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

Waller, Lashanda N.; Fox, Nyssa F.; Fox, Kevin M.; Fox, Alvin; Price, Robert L.

doi:10.1016/j.mimet.2004.02.012

Cited by 100 publications

(75 citation statements)

References 19 publications

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“…The exosporium was visible, but poorly stained (Figure 3 a). Ruthenium red staining significantly enhances the visibility of the exosporium (Figure 3 b) (Waller, Fox, Fox, Fox and Price, 2004). After CO 2 +H 2 O 2 treatment, protrusions of the exosporium were observed on a minority of the spores (Figure 3 d), indicating disruption.…”

Section: Tem Imagingmentioning

confidence: 94%

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Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

Zhang¹,

Dalal²,

Matthews³

et al. 2007

Journal of Microbiological Methods

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The present work examines chemical and structural response in B. anthracis spores killed by a mixture of supercritical carbon dioxide (SCCO 2 ) and hydrogen peroxide (H 2 O 2 ). Deactivation of 6-log of B. anthracis spores by SCCO 2 +H 2 O 2 was demonstrated, but changes in structure were observed in only a small portion of spores. Results from phase contrast microscopy proved that this treatment is mild and does not trigger germination-like changes. TEM imaging revealed mild damage in a portion of spores while the majority remained intact. Dipicolinic acid (DPA) analysis showed that <10% of the DPA was released from the spore core into the external milieu, further demonstrating only modest damage to the spores. Confocal fluorescent microscopy, assessing uptake of DNAbinding dyes, directly demonstrated compromise of the permeability barrier. However, the magnitude of uptake was small compared to spores that had been autoclaved. This work suggests that SCCO 2 +H 2 O 2 is quite mild compared to other sterilization methods, which has major implications in its application. These results provide some insight on the possible interactions between spores and the SCCO 2 +H 2 O 2 sterilization process.

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Section: Tem Imagingmentioning

confidence: 94%

“…The sections were then treated with Hanaichi lead citrate (0.15 % lead nitrate, 0.15 % sodium acetate, 1% sodium citrate dissolved in 41 mL water and 9 mL of 1N sodium hydroxide (Fisher Scientific)) for 2 min. Spores were observed by transmission electron microscopy (Waller et al, 2004).…”

Section: Ultra-structural Characterizationmentioning

confidence: 99%

Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

Zhang¹,

Dalal²,

Matthews³

et al. 2007

Journal of Microbiological Methods

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“…Use of a ruthenium red staining technique which has been used for visualization of carbohydrate capsules by electron microscopy resulted in the visualization of filaments on the surfaces of B. subtilis spores (Fig. 3) (48). Spore surface filaments are present and are possibly glycoprotein in nature, although this conclusion is drawn with caution, as ruthenium red staining is not totally glycoprotein specific.…”

Section: The Spore Crust Layermentioning

confidence: 99%

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

Stewart

2015

Microbiol Mol Biol Rev

138

155

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SUMMARYMuch of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacteriumBacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present onB. subtilisspores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of other aspects of the spore. Because the bacterial spore surface is important for the spore's interactions with the environment, as well as being the site of interaction of the spore with the host's innate immune system in the case of spore-forming bacterial pathogens, the exosporium is worthy of continued investigation. Recent exosporium studies have focused largely on members of theBacillus cereusfamily, principallyBacillus anthracisandBacillus cereus. Our understanding of the composition of the exosporium, the pathway of its assembly, and its role in spore biology is now coming into sharper focus. This review expands on a 2007 review of spore surface layers which provided an excellent conceptual framework of exosporium structure and function (A. O. Henriques and C. P. Moran, Jr., Annu Rev Microbiol61:555–588, 2007,http://dx.doi.org/10.1146/annurev.micro.61.080706.093224). That review began a process of considering outer spore layers as an integrated, multilayered structure rather than simply regarding the outer spore components as independent parts.

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“…For additional purification of spores, primarily to remove vegetative debris, the procedure described by Nicholson and Setlow (20) was used with modification (33). Washed spores (25 mg) were suspended in a 70% Renografin (Sigma Aldrich, St. Louis, MO) solution to a final concentration of 50%.…”

Section: Methodsmentioning

confidence: 99%

“…The sections were then treated with Hanaichi lead citrate (0.15% lead nitrate, 0.15% sodium acetate, 1% sodium citrate dissolved in 41 ml water and 9 ml of 1 N sodium hydroxide [Fisher Scientific]) for 2 min. Spores were observed by transmission electron microscopy (33).…”

Section: Methodsmentioning

confidence: 99%

Identification of a Second Collagen-Like Glycoprotein Produced by Bacillus anthracis and Demonstration of Associated Spore-Specific Sugars

et al. 2005

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Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these sporespecific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.

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

Cited by 100 publications

References 19 publications

Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

Supercritical carbon dioxide and hydrogen peroxide cause mild changes in spore structures associated with high killing rate of Bacillus anthracis

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

Identification of a Second Collagen-Like Glycoprotein Produced by Bacillus anthracis and Demonstration of Associated Spore-Specific Sugars

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