86 THE BACTERIOPHAGE 



To a suspension of bacteria in bouillon is added 0.01 cc. of abacteriophage 

 culture and a drop of the material is spread on an agar slant. Frequently 

 the medium remains sterile, as has been shown above. Sometimes a slight 

 fringe of culture is secured, always at the upper margin where the medium 

 is somewhat dried out. Material from this fringe is planted on a tube of 

 sterile agar and after incubation a culture layer studded with plaques 

 is found. A third transfer is made from the upper portion of the tube, and 

 this is continued until an apparently normal culture is secured. That is 

 to say, until the growth develops without plaques. At that time, the 

 culture consists of resistant bacteria, free of bacteriophage, and appearing 

 coccoid in morphology. 



In such cultures resistance is maintained during a certain num- 

 ber of passages. Then it gradually decreases and it is observed 

 that this resistance is associated with the coccoid form, for the 

 lengthening of the bacterial elements affords an indication that 

 resistance to the bacteriophage is decreasing. 



The coccoid form may be secured in another manner. Certain 

 of the agar tubes seeded with a secondary culture show after a 

 very long time — one or two months, or even more — a colony 

 situated near the top of the agar. This colony increases in size 

 slowly and may attain a diameter greater than one centimeter. 

 It is formed solely of cocci. There can be no doubt but that it 

 consists of modified bacteria since successive subculturings yield 

 normal bacillary forms. 



Atypical colonies have been secured with B. dysenteriae (Shiga, 

 Flexner, and Hiss), with B. colt, B. typhosus, and the paraty- 

 phoids. 



As is seen, the coccoid form is most certainly a resistant form 

 of the bacterium, and the return to bacillary form taking place 

 gradually, affords an index of the decrease in resistance. 



This morphologic transformation is accompanied by a profound 

 change in the properties of the bacterium. Coccoid cultures are 

 not agglutinable by a specific serum. This is also true of secon- 

 dary cultures and mixed cultures in general. Restoration of 

 agglutinability is coincident with loss in resistance and with the 

 return to normal morphology. 



In this connection it has been shown that strains of B. typhosus, 

 inagglutinable when derived from the body, are at the same time 

 composed of bacilli which are resistant to the anti-typhoid bac- 



