ABSTRACT
Solid phase cytometry (SPC) in conjunction with fluorescent viability staining has been investigated as a tool to detect viable but non-culturable Campylobacter jejuni in drinking water. Inoculated water samples were filtered over a polyester membrane filter and the retained cells were stained using a carboxyfluorescein ester as a substrate for intracellular esterases. The number of green fluorescent bacteria was automatically counted by an Ar laser scanning device (ChemScan) in 3 min. In parallel, the plate count was determined on Columbia Blood Agar. The number of culturable cells decreased below the detection limit of plate counting in less than 50 days. In contrast, the number of fluorescent bacteria remained at its initial level for at least 85 days. The discrepancy between the two results can be attributed to the transition of culturable C. jejuni cells into VBNC C. jejuni cells. Furthermore, as SPC can distinguish between low numbers of dividing and non-dividing cells of Campylobacter it has the potential to monitor attempts to resuscitate VBNC cells.
Subject(s)
Campylobacter jejuni/isolation & purification , Flow Cytometry/methods , Microscopy, Fluorescence/methods , Water Supply , Bacteriological Techniques , Campylobacter jejuni/growth & development , Colony Count, Microbial , Culture Media , Filtration/methods , Fresh Water/microbiology , Micropore FiltersABSTRACT
Laser scanning cytometry has been investigated as a tool to detect viable but non-culturable C. jejuni in drinking water. After suspending the cells in sterile drinking water, a sample was taken every seven days, the (see text) were retained on a polyester membrane filter and labelled using a fluorescein derivative as a substrate for intracellular esterases. The number of green fluorescent bacteria was automatically counted by an Ar laser scanning device in three minutes. In parallel, the number of culturable cells was determined on a non-selective medium. The number of culturable cells decreased to below the detection limit for cultivation in less then 50 days. At the contrary, fluorescent bacteria remained at the initial level during the 71 days of incubation. The discrepancy between the two results can be assigned to the presence of VBNC C. jejuni cells. Therefore laser scanning cytometry can be used as a fast and sensitive tool to detect viable but non-culturable C. jejuni in drinking water.
