Detection Of Endospores Research Articles

Development of automated image processing algorithms for Bacillus spp. endospore detection

Development of automated image processing algorithms for <i>Bacillus</i> spp. endospore detection

Facilitated endospore detection for Bacillus spp. through automated algorithm-based image processing.

Bacillus spp. endospores are important dormant cell forms and are distributed widely in environmental samples. While these endospores can have important industrial value (e.g. use in animal feed as probiotics), they can also be pathogenic for humans and animals, emphasizing the need for effective endospore detection. Standard spore detection by colony forming units (CFU) is time‐consuming, elaborate and prone to error. Manual spore detection by spore count in cell counting chambers via phase‐contrast microscopy is less time‐consuming. However, it requires a trained person to conduct. Thus, the development of a facilitated spore detection tool is necessary. This work presents two alternative quantification methods: first, a colorimetric assay for detecting the biomarker dipicolinic acid (DPA) adapted to modern needs and applied for Bacillus spp. and second, a model‐based automated spore detection algorithm for spore count in phase‐contrast microscopic pictures. This automated spore count tool advances manual spore detection in cell counting chambers, and does not require human overview after sample preparation. In conclusion, this developed model detected various Bacillus spp. endospores with a correctness of 85–89%, and allows an automation and time‐saving of Bacillus endospore detection. In the laboratory routine, endospore detection and counting was achieved within 5–10 min, compared to up to 48 h with conventional methods. The DPA‐assay on the other hand enabled very accurate spore detection by simple colorimetric measurement and can thus be applied as a reference method.

Hybrid CARS spectroscopy based on a high-repetition-rate all-PM-fiber laser source

We demonstrate a robust, simple, and compact all polarization-maintaining (PM) fiber laser source with a repetition rate of 79 MHz for broadband coherent anti-Stokes Raman scattering (CARS) spectroscopy based on impulsive excitation and narrowband probing. The careful dispersion management during the generation of pump pulses ensures efficient impulsive excitation, which is verified to cover an ultrabroad bandwidth exceeding 4000 cm–1. The employment of PM fibers enables the laser source to withstand external disturbances. This turn-key configuration can potentially simplify the implementation of many applications of CARS, such as spectroscopic histopathology, weaponized endospore detection, and precise thermometry of gases.

Rapid and simple detection of endospore counts in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker

Spore counting in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker was studied for developing a rapid and simple detection method. The newly developed method is based on the fluorescence enhancement by a new chelating agent, which forms a complex with EuCl3 and DPA. The results showed that 1,2-cyclohexanediamine-N,N,N′N′-tetraacetic acid (CyDTA) greatly enhanced the fluorescence intensity in all selected chelating agents. The optimal composition of the fluorescence complex DPA-Eu-CyDTA had a detection limit of 0.3 nM of DPA. Metal ions in high concentrations, including Cu2+, Fe2+, Fe3+, Al3+, and Zn2+, might lower the detection sensitivity, which could be eliminated by diluting the sample with the metal ions below 10 μM. The maximum release of DPA was achieved by heating treatments at 121 °C for at least 10 min for two types of Bacillus endospores. The spore concentrations and corresponding released DPA fluorescence intensities were linearly associated (coefficient R2 = 0.9993 and 0.9995 for Bacillus subtilis MA139 and Bacillus licheniformis BL20386, respectively). The detection limit for both strains reached approximately 6800 spores/mL. The verification results showed that the DPA fluorimetry assay developed in the present study was fully consistent with the plate-counting assay. The study shows that the fluorescence complex DPA-Eu-CyDTA can be reliably used for the detection of endospores in Bacillus fermentation for the production of probiotics.

Endospore recovery from large volumes using fieldable ultrafiltration for the diagnosis of drinking water treatment plants

Anaerobic sulfite-reducing and aerobic spore-forming bacteria are used as indicators to verify the reliability of water treatment plant efficiency. The objectives of this study aimed to highlight the more accurate endospore indicator for water treatment monitoring and to select an efficient filtration system in order to detect low amounts of endospores from large volumes. The performance of two different ultrafiltration modules was assessed with two filtration configurations (tangential and dead-end). The Hemoflow™ and the Dizzer® filters present recovery yields of 73% and 77% for 100-liter samples of drinking and sand filtered water, respectively. Both systems enabled detection of endospores at a concentration level of 100 spores in 100 liters, by analyzing the totality of the volume, whereas the standard method did not highlight the presence of spores, analyzing only 100 mL subsamples. In addition, experiments on a sand filtration pilot confirmed that the concentration calculation using the standard method is not reliable, due to extrapolation from the small volumes analyzed. This study highlights that the collection of large volumes using a fieldable dead-end hollow-fiber ultrafiltration system in association with the detection of anaerobic sulfite-reducing spore-forming bacteria is an effective way to monitor drinking water production.

Evaluation of applicability of the Sartorius Airport MD8 sampler for detection of Bacillus endospores in indoor air

This study was designed to evaluate the measuring range and lowest limit of detection of Bacillus endospores in the ambient room air when the Sartorius MD8 sampler, and two different culture methods for bacterial enumeration were used. Different concentrations of bioaerosol were generated inside the test chamber filled with either the high-efficiency particulate air (HEPA)-filtered air or with the ambient room air. The detection of endospores in the HEPA-filtered air was achievable: (1) when they were aerosolized at a concentration above 7.56 × 103 CFU/m3 and analyzed with spread plate method, and (2) when they were aerosolized at a concentration above 4.00 × 102 CFU/m3 and analyzed with pour plate method. The detection of endospores in the ambient room air was possible: (1) when they were aerosolized at a concentration above 9.1 × 103 CFU/m3 and analyzed with spread plate method, and (2) when they were aerosolized at a concentration above 5.6 × 102 CFU/m3 and analyzed with pour plate method. The microorganisms present in the ambient room air interfere with precise quantification of Bacillus endospores when their concentration is relatively low. The results of this study may be helpful in critical assessment of the results obtained from monitoring the air for bacterial endospores.

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy. A short incubation of bacillus endospore suspensions with ThT, under ambient conditions, resulted in (i) an enhancement of the fluorescence of ThT and (ii) the accumulation of ThT in the endospores, affording fluorescence images with excellent contrast ratios. Fluorescence images revealed that ThT tends to accumulate in the surface regions of the endospores. The observed fluorescence enhancement and dye accumulation, coupled with the sensitivity of emission techniques, provide an effective and rapid means of staining endospores without the inconvenience of pre- or posttreatment of samples.

Differentiation of Bacillus endospore species from fatty acid methyl ester biomarkers

A simple method to detect and differentiate Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) endospores using biomarker compounds, including dipicolinic acid methyl ester (DPAME) and fatty acid methyl esters (FAMEs), has been developed. The method is based on thermochemolysis methylation (TCM) of the endospores and gas chromatography-mass spectrometry (GC-MS) of the reaction products. A suspension of the sample mixed with sulfuric acid (H2SO4) and tetramethylammonium hydroxide (TMAH) in methanol (MeOH) at room temperature is sampled using a coiled wire filament (CWF) device, which consists of a tiny platinum helical wire coil attached to a retractable plunger that moves the coil in and out of a syringe needle housing. Sampling is accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port. While DPAME can be used for the general detection of endospores, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME could be detected in samples containing as few as 6000 endospores, and the GC-MS peak area percent reproducibility for FAMEs varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples.

SYTO dyes and histoproteins—Myriad of applications

SYTO dyes and histoproteins—Myriad of applications

Endospore dipicolinic acid detection during Bacillus thuringiensis culture

The aim of this work was to detect Bacillus thuringiensis endospore production during fermentation under conditions hindering endospore detection, i.e. in a complex undefined industrial medium with a high content of solids in suspension. Bacterial endospore production was measured using the photoluminescence of dipicolinate (DPA) with Tb3+. The high temperature and pressure of a conventional autoclave was used to release DPA from the endospores. The endospore was obtained from B. thuringiensis var. kurstaki HD-73 fermentations in industrial-type media with 25.1 and 54.1 g l(-1) glucose, 4.4 and 35.3 g l(-1) soybean meal, 5.8 g l(-1) yeast extract, 9.2 g l(-1) corn steep solids and mineral salts. In this study, we successfully determined the DPA concentrations during the culture of B. thuringiensis in high-concentration soybean meal media. A good correlation was found between microscope endospore counting and DPA measurements in the cultures. Because of synergy between Cry protein and endospore in B. thuringiensis bioinsecticides formulation, it is important to be able to determine endospore development during B. thuringiensis industrial-type fermentation, in order to ascertain the beginning of sporulation.

An automated front‐end monitor for anthrax surveillance systems based on the rapid detection of airborne endospores

A fully automated anthrax smoke detector (ASD) has been developed and tested. The ASD is intended to serve as a cost effective front-end monitor for anthrax surveillance systems. The principle of operation is based on measuring airborne endospore concentrations, where a sharp concentration increase signals an anthrax attack. The ASD features an air sampler, a thermal lysis unit, a syringe pump, a time-gated spectrometer, and endospore detection chemistry comprised of dipicolinic acid (DPA)-triggered terbium ion (Tb(3+)) luminescence. Anthrax attacks were simulated using aerosolized Bacillus atrophaeus spores in fumed silica, and corresponding Tb-DPA intensities were monitored as a function of time and correlated to the number of airborne endospores collected. A concentration dependence of 10(2)-10(6) spores/mg of fumed silica yielded a dynamic range of 4 orders of magnitude and a limit of detection of 16 spores/L when 250 L of air were sampled. Simulated attacks were detected in less than 15 min.

Medium Effects on the Stability of Terbium(III) Complexes with Pyridine-2,6-dicarboxylate

Systematic investigation of the stability of mono-, bis-, and tris-terbium(III) complexes with dipicolinic acid is reported. The formation constants were extracted from emission titration data. For this purpose, a mathematical formalism for two complexation models was developed. Comparative studies were conducted by variation of the acidity of the medium (from H0 = −2 to pH = 7), the buffer composition (i.e., containing seven different anions), and the salt concentration (from 0.04 to 1 M). It became evident that elevating the pH and lowering the salt concentration were important, not only for increasing the values of the formation constants, but also for forming polynuclear Tb−DPA complexes. Inhibition of the latter phenomenon was observed when acetate or sulfate was present. The main purpose of the present investigation was to establish optimum conditions for conducting terbium(III)-mediated emission assays for bacterial endospore detection.

Enhanced spore detection using dipicolinate extraction techniques

We have developed a method for bacterial endospore detection based on the presence dipicolinic acid (dpa), a substance unique to endospores. Since the sensitivity of this technique correlates directly with the amount of dpa extracted from the spores, we examined several types of extraction techniques for their dpa extraction efficiency. The three main categories investigated are physical, germination, and chemical methods for liberation of dpa from B. subtilis endospores. Attention is concentrated on the speed, efficiency, and simplicity of the extraction techniques for optimization of endospore detection using terbium–dipicolinate photoluminescence. Although methods from all categories succeeded in extracting dpa, the technique utilizing heated dodecylamine (dda) extracted the majority of the available dpa in less than 3min. Application of the dda extraction procedure to the terbium–dipicolinate photoluminescence method in conjunction with an increased detection capability resulted in a two-order of magnitude improvement in endospore detection. This combination of methods resulted the lowest reported limit of detection (LOD) (1000CFU/ml) for a terbium–dipicolinate photoluminescence method in the shortest reported time (5–7min) for the total procedure.

Mitigating phosphate interference in bacterial endospore detection by Tb dipicolinate photoluminescence

A new technique for the detection of bacterial endospores using terbium dipicolinate photoluminescence has been previously reported in the literature. The performance of this technique is adversely affected by the presence of substances that contain phosphate ions. This deficiency must be overcome before this method can be used as a viable means of detecting bacterial endospores. We have experimentally investigated methods for mitigating the phosphate problem and demonstrate that the addition of AlCl 3 is a solution to the problem. Proper experimental procedures for this method are also presented.

Bacterial Endospore Detection Using Terbium Dipicolinate Photoluminescence in the Presence of Chemical and Biological Materials

A determination of the viability of an endospore detection technique using terbium dipicolinate photoluminescence in the presence of other chemical and biological materials was performed. The compounds and organisms examined, possible environmental constituents, covered three broad categories: organic compounds, inorganic compounds, and biological materials. Each substance was tested for a false positive, which occurs if the intrinsic terbium photoluminescence is enhanced in the absence of a bacterial endospore. The detection technique was also investigated for false negatives, which occur if a known positive endospore signal is inhibited significantly. Although several materials may give rise to false negative signals, none caused a false positive signal to be observed.

Wavelength pair selection for bacterial endospore detection by use of terbium dipicolinate photoluminescence

Two regression techniques, a multivariate technique and a subtracted component technique, were applied to two-wavelength response functions of mixtures of terbium (III) dipicolinate and insoluble bacterial particles. Two wavelength pairs were analyzed: a pair of neighboring wavelengths (450/490 nm) and a pair of widely spaced wavelengths (490/622 nm). The analysis shows that two emission-wavelength spectra from terbium-treated endospores can be distinguished from the vegetative bacteria spectrum above a limit of detection for endospores, which depends on the regression algorithm used to analyze the spectra. The subtracted component method for the neighboring wavelength pair had far lower limits of detection than the other methods.