INTRODUCTION 



The quantitative assay of a number of biologically important 

 substances, by virtue of their ability to affect the metabolism of 

 certain microorganisms, is still relatively new. The principle 

 involved is the measurement of the influence of the substance on 

 the rate of formation of an end product of the metabolism, such as 

 the carbon dioxide developed by yeast or the acid formed by 

 Lactobacillus casei. In other instances the effect on the rate of growth 

 is measured directly by determining the mass or area of a colony, 

 as in the case of the assay of choline with a mutant of Neurospora 

 crassa. At the date of this writing, the microbiological methods have 

 been developed mainly for members of the vitamin B family and 

 for amino acids. The great sensitivity of these methods compensates 

 for the fact that in many cases the substances to be assayed occur 

 biologically in very high dilutions. The microbiological technique 

 has been employed almost exclusively on the macro scale. However, 

 no insurmountable problems are associated with the simple reduction 

 in volume and the use of the micro techniques already available, 

 which would be necessary for the adaptation of macro methods to 

 histological or cytological studies. The riboflavin method of Lowry 

 and Bessey ( 1944 ) marks a beginning in this direction. 



RIBOFLAVIN 



The method of Snell and Strong ( 1939) for the microbiological 

 determination of riboflavin in amounts of the order of 100 m/xg. was 

 modified by Lowry and Bessey (1944) so that measurements could 

 be performed in the range of 0.5-2.0 m/xg. with a probable error of 

 only about 1% for determinations, in triplicate, of pure riboflavin 

 solutions, and of about 3% for rat cornea. The modifications applied 

 were based on the observations that riboflavin is partially destroyed 

 during autoclaving in small tubes at pH 7.0, that air is inhibitory 

 to, while carbon dioxide stimulates the growth and acid production 

 of, the Lactobacillus casei used for the assay, that buffers in the pH 

 range of 4 to 6 stimulate the growth and acid production of the 

 organism, and that cysteine restores the basal medium if its effective- 

 ness deteriorates on standing after the autoclaving. 



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