I020 TUBERCULIN AND THE TUBERCULIN REACTION 



state that the active principle is made up of substance of low molecular weight cor- 

 responding to profound digestion of proteins, viz., the amino acids. A few investi- 

 gators like Bieling' claim to have isolated purified substances quite free from protein 

 and still highly active. 



RECENT CHEMICAL STUDIES 



Although it has been known since the earliest report of Koch that the active sub- 

 stance is present both in the bacterial bodies themselves and in the medium on which 

 they have been grown, the latter source has been until recently most commonly used 

 in investigations on the chemistry of the active material. A great step forward was 

 taken with the use of non-protein media on which the tubercle bacillus grows well. 

 After growth of tubercle bacilli these media always become potent tuberculins. It is 

 noteworthy that at the same time protein recognizable by the usual protein tests be- 

 comes apparent in these media. 



Formulae for a number of media of this character, so-called "synthetic media," 

 have been compiled by Calmette.^ I have devised one-' similar to a number of these, 

 with certain special advantages. This contains asparagine, ammonium citrate, acid 

 potassium phosphate, sodium carbonate, sodium chloride, magnesium sulphate, ferric 

 ammonium citrate, and glycerol. Acidic and basic radicles are present in such propor- 

 tion that the resultant mixture is neutral, and in such concentration that the medium 

 is well buffered against changes in reaction resulting from bacillary growth. The me- 

 dium furnishes a yield of 100-150 gm. of moist tubercle bacilli per liter. 



In an extensive investigation fostered by the National Tuberculosis Association, 

 Florence B. Seibert and I have studied the active products obtained from this medium 

 after removal of the bacillary growth. The greater part of the chemical study was 

 carried out by Dr. Seibert. Through the generous co-operation of Parke, Davis and 

 Company and the H. K. Mulford Company, larger quantities of material than hither- 

 to possible were made available. We had previously settled quite conclusively that 

 such media invariably contain protein after luxuriant growth of the tubercle bacillus. 

 The results of a number of investigators who had reported tuberculin activity in the 

 absence of protein in media of this type were explained partly by the great delicacy 

 of the tuberculin reaction, as compared with the relatively coarse chemical tests, and 

 partly by our finding^ that glycerol, a regular constituent of such media, interferes 

 markedly with the important biuret protein reaction. 



Having determined that protein gradually accumulated in media originally pro- 

 tein free, as bacillary growth on the medium progressed and tuberculin activity de- 

 veloped, our next concern was to determine with the unusually large quantity of 

 material available whether the activity resided in whole protein molecules or in prod- 

 ucts derived from them in the course of autolysis of the bacilli growing upon the me- 

 dium. 



In different experiments the clear, bacteria-free filtrate was subjected to dialysis 



' Bieling, R. : Cenlralbl. f. Bakteriol., Abt. I, Orig., 93, 56, 73. 1924. 



' Calmette, A.: loc. cit. 



^Long, E. R.: Tubercle, 6, 12%. 1924; Long, E. R., and Seibert, F. B.: Am. Rev.Tuhcrc.,\i,2>9?>■ 

 4 Seibert, F. B., and Long, E. R.: /. Biol. Chem., 64, 229. 1925. 



