The Viable but Nonculturable State

Microbial ecologists have long recognized that large proportions of microbial populations inhabiting natural habitats appear to be nonculturable. Indeed, plate counts of bacteria in soil, rivers and oceans typically indicate that far less than 1% of the total bacteria observed by direct microscopic examination can be grown on culture media. It has also long been known that certain portions of bacterial populations in natural environments seem to "disappear" during certain seasons, only to "reappear" at other times. We now understand that at least part of the explanation for these observations is not due to seasonal die-off of the cells, but to their entry into what is most commonly called the "viable but nonculturable" state.1

A bacterial cell in the viable but nonculturable (VBNC) state may be defined as one which fails to grow on the routine bacteriological media on which it would normally grow and develop into a colony, but which is in fact alive and metabolically active. Bacteria enter into this "dormant" state in response to one or more environmental stresses which might otherwise be ultimately lethal to the cell. Thus, the VBNC state should be considered a means of cell survival. Eventually, when the inducing stress is removed, these cells are able to emerge from the VBNC state, and again become culturable on routine media.

The typical VBNC response is illustrated in Figure 1.1, which shows the response of the human pathogen, V vulnificus, to exposure to low temperature (5°C). Such a temperature is below that at which this aquatic bacterium can grow and, if it were not for the VBNC response, is a temperature which would eventually lead to death of the population.

As is evident from Figure 1.1, cells lose their ability to be cultured (shown by the open squares) in a rather linear manner, eventually reaching a point where platings suggest a total lack of any living cells. However, whereas death of a bacterial population generally leads to lysis of the cells and loss of cell structure, direct examination of cells entering the VBNC state indicates that the cells remain intact (as shown by the open circles of Fig. 1.1). Such cells could, of course, have died, but simply not undergone lysis. The primary evidence that such cells are alive, even if nonculturable, is from data obtained when one of the "direct viability" assays is applied to such cultures. These assays (described below) allow the direct

Tracking Genetically-Engineered Microorganisms, edited by Janet K. Jansson, Jan Dirk van Elsas, Mark J. Bailey. ©2000 EUREKAH.COM.

Fig. 1.1. Entry of V vulnificus into the VBNC state in an artificial sea water microcosm at 5oC. Shown are plate counts (□) on HI agar in cfu/ml, total cell counts (♦) by the acridine orange staining method, and direct viable counts (O) by the substrate responsive method using yeast extract and nalidixic acid. Reprinted with permission from: Whitesides MD, Oliver JD. Appl Environ Microbiol 1997; 63:1002-1005.

Fig. 1.1. Entry of V vulnificus into the VBNC state in an artificial sea water microcosm at 5oC. Shown are plate counts (□) on HI agar in cfu/ml, total cell counts (♦) by the acridine orange staining method, and direct viable counts (O) by the substrate responsive method using yeast extract and nalidixic acid. Reprinted with permission from: Whitesides MD, Oliver JD. Appl Environ Microbiol 1997; 63:1002-1005.

determination of the viability of individual cells in a population, without the need for culture. As seen in Figure 1.1 (open circles), such assays often indicate that a large proportion of the apparently dead population is indeed alive.

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