The extraction of a Bacillus anthracis surrogate from pleated HVAC filter samples

Ian Solon

doi:10.17918/etd-3205

Understanding bioterrorist agents, and most specifically anthrax, has become a major concern of the Department of Homeland Security and the Environmental Protection Agency. After the 2001 attack through the United States Postal Service that occurred by sending letters which contained Bacillus anthracis, the causative agent of anthrax, it became essential to learn how severe another attack could be if it occurred and necessary to assess the efficiency of the clean up. Developing a quick and inexpensive method to extract bacteria from HVAC filters can provide valuable information on how to respond more effectively to different attack scenarios. The focus of this research has been to develop an extraction procedure of bacteria spores from a sample of a standard HVAC filter while gathering a better understanding of the bacteria once it is applied to the filter sample. The investigation consisted of applying the B. anthracis surrogate, Bacillus thuringiensis HD1011, to a standard pleated filter with a minimum efficiency reporting value (MERV) of 7. The use of an ultrasonic bath, a vortex device, and a shaker were analyzed individually and combined with one another to identify the preferred extraction approach. It was concluded that sonication followed by vortexing then shaking was the best approach; however, it did not seem reliable to base results from a direct colony count. Results were quantified based on presence versus absence using a most probable number method because the colony counts exceeded expected colony counts and were too numerous to count on some occasions. The vortex-shake extraction procedure was found to be significantly less efficient than when sonication preceded the vortex-shake sequence, where the vortex-shake sequence had a concentration mean of 0.49 (0.3-0.77) CFUs compared to 1.3 (0.8-2.1) CFUs for sonicate-vortex-shake agitation. Additionally, the controls were analyzed in a similar manner to the extraction results. The control data suggested that growth was occurring when the filter was present in the bacterial solution. With the filter present there was a spore concentration of approximately 1.0 CFU, nearly double the concentration when the filter was absent. Further investigation found that there was significantly more bacteria when the filter was present just for agitation, as well as for a full 24 hours prior to agitation compared to a series that never had any filter present. The data with no filter present ever produced a mean of 0.37 (0.14-0.47) CFU compared to means of 0.85 (0.52-1.35) CFU and 1.07 (0.65-1.74) CFU, respectively for when the filter was present for agitation and when it was present for 24 hours prior to agitation. The three different frequencies of agitation were used to identify the best method of extraction. It was found that the use of sonication improved recovery by a statistically significant amount, and that the order could play an important role in the effectiveness of bacterial removal. Going from the highest frequency of agitation to the lowest frequency in sequential order, proved to be the most efficient order for spore recovery. The ideal order was found to be sonication into vortexing followed by shaking.

The extraction of a Bacillus anthracis surrogate from pleated HVAC filter samples

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The extraction of a Bacillus anthracis surrogate from pleated HVAC filter samples

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