used by Gautier and Mourgues; and the extract was worked up by ordinary methods for the separation of organic bases. In the second case the oil was extracted with dilute sulfuric acid sol., and the phosphotungstate precipitate obtained was worked up by the acetone method-which has been used successfully in our work on yeast (5). Substances were separated in each fraction, but the quantity of each product was so small that the work will have to be repeated with more material.

EXPERIMENTAL.

I. The cod-liver oil was entirely soluble in ether, acetone, ligroin and chloroform, and partially so in benzene. With alcohol alone, or with alcoholic mercuric chlorid sol., a slight precipitate was noticed. About 23.5 k. of the oil were used. Each k. was extracted with a sol. of 660 cc. of abs. alcohol and 50 cc. of conc. hydrochloric acid made up to 2 1. with water. Each k. was extracted three times, a third part of the sol. having been used in each extraction. The extracts were isolated in a separatory funnel, and evaporated in vacuo. The residue, which had an agreeable smell, was dissolved in alcohol, filtered from a precipitate which consisted mainly of inorganic salts, and the sol. evaporated again. Oil separated, which was twice washed with water and then hydrolyzed with 5 percent sulfuric acid sol. The watery extract was precipitated with 5 percent sulfuric acid sol. containing 50 percent of phosphotungstic acid. The precipitate was filtered, washed with dilute sulfuric acid sol., and dried. It weighed 969 gm. when nearly dry.

Treatment of the phosphotungstic acid precipitate. The precipitate was decomposed with 2 k. of baryta in a mortar. The phosphotungstate of barium was suspended in water and shaken. The combined filtrates were freed from baryta with sulfuric acid and evaporated in vacuo. The residue did not give a precipitate with alcoholic mercuric chlorid sol. No precipitate was obtained with silver nitrate and baryta. The sol. was freed from silver and baryta, and was reprecipitated with phosphotungstic acid. The resulting precipitate amounted to only 304 gm., consequently considerable decomposition of the nitrogenous substances must have taken place. This view is strengthened by the fact that the precipitate, decomposed with neutral lead acetate, gave a very heavy precipitate with mercuric chlorid. Before using this reagent, others were tried, but without success. The decomposed phosphotungstate precipitate had a very pronounced smell of nitrogenous bases. The final sol. of free bases was precipitated with mercuric chlorid in alcoholic sol. Both the precipitate and the filtrate gave crystalline chlorids after elimination of mercury and evaporation.

Treatment of the phosphotungstic acid filtrate. The filtrate was freed from phosphotungstic and sulfuric acids by means of neutral lead acetate. The filtrate, freed from lead with hydrogen sulfid, was conc. in vacuo. It was thought likely that it contained a large amount of amino-acids derived from liver tissue. The whole sol. contained 4.5 gm. of nitrogen and only 0.24 gm. of amino-nitrogen, as determined by Van Slyke's method. The residue, after evaporation, was entirely soluble in alcohol with the exception of a small quantity of inorganic material. The liquid was freed from chlorid with silver acetate, and the silver removed with hydrogen sulfid. The residue, when dissolved in alcohol and slowly evaporated, gave a crystalline substance. This will be investigated in the near future. Treatment of the fatty residue obtained from the evaporated alcoholic-aqueous extracts. This oil was hydrolyzed with 5 percent sulfuric acid sol. for 2 hr. The filtered liquid, which smelt like herring, was precipitated with phosphotungstic acid; 19.5 gm. of dry precipitate were obtained.

Treatment of oil extracted with dilute alcohol. The extracted oil was hydrolyzed for 2 hr. with 5 percent sulfuric acid sol. The acid extracts were precipitated with phosphotungstic acid; 186 gm. of precipitate were obtained.

2. About 25 k. of the same supply of dark oil were extracted in portions of 2 k. each with 2 1. of 10 percent sulfuric acid sol. for 2 hr. on a shaking machine, and then left over night. The extracts were isolated in a separatory funnel and precipitated with phosphotungstic acid. The resulting precipitate (dry), which weighed 877 gm., was extracted with acetone and 57.2 gm. of insoluble fraction obtained. A second extraction of the oil, as above, yielded 240 gm. of phosphotungstate but only 4 gm. of the acetone-insoluble fraction. Treatment of the acetone-insoluble fraction. The 61.2 gm. of material insoluble in acetone, obtained by the above mentioned treat

ment, were treated in a mortar with 150 gm. of neutral lead acetate and shaken on a machine for I hr. Alcohol was added to render the precipitate more insoluble; the liquid was filtered. The filtrate was freed from excess of lead and evaporated. The resulting white residue was dissolved in water, and alcohol added. Gelatinous material separated out, which was filtered off; 1.3 gm. was obtained. The aqueous filtrate from this substance was evaporated in vacuo. the residue dissolved in alcohol and precipitated with alcoholic mercuric chlorid sol. The resulting precipitate was decomposed with hydrogen sulfid and the filtrate evaporated to dryness. The residue was redissolved in water, and the liquid freed from chlorid by means of silver acetate. The filtrate, freed from silver, gave 3 gm. of substance, which is now being carefully investigated.

The mercuric chlorid filtrate was freed from mercury with hydrogen sulfid, and evaporated; the residue was dissolved in water and freed from chlorid with silver acetate. The filtrate from the silver sulfid gave, after evaporation, o. I gm. of substance.

Treatment of the acetone-soluble fraction. The acetone sol., obtained by the above mentioned treatment, was diluted with water, decomposed with 2 k. of neutral lead acetate, shaken for 1 hr., and filtered. The precipitate was again suspended in 30 percent acetone and filtered. The combined filtrates were freed from excess of lead and evaporated in vacuo. The residue, which did not crystallize, was dissolved in water and precipitated with alcoholic mercuric chlorid sol.: 99 gm. of precipitate (dry) were obtained. Both the precipitate and the filtrate were freed from mercury and evaporated; both yielded small amounts of crystalline hydrochlorids. The filtrates from the hydrochlorids were fractioned in the usual manner with silver nitrate, and with silver nitrate and baryta. These fractions, which gave only exceedingly small amounts of different substances, will be investigated later when larger quantities of material are available.

SUMMARY. With the idea that the therapeutic action of codliver oil is not due to peculiar fatty constituents in the oil, but to the presence of nitrogenous substances, a separation of the latter from the oil was effected. The raw material used was crude cod-liver oil, since this is richer in organic bases than the purified variety.

Bibliography

1. GAUTIER and MOURGUES: Compt. rend. Acad. des Sciences, 1888, cvii, pp. 110 and 626.

2. ISCOVESCO: Compt. rend. Soc. de Biol., 1913, lxxvi, pp. 34, 74 and 117; 1914, 10th and 17th January.

3. FUNK and MACALLUM: Zeitschr. f. physiol. Chem., 1913, xcii, p. 13.

4. OSBORNE and MENDEL: Journ. Biol. Chem., 1914, xvii, p. 401. 5. FUNK: BIOCHEMICAL BULLETIN, 1916, ν. (Accepted for publica

tion, June 1, 1915; to be the opening paper in Vol. v.)

THE PROBLEM OF REJUVENESCENCE IN
PROTOZOA*

LORANDE LOSS WOODRUFF

It is a pleasure to accept the invitation to present a summary of the results derived from our genetic cultures of Paramecium at Yale, with special reference to the bearing of this and other work on the problem of rejuvenescence in protozoa; for, it seems to me, the problem has now passed successfully through the periods of youth and adolescence, and is approaching that of maturity, when we may confidently expect the production of some conclusions of general significance.

Although the problems of protoplasmic senescence and the function of conjugation have afforded the stimulus for investigations on the life history of infusoria since Ehrenberg, nearly a century ago, theorized on the potential 'immortality' of those forms, we may take, as the point of departure for our present brief review of the subject, the classical experimental studies of Maupas. As is well known Maupas' studies afforded a wealth of data, all of which indicated that continued reproduction by division results in degeneration and death, and seemed to place the conclusion, that conjugation is a sine qua non for the life of infusoria, upon a firm empirical basis.

A series of important investigations by Hertwig and Calkins confirmed Maupas' general conclusion that infusoria, after a more or less definite number of divisions, degenerate and finally die if conjugation is prevented. Calkins, however, made the significant discovery that artificial stimuli of different kinds may, for a time, be substituted with success for conjugation since, by the opportune use of artificial stimulation, he was able to prolong the life of one culture of Paramæcium caudatum to the 742d generation.