Table T16. Procedure for leucine 3H tracer experiments.

Table T16. Procedure for leucine ³H tracer experiments.


1) Using cut-off syringes, the sediment cores were subsampled cleanly while maintaining anoxic conditions in the cold room. The cleanly stoppered syringes were transported cold to the radioisotope van.
2) The ³H-leucine ([L-(3,4,5-³H[N])]; Perkin Elmer, NET-460A) was injected. Formalin was injected into one core prior to the radiotracer as a time-zero control.
3) After incubation, the sediment was extruded into a vial containing a 2% formalin solution and stored cold.
4) The fixed samples were transported to the Graduate School of Oceanography, University of Rhode Island, for processing for both protein production and microautoradiography.
5) Protein will be extracted using 5% trichloroacetic acid (TCA). After extraction, the samples will be rinsed with 5% TCA followed by 80% ethanol using centrifugation (Smith and Azam, 1992).
6) The samples will be radioassayed.
Microautoradiography:
7) Prokaryotes in the fixed samples will be separated from the bulk sediment by sonicating samples with sodium pyrophosphate and/or Tween-80.
8) The supernatant containing the cells will be filtered onto 0.2-µm polycarbonate filters and placed on a glass microscope slide covered with photographic emulsion (Kodak NTB-2).
9) The emulsion will be exposed to the radioactive samples for a period of weeks to months and processed.
10) The samples will be counterstained with acridine orange and observed with transmitted light and epifluorescence microscopy. The comparison of images for bacterial and silver grain distributions will show which cells have actively incorporated radiolabeled leucine.