MICROBIOLOGICAL ASSAY 



Yeast Fermentation Method 



In the methods of A. S. Schnltz et al.} of K. Heyns ^ and of H. H. 

 Bunzell,^ a sugar solution is fermented with yeast and the carbon 

 dioxide produced is measured. Schultz et al.,^ however, found that 

 other yeast-stimulating substances were present in urine and therefore 

 modified their original method by carrying out two fermentations, one 

 before and one after oxidation with potassium ferricyanide to convert 

 the aneurine into thiochrome ; the difference between the two results 

 was proportional to the true aneurine content. For the estimation of 

 aneurine in wheat, y^ast, bread, liver, milk and orange juice, they 

 used another modification,^ the aneurine being destroyed by treatment 

 with sodium sulphite solution at ioo° C. for thirty minutes at pM 5 to 

 6. Again, the difference between the results before and after this 

 treatment was claimed to be proportional to the aneurine content ; 

 interfering substances were said to be unaffected by sulphite, and the 

 sulphite degradation products were supposed not to stimulate the 

 growth of the yeast. ^ 



A modification of this method was used by R. J. Williams et al.,'' 

 who employed the " old process " strain of Saccharomyces cerevtsiae, 

 which was grown on a medium supplemented with yeast and liver 

 extracts freed from aneurine by adsorption on fuller's earth. This 

 method is said to be simpler than the original method and to be capable 

 of estimating as little as 0-00005 f^S- ^^ aneurine per 2-5 ml. of medium. 

 Unfortunately it is not specific, the thiazole moiety (5-j8-hydroxyethyl- 

 4-methyl-thiazole) of aneurine and the pyrimidine moiety (4-amino- 

 5-ethoxymethyl-2-methyl-pyrimidine) giving 60 and 30 % respectively 

 of the response given by aneurine hydrochloride. Cocarboxylase 

 does not stimulate the growth of the organism under these con- 

 ditions. 



N. S. Scrimshaw and W. B. Stewart ^ also found that the method of 

 Schultz et at. lacked specificity ; for instance, it gave poor results 

 when used for the assay of meat and egg products. They claimed to 

 have eliminated the sources of error in the method by first carrying 

 out a preliminary assay with graded amounts of aneurine added to 

 the blank to determine the range over which the response was linear 

 and then in the main assay using three tubes, one containing the 

 sample, another the blank and the third a blank plus a suitable 

 amount of aneurine. The aneurine content of the sample was calcu- 

 lated by comparing the amount of gas liberated in the first tube 

 (after correcting for the value obtained for the blank) with that pro- 

 duced in the third tube similarly corrected. The effectiveness of 

 sulphite cleavage of a known amount of aneurine added to the 

 blank was determined for each type of substance and, if necessary, 



3 33 



