Fish viruses: buffers and methods for plaquing eight agents under normal atmosphere

Abstract

A universal procedure was sought for plaque assay of eight fish viruses (bluegill myxovirus, channel catfish virus, eel virus, Egtved virus, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, lymphocystis virus, and the agent of spring viremia of carp (Rhabdovirus carpio), in dish cultures of various fish cells. Eagle minimal essential medium with sodium bicarbonate-CO(2) buffer (Earle's salt solution) was compared with minimal essential medium buffered principally with tris (hydroxymethyl)aminomethane or N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid at a pH or in the range of 7.6 to 8.0 depending upon temperature. Five fish cell lines collectively capable of replicating all fish viruses thus far isolated were tested and quantitatively found to grow comparably well in the three media. Two-phase (gel-liquid) media incorporating the various buffer systems allowed plaquing at 15 to 33 C either in partial pressures of CO(2) or in normal atmosphere, but greater efficiency and sensitivity were obtained with the organic buffers, and, overall, the best results were obtained with tris(hydroxymethyl)aminomethane. Epizootiological data, specific fish cell line response, and plaque morphology permit presumptive identification of most of the agents. At proper pH, use of organic buffers obviates the need for CO(2) incubators.