A rapid staining technique for the detection of the initiation of germination of bacterial spores

Hamouda, Tarek; Shih, Amy Y.; Baker, James R.

doi:10.1046/j.1472-765x.2002.01047.x

Cited by 76 publications

(53 citation statements)

References 24 publications

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“…To confirm spore germination, treated C. sordellii spores were smeared across a glass slide, air dried, and heat fixed over a flame. Cells were stained using the Wirtz-Conklin staining technique as described previously (25). Briefly, heat-fixed spore/bacterial smears were immersed in boiling malachite green stain (5 g/100 ml water) for 1 min.…”

Section: Methodsmentioning

confidence: 99%

Requirements for Germination ofClostridium sordelliiSporesIn Vitro

Ramírez

Abel-Santos

2010

J Bacteriol

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Clostridium sordellii is a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence of C. sordellii infection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment of Bacilli and Clostridia infections, we analyzed the requirements for C. sordellii spore germination in vitro. Our data showed that C. sordellii spores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case for Bacilli species, D-alanine had no effect on C. sordellii spore germination. C. sordellii spores germinated only in a narrow pH range between 5.7 and 6.5. In contrast, C. sordellii spore germination was significantly less sensitive to temperature changes than that of the Bacilli. The analysis of the kinetics of C. sordellii spore germination showed strong allosteric behavior in the binding of L-phenylalanine and L-alanine but not in that of bicarbonate or L-arginine. By comparing germinant apparent binding affinities to their known in vivo concentrations, we postulated a mechanism for differential C. sordellii spore activation in the female reproductive tract.

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Section: Methodsmentioning

confidence: 99%

Requirements for Germination ofClostridium sordelliiSporesIn Vitro

Ramírez

Abel-Santos

2010

J Bacteriol

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“…Cultures were grown in 50-ml aliquots contained in 500-ml plastic Erlenmeyer flasks fitted with Whatman Bug stoppers (Fisher Scientific, Hampton, NH) at 37°C with gentle shaking (180 rpm) for 48 h, after which 100 ml of sterile distilled water was added to dilute the medium and promote sporulation. After 10 to 11 days of continuous shaking, sporulation was confirmed at Ͼ99% via phase-contrast microscopy and a modified Wirtz-Conklin spore stain (13), and the spores were centrifuged at 630 ϫ g in a sealed-carrier centrifuge (Jouan Inc., Winchester, VA) at 4°C for 15 min. The spore pellets were then washed four times in Cellgro sterile water (Mediatech, Herndon, VA) and resuspended in sterile water.…”

Section: Methodsmentioning

confidence: 99%

Human Monoclonal Antibody AVP-21D9 to Protective Antigen Reduces Dissemination of the Bacillus anthracis Ames Strain from the Lungs in a Rabbit Model

et al. 2007

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Dutch-belted and New Zealand White rabbits were passively immunized with AVP-21D9, a human monoclonal antibody to protective antigen (PA), at the time of Bacillus anthracis spore challenge using either nasal instillation or aerosol challenge techniques. AVP-21D9 (10 mg/kg) completely protected both rabbit strains against lethal infection with Bacillus anthracis Ames spores, regardless of the inoculation method. Further, all but one of the passively immunized animals (23/24) were completely resistant to rechallenge with spores by either respiratory challenge method at 5 weeks after primary challenge. Analysis of the sera at 5 weeks after primary challenge showed that residual human anti-PA levels decreased by 85 to 95%, but low titers of rabbit-specific anti-PA titers were also measured. Both sources of anti-PA could have contributed to protection from rechallenge. In a subsequent study, bacteriological and histopathology analyses revealed that B. anthracis

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“…In a control experiment, B. cereus spores were prepared without the use of lysozyme. The purity of spore suspensions was checked using Wirtz-Conklin stain (5% aqueous malachite green and safranine O) (20) and transmission electron microscopy. Spores were Ͼ95% pure, with the remaining material being fragments of vegetative sporangium (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Photodynamic Inactivation of Bacillus Spores, Mediated by Phenothiazinium Dyes

Demidova

Hamblin

2005

Appl Environ Microbiol

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Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.

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A rapid staining technique for the detection of the initiation of germination of bacterial spores

Cited by 76 publications

References 24 publications

Requirements for Germination ofClostridium sordelliiSporesIn Vitro

Requirements for Germination ofClostridium sordelliiSporesIn Vitro

Human Monoclonal Antibody AVP-21D9 to Protective Antigen Reduces Dissemination of the Bacillus anthracis Ames Strain from the Lungs in a Rabbit Model

Photodynamic Inactivation of Bacillus Spores, Mediated by Phenothiazinium Dyes

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