BEHAVIOR OF ANURA TOWARD COLLOIDAL DYES 5 



2. METHODS 



The following species of amphibia have been studied by the 

 writer in connection with this investigation : 



Larvae and adult of Rana pipiens, Schreber; larvae and adult 

 of Rana sylvatica, Le Conte; year-old larvae and adult of Rana 

 catesbiana, Shaw; adult of Rana clamata, Daudin, and Rana 

 palustris, Le Conte; larvae of Acris (spec.?) ; advanced larvae of 

 Diemyctylus viridescens, Rafinesque, and larvae and adult of 

 Bufo lentiginosus, Shaw. 



Trypan blue (Griibler) and dye No. 161 (Niagara blue) 1 in 

 solutions of different concentrations as stated in the following 

 pages, were the principal dyes used. The action of these two dyes 

 on certain typical tissues was found to be identical. 



The young frog and toad larvae were placed in solutions of 

 trypan blue and dye No. 161 (Niagara blue), ranging in concen- 

 tration from 1: 800 to 1:2000. When treated in this manner a 

 considerable number of them usually die at about the time the 

 external gills begin to disappear. In dye solutions of low concen- 

 tration such as 1 :10,000 the larvae will live for an almost indefi- 

 nite time. The action of such solutions, although slower than 

 those of higher concentration, produces exactly the same results. 

 The younger larvae were in all cases allowed to develop in 

 solutions of dye which were made up fresh with ordinary tap 

 water every twenty-four hours. On account of the scarcity of 

 dye and number of experiments, it was often found necessary 

 later in the Summer to make use of previously used solutions and, 

 in the case of these older larvae, no difference in the action of the 

 dye was observed. 



In preparing larvae for section cutting which have remained 

 in solutions of dye, it has been found desirable to transfer them 

 from the dye solution to tap water, in which they should remain 

 for at least twenty-four hours before killing and fixation. The 

 object of this is to eliminate all of the dye from the alimentary 

 canal which, if present after killing, often dissolves in the fixing 



1 1 am indebted to Dr. Paul A. Lewis of the Henry Phipps Institute for the dye 

 No. 161. to whom niy thanks are due. 



