470 



VITAMIN Bi2 



assay pure vitamin B^b autoclaved in the medium is only 50 % to 70 % as 

 active as vitamin B12 whereas with the pad method the two forms have 

 equal activity. 



After approximately a year's use of the method it appears to have the 

 following major advantages: 



1. Other than the flat plates no special glassware or equipment such as 

 colorimeters is needed. 



2. Because of ease and simplicity large numbers of assays can be obtained 

 with a minimum of time and effort. 



3. Pads containing the standard amounts of vitamin B12 can be prepared 

 in advance. The standard curve covers a forty -fold range of concentration. 



Fig. 6. Vitamin Bio determination by the pad-plate method. The liver extracts 

 were diluted 1 to 100 before pipetting 0.01 and 0.02 ml. on the pads. Note diffuse edges 

 of the readily recognized methionine zones. 



4. Methionine is the only substance known to give a non-specific growth 

 response, and this interference when it occurs is visually apparent. 



5. Pure or crude forms of the vitamin need not be heated prior to assay, 

 in fact, aseptic conditions in general are not essential. 



6. Of greatest fundamental value is the equality of response of the organ- 

 ism to the various forms of the vitamin. 



Figure 6 shows an assay plate containing standard ('ur\'es, two liver 

 extracts, and methionine. The diameters of the zones due to pure vitamin 

 B12 in Fig. 6 are plotted against the logarithm of the dose and a straight 

 line ol)tained as shown in Fig. 7. 



4. Sample Treatment 



A suitable extraction procedure to release the \-arious forms of vitamin 

 B12 from natural materials has not been generally agreed upon. Various 



