206 THE BACTERIOPHAGE AND ITS BEHAVIOR 



7. THE BACTERIA OF SECONDARY CULTURES 



Spread a drop of a secondary culture upon the agar of a Petri dish 

 prepared in such a way that the agar layer has a depth of some 7 to 8 

 mm.* The result of seeding differs in accord with the virulence of the 

 bacteriophage present in the secondary culture, and consequently with 

 the resistance of the bacterium, since in a secondary culture there is an 

 equilibrium between the distinctive properties of the two antagonists. 



A secondary culture of B. dysenteriae Shiga or of the staphylococcus 

 obtained at 38°C. seeded upon a thin layer of agar usually gives no 

 colonies in spite of the presence of a bacteriophage of maximum viru- 

 lence. The agar is sterile (d'Herelle^^^) although the microscope 

 shows the turbidity of the secondary culture was due to the presence of 

 bacteria. 



We have seen that on agar the inhibitory products, resulting from the 

 attack on the bacteria by the bacteriophage, diffuse into the substrate. f 

 In a liquid medium these products freely exercise their inhibiting action. 

 Upon deep agar they diffuse as quickly as they are formed and no longer 

 interfere with the bacteriophage corpuscles so that the latter are able 

 to overcome the bacteria in spite of an acquired resistance. That this 

 is the true explanation is very well shown by the fact that on a thin agar 

 layer having a depth of only 1 or 2 mm. confluent colonies of resistant 

 bacteria develop when it is seeded with the same secondary culture which 

 gives no colonies whatever upon a thick layer of the medium. 



This question of the thickness of the substratum is also of significance 

 in cases where the spreading is made upon an agar slant; the lower part 

 of the medium may remain sterile while the upper portion is covered by 

 colonies, the latter being the more numerous as the layer of the agar 

 becomes thinner. 



If the bacteriophage has a high virulence, but not a maximal, the thick 

 layer of agar may still remain sterile although at times a few colonies 

 may be observed (d'Herehe^'^^). 



With a bacteriophage still less virulent the bacterial colonies are 

 always present and their number is the greater as the virulence of the 

 bacteriophage is the less. 



* We have seen that bacteriophagy does not take place upon solid media when 

 the products resulting from the action of the bacteriophage are not able to diffuse 

 into the medium. If a secondary culture is spread upon gelatin or even upon a 

 thin layer of agar a continuous layer of bacteria, quite naturally, develops. 



t We have likewise seen that these inhibiting products can not be the normal 

 secretory products of the bacteria since bacteriophagy takes place in filtrates of 

 old cultures. 



