SPECIAL PROPERTIES OF THE PRINCIPAL TOXINS 113 



and then of the fore extremities, a condition analogous to the post-diph- 

 theric paralysis occurring in man and ascribed to the effects of toxon. 



Method of Testing the Virulence and Toxicity of Diphtheria Bacilli. 

 Young guinea-pigs weighing from 250 to 300 grams are quite susceptible 

 to diphtheria toxin, and are used in determining the strength of a toxin 

 and in standardizing antitoxin. The test may be of great value in the 

 management of convalescent and " carrier" cases of diphtheria, harbor- 

 ing bacilli in the upper air-passages, in determining whether the 

 microorganisms are dangerous or merely harmless non-pathogenic sapro- 

 phytes. It is practically impossible, from the morphology of the organ- 

 ism alone, to decide whether or not a given culture is dangerous, and 

 prolonged quarantine may not only be irksome and inconvenient, but, 

 if the organisms are proved to be harmless, it is unnecessary as well. 



To be reliable, however, such a test must be carried out very care- 

 fully. In the case of a highly virulent culture, the mere introduction 

 of a few organisms beneath the skin will suffice to demonstrate their 

 dangerous character, but with cultures only slightly virulent, more care 

 is necessary, for although the patient may show no ill effects as a result 

 of the presence of the bacilli, in the throat of another and less immune 

 individual they may be highly dangerous. 



The following method has been used by the author in many hundreds 

 of such tests in the Philadelphia Hospital for Contagious Diseases, and 

 has proved of distinct value: 



1. Make a culture of the part harboring the bacilli on a tube of Loffler serum 

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

 smear and stain with Loffler'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 

 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). 



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