^+2^+ EXPERIMENTAL CHICK EMBRYOLOGY 



later blaatoderm 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 fil- 

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

 of the slide and into adecjuate (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 

 fixatives. 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 PBOCEDUKES : 



Fixatives : Klelnenberg' s Picro-sulphuric, Bouln, Michealis' fluid, or 0-5^ 

 acetic acid in 10^ formalin. (Given in order of preference.) Fixation 

 should be for at least h hours for the earlier stages to U8 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 satisfactorily by adding about 5^ by volume of NBi4.0H to the 

 70^ alcohol during dehydration. The alkaline ammonia decolorizes the 

 yellow picric acid. Bleaching of older stages may take 2h 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 embryos, in each of the graded alcohols, is generally indicated. The 

 alcohols should include 55^, 70^, 80^, 90^, 95^ and finally 100^. If the 

 yellow picric is not entirely removed, a small amount of LlCOi may be added 

 to each of the alcohols from 70^ to 90^- Make two changes in absolute 

 alcohol. 



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

 alcohol to pure cedar oil for 2k hours or more (i.e., until translucent). 

 Then transfer to xylol for JO minutes. 



Embedding : Embed in 56°C. paraffin to which has been added y^ Bayberiy 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 la Indicated. For 

 the large embryos with some cartilage a final embedding in a paraffin- rubber 

 mixture Is suggested (5O-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 microns. 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 ribbon on It. When the ribbon is properly oriented, 

 draw off the excess fluid with pipette and filter paper, and dry on a warming 

 plate at it-0°C. 



Staining : • 



1. Sectioned material: 



a. Heidenhaln's Iron Haematoxylln: Excellent for chromosome studies. 

 Mordant the sections for 12 hours in kio iron alum, rinse, stain 

 for 6 or more hours in Heidenhaln's Haematoxylln, then destaln in 

 2% iron alum while observing it under the dissection microscope. 

 Stop the destalnlng when the section has a uniform grayish appear- 

 ance, by placing It in slowly rxinnlng tap water. 



