210
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
ommend the use of the dark 14C02 uptake method for this purpose also
because an equally good alternative has still not appeared.
I tested this way of calibrating the thymidine method when working in the
meso trophic waters of the lagoon of the Great Barrier Reef, Australia. First I
calibrated the values of bacterial production (Pb) measured by the dark CO2
uptake method in its recent modification (sec Sect. 4.4.2.2.) and expressed
in units of mg CI- 1 h- 1 after radioactivity R; was measured in the cold TCAinsoluble fraction of bacterioplankton, incubated in the presence of 15 nmol
of eH)-TDR for 20 min, and expressed as cpml- 1 h- 1 . The PbIR; ratio thus estimated was then used during routine surveys on the distribution of bacterial
production in the lagoon of Heron Island atoll with the use of eH)-thymidine.
The control estimations made with the aid of the above reference method
usually gave quite acceptable coincidences; however, at several stations, the
thymidine method produced inexplicably high radioactivity counts (R;). This
feature of the thymidine method was observed by other users of thymidineand also leucine methods (Bell 1988; lellet et al. 1996; Witek et al. 1997). Later,
I used the thymidine method thus calibrated for mesotrophic oceanic waters
to measure the bacterial production in the productive waters of the Peruvian
upwelling area during the EI-Nino period when the waters of this area were
just mesotrophic. At each station in the surface samples the parallel estimations were also made with using the dark 14C02 uptake method, according to
which the thymidine method had been once calibrated. The results of both
methods at numerous stations demonstrated that, in general, values of bacterioplankton production obtained with the thymidine method calibrated after
the 14C-dark uptake method produced reasonably coin siding its values at
most stations. However, also in this case, at some stations the difference was
four to seven times without visible cause. These fluctuations most probably
reflected the change in composition of bacterioplankton, resulting in changes
of percentage of bacterial cells able to incorporate exogenous thymidine (Fig.
4.14).
Thus the only real way to use the R; values for estimating microbial
production (Pb) in situ is to calibrate this radioactivity in units of P b by some
reference method which produces absolute values, such as the dark 14C-uptake
method. In this case, P b and R; values are measured simultaneously in the
intact natural sample, and the conversion factor K j (~gCcpm-l) is calculated
as their ratio. Then by measuring R; values under standard conditions with the
same batch of isotopes, the microbial production P b can be calculated (Sorokin
1990b).
4.4.3.2 Practical Procedure
Below I describe the optimal procedure for estimating the R;. Then, especially
for the devoted adepts of the TDR method who want to use its official version
in any case, I advise an optimal technique.
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
ommend the use of the dark 14C02 uptake method for this purpose also
because an equally good alternative has still not appeared.
I tested this way of calibrating the thymidine method when working in the
meso trophic waters of the lagoon of the Great Barrier Reef, Australia. First I
calibrated the values of bacterial production (Pb) measured by the dark CO2
uptake method in its recent modification (sec Sect. 4.4.2.2.) and expressed
in units of mg CI- 1 h- 1 after radioactivity R; was measured in the cold TCAinsoluble fraction of bacterioplankton, incubated in the presence of 15 nmol
of eH)-TDR for 20 min, and expressed as cpml- 1 h- 1 . The PbIR; ratio thus estimated was then used during routine surveys on the distribution of bacterial
production in the lagoon of Heron Island atoll with the use of eH)-thymidine.
The control estimations made with the aid of the above reference method
usually gave quite acceptable coincidences; however, at several stations, the
thymidine method produced inexplicably high radioactivity counts (R;). This
feature of the thymidine method was observed by other users of thymidineand also leucine methods (Bell 1988; lellet et al. 1996; Witek et al. 1997). Later,
I used the thymidine method thus calibrated for mesotrophic oceanic waters
to measure the bacterial production in the productive waters of the Peruvian
upwelling area during the EI-Nino period when the waters of this area were
just mesotrophic. At each station in the surface samples the parallel estimations were also made with using the dark 14C02 uptake method, according to
which the thymidine method had been once calibrated. The results of both
methods at numerous stations demonstrated that, in general, values of bacterioplankton production obtained with the thymidine method calibrated after
the 14C-dark uptake method produced reasonably coin siding its values at
most stations. However, also in this case, at some stations the difference was
four to seven times without visible cause. These fluctuations most probably
reflected the change in composition of bacterioplankton, resulting in changes
of percentage of bacterial cells able to incorporate exogenous thymidine (Fig.
4.14).
Thus the only real way to use the R; values for estimating microbial
production (Pb) in situ is to calibrate this radioactivity in units of P b by some
reference method which produces absolute values, such as the dark 14C-uptake
method. In this case, P b and R; values are measured simultaneously in the
intact natural sample, and the conversion factor K j (~gCcpm-l) is calculated
as their ratio. Then by measuring R; values under standard conditions with the
same batch of isotopes, the microbial production P b can be calculated (Sorokin
1990b).
4.4.3.2 Practical Procedure
Below I describe the optimal procedure for estimating the R;. Then, especially
for the devoted adepts of the TDR method who want to use its official version
in any case, I advise an optimal technique.