630 APPENDIX. 



eggs may be preserved in 80 per cent, alcohol and are then ready for dehydration and 

 embedding. 



On account of the yolk, amphibian eggs are very brittle when embedded in paraffin, 

 thus making section cutting difficult. This difficulty can be overcome to a great extent 

 by the following method. After cutting a section, paint a coat of very thin celloidin over the 

 cut surface of the block of paraffin. The celloidin should be so thin that the surface does 

 not appear glossy after drying. Let the celloidin harden for a few seconds, and then cut 

 the next section. If this method is followed, it is possible to cut ribbons of sections as usual, 

 while at the same time each section is kept from breaking by the film of celloidin. The 

 sections are afterward treated as ordinary paraffin sections. 



Chick Embryos. — With an incubator, chick embryos are obtainable in any stage and 

 number, and afford, for the study of various structures, material which is otherwise difficult 

 or impossible to secure. Care should be taken to obtain eggs that are fresh and fertile. 

 The time should be marked on the eggs when they are put in the incubator. The student 

 should begin with the later stages since these are more easily handled. 



Removing the embryos from the eggs, especially the younger stages, requires great care, 

 but should be done as speedily as possible. Prepare a basin of normal salt solution warmed 

 to a temperature of 40 C. (104° F.). Take an egg from the incubator and allow it to lie in 

 one position for a minute or two in order to allow the side of the yolk containing the embryo 

 to come uppermost. Carefully break or cut a hole an inch in diameter in the shell. If any of 

 the white of the egg overflows, snip it off with scissors, otherwise it will cause the yolk to roll 

 over. The germinal area or embryo can be seen lying on the top of the yolk. Immerse the 

 egg in the warm salt solution. Insert one blade of the scissors into the yolk just beyond 

 the edge of the germinal area and cut rapidly around the area until a circular incision is 

 completed. Take up the edge of the germinal area with a pair of fine forceps and gently pull 

 it free from the yolk. A little yolk may cling to the lower surface, but this can usually be 

 removed by gentle shaking in the salt solution. Then with a glass slide transfer the 

 embryo to the fixing fluid. Care should be taken that the blastoderm is flat when it goes 

 into the fixative. 



For chick embryos of the first or second day of incubation, Zenker's or Flemming's fluid 

 is perhaps the best fixative. With Zenker's, fixation is complete in from 2 to 4 hours; with 

 Flemming's a little longer time is necessary. Wash in running water for an hour, or in several 

 changes of water for the same length of -time. Harden in graded alcohols (30 per cent., 

 50 per cent., and 70 per cent., an hour or more in each) and preserve in 80 per cent, alcohol. 



For embryos of more than two days' incubation, Zenker's or Bouin's fluid is a good 

 fixative. The time required for fixation (6 hours or more) varies with the size of the speci- 

 men. After Bouin's fluid, several changes of from 30 per cent, to 50 per cent, alcohol, 

 instead of water, should be used for washing. 



Mammalian Embryos.— Pig embryos can be obtained at an abattoir in considerable 

 numbers and with but little expense, and consequently yield some of the best mammalian 

 material for embryological purposes. It is necessary to ask merely that the man who removes 

 the viscera lay aside such uteri as appear distended. Owing to precocious development of 

 the chorionic vesicle, a uterus containing embryos of 6 mm. or larger will be noticeably 

 swollen. Cut the uterus open by a longitudinal incision as soon as possible. With the aid 

 of scissors, fine forceps and a horn spoon, carefully remove the embryos and transfer them 

 immediately to the fixing fluid. 



Embryos up to 35 or 40 mm. can be fixed in Mo in Zenker's or Bouin's fluid. They 

 should remain in the fixative from 8 hours (embryos of 6 mm.) to 48 hours (embryos of 



