450 GARY N. CALKINS 



the increased vitality of the individual recovered from the cyst 

 indicates that nuclear interchange or amphimixis is not directly 

 associated with rejuvenescence. Owing to the admixture of 

 relatively large quantities of nuclear material, the cytoplasms 

 of both the ex-conjugant and of the individual recovered from 

 the cyst must be different in make-up from these cytoplasms 

 prior to conjugation or encystment. The new micronucleus 

 likewise has undergone some change expressed by amphimixis 

 with conjugation, or, with encystment, some unidentified change 

 which may be vaguely characterized but not interpreted by the 

 mystical word 'purified.' 



To test this matter of rejuvenescence without amphimixis 

 further, and to eliminate the long period of rest necessary for 

 reorganization by encystment, a series of experiments was under- 

 taken in which the animals were allowed to unite in conjugation, 

 after which they were separated by cutting with a scalpel prior 

 to nuclear interchange. Controls for each experiment were: 

 1) one fixed and stained individual from each pair cut to determine 

 the stage in maturation of the pair when cut; 2) the isolation 

 culture series from which the conjugants were obtained, but in 

 which conjugation was not permitted; 3) normal ex-conjugants 

 from the same epidemic of conjugations and having the same 

 ancestry as the cut individuals. 



METHOD 



A series of Uroleptus is selected in which vitality is sufficiently 

 reduced to permit of unmistakable evidence of rejuvenescence. 

 Conjugation tests are repeated until a clearly defined epidemic 

 occurs. A pair of conjugating individuals is removed with a 

 pipette and transferred with a minimum of culture fluid to a clean 

 culture dish. The pair is then cut under a binocular micro- 

 scope with a fine scalpel. A dozen pairs may be cut before a 

 successful operation in the desired plane yields an individual 

 suitable for the experiment. Uroleptus, fortunately, is a most 

 satisfactory animal for this work, for its protoplasm is resistant 

 enough to be cut in any plane without disintegration and all cut 

 fragments regenerate perfectly. Furthermore, its method of 



