416 HISTOLOGICAL PROCEDURES 



possible to make a dissection to study the internal organs. inis is so oecause the car- 

 tilage and bone will not have developed. There is very little cartilage, even at 8 days. 



PERMANENT PREPARATIONS OF CHICK EMBRYOS 



The primitive streak stages can be best fixed while still on the egg, by dropping the fix- 

 ative onto the blastoderm gently from above. Since fixation renders the blastoderm 

 rather brittle, it is best to cut it out within a few minutes after fixation, and then trans- 

 fer it to a Syracuse dish with fresh fixative for the requisite time. (Use instruments 

 other than those for operating purposes. ) 



Later blastoderm and embryos may be excised in the manner described on preceding 

 page, and fixed either in Syracuse dishes or on slides (when they are held in place by the 

 filter paper rings). Within a minute of fixation gently wash the entire flat blastoderm 

 off of the slide and into adequate (similar) fixative by a gentle stream of fixative from a 

 pipette. Some of the yolk will remain adherent to the slide, and if the blastoderm is 

 allowed to remain long on the slide, it is apt to be torn during subsequent removal. 



Still later stages (up to 8 or 9 days) may be fixed "in toto" in any of the standard fixa- 

 tives. If it does not interfere with structures important in the examination, it is always 

 well to slit the abdomen to allow fixative to penetrate to the viscera the more readily. 



HISTOLOGICAL PROCEDURES: 



Fixatives : Kleinberg's Picro-sulphuric, Bouin, Michealis' fluid, or 0. 5% acetic 

 acid in 10% formalin. (Given in order of preference. ) Fixation should be for at 

 least 4 hours for the earlier stages to 48 hours for the 9 day embryos. 



Decoloration : The picric acid of the fixatives leaves the embryo stained a brilliant 

 yellow. This may be removed with lithium carbonate but more quickly and satis- 

 factorily by adding about 3% by volume of NH4OH to the 70% alcohol during dehydra- 

 tion. The alkaline ammonia decolorizes the yellow picric acid. Bleaching of older 

 stages may take 24 hours and several changes. 



Dehydration: Dehydration must be slow in order to avoid damage to the delicate 

 blastoderms and to insure complete dehydration of the larger, later embryos. One 

 hour periods for the early stages and as much as 12 hour periods for the older em- 

 bryos, in each of the graded alcohols, is generally indicated. The alcohols should 

 include 35%, 70%, 80%, 90%, 95% and finally 100%. If the yellow picric is not en- 

 tirely removed, a small amount of LiC03 may be added to each of the alcohols from 

 70% to 90%. Make two changes in absolute alcohol. 



Clearing: This is best accomplished by transferring the embryo from absolute 

 alcohol to pure cedar oil for 24 hours or more (i.e. , until translucent). Then trans- 

 fer to xylol for 30 minutes. 



Embedding : Embed in 56°C. paraffin to which has been added 5% Bayberry Wax and 

 5% Beeswax (measurements by weight). A total of 1 hour for the earliest stages to 

 as much as 5 hours for the 9 day chick embryo is indicated. For the large embryos 

 with some cartilage a final embedding in a paraffin-rubber mixture is suggested 

 (30-60 minutes). 



Sectioning : For cytological studies the sections should be no more than 10 microns 

 in thickness. For study of organs the sections may be as thick as 20 inicrons. Boil 

 some distilled water, cool, and to every 10 cc. add 1 drop of egg albumen, mix 

 thoroughly. Place a few drops of this albumen-water on the slide, and float the rib- 

 bon on it. When the ribbon is properly oriented, draw off the excess fluid with pi- 

 pette and filter paper, and dry on a warming plate at 40° C. 



