CELL-SPECIFIC RESPIRATORY ACTIVITY OF AQUATIC BACTERIA STUDIED WITH THE TETRAZOLIUM REDUCTION METHOD, CYTO-CLEAR SLIDES, AND IMAGE-ANALYSIS

We present an improvement of the INT -iodophenyl)-3-(p-nitrophenyl)-5- phenyltetrazolium chloride)] reduction method using Cyto-Clear slides, the fluorochrome DAPI (4',6'-diamidino-2 phenylindole), and an image analysis system, With this method we were able to simultaneously measu re cell dimensions and formazan crystals as indicators of the respirat ory activity of single bacteria, The method was tested on a natural ba cterioplankton community of an oligotrophic high mountain lake (Gossen kollesee, Tyrolean Alps, Austria, 2,417 m above sea level) in midwinte r (similar to 1-m-thick ice and snow layer; dissolved organic carbon, 0.51 mg liter(-1); water temperature, 2 degrees C). About 25% of plank tonic bacteria were respiratorily active, and a complex pattern of bac terial morphologies and specific respiratory activities was observed d uring a time series of INT incubation, Rod-shaped bacteria with cell l engths of between 1.6 and 4.8 mu m already showed visible activity aft er 0.5 h of INT incubation, Small cells (rods and cocci) in the size f raction <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 incuba tion, more than 90% of all cells between 3.2 and 6.4 mu m in cell leng th were respiratorily active, whereas only 5% of cells <1.6 mu m and 5 0% of filamentous bacteria contained formazan grains, We could disting uish five major bacterial phenotypes that showed distinct activity pat terns with respect to incubation period and numbers and sizes of forma zan crystals. There was no correlation between the total formazan volu me per active cell and bacterial cell volume, and for any size class o f active bacteria, total formazan volumes varied by about 2 orders of magnitude after 8 h of incubation, This indicates that cell-specific a ctivity is extremely variable and is not related to size and that a sm all portion of all cells may account for the overall activity.