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. 1992 Nov;58(11):3614–3621. doi: 10.1128/aem.58.11.3614-3621.1992

Assessment of [3H]Thymidine Incorporation into DNA as a Method To Determine Bacterial Productivity in Stream Bed Sediments

Louis A Kaplan 1,2,*, Thomas L Bott 1,2, John K Bielicki 1
PMCID: PMC183152  PMID: 16348806

Abstract

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25°C, but hydrolysis at 120°C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 μM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.

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Selected References

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  1. Amy P. S., Pauling C., Morita R. Y. Starvation-survival processes of a marine Vibrio. Appl Environ Microbiol. 1983 Mar;45(3):1041–1048. doi: 10.1128/aem.45.3.1041-1048.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell R. T. Further Verification of the Isotope Dilution Approach for Estimating the Degree of Participation of [H]thymidine in DNA Synthesis in Studies of Aquatic Bacterial Production. Appl Environ Microbiol. 1986 Nov;52(5):1212–1214. doi: 10.1128/aem.52.5.1212-1214.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bott T. L., Kaplan L. A. Bacterial biomass, metabolic state, and activity in stream sediments: relation to environmental variables and multiple assay comparisons. Appl Environ Microbiol. 1985 Aug;50(2):508–522. doi: 10.1128/aem.50.2.508-522.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chrzanowski T. H. Consequences of accounting for isotopic dilution in thymidine incorporation assays. Appl Environ Microbiol. 1988 Jul;54(7):1868–1870. doi: 10.1128/aem.54.7.1868-1870.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coveney M. F., Wetzel R. G. Experimental evaluation of conversion factors for the [h]thymidine incorporation assay of bacterial secondary productivity. Appl Environ Microbiol. 1988 Aug;54(8):2018–2026. doi: 10.1128/aem.54.8.2018-2026.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellenbroek Frank M., Cappenberg Thomas E. DNA Synthesis and Tritiated Thymidine Incorporation by Heterotrophic Freshwater Bacteria in Continuous Culture. Appl Environ Microbiol. 1991 Jun;57(6):1675–1682. doi: 10.1128/aem.57.6.1675-1682.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fallon R. D., Newell S. Y. Thymidine Incorporation by the Microbial Community of Standing Dead Spartina alterniflora. Appl Environ Microbiol. 1986 Nov;52(5):1206–1208. doi: 10.1128/aem.52.5.1206-1208.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Forsdyke D. R. Studies on the incorporation of [5-3H] uridine during activation and transformation of lymphocytes induced by phytohaemagglutinin. Dependence on the incorporation rate on uridine concentration at certain critical concentrations. Biochem J. 1968 Mar;107(2):197–205. doi: 10.1042/bj1070197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fuhrman J. A., Azam F. Bacterioplankton secondary production estimates for coastal waters of british columbia, antarctica, and california. Appl Environ Microbiol. 1980 Jun;39(6):1085–1095. doi: 10.1128/aem.39.6.1085-1095.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hobbie J. E., Daley R. J., Jasper S. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977 May;33(5):1225–1228. doi: 10.1128/aem.33.5.1225-1228.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hood M. A., Guckert J. B., White D. C., Deck F. Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae. Appl Environ Microbiol. 1986 Oct;52(4):788–793. doi: 10.1128/aem.52.4.788-793.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jeffrey W. H., Paul J. H. Underestimation of DNA synthesis by [h]thymidine incorporation in marine bacteria. Appl Environ Microbiol. 1988 Dec;54(12):3165–3168. doi: 10.1128/aem.54.12.3165-3168.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kirchman D., Ducklow H., Mitchell R. Estimates of bacterial growth from changes in uptake rates and biomass. Appl Environ Microbiol. 1982 Dec;44(6):1296–1307. doi: 10.1128/aem.44.6.1296-1307.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lovell C. R., Konopka A. Seasonal bacterial production in a dimictic lake as measured by increases in cell numbers and thymidine incorporation. Appl Environ Microbiol. 1985 Mar;49(3):492–500. doi: 10.1128/aem.49.3.492-500.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Morse M. L., Carter C. E. THE SYNTHESIS OF NUCLEIC ACIDS IN CULTURES OF ESCHERICHIA COLI, STRAINS B AND B/R. J Bacteriol. 1949 Sep;58(3):317–326. doi: 10.1128/jb.58.3.317-326.1949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nagata T. Production rate of planktonic bacteria in the north basin of lake biwa, Japan. Appl Environ Microbiol. 1987 Dec;53(12):2872–2882. doi: 10.1128/aem.53.12.2872-2882.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Paul J. H., Jeffrey W. H., DeFlaun M. F. Dynamics of extracellular DNA in the marine environment. Appl Environ Microbiol. 1987 Jan;53(1):170–179. doi: 10.1128/aem.53.1.170-179.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pollard P. C., Moriarty D. J. Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis. Appl Environ Microbiol. 1984 Dec;48(6):1076–1083. doi: 10.1128/aem.48.6.1076-1083.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robarts R. D., Wicks R. J., Sephton L. M. Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-H]Thymidine in a Hypertrophic Lake. Appl Environ Microbiol. 1986 Dec;52(6):1368–1373. doi: 10.1128/aem.52.6.1368-1373.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Servais P., Martinez J., Billen G., Vives-Rego J. Determining [H]Thymidine Incorporation into Bacterioplankton DNA: Improvement of the Method by DNase Treatment. Appl Environ Microbiol. 1987 Aug;53(8):1977–1979. doi: 10.1128/aem.53.8.1977-1979.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Winding A. [H]thymidine incorporation to estimate growth rates of anaerobic bacterial strains. Appl Environ Microbiol. 1992 Aug;58(8):2660–2662. doi: 10.1128/aem.58.8.2660-2662.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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