Sensitive determination of microbial growth by nucleic acid staining in aqueous suspension.
The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DAPI [4',6'-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and II) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 x 10(8) Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.
Martens-Habbena
Willm
W
Paleomicrobiology Group, Institute for the Chemistry and Biology of the Marine Environment, University of Oldenburg, D-26111 Oldenburg, Germany.
Sass
Henrik
H
D052160:Benzothiazoles; D003470:Culture Media; D003959:Diamines; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D004996:Ethidium; D007211:Indoles; D009696:Nucleic Acids / Q000032:analysis; D009930:Organic Chemicals; D020560:Proteobacteria / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D011804:Quinolines; D013194:Staining and Labeling / Q000379:methods*; D014867:Water
D052160:Benzothiazoles; D003470:Culture Media; D003959:Diamines; D007211:Indoles; D009696:Nucleic Acids; D009930:Organic Chemicals; C099476:PicoGreen; D011804:Quinolines; D014867:Water; C098022:SYBR Green I; C007293:DAPI; D004996:Ethidium