170
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
onto a 0.45-l1m pore size membrane filter. Further treatment is carried out as
described above for water samples. An example of the results of corresponding measurements in shown in Fig. 4.2.
5. Procedure for the use of TDR and 14C02
For the study of spatial distribution of specific heterotrophic microflora
decomposing complex organic compounds like hydrocarbons, pesticides, or
detergents, two different approaches can be applied, based on the use of
radioisotopic labels. One is measuring the decomposition rate of those compounds labeled with 14C, if they are available. The samples of water or of
bottom sediments are incubated with these compounds, and the 14C02 thus
formed on their oxidation by bacteria is extracted from the samples and
radioassayed as described below (see Sect. 4.3.2). The relative rate of its formation is used as an indicator of activity of the specific microflora, which is
able to decompose a given specific organic substrate. The technique of such
experiments, invented by Crawford et al. (1973), is described below in more
detail (see Sect. 4.3).
Another approach is based upon the use of some sensitive radioisotopic indicators of enhancement of bacterial growth in the presence of unlabeled specific organic substrates of the kind mentioned above added to
the samples. Dark 14C02 uptake by bacteria or their uptake of tritiated thymidine (TDR) can be used as such radioisotopic indicators. Bacteria which are
able to decompose a specific organic substrate use part of the energy liberated during its oxidation for their biosynthesis, which is accompanied by
uptake and incorporation of external CO2 or thymidine. This gives the possibility to measure the relative activity of these specific groups of bacteria, thus
exposing the localization of the active microbial populations, which are
involved in their decomposition in a water basin, and thus recording the
spatial distribution of the sources of pollution, because the above-mentioned
specific substrates are the pollutants. The experiments in this case are arranged
as follows. The samples of water (or silt), collected in duplicate during the
day-long field mission, are placed into the bottles (or beakers), and preserved in the refrigerator. Before the start of the experiment, their temperature is equalized. Then one half of them is charged with equal portions
of solution (or emulsion, if it is a hydrocarbon or oil) of the specific organic
substrate, containing some 3-5 mg of the substance. If the 14C02 dark uptake
is selected as an indicator of microbial activity, all the samples are kept
in the dark for half an hour and then charged with equal portions of 14C_
carbonate working solution as described below (see Sect. 4.4.2), for measuring the bacterial production. The samples are incubated at 18°C for 20 h
and then treated in the same way as described for the above analysis. The
difference in radioactivity of bacteria counted in parallel samples with and
without added organic substrate is used as evidence of relative activity of
corresponding specific microflora at the given point where they have been
sampled.
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
onto a 0.45-l1m pore size membrane filter. Further treatment is carried out as
described above for water samples. An example of the results of corresponding measurements in shown in Fig. 4.2.
5. Procedure for the use of TDR and 14C02
For the study of spatial distribution of specific heterotrophic microflora
decomposing complex organic compounds like hydrocarbons, pesticides, or
detergents, two different approaches can be applied, based on the use of
radioisotopic labels. One is measuring the decomposition rate of those compounds labeled with 14C, if they are available. The samples of water or of
bottom sediments are incubated with these compounds, and the 14C02 thus
formed on their oxidation by bacteria is extracted from the samples and
radioassayed as described below (see Sect. 4.3.2). The relative rate of its formation is used as an indicator of activity of the specific microflora, which is
able to decompose a given specific organic substrate. The technique of such
experiments, invented by Crawford et al. (1973), is described below in more
detail (see Sect. 4.3).
Another approach is based upon the use of some sensitive radioisotopic indicators of enhancement of bacterial growth in the presence of unlabeled specific organic substrates of the kind mentioned above added to
the samples. Dark 14C02 uptake by bacteria or their uptake of tritiated thymidine (TDR) can be used as such radioisotopic indicators. Bacteria which are
able to decompose a specific organic substrate use part of the energy liberated during its oxidation for their biosynthesis, which is accompanied by
uptake and incorporation of external CO2 or thymidine. This gives the possibility to measure the relative activity of these specific groups of bacteria, thus
exposing the localization of the active microbial populations, which are
involved in their decomposition in a water basin, and thus recording the
spatial distribution of the sources of pollution, because the above-mentioned
specific substrates are the pollutants. The experiments in this case are arranged
as follows. The samples of water (or silt), collected in duplicate during the
day-long field mission, are placed into the bottles (or beakers), and preserved in the refrigerator. Before the start of the experiment, their temperature is equalized. Then one half of them is charged with equal portions
of solution (or emulsion, if it is a hydrocarbon or oil) of the specific organic
substrate, containing some 3-5 mg of the substance. If the 14C02 dark uptake
is selected as an indicator of microbial activity, all the samples are kept
in the dark for half an hour and then charged with equal portions of 14C_
carbonate working solution as described below (see Sect. 4.4.2), for measuring the bacterial production. The samples are incubated at 18°C for 20 h
and then treated in the same way as described for the above analysis. The
difference in radioactivity of bacteria counted in parallel samples with and
without added organic substrate is used as evidence of relative activity of
corresponding specific microflora at the given point where they have been
sampled.