136 LABORATORY BACTERIOLOGY 
Films are made from the organs or blood and fixed in the 
usual manner. They are stained with alkaline methylene blue 
the same as those of bacteria. 
Staining blood films for animal parasites (protozoa). Several 
methods of staining blood films to demonstrate blood parasites 
are in use. Alkaline methylene blue stains most, if not all, 
parasitic protozoa, but the following (Nocht-Romanowsky stain) 
gives very satisfactory results. 
Preparation of the-stain. To 1 ounce of polychrome methy- 
lene blue (Griibler) add a 3% solution of acetic acid (U.S.P., 
33%), drop by drop, until it no longer turns red litmus paper 
blue above the zone coming into immediate contact with the 
dye. It usually requires about 5 drops. 
2. Make a saturated (1%) watery solution of methylene blue, 
preferably Ehrlich’s (Griibler) or Koch’s, dissolving the dye 
by gentle heat. This solution improves with age and should 
be at least 1 week old. 
3. Make a 1% watery solution of Griibler’s watery eosin. 
The mixture is prepared as follows : 
To 10 cc. of water add 4 drops of eosin solution, 6 drops 
of neutralized polychrome blue, and 2 drops of 1% methylene 
blue, mixing well. 
Wright’s stain is excellent for this work. It can be bought 
in the market. This is also true of Jenner’s stain. 
Preparation of films for the staining of protozoa in the blood. 
The blood should be drawn from the skin rather than from the 
larger veins. It is obtained by shaving the surface, cleansing 
it, and making a small incision with a scalpel or lance. Apply 
the edge of one end of a glass slide to a small drop of blood 
and place this edge on the surface of another slide resting on 
a firm surface. As soon as the drop of blood has spread along 
the line of contact, holding the slide at an angle of about 30°, 
draw it gently along the surface of the receiving slide, spread- 
ing the blood in a thin film. Allow the films to dry in the air 
before fixing. 
