usp31nf26s1_c62, General Chapters: <62> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: TESTS FOR SPECIFIED MICROORGANISMS

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 samples 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 Enumeration Tests

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

If surface-active substances are used for sample preparation, their absence of toxicity for microorganisms and their compatibility with any inactivators used must be demonstrated as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

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

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 systems) are used so that the viable microorganisms used for inoculation are not more than five passages removed from the original 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
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 ssp. enterica serotype typhimurium or, as an alternative,	such as ATCC 14028
Salmonella enterica ssp. enterica serotype 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 stable 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.

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
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 ssp. enterica serotype typhimurium or
		Salmonella enterica ssp. enterica serotype abony
	Inhibitory	S. aureus
Xylose Lysine Deoxycholate Agar	Growth promoting + Indicative	Salmonella enterica ssp. enterica serotype typhimurium or
		Salmonella enterica ssp. enterica serotype abony
		E. coli
Test for Pseudomonas aeruginosa
Cetrimide Agar	Growth promoting	P. aeruginosa
	Inhibitory	E. coli
Test for Staphylococcus aureus
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 number (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 obtained 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

), 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 shortest 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 appropriate 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

), 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 Products. 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 prescribed.

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 Antimicrobial Activity under Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

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.

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

, but using Soybean–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 Sample 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 directed 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 positive result and the largest quantity that gives a negative result. Determine from Table 2 the probable number of bacteria.

Table 2. Interpretation of Results

Results for Each Quantity of Product			Probable Number of Bacteria per g or mL of Product
0.1 g or 0.1 mL	0.01 g or 0.01 mL	0.001 g or 0.001 mL	Probable Number of Bacteria per g 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

, 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

, 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 Enrichment 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

, 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 mix. 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 Examination of Nonsterile Products: Microbial Enumeration Tests

) 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

, 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 Examination of Nonsterile Products: Microbial Enumeration Tests

) 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 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 the product to be examined as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

. Take two equal portions corresponding to not less than 1 g or 1 mL of the product to be examined. Heat one portion at 80

for 10 minutes, and cool rapidly. Do not heat the other portion.

Selection and Subculture— Transfer 10 mL of each of the mixed portions to two containers (38 mm × 200 mm) or other containers containing 100 mL of Reinforced Medium for Clostridia. Incubate under anaerobic conditions at 30

to 35

for 48 hours. After incubation, make subcultures from each tube on Columbia Agar, and incubate under anaerobic conditions at 30

to 35

for 48 hours.

Interpretation— The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the presence of Clostridia.

If no anaerobic growth of microorganisms is detected on Columbia Agar or the catalase test is positive, the product complies with the test.

Candida albicans

Sample Preparation and Pre-Incubation— Prepare the product to be examined as described in Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

, 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 if they have similar growth-promoting and inhibitory properties.

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.

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
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.

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

Adjust the pH so that after sterilzation 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
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 autoclave using a validated cycle.

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 autoclave 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 sterilization 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

Hydrate the agar, and dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilization 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.

(Official August 1, 2007)

Auxiliary Information— Staff Liaison : Radhakrishna S Tirumalai, Ph.D., Scientist

Expert Committee : (MSA05) Microbiology and Sterility Assurance

USP31–NF26 Page 81

Pharmacopeial Forum : Volume No. 29(5) Page 1722

Phone Number : 1-301-816-8339