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Germination of Bacillus anthracis spores is necessary for the transcription of plasmidic genes essential to the infection. Assessing germination potential is crucial to predict the risk associated with pathogenic Bacillus exposure. The aim of this study was to set up a viability assay based on membrane potential in order to predict the earliest germination event of spores. B. cereus and two strains of B. subtilis were used. The spores were isolated with a sodium bromide gradient. Approximately 107 spores were incubated at 37°C in tryptic soy broth (TSB). Aliquots were harvested at predetermined times and stained with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. Fluorescence characteristics were obtained using flow cytometry. The earliest detectable activation of membrane potential occurred after 15 min of incubation in TSB using DiOC6(3). Using DiBAC4(3), the earliest detectable signal was after 4 h of incubation. Control experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP)-treated spores did not show any change in the fluorescence intensity over time. Since no membrane potential and no germination were detected in CCCP-treated spores, the activation of membrane potential seems to be associated with germination. DiOC6(3) can be used as an early membrane potential indicator for spores. DiBAC4(3), by contrast, is not a early membrane potential marker.

Only a small number of studies have measured the plasmid copy number (PCN) variation during bacterial growth. Besides, information about the PCN in spores is still rare. In this work, we utilized a real-time PCR assay to evaluate the PCN of four different plasmids in Bacillus cereus. The PCN was measured in spores as well as during germination, active bacterial growth, and sporulation. Plasmid stability was also evaluated to ensure that plasmid loss does not affect the accuracy of the PCN measurement. We demonstrated that the PCN of low and high copy number plasmids varies with growth phase as well as culture media over B. cereus life cycle. The PCN was minimum during the germination and maximum during the stationary growth phase for all plasmids tested. We also demonstrated that the use of antibiotic in the culture media is not enough to ensure stable inheritance in spores of plasmids carrying antibiotic resistance genes. Moreover, we revealed that the PCN in spores is related to the PCN during endospores formation. Therefore, the plasmid partitioning during sporulation is not influenced by the unequal-size of the forespores and the mother cells, even for a plasmid distributed randomly.

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.

Aim: To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Methods and Results: Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0·02). Conclusion: It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. Significance and Impact of the Study: This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

By the end of the 90s, Defence Research and Development Canada (DRDC) initiated the investigation of a novel LIDAR concept which open the possibility of collecting all at once the detailed spectral information contained in the return signals. This 3-year project called SINBAHD (Stand-off INtegrated Bioaerosol Active Hyperspectral Detection) aimed at evaluating the capability of using UV LIF with intensified range-gated spectrometry to detect and characterize bioaerosol from stand-off position. Essentially, the LIDAR system monitors atmospheric volumes in which specific spectrally wide fluorescence signal can be generated from inelastic interactions with complex molecules forming the building blocks of most bioaerosols. This LIF signal is collected by the combination of a dispersive element and a range-gated ICCD that limits the spectral information within the selected volume. This technique has showed an important potential of detecting and discriminating different bioaerosol agent simulants in real time. Through the Standoff Biodetection Working Group (SBWG) under the CANUKUS CBR MOU, SINBAHD was invited to participate in the Joint Biological Standoff Detection System (JBSDS) increment II field demonstration trial. The purpose of this international trial, which took place at Dugway Proving Ground (DPG), Utah in June 2005, was to determine the benchmark sensitivity of different technologies to various biological simulants and interferents. SINBAHD demonstrated its high level of performance and the results made it possible to obtain new spectral signatures., The original document contains color images.

Aim: To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Methods and Results: Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0·02). Conclusion: It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. Significance and Impact of the Study: This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

Germination of Bacillus anthracis spores is necessary for the transcription of plasmidic genes essential to the infection. Assessing germination potential is crucial to predict the risk associated with pathogenic Bacillus exposure. The aim of this study was to set up a viability assay based on membrane potential in order to predict the earliest germination event of spores. B. cereus and two strains of B. subtilis were used. The spores were isolated with a sodium bromide gradient. Approximately 107 spores were incubated at 37°C in tryptic soy broth (TSB). Aliquots were harvested at predetermined times and stained with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. Fluorescence characteristics were obtained using flow cytometry. The earliest detectable activation of membrane potential occurred after 15 min of incubation in TSB using DiOC6(3). Using DiBAC4(3), the earliest detectable signal was after 4 h of incubation. Control experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP)-treated spores did not show any change in the fluorescence intensity over time. Since no membrane potential and no germination were detected in CCCP-treated spores, the activation of membrane potential seems to be associated with germination. DiOC6(3) can be used as an early membrane potential indicator for spores. DiBAC4(3), by contrast, is not a early membrane potential marker.

Improvements were made to a pyrolysis-gas chromatography-ion mobility spectrometry (Py-GC-IMS) stand-alone biodetector to provide more pyrolyzate compound information to the IMS detector module. Biological aerosols were disseminated at DRES, Alberta, Canada and the Py-GC-IMS was tested for its ability to detect the biological aerosols. Forty-two trials were conducted and a simple area calculation of the GC-IMS data domain biomarker peaks correlated with the correct bioaerosol challenge in 30 trials (71%). In another 7 trials, the status of an aerosol was determined to be biological in origin. Two additional trials had no discernible, unambiguous GC-IMS biological response, because they were blank water sprays. Reproducible limits of detection were at a concentration of less than 0.5 bacterial analyte-containing particles per liter of air (ACPLA). In order to realize this low concentration, an aerosol concentrator was used to concentrate 2000 liters of air in 2.2 minutes. The current series of outdoor trials has provided a platform to show that the Py-GC-IMS can provide information more specific than a biological or nonbiological analysis to an aerosol when the time of dissemination is unknown to the operator. The Py-GCIMS is shown to be able to discriminate between aerosols of a Gram-positive spore (BG), a Gram-negative bacterium (EH) and a protein (ovalbumin)., See also ADM001523., The original document contains color images. Presented at the Joint Service Scientific Conference on Chemical and Biological Defense Research (2002) Held 19-21 Nov 2002 at Hunt Valley, MD.

A previous generation of biological detection instruments, pioneered at DRES, used light sources in the 340-360 nm region. Although this wavelength region has been shown to excite important biomolecules related to live organisms, implementing them turned out to be extremely expensive due to complex technologies involved. An alternative light solution became imperative. In this paper, biological aerosol measurement was done with a laser diode emitting at 402 nm. It was chosen for its ability to excite a different class of biomolecules. There is a need to demonstrate that a fluorescence aerodynamic particle sizer (FLAPS) perform satisfactorily using these selected types of light sources when presented with a range of biological aerosol simulants. The findings indicated comparable fluorescence response from the select excitation sources in the presence of spore aerosol. Spores, by their physiological nature, contain very little biological material and the individual units are no larger than 0.7 %m and enclosed by a very refractile spore coat. Due to this refractile characteristic, conventional light microscopy of spores reveal very little of their cellular content. In contrast, vegetative cells are better subjects for examination in that they can easily be stained. Thus, performance of biological detectors must be rated by their ability to measure spore aerosols and in these experiments, it has been shown that 10 Agent Containing Particles Per Liter of Air (ACPLA) could be detected. In summary, we have shown that by using a small laser diode that emits at 402 nm, it is possible to replicate the performance of 340-60 nm light sources. The findings from this study have critical implications for the future of biological detection, both for military applications as well as in environmental monitoring. With this new light source, a detector will require no active cooling system, consume little power and weigh much less than current instruments., New replacement document scanned 15 Apr 04.

The history on slit to agar samplers shows that the first published description was on a system using 10 cm petri plates. At some point after the 1950's samplers that collected biological aerosol particles on 15 cm plates appeared. More recent patents have been filed that described slit samplers using 10 cm plates, usually citing economics as the prime motivation. Yet workers have cautioned that the smaller plates can become saturated when heavy aerosol clouds are encountered. In applications where the slit sampler is used as reference collector when biological detectors are under test, heavy clouds may often be encountered. This report will demonstrate how to determine performance characteristics of slit samplers designed for either 10 or 15 cm plates. It will also show the importance of controlled replicate measurements providing data suitable for rigorous statistical analysis. The results indicate that for measuring biological clouds of between 5 to 30 agent containing particles per liter of air (ACPLA) the 15 cm plate sampler design is more efficient than those targeted for the smaller collection surface. A statistical method has been employed to test difference between regression slopes.

The biological aerosol content of the air has been measured at Darwin and RAAF Base Tindal in the Northern Territory of Australia. The number of culturable bacteria and fungal spores together with organic material caught in a spore trap are reported. The total airborne particulate matter in the size range 0.5 to 30 microns was also measured at Tindal in October 1998 and April 1999. The ability of an aerodynamic particle sizer to act as an early warning device for the presence of biological warfare agents in this environment is discussed. It was concluded that such a device could only be successfully used as a trigger in the event of a military release of a biological agent in an environment with a well characterised aerosol background.

From observations done with flow cytometry, it was found that a single spore in liquid stream could be induced to fluoresce at 34060 run. In a prototype instrument, similar results were obtained when spore particles in air were excited with a CW UV laser. The current instrument, Fluorescence Aerodynamic Particle Sizer (second generation FLAPS2) measures particle size as well as intrinsic fluorescence characteristics of individual particles in an aerosol stream. Laser light at UV wavelength is used to preferentially excite aerosol material of a selected size range. The measured fluorescence signals represent intrinsic biological properties of the particles. By this method, it has been shown that inert particles like sand can be distinguished from biological particles like spores and vegetative bacteria. This instrument and its prototype have been demonstrated to effectively detect and characterise biological aerosols during joint field trial exercises in both 1995 and 1996. Integral with the FLAPS design is an aerosol concentrator that provides about 400-500 times normal ambient concentration. This makes it possible to sample at a rate of 3 seconds and within this period, about 300-3000 total particles are processed. To function effectively as a biological detector, the instrument compares background or ambient data to that of an unknown cloud. An ambient aerosol database from diverse localities will greatly enhance data analysis. To this end, the instrument has been use to collect data on ambient aerosols in different geographical sites, for example at DRES, Calgary, Dugway, Cornwall and a spot downwind from a cattle feedlot. The results show that FLAPS can distinguish between clean and "dirty" environments with respect to the fraction of fluorescent particles measured in a given aerosol population.