268 Mary Barber 



in survival of the species, but when the staphylococcus 

 became established in the human nares, until the onset of 

 penicillin therapy, penicillinase would be of no service and 

 the capacity to produce the enzyme might, therefore, have 

 declined. Although discussion of this type is somewhat 

 reminiscent of the argument about the hen and the egg, there 

 is much to be said for Fairbrother's point of view. 



Experimental attempts to isolate penicillin-destroying 

 variants from penicillin -sensitive strains of Staph, 

 aureus 



When a penicillin ditch-plate is flooded with a penicillin- 

 sensitive strain of Staph, aureus, there is an almost straight 

 lineof in hibition after 24 hours' incubation (Fig. la), although 

 resistant colonies are often seen in the zone of inhibition if the 

 cultures are incubated for 4-8 days. When, however, a 

 gradient plate (Szybalski, 1953) is similarly treated, a large 

 variety of resistant colonies are seen even after 24 hours' 

 incubation (Fig. lb and c). This difference is no doubt due to 

 the fact that penicillin gradient plates have a much larger 

 area of medium containing a concentration of penicillin only 

 just above the minimal bacteriostatic level than do penicillin 

 ditch-plates. Nevertheless, in experiments involving 26 peni- 

 cillin-sensitive strains of Staph, aureus, the present author 

 has failed to isolate penicillin-destroying cultures by flooding 

 gradient plates with a thick emulsion of bacteria, although 

 the presence of as small a proportion as one penicillinase- 

 producing staphylococcus to 10 million penicillin-sensitive 

 cells can be readily demonstrated by such means (Fig. Id). 



Staphylococci with very weak penicillin-destroying activ- 

 ity, however, have been isolated by cultivating penicillin- 

 sensitive strains of Staph, aureus for very long periods in 

 very low concentrations of penicillin. Initially the penicillin- 

 sensitive strains were inoculated into fluid medium con- 

 taining 0-005-0 -01 [ji/ml. penicillin. At long intervals (one 

 or more weeks) these were transferred to further broth tubes 



