Bacillus Endospores Isolated from Granite: Close Molecular Relationships to Globally Distributed Bacillus spp. from Endolithic and Extreme Environments

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain ~500 cultivable Bacillus spores and ~10⁴ total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted.     

Extreme Spore UV Resistance of <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Isolates Obtained from an Ultraclean Spacecraft Assembly Facility

Recent environmental microbial sampling of the ultraclean Spacecraft Assembly Facility at NASA Jet Propulsion Laboratory (JPL-SAF) identified spores of Bacillus pumilus as major culturable bacterial contaminants found on and around spacecraft. As part of an effort to assess the efficacy of various spacecraft sterilants, purified spores of 10 JPL-SAF B. pumilus isolates were subjected to 254-nm UV and their UV resistance was compared to spores of standard B. subtilis biodosimetry strains. Spores of six of the 10 JPL-SAF isolates were significantly more resistant to UV than the B. subtilis biodosimetry strain, and one of the JPL-SAF isolates, B. pumilus SAFR-032, exhibited the highest degree of spore UV resistance observed by any Bacillus spp. encountered to date.     

Cytotoxic Bacillus spp. belonging to the B. cereus and B. subtilis groups in Norwegian surface waters

AIMS: To investigate the presence and numbers of Bacillus spp. spores in surface waters and examine isolates belonging to the B. cereus and B. subtilis groups for cytotoxicity, and to discuss the presence of cytotoxic Bacillus spp. in surface water as hazard identification in a risk assessment approach in the food industry. METHODS AND RESULTS: Samples from eight different rivers with variable degree of faecal pollution, and two drinking water sources, were heat shocked and examined for the presence of Bacillus spp. spores using membrane filtration followed by cultivation on bovine blood agar plates. Bacillus spp. was present in all samples. The numbers varied from 15 to 1400 CFU 100 ml(-1). Pure cultures of 86 Bacillus spp. isolates representing all sampling sites were characterized using colony morphology, atmospheric requirements, spore and sporangium morphology, and API 50 CHB and API 20E. Bacillus spp. representing the B. cereus and B. subtilis groups were isolated from all samples. Twenty-one isolates belonging to the B. cereus and B. subtilis groups, representing eight samples, were screened for cytotoxicity. Nine strains of B. cereus and five strains belonging to the B. subtilis group were cytotoxic. CONCLUSIONS: The presence of cytotoxic Bacillus spp. in surface water represents a possible source for food contamination. Filtration and chlorination of surface water, the most common drinking water treatment in Norway, do not remove Bacillus spores efficiently. This was confirmed by isolation of spores from tap water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of food with water containing low numbers of Bacillus spores implies a risk for bacterial growth in foods. Consequently, high numbers of Bacillus spp. may occur after growth in some products. High numbers of cytotoxic Bacillus spp. in foods may represent a risk for food poisoning.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light.

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle

AIMS: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. METHODS AND RESULTS: Six feed concentrate samples from five different farms were analysed. High levels of spores (up to 10(6) spores g(-1)) were found. Identification of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, amplified ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identified to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80 degrees C were identified as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100 degrees C, were identified as members of the B. subtilis group (five isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. CONCLUSIONS: Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. SIGNIFICANCE AND IMPACT OF THE STUDY: The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community

Plasmid genes that are responsible for virulence of Bacillus anthracis are important targets for the DNA-based detection of anthrax. We evaluated the distribution of the Ba813 chromosomal DNA sequence (Ba813) within closely related Bacillus species. Ba813 was systematically identified from 47 strains or isolates of B. anthracis tested, thus indicating its reliability as a tracer for that species. From the 60 strains of closely related Bacillus spp. examined, three bona fide B. cereus and one bona fide B. thuringiensis were found to harbour Ba813. This marker was also detected in Bacillus sp. isolates that were present at high levels in soil samples collected in a place where an anthrax outbreak had occurred. The significance and the possible function of the Ba813 locus is discussed.     

Comparison of UV inactivation of spores of three encephalitozoon species with that of spores of two DNA repair-deficient Bacillus subtilis biodosimetry strains

When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m(2), respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.     

Manganese(II)-oxidizing Bacillus spores in Guaymas Basin hydrothermal sediments and plumes

Microbial oxidation and precipitation of manganese at deep-sea hydrothermal vents are important oceanic biogeochemical processes, yet nothing is known about the types of microorganisms or mechanisms involved. Here we report isolation of a number of diverse spore-forming Mn(II)-oxidizing Bacillus species from Guaymas Basin, a deep-sea hydrothermal vent environment in the Gulf of California, where rapid microbially mediated Mn(II) oxidation was previously observed. mnxG multicopper oxidase genes involved in Mn(II) oxidation were amplified from all Mn(II)-oxidizing Bacillus spores isolated, suggesting that a copper-mediated mechanism of Mn(II) oxidation could be important at deep-sea hydrothermal vents. Phylogenetic analysis of 16S rRNA and mnxG genes revealed that while many of the deep-sea Mn(II)-oxidizing Bacillus species are very closely related to previously recognized isolates from coastal sediments, other organisms represent novel strains and clusters. The growth and Mn(II) oxidation properties of these Bacillus species suggest that in hydrothermal sediments they are likely present as spores that are active in oxidizing Mn(II) as it emerges from the seafloor.     

Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168

Endospores of Bacillus spp. were purified from three Sonoran desert soil samples by Chelex extraction and NaBr density gradient centrifugation and their UV resistances compared with that of B. subtilis strain 168. Natural spore populations exhibited tight adherence to soil particles which was not readily overcome by the extraction and purification procedure. It was observed that spores purified from soil exhibited 2-3 fold higher resistance to UV (as measured by the 90% lethal dose, LD90) than did B. subtilis strain 168 grown on NSM, a standard laboratory sporulation medium, and purified by the same extraction procedure. Cultivation of spore-forming bacteria isolated from soil on NSM resulted in production of spores with essentially identical UV resistance as strain 168, suggesting that spore UV resistance is influenced by the environment in which spores are produced.     

Bacillus sporothermodurans and other highly heat-resistant spore formers in milk

A recent example of a micro-organism causing undesired growth in consumer milk is Bacillus sporothermodurans producing highly heat-resistant spores (HRS) which may survive ultra-high temperature (UHT) treatment or industrial sterilization. Molecular typing showed a heterogeneous group of farm isolates (non-HRS strains), but a clonal group of UHT isolates from diverse European countries and other continents (HRS-clone) suggesting a common source. During a survey of Belgian dairy farms for the presence of potentially highly heat-resistant spore formers, high numbers of these spores were detected in filter cloth, green crop and fodder samples. The strain collection showed a high taxonomic diversity with 18 potentially new species and with Bacillus licheniformis and Geobacillus pallidus as predominating species overall. Seventeen B. sporothermodurans isolates were identified, mainly originating from feed concentrate. Heat resistance studies showed the UHT resistance of B. sporothermodurans spores present in industrially contaminated UHT milk, but a lower heat resistance of laboratory-grown strains (HRS and non-HRS). Hydrogen peroxide, used as sanitizer in the dairy industry, was found to induce higher heat resistance of laboratory-grown B. sporothermodurans strains to a certain level. This indicates that sublethal stress conditions may affect the heat resistance. By transmission electron microscopy, structural differences at the spore level were found between HRS and non-HRS strains. The data indicate that the attainment of extreme heat resistance is rather multifactorial.     

Rope-producing strains of Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96 degrees C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10(4) rope-producing B. subtilis G1 spores per cm(2) on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.     

青海柴达木极端干旱沙地分离芽孢杆菌的分子鉴定及拮抗活性分析

【目的】研究高原极地环境微生物资源。【方法】采用rep-PCR指纹图谱分析、gyrB基因及16S rDNA基因序列分析等多项分子鉴定技术对分离自青海柴达木极端干旱沙地的8株芽孢杆菌菌株进行分类鉴定;通过平板对峙及接种离体叶片试验检测分离菌株的拮抗活性及对病原菌侵染的防效;采用MALDI-TOF-MS质谱分析生防菌株的活性成分。【结果】8株分离菌株鉴定为Bacillus amyloliquefaciens(6株)、Bacillus axarquiensis(1株)和Bacillus atrophaeus(1株);各菌株对油菜菌核病原真菌(Sclerotinia sclerotiorum)均具有显著的拮抗活性;接种离体叶片试验表明菌株对油菜菌核病菌的侵染具有较好防效;MALDI-TOF-MS质谱分析结果显示菌株DGL1(B.amyloliquefaciens)产生脂肽化合物Fengycin,菌株DGL6(B.axarquiensis)产生脂肽化合物Surfactin、BacillomycinsD和Fengycin,菌株DCD1(B.atrophaeus)产生脂肽化合物Surfactin、Fengycin。【结论】为高原干旱沙地极端环境微生物资源研究及生防菌资源开发和应用提供了研究材料。     

Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.     

Role of DNA repair in Bacillus subtilis spore resistance.

Wet-heat or hydrogen peroxide treatment of wild-type Bacillus subtilis spores did not result in induction of lacZ fusions to three DNA repair-related genes (dinR, recA, and uvrC) during spore outgrowth. However, these genes were induced during outgrowth of wild-type spores treated with dry heat or UV. Wet-heat, desiccation, dry-heat, or UV treatment of spores lacking major DNA-binding proteins (termed alpha-beta- spores) also resulted in induction of the three DNA repair genes during spore outgrowth. Hydrogen peroxide treatment of alpha-beta-spores did not result in induction of dinR- and rerA-lacZ but did cause induction of uvrC-lacZ during spore outgrowth. Spores of a recA mutant were approximately twofold more UV sensitive and approximately ninefold more sensitive to dry heat than were wild-type spores but were no more sensitive to wet heat and hydrogen peroxide. In contrast, alpha-beta- recA spores were significantly more sensitive than were alpha-beta- spores to all four treatments, as well as to desiccation. Surprisingly, RecA levels were quite low in dormant spores, but RecA was synthesized during spore outgrowth. Taken together, these data (i) are consistent with previous suggestions that some treatments (dry heat and UV with wild-type spores; desiccation, dry and wet heat, hydrogen peroxide, and UV with alpha-beta- spores) that kill spores do so in large part by causing DNA damage and (ii) indicate that repair of DNA damage during spore outgrowth is an important component of spore resistance to a number of treatments, as has been shown previously for UV.     

UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Determination of the most closely related bacillus isolates to Bacillus anthracis by multilocus sequence typing

There have been many efforts to develop Bacillus anthracis detection assays, but the problem of false-positive results has often been encountered. Therefore, to validate an assay for B. anthracis detection, it is critical to examine its specificity with the most closely related Bacillus isolates that are available. To define the most closely related Bacillus isolates to B. anthracis in our Bacillus collections, we analyzed by multilocus sequence typing (MLST) the phylogeny of 77 closely related Bacillus isolates selected from 264 Bacillus isolates. The selection includes all the Bacillus isolates that have been shown in our previous studies to produce false-positive results by some anthrax-detection assays. The MLST phylogenetic analyses revealed that 27 of the non-B. anthracis isolates clustered within the B. anthracis clade, and four of them (three sequence types, STs) had the highest degree of genetic relatedness with B. anthracis, 18 (11 STs) had the second highest, and five (five STs) had the third highest. We anticipate that the inclusion of the 19 ST isolates when analyzing B. anthracis detection assays will prove to be useful for screening for their specificity to detect B. anthracis.     

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Aims:  To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.
Methods and Results:  Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis , e.g. lack of β -haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis . PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis , but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an Alu I restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different.
Conclusions:  Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples.
Significance and Impact of the Study:  This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.     

Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis

Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells. Ruthenium red staining followed by electron microscopic analysis was used to demonstrate the presence of an external glycoprotein layer surrounding the spore of both Bacillus anthracis and Bacillus subtilis. This layer is less apparent with traditional staining methods used for electron microscopy. Renografin gradients were used to purify B. subtilis spores. These purified spores displayed greatly enhanced staining with ruthenium red, indicating nonspecific binding of renografin, which has a major carbohydrate constituent, methylglucamine. For B. anthracis, staining with ruthenium red was sufficiently intense that it was not significantly enhanced by renografin purification. In addition to demonstrating a previously undiscovered layer surrounding the spores of B. subtilis, the results help explain a long-standing controversy as to ultrastructural differences among these genetically closely related organisms. Ruthenium red staining provides an important addition to the identification of surface glycoproteins in studies to define similarities and differences in the exosporium layers of Bacillus species.