The AOAC Use Dilution method is a method of testing the efficacy of disinfectants, originally developed in 1955. Throughout its numerous revisions, it has become the standard for evaluating liquid and dilutable liquid disinfectants for hard surfaces. In particular, this method is specified by the U.S. EPA as the required method for disinfectant claim substantiation.
The Use Dilution method is performed, in brief, by soaking stainless steel carriers in bacteria, treating them with the disinfectant, and finally placing the carriers in growth broth to determine if any still have surviving bacteria. This makes the method a qualitative one, meaning that results are determined by whether or not bacteria grow, rather than by enumerating the bacteria.
One of the most recent revisions to the method, the result of several years of work by industry members and the EPA, was the lowering of the performance standard for Staphylococcus aureus and Pseudomonas aeruginosa, making the test’s pass/fail determination more reliable. This also had the benefit of making the test easier to pass, much to the delight of disinfectant developers.
Along with the change in performance standard, the EPA also added a requirement that, for some microorganisms, the different batches of test product be tested on different days.
Note: The information below is presented for educational purposes only. Current, detailed AOAC Methods can be obtained from AOAC.
Summary of Test Method and Important Parameters
- A 48 hour culture of an individual species of bacteria is dried onto a number of small, cylindrical, stainless steel test surfaces called penicylinders (coloquially referred to as “carriers”).
- If the product is to be marketed as a “one step” cleaner/disinfectant, organic soil is added to the culture prior to test initiation.
- The carriers (10, 30, or 60) are soaked in bacterial culture.
- Testing for initial product registration requires testing with 60 carriers per microorganism per lot of test product. Three lots of product, manufactured at or below the LCL, must be tested per microorganism.
- Testing for “supplemental organisms” requires testing with 10 carriers per microorganism per lot of test product. Two lots of product, manufactured at or below the LCL, must be tested per microorganism.
- Non-GLP screens for R&D purposes can also be performed with 10, 30, or 60 carriers.
- The carriers are dried.
- Three carriers are enumerated prior to test initiation to determine pre-test carrier microbial concentrations.
- Each dry, contaminated test carrier is transferred, individually, to a test tube filled with 10 mL of disinfectant, where they incubate for the specified contact time.
- The contact time used in the test will be the contact time the EPA allows on the product label.
- After the contact time has elapsed, the treated test carriers are transferred, individually, to test tubes containing a liquid neutralizer/growth medium.
- Three carriers are enumerated immediately following the conclusion of the test to determine post-test carrier microbial concentrations.
- The treated test surfaces are incubated in the neutralizing growth medium for 48 hours.
- After incubation, the number of tubes showing growth of the target microorganism is recorded.
- For Salmonella enterica to “pass” a 60 carrier test, at least 59 of the surfaces must demonstrate complete disinfection, for each batch tested.
- For Pseudomonas aeruginosa to “pass” a 60 carrier test, at least 54 of the surfaces must demonstrate complete disinfection, for each batch tested.
- For Staphylococcus aureus to “pass” a 60 carrier test, at least 57 of the surfaces must demonstrate complete disinfection, for each batch tested.
- To “pass” a 10 carrier test, complete disinfection must take place on all test surfaces.
Strengths of the AOAC Use-Dilution Test Method
- The AOAC Use-Dilution Test is approved by the EPA for data submission in conjunction with registration of dilutable disinfectants sold in the US. Now that the performance standard has been adjusted, the Use-Dilution Method is probably easier for most products to pass than the other method that can be used to register liquids as disinfectants, the AOAC Germicidal Spray Products as Disinfectants.
- The AOAC Use-Dilution Test is a “high-level” test for disinfectants, meaning that an antimicrobial solution must have appreciable biocidal activity on a relatively short time frame to pass the test.
Weaknesses of the Test Method
- The AOAC Use-Dilution Test Method carries some inherent variability, on the basis of statistics alone. For example, for Staphylococcus aureus, a product that produces a “passing” 3/60 on average will fail the test some appreciable percentage of the time.
- The AOAC Use-Dilution Test is subject to additional variability resulting from ambiguities of the official method. For example, the official test method does not address important parameters such as humidity level during drying of the carriers.
- The AOAC Use-Dilution Test is far removed from “real-life” use of disinfectants. The most glaring separation from “real life” product usage is that contaminated surfaces are submerged in excess disinfectant for the entire contact time (up to 10 minutes).
- In order to avoid false-positive results, the scientist performing the Use-Dilution Test must have experience and skill handling the wire hooks under considerable time pressure and must be able to transfer the contaminated penicylinders between test tubes without touching the sides of the tubes or contaminating the test system in other ways.
Microchem Laboratory has put a great deal of time and thought into perfecting the way we execute the AOAC Use-Dilution Test method. We take many measures to reduce test-to-test variability, have successfully submitted data to EPA for project registration, and are eager to answer questions our customers may have with regard to this test.