Open AccessDetection of viable, but non‐culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique
Author(s) -
Binnerup Svend Jørgen,
Jensen Dan Funck,
ThordalChristensen Hans,
Sørensen Jan
Publication year - 1993
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.1993.tb00021.x
Subject(s) - biology , pseudomonas fluorescens , acridine orange , microbiology and biotechnology , agar , bacteria , population , staining , fluorescence microscope , agar plate , incubation , pseudomonadaceae , pseudomonas , soil microbiology , fluorescence , biochemistry , genetics , demography , physics , quantum mechanics , sociology
Kanamycin‐resistant Pseudomonas fluorescens DF57‐3 cells (Tn5 modified) inoculated in soil microcosms rapidly lost their culturability, as defined by visible colony formation on Kings B agar supplemented with kanamycin. 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 suspension of bacteria from the soil was prepared in salt solution after a sonication procedure and a sample was filtered onto a 0.2 μm Nuclepore filter. The filter was then placed for 3–4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence microscopy. By staining and washing the filters carefully, disruption of microcolonies could be avoided. A majority of the microcolonies resulted from 2–3 cell divisions during the first 2 days of the incubation period, after which the cell divisions stopped. These microcolonies were taken to represent a population of viable, but non‐culturable cells and comprised about 20% of the initial inoculum. A similar recovery was obtained when the filters were incubated on the surface of citrate minimal medium or soil extract medium. A few microcolonies showed continued growth on the filters, however, and their number corresponded well with that of visible macrocolonies. Observation by microscopy of a few (2–3) cell divisions (microcolony epifluorescence technique) is proposed for determination of subpopulations of viable, but non‐culturable bacteria in soil.