ASSAY METHODS 49 



relatively potent concentrates or even for clinical examination of urine, 19 

 the colorimetric method is, however, probably superior to any other. 



Yeast Fermentation Method. This method, which is probably second 

 to the thiochrome method with respect to wideness of use, was developed 

 by Schultz, Atkin and Frey 20 - 21 ; it utilizes the fact, discovered by them, 

 that fermentation by suitable yeast is enhanced by the presence of free 

 thiamine. In its modified form the method takes advantage of the fact 

 that thiamine is cleaved quantitatively by sulfite, and the cleavage 

 products have no effect upon the fermentation. The difference between 

 the enhancement produced by an untreated solution and a sulfite-treated 

 one is used as a measure of the thiamine content. 



The application of this method requires the use of a special apparatus, 

 a Fermentometer, which is constructed so that 12 fermentations can be 

 run simultaneously. One-half gram of yeast is used in each bottle. The 

 carbon dioxide evolution after 3 hours of fermentation is measured and 

 the enhancement induced by 1 ng and 2 fig of thiamine are taken as 

 standards for comparison. Errors in assay under favorable conditions are 

 not more than a very few per cent. 



By the use of a Warburg apparatus thiamine can be assayed on an 

 ultramicro scale (0.005 fig to 0.025 fig) with errors no greater than ±5 

 per cent. 22 



Comparative studies have shown that the yeast fermentation method 

 gives results generally comparable in accuracy with those obtained by 

 the thiochrome method. 7 16> 23> 24 Details of procedure are described by 

 the originators. 21 



Microbiological Growth Methods. The yeast-growth method has not 

 been used extensively except in the laboratory where it originated, but 

 for certain types of investigation involving minute amounts of material 

 it had a distinct advantage. The growth of certain strains of yeast ("Old 

 Process") in an otherwise complete medium is greatly stimulated by 

 minute amounts of thiamine. 25, 26> 27 Thiamine degradation products are 

 likely to be effective, however, and these must be ruled out. 24 The effect 

 of thiamine is stimulatory rather than essential for growth, and differ- 

 ences in growth rates are therefore the basis of the response. By this 

 means it is possible to determine with satisfactory accuracy in un- 

 processed materials, amounts of thiamine as little as 0.0001 ^ig, and by 

 reducing the volume of the cultures, a lower limit can be reached. 



The use of the mold Phycomyces Blakesleeanus as a test organism 

 has been advocated by Schopfer 28 and was used, for example, by 

 Meiklej ohn 29 for the determination of thiamine in the blood. Sinclair, 

 however, has emphasized that the response is nonspecific. 30 Later this 

 investigator introduced a correction which was thought to take care of 



