BACTERIAL VACCINES 215 



A measured portion of the combined extracts is dried in vacuo and the solid 

 residue weighed to determine the amount of solids per cubic centimeter. 



Finally glycerin and water (equal parts) are mixed with the combined watery 

 extracts so that each cubic centimeter of the finished product contains 2 milli- 

 grams of solids. 



Tuberculin is made from the human tubercle bacillus and from the bovine 

 tubercle bacillus. 



In the preparation of all these tuberculins it is a customary precaution to 

 add 0.5 per cent, phenol just before placing in containers for storage or dispens- 

 ing and when diluting for use 0.5 per cent, phenol solution is the diluent 

 employed. 



B. F., B. E. and T. R., tuberculins not subjected to sterilization by heat, are 

 tested for sterility by planting on glycerin agar slants and guinea-pig injections 

 before dispensing. 



To determine whether grinding of dried bacteria has been adequate, sam- 

 ples are stained and examined microscopically and grinding continued until 

 such examinations reveal no bacteria that have escaped comminution. 



There are many tuberculins which are slight modifications of those intro- 

 duced by Koch, such as Maragliano's, basically and physiologically the same, 

 possessing slight if any advantages over the Koch products and not generally 

 employed. 



Radically different, scientifically of interest to the bacteriologist and im- 

 munologist and perhaps worthy of more extensive clinical investigation than 

 has been given it, is Von Ruck's tuberculin. 



In the Medical Record, vol. Ixxxii, Aug. 31, 1912, Karl Von Ruck describes the preparation, 

 physiological action and dosage of his tuberculin in the following words : 



"Method of Preparing. The culture of tubercle bacilli used is of human origin, grown on 

 bouillon, and has been perpetuated in my laboratory for the past 10 years; it was apparently 

 avirulent when first tested upon guinea-pigs and has continued so to the present time. On 

 the manufacture of the preparation heat has been avoided and the chemical effect of light 

 excluded. No chemicals have been introduced in kind or concentration that could injure, 

 split, reduce, or alter the several constituents. The cultures having reached their maximum 

 growth are collected upon a filter and washed free of adhering culture fluid until the filtrate 

 gives no further biurette reaction. The bacillary mass is then transferred to a glass container 

 immersed in distilled water containing 0.4 per cent, phenol, and with frequent stirring and 

 shaking it is macerated for several days, when the filtrate obtained contains the protein 

 designated as No. i; chemically examined, it shows primary proteose, 25 per cent.; secondary 

 proteose 70 per cent.; peptone, small amount; reaction acid. After further washing with dis- 

 tilled water, the bacilli are dried and powdered, when their fat extraction follows; after drying 

 they are again powdered and then partially extracted in distilled water yielding protein No. 2, 

 this showing coagulable protein, 0.03 per cent, (estimated by nitrogen) ; primary proteose and 

 deuterproteose in about equal amounts, total about 48 per cent., secondary proteose 50 per 

 cent., peptone trace, phosphorous content o. i per cent, and alkaline reaction. The bacillary 

 mass is again dried and powdered and suspended in 0.4 per cent, carbolized distilled water and 

 then ground wet in glass capsules with agate marble, until repeated microscopic examinations 

 no longer show a fragment or formed substance of the bacilli. Filtration through porcelain 

 gives protein No. 3, in solution, differing from No. 2 by absence of coagulable protein, the 

 relatively small amount of primary and increased amount of secondary proteose (75 per cent.) 



