Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line

Aim: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean‐in‐place regimes. Methods and Results: Exposing sessile spores to 1% NaOH at 65°C for 30 min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from ?20 to ?32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l^?1 KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log₁₀ CFU cm^?2) than untreated spores (3·6 log₁₀ CFU cm^?2). Conclusions: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. Significance of Study: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.     

Bacterial endospore inactivation caused by outgassing of vapourous hydrogen peroxide from polymethyl methacrylate (Plexiglas)

AIMS: To investigate the cause and to eliminate the inactivation of Bacillus anthracis strain Sterne spores settled onto agar and stainless steel surfaces in plastic holders. METHODS AND RESULTS: In an experimental chamber in which spores settled onto sampling surfaces, vapourous hydrogen peroxide (VHP) was used for decontamination between experiments. It was demonstrated that hydrogen peroxide (H(2)O(2)) absorbed into plastic (Plexiglas) surfaces and could outgas in the sample holders. Further experiments demonstrated that H(2)O(2) was released from Plexiglas sample holders in sufficient quantity to inactivate spores. High temperature degassing (30-35 degrees C) for several days or aluminum coating of the surfaces were two remedies found to be effective in preventing inadvertent spore inactivation. CONCLUSIONS: H(2)O(2) can be absorbed into plastic and released after an extended period of time (weeks), allowing a sufficient concentration to accumulate in small volumes to inactivate spores. Outgassing the plastic or coating the surface with an impermeable layer are potential solutions to reduce spore inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Many studies with bacilli and other organisms are carried out using small plastic containers that may have been sterilized using H(2)O(2) or other agents. This study presents a cautionary note to ensure elimination of H(2)O(2) or other sterilizing agents to prevent spurious results.     

Influence of Spore Moisture Content on the Dry-Heat Resistance of Bacillus subtilis var. niger

The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (z(D) = 21 C), but an unusually low z for spores on paper (z(D) = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (z(D) = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (a(w)) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below a(w) 0.2 or above a(w) 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature.     

Reaerosolization of fluidized spores in ventilation systems

This project examined dry, fluidized spore reaerosolization in a heating, ventilating, and air conditioning duct system. Experiments using spores of Bacillus atrophaeus, a nonpathogenic surrogate for Bacillus anthracis, were conducted to delineate the extent of spore reaerosolization behavior under normal indoor airflow conditions. Short-term (five air-volume exchanges), long-term (up to 21,000 air-volume exchanges), and cycled (on-off) reaerosolization tests were conducted using two common duct materials. Spores were released into the test apparatus in turbulent airflow (Reynolds number, 26,000). After the initial pulse of spores (approximately 10(10) to 10(11) viable spores) was released, high-efficiency particulate air filters were added to the air intake. Airflow was again used to perturb the spores that had previously deposited onto the duct. Resuspension rates on both steel and plastic duct materials were between 10(-3) and 10(-5) per second, which decreased to 10 times less than initial rates within 30 min. Pulsed flow caused an initial spike in spore resuspension concentration that rapidly decreased. The resuspension rates were greater than those predicted by resuspension models for contamination in the environment, a result attributed to surface roughness differences. There was no difference between spore reaerosolization from metal and that from plastic duct surfaces over 5 hours of constant airflow. The spores that deposited onto the duct remained a persistent source of contamination over a period of several hours.     

Reaerosolization of Fluidized Spores in Ventilation Systems

This project examined dry, fluidized spore reaerosolization in a heating, ventilating, and air conditioning duct system. Experiments using spores of Bacillus atrophaeus, a nonpathogenic surrogate for Bacillus anthracis, were conducted to delineate the extent of spore reaerosolization behavior under normal indoor airflow conditions. Short-term (five air-volume exchanges), long-term (up to 21,000 air-volume exchanges), and cycled (on-off) reaerosolization tests were conducted using two common duct materials. Spores were released into the test apparatus in turbulent airflow (Reynolds number, 26,000). After the initial pulse of spores (approximately 10¹⁰ to 10¹¹ viable spores) was released, high-efficiency particulate air filters were added to the air intake. Airflow was again used to perturb the spores that had previously deposited onto the duct. Resuspension rates on both steel and plastic duct materials were between 10⁻³ and 10⁻⁵ per second, which decreased to 10 times less than initial rates within 30 min. Pulsed flow caused an initial spike in spore resuspension concentration that rapidly decreased. The resuspension rates were greater than those predicted by resuspension models for contamination in the environment, a result attributed to surface roughness differences. There was no difference between spore reaerosolization from metal and that from plastic duct surfaces over 5 hours of constant airflow. The spores that deposited onto the duct remained a persistent source of contamination over a period of several hours.     

Evaluation of a wipe surface sample method for collection of Bacillus spores from nonporous surfaces

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of +/-0.12, and for painted wallboard it was 0.29 with a standard deviation of +/-0.15. Evaluation of a sonication extraction method for the polyester-rayon blend wipes produced a mean extraction efficiency of 0.93 with a standard deviation of +/-0.09. Wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel sample area and 105 CFU per unit of painted wallboard sample area. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling following the release of a biological agent such as Bacillus anthracis.     

Development of an aerosol surface inoculation method for bacillus spores

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.     

Evaluation of a Wipe Surface Sample Method for Collection of Bacillus Spores from Nonporous Surfaces

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of ±0.12, and for painted wallboard it was 0.29 with a standard deviation of ±0.15. Evaluation of a sonication extraction method for the polyester-rayon blend wipes produced a mean extraction efficiency of 0.93 with a standard deviation of ±0.09. Wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel sample area and 105 CFU per unit of painted wallboard sample area. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling following the release of a biological agent such as Bacillus anthracis.     

电解海水的抑菌活性及对食品加工表面材料的消毒效果

为了考察直接电解海水消除细菌污染的可能性,本文将海水及海水稀释成不同浓度后通过氧化电解水装置进行电解不同时间后,所得酸性电解海水、碱性电解海水和中性电解海水对病原菌[埃希氏大肠杆菌(Escherichina coli)、沙门氏菌(Salmonella)、单核细胞增生李斯特菌(Listeria moncytogene)、摩化摩根(Morganella morganii)、副溶血性弧菌(Vibrio parahaemolyticus)]以及食品加工表面接触材料(地板砖、不锈钢板、瓷砖、手套、抹布)的消毒效果进行分析研究.结果表明,酸性电解海水具有良好的杀菌效果,能将107 CFU/mL的病原菌悬液在1 min内几乎全部杀死.碱性电解海水和中性水无明显的杀菌效果.通过模拟食品加工过程,对食品加工表面接触材料人为染菌,研究电解海水对表面材料的消毒效果,结果表明酸性电解海水仍能将表面材料含有的107CFU/cm2病原菌在5 min之内几乎全部杀灭.由此说明电解海水对食品加工表面接触材料具有明显的消毒效果,能取代以淡水为原料的电解水杀菌效果是高效廉价和不浪费淡水资源的一种理想消毒剂.     

Contribution of Spores to the Ability of Clostridium difficile To Adhere to Surfaces

Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student''s t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species.     

Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel

Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis.     

Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces

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.     

Influence of physicochemical properties on the hygienic status of stainless steel with various finishes

The relative hygienic status of 16 stainless steel surfaces, characterised by topography and surface free energy was investigated. B. thuringiensis spores suspended in Bechamel sauce was chosen as the test fouling suspension. Surface topography was assessed using 10 standardised roughness parameters, along with scanning electron microscope observations. The number of residual adhering spores after a fouling and cleaning in place procedure was found to be influenced by the topography of the stainless steel surface, but not by the surface free energy. Among the various roughness parameters, R_A, R_RR R_PK and R_VK were shown to be related to the hygienic status. Microscopic observations demonstrated the influence of the shape and size of surface irregularities on the level of residual soil after cleaning. This confirms that the use of only one roughness parameter, usually R_A, is not sufficient in defining the hygienic status of stainless steel surfaces.     

Sporicidal effect of disinfectants on Bacillus cereus isolated from the milk processing environment

The sporicidal efficacy of sodium hypochlorite and a combination of peracetic acid and hydrogen peroxide on Bacillus cereus spores isolated from the milk processing environment was examined using the European Suspension Test and by a surface disinfection test on stainless steel and rubber. The results of the laboratory tests were compared with field trials in a milking installation. In general, it was difficult to obtain consistent results, as the repeatability and reproducibility of the tests were found to vary according to the test strain, spore suspension preparation, disinfectant test solution, organic load, contact time and temperature. The sporulation medium used to obtain spores influenced the sporicidal effect considerably. To overcome this problem a standard method for preparation of spore suspensions should be prescribed. The various disinfectants were more effective in suspensions than on surfaces and in field trials. For the suspension tests SE values ranging from 1.0 to 3.0 were reached within 10 min at 50°C, depending on the disinfectant used. Sodium hypochlorite-based products were most effective. The activity on spores on surfaces and in field trials was limited. In surface tests reductions of 0.4–0.8 were observed within 10 min at 50°C depending on the type of surface. The SE values obtained for rubber were lower compared with stainless steel. The decrease in spore levels found in the milking installation was comparable with the surface experiments, i.e. 0.4–1.0. It is important to develop standard test procedures to assess the sporicidal efficacy of disinfectants used in food hygiene. Surface tests should be included to reflect the in-use conditions more closely and minimum standards should be determined for both suspension tests and surface tests.     

Thermal Death of Bacillus subtilis var. niger Spores on Selected Lander Capsule Surfaces

Dry-heat sterilization of planetary lander capsules requires a knowledge of the thermal resistivity of microorganisms in the environment to which they will be subjected during sterilization of the space hardware. The dry-heat resistance of Bacillus subtilis var. niger spores on various lander capsule materials was determined at 125 C. Eight surface materials were evaluated, including a reference material, stainless steel. Survivor curves were computed, and decimal reduction times (D values) were obtained by a linear regression analysis. In four tests on stainless steel, the average value of D at 125 C was 17.07 min. The D values for the other seven materials tested ranged from 18.64 min on magnesium surfaces to 20.83 min on conversion-coated magnesium. Of the materials evaluated, the results indicate that there is only a significant difference in the thermal resistance of B. subtilis var. niger spores on conversion-coated magnesium and conversion-coated aluminum from that on the reference material, stainless steel. The differences in D values for all the test surfaces may be the result of variations in test procedures rather than the effect of the surfaces on the thermal resistivity of the spores.     

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra

Aims: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. Materials and Results: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair‐like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH‐sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore’s negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. Conclusions: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. Significance and Impact of the Study: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.     

Transport efficiency and deposition velocity of fluidized spores in ventilation ducts

Experiments with dry, fluidized spores were conducted in a test apparatus to delineate the extent of spore contamination and deposition behavior under normal airflow conditions within a ventilation system. The surrogate biological warfare agent used in experiments was the spore-forming bacterium Bacillus atrophaeus. Viable-spore-counting methods were used in the study because they provide the most important number for estimating human health effects. Three common ventilation duct materials were evaluated: flexible plastic, galvanized steel, and internally insulated fiberglass. Transport efficiency ranged from 9 to 13% in steel and fiberglass ducts; transport efficiency was far less (0.1–4%) in plastic duct. Results showed that the deposition of surrogate biological warfare agent was significantly different in the three duct materials evaluated. All experimentally determined, dimensionless deposition velocities were in the range of theoretical predictions for dimensionless roughness, k ⁺=10. All were 10–100 times greater than the velocities predicted for ducts with smoother surfaces, k ⁺=0.1. For plastic duct, greater dimensionless deposition velocities were likely the result of charge forces between spores and surface. However, for the steel duct, a relatively large dimensionless deposition velocity was unexpected. These findings imply that building contamination will likely vary, depending on the specific type of duct material used throughout an affected area. Results of this study may aid in refining existing particle-transport models and remediation activities.     

Recovery of Spores from Thermophilic Dairy Bacilli and Effects of Their Surface Characteristics on Attachment to Different Surfaces

Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of ~10⁷ spores ml⁻¹; spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from −10 to −20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 × 10⁴ spores cm⁻²), with fewer spores attaching to glass (3 × 10³ spores cm⁻²). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (~1 × 10³ spores cm⁻²), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.     

Implications of paper vs stainless steel biological indicator substrates for formaldehyde gas decontamination

Aims: This study was undertaken to determine the effectiveness of biological indicators currently being employed during formaldehyde decontamination. Data suggest that detectable amounts of formaldehyde are absorbed into the paper strips contained in currently used biological indicators. Absorbed formaldehyde has the potential to inhibit the growth of indicator spores, thus leading to false negative results. Indicators composed of either stainless steel carriers or paper strips were investigated to determine whether stainless steel carriers can be used as an alternative to paper strip indicators. Methods and Results: Biological indicators were exposed to formaldehyde gas and were tested for the presence of formaldehyde and any possible inhibition of spore growth. Absorbed formaldehyde was detected in the paper strip carriers while no formaldehyde was detected from any of the stainless steel carriers. Exposed paper strips were found to inhibit growth of up to 1 × 10⁶ spores while the stainless steel carriers did not inhibit the growth of spores. Conclusions: During decontamination, biological indicators composed of paper spore strips absorb formaldehyde and inhibit growth of any surviving spores. Stainless steel carriers do not absorb formaldehyde and are an ideal alternative substrate for biological indicators. Significance and Impact of the Study: The popular paper strip biological indicator can lead to false negative results during decontamination and is unsuitable for validating formaldehyde decontamination.     

Recovery Efficiency and Limit of Detection of Aerosolized Bacillus anthracis Sterne from Environmental Surface Samples

After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm²). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm²) or wipe or vacuum (929 cm²) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm²) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm² for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm² for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.Anthrax, the spectrum of diseases caused by infection with Bacillus anthracis, is not considered a communicable disease but is generally acquired via environmental exposures. Many anthrax cases through history have been the result of agricultural or industrial exposure to B. anthracis spores (33). The disease most often presents itself as a cutaneous infection; however, there are both gastrointestinal and inhalational forms of the disease. Inhalational anthrax is typically rapidly fatal, even with treatment. In general, inhalation exposures require specific conditions, such as poor ventilation and activities that disturb dust containing B. anthracis spores (13).Because diagnosing anthrax in its early stages in human and animal hosts is difficult and B. anthracis spores are extremely stable in the environment, this microorganism has been investigated, developed, and deployed as a biological weapon throughout the 20th century. Use of this microorganism has seen varied success during World War I (9) and subsequently. It is generally accepted that there was an accidental release of B. anthracis spores from a weapons manufacturing or development facility in 1979 in Sverdlovsk, USSR (now Yekaterinaburg, Russia) (10, 26). In 1993, an attempt by a civilian group, Aum Shinrikyo, to use this microorganism to attack a civilian population in a Tokyo suburb did not result in any casualties (22, 28).In 2001, envelopes containing a powder formulation of B. anthracis were mailed in the United States to several individuals. These letters were the presumed cause of 22 cases of clinical anthrax, 11 inhalational and 11 cutaneous, with 5 fatalities, all of whom suffered from inhalational disease (34). According to congressional testimony, the powdered spore suspension was “easily dispersed into the air” (29). Of the 11 individuals with inhalational disease, 2 had no history of handling mail or having any other direct contact with these threat letters (11, 21). Of the remaining nine individuals, eight were thought to have been exposed through handling or processing mail (20) but may never have picked up or directly handled the actual threat letters. Thus, some individuals who contracted inhalational disease may have been exposed to aerosols that were generated from residual spore material deposited on contaminated surfaces. This conclusion was borne out by a study conducted on the scene of one contamination incident, which demonstrated that spores could be reaerosolized from surfaces during simulated office activities—e.g., paper handling, foot traffic, moving containers—after a period of no entry and no ventilation for several days (38). McCleery et al. (25) found that reaerosolization of spores is possible in postal facilities.In the mail-related instance of 2001, aerosol exposures occurred. Since spore-contaminated surfaces can become sources for aerosol generation, nonporous surfaces (walls, desks, lockers, etc.) were decontaminated to reduce risk while porous surfaces (draperies and sofas) were removed. To determine the efficacy of decontamination, contaminated buildings were first sampled for the presence of B. anthracis spores followed by treatment by a variety of techniques. Postdecontamination sampling was used to determine efficacy (37) and to assess the safety for reoccupancy.The Government Accountability Office (GAO) reported that additional methodological validation of sampling collection and analytical methods should be conducted to enhance the interpretation of negative sampling results because initial samples from two postal facilities were negative, but later samples were positive (17). The GAO (17) report defined validation as “… a formal and independently administered empirical process. For validation, the overall performance characteristics of a given method must be certified as meeting the specified requirements for intended use and as conforming with applicable standards.” Currently, there is no preexisting standard for a presumable safe level of surface contamination with B. anthracis spores that may be assessed through sampling and analysis.Development of independent standards for assessing the requirements for surface sampling methods requires an understanding of the rate at which spores leave surfaces to become entrained in aerosols, the potential for aerosol exposure by humans, and the infectivity of inhaled spores. Inhalation infectivity has been researched, but estimates of a lethal dose vary (14, 15). Bartrand et al. (5) conducted a risk analysis on the mortality of guinea pigs and rhesus monkeys exposed to B. anthracis spores and found a 50% lethal dose (LD₅₀; i.e., the dose at which 50% of subjects die) of about 100,000 spores inhaled for 1-μm particles. Limitations of relating exposure to inhalation infectivity include quantification of the ability of spores to move from stasis on a surface to entrainment as an aerosol, quantification of exposures to the resultant aerosol, uptake by humans, room size and ventilation characteristics, and exposure time. Despite these limitations, it is necessary to standardize the performance of surface sampling methods.Brown et al. evaluated wipe (6), swab (7), and vacuum (8) spore collection methods with B. atrophaeus. These studies have added significant information to the understanding of recovery efficiencies for these three sampling methods; however, sampling performance was not evaluated at very low spore surface loading concentrations. Sampling performance measures at very low surface loading of B. anthracis are needed to aid in the decision making for decontamination and other interventions (31, 38).The goal of this study was to evaluate the current CDC environmental surface sampling methods for B. anthracis (12) as slightly modified based on subsequent CDC research (19, 30). We estimated B. anthracis Sterne sampling limit of detection (LOD), recovery efficiency (RE), and measurement precision for three sampling methods (swab, wipe, and vacuum) and two surfaces (steel and carpet) by allowing spores to settle from an aerosol in a controlled environment. In addition, we compared sample analyses performed at three laboratories to determine the level of interlaboratory variability.