Bacterial Growth and Productivity
263
to the method of 3H-thymidine incorporation into DNA; the two methods yielded
comparable rates of bacterial production (Simon and Azam, 1989). The bacterial
protein production method was, however, about ten times more sensitive than the
thymidine method and yielded bacterial carbon production directly without the need
to know the cell size of the bacterial community in the growth state.
Field Procedures
1. Fill three 60-ml sample bottles with water from each depth, stopper, and place
into sample bottle holders of a dedicated field box.
2. After all of the 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 ml of 37%
formaldehyde.
c. Wearing plastic gloves, and working with your partner to keep order, add
0.20 ml of [3,4,5- 3 H]-I-leucine to achieve ca. 10 nM final concentration 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 of the injections to the nearest minute.
d. Immediately incubate the samples at the depths from which they were collected
in situ. Incubate for at least 30 min but do not exceed 60min.
e. After the incubation period, retrieve the bottles and place them in the box.
f. Wearing gloves, inoculate each active bottle with 1.6 ml of 37% formaldehyde.
Record time to the nearest minute.
Laboratory Treatment and Assay of Field Samples
1. Soak the required number of filters in ultrapure water in a petri dish.
2. Wearing protective gloves, use a 30-ml disposable plastic syring with ca. 18 cm of
3-mm diameter Tygon tubing fitted to the syringe end to remove the samples.
a. Withdraw 5 ml of the sample and rinse the syringe; then discard it.
b. Withdraw another 5-ml sample and use it to remove air bubbles from the
syringe and tubing; then discard it.
c. Withdraw exactly 20 ml and dispense into a labeled test tube. Discard the
remainder from the tubing.
d. Repeat steps a to c for all of the samples.
e. Change the contaminated gloves and discard them in an appropriate manner.
f. With an automatic transfer pipet, pipet 6.5 ml of 15% TCA into each test tube
to expose the bacteria to a 5% TCA solution. Mix thoroughly (vortex).
g. Immediately lower the tubes in an appropriate rack into a water bath of 95
to 100°C; extract for 30 min.
h. Prepare a 12-port filtration apparatus.
I. After a 30-min exposure, vortex each sample and filter at a pressure differential
of 1 atm onto 0.45-llm pore size cellulose membrane filters (Millipore or
Sartorius). Each sample will fill the well of the Millipore manifold filtration
apparatus twice. Some wells filter more rapidly than others: stopper the wells
that have completed filtration while waiting for others.
J. Allow the wells to empty prior to rinsing. Rinse each tube with 5 ml of filtered
lake water, vortex, and pour into the appropriate well.
k. Carefully slide the filter off of the filtering unit with forceps and place it in a
263
to the method of 3H-thymidine incorporation into DNA; the two methods yielded
comparable rates of bacterial production (Simon and Azam, 1989). The bacterial
protein production method was, however, about ten times more sensitive than the
thymidine method and yielded bacterial carbon production directly without the need
to know the cell size of the bacterial community in the growth state.
Field Procedures
1. Fill three 60-ml sample bottles with water from each depth, stopper, and place
into sample bottle holders of a dedicated field box.
2. After all of the 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 ml of 37%
formaldehyde.
c. Wearing plastic gloves, and working with your partner to keep order, add
0.20 ml of [3,4,5- 3 H]-I-leucine to achieve ca. 10 nM final concentration 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 of the injections to the nearest minute.
d. Immediately incubate the samples at the depths from which they were collected
in situ. Incubate for at least 30 min but do not exceed 60min.
e. After the incubation period, retrieve the bottles and place them in the box.
f. Wearing gloves, inoculate each active bottle with 1.6 ml of 37% formaldehyde.
Record time to the nearest minute.
Laboratory Treatment and Assay of Field Samples
1. Soak the required number of filters in ultrapure water in a petri dish.
2. Wearing protective gloves, use a 30-ml disposable plastic syring with ca. 18 cm of
3-mm diameter Tygon tubing fitted to the syringe end to remove the samples.
a. Withdraw 5 ml of the sample and rinse the syringe; then discard it.
b. Withdraw another 5-ml sample and use it to remove air bubbles from the
syringe and tubing; then discard it.
c. Withdraw exactly 20 ml and dispense into a labeled test tube. Discard the
remainder from the tubing.
d. Repeat steps a to c for all of the samples.
e. Change the contaminated gloves and discard them in an appropriate manner.
f. With an automatic transfer pipet, pipet 6.5 ml of 15% TCA into each test tube
to expose the bacteria to a 5% TCA solution. Mix thoroughly (vortex).
g. Immediately lower the tubes in an appropriate rack into a water bath of 95
to 100°C; extract for 30 min.
h. Prepare a 12-port filtration apparatus.
I. After a 30-min exposure, vortex each sample and filter at a pressure differential
of 1 atm onto 0.45-llm pore size cellulose membrane filters (Millipore or
Sartorius). Each sample will fill the well of the Millipore manifold filtration
apparatus twice. Some wells filter more rapidly than others: stopper the wells
that have completed filtration while waiting for others.
J. Allow the wells to empty prior to rinsing. Rinse each tube with 5 ml of filtered
lake water, vortex, and pour into the appropriate well.
k. Carefully slide the filter off of the filtering unit with forceps and place it in a