Originally developed in 1961, the AOAC Germicidal Spray Test is a standard method for evaluating the efficacy of liquid disinfectants on hard, non-porous surfaces. It is one of the EPA-specified test methods for antimicrobial pesticide registration. This method is particularly appropriate for disinfectants which do not require dilution and are designed to be used with an aerosol or trigger spray.

The method is semi-quantitative, meaning that it relies on a series of qualitative observations (a test tube changing from clear to turbid as a result of microbial growth). The time-lapse photo below shows this process as it happens over the course of 48 hours.

Note: The information below is presented for educational purposes only. Current, detailed AOAC Methods can be obtained from AOAC.

Summary of the AOAC Germicidal Spray Test Method
  • A culture of a bacteria and sterile glass test surfaces (18 x 36 mm microscope slides) are prepared.
  • If the product is to be marketed as a "one step" cleaner/disinfectant, the test culture is supplemented with organic soil, usually fetal bovine serum.
  • Sterile test carriers are placed in petri dishes which have been lined with sterile filter paper.
  • Test carriers are inoculated with test culture by evenly spreading it across the surface of the slide and then dried.
  • Three carriers are enumerated prior to test initiation to determine pre-test carrier microbial concentrations.
  • The dry, contaminated test surfaces are then sprayed, individually, with test product.
  • Depending on the purpose of the test, either 10 or 60 contaminated test surfaces may be treated with the disinfectant product.
    • Testing for "supplemental organisms" requires testing with 10 contaminated test carriers per microorganism per lot of test product. Two lots of product, manufactured at or below the LCL, must be tested per microorganism.
    • Testing for initial product registration requires testing with 60 contaminated test carriers per microorganism per lot of test product. Three lots of product, manufactured at or below the LCL, must be tested per microorganism.
  • Contaminated test surfaces incubate in the disinfectant for a 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 surfaces are transferred, individually, to sterile test tubes or jars containing a neutralization/growth media.
  • Three carriers are enumerated prior to test initiation to determine post-test carrier microbial concentrations.*****The treated test surfaces are allowed to incubate in the neutralization media 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 Germicidal Spray Test Method
  • The AOAC Germicidal Spray Products Test is approved by the EPA for data submission in conjunction with registration of spray (aerosol/trigger-spray) disinfectants sold in the USA.
  • The AOAC Germicidal Spray Products Test is a "high-level" test for spray disinfectants, meaning that they must have appreciable biocidal (germ-killing) activity on a relatively short (<10 minutes) time frame to "pass" the test.
Weaknesses of the Test Method
  • The AOAC Germicidal Spray Products Test Method is fundamentally similar to the AOAC Use-Dilution Test and is 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 Germicidal Spray Products 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 glass test surfaces and standardization of the concentration of bacteria on the glass test surfaces.
  • While certainly less removed from "real-life usage" than the AOAC Use-Dilution Test, the AOAC Germicidal Spray Products Test is a somewhat unrealistic test model. Specifically, products undergoing testing are applied to the contaminated surfaces in large volumes relative to the amount of surface area, causing the product to "pool" on the test slides. If a consumer were to use the same volume of product per unit of surface area that is used in the test, then they would literally require gallons of product to disinfect their kitchen!
  • Additionally, usage directions specifying 10 minute "wet dwell" times (the required dwell time for most products) simply are not practical and are not adhered to by the vast majority of consumers of spray disinfectant products.

Microchem Laboratory has put a great deal of time and thought into perfecting the way we execute the AOAC Germicidal Spray Test method. We take many measures to reduce test-to-test variability, and often recommend the method as a high-level screen for spray disinfectant products being considered for registration with the EPA or claim optimization in the United States.

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