336 THE TUBERCLE BACILLUS 



the severity of the lesions or the virulence of the infecting organism. Serums 

 from cases of miliary tuberculosis often fail to produce the reaction. Tuber- 

 culous serous exudates (peritonitis, pleurisy) in most cases have the 

 property of agglutinating the tubercle bacillus. 



The technique of agglutination is as follows : 



Take four small sterile test-tubes 5-6 cm. long. A little of the pure culture 

 is put into the first and acts as a control : into the other three a mixture of 

 serum and culture is introduced in different proportions : thus, into the 

 second 1 drop of serum and 5 drops of culture, into the third 1 of serum and 

 10 of culture and into the fourth 1 of serum and 15 of culture. After shaking, 

 the tubes are placed at an angle of 45 and kept at laboratory temperature. 

 Agglutination only appears after the mixtures have been put up a few hours. 

 The tubes should be examined several times during the next 24 hours and 

 against a black ground. When the reaction is complete the fluid is clear 

 and there is a slight deposit at the bottom of the tube. A complete reaction 

 giving this appearance is alone of diagnostic value. The diagnosis should 

 always be completed by microscopical examination of the clumps (between a 

 slide and cover-glass and unstained). 



The power of agglutination possessed by the serum of persons suffering from 

 tuberculosis is of very limited diagnostic value for the following reasons : 



1. Homogeneous cultures are often agglutinated by the serum of healthy persons 

 26 times out of 100 examined (Arloing and Courmont) : 50 per cent, of cases according 

 to German statistics. 



2. The serum of persons suffering from febrile diseases other than tuberculosis 

 (enteric fever, puerperal fever, pneumonia, etc.) nearly always agglutinates homo- 

 geneous cultures of the tubercle bacillus. 



3. The serum of tuberculous subjects does not always agglutinate homogeneous 

 cultures of the infecting organism. A negative reaction is especially frequent in 

 cases of advanced phthisis, miliary tuberculosis, and " galloping " consumption. 

 The reaction is often absent in the very early stages of the disease, and is practically 

 never obtained in cases of lupus. Bezan9on, Griffon and Philibert obtained a 

 positive reaction in about 83 per cent, of cases of undoubted tuberculosis : Ivanova 

 observed agglutination in 66 per cent, of cases of tuberculosis and in 60 per cent, 

 of non-tuberculous persons. 



4. The method is very delicate in practice and the most minute precautions must 

 be taken in the preparation of the homogeneous cultures. 



(ii) Vasilescu describes a medium which he says gives homogeneous cultures 

 more easily than Arloing 's method. 



The medium has the following composition : 



Clear sterile calf serum, - 25 c.c. 



Distilled water, 75 ,, 



Neutral glycerin, - 3 



Heat the mixture to 100 C. in a water bath, distribute into tubes of 2 cm. diameter 

 in quantities sufficient to give a column 3 cm. high in each tube. Sterilize at 120 C. 

 for 15 minutes (the medium does not coagulate). Sow with a bacillus of the human 

 type. Incubate at 37 C. and shake the tubes every day. After two sub-cultures 

 in this medium the growth is homogeneous. 



(iii) Hawthorn suggests a simplification of Arloing 's technique. He sub- 

 cultures an homogeneous culture in glycerin-broth in 2 per cent, peptone 

 saline solution. By this means an homogeneous culture consisting of motile 

 bacilli is obtained in 48 hours without shaking the flask. These cultures are 

 more sensitive for the agglutination reaction than Arloing's. 



Vincent repeated Hawthorn's experiments and always found that the 

 cultures in the peptone saline medium were agglutinated even when the 

 serum of non-tuberculous persons was used (in dilutions of 1 in 30 and 1 in 



