Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ †
A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb3+ and d-alan...
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Online Access: | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067429 http://www.ncbi.nlm.nih.gov/pubmed/21296951 https://doi.org/10.1128/AEM.01966-10 |
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ftpubmed:oai:pubmedcentral.nih.gov:3067429 2023-05-15T16:26:16+02:00 Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ † Yang, Wan-Wan Ponce, Adrian 2011-04 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067429 http://www.ncbi.nlm.nih.gov/pubmed/21296951 https://doi.org/10.1128/AEM.01966-10 en eng American Society for Microbiology (ASM) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067429 http://www.ncbi.nlm.nih.gov/pubmed/21296951 http://dx.doi.org/10.1128/AEM.01966-10 Copyright © 2011, American Society for Microbiology Methods Text 2011 ftpubmed https://doi.org/10.1128/AEM.01966-10 2013-09-03T12:51:35Z A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb3+ and d-alanine triggers Clostridium spore germination and the concomitant release of ∼108 molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant green Tb3+-DPA luminescent spots as GCEs with time-gated luminescence microscopy. The intensity time courses of the luminescent spots were characteristic of stage I Clostridium spore germination dynamics. Micro-EVA was validated against traditional CFU cultivation from 0 to 1,000 total endospores/ml (i.e., phase-bright bodies/ml), yielding 56.4% ± 1.5% GCEs and 43.0% ± 1.0% CFU. We also show that d-alanine serves as a Clostridium-specific germinant (three species tested) that inhibits Bacillus germination of spores (five species tested) in that endospore concentration regime. Finally, GCE concentrations in Greenland ice cores and Atacama Desert soils were determined with micro-EVA, yielding 1 to 2 GCEs/ml of Greenland ice (versus <1 CFU/ml after 6 months of incubation) and 66 to 157 GCEs/g of Atacama Desert soil (versus 40 CFU/g soil). Text Greenland Greenland ice cores PubMed Central (PMC) Greenland Applied and Environmental Microbiology 77 7 2352 2358 |
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Methods Yang, Wan-Wan Ponce, Adrian Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ † |
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Methods |
description |
A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb3+ and d-alanine triggers Clostridium spore germination and the concomitant release of ∼108 molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant green Tb3+-DPA luminescent spots as GCEs with time-gated luminescence microscopy. The intensity time courses of the luminescent spots were characteristic of stage I Clostridium spore germination dynamics. Micro-EVA was validated against traditional CFU cultivation from 0 to 1,000 total endospores/ml (i.e., phase-bright bodies/ml), yielding 56.4% ± 1.5% GCEs and 43.0% ± 1.0% CFU. We also show that d-alanine serves as a Clostridium-specific germinant (three species tested) that inhibits Bacillus germination of spores (five species tested) in that endospore concentration regime. Finally, GCE concentrations in Greenland ice cores and Atacama Desert soils were determined with micro-EVA, yielding 1 to 2 GCEs/ml of Greenland ice (versus <1 CFU/ml after 6 months of incubation) and 66 to 157 GCEs/g of Atacama Desert soil (versus 40 CFU/g soil). |
format |
Text |
author |
Yang, Wan-Wan Ponce, Adrian |
author_facet |
Yang, Wan-Wan Ponce, Adrian |
author_sort |
Yang, Wan-Wan |
title |
Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ † |
title_short |
Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ † |
title_full |
Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ † |
title_fullStr |
Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ † |
title_full_unstemmed |
Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ † |
title_sort |
validation of a clostridium endospore viability assay and analysis of greenland ices and atacama desert soils▿ † |
publisher |
American Society for Microbiology (ASM) |
publishDate |
2011 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067429 http://www.ncbi.nlm.nih.gov/pubmed/21296951 https://doi.org/10.1128/AEM.01966-10 |
geographic |
Greenland |
geographic_facet |
Greenland |
genre |
Greenland Greenland ice cores |
genre_facet |
Greenland Greenland ice cores |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067429 http://www.ncbi.nlm.nih.gov/pubmed/21296951 http://dx.doi.org/10.1128/AEM.01966-10 |
op_rights |
Copyright © 2011, American Society for Microbiology |
op_doi |
https://doi.org/10.1128/AEM.01966-10 |
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Applied and Environmental Microbiology |
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77 |
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7 |
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2352 |
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2358 |
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1766015175947714560 |