Bacillus pantothenticus Spores activated by Dimethylformamide and Dimethylsulphoxide

Widdowson, Jean P.

doi:10.1038/214812a0

Cited by 11 publications

(3 citation statements)

References 6 publications

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“…An intrinsic limitation of the gentamicin protection assay, as with any assay based on quantifying CFU, is that information can be obtained only about viable organisms, e.g., there can be no accounting for B. anthracis spores that were taken up by macrophages but rapidly killed prior to serial plating. Germination-deficient spores (6,42,44,48,49) that are phagocytosed into a host cell vacuole but are unable to germinate also cannot be detected by assaying for CFU. Gentamicin protection assays are also limited by their ability to reveal only the average number of viable B. anthracis spores per macrophage within a population, but they cannot provide information at the single-cell level about the percentage of macrophages that become infected or how many spores are taken up into individual macrophages.…”

mentioning

confidence: 99%

High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently LabeledBacillus anthracisSpores

Stojkovic¹,

Torres²,

Prouty³

et al. 2008

Appl Environ Microbiol

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The engulfment of Bacillus anthracis spores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a highthroughput, nonsubjective manner, we labeled B. anthracis Sterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.

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confidence: 99%

High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently LabeledBacillus anthracisSpores

Stojkovic¹,

Torres²,

Prouty³

et al. 2008

Appl Environ Microbiol

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“…It has been suggested that activation by heat, reducing agents, and low pH may involve a reversible denaturation of proteins (e.g., by reduction of S-S bonds) resulting either in an "unblocking" of an enzyme system, or in a change in the permeability of a structure controlling the dormant state of the spore (14). Polar solvents which change protein conformation also activate spores (33). The high refractive index of spores may be attributable to dehydrated protein (30) which is not easily hydrated (32).…”

Section: Discussionmentioning

confidence: 99%

“…formamide and dimethylsulfoxide (33), by aging (23), by aqueous solvents (9), and by water vapor without heating (10).…”

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confidence: 99%

Water Vapor, Aqueous Ethyl Alcohol, and Heat Activation of Bacillus megaterium Spore Germination

Hyatt

Levinson

1968

J Bacteriol

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Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by L-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 2090

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Sterility Testing of Fat Emulsions Using Membrane Filtration and Dimethyl Sulfoxide

Placencia¹,

Oxborrow²,

Danielson³

1982

Journal of Pharmaceutical Sciences

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Bacillus pantothenticus Spores activated by Dimethylformamide and Dimethylsulphoxide

Cited by 11 publications

References 6 publications

High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently LabeledBacillus anthracisSpores

High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently LabeledBacillus anthracisSpores

Water Vapor, Aqueous Ethyl Alcohol, and Heat Activation of Bacillus megaterium Spore Germination

Sterility Testing of Fat Emulsions Using Membrane Filtration and Dimethyl Sulfoxide

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