A species-specific bacterial productivity method using immunomagnetic separation and radiotracer experiments

A species-specific bacterial productivity assay, combining radiotracer methods with immunomagnetic bead separation, was developed and tested in the marine environment. The capture method was optimized using cultures of the marine denitrifying strain, Pseudomonas stutzeri (ATCC 14 405). Immunocapture was optimal at a bead to target cell ratio of 10:1 using an indirect antibody technique,in which the target cell is first incubated with specific (primary) polyclonal antiserum and then with the secondary antibody-coated beads. Primary antibody concentration was less important than target cell concentration in determining the efficiency of target cell recovery. Reproducible recovery efficiencies of 75% could be obtained using cultures, but at natural seawater abundance levels, efficiency was much lower, around 20%. Estimates of total heterotrophic bacterial production and P. stutzeri production, based on radiotracer incorporation, were obtained for seawater samples from Monterey Bay, CA. To measure species-specific production, samples were incubated with radiotracers (methyl-[H-3]-thymidine and [C-14]-leucine), fixed, and concentrated. After separating P. stutzeri cells from the bacterial assemblage using immunomagnetic separation, target cell fraction radioactivity was measured. P. stutzeri abundance, estimated by immunofluorescence, represented less than 0.1% of the total bacterial abundance, whereas radiotracer incorporation by the target fraction represented 1-3% of the total assemblage tracer incorporation. (C) 1997 Elsevier Science Ireland Ltd.