204 DIFCO MANUAL 



lowest concentration of antibiotic capable of inhibiting growth of the test organ- 

 ism in the Penassay Broth. The approximate amount of antibiotic present in an 

 unknown is determined by comparing its activity in parallel with that of a 

 standard preparation of known potency. A quantity of the sample to be tested 

 is added to the first of a series of tubes containing broth previously inoculated 

 with the test organism. Further dilutions are made from this tube. Comparable 

 dilutions of the standard are also made. All tubes are thoroughly shaken and 

 incubated at 30°C. for 18 or 40 hours. More definite readings may be made in 

 40 hours than in 18. The highest dilution in which no growth appears is the end 

 point of the series, and the dilution of the unknown which gives such an end 

 point contains the same amount of antibiotic as that in the end point tube of 

 the standard series. 



It is sometimes difficult to obtain a sharp end point in the serial dilution 

 methods of penicillin or streptomycin assay. Also, the amount of antibiotic re- 

 quired is usually much larger in these methods than in the turbidimetric pro- 

 cedure. Foster* described a turbidimetric method for penicillin assay and claimed 

 that it was the most accurate method available. McMahan^ offered a 3-4 hour 

 turbidimetric procedure based on the same procedure used in the microbiological 

 assay of vitamins and considered it more precise than the cup plate method. Lee, 

 Foley, Epstein and Wallace^ modified the method of Foster and Woodruff^ and 

 obtained results in 90 minutes instead of four hours. 



The turbidimetric method is based on the proportional inhibition of the 

 growth of the test organism in the liquid medium as a function of the antibiotic 

 concentration. The growth measurements are made turbidimetrically and com- 

 pared with a standard curve obtained by running various concentrations of a 

 standard antibiotic in parallel with the unknown. 



To rehydrate the medium, dissolve 17.5 grams of Bacto-Penassay Broth in 

 1000 ml. distilled water. When used in the serial dilution method, distribute in 

 200 ml. quantities in 500 ml. Erlenmeyer flasks. For carrying tests organisms, as 

 described by the Food and Drug Administration,^ distribute in 10 ml. quantities 

 in tubes. For the turbidimetric procedure, distribute as required by the specific 

 procedure used. The medium is sterilized for 15 minutes at 15 pounds pressure 

 (121°C.). The final reaction of the medium will be pH 7.0. 



One pound of Bacto-Penassay Broth will make 25.8 liters of medium. 



ij. Bact., 47:199: 1944- * J- Biol. Chem., 144:285:1942. 



2 The Compilation of Tests and Methods of ^ j. giol. Chem., 153:249:1944. 

 Assay for Antibiotic Drugs, Federal Security e j. Biol. Chem., 152:485:1944. 

 Agency, Food and Drug Administration. 'J. Bact., 46:187:1943- 



3 Science, 1 09 : 305 : 1 949. 



BACTO 



PENASSAY BASE AGAR (B270) 



DEHYDRATED 



Bacto-Beef Extract 1.5 g. 



Bacto- Yeast Extract 3 g. 



Bacto-Peptone 6 g. 



Bacto-Agar 15 g. 



Bacto-Penassay Base Agar is prepared according to the formula specified in 

 "The Compilation of Tests and Methods of Assay for Antibiotic Drugs,"i 

 Federal Security Agency, Food and Drug Administration, for antibiotic assay. It 

 is recommended as a base layer in the cylinder assay of penicillin; for the cylinder 

 assay of aureomycin; for the plate assay of bacitracin and for maintaining cul- 



