274 GEORGE W. BARTELMEZ 



tions. In the study of the blastodiscs however, the important 

 objects are to preserve the form relations and to obtain sharp 

 differentiations in the cytoplasm. The sublimate-acetic mixture 

 above mentioned is best for this purpose. The optimum time of 

 fixation is between forty-five minutes and one and a half hours. 

 The whole yolk is dropped into the warm mixture in such a way 

 that the blastodisc floats down; it remains in this position owing 

 to the weight of the glass pin inserted to mark the orientation. 

 When the blastodisc is to be photographed enough 95 per cent 

 alcohol is added so that the egg just rests on the bottom of the 

 dish. After fixation the egg is washed in repeated changes of 35 

 per cent and 50 per cent alcohol and hardened in 70 per cent over- 

 night. The next day a pentagonal block is cut out as Blount 

 ('09) and Patterson ('09) direct. Material for sectioning is de- 

 hydrated in 95 per cent alcohol, gradually cleared in bergamot oil, 

 and imbedded in 55 to 58° paraffin. Most sections were cut 6| 

 micra thick and mounted with albumen fixative. 



After sublimate fixation the best stain is Bensley's ('11) neutral 

 gentian. The copper-chrom-hematoxylin method also gave good 

 results. Intra vitam staining with Janus green and neutral red 

 aided materially in studying the fresh primordial follicles under 

 the 3 mm. immersion lens. The blastodiscs of mature and fer- 

 tilized ova were studied and usually drawn in the life. They were 

 kept in the Patterson stage incubator ('09) in a mixture of physi- 

 ological salt solution and egg albumen, and observed with a 

 Zeiss binocular. Continuous observations could be made by this 

 means of stages between fertilization and the completion of the 

 first cleavage if proper precautions were taken to prevent chill- 

 ing the egg. For the first three hours and probably for much 

 longer the development is perfectly normal. 



In studying the yolk spherules of the younger oocytes the ovarj^ 

 was fixed for twenty-four hours in warm neutral formol (10 to 

 15 per cent) or in a 15 per cent solution of formol in 2.5 per cent 

 aqueous potassium bichromate. After washing, free hand or 

 frozen sections were made, stained in a saturated solution of 

 Sudan iii in 70 per cent alcohol, counterstained with a weak alco- 

 holic solution of methyl green, and mounted in glycerine. 



