182 MICROMETRY AND BLOOD PREPARATIONS 



and tri-acid stains (not equal to heat) is a modification of Zenker's fluid (Whitney). 

 To Muller's fluid, which is potassium bichromate 2 grams, sodium sulphate i gram, 

 and water 100 c.c., add 5 grams of bichloride of mercury and 5 c.c. of nitric acid 

 (C. P.). Fixation is obtained in five seconds. 



When using corrosive sublimate fixation one should after thorough washing in 

 water treat the film with Gram's iodine solution for about two minutes and then wash 

 with 70% alcohol until the yellow color of the film disappears. Methyl alcohol 

 for two minutes is satisfactory. 



Staining Blood-films. As separate staining with eosin and methyl- 

 ene blue rarely gives good preparations and as the modifications of the 

 Romanowsky stain recommended are easy to make and employ, and 

 give much greater information, the separate method of staining is not 

 recommended. The most satisfactory single stain is thionin. 



Rees' Thionin Solution. Take of thionin 1.5 gram, alcohol 10 c.c., aqueous 

 solution of carbolic acid (5%) 100 c.c. Keep this as a stock solution. It should 

 be at least two weeks old before using. For use, filter off 5 c.c. and make up to 

 20 c.c. with water. 



1. Fix films (a) by heat, (b) by alcohol and ether, or (c) preferably by i% 

 formalin in 95% alcohol for one minute. 



2. Stain for from ten to twenty minutes. Wash and mount. Malarial parasites 

 are stained purplish; nuclei of leukocytes, blue; red cells, faint greenish-blue. 



Ehrlich's Triacid or Triple Stain. There are required: 



1. Sat. aq. sol. orange G. (Dissolve 3 grams in 50 c.c. water.) 



2. Sat. aq. sol. acid fuchsin. (Dissolve 10 grams in 50 c.c. water.) 



3. Sat. aq. sol. methyl green. (Dissolve 10 grams in 50 c.c. water.) 



These three solutions may be kept as stock solutions. They keep well in the dark. 

 To make the stain, add 9 c.c. of No. 2 (acid fuchsin) to 18 c.c. of No. i (orange G.). 

 After they are mixed thoroughly, add 20 c.c. of No. 3 (methyl green). Then after 

 the first 3 ingredients are well mixed, add 5 c.c. of glycerin. Mix, then add 15 c.c. 

 of alcohol; again mix, and finally add 30 c.c. of distilled water. Keep the mixed 

 stain about one week before using. The best fixatives are heat and Whitneys' 

 modified Zenker. To use, stain films from two to five minutes; then wash and 

 mount. The triacid stain is a good tissue stain. The objections to the tri-acid 

 stain are that it does not stain malarial parasites or mast cells and that failure to 

 obtain good results is of frequent occurrence. 



Wright's Method. The stain is made by adding i gram of methylene blue (Grubler) 

 to 100 c.c. of a 1/2% solution of sodium bicarbonate in water. This mixture is 

 heated for i hour in an Arnold sterilizer. The flask containing the alkaline methyl - 

 ene-blue solution should be of such size and shape that the depth of the fluid does 

 not exceed 2 1/2 inches. When cool, add to the methylene-blue solution 500 c.c. 

 of a i to 1000 eosin solution (yellow eosin, water soluble). Add the eosin solution 

 slowly, stirring constantly until the blue color is lost and the mixture becomes 

 purple with a yellow metallic luster on the surface, and there is formed a finely 

 granular black precipitate. Collect this precipitate on a filter-paper and when 

 thoroughly dry (dry in the incubator at 38 C.) dissolve 0.3 gram in 100 c.c. of pure 



