2l8	PATHOGENIC BACTERIA.
The best method of obtaining a culture is to inoculate
a guinea-pig with tuberculous material, allow an artificial
tuberculosis to develop, kill the animal after a couple of
months, and make the cultures from the centre of one of
the tuberculous glands.
Of course many technical difficulties must be over-
come. The tuberculous material used for inoculaticm
may be sputum, injected beneath the skin by a hypo-
dermic syringe. The animal is allowed to live for a
month or six weeks, then killed. The autopsy is per-
formed according to directions already given. A large
lymphatic gland with softened contents or a nodule in the
spleen being selected for the culture, an incision is made
into it with a sterile knife, or a rigid sterile platinum
wire is introduced; some of the contents are removed
and planted upon blood-serum. After receiving the in-
oculated material the tubes are closed, either by a rub-
ber cap placed over the cotton stopper, which is cut off
and pushed in, or by a rubber cork above the cotton,
the idea of this rubber corking being simply to prevent
evaporation. The tubes must be kept in an incubator
at the temperature of 37-38° C.
Kitasato has published a method by which Koch has
been able to secure the tubercle bacillus in pure culture
from sputum. After carefully cleansing the mouth the
patient is allowed to expectorate into a sterile Petri dish.
By this method the contaminating bacteria from the
mouth and the receptacle are excluded, and the expecto-
rated material is made to contain only such bacteria as
were present in the lungs. The material is carefully
washed a great many times in renewed distilled sterile
water until all bacteria not enclosed in the muco-purulent
material are removed; it is then carefully opened with
sterile instruments, and the culture-medium—glycerin
agar-agar or blood-serum—is inoculated from the centre.
Kitasato has been able by this method to demonstrate
that many of the bacilli ordinarily present in tubercular
sputum are dead, although they continue to stain well.