Preparation of Labeled Food
109
subsequent centrifugation. A working suspension of necessary density is then
prepared from the sediment. This suspension of plant material can also be used
for preparing the mature detritus containing specific microfJ.ora and protozoa.
For this, the suspension of freshly labeled plant material is first heated in a test
tube in boiling water, then cooled and contaminated with a small piece of silt.
After 2-3 weeks of incubatIon at 25-3U vC, the plant detritus will be ready for
use.
Bacteria labeled with 14C can be used in the feeding experiments either in
a state of suspension of a single species grown in culture or in the state of the
natural labeled bacterioplankton assemblages. For the first, suspension of
labeled bacteria can be obtained by growing them on organic substrates
labeled with 14C, such as labeled glucose or protein hydrolyzate; but these are
rather expensive. Instead, acid hydrolyzate of labeled microalgae like
ChIarella can be easily prepared in the laboratory as follows. The suspension
of labeled algae with a Cr value of about 0.1-0.2 x 10- 3 ~g C cpm- 1 is placed into
a beaker and dried. Then 60% H 2S04 is added and it is heated in a boiling
water bath for 4-5 h. Then the hydrolyzate thus received is dissolved, neutralized, with Na2C03 heated again to ~lOO°C, cooled, and filtered first through
a GF filter, and then through a dense (0.2-~m pore size) membrane filter. The
hydrolyzate of algae thus prepared is distributed into ampules and sterilized
by boiling three times for 20min at I-day intervals.
To obtain labeled bacteria using these dissolved organic substrates,
first the cultures of bacteria are isolated from the water body and checked
for their ability (1) to grow on a given labeled substrate, and (2) to grow
homogeneously in the liquid medium. The strain of bacteria thus selected
grows in the liquid medium or in a small amount (2-3 ml) of the same medium
poured over the surface of 4 % solid agar in a petri dish. Into this medium,
before its contamination with the microbial culture, the labeled dissolved organic matter (LOM) is added (ca. 2-5~Ci per 100ml). If the specific
radioactivity of the LOM is too high (over 5 x lOOcpm/mgC) 3-5mgl- 1 of
nonlabeled glucose or peptone should be also be added to the medium
to enable the growth of bacteria with needed Cr. The culture thus grown
is washed from the remains of the label contained in the medium by
centrifugation-resuspension, thus making a working suspension of labeled
bacteria with a density of 0.2-0.5 mg Cml- 1 • The suspension is preserved at low
temperature.
To label natural bacterioplankton, ca. 0.3-0.5 mg C 1-1 of labeled protein
hydrolyzate (or hydrolyzate of algae) with inverse specific radioactivity of
not more than 0.1 1O-6 mg Ccpm- 1 is added to the natural surface water
prefiltered through a 1-~m pore size Nuc1eopore filter using the inverse filtration procedure described below (see Figs 6.1 and 6.2). The water is incubated
for 1-2 days. Then, to increase consumption by bacterioplankton of the
remains of the nonassimilated protein hydrolyzate, the nonlabeled yeast
hydrolyzate solution containing some 1-2mgC is added per 11 and the water
109
subsequent centrifugation. A working suspension of necessary density is then
prepared from the sediment. This suspension of plant material can also be used
for preparing the mature detritus containing specific microfJ.ora and protozoa.
For this, the suspension of freshly labeled plant material is first heated in a test
tube in boiling water, then cooled and contaminated with a small piece of silt.
After 2-3 weeks of incubatIon at 25-3U vC, the plant detritus will be ready for
use.
Bacteria labeled with 14C can be used in the feeding experiments either in
a state of suspension of a single species grown in culture or in the state of the
natural labeled bacterioplankton assemblages. For the first, suspension of
labeled bacteria can be obtained by growing them on organic substrates
labeled with 14C, such as labeled glucose or protein hydrolyzate; but these are
rather expensive. Instead, acid hydrolyzate of labeled microalgae like
ChIarella can be easily prepared in the laboratory as follows. The suspension
of labeled algae with a Cr value of about 0.1-0.2 x 10- 3 ~g C cpm- 1 is placed into
a beaker and dried. Then 60% H 2S04 is added and it is heated in a boiling
water bath for 4-5 h. Then the hydrolyzate thus received is dissolved, neutralized, with Na2C03 heated again to ~lOO°C, cooled, and filtered first through
a GF filter, and then through a dense (0.2-~m pore size) membrane filter. The
hydrolyzate of algae thus prepared is distributed into ampules and sterilized
by boiling three times for 20min at I-day intervals.
To obtain labeled bacteria using these dissolved organic substrates,
first the cultures of bacteria are isolated from the water body and checked
for their ability (1) to grow on a given labeled substrate, and (2) to grow
homogeneously in the liquid medium. The strain of bacteria thus selected
grows in the liquid medium or in a small amount (2-3 ml) of the same medium
poured over the surface of 4 % solid agar in a petri dish. Into this medium,
before its contamination with the microbial culture, the labeled dissolved organic matter (LOM) is added (ca. 2-5~Ci per 100ml). If the specific
radioactivity of the LOM is too high (over 5 x lOOcpm/mgC) 3-5mgl- 1 of
nonlabeled glucose or peptone should be also be added to the medium
to enable the growth of bacteria with needed Cr. The culture thus grown
is washed from the remains of the label contained in the medium by
centrifugation-resuspension, thus making a working suspension of labeled
bacteria with a density of 0.2-0.5 mg Cml- 1 • The suspension is preserved at low
temperature.
To label natural bacterioplankton, ca. 0.3-0.5 mg C 1-1 of labeled protein
hydrolyzate (or hydrolyzate of algae) with inverse specific radioactivity of
not more than 0.1 1O-6 mg Ccpm- 1 is added to the natural surface water
prefiltered through a 1-~m pore size Nuc1eopore filter using the inverse filtration procedure described below (see Figs 6.1 and 6.2). The water is incubated
for 1-2 days. Then, to increase consumption by bacterioplankton of the
remains of the nonassimilated protein hydrolyzate, the nonlabeled yeast
hydrolyzate solution containing some 1-2mgC is added per 11 and the water