Appendix I.
427
contents, broken at both ends, and dropped into fluid glycerine-bouillon
and shaken carefully.
In this bouillon a culture of bacilli, which we
believed to be Leprosy Bacilli for the reasons given below, developed,
a slight turbidity occurred about the third day and a pellicle subse-
quently formed on the surface, so that a ge ne r
a t
i
on good tenacious scum was
produced by the twelfth day. In the mode of formation of this
pellicle, minute particles appeared on the surface of the bouillon,
which daily increasing in number, joined together, so that the whole
growth could be seen steadily advancing from the centre to the peri-
phery. In some cases, a fortnight later this pellicle sank partly in the
fluid. From such original bouillon cultures, gelatine and glycerine-
agar media were inoculated. The growth liquefied the gelatine and
grew also well on agar. Generations up to the sixth were carried on
at Simla from April 26th to August 20th, 1891. Out of about twenty-
five experiments (some of which failed to give any culture in the capil-
lary tube), the following may be given as examples :—
1. Blister fluid over the tubercle of Puniya, and fluid taken from a blister over
normal skin of the back of same leper, mixed in a sealed capillary tube with an air
space. Taken on April 14th, 1891, kept at body temperature until May 2nd, when
part of the contents was examined. Leprosy Bacilli brightly stained were found.
The tube was resealed, and on May 14th this was broken and dropped into
bouillon. The tubes were kept at a temperature of 26°C. On May 25th a minute
white speck was seen floating on the surface of the liquid. Two days later, a thin
film, most dense in the centre, almost covered the surface of the liquid. A small
amount of this film subsided to the bottom of the test tube. Microscopic examina-
tion showed rods which were very feebly motile ; they did not change position, but
slowly bent and returned to the straight condition. Most of the bacilli were in
clusters. In measurement and shape these were undistinguishable from Leprosy
Bacilli. Specimens stained in warm Ziehl's solution for twenty minutes and decol-
orised in 25 per cent. nitric acid, were stained pink. No beaded appearance was
seen. On May 28th two specimens of this growth were stained for twenty-four
hours in Ziehl's solution, decolorised and found to show quantities of brightly
stained bacilli, separate and in clusters. On May 29th the bouillon was covered
with a coherent pellicle. By June 1st the pellicle had in great part subsided en
masse to the bottom of the tube.
Cultures in glycerine-bouillon, glycerine-gelatine, and glycerine-agar were made
from this original growth and carried on for several generations. The growth in
gelatine shows on the second day a minute white speck on the surface, and subse-
quently a liquefaction takes place along the needle track. This appearance is
represented in Fig. 2, Plate II. Subsequently two strata of growth form, separated "
by a clear liquid space. The microscopical appearance of these strata is identical
as far as staining with the Ziehl method is concerned ; both consist of bright red
rods, but those from the upper stratum are slightly thicker than those from the
lower one.
The growth on agar presents an irregular outline at the edge of the needle
stroke, and this extends rather rapidly, so that a great part of the surface is covered.
Its subsequent spread is, however, exceedingly slow.