COLICINS AND OTHER BACTERIOCINS 383 



tive if separated into two steps: the producing bacteria are 

 inoculated first, incubated, and then steriHzed by chloroform va- 

 pors (Fredericq, 1948) ; the sensitive bacteria are then seeded on 

 the plate. Whatever the method, the presence of a bacteriocin 

 results in an area where growth of the indicator is inhibited. In 

 order to distinguish producing from nonproducing bacteria in a 

 mixed population a triple agar layer is used. A suitable inocu- 

 lum of the bacteria under test is incorporated into a first layer, 

 which is then covered by a second layer of sterile agar. 

 When, after incubation, isolated colonies have appeared, a third 

 agar layer containing indicator bacteria is added. On further 

 incubation those colonies which produced bacteriocin are made 

 visible by a zone of inhibition in growth of the sensitive bacteria. 



As in the case of phage, the existence of a bacteriocin cannot be 

 demonstrated unless a suitable indicator is available. System- 

 atic search for the production of bacteriocins in any group of 

 bacteria therefore involves the same steps as a search for lyso- 

 genic strains, namely, collecting a wide variety of strains and 

 testing them against each other in all possible pairs. 



Such studies, mainly with enteric bacteria, have revealed that 

 about 20 per cent of the strains investigated produce colicins 

 active against a single selected strain oi Escherichia coli (Fredericq, 

 1948), or Shigella flexneri (Halbert, 1948). Seventeen different 

 groups of colicins have been distinguished by Fredericq using 

 such criteria as host range, morphology of the zone of inhibition, 

 and diffusibility. They are named A, B, C, D, E, etc. A single 

 bacterial strain may produce more than one type of colicin. 



The inhibition of growth of the sensitive indicator on agar may 

 be used for titration of bacteriocins. An arbitrary unit of bac- 

 teriocin activity may be defined as the highest dilution of the 

 product which still gives a clear zone of inhibition under stand- 

 ard conditions. The range of sensitivity of this method varies 

 greatly according to the type of colicin under study. A more 

 precise method, based on the bactericidal action of colicins, will 

 be described in Section 2b of this chapter 



