Activity assessment of microorganisms eluted from sediments using 5-cyano-2,3-ditolyl tetrazolium chloride: a quantitative comparison of flow cytometry to epifluorescent microscopy

Enhanced natural recovery may be successfully implemented at contaminated sediment sites, which are often characterized by large volumes of sediments with low to moderate levels of contamination to cost-effectively reduce human and ecological risks. In order to evaluate the potential for microbial c...

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Bibliographic Details
Published in:	Journal of microbiological methods Vol. 55; no. 3; pp. 865 - 874
Main Authors:	Gruden, Cyndee L., Khijniak, Ann, Adriaens, Peter
Format:	Journal Article Conference Proceeding
Language:	English
Published:	Shannon Elsevier B.V 01-12-2003 Elsevier Science
Subjects:	5-Cyano-2,3-ditolyl tetrazolium chloride Anaerobic Biological and medical sciences CTC DNA, Bacterial - analysis DNA, Bacterial - metabolism Flow cytometry Flow Cytometry - methods Fluorescent Dyes - metabolism Fundamental and applied biological sciences. Psychology Geologic Sediments - microbiology Microbiology Microscopy, Fluorescence Sediments Tetrazolium Salts - analysis Tetrazolium Salts - metabolism Flow cytometry CTC Sediments Anaerobic Microscopy Quantitative analysis
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Summary:	Enhanced natural recovery may be successfully implemented at contaminated sediment sites, which are often characterized by large volumes of sediments with low to moderate levels of contamination to cost-effectively reduce human and ecological risks. In order to evaluate the potential for microbial contribution to remediation strategies, physiological assessment of indigenous microorganisms is essential. We report here a method for rapid and accurate assessment of metabolically (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]) active microorganisms eluted from sediment, based on flow cytometry (FCM). Microorganisms eluted from sediment and suspended in estuarine medium were stained with CTC and counterstained with the DNA stain Picogreen (PG). Optimal stain concentrations and incubation times were employed. FCM quantification of the dual-stained microorganisms was not statistically different (paired t test; α=0.05; df=10) from enumeration (total or active numbers) by an established method (fluorescent microscopy) over two orders of magnitude (approximately 10 4–10 6/ml). This research suggests that FCM, which allows the collection and analysis of multiple parameters (light scatter and fluorescence emission), is a good candidate for microbial characterization in complex environmental matrices, such as sediments, across a broad range of activity levels (∼2% to 84% of total). Potential applications for this FCM-based method include the rapid assessment of changes in sediment microbial activity in response to enhanced bioremediation strategies.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0167-7012 1872-8359
DOI:	10.1016/j.mimet.2003.08.005