466 CORTNEBAGTERIUM 



iron content of the medium has to be carefully adjusted. (Pappenheimer and Johnson 

 1936). The initial reaction of the medium is of great importance, a fact which has been 

 emphasized by many workers (Bunker 1919, Hartley 1922, Andrewes et al. 1923, Watson and 

 LangstafF 1927). The most favourable starting reaction is at or just below pH 8-0, and it 

 is important that, during the 7 to 10 days which elapse between sowing the culture and 

 harvesting the toxin, the reaction should not swing far towards neutraMty. The range 

 over which growth of C. diphtherice takes place extends from about pH 5-7 to pH 8-7, 

 but the zone over which toxin production occurs appears to be Umited to pH 7-5-8-2. 

 The growth of the organism during its initial stages is associated with a slight production 

 of acid, probably derived in part from nitrogenous constituents of the medium. Later 

 there is a reversion in the alkaline direction, due to the spUtting-up of these organic 

 acids with the formation of carbonates. The balance between these metabolic activities 

 is in part determined by the oxygen pressure to which the culture is submitted. Partly 

 for this reason, partly perhaps for others, the shape of the flask in which the medium 

 is contained, the thickness of the layer of medium itself, and the type of plug used for 

 closing the mouth of the flask, all exert an influence on the grade of toxin produced. 

 It is also important to eliminate any movement which wiU prematurely break up the 

 veil of growth which forms at the surface of the medium. In practice it is found that 

 the best results are obtained by growing the organism in a shallow layer of medium, 

 in a cylindrical bottle which is kept lying on its side, and is plugged loosely with gauze 

 or cotton-wool (Bunker 1919, Hartley and Hartley 1922, Andrewes et al. 1923, Watson and 

 LangstafF 1927, Pope and Healey 19336). The cultures are incubated at 37° C. for 7 to 10 

 days, at the end of which time phenol, or preferably toluol, is added in sufficient strength 

 to ensure sterihzation, the flasks are allowed to stand for 24 hours, and the contents are 

 filtered. The crude toxin so obtained has now to be freed from the various non-toxic 

 substances, such as toxoids and substances derived from the bacterial cells and the medium 

 with which the pure toxin is contaminated. Progress has been made in the attempt to 

 isolate diphtheria toxin in a pure form, but complete success has not yet been achieved. 

 This is in part due to the fact that the toxin is very labUe, and is readily inactivated or 

 destroyed by heat or strong chemical reagents. It is probable that the specific material 

 of a toxic filtrate — toxin and toxoid — constitutes a very small fraction of the total con- 

 stituents of the crude filtrate (about 1 per cent, according to Glermy, 1925a). A con- 

 siderable concentration can be achieved by precipitation with weak acids, or ammonium 

 sulphate, or acetone, or by dialysis, or by fractional filtration through graded collodion 

 membranes, or by adsorption on to aluminium hydroxide followed by elution, or by a 

 combination of these methods (see Glenny and Walpole 1915, Watson and LangstafF 

 1926, Locke and Main 1928, LeuUer et al. 1931, Bunny et al. 1931, Schmidt 1931, Schmidt 

 et al. 1931, Tasman and Pondman 1931, Brandwijk and Tasman 1932, 1933, Zajdel 1932, 

 Tasman and van Waasbergen 1932, Wadsworth et al. 1932, Leonard and Holm 1933, 

 Wadsworth and Quigley 1934, Eaton and Bayne-Jones 1934, Goldie 1934). Eaton (1936), 

 however, has shown that the toxin is denatured by acid, and therefore recommends a 

 method of purification which does not necessitate the use of this reagent. 



The evidence so far obtained suggests that diphtheria toxin is a heat-coagulable 

 protein. The purest preparations contain about 16 per cent, nitrogen, 0-75 per cent, 

 sulphur, 9 per cent, tyrosine, and 1-4 per cent, tryptophan. The isoelectric point 

 is pH 4-1. The toxin is extremely sensitive to denaturation by solutions more 

 acid than pH 6 and by moderate heat. The amount of nitrogen per flocculating 

 (Lf) unit is 0-00045 mgm., and the M.L.D. for guinea-pigs is about 0-0001 mgm. 

 (Eaton 1936, Pappenheimer 1937, Pappenheimer and Eobinson 1937). It is 

 actively antigenic and, when injected into animals, stimulates the production of 

 a powerful antitoxin. It can be converted into toxoid by suitable treatment with 

 formalin ; in this state it will cause a precipitate when mixed with specific anti- 



