160
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
filtered onto the 0.2-f.lm Nucleopore filters, and subsequently rinsed twice with
portions of prefiltered water to eliminate the remains of substrate radioactivity. The filters are then instantly placed upside down with their filtering surface
on a gelatin film coating the microscopic slide. The gelatin film is prepared
according to Meyer Reil's procedure (1978): the slide is immersed in the
gelatin solution and then drained vertically for half a minute to make a fine
film. The filters are placed over the dampish film. This preparation is placed
first in a cold place to drain, and then into the dessicator to dry over silicagel.
The dry slide is exposed for some time in a humid atmosphere to make it possible to peel the filter from the preparation. After careful peeling, the bacteria remain safe in the preparation, being glued within the gelatin. These
preparations are again thoroughly dried and coated with Kodak NTB-2 photoemulsion diluted one third at 43°C. The coated preparations are placed in
the dessicator over silicagel and incubated at from 5 to 10°C for 3 days. Then
they are treated with the developer and fixer to process the photoemulsion
and are thoroughly washed with distilled water. The developed preparations
are immersed into 0.05% acridine orange solution for staining. Being
destained by subsequent passing through a series of citrate buffers with pH
values of 6, 5.5, 5 and 4, and then dried, the preparations are ready for microscope examination.
The above protocol is based on the experience of Tabor and Nijhof (1982).
It can be simplified by omitting the procedure of extracting the filter from the
preparation before coating with photoemulsion; but in this case black Nucleopore filters must be used for filtration. When it is completed, they should be
placed on the gelatin-covered microscope slides with their filtering surface
upwards (Meyer ReiI1978).
The percentage of actively metabolizing bacterial cells among the whole
bacterioplankton populations in marine waters varies between 20 and 80%
when using an individual amino acid (glutamate) or their mixture as the substrate. The active bacteria, which are able to incorporate TDR, comprise
usually only a minor part of total bacterioplankton, some 10-20%, rarely more.
Their share is greater in eutrophic waters of coastal embayments and less,
down to 6%, in the open sea (Petros-Alio and Newell 1989).
4.2.3 A Study of the Spatial Distribution of Metabolically Active
Heterotrophic Microbial Populations
4.2.3.1 General Remarks
The investigation of the spatial distribution of specific microbial populations
in water bodies is among the main fields of study in aquatic microbial ecology.
The use of radioisotopic methodology for this purpose largely simplifies
these studies and makes them immeasurably more adequate than traditional
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
filtered onto the 0.2-f.lm Nucleopore filters, and subsequently rinsed twice with
portions of prefiltered water to eliminate the remains of substrate radioactivity. The filters are then instantly placed upside down with their filtering surface
on a gelatin film coating the microscopic slide. The gelatin film is prepared
according to Meyer Reil's procedure (1978): the slide is immersed in the
gelatin solution and then drained vertically for half a minute to make a fine
film. The filters are placed over the dampish film. This preparation is placed
first in a cold place to drain, and then into the dessicator to dry over silicagel.
The dry slide is exposed for some time in a humid atmosphere to make it possible to peel the filter from the preparation. After careful peeling, the bacteria remain safe in the preparation, being glued within the gelatin. These
preparations are again thoroughly dried and coated with Kodak NTB-2 photoemulsion diluted one third at 43°C. The coated preparations are placed in
the dessicator over silicagel and incubated at from 5 to 10°C for 3 days. Then
they are treated with the developer and fixer to process the photoemulsion
and are thoroughly washed with distilled water. The developed preparations
are immersed into 0.05% acridine orange solution for staining. Being
destained by subsequent passing through a series of citrate buffers with pH
values of 6, 5.5, 5 and 4, and then dried, the preparations are ready for microscope examination.
The above protocol is based on the experience of Tabor and Nijhof (1982).
It can be simplified by omitting the procedure of extracting the filter from the
preparation before coating with photoemulsion; but in this case black Nucleopore filters must be used for filtration. When it is completed, they should be
placed on the gelatin-covered microscope slides with their filtering surface
upwards (Meyer ReiI1978).
The percentage of actively metabolizing bacterial cells among the whole
bacterioplankton populations in marine waters varies between 20 and 80%
when using an individual amino acid (glutamate) or their mixture as the substrate. The active bacteria, which are able to incorporate TDR, comprise
usually only a minor part of total bacterioplankton, some 10-20%, rarely more.
Their share is greater in eutrophic waters of coastal embayments and less,
down to 6%, in the open sea (Petros-Alio and Newell 1989).
4.2.3 A Study of the Spatial Distribution of Metabolically Active
Heterotrophic Microbial Populations
4.2.3.1 General Remarks
The investigation of the spatial distribution of specific microbial populations
in water bodies is among the main fields of study in aquatic microbial ecology.
The use of radioisotopic methodology for this purpose largely simplifies
these studies and makes them immeasurably more adequate than traditional