3. Results and discussion
The study was performed using an automatic microplate reader
to monitor Salmonella growth under various conditions. Using a
wavelength of 630 nm we were able to monitor bacterial growth
which is amplified by appearance of the violet formazan product.
As described by Tengerdy et al. (1967), in an actively growing culture,
the reducing activity is proportional to the number of bacteria
in the culture, assuming that all bacteria are in a similar metabolic
state. Thus the optical density signals obtained in presence of this
dye can be assimilated as an extrapolation of the usual bacterial
growth curve.
3.1. Impact of TV on Salmonella spp
As previously described (Weinberg, 1953) bacteria and among
them Salmonella are sensitive to TTZ under certain conditions. High
concentrations of TTC or TV can lead to total inhibition of bacterial
growth as shown for Salmonella with a TTC concentration of 5 g/l.
However, in these studies, initial bacteria concentrations were
relatively high (approximately 105 CFU/ml) and may not reflect
interaction of TTZ with very low Salmonella inocula (less than
100 CFU/ml). In liquid culture, a minimum concentration of 0.2 g/l
for TV was shown to be necessary to stain bacteria and to obtain a
noticeable coloration (Tachon et al., 2009). In our study, we decided
to observe the impact on Salmonella growth for TV concentrations
varying from 0.4 to 1.2 g/l. As we can observe in Fig.1, increase in TV
concentration led to a decrease in apparent bacterial growth rate
with total inhibition observed for Salmonella Enteritidis at a TV
concentration of 1.2 g/l. We also observe an increase of the lag
phase and a decrease of the final optical density (OD) obtained for
Salmonella Enteritidis which appears to confirm the toxic effect of
increasing TV concentration. The impact of TV on the eight Salmonella
strains tested is summarized in Table 1 which illustrates
the evolution of apparent growth rate and the apparent lag
depending on TV concentrations.We can observed in this Table that
growth curves of Salmonella strains are amplified, shown by the
increase of m* for the lower non inhibitory concentration of TV. For
all the Salmonella strains studied we observed the typical evolution
of l and m in presence of an inhibitor. Indeed, we can observe the
progressive decrease of m* and a limited evolution of the l* until
total inhibition is achieved. Strains show similar evolution except
for Salmonella London whichwas inhibited by TV even at the lowest
concentration tested. This result is not surprising when we consider
the large number of serotypes which form the genus Salmonella
enterica.
In Fig.1 and Table 1, we observe that toxicity of TV on Salmonella
strains seems to mainly impact the apparent growth rate than lag
compared to a previous study (Junillon and Flandrois, 2014) performed
on Listeria monocytogenes and another tetrazolium salt:
TTC. In Fig. 2A, we observed a proportional decrease of m* with
increasing of TV concentration up to 0.8 g/l. A threshold appears at