BLOOD EXAMINATIONS FOR PROTOZOA . 1141 



the spread is made by holding its narrow end in the drop until the 

 blood has flowed between the two, and then pushing the spreader, or 

 second slide, held at an angle of about 45, toward the opposite end 

 of the first slide. The drop of blood follows closely behind the 

 spreader, and the thickness of the film may be varied by changing the 

 angle of the spreader. The thin film of blood dries quickly in the 

 air. It is never fixed in the flame, but always in methyl alcohol or in 

 the stain itself. 



Many varieties of blood stains have been proposed, but the simplest 

 and best are those of Wright and MacNeal. Wright's stain is pre- 

 pared as follows: 



Dissolve 0.5 gm. of sodium bicarbonate in 100 c.c. distilled water, 

 and add 1 gm. of methylene blue (Gruebler). Any of the methylene 

 blues of Gruebler known as "BX," Koch's or Ehrlich's rectified may 

 be used. It seems to be important that the bicarbonate of soda be all 

 dissolved before adding the methylene blue. 



The mixture is next to be steamed in an ordinary steam sterilizer 

 for one hour, counting the time after "the steam is up." The heating 

 should not be done in a pressure sterilizer, or in a water-bath, or in 

 any other way than as stated. This steaming of the alkaline solution, 

 of methylene blue effects certain changes in the methylene blue where- 

 by a polychromatic quality is given to it, so that the compound with 

 eosin, which is later to be formed with it, has the property not only 

 of differentially staining the chromatin of the malarial parasite, but 

 also of differentiating and bringing out more sharply the nuclei and 

 granules of the white blood corpuscles. 



When the steaming is completed, the mixture is removed from the 

 sterilizer and allowed to cool, the flask being placed in cold water if 

 desired. When it is cold, without filtering, pour it into a large dish 

 or flask, and. add to it, stirring or shaking meanwhile, a sufficient 

 quantity of a 1:1000 solution of eosin (Gruebler, yellowish, soluble 

 in water) until the mixture, losing its blue color, becomes purplish, 

 and a scum with yellowish metallic luster forms on the surface, while 

 on close inspection a finely granular black precipitate appears in 

 suspension. This will require about 500 c.c. of the eosin solution for 

 100 c.c. of alkaline methylene blue solution. (The proper amount 

 of eosin to add may be determined by placing a drop of mixture on 

 white filter paper, and adding eosin until the bine spot shows a dis- 

 tinct halo of pink.) 



The precipitate is collected on a filter, and, without washing, is 



