The relation of the Parasite to the tissues of the Host.
10
SKIN.—1. An unulcerated papule was removed from the thigh of an advanced
case of the disease. The tissue was fixed in absolute alcohol, embedded in paraffin
and thin sections stained by the modification of Romanowski noted in the
first report.* Examination shewed in the deeper parts of the corium a layer of
small round celled infiltration which refused to take on the Romanowski stain
and appeared to be necrotic. Scattered throughout the dermis were isolated
parasitic bodies. There were rarely more than three or four to be seen in any
one field of the microscope. They occurred close beneath the epithelium in
the dermal papillæ and to the full depth of the tissue removed (about 2 m.m. ).
At first sight the bodies appeared to have no relation to the blood vessels and to
be situated in tissue cells. Close examination shewed that such bodies were in
every case in the cytoplasm of cells which appeared to be endothelial in nature
and which I believe to be the endothelium of very fine dermal capillaries. (Pl. I.
fig. 4, and Pl. II. fig. 13.) In most cases the bodies were not surrounded by a
vacuole. Occasionally a distinct vacuole was present.
2. Thin slices of ulcer margins from autopsy 7 were fixed in absolute alcohol
embedded in paraffin and sections treated with Romanowski's stain. The base of
the ulcers to the depth of 2 . 3 m.m. was formed of vascular tissue largely composed
of young fibrous tissue cells. Single parasites were seen in the endothelium of
* NOTE.—The method referred to is as follows.
Small slabs of tissue 1 to
2
m
.m. thick are placed on cover glasses to retain their shape and left in
absolute alcohol until complete dehydration has taken place. If necessary, the alcohol should be treated
with anhydrous copper sulphate freshly prepared and several changes of the alcohol should be made.
Embed in paraffin, using xylol (15 minutes) to displace the alcohol. Leave in melted hard paraffin, 20-30
minutes. Sections are cut as thin as possible and flattened by floating on water just hot enough not to
melt the paraffin. Float sections on to a slide, remove excess of water by tapping the end of the slide and
press firmly over the sections with a piece of clean filter paper. No fixative is used. Dry above the flame
taking care not to melt the paraffin. When quite dry, melt the paraffin and treat as usual with xylol,
absolute alcohol and distilled water. Stain for 10 minutes in 1-1,000 eosin (höchst B. A. Grubler).
Pour off the excess of eosin, rapidly pour over the section some distilled water, cover with an excess of
methylene blue solution prepared according to the following formula, and allow to stain for 15-20 minutes.
It is a good plan on first adding the mythylene blue stain to keep the stain moving by tilting the slide as
this tends to prevent deposit.
100 c.c. of 1 per cent. methylene blue (pure medicinal, Grubler) solution in distilled water.
5 c.c. of a 10) per cent. solution of chemically pure sodium carbonate.
Leave in the tropical sun until a strong red colour is seen on shaking.
Dilute 25 times for use.
Pour off excess of the methylene blue and wash momentarily in 70 per cent. alcohol. After the alcohol,
plunge as rapidly as possible into distilled water. If there is still any precipitate left, repeat the process,
until the section appears quite clean under a low power.
Wash in 1 to 400 acetic acid in distilled water, and place at once after washing in distilled water.
Repeat the acetic acid bath until the tissue becomes free from blue stain and the nuclei are a clear bright red.
Allow the section to dry upon the slide. Mount in Canada balsam or allow to remain uncovered as
in the case of a film. Sections mounted in Canada balsam last two or three months but eventually lose
their colour.
The method is applicable also to tissues such as areolar tissue or muscle which can be spread upon
a slide by the half-dry method.