To 1 l. of milk, from which the cream has been largely separated (Note 1), 0.05 M hydrochloric acid is slowly added, with stirring, through a capillary tube extending to the bottom of the beaker. The addition is continued until the solution attains a pH of 4.6 (Note 2). The end point is determined by withdrawing 5-cc. samples, diluting to 50 cc., adding methyl red, and matching against a buffered series (Note 3). Approximately 1 l. of acid is required; the separation of the casein is practically complete at this point. Three liters of water is then added, stirring is discontinued, and the flocculent precipitate of casein is allowed to settle in the refrigerator for twelve to twenty-four hours. The clear supernatant liquid which contains soluble proteins and salts is removed as completely as possible by siphoning; the precipitate is collected on a suction funnel and washed with cold distilled water until the washings are free of calcium (give no precipitate with ammonium oxalate).

The casein, which is contaminated with calcium phosphate and fats, is filtered to as small a volume as possible (about 500 cc.) and transferred to a 2-l. beaker. It is then treated with 0.1 M sodium hydroxide, the alkali being added slowly and with stirring through a capillary extending to the bottom of the beaker (Note 4). The addition of alkali is continued until the pH of the mixture reaches 6.3 (Note 5); 100–150 cc. of the alkali is required. The end point is determined by matching against a buffered series (Note 6), employing dibromo-o-cresolsulfonphthalein ("bromocresol purple"). At this pH the casein is completely in solution in the form of its sodium salt; fats, calcium phosphate, and any calcium caseinate remain undissolved. Care must be taken not to add more alkali than is necessary to bring the pH to the above point (Note 4). The milky solution is filtered through a thick layer (10–15 mm.) of filter paper pulp tightly packed upon a suction funnel. The filtrate may be slightly opalescent; if it is less clear it is again filtered through a fresh layer of pulp.

The filtrate is brought to a pH of 4.6 with 0.05 M hydrochloric acid just as in the original precipitation, the necessary amount of acid being determined by titration of an aliquot portion, diluted fivefold, with 0.01 M hydrochloric acid; 220–250 cc. of 0.05 M acid is required. As the reprecipitation progresses, the rate at which the acid is added is decreased in order to prevent precipitation at the tip of the capillary tube; vigorous mechanical stirring is, of course, essential. When the acidification is complete, 5 l. of cold distilled water is added and the flocculent precipitate allowed to settle in the refrigerator. After siphoning off the clear supernatant liquid, the casein is collected on a suction funnel, using hardened paper, washed with cold distilled water until free of chloride, sucked as dry as possible, and dried over calcium chloride in a vacuum desiccator. The yield is 23–29 g. of a colorless coherent product which may readily be pulverized in a mortar.