260
Exercise 19
up. Clean the cork borer and forceps (70~~ ethanol, or Isoclean, rinse, and dry)
between sample cuttings.
n. Freeze the vials at - 20°e.
o. Prepare five calibration vials as follows:
i. One (1.00) ml of unfiltered water from the well-mixed surface thymidine
sample bottle, plus 10 ml of scintillation cocktail for the liquid samples.
11. As above, but from a replicate sample bottle from the same depth.
111. As above, but from a replicate blank sample bottle from the same depth.
iv. One (1.00) ml offormalin-fixed lake water plus 10 ml of scintillation cocktail.
v. Internal standard vial with 1.0-ml formalin-fixed surface water plus 10 ml
of scintillation cocktail. 3H-hexadecane standard to be added later.
p. Waste disposal and decontamination:
1. Rinse the bottles with deionized water. Soak the bottles and stoppers for 2
days minimally in 2 to 4% alkaline detergent (e.g., isoclean) plus distilled
water.
11. Dispose of filtered waste in waste containers that can contain acids.
Acid Extraction of Thymidine Uptake Samples and Radioassay
1. Each sample is separated into two fractions: a perchloric acid soluble fraction for
DNA and RNA and the residue on the filter. Samples can be processed in groups
of ca. 18, which is a common capacity of a centrifuge rotor.
2. Perchloric acid soluble fraction:
a. Prepare and label a new liquid scintillation (LS) cap for each sample. Start
centrifuge refrigeration (4°C).
b. Add 50 III protein + DNA carrier solution (see "Apparatus and Supplies") to
each filter in LS vials.
c. Add 6.0ml of 0.5 M HCI0 4 .
d. Place into 70°C water bath and extract for 45 min; swirl to mix about every
15min.
e. Chill on ice.
r. Decent liquid to labeled centrifuge tube and cap. Centrifuge for 20 min at 4°C
and 10,000 rpm.
g. Carefully pipette 4.50 ml of supernatant to a LS vial with a labeled cap. Add
4.50 ml of appropriate scintillant (e.g., Packard Insta-Gel), cap, and mix
thoroughly. Keep undisturbed for 12 to 24 h before radioassay.
h. Include two background LS vials of 4.50ml 0.5 M HCI0 4 plus 4.50 ml scintillant.
i. Clean-up: Vortex the centrifuge tubes and decant suspension into a separate
perchloric waste container. Add a small amount of distilled water to each,
vortex, and decant. Wash the tubes thoroughly before reusing.
3. Residue on filter:
a. Make up two background samples with filters only.
b. Add 1.0ml of 1 N HCl and digest in an oven incubator at 90°C for 5 h. Retighten
the caps after 1 h to correct for expansion and loosening.
c. Add 2.0 ml of ethylacetate and wait several hours for dissolution of filters.
Dissolution is accelerated by affixing the sample tray to a rotary shaker (ca.
175 rpm).
d. Add 10.0 ml of colloidal scintillant (e.g., Safety-Solve) to each LS vial and shake
thoroughly. After several hours, shake well again.
Exercise 19
up. Clean the cork borer and forceps (70~~ ethanol, or Isoclean, rinse, and dry)
between sample cuttings.
n. Freeze the vials at - 20°e.
o. Prepare five calibration vials as follows:
i. One (1.00) ml of unfiltered water from the well-mixed surface thymidine
sample bottle, plus 10 ml of scintillation cocktail for the liquid samples.
11. As above, but from a replicate sample bottle from the same depth.
111. As above, but from a replicate blank sample bottle from the same depth.
iv. One (1.00) ml offormalin-fixed lake water plus 10 ml of scintillation cocktail.
v. Internal standard vial with 1.0-ml formalin-fixed surface water plus 10 ml
of scintillation cocktail. 3H-hexadecane standard to be added later.
p. Waste disposal and decontamination:
1. Rinse the bottles with deionized water. Soak the bottles and stoppers for 2
days minimally in 2 to 4% alkaline detergent (e.g., isoclean) plus distilled
water.
11. Dispose of filtered waste in waste containers that can contain acids.
Acid Extraction of Thymidine Uptake Samples and Radioassay
1. Each sample is separated into two fractions: a perchloric acid soluble fraction for
DNA and RNA and the residue on the filter. Samples can be processed in groups
of ca. 18, which is a common capacity of a centrifuge rotor.
2. Perchloric acid soluble fraction:
a. Prepare and label a new liquid scintillation (LS) cap for each sample. Start
centrifuge refrigeration (4°C).
b. Add 50 III protein + DNA carrier solution (see "Apparatus and Supplies") to
each filter in LS vials.
c. Add 6.0ml of 0.5 M HCI0 4 .
d. Place into 70°C water bath and extract for 45 min; swirl to mix about every
15min.
e. Chill on ice.
r. Decent liquid to labeled centrifuge tube and cap. Centrifuge for 20 min at 4°C
and 10,000 rpm.
g. Carefully pipette 4.50 ml of supernatant to a LS vial with a labeled cap. Add
4.50 ml of appropriate scintillant (e.g., Packard Insta-Gel), cap, and mix
thoroughly. Keep undisturbed for 12 to 24 h before radioassay.
h. Include two background LS vials of 4.50ml 0.5 M HCI0 4 plus 4.50 ml scintillant.
i. Clean-up: Vortex the centrifuge tubes and decant suspension into a separate
perchloric waste container. Add a small amount of distilled water to each,
vortex, and decant. Wash the tubes thoroughly before reusing.
3. Residue on filter:
a. Make up two background samples with filters only.
b. Add 1.0ml of 1 N HCl and digest in an oven incubator at 90°C for 5 h. Retighten
the caps after 1 h to correct for expansion and loosening.
c. Add 2.0 ml of ethylacetate and wait several hours for dissolution of filters.
Dissolution is accelerated by affixing the sample tray to a rotary shaker (ca.
175 rpm).
d. Add 10.0 ml of colloidal scintillant (e.g., Safety-Solve) to each LS vial and shake
thoroughly. After several hours, shake well again.