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Initial clearing 7. Enzyme method-If specimens were not processed for cartilage staining, soak them in saturated sodium borate solution for 2-12 hours to remove remaining formalin or alcohol and adjust body fluids to well above pH 7. Soak specimens in trypsin solution until 75-90% of the muscle tissue is cleared, typically 1-5 days at 20-300 C, pos- sibly longer depending on specimen volume relative to solution volume and activity or strength of trypsin. Use a volume of trypsin solution at least 10 to 40 times the volume of specimens. Completely change trypsin solution every 2-3 days. This method is preferred for all fish except embryos and larvae in which some critical bone mineral may be lost. For both freshly fixed and long preserved material, the enzyme method generally provides more consistent results with firmer whole specimens than the KOH method. or KOH method-Soak specimens in 2% KOH solution until muscle tissue begins to clear, typically 1 to 12 hours (use 1 % KOH for very small and delicate specimens). Monitor specimens closely-this method of clearing is simpler, less expensive, and tends to be faster than the enzyme method, but it is also more likely to result in fragile specimens with skin that literally splits at the seams ifthe specimens are inadequately fixed or if digestion of tissues is allowed to go too far. Results are usually better and more consistent if specimens are freshly fixed than if they were preserved and stored for a long time (Taylor and Van Dyke 1985). Bone staining procedure 8. Stain specimens in alizarin red stain solution until bones are adequately stained, a few hours to one day; monitor specimens closely. Rinse specimens briefly in distilled water. Final clearing and storage 9. Return specimens to clearing agent (trypsin solution or 1 or 2% KOH solution) until remainder of muscle is adequately trans- parent (some final clearing will take place in glycerin series if used for storage). Change solution after an hour or two to remove excess stain and continue clearing if necessary. If clearing in KOH solution, monitor specimens closely (this procedure is usually faster and less forgiving than the enzyme method). 10. Specimens may be stored in alcohol (e.g., 75% ethanol), in which they are easier to handle, but "to attain uniformity in clearing and avoid storage problems" (Taylor 1967), most researchers store cleared and stained specimens in pure or 1 00% glycerin. Gly- cerin also will reduce or eliminate cloudi- ness due to water in the remaining soft tissues. In either case, work specimens through at least a minimal graded series to the final concentration, 4-24 hours in each solution (e.g., 50% and 75% ethanol or 40%, 70%, and 100% glycerin). If speci- mens are not as transparent as desired at this point, try adding a 20% glycerin in 1 % KOH step to the beginning of the graded glycerin series. Add a few thymol crystals to containers with 100% glycerin to prevent fungus growth. 25