Fundamental Food Microbiology, Third Edition - Fuad Fathir

MICROBIAL STRESS RESPONSE IN THE FOOD ENVIRONMENT 115
Because of current controversy among researchers studying the VBNC phenomenon
of bacterial cells, the views and observation of the groups are summarized in
this chapter.
B. Proponent Views
The existence of the VBNC phenomenon was determined initially in starving Vib.
vulmificus cells kept in artificial seawater at 5�C and simultaneously determining
during storage the total microscopic cell counts (TMCs) following staining with
acridine orange; direct viable cell counts (TVCs) microscopically following exposure
either to an inhibitor that enables cells to elongate but prevents cell division or to a
tetrazolium salt that is metabolized to formazan (a fluorescence compound) by the
metabolically active cells; and enumerating CFUs in a selective agar media. 17,18
Figure 9.4 presents the results of a typical study. In such a study, the difference
between TMC and TVC is regarded as dead cells and that between TVCs and CFUs
is considered as VBNC cells. Researchers hypothesize that under unfavorable environmental
conditions, the VBNC cells (in the populations) enter a dormant state as
a survival strategy. As in sporulation in bacterial cells, this is an inducible, programmed
strategy and part of the life cycle of the cells. The cells remain metabolically
active but stopped multiplication. There are also suggestions that the formation
of the VBNC state occurs in two phases. In the initial state, the cells lose culturability
but retain cellular integrity and nucleic acid structures. Gradually, the integrity of
cells is lost and degradation of nucleic acid starts, leading to cell death.
Figure 9.4 Total cell counts (TMCs), direct viable counts (TVCs), and colony forming unit
(CFU) counts of a bacterial cell suspension in artificial seawater at 5�C. TMCs
and TVCs are obtained microscopically and CFUs are obtained by enumeration
on an agar medium. See text for other explanations. (Adapted from Colwell, R.R.,
Zentbl. Bakteriol., 279, 154, 1993.)
\
Log Cells/ml
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
CFU
TVC
TMC
0.0
0 2 4 6 8
Sample Time (d)