BIOASSAY OF SEAWATER: I. A 14C UPTAKE METHOD FOR THE DETERMINATION OF CONCENTRATIONS OF VITAMIN B12 IN SEAWATER

Abstract

A method is described employing 14CO2 uptake by the marine centric diatom Cyclotella nana (clone 13-1) for the bioassay of vitamin B12 in seawater. Seawater samples were filter sterilized, supplemented with sterile medium constituents, and diluted with a similarly supplemented, charcoal-treated filtered seawater. Samples or diluted samples, internal standards, and external standards were inoculated with the diatom such that the initial concentration of cells was approximately 1 × 104 per milliliter. The bioassay flasks were incubated for 47 hours, 14C as Na214CO2 was added, and 14C assimilation was measured after a 2-hour exposure. The rate of uptake of 14C could be related to B12 concentrations when these were in the range of 0.05 to 3.0 μμg B12 per milliliter. A 49-hour incubation time was selected since cells required time to adapt to seawaters collected from different locations. The concentration of B12 in a seawater sample was calculated from the radiocarbon uptake, the percentage of the internal standard recovered, and the dilution factor. The method is more sensitive than that described by Gold from which it was derived, and takes into account the serious inhibitory effects found in many samples of natural seawater. The limit of sure detection is about 0.05 μμg B12 per ml and the standard deviation of the method was about 0.3 μμg per ml when measuring 1.4 μμg B12 per ml in inhibitory seawater.