Evaluation of a Macrofoam Swab Protocol for the Recovery of Bacillus anthracis Spores from a Steel Surface

Abstract

A protocol to recover Bacillus anthracis spores from a steel surface using macrofoam swabs was evaluated for its accuracy, precision, reproducibility, and limit of detection. Macrofoam swabs recovered 31.7 to 49.1% of spores from 10-cm 2 steel surfaces with a <32.7% coefficient of variation in sampling precision and reproducibility for inocula of >38 spores.Thousands of swab samples of Bacillus anthracis spores were taken during investigations of the 2001 bioterrorist events (3,7,10,11,12,13), but no validated protocol was available at the time (8). The swab protocol used during the incident was modified from swab methods used by the food industry (1), National and Aeronautics and Space Administration (5), and hospital epidemiologists (4), but these methods did not involve sampling for spores. A recent study found that macrofoam swabs recover Ն30% more spores than rayon and polyester swabs, premoistening the swab increases recovery by Ն30%, and vortexing the swab recovers Ͼ25% more spores than extraction by sonication (9).We evaluated a protocol that recovers spores from steel surfaces with premoistened macrofoam swabs and vortex processing. Steel coupons with 10-cm 2 surface area, as recommended for environmental and medical-device sampling (6), were chosen for their smooth surfaces. Ethanol was used as the inoculation medium in order to spread the spores evenly over the coupon. These conditions may increase the difficulty of removing spores with a swab compared to the removal of weapons grade powder spores. Five analysts from two laboratories performed the protocol, and the data were evaluated to determine accuracy, sampling precision, reproducibility, and limit of detection (LOD).B. anthracis Sterne 34F2 spores (Colorado Serum, Denver, CO), previously stored in 50% ethanol to prevent germination, were added to 95% ethanol to attain ϳ10 6 spores ml Ϫ1 . This suspension was serially diluted 1:10 in 95% ethanol to attain inocula (I) ranging from 4.0 ϫ 10 0 to 6.0 ϫ 10 4 spores ml Ϫ1 (Table 1). For each experiment, 10 to 15 sterile 10-cm 2 steel coupons were placed in glass petri dishes and inoculated with 0.5 ml of the appropriate inoculum of spores, as described previously (9). Petri dish lids remained partially open as the coupons dried in a biological safety cabinet for 2 h. A macrofoam swab (Critical Swab; VWR, Suwanee, GA; catalog no. 10812-046) was dipped in phosphate-buffered saline plus 0.04% Tween-80 (PBST; pH 7.2), and excess fluid was expressed from the head. The swab was swept in horizontal, vertical, and diagonal sweeps to cover the coupon during each orientation. The swab was vortexed in 5 ml PBST for 2 min at 10-s intervals (V1), placed into a second tube of PBST, and vortexed again (V2).Ten sampled and 10 unsampled coupons were processed to determine the number of spores left after swabbing (L) and recovered from inoculated controls (C), respectively. Control coupon tests were performed prior to swab experiments to ensure that spores remained on the coupons during drying. The coupons were sonicat...

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax

“…These findings were consistent with those in our previous report, which demonstrated that the use of the SETS was optimal for detection of Francisella tularensis from spiked swabs (39). There are established protocols for recovery of B. anthracis spores from environmental swabs (13,14); however, this is the first report to evaluate procedures and develop a protocol for recovery of B. anthracis vegetative cells from clinical swabs. Further, this protocol was successfully employed for the identification of B. anthracis from cutaneous anthrax outbreak specimens.…”

Overview of Methodologies to Sample and Assess Microbial Burden in Low Biomass Environments

“…Macrofoam swabs were demonstrated to collect 30῎ more spores 8), 22) even at spore concentrations ῌ10 1 spores per preparation 8) . Recovery e$ciencies ῍90῎ were achieved from macrofoam swabs during direct inoculation studies (recovery e$-ciency from rayon and polyester was decidedly lower 22) ).…”

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

“…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%; .…”

“…Previous studies have primarily investigated sampling techniques by inoculating surfaces using a high-concentration liquid suspension of bacteria or spores (Rose et al 2004;Hodges et al 2006) or by evaluating environmental samples (Krischner and Puleo 1979;Sanderson et al 2004). These approaches either do not directly simulate the dry deposition of bacteria or spores from the aerosol state or may not have sufficient precision or accuracy to adequately differentiate between techniques.…”