Determination of Microbial Production
215
dating the accuracy of thymidine methodology, nobody who worked with sediments attached any importance to this. Then Carman et al. (1988) discovered
that both phenomena took place in samples of bottom sediments to an even
greater extent than in water. They found only about 2% of the 3H label incorporated into DNA in the macromolecular fraction insoluble in TCA. Moreover, with the use of double labeling ofTDR with 3H and with 14C in its methyl
group they were able to prove that thymidine added to the sediment is immediately catabolized by bacteria down to small molecules, which are rapidly
uptaken by them and via the de novo pathways of the pyrimidine and purine
synthesis it is incorporated into the DNA. Thus, even those 2% of label found
in the DNA originated not only from the incorporated CH)-TDR but also
from the labeled residues of its decomposed molecules. A major part of the
label added as CH)-TDR to the sediment samples was discovered in proteins,
in RNA, and also in lipids. The authors conclude that, faced with significant
participation of the de novo pathway in the labeling of DNA from exogenous
sources of CH)-TDR, the purification of DNA from the macromolecular fraction is itself of minor value, same as the values of original specific activity of
CH)-TDR, which are used to calculate its absolute incorporation rate into the
DNA. Therefore, also in the case of the bottom sediments, the TDR-method
cannot be used without calibration and estimation of the reliable empirical
conversion factor.
The detailed procedure of measuring R; in bottom sediments is given
below as modified by Fallon et al. (1983) and Thorn and Ventullo (1988).
The optimal nominal specific radioactivity of the CH)-TDR in the batch
destined for work with sediments should be within 2-4 Ci mmol. If it is impossible to obtain such a batch ready for use, there is good reason to decrease the
specific radioactivity in it by the injection of the necessary amount of nonlabe led thymidine. For example, 1-2ml of the unlabeled thymidine in 20%
ethanol solution should be added to the batch containing 5 m Ci of CH)-TDR
with the nominal specific radioactivity of around 40-60 Ci mmol, which contains Ij.lmol of this substance in 1 ml.
Sediment cores some 15-18mm in diameter are collected as shown above
(see Sect. 2.5.2) and closed at both ends while still in water. To prepare the
samples for the experiments, the column of sediment is pushed up with the
piston 1-2 cm above the edge of the tube and cut out. From this portion of
sediment approximately 2-3cm 3 is placed into the weighed 20-ml penicillin
bottle and weighed again to know the weight of the sediment subs ample. Then
0.5ml of filtered seawater and a portion of CH)-TDR containing 20mCi of
actual dpm radioactivity (previously measured in the working solution) are
added to the sediment subsample in the vial and its content is carefully mixed
with a fine glass stick fiat on its end. The bottle is closed with a serum cork
and placed for exposure into the darkened aquarium at a temperature nearing
that in situ. In the same way, a killed control subs ample is prepared to which
0.2mI20% formalin is added before the injection of the radioisotope. Because
the variability in R; estimamtions is caused mostly by deviations in the filtra-
215
dating the accuracy of thymidine methodology, nobody who worked with sediments attached any importance to this. Then Carman et al. (1988) discovered
that both phenomena took place in samples of bottom sediments to an even
greater extent than in water. They found only about 2% of the 3H label incorporated into DNA in the macromolecular fraction insoluble in TCA. Moreover, with the use of double labeling ofTDR with 3H and with 14C in its methyl
group they were able to prove that thymidine added to the sediment is immediately catabolized by bacteria down to small molecules, which are rapidly
uptaken by them and via the de novo pathways of the pyrimidine and purine
synthesis it is incorporated into the DNA. Thus, even those 2% of label found
in the DNA originated not only from the incorporated CH)-TDR but also
from the labeled residues of its decomposed molecules. A major part of the
label added as CH)-TDR to the sediment samples was discovered in proteins,
in RNA, and also in lipids. The authors conclude that, faced with significant
participation of the de novo pathway in the labeling of DNA from exogenous
sources of CH)-TDR, the purification of DNA from the macromolecular fraction is itself of minor value, same as the values of original specific activity of
CH)-TDR, which are used to calculate its absolute incorporation rate into the
DNA. Therefore, also in the case of the bottom sediments, the TDR-method
cannot be used without calibration and estimation of the reliable empirical
conversion factor.
The detailed procedure of measuring R; in bottom sediments is given
below as modified by Fallon et al. (1983) and Thorn and Ventullo (1988).
The optimal nominal specific radioactivity of the CH)-TDR in the batch
destined for work with sediments should be within 2-4 Ci mmol. If it is impossible to obtain such a batch ready for use, there is good reason to decrease the
specific radioactivity in it by the injection of the necessary amount of nonlabe led thymidine. For example, 1-2ml of the unlabeled thymidine in 20%
ethanol solution should be added to the batch containing 5 m Ci of CH)-TDR
with the nominal specific radioactivity of around 40-60 Ci mmol, which contains Ij.lmol of this substance in 1 ml.
Sediment cores some 15-18mm in diameter are collected as shown above
(see Sect. 2.5.2) and closed at both ends while still in water. To prepare the
samples for the experiments, the column of sediment is pushed up with the
piston 1-2 cm above the edge of the tube and cut out. From this portion of
sediment approximately 2-3cm 3 is placed into the weighed 20-ml penicillin
bottle and weighed again to know the weight of the sediment subs ample. Then
0.5ml of filtered seawater and a portion of CH)-TDR containing 20mCi of
actual dpm radioactivity (previously measured in the working solution) are
added to the sediment subsample in the vial and its content is carefully mixed
with a fine glass stick fiat on its end. The bottle is closed with a serum cork
and placed for exposure into the darkened aquarium at a temperature nearing
that in situ. In the same way, a killed control subs ample is prepared to which
0.2mI20% formalin is added before the injection of the radioisotope. Because
the variability in R; estimamtions is caused mostly by deviations in the filtra-