22 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



Solution A Solution B Solution C 



Acid fuchsln 0.5 gm. This la a 1^ Glacial acetic 2.0 cc. 



Ponceau de xylidine 0.7 gm. aqueous Distilled water 100.0 cc. 



Distilled water _ 100. cc. phosphomolyb- Aniline blue to 



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



Staining procedure: Masson A for 5O-60 seconds: Rinse in distilled water; 

 Masson B for 2-5 minutes; drain, do not rinse; Masson C for 10 minutes to 

 2 hours, depending on tissue; rinse in distilled water; I'jt acetic acid for 

 5 minutes to remove excess phosphomolybdi c acid; equal parts of 1^ acetic 

 and absolute alcohol for 1 minute or dropped directly onto the slide; abso- 

 lute 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. Gum damar is preferred. 



* Remove frog egg Jelly with 10^ Chlorox (see Shumway: I9I+2 Anat. Bee. 85:309). 



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 2l+ 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. 

 1. Put in 1^ KOH for 2k hours. 

 J. Put in Moll's solution for 2k hours, 

 k. Store in 100^ glycerine. 



BECCMMENDED BKFERENCES : 



Lee : "Vade Mecum" 



McClung: "Handbook of Mlcrascopical Technique" 



CLEANING AND BLEACHING OF VERTEBRATE SKELETONS 



A. Solution of Ammonia - 2%. 



B. Mixture of 50lt Benzene and 50^ Naptha. 



C. ^jrdrogen peroxide - 2^. 



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

 Place the skeleton in each of the above solutions for about 12 hours each. 

 Skeleton will come out clean and bleached. 



