Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water

Abstract

Direct microscopic quantification of respiring (i.e., viable) bacteria was performed for drinking water samples and biofilms grown on different opaque substrata. Water samples or biofilms developed in flowing drinking water were incubated with the vital redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and R2A medium. One hour of incubation with 0.5 mM CTC was sufficient to obtain intracellular reduction of CTC to the insoluble fluorescent formazan (CTF) product, which was indicative of cellular respiratory (i.e., electron transport) activity. This result was obtained with both planktonic and biofilm-associated cells. Planktonic bacteria were captured on 0.2-pm-pore-size polycarbonate membrane filters and examined by epifluorescence microscopy. Respiring cells containing CTF deposits were readily detected and quantified as red-fluorescing objects on a dark background. The number of CTC-reducing bacteria was consistently greater than the number of aerobic CFU determined on R2A medium. Approximately 1 to 10%o of the total planktonic population (determined by counterstaining with 4,6-diamidino-2-phenylindole) were respirometrically active. The proportion of respiring bacteria in biofilms composed of drinking water microflora was greater, ranging from about 5 to 35%, depending on the substratum. Respiring cells were distributed more or less evenly in biofilms, as demonstrated by counterstaining with 4,6-diamidino-2-phenylindote. The amount of CTF deposited in single cells of Pseudomonas putida that formed monospecies biofilms was quantified by digital image analysis and used to indicate cumulative respiratory activity. These data indicated significant cell-to-cell variation in respiratory activity and reduced electron transport following a brief period of nutrient starvation. The results of this study demonstrate that CTC reduction is a rapid and sensitive method for quantification and localization of viable bacteria in drinking water and other environmental samples. The method is particularly well suited for exploration of cellular activity in surface biofilms.

Evaluation of cell activity and of methods for the cultivation of bacteria from a natural lake community

“…Because the number of CTC-reducing cells exceeded plate counts by four orders of magnitude in some environmental samples [8], it has been assumed that the CTC reduction method provides a more sensitive indicator of viability than the plate count method. In Zwischenahner Meer, however, the maximum relative numbers of CTC-reducing cells (8% of the total cell count) were 10 times lower than the values detected in groundwater and drinking water (up to 88% [8,34]). Our low values were rather unexpected for such a eutrophic environment, since in some other aquatic systems the proportion of bacteria with active electron transport systems^at least in summer^correlated positively with the trophic state [35,36].…”

“…Our study demonstrates that the CTC reduction method can lead to a serious underestimation of the number of bacterial cells which are capable to divide at least once or twice. Further support for this conclusion comes from the low fraction of CTC-reducing cells that has also been reported for bacteria from drinking water samples or from soil [34,37]. One major reason for these low counts of CTC-reduc-ing bacteria may be the toxicity of CTC which inhibits glucose uptake and respiration, thymidine incorporation, and growth on agar plates [3].…”

Assessment of metabolic activity of single bacterial cells — new developments in microcolony and dehydrogenase assays

“…These techniques address different aspects of cell metabolism (see last section of this review) and it may consequently be difficult to relate results obtained by the different methods. A central requirement for viability assays therefore would be that they measure a process central to cellular energy metabolism [2] or to the maintenance of cellular integrity. It appears, that the term 'active but nonculturable' might be more precise than viable but nonculturable, but the latter term will be used here, with the above reservations in mind.…”

“…In recent years it has become evident that the physiological heterogeneity of bacterial populations is considerable, even for cells growing in pure culture [2]. In natural environments, heterogeneity may be larger as the growth conditions may vary over distances of few microns.…”

“…The fluorescence facilitates detection and enables the simultaneous determination of respiring cell number and total cell number after counter staining with the fluorescent DNA-binding stain 4'-6-diamidino-2-phenylindole (DAPI) [19]. Due to brief incubation periods, l-3 h, and detection by fluorescence microscopy or flow cytometry, CTC-reduction assays provide a rapid assessment of cell viability [2,19,20].…”