Washing and disinfecting fish gills: Preventing contamination by normal surface microflora of cell cultures inoculated with tissue for isolating intracellular pathogens

摘要

Contamination of cell cultures by the normal microflora of water, to which fish gills are exposed, is a major obstacle to in vitro culture of the pathogenic and presumptively antibiotic-sensitive rickettsia- and chlamydia-like organisms infecting gill epithelial cells. Therefore, a protocol was developed for removal or inactivation of these potential contaminating organisms. Gills of Atlantic salmon Salmo salar were vigorously mixed in several changes of 11 E PES-phosphate buffers and in solutions of glutaraldehyde and hydrogen peroxide and inoculated into cell cultures containing benzylpenicillin. The cultures were incubated for several weeks at 14degreesC. No contaminating organisms were observed by phase contrast microscopy in the cultures. Satisfactory results were also obtained by washing the gills 10 times in buffers only. No growth of rickettsia- and chlamydia-like organisms was detected in inoculated cell cultures, but infectious pancreatic necrosis virus was isolated, illustrating that the disinfection did not inactivate this particular pathogen that was present within the gill tissues.