THE SERUM TREATMENT OF MENINGOCOCCUS MENINGITIS 793 



unmistakable phagocytic activity in dilutions up to 1 : 5000. The method of Neuf eld 

 is used, as described on page 203. 



2. Complement-fixation Tests. The advantages of these tests are that the same 

 polyvalent antigen may be used as is employed for purposes of immunization; the 

 technic is simple, and the reactions are usually sharp and definite. According to 

 Sophian, in a series of comparisons with opsonic and complement-fixation tests the 

 results corresponded in every instance, a high opsonic content being accompanied by 

 a high complement-fixation reading. The latter indicates, at least, that the horse has 

 responded to immunization and that curative antibodies probably are present. Labor- 

 atories usually adopt their own standards in preparing antimeningococcic serum. In 

 the complement-fixation test Kolle requires complete inhibition of hemolysis with 

 0.1 c.c. of serum. 



Kitchens and Hansen 1 have advocated a dried meningococcus antigen prepared 

 as follows: 



1. The cultures are grown on salt-free agar at 37 C. for sixteen to eighteen hours. 



2. The growth is collected and suspended in distilled water; about 10 c.c. dis- 

 tilled water being used for each 20 square inches of agar surface. 



3. To this suspension is added an equal volume of 95 per cent, ethyl alcohol. 



4. This mixture is immediately centrifugalized. 



5. After the supernatant fluid has been removed the precipitated bacteria are 

 again suspended in 95 per cent, ethyl alcohol. 



6. This process of centrifugalization and resuspension is repeated a second and 

 a third time, but with ethyl ether instead of alcohol, the original volume of the 

 suspension being reduced one-half each time. 



7. The ether adhering to the bacteria after the precipitation and after removal 

 of the supernatant ether, is removed by vacuum. 



8. The bacterial mass now constituting the antigen is further dried over phos- 

 phorus pentoxid in vacuo for three days. 



9. The antigen is stored in tubes about 0.05 gram to each and kept in vacuo 

 over phosphorus pentoxid. 



For use, a small amount of the powder, 0.02 gram, is carefully ground in a mortar, 

 20 c.c. of normal salt solution being gradually added. 

 The technic of these tests is given on page 524. 



3. Agglutination Tests. These tests are readily conducted with the polyvalent 

 antigen used in immunization, a macroscopic technic, as that described on page 301, 

 being employed. Kolle regards a serum as satisfactory when it causes agglutination 

 of meningococci in dilutions. up to 1: 5000. 



4. Animal Inoculation Tests. These tests have been found quite irregular and 

 impracticable for general use. As stated by Jobling, not only does the pathogenicity 

 of the meningococcus vary considerably from day to day, but the resistance of animals 

 to this microorganism is also quite variable. By preparing a large quantity of bac- 

 terial emulsions and using sufficient controls to determine the fatal dose, and by 

 employing a standard lethal dose of emulsions mixed with varying quantities of 

 serum injected intraperitoneally into 250-gram guinea-pigs, some conception of the 

 protective value of a serum may be obtained. 



Hitchens and Robinson 2 have recently perfected an animal inoculation test which 

 promises the best means for testing the potency and standardizing a serum. The 

 M. L. D. of mixed cultures of meningococci is determined by washing off sixteen- 

 hour growths of each culture on slants of serum-dextrose agar with 1 c.c. of a 1 : 4 

 dilution of fresh guinea-pig serum. The emultions are mixed and 0.5, 0.25, 0.12, 

 0.06, and 0.03 c.c. injected intraperitoneally into white mice. The M. L. D. is the 

 smallest lethal dose at the end of forty-eight hours. In testing a serum 0.5 c.c. vary- 



1 Jour. Immunology, 1916, 1, 355. 2 Jour. Immunology, 1916, 1, 345. 



