Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration

Abstract

The electron transport system of respiring organisms reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Respiring bacteria deposit accumulated INT-formazan intracellularly as dark red spots. Corresponding to electron transport system activity, these deposits attain a size and a degree of optical density which allows them to be examined by light microscopy. If polycarbonate filters and epifluorescence microscopy are applied to analyze an INT-treated water sample, it is possible to differentiate between respiring and apparently nonrespiring bacteria. This differentiation, which permits determinations of the total number of bacteria and the proportion thereof involved in respiration, is realized directly within one and the same microscopic image. Initial applications of the present method for hydrobiological purposes showed that the proportion of respiring aquatic bacteria ranged between 6 to 12% (samples taken from coastal areas of the Baltic Sea) and 5 to 36% (samples taken from freshwater lakes and ponds). Cells of 1.6 to 2.4 ym (freshwater) and 0.4 ttm (Baltic Sea) account for the highest proportion of respiring bacteria.

Starvation survival of the fish pathogen Aeromonas salmonicida in seawater

“…Formazan crystals were observed as clear red spots when bright field illumination alone was used. Crystal formation was often observed near the inside of the cell wall as described by Zimmermann et al [13]. DAPI positive cells with formazan crystals were only registered as INT positive counts of A. salmonicida cells if the cells also exhibited a positive FA signal.…”

“…The number of respiring cells of A. was determined using a modified Iodophenyl)-3-(p-nitrophenyl)-5-phenyl chloride) (Sigma Chemical Co., St. method described by Zimmermann et salmonicida INT (2-(ptetrazolium Louis, MO) al. [13] and Ring and Parker [14]. The dehydrogenation of INT to red formazan crystals in cell cytoplasm was used in the combination with DAPI for the staining of all bacteria, and with monoclonal antibodies and fluorescein isothiocyanate-conjugated (FA) rabbit antibodies for the specific staining of A. salmonicida cells.…”

“…The dehydrogenation of INT to red formazan crystals in cell cytoplasm was used in the combination with DAPI for the staining of all bacteria, and with monoclonal antibodies and fluorescein isothiocyanate-conjugated (FA) rabbit antibodies for the specific staining of A. salmonicida cells. A final concentration of 0.2% INT was added to l-10 ml samples containing A. salmonicida cells as described by Zimmermann et al [13]. The samples were incubated on a rotary shaker for 35 min in the dark (Fig.…”

The polar lipid composition of walsby's square bacterium

“…To obtain information on the number of actively respiring cells, we examined the formation of intracellular formazan granules upon incubation with 2-( p-iodophenyll-3( p-nitrophenyll-5-phenyl tetrazolium chloride @NT) [14,15]. 10 ml portions of brine were incubated for 4-6 h at 35°C with 0.2 ml of a solution of 1 g 1-l INT and 0.1 ml of 0.1% glycerol.…”

Changes in viability, respiratory activity and morphology of the marine Vibrio sp. strain S14 during starvation of individual nutrients and subsequent recovery

“…Direct viable counts (DVC) were performed by counting 2-(p-iodophenyl)-3-(pnitrophenyl)-5-phenyl tetrazolium chloride (INT) (Sigma Fine Chemicals) reducing Vibrio sp. strain S14 cells, using a light microscope (Olympus) according to Zimmermann et al [18]. These parameters were determined throughout 120 h starvation as well as during 48 h of the subsequent nutritional upshift.…”