150 THE BACTERIOPHAGE AND ITS BEHAVIOR 



In view of the nature of the bacteriophage and considering the prin- 

 ciples governing its reproductive activity it would seem that the fol- 

 lowing technic should serve best for determining the rate of multi- 

 plication of a race of the bacteriophage. 



Take a series of 12 tubes, each containing 4.5 cc. of the bacterial 

 suspension with respect to which the virulence of the bacteriophage is 

 to be measured. This suspension should be prepared from a fresh 24- 

 hour agar culture of a known susceptible strain, and should contain 100 

 million bacteria per cubic centimeter. Introduce 0.5 cc. of the bacterio- 

 phage suspension (whether it be fecal filtrate, bacteriophage culture, or 

 what not) into the first tube of the series. Shake thoroughly. This will 

 give an initial dilution in which each cubic centimeter will contain 

 1-10~^ cc. of the original suspension. Remove* 0.55 cc. of this 10~^ 

 dilution and introduce 0.5 cc. into a second suspension, giving thus a 

 dilution of 10"^. Spread the 0.05 cc. remaining in the pipette over an 

 agar plate. t 



After carefully shaking the second dilution remove 0.55 cc. and spread 



lence. Thus, with 2 strains of B. pestis we could inoculate 2 guinea pigs with a 

 like quantity of the 2 cultures. After a given time, for example, 48 hours, if it 

 were possible to determine the exact number of plague bacilli present in each of 

 the 2 pigs, we would have 2 figures which would represent the relative virulences 

 of the strains. Such a determination would be infinitely more exact than the 

 usual method, based upon the time factor or upon the lethal dose. 



* Various authors have observed that it is necessary to use a fresh sterile 

 pipette for each successive dilution. This is self-evident, and I have always 

 followed this procedure, for it is obvious, since the bacteriophage is corpuscular, 

 that if but one single corpuscle remains adherent to the wall of the pipette it 

 will suffice to completely falsify the result. This would be much less significant 

 if the bacteriophage were soluble, like an antibody, for example. 



t It is essential that neither the surface of the agar nor the cover of the Petri 

 dish contain excess moisture which will mix with the drop of suspension and thus 

 form in effect a fluid medium in which the bacteriophage can develop. This would 

 give an entirely erroneous result. To avoid such an excess of water of condensa- 

 tion the agar should be cooled to 50 to 55° before it is poured into the plates, and 

 as additional precautions, when the agar has solidified the covers of the plates 

 should be replaced by fresh sterile covers, and the plates then placed in the 

 incubator until the next day. In this way the agar becomes well dried out and 

 the drop of fluid placed upon the surface evaporates rapidly, thus preventing 

 growth of the corpuscles in the drop of liquid which otherwise would persist for 

 a considerable time before evaporation. It is well to bear in mind the fact that 

 a liberal amount of agar should be used; the layer in the plates should have a 

 depth of at least 3 mm. If the agar is too thin the plaques will be too small, and 

 will form poorly. The reason for this has been discussed. 



