206
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
ity experiments. A definite part of the products of its catabolic decomposition
are incorporated by the microbial cells for biosynthesis and participate in the
nonspecific labeling of macromolecules. The catabolic pathway of nonspecific
labeling is evidenced by the dependence of the latter on the place of label in
the molecule of thymidine. For example, when CH)-6-methyl-CH)-thymidine
was used in experiments instead of the traditional CH)·mcthyl-thymidine, the
macromolecule fraction contained 0 to 35% of label in DNA and up to 60%
in the protein in different samples (Hollibaugh 1988).
Data evidenced that the nonspecific incorporation of CH)-TDR label was
responsible for the major part of radioactivity recorded in the macromolecule
fraction and then used by the numerous users of the thymidine method for
the calculation of microbial production; but, actually, it did not deal at all with
the synthesis of DNA and with microbial growth (Karl 1986). There were some
hopes that unspecific labeling, which causes errors in all the calculations made
with the thymidine method, depends upon the time of exposure and starts after
some 15 min of incubation. The discovery of the catabolic way for this process
also dashed these hopes, moreover as the latter was disproved experimentally
(Servais et al. 1987; Brittain and Karl 1990). Nonspecific labeling proves to be
even more pronounced in the samples of bottom sediments, where the CH)TDR added is readily utilized by local bacteria as the ordinary organic substrate (Carman et al. 1988).
Comparison of the DNA synthesis rate in cultures as estimated via CH)TDR incorporation and by its direct measurements with specific strains
showed that the former underestimated it by five to eight times even accounting for possible isotopic dilution (Jeffrey and Paul 1988). That CH)-TDR
cannot be accepted as a reliable measure of DNA synthesis can also be
deduced from the evidence of a large range of nonspecific labeling. Perhaps
even more serious is the indefinite and inestimable specific radioactivity of
CH)-TDR, as the precursor forming the thymidine nucleotide pool for DNA
synthesis (Karl 1986). First of all this concerns the initial specific radioactivity
of commercially supplied CH)-TDR preparations. For the users the specific
radioactivity as Ci mmot 1 (Re), written on the label of the batch preparation
is the only evidence of its level, and correspondingly it is introduced into the
formula for calculation of the CH)-TDR incorporation rate (Ir); but it cannot
be correct even in principle because this product is not stable in time. It is subjected to: (1) a relatively rapid radiolysis in a highly radioactive medium, thus
losing the label together with its methyl group. This process proceeds in a
refrigerated batch with a speed of approximately 4% a month. Its speed, being
dependent upon various factors, is immeasurable and unpredictable, because
the researcher can measure only the total dpm radioactivity. But it is not
changed due to the radiolysis. Only the content of thymidine is changed, but
it is inaccessible for direct analysis as it is too small. Therefore Moriarty (1990)
restricts the time of using a fresh batch to within 2 months on condition of its
storage in the refrigerator with 2 % ethanol added to slow down the selfradiolysis of CH)-TDR in the preparation.
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
ity experiments. A definite part of the products of its catabolic decomposition
are incorporated by the microbial cells for biosynthesis and participate in the
nonspecific labeling of macromolecules. The catabolic pathway of nonspecific
labeling is evidenced by the dependence of the latter on the place of label in
the molecule of thymidine. For example, when CH)-6-methyl-CH)-thymidine
was used in experiments instead of the traditional CH)·mcthyl-thymidine, the
macromolecule fraction contained 0 to 35% of label in DNA and up to 60%
in the protein in different samples (Hollibaugh 1988).
Data evidenced that the nonspecific incorporation of CH)-TDR label was
responsible for the major part of radioactivity recorded in the macromolecule
fraction and then used by the numerous users of the thymidine method for
the calculation of microbial production; but, actually, it did not deal at all with
the synthesis of DNA and with microbial growth (Karl 1986). There were some
hopes that unspecific labeling, which causes errors in all the calculations made
with the thymidine method, depends upon the time of exposure and starts after
some 15 min of incubation. The discovery of the catabolic way for this process
also dashed these hopes, moreover as the latter was disproved experimentally
(Servais et al. 1987; Brittain and Karl 1990). Nonspecific labeling proves to be
even more pronounced in the samples of bottom sediments, where the CH)TDR added is readily utilized by local bacteria as the ordinary organic substrate (Carman et al. 1988).
Comparison of the DNA synthesis rate in cultures as estimated via CH)TDR incorporation and by its direct measurements with specific strains
showed that the former underestimated it by five to eight times even accounting for possible isotopic dilution (Jeffrey and Paul 1988). That CH)-TDR
cannot be accepted as a reliable measure of DNA synthesis can also be
deduced from the evidence of a large range of nonspecific labeling. Perhaps
even more serious is the indefinite and inestimable specific radioactivity of
CH)-TDR, as the precursor forming the thymidine nucleotide pool for DNA
synthesis (Karl 1986). First of all this concerns the initial specific radioactivity
of commercially supplied CH)-TDR preparations. For the users the specific
radioactivity as Ci mmot 1 (Re), written on the label of the batch preparation
is the only evidence of its level, and correspondingly it is introduced into the
formula for calculation of the CH)-TDR incorporation rate (Ir); but it cannot
be correct even in principle because this product is not stable in time. It is subjected to: (1) a relatively rapid radiolysis in a highly radioactive medium, thus
losing the label together with its methyl group. This process proceeds in a
refrigerated batch with a speed of approximately 4% a month. Its speed, being
dependent upon various factors, is immeasurable and unpredictable, because
the researcher can measure only the total dpm radioactivity. But it is not
changed due to the radiolysis. Only the content of thymidine is changed, but
it is inaccessible for direct analysis as it is too small. Therefore Moriarty (1990)
restricts the time of using a fresh batch to within 2 months on condition of its
storage in the refrigerator with 2 % ethanol added to slow down the selfradiolysis of CH)-TDR in the preparation.