Determination of Microbial Production
207
Additionally the radiolysis the C-PH] bonds in the molecules of
labeled thymidine exhibits exchange reactions with the hydrogen of the molecules of the solvents like water or the ethanol, which is recommended to be
added to the batches. This exchange proceeds without chemical changes in the
labeled organic compounds, they only lose their label. This process proceeds
during the storage of the batches and is facilitated during extraction procedures of macromolecular purification under the acid or alkaline action (Evans
1974).
However, the instabiliy of TDR in the batch itself constitutes a minor
cause of the indefinite character of Rc with the CH)-thymidine method. One
of the major causes often is its initially wrong presentation on labels of the
batches by the supplier. The Rc written on the label cannot be controlled and
actually is never controlled by the users, or even the total dpm radioactivity
they contain, which does not change in time. This happens because for the
manufacturer it is profitable to make a large portion of labeled substance in
one large batch and after measuring its Rc once gradually distribute it into the
batches according to market demand. Thus, such a primary radioactive preparation may change its Rc even during storage before manufacturing fresh
batches from it. The discrepancies between the label and true Rc content are
a common phenomenon. The difference between them can be as much as five
times (Prescott 1970; Monks et al. 1971). A difference of several times can even
be in their dpm radioactivity. In the batch, naturally, it is always less than on
the label, because such a difference is profitable for the manufacturer. I myself
have discovered three times less dpm radioactivity than that written on the
label of the first Amersham CH)-TDR I dealt with. In this hopeless situation,
with the original specific radioactivity of CH)-TDR in its batch preparation,
with its dilution inside the cells of bacteria via the de novo synthesis of thymidine also being unknown, and accounting for its rapid catabolism by bacteria
and for nonspecific labeling, the correction procedure for some Rc percentages in ambiental samples for isotopic dilution recommended by Moriarty
(1986) is thwarted at every turn. It only creates the impression of safety in the
thymidine method, like the recommendation to use a higher concentration of
labeled thymidine to block the external and especially the de novo isotopic
dilution, where the results have been proved to be exactly the opposite (Karl
1986; Hollibaugh 1988). The former author has concluded that because of a
large nonspecific labeling, rapid decomposition of added CH)-thymidine in the
samples and the use of practically unknown values of its specific radioactivity,
the use of presumed DNA labeling from exogenous CH)-TDR sources as the
measure of microbial production is no more reliable than the general nonspecific labeling of all cellular macromolecules. Moreover, because of this a
situation with this method, also the absolute rates of thymidine incorporation
into DNA or even simply its uptake by bacteria in ambiental samples cannot
be estimated. Only the radioactivity (Rb) of the 3H label acquired by them in
the presence of CH)-TDR added and expressed as dpm [-I h- 1 could be measured. This conclusion automatically assumes that any reliable theoretical con-
207
Additionally the radiolysis the C-PH] bonds in the molecules of
labeled thymidine exhibits exchange reactions with the hydrogen of the molecules of the solvents like water or the ethanol, which is recommended to be
added to the batches. This exchange proceeds without chemical changes in the
labeled organic compounds, they only lose their label. This process proceeds
during the storage of the batches and is facilitated during extraction procedures of macromolecular purification under the acid or alkaline action (Evans
1974).
However, the instabiliy of TDR in the batch itself constitutes a minor
cause of the indefinite character of Rc with the CH)-thymidine method. One
of the major causes often is its initially wrong presentation on labels of the
batches by the supplier. The Rc written on the label cannot be controlled and
actually is never controlled by the users, or even the total dpm radioactivity
they contain, which does not change in time. This happens because for the
manufacturer it is profitable to make a large portion of labeled substance in
one large batch and after measuring its Rc once gradually distribute it into the
batches according to market demand. Thus, such a primary radioactive preparation may change its Rc even during storage before manufacturing fresh
batches from it. The discrepancies between the label and true Rc content are
a common phenomenon. The difference between them can be as much as five
times (Prescott 1970; Monks et al. 1971). A difference of several times can even
be in their dpm radioactivity. In the batch, naturally, it is always less than on
the label, because such a difference is profitable for the manufacturer. I myself
have discovered three times less dpm radioactivity than that written on the
label of the first Amersham CH)-TDR I dealt with. In this hopeless situation,
with the original specific radioactivity of CH)-TDR in its batch preparation,
with its dilution inside the cells of bacteria via the de novo synthesis of thymidine also being unknown, and accounting for its rapid catabolism by bacteria
and for nonspecific labeling, the correction procedure for some Rc percentages in ambiental samples for isotopic dilution recommended by Moriarty
(1986) is thwarted at every turn. It only creates the impression of safety in the
thymidine method, like the recommendation to use a higher concentration of
labeled thymidine to block the external and especially the de novo isotopic
dilution, where the results have been proved to be exactly the opposite (Karl
1986; Hollibaugh 1988). The former author has concluded that because of a
large nonspecific labeling, rapid decomposition of added CH)-thymidine in the
samples and the use of practically unknown values of its specific radioactivity,
the use of presumed DNA labeling from exogenous CH)-TDR sources as the
measure of microbial production is no more reliable than the general nonspecific labeling of all cellular macromolecules. Moreover, because of this a
situation with this method, also the absolute rates of thymidine incorporation
into DNA or even simply its uptake by bacteria in ambiental samples cannot
be estimated. Only the radioactivity (Rb) of the 3H label acquired by them in
the presence of CH)-TDR added and expressed as dpm [-I h- 1 could be measured. This conclusion automatically assumes that any reliable theoretical con-