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230Bacterial Fish Pathogens A simple chemical technique has been described which may readily delineate NocardiafromMycobacterium(Kanetsuna and Bartoni, 1972). Assuming that pure cultures are available, the bacteria are saponified in 2.5% (w/v) potassium hydroxide in a 1:1 (v/v) mixture of methanol and benzene at 37°C for 24 h. Crude mycolic acids frombona fidemycobacteria may be subsequently precipitated by addition of an equal volume of ethanol to an ethereal solution of the extracted lipids. Mycobacteria give rise to copious quantities of white precipitate of melting point between 45 and 70°C,whereas nocardias produce negligible amounts, which do not melt below 150°C (Kanetsuna and Bartoli, 1972). Aer. allosaccharophilaisolates may be identified by the examination of key phenotypic characters. In particular, the utilisation of L-arabinose and L-histidine as sole carbon sources, acid production from D-mannitol, D-melibiose, D-raffinose, L-rhamnose, salicin and sucrose, and the Voges Proskauer reaction were considered differential (Martinez-Murciaet al,1992). However, a word of caution is necessary, insofar as the organisms which clearly demonstrated genetic homogeneity were markedly heterogeneous phenotypically. This would complicate diagnoses. Aer. salmonicidamay be distinguished from other fish pathogens on the basis of a small number of phenotypic tests, notably the Gram-staining reaction (small Gram-negative rods), motihty (usually appears to be non-motile), growth at 37°C (usually a negative response), fermentative metaboHsm, catalase and oxidase production (both positive) and acid production from sucrose and xylose (both negative; recently, acid production from sucrose has been attributed to some isolates [Wiklundet ai,1992]). These tests will result in a provisional identification ofAer. salmonicida(McCarthy, 1976).In addition, it is recommended that pathogenic isolates should be examined for degradation of gelatin (positive), starch (positive) and urea (negative), arginine dihydrolase (positive), gluconate oxidation (negative) and ornithine decarboxylase production (negative). Unfortunately, this apparently simple state of affairs may be complicated by the increasing presence of "atypical" isolates, particularly in non-salmonid fish. In particular, these may be non- or slow-pigmenting.
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