Several factors affect the measurement of a test solution's antimicrobial activity, and these must be considered in the validation design. They include the nature of the microorganisms used as challenge organisms, the preparation of the inoculum of challenge organisms, the specific conditions of the test, and the conditions of recovery. These factors also affect the validation of recovery methods for aqueous or nonaqueous products, irrespective of their antimicrobial properties; thus, all test methods should be validated with these factors in mind.

The nature of the challenge microorganism exerts a strong effect upon the response to the antimicrobial agent, and so upon the neutralization required for recovery. Represented among these organisms in compendial tests are Gram-positive bacteria, Gram-negative bacteria, yeasts, and molds. Each organism to be used in the test must be included in the validation.

The preparation of the inoculum of challenge microorganisms also affects the testing of products having antimicrobial properties. The growth and preparation of the challenge organism determines the physiological state of the cell. This state has a direct influence on the results of any test of antimicrobial efficacy. Microbial tests do not use individual cells; rather, populations of cells are harvested for study. The data generated from these studies are less variable if the cell populations are homogeneous. Liquid cultures or confluent growths on solid medium are best suited for reproducible culture preparation. The conditions of organism preparation and storage must be standardized for the neutralizer evaluation and should reflect the conditions of the antimicrobial assay.

The specific conditions of the test, including buffers used, water, light conditions, and temperature, must be reproduced in the validation study. All test conditions also should be standardized and performed in the validation study exactly as performed in the test.

The conditions of microbial recovery are among the most crucial in accurately estimating the number of microorganisms present in a test solution. The first consideration is the recovery medium used to support the growth of survivors. This concern is discussed in detail below. The second consideration is the incubation conditions. Optimal conditions for growth must be present to ensure complete growth and reproducible results.

Three common methods are used to neutralize antimicrobial properties of a product: (1) chemical inhibition, (2) dilution, and (3) filtration and washing.

A validated method for neutralizing the antimicrobial properties of a product must meet two criteria: neutralizer efficacy and neutralizer toxicity. The validation study documents that the neutralization method employed is effective in inhibiting the antimicrobial properties of the product (neutralizer efficacy) without impairing the recovery of viable microorganisms (neutralizer toxicity). Validation protocols may meet these two criteria by comparing recovery results for treatment groups.

The first is the test group, in which the product is subjected to the neutralization method, then a low level of challenge microorganism [less than 100 colony-forming units (cfu)] is inoculated for recovery. The second is the peptone control group, in which the neutralization method is used with peptone, or diluting Fluid A (see Sterility Tests 71), as the test solution. The third is the viability group, in which the actual inoculum is used without exposure to the neutralization scheme. Similar recovery between the test group and the peptone group demonstrates adequate neutralizer efficacy; similar recovery between the peptone group and the viability group demonstrates adequate neutralizer toxicity.

In principle, the protocol must show that recovery of a low inoculum (less than 100 cfu) is not inhibited by the test sample and the neutralization method. Validation protocols may meet these two criteria by comparing recovery among three distinct test groups: (1) neutralized product with inoculum, (2) challenge inoculum control in buffered solution, and (3) inoculum in the absence of product or neutralizer. This can be established by directly comparing the result in the treated solution (1) to the inoculum (3) above. If the growth on the treated solution is not comparable to the growth on the inoculum group, it should be determined whether the neutralization method itself is toxic to the microorganisms.

The validation studies described above use challenge microorganisms that have never been exposed to antimicrobial agents, and thus are not identical to organisms seen in antimicrobial effectiveness testing or when a sterility test is performed on a preserved product. If the use of alternative media is desired, the recovery of injured microorganisms should be addressed in the validation study. This may be done by directly comparing the recovery of each challenge microorganism on the preferred medium and on the alternative medium, after exposure to the product. This exposure should include at least two time periods showing survival of less than 100 cfu per mL, unless the rate of kill of the antimicrobial agent is such that no recovery is possible even if the microorganism is plated within minutes of exposure. This comparison should be performed at least three times. The alternative medium is validated if the recovery seen on that medium is no less than that seen on the preferred medium, within an error of 0.5 log units.

The accuracy of any estimate of viable cfu is affected by the number plated. As the number of viable cells plated increases, crowding effects decrease the accuracy of the count, reducing the estimate. As the number decreases, random error plays an increasing role in the estimate.

The accepted range for countable colonies on a standard agar plate is between 25 and 250 for most bacteria and Candida albicans. This range was established in the food industry for counting coliform bacteria in milk. This range is acceptable for compendial organisms, except for fungi. It is not optimal for counting all environmental isolates. The recommended counting range for Aspergillus niger is between 8 and 80 cfu per plate. The use of membrane filtration to recover challenge microorganisms, or the use of environmental isolates as challenge microorganisms in antimicrobial effectiveness testing, requires validation of the countable range. This validation may be performed by statistical comparison of estimated cfu from successive pairs in a dilution series. Prepare a suspension so that plating will provide approximately 1000 cfu per plate, and then dilute twofold to a theoretical concentration of approximately 1 cfu per plate. Plate all dilutions in the series in duplicate, and incubate for recovery under the conditions of the Antimicrobial Effectiveness Testing 51. Compare the estimates of cfu per mL from paired tubes in the dilution series by the formula:

in which Lcfu is the number of colonies on the plate with the lower count (greater dilution), and Hcfu is the number of colonies on the plate with the higher count (lesser dilution). The estimates of the cfu per mL provided by Lcfu and Hcfu should agree within the limits of the formula with a critical value of 1.96. The upper limit of plate counts is then defined as the number (Hcfu) that reproducibly passes this test. This study should be independently repeated a sufficient number of times to establish an upper limit of cfu for the particular plating conditions.

There is a lower limit at which the ability of the antimicrobial effectiveness test to recover microorganisms becomes untenable. If the first plating is performed with 1 mL of a 10–1 dilution, cfu in the range of 1 to 10 per mL would not be seen. On this dilution plating, only the lower number of cfu may be reduced to 3, allowing as few survivors as 30 cfu per mL to be reported.

Lower counting thresholds for the greatest dilution plating in series must be justified. Numbers of colonies on a plate follow the Poisson distribution, so the variance of the mean value equals the mean value of counts. Therefore, as the mean number of cfu per plate becomes lower, the percentage error of the estimate increases (see Table 2). Three cfu per plate at the 10–1 dilution provide an estimate of 30 cfu per mL, with an error of 58% of the estimate.