Radiolabeled tracer method

• Page authors: Anabel von Jackowski, Hagen Buck-Wiese
• Responsible curator: Hagen Buck-Wiese

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Bacterial production
Approach: radiolabeled tracer incorporation
Context: incubation, lab
Spatial scale: point sample
Temporal scale: hours
Units: mol incorporated tracer L-1 h-1
Community captured: bulk, size-fractionated
Co-measurements: cell abundance

Method Overview

Bacterial secondary production is estimated by measuring the incorporation of radiolabeled precursors — most commonly ³H-leucine (into protein) or ³H-thymidine (into DNA) — into microbial biomass during short, dark incubations. Aliquots of seawater are amended with tracer concentrations of the radiolabeled substrate, incubated for a defined period, and then filtered or precipitated to collect macromolecular material. Radioactivity retained on the filter is measured by scintillation counting and converted to a production rate.

The leucine incorporation method is the most widely applied variant. Leucine is assumed to be incorporated exclusively into protein, and a theoretical or empirically determined conversion factor is used to translate leucine incorporation into units of carbon production. Size-fractionated filtration can separate bacterial from eukaryotic production.

Classic examples

• Kirchman (2001) Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments
• Kirchman et al. (2009) Standing stocks, production, and respiration of phytoplankton and heterotrophic bacteria in the western Arctic Ocean

Scale of measurement

As a bottle-based incubation, the method yields a point measurement in space. Incubation durations are typically a few hours, aimed at keeping the measurement close to in situ rates while minimising bottle effects and isotope dilution.

Data generated

The method yields bacterial carbon production rates, i.e., the rate at which heterotrophic bacteria synthesise new biomass. When combined with standing stock estimates (bacterial biomass), specific growth rates (d^-1) can be derived.

Units & currency

Units are mol incorporated tracer L^-1 h^-1.

Sample size

Replicate small-volume subsamples (1–5 mL) are typically processed per station.

Limitations

Leucine is assumed to be incorporated exclusively into protein and not re-mineralised during the incubation. Intracellular isotope dilution from unlabeled leucine pools can cause underestimation, and empirical conversion factors between leucine incorporation and carbon production are variable across environments and must be determined locally for highest accuracy. Bottle incubation can alter community composition and substrate availability relative to in situ conditions.

Step-by-Step Protocol

1. Dilute Leucine Stock: Dilute the stock solution (50 µl will be pipetted per sample to reach a final concentration of 20 nM). Store at 4 °C for short-term use, or at -20 °C for long-term storage.
   • Example: 90 µl of 1.0 mCi/ml Leucine stock + 1.410 ml sterile Milli-Q water.
   • Note: A 20 nM Leucine concentration in the sample is appropriate for a bacterial cell density of approx. 1 × 10⁶ cells/ml.
   • High Density Note: For samples with cell densities of 1 × 10⁷ to 1 × 10⁸ cells/ml, mix with unlabeled ("cold") Leucine to reach a final concentration of approx. 200 nM.
2. Label Tubes: Label 2 ml microcentrifuge tubes with 3 replicates per sample and mark a small dot on the outside of each tube. This will help orient the pellet during supernatant aspiration.
3. Set Incubator: Set the incubation chamber to the desired target temperature and place the incubation racks inside.
4. Pre-chill Equipment and Reagents:
   • Pre-chill the centrifuge to 4 °C.
   • Store one tube rack at 4 °C.
   • Keep both 5% and 100% Trichloroacetic Acid (TCA) stored at 4 °C.
   • Batch Limit Note: Process a maximum of 20 samples per centrifugation run. Processing more samples prolongs supernatant aspiration, risking pellet detachment.
5. Aliquot Water Samples: Pour 50 ml to 200 ml of your water sample into a processing container and record the exact timestamp.
6. Add Leucine Substrate: Pre-load 50 µl of the working Leucine solution into the 2 ml tubes (final concentration = 20 nM, approx. 3.5 µCi). Keep them at 4 °C.
7. Add Sample: Add 1.5 ml of your water sample (pipette 2 × 750 µl) to each tube. Record the exact timestamp.
8. Prepare Killed Controls (Blanks): Immediately add 80 µl of 100% TCA directly to the designated blank control tubes to arrest biological activity.
9. Incubation: Place the sample tubes into the temperature-controlled racks inside the incubator. Wrap the racks in aluminum foil and incubate for 15 minutes to 6 hours, depending on the oceanic region.
10. Pre-cooling Transfer: Just before the incubation period ends, transfer the tubes into the pre-chilled rack (4 °C).
11. Terminate Reaction: Add 80 µl of 100% TCA to all active sample tubes to terminate incubation (final concentration approx. 5% TCA). Do not add TCA to the already killed controls! Record the exact termination timestamp.
12. First Centrifugation: Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes. Ensure the marker dots face outward.
13. Aspirate Supernatant: Carefully remove the supernatant using a pipette (fitted with 1250 µl tips) or an aspiration setup (connected via tip, tubing, vacuum flask, and pump).
14. First Wash Step: Add 1.5 ml of ice-cold 5% TCA to each tube and shake the tubes thoroughly to resuspend/wash the precipitate.
15. Second Centrifugation: Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes (ensure the marker dots face outward).
16. Aspirate Supernatant: Carefully remove and discard the supernatant using a pipette or vacuum aspiration device as described in previous steps.
17. Second Wash Step: Add another 1.5 ml of ice-cold 5% TCA to each tube and shake thoroughly.
18. Third Centrifugation: Centrifuge the tubes at maximum rpm at 4 °C for 10 to 12 minutes.
19. Final Aspiration: Carefully remove and discard the final supernatant using a pipette or vacuum aspiration setup.
20. Drying the Pellet: Allow the protein pellet to air dry. For TCA treatments, a drying window of approximately 10 minutes is sufficient; the pellet does not need to be completely dehydrated.
21. Add Scintillation Cocktail: Add 1.5 ml of scintillation cocktail to each tube. Vortex the tubes thoroughly immediately prior to measurement to ensure complete mixing.
22. Load and Measure: Insert the microcentrifuge tubes into the 1.5 ml microtube adapters and place them inside the liquid scintillation counter cassettes.
    • Note on Timing: For high-accuracy results, samples should be measured on the following day. Allowing them to stand for 2 to 3 days is even better to achieve stable counting conditions.
    • Expected Yield: The resulting dpm (disintegrations per minute) values should ideally fall within the range of 1,000 to 999,000 dpm.

Common calculations/conversions

• Leucine-to-carbon conversion: theoretical factor is 3.1 kg C mol^-1 leucine (Simon & Azam 1989); empirical factors should be determined when possible.
• Thymidine-to-cell conversion requires an estimate of cells produced per mole of thymidine incorporated (typically ~2 × 10¹⁸ cells mol^-1).