84 M. R. Pollock 



concentrates of the supernatant fluid obtained after spinning 

 off cells from cultures of strain 5 were found to contain traces 

 of penicillinase activity. A technique for micro-assay of peni- 

 cillinase in untreated culture supernatant fluid was eventually 

 evolved which permitted accurate measurement of activity 

 with a sensitivity up to 100 times that of the manometric 

 method. Since this method has not been published before, 

 it is described here in detail. 



Micro -assay of Penicillinase Activity 



Two-ml. samples of culture supernatant fluid were diluted 1 : 5 in 1 % 

 gelatin broth containing 20 units penicillin/ml. and incubated at 30°. 

 One-ml. quantities were withdrawn every 15 min. and added to 9 ml. 

 ice-cold 0-01 M potassium phosphate buffer (pH 7-0). The residual 

 penicillin in the diluted sample was assayed in octuplicate by cup-plate 

 assay using the ICI strain of B. subtilis as indicator organism, by com- 

 parison with penicillin standards of from 0-8 to 2-0 units/ml. (Hum- 

 phrey and Lightbown, 1952). After addition of samples, the plates were 

 stored for 2 to 4 hr. at 0° to allow diffusion of the penicillin (and so 

 increase the sensitivity of the test) before incubation overnight at 35°. 

 The amount of enzyme preparation added was adjusted such that not 

 more than 50% of the penicillin had been destroyed before at least 

 3 samples had been taken. 



Zero-point estimation of penicillin made in this way proved that it 

 was possible to make accurate assays in the presence of enzyme. This 

 was feasible because of the decrease in enzyme activity on sampling, 

 due to a combination of 2 factors: (1) the lowering of temperature to 0° 

 until the antibiotic had time to diffuse away from the enzyme in the 

 cups and (2) the 1 : 10 dilution of substrate from a concentration which 

 was, initially, well below enzyme saturation level; this must have 

 caused a nearly proportional decrease in enzyme activity. 



For the purposes of this particular assay, the two following assump- 

 tions had to be made: (1) The activity in the culture supernatant fluid 

 corresponded approximately with total activity — as with all the other 

 strains of B. cereiis so far tested. (2) The Michaelis affinity constant of 

 the strain 5 enzyme was identical with that of the 5/B penicillinase. In 

 view of the other similarities between 5 and 5/B penicillinases (to be 

 discussed later) this seems an entirely reasonable assumption. 



The enzyme activity was finally calculated by plotting the residual 

 penicillin concentration against time, measuring the initial rate of peni- 

 cillin destruction and adjusting the value to what it would be were the 

 enzyme saturated with substrate. An accurate figure for the Km of 

 penicillinase is thus required — and can in fact be calculated, using the 

 same technique, by varying substrate concentration and having samples 

 with rather higher enzyme activities than those available from cultures 

 of strain 5. 



