HISTOLOGICAL PROCEDURES 417 



Staining: 



1. Sectioned material: 



a. Heidenhain' s Iron Haematoxylin: Excellent for chromosome studies. 

 Mordant the sections for 12 hours in 4% iron alum, rinse, stain for 6 or 

 more hours in Heidenhain' s Haematoxylin, then destain in 2% iron alum 

 while observing it under the dissection microscope. Stop the destaining 

 when the section has a uniform grayish appearance, by placing it in 

 slowly running tap water. 



b. Conklin's Haematoxylin: Add 1 drop of Kleinberg's Picro Sulphuric to 

 each 1 cc. of seasoned Delafield' s Haematoxylin. Stain sections for 6 to 

 10 minutes, rinse in (alkaline) tap water. Counterstain with 1 dip in 



0. 5% eosin in 95% 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 be 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. 



LUNDVALL TECHNIQUE FOR STAINING OF CHICK EMBRYO CARTILAGE: (Anat. 

 Anz. 25 and 27, 1905 and 1906) 



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

 age (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 transparencies. 



1. Fix in Bouin's or Kleinberg's Picro-sulphuric for 24 hours. 



2. Transfer to 70% alcohol containing 2% NH^OH to decolorize. Several changes 

 over a period of several hours may be necessary. 



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



4. Stain for 2 to 3 days in 0. 25% methylene blue (or toluidine blue) made up in 70% 

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



5. Destain in several changes of 70% alcohol for about 48 hours. 



6. Dehydrate for 4 hours in 95% alcohol. The softer tissues will become destained 

 and somewhat transparent. 



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

 added 25% (by volume) of benzyl benzoate. In this the embryo will clear com- 

 pletely and may be stored. (See Lundvall, 1904: Anat. Anzeiger 25 and 1906, 

 Anat. Anzeiger 27.) 



MODIFIED SPALTEHOLZ' METHOD FOR STAINING SKELETAL ELEMENTS: 



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

 bryos beyond the 10th day of incubation. 



1. Fix in 9 5% alcohol two weeks to harden. 



2. Transfer to 1% KOH for 24 hours. 



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

 pos sible. 



4. 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 24 hours. 



10. Put in Moll's solution for 24 hours^ 



11. Store in 100% glycerine. 



