AOAC Use Dilution Test (AOAC 955.14; 955.15; 964.02)
The AOAC Use Dilution method is specified by US EPA as a primary method for substantiation of efficacy claims for disinfectants. It is particularly appropriate for dilutable disinfectants.
It is semi-quantitative, meaning that it relies on a series of 60 qualitative observations (a test tube changing from clear to cloudy as a result of microbial growth).
Recently, as the result of several years of work by industry members and EPA, AOAC lowered the performance standard for Staphylococcus aureus and Pseudomonas aeruginosa, making the test's pass/fail determination more reliable and - to the delight of disinfectant makers - making the test easier to pass.
Along with the change in performance standard, EPA added a requirement that the different required 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.
- If the test includes "organic soil load," then the culture is amended with some percentage of organic matter (animal serum), before application to the test surfaces.
- For a valid test, at least 1 million cells per penicylinder (after drying) are required for Staphylococcus aureus and Pseudomonas aeruginosa, and at least 100,000 cells per penicylinder are required for Salmonella enterica.
- Depending on whether the test is for initial product registration or addition of "supplemental organisms," either 10 or 60 contaminated test surfaces are prepared.
- Each dry, contaminated test surface is transferred, individually, to a test tube filled with 10 mL of disinfectant using a wire hook, where they incubate for a specified contact time, typically near ambient temperature (~20-25 C). The contact time used in the test will be the contact time EPA allows on the product label.
- After the contact time has elapsed, the treated test surfaces are transferred, individually at intervals, to test tubes containing a liquid growth medium that has been amended with chemical agents to immediately neutralize the action of the disinfectant.
- After transfer from the disinfectant, 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 Method.
- 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.
Antimicrobial Test Laboratories 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.
For more information about the AOAC Use-Dilution Test, Contact the Lab Today!