Sensitive determination of microbial growth by nucleic acid staining in aqueous suspension

Applied and Environmental Microbiology
Willm Martens-Habbena, Henrik Sass

Abstract

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...Continue Reading

References

Apr 3, 2001·Applied and Environmental Microbiology·D K Button, B R Robertson
Jul 31, 2002·Applied and Environmental Microbiology·Stephanie A Connon, Stephen J Giovannoni
Aug 9, 2002·Nature·Michael S RappéStephen J Giovannoni
Mar 1, 2003·Journal of Microbiological Methods·Jeffrey D Brewster
Mar 1, 1993·Applied and Environmental Microbiology·D K ButtonB R Robertson

Citations

Sep 18, 2007·Microbial Ecology·Jacqueline SüssHenrik Sass
Feb 2, 2010·Applied and Environmental Microbiology·Hidetoshi UrakawaDavid A Stahl
Aug 17, 2006·Letters in Applied Microbiology·K-I InatomiS-S Lee
Jul 17, 2009·International Journal of Systematic and Evolutionary Microbiology·Henrik SassR F Rosenzweig
Mar 6, 2008·International Journal of Systematic and Evolutionary Microbiology·Ju Hyoung LimSung-Taik Lee
Jan 22, 2013·Cell·Corinne Ferrier MauricePeter James Turnbaugh
May 7, 2009·FEMS Microbiology Ecology·Heike BrielmannTillmann Lueders
Mar 5, 2013·Journal of Environmental Science and Health. Part. B, Pesticides, Food Contaminants, and Agricultural Wastes·Dolar PakSteven C Ricke
Mar 3, 2018·Frontiers in Cellular and Infection Microbiology·Stefanie HoffmannRoman G Gerlach
Aug 28, 2020·Frontiers in Microbiology·Ewa A PonieckaMartyn Tranter
Dec 30, 2018·Water Research·Hannah R Safford, Heather N Bischel

Related Concepts

DAPI hydrochloride
SYBR Green I
PicoGreen
Alkalescens-Dispar Group
Novidium
Indoles
Nucleic Acids
Organic Chemicals
Staining and Labeling
Proteobacteria

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