![]() ![]() 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
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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
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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
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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 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
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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
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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
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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
![]() ![]() ![]() ![]() 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
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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
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
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Selection and Subculture—
Shake the container, transfer 1 mL of Soybean–Casein Digest Broth to 100 mL of MacConkey Broth, and incubate at 42
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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
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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
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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
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Selection and Subculture—
Subculture on a plate of Cetrimide Agar, and incubate at 30
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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
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Selection and Subculture—
Subculture on a plate of Mannitol Salt Agar, and incubate at 30
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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
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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
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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
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Selection and Subculture—
Subculture on a plate of Sabouraud Dextrose Agar, and incubate at 30
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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
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Phosphate Buffer Solution pH 7.2—
Prepare a mixture of Purified Water and Stock Buffer Solution (800:1 v/v), and sterilize.
Sterilize in an autoclave using a validated cycle.
Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25
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Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25
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Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25
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Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25
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Adjust the pH so that after sterilization it is 5.6 ± 0.2 at 25
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Adjust the pH so that after heating it is 7.2 ± 0.2 at 25
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Adjust the pH so that after heating it is 7.4 ± 0.2 at 25
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Adjust the pH so that after sterilization it is 7.3 ± 0.2 at 25
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Adjust the pH so that after sterilization it is 7.1 ± 0.2 at 25
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Dissolve, warming slightly. Sterilize in an autoclave using a validated cycle, at a temperature not exceeding 115
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Adjust the pH so that after heating it is 7.4 ± 0.2 at 25
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Heat to boiling for 1 minute with shaking. Adjust the pH so that after sterilization it is 7.2 ± 0.2 at 25
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Heat to boiling for 1 minute with shaking. Adjust the pH so that after sterilization it is 7.4 ± 0.2 at 25
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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
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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
![]() ![]() ![]() (Official May 1, 2009)
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
USP32–NF27 Page 75
Pharmacopeial Forum: Volume No. 29(5) Page 1722
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