54 THE PRINCIPLES OF IMMUNOLOGY 



cated by demonstrating antitoxin in the blood. The methods of 

 demonstration were not easily applicable until the development 

 of the Schick test. This test is performed by injecting intra- 

 cutaneously one-fiftieth of the minimum lethal dose of a specially 

 prepared toxin contained in 0.2 c.c. of salt solution. Injection is pref- 

 erably on the flexor surface of the arm or forearm. Six-day broth 

 cultures of the organism are killed with phenol, sedimented in the 

 ice-box for two or three days, the supernatant fluid filtered through 

 a Berkefeld candle, and the clear filtrate accurately standardized. It 

 is well to keep this filtrate for several months or a year, so that its 

 rate of deterioration is reduced to a minimum. A control injection 

 is given with the same quantity of toxic broth heated to 75 C, so 

 as to destroy the toxin. 



The reactions to injections may be as follows: 



A. Positive reaction. This indicates that no antitoxin is present in the 

 body, thereby permitting the toxin to act upon the unprotected cells. Slight 

 reaction appears in from twelve to twenty-four hours in the form of redness, 

 which becomes more distinct in from twenty-four to forty-eight hours, reach- 

 ing its maximum on the third or fourth day, then gradually disappearing and 

 leaving an area of scaling and brown pigmentation. The area attains a 

 diameter of 10 to 20 mm., and varies in intensity, depending on the sensitive- 

 ness of the individual. 



B. Negative reaction. If no distinct reaction appears any more than is 

 seen in the control area the failure to react indicates that an amount of anti- 

 toxin is present in the body sufficient to neutralize the introduced toxin. Such 

 a reaction in a child of about three years of age probably indicates perma- 

 nent immunity. By varying the quantity of toxin injected, the amount of 

 antitoxin can be titrated. 



C. The pseudo-reaction. This is usually urticaria] in nature, appearing some- 

 times immediately and sometimes in from six to eighteen hours, reaching its 

 maximum on the third or fourth day. It fails to leave pigmentation after it 

 subsides. This is a reaction of hypersusceptibility to the protein substances 

 present in the toxic broth as the result of the autolysis of the diphtheria 

 bacilli, and is in nature the same as other reactions of hypersusceptibility 

 described subsequently (see page 236). Such a pseudo-reaction may intensify 

 the true reaction and represent a summation of the protein reaction and a 

 reaction to the toxin. This must be taken to indicate that an individual may 

 be hypersensitive to the proteins of diphtheria bacilli but at the same time 

 not possessing in his circulating blood any antitoxin. The differentiation 

 depends upon the difference between the reaction at the site of the test 

 injection and at the site of the control injection. 



Zingher divides the positive reactions as follows: -f -f indicates a strong 

 positive reaction with marked local redness, infiltration, and occasionally super- 

 ficial vesiculation ; -|- indicates positive reaction with redness but little or no 

 infiltration; indicates moderately positive reaction with moderate degree of 

 redness and no local infiltration ; i indicates a faintly positive reaction with only 

 slight redness and no local infiltration. 



The test has been found to be of great value in determining the 

 immunity of groups of individuals, particularly in institutions 

 where there has been exposure to diphtheria. It has also given con- 

 siderable information as to the incidence and duration of this variety 

 of active immunity. Immunity to diphtheria may be derived from 

 the mother and lasts for about six months after birth. The largest 

 number of positives is found from the ages of six to eighteen months. 

 This gradually decreases throughout life. 



Another method for determining the presence of antitoxin in the 

 blood is that of Romer. This depends upon the well-known fact 



