VII. ESTIMATION 457 



1. Tube Methods for Assay of \''itamix Bij 

 a. Laciohacillus lactis Dorner (8000) 



There are three distinctly different procedures for measurement of mi- 

 crobial orowlh due to vitamin Bi... The lirst is the usual test-tube method 

 in which .u;ro\vth is measured by turbidity readings or titration of acid 

 produced. The newer cup-plate methods already mentioned depend on 

 measurements of the circular areas of growth ai)Out a metal cylinder or 

 depression in the agar containing the sample. The third method is the pad 

 technique in which a small volume of the sample extract is pipetted on a 

 small paper i)ad after which the pad is placed on the solidified test medium. 

 The pad technique has been used with notable success in the assay of 

 vitamin Bi-., pyridoxine, inositol, biotin, and protogen, a growth factor 

 for protozoans and lactobacilli.-* L. laclis Dorner has been compared with 

 strams of L. leichmannii as to differences in response to vitamin B12 

 and vitamin Bi2a and to crude concentrates of \-itamin Bi.. such as liver 

 extracts.'-^' ^^ Hendlin and Soars'-^ found good agreement between assays 

 with L. lactis Dorner and Euglena gracilis whereas the values obtained 

 with L. leichmamiii (4797) were significantly lower. No reducing agents 

 were added to the medium. For L. lactis Dorner, \'itamin Bi2a autoclaved 

 in the medium was 70 % as active as ^^tamin B12 whereas with L. leichmannii 

 (4797) for liver concentrates were approximately one-third of the values 

 obtained with L. lactis Dorner. The addition of O.Ol % or 0.02% thiogly- 

 collic acid to the L. leichmannii (4797) medium brought the assay values 

 for liver extract and vitamin Bija obtained with this organism up to those 

 obtained with L. lactis Dorner. 



Cooperman et al.^° compared assay results obtained with a L. lactis 

 Dorner (8000) tube method and with the recently adopted U.S.P. method'^^ 

 in which L. leichmannii 313 (7830) is used. The values for purified and 

 crude liver extracts and for concentrates of fermentation origin obtained 

 with L. lactis Dorner were consistently less than one half the \'alues obtamed 

 with L. leichmannii 313. This discrepancy' could he eliminated by adding 

 0.25 7 of KCN per milliliter to the L. lactis Dorner medium, thus increasing 

 the values to those obtained with L. leichmannii 313. The L. lactis Dorner 

 medium did not contain reducing agents. With L. leichmannii 313 a suffi- 

 ciently high level of KCN (0.25 7 per milliliter) was interchangeable with 

 reducing agents in increasing the assay values over those obtained in the 



"W. L. Williams, R. G. Esposito, and J. V. Pierce, Federation Proc. 11, 458, 1952. 

 " D. Hendlin and M. H. Soars, J. Biol. Chem. 188, 603 (1951). 



'» J. M. Cooperman, R. Drucker, and B. Tabenkin, J. Biol. Chem. 191, 135 (1951). 

 " U. S. Pharmacopeia, 14th revision, 3rd Supplement, p. 15, 1951. 



