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The microbiologic flora of the intestine of the breast-fed infant is characterized by the predominance of Lactobacillus bifidus. Investigations of the factors responsible for the establishment of this organism in the intestine of the nursling have been hampered by the lack of suitable isolation and cultivation procedures. Recently Norris et al. (1950) described a semisynthetic medium, supplemented with most of the factors known to stimulate the growth of lactobacilli, that will support the growth of L. bifidus indefinitely in the bifid phase. In the present study, the growth stimulatory activity of each of the constituents of this medium was investigated in order to determine the minimal requirements of these organisms. A knowledge of these requirements may offer an explanation of the unique predominance of L. bifidus in the nursling's intestine. METHODS AND MATERIALS The medium, developed by Norris et at. (1950) for the cultivation of Lactobacillus bifidus, is a modification of the medium described by Teply and Elvehjem (1945). The composition of the modified medium is presented in table 1. The growth promoting activity of the constituents of the medium was determined by titrimetric assay. The preparation of the inoculum and details of the assay were similar to those reported in an earlier publication (Tomarelli et al., 1949). Unless stated otherwise all supplements were autoclaved with the medium. The strains of L. bifi4us' used in the present investigation were the four strains designated as Birch, Timberlain, Lockhart, and Perrish by Norris et al. (1950). These strains were anaerobic, possessed the characteristic bifid morphology and, as established by the present study, were identical in so far as nutritional requirements were concerned. Tests for catalase and nitrate reduction were made by standard procedures (Wilson and Miles, 1946). The determination of volatile acids was accomplished by the Duclaux method (Kamm, 1932) and by the partition method (Delwiche, 1949). RESULTS Vitamin requirements. The vitamin requirements of the strains of L. bifidus were determined by growth experiments in which single vitamins were omitted from the otherwise complete medium of table 1. It was demonstrated that only biotin and calcim pantothenate were essential for growth of these strains We are indebted to Dr. Paul Gy6rgy for cultures of the strain.

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