Germ Cells of Leptinotarsa Signaticollis. 137 



not give as perfect fixation, but enables one to study chemical 

 transformations more satisfactorily. This mixture was used al- 

 most exclusively for eggs in later stages of development. The 

 eggs were allowed to remain for about ten minutes in the reagent 

 which was maintained at a temperature of 55°C., and then trans- 

 ferred to 70 per cent alcohol. At the end of a day or two the egg 

 envelopes stand out from the egg, and can be readily dissected 

 off with the aid of needles. Good results were also obtained with 

 Hermann's platino-aceto-osmium mixture. The killing fluids 

 were used in the cold except in the case of the eggs as noted above. 

 The most satisfactory stains were found to be safranin (basic), 

 and lichtgriin (acid) . Others were used in various combinations, 

 but without such good results. Even for the study of chromo- 

 somes I have found these dyes superior to Heidenhain's iron- 

 alum-haematoxylin. Grubler's ''Safranin 0" was made up 

 according to the following formula: 



Safranin 1 gram 



Anilin water (4cc. anilin oil + 90 cc. water) 90 cc. 



Alcohol (95 per cent) 10 cc. 



Sections were left in the safranin for four to six hours, passed 

 through graded alcohols and immersed for a few seconds in the 

 acid stain. After washing in 95 per cent alcohol, the material 

 was transferred successively to absolute alcohol, clove oil and 

 xylol ; and then mounted in balsam. These two stains work to- 

 gether perfectly, and give sharp and clear contrast at every stage. 



Iron-alum-haematoxylin with or without counterstain (orange 

 G or lichtgriin) and Gram's gentian violet method were also em- 

 ployed, though to a less extent. 



For embedding, Johnston's parafiin-asphalt-rubber mixture was 

 used in various degrees of dilution with pure paraffin. This 

 method is especially helpful in working with mature eggs, which 

 show a great tendency to crumble when cut in pure paraffin. 

 Sections were as a rule cut 61^ in thickness. 



