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bone mineral is usually indicated by staining with alizarin Red S. This color may be faint, pink, or bright red in larval fishes with a~es from unhatched embryos, through those WIth complete absorption of the 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 examination of tissue structure because bone may be in an early stage of development or the mineral may have been removed during fIXation, clearing 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 differen- tially 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 hazar- dous and should be handled and disposed of accordingly. Chemicals: (applicable procedures: FP = fIX- ation and preservation; BL = bleaching; CL = clearing, protein digestion; CS = cartilage staining; BS = bone staining.) Alcian blue (powder) Alizarin red S (powder) Distilled water Ethanol (absolute ethanol preferred, denatured or 95% will suffice) CS If used as preservative FP, CL Formalin (saturated formaldehyde sol.) FP Glacial acetic acid CS Glycerin (glycerol) If used as preservative CL Hydrogen peroxide, 3% solution BL Potassium hydroxide (KOH) BL, CL, BS Sodium borate (powder) CS, CL Sodium phosphate monobasic FP Sodium phosphate dibasic ( anhydrous) FP Thymol (crystals) If glycerin is used CL Trypsin powder (pancreatic protease, pancreatin; 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 solution, but CS BS ALL other trypsin preparations with activ- ities as low as 80 units/mg have also been used successfully) CL Stock Solutions: (applicable procedures as above.) 10% buffered formalin solution In distilled water; buffer to pH 7.0 with 4.0 g sodium phosphate mono- basic and 6.5 g sodium phosphate dibasic per liter of formalin solution (recommended by Taylor and Van Dyke 1985), or to pH 6.8 with 4 g each of monobasic and dibasic sod- ium 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 solu- tions. Formalin solutions can be buffered with excess marble or lime- stone chips or limestone powder to near neutral, but phosphate buffering is more precise and reliable; borax (sodium borate) buffered formalin is not recommended (Taylor and Van Dyke). 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). Alternatively, fIXed specimens can be stored in alcohol (e.g, 75% ethanol via a graded series of concentra- tions), but expect greater shrinkage, deformation, and, if examined peri- odically, fading of melanophore pig- mentation than if stored in dilute formalin solutions. FP 50% ethanol solution In distilled water. CS If used in graded series for specimen preservation (storage). FP, CL 75% (or 70%) ethanol solution . In distilled water if used for specI- men preservation. FP, CL Alcian blue stain solution 20 mg alcian blue per 100 ml of 30% glacial acetic acid in ethanol (solution will keep at room temperature for 3-4 weeks). CS Saturated sodium borate solution Excess sodium borate powder m I j 20