Microbial limit test method suitability: a step-by-step guide (USP <61> and <62>)

A microbial limit test (MLT) is only meaningful if the method can actually recover organisms from your product. Method suitability, sometimes called method validation or "verification of recovery", is the experiment that proves it. You do it once for each product and repeat it when the formulation, the method or the supplier of a critical material changes.

What the two chapters cover

• USP <61> / Ph. Eur. 2.6.12: total aerobic microbial count (TAMC) and total combined yeast and mould count (TYMC).
• USP <62> / Ph. Eur. 2.6.13: tests for specified organisms such as E. coli, Salmonella, P. aeruginosa, S. aureus, Clostridia, C. albicans and bile-tolerant Gram-negative bacteria.
• USP <1111>: the acceptance criteria that tell you which tests apply to which dosage form.

Test organisms for <61>

Organism	Strain	Used for
Staphylococcus aureus	ATCC 6538	TAMC
Pseudomonas aeruginosa	ATCC 9027	TAMC
Bacillus subtilis	ATCC 6633	TAMC
Candida albicans	ATCC 10231	TAMC and TYMC
Aspergillus brasiliensis	ATCC 16404	TAMC and TYMC

Use cultures no more than five passages from the reference culture, and an inoculum of not more than 100 CFU.

Choosing the counting method

Method	Best for	Watch out for
Pour plate	Most soluble products, 1 mL of a 1 in 10 dilution per plate	Molten agar must be cooled to not more than 45 °C
Spread plate	Products where heat could harm organisms	Smaller volume (0.1–0.5 mL), so lower sensitivity
Membrane filtration	Antimicrobial products, large volumes, low limits	Membrane must suit the product; rinse validated
Most probable number (MPN)	Only when no other method works	Least precise; used for TAMC only

Most tablets, capsules and oral liquids are tested by pour plate. Antimicrobial products and those with low limits usually need membrane filtration.

The experiment

1. Prepare the product as you would for routine testing, usually a 1 in 10 dilution in buffered sodium chloride–peptone solution pH 7.0 or phosphate buffer pH 7.2.
2. Spike the prepared product with not more than 100 CFU of each organism, separately.
3. Run the method exactly as written: pour plate, spread plate or membrane filtration.
4. Run a control with the same inoculum in diluent without product.
5. Count and compare. The mean count from the product test must be within a factor of 2 of the control (50% to 200% recovery).

If recovery fails: neutralise

Many products, such as preserved liquids, antibiotics and some herbal extracts, inhibit growth. Try these in order, and document each attempt:

• Dilution: move from 1 in 10 to 1 in 100 or more, as long as you can still meet the acceptance criterion.
• Chemical neutralisers: polysorbate 80 and lecithin for preservatives such as parabens and quaternary ammonium compounds; sodium thiosulfate for halogens; histidine for aldehydes.
• Membrane filtration with rinsing: filter the product and rinse the membrane with several portions of diluent to wash the inhibitor away.
• Combination: dilution plus neutraliser plus filtration for strongly antimicrobial products.

Also show that the neutraliser itself is not toxic to the organisms by running a neutraliser-only control.

If you still cannot recover an organism

If no method recovers a particular organism, the pharmacopoeia accepts that the product's antimicrobial activity makes it unlikely to be contaminated with that species. Document the attempts and justify the conclusion; this is a scientific argument, not a shortcut.

Incubation for routine counts

• TAMC: soybean-casein digest agar at 30–35 °C for 3–5 days.
• TYMC: Sabouraud dextrose agar at 20–25 °C for 5–7 days.

Count plates with up to 250 colonies for TAMC and up to 50 for TYMC, and calculate CFU per gram or mL from the dilution. If TYMC organisms also grow on SCDA, they count in the TAMC; if bacteria grow on SDA, they count in the TYMC. This is why some sites use SDA with antibiotics where justified.

Specified organisms (<62>)

For each specified organism, show that it can be detected in the presence of the product at not more than 100 CFU, using the enrichment and selective media in the chapter.

Organism	Enrichment	Selective step	Selective plate
E. coli	Soybean-casein digest broth, 30–35 °C, 18–24 h	MacConkey broth, 42–44 °C, 24–48 h	MacConkey agar, 30–35 °C, 18–72 h
Salmonella	Soybean-casein digest broth, 30–35 °C, 18–24 h	Rappaport Vassiliadis broth, 30–35 °C, 18–24 h	Xylose lysine deoxycholate agar, 30–35 °C, 18–48 h
P. aeruginosa	Soybean-casein digest broth, 30–35 °C, 18–24 h	–	Cetrimide agar, 30–35 °C, 18–72 h
S. aureus	Soybean-casein digest broth, 30–35 °C, 18–24 h	–	Mannitol salt agar, 30–35 °C, 18–72 h

Typical colonies on the selective plate are confirmed by identification tests. Run growth promotion, inhibitory and indicative checks on each media lot, as described in the GPT guide.

Acceptance criteria (USP <1111>)

Dosage form	TAMC (CFU/g or mL)	TYMC (CFU/g or mL)	Specified organisms
Non-aqueous oral	10³	10²	Absence of E. coli (1 g or 1 mL)
Aqueous oral	10²	10¹	Absence of E. coli (1 g or 1 mL)
Rectal	10³	10²	–
Oromucosal, gingival, cutaneous, nasal, auricular	10²	10¹	Absence of S. aureus and P. aeruginosa
Vaginal	10²	10¹	Absence of P. aeruginosa, S. aureus and C. albicans
Inhalation	10²	10¹	Absence of S. aureus, P. aeruginosa and bile-tolerant Gram-negative bacteria

When a limit is written as 10¹, the maximum acceptable count is 20; for 10², it is 200; for 10³, it is 2,000. Your product specification may be tighter, and products with natural raw materials often have specific limits.

When to repeat method suitability

• A new product, or a change in formulation
• A change in the test method, dilution, membrane or neutraliser
• A change in the supplier of a raw material that could affect antimicrobial activity
• Unexpected results, such as repeated zero counts on a product that historically showed low counts

Common audit findings

• Method suitability done on one batch with no plan for formulation changes
• No neutraliser toxicity control
• Recovery calculated against the theoretical inoculum instead of a parallel control
• Passage numbers of cultures not traceable
• Specified organism tests not covered for every organism in the specification
• Plates counted outside the countable range without justification

Method suitability is a small study with a big job: it proves that every routine "complies" result for that product means something. Treat it with the same care as any validation.