Specific and rapid enumeration of viable but nonculturable and viable-culturable gram-negative bacteria by using flow cytometry

Abstract

An issue of critical concern in microbiology is the ability to detect viable but nonculturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods) are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium cells after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40, and propidium iodide (PI). The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

FIG. 1.

Histograms of the most suitable dyes in terms of the highest culturable and nonculturable E. coli O157:H7 (a and b), P. aeruginosa (d and e), P. syringae (g and h), and S. Typhimurium (j and k) counts without fluorescent beads. The agarose gel images of extracted DNA from the populations in selected gates P1 and P2 are shown on the top of corresponding gates in the histograms. The arrow indicates the location of the DNA band in the agarose gel image. The dots in gate P3 (c, f, i, and l) show the liquid counting beads for each organism.

FIG. 2.

Cell counts using the FCM for E. coli O157:H7, P. aeruginosa, P. syringae, and S. Typhimurium stained with the best two dyes, SYTO 9 and SYTO 13, versus culturable cell numbers using standard nutrient agars. The error bars along the x and y axes indicate the standard error of mean values of the respective measurements.

FIG. 3.

The number of propidium iodide-positive cells (cells with damaged cell membranes or theoretically dead) and culturable cells after heat shock at 72°C for 0, 5, 10, and 15 min. The error bars are ± the standard error of means calculated from true independent replications (three or four separately grown cultures).