The measurement of microbial kill requires the ability to measure the number of surviving microorganisms with time after exposure to the antimicrobial agent. Bioburden determinations have the same requirement as they depend on the ability to recover viable microorganisms in the presence of potentially antimicrobial products or raw materials. However, carryover of residual disinfectant from the test could inhibit growth in the recovery medium, leading to poor microbial recovery. This potential residual activity must be neutralized and it is necessary to demonstrate the adequacy of neutralization for these tests (1-4). This demonstration of neutralization in compendial microbiological tests is known as demonstration of method suitability.
The demonstration of method suitability in microbial assays is now well established. The expectation that the test would allow for, and overcome, bacteriostatic properties of the material to be tested first appeared with the first version of the United States Pharmacopeia (USP) Sterility Test (5) of USP XI. A description of the test appeared in USP XIV (6) and was detailed in USP XV (7) where it was first identified as Bacteriostasis and Fungistasis. This name was changed in the early 2000s to method suitability as part of the international harmonization process (8).
In addition to the sterility test, harmonization of the microbial limits tests (USP Chapters <61> and <62>) (9) increased the compendial expectations for demonstration of method suitability (10). Finally, although not specifically required by the compendial test for preservative activity (11), USP <51> antimicrobial effectiveness testing is mentioned specifically in the informational chapter <1227> (12) as expected to demonstrate method suitability (validation).
It is important at the outset to clearly define what the goal of the method suitability study (as described in the compendia). This goal is not to demonstrate the ability to recover microorganisms present in the product. If that were the case, then the challenge organisms would be inoculated directly into the product– they are not. This design will not work if the antimicrobial properties of the product are strong. In this case, the product could well kill off all challenge organisms before it was possible to plate the test organisms. The goal of a microbiological method suitability test is to demonstrate that any residual antimicrobial properties of the product or the recovery method have been neutralized using the challenge microorganisms as a kind of biological indicator of neutralization.
Three published methods are illustrative of different designs to method suitability studies. These methods have different goals and strengths. Dey and Engley describe a procedure that measures survival with time using Staphylococcus aureus as the index organism. The challenge organism is inoculated directly into the disinfecting solution and then sampled with time (13). The efficacy of the neutralizer was measured by increased recovery of the challenge organism among different treatments. This protocol is useful in identifying neutralizers. However, it lacks the ability to distinguish between improved neutralization of the disinfectant and improved recovery of the index organism.
Terleckyj and Axler describe a second method that uses Candida albicans as the index organism
(14). This protocol was designed as a control procedure to demonstrate neutralization for a fungicidal experiment. The initial step of this method was a neutralization period. The biocide was exposed to the neutralizer before addition of the challenge microorganism. The challenge organism was then added at a concentration of approximately 106 colony-forming units (cfu)/ml to the suspension after this initial incubation. Survival of the challenge organism was assayed after an additional 15-minute incubation. The basic design was first described in 1972 by Bergan and Lystad (15). An assumption of these methods is that all index organisms will behave identically, and so only one organism needs to be tested. This is not a valid assumption as different organisms will differ in their sensitivity to biocides, and it is this sensitivity that is of concern in a neutralizer evaluation study. In addition, the method of Terleckyj and Axler involves a centrifugation step after exposure to the biocide. The cells are resuspended before plating, diluting the biocidal agent. Further dilution occurs because of the high number of cells inoculated in the solution. This high concentration (106 cfu/ml) requires dilution for the determination of viable colony counts. These dilutions compromise the stringency of the procedure.
The final method is based on the compendial design and was described by the author and colleagues (1, 16). This method is similar in overall design to that described by Bergan and Lystad. However, it employs a smaller inoculum size, statistical analysis, and evaluation of the neutralizer with all index organisms. This procedure lends itself to specific modifications of the general procedure that are included to mimic the different biocidal assays. Details of these methods are described in Section V.
A good overall reference for microbial recovery studies can be found in USP <1227> “Validation of Microbial Recovery from Pharmacopeial Articles” (12). One specific function of a neutralizer evaluation is to serve as a control experiment to the biocidal evaluation. Therefore, replication of all critical parameters of the biocidal experiment is critical to the neutralizer evaluation. The importance of this point cannot be overstated. USP <1227> provides some points on critical parameters to these test designs (also see “Influential Factors” below).