216
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
tion procedure, it is more practical to make several (two to three) parallel
filtrations from one bottle - from both the experimental and the control -
rather than make the same number of parallel incubations.
The exposure time depends on the temperature of incubation: 20-40min
at >20°C; 40-60min at lS-20°C and 2 to 3h at <1S°c. The incubation time
may be preselected during the time course experiment with estimation of R;
values. It should be placed within the linear part of the corresponding curve.
Incubation is ended by injection of O.4ml 20% formalin containing 20mg of
unlabeled thymidine per SOml. The samples are preserved in the refrigerator
until the extraction procedure, which is accomplished as follows: the penicillin
incubation bottle is opened and weighed, then its content is mixed with a glass
stick and approximately O.S cm 3 subsamples are withdrawn from it and transferred into the preweighed centrifuge test tubes, which are weighed again to
know what parts of the subsample were taken for the extraction of the macromolecular fraction insoluble in cold TCA; 3% formalin solution is added into
a 4-ml test tube. The test tubes are closed, shaken and centrifuged for S min at
3000 rpm. The supernatant is discharged to remove the remains of nonassimilated labeled thymidine from the sediment. This operation is repeated
three times. Finally, the pellet is mixed with loS ml of 0.4 N N aO H + 2 mm
EDTA + 0.1 % SDS (Na-dodecylsulfate) and left for 12h at 2SoC to hydrolyze
the DNA, when the mixture is centrifuged for 10 min at SOOO g and weighed
again to know the total amount of liquid. One-ml portions of supernatant are
removed and placed into a small calibrated for volume test tubes, then neutralized with 2 N HCI in the presence of trace amounts of phenolphthalein
indicator. Then 0.02ml of Smgml- 1 aqueous DNA solution and O.lml of 1mg
ml- 1 thymidine aqueous solution are added into them as the carriers. After
mixing, ice-cold 10% TCA solution equal to the volume of liquid in the test
tubes is added into them, their content is mixed, and they are chilled on ice
for 1 h. Then the volume of liquid in the test tubes is recorded, its content is
mixed and loS ml is instantly withdrawn from them and filtered onto the Sartorius membrane filters 0.2-mm pore size presoaked in lOmg ISOml of the nonlabeled thymidine solution. The test tubes are washed with 2ml of ice cold S%
TCA. The liquid is filtered also through the same filter being sequenced with
additional three 1-ml portions of the same TCA solution. The filters are dried,
their edges are cut off, only their working area remaining, and they are radioassayed as described above (see Sect. 4.4.3.2). The dead control subsamples are
treated in the same way, so the mean value of radioactivity counted on experimental filters is corrected per the control one to estimate the R; value of the
radioactivity of 3H incorporated into the cold TCA insoluble macromolecular
fraction calculated as cpm per 1 g of wet sediment h- 1 of incubation.
The values of R; thus measured usually are corrected for the recovery percentage of labeled DNA during the extraction procedure and are expressed
in dpm units using an interval or external standard for quenching correction.
The recovery varies roughly between 20 and 70% (Fallon et al. 1983; Finlay et
al. 1984; Thorn and Ventullo 1988; Robarts and Zohary 1993). It is estimated
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
tion procedure, it is more practical to make several (two to three) parallel
filtrations from one bottle - from both the experimental and the control -
rather than make the same number of parallel incubations.
The exposure time depends on the temperature of incubation: 20-40min
at >20°C; 40-60min at lS-20°C and 2 to 3h at <1S°c. The incubation time
may be preselected during the time course experiment with estimation of R;
values. It should be placed within the linear part of the corresponding curve.
Incubation is ended by injection of O.4ml 20% formalin containing 20mg of
unlabeled thymidine per SOml. The samples are preserved in the refrigerator
until the extraction procedure, which is accomplished as follows: the penicillin
incubation bottle is opened and weighed, then its content is mixed with a glass
stick and approximately O.S cm 3 subsamples are withdrawn from it and transferred into the preweighed centrifuge test tubes, which are weighed again to
know what parts of the subsample were taken for the extraction of the macromolecular fraction insoluble in cold TCA; 3% formalin solution is added into
a 4-ml test tube. The test tubes are closed, shaken and centrifuged for S min at
3000 rpm. The supernatant is discharged to remove the remains of nonassimilated labeled thymidine from the sediment. This operation is repeated
three times. Finally, the pellet is mixed with loS ml of 0.4 N N aO H + 2 mm
EDTA + 0.1 % SDS (Na-dodecylsulfate) and left for 12h at 2SoC to hydrolyze
the DNA, when the mixture is centrifuged for 10 min at SOOO g and weighed
again to know the total amount of liquid. One-ml portions of supernatant are
removed and placed into a small calibrated for volume test tubes, then neutralized with 2 N HCI in the presence of trace amounts of phenolphthalein
indicator. Then 0.02ml of Smgml- 1 aqueous DNA solution and O.lml of 1mg
ml- 1 thymidine aqueous solution are added into them as the carriers. After
mixing, ice-cold 10% TCA solution equal to the volume of liquid in the test
tubes is added into them, their content is mixed, and they are chilled on ice
for 1 h. Then the volume of liquid in the test tubes is recorded, its content is
mixed and loS ml is instantly withdrawn from them and filtered onto the Sartorius membrane filters 0.2-mm pore size presoaked in lOmg ISOml of the nonlabeled thymidine solution. The test tubes are washed with 2ml of ice cold S%
TCA. The liquid is filtered also through the same filter being sequenced with
additional three 1-ml portions of the same TCA solution. The filters are dried,
their edges are cut off, only their working area remaining, and they are radioassayed as described above (see Sect. 4.4.3.2). The dead control subsamples are
treated in the same way, so the mean value of radioactivity counted on experimental filters is corrected per the control one to estimate the R; value of the
radioactivity of 3H incorporated into the cold TCA insoluble macromolecular
fraction calculated as cpm per 1 g of wet sediment h- 1 of incubation.
The values of R; thus measured usually are corrected for the recovery percentage of labeled DNA during the extraction procedure and are expressed
in dpm units using an interval or external standard for quenching correction.
The recovery varies roughly between 20 and 70% (Fallon et al. 1983; Finlay et
al. 1984; Thorn and Ventullo 1988; Robarts and Zohary 1993). It is estimated