266
Exercise 19
6. As the hypolimnion of a productive lake becomes anaerobic during seasonal stratification,
how might these conditions alter the metabolism and total bacterial productivity?
7. If inorganic turbidity in a lake or reservoir were to increase markedly, such as following a
rainstorm, how would this particulate loading influence bacterial productivity?
8. When a lake contains a major development of littoral plants and attached algae, how might
the productivity and development of planktonic bacteria be altered?
9. Fresh water draining from forested and extensive wetland areas often contains high
concentrations of dissolved organic matter. How might these dissolved organic compounds
influence the productivity of bacterioplankton? Of phytoplankton? Of zooplankton?
Apparatus and Supplies
1. Boats, anchors, a Van Dorn or similar nonmetallic water sampler, and suspension buoy for
the bottles.
2. Thymidine-DNA change method:
a. A sturdy box for thymidine uptake assay bottles that has holders for each bottle
separately and with a firm lid to secure the bottles even if the box were dropped or
inverted.
b. 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).
c. A calibrated line with clip rings at appropriate depths.
d. A thymidine kit for field, consisting of:
i. Formaldehyde auto pipet, adjusted to 1.6 ml.
11. Water withdrawal autopipet, adjusted to 2.0 ml.
111. 3H-thymidine ampoule (see procedure) in a capped vial with padding.
IV. Thymidine styringe, adjusted to 0.20 m!.
v. 50 ml of 37% formaldehyde.
vi. Six pairs of disposable vinyl gloves.
vi. Absorbent laboratory paper wipes.
V111. Sealable plastic bags for radioactive solid wastes.
3. 3H-Thymidine stock preparation:
a. [Methyl-3HJthymidine of highest purity and high specific actIvIty (e.g., Amersham
TRK.686) at ca. 80 Ci/mmo!. The stock solutions should be prepared (see stock
preparation example) to yield a 10 nM solution when adding 200 fll thymidine to a
63-ml sample in the field. Because ca. 4 ml of working solution are needed to inoculate
the 20 samples (200 fll each), ca. 1 ml, depending upon the specific concentrations, of the
stock solution should be diluted to ca. 4.2 ml for the working field solution.
b. Whatman filter paper, fine (e.g., No. 42 or 50).
c. Lyophilizer.
d. Polycarbonate centrifuge tube; glass jar.
e. Clean 2-ml and 5-ml ampoules, combusted at 500"C, capped with aluminum foil, and
autoclaved.
f. Laboratory radioisotope-absorbent bench pads.
g. On the day of the in situ assay:
I. Millex GV 0.22-flm filter unit (25-mm diameter) or equivalent.
11. A 5-ml disposable syringe and two l1-in., 22-gauge needles.
111. Lyophilized stock 3H-thymidine vial.
IV. A clean 5-ml ampoule, combusted at 500'C; glass annealing torch.
v. Disposable vinyl gloves.
vi. Disposable radioisotope-absorbent bench pads.
4. Laboratory assays:
a. Volumetric and Erlenmeyer flasks, graduated cylinders, syringes, and pipets as noted.
b. A lO-ml automatic transfer pipet with a 250-ml base flask, if available.
Exercise 19
6. As the hypolimnion of a productive lake becomes anaerobic during seasonal stratification,
how might these conditions alter the metabolism and total bacterial productivity?
7. If inorganic turbidity in a lake or reservoir were to increase markedly, such as following a
rainstorm, how would this particulate loading influence bacterial productivity?
8. When a lake contains a major development of littoral plants and attached algae, how might
the productivity and development of planktonic bacteria be altered?
9. Fresh water draining from forested and extensive wetland areas often contains high
concentrations of dissolved organic matter. How might these dissolved organic compounds
influence the productivity of bacterioplankton? Of phytoplankton? Of zooplankton?
Apparatus and Supplies
1. Boats, anchors, a Van Dorn or similar nonmetallic water sampler, and suspension buoy for
the bottles.
2. Thymidine-DNA change method:
a. A sturdy box for thymidine uptake assay bottles that has holders for each bottle
separately and with a firm lid to secure the bottles even if the box were dropped or
inverted.
b. 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).
c. A calibrated line with clip rings at appropriate depths.
d. A thymidine kit for field, consisting of:
i. Formaldehyde auto pipet, adjusted to 1.6 ml.
11. Water withdrawal autopipet, adjusted to 2.0 ml.
111. 3H-thymidine ampoule (see procedure) in a capped vial with padding.
IV. Thymidine styringe, adjusted to 0.20 m!.
v. 50 ml of 37% formaldehyde.
vi. Six pairs of disposable vinyl gloves.
vi. Absorbent laboratory paper wipes.
V111. Sealable plastic bags for radioactive solid wastes.
3. 3H-Thymidine stock preparation:
a. [Methyl-3HJthymidine of highest purity and high specific actIvIty (e.g., Amersham
TRK.686) at ca. 80 Ci/mmo!. The stock solutions should be prepared (see stock
preparation example) to yield a 10 nM solution when adding 200 fll thymidine to a
63-ml sample in the field. Because ca. 4 ml of working solution are needed to inoculate
the 20 samples (200 fll each), ca. 1 ml, depending upon the specific concentrations, of the
stock solution should be diluted to ca. 4.2 ml for the working field solution.
b. Whatman filter paper, fine (e.g., No. 42 or 50).
c. Lyophilizer.
d. Polycarbonate centrifuge tube; glass jar.
e. Clean 2-ml and 5-ml ampoules, combusted at 500"C, capped with aluminum foil, and
autoclaved.
f. Laboratory radioisotope-absorbent bench pads.
g. On the day of the in situ assay:
I. Millex GV 0.22-flm filter unit (25-mm diameter) or equivalent.
11. A 5-ml disposable syringe and two l1-in., 22-gauge needles.
111. Lyophilized stock 3H-thymidine vial.
IV. A clean 5-ml ampoule, combusted at 500'C; glass annealing torch.
v. Disposable vinyl gloves.
vi. Disposable radioisotope-absorbent bench pads.
4. Laboratory assays:
a. Volumetric and Erlenmeyer flasks, graduated cylinders, syringes, and pipets as noted.
b. A lO-ml automatic transfer pipet with a 250-ml base flask, if available.