402 THE DIPHTHERIA BACILLUS GROUP 



swab while doing so to bring every part of it in contact with the 

 medium. The serum is incubated at 37 C. for twelve to eighteen 

 hours. It is customary in many laboratories to make a preliminary 

 examination of the growth on the serum after five hours' incubation, 

 and also to make a smear from the swab itself after the serum has 

 been inoculated with it. By these preliminary examinations from 30 

 to 60 per cent, of diagnoses may be correctly anticipated. During the 

 first eighteen hours of incubation diphtheria bacilli outgrow practically 

 all other organisms. After this time the other organisms tend to out- 

 grow the diphtheria bacillus. 



Results. 1. Negative. Negative results may be due to several 

 factors: (a) the absence of diphtheria bacilli; (b) lack of care in 

 taking the culture, either failure to touch the infected membrane, or 

 making preparations immediately after the use of antiseptic gargles; 

 (c) improper smears and improper stains; (d) poor media; (e) 

 improper interpretation. 



2. Positive. Positive results do not necessarily prove that the 

 patient has diphtheria for carriers of diphtheria bacilli are fairly 

 numerous and appear to be responsible, in part at least, for the 

 spread of the disease. From 1 to 3 per cent. 1 of healthy people harbor 

 fully virulent bacilli in their mouths, and about 2 per cent, of all 

 school children in large cities have them. Positive results may also 

 be obtained with avirulent strains of diphtheria bacilli. In order to 

 determine the virulence it is necessary to isolate the organism in 

 pure culture and to inject two guinea-pigs respectively with a forty- 

 eight-hour broth culture. The isolation is best made from cultures 

 on Loffler's blood serum which microscopic examination has shown 

 to contain diphtheria bacilli. Such a culture is emulsified in broth 

 and streaked out on an agar plate, or, better, upon blood agar plates. 

 After twenty-four hours' incubation diphtheria colonies are removed 

 to plain (sugar-free) broth and incubated two days. One-half a cubic 

 centimeter of this forty-eight-hour broth culture per 100 grams weight 

 of guinea-pig is injected into Pig A, and a similar amount of the broth 

 culture, mixed prior to inoculation with an excess of antitoxin, allow- 

 ing half an hour for the antitoxin to unite with the toxin prior to 

 inoculation, injected into guinea-pig B. Guinea-pig A should die 

 in from one to five days, and an autopsy should present a typical 



1 Recent observations by Moss, Guthrie, and Gelien (Tr. XV Congress on Hyg. 

 and Demog., 1912, iv, 156) indicate that the number of carriers of virulent diphtheria 

 bacilli may greatly outnumber the actual cases of the disease. Their observations 

 showed that carriers were about four times as numerous as the cases. 



