VII. ESTIMATION 



469 



of 1.8 to 2 mm. in zone diameter. The zones are measured with pointed 

 calipers with an accuracy of ±0.1 mm. With the larger pads, standard 

 and sample solutions can l)e five times as dilute as with the smaller pads, 

 since a larger volume of liquid can be pipetted on the large pads. The liquid 

 added to the pad is allowed to dry before the pad is placed on the surface 

 of the agar. This permits the use of solvents other than water. The basal 

 medium immediately after inoculation is poured into 35 X 16-cm. flat 

 plates. The plates which have stainless steel edges cemented to plate glass 

 need only to be thoroughly cleaned, not sterilized. Large Pyrex plates may 



TABLE XV 



Comparison of Vitamin B12 Assays by the E. coli Pad Method and 

 L. leichmannii (4797) Tube Method 



Sample 



Pad method, 

 7/ml. or g. 



Tube method (Skeggs el 

 «'.*')• 7/nil. or g. 



Commercial liver extracts 

 1 

 2 

 3 

 4 

 5 

 6 

 7 

 8 

 9 

 10 

 11 



Antibiotic feed supplement A 



Antibiotic feed supplement B 



Fermentation solids 



also be used, but usuall}'' these give variations in depth of medium which 

 affect zone size. About eighteen samples at two levels in duplicate can be 

 assayed on one plate together with a standard curve in duplicate. 



The plates are incubated at 37", and zones can be measured at any time 

 from 5 hours to 4 days of incubation. 



Test solutions containing, after dilution, 1 mg. per milliliter of methio- 

 nine, or more, cannot be wholly satisfactorily assayed. The methionine 

 interference can be readih' detected by the diffuse zone of growth which 

 surrounds the vitamin B12 zone, but at this high concentration of methio- 

 nine the vitamiji-Bi2-zone size is affected. Table XV shows several com- 

 parative assays done by the pad method and the L. leichmannii (4797) 

 method ."^ The higher values for the fermentation samples reflect the pre- 

 ponderance of vitamin Bi2b in these samples. In the L. leichynannii (4797) 



