DETECTION OF VIABLE, BUT NON-CULTURABLE PSEUDOMONAS-FLUORESCENS DF57 IN SOIL USING A MICROCOLONY EPIFLUORESCENCE TECHNIQUE

Kanamycin-resistant Pseudomonas fluorescens DF57-3 cells (Tn5 modified ) inoculated in soil microcosms rapidly lost their culturability, as d efined by visible colony formation on Kings B agar supplemented with k anamycin. Thus, after 40 days only 0.02-0.35% of the initial inoculum was culturable. A microcolony epifluorescence technique was developed to determine the viable, but non-culturable subpopulation. A suspensio n of bacteria from the soil was prepared in salt solution after a soni cation procedure and a sample was filtered onto a 0.2 mum Nuclepore fi lter. The fitter was then placed for 3-4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence micros copy. By staining and washing the filters carefully, disruption of mic rocolonies could be avoided. A majority of the microcolonies resulted from 2-3 cell divisions during the first 2 days of the incubation peri od, after which the cell divisions stopped. These microcolonies were t aken to represent a population of viable, but non-culturable cells and comprised about 20% of the initial inoculum. A similar recovery was o btained when the filters were incubated on the surface of citrate mini mal medium or soil extract medium. A few microcolonies showed continue d growth on the filters, however, and their number corresponded well w ith that of visible macrocolonies. Observation by microscopy of a few (2-3) cell divisions (microcolony epifluorescence technique) is propos ed for determination of subpopulations of viable, but non-culturable b acteria in soil.