Usually one of the most important considerations during any test method is adequate neutralization. Some methods, such as an MIC test that just determines the concentration of a microbicide to inhibit the growth of microorganisms, are not time dependent. But these types of tests have limited application, except for the examination of preservation systems (see chapters 37, 38, 39, 40). Most other antimicrobial efficacy tests look at the ability of a disinfectant to kill microorganisms over time. Whether defining the amount of time required to kill a specified population of a certain microorganism or determining the time required to destroy a single log₁₀ of a microbial population (eg, to determine the D-value), the importance of stopping a disinfectant’s activity at the proper contact time cannot be overstated. Failure to properly neutralize a sample can cause overestimation of activity and a gross mischaracterization of the potency of a product, active ingredient, or formula.

Neutralization in efficacy testing is simply the quenching of antimicrobial activity of a disinfectant at a specified time point. This is typically accomplished through chemical neutralization, although filtration (separation of the microorganisms from the disinfectant) and other methods (eg, rapid cooling in heat processes) can be used. Chemical neutralization is the addition of a single quenching agent (or combination of agents) to the study that inactivates the antimicrobial being tested. When chemical neutralization is selected, the two variables to consider are the ability of the neutralizer to immediately stop the antimicrobial activity upon addition to the test system and to not have a deleterious or toxic effect on the microorganisms being tested. For the first, there are many references to neutralizers to consider based on active class.¹,² Common chemical neutralizers use lecithin, TWEEN^®, reducing agents, or simple dilution. Examples include both prepared media such as Letheen broth or Dey and Engley neutralizing broth as well as other media supplemented with additional TWEEN^®, lecithin, or other specific ingredients to address a particular chemistry. These include chemical (eg, sodium thiosulphate, a reducing agent) or enzymatic (catalase) neutralizers used for oxidizing agents such as hydrogen peroxide. With a complicated formula, often the efficacy of an active is not solely related to just the defined active ingredient alone (see chapter 6). Neutralization effectiveness testing must be incorporated using the final formula and the microorganism(s) being tested. A neutralization effectiveness test such as ASTM E1054, Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents, provides great detail on how to perform the evaluation and what the acceptance criteria should look like, but this can grow increasingly complicated during the research of multiple disinfectants or formulations or the evaluation of a single product with multiple microorganisms.³ A basic overview of the ASTM test includes adding the active product to the neutralizing solution at the proper test ratios, then inoculating this neutralized mixture with a microbial challenge and determining whether any disinfectant activity still persists when compared to a control, such as using an isotonic buffer in place of the active material. The microbial challenge must be at a low enough titer that even a small amount of residual activity is detected. It is of no value to challenge a neutralization system with relatively high levels (eg, 1.5 × 10⁷ colonyforming units [CFUs]) of bacteria because there may be sufficient residual activity to kill a half a million bacteria and not have the sensitivity to detect this when comparing diluted plate counts (eg, detecting 1.0 × 10⁷ CFUs). The strain of microorganism tested can also be important and is another key consideration. A neutralized product may have a small amount of residual active or excipients that do not impact a particular microorganism, for example, the gram-negative bacteria Pseudomonas aeruginosa, but might inhibit another bacteria such as Staphylococcus aureus. When testing multiple microorganisms or strains of similar species, it is sufficient to choose the most sensitive strains to a particular active as long as the experimenter understands that there remains some risk inherent in any scientific assumption. For example, it would not be required to confirm the neutralization of 15 different Burkholderia cepacia strains, when one or two would suffice, but it shouldn’t be assumed that neutralization will be complete for other Pseudomonas species, gram-positive bacteria, or even harder to kill microorganisms such as filamentous fungi or spores. It is typically safe to assume that the ability to neutralize a product at a higher concentration will ensure adequate neutralization for more dilute forms; however, even slight changes in a formula or a product, other than dilution, can impact the activity of the system and neutralization may need to be periodically confirmed during disinfectant formulation optimization.

In the absence of a good neutralization process and control, it will not be possible to understand whether the antimicrobial reaction has stopped at the desired time. The test article simply keeps killing as further microbial handling (eg, dilutions) is performed and will likely also continue to kill or inhibit growth of the microorganism during culturing. Occasionally, microbiological indications of inefficient neutralization can be detected on close inspection, such as a nondilutional plate series. This is when the serially diluted plates do not have counts that correspond in a serial manner. For example, with bacterial culturing, a 10^-1 plate with no colonies followed by a 10^-2 plate with 100 and a 10^-3 plate with 10 will give an instant signal that antimicrobial activity was still present in the first plate and subsequent dilution finally stopped this. Although ensuring that neutralizing ingredients are
present in the recovery agar as well as in a neutralizing broth will help, any indication of a neutralization issue should be investigated. When a neutralization problem is noted, either through a control or a failed test, alternatives must be considered. These include the use of a different neutralizer, the use of greater neutralizer volumes (and potential change in limits of detection), or the use of neutralization through filtration. Filtration can be a powerful tool when chemical neutralization is improbable. It is possible to pass the antimicrobial chemicals through an inert filter while trapping viable microorganisms that can be subsequently rinsed and plated. Other creative solutions are certainly possible when finding and confirming neutralizers, and neutralization should be at the forefront of factors to consider in order to ensure antimicrobial tests are reliable.

Whether qualitative or quantitative, the number of microorganisms in the initial challenge is a key piece of information to understand. Although it seems basic to have a good enumeration of your starting point, there are challenges. Most microbiologists investigating bacteria and fungi use plate counts to enumerate colonies and assume that each colony arose from a single cell. With this information, the number of survivors can be determined. Microbiologists will understand that colonies form from CFUs that can contain single or small groups of cells, and therefore, quantitation can only be an estimate. Bacteria like to clump and they like to associate. Proper vortexing, sonication, and accurate volume usage during serial dilution can mitigate this effect, but the imperfections in the test system must be understood. Vortex speed and duration can be important, and sonication intensity as well as the position and composition of the tubes being sonicated can matter.⁴ Also, something as basic as ensuring 10-fold serial dilutions can make a difference and often go unnoticed. Tenfold serial dilutions are typically done by adding 1 to 9 mL diluent (or equivalent) repeatedly until the bacterial numbers are low enough to count. If errors occur during media preparation and the volumes are not exact, miscalculations can magnify and give dramatically inaccurate counts. Just a half of a milliliter less diluent than projected can result in baseline counts that are many cells away from the actual number after these compounding effects are considered.

Growth media selection in the preparation of a challenge inoculum is another overlooked variable. Although the number of passes a culture undergoes may result in some genetic drift, it is the final incubation conditions that can really impact results. There is much discussion, appropriately, over the phase of growth and incubation time. An actively growing culture at 6 hours may be more vulnerable to the uptake of some antimicrobial agents than a dormant or dying culture that is 35 hours old. There is also a dramatic impact in the preparation of a test culture on solid agar-based media or in liquid broth conditions. Solid agar cultures can be generally more difficult to kill than their liquid equivalents (D.A.K., unpublished data, December 2007). It is not just the nature of the media but also the ingredients and constituents of the media that matter. Microorganisms will naturally uptake different growth elements depending on the medium to which they are subjected. This can occur regardless of the total titer achieved, impacting the susceptibility of the isolate. Examples of these effects can be seen in experiments using the AOAC International use-dilution method and a marketed disinfectant at a shortened contact time with S aureus.⁴ The only variable studied was the change in the media used to cultivate the challenge microorganism. As can be seen in Figure 61.1, the two medias selected produced nearly identical carrier populations. But when these carriers were tested in a qualitative (growth or no growth) disinfectant assay, the experimental outcome was dramatically different (Table 61.1). This difference would cause the test product to fail this EPA-required method with tryptic soy broth and pass with nutrient broth.

When discussing neutralization, it was noted that care should be taken in the selection of species to test for adequate neutralization. When cultivating a microorganism for disinfectant evaluations, the particular strain of a species can also matter. Variability can exist even in those cases where identical culture conditions are used. Notorious for this intraspecies heterogeneity is the spore-forming organism, Bacillus cereus (Figure 61.2). Comparisons of three different strains classified as the same species showed dramatically different disinfectant efficacy results (D.A.K., unpublished data, December 2007).

The variability between strains (or isolates), species, and genus of microorganisms is well described for bacteria and fungi but also in comparisons of virus types.⁵ Variability has been particularly reported in comparison between standardized, type culture strains, and more recent environmental isolates. Therefore, a challenge microorganism should be carefully selected that is representative of the goals of the experiment, whether it is for regulatory registration, research, or validation purposes.

Once a microorganism is selected, neutralization confirmed, and the protocol is established for enumeration and growth, there are many different types of methods to which these principles can be applied. Earlier, three tiers of disinfectant tests were referenced. These include studies to characterize active ingredients, simple studies to test formulations, and tests using critical surface materials that are used to predict product performance in the real world. Further, new methods continue to emerge as new microbial knowledge is uncovered and threats determined, such as bioreactor testing and evolving visualization studies.

The first, and simplest, approaches are tests designed to generally screen an active ingredient to determine its ability to kill certain microorganisms. This type of testing can take several forms. One such approach is the MIC or the
modification that creates the minimum bactericidal concentration (MBC) test. Although the MIC is much more common in antibiotic testing than disinfectant testing, there is value in using this method for other antimicrobials because of the ease of testing and the ability to screen multiple concentrations, actives, and microorganisms. This method does not apply well to formulated products because the dilution can dramatically impact the ability of the formulation’s excipients to work together. The MIC test will typically use a multi-well plate with increasing dilutions of the active that are then each inoculated with a standard test culture. Following incubation, the concentration that inhibits the challenge microorganism is considered the MIC. Note that in these tests, growth culture media will be present, which will limit the availability of the antimicrobial to act on the test microorganism and thereby overestimating the true MIC; this is particularly important with certain types of reactive microbicides such as iodine and hydrogen peroxide. By modifying this test to check the viability of the test microorganism from the MIC, it is easy to determine the MBC to differentiate preservative (microbiostatic) and microbicidal (kill) activity. Although the information is limited, MIC/MBC analysis can be a good first step when screening multiple actives.