DNA synthesis and tritiated thymidine incorporation by heterotrophic freshwater bacteria in continuous culture

Abstract

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 1018 to 1.31 x 1018 cells mol of tritiated thymidine-1 (mean, 0.60 x 1018 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 assumptions, were between 0.26 x 1018 and 0.34 x 1018 cells mol of thymidine-1 (mean, 0.30 x 1018 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.