V. Besnard et al., Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes, LETT APPL M, 31(1), 2000, pp. 77-81
A viable but non-culturable (VBNC) bacterial state was originally detected
in studies in environmental microbiology. In particular, this state has bee
n demonstrated for a number of human pathogens (Escherichia coli, Salmonell
a enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter je
juni). The presence of VBNC cells poses a major public health problem since
they cannot be detected by traditional culturing methods and the cells rem
ain potentially pathogenic under favourable conditions. But, as far as we k
now, the VBNC state has not been yet described in Listeria monocytogenes. I
n most studies, this has been assessed by the Kogure procedure based on cel
lular elongation in the presence of DNA gyrase inhibitors. The antibiotic u
sed was nalidixic acid in order to prevent DNA replication, only efficient
in Gram-negative bacteria studies. In this study, we describe a new DVC pro
cedure to detect and count viable of L. monocytogenes suspended in filtered
, sterilized distilled water. We used different concentrations of ciproflox
acin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria
cells were removed and resuspended in BHI broth, with yeast extract and cip
rofloxacin. The mixture was incubated at different incubation times at 37 d
egrees C. After different incubation times, cells were filtered through an
isopore polycarbonate black membrane filter and covered with a DAPI solutio
n or orange acridine. The filters were prepared and examined by epifluoresc
ence microscopy. Elongated cells were counted as viable cells, whereas norm
al size was regarded as nonactive ones. This method allows determination of
ciprofloxacin concentration and incubation time optimal to detect maximum
viable cells percentage in L. monocytogenes.