DNA-SYNTHESIS AND TRITIATED-THYMIDINE INCORPORATION BY HETEROTROPHIC FRESH-WATER BACTERIA IN CONTINUOUS CULTURE

Continuous cultivation of heterotrophic freshwater bacteria was used to assess the relationship between DNA synthesis and tritiated thymidine incorporation. The bacteria were grown on a yeast extract medium with generation times of 0.25 to 3.7 days. In six different continuous cultures, each inoculated with a grazer-free mixed bacterial sample from Lake Vechten (The Netherlands), tritiated thymidine incorporation into a cold trichloroacetic acid precipitate and bacterial cell production were measured simultaneously. Empirical conversion factors were determined by division of both parameters. They ranged from 0.25 x 10(18) to 1.31 x 10(18) cells mol of tritiated thymidine-1 (mean, 0.60 x 10(18) cells mol of tritiated thymidine-1). In addition, DNA concentrations were measured by fluorometry with Hoechst 33258. The validity of this technique was confirmed. Down to a generation time of 0.67 day, bacterial DNA content showed little variation, with values of 3.8 to 4.9 fg of DNA cell-1. Theoretical conversion factors, which can be derived from DNA content under several assumption, were between 0.26 x 10(18) and 0.34 x 10(18) cells mol of thymidine-1 (mean, 0.30 x 10(18) cells mol of thymidine-1). Isotope dilution was considered the main factor in the observed discrepancy between the conversion factors. In all experiments, a tritiated thymidine concentration of 20 nM was used. Control experiments indicated maximum incorporation at this concentration. It was therefore concluded that the observed difference resulted from intracellular isotope dilution which cannot be detected by current techniques for isotope dilution analysis.