[3H]Thymidine Incorporation To Estimate Growth Rates of Anaerobic Bacterial Strains
Applied and Environmental Microbiology
The incorporation of [3H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [3Hlthymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [3Hlthymidine during growth. It is concluded that the [3H]thymidine incorporation method underestimates bacterial growth in anaerobic environments. Measurements of in situ
... urements of in situ growth rates and activity of bacteria on the basis of incorporation of radiolabelled nucleosides have been applied to environmental samples for several years. Because the ability to incorporate thymidine is widespread among aerobic bacteria, the incorporation of tritiated thymidine is a widely used method, even though bacterial strains unable to incorporate [3H]thymidine are known (6). Studies of anaerobic communities incorporating [3H]thymidine are scarce. Gilmour et al. (1) studied the incorporation of [3H]thymidine into 10 strains of sulfatereducing bacteria and found very low rates of incorporation per cell produced (average, 3.3 x 1023 cells per mol of [3H]thymidine). Incorporation of [3H]thymidine in anaerobic environments has been shown (5, 7); the fermentative bacteria have been suggested as being the most important group of incorporating organisms (6). However, studies of incorporation of [3H]thymidine into fermentative and methanogenic strains are needed before this method can be used in anaerobic environmental samples. The purpose of this study was to examine the incorporation of [3H]thymidine in axenic cultures of fermentative, sulfate-reducing, and methanogenic bacteria, i.e., to evaluate the application of the method for quantification of growth rates in anaerobic environments. Strains. The following fermentative strains were used: Clostridium cellobioparum ATCC 15832, Clostridium formicoaceticum ATCC 27076, and Acetobacterium woodii ATCC 29683. The sulfate-reducing strain was Desulfovibrio vulgaris Gll. The methane-producing strains used were Methanococcus vannielii, Methanospirillum hungatei, and Methanobacterium formicicum. C. cellobioparum DSM 1351, C. formicoaceticum DSM 92, A. woodii DSM 1030, and M. vannielii DSM 1224 were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DSM); the other strains had been kept at the laboratory. Media. D. vulgaris Gll and the methanogenic strains kept in the laboratory were grown in a reduced mineral salts medium (10) with 2% yeast extract and buffered with t Present address: NaHCO3 under an atmosphere of N2 and CO2 (8:2). To D. vulgaris Gll, 20 mM sulfate was added. The M. hungatei, M. formicicum, and D. vulgaris Gll cultures were pressurized with H2. The strains from DSM were grown in the media described by DSM, with the following exceptions. The trace element solution was that of Worakit et al. (10); to the medium for C. for,nicoaceticum, CaCl2 (1 mg/liter) was added; to the medium for C. cellobioparum, clarified rumen fluid and cellobiose were added; and to the medium for A. woodii, another vitamin solution (3) was added. In order to evaluate any adsorption of thymidine and isotope dilution in nutrient-rich media, M. vannielii was grown in the reduced mineral salts media described above and in the rich media described by DSM, except that no a-methylbutyric acid was added and 5% (vol/vol) clarified rumen fluid was used instead of sewage sludge. All strains were shaken and grown at 37°C except for A. woodii, which was grown at 30°C.