Q-281. Optimal Growth Conditions and Viability of Mycobacterium bovis (BCG) for the Performance Evaluation for Disinfectants with Tuberculocidal Claims

J. A. Hasan, M. J. Duncan, S. F. Tomasino;
US EPA, Ft. Meade, MD.

Background: The U.S. EPA Office of Pesticide Programs regulates antimicrobial products used to control pathogenic microorganisms on inanimate surfaces. For disinfectants with tuberculocidal claims, AOAC INTERNATIONAL (AOAC) method 965.12 (II), the Confirmative in-vitro Test for Determining Tuberculocidal Activity, is accepted by the EPA to verify product efficacy. Mycobacterium bovis (BCG) is specified as a surrogate for M. tuberculosis. To generate the test culture, M. bovis (BCG) is grown stationary in Modified Proskauer Beck (MPB) broth for 21-25 days at 36±1°C in a slanted position. Previous testing indicated that there was a decline in inoculum viability as the culture aged over the 21-25 day incubation. To assess the optimal harvest period for the test culture, a study was conducted to evaluate culture viability across a 10 day period. Two recovery media (in house Middlebrook 7H9 and commercial Middlebrook 7H11) and incubation with reduced oxygen tension were included in the study design. Methods: M. bovis (BCG) was grown in MPB broth according to the method and harvested across a period of 15 to 25 days. Growth was harvested from randomly selected MPB tubes at each time point and standardized (i.e., homogenized and adjusted to 20% transmittance at 650 nm). Each standardized test culture was serially diluted and plated in triplicate on both media. One set of each medium was incubated under ambient air at 36 ±1°C, and under 5% CO2 at 36 ±1°C. At days 14 and 21 of incubation, colony counts were recorded for comparative enumeration. Results: Use of M7H9 and M7H11 agar resulted in comparable recovery of M. bovis (BCG) across the six sample periods. Incubation under 5% CO2 accelerated the colony development; however, counts with and without CO2 were comparable at Day 21. Culture harvested at Day 21 yielded the highest titer. Conclusion: Incubation and harvest parameters for M. bovis (BCG) specified in method 965.12 (II) are reasonable; however, based on the results of this study, method modifications to shorten the incubation period to 21±2 days and to employ either M7H11or M7H9 should be considered to further enhance the procedure.