468 VITAMIN Bi2 



3. Escherichia coli Pad-Plate Method 



As indicated in a preliminary report,^^ a method has been developed in 

 our laboratories which has proved of particular \'alue for the rapid assay 

 of large numbers of samples. The method utilizes the vitamin Bi2-requiring 

 mutant, E. coli 113-3, ''■ and a simple mineral salts-glucose medium solidified 

 with agar. The innovation which eliminates much of the time-consuming 

 mechanical manipulation usually involved in microbiological assay methods 

 is the use of paper pads originally developed for antibiotic assay** with 

 plates containing the solid assay medium. This technique possesses all the 

 general advantages of plate methods mentioned earlier and in addition 

 eliminates the boring of holes in the agar, removal of the plugs, and sealing 

 of the holes wdth hot agar. Besides the advances in technique the method 



TABLE XIV 

 Basal Medium For Pad-Plate Assay with E. coli 



has the fundamental advantage that vitamin B12, vitamin Bi2b, vitamin 

 Bi2c, amminocobalamin, and sulfite-treated cyanocobalamin all have equal 

 activity. 



The basal medium is given in Table XIV. The glucose and the salts 

 with the agar and most of the water are autoclaved separately for 5 to 10 

 minutes, combined, and inoculated at 45° to 48° with two 3-mm. loops of 

 E. coli slant culture per liter of medium. The inoculum slants which are 

 also used for stock cultures consist of Difco peptone 0.6 g., BBL trypticase 

 0.4 g., Difco yeast extract 0.3 g., beef extract 0.15 g., glucose 0.1 g., and 

 agar 1.5 g. per 100 ml. 



Standard solutions or extracts of test samples are pipetted on paper pads, 

 0.01 ml. or 0.02 ml. on the 6.5-mm. size, and 0.05 or 0.1 ml. on the 13-mm. 

 size (No. 740-E, available from Carl Schleicher and Schuell, New York). 



The following amounts of pure vitamin B12 give zone diameters which 

 form a straight line plotted against the log of the dose: 0.5, 1.0, 2.5, 5.0, 

 10, and 20 my. Over this range doubling the concentration gave an increase 



68 J. G. Vincent and H. W. Vincent, Proc. Soc. Exptl. Biol. Med. 55, 162 (1944). 



