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

Martens-Habbena, Willm and Sass, Henrik ORCID: https://orcid.org/0000-0001-8740-4224 2006. Sensitive determination of microbial growth by nucleic acid staining in aqueous suspension. Applied and environmental microbiology 72 (1) , pp. 87-95. 10.1128/AEM.72.1.87-95.2006

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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 108 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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Earth and Environmental Sciences
Additional Information:	Pdf uploaded in accordance with publisher's policy at http://www.sherpa.ac.uk/romeo/issn/0099-2240/ (accessed 24/02/2014)
Publisher:	American Society for Microbiology
ISSN:	00992240
Date of First Compliant Deposit:	30 March 2016
Last Modified:	09 May 2023 10:50
URI:	https://orca.cardiff.ac.uk/id/eprint/1261

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