BACTERIOLYSIS 33 



is secured. With a pipette a certain quantity of this concentrated 

 suspension is added, drop by drop, into a tube of bouillon until 

 the turbidity corresponds to that of the 250 million control sus- 

 pension. This approximation is adequate for routine practice, 

 giving a suspension which contains about 250 million bacilli per 

 cubic centimeter. This is the suspension to be used. A young 

 broth culture can be utilized, but the other suspension, more 

 accurately adjusted, is to be preferred. 



Experiment /. To a tube containing 10 cc. of a suspension of B. dysen- 

 teriae Shiga is added 0. 00,002 cc. of a culture of the bacteriophage ten days 

 old, i.e., a Shiga suspension that has been lysed for ten days. The tube is 

 shaken to ensure even distribution and with a tared platinum loop 0.01 cc. 

 of the liquid is removed and spread as uniformly as possible, by rubbing, 

 over the surface of an agar slant. This tube is incubated at 37C. After 

 eighteen hours it presents a Shiga culture studded with 51 plaques. 



The calculation is simple. The 10 cc. of suspension received 

 0.00,002 cc. of the bacteriophage culture, or 0.00,000,2 cc. for 

 each cubic centimeter of medium. The amount of material taken 

 for planting on agar was 0.01 cc. This contained, therefore, 

 0.00,000,002 cc. of the original bacteriophage culture. Upon agar 

 this 0.01 cc. gave 51 clear areas, or 51 colonies, each one of which 

 developed from a single bacteriophagous germ deposited upon 

 the surface. Fifty-one germs for 0.00,000,002 cc. represent 

 2,550,000,000 germs per cubic centimeter. This is, therefore, the 

 content of the culture of the bacteriophage which was used to 

 inoculate the bacterial suspension. 



The technic for counting the ultramicroscopic bacteriophage 

 hardly differs from that used in counting ordinary bacteria. 

 With the latter isolated colonies are secured on a surface other- 

 wise sterile. With the ultramicrobe, clear areas representing 

 colonies are obtained superimposed upon a layer of bacterial 

 growth. Counting can not be effected otherwise, since the bac- 

 teriophagous ultramicrobe is only able to develop upon the bac- 

 teria which constitute its culture medium. 



Each plaque represents a colony of the bacteriophage. This 

 is indisputable, for if the centre of such an area is touched with 

 the point of a drawn-out capillary pipette and this pipette is 

 dropped into a suspension of dysentery bacilli, the culture, when 



