Characteristic alterations of the budding process of Saccharomyces induced by ultraviolet treatment.

1951). These bodies usually occur with greatest frequency and staining intensity in smooth, virulent strains and diminish or disappear with colonial variation to rough, 0-inagglutinable forms. It was pointed out also that polar disposition of antigen would be more consistent with the observed pattern of end-to-end 0 agglutination (Pijper, J. Bact., 42, 345, 1941) than would an over-all surface distribution. Direct evidence for identity of the polar bodies with the 0 antigen haptene was sought by extracting the antigen complex by the diethyleneglycol procedure of Morgan (Biochem. J., 41, 2003, 1937). Even with repeated extraction, however, the yield of antigen from 12 hour acetone dried culture of Salmonella typhosa and Salmonella enteritidis was poor (ca 5 per cent of the dry weight). As evidence of incompleteness of antigen extraction, the extracted cells were agglutinated as readily as untreated cells by their homologous antisera; moreover, the polar bodies were diminished only slightly in number and staining intensity. However, the dialyzed, glycol-free cell extract precipitated strongly with specific antiserum (precipitation with as little as 1 jAg antigen per ml), and in confirmation of the observation of Goebel (J. Exptl. Med., 85, 499, 1947) it reacted with the periodate-Schiff spot test reagent (Hotchkiss, Arch. Biochem., 16, 131-141, 1948). Dry preparations of the antigen, obtained by fractional precipitation with acetone, also reacted strongly with the polysaccharide reagent and were biuret negative. A Ithrice washed specific precipitate of antigen and antibody also produced a red color following application of the periodate-Schiff reagent. Although it is possible that antibody could contribute to this reaction of the precipitate, the dialyzed serum proteins alone produced only a faint reaction. Treatment of the specific precipitate with trypsin, until biuret negative, did not alter its reaction to the periodate-Schiff reagent. Among those who have questioned the specificity of the periodate-Schiff reagent for polysaccharides, Wolman (Proc. Soc. Exptl. Biol. Med., 75, 583, 1950) pointed out that lipids containing unsaturated fatty acids are oxidized by periodate to Schiff-reacting aldehydes. This reaction is not blocked by prior application of acetylating reagents, whereas esterification of polysaccharide hydroxyls prevents their oxidation. The technique of McManus and Cason (J. Exptl. Med., 91, 651, 1950) for differentiating polysaccharide and unsaturated lipids has been applied to representative Enterobacteriaceae, with the result that prior acetylation prevented polar staining. Regeneration of the free hydroxyls by mild alkaline hydrolysis restored the staining properties to the cells. Moreover, no substance staining with Sudan Black B was found in the cells. Hence, it is likely that polar staining with the periodate-Schiff reagent is due to polysaccharide, rather than to lipids. Most of the evidence accumulated to date is in agreement with our previous suggestion that the stainable polar material represents, at least in part, the polysaccharide haptene of the 0 antigen complex.