28 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



d. Orange G - sat. solution in clove oil, use only 30-60 seconds. 



e. Mas son stain - differential stain; excellent for pituitary cell types. 



Solution A Solution B Solution C 



Acid fuchsin 0. 3 gm. This is a 1% Glacial acetic . . 2. cc. 



Ponceau de xylidine . 0. 7 gm. aqueous Distilled water. . 100. cc. 



Distilled water 1 00. cc. phosphomolyb- Aniline blue to 



Glacial acetic 1.0 cc. die acid saturation . . . .(3. gm. ) 



Staining procedure: Masson A for 30-60 seconds: Rinse in distilled water; 

 Masson B for 2-3 minutes; drain, do not rinse; Masson C for 10 minutes to 2 

 hours, depending on tissue; rinse in distilled water; 1% acetic acid for 5 minutes 

 to remove excess phosphomolybdic acid; equal parts of 1% acetic and absolute 

 alcohol for 1 minute or dropped directly onto the slide; absolute alcohol; xylol, 

 etc. 



PERMANENT MOUNTING 



While balsam dissolved in xylol is the standard mounting medium, dried balsam will chip 

 and often takes on a yellow tinge with age. Clarite is preferred. 



Special staining procedure for skeletons, particularly good for post-metamorphis stages 

 of amphibia and chick embryos beyond the 10th day. 



a. Fix in 95% alcohol two weeks to harden. 



b. Put in 1% KOH for 24 hours. 



c. Put in tap water and pick off as much of fleshy material as possible. 



d. Put in 95% alcohol; change once during 6 hour period. 



e. Put in ether for 1-2 hours to dissolve away any fat; use acetone if there is little 

 or no fat. 



f. Put in 95% alcohol for 6 hours; change once, 



g. Put in 1% KOH for 6 days. 



h. Put in Alizarin red "S" for 12 hours. 



i. Put in 1% KOH for 24 hours. 



j. Put in Moll's solution for 24 hours. 



k. Store in 100% glycerine. 



RECOMMENDED REFERENCES: 



Lee: "Vade Mecum" 



McClung: "Handbook of Micrascopical Technique" 



CLEANING AND BLEACHING OF VERTEBRATE SKELETONS 



1. Solution of Ammonia - 2%. 



2. Mixture of 50% Benzene and 50% Naptha. 



3. Hydrogen peroxide - 2%. 



Clean off all soft tissues, picking them away to the bone with scalpel. Place the skeleton 

 successively in each of the above solutions for about 12 hours each. Skeleton will come 

 out clean and bleached. 



