=IX. FAT.= When alcohol has been used in the preparation of the tissue, the fat-contents of the latter are dissolved out, and their presence can alone be told by the presence of vacuoles. When osmic acid is used as a fixing agent the oleates and oleic acid are blackened. The tissue should then be washed in running water and cut upon the freezing-microtome, or it may be imbedded in celloidin or paraffin if this is done as quickly as possible to prevent the loss of the fat. Chloroform or benzene should be used in place of xylol, as the last-named dissolves out the fat. Safranin should be used as a stain after fixation with any fluid containing osmic acid. Frozen sections are to be mounted in glycerin-gelatin; when balsam is used it should be warm melted Canada balsam without xylol. Formol fixation preserves fat, and tissues so fixed may be cut on the freezing-microtome and the sections stained with osmic acid, sudan III or scharlach R, with nuclear counterstaining when desired. For the demonstration of fat-embolism, fatty degeneration or fatty infiltration the following methods are advised:— 1. =Staining of Fat with Osmic Acid.= 1. Fix in formol for 24 hours. 2. Wash; freeze; cut. 3. Place sections in 1 per cent osmic acid, Flemming’s or Marchi’s fluid for 1-24 hours. 4. Wash in water, changing frequently. 5. 80 per cent alcohol ½-2 hours. 6. Wash in water. 7. Place section flat on slide; blot; add a drop of warmed glycerin-gelatin; cover quickly. Ringing or sealing is not necessary. Or, to mount section in balsam:— After 6, counterstain with hæmatoxylin or safranin; wash again; dehydrate quickly with absolute alcohol; clear in pure benzene; mount in pure melted Canada balsam (containing no xylol). 2. =Staining of Fat with Sudan III or Scharlach R.= Staining-solutions of these dyes may be made, as follows:— _a._ Dissolve stain in 70-80 per cent boiling alcohol, keep in the incubator over night, and use warm. _b._ Make a solution of absolute alcohol 70 cc., 10 per cent caustic soda solution 20 cc., water 100 cc. Saturate this with the stain, slightly heating. _c._ Make a mixture of 70 per cent alcohol 50 cc. and pure acetone 50 cc.; saturate this with the stain. All solutions of these dyes should be filtered before using, and should be kept covered to avoid evaporation and subsequent precipitation. 1. Formalin fixation 24 hours; cut on freezing-microtome. 2. Place sections in 70 per cent alcohol. 3. Stain in the simple solution 20-30 minutes; in the acetone or alkaline alcoholic solutions 2-3 minutes. 4. Wash in 50-70 per cent alcohol, differentiating as needed. 5. Transfer to water; thence to slide; blot, and mount in glycerin gelatin.