Biochemistry & Microbiology Methods

Sampling:

Ocean time series samples are collected in standard 8L Niskin bottles. For samples in and below the oxycline, a nitrogen line is attached to the upper air vent to prevent air from entering the bottle during subsampling. Samples for live analysis are first transferred without headspace to a 1L glass sample bottle with Teflon standard taper stopper. In the ship's lab, subsamples are transferred to 25 or 40 ml incubation vials, also under nitrogen. All vials are filled from the bottom with overflow of about 3 vial volumes and then sealed with no headspace.

Low molecular weight fatty acids: Volatile fatty acids are measured using the technique developed by Yang (1991), Yang et al. (1993) and Wu and Scranton (1994). Detection limits are about 1 (M for acetate). However, in some cases, deep water values are lower than 1 micromolar for acetate in which case we have estimated blanks in individual files.  Samples are poisoned with 1 ml 10N KOH per liter.

Fatty acid uptake rate constants: Acetate uptake rate constants are determined using radiolabeled tracers as described by Wu and Scranton (1994). Incubations are done anoxically in the dark in screw-top septum vials. Uptake includes both conversion of isotope to CO2 (respiration) and to biomass which can be filtered onto a 0.2 m Nuclepore filter (incorporation).

CH4:

CH4 is assayed by gas chromatography using the vial equilibration technique of Johnson et al. (1990) and a Carle 211AC gas chromatograph. Samples are poisoned by addition of 10N KOH solution at a rate of 200 l per 50 ml vial.

H2S:

Samples for sulfide analysis are taken in well-flushed glass syringes without bubbles and are injected into vials containing Zn-acetate. Upon return to the laboratory, the ZnS is dissolved and is analyzed spectrophotometrically by the method of Cline (1969).

Microbial census:

Abundances of remineralizers (bacteria) and regenerators (protozoa) are determined using microscopic censuses. Preserved samples (2% formaldehyde) are stained with a fluorochrome (DAPI or acridine orange) and captured on the appropriate porosity Nuclepore membrane (0.2 or 0.8 m). Filter-retained cells are enumerated and sized by epifluorescence microscopy according to Taylor et al. (1986). Larger, less abundant protozoa are enumerated on settled samples using inverted microscopy. Abundance and distribution of methanogens are determined by an autofluorescence microscopic technique whereby the fluorescence of coenzymes F420 and F350 in cells produced by two sets of excitation and barrier filters is considered presumptive identification of methanogens (Doddema and Vogels 1978).

Bacterial production: Bacterial incorporation is measured using 3H-leucine incorporation as described by Kirchman (1993). Triplicate samples are incubated for 10-12 h in gas-tight screw-top vials to minimized alteration of the redox potential. Time course experiments have confirmed that uptake is linear for at least 15 hours. Due to the fact that some important anaerobic bacteria appear to not take up exogenous thymidine under anoxic conditions (McDonough et al. 1986; Gilmour et al. 1990), the more common method of Fuhrman and Azam (1982) is inappropriate for this system.

NODC Data Submission by M.I. Scranton and G.T. Taylor, SUNY, Stony Brook
The CARIACO program - CARIACO 1 (November 1995)

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