WASH-OUT RECOVERY METHOD 



377 



WASHING 



tails about washing see the individual 

 fixatives. 



Wash-out Recovery Method (WO)— Written 

 by C. C. Macklin, Dept. of Histological 

 Research, The University of Western 

 Ontario, London, Canada. November 

 28, 1951 — A cannula loaded with physio- 

 logical saline, serum or other medium 

 is tied into the trachea of a mouse or 

 other mammal and the contents in- 

 jected into the fresh lung, withdrawn, 

 and examined as fresh or dried and 

 stained mounts. For most animals it 

 is convenient to wash out a part of a 

 lung through a cannulated bronchus. 

 Granular alveolar and phagocytic al- 

 veolar cells (pneumonocytes) are so 

 recovered. (Macklin, C. C, Proc. 6th 

 Intern. Cong. exp. C.ytology, Stock- 

 holm, 1947; published' 1949," 383-387). 

 See Dust Cells, Foam Cells, Vacuoloids. 



Wasserblau, see Brazilin-Wasserblau. 



Water Absorption bj' slices of liver. The 

 method has been standardized by Sperry 

 and Brand (W. M. and F. C, Proc. Soc. 

 Exp. Biol. & Med., 1939, 42, 147-150) 

 and may prove useful in the investiga- 

 tion of other tissues. 



Water Blue (Wasserblau), see Anilin Blue. 



Wear and Tear pigment, see Lipofuscin. 



Weigert Method. For myelin sheaths. 

 Kultschitzky modification (Romeis, B. 

 Taschenbuch der mikroskopischen tech- 

 nik, ii Auflage Section 999, p. 332). Fix 

 in 10% formalin and mordant in Miil- 

 ler's Fluid, or in Formalin Miiller or in 

 Weigert's Quick Mordant. Bring par- 

 affin or celloidin sections to water. Im- 

 merse in 3% aq. potassium bichromate or 

 in Miiller's fluid 12 hrs. Stain for 12- 

 24 hrs. in : 10% hematoxylin in abs. ale. 

 (1-6 months old), 10 cc; aq. dest., 100 

 CO. Wash and destain in: aq. lithium 

 carbonate, 100 cc. ; 1% aq. potassium 

 ferricyanide, 10 cc. until clear differen- 

 tiation appears between gray and white 

 matter. Wash, dehydrate and mount. 

 The following is provided by Dr. J. 

 L. O'Leary : Mordanting in the Weigert 

 procedure serves two purposes : (1 ) It 

 'renders the myelin sheath components 

 insoluble in the fat solvents necessary 

 to secure dehydration and imbedding. 

 (2) It distributes the chromate ion in 

 sufficient concentration in the myelin 

 sheaths to ensure the formation of an 

 adequate lake with hematoxylin in the 

 subsequent staining procedure. If par- 

 affin imbedding is to be used, it is abso- 

 lutely necessary to carry block mordant- 

 ing to the point where the myelin of all 

 fibers has been rendered insoluble. For 

 this reason paraffin imbedding of mate- 

 rial to be used for Weigert staining 

 should be restricted to small nerves and 

 thin pieces of spinal cord, otherwise 



overhardening results. Here excellent 

 results are to be achieved, the smallest 

 fibers staining as completely as by the 

 osmic acid method. Two methods are 

 applicable to paraffin imbedded sections, 

 the procedures for which are given sub- 

 sequently. These are : the Kultschitzky 

 modification of the Weigert method and 

 technique for routine diagnostic work 

 by Craig, p. 55. To make the stain 

 dissolve 1 gm. hematoxylin crystals in 

 300 cc. aq. dest. with aid of a little heat 

 and add 100 cc. sat. aq. ammonium alum 

 with a crystal of thymol. Allow to 

 ripen 10 days in flask stoppered with 

 cotton; then keep in dark. Fix smears 

 in Schaudinn's Fixative 5-10 min. 

 Wash thoroughly in several changes 

 aq. dest. Immerse in above hematoxy- 

 lin solution 3-5 min. Then pass 

 through 50, 60, 70, 90 and 95% alcohol, 

 at least 5 min. each. After immersing 

 in absolute 10 min. clear in xylol and 

 mount in xylol balsam. 



Warburg's Respiratory Enzyme, see Cyto- 

 chrome-Oxidase. 



Warthin-Starry method for spirochaetes in 

 sections has been modified by Faulkner, 

 R. R. and Lillie, R. D. Stain Techn., 

 1945, 20, 81-82 by the use of a buffered 

 solution. Use Walpole's buffer : 18.5 cc. 

 of solution of 11.8 cc. acetic acid in 

 1000 cc. aq. dest. + 1.5 cc. of solution 

 of 16.4 gm. sodium acetate in 1000 cc. 

 aq. dest. which gives pH of 3.6. 1. 

 Pass paraffin sections through xylol and 

 alcohols to aq. dest. buffered to pH 3.6 

 by addition of 20 cc. of above buffer to 

 480 cc. aq. dest. 2. Impregnate 45 min. 

 at 55-60°C. in paraffin oven in 1% aq. 

 silver nitrate similarily buffered. 3. 

 Place slides sections up on glass rods 

 pour on developer previously warmed 

 to 55-60°C. This developer is made by 

 heating 15 cc. 5% aq. gelatin in above 

 buffered aq. dest. and just before using 

 add 3 cc. 2% aq. silver nitrate and 1 cc. 

 3% aq. hydrochinone also made up in 

 the same buffered solution. While de- 

 veloping avoid direct sunlight and cold 

 drafts. Continue 3-5 min. until sec- 

 tions become golden brown or grayish 

 yellow and developer starts to turn 

 black. Pour off, rinse with warm 55- 

 60°C. tap water and then with aq. dest. 

 4. Dehydrate, clear and mount in xylene 

 clarite or balsam. Spirochaetes black. 

 Recommended for syphilitic lesions, 

 yaws and Vincent's stomatitis. 



Washing. The surplus of most aqueous 

 fixatives is removed by washing the tis- 

 sue in water. In the case of Zenker's 

 fluid, for example, wash for 12-24 hrs. 

 in running tap water. A convenient 

 way is to cover the wide mouth of a 

 bottle containing the tissue with gauze 



