Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores from Nonporous Surfaces

Abstract

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of ؎0.12, and for painted wallboard it was 0.29 with a standard deviation of ؎0. It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequences of an inappropriate public health response founded on information garnered by an ineffective sample collection method or procedure has the potential for undesired social and economic impact. Well-developed and validated procedures for the collection and analysis of biological environmental samples are required to provide the necessary level of confidence in agent characterization information provided.Researchers and investigators are aware that the Centers for Disease Control and Prevention (CDC)-recommended procedures for the collection of B. anthracis spores by swab, wipe, and vacuum filtration collection methods (6) underestimate the number of spores on surfaces, and attempts are being made to address the knowledge gap (4,5,18,19). Additionally, a number of studies have been conducted to determine the efficiency of the swab sample collection method (1,2,3,7,8,10,17,18), but limited studies have been conducted to examine the efficiency of the wipe spore collection method (10, 13). A recent study conducted by Sanderson et al. (19) that compared B. anthracis surface sampling results obtained by swab, wipe, and vacuum filtration methods suggests that the wipe collection method outperforms the swab method, but no independent recovery efficiency was established for the wipe method.The objective of this study was to empirically evaluate the wipe surface sample collection method for recovery efficiency with a polyester-rayon blend wipe material and estimate limits of detection for selected nonporous surfaces seeded with dry deposited Bacillus atrophaeus spores. Additionally, a sonication extraction method was evaluated for effectiveness in removing viable spores from the selected wipe collection material. MATERIALS AND METHODSSpore matrix. The material used as the test agent for this study was a powdered matrix containing B. atrophaeus spores (ATCC 9372; formerly Bacillus subtilis var. niger and subsequently "Bacillus globigii") (12) and silicon dioxide particles obtained from the U.S. Army Dugway Proving Ground Life Science Division. The spore material was prepared by cultivating B. atrophaeus in tryptic soy broth (Difco, Detroit, MI) containing 3 mg/liter MnSO 4 (Fisher Scientific, Pittsburgh, PA). After 80 to 90% sporulation, the spore suspension was centrifuged to obtain a spore suspension containing approximately 20% solids. Dry spore material was then prepared from the unwashed spore suspension with a laboratory spray dryer. The spore material was...

Development of an Aerosol Surface Inoculation Method for Bacillus Spores

“…One milliliter of a Bacillus subtilis culture grown overnight in TSB was used to inoculate 100 ml of 1:10-diluted Columbia broth (Difco, BD, Franklin Lakes, NJ) amended with 0.1 mM MnSO 4 . Following incubation at 35 Ϯ 2°C for 72 h, the spore suspension was washed 10 times by centrifugation and resuspension with chilled deionized water.…”

“…The spore surface concentrations are controlled by varying settling time and the initial aerosolized spore concentration in the chamber. Brown et al (4)(5)(6) tested various methods of collecting samples of Bacillus subtilis spores on porous and nonporous surfaces using this approach. In these studies, surface spore concentrations of 10 2 to 10 5 CFU/cm 2 were achieved.…”

Evaluating the long‐term persistence of Bacillus spores on common surfaces

“…Established microbiology laboratories can conduct these experiments, provided that careful documentation and training take place as laboratory staff turnover during multiyear periods may interfere with consistent methods. Although the efficiency of the recovery by wipe methods similar to the method used in this study has been well investigated by other researchers, it is variable as a function of initial inoculum dosage, surface type, humidity and time (GAO 2005, Brown et al ., 2007; Estill et al ., 2009; Rose et al ., 2011; Krauter et al . 2012, Herzog et al .…”

Development of an Aerosol System for Uniformly Depositing Bacillus Anthracis Spore Particles on Surfaces

“…The spores were allowed to settle onto flooring material placed on a bench. Brown et al (2007a;2007b;2007c) spores into a chamber and produced surface concentrations in the range of 10 2 to 10 5 colony forming units per cm 2 . In the anthrax attack of 2001, some of the material was believed to be in a "fluidized" form (defined here as having fumed silica added).…”

“…Further, because of the variability produced by having to deal with live organisms, it is not likely that the accuracy in measurement for spores will meet the NIOSH criteria. In studies of spore recovery from surfaces, the variability of CFUs per sample is not usually reported, only the recovery efficiency and its variability (Rose et al 2004;Hodges et al 2006;Brown et al 2007a;2007b;2007c). Recovery efficiency values reported in the Brown et al studies had a range of 28-90%; .…”