Calculation of Cell Production from [ 3 H]Thymidine Incorporation with Freshwater Bacteria

Abstract

The conversion factor for the calculation of bacterial production from rates of [H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20 degrees C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 x 10 cells mol of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 x 10; n = 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 x 10 cells mol of thymidine incorporated into TCA precipitate (standard error = 1.72 x 10; n = 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20 degrees C than at 15 and 10 degrees C. A detailed examination of the [H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 x 10 cells mol of thymidine incorporated is used.