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. They 
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 
injury 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 mid ventral 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 bcdy wall fell away from the viscera and opened 
