Handbook of Microbiological Culture Media

Handbook of Microbiological Culture Media
Dehydrated Culture Media
Culture Media Ingredients
Edition No. 10
Additives
Supplements
Reagents
TABLE OF CONTENTS
Scharlau Microbiology Introduction
Scharlau Microbiology Quality Policy
II
Safety (Regulations, Hazard Categories, Risk and Safety Phrases, Disposal)
Guidelines for correct manipulation in Microbiology
XIII
1-176
177-204
Additives
205-213
Supplements
215-229
Reagents
231-244
Index of products in this Manual
245-251
Scharlau Microbiology is part of the Scharlau Science Group

Introduction
It is a pleasure to introduce to you this edition no. 8 of
our Handbook of Microbiological Culture Media, revised
and extended, in a new and more attractive format.
The range of products has been increased, reafrming the
Scharlau Microbiology brand as an attractive alternative
to other manufacturers of culture media for Microbiology.
You will nd new products, according to the standards
of the European Pharmacopoeia, USP and other ofcial
organisms.
In this edition, we have separated additives, reagents
and selective supplements into different chapters, making it easier to locate each product.
We have also included additional safety information and
R+S sentences, following the EU guidelines 2001/59/
CE.
The company
In 1979, Dr. Jos Sancho Valls, Professor at the University of Barcelona, decided to start manufacturing
dehydrated culture media. A partnership was formed
with a local importer of research chemicals and the
products sold on the local market under ADSA=MICRO
brand name. In 1992 the company was integrated into
the Scharlau group. Thus we look back to more than 25
years of experience in the manufacture of culture media.
At present, a wide rage of products is offered under the
Scharlau brand, from standard laboratory chemicals
(inorganic salts, acids, buffers, volumetric solution,
solvents, etc.), to high purity specialized solvents for
HPLC and environmental analysis, up to culture media
for microbiology.
The technical supervision remains in the hands of Professor Sancho. Through his wide scientic knowledge,
we can offer good technical support.
Contact Scharlau
Dont hesitate. Contact Scharlau for any requirement of
culture media for microbiology.
The international presence of Scharlau Microbiology
grows day by day. A wider network of distributors and a
larger portfolio help to better penetrate the markets.
Scharlau Chemie, S.A., which was born 53 years ago to
manufacture exclusively organic chemicals for research
laboratories, has been increasing its products and processes to become a global manufacturer of laboratory
chemicals and culture media for microbiology.
If you work in Spain please contact:

SCHARLAB, S.L.
Tel.: 902 20 18 98
Fax: 900 50 29 35
E-mail: scharlab@scharlab.com
Post: Gato Prez, 33. Pol. Ind. Mas den Cisa
08181 Sentmenat, Barcelona, Spain
Customers outside of Spain please contact:
SCHARLAU CHEMIE, S.A.
Tel.: +34 93 715 18 11
Fax: +34 93 715 31 75
E-mail: export@scharlau.com
Post: Ctra. de Poliny a Sentmenat, Km. 8,2
08181 Sentmenat, Barcelona, Spain
Or ask for the address of our distributors in any of the
following countries:
ALBANIA
ANGOLA
ARGENTINA
AUSTRALIA
AUSTRIA
BAHRAIN
BANGLADESH
BARBADOS
BELGIUM
BENIN
BOLIVIA
BOSNIA & HERZEGOVINA
BOTSWANA
BRUNEI
BULGARIA
CHAD
CHILE
CHINA P.R.
COLOMBIA
COSTA RICA
IVORY COAST
CROATIA
CUBA
CYPRUS
CZECH REPUBLIC
DENMARK
DOMINICAN REPUBLIC
ECUADOR
EGYPT
EL SALVADOR
ERITREA
ESTONIA
ETHIOPIA
FINLAND
FRANCE
FRENCH POLYNESIA
GERMANY
GHANA
GREECE
GUINEA BISSAU
HAITI
HONG-KONG
HUNGARY
ICELAND
INDIA
INDONESIA
IRAN
IRAQ
IRELAND
ISRAEL
ITALY
JORDAN
KENYA
KOREA
KUWAIT
LATVIA
LEBANON
LIBYA
LITHUANIA
MADAGASCAR
MALAYSIA
MALTA
MAURITANIA
MAURITIUS
MOLDOVA
MOROCCO
MOZAMBIQUE
MYANMAR
NEW CALEDONIA
NEW ZEALAND
NIGERIA
NORWAY
PAKISTAN
PANAMA
PERU
PHILIPPINES
POLAND
PORTUGAL
QATAR
REUNION ISLAND
ROMANIA
RUSSIA
RWANDA
SERBIA & MONTENEGRO
SAUDI ARABIA
SENEGAL
SINGAPORE
SLOVAKIA
SLOVENIA
SOUTH AFRICA
SRI-LANKA
SUDAN
SULTANATE OF OMAN
SWEDEN
SWITZERLAND
SYRIA
TAIWAN
TANZANIA
THAILAND
THE NETHERLANDS
TOGO
TONGA
TRINIDAD & TOBAGO
TUNISIA
TURKEY
UGANDA
UKRAINE
UNITED ARAB EMIRATES
VENEZUELA
VIETNAM
YEMEN
ZAMBIA
ZIMBAWE
We stock over 700 different microbiological references in

our new 4.000 m2 warehouse in Sentmenat (Barcelona).

www.scharlau.com
Please use our website for the latest news and MSDS:
www.scharlau.com.
Our website has been redesigned recently to offer you a
much more modern and intuitive interface.
COA come with each bottle, which is why we dont publish them on the web.
The factory
The microbiological division of Scharlau Chemie, Scharlau Microbiology, manufactures a wide range of culture
media, reagents, stains, supplements and additives.
25 years experience in the production of dehydrated
culture media allows us to develop fast new formulations
to keep up with the latest developments in microbiology.
New production facilities have been inaugurated in the
year 2002, thus enabling us to further improve our processes and expand production capacity. The new factory
has been designed following the most latest directives to
assure the maximum quality of the nal product.
More than 700 references are available from Scharlau
Microbiology, ready to be delivered from our new 4000
m warehouse in Sentmenat, Barcelona. The strategic
situation, within less than 1000 km from most European
capitals, makes it possible to quickly restock our distributors. Barcelona, being one of the busiest ports in Europe,
boasts excellent world wide shipping connections.
2
II
Environmental care
One of Scharlau Chemies target regarding environmental issues was achieved in 2003, when we received ISO
14001 approval. Scharlau Chemie has always designed
its processes to minimize environmental impacts on the
rural area where it is located for about 30 years.
As a contribution to the packaging residue minimization

targets, Scharlau has replaced all its packing materials
to avoid the use of expanded polystyrene, which is difcult to recycle and expensive to dispose of. All internal
and external elements of our packaging are made from
recycled cardboard and are fully biodegradable and
recyclable.

Quality as standard
Our commitment to quality has always been very
important to keep our customers trust.
Scharlau Microbiology purchases raw materials and
transforms them into microbiological culture media,
using various processes and strict analytical controls.
Our target is the production of products of the highest
possible quality.
To achieve this, we have always worked under internal
quality standards, even before ISO regulations were
adopted. This fact not only allows us to manufacture
products of the highest quality, but also to maintain our
quality at a constant level.
The quality concept has evolved and nowadays, quality is not only understood as an attribute of the product,
but a feature which has to be present in everything
done in the company. It is useless to have an excellent
product, if delivery times or technical assistance are not
adequate.
In Scharlau Microbiology we work under these premises,
injecting quality into all our services, from the development of a product designed to cover real customer
requirements, to our after sales assistance.
Since 1997 Scharlau Microbiology is ISO 9002 accredited through a German TUV.
Certicate of Analysis
Every pack is sent together with its Certicate of Analysis,
which guarantees the quality of our products.
Together with the Certicate of Analysis we supply a second certicate to assure absence of Bovine Spongiform
Encephalopathy and Foot-and-Mouth Disease in our raw
materials of animal origin.
Each certicate includes the following information:
Physical-chemical specications:
1. Of the medium before reconstitution:
Colour
Appearance
Particle size / homogenization
2. Of the reconstituted medium ready to use:
Clarity
Precipitates
Gelifying or solidifying strength
Growth control
Results obtained with control strains under optimal
conditions.
III

Quality in our labels
We have changed the design of our label to make it
more useful and modern.
Product name and directions appear now in several
languages with medium specication and formula in
English. Our labels are printed according to CE regulations for dangerous goods.
IV
Expiry date and batch number

Good Laboratory Practices (GLP) which are followed
by many of our customers, indicate that all products in
the laboratory must be labelled showing batch number,
expiry date and other additional data (date of reception,
opening date, opened by...)
Our labels contain batch number and expiry date and
also a specic area is reserved for the user to write
down additional data.
Safety Regulations
Material Safety Data Sheets
MSDS include physical and chemical data, handling recommendations, toxicological information and considerations relative to the environment, disposal, storage and
transport of each product.
In some cases, MSDS are delivered with the products but
it is better to get them in advance. We edit MSDS of our
products on CD-ROM and they are also available from
our web:
www.scharlau.com > MSDS
Storage and Transport

Storage and transportation of dangerous goods is subjected to special regulations due to the specic risks they
involve. All parts handling dangerous goods must adopt
the appropriate safety measures to avoid damages.
Safe storage
Dangerous goods must be stored under special safety
conditions to avoid health or environmental injuries. It is
always important to keep in mind chemical in compatibilities and to separate the products to avoid its mixture in
case of accident.
Every substance can be classied into a group of substances having similar hazards, that can be stored
together. The same security measures will be applied to
all the substances belonging to the same classication.
If a substance has several hazard degrees, it should be classied as per its highest one:
Explosive > Oxidising > Flammable > Toxic > Corrosive > Harmful
There are three basic criteria that should be kept in mind to guarantee a safe storage of chemicals in the laboratories:
Stock of dangerous products must be limited to the minimum quantity needed.
As a general rule, purchases should never exceed the quantity needed for one year, and preferably should cover a
period of between 3 and 6 months.
Products must be stored in groups depending on its chemical compatibility, as shown below:
EXPLOSIVES
OXIDIZING
FLAMMABLE
TOXIC
CORROSIVES
HARMFUL
EXPLOSIVES
YES
NO
NO
NO
NO
NO
OXIDIZING
NO
YES
NO
NO
NO
NO
FLAMMABLE
NO
NO
YES
NO
YES*
YES
TOXIC
NO
NO
NO
YES
YES
YES
CORROSIVES
NO
NO
YES*
YES
YES
YES
HARMFUL
NO
NO
YES
YES
YES
YES
*COULD BE STORED TOGETHER IF CORROSIVE PRODUCTS ARE CONTAINED IN NON FRAGILE BOTTLES
Some products, that are specially dangerous, should be isolated from the rest.
Carcinogenic or high toxicity substances should be stored in specic cabinets conveniently marked and closed by
key.
Special attention must be paid to the safe storage of ammable liquids, due to the big amounts that are usually
needed in the laboratories. They should be stored in security cabinets and security drums.
Storage regulations of dangerous goods indicate minimum distances that should be kept between different classes
and special storage conditions for each class. Our new warehouse was built following this and other EC regulations
regarding safety and risk prevention.
Safety. Regulations
Safe transportation
Dangerous goods can be transported outside manufacturing sites only if they are packed, labelled and delivered in safe conditions.
Packaging must be ofcially approved to contain dangerous goods. Strict tests are performed to evaluate
physical resistance to break.
Packaging must be labelled according to specic
regulations for transport of dangerous goods.
Goods must be accompanied by several documents
describing its hazard and how to proceed in case of
damage during loading, transporting or downloading
operations.
Dangerous goods cannot be transported by unauthorized transport means. There are different international
regulations that are applied to the transport by road,
air or sea. The following classes apply to all means of
transport:
CLASS
1
2
3
4.1
4.2
4.3
5.1
5.2
6.1
6.2
7
8
9
DESCRIPTION
Explosive substances
Gases
Flammable liquids
Flammable solids
Spontaneously ammable solids
Substances that develop ammable gases in contact with water
Substances that promote combustion (oxidants)
Organic peroxides
Toxic substances
Substances that induce vomiting or infection
Radioactive substances
Corrosive substances
Various hazardous substances
Transport regulations
By road / railway
ADR (Accord europen rlatif au transport international
des merchandises Dangereuses par Route)
This European agreement on the international transport of dangerous goods by road changed last July 1st
2001 and all data in this catalogue have been updated
according to this new version.
The former ADR 1999 can be used until the end of
2002, but, from January 1st 2003, only ADR 2001 will
be applied.
RID (Reglement International concernant le transport
des marchandises Dangereuses par chemin de fer)
Regulates the international transport of dangerous
goods by railway. The new ADR2001 includes all
requirements described in RID and could be used
instead.
VI
By air
IATA (International Air Transport Association) / ICAO
(International Civil Aviation Organization)
IATA regulations for transport of dangerous goods include all requirements from ICAO as well as additional
technical instructions. All data in this catalogue refer to
the 43rd edition of Dangerous goods regulation from
IATA.
You will also nd in this catalogue the packing instructions for passenger aircraft (PAX) and cargo aircraft
(CAO). The Packing Instruction indicates the conditions for the packaging, as well as the quantity authorized on board the aircraft. If transport of the substance
is forbidden, an F is printed instead of the PAX or
CAO data.
The packages that comply PAX conditions, can be
always transported on cargo aircraft.
IATA issues an ID number that is an identication
number for hazardous goods. It is only used in cases
where there is no UN number.
By sea
IMDG (International Maritime
Code for Dangerous Goods)
has been drawn by the IMO (International Maritime Organization of the UN) and is the regulation applied to any transport of
dangerous goods by see.
Other transport / security data

included in this catalogue:
UN number
The Committee of Experts of
the United Nations (UN) issues
recommendations for all freight
forwarders on the assessment
of hazardous goods for transport purposes. They are
numbered consecutively. These UN numbers are given
to each product so that everyone knows how to handle
it, no matter if it is going to be sent by road, air or sea.
In case there is no UN number, IATA gives an ID number
for air transport of hazardous goods.
EC index number (EC = European Community)
This number is issued by the EC commission for the
classication, packaging and marking of dangerous substances. The marking with hazard symbols, risk warnings (R phrases) and safety precautions (S phrases), as
used in this catalogue, is linked with this number.
Safety. Hazard categories

E: EXPLOSIVE
Are considered all products and preparations, if they can explode through ignition or if
they are more sensitive than dinitrobenzene
towards blows
and friction.
Precautions: Avoid impact, knocks, friction,
sparks, re and heat.
O: OXIDISING
Are considered all products and preparations, which not being ammable, can produce
and enhance res in contact with ammable
products.
Precautions: Avoid all contact with ammable substances. Risk of ignition! The substance promotes res once started and impedes
re ghting.
F+: EXTREMELY FLAMMABLE

Are considered all products and preparations, with a ash point below 0C and a boiling
point of 35C or below.
Precautions: Keep away from naked ames,
sparks and sources of heat.
F: FLAMMABLE
Are considered all products and preparations, if:
a) They heat up and nally start burning
in contact with air at normal temperature
without any external energy supply
b) They can start burning in solid condition
ater short contact with a source of ignition
and continue burning after the source has
been taken away,
c) They have a ash point below 21C in
liquid condition,
d) They form in gaseous condition an explosive mixture with air under normal pressure,
e) They create in contact with water or wet air
highly ammable gases.
Precautions: Keep away from naked ames,
sparks and sources of heat.
T+: VERY TOXIC

are considered all products and preparations,
if they lead to death or to acute or cronical
health
injuries when inhaled, swallowed or absorbed
through the skin in very small quantities.
Precautions: All contact with the human
body must be avoided, as severe or even
lethal damage to health cannot be excluded.
Particular attention is drawn to the carcinogenic, teratogenic or mutagenic risks associated
with certain substances.
T: TOXIC
Are considered all products and preparations,
if they lead to death or to acute or cronical
health injuries when inhaled, swallowed or
absorbed through the skin in small quantities.
Precautions: All contact with the human
body must be avoided, as severe or even
lethal damage to health cannot be excluded.
Particular attention is drawn to the carcinogenic, teratogenic or mutagenic risks associated with certain substances.
Xn: HARMFUL
Are considered all products and preparations, that if inhaled, swallowed or absorbed
through the skin can cause death or serious
acute or chronic effects.
Precautions: Avoid contact with the human
body, including the inhalation of vapours.
Injury to health is possible with improper use.
With some substances, carcinogenic, teratogenic or mutagenic action cannot be fully
excluded, as well as possible sensitization.
C: CORROSIVE
Are considered all products and preparations,
if they can destroy skin by contact.
Precautions: Take special measures to
protect the eyes, skin and clothes. Do not
inhale vapours.
Xi: IRRITANT
Are considered all products and preparations, if they can produce irritation in a short,
prolonged or repetitive contact with the skin
or the respiratory tract.
Precautions: Avoid contact with eyes and
skin, do not inhale vapours.
N: DANGEROUS FOR THE

ENVIRONMENT
for the Environment can be considered those
products and substances, which can have
detrimental effects upon the ecosystem
of water, soil or air, climate, fauna, ora or
microorganisms in such a way, that they constitute an immediate or future danger for the
environment.
Precautions: According to the kind of hazard do not dispose to wastewater, soil or
environment. Observe special disposal regulations!
VII
Safety. Risk and Safety Phrases

R: Risk Phrases
58
1
2
59
60
61
62
63
64
65
67
66
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
VIII
Explosive when dry.

Risk of explosion by shock, friction, re or other
sources of ignition.
Extreme risk of explosion by shock, friction, re or
other sources of ignition.
Forms very sensitive explosive metallic
compounds.
Heating may cause an explosion.
Explosive with or without contact with air.
May cause re.
Contact with combustible material may cause re.
Explosive when mixed with combustible material.
Flammable.
Highly ammable.
Extremely ammable.
Extremely ammable liqueed gas.
Reacts violently with water.
Contact with water liberates extremely ammable
gases.
Explosive when mixed with oxidising substances.
Spontaneously ammable in air.
In use, may form ammable/explosive vapour-air
mixture.
May form explosive peroxides.
Harmful by inhalation.
Harmful in contact with skin.
Harmful if swallowed.
Toxic by inhalation.
Toxic in contact with skin.
Toxic if swallowed.
Very toxic by inhalation.
Very toxic in contact with skin.
Very toxic if swallowed.
Contact with water liberates toxic gas.
Can become highly ammable in use.
Contact with acids liberates toxic gas.
Contact with acids liberates very toxic gas.
Danger of cumulative effects.
Causes burns.
Causes severe burns.
Irritating to eyes.
Irritating to respiratory system.
Irritating to skin.
Danger of very serious irreversible effects
Limited evidence of a carcinogenic effect.
Risk of serious damage to eyes.
May cause sensitisation by inhalation.
May cause sensitisation by skin contact.
Risk of explosion if heated under connement.
May cause cancer.
May cause heritable genetic damage.
May cause birth defects.
Danger of serious damage to health by prolonged
exposure.
May cause cancer by inhalation.
Very toxic to aquatic organisms.
Toxic to aquatic organisms.
Harmful to aquatic organisms.
May cause long-term adverse effects in the
aquatic environment.
Toxic to ora.
Toxic to fauna.
Toxic to soil organisms.
Toxic to bees.
68
May cause long-term adverse eects in the

environment.
Dangerous for the ozone layer.
May impair fertility.
May cause harm to the unborn child.
Possible risk of impaired fertility.
Possible risks of harm to the unborn child.
May cause harm to breastfed babies
Harmful: may cause lung damage if swallowed.
Vapours may cause drowsiness and dizziness.
Repeated esposure may cause skin dryness or
cracking.
Possible risk of irreversible effects.
Combination of particulars risks

14/15
Reacts violently with water, liberating extremely

ammable gases.
15/29
Contact with water liberates toxic, extremely
ammable gas.
20/21
Harmful by inhalation and in contact with skin.
20/21/22 Harmful by inhalation, in contact with skin and if
swallowed.
20/22
Harmful by inhalation and if swallowed.
21/22
Harmful in contact with skin and if swallowed.
23/24
Toxic by inhalation and in contact with skin.
23/24/25 Toxic by inhalation, in contact with skin and if
swallowed.
23/25
Toxic by inhalation and if swallowed.
24/25
Toxic in contact with skin and if swallowed.
26/27
Very toxic by inhalation and in contact with skin.
26/27/28 Very toxic by inhalation, in contact with skin and if
swallowed.
26/28
Very toxic by inhalation and if swallowed.
27/28
Very toxic in contact with skin and if swallowed
36/37
Irritating to eyes and respiratory system.
36/37/38 Irritating to eyes, respiratory system and skin.
36/38
Irritating to eyes and skin
37/38
Irritating to respiratory system and skin
39/23
Toxic: danger of very serious irreversible effects
through inhalation.
39/23/24 Toxic: danger of very serious irreversible effects
through inhalation and in
contact with skin.
39/23/24/25
Toxic: danger of very serious irreversible
effects through inhalation, in contact with
skin and if swallowed.
39/23/25 Toxic danger of very serious irreversible effects
through inhalation and if swallowed.
39/24
Toxic: danger of very serious irreversible effects in
contact with skin.
39/24/25 Toxic: danger of very serious irreversible effects in
contact with skin and if swallowed.
39/25
Toxic: danger of very serious irreversible effects if
swallowed.
39/26
Very toxic: danger of very serious irreversible
effects through inhalation.
39/26/27 Very toxic: danger of very serious irreversible
effects through inhalation and in contact with skin.
39/26/27/28
Very toxic: danger ol very serious
irreversible effects through inhalation,
in contact with skin and if swallowed.
effects through inhalation and if swallowed.
39/27
effects in contact with skin.

effects in contact with skin and if swallowed.
39/28
effects if swallowed.
40/20
Harmful: possible risk of irreversible effects
through inhalation.
40/20/21 Harmful: possible risk of irreversible effects
through inhalation and in contact with skin.
40/20/21/22
Harmful: possible risk of irreversible
effects through inhalation, in contact
with skin and if swallowed.
40/21
Harmful: possible risk of irreversible effects in
contact with skin.
40/21/22 Harmful: possible risk of irreversible effects in
40/22
Harmful possible risk of irreversible effects if
swallowed.
42/43
May cause sensitisation by inhalation and skin
contact.
48/20
Harmful: danger of serious damage to health by
prolonged exposure.
48/20/21 Harmful: danger of serious damage to health by
prolonged exposure
48/20/21/22
Harmful: danger of serious damage to
health by prolonged exposure
through inhalation, in contact with skin
and if swallowed.
prolonged exposure through inhalation and if
swallowed.
48/21
prolonged exposure in contact with skin.
prolonged exposure in contact with skin and if
swallowed.
48/22
prolonged exposure if swallowed.
48/23
Toxic: danger of serious damage to health by
prolonged exposure through inhalation.
48/23/24 Toxic: danger of serious damage to health by
prolonged exposure through
inhalation and in contact with skin.
48/23/24/25
Toxic: danger of serious damage to
health by prolonged exposure through
inhalation, in contact with skin and if
swallowed.
prolonged exposure through
inhalation and if swallowed.
48/24
prolonged exposure in contact with skin.
prolonged exposure in
48/25
prolonged exposure if swallowed.
50/53
Very toxic to aquatic organisms, may cause longterm adverse effects in
the aquatic environment.
52/53
Harmful to acquatic organisms, may cause longterm adverse effects in the acquatic environment.
51/53
Toxic to acquatic organisms, may cause long-term
effects in the aquatic environment.
68/20
Harmful: possible risk of irreversible effects

through inhalation.
68/21
Harmful: possible risk of irreversible effects in
contact with skin.
68/22
Harmful: possible risk of irreversible effects if
swallowed.
68/21/22 Harmful: possible risk of irreversible effects in
68/20/21/22
Harmful: possible risk of irreversible
effects through inhalation, in contact
with skin and if swallowed.
S: Safety phrases
1
2
3
4
5
5.3
6
7
8
9
12
13
14
14.1
14.2
14.9
15
16
17
18
20
21
22
23
23.2
24
25
26
27
28
28.1
28.2
28.3
28.6
Keep locked up.

Keep out of reach of children.
Keep in a cool place.
Keep away from living quarters.
Keep contents under _ (appropriate liquid to be
specied by the manufacturer)
Keep contents under parafn oil.
Keep under (inert gas to be specied by the
manufacturer)
Keep container tightly closed.
Keep container dry.
Keep container in a well ventilated place.
Do not keep the container sealed.
Keep away from food, drink and animal feeding stuffs.
Keep away from _ (incompatible materials to be
indicated by the manufacturer)
Keep away from alkalis.
Keep away from oxidizing and acidic substances
as well as heavy metal compounds.
Keep away from ammable organic substances.
Keep away from heat.
Keep away from sources of ignition - No Smoking.
Keep away from combustible material.
Handle and open container with care.
When using do not eat or drink.
When using do not smoke.
Do not breathe dust.
Do not breathe gas/fumes/vapour/sray
(appropiate wording to be
specied by the manufacturer).
Do not breathe vapour.
Avoid contact with skin.
Avoid contact with eyes.
In case of contact with eyes, rinse immediately
with plenty of water and seek medical advice.
Take off immediately all contaminated clothing.
After contact with skin, wash immediately with
plenty of _ (to be specied by the manufacturer).
plenty of water.
soap and water.
soap and water, if possible
also with polyethylene glycol 400.
polyethylene glycol 400
(then rinse with plenty of water).
IX

29
30
33
34
35
36
37
38
39
40
41
42
43
43.1
43.3
43.6
43.7
43.8
44
45
46
47
48
49
50
50.1
51
52
53
54
55
56
57
58
59
60
Do not empty into drains.

Never add water to this product.
Take precautionary measures against static
discharges.
Avoid shock and friction.
This material and its container must be disposed
of in a safe way.
Wear suitable protective clothing.
Wear suitable gloves.
In case of insufcient ventilation, wear suitable
respiratory equipment.
Wear eye/face protection.
To clean the oor and all objects contaminated by
this material use _ (to
be specied by the manufacturer).
In case of re and/or explosion do not breathe
fumes.
During fumigation/spraying wear suitable
respiratory equipment
(appropriate wording to be specied)
In case of re, use _ (indicate in the space the
precise type of re-lighting
equipment. If water increases the risk add - Never
use water)
In case of re, use water.
In case of re, use powder extinguisher. Never
use water.
In case of re, use sand. Never use water.
In case of re, use metal-re powder. Never use
water.
In case of re, use sand, carbon dioxide or
powder extinguisher. Never use water.
If you feel unwell, seek medical advice (show the
label where possible)
In case of accident or if you feel unwell, seek
medical advice immediately
(show the label where possible)
If swallowed seek medical advice immediately
and show this container or label
Keep at temperature not exceeding _ C (to be
Keep wetted with _ (appropriate material to be
Keep only in the original container.
Do not mix with _ (to be specied by the
manufacturer)
Do not mix with acids.
Use only in well ventilated areas.
Not recommended for interior use on large
surface areas.
Avoid exposure - obtain special instruction before
use.
Obtain the consent of pollution control authorities
before discharging to wastewater treatment plants.
Treat using the best available techniques before
discharge into drains or
the aquatic environment.
Dispose of this material and its containes at
hazardous or special waste collection point.
Use appropriate containment to avoid
environmental contamination.
To be disposed of as hazardous waste.
Refer to manufacturer/supplier for information on
recovery/recycling
This material and its container must be disposed
of as hazardous waste.
61
62
63
64
Avoid release to the environment.Refer to special

instructions/Safety data sheets.
If swallowed, do not induce vomiting: seek
medical advice immediately
and show this container or label.
In case of accident by inhalation: remove casualty
to fresh air and keep at rest.
If swallowed, rinse mouth with water (only if the
person is conscious).
Combination of safety precautions

1/2
3/7
3/7/9
Keep locked up and out of reach of children.

Keep container tightly closed in a cool place.
Keep container tightly closed, in a cool well
ventilated place.
3/9
Keep in a cool well ventilated place
3/9/14 Keep in a cool, well ventilated place away from _
(incompatible materials
to be indicated by the manufacturer)
3/9/14.1 Keep in a cool, well ventilated place away from
alkalis.
3/9/14/49 Keep only in the original container in a cool, wellventilated place away from ... (incompatible
materials to be indicated by the manufacturer).
3/9/49 Keep only in the original container in a cool, wellventilated place.
3/14
Keep in a cool place away from ... (incompatible
materials to be indicated by the manufacturer).
7/8
Keep container tightly closed and dry.
7/9
Keep container tightly closed and in a wellventilated place.
20/21
When using do not eat, drink or smoke.
24/25
Avoid contact with skin and eyes.
27/28
After contact with skin, take off immediately all
contaminated clothing, and wash immediately with
plenty of ... (to be specied by the manufacturer).
29/35
Do not empty into drains; dispose of this material
and its container in a safe way.
36/37
Wear suitable protective clothing and gloves.
36/37/39 Wear suitable protective clothing, gloves and eye/
face protection.
36/39
Wear suitable protective clothing and eye/face
protection.
37/39
Wear suitable gloves and eye/face protection.
47/49
Keep only in the original container at temperature
not exceeding _ C (to be specied by the
manufacturer).
Safety. Disposal
Disposal of Laboratory Waste
Avoid contamination of the water
Elimination of residue through the drain is strictly forbidden. You have to bear in mind, that many chemicals
shall not be reduced by the waste water plants and shall
contaminate the environment.
Residue in the laboratory
We highly recommend naming a person in charge of the
residue of your company. This person should be informed of the laws and regulations regulating the waste
of residue in your own country.
In order to hand over the residue to a treatment plant,
it is necessary to organize the collection and storage
previously.
Collection of residue in the laboratory
The material of the residue containers has to comply
with the following conditions:
1. One has to be able to close the containers hermetically and the material has to be resistant to the contents. Plastic containers should not form toxic fumes
when disposed of through burning.
2. Generally, plastic containers are being used.
3. Corrosive products should be collected in metal
drums with inner plastic lining, like our Combi drum
(Ref. 055-0C0025).
4. Inammable or oxidizing products should be collected in metal or plastic drums resistant to the solvent.
5. For products that produce gases or vapours, special
containers with a security valve are required, in order
to avoid the danger of explosions.
If the collection containers are handed over to a transport agency, they have to carry the UN number that
approves them for transport by road.
Hazardous chemicals
In this handbook risk phrases (R-phrases), safety
phrases (S- phrases) and hazard symbols are added in
accordance with the EU Directive (67/548) and (83/467).
The absence of R- and/or S-phrases or hazard symbols
does not mean that those substances are harmless.
The normal safety precautions for handling chemicals
should always be observed.
General Waste Disposal procedures

The disposal methods outlined below are intended as a
guideline for the technician. We do not assume responsibility of their use. Careful consideration must be given
to the chemical and physical properties of the substance.
In addition, local laws and regulations may preclude the
use of these methods which were primarily designed
for the quantities we list. Observe all Federal, State and
local laws concerning health and pollution.
All procedures require the use of goggles or face shield,
gloves and apron. Gas masks or self- contained breathing units may also be required.
1. Neutralize the bases with diluted sulphuric acid, then
ush neutral solution down the drain with an abundance of water.
2. Treat the aldehydes or peracids with an excess of
sodium bisulte solution. Use starch iodide paper to
ensure complete reduction of any peracid. Flush the
resulting solution down the drain with an abundance
of water.
3. The compound should be burned. Ideally, all hydrocarbons and related solvents should be burned in an
approved presence of sodium carbonate and calcium hydroxide (slaked lime). The solid or the liquid
absorbed on vermiculite should be wrapped in paper
and burned in an approved incinerator.
4. Bury in an approved chemical and hazardous-waste
sand ll site, well away from streams, rivers or wells.
5. Azides and azo-compounds should be treated with
dilute ceric ammonium nitrate solution with cooling. Solutions may be discarded upon completion of
the reaction. Mixtures should be separated and the
organic portion burned.
6. Treat with an alcohol such as ethanol or butanol in
an appropriate solvent. The chemical reaction may be
vigorous and/or exotermic. Provisions must be made
for venting of large volumes of highly ammable
hydrogen and/or hydrocarbon gases. Neutralize the
solution with aqueous acid. Filter off any solid residue
for disposal as hazardous waste. Burn residual
organic materials in an incinerator equipped with an
afterburner and scrubber.
XI
Safety. Disposal
7. Neutralize the base with dry sodium bisulfate add
water slowly and ush the neutral solution down the
drain with an excess of water.
8. Carefully mix the acidic compound with dry sodium
bicarbonate. Dilute slowly and ush down the drain
with an excess of water.
9. Oxidize the mercaptan or nitrite with a weak aqueous
solution (up to 15%) of sodium or calcium hypoclorite.
Vigorous stirring may be required to ensure completion of the reaction. Neutralize the resulting mixture,
then discard down the drain with an excess of water.
10. Flush down the drain with an excess of water.
11. Bury in a site approved for disposal of poisonous or
hazardous wastes.
12. Refer to our Material Safety Data Sheet before handling or disposing.
Caution regarding hazardous

components
Some media contain components that are toxic or carcinogenic. Appropriate safety precautions must be taken
when using media with such components.
Basic fuchsin and acid fuchsin are carcinogens and
caution must be used in handling media with these compounds to avoid dangerous exposure that could lead to
the development of malignancies . Thalium salts, sodium
azide, sodium biselenite and cyanide are among the
toxic components found in some media. These compounds are poisonous and steps must be taken to avoid
ingestion, inhalation, and skin contact. Azides also react
with many metals, especially copper, to form explosive
metal-azides. The disposal of azides must avoid contact with copper or achieve sufcient dilution to avoid
the formation of such hazardous explosive compounds.
Media with sulphur containing compounds may result in
the formation of hydrogen sulphide which is a toxic gas.
Care must be used to ensure proper ventilation. Media
with human blood or human blood components must be
handled with great caution to avoid exposure to human immunodeciency virus and other pathogens that
contaminate some blood supplies. Proper handling and
disposal procedures must be followed with blood-containing as well as other media that are used to cultivate
microorganisms.
XII

Following instructions provide the correct guidelines for
the manipulations in microbiology. Every microbiological
laboratory is responsible for their proper implementation.
A. Personal techniques
A.1. Hygiene is the basic element and need for the safe
manipulation.
A.2. All manipulated microorganisms must be presumed
potentially pathogenic, regardless of their nature.
The following procedures or techniques must
therefore be avoided : oral pipetting, application
of cosmetics ,eating, drinking or smoking in the
laboratory. Wrappings and labels in the labortory
should be self-adhesive.
A.3. Manipulation of the microorganisms should be
conducted in such a way that will not provoke
any environmental contamination or hazard. For
example, sprays should always be avoided and
pipettes must be submerged immediately in the
disinfectant after use.
A.4. Manipulators (technicians) must be aware of the
risks and should know how to prevent them.
A.5. Manipulators must make sure that both, the user
and the equipment itself are perfectly safe from
any potential risk.
B. Equipment in general
B.1. A standard equipment for the sterilization of any
potentially hazardous material must be available
(autoclave, jars with disinfectant for the pipettes,
dry heat ovens etc).
B.2. Protective clothes must be used exclusively in the
working area, and should never be worn while in
public areas.
B.3.Toilets and other facilities must be available close
to the working areas, and separate from those of
use to the general public.
B.4. Any device which may probably produce the sprays
must be always handled in microbiological security cabins like Laminar Air Flow .
B.5. In case of any accident, all necessary substances
and equipments , either to minimize or neutralize
the risks must be readily available.
C. Basic characteristics of the

laboratory
C.1. The laboratory or plant should not be located in a
passage or corridor so that any outside person
would always remain away from it.
C.2. It must be equiped with an appropiate ventillation
system, mechanically regulated if possible, so as
to create a drift of owing air from the areas of
low risk to the contaminated areas, and then going outside, through the HEPA lters.
C.3. The laboratory must be built of easily washable,
waterproof and disinfectant-resistant surfaces.
D. Administrative procedures
D.1. Systems to conrm and assure the effectiveness of
the biological security code carried out by qualied personnel.
D.2. Training programmes regarding the security during
or at work, adapted to the level required by every
maninpulator / technician.
D.3. Adequate medical surveillance and facilities .
D.4. Procedures for the verication and maintenance
of the ventilation system and the equipment in
general.
D.5. Emergency procedures and systems for use in case
of any accident.
D.6. Strategies to restrain the freak entries and exits, to
maintain the security standards of laboratory and
the plant.
D.7. Procedures for the transportation, postage and
reception of the biological material (postage
regulations and related things).
D.8. Training of personnel to act as security ofcers.
D.9. Systems to eliminate any potentially hazardous
residues.
References
Report of Committee of Enquiry into Smallpox (1974)
Outbreak in March and April 1973. Cmnd.5626, Her
Majestys Stationery Ofce.
Report of the Working Party on the Laboratory Use of
Dangerous Pathogens. (The Godber Report) (1975).
Cmnd. 6054, Her Majestys Stationery Ofce.
Report of the Working Party on the Experimental Manipulation of the Genetic Composition of Micro-organisms.
(1975) Cmnd. 5880, Her Majestys Stationery Ofce.
PUBLIC HEALTH LABORATORY SERVICE (1980)
Safety Precautions-Notes for Guidance. Colindale,
MILLER, B.M. (1987) Laboratory Safety: Principles and
Practices. ASM. Whasington, D.C.
HARTREE, E., U. BROTH (1977) Safety in Biological
Laboratories. Biochem. Soc. Spec. Pub. n5. The Biochemical Society. London
COLLINS, C.H., E.G. HARTLEY, R. PILSWORT (1976)
The Prevention of Laboratory Adquired Infection. PHLS
Monograph n6. Her Magesty Stationery Ofce.
SMITH, J.A. (1996) Laboratory Safety in Clinical Microbiology. Cumitech n29. ASM. Whasington, D.C.
WHO (1983) Laboratory Biosafety Manual. Geneva.
The appearance and quality of the prepared medium
usually depends on the method used for rehydration
and storage. It is therefore very important to follow the
recommendations stated below.
XIII

Rehydration
Directions for the individual reconstitution are detailed
on the container of every media and must be strictly
followed.
The water used for reconstitution purposes must be
distilled or deionized, of pharmaceutical grade. The
solution or suspension must be completely homogeneous and the exposure to heat if necessary, should be
minimal.
In any case, it is always helpful to heat the water previously to 50-60C, to help dissolve the media.
For the preparation of the culture media the most recommended technique is the following:
Add necessary amount of powder to half the volume
of water needed. The water should have already been
warmed to 50-60 C. Stir the mixture thoroughly until the
powder is dissolved. Use rest of the water to dissolve the
powder sticking to the walls or sides of the container and
continue stirring and heating if necessary. Heat must
be applied directly and smoothly. Constant agitation will
prevent the components from sticking to the walls of the
container. Use a container with a capacity of approximately 2 to 3 times more the volume of the medium, to
allow plenty of space for manipulation.
Sometimes, It is necessary to boil the medium for more
than 1 minute. In such cases, the volume of evaporated
water must be restored.
Media not containing the agar or any solidifying agents
(broths and Nutrient Solutions) tend to dissolve easily and rapidly in preheated water, however sometimes
boiling is necessary to attain the complete dissolution.
These media have a tendency to form a concentrated
syrup or suspension which settles at the bottom of the
container. This thick suspension usually remains at the
bottom, risking the destruction of the medium contents
by hydrolysis, caramelization and pH drift when the heat
is applied. Therefore, thorough mixing should be assured before heating.
Media containing solidifying agents (Agars and Fluid
Media) must be preheated before sterilization. Media
containing agar must be brought to the boiling, since
agar is insoluble in water below 98-100 C. It is useful to
let the agar soak in preheated water for 5 to 10 minutes
before heating, in order to allow it to swell and to secure its
solubility and uniform distribution and dissolution.
A highly recommended
procedure can be practised
by removing the rehydrated
medium from the source of
heat when the mixture begins
to boil, letting it stand for a
while and then quickly bringing it to the boiling again. This
may be repeated 2 or 3 times
with constant agitation. The
complete dissolution of the
agar will be indicated by the
absence of granules in the
container.
Dissolution of culture media
directly during sterilization in
the autoclave is a frequent
and totally incorrect practice.
It alters the mediums qualities and frequently causes
the uneven dissolution of the
agar into layers with different
concentration gradients, pH
drifts and browning.
Nowadays, heating the rehydrated medium in a microwave
oven in order to melt the agar
XIV

or dissolve the medium components is a very common
procedure. In this case also, it is recommended to let
the agar-medium to soak for some time before using
the microwave and use the containers suitable for the
volume of medium to be prepared. Using a microwave
with 800 W for 4 minutes are enough to achieve the total
dissolution or melting of agar. However, it must be noted
that since this is a static procedure, a concentration gradient will appear which will create a stratication. Thus,
it may be necessary to shake the rehydrated medium
vigorously to homogenize the solidifying agent before
using or sterilizing it in the autoclave, in the same way
as if it were an autosterile medium. Microwave heating
can never be adopted as a substitute for autoclave
sterilization.
dium is introduced in the autoclave , and to let the temperature drop to 70-80C before removing the medium
so as to avoid severe temperature uctuations. Although
the use of cold water to cool media is widespread, it is
not recommended for media containing agar since it
causes akes and cloud formation.
Dehydrated culture media usually maintain their features

if reconstituted properly. Nevertheless, verication is
encouraged since some of them (colour, pH, etc) may
slightly change subject to the conditions of reconstitution
and the type of water used.
These recommendations are particularly important when
referring to uid media, since such media contain small
amounts of solidifying agent and are therefore very delicate. Cooling becomes as important as heating, as any
violent process may induce the formation of akes and
clouds. A slow and smooth cooling process is recommended.
Media containing phosphate buffers together with glucose or other carbohydrates may darken if overheated
as a concentrated solution. Precipitates may also appear
when using poor quality water.
Special attention must be paid to all media containing
agar at a pH under 5, since it hydrolyzes and looses the
ability to form gels. In those cases, unnecessary heating
must be avoided.
Sterilization
The indications given on every container must be followed, while bearing in mind that they refer to quantities
of up to 1 litre. As for bigger volumes, autoclaving and
heat penetration conditions of the medium will have to
be taken into consideration.
In all the cases autoclaving must be monitored regularly by the manometers and thermometers, as well
as the even distribution of heat. Nowadays this can be
done mechanically (thermocouples), chemically (thermal indicators) or biologically (thermoresistent spores).
Considering that overheating is one of the main causes
of culture media alteration, it is only natural to prevent,
whenever possible, the treatment of large quantities of
media and the prolonged exposure to heat. For large
autoclaves it is recommended to preheat before the meXV

Containers used for sterilization purposes must have a
large head space for air to allow foaming. If screw-cap
containers are used,the closures must be a half turned
or screwed to allow inner and outer pressure balancing.
All containers should be chemically inert to prevent alteration of the pH of contents. Borosilicate glass containers are highly recommended.
Media containing carbohydrates darken a little after heat
sterilization, therefore, sterilization should be carried out
at a temperature below 120C if possible.
Should autoclave not be available, sterilization can be
done in a pressure vessel (at 100C for 30 minutes). In
such case it is useful to resterilize the medium during
three days, if the medium allows the treatment.
Some media containing selective substances do not
need sterilization. The preparation process is quicker
since they are ready for use after dissolution. Since
these media are usually not very stable and preparation
is brief and easy, the exact quantities required should be
prepared.
Additions to the medium (if any) after sterilization must
be done aseptically, that is, additives should have been
previously sterilized. Additives are generally thermolabiles if not they would be part of the dehydrated mediums
composition. It is therefore necessary to allow the medium to cool to 50-60C before their addition, so as not to
harm the additive while permitting its correct distribution.
Once the addition is over, reheating must be avoided.
Distribution into nal containers must be completed
before sterilizing, to avoid any manipulation. Since it is
rarely possible to do so, manipulation should be done
in safety cabinets instead. The sterile cabinets or rooms
are not be exposed to any radiations and so, no any
kind of highly active reagent could affect the mediums
components. Excessive steam condensation can be
eluded by distributing the medium at temperatures close
to solidication, ranging from 45 to 50C.
Storage of dehydrated media

Dehydrated culture media should be stored safely, protected from humidity or moisture, light and heat, which
are the most frequent causes of their alteration.
SCHARLAU media are supplied in opaque, waterproof,
screw-cap plastic bottles, with an internal relief which
eliminates the need for an intermediate seal. Nevertheless a hermetic seal is assured by accurately tting
the cap and keeping both its sides and the border of its
mouth clean.
It is important to open the bottles in dry atmosphere
and close them immediately after their use. Refrigeration is not necessary even if low temperatures prolong
the mediums effectivity. The nature of the containers
of SCHARLAU make culture media are suitable for
prolonged storage in cool, dry places. If the media are
moist they become stiff and hard, lose their properties
XVI
and can even allow direct growth of microorganisms in

them. Under such circumstances they must not be used.
Although they usually have a dry and powdery appearance, the media may occasionally appear moist, oily but
never stiffened, without there being any alteration in their
composition (as e.g.in MRS, Tributyrin Agar, etc).
In any case, storage of dehydrated media is not indenite. It is not recommended to store large quantities
but to keep small amounts in stock, so as to maintain a
rotation of ready prepared dehydrated media. Registering the reception dates on the containers will avoid the
accumulation of older media.
Storage of ready
prepared culture media
Although it is best to prepare the media as you required,
it is common to store them ready prepared and sterilized
in order to spare preparation time.
Under these circumstances the biggest drawback is the
dehydration and every precaution taken to avoid it will
prolong the mediums useful life, which is usually between 4 and 6 weeks. We therefore recommend the use
of hermetically sealed screw-capped containers (either
bottles or tubes) instead of those plugged with cotton
wool. A moderate refrigeration (4C) usually prolongs
the life of the medium but it should also be noted that a
refrigerated environment is very dry and consequently
renders dehydration. In some cases, as regards in
thioglycolate media and almost all media recommended
for anaerobes, they are best stored at room temperature
for the less penetration of air. Direct light must always
be avoided, particulary when it is specically indicated
on the label of the medium.
Solid media for the plates are best stored in their original
containers than in the poured plates. Nevertheless,
poured plates may also be prepared and stored if the
indications below are followed:
a) Refrigerate the media immediately after solidication
while incubating controls to check the sterility of
the batch.
b) If the media are to be stored for a longer period,
plates should be individually sealed with a waterproof
seal and, if possible, stored in single containers.
c) If storage is to last more than a few days, the plates
should be wrapped in plastic bags to avoid dehydration of the media.
Since condensation water will inevitably appear, we suggest to store the poured plates inverted. Contamination
with condensation water can be avoided by pouring the
molten medium on the plates as cold as possible.
Media which have been stored in refrigeration should
be allowed to warm up gradually. It is recommended to
keep them at room temperature for a few hours before
inoculation, allowing misted containers to clear and
avoiding the delayed initiation of growth (or eventually its
total failure).

Stored ready prepared media which may involve a high
level of dehydration should never be used. Dehydration is indicated by excess condensation of water or
by a decrease in volume of liquid media; an increase of
concentration or precipitation of uid and liquid media;
or retraction, cracking and precipitation of solid media.
Plates with very dry surfaces or those which appear to
have changed colour should not be used either.
Remelting solid media

Although most solid culture media can be stored ready
prepared and sterilized, and can be remelted and poured
into plates whenever necessary, this procedure should
not be applied to media which do not need sterilization
or those with a pH 5 or less, since it would alter their
properties.
Remelting is done in a boiling water bath or by uent
steam autoclaving for 30 minutes. Do not apply direct
heat. However, it must be emphasized that remelted
media are prone to precipitation and darkening if held in
a molten state for more than 1 hour and at temperatures
ranging from 45 to 65C. Such media may also undergo
nutritional impairment to the desired microorganisms.
It is therefore recommended to avoid remelting of solid
media whenever possible.
One of the quickest and most recommended remelting methods for culture media is the use of microwave
ovens. In this case all the precautions exposed must be
carefully observed and practised in the media which are
labile and consequently radiation and minimum intensity
should be dosed for the shortest time intervals.
A usual and erroneous practice consists of imposing
strong radiation intensities for short periods, which results in partial remelting, sudden boiling and overowing
of the medium and an overall alteration of its properties.
Differentiation of culture media

SCHARLAU names for culture media are based on their
nal consistency or appearance. Thus, a medium is
named as:
Agar: Solid media with an agar content of 1% or more.
Fluid Medium (FM): Semisolid media with an agar content of less than 1%.
Broth: Liquid culture media with undened organic components (peptones, organ and tissue extracts,
etc...)
Nutrient Solution: Culture media with dened chemical
composition.
Selective Medium: Medium that allows the growth of a
biotype or a few biotypes between those that are
present in the inocule.
Differential Medium: Medium that allows to distinguish
or differentiate between similar culture types.
Enrichment Medium: Medium that aids the growth of a
dened biotype over the rest in the inocule.
General Medium: Medium that supports the growth of

a very wide range of microorganisms without
special nutrient needs.
Membrane Filter (MF): Media used when the Membrane Filter Technique is employed.
Membrane Filter Media

Utilization of the Membrane lter technquies has offered
some undoubted advantages to the Microbiology which
has certain specic problems. Membrane ltration allows
to examine big samples (in volume of liquid) with very
low microorganisms concentration or load, to separate
the microorganisms from the culture medium and even
the exchange of the microorganisms between two media
without affecting their growth.
Moreover, the MPN technique is more precise in these
media and also, the membrane with the colonial growth
may be stored and led.
The membrane ltration system is accepted by most
pharmacopeias as an alternative or the single method to
examine antimicrobial or with strong inhibitors substances, since a proper wash of the lter remove all the
substances that interfere with the microbial growth.
On other hand, the wide range of lters and qualities
available allows the examination of any product, since
you may nd membranes resistant to almost all the dissolvents.
Technique
Essentially, the technique consists of ltering the sample
through a lter with a suitable porosity (0,22 microns
for bacteria and 0,45 microns for fungi) with pressure
or suction, in such a way that the microorganisms are
retained on the membrane.
If the uid that has been ltered contains inhibitors, wash
the membrane several times with rinse liquid to remove
them. Membrane is removed aseptically and it is taken
and placed on the culture medium.
For sterility controls, the membrane is incubated directly
in the classical media. For these tests refer: Thioglycolate Broth (Ref. 2-186), Thioglicolate USP Fluid Medium
(Ref. 3-187), Tryptone Soy Broth (Ref. 2-200), Sabouraud USP Broth (Ref. 2-165).
Should an enumeration of colonies be desired, incubate
the lter on a solid medium or on a pad soaked in liquid
medium, but beware that the lower surface touches the
culture medium and that there are no air bubbles or air
gap in between.
Usually, general media may be used for these techniques. However, there are several culture media specically developed for these techniques, specially in the
water Microbiology.
XVII
Culture media manufacture process ow chart
Acetamide Medium
Ref. 03-428
Xn
R-40
S-36/37
Specication
Liquid medium for the enrichment and conrmative test

of presence of Pseudomonas aeruginosa in water acc.
EN 12780:2002 and ISO 16266 standard.
Formula (in g/L)

Acetamide ............................................ 2,0000
Magnesium sulfate, anhydrous ............ 0,2000
Sodium chloride .................................... 0,2000
Ferrous sulfate ..................................... 0,0005
Monopotassium phosphate .................. 1,0000
Sodium molybdate ................................ 0,0050
Final pH 7,0 0,5
Directions
Dissolve 3,4 g of powder in 1 L of distilled water. Heat
only if necessary. Sterilize in the autoclave at 121C for
15 minutes. Prepared medium may be opalescent and
with precipitate. The prepared medium remain active for
3 months if it is stored in the dark in a cool place.
Description
This nutrient solution has acetamide as the unique
carbon and nitrogen source, and it allows the growth of
only those microorganisms that are able to use acetamide. In water and in almost all the food materials, these
microorganisms are the non fermenting gramnegatives,
thus Pseudomonas aeruginosa is the only one that can
liberate ammonia by deaminating acetamide.
Some authors suggest the use of this nutrient solution as

a previous enrichment medium before the isolation medium, especially if you are studing very polluted samples
with companion ora.
Technique
Medium is inoculated with a couple of loops from the
culture or inoculum to be assayed and is incubated at
32-35C for 24-48 hours before proceeding for the isolation medium.
To conrm Pseudomonas aeruginosa, inoculate a loop
of culture inoculum in Asparagine Broth (Ref. 02-271)
and incubate at 35-37C for 24 hours. After this period,
pour 1 or 2 drops of Nesslers Reagent (Ref. 06-084) on
to the culture and observe for ammonia production: a
change in colour to yellow indicates ammonia production
and thus the presence of Pseudomonas aeruginosa .
References
EN 12780 Standard (2002) Water Quality-Detection and
enumeration of Ps.aeruginosa by membrane ltration.
CEN. Brussels.
DIN Standard 3841. Deutsche Einheitsverfahren zr
Wasser, Abwasser und Schlammuntersuchung Mikrobiologische Verfahren: Nachweiss von Pseudomonas
aeruginosa (K8).
KELLY, N.M., C.T. KEANZ (1983) Acetamide Broth for
Isolation of Pseudomonas aeruginosa from patients with
cystic brosis. J. Clin. Microbiol. 17:159-163.
ISO 16266 (2006). Water Quality. Detection and enumeration of Pseudomonas aeruginosa. Method by
membrane ltration.
Ref. 03-428 Acetamide Medium. Left: control; center:

Pseudomonas aeruginosa ATCC 27853; right: Pseudomonas aeruginosa ATCC 9027.
3
Actinomycete Media
Actinomycete Agar
Ref. 01-003
Specication
Solid culture medium for actinomycete maintenance and
propagation according to Ajello et al. formulation.
Formula (in g/L)

Meat peptone ......................................... 10,00
Casein peptone ........................................ 4,00
Heart extract ........................................... 10,00
Yeast extract ............................................. 5,00
Dextrose ................................................... 5,00
Potassium phosphate ............................. 15,00
Sodium chloride ........................................ 5,00
Starch ....................................................... 1,00
Ammonium sulfate .................................... 1,00
Cysteine ................................................... 1,00
Magnesium sulfate ................................... 0,20
Calcium chloride ....................................... 0,01
Agar ........................................................ 20,00
Final pH 7,0 0,2
Directions
Suspend 77 g of powder into 1 L distilled water and let it
soak. Bring to boiling with constant stirring and distribute
into suitable containers. Sterilize by autoclaving at 121C
for 15 minutes.
Actinomycete Broth
Ref. 02-003
Specication
Liquid culture medium, able to form a uid medium, for
the pathogenic actinomycete maintenance and propagation, according to Ajello et al. formulation.
Formula (in g/L)

Meat peptone ......................................... 10,00
Casein peptone ........................................ 4,00
Heart extract ........................................... 10,00
Yeast extract ............................................. 5,00
Dextrose ................................................... 5,00
Sodium chloride ........................................ 5,00
Starch ....................................................... 1,00
Cysteine ................................................... 1,00
Final pH 7,0 0,2
Directions
Dissolve 57 g of powder into1 L of distilled water. Heat if
necessary. Distribute into suitable containers and sterilize by autoclaving at 121C for 15 minutes.
Should a uid medium be required, incorporate 7 g of
Agar and follow the directions for the solid medium. After
the sterilization, cool it quickly but gently to avoid agar
occulation.
Description
Basically Pine and Watson formulated this medium for
the maintenance of pathogenic actinomycytes which was
then further modied by Ajello et al. Those media do not
possess any inhibitory agents, and so are not recommended for the isolation, but their slight reducing power
and their high nutritive capacity make them suitable for
facultative anaerobes also.
Technique
If growth conditions require strict anaerobiosis, the incubation must be done in special asks, but if the conditions are only moderate or not strict anaerobic, a good
anaerobiosis can be obtained by sealing the asks with
Sealing Anaerobic Agar (Ref. 01-174) or with the classic
alkaline pyrogallate caps.
Best results are obtained using an Anaerobic Jar with a
CO2 mixture generator.
The use of a liquid medium allows freezing of the cultures after their growth and the use also as a massive
production system for cellular preparations. In the uid
format, the Actinomycete Broth reaches a high reduction
potential allowing an easy growth to strict anaerobics.
There is only one difference between solid and broth
formulation which is the addition of agar 20,0 g/L to the
solid medium as a solidifying agent, providing the sufcient strength to support the surface growth.
References
AJELLO, GEORG, KAPLAN & KAUFFMAN (1963) CDC
Lab Manual for Medical Mycology, Publication n994 US
Govt. Prmtmg. Ofce. Washington.
LENNETTE, SPAULDING & TROULANT (1974) Manual
of Clinical Microbiology. ASM 2d. Ed. Washington.
PINE, L., A.HOWELL, S.J.WATSON (1960) Studies on
the morphological characters of Actynomyces bovis. J.
Gen. Microbiol. 23:403-424.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. London.
AFP Agar (Aspergillus avus/parasiticus Agar)

Ref. 01-405
T
R-45
S-45-53
Specication
Selective solid medium for the isolation and enumeration

of Aspergillus avus and Aspergillus parasiticus.
Formula (in g/L)

Peptone ................................................ 10,000
Yeast extract ......................................... 20,000
Ammoniun Iron Citrate .......................... 0,500
Dichloran ................................................ 0,002
Chloramphenicol .................................... 0,100
Agar ...................................................... 15,000
Final pH 6,3 0,2
Directions
Suspend 45,6 g of powder in 1 L of distilled water and
bring to the boil. Distribute in suitable containers and
sterilize in autoclave at 121C for 15 minutes.
Description
AFP Agar is a modication of the original medium developed by King, Hocking & Pitt for the enumeration and
isolation of moulds in foods. The inclusion of ammonium
iron citrate and the high temperature (30C) of incubation
enhances the quickly typical pigmentation in Aspergillus
avus and A. parasiticus.
Technique
The sample is inoculate on the surface of the AFP Agar
ad the plates are incubated at 30C for 42-48 hours.
Count the fungal colonies that shows the typical yelloworange pigmentation on the reverse. Express the results
as Aspergillus avus/parasiticus colonies per g or mL of
sample.
In this conditions Aspergillus orizae can produce a very
similar pigmentation than induce error in the counting.
Also, Aspergillus niger can be present but its pigment is
pale yellow and never turns to orange and after 48 hours
of incubation begin to produce the typical black conidiophora.
References
KING D.A. A.D. HOCKING & J.I. PITT (1979) Dichloranrose bengal mdium fot enumeration and isolation of
moulds from foods. Appl. Environm. Microbiol 37:959964
VANDERZANT, C. & D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbiological examination of foods. APHA. Washington.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. London
The elimination of glucose from the formula, with the

low value of pH and the presence of chloramphenicol
inhibits the bacterial growth and the dichloran repress
the spreading of the fungal colonies.
Ref. 01-405 AFP Agar. Left: control; right: Aspergillus ssp.
Algae Media
Algae Agar
Ref. 01-007
Specication
Solid culture medium for the isolation and cultivation of
algae from soil, water and waste water.
Formula (in g/L)

Sodium nitrate ........................................ 1,000
Dipotassium phosphate .......................... 0,250
Magnesium sulfate ................................. 0,513
Ammonium chloride ................................ 0,050
Calcium chloride ..................................... 0,058
Ferrous chloride ..................................... 0,003
Agar ...................................................... 15,000
Final pH 7,0 0,2
Directions
Suspend 17 g of powder into 1 L of distilled water and let
it soak . Bring to boiling with constant stirring. Distribute
into appropriate containers and sterilize in the autoclave
at 121C for 15 minutes.
Description
The balanced nutrient medium composition provide all
necessary nutrients for the good growth of the algae
, but it does not support the good growth of fungi and
bacteria which can not grow well or have difculties in
the medium.
It is also suitable for algicide testing, however it is essentially recommended for the algae-pattern maintenance
and cultivation or for the isolation of water contaminants.
Technique
For the maintenance of algal strains it is recommended
to incubate at room temperature, under a suitable
light source (natural, uorescent tube or incandescent
lamp) until a good growth is obtained (within one to two
weeks).
In these conditions, and without gel dehydration, cultures
can be maintained up to two months.
Algae Broth
Ref. 02-007
Specication
Nutritive solution for algae and cyanobacteria, suitable
for water algicide biotesting.
Formula (in g/L)

Sodium nitrate ........................................ 1,000
Ammonium chloride ................................ 0,050
Ferrous chloride ..................................... 0,003
Final pH 7,0 0,2
Directions
Dissolve 1,87 g of powder in 1 L of distilled water and
distribute into suitable containers. Sterilize in the autoclave at 121C for 15 minutes.
Description
This liquid medium is suitable for algae and cyanobacteria cultivation, and it is especially adapted for inocule
preparation and algicide biotesting, as per Fitzgeralds
technique.
Due to the less content of energy source, fungi and
bacteria do not grow or grow badly unlike algae, which
obtain all the necessary nutrients, except the energy
source.
Technique
Fitzgeralds procedure for the algicide ability verication
of chemical products is:
a) Inoculum preparation
Prepare the Algae Broth and distribute 20 mL. each in
the 50 mL capacity conical asks. Sterilize and keep
them cool until usage.
Regularly, one of the conical asks is inoculated with a
couple of loops from Chlorella emersonii culture from
slanted Agar (Ref 1-007) and it is incubated at room
temperature until good growth is observed.
This culture can be used as biotest inoculum, but for
only up to 30 days.
b) Biotest
1- Samples
Prepare one litre of pure distilled water and 1 litre of
distilled water containing the inhibitor. Add 120 mg of
Sodium nitrate and 2,5 g of Di-potassium phosphate to
each sample.
2- Technique of the Test
Prepare a double series of 50 mL capacity conical asks
and add 5, 12.5 and 25 mL respectively of water algicidal
mixture, and then rell with pure water to get 25 mL in
each conical ask.
Add only 25 mL of pure water in one or two conical
asks to use them for control purposes.
All the conical asks are inoculated with the same volume of inoculum, the necessary amount to get an algae
concentration about 300.000 cells/mL in each ask. As
a practice (not exactly) this concentration produces a
slight greenish tinge. If necessary, adjust the inoculum
by counting or photocolorimetry.
Algae Media
Incubate the inoculated asks at room temperature
under an homogeneous and standardized light (i.e. 20 W
uorescent light).
Countings of all the asks is carried out daily with a globule-cell count (Thoma, Neubauer type or similar).
The test is said to be over when the control conical
asks have an average concentration greater than 5x106
cells/mL, comparing the other asks to them.
3-Interpretation
Inhibitor concentration in the asks with equal growth
to the control is considered non toxic or ineffective. If
algal concentration is maintained or remains same as
at the starting of experiment, it is considered algistatic.
Concentrations that have reduced starting population are
considered as algicidic, with the different effectivity ratio
depending on the amount of loss.
References
CLESCERI, L., A.E. GREENBERG, A.D. EATON (1998)
Standard Methods for Examination of Water and Wastewater. APHA-AWWA-WEF. Washington, D.C.
ALLEN, (1952) Arch. Microbiol. 17:34
FITZGERALD (1962) Water and Sewage Works.
109:361.
Ref. 01-007 Algae Agar. Chlorella sp.
Antibiotic Media
While performing the antibiotic assays, the only methodology accepted universally is the microbiological
methodology. National pharmacopeia provides different
directives to adapt to new compounds. Nowadays, the
European Pharmacopeia (1996), following a path to
standardize all the countries in the EU, provides several
rules and recommendations but it does not impose strict
criteria, since they could interfere with the local legislation. Instead of this, the USP 25/NF 20 (2002) incorporates more concrete guidelines about the antibiotics that
are accepted for human consumption and their assay
methodology. However, many times the USP refers
to the US/FDA, which is the more detailed publication
about the antibiotics assay.
Kirshbaun and Arret, in 1959, published a report with all
the variations of this methodology. The success of their
rst attempt, and the discovery of new antibiotics made
the same authors revise their work and , in 1967, they
published a new report detailing 57 ofcial methods, and
in 1971 last edition they detailed 83 ofcial methods and
10 non ofcial methods. The XIX edition of the USP consider this last 1971 edition as its information source.
The present article simply pretends to resume the most
important characteristics of this methodology, following
the guidelines of the current pharmacopeia. There are
no detailed directions to perform the assay because it
is considered that this is going to be read by microbiologists who are familiar with the basic techniques.If you
are interested in this subject, we would ask you to read
the bibliography mentioned at the end of this publication,

especially 21CFR for the details about the preparation of
samples and the extraction of antibiotics for each pharmaceutical preparation.
The reference substances used in the assays are substances whose activity has been precisely determined
with references to the corresponding international standard or international reference preparation.
Microorganisms
Strains used in the antibiotics assay as well as the
preparation methods and inocula are resumed in the
Table I. Strains may be obtained from the following and
other collections:
ATCC: American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852-176. USA.
Fax +1 301-231-5826
CIP/CNCM: Collection de lInstitut Pasteur / Collection
Nationale de Cultures de Microorganismes,
Institut Pasteur, 25 rue du Docteur Roux, 75724
PARIS CEDEX 15. France. Fax +33 143 06 98
35
CECT: Coleccin Espaola de Cultivos Tipo, Dpto Microbiologia, Fac. Ciencias Biolgicas, Dr. Moliner
50, 46100 Burjasot (Valencia) Spain. Fax +34
63 86 43 72
7
Antibiotic Media
NCTC:National Collection of Type Cultures, PHLS
Central Public Health Laboratory, 61 Colindale
Avenue, LONDON NW9 5HT England.
NCIB: The National Collections of Industrial and Marine
Bacteria Ltd., 23 St Machar Drove, ABERDEEN
AB2 1RY, Scotland. U.K. Fax +4402 24 487
658
NCYC: National Collection of Yeast Cultures. Institute
of Food Research. Norwich Laboratory. Colney
Lane. NORWICH NR4 7UA. U.K.
Preparation methods
Method #1
Strains to be assayed are mantained in slanted tubes
of Antibiotic Medium 1 (Ref. 01-009), subculturing
to fresh tubes every week. The growth on a slanted
tube, incubated at 32-37 for 24 hours, is washed
with 3 mL of Ringer Solution (Ref. 06-073), and the
resulting suspension is used to inoculate a Roux
ask containing 250 mL of sterile and controlled
Antibiotic Medium 1 (Ref. 01-009).
For the maintenance of antibiotic resistent strains
prepare Antibiotic Medium 1 (Ref. 01-009) with the
addition of proper amounts of antibiotic, as described
in the Table II. The growth obtained in the Roux ask
after a 24 hours incubation at 32-37C is washed and
collected with 50 mL of Ringer Solution (Ref. 06-073)
and it is stored in refrigeration (4-6C)
# Strain
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
1
Method #2
Process is the same as the Method #1 but the
standardization is performed in the following way:
Centrifuge and collect the pellet. Sediment is suspended with 50-70 mL of Ringer 1/4 Solution (Ref.
06-073) and heat up to 70C for 30 minutes. Refrigerate the spore suspension obtained in this way.
Method #3
Proceed as per Method #2 performing the thermal
shock before the centrifugation. Wash the spore
suspension 3 consecutive times with distilled water
(25-50 mL each time) and centrifuge. Reconstitute
the nal suspension with 50-70 mL of sterile distilled
water and store it in refrigeration.
Method #4
Let the fungi grow for 6-8 weeks at room temperature
(20-25C) in several 3 L Erlenmeyer asks, containing each one about 200 mL of Antibiotic Medium 22.
After six weeks, test the sporulation regularly and,
when it reaches 80%, collect the spores of the aerial
micelium with a sterile spatula or any other suitable
instrument. The collected spores are suspended in
20 mL of Ringer 1/4 Solution (Ref. 06-073) and store
refrigerated.
Table I. Strains (Test organisms) for the Antibiotic Assays

Genus & species
Strain and Type culture collection #
Method
American Collection
European Collections
Staphylococcus aureus
Staphylococcus epidermidis
Micrococcus luteus
Micrococcus luteus (avus)
Enterococcus hirae
Bacillus pumilus
Bacillus cereus
Bacillus subtilis
Bacillus subtilis
Klebsiella pneumoniae
Escherichia coli
Escherichia coli
Bordetella bronchiseptica
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Pseudomonas aeruginosa
Mycobacterium smegmatis
Candida tropicalis
For candicidine, use method #1
ATCC 6538P
ATCC 29737
ATCC 9144
ATCC 12228
ATCC 9341
ATCC 10240
ATCC 10541
ATCC 11778
ATCC 6633
ATCC 10031
ATCC 10536
ATCC 9637
ATCC 4617
ATCC 9763
ATCC 2601
ATCC 25619
ATCC 607
CIP 53.156
CIP 53.134
CIP 68.21
CIP 53.45
CIP 53.160
CIP 58.55
CIP 76.18
CECT 193
CIP 52.62
CIP 1.83
CIP 53.153
CIP 54.127
CIP 2.83
CIP 53.157
CIP 1432.83
CECT 1324
NCTC 7447
NCIB 6751
NCIB 8853
NCTC 8340
NCTC 7743
NCIB 8192
NCTC 8241
NCTC 10320
NCTC 10400
NCTC 8236
NCTC 7427
NCIB 8879
NCIB 8666
NCTC 8344
NCYC 87
NCYC 853
NCIB 10817
NCTC 7017
CIP 1433.83 NCYC 1393
1
1
1
1
1
1
5
2
3
2
2
1
1
1
1
61
6
1
7
6
Antibiotic Media
Method #5
With the help of a loop inoculate a ask containing
100 mL of sterile and controlled Antibiotic Medium 3
(Ref. 02-011). from a slant culture. Incubate for 16-18
hours.
Method #6
Follow the directions stated in the Method #1 but incubate the slanted subcultures at 30C for 24 hours,
and the Roux asks at 30C for 48 hours.
Method #7
Organisms are maintained on Antibiotic Medium 36
in slanted tubes, subculturing them each week with
an incubation at 37C for 48 hours.
Collect the growth of a recent subculture with 3 mL
of Ringer 1/4 Solution (Ref. 06-073) and use it to
inoculate a 500 mL Erlenmeyer ask containing 100
mL of Antibiotic Medium 34. Add asseptically to the
ask 50 g of sterile glass balls and incubate at 27C
for 5 days with constant stirring (120 rpm and 3,5 cm
radius). Keep the suspension in refrigeration.
Inoculum Determination
For many years the ofcial US/FDA methods provided
specic instructions for the inoculum standardization. After 1985 the USA Federal Regulations and the USP XIX
adopted the criterion suggested by Kirshbaum, Kramer
and Gorth that recommends to dilute the microorganisms suspension until it provides a 25% transmitance at
580 nm in a 13 mm tube. However, even with this stand-
ardization it is necessary to test every batch of inoculum

to determine the exact volume that must be added to
each assay medium.
If the inoculum suspensions are prepared as described
in the above sections, they will be homogeneous enough
to determine the volumes that are going to be added,
with tests, without any other previous standardization
step.
Diffusion agar Assay
Prepare a series of asks with 100 mL of sterilized,
melted and cooled Seed Layer Medium (Ref. 01-009)
and add to each ask a different volume of microorganisms suspension, as per the volume pattern
suggested in Table IV.
Prepare Petri plates with the inoculated medium
according to the specicacions of each antibiotic and
put a steel or glass cylinder or a disc with the reference concentration of antibiotic. Incubate the plates
and measure the inhibition zones. The volume of
suspension that produces optimum zones, in denition as well as in diameter, is the one that is adopted
for the inoculum. In Table IV the optimum inhibition
zones are shown that are supposed to appear in the
different assays.
Turbidimetric Assay
Proceed in the same way as in the previous case but
instead of inoculating over the melted medium, inoculate in the asks containing 100 mL of the suitable
liquid medium, using the volumes pattern specied in
Table II. Buffer solutions and diluents

Formula (in g/L of distilled water)
Buffer1
Phosphate buffer 1% at pH 6,00,05
K2HPO4 ................................. 2,000

KH2PO4 ................................. 8,000
g
g
Phosphate buffer 0,1M at pH 7,90,1
K2HPO4 ............................... 16,730

KH2PO4 ................................. 0,523
g
g
KH2PO4 ............................... 13,600
Phosphate buffer 10% at pH 6,00,05
K2HPO4 ............................... 20,000

KH2PO4 ............................... 80,000
g
g
10
K2HPO4 ............................... 35,000

g
KOH 10N ............................... 2,000 mL
16
Phosphate buffer 0,1N at pH 7,0
K2HPO4 ............................... 13,600

KH2PO4 ................................. 4,000
g
g
17
Phosphate-methanol buffer at pH 6
Methanol .............................. 50,000

Buffer #1 ........................................
mL
q.s.
18
Sodium carbonate solution 1%
Sodium carbonate ............... 10,000
May require adjustment with phosphoric acid 18 N or KOH 10N before or after sterilization.
Antibiotic Media
Table III . With these volumes of inoculated medium
continue as if you were performing an assay, using
just the highest and lowest concentrations in relation
to the pattern. Incubate for 3-4 hours and read the
absorbances. With all these data you will be able to
establish the right inoculum that provides a better
answer between the high and low point, and so adopt
it for the assay.
Strain maintenance
Assay strains are maintained by vegetative propagation in slants and in duplicate. One of the duplicates is
used only for the next subculture, and the other one is
used for all the other operations. Medium, if there is no
other directive, is Antibiotic Medium 1 (Ref. 01-009) with
antibiotics or other additive if necessary. Details may be
obtained in Table II. Nonetheless, other media may be
used if considered necessary.
Buffer Solutions and diluents

Composition of the most common buffer solutions and
diluents is given in Table II. Dilutions of pure chemical
products as well as organic diluents have been ommited. In anycase, the compounds must be according
to the purity standards of the corresponding pharmacopeia. Water must always be distilled and be of reagent
grade. All buffer solutions and diluents have to be sterile.
Sometimes a little pH readjustment is necessary after
the sterilization.
Culture Media
The composition of the several SCHARLAU Microbiology Antibiotic Media is given below. Media still have the
nomenclature according to the US/FDA, which uses the
one described by Grove and Randall (media 1 to 13)
and Kirshbaum and Arret (media 18 to 21). The European Pharmacopeia names the media with alphabetical
letters, and the corresponding Scharlau media are as
follows:
Medium A..............Antibiotic Medium 1 and 11
Medium B .......................Antibiotic Medium 10
Medium C ........................ Antibiotic Medium C
Medium D ........................ Antibiotic Medium D
Medium E ........................ Antibiotic Medium E
Medium F........................Antibiotic Medium 19
Medium G .......................Antibiotic Medium 35
Note: Medium D is very similar to Antibiotic Medium 3
but with the addition of 2 g/L of potassium nitrate
in it.
SCHARLAU media described following are prepared
with the ingredients specied in the USP 23/NF 18, Eur.
Pharm. 3 and 21 CFR for these components, providing
an absolute reproducubility and reliability.
10
The components of the culture media accomplishes the

specications of the several pharmacopeia, however the
technician is responsible for the end use of dehydrated
culture media or any other variation in it, as long as they
still have the same characteristics described for the
above media. pH of the medium must be checked when
it is completely reconstituted and at 25C.
Antibiotic Medium 1
(Eur. Phar. Antibiotic Medium A
at pH 6,6)
Ref. 01-009
Specication
Antibiotic Medium 1 or Seed Layer is used for the antibiotic assays by the diffusion method in agar, that may
be performed in several ways (cylinder, punched-hole or
paper disc method).
Formula (in g/L)

Peptone ..................................................... 6,0
Casein Peptone .......................................... 4,0
Yeast extract ............................................... 3,0
Beef extract ................................................ 1,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 6,6 0,2
Directions
Add 30,5 g of medium to 1 L of distilled water. Heat to
the boiling and dispense into suitable containers. Sterilize in the autoclave at 121C for 15 minutes.
Antibiotic Media
Antibiotic Medium 2
Ref. 01-010
Specication
Antibiotic Medium 2 or Basal Layer is used for the antibiotic assays by the diffusion method in agar, that may
be performed in several ways (cylinder, punched-hole or
paper disc method).
Formula (in g/L)

Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Agar .......................................................... 15,0
Final pH 6,5 0,2
Directions
Antibiotic Medium 3
Ref. 02-011
Specication
Antibiotic medium 3 or Antibiotic Assay Broth may be
used in the inoculum preparation, serial dilutions or turbidimetric antibiotic assays.
Formula (in g/L)

Peptone ................................................... 5,00
Yeast extract ............................................. 1,50
Meat extract .............................................. 1,50
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Monopotassium phosphate ...................... 1,32
Dipotassium phosphate ............................ 3,68
Final pH 7 0,05
Directions
Antibiotic Medium 4
Ref. 01-012
Specication
Antibiotic Medium 4 is used in the antibiotic assays by
the diffusion method in agar, which may be performed
in several ways (cylinder, punched-hole or paper disc
method)
Formula (in g/L)

Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 6,5 0,1
Directions
Antibiotic Medium 5
Ref. 01-013
Specication
method).
Formula (in g/L)

Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Agar .......................................................... 15,0
Final pH 7,9 0,1
Directions
Antibiotic Medium 8
Ref. 01-014
Specication
method).
Formula (in g/L)

Peptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Agar .......................................................... 15,0
Final pH 5,9 0,1
Directions
11
Antibiotic Media
Antibiotic Medium 9
Ref. 01-015
Specication
method).
Formula (in g/L)

Casein peptone ........................................ 17,0
Soy peptone ............................................... 3,0
Sodium chloride .......................................... 5,0
Dipotassium phosphate .............................. 2,5
Dextrose ..................................................... 2,5
Agar .......................................................... 20,0
Final pH 7,2 0,2
Directions
Add 50 g of medium to 1 L of distilled water. Heat to the
boiling and dispense into suitable containers. Sterilize in
the autoclave at 121C for 15 minutes.
Antibiotic Medium 10
(Eur. Phar. Antibiotic Medium B)
Ref. 01-016
Specication
method).
Formula (in g/L)

Casein peptone ........................................ 17,0
Soy peptone ............................................... 3,0
Sodium chloride ......................................... 5,0
Dextrose ..................................................... 2,5
Agar .......................................................... 12,0
Final pH 7,2 0,2
Directions
Add 42 g of medium to 1 L of distilled water. After dissolution, add 10 mL of Polysorbate 80. Heat to the boiling
and dispense into suitable containers. Sterilize in the
autoclave at 121C for 15 minutes.
(Eur. Phar. Antibiotic Medium A
at pH 8,0)
Ref. 01-017
Specication
method)
Formula (in g/L)

Peptone ..................................................... 6,0
Casein peptone .......................................... 4,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 8,0 0,2
Directions
Ref. 02-544
Specication
Antibiotic Medium 13 is used in the turbidimetric antibiotic assays.
Formula (in g/L)

Peptone .................................................... 10,0
Dextrose ................................................... 20,0
Final pH 5,5 0,2
Directions
(Eur. Phar. Antibiotic Medium F)
Ref. 01-434
Specication
method)
12
Antibiotic Media
Formula (in g/L)
Directions
Peptone ..................................................... 9,4

Yeast extract ............................................... 4,7
Beef extract ................................................ 2,4
Sodium chloride ........................................ 10,0
Dextrose ................................................... 10,0
Agar .......................................................... 23,5
Final pH 6,0 0,1
Add 33 g of medium to 1 L of distilled water.Add 10 mL

of glycerol. Heat to the boiling and dispense into suitable
containers. Sterilize in the autoclave at 121C for 15
minutes.
Directions
Ref. 01-069
This medium is sold by Scharlay Microbiology under the
name Sporulating AK Agar.
Specication
method).
Formula (in g/L)

Peptone ................................................. 6,000
Casein peptone ...................................... 4,000
Yeast extract ........................................... 3,000
Meat extract ............................................ 1,500
Dextrose ................................................. 1,000
MnSO4.H20 ............................................. 0,003
Agar ...................................................... 15,000
Final pH 6,5 0,2
Directions
Antibiotic Medium 34 Base

Ref. 02-545
Antibiotic Medium 35 Base

(Eur. Phar. Antibiotic Medium G)
Ref. 01-545
Specication
method).
Formula (in g/L)

Peptone .................................................... 10,0
Meat extract .............................................. 10,0
Sodium chloride .......................................... 3,0
Agar .......................................................... 15,0
Final pH 7,0 0,1
Directions
Add 38 g of medium to 1 L of distilled water containing
10 mL glycerol. Heat to the boiling and dispense into
suitable containers. Sterilize in the autoclave at 121C
for 15 minutes.
Ref. 01-200
Sharlau Microbiology sold this medium as Tryptic Soy
Agar (TSA).
Specication
method)
Formula (in g/L)
Antibiotic Medium 34 is used in the turbidimetric antibiotic assays.
Casein peptone ........................................ 15,0

Soy peptone ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 0,1
Formula (in g/L)
Directions
Specication
Peptone .................................................... 10,0

Meat extract .............................................. 10,0
Sodium chloride .......................................... 3,0
Final pH 7,0 0,1
Add 40 g of medium to 1 L of distilled water.Heat to the

13
Antibiotic Media
Ref. 02-547
Specication
Antibiotic Medium 39 may be used in the inoculum
preparation, serial dilutions or turbidimetric antibiotic
assays.
Formula (in g/L)

Peptone ................................................... 5,00
Yeast extract ............................................. 1,50
Meat extract .............................................. 1,50
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Final pH 7,9 0,1
Directions
Ref. 01-546
Specication
metho
Formula (in g/L)

Polypeptone ............................................... 5,0
Dextrose ................................................... 10,0
Yeast extract ............................................. 20,0
Polysorbate 80 ........................................... 0,1
Monopotassium phosphate ........................ 2,0
Agar .......................................................... 12,0
Final pH 6,8 0,1
Directions
Ref. 02-548
Specication
the turbidimetric method.
14
Formula (in g/L)

Casein Peptone ....................................... 9,00
Yeast extract ............................................. 5,00
Sodium citrate ........................................ 10,00
Dextrose ................................................. 20,00
Final pH 6,8 0,1
Directions
Add 46g of medium to 1 L of distilled water. Heat to the
Antibiotic Medium C
(Eur. Phar. Antibiotic Medium C)
Ref. 02-601
Specication
Antibiotic Medium C may be used for the inoculum preparation, serial dilutions or turbidimetric antibiotic assays.
Formula (in g/L)

Peptone ................................................... 6,00
Yeast extract ............................................. 3,00
Beef extract .............................................. 1,50
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Final pH 7,0 0,2
Directions
boiling and dispense into suitable containers. Sterilize
in the autoclave at 121C for 15 minutes. If pH 8,0 is
desired, adjust with NaOH1N.
Antibiotic Medium D
(Eur. Phar. Antibiotic Medium D)
Ref. 02-549
Specication
Antibiotic Medium D may be used for the inoculum preparation, serial dilutions or turbidimetric antibiotic assays.
Formula (in g/L)

Heart extract ............................................. 1,50
Yeast extract ............................................. 1,50
Casein peptone ........................................ 5,00
Sodium chloride ........................................ 3,50
Dextrose ................................................... 1,00
Potassium nitrate ..................................... 2,00
Final pH 7,0 0,2
Antibiotic Media
Directions
Formula (in g/L)

Peptone ................................................... 5,00

Meat extract .............................................. 3,00
Disodium phosphate ............................... 26,90
Agar ........................................................ 12,00
Final pH 7,9 0,2
Antibiotic Medium E
(Eur. Phar. Antibiotic Medium E)
Ref. 01-430
Specication
Antibiotic Medium E is used in the antibiotic assays by
method).
Directions
the boiling and dispense into containers. Sterilize in the
autoclave at 121C for 15 minutes. Precipitates appearing after sterilization do not interfere in its normal
operation.
Table III. Specications for the antibiotic master standard solutions

Drying cond.
Amikacin
Amphotericin B1
Bacitracin
Bleomycin
Carbenicillin
Cephalothin
Cycloserine
Chloramphenicol
Chlortetracycline
Cloxacillin
Colistimethate Na
Colistin
Demeclocycline
Dihydrostreptomycin
Doxycycline
Erythromycin
Framicetine
Gentamicin
Gramicidin
Kanamycin SO4
Methacycline
Methylmicin
Nafcillin
Natamycin
Neomycin
Novobiocin
Nystatin
Oxytetracycline
Paromomycin
Penicillin G
Polymyxin B
Rifampin
Rolitetracycline
Sisomycin
Spectinomycin
Spiramycin
Streptomycin
Tetracycline
Ticarcillin
Thiostreptone
Tobramycin
Troleandomycin
Vancomycin
1
6
1
1
7
None
1
1
None
None
None
1
1
None
5
None
1
3
3
1
None
None
None
None
4
1
5
4
None
1
None
1
None
None
None
6
1
1
None
None
None
3
None
1
Initial solvent
Master soln
Distilled water
1,0
Dimethylsulfoxide
1,0
1
100,0
16
2,0
1
1,0
1
1,0
Distilled water
1,0
Ethanol
1,0
HCl 0,01N
1,0
1
1,0
Distilled water
1,0
Distilled water
1,0
HCl 0,01N
1,0
3
1,0
HCl 0,01N
1,0
Methanol
1,0
Distilled water
1,0
3
1,0
Ethanol
1,0
3
1,0
HCl 0,01 N
1,0
3
1,0
1
1,0
Dimethylsulfoxide
1,0
3
1,0
Absolute Ethanol
1,0
Dimethylformamide 1000,0
HCl 0,1N
1,0
3
1,0
1
1000,0
Distilled water
10000,0
Methanol
1,0
Distilled water
1,0
3
1,0
Distilled water
1,0
Methanol
1,0
3
1,0
HCl 0,1N
1,0
1
1,0
Dimethylsulfoxide
1,0
Distilled water
1,0
Isopropanol
1,0
Distilled water
0,4
mg
mg
ui
ui
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
ui
mg
mg
ui
ui
mg
mg
mg
mg
mg
mg
mg
mg
ui
mg
mg
mg
Days (cool) Final solvent

15
1
7
15
15
5
30
30
4
7
1
15
5
30
5
14
7
30
30
30
15
7
2
1
15
5
1
4
21
2
15
1
1
15
30
7
30
1
1
1
15
1
7
Distilled water
10
1
16
1
1
1
1
4
1
6
6
4
3
Distilled water
3
3
3
Ethanol
3
4
3
1
10
3
6
6
4
3
1
6
1
Distilled water
3
Distilled water
3
3
4
1
Dimethylsulfoxide
16
Distilled water
4
Use amber glass or work under red light

15
Antibiotic Media
Reference Standard
Each antibiotic must be assayed with a proper reference
standard or master standard pattern. Patterns may
be obtained from the USP NF Convention Inc. or other
organisms, such as:
European Pharmacopeia Secretariat, B.P. 907, F-67029
Strasbourg. CEDEX 1. Fax +33 388 41 20 36. e-mail:10
0536.3157@compuserve.com
USP-NF Reference Standards, 12601 Twinbrook Parkway, Rockville. Md. 20852, USA
British Pharmacopoeia Comission Laboratory, Government Buildings, Block 2, Honeypot Lane, Stanmore, Middlesex, HA7 1A4. England.
However, you may use domestic or working standards
if they have been compared to a reference pattern. In
Table III you may nd details about the reference standard: dessication conditions, initial diluent to obtain the
mother solution, activity period,etc...
Following are described the dessication methods for the
standards. US/FDA numeration have been maintained.
US/FDA Method #1
In vacuum oven, dry the pattern or master reference
standard at 60C for 3 hours, at pressure 5 mm Hg.
US/FDA Method #3
US/FDA Method #4
US/FDA Method #5
US/FDA Method #6
standard at 45C for 3 hours, at pressure 5 mm Hg .
US/FDA Method #7
standard at 35C for 4 hours, at pressure 5 mm Hg .
16
Diffusion Method in Agar

(Plate assay)
Once we decide the assay conditions (tabulated in
Table IV), we can prepare the plates as per the following
method .
Plate preparation
..Pour basal medium, melted and cooled to 50C, into
each plate, and let it solidify horizontally. Using 10 cm.
diameter plates, suggested volumes are 21 mL for each
basal layer and 4 mL for the seed layer. Nonetheless, in
the case of amphotericin and nystatin a single seed
layer of 8 mL is used, without any basal layer. Another
exception to this rule is bleomycin, for which a 10 mL of
basal layer and a 6 mL of seed layer is used. In practise,
when the standard 9 cm diameter plastic plates are used
it is advisable to use the volumes of about 15 mL for the
basal layer and 4 mL for the seed layer, in order to avoid
the contact between the cylinders and the plate cover.
While the basal layer solidies, adjust the temperature
for the medium of seed layer to 45-48C, and inoculate with the suggested (or tested) volume of inoculum
suspension. Homogenize by gentle stirring and carefully
pour the medium over the basal layer in 4 mL volumes,
except for the cases mentioned above. Be very careful
in this operation, since the nal results may be affected
if the layer is not very homogeneous, and forms bubbles
or clots. Once the agar solidies, place the cylinders or
make the holes. Put or make 6 cylinders or holes per
plate, centered in the plate at 60 angle from each other.
Reference pattern preparation and

standard curve
Master pattern is prepared according to the specications in Table IV, starting from the stock solution and
using the adequate diluent until the desired concentration is obtained. Reference level is always the medium
concentration among the 5 concentrations used to obtain
the standard curve (see Table IV).
Assay technique
To obtain the curve, 12 plates are used, three for each
level, except for the reference standard, which is included in all. In each batch of three plates, 3 alternate
holes or cylinders are lled with the reference concentration and the other three are lled with the corresponding
concentration. By proceeding in this way we can obtain
30 inhibition halos or inhibition zones corresponding to
the reference concentration and 9 halos for each one of
the other assay standards.
Three plates, with 6 holes or cylinders, are used in
each test. Holes or cylinders are lled alternatively with
the reference level and the diluted sample. Plates are
incubated at appropriate temperature (see Table IV) for
16-18 hours. After this period, inhibition halo readings
are performed using the proper measuring instrument.
Antibiotic Media
Table IV. Agar diffusion test specications
Assay
strain
(Table I)
Antibiotic
Amphotericin B
Bacitracin
Bleomycin
Carbenicillin
Cephalothin
Cloxacillin
Colistimethate sodium
Colistin
Dihydrostreptomycin
Doxycyclin
Erythromycin
Framicetin
Gentamicin
Nafcillin
Natamycin
Neomycin
Netilmycin
Novobiocin
Nystatin
Paromomycin
Penicillin G
Polymyxin
Rifamycin
Sisomycin
Spiramycin
Ticarcillin
Vancomycin
Maintenance Inoculum
Medium (mL/100 mL
(A.M. n)
medium)
P
F
S
R
A
A
O
O
J
I
E
J,H
D
A
P
A,H,J
D
D
Q
D
A
O
J
D
J
B
J
19
1
36
1
1
1
1
1
1
1
1
1
1
1
19
1
1
1
19
1
1
1
1
1
1
1
1
1,00
0,30
1,00
0,50
0,10
0,10
0,10
0,10
0,05
0,40
1,50
0,40
0,03
0,30
1,00
0,40
0,25
4,00
1,00
2,00
1,00
0,10
0,10
0,03
0,20
0,40
0,20
Medium
Basal
strain
Seed
strain
9
35
9
2
2
9
9
5
8
11
1
11
2
19
11
11
2
11
2
9
2
11
1
8
8
19
10
35
10
1
1
10
10
5
8
11
1
11
1
19
11
11
1
19
11
1
10
2
11
1
8
8
Potency calculation
To extrapolate from the standard curve, use the average
of the 36 diameters of the reference level and the several batches of 9 halos obtained in each assay level. The
average of the 36 halos of reference is the correction
value of the standard curve, and thus we can achieve
each point as the function of the average value of the
reference values of the corresponding standard.
Values obtained in this way are plotted over a semilogarithmic graph paper, drawing the antibiotic concentration
(in mcg/mL) on X axis, and the halo or zone diameters
on the Y axis. Standard curve is drawn by linking the
ve points or by tracing a straight line between the high
and low point, which are calculated using the following
statistical formulae:
3a+2b+c-e
A=
3e+2d+c-a
B=
where:
A: Halo diameter, calculated for the highest standard
concentration.
B: Halo diameter, calculated for the lowest pattern concentration.
c:
Reference halo diameter, average of the 36 halos
of the pattern plates.
a,b,d,e: Corrected average values of the other pattern
assay levels,from the highest to the lowest order.
Incubation
Temperat.
(C)
Final concentrations: assay levels for the standard curve in

Reference
IU/mL or in mcg/mL
halo
Ref.
(mm)
30 0,640
0,800
32-35 0,640
0,800
32-35 0,010
0,020
3712,800 16,000
32-35 0,640
0,800
32-35 3,200
4,000
37 0,640
0,800
37 0,640
0,800
37 0,640
0,800
30 0,640
0,800
32-35 0,640
0,800
35-37 0,640
0,800
37 0,640
0,800
32-35 1,280
1,600
28-30 3,200
4,000
32-35 6,400
8,000
32-35 0,064
0,080
35 0,320
0,400
3012,800 16,000
37 0,640
0,800
32-35 0,640
0,800
37 6,400
8,000
30 3,200
4,000
32-35 0,064
0,080
35-37 0,640
0,800
32-35 3,200
4,000
37 6,400
8,000
1,000
1,000
0,040
20,000
1,000
5,000
1,000
1,000
1,000
1,000
1,000
1,000
1,000
2,000
5,000
10,000
0,100
0,500
20,000
1,000
1,000
10,000
5,000
0,100
1,000
5,000
10,000
1,250
1,250
0,080
25,000
1,250
6,250
1,250
1,250
1,250
1,250
1,250
1,250
1,250
2,500
6,250
12,500
0,125
0,625
25,000
1,250
1,250
12,500
6,250
0,125
1,250
6,250
12,500
1,560
1,560
0,160
31,200
1,560
7,810
1,560
1,560
1,560
1,560
1,560
1,560
1,560
3,120
7,810
15,600
0,156
0,781
31,200
1,560
1,560
15,600
7,810
0,156
1,560
7,810
15,600
16-18
16-18
18-20
16-18
14-16
18-20
13-15
15-17
14-16
18-20
17-19
14-16
15-17
14-16
17-19
16-18
14-16
16-18
16-18
19-21
17-19
15-17
15-17
15-17
15-17
17-19
16-18
To estimate the potency of the test sample, calculate

average of the inhibition halo diameters and the reference and sample levels of the three plates, and perform
the corrections related to the correction point. Corrected
value of the problem is projected on the standard curve
and thus the theoritical concentration is obtained. Theoritical concentraion multiplied by the dilution factor gives
the real concentration of antibiotic in the sample.
Turbidimetric assay
Reference pattern preparation and
standard curve
Once the assay conditions are determined as per Table
III, prepare the pattern according to the specications in
Table V. At the time of assay experiment, take an aliquote of mother solution and prepare appropriate dilutions
to obtain the desired concentrations. Reference level is
the medium concentration between the ve proposed for
the standard curve.
17
Antibiotic Media
Table V. Turbidimetric assay specications
Assay strain Inoculum
(Table I)
(mL/100 mL
medium)
Antibiotic
Amikacin
Candicidin
Capreomycin
Chloramphenicol
Chlortetracycline
Cycloserine
Demeclocycline
Dihydrostreptomycin
Doxycyclin
Gramicidin
Kanamycin
Methacycline
Neomycin
Oxytetracycline
Rolitetracycline
Spectinomycin
Streptomycin
Tetracycline
Thiostreptone
Tobramycin
Troleandomycin
A
P
L
M
A
A
A
L
A
G
B
A
L
A
A
M
L
A
G
A
L
0,50
0,20
0,05
0,70
0,10
0,40
0,10
0,10
0,10
1,00
0,20
0,10
2,00
0,10
0,10
0,10
0,10
0,10
0,20
0,15
0,10
Assay
medium
3
13
3
3
3
3
3
3
3
3
3
3
39
3
3
3
3
3
3
41
3
Assay Technique
The assay is performed in the following way:
Prepare 3 tubes and put 1 mL (or 0,1 mL if the antibiotic
is gramicidin or tyrothricin) of every assay level of standard and sample. Thus, 15 tubes will be employed for the
pattern and 3 tubes for each problem.
Add asseptically 9 mL of inoculated assay broth to
each tube, and put them into a boiling water bath for
2-4 hours. The exact incubation time is determined by
observing the growth inside the tube, taking the standard
tube as the reference.
Tubes are removed from the boiling water bath and
0,5 mL of 12% formaldehyde is added in each tube in
order to stop the growth. Growth in the form of turbidity
is measured by a Spectrophotometer at the wavelength
530 nm. The zero is xed by the device and 100% transmission is provided by a blank containing only the sterile
medium and formaldehyde at the same proportion or
concentration as that of the assay.
Potency calculation
Statistical values corresponding to the highest and
lowest concentrations are determined according to the
following formulae:
3a+2b+c-e
A=
5
18
3e+2d+c-a
B=
5
Final concentrations: assay levels for the pattern curve in IU/mL or

mcg/mL
Incubation
temperature
(C)
32
29
37
32
32
32
32
37
32
37
32
32
37
32
32
32
37
32
37
32
32
Ref.
6,000
0,030
64,000
2,000
0,038
32,000
0,064
24,000
0,064
0,028
8,000
0,038
0,640
0,160
0,160
24,000
24,000
0,160
0,400
2,000
16,000
8,000
0,042
80,000
2,240
0,048
40,000
0,080
26,800
0,080
0,034
8,900
0,048
0,800
0,200
0,200
26,800
26,800
0,200
0,600
2,236
20,000
10,000
0,060
100,000
2,500
0,060
50,000
0,100
30,000
0,100
0,040
10,000
0,060
1,000
0,250
0,250
30,000
30,000
0,250
0,800
2,500
25,000
12,500
0,084
125,000
2,800
0,075
62,500
0,125
33,500
0,125
0,048
11,200
0,075
1,250
0,312
0,312
33,500
33,500
0,312
1,000
2,795
31,200
15,600
0,120
156,000
3,120
0,094
78,100
0,156
37,500
0,156
0,057
12,500
0,094
1,560
0,390
0,390
37,500
37,500
0,390
1,250
3,125
39,000
where:
A:
Absorbance value, calculated for the highest
value of the pattern curve.
B: Absorbance value, calculated for the lowest value of
the pattern curve.
a,b,c,d,e: Average values for each value in the triplicate
standard curve, from the highest to the lowest
concentration.
Potency estimation is performed by taking the average
value of each assay level in the pattern curve over a
semilogarithmic paper. Draw the absorbance over the
logarithmic scale and concentration over the abscissa.
If the calculated values are used, the result is a straight
line, but a curve may be drawn by linking the ve points.
The absorbance average value of the test sample is projected over the standard curve, obtaining in this way the
theoritical concentration which, multiplied by the dilution
factor, gives the real potency of the test sample.
Cross Contamination
with Penicillin
To perform this assay all the instrumentation and even
the environment must be free of penicillin. Plates are
prepared following the general method, using 10 mL of
Antibiotic Medium 1 (Ref. 01-009) in the basal layer and
4 mL of Antibiotic Medium 4 in seed layer and inoculating
the C strain of microorganism (see Table I).
Antibiotic Media
Reference standard is prepared according to the standard for penicillin, proposed in Table II, but taking the
mother solution of 100 units and the nal concentrations
at 0,0005;0,0125; 0,0050; 0,100 and 0,200 penicillin
units. Reference is the one with nal concentration 0,05
units (see Table III).
Sample is prepared by dissolving 1 g in 18 mL of distilled
water. From this initial solution, take 9 mL into a separating funnel, and add 20 mL of amyl acetate. Add then 1
mL of Solution #11 (see Table II) and stir it vigorously.
Let it rest and once the two layers are separated, take
the aqueous layer to another separating funnel. Check
the pH of the solution and if it is higher than 3, readjust
to 2,5 with HCl. Extract again with amyl acetate and
discard the aqueous layer.
Mix the two parts of amyl acetate, and wash them with
10 mL of Solution #2 (see Table II). Discard the aqueous
layer. Extract penicillin from the amyl acetate with 10 mL
of phosphate buffer pH 6 (Solution #1, Table II). Penicillin
presence is determined in this solution.
Use 15 plates to perform the assay: three for each
standard curve assay, except for the reference standard, which is present in all of them. Place 6 holes or
cylinders in each plate and ll them alternatively with the
reference solution and the corresponding assay level. In
this way, you will obtain 45 inhibition halos for the reference level and 9 halos more for each one of the other
levels.
A part of sample (2-5 mL) is treated with 0,1 mL of penicillinase and incubated at 37C for 1 hour. Use 3 plates
for each sample, and ll 2 holes or cylinders in each one
with the pattern reference level. Fill another 2 holes or

cylinders in each plate with the untreated sample, and
the last 2 holes or cylinders in each plate with the treated
sample.
Plates are incubated at 30C for overnight. Presence
of inhibition halos in the untreated sample, and the
absence of holes in the treated sample are a sign of
penicillin contamination. Should a qualitative estimation
of penicillin be desired, measure the halos and proceed
as in the normal penicillin assay.
Special lab requirements

The instruments used in all these assays must be
washed carefully before and after each test, in order to
remove all the traces of antibiotic.
To sterilize lab commodities, cover them well and use a
hot air oven at 200-220C for 2 hours. Volumetric asks ,
pipets, ... must be washed carefully.
Plate external
Cylinder ext.
Cylinder int.
Figure 1. Holes, discs or cylinders at 90
9 mm
6 mm
8 mm
Figure 2. Holes, discs or cylinders at 60
19
Antibiotic Media
Scharlau Chemie may supply all the necessary instruments to perform antibiotic assays, which is detailed
below:
Volumetric material
If it is possible, use guaranteed, checked and class
A glass.
Cylinders
Cylinders must be made of stainless steel. Exterior
diameter: 8 mm, interior diameter: 6 mm, height: 8-10
mm.
Holes
Holes should be made with a suitable punching
instrument of the cylinder dimension.
Discs
Use normalized paper lter discs, 6 mm diameter.
Plates
Use plastic or glass plates. If glass plates are employed, they must be washed and sterilized.
Tubes
All the tubes used in one assay must be equal and
uniform in their dimensions.
20
Colourimeter / Spectrophotometer
It must be able to read at 530 nm, have its own zero
adjustment and 100% transmission can be determined with a sterile medium solution at the same
conditions of assay.
References
ARRET, B. DIANA, P. JOHNSON y A. KIRSHBAUM
(1971) Outline of Details for Microbiological Assays
of Antibiotics: Second Revision. J. PHARM, Sci.
60,11,1689-1694.
EUROPEAN PHARMACOPEIA (1997) 3rd Ed. 2.7 Biological Assays. 2.7.2 Microbiological Assay of Antibiotics.
Council of Europe. Strasbourg.
SANCHO, GUINEA, PARES. (1980) Microbiologa
Analtica Bsica. Ed. JIMS. Barcelona,
U.S. PHARMACOPEIA XIX (1975) Antibiotic Assays.
U.S./F.D.A.: 21 CFR (1976 and following) 436.100 and
following.
U.S. PHARMACOPEIA XX/National Formulary XV
(1980) Antibiotic Assays.
U.S. PHARMACOPEIA 23/National Formulary 18 (1995)
Biological Tests and Assays. {81} Antibiotics Microbial
Assays.
U.S. PHARMACOPEIA 25/National Formulary 20 (2002)
Biological Tests and Assays. {81} Antibiotics Microbial
Assays.
EUROPEAN PHARMACOPEIA, Supplement (2002), 4th
Ed.,Council of Europe, Strasbourg,
APT Media
APT Agar
Ref. 01-026
Specication
Solid medium for general purposes, especially designed
for the cultivation of the heterofermentative lactic acid
bacteria that causes meat greening.
Formula (in g/L)

Casein Peptone .................................... 12,500
Yeast Extract .......................................... 7,500
Sodium Chloride ..................................... 5,000
Dipotassium Phosphate ......................... 5,000
Trisodium Citrate .................................... 5,000
Dextrose ............................................... 10,000
Magnesium Sulfate ................................. 0,800
Manganous Chloride .............................. 0,140
Iron Sulfate ............................................. 0,040
Thiamine HCl .......................................... 0,001
Polysorbate 80 ....................................... 0,200
Agar ...................................................... 15,000
Final pH 6,7 0,2
Directions
Suspend 61,2 g of powder into 1 L of distilled water and
let it soak . Heat to boiling with constant stirring. Distribute in suitable containers and sterilize in the autoclave at
121C for 15 minutes.
APT Broth
Ref. 02-026
Specication
Liquid version of the medium with the same name. It
is especially designed for the cultivation of lactic acid
bacteria.
Formula (in g/L)

Casein Peptone .................................... 12,500
Yeast Extract .......................................... 7,500
Sodium Chloride ..................................... 5,000
Dipotassium Phosphate ......................... 5,000
Trisodium Citrate .................................... 5,000
Dextrose ............................................... 10,000
Magnesium Sulfate ................................. 0,800
Manganous Chloride .............................. 0,140
Iron Sulfate ............................................. 0,040
Thiamine HCl .......................................... 0,001
Polysorbate 80 ....................................... 0,200
Final pH 6,7 0,2
Directions
Dissolve 46,2 g of powder into 1 L of distilled water,
heating up slightly if necessary. Distribute into suitable
containers and sterilize in the autoclave at 121C for 15
minutes.
Description
These two general purpose media (APT= All Purpose
with Tween), originally formulated by Evans and Niven,
have been used successfully for the isolation and cultivation of lactic acid bacteria that alter the food quality
and composition (especially meat) and require a high
thiamine level. For this reason, ignoring what peptone
and yeast extract could provide, the medium has been
complemented with an extra amount of thiamine.
Both versions, solid and liquid, have demonstrated their
efcacy for detection of lactobacilli that produce meat
greening. Moreover, if media are supplemented with
5% fruit juices, as APHA states, they are converted into
irreplaceable prospection media for any kind of food
biomodier.
However, without the inclusion of any inhibitory agent in
the formulation signies that the media have no selective
ability, and thus they can support the growth of almost all
the microbial types.
Technique
Usual detection technique for the bacteria those causes
the greening is as mentioned below:
Products to be examined are crushed carefully in Tryptone Water (Ref. 03-156), and with the same diluent a
dilution bank is prepared. From each dilution, APT Agar
plates are inoculated in mass and in triplicate, and they
are incubated at 32C for 48 hours. After the incubation
period, colonies are counted using usual technique, and
different types are selected. Every type is inoculated
in APT Broth, and is incubated at 32C for 24 hours or
more. From these pure cultures, streak on Frankfurt
sausages slices, and incubate those slices and also the
one without the culture as a Control, in a humid room
or atmosphere to verify its greening capacity. Final
identication is done by morphological and biochemical
characteristics.
References
EVANS, J.B. & C.F. NIVEN (1951) Nutrition of the heterofermentative lactobacilli that cause greening of cured
meat products J.Bact. 62:599
DEIBEL, R.H, J.B. EVANS & C.F. NIVEN (1957) Microbiological assay for the thiamin using Lactobacillus
viridescens. J. Bact. 74:818-821
DOWNES, F.P., K. ITO (2002) Compendium of methods
for the microbiological examination of food. 4rd. ed.
APHA. Washington.
21
Asparagine Broth
Ref. 02-271
Specication
Liquid medium for the presumptive assay and enumeration of Pseudomonas aeruginosa in bottled water by
MPN method.
Formula (in g/L)

Asparagine ................................................. 2,0
Magnesium sulfate ..................................... 0,5
Final pH 7,0 0,2
Directions
Dissolve 13,5 g of powder in 1 L of distilled water containing 8 mL of glycerol. Sterilize by ltration and distribute in tubes (10 mL/tube). To obtain broth of double
strength, dissolve 27 g of powder in 1 L of distilled water
containing 16 mL of glycerol.
Description
Asparagine medium is recommended for the microbiological analysis of bottled water. This is an excellent
enrichment medium for Pseudomonas aeruginosa, since
it is composed of a mineral base and the only carbon
source is asparagine. It may also be used in the multiple
tube technique in microbiological analysis of recreational
waters and as a presumptive test medium for the differentiation of non fermentative gramnegative bacteria.
Technique
Some standards suggests the viable enumeration by
MPN method with 5 tubes per series, inoculating 10 mL,
1 mL and 0,1 mL. All the tubes are incubated at 37C
for 48 hours. Growth, with or without pigmentation, is
estimated as a presumptive evidence of presence of
Pseudomonas aeruginosa. Enumeration is carried out
with MPN tables for 5 tubes (See MPN chapter in this
book).
Conrmation is performed subculturing a loop of each
tube in Acetamide Medium (Ref. 03-428).
References
PASCUAL ANDERSON, M.R. (1992) Microbiologa
Alimentaria. Diaz de Santos. Madrid.
22
Azide Dextrose Media

Azide Dextrose Broth
Ref. 02-343
Specication
Directions
Xn
R-22-32-52/53
S-7-46-61
Liquid and selective medium for the detection of streptococci in water, waste water and milk.
Meat extract ................................................ 4,5

Tryptose .................................................... 15,0
Dextrose ..................................................... 7,5
Sodium chloride .......................................... 7,5
Sodium azide .............................................. 0,2
Final pH 7,2 0,2
Directions
Dissolve 35 g of powder in 1 L of distilled water, heat if
necessary. Distribute into tubes and sterilize in the autoclave at 121C for 15 minutes. To make double strength
broth, dissolve 70 g of powder in 1 L of distilled water.
Description
Azide Dextrose Broth is formulated according to the
Standard Methods for the Examination of Water and
Wastewater.
Azide concentration in the medium restrains the growth
of gramnegative bacteria and lets a good growth of enterococci. This medium composition is very similar to the
Azide Dextrose Broth acc. to Rothe (Ref. 02-027), but
without phosphate buffering. Therefore, it is advisable to
use this medium with samples with a high salts concentration, to avoid any precipitation in the medium.
Azide Dextrose Broth acc. to

Rothe (Rothe Azide Broth)
Specication
Xn
R-22-32-52/53
S-7-46-61
Medium for the detection and enumeration of enterococci in water.
Formula (in g/L)

Meat peptone ........................................... 10,0
Casein peptone ........................................ 10,0
Dextrose ..................................................... 5,0
Sodium chloride .......................................... 5,0
Dipotassium hydrogen phosphate .............. 2,7
Potassium dihydrogen phosphate .............. 2,7
Sodium azide .............................................. 0,2
Final pH 7.0 0,2
Description
The Azide Dextrose Broth acc. to Rothe has been widely
used since 1948 for the detection of fecal streptococci. It
usually provides higher positive results than other similar
media. Its efciency is due to the Sodium azide, which
is both selective for enterococci and inhibitor of the accompanying ora through interference of the electron
transport chain.
This medium is also used for the primary enrichment of
food samples, particularly frozen vegetables.
Formula (in g/L)
Ref. 02-027
Dissolve 35,6 g in 1 L of distilled water. Heat if necessary to help dissolution. Divide into 10 mL portions and
pour into tubes. Sterilize by autoclaving at 121C for 15
minutes. As for the double strength medium, dissolve
71,2 g/L and proceed as indicated above.
Technique
Streptometry in Water
Add 10 mL of water to be examined to each one of three
tubes containing 10 mL of double strength medium.
Add 1 mL of sample to another three tubes containing
10 mL of medium each, of single strength. Then add 0.1
mL of water to each one of three tubes containing 10
mL of medium of single strength. Incubate at 37 C and
examine after 24 hours and 48 hours. All tubes which
show turbidity due to growth will be considered as PRESUMPTIVLY POSITIVE and will have to be conrmed on
EVA Broth (Ref.2-028). All tubes which result positive on
this second testing should be considered for the count
of the Most Probable Number (MPN).
When considering other type of samples, dilute them in
Ringer solution 1/4 or peptone water and then inoculate
the tubes as done before.
In case of the highly contaminated samples, dilutions
should be done before inoculation.
References
APHA-AWWA-WPCF (1980) Standard Methods for the
Examination of Water and Wastewater. 15th. Ed. APHA
Inc., Washington, D.C.
CLESCERI, L., A.E. GREENBERG, A.E. EATON (1998)
Standard Methods for the Examination of Water and
Wastewater. APHA-AWWA-WEA. Washington.
GUINEA, SANCHO y PARS. (1979) Anlisis
Microbiolgico de Aguas: Aspectos Aplicados. Ed.
Omega,Barcelona,.
ROTHE (1948) Illinois State Health Department.
DOWNES, F.C. & K.ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
23
Bacillus cereus Media

Bacillus cereus Agar (MYP Agar)
Ref. 01-262
Specication
Selective solid medium, according to Mossel, for the
isolation and identication of Bacillus cereus from food
samples, acc. ISO 7932 (2003) and ISO 21871:2006.
Formula (in g/L)

Peptone ................................................ 10,000
Mannitol ................................................ 10,000
Sodium chloride .................................... 10,000
Meat extract ............................................ 1,000
Phenol red .............................................. 0,025
Agar ...................................................... 15,000
Final pH 7,2 0,2
Directions
Suspend 46 g of powder in 900 mL of distilled water.

Let it soak and bring to boil. Sterilize in the autoclave at
121C for 15 minutes. Let it cool to 50C and then add
100 mL of Eggs Yolk Sterile Emulsion (Ref. 06-016) and
100 mg/L of Polymyxin (Ref. 06-021CASE). Homogenize
well and distribute into plates. Do not reheat or remelt
the complete medium.
Description
Mossels formulation is developed to detect and enumerate B.cereus in any kind of food, since it lets a good
differentiation and selection of these microorganisms.
Polymyxin addition inhibits most of accompanying
bacteria, but it does not affect the growth of B.cereus.
This bacteria do not ferment mannitol and thus there is
no change in the indicator around the colonies. On other
hand, due to lecithinase activity of B.cereus it produces
a halo or zone of white precipitate around the colonies.
A count of B.cereus over 100.000 cells/g of the food
sample is considered to be hazardous, since the accumulated phosphoril-choline may cause intoxication
symptoms in children. For this reason, besides common
isolation and identication, a viable enumeration must be
performed to evaluate the real population of cells.
Technique
According to the authors, dehydrated or dry samples

must follow a recovery process in the following way: 20
g of sample is mixed with 90 mL of Tryptone Water (Ref.
03-156) for a minimum period of 1 hour, at room temperature. Afterwards, add 90 mL more of Tryptone Water
and homogenize it. A dilution of 1:10 should be obtained.
If necessary, a decimal dilution bank using Tryptone water as the diluent can be prepared. With a Drigalsky loop
(Ref. 5-010), spread aliquotes of 0,1 mL over the surface
of the agar plates and let the agar medium absorb those
aliquots. Incubate the plates at 30C for 18-24 hours to
allow spore germination before giving denite results,
which are referred as B. cereus colonies per gram of
sample.
Suspected colonies show the following appearance:
irregular borders, pink colour and even purple in the
center, and with a halo of white precipitate. Colonies
with yellow halos must be discarded surely.
24
Sometimes,the confusion with other colonies of grampositive bacilli is possible, and hence to identity this,
conrmation has to be performed verifying the glucose
fermentation, gelatin degradation and nitrate reduction,
which are positive tests for Bacillus cereus.
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of

Microbiological Media. CRC Press. Londres.
CORRY, J.E.L., G.D.W. CURTIS & R.M. BAIRD. (2003)
Handbook of Culture Media for Food Microbiology. Elsevier
Sci. B.V. Amsterdam. The Netherlands.
DOWNES, F.P. & K. ITO (2001) Compendium of methods
for the microbiological examination of foods. 4th ed. APHA.
Washington DC. USA.
FIL-IDF 181:1998 Provisional Int. Standard. Dried Milk
Products. Enumeration of Bacillus cereus.- Most probable
number technique
ISO 7932 Standard (2004) 3rd ed. Microbiology of food
and animal feeding stuffs. Horizontal method for the
enumeration of presumptive Bacillus cereus. Colony count
technique at 30C.
ISO 21871 Standard (2006) Microbiology of food
and animal feeding stuffs.- Horizontal method for the
determination of low numbers of presumptive Bacillus
cereus.- Most probable number technique and detection
method.
MOSSEL, D.A.A., KOOPMAN. M.J. y JONGERIUS, E.
(1967) Enumeration of Bacillus cereus in foods. Appl.
Microbiol. 15:650-653.
PASCUAL ANDERSON, M.R (1992) Microbiologa
Alimentaria. Daz de Santos, S.A. Madrid.
Bacillus cereus Selective Agar (PEMBA)

Ref. 01-487
Specication
Selective solid medium for the enumeration of Bacillus
cereus in food, according to ISO 21871.
Formula (in g/L)

Peptone .................................................... 1,00
Mannitol .................................................. 10,00
Sodium chloride ........................................ 2,00
Disodium phosphate ................................. 2,50
Potassium phosphate ............................... 0,25
Brom thymol blue ..................................... 0,12
Sodium pyruvate .................................... 10,00
Agar ........................................................ 14,00
Final pH 7,2 0,2
Directions
Suspend 40 g of powder in 950 mL of distilled water. Let
it soak and bring to the boiling. Distribute into suitable
containers and sterilize by autoclaving at 121C for 15
minutes. Let it cool to 50C and then add 50 mL/L of
Eggs Yolk Sterile Emulsion (Ref. 06-016) and Polymyxin
B Sulfate (Ref. 06-021CASE) to reach a 100 U/mL concentration. Homogenize and pour into plates.
Bacillus cereus Media

Description
Bacillus Cereus Selective Agar is formulated according
to the Food Analysis Nordic Committee (NMLK) standards. This standard uses this medium and Blood Agar
Base (Ref. 01-352) simultaneously for the detection and
enumeration of B. cereus in any type of food. This medium can also be used to conrm doubtful colonies, and
in this last case Polymyxin addition may be ommitted.
determination of low numbers of presumptive Bacillus

cereus.- Most probable number technique and detection
method.
Ref. 01-262 Bacillus cereus Agar
Technique
NMLK proposes the simultaneous use of Bacillus cereus
Selective Agar and Blood Agar Base (Ref. 01-352). Both
media are inoculated by surface streaking with 0,1 mL
aliquotes which are spread with a Drigalsky loop. Both
series of plates are incubated at 30C for 24 hours.
Typical B.cereus colonies over Blood Agar are big, irregular, dirty white or grey-like colour with a hemolysis halo
surrounding them. In B.cereus Selective Agar, colonies
are blue, surrounded by a clear zone of egg yolk digestion (lecithinase positive).
If there is an equal amount of typical or similar colonies
in both the media, later conrmation is not necessary.
Control
References
ISO 21871 Standard (2006) Microbiology of food and
animal feeding stuffs.- Horizontal method for the
Bacillus cereus ATCC 10876
Baird Parker Agar Base (Eur. Phar. Medium O)

Ref. 01-030
Specication
Solid and selective culture medium for the screening of

staphylococci from a variety of samples, acc. to pharmacopeias and ISO standard.
Formula (in g/L)
Tryptone ................................................... 10,0

Sodium pyruvate ...................................... 10,0
Glycine ..................................................... 12,0
Meat extract ................................................ 5,0
Lithium chloride .......................................... 5,0
Yeast extract ............................................... 1,0
Agar ......................................................... 17,0
Final pH 7,0 0,2
Directions
Suspend 60 g in 950 mL of distilled water. Allow it to

soak and then bring to the boil with constant stirring.
Sterilize by autoclaving at 121 C for 15 minutes. Cool to
50 C and add 50 mL of Eggs Yolk Tellurite Sterile Emulsion (Ref. 06-026). Homogenize
and distribute into plates. Once prepared, the medium
must not be reheated nor sterilized again.
Description
The Baird Parker Agar Base is specially recommended

for the detection and enumeration of staphylococci in
food and other material, since it allows a good differentiation of coagulase-positive strains. The growth of the
accompanying bacteria is usually suppressed by the high

concentration in lithium, glycine and pyruvate. Lithium
and glycine enhances the growth of staphylococci. Even
if its high selectivity does not affect staphylococci it may
occasionally permit the growth of some Bacillus species,
yeast and very rarely, Proteus. The growth of Proteus
species can be suppressed by adding 50 mg/l of sulphamethazine.
The presence of tellurite and eggs yolk, which must
always be added to the medium after sterilization, allows
the differentiation of presumptly pathogenic staphylococcal colonies. A high correlation has been found between
the coagulase test and the presence of clearing zones
of lypolysis in this medium, which is due to the staphylococcal lecithinase. On the other hand, studies show that
almost 100% of coagulase-positive staphylococci are
capable of reducing tellurite, which produces black colonies, whereas other staphylococci can not always do so.
When using sterile reagents other than SCHARLAU
MICROBIOLOGY, the complete medium may be obtained by adding 50 mL sterile eggs yolk and 10 mL of
1% potassium tellurite solution. Plates should be used
on the same day of preparation or within 48 hours, to
avoid the loss of denition in the precipitated zones . The
basal medium, without the yolk or the tellurite, is perfectly stable and therefore can be repeatedly melted.
Technique
The inoculation is done by spreading 0,5 mL of sample

over each plate with a Drigalski loop (Ref. 5-010). After
18-24 hours of incubation at 35 C, select the colonies
25
Baird Parker Agar Base (Eur. Phar. Medium O)

which are black, shiny and convex with regular margins
surrounded by a zone of clearing. These can be presumptly identied as coagulase-positive Staphylococcus
aureus.
Colonial appearance after 24 h. at 35C:
Staphylococcus aureus: Black, shiny, convex, regular
margins, 1.0-1.5 mm diameter, surrounded by a
clearing zone of lipolysis 2-5 mm in width. May
produce wide opaque precipitate zones extending
into the cleared medium after 48 hours.
Other species of Staphylococcus: Black, usually dully,
with regular margins. Sometimes they are brown
with zones of clearing but these present wide
opaque zones.
Micrococcus spp: Brown, very small and without clearing.
Bacillus spp: Various shades of brown, big. May produce
clearing after 48 hours.
Yeasts: White, big and smooth.
The eggs yolk emulsion can be prepared by mixing
a fresh eggs yolk with an equivalent quantity (w/v) of
saline solution. Sterilize by ltration and aseptically add
to the medium. This reagentss reference, already sterilized, is SCHARLAU 6-016.
The potassium tellurite solution is prepared by dissolving 3,5 g potassium tellurite in 100 mL distilled water.
Sterilize by ltration. This sterile reagents SCHARLAU
MICROBIOLOGY reference is 6-011.
Although these solutions can be mixed to be added
to the Baird Parker Agar Base forming the commonly
known Egg Yolk Tellurite Sterile Emulsion (Ref. 06-026),
they are also stable as the separate supplement and can
be used in many other culture media.
References
ATLAS R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. Londres
BAIRD-PARKER, A.C. (1962) An improved diagnostic
and selective mdium for isolating coagulase-positive
staphylococci. J. Appl. Bact. 25:12.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 4rd
ed. APHA. Washington. USA
EUROPEAN PHARMACOPOEIA (2007) 5 ed. Suppl.
5.6 2.6.13 Microbiological examination of non-sterile
products. EDQM Council of Europe. Strasbourg.
ISO 5944:2001 Standard. Milk and Milk based products
Detection of coagulase positive staphylococci MPN
Technique. Geneva.
ISO 6888-1:1999 Standard.Microbiology of food and
animal feeding stuffs Horizontal method for the enumeration of coagulase-positive staphylococci Part 1
Technique using Baird-Parker Agar mdium. Geneva.
ISO 22718:2006 Standard. Cosmetics Detection of
Staphylococcus aureus.
FIL-IDF 60:2001 Standard. Lat et produits base de
lait Dtection des staphylocoques coagulase positive
Technique du nombre le plus probable. Brussels.
USP 29 NF 25 (2006) <61> Microbial Limit Tests. US
Phamacopoeial Conv. Inc. Rockville. Md, USA
ZANGERL, P. & H. ASPERGER. (2003) Media used
in the detection and enumeration of Staphylococcus
aureus in Handbook of Culture Media for Food Microbiology Corry et als. Eds. Elsevier Sci. BV msterdam
Bile Esculin Media

Bile Esculin Agar
Ref. 01-265
Specication
Solid culture medium for the identication of probiotic
streptococci in food samples.
Formula (in g/L)

Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Bile .......................................................... 40,0
Ferric citrate................................................ 0,5
Esculin ........................................................ 1,0
Agar .......................................................... 15,0
Final pH 7,0 0,2
Directions
let it soak. Heat to boiling and distribute into containers.
Sterilize in the autoclave at 121C for 15 minutes.
26
Description
This medium formulation is based on the modication by
Facklam and Moody of the original formulation by Swan
to verify the esculin hydrolysing capacity of streptococci
and their resistance to bile salts which inhibit gram positive bacteria.
In fact, this medium can substitute KAA Conrmative
Agar (Ref. 01-263), but it has not the same selectivity.
Hence, it is used just as a substrate to verify the two assays simultaneously in the biochemical tests that identify
enterococci.
Technique
Assay is performed by inoculating the surface of a slant
with the pure culture that is going to be veried. After the
24 hours incubation at 35C, it might produce translucid
colonies, surrounded by black halos or zones, due to
esculin hydrolysis. Resistance to bile salts is indicated
by the growth.
References
LEUCHNER, R.G.K., J.BEW, K.J.DOMIG, &
W.KNEIFEL. (2002) A collaborative study of a method
for enumeration of probiotic enterococci in animal feed J.
Appl. Microbiol. 93:781-786
Bile Esculin Media

DEIBEL, R.H., HARTMAN, P.A. (1976) The enterococci,
Compendium of Methods for the Microbiological Examination of Food. APHA.
FACKLAM, R.R., MOODY, M.D. (1970) Presumptive
identication of group D streptococci: The bile-esculin
test. Appl. Microbiol. 20:245.
PASCUAL ANDERSON, MR. (1992) Microbiologa
Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN.
SWAN,A. (1954), The use of bile-esculin medium and
Maxteds technique of Lanceeld grouping in the identication of enterococci. J.Clin.Pathol. 7:160.
FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev.
A. APHA International. Gaitherburg, VA
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Prss. London.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological examination of Foods. APHA.
Washington
Bile Esculin Azide Agar

Ref. 01-592
Specication
Xn
R-22-32-52/53
S-7-46-61
Solid medium for the enterococci conrmative test in

water by the membrane ltration method according to
ISO 7899-2.
Formula (in g/L)

Tryptone ................................................. 17,00
Peptone .................................................... 3,00
Yeast extract ............................................. 5,00
Bile ......................................................... 10,00
Sodium chloride ....................................... 5,00
Esculin ...................................................... 1,00
Ammonium-ferric citrate ........................... 0,50
Sodium azide ............................................ 0,15
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
Suspend 56.6 g of powder in 1 L distilled water and bring
to the boil. Distribute in suitable containers and sterilize
in autoclave at 121C for 15 minutes. Cool to 50-60C
and pour plates to 3-5 mm thickness. These plates can
be stocked at 2-8C until two weeks.
Description
The Bile Esculin Azide Medium is a modication of the
classical Bile Esculin proposed by Isenberg, Goldberg
and Sampson in 1970, with a reduction in the amount of
bile and the addition of the sodium azide. Brodsky and
Schieman shown that this medium, also know as Pzer
Enterococci Selective gave best results with the ltration
technique.
The actual formulation is according the ISO Standard
7899-2:2000 for the second step in the conrmation and
enumeration of enterococci in water by the membrane
ltration method. The colonies previously selected in
the Slanetz-Bartley Agar (Ref. 01-579 + 6-023) must
be conrmed by a short incubation on the Bile Esculin
Azide Medium that permits the verication of the esculin
hydrolysis in a selective environment.
Technique
After an incubation of 24-48 hours on Slanetz Bartley
Agar (Ref. 01-579 + 6-023), the membrane lter that
show typical colonies is transferred, with sterile forceps
and upright position, to a pre-warmed plate of Bile
Esculin Azide Agar. After two hours of incubation at 44
0,5C the membrane lter is inspected. All the typical colonies that show a brown to black colour in the surrounding medium are considered as positives and enumerate
as intestinal enterococci.
A heterogeneous distribution of the colonies or the presence of abundant and different micro organisms can
interfere with the differentiation of positive colonies.
References
ISO Standard 7899-2 (2000) Water Quality. Detection
and enumeration of intestinal enterococci. Part 2: Membrane ltration method.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Boca Raton. Fla.
ISENBERG, H.D., D. GOLDBERG & J. SAMPSON
(1970) Laboratory studies with a selective enterococcus
medium. Appl. Microbiol. 20:433
BRODSKY M.H. & D.A. SCHIEMANN (1976) Evaluation of Pzer Selective Enterococcus and KF media for
recovery of fecal streptococci from water by membrane
ltration. Appl. Environ. Microbiol. 31:695-699
Ref. 01-592 Bile Esculin Azide Agar. Enterococcus faecalis ATCC 29212
27
Blood Media
Blood Agar Base
Ref. 01-352
Specication
General purpose medium for the isolation and cultivation
of microorganisms.
Formula (in g/L)

Meat extract .............................................. 10,0
Tryptone ................................................... 10,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 0,2
Directions
Suspend 40 g of powder in 950 mL. of distilled water and
let it soak. Heat to the boiling and distribute into containers. Sterilize by autoclaving at 121C for 15 minutes. Let
it cool to 45-50C and then add debrinated blood in a
proportion about 7% or the desired enrichment.
Description
Blood Agar Base may be used for the cultivation of non
fastidious microorganisms, since it has a balanced nutrient base .
For the fastidious microorganisms, it is advisable to add
special enrichments, such as ascitic liquid, egg yolk,
etc..
This medium, with the addition of blood, is very suitable
for studies in hemolytic activity, but for the isolation of
pathogens Blood Agar Base Columbia type (Ref. 01034) is more suitable.
References
ATLAS, R.M. and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, London.
Blood Agar Base No. 2

Ref. 01-505
Specication
Improved base with high nutrient properties especially
adapted for the growth of very fastidious
microorganisms, acc. ISO 7932 (2003).
Formula (in g/L)

Proteose peptone ..................................... 15,0
Liver extract ................................................ 2,5
Yeast extract ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,0 0,2
Directions
Suspend 42,5 g in 950 mL of distilled water and bring to
boil. Distribute into asks and sterilize by autoclaving at
121C for 15 minutes. Cool to 45-50C and aseptically
add 7% of sterile debrinated blood. Mix gently and pour
into plates.
Note: Blood and medium should be mixed in a big ask
to assure proper blood oxidation and mixing.
Description
Blood Agar Base No. 2 allows a maximum recovery of
weak organisms without altering or interfering in their
hemolytic reactions. Compared to other Blood Agar
bases, this one shows an equal or higher stimulatory
growth ability, however it specially helps the formation of
pigment in the chromogenic bacteria.
References
CASMAN, E. (1947) A noninfusion blood agar base for
neisseriae, pneumococci and streptococci. Am. J. Clin
Path. 17:281-289
Washington
ISO 7932 Standard (2003). Microbiology of food and
animal feeding stuffs. Horizontal Methods for the enumeration of presumptive Bacillus cereus. Colony count
tecnique at 30C.
Blood Agar Base (Columbia)

Ref. 01-034
Specication
Medium especially rich in peptone, appropriate for blood
addition or to prepare Chocolate Agar.
Formula (in g/L)
Casein Peptone ........................................ 12,0
Meat peptone ........................................... 11,0
Starch ......................................................... 1,5
Sodium Chloride ......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 0,2
Directions
Add 44,5 g of powder to 950 mL of distilled water and
bring it to the boil. Distribute into suitable containers and
sterilize at 121C for 15 minutes. To obtain Blood Agar
cool it to 45-50C and aseptically add sterile debrinated
blood at 5% proportion.
Description
Blood Agar Base contains an equilibrated mixture of
meat and casein peptones, being suitable for preparing
selective and as diagnostic media with the addition of
28
Blood Media
blood or inhibitors. As it is presented, without additions, it
is also an excellent general culture medium.
Generally, Blood Agar base contains a casein peptone,
that aids big size colonies formation, or a meat petone,
that provides a well dened hemolysis halos or zones.
Blood Agar Base is prepared according to the Columbia
University formulation, and meets the two conditions
mentioned above.
Some applications for this base are:

Base Agar without either enrichment and inhibitors:
This medium supports growth of normal microorganisms as enterobacteriaand othyers more
strengh as Pasteurella, Brucella and Clostridium
perfringens.
Clostridium Selective Base Agar: Should a selective
clostridium medium be desired, add 240 mg/L
Sodium Azide and 180 mg/l Neomycin before the
sterilization, or alternatively the contents of SC
Inhibitor container (Ref. 06-012CASE).
Blood Agar: Aseptically add to the sterile medium 5%
sterile debrinated sheep blood and cool it to
45C. This way, medium is enriched and allows
the determination of typical hemolytic reactions
necessary for the identication of enterococci,
staphylococci and other microorganisms.
Selective Gram-positive cocci Blood Agar: Simultaneously at the time blood addition, also add 10
mg/L of colistine and 15 mg/L of Nalidxic Acid,
or, the contents of a CP Inhibitor container (Ref.
06-013CASE), to obtain an excellent selective
medium for gram-positive cocci.
Note: Some authors recommend Blood Agar Base as
the maintenance medium for Campylobacter.
References
CASMAN, E. (1947) A non infusion blood agar base for
neiseriae, pneumococci and streptococci. Am. J. Clin.
Path. 17:281-289.
Blood Azide Agar Base
Specication
Tryptose .................................................. 10,00

Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Sodium azide ............................................ 0,20
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
Technique
Ref. 01-242
Formula (in g/L)
Xn
R-22-32-52/53
S-7-46-61
Selective basal medium for staphylococci and streptococci isolation. With blood added can be used for the
determination of haemolytic reactions.
Suspend 33 g of powder in 1 L of distilled water and

sterilize in autoclave at 121C for 15 minutes. Cool to 4550C and aseptically add 5% of debrinated blood. Pour
in sterile plates.
Description
This medium is a nutrient base (Tryptose and Meat
extract) with a suitable osmotic value (Sodium chloride) that includes a selective agent (Sodium azide) to
suppressing the growth of gram-negative bacteria. The
addition of 5% debrinated blood supplies growth factors
for the fastidious microorganisms and is used for the
determination of the haemolytic patterns.
Technique
The plates are inoculated by the surface striking and
stabbing the agar several times to deposit inoculum beneath the agar surface, to show the hemolytic reaction of
both oxygen-stable and oxygen-labile streptolysins. After
an incubation of 18-24 and 48 ours in a suitable environment the hemolytic reactions are displayed:
Alpha-()-haemolysis is the reduction of haemoglobin to met-haemoglobin that sows a greenish
decolourisation of the medium surrounding the
colony.
Beta-()-haemolysis is the total lysis of erythrocytes that produce a clear zone surrounding the
colony.
Gamma-()-haemolysis means no haemolysis
and there is no change in the medium
Alpha-prime-()-haemolysis is a small zone of
complete haemolysis that is surrounded by area
of partial lysis around the colony.
In the haemolysis studies must be in mind that the
haemolytic reactions are affected by several conditions:
atmosphere (aerobic, anaerobic or CO2 enriched) of incubation, the composition of the culture media (presence
of sugars or growth factors) and the source of the blood
(horse rabbit, sheep, human)
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. London
ISENBERG, H.D. (1992) Clinical microbiology procedures handbook. Vol. I ASM. Washington DC.
PACKER, R.A (1943) The use of sodium azide as an
inhibition substance of gram-negative bacteria. J. Infect.
Dis. 67:113
29
Blood Media
RUOFF, K.L. (1995) Streptococcus p. 299-305, in P.R.
Murray et al. (ed.) Manual of Clinical Microbiology. 6th
ed. ASM Washington DC.
Blood Tryptose Agar Base

Ref. 01-551
Specication
Solid highly nutrient medium developed as Base for
Blood Agar for the isolation and cultivation of fastidious
microorganisms.
Formula (in g/L)

Tryptose ................................................. 10,00
Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Agar ........................................................ 15,00
Final pH 7,3 0,2
Directions
Suspend 33 g of powder in 1 L of distilled water and let
it soak. Bring to the boiling and distribute into suitable
containers. Sterilize autoclaving at 121C for 15 minutes.
Let it cool to 45-50C and then add 5-10% debrinated
blood or the suitable enrichment. Homogenize and pour
plates.
Description
Casman proposed Blood Tryptose Agar Base in 1947 as
alternative medium without tissue infusion components.
Their original formulations include dextrose that interferes with haemolytic reactions and it is omitted in the
present formulation.
This medium, with the addition of blood, is very suitable
for studies in haemolytic activity, but to isolate pathogens
Blood Agar Base Columbia type (Ref. 1-034) is more
suitable. This medium support the growth of a wide variety of fastidious microorganisms but several species of
Streptococcus and Neisseria require the addition of 1 g/L
of Yeast Extract for the optimal growth.
Technique
The plates are inoculate by the surface striking and stabbing the agar several times to deposit inoculum beneath
the agar surface, to show the hemolytic reaction of both
oxygen-stable and oxygen-labile streptolysins. After an
incubation of 18-24 and 48 ours in a suitable environment the hemolytic reactions are displayed:
Alpha- ()-haemolysis is the reduction of haemoglobin to met-haemoglobin that sows a greenish
decolourisation of the medium surrounding the
colony.
Beta- ()-haemolysis is the total lysis of erythrocytes that produce a clear zone surrounding the
colony.
30
Gamma- ()-haemolysis means no haemolysis

and there is no change in the medium
Alpha-prime- ()-haemolysis is a small zone of
complete haemolysis that is surrounded by area
of partial lysis around the colony.
In the haemolysis studies must be in mind that the
haemolytic reactions are affected by the environmental
conditions (aerobic, anaerobic or CO2 enriched) of incubation, the composition of the culture media (presence
of sugars or growth factors) and the source of the blood
(horse rabbit, sheep, human)
References
Microbiological Media. CRC Press. London.
BALLOWS, A. & W.J. HAUSLER (1981) Diagnostic Procedures for Bacterial, Mycotic and Parasitic Infections 6th
Ed. APHA Washington D.C.
CASMAN, E.P. (1947) A non-infusion blood agar base for
neisseriae, pneumococci and streptococci. Am. J. Clin.
Pathol. 17:281-289
HARMON, S.M. et al. (1998) FDA Bacteriological
Analytical Manual. 8th ed. AOAC International. Gaithersburg.
ISENBERG, H.D. (1992) Clinical Microbiology Procedures Handbook. Vol. I ASM. Washington DC.
BPRM Broth Base

Ref. 02-409
Specication
Base medium to cultivate Bacteroides fragilis and phage
recovery from the environmental samples according to
ISO Standard 10705-4.
Formula (in g/L)

Peptone .................................................. 10,00
Tryptone ................................................. 10,00
Yeast extract ............................................. 2,00
Sodium chloride ........................................ 5,00
Glucose .................................................... 1,80
L-Cysteine HCl ......................................... 0,50
Final pH 6,8 0,2
Directions
Dissolve 29,42 g of powder in 1 L of distilled water. Add
1 mL of a CaCl2 5% solution. Distribute in asks and
sterilize in the autoclave at 121C for 15 minutes. Cool
to 40C and add 1 mL/L of 1% sterile soution of hemin
prepared in 0,02% NaOH. Mix well. Just before the
utilization add 25mL/L of a sterile solution of 10,6% (w/v)
NaCO3. Adjust the pH to 7,0 with HCl.
Description
BPRM (Bacteroides Phage Recovery Medium) was
formulated according the specications by the Microbiology Department of the Barcelona University. Tartera
and cols. veried its efciency in the Bacteroides fragilis
phage recovery used as human fecal pollution indicators in environmental samples. Initially Kanamycin (100
mg/L) and Vancomycin (7,5 mg/L) were added to the
medium to prevent the growth of unwanted accompanying bacteria, but later the Vancomycin was replaced by
Nalidixic acid (100 mg/L).
BPRM Basal Broth can be used in double strengh and,
with the addition of adequate amounts of agar, in the
preparation of solid or semisolid media.
References
TARTERA, C., R. ARAUJO, T. MICHEL and JOFRE
(1992) Culture and decontamination methods affecting
enumeration of phages infecting Bacteroides fragilis in
sewage. Appl. Environm. Microbiol. 58:8:2670-2673.
ISO Standard 10705-4 (2001) Water Quality-Detection
and enumeration of bacterigophages. Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis.
31
Brain Heart Infusion Media (BHI Media)

Brain Heart Infusion Agar
(BHI Agar)
Ref. 01-599
Specication
General purpose solid medium for various fastidious
pathogenic microorganisms.
Formula (in g/L)

Brain Extract ............................................. 12,5
Heart Extract .............................................. 5,0
Sodium chloride .......................................... 5,0
Di-sodium phosphate ................................. 2,5
Dextrose ..................................................... 2,0
Agar .......................................................... 15,0
Final pH 7,4 0,2
Directions
bring to the boil . Distribute in tubes or asks and sterilize by autoclaving at 121C for 15 minutes.
Brain Heart Infusion Broth

(BHI Broth)
Ref. 02-599
Specication
Liquid culture medium for general purpose according to
ISO 5944.
Formula (in g/L)

Brain extract ............................................. 12,5
Heart extract ............................................... 5,0
Dextrose ..................................................... 2,0
Sodium chloride .......................................... 5,0
Final pH 7,4 0,2
Directions
Dissolve 37 g of powder in 1 L of distilled water, heating
up if necessary. Distribute into containers and sterilize in
32
Description
Brain Heart Infusion is used for the cultivation of fastidious bacteria (streptococci, pnemococci, meningococci,
etc.) and also is recommended for the cultivation of
pathogenic fungi.
Growth of the accompanying bacterial ora can be
almost completely suppresed by adding 20 I.U. penicillin
and 40 g streptomycin per mL culture medium.
If this medium is to be used for the selective isolation of
fastidious fungi (especially of Histoplasma capsulatum
and Blastomyces) add 10% sterile debrinated blood
and from mixinfected samples add also 0,05 g cycloheximide/mL and 0,5 g choramphenicol/mL.
This medium is not suitable for obtaining the characteristic hemolytic reactions even after addition of the blood
because of its glucose contents.
References
ROSENOW, E.C. (1919) Studies on elective localization.
J. Dent. Research. 1:205-209
HAYDEN R.L. (1923) Elective localization in the eye of
bacteria from infected teeth. Arch. Int. Med. 32:828-849
HOWELL(1948) The efciency of methods for the isolation of Histoplasma capsulatum Public. Health. Reports
63, 173, 178.
CONANT (1950) Diagnostic Procedures and Reagents
3rd Ed., APHA, Inc., New York, 452.
DIN Standard 10163. Bestimmung koagulase positiver
Staphylokokken.
APHA-AWWA-AWPC (1998) Standard methods for the
examination of Water and Wastewater. 20th ed. Washington.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Prss. London.
Washington
ISO 5944 Standard (2001) Milk and milk based products:
Detection of coagulase-positive staphylococci - MPN
Technique.
Brilliant Green Media

Brilliant Green Agar (BGA)
(Eur. Phar. Agar Medium L)
Ref. 01-203
Specication
Medium for Salmonella isolation, according to the European Pharmacopoeia
Formula (in g/L)

Meat peptone ....................................... 5,0000
Casein peptone .................................... 5,0000
Sodium chloride .................................... 5,0000
Yeast extract ......................................... 3,0000
Lactose ............................................... 10,0000
Sucrose .............................................. 10,0000
Phenol Red ........................................... 0,0800
Brilliant Green ....................................... 0,0125
Agar .................................................... 15,0000
Final pH 7,0 0,2
Directions
Suspend 53 g of powder in 1 L of distilled water and heat
to boiling with constant stirring. Dispense into containers
and sterilize at 121C for 15 minutes.
Description
BGA is a differential selective medium, able to detect
the presence of enteropathogenic bacteria in different
samples. This medium is a modication to Kauffmans
original formulation, and it complies with the ISO, HMO,
Eur. Phar., USP and APHA
specications.
Since it has a high brilliant green concentration, it inhibits
notably the growth of most bacteria, except Salmonella.
However, S. typhi and S. paratyphi are also inhibited.
Therefore, when their presence or Shigella is suspected,
it is recommended to use other media in parallel, as
Deoxycholate Lactose Agar (Ref. 01-057), MacConkey
Agar (Ref. 01-118), Salmonella Shigella Agar (Ref. 01171), Xylose Lysine Deoxycholate Agar (Ref. 01-211 or
1-552) or Endo Agar Base (Ref. 01-589) which are less
inhibitory.
Presence of lactose and sucrose allows a good differentiation between Salmonella, which produce pink
or colourless colonies with a red halo or zone, and the
companion ora, which produce smaller and green yellowish colonies with yellow halo, due to acid created by
lactose and/or sucrose fermentation.
Osborn and Stokes suggested the addition of 0,08 g/L of
sulfadiacine or 1 g/L of sulfapyridin in order to make this
medium more selective for Salmonella and provide suitable qualities to this medium to perform the examination
of food and eggs and their derivatives.
References
U.S.PHARMACOPOEIA (2002) 25 ed. Chap. <61> Microbial Limit Tests. USP/NF Conv.Inc. Rockville.MD
KAUFFMAN, F. (1935) Weitere Erfahrungen mit der
kombinierten Anreicherungsverfahren fr Salmonellabazillen Z. Hyg. Infekt. Krhn, 117; 26-32
EUROPEAN PHARMACOPOEIA,Supplement 4.2
(2002), 4th Ed., Council of Europe,Strasbourg, France
Brilliant Green Bile 2% Broth

Ref. 02-041
Specication
Liquid medium for the detection of coliforms in water,
as recommended by APHA and ISO 4831 and 9308-1
Standards.
Formula (in g/L)

Bile ....................................................... 20,000
Lactose ................................................. 10,000
Peptone ................................................ 10,000
Brilliant Green ......................................... 0,013
Final pH 7,2 0,2
Directions
Dissolve 40 g of powder in 1 L of distilled water and
bring to the boil. Distribute into containers containing
Durham tubes and sterilize by autoclaving at 121C for
15 minutes.
Description
Brilliant Green Bile 2% Broth has been widely used as
a medium for the assay of presumptive colimetries in
food, milk and water, through the Most Probable Number
Technique. This broth offers some advantages over
other similar broths as its balanced composition of bile
and Brilliant Green effectively suppresses the growth of
gram-positive bacteria, even that of the more fastidious
Clostridium perfringens.
It is recommended by the APHA for colimetries in water,
milk and food.
British and Australian methodology use the broth as an
intermediate stage between presumptive and conrmative colimetry, as it was an enrichment at 32C. Other
authors suggest it as an optimal base for the Eijkman
testing of gas production at 44C, for the identication
of E. coli.
This medium can be used as Presumptive broth for
E.coli (by uorescent reaction) if before sterilization
MUG (Ref. 06-102CASE) is added.
References
APHA (1985) Standard Methods for the Examination of
Water and Wastewater, 16th ed. Washington.
VANDERZANT, C., SPLITTSTOESSER, D.F.(1992)
Compendium of Methods for the Microbiological Examination of Food. 3rd. Ed. APHA. Washington.
33
Brilliant Green Media

DOWNES F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4rd. Ed.
APHA. Washington.
FDA (1998). Bacteriological Analitical Manual.8th ed.
Rev. A AOAC International, Gaithersburg MD..
PASCUAL ANDERSON, MR (1992) Microbiologia
Alimentaria. Diaz de Santos, S.A.,Madrid.
ISO 4831 Standard (1991) General guidance for the
enumeration of coliforms. MPN technique.
ISO 9308-1. Standard (1990) Water Quality-Detection
and enumeration of coliforms, thermotolerants coliforms
and presumptive E.coli. MPN Method.
Brilliant Green Modied Agar

Ref. 01-309
Specication
Solid culture medium for selective isolation of Salmonellae in food (except S. typhi) according ISO 6579, 6340,
6785 and IDF 93 Standards.
Formula (in g/L)

Peptone ................................................ 10,000
Meat extract ............................................ 5,000
Yeast extract ........................................... 3,000
Lactose ................................................. 10,000
Sucrose ................................................ 10,000
Sodium phosphate ................................. 0,600
Phenol red .............................................. 0,090
Brilliant green ......................................... 0,005
Agar ...................................................... 15,000
Final pH 6,9 0,2
Directions
Suspend 54,5 g of powder in 1 L of distilled water. Let it
soak and heat up to boiling with constant stirring. Distribute in plates. Do not autoclave.
This formulation was subsequently adopted by the ISO

and DIN ofcial method for detecting Salmonellae in
meat.
Technique
A previous enrichment in Tetrathionate Base Broth
(Ref.2-033) is recommended. Inoculate on the surface
of this plate medium in order to get separate colonies.
Incubate at 35-37C for a 18-24 hours period.
Salmonella colonies (except S.typhi) are red, pinkish or
white, but they are always surrounded by a red halo or
zone, which shows the non lactose or sucrose fermentation. Colonies of lactose and/or sucrose fermenting
bacteria produce yellow-green colonies surrounded by a
yellow halo. Sometimes, Proteus or Pseudomonas may
appear, and they produce red pointed colonies.
In very polluted samples, it is recommended to include
1 g/L of sodium sulfacetamide and 250 mg/L of sodium
mandelate.
References
DIN. 10181 Mikrobiologische Milchuntersuchung. Nachweis von Salmonellen. Referenzverfahren.
DIN 10160. Untersuchung von eisch und eischerzengnissen. Nachweis von Salmonellen. Referenzverfahren
ISO Standard 6579 Meat and meat Product. Detection
of Salmonellae. Reference Method. (1993)
PASCUAL ANDERSON MR (1992) Microbiologa Alimentaria. Diaz de Santos, S.A.Madrid,.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,Boca Raton, Fla.
ISO 6785 Standard (2001) Milk and milk products - Detection of Salmonella spp.
FIL-IDF Standard 93 (2001) Milk and milk based products - Detection of Salmonella spp.
ISO 6340 Standard (1995) Water Quality - Detection of
Salmonella spp.
Description
In this modication of the classical medium for Salmonellae, the concentration of brilliant green has been
reduced to obtain a less inhibitory medium. At the same
time, the nutrient basis has been enriched to enhance
the recovery of those microorganisms that are weakened
during the food production process.
Ref. 01-203 Brilliant Green Agar.
Salmonella
typhimurium
ATCC 14028
E. coli
ATCC 25922
Ref. 02-041 Brilliant Green Bile 2% Broth. Left: control;

center: E.coli ATCC 25922; right: Salmonella typhimurium ATCC 14028
Control
34
Bromcresol Purple Base Broth

Ref. 02-031
Specication
Liquid culture medium with indicator to carry out sugar
fermentation studies.
Formula (in g/L)

Gelatin peptone ...................................... 10,00
Sodium chloride ........................................ 5,00
Bromocresol purple .................................. 0,02
Final pH 6,8 0,2
Directions
Dissolve 15 g of powder in 1 litre of distilled water. Add
substrate to assay in the desired concentration and
distribute into containers provided with Durhams tubes.
Sterilize in the autoclave at 121C for 10 minutes. Heat
up the autoclave before putting in the tubes to avoid
sugar caramelization.
Addition of some kind of sugars may require a pH adjustment.
To study the fermentation of some sugars like Glucose
(Ref. 06-048), Lactose (Ref. 06-051), Maltose (Ref. 06052), Mannitol (Ref. 06-050) and Sucrose (Ref. 06-049)
it is advisable to add 10 g/L of each one.
Description
Bromcresol Purple Base Broth is the liquid version suitable to determine gas production (by Durhams tubes)
by enterobacteria which are sensitive to phenol red.
In the SCHARLAU formulation, meat extract has been
omitted as it was found unnecessary and it also provided low concentrations of fermentable sugars that could
change or give erroneous results.
The bacteria when ferment the carbohydrates, changes
the medium colour to yellow due to Bromcresol purple
pH indicator, and if they produce gas, it is retained in the
Durhams tube.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,Boca Raton, Fla.
Brucella Media
Brucella Agar
Brucella Broth
Ref. 01-042
Ref. 02-042
Specication
Specication
Solid medium for the cultivation of Brucella and other

fastidious microorganisms
Liquid culture medium for Brucella and other fastidious

microorganisms
Formula (in g/L)
Formula (in g/L)
Peptone of casein ..................................... 10,0

Meat Peptone ........................................... 10,0
Yeast Extract .............................................. 2,0
D(+) Glucose ............................................. 1,0
Sodium chloride .......................................... 5,0
Sodium bisulphite ...................................... 0,1
Agar .......................................................... 15,0
Final pH 7,0 0,2
Peptone of casein ..................................... 10,0

Meat Peptone ........................................... 10,0
Yeast Extract .............................................. 2,0
D(+) Glucose ............................................. 1,0
Sodium chloride .......................................... 5,0
Sodium bisulphite ...................................... 0,1
Final pH 7,0 0,2
Directions
bring to the boil. Distribute into suitable containers and
sterilize in autoclave at 121C for 15 minutes. If a high
selectivity medium is wanted, add 2 asks of Brucella
Selective Supplement (Ref. 06-025CASE). When Ethyl
Violet 1,25 mg/L is added to the culture medium, the
growth of biotype 2 of Brucella abortus is inhibited.
Some microbiologists promote this medium as suitable
for Campylobacter maintenance.
Directions
Dissolve 28 g of powder in 1 L of distilled water, heating if necessary. Distribute in suitable containers and
sterilize in autoclave at 121C for 15 minutes. If a highly
selectivity is wanted, add aseptically after autoclave,
Brucella Selective Supplement (Ref. 06-025CASE).
When Ethyl Violet 1,25 mg/L is added to the culture
medium, the growth of biotype 2 of Brucella abortus is
inhibited.
35
Brucella Media
Description
References
The Brucella Media are prepared from composition of

the APHAs Albimi Broth used for isolation of Brucella
species and the only difference between Broth and Agar
is the solidifying agent. Both media are suitable for the
isolation and cultivation of a lot of fastidious microorganisms including Streptococcus, Neisseria and Campylobacter, but they became selective with the addition of
antibiotics like polymyxin or bacitracin or chemical inhibitors like cycloheximide and ethyl violet. With some dyes
(fuchsin and thionin) the media became differential. See
the suitable reference for the technique in every case.
ALTON, G.G, L.M. JONES & D.E. PIETZ (1976) Las

tcnicas de laboratorio en la Brucelosis, 21 ed. Monograph n.55 FAO/WHO. Geneve.
CRUICKSHANK.(1965) Medical Microbiology. 11th ed.
E.S. Livingstone. Edimburgo.
ISENBERG H.D. (1992) Clinical Microbiology Procedures Handbook. ASM. Washington D.C.
MacFADDIN J.D. (1985) Media for Isolation-cultivationidentication-maintenance of medical bacteria. William &
Wilkins Baltimore MD.
VANDERZANT, C & D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbiological examination of food 3rd Ed. APHA. Washington D.C.
Caution
Brucella species are classied as Biosafety Level
3 pathogens. All manipulations with live cultures and
antigens must be conned to a Class II Biological Safety
Cabinet. Follow proper established laboratory procedures in handling and disposing of infectious materials.
Bryant & Burkey Media

Bryant & Burkey Lactate Broth
(BB-Lactate Broth)
Ref. 02-421
Specication
Liquid medium for the enumeration of spores of lactatefermenting clostridia in milk and dairy products
Formula (in g/L)

Peptone .............................................. 15,0000
Meat extract .......................................... 7,5000
Yeast extract ......................................... 5,0000
L-Cysteine HCl ..................................... 0,5000
Resarzurine .......................................... 0,0025
Sodium acetate .................................... 5,0000
Sodium lactate ...................................... 5,0000
Final pH 6,2 0,2
Directions
up only if necessary. Distribute in suitable containers for
every procedure and sterilize in the autoclave at 121C
for 15 minutes.
36
Description
BB medium is used to enumerate, by MPN technique,
the spores of gasogenic clostridia that are the producers of the swelling and rancidness of cheese in the dairy
industry (late blowing or butyric swelling). In normal
conditions of use,the medium allows the growth of other
microorganims also which are not directly related to the
cheese alteration, e.g. Cl. butyricum or Cl. sporogenes,
besides the main responsible organism Cl. tyrobutyricum, in presence of enough acetate, Clostridium tyrobutyricum ferments lactate, producing acetic and butyric
acids, CO2 and hydrogen.
Technique
Recommended technique is to enumerate the spores by
the MPN technique with fresh made medium, covered
with a vaspar layer of 2 mL that acts as a cap that assures a low redox potential and at the same time retains
gas produced in the reaction.
Sample must be previously decontaminated by heating up for 10 minutes at 75C in order to destroy all the
vegetative forms and only leaving the alive spores.
Incubation is performed for 7 days at 37C. Tubes will
be declared positive if they show a clear gas production. Despite the fact that results are often expressed as
butyric spores/mL, the limitations of the method suggest
that they should be expressed as lactate fermenting
clostridial spores.
Bryant & Burkey Media

Bryant & Burkey Modied
Fluid Medium
Ref. 03-557
Specication
Fluid medium for the enumeration of spores of lactatefermenting clostridia in dairy products
Formula (in g/L)

Tryptose .............................................. 15,0000
Meat Extract ......................................... 7,5000
Yeast Extract ........................................ 5,0000
Cystine HCl........................................... 0,6000
Resazurine ........................................... 0,0025
Sodium lactate ..................................... 3,0000
Sodium acetate .................................... 5,0000
Sodium thioglycollate ........................... 0,2000
Agar ...................................................... 0,7500
Final pH 5,9 0,2
maintaining its anaerobic condition by the thioglycollate

added. Also the amount of lactate is reduced because
the density of the medium retains easily the gas bubbles
produced.
Technique
Samples are processed by established procedures in
every product.
References
BERGRE, J. L. & S. SIVELA (1989) Detection and enumeration of clostridial spores related to cheese quality.
Classical and new method. FIL-IDF Bull. 51:18-23
BRYANT M.P. & L.A. BURKEY (1956) The characteristics of lactate-fermenting spore-forming anaerobes from
silage. J. Bacteriol. 71:43-46
ROSENBERGER, K.F. (1951) The development of methods for the study of obligate anaerobes in silage. Proc.
Soc. Appl. Bacteriol. 14:161-164
Directions
bring to the boil. Distribute in suitable containers for
every procedure and sterilize in autoclave at 121C for
15 minutes.
Description
This modication of the Bryant & Burkey Lactate Broth
(Ref. 02-421) add a little amount of agar that makes
the medium more thick to be used in greater volumes
Buffered Peptone Water Eur Phar (Buffered

Sodium Chloride-Peptone Solution pH 7.0)
Ref. 02-494
Specication
Diluent for the homogenization of samples according to
the European Pharmacopeia and ISO 21149.
Formula (in g/L)

Peptone .................................................... 1,00
Sodium chloride ........................................ 4,30
Final pH 7,0 0,2
Description
This solution is recommended by European Pharmacopoeia to dilute the sample for microbiological examination. Depending on the amount of fat in the sample to
examine it is the technicians decision regarding the kind
and quantity of emulsifying agent to be used.
References
EUROPEAN PHARMACOPOEIA (2007) 5 ed. Suppl.
5.6 2.6.13 Microbiological examination of non-sterile
products. EDQM Council of Europe. Strasbourg.
ISO 21149:2006 Cosmetics Enumeration and detection of aerobic mesophilic bacteria.
Directions
up if necessary. Add 1 to 10 mL of Polysorbate 80 (Ref.
06-088) or Polysorbate 20 depending on the type of food
to be diluted. Homogenize and distribute into containers.
Sterilize by autoclaving at 121C for 15 minutes.
37
Casein Lecithin Polysorbate Broth Base

Ref. 02-539
Specication
Liquid medium to dilute and neutralize samples of pharmaceutical, cosmetic, raw material or end-products for
the purpose of enumeration
Formula (in g/L)

Casein peptone ..................................... 20,00
Soya lecithin ............................................ 5,00
Final pH 7,3 0,2
Description
This medium is produced according the formulation of
the U.S. Pharmacopoeia. In the Section <61> Microbial
Limit Tests is proposed as alternative system to neutralize preservatives and didsinfectants before to proceed
with the enumeration process, specially by the membrane ltration method.
References
US PHARMACOPOEIA (2002) <61> Microbial Limits
Tests. 25th ed. US Pharmacopoeial Conv. Inc. Rockville.
MD
Directions
Dissolve 25 g of powder in 960 mL of distilled water prewarmed at 50C . Add 40 mL of polysorbate 20, homogenize and distribute in suitable containers. Sterilize in
Caseinate Agar
Ref. 01-569
Specication
Solid medium, acc. Standard Methods, for the detection
and enumeration of proteolytic organisms in dairy products
Formula (in g/L)

Tryptone ................................................... 5,00
Yeast extract ............................................. 2,50
Dextrose ................................................... 1,00
tri-Sodium citrate ...................................... 4,41
Sodium caseinate ................................... 10,00
Agar ........................................................ 15,00
Final pH 7,0 0,2
Directions
Description
Caseinate Agar is preferred to other media like Skimmed
Milk Agar for the detection of proteolytic organisms
because its better recovery of stressed cells and the
absence of false positives.
38
Technique
Pour the caseinate agar into plates so that the medium
is 2 mm thick (e.g. 12 mL for a 9-cm plate). After the medium is hardened, plate 0,1 mL quantities of the sample
or diluted sample on the agar surface and spread evenly
with a sterile Drigalski rod. To ensure absorption of the
sample allow inoculated plates to dry for 15 minutes,
and incubate them for 48 to 72 hours at 321C or for 72
hours at 211C. Colonies surrounded by a white or offwhite zone of casein precipitate are proteolytic. Highly
proteolytic bacteria will also produce a clear inner zone.
Report results as proteolytic count per gram or mL.
Designate the medium used and the incubation conditions.
References
MARTLEY, F.G., S.R. JAYASHANKAR & R.C. LAWRENCE. (1070) An improved agar medium for the detection of proteolytic organisms in total bacterial counts. J.
Appl. Bacteriol. 33:363-370
FRANK, J.F. G.L CHRISTEN & L.B. BULLERMAN
(1992) Test for groups of Microorganisms. In R. T.
Marshall (ed) Standard Methods for the examination of
Dairy Products. APHA. Washington D.C.
Cetrimide Agar (Pseudomonas Selective Agar)

(Eur. Phar. Agar Medium N)
Ref. 01-160
Specication
N
R-52/53
S-61
Solid culture medium for selective isolation of Pseudomonas aeruginosa acc. to ISO 22717.
Formula (in g/L)

Gelatin peptone ........................................ 20,0
Magnesium chloride ................................... 1,4
Potassium sulfate ..................................... 10,0
Cetyltrimethyl-Ammonium Bromide .......... 0,3
Agar .......................................................... 15,0
Final pH 7,2 0,2
Directions
add 10 mL of Glycerol. Bring to the boil and distribute
into suitable containers. Sterilize at 121C for 15 minutes.
Description
The Cetrimide Agar is based on the enormous resistance
of Ps. aeruginosa strains to the Quaternary Ammonium
Compounds (QACs). With regard to the CetyltrimethylAmmonium Bromide there has been growth at 1 g/L
concentrations, but in such cases it has been very poor
and slow.
An inhibitor concentration of 0,3-0,5 g/L does not seem
to affect the viability of the pyogenic species. Nevertheless, it does inhibit the rest of the fastidious accompanying bacteria, both gram-positive and gram-negative,
as well as other species of Pseudomonas which may
develop at lower inhibitory concentrations.
References
LOWBURY,E.J.L. & A.G. COLLINS (1955) The use of a
new cetrimide product in a selective medium for Pseudomonas aeruginosa J. Clin. Path. 8.47
BROWN, V.I. & J.L. LOWBURY (1965) Use of an improved Cetrimide Agar Medium and of culture methods
for Pseudomonas aeruginosa. J. Clin. Path. 18.752
FDA (1998) Bacteriological Analytical Manual. 8th ed.
Rev. A. AOAC International. Gaitherburg. VA.
Microbiological Media. CRC Press Inc.,Boca Raton,Fla.
EUROPEAN PHARMACOPOEIA,(2002) Supplement4.2
4th ed., 2.6.13 Microbiological examination of non-sterile
products. Council of Europe,Strasbourg,.
U.S. PHARMACOPOEIA (2002) 25 ed. <61> Microbial
limit tests. Us Pharmacopoeial Conv. Inc. Rockville, MD
ISO 22717:2006 Cosmetics Detection of Pseudomonas aeruginosa.
control
Pseudomonas aeruginosa ATCC

27853
Although Ps. aeruginosa prevails over any other fastidious bacteria after a 48 hour incubation at 35C, it is
recommended to rst isolate at 42C with an incubation
of 48 hours. By this method, almost complete inhibition
of other microorganisms is obtained.
39
Chapman-Stone Agar
Ref. 01-052
Specication
Solid and differential medium with a high selective ability,
for the isolation of staphylococci from food.
ment mannitol (biqger size) and the colonies that do not

ferment mannitol (smaller size) greater, since the latter
must grow solely depending on peptone as the nutrient
source.
Technique
Material under test is inoculated on the surface to produce separated colonies, and is incubated at 30C for
a 48 hours period. After this time, examine and select
colonies on the basis of these criteria:
White or non pigmented colonies are discarded, even if
they show a gelatin liquefaction halo (coagulase negative).
Golden yellow pigmented colonies, surrounded by a
clear zone of gelatin hydrolysis (positive Stones reaction) are selected to verify mannitol fermentation and
further, coagulase and hemolysis.
Formula (in g/L)

Peptone .................................................... 10,0
Yeast extract ............................................... 2,0
Gelatin ...................................................... 30,0
D-Mannitol ................................................ 10,0
Sodium chloride ........................................ 55,0
Agar .......................................................... 15,0
Final pH 7,0 0,2
Directions
heat up in boiling water bath until the total dissolution of
gelatin. Bring to the boiling. Distribute in tubes or asks
and sterilize at 121C for 15 minutes. Pour into plates
immediately. Avoid overheating.
Description
Chapman-Stone medium is according to a modication
of the classical 110 medium for Staphylococcus. The
main modication consists the inclusion of ammonium
sulfate, that allows the direct reading or observation of
gelatin hidrolysis, instead of adding reagents to the plate
medium. Another modication is the reduction in the
amount of sodium chloride. The saline content is selective itself, but it is reinforced by the ammonium sulfate,
which is also selective. Finally, lactose has been omitted
, making the difference between the colonies that fer-
Mannitol fermentation is checked by adding a few drops

of Bromcresol purple indicator (aq. sol. 0,04%) over the
colonies. Any colour change indicates positive mannitol
fermentation.
Coagulase production and hemolysis tests are performed in later subcultures on suitable media.
Chapman-Stone medium composition is very selective,
and so, medium can be employed without real sterilization. However, it is always advisable to sterilize it and
keep one control before use.
References
CHAPMAN (1948).An improved Stone Medium for the
isolation and testing of food-poisoning staphylococci
Food Research 13:100-105
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.London
Ref. 01-053 Chapman TTC Agar.
control
E. coli ATCC 25922

(MF technique)
40
E. coli ATCC 25922
Salmonella typhimurium ATCC 14028
Salmonella typhimurium
ATCC 14028
(MF technique)
Chapman TTC Agar (Tergitol 7 Agar)

Ref. 01-053
Specication
Medium for the detection of coliforms by membrane ltration technique in water analysis acc. ISO 9308-1:2000
standard.
Formula (in g/L)

Meat peptone ......................................... 10,00
Meat extract .............................................. 5,00
Lactose .................................................. 20,00
Yeast extract ............................................. 6,00
Bromothymol blue ................................... 0,05
Sodium heptadecyl sulfate ....................... 0,10
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
Suspend 56,2 g in 1 L of distilled water and bring to the
boil. Distribute into suitable containers and sterilize by
autoclaving at 121C for 15 minutes. Cool to 45-50C.
Add 2-3 mL/L of lter sterile 1% aqueous 2,3,5-triphenyltetrazolium chloride (TTC) (Ref. 06-023) and pour
plates. Do not reheat.
Description
This medium is formulated for the presumptive identication of coliforms in drinking water, by membrane
ltration technique.
Due to the instability the triphenyltetrazolium is provided
in a separate container, sterilized and ready to use.
Poured plates can be stored refrigerated for up to 8 days
without losing their effectiveness. They should not be
used if any dehydration or drying signs appear.
Technique
While using the membrane lter technique for the presumptive identication of coliforms in water, it should be
kept in mind that to every type of water corresponds a
minimum volume to be ltered and depends on the type
of water. Dilute with sterile phosphate buffer if necessary
to obtain the number of colonies on the membrane which
are easy to count.
For every water sample two volumes or double quantity

must be ltered over different membranes and incubated
on Tergitol 7 Agar at 35C and 44C respectively.
After 48 hours the typical colonies have the appearance
shown in the table.
Most coliform can not grow on this medium when incubated at 44C, except E. coli which forms a characteristic appearance.
Results are always expressed per 100 mL sample including any applied dilutions. Estimation is done by taking typical colonies which have grown at 35C as fecal
coliform, together with those grown at 44C as E.coli.
Nevertheless, according to the legislation and despite
the mediums selectivity, results can only be considered
as presumptive and all coliform colonies have to be conrmed by following the criteria stated below:
Typical appearance in EMB Agar (Ref.1-068) or Endo
Agar Base (Ref.1-589); characteristic reactions in Kligler
Iron Agar (Ref.1-103). For the conrmation of faecal E.
coli, verication of it is: a motile,gram-negative bacillus
and lactose fermentator with acid and gas production,
which gives negative results on the citrate test and
indole production positive.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc. London.
CHAPMAN G.H. (1951). A culture medium for detecting and conrming E. coli in ten hours. Am. J. Publ. Hlth
41:1381-1386.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 3rd
ed. APHA.Washington.
GUINEA, SANCHO,PARES (1979). Anlisis Microbiolgico de Aguas. Ed. Omega. Barcelona.
ISO 9309-1:2000 Standard. Water Quality. Detection and
enumeration of Escherichia coli and coliform bacteria.Part 1: Membrane ltration method.
SPECK, M (Ed.) (1982) Compendium of Methods for the
Microbiological Examination of Foods. 2nd. Ed. APHA.
Washington.
41
Chloramphenicol Glucose Media

Chloramphenicol Glucose Agar
(CGA) (Yeast Extract Dextrose
Chloramphenicol Agar
acc. FIL-IDF)
T
Ref. 01-366
Specication
Chloramphenicol Glucose Broth

(CGB) (Yeast Extract Dextrose
Chloramphenicol Broth
acc. FIL-IDF)
R-45
S-53-45
Solid and selective medium for the isolation and enumeration of fungi in milk and dairy products according
ISO 7954.
Formula (in g/L)

Dextrose ................................................... 20,0
Yeast extract ............................................... 5,0
Chloramphenicol ........................................ 0,1
Agar .......................................................... 15,0
Final pH 6,6 0,2
Directions
it soak. Bring to the boil and distribute into containers.
Sterilize in the autoclave at 121C for 15 minutes.
Description
This is the medium recommended by the Federation International Laitrere - International Dairy Federation (FILIDF) for the isolation and enumeration of fungi (moulds
and yeast) in milk and dairy products. This medium has
also been adopted by the DIN and ISO standard.
Medium relies its selectivity on the bactericidal action of
Chloramphenicol which, due to its thermostabity, may
be sterilized with the complete medium in the autoclave.
Moreover, pH may be adjusted near to neutrality, and
this fact allows the medium to be remelted several times
without affecting its stability, selectivity and efcacy.
Remeltings and overheatings may make the medium
darker.
Technique
Generally the mass seed method or poured plate
method is used to inoculate the medium, and an incubation at 22-25C for 4 to 5 days is carried out.
42
Ref. 02-366
Specication
T
R-45
S-53-45
Liquid and selective medium for the enumeration of fungi

in milk and dairy products using the MPN technique.
Formula (in g/L)

Dextrose ................................................... 20,0
Yeast extract ............................................... 5,0
Final pH 6,6 0,2
Directions
Dissolve 25 g of powder in 1 L of distilled water. Distribute in tubes and sterilize in the autoclave at 121C for 15
minutes.
Description
This is the liquid version of the medium with the same
name (Ref. 01-366) recommended by the Federation
International Laitrere - International Dairy Federation
(FIL-IDF) for the enumeration of fungi (moulds and
yeasts in liquid products).
Medium is specially adapted to enumerate yeast and,
eventually, moulds by the Most Probable Number technique (MPN).
References
FIL-IDF 94B Standard (1991). Enumeration of yeast and
moulds. Colony Count Technique at 25 C.
ISO 7954 Standard (1987) General guidance for enumeration of yeast and moulds - Colony count at 25C.
DIN Standard 10186. Mikrobiologische Milch Untersuchung. Bestimmung der Anzahl von Hefen und Schimmelpilzen. Referenzverfahren.
Citrate Azide Agar

Ref. 01-558
Specication
T
R-45-22-32-52/53
S-53-45-7-61
Selective differential solid medium for the detection and

enumeration of streptococci in dairy products.
Formula in g/L
Tryptone ................................................. 10,00
Yeast extract ........................................... 10,00
Sodium citrate ........................................ 20,00
Sodium azide ............................................ 0,40
Tetrazolium blue ....................................... 0,10
Agar ........................................................ 15,00
Final pH 7,0 0,2
Directions
Description
Citrate Azide Agar is the streptococci detection medium
preferred in dairy microbiology over the other media like
KF-Streptococcal Agar (Ref. 01-294) used in food microbiology because its high selectivity and security of use.
Technique
Pour the melted medium cooled to 45-50C over the
sample in a petri dish. Let it solidify and dose another
5-7 sterile volume of medium on the surface to facilitate
the micro-aerolic environment. The plates are incubated
at 37C for 48-72 hours and then the blue colonies are
counted. Express the results as streptococci per g or
mL of sample
References
MARSHALL, R.T. (1992) Standard Methods for the
Examination of Dairy Products. 16th Ed. APHA. Washington. DC
HARTMANN P.A, R.H. DEIBEL, & L.M. SIEVERDING
(1992) Enterococci. en Vanderzant & Splittstoesser:
Compendium of methods for the microbiological examination of foods. 3rd Ed. APHA. Washington DC.
EFTHYMIOU C.J., P. BACCASH, V.J. LABOMBARDI, &
D.S. EPSTEIN (1974) Improved isolation and differentiation of enterococci in cheese. Appl. Microbiol. 28:417422
SARASWAT, D.S. G.W. REINBOLD & W.S. CLARK
(1963) Selection of a medium for the isolation and enumeration of enterococci in dairy products. J. Milk Food
Technol. 26:114-118
43
CLED Agar (Brolacin Agar)

Ref. 01-047
Specication
Cystine,lactose,electrolyte decient medium, recommended for the isolation and identication of urinary
pathogenic bacteria.
Formula (in g/L)

Peptone ................................................. 4,000
Trypsic Peptone ..................................... 4,000
Meat extract ........................................... 3,000
Lactose ................................................. 10,000
L-Cystine ............................................... 0,128
Brom Thymol Blue ................................. 0,020
Agar ...................................................... 15,000
Final pH 7,4 0,2
Staphylococci coagulase(-ve): White or light yellow

colonies, with halo and the same size as of
Enterococci.
Proteus sp.: Blue, translucent and smaller than E.coli.
Pseudomonas aeruginosa: Plain, matt and wrinkled
colonies. Green colour and irregular boundary.
Corynebacteria: Pointed and grey colonies
Lactobacilli: Matt colonies, like Corynebacteria.
References
SANDYS, G.H. (1960) A new method of preventive
swarming of Proteus sp. J.Med.Lab.Tech. 17:224
MACKEY J.P. and G.H.SANDYS (1966) Diagnosis of
urinary tract infections. Brit. Med. J. 1.173
Directions
Add 36 g of powder to 1 L of distilled water and heat to
boiling. Sterilize by autoclaving at 121C for 15 minutes.
Description
This general purpose medium has been recommended
for bacteriological urine analysis. Current formulation is
a modication of the original one reported by Sandys,
that achieves an excellent colony differentiation without
inhibitors. This fact, and also the careful selection of nutritive components, makes this medium a substrate able
to support growth of most urinary
pathogenic bacteria.
Presence of lactose as a fermentable sugar allows classic differentiation and, at the same time, lack of electrolytes suppresses swarming waves on the members of
the Proteus species and sometimes growth of Shigella
sp. also.
The characteristics of colonies that grow on C.L.E.D.
Agar (after 18 hours of incubation):
Escherichia coli: Yellowish colonies, opaque, with core,
1,25 mm diameter. Non fermentative strains give
blue colonies.
Klebsiella sp.: Very mucuous colonies of variable colour,
from yellow to blue-white.
Salmonella sp.: Plain and blue colonies.
Enterococcus faecalis: Yellow colonies. 0,5 mm diameter.
Staphylococcus aureus: Convex yellow colonies. 0,75
mm diameter.
control
Proteus mirabilis ATCC 10975

44
CN Selective Agar Base for Pseudomonas

Ref. 01-609
N
R-52/53
S-61
Specications
Selective solid medium for the detection of Pseudomonas aeruginosa according the EN 12780-2002 and
ISO 16266 Standard.
Formula (in g/L)

Gelatine Peptone .................................... 16,00
Casein Peptone ...................................... 10,00
Potassium sulphate ................................ 10,00
Magnesium chloride ................................. 1,40
Cetrimide ................................................ 0,20
Agar ........................................................ 15,00
Final pH 7,1 0,2
Directions
Add 52,6 g of powder to 1 L of distilled water with 10
mL of glycerol. Heat to complete solution and sterilize in
autoclave at 121C for 15 min. Cool to 45-50C and add
to 500 mL of medium a ask of the Nalidixic Acid Selective Supplement (Ref. 06-124CASE). Homogenise and
pour plates.
Do not maintain the complete medium melted for more
than 4 hours. Do not re-melt. The nished plates can be
used without lose its efciency for one month if they are
refrigerated and in a dark place.
Description
The CN Selective Medium for Pseudomonas was progressively developed from the basic media of King, Ward
and Raney for the production of pigments. Browne and
Lowbury add the cetrimide as selective agent and Goto
and Enomoto improves efciency by adding nalidixic
acid. The presence of both inhibitors eliminates the contaminant microbiota from heavily polluted specimens and
was adopted by the CEN (Centre Europeen de Normalisation) in its EN Standard 12780 for the detection of Ps.
aeruginosa by ltering membrane in water.
control
Technique
A volume of the sample is ltered thorough a ltering
membrane of 0,45 m pore and the membrane is deposited on the surface of the CN medium. The plates are
incubated at 362C for a period of 444 h with a partial
examination at 222 h.
All the colonies producing a green or blue (pyocianin)
pigmentation in this period must be considered as Pseudomonas aeruginosa without any other conrmation.
All the colonies that produces uorescence under the
Woods light (without pyocianin production) era considered presumptive Ps. aeruginosa and must be conrmate on Acetamide Medium (Ref. 03-428).
All the colonies producing a brown-reddish pigment and
no produces uorescence nor pyocianine are considered
presumptive Ps. aeruginosa and must be conrmed by
the oxidase test and typical growth on Acetamide Medium (Ref. 03-428) and King B Agar (Ref. 01-029)
References
KING, E.O., M.K. WARD & E.E. RANEY (1954) Two
simple media for the demonstration of pyocianin and
uorescein. J. Lab. Clin. Med. 44:301
BROWN, V.L. & E.J.L. LOWBURY (1965) Use of an improved Cetrimide Agar Medium and of culture methods
for Ps. aeruginosa. J., Clin. Pathol. 18:752
GOTO S. & S. ENOMOTO (1970) Nalidixic acid cetrimide agar. A new selective plating medium for the selective isolation of Ps. aeruginosa. Jpn. J. Microbiol. 14:65
ROBIN, T. & J.M. JANDA (1984) Enhanced recovery of
Ps. aruginosa from diverse clinical specimens on a new
selective agar. Diag. Microbiol. Infect Dis. 2:207
EN STANDARD 12780 (2002) Water Quality. Detection
and enumeration of Ps. aeruginosa by membrane ltration. Brussels.
ISO 16266:2006 Standard. Water Quality Detection
and enumeration of Pseudomonas aeruginosa. Method
by membrane ltration.
Pseudomonas aeruginosa ATCC 27853

45
Cosmetic Diluent of Beerens

Ref. 02-257
Specication
Diluent to neutralize preservative systems in common
examination of cosmetic products.
Formula (in g/L)

Lecithine ................................................... 3,00
Sodium thiosulfate .................................... 5,00
L-Histidine HCl ......................................... 1,00
Peptone .................................................... 1,00
Sodium chloride ........................................ 8,50
Final pH 7,0 0,2
Directions
Dissolve 19,5 g of powder in 1 L of distilled water containing 30 mL of polysorbate 80.(Ref. 06-080). Distribute
into suitable containers and sterilize by autoclaving at
Let it cool to 50C and shake gently to redissolve polysorbate.
Description
Cosmetic diluent of Beerens has all the necessary
compounds to neutralize most of the chemical agents
included in cosmetic products to maintain and preserve it
free of microorganisms.
It complies with the EU recommendation that states that
before any microbiological examination, a treatment to
remove all the growth inhibitor systems in the cosmetics
must be performed.
However, this standard also declares that later dilutions
have to be performed in less aggressive media, that
may be considered as an enrichment and revitalization
system, and it suggests Letheen Broth (Ref. 02-236) or
Letheen Modied Broth (Ref. 02-237).
References
BEERENS, H., RAMONS, C., LEMAIRE, D. (1976). Rev.
Inst. Past. Lyon. 9:127.
BRIGIDI, P., MATTEUZZI, D. (1982) II Farmaco Ed. Pr.
37:8:260. Commission del Communautes Europeennes,
Groupe Special. Methodes de Controle Microbiologique
des Produits Cosmetiques: Limites Numeriques Applicables au controle Ofciel de la Qualit Microbiologique
des Produits Cosmetiques. XI/405A, ISPRA, 1976.
m-CP Agar Base

Ref. 01-513
Specication
Solid medium for the enumeration and isolation of
Clostridium perfringens in water acc. the European
Directive 12767/97
Formula (in g/L)

Tryptose .................................................. 30,00
Yeast extract ........................................... 20,00
Sucrose .................................................... 5,00
L-Cisteine HCl .......................................... 1,00
Magnesium sulfate.7H2O .......................... 0,10
Agar ........................................................ 15,00
Final pH 7,6 0,2
Directions
bring to the boil. Distributein suitable containers and
sterilize in autoclave at 121C for 15 minutes. Cool to
45-50C and aseptically add 2 asks of Selective Supplement m-CP (Ref. 06-125CASE) with the following
composition:
D-Cicloserine ............................................. 400 mg/L
Polymyxin B sulphate .................................. 25 mg/L
Indoxil-b-D-Glucoside .................................. 60 mg/L
Fenolftalein diphosphate ........................... 100 mg/L
Iron (III) Chloride ......................................... 90 mg/L
46
Homogenize avoiding bubble formation and pour in

plates.
Description
The m-CP Agar Base is a solid medium for counting
and isolating vegetative cells and spores of Clostridium
perfringens by the membrane ltration method. Its use is
compulsory in determining the quality of water for human
consumption in European Union by Directive 12767 (1207-1997) of the European Council.
Technique
A suitable volume of water is ltered through a membrane lter of 47 mm diameter and 0,45 mm pore. Put
the membrane on the surface of a plate of m-CP medium
freshly prepared and incubate in an anaerobic atmosphere at 441C for 213 hours. Expose the growth
obtained to amonium hydroxide vapours for 20-30 seconds. Count as Clostridium perfringens all the opaque
yellow colonies that turn to pink or red after the ammonium hydroxide exposure. Express the results as cfu/mL.
References
European Council (1998) Directive 98/83/CE on the
quality of the water destined to the human consuption.
EC Bull. 11-03-1998.
m-CP Agar Base
control
Clostridium perfringens
before the exposure
to Ammonia vapours (AM0257)
Clostridium perfringens
after the exposure
to Ammonia vapours (AM0257)
Cystine-Tryptone Fluid Medium (CTA Medium)

Ref. 03-045
Specication
Basic medium for strain maintenance or sugar fermentation assays. Its uidity makes it suitable to observe the
motility.
Formula (in g/L)

L-Cystine HCl ......................................... 0,500
Casein peptone .................................... 20,000
Sodium chloride ...................................... 5,000
Sodium sulte ......................................... 0,500
Phenol red .............................................. 0,017
Agar ........................................................ 2,500
Final pH 7,3 0,2
Directions
heat to boiling. Dispense and sterilize at 121C for 15
minutes. Addition of certain sugars may require a pH
readjustment.
Description
Cystine and Tryptone uid medium is appropiate for the
propagation and maintenance of bacterial strains, even
the fastidious ones, without additives.
The lack of fermentative sugars allows the long survival
of microorganims, and sometimes it has been used for
studies on severe microaerials fermentation, due to the
fact that these microorganisms do not grow on current
basal media. Medium has its maximum efcacy when
freshly prepared, but it can be stored for long periods
of time, taking care to avoid its dehydration. To achieve
this, screw caps or hermetic sealing are strongly recommended. In any case, if medium has been stored for a
long time, it is better to put it in a boiling water bath for a
few minutes before using it.
Technique
On this medium, we can maintain by deep stab a lot of
fastidious microorganisms like Neisseria, Enterococcus,
Pasteurella, Shigella and in most cases without needing
any additives or CO2. Even some light-sensitive anaerobic microorganisms can grow without special conditions
though in reduced atmopheres they give ideal growth on
this medium.
In the few cases as per the nutritive requirements of the
microorganisms like serum or ascitic liquid, which can
be added in an aseptic way to the molten medium, when
cooled to 45-50C.
Microorganism motility can be easily detected by growth
in the stab, and helped by the medium uidity.
Medium, with added sugars, may be used to study sugar
fermentation with microorganisms that do not grow on
phenol red classical media because of the additional
nutritional requirements. Acidication can be easily observed with the change in colour of phenol red indicator.
References
VERA, H.D. (1948) A simple medium for identication
and maintenance of the gonococcus and other bacteria.
J.Bact. 55:531
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.
47
Czapek-Dox Media
Czapek-Dox Agar
Ref. 01-051
Specication
Semisynthetic solid medium for the cultivation of fungi,
with sodium nitrate as the only nitrogen source.
Formula (in g/L)

Sucrose .................................................. 30,00
Sodium nitrate .......................................... 2,00
Magnesium glycerophosphate ................. 0,50
Potassium chloride ................................... 0,50
Ferrous sulfate ......................................... 0,01
Agar ........................................................ 15,00
Final pH 6,8 0,2
Directions
heat to boiling. Dispense and sterilize by autoclaving at
121C for 15 minutes. Should a lower pH be desired,
acidify the medium with sterile lactic acid after cooling to
45C.
Czapek-Dox Broth
Ref. 02-051
Specication
Semisynthetic liquid culture medium with sodium nitrate
as the only nitrogen source.
lactic acid 10% solution, producing a pH drop to 3,5. In

this medium, Warcup adds 5 g/L of Yeast extract (Ref.
07-079) and an antibiotic mixture (Streptomycin 30 mg/L
and Tetracycline 2 mg/L) to achieve total efcacy and
selectivity. In any case, bacterial growth is very poor.
Czapek-Dox medium has been adopted for the morphological studies of soil fungi, and it aids chlamydospora
formation by Candida albicans in shorter periods (Dawson, 1962). Temperatures and times of incubation always varies, and they can go from 1 to 5 weeks at room
temperature. Usually, it is 8-15 days at 15C. In case of
Candida, 28C for 48 hours seems to be optimum ; for
Penicillium, 22C and nonetheless, Aspergillus grows
better at 30-32C.
References
CZAPEK, F. (1903) Untersuchung uber die sticstoffgewinnungund einweissbildung der Panze.Beitr. Chem.
Physiol. Pathol. 1:540
DOX, A.W. (1910) The intracellular enzymes of Penicilliumand Aspergillus with special references to those of P.
camemberti. US Dept Agr. Bur. Animal Ind. Bull. 120:70
examination of Water and Wastewater. 18th ed. APHA.
Washington.
RAPER, K.B. & D.J.FENELL (1965) The genus Aspergillus. William & Wilkins Co. Baltimore.
THOM,C & M.B. CHURCH (1926 The aspergilli. William
& Wilkins Co. Baltimore.
WARCUP, J.H. (1950) The soil-plate method for isolation
of fungi from soil. Natur 166:117-118
Formula (in g/L)

Sucrose .................................................. 30,00
Sodium nitrate .......................................... 2,00
Potassium chloride ................................... 0,50
Magnesium glycerophosphate ................. 0,50
Ferrous Sulfate ........................................ 0,01
Final pH 6,8 0,2
Directions
Dissolve 33,5 g of powder in 1 L of distilled water. Dispense in suitable containers and sterilize in the autoclave at 121C for 15 minutes. Do not overheat
Description
Czapek-Dox medium, both liquid and solid version, is a
general cultivation medium of dened chemical composition, where the sole nitrogen source is sodium nitrate,
and the carbon source is sucrose. It has been employed
successfully in the isolation and cultivation of soil microorganisms especially fungi.
Its original version had a pH near neutrality, but it can be
rendered selective medium for the fungi by adding, (after
sterilization and before solidication), 10 mL of sterile
48
Fusarium sp.
D/E Neutralizing Agar

Ref. 01-610
Specication
Xi
R-43
S-24-37
Solid culture medium for the neutralization and testing of

antiseptics and disinfectants acc. ISO 22717 and 22718
standards.
Formula (in g/L)

Tryptone ................................................... 5,00
Yeast extract ............................................. 2,50
Dextrose ................................................. 10,00
Lecithin ..................................................... 7,00
Sodium thiosulphate ................................. 6,00
Sodium sulphite ........................................ 2,50
Sodium thioglycollate ............................... 1,00
Polysorbate 80 ......................................... 5,00
Bromcresol purple .................................... 0,02
Agar ........................................................ 15,00
Final pH 7,6 0,2
Directions
sterilize in autoclave at 121C for 15 minutes. The appearance of precipitates is normal and do not interferes
the results.
Description
Dey & Engley developed this medium in 1983 to recovery chemically damaged staphylococci, At the present
its use is generalized for testing by the contact method
(RODAC Plates) the efciency of antiseptics and disinfectants on impervious surfaces. The present formulation
incorporate neutralizing substances for almost all the
active products used as antiseptics and disinfectants.
Lecithin neutralizes quaternary ammonium compounds
(QACs); Polysorbate acts on phenolics and formalin;
thioglycollate neutralizes the organic-mercurial compounds; thiosulfate-sulte inactive halogen-compounds
and lecithin + polysorbate neutralizes ethanol and other
alcoholic compounds.
Technique
When the RODAC (Replicate Organisms Detection and
Counting) plates are lled in the laboratory be careful
with the meniscus of the agar: It should rise above the
rim of the plate to give a slightly convex surface to make
a proper contact with the surface to be sampled.
For sampling remove the cover of the RODAC plate and
carefully press the agar surface to the surface being
sampled. Make certain that the entire agar meniscus
contacts the surface. Replace the cover and incubate
in an inverted position under the time and temperature
conditions for the microorganisms in question. Express
the results as colonies per RODAC plate or Colonies
per cm2
References
DEY, B.P. & F.B. ENGLEY (1983) Methodology for
recovery of chemically treated Staphilococcus aureus
with neutralizing medium. Appl. Environm. Microbiol.
453:1533-1537
HICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW
(1992) Microbiological tests for equipment, containers,
water and air. In R.T. Marshall (Ed.) Standard Methods
for the examination of Dairy Products 16th ed. APHA
Washington.
EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F.
FRANK (2001) Microbiological Monitoring of the Food
Processing Environment. In DOWNES & ITO (Eds)
Compendium of Methods for the Microbiological Examination of Foods. 4th ed. APHA. Washington DC.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Culture Media. CRC Press. Boca Ratn, Fla.
Pseudomonas aeruginosa.
49
Decarboxylase Lysine Broth acc. to Taylor

Ref. 02-336
Technique
Specication
Liquid medium to differentiate enteric bacteria in the
L-Lysine decarboxylation assays according to ISO 6785,
21567 and IDF 93 Standards
Formula (in g/L)

Yeast extract ........................................... 3,000
D(+)Glucose ........................................... 1,000
Bromcresol purple .................................. 0,016
L-Lysine .................................................. 5,000
Final pH 6,8 0,2
It is advisable to use a vaseline seal to avoid spontaneous oxidation. The use of glucose in anaerobic conditions produces an acidication of the medium, the indicator will then turn to yellow if there is growth. The use of
the aminoacid will acidify the medium again, and then
it turns to grey and nally to violet, thereby showing the
positive reaction. The observations of these biochemical tests are performed after an incubation period of 24
hours at 37C.
References
Directions
Dissolve 9 g of powder in 1 L of distilled water. Distribute
in thin tubes in an amount of 2 or 5 mL per tube. Sterilize in the autoclave at 121C for 10 minutes.
Description
The capacity to decarboxylate some aminoacids has
been widely employed to classify Enterobacteriaceae.
Taylors formulation, including lysine, has been recently
included in several standards for the identication of Salmonella. This modication shows an improved performance, in comparison to Falkows formulation.
DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of foods. APHA.
Washington. DC.
TAYLOR, W.I. (1961) Isolation of Salmonellae from Food
Supplies. V Determination of the Method Choice for
Enumeration of Salmonella. App. Microbiol. 9, 487-490.
FIL-IDF 93 Standard (2001) Detection of Salmonella
spp.
ISO 21567:2004 Food and feeding stuffs Horizontal
method for the detection of Shigella ssp.
Ref. 01-057 Deoxycholate Lactose Agar
control

50
Escherichia coli ATCC 25922
Deoxycholate Media
Deoxycholate Citrate Agar
(Eur. Phar. Medium J)
Ref. 01-056
Specication
Differential and moderately selective plating medium for
enteric pathogenic bacteria, according the European
Pharmacopoeia.
Formula (in g/L)

Peptone ................................................. 10,00
Meat extract ............................................ 10,00
Lactose ................................................... 10,00
Ferric Citrate ............................................. 1,00
Sodium Citrate ........................................ 20,00
Sodium deoxycholate .............................. 5,00
Neutral red ............................................... 0,02
Agar ........................................................ 15,00
Final pH 7,3 0,2
Directions
bring to the boil. Immediatelly pour into plates. Plates
may be used at once or refrigerated for a few days. Do
not autoclave or overheat.
Description
The European Pharmacopoeia formulation is one of
these modications to rhr original medium developed by
Leifson in 1935. The inhibition of gram-positive microorganisms is due primarily to its content of sodium deoxycholate, although the two citrate compounds also are
active inhibitors. The lactose achieves differentiation of
enteric bacilli. Organisms that ferment lactose produce
acid that, in presence of neutral red indicator, results in
the formation of red colonies. Lactose non-fermenting
produces colourless colonies. The black centres due to
the ferric sulphide depot detect the production of SH2.
Technique
Inoculate the specimen as soon as possible directly onto
surface of medium. Incubate the plates at 35 2C for
18-24 hours. Plates can be incubated for an additional
24 hours if no lactose-fermenting are observed.
Typical colonial morphology on Deoxycholate Citrate
Agar is as follows:
Escherichia coli: Large, at, rose-red
Enterobacter / Klesiella: Large, mucoid, pale with pink
centre.
Proteus: Large, colourless to tan.
Salmonella: Large, colourless to tan.
Shigella: Colourless to pink
Pseudomonas: Irregular, colourless to brown
Gram-positive bacteria: No growth to slight growth
References
LEIFSON, E. (1935) New culture media based on sodium deoxycholate for the isolation of intestinal patgens
and for the enumeration of colon bacilli in milk and water.
J. Path.. Bact. 40:581-599.
HYNES, M. (1942) The isolation of intestinal pathogens
by selective media. J. Path. Bact. 54:193-207
EUROPEAN PHARMACOPOEIA (2002) 4th ed. Suppl.
4.2 . 2.6.13 Test for specied micro-organisms. Council
of Europe. Strasbourg.
MAC FADDIN, J.F. (1985) Media for isolation-cultivationidentication-maintenance of medical bacteria. William &
Wilkins, Baltimore, MD.
ATLAS, R.M. & L.C. PARKS (1991) Handbook of Microbiological Media. CRC Press London.
Deoxycholate Lactose Agar

Ref. 01-057
Specicacion
Differential solid medium for the isolation of enterobacteria according to APHA.
Formula (in g/L)

Peptone .................................................. 10,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Sodium citrate .......................................... 2,00
Sodium deoxycholate ............................... 0,50
Neutral red ................................................ 0,03
Agar ........................................................ 15,00
Final pH 7,1 0,2
Directions
heat to the boil. Do not autoclave and pour into sterile
petri plates. The medium loses its efciency if overheated and so avoid autoclaving and remelting.
Description
The Deoxycholate-Lactose Agar is very close to the
Deoxycholate Agar, differing only in the deoxycholate
amount and in its reduced inhibitory power. The present
formulation is made according to the recommendation of
APHA and AOAC.
References
GREENBERG, A.E., L.S. CLESCERI & A.D. EATON
(1995) Standard Methods for the examination of Water
and Wastwater. 19th ed. APHA-AWWA-WEF. Washington D.C.
SPECK, M.L (1984) Compemdium of methods for the
microbiological examination of food.2nd ed. APHA.
Washington D.C.
Microbiological Media. CRC Press, Boca Raton, Fla.
51
Dextrose Media
Dextrose Agar
Dextrose Broth
Ref. 01-089
Ref. 02-089
Specication
Specication
General purpose solid culture medium.
Liquid culture medium for general purposes.
Formula (in g/L)
Formula (in g/L)
Meat peptone ............................................. 5,0

Casein peptone .......................................... 5,0
Meat extract ................................................ 3,0
D(+)Glucose ............................................. 10,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 6,9 0,2
Casein peptone ........................................ 10,0

D(+)Glucose ............................................... 5,0
Sodium chloride .......................................... 5,0
Final pH 7,2 0,2
Directions
heat to boiling . Sterilize by autoclaving at 121C for
15 minutes. Should the acid pH is required, add sterile
tartaric acid solution when the medium is at 45C. Do
not reheat.
Description
This solid culture medium is suitable for many objectives
and it supports growth of most non fastidious microorganisms. Adding 5% sterile debrinated blood to this medium, makes it an excellent culture medium which satisfy
nearly all kinds of nutritive needs, even for meningococci
and pneumococci, however due to its high glucose content it is not suitable for hemolytic reaction studies.
This formulation is also in accordance with the one
suggested for the study of frozen food, and also for
fruit juices. In this latter case, it is suggested to use it in
duplicate, and one of the samples must be acidied in
order to facilitate the growth of moulds and yeast in such
a selective way.
Acidication can be easily achievevd by adding 7-8 mL
of 10% sterile tartaric acid to the sterile, and cooled medium at 45C,producing a pH drop to 3,5 0,2. In these
conditions, do not remelt the medium, as then agar
tends to get hydrolysed and do not solidify again.
References
APHA (1958) Recommended Methods for the Microbiological Examination of Food. APHA. Inc, New York.
SPECK, M. L. (1984) Compendium of Methods for the
Microbiological Examination of Food. 2nd ed. APHA.
Washington.
VANDERZANT & SPLITTSTOESSER (1992) Compendium of Methods for the Microbiological Examination of
Food. 3rd ed. APHA. Washington.
Directions
up if necessary. Dispense into containers and sterilize by
autoclaving at 121C for 10 minutes.
Description
This version of Dextrose Broth is formulated without
meat extract in order to make dextrose the single carbohydrate in the medium. It is a liquid culture medium for
the faster growth, since most microorganisms can use
glucose as their energy source, but it has one drawback that it is not buffered, and the strong acidication
produced by fermentation may damage its maintenance.
Hence, though it is used many times in blood cultures, it
has been replaced by other, more buffered media.
Due to the relatively high proportion of glucose, it is
advisable to use it freshly prepared, and with short
sterilization period, as otherwise toxic furfurales may be
developed.
On the other hand, its simple formulation makes it the
best medium for checking gas production from glucose
if Durhams tubes are used, as it has no indicator that
could interfere with it.
References
APHA (1958) Recommended Methods for the Microbiological Examination of Food. APHA. Inc, New York.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food.4th ed.
APHA. Washington.
MacFADDIN, J.D. (1985) Media for isolation-cultivationidentication-maintenance of medical bacteria. William &
Wilkins Co. Baltimore
Dextrose Purple Broth

Ref. 02-416
Specication
Liquid medium for E.coli detection in water, according to
German legislation (DEV).
52
Dextrose Media
Formula (in g/L)
Peptone .................................................. 10,00
Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Dextrose ................................................. 10,00
Final pH 7,1 0,2
of foods, like Bacillus coagulans (causing the typical

at-sour) other Bacillus and Sporolactobacillus and the
thermophilic Bacillus stearothermophilus.
Technique
Dissolve 28 g of powder in 1 L of distilled water. Distribute into tubes with Durhams tubes and sterilize by
The samples or its dilutions are inoculated in the medium, melted and cooled to 50C. Then are poured in
petri dishes and incubated for 72 h at 32C (mesophiles)
or for 48 hours at 55C (thermophiles). After incubation
the acid-producing colonies can be easily enumerated
because they show a yellow zone that contrast with the
purple medium.
Description
References
Directions
Medium can be used for any assay of degradation of

sucrose. It has been adopted by the German Federal
Government for detecting E.coli in water, according to
Eijkmans test, which is based on the production of acid
and gas (yellow change of indicator) from sucrose after
an incubation of 20 hours at 44C (0,5).
References
DIN Normative (Standards) 38411 (1991) Teil 6 (Juni
1991): Mikrobiologische Verfahren (Gruppe K): Nachweis von Escherichia coli und coliformen keimen (K6).
DEUTSCHE EINHEITSVERFAHREN zur Wasser-,
Abwasser- Und Schlammuntersuchung. VCH Verlagsgesellschaft D-6940 Weinheim.
VERORDNUNG ber Trinkwasser und ber wasser fur
Lebensmittelbetreibe vom 12/12/1990. Bundesgesetzbl.
Teil I 2613-2629 (1990)
Dextrose Tryptone PB Agar

Ref. 01-556
Specication
Solid medium for the cultivating the at-sour food spoiling microorganisms
Formula (in g/L)

Tryptone ................................................. 10,00
Dextrose ................................................... 5,00
Bromocresol Purple .................................. 0,04
Agar ........................................................ 15,00
Final pH 6,9 0,2
Directions
Description
This medium was adopted in 1930 by the National Canners Association for the detection of the microorganisms
causing the at-sour spoilage in the canned foods.
Latter it was used for the detection and enumeration
of all the micro-organisms related with acid spoilage
NATIONAL CANNERS ASSOCIATION (1968) Laboratory Manual for food canners and Processors. Vol. 1.
NCA. Washington
DOWNES, F.P. & K. ITO (2001) Compendium of methods for the Microbiological Examination of Foods. 4th ed.
APHA. Washington.
HORWITZ, W. (2000) Ofcial Methods of Analysis.
AOAC International, Gaithersburg. MD.
Dextrose Tryptone PB Broth

Ref. 02-556
Specication
Liquid medium for the microbiological examination of
canned foods
Formula (in g/L)

Tryptone ................................................. 10,00
Dextrose ................................................... 5,00
Bromocresol Purple .................................. 0,04
Final pH 6,9 0,2
Directions
if necessary. Distribute in suitable containers and sterilize in autoclave at 121C for 15 minutes.
Description
Baumgartner and Hersom proposed this medium, in
1956 for the microbial examination of canned foods and
at the present it is recommended for all the medium and
low acidity (pH 4,5) canned or heat-processed foods.
The microbial growth is supported by the peptone and
the pH indicator that turns from purple to yellow detects
the acid-producers from glucose.
Technique
Volumes of 10-20 mL of medium in duplicate are inoculated with 1-2 g or mL of sample to detect the aerobic
microorganisms. For the anaerobic ones it is convenient
inoculate other series of tubes with a more reducer medium like Liver Broth (Ref. 02-098). The incubation must
be at 35 and 55C for both series.
53
Dextrose Media
References
BAUMGARTNER, J.G. & A.C. HERSOM (1956) Canned
Foods. 4th ed. Churchill Ltd. London
DOWNES F.P. & K. Ito (2001) Compendium of methods
for the Microbiological Examination of Foods. 4th ed.
APHA. Washington. D.C.
Dichloran Glycerin Selective Agar (DG18 Agar)

Ref. 01-485
Specication
T
R-45
S-53-45
Solid differential and low water activity medium for the

determination of xerophilic fungi from low moisture food.
Formula (in g/L)

Peptone ................................................. 5,000
Dextrose ............................................... 10,000
Monopotassium phosphate .................... 1,000
Dichloran ................................................ 0,002
Chloramphenicol .................................... 0,100
Agar ...................................................... 15,000
Final pH 5,6 0,2
Directions
Suspend 31,7 g of powder in 820 mL of distilled water
and heat to the boiling. Add 180 mL of glycerin. Distribute into suitable containers and sterilize by autoclaving
Description
Among the culture media for xerophilic fungi, those that
have played a more successful role are the ones which
include any agent that restrains the continuous growth
of zygomicete fungal colonies. Dichloran (Dichlorebenzalkonium cloride) and Rose bengal are two of those
inhibitors.
DG18 Agar medium is according to the formulation proposed by Hocking & Pitt in 1980, and it includes Dichloran which limits the size of fungal colonies more efciently than Rose bengal. Chloramphenicol inhibits the
bacterial growth very well and its thermostability allows it
to be included in the medium before sterilization.
54
The inclusion of 18% (w/v) of Glycerine makes the

medium with a water activity (aw) of 0,995 without causing any problems that generally appear when this water
activity is reached with sodium chloride or sugar.
Technique
It is advisable to inoculate in mass and thus a surface
inoculation is recommended. This seed may be by
surface streaking as well as swab spreading or by Drigaslky loop. Never use inoculum more than 0,1 mL.
According to the standardized technique, plates must be
incubated at 22-25C, with partial readings after 3 and
5 days, and a denitive readings after 7-8 days. Results
are expressed in cfu xerophiles/g or mL of food sample.
References
HOCKING, A.D., J.I. PITT (1980) Dichloran-glycerol medium for enumeration of xerophilic funghi from low-moisture food. Appl. Environm. Microbiol. 39:488-492
PITT, J.I., A.D. HOCKING, D.R. GLENN (1983) An
improved medium for the detection of Aspergillus avus
and A. parasiticus. J. Appl. Bacteriol. 54:109-114.
PITT, J.I., A.D. HOCKING (1985) Fungi and Food Spoilage. Academic Press: Sydney
SAMSON, R.A., E.S. HOEKSTRA (2001) Introduction
to the Food Borne fungi. 6th. Ed. Centralbureau voor
Schimmelcultures: Baarn.
Differential Reinforced Clostridial Medium (DRCM)

Ref. 02-410
Specication
Liquid medium for the enumeration of clostridia in food
samples and other products by MPN technique.
Formula (in g/L)

Peptone ................................................ 10,000
Meat extract ............................................ 8,000
Yeast extract ........................................... 1,000
Starch ..................................................... 1,000
Glucose .................................................. 1,000
L-Cysteine HCl ....................................... 0,500
Sodium acetate ...................................... 5,000
Sodium bisulte ...................................... 0,500
Ferric-ammonium citrate......................... 0,500
Resazurine ............................................. 0,002
Final pH 7,0 0,2
Directions
Dissolve 27,5 g of powder in 1 L of distilled water. Bring
to the boil, distribute in tubes and sterilize in the autoclave at 121C for 15 minutes.
Description
This medium is a modication by Freame and Fitzpatrick
of the Gibbs classic medium, to easily detect the presence of sulte reducing clostridia. The modication is,
mainly, an addition of sodium bisulte and ferric citrate,
that make colonies black and thus more conspicuously visible. The current version of this medium has no
agar in order to facilitate the blackness of the medium.
Resazurine,the redox indicator allows the verication
of anaerobiosis in the medium in the same assay. LCysteine acts as reducing agent in this medium.
Technique
Sample to be examined is distributed in tubes as per the
MPN technique, and is covered with parafn or vaseline
oil to help the anaerobiosis. The bank of tubes is kept in
a boiling water bath at 75C for 30 minutes to remove
all the dissolved oxygen and vegetative cells. Then,
incubate at 30C up to 7 days before concluding any
negative results.
Generally, the spores of sulfate reducing clostridia
germinate between the second and fourth day, and the
medium turns black, in which case the test is positive.
The medium can be rendered selective by the addition of
70 IU/mL of Polymyxin sulftate.
Prepared tubes without the inoculation may be stored
up to 2 weeks provided the resazurine band does not
show an excessive oxidation (more than a 1/3 part of the
column).
References
GIBBS, B.M., FREAME, B. (1965) Methods for the recovery Clostridia from food. J.Appl. Bact. 36:23-33
FREAME, B., FITZPATRICK, B.W.F. (1972) The use of
DRCM for the isolation and enumeration of Clostridia
from Food. In Isolation of Anaerobics. Ed. Shapton AND
Board. Academic Press. London.
DIN Standard 38411 (1991) Teil 6 (Juni 1991): Mikrobiologische Verfahren (Gruppe K): Nachweis von Escherichia coli und coliformen keimen (K6).
55
DNAse Agar
Ref. 01-346
Mannitol fermentation may be simultaneously determined if 10 g of mannitol and 0,025 g of phenol red are
added to 1 L of DNAse Agar, before sterilization. Positive
results in both tests will determine with more certainty
that the microorganism is a pathogenic Staphylococcus
aureus.
This medium is also useful to identify Serratia marcescens in clinical specimens, since it is a DNAse producer.
Davis (1967) modied the medium by adding toluidine
blue and crystal violet, and stated that gramnegative
DNAse producing bacilli that grew on this medium may
be accepted as Serratia species.
Specication
Solid culture medium for the determination of the deoxyribonuclease activity of microorganisms, especially of
staphylococci and Serratia sp.
Formula (in g/L)

Tryptose .................................................. 20,00
DNA ......................................................... 2,00
Sodium chloride ........................................ 5,00
Agar ........................................................ 15,00
Final pH 7,3 0,2
Tehnique
Directions
to the boiling with constant stirring. Distribute into suitable asks and sterilize in the autoclave at 121C for 15
minutes. Cool it to 50C and pour it into the plates.
Description
Jeffries, Holtman and Guse (1957) incorporated DNA
into a general medium with agar to study bacterial and
fungal DNAse production. Microorganisms that are able
to produce DNAse, break DNA, reducing it to nucleotide
fragments.
This reaction is observed by the appearance of a clear
zone surrounding the growth, the rest of the plate remaining turbid. The process is as follows: Hydrochloric
acid reacts with DNA producing white precipitates that
make the medium turbid, although it does not react with
nucleotide fragments.
The same authors also observed that there is a correlation between coagulase production and DNase activity,
thus DNAse Medium may be used as a laboratory test to
diagnose pathogenic staphylococci.
DNAse Agar plates are inoculated with the microorganism to be studied by thick streak or inoculating at the
bottom and are incubated at 35-37C for a 18-24 hours
period.
To read, ood the plates with 1N chlorhydric acid and
observe if there are any clear or transparent zones surrounding the streak. If the plate becomes totally turbid
without any clear zone then the test is Negative however
if any clear zone developes around the growth, then the
test is accepted as Positive.
References
JEFFRIES, C.D., D.F. HOLTMAN y D.G. GUSE (1957).
Rapid Method for Determining the Activity of Microorganisms on NucleicAcids. J. Bacteriol. 73:590-591
DISALVO, J.W. (1958). Desoxyrribonuclease and
Coagulase Activity of Micrococci. Med. Tech. Bull. U.S.
Armed Forces. Med. J. 9:191
SMITH, P.B., G.A. HANCOCK & D.L. RHODEN (1969)
Improved mdium for detecting deoxyrribonuclease-producing bacteria. Appl. Microbiol. 18:991-993
control
Serratia marcescens ATCC 13880

56
Staphylococcus aureus ATCC 25923
EC Broth
Ref. 02-060
Specication
Selective medium for the detection of enterobacteria, in
water and foodstuff according to ISO 9308-2 and 7251
standards.
Formula (in g/L)

Peptone .................................................... 20,0
Bile Salt n.3 ................................................ 1,5
Lactose ....................................................... 5,0
Potassium dihydrogen phosphate .............. 1,5
Sodium chloride .......................................... 5,0
Final pH 6,9 0,2
Directions
Dissolve 37 g of powder in 1 L of distilled water. Distribute into tubes or containers with inverted Durham tubes
(for gas production). Sterilize at 121C for 15 minutes.
Description
EC Broth is a buffered medium containing lactose. It is in
the range of selective broths for Enterobacteriaceae. Its
efciency or selectivity is based on bile salts inhibitory
effect on other microorganisms.
This broth may be used for routine testing of water and
food, either alone or by using the Most Probable Number
method of enumeration.
The type of sample will determine how precise the
results are. If the incubation is at 35-37C for 48 hours,
gas formation may be interpreted as presumptive evi-
dence of coliform bacteria. Later conrmation will have

to be done using any of the classical methods.
Should the incubation take place at 44,5C, gas formation could be interpreted as a conrmation of the presence of Escherichia coli. Nevertheless, it must be taken
into account that the validity of this test is highly limited
by technical variations. A maximum incubation time of
24 hours in a water bath with a very precise temperature
regulation, is therefore recommended.
When using the samples more than 10 mL, the medium
must be reconstituted at a concentration equivalent
to that specied on the directions, once the sample is
added.
References
APHA-AWWA-WEF (1998). Standard Methods for the
Examination of Water and Wastewater, 20th ed. APHA,
Inc. Washington D.C., U.S.A.
PERRY and HAJNA, (1943). Am. J. Pub. Hlth. 33:550.
DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of foods. 4th ed.
APHA. Washigton DC
ISO Standard 9308-2 (1990) Water Quality Detection
and enumeration of coliform organisms, thermotolerant coliform organisms and pressumptive E. coli - MPN
method.
ISO Standard 7251 (1993). Microbiology General
Guidance for enumeration of presumptive E. coli. M.P.N.
Technique.
E. coli Direct Agar (ECD Agar)

Ref. 01-484
Specication
Solid culture medium for the detection of coliforms and
E.coli in water and food.
Formula (in g/L)

Tryptone ................................................. 20,00
Yeast extract ............................................. 5,00
Bile salts ................................................... 1,50
Sodium chloride ........................................ 5,00
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
Suspend 53 g of powder in 1 L of distilled water and add
5 g/L of carbohydrate. Heat to the boiling. Distribute into
suitable containers and sterilize by autoclaving at 121C
for 15 minutes. Cool to 50C and add 2 asks/L of MUG
Supplement (Ref. 06-102CASE)
Description
This medium is formulated according to the Swiss Standards for the detection of coliforms in the food. Although
the medium is complete by itself, it is recommended
to add 5 g/L of glucose or lactose in order to make the
colonial growth more evident.
Direct detection is achieved by adding to the preparation
the uorescent agent (MUG, Ref. 06-102). After incubation of the sample dilution or lter membrane, E.coli
colonies show a light blue uorescence when examined
under UV light.
References
ANONYMOUS. Schweizerisches Lebensmittelbuch. 5th.
Ed. Chap. 56A.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. Boca Raton. Fla.
57
EE Broth (Eur. Phar. Enrichment Broth Medium E)

Ref. 02-064
Specication
Liquid culture medium for the enrichment of enterobacteria from food samples according to ISO 8523 standard.
Formula (in g/L)

Gelatin peptone .................................... 10,000
Dextrose ................................................. 5,000
Ox bile .................................................. 20,000
Di-sodium phosphate ............................. 6,450
Brilliant green ......................................... 0,015
Final pH 7,2 0,2
Directions
heat at 100C for 30 min. and cool immediately.
Description
As the name suggests, this medium is for the enrichment
of enterobacteria, and is a modication by Mossel(1963)
of the classic Brilliant Green Bile 2% Broth (Ref. 02-041).
Substitution of lactose by glucose makes it more suitable
for enteric bacteria detection, whether gas or non-gasproducer, in food and different samples.
Usual recommended technique is as follows: sample

to be studied is added to sterile broth at a proportion of
10%. After strong homogenization, the mixture is incubated for a period of18-20 hours at 35-37C. Afterwards,
subcultures are performed on a solid media appropiate
for the selective enterobacteria isolation. For this step,
Violet Red Bile Agar (Ref. 01-164) is specially recommended, though there are also the MacConkey (Ref.
01-118), Deoxycholate or Brilliant green based media.
From the suspected colonies on this media, identication
can be performed following the common methodology.
References
MOSSEL, VISSER and CORNELISSEN (1963) The examination of foods for Enterobacteriaceae using a test of
the type generally adopted for the detection of salmonellae J. Appl.Bact. 26:444-452
Alimentaria. Daz de Santos, S.A. Madrid,.
EUROPEAN PHARMACOPOEIA,Supplement 4.2
(2002),2.6.13 Test for specied micro-organisms 4th
ed.,Council of Europe, Strasbourg.
detection of Enterobacteriaceae with pre-enrichment.
Elliker Broth
Ref. 02-288
Specication
Liquid culture medium for the enrichment of lactobacilli
and streptococci in the dairy industry.
Formula (in g/L)

Casein peptone ........................................ 20,0
Yeast extract ............................................... 5,0
Gelatin ........................................................ 2,5
Dextrose ..................................................... 5,0
Lactose ....................................................... 5,0
Sucrose ...................................................... 5,0
Sodium chloride .......................................... 4,0
Sodium acetate .......................................... 1,5
Ascorbic acid .............................................. 0,5
Final pH 6,8 0,2
Directions
Dissolve 48,5 g of powder in 1 L of distilled water, heating up to 50C to totally dissolve gelatin. Distribute into
suitable containers, and sterilize by autoclaving at 121C
for 15 minutes.
58
Description
Medium was formulated by Elliker, Anderson and Hannesson to cultivate lactobacilli and streptococci from milk
and other dairy products. Sodium acetate, in this medium at this concentration, gives two advantages: on one
hand it restrains gramnegative bacteria growth, and on
the other hand, it enhances lactic bacteria growth. The
latter produce massive growth due to the high amount of
sugars.
Technique
Readings are performed after 3-5 days of incubation at
37C. Should a solid medium be desired, add 15 g/L of
Agar Bacteriological (Ref. 07-004).
References
ELLIKER, P.R., ANDERSON, A.W., HANNESSON, G.
(1956) An Agar Culture Medium for Lactic Acid Streptococci and Lactobacilli. J. Dairy Sci. 39:1611.
HAUSLER, W.J. (1976) Standard Methods for the
Examination of Dairy Products, 14 Ed. (APHA). Washington, D.C.
Examination of Dairy Products, 16 Ed. (APHA). Washington, D.C
Endo Media
Endo Agar Base
Endo Dev Agar Base
Ref. 01-589
Ref. 01-606
Specication
Specication
Solid selective medium for the detection of coliform and

other enteric organisms, in milk and water, according to
the APHA specications.
Selective agar for the isolation and differentiation of

E.coli in water, according to the german legislation.
Formula (in g/L)
Formula (in g/L)
Peptone .................................................... 10,0

Lactose ..................................................... 10,0
Sodium sulte ............................................. 2,5
Di-potassium hydrogen phosphate ............. 3,5
Agar .......................................................... 15,0
Final pH 7,2 0,2
Meat extract ............................................ 10,00

Peptone .................................................. 10,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Sodium sulte ........................................... 2,50
Agar ........................................................ 20,00
Final pH 7,3 0,2
Directions
Directions
Suspend 41 g of powder in 1 L of distilled water. Bring

to the boil and add 2 vials of Basic Fuchsin 250 Supplement (Ref. 06-607CASE). Homogenize and distribute
into suitable containers. Sterilize at 121C for 15 minutes.Cool to 45-50C, homogenize and pour into plates.
Medium must appear slightly pinkish. If colour is very intense red, it can be decolourised by adding a few drops
of a sterile solution of sodium sulte 10% before pouring
it into the plates. Medium must be freshly prepared for
the use, and must not be used when it is red.

bring to the boil. Add 2 vials of Basic Fuchsin 250 Supplement (Ref. 06-607CASE). Homogenize and distribute
into suitable containers. Sterilize in the autoclave at
121C for 15 minutes. Cool it to 45-50C, homogenize
and pour into plates.
In these conditions, medium must appear slightly pinkish. If colour is very intense red, it can be decolourised
by adding a few drops of a sterile solution of sodium
sulte 10% before pouring it into the plates. Medium
must be freshly prepared for the use, and must not be
used when it is red.
Description
Endo Agar is used to conrm the detection of and to
count coliform bacteria following presumptive test of
drinking water, as well as for the detection and isolation
of coliforms and fecal coliforms from milk, dairy products
and other food.
Inoculate the plates by the streak-plate method and
incubate for 24 hours at 37C.
Colonies of coliform bacilli, which ferment lactose, are
pink to rose red, with or without green metallic sheen:
marked reddening of the medium may occur. Colonies
of other enteric bacilli, including Salmonella and of non
lactose fermentors are about the same colour as the
medium, being almost colourless to faint pink.
On exposure to oxygen, the plated medium gradually
becomes red due to the oxidation of sulte and can thus
no longer be used. It can only be kept for a few days
even if it is stored in the dark and at refrigerator temperature.
References
APHA /AWWA/WEF, (1985). Standard Methods for the
Examination of Water and Wastewater, 15th ed., Inc.
Washington D.C., U.S.A.
APHA (1967). Standard Methods for the Examination of
Dairy Products, 12th ed. , APHA Inc. Washington D.C.,
U.S.A.
ENDO, S (1904), ber ein Verfahren Zum Nachweis von
typhusbazillen. Zbl BaKt. Hyg. Abt. I, Orig, 35:109
Examination of Dairy Products, 16th ed. , APHA Inc.
Washington D.C., U.S.A.
Description
Medium is a modication over the classical ENDO (Ref.
01-589), according to the German legislation, to obtain a
better detection of damaged coliforms. Since the buffer
system is removed in this medium, this formulation
includes a more rich nutrient base and sodium chloride
to restore the osmotic balance. Agar concentration has
been increased to keep the strengh of gel after the water
sample is incorporated.
On this medium, E.coli colonies appear red, with metallic
sheen, meanwhile Klebsiella and Enterobacter only take
on the red colour. Colonies of other enteric bacteria are
colourless.
Technique
DEV standards recommends this medium to incubate
membrane lters used in the coliform detection and
in the re-seed on the surface of suspicious colonies
for their conrmation and isolation. However, the agar
strength allows the incorporation of the sample to be assayed in the medium mass, without any loss of consistency.
For times and temperatures of incubation, it is recommended to follow the standard for every purpose, or follow the technicians criteria. In any case, an incubation at
25-30C for 24-48 hours usually provides good results.
59
Endo Media
References
References

BUNDESGESUNDHEITSAMT: Amtliche Sammlung von
Untersuchungverfahren nach. 35 LMBG Beuth Verlag
Berlin Kln.
DIN 38411: Teil 6 (Juni 1991): Mikrobiologische Verfahren (Gruppe K): Nachweis von Escherichia coli und
coliformen keimen (K6).
APHA-AWWA-WEF (1985) Standard Methods for the

Examination of Water and Wastewater. 16th. Ed. Washington DC.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc.,London
Endo LES Agar Base
Endo MF Broth Base

Ref. 02-605
Specication
Ref. 01-604
Liquid medium especially formulated for the incubation

of membrane lters over the absorbent pads.
Specication
Formula (in g/L)
Medium for detecting and enumerating coliforms in water

and food, by the membrane ltration method.
Formula (in g/L)

Peptone .................................................. 15,00
Yeast extract ............................................. 1,20
Lactose ..................................................... 9,40
Sodium chloride ........................................ 3,70
Sodium deoxycholate ............................... 0,10
Sodium laurylsulfate ................................. 0,05
Sodium sulte ........................................... 1,60
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
bring to the boil. Add 4 vials of Basic Fuchsin 200 Supplement (Ref. 06-617CASE). Homogenize and distribute
in sterile plates. Do not autoclave. Final medium is
intense red in colour.
Description
Medium is a modication of the classical Endos medium, reformulated to use it with the membrane ltration
technique and a previous enrichment in Tryptose Lauryl
Sulfate Broth (Ref. 02-108) is always helpful and recommended for achieving in more growths and more brilliant
colonies.
Technique
Membrane(s) that have ltered the sample are incubated at 35C for 2-3 hours over a pad with Tryptose
Lauryl Sulfate Broth (Ref. 02-108), and after this they are
transferred to a plate with Endo LES Agar. Incubate at
35C for 24 hours. Coliform colonies appear red with a
characteristic metallic sheen.
60
Casein peptone ...................................... 10,00

Meat peptone ......................................... 10,00
Yeast extract ............................................. 1,50
Lactose ................................................... 12,50
Sodium chloride ....................................... 5,00
Potassium phosphate .............................. 5,75
Sodium lauryl-sulfate ............................... 0,05
Sodium deoxycholate .............................. 0,10
Sodium sulte .......................................... 2,10
Final pH 7,2 0,2
Directions
Suspend 47g of powder in 1 L of distilled water. Heat
to boiling. Add 4 vials of Basic Fuchsin 250 Supplement
(Ref. 06-607CASE) and homogenize. Optimum results
are obtained using the media on the same day it is prepared. Do not sterilize in the autoclave.
Description
Endo Broth for membrane ltration has the suggested
composition according to Standard Methods for detection and enumeration of coliforms by the membrane
ltration method.
Basically, it is the classical Endo medium, but selectivity
has been improved by adding lauryl-sulfate and sodium
deoxycholate and also the sodium sulte and basic
fuchsin. The mixture of peptones and yeast extract provide the medium with a nutritive substrate by which most
of the enteric bacteria can have the better recovery and
the colonies of coliforms take on a characteristic metallic sheen. So,the members of Enterobacter, Citrobacter
and Klebsiella types, after 20-22 hours of incubation,
take a metallic green-blue sheen, but not so intense as
Escherichia coli.
Technique
Endo Broth for membrane ltration may be used directly
or soaked in a pad as just by solidifying it with agar. In
some cases, it seems that a better results are obtained
if double-step method is used. That is, a short incubation of 2-3 hours on Tryptose Lauryl Sulfate Broth (Ref.
Endo Media
02-108) and a later incubation of 20-22 hours over Endo
Broth at 35C (see Endo LES Agar Base, Ref. 01-604).
The procedure suggested by APHA is ltration of minimal amounts of water (50 mL), but in any case, membranes with more than 400 colonies must be rejected for
enumeration. In the direct method, as in the double-step
method, lactose fermenting colonies show a characteristic sheen, and they have to be considered as presumptive coliforms, meanwhile the lactose non fermenting
colonies are colouress and transparent.
References
Examination of Water and Wastewater, 19th ed. APHA
Washington. D.C.
FIFIELD, C.W. & C.P. SCHAUFUS (1958) Improved
membrane lter medium for the detection of coliform
organisms.J. Amer.Water Works Assoc. 50:193

control
Eosin Methylene Blue Agar (EMB Agar)

Ref. 01-068
Specication
It has been repeatedly recommended for the detection, enumeration and differentiation of members of the
coliform bacteria.
Selective differential medium for the isolation of coliforms

from water acc. ISO 21150 Standard.
Technique
Formula (in g/L)

Peptone ................................................ 10,000
Lactose ................................................. 10,000
Dipotassium Hydrogen phosphate ......... 2,000
Yellowish Eosin ...................................... 0,400
Methylene Blue ....................................... 0,065
Agar ...................................................... 15,000
Final pH 7,1 0,2
Directions
Add 37,5 g to 1 L of distilled water. Bring to the boil and
distribute in suitable containers. Sterilize in the autoclave
Description
A very versatile medium originally developed for the
differentiation of E. coli and Enterobacter aerogenes. It
has also proved very effective in the rapid identication
of Candida albicans and presents a high correlation with
the coagulase test for staphylococci.
The Weld method for the identication of Candida albicans uses this medium with Chlortetracycline (100 mg/l)
in a 10% CO2 environment. The methods effectivity has
been tested with a variety of samples, such as sputum,
oral secretions, faeces, nails and vaginal secretions, all
of which provide denite results within 24-48 hours. staphylococci are also easily identied, particularly coagulase-positive strains. These have a very characteristic
appearance: small colourless colonies with a central red
nucleus.
Nevertheless, the mediums prevailing application is in
the differentiation of E. coli and E. aerogenes.
The medium should be sterilized once distributed into
tubes containing 20 mL of product each, and then be
refrigerated. Melt in a boiling water bath before use and
stir until it acquires a dark purple colour. Pour a tube into
each sterile plate and allow to solidify. It is advisable to
dry the mediums surface before use, leaving the plate
open but inverted on a heater.
For each doubtful lactose broth tube,inoculate one plate
by streaking , and incubate for 24 to 48 hours at 37C.
Examine afterwards.
61
Eosin Methylene Blue Agar (EMB Agar)

Escherichia coli and Citrobacter form at colonies of 2-3
mm in diameter and are dark violet in colour with
a black centre which produces a distinctive green
metallic glow when light is reected on it.
Enterobacter and Klebsiella form convex colonies
which are twice as big as the very smooth E. coli
, have no metallic glow and are pink in colour with
a dark blue centre. Non-lactose fermenting organisms produce colourless colonies.
Candida albicans colonies incubated in a CO2 atmosphere have a very peculiar cotton-like appearance which distinguishes them from other
Candida species that produce classical yeast like
colonies.
References
CLESCERI, L.S., A.E. GREENBERG & A.D. EATON.

(1998) Standard Methods for the Examination of Water
and Wastewater. 20th edition. APHA-AWWA-WEF.
Washington D. C.
HOLT-HARRIS, J. E. y TEAGUE O.A. (1916) A New Culture Medium for the Isolation of Bacillus typhosus from
Stools J. Infect. Dis. 18:596-600.
ISO 21150:2006 Cosmetics Detection of Escherichia
coli.
LEVINE, M (1918) Diferentation of E. coli and A. aerogenes on simplied Eosin-ethylene Blue Agar. J. Infect.
Dis. 23:43-47.
MENOLASINO, N.I., GRIEVES B. Y PAYNE P. (1960)
Isolation and Identication of Coagulase Positive Staphylococci on Levines Eosin-Methylene Blue Agar. J.
Lab. Clin. Med. 56(6) 908-910.
WELD, J. (1953) Candida albicans: Rapid Identication
in Cultures made directly from Human materials Arch.
Dermat. Syph. 67(5):473-478.
WINDLE TAYLOR, E. (1958)The Examination of Water
and Water Supplies. Churchill Ltd. 7th ed. Londres.
USP 29 NF 24 (2006) 2nd Suppl. <61> Microbial Tests.
USP Con. Inc. Rockville, MD, USA
Ethyl Violet Azide Broth (EVA Broth)

Ref. 02-028
Specication
Xn
R-22-32-52/53
S-7-46-61
Medium for the conrmation of enterococci in water.
Formula (in g/L)

Meat Peptone ..................................... 10,0000
Casein Peptone .................................. 10,0000
Dextrose ............................................... 5,0000
Sodium chloride .................................... 5,0000
Monopotasium phosphate .................... 2,7000
Dipotassium phosphate ........................ 2,7000
Sodium Azide ....................................... 0,3000
Ethyl Violet............................................ 0,0005
Final pH 6,8 0,2
Directions
Dissolve 35,6 g of powder in 1L of distilled water, heating
up slightly if necessary. Distribute in tubes or asks and
sterilize in the autoclave at 121C for 15 minutes.
Description
EVA Broth is a highly selective medium for some kinds
of enterococci, and it has been adopted by many Ofcial
Organisations, National and International.
Mediums high selectivity is due to the presence of
sodium azide and ethyl violet, as they inhibit other accompanying bacteria, blocking their respiratory chains,
leaving enterococci unaffected. In general, this medium
is always used as a conrmation medium in the second stage, recommending an inoculum from a suitable
medium such as Rothe Azide Broth (Ref. 02-027) to be
inoculated in this medium.
62
Technique
Each of the EVA Broth tubes is inoculated with one or
two loops from a presumed positive Rothe Azide Broth
(Ref. 02-027) ask, and are incubated for a 24-48 hours
period at 37C. Enterococcus presence is noted by the
turbidity in the medium.
Occasionally a slight turbidity may appear accompanied
by ample violet sediment at the bottom of the tube.
Commonly, growth conrmation in this medium is
considered enough to state Enterococcus presence.
However, conrmative identication must be carried out
by isolation in solid media and classication in one of the
four faecal enterococci species: Enterococcus faecalis,
Enterococcus faecium, Enterococcus bovis and Enterococcus equinum.
References
LITSKY, MALLMAN and FIFIELD (1953) Amer.J.Publ.
Hlth 43:873
Examination of Water and Wastewater. 19th. ed. APHA
Washington.
SPECK, APHA/Intersociety (1976). Compendium of
methods for the microbiological examination of food.
Washington.
GUINEA, SANCHO and PARES (1979). Anlisis microbiologicos de aguas: aspectos aplicados. Ed. Omega,
Barcelona.
FDA Broth (AATCC Bacteriostasis Broth)

Ref. 02-532
Specication
Liquid medium used in routine antibacterial testing of
antiseptics and disinfectants.
Formula (in g/L)

Meat peptone ......................................... 10,00
Meat Extract ............................................. 5,00
Sodium chloride ........................................ 5,00
Final pH 6,9 0,2
Directions
Dissolve 20 g of powder in 1 L distilled water, heating if
necessary. Distribute in suitable containers and sterilize
in autoclave at 121C for 15 minutes.
and disinfectants for antibacterial activities. It has the

formula required by the AOAC for Nutrient Broth used
in Phenol Coefcient and others determinations. The
AATCC uses this medium in the procedures for the detection of antibacterial activity of fabrics and in the production of the solid medium for this type of evaluations.
References
AATCC (1985) 1986 Technical Manual of the AATCC
Vol. 61. Research Triangle Park. N.C.
MACFADDIN J.F. (1985) Media for Isolation CultivationIdentication-Maintenance of Medical Bacteria. William &
Wilkins. Baltimore
ATLAS R.M, & LC PARKS (1993) Handbook of Microbiological Media. CRC Press. Boca Raton Fla.
HORWITZ, W. (2000) Ofcial Methods of Analysis. 17th.
Ed. AOAC International. Gaithersbourg, M.D.
Description
This medium is produced according the formulation
specied in U.S. Food ad Drug Administration (FDA),
Association of Ofcial Analytical Chemists (AOAC) and
American Association of Textile Chemists and Colourists (AATCC) procedures for the testing of antiseptics
Fecal Coliforms Media (FC Media)

Fecal Coliforms Agar (FC Agar)
Ref. 01-287
Specication
Solid, selective and differential medium for coliform enumeration by membrane lter technique.
Formula (in g/L)
Fecal Coliforms Broth

(FC Broth)
Ref. 02-287
Specication
Liquid, selective and differential medium for coliform
enumeration by membrane lter technique.
Tryptose .................................................... 10,0

Yeast extract ............................................... 3,0
Proteose peptone ....................................... 5,0
Bile salts # 3 ............................................... 1,5
Sodium chloride .......................................... 5,0
Lactose ..................................................... 12,5
Aniline blue ................................................. 0,1
Agar .......................................................... 15,0
Final pH 7,4 0,2
Formula (in g/L)
Directions
Directions

heat to boiling. Add two vials of Rosolic Acid Selective
Supplement (Ref. 06-085CASE). Mix and pour into sterile Petri plates. Do not autoclave nor overheat. Use
freshly prepared medium.
Tryptose .................................................... 10,0

Yeast extract ............................................... 3,0
Proteose peptone ....................................... 5,0
Bile salts # 3 ............................................... 1,5
Sodium chloride .......................................... 5,0
Lactose ..................................................... 12,5
Aniline blue ................................................. 0,1
Final pH 7,4 0,2

heat to boiling. Add two vials of Rosolic Acid Selective
Supplement (Ref. 06-085CASE). Do not autoclave nor
overheat. Use freshly prepared medium.
63
Fecal Coliforms Media (FC Media)

Description
FC Agar and Broth are formulated according to Geldreich et al., to detect the faecal coliforms in polluted water.
The bile salts included in these media make these
media selective for enterobacteria, and also selective
for coliforms due to the high temperature of incubation:
44,5C0,5C.
Freshly prepared medium has a red-garnet colour. Faecal coliform colonies are greenish-blue, and the medium
also turns to this colour. In case of other bacteria, when
they grow, show pinkish colonies, and then the medium
turns to uorescent red.
of coliforms. Should a total E.coli selectivity be desired,

incubate at 44,5C.
References
GELDREICH, E.E, H.F.CLARK, C.B. HUFF and
L.C.BEST (1965) Fecal-Coliform-organism medium for
the membrane lter technique. J. Am. Water Works Assoc, 57:208-214
APHA-AWWA-WPCF (1995) Standard Method for the
examination of water and wastewater. 19th ed. APHA
Washington DC.
Technique
Essentially, the technique consists of ltering the test
sample to be examined through a membrane lter of
suitable porocity (0,22-0,45 m), assisting the ltration
by pressure or suction, so that the microorganisms are
retained on the membrane. Remove the membrane
carefully and aseptically and take it to the culture medium.
Put the membrane over the agar,if using the solid medium, or over the impregnated pad if using the liquid version. Cover the Petri plates and incubate them at 37C
for 24 hours. After incubation, proceed with the counting
Without
Rosolic Acid
With
Rosolic Acid
Escherichia coli ATCC

25922
ATCC 14028
GE Motility Medium
Ref. 03-593
Specication
Semi-solid culture media for the motility test performance.
Formula (in g/L)

Gelatin .................................................... 52,00
Tryptose .................................................. 10,00
Heart extract ........................................... 10,00
Sodium chloride ........................................ 5,00
Agar .......................................................... 5,00
Final pH 7,2 0,2
Directions
Suspend 82 g of powder in 1 L distilled water, heating
until total solution. Distribute in suitable containers and
Description
In this medium, the presence of diffuse growth away
from the line or spot of inoculation evidences motility. Non-motile organisms growth only along the line of
inoculation. In the medium all the components supplies
nutrients and the little amount of agar introduces enough
strength to maintain solidity even at incubation temperatures, therefore, it is adaptable to use in both tubes and
64
plates for motility studies, improving the original formulation by Jordan et al.
Technique
After sterilization, cool the tubes by placing in cool
water bath up to the depth of the medium (in a 16x160
mm tube 8 cm depth or 15 mL of medium). For plates,
cool asks of medium to 50C and pour into sterile petri
dishes to a dept of 5 mm or more and allow solidifying.
From an overnight culture spot the inoculum on the
surface or stab just below the medium surface. If tubes
are used, inoculate by depth stab inoculation. Incubation
must be suitable in time and temperature to the suspected organism being tested. Periodically examine tubes or
plates for growth and signs of motility at last for 7 days.
References
ATLAS, R.M. & L.C. PARKS. (1993) Handbook of
Microbiological Media. CRC Press. London
MACFADDIN, J.F. (1985) Media for isolation-cultivationidentication-maintenance of medical bacteria. William &
Wilkins, Baltimore MD
DAMATO, R.F. & K.M. TOMFOHRDE (1981) Inuence
of media on temperature-dependant motility test for
Yersinia enterocolitica. J. Clin Microbiol. 14:347-348
JORDAN, E.O. M.E CALDWELL & D. REITER (1934)
Bacterial motility. J. Bacteriol. 27:165-173.
Giolitti-Cantoni Broth
Ref. 02-230
Specication
Liquid medium for the recovery and enumeration of low

numbers of coagulase-positive staphylococci in foods
acc. to the ISO 5944:2001, IDF 60:2001 and EN-ISO
6888-3:2003 Standards
Formula (in g/L)
Tryptone ................................................... 10,0

Meat extract ................................................ 5,0
Yeast extract ............................................... 5,0
Lithium chloride .......................................... 5,0
D-Mannitol ................................................ 20,0
Sodium chloride .......................................... 5,0
Glycine ....................................................... 1,2
Sodium pyruvate ........................................ 3,0
Polysorbate 80 ........................................... 1,0
Final pH 6,9 0,2
rst dilution (other products) may be inoculated in double

strength medium. MPN procedures need at least three
tubes for at least three dilution steps. If no anaerobic jar
is available, overlay with a layer of sterilized vaseline
(Ref. 6-077) or vaspar. Incubate anaerobically for 24-48
h at 37C.
After 24 hours, subculture any tubes showing blackening
or black precipitate by streaking onto Baird-Parker Agar
(Ref. 01-030). Incubate the remainder of the tubes for
a further 24 h and subculture all tubes showing growth
(irrespective of blackening) to Baird-Parker Agar.
When determining the bacterial count by the MPN
method, all tubes showing growth are considered as
presumptive positive for staphylococci and they are
conrmed only if they produce a positive result in the
coagulase test.
References
Directions
Dissolve 55,2 g of powder in 1 L of distilled water. The

medium can be prepared at single strength or double
strength using double quantity of powder Distribute into
tubes dispensing 10 mL/tube (Single strength) or 20
mL/tube (double strength). Sterilize by autoclaving at
121C for 15 minutes. Cool and add 1% Potassium Tellurite Sterile Solution (Ref. 06-089) using 0,1 mL/tube for
single strength and 0,2 mL/tube for double strength.
Description
This medium for the selective enrichment of staphylococci was formulated by Giolitti and Cantoni in 1966.
The growth of staphylococci is promoted by pyruvate,
glycine and above all by a high concentration of mannitol. Addition of Polysorbate 80 is necessary for the successful recovery of Staphylococcus aureus (Chopin et
al., 1985). Competitive microbiota is inhibited by lithium
chloride and potassium tellurite. Anaerobic growth conditions increase the selectivity of the medium. Generally,
growth of staphylococci can be recognized by a blackening or black precipitates in the culture medium due to
reduction of tellurite to metallic tellurium.
CHOPIN, A. et altri (1985) ICMSF Methods Studies XV.

Comparison of four media and methods for enumerating Staphylococcus aureus in powdered milk. J. Food
Protect. 48:21-27
EN-ISO 6888-3 Standard (2003) Microbiology of food
and animal feeding stuffs. Horizontal method for the enumeration of coagulase positive staphylococci (Staphylococcus aureus and other species). Part 3: Detection and
MPN technique for low numbers.
FIL-IDF (2001) Milk and milk based Products. Detection
of coagulase-positive staphylococci. MPN technique.
Standard 60:2001. Brussels.
GIOLITTI, G. A. CANTONI, C (1966) A medium for the
isolation of staphylococci from foodtuffs. J.Appl. Bact.
29, 395-398.
HARRIGAN, WF. a. McCANCE, M.E. (1976) Laboratory Methods in Food and Dairy Microbiology. Academic
Press. London.
ISO 5944 Standard (2001) Milk and milk based Products. Detection of coagulase-positive staphylococci.
MPN technique.
The prepared basal culture medium can be stored for

about 1-2 weeks in the refrigerator. The ready-to-use
medium must be used the same day of preparation. It is
advisable that the stored basal medium be degasied at
the moment of use by heating for 15 minutes at 100C,
cool rapidly and add sterile potassium tellurite solution.
Technique
Refer to the standard protocol for specic products

(Food and animal feeding stuffs EN-ISO 6888-3:2003;
Milk and milk based products ISO 5944:2001 and FILIDF 60:2001). As a general technique the following is
suggested:
Use food macerates or decimal dilutions and inoculate
1 mL to single strength medium. To lower the detection
limit 10 mL of the test sample (liquid products) or of the
Left: control; right: Staphylococcus
aureus ATCC 25923
65
Glucose Bromcresol Purple Agar

Ref. 01-502
Description
Specication
This medium is used to conrm enterobacteria by their

ability to ferment glucose. Glucose fermentation can be
detected by the production of acid that change the colour
of the medium to yellow.
Solid medium for the conrmation of enterobacteria

in diverse samples according to ISO 4702 and 8523
standards.
Formula (in g/L)

Tryptone ............................................... 10,000
Yeast extract ........................................... 1,500
Dextrose ............................................... 10,000
Sodium chloride ...................................... 5,000
Bromcresol purple .................................. 0,015
Agar ...................................................... 15,000
Final pH 7,0 0,2
References
ISO 4702 Standard (1993) Microbiology General Guidance for the enumeration of entrobacteriaceae without
ressucitation. MPN technique and colony count technique.
ISO 8523 Standard (1991) Microbiology General Guidance for the detection of Enterobacteriaceae with preenrichment.
Directions
bring to the boil. Distribute into containers and sterilize
by autoclaving at 121C for 15 minutes.
Glutamate Starch Pseudomonas Agar (GSP Agar)

Ref. 01-092
Specication
Solid, semiselective and differential solid medium for the
isolation of Pseudomonas and Aeromonas from very
contaminated samples.
Formula (in g/L)

Sodium L(+) glutamate ........................... 10,00
Soluble starch ......................................... 20,00
Phenol red ................................................ 0,36
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
bring to the boil. Dispense in tubes or asks and sterilize
by autoclaving at 121C for 15 minutes. Cool to 50C
and add 100.000 I.U. of sodium G penicillin and 0,01g of
pimaricine per litre. Pour into sterile plates.
Description
This formulation is according to Kielweins modication
to Korths medium, improving the latter because it supports growth of almost all types of Pseudomonas and
Aeromonas.
Selectivity is achieved due to the antibiotics (penicillin
and pimaricine) and glutamate, which is hard to metabolize by gram-negative bacteria. Differentiation between
Pseudomonas and Aeromonas is based on the utiliza66
tion of starch, which is degraded by Aeromonas with

acid production which causes change in colour of phenol
red to yellow. Pseudomonas also grows on this medium
but do not degrade starch and there is no acid production and thus their colonies remain blue-violet.
Technique
Antibiotics are added to this medium after sterilization
and cooling to 50C, in such a way that nal concentrations in the medium are 100 u/mL of penicillin and 10
mcg/mL of piramicine (it may be replaced by amphotericin or nystatin).
After solidication in plates, medium may be used by
surface inoculation or by leaving for the membrane
lters. Aeromonas colonies turn to yellow, and Pseudomonas ones do not.
The incubation is performed at room temperature (2025C) for 3 days. Sometimes, enterobacteria may also
grow but very slowly with pinpoint colonies.
References
KORTH, H. (1963) Ein Nhrboden zur Zchtung von
Pseudomonaden. Zbl.Backt.Parasit. Hyg. Abt. 190:225
STANIER, R., N. PALLERONI, M. DOUDOROFF. (1966)
The aerobic pseudomonads: A taxonomic study. J. Gen.
Microbiol. 42:159-271
KIELWEIN, G. (1971) Die Isolierung und Differenzierung
von Pseudomonaden aus Lebensmitteln. Arch G. Lebensmillelhyg. 22:29-37.
Gram Negative Broth (GN Broth)

Ref. 02-093
content of citrate and deoxycholate. On the other hand,

mannitol restrains the growth of Proteus and facilitates
the proliferation of Salmonella and Shigella.
The medium is strongly recommended for primary
enrichment, 14-16 rst hours, before going to selective
media such as EMB (Ref. 01-068) or MacConkey (Ref.
01-118). Its author, Hajna, declares an extraordinary
selectivity of the medium, whatever may the origin of the
sample, if everything is kept in a transport medium upto
the inoculation.
Specication
Liquid culture medium for enteric bacteria according
Hajnas formulation.
Formula (in g/L)

Peptone .................................................... 20,0
Dextrose ..................................................... 1,0
D-Mannitol .................................................. 2,0
Sodium citrate ............................................ 5,0
Sodium deoxycholate ................................. 0,5
Di-potassium phosphate ............................. 4,0
Monopotassium phosphate ........................ 1,5
Sodium chloride .......................................... 5,0
Final pH 7,0 0,2
References
Directions
Dissolve 39 g of powder in 1 L of distilled water. Dispense in tubes or asks and sterilize in the autoclave at
Description
HAJNA, A.A. (1955) A new enrichment medium for gramnegative organisms of the intestinal group Pub.Hlth.Lab
13:83
EDWARDS and EWING (1973). Identication of Enterobactericeae. Burgess Pub.Co. Minneapolis.
VANDERZANT & SPLITTSTOESSER (1992). Compendium of Methods for the Microbiological Examination of
Food. 3rd. Ed. APHA. Washington.
DOWNES, F.P. & K.ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
GN Broth (Gram Negative Broth) is an enrichment and

selective medium for enterobacteria, with a strong inhibitory action against gram-positive because of its high
HC Agar Base
Ref. 01-298
Specication
T
R-45
S-53-45
Selective solid medium for the enumeration of molds in

cosmetic products.
Formula (in g/L)

Tryptone ................................................... 2,50
Yeast Extract ............................................ 5,00
Dextrose ................................................. 20,00
Monopotasium phosphate ........................ 3,40
Ammonium chloride .................................. 1,40
Magnesium sulphate ................................ 0,06
Sodium carbonate .................................... 1,00
Chloramphenicol ...................................... 0,10
Agar ........................................................ 15,00
Final pH 7,0 0,2
Directions
bring to boil. Add 20 mL of polysorbate 80 and homogenize. Distribute in suitable containers and sterilize in
Description
The HC Agar was developed by Mead & ONeill in 1986
to attain reliable enumeration of moulds in cosmetic
products in short time. The nutrient basis of the medium
s the dextrose with the peptones and yeast extract that
supplies the energy, nitrogen and vitamins and growth
factors. The inorganic ions are given by ammonium chloride and magnesium sulphate and both phosphates acts
buffering the medium. Sodium carbonate and polysorbate are detoxiers and neutralising preservatives and
others toxic substances. The selectivity against bacteria
is due to the chloramphenicol.
Technique
Suitable sample is inoculated into surface of medium plates per duplicate and incubate aerobically at
27,50,5C for 72 hours. Count colonies of moulds from
duplicate plates and record average count of mould
count per g or mL of sample.
References
MEAD, C. & J. ONEILL (1986) A three-day mould assay for cosmetics and toiletries. J. Soc. Cosmet. Chem.
37:49-57
67
Heart Extract Broth

Ref. 02-564
Specication
Liquid medium for fastidious microorganisms cultivation
Formula (in g/L)

Heart Extract .......................................... 10,00
Tryptose .................................................. 10,00
Sodium Chloride ....................................... 5,00
Final pH 7,4 0,2
Directions
Description
The Heart Extract Broth is a very old and classical general purpose medium that can be used for the cultivation
of fastidious microorganisms. Its components supply a
very rich nutritive base which supplemented with suitable
additives can be used in all laboratory purposes, from
Blood Agar Base until Base for carbohydrate fermentation studies.
References
HUNTOON, F.M. (1918) Hormone Medium. A simple
medium employable as a substitute for serum medium.
J. of infect. Dis. 23:169-72
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Boca Ratn Fl.
MAC FADDIN, J.F. (1985) Media for Isolation-Cultivation-Identication-Maintenance of Medical Bacteria.
William & Wilkins. Baltimore.
Rev.A. AOAC International. Gaithersburg. MD.
Hektoen Enteric Agar

Ref. 01-216
Specication
Solid, selective and differential culture medium for isolation of pathogenic enterobacteria from very contaminated samples acc. ISO 21567.
Formula (in g/L)

Peptone .................................................. 12,00
Yeast extract ............................................. 3,00
Bile salts ................................................... 9,00
Lactose ................................................... 12,00
Sucrose .................................................. 12,00
Salicin ....................................................... 2,00
Sodium chloride ........................................ 5,00
Ammonium ferric citrate............................ 1,50
Acid fuchsin .............................................. 0,10
Bromothymol blue .................................... 0,06
Agar ........................................................ 15,00
Final pH 7,7 0,2
Directions
Suspend 77 g of powder in 1 litre of distilled water and
let it soak. Heat up by constant stirring until boiling.
Cool to 55-60C and pour into sterile plates. Do not
autoclave. This medium is very thermolabile and thus
overheating should be avoided.
Description
This culture medium, originally developed by King and
Metzger, has a high nutrient content like peptones,
fermentable sugars and combination of indicators which
makes this medium less toxic. All these characteristics
and the bile salts makes it a very selective and effective
medium.
68
Technique
In order to avoid the spreading of Proteus, it is necessary that the agar surface be perfectly dry at the moment
of inoculation. Inoculation must be carried out by surface
streaking, directly from rectal swabs or faeces dilutions.
If colonies are well separated after 18 hours of incubation, rst characteristic appearances or colony characters may be observed, as then those colonies become
more prominent after a longer period:
Shigella spp., Proteus inconstans: Raised colonies,
humid, green colour.
Salmonella sp.: Green-blue colonies, with or without
black core.
Pseudomonas spp.: Irregular colonies, plain, green or
brown.
Companion and non pathogenic bacteria: Salmon colour
colonies.
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media CRC Press. BocaRaton. Fla. USA
HORWITZ, W. (2000). Ofcial Methods of Analysis of the
AOAC Internacional 17th ed. Gaithersburg Md. USA
DOWNES, F.P. & K. ITO (2001) Compendium of
Methods for the Microbiological Examination of
Foods.4th ed. APHA. Washington DC. USA.
FORBES, B.A., D.F SAHM & A.S. WEISSFELD (Eds)
(1998) Bailey & Scotts Diagnostic Microbiology 10th ed.
Mosby. St Louis, Mo. USA
MURRAY, P.R., E.J. BARON, J.H. JORGENSEN, M.A.
PFALLER & R.H. YOLKEN (Eds) (2003) Manual of Clinical Mcrobiology 8th ed. ASM Press. Washington DC, USA
KING S.y METZGER W. Y. (1968). A new plating method
for the isolation of the enteric pathogens. Appl. Microbiol.
16:577.
US FDA (1998) Bacteriological Analytical Manual 8th
ed. AOAC Internacional. Gaithersburg, Md. USA.
ISO 21567. Standard (2004) . Horizontal method for the
detection of Shigella ssp.
m-HPC Agar
Ref. 01-593
Specication
Solid medium for the enumeration of heterotrophic micro-organisms from water by lter membrane technique
Formula (in g/L)

Peptone ................................................. 20,00
Gelatine .................................................. 25,00
Agar ........................................................ 15,00
Final pH 7,1 0,2
Directions
bring to the boil. Add 10 mL of glycerol and homogenize.
Distribute in suitable containers and sterilize in autoclave
Description
The membrane-Heterotrophic Plate Count Agar was
developed in 1979 by Taylor and Geldreich as an adaptation to the membrane ltration method of the Standard
Plate Count Medium and it is also know as m-SPC.
In this medium peptone supplies all he nutrients and
gelatine overcomes the trouble of liquefaction and overgrowth of the colonies. The m-HCP is recommended by
Standard Methods for Examination of Water and Wastewater in the ltration technique applied to the greats
volumes of water with a low microbial population.
Technique
Sampling
Refer to the Section 9060 of the Standard Methods or
the ISO 5667 Standard. In any case the time between
the sampling and analysis must exceed 8 hours (6 for
transfer and 2 for analysis). The samples can be refrigerated but never chilled. After 24 hours refrigeration the
samples must be rejected.
Procedure
A suitable volume to obtain 20-200 colonies on the 47
mm lter must be ltered. Grilled lter of 0,45 m pore
are preferred. The funnel and the lter are washed three
times with 20-30 mL of sterile water. The lter is placed
on the surface of a m-HPC Agar plate and incubated
for 48 h. at 352C. Express the results as Cfu/unity of
volume ltered
References
TAYLOR, R.H & E.E. GELDREICH (1979) A new membrane lter procedure for bacterial counts in potable water and swimming pool samples. J. Amer. Water Works
Assoc. 71:402-405
EATON, A.D., .S. CLESCERI & A.E. GREENBERG (Eds)
(1995) Standard Methods for the Examination of Water and
Wastewater. 19th Ed. APHA Washington DC.
Indole Nitrite Fluid Medium

Ref. 03-101
Specication
General purpose medium for indole production tests and
nitrite detection.
Formula (in g/L)

Casein peptone ........................................ 20,0
Dextrose ..................................................... 1,0
Potassium nitrate ........................................ 1,0
Agar ............................................................ 1,0
Final pH 7,2 0,2
Directions
Suspend 25 g of powder in 1 L of distilled water. Heat to
boiling and dispense in tubes. Sterilize at 121C for 15
minutes. If tubes are kept in refrigerator, heat them up in
boiling water bath for 2 minutes before using them.
Description
The extraordinary richness of this medium, due to high
quality and quantity of peptone, allows its usage as a
general medium. Even the fastidious microorganisms
such as Lactobacillus or Clostridium grow well on this

medium, and indole and nitrite tests can be carried out.
Adding 2 g/L of Bacteriological Agar to this medium can
make it suitable for motility test. Indole production can
be observed with Kovacs Reagent (Ref. 06-018) with or
without previous extraction with chloroform, and in another tube, nitrate assay can be carried out with Nitrate
reagents (Ref. 06-003 and Ref. 06-004).
This medium is not the most suitable one for indole
production detection in enterobacteria, because some
times there are some interferences. For that assay, SIM
Medium (Ref.3-176) or Nitrate Broth (Ref. 02-138) are
more suitable.
Nonetheless, Indole Nitrite Broth is the medium of choice
for nitrate reduction assays or denitrication assays with
any other bacterial biotype especially the grampositive
ones .
References
Food.3rd. Ed. APHA. Washington.
69
Inhibitory Substances Test Media

Specication
Solid media for the verication of antimicrobial inhibitor substances in food and packaging material.
Assay Agar at pH 6,0

Ref. 01-437
Formula (in g/L)
Casein peptone ........................................ 3,45
Meat peptone ........................................... 3,45
Sodium chloride ........................................ 5,10
Agar ........................................................ 15,00
Final pH 6,0 0,1
Directions
to the boil. Distribute in the suitable containers and sterilize in the autoclave at 121C for 15 minutes. Check the
pH and adjust it if necessary. Cool to 50C and inoculate
with the spore suspension of Bacillus subtilis to obtain a
conuent culture.
Homogenize well and pour the medium into sterile
plates, (15 mL per plate). Once the agar is solidied, put
the plates into the refrigerator until their use.

Ref. 01-438
Formula (in g/L)
Casein peptone ........................................ 3,45
Meat peptone ........................................... 3,45
Sodium chloride ........................................ 5,10
Trisodium phosphate ................................ 2,40
Agar ........................................................ 15,00
Final pH 8,0 0,1
Directions
heat to the boiling. Distribute into suitable containers and
sterilize in the autoclave at 121C for 15 minutes. Check
the pH and adjust it if necessary. Cool to 50C. Inoculate
a part of medium with the spore suspension of Bacillus
subtilis to obtain a conuent culture. Inoculate the other
part of medium with a cell suspension of Micrococcus
luteus ATCC 9341.
plates, (15 mL per plate). Once the agar is solidied, put
all the plates into the refrigerator until their use.
70

Ref. 01-439
Formula (in g/L)
Casein peptone ........................................ 3,60
Meat peptone ........................................... 3,60
Sodium chloride ........................................ 5,00
Trisodium phosphate ................................ 0,80
Agar ........................................................ 15,00
Final pH 7,2 0,1
Directions
heat to the boiling. Distribute into suitable containers and
sterilize in the autoclave at 121C for 15 minutes. Check
the pH and adjust it if necessary. Cool to 50C and add
50 mcg/L of Trimetoprim. Inoculate with the spore suspension of Bacillus subtilis to obtain a conuent culture.
plates, 15 mL per plate. Once the agar is solidied, put
the plates into the refrigerator until their use.
All the plates must be sealed with an adhesive and
water-proof tape, named, and packed in plastic bags
before storing them into the refrigerator until their use. If
they are stored at 3-6C, plates may be thus stored up
to 3 weeks. Do not freeze the plates, and never put them
back in the refrigerator once they have reached 10C.
Technique
Assay may be performed with 2 or 4 plates. If two plates
are used, one must be at pH 6.0 and the other one at pH
8.0, and both must be inoculated with Bacillus subtilis.
If four plates are used, include another plate at pH 7,2,
inoculated with Bacillus subtilis too, and another plate at
pH 8,0 inoculated by Micrococcus luteus.
If the sample is solid, use a punch to extract 8 mm x 2
mm diameter cylinders. If the sample is liquid, use antibiogram assay discs. Sample pieces or discs are placed
over the plates surface, 2 per plate, on all the plates.
As a control, put discs alternatively with the samples
in each plate, impregnated with the following standard
substances:
Assay Medium at pH 6,0: Penicillin 0,01 IU/disc
Assay Medium at pH 8,0: Streptomycin 0,5 mcg/disc
Assay Medium at pH 7,2: Sulfamidin 0,5 mcg/disc
Plates are incubated at 30C for 18-24 hours, except
those inoculated with M. luteus, which are incubated at
37C.
After the incubation period, read the inhibition halos
(zones of inhibition), but do not consider the diameter of
the discs or cylinders.
Assay is considered reliable when control discs provide
12 mm halos, and then samples with average diameter
greater than 4 mm are considered positive (with inhibitor presence), and samples with 1-4 mm halos must be
considered doubtful.
Inhibitory Substances Test Media

References
BAUR, E. (1975) Untersuchungen von Fleisch und
Wurstwaren mit dem Hemmstoff im Rahmen das Tierrtzlichen Lebensmittelberwachung. Fleischwirtschaft
55:843-845.
BOGAERTS, R., F. WOLF (1980) Eine standardisierte
Methode sum Nachweis von Rckstnden antibakterial
wirksauer substanzen in schen Fleich. Fleischwirtschaft
60: 667-675
DEUTSCHES FLEICHBESCHANGESETZ: Ausfhrungsbestimmungen A ber die Untersuchung und
Gesundheitspolizeiliche Behandlung der Schlachtiere
und des Fleisches bei Schlachtungen im Inland. Anlage
1 zu 20 Abs. 4: Vorschriften ber die Bakteriologische
Fleischuntersuchung.
Iron Sulte Agar

Ref. 01-328
Specication
Medium for detection and identication of sulte reducing clostridia.
Formula (in g/L)

Tryptone ................................................... 10,0
Sodium sulte ............................................. 0,5
Ferric citrate................................................ 0,5
Agar .......................................................... 15,0
Final pH 7,1 0,2
Directions
up to boiling with constantl stirring. Distribute in suitable
minutes.
Description
Although the medium was originally described by Wilson
Blair, it remained unused because it was not safe. There
have been many modications, and the one by Tanner
in 1944 for the National Canners Association of America
was the more lasting. However, it was also modied
because it was demonstrated that clostridia were highly
inhibited at concentrations of sulte over 0,1%, this being the reason why the Wilson Blair formulation was un-
suitable for clostridia. The actual formulation is according

to the one widely experimented by several authors, and
provides a relatively low false negative results.
Most of clostridia have sulte reductases in their citoplasm, but they are unable to expel them to the exterior. So, when H2S is produced from sulte, the colony
becomes dark due to the formation of precipitates of iron
sulde from citrate, which is less toxica than the alum
described by Wilson and Blair.
Note that there are many gram negative bacteria able to
reduce sulte with iron sulde production in this medium,
but in these cases the enzymes are extracellular and all
the medium becomes dark, rendering their enumeration
impossible.
Technique
To enumerate sulte reducing clostridia, vegetative
cells have to be eliminated by heating up the sample to
80C for 10 minutes. Sample is cooled under water and
mixed aseptically with an equal volume of sterile, double
concentrated, melted and cooled to 60C medium. Let it
solidify and incubate at 37C for 48 hours, with visual examination after 24 hours. Enumerate the black colonies,
which clearly contrast with the medium, and express
the result as spores of sulte reducing clostridium per
volume of sample.
Tubes with blackish medium, without discrete colonies,
must be rejected and the assay has to be restarted heat71
Iron Sulte Agar

ing up the sample at the same temperature but for additional 5 minutes more than the previous heating time.
Should thermophilic species are desired to be enumerated, incubate at 55C.
If the assay is performed by membrane ltration, it is
advisable to put the membrane over a layer of medium
solidied in the plate, and then cover it with another layer
of medium (20 mL) melted and cooled to 60C.
References
TANNER, F.W. (1944) The Microbiology of Food. 2 Ed.
Garrad Press U.S.A.
BUTTON, A.W.J. (1959) A note on the enumeration of
thermophilic sulfate-reducing bacteria. J. Appl. Bact.,
22(2) 278-280.
SCARR, M.P. (1959) Selective Media Used in the Microbiological Examination of Sugar Products. J. Sci. Food
Agri., 678-681.
Left and center: Clostridium perfringens
ATCC 13124; right: control.
Kanamycin Esculin Azide Media (KAA Media)

Kanamycin Esculin Azide Agar
(KAA Agar)
Ref. 01-263
Specication
Kanamycin Esculin Azide Broth

(KAA Broth)
Xn
R-22-32-52/53
S-7-46-61
Ref. 02-263
Specication
Xn
R-22-32-52/53
S-7-46-61
Solid medium for conrmative detection and isolation of

Lanceelds group D streptococci in food samples, according to Mossel et al.
Liquid medium for the presumptive detection of Lanceelds group D streptococci in food samples, according
to Mossel et al.
Formula (in g/L)
Formula (in g/L)
Tryptone ................................................. 20,00

Yeast extract ............................................. 5,00
Sodium chloride ........................................ 5,00
Disodium citrate ........................................ 1,00
Esculin ...................................................... 1,00
Ferric-Ammonium citrate .......................... 0,50
Sodium azide ............................................ 0,15
Kanamycin sulfate .................................... 0,02
Agar ........................................................ 15,00
Final pH 7,0 0,2
Tryptone ................................................. 20,00

Yeast extract ............................................. 5,00
Sodium chloride ........................................ 5,00
Disodium citrate ........................................ 1,00
Esculin ...................................................... 1,00
Ferric-Ammonium citrate .......................... 0,50
Sodium azide ............................................ 0,15
Kanamycin sulfate .................................... 0,02
Final pH 7,0 0,2
Directions
Suspend 48 g of powder in 1 L of distilled water and let it
soak. Heat to boiling and distribute into suitable containers. Sterilize in the autoclave at 121C for 15 minutes.
72
Directions
Dissolve 33 g of powder in 1 L of distilled water. Distribute in suitable containers and sterilize in the autoclave at
Kanamycin Esculin Azide Media (KAA Media)

Description
KAA Presumptive Broth and Conrmative Agar are the
two media that the Spanish National Center for Food
and Nutrition (CeNAN) recommend to detect, enumerate
and isolate Lanceelds group D streptococci in samples
of food and beverages like: bottled water, fresh, refrigerated, frozen or mashed meat, sh and molluscs, soft
drinks, pastries, spices and semiconserves. Kanamycin
and sodium azide are the selective inhibitory compounds.
Technique
Prepare tubes with 9 mL of broth, and Petri plates with
the agar. Make a decimal dilution bank from the sample
in duplicate, and inoculate 1 mL fractions in the tubes.
Incubate at 37C for 24 hours.
Presumptive presence of streptococci is indicated by the
development of a blackish-brown colour and the loss of
uorescence behind Woods light. These tubes are considered as positive, and then inoculate 0,1 mL aliquotes
from them over the surface of plates with Conrmative
Agar, spreading with a Drigalsky loop (Ref. 5-010).
Incubate those plates, in inverted position, at 37C for 24
hours. Colonies that appear surrounded by a black halo
are considered as group D streptoccoci, and are isolated
to conrm them biochemically and morphologically with
the following tests: microscopical examination, catalase
assay (that should be negative) in an azideless medium,
growth at 45C and resistance to a high saline concentration (6,5% of NaCl in BHI Broth (Ref. 02-102). Finally,
they have to grow in Bile Esculin Agar (Ref. 01-265) with
a similar appearance of the colonies in the Conrmative
Agar. Nonetheless, there are some exceptions to this
rule, i.e. Streptococcus equinus and S.bovis do not grow
in the hypersaline broth, and therefore, denitive identication has to be performed by serological methods.
This methodology does not allow the enumeration of
cells from the original sample, and as this is a necessary data, it is recommended to use the Most Probable
Number (MPN) technique with the presumptive broth,
doing it at double strength if necessary. For bottled
water, soft drinks and molluscs, CeNAN suggest the following technique:
Prepare broth tubes at normal concentration and at
double strength. Using a sterile pipet, inoculate ve broth
tubes of double strength with 10 mL of sample. Inoculate
ve tubes of normal concentration with 1 mL of sample
and ve tubes of normal concentration with 0,1 mL of
sample. Homogenize them well and incubate at 37C
for 48 hours. Tubes that show a blackish-brown colour
after the incubation period, are considered positive. Note
down the results and carry out the counting using the
MPN tables.
References
GUINEA, J., SANCHO, J., PARES, R. (1979). Anlisis
Microbiolgico de Aguas. Ed. Omega, Barcelona,.
MOSSEL, D.A.A., P.G.M. BUKER, J. ELDERING (1978)
Streptokokken der Lanceeld Gruppe D in Lebensmitteln
und Trinkwasser. Arch F. Lebensmittelhyg. 29:121-127
PASCUAL ANDERSON, MR (1992) Microbiologa
Alimentaria. Diaz de Santos, S.A. Madrid.
73
Kenner Fecal Media (KF Media)

Kenner Fecal Agar (KF Agar)
Ref. 01-294
Specication
Directions
Xn
R-22-32-52/53
S-7-46-61
Solid and selective medium for enterococci enumeration

and detection.
Formula (in g/L)

Proteose peptone ................................. 10,000
Yeast extract ......................................... 10,000
Sodium chloride ...................................... 5,000
Sodium glycerophosphate .................... 10,000
Maltose ................................................. 20,000
Lactose ................................................... 1,000
Sodium azide .......................................... 0,400
Bromocresol purple ................................ 0,015
Agar ...................................................... 20,000
Final pH 7,2 0,2
Description
Directions
heat to boiling by constant stirring. If it has to be used
immediately, it may not to be sterilized. Otherwise,
sterilize in the autoclave in small volumes, at 121C and
for 10 minutes maximum. In both the cases, let it cool to
50C and add 10 mL/L of TTC Sterile Solution 1% (Ref.
06-023). Homogenize well and distribute in sterile plates.
Note: Non homogeneous appearance is normal, and it
does not affect the mediums quality and efcacy.
Kenner Fecal Broth (KF Broth)

Ref. 02-294
Specication
Xn
R-22-32-52/53
S-7-46-61
Liquid and selective medium for enterococci enumeration by the MPN or membrane lter methods.
Formula (in g/L)

Proteose peptone ................................. 10,000
Yeast extract ......................................... 10,000
Sodium chloride ...................................... 5,000
Maltose ................................................. 20,000
Lactose ................................................... 1,000
Sodium azide .......................................... 0,400
Bromocresol purple ................................ 0,015
Final pH 7,2 0,2
74
Suspend 56,4 g of powder in 1 L of distilled water. If it

has to be used immediately, it may not to be sterilized,
just heat to boiling for one or two minutes, with constant
stirring. Otherwise, sterilize in the autoclave in small
volumes, at 121C and 10 minutes maximum. In both
cases, let it cool to 50C and add 10 mL/L of TTC Sterile
Solution 1% (Ref. 06-023). Homogenize well and distribute in sterile tubes.
Note: Non homogeneous appearance of medium is normal, and it does not affect the mediums quality
and efcacy.
Kenner, Clark and Kabler (1960,1961) discovered that

KF media were excellent for detecting enterococci in polluted water. Carbohydrates in this medium viz. lactose
and maltose, are utilised by most of enterococci, producing a big amount of acid and making the indicator turn
from violet to yellow. streptococci that do not belong to
D group may also grow in the medium, but they do not
produce enough acid to change the indicator colour.
Other microorganisms, are strongly inhibited by sodium
azide. enterococci reduce TTC to formazan and so their
colonies are red coloured.
Technique
Media can be used by following several techniques
depending on the sample. When a marginal contamination is thought, then often it is performed using the
membrane lter or MPN method. On the contrary, if a
high population of enterococci is suspected, it is more
advisable to make a plate count.
To use the membrane lter technique, incubate at 37C
for 48 hours on the surface of KF Agar or on an absorbent pad impregnated in KF Broth.
To use MPN technique, directly inoculate samples, of up
to 1 mL to tubes with 10 mL of KF Broth in normal concentration, and for larger samples, to tubes with 10 mL of
KF Broth in double strength.
If the sample is suspected of being highly contaminated,
prepare a decimal dilution bank and inoculate on the
surface 0,1 mL with a Drigalsky Loop (Ref. 5-010) or, if
desired, inoculate by stabbing 1 mL. Anyway, incubation
should be carried out at 37C and for a 48 hours period.
After the incubation, readings are performed by observing the indicator colour turning from violet to yellow, and
on the solid medium, colonies will be also pink or red
coloured.
It is very important to maintain the pH of the medium
over 7,0, or otherwise, false results may appear. Sterilization longer than the specied period could result in
caramelization and thereby a decrease in the pH.
Kenner Fecal Media (KF Media)

References
KENNER, B.A., CLARK, H.F., KABLER, P.W. (1961)
Fecal streptococcci I. Cultivation and Enumeration of
streptococci in Surface Waters. Appl. Microbiol. 9:15.
KENNER, B.A., CLARK, H.F., KABLER, P.W. (1960)
Fecal streptococci. II. Quantication of streptococci in
faeces. Am. I. Publ. Health, 50:1553.
Examination of Water an WasteWater. 20th. Ed. Washington.
King Media
King A Agar
Ref. 01-001
Specication
Solid Medium to enhance the pyocyanine production
by Pseudomonas aeruginosa acc. ISO 16266, 22717
standards.
Formula (in g/L)

Peptone .................................................... 20,0
Magnesium Chloride .................................. 1,4
Potassium Sulfate .................................... 10,0
Agar .......................................................... 15,0
Final pH 7,2 0,2
Directions
Suspend 46,4 g of powder in 1 L of distilled water with
glycerol 10 mL and let it soak . Heat with constant stirring
until it boils. Distribute into suitable containers and sterilize by autoclaving at 121C for 15 minutes. If tubes are
used, let them solidify with short slant and good butt.
Description
This A medium was formulated by King, Ward and
Raney in 1954 to enhance the pyocyanine production by
Pseudomonas aeruginosa. The blue pigment Pyocyanine
diffuses into the culture medium and its production varies
depending on the strains of Pseudomonas aeruginosa
and on the growth conditions.
Sometimes, although this medium enhances especially
blue pigment production, it is possible that green (pioverdine) or brown (piomelanine) pigments also appear and
mask the pyocyanine. Anyway, uorescence and other
pseudomonas pigments can be noticed on other more
suitable media, like King B Agar (1-029).
Technique
Slanted tubes or Petri dishes are inoculated by surface
inoculation by streaking and are then incubated at 3032C for 4-5 days. Petri plates usage has the disadvantage of the dehydration of the medium during incubation.
Therefore, it is better to use slanted tubes being careful
for the aeration by loosening the screw caps or replacing
them with cotton or aluminium caps.
In the recently isolated pathogenic strains from the
pathological material, pigment production is often shown
early i.e. after 24-48 hours of incubation, however if
the material is non pathogenic or if they come from
water, food or soil,then the pigmentation can be later or
delayed.
When pigment has not got the usual blue colour, it is due
to the production of two or more coloured substances.
At this time, and if it is not conrmed on other culture
medium, it is recommended to conrm by extraction:
on the culture slant, 0,5-1 mL chloroform is added,
and it is shaken for a few minutes until the pyocyanine
is diffused, which makes the solvent blue. After that,
chloroform is acidied with a few drops of HCl, obtaining
a rapid change in colour from blue to red,the fact that
conrms the presence of pyocyanine.
References
KING E.O., M. WARD y D.E. RANEY (1954) Two simple
media for the demonstration of pyocyanin and uorescein. J.Lab.Clin.Med. 44:301-307
US Pharmacopeia (2002) 25th ed. <61> Microbial Limit
Test. US Pharmacopoeial Conv. Inc. Rockville MD.
ISO 16266:2006 Standard. Water Quality. Detection
by membrane ltration
75
King Media
King B Agar (F Agar)
Ref. 01-029
Specication
Culture media for enhancing the uorescein production
by Pseudomonas species.acc. EN 12780:2002 and ISO
16266, 22717 standards.
Formula (in g/L)

Meat peptone ........................................... 10,0
Casein peptone ........................................ 10,0
Magnesium sulfate ..................................... 1,5
Agar .......................................................... 15,0
Final pH 7,2 0,2
Directions
Suspend 38 g of powder in 1L of distilled water with
10 mL of glycerol and let it soak . Heat to boiling and
distribute in suitable containers. Sterilize in the autoclave
at 121C for 15 minutes. Cool by solidifying in slanted
position with a long slant.
Description
F medium was formulated by King, Ward and Raney
in 1954 to enhance green uorescent pigment (pioverdine) production by Pseudomonas uorescens
and Ps.aeruginosa, in which pyocyanine production is
restricted.
Green-yellowish pigments, soluble and from uorescent
type, dene the Pseudomonas group I according to the
9th. edition of Bergeys Manual of Systematic Bacteriology, and therefore, detection of its productive capacity is
critical.
76
Technique
Slanted tubes are inoculated with Pseudomonas strains
and incubated at 30-31C for a 2-4 days period. If after
this time a green-yellowish colour does not appear on
the medium, the tubes should be kept under observation at room temperature for a period of 6-20 days more
before the culture can be rejected as negative. It can
be observed that Pseudomonas aeruginosa and Pseudomonas putida strains coming from water, soil or food,
produce pigments in a very slow way.
Pioverdine is not soluble in chloroform, so the conrmation of presence is usually done by characteristic uorescence verication under Woods light, comparing the
dubious tube to another uninoculated F medium , which
is considered as the control.
References
KING, E.O., M.WARD and D.E. RANEY (1954) Two
simple media for the demonstration of pyocyanin and
uorescein J.Lab.Clin.Med. 44:30-307
LENNETTE, E.H., E.H. SPAULDING and J.P. TROUANT
(1974) Manual of Clinical Microbiology. 2nd. Ed. ASM.
Washington.
US PHARMACOPOEIA (2002) <61> Microbial Limit
Tests. 25th ed. US Phamacopoeial Conv. Inc. Rockville.
MD.
DIN Standard 38411Teil 6 (Juni 1991): Mikrobiologische
Verfahren (Gruppe K): Nachweis von Escherichia coli
und coliformen keimen (K6).
PALLERONI, N. (1984) The genus Pseudomonas, in
Bergeys Manual of Systematic Bacteriology.
EUROPEAN STANDARD EN 12780:2002. Water quality.
Detection and enumeration of Pseudomonas aeruginosa
by membrane ltration. CEN. Brussels.
Kligler Iron Agar (KIA)

Ref. 01-103
Specication
Solid and differential medium for primary identication of
enterobacteria based on the fermentation of two sugars
and the hydrogen sulphide production according ISO
Standard 6340.
Formula (in g/L)

Meat extract .............................................. 3,00
Yeast extract ............................................. 3,00
Peptone .................................................. 20,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Dextrose ................................................... 1,00
Ammonium iron citrate ............................. 0,50
Sodium tiosulfate ...................................... 0,50
Phenol red ................................................ 0,03
Agar ........................................................ 15,00
Final pH 7,4 0,2
Directions
Add 58 g of powder to 1 L of distilled water and heat to
the boiling. Distribute in tubes and sterilize in the autoclave at 121C for 15 minutes. Let it solidify with short
slant and plenty of butt.
Description
Kligler Agar is a differential medium that has all the
characteristics of the 2-Sugar Russell Agar and the
Lead Acetate Medium for H2S detection. In this medium,
lactose fermentation and hydrogen sulphide production
can be detected, so it allows a presumptive diagnostic
of most enterobacteria. Glucose fermentation is shown
by acid production, which makes the indicator turn from
red to yellow, but since there is little sugar (dextrose),
acid production is very limited and then a reoxidation of
the indicator is produced on the surface of the medium,
and the indicator remains red. Otherwise, when lactose
is fermented, the large amount of acid produced avoids
reoxidation and then all the medium turns to yellow.
Hydrogen sulphide production is indicated by the medium turning black, due to the reaction of H2S (liberated
from tiosulfate) with the Fe ions from ammonium iron

citrate.
Technique
Kligler Iron agar is used in slanted tubes with short slant
and plenty of butt, that are inoculated on the surface as
much as in stab. Inoculum must be copious, it has to
come from a solid medium, because otherwise, readings
may be delayed (up to 2-3 days more). Normal incubation
is 18 hours at 37C.
It is recommended to use tubes with caps that allow
ventilation, like cotton caps, cellullose caps or cap-otest. Should screw caps be used, do not tighten them
because otherwise they can hinder the reoxidation of the
indicator.
Kligler medium provides excellent results if it is used
freshly prepared, however if it has been prepared before
a few days then, it is advisable to remelt it and solidify it
again to obtain more precision.
When H2 S production is more, it may make the readings difcult, and hence the early readings are strongly
recommended. Anyway, one can obtain more precise
readings if Three Sugar Iron Agar (Ref. 01-192) is used,
since this one has the sucrose that allows a greater differentiation between members of Proteus, Salmonella
and Shigella types.
References
KLIGLER (1918) Modication of Culture Media used in
the Isolation and Differentiation of Typhoid, Dyesentery
and allied Bacilli. J. Exper. Med. 28:319-332
KLIGLER (1917) A simple medium for the differentiation
of members of typhoid-paratyphoid groups. Am. J. Pub.
Hlth 7:1042-1044
DOWNES, F.P. & K.ITO (2001) Compendium of Methods
for the Microbiological Examination of Food. 4th. ed.
APHA. Washington.
Left: control; center: Escherichia coli

ATCC 25922; right: Salmonella typhimurium ATCC 14028.
77
Kligler Iron Agar (KIA)

Typical reactions of enterobacteria on Kligler Iron Agar
78
Lactose Media
Lactose Broth
(Eur. Phar. Broth Medium D)
Ref. 02-105
Specication
US PHARMACOPOEIA (2005) <61> Microbial limit test.

US Pharmacopoeial Conv. Inc. Rockville. Md USA
APHA-AWWA-WPCF (1998) Standard methods for the
examination of water and wastewater. 20th ed. APHA
Washington
Lactose Peptone Broth
Medium for the pre-enrichment and the detection of

enterobacteria and coliforms in milk and water according
ISO 9308-2 and 21150 standards.
Ref. 02-414
Formula (in g/L)
Specication
Peptone ...................................................... 5,0

Meat extract ................................................ 3,0
Lactose ....................................................... 5,0
Final pH 6.9 0,2
Formula (in g/L)
Directions
Add 13 g of powder to 1 L of distilled water, or in the
quantity required for the desired concentration. Dissolve
it and distribute into containers tted with Durham tubes.
Sterilize by autoclaving at 121C for 15 minutes. Avoid
any further reheating.
Description
Lactose Broth is a classical medium for use in the
presumptive testing for coliforms and for the enrichment
of Salmonella. This formulation is as per the standards
recommended by APHA, AWWA, USP-NF and European Pharmacopoeia.
It is commonly used with Durham fermentation tubes for
the examination of gas formation. If the volume of sample to inoculate is important, reconstitute the medium
at a concentration such, as to remain normal once the
sample has been added to it.
Although it is not the original formulation, this broth provides excellent results in Eijkman assays of gas production at 45C, which is a characteristic of Escherichia coli.
While preparing this medium it is important to avoid
overheating and to distribute it into tubes before sterilization.
References
FDA (1998) Bacteriological Analitical Manual 8th ed.
Rev A. AOAC International. Gaithersburg. Va. USA..
Foods. 3rd. Ed. APHA. Washington.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th ed.
APHA. Washington.
EUROPEAN PHARMACOPOEIA (2005) 5th ed. 2.6.13
Test for specied micro-organisms. EDQM. Council of
Europe. Strasbourg.
ISO 9308-2 Standard. (1990) Water Quality Detection
and enumeration of coliform organisms, thermotolerant
coliform and presumptive E. coli MPN technique.
Escherichia coli
Liquid medium for the enrichment and enumeration of

coliforms in water.
Casein peptone ...................................... 17,00

Soy peptone ............................................. 3,00
Lactose ................................................... 10,00
Sodium chloride ........................................ 5,00
Final pH 7,2 0,2
Directions
Dissolve 35 g of powder (or 70 g if double concentrated
medium is desired) in 1 L of distilled water. Distribute in
tubes provided with Durhams tubes and sterilize in the
Description
This medium is according to the German standards for
quality control of water.
Technique
German standards suggests the use of MPN technique
with 0,1, 1 and 10 mL of sample and an incubation at
361C for 444 hours. Tubes that change to yellow
and eventual gas production/accumulation in Durhams
tubes are considered positive.
References
Abwasser- Und Schlammuntersuchung. Mikrobiologie
Verfahren (Gruppe K) Nachweiss vom E. coli und coliformen keimen.
VERORDNNG ber Trinkwasser und Wasser fr Lebensmittelbetriebe vom 12- Dezember-1990- Bundesgesetzblatt; Teil I, 2613-2629 (1990)
MURRAY, PR, EJ BARON, MA PFALLER, FC TENOVER & RH YOLKEN (eds) (1995) Manual of Clinical
Microbiology, 6th ed. ASM Washington.
MaCFADDIN J.A. (1985) Media for Isolation-CultivationIdentication-Maintenance of Medical Bacteria. William&
Wilkins.Baltimore.
79
Lactose Media
Lactose Purple Modied Broth
Ref. 02-417
Specication
Liquid medium for coliforms and E. coli identication.
Formula (in g/L)

Meat peptone ......................................... 10,00
Meat extract .............................................. 3,00
Sodium chloride ........................................ 5,00
Lactose ................................................... 10,00
Final pH 7,2 0,2
Directions
Dissolve 28 g of powder in 1 L of distilled water. Distribute in tubes provided with Durhams tubes. Sterilize in
Description
This medium is according to the German standards for
quality control of water.
Technique
German standards suggests the use of MPN technique
with 0,1, 1 and 10 mL of sample and an incubation at
361C for 444 hours. Tubes that change to yellow
and eventual gas production/accumulation in Durhams
tubes are considered
positive.
References
Abwasser- Und Schlammuntersuchung. VCM Verlagsgesellschaft, D-6940. Weinhem.
VERORDNNG ber Trinkwasser und Wasser fr Lebensmittelbetriebe vom 12- Dezember-1990- Bundesgesetzblatt; Teil I, 2613-2629 (1990)
Lactose Sulte Broth Base

(Eur. Phar. Medium R)
Ref. 02-519
Specication
Liquid medium for the determination of H2S production by Clostridium perfringens according to ISO 7937
standard.
Formula (in g/L)

Peptone .................................................. 5,000
Yeast extract ........................................... 2,500
Sodium chloride ...................................... 2,500
Lactose ................................................. 10,000
L-Cysteine HCl ....................................... 0,300
Final pH 7,1 0,2
80
Directions
Dissolve completely 10,1 g of powder in 500 mL of
distilled water and sterilize in the autoclave at 121C
for 15 minutes. Cool and aseptically add a ask sodium
meta-bisulte sterile solution (Ref. 06-114CASE) and a
ask of sterile solution of ammonium ferric citrate (Ref.
06-113CASE). Mix well and distribute into sterile tubes
provided with Durhams tubes.
Description
This is a simple medium that selects Cl. perfringens
among other sulte reducing clostridia by their ability to
produce gas from lactose, at 46C. It has interferences
only with Cl. paraperfringens, however this microorganism is very rare in food samples.
Technique
All of the freshly prepared media tubes (or regenereated)
are inoculated in duplicate with 1 mL of sample dilution.
Sample dilution must be previously kept in boiling water
bath, boiling it for 10 minutes. Tubes are incubated in
anaerobic conditions at 46C for a period of 18-24 hours.
Cl. perfringens presence is observed by an iron sulde
precipitate appearing in the tubes. It indicates the sulte
reducing activity. Accumulation of gas in the Durhams
tubes is a sign of lactose fermentation.
References
Alimentaria. Diaz de Santos, S.A.,Madrid,.
EUROPEAN PHARMACOPOEIA (2002) Suppl. 4.2
(2001). Chap. 2.6.13 Tests for specied microo-organisms 4th Ed., Council of Europe, Strasbourg.
animal feeding stuff. Horizontal method for enumeration
of Cl. perfringens. Colony-count technique.
LB Media
Under this generic name there are several formulations
included which are derived from the base medium that
was originally described by Luria and lately modied by
different authors.
This nutrient base has become very popular among
culture media for the maintenance and propagation of
Escherichia coli in the assays about molecular genetics.
The nutrient richness and simplicity in their composition allow easy and quick modications, where the most
common are the addition of different antibiotics and
inhibitors as well as the variations in the sodium chloride
concentration.
The current SCHARLAU formulation program covers the
following formulations:
LB Agar
Ref. 01-384
Specication
Solid medium for general purposes recommended for
the molecular genetics studies with E. coli.
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Agar .......................................................... 15,0
Final pH 7,2 0,2
Description
Formulation of both the media, solid and liquid, are according to the Luria and Bartani base, in which sodium
chloride has been omitted to help in the variation of the
saline concentration with other additives.
LB Agar acc. to Lennox

Ref. 01-406
Specication
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,0 0,2
Directions
to the boil with constant stirring. Distribute into suitable
minutes.
LB Broth acc. to Lennox
Directions
Ref. 02-406

minutes.
Specication
Liquid medium for general purposes, recommended for
molecular genetics studies with E. coli.
LB Broth
Ref. 02-384
Specication
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Final pH 7,2 0,2
Directions
Dissolve 15 g of powder in 1 L of distilled water. Distribute into suitable containers and sterilize by autoclaving
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride .......................................... 5,0
Final pH 7,0 0,2
Directions
Description
Formulation of both the media, solid and liquid, are
according to the Luria and Bartani base modied by
Lennox.
81
LB Media
LB Agar acc. to Miller
Ref. 01-385
Specication
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride ........................................ 10,0
Agar .......................................................... 15,0
Final pH 7,2 0,2
Directions
minutes.
LB Broth acc. to Miller

Ref. 02-385
Specication
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 5,0
Sodium chloride ........................................ 10,0
Final pH 7,2 0,2
82
Directions
Description
Formulation of both the media, solid and liquid, are according to the Luria and Bartani base modied by Miller,
who has increased the sodium chloride concentration.
References
AUSUBEL, F.M., R. BRENT, R.E. KINGSTON, D.D.
MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL
(1994) Current protocols in molecular biology. Greene
Pub. Assoc. Inc. Brooklyn, N.Y.
GHERNA, R., P. PIENTA, R. COTE (Eds.) 1992. ATCC
Catalogue of Bacteria and Bacteriophages. Media
#1065, #1226, #1226, #1235, #1236, #1315, #1364.
American Type Culture Collection. Rockville MD. USA.
LURIA, S.E. & J.W. BURROUS (1955) Hybridization
between Escherichia coli and Shigella. J. Bacteriol.
74:461-476
LENNOX, E.S. (1955) Transduction of linked genetic
character of the host bacteriophage P1. Virology 1:190206.
MILLER, J.H. (1972) Experiments in Molecular Genetics.
Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.
SAMBROOK, J., E.F. FITSCH & T. MANIATIS (1989)
Molecular cloning: A laboratory manual. 2nd ed. Cold
Spring Harbor Laboratory. Cold Spring Harbor, N.Y.
Letheen Media
Letheen Agar
Ref. 01-236
Specication
Solid medium for assays of antimicrobial action of quaternary ammonium compounds (QACs).
Formula (in g/L)

Tryptone ................................................... 5,00
Meat extract .............................................. 3,00
Dextrose ................................................... 1,00
Lecithin ..................................................... 1,00
Agar ....................................................... 15,00
Final pH 7,0 0,2
Directions
Suspend 25 g of powder in 1 L of distilled water with 7
mL of Polysorbate 80 (Ref. 06-088). Let it soak and heat
to boiling with constant stirring. Distribute in suitable
minutes.
Description
Letheen Agar is formulated according to AOAC directions, that were taken from the research work of Weber
and Black for the assay of bactericidal action of quaternary ammonium compounds (QACs).
In fact, the medium is the classical one for the standardized counting with the addition of lecithin and polysorbate, which act as the neutralizers of the QACs.
Letheen Broth
Ref. 02-236
Specication
Liquid culture medium for the determination of germicidal
activity coefcients of cationic detergents.
Formula (in g/L)

Peptone .................................................... 10,0
Meat extract ................................................ 5,0
Lecithin ....................................................... 0,7
Sodium chloride .......................................... 5,0
Final pH 7,0 0,2
Directions
Dissolve 20,7g of powder in 1 L of distilled water with 5
mL of Polysorbate 80 (Ref. 06-088). Distribute in suitable
minutes.
Description
This is the liquid version of Letheen Agar (Ref. 01-236),
recommended by AOAC to verify the germicidal activity
coefcients in cationic soaps, although its formulation is
not the same.
Letheen Modied Agar

Ref. 01-237
Specication
Solid medium for the primary screening of microorganisms in cosmetics according to the FDA
Formula (in g/L)

Casein peptone ........................................ 10,0
Meat peptone ........................................... 10,0
Meat extract ............................................... 3,0
Yeast extract ............................................... 2,0
Dextrose ..................................................... 1,0
Lecithin ....................................................... 1,0
Sodium chloride .......................................... 5,0
Sodium bisulphite ....................................... 0,1
Agar .......................................................... 15,0
Final pH 7.0 0,2
Directions
Suspend 47 g of powder in 1 L of distilled water and add
7 mL of Polysorbate 80 (Ref. 06-088). Allow to soak and
heat to the boil with constant agitation.
Distribute into nal containers and sterilize by autoclaving at 121C for 15 minutes.
Letheen Modied Broth

Ref. 02-237
Specication
FDA recommended liquid medium for the primary recovery of stressed microorganisms in the microbial examination of cosmetics.
Formula (in g/L)

Casein Peptone ........................................ 15,0
Meat Peptone ........................................... 10,0
Meat extract ................................................ 5,0
Yeast extract ............................................... 2,0
Sodium chloride ........................................ 10,0
Lecithin ....................................................... 0,7
Sodium bisulte .......................................... 0,1
Final pH 7,0 0,2
Directions
Dissolve 43 g of the dry powder in 1 L of distilled water
with 5 mL of Polysorbate 80 (Ref. 06-088). Distribute
in suitable containers and autoclave at 121C for 15
minutes.
83
Letheen Media
Description
References
In the early 40s, Weber and Black recommended the

use of lecithin and polysorbates to neutralize the antimicrobial action of the quaternary amonium compounds
(QACs).
In 1965 the methodology was accepted by AOAC for the
antimicrobial assays and extendes their use to all the
cationic tensoactives (detergents).
The TAT (Tryptone Azolactin Tween) medium in the
Newburger Cosmetic Analysis Manual (2nd. Ed., 1977)
is closely similar in formulation and uses to the AOAC
recipe. In 1978 the FDA (Bacteriological Analytical
Manual, 1978) incorporated it as previous presumptive
and enrichment medium for every microbial examination
in cosmetics.
ASTM Standard E 640-78 (1991) Test method for preservatives in water-containing cosmetics. Philadelphia.
PA
FDA (1998) Bacteriogical Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg. MD
HORWITZ, W. (2000) Ofcial Methods of Analysis. 17th
ed. AOAC International. Gaithersburg. MD
LUCAS, J.P. (1977) Microbiological Examination of
Cosmetics. Newburguers Manual of Cosmetic Analysis.
AOAC
WEBER, G.R. & L.A. BLACK (1948) Relative efciency
of quaternary inhibitors. Soap and Sanit Chem. 24:134139
The SCHARLAU formulation of the Letheen Media

covers the old recipe of Lucas (1977) in the Newburger
Manual (Ref.1-236 Letheen Agar and Ref. 02-236
Letheen Broth) and the new recipe (Ref. 01-237 Letheen
Modied Agar and Ref. 02-237 Letheen Modied Broth)
of the FDA (B.A.M., 1979)
84
Liebermeister & Braveny Agar

Ref. 01-446
Specication
Solid medium for the selective isolation of -hemolytic
streptococci from throat samples.
Formula (in g/L)

Meat peptone ........................................... 1,00
Meat extract .............................................. 0,60
Yeast extract ............................................. 0,50
L(+)Lysine ................................................. 0,02
Sodium chloride ........................................ 6,00
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
Suspend 25 g of powder in 930 mL of distilled water and
heat to the boil. Distribute into suitable containers and
sterilize in an autoclave at 121C for 15 minutes. Cool
to 45C, then add 70 mL/L of sterile debrinated lambs
blood. Homogenize well and pour into sterile plates.
Description
Despite its simplicity, this medium has a better yield in
the recovery of -hemolytic streptococci than the commonly used Blood Agar.
Okamoto et al. and, later Bernheimer and Rodbart
demonstrated the strong stimulatory effect of the nucleic
acids on the hemolytic properties of streptococci. Liebermeister and Braveny formulated a medium with insufcient nutrients for the development of normal microorganisms but with an increased amount of nucleic acids
in the yeast extract. Moreover, they also included lysine,

which has an effect on the hemolysis similar to that of
the nucleic acids.
The result is that microorganims fail to grow or form only
small colonies that have hemolysis zones(-hemolysis)
of normal or greater than normal size. Viridans streptococci (alpha-hemolytic) virtually do not grow, and if
hemolysis zones form at all, they are minimal.
Technique
Plates are inoculated by surface seeding and incubated
at 37C for 24-48 hours. After the incubation small colonies form which are surrounded by a large, well dened
hemolytic zone. Staphylococci and enterococci are
almost viridans streptococci.
References
OKAMOTO, H., S. KYODA, R. ITO (1939) Jap. J. Med.
Sci. VI Pharmacol. 12:167.
BERNHEIMER, A.W., M. RODBART (1948) The effect
of nucleic acids and carbohydrates on the formation of
streptolysin. J. Exp. Med. 88:149.
LIEBERMEISTER, K., J. BRAVENY (1971) Ein Nhrsubstrat zur Isolierung von haemolytischen Streptokokken.
Z. Med. Mikrobiol. Inmunol. 156:149-153.
MILATOVIC, D. (1981) Comparison of ve selective
media for beta-haemolytic streptococci. J. Clin. Pathol.
34:556-558
85
Lingby Iron Agar Base

Ref. 01-584
Specication
Solid medium for the isolation and enumeration of
heterotrophic H2S producing bacteria from sh and sh
products.
Formula (in g/L)

Tryptone .................................................. 20,0
Meat extract ................................................ 3,0
Yeast extract ............................................... 3,0
Ferric citrate................................................ 0,3
Sodium thiosulfate ...................................... 0,3
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,4 0,2
Directions
Suspend 46,6 g of powder in 1L of distilled water and
heat to the boil. Distribute into suitable containers and
sterilize by autoclaving at 121C for 15 minutes. Cool to
45-50C and aseptically add 8 mL/L of a lter sterilized
solution of 5% L-Cysteine hydrochloride. Homogenize
well and pour into sterile plates.
Lingby Iron Agar Base has been formulated according to the Food Analysis Nordic Committee standard.
This standard states that cysteine addition must be
done separately in order to achieve better results in the
recovery of the H2S producing bacteria from sh and sh
products that have not been under heat treatment or with
any addition of preservatives.
Technique
For the total count of H2S producing bacteria, the Committee prescribes the following technique:
Transfer 1 mL of sample dilution to sterile Petri plates.
Add 10-12 mL of molten medium and cool to 45C. Homogenize by shaking it gently. When cooled, add a new
layer of medium and incubate at 201C for 3 days.
Select the adequate plates and proceed with total count.
H2S producing bacteria form black colonies.
If incubation temperature is too high or the covering
layer is too thin, black colonies loses the colour rapidly.
References
Nordisk Metodikkommit fr Livsmedil. (1994) UDC.
637.56:576.8.08-No96. 2ntg- Bacteriological examination of fresh and frozen seafood.
Description
Listeria Enrichment Media

Listeriosis
Listeriosis is the generic name for the whole group of
disorders caused by Listeria monocytogenes, which is
the only species of the Listeria genus that is important
as a human pathogen, howewer occasionally L.seeligeri,
L.welshimeri and L.ivanovii have been related with
human diseases. In anycase, all the species are pathogenic between the ovin and bovine cattle.
Positive diagnosis of listeriosis can be obtained only by
the isolation and cultivation of the responsible bacteria from blood or cerebrospinnal uid samples of the
affected organism. Faecal samples have a very debatable value, since actually it is estimated that between
1-10% of human population may be an intestinal carrier
of L.monocytogenes. This species of bacteria have been
isolated from 37 species of mammals, domestic or wild,
as well as from 17 species of birds and some species of
shes and shellshes.
Listeria monocytogenes bears a notorious tolerance to
heat, cold and desiccation, and it acts as a real psichrotrophic. These facts and the high morbidity when it
infects annimals and humans increase its interest as a
food-borne pathogen.
Its presence has been related with food as raw milk or
supposedly pasteurized milk, soft-ripped cheese, ice
creams, vegetables, raw meat, cured fermented meat,
raw or smoked sh, but it has been isolated also from
soil, silages and other environmental samples.
86
Detection and conrmation of

Listeria
Food control requires to culture the samples that use
to need 5-7 days before provide denitive results, and
although the commercial production of DNA probes to
help the diagnosis, they are not available yet.
The traditional technique of low temperature (4C) enrichment has been substituted for selective enrichments
at temperatures near to the optimum growth temperature. Isolation can be also performed over selective
and differential media, that most of the cases allow to
disregard the special Henry lighting technique, but nal
identication must be carried out by biochemical and
serological methods.
The type of sample restrains the technique of detection
because the companion ora is very important. If a population more than 100 Listeria cells/g is suspected, the
sample can be inoculated directly over the solid selective
media, but in other cases, an enrichment technique, in
one or two stages, must be adopted.
Specic identication
Directions
All the Listeria species are grampositive, oxidase- and

urease-negative. They produce catalase, do not reduce
nitrate and hydrolyze esculin. Provide +/+ result in the
O/F test for glucose and they also acidify the bottom
and surface in TSI Agar. Provide a positive result in the
Voges-Proskauer and Phenol Red tests. Differential
characteristics are shown in the table above.
Dissolve 54,5 g of powder into 1 L of distilled water. Distribute 500 mL in each container and autoclave at 121C
for 15 minutes. Cool to 50C and then aseptically add
the suitable selective supplement toeach 500 mL: UVM
I for primary enrichment (Ref. 06-106CASE) and UVM
II/Fraser for secondary enrichment (Ref. 06-111CASE).
Note: Prepared medium (broth+supplement) must be
kept away from light, since it helps the production of acriavine-oxidised photocomplexes that
repress Listeria growth.
References
DONNELLY, C.W., R.E. BRACHETT, S. DOORES, W.H.
LEE, J. LOVETT (1992)
Listeria, in Compendium of method for the microbiological examination of foods, Vanderzant & Splittstoesser
(eds) APHA. Washington.
ADAMS, M.R., M. O. MOSS (1995) Food Microbiology .
The Royal Society of Chemistry. Cambridge. U.K.
FDA/ Center for Food Safety & Applied Nutrition. (March
1999) Foodborne Pathogenic Microorganisms and
Natural Toxins Handbook: Listeria monocytogenes http://
vm.cfsan.fda.gov/mow
Following you will nd the Scharlau Microbiology production program in selective and differential media for the
enrichment and isolation of Listeria.
Listeria Enrichment Broth Base

(UVM)
Ref. 02-472
Specication
Liquid culture medium for the enrichment of Listeria sp.
Formula (in g/L)

Proteose Peptone ..................................... 5,00
Tryptone ................................................... 5,00
Meat extract .............................................. 5,00
Yeast extract ............................................. 5,00
Sodium chloride ...................................... 20,00
Esculin ...................................................... 1,00
Final pH 7,4 0,2
Description
This basal broth for the enrichment of listeria is made according the AOAC modications over the medium of the
Vermont University (UVM), since it was demonstrated
that a slight increase of acriavine concentration in the
secondary enrichment and a strong reduction in the
amount of nalidixic acid in all the stages allowed more
positive isolations.
Technique
Primary enrichment
Add 25 g or 25 mL of sample to 225 mL of primary
enrichment broth (Base Broth, Ref. 02-472, and UVM I,
Ref. 06-106CASE) and homogenize it all in a stomacher
for 2 minutes. Incubate the mixture at 30C for 24 hours,
but after the rst 4 hours take aliquots of 0,2 mL to plates
with Oxford Selective Agar (Ref. 01-471) in order to
make the isolation.
Secondary enrichment
After 24 hours of primary enrichment, inoculate the secondary enrichment broth (Base Broth, Ref. 02-472, and
UVM II/Fraser, (Ref. 06-111CASE) at the rate of 1:100.
Incubate at 30C. After 4 and 24 hours take aliquots of
0,2 mL to plates with Oxford Selective Agar (Ref. 01471) in order to make the isolation.
Isolation
Isolation is carried out on the Oxford Selective Agar (Ref.
01-471) plates prepared during the primary and secondary enrichment, after 24-48 of incubation at 30-37C.
Sometimes it is advisable to alkalinize the inoculum
before the seeding, by mixing 1 mL of enrichment broth
with 5 mL of 0,5% sterile KOH solution.
87

acc. to Lovett

acc. to Fraser
Ref. 02-498
Ref. 02-496
Specication
Specication
Liquid culture medium for the enrichment of Listeria, according Lovett et al.
Liquid culture medium for the enrichment and detection

of Listeria spp. according ISO standards 11290-1 and
11290-2.
Formula (in g/L)

Tryptone ................................................. 17,00
Yeast extract ............................................. 6,00
Soy peptone ............................................. 3,00
Sodium chloride ........................................ 5,00
Dextrose ................................................... 2,50
Final pH 7,3 0,2
Directions
Dissolve 36 g of powder into 1 L of distilled water and
distribute 500 mL per ask. Sterilize by autoclaving at
121C for 15 minutes. Cool to 50C and aseptically add
to each ask the content of one vial of Listeria Supplement for Selective Enrichment acc. to FDA/IDF (Ref.
06-107CASE). Homogenize and distribute into suitable
containers.
kept away from light, since it helps the production of acriavine oxidised photocomplexs that
repress Listeria growth.
Description
This medium according to Lovett et cols. formulation has
been adopted by the FDA for the analysis of food, and it
is recommended by the IDF/FIL for the selective enrichment of Listeria in milk samples, due to its good results
in the recovery of stressed bacteria, with only an enrichment stage.
Technique
Mix the sample (25 mL or 25 g) with 225 mL of complete
enrichment broth and incubate at 30C for 7 days. Make
subcultures after 24 hours, 48 hours and 7 days in the
following way:
Inoculate 0,5 mL of enrichment culture in a solid medium
for the Listeria isolation (Oxford Agar Base, Ref. 01-471,
or Palcam Agar Base, Ref. 01-470, with their respective
selective supplements).
Alkalinize 0,5 mL of enrichment culture by mixing with
4,5 mL of 0,5% sterile KOH solution and inoculate on a
solid medium for Listeria isolation.
88
Formula (in g/L)

Tryptone ................................................... 5,00
Meat extract .............................................. 5,00
Yeast extract ............................................. 5,00
Sodium chloride ...................................... 20,00
Esculin ...................................................... 1,00
Dissodium phosphate ............................. 12,00
Lithium chloride ........................................ 3,00
Final pH 7,2 0,2
Directions
Dissolve 57,4 g of powder into 1 L of distilled water.
Distribute 500 mL per ask and sterilize in the autoclave
at 121C for 15 minutes. Cool to 50C. Aseptically add
the Ferric Ammonium Citrate for Bacteriology (Ref.
06-112CASE) and Listeria Supplement for Secondary
Enrichment UVM II/Fraser (Ref. 06-111CASE) to each
ask and homogenize well.
kept away from light, since it helps the production
of acriavine oxidised photocomplex that repress
Listeria growth.
Description
This broth base for Listeria enrichment is made according to the modications of Fraser and Sparber over the
UVM medium, which have been adopted by the USDAFSIS. The inclusion of lithium chloride inhibits the development of enterococci which also may hydrolyze esculin
in the same way of Listeria. Thus, any darkness in the
medium produced by the reaction of esculetin coming
from esculin hydrolysis with iron present in the medium
can be taken as a presumptive presence of Listeria.
Moreover, it seems the ferric citrate helps L. monocytogenes development.
Technique
Although some authors use the Fraser Broth as the only
enrichment, it has been veried than better results are
obtained if it is employed as secondary enrichment, according the following methodology:
Inoculate the sample to examine in a primary enrichment
broth (UVM I, Ref. 02-472 or Lovett Broth, Ref. 02-498)
and incubate for 18-24 hours.

Take aliquots of 0,1 mL, inoculate them in tubes with 10
mL of Fraser Broth and incubate for 24-28 hours.
Tubes that become dark are considered presumptively
positive and must be subcultured over isolation and
conrmation solid media, such as Oxford Agar Base
(Ref. 01-471) or Palcam Agar Base (Ref. 01-470). Tubes
that remain clear are considered negative and can be
discarded or kept in incubation for 24 hours more to
clear the doubts if any.
Palcam Agar Base

Ref. 01-470
Specication
Solid, selective and differential medium for the detection,
enumeration and isolation of Listeria spp. according ISO
standards 11290-1 and 11290-2.
Formula (in g/L)

Tryptone ................................................. 23,00
Lithium chloride ...................................... 15,00
Mannitol .................................................. 10,00
Sodium chloride ........................................ 5,00
Yeast extract ............................................. 3,00
Starch ....................................................... 1,00
Esculin ...................................................... 0,80
Ferric Ammonium citrate........................... 0,50
Dextrose ................................................... 0,50
Phenol red ................................................ 0,08
Agar ........................................................ 13,00
Final pH 7,2 0,2
Directions
let it soak. Heat to boil and distribute 500 mL per ask.
Sterilize by autoclaving at 121C for 15 minutes. Cool
to 50C and aseptically add the Palcam Agar Selective
Supplement (Ref. 06-110CASE) to each ask. Mix well
and pour into sterile plates.
Listeria growth.
Description
Palcam Agar is based in the formulation described initially by van Netten et cols., which has a high selectivity and
a good colonial differentiation. Selectivity is achieved by
the inclusion of lithium chloride, acriavine, polymixin B
and ceftacidine, since they inhibit the growth of almost
all the gramnegative bacteria and most of grampositive
companion bacteria.
Listeria hydrolyze esculin to esculetin, which reacts with
ferric ammonium citrate producing a dark precipitate
colouring the colonies to green-grey with beige halos.
The colonies of enterococci or staphylococci that may
overpass the high selectivity of this medium can be
easily recognized, since they use mannitol and produce
yellow colonies and halos, contrasting with the cherry

red colour of medium.
However, when there are many Listeria colonies, the
entire medium turns dark, and the differentiation can be
interfered. In these cases it is advisable to perform a
more diluted inoculation.
Technique
Seed the Palcam Agar with a growth from a primary
enrichment broth (UVM I, Ref. 02-472 or Lovett, Ref.
02-498) or a secondary enrichment broth (UVM II, Ref.
02-472 or Fraser, Ref. 02-496). Incubate in a microaerophile atmosphere for 48 hours at 37C.
In these conditions, Listeria colonies have a size approx.
2 mm diameter, green-grey coloured with black core
and halo. Enterococcus and Staphylococcus colonies
are bigger, grey with green-brown halo if they do not
use mannitol or form yellow colonies with yellow halo if
they do. Anyway, suspicious colonies must be conrmed
biochemically and serologically.
Oxford Agar Base

Ref. 01-471
Specication
Xn
R-22-36/38
S-26-37/39-46
Solid, selective and differential medium for the detection,

enumeration and isolation of Listeria sp. according ISO
standards 11290-1 and 11290-2.
Formula (in g/L)

Tryptone ................................................. 10,00
Lithium chloride ...................................... 15,00
Proteose peptone ................................... 10,00
Sodium chloride ........................................ 5,00
Yeast extract ............................................. 3,00
Starch ....................................................... 1,00
Esculin ...................................................... 1,00
Ferric Ammonium citrate........................... 0,50
Agar ........................................................ 13,00
Final pH 7,2 0,2
Directions
let it soak. Heat to boil and distribute 500 mL per ask.
Sterilize by autoclaving at 121C for 15 minutes. Cool to
50C and aseptically add the Oxford Agar Selective Supplement (Ref. 06-109CASE) to each ask . Mix well and
pour into sterile plates.
Listeria growth.
89

Description
References
Oxford Agar is derivated from the original formulation by

Curtis et al, which worked a medium with a high nutritive
ability as the Columbia Agar and added inhibitor agents
to remove all the undesirable companion bacteria.
The current formulation keep the high capacity to support growth and restrain all the gramnegative ora and
most of grampositive, including yeast. Thanks to the
inhibitors incorporated in the selective supplement:
cycloheximide, acriavine, colystin, phosphomicyn
and ceftacidine. These inhibitors in addition to lithium
chloride restrain the growth of all other bacteria except
Listeria.
However, selectivity is not total, but Listeria colonies are
easily recognizable since as they hydrolyze esculin. Free
esculetin that reacts with the ferric ions and produce
a dark precipitate around the colonies, which typically
present a grey-blue colour with a very dark core.
McCLAIN, D., W.H. LEE (1988) Development of USDAFSIS Method for isolation of Listeria monocytogenes
from raw meat and poultry. JAOAC 71:3:660-664.
ATLAS, R.M. (1993) Handbook of Microbiological Media.
CRC Press. Boca Raton, Florida.
VANDERZANT, C., D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbiological examination of food. APHA, Washington D.C.
LOVETT, J., D.W. FRANCIS, J.M. HUNT (1988) Listeria
monocytogenes in raw milk; Detection, incidence and
pathogenicity. J. Food Protect. 50;188-192
LOVETT,J., A.D. HITCHINS (1989) Listeria isolation.
FDA Bacteriological Analytical Manual. 6th Ed. Supp.
Sept. 1987 (2nd Print):29.01
FRASER, J.A., W.H. SPERBER (1988) Rapid detection
of Listeria sp. in food and environmental samples by
esculin hydrolysis. J. Food Prot. 51:762-765.
van NETTEN, P., J.PERALES, A. van de MOODSDUCK,
G.D.W. CURTIS, D.A.A. MOSSEL (1989) Liquid and
solid selective differential media for the detection and
enumeration of Listeria monocytogenes. Int. J. Food
CURTIS, G.D., R.G. MITCHELL, A.F. KING, E.J. GRIFFIN (1989) A selective differential medium for the isolation of Listeria monocytogenes. Letters Appl. Microbiol.
8:95-98
ISO 11290 standard (1996) Microbiology of food and
animal feeding stuff. Horizontal method for the detection and enumeration of Listeria monocytogenes. Part 1
- Detection method. Part 2 - Enumeration method.
Technique
Although the selectivity of the medium is enough to allow
the isolation and differentiation by direct surface inoculation, a previous dilution of the inoculum is advisable, or
even more when the sample is highly polluted.
In anycase, most authors prefer one or two previous
cultures in any of the primary enrichment media (UVM I,
Ref. 02-472 or Lovett, Ref. 02-498) or secondary enrichment media (UVM II, Ref. 02-472 or Fraser, Ref. 02-496)
before inoculating in Oxford Agar.
Incubation is carried out at 37C, and after 24 hours
typical colonies of Listeria monocytogenes are visible.
However, it is recommended to extend incubation for
more 20-24 hours in order to evidence the slow growing strains even though this could allow staphylococci
or streptococci development, since they would grow
weakly.
Ref. 02-496 Listeria Enrichment Broth acc. Fraser.

Left: control; right: positive.
90
Lysine Media
Lysine Iron Agar (LIA)
Ref. 01-094
Specication
Differential medium for Enterobacteria, recommended by
Edwars and Ewing for Salmonella identication.
Formula (in g/L)

Gelatin peptone ........................................ 5,00
Yeast extract ............................................. 3,00
Dextrose ................................................... 1,00
L-Lysine .................................................. 10,00
Ammonium Ferric citrate .......................... 0,50
Agar ........................................................ 15,00
Final pH 6,7 0,2
Directions
heat to boiling . Dispense in tubes and sterilize by
autoclaving at 121C for 15 minutes. Allow to solidify in
slanted position, with copious depth and short slant.
Description
Lysine and Iron medium has been widely used for the
differentiation among different biotypes of Salmonella,
above all those corresponding to S. arizona, which, on
usual selective isolation in plate media, like MacConkey
or Deoxycholate, may give colour or colourless colonies
due to the fact that their lactose fermentative capacity is
quite variable.
If it is considered that these microorganisms, when
cultured in tubes with Kligler Iron Agar (Ref. 01-103) or
Triple Sugar Iron (Ref. 01-192), produce enough acid to
avoid sulde formation, it is comprehensible that they
are sometimes not recognized or
overlooked as pathogen, and wrong or negative results
are consequently obtained. On the other hand, Salmonella is the only genus of enterobacteria that normally
decarboxylates lysine and produces important amounts
of hydrogen sulde.
LIA works perfectly verifying these two characteristics,
and that is the reason why it is used at the same time of
use of Kligler Iron Agar (Ref. 01-103) and/or TSI (Ref.
01-192) in the second phase of isolation of pathogenic
enterobacteria.
Technique
From some suspicious colonies in the isolation media,
and with a needle, inoculate a Kliglers medium tube,
and without recharging the inoculation loop, pass it to
LIA by surface streaking and depth stab. Incubate them
closed, but at the same time sufciently ventilated, at
35-37C for 24 hours.
Microorganisms that decarboxylate the lysine, quickly,
produce a strong alkalinization in all the medium, that
is observed by the indicator turning to purple. Whereas,
those that have no lysine decarboxylase activity, acidify
the medium at the bottom producing a yellow colouration, meanwhile the surface of the medium remains with
the same original colour or with alkaline reaction.
Proteus type members are distinguished easily, since,
over the yellow bottom, they produce a typical red or
orange colour on the surface, due to oxidative deamination of lysine. The microorganisms which produces of
hydrogen sulde blacken the medium because of iron
sulfur precipitates.
Although the gas production may be observed, generally
this medium does not offer optimal conditions for this,
and gives very irregular results, even giving total inhibition in some cases.
References
EDWARDS, P.R. and M.A. FIFE (1961) Lysine-Iron Agar
in the detection of Arizona cultures. Appl. Microbiol. 99,
478-480.
EWING, J. (1982) Edwards and Ewings identication of
Enterobacteriaceae. 4th. Ed. Elsevier Sci. Pub. Co. Inc.
N.Y.
McFADDIN, J.F. (1985) Media for the isolation, cultivation, identication and maintenance of medical bacteria.
William & Wilkins. Baltimore
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press,London
DOWNES, F.P. & K. ITO (2001)) Compendium of Methods for the Microbiological Examination of Food. 4th ed
APHA. Washington
ed. AOAC International. Gaitherburg. MD.
MARSHALL R.T. (1992) Methods for the examination of
dairy products. APHA. Washington.
91
Lysine Media
92
M-17 Media
M-17 Agar
M-17 w/o Lactose Broth
Ref. 01-245
Ref. 02-580
Specication
Specication
Solid selective medium for Streptococcus thermophilus

in the common examination of yoghurt acc. to ISO 7889:
2003 and IDF 117: 2003.
Liquid selective medium for Streptococcus thermophilus

in the examination of yoghurt and other dairy products.
Formula (in g/L)

Tryptone ................................................... 2,50
Meat peptone ........................................... 2,50
Soya peptone ........................................... 5,00
Yeast extract ............................................. 2,50
Meat extract .............................................. 5,00
Sodium glycerophosphate ...................... 19,00
Ascorbic acid ............................................ 0,50
Lactose ..................................................... 5,00
Agar ....................................................... 15,00
Final pH 7,0 0,2
Directions
soak. Heat to boiling and dispense into suitable containers. Sterilize by autoclaving at 121C for 15 minutes.
Avoid unnecessary overheating and remelting.
M-17 Broth
Ref. 02-245
Specication
Liquid selective medium for Streptococcus thermophilus
in the common examination of yoghurt.
Formula (in g/L)

Tryptone ................................................... 2,50
Meat peptone ........................................... 2,50
Soya peptone ........................................... 5,00
Yeast extract ............................................. 2,50
Meat extract .............................................. 5,00
Ascorbic acid ............................................ 0,50
Lactose ..................................................... 5,00
Final pH 7,0 0,2
Directions
soak. Heat if necessary and dispense into suitable containers. Sterilize by autoclaving at 121C for 15 minutes.
Formula (in g/L)

Tryptone ................................................... 2,50
Meat peptone ........................................... 2,50
Soya peptone ........................................... 5,00
Yeast extract ............................................. 2,50
Meat extract .............................................. 5,00
Ascorbic acid ............................................ 0,50
Final pH 7,0 0,2
Directions
Disolve 37 g of powder in 1 L of distilled water, heating if
necessary. Dispense into suitable containers. Sterilize by
Description
M-17 Agar was developed by Terzaghi and Sandine for
the screening of bacteriophages in streptococci of dairy
industry. Afterward, Shankar and Davies demonstrated
the efcacy of this medium for selective isolation of
Streptococcus thermophilus in yoghurt. Medium combines a strong buffer, which aids development of streptococci, with a high concentration of glycerophosphate,
which inhibits the growth of lactobacilli.
Technique
Suggested technique to enumerate streptococci is to
seed in mass or by stabbing, with agar melted and
cooled to 50-55C, and then to incubate them at 42C for
a 24 hours period. With these conditions, all the colonies might be streptococci. Longer incubation periods or
lower temperatures may cause morphological changes
in the colonies which hinders in the the recognition of the
colonies.
FIL-IDF has adopted this medium for yoghurt examination, and uses it simultaneously with MRS Agar (Ref.
01-135).
Lactobacilli count is performed analogously in MRS Medium, at 30C and in CO2 enriched atmosphere.
Colonies of lactose positive streptococci are visible after
15 hours, and after 5 days they may reach a diameter of
about 3-4 mm, whereas those lactose negatives are 1
mm of diameter. However, longer incubation period may
hinder the observations, due to the growth of lactobacilli.
Bacteriophages presence is observed by the appearance of characteristic plaques over the bacterial growth.
93
M-17 Media
References
TERZAGHI, B.E. and SANDINE, W.E. (1975) Improved
medium for lactic streptococcacal phages from cheese
factories. Appl. Environm. Microbiol 29:807-813
SHANKAR, P.A. and DAVIES, F.L. (1977) Selective
Technique for yogurt Bacteria Enumeration. J. Soc. Dairy
Technol. 30:28.
FIL-IDF Standard 146A (1998) Identication of characteristic micro-organisma of yoghurt.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press,London
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th
ed.APHA. Washington.
ISO 7889 standard (2003). Yogurt - Enumeration of
characteristic microorganisms - Colony- count technique
at 37C.
94
MacConkey Media
In the production program of SCHARLAU you can nd
the following formulations of the MacConkey media:
MacConkey Agar ....................................... Ref. 01-118
(Eur. Phar. Medium H)
MacConkey Broth ...................................... Ref. 02-118
MacConkey Modied Broth ...................... Ref. 02-120
MacConkey Sorbitol Agar .......................... Ref. 01-541
MacConkey w/o Salt Agar ......................... Ref. 01-320
MacConkey WHO Broth ........................... Ref. 01-121
Mac Conkey G Broth ................................ Ref. 02-611
(Eur Pharm Medium G)
MacConkey Agar
(Eur. Phar. Medium H)
Directions
Dissolve 40 g of powder in 1 L of distilled water. Bring to
the boil and distribute into suitable containers tted with
Durham tubes. Sterilize by autoclaving at 121C for 15
minutes.
MacConkey G Broth
(Eur. Phar. Medium G)
Ref. 02-611
Specication
Ref. 01-118
Liquid medium for the detection and enumeration of

coliforms by MPN technique. It is a modication of the
classic medium, where neutral red is replaced by a less
aggressive indicator, according European Pharmacopoeia.
Specication
Formula (in g/L)
A selective and differential medium for the detection,

isolation and enumeration of coliforms from a variety of
samples according European Pharmacopoeia and ISO
standard.
Formula (in g/L)
Peptone .................................................. 20,00

Lactose ................................................... 10,00
Bile Salts .................................................. 5,00
Final pH 7,4 0,2
Peptone ................................................ 20,000

Lactose ................................................. 10,000
Bile Salts #3 ........................................... 1,500
Sodium chloride ...................................... 5,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,001
Agar ...................................................... 15,000
Final pH 7,2 0,2
Directions
Directions
Ref. 02-120
Suspend 51,5 g of powder to 1 L of distilled water. Bring

to the boil and sterilize by autoclaving at 121C for 15
minutes.
Specication
MacConkey Broth
Dissolve 35 g of powder in 1 L of distilled water. Heat

only if necessary to help the dissolution. Distribute into
suitable containers tted with Durham tubes. Sterilize by
MacConkey Modied Broth

coliforms by MPN technique according ISO standard.
Formula (in g/L)

coliforms by MPN technique.
Peptone .................................................. 20,00

Lactose ................................................... 10,00
Bile Salts .................................................. 5,00
Sodium chloride ........................................ 5,00
Final pH 7,4 0,2
Formula (in g/L)
Directions
Ref. 02-118
Specication
Peptone ................................................ 20,000

Lactose ................................................. 10,000
Bile Salts ................................................ 5,000
Sodium chloride ...................................... 5,000
Neutral Red ............................................ 0,075
Final pH 7,4 0,2
Dissolve 40 g of powder in 1 L of distilled water. Heat

only if necessary to help the dissolution. Distribute into
suitable containers tted with Durham tubes. Sterilize by
95
MacConkey Media
MacConkey Sorbitol Agar
Ref. 01-541
Specication
Selective and differential solid medium for the detection
of Enterohaemorrhagic Escherichia coli (EHEC O157:
H7) according ISO standard.
Formula (in g/L)

Peptone ................................................ 20,000
Sorbitol ................................................. 10,000
Bile salts ................................................. 1,500
Sodium chloride ...................................... 5,000
Neutral Red ............................................ 0,030
Crystal violet ........................................... 0,001
Agar ...................................................... 15,000
Final pH 7,1 0,2
Directions
Suspend 51,5 g of powder into 1 L of distilled water and
heat to boiling. Sterilize in the autoclave at 121C for 15
minutes. If the medium is used the same day of preparation autoclaving is not necessary but the boiling must be
for 3 minutes at least.
Description
The substitution of lactose by sorbitol for the isolation of
the enteropathogenic serotypes O111 y O55 of Escherichia coli was proposed in 1952 by Rappaport and
Hening. The usefulness of the medium was showed by
March and Ratman (1986) and Adas (1991) for the detection, differentiation and isolation of the enterohaemorrhagic (EHEC) and the verocytoxin-producing (VTEC)
strains of the serotype O17:H7 of E. coli.
The only modication on the typical MacConkey Media
formulations is the replacement of lactose with sorbitol.
The enterohaemorrhagic strains do not use this substrate and produce colourless colonies, but the other
serotypes can ferment sorbitol and produce red colonies
In all others aspects, MacConkey Agar with Sorbitol
works similarly as the other media in the MacConkey
group. Peptone supply the nitrogen and sodium chloride
the osmotic environment. Crystal violet and bile salts
inhibits the growth of grampositive bacteria. Neutral red
acts as the pH indicator.
Technique
Spread the inoculum on the dry surface of the medium
and incubate at 352C for 24 hours. Usually, the O157:
H7 serotype forms colourless colonies and the other
strains of E. coli red colonies. The results must be
recorded at 24 hours because an extended incubation
produce a decreasing colouring in the sorbitol-ferment-
96
ing colonies and some times there is some colony of the

o157:H7 serotype that begins to ferment sorbitol.
Some gramnegative bacteria like Pseudomonas, Proteus and Klebsiella can growth on the MacConkey Agar
with Sorbitol but their colonies are diverse and easy to
differentiate from E. coli.
Because the failure in the fermentation of sorbitol by
some strains on E. coli no-enterotoxigenic and the atypical colony production by some enterohaemorrhagic ones
it is recommended the use of some others media in concomitance with the MacConkey Agar with Sorbitol and to
conrm the suspect colonies by serological, biochemical
or molecular techniques.
MacConkey w/o Salt Agar

Ref. 01-320
Specication
A selective and differential medium for the detection,
isolation and enumeration of enterobacteria, especially
Proteus sp. from the clinical specimens.
Formula (in g/L)

Peptone ................................................ 20,000
Lactose ................................................. 10,000
Bile Salts #3 ........................................... 1,500
Neutral red .............................................. 0,075
Agar ...................................................... 15,000
Final pH 7,4 0,2
Directions
Add 46,5 g of powder to 1 L of distilled water. Bring to
the boil and distribute in suitable containers. Esterilize by
MacConkey WHO Agar

Ref. 01-121
Specication
Differential medium with moderate selectivity, without
crystal violet, according to WHO recommendation, for
the isolation of enterobacteria.
Formula (in g/L)

Peptone ................................................ 20,000
Lactose ................................................. 10,000
Bile Salts mixture .................................... 2,500
Sodium chloride ...................................... 5,000
Neutral Red ............................................ 0,075
Agar ...................................................... 15,000
Final pH 7,4 0,2
MacConkey Media
Directions
Suspend 53 g of powder in a litre of distilled water. Bring
to the boil, distribute in suitable containers and autoclave
Description
The MacConkey media are well known and popular
enrichment system for coliform bacteria.
At the beginning of the last century, MacConkey made
the original formulation and included ox bile as inhibitor
of the gram-positive bacteria and the litmus as the indicator of the acid production from lactose sugar. Lately
the litmus has been substituted by phenol red indicator
to make interpretations easier and more precise.
The media have been adapted to facilitate the coliform
detection with the advancement of knowledge of the
bacterial physiology. The most signicant modication to
the original formulation has been the substitution of the
ox bile by puried bile salts that improve the selectivity
and avoid the inherent turbidity which is due to the fat
material of the bile. The effectivity of the inhibition of the
bile salts is variable and depends on the relative concentration of cholate and taurocholate.
Another important modication was the inclusion of
supplementary inhibitors such as crystal violet and/or
brilliant green, that are the most popular formulations in
America, but not in Europe where the lower selectivity is
prefered.
In the 60s the toxicity of neutral red on the stressed
cells of coliforms was demonstrated, especially on
some strains of E. coli, and then the pH indicator was
changed to the Bromcresol purple, being less aggressive
than the neutral red.
However the most extensive use is in the liquid form,
nevertheless there are solids formulations with agar, for
every
modication.
At the moment, the several formulations available of the
MacConkey media offer a wide range from a low to high
selectivity and the lactose positive bacteria grown on this
medium form red colonies due to acid production due to
the lactose fermentation and thus E. coli colonies can be
easily distinguished as they also form a small precipitation zone of bile salts around them.
Eventually,some enterococci can also grow, but they
are easy to distinguish from the coliforms, as they form
smaller colonies and the absence of precipitation zone.
Medium can be used as Presumptive medium for E.coli
(by uorescent reaction) if before sterilization MUG (Ref.
06-102CASE) is added. In the medium with MUG the E.
coli colonies show a light blue uorescence under the
UV illumination. The formulation without salt offers a low
electrolyte content that almost suppresses the swarming
growth of Proteus.
Technique
From a decimal dilution bank, 1 mL samples are inoculated into empty sterile petri dishes in duplicate. Then ,15
mL of molten medium at 45C is poured into every plate
and mixed carefully . After the solidication, a second
layer of another 5 mL of sterile medium is poured into

every plate to seal the surfaceand improve enumeration of the colonies.
For the enumeration, after an incubation of 24 h. at
35C, select plates with 30-300colonies. The characteristic colonies must be conrmed for coliform identity by
gas production from lactose in a broth culture.
The MacConkey broth (code 2-118) can be used for the
enumeration of coliforms by the MPN technique, selecting the positive tubes that shows turbidity, colour turned
to red purple and with gas production.
The same above characteristics, but turning into a yellow
color are valid if the MacConkey broth code 2-120 is
used.
If the MUG medium is used a supplementary reading
under UV illumination must be carried out.
References
ADAMS, S. (1991) Screening for verotoxin-producing E.
coli. Clin Lab. Science 4:1:19-20.
CLESCERI, L.S., A.E. GEENBERG & A.D. EATON
and Wastewater. 20th ed. APHA-AWWA-WEF. Washington DC. USA
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods.4th. ed.
APHA. Washington
EUROPEAN PHARMACOPOEIA (2007) 5th ed.
(Supp.5.7) EDQM. Council of Europe. Strasbourg.
HITCHINS, A.D., P. FENG, W.D. WATKINS, S.R. RIPEY
y C.A. CHANDLER (1998). E. coli and coliform bacteria.
Bacteriological Analytical Manual. 8th ed. AOAC International. Gaitherburg. MD. USA
AOAC Intl. Gaithersburg. MD. USA
ISO 9308-2:1990 Standard. Water Quality. Detection and
enumeration of coliforms, thermotolerant coliforms and
presumptive E. coli. MPN Method.
Escherichia coli
MARCH, S.B. y S. RATMANN (1986) Sorbitol-McConkey
Medium for detection of E. coli O157:H7 associated with
hemorrhagic colitis. J. Clin. Microbiol. 23:869-872
McCONKEY, A.T. (1905) Lactose-fermenting Bacteria in
faeces. J. Hyg 5:333.
MURRAY, P.R., E.J. BARON, M.A. PFALLER, F.C. TENOVER, y R.H. YOLKEN (Eds) (1995) Manual of Clinical
Microbiology 6th ed. A.S.M. Washington D.C. USA
RAPPAPORT, F. y E. HENING (1952) Media for the isolation and differentiation of pathogenic E. coli (serotypes
O111 and O55) J. Clin. Pathology 5:361-362
USP 29-NF 25. (2006) <61> Microbial Limit Tests US
Pharmacopoeial Corp. Inc. Rockville. MD, USA
VANDERZANT, C. y D.F. SPLITTOESSER (Eds) (1992)
Compendium of methods for the microbiological examination of foods. 3rd ed. A.P.H.A. Washington D.C.
97
MacConkey Media
VARNAM, A.H. y M.G. EVANS (1991) Foodborne pathogens. Manson Publishing Ltd. London. U.K.
WHO (1963) International Standards for Drinking Waters, 7th ed., Churchill Ltd. London
WINDLE-TAYLOR, E.(1958) The Examination of Water
and Wastewater Supplies, 7th ed. Churchill Ltd. London
Malachite Green Broth

Ref. 02-467
Specication
Liquid culture medium for the selective enrichment of
Pseudomonas aeruginosa in water, according the DIN
38411 standard.
Formula (in g/L)

Meat peptone ......................................... 15,00
Meat extract .............................................. 9,00
Malachite green (Oxalate) ........................ 0,03
Final pH 7,0 0,2
Directions
To prepare concentrated broth: Dissolve 25 g of powder in 1 L of distilled water and distribute into suitable
containers.
To prepare diluted broth: Dissolve 8,3 g of powder in 1 L
of distilled water and distribute into suitable containers.
In both the cases, sterilize by autoclaving at 121C for
15 minutes.
Description
Habs and Kirschner rst described this medium in 1948
based on the resistance of Pseudomonas aeruginosa
to malachite green as a selective factor. Lately, in 1974
Schubert and Blum modied the medium composition
and proposed it as an enrichment step of Pseudomonas
in very polluted water since malachite green oxalate
at the concentration as above formulation inhibited the
growth of almost all gramnegative microorganisms but it
did not affect Pseudomonas growth.
Years after the proposal of Schubert and Blum, the
medium was adopted ofcially as an enrichment medium
98
by the German StandardizationInstitute (DIN) and other

legal bodies for the microbiological analysis of water and
food.
Technique
If the product to be examined is not restricted to specic
standard (as DIN 38411 for water), it is suggested that
the nal malachite green concentration for enrichment
should not exceed 0,01 g/L. Thus the concentrated or
diluted broth should be used in function of the size or
volume of the sample.
Carry out the incubation at 352C for 24-48 hours.
Cultures that show turbidity due to growth should be
selected for the later conrmation for the presence of
References
BUNDESGESUNDHEITSAMT: Amtliche Sammlung von
Untersuchungverfahren nach 35 LMBG Beuth Verlag
Berlin Kln.
DEUTSCHE EINHEITSVERFAHREN sur Wasser-,
Abwasser- und Schlammuntersuchung. VCH Verlagsgesellschaft D-6940 Weinheim.
DIN 38411; Teil 6: Mikrobiologische Verfahren (Gruppe
K):Nachweis von Pseudomonas aeruginosa (K8)
HABS, H., K.H. KIRSCHNER (1943) Der Pyocyaneus
Meerschweinchenhautversuch zur Prfung von Haut
desinfektionsmiteln. Z. Hyg. 124:557-578.
SCHUBERT, R., U.BLUM (1974) Zur Frage der Eignun
der Malachitgrn-Bouillon nach Habs u. Kirschner als
Anreicherungsmedium fur Pseudomonas aeruginosa
aus dem Wasser. Zbl. Bakt. Hyg. I Orig. B 158:583-587
Malt Extract Media

Media based on malt extract may be considered as general growth substrates due to their richness and nutrient
balance. They are very suitable for the cultivation of
fastidious microorganisms. Classically, with acidic pH,
they are used for the isolation, cultivation and maintenace of moulds and yeast, but with pH near to neutrality,
they support bacterial growth of bacteria with special or
fastidious nutritional needs.
then this will make the solidication of agar more difcult.

When acidication is below pH 5,0 do not remelt the
agar since the solidifying agent will be hydrolized.
The SCHARLAU manufacturing program covers all the

published formulations, and included in this manual are
some of the most commonly used. We propose that the
technicians choose the formulation most suited to their
requirements, or, alternatively, ask us for more details or
possible modications.
Specication
See also YM Media (Refs. 1-219, 2-219) and Wort Agar

(Ref. 01-132)
Malt Extract Agar No. 1

Ref. 01-573
Solid medium for the isolation and enumeration of fungi.
Formula (in g/L)

Malt extract ............................................... 30,0
Soy peptone ............................................... 3,0
Agar .......................................................... 15,0
Final pH 5,6 0,2
Directions
Ref. 01-111

soak. Bring to the boil. Distribute into suitable containers
and sterilize in the autoclave at 121C for 15 minutes.
Specication
Description
Culture medium for moulds and yeast.
Formula (in g/L)

Malt extract ............................................... 13,0
Dextrine ...................................................... 2,5
Gelatin peptone .......................................... 5,0
Agar .......................................................... 15,0
Final pH 5,5 0,2
Directions
heat gently with constant stirring until the boiling. Dispense in suitable containers and sterilize by autoclaving
at 115C for 15 minutes. Avoid overheating since the low
pH of the medium may hydrolize the agar.
Malt Extract Agar may support the growth of almost all

of the fungi very well, because of its balanced composition, and restrains most of the bacteria due to the strong
acidity.
Should more selection against the bacterial growth be
desired, readjust the pH to 3,5 by adding a sterile solution of 10% lactic acid or 5% tartaric acid to the molten
medium. After the additions do not reheat the medium.

Ref. 01-574
Specication
Solid medium for the isolation and enumeration of fungi.
Description
Formula (in g/L)
Malt Extract Agar is a classic culture medium for moulds

and yeast. Malt extract has enough sugar (maltose, glucose, sucrose) to allow a copious growth, and in extreme
cases, additional necessary growth factors are provided
by the gelatin peptone.
Malt extract ............................................... 30,0

Mycological peptone ................................... 5,0
Agar .......................................................... 15,0
Final pH 5,4 0,2
Technique
Malt Extract Agar has been widely used in maintenance,
isolation and identication of fungi, and it is also proposed in several pharmacopiea as a medium for the
control of sterility in pharmaceutical products, though it is
mostly used for comparative morphological studies.
Should more selectivity be desired, you can add a few
millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but
Directions
soak. Heat to the boil and distribute into suitable containers. Sterilize in the autoclave at 121C for 15 minutes.
99
Malt Extract Media

Description
Description
The balanced and rich nutrient composition of the medium makes it suitable for morphogenetic and structural
studies of fungi. Due to its low pH it restrains the bacterial growth to a greater extent, but the total supression
can be achieved by adding to the melted medium at
55C, 20 mL of sterile solution of 10% Lactic acid or 5%
tartaric acid, making the pH reduces to 3,5. In these
conditions, do not heat the medium to avoid the hydrolysis of agar.
This formulation of classic Malt Extract Broth is according to Reiss modication in order to achieve better
results for the cultivation of Aspergillus avus.
Malt Extract Broth No. 1

Ref. 02-111
Specication
Liquid culture medium for the moulds and yeasts.
Formula (in g/L)

Malt extract ............................................... 13,0
Dextrine ...................................................... 2,5
Gelatin peptone .......................................... 5,0
Final pH 5,5 0,2
Directions
Dissolve 20,5 g of powder in 1 L of distilled water, heating up if necessary. Distribute into suitable containers
and sterilize in the autoclave at 121C for 15 minutes.
Description
Malt Extract Broth is a classic culture medium for the
moulds and yeasts. Malt extract has sufcient sugar
(maltose, glucose, sucrose) to allow a copious growth,
and in more demanding cases, the necessary growth
factors are provided by the gelatin peptone.
Malt Extract Broth No. 2

Ref. 02-491
Specication
Liquid culture medium for the moulds and yeasts.
Formula (in g/L)

Mycological peptone ................................... 3,0
Malt extract ............................................... 17,0
Final pH 5,4 0,2
Directions
Dissolve 20 g of powder in 1 L of distilled water, heating up if necessary. Distribute in suitable containers and
sterilize by autoclaving at 121C for 15 minutes. Do not
overheat, since a browning by Maillard reaction can be
produced.
100
Technique
Malt Extract Broth has been widely used in maintenance, isolation and identication of fungi, and it is also
proposed in several pharmacopeia as a medium for the
control of sterility in pharmaceutical products, though it is
mostly used for comparative morphological studies.
Should more selectivity be desired, you can add a few
millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but this
makes the solidication of agar very difcult. When acidication is below pH 5,0, do not remelt the agar since the
solidifying agent is hydrolized below pH 5,0.
References
FDA (1998). Bacteriological Analytical Manual. 8th ed.
Revision A AOAC International Gaithersburg) MD.
DOWNES, F.P & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press,.
BALLOWS, HAUSLER, HERMAN, ISENBERG &
SHADOMY (eds.) (1991) Manual of Clinical Microbiology. ASM. Washington.
REIS, J. (1972) Ein selektives kulturmedium fr der
Nachweiss von Aspergillus avus. Zbl. Bakt. Hyg. I. Abt.
Orig. 220:564-566.
RAPP, M. (1974) Indikator-Zustze zur Keimdifferenzierung auf Wrze und Malzextrakt Agar Milchwiss.
29:341-344.
Mannitol Salt Agar (Chapman Agar)

Ref. 01-116
Specication
Selective medium for the isolation of staphylococci
according USP and ISO standard.
Formula (in g/L)

Meat extract ............................................ 1,000
Casein peptone ...................................... 5,000
Meat peptone ......................................... 5,000
Sodium chloride .................................... 75,000
D-Mannitol ............................................ 10,000
Phenol red .............................................. 0,025
Agar ...................................................... 15,000
Final pH 7,4 0,2
References
ATLAS, R.M. & L.C.PARKS (1993) Handbook of Microbiological Media. CRC Press. BocaRaton. Fla. USA
CHAPMAN (1945) The signicance of sodium chloride in
studies of staphylococci. J. Bact 50:201
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods.4th ed.
APHA. Washington DC. USA
Revision A. AOAC Internacional Inc. Gaithersburg. Md.
USA
USP 29- NF 25(2005) <61>Microbial Limit Tests. US
Pharmacopoeial Convention Inc. Rockville. Md. USA
Directions
bring to the boil. Dispense in tubes or asks and sterilize
Description
Mannitol Salt Agar is a classical medium for the detection and enumeration of staphylococci. It was described
by Chapman and has been adopted by many ofcial organisations. Several modications have been developed
from it with more or less similar effectivity.
This medium uses the advantage of high tolerance of
staphylococci to salinity, to use sodium chloride as a selective agent, since only the staphylococci and halophilic
enterobacteria are able to grow freely at this concentration of salt employed in this medium while other bacteria
are inhibited. It also exploits the correlation between
the pathogenic and fermentative capacity of mannitol
of staphylococci, to establish a presumptive diagnosis.
Mannitol fermentation with an accumulation of acid products is shown by the phenol red indicator turning yellow,
that produces a yellow halo surrounding the presumptive
pathogen colonies, meanwhile the rest of the medium
remains orange in colour.
Staphylococcus aureus ATCC 25923
Technique
A massive surface inoculation and an incubation at 37C
for 36 hours or at 32C for 3 days is recommended.
The typical appearance of the colonies after the correct
incubation is as follows: Presumptive pathogenic staphylococci (coagulase +) are mannitol positive and are big
colonies with a yellow halo. Non-pathogenic Staphylococci (coagulase -) are usually mannitol negative and are
small colonies without halo or change in colour.
In any case, coagulase presence must be tested by the
classical technique, after a pure culture in the liquid medium is obtained, in order to establish its true pahogenic
potential.
101
Maximum Recovery Diluent

Ref. 02-510
Specication
Isotonic diluent for the maximal recovery of stressed
microorganisms according ISO standard.
Formula (in g/L)

Peptone .................................................... 1,00
Sodium chloride ........................................ 8,50
Final pH 7,0 0,2
Directions
Disolve 9,5 g of powder in 1 L of distilled water and distribute into suitable containers. Sterilize by autoclaving at
Description
This formulation combines the osmotic pressure of the
physiological saline solution with the protective action
of the peptone to obtain a good recovery of stressed
microorganisms.
The sodium chloride assures the isotonic conditions and
the low concentration of the peptone does not allow the
cellular growth in the short period (2-4 hours) of time
required for the preparation of the dilution bank of the
sample.
102
Technique
According to the ISO method, the sample is diluted in a
ratio 1:10 with the Maximum Recovery Diluent and homogenized by a vortex mixer or stomacher. After a short
period (10-15 minutes) of rest, a decimal dilution bank
with the same diluent is released following the standard
procedures. Plates are inoculated from the different
concentration of the dilution bank.
Reference
ISO/DIS 6649 Meat and Meat Products. Detection and
Enumeration of Clostridium perfringens
Escherichia coli
Mayeux Agar
Ref. 01-223
Specication
Solid culture medium for the detection of Leuconostoc in
fermentation starters of mixed ora.
Formula (in g/L)

Peptone ................................................ 10,000
Yeast extract ........................................... 5,000
Sucrose .............................................. 100,000
Dextrose ................................................. 5,000
Sodium citrate ........................................ 1,000
Gelatine .................................................. 2,500
Sodium azide .......................................... 0,075
Agar ...................................................... 15,000
Final pH 6,0 0,2
Directions
heat up in boiling water bath at 50-55C, till the complete
liquefaction of the gelatine is obtained. Heat to boiling
and dispense into suitable containers. Sterilize in the
autoclave at 121C for 15 minutes. Avoid overheating,
since it may affect the solidication.
Description
This differential and selective medium for Leuconostoc
was originally described by Mayeux in 1961, and was
later modied by the same author to this formulation.
This allows a very specic separation of microorganisms
in the lactic fermentation starters with mixed ora.
Citrate, glucose and gelatine helps for the growth of Leuconostoc, and the large amount of sucrose allows a copious production of dextrane polymer by L.dextranicum.
Sodium azide avoids the growth of undesired ora, and

at the same time, hampers the colonial development of
lactic streptococci. Other authors state that the addition
of little amounts of tetracycline (15 mcg/mL) produces
the inhibition of lactic streptococci without affecting
Leuconostoc.
Technique
The plates are inoculated by surface inoculation, and
then incubated at 21C for 4 days. Most of the streptococcal strains, including Streptococcus lactis, S.cremoris
and S.diacetilactis do not grow or grow a little after the
third day of incubation. In those cases, their colonies are
small, opaque and cream or yellow coloured. Leuconostoc colonies have a bigger and earlier growth. Leuconostoc citrovorum form the colonies of 0,5 to 1 mm
diameter, which are translucent and iridescent.
L. dextranicum form big colonies (1-5 mm), which are
transparent and mucosal.
References
MAYEUX and COLMER (1961) J. Bact. 81:1.009.
MAYEUX, SANDINE and ELLIKER (1962) J. Dairy Sci.
45:665.
McDONOUGH, HARDGROVE and TITSLER (1962) J.
Dairy Sci. 45:656.
FIL-IDF Standard 149A (1997) Dairy starters of lactic
acid bacteria culture. Composition standard.
103
Meat Liver Agar

Ref. 01-562
Specication
reduced to H2S by some Clostridium spp and reacts with

the iron from ammonium iron citrate producing a dark
precipitate that blackening the medium.
Solid medium for the cultivation of anaerobic microorganisms
Technique
Formula (in g/L)

Meat Extract ........................................... 10,00
Liver Extract ........................................... 10,00
Dextrose ................................................... 0,75
Soluble starch ........................................... 0,75
Sodium sulte ........................................... 1,20
Ammonium Iron (III) citrate ....................... 0,50
Agar ........................................................ 13,00
Final pH 7,6 0,2
Directions
had to boiling with constantly stirring. Distribute in suitable containers and sterilize in the autoclave at 121C
for 15 minutes.
Description
The mixture of meat and liver extract is a highly reducing nutrient basis that provides the supply of nitrogen for
the growth of anaerobes. The energy source is provided
by the dextrose and the starch acts only as a metabolic
detoxier. The sulphite present in the culture medium is
104
The meat liver agar can be used in petri dishes or

in tubes. When the inoculum is in poured plates, the
reducing power of the medium permits the growth of the
anaerobes. If spreading plates are used the incubation in
an anaerobic environment is compulsory, but some times
is enough cover the plate with Sealing Anaerobic Agar
(Ref. 01-174). Temperature and time of the incubation
must be suitable to the sample, but it is recommended
more than 48 hours at 35-37C
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc. London.
CORETTI, C. (1962) Prfung eines leberpulvers auf
Eignung zur Herstellung von Leberbrhe und Leberagar
zur Anaerobenzuchtung. Berl. Mnch. Tierrztl. Wissch.
75:205.
VANDERZANT, C. & D.F. SPLITTSTOESSER (1992)
Compendium of methods for the microbial examination
of Foods. 3rd Ed. APHA. Washington.
Methyl Red Voges Proskauer Media (MRVP Media)

Methyl Red Voges Proskauer
Broth (MRVP Broth)
(Clarks Lubs Medium)

Modied Broth for Bacillus
(MRVP Modied Broth)
Ref. 02-207
Ref. 02-572
Specication
Specication
Classic liquid medium for differential tests (Voges

Proskauer and Methyl Red) in Enterobacteria according
ISO standards 6579 and 6585 and FIL - IDF 93 standard.
Liquid culture media to perform the Voges-Proskauer

test in Bacillus cultures according FIL-IDF 181 standard.
Formula (in g/L)

Peptone ...................................................... 7,0
Dextrose ..................................................... 5,0
Potassium phosphate ................................. 5,0
Final pH 7,0 0,2
Directions
Formula (in g/L)

Tryptone .................................................. 7,00
Dextrose ................................................... 5,00
Dipotasium phosphate .............................. 5,00
Sodium chloride ........................................ 5,00
Final pH 7,0 0,2
Directions

up only if necessary. Dispense in tubes and sterilize by

if it is necessary. Distribute in suitable containers (5 mL
in test tubes of 16 x 160 mm) and sterilize in autoclave
at 121C for 15 minutes
Description
Description
The classical Clark and Lubs medium which is used to

perform the tests of Methyl Red and Voges Proskauer,
that together with Indole and Citrate tests (IMViC) allow
the differentiation within the coliform group of bacteria.
The fundamentals of these reactions are as follows:
This medium is produced according the FIL-IDF formulation to perform the test of acetil-metil-carbinol production
in Bacillus cereus and other species of Bacillus.
Methyl Red Test (M.R. test)

Among the Enterobacteriaceae, the E.coli biotype ferments glucose by the mixed acid pathway, accumulating
acid, which reduces the initial pH. It can be detected by
the methyl red indicator, which turns yellow above the
pH 5,1 and becomes red below pH 4,4.
Voges Proskauer Test (V.P. test)
Enterobacteria of Klebsiella-Enterobacter biotype
ferment the glucose by the 2-3-butanediol pathway.
Although the acids are produced in this way, the neutral
or alkaline products are also formed and at the end the
reaction is neutral or alkaline. Due to this, the incubation
must be extended up to 3 days. After this period, the
methyl red reaction is negative.
Nonetheless, Voges Proskauer test is complementary
to Methyl Red test in some ways. It shows the 2-3-butanediol and acetoin production, that are substances
difcult to nd in the mixed acid pathway. It exploits the
fact that these two products, in alkaline medium, oxidize
themselves to diacetyl, which reacts with guanidine and
produces visibly coloured compounds.

Saline Broth
(MRVP Saline Broth)
Ref. 02-456
Specication
Liquid culture medium for the Mehyl Red and Voges
Proskauer tests.
Formula (in g/L)

Peptone ................................................... 7,00
Dextrose ................................................... 5,00
Sodium chloride ...................................... 30,00
Final pH 7,4 0,2
Directions
up only if necessary. Distribute into suitable containers
and sterilize by autoclaving at 121C for 15 minutes.
105
Methyl-red Voges Proskauer Media (MRVP Media)

Description
This medium is used to perform the tests of Methyl Red
and Voges Proskauer, that, together with Indole and Citrate tests allow the differentiation within the enteric bacteria. The fundamentals of these reactions are described
in the Methyl-Red Voges-Proskauer Broth (Ref. 02-207)
Technique
There are several techniques to carry out these tests.
One of them is as follows:
The tube with medium is inoculated with the microorganism to be studied and incubated at 30C for at least 3
days and up to 5 days maximum. Just before reading,
culture is separated in two portions, one for each test.
1) Methyl Red Test.
Add 4-5 drops of Methyl Red Reagent (Ref. 06-007) to
the culture, and shake in order to homogenize. Observe
for the colour development in the medium. The test is
considered positive if it turns to red and negative if it
remains yellow.
Positive (red colouration): E.coli, Edwardsiella, Shigella,
Salmonella, Citrobacter, Proteus, Klebsiella ozoenae, Klebsiella rhinoscleromatis, Yersinia.
Negative (yellow colouration): Enterobacter, Hafnia, Serratia, Klebsiella pneumoniae.
With Erwinia, this reaction has no signicance since it
gives variable reactions.
2) Voges Proskauer Test
Add Barrits Reagent to the medium (Ref. 06-027) until it
gets a milky appearance and then add OMearas Reagent (Ref. 06-006) until milky appearance disappears.
Shake vigorously.
Test is positive if the medium acquires a pink-violet colour, forming at the top of the tube. If the test is negative,
there is no colour formation. Relative amounts of each
reagent depend on initial volumes of the medium. Never
incubate above 30C.
Positive (pink-intense red): Enterobacter, Hafnia, Klebsiella pneumoniae, Serratia.
Negative (no colour change): Escherichia, Edwardsiella,
Citrobacter, Salmonella, Shigella, Yersinia, Klebsiella ozonae, Klebsiella rhinoscleromatis.
With Proteus and Erwinia types, this reaction has no
signicance since it gives variable reactions.
106
Voges Proskauer test may be performed in a quicker

way, using very little volumes of medium and massive inoculum. This allows the readings with short incubations
(18-20 hours), and also the readings may be accelerated
by gently heating the culture almost to boiling point after
adding the reagents. However, erroneous results are
more likely by using this method.
Refer to the FIL-IDF Standard for the specic technique
of MRVP Modied Broth.
References
VOGES, O, B. PROSKAUER (1898) Beitragzur
Ernhrungsphysiologic und zur Differentialdiagnose der
hmorrhagischen Septicemie. Z. Hyg.
CLARK, W., H. LUBS. (1915) The differentiation of bacteria of the colon-aerogenes family by the use of indicators. J. Inf. Dis. 17:160-173
BARRIT, M. (1936) The intensication of the Voges
Proskauer reaction by the addition of alpha-naftol. J.
Path. Bact. 42:441-452
OMEARA, R. (1931) A simple delicate and rapid
methods of detecting the formation of acetylmethyl/carbinol by bacteria fermenting carbohidrats. J. Path. Bact.
34:401-406
MOLLNDER, R., J. BHMANN, B. GREWING (1982)
Die Verstrkung der Voges-Proskauer Reaktion durech
fumarat. Zbt. Bakt. Hyg. I Alet. Orig. A 252:316-323.
SCHWEIZERISCHES LEBENSMITTELBUCH 5th Ed.
Ch. 56A. Berna.
Rev. A. AOAC International. Gaithersburg. MD.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,London.
ISO Standard 6579 (2002) Microbiology of Food and animal feeding stuffs- Horizontal method for the detection of
Salmonella species.
FIL-IDF (1998) International Provisional Standard 181.
Dried Milk Products: Enumeration of Bacillus cereus:
Most Probable Number Technique.
FIL-IDF (2001) Milk and Milk products Detection of
Salmonella.
ISO 6585 standard (2001) Milk and Milk products - Detection of Salmonella.
Microbial Content Test Agar (TSA Lecithin Polysorbate)

Ref. 01-613
Specication
Solid medium for sampling of surfaces of sanitary importance with RODAC plates technique.
Formula (in g/L)

Tryptone ................................................. 15,00
Soy peptone ............................................. 5,00
Sodium chloride ....................................... 5,00
Lecithin ..................................................... 0,70
Polysorbate 80 ......................................... 5,00
Agar ........................................................ 15,00
Final pH 7,3 0,2
Directions
The dehydrated medium has a characteristic brown
sugar appearance and may seem moist.
let it soak. Bring to the boil and dDistribute in suitable
containers and sterilize in autoclave at 121C for 15
minutes.
Description
This medium is a modication of the classical TSA for
the surface sampling by the RODAC (Replicate Organism Detection and Counting) plate technique. Collection
of samples from identical areas (replicate) before and
after treatment with disinfectant yields data useful in
evaluating cleaning procedures in environmental sanitation.
Lecithin is incorporated to neutralize quaternary ammonium compounds and polysorbate 80 is used to neutralize phenolic disinfectants, hexachlorophene, formalin
and, with lecithin, ethanol.
References
HICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW
(1992) Microbiological tests for equipment, containers,
water and air. In R.T. Marshall (Ed.) Standard Methods
for the examination of Dairy Products 16th ed. APHA
Washington.
EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F.
FRANK (2001) Microbiological Monitoring of the Food
Processing Environment. In Downes & Ito (Eds) Compendium of Methods for the Microbiological Examination of
Foods. 4th ed. APHA. Washington DC.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Culture Media. CRC Press. Boca Ratn, Fla.
107
Milk Agar
Ref. 01-514
Specication
Solid culture medium for the plate count test in dairy
products.
Formula (in g/L)

Peptone .................................................... 5,00
Yeast extract ............................................. 3,00
Powdered milk .......................................... 1,00
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
it soak. Bring to the boil and distribute into suitable containers. Sterilize by autoclaving at 121C for 15 minutes.
Description
Milk Agar is approved by the European Commission and
it is formulated according to the recommendations of the
European Association of Ice-Cream Producers (EuroGlace) for the microbiological examination of ice-creams.
Technique
Suggested technique is the standardized count on massinoculated plates. Inoculum is obtained from a decimal
dilution bank of the sample. Once inoculated, the plates
should be left undisturbed for 1-3 hours and then incubated at 30C for 3 days.
References
KLOSE, J. (1968) Harmonisierung des speisesrechtes
under EWC-Sbwaren 14:778-780.
KLOSE, J. (1968) Entwarf einer Riehlinie zar Aueichung
der Rechtvorschiften fur Speiseeis unden Mitiedsstaaten
der EWG. Sbwaren 14:780-782
108
Motility Media
Motility Indole Ornithine Fluid
Medium (MIO)
Ref. 03-422
Specication
Medium for the demonstration of motility, indole production and ornithine decarboxylase activity of enterobacteria.
Formula (in g/L)

Yeast extract ............................................. 3,00
Dextrose ................................................... 1,00
Gelatin peptone ...................................... 10,00
Casein peptone ...................................... 10,00
L-Ornithine HCl ......................................... 5,00
Agar .......................................................... 2,50
Final pH 6,6 0,2
Directions
Suspend 31,5 g of powder in 1 L of distilled water and let
it soak. Bring to the boil and distribute in tubes. Sterilize
Technique
Remove all the dissolved air in the medium by heating
up the tubes in boiling water bath and cooling them upto
room temperature. Taking the growth of the primary
isolation as the inoculum, inoculate the tubes by a single
deep stab. Incubate aerobically at 352C for a 18-24
hours period.
Motility can be observed by the diffuse growth at the
upper side of the stab, meanwhile the immotile bacteria
grow along the stab, producing a clear streak.
Ornithine decarboxylation is indicated by the presence of
a dark purple colour throughout the tube. Negative reaction produces only a single purple band at the top, and
the rest of the tube changes to yellow.
Indole production is veried after the addition of a few
drops of Kovacs Reagent (Ref. 06-018) (shake gently).
The presence of a red ring signies the positive reaction,
and if the colour is yellow, then the reaction is negative.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press,Boca Raton,Fla.
EDERER, G.M. and M. CLARK (1970) Motility-Indol-Ornithine Medium. Appl. Microbiol. 2:849.
FDA (1998) Bacteriological Analytical Manual 8th ed.
REvision A. AOAC International. Gaithersburg. MD.
Motility Nitrate Medium

Ref. 03-612
Specication
Medium for the motility determination and nitrate reduction of clostridia in food products acc. ISO 7937.
Formula (in g/L)

Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Disodiun phosphate .................................... 2,5
Galactose ................................................... 5,0
Agar ............................................................ 5,0
Final pH 7,3 0,2
Directions
Suspend 21,5 g of powder in 1 L of distilled water containing 5 mL of glycerol and heat to boiling. Dispense
Description
This semisolid medium has been made according to the
rules suggested by the US Food and Drug Administration for the identication of Clostridium perfringens in
food.
Technique
Prepared tubes regenerate themselves if kept in boiling
water bath for 10 minutes to eliminate the dissolved oxygen. Let them get cooled to 70-80C and then inoculate
them by stabbing the centre. Take a black colony grown
on TSN Agar (Ref. 01-195) as the inoculum. Incubate
the tubes at 37C for 18-20 hours without sealing nor in
anaerobic chamber.
In this medium, if the growth is stopped at 5-7mm. from
the surface, signies that there is anaerobiosis. Non-motile is evident as the growth is observed only inside the
stab.
To verify the nitrate reduction to nitrite, pour a few drops
of Nitrate A Solution (Ref. 06-003) and Nitrate B Solution
(Ref. 06-004) on the surface of the medium. If a pink or
red colour appears, reaction is positive.
Clostridium perfringens is an anaerobic , non-motile and
reducer of nitrate to nitrite microorganism.
References
FDA (1998) Baceriological Analytical Manual. 8th ed.
Revision A. AOAC International. Gaithersburg.
ISO 7937 Standard (2004). Microbiology of food and animal feeding stuffs. Horizontal methods for the enumeration of Clostridium perfringens. Colony count technique.
109
MRS Media
MRS Agar
Ref. 01-135
Specication
Solid culture medium for lactobacilli, according to de
Man, Rogosa and Sharpe and ISO standards 9332 and
15214.
Formula (in g/L)

Peptone Proteose ................................... 10,00
Meat extract .............................................. 8,00
Yeast extract ............................................. 4,00
D(+)Glucose ........................................... 20,00
Sodium acetate ........................................ 5,00
Triammonium citrate ................................. 2,00
Manganese sulfate ................................... 0,05
Polysorbate 80 ......................................... 1,00
Agar ........................................................ 14,00
Final pH 6,2 0,2
Directions
Suspend 66 g of powder in 1 L of distilled water. Bring to
the boil slowly with gentle stirring until complete dissolution. Dispense into suitable containers and sterilize by
MRS Broth
Ref. 02-135
Specication
Liquid culture medium for lactobacilli, according to de
Man, Rogosa and Sharpe and ISO standards 9332 and
15214.
Formula (in g/L)

Peptone Proteose ................................... 10,00
Meat extract .............................................. 8,00
Yeast extract ............................................. 4,00
D(+)-Glucose .......................................... 20,00
Sodium acetate ........................................ 5,00
Triammonium citrate ................................. 2,00
Manganese sulfate ................................... 0,05
Polysorbate 80 ......................................... 1,00
Final pH 6,2 0,2
110
Directions
Suspend 52 g of powder in 1 L of distilled water. Heat up
to complete dissolution and dispense into suitable containers. Sterilize by autoclaving at 121C for 15 minutes.
Description
MRS Agar and Broth are a modication of the previously
used media for the cultivation of lactobacilli, all of them
based on tomato juices nourishing properties. The addition of magnesium, manganese and acetate, together
with the Polysorbate, has provided an improved medium
for the growth of lactobacilli, including that of very fastidious species such as Lactobacillus brevis and Lactobacillus fermenti.
On the other hand, the quality of the peptones in addition
to the meat and yeast extracts, combine together all the
necessary growth factors that make the MRS media one
of the best media for the cultivation of lactobacilli.
Nevertheless, these media selectivity is low and the contaminants tend to grow in these media, which signies
a higher selectivity is needed. We therefore suggest the
use of subculture in solid medium, on double layer and
broth. In many cases,the growth is encouraged by a CO2
enriched atmosphere in the medium.
MRS media is particularly recommended for the enumeration and maintenance of lactobacilli either by the MPN
technique (in broth) or on the plate by massive inoculation, overlaying it with a second layer of molten medium.
This technique overcomes the need of a CO2 enriched
atmosphere.
References
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods.4th Ed.
FIL-IDF Standard 146 (2003) Yoghurt. Identication of
characteristic micro-organisms.
IFU Method No 5 (1996) Lactic Acid Bacteria Count Procedure. Schweizerischer Obstverband. CH-6302 Zug
IFU Method No 7 (1998) Sterility testing of aseptic lled
products, commercial sterile products and preserved
products. Schweizerischer Obstverband. CH-6302 Zug
IFU Method No 9 (1998) Microbiological examination of
potential spoilage micro-organisms of tomato products.
Schweizerischer Obstverband. CH-6302 Zug
ISO Standard 9232 (2003) Yoghurt Identication of
characteristic microorganisms (Lactobacillus delbrueckii
subsp bulgaricus and Streptococcus thermophilus)
ISO Standard 15214 (1998) Horizontal method for the
enumeration of mesophilic lactic acid bacteria Colony
count technique at 30C
MAN, J.C. de, ROGOSA, M. y SHARPE, M. Elisabeth
(1960) A mdium for the cultivation of lactobacilli. J.
Appl. Bact.; 23:130.
Mueller Hinton Media

Mueller Hinton Agar
Ref. 01-136
Specication
Widely recommended medium for antibiotic and sulfonamide susceptibility testing, according to the Kirby-Bauer
and the Ericsson methods.
Formula (in g/L)

Peptone .................................................... 17,5
Beef infusion solids .................................... 2,0
Starch ......................................................... 1,5
Agar .......................................................... 17,0
Final pH 7,3 0,2
Directions
Add 38 g of powder to 1 L of distilled water and let it
soak. Bring to the boil to dissolve the medium completely. Sterilize by autoclaving at 121C for 15 minutes.
Description
The Mueller Hinton Agar was originally designed for the
primary isolation of meningococci and gonococci. With
the addition of blood it becomes an optimal medium
for the growth of Neisseria. It is also more effective if
reheated and turned into a Chocolate Agar. It should
never be remelted or reheated once the blood has been
added to it.
Mueller Hinton Broth

Ref. 02-136
Specication
Liquid version of the agar with the same name, recommended for the studies about MIC of antibiotic.
Formula (in g/L)

Peptone .................................................... 17,5
Starch ......................................................... 1,5
Solids of meat infusion ............................... 2,0
Final pH 7,3 0,2
Directions
Add 21 g of powder to 1 L of distilled water and dissolve
it completely. Distribute in suitable containers. Sterilize
Description
Mueller Hinton Broth is the liquid version of the agar with
the same name, and can be used in parallel with the
agar when comparative studies are desired as well as
when a broth with a high nutritive capacity is required.
It is especially suggested for inoculum preparation for
sensitivity assays.
In this medium, and in its solid version, presence of the

starch is very important, since it acts as a detoxifying
agent against the toxic substances if present in the sample and it also acts as the cell regenerator.
Technique
For the culture of Neisseria the best results are obtained
if incubation is carried out in a humid chamber with a
CO2 enriched atmosphere, if an anaerobic jar is not
available. This environment can be obtained by placing
the plates in a hermetically sealed air-tight container,
a dessicator for instance, together with a cotton swab
soaked in water and a lighted candle end. Once the container is full the ame consume oxygen and by the time it
is extinguished, the atmosphere inside the container has
got 5 to 8% CO2 enrichment.
The Mueller-Hinton Agar has proved to be one of the
most efcient medium in the anti-bacterial susceptibility testing. Without the addition of blood it can even
be used for sulfonamide sensitivity testing since it is
free from most of its antagonists (nucleotides, etc.). If
this type of assay is conducted, the zones of inhibition
should be examined just after 12-18 hours, before the
usual overgrowth occurs, since after 24 hours it tends to
interfere with the examination of sulfonamides sensitivity.
For this purpose, a small inoculum will help the early
formation of zones of inhibition. It should amount to a
100 to 300 times smaller inoculum than that of the corresponding strain which is used in the antibiotic sensitivity testing.
In 1970 the WHO proposed this medium for antibacterial sensitivity testing, and it has been widely used since
then.
Sensitivity testing can be conducted by a variety of
techniques, both on solid and liquid media. The most
commonly used method in routine work is that derived
from Kirby-Bauer and recommended by the American
Association of Clinical Pathologists. It provides information on growth around a disk impregnated with antibacterial substance.
The Bauer-Kirby method is more precise and is semiquantitative by category. It uses the Mueller-Hinton Agar
and disks with high antibiotic concentration. The inoculum is rst standardized with a Mac-Farland nephlometer. Then the plate is inoculated with a swab dipped in
the standardized suspension, and nally the disks are
arranged properly and at the equidistance from each
other on the plate and then incubated.
Some authors suggest that the inoculum should be
modied by introducing a double layer of mass inoculated medium. This system undoubtedly provides sharper
and more dened zones of clearing or inhibition. Plates
are incubated at 37C
111
overnight and then the zones of inhibition are measured.

Results are reported in terms of Resistant, Moderately
Resistant and Sensitive strains (Table above).
The Ericsson technique, which has been adopted in
most European countries has already standardized the
culture medium (Mueller-Hinton) and the quantity per
plate (25 mL on 9 cm diameter plates). It has also standardized the inoculum concentration.
The fresh culture suspension to be examined (incubated
for 18 hours in liquid medium) must be diluted enough,
so as to ensure the presence of conuent growth on the
agar.
Suggested Dilutions:
Enterobacteria- Pseudomonas: dilution of 1/300.
Staphylococcus - Enterococcus: dilution of 1/300.
Streptococcus - Haemophilus: dilution of 1/10.
The plate is seeded by ooding its surface. The excess inoculum is removed with a sterile pipette and the
antibiotic disks are arranged properly on the plate. Allow
a pre-diffusion period of 30-60 minutes before incubation so that the antibiotic can slowly diffuse before the
growth. After the incubation at 37C for 12-18 hours,
measure the zones of inhibition and refer to the Assay
Regression Curves. Results are reported in terms of
112
Sensitive, Resistant or as Minimum Inhibitory Concentration (MIC).

This latest technique undoubtedly offers more precision
and reliability than the previous ones. Nevertheless, the
Kirky method, which is semiquantitative, is much more
simple and easy to adopt in everyday practice. On the
other hand, the Ericsson technique is highly recommended for the effectivity and the sensitivity studies.
The Mueller-Hinton medium plates can be stored refrigerated in plastic bags for a month without affecting their
results of sensitivity testing. However, they should not be
used if the medium shows any dehydration.
The Mueller Hinton Agar Scharlau fullls the WHO requirements for the conducting microbial sensitivity tests
and the basic characteristics are veried in every batch.
Nevertheless some variation in the results between
batches can be observed and technicians claims about
the origin of these variability. At this point must keep in
mind a lot of factors that are a source of variability:
1. Since the nutritional requirements of organisms vary,
some strains may be encountered that fail to grow or
grow poorly on these media.

2. Numerous factors can affect results: inoculum size,
rate of growth, medium formulation and pH, length of
incubation and incubation environment, disk content and drug diffusion rate, and measurement of
endpoints. Therefore, strict adherence to protocol is
required to ensure reliable results.
3. Disk diffusion susceptibility testing is limited to rapidly
growing organisms. Drug inactivation may result
from the prolonged incubation times required by slow
growers.
4. Media containing excessive amounts of thymidine or
thymine can reverse the inhibitory effects of sulfonamides and trimethoprim, causing zones of growth
inhibition to be smaller or less distinct.
5. Variation in the concentration of divalent cations,
primarily calcium and magnesium, affects results of
aminoglycoside, tetracycline, and colistin tests with
Pseudomonas aeruginosa isolates. A cation content
that is too high reduces zones sizes, whereas a
cation content that is too low has the opposite effect.
6. When Mueller Hinton Medium is supplemented with
blood, the zone of inhibition for oxacillin and methicillin may be 2 to 3 mm smaller than those obtained
with unsupplemented agar. Conversely, sheep blood
may markedly increase the zone diameters of some
cephalosporins when they are tested against enterococci. Sheep blood may cause indistinct zones or
a lm of growth within the zones of inhibition around
sulfonamide and trimethoprim disks.
7. Mueller Hinton Medium deeper than 4 mm may cause
false-resistant results, and agar less than 4 mm deep
may be associated with a false-susceptibility report.
8. A pH outside the range of 7,30,1 may adversely
affect susceptibility test results. If the pH is too low,
aminoglycosides and macrolides will appear to lose
potency; others may appear to have excessive activity. The opposite effects are possible if the pH is too
high.
9. When Mueller Hinton Medium is inoculated, no droplets of moisture should be visible on the surface or on
the petri dish cover.
10. Mueller Hinton Medium should be inoculated within
15 minutes after the inoculum suspension has been
adjusted.
11. The zone of inhibition diameters of some drugs, such
as the macrolides, aminoglycosides and tetracyclines, are signicantly altered by CO2. Plates should
not be incubated in increased CO2 atmosphere.
References
BAUER A.L., W.M.M. KIRBY, J.C.SHERRIS &
M.TURCK (1966) Antibiotic susceptibility testing by a
standardized single disc method. Am. J. Clin. Pathol 45:
493.
BARRY, A.L., M.D. COYLE, C. THORNBERRY, E.H.
GARLACH & R.W. HAWKINSON (1979) Methods of
measuring zones of inhibition with Bauer-Kirby disk susceptibility test. J. Clin. Microbiol. 10:885-889.
ERICSSON & SHERRIS (1971) Antibiotic sensitivity
testing. Report of an International Collaborative Study.
Acta Pathol. Microbiol. Scand Suppl. 217 p: 90.
HINDLER, J. (1998) Antimicrobial Susceptibility Testing
In Essential Procedures for Clinical Microbiology. ASM
Press. Washington D:C.
MUNRO, S. (1995) Disk Diffusion Susceptibility Testing.
In Clinical Microbiology Procedures Handbook. H.D.
Isenberg (Ed) APHA Whasington D.C.
MILLER, J.M., C. THORNBERRY & C.N. BAKER (1984)
Disk diffusion susceptibility test troubleshooting guide.
Lab. Med. 15:183-185.
NCCLS Standard M2-A6 (1997) Performance standards
for antimicrobial disk susceptibility tests. 6th ed. National
Committee for Clinical Laboratory Standards. Vilanova.
PA.
THORNSBERRY, C., W.G. GAVAN, E.H. GERLACH &
J.C. SHERRIS (1977) Cumitech 6. ASM. Washington.
WHO (1977) Requeriments for antibiotic susceptibility
tests. Technical Report Series No 610. Geneva.
WOODS, G.L. & J.A. WASHINGTON (1995) Antibacterial susceptibility tests: dilution and disk diffusion methods.
In P.R. Murray, E.J. Baron, M.A. Pfaller, P.C. Tenover
and R.H. Yolken (Eds.) Manual of Clinical Microbiology.
6th ed. APHA. Washington, D.C.
CFR (1972) Rules and Regulations. 37: 20525.
NEUMAN, M.A., D.F. SAMM, C. THORNSBERRY, I.E.
McGOWAN (1991) New developments in antimicrobial
agent susceptibility testing: A practical guide. ASM.
Washington, D.C.
For further information on the performance of the antibiotic disk susceptibility test refer to the M2-A6 NCCLS
Monograph.
113
Mycological Agar
Ref. 01-131
Specication
Solid culture medium for the maintenance, enumeration
and chromogenesis of fungi.
Formula (in g/L)

Soy peptone ............................................. 10,0
Dextrose ................................................... 10,0
Agar .......................................................... 17,0
Final pH 7,0 0,2
Directions
to boiling. Dispense in asks or tubes and sterilize in the
autoclave at 121C for 15 minutes. Should a selective
medium by the acidic pH be desired, adjust the pH to
4,0 with a sterile solution of 10% lactic acid, and do not
reheat the medium afterwards.
114
Description
Mycological Agar is a general medium that provides
enough nutrients for the development of most yeasts
and moulds.
It is employed in plates for the colonial isolation and
characterization, as it aids chromogenesis.
In the slants it is used for the maintenance of strains,
because its low content of glucose yields very slow acid
formation.
References
AJELLO, GEORG, KAPLAN and KAUFFMAN (1963)
CDC Lab Manual for Medical Mycology. PHS Pub. N
994, Washington DC.
ATLAS, M.R., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, London.
CTFA Microbiological Guidelines (1993) The Cosmetic
Toiletry and Fragance Association. Washington DC.
Neutralizing Fluid Eur. Phar.

Ref. 02-512
Specication
Liquid medium for neutralizing the antimicrobials according to the European Pharmacopoeia.
Formula (in g/L)

Peptone .................................................... 1,00
L-Histidine HCl ......................................... 1,00
Lecithin ..................................................... 3,00
Sodium chloride ........................................ 4,30
Final pH 7,0 0,2
Directions
Dissolve 20,1 g of powder in 1 L of distilled water containing 30 mL of Polysorbate 80 (Ref. 06-088). Distribute
121C for 15 minutes. Cool to 50C and homogenize the
solution.
Description
Neutralizing Fluid is formulated according to the European Pharmacopeia formulation for the microbiological
examination of non sterile products. Its composition is
the same as the general diluting solution for biological
assays with the addition of polysorbate and lecithin as
non toxic neutralizing agents.
However, the European Pharmacopoeia lets the technician to increase the concentration of polysorbate if
the original is not enough or to add other agents when
thepreservative type to be neutralized is not known.
This way, the European Pharmacopeia suggests the
compounds shown in the table below, which have to be
always aseptically added to the uid once sterilized and
cooled to 50C or below.
References
European Pharmacopoeia (2002) 4th ed. Supplement
4.2.2.6.13. Tests for specied microorganisms. Council
of Europe.Strasbourg.
115
Nickerson Agar (BiGGY)

Ref. 01-137
Specication
Solid medium for the isolation and identication of Candida sp.
Formula (in g/L)

Yeast extract ............................................... 1,0
Dextrose ................................................... 10,0
Glycine ..................................................... 10,0
Sodium sulte ............................................. 3,0
Ammonium Bismuth Citrate ........................ 5,0
Agar .......................................................... 15,0
Final pH 6,8 0,2
Directions
heat to boiling. Dispense in tubes or dishes, stirring the
precipitate before pouring. Do not autoclave. Avoid
overheating.
Description
Nickerson Agar is suitable for the isolation and identication of yeast of the Candida type. Medium is made
according to the general principles of Bismuth-Sulte
Agar, as inhibitor and differential agent, and completely
selective with the high concentration of glycine. This
medium is highly inhibitory, and does not allow bacterial
growth, however most Candida grow freely and quickly.
In some occasions, tiny colonies of the bacteria or highly
repressed moulds may appear. Bacterial development
may be totally prevented by adding neomycin sulfate to
the medium before pouring it into Petri dishes and its
concentration in the medium in this case must be around
2 mcg/mL, so that the antibiotic will not affect the development or appearance of yeast.
116
The appearance of the colonies in this medium after an

incubation of 48-72 hours at 30-35C is as follows:
Candida albicans: Creamy colonies, very convex, circular with very slight mycelial border and black or
dark brown colour. Neither it has metallic sheen
nor the diffused pigment, even after 72 hours of
incubation.
Candida tropicalis: Acuminated colonies, creamy, irregular and with slight mycelial borders. Dark brown
with black centre. After 72 hours of incubation it
may take on a metallic sheen and produce a diffussed zone of pigment.
Candida krusei: Big and plain colonies, with irregular
borders. Brown colour, darker in the centre. A yellow halo appears around the colony.
Candida parakrusei: Plain colonies, average size, irregular. Dark red centre and light red borders. Yellow
mycelial border.
Candida pseudotropicalis: Big and plain colonies, dark
red colour. Mycelial border.
Candida stellatoidea: Average size plain colonies, dark
brown colour, without mycelial development.
Rhodotorula: Creamy convex colonies, with irregular
border and colours ranging from pink to orange.
Moulds in general: Restricted colonial growth and cottony appearance.
To maintain these colony characteristics it is important
that the medium is freshly prepared and not reheated or
overheated.
References
NICKERSON, W.J. (1953) Reduction of inorganic
substance by yeast I. Extracellular reduction of sulte by
species of Candida. J.Inf.Dis 93:43.
Nitrate Broth
Ref. 02-138
Specication
A liquid culture medium, according to ISO 7932 standard, to determine the ability of enterobacteria to reduce
the nitrate to nitrites or free nitrogen gas.
Formula (in g/L)

Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Final pH 7,0 0,2
Directions
up only if necessary to help the dissolution. Distribute
into nal containers and sterilize by autoclaving at 121C
for 15 minutes.
Description
Technique
Inoculate 2-3 tubes of broth with one loop of pure culture
and incubate at 37C, reading after 18-24 hours, 2 days
and 5 days in each tube, adding some drops of Nitrate
A Reagent (Ref. 06-003) and of Nitrate B Reagent (Ref.
06-004). If the rst two readings are negative, it is recommended to investigate with the third one for the presence of nitrate by the method of zinc powder in order to
have quick nitrate reduction reaction .
References
DOWNES, F.P. & K. ITO (2001). Compendium of Methods for the Microbiological Examination of Food.4th ed.
APHA. Washington.
F.D.A. (1998) Bacteriological Analytical Manual 8th ede.
Rev. A. AOAC International, Gaithersburg.MD
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,London.
ISO standard 7932 (1993) General guidance for the
enumeration of B. cereus. Colony count at 30C.
The Nitrate Broth is prepared according to classical

formula for the assay of nitrate reduction by enterobacteria, although it can also be used with aerobic bacilli and
other bacterial types.
Nutrient Media
Nutrient Agar (APHA)
Nutrient Agar (Brit. Phar.)
Ref. 01-144
Ref. 01-140
Specication
Specication
Solid culture medium for the general purposes according

ISO standard.
Solid culture medium for general purposes and less

fastidious organisms acc. EN 12780:2002
Formula (in g/L)
Formula (in g/L)
Peptone ...................................................... 5,0

Meat extract ................................................ 3,0
Agar .......................................................... 15,0
Final pH 7,0 0,2
Meat extract ................................................ 1,0

Yeast extract ............................................... 2,0
Peptone ...................................................... 5,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,4 0,2
Directions
to boiling. Dispense into suitable containers and sterilize
in the autoclave at 121C for 15 minutes.
Directions
bring to the boil to dissolve completely. Sterilize by autoclaving at 121C for 15 minutes.
Description
The Nutrient Agar is a simple medium in the range of
meat infusions, complemented by a formulation which
reinforces its nutrient qualities as well as its growth factors by adding yeast extract. It is most suitable for general routine work and can support the growth of common
117
Nutrient Media
organisms, even those considered mildly fastidious with
regard to nutrient elements. Besides this, by incorporating sodium chloride it allows the addition of blood, even
though it is not an optimal medium for it.
Nutrient Broth (APHA)

Ref. 02-144
Specication
Liquid medium for the cultivation of non fastidious microorganisms according ISO standard.
Formula (in g/L)

Peptone ...................................................... 5,0
Meat extract ................................................ 3,0
Final pH 7,0 0,2
Directions
if necessary to help dissolve the medium. Distribute into
nal containers and sterilize by autoclaving at 121C for
15 minutes.
Description
The Nutrient Broth is the liquid version of the solid medium which bears the same name. It is a classical broth
in the range of meat infusions. It is useful for the routine
laboratory purposes since its yeast extract supplement
allows the growth of most common organisms. It is also
suitable for the preparation of inocula and for the efciency testing of bactericides, as well as for determination of the Phenol Coefcient and others.
Nutrient Broth No. 2
Directions
Ref. 02-561
Dissolve 8 g of powder in 1 L of distilled water heating up

only if necessary. Dispense into suitable containers and
sterilize by autoclaving at 121C for 15 minutes.
Specication
Description
Formula (in g/L)
Nutrient Broth is a modern version of the classical

general culture medium based on meat infusion. It is a
simple medium that may be used in general purposes
(i.e. maintenance of strains) as well as a base for other
specialized media. However, in this way, there are other
media with more nutrient capacity and better performance.
Nutrient broth is the liquid version of the Nutrient Agar,
and it is a classical medium for normal tasks with non
fastidious microorganisms. It is the ideal medium for
the subculture of general bacteria, especially staphylococci, to carry out later the coagulase and other biochemical tests. It may also be used to determine the Phenol
Coefcient by following the technique and microorganisms suggested by the AOAC.
Nutrient Broth (Brit. Phar.)

Ref. 02-140
Specication
A general purpose liquid culture medium for the less
fastidious microorganisms.
Formula (in g/L)

Meat extract ................................................ 1,0
Yeast extract ............................................... 2,0
Peptone ...................................................... 5,0
Sodium chloride .......................................... 5,0
Final pH 7,4 0,2
118
Liquid medium for general purposes.
Meat Extract .......................................... 10,00

Peptone .................................................. 10,00
Sodium chloride ........................................ 5,00
Final pH 7,5 0,2
Directions
Dissolve 25 g of the powder in 1 L of distilled water,
heating if necessary. Distribute in suitable containers and
Description
This medium in the classical way of the meat infusions,
presents a specially rich nutritional characteristics that
facilities the growth of very low inocula, even with fastidious microorganisms. Its formulation is according the
BSI for the determination of Rideal-Walker Coefcient of
disinfectants, where it is used at double concentration.
Nutrient DEV Agar

Ref. 01-451
Specication
Solid culture medium for the total enumeration of microorganisms in water, according to the German legislation
and ISO standard.
Nutrient Media
Formula (in g/L)
Meat peptone ........................................... 10,0

Meat extract .............................................. 10,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 18,0
Final pH 7,3 0,2
Directions
Suspend 43 g of powder in 1 L of distilled water. Bring

to the boil with constant stirring . Distribute into suitable
minutes.
Description
This medium is formulated according to the German

legislation but it differs from the other Anglo-Saxon
media with the same name in the concentration of its
compounds. This change intends to aid the recovery
and growth of damaged microorganisms.
Technique
German standards state a deep inocule of the water

sample, following the mass seed technique, directly in
the Petri plate. Incubation is performed at 202C for
444 hours in most of the cases, but incubations at
371C for the same period of time are also allowed. If
the water is chlorinated, the incubation time must last up
to 72 hours.
References
APHA (1948) Standard Methods for the Examination of

Dairy Products. Washington.
BRITISH PHARMACOPOEIA (1968), 357.
BRITISH STANDARD 541 (1934). Determining the Rideal-Walker Coefcient of Disinfectants. BSI London 9.
DOWNES F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food.4th ed
APHA. Washington.
EUROPEAN STANDARD EN 12780:2002 Water Quality.
Detection and enumeration of Pseudomonas aeruginosa
BUNDESGESUNDHEITAMT: Amtliche Sammlung von
Untersuchungsverfahren nach 35 LMBG. Beuth Verlag
Berlin- Kln.
VERORDNUNG von 12/12/1990 ber Trinkwasser und
ber Wasser fur Lebensmittelbetriebe. Bundesgesetzblatt: Teil I:2613-2629.
Abwasser- Und Schlammuntersuchung. VCH Verlagsgesellchaft, D-6940 Weinheim.
ISO 8523 standard (1991) General guidance for the
detection of enterobacteriaceae with pre-enrichment.
ISO 6785 standard (2001) Milk and milk-products - Detection of Salmonella spp.
ISO 6340 standard (1995) Water Quality - Detection of
Salmonella species.
ISO 6579 standard (2002) Horizontal method for the
detection of Salmonella spp.
ISO 10273 standard (1994) General guidance for the
detection of presumptive pathogenic Yersinia enterocolitica.
Nutrient Gelatin Media

Nutrient Gelatin
Ref. 03-088
Specication
Culture medium for determination of gelatin liquefaction.
Formula (in g/L)

Meat extract ................................................ 3,0
Gelatin peptone .......................................... 5,0
Gelatin .................................................... 120,0
Final pH 6,7 0,2
Directions
Suspend 128 g of powder in 1 L of cold distilled water
and heat gently in boiling bath up to 50C. Keep at that
temperature until total dissolution of gelatin. Dispense in
tubes or asks and sterilize in the autoclave at 121C for
15 minutes.
Description
Gelatin Nutrient is used, essentially, to identify pure
cultures that have no specic nutritional requirements.
On the other hand, gelatin liquefaction is considered
very important to differentiate enteric bacilli on the basis
of their proteolysis. Gelatin was one of the rst solidifying agents employed in bacteriology, and helped in the
development of the Plate Count Technique, performed
by Koch. Nonetheless, nowadays it is not used in that
way, since it was replaced by Agar, which bears incubations at higher temperatures and was not so attacked or
degraded as gelatin.
However, the Plate Count Method is still used with
gelatin. Standard Methods still recommend it for aerobic
counting at 20-22C.
119
Nutrient Gelatin Media

Technique
The gelatin liquefaction test may be performed in plates
or tubes, that are inoculated by stabbing,and incubated
at 20-22C or at the optimum temperature for the microorganism to assay. Because gelatin is liquid above 20C,
it is necessary to put the plates or tubes in the refrigerator before reading.
High temperature assays (35-37C) eliminate wrong
results from bacteriolysis, but they require more care in
readings and also inclusion of a control (uninoculated) in
order to verify effect of heat on the medium.
Gelatin tube readings must be carried out carefully, since
often they have diagnostic value. In the case of using
several plates, Stones reaction may be used to include
different strains in the same dish, seeding them by parallel streak. After incubation period, cool in the refrigerator
and then, over each streak, put a few drops of saturated
solution of Ammonium sulfate or Sulfosalicilic acid 20%
solution (freshly prepared).
Liquefaction (positive test) is shown by the presence
of a clear halo or zone around the growth, 10 minutes
after reagent addition. Incubation periods for gelatinase
activity assay vary, ranging from a few days to weeks,
at 20-22C. Some Klebsiella and Enterobacter strains
take up to 3 weeks before they show activity. Though
incubation times are not standarized, it is recommended
a maximum of 14 days with regular intermittent readings
every 3 days.
A modern version of this methodology is the usage of
photographic lm strips, not exposed, for the liquid media, but this te chnique has a drawback that sometimes
photosensitive particles included in the lm are toxic to
the microorganisms, and then they give false - negative
results.
References
APHA/AWWA (1995) Standard Methods for the Examination of Water and Wastewater. 19th. Ed. APHA Inc.
New York. 596-597.
ASM (1981) Manual of Methods for General Bacteriology, ASM, Washington, D.C.
120
Nutrient Gelatin DEV

Ref. 03-453
Specication
Culture medium for the enumeration of total bacteria
in not very polluted waters, according to the German
legislation.
Formula (in g/L)

Gelatin .................................................. 120,00
Meat extract ............................................ 10,00
Meat peptone ......................................... 10,00
Sodium chloride ........................................ 5,00
Final pH 7,3 0,2
Directions
Suspend 145 g of powder in 1 L of cold distilled water
and heat up, in boiling water bath, to 50-60C. Maintain
this temperature until total dissolution. Distribute into
minutes.
Description
This medium has the same applications as the Gelatin
Nutrient (Ref. 03-088) recommended by the APHA,
AWWA and Standard Methods. The only difference is the
concentration of nutrients and the inclusion of sodium
chloride.
Technique
German legislation states that water samples are inoculated by deep inoculum (in mass) in Petri plates. Incubation is performed at 202C for 444 hours. After the
incubation, carry out the counting of total bacteria.
If water is chlorinated, incubation must be 24 hours more
in order to let damaged cells recover and form visible
colonies.
References
Oxidation-Fermentation Fluid Medium (O/F Medium)

Ref. 03-037
Specication
Fluid medium according to the Hugh and Leifson formulation, for determining the oxidative and/or fermentative
metabolism of gram-negative bacilli.
Formula (in g/L)

Casein peptone ........................................ 2,00
Sodium chloride ........................................ 5,00
Bromothymol Blue .................................... 0,08
Agar .......................................................... 2,50
Final pH 7,1 0,2
Directions
bring to the boil. Add sugar in the desired concentration
and distribute in fermentation tubes. Add the vaseline
seals or vaspar to half of them. Sterilize by autoclaving
at 121C for 15 min.
Description
Hugh and Leifson obtained a clear differentiation of
gram-negative bacteria with this medium. They classied them into three categories: fermentative, oxidative
and inactive. The strain to be studied is inoculated in two
long narrow tubes (12x120 mm) by deep stab and one
is covered with a vaseline layer to induce an anaerobic
environment that forces the strain to carry out the fermentative metabolism.
Fermentative organisms give a copious production of
acid in both the tubes, and it is indicated by the yel-
low colouration of the bromothymol blue indicator. The

bacteria following the oxidative metabolism carry out this
reaction only in the tube without the vaseline but in the
other one, which is closed, they change insignicantlly
or simply do not grow. Inactive strains do not use sugars
and therefore do not induce any change in both the
tubes. However, some times there is a slight blue colouration in the open tube, probably due to alkalinization
by peptone degradation.
Some authors have proposed the usage of just one
tube for this assay, but in that case the medium must be
modied to be the solid (with 1,5% Agar) and with yeast
and/or cystine extract. In these tubes the stab must be,
at least, 8 cm.
Hugh and Leifson recommend simultaneous assay with
glucose, lactose and sucrose of 1% concentration, adding the sterilized sugars to the medium by ltration.
References
HUGH, R. and E. LEIFSON, (1953) The taxonomic
signicance of fermentative vs. oxidative metbolism of
cabohidrates by various gram negative bacteria J.Bact.
66:24
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological media. CRC Press, Inc.London
FDA (1998) bacteriological Analitycal Manual. 8th ed.
Rev. A. AOAC International. Gaithersburg. MD
DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of food. 4th ed.
APHA Washington
ISENBERG, H.D. (1992) Clinical Microbiology Procedures Handbook Vol I ASM Press. Washington
P Medium Agar
Ref. 01-500
Directions
Specication
Suspend 30,36 g of powder in 1 litre of distilled water

and bring to the boil. Distribute in suitable containers and
Solid culture medium used as seed agar in the inhibitory

substances test in milk.
Formula (in g/L)

Meat extract .............................................. 3,00
Peptone .................................................... 5,00
Soy peptone ............................................. 0,30
Dextrose ................................................... 5,25
Polysorbate 80 ......................................... 1,00
Sodium chloride ........................................ 0,50
Bromcresol purple .................................... 0,06
Agar ........................................................ 15,00
Final pH 7,8 0,2
Description
The present formulation is according the Food and Drug
Administration Bacteriological Analytical Manual for the
investigation of Inhibitory substances (Antibiotics and
preservatives) in milk. This medium that in previous editions of the BAM was called PM Indicator Agar, is used
as seed agar with B. stearothermophilus spores in the
qualitative method or disk assay.
References
MATURIN, L.J. (1998) Inhibitory substances in milk.
Qualitative Method II: B. stearothermophilus disk assay.
In FDA Bacteriological Analytical Manual. 8th Ed. Revision A. AOAC International Inc. Gaithersburg MD.
121
Peptone Agar
Ref. 01-570
Specication
Solid culture medium used for the enumeration of
contaminants in dairy products, according the FIL-IDF
standard
Formula (in g/L)

Casein Peptone ........................................ 7,50
Gelatine Peptone ...................................... 7,50
Sodium chloride ........................................ 5,00
Agar ........................................................ 15,00
Final pH 7,5 0,2
Directions
Description
Peptone Agar is produced according the formulation of
the FIL-IDF for its use in the detection and enumeration of non-lactic contaminants in butter, fresh cheese
and fermented milk. All the components of the medium
are sugar-free and the pH is adjusted to 7,5 thats is the
used for cheese and butter. If the sample is fermented
milk, the medium will be adjusted to 8.00 with 0,1N

NaOH sterile solution after the sterilization.
Technique
The inoculum or its dilution (in duplicate) is deposited
over the surface of the medium in the plate in volumes
of 0,1 mL. The inocula are spreaded quickly with a
Drigalski rod (Ref. 5-010) and let stand 15 minutes to
be absorbed in the medium. The plates are incubated
at 30C for 722 hours. Select plates with less than 150
colonies to the count. In the counting the needle-bite
colonies are not considered because they are probably
lactic bacteria. The contaminant must be conrmed by
its active catalase.
For the sampling, processing and dilution of the products
refer to the corresponding FIL-IDF standard.
References
FIL-IDF (1991) Provisional Standard 13: Butter, fresh
cheese and fermented milk. Enumeration of non-lactic
contaminants. Plate Count at 30C technique.
Peptone Water Phosphate-Buffered

Ref. 02-568
Specication
Liquid non-selective pre-enrichment medium for enterobacteria
Formula (in g/L)

Meat Peptone ......................................... 10,00
Sodium chloride ........................................ 5,00
Monopotasium phosphate ....................... 1,50
Final pH 7,2 0,2
Directions
Description
This medium is produced according the formulation of
the Schwezerisches Lebensmittelbuch and is recommended as non-selective pre-enrichment for sub-lethally
damaged cells of the enterobacteria group in food or in
others samples.
122
References
BEKERS, H.J. (1987) Studies with salmonellae. J. Appl.
Bact. 62:97-112
SCHWEIZERISCHES LEBENSMITTELBUCH (1992) 5th
ed. Chapter 56A
Phenol Red Broth Base

Ref. 02-032
Specication
Liquid culture media,suitable for the sugar and other
substrate fermentation studies according ISO 10273
standard.
Formula (in g/L)

Casein peptone .................................... 10,000
Sodium chloride ...................................... 5,000
Phenol red .............................................. 0,018
Final pH 6,8 0,2
Directions
Dissolve 15 g of powder in 1 L of distilled water. Add
sugar in the desired concentration and distribute into
suitable containers with Durhams tubes. Sterilize in
the autoclave at 121C for 10 minutes. Heat up the
autoclave before putting the tubes into it to avoid sugar
caramelization. Addition of some kinds of sugars may
need a pH adjustment.
Description
Phenol Red Base Broth is a liquid version of the agar
base for the fermentation studies, which is preferred by
many authors to use with Durhams tubes inclusion, to
verify the gas production.
Sugar addition can be done in sterile solution after autoclaving the medium, or by adding impregnated discs to 10 mL of
medium. Addition of some sugars may cause the acidication of the medium, in which case the original pH must be
maintained by adding a few drops of 0,1 N NaOH.
Should you be working with anaerobics, it is advisable to
use a freshly prepared medium, or put the medium in boiling
water bath for a few minutes, in order to eliminate dissolved
oxygen. Many authors recommend the addition of 0,04%
agar for these purposes to avoid convection streams and
subsequent incorporation of the air.
To study the sugar fermentations of enterobacteria, Bromcresol Purple Base Broth (Ref. 02-031) is more suitable, as
it is a better indicator of choice which is less toxic than the
phenol red.
References
ISO 10273 Standard (1994) General guidance for the detection of presumptive pathogenic Yersinia enterocolitica.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press,Boca Raton,Fla.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
Rev. A. AOAC International. Gaithersburg. MD
Phenylalanine Agar (PPA)

Ref. 01-083
Specication
Culture medium for Enterobacteria, according Ewing et
al. formulation.
Formula (in g/L)

Yeast extract ............................................... 3,0
DL-Phenylalanine ....................................... 2,0
Sodium chloride .......................................... 5,0
Agar .......................................................... 15,0
Final pH 7,3 0,2
Directions
to boiling. Dispense in tubes or asks and sterilize in the
Description
This formulation corresponds to the solid form, proposed
by Ewing et al. which is a modication of the medium
developed by Buttiaux et al. in order to achieve the
green colour that conrms the positive reaction which
lasts longer.
Capacity to deaminate the phenylalanine oxidatively to
convert it in phenylpiruvic acid is property of the Proteus
type in enterobacteria. Phenylalanine is revealed by
the presence of a characteristic greenish colour in the

medium when it reacts with iron. Nowadays, this test and
the urease production test, have a lot of importance in
the taxonomy of Proteus type .
Technique
A recommended technique is the following:
Inoculate the slant surface with plenty of inoculum, and
incubate it for 12-16 hours. Add 0,2 mL of 10% ferric
chloride solution so that the solution oods all over the
growth.
Phenylpiruvic acid presence (positive test) is shown by
the presence of a characteristic green-blue colour on the
surface, after approximately 1 minute.
References
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.London
BUTTIAUX,R., R. OSTEUX, R. FRESNOY & J. MORIAMEZ (1954) Les propiets biochimiques du genre
Proteus.Ann.Inst. Pasteur 87:357-386
EDWARDS and EWING (1973). Identication of Enterobacteriaceae. Burges Pub.Cod. Minneapolis.
ISENBERG, H.D. (1992) Clinical Microbiology Procedures Handbook. Vol I ASM Press Washington.
123
Plate Count Media

Plate Count Agar (PCA)
Ref. 01-161
Specication
Medium for the aerobic plate count by surface inoculation method (Standard Plate Count Agar) according ISO
4833 and 17410 standards.
Formula (in g/L)

Casein peptone .......................................... 5,0
Yeast extract ............................................... 2,5
Dextrose ..................................................... 1,0
Agar .......................................................... 15,0
Final pH 7,0 0,2
Directions
Suspend 23,5 g of powder in 1 L of distilled water.
Dissolve by bringing to the boil with frequent stirring. Distribute into nal containers and sterilize by autoclaving at
Description
The Plate Count Agar follows the directions given by
Buchbinder et al. in their study about media for the plate
count of microorganisms.
The original formulation of the standardized agar for
dairy microbiology has been modied in order to avoid
the addition of milk. This new composition allows the
growth of most microorganisms without any further additions.
This mediums formulation is equivalent to that prescribed by the Standard Methods for the Examination
of Dairy products, the USPs Tryptone Glucose Yeast
Agar, the Deutsche Landswirtchaft and to the APHA
and AOACs Plate Count Agar. Nowadays this is the
medium selected for the plate count of any type of the
sample.
Plate Count Modied Agar

Ref. 01-329
Specication
Modication of Plate Count Agar (Ref. 01-161), with a
lesser amount of agar, especially recommended for the
aerobic enumeration in plates, by the poured plates
method.
Formula (in g/L)

Casein peptone .......................................... 5,0
Yeast extract ............................................... 2,5
Dextrose ..................................................... 1,0
Agar ............................................................ 9,0
Final pH 7,0 0,2
124
Directions
Dissolve 17,5 g of powder in 1 L of distilled water. Heat
to the boiling by constant stirring. Distribute in the suitable containers and sterilize in the autoclave at 121C
for 15 minutes.
Description
Plate Count Modied Agar follows the same specications as Plate Count Agar, with the exception that of the
agar concentration has been reduced. This modication
provides a better growth of colonies if massive inoculation method is used, as the medium is softer and hence
the colony expansion is improved.
Plate Count Skim Milk Agar

Ref. 01-412
Specication
Solid medium for the plate counts of milk and dairy products, according to DIN and FIL/IDF standards.
Formula (in g/L)

Casein peptone .......................................... 5,0
Yeast extract ............................................... 2,5
Skimmed milk ............................................. 1,0
Dextrose ..................................................... 1,0
Agar .......................................................... 10,5
Final pH 7,0 0,2
Directions
it soak . Bring to the boil with constant stirring. Distribute
into suitable containers and sterilize in the autoclave at
Description
This medium, with the added milk, has a major nutrient richness than other standard media, however, the
opalescence of the medium makes early observations
sometimes difcult.
Due to its lesser agar concentration, it may be used by
the pouring plate method or by the surface inoculation
method.
Technique
Prepare a decimal dilution bank of the sample and take
1 mL in duplicate from each dilution and put them in
sterile Petri plates. Pour 20 mL approx. of sterile cooled
medium (around 47C) in each of the plates. Mix gently
by moving the plate in eight (8) shape. Leave the plates
undisturbed to solidify and incubate in inverted position.
Time and temperature of incubation depend on the type
of microorganism under study. For a general aerobic
count, incubate for 3 days at 30C, by observing also
after 24 and 48 hours.
The plate count method proposed by the APHA consists
of a massive inoculum by pouring the molten agar at
50C on plates containing the diluted samples. The nal
Plate Count Media

count is carried out after 48 hours of incubation at 3235C.
As for the microorganisms with other temperature
requirements, the following incubations have been suggested: 2 days at 32-35C, 2-3 days at 45C, 2 days at
55C, 3-5 days at 20C, 7-10 days at 5-7C.
Sample dilutions are prepared with solutions of 1/4 of
Ringer solution (Ref. 06-073), 1% of Peptone Water(Ref.
03-156) or 0,1M of phosphate buffer at pH 7,0; depending on their nature.
The poured plate count method is preferred to the
surface inoculation method, since it gives higher results.
Nevertheless, the latter gives a more appropriate isolation of the colonies.
References
Examination of Dairy Products, 16th Ed. APHA. Washington.
CLESCERI. L.S., A.E. GREENBERG, and A.D. EATON
and Wastewater, 20th ed.. APHA, AWWA, WEF. Washington.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th. Ed.
APHA. Washington.
HORWITZ, W. (2000) . Ofcial Methods of Analysis.
AOAC International. Gaithersburg
DIN 10192 Standard. Prfungesbestimmungen fr Milch

und Milcherzeugnisse (Deutsche Landwirtsachft, Fachbereit und Ernahrung). 1971.
FIL/IDF Standards 3 (1958), 100 (1981), 101 (1981),
109 (1982) and 132 (2004).
Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,Boca Raton,Fla.
BUCHBINDER, L., Y. BARIS, L. GOLDSTEIN (1953)
Further studies on new milk-free media for the standard plate count of dairy products. Am. J. Public Health
43:869-872.
ISO 4833 Standard (2003) Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of microorganisms. Colony count technique at 30C.
ISO 17410 Standard (2001) Horizontal Method for the
enumeration of psychrotrophic microorganisms.
ISO 8552 Standard (2004) Milk - Estimation of psychrotrophic microorganisms - Colony-count technique at
21C (Rapid method).
125
Potato Dextrose Media

Potato Dextrose Agar
Potato Dextrose Broth
Ref. 01-483
Ref. 02-483
Specication
Specication
Solid culture medium for the detection and enumeration

of yeast and moulds in food, specially recommended in
butter and other dairy products.
Liquid culture medium for the maintenance and multiplication of yeast.
Formula (in g/L)

Potato peptone ......................................... 4,00
Glucose .................................................. 20,00
Agar ........................................................ 15,00
Final pH 5,6 0,2
Directions
sterilize in the autoclave at 121C for 15 minutes. Do not
overheat.
Description
Potato Dextrose Agar is a weakly selective medium for
fungi due to its high sugar content and acidic pH. The
pigment production and aerial mycelium development
is enhanced by the potato peptone, specially in the
Fusarium, Aspergillus and Penicillium species.
The selectivity can be increased by adding antibacterial
antibiotics like chloramphenicol or tetracyclines, or by
simply decreasing the pH to an acidic level. At pH 3,5
the bacterial growth is almost totally inhibited without
signicant effect on fungi. This acidication can be
obtained by the aseptic addition of an adequate amount
of organic acid to the medium after sterillization: 10-15
mL/L of a 10% sterile solution of tartaric or lactic acid is
usually sufcient.
After its acidication the medium should not be overheated or reheated since it can hydrolyze the agar and
hence there can be a loss in solidication property of the
medium.
Technique
Distribute the diluted samples into sterile petri plates.
Pour the molten agar melted cooled to 45-50C and gently mix to homogenize the mixture. After the solidication,
plates are incubated for 5-7 days at 20-25C to permit
the complete development of the fungal colonies.
The weak consistency of the agar due to its original
acidity makes this medium inadequate for streaking.
126
Formula (in g/L)

Potato peptone ........................................... 4,0
Glucose .................................................... 20,0
Final pH 5,6 0,2
Directions
up only if necessary. Distribute into suitable containers
Description
Potato Dextrose Broth is the liquid version of the agar
with the same name. This broth is mainly used to detect
and enumerate yeast and moulds, since it does not
contain any solidifying agent it may be acidied without
altering its physical properties.
At pH 3,5, the bacterial growth is totally inhibited without
signicant inuence on fungi. This acidication may be
achieved by the aseptic addition of an adequate amount
of organic acid to the medium after sterilization:10-15
mL/L of a 10% sterile solution of tartaric or lactic acid.
This addition may also be made before sterilization, but
it must be considered that in acidic conditions Maillard
reactions are strong and hence the medium may turn
slightly brownish.
References
ATLAS, R.M. & PARKS,L.C. (1995) Handbook of
Microbiological Media for the Examination of Food. CRC
Press, London.
RICHARDSON, G. H. (1985) Standard Methods for the
examination of dairy products.15th Ed. APHA Washington.
DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of food. 4thEd.
APHA Washington
US PHARMACOPOEIA (2002) 25th ed. <61> Microbial
Limit Test. US Pharmacopoeial Convention Inc. Ltd.
Rockville. MD
FDA (1998) Bacteriological Analitycal Manual. 8th ed.
Rev. A. AOAC International. Gaithersburg. MD.
R2A Agar (Eur. Phar. Medium S)

Ref. 01-540
Specication
Solid medium for the enumeration of heterotrophic micro
organisms in treated waters
Formula (in g/L)

Yeast Extract .......................................... 0,500
Proteose peptone ................................... 0,500
Casein hidrolysate .................................. 0,500
Glucose .................................................. 0,500
Starch ..................................................... 0,500
Dipotassium hydrogen phosphate ......... 0,300
Magnesium sulphate, anhydrous .......... 0,024
Sodium pyruvate ................................... 0,300
Agar ...................................................... 15,000
Final pH 7,2 0,2
Directions
bring to the boil with constant stirring. Distribute into suitable containers and sterilize by autoclaving at 121C for
15 minutes.
Description
The R2A Agar was proposed in 1979 by Reasoner and
Geldenreich and few years later accepted by the APHA
as an alternative medium for stressed cells in treated
potable water.
The use of nutrient rich media like PCA or TSA allows to
the growth of normal microbiota, but do not permits the
recuperation of the stressed or chlorine resistant biota.
By the use of a medium like R2A of low nutrients in combination with a lower temperature and longer incubation
time it is possible induce the resuscitation of this damaged cells.
In the R2A Agar the source of nitrogen is the peptone
and the Yeast Extract supplies the vitamins and growth
factors. The source of carbon is the dextrose and magnesium sulphate and potassium phosphate maintains
the osmotic pressure. The starch is a detoxier and
sodium piruvate increases the recuperations of stressed
cells. The agar acts as gelling agent.
Technique
The water sample must be processed as quickly as
possible. If it is no possible within the rst 6 hours, the
sample must be refrigerate, but not for more than 30
hours: then the sample is rejected.
R2A Agar is used with pour plates, streak plates or
ltration but must be keep in mind that the pour plates
method can affect the recovery capacity of the medium
because the thermal shock. The incubation period at
35C is of 3-5 days but is more effective a incubation
temperature of 20-28C an a time of 5-7 days. In any
case the plates must be protected against an excessive
drying.
The fast-growing or non-stressed microorganisms in
these conditions of incubation produce different and
minute colonies than in the rich media.
References
ATLAS, R.M. (1995) Handbook of Media for Environmental Microbiology. CRC Press. Boca Raton USA.
EATON, A.D., A.E. GREENBERG and L.S. CLESCERI
(1995). Standard Methods for the Examination of Water
and Wastewater. 19 Ed. APHA Washington D.C. USA .
EUROPEAN PHARMACOPOEIA 4th Ed. Suppl. 4.6
(2004) 2.6.13 Test for specied Microorganisms (pg
2621)
GREENBERG, A.E., R.R TRUSSELL and L.S.
CLESCERI (1985). Standard Methods for the Examination of Water and Wastewater. 16 Ed. APHA-AWWAWPCF Washington D.C. USA
REASONER, D.J. and E.E. GELDREICH (1979) A new
medium for the enumeration and subculture of bacteria
from potable water. Abstracts of Annual Meeting . ASM
79th Meeting. Paper #N7.
Van SOETSBERGER, A.A. and C.H. LEE (1969) Pour
plates or streak plates? Appl. Microbiol 18:1092-1094.
127
Rappaport Vassiliadis Media

Rappaport Vassiliadis Broth
Ref. 02-379
Specication
Liquid medium for the selective enrichment of Salmonella in foodstuffs and other materials.
Formula (in g/L)

Soy peptone ........................................... 4,500
Sodium chloride ...................................... 7,200
Magnesium chloride ............................ 13,580
Malachite green ...................................... 0,036
Final pH 5,2 0,2
Directions
Dissolve 26,8 g of powder in 1 L of distilled water, heating if necessary to help dissolve the powder. Dispense
into test tubes or asks and sterilize by autoclaving at
Description
The Rappaport Vassiliadis medium complies with the
recommendations of the APHA for the examination of
food.
This culture medium is the modicaction of the R10 medium (from Rappaport et cols) or RV broth (from Vassiliadis et cols.)by van Schothort & Renaud. The modications are an adjustement in the magnesium chloride
concentration and a buffered reaction of the medium.
It shows a higher selectivity towards Salmonella and
produces better yields than other similar media, especially after preliminary enrichment and at an incubation
temperature of 43C.
Malachite green and magnesium chloride inhibit the
growth of the microorganisms normally found in the intestine but do not affect the proliferation of most Salmonellae. Malachite green inhibits the growth of Shigella.
Soy peptone improve the growth of Salmonella. The low
pH of the medium increases the selectivity.
Technique
Inoculate the culture medium with the sample or material from a pre-enriched culture in Buffered Peptone
Water (Ref. 02-277) and incubate for up to 18-24 hours
at 411C. Streak the sample material from the resulting
cultures onto selective culture media.
References
ATLAS, R.M., LC. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc.,London
VASSILIADIS,P (1983) The Rappaport-Vassiliadis(RV)
enrichment medium for the isolation of salmonellas: An
overview. J.Appl.Bact.54;54, 69-76.
128
VASSILIADIS, P, PATERAKI, EPAPAICONOMOU,N

, PAPADAKIS, J.A. A. TRICHOPOULOS, D (1976)
Nouveau procd denrichissement de Salmonella. Ann.
Microbiol. (inst. Pasteur) 127B (195-200)
RAPPAPORT, F. N. KONFORTI & B. NAVON (1956) A
new enrichment medium for certain salmonellae. J. Clin
Pathol. 9:261-266
VAN SCHOTHORST M. & A.M. RENAUD (1983)
Dynamics of salmonellae isolation with modied Rappaports medium (R10) J.Appl. Bact. 54: 209-215
FIL-IDF Standard 93B:1995. Milk and Milk products.
Detection of Salmonella. Brussels
Rev A. AOAC International. Gaithersburg. MD.
AOAC International. Gaithersburg. MD
DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of foods. 4th ed.
APHA Washington.
Rappaport Vassiliadis Modied

Semi-Solid Medium Base (MSRV)
Ref. 03-376
Specication
Semi-solid medium for the isolation of mobile strains of
Salmonella.
Formula (in g/L)

Tryptose ................................................. 4,590
Casein Peptone ...................................... 4,590
Sodium chloride ...................................... 7,340
Mono-Potassium phosphate .................. 1,470
Magnesium chloride ............................. 10,930
Malachite green ...................................... 0,037
Agar ........................................................ 2,700
Final pH 5,2 0,2
Directions
Suspend 31,6 g of powder in 1 L of distilled water. Heath
in a water bath until boil and complete dissolution. Cool
to 50C an add 20 mg/L of Novobiocin. Without autoclaving nor reheating, homogenize and pour plates. Keep
plates in a fresh place to settle the gel (1 hour minimum)
and handle it with care because the medium is only
semi-solid. It is recommended to keep MSRV plates in a
cooler at 2-8C at the dark.
Description
The Modied Semi-Solid Rappaport-Vassiliadis Medium
Base is formulated according DeSmedt and cols. That
shows its higher efciency over the traditional enrichment methodology.
Rappaport Vassiliadis Media

The rapid migration of mobile strains of Salmonella in
the semisolid medium allows to the early detection by
the production of an halo of growth around the inoculation zone.
The other competitive mobile organisms are inhibited
by the novobiocin, the malachite green and the high
concentration of magnesium chloride.
The low concentration of agar produces a very soft and
fragile gel but, at the temperature of incubation (42C), it
is an special environment in which the mobile strains of
Salmonella moves easy and quickly.
Technique
1. Three drops (~0,1 mL) of a pre-enrichment culture
are inoculated in a three different spots on the dry
surface of the medium in a room-temperate plate.
2. Incubate the plates aerobically in an upright position
for no longer than 24 hours at 42C.
3. The formation of a turbid or opaque halo around the
initial inoculation zone shows the presence of mobile
salmonellae.
4. To conrm the purity of the isolation and to follows
with the identication tests, samples of the external
border of the halo can be used.
5. To prevent false negatives results due to the absence of mobile strains of Salmonella is convenient
to performs simultaneously a traditional enrichment
in liquid medium.
References
De SMEDT, J.M., R. BOLDERDJIK, H. RAPPOLD and
D. LAUTENSCHLAEGER (1986). Rapid Salmonella
detection in foods by motility enrichment on a Modied Semi-Solid Rappaport-Vassiliadis Medium. J. Food
Protect. 49:510-514
De SMEDT, J.M. and R. BOLDERJIK (1987) Dynamics
of Salmonella isolation with Modied Semi-Solid Rappaport-Vassiliadis Medium. J. Food Protect. 50:658-661
HOLBROOCK, R., J.M. ANDERSON, A.C. BAIRDPARKER, L.M. DODDS, D. SAWHNEY , S.H. STRUCHBURY and D. SWAINE (1989) Rapid detection of Salmonella in food: A convenient two-day procedure. Lett. Appl.
Microbiol. 8:139-142
Reinforced Clostridial Media

Reinforced Clostridial Agar
Ref. 01-289
Specication
Solid medium for the cultivation and enumeration of
clostridia and other anaerobic bacteria.
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 3,0
Meat extract .............................................. 10,0
Dextrose ..................................................... 5,0
Sodium chloride .......................................... 5,0
Sodium acetate .......................................... 3,0
Soluble starch ............................................. 1,0
L-Cysteine HCl ........................................... 0,5
Agar .......................................................... 15,0
Final pH 6,8 0,2
Directions
heat to boiling with constant stirring. Distribute into suitable containers and sterilize in the autoclave at 121C
for 15 minutes.
Reinforced Clostridial Medium

(RCM) (Eur. Phar. Medium P)
Ref. 03-289
Specication
Fluid medium for the cultivation and enumeration of
clostridia by the MPN method.
Formula (in g/L)

Casein peptone ........................................ 10,0
Yeast extract ............................................... 3,0
Meat extract .............................................. 10,0
Dextrose ..................................................... 5,0
Sodium chloride .......................................... 5,0
Sodium acetate .......................................... 3,0
Soluble starch ............................................. 1,0
L-Cysteine HCl ........................................... 0,5
Agar ............................................................ 0,5
Final pH 6,8 0,2
Directions
to boiling with constant stirring. Distribute into suitable
minutes.
129
Reinforced Clostridial Media

Description
Reinforced Clostridial Agar was originally described by
Hirsch and Grinstead to initiate the growth of small inoculums and get a higher Clostridial count. Later, Barnes
and Ingram used the medium to develop vegetative cells
in assays of Clostridium perfringens. Barnes also used
this medium to count clostridia in food, moreover other
authors used this medium in enumeration assays of Cl.
thermoscharolyticum in sugar, study of intestinal ora,
and bacterial count in human or animal faeces, etc.
For the enumeration by the MPN method, the liquid version is the preferred one.
Technique
Material to be examined is ground in a Turmix or Stomacher, and a decimal dilution bank is prepared. From
each of the dilutions, take an aliquote to Petri plates or
tubes, and pour the molten medium at 50C over them.
Let it solidify. Incubate at 30-55C (depending on the
microorganism that is anticipated to be found) for 1-10
days. An anaerobic environment can be achieved if
tubes are used and they are covered with Sealing Anaerobic Agar (Ref. 01-174) immediately after the Reinforced
Clostridial Medium is solidied. If the plates are used,
they have to be incubated in the anaerobic jars.
Muoa and Pars added a lter sterilized solution of
Nalidixic acid 0,02 g/L, Polymyxin 0,025 g/L, Kanamy-
cin sulfate 0,05 g/L, Sodium iodoacetate 0,025 g/L and

triphenyl-tetrazolium HCl 0,025 g/L to obtain a selective
and differential medium for bidobacteria in water and
wastewater.
References
ATLAS, R.M., LC. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,Boca Raton,Fla.
INGRAM, M. and BARNES, E.M. (1956) A Simple Modication of the Deep Shake Tube for Counting Anaerobic
Bacteria. Lab. Practise 5, 4:145.
HIRSCH, A. and GRINSTEAD, E. (1954) Methods for
the Growth and Enumeration of Anaerobic Sporeformers
from Cheese, with Observations on the Effect of Nisin.
J.Dairy Res. 21:101.
MUOA, F.J., R. PARS (1988) Selective medium for
isolation and enumeration of Bidobacterium spp. Appl.
Environm. Microgiol 54:1715-1718.
EUROPEAN PHARMACOPOEIA,(2002) 4th ed. Supllement 4.2 Chap. 2.6.13.Test for specied micro-organisms. Council of Europe. Strasbourg.
Rinse Fluid K
Ref. 03-109
Specication
Medium for rinsing the membrane lters according to the
USP and European Pharmacopoeia specications.
Formula (in g/L)

Meat peptone ............................................. 5,0
Meat extract ................................................ 3,0
Final pH 6,9 0,2
Directions
Dissolve 8 g of powder in 1 L of distilled water with 10
mL of Polysorbate 80 (Ref. 06-088). Distribute into suitable containers and sterilize in the autoclave at 121C
for 15 minutes.
Description
This nutrient solution which is formulated according to
the USP (Rinsing Fluid K) and European Pharmacopoeial specications, removes all the fat and carobohydrate
residues, due to the surfactant effect of Polysorbate,
and at the same time, it avoids the osmotic shock to the
microorganisms.
130
Technique
After the ltration, wash the membrane by passing 3
volumes of 100 mL of solution through it.
When the sample has a high concentration of fats or
sugars it is recommended to double the concentration of
polysorbate
(2 mL/L).
References
US PHARMACOPOEIA (2002) 25th ed.<71> Sterility
Tests. US Pharmacopoeial Convention Inc. Rockville.
MD
European Pharmacopoeia. (2002) 4th Ed. V.2.18 Control
of microbial contamination in no sterile products
Rogosa Agar Base

Ref. 01-300
Specication
Selective solid medium for the isolation and enumeration
of lactobacilli.
Formula (in g/L)

Glucose ................................................ 20,000
Tryptone ............................................... 10,000
Sodium acetate .................................... 10,000
Monopotasium phosphate ...................... 6,000
Yeast extract ........................................... 5,000
Ammonium citrate .................................. 2,000
Sorbitan monooleate .............................. 1,000
Magnesium sulphate .............................. 0,575
Manganese sulphate .............................. 0,120
Ferrous sulphate .................................... 0,034
Agar ...................................................... 15,000
Final pH 5,5 0,2
Directions
add 1,32 mL of glacial acetic acid and the complementary amount of sodium acetate to t standard. Heat with
gently stirring until boiling. Pour plates without autoclaving nor overheating.
The low pH and high acetate concentration, that can be

adjusted to the sample confers to this medium a high
selectivity for the lactobacilli. Nevertheless, it is no suitable for the dairy samples, for which is recommended
the addition of 20% sterile tomato serum (Ref. 06-092)
or using the MRS Agar (Ref. 01-135) that offers a best
performance with the dairy lactobacilli.
Rogosa Agar, due to its high acidity, is not suitable for
the maintenance of the microorganisms.
References
ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951)
A selective medium for the isolation and enumeration of
oral and faecal lactobacilli. J. Bacteriol. 62:132.
ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951)
A selective medium for the isolation and enumeration of
oral and faecal lactobacilli. J. Dental Res. 30:682
DOWNES, F.P. & K. ITO (Eds) (1991) Compendium of
methods for the microbial examination of foods. 4th ed.
APHA. Washington D.C.
MACFADDIN J.D. (1985) Media for isolation-cultivationidentication-maintenance of medical bacteria. William &
Wilkins, Baltimore. MD.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. London.
Description
Rogosa Agar was developed in 1951 for the isolation
and enumeration of oral and faecal lactobacilli, but with
changes in the acetate concentration it was used for
several kinds of samples, including foods.
131
Rose Bengal Agar

Ref. 01-301
Specication
T
R-45
S-53-45
Solid and selective medium for the isolation of yeast and

moulds from the environment and food products.
Formula (in g/L)

Peptone .................................................... 5,00
Dextrose ................................................. 10,00
Rose bengal ............................................. 0,05
Chloramphenicol ...................................... 0,10
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
to boiling with constant stirring. Distribute in suitable
minutes.
Technique
After making a dilution bank, take 0,1 mL from each dilution and inoculate with a Drigalsky Loop (Ref. 5-010) or
glass spreader on Rose Bengal Agar plates. Should the
massive seed method be preferred, take 1 mL from each
dilution and put it in an empty plate. Pour the molten
medium at 50C and homogenize it by gently moving the
plate in an eight (8) shape. Incubate at 22C for 5 days
and proceed to enumerate the fungi.
References
ATLAS, R.M., & PARKS, L.C. (1993) Handbook of Microbiological Media. CRC Press, Inc.,Boca Raton,Fla.
DOWNES F.P. & K. ITO (2001) Compendium of Methods
for the Microbiological Examination of Food. 4th Ed.
APHA. Washington.
MARSHALL, R.T. (1993) Standard Methods for the examination of dairy products. 16th ed. APHA Washington.
examination of water and wastewater. 20th. ed. APHA
Washington.
Description
Rose Bengal Agar is a selective medium to detect
and enumerate moulds and yeast in food samples.
Apart from the nutritional requirements for moulds and
yeast,this medium also contains Rose Bengal, which
apart from tainting the yeast with a pink colour, also facilitates their count, avoiding massive growth of moulds
such as Rhizopus and Neurospora, therefore it is easier
to detect other moulds with slower growth.
Chloramphenicol and also the Rose bengal, restrains
bacterial growth, but does not interfere with fungi growth.
Yeast and moulds colonies after 5 days at 22C
132
Sabouraud Media
Sabouraud Chloramphenicol
Agar
Ref. 01-166
Specication
Description
T
R-45
S-53-45
Solid culture medium for the isolation of fungi.
Formula (in g/L)

Casein peptone .......................................... 5,0
Meat peptone ............................................. 5,0
D(+) Glucose ............................................ 40,0
Agar .......................................................... 15,0
Final pH 5,6 0,2
Directions
bring to the boil. Distribute into nal containers and
sterilize by autoclaving at 121C for 15 minutes. Do not
overheat or reheat the medium since it will affect the
solidication.
Description
This culture medium differs from the classical Sabouraud Agar only in the addition of Chloramphenicol. This
thermostable antibiotic has a wide antibacterial spectrum
which ensures the selective isolation of fungi from highly
contaminated samples, such as eudates, faeces, nails
and hair.
Sabouraud Dextrose Agar

(Eur. Phar. Agar Medium C)
Ref. 01-165
Specication
Medium for the enumeration and cultivation of fungi.
Formula (in g/L)

D(+) Glucose ............................................ 40,0
Casein peptone .......................................... 5,0
Meat peptone ............................................. 5,0
Agar .......................................................... 15,0
Final pH 5,6 0,2
Directions
Dissolve 65 g in 1 L of distilled water and bring to the
boil with frequent stirring. Distribute into nal containers
and sterilize by autoclaving at 121C for 15 minutes. Do
not overheat the medium as its acidic pH may partially
hydrolize the agar. Alternatively,if the European Pharmacopoeia formulation is desired, add before sterilization
50 mg/L of chloranphenicol (Ref. 06-118CASE)
Sabouraud Dextrose Agar is a modication of the classical Sabouraud medium for the cultivation of fungi. This
new formula helps to maintain the morphological aspects
of fungi and thus permits a reliable cultivation and differentiation.
Its selectivity is due to a low pH and a high glucose concentration, which together with incubation at a relatively
lower temperature (25-30C) favours the growth of fungi
while discouraging that of bacteria. Besides, the composition of this peptone has been studied to provide the
fungi with all their nitrogenated nutrient requirements.
Since the Sabouraud mediums strong acid reaction
partially hydrolyzes the agar, only the required amount
should be prepared and it should not be remelted. Any
overheating will considerably diminish its gelling capacity.
Should a higher selectivity be required, a variety of inhibitors or selective agents may be added after sterilization, while the medium is still in the molten form. It can
even be made differential by adding the indicator agents.
Some of the inhibitory and differential mixtures most
commonly used are listed below:
Penicillin: at 20,000 units/litre, encourages the selectivity
of the medium by inhibiting most of the bacteria.
Penicillin and Streptomycin: at 20,000 u/L and 40,000
u/l each, favours the isolation of Histoplasma in
dogs.
Penicillin and Neomycin: at 20,000 u/L and 40 mg/L
each, is used for the isolation of yeast.
Streptomycin and Chloramphenicol: at 40 mg/L and 500
mg/L each, for the isolation of Trichophyton verrucosum .
Colistin, Novobiocin and Cycloheximide: at 8 mg/L, 0.1
mg/L and 30 mg/l each, for the isolation of Candida albicans .
Potassium Tellurite: at 150 mg/L, is used for the primary
isolation of fungi from scales and scabs.
Cupric Sulfate, Crystal Violet and Brilliant Green: at 500
mg, 2 mg and 5 mg each, achieves considerable
bacterial inhibition.
Triphenyltetrazolium chloride (TTC): at 100 mg/L, it is the
basis of a Pagano-Levin medium for the isolation of Candida albicans, unpigmented, among
other pathogenic yeast which form pink coloured
colonies.
Sabouraud Broth
Ref. 02-165
Specication
Liquid medium for the sterility control.
133
Sabouraud Media
Formula (in g/L)
Casein Peptone .......................................... 5,0
Meat Peptone ............................................. 5,0
D (+) Glucose ........................................... 20,0
Final pH 5,8 0,2
Directions
up only if necessary. Dispense into suitable containers
and sterilize in preheated autoclave for 15 minutes at
121C. Avoid overheating, since it may caramelize the
glucose.
Description
This medium is especially adapted to fungi and acidophilic bacteria culture.
Sabouraud USP Broth is according to the formulations of
US Pharmacopeia, US NF and 21 CFR guidelines. In the
latest editions of these books it is also mentioned and
allowed to use Tryptone and Soy Broth for the sterility
checking in parenteral pharmaceutical products. This formulation is similar to the Antibiotic Medium 13 by Grove
and Randall and 21 CFR.
This medium is not a selective one, but the strong acidic
pH notably inhibits the growth of non acidophilic microorganisms. Nonetheless, special measures must be taken
while reconstituting and heating the medium due to this
strong acid reaction and the high content of glucose. It is
important to preheat the autoclave and thereby reach the
sterilization temperature as soon as possible and in a
regular way, since otherwise, glucose becomes caramelized turning the medium dark and effectiveless.
Technique
It has been recommended to use this medium in many
tests and assays, but for a long time it has been the medium of choice for the verication of sterility of the sterile
pharmaceutical products.
Efcacy of the medium and absence of fungistatic products is veried by checking if there is a growth from a
loop of inoculum of Candida albicans, from a 1:1000 dilution of a fresh 24 hours grown culture. Sterility assay or
test is carried out in controlled and veried medium. To
check the fungistatic activity of any product, prepare an
inoculum as mentioned above and inoculate two series
of tubes with the same medium as follows:
a) Add to one batch the specied amount of product.
This is the test series.
b) Add to the another batch only the inoculum and
simultaneously incubate with the test series. This is
the control series.
Incubation of both the series must be carried out at 22C
for 10 days. After this period compare the results or
observations of both the series. If the assay series has a
lesser growth than the control one, product has the fungistatic activity. If the growth is equal or more, then it has
not any fungistatic properties. For the quantitative assay
of the fungistatic activity, perform the assay with several
series of different concentrations (one lower than the
previous) until reaching an equal growth in both control
and test series.
134
Sabouraud Oxytetracycline
Agar Base (OGYEA)
Ref. 01-275
Specication
Solid culture medium for the total enumeration of moulds
and yeasts.
Formula (in g/L)

Glucose .................................................... 20,0
Yeast extract ............................................... 5,0
Agar .......................................................... 20,0
Final pH 7,0 0,2
Directions
it soak for a few minutes. Distribute into suitable containers and sterilize by autoclaving for 10 minutes at 115C.
Cool to 50C and then add oxytetracycline (Ref. 06115CASE) to reach a 0,1 mg/mL concentration.
Description
This formulation differs with others as it has no peptone
and has a neutral reaction or pH. Unlike the others, it
has a high oxytetracycline concentration that makes it
almost impossible for the growth of bacteria.
Technique
Some authors suggests an inoculum of 1 mL in each
dilution, in duplicate and in mass. Perform an incubation
at 22-25C for 5 days with the intermittent observations
or readings after 3 days of incubation.
References
AJELLO, L.(1957) Cultural Methods for Human Pathogenic Fungi J. Chron. Dis. 5:545-551.
PAGANO, J., LEVIN,J.D. and TREJO, W.(1957-58)
Diagnostic Medium for Differentiation of Species of Candida. Antibiotics Annual, 137-143.
SABOURAUD, R.(1910) Les Tignes. Masson, Paris.
HANTSCHKE, D.(1968) Mykosen, 11:769-778.
EUROPEAN PHARMACOPOEIA (2002), 2.6.13 Tests
for specied micro-organisms Supplement 4.2, 4th Ed.,
EDQM. Council of Europe, Strasbourg.
US PHARMACOPOEIA (2002) 25th Ed. <51>Antimicrobial efectiveness Testing; <61> Microbial Limit Tests. US
Pharmacopoeial Convention Inc. Rockville. MD
ISO 13681 Standard (1995) Enumeration of yeasts and
moulds - Colony count technique.
Salmonella Shigella Agar (SS Agar)

Ref. 01-171
Specication
Solid and highly selective medium for the isolation of
Salmonella and some Shigella species.
Formula (in g/L)

Meat extract ........................................ 5,00000
Peptone .............................................. 5,00000
Lactose ............................................. 10,00000
Bile salts ............................................. 8,50000
Sodium citrate .................................. 10,00000
Sodium thiosulfate .............................. 8,50000
Ferric citrate........................................ 1,00000
Brilliant green ..................................... 0,00033
Neutral red .......................................... 0,02500
Agar .................................................. 15,00000
Final pH 7,0 0,2
Directions
Suspend 63,1 g of the dehydrated medium in 1 L of
distilled water. Slowly bring to the boil, stirring until complete dissolution. Boil for 2 minutes. Do not autoclave.
Cool to 50C and pour into sterile Petri dishes.Do not
overheat.
Description
The SS Agar is a highly selective agar for the isolation of
Salmonella and Shigella species from very contaminated
samples.
Selectivity is obtained by a high concentration of bile
salts and brilliant green, which inhibits the growth of
gram-positive bacteria. As for the other gram-negative
ora, its growth is highly repressed by the presence of
citrate and thiosulfate. Nevertheless, some coliforms
may still grow on this medium. In such case, differentiation between pathogenic species and coliforms becomes
evident by the colour change of the pH indicator neutral
red. Lactose fermenters produce a pink or red colored
medium and colonies, while non-fermenting species
form colourless colonies and turn the medium yellow.
Should any species eventually produce H2S, it will be
easily detected by the ferrous suldes black precipitate,
which turns the colonies black.
The peptone and the meat extract are usually capable of
inducing the growth of most pathogenic species, nevertheless some Shigella are very fastidious and grow
poorly.
medium, such as Brilliant Green Agar (Ref. 01-203) or

MacConkey Agar (Ref. 01-118).
Incubate the inoculated plates at 37C for 18-24 hours.
The suspicious colonies should then be subcultured
on differential media to be identied biochemically or
serologically.
Appearance of the colonies after 24 hours on SS Agar:
Shigella: Colourless, transparent and at.
Salmonella (Non H2S producers): Colourless, transparent and at.
Salmonella (H2S producers): Black or black centered,
at, with transparent borders.
Proteus: Similar appearance as Salmonella colonies, but
smaller in size.
Escherichia coli: If they grow, they are small, convex and
pink or red coloured.
Coliforms (in general): Big, opaque, smooth and coloured in white or pink shade.
References
LEIFSON, E.(1935) New culture media based on sodium
deoxycholate for the isolation of intestinal pathogens and
for the enumeration of colon bacilli in milk and water. J.
Pathol. Bacteriol., 40.581.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th Ed.
APHA. Washington DC.
HORWITZ,W.(2000). Ofcial Methods of Analysis 17th
ed. AOAC International. Gaithersburg. MD.
ATLAS, R.M.,and L.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, London
GRAY, L.D. (1995) Escherichia, Salmonella, Shigella
and Yersinia. In Murray, Baron, Pfaller Tenover & Yolken
(eds) Manual Clinical Microbiology. 6th ed. ASM Washington DC.
Technique
While using the samples suspected of being exposed
to the treatments that might have damaged the viability
of microorganisms (processed food, faeces from the
patients under antibiotic treatment, etc.) it is advisable to
proceed with a previous enrichment in Selenite Cystine
Broth Base (Ref. 02-602) or Tetrathionate Base Broth
(Ref. 02-033/Ref. 02-335). Afterwards, inoculate SS
Agar plates heavily with the specimen and proceed in
the same way with other specimens of a less selective
135
Schaedler Media
Schaedler Agar
Ref. 01-231
Specication
Solid medium with high reducing and nutrient capacity
for the cultivation of fastidious anaerobic microorganisms.
Formula (in g/L)

Casein peptone ........................................ 5,60
Soy peptone ............................................. 1,00
Meat peptone ........................................... 5,00
Yeast peptone ........................................... 5,00
Glucose .................................................... 5,80
Sodium chloride ........................................ 1,70
Tris buffer ................................................. 3,00
L-Cysteine HCl ......................................... 0,40
Hemine ..................................................... 0,01
Agar ........................................................ 15,00
Final pH 7,6 0,2
Directions
heat to boiling. Dispense into suitable containers and
sterilize in the autoclave at 121C for 15 minutes. Pour
into sterile plates just before the use.
Schaedler Broth
Ref. 02-231
Specication
Liquid version of the agar with the same name, especially suitable for fastidious anaerobic microorganisms.
Formula (in g/L)

Casein peptone ........................................ 5,60
Soy peptone ............................................. 1,00
Meat peptone ........................................... 5,00
Yeast peptone ........................................... 5,00
Glucose .................................................... 5,80
Sodium chloride ........................................ 1,70
Tris buffer ................................................. 3,00
L-Cysteine HCl ......................................... 0,40
Hemine ..................................................... 0,01
Final pH 7,6 0,2
136
Directions
Dissolve 28,3 g of powder in 1 L of distilled water, heating up only if necessary. Distribute into suitable containers and sterilize by autoclaving at 121C for 15 minutes.
Description
These media viz. Schaedler Agar and Broth, were
developed to create the selective conditions to allow
the growth of fastidious anaerobic microrganisms from
a mixed ora, like gastrointestinal tract, where there are
many antagonistic activities
between fast growing facultatives and the delicate fastidious anaerobic organisms. For this aspect, the media
with thioglycolate are widely used, but this compound
seems to inhibit some delicate anaerobic organisms. On
the other hand, Schaedler media have L-Cystine as a
reducing agent, thus some gramnegative do not grow.
Effective separation or isolation in several biotypes is
achieved with the addition of selective agents to the nutrient base. For example, this medium can be rendered
selective for lactic bacteria by adding 10 g/L of sodium
chloride and 0,002 g/L of neomycin.
For the selection of Clostridium and Bacteroides, it is
more advisable to add 2 g/L of placenta powder and
0,002 g/L of neomycin. Should a selective medium for
Flavobacterium be desired, add 7 mL of alcoholic solution of tyrothricin 0,5% to 1 L of medium base. In any
case, incubation must be carried out at 37C and in an
anaerobic atmosphere.
References
SCHAEDLER, R.W., DUVOS, R. and COSTELLO, R.
(1965) The development of the bacterial ora in the gastrointestinal tract of mice. J. Exp. Med. 122:59.
ATLAS, R.M., LC. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,London
STALONS, D.R., C.THORNSBERRY and V.R. DOWELL
(1974) Effect of culture medium and CO2 concentration
of growth of anaerobic bacteria commonly encountered
in clinical specimens. Appl. Microbiol 27:1098-1104.
ISENBERG H.D. (1992) Clinical Microbiology Procedures Handbook. ASM. Washington DC.
MARSHALL, R.T. (1992) Standard Methods for the examination of Dairy Products. APHA. Washington
MacFADDIN, J.F. (1985) Media for Isolation-Cultivation- Identication and Maintenance of Medical bacteria.
William & Wilkins. Baltimore, MD, USA.
WILKINS, T.D. and S. CHALGREN (1976) Medium for
use in the susceptibility testing of anaerobic bacteria.
Antimicrob. Agents. Chemother 10:926:928.
Sealing Anaerobic Agar

Ref. 01-174
Specication
Xn
R-22-43
S-24-37-46
Solid substrate with high reducing capacity to cover and

seal the cultures anaerobically.
Formula (in g/L)

L-Cysteine HCl ......................................... 0,30
Sodium Thioglycolate .............................. 0,70
Resazurine ............................................... 0,01
Agar ........................................................ 20,00
Final pH 7,0 0,2
Directions
bring to the boil. Dispense into suitable containers and
sterilize in the autoclave at 121C for 15 minutes.
Description
This solid substrate, without the capacity to support any
growth but with strong reducing power, is made to cover
the tubes for anaerobic growth. Once liquied, let it cool
to 50-55C and pour into the tubes, taking care not to
mix it with the medium.
A minimum column of 1,5 cm over the medium is recommended to assure an anaerobic cover.
Should cultures be stored for a long period of time, it is
advisable to put a sterile vaseline layer over the cap to
avoid
desiccation.
References
SANCHO, J. (1977) Personal communication.
Selenite Media
Selenite Brilliant Green
Broth Base (SBG Broth Base)
Ref. 02-603
Specication
avoided. Is not advisable to store the prepared medium

for more than eight days, since it loses its selectivity
notably.
Selenite Broth Base
Selective enrichment medium for Salmonella types.
Ref. 02-598
Formula (in g/L)
Specication
Gelatine peptone .................................... 5,000

Yeast extract ........................................... 5,000
D-Mannitol .............................................. 5,000
Sodium taurocholate .............................. 1,000
Sodium sulfapyridine .............................. 0,500
Brilliant green ......................................... 0,005
Final pH 7,2 0,2
Directions
Dissolve 20 g of powder. in 1 L of distilled water and add
4 g of sodium biselenite (Ref. 06-615). Homogenize and
bring to the boil. Distribute in suitable containers. Termolabile medium: Use immediately. Do not autoclave.
Description
SBG Broth is a modication to the classical Osborne and
Stockes medium for enrichment of Salmonella from eggs
and egg derivative products.
The medium is maintained at a neutral pH, in spite of the
acid products are liberated from the mannitol fermentation, due to the strong phosphate buffer. On the other
hand, inhibitor effect of sulfamide in gram-negative
bacteria is helped by the classical selective agents for
salmonellae like brilliant green, selenite and bile salts.
Notwithstanding, presence of these substances, makes
the medium thermolabile and thus autoclaving must be
Liquid medium for Salmonella and Shigella enrichment.
Formula (in g/L)

Peptone .................................................... 5,00
Lactose ..................................................... 4,00
Final pH 7,0 0,2
Directions
Dissolve 19 g of powder. in 1 L of distilled water and add
4 g of sodium biselenite (Ref. 06-615). Homogenize and
bring to the boil. Distribute in suitable containers. Termolabile medium: Use immediately. Do not autoclave.
Description
Selenite Broth is formulated according to an original
formulation by Leifson for selective enrichment of Salmonellae from very contaminated samples.
Enrichment is especially effective during the rst 12
hours of cultivation, since in this period it seems that
only Salmonellae, some Proteus and some strains of
Pseudomonas grow easily. For this reason, it is advisable not to extend the enrichment phase and go quickly
for the selective medium, either liquid or solid. According
to Bnffer, the efcacy of the medium is improved notably if enrichment is performed at 43C. Presence of a red
precipitate in the medium before inoculation, indicates
137
Selenite Media
that there was a overheating in which case the selective
properties of the medium are reduced.
Selenite Cystine Broth Base

Ref. 02-602
Specication
Liquid enrichment medium for Salmonella sp. acc. USP
and ISO 6785 and 6340 standards
Formula (in g/L)

Peptone .................................................... 5,00
Lactose ..................................................... 4,00
L-Cystine .................................................. 0,01
Final pH 7,0 0,2
Directions
Dissolve 19,01 g of powder. in 1 L of distilled water and
add 4 g of sodium biselenite (Ref. 06-615). Homogenize and bring to the boil. Distribute in suitable containers. Termolabile medium: Use immediately. Do not
autoclave.
Description
Selenite Cystine Broth has been developed according
to Leifsons formulation with the addition of L-Cystine to
comply with FDA specications, since it was proved that
the medium was better in reduced CO2 atmosphere.
Essencially, it is an enrichment medium for Salmonella
coming from food or pathological materials, such as faeces or urine, as a previous step to isolation in selective
media plates such as SS Agar (Ref. 01-171) or Hektoen
Agar (Ref. 01-216).
Technique
For normal assays or experiments, an incubation at
37C for a period not exceeding 18 hours is recommended, since within this period a good nutrition of coliforms
and an enhancement of pathogens is achieved, but after
24 hours this effect seems to disappear and the growth
of accompanying organisms may mask the growth of
Salmonella.
Appearance of red precipitate before inoculation is the
indication of overheating the medium, in which case the
selective properties are signicantly reduced. Presence
of abundant sample residues may also inactivate the
selective property of the medium, if the sample is e.g.
faeces and or egg powder. In those cases, it is better
to make a dilution of 1:10 and let the bigger particles
separate by settling down the dilution tube, and then
inoculate Selenite Cystine Broth with an aliquot portion
of it. Maintain a proportion of 1:10 between the sample
and the medium.
It has been demonstrated that when it is desired to isolate Salmonella from faeces,the results are better if the
enrichment medium is incubated at 43C. However this
procedure does not work with the isolation of Salmonella
typhi.
138
When starting material is urine, the best procedure is to

use Selenite Cystine Broth in double concentration, and
to inoculate it with an equal volume of urine. Anyway,
subculturing must always be done after 6 hours of incubation but before 24 hours. Most authors recommend
the simultaneous use of another enrichment broth, such
as Tetrathionate Base Broth (Ref. 02-033).
References
US PHARMACOPOEIA (2002) 25th ed Chapter <61>
Microbial Limit Tests The U.S. Pharmacopoeial Convention. Rockville MD.
DOWNES F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th ed.
APHA. Washington.
Rev. A. A.O.A.C.International.Gaitherburg VA.
LEIFSON, E. (1936) A new Selenite Selective Enrichment media for the Isolation of Typhoid and Paratyphoid
(Salmonella) Bacilli Am.J.Hyg. 24:423-432.
US FDA (1962) The determination of Salmonellae in
Food.
BNFFER, J.R. (1971) Comparison of the isolation of
Salmonellae from human faeces by enrichment at 37C
and 43C Zbl. Bakt. I Orig. 217:(35-40)
STOCKES, J.L. and OSBORNE, W.W. (1955) A Selenite
Brilliant Green Medium for isolation of Salmonella. Appl.
Microbiol 3-4:217-227.
ATLAS, R.M., LC. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc London
DIN - Standard 10160: Untersuchung von Fleisch u.
Fleischerzneugissen. Nachweiss von Salmonella (Referenzverfahren).
ISO 6785 Standard (2002) Milk and Milk products - Detection of Salmonella spp.
ISO 6340 Standard (1995) Water Quality Detection of
Salmonella spp.
Ref. 02-602
Selenite Cystine
Broth Base
Left: Salmonella typhimurium ATCC 14028; right:

control.
Sellers Agar
Ref. 01-175
Solid differential medium for gram-negative non fermenting coccobacilli.
- Glucose oxidation and pH changes that can be followed with the colour variation of the pH indicators
included (phenol red and bromo thymol blue)
- Nitrogen gas release, indicated by the bubbles which
may eventually break the agar.
Formula (in g/L)
Technique
Specication
Gelatin peptone .................................... 20,000

Sodium chloride ...................................... 2,000
Sodium nitrate ........................................ 1,000
Sodium nitrite ......................................... 0,350
D-Mannitol .............................................. 2,000
L-Arginine ............................................... 1,000
Yeast extract ........................................... 1,000
Bromothymol Blue .................................. 0,040
Phenol red .............................................. 0,008
Agar ...................................................... 13,500
Final pH 6,7 0,2
Directions
bring to the boil. Distribute into tubes and sterilize by
autoclaving at 121C for 15 minutes. Let it solidify in
slanted position with short slant and good butt.
Just before the inoculation, allow 2 drops of sterile glucose 10% solution to slide down on the opposite side of
the slant.
Description
Sellers Agar is formulated to differentiate the gram negative bacilli that do not produce characteristic acidication
by the fermentation in the usual diagnostic media, such
as TSI (Ref. 01-192) or Kligler Iron Agar (Ref. 01-103).
Criteria for the differentiation among the microorganisms
that can be obtained from this medium are as follows:
- Fluorescence production enhanced by the magnesium
sulfate and mannitol.
The medium is inoculated with a pure culture by streaking on the surface and also by stabbing deeply. Incubation is performed at 35-37C for 24-48 hours. After
the incubation period, observe the uorescence under
Woods light and also observe the change in pH and
gas production. The yellow band on the surface of the
medium due to glucose oxidation may disappear within
24 to 48 hours.
On the slant, uorescence may appear under UV light
in some species of the uorescent Pseudomonas group,
especially Ps. aeruginosa. On the surface of the medium, most of Acinetobacter species produce a yellow
band due to glucose oxidation. This band may disappear
after 24 hours, and this phenomenon is very common
with some strains of A. calcoaceticus.
A blue colour at the bottom of the tube indicates arginine-dehydrolase positive or any other reaction like
anaerobic degradation of nitrate or the mannitol utilisation by some strains of Alcaligens faecalis. Nitrogen gas
released in the form of small bubbles or splitting of agar
indicates denitrication reaction from nitrate or from
nitrite. In the table below all these typical reactions are
tabulated.
References
ATLAS, R.M. and R.C. PARKS (1993) Handbook of
Microbiological Media. CRC Press, London
MacFADDIN, J. (1985) Media for isoltion-cultivation-idencation-maintenance of medical bacteria. Vol I. William &
Wilkins. Baltimore,MD.
SELLERS, W. (1964) J. Bact. 87:46
139
SF Medium
Ref. 02-072
Specication
T
R-45-22-32-52/53
S-53-45-7-61
Liquid selective medium for faecal streptococci detection

in several materials.
Formula (in g/L)

Casein peptone .................................... 20,000
Dextrose ................................................. 5,000
Di-potassium phosphate ......................... 4,000
Sodium azide .......................................... 0,500
Sodium chloride ...................................... 5,000
Brom cresol purple ................................. 0,032
Final pH 6,9 0,2
Directions
Dissolve 36 g of powder in 1 L of distilled water. Dispense in tubes or asks with Durhams tubes and sterilize in the autoclave at 121C for 15 minutes.
Description
The efcacy of this diagnostic medium for detecting the
faecal streptococci (enterococci) from several materials
has been widely proved since its publication by Hajna
and Perry, who utilized the advantage of the azide effect
and combined it with the fermentative ability of strepto-
cocci to ferment glucose at 44,5C. Inhibition of accompanying bacteria is achieved by the high concentration of
azide, which does not affect the growth of streptococci.
Enterococci growth is indicated by the indicator brom
cresol purple turning to yellow, when the tubes are incubated at 44,5C.
In this medium, the indicator turning yellow in presence of enterococci is evident after 18-20 hours, but to
proceed for the isolation, a supplementary incubation in
petri plates is recommended.
If the MPN method is used, do not excessively dilute the
medium with the sample.
References
HAJNA, A.A. and PERRY, C.A. (1943) Comparative
study of presumptive and conrmativemedia for bacteria
of the coliform group an fecal streptococci. Am.J.Pub.
Hlth.,33:550.
Microbiological Media. CRC Press, Inc.,London
examination of Water and Wastewater,20th Ed.,APHA,
Washington.
DOWNES, F.P. and K. ITO (2001)Compendium of Methods for the Microbiological Examination of Food,4th Ed.
American Public Health Association,Washington D.C.
SIM Medium
Ref. 03-176
Specication
Differential uid medium for detecting the motility, H2S
production and indole formation.
Formula (in g/L)

Yeast extract ............................................. 10,0
Casein Peptone ........................................ 10,0
Meat peptone ............................................. 6,0
Ferric-ammonium sulfate ............................ 0,2
Sodium thiosulfate ...................................... 0,2
Agar ............................................................ 3,7
Final pH 7,3 0,2
Directions
soak for a few minutes. Heat to boiling and dispense into
suitable containers. Sterilize by autoclaving at 121C for
15 minutes.
Description
This classical medium was originally developed to distinguish several types of enterobacteria, on the basis of
motility test , detection of indole and H2S production.
It is a semisolid or uid medium, and so the motile microorganisms can move freely. At the same time, richness
of sulfur containing amino acids and presence of thiosul140
fate allow those microorganisms that are able to produce

suldes, and then this reacts with iron and produces
black precipitates which in turn make the medium darker.
The amount of thiosulfate present in medium does not
affect the motility mechanisms, instead it assures H2S
production by those microorganisms that are not able to
produce it from cystine or cysteine.
Finally, the medium allows the production of indole from
Tryptophan present in the peptone, which can be easily
detected with the addition of Kovacs Reagent (Ref.
06-018) (directly or with extraction) or with paper stripes
impregnated with the reagent.
These three characteristics are common for enterobacteria, and on the basis of these properties, more differential or even selective media can be used.
Technique
Recommended technique is to inoculate by deep stab
from a pure culture (or from an isolated colony). After an
incubation period of 16-18 hours at 37C, observe for the
clarity in stab. Immotile microorganisms produce growth
only in the stab, whereas motile ones may be easily
detected by their displacement which is indicated by the
turbidity in the medium.
H2S production is indicated by the general blackening of
the medium when there is a great amount of FeS produced or by blackening of the stab when there is a little
amount of FeS produced.
SIM Medium
Indole formation is the last test to be performed if the
soaked or impregnated paper stripes are not used.
Despite the fact that many authors suggest a previous extraction of indole by stirring on the surface of the
culture medium with chloroform. If Kovacs Reagent
(Ref. 06-018) is employed then this is not necessary and
the observations can be done by pouring a few drops of
reagent on the surface of the medium. A positive test will
produce the interphase in the medium turning to purple red colour, whereas a negative test will produce no
colour change. Many a times, chloroform extraction may
give erroneous results, since the appearance of colour
must be observed immediately after the reagent addition. However if it is delayed by more than 30 seconds,
the test must be considered negative.
References
HARRIGAN, W.F. and McCANCE, M.E. (1966) Laboratory Methods in Microbiology. Academic Press, .
BLAZEVIC, D.J. (1968) Improved Motility Indole Medium. Appl. Microbiol. 16, (4),668.
BULLMASH, J.M. and FULTON, M (1964). Discrepant
Test for Hydrogen Sulde. J.Bact. 88(6)1813.
Simmons Citrate Medium

Ref. 01-177
Specication
Medium for verifying the citrate utilization by enterobacteria according ISO 10273 standard.
Formula (in g/L)

Monoammonium phosphate ..................... 1,00
Sodium citrate .......................................... 2,00
Sodium chloride ........................................ 5,00
Brom thymol blue ..................................... 0,08
Agar ........................................................ 15,00
Final pH 6,8 0,2
Directions
Dissolve 24 g of powder in 1 L of distilled water. Bring to
the boil. Dispense in tubes and sterilize by autoclaving at
121C for 15 minutes. Allow to solidify with long slant.
Description
Simmons Citrate Agar is the solid version of the classical
Koser citrate medium, and it can be used in the plates as
well as in slanted tubes. Slanted tubes can be inoculated
by surface streaking or by deep stab.
Although, originally, it was described as an isolation and
identication medium for certain fungi, Edwards and
Ewing had recommended it for the IMViC test since it
has the advantage over the Kosers medium that the
readings can be made by the indicator colour change,
instead of turbidity of the medium, which is sometimes
difcult to detect.
Technique
The techique is simple and one has to take care in using an inoculum as small as possible and the medium
should be freshly prepared, because if it is very dry, false
turning (colour change) may appear, even before the
inoculation, especially at the bottom of the slant.
The basis of this medium is in the capacity of microorganisms to use citrate as carbon source and ammonium
compounds as the nitrogen source for their growth.
Among enterobacteria, these properties are possessed
by the following genera: Enterobacter, Klebsiella, Serratia, Citrobacter and some species of Salmonella as
S.schottumelleri, S.typhimurium, S.arizona etc. whereas
Escherichia, Shigella, Salmonella typhi and S.paratyphi
are unable to grow.
Although, the test result must be read as the growth produced, the presence of an indicator makes it easy, as the
citrate degradation yields an alkaline reaction, which is
indicated by the turning of the indicator to intense blue.
This is evident even when the growth rate is high.
References
SIMMONS J.S. (1926) A culture medium for differentiating organisms of typhoid-colon aerogenes group and for
isolatig certain fungi.J.Inf.Dis. 39:209
FDA (1998) Bacteriological Analytical Manual. 8th Ed.
Revision A. AOAC International. Gaithersburg.
APHA-AWWA-WEC (1998) Standard Methods for the
examination of water and wastewater. APHA. Washington DC.
Ed. AOAC International. Gaithersburg. MD.
detection of presumptive pathogenic Yersinia enterocolitica.
141
Slanetz Bartley Media

Slanetz Bartley Agar
Ref. 01-178
Specication
Description
Xn
R-22-32-52/53
S-7-46-61
Differential and selective medium for the enumeration of

enterococci,
Formula (in g/L)
Technique
Tryptose .................................................... 20,0

Yeast extract ............................................... 5,0
Dextrose ..................................................... 2,0
Sodium azide .............................................. 0,4
TTC............................................................. 0,1
Agar .......................................................... 12,0
Final pH 7,0 0,2
Directions
bring to the boil. Cool to 50C and distribute into sterile
petri plates immediately. Do not autoclave or overheat.
Description
The Slanetz Bartley Agar is recommended for the
determination of faecal streptococci by the membrane
ltration technique. Although originally this medium was
described by the authors only for the above technique, it
gives such excellent results that it is now recommended
for viable enumeration of enterococci from food samples.
Slanetz Bartley Agar Base

Ref. 01-579
Specication
This formulation, without TTC allows its sterilization by

autoclaving without the development of the pink colour
due to the formazan which is formed as a result of the
partial-thermal reduction of TTC. This modication is
more tedious in its preparation but provides a colourless medium, making the results easier to read and the
colonies are more sharply dened.
For the membrane ltration technique, take 100 mL of

water sample which is previously homogenized, and
pass it through a sterile membrane lter. Then wash
it with 30 mL of sterile dilution water to wipe away the
things left on the funnel of the ltering system.
By using sterile forceps, transfer the membrane aseptically on to the culture medium contained in a Petri dish,
making sure that the lter surface faces upwards. Close
the lid and invert the plate. lncubate at 37C for 48
hours.
All the developed colonies which appear red or purple
coloured must be considered as enterococci, since those
bacteria reduce Triphenyltetrazolium-HCl to an insoluble formazan which is red in colour. The secondary or
accompanying gram negative bacteria are inhibited by
sodium azide.
As regards, food samples from a decimal dilution bank
of the product to be examined, incoculate 0,1 mL on the
surface of the petri dishes using the Drigalski technique.
lncubation and examination is carried out as same as in
the membrane ltration technique.
References
Xn
R-22-32-52/53
S-7-46-61
Differential and selective medium for the detection and

enumeration of enterococci acc. ISO Standard 78992:2000
Formula (in g/L)

Tryptose .................................................... 20,0
Yeast extract ............................................... 5,0
Dextrose ..................................................... 2,0
Sodium azide .............................................. 0,4
Agar .......................................................... 12,0
Final pH 7,0 0,2
SLANETZ L.W. & BARTLEY, C.H. (1957). Numbers of

Enterococci in Water, Sewage and Faeces Determined
by The
Membrane Filter technique with an lmproved Medium.
J.Bact., 74:591-596.
ATLAS, R.M., & LC. PARKS (1993) Handbook of Microbiological Media,CRC Press Inc.,London
ISO Standard 7899-2:2000 Water Quality. Detection
and enumeration of enterococci by membrane ltration
method.
LACHICA, L.V.F. & P.A., HARTMAN (1968) Two improved media for isolating and enumerating enterococci
in certain frozen foods
J. Appl. Bact. 31:151-156
Directions
bring to the boil. Sterilize by autoclaving at 121C for
15 minutes. Cool to 50C and add 10 mL/L of 1% TTC
sterile solution (Ref. 06-023). Mix well and distribute into
sterile petri plates immediately.
142
Ref. 01-579 SlanetzBartley Agar Base + Ref.

06-023 TTC 1% Sterile
Solution.
Enterococcus faecalis
ATCC 29212.
SOB Broth
Ref. 02-523
Specication
Liquid medium for the cultivation of recombinant strains
of Escherichia coli.
Formula (in g/L)

Tryptone .............................................. 20,000
Yeast Extract .......................................... 5,000
Sodium Chloride .................................... 0,500
Magnesium sulphate ............................. 2,400
Potasium chloride ................................... 0,186
Final pH 7,0 0,2
The addition of 4 g/L of dextrose to this medium produce the SOC Medium that is used in the nal phase of
transformation and in the recovery of electroporated E.
coli cells.
References
HANAHAN, D. (1983) Studies on transformation of Escherichia coli wth plsmids. J. Mol. Biol. 166:557
SAMBROOK, J., E.F. FRITSCH & T. MANIATIS (1989)
Molecular cloning: a laboratory manual. 2nd Ed. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Directions
Dissolve 28 g of powder in 1 L of distilled water heating
if it is necessary. Distribute in suitable containers and
Description
This medium was developed by Hanahan in 1983 as a
nutritionally rich base for growth preparation and transformation of competent cells that allows the introduction
of foreign DNA onto cells..
Sporulating Agar (AK Agar) (USP Antibiotic Medium 32)

Ref. 01-069
Specication
This medium is according to the Arret and Kirshbaum
formulation, adopted by US FDA, for the preparation
of spore suspension for antibiotic assay. It is the same
medium as the Antibiotic Medium 32.
Formula (in g/L)

Gelatin peptone .......................................... 6,0
Casein peptone .......................................... 4,0
Yeast extract ............................................... 3,0
Meat extract ................................................ 1,5
Dextrose ..................................................... 1,0
Manganous sulfate ..................................... 0,3
Agar .......................................................... 15,0
Final pH 6,6 0,2
Directions
Dissolve 30,8 g of powder in 1 L of distilled water. Bring
it to the boil with constant stirring . Dispense in suitable
containers and sterilize at 121C for 15 minutes.
in 10 mL of Ringer Solution the growth from a slant. With

this suspension, inoculate on the surface a Roux ask
(bottle) containing 300 mL of solidied and controlled
Sporulation Agar.
The roux ask must be incubated at 35C for 5 days.
Growth is harvested with 50 mL of sterile Ringer Solution, helping with a Drigalsky loop or sterile crystal
balls (pearls) if necessary. The suspension obtained is
carefully centrifuged at 5000 rpm for 15 minutes. Discard
the supernatant liquid and suspend the pellet in a fresh
volume of Ringer Solution. Put it in a boiling water bath
at 70C for 30 minutes. This suspension is active upto 6
months if stored refrigerated. Depending on the objective
of the experiment,it can be standardized by turbidimetry.
References
ARRET and KIRSHBAUM (1959) J.Milk. Food Tech.
22:329
SANCHO, GUINEA, PARES (1980) Microbiologa
Analtica Bsica. Ed. JIM. Barcelona,
US PHARMACOPOEIA (2002) 25th ed. <81> Antibiotic
Microbial Assays. US Pharmacopoeial Convention Inc.
Rockville. MD
Description
Sporulating Agar is made according to the original formulation of Arret and Kirshbaum, and later adopted by
FDA for the preparation of Bacillus subtilis ATCC 6633
spores suspension. To prepare this suspension, suspend
143
SPS Agar
Ref. 01-050
Specication
Solid medium for the detection of Clostridium perfringens in food.
Formula (in g/l)

Sodium sulte ........................................... 0,50
Polymyxin (B) sulfate ................................ 0,01
Sodium sulfadiazine ................................. 0,12
Casein peptone ...................................... 15,00
Yeast extract ........................................... 10,00
Ferric citrate.............................................. 0,50
Sodium thioglycolate ................................ 0,10
Polysorbate 80 ......................................... 0,05
Agar ........................................................ 15,00
Final pH 7,0 0,2
Directions
bring to the boil. Distribute into tubes or screw-cap
minutes. Cool the sterilized medium quickly by placing in
the refrigerator or in cold water.
Description
The SPS Agar (Sulte-Polymyxin-Sulfadiazine) is a
modication of the original Wilson & Blair medium for the
detection of clostridia. The present medium excels the
formulation of Mossel and also the later modication of
Angelotti et al. It achieves a higher selectivity for Cl. perfringens with the addition of sulfadiazine and polymyxin.
On the other hand, the nutritional substrates constituted
by the tryptone and the yeast extract are complemented
by the Polysorbate, which also allows the recovery of
the most delicate cells. The anaerobic conditions are improved by the thioglycolate, which permits the use of the
medium on the plates without the Miller-Prichett tubes,
used by Mossel and Wilson-Blair.
The differential system consists of sodium sulte and

ferric citrate. It allows the detection of sulte-reducing
organisms, which form black colonies due to the ferrous
sulde precipitate.
Technique
The usual technique for the use of this medium is as
follows:
The samples to be examined are ground or homogenized with a vortex in a stomacher and then a decimal
dilution bank is prepared. Take a sample aliquot from
each one of these dilutions and place in the petri dishes.
The medium, molten and cooled to 50C, is now poured
in the dishes and allowed to solidify. The dishes are incubated in an anaerobic system at 35C for 24-36 hours.
Usually, 90% of the black colonies which are formed can
be attributed to the Clostridium perfringens. Nevertheless, and since the medium is not extremely selective,
it is advisable to verify the black colonies formed by
gram-positive sporulated immotile organisms incapable
of reducing the nitrates to nitrites.
The Indole Nitrite Fluid Medium (Ref. 03-101) is suitable
for such purposes, still a small quantity of agar has to be
occasionally added in it.
Most clostridia are sulte reductors. Among them are Cl.
perfringens and the Cl. botulinum which along with Cl.
bifermentans are the species most frequently involved
in food poisoning.
References
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food,4th Ed.
APHA,Washington.
FDA (1998) Bacteriological Analytical Manual, 8th ed.
control
Clostridium perfringens ATCC 13124

144
Staphylococcus 110 Agar

Ref. 01-587
Technique

heat up to 55-60C in boiling water bath until total dissolution of gelatin. Bring to the boil. Dispense in suitable
minutes. Before pouring in the petri dishes, gently shake
to suspend possible precipitate. Should a blood addition be desired, cool to 45C and add sterile debrinated
blood and homogenize before pouring in the plates.
The sample is inoculated in the petri dish by wire loop to

obtain isolated colonies, and it is incubated for 48 hours
at 30C.
At 37C early reactions or results are obtained, but
pigmentation is not good,as the pigmented colonies form
a characteristic golden yellow colour, which in this case
are white.
Acid production from mannitol is veried by adding a few
drops of Bromthymol Blue 0,04% solution on suspected
colonies. Positive reaction produces a yellow colouration.
Gelatin hydrolysis (Stones reaction) is visualised by a
clear halo surrounding the colony which forms 10 minutes after a few drops of 20% aq. soln.of Sulfosalicylic
acid or sat. aq. solution of Ammonium sulfate are added
over them.
These last two reactions are helped by placing Oxford
cups around the discrete colonies, isolating them from
affecting the other colonies. Identication is completed
by verifying hemolysis tests, subculturing on Blood Agar
plates, and verifying plasmacoagulase activity on rabbit
serum.
Although the subcultures can be done from isolated
colonies in 110 medium, results are always better if a
short (4-5 hours) incubation on Tryptic Soy Broth (Ref.
02-200) is carried out.
Description
References
Specication
Selective medium for the isolation and presumptive identication of staphylococci.
Formula (in g/L)

Casein Peptone ...................................... 10,00
Yeast extract ............................................. 2,50
Gelatin .................................................... 30,00
Sodium chloride ...................................... 75,00
Di-potassium phosphate ........................... 5,00
Lactose ..................................................... 2,00
D-Mannitol .............................................. 10,00
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
This medium combines the basic criteria of staphylococci

identication i.e.: halotolerance, pigmentation, mannitol
fermentation and gelatin liquefaction as were marked by
Stone and Chapman. Smucker and Appleman suggests
the addition of sodium azide (5mg/L) to improve the
inhibition of Bacillus spp.
It is widely known that pathogenic staphylococci are coagulase positive, pigment former, highly halotolerant and
able to liquefy gelatin and produce acids by fermenting
mannitol. They are also strongly hemolytic.
In the 110 medium, the following reactions can be
observed: pigmentation, mannitol fermentation, gelatin
liquefaction and saline tolerance. From this medium,
hemolysis and coagulase production can also be proved
afterwards.
STONE, R.V. (1935) A cultural method for classifying

staphylococcias of the food poisoning type. Proc.Soc.
Exptl.Biol Med.33:185-187
CHAPMAN, G.H. (1945) The signicance of sodium
chloride in studies of staphylococci. J. Bacteriol 50:201205
Revision A. AOAC International. Gaithersburg
145
Starch Media
Starch Agar
Ref. 01-283
Specication
Solid medium to detect the starch hydrolysis by microorganisms,
Formula (in g/L)

Meat extract ................................................ 3,0
Peptone ...................................................... 5,0
Soluble starch ............................................. 2,0
Agar .......................................................... 15,0
Final pH 7,2 0,2
Directions
Suspend 25 g of powder into 1 L of distilled water and
let it soak. Heat to boiling and distribute into suitable
minutes.
Description
Although this medium was initially formulated to perform
the test for the identication of Bacillus cereus, it can
be applied to any kind of microorganism where starch
hydrolysis activity is required to be analyzed.
Technique
Over the medium plates with Starch Agar, inoculate in
straight streaks the strains to be examined, (maximum
four per plate). Incubate at 30-35C for 48 hours if the
strains are of Bacillus cereus and up to 5 days for dubious cases.
After the incubation, ood the plates with an alcoholic
iodine solution 2%. Starch hydrolysis is seen by the appearance of a clear halo surrounding the growth streak,
whereas the rest of the medium in the plate acquire a
dark blue colour.
The bigger the clear zone, the starch activity is considered higher of the strain under study.
References
COLLINS, C.H., LYNE, P.M. (1976) Microbiological
Methods. 4th Ed. Butterworths,London.
ISENBERG H.D. (1992) Clinical Microbiology Procedures Handbook. ASM Washington.
ATLAS, R.M., & LC. PARKS (1993) Handbook of Microbiological Media. CRC Press,Inc.,London
146
Stuart-Ringertz Transport Medium

Ref. 03-454
Specication
Xn
R-22-43
S-24-37-46
Medium for the conservation and transport of pathological specimens or fastidious microorganisms.
Formula (in g/L)

Sodium thioglycolate .............................. 1,000
Methylene blue ....................................... 0,002
Agar ........................................................ 8,000
Final pH 7,4 0,2
Directions
bring to the boil. Distribute in tubes or asks close with
air tight cap in such a way that the medium forms a
vertical column of 7-10 cm. Sterilize in the autoclave
at 121C for 15 minutes and cool quickly in the vertical
position.
Description
The growth of microorganisms in this medium is restricted by the total lack of nitrogen, but they remain alive and
inactive for a long period. Thanks to the buffering and
protective effect of glycerophosphate. Thioglycolate pro-
vides a reducing environment which is aided and maintained by to the low concentration of agar, that avoids
convection streams and restricts oxygen difussion.
Progressive oxidation of the medium can be seen by
the change of the methylene blue, which acts as an Eh
(redox) indicator.
Technique
Sample is placed directly inside the tube, taking care that
it is beneath the blue band. If the sample is taken with a
swab, it is advisable to impregnate it with a suspension
of active carbon (activated charcoal) before puting it into
the transport medium.
The sample must always be in the centre of the medium
and beneath the blue band that indicates oxidation. If
the depth of the blue band is bigger than the half of the
medium, do not use the tube.
References
STUART, R.D. (1959) Transport medium for specimens
in public health bacteriology. Publ. Hlth. Rep. 74:431438.
RINGERTZ, O. (1960) A modied Stuart medium for the
transport of gonococcal specimens. Acta Path. Microbiol.
Scand. 48:105-112
147
TCBS Media
TCBS Agar
Ref. 01-190
Specication
TCBS Agar is specially recommended for the selective
isolation of Vibrio that causes cholera or dysenteric diarrhoea, and for the examination of food that might have
Vibrio.
Formula (in g/L)

Casein peptone ........................................ 5,00
Meat peptone ........................................... 5,00
Yeast peptone ........................................... 5,00
Sodium citrate ........................................ 10,00
Sodium thiosulfate .................................. 10,00
Ox bile ...................................................... 5,00
Sodium Cholate ........................................ 3,00
Sucrose .................................................. 20,00
Sodium chloride ...................................... 10,00
Ferric citrate.............................................. 1,00
Thymol blue .............................................. 0,04
Bromo thymol blue ................................... 0,04
Agar ....................................................... 14,00
Final pH 8,6 0,2
Directions
bring to the boil. Boil it for 1 minute. Cool to 45-50C and
pour it into sterile petri plates. Do not autoclave it.
TCBS Modied Agar

Ref. 01-567
Specication
TCBS Agar is specially recommended for the selective
isolation of Vibrio parahaemolyticus according ISO 8914
standard.
Formula (in g/L)

Proteosa Peptone ................................... 10,00
Yeast Extract ............................................ 5,00
Sucrose .................................................. 20,00
Sodium citrate ........................................ 10,00
Sodium chloride ...................................... 10,00
Ox bile ...................................................... 8,00
Sodium thiosulphate ............................... 10,00
Iron citrate ................................................ 1,00
Thymol Blue ............................................. 0,04
Bromthymol blue ...................................... 0,04
Agar ........................................................ 14,00
Final pH 8,6 ( 0,2)
148
Directions
bring to the boil. Boil it for 1 minute. Cool to 45-50C and
pour it into sterile petri plates. Do not autoclave it.
Description
Nowadays TCBS Agar is universally accepted as the
medium of choice for differential isolation of enteropathogenic Vibrio, and it achieves a great inhibition of all the
accompanying organisms. This formulation allows a
high growth of Vibro cholerae and V.parahaemolyticus.
V.alginoliticus and NAG vibrios also grow well.
Enterobacteria are strongly inhibited by high concentrations of citrate, thiosulfate, bile and sodium chloride.
Although some enteric bacteria may also grow in this
medium, their colony morphology is quite different than
Vibrios.
The organisms that may be confused with vibrios are
some biotypes of Proteus and Pseudomonas. There are
some resistent enterococci which may form exceptionally small and yellow colonies on this medium.
Usually, in this medium, colonies are selected or chosen
and then identied with primary tests (oxidase reactions
in Kliger Iron Agar (Ref. 01-103), MRVP Broth (Ref.
02-207), and antibiogram) before performing serological
identication and phage typing.
Due to its high selectivity, the medium allows massive
inoculation of pathological material. Once solidied and
cooled, the medium is turbid, but the observations are
not affected.
This medium is very thermolabile and so it must not be
autoclaved, overheated or remelted.
Colonial appearance on TCBS Agar after 24 hours at
37C:
Vibrio alginolyticus: Big, yellow
Vibrio cholerae and sucrose fermentative strains of Vibrio parahaemolyticus: Average size, dirty yellow
with yellow halo in the center
Non sucrose fermentative strains
of Vibrio parahaemolyticus: Small, yellow, without halo
and with a green core.
Streptococcus faecalis: Very small and convex, yellow
with yellow halo
Enterobacteria: Small and transparent
Pseudomonas, Aeromonas, Proteus: Average size and
blue.
References
KOBAYASHI, T., ENOMOTO, S. SAKAZARI, R. and
KUWAHARA, S. (1963) A new selective medium for
pathogenic vibrios TCBS, (modied Nakanishi Agar)Jap.
J.Bact.18:387.
Revision A. AOAC INTERNATIONAL. Gaithersburg
detection of Vibrio parahemolyticus.
TCBS Media
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food,4th Ed.
American Public Health Association,Washington D.C.
BHATTACHARYA, M.K., S.K. BATTACHARYA, S.
GARG, P.K. SAHA, D.DUTTA, G.B. NAIR, B.C. DEB &
KP DAS (1993). Outbreak of Vibrio cholerae non-01 in

India and Bangladesh. Lancet, 341:1346-1347
HORWITZ, W. (2000) Ofcial Methods of Analysis of
AOAC International 17 ed. Gaithersburg. MD
Ref. 01-567
TCBS Modied Agar
Proteus mirabilis
ATCC 14273
Vibrio alginolyticus
control
Terric Broth
Ref. 02-474
Specication
Liquid medium for the cultivation of recombinant strains
of Escherichia coli.
Formula (in g/L)

Casein peptone ...................................... 12,00
Yeast extract ........................................... 24,00
Final pH 7,3 0,2
Directions
Dissolve 47,6 g of powder in 1 L of distilled water, heating up only if necessary. Distribute in suitable containers
Description
Terric Broth was developed by Tartoff and Hobbs to
improve yield in plasmid bearing E. coli. This medium
supports a high cellular density and mass and maintains
the growth in the logarithmic phase for a long time. Due
to this fact, it provides greater yields of recombinant
proteins and plasmid DNA. On many occasions it substitutes the classical LB Broth (Ref. 02-385, 2-384 and
2-406) therefore usually increases yields of plasmid DNA
and recombinant proteins.
References
SAMBROC, J., E.F. FRITSCH, T. MANIATIS (1989) Molecular Cloning: A laboratory Manual. 2 Ed.,Cold Spring
Harbor Press. Cold Spring Harbor,USA.
TARTOFF, K.D. & C.A. HOBBS (1987) Improved media
for growing plasmids and cosmid clones. Bethesda Research Laboratoires Focus 9:12
149
Tetrathionate Media
Tetrathionate Broth Base
Ref. 02-033
Specication
Medium for the selective enrichment of Salmonellae
(AOAC 17th, ICMSF 1968, USP 25th)
Formula (in g/L)

Meat peptone ............................................. 2,5
Casein peptone .......................................... 2,5
Bile salts ..................................................... 1,0
Calcium Carbonate ................................... 10,0
Sodium Thiosulfate ................................... 30,0
Directions
Suspend 46 g of powder to 1 L of distilled water, heat to
boiling and cool to 40-45C. Add 20 mL of iodine-iodide
solution and 2 vials of the Selective Supplement of Brilliant Green-Novobiocin ref. 06-017CASE and distribute
in sterile tubes.
Do not heat after adding the iodine solution. Medium
must be used immediately. Without the iodine solution,
medium can be stored in refrigeration for some days.
The appearance of white medium precipitate is normal,
and it comes from calcium carbonate.
Description
This is the version originally used, which has been modied or improved the Muller-Kauffmann formulation, since
the latter one has more efcacy.
Muller-Kauffmann Medium Base

Ref. 02-335
Specication
Medium for selective enrichment of Salmonellae, acc.
ISO standard.
Formula (in g/L)

Bile salts #3 .............................................. 4,78
Meat extract .............................................. 4,30
Casein peptone ........................................ 8,60
Sodium chloride ........................................ 2,60
Calcium carbonate ................................. 38,70
Directions
Add 107 g of powder to 1 L of distilled water. Heat to
boiling and let it cool to 40-45C. Add 20 mL of iodineiodide solution and 2 vials of the Selective Supplement
of Brilliant Green-Novobiocin Ref. 06-017CASE and
distribute into sterile tubes.
Do not heat after adding the iodine solution. Complete
medium must be used immediately; the base, without
iodine, may be stored in the refrigerator for some days.
150
White precipitate is due to calcium carbonate and it must

be considered as normal.
Description
Tetrathionate Broth is a classic medium for the enrichment of enteric or intestinal pathogens, and for all the
members of Salmonella type, from very polluted samples, like faeces, urine, waste water and others.
During the preparation, when iodine is added, tetrathionate is produced from the sulfate, and this salt together
with the bile salts in the medium, provoke a strong inhibition to most of the normal intestinal bacteria, except for
those which are capable of reducing tetrathionate, e.g.
Salmonellae. Reduction reaction liberate sulfuric acid,
which is neutralized by the carbonate, avoiding a decrease of the pH, which is harmful even for Salmonellae.
However, many Proteus species resist the bile salts
concentration and moreover, they may even reduce
tetrathionate. So, many authors recommend the addition
of other inhibitors simultaneously, such as 0,1% Brilliant
Green Solution (10 mL/L) which at the same time inhibits
gram-positive ora, or Novobiocin in a concentration
between 4 and 40 mg/L.
Basal medium can be kept indenitely in the refrigerator,
but after the addition of inhibitors, efcacy of the medium
decreases with time.
Sorting the inhibitors depending on their stability if they
are kept in the refrigerator produce the following list: brilliant green, novibiocine, which is effective for 2 months
in the refrigerator but only 48 hours at 37C, and nally
iodine solution, which is only effective for 40 hours once
the inhibitor is added to the medium.
Technique
It is recommended to prepare the Base Broth, distribute
it into tubes, sterilize and cool it. Add the Brilliant Green
Tetrathionate Media
solution and store it in the refrigerator. If you are going
to use the medium before 60 days, you may also add
novobiocin. Iodine-iodide solution has to be added just
before its use. Do not reheat the medium after one of
these additions.
Usual technique consists of adding the sample to the
medium (1:10) and then homogenizing it well. Incubate
at 37C for a period not longer than 48 hours, since
after this time the medium loses its selectivity and the
suppressed ora may also grow. Some authors suggest
to incubate at 43C and perform observations after 18,
24 and 48 hours, but one can get better results if the
sample from the surface of the broth is taken after 30-36
hours.
Take aliquotes with a loop and inoculate on the surface
of the selective media like SS Agar (Ref. 01-171) or
Hktoen Enteric Agar (Ref. 01-216), etc...
References
DIN Standard 10160 Untersuchung von Fleisch und
Fleischerzeugnissen: Nachweis von Salmonellen. Referenzverfahren.
DIN Standard 10181 Mikrobiologische Milchuntersuchung: Nachweis von Salmonellen. Referenzverfahren.
DOWNES, F.P. & K.ITO (2001) Compendium of methods fort he microbiological examination of foods. 4th ed.
Revision A. AOAC International. Gaithersburg. Md. USA.
FIL-IDF Standard 93. (2001) Milk and milk products:

Research of Salmonella.
ed. AOAC International. Gaithersburg. Md.USA
ISENBERG, H.D. (1992) Clinical Microbiology Procedures Handbook. Vol. 1. APHA. Washington DC. USA
ISO Standard 6579 (2002) Microbiology of food and animal feeding stuffs Horizontal method fort he detection
of Salmonella spp.
ISO Standard 6785 (2001) Milk and Milk Products Detection of Salmonella spp.
ISO (1975) Standard 3565. Meat Products: Reference
Method for detection of Salmonellae..
U.S. PHARMACOPEIA. (2002) 25th ed. <61> Microbial
Limits Test. US Pharmacopoeial Convention Inc. Rockville. Md. USA.
KAUFFMAN, F. (1931) Ein Kombiniertes Anreicherungus
verfahren fr Typhus und Paratyphus Bazillen.Zblt. Bakt
Microbiol. Hyg Abt. I. Orig. 119:148
MARSHALL, R.T. (1993) Standard methods for the examination of dairy products. 16th ed. APHA Washington
DC. USA.
MULLER, L. (1923) Un nouveau milieu
denrichiessement pour la recherche du bacille typhique
est des partyphyques. Comp. Rend. Soc. Biol. 89:434437.
Thioglycollate Media
Thioglycollate Broth (USP
Alternative Thioglycollate
Medium)
Ref. 02-186
Specication
Directions
Xi
R-43
S-24-37
A medium for sterility test and the cultivation of microaerophilic and anaerobic organisms. It is specially used for
viscous or turbid samples.
Formula (in g/L)

Peptone from casein ................................ 15,0
Yeast extract ............................................... 5,0
Dextrose ..................................................... 5,5
Sodium chloride .......................................... 2,5
Sodium thioglycollate ................................. 0,5
L-Cystine .................................................... 0,5
Final pH 7,1 0,2

if necessary to help dissolution. Distribute into suitable
minutes. This culture medium should always be freshly
prepared or heated at 100C for 10 minutes before use.
Description
The Thioglycolate broth is a standard medium, named
also Alternative Thioglycollate Medium, formulated and
recommended by USP, NF, NIH and FDA.
It is used for sterility testing of biological products or
samples of turbid appearance where Fluid Thioglycollate Medium (Ref.3-187) is not suitable because of its
viscosity.
The formula of Thioglycollate broth is the same as
Thioglycollate USP Fluid Medium without resazurin and
agar.
Media must be freshly prepared, boiled, sterilised,
cooled and used within 4 hours for its inoculation.
151
Thioglycollate Media
References
Description
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc. London
DOWNES, F.P. & K. ITO. (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th
Ed. APHA. Washington DC. USA
ed. AOAC International. Gaithersburg. Md. USA
US PHARMACOPOEIA (2002) <71> Sterility Test. 25th
ed. US Phamacopoeial Convention Inc. Rockville. Md.
USA.
The Thioglycollate Fluid Medium is a standard medium

formulated and recommended by European Pharmacopoeia, USP, NIH and FDA.
The reducing agents thioglycollate and L-cystine ensure
an anaerobiosis which is adequate even for fastidious
anaerobes. The sulfhydryl groups of these substances
also inactivate arsenic, mercury and other heavy metal
compounds. The Thioglycollate media are thus suitable
for the examination of materials which contain heavy
metals or heavy metal preservatives.
The higher viscosity of the uid thioglycollate medium
prevents rapid uptake of oxygen. Any increase in the
oxygen content is indicated by the redox indicator sodium resazurine which changes its colour to pink.
Thioglycollate Fluid Medium

Xi
Ref. 03-187
R-43
S-24-37
Specication
Fluid medium for sterility testing acc. to Eur. Phar., USP,

FDA, and ISO 7937:2004 for the cultivation of microaerophilic and anaerobic organisms. It is specially used for
viscous or turbid samples.
Formula (in g/L)

Peptone from casein ............................ 15,000
Yeast extract ........................................... 5,000
Dextrose ................................................. 5,500
Sodium chloride ...................................... 2,500
Sodium thioglycollate ............................. 0,500
L-Cystine ................................................ 0,500
Resazurine ............................................. 0,001
Agar ........................................................ 0,750
Final pH 7,1 0,2
Directions
Dissolve 30 g of powder in 1 L of distilled water, slowly
bring to the boil, stirring until complete dissolution. Distribute into nal containers and sterilize by autoclaving
at 121C for 15 minutes. Store in dark at room temperature for not more than one month. If after the storage the
medium is pink coloured (sign of oxidation) for more than
1/3 of its depth, recreate anaerobic conditions by heating
Technique
Inoculate the culture medium with the sample material
taking care that the sample reaches the bottom of the
tubes.
Incubate for at least 14 days at the optimal temperature.
Anaerobes grow in the lower part of the culture medium
container.
References
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media, CRC Press Inc., London
BREWER J.H (1940) Clear liquid medium for the
aerobic cultivation of anaerobes. J. Amer. Med. Assoc.
113:598-600
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 3rd ed.,
A.P.H.A., Washington D.C.
EUROPEAN PHARMACOPOEIA, (2005) 2.6.1 Sterility. 5th Ed., EDQM Council of Europe. Strasbourg.
FDA (1998).Bacteriological Analytical Manual, 8th ed.
Revision A., AOAC International. Gaithersburg. MD
HORWITZ, W., (2000) Ofcial Methods of Analysis, 17th
ed., A.O.A.C. International. Gaithersburg. MD
ISENBERG, H.D. (Ed), (1998) Essential Procedures for
Clinical Microbiology. ASM., Washington. USA
MacFADDIN, J.F. (1985) Media for Isolation-cultivationidentication-maintenance of medical bacteria. Vol. I.,
Williams & Wilkins. Baltimore. MD. USA
US PHARMACOPOEIA (2005) 25th Ed. <71> Sterility Test., US Pharmacopoeial Convention Inc., Rockville
MD.
Ref. 03-187 Thioglycollate

Fluid Medium
Left: anaerobical growth; center: control; right:

fermentative growth
152
Triple Sugar Iron Agar (TSI Agar) (Eur. Phar. Agar

Medium M)
Ref. 01-192
Specication
Differential medium for identication of enterobacteria,
according ISO standard.
Formula (in g/L)

Peptone ................................................ 20,000
Meat extract ............................................ 3,000
Yeast extract ........................................... 3,000
Lactose ................................................. 10,000
Sucrose ................................................ 10,000
Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Ferric ammonium citrate ......................... 0,500
Phenol red .............................................. 0,025
Agar ..................................................... 12,000
Final pH 7,4 0,2
Directions
Dissolve 64,8 of powder in 1 L of distilled water and
bring to boiling. Dispense into tubes and sterilize at
121C for 15 minutes. Leave to solidify with short slant
and good butts.
Description
TSI Agar is a modication of the classical Kligers agar.
1% of sucrose has been added to this medium to differentiate Proteus and Hafnia (sucrose positive) from
Salmonella and Shigella (sucrose negative).
Sugar degradation with acid formation is detected by the
turning of an indicator (phenol red) to yellow, whereas
if there is alkalinization, it turns to purple. When there is
only glucose degradation, the acid production is weak
and is evaporated on the surface, so indicator may be
reoxidised producing an alkaline surface (red) and an
acid butt (yellow). If lactose or sucrose are degradated,
acid production is intense and then all of the medium
(surface and depth) turns yellow. Gas production is
detected by the formation of bubbles and occasionally
cracks in the agar.
Hydrogen sulde production, from thiosulfate or sulfured
aminoacids of peptones, is detected by the formation
of black FeS precipitate when medium reacts with iron
salts.
Use the medium in slanted tubes with good depth
and short slant. Inoculate by streaking on surface and
stabbing deeply. It is advisable to use tubes with cotton
plugs, in order to allow a reoxidation of the indicator. If
screw caps are used, they must be loose.
Following will nd the table of reading (observations) and
interpretation of results in TSI Agar.
References
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the microbiological examination of Foods. 4th ed.
EDWARD, S.P. and EWING, W.H. (1962). Identication

of Enterobacteriaceae. Burgess. Pub. Co. Minneapolis.
EUROPEAN PHARMACOPOEIA.(2005) Supp. 5.8
2.6.13 Test for specied micro-organisms. EDQM. Strasboug E.U.
FIL-IDF (1991) International Standard 93A. Milk and Milk
Products. Detection of Salmonella species.
HAJNA, A.A. (1945) Triple Sugar-Iron medium for the
identication of the intestinal group of bacteria. J.Bact.
49:516-517
ed. AOAC International. Gaithersburg. Md. USA.
153
Triple Sugar Iron Agar (TSI AGAR) (Eur.Phar. Agar

Medium M)
ISO 3560 Standard. (1975) Reference Method for the
Detection of Salmonella in meat and meat products.
ISO 6579 Standard. (2002) Microbiology of foods and
animal feeding stuffs - Horizontal method for the detection of Salmonella spp.
ISO 6785 Stanbdard (2001) Milk and milk Products
Detection of Salmonella spp.
ISO 10272 Standard (1995) Microbiology of foods and
animal feeding stuffs - Horizontal method for the detection of thermotolerant Campylobacter.
KRUMWIEDE, C. & L. KOHN (1917) A triple sugar modication of the Russell Double Suigar Medium. J. Med.
Res. 37:225-229
US PHARMACOPOEIA (2002)<61> Microbial Limit
Tests. 25th ed. US Phamacopeial Convention Inc. Rockville. Md. USA
Todd Hewitt Broth

Ref. 02-191
Liquid culture medium for the propagation of beta-hemolytic streptococci and for studies about serologic typing.
as an alternative type in epidemiologic studies of group A

streptotocci as well as pathogenic microorganisms.
With the addition of 15g/L of agar, medium can be solidied and then it is an excellent substrate for the production of capsules in streptococci.
Formula (in g/L)
References
Specication
Meat extract .............................................. 10,0

Casein peptone ........................................ 20,0
Glucose ...................................................... 2,0
Sodium bicarbonate ................................... 2,0
Sodium chloride .......................................... 2,0
Disodium phosphate ................................... 0,4
Final pH 7,8 0,2
Directions
Dissolve 36,4 g of powder in 1 L of distilled water and
Description
This classical medium formulation has been modied
to achieve optimal results in the growth and production
of hemolysins, which are not inhibited due to the high
buffer composition of the medium. Many ofcial organisations, such as APHA, have recommended this medium
154
TODD, E.W. and HEWITT, L.F. (1932) A new culture

medium for the production of antigenic streptococcol
haemolysin.J. Path. Bact. 35:973-974
MacFADDIN, J. (1985) Media for isolation-cultivationidentication-maintenance of medical bacteria. Vol. 1.
Williams & Wilkins. Baltimore. USA.
Microbiological Media. CRC Press Inc.London
ISENBERG H.D. (1992) Clinical Microbiology Procedures Handbook. ASM. Washington DC.
Tryptic Soy Media

Tryptic Soy Agar (TSA)
(Eur. Phar. Agar Medium B)
(Casein Soybean Digest Agar)
Ref. 01-200
Specication
General purpose solid medium containing animal and
plant peptone according ISO 9308-1 standard.
Formula (in g/L)

Casein peptone ........................................ 15,0
Soy peptone ............................................... 5,0
Sodium chloride .......................................... 5,0
Agar ......................................................... 15,0
Final pH 7,3 0,2
Directions
Mix 40 g of powder in 1 L of distilled water. Let it soak
and bring to the boil to dissolve the agar. Sterilize by
Description
TSA is a widely used medium containing two peptones
which support the growth of a wide variety of organisms,
even that of very fastidious ones such as Neisseria,
Listeria, Brucella, etc. It is frequently used for routine
diagnostic purposes due to its reliability and its easily
reproducible results.
The following list includes some of its most common
applications:
1.- Sensitivity testing either by the Kirbky-Bauer system
or by following the WHO guidelines. Both the systems recommend the use of the Mueller Hinton Agar
(Ref. 01-136) for verication purposes.
2.- The medium provides, with added blood,perfectly dened hemolysis zones, while preventing the lysis of
the erythrocytes due to its sodium chloride content.
3.- It can be used for the preparation of an exceptionally
nutrient chocolate agar, thanks to the richness of its
peptones.
4.- In a reducing environment or with a CO2 enriched
atmosphere, its plates provides an excellent medium
for the isolation of Brucella and Neisseria . It may be
made selective by using certain additives.
5.- Most streptococci grow in this medium though clear
differences can be observed from one species to
another.
6.- The Tryptic Soy Agar is the selective medium for the
count of urine samples although the differentiation
must be done on selective differential media.
7.- Several tests for the differentiation and identication
of staphylococci can be obtained in this medium,
provided with suitable additives.
8.- Yeast, particularly Candida species, can grow in this
medium forming very characteristic colonies.
9.- Chromogenic pseudomonads frequently produce

pigmentation on the TSA and are therefore easily
recognized.
10.- It is widely used for testing contaminated samples.
A vast bibliography documents its applications in the
food industry.
11.- It has been frequently used in the Health industry to
produce antigens, toxins,etc...
12.- Its simple and inhibitors-free composition makes it
suitable for the detection of antimicrobial agents in
food and other products.
13.- A balanced and highly nutrient value together with a
lack of fermentable carbohydrates make this medium
one of the most recommended for the strain maintenance.
Tryptic Soy Broth (TSB)

(Eur. Phar. Broth Medium A)
(Casein Soybean Digest Broth)
Ref. 02-200
Specication
Highly nutrient liquid medium for the general purposes,
formulated according to USP, FDA and Eur. Phar. regulations.
Formula (in g/L)

Casein peptone ........................................ 17,0
Soya peptone ............................................. 3,0
Sodium chloride .......................................... 5,0
Dextrose ..................................................... 2,5
Final pH 7,3 0,2
Directions
Dissolve 30 g of powder in 1 L of distilled water and
Description
The Tryptic Soy Broth was initially developed for the
cultivation of very fastidious microorganisms without the
addition of serum, blood or any other enrichment agent.
As a general purpose culture medium it supports the
growth of most organisms, both aerobes and facultatives, even if their requirements are high. Due to its high
vitamin content the development of Brucella, Pasteurella and Streptococcus is perfectly viable, moreover a
CO2 enriched atmosphere can further favour it.
In anaerobic conditions this broth will easily bear the
growth of Bacteroides and Clostridium species. For this
purpose, the best results can be obtained by adding
0.3% agar and 0.05% sodium azide for Clostridium.
The Tryptic Soy Broths superior growth-promoting properties makes it particularly suitable for the tube dilution
method for antibiotic sensitivity testing. It also achieves
good results in the detection of gram-positive cocci. The
broth can be used for bile solubility testing in pneumo-
155
Tryptic Soy Media

cocci, and also used for catalase and coagulase assays
and for the preparation of hypersaline broths.
It is a most suitable medium for the preparation of antigens and toxins in bacteria, moulds and yeasts.
TSB is used as a primary enrichment medium for food
examination. In the dairy industry it is employed for testing resazurine reduction.
The medium is not suitable for maintenance purposes
since carbohydrate fermentation liberates many acids
which may threaten the organisms viability. Therefore,
though it allows the growth of streptococci and Neisseria, these species tend to die if repeatedly subcultured in
this medium. Such fastidious organisms are best maintained on Cystine Tryptone Fluid Medium (CTA) (Ref.
03-045) or even TSA (Ref. 01-200) if it is not suitable or
convenient to the use of solid media.
References
US PHARMACOPOEIA (2002) 25th ed. <61> Microbial
Limit Tests. US Phsarmacopoeial Convention. Rockville.
MD.
EUROPEAN PHARMACOPOEIA (2002) 2.6.13 Tests
for Specied Microorganisms. 4th Ed. Suppl.4.2 EDQM
Council of Europe Strasbourg.
ATLAS, R.M., & LC. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.London
DOWNES, F.P. & K. ITO (2001). Compendium of Methods for the Microbiological Examination of Food, 4th
ed,ASM,Washington,D.C.
Alimentaria. Diaz de Santos, S.A.Madrid,.
ISO 9308-1 Standard (2000) Water Quality - Detection
and enumeration of E.coli and coliform bacteria. Membrane ltration method.
156
Tryptic Soy Broth w/o Dextrose

(TSB w/o Dextrose)
Ref. 02-227
Specication
Liquid culture medium for the massive production of
spores of B. stearothermophilus for the inhibitory sustances in food acc. FDA-BAM
Formula (in g/L)

Tryptone ................................................. 17,00
Soy peptone ............................................. 3,00
Sodium chloride ........................................ 5,00
Dipotasium phosphate .............................. 2,50
Final pH 7,3 0,2
Directions
Disolve 27,5 g of powder in 1 L of distilled water, heating if necessary. Distribute in suitable containers and
Description
TSB w/o Dextrose is produced according the formulation
from Bacteriological Analitycal Manual of Food and Drug
Administration for the massive production of spores of
Bacillus stearothermophilus used to determine the presence of inhibitory sustances in milk and dairy products.
This medium is not recommended for sugars fermentation studies because the great amount of fermentable
carbohidrates in the soy peptone.
References
MATURIN, L.J. (1998) Inhibitory substances in milk.
Qualitative Method II: B. stearothermophilus disk assay.
en FDA Bacteriological Analitical Manual. 8th Ed. Revision A. AOAC International Inc. VA.
Tryptone Bile Agar

Ref. 01-526
Selective solid medium for the rapid enumeration of
Escherichia coli according ISO 9308-1 standard.
The indol producer microorganisms other than E. coli are

inhibited by the bile salts and the incubation temperature, but in sugar-rich samples the indol production can
be inhibited due to the sugar concentration interferes the
tryptophanase synthesis.
Formula (in g/L)
Technique
Specication
Tryptone ................................................. 20,00

Bile salts #3 .............................................. 1,50
Agar ........................................................ 15,00
Final pH 7,2 0,2
Directions
Suspend 36,5 g of powder in 1 L distilled water and bring
to the boil. Distribute into suitable containers and sterilize at 121C for 15 minutes.
Description
This medium is formulated according the Anderson &
Baird-Parker Direct Plating Method (DPM) for the rapid
enumeration of Escherichia coli in raw meats. The
method is based on the characteristically production of
indol from tryptophane when the bacteria growth at 44C
over a cellulose acetate membrane in the surface of the
Tryptone bile agar.
The International Commission on Microbiological Standards for Food noticed that he DPM was lesser variable
and offer a better recovery and rapidity than the MPN
method for frozen samples of meat. The ISO Standard
6391:1988 also uses this medium for the E. coli enumeration.
A cellulose acetate membrane of 0,45 m pore is extended on the surface of the medium. A 0,5 mL of the sample
dilution (with Tryptone Water Ref. 03-156) inoculum is
spreaded on the membrane and the plates are incubate
in upright position at 441C for 18-24 hours.
After incubation the membrane is immersed in indole
reactive using the petri dish cover as container and
exposed the direct sunlight for 5 minutes or to the Wood
lamp for 10 minutes. The indole positive colonies turns
to the reddish colours (pink to deep red). The results are
expressed as number of Escherichia coli per g or mL of
sample.
References
ANDERSON, J.M. & A.C. BAIRD-PARKER (1975) Appl.
Bact. 39:111-117
International Commission on Microbiological Specications for Foods (1979) Can. J. Microbiol. 25:1321-1327
and enumeration of E.coli and coliform bacteria. Membrane ltration method.
157
Tryptone Glucose Extract Media

Tryptone Glucose Extract Agar
(TGE Agar)
Ref. 01-082
Specication
Plate count medium for the milk and dairy products, according to Standard Methods for Dairy Products.
Formula (in g/L)
APHA-AWWA-WEF (1998) Standard Metods for the

Examinationof Water and Wastewater. 20th ed. APHA.
Washington
AOAC International. Gaitherburg. MD
Tryptone Glucose Extract Broth

for Membrane Filtration (m-TGE
Broth)
Meat extract ................................................ 3,0

Casein peptone .......................................... 5,0
D (+) Glucose ............................................. 1,0
Agar .......................................................... 15,0
Final pH 7,0 0,2
Ref. 02-082
Directions
Formula (in g/L)
Add 24 g of powder in 1 L of distilled water. Heat to the

boil by constantly stirring. Dispense in suitable containers and sterilize by autoclaving at 121C for 15 minutes.
Description
Solid medium Tryptone Glucose Extract was adopted
long ago as an alternative to Nutrient Agar acc. APHA
(Ref. 01-144) and Nutrient Agar acc. British Pharmacopoeia (Ref. 01-140) for milk bacteria enumeration, being
a complement to Plate Count Agar (Ref. 01-161).
Technique
For the enumeration purposes, it is suggested to use the
poured plate method, and incubation at 30-32C for 48
hours. If the dilutions in the plate is more than 10% it is
advisable to add milk to the medium. To do this, prepare
the supension of skimmed milk (Ref. 06-019) separately,
and sterilize it for 10 minutes at 118C. Autoclaving must
be as short as possible. Homogenize with the culture
medium which is sterilized and cooled to 50C. The use
of natural milk is not recommended due the wide variation .
Medium must be quickly poured into Petri dishes because if it remains hot for too long, occules and abnormal precipitates may appear. If the sample under study
is not diluted or the volume in the plate is more than 2
mL, it is not necessary to add the skimmed milk (Ref. 06019) because it is assumed that the sample provides the
required growth factors.
References
FDA.(1998) Bacteriological Analytical Manual 8th ed
Revision A. AOAC International,Gaitherburg MD
DOWNES, F.P. & K. ITO (2001) Compendium of methods for the Microbiological Examination of Foods, 4th
Ed. APHA, Washington.
MARSHAL, R.T.. (Ed.) (1992) Standard Methods for the
Examination of Dairy Products. 16th Ed. APHA. Washington.
158
Specication
Non-selective liquid medium for enumerating microorganisms by membrane ltration method.
Tryptone ................................................ 10,00

Meat extract .............................................. 6,00
Dextrose ................................................... 2,00
Final pH 7,0 0,2
Directions
Dissolve 18 of powder in 1 L of distilled water, heating
if it is necessary. Distribute in suitable containers and
Description
This medium is a liquid nutritive substrate that can be
used in the colony count by the membrane ltration
method for the absorbent pad impregnation The broth
has the same formulation as Tryptone Glucose Extract
Agar , except that the broth contains no agar and the
ingredients are at twice the concentration.
Technique
The sample is ltered through a membrane. In a plate
the absorbent pad is impregnated with the medium
avoiding any excess. The membrane is transferred on
the pad and the complete system is incubated at 352C
for 18-24 hours. After this time total colonies are counted
and results recorded.
References
DOWNES, FP. & K. ITO (2001) Compendium of methods
for the microbiological examination of foods 4th ed APHA
Washington
examination of water and wastewater.
Tryptone Phosphate Water (Buffered Peptone Water)

Ref. 02-277
Dilution and nonselective pre-enrichment liquid medium
acc. to ISO 6579, 8523, 8261 and 6785 standards.
water and the pH change absorbing capacity of the

phosphate buffer.
The composition of this diluent is made according the
specication of the ISO Standard 6579 for the detection
of Salmonella in foods.
Formula (in g/L)
References
Specication
Peptone .................................................... 10,0

Sodium chloride .......................................... 5,0
Final pH 7,0 0,2
Directions
Dissolve 25,5 g of powder in 1 L of distilled water.
Distribute into suitable containers and sterilize in the
Description
This formulation of Tryptone Phosphate Water has the
advantages of the two classical diluents for food samples: it has the property of revitalization of the peptoned
ATLAS, R.M.,& L.C. PARKS (1993) Handbook of Microbiological Media,CRC Press, Inc.,London
ISO 6579 Standard (2002). Microbiology of food and
animal feeding stuffs. Horizontal method for the detection of Salmonella spp.
ISO 8261 Standard (2001) Milk and milk products General guidance for the preparation of test samples for
microbiological examination.
Tryptone Sulte Neomycin Agar (TSN Agar)

Ref. 01-195
Specication
Solid selective medium for Clostridium perfringens isolation.
Formula (in g/L)

Casein peptone ...................................... 15,00
Sodium sulte ........................................... 1,00
Neomycin sulfate ...................................... 0,05
Polymyxin B .............................................. 0,02
Yeast extract ........................................... 10,00
Ferric citrate.............................................. 0,50
Agar ........................................................ 13,50
Final pH 7,2 0,2
Directions
bring to the boil. Dispense in suitable containers and
To obtain better results, add 20 mL/L of a solution containing 1 g/L dipotassium phosphate, 0,5 g/L sodium carbonate and 1 g/L sodium thioglycollate just before use.
Description
This culture medium was formulated by taking the
advantage of the tolerance of Cl. perfringens to the
high concentration of sulte, which apart from being an
inhibitor agent, provides a strong reducing environment.
Selection of Cl.perfringens is almost complete when it
is incubated at 46C, since neomycin and polymyxin
included in the medium restrain the development of

Cl.bifermentans and all the accompanying gram negative bacteria.
The medium is especially suitable for the investigation of
food products, and it may be used in tubes as well as in
plates. If the incubation is not performed in an anaerobic
jar, thioglycolate buffered solution must be added or the
inoculated surface must be covered with a sterile layer of
medium.
Colonies of Cl.perfringens form very characteristic black
colonies that, if exposed to air, become decolourised by
oxidation. TSN have a very short storage period once
prepared, so it is advisable to rehydrate or reconstitute it
in small amounts and use it on the day of its preparation.
References
MARSHALL, R.S., STEENBERGEN, J.F., MacCLUNG,
L.S. (1955) Rapid Technique for the enumeration of
Clostridium perfringens. Appl. Microbiol. 13:559-563.
MOSSEL, D.A.A (1959) Enumeration of sulte reducing
clostridia occurring in nfoods. J. sci. Food Agr. 10:662669
MacFADDIN, J.F. (1985) Media for Isolation-CultivationIdentication-Maintenance of Medical Bacteria,Williams
& Wilkins. Baltimore,USA.
ATLAS, R.M., & L.C. PARK (1993) Handbook of Microbiological Media,CRC Press Inc.,London
159
Tryptone Water (Peptone Water)

Ref. 03-156
Specication
Substrate with low nutrient capacity, for the research of
indole production in coliform micro-organisms according
ISO 7251 standard.
Formula (in g/L)

Casein peptone ........................................ 10,0
Sodium chloride .......................................... 5,0
Final pH 7,2 0,2
Directions
Dissolve 15 g of powder in 1 L of distilled water and dispense into suitable containers. Sterilize by autoclaving at
Description
The standard protocol requires to reinoculate one loop
from each suspected tube in 10 mL of Tryptone Water.
Incubate for 48 hours at 44C before investigating the
indole production with the Kovacs Reagent for indole
(Ref. 06-018).
As an alternative method, the Ehrlichs Reagent (Ref.

06-024) could also show indole production. After 48
hours of incubation at 37C, take 0,5 mL of growth and
mix it with 0,5 mL of Ehrlichs Reagent. Let them settle
a few minutes. If it takes on a pink colour,then the test is
positive. Colour appearance is accelerated if a few drops
of saturated solution of potassium persulfate is added.
Other authors prefer extraction and concentration of indole with 1 mL of ether, and then observe on the extract
with any of the reagents mentioned above.
References
ATLAS, R.M.& L.C. PARKS (1993) Handbook of Microbiological Media,CRC Press Inc.,London
DOWNES, F.P. & K. ITO (2001). Compendium of Methods for the Microbiological Examination of Food. 4th Ed.
APHA,Washington
APHA-AWWA-WEP (1998) Standard Methods for the
examination of water and wastewater. 20th ed. APHA.
Washington, DC.
enumeration of E.coli by the MPN technique.
Tryptone Yeast Extract Agar

Ref. 01-590
Specication
Solid medium for the enumeration of water microorganisms acc. ISO Standard 6222.
Formula (in g/L)

Tryptone ..................................................... 6,0
Yeast extract ............................................... 3,0
Agar .......................................................... 15,0
Final pH 7,2 0,2
Directions
bring to the boil. Distribute into containers and sterilize
Description
This medium, formulated according to ISO Standard
6222, is for the enumeration of heterotrophic microorganisms from water.
Technique
From the water sample, obtained according the ISO
Standard 5667-2 and 5667-3, make a decimal dilution
bank (see ISO Standard 6887) with Ringer Solution (Ref.
6-073) and take aliquots to 2 parallel series of plates.
Pour the Tryptone Yeast Extract Agar melted and cooled
to 45C, and homogenize with sample (see ISO Standard 8199). Once solidied, incubate one of the series at
160
362C for 442 hours and the other one at 22C for 3
days (684 hours).
In order to achieve a good count, select plates with
30-300 colonies. Express the results as number of
colony forming units per milliliter (cfu/mL) of the sample
for each temperature of incubation. If there are no colonies with the undiluted sample express the results as no
detected in one mL. If there are more than 300 colonies
in the highest dilution express the results as >300/mL
References
ISO Standard 6222 Watewr Quality Enumeration of
culturable microorganisms. Colony count by inoculation
in a nutrient agar culture.
ISO Standard 5667-2 (1991) Water Quality-Sampling
Guidance on sampling techniques
ISO Standard 5667-3 (1996) Water Quality Sampling.
Guidance on the presevation and handling of samples
ISO Standard 6887 (1999) Microbiology- General
Guidance for the preparation of dilutions for microbiological examination.
ISO Standard 8199 (1988) Water Quality General
guide to the enumeration of microorganisms by culture.
Tryptophan Broth
Ref. 02-418
Specication
Liquid medium for the indole production according to
ISO standard.
Formula (in g/L)

Meat peptone ........................................... 10,0
L-Tryptophan .............................................. 1,0
Sodium chloride .......................................... 5,0
Final pH 7,2 0,2
Directions
Dissolve 16 g of powder in 1 L of distilled water. Distribute into suitable containers and sterilize in the autoclave
Description
This broth allows the indole production from the tryptophan, and therefore it is suitable for the differentiation
and identication of coliforms from water and food.
Its formulation is according to the German standards for
waters and foods.
Technique
Medium is inoculated with the previously isolated culture,
and then incubated at 30-32C for 24-48 hours.
Indole production is observed adding a few drops of
Kovacs Reagent (Ref. 06-018) over the broth (with or
without previous extraction) and shaking gently. Formation of a red ring indicates indole presence.
References
VERORDNUNG ber Trinkwasser und ber Wasser fr
Lebensmittelbetriebe vom 12-12-1990. Bundesgesatbl. I.
2613-2619.
BUNDESGESMELHEITSAMT: Amtliche Samnulung von
Untersuchungs verfahren nach #35LMBG. Beuth Verlag.
Berlin-Kln.
ISO 9308-1 Standard (2000) Water Quality. Detection
and enumeration of Escherichia coli and coliform bacteria. Part 1: Membrane ltration method.
ISO 6785 Standard (2001) Milk and milk products. Detection of Salmonella spp.
Tryptose Media
Tryptose Agar
Tryptose Broth
Ref. 01-197
Ref. 02-197
Specication
Specication
Solid medium for isolation, cultivation and differentiation

of Brucella, streptococci and fastidious pathogens.
Liquid culture medium for massive culturing of fastidious

microorganisms.
Formula (in g/L)
Formula (in g/L)
Tryptose ................................................ 20,000

Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Thiamine HCl .......................................... 0,005
Agar ...................................................... 15,000
Final pH 7,2 0,2
Tryptose ................................................ 20,000

Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Thiamine HCl .......................................... 0,005
Final pH 7,3 0,2
Directions
Suspend 41g of powder in 1 L of distilled water and heat
to boiling. Dispense in tubes or asks and sterilize it in
To obtain better results, add to the molten medium, 20
mL/L of a solution composed by dipotassium phosphate
1 g/L, sodium carbonate 0,5 g/L and sodium thioglycolate 1 g/L just before using the medium.
Directions
up if necessary. Dispense into suitable containers and
Description
Tryptose culture media are suitable, essencially for the
growth of fastidious strains, such as Listeria, Pasteurella,
Brucella, etc. despite the fact that nowadays it seems
there is a trend towards the use of more dened media
such as Brucella Agar (Ref. 01-042) and Broth (Ref.
161
Tryptose Media
02-042), which provide better results. Tryptose Agar and
Broth have been recommended by several National and
International Organisations for Brucelosis Control and for
the maintenance, propagation and cultivation of standardized strains. They may be successfully used to differentiate the several types of Brucella with the addition
of suitable indicators. In this case, its use is analogous to
the Brucella Broth (Ref. 01-042).
With the adequate additives (crystal violet, sodium azide
etc.) the media may become very selective and efcient,
and their nutrient conditions may be improved if citrate
and blood are added, although glucose is not very suitable for observing hemolysis.
References
CASTAEDA, M.R. (1947) A practical method for rutine
blood cultures in brucellosis. Proc. Soc. Exp.Biol. Med.
64:114-115
HAUSSLER, W.J. (1976) Standard Methods for the
Examination of Dairy Products,9th Ed. APHA.
MARSHALL, R.T.. (1992) Standard Methods for the Examination of Dairy Products. 16th Ed. APHA. Washington.
RENNER, E.D., K.J. McMAHUN (1981) Brucellosis in Diagnostic Procedures for Bacterial Mycotic and Parasitic
Infections. 6th Ed.,APHA, Washington.DC
ATLAS, R.M. & LC. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc.,London.
DOWNES,F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4rdEd.
APHA.Washington
MURRAY,P.R., E.J. BARON, M.A PFALLER, F.C. TENOVER, & R.H. YOLKEN (1995) Manual of Clinical Microbiology. 6th ed. APHA. Washington DC .
Tryptose Phosphate Broth

Ref. 02-199
Specication
Liquid culture medium, with glucose and buffer, for
cultivation of fastidious microorganisms and for tissues
culture media.
Formula (in g/L)

Tryptose .................................................... 20,0
Dextrose ..................................................... 2,0
Sodium chloride .......................................... 5,0
Final pH 7,3 0,2
Directions
Dissolve 29,5 g of powder into 1 L of distilled water. Dispense into suitable containers and sterilize by autoclaving at 121C for 15 minutes.
162
Description
Tryptose Phosphate Broth is a recommended medium
for the cultivation and propagation of microorganisms
that have strong needs, such as streptococci, meningococci, and Brucella. It has also been also used to
determinate antibiotic sensitivity testing by the dilution in
tube method.
This medium has been used as primary diluent and
emulsier in dairy products for determination of Brucella,
but it is really effective for the cultivation of many streptococci and to test the bile solubility of these organisms.
When it is used to isolate streptococci, it is suggested to
add 0,1% Agar to render it into a uid medium. Should a
very selective medium be desired, add 2,5% of sodium
azide. To get a solid medium, add 1,5% of Agar.
References
GINSBERG, H.S. (1955) Tryptose Phosphate Broth as
Supplementary Factor for Maintenance of Hella Cell Tissue Cultures. Proc. Soc. Exper. Biol. Med. 89(1):66-71.
WAISBREN, B.A. (1951). The Tube Dilution Method of
Determining Bacterial Sensitivity to Antibiotics. Am. J.
Clin. Path 21:884.
BALOWS, A., W.J. HAUSSLER (1981) Diagnostic Procedures for Bacterial Mycotic and Parasitic Infections.
6thEd,APHA. Washington.
ATLAS, R.M., & J.W. SNYDER (1995) Handbook of Media for Clinical Microbiology. CRC Press. London
Revision A. AOAC International. Gaithersburg. MD.
Tryptose Lauryl Sulfate Media

Tryptose Lauryl Sulfate Broth
Ref. 02-108
Specication
coliform bacteria according IDF-FIL 73B and ISO 4831
and 7251 standards.
Formula (in g/L)

Tryptose .................................................. 20,00
Sodium Lauryl sulfate ............................... 0,10
Lactose ..................................................... 5,00
Sodium chloride ........................................ 5,00
Final pH 6,8 0,2
Directions
Dissolve 35,6 g of powder in 1 L of distilled water. Distribute into tubes or containers tted with the inverted
Durham tubes (for gas). Sterilize at 121C for 15 minutes. As for the double concentration medium, dissolve
71,2 g/L and proceed as indicated above.
Preferably store the broth at room temperature, and use
screw-capped bottles to prevent evaporation of water.
Refrigerated broth generally becomes cloudy or forms
precipitates but clears at incubation at room temperature. However, clarity is not important as only the gas
production is signicant criterion.
Description
Laurylsulfate broth is used for MPN Presumptive Test
of coliforms in water and sewage, conrmatory test of
lactose fermentation with gas production for milk and
detection of coliforms in food.The high nutrient quality
and the presence of phosphate buffer in this medium ensures rapid growth and increased gas production, even
by slow lactose-fermenting coliforms.
This medium can be used as Presumptive broth for
Technique
If the volume of sample to inoculate is substantial, then
reconstitute the medium at such a concentration which
would remain normal, once the sample has been added
to it.
Incubate at 37C for 24-48 hours. Lactose fermentation within 48 hours, shown by the appearance of gas in
the Durham tubes , indicates the presence of coliform
bacteria.
Verication can be done by the isolation and identication of coliforms on an appropiate medium.
References
F.D.A. (1998) Bacteriological Analytical Manual. 8th ed.
Revision A. AOAC International Gaitherburg, MD.
FIL-IDF Standard 73B (1998) Milk and milk products.
Enumeration of coliforms. IDF. Brussels.
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th. ed.
APHA. Washington.
MARSHALL R.T. (1992) Standard Methods for the examination of dairy products. 16th ed. APHA. Washington
examination of water and wastewater. APHA. Washington
HORWITZ, W. (2000) Ofcial methods of Analysis.17th
ed. AOAC International. Gaithersburg. MD.
enumeration of coliforms - MPN technique.
ISO 7251 Standard (1993) General guidance for enumeration of E.coli by MPN technique.
Tryptose Lauryl Sulfate

Mannitol Tryptophan Broth
Ref. 02-460
Specication
Liquid medium for the production of indole and gas in
a single tube, according to the ISO 9308-1 and 9308-2
standards.
Formula (in g/L)

Tryptose .................................................. 20,00
Mannitol .................................................... 5,00
Sodium chloride ........................................ 5,00
Sodium Lauryl sulfate ............................... 0,10
L-Tryptophan ............................................ 0,20
Final pH 6,8 0,2
Directions
Dissolve 35,8 g of powder in 1 L of distilled water,
heating up if necessary. Distribute in tubes containing
Durhams tubes and sterilize by autoclaving at 121C for
15 minutes. Do not overheat.
Description
This broth is proposed in the ISO 9308-1 standard as an
alternative medium for the production of indole and gas
in a single tube and to conrm the presence of thermotolerant coliforms and the presumptive presence of E.coli
in the water sample.
163
Tryptose Lauryl Sulfate Media

Technique
Tubes with medium are inoculated from suspicious
colonies on the already incubated membrane and then
are incubated at 44C for 24 hours. Gas production, that
appears in the Durham tubes, conrms the presence of
thermotolerant coliforms.
If after the addition of 0,2-0,3 mL of Kovacs Reagent
(Ref. 06-018) a cherry red colour appears on the top
surface of the medium (Indole +), a presumptive presence of E.coli is considered and it should be conrmed
with other tests.
References
ISO Standard 9308-1 (1990) Water Quality Detection
and Enumeration of coliform organisms, thermotolerant
coliform organisms and presumptive E.coli. Part 1. Membrane ltration method. Part 2. MPN method.
164
Tryptose Sulte Cycloserine Agar Base (TSC Agar Base)

Ref. 01-278
Specication
Solid selective and differential medium for isolation and
presumptive identication of Clostridium perfringens according to ISO 7937 Standard.
Formula (in g/L)

Tryptose .................................................. 15,00
Soya peptone ........................................... 5,00
Yeast extract ............................................. 5,00
Sodium metabisulte ................................ 1,00
Ferric ammonium citrate ........................... 1,00
Agar ....................................................... 20,00
Final pH 7,6 0,2
Directions
soak . Heat to boiling and distribute into suitable containers, but not more than 250 mL in each one. Sterilize in
the autoclave at 121C for 15 minutes. Let it cool to 60C
and add 1 ask of CycloserineSelective Supplement
(Ref. 06-116CASE) to every 250 mL of medium. Mix well
and pour it into plates. If it is desired to include egg yolk,
then add Egg Yolk Sterile Emulsion (Ref. 06-016) in a
concentration of 80 mL/L, simultaneously to the antibiotic,
Description
The medium is a modication of the classical TSN Agar
(Ref. 01-195) in which the traditional antibiotics, polymixin and neomycin have been replaced by cycloserine.
Cycloserine has been found more selective for Clostridium perfringens, and it seems also to reduce the trend to
produce diffuse blackening. On the other hand, Clostridium perfringens is more resistant to cycloserine than to
sulfadiazine, polymixin and neomycin, which permits a
better dosage.The medium has sodium metabisulte and
ferric ammonium citrate to manifest the reducing capacity of sulte, and in this way, three
differential characteristics of this anaerobic species may
be veried with just one assay. These characteristics are
sute reduction, growth at 46C and cycloserine resistance.

Nevertheless, it has to be noted that cycloserine does
not tolerate temperatures above 100C and its stability
in a solution is very restrained, even then it is used in
alkaline media. Therefore, it is advisable to prepare the
exact number of plates that are going to be used.
Anyway, if it is desired, an active solution of cycloserine
in phosphate buffer at pH 8,0 may be prepared (Dipotassium phosphate 16,73 g/L and monopotassium phophate
0,52 g/L) and if it is maintained refrigerated, it can be
used for approx. 5 days.
Technique
The standard procedure recommends surface inoculation of the samples or their dilutions, and once absorbed,
to pour a second layer as cover and seal for anaerobiosis. After an incubation at 46C for 18-20 hours, proceed
to enumerate the black colonies that appear in the plate.
References
SMITH, L.D. (1981) Clostridial Anaerobic Infections, in
Diagnostic Procedures for Bacterial,Mycotic and Parasitic Infections. 6thEd. APHA,Washington.
ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc.,London
DOWNES, F:P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4rd ed.
APHA Washington
animal feeding stuffs- Horizontal method for enumeration
of Clostridium perfringens - Colony-count technique.
DIN Standard 10165. Referenz Verfahren fur Bestimmung von Clostridium perfringens. Fleisch und
Fleischerzeugnissen.
Revision A. AOAC International. Gaithersburg. MD.
ISO 6461-2:1986 Standard Water Quality - Detection
and enumeration of the spores of sulte - reducing
anaerobes (Clostridia) - Part 2: Method by MF.
Left and center: Clostridium perfringens

ATCC 13124; right: control.
165
Urea Media
Urea Agar Base
acc. to Christensen
Ref. 01-261
Specication
Solid medium for detection of urea lysis, according to
ISO 6579, 6340 and 6785 standards and DIN 10160
standard.
Formula (in g/L)

Salmonella spp.
ISO 6579 Standard (2002) Microbiology of food and animal feeding stuffs. Horizontal method for the detection of
Salmonella spp.
DIN Standard 10160. Untersuchung von Fleisch und
Fleischerzeugnissen. Nachweiss von Salmonellen. Referenzverfahren.
FIL-IDF 93 Standard (2001) Detection of Salmonella.
Urea Broth Base
Gelatin peptone ...................................... 1,000

Dextrose ................................................. 1,000
Sodium chloride ...................................... 5,000
Phenol red .............................................. 0,012
Agar ...................................................... 15,000
Final pH 7,0 0,2
Ref. 02-202
Directions

Yeast extract ............................................. 0,10
Phenol red ................................................ 0,01
Final pH 6,8 0,2
Suspend 24 g of powder in 950 mL of distilled water and

bring to the boil. Sterilize in the autoclave at 121C for
15 minutes. Let it cool to 50-55C. Add 50 mL of Urea
Sterile Solution 40% (Ref. 06-083) and mix well. Distribute aseptically in tubes and let them solidify in slanted
position.
Description
Urea Agar complies with Christensens specications,
and it is recommended for the detection of urolytic or
urea degrading microorganisms, especially Enterobacteriaceae, although it can be used also with gram positive
bacteria.
Technique
Pure culture is inoculated by surface streaking, and then
incubated at 37C. Generally, organisms with strong
urease activity can be read after 3-5 hours.
Reaction is evident as the medium changes its colour. It
turns form orange to pink-fuchsia, due to a strong alkalinization produced by ammonia release.
References
CHRISTENSEN, W.B. (1946) Urea decomposition as
means of differentiating Proteus and Paracolon cultures
from each other and from Salmonella and Shigella types.
J.Bact. 52:461.
EDWARDS and EWING (1962) Identication of Enterobacteriaceae. Burgess Pub. Co.
ATLAS, R.M., & L.C. PARK (1993) Handbook of Microbiological Media. CRC Press Inc.London
DOWNES, F.P. & K. ITO (2001) Compendium of methodscfor the Microbiological Examination of Foods 4th ed.
APHA. Washington
MARSHALL, R.T. (1992) Standard Methods for the examination of Dairy Products. 16th ed APHA. Washington
DC
166
Specication
Liquid diagnostic medium according to Rustigian and
Stuart formulation.
Formula (in g/L)
Directions
Dissolve 19 g of powder into 950 mL of distilled water
and sterilize by autoclaving at 121C for 15 minutes. Let
it cool to 50-55C and then add 50 mL of Urea Sterile
Solution 40% (Ref. 06-083). Mix well and dispense in
hemolysis tubes (3,0 mL/tube).
Description
According to Rustigian and Stuart, this Urea Broth is
excellent for diagnosing enterobacteria, since within this
family, only Proteus may alkalinize the medium over pH
8,1. Despite the fact that some authors prefer a buffer of
potency 10 or 100 times lower to obtain faster results for
saving the time (about 2 hours) does not compensate for
the instability of the medium.
Urease production is shown by the indicator turning to
dark pink, produced by strong alkalinization by ammonium. With plenty of inoculum (2-3 loops in 3-5 mL of
medium), Proteus produces the colour change after 6-8
hours, meanwhile other positive enterobacteria need up
to 24-48 hours.
References
RUSTIGIAN, R., STUART, C.A. (1941) Decomposition of
urea by Proteus. Proc. Soc. Exp. Biol. Med. 47:108
DOWNES, FP & K ITO (2001) Compendium of Methods for the Microbiological Examination of Food, 4rd ed.
APHA,Washington.
Violet Red Bile Media

Violet Red Bile Agar (VRB Agar)
Ref. 01-164
Specication
Selective and differential agar medium for the detection
and enumeration of coliforms in milk and other dairy
products, according to APHA, ICMSF, FIL-IDF and ISO
standard.
Formula (in g/L)

Yeast extract ........................................... 3,000
Gelatin peptone ...................................... 7,000
Bile salts #3 ............................................ 1,500
Lactose ................................................. 10,000
Sodium chloride ...................................... 5,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,002
Agar ...................................................... 13,000
Final pH 7,4 0,2
Directions
Suspend 39,5 g in 1 L of distilled water. Bring to the
boil and distribute into nal containers. Sterilization at
121C for 15 minutes is optional, but If the medium is
to be used on the same day of preparation it need not
be sterilized. No sterilization improves the recovery of
stressed microorganisms.
Description
The Violet Red Bile Agar corresponds to the classic
formulation of standardized media for the screening of
coliforms in milk and other dairy products.This medium
has been adopted for the enumeration of coliforms as
well as for differentiating between lactose fermenting and
non-lactose fermenting organisms, due to its contents of
crystal violet and bile salts, whose inhibiting or selecting
properties have been widely conrmed.
This medium can be used as Presumptive medium for
Technique
The recommended procedure is the massive inoculation directly on Petri dishes, with the molten agar cooled
to 47C. Observations can be read after 24 hours of
incubation at 37C.
The size of the colonies ranges from 2 to 5 mm, depending on the amount per plate. The enterococci that might
eventually develop will appear small in size and pink
coloured. Lactose fermenting enterobacteria acquire a
dark red colour with a clearing zone around them, while
lactose non-fermenting ones form colourless colonies.
References
DOWNES, F.P. & K. ITO (2001). Compendium of Methods for the Microbiological Examination of Food. 4rd ed.
APHA, Washington. DC
MARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products,16thEd. APHA, Washington.
DC
ICMSF (1978). Microorganisms in Food, University of
Toronto Press.
ISO (1986) Standard 5541-1 Milk and Milk Products.
enumeration of coliforms. Colony count technique at
30C
FIL-IDF. (1998) Standard 73B. Enumeration of coliform
bacteria.
Violet Red Bile Dextrose Agar

(VRBD Agar)
Ref. 01-295
Specication
Solid medium for the enumeration of enterobacteria according ISO 8523 standard.
Formula (in g/L)

Yeast extract ........................................... 3,000
Gelatin peptone ...................................... 7,000
Bile salts #3 ............................................ 1,500
D (+) Glucose ....................................... 10,000
Sodium chloride ...................................... 5,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,002
Agar ...................................................... 13,000
Final pH 7,4 0,2
Directions
Suspend 39,5 g in 1 L of distilled water and let it soak.
Bring to the boil and sterilize by autoclaving at 121C for
15 minutes. If the medium is to be used on the same day
of preparation it need not be sterilized. Prolonged heating in thermostatic bath could cause slight precipitates.
Description
This medium is a modication of the Violet Red Bile Agar
(Ref.1-164) and the MacConkey Agar (Ref.1-118) as
described by Mossel et al. These authors proved that the
addition of glucose to the Violet Red Bile Agar favoured
both the growth of the most fastidious enterobacteria
and the recovery of those having suffered from adverse
conditions. Later on, Mossel himself realized that by
removing the lactose and keeping the glucose, the
mediums efciency remained stable. Furthermore, an
economic improvement occurred since the same amount
of product allows the reconstitution of more litres of the
medium.
167
Violet Red Bile Media

This medium can be used as presumptive medium for
Technique
The Violet Red Bile Dextrose Agar is widely used in the
analysis of food, medicines and cosmetics. It is particularly indicated for the recovery of bacteria which have
been damaged during preparation. In such cases, a
progressive enrichment is recommended in TSB (Ref.
02-200) rst and in EE Broth (Ref. 02-064) next. Once
the enriched culture is ready it can be inoculated by
profound inoculation in tubes or by isolation in Violet Red
Bile Dextrose Agar plates.
For the count of enterobacteria, the technique to use will
be the massive inoculum described for the Violet Red
Bile Agar.
Observations can be read after 24 hours of incubation
at 31C. Enterobacterial colonies form an intense purple
colouring surrounded by a clearer zone . If enterococci
colonies eventually develop, then they will be small and
pink coloured.
Violet Red Bile Lactose

Dextrose Agar (VRBLD Agar)
(Eur. Phar. Medium F)
Ref. 01-220
Specication
Solid selective medium for the detection of Enterobacteriaceae according the European Pharmacopoeia.
Frmula (in g/L)

Yeast extract ........................................... 3,000
Peptone .................................................. 7,000
Sodium chloride ...................................... 5,000
Bile salts # 3 ........................................... 1,500
Lactose monohydrate ........................... 10,000
Glucose monohydrate .......................... 10,000
Neutral red .............................................. 0,030
Crystal violet ........................................... 0,002
Agar ...................................................... 15,000
Final pH 7,4 0,2
Directions
Suspend 51.5 g of powder in 1 L of distilled water and
heat to the boil. Pour into Petri dishes inmediately. Do
not sterilize in autoclave nor overheat.
Description
This medium developed in 1962 by Mossel et al. as
more effective than MacConkey Agar for the detection of
Enterobacteriaceae in foods, has been ofcially adopted
by the European Pharmacopoeia for the microbiological
examination of non-sterile products. The medium is spe-
168
cially used in the recovery of process-stressed bacteria

using a progressive enrichment technique.
This medium can be used as presumptive medium for
Technique
Sample is diluted 1:10 in Lactose Broth (Ref. 02-105)
and incubate 2-5 hours at 35-37 C. Then a volume of
this pre-enrichment is ten fold dilute in EE Broth (Ref.
02-064) and incubate at 35-37C for 18-24 hours. From
this enrichment the surface of several plates of VRBDL
Agar are inoculated. The product passes the test if after
18-24 hours of incubation at 35-37C there is no growth
of gram negative bacteria in any plate.
In the surface of the VRBDL Agar the Enterobacteriaceae colonies are deep purple in colour surrounded by a
clearing zone. Sometimes are present little colonies from
Pseudmonas or Aeromonas that can be easy differentiated by the oxidase test.
References
MOSSEL, D.A.A., MENGERINK and SCHOLTS H.H.
(1962) Use of a modied MacConkey Agar medium for
the selective growth and enumeration of all Enterobacteriaceae. J. Bact. 84:381.
MOSSEL, D.A.A., VISER, M. and CORNELISSEN,
A.M.R. (1963) The examination of food for Enterobacteriaceae using a test of the type generally adopted for the
detection of Salmonellae. J. Appl. Bact.(26) 444-452.
MOSSEL, D.A.A. (1985) Media for Enterobacteriaceae.
Int. J. Food Microbiol. 2:27-35
ISO 5552 Standard (1997) Meat and Meat Products.
Detection and enumeration of Enterobacteriaceae
without resuscitation. MNP technique and colony-count
technique.
MOSSEL, D.A.A. and M.A. RATTO (1970) Rapid detection of sub-lethally impaired cells of Enterobacteriaceae
in dried foods Appl. Microbio 20: 273-275.
EUROPEAN PHARMACOPOEIA 3 Edicin (Suppl.
1999) Cap. 2.6.13 Microbiological examination of non
sterile products. Tests for specied organisms. Council
of Europe. Strasbourg
Ref. 01-164 Violet Red Bile Agar
ATCC 14028
control
Vogel Johnson Agar (VJ Agar)

Ref. 01-206
Specication
Solid and very selective medium for isolation and identication of staphylococci according ISO 22718 standard.
Formula (in g/L)

Casein Peptone .................................... 10,000
Yeast Extract .......................................... 5,000
Mannitol ................................................ 10,000
Litium chloride ........................................ 5,000
Glycine ................................................ 10,000
Phenol Red ............................................. 0,025
Agar ...................................................... 15,000
Final pH 7,2 0,2
Directions
bring to the boil. Dispense in suitable containers and
sterilize at 121C for 15 minutes. Cool it to 50C approx.
and add aseptically 20 mL of Potassium Tellurite Solution 1% (Ref. 06-089) or 6,0 mL of Potassium Tellurite
Solution 3.5% (Ref. 06-011). Do not reheat after tellurite
addition.
Description
VJ Agar is a selective medium for detection and enumeration of pathogenic staphylococci.
The mediums strong selective action is due to lithium
chloride, glycine and potassium tellurite presence. They
inhibit almost all the accompanying organisms, meanwhile staphylococci are not affected. Although staphy-
lococci may reduce tellurite to tellurium, lithium may perform some action that is compensated by glycine.
Moreover a high correlation between tellurite reduction
and mannitol fermentation has been proved, and this is
shown in the medium by the indicator turning to yellow
due to the amount of acid produced.
The mediums selectivity avoids, in the rst 24 hours, the
development of any other bacteria, so massive inoculation is permited. Nonetheless, after this period, it is
possible that other bacteria may appear like micrococci,
which produce tiny colonies, and staphylococci that
ferment mannitol and coagulase negative, therefore it is
recommended to verify this last test separately.
Due to reduced tellurite, staphylococci generally appear
as black colonies over red medium (if they do not ferment mannitol) or yellow medium (if they do, and these
are presumptive pathogen). Saprophytic staphylococci
(S.epidermidis, S.saprophiticus and S.intermedius) have
a grey-black colour and are mannitol negative. Complete
medium may be stored up to 1 week in the refrigerator.
Do not remelt it after tellurite is added.
References
VOGEL and JOHNSON (1960) A modication of the
tellurite-glycine medium for the use in the identication of
Staphylococcus aureus. Pub. Health. Lab. 18:131-133.
US PARMACOPOEIA (2002) 25th ed. <61> Microbial Limit
Tests. Pharmacopoeial Convention. Rockville. MD
ATLAS, R.M.,& L.C. PARK (1993) Handbook of Microbiological Media. CRC Press Inc.,London
169
Wilkins-Chalgren CN Modied Fluid Medium

(WCCN Modied Fluid Medium)
Ref. 03-408
Specication
Fluid medium for the cultivation and enumeration of
anaerobic bacteria by the impedometry technique.
Formula (in g/L)

Tryptone ............................................. 10,0000
Gelatin peptone .................................. 10,0000
Gelatin .................................................. 8,0000
Dextrose ............................................. 41,0000
Yeast extract ......................................... 5,0000
Sodium chloride .................................... 5,0000
Ammonium sulfate ................................ 5,0000
L-Arginine ............................................. 1,0000
Sodium pyruvate .................................. 1,0000
Sodium bicarbonate ............................. 1,0000
Ferrous sulfate ..................................... 0,1000
Calcium chloride ................................... 0,1000
Hemine ................................................. 0,0050
Menadione ............................................ 0,0005
Agar ...................................................... 0,3000
Final pH 7,1 0,2
Directions
Suspend 87,5 g of powder in 1 L of distilled water. Bring
to the boil. Sterilize in the autoclave at 121C for 10
minutes.
Prepared medium takes on a dark colour due to the high
concentration of sugar. Do not reheat.
Description
This medium is a modication of the Wilkins-Chalgren
Anaerobic Medium for impedometric methods.
References
WILKINS, T.D. and S. CHALGREN (1976) Medium for
use in antibiotic susceptibility testing of anaerobic bacteria. Antimicrob. Agents Chemorther 10:6:926.
WL Nutrient Media
WL Nutrient Agar
Ref. 01-210
Specication
Solid medium for the cultivation and enumeration of
yeast and bacteria for microbiological control in brewing
and other fermentation industries.
Formula (in g/L)

Yeast extract ......................................... 4,0000
Tryptone ............................................... 5,0000
Dextrose ............................................. 50,0000
Magnesium sulfate ............................... 0,1250
Potassium chloride ............................... 0,4250
Iron (III) chloride ................................... 0,0025
Manganese sulfate ............................... 0,0025
Bromcresol green ................................. 0,0220
Agar .................................................... 20,0000
Final pH 5,5 0,2
170
Directions
Suspend 80 g of the powder in 1 L of distilled water.
Mix thoroughly. Heat with frequent agitation and boil for
one minute. If a nal pH of 6,5 is desired, the pH may be
adjusted with one percent aqueous sodium carbonate,
using about 30 mL per litre of medium.
Dispense and sterilize the medium by autoclaving at
The WL Differential Agar has the same formula as the
WL Nutrient Agar with the addition of 2 asks/L of Cycloheximide Selective Supplement Ref. 06-022CASE.
Description
WL Nutrient Agar was formulated by Green and Gray in
the Wallerstein Laboratory for use in the control of industrial fermentations, particularly the processing of beer. It
is recommended for examination of worts, beers, liquids
containing yeast and other materials.
WL Nutrient Agar has a pH of 5,5 which is optimal for
the enumeration of brewersyeast . If bakers or distillers
yeast is to be examined, the pH should be adjusted to
6,5 (better yields). When cultivating the microorganisms
from an alcoholic mash, tomato juice should be added to
the medium.
WL Nutrient Media
WL Differential Agar contains cycloheximide to suppress
yeast and any other moulds which may be present; this
medium allows reliable counting of all bacteria which
may be encountered in the tests performed in brewery
laboratories.
Technique
Dilute the sample material and spread 0,1 mL onto WL
Nutrient Agar or WL Differential Agar.
The WL Nutrient Agar and WL Differential agar are used
together, one plate with nutrient agar and two with the
differential agar.
The WL Nutrient Agar plate is incubated aerobically to
obtain a total count, mainly of yeast colonies. One WL
Differential Agar plate is incubated aerobically for growth
of acetic acid bacteria, Flavobacterium, Proteus, and
other organisms; the second plate is incubated anaerobically for detection of such organisms as lactic acid bacilli
and Pediococcus species.
Plates prepared with both the media are generally incubated at 25C, if brewing materials are being studied,
and at 30C for bakers yeast and alcohol mash samples. Incubation may be continued for a week, or even
for ten days to two weeks, depending upon the ora
present. Counts are made at the intervals during the
incubation period.
Description
WL Medium was developed in the Wallerstein Laboratories for industrial uses, since it allows to differentiate
between beer yeast and wild yeasts contaminants.
Adjusting pH to 6,5 is very advisable to enumerate bread
and alcohol yeast. The medium also allows bacterial
growth and it is possible then to count the contaminant
bacteria of fermented liquors, but it is recommended to
use WL Differential Broth for this assay, since it inhibits
yeast growth.
The WL Nutrient Broth is useful to enumerate cells by
the MPN technique. Alternatively, it can be used as an
enrichment broth previous to the colony plate count.
Technique
Liquid medium for the microbial control of industrial fermentations and massive cultivation of yeast.
Usual the technique is to inoculate one plate of WL Nutrient Broth and two plates of WL Differential. Incubate all
the plates at 25C for 5-15 days. It is advisable to incubate one of the plates with differential agar anaerobically
to enhance development of contaminants that produce
lactic acid. Green and Gray stated that to perform the
observations of viable yeast in bread, WL Nutrient broth
at pH 5,5 may be used, but to do it in distilleries, the pH
has to be adjusted to 6,5.
Analogously, time and temperature of incubation vary
depending on material to be analysed. Beer samples
are incubated at 35C, but bread, and alcohol fermentation ones are incubated at 30C. Incubation time varies
between 2 and 7 days, and in some cases, depending on the ora found, it may be up to 14 days. When
Differential type (with cycloheximide) is employed for a
bacterial count, it should be incubated anaerobically to
detect cocci in beer and lactic bacilli, and it should be
incubated aerobically to detect acetic acid bacteria and
thermobacteria.
Formula (in g/L)
References
WL Nutrient Broth
Ref. 02-210
Specication
Yeast extract ......................................... 4,0000

Tryptone ............................................... 5,0000
Dextrose ............................................. 50,0000
Magnesium sulfate ............................... 0,1250
Potassium chloride ............................... 0,4250
Ferric chloride ....................................... 0,0025
Manganous sulfate ............................... 0,0025
Bromcresol green ................................. 0,0220
Final pH 5,5 0,2
Directions
Dissolve 60 g of powder in 1 L of distilled water and dispense into suitable containers. Sterilize by autoclaving at
121C for 15 minutes. Should a pH 6,5 be desired, adjust it by adding 30 mL of Sodium carbonate solution 1%.
To obtain 1 L of WL Differential Broth just add, aseptically, 2 asks of Cycloheximide Selective Supplement
(Ref. 06-022CASE) to 1 L of WL Nutrient Broth, after
sterilization.
GREEN, S.R. & GRAY, P.P. (1950) A differential procedure applicable to bacteriological investigation in brewing. Wallerstein Lab. Comm. 13:357
GREEN, S.R. & GRAY, P.P. (1950) Paper read at
Am.Soc. of Brewing Chemists Meeting; Wallerstein Lab.
Comm. 12:43
GREEN, S.R. & GRAY, P.P. (1951) A differential procedure for bacteriological studies useful in the fermentation
industries. Wallerstein Lab. Comm. 14:289
GRAY, P.P. (1951) Some advances in microbiological
control for beer quality. Wallerstein Lab. Comm. 14:169
ATLAS, R.M., & L.C. PARK (1993) Handbook of Microbiological Media for the examination of Food. CRC Press
Inc. Boca Ratn,Fla.
MASTERS BREWERS ASSOCIATION OF THE AMERICAS (2002) The Practical Brewer 3rd ed. St. Paul. Minnesota
171
Wort Media
Wort Agar
Ref. 01-132
Specication
Solid medium for the cultivation, isolation and enumeration or enrichment of fungi, especially of yeast.
Formula (in g/L)

Malt extract ............................................... 15,0
Casein Peptone .......................................... 1,0
Maltose ..................................................... 12,5
Dextrine ...................................................... 2,5
Ammonium chloride .................................... 1,0
Agar .......................................................... 17,0
Final pH 4,8 0,2
Directions
add 2-3 mL of glycerol and bring to the boil to dissolve
completely. Distribute into nal containers and sterilize
by autoclaving at 121C for 15 minutes. Do not overheat. Prolonged heating will diminish the gelling strength
of the medium.
Description
Wort Agar is used for the cultivation, isolation and enumeration of yeast and moulds.
It is particulary well adapted for counting osmophilic
yeast in butter, sugar and syrups, in lemonade and more
generally in sweet or soft drinks.
For a more selective utilization it is possible to adjust the
pH to 4,5 or 3,5. Never heat the medium after adding
acid, in order to prevent the loss of solidifying properties
of the agar. The acid pH inhibits the growth of bacteria
and favours that of yeast.
Wort Broth
Ref. 02-132
Specication
This medium is the liquid version of the classical Wort
Agar (Ref. 01-132).
Formula (in g/L)

Malt extract ............................................... 15,0
Casein Peptone .......................................... 1,0
Maltose ..................................................... 12,5
Dextrine ...................................................... 2,5
Ammonium chloride .................................... 1,0
Final pH 4,8 0,2
172
Directions
add 2-3 mL of glycerol and bring to the boil to dissolve
completely. Distribute into nal containers and sterilize
Description
It is especially designed to propagate the multiplication
of yeast, and often it has been employed as a semiselective or enrichment medium, due to its high acidity,
which makes it inhibitory for bacteria. This effect may
be more enhanced by adding, before sterilization, 10
mL/L of a 10% solution of lactic or tartaric acid. To avoid
precipitate it is recommended to sterilize by ltration.
References
SCARR, M.P. (1959) Selective media used in the microbiological examination of sugar products. J. Sci. Food
Agric. 10:678-681
RAPP, M (1974) Indikator-zustze zur Keimdifferenzierung auf Wrze und Malzextrakt-Agar. Milchwiss
29:341-344
ATLAS, R.M.,& L.C. PARKS (1993) Handbook of Microbiological Mediafor the examination of Food. CRC Press
Inc.London
MASTERS BREWERS ASSOCIATION OF THE AMERICAS (2002) The Practical Brewer. 3rd ed. St. Paul. Minnesota
Xylose Lysine Deoxycholate Media

Xylose Lysine Deoxycholate
Agar (XLD Agar) (Eur. Phar.
Agar Medium K)
Ref. 01-211
Specication
Solid medium for the isolation of enteropathogenic
species, especially Salmonella according to ISO 6340
standard.
Formula (in g/L)

Xylose ....................................................... 3,50
L-Lysine .................................................... 5,00
Lactose ..................................................... 7,50
Sucrose .................................................... 7,50
Sodium chloride ........................................ 5,00
Yeast extract ............................................. 3,00
Phenol red ................................................ 0,08
Sodium Deoxycholate .............................. 2,50
Ammonium ferric citrate............................ 0,80
Agar ........................................................ 15,00
Final pH 7,4 0,2
Directions
Suspend 56,68 g of powder in 1 L of distilled water. Heat
up constantly with stirring until boiling. Pour it immediately into plates. Do not autoclave and avoid remelting.
Description
Xylose Lysine Deoxycholate Agar is a differential medium, slightly selective, very suitable for the detection
of pathogenic enterobacteria, especially Shigella. Gram
negative ora is inhibited by the low amount of deoxycholate, but Shigella grows easier in this medium than in
any other selective media.
Xylose, lactose or sucrose fermentation produce the
acidication of the medium, and this is seen by an
indicator turning to yellow, surrounding the colonies. This
colour disappears after 24 hours, so observations must
be carried out between 18 and 20 hours.
Hydrogen sulde production from thiosulfate is easily detected because colonies become darker, due to the ferric
sulfure precipitate. Lysine decarboxylation to cadaverine
may also be observed in the medium, since it produces
alkalinization and consequently the indicator turns to red.
All these reactions allow a good differentiation of Shigella, which besides Edwardsiella and Proteus inconstans
are the single enterobacteria that do not ferment xylose
and therefore show negative fermentation reaction.
Salmonella type members do ferment xylose, but it is
consumed quickly and then alkalinization of the medium,
due to lysine decarboxylation, may mask the reaction.
The difference between Shigella and Salmonella is that
with the latter colonies become darker due to ferrous
sulfure precipitates, and this is a common property with
Edwardsiella. The other types of enterobacteria do not
suffer this phenomenon, since acid acumulation due to
lactose and sucrose fermentation is so high that it avoids
pH reversion by decarboxylation and even ferrous sulfure precipitate in the rst 24 hours.
In the table below, typical colonial appearances on XLD
medium after 24-36 hours of incubation at 37C are
described.
References
TAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose
Lysine Agars: New media for isolation of enteric pathogens. Am. J.Clin. Path 44:471-475
DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food,4th ed.
APHA,Washington.
ICMSF (1978) Microorganisms in Food 1. Univ. Toronto
Press.
Revision A. AOAC International Gaithersburg. MD.
ATLAS, R.M.,& L.C. PARK (1993) Handbook of Microbiological Mediafor the examination of Food. CRC Press
Inc. London
Alimentaria. Diaz de Santos, S.A. Madrid, .
EUROPEAN PHARMACOPOEIA, (2002) 2.6.13 Test
for specied micro-organisms 4th Ed.,Suppl. 4.2 EDQM
Council of Europe, Strasbourg,
US PHARMACOPOEIA (2002) <61> Microbial Limit
Tests. 25th Ed. US Pharmacopoeial Convention. Rocville.
MD.
Salmonella spp.
Xylose Lysine Deoxycholate

Modied Agar (XLD Modied
Agar)
Ref. 01-552
Specication
Medium for isolation of enteropathogenic species, especially Salmonella in food and animal feeding stuffs, acc.
ISO Standard 6579:2002
Formula (in g/L)

Xylose ...................................................... 3,75
L-Lysine ................................................... 5,00
Lactose .................................................... 7,50
Sucrose ................................................... 7,50
Sodium chloride ....................................... 5,00
Yeast extract ............................................ 3,00
Phenol red ............................................... 0,08
Sodium Deoxycholate .............................. 1,00
Sodium thiosulfate ................................... 6,80
Ammonium iron(III) citrate ....................... 0,80
Agar ....................................................... 15,00
Final pH 7,4 0,2
173
Xylose Lysine Deoxycholate Media

Directions
precipitates, and this is a common character with Edwarsiella. The other types of enterobacteria do not suffer this
phenomenon, since acid accumulation due to lactose
and sucrose fermentation is so big that it avoids pH
reversion by decarboxylation and even ferrous sulde
precipitate in the rst 24 hours.
Suspend 55,43 g of powder in 1 L of distilled water. Heat

up constantly stirring until boiling. Pour it immediately
into plates. Do not sterilize and avoid remelting.
Description
Xylose Lysine Deoxycholate Agar is a differential medium, slightly selective, very suitable for the detection
of pathogen enterobacteria in food, especially Shigella,
with a modication in the original formulation of Taylor to perform the specications of the ISO standard
6579:2002
Gram negative ora is inhibited by the low amount of
deoxycholate, but Shigella grows easier than in other
selective media.
Xylose, lactose or sucrose fermentation produce medium
acidication, and this is shown by an indicator turning to
yellow, surrounding the colonies. This colour disappears
after 24 hours, so readings must be carried out between
18 and 20 hours.
Sulfhydric production from thiosulfate is easily detected
because colonies become darker, due to the ferric
sulde precipitate. Lysine decarboxylation to cadaverine
may also be observed in the medium, since it produces
alkalinization and consequently the indicator turns to red.
All these reactions allow a good differentiation of Shigella, which besides Edwarsiella and Proteus inconstans
are the single enterobacteria that do not ferment xylose
and therefore show negative fermentation reaction.
Salmonella-type members do ferment xylose, but it is
consumed quickly and the medium alkalinization, due
to lysine decarboxylation, may hide the reaction. The
difference between Shigella and Salmonella is that with
the latter colonies become darker due to ferrous sulde
In the table below, typical colonial appearances on XLD

Agar after 24-36 hours of incubation at 37C are describe.
References
TAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose
Lysine Agars: New media for isolation of enteric pathogens. Am. J. Clin. Path 44:471-475
ICMSF, ( 1978) Microorganisms in Food 1. University of
Toronto Press.
FDA (1990) Bacteriological Analytical Manual AOAC
International Arlington. VA. USA.
ATLAS, R.M., L.C. PARK (1993) Handbook of Microbiological Media for the examination of Food. CRC Press
Inc. Boca Ratn.
PASCUAL ANDERSON, MOR. (1992) Microbiologa
Alimentaria. Diaz de Santos, S.A. Madrid.
ISO Standard 6579 (2002) Microbiology of foods and
animal feeding stuffs. Horizontal method for the detection of Salmonella spp.
Colony appearance
Microorganism
Red colonies, transparent
Shigella sp., Proteus incontans, Salmonella paratyphi A.,

sometimes S.cholerasuis and S. Pullorum
Edwardsiella and most of biotypes of Salmonella
Salmonella typhi
Pseudomonas, Proteus rettgeri.
Escherichia when it grows, Enterobacter, Aeromonas, Citrobacter.
Klebsiella, Citrobacter intermedius when it grows
Most of Proteus mirabilis, P.vulgaris.
Serratia, Hafnia.
Red colonies, transparent with black core.

Orange and slightly opaque colonies
Red and translucent colonies, without halo.
Yellow and opaque
Yellow, opaque, mucose and with black core.
Yellow, transparent and with black core.
Yellow, opaque and without halo
Ref. 01-552
XLD Modied Agar
control
174
Yeast Extract Media

Yeast Extract Agar
Ref. 01-465
Specication
Solid medium for the enumeration of microorganisms
from water.
Formula (in g/L)

Tryptone .................................................... 5,0
Yeast extract ............................................... 3,0
Agar .......................................................... 15,0
Final pH 7,2 0,2
Directions
Description
This medium, formulated according to Windle Taylor, is
the most used in the UK for the enumeration of heterotrophic microorganisms from water. Distinction between
bacteria, yeast and lamentous fungi must be carried out
by morphology after differential incubations at 35 and
20C.
Technique
From the water sample, make a decimal dilution bank
with Ringer Solution (Ref. 06-073) and take aliquotes to
2 parallel series of plates. Pour the Yeast Extract Agar,
molten and cooled to 45C, and homogenize with sample. Once solidied, incubate one of the series at 35C
for 24 hours and the other one at 20C for 3 days.
In order to achieve a good count, select the plates with
30-300 colonies.
References
WINDLE TAYLOR, E. (1958) The examination of water
and water supplies. 7th Ed. Churchill Ltd. London.
ATLAS, R.M., & L.C. PARKS (1993) Handbook of microbiological media, CRC Press, London
Yeast Extract Peptone Dextrose

Agar (YPD Agar)
Ref. 01-473
Specication
Solid medium for the cultivation of yeast in molecular
biology procedures.
Formula (in g/L)

Peptone .................................................... 20,0
Yeast extract ............................................. 10,0
Dextrose ................................................... 20,0
Agar .......................................................... 20,0
Final pH 6,8 0,2
Directions
Yeast Extract Peptone Dextrose

Broth (YPD Broth)
Ref. 02-473
Specication
Liquid medium for the cultivation of yeast in molecular
biology procedures.
Formula (in g/L)

Peptone .................................................... 20,0
Yeast extract ............................................. 10,0
Dextrose ................................................... 20,0
Final pH 6,8 0,2
Directions
up if necessary. Distribute into suitable containers and
Description
These media support the growth of most heterotrophic
microorganisms, but due to their simple composition
they have been adopted as the basal media for the routine cultivation of yeasts for molecular biology studies.
References
SHERMAN, F. (1991) Studies on the phenotype switching with Candida albicans. Meth. Enzimol 194:3-17.
MARTINEZ, J.P., M.L. GIL, M. CASANOVA, J.L. LOPEZRIBOT, J. GARCIA DE LOMAS, R. SENTANDREU
(1990)
Wall mannoproteins in the cells from colonial phenotypic
variants. J. Gen. Microbiol. 136:2421-2432.
ATLAS, R.M., L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. London
AUSUBEL, F.M., R.BRENT,R.E. KINGSTON, D.D.
MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL
(1994) Current Protocols in Molecular Biology. Current
Protocols. Brooklyn. N.Y.
175
Yeast Malt Media

Yeast Malt Agar (YM Agar)
Yeast Malt Broth (YM Broth)
Ref. 01-219
Ref. 02-219
Specication
Specication
Solid medium for the cultivation of fungi and actinomycete.
Liquid medium for the cultivation of fungi and actinomycetes.
Formula (in g/L)
Formula (in g/L)
Dextrose ................................................... 10,0

Peptone ...................................................... 5,0
Malt extract ................................................. 3,0
Yeast extract ............................................... 3,0
Agar .......................................................... 20,0
Final pH 6,2 0,2
Dextrose ................................................... 10,0

Peptone ...................................................... 5,0
Malt extract ................................................. 3,0
Yeast extract ............................................... 3,0
Final pH 6,2 0,2
Directions
let it soak. Bring to the boil and distribute into suitable
minutes.
Directions
Description
This is a classical culture medium for the cultivation of
moulds, yeasts and acidophilic actinomycetes. Medium
may become selective to one or other group of microorganisms by adding antibiotics when the medium is at
50C.
References
ATLAS, R.M.,& L.C. PARK (1993) Handbook of Microbiological Mediafor the examination of Food,CRC Press
Inc.London.
SAMSOM, R.A., E.S. HOEKSTRA, J.C. FRISVAD, O.
FILTENBORG (2002) Introduction to food- and airborne
fungi. 6th. Ed. CBS. Utrech.
176

Denitions
Production Process
EXTRACTS are concentrated preparations of liquid,

solid or intermediate consistency, usually obtained from
dried vegetable or animal matter. For some preparations,
the matter to be extracted may undergo a preliminary
treatment, for example, inactivation of enzymes, grinding or defatting. Extracts are prepared by maceration,
percolation or other suitable, validated methods, using
water, ethanol or another suitable solvent. After extraction unwanted matter is removed if necessary.
The general production process is eschematized in the

following ow-chart.
PEPTONES are complex water-soluble mixes of free

amino acids, peptides, sugars, mineral salts and other
components obtained by acid, alkaline or enzymatic
hydrolysis of protein substrates. Their very variable characteristics depend on:
- The nature of the substrate(s).
- The nature of the hydrolysis: enzyme(s), alkali(s),
acid(s), term of hydrolysis.
- The technique of purication (ltration, ultraltration,
).
- The other operating conditions used in the production
process.
The term peptone is more commonly applied to the
hydrolisates obtained by enzymatic digestion. The enzymes more frequently used are:
Papain that acts on the links adjacent to arginine, lysine,
phenylalaline and glycine. Bromelain and Ficin are
also used because are similar to papain but from
other plants and with pH-range and temperaturerange different.
Pepsin, that acts on the links adjacent to phenylalanine
or leucine.
Pancreatin (a variable mixture of trypsin and chimotrypsin) that acts on the links adjacent to arginine,
lysine, tyrosine, tryptophane, phenylalanine and
leucine.
Microbial Proteases are obtained from microbial
cultures as extracellular enzymes and used for the
peptones production. Acid and Neutral microbial proteases works in similar way as papain and alkaline
microbial proteases as pancreatin.
The papain is obtained from plant material and pepsin
and pancreatin, that are animal enzymes, are of swine
origin.
179

Warranty of health and origin
All the animal tissue raw materials used in the elaboration of the Scharlau Microbiology Peptones come from
approved slaughterhouses and are covered by certicates obtained from the veterinary authorities.
The country of origin of bovine animal tissues and casein
used in the manufacture of each batch of peptone is
specied in the health certicate.
These documents certify that animals from which tissues
have been taken were in good health and t for human
consumption. They are used by Scharlau Microbiology
Quality Control Department to edit a health certicate for
each batch of product manufactured: a copy of this certificate is submitted to our customers upon each delivery.
Analytical data and control

methods
All data guring in the following documentation result
from the analysis of a signicant number of batches of
every product. These data may be:
Typical data, which are in fact average values (comparative tables and technical data sheets).
Norms of acceptability (Technical data sheets).
Bacteriological controls as well as most general physicochemical controls are carried out each batch systematically. The other characters are veried according to
routine.
For the control methodology, a norm is dened specically for each character. When a pharmacopoeial
monograph (Eur. Phar. 4th ed. 2002; USP 25th ed 2002)
is available, it is adopted as routine method. If it is not
possible an other documented methodology is accepted.
The typical data are show in the following Tables 1 to
6 and in the specic description of any product. The
information contained in this publication is based in own
research and development work and is the best of our
knowledge true and accurate. Users should however
conduct their own tests to determine the suitability of our
products for their own purpouses. Statements contained
herein should not be considered as a warranty of any
kind, expressed or implied, and no liability is accepted
for the infringement of any patents.
References
ATLAS, R.M. & L.C. PARKS ( 1993) Handbook of Microbiological Media CRC Press London.
BRYDSON, E.Y. (1978) Natural and Synthetic Culture
Media for Bacteria. In M. Rechcigl Jr. (ed) Handbook
180
Series in Nutrition and Food. Section G. Vol III. CRC

Press. Cleveland
SYKES, J. (1956) Constituents of Bacteriologic Culture
Media. Cambridge University Press. Cambridge.
181
182
183
184
185
186
Agar-Agar
Ref. 07-490
Agar is the dried, hydrophilic, colloidal substance extracted from the algae known as Agarophytes (several
species and genera of the Class Rodophyceae). It consists of two polysaccharides, agarose and agaropectine,
in a variable proportion depending on the geographical
origin zone.
The Agar-Agar is a solidifying agent with the same gelling strength as the Agar Technical but with an inferior
grade of purication, and shows a greater opacity and
a higher salts content. Its use among the culture media
is recommended only when the brightness and clarity is
not a critical requirement.
The most important characteristics are shown in the following tables. Data are average values, which may vary
from batch to batch.
Agar Bacteriological
Ref. 07-004
Agar is the dried, hydrophilic, colloidal substance extracted from the algae known as Agarophyites (several
origin zone.
The Agar Bacteriological is a solidifying agent selected
and prepared by mixing different agar from several
zones of origin and is especially recommended for
gelling the microbiological culture media where a great
transparency and brightness is required.
187
Agar Technical
Ref. 07-521
Agar is the dried, hydrophilic, colloidal substance extracted from the algae known as agarophyites (several
origin zone.
The Agar Technical is a solidifying agent with a gel
strengh higher than Agar Bacteriological, especially suggested when the culture medium does not require a total
brightness, since it shows a slight turbidity.
Beef Extract
Ref. 07-515
For a long time beef extract has been the basic component of culture media, and initially it substituted meat
infusions due to its easy usage. Now, there is a trend to
substitute it by peptones and different mixtures with a
more dened composition, because they allow a greater
reproducible result.
Scharlau Microbiology Beef Extract is obtained from free
tendons and fat beef muscle, enzymatically predigested.
Its production also includes the elimination of fermentable sugars.
Among the raw materials and auxiliaries used in its
preparation, the bovine constituents belong to the category 4 of the WHO classication. The bovine tissues
are sourced from New Zealand, and come from herds
free from Bovine Spongiform Encephalopathy virus and
foot-and-mouth disease after examination by the Veterinary Authorities. The enzymatic preparation is of porcine
origin.
The product does not contain and is not derived from
specied risk material as dened in the European Commission Decision 97/534/EC.
The manufacturing process includes boiling at 100C for
a minimum of 5 minutes and instantaneous heating at
200C on spray drying.
188
The totally desiccated version is easier to use than the

paste form, and require less quantity in order to obtain
the same effects. Beef extract solutions are clear, slightly
coloured and with pH near to neutral. In culture media
they are used in concentrations varying from 0,3-0,5%.
The most important characteristics are shown in the following gures and tables and in Tables 1 to 6 at the beggining of this chapter. Data are average values, which
may vary from batch to batch.
Beef Extract
07-515 Beef Extract
Molecular Weigth Distribution
07-515 Beef Extract

Amino Acids (Free/Total) x 100
Bile
Ref. 07-039
Ox bile powder is obtained by spray drying the fresh bile
at high temperature, assuring the maintenance of the
most important characteristics or properties of fresh bile.
1g of ox bile powder is corresponds to approx. 10 g of
fresh bile.
The bile used in its preparation is naturally of bovine
origin. It comes from animals which are raised and
slaughtered in Holland, and are in good health and suitable for human comsumption on ante- and post- mortem
examination by the Veterinary Authorities. These animals
come from herds free from Bovine Spongiform Encephalopathy and Foot-and-Mouth disease.
Dry powder has a colour varying form yellow-beige to

yellow-green, and it allows transparent 5% solutions in
water, that have a pH between 6 and 7,5. In alcoholic
solutions (ethanol 84%) it provides less than 0,1% of
insoluble substances.
The most important characteristics are shown in the following tables and in Tables 1 to 6 at the beggining of this
chapter. Data are average values, which may vary from
batch to batch.
The manufacturing process includes the rapid heating at

In the culture media it is employed at concentrations
varying from 1-2%. Ox bile act as a non enteric microbial ora inhibitor, and it is used as selective agent in
enterobacterial isolation media. Ox bile solutions at low
concentrations are clear and have a slight colour, but
at higher concentrations they are opalescent and have
darker colours.
189
Bile Salts #3
Ref. 07-525
The bile salts for microbiological applications are obtained from fresh animal (sheep, pig) bile by precipitation
with chlorhidric acid, in a process that removes pigments
and other toxic substances and concentrate the bile
salts. Nevertheless the standardization of the results is
very difcult because the composition of the nal mixture
depends not only on the process but on the raw material
that is very variable in origin.
In the normal preparation of bile salts several components like gluconate, taurocholate, cholate and deoxycholate, and others, can be identied, and the inhibitory
character of the mixture depends on the relative rate
between all these substances. Usually, in the microbiological culture media, the bile salts are dosed in a
concentration of 0,5% (w/v) to inhibit the growth of grampositive bacteria.
There are other type of preparations of mixture o bile
salts, with a higher level of purication and concentration
of actives substances. These ones are used, in microbiological applications, in lower concentration, that is,
a third o the concentration of the normal preparations.
Because its efcacy, they are called Bile Salts #3 and its
usual concentration in culture media is 0,15%.
Brain Extract
Ref. 07-076
For a long time animal brain infusions have been one of
the basic components of some culture media for fastidious microorganisms, and nowadays, in most cases they
are still necessary.
Scharlau Microbiology Brain Extract produces clear,
clean and stable solutions at 121C, and provide the culture medium with very complex nutrients. It is obtained
from brains of healthy pigs with ante- and post-mortem
sanitary certication.
Among the raw materials and auxiliaries used in its preparation, the bovine constituents belong to the category 4
of the WHO classication. The product does not contain
and is not derived from specied risk material as dened
in the European Commission Decision 97/534/EC. All
the constituents are of swine origin.
batch to batch.
190
The Scharlau Bile Salts #3 is standardized batch by

batch intending to supply a product as homogeneous as
possible.
Dry powder has a owable blurred white aspect, with a
bitter in odour and taste, but provides clean, transparent
and pale yellow 2% solutions in water, which present
an alkaline reaction (pH 8,0) and therefore its addition
to culture media may require pH adjustment. When this
product is used as culture media component it is advisable do nod exceed never the 0,3 % (w/v) concentration.
batch to batch.
Casein Acid Hydrolysate

Ref. 07-151
Casein Acid hydrolysate is a protein hydrolysate obtained by acid digestion, where all the casein compounds reduced to their aminoacids, except tryptophane which almost disappears. Vitamines also almost
disappear due to the acid digestion process.
Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the category
4 of the WHO classication. The lactic casein from cow
milk is sourced from New-Zealand, and come from herds
free from Bovine Spongiform Encephalopathy and footand-mouth disease after examination by the Veterinary
Authorities.
07-151 Casein Acid Hydrolisate

07-151 Casein Acid Hydrolisate

191
Casein Pancreatic Peptone

Ref. 07-154
Casein Pancreatic Peptone is a protein hydrolysate,
obtained by digestion with pancreatic extracts. It differs
from Tryptone (Ref. 07-119) only in the way it is obtained, which produces a different aminoacid composition and a lesser molecular size. It is the most used type
of peptone in industrial fermentation operations.


preparation the bovine constituents belong to the category 4 of the WHO classication. The Milk casein from
cow milk is sourced from New-Zealand, and come from
herds free from Bovine Spongiform Encephalopathy and
foot-and-mouth disease after examination by the Veterinary Authorities.
The enzymatic preparation is of porcine origin.
07-154 Casein Pancreatic Peptone

192
07-154 Casein Pancreatic Peptone

Casein Trypsic Peptone (Tryptone)

Ref. 07-119
The Casein Trypsic Peptone or Tryptone, is a protein
hydrolysate obtained by digestion of casein with an
especially tripsin-enriched pancreatic enzymatic preparation.
Both, its nitrogen content and balanced amino-acid composition makes it more suitable for the manufacturing of
culture media, producing exceptionally clear solutions.


preparation the bovine constituents belong to the category 4 of the WHO classication. The lactic casein from
cow milk is sourced from New-Zealand, and come from
herds free from Bovine Spongiform Encephalopathy
and foot-and-mouth disease after examination by the
Veterinary Authorities. The enzymatic preparation is of
porcine origin.
07-119 Casein Trypsic Peptone

07-119 Casein Trypsic Peptone

193
Gelatin Pancreatic Peptone

Ref. 07-153
gining of this chapter. Data are average values, which

Gelatin peptone is a cream coloured powder, with

characteristic odour, obtained by pancreatic digestion of
gelatin.
The gelatin is of porcine origin. None of the raw materials and auxiliaries used in its preparation are of bovine
origin. The manufacturing process includes boiling at
100C for a minimum of 5 minutes and instantaneous
heating at 200C on spray drying.
It has a low content of Tryptophan, and has no fermentable sugars. Their solutions, even at high concentrations
(10%), take a light colours, without precipitate, due to
their elaborated manufacturing process. It produces a
slight acid reaction (after sterilization) and has no indole.
Its nutritional capacity is low, but it may be used for non
fastidious microorganisms, and complies with the peptone specications for fermentation studies.
The most important characteristics are shown in the following gures and tables and in Tables 1 to 6 at the beg-
07-153 Gelatin Pancreatic Peptone

194
07-153 Gelatin Pancreatic Peptone

Heart Extract
Ref. 07-077

Bovine heart extract has been widely used as an alternative to meat extract where very special nutrient requirements are needed. Scharlau Microbiology Heart Extract
is obtained from the bovine cardiac muscle of healthy
animals with explicit sanitary certication.
preparation, the bovine constituents belong to the category 4 of the WHO classication. The bovine tissues
are sourced from New Zealand, and come from herds
Authorities.
specied risk material as dened in the European Commission Decision 97/534/EC. The manufacturing process
includes boiling at 100C for a minimum of 5 minutes
and instantaneous heating at 200C on spray drying.
07-077 Heart Extract

07-077 Heart Extract

195
Lecithin
Ref. 07-342
Phosphatidylcholine, a mixture of diglycerides of the
stearic, palmitic and oleic acids, linked to the cholic ester
of the phosphoric acid.
The SCHARLAU Lecithin is a clear brown powder,
obtained from soy beans by extraction. It is especially
treated to be included in culture media as an emulsier
or as a nutrient factor for fastidious microorganisms.
Liver Peptone
Ref. 07-614
Liver peptone is a proteic hydrolized by enzymatic digestion of fresh swine liver, followed by a careful desiccation
process to maintain its fundamental characteristics.
Liver peptone is very employed in culture media for Trichomonas and other fastidious protozoa, and for some
pathogen and saprotic fungi, micoplasms and anaerobic bacteria.
Liver peptone provides clear solutions of a dark colour,
and is perfectly compatible with other medium components. It accepts sterilization and does not loose its
characteristics. Generally it uses to replace peptones, at
the same weight. In culture media for protozoa, its concentration uses to be high (25-30 g/L), but for bacteria
it uses to vary from 0,5 to 1 %, except for anaerobics
enrichment media, where concentration may be higher.
196

Liver Peptone
07-614 Liver Peptone
07-614 Liver Peptone

Malt Extract
Ref. 07-080
Malt extract is used in the culture media for fungi, as
much as enrichment as a true nutritive base, because
very often it substitutes the peptone. It is obtained by
extraction of soluble fraction of malted barley, followed
by a drying proccess at low tempertature so that there
is only minimal alteration in its nitrogenated composition
and high sugar content, especially maltose. All the raw
materials and auxiliaries are of plant origin. It has no
diastatic activity. Very hygroscopic product.
Malt extract solutions are usually opalascent or turbid.
Should clear solutions are required, ltration is necessary.
197
Meat Extract
Ref. 07-075
Meat extract has been the basic component of culture
media for a long time, and initially it substituted meat
infusions due to its easy usage. Now, there is a trend to
substitute it by peptones and different mixtures with a
more dened composition, because they allow a greater
reproductivity result.
Meat extract is obtained from free tendons and fat
tissues of the animals (sheep and pork), which are
enzymatically predigested. Its production involves the
elimination of fermentable sugars.
The totally desiccated (dried) version is easier to use

and requires less quantity in order to obtain the same
effects. Meat extract solutions are clear, slightly coloured
and with pH near to neutrality. In the culture media they
are used in concentrations varying from 0,3-0,5%.

preparation the bovine constituents belong to the category 4 of the WHO classication. The bovine tissues are
sourced from New-Zealand, and come from the herds
Authorities.
specied risk material as dened in the European Commission Decision 97/534/EC. The other constituents are
of porcine origin. The manufacturing process includes
boiling at 100C for a minimum of 5 minutes and instantaneous heating at 200C on spray drying.
07-075 Meat Extract

198
07-075 Meat Extract

Meat Peptone
Ref. 07-152
Meat peptone is an hydrolysate obtained by a partial
digestion of meat by pepsine. It complies with the USP/
NF25 and Eur. Phar. 4th. Ed. specications for the peptic
digestion of animal tissues.
It is a ne powder, cream or brown coloured, that gives
very clear and light solutions and is specially prepared
for using in the culture media.


preparation the bovine constituents belong to the category 4 of the WHO classication. The bovine tissues
are sourced from New-Zealand, and come from the
Foot-and-Mouth Disease after the examination by the
Veterinary Authorities.
of porcine origin.
07-152 Meat Peptone

07-152 Meat Peptone

199
Peptone from Casein

Ref. 07-489
The Peptone from casein is a protein hydrolysate obtained from trypsin-digested casein according to the USP
specications. Both its nitrogen rate and its balanced
amino-acid composition provide an adequate support for
the production of culture media, producing exceptionally
clear solutions.
The digestion process by an exceptionally trysin-rich

enzymatic preparation produces a high content of tryptophane and assures the absence of fermentable sugars
and enzymatic activity.

are sourced from New-Zealand, and come from the
Foot-and-Mouth Disease after the examination by the
07-489 Peptone from Casein

200
07-489 Peptone from Casein

Proteose Peptone
Ref. 07-213
This peptone is obtained after a partial enzymatic (peptic) digestion process of animal tissues. It is obtained in
such a way that there is a high proportion of peptides
of low molecular weight, with free amino acids and
other growth factors. Although all these things make its
denition very difcult, it has a high nutritive capacity
that makes it suitable for obtaining toxins and as a basic
growth support for very fastidious microorganisms.

Among the raw materials and auxiliaries used in its production, the bovine constituents belong to the category
4 of the WHO classication. The bovine tissues are
sourced from New-Zealand, and come from herds of cattle which are free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the
of porcine origin.
07-213 Proteose Peptone

07-213 Proteose Peptone

201
Soy Peptone
Ref. 07-155
Soy peptone is a proteic hydrolysate obtained by papaic
digestion of soy our. It complies with the USP/NF25 and
Eur. Phar. 4th. Ed. specications for these type of products, and it is a useful compound in laboratory culture
media. However, due to its high content of sugar it is not
recommendable for fermentation assays.
07-155 Soy Peptone

202
07-155 Soy Peptone

Tryptose
Ref. 07-197
Tryptose is a mixed peptone with high nutrient properties
that make it appropiate for the use in culture media for
very fastidious microorganisms.
are sourced from New-Zealand, and come from herds
free from Bovine Spongiform
Encephalopathy and foot-and-mouth disease after examination by the Veterinary Authorities.


of porcine origin.
07-197 Tryptose
07-197 Tryptose
203
Yeast Extract
Ref. 07-079
A water soluble extract of fresh autolyzed yeast cells.
Prepared and standardized for use in microbiological
culture media.
It is commonly added to culture media in concentrations
between 0.2% and 1%.
07-079 Yeast Extract

204
07-079 Yeast Extract

Additives
Dextrose Powder (D(+)- Glucose Powder)

Ref. 06-048
Specications
Specic rotation ([]20C/D, c=10, H O) ...... +52,6 - +53,2
Molecular weight .............................. 180,16 g/mol
Acidity/alkalinity ................................ passes test
Insoluble in water ................................. max. 0,01
%
Chlorides (Cl)........................................ max. 0,01
%
Sulfates (SO4) ...................................... max. 0,02
%
Sulte (as SO2) .................................... max. 0,001 %
Arsenic (As) .......................................... max. 0,0001 %
Calcium (Ca)......................................... max. 0,02
%
Heavy metals (as Pb) ........................... max. 0,0005 %
Iron (Fe) ................................................ max. 0,0005 %
Lead (Pb) .............................................. max. 0,00005%
Sulfated ash ......................................... max. 0,1
%
Water (K.F.) .......................................... max. 1
%
Foreign sugars,starchs,dextrines ..... passes test
Residual solvents (Eur. Phar./ICH) ...... excluded
2
Presentation
500 g Flask
Description
Puried and standardized carbohydrate for the use in
microbiological cultrure media as energy source for
bacteria. Carbohydrate are adapted to the suitable basic
media.
Scharlau carbohydrates are pure and without mixtures,
and this important characterstic is assured in order to
get always right results.
Physical Data
Bulk density............................................... ~ 630kg/m3
Solubility in water (20C) .......................... ~ 470g/L
Melting point ............................................. ~ 146C
Ignition temperature ................................. ~ 500C
pH (100 g/L H2O, 20C)................................ 6-7
Eggs Yolk Sterile Emulsions

Eggs Yolk Sterile Emulsion
Ref. 06-016
Presentation
100 mL Flask
Description
Sterile eggs yolk emulsion stabilized for use in bacteriology, especially with Bacillus cereus Agar Base (Ref.
01-262), Bacillus cereus Selective Agar Base (Ref. 01487) and Tryptose Sulte Cycloserine Agar Base (Ref.
01-278).
It is also used for the detection of lecithinase in species such as Bacillus, Clostridium and Staphylococcus,
and in all processes related with this enzyme which are
present in dairy microorganisms and most psychrotrophics.
Lecithinase Assay
The medium is prepared by aseptically adding 0,5-1,0
mL of yolk emulsion to 10 mL of sterile melted solid
medium, cooled to 55-60C. Tryptic Soy Agar (Ref. 01200), Nutrient Agar (APHA) (Ref. 01-144) and Nutrient
Agar (B. Ph.) (Ref. 01-140) are very adequate for these
purposes. The solid medium is inoculated with the assay
strain and incubated at 35-37 C for 5 days. If there is
lecithinasic activity the broths will turn opalescent and
solid media will present an opaque zone of clearing
around the colonies.
Bacillus cereus, with a strong lecithinase, produces visible results in just a few hours.
The Eggs Yolk Sterile Emulsion has been widely used

as bactericide neutralizer on sampling liquids. Such an
effect can be considerably intensied by adding a bit of
Polysorbate with the yolk in the formulation. 1% (v/v) is
usually enough.
Eggs Yolk Tellurite Sterile

Emulsion
Xn
Ref. 06-026
Presentation
R-22
S-46
100 mL Flask
Description
This emulsion has been especially formulated for its addition to the Baird Parker Agar Base (Ref. 01-030)
Aseptically add 50 mL of Eggs Yolk Tellurite Sterile
Emulsion to 1 litre of sterile Baird Parker Agar Base
(Ref. 01-030), melted and cooled down to approximately
55-60C. Mix uniformly avoiding bubbles and foam, and
pour into Petri dishes.
The presumptive Staphylococcus aureus colonies show
the lecithinase activity by the halo digestion around the
colonies and simultaneously a black center due to the
tellurite reduction.
207
Lactose Powder
Ref. 06-051
Specications
Appaerance of solution (10% water) ....... passes test
Proteins ............................................ passes test
Arsenic (As)...........................................max. 0,00005 %
Copper (Cu) .......................................... max. 0,0025 %
Lead (Pb)...............................................max. 0,00005 %
Zinc (Zn) ............................................... max. 0,0025 %
Sulfated ash ......................................... max. 0,1
%
Water (K.F.)............................................4,5 - 5,5
%
Residual solvents (Eur. Phar./ICH)...........excluded
2
Presentation
500 g Flask
Description
media.
Physical Data
Bulk density.............................................~ 500
Solubility in water (20C) ............freely soluble
Melting point ...........................................~ 223
pH (50 g/L H2O, 20C) ................................4-6
kg/m3
C
Maltose Powder
Ref. 06-052
Presentation
500 g Flask
Description
media.
208
Physical Data
Bulk density.............................................~ 320
Melting point ..................................~ 160 - 165
pH (50 g/L H2O, 20C) ..........................4,5-6,0
kg/m3
C
Specications
Specic rotation ([]20C/D, c=10, H O) ...... +137 - +139
Proteins ............................................ passes test
Arsenic (As).......................................max. 0,00005
Copper (Cu).......................................max. 0,0005
Heavy metals (as Pb)........................max. 0,002
Barium (Ba).......................................max. 0,0005
Calcium (Ca)......................................max. 0,005
Sulfated ash.......................................max. 0,1
Water (K.F.)........................................4,5 - 5,5
Residual solvents (Eur. Phar./ICH) ...... excluded
2
%
%
%
%
%
%
%
Mannitol Powder
Specications
Ref. 06-050
Presentation
500 g Flask
Description

media.
Physical Data
Bulk density...................................~ 400 - 500
Spec. density.................................................1,49
Solubility in water (25C)...........................213
Melting point ...................................~ 164 -169
Boiling point (4 hPa) ......................~ 290 - 295
pH (100 g/L H2O, 20C) ..............................5-7
Assay (iodometric) ............................ min. 98

%
Conductivity (20C, 20% water)........ passes test
Specic rotation ([]20C/D, c=8, Na B O ) ... +23 - +25

Molecular weight ............................. 182,17 g/mol
Chlorides (Cl)........................................ max. 0,005 %
Sulfates (SO4) ....................................... max. 0,01
%
Arsenic (As) .......................................... max. 0,0001 %
Copper (Cu) .......................................... max. 0,001 %
Lead (Pb)...............................................max. 0,00005 %
Nickel (Ni) ............................................. max. 0,0001 %
Zinc (Zn) ............................................... max. 0,0025 %
Related substances (sorbitol) ............... max. 2
%
Red. impurities (as glucose) ................. max. 0,05
%
Sulfated ash ......................................... max. 0,1
%
Loss on drying (105C, 4h) ................... max. 0,3
%
Residual solvents (Eur. Phar./ICH)...........excluded
kg/m3
g/m3
g/L
C
C
Polysorbate 80 (Polyoxyethylene sorbitan monooleate)

Ref. 06-088
Presentation
100 mL Flask
1 L Flask
Description
Under the name of Polysorbate 80 or polyoxyethylene
sorbitanmonooleate are included a serie of derivates of
polyose-1,2-ethanodiol sorbitan-mono-9-octodecenoate.
The product supplied by SCHARLAU is veried to be a
nutrient in some cases and an emulsionant in others, but
it is always compatible with the rest of components of
the culture medium.
It is a very thick liquid, amber colour, and density 1,08
approx. It is very soluble in water, it has an average solubility in organic diluents but it is not soluble in mineral
lipids.
The incorporation of Polysorbate 80 to culture media may slightly affect the nal pH, if the SCHARLAU
medium is not originally formulated to be composed by
Polysorbate.
Although it may bear sterilization in the autoclave when
it is more than 1% in the medium, it is usual to homogenize the medium after the sterilization, since with the
autoclaving sometimes the polysorbate is separated
from the medium.
Polysorbate is a tensioactive agent that makes decrease
the supercial tension of the cell, modifying at the same
time the cellular exchange speed. The response use to
be a quicker growht or the increase of some bacterial
activities.
209
Potassium Tellurite Solutions

Potassium Tellurite Solution 1%
Ref. 06-089
Presentation
Xn
R-22
S-46
100 mL Flask
There is high relation between the ability to reduce potassium tellurite to tellure and the staphylococcis pathogenity. Therefore, the presence of potassium tellurite in
a medium helps to determine staphylococci of clinical
interest, together with other tests.
Potassium Tellurite
Solution 3,5%
Xn
Ref. 06-011
Presentation
It is used in media such as Giolitti Cantoni Broth (Ref.

02-230), Vogel-Johnson Agar (Ref. 01-206) and other
selective media for staphylococci. This solution is also
contained in selective media for corynebacteria, streptococci and vibrios.
R-22
S-46
100 mL Flask
Description
The Potassium Tellurite Solution should be stored at

room temperature, since low temperatures will cause
the crystallization and later precipitation of the product.
Should this occur, intense agitation will help redissolve
the precipitate. Due it its thermolabile qualities, the potassium tellurite is supplied sterile ltered.
Aquouse solutions of potassium tellurite at 1% or 3,5%,

sterilized by ltration and suitable to be used as an
inhibitor additive in culture media.
The Potassium Tellurite Solution is added to culture media as an inhibitor. Its purpose is to prevent the growth of
most gram-negatives and of those gram-positives unable
of reducing it.
Ringer Powder
Ref. 06-073
Description
Presentation
Ringer saline solution is an isotonic medium which is

more balanced than the simple sodium chloride saline
solution, and its formulation permits the autoclaving
without producing any precipitation.
500 g Flask
100 g Flask
Specication
Isotonic solution for the cellular suspensions.
Formula (in g/L)

Sodium chloride ...................................... 2,250
Potassium chloride ................................. 0,105
Sodium bicarbonate ............................... 0,050
Directions
To obtain an isotonic solution for eukaryotic cells, dissolve 10 g of powder in 1 L of distilled water. To obtain
an isotonic solution for prokaryotic cells, dissolve 2,5 g of
powder in 1 L of distilled water.
Distribute into suitable containers and sterilize in the
210
For the routine work with bacteria the solution is diluted

one fourth (to the fourth part) (Ringer 1/4), and is employed to get cell suspensions or to prepare the dilution
banks.
Nonetheless, to dilute the food samples or substances
that have undergone thermal treatment, it is more advisable to use Peptone Water (Ref. 3-156) for the dilutions,
since the Peptone Water acts as a revitalizer also.
References
DAVIS, J.G. (1956) Laboratory Control of Dairy Plant.
Dairy Industries Ltd. London.
ANONYMOUS (1937) Bacterial Tests for Graded
Milk. Memo 139-Food. Dept. of Health and Social
Security,London.
Skimmed Milk
Ref. 06-019
Presentation
500 g Flask
Description
Powder of skimmed milk for bacteriology is obtained
after a depurated atomization process that keep it free
from thermophil organismsm that use to interfere with its
use.
100 g of powder produce 1 L of skimmed milk. Water
addition must be gradual, until getting an homogeneous paste. Then fulll with water to the desired volume.
Sterilization may be under uent vapor for 30 minutes
and three consecutive days or in the autoclave at 121C
for 15 minutes or at 114C for 15-20 minutes (this last
way is the better). Anycase, do not overheat prepared
milk since natural sugars may become caramelizated
and produce toxic compounds.
Skimmed milk may be use alone or as an additive to

other culture media. It is a very suitable medium for the
culture of acid lactic bacteria and for identication in
general, in base to its capacity to coagulate o peptonize
milk. With the additon of suitable indicators as bromcresol purple at 0,004% the pH variations in the transformation may be screened. It also accepts oxid-reduction
indicators like methylen blue, resazurine or TTC (Ref.
6-023) to verify microorganisms development.
Skimmed milk may be added to media as Tryptone Soy
Agar (Ref. 01-200) or Nutrient Agar (Ref. 01-144 and 01140) to detect caseolytic activity.
Fat ............................................................. 0,5
Protein ..................................................... 33,0
Ash ............................................................ 8,0
Moisture ..................................................... 5,0
Lactic acid .................................................. 1,5
Antibiotic test .....................................Negative
%
%
%
%
%
Sodium Biselenite
Ref. 06-615
Presentation
R-23/25-33-50/53
S-20/21-28-45-60-61
100 g Flask
Description
Chemical compound to be added to selenite based
culture media.
The toxic and potetially theratogenicity of this product
recommend its exclusion from the dehydrated powder
mixture of culture media, to minimize the hazard of accidental inhalation or contact. The supply of this product in
a separate form from the base medium, with all the risk
considerations, enhances its safe and responsible use.
The intended use of this product is to complete the following culture media by adding the specied amounts:
02-602 Selenite Cystine Broth Base .....................4 g/L
02-603 Selenite Brilliant Green Broth Base ...........4 g/L
02-598 Selenite Broth Base ..................................4 g/L
The use of this product is restricted to technically qualied personnal. Keep attention to the job limitations in
inexpert personnal.
211
Sucrose Powder (D(+) Saccharose Powder)

Specications
Ref. 06-049

Conductivity ..................................... max. 35 S/cm
Chlorides (Cl)........................................ max. 0,0035 %
Sultes (as SO3) ................................... max. 0,0015 %
Sulfates (SO4) ....................................... max. 0,005 %
Dextrines .......................................... passes test
Glucose and invert sugar ................. passes test
Calcium (Ca)......................................... max. 0,001 %
Lead (Pb) .............................................. max. 0,00005
%
Sulfated ash ......................................... max. 0,01
%
Organic volatile impurities (NF) ......... passes test
Residual solvents (Eur. Phar./NF) ....... max. 0,5
%
2
Presentation
500 g Flask
Description
media.
Physical Data
Bulk density....................................~ 800 - 950
Melting point ...................................~ 169 -170
pH (100 g/L H2O, 20C) ..............................~ 7
212
kg/m3
C
TTC Sterile Solution 1%

Ref. 06-023
The general structure is the following:
Presentation
100 mL Flask
Description
Sterile solution at 1% of 2-3-5-triphenyl-2H-tetrazolium
chloride. It is used as an additive for culture media to
show biological activity, since the colourless form gets
hydrogenizated or reduced to a red insoluble pigment:
triphenylformazan, which may be easily observed.
Despite of TTC decomposes at 243C, it is not advisable to incorporate it to culture media before sterilization, because it lose efcacy. Very good results may be
achieved when the addition is carried out asseptically
with cold medium at 60C maximum. TTC is photolabile
and becomes yellow by the effect of light, therefore keep
it in the refrigerator and avoid direct light.
Concentration of use vary depending on the medium, but
generally it goes between 0,3 and 1% (v/v).
This product is especially produced to be added to

the following media: Chapman TTC Agar Base (Ref.
01-053), KF Media (Ref. 01-294 and Ref. 02-294) and
Slanetz Bartley Agar Base (Ref. 01-579).
Urea Sterile Solution 40%

Ref. 06-083
Presentation
100 mL Flask
Description
It is supplied to be used with the dehydrated media Urea

Agar acc. to Christensen (Ref. 01-261) and Urea Broth
(Ref. 02-202). It must be added to these media after the
sterilization and with the media cooled below 55C.
Once it is added, do not reheat the media.
Aquose urea solution 40%, sterilized by ltration and

suitable to be used as an additive in culture media.
Vaseline Sterile
Ref. 06-077
Presentation
100 mL Flask
Description
Liquid media may be kept in anaerobic conditions if
sterile compounds, as vaseline, are used to grant an
hermetic lock.
To achieve an hermetic lock in tubes with liquid medium,
heat them up in boiling bath for 10 minutes to remove
the oxygen and add after the vaseline to avoid the
incorporation of air to the medium. To seed the tubes,

use a capilar pipette or inoculate them previously to the
vaseline addition.
Vaspar is another method used to achieve an hermetic
lock. It is prepared melting vaseline and solid parane
toghether in equal parts.
Vaseline sterile is especially suggested for the addition
to O/F Medium (Ref. 03-037). For solid media is more
suitable to use the Sealing Agar (Ref. 01-174).
213
Supplements
Improved NEW presentation

PRESENTATION:
10 VIALS INSIDE A RESISTANT CASE
REFERENCE DESCRIPTION
06-012CASE SC Selective Supplement for Clostridium
VIAL CONTENTS: 120 mg of Sodium azide, 90 mg of Neomycin
sulphate and solvent.
Each vial is sufcient to supplement 500 ml of Blood
Columbia Agar Base (Ref. 01-034) or Blood Agar Base
(Ref. 01-352) in order to prepare Clostridium spp. Selective Agar.
06-013CASE CP Gram-positive cocci in Blood Agar Selective Supplement
VIAL CONTENTS: 5 mg of Colistin sulphate, 7,5 mg of Nalidixic acid
and solvent.
Each vial is sufcient to supplement 500 ml of Blood Columbia
Agar Base (Ref. 01-034) or Blood Agar Base (Ref. 01-352) and
obtain Staphylococcus and Streptococcus Selective Agar.
06-017CASE Brilliant Green + Novobiocin Selective Supplement
VIAL CONTENTS: 5 mg of Brilliant green, 20 mg of Novobiocin
and solvent.
Each vial is sufcient to supplement 500 ml of Tetrationate
Base Broth (Ref. 02-033) and Muller-Kauffmann Medium
(Ref. 02-335).
06-021CASE Polymyxin B Sulphate Selective Supplement
VIAL CONTENTS: 50 mg Polymyxin B sulphate and solvent.
Each vial is sufcient to supplement 500 ml of Bacillus cereus
Agar Base (Ref. 01-262).
06-022CASE Cycloheximide Selective Supplement
VIAL CONTENTS: 2 mg of Cycloheximide and solvent.
Each vial is sufcient to supplement 500 ml of WL Nutrient
Agar (Ref. 01-210) or WL Nutrient Broth (Ref. 02-210)
and converted in WL Differential Agar or Broth.
06-025CASE Brucella Selective Supplement
VIAL CONTENTS: 50 mg of Cycloheximide, 3000 u.i. of
Polymyxin B Sulphate, 12500 u.i. of Bacitracin sulphate
and solvent.
Each vial is sufcient to supplement 500 ml of Brucella Selective
Agar (Ref. 01-042) and Brucella Selective Broth (Ref. 02-042).
06-085CASE Rosolic Acid Selective Supplement
VIAL CONTENTS: 50 mg of Rosolic acid and solvent.
Each vial is sufcient to supplement 500 ml of Faecal Coliforms
Agar (m-FC Agar) (Ref. 01-287) or Faecal Coliforms Broth
(m-FC Broth) (Ref. 02-287).
06-091CASE CPB Selective Supplement for Campylobacter
VIAL CONTENTS: 2,5 mg of Trimethoprim, 5 mg of Vancomycin,
7,5 mg of Cephalotin, 1250 u.i. of Polymyxin B sulphate,
1 mg of Amphotericin B and solvent.
Each vial is sufcient to supplement 500 ml of Blood Columbia
Agar Base (Ref. 01-034) and obtain 500 ml of Campylobacter
Selective Agar acc. Blaser-Wang.
06-102CASE MUG Supplement. Fluorescent Agent for Escherichia coli
VIAL CONTENTS: 50 mg of MUG
(4-methilumbeliferil--D glucuronide) and solvent.
Each vial is sufcient to supplement 500 ml of Coliforms Agar
or Broth.
06-106CASE Listeria Selective Supplement for Primary Enrichment (UVM I)
VIAL CONTENTS: 10 mg of Nalidixic acid, 6 mg of Acriavine and
solvent.
Each vial is sufcient to supplement 500 ml of Listeria Enrichment
Broth Base (UVM) (Ref. 02-472) in order to prepare 500 ml
of Listeria Primary Enrichment Medium (UVM I formulation).
06-107CASE Listeria Selective Supplement for Enrichment acc. FDA
and IDF/FIL
VIAL CONTENTS: 20 mg of Nalidixic acid, 25 mg of Cycloheximide,
7,5 mg of Acriavine and solvent.
Each vial is sufcient to supplement 500 ml of Listeria
Enrichment Broth Base acc. Lovett (Ref. 02-498).
06-109CASE Oxford Agar Selective Supplement for Listeria
VIAL CONTENTS: 5 mg of Phosphomycin, 1 mg of Sodium
Cephotaxim, 10 mg of Colistin, 200 mg of Cycloheximide,
2,5 mg of Acriavine and solvent.
Each vial is sufcient to supplement 500 ml of Oxford Agar
Base (Ref. 01-471) in order to prepare Listeria Selective Agar
(Oxford formulation).
06-110CASE Palcam Agar Selective Supplement for Listeria
VIAL CONTENTS: 5 mg of Polymyxin B sulphate, 10 mg of Sodium
ceftazidime, 2,5 mg of Acriavine and solvent.
Each vial is sufcient to supplement 500 ml of Palcam Agar
Base (Ref. 01-470) in order to prepare Listeria Selective Agar
(Palcam formulation).
216
REFERENCE DESCRIPTION
06-111CASE Listeria Selective Supplement for Secondary Enrichment
(UVM II/Fraser)
VIAL CONTENTS: 10 mg of Nalidixic acid, 12,5 mg of Acriavine
and solvent.
Enrichment Broth Base (UVM) (Ref. 02-472) in order to prepare
500 ml of Listeria Secondary Enrichment Medium (UVM II
formulation); or to supplement 500 ml of Listeria Enrichment
Broth acc. Fraser (Ref. 02-496) in order to prepare 500 ml
of Fraser Broth.
06-112CASE Ferric Ammonium Citrate for Bacteriology
VIAL CONTENTS: 250 mg of Ferric ammonium citrate and solvent.
Enrichment Broth Base acc. Fraser (Ref. 02-496)
06-113CASE Ferric Ammonium Citrate for bacteriology
VIAL CONTENTS: 312 mg of Ferric ammonium citrate and solvent.
Each vial is sufcient to supplement 500 ml of Lactose Sulphite
Broth Base (Ref. 02-519).
06-114CASE Disodium disulphite Selective Supplement for bacteriology
VIAL CONTENTS: 375 mg of Disodium disulphite and solvent.
Each vial is sufcient to supplement 500 ml of Lactose Sulphite
Broth Base (Ref. 02-519).
06-115CASE Oxytetracycline Selective Supplement
VIAL CONTENTS: 50 mg of Oxytetracycline and solvent.
Each vial is sufcient to supplement 500 ml of Sabouraud with
Oxytetracycline Agar Base (OGYEA) (Ref. 01-275).
06-116CASE Cycloserine Selective Supplement
VIAL CONTENTS: 100 mg of Cycloserine and solvent.
Each vial is sufcient to supplement 250 ml of Tryptose Sulphite
Cycloserine (TSC) agar Base for Clostridium perfringens
Ref. 1-278).
06-118CASE Chloramphenicol Selective supplement
VIAL CONTENTS: 25 mg of Chloramphenicol and solvent.
Each vial is sufcient to supplement 500 ml of Sabouraud
Dextrose Agar (Eur. Phar. Agar Medium C), (Ref. 01-165).
06-124CASE Nalidixic Acid Selective Supplement
VIAL CONTENTS: 7,5 mg of Nalidixic acid and solvent.
Each vial is sufcient to supplement 500 ml of CN Selective
Agar Base for Pseudomonas (Ref. 01-609).
06-125CASE m-CP Selective Supplement
VIAL CONTENTS: 200 mg of D-Cycloserine, 12,5 mg of Polymyxin
B sulphate, 30 mg of 3-indoxyl--D-glucopyranoside, 50 mg
of Phenolphthalein diphosphate, 45 mg of Iron III Chloride,
and solvent.
Each vial is sufcient to supplement 500 ml of m-CP Agar Base
(Clostridium perfringens Agar) (Ref. 01-513).
Storage conditions:
Should be stored at 2-8C in the dark.
Shelf life:
18-24 months depending the reference. Larger vial-larger variety of supplements.
MORE INFORMATION
AVAILABLE IN OUR WEB
AND CD-ROM CATALOGUE
Selective Supplements
Culture media supplements in a practical presentation:
an extemporaneous solution.
Main advantages:
fast
simple
easy
safe
ready to add
easy storage
longer shelf life
less risk of contamination
With this 10 vial-case format you no longer have to worry
about things like sterile solvents, sterile syringes, sterilising the supplements that must be added to the medium
by ltration ...
With one simple pressure on the lid you obtain the sterile
supplement solution, ready to add to the medium base.
Selective supplements should be stored at 2-8C in
the dark. When stored as directed the reagents remain
stable until the expiry date shown on the label.
Method of use: 4 simple steps
Press on the cap breaking the container that

holds
the solid.
The supplement is now

ready. Open the vial
aseptically, close to a ame or in a safety cabinet.
Pour over the medium
base, which has been
cooled down to 45-50C.
The solid then falls

into the solvent. Shake
vigorously
for total dissolution.
Homogenize and
distribute into the suitable
container: asks, tubes or
plates.
217
Basic Fuchsin 200 Selective Supplement

Ref. 06-617CASE
Contents
Precautions
This product should be for laboratory use only.
Do not use beyond stated expiry date.
R-11
S-7-16
The box contains 10 vials. Each vial is sufcient to supplement 250 mL of Endo LES (Ref. 01-604).
Applicable media
Ref. 01-604 Endo LES Agar Base
Vial contents
Necessary amount for 250 mL of medium.
Basic Fuchsin ............................................ 200 mg
Ethanol .......................................................... 5 mL
Directions
Mix the liquid with the powder by pressing down on the
cap. Shake to dissolve and aseptically add the solution
to 250 mL of Endo LES Agar Base.
Note: Dont heat the media once the supplement has
been added.
Basic Fuchsin 250 Selective Supplement

Ref. 06-607CASE
Contents
F
R-11
S-7-16
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Endo Agar Base (Ref. 01-589), Endo
DEV Agar Base (Ref. 01-606) and only 250 mL of Endo
Base Broth (Ref. 02-605).
Vial contents
Necessary amount for 500 mL of solid medium or 250
mL of liquid broth.
Basic Fuchsin ............................................ 250 mg
Ethanol .......................................................... 5 mL
Directions
to the boiled medium. Homogenize and use as per each
monography medium. Do not remelt the solid media.
218
Precautions
Applicable media
Ref. 01-589 Endo Agar Base
Ref. 01-606 Endo DEV Agar Base
Brilliant Green + Novobiocin Selective Supplement

Ref. 06-017CASE
Precautions
Contents

The box contains 10 vials. Each vial contains inhibitors

sufcient to add to 500 mL of Tetrathionate Broth Base
Ref. 02-033 or 02-335 Muller Kauffmann Medium.
Applicable media
Ref. 02-033 Tetrathionate Base Broth
Ref. 02-335 Muller-Kauffmann Medium
Vial contents
Brilliant Green ................................................ 5 mg
Novobiocin, sodium salt .............................. 20 mg
Distilled water ................................................ 5 mL
Directions
Mix the liquid with the powder by pressing down on
the cap. Shake to dissolve and aseptically add the vial
contents to 500 mL of boiled broth base cooled to 50C.
Homogenize and use as per each monography medium.
Note: Dont heat the media once the supplements have
been added.
219
Chloramphenicol Selective Supplement

Ref. 06-118CASE
Contents
Precautions
R-45
S-53-45
The box contains 10 vials. Each vial is sufcient to

supplement 500 mL of Sabouraud Dextrose Agar (Ref.
01-165).
Applicable media
Ref. 01-165 Sabouraud Dextrose Agar (Eur. Phar. Agar
Medium C)
Vial contents
Chloramphenicol ......................................... 25
Distilled water ................................................ 5
mg
mL
Directions
to 500 mL of sterile agar base cooled to 50C. Homogenize and distribute the complete medium into the
containers.
been added.
m-CP Selective Supplement

Ref. 06-125CASE
Precautions
Contents


supplement 500 mL of m-CP Agar Base, Ref. 01-513,
in order to prepare 500 mL of m-CP (Clostridium perfringens) Agar.
Vial Contents
Necessary amount for 500 mL of complete medium.
D-Cycloserine .......................................200,0 mg
Polymixin B sulfate .................................12,5 mg
3-Indoxyl--D-Glucopyranoside .............30,0 mg
Pehnolphthalein di-phosphate ................50,0 mg
Iron III Cloride .........................................45,0 mg
Distilled water ...........................................5,0 mL
Directions
to 500 mL of sterile agar base cooled to 50C.
been added.
220
Applicable media
Ref. 01-513 m-CP Agar Base
CP Gram-positive cocci in Blood Agar Selective

Supplement
Ref. 06-013CASE
Contents
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Blood Columbia Agar Base Ref. 01034 or Blood Agar Base Ref. 01-352 in order to prepare
500 mL of Staphylococcus and Streptococcus selective
blood agar.
Precautions
Applicable media
Ref. 01-034 Blood Agar Base (Columbia)
Ref. 01-352 Blood Agar Base
Vial contents
Colistin sulfate ........................................ 5,00 mg
Nalidixic Acid, sodium salt ...................... 7,50 mg
Distilled Water ........................................ 5,00 mL
Directions
to 500mL of blood agar. Homogenize and use as per
each monography medium.
been added.
221
Cycloheximide Selective Supplement

Ref. 06-022CASE
Precautions
Contents


supplement 500 mL of WL Nutrient Agar or Broth Ref.
01-210 or 2-210 in order to prepare 500 mL of WL Differential Agar or Broth.
Applicable media
Ref. 01-210 WL Nutrient Agar
Ref. 02-210 WL Nutrient Broth
Vial contents
Cycloheximide ............................................. 2 mg
Sodium chloride ........................................... 8 mg
Distilled water .............................................. 5 mL
Directions
been added.
Cycloserine Selective Supplement

Ref. 06-116CASE
Precautions
Contents

The box contains 10 vials. Each vial is sufcient to supplement 250 mL of Tryptose Sulte Cycloserine Agar
Base (TSC Agar) Ref. 01-278.
Vial contents
D-Cycloserine ..........................................100 mg
Distilled water ..............................................5 mL
Directions
cap. Shake to dissolve and aseptically add the solution to 250 mL of sterile agar base cooled to 50C. If
it is desired, add 20 mL of Egg Yolk Sterile Emulsion
(Ref. 06-016). Homogenize and distribute the complete
medium into the plates.
Note: Dont heat the media once the supplements has
been added.
222
Applicable media
Ref. 01-278 Tryptose Sulte Cycloserine Agar (TSC
Agar)
Ferric Ammonium Citrate for Bacteriology

Ref. 06-112CASE
Ref. 06-113CASE
Contents
Contents
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Listeria Enrichment Broth acc. Fraser
Ref. 02-496 in order to prepare 500 mL of Fraser broth.
Vial contents
The box contains 10 vials. Each vial is sufcient to supplement 500 ml of Lactose Sulte Broth Base Ref. 02-519 in
order to prepare 500 ml of Lactose Sulte Broth.
Vial contents

Ferric ammonium citrate ......................... 250 mg
Distilled water ............................................. 5 mL
Necessary amount for 500 ml of complete medium.

Ferric ammonium citrate ......................... 312 mg
Directions
to 500 mL of sterile broth base cooled to 50C.
been added.
Precautions
Directions
cap. Shake to dissolve and aseptically add the solution to
500 ml of sterile broth base cooled to 50C.
Note: Dont heat the media once the supplement has been
added.
Precautions


Applicable media
Applicable media
Ref. 02-496 Listeria Enrichment Broth Base

acc. to Fraser
Ref. 02-519 Lactose Sulte Broth Base
Listeria Selective Supplement for Enrichment

acc. FDA/IDF
T
Ref. 06-107CASE
R-61-25-68
S-36/37-45-53
Contents
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Listeria Enrichment Broth Base acc.
Lovett Ref. 02-498 in order to prepare 500 mL of Listeria
enrichment broth according FDA and IDF/FIL.
Vial contents
Nalidixic acid, sodium salt ...................... 20,0 mg
Cycloheximide ........................................ 25,0 mg
Acriavine ................................................. 7,5 mg
Distilled water ........................................... 5,0 mL
Directions
been added.
Precautions
Applicable media
Ref. 02-498 Listeria Enrichment Broth Base acc. Lovett.
223
Listeria Selective Supplement for Primary

Enrichment (UVM I)
Precautions
Ref. 06-106CASE
R-61-25-68
S-36/37-45-53

Contents
supplement 500 mL of Listeria Enrichment Broth Base
(UVM) Ref. 02-472 in order to prepare 500 mL of Listeria primary enrichment medium.
Applicable media
Ref. 02-472 Listeria Enrichment Broth Base (UVM)
Vial contents
Nalidixic acid, sodium salt ......................... 10 mg
Acriavine .................................................... 6 mg
Distilled water .............................................. 5 mL
Directions
been added.
Listeria Selective Supplement for Secondary Enrichment

(UVM II / Fraser)
Ref. 06-111CASE
Contents
T
R-61-25-68
S-36/37-45-53

supplement 500 mL of Listeria Enrichment Broth Base
(UVM) Ref. 02-472 in order to prepare 500 mL of Listeria secondary enrichment medium (UVM II formulation);
or to supplement 500 mL of Listeria Enrichment Broth
acc. Fraser Ref. 02-496 in order to prepare 500 mL of
Fraser Broth.
Vial contents
Nalidixic acid, sodium salt ..................... 10,0 mg
Acriavine ..............................................12,5 mg
Distilled water ..........................................5,0 mL
Directions
been added.
224
Precautions
Applicable media
Ref. 02-472 Listeria Enrichment Broth Base (UVM)
Ref. 02-496 Listeria Enrichment Broth Base acc. to
Fraser
MUG Supplement
Ref. 06-102CASE
Precautions
Contents

The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Coliforms Agar or Broth.
Applicable media
Vial contents
MUG (4-methilumbeliferil--D-glucuronide) .....50 mg
Distilled water ..............................................5 mL
Directions
to 500 mL of agar or broth cooled to 50C.
MUG supplement may be added to almost all the media
that allow the growth of Escherichia coli for its identication. However, results will be more reliable in all those
media that are selective for coliforms. Attached is a list of
the most currently used. In our Culture Media Handbook
you will nd a table indicating the aspect of Escherichia
coli in each medium.
been added.
Ref. 01-047 CLED Agar (Brolacin Agar)

Ref. 01-118 MacConkey Agar (Eur. Phar. Medium H)
Ref. 01-164 Violet Red Bile Agar (VRB Agar)
Ref. 01-220 Violet Red Bile Dextrose Lactose Agar
(VRBDL Agar)
Ref. 01-484 E.coli Direct Agar (ECD Agar)
Ref. 02-041 Brilliant Green Bile 2% Broth
Ref. 02-060 EC Broth
Ref. 02-105 Lactose Broth (Eur. Phar. Broth Medium D)
Ref. 02-108 Tryptose Lauryl Sulfate Broth
Ref. 02-118 MacConkey Broth
Ref. 02-120 MacConkey Modied Broth
Ref. 02-611 MacConkey G Broth (Eur. Phar. Medium G)
Nalidixic Acid Selective Supplement

Ref. 06-124CASE
Directions
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of CN Selective Agar for Pseudomonas
(Ref. 01-609).
Mix the liquid with the powder by pressing downd on the

cap. Shake to dissolve and asseptically add the solution
been added.
Vial Contents
Precautions
Contents
Necessary amount for 500 mL of complete medium:

Nalidixic acid sodium salt ........................ 7,5 mg
Distilled water .......................................... 5,0 mL

Applicable media
Ref. 01-609 CN Selective Agar Base for Pseudomonas.
225
Oxford Agar Selective Supplement

T
Ref. 06-109CASE
R-25-52/53-61/68
S-36/37-45-53-61
Contents
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Oxford Agar Base Ref. 01-471 in
order to prepare 500 mL of Listeria selective agar (Oxford
formulation).

Applicable media
Ref. 01-471 Oxford Agar Base
Vial contents
Acriavine ................................................... 2,5
Fosfomicyn ................................................. 5,0
Sodium cefotaxim ....................................... 1,0
Colystin ..................................................... 10,0
Cycloheximide ........................................ 200,0
Distilled water ............................................. 5,0
Precautions
mg
mg
mg
mg
mg
mL
Directions
500 mL of sterile agar base cooled to 50C.
been added.
Oxytetracycline Selective Supplement

Ref. 06-115CASE
Contents
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Sabouraud with Oxytetracycline Agar
Base Ref. 01-275.
Vial contents
Oxytetracycline HCl .................................. 50 mg
Directions
to 500 mL of sterile agar base cooled to 50C. Homogenize and distribute the complete medium into the plates.
been added.
226
Precautions
Applicable media
Ref. 01-275 Saboraud with Oxytetracycline Agar (OGYEA)
Palcam Agar Selective Supplement

Ref. 06-110CASE
Precautions
Contents


supplement 500 mL of Palcam Agar Base Ref. 01-470
in order to prepare 500 mL of Listeria Selective Agar
(Palcam formulation).
Applicable media
Ref. 01-470 Palcam Agar Base
Vial contents
Acriavine ............................................... 2,5 mg
Polymixin B sulphate .............................. 5,0 mg
Sodium ceftazidime ............................. 10,0 mg
Distilled water ......................................... 5,0 mL
Directions
been added.
Polymixin B Sulfate Selective Supplement

Ref. 06-021CASE
Precautions
Contents


supplement 500 mL of Bacillus cereus Agar Base Ref.
01-262.
Applicable media
Ref. 01-262 Bacillus cereus Agar
Ref. 01-487 Bacillus cereus Selective Agar
Vial contents
Polymixin B sulphate ................................ 50 mg
Directions
cap Shake to dissolve and aseptically add the solution to
450 mL of sterile agar base cooled to 50C.
Add also 50 mL of sterile Egg Yolk Emulsion. Homogenize and distribute the complete medium into the plates.
been added.
227
Rosolic Acid Selective Supplement

Ref. 06-085CASE
Contents
F
R-11
S-7-16
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Fecal Coliforms Agar or Broth (m-FC)
Ref.1-287 or 2-287 in order to prepare 500 mL of m-FC
complete medium.
Vial contents
Rosolic Acid ............................................. 50 mg
Ethanol ...................................................... 5 mL
Directions
cap. Shake till total dissolution and aseptically add the
solution to 500 mL of agar or broth cooled to 50C.
Use medium newly made.
been added.
228
Precautions
Applicable media
Ref. 01-287 Fecal Coliforms Agar (FC Agar)
Ref. 02-287 Fecal Coliforms Broth (FC Broth)
SC Selective Supplement
Ref. 06-012CASE
Contents
R-25-32-42/43-51/53
S-7-22-24-37-45-61
The box contains 10 vials. Each vial is sufcient to supplement 500 mL of Blood Columbia Agar Base Ref. 01034 or Blood Agar Base Ref. 01-352 in order to prepare
500 mL of Clostridium ssp. selective agar.
Precautions
Applicable Media
Ref. 01-034 Blood Columbia Agar Base
Ref. 01-352 Blood Agar Base
Vial contents
Sodium Azide .......................................120,0 mg
Neomycine sulfate ..................................90,0 mg
Directions
to 475 mL of sterile agar base cooled to 50C. Add 25
mL of debrinated blood . Homogenize and distribute
into the plates.
been added.
Disodium disulte (meta-bisulte) for Bacteriology

Ref. 06-114CASE
Contents
The box contains 10 vials. Each vial is sufcient to supplement 500 ml of Lactose Sulte Broth Base Ref. 02-519 in
order to prepare 500 ml of Lactose Sulte Broth.
Precautions
Reagent for laboratory use only.
Applicable Media
Vial contents
Ref. 02-519 Supplement for Lactose Sulte Broth

Disodium sulte ....................................375,0 mg
Directions
500 ml of sterile broth base cooled to 50C.
been added.
229
Reagents
Barrits Reagent
Ref. 06-027
Presentation
F
R-11-36
S-7-16-26
100 mL dropper ask
Specication
Reagent for the Voges-Proskauer test in enterobacteriaceae.
Description
All the enterobacteria ferment dextrose, but some species like Klebsiella, Enterobacter, etc..., do it following
the 2-3-butanediol path and other species like E.coli,
Salmonella, etc..., do it by the mix acid path.
Voges Proskauer test shows the production of 2-3butanediol and acetoine, that are only produced in big
amounts in the 2-3-butanediol path. The basis of the test
is that these compounds, in alkaline medium and with
air, bear an oxidation and become diacethyl, which at the
same time reacts with guanidine producing very visible
coloured compounds.
OMeara, in 1931, observed that adding creatine to the
alkaline solution (OMearas Reagent, Ref. 06-006) aids
diacethyl reaction with guanidine, and then it was easier
to detect the red coloured compounds.
Later, in 1936, Barrit demonstrated that the addition
of an alcoholic solution of alpha-naphtol 5% (Barrits
Reagent, Ref. 06-027) increased very much sensibility,
and it was possible to obtain positive reaction even when
the nal concentration of diacethyl was very low. It is
important to add the Barrits Reagent before the alkaline
solution.
Technique
Microorganism to be assayed is inoculated in MRVP
Broth (Ref. 02-207) and is incubated at 30C for a period
between 3 and 5 days maximum.
Just before read, add Barrits Reagent (Ref. 06-027)
until all the medium gets a milky look. Following, add
OMearas Reagent (Ref. 06-006) until the milky look
disappears and then shake chiey. Relative volumes of
each reagent depend on the initial volume of inoculated
medium.
When test is positive a violet pinked colour appears before 5 minutes, starting from top. When test is negative
there is no change ol colour.
There is a quicker way to perform the Voges-Proskauer
test, with very little volumes of medium and massive
inocules. This way allows very short incubations (18-20
hours) and the read may be accelerated by heating up
the culture almost to boiling after adding the reagents.
However, this method increases the possibility of getting
wrong results.
References
BARRY, A.L., K.L. FEENEY (1967) Two Quick Methods
for the Voges Proskauer Test. Appl. Microbiol. 15:11381141.
BARRIT, M.M. (1936) The intensication of The Voges
Proskauer Reaction by the Addition of alpha-Naphtol. J.
Pathol. Bacteriol. 42:441-453.
BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles
of Biochemical Tests in Diagnostics Microbiology. John
Wiley Sons. N.Y.
OMEARA, R.A.Q. (1931) A simple, Delicated and Rapid
Method of Detecting the Formation of Acetylmethyl-carbinol by Bacteria Fermenting Carbohydrate. J. Pathol.
Bacteriol. 34:401-406.
McFADDIN, J.E. (2000) Biochemical tests for identication of medical bacteria. 3rd. Ed. Cippincott William &
Wilkins. Philadelphia.
233
Crystal Violet Dye Solution for Gram Staining

Ref. 06-029
Presentation
Xn
R-40-52/53
S-36/37-61
100 mL dropper ask

1 L dropper ask
Description
This solution has been prepared according to the specications by Hucker for the Gram staining, and it is very
stable though, when it is too old, it may require ltration
immediately before the use. Elderliness does not affect
the staining properties but may force to make decouloring times longer.
Technique
Fix the smear following the habitual method and let it
cool or dry.
Cover the extension with Crystal Violet Dye Solution
(Ref. 06-029) and let it act for 1 minute
Wash the exceed of colouriser. The best way is to put
the preparation in a precipitate glass with uent water.
Do not wash excessively. This step may be critical for
the rest of the test.
Cover the preparation with Lugol Solution (Ref. 06-030)
and drain immediately. Cover again with new solution
and let it act for 1 minute.
Wash softly again with water. To put the preparation in
water uent for 5 seconds will be enough.
Decolourate, pouring the Gram Decoluriser (Ref. 06031), drop to drop, over the slanted microscopical slide
until total decolourising. Anycase, this step may not be
longer than 60 seconds.
Wash with water to stop the decolouring action.
234
Cover the preparation with Safranine Dye Solution (Ref.

06-032) and let it act for 1 minute.
Wash gently to remove the excess of colouriser, putting
the preparation in uent water for 1-2 seconds.
Dry and observe under microscope in homogeneous
inmersion.
Microorganisms that get coloured by the rst colouriser,
Crystal Violet, become dark blue coloured and it is said
that they take the gram, and they are call grampositive
(G+). Those microorganisms that just get coloured by
the contrast colouriser become red and they are called
gramnegative (G-).
Most of eukariote cells, except yeasts, are coloured as
gramnegative and thus the staining is not very signicative. In spite of, it is one of the rst levels in the systematic identication of prokariote: between the bacteria, all
the coci, except Neisseria and Veillonella, are grampositive, and all the sporogen bacilli and some part of the
other bacilli are grampositive too. Spiriles, vibria, rikettsia, clamidia and most bacilli are gramnegative.
Should actinomycete presence is suspected or microorganisms are not well coloured as grampositives, it is advisable to use Crystal Violet with Anilin for Actinomycete.
References
BARTHOLOMEW, J.W. (1962) Variables Inuencing Results, and the Precise Denition of Steps in Gram Staining as a Means of Standardizing the Results Obtained.
Stain Technol. 37:139-155.
PAIK, G. (1980) Reagents, Stain and Miscellaneous
Procedures, in Manual of Clinical Microbiolgy by Lenette,
Balows, Hausler and Truant (eds.). ASM, Washington.
CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed.
Decolouriser for Gram Staining

Ref. 06-031
Presentation
Xi
R-11-36-66-67
S-7-9-16-26
100 mL dropper ask

1 L dropper ask
Description
Grams decolouriser is a mixture of alcohol and acetone
especially adapted to act softly and quickly over base
colourings. It use to be enough 15 or 20 drops to achive
an total decolourising of a correctly coloured smear.
Technique
cool.

inmersion.
gramnegative (G-).
References

235
Kovacs Reagent
Ref. 06-018
Presentation
C
R-10-20-34
S-26-36/37/39-45
100 mL ask
1L in ask
Description
Many microorganisms can produce indole (=benzopyrrole) from Tryptophane thanks to a Tryptophanase, in
a process favored by oxygen and inhibited by glucose.
We therefore recommend that media used in this test
contain no glucose, present a high Tryptophane content
and are incubated aerobically.
The indole production property constitutes a classical
test for the differentiation of Escherichia and Enterobacter, integrated in the IMViC. It is also widely used for
the differentiation of other non enteric microorganisms.
The indole test can be conducted by various means, still
the biochemical basis of the reaction remain the same.
When a Pyrrol is mixed with a heated alcoholic p-dimethylaminobenzaldehyde solution, a peculiar cherry-red
coloring develops (Rosindole). If the reagents solution
is prepared with concentrated hydrochloric acid it will not
be necessary for it to progress in hot, as is the case with
our reagents.
The reagent which was initially used was that of EhrlichBhme (Ref. 06-024) with a previous extraction and concentration in Xylene, Ether, Chloroform or Toluol. Later
on Kovacs modied the original reagent by replacing
the ethanol with amyl-alcohol, so that a previous extraction was no longer necessary. In 1956 Gadenbusch and
Gabriel proved that the Kovacss reagent was much
stable if instead of Amyl alcohol, Butilic or Isoamyl were
used.
Nevertheless, the indole test reaction with paradiaminobenzaldehyde is not very specic since at least 17
compounds close to Indole are known to react similarly.
Although other reagents such as Oxalic acid and Hydroxilamine HCI have been proposed, their use has not
been widespread.
Since indole is one of the most volatile compounds of

the group, some authors choose to conduct the test
using strips impregnated in reagent. Others suggest
placing the reagent directly on the caps swabs, avoiding
all contact with the culture medium so that the reaction
will occur with just the indoles steam. Isenberg and
Suddenheim demonstrated that if a previous extraction
with Toluene was performed, only indole and AlphaMethylindole (=indoleacetic acid) were detected. Such
is the most common practice nowadays when using the
Ehrlich-Bhme reagent.
Technique
When conducting the indole production test on various groups of bacteria, an appropriate reagent for each
group must be considered. Kovacss reagent (Ref.
06-018) is recommended for enterobacteria while the
Ehrlich reagent is for non-fermentating and anaerobes in
general.
The directions to follow during the assay are:
Inoculate the pure culture to be veried in a high Tryptophane content medium, as for example the Indole-Nitrite Fluid Medium (Ref. 03-101), the SIM Medium (Ref.
03-176) or a non-glucose tryptone broth. Incubate at
35C for 48 hours. Incubation time can be reduced to 4
hours if a massive inoculum in solid medium is done followed by seeding of a small volume (0,5 mL) of culture
medium.
In both cases, examination after incubation in the following way:
a) Kovacss Reagent (Ref. 06-018, for enterobacteria)
Add 0,5 mL of reagent to the broths surface, shaking
lightly to help extraction. If a cherry-red color develops in
less than a minute it will be considered a POSITIVE REACTION. No change in the original coloring constitutes a
negative reaction.
b) Ehrlich-Bhme Test
Add 1 mL of Xylene or Toluene to the broth and shake
energically to help extraction. Allow to stand for 2 minutes until both layers separate. Then slide 0.5 mL of the
reagent carefully down the sides of the tube, making
sure there is no agitation. Should a dark red colored ring
appear in the interface, it will be considered a POSITIVE
REACTION.
Storage
Reagents must be stored refrigerated and avoiding
direct light.
References
BHME, A. (1905) Die Anwendung der Ehrlichschen Indolreaktion fr bakteriologische zwecke. Zentralbl. Bakt.
Parasit. Abt 1, Jena 40:129-133
236
Kovacs Reagent
KOVACS, N. (1928) Eine vereinfachte Methode zum
Nachweis der Indolbihdung duch bakterien. Z. Immunitats. Forsch. Exp. Ther. 55:311-315
GADEBUSCH, H.H. and GABRIELS, S. (1956) Modied
Stable Kovacss Reagent for the detection of Indol. Am.
J. Clin. Pathol. 26:1373-1375
ISENGERG, H.D. and SUNDHEIM, L.H. (1958) Indole
Reactions in Bacteria J. Bact. 75:682-690
CENTER FOR DISEASE CONTROL (1968)Identication of Unusual Pathogenic Bacteria Atlanta G.
VIRGINIA POLYTECHNICAL INSTITUTE (1972) Anaerobe Laboratory Manual Blaksburg,Va.
EDWARDS, P.R. and EWING, W.H. (1972) Identication of Enterobacteriaceae 3rd Ed. Burgess
Pub. Co. Minneapolis
McFADDIN, J.F. (2000) Biochemical tests for identication of medical bacteria. 3rd. Ed. Lippincott William &
of coliforms thermotolerant coliforms and presumptive
E.coli - MPN method.
Kovacs reaction on: Ref. 02-277 Tryptone Phosphate

Water, Ref. 02-460 Tryptose Lauryl Sulfate Mannitol
Tryptophan Broth, Ref. 02-494 Buffered Peptone Water
Eur. Phar., Ref. 03-156 Tryptone Water.
Left: control; center: Salmonella typhimurium ATCC

14028 (negative reaction); right: Escherichia coli ATCC
25922 (positive reaction).
Lactophenol Blue
Ref. 06-037
Presentation
C
R-21/22-34-41
S-26-36/37/39-45
100 mL ask
1 L ask
Specication
Reagent for staining of fungi in fresh and xed preparations.
Description
Lactophenol Blue is an excellent colouriser for fresh
preparation of fungi, since it has, in a single solution, the
propierties of a mordant, a xer and a colouriser.
In fungi preparations for microscopical examination is
not usual to use water neither aquose colouriser solutions, since most of the moulds expel water and remain
trapped in air microbubbles. This reason makes Lactophenol Blue an idoneous medium for the examination,
becase it moistens the structures at the same time that it
acts like a xer and soft mordant.
In other hand, its nature makes the preparations useful
longer because evaporation is smaller. This effect can be
enhanced if the preparations are sealed with vaspar or
nails varnish.
The Cotton Blue, China Blue or Soluble Anilin Blue is
probably, an impuried and complex colouriser but, its
efcacy in the staining of moulds and plants material has

been demonstrated.
Technique
Put a bit of mould to be assayed in a clean microscopical
slide and pour one or two drops of Lactophenol Blue.
Disperse the material with two needles, mixing it with the
colouriser.
Add a couple of drops of water and homogenize all
before putting the overglass.
Heat slightly the preparation over a ave until it will be
almost boiling. In that precise moment, press to remove
all the excess of liquid and seal the borders with vaspar
or nails varnish.
Preparation is ready for the microscopical examination.
References
HARRIGAN, W.F. and McCANCE, M.E. (1976) Laboratory Methods in Food and Dairy Microbiology. Academic
Press. London.
William and Wilkins. Baltimore
LARONE, D.H. (2002) Medically important fungi. ASM
Press. Washington, DC.
237
Lugol Solution for Gram Staining

Ref. 06-030
Presentation
100 mL dropper ask
1 L dropper ask
Description
Iodine solution has been prepared according to the
specications by Burke, therefore it is more stable than
the classical Lugol formulation, and it does not affect
the colouring. The solution may be stored for months at
room temperature, but if a characteristic amber colour is
observed, it must be discarded.
Technique
cool.
238

inmersion.
gramnegative (G-).
References
William and Wilkins. Baltimore.
Malachite Green
Ref. 06-038
Presentation
100 mL ask
1 L ask
Description
Malachite Green for Spores has been prepared according to the formulation by Barholomew and Mittwer, in
1950, which was a modication over the procedure by
Schaeffer and Fulton, in 1933, that was based in the
original one by Wirtz.
Essencially, the modication is the supression of the step
where the preparation was heated up. To achieve this
without affecting the test, they prepared a more concentrated colouriser and let it act longer, and afterwards they
did the contrast with a softer colouriser, Safranine Dye
Solution (Ref. 06-032).
This stain is a saturated and stabilized aquose solution
of malachite green. To use it with the Schaeffer technique it has to be diluted at 50% to avoid the formation
of precipitates.
Technique
Prepare a smear of microorganism, in the habitual way,
and let it dry. Fix strongly by passing the microscopical
slide over a ame about 20 times.
Before doing the staining, let cool the microscopical
slide. Cover all the smear with Malachite Green for
Spores Stain and let it act for 10 minutes.
Wash with water to remove the excess of colouriser.
Contrast by covering the smear with Safranine Dye Solution (Ref. 06-032) and letting it act for 15-30 seconds.
Wash again, dry and perform the microscopical examination in homogeneous inmersion.
Cellular bodies appear red or pinked coloured, whereas
spores are green.
Should the Schaeffers technique is wanted to be used
diluting the colouriser, once the smear is covered with
Malachite Green, do not let it act for 10 minutes and
instead of this, bring it to the boiling 3 consecutive times.
Then follow the described technique.
References
BARTHOLOMEW, J.W., MITTWER, T. (1950) A Simplied Bacterial Spore Stain. Stain Technol. 24:153-156.
SCHAEFFER, A.B., M. FULTON (1933) A Simplied
Method of Staining Endospores. Science, 77, 194.
239
Methyl Red
Ref. 06-007
Presentation
F
R-11
S-7-16
100 mL ask
1 L ask
Specication
Indicator solution for the fermentation test in enterobacteria.
Description
Clark and Lubs, in 1915, described the Methyl Red test
to distinguish between the E. coli group and Enterobacter.
All the enteric bacteria ferment dextrose, some do it
following the 2-3-butanediol path, like Klebsiella, Enterobacter, etc..., and other follow the mix acid path,
like E.coli, Salmonella, etc...In this last case, there is an
accumulation of acid products produced by the sugar
unfoldment. This causes a decrease of pH, reaching a
value up to 4 after the incubation. Adding the methyl red
alcoholic solution the change of the indicator may be detected: it remains yellow over pH 5,1 and red below pH
4,4. If the change is positive it means that the dextrose
fermentation followed the mix acid path, since in the 23-butanediol path, after 3 days of incubation, predominate the neutral or alkaline products.
Too early reads may produce false positive results.
240
Technique
Inoculate a tube of MRVP Broth (Ref. 02-207) and
incubate at 30C for 3-5 days. Take 5 mL of culture and
transfer them to a clean tube and then add 5 or 6 drops
of indicator.
Positive reaction is shown by the presence of a red
colour, whereas the negative reaction produce a yellow
or orange colour.
Most cases, a 48 hours incubation at 37C is enough,
but if the results are doubtous, the assay must be repeated incubating at 30C for 5 days.
There is a quicker way to perform the test: suspend a
loop of bacterial growth from a solid medium in 0,5 mL
of Azide Dextrose Broth acc. to Rothe (Ref. 02-027) and
incubate at 37C for 18 hours. Add a couple of drops of
indicator and read the results as in the last case.
References
BARRY, A.L., K.L. BERNSOHN, A.P. ADAMS, L.D.
THRUPP (1970) Improved 18-hour methyl red test. Appl.
CLARK, W.M. and LUBBS, H.A. (1915) The Differentiation of Bacteria of the Colon-Aerogenes Family by the
use of indicators. J. Infect. Dis. 17:161-173.
EDWARDS, P.R., W.H. EWING (1972) Identication of
Enterobacteriaceae. 3rd. Ed. Burgess Pub Co. Minneapolis.
Wiley Sons. N.Y.
McFADDIN, J.F. (2000) Biochemical tests for identication of medical bacteria. 3rd. Ed. Lippincott William and
Nitrates Reduction Reagents

Nitrates A Solution
Ref. 06-003
Presentation
The scheme for the global process is the following:

C
R-10-35
S-23.2-51-26-36/37/39-45
100 mL dropper ask
Nitrates B Solution
Ref. 06-004
Presentation
C
R-10-35
S-23.2-51-26-36/37/39-45
100 mL dropper ask
Specication
Generally, the Griess-Ilosvays reagents detect the

presence of nitrites with bacterial origin in a medium that
initially has no nitrites (Indole Nitrite Fluid Medium, Ref.
03-101 and Nitrate Broth, Ref. 02-138).
The scheme for the complete reaction is the following:
Griess-Ilosvays Reagents for the verication of the

nitrates reduction through nitrites detection.
Description
To use them, mix equal parts of the solutions A and B.
Once they are mixed, the reagents are stable just for a
few hours. Alone they may be stored for several months
at room temperature. Nitrates B solution may produce a
slight cristalization that does not affect its efcacy. This
process is accelerated with refrigeration, therefore it is
recommended not to store them in the refrigerator.
Nitrates reduction in bacteria is performed through several ways and it obeys different procedures.
Nitrates Assimilation involves a reduction to ammonia
in several steps where nitrite may be detected. The ammonia that is produced is nally incorporated to the cellular material. However, in the Deassimilation process,
nitrite is used as the nal receiver of electrons, and thus,
more than an assimilation process it is an energetic reaction of respiration without oxygen, and this fact allows
the facultative growth of many aerobic in anaerobiosis.
In this case, it is usual the presence of nitrite acumulations, which may be toxical for the microorganism. In
other cases, nitrate may reduce itself to gas states and it
is expeled as free nitrogen bubles. This process is called
Denitrication, since it makes the active ion (NO3) an
inert gas (N2).
References
WALLACE, G.I., S.L. NEAVE (1927) The nitrite test as
applied to bacterial cultures. J.Bact. 14:377-384.
BLAZEVIC, D.J., G.M. EDERER (1975) Principles of
Biochemical Tests in Diagnostics Microbiology. John
Wiley Sons. NY.
FORBES, B.A., D.F. SAMM, A.S. WEISSFELD (1998)
Bailey & Scotts Diagnostics Microbiology. 10th. Ed.
Mosby. St. Louis.
GRIESS, P. (1879) Liebereinige Azoverbindungen. Ber.
Deutsch. Chem. Geselkch. 12:426-427.
McFADDIN, J.F. (2000) Biochemical Tests for identication of medical bacteria. 3rd. Ed. Lippincott William and
241
OMearas Reagent
Ref. 06-006
Presentation
C
R-22-35
S-26-36/37/39-45
100 mL dropper ask
Specication
Reagent for the Voges-Proskauer test in enterobacteriaceae.
Description
All the enterobacteria ferment dextrose, but some species like Klebsiella, Enterobacter, etc..., do it following
the 2-3-butanediol path and other species like E.coli,
Salmonella..., do it by the mix acid path.
Voges Proskauer test shows the production of 2-3butanediol and acetoine, that are only produced in big
amounts in the 2-3-butanediol path. The basis of the test
is that these compounds, in alkaline medium and with
air, bear an oxidation and become diacethyl, which at the
same time reacts with guanidine producing very visible
coloured compounds.
OMeara, in 1931, observed that adding creatine to the
alkaline solution (OMearas Reagent, Ref. 06-006) aids
diacethyl reaction with guanidine, and then it was easier
to detect the red coloured compounds.
Later, in 1936, Barrit demonstrated that the addition
of an alcoholic solution of alpha-naphtol 5% (Barrits
Reagent, Ref. 06-027) increased very much sensibility,
and it was possible to obtain positive reaction even when
the nal concentration of diacethyl was very low. It is
important to add the Barrits Reagent before the alkaline
solution.
242
Technique
Microorganism to be assayed is inoculated in MRVP
Broth (Ref. 02-207) and is incubated at 30C for a period
between 3 and 5 days maximum.
Just before read, add Barrits Reagent (Ref. 06-027)
until all the medium gets a milky look. Following, add
OMearas Reagent (Ref. 06-006) until the milky look
disappears and then shake chiey. Relative volumes of
each reagent depend on the initial volume of inoculated
medium.
When test is positive a violet pinked colour appears before 5 minutes, starting from top. When test is negative
there is no change ol colour.
There is a quicker way to perform the Voges-Proskauer
test, with very little volumes of medium and massive
inocules. This way allows very short incubations (18-20
hours) and the read may be accelerated by heating up
the culture almost to boiling after adding the reagents.
However, this method increases the possibility of getting
wrong results.
References
BARRY, A.L., K.L. FEENEY (1967) Two Quick Methods
for the Voges Proskauer Test. Appl. Microbiol. 15:11381141.
BARRIT, M.M. (1936) The intensication of The Voges
Proskauer Reaction by the Addition of alpha-Naphtol. J.
Path. Bact. 42:441-453.
Wiley Sons. N.Y.
OMEARA, R.A.Q. (1931) A simple, Delicated and Rapid
Method of Detecting the Formation of Acetylmethyl-carbinol by Bacteria Fermenting Carbohydrate. J. Pathol.
Bact. 34:401-406.
McFADDIN, J.F. (2000) Biochemical tests for identication of medical bacteria. 3rd. Ed. Lippincott William and
Oxidase Reagent
Ref. 06-057
Presentation
5 g ask
Specication
Reagent for the detection and verication of bacterial
citochrome-oxidase.
Description
The chemical formulation for the Oxidase Reagent is
N,N-dimethyl-P-phenyldiamine-2-HCl, and it is also
designated as 4-amino-N,N-dimethylaniline-2-HCl. It is
advisable to store it in powder at 4C since it has a very
short life time when is dissolved.
Directions
Prepare an aquose solution of Oxidase Reagent 1% immediately before the use. It is recommended to prepare
just the amount that is going to be used, since once
diluted it will work just for a week, even if it is kept at
4C and avoiding direct light. Althought self oxidation is
restrained with the addition of ascorbic acid 0,01%, if the
liquid is dark it must not be used. The normal colour for
the solution is transparent or slightly pinked.
Technique
There are several techniques to determinate citrocromeoxidase in the different genus. The more standardized
are the following:
a) Soak a ltration paper disc with the reagent and put it
over a clean Petri plate. Take a colony and spread
it over the paper. This step must be performed with
a Platinum-Iridium loop (Ref. 5-006) or a Pasteur pipette. The use of metallic objects (nicrom loop, etc...)
may produce wrong positive results.
b) Flood the colony with reagent directly in the plate. Following this way, colonies are not able to subculture,
but the test does not interfere with the Gram staining
and the colony may be observed at the microscope.
Positive reaction is shown by the presence of a pink
colouring, that becomes dark red and nally black after
10 minutes.
The basis of the test is the following:

Citochromes are hemoproteins that act as oxidant enzimes in the transportant chains of oxidative phosphorilation electrons. Generally, citochromes are found just in
the aerobic bacteria, whereas the strict anaerobic does
not present them.
Citochrome-oxidase found even in the enterobacteriaceae is composed by a3 citochrome. However, the
substance that the Oxidase Reagent is able to reduce is
c citochrome, thus the test just determinates the presence of c citochrome and therefore test is positive only
for the bacteria that have c citochrome in their respiratory chains.
Reagent is very unstable to oxygen, and light acts as a
catalyst in the self oxidation and so the reagent may be
stored in amber ask. When the reaction in the colony is
very slow, it has to be considered negative or very weak,
since the lately appaerance of colour is more attributable
to the spontaneous oxidation than to the truly presence
of c citochrome.
Generally, except a few cases, oxidase production is
linked to agelation in the following way: Polar agelation is related with oxidase positive; peritrichous agelation is related with oxidase negative.
The oxidation of c citochrome for the positive reaction
of oxidase is the following and it may be observed that
molecullar oxygen is absolutely necessary:
References
GABY, W.L., C. MARTLEY (1957) Practical laboratory
test for the identication of Pseudomonas aeruginosa. J.
Bact. 74:356-358.
BLAZEVIC, D.J., G.M. EDERER (1975) Principles of
biochemical test in diagnostic Microbiology. John Wiley
Sons. NY.
FORBES, B.A., D.F. SAHM, A.S. WEISSFELD (1998)
Bailey & Scotts Diagnostic Microbiology. 10th. Ed.
Mosby. St. Louis.
of coliforms, thermotolerant coliforms and presumptive
E.coli - MPN method.
243
Safranin Dye Solution for Gram Staining

Ref. 06-032
Presentation
100 mL dropper ask
1 L dropper ask
Description
The contrast colouriser is composed by the classic
Safranine solution 0,25%. It is demonstrated that this
solution is more effective than the fuchsine. This contrast colouriser is employed also in many other staining
methods.
Technique
cool.
244

inmersion.
gramnegative (G-).
References

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Ref. 03-428 Acetamide Medium. Left: control; center:

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AFP Agar (Aspergillus avus/parasiticus Agar)

Selective solid medium for the isolation and enumeration

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