202
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
lowing decade on in situ microbial production in various aquatic habitats. The
results of these studies and all the aspects of thymidine method application
were thoroughly reviewed (Staley and Konopka 1985; Moriarty 1984, 1986,
1990; Karl 1986; Riemann and Bell 1990; Ducklow and Carlson 1992; Robarts
and Zohary 1993). I do not intend repeat here all the points and conclusions
these reviews contain, moreover many of them are still d matter of controversy and discussion. My goal is to give a necessary understanding of the
modern situation with the applicability of this method and its real possibilities in order to avoid its uninformed use and to avoid useless expectations or
mistakes concerned with it.
The problems with the use of the thymidine method arose soon after the
first experience of its practical implementation. They appeared during the
attempts to compare this new method with the existing, more direct, methods
like microscopic or CO2 dark uptake methods (Kirchmann et al. 1982). The
results definitely indicated that the thymidine method, when applied with
the use of a theoretically deduced conversion factor, inevitably results in grave
underestimations of bacterial production. Moreover, it was stated that these
underestimations were very variable in various kinds of aquatic environments,
and even in samples taken on vertical profiles, or in the same basin but during
vanous seasons.
It became clear that the CH)-TDR technique could not be used with these
theoretically derived conversion factors. More and more researchers became
convinced that it was possible to obtain reliable data with this method only
after its thorough calibration against some older but safer methodology, like
those listed above. Such a calibration, with parallel measuring of the growth
rate of bacteria by both methods gave the possibility to calculate empirical
conversion factors, which could then be used for translation of the amounts of
the thymidine incorporated per time and per volume of the sample into the
number of bacteria divided, e.g., to the amounts of bacteria thus produced.
Numerous estimations of these empirical conversion factors disclosed that
they may range within about two orders of values between 0.5 up to 60 X 10 9
cells mmol- 1 of incorporated thymidine, depending on numerous ambient
factors (Scavia et al. 1986; Bell 1988; Ducklow and Carlson 1992; Tuomi 1997).
These data evidently dash the hopes that the thymidine method can become
a universal one for measuring microbial production (Karl 1986), although
some optimists still manage to keep these hopes high (Moriarty 1990; Robarts
and Zohary 1993); but the very fact of the invalidity of the theoretical conversion factor quite clearly pointed out that something was wrong with the
assumptions which created the theoretical background of the method and
promised its accuracy. The researchers followed this line, and during the
second half of the decade carefully studied the possible sources of large conversion factor deviations, starting from biochemical mechanisms CH)-TDR
uptake and incorporation, distribution of labels in macromolecular fractions,
ability of ambiental microflora to incorporate this label, problem of catabolism of TDR added into the samples by microbia, etc. As a result, most of the
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
lowing decade on in situ microbial production in various aquatic habitats. The
results of these studies and all the aspects of thymidine method application
were thoroughly reviewed (Staley and Konopka 1985; Moriarty 1984, 1986,
1990; Karl 1986; Riemann and Bell 1990; Ducklow and Carlson 1992; Robarts
and Zohary 1993). I do not intend repeat here all the points and conclusions
these reviews contain, moreover many of them are still d matter of controversy and discussion. My goal is to give a necessary understanding of the
modern situation with the applicability of this method and its real possibilities in order to avoid its uninformed use and to avoid useless expectations or
mistakes concerned with it.
The problems with the use of the thymidine method arose soon after the
first experience of its practical implementation. They appeared during the
attempts to compare this new method with the existing, more direct, methods
like microscopic or CO2 dark uptake methods (Kirchmann et al. 1982). The
results definitely indicated that the thymidine method, when applied with
the use of a theoretically deduced conversion factor, inevitably results in grave
underestimations of bacterial production. Moreover, it was stated that these
underestimations were very variable in various kinds of aquatic environments,
and even in samples taken on vertical profiles, or in the same basin but during
vanous seasons.
It became clear that the CH)-TDR technique could not be used with these
theoretically derived conversion factors. More and more researchers became
convinced that it was possible to obtain reliable data with this method only
after its thorough calibration against some older but safer methodology, like
those listed above. Such a calibration, with parallel measuring of the growth
rate of bacteria by both methods gave the possibility to calculate empirical
conversion factors, which could then be used for translation of the amounts of
the thymidine incorporated per time and per volume of the sample into the
number of bacteria divided, e.g., to the amounts of bacteria thus produced.
Numerous estimations of these empirical conversion factors disclosed that
they may range within about two orders of values between 0.5 up to 60 X 10 9
cells mmol- 1 of incorporated thymidine, depending on numerous ambient
factors (Scavia et al. 1986; Bell 1988; Ducklow and Carlson 1992; Tuomi 1997).
These data evidently dash the hopes that the thymidine method can become
a universal one for measuring microbial production (Karl 1986), although
some optimists still manage to keep these hopes high (Moriarty 1990; Robarts
and Zohary 1993); but the very fact of the invalidity of the theoretical conversion factor quite clearly pointed out that something was wrong with the
assumptions which created the theoretical background of the method and
promised its accuracy. The researchers followed this line, and during the
second half of the decade carefully studied the possible sources of large conversion factor deviations, starting from biochemical mechanisms CH)-TDR
uptake and incorporation, distribution of labels in macromolecular fractions,
ability of ambiental microflora to incorporate this label, problem of catabolism of TDR added into the samples by microbia, etc. As a result, most of the