A Common Misreading of the Standard

A quality manager at a contract manufacturer recently told us that their microbial testing program consisted of “testing to USP <61>.” When we reviewed their procedure, we found they were performing total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) — but not testing for any of the specified organisms covered by USP <62>. Their CoAs showed passing microbial counts. They had no data on Salmonella, E. coli, or Staphylococcus aureus.

This is a common gap. USP <61> and <62> are complementary chapters that together define the microbial quality requirements for non-sterile pharmaceutical and dietary supplement products. Using only one without the other leaves a significant portion of the required testing unaddressed.

This post explains what each chapter covers, how they work together, and where manufacturers most commonly make procedural errors.

USP <61>: Microbial Enumeration Tests

USP <61> covers the quantitative enumeration of aerobic microorganisms in non-sterile products. The two primary counts are:

Total Aerobic Microbial Count (TAMC): Enumerates aerobic bacteria using Tryptic Soy Agar (TSA) or an equivalent validated medium. Incubation at 30–35°C for 3–5 days. Results reported in CFU/g (for solids) or CFU/mL (for liquids).

Total Yeast and Mold Count (TYMC): Enumerates yeasts and molds using Sabouraud Dextrose Agar (SDA) or an equivalent validated medium. Incubation at 20–25°C for 5–7 days. Results reported in CFU/g or CFU/mL.

Acceptance Criteria

USP <2023> (Microbiological Attributes of Non-Sterile Pharmaceutical Products) provides the acceptance criteria for dietary supplements by dosage form. For oral dietary supplements (tablets, capsules, powders), the typical limits are:

TAMC: ≤10³ CFU/g (10,000 CFU/g) for most solid oral forms
TYMC: ≤10² CFU/g (1,000 CFU/g) for most solid oral forms

Note that these are the USP acceptance criteria — your product specification may be more stringent, and some ingredient categories (e.g., probiotics, fermented ingredients) require special consideration. Consult your regulatory affairs team on the appropriate limits for your specific product category.

Method Suitability Testing

Before using a USP <61> procedure for a specific product, you must demonstrate method suitability — that the product matrix does not inhibit or enhance microbial growth in a way that would invalidate the count. This is done by performing a recovery test using specified challenge organisms at defined inoculum levels.

Method suitability must be performed for each product or product category, and must be repeated if the product formulation changes significantly. A CoA from a lab that cannot provide method suitability data for your specific matrix should be questioned.

USP <62>: Tests for Specified Microorganisms

USP <62> covers qualitative absence testing for specific pathogenic or indicator organisms. Unlike USP <61>, which gives you a count, USP <62> gives you a presence/absence result for each specified organism.

The organisms tested under USP <62> depend on the product category and route of administration. For oral dietary supplements, the standard specified organisms are:

Bile-tolerant gram-negative bacteria (BTGN): Indicator organisms for fecal contamination. Tested by enrichment in Mossel broth followed by plating on selective media.
Escherichia coli: Absence required in 1 g or 1 mL for most oral products.
Salmonella spp.: Absence required in 10 g or 10 mL for most oral products.
Staphylococcus aureus: Absence required in 1 g or 1 mL for some product categories.
Pseudomonas aeruginosa: Typically required for topical and some inhalation products; less commonly required for oral supplements unless the product is applied to mucous membranes.
Candida albicans: Required for some oral and topical products.

The specific organisms required for your product should be defined in your product specification and should be consistent with USP <2023> acceptance criteria for your dosage form and route.

Pre-Enrichment and Selective Media

USP <62> methods use a multi-step approach: pre-enrichment in a non-selective broth, followed by selective enrichment, followed by plating on selective/differential media. This approach is designed to detect low levels of target organisms that might be present in a large sample.

The pre-enrichment step is critical and is sometimes omitted in abbreviated testing programs. Skipping pre-enrichment reduces sensitivity and can produce false-negative results for stressed or injured organisms. If your contract lab’s method does not include a pre-enrichment step for Salmonella or E. coli, ask them to justify the omission with validation data.

Common Procedural Errors

In our laboratory work, we see the following errors most frequently in microbial testing programs:

1. Testing only <61> without <62> As described above, this leaves specified organism testing unaddressed. Both chapters are required for a complete microbial quality program.

2. Using acceptance criteria from the wrong USP chapter USP <61> provides the test procedure; USP <2023> provides the acceptance criteria. Some quality programs reference <61> limits that do not exist — the limits are in <2023>.

3. Failing to perform method suitability testing Method suitability is not optional. Without it, you cannot demonstrate that your test results are valid for your specific matrix.

4. Inadequate sample size USP <61> and <62> specify minimum sample sizes. Testing a 0.1 g sample when the method requires 1 g produces results that are not comparable to the acceptance criteria.

5. Incubation time and temperature deviations TAMC and TYMC require different incubation conditions. Incubating TYMC plates at 35°C instead of 20–25°C will suppress mold growth and produce falsely low counts.

6. Not testing raw materials separately from finished product Finished product testing is required, but it does not substitute for raw material testing. A contaminated raw material may be diluted in the finished product to below the detection limit — but the contamination is still present.

Rapid Methods and Alternative Technologies

Alternative microbial testing technologies — including ATP bioluminescence, PCR-based methods, and automated impedance systems — are increasingly used for rapid screening and in-process monitoring. These methods can provide results in hours rather than days.

However, rapid methods must be validated as equivalent to the compendial methods before they can be used for batch release decisions. USP <1223> (Validation of Alternative Microbiological Methods) provides the framework for this validation. Results vary by matrix and organism, and validation data from one product type may not be transferable to another.

Practical Checklist: Microbial Limits Testing for Dietary Supplements

Product specification includes both TAMC/TYMC limits (from USP <2023>) and specified organism requirements (from USP <62>)
Method suitability testing completed for each product or product category
Sample size meets USP minimum requirements for each test
TAMC incubated at 30–35°C for 3–5 days; TYMC incubated at 20–25°C for 5–7 days
All required specified organisms tested per USP <62> for the product’s dosage form and route
Pre-enrichment step included in Salmonella and E. coli testing procedures
Raw material microbial testing performed separately from finished product testing
Contract lab can provide method suitability data for your specific matrix on request
Any out-of-specification microbial result triggers a documented investigation before lot disposition

Microbial Limits Testing for Dietary Supplements — USP <61> and <62> Explained