EXPERIMENTAL CHICK EMBRYOLOGY U25 



b. Conklln's Haematoxylin: Add 1 drop of ELinenterg' 3 Picro Sul- 

 phuric to each 1 cc. of seasoned Delafleld's Haematoxylin. Stain 

 sections for 6 to 10 minutes, rinse in (alkaline) tap water. 

 Counterstain with 1 dip in 0.5^ eoaln in 951^ alcohol (counterstain 

 not recommended for photographs ) . 



c. Harris' Haematoxylin: Stain for 6 minutes, blue in alkaline tap 

 water, and counterstain if desired. 



2. Whole mounts: These may he stained for from 6 hours to 2 days in either 

 of the (above) Haematoxylins, depending upon the size and stage of the 

 embryo. Large embryos (72 hours and older) may thus be stained in toto 

 and later sectioned. If the stain does not penetrate adequately,, the 

 sections may be further stained after mounting. 



LUJTOVALL TECHNIQUE FOB STAINIIC OF CHICK EMBKfO CABTILAGE: 



This technique may be used either on a chorio-allantoic graft which has developed 

 cartilage (and bone) or it may be used with the whole embryo of 9 to 10 days Incubation 

 age. The Spalteholz technique may be used even for later stages for complete trans- 

 parencies. 



1. Fix in Bouin's or ELlnenberg's Picro- sulphuric for 2k hours. 



2. Transfer to 70^ alcohol containing 2^ NHi^OH to decolorize. Several changes over 

 a period of several hours may be necessary. 



5. Using forceps, remove skin, feathers, and all fatty tissue. 



k. Stain for 2 to 5 days in 0.25^ methylene blue (or toluidlne blue) made up in 70^ 

 alcohol to which is added 3% HCl by volume. This will overstaln. 



5. Destaln in several changes of 70^ alcohol for about k8 hours. 



6. Dehydrate lor k hours in 95^ alcohol. The softer tissues will become destalned 

 and somewhat transparent. 



7. Transfer the embryo to Methyl Salicylate (oil of wlntergreen) to which has been 

 added 2'y^ (by volume) of benzyl benzoate. In this the embryo will clear conplete- 

 ly and may be stored. (See Lundvall, 190*1: Anat. Anzeiger 25 and I906, Anat. 

 Anzelger 27. ) 



MODIFIED SPALTEHOLZ' METHOD FOB STAINING SKELETAL ELEMENTS : 



The following procedure is excellent for post-metamorphic amphibia and for chick 

 embryos beyond the 10th day of Incubation. 



1. Fix In 95^ alcohol two weeks to harden. 



2. Transfer to 1^ KOH for 2h hours. 



5. Transfer to tap water and, with forceps, pick off as much fleshy material as 



possible. 

 k. Transfer to 95^ alcohol, change once in 6 hour period. 



5. Transfer to ether for 1 to 2 hours to dissolve away any fat, or use acetone if 



there is little or no fat. 



6. Transfer to 95^ alcohol for 6 hours, change once. 



7. Transfer to 1^ KOH for 6 days. 



8. Put In Alizarin red "S" for 12 hours. 



9. Transfer to 1^ KOH for 2k hours. 



10. Put in Moll's solution for 2k hours. 



11. Store In 100^ glycerine. 



METHODS FOR OBSERVING THE DEVELOPMENT OF THE CHICK EMBRYO 



The hen's egg is fertilized at the upper end of the oviduct so that by the time It is 

 layed the embryo has reached the stage of gastrulation, at least. The blastoderm of such 

 an egg shows no visible structures at this time but after 18 hours of incubation the 

 primitive streak may be discerned. Candling will not generally indicate einy development 

 before the 53 hour stage. 



It Is now possible to replace a portion of the shell with a cover-glass window 

 through which development can be observed from day to day. A second method is to remove 



