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At least 19 records

The spore coat is essential for Bacillus subtilis spore resistance to pulsed light, and pulsed light treatment eliminates some spore coat proteins

Microbial surface contamination of equipment or of food contact material is a recurring problem in the food industry. Spore-forming bacteria are far more resistant to a wide variety of treatments than their vegetative forms. Understanding the mechanisms underlying decontamination processes is needed to improve surface decontamination strategies against endospores potentially at the source of foodborne diseases or food-spoilage. Pulsed light (PL) with xenon lamps delivers high-energy short-time pulses of light with wavelengths in the range 200 nm-1100 nm and a high UV-C fraction. Bacillus subtilis spores were exposed to either PL or to continuous UV-C. Gel electrophoresis and western blotting revealed elimination of various proteins of the spore coat, an essential outer structure that protects spores from a wide variety of environmental conditions and inactivation treatments. Proteomic analysis confirmed the elimination of some spore coat proteins after PL treatment. Transmission electron microscopy of PL treated spores revealed a gap between the lamellar inner spore coat and the outer spore coat. Overall, spores of mutant strains with defects in genes coding for spore coat proteins were more sensitive to PL than to continuous UV-C. This study demonstrates that radiations delivered by PL contribute to specific damage to the spore coat, and overall to spore inactivation. Keywords Decontamination, UV, proteins, proteomics, microscopy Journal Pre-proof

59 BASIC BIOLOGICAL SCIENCES↗

Comparison of Bacillus atrophaeus spore viability following exposure to detonation of C4 and to deflagration of halogen-containing thermites

Energetic materials are being considered for the neutralization of spore-forming bacteria. In this study, the neutralization effects of a monomolecular explosive were compared to the effects of halogen-containing thermites. Bacillus atrophaeus spores were exposed to the post-detonation environment of a 100 g charge of the military explosive C-4 at a range of 50 cm. These tests were performed in the thermodynamically closed environment of a 506-l barometric calorimeter. Associated temperatures were calculated using a thermodynamic model informed by calculations with the Cheetah thermochemical code. Temperatures in the range of 2300–2800 K were calculated to persist for nearly the full 4 ms pressure observation time. After the detonation event, spores were characterized using optical microscopy and the number of viable spores was assessed. Results showed live spore survival rates in the range of 0.01%–1%. For the thermite tests, a similar, smaller-scale configuration was employed that examined the spore neutralization effects of two thermites: aluminum with iodine pentoxide and aluminum with potassium chlorate. Only the former mixture resulted in spore neutralization. These results indicate that the detonation environment produced by an explosive with no chemical biocides may provide effective spore neutralization similar to a deflagrating thermite containing iodine.

36 MATERIALS SCIENCE↗

Computational Fluid Dynamics Modeling of Bacillus anthracis Spore Deposition in Rabbit and Human Respiratory Airways

Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.

bacillus anthracis↗

Ice Nucleation of Fungal Spores from the Classes Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes, and the effect on the Atmospheric Transport of these Spores

Ice nucleation on fungal spores may affect the frequency and properties of ice and mixed-phase clouds. We studied the ice nucleation properties of 12 different species of fungal spores chosen from three classes: Agaricomycetes, Ustilagomycetes, and Eurotiomycetes. Agaricomycetes include many types of mushroom species and are cosmopolitan all over the globe. Ustilagomycetes are agricultural pathogens and have caused widespread damage to crops. Eurotiomycetes are found on all types of decaying material and include important human allergens. We focused on these classes since they are thought to be abundant in the atmosphere and because there is very little information on the ice nucleation ability of these classes of spores in the literature. All of the fungal spores investigated were found to cause freezing of water droplets at temperatures warmer than homogeneous freezing. The cumulative number of ice nuclei per spore was 0.001 at temperatures between -19 °C and -29 °C, 0.01 between -25.5 °C and -31 °C, and 0.1 between -26 °C and -36 °C. On average, the order of ice nucleating ability for these spores is Ustilagomycetes > Agaricomycetes ≅ Eurotiomycetes. We show that at temperatures below -20 °C, all of the fungal spores studied here are less efficient ice nuclei compared to Asian mineral dust on a per surface area basis. We used our new freezing results together with data in the literature to compare the freezing temperatures of spores from the phyla Basidiomycota and Ascomycota, which together make up 98 % of known fungal species found on Earth. The data show that within both phyla (Ascomycota and Basidiomycota) there is a wide range of freezing properties, and also that the variation within a phylum is greater than the variation between the average freezing properties of the phyla. Using a global chemistry-climate transport model, we investigated whether ice nucleation on the studied spores, followed by precipitation, can influence the atmospheric transport and global distributions of these spores in the atmosphere. Simulations show that inclusion of ice nucleation scavenging of fungal spores in mixed-phase clouds can decrease the surface annual mean mixing ratios of fungal spores over the oceans and polar regions and decrease annual mean mixing ratios in the upper troposphere.

fungal spores↗

High-Resolution Spore Coat Architecture and Assembly of Bacillus Spores

Elucidating the molecular architecture of bacterial and cellular surfaces and its structural dynamics is essential to understanding mechanisms of pathogenesis, immune response, physicochemical interactions, environmental resistance, and provide the means for identifying spore formulation and processing attributes. I will discuss the application of in vitro atomic force microscopy (AFM) for studies of high-resolution coat architecture and assembly of several Bacillus spore species. We have demonstrated that bacterial spore coat structures are phylogenetically and growth medium determined. We have proposed that strikingly different species-dependent coat structures of bacterial spore species are a consequence of sporulation media-dependent nucleation and crystallization mechanisms that regulate the assembly of the outer spore coat. Spore coat layers were found to exhibit screw dislocations and two-dimensional nuclei typically observed on inorganic and macromolecular crystals. This presents the first case of non-mineral crystal growth patterns being revealed for a biological organism, which provides an unexpected example of nature exploiting fundamental materials science mechanisms for the morphogenetic control of biological ultrastructures. We have discovered and validated, distinctive formulation-specific high-resolution structural spore coat and dimensional signatures of B. anthracis spores (Sterne strain) grown in different formulation condition. We further demonstrated that measurement of the dimensional characteristics of B. anthracis spores provides formulation classification and sample matching with high sensitivity and specificity. I will present data on the development of an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures on the B. anthracis surfaces. These studies demonstrate that AFM can probe microbial surface architecture, environmental dynamics and the life cycle of bacterial and cellular systems at near-molecular resolution under physiological conditions.

59 BASIC BIOLOGICAL SCIENCES↗

Rupturing of Biological Spores As a Source of Secondary Particles in Amazonia

Airborne biological particles, such as fungal spores and pollen, are ubiquitous in the Earth’s atmosphere and play an important role in the atmospheric environment and climate, impacting air quality, cloud formation, and the Earth’s radiation budget. The atmospheric transformations of airborne biological spores at elevated relative humidity remain poorly understood and their climatic role is uncertain. Using an environmental scanning electron microscope (ESEM), we observed rupturing of Amazonian fungal spores and subsequent release of nanometer to submicron size fragments after exposure to high humidity. We find that fungal fragments contain elements of inorganic salts (e.g., Na and Cl). They are hygroscopic in nature with a growth factor up to 2.3 at 96% relative humidity, thus they may potentially influence cloud formation. Due to their hygroscopic growth, light scattering cross sections of the fragments are enhanced by up to a factor of 10. Furthermore, rupturing of fungal spores at high humidity may explain the bursting events of nanoparticles and may provide insight into new particle formation in Amazonia.

Environmental Molecular Sciences Laboratory↗

A microfluidic spore chamber for long-term imaging of single-spore hyphal development.

Understanding the life cycle of fungal spores is essential for elucidating their roles in pathogenesis, dispersal, and survival. However, studying spore development under controlled, spatially defined conditions remains challenging. Here, we present the Spore Chamber, a custom-built microfluidic platform engineered for parallel trapping and long-term imaging of individual spores under defined media conditions, enabling real-time visualization of hyphal development. Using Aspergillus fumigatus as a model organism, we demonstrate that sparse trapping of individual spores within size-matched trap geometries enables long-term time-lapse imaging of key developmental stages, including germination, polarized hyphal elongation, branching, and conidiophore formation. To assess the device's capacity to resolve morphogenetic responses to exogenous signals, we introduced lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (COs). Rhizobium-derived, non-sulfated LCO (nsLCO) mixtures induced enhanced secondary branching (hyperbranching), a response not previously reported in A. fumigatus under these signal conditions, to our knowledge, whereas sulfated LCOs and CO4 did not significantly alter branching patterns. In addition, long-term confinement and imaging revealed rare developmental morphologies previously described primarily in mutant strains, including split conidiophore formation, elongated phialides, and stress-associated phenomena such as microcyclic conidiation, and chlamydospore development. Together, these results establish the Spore Chamber as a targeted microfluidic platform for single-spore phenotyping and long-term developmental analysis, with applications in fungal biology, chemical signaling studies, and host–microbe interaction research.

Antifungal screening↗

Dataset for "A Microfluidic Spore Chamber for Long-Term Imaging of Single-Spore Hyphal Development"

Understanding the life cycle of fungal spores is essential for elucidating their roles in pathogenesis, dispersal, and survival. However, studying spore development under controlled, spatially defined conditions remains challenging. Here, we present the Spore Chamber, a custom-built microfluidic platform engineered for parallel trapping and long-term imaging of individual spores under defined media conditions, enabling real-time visualization of hyphal development. Using Aspergillus fumigatus as a model organism, we demonstrate that sparse trapping of individual spores within size-matched trap geometries enables long-term time-lapse imaging of key developmental stages, including germination, polarized hyphal elongation, branching, and conidiophore formation. To assess the device’s capacity to resolve morphogenetic responses to exogenous signals, we introduced lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (COs). Rhizobium-derived, non-sulfated LCO (nsLCO) mixtures induced enhanced secondary branching (hyperbranching), a response not previously reported in A. fumigatus under these signal conditions, to our knowledge, whereas sulfated LCOs and CO4 did not significantly alter branching patterns. In addition, long-term confinement and imaging revealed rare developmental morphologies previously described primarily in mutant strains, including split conidiophore formation, elongated phialides, microcyclic conidiation, and chlamydospore development. Together, these results establish the Spore Chamber as a targeted microfluidic platform for single-spore phenotyping and long-term developmental analysis, with applications in fungal biology, chemical signaling studies, and host–microbe interaction research. Videos of the observed phenomena are included in this data set.

59 BASIC BIOLOGICAL SCIENCES↗

Scalable fabrication of a tough and recyclable spore-bearing biocomposite thermoplastic polyurethane

Thermoplastic polyurethanes (TPUs) are a class of versatile thermoplastic elastomers, but most of their products lack a proper recycling strategy or have no end-of-life solutions. To pursue a sustainable end-of-life solution for TPU-based products, self-disintegrating biocomposite TPUs have recently been developed by embedding spores of TPU-degrading bacteria into TPUs via melt extrusion. Herein, we improve upon spore-bearing biocomposites and demonstrate industrially relevant manufacturing conditions for fabricating biocomposite TPUs. To minimize the coloration of biocomposite TPUs, spore production was modified. The innate brown color of the resulting materials was diminished by reducing FeSO 4 in sporulation media, generating white spores without compromising spore productivity, viability, morphology or heat-shock tolerance. Biocomposite TPUs containing white spores displayed a 45 % increase in toughness compared to TPUs without spores, while retaining ∼ 90 % spore viability post processing. Furthermore, biocomposite TPU fabrication was demonstrated using a scalable continuous extruder followed by injection molding. Biocomposite TPUs generated by these industry-relevant processes exhibited comparable toughness improvement and spore viability to biocomposite TPU prepared using a lab scale microcompounder, while enhancing productivity by 30-fold. Finally, spore addition significantly improved the recyclability of biocomposite TPUs, enabling 80 % toughness retention after 5 rounds of iterative melt processing. Additionally, no negative effect on the lifespan of the generated TPUs was observed over 1 year of storage. Overall, this study confirms that spore-bearing biocomposite TPUs are promising for practical applications, offering an accessible method to enhance toughness and sustainability of commercial TPUs through the incorporation of spore-based living fillers.

36 MATERIALS SCIENCE↗

Optical and structural properties of plasma-treated Cordyceps bassiana spores as studied by circular dichroism, absorption, and fluorescence spectroscopy

To understand the killing mechanism of fungal spores by plasma treatment, the optical, structural, and biological properties of the insect pathogenic fungus Cordyceps bassiana spores were studied. A nonthermal atmospheric-pressure plasma jet (APPJ) was used to treat the spores in aqueous solution. Optical emission spectra of the APPJ acquired in air indicated emission peaks corresponding to hydroxyl radicals and atomic oxygen. When the APPJ entered the aqueous solution, additional reactive species were derived from the interaction of plasma radicals with the aqueous solution. Fluorescence and absorption spectroscopy confirmed the generation of hydroxyl radicals and hydrogen peroxide in the plasma-activated water (PAW). Spore counting showed that plasma treatment significantly reduced spore viability. Absorption spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis of the DNA extracted from plasma-treated spores showed a reduction in spore DNA content. The magnitude of the dip in the CD spectrum was lower in the plasma-treated spores than in the control, indicating that plasma treatment causes structural modifications and/or damage to cellular components. Tryptophan fluorescence intensity was lower in the plasma-treated spores than in the control, suggesting that plasma treatment modified cell wall proteins. Changes in spore viability and DNA content were attributed to structural modification of the cell wall by reactive species coming from the APPJ and the PAW. Our results provided evidence that the plasma radicals and the derived reactive species play critical roles in fungal spore inactivation.

60 APPLIED LIFE SCIENCES↗

A Stochastic Intracellular Model of Anthrax Infection With Spore Germination Heterogeneity

We present a stochastic mathematical model of the intracellular infection dynamics of Bacillus anthracis in macrophages. Following inhalation of B. anthracis spores, these are ingested by alveolar phagocytes. Ingested spores then begin to germinate and divide intracellularly. This can lead to the eventual death of the host cell and the extracellular release of bacterial progeny. Some macrophages successfully eliminate the intracellular bacteria and will recover. Here, a stochastic birth-and-death process with catastrophe is proposed, which includes the mechanism of spore germination and maturation of B. anthracis . The resulting model is used to explore the potential for heterogeneity in the spore germination rate, with the consideration of two extreme cases for the rate distribution: continuous Gaussian and discrete Bernoulli. We make use of approximate Bayesian computation to calibrate our model using experimental measurements from in vitro infection of murine peritoneal macrophages with spores of the Sterne 34F2 strain of B. anthracis . The calibrated stochastic model allows us to compute the probability of rupture, mean time to rupture, and rupture size distribution, of a macrophage that has been infected with one spore. We also obtain the mean spore and bacterial loads over time for a population of cells, each assumed to be initially infected with a single spore. Our results support the existence of significant heterogeneity in the germination rate, with a subset of spores expected to germinate much later than the majority. Furthermore, in agreement with experimental evidence, our results suggest that most of the spores taken up by macrophages are likely to be eliminated by the host cell, but a few germinated spores may survive phagocytosis and lead to the death of the infected cell. Finally, we discuss how this stochastic modelling approach, together with dose-response data, allows us to quantify and predict individual infection risk following exposure.

59 BASIC BIOLOGICAL SCIENCES↗

Fungal Spore Seasons Advanced Across the US Over Two Decades of Climate Change

Abstract Phenological shifts due to climate change have been extensively studied in plants and animals. Yet, the responses of fungal spores—organisms important to ecosystems and major airborne allergens—remain understudied. This knowledge gap limits our understanding of their ecological and public health implications. To address this, we analyzed a long‐term (2003–2022), large‐scale (the continental US) data set of airborne fungal spores collected by the US National Allergy Bureau. We first pre‐processed the spore data by gap‐filling and smoothing. Afterward, we extracted 10 metrics describing the phenology (e.g., start and end of season) and intensity (e.g., peak concentration and integral) of fungal spore seasons. These metrics were derived using two complementary but not mutually exclusive approaches—ecological and public health approaches, defined as percentiles of total spore concentration and allergenic thresholds of spore concentration, respectively. Using linear mixed‐effects models, we quantified annual shifts in these metrics across the continental US. We revealed a significant advancement in the onset of the spore seasons defined in both ecological (11 days, 95% confidence interval: 0.4–23 days) and public health (22 days, 6–38 days) approaches over two decades. Meanwhile, total spore concentrations in an annual cycle and in a spore allergy season tended to decrease over time. The earlier start of the spore season was significantly correlated with climatic variables, such as warmer temperatures and altered precipitations. Overall, our findings suggest possible climate‐driven advanced fungal spore seasons, highlighting the importance of climate change mitigation and adaptation in public health decision‐making.

Environmental Sciences & Ecology↗

Advancing Protein Display on Bacterial Spores through an Extensive Survey of Coat Components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 types of proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SPs investigated, 26 formed functional fusions with β-glucuronidase (GUS)─a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed 1) SP promoter activation dynamics, 2) GUS activity on spores, 3) surface availability, and 4) protection from thermal and proteolytic degradation. Multicopy expression and pairwise coexpression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and the difficulty in predicting compatibility between different SP fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface-exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (small spore coat assembly protein A) as an effective spore display carrier. SscA supported enzyme activity at least 4-fold higher than any other SP, including the well-established anchor, CotY. We attribute this to its promoter, which demonstrated early and sustained activation relative to other SPs and its small size (∼3 kDa), which likely minimally interferes with enzyme folding, oligomerization, and activity. Labeling and genetic studies, its hydrophobic nature, and low surface availability suggest that SscA assembles within the inner spore coat, which makes it stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledge base to advance the biotechnological utility of B. subtilis spores.

Bacillus subtilis↗

Development of a high-throughput method for processing sponge-stick samples to detect viable Bacillus anthracis spores

Since the national validation of the sponge-stick based method for detection of Bacillus anthracis spores in environmental samples, there have not been focused efforts to address the low throughput nature of the method, which processes only one sample at one time. Sample processing remains a serious bottleneck for rapidly analyzing large numbers of samples expected from a biological warfare attack. Therefore, we developed a high-throughput method to simultaneously process multiple sponge-stick samples to be better prepared for rapid response and recovery after wide area anthrax incidents. In this method, sponges are placed in 50 mL tubes containing 25 mL extraction buffer and shaken to release spores, after which the suspension is recovered for analysis. Here, we determined that an additional extraction step, conducted in the same tubes with 10 mL buffer, further increased spore recovery from sponge-stick by approximately 10 %. We determined that orbital shaking and multi-tube vortexing were both more effective than reciprocating shaking for recovering spores. We conducted simultaneous processing of up to 12 sponge-stick samples and demonstrated comparable spore recovery efficiencies to the traditional low-throughput stomacher-based method (approximately 60 % recovery at 10 2 -spore level and 75 % recovery at 10 4 -spore level for both methods in three replicate experiments, P > 0.05 for two-tailed t-tests for each experiment and spore level). We also demonstrated that our high-throughput method could be integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis and could detect levels as low as 40 spores per sponge even when challenged by a PCR particulate contaminant.

Anthrax↗

Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores

Abstract Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40–72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20 min) and lysozyme (100 μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.

Biotechnology & Applied Microbiology↗

Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation

Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro , we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis . We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.

59 BASIC BIOLOGICAL SCIENCES↗

Evaluation of surface sampling method performance for Bacillus Spores on clean and dirty outdoor surfaces.

Recovery of Bacillus atrophaeous spores from grime-treated and clean surfaces was measured in a controlled chamber study to assess sampling method performance. Outdoor surfaces investigated by wipe and vacuum sampling methods included stainless steel, glass, marble and concrete. Bacillus atrophaeous spores were used as a surrogate for Bacillus anthracis spores in this study designed to assess whether grime-coated surfaces significantly affected surface sampling method performance when compared to clean surfaces. A series of chamber tests were carried out in which known amounts of spores were allowed to gravitationally settle onto both clean and dirty surfaces. Reference coupons were co-located with test coupons in all chamber experiments to provide a quantitative measure of initial surface concentrations of spores on all surfaces, thereby allowing sampling recovery calculations. Results from these tests, carried out under both low and high humidity conditions, show that spore recovery from grime-coated surfaces is the same as or better than spore recovery from clean surfaces. Statistically significant differences between method performance for grime-coated and clean surfaces were observed in only about half of the chamber tests conducted.

36 MATERIALS SCIENCE↗