THE DIPIirilERIA BACILLUS 429 
viiicinfi'. Small amounts of toxin have been detected in these cultures, 
but the well-recognized synthetic powers of bacteria make this line 
of evidence inconclusive. There are two current theories which receive 
serious consideration. One theory maintains that diphtheria toxin is 
enzymic in nature, the other theory assumes that the toxin is related 
to the proteins, particularly the globulins. The toxin is readily 
destroyed by exposure to light, heat, protoplasmic poisons and to peptic 
digestion, consequently moderate amounts of it may be swallowed 
without apparent harm. Acids destroy the toxin slowly, and oxidizing 
agents, as hydrogen peroxide, iodine and iodine trichloride, reduce the 
toxicity very materially. An exposure to 60° C. for ten hours, or at 
70° C. for two hours, attenuates the toxin, and it is rapidly inactivated 
or destroyed at 100° C. 
Protein precipitants, as ammonium sulphate and alcohol, precipitate 
the toxin from the broth in an insoluble state with but little reduction 
in potency. The precipitate, after dialysis to remove the salts, is 
soluble in water. A further reduction in volume and partial puri- 
fication can be attained by evaporating the broth to one-tenth its 
original volume in vacuo at a temperature not exceeding 25° C. (in 
the dark), precipitating with alcohol, filtering and dissolving in water 
and again precipitating, then drying the precipitate in racvo. The 
toxin may be precipitated with weak acetic acid also.^ Upon dialysis, 
part of the toxin passes into the dialysate. This fraction contains most 
of the amino-nitrogen, but it is low in toxic units.- A most important 
observation by Ramon, ^ that formaldehyde deprives diphtheria 
toxin of its poisonous properties, without materially lessening its 
antigenic properties, promises to change very considerably current 
methods of immunization, both of persons, and experimental animals 
as well. Toxin that is thus changed by formaldehyde is called "Ana- 
toxin." 
Physiological Action.— The chemical constitution of the toxin mole- 
cule is unknown, and toxin cannot be detected or assayed chemically. 
It provokes, however, a definite physiological response in susceptible 
animals, as guinea-pigs, and its presence is detected and its strength 
determined by injecting graduated doses into them, as mentioned 
above. From its physiological action the toxin molecule appears to 
consist of three components in varying amounts: (a) Toxin, which 
causes the acute symptoms of intoxication, parenchymatous degenera- 
tion and death when injected into susceptible animals. This fraction 
of the toxin, according to P^hrlich, has a special affinity for the antitoxin. 
(6) Toxone: the toxone causes edema at the site of inoculation and 
the postdiphtheritic paralyses. It combines with antitoxin more 
slowly than the toxin, (c) Toxoid: diphtheria toxin rather readily 
loses its toxic properties on standing, retaining its power of combining 
1 Watson and \Vallace: Jour. Pathol, and Bacteriol., 1924, 27, 289. 
' 2 Sbarskii and Yeimolcva: Bioehcm. Ztschr., 1927, 85, 419. 
^ Ann. Inst. Pasteur, 1925, 39, 1; 1926, 40, 1, 
