AOAC 955.15, 964.02, 955.14

Use Dilution Methods

Summary

AOAC Use Dilution Methods 955.15 (Staphylococcus aureus), 964.02 (Pseudomonas aeruginosa) and 955.14 (Salmonella enterica) are the standard test methods required by the U.S. Environmental Protection Agency for validating disinfectant claims. The tests are designed to evaluate the antimicrobial efficacy of water-soluble powders and non-volatile liquid products (both dilutable and ready to use liquids) on hard, non-porous surfaces contaminated with bacteria. 

Testing for basic efficacy claims is divided into the following categories:

• Limited Spectrum: for products with limited efficacy; effective against Gram-negative (Salmonella enterica) or Gram-positive bacteria (Staphylococcus aureus), but not both.
• General or Broad Spectrum: for products having efficacy against both Gram-negative (Salmonella enterica) and Gram-positive bacteria (Staphylococcus aureus)
• Hospital or Healthcare Disinfectants: for testing of products recommended for use in hospitals, clinics, dental offices, nursing homes, or any other healthcare-related facility; products must have efficacy against both Staphylococcus aureus and Pseudomonas aeruginosa.

Once a product establishes efficacy as a broad spectrum or hospital/healthcare disinfectant, the product may be tested against additional microorganisms (any bacteria claimed on the label in addition to the base claim) including fungi, Mycobacterium, and viruses. 

Test Overview

• Stainless steel penicylinders (“carriers”) are contaminated with microorganisms.
• Inoculated carriers are submerged in the prepared disinfectant and held for the appropriate exposure time (this will be the time the EPA allows on the product label).
• At the completion of the exposure period, the treated carriers are transferred to recovery broth with neutralizers. 
• The treated/neutralized carriers are incubated and observed for the presence of growth.

• 60 replicates per microorganism are tested against three batches of the disinfectant for initial product registration.
  • For Staphylococcus aureus: growth must be observed in ≤3 replicates out of 60 for each lot to demonstrate efficacy.
  • For Pseudomonas aeruginosa: growth must be observed in ≤6 replicates out of 60 for each lot to demonstrate efficacy.
  • For Salmonella enterica: growth must be observed in ≤1 replicate out of 60 for each lot to demonstrate efficacy.
• 10 replicates per microorganism are tested against two batches of the disinfectant for confirmatory testing and testing of additional organisms. 
• Growth must not be observed in any replicate out of 10 for each lot to demonstrate efficacy.
• Non-GLP screening for R&D AND MARKETING purposes can be tested using 10, 30, or 60 carriers against multiple batches at various exposure times. 

References and Guidelines

1. Official Methods of Analysis (2013) 19th Ed., AOAC INTERNATIONAL, Gaithersburg,
2. MD, AOAC Use-dilution Method 955.15
3. Official Methods of Analysis (2013) 19th Ed., AOAC INTERNATIONAL, Gaithersburg,
4. MD, AOAC Use-dilution method 964.02
5. Official Methods of Analysis (2013) 19^th Ed., AOAC INTERNATAIONAL, Gaithersburg, MD, AOAC Use-dilution method 955.14
6. Office of Chemical Safety and Pollution Prevention Test Guidelines: Series 810 –
7. Product Performance Test Guidelines. See:
8. https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-810-product-performance-test-guidelines
9. 5. Tomasino, S.F., Parker, A.E., Hamilton, M.A., & Hamilton, G.C. (2012) Performance of
10. the AOAC Use-dilution method with Targeted Modifications: Collaborative Study. J.
11. AOAC Int. 95 (6), 1618-1628
12. 6. Parker, A.E., Hamilton, M.A., & Tomasino, S.F. (2013) A Statistical Model for
13. Assessing Performance Standards for Quantitative and Semi-quantitative Disinfectant
14. Test Methods. J. AOAC Int. (accepted for publication)
15. 7. Tomasino, S.F., Parker, A.E., & Hamilton, M.A. (2013) Use of Statistical Modeling to
16. Reassess the Performance Standard for the AOAC Use-dilution Methods (955.15 and
17. 964.02). J. AOAC Int. (accepted for publication)
18. 8. Standard Operating Procedure for AOAC Use Dilution Method for Testing Disinfectants
19. (MB-05), EPA Office of Pesticide Programs, Microbiology Laboratory, Environmental
20. Science Center, Ft. Meade, MD, http://www.epa.gov/pesticides/methods/atmpa2z.htm