11

          placing the films first in the eosin solution and then in the methy-
          lene blue solution as by mixing the two solutions together before
          pouring them on the slide.

     So far as I am aware, it has not yet been found possible to stain organisms
in sections by Romanowsky's method. Treated in the ordinary way, sections
stain merely with the methylene blue, and the nuclei show no trace of the charac-
teristic red body of the Romanowsky stain. Films treated first by alcohol, xylol,
and the paraffin bath, still stain quite readily, so that the reason why sections do
not stain appears to be a physical one. Stephens found that Romanowsky staining
was developed when films were placed first in eosin and then in the methylene
blue solution, and we were accustomed to use this method when dealing with large
numbers of films. By employing this method of saturating sections first with eosin
and then placing them in the methylene blue solution I have been able to stain the
nuclei and included parasites with the characteristic red of Romanowsky's stain.
The following is an account of the method.

     Fix the tissues in absolute alcohol or saturated aqueous solution of perchl-
oride of mercury. Use small slabs 1 to 2 millimetres thick placed upon paper so
that they may harden flat. Remove carefully all adherent paper fibre and embed
in paraffin, using, after complete dehydration (1 to 4 hours in absolute alcohol)
only 15 minutes in xylol and at most 20 minutes in hard paraffin.

     Cut thin sections. Flatten by floating out on water not quite hot enough
to melt paraffin. Float on to a slide. Remove the excess of water and press firmly
with filter paper. No fixative is necessary if this be done. Dry a few seconds
at a height above the flame just sufficient not to melt the paraffin. Melt the
paraffin and treat as usual with xylol, absolute alcohol, and water.

     Stain for 10 to 15 minutes in 1—1,000 eosin (Höchst, B. A. Grübler).

     Pour off the excess of eosin and press the section with filter paper. Add
an excess of the methylene blue solution prepared according to the following
formula and allow to stain for 15-20 minutes.

     100 c. c. of methylene blue (pure medicinal, Grübler) solution in distilled
          water.

     5 c. c. of a 10 per cent. solution of chemically pure sodium carbonate.
     Leave in tropical sun until a strong red colour is seen on shaking.
     Dilute 25 times for use.

     Pour off the excess of stain and wash rapidly in 70 per cent. alcohol. The
washing in alcohol should be quite momentary. If the washing is too prolonged
too much red colour will be removed, and if too short some precipitate will be left,
Transfer as rapidly as possible to water.

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