL-6 ............ Mix thoroughly...........................0................................................... Dissolve by adding KOH and adjust pH to 6.....................................5..........................0mL of distilled/deionized water..........0.............. 2..............0 ± 0....................................... except fer- ric ammonium citrate solution..0mL of washed cells and 250...5g Fe(NH4) citrate.........................68g K2HPO4 .................. Mix thoroughly................0mL: Carbohydrate........2 at 25°C Ferric Ammonium Citrate Solution: Composition per 20.................................. Use: For the cultivation of freshwater Myxobacterium spe- cies.... Autoclave for 15 min at 15 psi pressure–121°C........................ Cool to 45°– 50°C............................5. Mix thoroughly..... Pour into sterile Petri dishes......................3...............................01g Na2MoO4·2H2O . In a separate flask. Bacterial Cell Agar (BCA) Composition per liter: Tryptose ............................................................0..10..............................20.............................................................15g MgSO4·7H2O.. 8. Aseptically combine 25................................... 0... Mix thoroughly......... Wash the cells four times in sterile 0.... Inoculate with a culture of Aeromonas hydrophila.....................................................0g NaCl ............................02g Resazurin ....................................... 0....................3 ± 0..................................2 at 25°C 57 Preparation of Medium: Add components........................ Mix thoroughly.......0mg Nicotinic acid ............18 g FeSO4·7H2O...............................................0mg Trace elements solution............1g NiCl2·6H2O ...............................................................0.......................0mL..0mg p-Aminobenzoic acid ...................0....3 mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500........... 17.... Autoclave for 15 min at 15 psi pressure–121°C.0g MgSO4·7H2O ................ Carbon Source: Composition per 10.......0g NaCl ..................5 g CoSO4·7H2O .......... 0............................... Centrifuge culture in 40...0......................2..... Gently heat and bring to boiling..... Filter sterilize...................................................0mL................0............ and 5.................0g Na2HPO4·2H2O............... except agar.0mL..................0mL pH 7..0 with KOH....01g H3BO3 .. 0............................ 0.............0L with additional distilled/deionized water.................... 2.....................7g pH 7... 0..........................0mg Lipoic acid.0g or organic acid ........ Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O............... Incubate with shaking at 30°C for 72 hr................... 0...........0mg Thiamine·HCl .......... 0.......... trace elements solution SL6.0............. Autoclave for 15 min at 15 psi pressure– 121°C.....05g Preparation of Ferric Ammonium Citrate Solution: Add ferric ammonium citrate to distilled/deionized water and bring volume to 20.............................36g Agar .... Mix thoroughly............................3g NH4Cl.....................................85% saline........................................................ Cool to 45°–50°C..0mL: Ferric ammonium citrate....................................... Aseptically distribute into sterile tubes or flasks...0g NH4Cl .......1...0mg Calcium DL-pantothenate ..................5...........................0...... 5........................025 g KAI(SO4)2·12H2O ........ Bacteroides cellulosolvens Medium Composition per liter: Cellobiose or cellulose .3g CoCl2·6H2O ..... 1..... and carbon source.... Cool to 50°–55°C....0L......... 2. Bring volume to 1......................................0mL volumes at 10..........01g Ferric ammonium citrate solution........................................ to distilled/deionized water and bring volume to 1.....0mg ...0g Nitrilotriacetic acid.............................01g MnSO4·H2O .... Preparation of Medium: Add components.............. 0.. Vitamin Solution: Composition per liter: Pyridoxine·HCl.............0g of agar to 1............................ 0.......... to distilled/deionized water and bring volume to 965....0g MnSO4·2H2O ......0g NaHCO3 ........25g Na2S·9H2O ............................................................. Autoclave for 15 min at 15 psi pressure–121°C........................1......5...... 0...............0L.10.0.........5.................................................18g (NH4)2SO4 ......18 g ZnSO4·7H2O .......................................................................... Adjust pH to 7......................................0mL Carbon source ........................0mL of sterile trace elements solution SL-6................................................................................................................5 g CaCl2·2H2O................. Autoclave for 15 min at 15 psi pressure–121°C.........................................................................0................................01g Na2SeO3·5H2O ............12g CaCl2·2H2O .......0.................................................................0mL Vitamin solution ....................... 10....................5.......... sterile agar solution..............................5.............................................. Mix thoroughly...68g Beef extract ..........................0L of distilled/deionized water.01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.......... 1.02g CuCl2·2H2O ..3g L-Cysteine·HCl·H2O................ Adjust pH to 4.......... Resuspend the cell pellet in 25................................................ Mix thoroughly....................0mL of cooled..................... 0................... Autoclave for 15 min at 15 psi pressure– 121°C...................03g NiCl2·6H2O .....0mL distilled/ deionized water... 15.................5............................. Gently heat and bring to boiling.............. Use: For the heterotrophic growth of Bacillus tusciae.............. 10........................................ 1...5g NaHCO3 .............0..............9g KH2PO4 ............................................. add 15........1..............................................................0 ± 0........5g H3BO3 .1g Na2MoO4·2H2O ......................................Bacteroides cellulosolvens Medium Bacillus tusciae Medium Composition per liter: Agar ....... Cool to 30°C........................................ Aseptically add 20.......................... 0.......................................

............................ 1................0mL.................................................................... Gently heat and bring to boiling............0.. except agar.....5g CaCl2·2H2O .0...................................................... Use: For the cultivation and maintenance of Bacteroides cellulosolvens...............................3.....2 at 25°C Trace Elements: Composition per liter: CaCl2·2H2O ............ 5.......... 25.....................................0mg Vitamin B12 ............................0g Preparation of Medium: Add components..18g CuSO4·5H2O................... and other materials................... except RPF supplement........................................... Supplemented Composition per liter: Agar .................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................................... Autoclave agar separately to avoid acid hydrolysis..............0 ± 0........................................... 5...................0mg Folic acid..................................0g Beef extract ...........0....................................................0g LiCl ............. lase-positive staphylococci from soil..2g Trace elements.................. Pour into sterile Petri dishes.. Mix thoroughly........... 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...............25............................1............................................. Mix thoroughly....... Mix the two solutions together..............................0L....................................... Mix thoroughly.........58 Baird-Parker Agar Biotin ....................................... Preparation of Medium: Add components............25g (NH4)2SO4 .0L........ 12. Mix thoroughly but gently......................... Filter sterilize... Preparation of Cellobiose Solution: Add cellobiose Use: For the selective isolation and enumeration of coagu- Cellobiose Solution: Composition per 50.................................. distilled/deionized water and bring volume to 100...................................0mL pH 4..........................0 ± 0..............5.. 2.0g KH2PO4 ... to distilled/deionized water and bring volume to 900.....25g (NH4)2SO4 ............... 17............ Pour into sterile Petri dishes.... Autoclave for 15 min at 15 psi pressure–121°C....... to distilled/deionized water and bring volume to 950......... 5............. except cellobiose solution...................................................................................0mL Caution: Potassium tellurite is toxic..............................0mL of filter-sterilized RPF supplement..........................................6............... Aseptically distribute into sterile tubes or flasks... Adjust medium to pH 4. Pour into sterile Petri dishes or distribute into sterile tubes..... Mix thoroughly......................100......0g KH2PO4 ..............0mL..................0mg Rabbit plasma ..............0g Beef extract ..............................1mg Preparation of RPF Supplement: Add components to Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...0g Yeast extract.........2g Trace elements....2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath and BD Diagnostic Systems....................0 ± 0................................................................... Preparation of Medium: Add components.................30.................................... 10........................ 12......................... Filter sterilize.........3.......... 17.......... Autoclave for 15 min at 15 psi pressure–121°C....................15g H3BO3 ....................... Autoclave for 15 min at 15 psi pressure–121°C.. BAM SM Agar (ATCC Medium 1656) Composition per liter: Agar ......... 1...................... 1...... BAM Agar (ATCC Medium 1655) Composition per liter: Agar .0 with H2SO4.............................................. 10........................................ 5.........................................................0mL pH 4....20..... Baird-Parker Agar Composition per liter: Agar .................... 10................................0mL.0g MgSO4·7H2O........0mg K2TeO3 ...........................................................0mL of distilled/deionized water................. 3..................................0............................................................................0.... Colonies surrounded by an opaque zone of coagulated plasma are diagnostic for Staphylococcus aureus........................................................ Aseptically add 100.....16g MnSO4·4H2O............0g LiCl ..................................75g Trypsin inhibitor ............. to distilled/deionized water and bring volume to 800............................. 1..0. 0.5..............0mL: (or cellulose) to distilled/deionized water and bring volume to 50................ Mix thoroughly.......0g Glycine................0..0g Yeast extract ...........5g CaCl2·2H2O ................................... Cool to 45°–50°C......0L of distilled/deionized water...........................................0g Sodium pyruvate ......0......... and other materials... 2..... D-Cellobiose (or cellulose) .. 10................0mL of sterile cellobiose (or cellulose) solution.......0................2 at 25°C . Add agar to 200.....................0g Glucose...............................................0mL: Bovine fibrinogen ... Cool to 45°–50°C....66g Na2MoO4·2H2O...........................0g Glucose.......................................................................................................................18g CoCl2·6H2O.......................... Use: For the cultivation and maintenance of Bacillus acidoterrestris... 5.................. Aseptically add 50....0...3g ZnSO4·7H2O.0...0 ± 0.....................0g Glycine...................................0...0g Sodium pyruvate ...........0g RPF supplement.......2 at 25°C RPF Supplement: Composition per 100....... For the differentiation and identification of staphylococci on the basis of their ability to coagulate plasma....0g Yeast extract ............. Mix thoroughly..0g pH 7.......... Gently heat and bring to boiling....0....1g Preparation of Trace Elements: Add components to 1...... Mix thoroughly............ Gently heat and bring to boiling.............................................................................0g MgSO4·7H2O........................................0g Pancreatic digest of casein .. air..0g Pancreatic digest of casein .........0mL........0mL.... Mix thoroughly...................... Mix thoroughly.......... Autoclave for 15 min at 15 psi pressure–121°C.....0mL pH 7.... Baird-Parker Agar.........0g Yeast extract................................... air..

.......1mg 59 MnSO4·4H2O..............0g Preparation of Solution 2: Add yeast extract to distilled/deionized water and bring volume to 100............................................... Mix thoroughly............1............................... 0........................................... 2....18g CuSO4·5H2O ................ 0....................................0L......................................................0 ± 0............................... Add remaining components and bring volume to 1......................................................0....................................0mL Solution 2 .............. 0.........1g ZnCl2 ..........................66g Na2MoO4·2H2O ................01g Na2MoO4·2H2O....... Basal Thermophile Medium Composition per liter: Solution 1 .. Trace Elements Solution: Composition per liter: Nitrilotriacetic acid......0.................5g CaCl2·2H2O................3............................... Preparation of Medium: Add components...........0mg ..................................16g MnSO4·4H2O .......2g Trace elements solution... 0........................................................ 0. Mix thoroughly..10g Preparation of Trace Elements: Add components to 1........................................... 0....0........................................Basal Thermophile Medium Trace Elements: Composition per liter: CaCl2·2H2O........................................................................ 50............2g MnCl2·4H2O ... 0................5µg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....................................................0mL Resazurin (0...........................2 at 25°C Trace Elements: Composition per liter: CaCl2·2H2O................ 9.............................2% solution) ..02g CoCl2·6H2O.0 with H2SO4.............0..0............................................. 9....... 850.............8g K2HPO4 .....................0mL................... 3............................1g Preparation of Trace Elements: Add components to 1. Cool to 45°–50°C... Solution 3: Composition per 50............................. Use: For the cultivation and maintenance of Bacillus cycloheptanicus......... 1.......................................02mg Co(NO3)2 .................................... Use: For the cultivation of Beggiatoa species......................... Mix thoroughly..................... 0.......................16g MnSO4·4H2O ..0mL Vitamin solution .25g (NH4)2SO4 ............0L....... 0....... Filter sterilize........ 0........... Pour into sterile Petri dishes or distribute into sterile tubes................................. 1.............. Autoclave for 15 min at 15 psi pressure–121°C.....................................0.....................0mL: Yeast extract ............................. 0................................................................................... Autoclave for 45 min at 15 psi pressure–121°C....... Mix the two solutions together.................0g K2HPO4 ..0.......... Modified Composition per liter: Agar ...................0mL pH 4..................15g H3BO3 .......0mL........75g MgCl2·6H2O .............................1mg ZnSO4·7H2O .............................. Mix the two solutions together.........0g Yeast extract.................................................... 0.........0mL of distilled/deionized water............................................. Use: For the cultivation and maintenance of Bacillus cycloheptanicus...............10.... 0.0mL: Glucose........ Adjust pH to 6....................5...................1g CuCl2 ............ 0...............01g Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 100.... Adjust medium to pH 4....... Cool to 45°–50°C.....0mL Solution 1: Composition per 850....... 0....................................66g Na2MoO4·2H2O ...................03mL Preparation of Solution 1: Add components to dis- tilled/deionized water and bring volume to 850..0g FeCl3·4H2O........... Autoclave for 45 min at 15 psi pressure–121°C..........0g Preparation of Solution 3: Add glucose to distilled/ deionized water and bring volume to 50. 0....................................................................................... to distilled/deionized water and bring volume to 800..... Solution 2: Composition per 100.................................................0.................................18g CoCl2·6H2O ................................ Pour into sterile Petri dishes or distribute into sterile tubes.......15g H3BO3 .............. Autoclave agar separately to avoid acid hydrolysis....... Add agar to 200...................................................... Gently heat and bring to boiling...........18g CoCl2·6H2O ........ except agar. 5...................2g Trace elements ......0g KH2PO4 ................................0mg H3BO3 ......................01mg CuSO4·5H2O.............................0mL: Pancreatic digest of casein .. Mix thoroughly.....................................................................18g CuSO4·5H2O ....... 0..................................................02g Na2SeO3.... Autoclave agar separately to avoid acid hydrolysis...................................0g MgSO4·7H2O ................................ Autoclave for 45 min at 15 psi pressure–121°C..........0mL Solution 3 ...... BAM SM Agar....... 100...12..1g CaCl2·2H2O ..01g Disodium EDTA ..........9g KH2PO4 .............................. 0......0............................................................ Add agar to 200.......................017g H3BO3 ....................1.................................... Mix thoroughly.........0 with H2SO4. 0........30g ZnSO4·7H2O ..............................0g Glucose .......................................................................................................................0mL of distilled/deionized water................0L of distilled/deionized water........................................................................ Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl.... Gently heat and bring to boiling.................... Mix thoroughly...0...............0...3g ZnSO4·7H2O ............................................5 with KOH..................................................................................................................................... Cool to 45°–50°C...............0mL................................. 0..................0................................................... to distilled/deionized water and bring volume to 800..................................................................10.......... Preparation of Medium: Add components................... Mix thoroughly................................................ 30........ Autoclave for 15 min at 15 psi pressure–121°C...2mg FeSO4·7H2O...............0mg CaSO4·2H2O.......... Mix thoroughly.................0.......................... except agar.........1.........0........0.................... 7...........0L of distilled/deionized water..........................7g MgSO4·7H2O .................. 0........... 0........5g NaCl ........ 0......... 5............... 0........ Basal Mineral Medium Composition per liter: NH4Cl.0mL of distilled/deionized water............0mL FeSO4·7H2O (10% solution) .0mL........... Adjust medium to pH 4.................0mL.................................. Mix thoroughly..........01mg NaMoO4·2H2O .........................................................................5g NH4Cl ........

............................................0mg Biotin .................... 2......................0.5mg Na2S·9H2O Solution: Composition per 100..0mg Folic acid....................................... Store below −20°C.......................... 10.0mg Cyanocobalamin ............................................... 0......................................... Quickly adjust pH to 10 with fresh 3N NaOH and flush under 100% N2............ Cool and continue flushing under 90% N2 + 10% CO2.......... Prepare in a chemical fume hood..................0mL Mineral solution 2 ... 0...................................................60 BC Medium Thiamine·HCl ...... Mix thoroughly.....8g Preparation of Mineral Solution 2: Add components to distilled/deionized water and bring volume to 1........................0mg Thioctic acid ...................0mL of distilled/deionized water..........0mL volumes under 100% N2..........0L with distilled/deionized water........... Mineral Solution 2: Composition per liter: NH4Cl.01g AlK(SO4)2·12H2O .............................0mg Nicotinic acid .................. and other cellulose-degrading microorganisms...............................10....0mg p-Aminobenzoic acid ..12..0L....... Autoclave for 15 min at 15 psi pressure–121°C.................... 2............................................................ 5...................................................................................3..1% solution).....................0mL of distilled/deionized water and adjust to pH 6................0g FeSO4 ·7H2O ..................0mL with distilled/deionized water............................10..................0g .....6 ± 0................ and solution 3 under 100% N2................................................. Mix thoroughly................................................0L.....10.... Bring volume to 200... 3......................1g ZnSO4·7H2O..................9g Preparation of Medium: Add cellulose and NaHCO3 Caution: This medium contains sodium sulfide and may produce toxic H2S gas.................. Transfer anaerobically to tubes or flasks and stopper.......... 5.................... Preparation of Medium: Aseptically combine solution 1.......0L................... 5....... Preparation of Na2S·9H2O Solution: Add Na2S·9H2O Wolfe’s Mineral Solution: Composition per liter Na2S·9H2O.......0mg Cyanocobalamin........................ 2...........0.............................0mL of cysteine-sulfide reducing solution............................. Add all other components except cysteine-sulfide reducing solution.1...........6 at room temperature.........01g H3BO3 ................0.......0.........1g CoCl2·6H2O.... do not adjust....0mL................................................................... 3.0mL... 2.......... 5................... Fervidobacterium nodosum.... Add Na2S·9H2O.......... solution 2....01g Na2MoO4·2H2O.................................5g Na2S·9H2O ........5 with KOH to dissolve....................0mL pH 7.................5g KH2PO4 ..0mL Cysteine-sulfide reducing solution .. Use: For the cultivation and maintenance of Clostridium species...................................................................... Boil under 100% N2.. The pH should be 7.1............... Mix thoroughly................ Acetivibrio cellulosolvens.........................2g to distilled/deionized water and bring volume to 800......8mL more of cysteine-sulfide reducing solution..........................................2............................1..................10............10......................... 0.. Add three drops of concentrated HCl... Add remaining components one at a time............... Mix thoroughly.......... Mix thoroughly............................ 2................................. Autoclave for 15 min at 15 psi pressure– 121°C...................................................0mL of distilled/ Mineral Solution 1: Composition per liter: deionized water.......... Cysteine-Sulfide Reducing Solution: Composition per 200.........5g Preparation of Cysteine-Sulfide Reducing Solution: Add L-cysteine·HCl·H2O to 50........0g NaHCO3 ............................................................................................10............................0g Nitriloacetic acid ....1mL of sterile Na2S·9H2O solution to each tube.............0mL volumes and cap....................5....0mg Riboflavin ... Bacteroides cellulosolvens................. Vitamin Mixture: Composition per liter: Pyridoxine·HCl..... Bring volume to 1.......... 1........................0....................................0mL: Preparation of Vitamin Mixture: Add components to distilled/deionized water and bring volume to 1...................1g CuSO4·5H2O............. Add 8...........................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.................................2g CaCl2·2H2O...................................0....... 5.......................2.....................................................................2 at 25°C MgSO4·7H2O...................0mg Thiamine·HCl .0..........0mg Biotin .75.................................................. Immediately prior to inoculation....0mg Lipoic acid (thioctic acid) .2........................................8mL FeSO4·7H2O solution ..... Heat and boil under 90% N2 + 10% CO2..... Distribute into tubes in 10........................0mL Resazurin (0..................0mL Vitamin mixture ................... and Thermoanaerobium brockii....................0mg p-Aminobenzoic acid ...............................5................................ aseptically add 0........................ Preparation of Mineral Solution 1: Add K2HPO4 to distilled/deionized water and bring volume to 1........... Use: For the cultivation and maintenance of Acetivibrio cellulolyticus.....1g CaCl2 ...... 5........................................0mg Calcium pantothenate ...............5g NaCl .......................0g to distilled/deionized water and bring volume to 100.. BC Medium (Medium for Acetivibrio cellulolyticus) Composition per liter: Cellulose powder ............................ BCP Azide Broth (Bromcresol Purple Azide Broth) Composition per liter: Casein peptone ....0g Yeast extract ........................0.01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500...........0g Mineral solution 1 .................5.. 12........................................... K2HPO4 ....................... Add 4.........0..0mL: L-Cysteine·HCl·H2O............... FeSO4·7H2O Solution: Composition per 100.............5g MnSO4·H2O................. Distribute anaerobically into tubes in 7...........0g Na2SO4 ...............0mL Wolfe’s mineral solution ...........75.........0.......4g MgSO4·7H2O ........0L.............................................. Preparation of FeSO4·7H2O Solution: Dissolve FeSO4·7H2O in 100.....................0mL: FeSO4·7H2O.

....................................0g Yeast extract.............. Mix thoroughly.......................7g NaN3...... Mix thoroughly........................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems.........2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems............. Mix thoroughly...................................... BCYE Differential Agar (Buffered Charcoal Yeast Extract Differential Agar) Composition per liter: Agar ........ Add 4........................ Filter sterilize.......................0L....... Filter sterilize.......0mL Cysteine·HCl·H2O solution .... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension..... except Lcysteine and antibiotic solutions............... activated................. activated .......16........................10....25g pH 6.............. Preparation of Medium: Add components......... Preparation of Medium: Add components.. 2.. Adjust medium to pH 6....... Cool to 50°–55°C.......0g α-Ketoglutarate..................0mL: Agar .0mL of sterile antibiotic solution.0L. 4......0mL................... 1.... 0........ Vancomycin..........9 ± 0.............................. 0..................... 5... except cys- teine.................. to distilled/deionized water and bring volume to 1.. 10................................ BCYE Agar (BCYE Alpha Base) (Buffered Charcoal Yeast Extract Agar) Composition per liter: Agar ........ Legionella pneumophila appears as light blue/green colonies...0mL of L-cysteine·HCl·H2O solution and 10........... 15.. Heat gently and bring to boil for 1 min..........9 ± 0...0L..............................................0g L-Cysteine·HCl·H2O ...........4g Fe4(P2O7)3·9H2O .............................0g Yeast extract....................25g Bromcresol Purple ................. Azides also react with metals and disposal must be highly diluted........................... except Lcysteine·HCl·H2O...........0g L-Cysteine·HCl·H2O ..................9 ± 0................... 0.................................... Mix thoroughly..... to distilled/deionized water and bring volume to 1..... Use: For the isolation....... Preparation of Medium: Add components distilled/ deionized water to 1..................................80...................................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid).........................9 61 with 1N KOH.............. Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly......... Use: For the isolation........................................ cultivation.... Adjust medium to pH 6.............032g pH 6...........................0g α-Ketoglutarate.........0g K2HPO4 .. 10.................. 0.... Mix thoroughly...... Legionella micdadei appears as blue/gray or dark blue colonies... ..................................................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ......... cultivation.............0mL of a 10% solution of L-cysteine·HCl·H2O that has been filter sterilized.........0g Yeast extract ..................0mL........ Distribute into tubes or flasks...................... Antibiotic Solution: Composition per 10........ cultivation..................................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) . 10.0g Charcoal..... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension..... Mix thoroughly.0.........................................0mg Vancomycin ....................4... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension................................................ Add 4............................ Mix thoroughly.... Autoclave for 15 min at 15 psi pressure–121°C................................... Cool to 50°– 55°C.... and maintenance of Legionella pneumophila and other Legionella species from environmental water samples...........0mg Cephalothin ..4g Fe4(P2O7)3·9H2O ...............7g KH2PO4 .... BCYE Selective Agar with CCVC (Buffered Charcoal Yeast Extract Selective Agar with Cephalothin...... For the presumptive differential identification of Legionella species based on colony color and morphology........ 0......................... 10........ Use: For the isolation...... Preparation of Medium: Add components.............. Cool to 50°–55°C........BCYE Selective Agar with CCVC D-Glucose............................ activated....... 5.01g pH 6......... 0........ 2..............5g Bromcresol Purple .............. 2.... Autoclave for 15 min at 15 psi pressure–121°C.. 0............0g α-Ketoglutarate .....................9 with 1N KOH........ L-Cysteine·HCl·H2O Solution: Composition per 10................0........................................ 15....25g Antibiotic solution..... 10.....1..........2.... Gently heat to boiling................9 with 1N KOH.0mg Colistin .......2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems.0mL pH 6........................0g NaCl ............... Adjust medium to pH 6.. Mix thoroughly.............................01g Bromothymol Blue .2 at 25°C Caution: Sodium azide is toxic..................... to distilled/deionized water and bring volume to 1... Add 4.. Autoclave for 15 min at 15 psi pressure–121°C..................0mL of a 10% solution of L-cysteine·HCl·H2O that has been filter sterilized......... 0.... and maintenance of Le- gionella pneumophila and other Legionella species from environmental water samples................ Heat gently and bring to boil for 1 min....0g Fe4(P2O7)3·9H2O .... 1.....0mL: L-Cysteine·HCl·H2O.................. Heat gently and bring to boil for 1 min......0g Charcoal...... 2......................0mL: Cycloheximide . For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from environmental water samples....... Colistin......... and Cycloheximide) Composition per 1014....................5mg Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10.................9 ± 0.0L...........15........0g Charcoal..................................................................... Use: For use in the confirmation test for the presence of fecal streptococci in water and wastewater.....10............0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10............ and maintenance of Legionella pneumophila and other Legionella species from environmental samples................................1..............

......4........... 0....................................................9 with 1N KOH.... and Cefamandole) Composition per 1014..........0mL............0mL pH 6...10.. and Cycloheximide) Composition per 1014.. Cool to 50°–55°C.. 3.....1..0g Yeast extract ..... Antibiotic Solution: Composition per 10........................80...... Polymyxin B...0mL L-Cysteine·HCl·H2O solution ..0g Charcoal........ 0..... 2...... Antibiotic Solution: Composition per 10.......0g α-Ketoglutarate .......08g Vancomycin ...................... Heat gently and bring to boil for 1 min....000U Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10.......... L-Cysteine·HCl·H2O Solution: Composition per 10.................................................................9 ± 0.................................9 with 1N KOH................... except Lcysteine·HCl·H2O solution and antibiotic solution.0g Charcoal.......... Heat gently and bring to boil for 1 min...................... 100....0g Yeast extract.......................................... Preparation of Medium: Add components.. activated ................................... Mix thoroughly.......................................... 10........................................................... Use: For the isolation. Preparation of Medium: Add components...... Preparation of Medium: Add components.....0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid)..............................................25g Antibiotic solution ......................................... to distilled/deionized water and bring volume to 1....................................... Vancomycin........ 0.................................................... Mix thor- ...9 ± 0................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems................. 2........ Polymyxin B..............1...................................79.....................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) .............0mL pH 6............ Mix thoroughly....0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10....0mL: Agar ......9 ± 0................ Filter sterilize..........................................4....10..... 1..... For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples.... except Lcysteine·HCl·H2O solution and antibiotic solution...0g Fe4(P2O7)3·9H2O ....0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ...................0mg Polymyxin B...... Mix thoroughly.........0mL: Glycine... Antibiotic Solution: Composition per 10....... 10....0...25g Antibiotic solution... Filter sterilize.................. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.... Mix thoroughly..................................................... cultivation..................... Vancomycin..0g α-Ketoglutarate....0mg Polymyxin B .......................0mL of Lcysteine·HCl·H2O solution and 10................... Mix thoroughly..................................0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10................1....... Add 4...........................0mL: L-Cysteine·HCl·H2O.0g Charcoal...0g α-Ketoglutarate... 1...2.... For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples.....10.0mg Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10..3... cultivation........................... Anisomycin............ Mix thoroughly................. to distilled/deionized water and bring volume to 1....62 BCYE Selective Agar with GPVA BCYE Selective Agar with GPVA (Buffered Charcoal Yeast Extract Selective Agar with Glycine...0mL L-Cysteine·HCl·H2O solution ............................0mL: Glycine ........................... and maintenance of Legionella pneumophila and other Legionella species from environmental samples..................0mL....................0mL. Adjust medium to pH 6.0mL of L-cysteine·HCl·H2O solution and 10............ Add 4................ Adjust medium to pH 6.. activated......... and maintenance of Le- gionella pneumophila from environmental samples..............10.................. Mix thoroughly................................0L...................................................................15......0mL: Agar .0mL: L-Cysteine·HCl·H2O..........08g Vancomycin .............0g Fe4(P2O7)3·9H2O ..................................0mL: L-Cysteine·HCl·H2O .....2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath..................... Cool to 50°–55°C...........................................1.................. BCYE Selective Agar with GVPC (Buffered Charcoal Yeast Extract Selective Agar with Glycine.. 10.. Use: For the isolation............. and Anisomycin) Composition per 1014......................................2..........................0mL L-Cysteine·HCl·H2O solution .................. Filter sterilize.0mL.0g Fe4(P2O7)3·9H2O ................0mL of sterile antibiotic solution... 15................ activated.0L............ 10....0L.0mg Cefamandole......200U Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10....................0....... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension... BCYE Selective Agar with PAC (Buffered Charcoal Yeast Extract Selective Agar with Polymyxin B. 1......0mL pH 6............. Mix thoroughly..... Autoclave for 15 min at 15 psi pressure–121°C......0mL........ 10.................... 15...................................0g Anisomycin ...0mL..... Autoclave for 15 min at 15 psi pressure–121°C.............80.................. 5............ 4.............0mL: Polymyxin B.25g Antibiotic solution ................. except Lcysteine·HCl·H2O solution and antibiotic solution.......000 units Anisomycin ............ Filter sterilize.....2 at 25°C L-Cysteine·HCl·H2O Solution: Composition per 10.........0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10........0mL: Agar .......... Filter sterilize.... to distilled/deionized water and bring volume to 1. Filter sterilize...............0g Cycloheximide ......0g Yeast extract.................... L-Cysteine·HCl·H2O Solution: Composition per 10. Mix thoroughly......0mL of sterile antibiotic solution........................................ Mix thoroughly.

.......... Fe4(P2O7)3·9H2O solution....................0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10........ Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.......... Heat gently and bring to boil for 1 min........................... Add 4... 10... 1. Antibiotic Solution: Composition per 10............. Mix thoroughly............. Mix thoroughly................. Cool to 50°–55°C.. BCYEα with Alb (Buffered Charcoal Yeast Extract Agar with Albumin) Composition per liter: Agar .9 ± 0.. activated .........2......0mL............. Heat gently and bring to boil for 1 min..000 units Anisomycin .... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.......... Aseptically add the L-cysteine·HCl·H2O solution.... L-Cysteine·HCl·H2O Solution: Composition per 10.... Use: For the isolation.....................9 ± 0....................10..............................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid).........0mL L-Cysteine·HCl·H2O solution .................... Anisomycin............0........ and maintenance of Legionella pneumophila and other Legionella species from environmental samples.... Cool to 50°–55°C...................... Filter sterilize.......0mL.................................. BCYEα without L-Cysteine (Buffered Charcoal Yeast Extract Agar without L-Cysteine) Composition per liter: Agar ........... 2................... Use: For the isolation......................0mL pH 6.................................0mL: L-Cysteine·HCl·H2O.....5mg Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10.......... except Fe4(P2O7)3·9H2O solution...........25g Preparation of Fe4(P2O7)3·9H2O Solution: Add Fe4(P2O7)3·9H2O to distilled/deionized water and bring volume to 10................... activated.............0mL pH 6................9 with 1N KOH. Filter sterilize......... Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C....0mL......0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid)......... 10... Use: For the isolation..10.........0mL: Fe4(P2O7)3·9H2O ...9 with 1N KOH.............................0g Charcoal............................................ Mix thoroughly..10.0mL of sterile antibiotic solution..............0mL: Agar ....... and bovine serum albumin solution—to distilled/deionized water and bring volume to 970... 80.............0mL of sterile bovine serum albumin solution.....9 with 1N KOH......................0g Yeast extract ............... Mix thoroughly......... Heat gently and bring to boil for 1 min.... Autoclave for 15 min at 15 psi pressure– 121°C..9 ± 0.... Mix thoroughly.... and Vancomycin) (Wadowsky–Yee Medium) Composition per 1014............. and 10.................................0mL: Bovine serum albumin ......... Adjust medium to pH 6........... 15............... and maintenance of Legionella pneumophila and other Legionella species from environmental samples..................0mL of L-cysteine·HCl·H2O solution and 10... Fe4(P2O7)3·9H2O solution................. Mix thoroughly.25g Antibiotic solution .. Preparation of Medium: Add components—except Lcysteine·HCl·H2O solution...... Mix thoroughly........ Mix thoroughly........ Adjust medium to pH 6.... Adjust medium to pH 6.................................1...0L...............................0mL of L-cysteine·HCl·H2O solution and 10..... Mix thoroughly............0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) .....BCYEα without L-Cysteine oughly............0g Fe4(P2O7)3·9H2O solution ..0mL.......................2........................0........................ 0......0mL....1g Preparation of Bovine Serum Albumin Solution: Add bovine serum albumin to distilled/deionized water and bring volume to 10........... Preparation of Medium: Add components...0g 63 Yeast extract ............... Fe4(P2O7)3·9H2O Solution: Composition per 10...0mL of sterile antibiotic solution.................................2 at 25°C Fe4(P2O7)3·9H2O Solution: Composition per 10............. Filter sterilize........0mg Vancomycin ........ cultivation........0g Charcoal..... cultivation.................4g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10...... and maintenance of Legionella pneumophila and other Legionella species from environmental samples.0mL pH 6... Filter sterilize.......... to distilled/deionized water and bring volume to 990........ except Lcysteine and antibiotic solution......0mL........... 1.............. Autoclave for 15 min at 15 psi pressure–121°C............0g Fe4(P2O7)3·9H2O .. Filter sterilize............ Filter sterilize................1.......................0g α-Ketoglutarate .. Adjust medium ..... Preparation of Medium: Add components......................0g α-Ketoglutarate.........0... 10...........0mL: L-Cysteine·HCl·H2O .......2 at 25°C Bovine Serum Albumin Solution: Composition per 10.... For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples. activated .....15... 10.....0............................25g Preparation of Fe4(P2O7)3·9H2O Solution: Add Fe4(P2O7)3·9H2O to distilled/deionized water and bring volume to 10.........0mL..... Mix thoroughly........ to distilled/deionized water and bring volume to 1... 0...............0mL L-Cysteine·HCl·H2O solution ............ 15... Cool to 50°–55°C. Mix thoroughly.............. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension...........10......0mL: Polymyxin B .......................0mL....0mL: Fe4(P2O7)3·9H2O ........0g Bovine serum albumin solution..............0g Charcoal....2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems............... BCYE Selective Agar with PAV (Buffered Charcoal Yeast Extract Selective Agar with Polymyxin B......................0mL Fe4(P2O7)3·9H2O solution ......... 10...0g α-Ketoglutarate . 40...................10.. For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from potable water samples.......... L-Cysteine·HCl·H2O Solution: Composition per 10................... cultivation...0g Yeast extract......... Add 4.............. 10.........4..............

........ 0........4 ± 0.1% solution) .. Use: For the cultivation and maintenance of a wide variety of microorganisms......... Bdellovibrio Medium Composition per Petri dish: Base layer agar............................................. 1..............0mL Co(NO3)2 (0........... 1................0g pH 7........................................... 5............01% solution) . 15..... Heat gently and bring to boil for 1 min...... Allow base layer agar to solidify...................... Hosts include Erwinia amylovora....... Heat gently and bring to boiling........ Mix thoroughly...............5g CaCl2 ........... 1...........5......2 ± 0...........0L................ Mix thoroughly................... Autoclave for 15 min at 15 psi pressure–121°C...10.............10............. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......0mL MgSO4·7H2O (1% solution).............................. Beggiatoa and Thiothrix Medium Composition per liter: CaSO4·2H2O (saturated solution)...........................................2........... Cool the semisolid agar to 40°–45°C....6g Preparation of Host Medium: Add components to distilled/deionized water and bring volume to 1... Mix thoroughly............... Adjust pH to 7...............00005% solution)......................... Gently heat and bring to boiling..0L............... Semisolid Agar: Composition per liter: Agar ....................... 0........................................ 10. 10.................1....0L.......... Beef Extract Agar Composition per liter: Agar .......... Use: For the cultivation of Bdellovibrio bacteriovorus and Bdellovibrio starrii........... Incubate host culture for 24–48 hr at 30°C............................0mL MnSO4·4H2O (0..9 with 1N KOH..........0mL ZnSO4·7H2O (0..0L...1% solution)...0mL of host medium..... Recommended for the culture of microorganisms from water..............................................0g Peptone.....2 at 25°C ....0............................0mL Host medium..................... 10........... Pour the base layer agar into a sterile Petri dish....6g Preparation of Semisolid Agar: Add components to distilled/deionized water and bring volume to 1..... Use: For the cultivation and maintenance of a wide variety of microorganisms....0g pH 7................ Adjust pH to 7............................ 5.........0L.........2 Distribute into tubes in 10..........................0mL of the previously grown host culture.. Distribute into tubes or flasks....0mL volumes....0L.............. or Pseudomonas putida............... Use: For the cultivation of Beggiatoa species and myxotrophic Thiothrix species.......2 Distribute into tubes in 10...0g Sodium acetate .....2 Distribute into tubes in 10........................... Autoclave for 15 min at 15 psi pressure–121°C...............0g Peptone.........3........0mL Preparation of EDTA Solution: Add EDTA and FeSO4 to concentrated HCl.....................0mL K2HPO4 (1% solution) .................4 ± 0........ Melt the base layer agar and semisolid agar.........0mL................0mL Na2MoO4·2H2O (0......................1g Catalase ......0mL: FeSO4 . 3.............. 3.0g Peptone......................0..............0g Beef extract ................. Mix thoroughly........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............7.......... Add 1.. Autoclave for 15 min at 15 psi pressure–121°C.............. Base Layer Agar: Composition per liter: Agar ................................ Escherichia coli... Pour over the solidified base layer agar................0g EDTA..0g Peptone..0mL Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 1...... Distribute into tubes or flasks...........2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................0mL Trace Elements: Composition per liter: EDTA solution... 10... Pour into Petri dishes or leave in tubes................. Autoclave for 15 min at 15 psi pressure–121°C......................... Mix thoroughly.. 3........... Autoclave for 15 min at 15 psi pressure–121°C.....0g HCl............................000U pH 7..... Distribute into tubes or flasks.. Beggiatoa Medium (ATCC Medium 138) Composition per liter: Yeast extract ..0mL H3BO3 (0....................0g Agar .............................. Beef Extract Broth Composition per liter: Peptone .....0mL Semisolid agar................................ Heat gently and bring to boiling..... Mix thoroughly.....2. Mix thoroughly............ 20............................................. Use: For the isolation......... Carefully add to distilled/deionized water and bring volume to 100......0mL Host Medium: Composition per liter: Yeast extract................... 6................. 20..... Mix thoroughly.......0mL Trace elements.64 Bdellovibrio Medium to pH 6....................................6g Preparation of Base Layer Agar: Add components to distilled/deionized water and bring volume to 1.............. 10................. EDTA Solution: Composition per 100..................................... and maintenance of Legionella pneumophila and other Legionella species from environmental samples....................... Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.... 3...10...... Mix thoroughly............................. Adjust pH to 7.......... concentrated............. Autoclave for 15 min at 15 psi pressure–121°C..... Mix thoroughly. cultivation................................. Serratia marcescens..............0mL of sterile Fe4(P2O7)3·9H2O solution............. 5...................0mL CuSO4·5H2O (0.......0mL NH4Cl (4% solution) ....... Aseptically add 10...............................................................02% solution)......01% solution) .....0g Beef extract ......................................................0g Yeast extract..... Autoclave for 15 min at 15 psi pressure–121°C..........0mL volumes............ 10............ 19........................ 0.. Recommended for the culture of microorganisms from water.................. Cool to 50°–55°C..0mL volumes........0L..... Preparation of Medium: Inoculate appropriate bacterial host into 10.....................2........ Gently heat and bring to boiling........................................0g Yeast extract....................................

......................0g NaCl .........................0mL of sterile NaHCO3 solution...3.........0mL.................................................0mL: D-Glucose.... Aseptically add 100................................ 100... NaHCO3 Solution: Composition per 10........0L...........0mL..5g pH 5.. and Clostridium barkeri.......5 ± 0............. Mix thoroughly... 5........... Use: For the cultivation and maintenance of Beggiatoa alba and Vitreoscilla species................. 1.................... 0...0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10............ Modified Composition per liter: Agar ................ Mix thoroughly....0mg pH 6................ Beijerinckia species...........................1..................20....... Preparation of Medium: Add components..................................................... Beijerinckia indica............. 25...........8g KH2PO4 ........ Autoclave for 15 min at 15 psi pressure–121°C............ Beijerinckia indica............. Autoclave for 15 min at 15 psi pressure–121°C........................... Benzoate Medium Composition per liter: Noble agar .2g Benzoate solution ..........0g Sodium benzoate ........... Pour into sterile Petri dishes or distribute into sterile tubes Use: For the cultivation and maintenance of Beijerinckia derxii...........0......2g MgSO4·7H2O...0mL......0g K2HPO4 ...................... 0...................... 5.2 at 25°C Preparation of Medium: Add components to distilled/ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....0g KH2PO4 ...... Gently heat and bring to boiling.....................5g MgSO4·7H2O............ 0.2 at 25°C Na2S2O3 Solution: Composition per 100...................................0mg Na2MoO4·2H2O .............1g FeSO4·7H2O ..................... Use: For the cultivation of Beggiatoa alba...........20............0................. Autoclave for 15 min at 15 psi pressure–121°C...........0mg CuSO4·5H2O...... Use: For the cultivation and maintenance of Thiobacillus thermophilica......0mL: Na2S2O3 .......5 ± 0....... Mix thoroughly..................1..................5..................................................... Mix thoroughly...............0g Glucose.............1........................ Autoclave for 15 min at 15 psi pressure–121°C......................5.................2 at 25°C Glucose Solution: Composition per 50......5. 15......0mg Na2MoO4·2H2O..................4.......................... Autoclave for 15 min at 15 psi pressure–121°C...... to tap water and bring volume to 1. 20....................... except glu- cose solution..... 0..1g FeSO4·7H2O..................... Mix thoroughly................................ 0........15.........2g MgCl2 ......... except cat- alase..................0 ± 0...................................................0mL pH 6. Use: For the isolation and cultivation of Beijerinckia derxii.......................0g MgSO4·7H2O ....... Use: For the cultivation of Beijerinckia species...5.............10.......................0mg MnSO4·H2O.....................0...........5g Sodium acetate ...0mL.................. Beijerinckia fluminensis.........................0mg ZnSO4·6H2O ..............0g KH2PO4 ...................................................................................0... deionized water and bring volume to 1........... Autoclave for 15 min at 15 psi pressure–121°C...................................... Cool to 45°–50°C................0g Na2HPO4 .......................0mL pH 7... and Beijerinckia mobilis......................... Pour into sterile Petri dishes or leave in tubes................................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 50....2 at 25°C 65 Beijerinckia Medium............................................................ Beijerinckia fluminensis..................................................................................................0L...............1g NH4Cl .........50....................................... Filter sterilize...... Mix thoroughly..... Beijerinck’s Thiobacillus Medium Composition per liter: Noble agar ............ Beijerinckia mobilis............................................................... Distribute into tubes or flasks........0mL: NaHCO3 ................0mg Glucose solution .............................. 20......1g Na2S2O3 solution ................................. Aseptically add 10....... 2...................... Beijerinckia Medium Composition per liter: Glucose .......... Distribute into tubes or flasks..................................................................0mL NaHCO3 solution.................................................... Preparation of Medium: Add components.............................................................................................................................01g pH 7. Autoclave for 15 min at 15 psi pressure–121°C.3mg ZnSO4·7H2O......0mg CuSO4·6H2O ....................20............... 4.....0L....... Beggiatoa Medium (ATCC Medium 1193) Composition per liter: Sodium sulfide ................0...........2g Yeast extract ..... Mix thoroughly... except Na2S2O3 solution and NaHCO3 solution......... 10.... Mix thoroughly..01g Yeast extract.......0........................ to distilled/deionized water and bring volume to 890.......................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........0mL ..............0g Preparation of Na2S2O3 Solution: Add Na2S2O3 to distilled/deionized water and bring volume to 100...........000 units of sterile catalase..........0mg MnSO4·6H2O ................... 0........................................ Distribute into tubes or flasks...........................................0g K2HPO4 ..........3..............5 ± 0......................................01g Nutrient broth.................. 0................................ Beijerinckia Agar Composition per liter: Agar .....................0mL............0............................. 0.................... Gently heat and bring to boiling..............8g KH2PO4 ..................0L................0g (NH4)2HPO4 ...... Mix thoroughly.......... 10.0–7......... to distilled/deionized water and bring volume to 950................................ Mix thoroughly.......... Filter sterilize..... Aseptically add 10........................ Mix thoroughly............................0.....0mL of sterile Na2S2O3 solution and 10..............................0mL of sterile glucose solution.....2g MgSO4·7H2O ...................... Pour into sterile Petri dishes or leave in tubes...................Benzoate Medium Preparation of Medium: Add components.....

..............0mL Preparation of Solution B: Add components to distilled/deionized water and bring volume to 300..... Use: For the cultivation of Pseudomonas putida and other microorganisms that can utilize benzoate as a carbon source.. 3... 0.......3............................0mg Trace metals solution.......................0L. Gently heat and bring to boiling.................... Mix thoroughly..0mL: Sodium benzoate.......................................0................. Mix thoroughly.................................. Heat gently to boiling... Mix thoroughly......0mL Phosphate Buffer Solution: Composition per 200................. Mix thoroughly....... Preparation of Medium: Add components................... 1......... Pour into sterile Petri dishes or leave in tubes..........0 with KOH............0g Sodium benzoate ... to distilled/deionized water and bring volume to 600....... Benzoate Medium II Composition per 1................................................0mL pH 8...................0mL..................0mL......... Aseptically add the sterile benzoate solution...0g Preparation of Benzoate Solution: Add sodium ben- zoate to distilled/deionized water and bring volume to 25..........23g Preparation of Ferric EDTA Solution: Add EDTA and KOH to distilled/deionized water and bring volume to 186......0.........0mL: MgSO4·7H2O.... 200.............. 1................2g FeSO4·7H2O.... Mix thoroughly........................... 0.......... 1........ 700...0mL.....................2g Yeast extract..... Use: For the cultivation of Pseudomonas putida and other microorganisms which can utilize benzoate as a carbon source.......................... Cool to 45°–50°C.......................0mg Na2MoO4·2H2O................ Solution B: Composition per 300........................................................... Autoclave for 15 min at 15 psi pressure–121°C......................7g KOH ........... anhydrous ................................... Mix thor- oughly......3g Phosphate buffer solution........... 3.................0mL: MnSO4·H2O..0mL............0mL...0mL Preparation of Trace Metals 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 950... Aseptically add 25........0g (NH4)2HPO4 ....0mg Na2B4O7·10H2O .......1. Cool to room temperature....... Autoclave for 15 min at 15 psi pressure–121°C.0mL: Sodium benzoate........................0mL..................2 at 25°C Solution A: Composition per 700.............0mL: EDTA........... Mix thoroughly... Mix thoroughly..............5g Preparation of Benzoate Solution: Add sodium ben- zoate to distilled/deionized water and bring volume to 25.............2g Citric acid.......0mL sterile benzoate solution.............................5L: Noble agar.... Cool to room temperature.......... Cool to 45°–50°C............. Autoclave for 15 min at 15 psi pressure–121°C.................0mL: K2HPO4 ....0mL............................5g MgSO4·7H2O .....2mg Ferric EDTA solution ..........2 at 25°C Benzoate Solution: Composition per 25...............................2g CaCl2 ............ Benzoate Minimal Salts Medium Composition per liter: K2HPO4 ......... Filter sterilize................. 10............10....0mL pH 7.... add FeSO4·7H2O to distilled/deionized water and bring volume to 364.0mL Solution B................... 300.1..10..............25............ Trace Metals Solution: Composition per 300..................0mg ZnSO4·7H2O.................0g NaNH4HPO4·4H2O ..................................... 0.............. 0. Mix thoroughly................ Use: For the cultivation of microorganisms that can utilize benzoate as a carbon source........ Cool to 45°C............. In a separate flask.13.............. Combine the two solutions...........0mL...... 3..................................................5g MgSO4·7H2O .................5g Preparation of Phosphate Buffer: Add components to distilled/deionized water and bring volume to 200.. Sparge with air overnight to oxidize the Fe2+ to Fe3+....0mL............. Mix thoroughly.0 ± 0...........2...................................................... Mix thoroughly.0mL............. Preparation of Solution A: Add components to distilled/deionized water and bring volume to 700..................... Mix thoroughly..... 1............................................ 30.....................67g KH2PO4 ... Adjust pH to 9........... Aseptically distribute into sterile tubes or flasks..0...........0mL: Sodium benzoate..................... Autoclave for 15 min at 15 psi pressure–121°C.. Preparation of Medium: Add components except ben- zoate solution to distilled/deionized water and bring volume to 975.....................0g NH4Cl ......... except agar and sodium benzoate........ Mix thoroughly....................50............. Mix thoroughly and pour into sterile Petri dishes or leave in tubes... In a separate flask....10................17.......0mL..10...... Autoclave for 15 min at 15 psi pressure–121°C.....................0mL Benzoate Solution: Composition per 25............................0mL of sterile benzoate solution....................................................... 10....................................0mg CuSO4 ...12g KH2PO4 ..................... Autoclave for 15 min at 15 psi pressure–121°C.............. Mix thoroughly........0g Preparation of Benzoate Solution: Add sodium ben- zoate to distilled/deionized water and bring volume to 25......................0mg Co(NO3)2·6H2O..........................................0mL.... 0......................... 2............................. Aseptically combine the two autoclavesterilized solutions......................2g Benzoate solution..............25...............66 Benzoate Medium II Benzoate Solution: Composition per 25.............. Benzoate Nitrate Salts Medium (BNS) Composition per liter: Solution A ......................................................... Cool to 45°– 50°C...........5............... Adjust pH to 7........... add agar to distilled/deionized water and bring volume to 375.......... Store in the dark...... Mix thoroughly.....................0mL: KNO3 ....... Ferric EDTA Solution: Composition per 550...........0..............................................50............9g FeSO4·7H2O ...1g Benzoate solution..........................2 ± 0.. Aseptically add 25..... ...................................................... Filter sterilize........ Mix thoroughly........ Filter sterilize.0g NaCl ...........................................

................. 0.............................................................. For the isolation of cyanobacteria from freshwater habitats...................................................... 2.........0............... BG 11 Uracil Agar Composition per liter: Agar ............................................... 0.. Distribute into tubes or flasks.. 10............. Aseptically add 100.......1.......0.........079g Co(NO3)2·6H2O .....10......................................... 67 Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1.......222g CuSO4·5H2O.............0................075g K2HPO4 ................ 10....2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 .................0mg EDTA disodium salt ..........................................................036g Na2CO3 ........0L..................2.....1.... Use: For the cultivation and maintenance of Synechococ- cus species. Mix thoroughly..................... Fill tubes completely..................1.....0.... Preparation of Medium: Add components......... Autoclave for 15 min at 15 psi pressure–121°C............... Filter sterilize.............81g Na2MoO4·2H2O............... Mix thoroughly..............0..............0L..0g NaNO3 .......................075g K2HPO4 .................................. 100.......049g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1.............049g Vitamin B12.............. Mix thoroughly........................... Mix thoroughly................0mL of sterile Vitamin B12 solution..........................0mg Trace metal mix A5..........................075g K2HPO4 ..............................0mg Ferric ammonium citrate ....................................... Heat gently to boiling...................... 0...........39g ZnSO4·7H2O ..........0mL....... Filter sterilize..BG 11 Uracil Agar Preparation of Medium: Aseptically combine 700.5g MgSO4·7H2O............ 6...............................0mL............. except Vi- tamin B12 solution....................................079g Co(NO3)2·6H2O ......................... 0....... Adjust pH to 8..............0mL of sterile solution A with 300...........0mL: Vitamin B12. 0.....39g ZnSO4·7H2O .......86g MnCl2·4H2O................86g MnCl2·4H2O.02g Citric acid.........................1 ± 0...6.2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 ......................................................................... Distribute into sterile tubes or flasks....049g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1............0mg Vitamin B12 solution ......0mL pH 7................ 0....1..........................0.... Agar .02g Citric acid.......................... 6..........................................................................81g Na2MoO4·2H2O ...... For solid medium............................................. 6...................036g Na2CO3 ................ except Vi- tamin B12 solution.....................2................................................... 0............ 1.............0mg Ferric ammonium citrate. 0............................................ Use: For the cultivation and maintenance of a wide variety of cyanobacteria..0L.................0mg Ferric ammonium citrate. Mix thoroughly................ Autoclave for 15 min at 15 psi pressure–121°C. to distilled/deionized water and bring volume to 900... Mix thoroughly......................... Vitamin B12 Solution: Composition per 100............0mL of sterile Vitamin B12 solution.......................................................... 0.0g ........................... Pour into sterile Petri dishes or leave in tubes............ Mix thoroughly........................ to distilled/deionized water and bring volume to 900..........................................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...............0µg Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 100........... 1...................... Aseptically add 100...................................... 0... 0....... Vitamin B12 Solution: Composition per 100...............222g CuSO4·5H2O ............... Heat gently to boiling.. 0....................02g Citric acid .......................0mg EDTA disodium salt.....................................................0mL....... pour into sterile Petri dishes or leave in tubes...........079g Co(NO3)2·6H2O..................................100............ Use: For the cultivation and maintenance of Synechococcus species..0mg Vitamin B12 solution.........................0mL pH 7...... Heat gently to boiling.. BG 11 Marine Broth (Medium BG 11 for Marine Cyanobacteria) Composition per liter: NaCl ............................................0mg Disodium EDTA .........04g CaCl2·2H2O ..............................................39g ZnSO4·7H2O.......... Mix thoroughly.........................................5g MgSO4·7H2O ............................... 0........................................................................... Aseptically distribute into sterile screwcapped tubes...................................................0L....0mL of sterile solution B..................................... 1................................................................................................................. 10............. Mix thoroughly..................... 0.............86g MnCl2·4H2O ..........0..........0g NaNO3 .............................................................2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 ..........................0mL Trace metal mix A5.......................1 ± 0.............. 0............................036g Na2CO3 ...................0.......... 1.....6........................................................................5g MgSO4·7H2O ..0mL pH 7.........................................................04g CaCl2·2H2O....................222g CuSO4·5H2O .... 0...................................................................................................................................04g CaCl2·2H2O................. 1......0g NaNO3 ............0mL Trace metal mix A5 ................................................0g NaCl ................. Mix thoroughly................ 1................1......... For the isolation of cyanobacteria from freshwater habitats...................0µg Preparation of Medium: Add components.... BG 11 Marine Agar (Medium BG 11 for Marine Cyanobacteria) Composition per liter: Agar ...............81g Na2MoO4·2H2O .................. Use: For the cultivation of Alcaligenes xylosoxydans...... 1......... Autoclave for 15 min at 15 psi pressure–121°C...............10.............................1 ± 0.... 2.........................................0mL........................ 1.. 1......... 6..... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....................................

................10....81g Na2MoO4·2H2O .................................. to 1..............................................0.......075g K2HPO4 ..........68 Bile Salts Gelatin Agar Uracil ......... Pour into sterile Petri dishes................ Biphenyl Agar (DSMZ Medium 457d) Composition per liter: Agar ....0.... 1...0L.....2g ZnSO4·7H2O........................................................0g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1........036g Na2CO3 .....8g NaNO3 ....................... and let the ethanol evaporate so that the crystals of biphenyl coat the lid of the Petri dish.............. 1................0................................ 1.5 ± 0........................02g Citric acid........................... Use 2........0mL pH 6.. Mix thoroughly. Preparation of Medium: Add components to distilled/ Preparation of Medium: Add components............1 ± 0............... 2....5g MgSO4·7H2O .........03g Na2MoO4·H2O.......................................................................... Blastobacter denitrificans Agar (LMG Medium 157) Composition per liter: Agar ................03g NiCl2·6H2O.......................... 0..............5g FeSO4·7H2O. Distribute into tubes or flasks.......................... Use: For the selective isolation of Salmonella typhi and other enteric bacilli and for the detection of Salmonella by the membrane filter method............ 0..... 1..........................................0L distilled/deionized water..............................0mg Trace metal mix A5...................................0L....................................................................................................5g Yeast extract ...0........................................ 2...........................0.......................10............0L.........................0L ethanol.........................0....................................0mL pH 7................ 0...0....... 0............. Autoclave for 15 min at 15 psi pressure–121°C.... 0.. Heat gently to boiling. Mix thoroughly.................................... 3..... Filter sterilize using a cellulose filter membrane....0g FeSO4·7H2O ........0g Pancreatic digest of casein .0g Na2HPO4 ...2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........................52g (NH4)2SO4 ........................0g NaCl .......................................................... 0..........2 at 25°C .............. 10.........0g Tryptone .......... 2.............3g CoCl2·6H2O.............................................. except bi- deionized water and bring volume to 1............... Heat and gently bring to boiling..........5g Na2CO3 ......... 1...2g Glucose solution ...... Mix thoroughly...... Distribute into tubes or flasks................................ Mix to disperse the precipitate and aseptically distribute into sterile tubes or flasks................... 0......................... Bismuth Sulfite Broth (m-Bismuth Sulfite Broth) Composition per liter: Bi2(SO3)3 ............................................................ Use: For the cultivation and maintenance of Anabaena variabilis......................5g Sodium acetate .............................. 0......................................................7 ± 0..................100......0mL: Gelatin..049g Trace Elements Solution SL-6: Composition per liter: H3BO3 ..................... 0..........................................2g Trace elements solution SL-6 ..... 1.....................................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.......04g CaCl2·2H2O...............2g CaCl2·2H2O................. Mix thoroughly.............................. Boil for 1 min...............6g pH 7..........................................................................10.................................... 0..................... 0.......10... Do not autoclave................10.................0g Sodium taurocholate .0L................. 1...................100............. Autoclave for 15 min at 15 psi pressure–121°C.............................................02g CuCl2··2H2O ................................39g ZnSO4·7H2O .............1........ Mix thoroughly. 0.....0g Glucose................................ 0......2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 ......0g Lab Lemco beef extract....16....1g Water ..... Use: For the cultivation of Vibrio cholerae................................... Bile Salts Gelatin Agar Composition per 100.......................0L........... 15..........................................................................0L........0........................................................5g Biphenyl...........1g MnCl2·4H2O .. Aseptically add sterile agar medium to the crystal-layered Petri dish.2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......................................0g Na2HPO4 ...............................0g Beef extract ............................25g MgSO4·7H2O ................................................0mg Ferric ammonium citrate........ Use: For the cultivation of biphenyl-utilizing bacteria...........................15..............0mL phenyl solution.......0mL pH 8......... Pour into sterile Petri dishes or leave in tubes.......................................................44g KH2PO4.............222g CuSO4·5H2O .......... Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1..................5g Pancreatic digest of casein ...9........ Add an aliquot of the biphenyl solution to the lid of a sterile Petri dish so that the final concentration will be approximately 0.....2mL of medium for each membrane filter.........................................3 ± 0.10............05g Trace elements solution SL-4 . 6................................................4...2............................86g MnCl2·4H2O.........0mL pH 7........................................... Mix thoroughly and heat with frequent agitation until boiling...................... Gently heat and bring to boiling.. Cool to 45°–50°C.8.................................... Adjust pH to 3...................0–2...................9 ± 0...................... 0.............................................................. 0................ Cool to 50°C....... Mix thoroughly.............................................. 6..............................................................................................0... 0........................0mg EDTA disodium salt.............................0................................ Adjust pH to 6.....2 at 25°C Trace Elements Solution SL-4: Composition per liter: EDTA ...................... Biphenyl Solution: Composition per liter: Biphenyl .........................................079g Co(NO3)2·6H2O .0g Agar ..................................................... Preparation of Biphenyl Solution: Add biphenyl to 1..................................0................0g Peptic digest of animal tissue ........25g/ L biphenyl.............................................. Autoclave for 15 min at 15 psi pressure–121°C..............................................

..........5g MgSO4·7H2O.....0L........................0g tamin B12 solution.....................................6.....1....................04g CaCl2·2H2O ....Blue-Green Nitrogen-Fixing Agar Glucose Solution: Composition per 10................................. Mix thoroughly...... Autoclave for 15 min at 15 psi pressure–121°C.............................................0.....0mL: Vitamin B12.................. Aseptically pour into sterile Petri dishes or distribute into sterile tubes... 500............................10......................................................5g Preparation of Glucose Solution: Add glucose to 10.8 ± 0.........2.............. Use: For the cultivation and maintenance of Synechococcus species.....0L...................................................... except Vi- Nutrient agar .......................................079g Co(NO3)2·6H2O.......................... 1.......................................0L..........5g L-Cystine·HCl ............02g Citric acid ........................0mL............................ defibrinated blood to a final concentration of 5%... Use: For the cultivation of Blastobacter denitrificans..0mL of sterile glucose cystine solution and 5.........0mL........0g pH 6............................. Preparation of Medium: Add compents........................... ..................... Aseptically add 10...... 12.....0mL Human blood........ Gently heat while stirring and bring to boiling. 0........... Autoclave for 15 min at 15 psi pressure–121°C......0..............0.. Vitamin B12 Solution: Composition per 50......... Mix thoroughly and pour into sterile Petri dishes.. Mix thoroughly. Cool to 45°–50°C.............. Cool to 45°-50°C..2 at 25°C Nutrient Agar: Composition per liter: Agar .................................... Cool the basal medium to 45°–50°C.......0.. Autoclave for 15 min at 15 psi pressure–121°C......................... Mix thoroughly......... Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1............................................................. Mix thoroughly........ 69 Blue-Green Agar Composition per liter: Agar ............6.................... Heat gently and bring to boiling.....0........................ except glucose solution............. Mix thoroughly...0mL.................................................................. 5...............................036g Na2CO3 ................................... Filter sterilize........ Mix thoroughly...... Blood Glucose Cystine Agar Composition per 100..........0L.......................0g MgSO4·7H2O.......................................1.....0mg Ferric ammonium citrate ....... Heat with frequent agitation and boil for 1 min to completely dissolve...0mL pH 6...1 ± 0.0mL Glucose cystine solution .......2...39g ZnSO4·7H2O..................0mL pH 7................................... Mix thoroughly......................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....0mL......................................................075g K2HPO4 .........................................................0................................075g K2HPO4 .......... Mix thoroughly.............................................................................................................04g CaCl2·2H2O ...................... ponents to distilled/deionized water and bring volume to 50................. Filter sterilize..0mL Trace metal mix A5 ........................... Blood Agar Base with 2....................... Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 50...5g Trace Metal Mix A5: Composition per liter: Preparation of Glucose Cystine Solution: Add com- H3BO3 ................ to distilled/deionized water and bring volume to 990. Autoclave for 15 min at 15 psi pressure–121°C........... infusion from ..................2 at 25°C Trace Metal Mix A5: Composition per liter: H3BO3 ........... aseptically add 10....0mL: Preparation of Medium: Add components........02g Citric acid .................0.0g Tryptose .. Preparation of Medium: To 85.............................0mL of human blood.....1.....8 ± 0... fresh...0mg Trace metal mix A5 .....0mg Vitamin B12 solution................ Filter sterilize.....0g NaCl ......0.................0.... Preparation of Nutrient Agar: Add components to distilled/deionized water and bring volume to 1. 2...........85....................0............10...... to glass-distilled water and bring volume to 950...........................0g NaNO3 ........1.................................................................................. 3......................................0mg EDTA disodium salt ........................1......39g ZnSO4·7H2O............................ Mix thoroughly......0mL: Glucose ................5..................................................................... Aseptically add sterile...222g CuSO4·5H2O.............0g Agar .....049g Preparation of Trace Metal Mix A5: Add components to distilled/deionized water and bring volume to 1........... 30...6.....5% NaCl Composition per liter: Beef heart..................... Add Vitamin B12 solution.0g Pancreatic digest of gelatin ...................1 ± 0.81g Na2MoO4·2H2O........................... Pour into sterile Petri dishes or distribute into sterile tubes....81g Na2MoO4·2H2O.... Distribute into tubes or flasks......................................... sterile agar solution.........................0g Beef extract ........0mL of distilled/deionized water..... Use: For the cultivation of Francisella tularensis..........0mL glucose solution........................... 10........... Mix thoroughly...............................10... Mix thoroughly.....0mL of cooled............................... 15......................... 15...01g Use: For the cultivation of Paracoccus halodenitrificans.......0.....................0..........................036g Na2CO3 ............0mL pH 7............. Mix thoroughly......2 at 25°C Glucose ...........................0...... Pour into sterile Petri dishes or distribute into sterile tubes.......................6... Blue-Green Nitrogen-Fixing Agar Composition per liter: Glucose Cystine Solution: Composition per 50.............................0mg Ferric ammonium citrate .................................86g MnCl2·4H2O .....0mg EDTA disodium salt .............................0mL: Noble agar ... 1... 50...................049g Source: Nutrient agar is available as a premixed powder from BD Diagnostic Systems........222g CuSO4·5H2O... Cool the basal medium to 45°–50°C.................0....86g MnCl2·4H2O ......................................................0...............................0................................0.......079g Co(NO3)2·6H2O..........................................

........0 ± 0........................................20..................0mL..5g of sucrose........0mL Bromothymol Blue ....................................................0L...1.............0......70 BMPA-α Medium Preparation of Medium: Add components to glass dis- Preparation of Potato Extract Solution: Wash......0g KH2PO4 ... Aseptically add 1..0g KH2PO4................ Mix thoroughly... Add 1.. 10.................1....................0g Pyridoxine . BMPA-α Medium (Edelstein BMPA-α Medium) Composition per liter: Agar ................0mL L-Cysteine·HCl·H2O solution ......... 0..... Use: For the selective isolation and cultivation of Legionella pneumophila and other Legionella species.........000U Preparation of Antibiotic Inhibitor: Add compo- and slice several large potatoes.......0g NaCl ..... Bovine Albumin Tween 80 Medium......0g Na-succinate ... Adjust medium to pH 6...................... 2....0L of distilled/deionized water.10............................................ 1.9 ± 0............... Add 20................................0g of agar and 2.....5g of L-malic acid in 50.............................1....1. Filter sterilize..... Mix thoroughly...........1g Na-glutamate ......5......... Mix thoroughly.. Autoclave for 15 min at 15 psi pressure–121°C........... 200..0g Potato extract solution .. 2...0mL pH 6................0...0mL of sterile vitamin solution................................. Mix thoroughly............................................. Place the bag with the potatoes in 1.....................0mL of the sterile L-cysteine·HCl·H2O solution and 10.......................................... Mix thoroughly............................. Add 950... Antibiotic Inhibitor: Composition per 10.............. Mix thoroughly.............0mL ....950................. except cys- teine and antibiotic inhibitor............... Use: For the cultivation of Bosea thiooxidans.........................1g pH 7............5g KOH........ anhydrous................ peeled..........5............ 20................................................................................................ anhydrous.... Mix thoroughly................... activated............................ Mix thoroughly........ 13...............0mL Preparation of Bromothymol Blue Solution: Add Bromothymol Blue to 100............0mL pH 7.........0mL.................... Mix thoroughly............................. 1............... Heat gently and bring to boiling..5g MgCl2 ............ 2..... Modified (Albumin Fatty Acid Broth.............0mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 100..... nents to distilled/deionized water and bring volume to 10..........0g L-Malic acid ................ 2.........2 at 25°C Potato Extract Solution: Composition per liter: Potatoes....0mL: Anisomycin .........................................................0L..........0g lipoferum...0. Adjust pH to 7..0g Yeast extract................ 100.......0mL......0mL: tilled water and bring volume to 1.10..........................08g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10............................ Filter sterilize............................0mg Polymyxin B .... Cool to 50°–55°C... washed.....................0g Na2HPO4 ................ 0........................ 80...........4....... Gently heat and bring to boiling.............0mL of the sterile antibiotic solution................5-8............ Pour into sterile Petri dishes or distribute into sterile tubes...........0mL: Bosea thiooxidans Medium (DSMZ Medium 763) Composition per liter: L-Cysteine·HCl·H2O ........................................... Mix thoroughly..0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ....2 at 25°C Source: This medium is available as premixed vials from Oxoid Unipath...........5g Sucrose.....0mL of potato extract solution.........0g α-Ketoglutarate............... Pour into sterile Petri dishes or distribute into sterile tubes. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension....... cherella.......................................................5 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... Vitamin Solution: Composition per 100............0...................................2g Fe4(P2O7)3·9H2O ........... 4.0.............................. Add 10.......................................9 with 1N KOH..................0mL of 95% ethanol.............0mL of distilled/deionized water.......08g Cefamandole .....5g Yeast extract ... 2.......0 by adding KOH so that the solution is green. 900.....................0mL Albumin fatty acid supplement ..... Leptospira Medium) Composition per liter: Basal medium ..............................................0mL Glycerol (10% solution) .....0mL of Bromothymol Blue solution................. peel........... Autoclave for 15 min at 15 psi pressure–121°C.............0mL: Biotin ..................... Na2S2O3·5H2O.05g Antibiotic inhibitor ... Autoclave for 15 min at 15 psi pressure– 121°C.....10..... Heat gently and bring to boiling for 1 min..................3g NH4Cl (25% solution) ........................................... and Nostoc species.0mL Vitamin solution.... Bromothymol Blue .... BMS Agar Composition per liter: Agar . Mix thorougly.. Use: For the cultivation and maintenance of Azospirillum L-Cysteine·HCl·H2O Solution: Composition per 10......0mL................. 100....... Check pH after autoclaving and readjust if necessary.. Filter sterilize......................... Preparation of Medium: Add components....................... Use: For the cultivation and maintenance of Calothrix...............................................................0g Charcoal........................... Filter through cotton..... 0................................. Cool to 50°–55°C. Preparation of Medium: Dissolve 2............ to distilled/deionized water and bring volume to 980........... Place the poltato slices in a gauze bag.5g Ethanol...... 95% .... and sliced ...... 0... Boil for 30 min......1.....0mL Basal Medium: Composition per liter: Na2HPO4... Ellinghausen and McCullough....... Distribute into tubes or flasks....... Fis- Bromothymol Blue Solution: Composition per 100.............. Autoclave for 15 min at 15 psi pressure–121°C..............................

.. Bovine Albumin Tween 80 Semisolid Medium.....................................0L....1.........2..0mL: Bovine albumin fraction V ....1..0mL with distilled/deionized water.....................2 at 25°C Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 1......... 1....5% solution)...0mL FeSO4·7H2O (0. Bring volume to 200........................0............. Adjust pH to 7.................................................................................................25......... 10..........0mL MgCl2·2H2O (1..........................0g Tween 80 (10% solution) .2g Na2HPO4......0mL Cyanocobalamin (0.. Filter sterilize..... anhydrous ............ 1.....0mL.......0g Na2HPO4 ....2.... 1..................0mL CuSO4·5H2O (0................2....5% solution)................0mL Basal Medium: Composition per liter: Agar ............... Modified (Albumin Fatty Acid Semisolid Medium......100.....................................5% solution) .............4 ± 0....... Mix thoroughly..........0g KH2PO4.......................0...................0mL CaCl2·2H2O (1.................................. Cool to 25°C.... Albumin Fatty Acid Supplement: Composition per 200..0mL MgCl2-CaCl2 solution ...1................0mL: Bovine albumin fraction V ........ Store at −20°C.. Use: For the cultivation and maintenance of Leptospira species............5% solution) ............................... sterile basal medium..0g Polysorbate (Tween) 80 (10% solution) ..........................1..........2...... Mix thoroughly.0g KH2PO4 ..............0L...........0mL ZnSO4 (0.0mL Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 1......Brackish Acetate Sodium pyruvate (10% solution) .........4% solution) .....................3% solution) ......................3% solution) ...................2... Use: For the cultivation of Leptospira species.................................................................................0mL of sterile albumin supplement....................................... Adjust pH to 7........... Adjust pH to 7.....20.........4% solution) .......................................5% solution)..........0g NaCl ................... Modified) Composition per liter: Basal medium ....... Gently heat and bring to boiling............. 1.......0mL Preparation of Albumin Supplement: Add compo- nents to distilled/deionized water and bring volume to 100...................2.......... Albumin Fatty Acid Supplement: Composition per 200...................................0mL of sterile albumin fatty acid supplement and 900.................2mL Preparation of Albumin Fatty Acid Supplement: Add bovine albumin to 100... Mix thoroughly..................0mL: CaCl2·2H2O .......5g Preparation of MgCl2-CaCl2 Solution: Add components to distilled/deionized water and bring volume to 100............. Aseptically distribute into sterile tubes or flasks.... Gently heat and bring to boiling............... Aseptically distribute into sterile tubes or flasks........0mL FeSO4·7H2O (0................ Adjust pH to 7............0mL Albumin supplement .........4% solution) ............ 1.02% solution) ..............0mL......0mL Sodium pyruvate (10% solution)....................3g Glycerol (10% solution) .. 250........................ 2.................... Add remaining components while stirring...........0mL ZnSO4·7H2O (0............2mL 71 Preparation of Albumin Fatty Acid Supplement: Add bovine albumin to 100........0mL Thiamine·HCl (0.......................20...............0L...... 1.........................................0mL: Bovine albumin ....... Mix thoroughly......0mL Albumin fatty acid supplement...................... Bovine Serum Albumin Tween 80 Agar (BSA Tween 80 Agar) Composition per liter: Basal medium .....11.1.0mL of distilled/deionized water...............0mL CuSO4·5H2O (0............. Autoclave for 15 min at 15 psi pressure–121°C..0mL Glycerol (10% solution)....... Add remaining components while stirring... 1..............0g NaCl ...0g Polysorbate (Tween) 80 (10% solution) .................4. Filter sterilize.......0mL pH 7......0mL Vitamin B12 (0..... Albumin Supplement: Composition per 100.. Autoclave for 15 min at 15 psi pressure–121°C....0mL Basal Medium: Composition per liter: Agar ........ Adjust pH to 7...................................................1............. 1.......................4...... Mix thoroughly............... Preparation of Medium: Aseptically combine 100..10....0mL MgCl2·2H2O (1..1.................... Mix thoroughly...5% solution). 100................................ Store at −20°C........4 ± 0...........05g Artificial seawater .... 20....0....................2% solution) ..........................0mL of sterile basal medium..............4.... Cool to 25°C.............25.......1..................3g NH4Cl (25% solution).....5mL FeSO4 (0...2 at 25°C Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 1... Filter sterilize.....................0mL of sterile basal medium.................................4............... aseptically add 100........4.......... Aseptically distribute into sterile tubes or flasks...4.....0mL Sodium pyruvate (10% solution) ... Preparation of Medium: To 900..............1...................................... MgCl2-CaCl2 Solution: Composition per 100........... 0.....0mL Thiamine (0.1.. Mix thoroughly.................. Use: For the cultivation of Leptospira species..................5g MgCl2·6H2O ....0g NaH2PO4·2H2O ...2..................... Mix thoroughly........ Preparation of Medium: Aseptically combine 100..........0..................5% solution .........5% solution).......1..5% solution).............. Adjust pH to 7............0g KNO3 .........0mL ZnSO4·7H2O (0.......0mL ..... 12..... Ellinghausen and McCullough............... Cool to 25°C.................1.....0mL with distilled/deionized water.............0mL pH 7.............. anhydrous ...900.0mL NH4Cl (25% solution) ................................. Bring volume to 200.0mL of distilled/deionized water.........0mL of sterile albumin fatty acid supplement and 900..................... 1.... Mix thoroughly......................0mL of cooled.... 20....... Brackish Acetate Composition per liter: Sodium acetate ...2. Mix thoroughly....................................5% solution).. Autoclave for 15 min at 15 psi pressure–121°C...0mL Thiamine·HCl (0.........................2% solution) ................. 900................0mL CaCl2·2H2O (1...0mL Vitamin B12 (0.........

0g CaCl2·2H2O.........................04g Co(NO3)2·6H2O...................2.................0mL Vitamins ...024g NaF..........7g Nitrilotriacetic acid ........... 5...........................0mL pH 7..7g Nitrilotriacetic acid...............................1................................0mL................5..............25mg “Metals 44” ....................................................................................................................................... 50....66g NaHCO3 ..................0... 1......................................... Distribute into tubes or flasks.................25g MnSO4·7H2O .... Add aseptically to sterile basal medium.........0.......................... Aseptically distribute into sterile tubes or flasks.0mg Biotin .................102g KCl ... 3....................................................................0mL Preparation of Modified Hutner’s Basal Salts: Dissolve the nitrilotriacetic acid first and neutralize the solution with KOH.....................................3..0mg Preparation of Artificial Seawater: Add components to distilled/deionized water and bring volume to 100.........0mL: ZnSO4·7H2O... Vitamin Solution: Composition per liter: Thiamine·HCl ...0mL Modified Hutner’s basal salts..........5g EDTA ............0........... Preparation of Medium: Add a few drops of H2SO4 to the distilled water to retard precipitation of the metal salts...... Mix thoroughly............................1g FeSO4·7H2O ................0mL...........1......................0g Peptone .......................... Mix thoroughly..............018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100..664g NaHCO3 ........... Add components........72 Brackish Prosthecomicrobium Medium Modified Hutner’s basal salts ......... Autoclave for 15 min at 15 psi pressure–121°C. 2........................................................................................................ 0.................................29...........................2.....................................................0. 0......34g FeSO4·7H2O ............ 3...............................................0mg Folic acid....................25g Glucose...............0g CaCl2·2H2O ...................25g Yeast extract ................ 2..0........................... 0...............................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................. 5..................................025g Na2B4O7·10H2O..................23..........0......0.......018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100...................................1mg ...0mg Riboflavin....................3............026g SrCl2 ....154g CuSO4·5H2O....... 5............1g H3BO3 ............................0mg Riboflavin ...................... 0........0......................... 29...0mL.............. 10.................... Aseptically add “Metals 44” and vitamin solutions...........................0mL of artificial seawater and 720............................0mg D-Calcium pantothenate .......................... Mix thoroughly........................ Mix thoroughly................ Mix thoroughly...............477g MgCl2 ........................................04g Co(NO3)2·6H2O .....0..... Store at 5°C............................2.0mg Vitamin B12 ..................917g CaCl2 ....0mg D-Calcium pantothenate...................................................... Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O............................................................................................ 9......................................15.................. 1....................1g (NH4)2MoO4 .. “Metals 44”: Composition per 100........................2 with KOH or H2SO4............0................... 10. 0.................................. Add other ingredients and readjust the pH with KOH and/or H2SO4 to 7..............1g (NH4)2MoO4 ..............................................5g MgCl2 ........10.............. Add aseptically to sterile basal medium..................... 23....0mg Folic acid ........................... 0............................ Adjust pH to 7........ Filter sterilize and add aseptically to sterile basal medium......................................2 at 25°C Artificial Seawater: Composition per liter: NaCl ...................................... 5.....................................................................................1g FeSO4·7H2O...... Sterilize by autoclaving for 15 min at 15 lbs pressure–121°C............................................................... Autoclave for 15 min at 15 psi pressure–121°C. 20.................. 0......0mL: ZnSO4·7H2O ......................25g Artificial seawater ..0mL.....................3.................0mg Biotin .................0mL of distilled/deionized water...............................................0mL pH 7........................................... Brackish Prosthecomicrobium Medium Composition per liter: Agar .......... Mix thoroughly...... except for “Metals 44” and vitamin solutions.............................................................2. 3.2 at 25°C Artificial Seawater: Composition per liter: NaCl .........................................5g EDTA...........10. There may be a slight precipitate................................ 250.............. 0........................................... 0....096g H3BO3 ...........................1g KCl.....0L................................0......... Use: For the cultivation of Filomicrobium fusiforme......................................................................25mg “Metals 44” ............................................................192g KBr ..................................................2 ± 0.... 0.........026g SrCl2 ........................................ to 250.......................................0mL Vitamin solution..................0..........................................................................50....................0mg Preparation of Artificial Seawater: Add components to distilled/deionized water and bring volume to 100....2 ± 0.................9g CaCl2 ............................. Add other components and adjust the pH to 7.......................................................34g FeSO4·7H2O...... Vitamin Solution: Composition per liter: Thiamine·HCl ......................................................... 0.....................................................................................................................154g CuSO4·5H2O ................................................981g Na2SO4 ..........................19g KBr.....9.................0.... “Metals 44”: Composition per 100..................................................................................025g Na2B4O7·10H2O ..........5................. Modified Hutner’s Basal Salts: Composition per liter: MgSO4·7H2O .........................................................0........................................................ Store at 5°C...........................................0...................................................... There may be a slight precipitate..................................................................................0mL Preparation of Modified Hutner’s Basal Salts: Dissolve the nitrilotriacetic acid first and neutralize the solution with KOH........ 0........ 0.......................................0mg Vitamin B12..........................................................................................................................................5.........................................................................25g MnSO4·7H2O..........................................................024g NaF ....0g Na2SO4 ............4........................0.....20..........

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

........... Escherichia coli appears as dark red colonies with a pink halo...... Medium is sensitive to light...................0g Lactose .................. Boil for 1 min.......0g Preparation of Carbohydrate Solution: Add carbo- hydrate to distilled/deionized water and bring volume to 10......... 0..0L.... 10.........5....0mL amounts for testing 1........... Cool to 25°C.................... as well as in other materials of sanitary importance.. Enterobacter species appear as pink colonies............................... Autoclave for 12 min (not longer than 15 min) at 15 psi pressure– 121°C.........9 ± 0.......2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems and Oxoid Unipath................... the gas is trapped in the Durham tube........013g pH 7....0mL or less of sample........ 0........................ 10..........5.......25g Lactose ...................2 at 25°C Carbohydrate Solution: Composition per 10.......3...........................16g Brilliant Green...................... For plating 10......9g Na2SO3 .0g Phenol Red .. 0.................. 20. cool the broth rapidly...0mL sterile riboflavin. 10. 2...... Add 2........ Gently heat and bring to boiling.......0g Yeast extract .......205g Basic Fuchsin .....................0g Lactose .. Mix thoroughly......... 10....... 20................................................ Gently heat with frequent mixing....................15g Pancreatic digest of gelatin ................ Brilliant Green Bile Agar Composition per liter: Noble agar............... in 10.................0g Beef extract ................. cool the broth rapidly.......... Mix thoroughly.....20...... Bacteria that ferment the specific carbohydrate turn the medium yellow...013g pH 7. Distribute into tubes or flasks.........065g FeCl3 ......2 ± 0............... Filter sterilize............ Distribute into tubes containing inverted Durham tubes..................................0................................... dehydrated.........6........................ After sterilization..... in 10....04g Carbohydrate solution ...0L.. 1............................... Autoclave for 12 min (not longer than 15 min) at 15 psi pressure– 121°C.............20................ Use: For the detection of coliform microorganisms in water and wastewater. Gently heat and bring to boiling.............10....0g NaCl ...................74 Brilliant Green Bile Agar 45°–50°C... Preparation of Medium: Add components to distilled/ deionized water and bring volume to1......025g pH 6.........................0L.........0 ± 0.....2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems..........0.......................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to1.......................... and Caulobacter henricii............20.......... Aseptically add 5........0mL to each sterile absorbent filter used... Distribute into tubes containing inverted Durham tubes.................0L. Mix thoroughly............0g based on their fermentation of specific carbohydrates.. 10......... dehydrated.......... Caution: Basic Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contamination of the skin.......................... Mix thoroughly...............95mg Brilliant Green .............................0g Brilliant Green ...............0g NaCl .0g Pancreatic digest of gelatin ..................................................... Autoclave for 10 min at 15 psi pressure–121°C...... Mix thoroughly....................0g Lactose .......... 0.....078g Erioglaucine ....... 0......... Brilliant Green Bile Broth with MUG Composition per liter: Use: For the differentiation of a variety of microorganisms Oxgall................................................... Brilliant Green Broth (m-Brilliant Green Broth) Composition per liter: Proteose peptone No....... Mix thoroughly........................ prepare the medium double strength................. 0........... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... 3... 10..0............... 8............................ Distribute into test tubes that contain an inverted Durham tube. The presence of Eschrichia coli and other coliforms is determined by the presence of fluorescence in the tube....2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems..0mL.........................................0mL pH 7..........0mL: Carbohydrate ...0............. After sterilization................. Use: For the detection and enumeration of coliform bacteria in materials of sanitary importance such as water and sewage..................................0mL samples................. Bromcresol Purple Broth Composition per liter: Peptone .............................................................................0mL amounts for testing 1. Care should be taken to avoid exposure of the prepared medium to light..............0g Sucrose ....................03mg pH 6............................................... Pour into sterile Petri dishes.......0g Pancreatic digest of gelatin ..... 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.......... Mix thoroughly.. Use: For the cultivation of Brevundimonas spp...............0g Bromcresol Purple................... MUG (4-Methyl umbelliferylβ-D-glucuronide) ...015g Oxgall............................................................................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems............... Use: For the selective isolation and differentiation of Sal- monella from polluted water by the membrane filter method..........05g Brilliant Green...... 0........9 ± 0.......................... .. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................0L. Pour into sterile Petri dishes or distribute into sterile tubes... deionized water and bring volume to 1.......................................................2 ± 0...... dehydrated.................0mL or less of sample... Turbidity in the broth and gas in the Durham tube are positive indications of Escherichia coli.... Autoclave for 15 min at 15 psi pressure– 121°C............ When bacteria produce gas...10............................0.0295g KH2PO4 ......... Brilliant Green Bile Broth (Brilliant Green Lactose Bile Broth) Composition per liter: Oxgall..

..2 at 25°C Bromcresol Purple Solution: Composition per 10...................................................... Autoclave for 20 min at 15 psi pressure–121°C............................................... Autoclave for 15 min at 15 psi pressure–121°C............... Filter sterilize......... except bo- vine serum............. 3................ except horse serum.............1.................................................... Mix thoroughly.................... Heat gently and bring to boil for 1 min.... glucose solution.............................. sodium thiosulfate solution......................2mL Maranil solution (5% solution) ..................................................................2mL of sterile glucose solution and1.............. Autoclave for 15 min at 15 psi pressure–121°C...0g Yeast extract. to distilled/deionized water and bring volume to 900..................0mL of sterile lactose solution......0mL of horse serum...........................0g Sucrose.............. NaCl..0mL of sterile sodium thiosulfate solution................ and yeast extract to distilled/deionized water and bring volume to 1...2 at 25°C Preparation of Medium: Add components..........0mL pH 7.........0mL Bromothymol Blue (1% solution).................0mL of sterile L-cysteine·HCl·H2O solution and 10.................................9 with 1N KOH..... 1....0mL pH 6.....................................15....... 500...0g Agar .....0mL pH 7.................0mg Polymyxin B...................................3.5g Glucose.......0........ Cool to 45°–50°C................................................ Mix thoroughly...............0L................................10............ Aseptically distribute into sterile tubes or flasks............... Use: For the cultivation of Spiroplasma citri..1...100..... Cool to 50°–55°C.................... Mix thoroughly...5g Bovine serum..... activated ...0g Peptone.. sterile agar solution aseptically add 27.......0mL L-Cysteine·HCl·H2O solution ..........................16g Ethanol (95% solution) ......0g Tryptose ....................0g Fructose..0mL Glucose (33% solution) ......................... Autoclave for 15 min at 15 psi pressure–121°C...........Buffered Charcoal Yeast Extract Differential Agar Bromcresol Purple Dextrose Broth (BCP Broth) Composition per liter: Glucose ............... Cool to 45°–50°C..01g Bromothymol Blue ..0mL: Bromcresol Purple ...........0mL of ethanol....................... 2........................ 10..................0mL: Agar ............ Filter sterilize.................... 5...............0mL............................................0mL of sterile antibiotic solution..........17............0g Bromcresol Purple solution ...............5g Fe4(P2O7)3·9H2O ..................... Use: For the isolation and cultivation of Simonsiella species and Alysiella species.....50.0g K2HPO4 ...... Adjust medium to pH 6...........0mL: Vancomycin . Mix thoroughly........ to distilled/deionized water and bring volume to 1.0g Papaic digest of soybean meal .... Aseptically add sterile bovine serum.........10......... Pour into sterile Petri dishes or distribute into sterile tubes.................. Cool to 45°–50°C...... 5....... 1..0g ACES (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) buffer ..................................................................................................0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10............. 5......... Mix thoroughly.. Mix thoroughly.................. Mix thoroughly............ 0.10. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension.... 4.....................0g Glucose ................0g Peptone.0mL pH 7... 10.....0g NaCl ......................................25g Bromcresol Purple... ............. 10.......................................... 5..... Adjust pH to 7..........0g Phenol Red ..2...........................8........ Add 4.........0mL Sodium thiosulfate (50% solution) ........0mL of sterile Bromothymol Blue solution.............................0L.......0g NaCl .....................0L... 1................... solids from infusion........................ 10.................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..............................1... Bromothymol Blue Agar Composition per liter: Agar .... Mix thoroughly...............6 ± 0. except Lcysteine·HCl·H2O solution and antibiotic solution....................................0g Lactose (33% solution) .....2... To the cooled.7–7........0g Charcoal....... and maranil solution...........2................................. Gently heat and bring to boiling......0mL....... 11....2 at 25°C Antibiotic Solution: Composition per 10.....................................000U Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10..10.......... Mix thoroughly......... Distribute into tubes in 12–15mL volumes.......................... Use: For the cultivation and differentiation of bacteria based on their ability to ferment glucose..... Use: For the selective isolation and cultivation of members of the Enterobacteriaceae.............02g Horse serum ................7–7.........................01g Antibiotic solution.............. to distilled/deionized water and bring volume to 900..0mL pH 7...........................0.. Pour into sterile Petri dishes or distribute into sterile tubes......................0mL................. Preparation of Medium: Add components......... 100....0g Sorbitol.....0g Beef extract ....................... Bromothymol Blue solution.3 ± 0.......... 70.................1...................................0g Yeast extract .........4....0g NaCl ......................0mL Preparation of Bromcresol Purple Solution: Add Bromcresol Purple to 10..2 at 25°C Preparation of Medium: Add components.. 10..0mL................................... peptone..0g Yeast extract .......................17.................5................................... BSTSY Agar Composition per liter: Pancreatic digest of casein ................. Autoclave for 15 min at 15 psi 75 pressure–121°C..........9 ± 0........... Mix thoroughly.......................................................0 ± 0......0mL: L-Cysteine·HCl·H2O..................0........... Mix thoroughly............ L-Cysteine·HCl·H2O Solution: Composition per 10..0mL of sterile maranil solution.1... Bacteria that ferment glucose turn the medium yellow......... 10...8 at 25°C Preparation of Medium: Add agar....0............ Adjust pH to 8.....27. Buffered Charcoal Yeast Extract Differential Agar (DIFF/BCYE) Composition per 1014................... Filter sterilize separately the lactose solution.............. BSR Medium Composition per liter: Beef heart..................... Mix thoroughly..................................................... Aseptically add 100...........2......... 0...

..........0L........ 0...............4-Butanediol ......................................................... Mix thoroughly...2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1......15................... Autoclave for 15 min at 15 psi pressure–121°C.............................. 0................. Mix thoroughly....025g Na2B4O7·10H2O........0mL Hutner’s mineral base .0.................. 0................................01g (NH4)2MoO4 ................011g SrCl2·6H2O .........................01g NaCl ................................0mg Na2MoO3 . 9.. Burke’s Modified Nitrogen-Free Medium with Benzoate Composition per liter: Sodium benzoate ................................... and maintenance of Le- gionella pneumophila and other Legionella species from environmental samples..................................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............. 0.......0L...........02g pH 7.................................... 20............................................. Butanediol Medium Composition per liter: NaH2PO4·H2O ................................................ 0......................76 Buffered Marine Yeast Medium Use: For the isolation...........25mg “Metals 44” ..........................6...............................................2g Na2HPO4 .......................1.............................25g MnSO4·7H2O ....0..... Use: For examining fuels for microbial contamination and for studying hydrocarbon utilization by microorganisms...................0g KH2PO4 .....................................20......011g SrCl2·6H2O .01g FeSO4·7H2O...........................................................0g NH4NO3............2g Na2HPO4 ......... Autoclave for 15 min at 15 psi pressure–121°C.............34g FeSO4·7H2O......0mg Na2MoO3 .............189g NaHCO3 .............8 ± 0......0...................................... 24............. 0.............................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...50....................... Mix thoroughly..0..0 ± 0.............................................................. 0................................0......................................1g FeSO4·7H2O...................0..............1g 1.................................0g Agar ..............................0.....5mg pH 7................................8 ± 0......... Pour into sterile Petri dishes or leave in tubes...............................1-1................0g CaCl2·2H2O ......................0mL: ZnSO4·7H2O .......... 70.........5mg pH 7.................................. Autoclave for 15 min at 15 psi pressure–121°C...............................................20...........2g FeCl3 ...0......................................... Autoclave for 15 min at 15 psi pressure–121°C...............................5g MgSO4·7H2O.....2........... Mix thoroughly.................. Use: For the cultivation of Pseudomonas species and other microorganisms which can utilize benzoate as sole carbon source......... 0..... 29............ Mix thoroughly.. There may be a slight precipitate. Hutner’s Mineral Base: Composition per liter: MgSO4·7H2O..................................5g ................................................... pH 6................1.0g NH4Cl ............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........................................................................018g Preparation of “Metals 44”: Add components to distilled/deionized water and bring volume to 100........................................................02g KH2PO4 .......0...........05g CaSO4·2H2O........ Buffered Marine Yeast Medium Composition per liter: NaCl ............. Dissolve the nitrilotriacetic acid first and neutralize the solution with KOH..........................................05g CaCl2·2H2O .................. Bushnell-Haas Agar Composition per liter: Agar ................................. layer 0........... “Metals 44”: Composition per 100........01g Adenine ............... 0............0g 1M Phosphate buffer........5g EDTA ....8 ..........0mL KOH (1N ) ..............154g CuSO4·5H2O .....0.........1................................................................... 1.........0L...................... 0...01g NaCl ...................................0g MgSO4·7H2O..................0....0......... Gently heat and bring to boiling..........................................................0...............0...............0.....8.................. cultivation................. For the selective recovery of Legionella pneumophila while reducing contaminating microorganisms from environmental water samples.......................72g MgSO4·7H2O... Distribute into tubes or flasks...................0...........................0g K2HPO4 ................................ Also for the cultivation of Nocardia species.......................4g Preparation of 1M Phosphate Buffer......0L...................7g Nitrilotriacetic acid ............................................................................... 3....... Store at 5°C.. Distribute into tubes or flasks............ Adjust pH to 6................................0............0mL Preparation of Hutner’s Mineral Base: Initially add a few drops of H2SO4 to the distilled water to retard precipitation.1...................0....................................0mL pH 6.................................. Use: For the cultivation of Azotobacter vinelandii......................0L.......................... Add other ingredients and adjust the pH to 7....................................................................... 85............................. Autoclave for 15 min at 15 psi pressure–121°C....................................... 10....0............02g KH2PO4 ..........................................................0g CaCl2·2H2O........19g NaHCO3 ....... pH 6.................. 0.......... Distribute into tubes or flasks..............................................7.....04g Co(NO3)2·6H2O .........................................6.................0g Yeast extract......0% hydrocarbon on agar surface or aseptically add sterile hydrocarbon to cooled agar prior to pouring plates........1......................................... Use: For the cultivation and maintenance of Pseudomonas species.................05g CaSO4·2H2O .....................1.................................................... For use in cultivating hydrocarbon-utilizing bacteria........ Burke’s Modified Nitrogen-Free Medium Composition per liter: MgSO4·7H2O ................. Add aseptically to sterile basal medium...........01g Adenine ...8: Add components to distilled/deionized water and bring volume to 1... Mix thoroughly...................4g NaH2PO4·H2O.................. 5............0mL...... 0..................................................................... Distribute into tubes or flasks.....................0g NaCl ........... pH 6........................01g FeSO4·7H2O.............................................2 at 25°C 1M Phosphate Buffer..8: Composition per liter: K2H2PO4 .........................................................8 ± 0.................................................................2 with KOH and/or H2SO4.....................

. and Na2S·9H2O solution............01g NaWO4·2H2O.......BY Agar Medium K2HPO4 ..............10......................... 0......... 4......................25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10...... Gently heat and bring to boiling.75mL: Acetic acid.....0g Cellobiose.......................................0..... 0...................... Gently heat and bring to boiling.8........0mL pH 7.................... 0................................................................2....0........0mL Minerals solution .............. Distribute into rubber-stoppered tubes under 100% CO2............01g Preparation of Modified Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500............................5 with KOH. Aseptically and anaerobically add 20...................... Sparge under 100% CO2............7 ± 0.......... 0............1g FeSO4·7H2O.. 1..0 ± 0.................................. 1.......... Add components.... 0....25mL n-Valeric acid.....0g MnSO4·H2O ........................ Preparation of Medium: Prepare and dispense medium under 100% CO2......................0mL Volatile fatty acid mixture........................ except carbohydrate solution........01g H3BO3 .................................................1g ZnSO4·7H2O ............................. Distribute into tubes or flasks........................... 2...0mL: Na2CO3 ....... Cool to room temperature while sparging with 100% CO2........................................... Add Na2CO3.................. Adjust pH to 6................................. 0....0g KH2PO4 .................................... 1........ 1......................0mL................ 0......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......2 ± 0....0g Nitrilotriacetic acid .................. 3............ 2......50mL Butyric acid ... 0....... Continue boiling for 5 min.......... 6..............0L..................0g K2HPO4 .0g Yeast extract....... 10.......................................0L...............25mL Preparation of Volatile Fatty Acid Mixture: Com- bine components......... 2..... Autoclave for 15 min at 15 psi pressure–121°C........................0mL L-Cysteine·HCl·H2O solution ......................150........1g CoCl2·6H2O .............................. Use: For the cultivation of Butyrivibrio species....... clarified ................... L-Cysteine·HCl·H2O Solution: Composition per 10....................................................................................................................0mg Resazurin .....................0mL Na2S·9H2O solution..........05g pH 7...............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......0mL.......................... 1..... Sparge with 100% CO2....1................................... Butyrivibrio species Medium Composition per 1001.......0mg Rumen fluid........0mL of sterile carbohydrate solution........................0....................01g NiC12·6H2O ..... 0......................... Autoclave for 15 min at 15 psi pressure–121°C.......... 0.... Continue sparging with 100% CO2 until pH reaches 6...0g Beef extract ...........................25g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10................................................1......0mL of sterile L-cysteine·HCl·H2O solution........................1g MgSO4·7H2O...................... Autoclave for 15 min at 15 psi pressure–121°C..... 6........... 4......8..........5............. 12...........0mL..................................................0L.........10..01g Na2SeO3 .................................................................1mL pH 6...............5g NaCl ....5g CaCl2·2H2O... Add remaining components one at a time...6g Preparation of Minerals Solution: Add components to distilled/deionized water and bring volume to 1........................................................ 0.........................................2 at 25°C Modified Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ...............0....................................5g CaCl2 ............... Mix thoroughly............ Use: For the cultivation of Pseudomonas putida..............15....0..20.......0g (NH4)2SO4 ...................1.......3g Hemin..0g Pancreatic digest of casein ...............................0mL: Na2S·9H2O .......................... 77 Na2S·9H2O Solution: Composition per 10.......10........................................................................... 1. soluble ........................................5..... Add distilled/deionized water to 1.......................................... .................................................. Sparge with 100% CO2..........01g CuSO4·5H2O ......................................................... Na2CO3.....2 at 25°C Minerals Solution: Composition per liter: NaCl .......... 0........................................................................... and 10.................... BY Agar Medium (ATCC Medium 2038) Composition per liter: Agar ............75.......0g Preparation of Carbohydrate Solution: Add components to distilled/deionized water and bring volume to 20................... 0............................................................................0mL Carbohydrate solution..........01g Na2MoO4·2H2O .......0g Yeast extract .. Mix thoroughly..................................... 0..........25mL iso-Valeric acid ................................0L.......... Carbohydrate Solution: Composition per 20........................ L-cysteine·HCl·H2O solution...... Autoclave for 15 min at 15 psi pressure–121°C.. to distilled/deionized water and bring volume to 960.0g Pancreatic digest of casein ................1g AlK(SO4)2·12H2O...............................1................ Mix thoroughly.. Adjust pH to 6........... 0.....2g Modified Wolfe’s mineral solution ....0mL Mix thoroughly.0mL: Glucose..................... Autoclave for 15 min at 15 psi pressure–121°C..0g Starch........... Adjust pH to 7..................................................3g Yeast extract........ Mix thoroughly........3............25mL Propionic acid. Distribute into tubes or . 0....0mL of sterile Na2S·9H2O solution or...... 0...... Mix thoroughly.................. inject the appropriate amount of sterile carbohydrate solution..................................................... 0...............0g Glycerol ................................ 0........................................... Mix thoroughly................................ Autoclave for 15 min at 15 psi pressure–121°C.......0g Maltose ...5..................................0g MgSO4·7H2O ................... sterile Na2S·9H2O solution.................................................................................................................................................................................................... Volatile Fatty Acid Mixture: Composition per 7..................................................5g K2HPO4 ........0mL DL-2-Methyl butyric acid .1........25mL iso-Butyric acid ...... Mix thoroughly...0g NaCl ......................................... 1...........................................0mL: L-Cysteine·HCl·H2O ...... and sterile L-cysteine·HCl·H2O solution into individual tubes containing medium....................... using a syringe....0mL of distilled/ deionized water.........................................

...........0.................0g Pancreatic digest of casein .......................025mg Niacinamide .................0...... Distribute into tubes or flasks...................................... 1........025g L-Serine .......01g Nicotinamide adenine dinucleotide ......1...........................................................0..............................0g KCl ...........025g L-Valine ................................0..........5........ Mix thoroughly.....2........0.......... live.......................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.... reduced .............................................05g Ascorbic acid................0mL: Beef heart........0L..........025g L-Cystine ...........52g Peptone.......03g L-Alanine ..........................5 ± 0. Filter sterilize............0.....................0g Yeast extract... Adjust pH to 6.............0mL Fresh yeast extract solution..0.......0........................................................................................................ Autoclave for 15 min at 15 psi pressure–121°C.......... 25........................................ ............025g L-Phenylalanine ................ Aseptically distribute into sterile tubes or flasks.....................................0mL: Baker’s yeast.................... Adjust pH to 7.......................................0.........0......8.........................075g L-Arginine·HCl.........32g NaCl .... 1...................................0mg PPLO broth without Crystal Violet..............9..............................................0..............0............0mL of distilled/deionized water...................................................................................... 0.01g Hydroxy-L-proline.... 2.......................4g Fe4(P2O7)3·9H2O .2 ± 0... 10.................5mg Cholesterol ................................................................0...... Pour into sterile Petri dishes or leave in tubes..............................................0.....................................05mg Niacin ...02g L-Isoleucine .... Allow to stand....... Mix thoroughly.........0mg Nicotinamide adenine dinucleotide phosphate...............0.................................................0.......... 11.............................................0.0..................................... Use: For the cultivation of Legionella pneumophila.... Gently heat and bring to boiling.............................................. BYEB (Buffered Yeast Extract Broth) Composition per liter: ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid) ....................... Mix thoroughly.......................................0mL..... Adjust pH to 6...........05mg p-Aminobenzoic acid . Use: For the cultivation of Cytophaga flevensis............................04g L-Aspartic acid ......................... 10..............1.......................083g L-Glutamic acid .0....7.......................................................................................................2g MgSO4·7H2O............0...........................................025mg Biotin .....5mg Cocarboxylase .....................................1.............................................2g NaH2PO4·H2O .........0mL pH 7........................................0..05g L-Proline .....................................0..........0...............02g Phenol Red ........................................................................04g L-Tyrosine...........0g L-Cysteine·HCl·H2O ....6.....25g pH 6........14g L-Glutamine...........01g L-Tryptophan ................ Fresh Yeast Extract Solution: Composition per 100............................025mg Pyridoxine ..............0L............................ Autoclave for 90 min at 15 psi pressure–121°C........01g Thymidine .............................0............. 15........................................0......................................................... PPLO Broth without Crystal Violet: Composition per 500......... 100.....................07g L-Lysine·HCl .............................................................................02g L-Methionine ........... 0............... anhydrous....................2mg 5-Methyldeoxycytidine ....5......................................78 BYEB flasks.....01mg Thiamine·HCl . starch-free........26g CaCl2.............................. Use: For the cultivation and maintenance of Paracoccus thiocyanatus.......0..........0g Phenol Red ......... Autoclave for 15 min at 15 psi pressure–121°C.........01g Deoxycytidine .... Filter sterilize....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................................0.....01mg D-Calcium pantothenate ....................2...............................................0....................... C/10 Agar Composition per liter: Agar .........1............ Adjust pH to 7...015g Deoxyadenosine ...............0g Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100..................0.......... Myxococcus fulvus............................. pressed......................................... 10...................................................................................01mg Riboflavin..............01g Deoxyguanosine ............2.............9 ± 0....... Remove supernatant solution.......................0mg Choline chloride ..........................................................025mg Pyridoxal·HCl.....................................................0....................................... and Myxococcus xanthus..............0mg Uridine triphosphate ........ Filter sterilize.............................1g Sodium acetate·3H2O .................................................................0.........0.........4g L-Cysteine·HCl·H2O..............................0...................0mg Tween 80 ......... C 3G Spiroplasma Medium Composition per liter: Sucrose...............................................................0g CaCl2·2H2O............500..........................6– 6............................ Preparation of CMRL-1066 Medium: Add components to distilled/deionized water and bring volume to 1......0.0...... 3........... Mix thoroughly.....03g L-Threonine .8g NaHCO3 ....0...........................01mg Source: CMRL-1066 medium is available as a premixed powder from BD Diagnostics............0..........................................0L.........0....................0.................0.........................................................................0............................................................................... 1.......2g D-Glucose .....................................................................150....0g α-Ketoglutarate........02g L-Histidine·HCl·H2O .....................0....................0..0mg Sodium glucoronate·H2O ..........................01g Glutathione...........15g Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 500..................................................................................2...................................0.....................................................................1.........................2mg Coenzyme A ..... CMRL-1066 Medium: Composition per liter: NaCl .1mg Inositol.............................................4....0............07g L-Leucine...........................0mL CMRL-1066 medium............2 at 25°C Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems......0mg Flavin adenine dinucleotide ............................0......................................01mg Folic acid ....0.............. Pour into sterile Petri dishes or leave in tubes...........................................06g Glycine ..........0mL Horse serum ...........36g pH 7...... Flexi- bacter filiformis.....................50......................................... infusion from .................................................................

........ and CMRL medium—to distilled/deionized water and bring volume to 75..........025mg Pyridoxal·HCl.. Mix thoroughly...........................01g Hydroxy-L-proline......................... reduced ....5.........................5mg Cholesterol ...025g L-Serine ....0............................................025mg Niacinamide ..................... 0........... CMRL-1066 Medium: Composition per liter: NaCl ..................................................................05g L-Proline.52g Peptone............................ and 5................. and 0...............02g Phenol Red ...................0....................CAL Agar Preparation of Medium: Add components—except horse serum...0............................ Mix thoroughly........................0...............0........ Preparation of CMRL-1066 Medium: Add components to distilled/deionized water and bring volume to 1.. Adjust pH to 6...............2g MgSO4·7H2O ..... 0...............0mL Fresh yeast extract solution.........5..... Aseptically add 20......01mg D-Calcium pantothenate ........................................0..........................0.......0g Cellobiose....0.............. 0.........0mg Uridine triphosphate ..0........................................................................ 0.........................................................2g NaH2PO4·H2O.......0mL of sterile CMRL medium................................. 0................................0.. Pour into sterile Petri dishes...5g L–Lysine·HCl..........15g Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems...........0mg Tween 80 .......0..............................................................................04g L-Tyrosine ....0mL...2g D-Glucose.......... 50...............01g Thymidine .........................2 at 25°C PPLO Broth without Crystal Violet: Composition per 500............. 0.............................................................................. Adjust pH to 7..... 0.................................................................0mg PPLO broth without Crystal Violet..... Mix thoroughly...025mg Pyridoxine ................... Autoclave for 90 min at 15 psi pressure–121°C.........................................................................5g NaCl ..0.........5mg Cocarboxylase ................... 5...........................................01mg Source: CMRL-1066 medium is available as a premixed powder from BD Diagnostics...............................07g L-Lysine·HCl...........5g Yeast extract ............. 0......................................26g CaCl2............................6................0mL: Baker’s yeast.................................0................................ Distribute into sterile tubes or flasks....................... and CMRL medium—to distilled/deionized water and bring volume to 795.......................32g NaCl .... 0..0mL..05g Ascorbic acid ......................... Filter sterilize...................................................1...... live.0mL of sterile horse serum......................1.......... Autoclave for 15 min at 15 psi pressure– 121°C.............025mg Biotin ........................... Preparation of Medium: Add components—except horse serum.....0mL of sterile horse serum...........01mg Riboflavin.....................075g L-Arginine·HCl ...4............................0mL of sterile yeast extract.01g Deoxyguanosine ...................................................................................0g Phenol Red ...........02g L-Methionine ....20.................083g L-Glutamic acid................................. 0..............0mL Horse serum ..............................1......................... Use: For the cultivation and maintenance of Spiroplasma species.............................. Allow to stand............................ 0.........015g Deoxyadenosine ... Adjust pH to 7.......................................03g L-Alanine...............0mg Nicotinamide adenine dinucleotide phosphate.................................... 0...0........................ Fresh Yeast Extract Solution: Composition per 100.................................................8..............................025g L-Phenylalanine................................... 0........................................01g Nicotinamide adenine dinucleotide .................................0...................0mL CMRL-1066 medium....... 0.......6... 0....01mg Thiamine·HCl ............. Adjust pH to 7........05mg p-Aminobenzoic acid ................................................................. 0...............................................................5mL pH 7......................................3............02g L-Isoleucine ...04g L-Aspartic acid ..............................0.0g Sodium deoxycholate .............2mg 5-Methyldeoxycytidine ........................ fresh yeast extract... CAL Agar (Cellobiose Arginine Lysine Agar) (Yersinia Isolation Agar) Composition per liter: Agar ...............0.................................................... Use: For the cultivation and maintenance of Spiroplasma kunkelii..........1mg Inositol....................5 ± 0........................................0.0L............................................... infusion from ................3.................................................... .....5. 25.......................................................0mL: Sucrose.................03g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................. anhydrous .................01mg Folic acid .... Distribute into sterile tubes or flasks..................................... Aseptically add 150...........06g Glycine..........................................................................1g Sodium acetate·3H2O...........0.............................0..................07g L-Leucine ......... 10.................. 0.......0................................................ 0.................................... fresh yeast extract..... 0....01g Glutathione.0mg Sodium glucoronate·H2O .......... pressed.......01g Deoxycytidine ..............................................0mL of sterile fresh yeast extract solution......................... 11.............................03g L-Threonine..................................0g L–Arginine·HCl .......0..............0.......2mg Coenzyme A ..................................0mL: Beef heart... Heat to boiling............................................................0g KCl.....................5................. 12...................................................025g L-Valine ... 0.0............................20.. C 3N Spiroplasma Medium Composition per 100.......... 2........0mL................025g L-Cystine ............................5g Neutral Red ............14g L-Glutamine .......0..................... 1....................... Filter sterilize.....50........................... Remove supernatant solution............0mL of distilled/deionized water.............................. 2......... Autoclave for 15 min at 15 psi pressure– 121°C.................................0.....................................................5........................................................... Do not autoclave.2.......................................02g 79 L-Histidine·HCl·H2O .......................................................................................................................................................................................................... starch-free....................0mg Choline chloride ...........1........7.... Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 500..0............5mL of sterile CMRL medium.......0g Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100.................. 6..0L.......0mg Flavin adenine dinucleotide .................1.........................................................................................................................2.... 1.......................8g NaHCO3 .......6– 6.................4g L-Cysteine·HCl·H2O ................ 0.......01g L-Tryptophan ...05mg Niacin ....................................................... 0..

.... 1........................................................1...................... Mix thoroughly.0......................4g FeCl3·6H2O .20..........0mL: Acid Fuchsin .......10............................................. 0............5....... Preparation of Medium: Prepare and dispense medium under 100% N2................................................ Preparation of Medium: Prepare and dispense medium under 100% N2...............................................0mL Caldicellulosiruptor Medium Composition per liter: Preparation of Trace Elements Solution SL-10: Pancreatic digest of casein ............................... Sparge with 100% N2................0g CaCl2·2H2O ....1 g NaOH (1N solution) ...........0mg Na2MoO4·2H2O ..............1 ± 0...........0mg CuCl2·2H2O ............7g 100× salt solution ........ Carbohydrate Solution: Composition per 100...........0g Ascorbic acid...... Add components to distilled/deionized water and bring volume to 1........................................................................................... 6..... 10......................2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .............5g Cellulose ..........2 ± 0..............................80 Caldicellulosiruptor Medium Use: For the isolation and characterization of Yersinia enterocolitica and enumeration of Yersinia enterocolitica from water and other liquid specimens.................................0g K2HPO4 .5mg pH 7.......................................................0g Preparation of Carbohydrate Solution: Add carbo- hydrate to distilled/deionized water and bring volume to ............................................................................................................19................................. Add remaining components.................0mg CuCl2·2H2O.... Add distilled/deionized water and bring volume to 1.....................................0mg HCl (25% solution) ......................................... 70................... 0........... 1......................................... 100..........0g MnSO4·H2O..... 190.. Mix thoroughly......................0mg ZnCl2 ............. Invert Petri dishes......2...............1................. 1...........0mL: Carbohydrate ...................................................... 0..........0mL Preparation of Andrade’s Indicator: Add compo- nents to distilled/deionized water and bring volume to 100..... 24......... 1................................................................. 1......................5mg Trace elements solution SL-10 ..................0mg HCl (25% solution) ......................6............................ 0.....................................................................................0mL pH 7........................................................ Caldicellulosiruptor Medium Composition per 1001.....................................75g MgCl2·6H2O.........................0mg MnCl2·4H2O............................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10..9g KH2PO4 ..................................... 1...... Gently heat and bring to boiling............ 2.............. 70..................4....................................10..........0mg Na2MoO4·2H2O .......................0g NaCl ...................... 2.................... Autoclave for 15 min at 15 psi pressure–121°C............. 0.... Mix thoroughly..................1........................3g Preparation of 100× Salt Solution: Add components to distilled/deionized water and bring volume to 1......................0mL pH 7..............0mL of HCl solution........ 0.........0g Meat extract................. 0...........9g NH4Cl.............. Anaerobically distribute into tubes or flasks......... 1................................. 10.. Use: For the cultivation of Caldicellulosiruptor saccharolyticus..................... Autoclave for 15 min at 15 psi pressure–121°C................0mg MnCl2·4H2O.5g FeSO4·7H2O .................................................................. 10......................................... 0................................... Sparge with 100% N2......................... Carbohydrate Fermentation Broth Composition per liter: Peptone .... 1..................................................... 0.............................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................................0g K2HPO4 .......... Mix thoroughly............................................................................0mg ZnCl2 .......................... 190... Anaerobically distribute into tubes or flasks......0L.......................................................................................0L..3.................9g NH4Cl...................... 2................. Autoclave for 15 min at 15 psi pressure–121°C............5...........................................0mL of HCl solution.................0mL Andrade’s indicator ..............0g Yeast extract..9g KH2PO4 ................................... H3BO3 ........ Autoclave for 15 min at 15 psi pressure– 121°C............................................................ Mix thoroughly...............75g Resazurin .........................4g NH4Cl ............0L................................0....5g CoCl2·6H2O .... 36... 50.75g MgCl2·6H2O..........................0L........... Mix thoroughly.............1g KH2PO4 ................................................................ Pour into sterile Petri dishes................0L.0g NaCl ....75g L-Cysteine·HCl............. 1.. 0.........................................................................................................................0mL....................... 0.....................0mg H3BO3 ...................5g CoCl2·6H2O ............................... Add components to distilled/deionized water and bring volume to 1... Autoclave for 15 min at 15 psi pressure–121°C.............. Spread 0..................5.......................................0L.......0g Carbohydrate solution ......................................................... Add distilled/deionized water and bring volume to 1..5mg Resazurin ..............5g Cellobiose . 36.............................0g NaCl ...........................10................... Sparge with 100% N2. Use: For the cultivation of Caldicellulosiruptor saccharolyticus..........0mg NiCl2·6H2O .. 2. 2..... Add remaining components................................................................2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ............ Sparge with 100% N2.........0mL 100× Salt Solution: Composition per liter: MgSO4 ................................................................0g Yeast extract.............................................. Mix thoroughly....... 0............... 24.. Mix thoroughly.........2 at 25°C Andrade’s Indicator: Composition per 100............................................2 ± 0.................4g FeCl3·6H2O .. 16.........................0g Cellobiose ...........................................................................2.. Use: For the cultivation and maintenance of Pseudomonas putida.....0mg Add FeCl2·4H2O to 10.............0mL: Pancreatic digest of casein ................2mL of 2m D-(+) camphor solution in methylene chloride (CH2Cl2) on the inside cover of each plate.............0mg NiCl2·6H2O .....0g K2HPO4 ........5mg L-Cysteine·HCl. 100............................................... Allow to cool to room temperature............................ Camphor Minimal Medium Composition per liter: Agar ............

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

....................... 0........... Autoclave for 15 min at 15 psi pressure–121°C............... incubate in an atmosphere of 50% CO + 50% air....................0mg CuCl2·2H2O.... After inoculation............0mg Ferric ammonium citrate .......0L.......................................... 0............0L............... 5.....................................................02mL of sterile sulfide-calcium solution to each tube.... 0.0mg Folic acid................. 20........................................0mL Vitamin solution .0mg Ferric ammonium citrate.........5g MgSO4·7H2O............... Mix thoroughly....................................9g H3BO3 ...... 0...............................01g NaWO4·2H2O...........................................................................0mL Yeast extract solution ........0mL TS2 Trace Elements Solution: Composition per liter: Na2MoO4·2H2O ....0mL volumes into anaerobic tubes................................2mg TS2 trace elements solution ............................................ After inoculation......... 100..0L................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................. 2.33g CaCl2·2H2O ........................... Adjust pH to 7.........................0mL pH 6............... Mix thoroughly........................................... Preparation of Medium: Add components to distilled/ deionized water.............52g KCl ........................ Add components..... 30...................................................................................................... 0.........................0...................................2g CaCl2·2H2O.............................2 at 25°C Trace Elements Solution SL-4: Composition per liter: EDTA................ Distribute into tubes or flasks................ 36....0..... 10......................... Add NaHCO3.....................0............................0L............. Mix thoroughly.........20..........0mg NiCl2·6H2O .....................................................10................0mg Riboflavin . 5.......2g ZnSO4·7H2O ......................0...................................................................................................0.................................0mg Biotin .............. Adjust pH to 6.... Mix thoroughly....0L...........0mL of distilled/ Caution: Carbon monoxide (CO) is a toxic gas.......................0g Preparation of Sulfide-Calcium Solution: Add components to distilled/deionized water and bring volume to 1....................................................0L.................................. 10..........................................01g NiC12·6H2O ..1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1. 5................... 0...30.......................................0mg p-Aminobenzoic acid ........9g H3BO3 .......................... 1.. Use: For the cultivation of Spirochaeta thermophila.. 10... 20............. Mix thoroughly...... Cool to room temperature while sparging with 80% N2 + 20% CO2.....0mg CuCl2·2H2O .................... 9......... 10...............5 with KOH.......0mg components to distilled/deionized water and bring volume to 1..........................1.......................................................0mg Vitamin B12 ....................... 10........... 20.............. 0............0g KH2PO4 ................................................3g CoCl2·6H2O..............................33g NH4Cl .............2g CaCl2·2H2O ................0..0............................. Anaerobically distribute 10. 0.......0............... 5.......................... Mix thoroughly............................................... to distilled/deionized water and bring volume to 1........01g Preparation of TS2 Trace Elements Solution: Add Preparation of Modified Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500................0mg Thiamine·HCl ............................................ 1............................. incubate in air.......................................................................................... Mix thoroughly....... Sparge with 100% N2............................................................... Use: For the organotrophic cultivation of Oligotropha carboxidovorans............................................................. Add remaining components one at a time.....0mL TS2 Trace Elements Solution: Composition per liter: Na2MoO4·2H2O................................................20..............................0mL Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1................ Sulfide-Calcium Solution: Composition per liter: Na2S·9H2O.........................1g MnCl2·4H2O...... Mix thoroughly..... Gently heat and bring to boiling.. Carboxydobacterium Medium Composition per liter: Na2HPO4·12H2O........0......20............................................................................................................2.. Autoclave at 121°C for 15 min.. Adjust pH to 6................... Carboxydothermus Medium Composition per 1030....... Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ................29g Resazurin ..... 2......2g Trace elements solution SL-6...5g NH4Cl ............. Autoclave for 15 min at 15 psi pressure–121°C....................................33g KH2PO4 ............................... Anaerobically distribute into tubes.......... Continue boiling for 3 min....0L..................... Mix thoroughly..........................................................3.0mg Na2SeO3 ...........................82 Carboxydobacterium Medium Na2SeO3 ...0g KH2PO4 ..... 15.5g MgSO4·7H2O .........0L.............0g Sodium acetate .....................8..................0mg Na2SeO3...............................................5g NH4Cl....... 10.......... Mix thoroughly........................................... Carboxydobacterium Medium Composition per liter: Na2HPO4·12H2O ...... .............. Distribute into tubes or flasks..................... Add distilled/deionized water to 1..... except NaHCO3 and sulfide-calcium solution....................................................0mg Calcium DL-pantothenate....................................1..0mg Preparation of TS2 Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.......................................................... 0..................3g CoCl2·6H2O ..........................2mg TS2 trace elements solution ..............9 ± 0..............5g FeSO4·7H2O .................0mg NiCl2·6H2O...................9.......0mL: MgCl2·6H2O ... 5................9...............1..................0mL Na2S·9H2O solution .......................... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2........0.. 5.......................................5mg Trace elements solution SL-4......0...1............... 1......................................................... deionized water and bring volume to 1...1g MnCl2·4H2O ............. 0..........................0.........2g ZnSO4·7H2O...............................0g CaCl2·2H2O..... Aseptically and anaerobically add 0.. Adjust pH to 6.....................................0mg Lipoic acid ... 1......0mg Nicotinic acid ........... Use: For the autotrophic cultivation of Oligotropha carboxidovorans...... Autoclave for 15 min at 15 psi pressure–121°C...................0L..........

..................................................................................................0mL of sterile Na2S·9H2O solution.... 0..............Carcboxydobrachium pacificum Medium Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O............................................................................ 2..............0mg Lipoic acid ................ Mix thoroughly. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C............................................... 20.......5mg Vitamin solution ...... FeCl2·4H2O...........................................3g CoCl2·6H2O ...0mg Riboflavin ...0.0mg HCl (25% solution) ..5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10....0mg H3BO3 ......... Autoclave for 15 min at 15 psi pressure–121°C.............. 1.0....0mL NaHCO3 solution..................6...........0mL..0mL L-Cysteine solution..............0 ± 0......0mg Calcium DL-pantothenate...................................0mL: Na-pyruvate .. Na2S·9H2O Solution: Composition per 10...2................... 5..................0.... 0................................................... Use: For the cultivation and maintenance of Carboxydot- Add FeCl2·4H2O to 10.........36......................................... 0.......................... Adjust pH to 7............................................................................0mL Caution: Carbon monoxide is a toxic gas........5.............3g Na2HPO4 ...... 5................. and Na2S·9H2O solution.......................0mL of HCl solution..... to distilled/deionized water and bring volume to 1........... Mix thoroughly..............................4g Preparation of Trace Elements Solution SL-10: Vitamin Solution: Composition per liter: Pyridoxine-HCl ............... Sparge with 100% N2......0mg Thiamine·HCl .....45g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10...7g Preparation of Na2S·9H2O Solution: Prepare and dis- pense solution anaerobically under 100% N2.................... Autoclave for 15 min at 15 psi pressure–121°C......0mg D-Ca-pantothenate ... 0.. Autoclave for 15 min at 15 psi pressure–121°C.................................................................0g MgSO4·7H2O .........10............................................................... 0...0mL....................................... Mix thoroughly...0mg NiCl2·6H2O...........03g Resazurin .. Vitamin Solution: Composition per liter: Pyridoxine·HCl ..0mg Vitamin B12 ............................2g ZnSO4·7H2O ......... Sparge under 100% N2 for 10 min.....0mL: Na2S·9H2O ....7g CaCl2·2H2O.........5g CoCl2·6H2O.....03g NiCl2·6H2O ..0mL Na2S·9H2O solution ......... 10...............................0mg Biotin .0..................... Yeast Extract Solution: Composition per 10..................... 10................... 10.................5..................... 5..................................... 2.....5.........................0mg Nicotinic acid ....0mL of sterile yeast extract solution.....................0mg Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.. 10..........................0L..................15g Yeast extract .. 5............................................................. 0.... Pressurize inoculation flask with CO (carbon monoxide) gas at 2 bar pressure........................................................0mg Na2MoO4·2H2O....................... 3............ Sparge with 100% N2.................... 10..... Add distilled/deionized water and bring volume to 1.... Add remaining components.................... 0................................ Aseptically and anaerobically combine with 10.......................0mg CuCl2·2H2O. Mix thoroughly.......0mL of sterile vitamin solution...... 5.....0mL.............................. yeast extract solution..... Mix thoroughly.0........... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C..... 0.1g Na2MoO4·2H2O ...100.................... 83 NH4Cl ...................................0mL pH 7.................01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.... Mix thoroughly.....................................0............ Autoclave for 15 min at 15 psi–121°C........................................0mL.........9g KCl..........0mg p-Aminobenzoic acid .......................... Trace Elements Solution SL-10: Composition per liter: Preparation of Medium: Add components...........................02g CuCl2·2H2O ................................................................................ Sparge with 100% N2.....0mL: Na2S·9H2O............0mL.........0L................................................5.1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1....5g Preparation of Pyruvate Solution: Add pyruvate to distilled/deionized water and bring volume to 10......................................... Mix thoroughly..................... Mix thoroughly....................... 5........................... Autoclave for 15 min at 15 psi pressure–121°C..................0mg Nicotinic acid ................................... 10... Na2S·9H2O Solution: Composition per 10....... 0..................................... NaHCO3 Solution: Composition per 10.........................................................................05g Na2SiO3 .................................. Sparge with 100% N2....................................2......................... Mix thoroughly... hermus hydrogenoformans......... Must be prepared freshly.................................................................... Mix thoroughly.............................0... except vitamin solution..........0mL Trace elements solution SL-10...............................0L...........1....................................0L......................70..................... Autoclave for 15 min at 15 psi pressure–121°C....0mL Pyruvate solution.............................. Mix thoroughly..... 0..0mg MnCl2·4H2O ................................................ Sparge with 100% N2.45g Preparation of L-Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10............................24................2 at 25°C Pyruvate Solution: Composition per 10.................0mg ..................................... L-Cysteine Solution: Composition per 10...................... and 10........5g H3BO3 ...................................... Filter sterilize.......0mg Folic acid....0mL: Yeast extract.......0mL: NaHCO3 .. Carcboxydobrachium pacificum Medium (DSMZ Medium 902) Composition per 1050mL: NaCl .................................................. Cool to 25°C...........0mg Thiamine-HCl·2H2O .0mg ZnCl2 ...................0mL..... 10..0.........................................190.. 0..........................................................0................... 10......... 10............... Add Na2S·9H2O to distilled/deionized water and bring volume to 10..........0mL: L-Cysteine·HCl·H2O.0mg Riboflavin...................................................... Mix thoroughly...............

Adjust pH to 7.................5.......0..........0L..10............... Distribute into tubes or flasks.....1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.....................................1g Solution B (Metallic salts)..............0mL.......7g NaMoO4·2H2O .. Mix thoroughly............27g Sodium glutamate . and 10...............................0mg Vitamin B12 ................0L......... Solution B (Metallic Salts): Composition per 100.......... Use: For the cultivation of myxobacteria.............. The final pH should be 7..................... L-Cysteine-HCl·H2O solution.....................2..............................................................5g Ethylenediaminetetraacetic acid................. 0.....0..........................0L.............0g Solution A (Mineral Salts) ....... 5...................................01g Calcium pantothenate .......................................0......0L.............0g Yeast extract... 5.......................05mg Tris/HCl-buffer 10mM.........0g Sodium acetate .......................0mg Biotin .........0.......... Use: For the cultivation and maintenance of Caryophanon Preparation of Medium: Add components to distilled/ latum and Vitreoscilla stercoraria......................................... Filter sterilize...1g Thiamine·HCl ........1...0mL Na2S·9H2O.0mg Biotin ..................0g Yeast extract ......... 1............................ Pour into sterile Petri dishes or leave in tubes......4.................3g MnSO4·H2O......0g K2HPO4 ................. Autoclave for 15 min at 15 psi pressure– 121°C..................8 ± 0.....0g Casein hydrolysate ..........................0g Agar .20... Gently heat and bring to boiling...................................0mL Preparation of Solution A (Mineral Salts): Add nitrilotriacetic acid to 500............... 1........ CAS Medium Composition per liter: Pancreatic digest of casein ...................................2 with H2SO4 or KOH....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 950......5....0..... Autoclave for 15 min at 15 psi pressure–121°C........................84 Caryophanon latum Medium p-Aminobenzoic acid ..................................................... Mix thoroughly......................................................34g FeSO4·7H2O . Sparge with 80% H2 + 20% CO2.........0mL: ZnSO4·7H2O............... 2.............................................0g K2HPO4 ........................................................................................ Autoclave for 15 min at 15 psi pressure–121°C............... Caryophanon latum Medium Composition per liter: Papaic digest of soybean meal .......0mg Folic acid . Sparge with 100% N2 of at least 30 min........................... Add distilled/deionized water to 1................................ Distribute into anaerobe tubes or bottles.2..0L with distilled/deionized water...................... 10................0g KCl ........ NaHCO3 solution...... 10.....04g CoCL2·6H20 . Distribute into tubes or flasks.. Preparation of Medium: Prepare and dispense medium under 100% N2....2g pH 7............ except vitamin solution................. 0............................................................ 10...................................................... 50... 2.....................................0g MgCl2·6H2O .......................250..............0...........2 ± 0..................10...2 at 25°C deionized water and bring volume to 1....................... Dissolve by adjusting pH to 6.........................................0mL Vitamin solution.................. 2.............. 0..........1mg Preparation of Medium: Combine components.........0mL pH 7..........................29.... pyruvate solution...................0mg Thiamine·HCl ............. 1..................................67g Nitrilotriacetic acid..................................................................................20......5..............3................................................................... Autoclave for 15 min at 15 psi pressure–121°C.....0g CaCl2·2H20 ..... Vitamin Solution: Composition per liter: Pyridoxine·HCL ..........................5................................... Mix thoroughly................. Use: For the cultivation of Carboxydibrachium pacificum (Carboxydobrachium pacificum)....................................................... 2............................. .............................0L................0mg Folic acid............. Readjust pH to 7...............02g Na2B4O7·10H20 ........................0mL of distilled/deionized water...........0mL pH 7................................................ Aseptically and anaerobically add per liter.......3g CuSO4·5H2O...........................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............ Add remaining components.........2.0.0.................0mL pyruvate solution. 10........ 0... Mix thoroughly...0mg Riboflavin..0mL L-Cysteine-HCl·H2O solution.0mL vitamin solution........... Pour into sterile Petri dishes or leave in tubes.........0mg Nicotinamide ..................... Mix thoroughly..... 0....................... and Na2S·9H2O solution......0mg Vitamin B12.......................4 ± 0....0g Vitamin-free casamino acids..............................0g Pancreatic digest of casein ................................5 with KOH....0mL NaHCO3 solution.................... Use: For the cultivation and maintenance of Microcyclus aquaticus....0.... Casamino Acids Glucose Medium (CAGV Medium) Composition per liter: Agar ............................0g MgSO4·7H2O .................... Gently heat to boiling...1000..................2mg Biotin ...... Distribute into tubes or flasks.................... 0..... Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.8 .......................0mL......... 2.........................................................0g Glucose ..................... Mix thoroughly.............. 10...................02g Preparation of Solution B (Metallic Salts): Add a few drops of H2SO4 to distilled/deionized water to inhibit precipitate formation.... Distribute into tubes or flasks.......... pH 7................................ 1................. Filter sterilize.0. Adjust pH to 7............. Mix thoroughly........................25g pH 6........0g CaCl2·2H2O .................. Add components to acidified distilled/deionized water and bring volume to 100...................................5..... Casein Medium Composition per liter: NaCl .....................0mg Lipoic acid ..............1g FeSO4·7H20 ....0g MgSO4·7H2O ............ Autoclave for 15 min at 15 psi pressure–121°C...........................5.........................20..0.................... to distilled/deionized water and bring volume to 1...........12..............0L........ 1.............. Mix thoroughly.......0............. Add components....... 20.................... Bring volume to 1....2 ± 0......2 at 25°C Solution A (Mineral Salts): Composition per liter: MgSO4·7H2O............................ Mix thoroughly...............

.......................................... Readjust pH to 7....0.................................................2....................................................... 2.......................................................0g Potassium phosphate buffer (0......0....... Mix thoroughly...................1g MgSO4·7H2O......... Distribute into tubes or flasks... Mix thoroughly..0mL of distilled/deionized water.................5 ± 0................... Dissolve by adjusting pH to 6....................5g Co(NO3)2·6H2O......1g Nitrilotriacetic acid....0.........................................025g H2SO4 .....................2 at 25°C Preparation of Medium: Combine components.......... 0.........7g Na2HPO4 ............ Use: For the isolation of cyanobacteria................ Castenholz .....5mL pH 7.....................025g CuSO4·5H2O...................... Autoclave for 15 min at 15 psi pressure–121°C...0..0L............5 with KOH..........................................2........................ Mix thoroughly................................ Autoclave for 15 min at 15 psi pressure– 121°C...............28g Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1..1...........................................0.0mL pH 7........5.......................................................06g NaCl .......................5 ± 0.......... 0................................................0...............................5g ZnSO4·7H2O............0g Yeast autolysate..0mL Micronutrient solution ......8............................. Mix thoroughly....................... .......... 0....0g Glycerol ...................................0mg FeCl3 solution................... 0.......................... Mix thoroughly...........025g Na2MoO4·2H2O ....025g H2SO4 . Use: For the isolation of halophilic cyanobacteria. and enumeration...................103g MgSO4·7H2O................... Mix thoroughly........................... Mix thoroughly................. For the cultivation of Chloroflexus species and Fischerella species. Autoclave for 15 min at 15 psi pressure–121°C...... Castenholz: Add components to distilled/deionized water and bring volume to 1.5mL Preparation of Trace Metal Solution....0g NaNO3 ... 85 Preparation of Medium: Add nitrilotriacetic acid to 500..............................................................111g KNO3 .........................................................................0L...... 1.......11g KNO3 ... 0................................0......2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O........... Add remaining components.............10............ 0..........................................................................................1g Nitrilotriacetic acid.. 1.. 0.....................1g CaSO4·2H2O .................0g MgSO4·7H2O .................160.0.............................................................0mL of distilled/deionized water.... Autoclave for 15 min at 15 psi pressure–121°C.................................................... Pour into sterile Petri dishes or leave in tubes.......3mg Trace metal solution................0mL Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......2. Adjust pH to 7..........2) ......................0.....5 with KOH..1g Nitrilotriacetic acid ..... 0........................0L with distilled/deionized water.................................. of iron and sulfur bacteria from the Sphaerotilus group.......1..... Bring volume to 1................... 0...............0......0... including thermophilic species....................................................................................... 15..........5g ZnSO4·7H2O . Preparation of Medium: Add nitrilotriacetic acid to 500..0....0L...... Distribute into tubes or flasks..........28g H3BO3 .................................... Modified (Medium D..1g CaSO4·2H2O.......................................................0.................................. 2............................. Readjust pH to 7..........................................2 ± 0.................................... 8.................................. Mix thoroughly.......... Modified) Composition per liter: NaCl ................5g CoCl2·6H2O .................................................................... Castenholz D Medium... 1.................0.. 0.... Use: For the cultivation and maintenance of Myxococcus species...............................06g NaCl ............................... Gently heat to boiling.....0......... Castenholz DG Medium (Medium DG) Composition per liter: Glycyl-glycine buffer ....... Add remaining components......................................................0g Agar ..........0................11g KNO3 .................................... Bring volume to 1............5.....0L pH 7..... Mix thoroughly..........................................06g FeCl3 ........0........025g CuSO4·5H2O ....28g H3BO3 ...2 at 25°C Trace Metal Solution.5 ± 0..0L with distilled/deionized water....... Mix thoroughly.01M solution................................................... Castenholz: Composition per liter: MnSO4·H2O.....................0L. pH 7.....................................................0L........2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O ...............0mg FeCl3 solution .0....... 0..................... Distribute into tubes or flasks.......025g Na2MoO4·2H2O..............5mL pH 7.................0............... Micronutrient Solution: Composition per liter: MnSO4·H2O ............... Use: For the isolation........................................................1g MgSO4·7H2O ......................28g Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1....... 0...........0mL Micronutrient solution.... 0.........................7g Na2HPO4 ...................................................................... Dissolve by adjusting pH to 6................... 5.......................0................................. 1......... 20.........................8g NaNO3 .............................. Castenholz D Medium (Medium D) Composition per liter: NaNO3 .................69g Na2HPO4 .......................... Casitone Glycerol Yeast Autolysate Broth (CGY Autolysate Broth) Composition per liter: Pancreatic digest of casein .................................Castenholz DG Medium Use: For the cultivation and maintenance of Halobacterium species and other halophilic bacteria... 0...................................... Distribute into screw-capped tubes or flasks.........5mL Preparation of Micronutrient Solution: Add com- ponents to distilled/deionized water and bring volume to 1......... Casitone Agar Composition per liter: Pancreatic digest of casein ............................................1g CaSO4·2H2O........ cultivation........

........................................................................... Dissolve by adjusting pH to 6....2. 500....5mL Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1...................1g Nitrilotriacetic acid......1.................................... Distribute into tubes or flasks.0mg Nitrilotriacetic acid.. that require reduced nitrogen concentrations...........................5mL pH 7........... Autoclave for 15 min at 15 psi pressure– 121°C............................................................... Readjust pH to 8................................0mL Micronutrient solution........28g Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1................8g NaNO3 .......1g CaSO4·2H2O ................................................2 at 25°C Preparation of Medium: Combine components....025g CuSO4·5H2O .........0µg CuSO4·5H2O.....................0mg CaSO4·2H2O....................................................................................... Add remaining components..........................................................................................7g NH4Cl.........5mL pH 7..............................................0..... Bring volume to 1...................... Mix thoroughly.. Mix thoroughly................103.5g CoCl2·6H2O..2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O..........28g Castenholz Medium Composition per liter: Tryptone ................................ Use: For the isolation of cyanobacteria....28g H3BO3 ..........025g Na2MoO4·2H2O............ 0.........0mg NaCl ............... 46................... Add remaining components..........0L.............................. 0....................................0g NaNO3 ............1g CaSO4·2H2O.28g H3BO3 .......5g ZnSO4·7H2O .................. Mix thoroughly...1....0L with distilled/deionized water...............................0.. 0........5 ± 0.....................100..........025g Na2MoO4·2H2O .....................140........ Distribute into tubes or flasks.............. 0..................0.. Mix thoroughly.....................................0mL of distilled/deionized water..... 0. Add remaining components..025g H2SO4 .2 ± 0...0.... Autoclave for 15 min at 15 psi pressure– 121°C.......0.........0mg MnSO4·H2O........................5mg FeCl3·6H2O.............2...0.. Mix thoroughly........................................ Preparation of FeCl3 Solution: Add FeCl3·6H2O to distilled/deionized water and bring volume to 1........2 with NaOH................ 0.................................... 8.......................................................................0.. Use: For the isolation of cyanobacteria................................................0L with distilled/deionized water.............5g ZnSO4·7H2O...................... Autoclave for 15 min at 15 psi pressure– 121°C........... Mix thoroughly................................0..............0........ 0..............................5g CoCl2·6H2O ........................................... ..................................................0L.......... 0....0mg FeCl3 solution..... Mix thoroughly...............0L...........0L with distilled/deionized water..0...........................................1..........0mL Micronutrient solution .. Mix thoroughly............ Adjust pH to 8...................0g Yeast extract ....04µg Na2MoO4·2H2O..............................025g CuSO4·5H2O...................................... 0.........................................................................11g KNO3 .....0mg MgSO4·7H2O........................ 0....... 0........................1g MgSO4·7H2O .........5 with KOH...................... 2.......................................................100.................0mg KNO3 ...................0µg pH 8.............. Mix thoroughly.5 with KOH............... 2....... Micronutrient Solution: Composition per liter: Micronutrient Solution: Composition per liter: MnSO4·H2O ...........................................................................1................... 0.........................................8................ 0............2mg H3BO3 ........................... 1..........................8.. 2............................................ 25................... including thermophilic species............................ Readjust pH to 8...................5g ZnSO4·7H2O ..........................................................................................025g H2SO4 ..... Use: For the isolation of cyanobacteria........ 25...........0...................................5mL MnSO4·H2O............................ Dissolve by adjusting pH to 6..... Autoclave for 15 min at 15 psi pressure– 121°C.....................5 ± 0................... including thermophilic species............0mL of distilled/deionized water............. 0.....................47mg CoCl2·6H2O................. Mix thoroughly..........................025g H2SO4 ......689.. Use: For the cultivation of Thermus aquaticus....... Preparation of Medium: Add nitrilotriacetic acid to 500....................... 0............................................... Mix thoroughly..............86 Castenholz DGN Medium Micronutrient Solution: Composition per liter: MnSO4·H2O .........025g CuSO4·5H2O ...........0...0L. Bring volume to 1............ 0............. Mix thoroughly............28g H3BO3 .....................................................................0mL of distilled/deionized water............... Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1....2g Na2HPO4 ..........................5mL Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1........................0..........11g MgSO4·7H2O..............................06g NaCl ..025g Na2MoO4·2H2O .60............... Preparation of Medium: Add nitrilotriacetic acid to Preparation of Medium: Add nitrilotriacetic acid to 500..0. Readjust pH to 8........................................................................1g Nitrilotriacetic acid ...................................................................................................... 0.2........................ Castenholz ND Medium (Medium ND) Composition per liter: Na2HPO4 ..................................................................... Bring volume to 1....5 with KOH.................0mg Na2HPO4·2H2O ..............2 at 25°C FeCl3 Solution: Composition per liter: FeCl3·6H2O .................................................................................................................................................... 0................. Distribute into tubes or flasks..................... Distribute into tubes or flasks.......2............................2.......................................................0L..0mg FeCl3 solution ........0......................06g NaCl ............5g CoCl2·6H2O ................... 0.................... Dissolve by adjusting pH to 6................... including thermophilic species............ 0.. Castenholz DGN Medium (Medium DGN) Composition per liter: Glycyl-glycine buffer.......5mg ZnSO4·7H2O...

..........0mL of sterile distilled/deionized water........6.................................6...............0L............. 0........... 0.....0mL pH 7.......................... Use: For the cultivation of Caulobacter species from marine isolates........................................10.... 2×: Composition per liter: Agar ......................0L.............................................20..............................500......... 0.........................................................................................2.......... 2............... 100.... 1..0 ± 0..............................................................025g CuSO4 ......................................4g Na2HPO4 .22g KNO3 ............... 2×.. Preparation of Medium: Aseptically combine 500....... Castenholz TYE Medium with 2% Trypticase Yeast Extract Composition per liter: Castenholz salts.................. 0..............................0...............................5mL Preparation of Nitsch’s Trace Elements: Add components to distilled/deionized water and bring volume to 1........ Mix thoroughly..... and 400........ Preparation of Medium: Aseptically combine 500..................................6 ± 0......... Caulobacter Medium Composition per liter: Agar ...............................0mL of sterile 2% TYE.....................................0g NaNO3 ..........5g CoCl2·6H2O ...............................................................................0mL of sterile distilled/deionized water.................... 30.....0mL 2% TYE ................0g NaNO3 ................................................................ Distribute into tubes or flasks.....................5mL Preparation of Nitsch’s Trace Elements: Add components to distilled/deionized water and bring volume to 1............0mL of sterile Castenholz salts.................. 0.......................................................................................................................0......................... 0...........................100........... Mix thoroughly...........................016g FeCl3 solution (0..........................0................... 0.016g H2SO4 .............................. Use: For the cultivation and maintenance of Thermus species........................ Autoclave for 15 min at 15 psi pressure–121°C.................................0.................2.............................. 2.............................. and 400.....2g H3BO3 .. 1....... Autoclave for 15 min at 15 psi pressure–121°C..046g Na2MoO4 ...... 0........................................ 0..... Adjust pH to 8....................................... 0..............................................................0............................................ 1% TYE: Composition per liter: Pancreatic digest of casein ..2g CaSO4·2H2O .......... filtered.......... Mix thoroughly.....0mg pH 7..............0L....2g MgSO4·7H2O .......................................................2g H3BO3 ...21g Nitrilotriacetic acid ............. Nitsch’s Trace Elements: Composition per liter: MnSO4 ....2 at 25°C Castenholz Salts.................046g Na2MoO4... 0...2 at 25°C Preparation of Medium: Add components to tap water and bring volume to 1..... Pour into sterile Petri dishes or leave in tubes.....0mL Nitsch’s trace elements ................................................................2........2g Nitrilotriacetic acid ..............................................4g Na2HPO4 .............................0g MgSO4·7H2O. 10.....................0..0........................................016g H2SO4 ...... Mix thoroughly.0g Yeast extract .....100...................... Mix thoroughly.... 1..........0mL 1% TYE ..2 at 25°C Preparation of Medium: Combine components....................... Gently heat and bring to boiling.....016g FeCl3 (0.....2 at 25°C Castenholz Salts............22g KNO3 .....................................................................................0mL Preparation of Castenholz Salts..................20...03% solution) .............................12g NaCl .... 2×: Add components to distilled/deionized water and bring volume to 1.. 0..................... Gently heat and bring to boiling................................21g MgSO4·7H2O ................................................................................ 0.0g Peptone ... 10..................................025g CuSO4 ....0g Preparation of 1% TYE: Add components to distilled/ deionized water and bring volume to 1.............2........................0....................................... Autoclave for 15 min at 15 psi pressure–121°C....0mL Nitsch’s trace elements..... Distribute into tubes or flasks. Adjust pH to 7..... 0............. Autoclave for 15 min at 15 psi pressure–121°C........03% solution) .. 2×: Add components to distilled/deionized water and bring volume to 1................................................... 2× ....2g Riboflavin................ Adjust pH to 8.........5g Seawater....... 2×: Composition per liter: Agar ................0L.. Pour into sterile Petri dishes or leave in tubes..................................0mL pH 7............ 2............. Gently heat and bring to boiling....................... Nitsch’s Trace Elements: Composition per liter: MnSO4 ............0g Preparation of 2% TYE: Add components to distilled/ deionized water and bring volume to 1.......2g CaSO4·2H2O ..... Mix thoroughly......0mL Preparation of Castenholz Salts..... 2×.....500.............................................0L...0....... 30..................2....0mL of sterile Castenholz salts...........................................................6 ± 0........ 0...0g Yeast extract..Caulobacter Medium Castenholz TYE Medium (Castenholz Trypticase Yeast Extract Medium) Composition per liter: Castenholz salts....... 2× .......................... 100.....0L................ Mix thoroughly......... Autoclave for 15 min at 15 psi pressure– 121°C..................10...... Autoclave for 15 min at 15 psi pressure–121°C.........5g ZnSO4 .................................................... Adjust pH to 7.........................................12g 87 NaCl ....... 0...............0L.5g CoCl2·6H2O........................................................................2.................... Use: For the cultivation and maintenance of Thermonema lapsum and Thermus species..................0g Yeast extract ..................1....5g ZnSO4.........0g Peptone . Gently heat and bring to boiling...0mL of sterile 1% TYE............... 2% TYE: Composition per liter: Pancreatic digest of casein ...................... Mix thoroughly..........1................0L pH 7........................ ...................................... Caulobacter Medium Composition per liter: Agar ..........0 ± 0................ Use: For the cultivation of Caulobacter species from fresh water................. 0.

.................................0L pH 7.0mL of L-cysteine·HCl·H2O solution............ Preparation of BCYE-Alpha Base: Add components...........0g Yeast extract.....................................0g ACES buffer (2-[(2-Amino-2-oxoethyl)amino]-ethane sulfonic acid)...... Mix thoroughly................. 2......... 1............................... 1..........................100............................ 0........0mL of distilled/deionized water......... 1. 10.........9 ± 0.................................1......0g Charcoal.......0mL sterile antibiotic supplement.1g K2HPO4 ..................................0mL........................ CCVC Medium (Cephalothin Cycloheximide Vancomycin Colistin Medium Composition per liter: BCYE-alpha base ................. Mix thoroughly..... 10.... Mix thoroughly......... Use: For the selective isolation and cultivation of Legionella species from environmental samples..... Adjust pH to 6........................4 at 25°C Preparation of Medium: Add components to filtered aged seawater and bring volume to 1..................0g Riboflavin..... Use: For the cultivation and maintenance of Asticcacaulis excentricus...................... 990...3..................... Distribute into tubes or flasks......... activated ...0g Yeast extract .....81mg CaCl2 ...0g α-Ketoglutarate .....88 Caulobacter Medium Caulobacter Medium Composition per liter: Glucose ......................................0....................................................... Filter sterilize.....................9 with sterile 1N KOH....................0L.............................................................. Gently heat to boiling.........22mg CuSO4·5H2O ............................. Mix thoroughly............ 10............. Antibiotic Supplement Solution: Composition per 10... Autoclave for 15 min at 15 psi pressure–121°C..........5mg Preparation of Antibiotic Supplement Solution: Add components to 10..................................0mL L-Cysteine·HCl·H2O Solution: Composition per 10...............63mg MgCl2·4H2O...0L pH 7............1.....4g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10............................... Add 4.... 2...0mg Vancomycin .0mL.........................0L..0mL pH 6.............................0g K2HPO4 ......20...0mL Salt Solution: Composition per 100. 0...........16.............................2mg (NH4)6Mo7O24·7H2O . Use: For the enrichment of Stella species from polluted waters.... Mix thoroughly.. Autoclave for 15 min at 15 psi pressure–121°C................................ 3.. Adjust medium to pH 6........................................ 9................................................................................... 0....10....................0g NH4Cl ............. Preparation of Medium: To cooled BCYE-alpha base............................9 with 1N KOH. 1............................................. Distribute into tubes or flasks...........................................................................033g FeSO4·7H2O................4..............................2g Preparation of Medium: Add components to tap water and bring volume to 1...... 0................................................ to distilled/deionized water and bring volume to 990....1................066g MgSO4 ......... 0.........0g Yeast extract............................. add 10.........................................................................15........0g MgSO4·7H2O ...............................0g Peptone...........................................................................0g Seawater...80...... Mix thoroughly......... 0.................................10........................0mL: L-Cysteine·HCl·H2O. 1.............. Pour into sterile Petri dishes with constant agitation to keep charcoal in suspension........ Pseudomonas carboxydohydrogena...........................1g KNO3 . 1................ Distribute into tubes or flasks......... Autoclave for 15 min at 15 psi pressure–121°C..079mg Co(NO3)2·H2O .........0mg Colistin .................. Use: For the cultivation of Caulobacter halobacteroides and Caulobacter maris..0mg ZnSO4·7H2O ..0mL Antibiotic supplement .. Autoclave for 15 min at 15 psi pressure–121°C........................... 3.............. Pour into sterile Petri dishes or leave in tubes....... Pedomicrobium species.............................................................65g ......................................0g Yeast extract...0g Fe4(P2O7)3·9H2O ....0mg Seawater .... Filter sterilize....................... Caulobacter Medium II Composition per liter: Peptone...0g Peptone.2–7......................................0...................0mL: EDTA ...................................................0mg Cephalothin . except L-cysteine·HCl·H2O solution.......................4...25g L-Cysteine·HCl·H2O solution ................................................. Distribute into tubes or flasks.........02mg Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 100........ 10.................. Caulobacter Medium with Riboflavin Composition per liter: Peptone............................ 0........... 10.......................................0mL....................... Pirellula staleyi................2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems...................................... Cool to 50°–55°C..............0L.............4 at 25°C Preparation of Medium: Add components to filtered aged seawater and bring volume to 1....... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.... Mix thoroughly.... 0.......... Mix thoroughly.... Mix thoroughly......................0L............ Caulobacter Medium Composition per liter: Agar ..................... Heat gently and bring to boiling for 1 min..........1...... Adjust pH to 7......................... Cellulolytic Agar with Sea Salts Composition per liter: Agar ............................ 1......... Adjust pH to 7........0mL: Cycloheximide ....... Autoclave for 15 min at 15 psi pressure–121°C... Labrys monachus.......... BCYE-Alpha Base: Composition per liter: Agar ........................................................................................................................... and Stella species.... Caulobacter species.....................................0... Use: For the cultivation of Caulobacter vibrioides. 1...........0g Salt solution ................................0g Yeast extract............................2–7....2–7................................3mg NaBO3·4H2O.....2.2.........

........... Add components to distilled/ deionized water and bring volume to 1..............................................61............... 1.....1. Autoclave for 15 min at 15 psi pressure–121°C........................1% solution).................2 at 25°C Mineral Solution: Composition per liter: NaCl .......0g (NH4)2SO4 ............0g CaCl2 ............ 1 filter paper.. 6...........................0mg p-Aminobenzoic acid .................... 8...1mg KAl(SO4)2·12H2O ............1............................2 ± 0....................................0L................0mL: Cellulose powder................................................ Autoclave for 15 min at 15 psi pressure– 121°C..........6....................................................0mg Folic acid .......................................................................................0mL Filtered seawater ..........0mL pH 7....................................096g Cellulose suspension............................0g Yeast extract....................................... Add components to distilled/deionized water and bring volume to 1..........10........ Preparation of Medium: Prepare and dispense medium anaerobically in 100% N2 atmosphere................5.....0g NaCl ..........65g NH4Cl............... Cellulolytic Broth with Sea Salts Composition per liter: K2HPO4 .......................5g 89 Filtered seawater .........................0g CaCl2 .10....................2g L-Cysteine·HCl·H2O ...0g K2HPO4 .......................................................6g Yeast extract......................... 0...........2 with 5M NaOH. 30.......................0.......................... Mix thoroughly............0mg FeSO4·7H2O ........... Use: For cultivation of Clostridium stercorarium and other bacteria that can utilize cellulose as a carbon source..................1% solution)........5.............................0g (NH4)2SO4 ..............0mg ZnSO4·7H2O.................................1............. Distribute into tubes or flasks that contain cellulose as a strip (4................. 1..........................0mL of distilled/deionized water and mix thoroughly.............6g MgSO4 .......... Cellulolytic Medium Composition per liter: NaHCO3 .................................................. 1.............0mg Thioctic acid ..0L............ 0.....5.0mL pH 7............................................................0mg Cyanocobalamin................... Preparation of Medium: Prepare and dispense medium anaerobically under 100% N2..................0mL Resazurin (0..........................0...............................0...... 0.0mL Resazurin (0...2 ± 0......296g KH2PO4 ...................... Distribute into tubes or flasks......................................0mL L-Cysteine·HCl·H2O solution ...................0....150........2 at 25°C Cellulose Suspension: Composition per 200................................................. Mineral Solution: Composition per liter: NaCl ................... Mix thoroughly........0L...................................... Filter sterilize.....12g CaCl2·2H2O .................0....................2 with 5m NaOH........................................0mg Thiamine·HCl . 200...................0mg CuSO4·5H2O............6g MgSO4 ..................... 0....................0mg Nitrilotriacetic acid...................5g CaCl2 ............................6g L-Cysteine·HCl·H2O ............. Distribute into tubes or flasks.......200..0mL: Cellulose powder..................................0...............................................0L.........2......................................0µg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...........................5.........21.................................................................................0mg Biotin ....................................0............5.........................................................................1................................................0mg Riboflavin......................5..... 8............................................................................ 0......................... Adjust pH to 7...... Use: For the cultivation and maintenance of Clostridium papyrosolvens and other marine bacteria that can utilize cellulose as a carbon source.........................15.........................0mL Mineral solution ...................................................Cellulolytic Medium Yeast extract................ Adjust pH to 7...........1% solution) .............................................. ....... Add components to distilled/ deionized water and bring volume to 1... Whatman CF11 ..........................................2.......................................................... Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C..................... Use: For the cultivation and maintenance of Clostridium papyrosolvens and other marine bacteria that can utilize cellulose as a carbon source............................................... Mix thoroughly.............0.......1mg Wolfe’s vitamin solution .. 1..................200..............................................................0cm) of Whatman No..2......................................... Preparation of Medium: Prepare and dispense medium anaerobically in 100% N2............................ 6.....................5 ± 0.........................06g Cellulose................................0mL Resazurin (0......................................0mg NaCl .............2.......................... Mix thoroughly.........0mg CoCl2·H2O.........1.........0mL of distilled/deionized water and mix thoroughly...................... 1...........0mg Nicotinic acid ......................... 0....0...................................................5cm × 1.......... 100..18g (NH4)2SO4 .96g L-Cysteine·HCl·H2O ......................................0g NH4Cl ........................................................ 0......................... Cellulolytic Agar for Thermophiles Composition per liter: Agar .....0g Preparation of Cellulose Suspension: Add cellulose powder to 200.......................0mL pH 7..................2 with 5m NaOH....2 ± 0.............................................................................................096g MgSO4 ......5g Cellulose suspension...............................0L...............................6g Preparation of Mineral Solution: Add components to distilled/deionized water and bring volume to 1..........200.........................0.......................................15g MgSO4·7H2O.............0mL pH 7....0....................6g Preparation of Mineral Solution: Add components to distilled/deionized water and bring volume to 1...................................................... 5.......0mg Calcium DL-pantothenate ....68g K2HPO4 ....0mg Resazurin ...............................2 at 25°C Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl..............................0L...................0mg MnSO4·H2O ......................... 0.. 10...........6......0mL Mineral solution ................................ 1.........2 at 25°C Cellulose Suspension: Composition per 200.......... 0................................................................................................65g NH4SO4 .............0g Preparation of Cellulose Suspension: Add cellulose powder to 200....... Adjust pH to 7..1mg Na2MoO4·2H2O........................................................ 1.........1mg H3BO3 . Whatman CF11 ...............5. 150..............................

. 0...... Distribute into tubes using anaerobic techniques under a gas phase of 98% CO2 + 2% H2.............0mL of sterile L-cysteine·HCl solution........5.45g MgSO4·7H2O ...............................................................1g CaCl2·2H2O...................................0mL of freshly prepared alkaline solution................0mL of sterile NaHCO3 solution......... 1.................................................... Autoclave for 15 min at 15 psi pressure–121°C.............. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C... Gently heat and bring to boiling under a gas phase of 98% CO2 + 2% H2................................0g Glucose........4 ± 0.........................25g Na2S·9H2O...................0mg Cellobiose solution .... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C......................8g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.........2.9g K2HPO4 ...................... Use: For the cultivation and maintenance of Cellulomonas fermentans........................ and L-cysteine·HCl solution.............. 0................................... Use: For the cultivation and maintenance of Cellulomonas species...1..............................25g Preparation of Alkaline Solution: Add components to 25..............................9g (NH4)2SO4 ... Preparation of Medium: Add components...... 5.... Autoclave for 15 min at 15 psi pressure– 121°C.............. 10.. Mix thoroughly... 0.............5g (NH4)2SO4 ................ Aseptically distribute into sterile tubes or flasks... Cellulolytic Medium with Rumen Fluid and Soluble Starch Composition per liter: Basal medium ...................................2.............0mL of sterile Wolfe’s vitamin solution and 5........................................................ Prepare freshly........................0mL.................................975...............0mL of sterile L-cysteine·HCl·H2O solution....................0L...........................18g CaCl2 ...0g Preparation of Cellobiose Solution: Add cellobiose (or cellulose) to distilled/deionized water and bring volume to 50..... 15.....1..................09g Resazurin ........................ Preparation of Medium: Add components..........................................0g NaCl ................ 10.0g Pancreatic digest of casein ........................................1g Cellulose........................ Mix thoroughly...............0mL: L-Cysteine·HCl·H2O ...0mL: NaHCO3 ................ Mix thoroughly................ Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.0mL...37g Pancreatic digest of casein .................0mL: D-Cellobiose (or cellulose).........................................2 at 25°C Cellobiose Solution: Composition per 50....... Cellulomonas PTYG Medium (Cellulomonas Peptone Tryptone Yeast Extract Glucose Medium) Composition per liter: Agar ...............................................................................0mL pH 7.......................0g Soluble starch........................................................... 0....................................0mL pH 6................ Mix thoroughly................................................ Cellulomonas fermentans Medium Composition per liter: Yeast extract. Mix thoroughly.......... L-Cysteine·HCl Solution: Composition per 10..8 ± 0................................0mL.18g .......0mg Preparation of Basal Medium: Add components to distilled/deionized water and bring volume to 975..................25.................0g Cellobiose ................0g Peptone ........................................ to distilled/deionized water and bring volume to 985...0........ Aseptically and anaerobically add 50.0mL.......0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............................................ Use: For the cultivation of cellulolytic bacteria........................ NaHCO3 solution....... Filter sterilize.... Distribute into tubes or flasks..................... Pour into sterile Petri dishes or leave in tubes...... Alkaline Solution: Composition per 25...5g Preparation of L-Cysteine·HCl Solution: Add Lcysteine·HCl to distilled/deionized water and bring volume to 10...........3g MgCl2·6H2O........................02g FeSO4·7H2O ...... 10................. 5.................. Autoclave for 15 min at 15 psi pressure–121°C...........................21g KH2PO4 .........0g K2HPO4 ....................................25mg Resazurin ..5.......0.......................................... 0.....0g Yeast extract ....... Heat to boiling and cool to 25°C........ Cool slightly........................... Mix thoroughly......68g KH2PO4 .............. 5.................... Mix thoroughly............0mL L-Cysteine·HCl solution .......................... and 10...................................0g NH4Cl .......0mL: Agar ....... Cellulose Anaerobe Medium (LMG Medium 94) Composition per liter: NaHCO3 ...... Mix thoroughly........ Mix thoroughly..........0mL.............................2................15.............................0...2 at 25°C Basal Medium: Composition per 975.... Aseptically add 10........5.................0mL. 5.............45g KH2PO4 .8..............................................................................0mL Alkaline solution............................................ 0...... NaHCO3 Solution: Composition per 10. except cel- lobiose solution...................... Sparge under 100% N2 for 10 min........ Adjust pH to 6.. 50... 1...........0mL of sterile cellobiose solution.......0mL of distilled/deionized water.... 1.............. 6..................... ................. to distilled/deionized water and bring volume to 930..................5.....0mL...........0mL: L-Cysteine·HCl·H2O .5..............................................0. 0......... Add 25......................................................................... Mix thoroughly............0mL of basal medium...................................90 Cellulolytic Medium with Rumen Fluid and Soluble Starch L-Cysteine·HCl·H2O Solution: Composition per 20................. Use: For the cultivation of Selenomonas ruminantium and Succinimonas amylolytica.........0mL NaHCO3 solution.........0g NaHCO3 ... Preparation of Medium: Prepare 975........... Gently heat and bring to boiling............ Mix thoroughly..0g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 20....................... 0... 1.0mL: L-Cysteine·HCl ... except Wolfe’s vitamin solution and L-cysteine·HCl·H2O solution... 0. 0........................ Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.

.............................. 5..............0mL of distilled/deionized water....1............................................................. 21..........................0mg Folic acid....................... 350..............0mg ZnSO4·7H2O ............. 10.........................0mL Solution B................................10......................1mg H3BO3 ....... sterile solution B......0g MgSO4·7H2O............................................................................... 5..1...................... 2........................................0............ 5..............1.....0g MgSO4·7H2O................................5...................... 0............................................................0g Preparation of Solution B: Add components to distilled/deionized water and bring volume to 650............. 0.................... Herpetosiphon species................. 1..0mg Nicotinic acid .......................................................0mg CoCl2·6H2O ............ Aseptically add 10........................................................... Saprospira species.......... Auto- 91 clave both solutions separately for 15 min at 15 psi pressure–121°C...... Autoclave for 15 min at 15 psi pressure–121°C.01g NaMoO4·2H2O . Adjust pH to 6............................................ 2.... sterile solution A and 650... Mix thoroughly.............................Cellulose Overlay Agar (NH4)2SO4 .......15g MgSO4·7H2O ..0mL Solution A: Composition per 350.................. add remaining components to distilled/deionized water and bring volume to 900...........5..........................................1................0mL Cysteine hydrochloride solution ...................................0–7.......................... 0....... Cellulose Broth Composition per liter: Cellulose...................................................01g BaCl2 .................................................................... 5............. 1........................................................ Mix thoroughly................0mg NaCl ...... 650.... 5............5 at 25°C Preparation of Medium: Add cellulose to 100..... Mix thoroughly...... Mix thoroughly. Cellulose Overlay Agar: Composition per liter: Solution A .............0mL sterile cysteine hydrochloride solution...... 0.....................0mg Resazurin .............0mL Preparation of Solution A: Add components to distilled/deionized water and bring volume to 350............................. Mix thoroughly.............8......0.......................................... Add 485......................................0mL..............0mg MnSO4·H2O ............................................................................................... Adjust pH to 7.....5 with KOH..................... 0....................................................0mL Solution A .... 1......0mL Cellulose overlay agar ............2g CaCl2·2H2O. L-Cysteine Solution: Composition per 10... 5......................................................0mg FeSO4·7H2O.............................0L.0............................. 1......1. Mix thoroughly...................0g Trace elements solution..... powdered....... 0.................................0mL Stan 5 Agar: Composition per liter: Solution B...............................................0... Cool to 45°–50°C..........0mL: CaCl2·2H2O .................................................. Use: For the cultivation of cellulose-utilizing anerobic bacteria............... Gently heat and bring to boiling..................... Preparation of Medium: Add nitrilotriacetic acid to 500.... 5.............0mg Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 1............. 15........... Cool to 45°–50°C..............0mL: CaCl2·2H2O .................... Add remaining components except vitamin solution and cysteine hydrochloride solution..0mg CuSO4·5H2O ................................................... In a separate flask.........12g K2HPO4 ..........................................5.............1g FeCl3 ....................................................................................0mL Solution A: Composition per 350.........................1mg KAl(SO4)2·12H2O.....................1mg Vitamin solution......................... and Flexithrix species...................................0............................ 0.............. 0................0mg Lipoic acid ........................................... Trace Elements Solution: Composition per liter: EDTA.........................................................................1mg Na2MoO4·2H2O . 1..................................................02g KBr .............................. Aseptically combine the two sterile solutions........................................... Mix thoroughly................02g CuSO4 ....................................0mg Thiamine·HCl .............................................0mL of cooled................................................. Use: For the isolation and cultivation of Cytophaga spe- cies...............................0mL. 10................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1..............................................0g (NH4)2SO4 ................................................... Cellulose Overlay Agar Composition per plate: Stan 5 agar ...0mL distilled/deionized water.......0mg Vitamin B12 .......2 at 25°C Vitamin Solution: Composition per liter: Pyridoxine·HCl ........................... 61..............0mg SnCl2·2H2O ....0g K2HPO4 ........... Filter sterilize................. Autoclave for 15 min at 15 psi pressure–121°C...........0mL: L-Cysteine·HCl·H2O ..............0g Trace elements solution.1........0mL: Agar ..............0g MgSO4·7H2O ............... Mix thoroughly.................. 15.......0mL of sterile vitamin solution and 5.. Aseptically distribute into sterile tubes or flasks.... Cool to 50°C.................................................. 296..........02g ZnCl2 ....0g MnCl2·4H2O ..0mg Riboflavin .......................5........ Aeptically distribute to tubes or flasks... 1........ 1.........1....5........................................................0mg p-Aminobenzoic acid ................. Autoclave for 15 min at 15 psi pressure–121°C....................1 ± 0.................................0mL .........0..................................01g H3BO3 ............................... Sparge with 100% N2....................0mg Calcium DL-pantothenate............ 0...5g Preparation of L-Cysteine Solution: Add L-cys- teine·HCl·H2O to distilled/deionized water and bring volume to 10.................. Cool to 45°–50°C.............................................................................1g CoCl2 ....................0mg Biotin ..02g pH 7.. Mix thoroughly..... 0... 0. 650......0..........0mL. 350....................................0L.. Solution B: Composition per 650.........0mg LiCl.............................................. 1..... Autoclave for 15 min at 15 psi pressure–121°C...........................................................................0g (NH4)2SO4 .................0mL of cooled.......................0mL pH 7..0mL of distilled/deionized water.1...............0mg CaCl2·2H2O................0g (NH4)2SO4 ..............................0mg Nitrilotriacetic acid ....................... Preparation of Stan 5 Agar: Aseptically combine 350.. Mix thoroughly................... Gently heat and bring to boiling......................................0mL..............0g K2HPO4 ....... 5.............

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

...0........................Chlorinated Fatty Acid Medium Use: For the cultivation and maintenance of Rhodospirillum species............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.........................................................................20.................. 2... and Flexithrix species........... 3........... Pour into sterile Petri dishes or leave in tubes........................1g CaCl2·2H2O.. concentrated..............................0g Na2HPO4 ..........................0L...........0g Sodium acetate...............................0g Agar ...........3g FeSO4·7H2O .........................0g Yeast extract .........0mL: (NH4)2SO4 .......2...... CHCA Salts Medium (Cyclohexane Carboxylic Acid Salts Medium) Composition per liter: K2HPO4 ..........0g Agar .............. Use: For the cultivation of Chlamydomonas reinhardtii..........001g ZnSO4·7H2O.................................. 1...0g K2HPO4 ......................................0......4......0....... 0.................. Add this solution to 2....0......001g pH 8...0L.........0g NH4NO3 ............0mL of cold concentrated HCl..... Gently heat and bring to boiling.........0L............... 1.................................. Adjust the pH to 7........ Cetrimide Agar.......40................... Filter the solution through Whatman #1 filter paper.....0g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....... Mix thoroughly..................................... solids from infusion..................5g ................. Cetrimide Agar............0 with KOH...................... Herpetosiphon species...........01g MnCl2·4H2O . 0...... USP (Pseudosel® Agar) Composition per liter: Pancreatic digest of gelatin .......... cultivation................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.... Gently heat and bring to boiling.... Chitin Agar Composition per liter: Agar ....... Use: For the selective isolation.............1.................................... cultivation..0g MgSO4·7H2O ...0L..... For the cultivation and maintenance of Arthrobacter globiformis....... Pour into sterile Petri dishes or leave in tubes....... Use: For the cultivation and maintenance of bacteria that can utilize cyclohexane carboxylic acid as a carbon source.........................0g Chitin ..0g MgCl2 ........... 2...... Distribute into tubes or flasks......1g Yeast extract......... 10.......9g pH 7............... Distribute into tubes or flasks................................... Use: For the selective isolation and cultivation of streptomycetes........................................................... Pour into sterile Petri dishes or leave in tubes........................5g KH2PO4 ......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..................... Mix thoroughly..............................6g K2SO4 ...01g pH 7..........0mL pH 7..........2................................ 1.. Adjust pH to 7.........................7g MgSO4·7H2O......... Distribute into tubes or flasks................5g FeSO4·7H2O.................. 10....................... Precipitated: Composition per 2... Mix thoroughly....0 ± 0.....0g Tryptose.... 93 Chitin Agar Composition per liter: Agar ...................... Mix thoroughly....................................................................0g NaCl .................0 ± 0....0............................................2 ± 0...............................................................................................0L of distilled/deionized water at 5°C...............2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems.... 0......... Chlamydomonas Enriched Medium Composition per liter: Agar ....... Use: For the selective isolation............. Pour into sterile Petri dishes or leave in tubes............. 20.... nonfermentative bacteria........1................1g KH2PO4 ................................. 13....... Autoclave for 15 min at 13 psi pressure–118°C............... Distribute into tubes or flasks............ Precipitated: Add chitin to 400............................................................................................................. 1.......01g Na2MoO2·2H2O ...................................... Gently heat and bring to boiling................................................ Autoclave for 15 min at 15 psi pressure–121°C.......... and identifica- tion of Pseudomonas aeruginosa and other Gram-negative........................................................................................................................................................................................................4g Cetrimide .....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................... Autoclave for 20 min at 15 psi pressure–121°C.................. Chlorinated Fatty Acid Medium Composition per 1001. and identifica- tion of Pseudomonas aeruginosa and other Gram-negative............ precipitated ................................0g (NH4)2SO4 ......0g Cetrimide ..........15.....0g HCl........0.........................................0.... Use: For the isolation and cultivation of Cytophaga spe- cies....................................15..... 0......................0mL Preparation of Chitin........ 15.................. 0................0g Tryptose ....... Distribute into tubes or flasks....... Mix thoroughly..1...........3........ Autoclave for 15 min at 15 psi pressure–121°C..........................10..............2 ± 0...............3g Glycerol ............................................. Autoclave for 15 min at 13 psi pressure–118°C............................... Distribute into tubes or flasks....... Gently heat and bring to boiling... Pour into sterile Petri dishes or leave in tubes.........................2g FeSO4 ....................................0............. 400.....0L.................7g MgSO4·7H2O............. Non-USP Composition per liter: Beef heart..... 0............................5L: Chitin ................................... 0.....01g ZnSO4·7H2O ....................5g Cyclohexane carboxylic acid ....... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................................................... Gently heat and bring to boiling............0.0L..................................... nonfermentative bacteria............ 5.................0g Chitin........ 0........ Autoclave for 15 min at 15 psi pressure–121°C............................ anhydrous......... Pour into sterile Petri dishes or leave in tubes....................5g KH2PO4 .... Mix thoroughly........................0...........0mg Chitin........ Gently heat and bring to boiling.0mg MnSO4 .... Saprospira species........ 500............. Dialyze the precipitated chitin against tap water for 12 hr.......

............0L: MgSO4·7H2O .................19g MnCl2·4H2O ....................... Tightly close cap................................ Chlorobiaceae Medium 1 Composition per 4990................. Cool to 25°C under 100% N2...0mL bottles using positive pressure of 95% N2 + 5% CO2...8 ± 0....................0mL: Solution 1 ...25g CuCl2·2H2O .................1g ZnCl2 ...0mL pH 6........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.........................25g Na2MoO4·2H2O .........................1..........................................................................................................0mL Na2S·9H2O solution ........... 10. Mix thoroughly.......0mL Na2S·9H2O solution.............................0......................0.............................................................. Cool to 45°–50°C......0L of sterile.....................1..........5g KCl...........................0g Disodium EDTA.............................. Use: For the isolation and cultivation of members of the Chlorobiaceae.................1g Ca(NO3)2 ........... Cool to 25°C under 100% N2.. Mix thoroughly................................................................................................... 1........................................0mL: Use: For the cultivation of Alcaligenes species........... 1........ Mix thoroughly...1...860..........................0mL H2O .....................0mg Preparation of Medium: To 4............................25g ZnCl2 .........04g CuCl2·2H2O................................................... Filter sterilize......... Filter sterilize with positive CO2 pressure......... 0.... Preparation of Vitamin B12 Solution: Add Vitamin B12 to distilled/deionized water and bring volume to 100............................................................................. Preparation of Medium: Combine components. 40............................0L........2 at 25°C Trace Elements Solution: Composition per liter: FeSO4·7H2O.........0...............2 at 25°C Solution 1: Composition per 4.................... 0........................................................................... 6........................................................8............ Vitamin B12.............. pH 7........... Autoclave for 15 min at 15 psi pressure–121°C..........................................................0L Trace elements solution .........1............... 0........0......... Cool to 25°C......0mL: Solution 1 ................. 1....................................................... 0...0.........04g CuCl2·2H2O.....................0L................................1.............................0mL Trace elements solution ......... NaHCO3 Solution: Composition per 100.............................................0L.........0mL....100......................... 1........ Gas with 100% CO2 for 20 min.....................................................1g ZnCl2 .............................................................. 1................................................................ Mix thoroughly.............................................................0mL pH 6.......................05g Trace elements solution SL-8..02g NiCl2·6H20 ..........1g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water............0mL: KH2PO4 .................0. Mix thoroughly.................................... Cool to 25°C. ponents to distilled/deionized water and bring volume to 1...... tilled/deionized water and bring volume to 950...........................................0mL: Na2S·9H2O................... Autoclave for 15 min at 15 psi pressure–121°C..........................................20.............................0.................................0g Co(NO3)2·6H2O ............................4g CaCl2·2H2O .........................02g HCl (25% solution) ................... Autoclave for 15 min at 15 psi pressure–121°C...................0.. 7..... Mix thoroughly.................0.............06g NaMoO4·2H2O .0L O2-free water...02g NiCl2·6H20 .............................25g H3BO3 ... 60.......2) ...................................0..............................................................................................................5g MgSO4·7H2O........5............................0...................0................. Mix thoroughly.. Vitamin B12 Solution: Composition per 100.. 1................0mL NaHCO3 solution .......................0g NH4Cl ....................7g KH2PO4 ... Saturate with CO2 by stirring under 100% CO2 for 30 min.........................25g Preparation of Solution 1: Add components to dis- tilled/deionized water and bring volume to 4........94 Chlorobiaceae Medium 1 MgSO4·7H2O ................ Trace Elements Solution: Composition per liter: FeCl2·4H2O.. 0....0mL: O2-Free Water: Composition per 860.......................02g ...........................................................................4.........0mL pH 7....... Gas with 100% N2 for 15 min in a screw-capped bottle.......................5g CoCl2·6H2O................... 0......................2.....................................19g MnCl2·4H2O ...........................8 ± 0........07g H3BO3 .............................................06g NaMoO4·2H2O ...5g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 100............. Chlorobiaceae Medium 2 Composition per 1051...........................0g MnSO4·H2O ...... Aseptically distribute into sterile 100.. Completely fill bottles with medium except for a pea-sized air bubble.. Autoclave for 45 min at 15 psi pressure–121°C................. 2...... Adjust pH to 6.5mL Preparation of Trace Elements Solution: Add com- Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to 1................... 950.................................54g Phosphate buffer (20mM solution...........2 ± 0.........................................0mL: Trace Elements Solution SL-8: Composition per liter: NaHCO3 ..... 0.......0mL Vitamin B12 solution..........0.......5g CoCl2·6H2O.........5............ Mix thoroughly.860......................................................... 0........ 0....... Na2S·9H2O Solution: Composition per 100............0............... 1......0mL Vitamin B12 solution .....................0........... CO2-satu- rated solution 1................075g 2-Chloropropionate ..... Distribute into tubes or flasks.....................................7g NH4Cl..1g NiSO4·6H2O....5...0................... Autoclave for 15 min at 15 psi pressure–121°C.............................................07g H3BO3 ..................................0mL..................1g NH4VO3 ....0........2g FeCl2·4H2O.................... aseptically add the remaining components..........0mL NaHCO3 solution............. 1...... Mix thoroughly...........2 at 25°C Solution 1: Composition per 950.......0mL.......7g CaCl2·2H2O................................................................................................ 0.....

.. .............4 at 25°C FeCl3 Solution: Composition per liter: FeCl3 .....0mL of sterile 1-chlorobutane..7g H3BO3 ..0mg MgSO4·7H2O..... Aseptically add 0........................................................................................... NaHCO3 Solution: Composition per 100... Preparation of Medium: Add components.... except 1- chlorobutane...................... Mix thoroughly.........................5.....................0mL of sterile Na2S·9H2O solution....................................0g Preparation of Solution B: Add Na2S·9H2O to distilled/deionized water and bring volume to 100..........................................0mL of sterile NaHCO3 solution.........5g Na2S..0g Glycyl-glycine ............0mL: Vitamin B12 ............................15............................. Use: For the cultivation of Corynebacterium species..................... Autoclave for 15 min at 15 psi pressure–121°C.................................0mL of cooled......0mL pH 8...8....................................0mL: NaHCO3 .........29g Preparation of FeCl3 Solution: Add FeCl3 to distilled/ deionized water and bring volume to 1.........0–7. Mix thoroughly.......... 10............................0g MgCl2·6H2O.5g Solution A .....................0mL Micronutrient solution.. Preparation of Medium: Add components..............................0mL: KH2PO4 .0.....................0............................... Filter sterilize..0mL: Na2S·9H2O.0g Preparation of Solution C: Add Na2S2O3·9H2O to distilled/deionized water and bring volume to 100........................................... Chloroflexus Agar Composition per liter: Agar ........ NH4NO3................8 with sterile H2SO4 or Na2CO3............................................... Autoclave for 15 min at 15 psi pressure–121°C............. Vitamin B12 Solution: Composition per 100..... 1........ Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C.....1g Nitrilotriacetic acid........2mL of sterile solution B...........0 ± 0............ Mix thoroughly...................0............................................1.................0mg MnCl2·6H2O ..............0mg FeCl3 solution.......0L Mix thoroughly....................... except so- lution A..........2...........0mL or 100.0mL of medium....................5g Yeast extract ...........0mL.......5g Na2HPO4·12H2O ............ and 1..Chloroflexus Agar 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.......0....5....103g CaSO4·2H2O.............................. 1..............................................0L..0L..0mg CuSO4·5H2O..................................................2 with H3PO4........................................689g Na2HPO4 .... 2..................0mL: Na2S2O3·9H2O ...... Aseptically distribute into sterile tubes or flasks...................................................................0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1............... Distribute into tubes or flasks..0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100.........................10..................0mL....... 1.. Adjust pH to 6......0mL: NaHCO3 .......................0mL bottles with metal screwcaps and rubber seals.. to distilled/deionized water and bring volume to 1..............5....................... to distilled/deionized water and bring volume to 1................0L........111g KNO3 ..................0mg Preparation of Vitamin B12 Solution: Add Vitamin 95 Trace Elements Solution: Composition per liter: FeCl3·6H2O............... Mix thoroughly........1............. Completely fill bottles with medium except for a pea-sized air bubble.............0mL............................ solution B..0mL................10......... Use immediately.......0g NH4Cl.. Chlorobium thiosulfatophilum Medium Composition per 1050....... Solution C: Composition per 100.................. Preparation of 1-Chlorobutane: Filter sterilize..... Mix thoroughly.....................0.... Chlorobutane Medium Composition per 1002..0...............................20....................0g Preparation of Solution A: Add NaHCO3 to distilled/ deionized water and bring volume to 100.0...............10................... Bring pH to 7....... Autoclave for 15 min at 15 psi pressure–121°C................06g NaCl ..0mg 1-Chlorobutane.......................0mL Solution B ..................0................... B12 to distilled/deionized water and bring volume to 100............. Mix thoroughly.......0mL..........0...........1g NaNO3 ..2mL of sterile solution A. 0........................ 0.................. Aseptically add 2..............1mL of sterile solution C for each 10..................................................................0mL pH 7..................0........................................... Aseptically distribute into sterile 50..................2 at 25°C Solution A: Composition per 100....0 ± 0.5..1g Co(NO3)2·6H2O............................................... 10...1g ZnSO4·7H2O...... Mix thoroughly...... sterile solution 1..... Autoclave for 15 min at 15 psi pressure–121°C...0mL Trace elements solution .............0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100...............4.........0mL................... 10..... Mix thoroughly.......... Na2S·9H2O Solution: Composition per 100.................0mL: Na2S·9H2O............................. 1...........................................................2g MgSO4·7H2O......................................... aseptically add 60..5g CaSO4·2H2O....................1.................50.................................................... 40....0mL pH 7....... Mix thoroughly... 5....5g NH4Cl ..................................................................................... Mix thoroughly.............. Use: For the cultivation and maintenance of Chlorobium limnicola...0..0mL of sterile Vitamin B12 solution.....................0mg FeSO4·7H2O ................................................. Adjust pH to 7........ 2..0..................................... Solution B: Composition per 100......0g KH2PO4 ......2–8.......................20.......................... 5..............0mg Yeast extract ...... Filter sterilize...2 at 25°C Use: For the isolation and cultivation of freshwater and soil members of the Chlorobiaceae. and solution C..........0mL Solution C ....................... and 0...........0mL: Preparation of Medium: To 950............0L.......................................0................ 0................. Cool to 45°–50°C. Mix thoroughly........................

......... 0..........1g KNO3 .................................0mL of medium into 100mL screw-capped bottles with rubber septa under 100% N2............. Pour into sterile Petri dishes or leave in tubes........................................... 1.........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........... 0....... use a sterile syringe..................................0mL................................................1g NaCl ...1g CaCl2·2H2O ..................0 mL: Glycyl-glycine .......... Add a magnetic stir bar................................................. Use: For the cultivation of Chloroflexus aurantiacus... 1..... Gently heat and bring to boiling.1g MgSO4·7H2O..................................... Use: For the growth and maintenance of Chloroflexus aggregans and Roseiflexus castenholzii.2g ZnSO4·7H2O ....0mL of neutralized sulfide solution into each bottle.... Stir the solution continuously to avoid precipitation of elemental sulfur............................1........................ 1.0mL... Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O ........................................................0.....045g CuSO4·2H2O .......... Adjust pH to about 7...........................................0mL Mix thoroughly.............................................1g NaCl .. Trace Elements Solution SL-6: Composition per liter: MnCl2·4H2O...................... 0.......................1g Na2MoO4·2H2O.0mL Trace elements solution SL-6....1g MgSO4·7H2O ......5g Na2HPO4·7H2O..........0.....................2 at 25°C Ferric Citrate Solution: Composition per 100......................................................1....... 0..... 1........1mg Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 100...... Gently heat and bring to boiling. Adjust pH to about 7... Chloroflexus aggregans Medium (DSMZ Medium 87a) Composition per 1061................0...... Neutralized Sulfide Solution: Composition per 100.....................................1.............................. Stir the solution continuously to avoid precipitation of elemental sulfur.......... The final solution should be clear and yellow in color..............0L.......... Mix thoroughly.................................3g CoCl2·6H2O .......................5mL Preparation of Micronutrient Solution: Add components to distilled/deionized water and bring volume to 1..............025g H2SO4................. The final solution should be clear and yellow in color.......... 0...... Autoclave for 15 min at 15 psi pressure–121°C.5.......0mL.....5g H3BO3 .. Mix thoroughly. Sparge under 100% N2 gas for 3 min....01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1..................................1g Na2MoO4·2H2O .....0............. Chloroflexus Medium................. Distribute into tubes or flasks..3 with sterile 2M H2SO4.... Modified Composition per 1001.....0.........5g CoCl2·6H2O .....11....................................4............... 0...................................................0.....05g Neutralized sulfide solution .............. except neu- tralized sulfide solution.... Incubate the culture at 50°C at a light intensity of 300–500 lux..... Adjust pH to 8.. 0.0L.................... 0.............0...... Mix thoroughly. Cool to room temperature. 0... to distilled/deionized water and bring volume to 1050....... 0....................... 0.......................................................... to distilled/deionized water and bring volume to 1.......... 0........................ Do not open the bottle to add H2SO4..................................................................... Sparge under 100% N2 gas for 3 min..................... Cool to room temperature...05g Neutralized sulfide solution ............... inject 1........ 0................... concentrated .. use a sterile syringe...................0mL pH 8......................................................................... 0......... Distribute 90..2–8.......................2 ± 0......................... Mix thoroughly.........................3g CoCl2·6H2O..............................................0g NaNO3 ..................0mL: Na2S·9H2O.......................0................................ Preparation of Medium: Add components........... 0............................96 Chloroflexus aggregans Medium Micronutrient Solution: Composition per liter: MnSO4·7H2O ..................0......................... except Na2S............... Readjust pH to 8.. Mix thoroughly................... 0. Continue boiling for 3–4 min under 100% N2.............0g Glycyl-glycine ............................................ For heavy cell suspension supplement periodically with sterile yeast extract solution to yield a final concentration of 0...................... Autoclave for 15 min at 15 psi pressure–121°C...........................5g KNO3 .....................0mL Ferric citrate solution ....................... Do not open the bottle to add H2SO4......28g H3BO3 ........................................02g CuCl2·2H2O ................................ 0................. 0..................... Sparge under 100% N2 gas for 3 min.....2–8. Ferric Citrate Solution: Composition per 100.... Mix thoroughly........0L........1g CaCl2·2H2O..........0...03g NiCl2·6H2O ............2g ZnSO4·7H2O... Cool to room temperature.....01g Preparation of Trace Elements Solution SL-6: 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 Neutralized Sulfide Solution: Composition per 100..... Mix thoroughly......0mL: Ferric citrate ..................0mL: Na2S·9H2O ................ Add Na2S........................................1%.......... Adjust pH to 8................... 11.....2 ± 0...........................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............1g Preparation of Ferric Citrate Solution: Add ferric citrate to distilled/deionized water and bring volume to 100.................................0mL pH 8........03g NiCl2·6H2O................. 0........................................................ Add a magnetic stir bar......................................................................1g Na2HPO4·2H2O ...... 2...0mL: Yeast extract.0g NaNO3 ....................................0mL.........2....................... 1.02g CuCl2·2H2O...... Preparation of Medium: Add components.......................................................................................................5g H3BO3 .................... Autoclave for 15 min at 15 psi pressure–121°C............................0........3 with sterile 2M H2SO4..... Mix thoroughly........................................0g Yeast extract ...5g ZnSO4·7H2O . 0.................4............... Using a sterile syringe..1..............0mL Ferric citrate solution ........................0mL: Ferric citrate .......... ......... 0........0L.....................0mL Trace elements solution SL-6 ................ 0........025g Na2MoO4·2H2O ..

...................0g Glucose .....5g MgSO4·7H2O .......................................... Filter.0g K2HPO4 ....0mL of neutralized sulfide solution into each bottle.................... 1.....0mL Resazurin (0.....5................... Mix thoroughly.......0g Starch.......500................ Eubacterium species............ Cool to 25°C.. add the remaining components................... Add meat and NaOH to distilled/deionized water and bring volume to 1...............0mL Preparation of Chopped Meat Extract: Use lean beef or horse meat................... Add the L-cysteine·HCl·H2O................ 0.......... Autoclave for 15 min at 15 psi pressure–121°C.......................................... Cool to room temperature............................ 1.............. 4.........0g NaOH (1N solution) ....0L of chopped meat extract filtrate.............................. For the cultivation of ATCC strain 35944.......0mL pH 7......5................................................ Vibrio parahaemolyticus..........2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath.. Add meat and NaOH to distilled/deionized water and bring volume to 1...............0 ± 0..........0L.................0g Bromothymol Blue ............0mL pH 7....................... Filter..................................................... Chlorohydroxybenzoic Acid Medium Composition per liter: K2HPO4·3H2O........................ 8......... Use: For the cultivation of anaerobic bacteria... Distribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by .. Use: For the growth of Vibrio cholerae..............0.......................... Gently heat to boiling..........................................04g Thymol Blue ....................0 with NaOH........................... Mix thoroughly..0L.... Cap with rubber stoppers and place tubes in a press.......................................... Chopped Meat Carbohydrate Medium with Rumen Fluid Composition per 1390............................................ Preparation of Medium: To 1..............0-7.... Reserve ground meat particles and filtrate... Remove fat from surface.....25g NH4Cl......................0g 5-Chloro-2-hydroxybenzoic acid ......... Use: For the growth and maintenance of Chloroflexus au- rantiacus.... 10. 4.....0 ± 0................0mL Rumen fluid...2 at 25°C Chopped Meat Extract: Composition per liter: Beef or horse meat...................0g Peptone..... Remove fat and connective tissue................... 0....... add the remaining components.....................0g Maltose .0L of chopped meat extract filtrate........ except L-cysteine·HCl·H2O and chopped meat solids........................... Dry agar plates before using... 0............... Add distilled/deionized water to filtrate and bring volume to 1..... Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust..................................................................0g Cellobiose.................0g 97 Maltose .......... Cholera Medium TCBS Composition per liter: Sucrose................................. Gently heat to boiling............................ 0.....................................0mL of distilled/deionized water.......... Cool to room temperature...................0.500.............. ...................04g pH 8............................. Remove fat and connective tissue.............................................. Add the L-cysteine·HCl·H2O................ Add remaining components and bring volume to 1..0....Chopped Meat Carbohydrate Medium with Rumen Fluid Preparation of Medium: Add components......... 0....... 150..................................................... Add distilled/deionized water to filtrate and bring volume to 1......5... 3..............................1.............0L....1.....0mL: Peptone.0mg ZnSO4·7H2O ......... Remove fat from surface... Distribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2................0g L-Cysteine·HCl·H2O...0..... 14..0g Na2S2O3·5H2O .............. Mix thoroughly............................................0g Yeast extract..................... and other Vibrio species..... 10.................. Continue boiling for 3–4 min under 100% N2.....0mg pH 7............0g NaH2PO4·H2O......0g Ferric citrate ................0mL Rumen fluid.................. Reserve ground meat particles and filtrate..................... Gently heat and bring to boiling while stirring........................ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................... to distilled/deionized water and bring volume to 990................ Grind...0g Cellobiose ............................................. 25.................. 4.......0mL Preparation of Chopped Meat Extract: Use lean beef or horse meat...................... 200......... 1........0mL: Peptone ...............1......... 200................................... Pour into sterile Petri dishes............. Gently heat and bring to boiling............................................................................. Distribute 90................ inject 1.....1.0L Chopped meat extract solids .... except L-cysteine·HCl·H2O and chopped meat solids........................0g Starch................... 5. Do not autoclave....... 1................................... 25...... Using a sterile syringe.....................0g Ox bile..................... Autoclave for 15 min at 15 psi pressure–121°C....................4 at 25°C Preparation of Medium: Add 5-chloro-2-hydroxybenzoic acid to 800........ 150..... Cool to room temperature........ Cool to 25°C...................................................................... Use: For the cultivation of bacteria that can utilize 5-chloro-hydroxybenzoic acid......... 20..........30.... 3...0L.............0g NaCl ...........................6 ± 0................... 10.......... Preparation of Medium: To 1...............0g NaOH (1N solution) ....... Gently heat and bring to boiling.... Adjust pH to 7.......... 1............0g Agar .............5g Chopped meat extract filtrate ............... Adjust pH to 7.0mL Resazurin (0..........................1.......0g Yeast extract ......................................0g L-Cysteine·HCl·H2O............... except neu- tralized sulfide solution..0g K2HPO4 ..........0mg CoSO4 ..................................................012g MnSO4·H2O ...................0L..............1g FeSO4·7H2O........................0g Yeast extract...........0g Sodium citrate ...........025% solution) .................... 5................ 10........................................0mL of medium into 100mL screwcapped bottles under 100% N2...............2g Nitrilotriacetic acid ............... Distribute into tubes or flasks.................... Gently heat and bring to boiling while stirring.................... 0...... 1.............2 at 25°C Chopped Meat Extract: Composition per liter: Beef or horse meat..... including Fusobacterium prausnitzii..........0L........5g Chopped meat extract filtrate . Adjust pH to 7.... 30.....0L Chopped meat extract solids .................... 4.......................... Grind.................. 2............................025% solution) .......................... and Prevotella ruminicola......... Chopped Meat Carbohydrate Medium with Rumen Fluid (ATCC Medium 1016) Composition per 1390................0mL Mix thoroughly.

....5g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 100...... Cool to 25°C under 100% N2.......0mL NaHCO3 solution..1...................... Do not overheat........... Use: For the differentiation and presumptive identification of Escherichia coli which forms blue colonies.......... Tightly close cap....1...........................................19g MnCl2·4H2O ........................0L: MgSO4·7H2O................................. NaHCO3 Solution: Composition per 100...................................0.2...... Vibrio cholerae form turquoise blue colonies and Vibrio alginolyitcus colonies are colorless. Cool to 45-50°C... Eubacterium species............. CHROMagar O157 Composition per liter: Proprietary.........0mL Trace elements solution.................10...0mL pH 7.................5mL Preparation of Trace Elements Solution: Add com- ponents to distilled/deionized water and bring volume to ...... Heat long enough with shaking every 5 min to ensure complete dissolution................................... Mix thoroughly...... Use: For the differentiation and presumptive identification of Escherichia coli and other coliform bacteria which form red colonies......... Mix thoroughly. Cool to 25°C....2 at 25°C Solution 1: Composition per 4.......................... of Vibrio parahaemolyiticus which form mauve colonies..... Source: CHROMagar EEC is available from CHROMagar Microbiology....0mL Na2S·9H2O solution ............ Filter sterilize with positive CO2 pressure...0.............................. Gently heat in a boiling water bath or steam bath........... FeCl2·4H2O................... Adding tellurite can increase specificity...............7g CaCl2·2H2O ..0............................0L O2-free water .. Heat long enough with shaking every 5 min to ensure complete dissolution....... Source: CHROMagar Vibrio is available from CHROM- Use: For the cultivation of anaerobic bacteria............ Adding tellurite can increase specificity...........0mL: H2O.... Cool to 45-50°C........ Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust....... Pour into sterile Petri dishes.................................. Do not overheat........0.....0mL.. Shake periodically during heating to dissolve components........ Source: CHROMagar E...... Gently heat in a boiling water bath or steam bath........ Autoclave for 15 min at 15 psi pressure–121°C.... coli volume) into tubes under O2-free 97% N2 + 3% H2............ 5............0mL: NaHCO3 ..................... Autoclave for 45 min at 15 psi pressure–121°C. Pour into sterile Petri dishes... Cool to 45-50°C... Shake periodically during heating to dissolve components....... CHROMagar Vibrio Composition per liter: Proprietary.......................... Mix thoroughly................ Cool to 25°C under 100% N2........................ Gas with 100% N2 for 15 min in a screw-capped bottle........0mL: Proprietary..04g CuCl2·2H2O.... Mix thoroughly.............98 CHROMagar E...............................5g CoCl2·6H2O........1....0g deionized water and bring volume to 1................. Heat long enough with shaking every 5 min to ensure complete dissolution............................... including Butyrivibrio crossotus............ Use: For the differentiation and presumptive identification Solution 1 .......0mL Preparation of O2-Free Water: Autoclave H2O for 15 min at 15 psi pressure–121°C.....1g ZnCl2 ........................ 6...7...... Cap with rubber stoppers and place tubes in a press...................... Shake periodically during heating to dissolve components.0L......... Saturate with CO2 by stirring under 100% CO2 for 30 min. coli Composition per liter: Chromatiaceae Medium 1 Composition per 4990...................................... Adding tellurite can increase specificity......... Na2S·9H2O Solution: Composition per 100.......... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...........07g H3BO3 .. Do not overheat.................................0L............... 20.... O2-Free Water: Composition per 860.......1...............5g KCl ..7g KH2PO4 ..............3 ± 0...... Use: For the differentiation and presumptive identification Trace Elements Solution: Composition per liter: of Escherichia coli O157. Mix thoroughly.........06g NaMoO4·2H2O . and Ruminococcus species........ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.... Preparation of Medium: Add components to distilled/ agar Microbiology....0mL: Na2S·9H2O ...............1.......................... Do not overheat.......... 5............ Mix thoroughly...................... 100...... Mix thoroughly.... 860.................... 860........... Adding tellurite can increase specificity........................0...................... CHROMagar ECC Composition per liter: Proprietary... Gently heat in a boiling water bath or steam bath...... Pour into sterile Petri dishes. Shake periodically during heating to dissolve components.........02g HCl (25% solution) ... Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water....... Gas with 100% CO2 for 20 min.......25g Preparation of Solution 1: Add components to dis- tilled/deionized water and bring volume to 4.......7g NH4Cl ...........................0.......... coli is available from CHROM- agar Microbiology.......................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1............ 4............................................................ Gently heat in a boiling water bath or steam bath.. Pour into sterile Petri dishes... CHROMagar E.0L..................... Heat long enough with shaking every 5 min to ensure complete dissolution.. Cool to 45-50°C..02g NiCl2·6H20 ..........0L...................0mL Vitamin B12 solution.... Source: CHROMagar O157 is available from CHROMagar Microbiology....0................0L...

............0 ± 0...... Mix thoroughly............ Mix thoroughly........... 5.......05g Trace elements solution SL-8 ......................2g CaCl2·2H2O ......0mg CuCl2·2H2O.............................................. Mix thoroughly............................................................... Mix thoroughly...................0g NH4Cl........ Completely fill bottles with medium except for a pea-sized air bubble.......................0..........0mL bottles using positive pressure of 95% N2 + 5% CO2.950.............. aseptically add the remaining components....................5g ...... 0.................0..0mL............................................. 5...............19g MnCl2·4H2O.........0mL..........0L............2 at 25°C Solution 1: Composition per 950...............................................0L of sterile.........................................................2g FeCl2·4H2O ........ NaHCO3 Solution: Composition per 100................0mL: Na2S·9H2O Solution: Composition per 100........................... 0....................................... NaHCO3 Solution: Composition per 50....................0......................................0mL of sterile Vitamin B12 solution.......0mL Na2S·9H2O solution......06g NaMoO4·2H2O...................5g CoCl2·6H2O...................0....07g VOSO4·2H2O ..... 0....... Mix thoroughly...............................................2............0mg Na2SeO3..................2.........1........................................................... Preparation of Vitamin B12 Solution: Add compo- Preparation of Vitamin B12 Solution: Add Vitamin rated solution 1................2......................02g KH2PO4 ................... Use freshly prepared solution................ 1....................0mL Sodium pyruvate solution...........0........... 0.... 0... Mix thoroughly......................... 0.0mL: Chromatium Medium (ATCC Medium 1449) Composition per liter: nents to distilled/deionized water and bring volume to 100.... 1.............. Mix thoroughly.........................Chromatium Medium 99 1................................0........0mL pH 7..................................................1..................................07g H3BO3 ............................... Use: For the isolation and cultivation of members of the Use: For the isolation and cultivation of freshwater and soil Chlorobiaceae.............5g NH4Cl ......... Vitamin B12 Solution: Composition per 100........................................ Use freshly prepared solution...01g Trace elements................................ Na2S·9H2O ..... Aseptically distribute into sterile 100.......................2g FeCl2·4H2O...............1g ZnCl2 ................................03g NiCl2·6H2O. sterile solution 1.....0mL of cooled.............2.............0mL Vitamin B12 solution .............. Autoclave for 15 min at 15 psi pressure–121°C................................................................................................. Autoclave for 15 min at 15 psi pressure–121°C........1........................... Filter sterilize......................... NaHCO3 .................0mL NaHCO3 solution.............. Filter sterilize........... Preparation of Medium: To 950....... CO2-satu- Solution 1 .......4g MgSO4·7H2O......................0mL: Vitamin B12 Solution: Composition per 100.................... Cool to 25°C........ Chromatiaceae Medium 2 Composition per 1051.......................................... Mix thoroughly.....................025g H3BO3 .....188g MnCl2·4H2O ........... Preparation of Trace Elements Solution SL-8: Add components to distilled/deionized water and bring volume to 1..........0mg Vitamin B12 .................0mL Na2S·9H2O solution .............0...0g distilled/deionized water and bring volume to 50........................3 with sterile H2SO4 or Na2CO3....... 1...0...................1..... Autoclave for 15 min at 15 psi pressure–121°C............0mL: Sodium pyruvate ...........0...................................................................................... 50...............2 at 25°C Trace Elements: Composition per liter: Disodium EDTA.. 0..0...... 2........................................................................................................................0mL: NaHCO3 ..............5....0mL Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950..... Completely fill bottles with medium except for a pea-sized air bubble....0mL.............................................. Cool to 45°–50°C..................04g CuCl2·2H2O ..0g to distilled/deionized water and bring volume to 100.............02g NiCl2·6H20 .........0mL: Vitamin B12......0mg Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 1.......................................0mL bottles with metal screwcaps and rubber seals............ Preparation of Medium: To 4....1g ZnCl2 ................ 5................................................60................. aseptically add 60.......3........................0mL pH 7......... Aseptically distribute into sterile 50..................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 50.....0mL: KH2PO4 ..................0.......... members of the Chromatiaceae.......................5g MgSO4·7H2O .40......0mL.......05g Disodium EDTA........... Sodium Pyruvate Solution: Composition per 50..............0mL of sterile Na2S·9H2O solution....5g CoCl2·6H2O ...0mL: Preparation of NaHCO3 Solution: Add NaHCO3 to Preparation of Na2S·9H2O Solution: Add Na2S·9H2O Na2S·9H2O Solution: Composition per 50...... 40.................................................... Cool to 45°–50°C... 50......0mL. Autoclave for 15 min at 15 psi pressure–121°C.....0mL NaHCO3 solution .........0...0mL............................... Filter sterilize..... 0. Adjust pH to 7........ 0........... 0....19g Na2MoO4·2H2O............. Trace Elements Solution SL-8: Composition per liter: Disodium EDTA ........................4g CaCl2·2H2O................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100......................... 50...................0mL: Na2S·9H2O.6............. and 1............0L........................................... Adjust pH to 7..3 ± 0.......................................... Mix thoroughly.0L.0mL of sterile NaHCO3 solution................0mg B12 to distilled/deionized water and bring volume to 100.............0mL..................... Filter sterilize......0mL or 100............................

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

.. Autoclave for 15 min at 15 psi pressure–121°C............... Preparation of Medium: Add components.............................. Aseptically add 20.......................20.......0mg H3BO3 ...........0.......5mg KH2PO4 ....................1 atm pressure.............1g H3BO3 .. Filter sterilize under 100% CO2 into a sterile....... 20...... Mix thoroughly.3g CoCl2·6H2O ............. 0...................................................0mg VOSO4·2H2O ................................................... Autoclave for 15 min at 15 psi pressure–121°C................2..............1 atm pressure for 30 min with magnetic stirring. Chromatium tepidum Medium Composition per 1001......................... Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100...........0mL..........................0mg Disodium ethylendiamine-tetraacetate (Disodium EDTA) ............................................................. 200........................................................................................0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20...... Add 860................................................ 3.......0mL.0mL: NaHCO3 ............... 400. Solution F: Composition per 100.........................0mL Trace elements solution..5g Solution A .....................1.........0g FeSO4·7H2O.................... Use: For the growth and maintenance of Chromatium salexigens...................188..............0L.0mL of solution B.......................... Autoclave for 15 min at 15 psi pressure–121°C...........0mg NiCl2·6H2O .............. Mix thoroughly....................... Mix thoroughly...........2..........0mg Na2MoO4·2H2O ........2g FeCl2·4H2O............................................ Solution E: Composition per 100...........20.....5g CoCl2·6H2O... gas-tight 100..0g Preparation of Solution A: Add NaHCO3 to distilled/ deionized water and bring volume to 100......0mL.......30..........0mg Preparation of Solution D (Trace Elements Solution SL-12B): Add components to distilled/deionized wa- ter and bring volume to 1.................10......0mg Na2WO4·2H2O...................... to distilled/ deionized water and bring volume to 960.......... 10. 2.........0mL................. Mix thoroughly...........0........ 100...25.............................................190.................05–0....... Adjust pH to 7...................................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20... the iron in the medium will precipitate out of solution as black flocs........0mL pH 7....0mL: Na2S·9H2O ...................................1.........5....0mL: NH4Cl.............0mg Na2MoO4·2H2O.....0mL: Na2S·9H2O .......0mL of solution D......... 50... Mix thoroughly.....19g MnCl2·2H2O...........2 at 25°C Solution A: Composition per 100...............................................0L.................0mg NiCl2·6H2O................... Mix thoroughly......... 50............. Preparation of Medium: Saturate cooled solution A under 100% CO2 at 0............. Solution B: Composition per 100.......................... 10..............0mg NaCl .6...............05–0.........0mL: Na2S·9H2O...................................................................5mg NaHCO3 solution ............. Sparge with 100% CO2 until saturated.... Trace Elements Solutions: Composition per liter: Disodium ethylendiamine-tetraacetate (Disodium EDTA) ............................0mL of solution C.........0mL screw-capped bottle........... Adjust pH to 7........................... 0........................ 400............................ Sparge with 100% N2 for 3–4 min.............100.............0 ± 0..............0mL Solution D . Mix thoroughly......................10.............0mg MgSO4·7H2O ........ Na2S·9H2O Solution: Composition per 20..................3 with sterile 2M HCl or sterile 2M Na2CO3 solution.............................................0g MgCl2·6H2O .................0mL: NaHCO3 ............ 24...........................70...2..............5g Preparation of Solution E: Add NaHCO3 to distilled/ deionized water and bring volume to 100...........................................0g Preparation of Solution F: Add Na2S·9H2O to dis- tilled/deionized water and bring volume to 100........... Use: For the growth and maintenance of Chromatium tepi- dum..................... 20.... Chromatium/Thiocapsa Medium Composition per 1060..........0mg MnCl2·4H2O ...................................... Distribute into sterile screw-capped tubes or bottles so that medium completely fills container........................ Dispense into a screw-capped bottle....... 0........................................0mL: NaHCO3 ...................................................................... Autoclave for 15 min at 15 psi–121°C....... 18......................................0mL.............................0mL: KH2PO4 ............0g NH4Cl ...... Leave a small gas bubble in each bottle to accommodate pressure changes.............0mL Na2S·9H2O solution....................... After 24 hr.. 1.......... Mix thoroughly............. 42......................0mL...................... 0.......................................0mg Preparation of Trace Elements Solutions: Add components to distilled/deionized water and bring volume to 1...0 ± 0.....................................................0mg CuCl2·2H2O.................................................0mL Trace elements solution .........0mg ZnCl2 ......Chromatium/Thiocapsa Medium Solution D (Trace Elements Solution SL-12B): Composition per liter: Disodium ethylendiamine-tetraacetate (Disodium EDTA) ...................................... 7......................0mL bottles under 95% N2 and 5% CO2 at 0.. Autoclave for 15 min at 15 psi pressure–121°C......0mg ZnCl2 ................... 5....0g .......................... 10....................0mL of solution E...... Autoclave for 15 min at 15 psi pressure– 121°C.....................0mg CuCl2·2H2O ...2 at 25°C 101 NaHCO3 Solution: Composition per 20.0mL of solution F through one of the screw-capped openings under 95% N2 and 5% CO2 with magnetic stirring.... and 20...........................1...0mL of sterile Na2S·9H2O solution.0mL of sterile NaHCO3 solution and 20..................................0mL pH 7..................... Mix thoroughly..................0mg CaCl2·2H2O.......... except NaHCO3 solution and Na2S·9H2O solution..............0mL Solution B..............................1......0mL Solution C...............0mg Ammonium acetate (or sodium pyruvate) .................. 1... 5...........................1................. 10...

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

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

... Autoclave for 15 min at 15 psi pressure–121°C.........104 Clostridium beijerinckii Agar K2HPO4 Solution: Composition per 10......0mL: Ground meat.....1.. Mix thoroughly............ Autoclave for 15 min at 15 psi pressure–121°C..0mL: Thiamine·HCl . Adjust pH to 7.......... and 1....0mg H3BO3 ...............................0g Yeast extract... Pour into sterile Petri dishes or leave in tubes..................................... Autoclave for 15 min at 15 psi pressure–121°C.4............................................... Sparge with 80% N2 + 20% CO2. Cool to 25°C. 20....................30.....................5 ± 0..............1........6............... Sparge with 80% N2 + 20% CO2..........0g Cellobiose........ Mix intensively.........................0L...........0mL Vitamin solution...0mg NaHCO3 solution ........ Aseptically and anaerobically add 20................................0mL.............. Distribute into tubes or flasks............... 4...2 at 25°C Polygalacturonic Acid Solution: Composition per liter: Polygalacturonic acid (or pectin) ....24..0g K2HPO4 ........................ Sodium caproate ................................... Mix thoroughly...............0g Proteose peptone No........ Na2S·9H2O solution................. Clostridium cellobioparum Agar Composition per 1025..................... 3 . Mix thoroughly.0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10................... to distilled/ deionized water and bring volume to 919.................... Aseptically distribute into sterile tubes or flasks............100..................... fat free ...... Preparation of Medium: Add components...........20.0mL................................... except glu- Sodium Caproate Solution: Composition per 20............... Bring volume to 50................. Mix thoroughly...... Use: For the cultivation and maintenance of Syntrophospora bryantii.. 5...............................................................................................................0µg p-Aminobenzoic acid ....................... 5........................... 100.......0mL of sterile trace elements solution SL10.........................................0mL Na2S·9H2O solution............. Add distilled/deionized water and bring volume to 1............ 3...... and trace elements solution SL-10... Add remaining components...0mL..... Cool to 25°C. 5............. Aseptically add sterile glucose solution and K2HPO4 solution....0µg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 20....................... Autoclave for 15 min at 15 psi pressure–121°C...............0g Agar ......................15............... Preparation of Medium: Add components.....0g Sodium thioglycolate ....... 0........................................ Autoclave for 15 min at 15 psi pressure–121°C.................................. Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20........5g CoCl2·6H2O...0mL.20............................................................. Filter sterilize..0mL pH 7.. Sparge with 80% N2 + 20% CO2............ 1...4g Preparation of K2HPO4 Solution: Add K2HPO4 to distilled/deionized water and bring volume to 10................... 2.................................0mL.....................0µg D(+)-Biotin ..............0mg Na2MoO4·2H2O...... 0............................................. Mix thoroughly...................................... Clostridium bryantii Medium Composition per liter: NaCl ......2.........................0mL.... Mix thoroughly...............0g MgCl2·6H2O........ 10................. Gently heat and bring to boiling...5........................20... 40. 10.........2 ± 0............ Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...............0g KCl................................. Autoclave for 15 min at 15 psi pressure–121°C.........0g K2HPO4 ................0mg HCl (25% solution) ..............0mL of sterile sodium caproate solution.....0mL of HCl solution......... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O...........0mL with distilled/deionized water.........0g Polygalacturonic acid solution.................................................................5.....................................0mg MnCl2·4H2O ..................4g Preparation of Sodium Caproate Solution: Add sodium caproate to distilled/deionized water and bring volume to 20................ sodium caproate solution................................. 20........ Mix thoroughly.....70......0g Pancreatic digest of casein ...........0mg NiCl2·6H2O. 5..........0..........................0mL: K2HPO4 ...0mL: Na2S·9H2O Solution: Composition per 20.......................... Sparge with 80% N2 + 20% CO2.........2..........0mL of sterile Na2S·9H2O solution............................ Sparge with 80% N2 + 20% CO2.....0mL pH 7......................... Clostridium beijerinckii Agar Composition per liter: Agar .........1.....3g KH2PO4 .... 5........... 0...........5g NH4Cl.. 3.....................2g CaCl2·2H2O.......20............0L....................1g Na2SO4 ............................190.......0g Maltose .....0g Yeast extract ..........36............................................. Sparge with 80% N2 + 20% CO2........0mg ZnCl2 . Mix thoroughly.......50....................0g Glucose........... Autoclave for 15 min at 15 psi pressure–121°C...2 at 25°C NaHCO3 Solution: Composition per 20..5mg Na2S·9H2O ...0g .15g Resazurin ................ 0........................................ Use: For the cultivation of myxobacteria................0mL Trace elements solution SL-10 .......................................................................5...............................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20...... Mix thoroughly............................................................................ Autoclave for 15 min at 15 psi pressure–121°C......................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.... Aseptically and anaerobically distribute into sterile screw-capped bottles............0mL Sodium caproate solution............................................................................1..... Vitamin Solution: Composition per 20.............................. 21...0mg CuCl2·2H2O....................1................................... except NaHCO3 solution...........0mL............... to distilled/deionized water and bring volume to 890............500................. Use: For the cultivation and maintenance of Clostridium beijerinckii.......0mL of sterile NaHCO3 solution.................................. Mix thoroughly.............................. Mix thoroughly.....0mL: cose solution and K2HPO4 solution........................0mL: NaHCO3 ... 20...... Mix thoroughly...........

......... Add L-cysteine·HCl·H2O........3g FeSO4 .............. 0...............0g Avicel) ............ Distribute 7.......10...................... 1............ Adjust pH to 7............................0mL..20......... Cool to 50°–55°C....................................0mg pH 7..........0mL: Na2S·9H2O............... Cap tubes with rubber stoppers...................................25g L-Cysteine·HCl·H2O........25g Preparation of L-Cysteine·HCl Solution: Add L- cysteine·HCl to distilled/deionized water and bring volume 105 to 10.................................0g CaCl2·2H2O ..... Mix thoroughly...................2......... Aseptically and anaerobically distribute into sterile screw-capped bottles................. 10................... 1.0mL of sterile L-cysteine·HCl solution..0mL: D-Cellobiose..........................................0g Na2CO3·10H2O.....5................................. 20.............. Filter suspension and retain the filtrate and the meat particles......................................................... Use: For the cultivation and maintenance of Clostridium cellulolyticum and other bacteria that can degrade cellulose..................4............................ except cel- lobiose solution........... Clostridium Cellulose Medium Composition per liter: Agar ..........0L................. Mix thoroughly...........2g Yeast extract .............0L with distilled/deionized water.0...........................50.... Store under N2 gas..0g MgCl2·6H2O ........2......5g (NH4)2SO4 ..............0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50..............0mL of 1N NaOH...................... Autoclave for 15 min at 15 psi pressure–121°C....................... Distribute into tubes under 100% N2.....0g CaCO3.........20............................0mL L-Cysteine·HCl solution....1g MgSO4·7H2O..............0g K2HPO4 ......0........ Gently heat and bring to boiling.........................1............................ Autoclave for 15 min at 15 psi pressure–121°C................................ Clostridium Cellulolytic Medium Composition per liter: Agar .......... Add ground meat to 25..... Heat to boiling... Autoclave for 15 min at 15 psi pressure–121°C.......................................1......5g KH2PO4 ................... Add L-cysteine·HCl·H2O......................... Sparge under 100% N2 gas for 3 min. Mix thoroughly............ Na2S·9H2O Solution: Composition per 20....................25..... 0..................5........................0mL: L-Cysteine·HCl.....0mL......................................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 30....5............... Mix thoroughly.... Adjust pH to 7.... Aseptically and anaerobically add 50.................0mg pH 7. Gently heat and bring to boiling.............. Skim fat off surface............5 ± 0.5g CaCl2 .......................7....................1................................0g L-Cysteine·HCl·H2O ............0g Filter paper (or 5......................................................................... 0..............9g Yeast extract ............. Mix thoroughly........... and L-cysteine·HCl solution.....................................................1g Resazurin ...5g CaCl2·2H2O . Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C..................... Bring volume of filtrate to 1....................................2....0mL: NaCl ................................................................. Use: For the cultivation and maintenance of Clostridium cellulolyticum and other bacteria that can degrade cellulose. 0....................... Prereduce under 100% N2..2 at 25°C Preparation of Medium: Add components....1...................... Sparge with 100% N2..........1... to distilled/deionized water and bring volume to 1.....................1...............0mL: NaHCO3 ..........1.......... L-Cysteine·HCl Solution: Composition per 10......................0g KH2PO4 ....................0L of distilled/ deionized water..............10.....................................................1..0mL of agar into tubes containing meat particles (use 1 part meat particles to 4–5 parts fluid)..............0mL Cellobiose solution ....... Add remaining components............................................................... 0.........2.300......................0L. Add 1........5g Resazurin .....................0mL of sterile cellobiose solution...........................30.......... 30....................... to distilled/deionized water and bring volume to 900.....0........ 1........ Sparge with 100% N2..............Clostridium cellulovorans Medium Soluble starch................0 ± 0.................15g Resazurin ............. Use: For the cultivation of Clostridium cellobioparum.......0mg Rumen fluid............. Autoclave for 15 min at 15 psi pressure– 121°C. Grind meat finely.............. clarified ..................0mg NaOH (1N solution)................................2 at 25°C Preparation of Medium: Remove fat and connective tissue from lean beef or horse meat................0mL............................0g KH2PO4 ......0g L-Cysteine·HCl·H2O..... 0.......... Clostridium cellobioparum Medium Composition per 1010................1......................................0g Polypeptone™ ................... Pour into sterile Petri dishes or distribute into sterile tubes..0mL NaHCO3 solution ...................... Preparation of Medium: Add components......... Use: For the cultivation and maintenance of Clostridium cellobioparum and Clostridium polysaccharolyticum..0mL pH 7...............0mL of sterile NaHCO3 solution. and 10... NaHCO3 solution.......15g FeSO4·7H2O ...2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................8 ± 0....................... Clostridium cellulovorans Medium Composition per liter: K2HPO4·3H2O ........................25g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20........6........... except L-cysteine·HCl·H2O...20............................0mL................................3g MgCl2·6H2O ... Mix thoroughly........................................ Na2S·9H2O solution.....0......... Autoclave for 15 min at 15 psi pressure–121°C.5g (NH4)2SO4 ........ Continue boiling for 15 min while stirring....................... 1........ 5............. Filter sterilize........... 0.....0g K2HPO4 ....2 at 25°C Cellobiose Solution: Composition per 50..0mL of sterile Na2S·9H2O solution............. Autoclave for 15 min at 15 psi pressure–121°C.........................0g Cellulose....... 0.... Cool to room temperature................................................. Mix thoroughly..5g K2HPO4·3H2O ................................... Mix thoroughly...... except Lcysteine·HCl·H2O.......0 ± 0....0mL................... Mix thoroughly......5g (NH4)2SO4 ................. Sparge with 100% CO2..............2.............................................. Heat to boiling.........0mL pH 6............0g ....................................0mL Na2S·9H2O solution....................... NaHCO3 Solution: Composition per 30.................................................

...............................................0mL Trace Elements Solutions SL-6: Composition per liter: MnCl2·4H2O ...........03g NiCl2·6H2O.................. Cool to 50°C while sparging with 100% CO2........................0 ± 0.................. 100.0mL Rumen fluid........0g K2HPO4 .... Mix thoroughly.0......................5g FeSO4·7H2O ..............................1..............3g CoCl2·6H2O........0L....... to distilled/deionized water and bring volume to 900................................ 30...... Mix thoroughly.... 190... 1.......... 70.... 0......................0mL: Cellulose.......... MN 300 or D-cellobiose...0g KCl.......................02g CuCl2·2H2O... 10....... Autoclave for 15 min at 15 psi pressure–121°C................0mg Resazurin .......... Mix thoroughly.................0g Yeast extract ....... 80...... 0...................0g Preparation of Cellobiose Solution: Add cellulose or cellobiose to distilled/deionized water and bring volume to 50..0mg NiCl2·6H2O ....... Filter sterilize............................................5.0mg MnCl2·4H2O... Autoclave for 15 min at 15 psi pressure–121°C................................0mg HCl (25% solution) ...................20.... 0...............................0mL of sterile Na2CO3 solution... MN 300 or cellobiose solution....................................... Sparge with 100% N2........................10.......0mL.0g Preparation of Fructose Solution: Add fructose to distilled/deionized water and bring volume to 50................................ Mix thoroughly.........0............ Preparation of Medium: Add components..... to distilled/deionized water and bring volume to 920......................0mg Fructose solution .........0mL Trace elements solution SL-4................. Mix thoroughly............................ Sparge under 100% N2 gas for 3 min................................... 100...10........................ Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ...................................15g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 20...... Fructose Solution: Composition per 50......................... 36..................... Preparation of Medium: Add components.....0mL Solution B.1.0mL of sterile fructose solution and 30......................................................0mL: Na2CO3 .................................. 900............0mL.........0mL pH 8......................... Autoclave for 15 min at 15 psi pressure–121°C.................... Use: For the cultivation and maintenance of Clostridium cellulovorans..... Na2CO3 solution................... Aseptically and anaerobically pour into sterile Petri dishes or distribute into sterile screw-capped bottles under 100% CO2...... Clostridium formicoaceticum Agar Composition per liter: Agar ........................0mL................ 0............................5g L-Cysteine·HCl·H2O ................................ Gently heat and bring to boiling........ 1... 0.. Mix thoroughly............................0mg CuCl2·2H2O ................................................. 6............................ 1.0mL pH 7...........0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10....................................... Store under N2 gas........... Add distilled/deionized water and bring volume to 1....... Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly.................................0mL Trace elements solution SL-10 ....2g Trace elements solution SL-6................. and 20........................0mL of sterile Na2S·9H2O solution........ Mix thoroughly....0mL of sterile NaHCO3 solution........................................................................................... 24.............................0mL of HCl solution.......................75g Pyridoxine·HCl......... Aseptically and anaerobically distribute into sterile screw-capped bottles......... Mix thoroughly..... Na2S·9H2O Solution: Composition per 20.....15................ Sparge with 100% N2.......................... Use: For the cultivation and maintenance of Clostridium formicoaceticum.............0mg Cellulose........ Add remaining components..............0........................................ Clostridium halophilum Medium Composition per liter: Solution A ..................................... 2............0mL..................5g H3BO3 ................................................15g Resazurin ...10.................................... Na2CO3 Solution: Composition per 30.0....................0mL of sterile cellobiose solution.....0mL: Fructose ........................................................................... Store under N2 gas.. clarified ......................0..................0..2g ZnSO4·7H2O....2 at 25°C Trace Elements Solution SL-4: Composition per liter: EDTA..........0L....... 0.0mL: Na2S·9H2O..............5g MgSO4·7H2O ............0mL Na2CO3 solution..... Aseptically and anaerobically add 50.. except cel- lobiose solution....0mL..........50.. Mix thoroughly................. and Na2S·9H2O solution.............0mL Na2S·9H2O........................ Filter sterilize.............. Sparge under 100% N2 gas for 3 min... Adjust pH to 8..............0g Preparation of Na2CO3 Solution: Add Na2CO3 to distilled/deionized water and bring volume to 30..............5g CoCl2·6H2O ........0mg ZnCl2 .........0..................................................... 50....0.5..........................0mL NaHCO3 solution.....................0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 30. 30.................. except fructose solution and NaHCO3 solution....20...........0mL..........30..... Sparge with 100% CO2........ Mix thoroughly................5g Yeast extract.................................... 0........................ Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1..........................................................2 at 25°C Cellobiose Solution: Composition per 50............0 ± 0................ Autoclave for 15 min at 15 psi pressure–121°C... Mix thoroughly............................1g Na2MoO4·2H2O........................ Aseptically and anaerobically add 50... Sparge with 100% N2...........0L............................................0mL.................... Mix thoroughly..5g Pancreatic digest of casein ...................... 1............ 5..........................0mL ...106 Clostridium formicoaceticum Agar NH4Cl..................................................................................1............01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.......0mL: NaHCO3 ................... 0...0mg Na2MoO4·2H2O ..............0mg H3BO3 .......0g Sodium thioglycolate...................................... 0............. NaHCO3 Solution: Composition per 30...........

............ 1.......................3g Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10.................................223g 107 Preparation of Solution C: Add components to distilled/deionized water and bring volume to 10.0 ± 0................... 5..2 at 25°C Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl............................................................................................0.....0g Nitrilotriacetic acid...................................................0................................................... Gently heat and bring to boiling.........0mL..........0mL............1g CaCl2·2H2O .............................3......................0mg Nicotinic acid ......................................Clostridium hydroxybenzoicum Medium Solution C ................ Solution D: Composition per 10....0mg Na2SeO3·5H2O ......................5.....1............................................................................. Use: For the cultivation of Clostridium halophilum..............................0mg NiCl2·6H2O ........1g CaCl2·2H2O ..........0mg Vitamin B12............................0mg Na2WO4·2H2O. Mix thoroughly................5g CoCl2·6H2O ..0) ....... Autoclave for 15 min at 15 psi pressure–121°C................ Continue boiling for 3 min. 5.......................................8mL volumes into anaerobe tubes. 50..............0mg Folic acid..........0mL: Na2S·9H2O ......................................................0g Yeast extract ..........0mg HCl (25% solution) ..0mg Lipoic acid . Sparge with 100% N2 for 20 min..5............................................................................10...................... Sparge with 100% N2.....0mL.................0mL pH 7.................0mg Biotin ..................................................................... 190......................5..0L........0mL Trace elements solution SL-10 ............................. 2...............0mg Thiamine·HCl ....... 1.........0mL of sparged solution B..... 5...........................................1...0mL....................................................... 0..............0µg Wolfe’s vitamin solution ...............0g CaC12·2H2O.......... 70..........................................358g KH2PO4 .............................................................0.............. Autoclave for 15 min at 15 psi pressure–121°C.. pH 7......10..................... Solution B: Composition per 80...........................................0mL: NaCl ...............................................1g L-Lysine·HCl .......1. Clostridium hydroxybenzoicum Medium Composition per 1055.0mL: NaHCO3 ........ 5.. 5........ Anaerobically combine 900..0mL: K2HPO4 .........................0mg Riboflavin ..................5.............................10.................................. Preparation of Solution A: Prepare and dispense medium under 80% N2 + 20% CO2......0mg CuCl2·2H2O .........0L......................................... Sparge with 100% N2.........2g NH4Cl....0............................0........0. Mix thoroughly..............0......10.........1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.....................3......................................................... Mix thoroughly.......................................................................0mg Na2MoO4·2H2O ............................................0mg Thiamine·HCl ............02M solution................ Add components to distilled/ deionized water and bring volume to 900............5................................0g MnSO4·2H2O........................................................... Solution C: Composition per 10.................................1mL of sterile solution D to each tube...........................5g NaCl .....................................0mg H3BO3 ............ Mix thoroughly....................15................................75g NH4Cl ....................................0mg Riboflavin.... 36.................................. 10............................... Mix thoroughly......................4g Na2S·9H2O ............................0mg Biotin .. 5...........025g Resazurin ......................0g Yeast extract.....2............................... Adjust pH to 8........8g Glycine ......................10.............5...0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10....... 24............................... 5.....0g L-Alanine..................4g MgCl2·7H2O .........10................................................ 6.........................2..........................0mg p-Aminobenzoic acid ............................................................2..............0mL of HCl solution..... 60...........................0mg Resazurin .......1mL of sterile solution C and 0..................................................................................... 5.......................... Mix thoroughly................. 2......0mg Lipoic acid............................ Anaerobically distribute 9...0mg Calcium DL-pantothenate ..................0.........................0mg ZnCl2 .................................. Mix thoroughly...............86g MgSO4·7H2O ......... 5..................................................3 ± 0............................................................. 1.................... 25.....................10...... 100....0g L-Arginine·HCl..0................................0..............................................................0L.....0mL Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl ...........................................0mg Vitamin B12 ................. Cool to room temperature while sparging with 80% N2 + 20% CO2.........................................2 at 25°C Solution A: Composition per 900...................................... Mix thoroughly..............05mg Sodium/potassium phosphate buffer (0............................. 5..........1g ZnSO4·7H2O.............. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2............0L Wolfe’s vitamin solution .. 2................................ 2......................................... 1....................... Aseptically and anaerobically add 0........................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1............................................... 0..5g CoCl2·6H2O... Mix thoroughly............................ Add remaining components........0.....................................................0......0g Betaine ........................................0mg Calcium DL-pantothenate......................................1........................... 0..... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ...................................................................................................1g FeSO4·7H2O ...............................................0mL Solution D ...0mg Folic acid .......0.0g Preparation of Solution B: Add NaHCO3 to distilled/ deionized water and bring volume to 80...............................................5g L-Cysteine·HCl ........................0mL: NaCl ...0mg MnCl2·4H2O................... Add distilled/deionized water and bring volume to 1... Mix thoroughly.......0mL Wolfe’s mineral solution ...............................1..........1g ................................................... Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O....0..................................................... Autoclave for 15 min at 15 psi pressure–121°C................0mg Nicotinic acid .............0mL pH 8............................................................0mL of cooled solution A with 80..................... 1......0mg p-Aminobenzoic acid ............................

..........1... Use: For the cultivation of Clostridium hydroxybenzoicum...........................................0............ Mix thoroughly.......3mg Preparation of Wolfe’s Mineral Solution: Add ni- trilotriacetic acid to 500............... 0.........0mg Vitamin B12........................ Adjust pH to 7................. to distilled/deionized water and bring volume to 100........................... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O...0mg Riboflavin................................. Gently heat and bring to boiling................5...................................... 4...0L....................0mg Nicotinic acid ..........0mg H3BO3 ..............5..1% solution)............. 15.2 ± 0..................................................... Distribute into tubes or flasks...70.........................0mg Resazurin ...0 ± 0.......................... 20...025g KAl(SO4)2·12H2O................3 ± 0..............24......0mL pH 8.0mL: Potassium acetate ............5....................................................................... Clostridium litorale Medium Composition per liter: Solution A .........................5........................ 2......................................................................5............... Mix thoroughly.0mL of distilled/deionized water................. Add sodium thioglycolate and ethanol...2. Cool rapidly to 50°C..............................................0mg MgSO4 ......0µg Biotin .....................50.0mg MgSO4·7H2O .............................................................0mg Folic acid ...20........ Autoclave for 15 min at 15 psi pressure–121°C....... Sparge with 100% N2......................................... Mix thoroughly.......0mg Cellulose suspension..............0mg FeSO4·7H2O........0g NaCl .........................10............0mL....... ethanol................0mg MnSO4·H2O ...........2........................ 80.......... Distribute into tubes containing a small amount of CaCO3...............5g CoCl2·6H2O..................................0mL pH 7......................5g CaC12·2H2O ........................................... Adjust pH to 6.................................5............................6. 0.............0mL Solution B................................................................................. Add remaining components................... Gently heat and bring to boiling.....2...................0mL pH 7..0mL Solution D ..... Combine components...0g Yeast extract ........ Clostridium kluyveri Agar Composition per 100...................................... 23.................................0mL.5g CaCl2 ................ Use: For the cultivation of Clostridium lentocellum and other Clostridium species..................................0mg Thiamine·HCl .....2 at 25°C Preparation of Medium: Add components.0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10....01g H3BO3 ........................................................................................... .... Clostridium lentocellum Agar Composition per 1201.... Store anaerobically................0mg Na2MoO4·2H2O........................50......... Mix thoroughly.................1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1...0g Sodium thioglycolate .......... 25.........0mg MnCl2·4H2O .................................. 2......................02g CuSO4·5H2O ..................... 900...................6g Yeast extract...........25.......2............................. 96...........0µg Resazurin ................ 0......................................0mg K2HPO4 ..0mL of HCl solution....................................... Mix thoroughly.... 1.................. 1................. Preparation of Medium: Prepare and dispense medium under 100% N2..... Add remaining components... 96................190.... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........................0mg CaCl2·2H2O....................96g L-Cysteine·HCl.... 31..........................................2......................5 with KOH............... 50........................................ Mix thoroughly............................8...................................0mg Biotin ..............5.......................0mg CuCl2·2H2O........1.....0mL: Whatman CF cellulose powder......200.............................0L......................................... 1.....................................................65g NH4SO4 .0µg Wolfe’s vitamin solution ....0mg NH4·Cl................100.................................0mg HCl (25% solution) .............. 0.......................0g MgSO4·7H2O..................0mg Na2MoO4·2H2O ......................... 0...................................................................0mL Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl...............0mL.....................0g K2HPO4 ... Autoclave for 15 min at 15 psi pressure–121°C............... variable Ethanol ...36.....................................................1....2mg Agar ........0...86g L-Alanine . Autoclave for 15 min at 15 psi pressure–121°C..................................... Gently heat and bring to boiling.......0µg CaCO3 ..................0mg Na2SeO3·5H2O .0µg p-Aminobenzoic acid .............1....................................................................................... 10..................................................0g Preparation of Cellulose Suspension: Add components to distilled/deionized water and bring volume to 100. 10. Add distilled/deionized water to 1......................01g Na2SeO3·5H2O ........................................................ 0.... Pour into sterile Petri dishes or leave in tubes.............. Continue boiling for 3 min..................0mL Solution C..........................................................................0mg Calcium DL-pantothenate ..........0mg KH2PO4 ......0mL Trace elements solution SL-10................................................................................................. Anaerobically distribute into tubes or flasks.......................................................... 30. and CaCO3.................................... Add components to distilled/ deionized water and bring volume to 900..................................................................................10. Add distilled/deionized water and bring volume to 1.......0mL: Agar .....................108 Clostridium kluyveri Agar NiCl2·6H2O ........................................................1...............0mg ZnCl2 .10...................................01g Na2MoO4·2H2O ...................... 0..........................0g Betaine..... 1............................................................ Adjust pH to 6......0mg p-Aminobenzoic acid .......2....... Use: For the cultivation and maintenance of Clostridium kluyveri...................0mg NiCl2·6H2O.................................................................... Mix thoroughly........ 0....2 at 25°C Cellulose Suspension: Composition per 100........................0L....................................0mL Resazurin (0........ 10............................ 0.......... except so- dium thioglycolate.. 1... Preparation of Solution A: Prepare and dispense medi- um under 80% N2 + 20% CO2............. Mix thoroughly................................... Continue boiling for 5 min................................................................................................................. Cool to room temperature while sparging with 80% N2 + 20% CO2....................................................................................................... 10.......................0mg Lipoic acid............................. 1........................................................0L..............2 at 25°C Solution A: Composition per 900.................0mL: NaCl ...........2g NH4Cl .....

........................................... Autoclave for 15 min at 15 psi pressure–121°C.. 1.5....................................................5 with KOH........................0L. Mix thoroughly..................... Aseptically and anaerobically add 50... Cool to room temperature while sparging with 80% N2 + 20% CO2....................................10.................0mg D-Ca-pantothenate ......... Sparge with 100% N2....1........................ Filter sterilize........ Mix thoroughly....... Gently heat and bring to boiling..................................0.......................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0g Preparation of Fructose Solution: Add fructose to distilled/deionized water and bring volume to 50...............................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.........5.......................0g Nitrilotriacetic acid........ Filter sterilize............................................02g H3BO3 ......................0mL: Fructose......358g KH2PO4 ..... Store anaerobically......... 10............... Mix thoroughly................................. teine·HCl·H2O to distilled/deionized water and bring volume to 10.........20mg Fructose solution....... Mix thoroughly. Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere......0g Na2S·9H2O .........0g Yeast extract.......... Mix thoroughly..........................0mL Cysteine solution... to distilled/deionized water and bring volume to 900.......... and 10......0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.........1g CaCl2·2H2O............5....................................... Solution C: Composition per 10..0mL of sparged solution B.......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...........................0mL...............................0mg Vitamin B12........ 10..........0mL Vitamin solution....01g Na2SeO3·5H2O .....0........2.......................0mL fructose solution........ 1. Boil for 10 min........50...........0g NaCl ... Add remaining components.....2...........................................0mg Nicotinic acid ..................0mL: K2HPO4 ...... Aseptically and anaerobically add 0........................... cysteine solution.................01g Na2MoO4·4H2O..........0mL pH 5.... Solution D: Composition per 10.............................10..........0mL Na2S·9H2O solution............ 0...................................................0L.02g Na2WO4·2H2O ..8mL volumes into anaerobe tubes.............................9 ± 0.. 0....0....... Autoclave for 15 min at 15 psi pressure–121°C............... 0...................0mg Biotin ......................................0mL..... Anaerobically distribute 9....10.........................0mg Thiamine-HCl·2H2O ...0mL: L-Cysteine Solution: Composition per 10...................10.....................025g KAl(SO4)2·12H2O ........ except NaHCO3 solution................ and trace elements solution SL-10..................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...... Add components.0mL: Trace Elements Solution: Composition per liter: Na2S·9H2O..........0..................... 0................. Add distilled/deionized water to 1..........................0mL..................0mL: NaHCO3 ..................0.. deionized water and bring volume to 80.............................1...........................1mL of sterile solution C and 0............ Autoclave for 15 min at 15 psi pressure–121°C....0..................... 0.... Mix thoroughly...0..0mL vitamin solution... 5........10.3...0mL..... Cool to 25°C............................0mL Trace elements solution ........ Anaerobically combine 900.......................0...... MgSO4·7H2O... Sparge with 100% N2.................. Mix thoroughly...........0................................. 5...........................01g CuSO4·5H2O..................5...................0.....0mL cysteine solution......0mL of cooled solution A with 80.. Mix thoroughly..............................................0mL of distilled/deionized water...............0mg Folic acid ..........0mg Riboflavin....................0g MnSO4·2H2O.0mg Lipoic acid......... Sparge with N2..............................0mL......1g FeSO4·7H2O ..............5................0..3g Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10........................................ 0.......................... Mix thoroughly... Mix thoroughly.......1g NiCl2·6H2O.............0..10................................................... Autoclave for 15 min at 15 psi pressure– 121°C..... Sparge with 80% H2 + 20% CO2................................. Vitamin Solution: Composition per liter: Pyridoxine-HCl ...... 1.... Na2S·9H2O solution............. Sparge with 80% N2 + 20% CO2.............2g KCl.............................223g L-Cysteine·HCl·H2O.....5g NaCl ............... Autoclave for 15 min at 15 psi pressure–121°C.................................... 10..0....... fructose solution.....0mg p-Aminobenzoic acid .............................. Sparge with 100% N2 for 20 min.........................0mL......................0mL: 109 Na2S·9H2O Solution: Composition per 10... Adjust pH to 8................................ 0...................0mL.......... Use: For the cultivation of Clostridium litorale...............................5g CoSO4·7H2O.........0mL: NaHCO3 .....Clostridium ljungdahlii Medium Solution B: Composition per 80.................18g CaCl2·2H2O ...............0mL Na2S·9H2O solution....................... Sparge with 100% N2............8g MgSO4·7H2O ........ Sparge with 100% N2......................3..................... Mix thoroughly...................... 0.....5.............................. 10.......0mL.....1mL of sterile solution D to each tube...................................... 0....................... Clostridium ljungdahlii Medium (DSMZ Medium 879) Composition per liter: NH4Cl..........................0mL NaHCO3 solution ................. Filter sterilize............ Autoclave for 15 min at 15 psi pressure–121°C...................................0mL trace elements solu- .....................18g ZnSO4·7H2O......0mL NaHCO3 solution.............................. Dissolve by adjusting pH to 6........................................................ Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2....................................................................................................1g KH2PO4 ............ vitamin solution....................... Fructose Solution: Composition per 50.....................................................0.................2 at 25°C NaHCO3 Solution: Composition per 10.......

..............0g NaCl ...........0mL: Solution A ........ Sparge with 100% N2........................................0mg Riboflavin ... Autoclave for 15 min at 15 psi pressure–121°C...0mL........2.....0mg ....... Mix thoroughly..................................................0mL pH 7... Aseptically and anaerobically add 100................................................................................1................ 1..........................................................................5............................0mL Trace elements solution SL-10 ..........0mg Thiamine·HCl ...........0mL: K2HPO4 ..........0g MgSO4·7H2O..............................0mg Calcium DL-pantothenate................0mL pH 7.1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........... filtered..................0mg Na2SeO3·5H2O ................ 0. Sparge with 100% N2.......................................... Cellobiose Solution: Composition per 50. Mix thoroughly..............0mL Solution C....... 5..............3 ± 0................................... Aseptically and anaerobically distribute into sterile screw-capped bottles under 80% N2 + 20% CO2..0mL Na2S·9H2O solution......... Use: For the cultivation and maintenance of Clostridium papyrosolvens..... 10...................................... Sparge with 80% N2 + 20% CO2....................................... Preparation of Medium: Add components..... Autoclave for 15 min at 15 psi pressure–121°C.................0mL of sterile phosphate solution and 10......... 6........................................... 200......................0g Yeast extract ......... 190.................................2 at 25°C Phosphate Solution: Composition per 100........6....................0mL: Sodium vanillate............... Add distilled/deionized water and bring volume to 1......0L.........110 Clostridium M1 Medium tion SL-10.............................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10...............0mg H3BO3 ..........................................10..........0. Clostridium pfennigii Medium Composition per 1001.....0g Betaine·H2O .............8g Preparation of Mineral Salt Solution: Add components to distilled/deionized water and bring volume to 1........................ 2.................................................................5g Resazurin .................... Mix thoroughly..................0mL............................................................ Sparge with 100% N2............0g Yeast extract ............................0mL Vitamin solution...............0L.............0g Yeast extract.. Autoclave for 15 min at 15 psi pressure–121°C.... Vitamin Solution: Composition per liter: Pyridoxine·HCl ...............................0g NaHCO3 ..0mL of sterile Na2S·9H2O solution...................................................10......... 50.........0mL Cellobiose solution ......................................2g NH4Cl.........0mg ZnCl2 ........ ly............0mL........1..........................................0mg CuCl2·2H2O ....0mg MnCl2·4H2O......... 25.. to distilled/ deionized water and bring volume to 890................................................................................0g MgSO4·7H2O .......................... Mix thoroughly.............0mL... 5........................................1..........................0mL Mineral salt solution............................... Sparge under 100% N2 gas for 3 min..............2 with 5N NaOH...............0mg Na2MoO4·2H2O ........................................0mg Biotin ........................... 0....... Adjust pH to 7................................5g CaCl2·2H2O.........................................0g L-Alanine............................. 1.................................................2g CaCl2·2H2O ..0µg Phosphate solution ................... Mix thoroughly.......................... 70............... Clostridium papyrosolvens Medium Composition per liter: K2HPO4 ............. 100...........0mg HCl (25% solution) ....... Sparge with 100% N2............................................. 0..............65g NH4Cl ............................................... Add remaining components......................................... Filter sterilize.................2...................................................................... Mix thorough- Use: For the cultivation and maintenance of Clostridium halophilum and Clostridium litorale...6.................... Na2S·9H2O Solution: Composition per 10..... Preparation of Medium: Add components.... 6.. 1........6g L-Cysteine·HCl ...... Aseptically and anaerobically add 50.0–7...0mg p-Aminobenzoic acid ..............5g CoCl2·6H2O ..358g KH2PO4 ............... 36....................................1.................................... 5........... Aseptically and anaerobically distribute into sterile tubes or bottles...............0mg Folic acid..................0mL: Na2S·9H2O........................................ Mix thoroughly..............2 ± 0.......................................... to distilled/deionized water and bring volume to 950.......0mg Seawater.................. 1.... Clostridium M1 Medium Composition per liter: NaCl ..10..........................0mL Solution B.... Mix thoroughly................ 10..... 2..........0mL pH 7.................0L..................... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .......9......................1.. 5.................3g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............................ Mix thoroughly............ 5...................2 at 25°C Mineral Salt Solution: Composition per liter: (NH4)2SO4 .............0mL of HCl solution................................223g Preparation of Phosphate Solution: Add components to distilled/deionized water and bring volume to 100........................................ Mix thoroughly...............0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50.................................................. Final pH is 5..........................0mg NiCl2·6H2O ...........0mL....... Aseptically and anaerobically distribute into sterile screwcapped bottles under 100% N2................ 2...................................................................................... Filter sterilize.................................................... 2....................................15..............1................... 0..... except phosphate solution and Na2S·9H2O solution.......................0mL Solution D .................. 890................................. 24......0...........................0.. Autoclave for 15 min at 15 psi pressure–121°C...........0mg Nicotinic acid . Mix thoroughly............... Mix thoroughly..........2 at 25°C Solution A: Composition per 890...0mg Lipoic acid ................................0mg Vitamin B12 ............. 10.............. Use: For the cultivation of Clostridium ljungdahlii............... 150....... 1................ except cellobiose solution...............0mL: D-Cellobiose .......... 5................. 0..100........................................0mg Resazurin .................. 5.............0g Resazurin ................................ 100.....0mL of sterile cellobiose solution..........

.0g Na2S·9H2O ..0mL Vitamin solution......... Use: For the cultivation and maintenance of Clostridium pfennigii.........................................4.0 ± 0........1 ............0mL pH 7.....0mL................. Use: For the cultivation and maintenance of Clostridium propionicum............................................. Mix thoroughly.......5......................................0mL. Bring pH to 7................................................................. Mix thoroughly..... Na2S·H2O solution.....0g K2HPO4 .................. 0................1........... 6.5mL Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1..........................0mL.0mg HCl (25% solution) ...........2..........................................0mL: Na2S·9H2O Solution: Composition per 10.....0mg Na2S·H2O solution.25mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.... 24.....2g CaCl2·2H2O .......................................Clostridium sticklandii Medium Rumen fluid.....1g FeSO4·7H2O ............................................ .. 2............ Adjust pH to 7..........................0mL of medium. Distribute into tubes or bottles under 100% N2....... pH 7.........6g CaCl2·2H2O....0mL CaSO4.................................................... Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C..............0L........10... 1.........................2........0.... Mix thoroughly.......... 0........... Mix thoroughly.......... 5.....0mL Mineral solution ....0.......... 36......................2 at 25°C Solution B: Composition per 100...............................................................................0mL: Na2S·9H2O ........0mL: Preparation of Medium: Add components................0g NaCl ...........................2mg Nicotinic acid .. Autoclave for 15 min at 15 psi pressure– 121°C.............................. Mix thoroughly........................................... Mix thoroughly.......0g L-Lysine·HCl ..............0mL.............0.................0mg FeSO4·7H2O ........................ 0............ Mineral Solution: Composition per liter: KH2PO4 ........0mL Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 890...............267........................ 0...0g Preparation of Mineral Solution: Add components to distilled/deionized water and bring volume to 1.......... 1........0g NH4Cl ..... 70.0g MgCl2·6H2O..........................................................................1..........................0mL: Preparation of Trace Elements Solution SL-10: NaHCO3 ...................0g Sodium formate ............ Distribute 8..............25mg Pantothenic acid ......... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ..... 2......0............0mg Preparation of Solution D: Add Na2S·9H2O to distilled/deionized water and bring volume to 1........................................................ 8....0mg CuCl2·2H2O ..0mL................................................................5mg p-Aminobenzoic acid ....................0.......62mg Biotin ..................................................... Add remaining components...................... Clostridium propionicum Medium Composition per 1007................3........0L............. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.0g L-Arginine·HCl........0mg Potassium phosphate buffer solution.............. Mix thoroughly....................9mL of sterile solution A........................... and 0..........0g L-Alanine ................ 1M..........0g Peptone ............. Adjust pH to 6......... 10................. Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10...........................................................0mL of sterile solution B....... Mix thoroughly..............01mL of sterile solution D......1mL of sterile Na2S·H2O solution to each 10............ 100.. Yeast extract .................... 190.................0L...........10....... Mix thoroughly.............0................. 2............................................ add (using a syringe) 1...3...........................................0mg ZnCl2 .................................... Filter sterilize..9............5g CoCl2·6H2O ..........75g MgSO4·7H2O..... Sparge with 100% N2 for 20 min........ Autoclave for 15 min at 15 psi pressure–121°C..........................................................................................0g L-Cysteine·HCl ...............................................................................................50....................................... 1....................................0mg H3BO3 ......................... Sparge with 80% N2 + 20% CO2 for 20 min.... Sparge with 80% N2 + 20% CO2 for 20 min. Add distilled/deionized water and bring volume to 1....................... Vitamin Solution: Composition per liter: Pyridoxine·HCl ..........................................................................0L..............0mL 111 Solution D: Composition per 1......................5mL: Yeast extract .... Clostridium sticklandii Medium Composition per liter: Add FeCl2·4H2O to 10.... 8...............1....9mL into anaerobic tubes under 80% N2 + 20% CO2. except L-Cysteine ........ saturated solution...10........................ clarified .... Mix thoroughly...3g MgSO4·7H2O...................0mg NiCl2·6H2O ......24g Preparation of Solution C: Add L-cysteine to distilled/ deionized water and bring volume to 10............0g NH4Cl..... 1........ to distilled/deionized water and bring volume to 1..................................0mL of HCl solution.... Preparation of Medium: To each tube containing 8.................78........................... 5.............................1mL of sterile solution C...2........................... Aseptically add 0.3g Preparation of Solution B: Add NaHCO3 to distilled/ deionized water and bring volume to 100. Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C................................................................... Use: For the cultivation of Clostridium sticklandii.1........... 6........ Mix thoroughly.....................25mg Thiamine·HCl ........... Adjust pH to 7............................018g Resazurin ....0L......................... 10..............................0mg MnCl2·4H2O...................0....0mg Na2MoO4·2H2O ........ 6...................................5................................. Distribute into tubes or flasks......... Autoclave under 80% N2 + 20% CO2 for 15 min at 15 psi pressure–121°C...0mL Trace elements solution SL-10 ...2....... Solution C: Composition per 10..............

.............2 at 25°C Vitamin Solution: Composition per liter: Pyridoxine-HCl ....1... Autoclave for 15 min at 15 psi pressure–121°C...0....0mL: Na2S·9H2O..............................................8g NH4Cl .... 70........................................2.......0mg Trace elements solution. 10.... 20...................0.......0mL.....10........................0mL Na2S·9H2O solution ...2g CaCl2·2H2O............................ Sparge with 80% N2 + 20% CO2 until pH reaches below 6.....5.. Mix thoroughly.......... 2...........................0mg Lipoic acid..................5 with KOH...............0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50..0mg CuCl2·2H2O ........0................................... to distilled/deionized water and bring volume to 985........................50.................................................................................0mL of sterile cellobiose solution...................... 0........... 0...5g CoCl2·6H2O ...3g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.... except vitamin solution...................................................................................... Autoclave for 15 min at 15 psi pressure–121°C......................................................................................................................5g NaCl ......0mg Riboflavin.................................... Mix thoroughly..................... Use: For the cultivation and maintenance of Clostridium termitidis..........................3g MgCl2·6H2O ....................................................................... Mix thoroughly....................................5..................................0mL: NaHCO3 ..5...10..........01g Na2SeO3·5H2O ..0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.. Aseptically and anaerobically add 50....................0.... 1...0g Yeast extract ....................................................... 10..................0mL Vitamin solution ................... and bring volume to 920....0mL of HCl solution.............................................15g Resazurin ............0........0mg D-Ca-pantothenate ....0mg Nicotinic acid ........................................................5.0mg Folic acid ............ Filter sterilize..............0mL.................................0mL of distilled/deionized water...............5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 20..................0mg MnCl2·4H2O.......0..........0mg H3BO3 ........................1g NiCl2·6H2O............18g ZnSO4·7H2O.......................... Mix thoroughly...0mL.......................................................... Cool to 25°C while sparging with 100% ....... Mix thoroughly..................................................0mL....................0mL of sterile Na2S·9H2O solution............... 5....... Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O ...0mg Vitamin B12.0mL..........................................0mL: D-Cellobiose.................................5g Yeast extract..................................................... 0.......0g Na2HPO4·12H2O . and Na2S·9H2O solution.........0mL of sterile NaHCO3 solution................... Mix thoroughly................................0mg Thiamine-HCl·2H2O .................0... Mix thoroughly....................... Sparge with 100% N2................................. 0...........................................................0g Nitrilotriacetic acid.18g CaCl2·2H2O ...................... NaHCO3 solution.................................................3. Cellobiose Solution: Composition per 50......................................................... Add remaining components..................................................................5..............................0...........0mL Na2S·9H2O solution..........5g MgCl2·6H2O............... and 10............ Preparation of Medium: Add components.....10..................................6g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.......................0mL..................................... Autoclave for 15 min at 15 psi pressure–121°C........ Filter sterilize... 5............... and Na2S·9H2O solution.... Clostridium thermoaceticum Medium (TYE-CO) (DSMZ Medium 316) Composition per liter: Trypticase ...................................................................1.....0g KH2PO4 .. Na2S·9H2O Solution: Composition per 10................................. Mix thoroughly......................................3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500...................................................0..........................0mg HCl (25% solution) ..0L........... 4........2................................0L.......................................................... Cool to room temperature.............. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C..................................................1..........0g KCl..................................... except cellobiose solution.........................................0mL NaHCO3 solution .............................0...................0mL pH 7.............112 Clostridium termitidis Medium Clostridium termitidis Medium Composition per liter: NaCl ..............0..........................................2g FeSO4·7H2O ...................... 1........... 1........................................ Add remaining components.....0....025g KAl(SO4)2·12H2O .....0mg Na2MoO4·2H2O ..... NaHCO3 Solution: Composition per 20.. 0............0mg Biotin ................4g NH4Cl.....0.....0mL Cellobiose solution .. 0........................................... Add distilled/deionized water and bring volume to 1..................................................................10. Sparge with 80% H2 + 20% CO2.........................0mg Resazurin ......1g FeSO4·7H2O ....0L..............................1............ Add distilled/deionized water to 1................................................. 190.....0.......................5......................0mg Trace elements solution SL-10 ........................3...........................1.................................... 100.............0 ± 0.............. Dissolve by adjusting pH to 6..0........... Sparge under 100% N2 gas for 3 min...............1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........ Trace Elements Solution: Composition per liter: MgSO4·7H2O....0 ± 0.................................................0mg NiCl2·6H2O ......... Sparge with 100% CO2..................................................2........0mg ZnCl2 ..................... 0...........02g H3BO3 ............2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O .......0g MnSO4·2H2O...........................................3g KH2PO4 .............................................0mL pH 7.......................... 24....................01g CuSO4·5H2O.... Aseptically and anaerobically distribute into sterile screw-capped bottles under 80% N2 + 20% CO2... Mix thoroughly................................................ Mix thoroughly.............20.................1................ Preparation of Medium: Add components. 36...0.................................. Sparge with 100% N2.. Mix thoroughly..5g CoSO4·7H2O.................0mg p-Aminobenzoic acid .. Autoclave for 15 min at 15 psi pressure–121°C.......01g Na2MoO4·4H2O... 6................

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

..............0mg ZnCl2 ............ 1....5mg Resazurin ...... Add distilled/deionized water to 1..........1... 0..5...........................0...0mg Folic acid ...2.......... Mix thoroughly...........0mL: Na2S·9H2O ............1..... Autoclave for 15 min at 15 psi pressure–121°C...0mg Thiamine·HCl .............. 24................................... 12................................................. Autoclave for 15 min at 15 psi pressure–121°C.......... 0................................ Vitamin Solution: Composition per liter: Pyridoxine·HCl............3g NaCl ........ 0....................... Distribute into tubes or flasks under 97% N2 + 3% H2.........................3g MgCl2·6H2O.. Na2S·9H2O Solution: Composition per 10............................. Adjust pH to 7. 0....................0mg Calcium DL-pantothenate ... Use: For the cultivation and maintenance of Thermoanaerobacterium thermosulfurogenes.............................0g Yeast extract .............................................2mL FeSO4 (1................................ NaHCO3 solution.........................................................0mg Vitamin B12.................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1. Adjusting pH to 6....0mL.................. .....................................2..... Sparge with 100% N2.....0....................0mL of HCl solution................. Na2S·9H2O solution.... Mix thoroughly...... and bring volume to 940.....................0mL........0mg NiCl2·6H2O ..................................................0 ± 0.114 Clostridium thermosulfurogenes Medium MnCl2·4H2O..........................................0g Yeast extract............ 10..................................... 1................................................0mL of sterile glucose solution and 10....2 at 25°C Glucose Solution: Composition per 50...... 0.........0...........0% solution) .5...............0mL of sterile casamino acids solution............ yeast extract solution........ Mix thoroughly......1mL Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.. 0.............................. 0......0g K2HPO4 . Aseptically and anaerobically distribute into tubes or bottles....................................................... and trace elements solution SL-10........................................................................................................... except Na2HPO4 solution........02g CuCl2 ...........................................6.....5 with KOH.....................0mL Trace elements solution ..0mg HCl (25% solution) ..... Autoclave for 15 min at 15 psi pressure–121°C....................... Preparation of Medium: Add components.0mL Vitamin solution............ Add remaining components..........0mg Biotin .......................0mg p-Aminobenzoic acid . 10.................5........... Mix thoroughly................................. 70...........10..........0mL of distilled/deionized water.................... 2.................................25% solution)..........................................................0mL of sterile Na2HPO4 solution..............................5..........01g Na2MoO4·2H2O... Boil until color changes from red to colorless....... Pour into sterile Petri dishes or leave in tubes.........0mg Na2MoO4·2H2O .............0 with 1N HCl before use... Autoclave for 15 min at 15 psi pressure–121°C.........0g FeCl3·4H2O.5g CaCl2 .......................1g CaCl2·2H2O ......02g CoCl2·6H2O ......... to distilled/ deionized water and bring volume to 930....017g H3BO3 ..2................................................. Sparge with 100% N2........................................... Sparge with 80% N2 + 100% CO2....5...................5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10................02g Na2SeO3 .......2g MnCl2·4H2O ...0mL: Na2HPO4·12H2O.......10.............. Cap with rubber stoppers.....................0mg Riboflavin............................................................. 100....................................................5........... Mix thoroughly............................................................................................ 0..... 36............ Use: For the cultivation and maintenance of Clostridium species and other bacteria that can utilize cellobiose as a carbon source........... 0............... 10..................................... Sparge with 80% N2 + 100% CO2.............................2g FeSO4·7H2O.........0mg Glucose solution .............. except glucose solution and Na2S·9H2O solution.................................... 0.................. Clostridium thermosulfurogenes Medium Composition per 1015............................ Sparge with 100% N2....1g ZnCl2 ...0mL: D-Glucose....0mL of sterile Na2S·9H2O solution.......50......... 1.......................9g KH2PO4 ............... 10..........0mg H3BO3 ..0mL.............................. and 1.............. Sparge with 100% N2........................................................................................................... Autoclave for 15 min at 15 psi pressure–121°C.. casamino acids solution.0mL of sterile NaHCO3 solution............................................... CM4 Medium Composition per liter: Cellobiose...........................0mL Na2S·9H2O solution............ Add distilled/deionized water and bring volume to 1....0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 50..................5................ 1..........................0g MgCl2 .. Aseptically and anaerobically add 20. 1...................0L... 5.5g NaCl ..............0............. vitamin solution........0mL pH 6............. Mix thoroughly............10..... Mix thoroughly..........0................ Add remaining components.............5g (NH4)2SO4 .........................................0mL of sterile trace elements solution SL-10.....................................0L..... Autoclave for 15 min at 15 psi pressure–121°C......0............................... indicating a reduced state....... Mix thoroughly........................ Trace Elements Solution: Composition per liter: Nitrilotriacetic acid .. 0..3g NH4Cl..0L.......... 10....................... 6.........5.............. Aseptically and anaerobically add 50..............................0mL..........0mL of sterile vitamin solution...... Aseptically and anaerobically distribute into tubes or bottles.... Mix thoroughly..................... Cool................................................................................01g Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.....................................0mg CuCl2·2H2O ....0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10... 0............................. Use: For the cultivation and maintenance of Clostridium thermosuccinogenes.........0132g Resazurin (1.0L..0mL of sterile yeast extract solution............................ Adjust pH to 7................10....... 0...0mg Lipoic acid......................0mL of sterile Na2S·9H2O solution......................75g Sodium thioglycolate...... Gently heat until boiling..............0g KH2PO4 ................. Preparation of Medium: Add components......1.........................................................0mg Nicotinic acid ................................ 5.

.............................................0g MgCl2·6H2O...................................... 20........... Autoclave for 15 min at 15 psi pressure–121°C......................................... Mix thoroughly......... 8....0g Ammonium tartrate ........... Thiamine Solution: Composition per 100mL: Thiamine......................................... 1.....................................0L....................................... Hyphomicrobium species......................................... Hyphomicrobium variabile........................ Autoclave for 15 min at 15 psi pressure–121°C.......0 ± 0........................................................................................ 29.............0L...0mg FeCl2·4H2O......... Gently heat and bring to boiling...... Mix thoroughly.......................... Filter sterilize................................................................. Distribute into tubes or flasks...0mL.... 1......................... Preparation of Medium: Add components...1g CaCl2·6H2O..... 0............................. Cool to 45°–50°C........................ and Methylophilus methylotrophus. 20............ Cook’s Cytophaga Agar Composition per liter: Agar .... Filter sterilize........................... Pour into sterile Petri dishes or leave in tubes.........0mg Preparation of Thiamine Solution: Add thiamine to distilled/deionized water and bring volume to 100.......75g Malt extract ..0mL pH 6......0mL Thiamine solution................... Mix thoroughly.........0 ± 0.........................2g KH2PO4 ........... Lysobacter enzymogenes........75g Yeast extract . Hyphomicrobium zavarzinii...................0g Glucose.......... to distilled/deionized water and bring volume to 990.....0µg MnSO4·5H2O ......0g Chitin.............................0g KH2PO4 .............2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...3 ± 0............0mL of sterile trimethylamine solution.........20.................4g 115 CaCl2·6H2O .0L..0g Pancreatic digest of casein .0g KH2PO4 ...2.............6g Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 500mL.......... 1.............. and Methylophilus methylotrophus........................................................................... except salt solution and thiamine solution.0mg ZnSO4. Gently heat and bring to boiling................... Use: For the cultivation of Aminobacter aminovorans.......... Ba- cillus species.................10..................45.................5..... Filter sterilize....................... 0....................................................2..0 ± 0.............0µg CoCl2·6H2O ..................... noble......... Coprinus Medium Composition per 1026mL: Agar ...... Methylobacterium extorquens.. Pour into sterile Petri dishes or distribute into sterile tubes..... colloidal .........................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.................................................. Lysobacter gummosus..........0mL pH 7............10............. Mix thoroughly....................................................................0mL: Trimethylamine............ to distilled/deionized water and bring volume to 1............20........................................... except tri- methylamine solution..... Colwella psychroerythrus Medium Composition per liter: NaCl ............... Mix thoroughly.60g Salt solution.......... Aseptically add 10.10..................................................................... Preparation of Medium: Add components................ Methylobacterium species.........7g MgSO4·5H2O ...... Colloidal Chitin Agar Composition per liter: Agar ..............5g MgSO4·7H2O ........0g Pancreatic digest of casein .... Aseptically add 25...... Distribute into tubes or flasks.0µg H3BO3 ...................... Ba- cillus species. 4........ Use: For the cultivation of Lysobacter antibioticus.............. 1......... 0....10.............. 1............... Pour into sterile Petri dishes or leave in tubes........ Mix thoroughly..........70................... 1...................0µg FeCl3·6H2O ........................0mL of sterile thiamine solution..........0g Asparagine...................................... Hyphomicrobium species............................................. For the germination of spores of Micromonospora species. Mix thoroughly....2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.... Hyphomicrobium facilis.......................................01g MnCl2 ..............10..... ...................0g Preparation of Trimethylamine Solution: Add trim- ethylamine to distilled/deionized water and bring volume to 10............................................ Gently heat and bring to boiling........................0mL of sterile salt solution and 1...0g K2HPO4 ............ soil....................0g K2HPO4 ......................0........... 0.....................................0g Pancreatic digest of casein . 20...............0g Na2SO4·10H2O ........ Use: For the cultivation of Aminobacter aminovorans.............0mL......... 0.................... 0.......... 25..........................10......................2 at 25°C Salt Solution: Composition per 500mL: Na2HPO4 .....5..2 at 25°C Trimethylamine Solution Composition per 10........ Hyphomicrobium facilis.................... 0.................................. Mix thoroughly................ Cool to 50°C........0.........0L....0........ Pour into sterile Petri dishes or distribute into sterile tubes..........................................33......................... Gently heat and bring to boiling........... 0..5g KH2PO4 ...................................... Mix thoroughly..2..................................................................... or sediment.............................................05g ZnSO4·7H2O ... and other Lysobacter species....0mg pH 7. Autoclave for 15 min at 15 psi pressure– 121°C......................0µg CuSO4·5H2O ...... Methylobacterium species............. Autoclave for 15 min at 15 psi pressure–121°C..Coprinus Medium Colby and Zatman Agar Composition per liter: Agar.......... 5..................2g NaCl ....................... 0........................ Hyphomicrobium aestuarii.............8 ± 0...... Hyphomicrobium aestuarii................................ Hyphomicrobium variabile........... Methylobacterium extorquens............................ Adjust pH to 7.. Distribute into tubes or flasks................ Mix thoroughly.........62g (NH4)2SO4 ......................0mg Trimethylamine solution ....0g pH 7........0µg Na2MoO4·2H2O ....................... 8.....0......... Hyphomicrobium zavarzinii......................... Lysobacter brunescens.............10....0mg pH 7........... Use: For the isolation and cultivation of Micromonospora species from water..5....3g FeSO4·7H2O........................0mL.

........ 10mg NaMoO4·2H2O......................... Add the Na2S..6mL Solution A ..................................................... Gently heat and bring to boiling.....10........ Add the gelatin........................... Gelatin Solution: Composition per 100................. Dendrophoma obscurans.........22.......0g of Trypticase.......................... Add agar to filtrate.............. and Trichophyton violaceum.......... Gently heat and bring to boiling.0L... Na2S Solution: Composition per 100........ Leave overnight in refrigerator...... 0....2....0mL Solution B ........... 30mg CuCl2·2H2O ...1g ZnCl2 ..........................................0L.................0 ± 0.......................0L with distilled/deionized water........................116 Coprothermobacter proteolyticus Medium Use: For the cultivation and maintenance of Coprinus cinereus..0g NaOH solution ..50....10................................... Autoclave for 15 min at 15 psi pressure–121°C.......................................2 at 25°C Preparation of Medium: Add cornmeal polenta to distilled/deionized water and bring volume to 1.....................0mL of medium.................................... Continue boiling for 30 min.............. 0..0mL Mineral salts solution.. Use: For the cultivation and maintenance of many filamentous fungi.... 20mg NiSO4·H2O.... Solution A: Composition per liter: NH4Cl.............. Add 50............................ Mix thoroughly................ Anaerobically distribute 9........0L..........................................................0g tion with 100% CO2 for 30 min........... Cornmeal Agar (CMA) Composition per liter: Agar ............... Boil for 5 min....0.... 200...........7......................... Preparation of Medium: Add cornmeal to distilled/ deionized water and bring volume to 800..................5mL of resazurin solution...... Distribute into tubes or flasks..0mL Na2S solution ........... 10mg H3BO4 .............................. Pour into sterile Petri dishes or leave in tubes........................... 40.....0L......................15g MnCl2·4H2O..... 100...............................0g Na2CO3 ..... Heat to 60°C for 1 hr...... Add agar..5g Soil extract.... add 1..............................0mL Resazurin solution...........0mL: Resazurin .......................0g Preparation of NaOH Solution: Add NaOH to distilled/deionized water and bring volume to 1...........0mL............................................. 40mg Na2WO4·2H2O ...........0mL of mineral salts solution with pipets which have been flushed for a few times with 100% N2.................................................................................................. Mineral Salts Solution: Composition per liter: EDTA·2H2O ..........5mL Solution B: Composition per liter: K2HPO4·3H2O .........................................................20........................... 0.......................................... 2..0mL: 100....2mL of sterile Na2S solution to each 9.......................... Filter through paper and reserve filtrate..............................0mL volumes into anaerobic tubes fitted with butyl rubber stoppers... Resazurin Solution: Composition per 100.........5g Distilled water... 0....0g pH 7..........0mL of solution A................. Mix thoroughly...................................... 20mg H2SeO3 .........2g Preparation of Soil Extract: Add components to 200...........0L of NaOH solu- Gelatin............... Mix thoroughly.......... 4............. Sparge with 100% N2 for 15 min................. and 10.......0g Cornmeal polenta .... One hour prior to inoculation............0....................0g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1...................... Gently heat and bring to boiling. Coprothermobacter proteolyticus Medium Composition per 1168...... Autoclave for 15 min at 15 psi pressure–121°C...5g CoCl2·H2O .................... 0.........................0mL of sterile gelatin solution and 0............................ . Add 10.................................................15...........................................0mL of soil extract........... Autoclave for 10 min at 15 psi pressure–121°C.... Preparation of Medium: Sparge 1...........0.................0g Preparation of Solution B: Add K2HPO4·3H2O to distilled/deionized water and bring volume to 1...........77............................... 100 ml Preparation of Na2S Solution: Gently heat 100.0mL Soil Extract: Composition per 200............... Preparation of Gelatin Solution: Gently heat Use: For the cultivation of Coprothermobacter proteolyti- NaOH Solution: Composition per liter: NaOH .............................. Adjust pH to 4 with HCl.............. Mix thoroughly........ Autoclave for 15 min at 15 psi pressure–121°C................0g CaCl2·2H2O...................................................................0mL of distilled/deionized water to 100°C.. Mix thoroughly............ 50.... 100...........................0mL of solution B...... Sparge with 100% N2 for 10 min.......... 2.0g Agar ....... 2................... Mix thoroughly..0mL: Na2S ............ 2.............. Mix thoroughly..........0g MgCl2·H2O......................0mL....0mL of distilled/deionized water..............................1g FeSO4·7H2O.......................0mL of distilled/deionized water to 80°C.........2g Preparation of Resazurin Solution: Add resazurin to distilled/deionized water and bring volume to 100........................... Mix thoroughly....................... Filter through Whatman #1 filter paper...0mL: African Violet soil ...................................... Autoclave for 60 min at 15 psi pressure–121°C...0g of yeast extract and 2................................................. Mix thoroughly.0L Gelatin solution.......113.... cus............. Pour into sterile Petri dishes or distribute into sterile tubes....................1mL: Yeast extract............... Bring volume to 1..... Mix thoroughly............ Sparge with 100% N2 for 15 min....1g AlCl3·H2O ....1..... Adjust pH to 3 with HCl............................... 10mg Preparation of Mineral Salts Solution: Add components to distilled/deionized water and bring volume to 1.... 3.................0L................. Cornmeal Agar with Soil Extract Composition per liter: Cornmeal ..... Add 2.... Mix thoroughly............ Autoclave for 15 min at 15 psi pressure–121°C..................... Sparge with 100% N2 for 10 min................0g Trypticase............. 0........

.......0g Glucose..........2g Pentachloronitrobenzene (PCNB)..CP Medium for Coprothermobacter proteolyticus 117 Use: For the cultivation and maintenance of Helicodendron tubulosum..... agan...........0L with tap water... 1............................ other Corynebacterium species............. Use: For the cultivation of Streptomyces species.............2........... Boil for 1 hr..... Return volume to 1................... Simmer for 10 minutes........400......0g Vancomycin ..................0... Use: For the cultivation and maintenance of Brevibacteri- um helvolum....................4g Resazurin . Gently heat until boiling.................... Use: For the cultivation of Phytophthora species...0mL pH 7............... Use: For the cultivation of Phytophthora species.... Mix thoroughly... 10......... Gently heat and bring to boiling....65. Filter through cheesecloth.. Corynebacterium Medium with Salt (DSMZ Medium 229) Composition per liter: NaCl ........8... Add agar..... Cornmeal Yeast Extract Seawater Agar (ATCC Medium 2422) Composition per liter: Instant Ocean .4g Pancreatic digest of casein ..................... tryptic digest .........................0g Preparation of Medium: Add cow manure to tap water and bring volume to 1........................ Rhodococcus equi........... 2........0mL Wolfe’s mineral solution ..........0g Yeast extract...............5...................................................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........................ to distilled/deionized water and bring volume to 1.......0g NaCl ............... and yeast extract to 400....................................... Distribute into tubes or flasks........ CP Medium for Coprothermobacter proteolyticus Composition per 1010.. 15....0g Cornmeal.. Pour into sterile Petri dishes or leave in tubes........... Gently heat and bring to boiling.........0g Yeast extract ...........................2 at 25°C .... Aseptically add pimaricin and vancomycin............15....................2 at 25°C Cornmeal Phytophthora Isolation Medium No............... Mix thoroughly.......0L..... 17. Mortierella minutissima..0mL: NaHCO3 ...........................0g NH4Cl ..................... Gently heat and bring to boiling...... Brevibacterium linens.......... Gently heat until boiling...1.......2............................ Sporolactobacillus inulinus....................5. 0.50................................15...................2–7............... Cellulomonas cellasea.0g deionized water and bring volume to 1.. to distilled/deionized water and bring volume to 1..............................0g pH 7.................... Pour into sterile Petri dishes............................ Pour into sterile Petri dishes or distribute into sterile tubes.........10.. Curtobacterium flaccumfaciens....0.......... Microbacterium laevaniformans..............0g pH 7.........5g Agar .....3g Pentachloronitrobenzene (PCNB).................................... 0................0L................. Pour into sterile Petri dishes.0g Casein peptone.... Filter through Whatman filter paper............ Beef extract ........ except pimaricin and vancomycin......... Rhodococcus fascians............ Filter through cheese cloth...................................................5 at 25°C Cornmeal Infusion: Composition per liter: Yellow cornmeal . 2 Composition per liter: Agar ....................................0L............... Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly........ 10....2.....................6–6.... Use: For the cultivation and maintenance of fungi.. Autoclave for 15 min at 15 psi pressure–121°C................................5..... 15...................................0mg pH 5................ Corynebacterium ammoniagenes.... Mix thoroughly................ Corynebacterium callunae................0g CaCl2·2H2O ..0g Vancomycin .025g Pimaricin ......4g K2HPO4·3H2O ... 15.............................. Gently heat and bring to boiling.... Distribute into tubes or flasks.................. and Streptococcus mutans.............. Gently heat and bring to boiling.... 2.......... 0......... Deinococcus radiodurans.. Mortierella hygrophila....... Pour into sterile Petri dishes or leave in tubes.. Autoclave for 15 min at 15 psi pressure– 121°C.....0g MgCl2·6H2O ... solids from infusion ..... 50............................... 5......................... Brochothrix thermosphacta...........1g Pimaricin ..........................0g Agar ............ Corynebacterium glutamicum...............................3 ± 0... 1 Composition per liter: Preparation of Medium: Add components to distilled/ Agar ...... Bring volume to 1........... except pimaricin and vancomycin...................................... Mortierella humilis.......... 15................................................... Autoclave for 15 min at 15 psi pressure–121°C............2 ± 0... Use: For the cultivation and maintenance of Nesterenkonia halobia=Micrococcus halobius... solids from infusion .... Mix thoroughly...........0 at 25°C Preparation of Medium: Add components..... Adjust pH to 7.......0 ± 0...................10............0g Cornmeal............................................. Mix thoroughly................ Aseptically add pimaricin and vancomycin.....0g Yeast extract .................0.............. Microsporum distortum...........................................0g Peptone ..................................0mL pH 7........ 0................................................ Autoclave for 15 min at 15 psi pressure–121°C.......................... 100.. Mycobacterium vaccae...........6–6.... Corynebacterium Agar Composition per liter: Agar .... Pour into sterile Petri dishes or leave in tubes...0L.................0L with distilled/deionized water.............................................................0g Cornmeal infusion.............................01g pH 5....0 liter........................................ Cow Manure Agar Composition per liter: Cow manure ........10.. Cornmeal Phytophthora Isolation Medium No.....................0mL Na2S·9H2O solution .....................0mL cornmeal infusion and bring volume to 1.................1..................0L................0 at 25°C Preparation of Medium: Add components....... Distribute into tubes or flasks...... Preparation of Medium: Add instant ocean. Autoclave for 15 min at 15 psi pressure–121°C........... Gently heat and bring to boiling.......... and Nigrospora sphaerica.... Mix thoroughly..........0L. 0........................5mg Gelatin solution ............0g Preparation of Cornmeal Infusion: Add cornmeal to distilled/deionized water and bring volume to 1.........0g Agar .

.............. 0.........5g CYC Medium Composition per liter: ... Distribute into tubes or flasks.8.... Mix thoroughly.............................................................. Autoclave for 15 min at 15 psi pressure–121°C......... CTT Medium Composition per liter: Agar .1....... Cool to 25°C... Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O ...0mL of distilled/deionized water........................0 with sterile HCl........................................................0g Casamino acids...................................................0g Preparation of Magnesium Sulfate Solution: Add MgSO4·7H2O to 10............0g Yeast extract ...... Mix thoroughly......0L Magnesium sulfate solution ......................... Preparation of Medium: Prepare medium anaerobically under 100% CO2..... vitamin free ............01g Na2SeO3·5H2O ...... 0..............5g NaCl ........................................ 1......... Use: For the cultivation and maintenance of Stigmatella aurantiaca........................ Use: For the cultivation of myxobacteria............ Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly................ 0..........................0g Potassium phosphate buffer (0............... to distilled/deionized water and bring volume to 880. Aseptically add sterile components.. Mix thoroughly....0g K2HPO4 ............6) .0L......... 0.......0mL pH 7.. 0............................ Gently heat and bring to boiling..... Sparge with 100% N2............. 3. 20...............................025g KAl(SO4)2·12H2O..................... Mix thoroughly.....0mL of sterile Wolfe’s mineral solution to each tube.... 0........................................................... Cool to room temperature while sparging with 100% CO2.................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................................................................0g Cyanocobalamin. Pour into sterile Petri dishes or leave in tubes.......................... Sparge with 100% CO2 for 20 min.5mg pH 7................0g MgSO4·7H2O........500...........2 at 25°C Use: For the cultivation of myxobacteria....0mL pH 7.............................. and MgSO4·7H2O to distilled/deionized water and bring volume to 500..... Autoclave for 15 min at 15 psi pressure–121°C........0mL.......1g NiCl2·6H2O ....... Distribute into tubes or flasks.............. Adjust pH to 7......................0g NaNO3 ................ Before use....................0L.............................. Distribute into tubes or flasks........... 10.......3mg Preparation of Wolfe’s Mineral Solution: Add ni- trilotriacetic acid to 500.......... pH 7...... Gently heat and bring to boiling.96g Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................0mL: MgSO4·7H2O.01g Na2MoO4·2H2O ..0mL of sterile Na2S·9H2O solution..............0mL: Gelatin..............15................. Autoclave for 15 min at 15 psi pressure–121°C........1g FeSO4·7H2O....0g Yeast extract ................... Use: For the cultivation of myxobacteria........ 0...................3... Add components...............0g MnSO4·2H2O ............ Add distilled/deionized water to 1................. Agar ...118 CT Agar Gelatin Solution: Composition per 100.... Gently heat until boiling...0.....................................0g MgSO4·7H2O .. Na2S·9H2O Solution: Composition per 10.........6..0g Pancreatic digest of casein ..2 ± 0.................................................................... 0......1g CaCl2·2H2O..........................15.0mL. except gelatin solution............15...... and 10..............................21g Potassium phosphate buffer (1 mM........................... Add remaining components.....................1....10....0mL of distilled/deionized water........ and Wolfe’s mineral solution.................... 0......................................6................. 2........... 10.............0g Yeast extract ......................................30...............0...........02g CuSO4·5H2O .....0g Nitrilotriacetic acid ............................ Sparge with 100% N2.... Mix thoroughly............................10... Use: For the cultivation of Coprothermobacter proteolyti- cus................. Adjust pH to 6. Pour into sterile Petri dishes or distribute into sterile tubes............................................0mL.................................. Preparation of Medium: Add agar............. Mix thoroughly................................ Mix thoroughly.........1................................ neutralize to pH 7...................0mL........2......................... Aseptically and anaerobically add 100..... Autoclave for 15 min at 15 psi pressure–121°C................................2.....0......5 with KOH.....0g Pancreatic digest of casein ........ Gently heat and bring to boiling.........0g CaCl2·2H2O ..... 1..........5g MgSO4 ........ Na2S·9H2O solution.........5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....0..1g ZnSO4·7H2O ..0L...6 ± 0.........5g CoCl2·6H2O .......6)............ Pour into sterile Petri dishes or leave in tubes.......0g Pancreatic digest of casein ........................................................................................... Mix thoroughly.01g H3BO3 ............ Mix thoroughly.. Preparation of Medium: Combine components.............................. 20................0g Preparation of Gelatin Solution: Add gelatin to distilled/deionized water and bring volume to 100...... 0................ CY Agar Composition per liter: Agar ...............0g Pancreatic digest of casein ....5g KCl ......................3...................... 3.............. Gently heat and bring to boiling..................0...............1.......0mL of sterile gelatin solution................. Aseptically combine the two solu- Sucrose ... 1.............. Pour into sterile Petri dishes or leave in tubes................. Adjust pH to 7............... Autoclave agar–pancreatic digest of casein-MgSO4·7H2O solution and potassium phosphate buffer solution separately for 15 min at 15 psi pressure– 121°C................0mL: Na2S·9H2O............. Autoclave for 15 min at 15 psi pressure–121°C.................................. CT Agar Composition per liter: tions............... CT Medium Composition per liter: Agar ............. 0.........2 at 25°C Magnesium Sulfate Solution: Composition per 10.....6 ± 0.. Adjust pH to 6....................................02M solution...........3................................ pancreatic digest of casein. pH 7... 0........................................0g Tris(hydroxymethyl)aminomethane buffer ..................................

........... 0......2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.....0g Agar .........................................01g Antibiotic solution ...........0L.......................0mL.. Cross and Attwell Modification (DSMZ Medium 550) Composition per liter: Sucrose............0mg pH7......10....................................................... Adjust pH to 7........................ 3....................3.......... 490..025g Preparation of Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10............... 0........................ Mix thoroughly.......0g MgSO4·7H2O........2 at 25°C Antibiotic Solution: Composition per 10..........................0.......................................0g ACES (N-2-acetamido-2-aminoethanesulfonic acid)....... Filter sterilize cyclohexanone.....................0g Cyclohexanecarboxylic acid ...........0mL.........0mg ZnSO4·7H2O ..................................2.................................................. Mix thoroughly.....2 ± 0.0g Casamino acids ......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.. Thermobifida fusca...... 30.... Mix thoroughly....................................02g FeSO4·7H2O...................................0mL pH 6................................... 490.................. 5......... Autoclave for 20 min at 15 psi pressure–121°C. 15..............15...................1.......... Mix thoroughly..............1g CaCl2·2H2O ..... except an- tibiotic solution........ Preparation of Medium: Add components................. 0.................... Aseptically distribute into sterile tubes.................................................................... Autoclave for 15 min at 15 psi pressure–121°C....01g pH 7................. 0.....0..................0g KH2PO4 ..................... noble......................... CYC Medium.................... Pour into sterile Petri dishes or leave in tubes..2 ± 0............. Saccharopolyspora thermophila.....25g MgSO4·7H2O................0............0mL Solution B... to distilled/deionized water and bring volume to 999. 119 Use: For the cultivation and maintenance of Corynebacterium cyclohexanicum and Saccharomyces cerevisiae................ Solution B: Composition per 490mL: Agar ...................... Distribute into tubes or flasks..........................0mL: Cycloheximide ....... 1.... to distilled/deionized water and bring volume to 990............0g K2HPO4 ...0................. Distribute into tubes or flasks........................0......0 ± 0....1 at 25°C Solution A: Composition per 490mL: Yeast extract ......3.....01g pH 7........................ Cyclohexanecarboxylic Acid Agar Composition per liter: Agar.................................................................................................. Gently heat and bring to boiling..01g FeCl3·6H2O.................0g (NH4)2SO4 ............5g Cyclohexanecarboxylic acid ............0g K2HPO4 ....................0g . 2....................................... Distribute into tubes or flasks...0g Yeast extract......... Aseptically add sterile antibiotic solution................................ 1.... Use: For the isolation and cultivation of Thermoactinomy- ces species... 15........0..............0mL Solution C..... Distribute into tubes or flasks...........................0mg MnSO4·4H2O .........0...................................CYE-ACES Agar FeSO4·7H2O.10.................................................. 1.....................1g FeSO4·7H2O..... Autoclave for 15 min at 15 psi pressure–121°C........................ 0............ Cyclohexanone Medium Composition per liter: NH4NO3..............5g NH4NO3..................0mL Solution D ....5g KCl............. 10.......................0L.........0g Activated charcoal...... Mix thoroughly.. 10..9 ± 0.... Cool to 45°–50°C................01g FeCl3·6H2O................................. 1...........................................0g MgSO4·7H2O .................................2.............................................................0..........................................................0mg CaCl2·2H2O....................1..........................................................2g Yeast extract.................01g NaMoO4·7H2O ... 2..................1...................... Gently heat and bring to boiling............................0g MgSO4·7H2O ..2g CaCl2·2H2O ...................................................................................... Gently heat and bring to boiling.... Use: For the cultivation and maintenance of Thermomonospora curvata..... Autoclave for 15 min at 15 psi pressure–121°C........... Use: For the cultivation and maintenance of Alcaligenes faecalis and other bacteria that can utilize cyclohexanecarboxylic acid as a carbon source...0....................... Use: For the cultivation and maintenance of Nocardia species and other bacteria that can utilize cyclohexanone as a carbon source... Cyclohexanecarboxylic Acid Medium Composition per liter: K2HPO4 ........1g NaNO3 .......................................................................................................2............................................ Mix thoroughly....................... Cool to 50°C..................................0mL pH 7........................ Thermoactinomyces vulgaris..... 2.0L............................................................................................... Mix thoroughly..0mL Preparation of Medium: Add components.............................. Filter sterilize............... CYE-ACES Agar (DSMZ Medium 585) Composition per liter: Solution A .......5g Tryptophan ...... 0... 0.........................................................................................................0mL.................................. 2.... 10.................................................. and Thermobifida alba................5g Yeast extract ..... 1..................... except cyclohexanone........... Autoclave for 15 min at 15 psi pressure–121°C..............01g ZnSO4·7H2O.... 0..................0mL of cyclohexanone.........05g Novobiocin.......0g K2HPO4 .... 0.......0g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 490............................................................. Pour into sterile Petri dishes or leave in tubes.... Mix thoroughly. Aseptically add 1......10................0mL........................0g KH2PO4 .2 ± 0....................0mg Cyclohexanone ............................ 6......................2 at 25°C Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.................................................. Mix thoroughly.............................................................................

....0mL: L-Cysteine·HCl·H2O. except L- cysteine·HCl·H2O solution and Fe4(P2O7)3 solution......0mL: L-Cysteine·HCl·H2O ....25g Preparation of Fe4(P2O7)3 Solution: Add soluble Fe4(P2O7)3 to distilled/deionized water and bring volume to 10........................ Do not use if brown or yellow... Filter sterilize..............0 N KOH.......... Preparation of Medium: Add components................................. Store in the dark.... Pour in 20......17...25g Preparation of Fe4(P2O7)3 Solution: Add soluble Fe4(P2O7)3 to distilled/deionized water and bring volume to 10. Filter sterilize........................ This is a critical step......................................... Preparation of Medium: Add components................ Do not heat over 60°C to dissolve.. activated. Mix thoroughly..0g Preparation of Glucose Solution: Add D-glucose to distilled/deionized water and bring volume to 100.. Afipia felis.. except oughly.................2 at 25°C Glucose Solution: Composition per 100........... Filter sterilize... Fe4(P2O7)3 Solution: Composition per liter: Fe4(P2O7)3............. 0.5mg Glucose solution ... Afipia broomeae.0mL of sterile L-cysteine·HCl·H2O solution and 10...... Adjust pH to 6..... Mix thoroughly.........25g Preparation of Solution D: Add Fe4(PO4)2 to distilled/ deionized water and bring volume to 10......0g Cyanocobalamin..................1.................. Gently heat and bring to boiling.... Finally.................10. CYE Agar.............0N KOH.............0mL............................. Add 10...............................0mL solution D to 490............ Solution C: Composition per 10mL: Use: For the cultivation and maintenance of Legionella species and Tatlockia micdadei.... 0.0g Charcoal..15.... Cool to 50°C..................0mL.............................. Add 10.......10. L- cysteine·HCl·H2O solution and Fe4(P2O7)3 solution... 10... Mix thoroughly................0g ACES buffer (N-2-acetamido2-aminoethane sulfonic acid) .9 ± 0................ Use: For the cultivation and maintenance of Legionella species and Xylella fastidiosa.. 17.......... Use: For the cultivation of Afipia clevelandensis..0 to 4............. Swirl medium while pouring to keep charcoal in suspension....... Do not heat over 60°C to dissolve.. acid-washed.................0mL solution C and then 10...... CYE Agar (Charcoal Yeast Extract Agar) Composition per liter: Agar ............. Solution D: Composition per 10mL: Fe4(PO4)2.....0mL volumes into sterile Petri dishes.... Autoclave for 15 min at 15 psi pressure–121°C............5mL of 1.....0g CaCl2·2H2O ........... Autoclave for 15 min at 15 psi pressure– 121°C.................0mL..0g L-Cysteine·HCl·H2O solution .... Autoclave for 15 min at 15 psi pressure–121°C.......0mL...0mL......... 0.......................... Mix thoroughly.... The pH of the medium is critical....0mL volumes into sterile Petri dishes...0mL.....4g Preparation of L-Cysteine·HCl·H2O solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10..05 at 50°C L-Cysteine·HCl·H2O Solution: Composition per 10.... Fe4(P2O7)3 Solution: Composition per liter: Fe4(P2O7)3 ... acid-washed . Adjust the pH 6...........5.1..0g Yeast extract .......0mL of sterile Fe4(P2O7)3 solution... Mix thoroughly..................0..9 at 50°C by adding 4. Pour in 20...0mL solution B........0g Pancreatic digest of casein .... L-Cysteine·HCl·H2O ... 10...........0mL.....120 CYE Agar Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 490..............5mL of 1....... activated.0 N KOH... Buffered (Charcoal Yeast Extract Agar...........0mL solution A.................... Mix thoroughly...............2 ± 0...... Filter sterilize.. Heat to 5055°C to dissolve Fe4(PO4)2............... to distilled/deionized water and bring volume to 980... Buffered) Composition per liter: Agar .......... Do not use if chemical loses its green color and becomes brown or yellow.................................. The soluble Fe4(P2O7)3 must be kept dry and in the dark. add 490..............0mL pH 6...... 100.................0g Yeast extract ............0....... Mix thoroughly................0–4................. Prepare solutions freshly.0g L-Cysteine·HCl·H2O solution ...05 at 50°C by adding 4.........10... Mix thor- CYG Agar Composition per liter: Agar ..9 at 50°C by adding 4.....0mL....................... 2........4g Preparation of L-Cysteine·HCl·H2O Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10......................0mL................. Pour into Petri dishes or aseptically distribute into sterile tubes....0. Mix thoroughly....... Autoclave for 15 min at 15 psi pressure–121°C...0mL Fe4(P2O7)3 solution ................. Cool to 50°C.................... The soluble Fe4(P2O7)3 must be kept dry and in the dark... Mix thoroughly........9 ± ... Preparation of Medium: Add 10.......................... 10.. Prepare solutions freshly.....0mL...............2..... Gently heat to boiling. Swirl medium while pouring to keep charcoal in suspension..... Filter sterilize.....0mL Fe4(P2O7)3 solution...........0g Charcoal....4g Preparation of Solution C: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.10. Cool to 50°C...... Filter sterilize...... Do not use if brown or yellow............ Adjust pH to 6.5 ml of sterile 1......0mL pH 7...... Mix thoroughly..9 ± .......... and Xylella fastidiosa. The mixture dissolves readily in a 50°C water bath............................. Mix thoroughly...3......0mL of sterile Fe4(P2O7)3 solution... Legionella pneumophila.... to distilled/deionized water and bring volume to 980.... This is a critical step.......0–4.... Cool to 25°C........ Legionella longbeachae.... The mixture dissolves readily in a 50°C water bath........ ........... Gently heat to boiling.......0g Yeast extract.........0mL pH 6............ Mix thoroughly.0mL of sterile L-cysteine·HCl·H2O solution and 10....05 at 50°C L-Cysteine·HCl·H2O Solution: Composition per 10.............................. Swirl medium in flask during dispensing to petri dishes or tubes to keep charcoal suspended.0mL: D-Glucose .. 0.

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

................................... 100............. and Cellulophaga lytica...................... Modified Composition per liter: Sucrose ..........................................................................0g Agar ...........0g MgSO4 ..................0mL.................................. Autoclave for 15 min at 15 psi pressure–121°C.............. 1......1.......... Na2S·9H2O Solution: Composition per 100.....0mL pH 7............ 24......... NaHCO3 Solution: Composition per 100... 15............. Use: For the cultivation and maintenance of Streptomyces species.......... Cytophaga marinoflava........................... For the cultivation of Actinoplanaceae........................................................ Aseptically add sterile NaHCO3 solution.. Use: For the cultivation of Cytophaga agarovorans.........1............................................. to distilled/deionized water and bring volume to 950................................2g Preparation of Sodium Bicarbonate Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.................. Mix thoroughly........................ Mix thoroughly........................0mL....... Flexithrix dorotheae........... Saprospira grandis.................. Cool to 50°–55°C......................... Mix thoroughly.....0mL of sterile bicarbonate solution and 10............................ Aseptically add 50. Preparation of Medium: Add components............ Distribute into tubes or flasks.... Cool to 50°C..... Mix thoroughly..1. Pour into sterile Petri dishes or distribute into sterile tubes.............. except cellobiose solution........ Gently heat and bring to boiling.01g pH 7.. Mix thoroughly....... 0..................0.............................0mL..... Cytophaga Medium Composition per liter: NaCl ..... Microscilla sp.....................................0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100......... Autoclave for 15 min at 15 psi pressure–121°C............ Autoclave for 15 min at 15 psi pressure–121°C..................... Mix thoroughly...........................0mL: NaHCO3 ........................1. Mix thoroughly...2g Yeast extract........0mL................... Calcium Chloride Solution: Composition per 10.....................0mL............ except NaHCO3 solution.....7g Agar ....... Autoclave for 15 min at 15 psi pressure–121°C.......0g Preparation of Cellobiose Solution: Add cellobiose to distilled/deionized water and bring volume to 50.....................................0mL Glucose Solution: Composition per 100......................25mg NaHCO3 solution..0g MgSO4·7H2O ........0g Preparation of NaHCO3 Solution: Add the NaHCO3 to distilled/deionized water and bring volume to 100...............10....................................................................... 6............................ Cool to 50°C...............0.......122 Cytophaga Marine Medium Cellobiose Solution: Composition per 50............................... 1.5g CaCl2 . Czapek Dox Agar........................ to distilled/deionized water and bring volume to 900................. Marinilabilia salmonicolor...0mL.................. Use: For the cultivation and maintenance of Cytophaga aurantiaca and Cytophaga hutchinsonii...........................................................................15..................................................0mL... 0......0g Agar .....................................04g FeCl3·6H2O........ and sterile Na2S·9H2O solution..0g KH2PO4 .. 1.......... Pour into sterile Petri dishes or distribute into sterile tubes....... Gently heat and bring to boiling... Use: For the cultivation and maintenance of Cellulophaga lytica.. Flammeovirga aprica....................0g NH4Cl .................0.......0mL. Persicobacter diffluens..3g MgCl2·6H2O.0mL Glucose solution ...................10... 1.... except so- dium bicarbonate and calcium chloride solution to distilled/ deionized water and bring volume to 900...5g MgSO4·7H2O.....................0g Tryptone ................2.... and Na2S·9H2O solution... 30................ Autoclave for 15 min at 15 psi pressure–121°C..............1.............. Marinilabilia salmonicolor.................... 10.......... Mix thoroughly.................0............0g K2HPO4 ............................0mL......... 4......... Autoclave for 15 min at 15 psi pressure–121°C..... Flexibacter tractuosus....... Cytophaga latercula....... 1...................0g Yeast extract .................... Flexibacter litoralis.2g Preparation of Calcium Chloride Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10.....0mL of sterile cellobiose solution.0 ± 0....0g ......... Filter sterilize.....0mL: D-Cellobiose........0mL............................. Autoclave for 15 min at 15 psi pressure– 121°C...10.......0g NaNO3 . Mix thoroughly....2 at 25°C Preparation of Medium: Add components......................0mL: NaHCO3 ..3.......................... sterile glucose solution............... Aseptically add 10.......................................0g KCl .. Combine the sterile solutions...................0mL..............30....................... Mix thoroughly..... to distilled/deionized water and bring volume to 889.... Mix thoroughly........... Adjust pH to 7.............................. Mix thoroughly............0mL sterile calcium chloride solution..................0. Preparation of Medium: Add components...................6g CaCl2·2H2O... Mix thoroughly.......10........................ Cytophaga Marine Medium (DSMZ Medium 172) Composition per liter: NaCl ...... Preparation of Medium: Add components.............0mL: Na2S·9H2O .. 6.0mL: CaCl2·2H2O....3 ± 0.......... Mix thoroughly........................... Pour into sterile Petri dishes or distribute into sterile tubes.....7g Sodium bicarbonate solution...0mL: Glucose.................................... add sucrose to distilled/deionized water and bring volume to 100.............30.0g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 100......... glucose solution... Filter sterilize........................................................5................5g FeSO4·7H2O . 15.................0mL Na2S·9H2O solution ............. Cool to 45-50°C............... except su- crose.....................0mL Calcium chloride solution . Czapek Agar (ATCC Medium 312) Composition per liter: Sucrose ....2 at 25°C Sodium Bicarbonate Solution: Composition per 10........................................... Autoclave both solutions separately for 15 min at 15 psi pressure– 121°C................... In a separate flask.0g KCl.................... Pour into sterile Petri dishes or distribute into sterile tubes.

...... Distribute into screw-capped tubes...........................................2g Ethanol .................0g Yeast extract ................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1................................ For amino acid....... Mix thoroughly........................................................ L-lysine..........................0mg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1....... 200....... Mix thoroughly....................................................0g Peptone ...................0mL pH 5............0g Glucose ......................Decarboxylase Medium............3 ± 0.... 5.......... Distribute into screw-capped tubes in 5........ Use: For the cultivation and maintenance of osmophilic fungi................... 0....... 0....0g Bromcresol Purple.......5 ± 0.... Distribute into tubes or flasks................0mL Preparation of Bromcresol Purple Solution: Add Bromcresol Purple to ethanol...............5............. Adjust pH to 5..........5g Bromcresol Purple ......5g FeSO4·7H2O.....2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems..2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems.......... use L-arginine........... except min- Use: For the cultivation and maintenance of a variety of eral oil..0........................ Autoclave for 15 min at 15 psi pressure–121°C...........................0g Glucose.......................35g FeSO4 ....................5.................... Falkow Composition per liter: gal species........................0mL pH 6...........................0g Beef extract ...5g KCl........ Mix thoroughly..................................... Autoclave for 15 min at 15 psi pressure–121°C... 30......... 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.................... deionized water and bring volume to 1........... 12........................0mg Mineral oil......... overlay medium with 1.0L.............01g pH 6..............................................0mg Pyridoxal ................. Distribute into tubes or flasks.....02g Mineral oil ....8 ± 0............... fungal and bacterial species that can use nitrate as sole nitrogen source................................. Ornithine Modified Composition per liter: L-Ornithine ..............0mL volumes.......................... 0... 1.. After inoculation........... Use: For the cultivation and differentiation of bacteria based on their ability to decarboxylate the amino acid................... 5.. 5................................................ Decarboxylase Base............................ Filter sterilize. 0..............................................0 ± 0.......................................................5.................. 0...0L................ 20.......... For amino acid...8 ± 0.................................. to distilled/deionized water and bring volume to 1...............................0g K2HPO4 ...... 2........0mL volumes. 200..0g Bromcresol Purple solution ....0g Agar ................................. 10............. lysine...........................0mL with distilled/deionized water..... 3................ 0...2 at 25°C Bromcresol Purple Solution: Composition per 100... 0.............. Autoclave for 15 min at 15 psi pressure–121°C...................... Mix thoroughly... Mix thoroughly.............................1.............. Preparation of Medium: Add components..................................................... use L-arginine.....0.....0g Meat peptone .......... to distilled/deionized water and bring volume to 1............................ Czapek Dox Broth Composition per liter: Sucrose.. 0............... Mix thoroughly. Mix thoroughly............................ or ornithine turn the medium turbid purple.....0g NaNO3 ...... or L-ornithine....0L.....................................................................0mL of sterile mineral oil per tube....... Use: For the cultivation and differentiation of bacteria based on their ability to decarboxylate ornithine... overlay medium with 1........................................5 with HCl or NaOH........................... 0............. Autoclave medium and mineral oil separately for 15 min at 15 psi pressure–121°C..3.0g K2HPO4 ............ 1.................................. Gently heat until dissolved................. Amino acid ... 5..... except mineral oil.0g Yeast extract ......... Autoclave medium and mineral oil separately for 15 min at 15 psi pressure–121°C....... or L-ornithine... Use: For the cultivation and maintenance of numerous fun- Decarboxylase Medium Base............ Møller Composition per liter: Amino acid... Preparation of Medium: Add components to distilled/ 123 Source: This medium is available as a premixed powder from BD Diagnostic Systems.......................0g Magnesium glycerophosphate ................................................. Bacteria that decarboxylate arginine......5g K2SO4 .........10.......0g KCl..... Bring volume to 100.................. 10..................2 at 25°C Decarboxylase Medium........... Distribute into tubes or flasks.......................... lysine............................ 5........ Bacteria that decarboxylate ornithine turn the medium turbid purple............................. Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1........... ....5g MgSO4·7H2O ......... After inoculation..3............. 3.........0g Peptone....5g KCl............... Pour into sterile Petri dishes or leave in tubes........2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath........ or ornithine turn the medium turbid purple.......0L................... 0........ 50.................. Preparation of Medium: Add components.... L-lysine...01g Cresol Red............................................. Mix thoroughly.......................... Autoclave for 15 min at 15 psi pressure–121°C.............................. Distribute into screw-capped tubes in 5........................................... Gently heat and bring to boiling....................0L..... Bacteria that decarboxylate arginine................................ 0. Ornithine Modified Agar .0g NaNO3 ........................................................0mL of sterile mineral oil per tube...........0L....................... Sucrose.......................01g pH 7...........5g FeSO4·7H2O.................................................0g MgSO4·7H2O ........................0mL pH 6.........0g NaNO3 ...........0mL: Bromcresol Purple........... Pour into sterile Petri dishes or leave in tubes...................................200........

.........................31g H3BO3 ........ 100..................01g Na2SeO3·5H2O .................... 2....................................................17g KI..0... Use: For the cultivation of Deferribacter desulfuricans and Deferribacter thermophilus. 0....................................... 0........0mL Preparation of TK6-3 Solution: Add components to distilled/deionized water and bring volume to 1.......0.........................0mL Vitamin solution........5g NaCl .................10..........0mL of distilled/deionized water..........................................04g H2SO4 (1N solution)............ Preparation of Medium: Add components............... Mix thoroughly..........1.....0mL pH 7.... 0........................................................................................ Cool to room temperature while sparging with 100% N2...................5g NH4Cl .........19g Na2MoO4·2H2O..................025g KAl(SO4)2·12H2O........... 0........ Aseptically distribute into sterile tubes or flasks. except glucose solution and yeast extract solution.43g MgSO4·7H2O.................................0mL............0mL ...................... Mix thoroughly...................2 at 25°C Glucose Solution: Composition per 100.........................................................................5g CoSO4·7H2O ............ 0..................................0....0mL of sterile yeast extract solution...... 5.1g FeSO4·7H2O.............................................2 ± 0.....0mL Yeast extract solution ............................ to distilled/deionized water and bring volume to 890......... Mix thoroughly........................ 0.............. 0........................................ Filter sterilize.......... Preparation of Medium: Add components..........................................0g Na-acetate ......... Dissolve by adjusting pH to 6..1g sulfur per 10mL medium)...................................................0mL: Yeast extract ..........................4............................. Mix thoroughly.......................................................... 1. 0........0g Sulfur......0mL.................................................................................................. 0...........................................5g NH4Cl. Adjust pH to 7..1..................0g KNO3 .3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500..... 0............... 10....................... Trace Elements Solution: Composition per liter: MgSO4·7H2O ......0mL Solution A .....01g Na2MoO4·4H2O ..02g H3BO3 .....................................124 Deferribacter Medium Deferribacter Medium (DSMZ Medium 935) Composition per liter: NaCl ............................................0.... 5...... NaHCO3 solution................................0L.................... Autoclave for 20 min at 110°C....... 10............................. 10....................................................5 with KOH...................................................................... Anaerobically under 100% N2 distribute into tubes of bottles containing the sulfur (0..................................................0mL: Basal solution ...........10. Must be prepared freshly.............................0mL: NaHCO3 ..................84mg Glucose solution ......................... Aseptically add 100......0mL sterile NaHCO3 solution..............0mg Vitamin B12 .....3g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10. 0.................................................33g Yeast extract........................... Aseptically and anaerobically add 10......................................................0 ± 0...... Defined Glucose Medium EMSY-1 Composition per liter: Na2HPO4 .........0L Solution B........................ 0............0mg Biotin ...0mL TK6-3 solution ...........0mg Folic acid............................................................... 1...0.......... Autoclave for 15 min at 15 psi pressure–121°C...........................45g CuSO4·5H2O...................................................................................................................41g CaCl2·2H2O ................. Mix thoroughly...........................0....................0mg p-Aminobenzoic acid .................................................................... Mix thoroughly.......... 10....................2 at 25°C NaHCO3 Solution: Composition per 10.................... 5....... Vitamin Solution: Composition per liter: Pyridoxine-HCl ....1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1....................... 25....................0mL Trace elements solution ........................................................33g KH2PO4 ............................. 0..... Cool to 25°C................ Boil for 3 min. Cool rapidly to 25°C............................. 0.........................................0L................................0g Nitrilotriacetic acid .............................0mL distilled/deionized water.. 0.... 3..............................0mg Riboflavin ........04g NaCl .................33g MgCl2·6H2O...........................0mg Lipoic acid .......... Gently heat and bring to boiling.........15g NaHCO3 solution . 0... Yeast Extract Solution: Composition per 10.33g CaCl2·2H2O........... Defined Medium with Povidone Iodine Composition per 1025....0g MnSO4·2H2O ......... Use: For the cultivation and maintenance of Xanthomonas campestris......... 0................... Sparge with 80% H2 + 20% CO2.76g MnSO4·H2O....1g NiCl2·6H2O .....................................................03g FeCl3·6H2O......................... 0...................0.................. Add remaining components............0mg Nicotinic acid ................. 2..................0mL pH 7.. except vitamin solution..... 10..................................................... 1.0................................................................ 5..... 5.................. 0.............0g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 100.....................0mL............10........... 5.......................1.. powdered......................0mL Solution C...0.................79g KH2PO4 ............ The final pH should be 7. 0..............................0....................... 1. Autoclave for 15 min at 15 psi pressure–121°C.......................... 5..0mL sterile vitamin solution and 10...............0L...................................................... Mix thoroughly.. TK6-3 Solution: Composition per liter: ZnSO4·7H2O..............................0mL of sterile glucose solution and 10.............................. Gently heat and bring to boiling..........................0.......................... Add distilled/deionized water to 1........ Filter sterilize........0.....................7g Citric acid ..........................................................0mL............ Mix thoroughly.............0...................0mg Thiamine-HCl·2H2O ....4g Preparation of Yeast Extract Solution: Add yeast extract to distilled/deionized water and bring volume to 10.... and sulfur to 980...........................................................................................................10...................................18g CaCl2·2H2O.......................................0mL: Glucose...........................01g CuSO4·5H2O ........................................ Mix thoroughly............................. Filter sterilize...................33g KCl.. 2.......................................18g ZnSO4·7H2O ..................................... 1......0mg D-Ca-pantothenate..........................

......... Solution A: Composition per 100.............0mL..... 4.......................................................... 0............ 100.0mL Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O...................... 1..... Pour into sterile Petri dishes or distribute into sterile tubes..........................0mg Biotin ..............................0mg Na2MoO4·2H2O........................0g CaCl2·2H2O............ 1.................................0mL Solution G ........ Solution B: Composition per 100mL: NaHCO3 ......0 L Mix thoroughly.................................0mL: Ferric ammonium citrate.....70.01g Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 250............................................................0mL Solution E ..... 0..6g MgSO4·7H2O .............................. 0...............................11g Preparation of Solution C: Add components to dis- tilled/deionized water and bring volume to 10...........................................................................173g Peptone .........................................................4g Na2MoO4·2H2O ....0.......0 mL in bottles.................. Adjust pH to 6.................... Pressurize closed bottles with H2 + CO2 gas to 0.......1g Resazurin .0mL pH 7.................................... Solution C: Composition per 100..0mL: H3BO3 ..................1mg Trace elements solution SL-10........... Filter sterilize................. Mix thoroughly............................... 0..............1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 100..9g KH2PO4 ............................0mL: D-Glucose............................................015mg Trace elements . 900........................ 4........ 1. Autoclave for 15 min at 15 psi pressure–121°C.................5 bar overpressure....... Solution D: Composition per 10mL: Na2-EDTA .................. 0.................. 10............................................. 1...36............................................... Gently heat and bring to boiling......................... Autoclave for 15 min at 15 psi pressure– 121°C.....................................................................0g (NH4)2SO4 .........2 ± 0........0mg MnCl2·4H2O .....0mg AlCl3........ 1....06g CoCl2·6H2O .. Mix thoroughly............................................................................ 0............................................................................................................... Close bottles.............................................................................1g Preparation of Solution C: Add povidone-iodine to distilled/deionized water and bring volume to 100...1...............................0................ Use: For the cultivation and maintenance of Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia..................................... Filter sterilize.. Autoclave for 15 min at 15 psi pressure–121°C.0L of cooled......... Flush solution with 80% N2 + 20% CO2 gas......... 0.................0mL of sterile solution A..........................0mL of sterile solution C................... Cool to 45°–50°C...........................02g FeSO4·7H2O.............. 10.........0mg FeCl2·4H2O...0mg ZnCl2 . 0...........0mL......................012g Thiamine ......075g EDTA ...5....................................................................................................19g ZnSO4·7H2O ......................... Gently heat and bring to boiling..................................0mL of sterile solution B................... Mix thoroughly.0....................................................................................8g KH2PO4 ........0mL pH 6..0mL......2 at 25°C Solution A: Composition per liter: K2HPO4 ............0........................................0... Preparation of Medium: To 1............. and 10....................................................................................12g CaCl2·2H2O ................... 15.................. Mix thoroughly...........................1.....0...............0mL..0L.....................................5g CoCl2·6H2O............ 0.....0g Na2HPO4 ............. 0............................................................................................... 125 Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.......................... Mix thoroughly.............................0g K2HPO4 .......................... Autoclave for 15 min at 15 psi pressure–121°C.......................7g MnSO4·H2O ........... 10.............. aseptically add 5... Filter sterilize...............460g NaH2PO4·H2O .. Close bottles............0mL Solution D ...... 4.730g NH4HCO3 ....0g Preparation of Solution B: Add glucose to distilled/ deionized water and bring volume to 100......................................0g MgSO4·7H2O ... Autoclave for 15 min at 15 psi pressure– 121°C.........0mL: Povidone-iodine ....................................... Defined Medium for Rhodopseudomonas Composition per liter: Malic acid .................0................ Cool to room temperature under 80% H2 + 20% CO2 gas..........................................................................................653g Na-acetate....... Flush solution with 100% N2 for 20 min....... 10........................2g CaCl2·2H2O.................................... Solution B: Composition per 100............8...................3.... Distribute 9 ml volumes into 50 mL serum bottles under 80% H2 + 20% CO2 gas.........0....... Mix thoroughly.....443g Preparation of Solution B: Add components to distilled/deionized water and bring volume to 100................0mL Solution B..0mL Solution F ... 10.......................................... 0...................190........................... Solution C: Composition per 10mL: MgCl2·6H2O ......0L........Dehalobacter restrictus Medium Basal Solution: Composition per liter: Agar ..........................................................................05g Cu(NO3)2·3H2O ...0mg .................0 mL in bottles... 0.... sterile bas- al solution.....2 at 25°C Trace Elements: Composition per 250................ Dehalobacter restrictus Medium (DSMZ Medium 732) Composition per 1046mL: Solution A .100...........8 ± 0........................5mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 1.................................. 0. Mix thoroughly......... 20..................... Mix thoroughly............................................. 10.... 0.................... 0.......0mL Solution C..... Use: For the cultivation and maintenance of Rhodobacter capsulatus................. Distribute into tubes or flasks.0..........5g Preparation of Basal Solution: Add components to distilled/deionized water and bring volume to 1................................................. 0..............4g NH4Cl........................5................................. Mix thoroughly.....

..................... Preparation of Medium: Add components.....900........ 0.....0mg p-Aminobenzoic acid ................. Autoclave for 15 min at 15 psi pressure–121°C....1mL sterile solution E..........5mg Nalidixic acid ................................ 2................. Aseptically distribute into sterile tubes or flasks.....0mg Lipoic acid .........0mL sterile solution B.............................................0mg HCl (25% solution) ............... Mix thoroughly.......................................................0mg D-Ca-pantothenate.. Autoclave for 15 min at 15 psi pressure–121°C................ 5.............................................................. Tetrachloroethene: Tetrachloroethene .....0mg Vitamin B12 .........................20.................4........ to distilled/deionized water and bring volume to 990........3g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.. Solution E: Composition per liter: Vitamin solution.............................................. Mix thoroughly...................................... Sparge with 100% N2...... Sparge with 100% N2... 5........2 at 25°C Source: This medium is available as a premixed powder and supplement from BD Diagnostic Systems.....................................1........................................... Preparation of Solution D: Add Na2-EDTA.0g Na2HPO4 ..........35g Esculin .........0mL: Na2S·9H2O....... Solution F: Composition per 10.................................. Sparge with 100% N2. 200................0g MgSO4 .......... Mix thoroughly.............................. Mix thoroughly....... Use: For the cultivation of Dehalobacter restrictus and Sulfurospirillum halorespiran.....0g Proteose peptone No................. 100....0mL....................0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10...............1.......................................10.............0L....5............... Add remaining components....................5....0mL of HCl solution...........15mL sterile solution G...................0mg Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.... Close bottles..... Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ..... Adjust pH to 7. 10.0mL sterile tetrachloroethene to the 45.............................12...........0L.... Hexadecane: Hexadecane .........................0mL pH 7..............................................0g LiCl........0mL Preparation of Solution G: Using a syringe.......0mg H3BO3 ......0 mL trace solution SL-10 in a Hungate bottle.......0mL of sterile NaHCO3 solution.....................0g Tryptose.......................... Preparation of Medium: Add 9mL sterile solution A to a 50 mL sterile bottle.............................................. 80... Inoculate the culture into the medium...... Add distilled/deionized water and bring volume to 1..................................0mg Thiamine-HCl·2H2O ...............81...................0mL Tetrachloroethene........... 0........................................5 ± 0...............0..... 300..............06g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10....................................................10........ Mix thoroughly............0g Glucose. and 0.........................................2................0mL Preparation of Tetrachloroethene: Add tetrachloro- ethene to a 10mL serum bottle....0mg Riboflavin ..................... 6..0mg Calcium pantothenate ................................................................0mg Nicotinic acid .................... 10..................... 10.......2 ± 0. Aseptically add 10...........0mL: NaHCO3 ...........0g Acriflavin·HCl..........................01mL sterile solution D............................... Autoclave for 15 min at 15 psi pressure–121°C........................................... Use: For the cultivation of Deleya halophila... Mix thoroughly.......... Solution G: Hexadecane ..............0.........0mg CuCl2·2H2O .............................. 0........................... aseptically inject 5..............5............................................................................0mg Thiamine-HCl·2H2O .............. Filter sterilize.....0mL Vitamin Solution: Composition per liter: Pyridoxine-HCl ..................... and AlCl3 to 10............. Deleya halophila Medium Composition per liter: NaCl ...........0mg Biotin ....0mL Preparation of Hexadecane: Add hexadecane to a 100mL serum bottle........................19...........0mg D(+)-Biotin .0mL......................0mL Seven vitamin solution............10......................0mL vitamin solution and 100.....1mL sterile solution F. Filter sterilize..................... Then add by injection 0.5.......................................... 100..................................0g KCl .............................................. Autoclave for 15 min at 15 psi pressure–121°C..........................3.. 24............................................ Cool to 50°C........................... Sparge with 100% N2............... 5...........1.................. Autoclave for 15 min at 15 psi pressure–121°C.......................................... Mix thoroughly................... 5......... 5.................... 2............0g Yeast extract ....... 5.............0L..................... 10...........................47g NaBr ...... 0..0g CaCl2 ............................. 200............................... Autoclave for 15 min at 15 psi pressure–121°C..0mL sterile hexadecane in the 100mL serum bottle............0mg Vitamin B12 ......................... 2.0mg Nicotinic acid .............. except NaHCO3 solution.............0mg Ferric ammonium citrate supplement..................... Demi-Fraser Broth Composition per liter: NaCl .3...............126 Deleya halophila Medium NiCl2·6H2O ........................9............ Mix thoroughly............... Flush solution with 100% N2 for 20 min.... Sparge with 100% N2.6g Yeast extract .. Sparge with 100% N2................... 45.............0g KH2PO4 .................................................................026g NaHCO3 solution..................................0mL seven vitamin solution.. Autoclave for 15 min at 15 psi pressure–121°C.................. Sparge with 100% N2....6g Beef extract ..........................................1mL sterile solution C.............100............. Preparation of Solution E: Combine 900........0mg p-Aminobenzoic acid .............................................................2 at 25°C NaHCO3 Solution Composition per 10.......................................0g MnCl2 ............. FeCl2·4H2O........ 20...........5...........0mg Folic acid........ Mix thoroughly..............................0mL pH 7.....45....0................ Then add by injection 1....1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1................... .. Mix thoroughly....................10..........0mL.. 0..

.....5g water and bring volume to 10............................. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C....................0mg ZnCl2 .64g Preparation of Na-Acetate Solution: Add Na-acetate p-Aminobenzoic acid .....................24.........0mL of sterile ferric ammonium citrate supplement........ seven vitamin solution..................0mg H3BO3 ................................. Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O. pale colonies with a pink center............2.16....... Aseptically distribute into sterile tubes or flasks................. Preparation of Medium: Prepare and dispense medium under 100% N2 gas atmosphere......................................................... Cool to 45°–50°C................ Cool to room temperature........................ Pour into sterile Petri dishes............................ Aseptically add 10............................ 10....0L... Mix thoroughly.... 1.........3 ± 0..0mL...............Deoxycholate Agar 127 Ferric Ammonium Citrate Supplement: Composition per 10.....................0mL trace elements solution SL-10.............................................................. to distilled/deionized water and bring volume to 10.10..................8-7..7g KCl..0....................................................... flat......... Do not autoclave........033g pH 7........... colorless to tan colonies........... Aseptically and anaerobically distribute into sterile tubes or bottles......................02g Resazurin ......2 at 25°C Na-Acetate Solution: Composition per 10.... rose-red colonies.................................... Filter sterilize.........10....................1.............. Sparge with 80% N2 + 20% CO2..................................................8-7............... 200....0mg Na2MoO4·2H2O..1... 10.............................0g Neutral Red ............... Sparge with 80% N2 + 20% CO2... 0. Mix thoroughly........5g CoCl2·6H2O......0mL seven vitamin solution........................ Mix thoroughly......................... cultivation.... Agar .................15g Na2SO4 ................ Add components................................. Autoclave for 15 min at 15 psi pressure–121°C..................... 0..0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10.... Preparation of Medium: Add components..............0mL: Source: This medium is available as a premixed powder from BD Diagnostic Systems..... Aseptically and anaerobically add 10....0mL Trace elements solution SL-10 ............... Adjust pH to 6. Na-acetate solution..... Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride .......................0mg Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1... Na2S·9H2O solution.................0mL Na2S·9H2O solution.. 0........1...............0mL Na-acetate solution...............100....0mg NiCl2·6H2O............................ Mix thoroughly...............0mL..............0g MgCl2·6H2O................0mg MnCl2·4H2O ...................0mL of HCl solution...................0mL..0mL........................ 0..........190....... Sparge with 80% N2 + 20% CO2..... Use: For the cultivation of Listeria species from environmental samples.............. to distilled/deionized water and bring volume to 949........5g Preparation NaHCO3 ................................ 0.......2 at 25°C NaHCO3 Solution: Composition per 10.0mL Seven vitamin solution.......... and trace elements solution SL-10......70.0g NaNO3 ...................... Mix thoroughly....0mL....5.......................................... 0..........5............................2......... 3.......0mL pH 6..................................... Filter sterilize.... Mix thoroughly..0g Sodium deoxycholate .. Mix thoroughly...................................... Sparge with 100% N2. Add remaining components.............. 0...................... 100......... 2............................................ Filter sterilize...............0g Peptic digest of animal tissue ................................................ to distilled/deionized water and bring volume to 990.........................10.........10.....0mg Thiamine-HCl·2H2O ........0g K2HPO4 ..0mg Calcium pantothenate .........0g Pancreatic digest of casein ................... and 1........................... enumeration............ 100.............................................0mL NaHCO3 solution.... 20..........5g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.................1..........0mL NaHCO3 solution ............................80.... except NaHCO3 solution............ and differentiation of Gram-negative enteric microorganisms from a variety of clinical and nonclinical specimens.........6........ Filter sterilize.. mucoid....................................0g Ferric citrate .. Add distilled/deionized water and bring volume to 1..................................................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 1.5.. except fer- ric ammonium citrate supplement......0mg Nicotinic acid .................... Autoclave for 15 min at 15 psi pressure–121°C...................................... 200. Mix thoroughly........ Mix thoroughly........................... Ferric ammonium citrate..... Mix thoroughly...............................................................0mg HCl (25% solution) ............................... Use: For the selective isolation..0g NaCl . Autoclave for 15 min at 15 psi pressure–121°C...........0g Lactose .......25g CaCl2·2H2O............. 300........................1...................................... 1............................ Proteus and Salmonella species appear as large........36.....0mg CuCl2·2H2O............................ 1..........................................20......0L..1...0mL: D(+)-Biotin ......... Mix thoroughly........ Mix thoroughly... Escherichia coli appears as large............................ Deoxycholate Agar Composition per liter: of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10..................0mL Na2S·9H2O solution........................0mL........ Shigella species appear as colorless to pink colonies........0g Sodium citrate .........................0mg Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O................2.5mg Na-acetate solution .. Use: For the cultivation of Denitrovibrio acetiphilus....... 10.................... ...............0g KH2PO4 .................... Enterobacter and Klebsiella species appear as large..........................0L.. Sparge with 100% N2...........................................5g NH4Cl...... 0................... Gently heat and bring to boiling......0mL: Na-acetate .... Denitrovibrio Medium (DSMZ Medium 881) Composition per 1032mL: NaCl .................................0mg Vitamin B12 ......

..................................................0........................................................... Mix thoroughly............ 5..........................0mg Vitamin B12................................0mg Calcium pantothenate ....................0......................300...........0g Lactose ...........190.........................0mL of HCl solution..........70............................................... Solution C (NaHCO3 Solution): Composition per 100....................... Use: For the selective isolation............................0-7....................0g Na2S2O3·5H2O . 10.............. rose-red colonies...................................... Pour into sterile Petri dishes......0mg HCl (25% solution) .................................................5......... 1....................................... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1...................... 10.........5g CoCl2·6H2O...................3 at 25°C Solution A: Composition per 920mL: NaCl .6................................................................ 15............. 5........4 ± 0......................36.......... Mix thoroughly............. Autoclave for 15 min at 15 psi pressure–121°C............ Cool to 50°C....... Preparation of Medium: Add components to distilled/ deionized water and bring volume to 1.0mL pH 7............... 2.....................................0mg Nicotinic acid ............ 5....... Gently heat and bring to boiling........................................2.................... 15........................................................ 10..... Add remaining components........0mL........................ 1.....0mg CuCl2·2H2O...................................................5mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 920.........................0mg p-Aminobenzoic acid ................ 5............ 50......0L......................... colorless to tan colonies..........................29g NH4Cl ..........0mg Thiamine-HCl·2H2O ...................0g Beef extract ................................................................3 ± 0... Pour into sterile Petri dishes.............. Autoclave for 15 min at 15 psi pressure–121°C...2.............1g KH2PO4 ..... Sparge with 100% N2...0g Yeast extract.................................... Autoclave for 15 min at 15 psi pressure–121°C.......0g Lactose ..................... 1............. Autoclave for 15 min at 15 psi pressure–121°C..................0mg H3BO3 .... 920........................... Sparge with 100% N2...... especially Salmonella and Shigella species..............2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath................................. Use: For the cultivation and maintenance of Dermabacter hominus.............100.......0g Sodium citrate ..................................................0g Glucose .025g pH 7.................0mL Solution C (NaHCO3 solution)..................2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath and BD Diagnostic Systems..0mg Na2MoO4·2H2O........... Do not autoclave.... Add distilled/deionized water and bring volume to 1......................... pale colonies with a pink center...........................................200.................... 10..0L................................4g MgSO4·7H2O...............................24g CaCl2·2H2O ........ 10.. 50...100......100...... enumeration..... Do not autoclave............................................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 ..... Deoxycholate Agar (Desoxycholate Agar) Composition per liter: Agar .................0g Preparation of Solution C (NaHCO3 Solution): Add NaHCO3 to distilled/deionized water and bring volume to 100........ mucoid....... 10....................0g Sodium deoxycholate..................... .........................72g MgCl2·6H2O ... flat...........0mg D(+)-Biotin ..............................0............ Mix thoroughly.............. Distribute into tubes or flasks...........................0mL Solution F ......56g KCl ........................................................................ 5................... 5.24.......... 1..................20............ 13............ 1.0g Neutral Red .....................0g Sodium deoxycholate........ 0..............0mg ZnCl2 ..........................................................0mL Solution E ............. Deoxycholate Citrate Agar Composition per liter: Sodium citrate ......0g Peptone.........................200.0L............0g NaCl ............0mL.......................128 Deoxycholate Agar Pseudomonas species appear as irregular colorless to brown colonies.. 2........................................................ 10..........0mg NiCl2·6H2O.. Cool to 45°–50°C...........10.............. Gently heat and bring to boiling............ Sparge with 80% N2 + 20% CO2...................... Desulfacinum hydrothermale Medium (DSMZ Medium 875) Composition per 1004mL: Solution A ......................0mL Solution B (Trace elements solution SL-10) ...08g Resazurin .................. Mix thoroughly..........0g Peptone................................ Proteus and Salmonella species appear as large............0g K2HPO4 ........... Dry the agar surface before use.0...................................................................................................... Shigella species appear as colorless to pink colonies..............0mL: NaHCO3 ........ 5......0mL Solution D (Seven vitamin solution) ............................................... Mix thoroughly...........................................................0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10..............0mg MnCl2·4H2O ............................ Pseudomonas species appear as irregular colorless to brown colonies............... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O.............................03g pH 7.................. 0............ Mix thoroughly............0g Agar ......5g Ferric citrate ...........2.....................1..........80...............................................3 ± 0....... Use: For the selective isolation and cultivation of enteric pathogens............0................................................................................... and differentiation of Gram-negative enteric microorganisms from a variety of clinical and nonclinical specimens.......................... Mix thoroughly.........0g Neutral Red ......... Enterobacter and Klebsiella species appear as large.0g pH 7...................... cultivation............................................0g Ferric citrate .....0L................. Solution D (Seven Vitamin Solution): Composition per liter: Dermabacter Medium Composition per liter: Pyridoxine hydrochloride ...........................................0g NaCl ......... Escherichia coli appears as large................2 at 25°C deionized water and bring volume to 1.....................................................

..................... 10...........................................5. Sparge with 100% N2..................... 0......... Sparge with 100% CO2.0mL........ Autoclave for 15 min at 15 psi pressure–121°C.......................................0mL sterile solution E........7g NH4Cl.......... Desulfitobacterium dehalogenans Medium Composition per liter: Solution A ..... 0.... 10...... Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 20...... Preparation of Medium: Aseptically and anaerobically Use: For the cultivation of Desulfacinum hydrothermale.................. Preparation of Solution A: Add components...........................................0mL......................5g FeSO4·7H2O ...........0mg Thiamine·HCl ...0mL: Na2HPO4 .......... 2.....................................................3g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10........................................................5 ± 0.......5g Preparation of Sodium Pyruvate Solution: Add sodium pyruvate to distilled/deionized water and bring volume to 10.................. Add remaining components one at a time........ 0............ 2............0mL............. 1.......... Autoclave for 15 min at 15 psi pressure– 121°C.....................0mL Trace elements solution SL-10 with EDTA ........0mg Nicotinic acid ..............0mL Na2S2O3 solution ...3........... Mix thoroughly......0mL...6g NH4Cl ....0-7...................... Sparge with 100% N2. Filter sterilize....................1.... Mix thoroughly.................. 0.........2 at 25°C Na2S·9H2O Solution: Composition per 10. 3........................................................0mL... Sodium Pyruvate Solution: Composition per 10.....0mL Solution A: Composition per 955...0g 3-Chloro-4-hydroxy-phenylacetic acid..................... 1... 0.................955.......0mL....................1g CaCl2·2H2O .............................Desulfitobacterium hafniense Medium bring volume to 1..0mL of sterile solution C............ 0..10.1g CoCl2·6H2O .......................................... 0........0mL pH 7................5 with KOH......0g Preparation of Solution F: Add Na2S·9H2O to dis- 129 to boiling............ 3................ Mix thoroughly.................1g CuSO4·5H2O... 0..5.........5mg Na2S·9H2O solution ...................... 2. 10........5..........................................................0mL Selenite-tungstate solution ........................0........0mL: Sodium pyruvate .................... and 1....................... Mix thoroughly................. Sparge with 100% N2......................0g MnSO4·H2O..... Mix thoroughly........ Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl......................................0mL of sterile solution A with 25....... Autoclave for 15 min at 15 psi pressure–121°C.. 25................................................5g NaCl ...................... to distilled/deionized water and bring volume to 955........ Sparge with 100% N2.....0mL: Na-lactate .....0mL of distilled/deionized water....... Desulfitobacterium hafniense Medium Composition per 1005...2.......................25................................................ 0........................................0mL: Sodium pyruvate .................................. Solution B: Composition per 25.............. Autoclave for 15 min at 15 psi pressure–121°C........................1g CaCl2 .......................0mL................. Mix thoroughly.... 1.....................................................................................................................................0g K2HPO4·3H2O .........0...................................................................... Mix thoroughly..............5g Preparation of Solution E: Add Na-lactate to distilled/ deionized water and bring volume to 10....20..............................1......... Gently heat and bring combine 955.........5g KH2PO4 .0L.................................................. Aseptically and anaerobically distribute into sterile tubes or bottles...............01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500...... Sparge with 100% N2....................................44g MgCl2·6H2O ........ Aseptically and anaerobically distribute into sterile tubes or flasks....................0g Nitrilotriacetic acid .............0mL of sterile solution B and 20......................... 0.......01g H3BO3 .......01g Na2MoO4·2H2O..4g MgCl2·6H2O ........05g Resazurin ...............................0mL Solution C ... Solution F: Composition per 100.............0mL: NaHCO3 ...................... Add 50..........................1............0mL sterile solution C.................................................01g A1K(SO4)2·12H2O .......................10..... Add distilled/deionized water to 1.....0.0mL: NaHCO3 .............................2.................................. 0... Solution E: Composition per 10..........0mL Wolfe’s Mineral Solution: Composition per liter: MgSO4·7H2O..0mL Solution B ............. Filter sterilize...............................................0mL: Na2S·9H2O .................0mg Wolfe’s mineral solution .................... Adjust pH to 7............... to 920......................... except Lcysteine·HCl·H2O..............0mL sterile solution D......0mL: Na2S·9H2O....... 13.0mg Riboflavin...... 0....0mL Wolfe’s vitamin solution ......................... The pH of the completed medium should be 7................... Solution C: Composition per 20..............................3.... Mix thoroughly.............2g CaCl2 .0mL sterile solution B...0.5g L-Cysteine·HCl·H2O ................0g tilled/deionized water and bring volume to 100.................. 0......... 10......................................................................0... Sparge with 100% N2.... Adjust pH to 6......0mL sterile solution A................ 1...........0mg Lipoic acid.....................0mL Sodium pyruvate solution......................................................................0mL sterile solution F........................................ Autoclave for 15 min at 15 psi pressure–121°C.................5.... 1.............2g Yeast extract..........1g NaCl ..................................1g ZnSO4·7H2O ........0L...............5................. Mix thoroughly.............. 5................................0... Mix thoroughly............0mg ...................... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere.......................................... 10.........0mg p-Aminobenzoic acid ...... Cool to room temperature while sparging with 90% N2 + 10% CO2.............2g Preparation of Solution B: Add sodium pyruvate to distilled/deionized water and bring volume to 25......... Use: For the cultivation of Desulfitobacterium dehalogen- ans.................2........... Add L-cysteine·HCl·H2O.............................. Filter sterilize.........................0g Yeast extract .................

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

.200.............................................................25.........................0mL. Sparge with 100% N2................................ Autoclave for 15 min at 15 psi pressure–121°C.............. 0..................0mg NiCl2·6H2O......70. Aseptically and anaerobically add 20.........0mg Folic acid........................................ 5..............................0mL: NaHCO3 .. Filter sterilize........................................................ 4........................ Cool to room temperature...0mg Vitamin B12 ........................................................0mL of sterile cysteine solution 10.......0mg D(+)-Biotin ......... Filter sterilize..100.....3........5g to distilled/deionized water and bring volume to 10....................190...................200................................ KOH Solution: Composition per 100.1.................20.0mL: KOH ..........................24...... Mix thoroughly.......... Mix thoroughly...... 131 Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100..............20.0L................... 10............................... Desulfitobacterium PCE Medium (DSMZ Medium 717) Composition per liter: (NH4)H2PO4 ........ Mix thoroughly................................................ Mix thoroughly..........1g Ca(NO3)2·4H2O.................................................................0mg Na2SeO3·5H2O ....................0L................................... Filter sterilize.0g Preparation of KOH Solution: Add KOH to distilled/ deionized water and bring volume to 100..........0mg Nicotinic acid ..............................................0mL of HCl solution........................................................80.................5....................................... Na-pyruvate solution..................................... 0......................................... Na-pyruvate solution.......................0L................36.................................20....... 10.......0mg p-Aminobenzoic acid .. 0........1..... 10......0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.......................................0mg H3BO3 ...............................................5g CoCl2·6H2O...........0mL sterile Na-thiosulfate solution.................0mg Folic acid ....................................... Filter sterilize............ 0.................5.................................5g Na2WO4·2H2O ......50............................................................................................. Filter sterilize.............................................10..5..................................... Cool to room temperature................. Sparge with 100% N2.........................6................ 2....... 0...................................... Mix thoroughly........... 5........... and Na2S·9H2O solution..0mg CuCl2·2H2O.......0mL Selenite-tungstate solution .................. Mix thoroughly............. Sparge with 80% N2 + 20% CO2.........................0mL KOH solution ......................................0mL Vitamin solution.............................0mg Na2S·9H2O Solution: Composition per 10mL: Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......................0mL sterile vitamin solution.......100..........0g Na-lactate ............ 0.... to distilled/deionized water and bring volume to 940...0mg Thiamine-HCl·2H2O .......Desulfitobacterium PCE Medium p-Aminobenzoic acid .10..............05g Resazurin ...............................................................................................................2.. 5.......0mg Lipoic acid........ Sparge with 80% H2 + 20% CO2.......0mg Na2MoO4·2H2O.1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0mg Calcium pantothenate ..............0mg MnCl2·4H2O ........................................ Add components.....................................................0...0mg Lipoic acid .......0L.............. Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ......1... Adjust pH to 7..............100................ Vitamin Solution: Composition per liter: Pyridoxine-HCl . Cool to 25°C..... Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C............................... Preparation of Na2S·9H2O Solution: Add Na2S·9H2O Na-lactate Solution: Composition per 100..................0mg Vitamin B12.....0mg Riboflavin............5........0mg HCl (25% solution) ............1g Yeast extract....0mL pH 7......................................88g MgSO4·7H2O ...........20......0mL........0L................. and 10............0mL of sterile Na2S·9H2O solution.....................0mg Biotin ............... ........................... Sparge with 100% N2......................................................... Filter sterilize............ Sparge with 100% N2............2....0mg ZnCl2 ..0mg Nicotinic acid .....0mL......... 2.............1mg NaHCO3 solution .................... Preparation of Medium: Prepare and dispense medium under an oxygen-free 100% N2............................................................................0mg D-Ca-pantothenate ..........0mL Na2S·9H2O solution.... Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O...... except vitamin solution........................0mL..................1................0mL Na-lactate ...0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10. Use: For the cultivation of Desulfitobacterium dehalogen- ans.....0mg Vitamin B12...............300............ Mix thoroughly... Aseptically and anaerobically distribute into sterile tubes or flasks........ Mix thoroughly...0mL.. 2......................... Mix thoroughly............................3mL Seven vitamin solution........5..............0g Preparation of Na-lactate Solution: Add Na-lactate to distilled/deionized water and bring volume to 100...........5. Mix thoroughly......... Add remaining components................ NaHCO3 Solution: Composition per 100.....0mL Trace elements solution ....................0mg Biotin .......... Mix thoroughly.................................... Sparge with 80% N2 + 20% CO2..1 ± 0...... 3.... Na-thiosulfate solution...0mg Thiamine-HCl·2H2O ..........0mg p-Aminobenzoic acid ...2.......... Autoclave for 15 min at 15 psi pressure–121°C....0mL Na-fumarate ..................................10.. 0....... Sparge with 80% H2 + 20% CO2................................0mL sterile........................................................... Add distilled/deionized water and bring volume to 1..... Autoclave for 15 min at 15 psi pressure–121°C........................1mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 1......5...0mL: Na2S·9H2O.................... Sparge with 100% N2.... Mix thoroughly..... cysteine solution....2 at 25°C Selenite-Tungstate Solution Composition per liter: NaOH ..............................................

.............64g Na2HPO4·2H2O...............................................2...............1g Resazurin .................. 100...................... 1......... to distilled/deionized water and bring volume to 977...... Sparge with 80% N2 + 20% CO2.... 0............................................ Mix thoroughly.............................0-7........ Aseptically and anaerobically add 10................................................ Adjust pH to 7... and trace elements solution SL-10..................11g MgCl2·6H2O.............41g NH4Cl.....0mg Calcium pantothenate .....................0L...................................0mL CaCl2 solution......... 200........2mL pH 7....................................0mL: NaCl .......10.......................................................................3......... 20........... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere..... except NaHCO3 solution...... and 1.............................1g Na2SO4 ..........3...........0g Preparation of Na-fumarate Solution: Add Na-fumarate to distilled/deionized water and bring volume to 100..... Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 gas atmosphere.....................0mg HCl (25% solution) .....0mL. 10.. 0................................................ Sparge with 80% N2 + 20% CO2...............................0mL.....132 Desulfobacca Medium Na-fumarate Solution: Composition per 100...................................0mL selenite-tungstate solution............. Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C.....0g Na-acetate ...................................0L.......................... 0....0mL vitamin solution.................... Sparge with 100% N2....................0mL Solution C..... to distilled/deionized water and bring volume to 873........................................ Use: For the cultivation of Desulfobacca acetoxidans.... Mix thoroughly..0mL seven vitamin solution..........0mL: Na-fumarate ................... 36............0mg Vitamin B12 ...0-7..........2 at 25°C Seven Vitamin Solution: Composition per liter: Pyridoxine hydrochloride ..................................................... 20... Na2S·9H2O Solution: Composition per 10mL: Na2S·9H2O ......0mL.............................0mg Preparation of Seven Vitamin Solution: Add components to distilled/deionized water and bring volume to 1........................... 80........0mL Na-fumarate solution. 100............ Autoclave for 15 min at 15 psi pressure–121°C.............. Adjust pH to 7.........................0mg CuCl2·2H2O....... Na-lactate solution....................0.................................. Autoclave for 15 min at 15 psi pressure–121°C...... 10..... Aseptically and anaerobically distribute into sterile tubes or bottles.....2.................... 10.1–7......0.. 1....0mg NiCl2·6H2O.................0mg Na2SeO3·5H2O ...................0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1..... Mix thoroughly..... 1...........0mL Seven vitamin solution....... Mix thoroughly... 3........................0mL: CaCl2·2H2O ................0....2mL seven vitamin solution............ Mix thoroughly...............................0mL Nalactate solution.. 10................................................ Desulfobacter Medium Composition per 1001........................4...................... selenite-tungstate solution......0mL trace elements solution SL-10......0mL Solution E (Vitamin solution) .......................................... 0.0mg MnCl2·4H2O...................................... CaCl2 Solution: Composition per 10....... 0............ 190..................................3mL Na2S·9H2O solution........0mg p-Aminobenzoic acid .........24............................................................. Add components.................................. Mix thoroughly....0mL Na2S·9H2O solution... 70.... seven vitamin solution... Add components......................10...........0mL selenite-tungstate solution............................ CaCl2 solution................. Use: For the cultivation of Desulfitobacterium sp.................................... and 1................ 20..5g CoCl2·6H2O ...........................7mL..................... Sparge with 80% N2 + 20% CO2............... KOH solution......... Aseptically and anaerobically add 50. and trace elements solution SL-10.0mL NaHCO3 solution.... Add distilled/deionized water and bring volume to 1....0mL pH 7...... 1........11g Preparation of CaCl2 Solution: Add CaCl2·2H2O to distilled/deionized water and bring volume to 10......0mL CaCl2 solution .............. 0.........4 at 25°C Solution A: Composition per 870...... Na-fumarate solution................53g KH2PO4 ..................... Mix thoroughly..........5g Na2WO4·2H2O. Trace Elements Solution SL-10: Composition per liter: FeCl2·4H2O ........... Mix thoroughly. Na2S·9H2O solution........... Filter sterilize............. 300.... Mix thoroughly....... Autoclave for 15 min at 15 psi pressure–121°C......... Mix thoroughly......... Filter sterilize.............. 1..0mL trace elements solution SL-10. 10...5mg Na2S·9H2O solution.... 1......................... 10..........0mL of HCl solution...................... except Na2S·9H2O solution..3g NaCl .............0mL Solution B (Trace elements solution SL-10) .......... and Desulfitobacterium hafniense....... 200................ Mix thoroughly...0..0mL: Solution A .............0mL Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O to 10............. Sparge with 100% N2.................. 3....... Selenite-Tungstate Solution Composition per liter: NaOH . 0...........................21........................ 100................................... Filter sterilize......................0mg Nicotinic acid ......................... 16.......... Cool to room temperature.0L............................... 870.... Cool to room temperature....0mg Na2MoO4·2H2O ..0g Na2SO4 ...0mg ZnCl2 ....3.......1.. Autoclave for 15 min at 15 psi pressure–121°C.......................0mL Selenite-tungstate solution .... selenite-tungstate solution......6............. vitamin solution.3g CaCl2·2H2O...............0g .............. Sparge with 100% N2... 20....................2.......................................................... Sparge with 80% N2 + 20% CO2.........0mg Thiamine-HCl·2H2O ..1 ± 0...0mL KOH solution....... 100.............0g MgCl2·6H2O ................0mg D(+)-Biotin ....... Aseptically and anaerobically distribute into sterile tubes or bottles................8mL.................. 4............................................1.. seven vitamin solution........ Add remaining components..............0mg H3BO3 ............... 0...0mL Trace elements solution SL-10 . Desulfobacca Medium (DSMZ Medium 728) Composition per liter: NaHCO3 ........5g Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.............0mL Solution F .........0mL Solution D ..............

.......10....................................0mL: Sodium acetate·3H20......0mL......2g CaCl2·2H2O ..............................................0mL: Na2S·9H2O.......................... 2.... 10...5g NH4Cl..............0mg Vitamin B12 .............. Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O .........................0.............................0g MgCl2·6H2O ......................0g distilled/deionized water and bring volume to 10........3............... Mix thoroughly.......................................... 5................................................ 2...............Desulfobacter postgatei Medium KCl............... 2.............0mL Solution E (Vitamin solution) .....................15g Resazurin .. Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100.......... Solution D: Composition per 10.....0.......0mg CuCl2·2H2O .............. 0......0mg Nicotinic acid ....................... 2...... 1..............................................5g NH4Cl ....70.......... Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2...................... 5............................... 0..................0.............. 0...........0mg ZnCl2 .... 5............. 5..............................................................0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870.......0mg p-Aminobenzoic acid ......0mg Na2MoO4·2H2O .... solution E.2......................... Solution C: Composition per 100...................................0mL...................................................... and solution F..............0mL: NaHCO3 .2g CaCl2·2H2O.....0........ Use: For the cultivation and maintenance of Desulfobacter species and Malonomonas rubra................ Gently heat and bring to boiling. Seal the flask under 80% N2 + 20% CO2.... in that order.............. solution C................ 870...........1..........0mL of HCl solution..0mL Solution C................................................. 10.............36.........0mg HCl (25% solution) ..................................................... 5..................................0.....0mg Riboflavin .............. Mix thoroughly.................0mL pH 7.........0L.............................................. 1............................. Mix .......... Continue boiling for 3-4 min............................................100.............0mL Mix thoroughly..........0mL..............0mL...........................0mg MnCl2·4H2O ...0mg NiCl2·6H2O..........0mL.......0mg Biotin ............................................................................. Autoclave for 15 min at 15 psi pressure–121°C....................................0mg MnCl2·4H2O..........................0mL: Solution D: Composition per 10......0mL of HCl solution.... Mix thoroughly.............................. Gas under 100% N2...........................................0mg HCl (25% solution) ......................................2. Filter sterilize...................... Sparge with 100% N2.............................. Gas under 100% N2........0mg H3BO3 ....... Flush with 80% N2 + 20% CO2 to remove dissolved oxygen................ Mix thoroughly.........................5g Preparation of Solution D: Add sodium acetate to distilled/deionized water and bring volume to 10............................. 5............. 0.......0L........................ 190.....24..... Solution C: Composition per 100.0mg ZnCl2 ..........0mL: Solution A . 6..................0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10..............................................................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..15g Resazurin ...............1................3g KH2PO4 .....................0mL Solution B (Trace elements solution SL-10) ................. 36............. Mix thoroughly......................................................................5g CoCl2·6H2O............ 100....................... Add remaining components................ 1..............................1..... Mix thoroughly...................... 100.......................1mg Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10...................... Autoclave for 15 min at 15 psi pressure–121°C......... 10...7....... Mix thoroughly............................ Autoclave for 15 min at 15 psi pressure–121°C................0mg CuCl2·2H2O............................. Solution F: Composition per 10... Add distilled/deionized water and bring volume to 1.........0mg Thiamine·HCl .................................0g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100........ 0........................... 10................................................................................................................................. Gas under 100% N2... Autoclave for 15 min at 15 psi pressure–121°C.........190...0mL: NaCl .0mg Na2MoO4·2H2O............. Mix thoroughly........................ Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1............................0mg Lipoic acid ............5.................................... Preparation of Medium: Aseptically and anaerobically combine solution A with solution B................................................ Add remaining components.........0mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870.............................. Autoclave for 15 min at 15 psi pressure–121°C.......................0L............................................................. Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O........0mg Folic acid..........5g CoCl2·6H2O ....... Autoclave for 15 min at 15 psi pressure–121°C................................. Allow to cool to room temperature while gassing under 80% N2 + 20% CO2......................3g KCl ................4 at 25°C Solution A: Composition per 870............ 10.......0mg H3BO3 ................................. Gas under 100% N2.............. Add distilled/deionized water and bring volume to 1........... 24.................3g KH2PO4 ........... solution D..........0mL Solution D ...........0g Na2SO4 ....................0mL Solution F ..................................................0mL: NaHCO3 ..0mg NiCl2·6H2O ....... Gas under 80% N2 + 20% CO2......0mg Calcium DL-pantothenate..................... Filter sterilize........ Mix Mix thoroughly.....0mL Mix thoroughly.......... Solution E (Vitamin Solution): Composition per liter: 133 thoroughly.........0mL Pyridoxine·HCl ................... 70..... Continue gassing until pH reaches below 6........................................................ Desulfobacter postgatei Medium (DSMZ Medium 193) Composition per 1001...............4g Na-acetate·3H2O..6...................1-7........................................................................................................... 5........ 0.

.... Sparge with 100% N2...................0mg Thiamine-HCl·2H2O ............... Use: For the cultivation of Desulfobacter postgatei....................................................... Addition of 10–20mg sodium dithionite per liter from a 5% (w/v) solution......1-7............................70..................... Cool to room temperature. Add remaining components...........................0mL solution E................0mL solution B.............0mg Biotin ..5g Preparation of Solution D: Add Na-acetate·3H2O to distilled/deionized water and bring volume to 10......... Autoclave for 15 min at 15 psi pressure–121°C....... Autoclave for 15 min at 15 psi pressure–121°C.................... Mix thoroughly...0mg Riboflavin......... Cool to room temperature.................................1g Na2SO4 ........ Filter sterilize..................0mg D-Ca-pantothenate..............................10mg Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1....................... Desulfobacter sp........ freshly prepared under N2 and filter-sterilized......... Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels.........................................................................0mL solution C................0....................................... 0......................36..... Cool to room temperature....... Add distilled/deionized water and bring volume to 1........0L..5...........0L................ 0.................. 10....................0mg p-Aminobenzoic acid .....................15g Resazurin ..... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O ........ 5.. Autoclave for 15 min at 15 psi pressure–121°C....0mL. Medium thoroughly.................0mg Nicotinic acid ................... CoCl2·6H2O....5..0mg Lipoic acid............................................. 10....................... 0.......................... Preparation of Solution F: Add Na2S·9H2O to dis- Solution D: Composition per 10....0mg NiCl2·6H2O....................0mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870.................0mg Lipoic acid ..0mg Vitamin B12.....0mg MnCl2·4H2O ....................0mg Folic acid ............ Autoclave for 15 min at 15 psi pressure–121°C....0mL Solution E (Vitamin solution) .....0mL Solution B (Trace elements solution SL-10)..............................0mL Mix thoroughly..............0mL: NaCl ......0mL solution F...... Distribute anaerobically under 80% N2 + 20% CO2 into appropriate vessels..................0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10..................................................... may stimulate growth.................... Sequentially add 1....... Autoclave for 15 min at 15 psi pressure–121°C....... Addition of 10–20mg sodium dithionite per liter from a 5% (w/v) solution....................0mg HCl (25% solution) ............ 5........ Sparge with 100% N2........................6......................... Boil solution A for a few minutes........ Mix thoroughly............ and 10.....4 at 25°C Solution A: Composition per 870.10mg Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1.0mL solution D.....................0mL: Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100....0mL solution B.....................................10........................0mL: NaHCO3 ...... Medium (DSMZ Medium 195) Composition per 991.................................................... 21..............0mL Solution D ............ Sparge with 100% N2................................... freshly prepared under N2 and filter-sterilized...4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10......... Sparge with 100% N2....................0mL solution E................... 10. Solution C: Composition per 100............................................................... 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.......10....................................... Solution E (Vitamin Solution): Composition per liter: Pyridoxine-HCl .....5.......................... 0............... 1.............. Use: For the cultivation of Desulfobacter species....0mg ZnCl2 ........ .................................. 0......4g tilled/deionized water and bring volume to 10....................................................... Sparge with 100% N2.2............................0mL: Solution A ..............................................................5................ 5...........0mL Solution C .........10........0mg Thiamine-HCl·2H2O ....0mL..............................190...0mL solution F......0mL: Na2S·9H2O ..................................134 Desulfobacter sp........... 100..................0g Solution F: Composition per 10.......3g KH2PO4 .......................................................................................... Solution F: Composition per 10.............. 2.2g CaCl2·2H2O......................0mg Riboflavin ..0mg Biotin ................. 10............. 2..................2..............................................0g KCl... Mix thoroughly...... Mix thoroughly......... Solution E (Vitamin Solution): Composition per liter: Pyridoxine-HCl .....100......0mg H3BO3 .. 3...5.................24.... Cool to room temperature............0mL pH 7........................................ 100......5g Na-acetate·3H2O..5g NH4Cl...........0L......2. 10. Paracoccus solventivorans..................100..............0mg Vitamin B12 ............................. 1.........0mL: Na2S·9H2O.......... Preparation of Medium: Gently heat solution A and bring to boiling........................... Flush with 80% N2 + 20% CO2 to remove dissolved oxygen........................870...........0mg Nicotinic acid ........5.............. Preparation of Medium: Gently heat solution A and bring to boiling.....0mL Mix thoroughly.0mg Na2MoO4·2H2O................... Mix thoroughly...........0mg Folic acid.......................0g MgCl2·6H2O...... Sequentially add 1.........5.........................0mg p-Aminobenzoic acid .................. Boil solution A for a few minutes............... 5..............................1.....0mL solution D.....................0mL.........2...........................0. Mix thoroughly.. Mix thoroughly...................................... may stimulate growth........ 0. Gas with 80% N2 + 20% CO2 gas mixture to reach a pH below 6.. 5........................ Sparge with 100% N2..............................................................0mg CuCl2·2H2O................. and Desulfotomaculum sp. Sparge with 100% N2............. Gas with 80% N2 + 20% CO2 gas mixture to reach a pH below 6................... Autoclave for 15 min at 15 psi pressure–121°C.......... 5................ 3....... 10.0mg D-Ca-pantothenate ..........0mL of HCl solution..........0mL solution C............................... and 10.....

........ 2.......... Add remaining components.................................0....10.............. 1........0mL Solution E (Vitamin solution) ....0g MgCl2·6H2O.........5g NH4Cl.......0mL Solution F...............15g Resazurin ......................................1–7........0mg p-Aminobenzoic acid ................... Allow to cool to room temperature while gassing under 80% N2 + 20% CO2.................0mg Calcium DL-pantothenate........................ Continue boiling for 3-4 min......................0mL: NaHCO3 .................... Continue gassing until pH reaches below 6...................................2..........................................5... Mix thoroughly.............. 1............................................................ Mix thoroughly....0mg Vitamin B12......0g Na2SO4 .......................................... solution E...................0g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 100.............................7........................................ 10...... and solution G...............0mL Solution B (Trace elements solution SL-10) ......... 0.....................................0mg CuCl2·2H2O ..................0mL Solution C.......... Autoclave for 15 min at 15 psi pressure–121°C...0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10..................................................................0mg 135 Riboflavin.................. 1...3g KH2PO4 .............. 2........... Seal the flask under 80% N2 + 20% CO2.....................0............ 10...................................... Filter sterilize....... Autoclave for 15 min at 15 psi pressure–121°C......................................... 6...2g CaCl2·2H2O.................................. 1.0mg MnCl2·4H2O ..................0...................... Gas under 80% N2 + 20% CO2........... 870.... Continue boiling for 3-4 min...............94..........0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870.....................................5g CoCl2·6H2O ...................................... Gas under 100% N2.. Gas under 80% N2 + 20% CO2.................................... in that order..0mg H3BO3 ............ 190...............3g KCl ......................0mL: Solution A ..................100....................................................0mg NiCl2·6H2O ...............................................190............................................0L............. Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O...0................ Allow to cool to room temperature while gassing under 80% N2 + 20% CO2.....................0mL Solution C ........0mL Solution B (Trace elements solution SL-10).....0mL.. 10.......... 3.............................0mL Solution D .............1.............................0mL...................................................................15g Resazurin ............. Mix thoroughly............................ Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2...................................0mg Preparation of Solution G: Add phenol to distilled/ deionized water and bring volume to 10............................ Gas under 100% N2................... 100............................... 24....................0mg Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 870...........................870..................0mL pH 7.......... 1................................. 7.........................................................................0mL: NaCl ..............................70.... Autoclave for 15 min at 15 psi pressure–121°C.............0mL Solution D .....0mg Biotin ............. Use: For the cultivation of Desulfobacterium anilini................................................... Autoclave for 15 min at 15 psi pressure–121°C............10...........0....0mL.............0mL: Sodium acetate·3H20................ 0..................Desulfobacterium anilini Medium Desulfobacterium anilini Medium Composition per 1011.......0mL Solution F ......................... 10........3g KCl..0mL: Phenol...........0mg ZnCl2 .....................10.......3g KH2PO4 ......................... Mix thoroughly....2....... 5...0mg Folic acid ........ Solution C: Composition per 100.... Autoclave for 15 min at 15 psi pressure–121°C................ Gas under 100% N2.........................1........................5.............................................. 5..........................................................................0L..........4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10................ 100..................................0mL Solution G ......100......................... Filter sterilize.. Seal the flask under 80% N2 + 20% CO2........................0g Na2SO4 .......... Autoclave for 15 min at 15 psi pressure–121°C.......... Mix thoroughly...................................................0mg HCl (25% solution) ............0mg Nicotinic acid .....................1.... 0.................... Solution E (Vitamin Solution): Composition per liter: Pyridoxine·HCl ..0... solution D.....................................................................................1... solution C........... Mix thoroughly..........0mg Thiamine·HCl ..0mg ....... Solution G: Composition per 10.......................... Add distilled/deionized water and bring volume to 1...................0mL pH 7................ solution F... Gently heat and bring to boiling.... 70.........0mL............................1–7...................... Mix thoroughly............................5g Preparation of Solution D: Add sodium acetate to dis- tilled/deionized water and bring volume to 10.........................................................10.......................... Preparation of Medium: Aseptically and anaerobically combine solution A with solution B............................. Mix thoroughly....... 36.........5g CoCl2·6H2O........................ Desulfobacterium anilini Medium Composition per 1002...................0mL: Solution A ..........0mL: NaCl ....1mg Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1..........................0mL: Na2S·9H2O .......0g MgCl2·6H2O .....................................0mL Solution E (Vitamin solution) ..............................5g NH4Cl .0mL Solution G ..3............. Mix thoroughly........ Gently heat and bring to boiling.....4 at 25°C Solution A: Composition per 870......................... 0.....................................0mg ZnCl2 ................................................. Gas under 100% N2................0.0mg Lipoic acid ........................................................................0mg MnCl2·4H2O....0mL of HCl solution..............................................2g CaCl2·2H2O ...........0.......................... Mix thoroughly................ Continue gassing until pH reaches below 6. 5...... Prepare solution freshly..... Solution D: Composition per 10........................0mL............................0mL............ 5............................... 5...... Solution F: Composition per 10.0mg Na2MoO4·2H2O ................... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O .........................................4 at 25°C Solution A: Composition per 870.10............

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

.... Autoclave for 15 min at 15 psi pressure–121°C........... Use: For the cultivation of Desulfobacterium cetonicum.0mL Solution E ................................................................................................ Solution D: Composition per 40.........2g Preparation of Solution C: Add sodium butyrate to distilled/deionized water and bring volume to 10. Continue boiling until resazurin turns colorless.................. 0........................................... Check that final pH is 7........................5g CoCl2·6H2O .. 0............0mL Solution B (Trace elements solution SL-10) . 10.................................................................... 2...........................................0g KCl ..............3.3g CaCl2·2H2O....0mg Folic acid...5g NH4Cl ...3g combine solution A with solution B.....2 at 25°C Solution A: Composition per 900....................... Mix thoroughly................. and solution F.0mg HCl (25% solution) .......21....................................0g Preparation of Solution D: Add NaHCO3 to distilled/ deionized water and bring volume to 40.......0mL pH 7...........................4...0......................... 36...0mL Solution F ..0mg Biotin .... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O.... 2............0mL Solution C .......................... 1................ 5...... 2...........................................0mg CuCl2·2H2O ..............10.............4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10......8g KH2PO4 ...0mg Thiamine·HCl .................................................................................... Mix thoroughly........... Mix thoroughly......6 ± 0..........2–7..........0mg Na2MoO4·2H2O .................0g MgCl2·6H2O ........0g Na2SO4 ...............0mL of sterile solution C....2–7..................................................................................2g CaCl2·2H2O ....................... Autoclave for 15 min at 15 psi pressure–121°C....................................0mL: Na2S·9H2O................ Gas under 100% N2. Aseptically and anaerobically combine 950................................................1g ZnCl2 ............................................ Autoclave for 15 min at 15 psi pressure–121°C.......................0mL: Solution A .....10....... Add remaining components...................... Gas under 100% N2......0mg MnCl2·4H2O.. Sparge with 100% N2....40.................0............19g MnCl2·4H2O ............ 190......... Autoclave for 15 min at 15 psi pressure–121°C......................... 10......7g MgCl2·6H2O....................... 0.......................0mL.........0mL: Na2S·9H2O ............ Mix thoroughly......................0mL of HCl solution................... 6................... 0.................................0..... 0......0mL Solution H ........ Autoclave for 15 min at 15 psi pressure–121°C..................................0mL............ 5............................0mg ZnCl2 ...................0mL....................... Anaerobically distribute into sterile tubes or flasks under 80% N2 + 20% CO2.0mL Solution D . 10..... 900...........................2............0..................................................................... 10...... Desulfobacterium indolicum Medium Composition per 1002..........0mg Vitamin B12 ......0mL Solution C............................ 2......................... Autoclave for 15 min at 15 psi pressure–121°C...........................0g Na2SO4 .............................0mL Solution D ......0............................................................................................0mL Solution E (Wolfe’s vitamin solution)....0...................................... under 80% N2 + 20% CO2....................1.......1............................950......................0mL: NaCl ............... 100.... tilled/deionized water and bring volume to 10.... Autoclave for 15 min at 15 psi pressure–121°C.................................... Add components to distilled/deionized water and bring volume to 900................. Mix thoroughly.................................................................................................... 50...................3g NH4Cl....15g Resazurin ..5g CoCl2·6H2O................... solution D.. indicating reduction.......................... Cool to 45°–50°C............. 1.0.... Sparge with 100% N2.................0mL of sterile solution E........ in that order... Mix thoroughly............. 1...................................3g KH2PO4 ..................................0mL of sterile solution B...............1........0mL Solution G .. Sparge with 100% N2.. Autoclave for 15 min at 15 psi pressure–121°C........................................... 0........ 24............... Mix thoroughly... Solution B (Trace Elements Solution SL-10): Composition per liter: FeCl2·4H2O .........4mL pH 7...................0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10............................. 70........4mL: Solution A ................................. Add distilled/deionized water and bring volume to 1...........05g Preparation of Solution A: Add components to dis- tilled/deionized water and bring volume to 950........0mL of sterile solution A with 1.0mL of sterile solution D...1.............................. solution C......................0L.... Sparge with 100% N2............................ Mix thoroughly... Solution F: Composition per 10.................... 0......................................3..................0mL: NaHCO3 .0mg H3BO3 ............. Preparation of Medium: Prepare and dispense medium Desulfobacterium cetonicum Medium Composition per 1011.........................0mL...Desulfobacterium indolicum Medium Riboflavin ....... Mix thoroughly.4 at 25°C Solution A: Composition per 950.....0mL: NaCl ......... Sparge with 80% N2 + 20% CO2.................................. Gently heat and bring to boiling.................................................................................................. Preparation of Solution E: Add Na2S·9H2O to dis- Use: For the cultivation and maintenance of Desulfobacterium catecholicum.............. solution E.. Mix thoroughly............................... 4........0mL.........0mg NiCl2·6H2O .........0mL: Sodium butyrate . Cap with rubber stoppers..................07g ............................ Solution E: Composition per 10.................................. Preparation of Medium: Aseptically and anaerobically 137 Solution C: Composition per 10................ and 10................10......... 30.............0mg Preparation of Solution A: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2..0L.....................................................0mL...1mg Preparation of Solution E (Vitamin Solution): Add components to distilled/deionized water and bring volume to 1.......0mL Solution B (Trace elements solution SL-10).. Mix thoroughly........0........................................

......3....................0mL....... 0.......... bring volume to 1......................0mg NaOH (0...................... 1............................ Mix thoroughly.. 5.. 10....... Prepare solution freshly.......................0mL of sterile solution E............ Solution H: Composition per 10............................. 0......................................... 0......0mL.... Filter sterilize......................... Adjust pH to 6....7mL: Indole ..............0mg CuCl2·2H2O ................... 1.....6.............................5g NH4Cl ............. Aseptically and anaerobically distribute into sterile tubes or flasks....0mL Solution C....................................... Bring volume to 1......................... 6......0....0mg Thioctic acid ........ 5.........036g NiCl2·6H2O.........................4mL pH 7......0.................................... Mix thoroughly................. Use: For the cultivation and maintenance of Desulfobacterium indolicum.......... Adjust pH to 6.. 10.0mL Solution F ............... To 100.................. Aseptically gas under 100% N2 for 20 min...............01g Thiamine·HCl ................1.....0mL of sterile NaCl solution and 0........................1. Add indole to distilled/deionized water and bring volume to 100.... Preparation of Solution B (Trace Elements Solution SL-10): Add the FeCl2·4H2O to 10.........0mL of sterile solution G.............. Autoclave for 15 min at 15 psi pressure–121°C.............2 at 25°C Solution A: Composition per 930........0mg Calcium pantothenate ... Mix thoroughly........ 5...0mL of HCl solu- tion. Bring volume to 1..........................0mL of sterile solution D and 0...................... Mix thoroughly.... Continue boiling until resazurin turns colorless. Mix thoroughly..... Mix thoroughly. Filter sterilize......3g NaCl (30% solution) ..... Gas under 100% N2 for 20 min....................4g Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10....0mL of sterile indole solution......................0mg p-Aminobenzoic acid ....... Solution B (Trace Elements Solution SL-10): Composition per liter: Solution F: Composition per liter: FeCl2·4H2O.......0mg Riboflavin . Aseptically gas under 80% N2 + 20% CO2 for 20 min....... Mix thoroughly.................0g Na2SO4 .............21..................7mL Preparation of Solution D: Prepare and dispense all solutions anaerobically under 100% N2... Cap with a rubber stopper...............................0....................3g KH2PO4 ..................................5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50..................0mL MgCl2·6H2O (40% solution)................... Aseptically gas under 100% N2 for 20 min.............. 3...... Gently heat and bring to boiling.............0.......5g Preparation of Solution H: Add Na2S2O4 to distilled/ deionized water and bring volume to 10......................................0...................0 with NaOH........... and 10..........................0mL of sterile solution F......... Cool to 25°C.................... 2..............138 Desulfobacterium Medium Na2MoO4·2H2O .................................7mL of sterile MgCl2·6H2O solution...... 50..........................................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..... Solution G: Composition per 10..... 10...............................0...................1.......0..................................... 1................................0mL of sterile solution B...............0mL....................... aseptically and anaerobically add 30...........0mg Cyanocobalamin .............................01M solution) ............0mg Preparation of Solution A: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2..................2.......0L with .......................................................................... Mix thoroughly......... Cool to 45°–50°C...0mL with distilled/deionized water... sterile solution A......0mL: Na2S·9H2O............................ Solution D: Composition per 107.................4mL of sterile solution H..............0mL Preparation of Solution B (Trace Elements Solution SL-10): Add the FeCl2·4H2O to 10...........0 with NaOH.......................0. Cap with rubber stoppers............ 0.................................. Preparation of Medium: To 900............................................... 2......... aseptically and anaerobically add in the following order: 1..........0mg Folic acid......... Filter sterilize...........0mL of HCl solu- tion.................................................0mL with distilled/deionized water.....................0mL Solution E .0mL: NaHCO3 .................... 0... Aseptically gas under 100% N2 for 20 min........ Mix thoroughly.........0mL of sterile solution C.........................................................................0mg CuCl2·2H2O. 2.. Mix thoroughly...........0L with distilled/deionized water.................................... Aseptically gas under 100% N2 for 20 min........... Solution C: Composition per 50...................... Solution E (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl .................0mL Solution B (Trace elements solution SL-10) .............. indicating reduction.................4mL: Solution A ..............0mL.....19g MnCl2·4H2O ...... Gently heat while stirring until dissolved......... Mix thoroughly...............................................................0g KCl ... aseptically and anaerobically add 7............................................................................. Immediately prior to inoculation....................0mL....................2g CaCl2·2H2O ........0g MgCl2·6H2O ......................... 5......................................... Filter sterilize............0L Preparation of Solution F: Add Na2SeO3·5H2O to 1.........5g CoCl2·6H2O...... Filter sterilize.. Autoclave the three solutions separately for 15 min at 15 psi pressure–121°C........ Mix thoroughly..3.10... 50.7................ 0... 0....0mL Solution G .............................024g H3BO3 ............0........................................0mL Na2SeO3·5H2O ...............................0mg HCl (25% solution) .......................15g Resazurin ....................... Add components to distilled/deionized water and bring volume to 930..............0 ± 0........................................ Bring volume to approximately 900.............0mL of cooled........ 10.............0L...................................... 930...................................0mL Solution D (Wolfe’s vitamin solution) ....... Desulfobacterium Medium Composition per 1002............0L of NaOH solution.0mL: NaCl ...... 5..07g Na2MoO4·2H2O..0mg Biotin .0mg Nicotinic acid ... 2.. Bring volume to approximately 900....0........... Cool to 45°–50°C................... 5..... Prepare the NaCl solution and the MgCl2·6H2O solution separately....024g H3BO3 ...................................1g ZnCl2 ................. Mix thoroughly.0mg HCl (25% solution) .................0mL: Na2S2O4 ......................................................0........036g NiCl2·6H2O .........

....................................0mg NaOH (0..........0.................................................................. Use: For the cultivation and maintenance of Desulfobacterium autotrophicum..........0........................... 10.........................3g KH2PO4 .......................0mg Biotin .............................0mL: NaHCO3 ................ Mix thoroughly.......4mL pH 7.......0L of NaOH solution. Immediately prior to inoculation..... 0..5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50..0.................. 10..... 1...........................024g H3BO3 ...........0mL... 2.........................0mg p-Aminobenzoic acid ...... Mix thoroughly...... Continue boiling until resazurin turns colorless..... Mix thoroughly.................21............ Solution C: Composition per 50..........................0mL: Na2S2O4...... Aseptically gas under 100% N2 for 20 min...... aseptically and anaerobically add in the following order: 1.......... Mix thoroughly.............2.............0L........................0mL of sterile solution D.................................................................................... sterile solution A...............................................1...... 5...... Na2S·9H2O......................... 5.................0..19g MnCl2·4H2O .................... Filter sterilize................................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.......0mg HCl (25% solution) ......... 5....0mL Solution E ......... Autoclave for 15 min at 15 psi pressure–121°C........2.............. Desulfobacterium Medium with Lactate Composition per 1002.......... 2....01M solution) ................................................................0mL Solution B (Trace elements solution SL-10) ...............................0mg Cyanocobalamin .............................4mL: Solution A ...........................................................................5.........1mg Preparation of Solution D (Wolfe’s Vitamin Solution): Add components to distilled/deionized water and ................1................ Aseptically gas under 100% N2 for 20 min................. Aseptically and anaerobically distribute into sterile tubes or flasks.............0........................0....... Solution C: Composition per 50... Cap with a rubber stopper............ 0.................................................................10g ZnCl2 ..... Solution D (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl .........................................................0mg Thioctic acid .036g NiCl2·6H2O..0mL of sterile solution E....5............0mL Solution F ............................. Aseptically gas under 100% N2 for 20 min...0mg Folic acid ......................................Desulfobacterium Medium with Lactate distilled/deionized water............. 10..... Preparation of Medium: To 900..................0mL of sterile solution B.....................5g CoCl2·6H2O................................2......... 1..... Cool to 45°–50°C..... Gently heat and bring to boiling.............0mL Solution D (Wolfe’s vitamin solution) ..................0g MgCl2·6H2O ......930....................01g Thiamine·HCl ......5.0mL tion.................. 5.............. 50...... Aseptically gas under 100% N2 for 20 min.................... 0......................... Bring volume to 1.............5..01g Thiamine·HCl ............................1......................................... Solution D (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl...........2g CaCl2·2H2O .......................................0.5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50............. 5...... Filter sterilize.............................2 at 25°C Solution A: Composition per 930.......... Mix thoroughly........... Mix thoroughly..3..................... Mix thoroughly........................................... Aseptically gas under 100% N2 for 20 min...............0mg Calcium pantothenate ................. sodium salt ..... Adjust pH to 6......0mL.........................15g Resazurin .1g KCl ............0...............................................................0L Preparation of Solution E: Add Na2SeO3·5H2O to 1................ Solution B (Trace Elements Solution SL-10): Composition per liter: Solution F: Composition per 10..............0mg Nicotinic acid ...... 50.................... Filter sterilize...........0mg Biotin ..................................... Aseptically gas under 80% N2 + 20% CO2 for 20 min.......0mg Thioctic acid ....................... 0........ Cool to 25°C..............................................0L with distilled/deionized water.......... Solution E: Composition per liter: 139 Solution C........0mL.........4mL of sterile solution G..................0mL of sterile solution F........................... and 10..................3............................................. 2.2..................0 with NaOH.....0mg Riboflavin....... Gas under 100% N2 for 20 min...... Mix thoroughly...... 10...0mg p-Aminobenzoic acid ..................................................... Filter sterilize.0mL..............................0mg Folic acid................................................ 1................................0mL of cooled.......0.............................. Filter sterilize..........0mL with distilled/deionized water..........0mL.............................5g NH4Cl ...............0mg Cyanocobalamin........... indicating reduction............................1.......................0....... Cap with rubber stoppers.0mg Nicotinic acid ....................... Filter sterilize...........070g Na2MoO4·2H2O..........0mg Riboflavin ...................................1mg Preparation of Solution D (Wolfe’s Vitamin Solution): Add components to distilled/deionized water and bring volume to 1...6.......0mL of sterile solution C...................... Solution G: Composition per 10..................................... Prepare solution freshly..........................0mL Solution G ............0mg CuCl2·2H2O................. Add components to distilled/deionized water and bring volume to 930............ Filter sterilize..................................... 0......................................... Aseptically gas under 80% N2 + 20% CO2 for 20 min.....................4g tilled/deionized water and bring volume to 10... 3.......0mL: NaCl ............0g Lactic acid..................................5............................................. Mix thoroughly........0mL: FeCl2·4H2O.. Bring volume to approximately 900.....................0mL of HCl solu- Na2SeO3·5H2O .............0mL: NaHCO3 ..........0g Na2SO4 ...0 ± 0.....................0mg Calcium pantothenate ...0mg Preparation of Solution A: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2.......... aseptically and anaerobically add 0............ 5...... Mix thoroughly........5.........................0............. Autoclave for 15 min at 15 psi pressure–121°C.........5g Preparation of Solution G: Add Na2S2O4 to distilled/ deionized water and bring volume to 10.............

.... reduction.. Cap with rubber stopper............... 3..............0mL Solution C .....................0mg HCl (25% solution) .. Mix thoroughly............ Aseptically gas under 100% N2 for 20 min.......... Continue boiling until resazurin turns colorless.....0mL...................0L Preparation of Solution E: Add Na2SeO3·5H2O to 1.......... Solution E: Composition per liter: Solution B (Trace Elements Solution SL-10): Composition per liter: Na2SeO3·5H2O .............................01M solution)..................0L of NaOH solution..... Mix thoroughly..0mg CuCl2·2H2O............................................... sterile solution A...1.0mL..........................................10...........50............0g MgCl2·6H2O................ Gently heat and bring to boiling..........0mL Preparation of Solution B (Trace Elements Solution SL-10): Add the FeCl2·4H2O to 10...........0...... indicating FeCl2·4H2O.......... Adjust pH to 6................................3. 21..................0 ± 0.....0mg Riboflavin........................ Solution G: Composition per 10..0...... Bring volume to 1......................... in the following order.... Mix thoroughly........ Filter sterilize................................................. Cap with a rubber stopper...... Solution C: Composition per 50.................................................0mg Cyanocobalamin............................0mL of sterile solution E.......0mL of HCl solu- tion.........2................... Mix thoroughly...............................................0g Na2SO4 ........ Cool to 45°– 50°C.........................................10......0mg Biotin ........................0...5g Preparation of Solution D: Prepare and dispense solution anaerobically under 80% N2 + 20% CO2.5g NH4Cl........ Cap with rubber stoppers....0mL: NaHCO3 ........0mg Thioctic acid ............... Prepare solution freshly....................0mL Solution E (Wolfe’s vitamin solution) .................... Filter sterilize..........................................024g H3BO3 ..... Mix thoroughly.............5.......4mL: Solution A ...........................0....... Aseptically and anaerobically distribute into sterile tubes or flasks..0mL..01M solution) ..15g Resazurin ..............4mL of sterile solution G...................... Solution G: Composition per 10........... 50....0mg Folic acid ..2..... Cool to 25°C......................... Filter sterilize...0mL Solution H ... 1..................................................... 1........0mL Solution D ..............................07g Na2MoO4·2H2O......... Desulfobacterium Medium............2.............. Aseptically gas under 100% N2 for 20 min....................................................................................2. Filter sterilize...... 0............0mL...0L Preparation of Solution F: Add Na2SeO3·5H2O to 1.. aseptically and anaerobically add... 0.. Modified Composition per 1002....0.........................0L of NaOH solution.... Filter sterilize......5......4g ........................0mg Preparation of Solution A: Prepare and dispense solu- tion anaerobically under 80% N2 + 20% CO2.....................036g NiCl2·6H2O.............1.....................0mg NaOH (0............. Solution F: Composition per liter: Na2SeO3·5H2O ....2g CaCl2·2H2O.................. 0...... Filter sterilize.........................0mL: Na2S·9H2O..............................................0L with distilled/deionized water.0 with NaOH.................................5g CoCl2·6H2O.........................................0mL Solution B (Trace elements solution SL-10)............... Autoclave for 15 min at 15 psi pressure–121°C........................................ Mix thoroughly...........0mL: Na2S·9H2O ....................................... Solution F: Composition per 10..........0mL of sterile solution B....... 0............... 3........... Immediately prior to inoculation............. Aseptically gas under 80% N2 + 20% CO2 for 20 min........0........................ Autoclave for 15 min at 15 psi pressure–121°C................01g Thiamine·HCl ......................920.................................................5........................................... Aseptically gas under 100% N2 for 20 min.......................... Mix thoroughly........ and 10......5g Preparation of Solution G: Add Na2S2O4 to distilled/ deionized water and bring volume to 10.......0..................................... Solution D: Composition per 10............10........ Modified bring volume to 1..........0mg Nicotinic acid ....................2 at 25°C Solution A: Composition per 920........... Autoclave for 15 min at 15 psi pressure–121°C............ Preparation of Medium: To 900....................0mL..... aseptically and anaerobically add 0..............0mL of cooled..3g KH2PO4 ........ Aseptically gas under 100% N2 for 20 min..........0mL of sterile solution C.................. Add sodium acetate to distilled/deionized water and bring volume to 10...1....... Solution E (Wolfe’s Vitamin Solution): Composition per liter: Pyridoxine·HCl................ Mix thoroughly....... Aseptically gas under 100% N2 for 20 min...4mL pH 7...2...............5............................................................0mg Calcium pantothenate .........0...5........................1mg Preparation of Solution E (Wolfe’s Vitamin Solution): Add components to distilled/deionized water and bring volume to 1.................................................19g MnCl2·4H2O ...........0mg NaOH (0........0mL of sterile solution D................. Mix thoroughly.............. Aseptically gas under 100% N2 for 20 min...............6...1g ZnCl2 .5................................................... Add components to distilled/deionized water and bring volume to 920...................................................... Use: For the cultivation of Desulfobacterium autotrophi- cum........ Mix thoroughly. 1............. 3. Mix thoroughly..........0mL: Na2S2O4....0L............. Filter sterilize.1...0mL: NaCl ....... Bring volume to approximately 900.................... Cool to 45°–50°C...................... 0...........0L.0........0g KCl..................... Mix thoroughly......0mL Solution F. 0...................... 10...0mL Solution G ...4g Preparation of Solution F: Add Na2S·9H2O to distilled/deionized water and bring volume to 10.................................0mL: Sodium acetate·3H2O .............................0mL of sterile solution F......................140 Desulfobacterium Medium................................ 1.......................................... 10.......0mL with distilled/deionized water.....0mg p-Aminobenzoic acid ......5g Preparation of Solution C: Add NaHCO3 to distilled/ deionized water and bring volume to 50. Gas under 100% N2 for 20 min................

..0mL Solution F......2 at 25°C Solution A: Composition per liter: Na2SO4 ... aseptically and anaerobically add in the following order: 1...0mL Solution E .......... Autoclave for 15 min at 15 psi pressure–121°C..................... 0....... Solution H: Composition per 10........10...........................0mg NiCl2·6H2O . Mix thoroughly..............................................7g CaCl2·2H2O .....0L....................................0mL of sterile solution D.............4.Desulfobacterium oleovorans Medium Preparation of Solution G: Add Na2S·9H2O to distilled/deionized water and bring volume to 10............... Gas under 100% N2.. 1.0mL...... 2...1..........................................................0mL Solution I.........0mL of sterile solution E.....................0g NaCl ........... Shake until stearic acid dissolves.............2....................... Filter sterilize. 100....0mL of HCl solution...................2 ± 0............... Gas under 100% N2 for 20 min... 24..... Solution D: Composition per 50.....0mg Folic acid ........................................................................................ Filter sterilize.....................................................0L......... Autoclave for 15 min at 15 psi pressure–121°C......................5mL Preparation of Solution G: Add components to distilled/deionized water and bring volume to 80.... 36.0mL..... Mix thoroughly................... 190.................... aseptically and anaerobically add 0.............. Cap with a rubber stopper...................... Use: For the cultivation and maintenance of Desulfobacter curvatus and Desulfobacter latus........50........0mg CuCl2·2H2O ....10.... Continue boiling for 3-4 min........0mL pH 7......................0mL Solution C (Selenite-tungstate solution) ...........0............................................................10....85g NaOH (4....... Gas under 100% N2...2..................0mg ZnCl2 ................................................................ Gas under 80% N2 + 20% CO2.................................... Autoclave for 15 min at 15 psi pressure–121°C.....0mL Preparation of Solution B (Trace Elements Solution SL-10): Add FeCl2·4H2O to 10............0mg Calcium DL-pantothenate ..0mL Solution D ...................................... ...............................0mL: Solution A ........ Mix thoroughly....0mg Thiamine·HCl .... Gently heat and bring to boiling........................................................................5.0M solution)..... Mix thoroughly.67..................................................... Add distille