Activity of an Attached and Free-Living Vibrio sp. as Measured by Thymidine Incorporation, p -Iodonitrotetrazolium Reduction, and ATP/DNA Ratios

Abstract

Three independent techniques, [ 3 H]thymidine incorporation, the reduction rate of p -iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica , nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [ 3 H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [ 3 H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [ 3 H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells.