Determination of Microbial Production
199
Concerning the upper layer of reduced coastal bottom sediments, the
technique of estimation of anoxic bacterial photosynthesis in general is
the same as described above for microphytobenthos photosynthesis with
the application of corer tubes shown in Fig. 2.24. The solution of labeled
14C-carbonate is injected into the water column together with Na2S9H20
solution to create a H 2S content of some 3-5mgSI- 1 .After mixing, the plug in
the lower stopper is taken out until one third of the water passes out through
the column of sediment. The plug is fixed in place, the water in the corer is
replenished with water containing the same concentration of 14C-carbonate
and H 2S closed hermetically without air bubbles and incubated in situ being
inserted into the native sediment (Fig. 2.25). The background dark uptake of
14C02 is measured in the same parallel core incubated in complete darkness.
After incubation for half the light day the supernatant water from both
cores is drained out, the cores frozen in vertical position, and the frozen core
pushed up and fine 2-3-mm thick layers cut from it down to 6cm depth.
These subsamples are processed and photosynthetic production by anaerobic
photobacteria is calculated as described for microphytobenthos (see Sect.
2.5.5.5).
4.4.3 The Thymidine Method
4.4.3.1 General Considerations
In situ bacterial production according to this method is evaluated by measuring of the rate of incorporation of CH)-thymidine nucleoside into the
DNA of microbial cells as evidence of its synthesis, accompanied by their
growth and division. The labeled thymidine used for this purpose has tritium
labeled only in its methyl group. Therefore it is named [3H] methyl thymidine
or [3H] TDR. When it is transformed to uracil during cellular metabolism, it
loses its label together with the methyl group. This illustrates the specific
nature of thymidine labeling of DNA. Preparations of CH)TDR need to have
an extremely high specific radioactivity of 20 to 100Cimmol- 1 added into the
samples to finally achieve TDR concentrations of 5 to 25 nmoll- 1 with a dpm
radioactivity of about 1-2IlCiml-l.
The idea of using this nucleoside for measuring bacterial growth via DNA
synthesis emerged as a result of progress in the biochemistry of nucleic acids,
when it became known that thymidine is unique among other nucleosides,
because its major function in the cell is its participation in DNA synthesis.
Before this method was proposed in 1980-1982 for measuring bacterial production in their natural populations, it had also been known that at least some
heterotrophic bacteria, such as E. coli, possess a special transport mechanism,
which enables the transfer of large molecules of this nucleoside inside their
cells from exogenous sources. To transform thymidine thus taken up into the
nucleoside persecutor of DNA, these bacteria have a special phosphorylase
199
Concerning the upper layer of reduced coastal bottom sediments, the
technique of estimation of anoxic bacterial photosynthesis in general is
the same as described above for microphytobenthos photosynthesis with
the application of corer tubes shown in Fig. 2.24. The solution of labeled
14C-carbonate is injected into the water column together with Na2S9H20
solution to create a H 2S content of some 3-5mgSI- 1 .After mixing, the plug in
the lower stopper is taken out until one third of the water passes out through
the column of sediment. The plug is fixed in place, the water in the corer is
replenished with water containing the same concentration of 14C-carbonate
and H 2S closed hermetically without air bubbles and incubated in situ being
inserted into the native sediment (Fig. 2.25). The background dark uptake of
14C02 is measured in the same parallel core incubated in complete darkness.
After incubation for half the light day the supernatant water from both
cores is drained out, the cores frozen in vertical position, and the frozen core
pushed up and fine 2-3-mm thick layers cut from it down to 6cm depth.
These subsamples are processed and photosynthetic production by anaerobic
photobacteria is calculated as described for microphytobenthos (see Sect.
2.5.5.5).
4.4.3 The Thymidine Method
4.4.3.1 General Considerations
In situ bacterial production according to this method is evaluated by measuring of the rate of incorporation of CH)-thymidine nucleoside into the
DNA of microbial cells as evidence of its synthesis, accompanied by their
growth and division. The labeled thymidine used for this purpose has tritium
labeled only in its methyl group. Therefore it is named [3H] methyl thymidine
or [3H] TDR. When it is transformed to uracil during cellular metabolism, it
loses its label together with the methyl group. This illustrates the specific
nature of thymidine labeling of DNA. Preparations of CH)TDR need to have
an extremely high specific radioactivity of 20 to 100Cimmol- 1 added into the
samples to finally achieve TDR concentrations of 5 to 25 nmoll- 1 with a dpm
radioactivity of about 1-2IlCiml-l.
The idea of using this nucleoside for measuring bacterial growth via DNA
synthesis emerged as a result of progress in the biochemistry of nucleic acids,
when it became known that thymidine is unique among other nucleosides,
because its major function in the cell is its participation in DNA synthesis.
Before this method was proposed in 1980-1982 for measuring bacterial production in their natural populations, it had also been known that at least some
heterotrophic bacteria, such as E. coli, possess a special transport mechanism,
which enables the transfer of large molecules of this nucleoside inside their
cells from exogenous sources. To transform thymidine thus taken up into the
nucleoside persecutor of DNA, these bacteria have a special phosphorylase