894 EXPERIMENTAL INFECTION AND IMMUNITY 



5. Observe the animals for at least four days, especially for the development of a 

 characteristic edema about the site of injection. 



6. After death perform a careful autopsy, paying particular attention to the 

 bloody edema at the site of injection and marked hyperemia of the suprarenal glands. 

 Make cultures on Loeffler's blood-serum medium of edematous area, peritoneum, and 

 heart blood. 



(a) To what has death been due? 



(b) Has diphtheria toxin a selective affinity for any particular tissue? 



(c) Has heat any effect upon diphtheria toxin? 



(d) Is there a period of incubation before symptoms develop and why? 



EXPERIMENT 7. METHOD OF TESTING THE VIRULENCE AND TOXICITY 

 OF DIPHTHERIA BACILLI 



1. Make a culture of a patient harboring the bacilli on a tube of Loeffler's serum 

 medium. Inoculate at 35 C. for from eighteen to twenty-four hours; prepare a 

 smear and stain with Loeffler's methylene-blue. If diphtheria bacilli are present, 

 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 speci- 

 fied are more resistant and less reliable for 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 production 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. 



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

 peritoneum, and heart blood. Observe whether acute hyperemia of the suprarenal 

 glands is present. 



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

 of toxemia develop paralysis of the hindquarters two or three weeks later. According 

 to Ehrlich, this paralysis is due to the action of "toxon," a toxic substance secreted, 

 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 

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

 in the median abdominal line of a 250- to 300-gram guinea-pig. 



(a) Is the diphtheria bacillus aggressive? 



(b) What evidence have you that the lesions and death are due to a 

 toxin? 



