bacteria

Bacterial Production (Thymidine Uptake)

Abstract: 

An important part of the McMurdo Long Term Ecological Research (LTER) is monitoring of spatial and temporal patterns, and processes that control bacterial production in perennial ice-covered lakes. This data set quantifies thymidine uptake by bacteria, which can be used to estimate bacterial production.

Core Areas: 

Dataset ID: 

42

Associated Personnel: 

6
199
200
799

Short name: 

limno_bacteria

Methods: 

Lake water samples were collected at specific depths with a five-liter Niskin bottle during normal LTER limnological sampling. Sub-samples were decanted into three 1-L Nalgene bottles (2-light and 1-amber), two-500 mL amber Nalgene bottles, three-150 mL borosilicate glass bottles, two-20 mL scintillation vials, and one-30 mL serum vial. Five-20 mL scintillation vials (3-live treatments, 2-kill treatments) were prepared at each depth for TDR analysis. TDR samples (10 mL) were taken from the one-liter amber Nalgene bottle and placed in each scintillation vial. 0.5 mL filtered formalin (0.2 micro m) was pipetted into each killed treatment vial. 3H Thymidine was pipetted into each vial (final concentration of 20 nM thymidine), first the live treatments and then the kill treatments. Samples were incubated in the dark at 1-4 degC for 20 hours (thymidine incorporation has been shown to be linear for 20 hours under these conditions, Priscu unpublished). At the end of the incubation period 10 mL of ice cold 10 percent TCA was added to stop thymidine uptake and precipitate nucleotides. Vials were stored at 4 degC until filtered. Each sample was filtered through a 0.2 micro m Nucleopore polycarbonate membrane filter. Both the scintillation vial and filter tower were rinsed three times with 5 percent TCA. Each filter was placed into a new scintillation vial and 15 mL of Cytoscint cocktail was added. Samples were counted with a pre-calibrated (3H) liquid scintillation counter. Thymidine uptake rate (TDR nM d-1) was calculated using the following equation:

nM TDR/day = (DPML - DPMK) * a /(alpha * t)
 where DPML is the average dpm for the live treatment,
 DPMK is the average dpm for the kill treatment,
 a is the final concentration of thymidine in each vial (20nM),
 alpha is the total dpm added to each vial, and t is the incubation period.

The thymidine uptake rate was adjusted to the ambient lake temperature using the Arrhenius equation:

TDRadj = TDR * e^(Ea (( 1 / (CI + 273 K)) - ( 1 / (CA + 273K))) / R )
 

where Ea is the energy of activation (12,600 cal mol-1, Q10 = 2.2),
degCI is the incubation temperature (degC),
degCA is the ambient lake water temperature at specific depth,
and R is a gas constant (1.987 cal mol-1 K-1).

During the 0607 season, the Priscu group did a comparison of the "filtration method" used in previous seasons (see above and also Appendix 6.11 "Previously used Methods" of the Limnological Methods Manual; Takacs, C.T. and J.C. Priscu. 1998. Bacterioplankton dynamics in the McMurdo Dry Valley lakes: Production and biomass loss over four seasons. Microbial Ecology 36:239-250), with the "centrifugation method" used by the Palmer (PAL) LTER (Fuhrman, J. A. and F. Azam. 1982. Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: evaluation and field results. Marine Biology 66:109-120). Results showed no significant difference between the two methods (ELB 0-20 m: mean and standard deviation of nM TDR/d determined via filtration method, 0.0114 and 0.0097, respectively; mean and standard deviation of nM TDR/d determined via centrifugation method, 0.0129 and 0.0131, respectively; paired t-test T value, -1.063; P value, 0.328).

We therefore continued measurement of bacterial production in seasons following the 0607 season using the centrifugation method. We also complemented our thymidine data with 3H-leucine incorporation data, as is done in the PAL LTER, for selected seasons for comparative purposes. The new method via centrifugation is as follows: Lake water samples were collected at specific depths with a five-liter Niskin bottle during normal LTER limnological sampling. Sub-samples were decanted into two 1-L Nalgene bottles (amber). Five-2 mL microcentrifuge tubes (3-live treatments, 2-kill treatments) were prepared at each depth for TDR or Leu analysis. 3H Thymidine or Leucine was pipetted into each live treatment tube (final concentration of 20 nM). Samples (1.5 mL) were taken from a one-liter amber Nalgene bottle and placed in each tube (both live and kill treatments). Live tubes were capped, inverted several times, and incubated in the dark at 1-4 deg C for 20 hours (thymidine incorporation has been shown to be linear for 20 hours under these conditions, Priscu unpublished). 100 micro L of ice cold 100 percent TCA was added to each kill treatment tube. Tubes were capped, inverted several times, and placed on ice for 15 minutes. 3H Thymidine or Leucine was pipetted into each kill treatment tube (final concentration of 20 nM). Tubes were capped and incubated in the dark at 1-4 deg C for 20 hours. At the end of the incubation period, 100 micro L of ice cold 100 percent TCA was added to the live treatment tubes to stop thymidine or leucine uptake and precipitate nucleotides. Samples were inverted and placed on ice for 15-30 minutes. All tubes were stored at 4 deg C until processing. Each sample was centrifuged at 14,000 (~15,000 x g) for 15 minutes. Supernatant was poured out and tube was tapped to get the last drops out of the tube. 1 ml of cold 5 percent TCA was added to each tube. Tubes were re-spun for 5 minutes, supernatant poured out, and tube tapped to get the last drops out. 1 ml of cold 80 percent ethanol was added to each tube. Tubes were re-spun for 5 minutes, supernatant poured out, and tube tapped to get the last drops out. Tubes were dried overnight in fume hood with caps off. 1 ml of Cytoscint ES scintillation cocktail was added to each tube, tubes were vortexed, and samples were counted with a pre-calibrated (3H) liquid scintillation counter. Thymidine or Leucine uptake rate (TDR or Leu nM d-1) was calculated using the following equation:

nM TDR or Leu day -1 = ((DPML - DPMK) * a) / (alpha * t)

where DPML is the average dpm for the live treatment,
DPMK is the average dpm for the kill treatment,
a is the final concentration of thymidine in each vial (20nM),
alpha is the total dpm added to each vial, and t is the incubation period.

The thymidine or leucine uptake rate was adjusted to the ambient lake temperature using the Arrhenius equation:

TDR or LEU adj = TDR or Leu * e^(Ea (( 1 / (CI + 273 K)) - ( 1 / (CA + 273 K))) / R )

where Ea is the energy of activation (12,600 cal mol-1, Q10 = 2.2),
degCI is the incubation temperature (degC),
degCA is the ambient lake water temperature (degC) at specific depth,
and R is a gas constant (1.987 cal mol-1 K-1).

Data sources: 

LIMNO_BACT_PROD

Maintenance: 

Data from this table was submitted to INSTAAR by John Priscu's team at Montana State University. The raw data files listed  under 'file name' are the names of the original files submitted. The 1993/94 and 1994/95 datasets are Microsoft Excel version 6.0 files, and  the 1995/96, 1996/97 and 1997/98 datasets are ascii text files. Upon arrival at INSTAAR, the data manager fine-tuned the location codes and  limno runs to match those provided in the "locations, dates, codes for lake chemistry, biology samples" file. The file was imported into Microsoft Access on INSTAAR's Unix system, and can currently be found there. The file was then exported in ascii, comma delimited text and MS-DOS text (table layout) on the MCM LTER web site. Both of these files are linked to this web page above. Information for the metadata was  obtained from the Metatdr9697.rtf file. The file was called up using  Microsoft Word version 6.0. Text from this file was used to create this page in html format.
 
In 2006, this metadata was standardized by Chris Gardner and Inigo San Gil using the EML format.  
 
In 2014, this metadata was enhanced using the Drupal Ecological Information Management System (Inigo San Gil)
 
Per A. Chiuchiolo and J. Priscu suggestion, 4 columns were added to the data table to reflect new methodologies: Starting season 06/07, we included Leucine measurements in our bacterial production assay, and we also started a new method and did comparisons with the old method.

Additional information: 

The energy of activation was experimentally derived for Lake Bonney (Priscu, unpublished). If the average of the kill treatment uptake exceeds live treatment uptake, uptake is reported as zero.

Pages

Subscribe to RSS - bacteria