Antimicrobial Effectiveness Testing: Purpose and Legacy Method Notes

In short: Antimicrobial effectiveness testing (AET), also called preservative effectiveness testing, evaluates whether a product’s preservative system controls challenge microorganisms under the applicable compendial method. The numeric method details below are retained from a 2020 FDA laboratory manual; compare them with the current official USP <51> before use as a procedure.

What the test is for

USP General Chapter <51> is titled Antimicrobial Effectiveness Testing. FDA explains that this type of study can be used to determine a minimally effective preservative level, followed by suitable preservative-content testing at batch release and during stability studies. Preservatives are not a substitute for good manufacturing practices or a way to bring an initially failing microbial result into specification; FDA’s CGMP Q&A specifically warns against that practice (FDA Q&A 19 and 21, dated 11 June 2015; 21 CFR §§211.113(a) and 211.165(f)).

The legacy article closely follows FDA Office of Regulatory Affairs document ORA.007, Pharmaceutical Microbiology Manual, Revision 02 (25 August 2020). That public manual says to check QMiS for the most current official copy. The public USP preview identifies a 2017 chapter text, but does not expose the full current chapter. The details below therefore describe the legacy method for review; they are not an approved test procedure.

For related reading, see the microbiological analytical data checklist, microbiological laboratory practice SOP and culture-dilution preparation SOP.

Legacy method details to verify against current USP <51>

Media, growth promotion and method suitability

The legacy text lists Soybean-Casein Digest (SCD) and Sabouraud Dextrose (SD) media for culturing the challenge organisms. It calls for growth-promotion testing of the medium and for checking that the product’s antimicrobial properties do not prevent recovery of the organisms. Where needed, it describes adding a suitable neutralizer to the medium or diluent.

It also gives a solid-medium growth-promotion recovery of at least 50% of the calculated inoculum value. The copied method says one suitability sample may cover multiple samples of the same product from the same manufacturer when the product form and amount are the same.

Current-chapter note. The full current text of USP <51> is subscriber-only and could not be read for this review, so the media, neutralizer, growth-promotion recovery and pooling statements above are reported as legacy FDA ORA.007 details, not as confirmed current compendial requirements. The general principle is stable: if the product’s preservative or formulation inhibits growth, that activity must be neutralized (by a specific neutralizer, dilution, membrane filtration with rinsing, or a combination), and the method must be shown to recover the challenge organisms. USP <1227>, Validation of Microbial Recovery from Pharmacopeial Articles, is the chapter that describes validating such recovery and neutralization. Its public preview does not show a numeric recovery criterion, so confirm the exact recovery acceptance value (including the legacy “at least 50%” figure), the permitted media and neutralizers, and whether one suitability study may cover several samples in the current USP <51>, <1227> and your approved method. For EU or Ph. Eur. markets, the equivalent test is Ph. Eur. 5.1.3, Efficacy of antimicrobial preservation, which has its own criteria (A and B) and must be read separately; do not assume the USP values apply.

Challenge organisms and culture preparation

The five organism-strain pairs retained in the legacy method are:

That method says cultures should be no more than five passages from the original stock. It specifies SCD agar at 32.5 ± 2.5°C for 3–5 days for the listed bacteria; SD agar at 22.5 ± 2.5°C for 3–5 days for C. albicans and 3–7 days for A. brasiliensis. The legacy suspension target is about 1 × 10⁸ CFU/mL; it lists 0.05% polysorbate 80 in saline for the A. brasiliensis suspension. It also describes broth preparation as an alternative for the listed organisms except A. brasiliensis.

The same source gives the following storage and use windows for prepared suspensions: refrigerate at 2–8°C if not used within 2 hours; use bacterial and yeast suspensions within 24 hours; and permit refrigerated mold suspension storage for up to 7 days.

Product groups, inoculation and sampling schedule

The legacy source divides aqueous-based products into four groups: (1) injections and other parenterals, including emulsions, otic products, sterile nasal products and ophthalmic products; (2) topical products, nonsterile nasal products and emulsions, including products for mucous membranes; (3) oral products other than antacids; and (4) antacids. The source’s category scope should be checked against the current compendial text before assigning a product to a category.

The copied method further specifies a 0.5–1.0% inoculum suspension volume relative to product volume and incubation at 22.5 ± 2.5°C. If neutralization requires substantial dilution, it gives a 10⁷–10⁸ CFU/mL inoculum as an example to allow a three-log reduction to be measured. It recommends starting with at least 20 mL of product and, where product is transferred, using five sterile capped containers.

Example in-house criterion (not a pharmacopoeial requirement): the legacy article recommends 20 mL in each of five tubes for laboratory-to-laboratory consistency. It also proposes duplicate plates per dilution and carrying dilutions through 10⁻³ as sufficient in most cases. Treat these as laboratory-specific examples that require method-suitability support, not as universal USP requirements.

Legacy acceptance criteria

The table below preserves the acceptance values in the imported article. Log reductions are measured against the initial calculated count unless the row states a comparison with the 14-day count. The legacy text defines “no increase” as no more than 0.5 log₁₀ above the previous result.

Records and retention

Document the approved method, product-specific suitability, challenge preparation, counts, calculations, observations and investigations in the applicable controlled records. Retain production, control or distribution records specifically associated with a product batch for at least 1 year after the expiry date of that batch (21 CFR §211.180(a)). EU GMP Part I Chapter 4 §4.11 also sets a minimum of 5 years after Qualified Person certification when that is longer. Keep records longer when local law or the marketing authorization requires it.

Frequently asked questions

Can a preservative replace microbial controls during manufacture?

No. FDA’s CGMP Q&A cites USP <51> and 21 CFR §§211.113(a) and 211.165(f) to explain that preservative action cannot be used to mask an initially failing microbial result or replace manufacturing controls.

Are the numerical tables above ready to use as a laboratory procedure?

No. They are retained from the 2020 FDA ORA manual and need comparison with the current official USP <51>, product monograph and approved method before use.

Does every product use the same challenge concentration and schedule?

The legacy method divides products into groups and gives different values. The current product category and method must be confirmed from the applicable official compendial and regulatory sources.

References

• United States Pharmacopeia (USP), USP-NF General Chapter <51>, Antimicrobial Effectiveness Testing, public preview identifying 2017 text: official USP preview. The full current chapter was not available in the public preview.
• U.S. Food and Drug Administration (FDA), Office of Regulatory Affairs, Pharmaceutical Microbiology Manual, document ORA.007, Revision 02, revised 25 August 2020: official FDA PDF. The public copy instructs users to check QMiS for the current official version.
• FDA, Questions and Answers on Current Good Manufacturing Practice Regulations: Production and Process Controls, Q&A 19 and 21 (11 June 2015): official FDA page.
• United States, 21 CFR §§211.113(a), 211.165(f) and 211.180(a), current eCFR accessed 16 October 2026: 21 CFR Part 211.
• USP-NF General Chapter <1227>, Validation of Microbial Recovery from Pharmacopeial Articles, public preview: official USP preview. Full text is subscriber-only.
• European Pharmacopoeia (EDQM), general chapter 5.1.3, Efficacy of antimicrobial preservation (current edition; not reviewed in full text): edqm.eu.
• European Commission, EudraLex Volume 4, Part I, Chapter 4, Documentation, Revision 1 (January 2011), §4.11: official PDF.