362 PROTECTIVE INOCULATIONS. 
are redissolved by hot water containing sodium chloride and very 
dilute potash solution. The elementary analysis gave N 5.90 per 
cent, C 35.19 per cent, and H 7.02 per cent. The results obtained 
are believed to show that the active substance present in the lymph 
is a toxalbumin. In experiments made with Koch’s lymph in Pas- 
teur’s laboratory by Bardach, a very decided elevation of temperature 
was produced in tuberculous guinea-pigs by the subcutaneous injection 
of 0.1 gramme, and a fatal result by the injection of 0.2 to 0.5 gramme. 
In man a decided febrile reaction is produced in tuberculous patients 
by very much smaller doses—0.001 cubic centimetre. 
Hammerschlag, in his chemical researches, found that the tubercle 
bacillus yields a larger proportion of substances soluble in alcohol and 
ether than any other bacilli tested (twenty-seven per cent). The alco- 
holic extract contains fat, lecithin, and a toxic substance which pro- 
duces convulsions in rabbits and guinea-pigs. The portion insoluble 
in alcohol and ether contains cellulose and an albuminoid substance. 
No ptomains were found, but a toxalbumin was isolated, which 
caused an elevation of temperature in rabbits of 1° to 2° C., lasting 
for a day or two. 
Koch (1891) has given a full account of his method of preparing 
crude tuberculin, and also the process by which he obtains from this 
a tuberculin which appears to be pure, or nearly so. To obtain con- 
siderable quantities of the crude product the tubercle bacillus is culti- 
vated in an infusion of calves’ flesh, or of beef extract to which one 
per cent of peptone and four or five per cent of glycerin have been 
added. This culture liquid must be made slightly alkaline, and it 
is placed in flasks with a flat bottom, which should not be more than 
half filled—thirty to fifty cubic centimetres. The inoculation is made 
upon the surface with small masses from a culture upon blood serum 
or glycerin agar. By accident Koch discovered that these masses 
floating upon the surface give rise to an abundant development, and 
to the formation of a tolerably thick and dry white layer, which finally 
covers the entire surface. At the end of six to eight weeks develop- 
ment ceases, and the layer after a time sinks to the bottom, breaking 
up meanwhile into fragments. These cultures, after their purity has 
been tested by a microscopical examination, are poured into a suitable 
vessel and evaporated to one-tenth the original volume over a water 
bath. The liquid is then filtered through procelain. The crude tu- 
berculin obtained by this process contains from forty to fifty per cent 
of glycerin, and consequently is not a suitable medium for the develop- 
ment of saprophytic bacteria, if they should by accident be introduced 
into it. It keeps well and preserves its activity indefinitely. 
