LUMINESCENCE-BASED DETECTION OF ACTIVITY OF STARVED AND VIABLE BUT NONCULTURABLE BACTERIA

A naturally luminescent bacterium, Vibrio harveyi, and two bacteria, E scherichia coli and Pseudomonas fluorescens, which had been geneticall y marked with luminescence were starved in liquid medium at 4 and 30-d egrees-C for 54 days. Total cell concentrations and concentrations of culturable and viable cells were determined by acridine orange stainin g, dilution plate counting, and direct viable counting, respectively, and population activity was measured by luminometry. V. harveyi became nonculturable but maintained viability during starvation at 4-degrees -C and maintained both culturability and viability at 30-degrees-C. In contrast, E. coli became viable but nonculturable during starvation a t 30-degrees-C but not at 4-degrees-C. Luminescence of nonculturable c ells of both strains, and culturable cells of V. harveyi, decreased to background levels during starvation. Luminescence of starved culturab le cells of E. coli also fell below background levels but occasionally increased to detectable values. Viable, nonculturable forms of P. flu orescens were not detected at either temperature, and cells starved at 4-degrees-C showed no decrease in luminescence measured during incuba tion of samples at 25-degrees-C. Following incubation of late-log-phas e cells with yeast extract and nalidixic acid, changes in light output directly paralleled changes in cell length, as observed during direct viable counting. Quantification of changes in luminescence following incubation of starved cells with yeast extract enabled measurement of the activity of both culturable and viable but nonculturable cells. Me asurement of luminescence was significantly more sensitive, rapid, and convenient in quantifying activity following nutrient amendment than measurement of changes in cell length. Luminescence-based marker syste ms potentially provide a selective means of detecting the presence and activity of viable but nonculturable cells in the soil and freshwater environments, where indigenous luminescent populations are negligible , and enable assessment of the activity and environmental impact of su ch populations.