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presence and time and/or degree of osteogenesis is more difficult because newly deposited bone mineral is much more labile than mineral that has been deposited for some time. The pre- sence of bone mineral is usually indicated by staining with alizarin Red S. This color may be faint, pink, or bright red in larval fishes with ages from unhatched embryos through those with complete absorption ofthe yolk sac or even older. To state in the absence of the red color that osteogenesis or bone development is not present at any of these developmental stages may be incorrect without microscopic examin- ation of tissue structure because bone may be in an early stage of development or the mineral may have been removed during fixation, clear- ing or staining steps." They further observed that fish larvae with obvious bone, often lose bone stain while in enzyme clearing solution, but that much of it remains in specimens cleared in potassium hydroxide solutions. Accordingly, they recommend that fish larvae be fixed in neutral-buffered (pH 6.5-7.2) formalin and that some be differentially stained for bone and cartilage, while others are stained for bone only and cleared with potassium hydroxide (instead of enzymes) soon after fixation. Safety Many of the chemicals and solutions used in clearing and staining can be hazardous and should be handled and disposed of accordingly. Chemicals Abbreviations for applicable procedures are: FP = fixation and preservation; BL = bleaching; IC = initial clearing, protein digestion; CS = cartilage staining; BS = bone staining; FC = final clearing and storage (fmal preservation). Alcian blue (powder) Alizarin red S (powder) Distilled water Ethanol (absolute ethanol preferred, denatured or 95% will suffice) CS if used as preservative FP, FC Formalin (saturated formaldehyde sol.) FP Glacial acetic acid CS Glycerin (glycerol), if used for storage FC Hydrogen peroxide, 3% solution BL Potassium hydroxide (KOH) BL, IC, FC, BS CS BS ALL CS,IC FP FP Sodium borate (powder) Sodium phosphate monobasic Sodium phosphate dibasic (anhydrous) Thymol (crystals), if glycerin is used for storage Trypsin powder (pancreatic protease, pan- creatin; sufficiently purified to be free of collagenase and elastase; trypsin from pig pancreas with an activity of 300 units/mg produces a clear, highly effective solu- tion, but other trypsin preparations with activities as low as 80 units/mg have also been used successfully) FC IC Stock solutions Abbreviations for applicable procedures are the same as for chemicals. 10% buffered formalin solution- In distilled water; buffer to pH 7.0 with 4.0 g sodium phosphate monobasic and 6.5 g sodium phosphate dibasic per liter of formalin solution (recommended by Taylor and VanDyke 1985), or to pH 6.8 with 4 g each of monobasic and dibasic sodium phosphate per liter of formalin solution. The latter is about twice the 1.8 g each of monobasic and dibasic per liter recommended by Markle (1984) for 5% formalin solutions. Formalin solutions can be buffered with excess marble or limestone chips or limestone powder to near neutral, but phosphate buffering is more precise and reliable; borax (sodium borate) buffered formalin is not recom- mended (Taylor and Van Dyke 1985). FP 3-5% buffered formalin solution- In distilled water; buffer with 1.8 g sodium phosphate monobasic and 1.8 g sodium phosphate dibasic per liter of formalin solution (Markle 1984). Altematively,fixed specimens can be stored in alcohol (e.g., 75% ethanol via a graded series of concen- trations), but expect greater shrinkage, deformation, and, if examined periodically, fading of melanophore pigmentation than if stored in dilute formalin solutions. FP 50% ethanol solution- In distilled water. CS And if used in graded series for spec- imen preservation (storage). FP, FC 23