VITAMIN Bjo (KRYTHROTIN) 



3. ESTIMATION OF VITAMIN B12 



Microbiological Methods 



It has already been stated that vitamin B^g is identical with one of 

 the factors necessary for the growth of Lactobacillus lactis Dorner, and 

 this organism was the first to be used for the assay of vitamin B12/ 

 the growth response being measured either turbidimetrically or by 

 titration of the lactic acid produced. L. lactis responds to o-oi m/xg. 

 per ml. of vitamin B^g- This organism is not entirely satisfactory, 

 however. In the first place, it responds to thymidine in the absence 

 of vitamin B^g,^ and in the second place it readily produces mutants 

 that grow without vitamin B12, and the composition of the medium 

 and the conditions of growth have to be carefully standardised if 

 consistent results are to be obtained.^ The first of these objections is 

 avoided by carrying out a preliminary separation of the vitamin B^g 

 from other growth factors by paper partition chromatography (page 

 532). The filter paper strip can either be laid on an agar plate seeded 

 with L. lactis and the zones of stimulation measured after incubation* 

 or it can be cut into small pieces and each one separately assayed in 

 test tubes in the ordinary way.^ 



A variety of media have been employed for the growth of L. lactis 

 as several laboratories failed to obtain satisfactory results with 

 Shorb's medium ; ^» ^ the addition of tomato-juice and Tween 80 is 

 said to be necessary.^* ^ Inconsistent results can also be obtained if 

 the amount of air in the tubes varies from one experiment to another. 

 Thus under anaerobic conditions, produced by the addition of reducing 

 substances or by the removal of oxygen, L. lactis will grow in the 

 absence of vitamin B^g, whereas on aeration or addition of oxidising 

 substances growth is inhibited and the inhibition can be overcome by 

 vitamin B^g ; carbon dioxide is essential for growth in any event. ^ 

 Thus, assuming sufficient carbon dioxide to be present, the response 

 will increase with the amount of oxygen in the atmosphere of the 

 tubes or with the amount of oxidising substances, e.g. peroxides, in the 

 tube itself. This presumably explains why in the absence of vitamin 

 B12 the amount of growth varies with the diameter of the tubes, ^ and 

 why the cup-plate method of assay gives more consistent results than 

 assays using test-tubes ; «, 10 ^ith the former the standard deviation 

 (66 % confidence limits) is said to be dz 10 % and with the titrimetric 

 method ±21 %. Using the cup technique, L. lactis gave no response 

 to thymidine, desoxy ribonucleic acid or ascorbic acid.^^ 



On the whole, more satisfactory results have been obtained with 

 another Lactobacillus, L. leichmannii }'^ which is more stable and less 

 exacting in its requirements than L. lactis .^"^ This organism has also 

 been used in conjunction with paper chromatography, the paper strips 



534 



