A signature-based method to distinguish time-of-flight secondary-ion mass spectra from biological samples

Time-Of-Flight Mass Spectrometry (TOF-SIMS) was used to determine elemental and biomolecular ions from isolated protein samples. We identified a set of 23 mass-to-charge ratio (m/z) peaks that represent signatures for distinguishing biological samples. The 23 peaks were identified by Singular Value Decomposition (SVD) and Canonical Analysis (CA) to find the underlying structure in the complex mass-spectra data sets. From this modified data, SVD was used to identify sets of m/z peaks, and we used these patterns from the TOF-SIMS data to predict the biological source from which individual mass spectra were generated. The signatures were validated using an additional data set different from the initial training set used to identify the signatures. We present a simple method to identify multiple variables required for sample classification based on mass spectra that avoids overfit. This is important in a variety of studies using mass spectrometry, including the ability to identify proteins in complex mixtures and for the identification of new biomarkers.     

Differentiation of Clostridium botulinum Types A, B, and E by Pyrolysis-Gas-Liquid Chromatography

Vegetative cells and spores of 10 strains of Clostridium botulinum representing types A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY) medium. Five type E strains were also grown in Multipeptone-sucrose-Nutramino acids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquid chromatography (PGLC) analysis, and the resulting pyrograms were examined for variations in elution patterns between spores and vegetative cells of types A, B, and E grown in the TPSY medium and spores and vegetative cells of type E grown in the TPSY medium and spores and vegetative cells of type E grown in TPSY and MSN media. Growth and toxin production of all 10 strains of C. botulinum were investigated by using a modified dialysis sac culture technique. The dialysate supernatant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from the dialysate was also subjected to PGLC analysis. Control samples consisting of (i) noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii) 1.0 mg of partially purified toxin were also analyzed by PGLC. Differences between pyrograms of cultures were suitable for positive identification at the type level but not at the strain level. Pyrograms permitting differentiation were also obtained between spores and vegetative cells as well as between the same cultures grown in different media. The dialysis sac technique was useful in detecting growth but not toxin production of C. botulinum.     

Levels of glycine betaine in growing cells and spores of Bacillus species and lack of effect of glycine betaine on dormant spore resistance

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.     

Recovery Patterns of Spores of Putrefactive Anaerobe 3679 in Various Subculture Media after Heat Treatment

A comparative study was made of the heat resistance of spores of putrefactive anaerobe 3679 grown in two different sporulation media and of the recovery pattern of these spores in several subculturing media after treatment with moist and dry heat. The heat resistance of the spores was characterized in the form of D and z values. The D values were determined by the modified Schmidt method. The z values were established by the graphic method. The results revealed significant differences in D and z values, depending on the type of heat and sporulation and subculture media. Spores grown in beef heart infusion showed higher heat resistance than those grown in Trypticase. Among the seven subculture media used, the largest number of spores was recovered in beef infusion. The magnitude of the D values at 121.1 C obtained with spores heated in moist heat decreased, depending on the subculture medium used, in the following order: beef infusion, pea infusion, yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. With spores subjected to dry heat, D values at 148.9 C decreased with the subculture medium in the following order: beef infusion, yeast extract, pea infusion and liver infusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained with spores subjected to dry heat were approximately double those obtained with moist heat. Their relative magnitude varied slightly, depending on the type of subculture medium used. However, the relative magnitudes of the D values and z values with reference to the subculture media used were different with moist heat from those obtained with dry heat. Two theories are discussed as possible explanations for the logarithmic order of death of bacterial spores. The results obtained in these experiments, together with the findings of other workers, are most compatible with the theory that heat treatment of spores results in an increased rate of random injury to the genetic material of the spores.     

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.     

Effect of Cell Moisture on the Thermal Inactivation Rate of Bacterial Spores

Thermal inactivation rates were determined for two strains of Bacillus subtilis var. niger spores after equilibration to various relative humidity (RH) levels. In these tests, small thin stainless-steel squares were each inoculated with a drop of spore suspension and equilibrated to 11, 33, or 85% RH. Following equilibration, the squares were placed on a hot plate preheated to 108, 125, 136, 164, or 192 C for various exposure times and then assayed for surviving organisms. The results revealed that spores of the A strain of B. subtilis were least resistant if preequilibrated to 11% RH and most resistant if preequilibrated to 85% RH. The same trend was obtained at all temperatures except 192 C, at which, no difference was noted, probably because the rapid kill time approaches the heat-up time of the stainless-steel square. The B strain of B. subtilis spores showed an opposite RH effect; that is, the cells preequilibrated to 11% RH were the most resistant. Because the two strains of spores were grown on different media, further studies were conducted at 136 C after subculturing the cells on different media. When the B strain was subcultured on the A strain medium, the pattern was reversed; the cells preequilibrated to low RH were then least resistant. Although it was not possible to reverse these cells to the original pattern by subculturing on the original B strain medium again, the pattern was altered to the point that there was no significant difference in heat resistance of these cells regardless of the preequilibration RH. The same result was obtained when the A strain was grown on the B strain medium; that is, the thermal resistance could not be reversed, but it was altered from the point where the low RH equilibrated cells were least resistant initially to the point where there was no significant difference in any of the cells regardless of what RH was used for preequilibration. The thermal resistance of spores seemed to be dependent on (i) the medium on which the spores are grown, (ii) the RH on which they are exposed before heating, and (iii) some genetic characteristic of the cell.     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

Elemental composition of arbuscular mycorrhizal fungi at high salinity

We investigated the elemental composition of spores and hyphae of arbuscular mycorrhizal fungi (AMF) collected from two saline sites at the desert border in Tunisia, and of Glomus intraradices grown in vitro with or without addition of NaCl to the medium, by proton-induced X-ray emission. We compared the elemental composition of the field AMF to those of the soil and the associated plants. The spores and hyphae from the saline soils showed strongly elevated levels of Ca, Cl, Mg, Fe, Si, and K compared to their growth environment. In contrast, the spores of both the field-derived AMF and the in vitro grown G. intraradices contained lower or not elevated Na levels compared to their growth environment. This resulted in higher K:Na and Ca:Na ratios in spores than in soil, but lower than in the associated plants for the field AMF. The K:Na and Ca:Na ratios of G. intraradices grown in monoxenic cultures were also in the same range as those of the field AMF and did not change even when those ratios in the growth medium were lowered several orders of magnitude by adding NaCl. These results indicate that AMF can selectively take up elements such as K and Ca, which act as osmotic equivalents while they avoid uptake of toxic Na. This could make them important in the alleviation of salinity stress in their plant hosts.     

Use of Fatty Acid Methyl Ester Profiles for Discrimination of Bacillus cereus T-Strain Spores Grown on Different Media

The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 ω9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.In September 2001, letters containing spores of Bacillus anthracis, the causative agent for anthrax, were mailed to television and print media outlets, as well as two U.S. congressional offices, in an act of bioterrorism. Genetic tests identified a single strain of B. anthracis, Ames, in all evidence samples. Since then, considerable effort has gone into developing techniques that can be used to analyze microbiological evidence recovered from a crime scene. Because the Ames strain used in the 2001 attacks was difficult to distinguish genetically from several commonly used Ames strains (20), many recently developed techniques have concentrated on nongenetic signatures associated with the cell that are unique to the methods that were used to culture an organism. Examples include assays that detect the presence of residual agar on spores (59), C/N isotope ranges for different medium components (22, 30, 31), and detection of heme in spores grown on blood-containing media (56). Phenotypic signatures such as these that indicate specific metabolic substrates, characteristic compounds, or defining features of an organism''s production process could help in the attribution of a biocrime by providing leads or excluding suspects during an investigation (40, 59).One phenotypic system that has not been fully tested in a forensic context is fatty acid methyl ester (FAME) analysis. FAMEs have long been recognized as useful biochemical markers for bacterial classification and characterization (38, 53, 54). The types and relative abundances of fatty acids produced within a cell are largely determined by an organism''s genotype and can be used for identification of different species (50) and strains (34, 51) and for discriminating between free spores and vegetative cells (43, 47). Commercial systems that streamline fatty acid extraction and detection procedures (42) have facilitated the widespread use of fatty acid profiling to identify bacteria in clinical, agricultural, and biodefense settings (29, 53, 55).Besides aiding in the identification of bacterial species, FAME profiling can potentially provide information on the methods used to grow microorganisms of forensic interest. Within the Bacillus group, the amino acid content or the type of complex additives used in the cultivation media can significantly affect the fatty acid composition of bacterial cultures. The relative proportions of branched fatty acids (iso-odd, iso-even, and anteiso), which are prevalent in Bacillus spp. (33, 36a), are heavily dependent on the ratio of amino acid precursors (leucine, valine, and isoleucine) and the corresponding α-keto acids present in the growth media (12, 27, 28, 32). Accordingly, the complex additives and protein sources that supply these amino acid precursors in growth media also affect the fatty acid compositions of Bacillus cultures. For example, it has been reported that inclusion of components such as yeast extract, beef extract, or casein hydrolysate in growth medium formulations can change the relative ratios of iso and anteiso fatty acids in Bacillus cereus cultures (24, 25). Brain heart infusion (BHI) has been observed to have a similar effect on the relative proportions of branched fatty acids in Bacillus caldolyticus (52). Despite the clear relationship between the fatty acid compositions of vegetative cells and the formulations of growth media, no study has tested whether this could be exploited for investigative purposes by determining whether diagnostic FAME signatures for growth media exist within spores of a forensically relevant organism.To test whether FAME signatures can be used to infer the compositional characteristics of the sporulation medium, we examined fatty acid profiles among Bacillus cereus T-strain (BcT) spores grown on 10 different media, spanning nutrient formulations that varied primarily in the source of protein, either in the form of complex additives (yeast extract, beef extracts, brain-heart solids, etc.) or direct protein supplements (peptone, tryptone, or gelatin digest). Formulation pairs that differed in other medium attributes, such as the presence of supplemental sugars, the physical state (agar versus broth), or blood supplements, were included to compare the resulting FAME profile differences with the variation that derives from complex additive/protein components.The effects that protein components in the sporulation medium have on FAME profiles were specifically targeted in the experimental design because of the direct biosynthetic relationship between amino acids and the three structure classes of branched fatty acids in Bacillus (24, 27). In addition, there are a limited number of common or commercially available complex additive and protein sources used for microbiological media. Identifying forensic signatures based on a reduced number of defining components rather than the myriad of possible medium formulations makes comprehensive surveys feasible, an added advantage for any potential forensic marker.To further frame our study in the context of forensic or investigative applications, we chose B. cereus as a target organism because of its genetic, structural, and biochemical similarities to B. anthracis (6, 19, 26). Also, since evidence from the 2001 anthrax mailings was predominantly composed of Bacillus spores (3), we used spore preparations of B. cereus for all FAME analyses.Lastly, analysis of forensic signatures from fatty acid profiles is complicated by the large number of variables (typically >15) and complex interactions among different fatty acid structures during cellular biosynthesis (28). Therefore, FAME profiles were analyzed with orthogonal multivariate statistical techniques that first considered all variables simultaneously and analyzed the overall dissimilarities among spore FAME profiles and, second, maximized differentiation among groups using a subset of variables and extracted patterns in fatty acid differences that are diagnostic for specific medium formulations.     

Induction of multispored asci in two-spored strains of Saccharomyces cerevisiae by amitrole.

Although growth of two yeast strains characterized by consistent production of two diploid spores per ascus was inhibited in complex presporulation media containing amitrole, a fraction of the cells produced were able to form asci with more than two spores after transfer to acetate sporulation medium. Cells grown in a defined presporulation medium containing amitrole did not acquire this ability. The increase in spore numbers per ascus is attributed either to the induction by amitrole in growth medium of cells with more than one nucleus or to the restoration of normal meioses in the multispored asci.     

Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.     

Oxidation of Elemental Sulfur by Fusarium solani Strain THIF01 Harboring Endobacterium Bradyrhizobium sp.

Nineteen fungal strains having an ability to oxidize elemental sulfur in mineral salts medium were isolated from deteriorated sandstones of Angkor monuments. These fungi formed clearing zone on agar medium supplemented with powder sulfur due to the dissolution of sulfur. Representative of the isolates, strain THIF01, was identified as Fusarium solani on the basis of morphological characteristics and phylogenetic analyses. PCR amplification targeting 16S rRNA gene and analyses of full 16S rRNA gene sequence indicated strain THIF01 harbors an endobacterium Bradyrhizobium sp.; however, involvement of the bacterium in the sulfur oxidation is still unclear. Strain THIF01 oxidized elemental sulfur to thiosulfate and then sulfate. Germination of the spores of strain THIF01 was observed in a liquid medium containing mineral salts supplemented with elemental sulfur (rate of germinated spores against total spores was 60.2%), and the culture pH decreased from pH 4.8 to 4.0. On the contrary, neither germination (rate of germinated spores against total spores was 1.0%) nor pH decrease was observed without the supplement of elemental sulfur. Strain THIF01 could also degrade 30 ppmv and ambient level (approximate 500 pptv) of carbonyl sulfide.     

Sporulation in Bacillus subtilis is independent of membrane fatty acid composition.

Growth and sporulation of a Bacillus subtilis mutant deficient in branched fatty acid synthesis (gene symbol bfmB) were examined. The mutant, which produces an acyl-coenzyme A:acyl carrier protein transacylase with reduced affinity for branched fatty acid primers, could grow in media containing any one of a wide range of low-molecular-weight fatty acids having branched, cyclic, saturated, or unsaturated carbon chains. The fatty acid composition of cellular lipids depended on the compound used to support growth. Cultures of the bfmB mutant grown in the presence of 3-methylcrotonate contained an unusually high fraction (73%) of straight-chain fatty acids in the cellular lipids. The mutant sporulated with any one of the precursors of branched fatty acids in the medium; isolated spores contained mainly this branched fatty acid and only 10% or less straight-chain fatty acids regardless of the straight-chain fatty acid content of vegetative cells. Exceptional were spores grown in the presence of cyclobutane-carboxylic acid, which contained 28% straight-chain fatty acids. The branched fatty acid composition of spores could be modified greatly by changing the supply of precursors in the medium.     

金霉菌的生理与金霉素的生产 I.接种培养基对于金霉菌的代谢及抗生素产量的影响

将金莓菌的芽孢接种到不同的接种培养基中,24小时后再接种到同一的酦酵培养基上,发现金霉素的产量有显著不同。一个好的接种培养基比一个坏的接种培养基所生的菌体在酦酵过程中产生的金霉素可以大出7至8倍。接种培养基的 pH 值对于以后菌体在酦酵培养基中金霉素的生产也有显著的影响。接种培养基并不显著地影响菌体在酦酵培养基上的生长,但已显然变更了它的代谢作用。由不同接种培养基中萌生的菌体,在同一酦酵培养基中醣的利用率不同,氧化的能力不同,有机酸的形成和堆积也不同,它们对于一些金属元素的反应也不一样。这个研究证明了金霉菌的初期的培养环境,对于以后菌体的发育和金霉素的生产有极大的影响。同时亦指出了金霉菌的发育在不同时期需要不同的环境,有着不同的代谢类型。去掌握这个生物规律——环境条件和发育的过程,是正确的研究方向,是提高抗生素产量的必要途径。     

Stable isotope ratios and forensic analysis of microorganisms

In the aftermath of the anthrax letters of 2001, researchers have been exploring various analytical signatures for the purpose of characterizing the production environment of microorganisms. One such signature is stable isotope ratios, which in heterotrophs, are a function of nutrient and water sources. Here we discuss the use of stable isotope ratios in microbial forensics, using as a database the carbon, nitrogen, oxygen, and hydrogen stable isotope ratios of 247 separate cultures of Bacillus subtilis 6051 spores produced on a total of 32 different culture media. In the context of using stable isotope ratios as a signature for sample matching, we present an analysis of variations between individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times. Additionally, we correlate the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen for growth medium nutrients or water with those of spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures.     

Identification of Bacillus spores by matrix-assisted laser desorption ionization-mass spectrometry.

Unique patterns of biomarkers were reproducibly characterized by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry and were used to distinguish Bacillus species members from one another. Discrimination at the strain level was demonstrated for Bacillus cereus spores. Lipophilic biomarkers were invariant in Bacillus globigii spores produced in three different media and in B. globigii spores stored for more than 30 years. The sensitivity was less than 5,000 cells deposited for analysis. Protein biomarkers were also characterized by MALDI analysis by using spores treated briefly with corona plasma discharge. Protein biomarkers were readily desorbed following this treatment. The effect of corona plasma discharge on the spores was examined.     

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.     

Metabolic adaptation of Pteris vittata L. gametophyte to arsenic induced oxidative stress

The sporophyte and gametophyte of Pteris vittata are arsenic hyperaccumulators, however, little is known about the mechanism by which the gametophyte deals with this toxic element. An in vitro system (spores grown in arsenic amended nutrient media) was used to investigate the impact of arsenic on growth of the gametophyte and the role of antioxidative systems in combating As-stress. When mature spores of P. vittata were grown in medium amended with 0-50 mg kg(-1) of arsenic (as arsenate), the arsenic concentration in the gametophyte increased, with increasing arsenate in the media, but did not inhibit the spore germination and biomass development. Increases in the level of antioxidant enzymes, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione-Stransferase) and of ascorbic acid and glutathione probably enabled the gametophyte to withstand the oxidative stress caused by arsenate.     

Effect of Pyroquilon, an Inhibitor of Melanin Synthesis, on Sporulation and Secondary Infection of Magnaporthe grisea

The effect of pyroquilon. an inhibitor of meianin synthesis. on the sporulation and secondary infection of Magnaporthe grisea spores was investigated. Spore formation of M. grisea was significantly inhibited on the pyroquilon-containing medium. but mycelial growth was not impaired. Moreover, although the colour of the spore suspension obtained from control medium without pyroquilon was black, the suspension prepared from spores which had grown on the pyroquilon-containing medium was red-brown. The cell walls of the spores consisted of two layers. the outer of which was highly electron-dense and saw-like in cross section, regardless of treatment. Both the outer and the inner layers of the cell walls of spores which had grown on pyroquilon-containing medium were thin compared with those of control spores. When M. grisea spores which had formed on the pyroquilon-containing medium were inoculated onto rice leaf sheaths, red brown appressoria were formed. Compared with the control, appressorial penetration and hyphal growth in the host cells were inhibited. The inhibitory effect pyroquilon exerted upon the infection behavior of M. grisea spores was dependent on the dose of the chemical.     

Stable Isotope Ratios and Forensic Analysis of Microorganisms

In the aftermath of the anthrax letters of 2001, researchers have been exploring various analytical signatures for the purpose of characterizing the production environment of microorganisms. One such signature is stable isotope ratios, which in heterotrophs, are a function of nutrient and water sources. Here we discuss the use of stable isotope ratios in microbial forensics, using as a database the carbon, nitrogen, oxygen, and hydrogen stable isotope ratios of 247 separate cultures of Bacillus subtilis 6051 spores produced on a total of 32 different culture media. In the context of using stable isotope ratios as a signature for sample matching, we present an analysis of variations between individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times. Additionally, we correlate the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen for growth medium nutrients or water with those of spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures.