In a previous report1 the fact that substances are excreted in the urine which stimulate and inhibit the division rate of the protozoan, Colpidium campylum, was recorded. It was shown, also, that in urine obtained from persons not having within the body a focus of proliferating cells the inhibiting factor dominates and that in persons having within the body a focus of proliferating cells the stimulating substances dominate. The present paper records observations made on various types of tissue, normal and neoplastic, from which both the cell-division stimulating and cell-division inhibiting fractions were isolated.

In the paper referred to previously an extremely crude method of separation for inhibitor and stimulator in urine was described. For the present work an improved technic was developed, which is described below.

Isolation of Inhibiting Fraction: Fifty grams of a selected tissue are reduced to a fine pulp. This pulp is extracted three times, 100 c.c. of acetone being used for each extraction, and each extraction continuing for one hour. After each extraction the suspension is filtered and the tissue residue is set aside for the isolation of the stimulating fraction. The three acetone extractions are then combined and evaporated to dryness on the steam bath, the resultant residue being re-extracted with 25 c.c. of acetone. After filtration, the acetone is placed in the icebox over night and the residue is discarded. After twelve hours in the icebox a precipitate forms which is filtered off and discarded. The clear acetone solution is again evaporated to dryness and taken up in 20 c.c. of 70 per cent alcohol and filtered. The alcoholic solution is placed in the icebox over night and a precipitate which settles out is filtered off and discarded. The alcoholic solution is evaporated to dryness and the residue is taken up in distilled water in the proportion of 5 c.c. for every 50 grams of tissue originally used. The fatty emulsion which forms is sterilized by steam under pressure.

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