CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 173 



washed in running tap-water. The larvae from which the gill- 

 plates were taken were fixed for other purposes, and no special 

 effort was made to insure good fixation of the plates. Thej^ 

 were preserved in position in 5 per cent formalin, which was 

 later gradually replaced with 80 per cent alcohol. 



The fixed gill-plates were carefully removed from the larvae in 

 80 per cent alcohol and were left attached to the gill arches, 

 which, in subsequent handling, were grasped by forceps to prevent 

 injuiy of the plates. Hydrogen peroxide was added to the 80 

 per cent alcohol drop by drop through a fine glass capillary 

 siphon until the solution amounted to equal parts of each. 

 In this the plates were bleached for four to twelve hours and then 

 transferred to the mordant by the above-mentioned drop-process 

 and stained in iron haematoxylin. They were dehydrated by 

 this drop method, cleared in cedar-wood oil followed by xylol, 

 cut from gill arches, after being transferred to the slide, and 

 then covered with damar and a thin cover-glass. While the 

 damar was hardening they were kept for twenty-four hours or 

 more under slight pressure to insure flat preparations. 



The peritoneum, mesentery, and lungs 



These preparations were made from larvae 3 to 4 inches long. 

 All the tissues of a given individual were not only fixed together 

 in the same fixative for the same length of time, but also received 

 the same treatment in all subsequent processes. They were put 

 into the fixatives within an estimated maximum of two minutes 

 after the first incision. Two methods of procedure were used in 

 preparing these tissues for fixation: 



1. In order to avoid any possible unfavorable effect of cap- 

 tivity, the tissues were fixed in situ in the field as soon as the 

 larvae were taken from the net. The animals were prepared for 

 fixation as follows : With sharp scissors the body wall was cut 

 open along the midventral line and also lateral incisions were 

 made on each side at right angles to the first incision behind the 

 pectoral girdle and in front of the pelvic girdle, so that the two 

 halves of the body wall fell away from the viscera and opened 



