Membrane filtration – Fluorescent antibody staining procedure for detecting and quantifying Renibacterium salmoninarum in coelomic fluid of Chinook Salmon ( Oncorhynchus tshawytscha )

Abstract

We developed a rapid method for detecting and quantifying the pathogen Renibacterium salmoninarum in coelomic fluid of spring chinook salmon ( Oncorhynchus tshawytscha ) by concentrating the bacteria on 0.2-μm polycarbonate filters and staining them with specific fluorescein-labeled antibody. Centrifugation of samples and resuspension of the sedimented material in phosphate-buffered saline containing Triton X-100 increased the ease of filtration. Background fluorescence was reduced by counterstaining filters with Eriochrome black T. Postfiltration staining, rinsing, and counterstaining were done in the syringe-mounted filter holders, reducing handling of the filters and possible loss of bacteria. The number of bacteria detected by the filtration – fluorescent antibody technique in a broth culture of R . salmoninarum ranged from 6.7 × 10 7 to7.6 × 10 7 /mL and was slightly higher than that determined by plate count (9.6 × 10 6 /mL). Increasing the sample dilution or decreasing the number of microscope fields examined generally increased the variability of filter counts of R . salmoninarum . Using the filtration – fluorescent antibody technique, we detected the bacterium in the coelomic fluid of 85% of spawning female spring chinook salmon sampled from a hatchery population.