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 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.

FIG. 1.

Comparison of six nucleic acid dyes for the detection of bacterial cells in a microplate assay. Each data point represents the mean of four (PicoGreen) or eight replicates. Standard deviations were omitted for clarity. AO, acridine orange; EthBr, ethidium bromide; PG, PicoGreen; SG II, SYBR green II; SG I, SYBR green I.

FIG. 2.

Quantification of washed E. coli cells at low cell densities by SYBR green I staining. Error bars represent standard deviations for eight replicates.

FIG. 3.

Quantification of washed E. coli cells (open circles) and lambda phage DNA (closed circles) in a microplate assay. Samples were serially diluted, and fluorescence intensities were measured after staining with SYBR green I. Linear regression was calculated from log-transformed data. Error bars represent standard deviations for eight replicates.

FIG. 4.

Interference of rRNA with SYBR green I staining of double-stranded DNA. Increasing amounts of E. coli rRNA were added to 100 ng ml⁻¹ lambda phage DNA and stained with SYBR green I. The fluorescence was measured and is displayed as a relative increase compared to that of pure DNA (defined as 100%). Linear regression was calculated from two parallel experiments.

FIG. 5.

Effects of different glutaric dialdehyde (circles) and formaldehyde (triangles) concentrations on fluorescence emission of SYBR green I-stained DNA. Different DNA concentrations (dotted lines, 10 ng ml⁻¹; dashed lines, 100 ng ml⁻¹; solid lines, 1,000 ng ml⁻¹) were incubated with fixative for 1 hour prior to staining. The resulting fluorescence was recorded and is displayed as a relative decrease compared to that of pure DNA (defined as 100%). Each data point represents the mean of three replicates. Standard deviations were negligible and were omitted for clarity.

FIG. 6.

Growth curves of Muricauda ruestringensis (A), Desulfovibrio acrylicus (B), and Oceanospirillum sp. strain GM1 (C) obtained by determining the optical density at 436 nm (filled squares) and by fluorescence measurement after SYBR green I staining (circles). The resulting growth rates of the strains in panels A to C and the other strains tested are given in Table 2.

FIG. 7.

Analysis of most-probable-number dilution series inoculated with water from Buzzards Bay. MPN series were set up in 96-well plates using three different media with different substrate additions. To 800 μl medium in the first dilution, 200 μl of sample was added and consecutively diluted into the following 11 wells. Subsamples were taken after 3, 17, and 141 days of incubation and were analyzed by SYBR green I staining and fluorescence determination. Different symbols represent seven parallel dilutions, and open diamonds represent uninoculated controls (by accident, the least diluted control of series C was inoculated but not further diluted). Medium A (top) contained peptone, yeast extract, and a substrate mix, while medium B (middle) received only the substrate mix and medium C (bottom) received no organic substrates.