Edwards, West and Bruner (Jour. Bact., 55, 1948, 711), Bruner, Edwards and Hopson (Jour. Inf. Dis., 85, 1949, 290) and West and Edwards (U. S. Pub. Health Service Mono- graph 22, 1954), 32 O groups and 87 H anti- gens have been established. When the sera used in the classification of Bethesda- Bellerup cultures were used in the examina- tion of normal strains of E. freundii, it was found that either or both the O and H anti- gens of the majority of E. freundii cultures could be recognized. Thus, in view of the similarity of their biochemical reactions and their close serological relationships, it seems better to regard the Bethesda-Bal- lerup group as a variety of the species E. freundii. Subsequent biochemical investiga- tions have confirmed this view. Comments: In 1932, Werkman and Gillen (Jour. Bact., 23, 1932, 177), following the custom prevalent at that time, established the citrate-positive coliform organisms in a separate genus, Citrobacter, and subdivided this genus into seven species on the basis of action on gelatin and on differences in their fermentation of sucrose, esculin, salicin, dulcitol and similar compounds. Five of the seven species described by Werkman and Gillen produced H2S in proteose peptone- ferric citrate agar and, following Vaughn and Levine (Jour. Bact., U, 1942, 502), are all regarded here as belonging to Esch- erichia freundii. Source: Isolated from canal water in Holland. Habitat: Normally found in soil and water and, to a varying degree, in the intes- tinal canals of man and other animals. Widely distributed in nature. 4. Escherichia intermedia (Werkman and Gillen, 1932) Vaughn and Levine, 1942. (Citrobacter intermedium Werkman and Gillen, Jour. Bact., 23, 1932, 178; Vaughn and Levine, Jour. Bact., U, 1942, 498.) in.ter.me'di.a. L. adj. iniermedius inter- mediate. Short rods, with rounded ends, occur- ring singly, in pairs and in short chains in young nutrient agar or broth cultures. Activel}^ motile by means of peritrichous fiagella or non-motile. Gram-negative. Gelatin stab: No liquefaction after 60 days at 20° C. Agar slant: Smooth to wrinkled surface; grayish white, abundant, raised, butyrous growth. Levine's eosine-methylene blue agar: Well isolated colonies vary from 1 to 4 mm in diameter. No confluence of neighboring colonies. Colonies are slightly to moderately raised with surfaces varying from flat to conve.x and usually smooth and glistening but sometimes dull, rough and granular. By transmitted light two types of colonies have been observed: (1) colonies having almost the same appearance throughout but with a distinctly lighter center, the color being similar to the medium; (2) colonies having a dark brownish central area which diffuses out to a lighter margin. By reflected light three types of colonies have been observed: (1) dark, button-like, concentrically ringed colonies possessing a strong, greenish metallic sheen so char- acteristic for Escherichia coli; (2) colonies with dark, purplish, wine-colored centers surrounded by a light pink zone; some colonies are concentrically ringed; (3) pink colonies with no suggestion of sheen but sometimes concentrically ringed. Nutrient broth: Turbid; slight ring at surface. Litmus milk : Acid ; sometimes coagulated and reduced; no proteolysis. Potato: Abundant, white to ivory- col- ored growth. Indole maj' or may not be produced. Hj'drogen sulfide not detected in proteose peptone ferric citrate agar. Acid or acid and gas produced from xj'lose, arabinose, rhamnose, glucose, fruc- tose, mannose, galactose, lactose, maltose, trehalose and mannitol. No acid or gas from melezitose, amygdalin or erj'thritol. Su- crose, raffinose, cellobiose, a-methyl-gluco- side, adonitol, dulcitol, glycerol, inositol, sorbitol, starch, aesculin, salicin and sodium malonate may or may not be fermented. Fermentation of glucose: The end prod- ucts characteristic for the genus Escherichia are produced. Carbon dioxide and hydrogen gases are produced in approximately equi- molar proportions (gas ratio, 1:1) besides
