Composite sampling of a Bacillus anthracis surrogate with cellulose sponge surface samplers from a nonporous surface

Abstract

A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas), larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC) and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p = 0.261). Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p = 0.720) for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001). The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

Figure 1. Sponge Stick Sampler.

Sides of the sponge sampler used for sampling. Sides A and C correspond to the approximate 38.1 × 31.75 mm front and rear portions, Sides B and D to the 12.7 × 38.1 mm edge portions, and E and F to the 6.35 × 38.1 mm angled tip portions of the sponge sampler. The adapted CDC approach (Test A) utilized the entire sponge surface, while the modified protocol (Test B) used only Sides A–D.

Figure 2. Test Setup Diagram.

Shaded areas indicate inoculated coupons. Under the adapted CDC approach (Test A), all sides of the sponge (except one edge, B or D) were used for sampling each coupon in a series. Under the modified protocol (Test B) sponge-stick Sides A–D were used to individually sample each coupon. For Condition 1, the inoculated coupon was located at Coupon 1 and was the first coupon sampled; for Condition 2, the inoculated coupon was located at Coupon 4 and was the last coupon sampled. For each test, a positive (inoculated) and negative (sterile) coupon were sampled.

Figure 3. Mean Spore Recoveries.

Data show means (± SD) from all tests, calculated from three replicates of each test.

Figure 4. Comparison of Contaminant Transfer between the Two Sampling Protocols.

White points connected by the dotted line represent mean contaminant transfer (±SD) resulting from the adapted CDC approach (Test A), and solid points connected by the solid line represent mean contaminant transfer (± SD) resulting from the modified sampling protocol (Test B). Means were calculated from three replicates of each test.

Figure 5. Impact of Sponge Sampler Moisture Loss on the Number of Spores Recovered.

White bars represent mean % moisture loss per area sampled (±SD) using the adapted CDC approach (Test A); grey bars represent mean % moisture loss per area sampled (±SD) using the modified sampling protocol (Test B). White points represent the mean number of spores recovered per area sampled (±SD) using the adapted CDC approach (Test A), and black points represent the mean number of spores recovered per area sampled (±SD) using the modified sampling protocol (Test B). Means were calculated from three replicates of each test.

Figure 6. Impact of Moisture Loss on Contaminant Transfer.

The mean cumulative percent of contamination transfer (±SD) versus mean cumulative percent moisture loss (±SD) for both the adapted CDC approach (Test A) and the modified protocol (Test B). White points with the dashed regression line represent the adapted CDC approach (Test A). Solid points with the solid regression line represent the modified sampling protocol (Test B). Cumulative means were calculated from three replicates of each test.