The HiMedia Manual

Dehydrated Culture Media
ost bacteria reproduce by an asexual
process called Binary Fission. In this
sequence of events, the chromosome
duplicates, the cell elongates, and
the plasma membrane pinches inward at the centre of
the cell. When the nuclear material has been evenly
distributed, the cell wall thickens and expands
inward to separate the dividing cells. Once the
division is complete, bacteria grow and
develop the features that make each
species unique.
Bacteria must meet certain nutritional
requirements in order to grow. Most
bacteria have relatively simple
requirements, with water an
absolute necessity. In addition
bacteria need foods that can serve
as energy sources and raw
materials for the synthesis of cell
components. These foods
generally include proteins for
structural compounds and
enzymes, carbohydrates for
energy, and a series of vitamins,
minerals, and inorganic salts.
A nutrient material prepared for
the growth of microorganisms in a
laboratory is called a culture
media, and the microbes that grow
and multiply in or on a culture
medium are referred to as a culture.
Since the time of Pasteur and Koch,
microbiologists have used broth media
containing beef extract for the cultivation
of bacteria. Nutrient Broth is the modern
form of this broth. When agar is used to
solidify the medium, the product is called
Nutrient Agar.
One of the major developments in microbiology was
Robert Koch's use of a solid culture medium on which

AD
bacteria would grow. He accomplished this by solidifying beef
broth by gelatin. When inoculated onto the surface of the
nutritious medium, bacteria grew vigorously at room
temperature and produced discrete, visible mounds of cells.
However, Koch was dismayed to find that certain bacteria
produced a chemical substance that was able to digest
gelatin. Also, gelatin liquefied at higher temperatures,
normally used to grow bacteria. Fanny Eilshemius, wife of
Koch's associate, Walter Hesse, suggested the solution to this
problem. For years, Fanny had been using a seaweed-derived
powder called agar to solidify her jams and jellies. Her mother,
who learned it from Dutch friends living in Java, had passed
the formula onto her. The use of agar in media preparation
was more valuable because it mixed easily with most liquids,
and once gelled, didn't liquefy even at higher temperatures.
Over the years many types of nutrient media were developed
for the cultivation of bacteria. These can be broadly classified
as Broth Media, Agar Media and Semisolid Media.
Bacteria grown in liquid broth medium usually forms colloidal
suspensions. However, agar media provide a surface for the
bacteria to grow and form colonies. Of the various types of
agar media Nutrient Media is the simplest. Nutrient media is
an undefined medium that contains complex ingredients, such
as yeast extract or casein hydrolysate that consists of a
mixture of many chemicals in unknown proportions. Nutrient
Agar is the most widely used nutrient media.
In complete contrast to the nutrient (undefined) media lies the
Chemically Defined Media (defined media). These media are
also referred to as Synthetic Media. These media do not
contain any animal, yeast or plant tissues. Also all the
ingredients used are of known composition and fill a specific
need. Media used to carry out assays (Vitamin Assay Media)
are examples of chemically defined media.
Another type of media are the Differential Media. This medium
helps to distinguish different organisms on a single plate.
MacConkey Agar is the typical example of a differential
medium. It contains the dyes neutral red and crystal violet in
addition to the carbohydrate lactose. Lactose fermenters
utilize lactose, take up the dyes and form red colored colonies.
Non-lactose fermenters grow to form colourless colonies.
Selective Media are used to grow only the selected organisms.
These media contain ingredients to inhibit the growth of
certain bacteria while selecting or allowing the growth of
others. MacConkey Agar contains bile salts to inhibit the
growth of Gram-positive organisms. Therefore in addition to
being a differential medium it is also a selective medium.
Other examples of selective media are EMB Agar, Hektoen
Enteric Agar, XLD Agar, and Mannitol Salt Agar etc.
Certain fastidious bacteria require Enriched Media containing
special nutrients to grow. Neisseria species are cultured on
Chocolate Agar, a heme protein-containing agar. This is
because these species require heme proteins for their growth.
Blood Agar is another example of an enriched media that is
used to culture streptococci.
Specialized media are generally required to culture
recombinant strains of certain bacteria used in molecular
biology studies. Minimal Media are employed for this purpose.
These media essentially contain a carbon source such as
glucose and various salts that provide essential elements like
magnesium, nitrogen etc. that allow the bacteria to synthesize
protein and nucleic acids.
Semisolid Media are employed to check for bacterial motility.
In this case, a small portion of agar is added to the growth
medium to make it stiff but not as solid as an agar medium.
The various motility agar media are examples of semisolid
media. Transport Media that are used in the transport of
specimens to the laboratory for cultivation are also an
example of semisolid media. Stuart Transport medium is a non-
nutrient soft agar gel containing a reducing agent to prevent
oxidation and charcoal to neutralize certain bacterial
inhibitors for gonococci.
Reference :
Alcamo E. I, 2001, Fundamentals of Microbiology, 6th Edition. Jones and
Bartlett Publishers.

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DehydratedCultureMedia
®
A-1 Broth M874
A-1 Broth is used for detecting faecal coliforms in water samples waste water, seawater and foods by MPN Method.
Composition **
Ingredients Gms/Litre
Casein enzymic hydrolysate 20.000
Lactose 5.000
Sodium chloride 5.000
Salicin 0.500
Polyethylene glycol p-isooctylphenyl ether (Triton 100) 1.000
Final pH ( at 25°C) 6.9±0.1
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 31.5 grams in 1000 ml distilled water. Heat if necessary
to dissolve the medium completely. Distribute 10 ml amounts into
tubes containing inverted Durhams tubes. Sterilize by autoclaving
at 15 lbs pressure (121°C) for 10 minutes.
Principle and Interpretation
Escherichia coli is used as the indicator organism to detect the
faecal contamination of water.Andrews and Presnell (1) devised
A-1 Medium, which was capable of recovering Escherichia
coli from estuarine waters in 24 hours instead of 72 hours by
avoiding the pre-enrichment step as recommended by APHA
(2).This greatly reduced the time required for the complete
identification of E. coli (3) by the elevated temperature and
most probable number (MPN) methods, routinely used for water
analysis.A-1 Medium substantially reduces the incidence of false
positive cultures.Also, Stanbridge and Delfino found that the
results obtained by using 3-hours pre-incubation step (using A-1
Medium) were statistically comparable with the two-step MPN
technique for the enumeration of E. coli in chlorinated waste-
water (4). Fast recovery of faecal coliforms from shell fish (5)
and sea water (6) was also reported.A-1 Medium also conforms
to the standard methods identified for the isolation of faecal
coliforms in food, water and wastewater (2, 7).
Casein enzymic hydrolysate provides carbonaceous and
nitrogenous substances required for bacterial metabolism. Lactose
and salicin act as energy sources and sodium chloride maintains
osmotic equilibrium. Polyethylene glycol p-isooctylphenyl ether
acts as a surfactant. Presence of gas bubbles in the inverted
Durhams tubes is a positive indication of presence of faecal
coliforms.The density of faecal coliform can be calculated by the
standard methods using the MPN table.
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder
Colour and Clarity
Light amber coloured clear solution after cooling to room temperature.
Reaction
Reaction of 3.15% w/v aqueous solution at 25°C. pH : 6.9±0.1
Cultural Response
M874: Cultural characteristics observed after an incubation at different
temperatures for 18-24 hours.
Organism Inoculum
(CFU)
Growth at 35°C Growth at 44.5°C
Bacillus subtilis ATCC 6633 50-100 none none
Enterobacter aerogenes ATCC
13048
50-100 luxuriant (may or
may not produce
gas)
poor-fair
Escherichia coli ATCC 25922 50-100 luxuriant with gas luxuriant with gas
Salmonella Typhimurium ATCC
14028
50-100 luxuriant without
gas
good without gas
Enterococcus faecalis ATCC 19433 50-100 poor none - poor
Reference
1. Andrews and Presnell, 1972,Appl. Microbiol., 23:521.
2. EatonA. D., Clesceri L. S., and GreenbergA.W., (Eds.), 2005, Standard Methods for the Examination of
Water andWastewater, 21st Ed.,APHA,Washington, D.C.
3. Andrews, Diggs andWilson, 1975,Appl. Microbiol., 29:130.
4. Standridge and Delfino, 1981,Appl. Environ. Microbiol., 42:918.
5. Hunt and Springer, 1978, J.Assoc. Off.Anal. Chem., 61:1317
6. Miescier et al, 1978, J.Assoc. Off.Anal. Chem., 61:772.
7. Downes F. P. and Ito K., (Eds.), 2001, Compendium of Methods for the Microbiological Examination of
Foods, 4th Ed.,American Public HealthAssociation,Washington, D.C.
Product Profile
Reconstitution 31.50 g/l
Quantity on Preparation (500g) 15.87 L
pH (25°C ) 6.9±0.1
Supplement None
Sterilization 121°C / 10 minutes
Storage Dry Medium : Below 30°C
Prepared Medium : 2-8° C
Note :Veg Medium is also available.

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®
AATCC Bacteriostasis Broth (FDA Broth) /
AATCC Bacteriostasis Agar M221/M231
AATCC Bacteriostasis Agar is used for the detection of antibacterial activity of fabrics while,AATCC Bacteriostasis Broth (FDA Broth) is
used for routine antibacterial testing of antiseptics and disinfectants.
Composition **
M221 M231
Ingredients Gms/Litre Gms /Litre
Peptic digest of animal tissue 10.000 10.000
Beef extract 5.000 5.000
Sodium chloride 5.000 5.000
Agar - 15.000
Final pH ( at 25°C) 6.8±0.2 7.2±0.2
Directions
Suspend 35 grams of M231 or 20 grams of M221 in 1000 ml
distilled water. Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Mix well and dispense as desired.
AATCC Bacteriostasis Agar is used in accordance with the
standard procedure (1, 2, 3).AATCC Bacteriostasis Broth (FDA
Broth) is useful for subcultures in phenol coefficient and dilution
tests of bacteriostatic, germicidal, sporicidal activity (1) and also
as a base for the preparation of AATCC Bacteriostasis Agar(2).
AATCC Bacteriostasis Agar may be used to carry stock cultures of
Escherichia coli and Staphylococcus aureus.Also, it is used for the
detection of antibacterial activity of fabrics.
Peptic digest of animal tissue and beef extract are sources of
carbon, nitrogen, vitamins and minerals. Sodium chloride provides
essential ions.
The test cultures of Escherichia coli and Staphylococcus aureus
are grown in AATCC Bacteriostasis Broth for 24 hours. 1 ml of
this culture is mixed with 150 ml of AATCC Bacteriostasis Agar
and poured into the plate. After the agar solidifies, a circular
sterile test fabric of 28.6 mm diameter is applied onto the plate.
Incubation is carried out at 35°C for 18 - 24 hours and inhibition
of growth around the test fabric is observed.
Quality Control
Appearance
M221: Cream to yellow homogeneous free flowing powder
Gelling
M231: Firm, comparable with 1.5% Agar gel
Colour and Clarity
M221:Amber coloured clear solution in tubes.
M231:Amber coloured clear to slightly opalescent gel forms in
Petri plates.
Reaction
M221:Reaction of 2.0% w/v aqueous solution at 25°C. pH : 6.8±0.2
M231: Reaction of 3.5% w/v aqeous solution at 25°C. pH : 7.2±0.2
Cultural Response
M221: Cultural characteristics observed after an incubation at 35-37°C
for 18-24 hours.
for 18-24 hours.
Organism Inoculum
(CFU)
Growth Recovery
M221
Escherichia coli ATCC 25922 50-100 good-luxuriant —
Pseudomonas aeruginosa ATCC 27853 50-100 good-luxuriant —
Staphylococcus aureus ATCC 6538 50-100 good-luxuriant —
Salmonella Typhi ATCC 6539 50-100 good-luxuriant —
M231
Escherichia coli ATCC 25922 50-100 good-luxuriant >=70%
Pseudomonas aeruginosa ATCC 27853 50-100 good-luxuriant >=70%
Salmonella Typhi ATCC 6539 50-100 good-luxuriant >=70%
Staphylococcus aureus ATCC 6538 50-100 good-luxuriant >=70%
Reference
1. Williams, (Ed.), 2005, Official Methods ofAnalysis of theAssociation of OfficialAnalytical Chemists, 19th
Ed.,AOAC,Washington, D.C.
2. Tech. Manual ofAATCC, 1985,Vol. 61,AATCC, ResearchTriangle Park,N.C.
3. Ruhle and Brewer, 1931, USFDA Methods ofTestingAntiseptics and Disinfectants, USDA Circ.:198.
Product Profile
Reconstitution (M221) : 20.00 g/l
(M231) : 35.00 g/l
Quantity on Preparation (500g) (M221) : 25.00 g/l
(M231) : 14.29 g/l
pH (25°C ) (M221) : 6.8±0.2
(M231) : 7.2±0.2
Supplement None
Storage Dry Medium : Below 30°C Prepared
Medium : 2-8° C

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®
AATCC Mineral Salts Agar M232
AATCC Mineral Salts Agar is recommended for evaluation of fungicides used on textiles and to study the resistance of textiles to mildew
and rot.
Composition **
Ammonium nitrate 3.000
Monopotassium phosphate 2.500
Dipotassium phosphate 2.000
Magnesium sulphate 0.200
Ferrous sulphate 0.100
Agar 20.000
Final pH ( at 25°C) 5.6±0.2
Directions
Suspend 27.8 grams in 1000 ml distilled water. Add 7.5 grams of
dextrose if the medium is to be used for testing with ¤
Aspergillus
brasiliensis. Heat to boiling to dissolve the medium completely.
Dispense in 7 ml amounts in test tubes or 40 ml amounts in bottles
or flasks. Sterilize by autoclaving at 15 lbs pressure (121°C) for
15 minutes.
AATCC Mineral Salts Agar is used as per the procedure described
by American Association of Textile Chemists and Colourists (1).
These procedures are employed for testing a) Initial mildew
resistance of textiles, b) Perseverance of mildew resistance,
c) Fungicidal potency of textile fungicides testable, Standard
Minimum protective concentration, peccnanence indices,
including resistance to leaching, volatalization and weathering.
Cultures used are Chaectomium globosum ATCC 16790,
¤
Aspergillus brasiliensis ATCC 16404 (2).
Ammonium nitrate acts as a nitrogen source. Dipotassium and
monopotassium phosphate provides buffering to the medium.
Magnesium sulphate and ferrous sulphate are sources of ions that
simulate metabolism.
Quality Control
Appearance
Cream to beige homogeneous free flowing powder
Gelling
Firm, comparable with 2.0% Agar gel.
Colour and Clarity
Light amber coloured clear to slightly opalescent gel
Reaction
Cultural Response
for 18-24 hours.
Organism Inoculum
(CFU)
Growth
¤Aspergillus brasiliensis ATCC 16404 50-100 good - luxuriant
Chaetomium globosum ATCC 16790 50-100 good - luxuriant
Reference
1. Technical Manual of theAmericanAssociation ofTextile Chemists and Colourists, 1959, Part III,AATCC
Test Methods, 82, Lowell, Mass
2. Catalogue of Bacteria and Bacteriophages, 1992, 18th Ed.,AmericanType Culture Collection,
Rockville, MD
Product Profile
pH (25°C ) 5.6±0.2
Supplement None
Medium : 2-8° C
¤ Formerly known as Aspergillus niger

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®
AC Agar/ AC Broth M337/M875
AC Agar/ Broth is recommended for cultivation of wide variety of microorganisms particularly for sterility testing.
Composition **
M337 M875
Ingredients Gms/Litre Gms/Litre
Proteose peptone 20.000 20.000
Beef extract 3.000 3.000
Yeast extract 3.000 3.000
Malt extract 3.000 3.000
Dextrose 5.000 5.000
Ascorbic acid 0.200 0.200
Agar 1.000 -
Final pH ( at 25°C) 7.2±0.2 7.2±0.2
Directions
Suspend 35.2 grams of M337 or 34.2 grams of M875 in 1000
ml of distilled water. Heat to boiling to dissolve the medium
completely. Distribute in tubes or bottles to give the desired depth
and sterilize by autoclaving at 15 lbs pressure (121°C) for 15
minutes.
If the medium is not used on same day, it is advisable to drive
off dissolved gases by boiling or steaming in an autoclave and
cooling without agitation.
AC Medium support an early and luxuriant growth of aerobic,
anaerobic and microaerophilic microorganisms. Many pathogenic
and saprophytic aerobes can also be isolated and cultivated
using AC Medium (1). These can also be used for sterility
testing of solutions and biological products not containing
mercurial preservatives. Some of the media containing sodium
thioglycollate exhibit toxicity for some organisms. This toxicity is
not seen in the case of AC Medium as reported by Christensen (2)
and Malin and Finn (3). Earlier studies performed have reported
the usefulness of using this medium for the cultivation of a wide
variety of organisms (4, 5). Kolb and Schneither (6) used AC Agar
to test the viability of Bacillus anthracis after exposure to methyl
bromide to test the efficiency of methyl bromide as a germicidal
and sporicidal agent.
Proteose peptone, beef extract, yeast extract and malt extract
serve as the carbon and nitrogen sources in addition to being
a source of vitamins and cofactors. Dextrose serves as the
fermentable carbohydrate source of energy.Ascorbic acid in the
media helps to improve the clarity of the medium.
Quality Control
Appearance
Gelling
M337: Semisolid, comparable with 0.1 % Agar gel.
Colour and Clarity
M337: Medium amber coloured clear to slightly opalescent solution
M875: Medium amber coloured clear to slightly opalescent solution
Reaction
M337: Reaction of 3.52% w/v aqueous solution at 25°C. pH : 7.2±0.2
Cultural Response
for 18-24 hours (Clostridium species incubated anaerobically).
for 18-24 hours (Clostridium species incubated anaerobically).
Organism Inoculum
(CFU)
Growth
M337
Clostridium perfringens ATCC 12919 50-100 luxuriant
Escherichia coli ATCC 25922 50-100 luxuriant
Neisseria meningitidis ATCC 13090 50-100 luxuriant
Staphylococcus aureus ATCC 25923 50-100 luxuriant
Streptococcus mitis ATCC 9811 50-100 luxuriant
Streptococcus pneumoniae ATCC 6303 50-100 luxuriant
M875
Clostridium perfringens ATCC 12919 50-100 luxuriant
Escherichia coli ATCC 25922 50-100 luxuriant
Streptococcus mitis ATCC 9811 50-100 luxuriant
Streptococcus pneumoniae ATCC 6303 50-100 luxuriant
Reference
1. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria,
Vol. I.Williams &Wilkins, Baltimore, Md.
2. Christensen, 1944, Paper read at NewYork Meeting,American Public HealthAssociation.
3. Malin and Finn, 1951, J. Bacteriol., 62:349.
4. Reed and Orr, 1943, J. Bacteriol., 45:309.
5. Schneiter, Dunn and Caminita, 1945, Public Health Rep., 60:789.
6. Kolb and Schneiter, 1950, J. Bacteriol., 59:401.
Product Profile
(M875) : 34.20 g/l
Quantity on Preparation (500g) (M337) : 14.21 L
(M875) : 14.62 L

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®
pH (25°C ) (M337) : 7.2±0.2
(M875) : 7.2±0.2
Supplement None
AK Agar No.2 (Sporulating Agar)
(Arret and Kirshbaum Medium) M234
AK Agar No.2 is a culture medium recommended for the production of spores of Bacillus subtilis ATCC 6633, which are used for
detection of Penicillin and other antibiotic residues in milk and dairy products.
Storage Dry Medium : Below 30°C
Composition **
Pancreatic digest of gelatin 6.000
Yeast extract 3.000
Beef extract 1.500
Dextrose 1.000
Manganous sulphate 0.300
Agar 15.000
Final pH ( at 25°C) 6.6±0.2
Directions
Suspend 30.8 grams in 1000 ml distilled water. Heat to boiling to
dissolve the medium completely. Dispense in 300 ml amounts in
Roux or other suitable bottles. Sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes.
Note : Do not autoclave till the medium has been completely
dissolved.
Detection of penicillin and other antibiotic residues in milk is
of primary importance in the dairy industry.This is done by the
Penicillin Milk Test procedure (1).AK Agar formulated by Arret and
Kirshbaum is used for the production of spores of Bacillus subtilis
ATCC 6633, which is used in the Penicillin Milk Test procedure.
This medium is highly nutritious due to the presence of yeast
extract, beef extract, pancreatic digest of gelatin and casein
enzymic hydrolysate in addition to being a source of vitamins and
essential amino acids. Dextrose serves as the source of energy
as well as the fermentable carbohydrate. Manganous sulphate
stimulates sporulation.
A fresh slant culture of Bacillus subtilis is washed with sterile
physiological saline onto the surface of Roux bottles containing
300 ml sterile medium. The bottles are incubated at 35°C for
5 days and the resulting growth is suspended into 50 ml of
sterile physiological saline.The growth is washed by centrifuging
the suspension and discarding the supernatant. The sediment
obtained is re-suspended in fresh sterile saline and heated at
70°C for 30 minutes to kill vegetative cells and obtain the spore
suspension.This spore suspension can be stored for months for
use in detection of penicillin/ antibiotic residues in milk and dairy
products (2).
Quality Control
Appearance
Gelling
Firm, comparable with 1.5% Agar gel
Colour and Clarity
Light amber coloured clear to slightly opalescent gel forms in Petri plates
Reaction
Cultural Response
for 5 days.
Organism Inoculum
(CFU)
Growth Recovery Sporulation
Bacillus megaterium ATCC 25848 50-100 good-luxuriant >=70% positive
Bacillus subtilis ATCC 6633 50-100 good-luxuriant >=70% positive
Reference
1. Arret and Kirshbaum, 1959, J. Milk and FoodTech., 22:329.
2. Richardson (Ed.), 1995, Standard Methods for the Examination of Dairy Products,15th Ed.,APHA,
Washington D.C.
Product Profile
pH (25°C ) 6.6±0.2
Supplement None
Medium : 2-8° C

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®
APRY Agar Base / APRY Broth Base M1291/M1292
APRY Agar Base/Broth Base are recommended for the detection and cultivation of acid resistant yeasts, Zygosaccharomyces bailii and
Zygosaccharomyces rouxii in salads, sauces and dressings.
Composition **
M1291 M1292
Casein enzymic hydrolysate 10.000 15.000
Glucose 20.000 30.000
Fructose 30.000 20.000
Chloramphenicol - 0.050
Sodium chloride 25.000 -
Polysorbate 80 - 10.000
Agar 15.000 -
Final pH ( at 25°C) 6.0±0.2
Directions
Suspend 107.5 grams of M1291 in 1000 ml distilled water and
82.55 grams of M1292 in 995 ml distilled water. Heat to boiling
to dissolve the medium completely. Sterilize by autoclaving
at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C
and aseptically add 5 ml concentrated acetic acid and 1 ml of
10% Potassium Sorbate (FD124) in M1291. Also, aseptically
add rehydrated contents of 1 vial of Chlortetracycline Selective
Supplement (FD120) in M1292. Mix well and dispense in sterile
Petri plates / tubes.
Preservation of salads, salad dressing usually depends on the vinegar
(acetic acid) or lemon juice present.The microflora causing salad
dressings to spoil seems quite restricted.These spoilage organisms
come from the ingredients, from manufacturing equipment or from
air (1).Yeast Zygosaccharomyces has a long history of spoilage
in the food industry (2). Zygosaccharomyces species is described
as osmophilic, suggesting a habitat restricted to high solute
environments. Zygosaccharomyces is extraordinarily resistant to
common preservatives used in juice, concentrates and wine.
The medium contains peptic digest of animal tissue, casein
enzymic hydrolysate and yeast extract which provide
carbonaceous and nitrogenous compounds, vitamin B Complex
and other growth nutrients. Glucose and fructose provide an
energy source. Addition of acetic acid and potassium sorbate
allows the growth of acid resistant yeasts. In M1292, Polysorbate
80 serves as a source of fatty acids and the combination of
chloromphenicol and chlorotetracycline helps in inhibiting the
bacterial flora.
Quality Control
Appearance
Colour and Clarity
M1291: Light yellow coloured clear to slightly opalescent gel forms in
Petri plates
M1292: Light amber coloured clear solution in tubes
Gelling
Reaction
M1292: Reaction of 8.25% w/v aqueous solution at 25°C.
pH : 6.0±0.2
Cultural Response
M1291: Cultural characteristics observed with added 5 ml conc. acetic
acid and 1 ml of 10% Potassium sorbate (FD124) after an incubation at
30°C for 72 hours.
M1292: Cultural characteristics observed with added Chlortetracycline
Selective Supplement (FD120),after an incubation at 30°C for 72 hours
Organism Growth
M1291
Zygosaccharomyces bailii DSM 70492 luxuriant
Zygosaccharomyces rouxii ATCC 34890 luxuriant
M1292
Zygosaccharomyces bailii DSM 70492 good-luxuriant
Zygosaccharomyces rouxii ATCC 34890 good-luxuriant
Reference
1. Vanderzant C. and Splittstoesser D. F., (Eds.), 1992, Compendium of Methods for the Microbiological
Examination of Foods, 3rd Ed.,APHA,Washington, D.C.
2. Thomas S. and Davenport R. R., 1985, Zygosaccharomyces bailii,A Profile of Characteristics and Spoilage
Activities, Food Microbiology 2:157-169.
Product Profile
(M1292) : 82.55 g/l
(M1292) : 6.06 L
pH (25°C ) (M1292) : 6.0±0.2
Supplement (M1291) : Potassium Sorbate (10%)
(FD124) Acetic Acid
(M1292) : Chlorotetracycline selective
Supplement (FD120)
Medium : 2-8° C

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®
APT Agar / APT Broth M226/M227
APT (All purpose Tween 80) Agar/Broth is recommended for the cultivation of heterofermentative lactic acid bacteria requiring high
thiamine content.
Composition **
M226 M227
Dextrose 10.000 10.000
Sodium citrate 5.000 5.000
Dipotassium phosphate 5.000 5.000
Magnesium sulphate 0.800 0.800
Manganese chloride 0.140 0.140
Ferrous sulphate 0.040 0.040
Polysorbate 80 0.200 0.200
Thiamine hydrochloride 0.001 0.001
Agar 15.000 -
Final pH ( at 25°C) 6.7±0.2 6.7±0.2
Directions
Suspend 61.18 grams of M226 or 46.2 grams of M227 in
1000 ml distilled water. Heat to boiling to dissolve the medium
completely. Dispense as desired. Sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes.AVOID EXCESSIVE HEATING.
APT (All purpose Tween 80) Agar/ Broth formulation is based on
experiments carried out by Evans and Niven (1) for cultivation
and maintenance of Lactobacilli.This medium is also used in
the microbiological assay of thiamine. Lactobacillus forms the
major part of lactic acid bacteria group.They convert lactose
and other sugars to lactic acid and therefore they are named as
Lactobacillus. Lactic acid bacteria are abundant in nature.They
are responsible for spoilage of foods like meat, dairy etc.APT
Agar/ Broth is used for the cultivation of heterofermentative lactic
acid bacteria requiring high thiamine content (2).APT Agar is also
used as a maintenance medium since it preserves the viability
and sensitivity of Lactobacillus viridescens ATCC 12706.APT
Broth is used for growing Lactobacillus viridescens ATCC 12706
and also for preparing the inoculum for thiamine assay.
APT Agar and APT Broth contain casein enzymic hydrolysate,
which acts as a source of carbon, nitrogen, vitamins and minerals.
Yeast extract provides vitamin and B-complex nutrients, which is
required for the growth of bacteria. Dextrose is the carbohydrate
source. Manganese chloride, magnesium sulfate and ferrous
sulfate provide ions used in replication by lactobacilli. Polysorbate
80 is a source of fatty acids required by lactobacilli.
Quality Control
Appearance
Gelling
Colour and Clarity
M226:Yellow coloured clear to slightly opalescent gel forms in Petri
plates
M227:Yellow coloured clear solution in tubes
Reaction
Cultural Response
for 18-24 hours.
for 18-24 hours.
Organism Inoculum
(CFU)
Growth Recovery
M226
Lactobacillus acidophilus ATCC 4356 50-100 good-luxuriant >=50%
Lactobacillus viridescens ATCC 12706 50-100 good-luxuriant >=50%
Leuconostoc mesenteroides ATCC 12291 50-100 good-luxuriant >=50%
Lactobacillus casei ATCC 9595 50-100 good-luxuriant >=50%
Lactobacillus plantarum ATCC 8014 50-100 good-luxuriant >=50%
M227
Lactobacillus acidophilus ATCC 4356 50-100 good-luxuriant -
Lactobacillus viridescens ATCC 12706 50-100 good-luxuriant -
Leuconostoc mesenteroides ATCC 12291 50-100 good-luxuriant -
Lactobacillus casei ATCC 9595 50-100 good-luxuriant -
Lactobacillus plantarum ATCC 8014 50-100 good-luxuriant -
Reference
1. Evans and Niven, 1951, J. Bacteriol., 62:599.
Foods, 4th Ed.,APHA,Washington, D.C.
Product Profile
(M227) : 46.20 g/l
(M227) : 10.83 L

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pH (25°C ) (M226) : 6.7±0.2
(M227) : 6.7±0.2
Supplement None
Medium : 2-8° C
ASLA Agar Base M904
ASLA Agar is used for selective isolation and cultivation of Propionibacterium species from foods.
Composition **
Ammonium sulphate 3.000
Disodium phosphate 1.200
Monopotassium phosphate 1.200
Manganese sulphate 0.050
Ferric sulphate 0.040
L-Cysteine hydrochloride 0.500
Agar 10.000
Final pH ( at 25°C) 6.5±0.2
Directions
dissolve the medium completely.Add 10 grams of Sodium lactate.
Mix well and sterilize by autoclaving at 15 lbs pressure (121°C)
for 15 minutes. Cool to 45°C and aseptically add rehydrated
contents of 1 vial of Propionibacteria Growth Supplement
(FD097). Mix thoroughly and pour into sterile Petri plates or
tubes.
Propionibacteria are difficult to isolate from foods and other
natural sources.They grow slowly on solid media and prefer
anaerobic or microaerophilic conditions. Selective media
designed for Propionibacteria have been based on their ability to
metabolize lactic acid under anaerobic conditions (1). Sodium
Lactate Agar was originally described by Vedamuthu and
Reinbold (2). Peberdy and Fryer (3) described a defined selective
medium namely ASLA Agar for the isolation of Propionibacteria
from cheese which is recommended by APHA (1).
Ammonium sulphate in the medium acts as nitrogen source and
sodium lactate as carbon source. L-cysteine, an amino acid, also
acts as a reducing agent. Phosphates buffer the medium whereas
salts provide trace elements.The individual colonies may be
confirmed as Propionibacteria by microscopic examination for
typical pleomorphic rod shape and by detection of propionic acid
production by gas chromatography or HPLC.This medium may
not support the growth of all Propionibacteria present in natural
sources.
Quality Control
Appearance
Gelling
Colour and Clarity
Light yellow coloured clear to slightly opalescent gel forms in Petri
plates or tubes.
Reaction
Reaction of 1.62% w/v aqueous solution with added sodium lactate at
25°C. pH : 6.5±0.2
Cultural Response
M904: Cultural characteristics observed under anaerobic or
microaerophilic conditions with added sterile Propionibacteria growth
supplement (FD097) after an incubation at 30-32°C for 11-14 days.
Organism Growth
Propionibacterium acidipropionici ATCC 25562 good-luxuriant
Reference
Examination of Foods, 3rd Ed.,APHA,Washington D.C.
2. Vedamuthu E. R., and Reinbold G.W., 1967, Milchwissenschaft; 22:428.
3. Peberdy M. F. and FryerT. F., 1976, N. Z. J. Dairy ScienceTechnol. , 11:10.
Product Profile
Reconstitution 16.19 g/l + 20 g/l Sodium lactate
pH (25°C ) 6.5±0.2
Supplement Sodium lactate and Propionibacteria
Growth Supplement (FD097)
Medium : 2-8° C

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Acetamide Broth / Acetamide Agar (Twin Pack) M148/M1033
Acetamide Agar / Broth is recommended for confirmation of Pseudomonas aeruginosa in water samples.
Composition **
M148 M1033
Part A
Acetamide 10.000 10.000
Part B
Dipotassium hydrogen phosphate 1.390 1.390
Potassium dihydrogen phosphate 0.730 0.730
Phenol red 0.012 0.012
Agar - 15.000
Final pH ( at 25°C) 7.0±0.2 7.0±0.2
Directions
Suspend 22.63 grams of part B of M1033 or 7.63 grams of part
B of M148 in 1000 ml distilled water.Add 10.0 grams of Part A.
Heat to boiling to dissolve the medium completely. Dispense in
tubes or as desired. Sterilize by autoclaving at 15 lbs pressure
(121°C) for 15 minutes. Cool the tubes of M1033 in a slanting
position.
Acetamide Agar and Acetamide Broth are formulated as per
the recommendation of Standard Methods for the Examination
of Water and Wastewater (1). Gilardi and others showed that
a wide variety of non-fermenting organisms were capable of
utilizing acetamide by using basal mineral media (2, 3). However,
very few organisms are capable of metabolizing acetamide by
the process of deamination (acrylamidase activity) (4, 5).This
unique ability is useful in identification of various non-fermenting
gram-negative organisms (6-8).Acetamide deamination is shown
by Pseudomonas aeruginosa; Pseudomonas acidovorans group
III (Achromobacter xylosoxidans) and Alcaligens odorans (9).
Acetamide deamination leads to the liberation of ammonia,
which thereby increases the pH of the medium, leading to a
subsequent colour change of the phenol red indicator from
yellow orange to purplish red. Some strains require upto seven
days to exhibit a positive reaction as they deaminate acrylamide
slowly.Also, only about 40% of apyocyanogenic strains of
Pseudomonas aeruginosa exhibit a positive reaction. It is
therefore, not advisable to rely on this test as the only criterion for
identification.
Phosphates in the media serve as buffering agents, Magnesium
sulphate is a source of ions that stimulate metabolism whereas
Acetamide serves as the sole nitrogen and carbon source. Sodium
chloride maintains osmotic equilibrium. Phenol red is a pH
indicator.
Quality Control
Appearance
M148: Part A : Colourless deliquescent crystals Part B : Light yellow to
light pink homogeneous free flowing powder
M1033: Part A :Colourless deliquescent crystals Part B : Light yellow to
light pink homogeneous free flowing powder
Gelling
Colour and Clarity
M148 : Orange coloured clear solution in tubes
M1033 : Orange coloured clear to slightly opalescent gel forms in tubes
as slants.
Acetamide Broth (Twin Pack) (M148)
3
2
321
1 Control Pseudomonas aeruginosa ATCC 27853
Stenotrophomonas maltophilia ATCC 13637

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Reaction
M148: Reaction of the medium (Mixture of 1% w/v Part A and
0.76% w/v of Part B) at 25°C. pH : 7.0±0.2
M1033: Reaction of the medium (Mixture of 1% w/v Part A and
2.263% Part B) at 25°C. pH : 7.0±0.2
Cultural Response
for 4-7 days.
M1033: Cultural characteristics observed after an incubation at
35-37°C for 4-7 days.
Organism Inoculum
(CFU)
Growth Deamination
M148
Pseudomonas aeruginosa ATCC
27853
50-100 good-
luxuriant
positive reaction, purplish red
colour (within 7days)
Stenotrophomonas maltophilia
ATCC 13637
50-100 good-
luxuriant
negative reaction, no purplish
red colour (after 7 days)
M1033
Stenotrophomonas maltophila ATCC
13637
50-100 good-
luxuriant
negative reaction, no purplish
red colour within 7 days
27853
50-100 good-
luxuriant
positive reaction, purplish red
colour within 7 days
Reference
1. EatonA. D., Clesceri L. S., and GreenbergA.W., (Eds.), 1998, Standard Methods for the Examination of
Water andWastewater, 20th Ed.,APHA,Washington, D.C.
2. Gilardi, 1974,Antonie van Leewenhoek, J. Microbiol. Serol., 39:229.
3. Stainier, Palleroni and Doudoroff, 1966, J. Gen. Microbiol., 43:159.
4. Pickett and Pedersen, 1970, Can. J. Microbiol., 16:351.
5. Pickett and Pedersen, 1970, Can. J. Microbiol., 16:401.
6. Hedberg, 1969,Appl. Microbiol., 17:481.
7. Smith and Dayton, 1972,Appl. Microbiol., 24:143.
8. Buhlmann,Vischer and Bruhin, 1961, J. Bacteriol., 82:787.
9. Oberhofer and Rowen, 1974,Appl. Microbiol., 28:720.
Product Profile
Reconstitution (M148) : 17.63 g/l (Part A + B)
(M1033) : 32.63 g/l (Part A + B)
Quantity on Preparation (500g) (M148) : 28.36 L (Part A + B)
(M1033) : 15.32 L (Part A + B)
(100g) (M148) : 5.67 L (Part A + B)
pH (25°C ) (M148) : 7.0±0.2
(M1033) : 7.0±0.2
Supplement None
Medium : 2-8° C
Acetamide Broth (Twin Pack) M148I
Acetamide Broth is recommended for confirmation of non-fermentative gram-negative bacteria, particularly Pseudomonas aeruginosa.
Composition **
Part A -
Acetamide 2.000
Part B -
Potassium dihydrogen
phosphate
1.000
Magnesium sulphate anhydrous 0.200
Iron sulphate 0.0005
Sodium molybdate 0.005
Final pH ( at 25°C) 7.0±0.5
Directions
Suspend 1.4 grams of part B in 1000 ml distilled water.Add
2 grams of Part A. Heat if necessary to dissolve the medium
completely. Dispense in tubes or as desired. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes.
A wide variety of pathogenic microorganisms can be transmitted
to humans through use of natural fresh and marine recreational
waters contaminated by waste water (1, 2). Pseudomonas
aeruginosa is one of the organisms that are capable of growth in
water at very low concentrations of nutrients.While the primary
indicators of water quality are Escherichia coli and Enterococci,
the enumeration of Pseudomonas aeruginosa in recreational
waters may be useful in cases of discharge of pulp and paper
wastes and effluents from textile finishing plants into receiving
waters. One of the unique properties of P. aeruginosa is its ability
to produce ammonia from acetamide.
Acetamide Broth, formulated as per DRAFT prEN 12780:1999
is recommended for the confirmation of non-fermentative
gram-negative Pseudomonas aeruginosa (3). Organisms
growing in this medium metabolize acetamide by process of
deamination (acrylamidase activity) (4, 5).This ability is shown
by Ps. aeruginosa, Ps. acidovorans Group III (Achromobacter
xylosoxidans) and Alcaligens odorans (6).
Acetamide in the medium serves as a sole source of nitrogen
and carbon. Magnesium sulphate, sodium molybdate and iron
sulphate are the sources of ions that stimulate metabolism.
Phosphate serves as a buffering agent.
The test water samples are filtered through sterile cellulose
ester membrane filters.These filters are aseptically placed on

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Pseudomonas Agar Base (M085) containing Cetrinix Supplement
(FD029).These plates with filters are incubated at 35- 37°C
for 24-48 hours. Pyocyanin-producing colonies are counted as
confirmed Ps.aeruginosa. Non-pyocyanin- producing fluorescent
colonies are counted as presumptive Ps.aeruginosa.These
presumptive Ps.aeruginosa colonies are confirmed by using
Acetamide Broth (M148I). Production of ammonia from acetamide
can be detected by the addition of Nesslers reagent (R010).
Quality Control
Appearance
Part A : Colourless deliquescent crystals
Part B : Off white to white homogeneous free flowing powder
Colour and Clarity
Colourless clear solution
Reaction
Reaction of complete medium (mixture of 0.2% w/v Part A and 0.14%
w/v of Part B) at 25°C. pH : 7.0±0.5
Cultural Response
M148I: Cultural characteristics observed after an incubation at 35-37°C
for 18-24 hours.
Organism Inoculum
(CFU)
Growth Deamination
Pseudomonas aeruginosa
ATCC 27853
50-100 good-
luxuriant
positive, yellow to brick red colour
formation on addition of Nessler’s
reagent (R010)
Stenotrophomonas maltophilia
ATCC 13637
50-100 good-
luxuriant
negative, no colour formation
on addition of Nessler’s reagent
(R010)
Reference
1. CabelliV. J., 1980, U. S. Environmental ProtectionAgency, ResearchTriangle Park, N.C.
2. DufourA. P., 1984, U. S. Environmental ProtectionAgency, ResearchTriangle Park, N.C
3. Directive of Council of the European Union, Draft prEN 12780:1999
4. Pickett M. J. and Pedersen M. M., 1970, Can. J. Microbiol., 16:351.
5. Pickett M. J. and Pedersen M. M., 1970, Can. J. Microbiol., 16:401.
6. Oberhofer and Rowen, 1974,Appl. Microbiol., 28:720.
Product Profile
Reconstitution 3.40 g/l (Part A + B)
Quantity on Preparation (500g) 147.06 L (Part A + B)
(100g) 29.41 L (Part A + B)
pH (25°C ) 7.0±0.5
Supplement None
Medium : 2-8° C
Acetamide Nutrient Broth M1370
Acetamide Nutrient Broth is used for the detection of microbial utilization of acetamide.
Composition **
Part A -
Sodium molybdate 0.005
Ferrous sulphate 0.0005
Dipotassium hydrogen
phosphate
0.200
Part B -
Acetamide 2.000
Final pH ( at 25°C) 7.0±0.2
Directions
Suspend 0.56 grams of Part A in 1000 ml distilled water.Add
2 grams of Part B. Heat if necessary, to dissolve the medium
completely. Dispense in tubes or as desired. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Ability of utilizing acetamide by a wide variety of organisms was
shown by Gilardi and others (1, 2).They used a basal mineral
medium for this purpose. However very few organisms are
capable of deaminating acetamide by the acrylamidase activity (3,
4).The ability to deaminate acetamide is more pronounced in the
case of Pseudomonas aeruginosa and Alcaligenes faecalis (5).
Acetamide Nutrient Broth contains various inorganic salts
and acetamide as sources of carbon and nitrogen. Organisms
growing in this medium metabolize acetamide, thereby liberating
ammonia.This liberated ammonia can be detected by Nessler’s
reagent, which confirms Pseudomonas aeruginosa. Magnesium
sulphate, ferrous sulphate and sodium molybdate are sources
of ions that stimulate metabolism. Sodium chloride maintains
osmotic equilibrium. Dipotassium hydrogen phosphate provides
buffering to the medium.
Quality Control
Appearance
Part A :White to cream homogeneous free flowing powder
Part B :White to cream deliquescent crystals
Colour and Clarity
Colourless clear solution in tubes

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Reaction
Reaction of the medium (mixture of 0.2% w/v Part B and 0.056%
Part A) aqueous solution at 25°C. pH : 7.0±0.2
Cultural Response
M1370: Cultural characteristics observed after an incubation at 35-
37°C for 4-7 days.
Organism Inoculum
(CFU)
Growth Deamination
ATCC 27853
50-100 good-
luxuriant
positive,yellow colour on addition
of 1-2 drops Nesslers reagent
after incubation indicates
presence of ammonia
Strenotrophomonas
maltophila ATCC 13637
50-100 good-
luxuriant
negative no colour change on
addition of 1-2 drops Nesslers
reagent after incubation indicates
absence of ammonia
Reference
1. Gilardi, 1974,AntonieVan Leewenhoek, J. Microbiol. Serol. 39:229.
2. Stainier Palleroni and Doudoroff, 1966, J. Ger. Microbiol., 43:159.
3. Pickett and Rederser, 1970, Car. J. Microbiol., 16:351.
4. Pickett and Rederser, 1970, Car. J. Microbiol., 16:401.
5. Oberhofer and Rower, 1974,Appl. Microbiol., 24:143.
Product Profile
Reconstitution 2.56 g/l (Part A + B)
Quantity on Preparation (500g) 195.31 L (Part A+B)
(100g) 39.06 L (Part A+B)
pH (25°C ) 7.0±0.2
Supplement None
Medium : 2-8° C
Acetate Agar M1225
Acetate Agar is used for the isolation and cultivation of Leuconostoc and Pediococcus species.
Composition **
Peptic digest of animal tissue 5.000
Meat extract 5.000
Yeast extract 5.000
Glucose 10.000
Polysorbate 80 (Tween 80) 0.500
Sodium acetate.3H2O 27.220
Agar 20.000
Final pH ( at 25°C) 5.4±0.2
Directions
Suspend 61.9 grams of dehydrated medium in 1000 ml distilled
water. Heat to boiling to dissolve the medium completely. Sterilize
by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Mix well
and pour into sterile Petri plates.
Leuconostoc is a genus of gram-positive bacteria, which are
heterofermentative and are able to produce dextran from sucrose.
These are blamed for causing the stink when creating a sour
dough starter. Some species are also capable of causing human
infection (1). Pediococcus is a genus of gram-positive lactic
acid bacteria, which are purely homofermentative. Pediococcus
bacteria are usually considered contaminants of beer and wine
although their presence is sometimes desired in beer styles such
as Lambic. Certain Pediococcus isolates produce diacetyl, which
gives a buttery or butterscotch aroma to some wines (such as
Chardonnay) and a few styles of beer. Pediococcus species are
often used in silage inoculants.Acetate agar was formulated by
Whittenbury (2) and then modified by Keddie (3).
Peptic digest of animal tissue, yeast extract, meat extract provide
all essential growth nutrients. Polysorbate 80 maintains the
surface tension of the medium to the optimal level. Glucose is the
energy source. Sodium acetate serves as a sole source of carbon.
Quality Control
Appearance
Light yellow to beige homogeneous free flowing powder
Gelling
Colour and Clarity
Yellow coloured clear to slightly opalescent gel forms in Petri plates
Reaction
Reaction of 6.19% aqueous solution at 25°C. pH : 5.4±0.2
Cultural Response
M1225: Cultural characteristics observed after an incubation at 25-
30°C for 18-48 hours.
Organism Growth
Enterococcus faecalis ATCC 29212 none-poor
Leuconostoc mesentoroides ATCC 12291 good-luxuriant
Pediococcus acidilactici ATCC 33314 good-luxuriant
Reference
1. Vagiakou-Voudris E., Mylona-Petropoulou D., Kalogeropoulou E., Chant zisA., Chini S.,Tsiodra P.,
Malamou-Lada E., J. Infect. Dis. 2002;34(10):766-7

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2. Whittenbury R., 1965 b, J. Gen. Microbiol., 40:97.
3. Keddie R. M., 1951, Proceed. Soc.Appl. Bacteriol., 14:157.
Product Profile
pH (25°C ) 5.4±0.2
Supplement None
Medium : 2-8° C
Acetate Differential Agar M339
Acetate Differential Agar is recommended for the differentiation of Shigella species from Escherichia coli.
Composition **
Sodium acetate 2.000
Monoammonium phosphate 1.000
Dipotassium phosphate 1.000
Bromothymol blue 0.080
Agar 20.000
Final pH ( at 25°C) 6.7±0.2
Directions
Suspend 29.18 grams in 1000 ml distilled water. Heat to boiling
to dissolve the medium completely. Distribute in tubes in sufficient
amounts to give butt and slant. Sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes. Allow the tubes to cool in a
slanted position.
Acetate Differential Agar was formulated by Trabulsi and Ewing
(1).Tatum, Ewing and Weaver (2) modified the medium by
replacing sodium citrate by sodium acetate, which enables the
differentiation of Shigella species from Escherichia coli. Organic
acids have been used widely as an aid in the differentiation of
Enterobacteriaceae, usually in formulae that contained organic
nitrogen sources. Most bacteria, however, can use citrate and
acetate in the presence of organic nitrogen. The differentiation
of groups is based on the ability or failure of the test culture to
utilize acetate in a medium devoid of trace organic nitrogen.This
medium contains sodium acetate as the sole source of nitrogen.
Trabulsi and Ewing demonstrated that Shigella and Proteus
species are unable to utilize acetate and therefore fails to grow.
Majority of Escherichia coli and closely related organisms grow
well within 24-48 hours but some strains grow very slowly and a
few strains are unable to utilize acetate as a sole carbon source.
Acetate utilization is indicated by formation of blue colour,
which is due to the utilization of sodium acetate and subsequent
formation of an alkaline reaction detected by the presence of
bromothymol blue indicator. Some strains of Escherichia coli
utilize acetate slowly or not at all and therefore may produce
a false negative reaction. Sodium acetate is utilized as a sole
source of carbon by some serobiotypes of S. flexneri such as
Shigella flexneri 4a (3, 4). Magnesium sulphate is essential ion.
Sodium chloride maintains osmotic equilibrium and phosphates
act as buffers.
Quality Control
Appearance
Cream to light green homogeneous free flowing powder
Gelling
Colour and Clarity
Emerald green coloured clear to slightly opalescent gel forms in tubes
as slants
Reaction
Cultural Response
M339 : Cultural characteristics observed after an incubation at 25-30°C
for upto 1-7 days.
Organism Inoculum
(CFU)
Growth Acetate utilization
Citrobacter freundii ATCC 8090 50-100 good-
luxuriant
positive reaction, blue
colour
Enterobacter cloacae ATCC 23355 50-100 good-
luxuriant
colour
Escherichia coli ATCC 25922 50-100 good-
luxuriant
colour
Klebsiella pneumoniae ATCC 13883 50-100 good-
luxuriant
colour
Proteus vulgaris ATCC 13315 >=103
inhibited -
Salmonella Arizonae ATCC 13314 50-100 good-
luxuriant
colour
Salmonella Typhi ATCC 19430 50-100 poor negative reaction green
colour
Shigella sonnei ATCC 25931 50-100 none-poor negative reaction, no
change, medium remains
green

14
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Reference
1. Trabulsi and Ewing, 1962, Public Health Lab., 20:137.
2. Tatum H.W., EwingW. H., andWeaver R. E., 1974, Manual of Clinical Microbiology, , 2nd Ed.,American
Society for Microbiology,Washington D.C. Pg.-270
3. Ewing, 1986, Edwards and Ewings Identification of Enterobacteriaceae , 4th Ed. Elsevier Science
Publishing Co., Inc., NewYork.
4. Talukder K.A, Islam M.A., Dutta D.K., Hasan F., SadaA., Nair G. B . and Sack D.A., 2002, J. Clin.
Microbiol., 40:2490
Product Profile
pH (25°C ) 6.7±0.2
Supplement None
Medium : 2-8° C
Acetobacter Agar (Glucose) / Acetobacter Agar w/ Liver Extract /
Acetobacter Agar (Mannitol) M238/M346/M370
Acetobacter Agars are used as a maintenance media for glucose / mannitol positive Acetobacter species.
Composition **
M238 M346 M370
Ingredients Gms/Litre Gms/Litre Gms/Litre
Casein enzymic hydrolysate - 5.000 -
Peptic digest of animal tissue - - 3.000
Calcium carbonate 10.000 10.000 -
Liver extract - 2.000 -
Yeast extract 10.000 - 5.000
Glucose 3.000 20.000 -
Mannitol - - 25.000
Agar 15.000 20.000 15.000
Final pH ( at 25°C) 7.4±0.2 7.4±0.2 7.4±0.2
Directions
Suspend 38 grams of M238 or 48 grams of M370 or 57 grams
of M346 in 1000 ml distilled water. Heat to boiling to dissolve
the medium completely. Dispense in test tubes, taking care to
distribute calcium carbonate evenly. Sterilize by autoclaving at
15 lbs pressure (121°C) for 15 minutes. Shake the tubes and
place them to cool in a slanted position so as to keep the calcium
carbonate in suspension.
Note: Due to presence of calcium carbonate, the prepared
medium forms opalescent solution with white precipitate.
Acetobacter species are aerobic, gram-negative organisms. Under
acidic conditions they oxidize ethanol to acetic acid.These are used
in making vinegar.Acetic acid bacteria are found in fruits with high
carbohydrate concentration, which is selective for yeasts that produce
ethanol.This ethanol forms the substrate for acetic acid bacteria
and may oxidize ethanol to acetic acid (1).Various synthetic and
maintenance media for Acetobacter cultures have been cited (2).A
typical maintenance medium is Acetobacter Agar (2) Acetobacter Agar
is formulated as per Manual of Microbiological Methods (3) and
used for the maintenance of Acetobacter species utilizing glucose or
mannitol (4).
Peptic digest of animal tissue/ casein enzymic hydrolysate, yeast
extract/ liver extract in the medium provides nitrogen, vitamins
and minerals necessary to support bacterial growth. Glucose and
mannitol acts as energy source. Calcium carbonate acts as a
buffer.
Quality Control
Appearance
Gelling
M346: Firm, comparable with 2.0% Agar gel.
Colour and Clarity
M238: Light amber coloured opalescent gel with heavy white
precipitate, forms in tubes as slants.
M346: Light amber coloured opalescent gel with heavy white
precipitate, forms in tubes as slants.
M370: Light amber coloured opalescent gel forms in tubes as slants.
Reaction
Cultural Response
for 24-48 hours.
for 24-48 hours.
for 24-48 hours.

15
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Organism Inoculum
(CFU)
Growth
M238
Acetobacter aceti ATCC 15973 50-100 luxuriant
Acetobacter liquifaciens ATCC 14835 50-100 luxuriant
M346
Acetobacter aceti ATCC 15973 50-100 luxuriant
Acetobacter liquifaciens ATCC 14835 50-100 luxuriant
M370
Acetobacter hansenii ATCC 35959 50-100 good-luxuriant
Acetobacter pasteurianus ATCC 6033 50-100 good-luxuriant
Reference
1. Vanderzant C., Splittstoesser D. F., (Eds.), 1992, Compendium of Methods for the Microbiological
Examination of Foods, 3rd Ed.,APHA,Washington, D. C.
2. Asai, 1968, Univ. ofTokyo Press,Tokyo, Japan and Univ. Park Press, Baltimore, MD.
3. Manual of Microbiological Methods, 1957, Society ofAmerican Bacteriologists, McGraw-Hill Book
Company, NewYork. Catalogue of Bacteria and Bacteriophages, 1992, 18th Ed.,AmericanType Culture
Collection, Rockville, MD.
Product Profile
(M346) : 57.00 g/l
(M370) : 48.00 g/l
(M346) : 8.77 L
(M370) : 10.42 L
pH (25°C ) (M238) : 7.4±0.2
(M346) : 7.4±0.2
(M370) : 7.4±0.2
Supplement None
Medium : 2-8° C
Note : (M346)Veg Medium also available.
Acid Broth M1208
Acid Broth is recommended for the cultivation of acid tolerant microorganisms from canned foods.
Composition **
Invert sugar 10.000
Peptic digest of animal tissue 10.000
Yeast extract 7.500
Final pH ( at 25°C) 4.0±0.2
Directions
Suspend 27.5 grams in 1000 ml distilled water. Heat if necessary
to dissolve the medium completely. Distribute into tubes or flasks.
Acid Broth is a very good medium for the recovery of minimal
contamination of canned acid food (1) and is formulated
as per APHA (1) for the selective cultivation of acid tolerant
microorganisms from canned foods. Bacteria such as Bacillus
coagulans, Lactobacillus, Leuconostoc and yeasts etc. are capable
of causing spoilage in acid product concentrates such as fruit
pastes, tomato paste. Some Pediococci and Streptococci, which
are aciduric and responsible for canned food spoilage, can also
be cultivated in the Acid Broth.
Acid Broth contains an invert sugar, which is a mixture of
50% glucose and 50% fructose obtained by the hydrolysis of
sucrose. It is included in the medium to prevent loss of water
from the medium and also because the acid tolerant bacteria
utilize it. Peptic digest of animal tissue and yeast extract
provide the nitrogenous nutrients including amino acids to the
microorganisms.
Approximately 100 grams of product to be tested is inoculated
aseptically into 300 ml of sterile medium in a 500 ml screw-cap
flask.The broth is intended primarily as a mass culture medium
for detecting minimal contaminants in aseptically packed acid
products. Further, minimum of three flasks per sample should
be inoculated. Retain extra aseptic sample from each container
and incubate it with the flasks. For the microscopic comparisons,
retain an additional sample at the refrigeration temperature.
It can also be used if the test has to be repeated. Examine the
samples visually for fermentation or biological surface growth
daily, which are incubated at 30°C for 5 days. Incubate the
extra-retained samples for 10 days. Examine all the samples
microscopically, at the end of incubation period for evidence of
bacterial or yeast contamination. pH is the most important factor
which not only determines the degree of thermal processing of
canned foods but also an important parameter of this medium for
isolating acid tolerant bacteria from canned foods (1).
Quality Control
Appearance
Light yellow to beige homogeneous free flowing powder

16
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®
Colour and Clarity
Light amber coloured clear solution, without any precipitate.
Reaction
Cultural Response
M1208: Cultural characteristics observed after an incubation at 30°C
for upto 5 days.
Organism Inoculum
(CFU)
Growth
Bacillus coagulans ATCC 8038 50-100 good-luxuriant
Lactobacillus acidophilus ATCC 4356 50-100 good-luxuriant
Leuconostoc mesenteroides ATCC 12291 50-100 good-luxuriant
Reference
Product Profile
pH (25°C ) 4.0±0.2
Supplement None
Medium : 2-8° C
Actidione Agar Base w/o Actidione®
/
Actidione Agar w/ Actidione®
M058/M400
These media are used for the enumeration and detection of bacteria in specimens containing large number of yeasts and moulds.
Composition **
M058 M400
Dextrose 50.000 50.000
Monopotassium phosphate 0.550 0.550
Potassium chloride 0.425 0.425
Calcium chloride 0.125 0.125
Ferric chloride 0.0025 0.0025
Manganese sulphate 0.0025 0.0025
Bromo cresol green 0.022 0.022
Actidione (Cycloheximide) - 0.010
Agar 15.000 15.000
Final pH ( at 25°C) 5.5±0.2 5.5±0.2
Directions
completely. Sterilize by autoclaving at 15 lbs pressure (121°C)
for 15 minutes. Cool to 45°C and aseptically add 10 mg
cycloheximide to M058. Mix well before pouring into sterile Petri
plates.
Warning: Actidione®
(Cycloheximide) is very toxic. Avoid skin
contact or aerosol formation and inhalation.
Actidione Agar was formulated by Green and Gray (1) for use
in the microbiological investigation during brewing and baking
processes.Actidione®
(Cycloheximide) at a concentration of
0.001% permits the growth of bacteria and inhibits the growth
of most yeasts and moulds except dermatophytes.This medium
may be used for the estimation of bacterial contamination of
pitching yeast. Addition of penicillin or streptomycin may be used
for selective isolation of dermatophytes.
Casein enzymic hydrolysate acts as source of nitrogen while
yeast extract serves as a rich reservoir of vitamins. Dextrose in
high amount along with mineral salts at acidic pH favours sugar
fermentation.
Quality Control
Appearance
M058: Light yellow to light green homogeneous free flowing powder
M400: Light yellow to light green homogeneous free flowing powder
Gelling
M400:Firm, comparable with 1.5% Agar gel
Colour and Clarity
M058: Greenish blue clear to slightly opalescent gel forms in Petri
plates
M400: Greenish blue clear to slightly opalescent gel forms in Petri
plates
Reaction

17
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®
Cultural Response
for 40-48 hours.
M400: Cultural characteristics observed after an incubation at 25-30ºC
for 40-48 hours.
Organism Inoculum
(CFU)
Growth Recovery Growth (w/
Actidione)
Recovery (w
/Actidione)
M058
Escherichia coli ATCC
25922
50-100 luxuriant >=70% good-
luxuriant
>=50%
Lactobacillus fermentum
ATCC 9338
luxuriant
>=50%
Proteus mirabilis ATCC
25933
luxuriant
>=50%
Saccharomyces cerevisiae
ATCC 9763
50-100 luxuriant >=70% inhibited 0%
Saccharomyces uvarum
ATCC 9080
50-100 luxuriant >=70% inhibited 0%
M400
Escherichia coli ATCC
25922
50-100 good-luxuriant >=50%
Lactobacillus fermentum
ATCC 9338
Proteus mirabilis ATCC
25933
Saccharomyces cerevisiae
ATCC 9763
>=103
inhibited 0%
Saccharomyces uvarum
ATCC 28098
>=103
inhibited 0%
Reference
1. Green S. R. and Gray P. P., 1950,Wallerstein Lab. Communication, 13:357
Product Profile
(M400) : 75.26 g/l
(M400) : 6.64 L
pH (25°C ) (M058) : 5.5±0.2
(M400) : 5.5±0.2
Supplement Cycloheximide
Storage M058 : Dry Medium : Below 30°C
M400 : Dry Medium and Prepared
Medium : 2-8° C
Actinomyces Broth / Actinomyces Agar /
Actinomycete Isolation Agar M233/M341/M490
Actinomyces Agar/Broth is recommended for the cultivation and maintenance of anaerobic Actinomyces species.Actinomycete Isolation
Agar is also used for the isolation and propagation of Actinomycetes from soil and water.
Composition **
M233 M341 M490
Ingredients Gms/Litre Gms/Litre Gms/Litre
Beef heart infusion, solids 10.000 10.000 -
Sodium caseinate - - 2.000
Tryptose 10.000 10.000 -
L-Asparagine - - 0.100
Casein enzymic hydrolysate 4.000 4.000 -
Sodium propionate - - 4.000
Dipotassium phosphate - - 0.500
Yeast extract 5.000 5.000 -
Dextrose 5.000 5.000 -
Ferrous sulphate - - 0.001
L-Cysteine hydrochloride 1.000 1.000 -
Starch, soluble 1.000 1.000 -
Sodium chloride 5.000 5.000 -
Monopotassium phosphate 15.000 15.000 -
Ammonium sulphate 1.000 1.000 -
Magnesium sulphate 0.200 0.200 0.100
Calcium chloride 0.020 0.020 -
Agar - 20.000 15.000
Final pH ( at 25°C) 7.2±0.2 6.9±0.2 8.1±0.2
Directions
Suspend 21.70 grams of M490 in 1000 ml distilled water
containing 5 ml glycerol or suspend 77.22 grams of M341 or
57.22 grams of M233 in 1000 ml distilled water. Heat to boiling
to dissolve the medium completely.Distribute into tubes or flasks.
Actinomycetes are gram-positive bacteria, which show marked
chemical and morphological diversity but form a distinct
evolutionary line of organisms that range from coccoid and
pleomorphic forms to branched filaments (1). Actinomycetes
form an integral part of soil, water and vegetation. Actinomycete
development leads to the formation of volatile metabolites
(2).Traces of these volatile metabolites are sufficient to impart
disagreeable odour to water or a muddy flavour to fish (3).
Actinomycetes also cause disruptions in wastewater treatment by
forming massive growths, which are capable of producing thick
foam in the activated sludge process (4, 5).Actinomyces Broth is
a modification of the Maintenance Medium formulated by Pine
and Watson (6).Actinomyces Agar/Broth is further modified and
is recommended for the cultivation and maintenance of anaerobic

18
A
®
Actinomycete species (7).Actinomyces Isolation Agar used for
isolation and propagation of Actinomycetes from soil and water
was formulated by Olsen (8).
Actinomyces Agar/ Broth contains beef heart infusion, casein
enzymic hydrolysate, yeast extract, starch and dextrose, which
act as sources of carbon, nitrogen, sulphur, vitamins and other
essential growth factors.The metallic salts provide essential
electrolytes and minerals. Actinomycete Isolation Agar contains
sodium caseinate as nitrogen source.Asparagine in addition
to being an amino acid is also a source of nitrogen. Sodium
propionate is used as a substrate in anaerobic fermentation.
Dipotassium phosphate provides the buffering system.The
sulphates serve as source of sulphur and metallic ions. Glycerol
serves as an additional source of carbon.
Inoculate M490 plates with 1 drop of diluted culture or specimen
and spread over the surface using a sterile bent glass rod.
Incubate at 35°C for 40-72 hours.The media can be used for long
term storage after sufficient growth is obtained.Agar slants are
used for maintenance of cultures over a shorter period of time.
Quality Control
Appearance
Gelling
M341: Firm, comparable with 2.0% Agar gel.
M490:Firm, comparable with 1.5% Agar gel
Colour and Clarity
M233:Yellow to light amber coloured clear solution in tubes.
M341:Yellow to light amber coloured clear to slightly opalescent gel
forms in Petri plates.
M490:Yellow to light amber coloured clear to slightly opalescent gel
forms in Petri plates
Reaction
M490:Reaction of 2.2% w/v aqueous solution containing 0.5% v/v
glycerol at 25°C. pH : 8.1±0.2
Cultural Response
for 40-72 hours.(*- incubated anaerobically)
for 40-72 hours (*- incubated anaerobically)
for 40-72 hours.
Organism Growth
M233
*Actinomyces israelii ATCC 10049 luxuriant
Streptomyces achromogenes ATCC 12767 good
Streptomyces albus subsp albus ATCC 3004 good
Streptomyces lavendulae ATCC 8664 good
*Actinomyces bovis ATCC 13683 good
M341
*Actinomyces israelii ATCC 10049 luxuriant
Streptomyces achromogenes ATCC 12767 good
Streptomyces albus subsp albus ATCC 3004 good
Streptomyces lavendulae ATCC 8664 good
*Actinomyces bovis ATCC 13683 good
M490
Nocardia asteroides ATCC 19427 good-luxuriant
Escherichia coli ATCC 25922 inhibited
Streptomyces albus subsp albus ATCC 3004 good-luxuriant
Streptomyces lavendulae ATCC 8664 good-luxuriant
Reference
1. Collee J. G., FraserA. G., Marmion B. P., SimmonsA., (Eds.), Mackie and McCartney, Practical Medical
Microbiology, 1996, 14th Edition, Churchill Livingstone.
2. Adams B.A., 1929,Water and Water Eng., 31:327.
3. EatonA. D., Clesceri L. S. and GreenbergA.W., (Eds.), 2005, Standard Methods for the Examination of
Water andWastewater, 21st Ed.,APHA,Washington, D.C.
4. Lechevalier H.A., 1975, Environ. ProtectionTechnol. Ser., EPA-600/ 2-75-031, U. S. Environmental
ProtectionAgency, Cincinnati, Ohio.
5. Lechevalier M. P., and Lechevalier H.A., 1974, Int. J. Syst.Bacteriol., 24:278.
6. Pine L., andWatson S. J., 1959, J. Lab. Clin. Med., 54(1), 10
7. Ajello L., Georg L. K., KaplanW. and Kaufman L., 1963, CDC Lab Manual Med. Mycology, PHS Publication
No. 994, CDC,Washington D.C.
8. Olsen, 1960, Personal Communication.
Product Profile
(M341) : 77.22 g/l
(M490) : 21.70 g/l
(M341) : 6.48 L
(M490) : 23.04 L
pH (25°C ) (M233) : 7.2±0.2
(M341) : 6.9±0.2
(M490) : 8.1±0.2
Supplement (M490) : Glycerol
Medium : 2-8° C

19
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®
Adams Agar M855
Adams Agar is recommended for examining sporulation in yeasts.
Composition **
Dextrose 0.400
Sodium acetate 2.300
Agar 20.000
Directions
dissolve the medium completely. Dispense in test tubes. Sterilize
by autoclaving at 108-112°C for 15 minutes.Allow the tubes to
solidify in a slanted position.
Sporulation is one of the most important characteristics for yeast
taxonomic and genetic studies and makes possible the controlled
hybridization of new strains. Sporulation depends on the state
of the culture, the suitability of the medium employed and
environmental factors (1).The formation of adequate numbers of
4-spored asci in yeasts is essential for genetical analysis, and, as
spore viability decreases with age, it is advisable to induce rapid
sporulation and transfer spores as soon as possible to a nutrient
medium containing sugar.Adams (2) has described a convenient
way of obtaining ascospores from Bakers yeast. He described
a modified Stantial (1935) acetate medium consisting of low
concentrations of glucose, sodium acetate, and agar upon which
he obtained high yields of asci with a large number of yeast
cultures.Although, in his original experiments,Adams (1949)
tested a variety of acetate salts, including potassium acetate, he
found none of them superior to sodium acetate in about 0.24 per
cent concentration.
Dextrose in the medium stimulates sporulation (3).Acetate and
dextrose are used as carbon sources.
Quality Control
Appearance
Off-white to light yellow homogeneous free flowing powder
Gelling
Firm,comparable with 2.0% Agar gel.
Colour and Clarity
Yellow coloured clear gel forms in tubes as slants
Cultural Response
M855: Cultural characteristics observed after an incubation at 30°C for
18-48 hours.
Organism Inoculum
(CFU)
Growth Sporulation
Saccharomyces cerevisiae ATCC 9763 50-100 luxuriant positive
¤Aspergillus brasiliensis ATCC 16404 50-100 luxuriant negative
Candida albicans ATCC 10231 50-100 luxuriant negative
Penicillium notatum ATCC 10108 50-100 luxuriant negative
Reference
1. Yishan L. in. 1979, ModifiedYeast Sporulation Media.American Society of Brewing Chemists Inc.Vol. 37,
66-69.
2. AdamsA. M., 1949, Can. J. Res., 27, 179.
3. Stantial H., 1935,The Sporulation ofYeast,Trans. Roy. Soc. Can., III, 29, 175-188.
Product Profile
pH (25°C ) —
Supplement None
Sterilization 108-112°C / 15 minutes
Medium : 2-8° C
¤ Formerly known as Aspergillus niger

20
A
®
Aeromonas Isolation Medium Base M884
Aeromonas Isolation Medium Base with added Ampicillin supplement is recommended for selective and differential isolation of
Aeromonas hydrophila from clinical and environmental specimens.
Composition **
Peptone, special 5.000
Yeast extract 3.000
L-Lysine hydrochloride 3.500
L-Arginine hydrochloride 2.000
Inositol 2.500
Lactose 1.500
Sorbose 3.000
Xylose 3.750
Bile salts 3.000
Sodium thiosulphate 10.670
Ferric ammonium citrate 0.800
Bromo thymol blue 0.040
Thymol blue 0.040
Agar 12.500
Final pH ( at 25°C) 8.0±0.2
Directions
to dissolve the medium completely. DO NOT AUTOCLAVE. Cool
to 45-50°C and aseptically add rehydrated contents of 1 vial of
Aeromonas Selective Supplement (FD039). Mix well and pour
into sterile Petri plates.
Aeromonas species occur widely in soil and water where these
species cause disease in fish and amphibians.Also found in
untreated and chlorinated drinking water, raw food and raw milk
(9, 10). It is observed that the major cause of gastrointestinal
infections by Aeromonas species (10, 11) is because of ingesting
infected water (12, 13).This medium therefore, may be
considered as a useful diagnostic aid for investigating diarrhoeal
disease (5, 14).Aeromonas medium was found to be superior
over some other formulae for detection of Aeromonas species in
tap water, bottled water and foods including meat, poultry, fish
and seafood (6, 7, 8).Aeromonas Isolation Medium is based on
the formulation of Ryan (1). It is a modification of XLD Medium,
which supports the growth of Aeromonas, Plesiomonas, Proteus,
as well as Enterobacteriaceae so the medium is used as universal
medium in the investigation of enteric disease.The selectivity of
the medium is increased by the addition of Ampicillin (FD039).
The effectiveness of Ampicillin as a selective agent has been
reported by several workers (2, 3, 4, 5).
It was noted that the recovery of Aeromonas species was very
low from fresh foods of animal origin when cultivated on clinical
media.Also difficulties were encountered in distinguishing the
Aeromonas hydrophila group from the background microflora.
Polumbo et.al formulated Starch Ampicillin (SA) Agar with starch
hydrolysis as the differential trait and ampicillin to suppress the
background microflora (15).
Peptone special and yeast extract provide essential nitrogenous
compounds. The salts provide the essential minerals and
electrolytes. Sodium chloride maintains osmotic equilibrium.
Lactose, sorbose, inositol and xylose are sources of carbon and
energy.Ampicillin, bile salts and sodium thioglycollate makes the
medium selective. Bromothymol blue and thymol blue acts as
indicators giving the characteristic colony colour.
Aeromonas Isolation Medium Base (M884)
Aeromonas hydrophila ATCC 7966

21
A
®
Quality Control
Appearance
Light yellow to light tan homogeneous free flowing powder
Gelling
Colour and Clarity
Dark green coloured clear to slightly opalescent gel forms in Petri plates.
Reaction
Cultural Response
M884 : Cultural characteristics observed with added Aeromanas Selective
Supplement (FD039) after an incubation at 35-37°C for 18-24 hours.
Organism Inoculum
(CFU)
Growth Recovery Colony
characteristics
Aeromonas hydrophila ATCC
7966
50-100 luxuriant >=50% dark green, opaque
with dark centre
Escherichia coli ATCC 25922 >=103
inhibited 0% -
ATCC 27853
50-100 good-
luxuriant
>=50% blue/grey, translu-
scent pinpoint
Salmonella Typhi ATCC 6539 >=103
inhibited 0% -
Shigella flexneri ATCC 12022 >=103
inhibited 0% -
Reference
1. Ryan N., 1985, Personal Communication.
2. Richardson C. J., Robinson J. O.,Wagener L. B., BurkeV. J., 1982,Antimicrob., Chemother., 9:267.
3. Moulsdale M.T., 1983,The Lancet, 1:351.
4. Rogol M., Sechter I., Grenber L., Gerichter Ch. B., 1979, J. Med. Microbiol., 12:229.
5. Atkinson M., 1986, Culture,Vol. 7, No. 2.
6. Holmes P. and Sartory D. P., 1993, Letters inApplied Microbiol., 17: 58.
7. C. Pin M. L., Marin M. L., Garcia J. et al, 1994, Letters inApplied Microbiol., 18:190.
8. Warburton D.W., McCormick J. K., and Browen B., 1994, Can. J. Microbiol., 40:145.
9. Steering Group on the Microbiological Safety of Foods (SGMSF) in Methods for Use in Microbiological
Superveillance, 1994, MAFF, Ergon House, London SWIP3TR.
10. Buchanan R. L. and Palumb S.A., 1985, J. Food Safety, 7:15.
11. BurkeV. et al 1984,Appl. Environ. Microbiol., 48:361.
12. GeorgeW. L., 1987, Clin. Microbiol., Newsletter 9, 121.
13. Holmberg S. D., et al, 1986,Ann. Intern. Med., 105:683.
14. Moyer N. P., 1987, J. Clin. Microbiol., 25:2044.
15. Palumbo S.A., Maxino F.,WilliamsA. C., Buchanan R. L., andThayer D.W., 1985,Appl. Environ. Microbiol.,
50:1027.
Product Profile
pH (25°C ) 8.0±0.2
Supplement Aeromonas Selective Supplement
(FD039)
Sterilization DO NOT AUTOCLAVE
Medium : 2-8° C
Aero Pseudo Selective Agar M1620
Aero Pseudo Selective Agar is used for detecting Pseudomonas and Aeromonas in food stuffs as well as in waste water and equipment
of the food industry.
Composition **
Sodium glutamate 10.000
Starch, soluble 20.000
Potassium dihydrogen
phosphate
2.000
Magnesium sulfate 0.500
Phenol red 0.360
Agar 12.000
Final pH (at 25°C) 7.2±0.2
Directions
to dissolve the medium completely. Sterilize by autoclaving at
15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C.Add
100,000 IU Penicillin G sodium salt, 0.01 g Pimaricin, if desired.
Mix well and pour into sterile Petri plates.
Aeromonas may not be truly indigenous to the marine
environment, but may have a transient existence after entering
salt water via rivers or sewage inputs (1). Foods that come in
direct contact with water are likely sources of motile aeromonads,
with fish and seafood products most often contaminated
(2). Motile aeromonads can survive at low temperatures and
therefore have been associated with refrigerated animal products
such as chicken, dairy products, raw milk and vegetables (3, 4).
The predominant organism found in these foods is Pseudomonas
species with the motile aeromonads present in lower numbers.
Pseudomonas are capable of causing spoilage because they are
psychrotrophic and thus multiply at refrigeration temperatures
(2).Also they attack various substances in the food to produce
compounds associated with off-flavour and off-odours.Aero
Pseudo Selective Agar medium has been proposed by Kielwein for
detecting Pseudomonas and Aeromonas in foodstuffs, waste water
and equipments used in the food industry (5, 6, 7, 8).
The medium contains sodium glutamate and starch as the only
sources of nutrients. Organisms other than Aeromonas and
Pseudomonas are unable to metabolize these nutrients sources (9).
Aeromonas degrades starch, producing acid.The acid produced

22
A
®
causes the phenol red indicator to change from red to yellow.
This reaction is not exhibited by Pseudomonas. Added Penicillin G
improves the selectivity of the medium.The medium is made more
selective by the addition of antimycotic agent namely Pimaricin.
Quality Control
Appearance
Light yellow to pink homogeneous free flowing powder
Gelling
Firm comparable with 1.2% Agar gel.
Colour and Clarity
Red coloured, clear to slightly opalescent gel forms in Petri plates.
Reaction
Cultural Response
M1620: Cultural characteristics observed with added Penicillin G
sodium salt, after an incubation at 35-37°C for 18-24 hours.
Organism Inoculum
(CFU)
Growth Recovery Colour of colony
Escherichia coli ATCC 25922 50-100 none-poor <=10% -
Staphylococcus aureus ATCC
25923
>=103
inhibited 0% -
27853
50-100 good-
luxuriant
>=50% red-violet
surrounded by a red
violet zone
ATCC 9027
50-100 good-
luxuriant
>=50% red-violet
surrounded by a red
violet zone
10145
50-100 fair-good 30-40% red-violet
surrounded by a red
violet zone
Aeromonas hydrophila ATCC
7966
50-100 good-
luxuriant
>=50% yellow surrounded
by a yellow zone
Aeromonas caviae ATCC
15467
50-100 good-
luxuriant
>=50% yellow surrounded
by yellow zone
Reference
1. Rippey S. R. and CabelliV. J., 1979,Appl. Environ. Microbiol.,38:108
3. Callister S. M., and AggerW.A., 1987,Appl. Environ. Microbiol., 5 3:249
4. Hunter P. R. and Burge S. H., 1987, Lett.Appl. Microbiol., 4:45
5. Kielwein G., Gerlach R. U., Johne H., 1969,Arch. F. Lebensmittelhyg., 20; 34-38 .
6. Kielwin G., 1969,Arch. F Lebensmittelhyg., 20; 131-133.
7. Kielwin G., 1971,Arch. F. Lebensmittelhyg., 22; 15-19.
8. Kielwin G., 1971, 22; 29-37.
9. Stanier R.Y., Palleroni N. J., 1966, J. Gen. Microbiol., 42; 159-271.
Product Profile
pH (25°C ) 7.2±0.2
Supplement Penicillin G Sodium Salt
Pimaricin (if desired)
Medium : 2-8° C
Algae Culture Broth / Algae Culture Agar M342/M343
Algae Culture Agar / Broth is recommended for the isolation and cultivation of algae from soil, water and sewage.The broth medium
is used for preparing inoculum whereas the agar medium is used for carrying stock cultures of algae used in the bioassay of algicidal
chemicals.
Composition **
M342 M343
Sodium nitrate 1.000 1.000
Dipotassium phosphate 0.250 0.250
Ammonium chloride 0.050 0.050
Ferric chloride 0.003 0.003
Agar - 15.000
Final pH ( at 25°C) 7.0±0.2 7.0±0.2
Directions
completely. Dispense as desired. Sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes.
Algae (singular alga) encompass several groups of relatively
simple living aquatic organisms that capture light energy through
photosynthesis, using it to convert inorganic substances into
organic matter.Algae range from single-cell organisms to
multicellular organisms, some with fairly complex differentiated
form and (if marine) called seaweeds.Algae are usually found in
damp places or water bodies and thus are common in terrestrial
as well as aquatic environments. Various algae play significant
roles in aquatic ecology.Algae are used by humans in a number
of ways. Because many species are aquatic and microscopic, they
are cultured in clear tanks or ponds and either harvested or used
to treat effluents pumped through ponds (1, 2).Algae Culture
Agar / Broth are recommended for the isolation and cultivation of
algae from soil, water and sewage.Algae Culture Broth is used to
prepare the inoculum for the bioassay of algicidal chemicals.Algae

23
A
®
Culture Agar is used for maintaining stock cultures of algae used
in the bioassay of algaecide chemicals. It is a slight modification
of the formula of Allen (3). Fitzgerald (4) recommended it for the
cultivation of algae.
The medium provides all necessary nutrients for good growth
of Algae but does not provide for other than minimal growth of
bacteria and fungi. Stock cultures are prepared by inoculating the
surface of slants with the algal culture and incubation at room
temperature under a suitable light source.These stock cultures
can be maintained for several months.
Quality Control
Appearance
M342:White to light yellow homogeneous free flowing powder
M343: Off-white to light yellow homogeneous free flowing powder
Gelling
Colour and Clarity
M342:White coloured clear to slightly opalescent solution in tubes.
M343:White coloured clear to slightly opalescent gel forms in Petri plates.
Reaction
Cultural Response
M342: Cultural characteristics observed under suitable light source after
an incubation at 20-25°C within 1 week.
M343: Cultural characteristics observed under suitable light source after
an incubation at 20-25°C within 1 week.
Organism Growth
M342
Chlorella pyrenoidosa ATCC 50476 good-luxuriant
M343
Chlorella pyrenoidosa ATCC 50476 good-luxuriant
Reference
1. Lembi C.A. andWaaland J. R., (Ed.),Algae and HumanAffairs, 1988,Cambridge University Press.
2. Guiry M. D. and Blunden G., (Ed.), 1991, Seaweed Resources in Europe : Uses and Potential. JohnWiley
and Sons Ltd.
3. Allen, 1952,Arch. Microbiol., 17:34.
4. Fitzgerald, 1962,Water and SewageWorks, 109:361.
Product Profile
(M343) : 16.87 g/l
(M343) : 29.64 L
(100g) (M342) : 53.47 L
pH (25°C ) (M342) : 7.0±0.2
(M343) : 7.0±0.2
Supplement None
Medium : 2-8° C
Alicyclobacillus Medium / Alicyclobacillus Agar M1561/M1650
Alicyclobacillus Agar/medium is used for the isolation, cultivation and detection of Alicyclobacillus species from fruit juice.
Composition **
M1561 M1650
Glucose 5.000 -
Dextrose - 5.000
Ammonium sulphate 0.200 0.200
Potassium dihydrogen phosphate 3.000 3.000
Zinc sulphate 0.00018 0.00018
Copper sulphate 0.00016 0.00016
Manganese sulphate 0.00015 0.00015
Cobalt chloride 0.00018 0.00018
Boric acid 0.00010 0.00010
Sodium molybdate 0.00030 0.00030
Agar - 18.000
Final pH ( at 25°C) 4.0±0.2 4.0±0.2
Directions
completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for
15 minutes. Mix well and dispense as desired.
Note: Adjust the pH of the medium to 4.0 ± 0.2 (after
sterilization) using 1N H2
SO4
or 1N NaOH.
Alicyclobacillus species are gram-positive aerobic thermophillic,
and spore forming acidophilic bacteria. Alicyclobacillus are
sometimes called Acidophilic Thermophilic Bacteria (ATB).These
spore-forming organisms are able to survive the relatively mild

24
A
®
pasteurization temperatures used for fruit juices and drinks. Some
are even able to grow out and cause spoilage of the beverage. Even
very low numbers of Alicyclobacillus are able to cause spoilage and
produce objectionable flavours and odours specially affecting the
quality of fruit juice (1, 2) and in the beverages, damaging the brand.
These bacteria are able to grow at pH values as low as 2.5 and also
at elevated temperatures as high as 60°C.
These media have a pH of 4.0 ± 0.2 which inhibits most of the
microbial flora. Rest of the microbial flora is inhibited at 60°C,
which is the optimum growth temperature for Alicyclobacillus
species.Alicyclobacillus Agar is a slight modification of the
medium recommended for the cultivation of Alicylobacillus
acidoterrestris (3).
Quality Control
Appearance
Gelling
Colour and Clarity
M1561:Light amber coloured clear solution in tubes
M1650:Light amber coloured clear to slightly opalescent gel forms in
Petri plates
Reaction
M1650: Reaction of 2.89% w/v aqueous solution at 25°C.
pH : 4.0±0.2
Cultural Response
for 48-72 hours.
for 48-72 hours.
Organism Growth
M1561
Alicyclobacillus acidocaldarius ATCC 27009 good-luxuriant
Candida albicans ATCC 10231 inhibited
Staphylococcus aureus ATCC 25923 inhibited
Saccharomyces cerevisiae ATCC 9763 inhibited
M1650
Alicyclobacillus acidocaldarius ATCC 27009 good to luxuriant
Staphylococcus aureus ATCC 25923 inhibited
Saccharomyces cerevisiae ATCC 9763 inhibited
Candida albicans ATCC 10231 inhibited
Reference
1. Ceny G., HennlishW. and K Rocallia-Furchtsaftwerb ducrh Baciilen. Isobioerung and Charakteriseeuing des
Verdebserregens-Z hebers Utres Forsch 179: 224-227, 1984.
2. Baungart and Merve S.,The Impact ofAlicyclobacillus acidoterstris on the Quality of Juices and Soft Drinks
Fruit processing 7: 251-254 (2000).
3. Atlas R. M., 2004, Handbook of Microbiological Media, 3rd Ed, CRC Press.
Product Profile
(M1650) : 28.95 g/l
(M1650) : 17.27 L
pH (25°C ) (M1561) : 4.0±0.2
(M1650) : 4.0±0.2
Supplement None
Medium : 2-8° C
Alkaline Peptone Water M618/M618I
Alkaline Peptone Water is recommended for enrichment of Vibrio species.
Composition **
M618 M618I
Final pH ( at 25°C) 8.4±0.2 8.6±0.2
Directions
Suspend 20 grams of M618 or 50 grams of M618I in
1000 ml distilled water. Heat if necessary to dissolve the medium
completely. Dispense as desired and sterilize by autoclaving at
15 lbs pressure (121°C) for 15 minutes.
Clinical materials containing small numbers of Vibrio should be
inoculated into an enrichment medium prior to plating onto a
selective medium, such as TCBS Agar (M189).Alkaline Peptone
Water is a suitable enrichment broth for this purpose (1-3).The
relatively high pH of the medium (approximately 8.4) provides a
favorable environment for the growth of Vibrios.This medium is

25
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recommended by APHA (4) for enrichment of Vibrio species from
seafood, infectious materials and other clinical specimens such as
faeces (5). A slight modification of this medium has recently been
approved by the ISO Committee (6) for detection of Vibrio species.
Peptic digest of animal tissue provides amino acids and other
nitrogenous substances. Sodium chloride maintains osmotic
equilibrium.
Add 10 grams of seafood to 90 ml of Alkaline Peptone Water and
incubate for upto 18-20 hrs at 37°C. Prolonged incubation will
result in growth of the suppressed contaminating organisms to
develop (7). Growth in tubes is indicated by turbidity compared
to an un-inoculated tube (control). Growth from the enrichment
broth is used for plating on selective media. For biochemical
identification a pure culture is recommended.
Quality Control
Appearance
M618I: Cream to yellow homogeneous free flowing powder
Colour and Clarity
M618: Light yellow coloured clear solution without any precipitate
M618I: Light yellow coloured clear solution without any precipitate
Reaction
M618: Reaction of 2% w/v aqueous solution at 25°C. pH : 8.4±0.2
M618I:Reaction of 5% w/v aqueous solution at 25°C. pH : 8.6±0.2
Cultural Response
for 18-24 hours.
M618I: Cultural characteristics observed after an incubation at 35-37°C
for 18-24 hours .
Organism Inoculum
(CFU)
Growth
M618
Vibrio cholerae ATCC 15748 50-100 luxuriant
Vibrio parahaemolyticus ATCC 17802 50-100 luxuriant
M618I
Vibrio cholerae ATCC 15748 50-100 luxuriant
Vibrio parahaemolyticus ATCC 17802 50-100 luxuriant
Reference
1. Gilligan, Janda, Karmali and Miller, 1992, Cumitech 12A, Laboratory Diagnosis of Bacterial Diarrhea,
Coord. Ed., Nolte,American Society for Microbiology,Washington, D.C.
2. Forbes B.A., SahmA. S., and Weissfeld D. F., Bailey & Scotts Diagnostic Microbiology, 10th Ed., 1998,
Mosby, Inc., St. Louis, Mo.
3. Isenberg, (Ed.), 1992, Clinical Microbiology Procedures Handbook,Vol. I, American Society for
Microbiology,Washington, D.C.
Foods, 4th Ed.,APHA,Washington, D.C.
5. Cruikshank R., 1968, Medical Microbiol., 11th Ed., Livingstone Ltd., London.
6. International Organization for Standardization (ISO), 1990, Draft ISO/DIS 8914.
7. Finegold S. M. and MartinW. J., 1982,W. J. Bailey and Scotts Diagnostic Microbiol, 6th Ed., C.V. Mosby
Co., St. Louis, p. 242.
Product Profile
(M618I) : 50.00 g/l
(M618I) : 10.00 L
(100g) (M618) : 5.00 L
pH (25°C ) (M618) : 8.4±0.2
(M618I) : 8.6±0.2
Supplement None
Medium : 2-8° C
Note : (M618) :Veg Medium is also available.
Alternative Thioglycollate Medium (NIH Thioglycollate Broth) M010
Alternative Thioglycollate Medium is recommended for sterility testing of turbid or viscous biological products.
Composition **
Yeast extract 5.000
Dextrose 5.500
L-Cystine 0.500
Sodium thioglycollate 0.500
Final pH ( at 25°C) 7.1±0.2
Directions
Suspend 29 grams in 1000 ml distilled water. Heat if necessary
to dissolve the medium completely. Sterilize by autoclaving at 15
lbs pressure (121°C) for 15 minutes. Mix well and dispense as
desired.
Note: It is preferable to use freshly prepared medium,
alternatively it should be boiled and cooled just once prior to use
as on reheating, toxic oxygen radicles are formed.
Alternative Thioglycollate Medium is formulated as described
in the N.I.H. memorandum (1). It is used for the sterility testing
of certain biological products which are turbid or viscous and

26
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can’t be tested using Fluid Thioglycollate Medium (M009). Both
the media have similar composition, except agar and resazurin
that are not included in Alternative Thioglycollate Medium.This
deletion makes it suitable for sterility testing of viscous products.
Casein enzymic hydrolysate and yeast extract serve as source
of essential nutrients to the contaminants, if present. Dextrose
serves as the energy source. Sodium chloride maintains the
osmotic equilibrium of the medium whereas L-cystine, an amino
acid, also serves as source of essential growth factors. Sodium
thioglycollate and L-cystine lower the oxidation-reduction
potential of the medium by removing oxygen to maintain a low
Eh. Sodium thioglycollate also helps to neutralize the toxic effects
of mercurial preservatives (2, 3).
Quality Control
Appearance
Colour and Clarity
Yellow coloured clear solution without any precipitate
Reaction
Cultural Response
for 24-72 hours.
Organism Inoculum
(CFU)
Growth
* Bacteroides vulgatus ATCC 8482 50-100 luxuriant
* Clostridium sporogenes ATCC 11437 50-100 luxuriant
Candida albicans ATCC 10231 50-100 luxuriant
Bacillus subtilis ATCC 6633 50-100 luxuriant
* Bacteroides fragilis ATCC 25285 50-100 luxuriant
Micrococcus luteus ATCC 10240 50-100 luxuriant
Streptococcus pyogenes ATCC 19615 50-100 luxuriant
Candida albicans ATCC 2091 50-100 luxuriant
Candida albicans NCYC 854 50-100 luxuriant
Bacillus subtilis NCTC 8236 50-100 luxuriant
Clostridium sporogenes ATCC 19404 50-100 luxuriant
Clostridium sporogenes NCTC 532 50-100 luxuriant
Clostridium sporogenes CIP 79.3 50-100 luxuriant
Staphylococcus aureus ATCC 6538p 50-100 luxuriant
Staphylococcus aureus NCIMB 9518 50-100 luxuriant
Micrococcus luteus ATCC 9341 50-100 luxuriant
Micrococcus luteus NCTC 7743 50-100 luxuriant
Key : * : Incubated anaerobically.
Reference
1. N.I.H. Memorandum, 1955: Culture Media for SterilityTests, 4th Revision.
2. Nungester, Hood andWarren, 1943, Proc. Soc. Exp. Biol. Med., 52: 287
3. Portwood, 1944, J. Bacteriol., 48: 255
Product Profile
(100g) 3.45 L
pH (25°C ) 7.1±0.2
Supplement None
Medium : 2-8° C
Alternative Thioglycollate Medium (NIH Thioglycollate Broth) (M010)
1 2 3 4 5 6 7
1 Control 2 Streptococcus pyogenes ATCC 19615 3 Staphylococcus aureus ATCC 25923
4 Bacillus subtilis ATCC 6633 5 Bacteroides vulgatus ATCC 8482 6 Candida albicans ATCC 10231
7 Bacteroides fragilis ATCC 25285 8 Clostridium sporogenes ATCC 19404
8

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