Pharmaceutical microbiology: Harmonization of microbial limits test for nonsterile products

Revisions to USP <61> are designed to harmonize with European Pharmacopeia

By Fran McAteer, Microbiology Research Associates

The United States Pharmacopeia (USP) is significantly changing the USP <61> Microbial Limits Test, effective August 1, 2007. The updated test can be found in the second supplement USP-NF pages 3757-3765. Among the revisions, the test is now divided into two parts as follows:

  1. USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
  2. USP <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms

Many of the changes are designed to harmonize with the European Pharmacopeia methods. A brief summary of the changes is listed below.

  • The Method Suitability test replaces the Preparatory Test for product inhibition. The growth promotion organisms and the methodology have been significantly updated to include more types of organisms and different growth media.
  • USP <61> Microbial Enumeration Tests includes changes in pass/fail criteria and includes longer incubation durations than in previous editions.
  • The tests for specified microorganisms are included in USP <62>. The modifications change many microbiological media utilized in testing for specific pathogens. Critical changes also are being made to incubation temperatures and duration.
  • More organisms have been specified in the new USP <62> chapter than in previous USP editions. Organisms such as Candida albicans, Clostridia species, and bile-tolerant Gram-negative bacteria may be required to be tested depending on specifications regarding product formulation.
  • No retests are allowed.

Bio/pharmaceutical companies are advised to review the status of their nonsterile product testing in light of the upcoming changes to USP <61> and <62> microbial limits testing. It is also recommended that these products be revalidated to conform to the new USP <61> Suitability Test. Companies must now specify which microorganisms are required to be absent.

The suitability of the test method demonstrates that the test specimen to which the testing is applied does not, in and of itself, inhibit the recovery of the microorganisms that may be present. The performance of the Suitability Test ensures that any antimicrobial activity inherent in the sample to be tested does not adversely affect the reliability of the test and that the test procedure to be routinely utilized is otherwise suitable for use with the sample.

Testing is performed as described in USP 29 <61> and <62> 2nd supplement, using an inoculum of not more than (NMT) 100. Test organisms include: Aspergillus niger (ATCC #16404), Candida albicans (ATCC #10231), Escherichia coli (ATCC #8739), Bacillus subtili (ATCC #6633), Pseudomonas aeruginosa (ATCC #9027), Staphylococcus aureus (ATCC #6538), and Salmonella typhimurium (ATCC #14028).

Media and solutions utilized are: cetrimide agar (PSD); MacConkey agar (MAC); mannitol salt agar (MSA); Sabouraud dextrose agar (SAB); tryptic soy agar (TSA); tryptic soy broth (TSB); phosphate buffer (PB); phosphate buffer w/tween 80; xylose-lysine-desoxycholate agar (XLD); MacConkey broth (MAC broth); Rappaport vassiliadis salmonella enrichment broth (RVSE); Sabouraud dextrose broth (SAB broth); enterobacteria enrichment broth-Mossel (EEBM); and violet red bile glucose agar (VRBG). See Table 1.

Acceptance criteria include the following:

  • The average plate counts obtained from the test plates, for the total aerobic count and the total yeast and mold count, must be > 50% and < 20% of those obtained from the positive control inoculum verification plates.
  • For verification of specified organisms, typical growth in the presence of the sample must be received for all test organisms on selective media as described in USP 29 <62> 2nd supplement.
  • Positive control inoculum must be performed for each organism preparation and have an average value of NMT 100 CFU per total inoculated volume for the specified organism.

Overall, USP <61> and <62> provide harmonization to the existing European Pharmacopeia method for testing nonsterile pharmaceuticals. The new USP methods are now more inclusive for more organisms. These are major impact changes for microbial limits testing, and in the upcoming months, many companies will have to evaluate their nonsterile products to ensure they adhere to the updated methods. Bio/pharmaceutical companies are advised to factor these USP methods into their testing schedules.

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Author’s Note: Please be advised that the United States Pharmacopeia announced on November 14, 2006, that the effectivity date for USP <61> and <62> compliance has changed from August 1, 2007, to May 1, 2009.


1. USP 29, 2nd supplement, August 2006.

Fran McAteer is vice president of quality at Microbiology Research Associates (Acton, MA), an FDA-registered contract microbiology testing laboratory specializing in USP testing for pharmaceuticals, biologics, and medical devices. He has expertise and experience in pharmaceutical microbiology and acts as a consultant for many companies. He can be reached via e-mail at [email protected]


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