CHROMOSOME NUMBER AND PAIRS IN AMBYSTOMA 175 



close to the spine. Under the binocular lens, in water, the 

 peritoneum was carefully loosened from the underlying tissue 

 by scraping it with a sharp scalpel, first along the edge cut 

 from the back. Sections of this loosened edge were then grasped 

 by the forceps and relatively large sheets were easily pulled off 

 from the underlying tissue. The peritoneum covering the dorsal 

 and lateral portion of the body wall is deeply pigmented and to 

 it adhere considerable muscle and connective tissue when the 

 peritoneum is removed. This portion was grasped with the 

 forceps in removing the peritoneum from the body wall, as well 

 as in all subsequent handling. Consequently the cells in the 

 ventral transparent region available for study have been undis- 

 turbed by instruments. However, there still remains a possi- 

 bility that the strain of pulling the peritoneum loose might 

 disturb some cells. 



Peritoneum fixed in Flemming's and Hermann's solutions was 

 stripped from the body wall after four hours of fixation and then 

 fixed twenty hours longer. That treated with the various picric 

 acid mixtures was stripped immediately after fixation. However, 

 the peritoneum fixed in the chromic acid modification of Bouin's 

 solution may be preserved in alcohol for as much as a year before 

 stripping. That of Ambystoma punctatum, fixed in Flemming's 

 stronger solution and preserved in 5 per cent formahn, can be 

 stripped at least six months after fixation. 



Material fixed in osmic acid fluids was w^ashed five to fourteen 

 hours in frequent changes of tap-water. Picric acid preparations 

 were gradually transferred to 70 per cent alcohol, beginning with 

 10 per cent and progressing through successively stronger grades 

 differing by 10 per cent. They remained in each grade five to 

 ten minutes. The tissues remained in 70 per cent alcohol con- 

 taining a few drops of saturated aqueous lithium carbonate 

 solution until the picric stain was removed, and before staining 

 they were returned to water by reversing the above process. 

 All of the material w^as stained in Heidenhain's haematoxylin after 

 mordanting in a 2^ per cent solution of iron alum for four to six 

 hours. No counterstains were used. 



JOURNAL OF MORPHOLOGY, VOL. 33, NO. 1 



