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

Abstract

An issue of critical concern in microbiology is the ability to detect viable but non-culturable (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 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 typhimurium after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40 and propidium iodide (PI). The FCM data were compared with 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 non-culturable, 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 illustrates the usefulness of the approach for the rapid and unbiased detection of dead vs. live organisms.