Measurement of Active Cells in Environmental Samples

Several approaches have been considered for assessing microbial viability/activity in environmental samples. Epifluorescence microscopy, in combination with the use of oxido-reduction dyes, is used to determine the percent of active cells in aquatic environments. The most popular oxido-reduction dyes are INT (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (cyanoditolyl tetrazolium chloride) (Poschet al., 1997; Pyle et al., 1995a). A good correlation was found between

the number of CTC-positive E. coli cells and the CFU (colony forming units) count, regardless of the growth phase (Cre´ach et al., 2003).

The direct viable count (DVC) method was pioneered by Kogure and his collaborators in Japan (Kogure et al., 1984). The sample is incubated with trace amounts of yeast extract and nalidixic acid. The latter blocks DNA replication but not RNA synthesis. This leads to cell elongation of active cells, which are counted using epifluorescence microscopy. Some methods allow the detection in aquatic samples of specific bacterial pathogens, including those in the VBNC state. One such method combines fluorescent in situ hybridization (FISH; see Chapter 1 for more details) with DVC, followed by cell enumeration using a laser scanning cytometer.

This approach gives information on the identity of the bacteria (Baudart et al., 2002). To detect specific bacteria in aquatic environments, the cells can be simultaneously labeled with a fluorescent antibody (FA technique) in combination with viability/activity markers such as cyanoditolyl tetrazolium chloride or propidium iodide, which is an indicator of membrane integrity (Caruso et al., 2003).

Fluorescein diacetate (FDA) is transformed by esterase enzymes to a fluorescent compound, fluorescein, which accumulates inside the cells (FDA is a nonpolar compound and fluorescein a polar compound). The active fluorescent cells are counted under a fluorescent microscope. This method is best suited for active fungal filaments but can be applied to bacterial cells.