| Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage using EMA, sodium deoxycholate and real-time PCR. | |
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MedLine Citation:
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PMID: 19733199 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The effect of refrigerated and frozen storage on the viability of Vibrio vulnificus was evaluated using cell suspensions (1x10(8) CFU/ml). Ethidium bromide monoazide (EMA) was utilized to selectively allow real-time (Rti) PCR amplification of target DNA from viable but not dead cells. Bacterial survivors from the EMA Rti-PCR were evaluated by comparison with the plate count assay following different temperature exposures (-20 and 4 degrees C) every 24 h for 72 h. The log CFU values from the EMA Rti-PCR assays were erroneously higher than that from plate counts. DNA amplification was not completely suppressed by EMA treatment of low temperature destroyed cells suggesting that membrane damage was not sufficient to allow effective EMA penetration into the cells. The optimal concentration of sodium deoxycholate (SD) was also determined to enhance discrimination of viable and dead cells following exposure of cells to low temperatures. The use of 0.01% or less of SD did not inhibit the Rti-PCR amplification derived from viable bacterial cells. A rapid decrease of the log CFU was observed with cell suspensions subjected to frozen storage and a slow decline in the log CFU occurred at 4 degrees C. The combination of SD and EMA treatments applied to cells of V. vulnificus held at -20 degrees C and 4 degrees C resulted in a high level of correlation between the log of CFU (plate counts) and the log of the number of viable cells determined from SD+EMA Rti-PCR. |
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Authors:
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Jung-Lim Lee; Robert E Levin |
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Publication Detail:
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Type: Comparative Study; Evaluation Studies; Journal Article; Research Support, U.S. Gov't, Non-P.H.S. Date: 2009-09-03 |
Journal Detail:
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Title: Journal of microbiological methods Volume: 79 ISSN: 1872-8359 ISO Abbreviation: J. Microbiol. Methods Publication Date: 2009 Nov |
Date Detail:
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Created Date: 2009-10-20 Completed Date: 2009-12-16 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8306883 Medline TA: J Microbiol Methods Country: Netherlands |
Other Details:
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Languages: eng Pagination: 184-8 Citation Subset: IM |
Affiliation:
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Department of Food Science, Massachusetts Agricultural Experiment Station, University of Massachusetts, Amherst, MA 01003, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Colony Count, Microbial Deoxycholic Acid / pharmacology* Detergents / pharmacology Diagnostic Errors Ethidium / pharmacology* Fluorescent Dyes / pharmacology Freezing* Humans Microbial Viability / radiation effects* Polymerase Chain Reaction / methods* Refrigeration* Statistics as Topic Vibrio vulnificus / physiology*, radiation effects |
| Chemical | |
Reg. No./Substance:
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0/Detergents; 0/Fluorescent Dyes; 3546-21-2/Ethidium; 83-44-3/Deoxycholic Acid |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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