AOAC Use Dilution Test

AOAC Use Dilution Test (AOAC 955.14; 955.15; 964.02)

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Welcome to Antimicrobial Test Laboratories' AOAC Use Dilution Test Method Resource Page.

Below, you will find a summary of the AOAC Use Dilution method, along with some of its strengths and weaknesses. The AOAC Use Dilution method is specified by US EPA as a method which can be used to substantiate efficacy claims for disinfectants. It is particularly appropriate for dilutable disinfectants.

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.
Each dry, contaminated test surface is transferred, individually, to a test tube filled with 10 mL of disinfectant using a wire hook. Depending on the purpose of the test, either 10 or 60 contaminated test surfaces (and corresponding tubes filled with disinfectant) are required.
Contaminated test surfaces incubate in the disinfectant for a specified contact time, typically near ambient temperature (~20-25 C).
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.
To "pass" a 60 carrier test, at least 59 of the 60 surfaces tested must demonstrate complete disinfection (no detectable growth of the target microorganism in the test tubes containing neutralizing growth medium). 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. bullet 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 (<10 minutes) time frame to "pass" the test.

Weaknesses of the Test Method:

The AOAC Use-Dilution Test Method is notoriously variable, on the basis of statistics alone (a product that produces a "passing" 1+/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 and standardization of the concentration of bacteria on the test surfaces.
The AOAC Use-Dilution Test is far removed from "real-life" use of disinfectants. The most glaring disconnect is that contaminated surfaces are literally submerged in disinfectant for the entire contact time (up to 10 minutes). Unless end users of dilutable disinfectants are literally submerging their contaminated surfaces for the full contact time specified on product labels, then they may not be getting the reductions in microorganisms they expect from the disinfectant products they purchase.
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, and are so confident in our capabilities that we often recommend the method as a high-level screen for disinfectant products being considered for registration with the EPA or claim optimization in the United States.

For more information about the AOAC Use-Dilution Test, Contact the Lab Today!