Chapter XVI : Some Embryological Methods 123 



olabrus) 10 minutes after fertilization the formation of blastodisc and 

 polar bodies may be observed; 30 to 33 minutes after fertilization the 

 two pronuclei may be found in close approximation. 



If other than the very early stages are required, the fertilized eggs 

 must be transferred to a hatching -box. This is best done by means of a 

 horn spoon and a feather. The hatching-box must be provided with a 

 very gentle (drop by drop) stream of running water. According to Exner 

 the eggs are best placed in the box on a layer of glass rods which are 

 from one-twelfth to one-sixth of an inch apart. Three-eighths of an 

 inch below the rods there should be a layer of pebbles covering the floor 

 of the box. Dead eggs, recognizable by their opacity, should be removed 

 at least once a day. See also memorandum 4. 



10. For the Study of Early Cleavage in Living Material the eggs of some 

 of the water snails afford an abundance of excellent material. By watch- 

 ing aquaria which contain snails the fresh material can easily be obtained 

 during the spring and summer. Twigs and bits of board to which the 

 egg-masses may be attached should be placed in the aquaria. 



11. For the Study of the Formation of Polar Bodies, Fertilization, and Early 

 Cleavage in Sections nothing surpasses the eggs of Ascaris. The Ascaris 

 (A. megalocephala) from the horse is preferable although A. lumbri- 

 coides from the pig will answer. 



The ovisacs, two in number, are very long convoluted tubes. Differ- 

 ent regions contain eggs in different stages of development. The 

 thicker tubes toward the anterior end of the animal contain cleavage 

 stages; back of these are cells showing extrusion of the polar bodies and 

 fertilization stages. The material must be fresh; either bring the live 

 Ascaris to the laboratory or take the fixing fluid to the place for obtain- 

 ing the material. Slit open the abdominal wall of the worm and remove 

 the ovisacs and after separating the numerous convolutions somewhat, 

 fix them entire for 24 hours in picro-acetic acid (reagent 23, Appendix 

 B), or for 15 to 25 minutes in acetic-alcohol-chloroform (reagent 26) satu- 

 rated with corrosive sublimate. Preserve in 80 per cent, alcohol. To 

 locate eggs of the desired stage tease out eggs at intervals along the 

 ovisacs, stain with acid carmine (reagent 37) and examine. The proper 

 region once located, cut out small lengths of the tube, imbed it in par- 

 affin and make thin transverse sections. In order to keep the eggs from 

 shriveling, the bath in hot paraffin must be curtailed. Use the method 

 for delicate objects (chap, vi, vii). Stain by the iron-hematoxylin method 

 (reagent 51, Appendix B). 



12. Directions for Orienting Serial Sections. a) In mounting transverse 

 sections (sections across the main axis of the object), the sections, begin- 

 ning at the anterior end of the object, are laid on the slide in the same 

 sequence as the reading on the page of a book. In order to have right 



