Bacterial Growth and Productivity
267
c. A multiple filtration manifold unit (e.g., Millipore 122S) to permit simultaneous vacuum
filtration of up to 12 samples, 2S-mm diameter, if available; 12 stoppers (#6).
d. Polypropylene test tubes, SO m!.
e. Cellulose acetate membrane filters (e.g., Millipore), 2S-mm diameter, 0.22-llm pore size.
f. Cork borer, size # 12 (20.2-mm diameter).
g. Vortex mixer.
h. Trichloroacetic acid (TCA), (Caution: TCA is caustic, adheres to skin, and is very
corrosive):
i. Measure 30.0 g of TCA into 200-ml volumetric flask, bring up to mark with pure
water, and mix thoroughly. Carefully transfer to a 2S0-ml automatic transfer pipet.
Label IS% TCA.
11. Measure 10.0 g of TCA into a 200-ml volumetric flask, bring to mark with pure
water, and mix thoroughly. Carefully transfer to a 2S0-ml glass-stoppered Erlenmeyer
flask. Label S% TCA.
I. Algal marker, if needed:
i. An aliquot of dense algal culture is harvested by centrifugation and then extracted in
SO% ethanol and recentrifuged until the supernatant is clear.
11. The pellet of algal cell fragments then is suspended in ca. 2S ml of SO% ethanol and
retained for dilution and resuspension for marking filtration areas in samples from
oligotrophic waters (see procedures).
S. Acid extraction of thymidine uptake samples:
a. Water bath, 70°C, with rack for ca. 20 LS vials.
b. Ice bath with rack for LS vials.
c. O.S M HCI04; keep refrigerated, discard after one week.
d. Refrigerated centrifuge WC), chilled rotor; polypropylene centrifuge tubes (lS-ml) with
caps.
e. Transfer pipettes, automatic pipettes, vortex mixer, Pasteur pipettes, LS vials and caps,
and ice.
f. Oven incubator, 90°C.
g. Protein-DNA carrier solution:
I. Transfer SOmg of deoxyribonucleic acid (e.g., DNA, sodium salt, Sigma D-lS01 from
calf thymus) to a SO-ml flask.
11. Add SO mg of purified borine serum albumin.
111. Add SO ml of 0.0 1 M NaOH; cover and stir with a magnetic stir bar until dissolved.
IV. Store refrigerated in tightly capped vials. Stable for at least six months.
Bacterial Production of Protein Method
I. Sturdy box for leucine uptake assay bottles with holders for each bottle separately and with
a firm lid to secure bottles even if the box were dropped or inverted.
2. Ca. 20 60-ml ground-glass-stoppered reagent bottles with spring clips at the necks. Label
three bottles for each depth (two experimental measures and one control).
3. A calibrated line with clip rings at appropriate depths.
4. Leucine kit for field (identical to the thymidine kit discussed above; see p. 266).
S. 3H-Leucine stock preparation:
a. [3,4,S-3H]-I-leucine of highest purity and high specific activity at ca. 140 Ci/mmol. The
stock solutions should be prepared to yield a 10 nM solution in final concentration
when adding 200 III of leucine to a 63-ml sample in the field. Because ca. 4 ml of working
solution are needed to inoculate the 20 samples (200 III each), ca. 1 ml, depending on the
specific concentrations of the stock solution should be diluted to ca. 4.2 ml for the working
field solution.
b. Laboratory radioisotope-absorbent bench pads.
6. Laboratory assays:
a. Volumetric and Erlenmeyer flasks, graduated cylinders, syringes, and pipets as noted.
267
c. A multiple filtration manifold unit (e.g., Millipore 122S) to permit simultaneous vacuum
filtration of up to 12 samples, 2S-mm diameter, if available; 12 stoppers (#6).
d. Polypropylene test tubes, SO m!.
e. Cellulose acetate membrane filters (e.g., Millipore), 2S-mm diameter, 0.22-llm pore size.
f. Cork borer, size # 12 (20.2-mm diameter).
g. Vortex mixer.
h. Trichloroacetic acid (TCA), (Caution: TCA is caustic, adheres to skin, and is very
corrosive):
i. Measure 30.0 g of TCA into 200-ml volumetric flask, bring up to mark with pure
water, and mix thoroughly. Carefully transfer to a 2S0-ml automatic transfer pipet.
Label IS% TCA.
11. Measure 10.0 g of TCA into a 200-ml volumetric flask, bring to mark with pure
water, and mix thoroughly. Carefully transfer to a 2S0-ml glass-stoppered Erlenmeyer
flask. Label S% TCA.
I. Algal marker, if needed:
i. An aliquot of dense algal culture is harvested by centrifugation and then extracted in
SO% ethanol and recentrifuged until the supernatant is clear.
11. The pellet of algal cell fragments then is suspended in ca. 2S ml of SO% ethanol and
retained for dilution and resuspension for marking filtration areas in samples from
oligotrophic waters (see procedures).
S. Acid extraction of thymidine uptake samples:
a. Water bath, 70°C, with rack for ca. 20 LS vials.
b. Ice bath with rack for LS vials.
c. O.S M HCI04; keep refrigerated, discard after one week.
d. Refrigerated centrifuge WC), chilled rotor; polypropylene centrifuge tubes (lS-ml) with
caps.
e. Transfer pipettes, automatic pipettes, vortex mixer, Pasteur pipettes, LS vials and caps,
and ice.
f. Oven incubator, 90°C.
g. Protein-DNA carrier solution:
I. Transfer SOmg of deoxyribonucleic acid (e.g., DNA, sodium salt, Sigma D-lS01 from
calf thymus) to a SO-ml flask.
11. Add SO mg of purified borine serum albumin.
111. Add SO ml of 0.0 1 M NaOH; cover and stir with a magnetic stir bar until dissolved.
IV. Store refrigerated in tightly capped vials. Stable for at least six months.
Bacterial Production of Protein Method
I. Sturdy box for leucine uptake assay bottles with holders for each bottle separately and with
a firm lid to secure bottles even if the box were dropped or inverted.
2. Ca. 20 60-ml ground-glass-stoppered reagent bottles with spring clips at the necks. Label
three bottles for each depth (two experimental measures and one control).
3. A calibrated line with clip rings at appropriate depths.
4. Leucine kit for field (identical to the thymidine kit discussed above; see p. 266).
S. 3H-Leucine stock preparation:
a. [3,4,S-3H]-I-leucine of highest purity and high specific activity at ca. 140 Ci/mmol. The
stock solutions should be prepared to yield a 10 nM solution in final concentration
when adding 200 III of leucine to a 63-ml sample in the field. Because ca. 4 ml of working
solution are needed to inoculate the 20 samples (200 III each), ca. 1 ml, depending on the
specific concentrations of the stock solution should be diluted to ca. 4.2 ml for the working
field solution.
b. Laboratory radioisotope-absorbent bench pads.
6. Laboratory assays:
a. Volumetric and Erlenmeyer flasks, graduated cylinders, syringes, and pipets as noted.