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Keywords:

  • sample recovery efficiency;
  • spore sampling;
  • surface sampling

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

Aim:  To evaluate US Centers for Disease Control and Prevention recommended swab surface sample collection method for recovery efficiency and limit of detection for powdered Bacillus spores from nonporous surfaces.

Methods and Results:  Stainless steel and painted wallboard surface coupons were seeded with dry aerosolized Bacillus atrophaeus spores and surface concentrations determined. The observed mean rayon swab recovery efficiency from stainless steel was 0·41 with a standard deviation (SD) of ±0·17 and for painted wallboard was 0·41 with an SD of ±0·23. Evaluation of a sonication extraction method for the rayon swabs produced a mean extraction efficiency of 0·76 with an SD of ±0·12. Swab recovery quantitative limits of detection were estimated at 25 colony forming units (CFU) per sample area for both stainless steel and painted wallboard.

Conclusions:  The swab sample collection method may be appropriate for small area sampling (10 –25 cm2) with a high agent concentration, but has limited value for large surface areas with a low agent concentration. The results of this study provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure.

Significance and Impact of the Study:  It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequence of an inappropriate public health response founded on information gathered using an ineffective or unreliable sample collection method has the potential for undesired social and economic impact.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

Following a biological agent release, such as, the Bacillus anthracis incidents of October 2001, environmental samples are collected and analysed to provide information on initial agent concentration, location and extent of contamination, and ultimately confirmation that clean-up goals are achieved (Small et al. 2001). 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 from 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 procedure for the collection of B. anthracis spores using the swab collection method (Centers for Disease Control and Prevention 2002) underestimates the number of Bacillus spores on surfaces, but only a few studies have been conducted to examine actual swab recovery efficiencies and limits of detection (Barnes 1952; Angelotti et al. 1958; Angelotti et al. 1964; Favero et al. 1968; Favero 1971; Puleo et al. 1973Kirschner and Puleo 1979; Rose et al. 2004).

Data to adequately assess spore collection and recovery efficiencies, and limit of detection for the recommended swab sample collection method are especially limited for spores deposited as dry particles. Research efforts have been hampered because of the difficulty in quantifying the number of viable spores actually deposited on a given surface. Without reliable initial surface loading data, sampling method efficiency cannot be accurately determined.

The objective of this study was to empirically evaluate the swab surface sample collection method for recovery efficiency using a rayon swab 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 swab collection material.

Materials

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

Spore matrix

The material used as the test agent for this study was a powdered matrix containing B. atrophaeus spores (ATCC# 9372, formerly B. subtilis var. niger and subsequently B. globigii) 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, USA) containing 3 mg l−1 MnSO4 (Fisher Scientific, Pittsburgh, PA, USA). After 80–90% 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 using a laboratory spray dryer. The spore material was dry blended with Aerosil R812S fumed silica particles (Degussa, Frankfurt am Main, Germany) at 80% dry spore material to 20% silica, and jet milled to a uniform particle size. The final powdered matrix contained approximately 1011 viable spores per gram. The B. atrophaeus spore material was extensively designed to enhance aerosol suspension and inhalation characteristics, and the removal, extraction and recovery characteristics of a different Bacillus species, native spore material or spore material prepared using a different method may differ.

Reference surface material

Stainless steel coupons measuring 1·25 cm × 5 cm (6·25 cm2) were used as reference surfaces. The coupons were cut from 1·2 mm thick 316-l stainless steel (Neeley Plastic Fabrication Inc., Albuquerque, NM, USA). The stainless steel coupons were washed with Alcojet powdered detergent (Alconox Inc., New York, NY, USA), rinsed in de-ionized water, air dried, and autoclave sterilized at 121°C and 1500 kPa for 40 min.

Sample surface material

Stainless steel and painted wallboard coupons measuring 2·5 cm × 10 cm (25 cm2) were used for representative sample collection surfaces. The stainless steel sample coupons were prepared in the same manner as the stainless steel reference coupons. The painted wallboard coupons were cut from 6-mm smooth wallboard and painted with white interior latex semi-gloss paint (Glidden, Cleveland, OH, cat #HM1420). To minimize paint surface irregularities, the paint was applied to the wallboard surface using a pressurized paint sprayer. The painted wallboard coupons were air dried, and UV-C (254 nm) sterilized at 200 μW cm−2 for 20 min.

Swab material

The swab material evaluated in this study was sterile rayon (Starplex Scientific, Inc., Etobicoke, Ontario, Canada, cat #S09). While it is recognized that the addition of a surfactant to the wetting agent potentially enhances particle removal from surfaces (Petersen et al. 1973; Buttner et al. 2001; Rose et al. 2004), the objective of this study was the evaluation of the current CDC recommended wipe collection method. Wetting agents currently recommended by the CDC include sterile de-ionized water, sterile saline and sterile Phosphate Buffered Saline (Centers for Disease Control and Prevention 2002). For this reason, sterile de-ionized water was selected for use as the wetting agent in this study and because a recent comparative evaluation of the swab method by Sanderson et al. (2002) also utilized sterile de-ionized water as the wetting agent.

Aerosol deposition system description

Components of the aerosol deposition system include a TSI Model 3400A Fluidized Bed Aerosol Generator (TSI Inc., Minneapolis, MN, USA), an aerosol mixing chamber and an aerosol deposition chamber. The mixing chamber is designed to receive spore material from the aerosol generator and provide a confined volume allowing for concentration equilibration before transfer to the aerosol deposition chamber. The aerosol deposition chamber is designed to receive the aerosolized spore material from the mixing chamber, provide uniform mixing, and allow undisturbed settling of spore material onto reference and sample coupon surfaces.

The aerosol mixing chamber is a cylindrical containment vessel with a diameter of 45 cm, a height of 30 cm, and a volume of 0·048 m3. The chamber is constructed of carbon steel with an enamel coated surface. Valved feed-through ports are provided for injecting spores into the chamber from the fluidized bed aerosol generator, transferring spore material to the aerosol deposition chamber, and collecting samples for concentration analysis by a TSI 3110A Aerodynamic Particle Sizer (TSI Inc., Minneapolis, MN, USA).

The aerosol deposition chamber is a cubic containment vessel with dimensions of 90 cm × 90 cm × 90 cm providing an interior volume of 0·73 m3. The chamber is constructed of polypropylene sheets welded at the seams to make the chamber watertight. Access doors are located at the front and rear of the chamber, and windows, constructed from 12-mm clear static-free polyvinyl chloride, are located on side walls and both doors. A feed-through port is located in the chamber top to receive aerosol from the mixing chamber. Two muffin fans provide convective airflow and circulation for aerosol dispersion and mixing. A sliding tray is located in the bottom of the chamber for reference and sample coupon placement and access.

Extraction efficiency methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

Extraction efficiency, which is a measure of material transfer effectiveness from the collection medium, such as swabs, to the extraction solution, was determined. Several extraction methods are currently used to extract micro-organisms from environmental sample collection media, such as shaking, vortexing and sonication, but the most widely used are vortexing and sonication (Angelotti et al. 1964; Puleo et al. 1967a, 1967b; Kirschner and Puleo 1979; Rose et al. 2004). A sonication method for extracting spores from the wipe collection medium into a buffer solution containing surfactant was evaluated by the following method.

Swab inoculation

A spore suspension was prepared by suspending 1·0 mg of the spore stock (1011 CFU g−1) in 10 ml Butterfield Buffer solution (3 mmol l−1 KH2PO4, pH 7·2) to produce a nominal 107 CFU ml−1 suspension. Twenty swabs were directly inoculated with 100 μl (106 CFU) of the suspension. Following inoculation, the swabs were immediately placed into tubes containing 10·0 ml Butterfield Buffer with 0·01% Tween 80 (Fisher Scientific, Pittsburgh, PA, USA; cat# BP-338-500). Five additional tubes containing 10·0 ml Butterfield Buffer with 0·01% Tween 80 (BBT) were directly inoculated with 100 μl (106 CFU) of the same spore suspension as references.

Extraction and enumeration

Spores were extracted from the swabs into the BBT by sonicating in a VWR Model 250T ultrasonic bath (VWR International, Tempe, AZ, USA) for 15 min at sweeping frequencies between 38·5 and 40·5 kHz and an average power of 180 W. The extraction suspension was then heat treated at 65°C for 60 min to kill any bacterial vegetative cells and fungal spores which may be present in the suspension, and to activate the Bacillus spores for rapid germination (Foerster and Foerster 1966; Preston and Douthit 1984). While an estimated 5% of viable spores were killed by the heat treatment (Montville et al. 2005), the same relative numbers of viable spores were killed in both the reference and sample suspensions. Following heat treatment, the suspension was vortexed for 15 s and five log serial dilutions of the extracted spore suspension were then prepared in sterile de-ionized water. A 1·0-ml aliquot of the suspension and 1·0 ml of each dilution were spread to Petrifilm Aerobic Plate Count Media (3M Microbiology products, St Paul, MN, USA) in triplicate. The Petrifilm plates were then incubated at 37°C for 24 h. Following incubation, colonies with distinct margins were counted by eye. Only plates with counts between 30 and 300 CFU were included with counts logged into a laboratory notebook. Total CFU per sample were determined as a function of dilution factor and extraction volume. References were subjected to the same procedure.

Calculations

Extraction efficiency was calculated as the number of CFU enumerated from the swab extraction suspension, relative to the mean number of CFU enumerated from the reference suspensions. The mean extraction efficiency was calculated using the equation:

  • image(1)

where ηe is the mean extraction efficiency, Ci is the average wipe count for three replicates, inline imageis the mean reference count, i is the sample number and n is the sample size.

Standard deviation (SD) was calculated using typical statistical methods for normally distributed data.

Recovery efficiency methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

For this study, two surface coupons were positioned side-by-side or co-located in the aerosol deposition chamber and seeded with the dry aerosolized B. atrophaeus spore matrix. One of the surfaces, a stainless steel reference coupon (1·25 cm × 5 cm), was sized to fit into a sample vial for direct spore removal, while the other surface, a sample surface coupon (2·5 cm × 10 cm), was sized for a typical swab application. Deposited spore material was directly removed from the reference coupon surface and cultured for enumeration of CFU, whereas deposited spore material was collected from the sample coupon by swab and extracted by sonication for enumeration by culture. Recovery efficiency, which is a measure of overall transfer effectiveness from surface to culture, was calculated as the number of CFU enumerated from the swab sample per unit area relative to the number of CFU enumerated from the co-located reference coupon per unit area.

Surface material layout

Reference and sample surface coupons were placed side-by-side in the sliding tray at the bottom of the aerosol deposition chamber with a separation of 1 cm. The tray was designed to accommodate 20 sets of the co-located coupon pairs. A temporary spray adhesive (J. T. Trading Corporation, Newton, CT, USA; cat #SF202) was applied to the bottom of the tray prior to placement of the reference and surface coupons to prevent extraneous spore re-aerosolization and re-deposition during sample collection.

Surface seeding

Reference and surface sample coupons were seeded with the spore mixture by dry aerosol deposition. The spore mixture was aerosolized by the Fluidized Bed Aerosol Generator, injected into the mixing chamber and monitored for volumetric concentration. After the volumetric concentration correlating to the desired surface loading was achieved, the mixing chamber contents were rapidly flushed into the deposition chamber where they were mixed by circulating fans for 15 min and then allowed to settle onto the coupons for 24 h. Surface loadings in the ranges of 100–1000 and 10 000–100 000 CFU cm−2 were evaluated for each surface type. For each surface loading and surface type, 20 sample coupons and 20 co-located reference coupons were seeded. During the surface seeding and sample collection process, temperature was maintained at 25 ± 2°C and relative humidity was maintained at 30% ± 10%.

Reference collection

Spores were collected from the reference coupons by gently misting the coupon surface with sterile de-ionized water to mitigate spore re-aerosolization, carefully placing the reference coupon in a prelabelled sterile 50-ml Blue Falcon screw-top tube (Becton Dickinson Labware, Franklin Lakes, NJ, USA) containing 30·0 ml of sterile BBT, and sealed with a cap.

Surface sample collection

Using aseptic procedures, spores were collected from the sample coupons by moistening a sterile polyester swab with 0·05-ml (50 μl) sterile de-ionized water, swabbing the sample surface by moving the swab back and forth across the surface with horizontal and vertical strokes covering the entire sample surface. After sample collection, the swab was placed in a prelabelled, sterile 50-ml Blue Falcon screw-top tube (Becton Dickinson Labware, Franklin Lakes, NJ, USA) containing 30·0 ml sterile BBT, and sealed with a cap. Samples were collected by two researchers, each collecting half of the samples. Following sample collection the aerosol deposition chamber was cleaned and sterilized using DF-200, a Sandia National Laboratories developed spore sterilizing agent (Tucker 2003; Tucker et al. 2003).

Extraction and enumeration

Spores were removed from the reference coupon and extracted from the sample swab using the process described in the Extraction Efficiency Methods Section of this paper.

As recovery efficiency values are calculated relative to the reference coupon count, the effectiveness of the spore removal process from the reference surface was also measured. The effectiveness of the sonication process for the removal of spores from the reference surface was evaluated for 24 reference coupons by seeding the coupons at approximately 200 000 CFU cm−2, sonicating in BBT for 15 min, heat treating at 65°C for 60 min, removing the coupon from the buffer, gently rinsing with de-ionized water, contact plating to Brain Heart Infusion agar, and incubating at 37°C for 24 h. Following incubation, colonies forming on the reference surface with distinct margins were counted by eye.

Calculations

Recovery efficiency was calculated as the number of CFU enumerated from the swab surface sample per unit area relative to the number of CFU enumerated from the co-located reference coupon per unit area. The mean recovery efficiency was calculated using the equation:

  • image(2)

where ηr is the mean recovery efficiency, Si is the average sample count for three replicates, Ri is the average reference count for three replicates, i is the co-located sample and reference number and n is the sample size.

Standard deviation, standard error and confidence interval were calculated using typical statistical methods for normally distributed data.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

The extraction efficiency using the sonication method from rayon swabs ranged from 0·532 to 0·942 with a mean extraction efficiency of 0·756 and an SD of ±0·118 (n = 20). The removal efficiency from the reference coupons was 0·999 with an SD of ±0·001 (n = 24).

Observed recovery efficiencies for the stainless steel surface ranged from 0·146 to 0·747 with a mean recovery efficiency of 0·414 and an SD of ±0·167 (n = 40). Differences in recovery efficiency were noted between low and high surface loading conditions. The mean recovery efficiency from stainless steel was 0·395 (n = 20) at low surface loading, and 0·429 (n = 20) at high surface loading. However, the differences were not statistically significant at the 0·05 significance level (P = 0·125). Recovery efficiency statistical analysis data for stainless steel are presented in Table 1.

Table 1.   Recovery efficiency statistics for rayon swabs from stainless steel
Surface loadingMeanMedianStandard deviationStandard errorRange95% Confidence interval
100–1000 CFU cm−2 (n = 20)0·3950·363±0·198±0·0460·151–0·6590·305–0·486
10 000–100 000 CFU cm−2 (= 20)0·4290·423±0·145±0·0310·146–0·7470·369–0·489
overall (n = 40)0·4140·395±0·167±0·0270·146–0·7470·362–0·466

The range of recovery efficiencies for the painted wallboard surface was wider at 0·030 to 0·887 producing a greater SD of ±0·232 about a mean of 0·405 (n = 40). Differences in recovery efficiency were also noted between low and high surface loading conditions for painted wallboard. The mean recovery efficiency for painted wallboard was 0·355 (n = 20) at low surface loading and 0·456 (n = 20) at high surface loading. However, the differences were not statistically significant at the 0·05 significance level (P = 0·497). Recovery efficiency statistical analysis data for painted wallboard are presented in Table 2.

Table 2.   Recovery efficiency statistics for rayon swabs from painted wallboard
Surface loadingMeanMedianStandard deviationStandard errorRange95% Confidence interval
100–1000 CFU cm−2 (n = 20)0·3550·293±0·239±0·0540·030–0·8870·250–0·460
10 000–100 000 CFU cm−2 (n = 20)0·4560·430±0·219±0·0490·072–0·8080·359–0·552
overall (n = 40)0·4050·375±0·232±0·0350·030–0·8870·336–0·474

No statistical difference in recovery efficiency means was noted between stainless steel and painted wallboard; however, the results of a Two-Sample F-Test of Variances indicate a significant difference in recovery efficiency variability between the stainless steel and painted wallboard surface sample types (P = 0·023).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

It must be reiterated that the spore material used in this study was a powdered form extensively designed to enhance aerosol suspension and inhalation characteristics, and the removal, extraction, and recovery characteristics of a different Bacillus species, native spore material, or spore material prepared using a different method may differ. It is recognized that the Bacillus species investigated in this study, B. atrophaeus, does not possess an exosporium such as that seen in B. anthracis; however, the reported sample recovery efficiency results provide valuable information for the interpretation of swab environmental sample analytical results. In addition, while the spore preparation contained nonspore material, there was no attempt to evaluate method efficiency in the presence of dust, bacterial vegetative cells, fungal spores, detritus, or other native background material which might interact with removal, extraction or plating efficiency.

Recovery efficiency for Bacillus spores from stainless steel surfaces using rayon swab collection media is reported in the literature. Rose et al. (2004) reported a mean recovery efficiency value for rayon swabs of 0·085 from stainless steel using sonic extraction, as compared with the 0·414 value observed in this study. The higher recovery efficiency reported in this study may be attributable to differences in surface seeding methods or spore matrix composition. Rose et al. (2004) used an ethanol liquid suspension of native purified B. anthracis Sterne spores applied to the surface and allowed to dry, whereas the B. atrophaeus spores in this study were applied to the surface as a dry powder. Upon evaporation of the ethanol from the liquid suspension, the spores may become fixed to the surface and consequently more difficult to remove by swab, as suggested by Rose et al. (2004), whereas spores applied to the surface in a dry powder form, specifically designed as a respirable aerosol, are more readily removed by the mechanical forces applied by swab action.

Results of this study also reveal a high variability in recovery efficiency values for the swab sample method using rayon swab collection material as evidenced by the relatively high SD values relative to the mean recovery efficiency for stainless steel at ±40·3%, and for painted wallboard at ±57·3%. This is consistent with previous research as Rose et al. (2004) reported SD values relative to the mean recovery efficiency of ±51·8% for rayon swabs from stainless steel surfaces. Angelotti et al. (1958) noted the generally low precision of swab sampling methods, and suggested that not only these methods are subject to errors inherent to the sampling mechanism itself, such as, swab material composition, surface composition, and mechanical removal action, but are also subject to collection and processing errors. Possible sources of collection and processing error contributing to low precision cited in the literature include operator collection technique, such as angle and pressure applied to surface, variations in extraction method, and processing errors, such as, pipetting and counting (Angelotti et al. 1958; Rose et al. 2004). Additional sources of error, specific to this study, are the potential for nonhomogeneous surface deposition of spore material resulting in unequal surface loading of reference and sample coupons, and potentially incomplete removal of spores from the reference coupon.

No statistical difference in recovery efficiency means was noted between stainless steel and painted wallboard; however, the results of a Two-Sample F-Test of Variances indicate a significant difference in recovery efficiency variability between the stainless steel and painted wallboard surface sample types (P = 0·023). While minor differences in porosity between stainless steel and painted wallboard may contribute to the recovery efficiency differential, differences in surface textural and physiochemical adhesive properties are more likely the cause.

For quantitative culture analysis, small aliquots of extraction suspension or serial dilutions are plated to growth medium, incubated and CFU counted. Using this analytical method, the quantitative limit of detection for rayon swabs was calculated from recovery efficiency values.

The following parameters were used for the limit of detection calculation: (i) at least 1 CFU ml−1 required for culture determination, and (ii) 10 ml of extraction suspension. While the surface area evaluated in this study was 25 cm2, the assumption is made that the number of CFU required for detection is independent of sample surface area and primarily a function of recovery efficiency.

If 1 CFU ml−1 is required in the extraction suspension for a positive culture, then 10 CFU are required in the total extraction suspension of 10 ml. Using the mean recovery efficiency values and the preceding assumptions, the estimated quantitative limit of detection per sample area for swabs is approximately 25 CFU for both stainless steel and painted wallboard surfaces. Thus, for a sample area of 25 cm2, a surface loading of approximately 1·0 CFU cm−2 is required to recover 1 CFU from swab samples. If smaller surface areas are sampled, a surface loading greater than 1·0 CFU cm−2 will be required to recover 1 CFU. Additionally, while the 1 CFU ml−1 detection requirement proposed by Buttner, et al. (2004) is theoretically detectable, under actual conditions more than 1 CFU ml−1 may be required for reliable culture which would increase the number of CFU required for detection. The 25 CFU detection limit also represents the method sensitivity, that is, an additional 25 CFU per sample area is required for each incremental increase in recovered CFU.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

The method efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling for a spore forming biological agent, such as, B. anthracis. While the swab sample collection method may be appropriate for small area sampling (10–25 cm2) with high agent concentration, other methods, such as, wipe or vacuum, may be more appropriate for large area sample collection with lower surface loading. The results of this study also provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure. This study also emphasizes the need for well-developed and validated procedures for the collection, extraction and analysis of biological environmental samples to provide the necessary level of confidence in information provided to public health decision makers.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References

Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000. This work was supported by the Department of Homeland Security under WFO-081030926. We thank Dr. Lloyd Larsen of the U.S. Army Dugway Proving Ground Life Science Division for generously providing the powdered B. atrophaeus spore material.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials
  5. Extraction efficiency methods
  6. Recovery efficiency methods
  7. Results
  8. Discussion
  9. Conclusions
  10. Acknowledgements
  11. References
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