166 . CM. CHILD 



5. The technique of the indirect method 



This method is chiefly useful where it is desired to follow the 

 morphological features as well as to compare the rates of reaction. 

 As stated on page 158 above, the results with this method are the 

 inverse of those obtained by the direct method. There the resist- 

 ance varies inversely, here it varies directly as the rate of reac- 

 tion. The results by this method really represent the degrees of 

 acclimatization to the reagent used. 



In my earlier experiments, where the morphological changes 

 were followed in animals and pieces, this method, usually with 

 1 to 1.5 per cent absolute alcohol as the reagent, was used exclu- 

 sively. It was only after the relation between the rate of reaction 

 and the resistance was discovered that the direct method was 

 developed. 



The data in my 'Study of senescence and rejuvenescence'. 

 (Child '11a) were all obtained by this method. The procedure 

 used at that time is described on pages 538 to 540 of that paper. 

 Since then I have found it more convenient to use 1-liter Erlen- 

 meyer flasks instead of the Stender dishes in sealed jars, as there 

 described. The lots of worms or pieces, ten each in most cases, 

 are placed in the flasks, which are filled with the reagent and 

 corked, leaving an air space beneath the cork of some 10 mm. in 

 depth. The fluid is renewed every four days or oftener, prelimi- 

 nary experiments having shown that in such a flask containing 

 well aerated water twenty-five large worms would live for a week 

 or ten days at a temperature of 20°C. without showing any bad 

 efl"ects. In the experiments with depressing agents the liquid is 

 always well aerated at the time the worms are added because of 

 the thorough shaking necessary for a uniform mixture of the 

 water and the reagent, moreover the rate of reaction in the ani- 

 mals is much lower in the depressing medium than in water, so 

 that it is impossible that lack of oxygen or harmful accumula- 

 tion of metabolic products should occur at ordinary temperatures 

 within four days. 



In these experiments the lots are examined each day, or in 

 many cases every forty-eight hours and the number of worms or 

 pieces remaining intact is recorded. Attempts to follow stages 



