SPECIAL PROPERTIES OF THE PRINCIPAL TOXINS 115 



they must be isolated in pure culture. Never attempt a guinea-pig test with an 

 impure culture! 



2. Isolate by the "streak" method, on plates of blood-serum. 



3. Inoculate a tube of 1 per cent, glucose bouillon, which is neutral or slightly 

 alkaline, with several different colonies. 



4. Incubate at 35 C. for three days, keeping the tube in a slanted position in 

 order to give the culture as much oxygen as possible. If a good growth does not 

 appear in twenty-four hours, transplant to another tube of bouillon until the bacilli 

 have been "educated" to grow on the medium. 



5. Examine for purity. ' Select a 250- to 300-gram guinea-pig and inject 2 c.c. 

 of the unfiltered culture in the median abdominal line. Animals over the weight 

 specified are more resistant and less reliable for the test. The unfiltered culture is 

 used, since toxin is but one element of the disease-producing power of diphtheria 

 bacilli, and toxin production in bouillon may not be a true index of the toxin produc- 

 tion in mucous membranes. 



6. Carefully observe the animal for at least four days. Even slight toxemia, 

 especially if accompanied by edema at the site of injection, should be regarded as a 

 positive result (Fig. 37). 



7. After death perform a careful autopsy. Make cultures of the edematous 

 area, peritoneum, and heart blood. Diphtheria bacilli may be found in the edema- 

 tous fluid, but will rarely be found in the peritoneum or in the blood. Observe whether 

 acute hyperemia of the suprarenal glands is present (Figs. 37 and 38). 



8. Not infrequently animcls showing mild or even an absence of the symptoms 

 of toxemia develop paralysis cf the" hind quarters two or three weeks later. Accord- 

 ing to Ehrlich, this paralysis is due to the action of "toxon, " a toxic substance se- 

 creted by the bacillus or, as believed by others, a modified form of toxin. 



9. To prove that diphtheria was the cause of the toxemia or death mix 2 c.c. of 

 the culture in a test-tube with 1 c.c. of diphtheria antitoxin (500 units) . After stand- 

 ing aside for an hour at room temperature, inject the mixture subcutaneously in the 

 median abdominal line of a 250 to 300 gram guinea-pig. Symptoms of toxemia do 

 not develop. 



In a comparative study of the above and other methods Kolmer 

 and Moshage 1 found that washing off a pure culture from a slant of 

 Loeffler's blood-serum media with 10 c.c. of sterile salt solution, emulsi- 

 fying and injecting 4 c.c. subcutaneously in the medium abdominal line 

 of a 250- to 300-gram guinea-pig, yielded equally delicate results. The 

 intracutaneous method of Neisser which has also been advocated by 

 Zingher and Soletsky, 2 while being more economical in that 2 pigs suffice 

 for 4 or even 6 tests, was found to yield somewhat indefinite reactions 

 with bacilli of low virulence. 



Standardizing Diphtheria Toxin. The strength of a diphtheria toxin 

 is estimated by injecting subcutaneously a series of guinea-pigs weighing 

 approximately 250 grams, with decreasing amounts of toxin. How 

 many dilutions will be necessary it is impossible to state; for exact results 

 several pigs of the same weight should be inoculated with the same dose, 

 and the effects should show various gradations, dependent upon the 



1 Jour. Infect. Dis., 1916, 19, 1. 2 Jour. Infect. Dis., -1915, 17, 454. 



