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Exercise 19
a macromolecule such as DNA. Thymidine generally is efficiently transported across
the cell membrane and converted to thymidine monophosphate by the enzyme
thymidine kinase (Moriarty, 1986). Cyanobacteria, eukaryotic algae, and fungi
generally lack thymidine kinase or the transport mechanism needed to assimilate
thymidine. This characteristic is important in that it restricts, in short incubation
times, uptake to direct DNA synthesis by the heterotrophic bacteria. Adenine and
adenine nucleotides, in contrast, are less frequently used as measures of bacterial
growth rates.
In practice, natural bacterial communities are incubated at in situ conditions with
a known quantity and specific activity of tritiated thymidine for a brief period
( < 45 min). The macromolecules then are separated chemically to evaluate synthesis
into different constituents. The measures of changes in DNA then are converted to
rates of bacterial productivity in carbon units.
Laboratory Preparation for Field Operations:
Stock 3H-Thymidine Solution
1. Disperse 1.05 ml of the purchased [methyPH]thymidine (usually stored in 2%
ethanol) solution into clean 2-ml ampoules. Seal the ampoules immediately and
autoclave for 15 min (SLOW EXHAUST). Label well and store and 4°C.
2. On the day prior to the in situ assays:
a. Freeze one of the stock ampoules period above.
b. Open the ampoule and carefully place the vial in a centrifuge tube; fasten the
filter paper over the tube mouth with a rubber band. Place into a beaker and
similarly cover with a filter paper. Place into a third container and similarly
cover with a filter paper.
c. Lyophilize according to freeze-dryer procedures to remove solvents (e.g.,
ethanol) and tritiated water. Thymidine should not be stored in the dried state for
any appreciable time because this condition can accelerate self-decomposition.
d. Remove the filter paper covers carefully with plastic gloves, as they may be
slightly contaminated with radioactivity.
e. Add 4.2 ml of ultrapure water and transfer to a clean 5-ml ampoule (working
stock solution).
f. Remove a 100-,u1 sample for calibration (see below).
g. Immediately seal the 5-ml ampoule of working stock solution and autoclave
15 min (SLOW EXHAUST). Store well labeled at 4°C.
3. Calibration procedure (needed for each series):
a. Remove a 100-,u1 sample from an ampoule containing the working stock solution
and add it to 5.00 ml ultrapure water. After thorough mixing, remove two 100-,u1
aliquots and add to two 900-,u1 aliquots of water in liquid scintillation (LSC)
vials. Prepare a third LSC vial with 1.00 ml of pure water for later internal
standardization.
b. Add 9.0ml of LSC cocktail for aqueous solution (e.g., Instagel or Safety-Solve)
to each vial. Radioassay.
Field Procedures
1. The assay is better accomplished with two persons, and both are needed when
inoculation and fixation are to be completed within 1 min.
2. Fill three 60-ml sample bottles with water from each depth (see "Apparatus and
Exercise 19
a macromolecule such as DNA. Thymidine generally is efficiently transported across
the cell membrane and converted to thymidine monophosphate by the enzyme
thymidine kinase (Moriarty, 1986). Cyanobacteria, eukaryotic algae, and fungi
generally lack thymidine kinase or the transport mechanism needed to assimilate
thymidine. This characteristic is important in that it restricts, in short incubation
times, uptake to direct DNA synthesis by the heterotrophic bacteria. Adenine and
adenine nucleotides, in contrast, are less frequently used as measures of bacterial
growth rates.
In practice, natural bacterial communities are incubated at in situ conditions with
a known quantity and specific activity of tritiated thymidine for a brief period
( < 45 min). The macromolecules then are separated chemically to evaluate synthesis
into different constituents. The measures of changes in DNA then are converted to
rates of bacterial productivity in carbon units.
Laboratory Preparation for Field Operations:
Stock 3H-Thymidine Solution
1. Disperse 1.05 ml of the purchased [methyPH]thymidine (usually stored in 2%
ethanol) solution into clean 2-ml ampoules. Seal the ampoules immediately and
autoclave for 15 min (SLOW EXHAUST). Label well and store and 4°C.
2. On the day prior to the in situ assays:
a. Freeze one of the stock ampoules period above.
b. Open the ampoule and carefully place the vial in a centrifuge tube; fasten the
filter paper over the tube mouth with a rubber band. Place into a beaker and
similarly cover with a filter paper. Place into a third container and similarly
cover with a filter paper.
c. Lyophilize according to freeze-dryer procedures to remove solvents (e.g.,
ethanol) and tritiated water. Thymidine should not be stored in the dried state for
any appreciable time because this condition can accelerate self-decomposition.
d. Remove the filter paper covers carefully with plastic gloves, as they may be
slightly contaminated with radioactivity.
e. Add 4.2 ml of ultrapure water and transfer to a clean 5-ml ampoule (working
stock solution).
f. Remove a 100-,u1 sample for calibration (see below).
g. Immediately seal the 5-ml ampoule of working stock solution and autoclave
15 min (SLOW EXHAUST). Store well labeled at 4°C.
3. Calibration procedure (needed for each series):
a. Remove a 100-,u1 sample from an ampoule containing the working stock solution
and add it to 5.00 ml ultrapure water. After thorough mixing, remove two 100-,u1
aliquots and add to two 900-,u1 aliquots of water in liquid scintillation (LSC)
vials. Prepare a third LSC vial with 1.00 ml of pure water for later internal
standardization.
b. Add 9.0ml of LSC cocktail for aqueous solution (e.g., Instagel or Safety-Solve)
to each vial. Radioassay.
Field Procedures
1. The assay is better accomplished with two persons, and both are needed when
inoculation and fixation are to be completed within 1 min.
2. Fill three 60-ml sample bottles with water from each depth (see "Apparatus and