MODES OF ASSAY 181 



is three-fold. In the first place, penicillin must be guarded against their action, during 

 its production and use, by the careful exclusion of all contaminating bacteria. In the 

 second place, they may be employed for the destruction of penicillin in mixtures of iienicillin 

 and bacteria, as, for example, in the cultivation of material from penicillin-treated tissues. 

 Finally, their immediate interest lies in the relation of penicillinase production to penicillin 

 resistance. Though only a few observations have been made, it appears that most naturally 

 resistant bacteria of medical importance are not penicillinase-producers ; and as regards 

 induced resistance. Rake and his colleagues (1944) could find no evidence of penicillinase 

 in Staph, aureus habituated to growth in penicillin broth. 



The acquisition of penicillin resistance is not accompanied by increased resistance to 

 certain other antibacterial agents, and vice versa. This holds for sulphonamides (see 

 above) acridines (Mcintosh and Selbie 19436) and helvolic acid (Chain, Florey, Jennings 

 and WiUiams 1943). Schnitzer and his colleagues (1943), however, found that Stajih. 

 albus strains grown on medium containing BaCl2 had increased in penicillin-resistance 

 to the same extent as peniciUin-trained strains. 



Preparations of actinomycin A (Waksman and Tishler 1942), notatin (Coult- 

 hard et al. 1942, Kocholaty 19436), and gramicidin (McKee, Rake and Menzel 1944) 

 are, like penicillin, inhibitory in dilutions greater than 1/10^, but all of them are 

 considerably more toxic than penicillin to animal tissues. None of the other 

 antibacterial agents, including the sulphonamides, has an in vitro activity of this 

 order. 



Apart from penicillinase and destructive chemical agents, there are no reports 

 of any substances that antagonize penicillin to any conspicuous extent. It retains 

 its in vitro activity in the presence of peptone, blood, serum, pus and tissue auto- 

 lysates (Abraham et al. 1941, Hobby, Meyer and Chaffee 1942a, Dawson et al. 1943), 

 though according to Bigger (1944a), some destruction occurs in blood (see also 

 Humphrey 1944). 



Modes of Assay. In the absence of chemically defined penicillin, any measure 

 of penicillin must be arbitrary. The measure in general use, the Oxford unit, is 

 arbitrarily based on the activity of a certain buffered preparation of the substance. 



The usual method of assay, which has proved sufiiciently accurate for both research 

 and therapeutic purposes, consists in placing measured volumes of a series of dilutions 

 of penicillin into cups formed b}' open, upright glass cylinders, the lower rims of which 

 are embedded and sealed in the surface of an agar plate inoculated with a standard strain 

 of Staph, aureus. The penicillin diffuses through the agar forming the floor of the cup, 

 and spreads outwards from under the lower edges of the cylinders. Incubation of the 

 agar plate reveals clear circular areas where growth of the Staph, aureus has been inhibited 

 round and under the cup. Within certain limits, the diameter of the zone of inhibition 

 is independent of the number of organisms inoculated ; and under standardized con- 

 ditions the diameter of the inhibition zone produced by high dilutions of j^enicillin is propor- 

 tional to the concentration of i^enicillin (Fig. 28). By this means, preparations of luoknown 

 strength may be titrated against a standard preparation. Modifications of the method 

 include cutting circular discs out of the agar plate, and sealing the bottom of the cup 

 so formed with a little molten agar (Fleming 1942) ; and the use of a sensitive strain of 

 spore-bearing baciUus, which can be preserved more readily than Staph, aureus as a standard 

 dried culture (cf. the cup-plate method of Rose and Miller 1939 ; see also Wilkins and 

 Harris 19436, Foster and Woodruff 1944, Heatley 1944). 



Potency may also be measured in fluid cultures or in body fluids containing penicillin 

 by noting the dilution that inhibits growth of the test bacterium. The inhibition may 

 be judged by inspection (see Fleming 1942, 1943, Florey and Florey 1943) ; nephelometric 

 measurements of growth-turbidity (Foster and Woodruff 1943, Foster and Wilker 1943) ; 



