The AOAC Tuberculocidal Activity of Disinfectants method is specified by US EPA as a method which can be used to substantiate tuberculocidal efficacy claims for disinfectants. It is a modified version of the AOAC Use-Dilution test method and particularly appropriate for dilutable disinfectants.


Summary of the AOAC Tuberculocidal Activity of Disinfectant Test Method

  • A culture of Mycobacterium bovis BCG, an EPA recommended surrogate of Mycobacterium tuberculosis, is cultured for 21 days. This is a much longer incubation time compared to other bacteria such as E. coli, as M. bovis BCG is a slow growing bacterium with a doubling time of 12-24 hours (E. coli has a doubling time of 20 minutes).
  • The bacteria are dried onto a number of small, cylindrical, porcelain 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. 10 contaminated test surfaces 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 medium that has been amended with chemical agents to immediately neutralize the action of the disinfectant.
  • Immediately after transfer from the disinfectant into the neutralizer, the treated test surfaces are transferred into bacterial growth medium and incubated for 60 days.
  • After the 60 day incubation, the number of tubes showing growth of the target microorganism is recorded.
  • To "pass" a 10 carrier test, complete disinfection (no detectable growth of the target microorganism in the test tubes containing growth medium) must take place on all test surfaces. If no growth is detected after 60 days, the test tubes are incubated for an additional 30 days.

Strengths of the AOAC Tuberculocidal Activity of Disinfectants Test Method

  • The AOAC Tuberculocidal Activity of Disinfectants Test is approved by the EPA for data submission in conjunction with registration of tuberculocidal disinfectants sold in the US.
  • The AOAC Tuberculocidal Activity of Disinfectants 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 AOAC Tuberculocidal Activity of Disinfectants Test Method

  • The AOAC Tuberculocidal Activity of Disinfectants Test is subject to 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 Tuberculocidal Activity of Disinfectants Test is far removed from "real-life" use of disinfectants. The most glaring example of this is that contaminated surfaces are literally submerged in disinfectant for the entire contact time (up to 10 minutes). End users of dilutable disinfectants are not likely to be literally submerging their contaminated surfaces for the full contact time specified on product labels.
  • In order to avoid false-positive results, the scientist performing the AOAC Tuberculocidal Activity of Disinfectants Test must have experience and skill handling the wire hooks under 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.
  • Because of the slow growth rate of the test microorganism and the long incubation time of the test tubes, the AOAC Tuberculocidal Activity of Disinfectants Test is susceptible to contamination.

Microchem Laboratory specializes in testing disinfectant and other antimicrobial agents, such as tuberculocides.

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