36 



ONTOGENY AND SYSTEMATICS OF FISHES-AHLSTROM SYMPOSIUM 



Table 5. Method of Clearing and Staining Cartilage and Bone in Larvae, Juvenile and Adult Fish. 



Dehydration: 



1. 50% distilled H,0, 

 50%of95%ethanol. 



2. Absolute ethanol 

 (95% ethanol may 

 be substituted). 



-1 day ►[- 



-1 day ►(- 



h 



2 days • 

 2 days 



>h- 



3 days  



►h 3 days 



-one intermediate change 



-►h • 



-5 days- 

 -7 days- 



-► 

 ■-► 



Staining cartilage: 

 100 ml solution: 



A. 70 ml absolute ethanol, 

 30 ml acetic acid, 



20 mg alcian blue. 

 100 ml solution: 



B. 60 ml absolute ethanol, 

 40 ml acetic acid, 



30 mg alcian blue. 



— I day 



-Solution A- 



-►h I'/idays ►h"2 days->- 



-►H Solution B ► 



Neutralization: 

 saturated sodium 

 borate solution. 



'/2 day - 



-►I-- 

 I- 



2 days 



-one intermediate change - 



-► 

 -► 



Bleaching: 

 pigmented 

 specimens only. 

 100 ml solution: 

 15 ml 3% H,0„ 

 85 ml 1% KOH. 



-20 min. 



-►!-■ 



-40 min. 



-► h - 1 hour ► I 1 1/2 hours - 



Trypsin digestion: 

 100 ml solution: 

 35 ml saturated sodium 

 borate. 65 ml distilled 

 H,0. trypsin powder. 



-Keep in solution until 60% clear, change to fresh solution every 10 days- 



Staining bone: 



1% KOH solution with 

 alizarin red stain. 



I day - 



-►[-■ 



2 days • 



-►h 



-4 days 



Destaining: 



100 ml solution: 

 35 ml saturated sodium 

 borate, 65 ml distilled 

 HjO, trypsin powder. 



-2 days — ►!-  



Change to fresh solution every 10 days until solution remains - 

 unstained and specimen is clear 



Preservation: 

 30% glycerin and 

 70% of 1% KOH. 

 60% of glycerin 

 and 40% of I % KOH. 

 1 00% glycerin with thymol 

 as final preservative*. 



1 week — ►!- - -2 weeks- — ►!- 4 weeks- 



* Direct sunlight and 100% glyceiine help to clear and destain difficult specimens. 



15 parts of 3% hydrogen peroxide solution with 85 parts of 

 1% potassium hydroxide solution. Bleach larvae and small ju- 

 veniles up to 80 mm SL for 20 to 40 minutes depending on 

 size. Larger juvenile fish and adults may be bleached 1 to 1 Vi 

 hours. 



Trypsin digestion and alizarin red staining. — The clearing and 

 alizarin staining process has been well described by Taylor ( 1 967) 

 and need not be repeated here. Simply continue after bleaching 



with the Trypsin digestion, which are Taylor's steps 4 and 5. 

 We saw no need in modifying Taylor's method. 



Removal of semitransparent tissue. ~^\\ex\ studying cleared and 

 stained material of large fish, the structures studied (caudal com- 

 plex, pectoral fin supports, pterygiophores, vertebral column, 

 etc.) may have to be dissected out and adhering tissue removed. 

 This can be accomplished by time consuming picking with 

 tweezers or by placing the material in a two-phase phenol so- 



