Bone Staining 17.") 



solutions: 



Potassium hydroxide, 2%: 



potassium hydroxide, white sticks 20.0 gm. 



disdlled water 1000.0 ml. 



Alizarine stock soltition: (HoUister, 193-f) 



alizarine red S, C.I. 58005, saturated solution in 



50% acetic acid 5.0 ml. 



glycerol 10.0 ml. 



chloral hydrate, 1% aqueous 60.0 ml. 



Alizarine working soltition: 



alizarine stock solution 1.0 ml. 



1-2% potassium hydroxide in distilled water . 1000.0 ml. 



Make up at least 500 ml.; use at room temperattire. 



Clearing solution #\: [Hood and Neill, 1948) 



2% potassium hydroxide 150.0 ml. 



0.2% formalin 150.0 ml. 



glycerol 150.0 ml. 



Clearing solution #2: {Hood and Neill, 1948) 



2% potassium hydroxide 100.0 ml. 



glycerol 400.0 ml. 



procedure: 



1. From hardening solution, rinse fe^v minutes in distilled water. 



2. Leave in 2% potassium hydroxide luitil skeleton shows throtigh 

 musculature: 2-4 hours for small embryos, 48 hours or longer for 

 larger forms. {St. Amand and St. Amand, 1951 , warmed solution to 

 38°C for qtiicker action.) 



3. When clear, transfei" to alizarine working solution: 6-12 hours or 

 longer depending on specimen. Skeleton shotdd be deep red. 

 Large specimens may require fresh changes of dye solution. 



4. Transfer directly to 2% potassium hydroxide: 1 day or longer im- 

 til soft tissues are destained. The KOH may be 1.0-0.5% for small 

 specimens. Sunlight or lamp speeds up process. 



5. Clear in solution #1:2 days, room temperature. Then in soliuion 

 #2: 1 day. 



6. Transfer to pure glycerol Avith thymol added as preservative. 



7. Store in sealed tubes or bottles. Mount on olass rods and seal in 

 musetnn jars, or embed in plastic. 



rt" 



