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Applied and Environmental Microbiology, January 2009, p. 39-44, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01563-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Surface Sampling of Spores in Dry-Deposition Aerosols

Jason M. Edmonds,^* Patricia J. Collett, Erica R. Valdes, Evan W. Skowronski, Gregory J. Pellar, and Peter A. Emanuel

Edgewood Chemical Biological Center, U.S. Army, Department of Defense, 5183 Blackhawk Road, Aberdeen Proving Ground, Maryland 21010

Received 9 July 2008/ Accepted 24 October 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ability to reliably and reproducibly sample surfaces contaminated with a biological agent is a critical step in measuring the extent of contamination and determining if decontamination steps have been successful. The recovery operations following the 2001 attacks with Bacillus anthracis spores were complicated by the fact that no standard sample collection format or decontamination procedures were established. Recovery efficiencies traditionally have been calculated based upon biological agents which were applied to test surfaces in a liquid format and then allowed to dry prior to sampling tests, which may not be best suited for a real-world event with aerosolized biological agents. In order to ascertain if differences existed between air-dried liquid deposition and biological spores which were allowed to settle on a surface in a dried format, a study was undertaken to determine if differences existed in surface sampling recovery efficiencies for four representative surfaces. Studies were then undertaken to compare sampling efficiencies between liquid spore deposition and aerosolized spores which were allowed to gradually settle under gravity on four different test coupon types. Tests with both types of deposition compared efficiencies of four unique swabbing materials applied to four surfaces with various surface properties. Our studies demonstrate that recovery of liquid-deposited spores differs significantly from recovery of dry aerosol-deposited spores in most instances. Whether the recovery of liquid-deposited spores is overexaggerated or underrepresented with respect to that of aerosol-deposited spores depends upon the surface material being tested.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Since 2001, the biological defense community has made great strides in improving our readiness to respond to and recover from an attack with a biological pathogen. Lessons learned in restoring operations within large building complexes contaminated with Bacillus anthracis spores or ricin toxin have led to the development of more-efficient tools for the detection of biological pathogens and means of decontaminating affected facilities. A key step in determining the extent of contamination during the initial stages of a bioterrorist attack and in ascertaining if a building can be reoccupied in the final stages of cleanup is our ability to effectively sample an area and deliver a specimen for testing.

Currently there are several commercially available sampling products (2, 3, 8, 9, 16, 23), but test data demonstrate that they are limited in their ability to detect and recover biological samples, with estimated recovery efficiencies ranging from approximately 10% to 40% (8, 11, 19). Studies have also shown that a significant proportion of the biological agent that can be sampled from a solid surface remains adhered to the sampling materials despite efforts to wash it off for testing or is lost during the sample processing (1, 7, 19).

Historically, recovery efficiencies of swabs found in sampling kits have been calculated based on their ability to recover a liquid bacterial suspension deposited on hard, smooth, and nonporous surfaces which have then been allowed to air dry prior to the surface sampling tests. Application of a liquid sample to a test coupon was undertaken because it allowed the researchers to accurately and reproducibly apply a known amount of biological agent to the test coupon. However, in every bioterrorist incident since 2001, dried aerosols of the agent were abruptly released into the air, followed by a period in which they gradually settled onto surfaces. As part of this study, we sought to determine if liquid application of test solutions altered the sampling data in comparison to aerosol deposition on surfaces. To accomplish this task, we constructed a dry-deposition chamber, which more closely simulates a real-world encounter. Using this new approach, we have found that on some surfaces, the liquid-deposition method leads to recovery rates significantly different from that of dry deposition. Additionally, the variation in recovery efficiency across all surface materials with aerosol-deposited spores is significantly smaller than that with liquid-deposited spores. The combination of a smaller standard deviation and a more real-world-like deposition method better aids in identifying materials best suited for sampling procedures.

In addition to comparing the recovery of liquid-deposited and aerosol-deposited Bacillus spores from test surfaces, this study was undertaken in order to determine what sampling swab materials were most effective at surface sampling and whether methods or buffers could be developed which would reduce the amount of residual biological material which remained adhered to the current swab-based sampling kits.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Spore preparation.
Two grams of previously harvested Bacillus atrophaeus subspecies globigii (14) Dugway 1088 spores (BG spores) were washed, pelleted, and resuspended six times in sterile distilled water. After the final wash, spores were suspended in 20 ml of water, and the concentration of spores measured 4.1 x 10¹⁰ CFU/ml. Spore stocks were periodically checked by performing a spore staining procedure to verify the lack of progression into the vegetative stage. This suspension was used for the dry-deposition experiments. A further 1:100 dilution was performed using 100 µl of the stock suspension to attain a 4.1 x 10⁸ CFU/ml suspension to be used for the liquid-deposition portion of this experiment. All spore suspensions were stored at 4°C when not in use.

Swab description.
In this study, four different swab materials were utilized in determining rates of recovery from a variety of surface materials. The swabs used were cotton-tipped (Puritan; Fisher Scientific, Suwanee, GA; catalog no. 14-959-102), Dacron-tipped (FisherBrand; Fisher Scientific, Suwanee, GA; catalog no. 14-959-97A), rayon-tipped (Starplex Scientific Inc.; VWR, Suwanee, GA; catalog no. 14211-774), and a polyurethane macrofoam-tipped swab (Critical Swab; VWR, Suwanee, GA; catalog no. 10812-046).

Coupon description.
Four unique surface materials served as coupons on which the BG spores were to be deposited: glass, chemical agent-resistant coating (CARC)-painted steel, polycarbonate, and vinyl tile. All coupons were cut 1/8-in. thick, 2 cm by 5 cm, by the machine shop on the Aberdeen Proving Grounds, Edgewood Area. Prior to any deposition, all coupons were sterilized with the use of an autoclave (BetaStar Corporation, Telford, PA).

Deposition chamber.
In order to determine if aerosol deposition would yield differences from liquid-deposition techniques, it was necessary to design an aerosol-deposition chamber that could reliably and uniformly deposit the biological test specimen throughout the testing chamber. Two ionizing fans were installed to decrease the static charges within the circular deposition chamber and to continually mix the air during the aerosolization of the spores. The rotating base of the platform was rotated at a speed such that an individual coupon would not be exposed to any single point in the chamber for an extended period of time and which would not create turbulent airflow within the chamber.

Coupons were placed on the circular deposition platform, which was divided into three separate zones. The center zone was defined as 0 to 12 in. from the axis of rotation, the middle zone was 12 to 24 in. from the axis, and the edge zone was 24 to 40 in. from the center point of the platform. Deposition of spores was performed in triplicate with a combined total of 90 test coupons per zone. The concentration of spores deposited on each zone was relative to that of the neighboring areas of the deposition platform. For this experiment, the numbers of spores deposited on the middle and edge zones were reported relative to deposition on the center.

Preparation of dry coupons.
The dry deposited coupons were laid inside a circular aerosol chamber consisting of a rotating platform and removable lid (manufactured in-house with 12-in. turntable; Barnard LTD, Chicago, IL). Ten coupons of each material per swab type and 10 glass control coupons were laid out on the platform in a predetermined deposition zone. When this was completed, the lid was replaced, the rotating platform was plugged in, and ionizing fans (3M Mini air ionizer, model 960) were turned on during the deposition process. One ml of a 10¹⁰ stock solution of BG spores was loaded into a nebulizer (Aeroneb Go 7070 micropump nebulizer; Active Forever, Scottsdale, AZ) and aerosolized onto the coupons through a slit in the top on the chamber lid. After the deposition was complete, the fans were turned off and the platform was allowed to continuously rotate overnight.

Preparation of liquid coupons.
For each surface material, 10 coupons per swab type and 10 glass control coupons were laid down inside a class II type B2 biosafety cabinet. Each coupon received five 20-µl drops of a 10⁷/ml stock of BG spores. The coupons were allowed to dry inside the biosafety cabinet for a minimum of 3 h until all liquid had completely evaporated.

Sampling.
The sampling process was performed identically for the dry-deposition and liquid-deposition samples. Each of the 10 glass control coupons was placed in a 50-ml conical tube containing 10 ml of 0.1% phosphate-buffered saline (PBS)-Triton X-100. The additional coupons were broken down into 10 coupons per swab type. All swabs were premoistened with 100 µl of sterile water prior to sampling. Each coupon was swabbed with a single swab methodically, 5 times along the length, rotated 90 degrees, swabbed 12 times along the width, rotated again 90 degrees, and swabbed an additional 5 times along the length. After swabbing, the swab heads were snipped off with sterile wire cutters into individual 50-ml conical tubes, each containing 10 ml of 0.1% PBS-Triton X-100.

After the sampling was complete, all of the tubes containing either swab samples or the submerged glass coupon controls were subjected to 10 min of vortexing using a large area mixer (catalog no. 099A-LC1012; Glas-Col, Terra Haute, IN). After vortexing, the tubes were placed in a sonic bath (Branson 5510; Branson Ultrasonics Corporation, Danbury, CT) for an additional 10 min. At the completion of processing, the samples were plated in triplicate, using a spiral plater (Spiral BioTech Autoplate 4000; Advanced Instruments, Norwood, MA). Plates were incubated overnight at 37°C, and colonies were counted the next day using a Q-Count instrument (Advanced Instruments, Norwood, MA). The CFU were recovered and recorded, and coefficients of variation (CVs) were calculated. CFU obtained by this process are the basis for the definition of recovery throughout this paper and are used in all recovery comparisons.

Analysis and statistics.
Ten coupons were used in each experimental replicate, and three or four experimental replicates were performed for each swab and deposition set. Samples which were contaminated with non-BG growth or were inaccurate due to mechanical error of the spiral plater were omitted from statistical analysis. Samples from each coupon were collected and plated in triplicate and the mean of each triplicate recorded. Mean CFU counts for each data set were calculated by averaging samples kept for statistical analysis. Percent recovery was calculated by dividing the mean CFU count from the swab material by the mean CFU count of the control samples. Only culturable spores were recorded, and no attempt to determine the number of nonculturable viable spores was made. Pairwise comparisons between deposition methods was done by performing Welch's t test, which allows for unequal sample sizes, data which are not normally distributed, and variances which are not equal.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Dry-agent aerosol-deposition chamber.
Analysis of the deposition showed spore concentrations of 100% and 100.9% of the amount of BG spores in the middle and edge zones, respectively, compared to the center zone. The combined CVs of all three replicates within the center, middle, and edge zones were 12.2%, 10.3%, and 11.9%, respectively. A two-tailed Welch's t test of the three zones was performed, and no statistical difference in deposition among the three zones was observed.

Scanning electron microscopy (SEM) was used to ascertain if deposition methods resulted in an even distribution of individual spores across a sampling surface. SEM visualization revealed consistent coating of the sampling surface with individual spores across the entire contaminated coupon for aerosol-deposited spores. Coupons contaminated with liquid-deposited spores showed large clumps of spores in distinct areas of the coupon.

Nonionic surfactants.
Nonionic detergents were used to supplement sampling buffers to determine if recovery efficiencies would be improved. Glass coupons were selected for these tests because they consistently demonstrated the highest recoveries. No statistical difference in recovery efficiency of dried spores was determined using 1x PBS extraction buffer with 0.1% of Triton X-100, Tween 80, SilwetL-77, Tyloxapol, or hydroxyethyl cellulose. Each of the five surfactant buffers recovered approximately 98% of the spores deposited on glass coupons based on the known concentration of the spore stock. Double-distilled deionized water led to a recovery of 25% of the spores deposited on the coupons, and a 1x PBS buffer resulted in a recovery of only 11% of the dried spores.

Recovery of biological agents deposited by liquid and dry aerosol methods.
Aerosol and liquid deposition of BG spores was conducted on test coupons using the four swabbing materials. All four swab types recovered a higher percentage of liquid-deposited spores than with aerosol-deposited spores on glass coupons (Table 1). Cotton swabs recovered 88.7% of liquid-deposited spores, compared to 62.4% of the aerosol-deposited spores. Dacron swabs recovered 82.1% and 64.9% of spores deposited by liquid and aerosol means, respectively. Similarly, rayon swabs recovered 87.5% of the liquid-deposited spores and 65.2% of the aerosol-deposited spores on glass. Macrofoam swabs recovered 89.1% of the liquid-deposited spores and only 61.2% of the aerosol-deposited spores.

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TABLE 1. Evaluation of recovery efficiencies of cotton, dacron, rayon, and macrofoam swabs for glass, CARC-painted steel, polycarbonate, and vinyl coupons with liquid or dry aerosol BG spore deposition

There was no statistical difference in recovery between the two deposition methods with cotton and macrofoam swabs on CARC-painted coupons (Table 1). Cotton swabs recovered 47.0% of the liquid-deposited spores and 51.7% of the aerosol-deposited spores. Macrofoam swabs recovered 55.7% of the liquid-deposited spores, while recovering 51.5% of the aerosol-deposited spores. Both Dacron and rayon swabs recovered more aerosol-deposited spores, 57.6% and 53.1%, respectively, than liquid-deposited spores, 42.5% and 43.6%, respectively.

The only swab material which recovered a statistically different amount of spores between the two deposition methods from vinyl floor tile coupons was cotton, which recovered 60.3% of the aerosol-deposited spores compared to 49.0% of the liquid-deposited spores (Table 1). Dacron swabs recovered 62.2% and 68.7% of the liquid- and dry-deposited spores, respectively. Vinyl tiles sampled with rayon swabs recovered 58.3% and 60.2% of the liquid- and dry-deposited spores, while macrofoam swabs recovered 72.0% of the liquid-deposited spores and 67.0% of the dry-deposited spores.

All four swab types recovered significantly more spores from liquid-deposited spores on polycarbonate coupons than from aerosol-deposited coupons (Table 1). Cotton recovered 74.9% of the liquid-deposited spores, compared to 65.1% of the aerosol-deposited spores. Dacron swabs recovered 83.4% and 71.9% of the liquid- and aerosol-deposited spores, respectively. Rayon recovered 75.4% and 68.9% of the liquid- and aerosol-deposited spores, respectively, and macrofoam recovered 88.3% and 75.5%, respectively.

In addition to observing statistically different surface recovery rates for spores deposited by liquid versus aerosol methods, the variation of individual swab replicates was smaller in sampling of the aerosol-deposited spores. The standard deviation of all four swab types on all four liquid-deposited surface materials was 21.4%. Sampling of aerosol-deposited spores produced a standard deviation of 13.8% across all four surface types sampled with all four swab materials. The standard deviations of individual swabs across all surface types were also significantly smaller on aerosol-deposited spores. The standard deviations of cotton, Dacron, rayon, and macrofoam across all four surface types of liquid-deposited spores were 20.2%, 21.7%, 21.2%, and 21.0%, respectively, compared to only 13.1%, 12.1%, 14.3%, and 14.2% when aerosol-deposited spores were sampled.

Concentration-dependent recovery of liquid-deposited spores.
An additional test was performed in which glass coupons were seeded with spore concentrations ranging from 10⁴ to 10⁷ spores and swabbed with Dacron swabs. It was found that recovery efficiency does increase as the concentration of seeded spores increases (Table 2).

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TABLE 2. Percent recovery of liquid-deposited spores on glass coupons over a range of four logs, utilizing a dacron swab

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sampling methodology studies have traditionally relied on liquid deposition of biological agents, which were then allowed to dry prior to sampling. Reproducibility studies of glass coupons in three experimental replicates consisting of 90 samples each demonstrated less than a 1% difference in spores recovered between any two of the three zones within the deposition chamber and resulted in replicate CVs of less than 9%. The consistency of the deposition clearly demonstrates the utility of the deposition test chamber for surface sampling recovery studies.

Coupon sampling procedures were selected to closely mimic procedures which originated with the food industry (2, 10, 13, 15, 18) and were later modified in response to the resulting contamination of mail facilities after the 2001 anthrax attacks. While individual procedures used a range of buffer components, coupon size, and swab agitation, the sampling assays consistently used bacterial loads of 10⁴ CFU per coupon (5, 6, 11, 19). The aim of this study was to develop methods to sample highly contaminated areas such as those encountered by first responders in the Hart Senate Office Building in 2001. While no official quantification of the material present in the Daschle letter has been published, it is estimated that the letters each contained 2 g of "weapons-grade" Bacillus anthracis at a concentration of 10¹¹ to 10¹² CFU/g (12). Letters and sorting equipment which came into contact with the Leahy letter were found to be contaminated with as high as 8 x 10⁶ CFU/100 cm² (4, 20, 21). These levels are well above those which have historically been used in the food industry sampling studies (10, 17, 22) and in several other surface sampling studies (5, 6, 8, 11, 19-21).

The recovery efficiencies reported in this study are higher than those in previously reported research which used similar methods. Rose et al. (19) reported recovery efficiencies of 27.7%, 30.7%, and 10.0% with cotton, macrofoam, and rayon swabs, respectively, on liquid-deposited stainless steel coupons, and Hodges et al. reported a macrofoam efficiency of 49.1% on liquid-deposited stainless steel coupons. In contrast, the recovery efficiencies reported in this study of liquid-deposited glass coupons exceed 88% for these same materials. A key difference is that the coupon seeding densities employed in this study were 10⁶ CFU, which is 100 times higher than the 10⁴ CFU used in previously mentioned studies. We have expanded on inferences made in those papers and thoroughly demonstrated that recovery efficiencies are dependent upon the initial seeding concentrations. Decreased efficiencies at lower spore densities could be explained by residual levels of biological material that remained adhered to the swabbing materials. Once that baseline of irreversibly bound material had been exceeded, the remaining spores may be more easily dislodged during washing steps, leading to higher percent recoveries. When evaluating the efficiency of any material to be used as a tool for recovery, the concentration of the seeding material is a critical component in performing a study which is relevant to addressing the question being asked. In the case of evaluation of sampling materials to be used in determining the threat of a biological act of terrorism, seeding concentrations larger than what has historically been used need to be chosen for this purpose.

The data presented here demonstrate that 11 of the 16 swab and surface material combinations show a statistical difference in recovery between aerosol and liquid deposition. Whether the percent recovery of aerosol-deposited spores was greater or smaller than the recovery of liquid-deposited spores was dependent upon the surface material being sampled. On glass and polycarbonate coupons, both of which are smooth surfaces, recovery of aerosol-deposited spores was less than the recovery of liquid-deposited spores. SEM visualization of coupons seeded with liquid solutions of spores and then subjected to air drying suggests that as the liquid evaporates, surface tension gradually draws large numbers of spores into contact with one another, creating clumps of spores consisting of hundreds or thousands of spores (Fig. 1). These clumps create an environment where the swab heads can easily recover large amounts of spores which do not necessarily require direct interaction with the swabs themselves. In contrast, the aerosol-deposited spores are more uniformly spread across the entire coupon surface. The recovery of these spores relies on the direct interaction of individual spores sticking to the swab fibers, leading to lower recovery rates.

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FIG. 1. Scanning electron micrographs of glass coupons seeded by liquid deposition (left) or dry aerosol deposition (right).

Two of the surface materials, the CARC-painted steel and vinyl tile, are not flat and smooth surfaces. The interactions between the spores and these coupons differ from what is observed with glass and polycarbonate coupons. Thus, the recovery efficiencies between the two deposition methods also differ. Both CARC-painted steel and vinyl tile have crevices and depressions, and CARC-painted steel is also composed of peaks and elevated surface features, which can act to catch and tear fibers on swabs. Similarly to what is observed with the flat surfaces, the liquid-deposited spores congregate into clumps as the water evaporates. On CARC-painted steel and vinyl tile, these clumps are drawn down into crevices, making them inaccessible to the swab heads during sampling. In contrast the dry-deposited spores appear to stay on whatever surface feature they fall onto, including the tops of the peaks (Fig. 2). These spores are more easily accessible to the swab heads and recovered from the coupon.

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FIG. 2. Scanning electron micrographs of CARC-painted steel coupon seeded by aerosol deposition.

Of the four swab types tested, no single swab outperformed all others on all four surfaces. On glass and CARC-painted steel surfaces, all four swab materials recovered statistically similar amounts of spores from the aerosol-deposited coupons on their respective surface types. On polycarbonate coupons, macrofoam outperformed both cotton and rayon and was statistically identical to Dacron. However, Dacron recovered statistically more spores than cotton, making macrofoam the better choice for this surface. Swabbing of vinyl tile produced a similar result to that with polycarbonate. Dacron and macrofoam swabs recovered statistically identical amounts of BG spores from the vinyl coupons while recovering statistically more spores than both cotton and rayon.

Identifying an optimal methodology for sampling contaminated surfaces requires accurately recreating the contamination event under controlled circumstances in a laboratory setting in order to measure variations in the method and then assaying an array of sampling materials. While the recovery of liquid-deposited spores averaged nearly 71% and aerosol-deposited spores averaged almost 62% recovery, the recovery of individual swabs between the two deposition methods varied greatly. Dacron was statistically the worst universal material at recovering liquid-deposited spores, picking up 64% across all four surface materials while also having the worst standard deviation at 22%. However, the Dacron swab was the most efficient material in recovering aerosol-deposited spores, averaging a recovery of just above 65% across all four surface materials, and also had the smallest standard deviation, 12%. The material which showed the greatest difference in recovery between the two deposition methods was a polyurethane macrofoam, which recovered 78% of liquid-deposited spores across all four surface materials while only recovering 63% of aerosol-deposited spores. Without the proper real-world simulation of an aerosol deposition, an inferior sampling material could potentially be used and could give an inaccurate estimation of the contamination level and place the health of many individuals at risk.

 
Thanks to Dan Carmany, Julia Collins, and Angela Buonaugurio at the Edgewood Chemical Biological Center for technical contributions. We thank Jason Wojcik for graphic support.

We acknowledge the contribution of Brian Reinhardt and Sean Nolan at the Defense Threat Reduction Agency for funding of this study. Postdoctorate fellowship support for Jason Edmonds was provided by the National Research Council.

 
* Corresponding author. Mailing address: Edgewood Chemical Biological Center, U.S. Army, Department of Defense, 5183 Blackhawk Road, Aberdeen Proving Ground, MD 21010. Phone: (410) 436-7348. Fax: (410) 436-2081. E-mail: jason.edmonds1{at}us.army.mil

Published ahead of print on 7 November 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, January 2009, p. 39-44, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01563-08
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