Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

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 ؋ 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.

High Abundance of Ammonia-OxidizingArchaeain Coastal Waters, Determined Using a Modified DNA Extraction Method

“…An alternative fluorescence-based assay using SYBR green I dye-which is less affected by RNA and extracted cocontaminants than other commonly employed fluorescent dyes (29) and which has a linear relationship between 10 and 500 pg of DNA l Ϫ1 (R 2 , 0.99)-was subsequently used to determine DNA concentrations. The DNA yield obtained with the UltraClean DNA extraction kit (0.52 Ϯ 0.04 ng ml Ϫ1 [mean Ϯ standard error {SE} for four replicates]) was significantly lower than those obtained with the FastDNA kit (1.40 Ϯ 0.15 ng ml Ϫ1 [mean Ϯ SE for four replicates]) and phenol-chloroform extraction (1.56 Ϯ 0.22 ng ml Ϫ1 [mean Ϯ SE for four replicates]) (P, Ͻ0.0001) ( Table 1).…”

Three adapted methods to quantify biomass and activity of microbial leaching cultures

“…Unfortunately cheaper dyes like ethidium bromide (2.0 Â 10 7 cells/mL) or DAPI (1.0 Â 10 8 cells/mL) have been shown to be less sensitive by Martens-Habbena and Sass (2006). Acridine Orange was not suitable at all.…”

“…The nucleotide-specific fluorophor PicoGreen was used for the subsequent staining procedure. Martens-Habbena and Sass (2006) have used this dye for the detection of 0.5 Â 10 5 cells/mL, and due to its lower background fluorescence they could demonstrate it to be superior to Acridine Orange (Martens-Habbena and Sass, 2006). Total fluorescence of the stained sample was measured by means of a microplate reader in order to keep the approach as simple as possible.…”

“…Martens-Habbena and Sass (2006) applied this technique for the biomass quantification on sediment samples. Using the fluorophors PicoGreen and SYBRGreen detection limits as low as 0.5 Â 10 5 cells/mL were reached.…”

Burkholderia sediminicola sp. nov., isolated from freshwater sediment

“…The plates were incubated in a GasPak EZ anaerobic container (BD) for 3 months at ambient temperature in a cycle consisting of 12 h light and 12 h darkness. In order to detect nonturbidity growth in extinction wells (Martens-Habbena & Sass, 2006), portions of cell suspensions were stained with 49,69-diamidino-2-phenylindole (DAPI) and fluorescence was measured by a microplate reader Flx 800 (BioTek). One single colony was isolated on a 1/10 R2A agar plate to which bacterial suspension was transferred from a well showing bacterial growth.…”