BG Spores Research Articles

Fully integrated digital microfluidics platform for automated immunoassay; A versatile tool for rapid, specific detection of a wide range of pathogens

With the tangible threat posed by the release of chemical and biological warfare (CBW) agents, detection of airborne pathogens is a critical military and security concern. Recent air sampling techniques developed for biocollection take advantage of Electrowetting on Dielectric (EWOD) to recover material, producing highly concentrated droplet samples. Bespoke EWOD-based digital microfluidics platforms are very well suited to take full advantage of the microlitre concentrated droplet resulting from this recovery process. In this paper we present a free-standing, fully automated DMF platform for immunoassay. Using this system, we demonstrate the automated detection of four classes of CBW agent simulant biomolecules and organisms each representing credible threat agents. Taking advantage of the full magnetic separation process with antibody-bound microbeads, rapid and complete separation of specific target antigen can be achieved with minimal washing steps allowing for very rapid detection. Here, we report clear detection of four categories of antigens achieved with assay completion times of between six and ten minutes. Detection of HSA, Bacillus atrophaeus (BG spores), MS2 bacteriophage and Escherichia coli are demonstrated with estimated limit of detection of respectively 30 ng ml-1, 4 × 104 cfu ml-1, 106 pfu ml-1 and 2 × 107 cfu ml-1. The fully-integrated portable platform described in this paper is highly compatible with the next generation of electrowetting-coupled air samplers and thus shows strong potential toward future in-field deployable biodetection systems and could have key implication in life-changing sectors such as healthcare, environment or food security.

Sampling efficiencies of two modified viable cascade impactors

ABSTRACTPrevention of airborne contagious diseases depends on successful characterization of aerosols in the environment. The use of cascade impactors to characterize ambient aerosols is one of the most commonly used methods, providing data on both particle size and concentration. In this study, the use of a cascade impactor recently described in the literature using 8 mL of liquid in Petri dishes (CI-L) was compared with a new method that uses wet membrane filters on top of wax filled Petri dishes (CI-WWMF). Sampling efficiencies of the cascade impactors were evaluated using 0.5, 1, 3, and 5 μm polystyrene latex (PSL) microspheres and aerosol consisting of single spores of Bacillus atrophaeus var. globigii (BG). The sampling efficiency of the CI-L was 6%, 11%, 17%, 21%, and 58% for 0.5, 1, 3, 5 μm PSL microspheres and BG spores, respectively. Higher overall sampling efficiencies of 71%, 91%, 60%, 64%, and 104% were observed for the same size and type of particles for the CI-WWMF. This study indicates tha...

Modeling ultraviolet dose–response ofBacillusspore clusters in air

ABSTRACTThis article presents a new way to model the narrow band ultraviolet (UV-C, 254.7 nm) radiation dose–response behavior of Bacillus spores in clusters based on knowledge of the UV-C dose–response behavior of the single spores. The approach is demonstrated using experimentally obtained survival rates for Bacillus atrophaeus var. globigii (BG) spores and BG spore clusters of several sizes exposed to UV-C fluence when aerosolized and when deposited on dry surfaces. These results are modeled to derive predicted survival rates for spores in clusters of arbitrary cluster diameter under similar conditions. The essential feature of the approach is accurate accounting for attenuation of incident UV fluence within the spore cluster to derive the fluence incident on individual spores within the cluster. The results suggest that UV fluence attenuation by bacterial spores may increase with increasing fluence, a phenomenon that has not been previously reported for bacterial spores. The results are of utility in ...

Quantitative Analysis of Effects of UV Exposure and Spore Cluster Size on Deposition and Inhalation Hazards ofBacillusSpores

Reviews of the effects of solar UV radiation on the survival rate of aerosolized biological material found that the current understanding of environmental viability degradation in response scenarios is insufficient to inform appropriate emergency response measures. We evaluated the effects of UV degradation, in terms of the number of viable, culture forming units as a function of spore cluster size on the downwind hazard presented by a release of a biological organism such as Bacillus anthracis into the environment. We used experimentally derived survival rates for B. atrophaeus var. globigii (BG) spores and BG spore clusters (as a surrogate for Bacillus anthracis) of various sizes exposed to UVC fluences to derive predicted survival rates for single spores and spore clusters of up to 10 µm. For the range of weather conditions encountered in hazard estimates, as characterized by Pasquill-Gifford-Turner classes, we calculated and compared the downwind inhalation and deposition hazards for single spores ver...

Operational Evaluation of the Rapid Viability PCR Method for Post-Decontamination Clearance Sampling

The Rapid Viability Polymerase Chain Reaction (RV-PCR) method was evaluated during the Bio-Response Operational Testing and Evaluation (BOTE), an interagency project to evaluate field-level facility biological remediation, using leading decontamination technologies. The tests were performed using an intentional release (aerosolization) of spores of Bacillus atrophaeus subspecies globigii (BG), as a surrogate for Bacillus anthracis, the etiologic agent for anthrax. Three decontamination methods were assessed including fumigation with vaporized hydrogen peroxide (VHP), fumigation with chlorine dioxide (CD), and a surface treatment process using pH-adjusted bleach.The RV-PCR method was developed to rapidly detect live B. anthracis spores during a bioterrorism event. The method uses a change in realtime PCR response before and after a nine hour incubation step, to determine the presence of viable bacterial spores in the sample; the method was recently verified for air filter, wipe and water samples at the 10-spore level for B. anthracis Ames spores, and was also developed for swab, sponge-stick, and vacuum sock/filter samples. In the method, high throughput sample processing is combined with PCR-based analysis before and after a rapid culture step to speed viability determination, especially for complex surface and environmental samples that present challenges to current culture-based methods. In the BOTE project, a total of 159 surface wipe samples from post-decontamination events were analyzed by splitting the suspension after spore recovery into two equal parts, with one part analyzed by RV-PCR and the other part by culture after concentrating to the same volume. In the BOTE project, the RV-PCR method provided rapid results for post-decontamination samples that were 98% (156/159 samples) consistent with results from culture analysis. The percentage agreement was noteworthy, given the large number of samples containing low spore levels. For the Post-VHP, Post-Bleach, and Post-CD event samples, the percentage agreement was 93% (41/44 samples), 100% (47/47 samples), and 100% (68/68 samples), respectively. The RV-PCR method performed well for the surrogate BG spores exposed to decontaminants at real-world application levels, and with wipe samples containing background debris and indigenous microbial populations.

Passive Detection of Biological Aerosols in the Atmosphere with a Fourier Transform Instrument (FTIR)—the Results of the Measurements in the Laboratory and in the Field

Fourier Transform Infrared Radiation (FTIR) spectroscopy is one of the most powerful methods for the detection of gaseous constituents, aerosols, and dust in planetary atmospheres. Infrared spectroscopy plays an important role in searching for biomarkers, organics and biological substances in the Universe. The possibility of detection and identifications with FTIR spectrometer of bio-aerosol spores (Bacillus atrophaeus var. globigii=BG) in the atmosphere is discussed in this paper. We describe the results of initial spectral measurements performed in the laboratory and in the field. The purpose of these experiments was to detect and to identify bio-aerosol spores in two conditions: 1) In a closed chamber where the thermal contrast between the background and aerosols was large, and 2) In open air where the thermal contrast between the background and aerosols was small. The extinction spectrum of BG spores was deduced by comparing our measurements with models, and other measurements known from the literature. Our theoretical and experimental studies indicate that, during passive remote sensing measurements, it is difficult—but possible to detect and to identify bio-aerosol clouds by their spectral signatures. The simple spectral analysis described in the paper can be useful for the detection of various kinds of trace aerosols—not only in the Earth’s atmosphere, but also during planetary missions in the environments of other astronomical objects such as planets, comets etc. We expect that the interpretation of data from spectrometric sounding of Venus and Mars during the current missions Mars and Venus Express, and later during the Rosetta mission will benefit from our experimental work and numerical modelling.

On-Column Labeling of Gram-Positive Bacteria with a Boronic Acid Functionalized Squarylium Cyanine Dye for Analysis by Polymer-Enhanced Capillary Transient Isotachophoresis

A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed "PectI." It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ(ex) = 630 nm, λ(em) = 660 nm) with a high affinity constant (K = ~10(2.80) M(-1)) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, Bacillus globigii (Bg), which serves as a simulant of B. anthracis (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer's previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as "PectI" dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, Saccharomyces cerevisiae (resolution, R(s) = 6.09, and apparent plate number, N = 2.7-3.3 × 10(5)), were successfully achieved.

EWOD driven cleaning of bioparticles on hydrophobic and superhydrophobic surfaces

Environmental air monitoring is of great interest due to the large number of people concerned and exposed to different possible risks. From the most common particles in our environment (e.g. by-products of combustion or pollens) to more specific and dangerous agents (e.g. pathogenic micro-organisms), there are a large range of particles that need to be controlled. In this article we propose an original study on the collection of electrostatically deposited particles using electrowetting droplet displacement. A variety of particles were studied, from synthetic particles (e.g. Polystyrene Latex (PSL) microsphere) to different classes of biological particle (proteins, bacterial spores and a viral simulant). Furthermore, we have compared ElectroWetting-On-Dielectric (EWOD) collecting efficiency using either a hydrophobic or a superhydrophobic counter electrode. We observe different cleaning efficiencies, depending on the hydrophobicity of the substrate (varying from 45% to 99%). Superhydrophobic surfaces show the best cleaning efficiency with water droplets for all investigated particles (MS2 bacteriophage, BG (Bacillus atrophaeus) spores, OA (ovalbumin) proteins, and PSL).

Identification and Quantitation of Bacillus globigii Using Metal Enhanced Electrochemical Detection and Capillary Biosensor

Presented herein are two detection strategies for the identification and quantification of Bacillus globigii, a spore forming nonpathogenic simulant of Bacillus anthracis. The first strategy involves a label-free, metal-enhanced electrochemical immunosensor for the quantitative detection of Bacillus globigii (atrophaeus). The immunosensor comprises of antibacillus globigii (BG) antibody self-assembled onto a gold quartz crystal electrode via cystamine bond. A solid-phase monolayer of silver underpotentially deposited onto the cystamine modified-Au-electrode surface is used as the redox probe. The monolayer was also generated by adsorbing silver nanoparticles on the gold electrode. When the antibody-modified electrode is exposed to BG spores, the antibody-antigen (Ab-Ag) complex formed insulated the electrode surface toward the silver redox probe. The variation of redox current was found to be proportional to the concentration of the BG spores between 1 x 10(2)-3.5 x 10(4) spores/mL. A detection limit of 602 spores/mL was obtained, which is well-below the infectious dose of anthrax spores at 2.5 x 10(5) spores/mL. The second approach involves the use of ultrasensitive portable capillary biosensor (UPAC) to detect the spores. The capillary is an enclosed system that acts as the flow cell, the waveguide, and the solid support for immobilized bimolecular probes. An evanescent excitation generates a signal from an antigen-antibody-fluorophore complex, which propagates along the capillary and is guided to the detector. A limit of detection of 112 spores/mL was reported using the UPAC sensor. Both methods showed lower detection limits compared to the conventional ELISA. The effect of potential interferants tested using Bacillus pumilus confirmed the selectivity for the analyte. This work should allow the first responders to rapidly detect and quantify Bacillus globigii spores at concentrations that are well-below the infectious dose.

THE STANDOFF AEROSOL ACTIVE SIGNATURE TESTBED (SAAST) AT MIT LINCOLN LABORATORY

Standoff LIDAR detection of BW agents depends on accurate knowledge of the infrared and ultraviolet optical elastic scatter (ES) and ultraviolet fluorescence (UVF) signatures of bio-agents and interferents. MIT Lincoln Laboratory has developed the Standoff Aerosol Active Signature Testbed (SAAST) for measuring polarization-dependent ES cross sections from aerosol samples at all angles including 180° (direct backscatter) [1]. Measurements of interest include the dependence of the ES and UVF signatures on several spore production parameters including growth medium, sporulation protocol, washing protocol, fluidizing additives, and degree of aggregation. Using SAAST, we have made measurements of the polarization-dependent ES signature of Bacillus globigii (atropheaus, Bg) spores grown under different growth methods. We have also investigated one common interferent (Arizona Test Dust). Future samples will include pollen and diesel exhaust. This paper presents the details of the apparatus along with the results of recent measurements.

Estimation of Viable Spores in Bacillus atrophaeus (BG) Particles of 1 to 9 μm Size Range

AbstractThis study examined the viable organism content in a bioaerosol disseminated from a letter contaminated with Bacillus atrophaeus (BG) (anthrax simulant). In addition to using nutrient medium based slit samplers to sample the bioaerosol in the air space where the dispersion occurred, the individual who opened the contaminated letter was also used as a bioaerosol sampler by incorporating an external filter trap positioned over the inlet of the respirator filter canister worn by the individual. The reasons for this latter approach should be clear: it would provide actual quantitative information on the inhalation exposure hazard to an individual faced with this scenario. Where bioaerosol particles may be comprised of aggregates of many smaller particles, organism viability within the aggregate is important and may have an impact on the current definition of ‘infectious dose’ or ‘lethal dose’. Both terms assume single organism counts. Results from two different methods of measuring aerosols due to opening contaminated letters are presented: slit samplers and glass microfibre filter medium placed on the inlet port of a respirator cartridge. The total number of viable bioaerosol particles measured at the inhalation port of the filter canister was 40 times greater than measured by the slit samplers. Accordingly, this may be accounted for by a distribution of bioaerosol particles comprised of aggregates of single BG spores. A model has been proposed that estimates the number of individual viable spores in a distribution of aerosol particles through a relatively simple probability density function distribution for particle size, and which incorporates realistic assumptions regarding particle diameters, packing density and spore viability.

Analysis of Bacillus globigii spores by CE

It is imperative in today's world that harmful airborne or solution-based microbes can be detected quickly and efficiently. Bacillus globigii (Bg) spores are used as a simulant for Bacillus anthracis (Ba) due to their similar shape, size, and cellular makeup. The utility of CE to separate and detect low levels of Bg spore concentrations will be evaluated. To differentiate spores from background particulates, several dyes, including fluorescamine, C-10, NN-127, Red-1c, and indocyanine green (ICG), were utilized as noncovalent labels for proteins on the Bg spore surface, as well as for HSA and homoserine standards. On-column labeling, with dye present in the running buffer, was utilized to obtain greater sensitivity and better separation. CE with LIF detection enables interactions between the dye and spore surface proteins to be observed, with enhanced fluorescence occurring upon binding of the dye to surface protein. Resulting electropherograms showed unique fingerprints for each dye with Bg spores. Migration times were under 10 min for all dye-spore complexes, with net mobilities ranging from 3.5x10(-4) to 6.9x10(-4) cm(2) V(-1) s(-1), and calibration curves yielded correlation coefficients of 0.98 or better for four of the dyes studied.

Waterborne Pathogen Detection Using Raman Spectroscopy

Raman spectroscopy is being evaluated as a candidate technology for waterborne pathogen detection. We have investigated the impact of key experimental and background interference parameters on the bacterial species level identification performance of Raman detection. These parameters include laser-induced photodamage threshold, composition of water matrix, and organism aging in water. The laser-induced photodamage may be minimized by operating a 532 nm continuous wave laser excitation at laser power densities below 2300 W/cm(2) for Grampositive Bacillus atrophaeus (formerly Bacillus globigii, BG) vegetative cells, 2800 W/cm(2) for BG spores, and 3500 W/cm(2) for Gram-negative E. coli (EC) organisms. In general, Bacillus spore microorganism preparations may be irradiated with higher laser power densities than the equivalent Bacillus vegetative preparations. In order to evaluate the impact of background interference and organism aging, we selected a biomaterials set comprising Gram-positive (anthrax simulants) organisms, Gram-negative (plague simulant) organisms, and proteins (toxin simulants) and constructed a Raman signature classifier that identifies at the species level. Subsequently, we evaluated the impact of tap water and storage time in water (aging) on the classifier performance when characterizing B. thuringiensis spores, BG spores, and EC cell preparations. In general, the measured Raman signatures of biological organisms exhibited minimal spectral variability with respect to the age of a resting suspension and water matrix composition. The observed signature variability did not substantially degrade discrimination performance at the genus and species levels. In addition, Raman chemical imaging spectroscopy was used to distinguish a mixture of BG spores and EC cells at the single cell level.

REAGENTLESS BIO-SAMPLING METHODS FOR IR DETECTION

Currently there exists a critical need within the military and homeland defense for highly sophisticated yet, small, lightweight portable sensors and detection systems for identifying and quantifying biological and biowarfare agents (BWA) in both liquid and aerosolized form. Our proposed BWA detection system is based upon Fourier Transform Infrared Spectroscopy (FTIR), where the main advantages of this approach are that it is reagentless, operates in heterogeneous aqueous environments, and provides fast detection and high sensitivity/selectivity to bacterial spores with minimal false alarms. The key enabler to using FTIR for BWA detection is to develop selective and robust sampling protocols coupled to a miniaturized, portable FTIR unit. To that end, we have developed front-end liquid flow cells which incorporate electric field (E-Field) concentration methods for spores onto the surface of an Attenuated Total Reflection (ATR) IR crystal. IR spectra are presented which show collection and detection results with BG spores in water. The approaches we have developed take advantage of the fact that all spores are negatively charged in neutral pH solutions. Therefore, E-Field concentration of spores directly onto an ATR sampling element enables low level concentration measurements to be possible.

Single particle multichannel bio-aerosol fluorescence sensor

We describe a prototype low-cost multi-channel aerosol fluorescence sensor designed for unattended deployment in medium to large area bio-aerosol detection networks. Individual airborne particles down to ~1mum in size are detected and sized by measurement of light scattered from a continuous-wave diode laser (660nm). This scatter signal is then used to trigger the sequential firing of two xenon sources which irradiate the particle with UV pulses at ~280 nm and ~370 nm, optimal for excitation of bio-fluorophores tryptophan and NADH (nicotinamide adenine dinucleotide) respectively. For each excitation wavelength, fluorescence is detected across two bands embracing the peak emissions of the same bio-fluorophores. Current measurement rates are up to ~125 particles/s, corresponding to all particles for concentrations up to 1.3 x 104 particles/l. Developments to increase this to ~500 particles/s are in hand. Device sensitivity is illustrated in preliminary data recorded from aerosols of E.coli, BG spores, and a variety of non-biological materials.

Angularly-resolved elastic scatter from single particles collected over a large solid angle and with high resolution

Elastic light scattering from a single non-spherical particle of various morphologies has been measured simultaneously with a large angular range (90° < θ < 165° and 0° < ϕ < 360°) and with high angular resolution (1024 pixels in θ and 512 pixels in ϕ). Because the single-shot laser pulse is short (pulse duration of 70 ns), the tumbling and flowing particle can be treated as frozen in space. The large angle two-dimensional angular optical scattering (hereafter referred to as LA TAOS) intensity pattern, I(θ,ϕ), has been measured for a variety of particle morphology, such as the following: (1) single polystyrene latex (PSL) sphere; (2) cluster of PSL spheres; (3) single Bacillus subtilis (BG) spore; (4) cluster of BG spores; (5) dried aggregates of bio-aerosols as well as background clutter aerosols. All these measurements were made using the second harmonic of a Nd:YAG laser (0.532 μm). Islands structures in the LA TAOS patterns seem to be the prominent feature. Efforts are being made to extract metrics from these islands and compare them to theoretical results based on the T-matrix method.

The effect of filter material on bioaerosol collection of Bacillus subtilis spores used as a Bacillus anthracis simulant

The objective of this study was to determine filter materials and extraction methods that are appropriate to use for environmental sampling of B. anthracis. Four types of filters were tested: mixed cellulose ester (MCE) with a pore size of 3 microm, polytetrafluoroethylene (PTFE) with pore sizes of 1 and 3 microm, and gelatin with a pore size of 3 microm. Bacillus subtilis var. niger endospores (also known as Bacillus globigii[BG]) were used as a surrogate for B. anthracis. Endospores were collected into Button Inhalable Aerosol Samplers with sampling times of 15 minutes, 1 hour, and 4 hours. Physical collection efficiency was determined by measuring upstream and downstream B. subtilis concentrations with an optical particle counter. Vortexing with ultrasonic agitation and vortexing with shaker agitation extraction methods were evaluated. The MCE, 1 microm PTFE, and gelatin filters provided physical collection efficiencies of 94% or greater. The 3 microm PTFE filter showed inconsistent physical efficiency characteristics between filters. Epifluorescence microscopic analysis of the gelatin filter extraction fluid revealed the presence of contamination by non-culturable bacteria. Mean differences for microbial culturability were not statistically significant for filter materials and extraction methods. However, the vortexing with shaker agitation extraction method resulted in higher total microbial counts in the extraction fluids for MCE and 1 microm PTFE filters when compared to vortexing with ultrasonic agitation. In summary, the MCE and 1 microm PTFE filters in combination with vortexing and shaker extraction demonstrated the best performance for the filter collection and extraction of BG spores.

Bacillus globigii Bugbeads: A Model Simulant of a Bacterial Spore

Nonpathogenic microorganisms are often used as simulants of biological pathogens during the initial phase of detection method development. While these simulants approximate the size, shape, and cellular organization of the microorganism of interest, they do not resemble its surface protein content, a factor particularly important in methods based on immunorecognition. Here, we develop and detect an artificial bacterial spore--B. globigii (BG) Bugbead-a particle mimicking the antigenic surface of BG spores. Two methods of spore protein extraction were compared both quantitatively (by protein concentration assay) and qualitatively (by SDS-PAGE and Western blot): extraction by mechanical disruption and extraction by chemical decoating. The former method was more efficient in producing more protein and a greater number of antigens. BG Bugbeads were made by conjugating the extracted proteins to 0.8-microm carboxyl-coated polystyrene particles via carbodiimide coupling. BG Bugbeads were successfully detected by a bead-based enzyme-labeled immunoassay with fluorescence detection with a detection limit of 6.9 x 10(3) particles/mL. Formation of the Bugbead-capture bead complex was confirmed by ESEM. The concept of a harmless artificial spore can be applied to developing improved simulants for pathogenic spore-forming microorganisms such as B. anthracis, C. botulinum, and B. cereus, which can to be used for method validation, instrument calibration, and troubleshooting.

An Integrated Metal Clad Leaky Waveguide Sensor for Detection of Bacteria

An integrated optical metal clad leaky waveguide (MCLW) sensor device has been developed for the detection of bacteria. This is more sensitive than waveguide sensors currently in use. The MCLW device has been fabricated to extend the evanescent field to provide significant light intensity over the entire volume of the bacteria bound on the chip surface within this field. This in turn increases the interaction of the light with the entire volume of the bacteria. MCLW devices have been used for detecting refractive index changes, scattering, and fluorescence from bacterial spores captured on an immobilized antibody. The detection limit of Bacillus subtilis var. niger bacterial spores using refractive index detection was 8 x10(4) spores/mL. The scattering intensity of the BG spores was found to be three times greater than the scattering intensity generated using surface plasmon resonance. The extended light propagation along the direction of flow for a few millimeters provides an effective interrogation approach to increase the area of detection to detect low concentrations down to 1 x 10(4) spores/mL. The sensor was then optimized by studying the key factors affecting sensor performance including changing the pH of the medium, type of antibody immobilization matrix, sensor surface regeneration approaches, and longevity of the sensor.

Evaluation of a high-volume portable bioaerosol sampler in laboratory and field environments

This study investigated the physical and biological performances of a portable centrifugal sampler for viable bioaerosols, RCS High Flow. The performance of the test sampler in the laboratory and field environments was compared with that of a reference sampler, BioSampler. The laboratory experiments with non-biological particles of KCl, oleic acid, and polystyrene latex showed that the test sampler's collection efficiency is about 22% for 0.5-microm particles, 48% for 1.0-microm particles, and approximately 100% for particles of 2.5 microm and larger. These tests indicated that the sampler's cut-off size (d50) was 1.1 microm. The test sampler's physical performance when collecting the spores and vegetative cells of Bacillus subtilis var. niger (BG) was similar to that when collecting non-biological particles of the same size. In the laboratory tests, the RCS High Flow sampler was found to enumerate approximately 40% of BG spores and cells relative to the reference sampler, BioSampler. A similar ratio was found during testing in an indoor environment. This ratio decreased to below 10% when testing was performed in an outdoor environment. We hypothesize that the test sampler's underperformance compared with the BioSampler could be caused by the damage to sensitive microorganisms during the collection process, test sampler's sensitivity to wind direction and speed as well as break-up of particle aggregates during the impingement process in BioSampler, which resulted in more colony-forming units (CFUs) being counted by the reference sampler than by the test sampler. Overall, when the RCS High Plus is used to sample culturable airborne microorganisms, the results obtained may have to be adjusted to avoid potential underestimation of microorganism concentration in the air. The laboratory testing of the RCS High Flow bioaerosol sampler showed that the sampler collects 1 microm particles and larger with an efficiency of 50% and higher; the efficiency reaches approximately 100% for particles of 2.5 microm and larger. When considering this result, most of the airborne fungal spores would be collected with an efficiency between 50 and 100%. The field testing, however, indicated that the RCS High Flow sampler recovered from 41 to 71% of microorganisms collected relative to the reference sampler, Biosampler, and this ratio dropped to below 5% during outdoor testing. Thus, while the RCS High Flow sampler offers certain advantages over other samplers for viable bioaerosols--it is lightweight, battery operated, and collects viable microorganisms at a high flow rate directly on agar media, the results obtained may have to be adjusted to avoid potential underestimation of microorganism concentration in the air, especially if sampling is performed outdoors.