Bacterial Growth and Productivity
259
Supplies," p. 266), stopper, and place into sample bottle holders of a dedicated field
box.
3. After all depths have been sampled and the bottles filled:
a. Remove 2 ml of water from each bottle.
b. Preserve one bottle from each depth (killed blank) with 1.6-mI37% formaldehyde.
c. Wearing plastic gloves, and working with your parther to keep order, add
0.20-ml 3H-thymidine to each of the active experimental bottles and then to
the formaldehyde-fixed blank bottles to avoid contaminating the active bottles.
Record the beginning and ending times ofthe injections to the nearest minute.
d. Immediately incubate the samples at the depths from which they were collected
in situ. The incubation should be at least 30 min but should not exceed 45 min.
e. After the incubation period, retrieve and place the bottles in the box.
f. Wearing plastic gloves, inoculate each active bottle with 1.6-ml of 37% formaldehyde. Record the time to nearest minute.
Laboratory Treatment and Assay of Field Samples
1. Place the sample bottles on ice until the analyses are undertaken.
2. Soak the required number of filters in ultrapure water in a petri dish.
3. Wearing plastic gloves, use a 30-ml disposable plastic syringe with ca. 18 cm of
3-mm diameter Tygon tubing fitting to the syringe end to remove the samples.
a. Withdraw 5 ml of the sample and rinse the syringe; discard.
b. Withdraw another 5-ml sample and use it to remove the air bubbles from the
syringe and tubing. Discard.
c. Withdraw exactly 20 ml and dispense into a labeled test tube. Discard the
remainder in tubing.
d. Repeat steps a to c for all of the other samples.
e. Change contaminated gloves.
f. With an automatic transfer pipet, pipet 10ml of 15% TCA into each of the first
12 tubes. Mix thoroughly (Vortex). Allow 10 min of exposure to TCA.
g. Prepare a 12-port filtration apparatus.
h. After 10 min of exposure, vortex each sample and filter at a pressure differential
of 1 atm. Each sample will fill the well of the Millipore manifold filtration
apparatus twice. Some wells filter more rapidly than others; stopper wells that
have completed filtration while waiting for the others.
i. Allow the well to empty prior to rinsing. Rinse each tube with 5 ml of 5% TCA;
vortex and pour into well.
j. Add 5 ml of ice-cold 80% ethanol and filter in order to remove thymidine taken
into the cells but not incorporated into the DNA (Robarts and Wicks 1989).
k. Rinse each filter four additional times: (1) Add 1 ml of 5% TCA and filter; (2)
repeat the first step twice; and (3) add 1 ml of 50% ethanol/algal mixture for
marking the filtration area on the filters when the waters are oligotrophic and
require visualization (see "Apparatus and Supplies," p. 267).
1. Carefully slide the filter off the filtering unit with forceps and place it in a
labeled vial with the filtered surface up. Filter the remainder of the samples as
above.
m. Excise the filter margins with a cork borer of the same diameter as the filtered
area by placing the filters on cardboard. Remove the contaminated edge and
discard it in radioactive waste. Place the filter into a vial with the filtered side
259
Supplies," p. 266), stopper, and place into sample bottle holders of a dedicated field
box.
3. After all depths have been sampled and the bottles filled:
a. Remove 2 ml of water from each bottle.
b. Preserve one bottle from each depth (killed blank) with 1.6-mI37% formaldehyde.
c. Wearing plastic gloves, and working with your parther to keep order, add
0.20-ml 3H-thymidine to each of the active experimental bottles and then to
the formaldehyde-fixed blank bottles to avoid contaminating the active bottles.
Record the beginning and ending times ofthe injections to the nearest minute.
d. Immediately incubate the samples at the depths from which they were collected
in situ. The incubation should be at least 30 min but should not exceed 45 min.
e. After the incubation period, retrieve and place the bottles in the box.
f. Wearing plastic gloves, inoculate each active bottle with 1.6-ml of 37% formaldehyde. Record the time to nearest minute.
Laboratory Treatment and Assay of Field Samples
1. Place the sample bottles on ice until the analyses are undertaken.
2. Soak the required number of filters in ultrapure water in a petri dish.
3. Wearing plastic gloves, use a 30-ml disposable plastic syringe with ca. 18 cm of
3-mm diameter Tygon tubing fitting to the syringe end to remove the samples.
a. Withdraw 5 ml of the sample and rinse the syringe; discard.
b. Withdraw another 5-ml sample and use it to remove the air bubbles from the
syringe and tubing. Discard.
c. Withdraw exactly 20 ml and dispense into a labeled test tube. Discard the
remainder in tubing.
d. Repeat steps a to c for all of the other samples.
e. Change contaminated gloves.
f. With an automatic transfer pipet, pipet 10ml of 15% TCA into each of the first
12 tubes. Mix thoroughly (Vortex). Allow 10 min of exposure to TCA.
g. Prepare a 12-port filtration apparatus.
h. After 10 min of exposure, vortex each sample and filter at a pressure differential
of 1 atm. Each sample will fill the well of the Millipore manifold filtration
apparatus twice. Some wells filter more rapidly than others; stopper wells that
have completed filtration while waiting for the others.
i. Allow the well to empty prior to rinsing. Rinse each tube with 5 ml of 5% TCA;
vortex and pour into well.
j. Add 5 ml of ice-cold 80% ethanol and filter in order to remove thymidine taken
into the cells but not incorporated into the DNA (Robarts and Wicks 1989).
k. Rinse each filter four additional times: (1) Add 1 ml of 5% TCA and filter; (2)
repeat the first step twice; and (3) add 1 ml of 50% ethanol/algal mixture for
marking the filtration area on the filters when the waters are oligotrophic and
require visualization (see "Apparatus and Supplies," p. 267).
1. Carefully slide the filter off the filtering unit with forceps and place it in a
labeled vial with the filtered surface up. Filter the remainder of the samples as
above.
m. Excise the filter margins with a cork borer of the same diameter as the filtered
area by placing the filters on cardboard. Remove the contaminated edge and
discard it in radioactive waste. Place the filter into a vial with the filtered side