ESTIMATION 



Wright 2* recommended the general procedure of Snell and Strong 

 with either of two modified media. For routine work he advocated 

 a relatively simple medium, containing photolysed peptone, cystine, 

 glucose, xylose, riboflavine-free yeast, nicotinic acid, calcium panto- 

 thenate and inorganic salts. The second medium, which was claimed 

 to give a steeper standard curve and a slignt extension of the 

 assay range, was more complicated, and contained, in addition to 

 the constituents listed above, tryptophan, adenine, guanine, uracil, 

 xanthine, pyridoxine, and ^-aminobenzoic acid. In both instances, 

 the growth response was measured by titration of the lactic acid 

 produced. 



The use of Leuconostoc mesenteroides was advocated by Romberg 

 et al. ; ^5 it was said to be sensitive to o-oooi /xg. per ml. of riboflavine 

 as against o-02 /xg. per ml. for L. helveticus. 



Chemical Assays 



Unlike other members of the vitamin B complex, riboflavine solu- 

 tions are strongly fluorescent, and the intensity of the fluorescence is 

 proportional to the concentration of riboflavine. Assays based on 

 measurement of the fluorescence have been widely used, perhaps 

 more widely than the microbiological method, which is of more recent 

 date. The fluorimetric method appears to have been first used by 

 M. van Eekelen and A. Emmerie,^^ who eliminated interfering sub- 

 stances by oxidation with potassium permanganate (excess of which 

 was removed with hydrogen peroxide), which is without effect on 

 riboflavine. The intensity of the fluorescence of the purified solution 

 was measured in a step-photometer. They found that the riboflavine 

 could be adsorbed on lead sulphide and eluted with pyridine-acetic 

 acid without loss, and in later work this was introduced as an addi- 

 tional purification stage. The fluorometric method was also used by 

 Supplee et al.,^"^ S. M. Weisberg and I. Levin ^s and A. Z. Hodson and 

 L. C. Norris.29 The last-named workers reduced all the pigments 

 with sodium dithionite and stannous chloride and then reoxidised 

 the riboflavine by shaking with air ; this procedure did not reoxidise 

 the interfering pigments. The fluorescence of the resulting solution 

 was then measured in a fluorimeter (reading A). A second reading 

 (B) was taken after the addition of a known amount of riboflavine, 

 and a third reading (C) after reduction of the test solution with sodium 

 dithionite. Finally, the reading (D) of a pure solution containing 

 the same amount of riboflavine as that added to the test solution was 

 recorded. The true riboflavine content of the unknown solution was 



D 



calculated from the expression (A — C) x -p _ . . 



159 



