Carbohydrate Fractions from Vibrio Cholerae,and Related Organisms. ∗

We have shown 1 that the carbohydrate substances extracted from Vibrio cholerae, and related organisms, are so closely allied that cross-reactions will occur between them and immune sera at high dilutions. This cross-relationship was independent of the agglutination reaction, to differentiate the presumably pathogenic from non-pathogenic vibrios, since carbohydrate from a water vibrio, for example, would precipitate with serum against a pathogenic form, or against a rough non-agglutinable form. The vibrios included pathogenic smooth agglutinating forms, rough forms, some of which agglutinated and some of which did not, from human sources and from water, and non-agglutinating smooth water vibrios. These vibrios were characterized by the possession of a carbohydrate which was very similar if not identical in all.

A closer study has now been made of the carbohydrates of these organisms. The method of carbohydrate extraction will be given in detail subsequently. 2 The 48-hour growth of 300 to 500 Roux flasks is washed off in normal saline, of which 10 cc. or less is used for each bottle. Glacial acetic acid is added to the washings to make a N/20 solution, and the organisms are then boiled under reflux until coagulation. Upon cooling, clumps fall to the bottom and leave a clear yellow supernatant fluid. Coagulation time varies with the type of organism. With rough organisms coagulation and clumping may take place between 10 minutes and one hour; smooth organisms, from 3 to 6 hours. The organisms are removed from the extract in a Sharpies super centrifuge, and the extract filtered to a water clearness in Seitz filters. After concentration to approximately 500 cc, the extract is precipitated with 3 volumes of absolute alcohol. The alcohol precipitation is repeated at least 6 times and the usual protein reactions become negative after the second or third precipitations.