Estimation of Relative Microbial Activity in Aquatic Habitats
159
of their multiplication. It might indicate at least the location of specific
microbial activity in water basins.
4.2.2 The Technique of Microradioautography
As mentioned above, this method was used for the estimation of the percentage of active bacteria within bacterioplankton populations (Ramsay 1974;
Hoppe 1976, 1978; Meyer Reil 1978; Tabor and Nijhof 1982), for the distinction between the algal and bacterial uptake of organic substances in natural
waters (Munro and Brock 1968), and for estimation of the percentage of bacterioplankton cells able to uptake exogenous thymidine and individual amino
acids (Riemann et al. 1984; Douglas et al. 1987; Petros-Alio and Newell 1989).
The latter problem is combined with evaluating the validity of the TDRuptake method for measuring bacterial production (see below).
To evaluate the percentage of active microbial cells in the whole bacterioplankton population, a sample of natural water is incubated with a portion
of 3H-Iabeled dissolved organic substrate with a very high specific radioactivity. For this purpose, only tritium can be used as a label; because of the microscopic size of the objects, only this isotope with a very low radiation energy
may activate the silver grains of photoemulsion sufficiently close to the bacterial cells. Therefore either 3H-Iabeled glucose and acetate or 3H-Iabeled
mixture amino acids are used as substrates for this purpose. The specific
radioactivity of these substrates should be not less than 1 Cimmol- 1 • Frequently, the preparations with the 5 to 15 Cimmol- I specific radioactivity are
used to achieve in a relatively short incubation time the radioactivity in the
individual cells of bacteria sufficient to be detected by radioautography. Once
the incubation with the radioactive substrate is terminated, the bacterioplankton cells are filtered through black Nucleopore filters, which are then
placed on microscopic slides and coated with special fine-grain photoemulsion. After several days of incubation under cool conditions, the photoemulsion on the slides is developed and bacteria are stained with the fluorochrome
archidine orange according to Hobbie et al. (1977). The preparation is examined under the epifluorescence microscope by counting the total number of
bacteria shining blue-green on it, and the active bacteria among them, which
are surrounded by black grains of silver. This gives the immediate possibility
to estimate the share of active bacteria in the whole bacterioplankton.
The exact procedure for such a microradioautography varies in different
publications. The most feasible among them is as follows: 0.3-1 mCi of 3H_
glucose with nominal specific radioactivity 10 to 20Cimmol- 1 is injected into
lOml subsamples of natural water. The subs ample is incubated for 3-6h at the
in situ temperature. Once the incubation is terminated, bacteria are fixed
with 3% formalin (end concentration) and diluted three to five times with
prefiltered natural bacteria-free water. The 1-3-ml portions of this dilution are
159
of their multiplication. It might indicate at least the location of specific
microbial activity in water basins.
4.2.2 The Technique of Microradioautography
As mentioned above, this method was used for the estimation of the percentage of active bacteria within bacterioplankton populations (Ramsay 1974;
Hoppe 1976, 1978; Meyer Reil 1978; Tabor and Nijhof 1982), for the distinction between the algal and bacterial uptake of organic substances in natural
waters (Munro and Brock 1968), and for estimation of the percentage of bacterioplankton cells able to uptake exogenous thymidine and individual amino
acids (Riemann et al. 1984; Douglas et al. 1987; Petros-Alio and Newell 1989).
The latter problem is combined with evaluating the validity of the TDRuptake method for measuring bacterial production (see below).
To evaluate the percentage of active microbial cells in the whole bacterioplankton population, a sample of natural water is incubated with a portion
of 3H-Iabeled dissolved organic substrate with a very high specific radioactivity. For this purpose, only tritium can be used as a label; because of the microscopic size of the objects, only this isotope with a very low radiation energy
may activate the silver grains of photoemulsion sufficiently close to the bacterial cells. Therefore either 3H-Iabeled glucose and acetate or 3H-Iabeled
mixture amino acids are used as substrates for this purpose. The specific
radioactivity of these substrates should be not less than 1 Cimmol- 1 • Frequently, the preparations with the 5 to 15 Cimmol- I specific radioactivity are
used to achieve in a relatively short incubation time the radioactivity in the
individual cells of bacteria sufficient to be detected by radioautography. Once
the incubation with the radioactive substrate is terminated, the bacterioplankton cells are filtered through black Nucleopore filters, which are then
placed on microscopic slides and coated with special fine-grain photoemulsion. After several days of incubation under cool conditions, the photoemulsion on the slides is developed and bacteria are stained with the fluorochrome
archidine orange according to Hobbie et al. (1977). The preparation is examined under the epifluorescence microscope by counting the total number of
bacteria shining blue-green on it, and the active bacteria among them, which
are surrounded by black grains of silver. This gives the immediate possibility
to estimate the share of active bacteria in the whole bacterioplankton.
The exact procedure for such a microradioautography varies in different
publications. The most feasible among them is as follows: 0.3-1 mCi of 3H_
glucose with nominal specific radioactivity 10 to 20Cimmol- 1 is injected into
lOml subsamples of natural water. The subs ample is incubated for 3-6h at the
in situ temperature. Once the incubation is terminated, bacteria are fixed
with 3% formalin (end concentration) and diluted three to five times with
prefiltered natural bacteria-free water. The 1-3-ml portions of this dilution are