ESTIMATION 



riboflavine had been removed by adsorption on norite at pR 5. Satis- 

 factory assays of riboflavine in milk were obtained by this method. 

 A fraction prepared from liver extract was used by Wagner et at. ,^ 

 whilst others have used rice polishings * and rice bran,^ both of which 

 were claimed to be more reliable sources of the vitamin B complex 

 than whole wheat or yeast. 



Chicks were used for riboflavine assays by T. H. Jukes,® but these 

 appear to give less satisfactory results than rats. 



Microbiological Assays 



The biological method of assay has now been completely super- 

 seded by the microbiological method, introduced by E. E. Snell 

 and F. M. Strong. ^ They used a lactic acid-producing organism, 

 Lactobacillus casei e, now generally referred to in this country as 

 L. helveticus ; this will not grow in the absence, inter alia, of ribo- 

 flavine. Using a suitable basal medium, the amount of lactic acid 

 produced is proportional to the concentration of added riboflavine. 

 This method and its modifications have been extensively used in the 

 assay of foodstuffs. The original mediiun employed by Snell and 

 Strong consisted of alkali-treated peptone, cystine, yeast supplement, 

 glucose and inorganic salts, but various modifications were made by 

 later workers in order to improve the specificity of the medium and 

 the growth response. 



E. C. Barton-Wright, 8 for example, advocated the addition of 

 xylose, asparagine, nicotinic acid and pantothenic acid to Snell and 

 Strong's medium, whilst R. D. Greene and A. Black, ^ on the other 

 hand, supplemented the original medium with ammonium sulphate, 

 sodium acetate and photolysed yeast ; with this modified medium, 

 they obtained results in good agreement with those given by the rat 

 growth method. M. Landy and D. M. Dicken ^^ evolved a completely 

 new medium, which they used for the assay, not only of riboflavine, 

 but also of pantothenic acid, nicotinic acid, folic acid, pyridoxine and 

 biotin. It was a complex medium, containing casein hydrolysate, 

 sodium acetate, glucose, asparagine, tryptophan, cystine, inorganic 

 salts, guanine, adenine, xanthine and uracil, aneurine, biotin, folic 

 acid, calcium pantothenate, nicotinic acid and pyridoxine. For the 

 assay of other vitamins, riboflavine was included and the particular 

 vitamin being assayed was omitted. 



Other workers showed that various substances might interfere 

 with the microbiological estimation of riboflavine, and suggested 

 methods of overcoming the interference. Urea, for instance, inhibits 

 the growth-promoting action of ribofiavine,^i so that to obtain satis- 

 factory assays of urine a correction must be applied for the urea 



157 



