430 THE DIPHTHERIA BACILLUS GROUP 
Results.— 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) im- 
proper smears and improper stains; (d) poor media; (e) improper inter- 
pretation. 
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^ of healthy people harbor fully 
virulent bacilli in their mouths, and about 2 per cent of all school chil- 
dren in large cities have them. Guthrie, Gelien and Moss' studying an 
outbreak of diphtheria very carefully, conclude that the diphtheria 
bacilli present in the naso-pharynx of a majority of healthy carriers are 
avirulent; that these avirulent bacilli cannot produce diphtheria, and 
that there is no proof that avirulent diphtheria bacilli become virulent. 
Positive results may also be obtained with avirulent strains of diphtheria 
bacilli. In order to determine the \'irulence 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 of the organism 
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 tw^enty-four hours' incubation diphtheria 
colonies are removed to plain (sugar-free) broth reinforced with 0.1 
per cent glucose, and incubated two days. One half a cubic centimeter 
of this forty-eight-hour broth culture per 100 gm. weight of guinea-pig 
is injected into pig A, and a similar amount of the broth culture, 
mixed before inoculation with an excess of antitoxin (allowing one- 
half hour for the antitoxin to unite with the toxin prior to inocula- 
tion) is injected into guinea-pig B. Guinea-pig A should die in from 
one to five days, and an autopsy should present a typical picture of 
diphtheria poisoning. Guinea-pig B should live because the diphtheria 
toxin is neutralized by the antitoxin. 
The diagnosis of diphtheria by serological methods is not practical. 
Dissemination and Prophylaxis.— Diphtheria bacilli are spread chiefly 
by contact or by carriers. Occasionally milk appears to be a vehicle 
of transmission. As a prophylactic agent for destroying diphtheria 
bacilli, antitoxin is one of the greatest blessings which bacteriology has 
conferred on medicine. Diphtheria antitoxin is used in two ways: (a) 
prophylactically; (6) curatively. If diphtheria breaks out in a house- 
hold or a hospital, those in immediate contact with the patient should 
' Observations by Moss, Guthrie and Gelien (Trans. XV Congress on Hyg. and 
Demog., 1912, 4, 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 numero us as the cases. 
2 Johns Hopkins Hosp. Bull., 1920, 31, 381. 
