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Interne urals I // Anterior-Dorsal Projection Fig. 6. Location of selected skeletal features 01 metalarval and early-juvenile catostomids. Top-- lateral view. Middle--dorsal view. Bottom--ventral view. Clearing and Staining Procedures for Skeletal Study of Small Fish These instructions are modified from Snyder and Muth (1988) and based on proce- dures detailed by Fish (1932, Method III), Taylor (1967), Potthoff (1984), and Taylor and Van Dyke (1985). See Taylor (1967) and Taylor and Van Dyke (1985) for detailed explanations and discussions of the various steps, factors affecting them, and alternatives. The procedures that follow are for differential staining of cartilage and bone beginning with living specimens. To begin with previously preserved specimens, stain only for cartilage, only for bone, or clear ( make transparent) without staining, skip the irrelevant steps. Minimum and maximum times given in the procedures are approximate for single specimens measuring 10 and 25 mm total length, respectively, and processed in 20 ml vials. Times for other sizes and numbers of specimens can be approximated accordingly. Vertebrates as large as 500 mm have been cleared and stained by these procedures but time requirements are considerably greater; clearing alone can take several weeks. Potthoff (1984) provides a diagram of approx- imate times for specimens 10 to 500 mm standard length. Specimens larger than 30 mm with scales or thick skin may need to be scaled or skinned, or selectively and carefully punctured over the body with a sharp needle, prior to clearing and staining. Some larger specimens may need to be eviscerated. Fatty or oily specimens may need "degreasing" in xylene before staining or clearing and speci- mens with large amounts of guanine or simi- lar white or silvery substances may needed soaking in 2% or stronger potassium hydrox- ide solution after clearing by the enzyme method (Taylor and Van Dyke 1985). Specimens should never occupy more than 25% of solution volume during ftxation; lesser percentages (e.g., ::510%) are recom- mended. During clearing and staining, results will be better and time requirements may be less if specimens occupy much less than 10% of solution volumes (e.g., down to 2% of solution volume during neutralization and clearing). For specimens 30 mm total length or less, most or all steps can be carried out conveniently in 20 ml or similar-size vials. During each step, periodically turn or move specimens to minimize solution stratification and aid penetration of solutions into tissues of specimens being processed. For the most reliable results begin with freshly fIXed and preserved specimens. Older museum specimens mayor may not clear and stain properly depending on original ftxative, preservative, and subsequent care. However, properly fIXed and preserved specimens should clear and stain nearly as well as fresh material, even after a few decades. With specific regard to ftsh embryos and larvae, Taylor and Van Dyke (1985) made the following observations. "The presence of cartilage in embryos and larval ftshes is readily determined by this method [differ- ential staining with enzyme clearing]. But, determining the presence and time and/or degree of osteogenesis is more difftcult because newly deposited bone mineral is much more labile than mineral that has been deposited for some time. The presence of 19