The purpose of this study was to establish a set of descriptive data which could be used as a reference or a standard in the identification of Bacterium salmonicida, the cause of furunculosis in fish. Since a complete description of B. salmonicida was not available, bacteriologists were not in a position to identify this microorganism in a manner which would meet the rigorous requirements of present-day bacteriology.Ten strains of B. salmonicida were employed in these studies. A detailed description is presented which includes the morphological, cultural, and physiological characteristics of B. salmonicida. In addition, various factors such as oxygen, pH, temperature, and composition of media are discussed in terms of their influence on the formation of the melanin-like pigment which is characteristically produced by this microorganism.

An extensive literature exists concerning the role of tyrosine and tyrosinase in the formation of melano-pigments. This interest reflects the widespread distribution of the enzyme and the conspicuousness of such pigments in man, animals, and plants. Several comprehensive reviews exist on these pigments and the various factors which influence their formation (Raper, 1928; Lerner and Fitzpatrick, 1950; Dawson and Tarpley, 1951). Much of the emphasis has been directed to the metabolism of tyrosine, an amino acid which plays an important role in melanopigmentation. The formation of these darkly colored products from their precursors is a complex phenomenon, and detailed information concerning their identity and the sequence of chemical reactions which transform tyrosine to pigmented products is also incomplete. Bacterium salmonicida, a pathogen of salmonoid fishes, produces a dark amber to brown-black pigment especially when grown on a tryptic digest of casein-yeast extract medium (Gutsell and Snieszko, 1949). Griffin (1952) reported that only a light brown color was produced in nutrient agar and that the addition of L-phenylalanine or L-tyrosine resulted in the formation of a deep amber pigment. The present report deals largely with the effect of compounds structurally related to tyrosine and phenylalanine on pigment formation. These studies suggest that B. salmonicida may be a convenient tool for investigating the nature and formation of pigments derived from phenylalanine and tyrosine.

Outbreaks of ulcer disease among hatchery brook trout (Salvelinus fontinalis) have been described by Fish (1934) and Wolf (1938, 1940). Fish frequently isolated Psudomonas hydrophila from the infected trout. This common water bacterium was also isolated from ulcer disease in pike (Esox lucius) and brook trout by Reed and Toner (1942). Bacterium salmonicida, the cause of fish furunculosis, also has been isolated from trout with ulcer disease, as reported by Davis (1946). It seems likely that the upper picture in the color plate (Taf. [XXII XXXI) in Plehn's book (1924) depicts ulcer disease rather than furunculosis. In an outbreak of ulcer disease among brook trout at Kearneysville, West Virginia, Snieszko and Friddle (1950) regularly isolated a Hemophilus-like bacterium. Subsequently these authors isolated the same bacterium from outbreaks of ulcer disease in Maryland, Pennsylvania, Massachusetts, and Rhode Island. Some of the epizootics in trout were complicated by furunculosis, and in such cases B. salmonicida was also isolated. Trout artificially inoculated with this Hemophilus-like microorganism developed typical lesions and usually died. From such trout the same bacterium was then recovered. This rendered more probable an etiological relationship between this hitherto unknown bacterium and the ulcer disease of trout. Diagnosis of ulcer disease is often complicated by the occurrence in the trout of other easily isolated bacteria such as B. salmonicida, P. hydrophila, and water saprophytes. Therefore methods are here described for the isolation, cultivation, and identification of the Hemophilus-like bacterium believed to be the etiological agent of ulcer disease. Some studies on growth factor requirements are also described. METHODS OF ISOLATION AND CULTIVATION In the first isolations of the Hemophilus-like bacterium from brook trout with ulcer disease, the minute colonies, seen only under magnification, appeared only on media abundantly inoculated with blood or minced tissues of the examined fish. Therefore additional, and more successful, isolations were made on media containing trout or rabbit blood, or tissue extracts. The standard basal medium that was used in these experiments, unless otherwise stated, contained 5 grams of each of the following components per liter of distilled water: tryptic digest of casein, yeast extract, proteose peptone no. 3 (Difco), maltose, and sodium chloride. The medium was usually sterilized for 15 minutes at 115 to 118 C. Solid

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