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all fish except embryos and larvae in which some critical bone mineral may be lost. For both freshly fixed and long pre- served material, the enzyme method gen- erally 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 speci- mens). Monitor specimens closely -- this method of clearing is simpler, less expen- sive, 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 if the specimens are inadequately fixed or if di- gestion of tissues is allowed to go too far. Results are usually better and more con- sistent 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 solu- tion until bones are adequately stained, a few hours to one day; monitor specimens closely. Rinse specimens briefly in dis- tilled water. Final Clearing and Storage: 9. Return specimens to clearing agent (tryp- sin solution or 1 or 2% KOH solution) until remainder of muscle is adequately transparent (some final clearing will take place in glycerin series if used for stor- age). 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 for- giving 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 clear- ing and avoid storage problems" (Taylor 1%7), most researchers store cleared and stained specimens in pure or 100% gly- cerin. Glycerin also will reduce or eliminate cloudiness due to water in the remaining soft tissues. In either case, work specimens through at least a mini- mal graded series to the final concentra- tion, 4-24 hours in each solution (e.g., 50% and 75% ethanol or 40%, 70% and 100% glycerin). If specimens 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. 22