406 BACILLI IN CHRONIC INFECTIOUS DISEASES. 



3. Its active ingredients are of the nature of albumoses, alkaloidal sub- 

 stances, and extractives. The action of these is in certain instances antag- 

 onistic. 



4. Its remedial and inflammatory actions are connected with the presence 

 of certain of its albumoses, while its fever producing properties are chiefly 

 associated with substances of non-albuminous nature. 



5. The albumoses are not lost by dialysis ; the latter are. By the adoption 

 of suitable methods it is thus possible to remove the substances which cause 

 the fever, while retaining those which are beneficial in their action. 



6. The fever produced by tuberculin is thus absolutely unessential to its 

 remedial action. 



In a communication dated October, 1891, Koch 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 considerable quantities of the crude 

 product the tubercle bacillus is cultivated in an infusion of calves' 

 flesh or of beef extract to which one per cent of peptone and four to 

 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 centi- 

 metres. 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 development 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 fil- 

 tered through porcelain. The crude tuberculin obtained by this pro- 

 cess contains from forty to fifty per cent of glycerin, and consequently 

 is not a suitable medium for the development of saprophytic bacteria, 

 if they should by accident be introduced into it. It keeps well and 

 preserves its activity indefinitely. 



From this crude tuberculin Koch has obtained a white precipitate, 

 with sixty-per-cent alcohol, which has the active properties of the 

 crude tuberculin as originally prepared. This is fatal to tuberculous 

 guinea-pigs in doses of two to ten milligrammes. It is soluble in 

 water and in glycerin, and has the chemical reactions of an albu- 

 minous body. In preparing it one and a half volumes of absolute 

 alcohol are added to one volume of the crude tuberculin, and, after 

 stirring it to secure uniform admixture, this is put aside for twenty-four 

 hours. At the end of this time a flocculent deposit will be seen at the 

 bottom of the vessel. The fluid above this is carefully poured off, 

 and an equal quantity of sixty-per-cent alcohol poured into the vessel 



