Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis

Abstract

The rate of tritiated thymidine incorporation into DNA was used to estimate bacterial growth rates in aquatic environments. To be accurate, the calculation of growth rates has to include a factor for the dilution of isotope before incorporation. The validity of an isotope dilution analysis to determine this factor was verified in experiments reported here with cultures of a marine bacterium growing in a chemostat. Growth rates calculated from data on chemostat dilution rates and cell density agreed well with rates calculated by tritiated thymidine incorporation into DNA and isotope dilution analysis. With sufficiently high concentrations of exogenous thymidine, de novo synthesis of deoxythymidine monophosphate was inhibited, thereby preventing the endogenous dilution of isotope. The thymidine technique was also shown to be useful for measuring growth rates of mixed suspensions of bacteria growing anaerobically. Thymidine was incorporated into the DNA of a range of marine pseudomonads that were investigated. Three species did not take up thymidine. The common marine cyanobacterium Synechococcus species did not incorporate thymidine into DNA.

Fatty acid composition and microbial activity of benthic marine sediment from McMurdo Sound, Antarctica

“…The average rate for the 4 sites was found to be 7.6 × 10 4 bacterial cell divisions per h per g sediment. It is known that acetate-utilizing SRB do not incorporate [3H]thymidine [21]. Although this group of organisms was present in the Antarctic sediments studied (section 4.2.2), their relative low abundance (approx.…”

“…The metabolic activity of the East Sound sites were on the average 45 and 73% higher in their incorporation of thymidine and sodium acetate, respectively. The increase in lipid synthesis suggested by the data in Table 5 assumes that the acetate pool size at these sites parallels the thymidine pool measured by isotope dilution [21]. Unfortunately, the acetate pool size at each site was not measured.…”

“…Freshly collected 1-g sediment samples were added to disposable 15 ml centrifuge tubes with 30/xCi [SH]thymidine (SA 79 Ci/mmol), 40/L1 of 10/~M thymidine and 200/~1 of a 0.3 mM solution of cycloheximide (eukaryotic inhibitor) [21] in filter-sterilized seawater (0.22 /xm millipore) at -2°C. The sediment was incubated aerobically for 8 h at -2°C under low light (35 /xE-m -2. s-l).…”

“…The sediment was incubated aerobically for 8 h at -2°C under low light (35 /xE-m -2. s-l). These incubation conditions were found to be optimal by isotope dilution and time-course experiments [21]. In addition, 80% ethanol-killed controls were incubated in parallel with all samples to determine any unincorporated [3H]thymidine carried through the DNA purification procedure.…”

Big Soda Lake (Nevada). 1. Pelagic bacterial heterotrophy and biomass1

“…Most studies have focused on the assimilation of thymidine in aerobic systems. Recently, Pollard and Moriarty (1984) have shown that some anaerobic bacteria also assimilate thymidine and suggested that the method was applicable to anaerobic systems.…”