88 STAINING TECHNIQUE 
steamed in an Arnold steam sterilizer for one hour. The steaming 
of the alkalin solution of methylene blue effects certain changes in 
the methylene blue whereby a polychromatic property is given to it 
so that the compound with eosin, which is later to be formed with it, 
has not only the property of differentially staining the chromatine 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. When it is cold, without filtering, pour 
it into a large dish or flask and add to it shaking or stirring meanwhile 
a 1 to 1000 solution of eosin (Grubler, yellowish, soluble in water suffi- 
cient in quantity, until the mixture losing its blue color becomes pur- 
ple in color, and a scum with yellowish metallic luster form 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 the alkalin methylene blue solution. 
The precipitate is collected on a filter and allowed to dry thereon 
without washing. When thoroughly dry a saturated solution in pure 
methyl alcohol is made. Thrce-tenths of a gram of the dry substance 
will thoroughly saturate 100 c.c. of the methyl alcohol in a few minutes. 
The saturated alcoholic solution of the precipitate is next filtered 
and to the filtrate is then added 25 per cent of methyl alcohol; e.g., 
to 80 c.c. of the saturated alcoholic solution 20 ¢c.c. of methyl alcohol 
are added. 
This somewhat diluted solution of the precipitate is the staining 
fluid. It is permanent and may be kept on hand. Care should be 
taken to prevent evaporation of the alcohol so that the fluid may not 
become too saturated. The object of the dilution is to prevent precipi- 
tations on the blood film in the process of staining. , 
II. The Staining of the Blood Films. The films of blood which are 
spread thinly are allowed to dry in the air. As much of the staining 
fluid is poured upon the film as the cover glass or slide will readily 
hold without draining off. Allow the staining fluid to remain in con- 
tact with the film for one minute. Next, add to the staining fluid 
on the cover glass or slide, sufficient water drop by drop, until it becomes 
semi-translucent and a reddish tint becomes visible at its margin, 
while a metallic scum forms on its surface. The amount of water 
required will vary with the amount of staining fluid on the preparation. 
Generally 8 or 10 drops will be required if « seven-eighth square cover 
glass is used. The staining fluid thus diluted should be allowed to 
remain on the preparation for 2 or 3 minutes, during which time the 
