1. An investigation was made of the effect on the casein number of (a) advanced lactation, (b) storage of the milk sample at room temperature and in the refrigerator, and (c) storage of the sample at room temperature after the addition of the following preservatives: potassium dichromate, mercuric chloride, formaldehyde, crystal violet and sodium azide.

2. The casein number method of mastitis diagnosis is not applicable to cows in advanced lactation. It did not distinguish between infected and uninfected quarters, as all quarters in the drying-off stage produced milk with a number below normal. At the end of lactation the mean casein, soluble protein plus non-protein, and total nitrogen contents were 21, 86, and 36% greater respectively than when the cows were in full milk.

3. There was a decrease in the casein number of the milk samples on keeping at 16–18° C. or with preservatives owing to the hydrolysis of casein to proteose-peptone and non-protein nitrogenous substances. For the diagnosis of mastitis from the casein number, samples should not be kept at room temperature for more than a few hours before analysis. Keeping at 0–2° C, and at room temperature with the addition of 0·2, 0·05, and 0·01 g. potassium dichromate, formalin or mercuric chloride respectively per 100 ml. of milk, prevented significant reduction of the casein number for 24–48 hr.