Influence of saline media on the fluorescence emission of Bacillus spores

Single-particle levitation in conjunction with 264.3-nm laser excitation is used to measure the fluorescence emission of individual particles of Bacillus globigii spores. With precise humidity control, the fluorescence emission of wetted and desiccated Bacillus spore particles is measured from 300 to 450 nm. Comparison of spectra for Bacillus spores suspended in a standard buffer aqueous solution and for a desiccated 10-mum-diameter aggregate Bacillus spore particle shows that the spectra is virtually indistinguishable. However, at 85% relative humidity, corresponding to a 4.5M sodium chloride solution, the spore spectra redshifts by approximately 25 nm. It is postulated that the spectra redshifting is a result of specific interactions between the tyrosine fluorophore of the Bacillus spore and the phosphate moieties in the buffer solution.     

Detection of minute chemical species by principal-component analysis

A novel analytical technique based on the detection of minute bands in a mixture spectrum with the use of principal-component analysis (PCA) is presented. This new aspect of PCA indicates that overlapped spectra of some components can be separated with no a priori knowledge of the components when the absorbances of the components vary greatly. This technique can be used for the detection of minute chemical species. The concept was confirmed by computer simulations. In the simulations, abstract spectra (loading vectors) were successfully obtained, and the changes of the component absorbances were also successfully followed semiquantitatively by calculating their scores. The method developed with PCA was applied to the analysis of infrared reflection-absorption (RA) spectra to study molecular interaction mechanism between alkyl-deuterated dipalmitoylphosphatidylcholine (DPPC-d(62)) monolayer and sucrose. The samples were Langmuir-Blodgett (LB) films of the DPPC-d(62) monolayer that was prepared on a sucrose solution. The LB films consisted of the following phases: air/DPPC-d(62) + sucrose/sucrose/substrate (gold). The abstract spectra corresponding to "DPPC-d(62) + sucrose" and "sucrose" phases were successfully separated by PCA, and the absorbance change of sucrose in each phase was semiquantitatively calculated from the score. The absorbance change was experimentally confirmed with quartz-crystal microbalance (QCM) experiments. In addition, minute water molecules that remained in the LB films after drying were readily detected from an abstract spectrum, and their binding site was found to be the phospholipid moiety in the head group of DPPC-d(62).     

Discrimination between Bacillus species by impedance analysis of individual dielectrophoretically positioned spores

We combine the use of dielectrophoretic positioning with electrical impedance measurements to detect and discriminate between individual bacterial spores on the basis of their electrical response. Using lithographically defined microelectrodes, we use dielectrophoresis to manipulate individual bacterial spores between the electrodes. The introduction of a single spore between the microelectrodes produces a significant change in electrical response that is species-dependent. When positioned between two electrodes and an AC voltage was applied, single spores caused current increases averaging 6.8 (+/-2.4) pA for Bacillus mycoides to 1.18 (+/-0.37) pA for Bacillus licheniformis. Using a mixture of spores of two different species, we demonstrate the ability to distinguish the species of individual spores in real time. This work demonstrates the feasibility of using impedance measurements for real-time detection and discrimination between different types of spores.     

Concentration of Bacillus spores by using silica magnetic particles

Silica particles are mainly used for the concentration of nucleic acid for diagnostic purposes. This is usually done under acidic or chaotropic conditions that will demolish most of the living organisms and prevent the application of other diagnostic tests. Here we describe the development of a method for the capturing and concentration of Bacillus spores using silica magnetic particles to enable fast and sensitive detection. We have shown that capturing various Bacilli spores via silica magnetic particles is limited, with large differences between spore batches (42 +/- 25%). The hydrophobic exosporium layer of spore limits the capture by the hydrophilic silica beads. Partial removal of Bacillus exosporium increases capture efficiency. To increase capturing efficiency without harming the spores' viability, a cationic lipid, didecyldimethylammonium bromide (DDAB), was used as a coat for the negatively charged silica particles. DDAB treatment increased capture efficiency from 42% to more than 90%. Using this method, we were able to capture as few as 100 Bacillus anthracis spores/mL with 90% efficacy. Release of captured spores was achieved by the addition of albumin. The capture and release processes were verified by plating and by flow cytometry using light scatter analysis. The method is simple, efficient, easy to operate, and fast.     

Rapid chemical digestion of small acid-soluble spore proteins for analysis of Bacillus spores

A method for the rapid identification of Bacillus spores is proposed, based on the selective release and chemical digestion of small, acid-soluble spore proteins (SASPs). Microwave-assisted acid hydrolysis of SASPs from B. anthracis str. Sterne and B. subtilis str. 168 was accomplished in a single step requiring only 90 s of heating. The peptide products of the chemical digestion were identified by postsource decay sequencing with a MALDI-TOF-MS equipped with a curved-field reflectron. The specificity of the observed SASP peptides was evaluated using a cross-species sequence search. The incomplete nature of the acid digestion under these conditions allowed detection of the digest products along with the proteins from which they originated, which increased species identification confidence. The feasibility of this approach for the rapid identification of Bacillus species was further demonstrated by analyzing a mixture of B. subtilis str. 168 and B. anthracis str. Sterne spores.     

Principal component analysis of fluorescence changes upon growth conditions and washing of Pseudomonas aeruginosa

We have measured the autofluorescence from suspensions of Pseudomonas aeruginosa in the growth medium and after one, two, and three washes. The bacterium was grown in two different media, nutrient broth and King's B broth. The bacterium was harvested after 12, 24, and 48 h of growth. The fluorescence was measured with excitation every 10 nm from 200 nm to 600 nm. The fluorescence profiles were analyzed using principal component analysis. We found that most of the information is in the first three principal components. Stark differences in the value of the first principal component were noted between the samples in broth and those with one, two, or three washings. The second and third principal components noted differences between the samples washed once and those washed two or three times. There was no significant difference between samples washed two and three times. There are small differences noted between the samples grown in the two different broths, and no differences were noted among the samples harvested at different times.     

Nondestructive varnish identification by ultraviolet fluorescence spectroscopy

The identification of the chemical nature of varnish is essential for art restorers in order to choose a suitable solvent during its removal. Until today, such identification has been performed using chemical analysis after sampling. An innovative technique is presented here, using ultraviolet (UV) fluorescence spectroscopy. The method is nondestructive, workable in situ, and leads to results in real time. It is based on the comparison between the emission spectrum of an unknown varnish with those of fresh, artificially aged, or old reference resins and varnishes, for different monochromatic excitation wavelengths. The resin and the nature of the varnish as spirit, oil, or mixed can thus be identified. Various examples are presented on home-made samples applied on fluorescent backgrounds and on real works of art.     

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.     

Single-molecule identification by spectrally and time-resolved fluorescence detection

A method to identify single molecules rapidly and with high efficiency based on simple probability considerations is proposed. In principle, any property of a detected photon in a single-molecule fluorescence experiment, e.g., emission wavelength, arrival time after pulsed excitation, and polarization, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dyes embedded in a polymer film can be identified with time-correlated single-photon counting spectrally resolved in two channels.     

Detection of anthrax simulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores

Recent advances in the development of ultrasensitive micromechnical thermal detectors have led to the advent of novel subfemtojoule microcalorimetric spectoscopy (CalSpec). On the basis of principles of photothermal IR spectroscopy combined with efficient thermomechanical transduction, CalSpec provides acquisition of vibrational spectra of microscopic samples and absorbates. We use CalSpec as a method of identifying nanogram quantities of biological micro-organisms. Our studies focus on Bacillus subtilis and Bacillus cereus spores as simulants for Bacillus anthracis spores. Using CalSpec, we measured IR spectra of B. subtilis and B. cereus spores present on surfaces in nanogram quantities (approximately 100-1,000 spores). The spectra acquired in the wavelength range of 690-4000 cm(-1) (2.5-14.5 microm) contain information-rich vibrational signatures that reflect the different ratios of biochemical makeup of the micro-organisms. The distinctive features in the spectra obtained for the two types of microorganism can be used to distinguish between the spores of the Bacillus family. As compared with conventional IR and Fourier-transform IR microscopic spectroscopy techniques, the advantages of the present technique include significantly improved sensitivity (at least a full order of magnitude), absence of expensive IR detectors, and excellent potential for miniaturization.     

Method of measuring Bacillus anthracis spores in the presence of copious amounts of Bacillus thuringiensis and Bacillus cereus

A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 mm2. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 +/- 31 (n = 11), 2742 +/- 38 (n = 3), 3053 +/- 19 (n = 2), 2777 +/- 26 (n = 2), 2953 +/- 24 (n = 2), and 3105 +/- 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 min-1. A hindrance factor (alpha) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x 105 BT + BC spores/mL, suggesting that alpha is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.     

Photocatalytic inactivation of spores of Bacillus anthracis using titania nanomaterials

Studies on photocatalytic inactivation of spores of Bacillus anthracis have been carried out using nanosized titania materials and UVA light or sun light. Results demonstrated pseudo first order behaviour of spore inactivation kinetics. The value of kinetic rate constant increased from 0.4h(-1) to 1.4h(-1) indicating photocatalysis facilitated by addition of nanosized titania. Nanosized titania exhibited superior inactivation kinetics on par with large sized titania. The value of kinetic rate constant increased from 0.02 h(-1) to 0.26 h(-1) on reduction of size from 1000 nm to 16 nm depicting the enhanced rate of inactivation of Bacillus anthracis Sterne spores on the decrease of particle size.     

Identification of bacterial spores using statistical analysis of Fourier transform infrared photoacoustic spectroscopy data

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) has been applied for the first time to the identification and speciation of bacterial spores. A total of forty specimens representing five strains of Bacillus spores (Bacillus subtilis ATCC 49760, Bacillus atrophaeus ATCC 49337, Bacillus subtilis 6051, Bacillus thuringiensis subsp. kurstaki, and Bacillus globigii Dugway) were analyzed. Spores were deposited, with minimal preparation, into the photoacoustic sample cup and their spectra recorded. Principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations were used on this spectral library to develop algorithms for step-wise classification at three levels: (1) bacterial/nonbacterial, (2) membership within the spore library, and (3) bacterial strain. Internal cross-validation studies on library spectra yielded classification success rates of 87% or better at each of these three levels. Analysis of fifteen blind samples, which included five samples of spores already in the spectral library, two samples of closely related Bacillus globigii 01 spores not in the library, and eight samples of nonbacterial materials, yielded 100% accuracy in distinguishing among bacterial/nonbacterial samples, membership in the library, and bacterial strains within the library.     

Whole-surface analysis of semiconductor wafers by accumulating short-time mapping data of total-reflection X-ray fluorescence spectrometry

Total-reflection X-ray fluorescence (TXRF) spectrometry with no chemical preconcentration, often called "straight-TXRF", is now widely used in the semiconductor industry. The small detection area of TXRF enablesmapping measurement of contamination of the semiconductor surface, which is very useful in process characterization. However, the small detection area had been believed to limit rapid whole-surface analysis. Contrary to this general understanding, in this study we demonstrated that a new method, called "sweeping-TXRF", which is essentially short-time multipoint mapping by straight-TXRF, can rapidly provide an average concentration. A considerable problem of this method is the contribution of errors in glancing angle and areal element distribution to the fluorescence. Using statistics, we examined the errors and demonstrated that most of them are canceled and are not significant in actual semiconductor applications. The results of an experiment that measured localized 6 x 10(10) atoms cm(-2) nickel contamination supported the above conclusion. Applying sweeping-TXRF to existing TXRF instruments is easy-the only requirement is a small software modification. We believe that sweeping-TXRF will be utilized for rapid whole-surface analysis in many fields, especially in the semiconductor industry.     

Two-photon luminescence imaging of Bacillus spores using peptide-functionalized gold nanorods

Bacillus subtilis spores (a simulant of Bacillus anthracis) have been imaged by two-photon luminescence (TPL) microscopy, using gold nanorods (GNRs) functionalized with a cysteine-terminated homing peptide. Control experiments using a peptide with a scrambled amino acid sequence confirmed that the GNR targeting was highly selective for the spore surfaces. The high sensitivity of TPL combined with the high affinity of the peptide labels enables spores to be detected with high fidelity using GNRs at femtomolar concentrations. It was also determined that GNRs are capable of significant TPL output even when irradiated at near infrared (NIR) wavelengths far from their longitudinal plasmon resonance (LPR), permitting considerable flexibility in the choice of GNR aspect ratio or excitation wavelength for TPL imaging.      

Real-time detection of kinetic germination and heterogeneity of single Bacillus spores by laser tweezers Raman spectroscopy

Germination is the process by which a dormant spore returns to its vegetative state when exposed to suitable conditions. We report on the real-time detection of kinetic germination and heterogeneity of single Bacillus thuringiensis spores in an aqueous solution by monitoring the calcium dipicolinate (CaDPA) biomarker with laser tweezers Raman spectroscopy (LTRS). A single B. thuringiensis spore was optically trapped in a focused laser beam, and its Raman spectra were recorded sequentially in time after exposure to a nutrient-rich medium, so that the CaDPA amount inside the trapped spore was monitored during the dynamic germination process. The CaDPA content in an individual spore was observed to remain almost constant in the first period and then decrease very rapidly due to its release into the medium (within approximately 2 min). The time-to-germination (t(germ)), defined as the time required for the CaDPA band intensity to decrease to the midpoint from its initial value, was found to be stochastic for individual spores with a typical value of approximately 30 min under the experimental conditions. The distribution of the time-to-germination was measured from a time lapse measurement of a population of spores. The results demonstrated that LTRS can be used to noninvasively detect the kinetic germination process at the single-cell level and explore cellular heterogeneity.     

Emission wavelength dependence of fluorescence lifetimes of bacteriological spores and pollens

Concern about biological terrorism has greatly increased in the 21st century, and correspondingly, so has the need for accurate detection and identification of biological hazards, such as Bacillus anthracis. Optical techniques have been shown to be useful for this purpose. Use of fluorescence lifetimes as a function of emission wavelength for different materials using point- detection methods appears to be an additional viable option. Although the lifetimes range only between 2 and 6 ns, most biological materials tested in this study were distinguishable. A preliminary database has been compiled for use in a possible future detection system.     

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.     

Optical properties of Bacillus subtilis spores from 0.2 to 2.5 num

We have used spectral reflectance and transmittance measurements combined with Kramers-Kr?nig analyses to obtain the real (n) and imaginary (k) parts of the complex refractive index, N = n + ik, of Bacillus subtilis spores over a wavelength interval from 0.2 to 2.5 mum. Samples were in the form of thin solid films, pressed pellets, and suspensions in water and glycerol. The optical constants of spores suspended in water were found to differ from those of spores suspended in glycerol. In addition, spores previously exposed to water in earlier experiments and subsequently dried exhibited different optical constants from spores that had not been exposed to water.