206 DIFCO MANUAL 



as a seed layer and for carrying stock cultures of Micrococcus pyogenes var. 

 aureus (PCI 209P) in penicillin cylinder assay; for maintaining test organism, 

 Bacillus suhtilis (ATCC ?^6633) in streptomycin assay; for seed layer in aureo- 

 mycin cylinder assay and for maintaining cultures of M. pyogenes var. aureus 

 (PCI 209P) for the turbidimetric assay of aureomycin; for seed and base layer 

 and for maintaining cultures of Sarcina lutea (PCI 1001) in chloramphenicol 

 cylinder assay; for seed layer in bacitracin plate assay. 



In the assay of antibiotics by the cylinder method it has been found that for 

 penicillin, aureomycin and bacitracin, better results are obtained with a small 

 amount of dextrose in the seed layer and no dextrose in the medium used for the 

 base layer. For the assay of these antibiotics the use of Bacto-Penassay Seed Agar 

 has resulted in more clear-cut zones of inhibition. A layer of Bacto-Penassay Base 

 Agar is allowed to solidify in plates, and is overlaid with about 4 ml. of Penassay 

 Seed Agar (100 x 20 mm. plate) containing a carefully standardized inoculum 

 of the test organism. The plate is tilted to obtain an even distribution of the 

 inoculum on the surface. Following solidification of the seed layer, the plate is 

 ready to receive the cylinders and antibiotic solutions under assay. 



Formerly, the base agar was used for both the base and seed layers; however, 

 it was found that the zones of inhibition were not as sharply defined as was 

 desired. The use of Bacto-Penassay Seed Agar as the seed layer has resulted in a 

 more clear-cut zone of inhibition. 



Plates are prepared the day samples are to be tested by adding 21 ml. of 

 Penassay Base Agar to sterile petri dishes (100 x 20 mm.). After the base layer 

 has solidified it is overlaid with 4 ml. of Bacto-Penassay Seed Agar, inoculated 

 with the test organism. 



To rehydrate the medium, suspend 30.5 grams of Bacto-Penassay Seed Agar 

 in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium 

 completely. Distribute in 100 ml. quantities in 300 ml, Erlenmeyer flasks. Sterilize 

 in the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reac- 

 tion of the medium will be pH 6.6. 



One pound of Bacto-Penassay Seed Agar will make 14.8 liters of medium. 



1 The Compilation of Tests and Methods of 

 Assay for Antibiotic Drugs, Federal Security 

 Agency, Food and Drug Administration. 



BACTO 



YEAST BEEF AGAR (B244) 



DEHYDRATED 



Bacto-Beef Extract 1.5 g. 



Bacto- Yeast Extract 3 g. 



Bacto-Peptone 6 g. 



Bacto-Dextrose 1 g. 



Bacto- Agar 15 g. 



Bacto-Yeast Beef Agar is a slight modification of the medium recommended 

 by Schmidt and Moyer^ for use in the cylinder plate procedure for assaying 

 penicillin as described by Abraham, et al.^ 



As a result of their extensive use of the cylinder plate method, Schmidt and 

 Moyer made a number of observations and refinements which improved the 

 technique of penicillin assay by this method. They claimed that the cylinder 

 plate method was a more practical procedure than the serial dilution method, 

 that it is more adaptable for assay of a large number of specimens, that samples 

 need not be sterile, as is necessary in the serial dilution method, and that more 



