ESTIMATION 



either being laid on agar plates seeded with the organism ^^ or cut into 

 pieces and each separately assayed.^* The cup-plate assay method 

 also gives good results with L. leichmannii}^ 



A study of the nutritional requirements of two strains of L. leich- 

 mannii was made by Hoffmann et al}^ They used a basal medium 

 containing glucose, sodium acetate, sodium citrate, trypsin-digested 

 casein, acid-hydrolysed casein, salts, cystine, asparagine, tryptophan, 

 Tween 80, pyrimidines and members of the vitamin B complex. It 

 was found that a growth factor was formed on autoclaving and that 

 some vitamin B12 was destroyed. The first difficulty was overcome 

 either by replacing the glucose by sucrose or by the addition of thio- 

 glycolic acid or asparagus extract. Thioglycolic acid also protected 

 the vitamin B12 from destruction during autoclaving. The growth- 

 stimulating effect of thymidine and other desoxyribosides was 

 measured and corrected for by assaying the samples before and 

 after heating with o-2N sodiiun hydroxide at 100° C. for 30 minutes, 

 which destroys vitamin B12 but does not affect desoxyribosides. 



An organism of a different type used for the assay of vitamin B^g 

 was Euglena gracilis var. bacillaris which exhibited a quantitative 

 response to the vitamin but was not stimulated by thymidine.^® 

 Whereas L. leichmannii required a concentration of at least o-i m/xg. 

 per ml. to produce a measurable growth response, Euglena required 

 one-tenth of this amount. 



Animal Assays 



Vitamin B^g appears to be a component of the animal protein 

 factor (page 539), which can be assayed by measuring the growth 

 response of chicks or rats fed diets containing soya bean meal as the 

 sole source of protein. Attempts have been made to use these methods 

 for the assay of vitamin B^g concentrates, but they do not seem to be 

 specific for vitamin B^g, as the results were sometimes inconsistent 

 with those obtained by microbiological assay or clinical tests on per- 

 nicious anaemia patients. More consistent results were obtained when 

 a thyrotoxic condition was first induced in the experimental animals 

 by feeding iodinated casein, but it has been claimed that this test also 

 is not specific for vitamin B^g (page 541). Thus liver extracts active 

 in pernicious anaemia failed to stimulate the growth of chicks and 

 crude liver extracts discarded from anti-pernicious anaemia fractions 

 were highly active,^ ^ whereas vitamin Bjg replaced the animal protein 

 factor activity of injectible liver preparations when tested on thyro- 

 toxic chicks.^® With normal chicks, maximum growth was not 

 obtained until other supplements were added. ^^ 



Mice have also been used for the assay of animal protein factor, 



