638 APPENDIX: LABORATORY METHODS 



of 95 per cent alcohol were added with constant stirring. The polysac- 

 charide separated as a white curdy mass which settled rapidly. After 

 standing overnight the supernatant fluid was poured off and the pre- 

 cipitate centrifuged. Usually a three-layer separation was obtained, but 

 in cases in which only a single volume of alcohol was used, the phos- 

 phates and other salts were not thrown out and no syrupy layer was 

 found. The three-layer separation was also not obtained in the absence 

 of phosphates. If the broth used is phosphate-free, purification of the 

 polysaccharides may often be greatly facilitated by addition of sufficient 

 phosphate to bring about the three-layer separation. A solution of 10 

 grams of sodium acetate in 250 cc. of water was made acid to litmus 

 with acetic acid and the alcohol precipitate (or middle layer) was dis- 

 solved in this and reprecipitated with 300 cc. of 95 per cent alcohol. 

 The centrifuged precipitate was dissolved in 250 cc. of water contain- 

 ing 10 grams of sodium acetate and 5 cc. of glacial acetic acid, and the 

 turbid solution was shaken with 50 cc. of chloroform and 10 cc. of 

 n-butyl alcohol for 30 minutes. On centrifugation a semi-solid emulsion 

 separated between the aqueous layer and the chloroform. The solution 

 and the chloroform were poured off and the layer of emulsion was 

 washed with two 50 cc. portions of water which were saved for washing 

 later emulsion layers. 50 cc. of chloroform and 10 cc. of butyl alcohol 

 were added to the aqueous layer and the shaking was repeated. On cen- 

 trifugation a smaller semi-solid emulsion layer was formed and this 

 was treated as before. Shaking with chloroform was repeated as long as 

 an emulsion layer formed. Seven shakings were usually required. The 

 washings of the emulsion layers were combined and shaken with fresh 

 additions of chloroform as long as an emulsion layer formed, and were 

 then combined with the main aqueous solution, the total volume now be- 

 ing 350 cc. The polysaccharide was precipitated with 500 cc. of 95 per 

 cent alcohol, redissolved in 250 cc. of water, and the solution tested for 

 phosphate and glycogen. Phosphate may be removed either by repeated 

 precipitations with alcohol in the presence of sodium acetate and acetic 

 acid or with glacial acetic acid in the presence of sodium acetate. Glyco- 

 gen may be left behind by precipitating the SI from a salt-free aqueous 

 solution with copper acetate. In the instance quoted the solution was 

 free from phosphate but gave a strong iodine test for glycogen. 20 cc. 

 of a saturated solution of copper acetate slightly acidified with acetic 

 acid were added and the resulting bluish precipitate was centrifuged 

 off. The supernatant fluid remained clear when more copper acetate was 

 added. The precipitate was dissolved in 50 cc. of 20 per cent sodium 



