Characterization of bacteria by multiparameter flow cytometry.

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.     

Characterization of bacteria by multiparameter flow cytometry

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.     

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.     

Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment.     

Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples

Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10(0) target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis.     

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.     

Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors

The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e., Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent L. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.     

Microbial analysis at the single-cell level: tasks and techniques

The heterogeneity of microorganisms themselves is orders of magnitude greater than the heterogeneity of perspectives from which they are contemplated by human observers. Even closely related species may exhibit marked differences in biochemistry and behavior, and, under many conditions, similar, striking heterogeneity may exist within a clonal population of organisms which, in the aggregate, occupy too small a region of space to be visible to the unaided human eye. Using methods of microscopy, microspectrophotometry, and cytometry developed and refined since the 1960s, it is now possible to characterize the physiology and pharmacology of individual microorganisms, and, in many cases, to isolate organisms with selected characteristics for culture and/or further analysis. These methods include fluorescent and confocal microscopy, scanning and image cytometry, and flow cytometry. Fluorescence measurements are particularly important in single-cell analysis; they allow demonstration and quantification of cells' nucleic acid content and sequence, of the presence of specific antigens, and of physiologic characteristics such as enzyme activity and membrane potential. Multiparameter cytometry, combined with cell sorting, provides insight into population heterogeneity and allows selected cells to be separated for further analysis and culture. The technology is applicable to a wide range of problems in contemporary microbiology, including strain selection and the development of antimicrobial agents.     

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.     

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.     

New detection systems of bacteria using highly selective media designed by SMART: selective medium-design algorithm restricted by two constraints

Culturing is an indispensable technique in microbiological research, and culturing with selective media has played a crucial role in the detection of pathogenic microorganisms and the isolation of commercially useful microorganisms from environmental samples. Although numerous selective media have been developed in empirical studies, unintended microorganisms often grow on such media probably due to the enormous numbers of microorganisms in the environment. Here, we present a novel strategy for designing highly selective media based on two selective agents, a carbon source and antimicrobials. We named our strategy SMART for highly Selective Medium-design Algorithm Restricted by Two constraints. To test whether the SMART method is applicable to a wide range of microorganisms, we developed selective media for Burkholderia glumae, Acidovorax avenae, Pectobacterium carotovorum, Ralstonia solanacearum, and Xanthomonas campestris. The series of media developed by SMART specifically allowed growth of the targeted bacteria. Because these selective media exhibited high specificity for growth of the target bacteria compared to established selective media, we applied three notable detection technologies: paper-based, flow cytometry-based, and color change-based detection systems for target bacteria species. SMART facilitates not only the development of novel techniques for detecting specific bacteria, but also our understanding of the ecology and epidemiology of the targeted bacteria.     

Chromosome mapping in lactic acid bacteria

The chromosome structure of lactic acid bacteria has been investigated only recently. The development of pulsed-field gel electrophoresis (PFGE) combined with other DNA-based techniques enables whole-genome analysis of any bacterium, and has allowed rapid progress to be made in the knowledge of the lactic acid bacteria genome. Lactic acid bacteria possess one of the smallest eubacterial chromosomes. Depending on the species, the genome sizes range from 1.1 to 2.6 Mb. Combined physical and genetic maps of several species are already available or close to being achieved. Knowledge of the genomic structure of these organisms will serve as a basis for future genetic studies. Macrorestriction fingerprinting by PFGE is already one of the major tools for strain differentiation, identification of individual strains, and the detection of strain lineages. The genome data resulting from these studies will be of general application strain improvement.     

Diversity, biological roles and biosynthetic pathways for sugar-glycerate containing compatible solutes in bacteria and archaea

A decade ago the compatible solutes mannosylglycerate (MG) and glucosylglycerate (GG) were considered to be rare in nature. Apart from two species of thermophilic bacteria, Thermus thermophilus and Rhodothermus marinus, and a restricted group of hyperthermophilic archaea, the Thermococcales, MG had only been identified in a few red algae. Glucosylglycerate was considered to be even rarer and had only been detected as an insignificant solute in two halophilic microorganisms, a cyanobacterium, as a component of a polysaccharide and of a glycolipid in two actinobacteria. Unlike the hyper/thermophilic MG-accumulating microorganisms, branching close to the root of the Tree of Life, those harbouring GG shared a mesophilic lifestyle. Exceptionally, the thermophilic bacterium Persephonella marina was reported to accumulate GG. However, and especially owing to the identification of the key-genes for MG and GG synthesis and to the escalating numbers of genomes available, a plethora of new organisms with the resources to synthesize these solutes has been recognized. The accumulation of GG as an 'emergency' compatible solute under combined salt stress and nitrogen-deficient conditions now seems to be a disseminated survival strategy from enterobacteria to marine cyanobacteria. In contrast, the thermophilic and extremely radiation-resistant bacterium Rubrobacter xylanophilus is the only actinobacterium known to accumulate MG, and under all growth conditions tested. This review addresses the environmental factors underlying the accumulation of MG, GG and derivatives in bacteria and archaea and their roles during stress adaptation or as precursors for more elaborated macromolecules. The diversity of pathways for MG and GG synthesis as well as those for some of their derivatives is also discussed. The importance of glycerate-derived organic solutes in the microbial world is only now being recognized. Their stress-dependent accumulation and the molecular aspects of their interactions with biomolecules have already fuelled several emerging applications in biotechnology and biomedicine.     

Rapid electrochemical detection and identification of catalase positive micro-organisms

The rapid detection and identification of bacteria has application in a number of fields, e.g. the food industry, environmental monitoring and biomedicine. While in biomedicine the number of organisms present during infection is multiples of millions in the other fields it is the detection of low numbers of organisms that is important, e.g. an infective dose of Escherichia coli O157:H7 from contaminated food is less than 100 organisms. A rapid and sensitive technique has been developed to detect low numbers of the model organism E. coli O55, combining Lateral Flow Immunoassay (LFI) for capture and amperometry for sensitive detection. Nitrocellulose membranes were used as the solid phase for selective capture of the bacteria using antibodies to E. coli O55. Different concentrations of E. coli O55 in Ringers solution were applied to LFI strips and allowed to flow through the membrane to an absorbent pad. The capture region of the LFI strip was placed in close contact with the electrodes of a Clarke cell poised at +0.7 V for the detection of hydrogen peroxide. Earlier research identified that the consumption of hydrogen peroxide by bacterial catalase provided a sensitive indicator of aerobic and facultative anaerobic microorganisms numbers. Modification and application of this technique to the LFI strips demonstrated that the consumption of 8 mM hydrogen peroxide was correlated with the number of microorganisms presented to the LFI strips in the range of 2 x 10(1)-2 x 10(7) colony forming units (cfu). Capture efficiency was dependent on the number of organisms applied and varied from 71% at 2 x 10(2) cfu to 25% at 2 x 10(7) cfu. The procedure was completed in less than 10 min and could detect less than 10 cfu captured from a 200 microl sample applied to the LFI strip. The approached adopted provides proof of principle for the basis of a new technological approach to the rapid, quantitative and sensitive detection of bacteria that express catalase activity.     

Flow cytometry, microscopy, and DNA analysis as complementary phytoplankton screening methods in ballast water treatment studies

Ballast water is the main vector for marine invasions. To minimize the spread of invasive species, the International Maritime Organization (IMO) has adopted the Ballast Water Management Convention which requires the installation of shipboard ballast water treatment systems (BWTS). During BWTS tests, the phytoplankton abundance and species composition were followed after treatment with both filtration and ultraviolet radiation. Although the installation fulfilled the IMO criteria after a 5-day holding time in a model ballast tank, the ultimate effectiveness of the treatment was further tested in long-term (20 days) incubation experiments under optimal phytoplankton growth conditions. Application of flow cytometry, microscopy, and DNA sequencing to these incubation samples gave an indication of the phytoplankton species that might be introduced by ballast water discharge—despite treatment. Phytoplankton was reliably quantified using flow cytometry, while fast identification was best done using microscopy. Some groups that contained potentially toxic species could not be identified at species level using microscopy; for these species, identification using genetic techniques was necessary. It is concluded that if long-term incubation experiments are used as an additional tool in testing BWTS effectiveness, a combination of phytoplankton screening methods can be applied depending on the detail of information that is required.     

Survival curves for microbial species stored by freeze-drying

The survival of a variety of species of microorganism following storage for up to 20 years has been analyzed. The organisms were freeze-dried, sealed in ampoules under vacuum (<1 Pa) and stored in the dark at 5 degrees C. The yeast that was tested, Saccharomyces cerevisiae, showed only 8% survival when recovered shortly after freeze-drying, but subsequent loss during storage was the least among all the tested microorganisms. The decrease in the logarithm of survival per year (log survival) was -0.010, which corresponds to a survival rate of 97.7% per year. The Gram-negative bacteria tested, Escherichia coli, Pseudomonas putida, and Enterobacter cloacae, showed 42.6, 33.5, and 50.8% survival shortly after freeze-drying, which was higher than the corresponding survival of S. cerevisiae, but the subsequent loss during storage was greater than S. cerevisiae, the log survival figures being -0.041, -0.058, and -0.073 per year. These values correspond to survival rates of 91.0, 87.5, and 84.5% each year. The Gram-positive bacteria tested, Lactobacillus acidophilus and Enteroccoccus faecium, showed 62.5 and 85.2% survival shortly after freeze-drying, which was even higher than that of the Gram-negative species, and these organisms also showed better survival during storage than Gram-negative bacteria; their log survival rates were -0.018 and -0.016 per year, which corresponded to survival rates of almost 96% per year. Comparison of these results with other published data for different drying conditions suggests that survival during storage is strongly influenced by the degree of vacuum under which the ampoules were sealed. The excellent survival after freeze-drying of each species might be attributable to the high level of desiccation and to sealing under vacuum.     

Production of precise microbiology standards using flow cytometry and freeze drying.

BACKGROUND: Quality control standards provide a quantity of microorganisms for routine use in microbiology to demonstrate the efficacy of testing methods and culture media. Standards are normally prepared by diluting a culture of microorganisms to obtain a suspension that contains an estimated number of colony-forming units per milliliter. The variability and inaccuracy of these standards increase the potential for false results. Flow cytometry has been used extensively to prepare precise standards of Cryptosporidium and Giardia that contain exact numbers of organisms in a volume of liquid (1). However, the same levels of accuracy have yet to be obtained for bacterial quality control standards. METHODS: A modification of a Becton Dickinson FACScalibur flow cytometer enabled 30 bacterial cells to be sorted into a single droplet, mixed with a cryoprotective solution within the droplet, and frozen in liquid nitrogen. The frozen droplets were then freeze dried for stable preservation of the viable bacterial cells. RESULTS: A freeze-dried sphere 3 mm in diameter was produced, which contained 30 microorganisms. The within-batch variation for these freeze-dried spheres was no greater than two standard deviations, and the between-batch variation was less than one standard deviation. CONCLUSIONS: Bacterial reference controls can now be produced with consistent accuracy and unparalleled precision, thus enabling harmonization across the microbiological testing industry.     

Interactions of Nitrifying Bacteria and Heterotrophs: Identification of a Micavibrio-Like Putative Predator of Nitrospira spp.

Chemolithoautotrophic nitrifying bacteria release soluble organic compounds, which can be substrates for heterotrophic microorganisms. The identities of these heterotrophs and the specificities of their interactions with nitrifiers are largely unknown. In this study, we incubated nitrifying activated sludge with ¹³C-labeled bicarbonate and used stable isotope probing of 16S rRNA to monitor the flow of carbon from uncultured nitrifiers to heterotrophs. To facilitate the identification of heterotrophs, the abundant 16S rRNA molecules from nitrifiers were depleted by catalytic oligonucleotides containing locked nucleic acids (LNAzymes), which specifically cut the 16S rRNA of defined target organisms. Among the ¹³C-labeled heterotrophs were organisms remotely related to Micavibrio, a microbial predator of Gram-negative bacteria. Fluorescence in situ hybridization revealed a close spatial association of these organisms with microcolonies of nitrite-oxidizing sublineage I Nitrospira in sludge flocs. The high specificity of this interaction was confirmed by confocal microscopy and a novel image analysis method to quantify the localization patterns of biofilm microorganisms in three-dimensional (3-D) space. Other isotope-labeled bacteria, which were affiliated with Thermomonas, colocalized less frequently with nitrifiers and thus were commensals or saprophytes rather than specific symbionts or predators. These results suggest that Nitrospira spp. are subject to bacterial predation, which may influence the abundance and diversity of these nitrite oxidizers and the stability of nitrification in engineered and natural ecosystems. In silico screening of published next-generation sequencing data sets revealed a broad environmental distribution of the uncultured Micavibrio-like lineage.     

The flow cytometry of Bacillus anthracis spores revisited

BACKGROUND: The potential use of Bacillus anthracis spores as a weapon of terror has rekindled interest in the rapid detection and identification of the spores of these bacteria. Prior efforts to utilize flow cytometry (FCM) for this purpose resulted in tedious and time-consuming protocols. Advances in rapid immunoassays suggest a reinvestigation of the use of FCM because this may allow for the development of a rapid and sensitive system for detection and/or identification of spores in suspect samples. METHODS: In this study, antiserum was raised in goats using three different strains of B. anthracis spores as the immunogen. The resultant antibodies were purified, labeled with fluorescein, and evaluated for use in an immunoassay on a Coulter Epics XL flow cytometer. In the protocol that was developed, fluorescein-labeled antibodies are simply mixed with the sample, allowed to incubate, and then analyzed on the flow cytometer. Washes and centrifugation were eliminated. RESULTS: The results showed that a rapid (5 min) and sensitive immunological analysis was feasible. The detection limit (approximately 10(3) colony-forming units [CFU]/ ml) varied with strain, but there was no difference in the detection limit between live and irradiated spores. In addition, the power of FCM was utilized to minimize false-positive reactions among similar species of Bacillus by placing constraints on scatter and fluorescence intensity. The data also suggest that scatter might be useful to determine spore viability. CONCLUSION: This study shows that FCM may be an effective platform on which to perform immunological analysis for the detection and/or presumptive identification of B. anthracis spores. Published 2000 Wiley-Liss, Inc.