AOAC 966.04 Sporicidal Activity Test

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The AOAC 966.04 Sporicidal Activity Test contrasts with the more R&D approach of an ASTM E2315 Suspension Time Kill using endospores, giving a more robust challenge for the rigor of regulatory claims.

An ASTM E2315 gives the disinfectant a “best case” scenario of total emersion and total contact of spore to disinfectant; the Sporicidal Activity Test has the spores dried onto a surface.

AOAC 966.04 Sporicidal Activity Test: Procedure at a Glance

Essential for the registration and verification of sporicidal claims with the EPA as a countertop disinfectant and the FDA as a high-level disinfectant, the AOAC 966.04 Sporicidal Activity Test uses either Bacillus subtilis or Clostridium sporogenes as the model organism for endospore-forming bacteria. The test begins with the dessication of 105-106 colony-forming units of the specified species onto each individual carrier. The starting number of spores per carrier is enumerated to ensure a robust challenge for the disinfectant.

Following enumeration, the testing begins by transferring dried, inoculated carriers into the testing disinfectant. The carriers are exposed to the disinfectant for a specified contact time after which they are transferred to a recovery media supplemented with a neutralization compound appropriate to the disinfectant’s active ingredient. The recovery broth allows any spores that were not killed in the disinfection process to retain viability prior to incubation. A 21 day period of incubation—anaerobic incubation in the case of C. sporogenes—gives the microbe the chance to grow to levels perceivable for qualitative assessment of growth.

Following incubation, tubes positive for growth are counted and their population is verified as being comprised of the original microbe by plating on tryptic soy agar and/or gram staining and subsequent visualization.

AOAC 966.04 Sporicidal Activity Test: Microchem’s Expertise

With Microchem Laboratory, both tests can be personalized to the study sponsor’s needs with variation on the microbial load present in the test as well as variation in contact times—perfect for zoning-in on an effective minimum specific to the disinfectant. Similarly, some high-level disinfectants require a mixing prior to use. This period has an inherent “dwell time” or the space between inital mixing and use. Microchem has experience varying this parameter in tests, including very minute dwell times designed to mimic the mixing of base and activator en route to the nebulizer of a spray bottle. Beyond that, many other parameters can be varied within the test, such as the contact temperature and the “soil load” or presence of organic matter intended to increase the rigor of the test.

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