The United States Pharmacopeia (USP) significantly changed the USP XXXI <61> Microbial Limits Test. The updated USP test is now divided into two parts:
- USP <61> Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests
- USP <62> Microbiological Examination of Non-Sterile Products: Tests for Specified Microorganisms
The effective date is now May 1, 2009. Many of the changes are designed to harmonize with the European Pharmacopeia methods. A brief summary of the changes is listed below.
- USP <61> Microbial Enumeration Tests includes changes in pass/fail criteria and includes longer incubation durations than in previous editions.
- The Preparatory Test is replaced by the Method Suitability for product inhibition. The growth promotion organisms and the methodology had been significantly updated to include more types of organisms and different growth medias.
- The tests for specified microorganisms are included in USP <62>, the modifications change many microbiological medias 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.
Clients are advised to review the status of their non-sterile product testing in light of the upcoming changes to USP <61>, <62> Microbial Limits Testing. It is also recommending that these products be revalidated to conform to the new USP <61> and <62> Suitability Test. Clients must now specify which microorganisms are required to be absent. This requirement is based on the unique characteristics of the product based on formulation process, raw materials, etc.
Testing is performed as described in USP XXXI <61> and <62> an inoculum of not more than (NMT) 100 CFU.
Test organisms include:
- Aspergillus niger (ATCC #16404)
- Bacillus subtilis (ATCC #6633)
- Candida albicans (ATCC #10231)
- Escherichia coli (ATCC #8739)
- Pseudomonas aeruginosa (ATCC #9027)
- Salmonella typhimurium (ATCC #14028)
- Staphylococcus aureus (ATCC #6538)
- Clostridium sporogenes (ATCC #19404)
The new media and solutions recommended are:
- Cetrimide Agar (PSD)
- Enterobacteria Enrichment Broth Mossel (EEBM)
- MacConkey Agar (MAC)
- MacConkey Broth (MAC Broth)
- Mannitol Salt Agar (MSA)
- Phosphate Buffer (PB)
- Phosphate Buffer w/Tween 80
- Rappaport Vassiliadis Salmonella Enrichment Broth (RVSE)
- Sabouraud Dextrose Agar (SAB)
- Sabouraud Dextrose Broth (SAB Broth)
- Tryptic Soy Agar (TSA)
- Tryptic Soy Broth (TSB)
- Violet Red Bile Glucose Agar (VRBG).
- Xylose Lysine Deoxycholate Agar (XLD)
- Reinforced Medium for Clostridia (RM)
- Columbia Agar (CA)
Typical Morphology Characteristics on Selective Agar Media and Biochemical Assays
|Microorganism||S. aureus||P. aeruginosa||Salmonella spp.||E. coli||E. coli (Bile Tolerant)||C. albicans|
Salt Agar (MSA)
|Cetrimide Agar (PSD)||Xylose Lysine Deoxycholate Agar (XLD)||MacConkey Agar (MAC)||Violet Red Bile Glucose Agar (VRBG)||Sabouraud Dextrose Agar (SAB)|
|Characteristic Colonies Morphology||Yellow or White Colonies with a Yellow Zone||Green||Red, Black Precipitates||Brick-Red||Red Colonies||White Colonies|
|Gram Stain||Positive Cocci (In Clusters)||Negative Rods||Negative Rods||Negative Rods (Cocco-bacilli)||Negative Rods (Cocco-bacilli)||Round to Oval Buds|
|Biochemical Assays||Coagulase Positive||Oxidase Positive||NA||NA||NA||NA|
The Suitability of the Test Method demonstrates that the test specimen to which the testing is applied does not, 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.
Acceptance Criteria include the following:
(a) 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 ≤ 200% of those obtained from the positive control inoculum verification plates.
(b) For verification of specified organisms typical growth in the presence of the sample must be received for all test organisms on selective medias as described in USPXXXI <62> Supplement.
(c) Positive control inoculums 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 existing European Pharmacopeia method for testing non-sterile pharmaceuticals. The new USP methods are now more inclusive for more organisms. These are major impact changes for microbial limits testing. In the upcoming months, many companies will have to evaluate their non-sterile products to these updated methods. Clients are being advised to factor these USP methods into their testing schedules.
- USP XXXI <61> and <62>
Corresponding author Fran McAteer is vice president of quality at Microbiology Research Associates, Inc., 33 Nagog Park, Acton, MA 01720; 1-978-263-2624, fax 1-978-263-2786, an FDA-registered contract microbiology testing laboratory specializing in USP testing for pharmaceuticals, biologics, and medical devices. The author has expertise and experience in Pharmaceutical microbiology and acts as a consultant for many companies. The article was edited by Ms. Elaine Cung, Head Microbiologist at Microbiology Research Associates, Inc.