The Use of Density Gradient Centrifugation for the Separation of Germinated from Nongerminated Spores

Prentice, G. A.; Wolfe, F. H.; Clegg, L. F. L.

doi:10.1111/j.1365-2672.1972.tb03706.x

Cited by 19 publications

(13 citation statements)

References 5 publications

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“…The relative order in wet density that we observed for B. anthracis: dormant spores > vegetative cells > germinated spores, agrees with previous reports on other Bacillus (Black and Gerhardt 1961;Prentice et al 1972).…”

Section: Discussionsupporting

confidence: 92%

Wet and dry density ofBacillus anthracisand otherBacillusspecies

Carrera

Zandomeni

Sagripanti

2008

Journal of Applied Microbiology

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Aims: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. Methods and Results: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1AE162 and 1AE165 g ml )1 and the other group included strains whose spores showed higher density values between 1AE174 and 1AE186 g ml )1 . Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R 2 = 0AE95) between density and volume for the spores of all strains and species studied. Conclusions: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.

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Section: Discussionsupporting

confidence: 92%

Wet and dry density ofBacillus anthracisand otherBacillusspecies

Carrera

Zandomeni

Sagripanti

2008

Journal of Applied Microbiology

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“…1965 as described in ‘Materials and methods’) able to sustain efficient growth and sporulation of all Bacillus species studied. A series of techniques involving a variety of reagents, including lysozyme (Prentice et al. 1972) and renographin (Tamir and Gilvarg 1966), have been used in other studies to purify spores from their plate or liquid cultures, separating the cells and the germinated spores from the dormant ones.…”

Section: Resultsmentioning

confidence: 99%

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

Jl¹,

Carrera²,

Insalaco³

et al. 2007

J Appl Microbiol

103

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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.

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“…After each wash, the spores were collected by low‐speed centrifugation (2000 g). The spores used in this study appeared clean by microscopic examination, and did not need additional purification steps, such as lysozyme digestion (Prentice et al. 1972) or gradient centrifugation (Tamir and Gilvarg 1966).…”

Section: Methodsmentioning

confidence: 97%

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

et al. 2007

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Aims: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. Methods and Results: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0·81 ± 0·08 μm and 0·86 ± 0·08 μm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1·26 ± 0·13 μm or shorter, and another group of strains with mean spore lengths between 1·49 and 1·67 μm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. Conclusions: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.

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The Use of Density Gradient Centrifugation for the Separation of Germinated from Nongerminated Spores

Cited by 19 publications

References 5 publications

Wet and dry density ofBacillus anthracisand otherBacillusspecies

Wet and dry density ofBacillus anthracisand otherBacillusspecies

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

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

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