Determination of Microbial Production
213
into three cold plastic test tubes which have 20-ml marks, thus having two subsamples from each experimental bottle and one from the control; 2.3 ml ice
cold 50% TCA solution is added into each test tube. After subsequent 10min
chilling on ice, all three subsamples are filtered onto 0.20-f-lm Sartorius
membrane filters with a working area diameter of 20mm. The filters should
be presoaked for several hours in the same carrier solution of nonlabeled
thymidine which was used before to terminate the incubation. The filters, after
filtration, are rinsed in the funnel five times with 1-ml portions of 5% cold
TCA solution and finally with 1 ml of the above-mentioned carrier solution
diluted 1 : 15 with distilled water. The filters are dried, their external edges are
cut out. Then they are placed into the scintillation vials, dissolved with 1 ml of
ethylene glycol, filled with 10 ml of scintillation cocktail, and radioassayed after
previous storage for some 10h.
The R; values are calculated as mean cpm radioactivity of filters
from two parallel filtrations minus the dead control, and are expressed as
cpml-1h- 1 .
3. Calculation of the (presumed) thymidine incorporation rate into the DNA
(T;)
It was also mentioned that this value cannot, in principle, be estimated with
reasonable accuracy when using the thymidine method; but in practice, it
is used for calculating during routine hydrobiological works. It can be done
according to corresponding recommendations (Moriarty 1986, 1990; Bell
1986). The T; values are calculated even by users of the thymidine method who
employ empirical conversion factors for computation of microbial production.
However, in this case, this step seems to be quite useless, because all other
members of the equation remain constant except the radioactivity of 3H measured in dpm units in the macromolecular fraction (R;). This equation looks
R. xl.35x10 4
as follows: N = ----',----- where N is the number of bacteria divided (or
Rcxt
produced) h- I , 1.35 is the joint constant assuming the conversion factor
K f = 0.5 X lOis cells mol-I of thymidine incorporated, Rc is the specific radioactivity of eH)-TDR used and t is the incubation time, h. Users of this method
calculate the T; as follows: T; = : mmoll- 1 , where R; is the radioactivity of
c
TDR incorporated by bacterioplankton into the cold TCA-insolible fraction,
dpm l-l_h-l, and Rc is the specific radioactivity of TDR preparation used, dpm
mmol- I .
4. Calibration of R; after the bacterial production (Pb) measured with the dark
14C02 uptake method
Both parallel experiments for measuring of R; and P b are performed with the
use of standard protocols, derived for both methods to accord with the environmental conditions. When the thymidine method is used to facilitate scan-
213
into three cold plastic test tubes which have 20-ml marks, thus having two subsamples from each experimental bottle and one from the control; 2.3 ml ice
cold 50% TCA solution is added into each test tube. After subsequent 10min
chilling on ice, all three subsamples are filtered onto 0.20-f-lm Sartorius
membrane filters with a working area diameter of 20mm. The filters should
be presoaked for several hours in the same carrier solution of nonlabeled
thymidine which was used before to terminate the incubation. The filters, after
filtration, are rinsed in the funnel five times with 1-ml portions of 5% cold
TCA solution and finally with 1 ml of the above-mentioned carrier solution
diluted 1 : 15 with distilled water. The filters are dried, their external edges are
cut out. Then they are placed into the scintillation vials, dissolved with 1 ml of
ethylene glycol, filled with 10 ml of scintillation cocktail, and radioassayed after
previous storage for some 10h.
The R; values are calculated as mean cpm radioactivity of filters
from two parallel filtrations minus the dead control, and are expressed as
cpml-1h- 1 .
3. Calculation of the (presumed) thymidine incorporation rate into the DNA
(T;)
It was also mentioned that this value cannot, in principle, be estimated with
reasonable accuracy when using the thymidine method; but in practice, it
is used for calculating during routine hydrobiological works. It can be done
according to corresponding recommendations (Moriarty 1986, 1990; Bell
1986). The T; values are calculated even by users of the thymidine method who
employ empirical conversion factors for computation of microbial production.
However, in this case, this step seems to be quite useless, because all other
members of the equation remain constant except the radioactivity of 3H measured in dpm units in the macromolecular fraction (R;). This equation looks
R. xl.35x10 4
as follows: N = ----',----- where N is the number of bacteria divided (or
Rcxt
produced) h- I , 1.35 is the joint constant assuming the conversion factor
K f = 0.5 X lOis cells mol-I of thymidine incorporated, Rc is the specific radioactivity of eH)-TDR used and t is the incubation time, h. Users of this method
calculate the T; as follows: T; = : mmoll- 1 , where R; is the radioactivity of
c
TDR incorporated by bacterioplankton into the cold TCA-insolible fraction,
dpm l-l_h-l, and Rc is the specific radioactivity of TDR preparation used, dpm
mmol- I .
4. Calibration of R; after the bacterial production (Pb) measured with the dark
14C02 uptake method
Both parallel experiments for measuring of R; and P b are performed with the
use of standard protocols, derived for both methods to accord with the environmental conditions. When the thymidine method is used to facilitate scan-