Double staining (CTC-DAPI) for detection and enumeration of viable but non-culturable Campylobacter jejuni cells

Direct microscopic enumeration of viable Campylobacter jejuni cells (ie, respiring bacteria) were performed in both culturable and non-culturable states. Five different C jejuni strains were used, including a reference strain, ATCC 33291. Cells from all five strains were incubated alone with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a redox dye. It was reduced by an electron transport chain to an insoluble red fluorescent CTC formazan salt, which accumulated intracellularly. The presence of these red CTC crystals in the bacteria cells was indicative of cellular respiratory activity. Counterstaining with 4'-6 diamino-2 phenylindole (DAPI), which fluoresces in blue, made a suitable contrast and allowed simultaneous enumeration of total and viable bacteria on a single filter. Four hours of incubation with 5 mM CTC under a microaerobic atmosphere was found to be the optimal condition yielding the maximum number of respiring cells (both culturable and non-culturable). When used in combination with standard culture techniques, double staining makes it possible to monitor the viable but non-culturable cells of jejuni obtained by starvation more easily than with the direct viable count procedure.