Preparation and Properties of a Specific Polysaccharide from a Strain of Vibrio Cholerae. ∗

Abstract

After a number of trials the method described below gave satisfactory results in the preparation of a specific polysaccharide of the strain known as Inaba variant, which belongs to Group VI in the chemical classification of the vibrios (Linton, Shrivastava and Mitra 1 ). The method is based on that of Heidelberger, Kendall and Scherp. 2

To the 72-hour growth of Inaba variant in 21 liters of 1% peptone water (pH 8,0), phenol was added to make a final concentration of 0.5% and the mixture allowed to stand for an hour. The bacterial growth was then removed in a Sharpies supercentrifuge. The peptone water was neutralized with acetic acid and concentrated in vacuo to 2 liters. To the concentrate 60 gm. of sodium acetate and 12 cc. of glacial acetic acid were added and the mixture precipitated with 2 volumes of absolute alcohol, or if necessary for complete precipitation, 3 volumes. Larger amounts of alcohol were found to bring down peptone, inorganic salts, etc., in quantities which greatly interfered with the subsequent purification. The precipitate was taken up in 150 cc. of water, 3 or 4 cc. of acetic acid and 10 cc. of saturated sodium acetate solution, and centrifuged; if the material thrown down gave only a faintly Molisch positive reaction it was discarded. If it gave a strong reaction, it was again dissolved in water, treated as above, and the 2 centrifuged solutions were mixed.

The solution was precipitated ivith a minimuni amount of alcohol, one-ha1 f volume usually being found sufficient. This precipitate was taken off antl further addition oi alcohol to the supernatant usually yielded only a very small precipitate which could lx neglected. The above process was repeated and the final precipitate was taken up in a niininivm amount oi saturated sodium acetate (20 cc.) and aliout 10 cc. of acetic acid, centrifuged. and the supernatant precipitated with chilled acetic acid. Usually 1.5 to 20 volumes were required to give a flocculent precipitate.