We present an improvement of the INT [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride)] reduction method using Cyto-Clear slides, the fluorochrome DAPI (4(prm1),6(prm1)-diamidino-2 phenylindole), and an image analysis system. With this method we were able to simultaneously measure cell dimensions and formazan crystals as indicators of the respiratory activity of single bacteria. The method was tested on a natural bacterioplankton community of an oligotrophic high mountain lake (Gossenkollesee, Tyrolean Alps, Austria, 2,417 m above sea level) in midwinter ((symbl)1-m-thick ice and snow layer; dissolved organic carbon, 0.51 mg liter(sup-1); water temperature, 2(deg)C). About 25% of planktonic bacteria were respiratorily active, and a complex pattern of bacterial morphologies and specific respiratory activities was observed during a time series of INT incubation. Rod-shaped bacteria with cell lengths of between 1.6 and 4.8 (mu)m already showed visible activity after 0.5 h of INT incubation. Small cells (rods and cocci) in the size fraction <1.6 (mu)m and long filamentous bacteria (up to 120 (mu)m) were visibly active only after a 2-h incubation period. After 8 h of incubation, more than 90% of all cells between 3.2 and 6.4 (mu)m in cell length were respiratorily active, whereas only 5% of cells <1.6 (mu)m and 50% of filamentous bacteria contained formazan grains. We could distinguish five major bacterial phenotypes that showed distinct activity patterns with respect to incubation period and numbers and sizes of formazan crystals. There was no correlation between the total formazan volume per active cell and bacterial cell volume, and for any size class of active bacteria, total formazan volumes varied by about 2 orders of magnitude after 8 h of incubation. This indicates that cell-specific activity is extremely variable and is not related to size and that a small portion of all cells may account for the overall activity.
