ブリ貪食細胞のPasteurella piscicidaに対する細胞内殺菌能測定法の検討

概要

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Applicability of some methods for determination of intracellular bacterial killing by yellowtail phagocytic cells against Pasteurella piscicida was investigated. The following method was found applicable. A mixture of phagocytic cells prepared from yellowtail kidney, viable Pasteurella piscicida suspension and normal yellowtail serum was prepared, and incubated at 25°C for 30min. Then, 100μg/ml streptomycin and 1, 500units/ml penicillin G were added to the mixture, and incubated again at 25°C for 15min. The phagocytic cells and the remaining extracellular bacteria were separated by centrifuging at 5, 900×g for 5min on Percoll at 1.070 specific gravity. Phagocytic cells were washed three times by modified L-15, and resuspended in modified L-15 at a density of 1×107 cells/ml. After incubation at 25°C for 60min, the phagocytic cells were destroyed by 15% sodium chloride solution containing 0.2% non-ionic detergent, BL-25, at 0°C for 15min. The number of viable bacteria in destroyed phagocytic cells was counted by the pour plate method using BHI agar containing 1.0% sodium chloride.