Microbial Stress Response in the Food Environment ◾ 111
Opponent Views
Several questions have been raised by some researchers regarding the existence of the bacterial
VBNC phenomenon. One is based on the hypothesis of the proponent group that the VBNC
state is a survival strategy programmed into the life cycle, induced under unfavorable conditions
(as in the sporulation process), and thus genetically determined. Although the genetic basis of
bacterial sporulation is well understood, there is no information to prove directly that the VBNC
state in bacteria is programmed and has a genetic basis. This information is necessary to resolve
the hypothesis. Another question has been raised on the study methods used to determine the
VBNC phenomenon. In the direct method, VBNC cells in a population are determined from the
differences in TVCs, determined microscopically, and CFUs, determined by agar media plating
methods. Vital staining and optical methods used in TVC studies are not accepted by many in
the scientific community as effective methods to differentiate between dead cells and viable cells.
In the indirect methods used to study cell volume, membrane permeability, membrane potential,
the total population consisting of a mixture of dead cells (in large numbers), VBNC cells, and
cells capable of forming CFUs were used instead of using only the VBNC cells. Currently, no
method is available to separate the three cell groups in a population and conduct studies on the
cellular changes only in the VBNC cells. Therefore, the validity of these indirect studies is open
to question. Finally, some have questioned the validity of the resuscitation method used by proponent groups to prove the existence of the VBNC phenomenon. An increase in CFUs, following
incubation of a total population of stressed cells in a suitable environment for one or more days,
was suggested because of the regain of culturability of the VBNC cells. Some researchers have
suggested that this large increase in CFUs (as much as six or more log cycles) can be a result of the
multiplication of a few viable cells during prolonged incubation. Determining simultaneously the
TMCs, TVCs, and CFUs during a resuscitation period, as was conducted during the determination of VBNC cells (Figure 10.5), would have proven that the increase in CFUs during resuscitation was not from growth of a few viable cells but from the transformation of VBNC cells to the
culturable state. Similarly, VBNC cells regain virulence following resuscitation, as determined by
the resuscitated cells attaching to HeLa cells, colonizing egg embryo, and causing intestinal infection, resulted from the growth of a few viable cells in the original population.
Current Views
The results of several recent studies have provided a better insight on bacterial cells exposed to an
unfavorable environment for a prolonged period. These results show that the so-called VBNC cells
are really viable as well as culturable but are not recovered unless they are exposed to appropriate
culture conditions. Many of the starved or stressed cells have imbalanced metabolic pathways.
When these cells are exposed to a nutritionally rich medium, they start metabolism and generate
large quantities of superoxides and free radicals. However, because of defective metabolic processes, they cannot detoxify these products and are killed. This death can be prevented or greatly
reduced by exposing the cells in a medium that either reduces the production of the superoxides
and free radicals or increases their degradation. Supplementation of pyruvate or catalase to resuscitation media was thus found to transform the cells to a culturable state very rapidly, as determined
by an almost immediate increase in CFUs without any change in TMCs. Several studies in the
1970s and 1980s on bacterial sublethal injury have shown that following environmental stress of
a bacterial population, the recovery of the survivors can be increased either by incubating the cells
in a minimal medium or in a nutritionally rich medium supplemented with pyruvate or catalase.
Opponent Views
Several questions have been raised by some researchers regarding the existence of the bacterial
VBNC phenomenon. One is based on the hypothesis of the proponent group that the VBNC
state is a survival strategy programmed into the life cycle, induced under unfavorable conditions
(as in the sporulation process), and thus genetically determined. Although the genetic basis of
bacterial sporulation is well understood, there is no information to prove directly that the VBNC
state in bacteria is programmed and has a genetic basis. This information is necessary to resolve
the hypothesis. Another question has been raised on the study methods used to determine the
VBNC phenomenon. In the direct method, VBNC cells in a population are determined from the
differences in TVCs, determined microscopically, and CFUs, determined by agar media plating
methods. Vital staining and optical methods used in TVC studies are not accepted by many in
the scientific community as effective methods to differentiate between dead cells and viable cells.
In the indirect methods used to study cell volume, membrane permeability, membrane potential,
the total population consisting of a mixture of dead cells (in large numbers), VBNC cells, and
cells capable of forming CFUs were used instead of using only the VBNC cells. Currently, no
method is available to separate the three cell groups in a population and conduct studies on the
cellular changes only in the VBNC cells. Therefore, the validity of these indirect studies is open
to question. Finally, some have questioned the validity of the resuscitation method used by proponent groups to prove the existence of the VBNC phenomenon. An increase in CFUs, following
incubation of a total population of stressed cells in a suitable environment for one or more days,
was suggested because of the regain of culturability of the VBNC cells. Some researchers have
suggested that this large increase in CFUs (as much as six or more log cycles) can be a result of the
multiplication of a few viable cells during prolonged incubation. Determining simultaneously the
TMCs, TVCs, and CFUs during a resuscitation period, as was conducted during the determination of VBNC cells (Figure 10.5), would have proven that the increase in CFUs during resuscitation was not from growth of a few viable cells but from the transformation of VBNC cells to the
culturable state. Similarly, VBNC cells regain virulence following resuscitation, as determined by
the resuscitated cells attaching to HeLa cells, colonizing egg embryo, and causing intestinal infection, resulted from the growth of a few viable cells in the original population.
Current Views
The results of several recent studies have provided a better insight on bacterial cells exposed to an
unfavorable environment for a prolonged period. These results show that the so-called VBNC cells
are really viable as well as culturable but are not recovered unless they are exposed to appropriate
culture conditions. Many of the starved or stressed cells have imbalanced metabolic pathways.
When these cells are exposed to a nutritionally rich medium, they start metabolism and generate
large quantities of superoxides and free radicals. However, because of defective metabolic processes, they cannot detoxify these products and are killed. This death can be prevented or greatly
reduced by exposing the cells in a medium that either reduces the production of the superoxides
and free radicals or increases their degradation. Supplementation of pyruvate or catalase to resuscitation media was thus found to transform the cells to a culturable state very rapidly, as determined
by an almost immediate increase in CFUs without any change in TMCs. Several studies in the
1970s and 1980s on bacterial sublethal injury have shown that following environmental stress of
a bacterial population, the recovery of the survivors can be increased either by incubating the cells
in a minimal medium or in a nutritionally rich medium supplemented with pyruvate or catalase.