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...

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This work was initiated to address the gaps identified by Congress regarding validated biothreat environmental sampling and processing methods. Nine Laboratory Response Network-affiliated laboratories participated in a validation study of a cellulose sponge wipe-processing protocol for the recovery, detection, and quantification of viable Bacillus anthracis Sterne spores from steel surfaces. Steel coupons (645.16 cm 2 ) were inoculated with 1 to 4 log 10 spores and then sampled with cellulose sponges (Sponge-Stick; 3M, St. Paul, MN). Surrogate dust and background organisms were added to the sponges to mimic environmental conditions. Labs processed the sponges according to the provided protocol. Sensitivity, specificity, and mean percent recovery (%R), between-lab variability, within-lab variability, and total percent coefficient of variation were calculated. The mean %R (standard error) of spores from the surface was 32.4 (4.4), 24.4 (2.8), and 30.1 (2.3) for the 1-, 2-, and 4-log 10 inoculum levels, respectively. Sensitivities for colony counts were 84.1%, 100%, and 100% for the 1-, 2-, and 4-log 10 inocula, respectively. These data help to characterize the variability of the processing method and thereby enhance confidence in the interpretation of the results of environmental sampling conducted during a B. anthracis contamination investigation.