Determination of Microbial Production
191
The samples thus charged are placed for exposure into the dark incubator, which makes their light isolation safer. Simulation of the in situ temperature on vertical profiles is always a problem. The most practical way to solve
it on board ship or in the field laboratory is to make two dark aquariaincubators, one for the upper mixed layer temperature, and another for the
subthermocline layer. The 14C02 uptake rates measured in samples taken from
depths with intermediate temperature records can be corrected with the use
of the QlO° coefficient, which for natural bacterioplankton is close to 2.4. After
the samples are put into the incubators, two to three extra samples are killed
with a weak Lugol solution and charged with the same portions of working
isotope solution which serve as dead controls. The optimal exposure time for
a definite water basin and for a given season is best selected by making a time
course curve of 14COZ dark uptake in a surface sample. The incubation time
should fit within the linear part of this curve (Fig. 4.7). For example, the incubation times used in my practice were: in Antarctic waters (2°C) 60 h, in subarctic seas in summer «10 0c) 36 h, in temperate waters in summer (>15 DC)
18-20h, in tropical waters (>20°C) 10-12h.
After incubation, the samples are transferred again into a darkened place.
One by one the bags are opened and the samples still inside are fixed with a
weak (yellowish color) Lugol solution. Then the content of each bottle is
prefiltered through 3-mm pore size Nucleopore filters in the funnel of a kind
shown in Fig. 2.7 with a working area diameter of -40mm. The set for filtration is arranged as shown in Fig. 4.8. The draft is created by the weight of water
column in the long tube. The Nucleopore filters are kept in a weak detergent
solution. After the filter is fixed in the funnel, a portion of distilled water is
passed first through the system to fill it. Then the tap is closed and the sample
is poured into the funnel. By opening the tap the sample is filtered. The filtrate
is collected and then refiltered through a 0.2--O.4-llm pore size membrane filter
retaining bacteria. Should it be difficult to filter the whole sample, a part of it
thus may be filtered, recording its total volume and the volume filtered. The
filters are treated in the funnel as in estimations of photosynthesis (see Sect.
2.3.2.5) and are placed still wet into the scintillation vials for counting their
cpm radioactivity R i . The same operation is carried out with the dead controls.
Then, mean Ri values measured in each sample are corrected by subtracting
the mean control count. No quench corrections are not needed because the
radioactivity of 14C02 in its working solution has been measured under the
same counting conditions as that of Ri (see Sect. 2.3.2.2). The dark uptake
Ri x Ck x 103 x 1.06 x 24
values Ac are calculated as follows: A =
, mgCm- 3
c
Rt x t
day-I, if: Ri is the mean radioactivity of parallel filters corrected for control,
cpm per whole volume of the sample; Ck is TCOrcarbon content in water mg
1-1, and R, cpm radioactivity of a portion of working solution added into the
sample. Then the production of heterotrophic bacteria (Ph) will be equal to:
Ph = Ac 12 mg C m- 3 day in the units of carbon, or Ph = Ac 60 mg m- 3 day-l in the
191
The samples thus charged are placed for exposure into the dark incubator, which makes their light isolation safer. Simulation of the in situ temperature on vertical profiles is always a problem. The most practical way to solve
it on board ship or in the field laboratory is to make two dark aquariaincubators, one for the upper mixed layer temperature, and another for the
subthermocline layer. The 14C02 uptake rates measured in samples taken from
depths with intermediate temperature records can be corrected with the use
of the QlO° coefficient, which for natural bacterioplankton is close to 2.4. After
the samples are put into the incubators, two to three extra samples are killed
with a weak Lugol solution and charged with the same portions of working
isotope solution which serve as dead controls. The optimal exposure time for
a definite water basin and for a given season is best selected by making a time
course curve of 14COZ dark uptake in a surface sample. The incubation time
should fit within the linear part of this curve (Fig. 4.7). For example, the incubation times used in my practice were: in Antarctic waters (2°C) 60 h, in subarctic seas in summer «10 0c) 36 h, in temperate waters in summer (>15 DC)
18-20h, in tropical waters (>20°C) 10-12h.
After incubation, the samples are transferred again into a darkened place.
One by one the bags are opened and the samples still inside are fixed with a
weak (yellowish color) Lugol solution. Then the content of each bottle is
prefiltered through 3-mm pore size Nucleopore filters in the funnel of a kind
shown in Fig. 2.7 with a working area diameter of -40mm. The set for filtration is arranged as shown in Fig. 4.8. The draft is created by the weight of water
column in the long tube. The Nucleopore filters are kept in a weak detergent
solution. After the filter is fixed in the funnel, a portion of distilled water is
passed first through the system to fill it. Then the tap is closed and the sample
is poured into the funnel. By opening the tap the sample is filtered. The filtrate
is collected and then refiltered through a 0.2--O.4-llm pore size membrane filter
retaining bacteria. Should it be difficult to filter the whole sample, a part of it
thus may be filtered, recording its total volume and the volume filtered. The
filters are treated in the funnel as in estimations of photosynthesis (see Sect.
2.3.2.5) and are placed still wet into the scintillation vials for counting their
cpm radioactivity R i . The same operation is carried out with the dead controls.
Then, mean Ri values measured in each sample are corrected by subtracting
the mean control count. No quench corrections are not needed because the
radioactivity of 14C02 in its working solution has been measured under the
same counting conditions as that of Ri (see Sect. 2.3.2.2). The dark uptake
Ri x Ck x 103 x 1.06 x 24
values Ac are calculated as follows: A =
, mgCm- 3
c
Rt x t
day-I, if: Ri is the mean radioactivity of parallel filters corrected for control,
cpm per whole volume of the sample; Ck is TCOrcarbon content in water mg
1-1, and R, cpm radioactivity of a portion of working solution added into the
sample. Then the production of heterotrophic bacteria (Ph) will be equal to:
Ph = Ac 12 mg C m- 3 day in the units of carbon, or Ph = Ac 60 mg m- 3 day-l in the