Limitations of 3 H-thymidine incorporation in measu- ring bacterial production in marine systems (original) (raw)
Bacterial production estimates are essential in order to assess the role of bacterioplankton in the flux of carbon in the ocean. Several methods have been developed to quantify bacterial production in marine systems, but the estimation of DNA synthesis rate from measurements of the rate of tritiated thymidine incorporation has been considered the most promising since its publication in 1980. Due to its high sensitivity and apparent simplicity it has been extensively used and modified. However, our current knowledge of thymidine bacterial metabolism is still insufficient and moreover, there are some methodological issues that need to be reviewed and clarified. The accurate application of the method in natural waters requires several precautions like the extraction and purification of the DNA, the measurement of isotope dilution by extracellular and/or intracellular non-labelled thymidine, and the estimation of a conversion factor to transform thymidine incorporation into bacterial production. All these methodological requirements make the thymidine method not appropriate for routine measurements. Its application to field studies without considering these prerequisites leads to erroneous estimations of bacterial production. Implications in our understanding of the microbial ecology of marine systems are discussed. RESUMEN: LIMITACIONES DEL MÉTODO DE LA INCORPORACIÓN DE 3 H-TIMIDINA EN LA MEDIDA DE PRODUCCIÓN BACTERI-ANA EN SISTEMAS MARINOS.
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Marine Ecology Progress Series, 1992
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Microbial Ecology, 2001
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Validity of the tritiated thymidine method for estimating bacterial growth rates: …
Applied and Environmental …
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.
Aquatic Microbial Ecology, 1996
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Applied and Environmental Microbiology
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.
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