4

     This solution should be allowed to stand, in a corked bottle in a hot sun
for one or two days (or in an incubator for a week), until the blue has acquired
a red tinge when looked at through the bottle held up to the light.

     Stock solution, B :—

     This solution should be kept in a dark place; if exposed to the sun it
will fade.

     A and B are stock solutions. For use each of them has to be diluted
twenty-five times, that is, 4 C. C. of each should be added to 100 C. C. of
distilled water. These diluted solutions (C and D) are kept in separate bottles.

     The slides to be stained are placed, after previous fixing in absolute alcohol,
in a flat porcelain or glass dish, film surface upwards. Equal parts of the
dilute solutions (C and D) are taken (10 C. C. of each are usually sufficient for
two or three slides in a flat dish), and poured simultaneously over the slides so
that they mix at once. The dish is then rocked backwards and forwards, as is
done in the development of photographic plates, for from fifteen minutes to one
hour, during which time a red precipitate will form on the sides of the porcelain
dish. The time required for staining varies greatly with different solutions, but
a few trials will indicate the time which any particular solution takes.

     In order to ascertain when the films are sufficiently stained they should be
removed from the solution from time to time and examined while wet (without
previous washing in water) with a ¼th or 1/6th inch objective. If the blood plate-
lets and the nuclei of the leucocytes are a deep ruby red, the slides are sufficiently
stained, and the parasites will show up well. They are then washed for a few
moments in distilled water and allowed to dry. Too much washing decolourizes
the film. When dry, the slides are examined under the oil-immersion lens with-
out applying a cover-glass.*

     In well stained films the red blood corpuscles should be a transparent
pink colour, the body of the parasites a light blue, and the chromatin a deep red.
To obtain correct staining it is sometimes necessary to alter the proportions of
the stains; for example, better results are often obtained by using three or even
four parts of the eosin solution to two of the methylene blue, but these details
can be easily worked out by a few trials.

     It happens, however, with some solutions that in spite of alterations in the
relative proportions of the stains, and alterations in the time of staining, the
red blood corpuscles remain stained a more or less deep blue colour. This,
however, is of little or no consequence, provided that the nuclei of the leucocytes

     * An exceedingly useful and easily worked "Romanowsky" stain in a single solution is that recom-
mended by Major W. B. Leishman, R.A.M.C., in the "British Medical Journal" for September 21st, 1901,
page 757.