342 VITAL STAINING 



cent, alcohol for ten minutes. (5) Pass through graded mixtures of 95 

 per cent, alcohol and benzene in the following proportions : 9:1,8:2, 

 7 : 3, 5 : 5, 3 : 7, 2 : 8, and 1 : 9, leaving ten minutes in each. (6) Clear 

 in benzene, one or more changes for an hour or two. (7) Imbed in 

 benzene saturated with wax at 37° C. for an hour, followed by paraffin 

 at 56° C. (four changes in thirty minutes). Sections can be mounted 

 unstained or counterstained with Harris's haematoxylin. 



Silver Impregnation Method of Yamasaki {Arbeit, anat. 

 Institut kaiserlich.-jap. Univ. Seiidai, xv, 1933, p. 7). According 

 to Yamasaki neutral red droplets after fixation in alkaline-MiJller- 

 formalin are argentophilc, while trypan blue droplets are not 

 {ibid., XV, 1938, p, 19). He demonstrates neutral red droplets by 

 the following method : — 



(1) Fix in Midler's fluid, 18 c.c. ; formalin, 2 c.c. ; 40 per cent, 

 KOH, 8 drops — for twelve to twenty-four hours. (2) Wash with 

 repeated changes of distilled water for twelve to twenty-four hours. 



(3) Transfer to 0-75 per cent, silver nitrate solution for twenty-four 

 hours. (4) Wash in distilled water, then reduce for twenty-four hours 

 in hydroquinone, 1-5 gm. ; formol, 5 c.c. ; distilled water, 100 c.c. 

 (5) Wash rapidly and dehydrate in alcohols and imbed. Sections 

 should be rapidly differentiated in a 5 per cent, watery solution of 

 sodium thiosulphate. Then well washed and stained with alum 

 carmine. 



774. Hu's Technique (Proc. Soc. Expt. Biol. Med., -axi^s., 1931, p. 258). 

 Hu has taken advantage of the fact that neutral red is only slightly 

 soluble in aqueous or alcoholic solutions containing corrosive sub- 

 limate, and devised the following technique. Four solutions are first 

 prepared. 



(A) The fixing solution. Dissolve a little neutral red in 15 c.c. of 

 40 per cent, formalin in a flat dish. Add 85 c.c. of Zenker's fluid. Stir 

 with a glass rod and filter off the precipitated dye. 



(B) Dehydrating solution. To 100 c.c. of absolute alcohol add 12 c.c. 

 of the same reagent saturated with HgCla, then sufficient neutral red 

 until some of it remains undissolved at the bottom of the bottle. 



(C) Aqueous sohition. To 100 c.c. of distilled water add 2 c.c. of a 

 saturated aqueous solution of HgClg, and then saturate with neutral 

 red. Filter. 



(D) Counterstaining solution. Saturate some of solution (C) with 

 methylen blue. Filter. This must be prepared fresh each time it is 

 required. 



Method of Procedure. — (1) Fix in solution (A) blocks of tissue about 

 1 cm. square and 3-4 mm. thick for twenty-four hours. (2) Dehydrate 

 in three changes of solution, (B) one or more hours each time, according to 

 the size of the pieces. (3) Clear in two changes of xylol, three to four 

 hours in each. (4) Imbed in paraflin and mount sections in the usual 

 manner, after removing wax with xjlol. 



Counterstaining Sections. — (1) Remove wax in xylol. (2) Wash off 

 xylol with solution (B). (3) Wash off alcohol rapidly with solution (C). 



(4) Stain in solution (D) for about thirty seconds. (5) Remove excess 

 of stain with solution (C). (6) Dehydrate with solution (B). (7) Clear 

 in xylol and mount. 



768. Demonstration of New Formation in the Cytoplasm of 

 Cells after Neutral Red Staining. The Krinom (see § 745). After 

 intense staining with neutral red, a substance is formed which 



