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112 á61ñ Microbiological Examination / Microbiological Tests USP 38

PLATE-COUNT METHODS

Pour-Plate Method—Prepare the sample using a method that has been shown to be suitable as described in Growth Pro-
motion Test and Suitability of the Counting Method. Prepare for each medium at least two Petri dishes for each level of dilution.
Incubate the plates of Soybean–Casein Digest Agar at 30° to 35° for 3 to 5 days and the plates of Sabouraud Dextrose Agar at
20° to 25° for 5 to 7 days. Select the plates corresponding to a given dilution and showing the highest number of colonies less
than 250 for TAMC and 50 for TYMC. Take the arithmetic mean per culture medium of the counts, and calculate the number
of cfu per g or per mL of product.
Surface-Spread Method—Prepare the sample using a method that has been shown to be suitable as described in Growth
Promotion Test and Suitability of the Counting Method. Prepare at least two Petri dishes for each medium and each level of dilu-
tion. For incubation and calculation of the number of cfu, proceed as directed for the Pour-Plate Method.

MOST-PROBABLE-NUMBER METHOD

Prepare and dilute the sample using a method that has been shown to be suitable as decribed in Growth Promotion Test and
Suitability of the Counting Method. Incubate all tubes for 3 to 5 days at 30° to 35°. Subculture if necessary, using the procedure
shown to be suitable. Record for each level of dilution the number of tubes showing microbial growth. Determine the most
probable number of microorganisms per g or mL of the product to be examined from Table 3.

Interpretation of the Results

General Chapters

The total aerobic microbial count (TAMC) is considered to be equal to the number of cfu found using Soybean–Casein Digest
Agar; if colonies of fungi are detected on this medium, they are counted as part of TAMC. The total combined yeasts and
molds count (TYMC) is considered to be equal to the number of cfu found using Sabouraud Dextrose Agar; if colonies of bacte-
ria are detected on this medium, they are counted as part of TYMC. When the TYMC is expected to exceed the acceptance
criterion due to the bacterial growth, Sabouraud Dextrose Agar containing antibiotics may be used. If the count is carried out
by the MPN Method, the calculated value is TAMC.
When an acceptance criterion for microbiological quality is prescribed, it is interpreted as follows:
— 101 cfu: maximum acceptable count = 20;
— 102 cfu: maximum acceptable count = 200;
— 103 cfu: maximum acceptable count = 2000;
and so forth.
The recommended solutions and media are described in Tests for Specified Microorganisms á62ñ.

á62ñ MICROBIOLOGICAL EXAMINATION OF NONSTERILE

PRODUCTS: TESTS FOR SPECIFIED MICROORGANISMS

INTRODUCTION

The tests described hereafter will allow determination of the absence of, or limited occurrence of, specified microorganisms
that may be detected under the conditions described.
The tests are designed primarily to determine whether a substance or preparation complies with an established specification
for microbiological quality. When used for such purposes, follow the instructions given below, including the number of sam-
ples to be taken, and interpret the results as stated below.
Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the
Pharmacopeial method has been demonstrated.

GENERAL PROCEDURES

The preparation of samples is carried out as described in Microbiological Examination of Nonsterile Products: Microbial Enumer-
ation Tests á61ñ.
If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralized as described in
Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ.
If surface-active substances are used for sample preparation, their absence of toxicity for microorganisms and their compati-
bility with any inactivators used must be demonstrated as described in Microbiological Examination of Nonsterile Products: Micro-
bial Enumeration Tests á61ñ.

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USP 38 Microbiological Tests / á62ñ Microbiological Examination 113

GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY OF THE

TEST AND NEGATIVE CONTROLS

The ability of the test to detect microorganisms in the presence of the product to be tested must be established. Suitability
must be confirmed if a change in testing performance or a change in the product that may affect the outcome of the test is
introduced.

Preparation of Test Strains

Use standardized stable suspensions of test strains as stated below. Seed-lot culture maintenance techniques (seed-lot sys-
tems) are used so that the viable microorganisms used for inoculation are not more than five passages removed from the origi-
nal master seed-lot.

AEROBIC MICROORGANISMS

Grow each of the bacterial test strains separately in containers containing Soybean–Casein Digest Broth or on Soybean–Casein
Digest Agar at 30° to 35° for 18 to 24 hours. Grow the test strain for Candida albicans separately on Sabouraud Dextrose Agar or
in Sabouraud Dextrose Broth at 20° to 25° for 2 to 3 days.
Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83, or NBRC 13276

General Chapters
Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626, CIP 82.118, or NBRC 13275
Escherichia coli such as ATCC 8739, NCIMB 8545, CIP 53.126, or NBRC 3972
Salmonella enterica subsp. enterica serovar Typhimurium or, as an alterna- such as ATCC 14028
tive,
Salmonella enterica subsp. enterica serovar Abony such as NBRC 100797, NCTC 6017, or CIP 80.39
Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72, or NBRC 1594

Use Buffered Sodium Chloride–Peptone Solution pH 7.0 or Phosphate Buffer Solution pH 7.2 to make test suspensions. Use the
suspensions within 2 hours or within 24 hours if stored at 2° to 8°.

CLOSTRIDIA

Use Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or
CIP 79.3). Grow the clostridial test strain under anaerobic conditions in Reinforced Medium for Clostridia at 30° to 35° for 24 to
48 hours. As an alternative to preparing and then diluting down a fresh suspension of vegetative cells of Cl. sporogenes, a sta-
ble spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2° to 8° for a validated
period.

Negative Control

To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There
must be no growth of microorganisms. A negative control is also performed when testing the products as described under
Testing of Products. A failed negative control requires an investigation.

Growth Promotion and Inhibitory Properties

of the Media

Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from
ingredients. Verify suitable properties of relevant media as described in Table 1.
Table 1. Growth Promoting, Inhibitory, and Indicative Properties of Media
Test/Medium Property Test Strains
Test for bile-tolerant Gram-negative bacteria
Enterobacteria Enrichment Broth Mossel Growth promoting E. coli
P. aeruginosa
Inhibitory S. aureus
Violet Red Bile Glucose Agar Growth promoting + Indicative E. coli
P. aeruginosa
Test for Escherichia coli

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114 á62ñ Microbiological Examination / Microbiological Tests USP 38

Table 1. Growth Promoting, Inhibitory, and Indicative Properties of Media (Continued)

Test/Medium Property Test Strains
MacConkey Broth Growth promoting E. coli
Inhibitory S. aureus
MacConkey Agar Growth promoting + Indicative E. coli
Test for Salmonella
Rappaport Vassiliadis Salmonella Enrichment Broth Growth promoting Salmonella enterica subsp. enterica serovar
Typhimurium or
Salmonella enterica subsp. enterica serovar
Abony
Inhibitory S. aureus
Xylose Lysine Deoxycholate Agar Growth promoting + Indicative Salmonella enterica subsp. enterica serovar
Typhimurium or
Salmonella enterica subsp. enterica serovar
Abony
Test for Pseudomonas aeruginosa
Cetrimide Agar Growth promoting P. aeruginosa
Inhibitory E. coli
Test for Staphylococcus aureus
General Chapters

Mannitol Salt Agar Growth promoting + Indicative S. aureus

Inhibitory E. coli
Test for Clostridia
Reinforced Medium for Clostridia Growth promoting Cl. sporogenes
Columbia Agar Growth promoting Cl. sporogenes
Test for Candida albicans
Sabouraud Dextrose Broth Growth promoting C. albicans
Sabouraud Dextrose Agar Growth promoting + Indicative C. albicans

Test for Growth-Promoting Properties, Liquid Media—Inoculate a portion of the appropriate medium with a small num-
ber (not more than 100 cfu) of the appropriate microorganism. Incubate at the specified temperature for not more than the
shortest period of time specified in the test. Clearly visible growth of the microorganism comparable to that previously ob-
tained with a previously tested and approved batch of medium occurs.
Test for Growth-Promoting Properties, Solid Media—Perform Surface-Spread Method (see Plate-Count Methods under
Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ), inoculating each plate with a small number
(not more than 100 cfu) of the appropriate microorganism. Incubate at the specified temperature for not more than the short-
est period of time specified in the test. Growth of the microorganism comparable to that previously obtained with a previously
tested and approved batch of medium occurs.
Test for Inhibitory Properties, Liquid or Solid Media—Inoculate the appropriate medium with at least 100 cfu of the ap-
propriate microorganism. Incubate at the specified temperature for not less than the longest period of time specified in the
test. No growth of the test microorganism occurs.
Test for Indicative Properties—Perform Surface-Spread Method (see Plate-Count Methods under Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests á61ñ), inoculating each plate with a small number (not more than 100 cfu) of
the appropriate microorganism. Incubate at the specified temperature for a period of time within the range specified in the
test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and
approved batch of medium.

Suitability of the Test Method

For each new product to be tested perform sample preparation as described in the relevant paragraph under Testing of Prod-
ucts. At the time of mixing, add each test strain in the prescribed growth medium. Inoculate the test strains individually. Use a
number of microorganisms equivalent to not more than 100 cfu in the inoculated test preparation.
Perform the test as described in the relevant paragraph under Testing of Products using the shortest incubation period pre-
scribed.
The specified microorganisms must be detected with the indication reactions as described under Testing of Products.
Any antimicrobial activity of the product necessitates a modification of the test procedure (see Neutralization/Removal of An-
timicrobial Activity under Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ).
For a given product, if the antimicrobial activity with respect to a microorganism for which testing is prescribed cannot be
neutralized, then it is to be assumed that the inhibited microorganism will not be present in the product.

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USP 38 Microbiological Tests / á62ñ Microbiological Examination 115

TESTING OF PRODUCTS

Bile-Tolerant Gram-Negative Bacteria

Sample Preparation and Pre-Incubation—Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to
be examined as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ, but using Soy-
bean–Casein Digest Broth as the chosen diluent, mix, and incubate at 20° to 25° for a time sufficient to resuscitate the bacteria
but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours).
Test for Absence—Unless otherwise prescribed, use the volume corresponding to 1 g of the product, as prepared in Sam-
ple Preparation and Pre-Incubation, to inoculate Enterobacteria Enrichment Broth Mossel. Incubate at 30° to 35° for 24 to 48
hours. Subculture on plates of Violet Red Bile Glucose Agar. Incubate at 30° to 35° for 18 to 24 hours.
The product complies with the test if there is no growth of colonies.
Quantitative Test—
Selection and Subculture—Inoculate suitable quantities of Enterobacteria Enrichment Broth Mossel with the preparation as di-
rected under Sample Preparation and Pre-Incubation and/or dilutions of it containing respectively 0.1 g, 0.01 g, and 0.001 g (or
0.1 mL, 0.01 mL, and 0.001 mL) of the product to be examined. Incubate at 30° to 35° for 24 to 48 hours. Subculture each of
the cultures on a plate of Violet Red Bile Glucose Agar. Incubate at 30° to 35° for 18 to 24 hours.
Interpretation—Growth of colonies constitutes a positive result. Note the smallest quantity of the product that gives a posi-
tive result and the largest quantity that gives a negative result. Determine from Table 2 the probable number of bacteria.
Table 2. Interpretation of Results

General Chapters
Results for Each Quantity of Product
Probable Number
0.1 g or 0.01 g or 0.001 g or of Bacteria per g
0.1 mL 0.01 mL 0.001 mL or mL of Product
+ + + more than 103
+ + – less than 103 and more than 102
+ – – less than 102 and more than 10
– – – less than 10

Escherichia coli

Sample Preparation and Pre-Incubation—Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to
be examined as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ, and use 10 mL
or the quantity corresponding to 1 g or 1 mL, to inoculate a suitable amount (determined as described under Suitability of the
Test Method) of Soybean–Casein Digest Broth, mix, and incubate at 30° to 35° for 18 to 24 hours.
Selection and Subculture—Shake the container, transfer 1 mL of Soybean–Casein Digest Broth to 100 mL of MacConkey
Broth, and incubate at 42° to 44° for 24 to 48 hours. Subculture on a plate of MacConkey Agar at 30° to 35° for 18 to 72 hours.
Interpretation—Growth of colonies indicates the possible presence of E. coli. This is confirmed by identification tests.
The product complies with the test if no colonies are present or if the identification tests are negative.

Salmonella

Sample Preparation and Pre-Incubation—Prepare the product to be examined as described in Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests á61ñ, and use the quantity corresponding to not less than 10 g or 10 mL to
inoculate a suitable amount (determined as described under Suitability of the Test Method) of Soybean–Casein Digest Broth, mix,
and incubate at 30° to 35° for 18 to 24 hours.
Selection and Subculture—Transfer 0.1 mL of Soybean–Casein Digest Broth to 10 mL of Rappaport Vassiliadis Salmonella En-
richment Broth, and incubate at 30° to 35° for 18 to 24 hours. Subculture on plates of Xylose Lysine Deoxycholate Agar. Incubate
at 30° to 35° for 18 to 48 hours.
Interpretation—The possible presence of Salmonella is indicated by the growth of well-developed, red colonies, with or
without black centers. This is confirmed by identification tests.
The product complies with the test if colonies of the types described are not present or if the confirmatory identification
tests are negative.

Pseudomonas aeruginosa

Sample Preparation and Pre-Incubation—Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to
be examined as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ, and use 10 mL
or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under Suitability of the

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116 á62ñ Microbiological Examination / Microbiological Tests USP 38

Test Method) of Soybean–Casein Digest Broth, and mix. When testing transdermal patches, filter the volume of sample corre-
sponding to one patch of the preparation (see Transdermal Patches under Preparation of the Sample in Microbiological Examina-
tion of Nonsterile Products: Microbial Enumeration Tests á61ñ) through a sterile filter membrane, and place in 100 mL of Soybean–
Casein Digest Broth. Incubate at 30° to 35° for 18 to 24 hours.
Selection and Subculture—Subculture on a plate of Cetrimide Agar, and incubate at 30° to 35° for 18 to 72 hours.
Interpretation—Growth of colonies indicates the possible presence of P. aeruginosa. This is confirmed by identification
tests.
The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.

Staphylococcus aureus

Sample Preparation and Pre-Incubation—Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to
be examined as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ, and use 10 mL
or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under Suitability of the
Test Method) of Soybean–Casein Digest Broth, and homogenize. When testing transdermal patches, filter the volume of sample
corresponding to one patch of the preparation (see Transdermal Patches under Preparation of the Sample in Microbiological Ex-
amination of Nonsterile Products: Microbial Enumeration Tests á61ñ) through a sterile filter membrane, and place in 100 mL of
Soybean–Casein Digest Broth. Incubate at 30° to 35° for 18 to 24 hours.
Selection and Subculture—Subculture on a plate of Mannitol Salt Agar, and incubate at 30° to 35° for 18 to 72 hours.
Interpretation—The possible presence of S. aureus is indicated by the growth of yellow or white colonies surrounded by a
General Chapters

yellow zone. This is confirmed by identification tests.

The product complies with the test if colonies of the types described are not present or if the confirmatory identification
tests are negative.

Clostridia

Sample Preparation and Heat Treatment—Prepare a sample using a 1 in 10 dilution (with a minimum total volume of 20
mL) of not less than 2 g or 2 mL of the product to be examined as described in Microbiological Examination of Nonsterile Prod-
ucts: Microbial Enumeration Tests á61ñ. Divide the sample into two portions of at least 10 mL. Heat one portion at 80° for 10
minutes, and cool rapidly. Do not heat the other portion.
Selection and Subculture—Use 10 mL or the quantity corresponding to 1 g or 1 mL of the product to be examined of
both portions to inoculate suitable amounts (determined as described under Suitability of the Test Method) of Reinforced Medi-
um for Clostridia. Incubate under anaerobic conditions at 30° to 35° for 48 hours. After incubation, make subcultures from each
container on Columbia Agar, and incubate under anaerobic conditions at 30° to 35° for 48 to 72 hours.
Interpretation—The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reac-
tion indicates the presence of Clostridia.
This is confirmed by identification tests. The product complies with the test if colonies of the types described are not present
or if the confirmatory identification tests are negative.

Candida albicans

Sample Preparation and Pre-Incubation—Prepare the product to be examined as described in Microbiological Examination
of Nonsterile Products: Microbial Enumeration Tests á61ñ, and use 10 mL or the quantity corresponding to not less than 1 g or 1
mL, to inoculate 100 mL of Sabouraud Dextrose Broth, and mix. Incubate at 30° to 35° for 3 to 5 days.
Selection and Subculture—Subculture on a plate of Sabouraud Dextrose Agar, and incubate at 30° to 35° for 24 to 48
hours.
Interpretation—Growth of white colonies may indicate the presence of C. albicans. This is confirmed by identification tests.
The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.

RECOMMENDED SOLUTIONS AND

CULTURE MEDIA

NOTE—This section is given for information.

The following solutions and culture media have been found satisfactory for the purposes for which they are prescribed in the
test for microbial contamination in the Pharmacopeia. Other media may be used provided that their suitability can be demon-
strated.
Stock Buffer Solution—Transfer 34 g of potassium dihydrogen phosphate to a 1000-mL volumetric flask, dissolve in 500
mL of Purified Water, adjust with sodium hydroxide to a pH of 7.2 ± 0.2, add Purified Water to volume, and mix. Dispense in
containers, and sterilize. Store at a temperature of 2° to 8°.
Phosphate Buffer Solution pH 7.2—Prepare a mixture of Purified Water and Stock Buffer Solution (800:1 v/v), and sterilize.

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USP 38 Microbiological Tests / á62ñ Microbiological Examination 117

Buffered Sodium Chloride–Peptone Solution pH 7.0

Potassium Dihydrogen Phosphate 3.6 g
Disodium Hydrogen Phosphate Dihydrate 7.2 g (equivalent to 0.067 M phosphate)
Sodium Chloride 4.3 g
Peptone (meat or casein) 1.0 g
Purified Water 1000 mL

Sterilize in an autoclave using a validated cycle.

Soybean–Casein Digest Broth
Pancreatic Digest of Casein 17.0 g
Papaic Digest of Soybean 3.0 g
Sodium Chloride 5.0 g
Dibasic Hydrogen Phosphate 2.5 g
Glucose Monohydrate 2.5 g
Purified Water 1000 mL

Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle.
Soybean–Casein Digest Agar

General Chapters
Pancreatic Digest of Casein 15.0 g
Papaic Digest of Soybean 5.0 g
Sodium Chloride 5.0 g
Agar 15.0 g
Purified Water 1000 mL

Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle.
Sabouraud Dextrose Agar
Dextrose 40.0 g
Mixture of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein (1:1) 10.0 g
Agar 15.0 g
Purified Water 1000 mL

Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle.
Potato Dextrose Agar
Infusion from potatoes 200 g
Dextrose 20.0 g
Agar 15.0 g
Purified Water 1000 mL

Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle.
Sabouraud Dextrose Broth
Dextrose 20.0 g
Mixture of Peptic Digest of Animal Tissue and Pancreatic Digest of Casein (1:1) 10.0 g
Purified Water 1000 mL

Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle.
Enterobacteria Enrichment Broth Mossel
Pancreatic Digest of Gelatin 10.0 g
Glucose Monohydrate 5.0 g
Dehydrated Ox Bile 20.0 g
Potassium Dihydrogen Phosphate 2.0 g
Disodium Hydrogen Phosphate Dihydrate 8.0 g
Brilliant Green 15 mg
Purified Water 1000 mL

Adjust the pH so that after heating it is 7.2 ± 0.2 at 25°. Heat at 100° for 30 minutes, and cool immediately.

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118 á62ñ Microbiological Examination / Microbiological Tests USP 38

Violet Red Bile Glucose Agar

Yeast Extract 3.0 g
Pancreatic Digest of Gelatin 7.0 g
Bile Salts 1.5 g
Sodium Chloride 5.0 g
Glucose Monohydrate 10.0 g
Agar 15.0 g
Neutral Red 30 mg
Crystal Violet 2 mg
Purified Water 1000 mL

Adjust the pH so that after heating it is 7.4 ± 0.2 at 25°. Heat to boiling; do not heat in an autoclave.
MacConkey Broth
Pancreatic Digest of Gelatin 20.0 g
Lactose Monohydrate 10.0 g
Dehydrated Ox Bile 5.0 g
Bromocresol Purple 10 mg
Purified Water 1000 mL
General Chapters

Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle.
MacConkey Agar
Pancreatic Digest of Gelatin 17.0 g
Peptones (meat and casein) 3.0 g
Lactose Monohydrate 10.0 g
Sodium Chloride 5.0 g
Bile Salts 1.5 g
Agar 13.5 g
Neutral Red 30.0 mg
Crystal Violet 1 mg
Purified Water 1000 mL

Adjust the pH so that after sterilization it is 7.1 ± 0.2 at 25°. Boil for 1 minute with constant shaking, then sterilize in an
autoclave using a validated cycle.
Rappaport Vassiliadis Salmonella Enrichment Broth
Soya Peptone 4.5 g
Magnesium Chloride Hexahydrate 29.0 g
Sodium Chloride 8.0 g
Dipotassium Phosphate 0.4 g
Potassium Dihydrogen Phosphate 0.6 g
Malachite Green 0.036 g
Purified Water 1000 mL

Dissolve, warming slightly. Sterilize in an autoclave using a validated cycle, at a temperature not exceeding 115°. The pH is
to be 5.2 ± 0.2 at 25° after heating and autoclaving.
Xylose Lysine Deoxycholate Agar
Xylose 3.5 g
L-Lysine 5.0 g
Lactose Monohydrate 7.5 g
Sucrose 7.5 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g
Phenol Red 80 mg
Agar 13.5 g
Sodium Deoxycholate 2.5 g

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USP 38 Microbiological Tests / á62ñ Microbiological Examination 119

Xylose Lysine Deoxycholate Agar

Sodium Thiosulfate 6.8 g
Ferric Ammonium Citrate 0.8 g
Purified Water 1000 mL

Adjust the pH so that after heating it is 7.4 ± 0.2 at 25°. Heat to boiling, cool to 50°, and pour into Petri dishes. Do not heat
in an autoclave.
Cetrimide Agar
Pancreatic Digest of Gelatin 20.0 g
Magnesium Chloride 1.4 g
Dipotassium Sulfate 10.0 g
Cetrimide 0.3 g
Agar 13.6 g
Purified Water 1000 mL
Glycerol 10.0 mL

Heat to boiling for 1 minute with shaking. Adjust the pH so that after sterilization it is 7.2 ± 0.2 at 25°. Sterilize in an auto-
clave using a validated cycle.

General Chapters
Mannitol Salt Agar
Pancreatic Digest of Casein 5.0 g
Peptic Digest of Animal Tissue 5.0 g
Beef Extract 1.0 g
D-Mannitol 10.0 g
Sodium Chloride 75.0 g
Agar 15.0 g
Phenol Red 0.025 g
Purified Water 1000 mL

Heat to boiling for 1 minute with shaking. Adjust the pH so that after sterilization it is 7.4 ± 0.2 at 25°. Sterilize in an auto-
clave using a validated cycle.
Reinforced Medium for Clostridia
Beef Extract 10.0 g
Peptone 10.0 g
Yeast Extract 3.0 g
Soluble Starch 1.0 g
Glucose Monohydrate 5.0 g
Cysteine Hydrochloride 0.5 g
Sodium Chloride 5.0 g
Sodium Acetate 3.0 g
Agar 0.5 g
Purified Water 1000 mL

Hydrate the agar, and dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterili-
zation it is about 6.8 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle.
Columbia Agar
Pancreatic Digest of Casein 10.0 g
Meat Peptic Digest 5.0 g
Heart Pancreatic Digest 3.0 g
Yeast Extract 5.0 g
Maize Starch 1.0 g
Sodium Chloride 5.0 g
Agar, according to gelling power 10.0–15.0 g
Purified Water 1000 mL

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Hydrate the agar, and dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterili-
zation it is 7.3 ± 0.2 at 25°. Sterilize in an autoclave using a validated cycle. Allow to cool to 45° to 50°; add, where necessary,
gentamicin sulfate corresponding to 20 mg of gentamicin base, and pour into Petri dishes.

á63ñ MYCOPLASMA TESTS

INTRODUCTION

The genus Mycoplasma represents a group of minute bacteria which have no cell walls. The genus comprises more than 120
species. They are the smallest self-replicating prokaryotic organisms. The cells vary in size and morphology and cannot be
Gram stained, but impressions of colonies on solid agar can be stained with methylene blue or equivalent stain. Mycoplasma
are parasites and commensals, and some may be pathogenic to a variety of animal and plant hosts. In humans, Mycoplasma
are usually surface parasites that colonize the epithelial lining of the respiratory and urogenital tracts. Mycoplasma are com-
mon and may cause serious contamination in cell and/or tissue cultures used to generate compendial articles. They may also
cause contamination of filtered sterilized soybean casein digest broth. A cell culture infection may persist for an extended peri-
od of time without causing apparent cell damage. Infection of cells in a culture can affect nearly every pathway of cell metabo-
lism, including alteration of the cells' phenotypical characteristics and normal growth. The presence of Mycoplasma species
General Chapters

does not always result in turbid growth in cultures or visible alteration of the cells.
Testing for Mycoplasma is a necessary quality control requirement to assure reliably pure biotechnological products and al-
lied materials used to generate these products. This general test chapter describes two methods required to detect Mycoplas-
ma contamination of test articles, tissues and/or cell cultures used to produce test articles, digest broth, or any other material
in which Mycoplasma contamination is suspected. These are: (A) the agar and broth media procedure and (B) the indicator
cell culture procedure. These tests require careful aseptic technique and suitable laboratory conditions. In order to ensure ap-
propriate testing and interpretation of results, personnel should be properly trained and qualified. A validated nucleic acid am-
plification technique (NAT) or an enzymatic activity based method may be used to detect Mycoplasma, provided such a meth-
od is shown to be comparable to both methods (A) and (B). Alternative methods must be suitably validated. Validation re-
quirements for alternate methods will not be addressed in this chapter.

CULTURE METHOD

Choice of Media

The test is carried out using a sufficient number of both solid and liquid media to ensure growth in the chosen incubation
conditions of small numbers (approximately 100 colony-forming units, cfu; or 100 color-changing units, ccu) of Mycoplasmas
that may be present in the test article/material. Liquid media must contain phenol red. The range of media chosen is shown to
have satisfactory nutritive properties for at least the microorganisms shown in Quality Control Test Strain Organisms (below).
The nutritive properties of each new batch of medium are verified for the appropriate microorganisms in the list. When testing
for Mycoplasmas include in each test at least two known Mycoplasma species or strains (listed in Quality Control Test Strain
Organisms) as positive controls, one of which should be a dextrose fermenter (i.e., M. pneumoniae or equivalent species and
strain) and one of which should be an arginine hydrolyzer (i.e., M. orale or equivalent species and strain). Only when testing
insect cell lines should one include a Spiroplasma control strain (e.g., S. citri ATCC 29747, S. melliferum ATCC 29416, or equiva-
lent species and strains). Additionally, these strains may be a little more fastidious in their nutritional requirements. They re-
quire lower incubation temperatures (as do insect cell lines).

Quality Control Test Strain Organisms

Positive control cultures should be not more than 15 passages from isolation. Mycoplasma species or strains suitable for use
are listed below:
— Acholeplasma laidlawii (vaccines and/or cell-derived materials/cultures for human and veterinary use when an antibiotic
has been used during production)
— M. gallisepticum (when avian material has been used during production or when the vaccine or cell culture is intended for
use in poultry)
— M. hyorhinis (nonavian veterinary vaccines or cell cultures)
— M. orale (vaccines for human and veterinary use)
— M. pneumoniae (vaccines or cell banks for human use) or another suitable species of D-glucose fermenter such as M. fer-
mentans

Official from December 1, 2015

Copyright (c) 2015 The United States Pharmacopeial Convention. All rights reserved.