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 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; alpha=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 (approximately 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.
Gruden
Cyndee L
CL
Environmental and Water Resources Engineering, University of Michigan, Ann Arbor, MI 48104, USA.
Khijniak
Ann
A
Adriaens
Peter
P
D004269:DNA, Bacterial / Q000032:analysis / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D019015:Geologic Sediments / Q000382:microbiology*; D008856:Microscopy, Fluorescence; D013778:Tetrazolium Salts / Q000032:analysis / Q000378:metabolism*
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D013778:Tetrazolium Salts; C075453:5-cyano-2,3-ditolyltetrazolium