Microbial Growth

Advances in Microbiology

The Viable but Nonculturable State
Some bacteria can enter a state known as viable but nonculturable. These organisms, while still
alive, cannot be cultured by normal methods used in the laboratory. It was previously thought that
all viable cells were culturable in the laboratory and would form colonies when grown on nutrient
rich agar media. However, in 1982, scientists discovered Vibrio cholerae, the causative agent of
cholera, in the Chesapeake Bay in Maryland. Even more cause for worry was that they showed that
this bacterium, when incubated in artificial sea water, remained viable but lost the ability to form
colonies on culture medium normally used for the growth of this organism in the laboratory. It was
from this discovery that the concept of the viable but nonculturable state emerged. A number of
microorganisms, including pathogens such as Helicobacter pylori, the causative agent of ulcers,
have now been shown to be able to enter this state.

What does the term "viable but nonculturable" really mean? It means that the conditions for these
organisms to resume growth are not being met using the normal laboratory culture conditions.
Bacteria that have been semi-starved will immediately resume growth when provided with the
appropriate nutrients and conditions. Why don't viable but nonculturable bacteria also resume
growth when nutrients are provided? Evidence suggests that these cells have become too starved
to be able to grow on rich medium initially. This phenomenon was observed over three decades ago
and was known then as "substrate accelerated death." It was found that substrates that normally
supported growth of certain Gram negative bacteria hastened their death when introduced into
starved populations of these bacteria. These substrates were considered "lethal" substrates. These
starved cells require an adjustment period to allow phenotypic adaptation back to the normal growth
state. A sudden shift to nutrient rich agar creates a metabolic imbalance that results in the formation
of superoxide and free radicals, causing DNA damage that can result in cell death.

How can viable but nonculturable cells be resuscitated? Nutritional restriction appears to be a key in
maintaining viability of these cells and allowing them time to adapt to growth on rich medium again.
By cultivating the cells on nutritionally limited medium, they will slowly adapt to growth on richer
medium. In some cases, it is thought that the bacteria may also require specific extracellular signals
before growth is possible. Although some knowledge has been gained about how cells can be
resuscitated, little is known about the physiological changes a cell undergoes when entering and
exiting the viable but nonculturable state. This is likely to remain a subject of active research for
years to come.

1
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How does the viable but nonculturable state benefit a microbe? Why might
an organism choose this over sporulation?




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