A Microbiological Assay Method for Thiamine

It is generally recognized that the most useful and convenient means of quantitatively determining members of the vitamin B complex involve microbiological methods. Several methods of this type have been proposed for the assay of thiamine but each one in use today possesses certain inherent disadvantages that limit its value. The fermentation method of Schultz, Atkin, and Frey (1,2), based on the increased rate of alcoholic fermentation by yeast upon addition of thiamine increments, is not adaptable to large scale work. The number of assays that can be carried out is limited by the apparatus. In addition, the “pyrimidine” cleavage product is active, thus requiring two determinations for each product in order to obtain a true picture of the thiamine content of certain food materials. The method in which Phycomyces blakesleeanus is used as the test organism (3,4) is time-consuming and is subject to error, since the organism is able to utilize the two moieties of the thiamine molecule. Both the “pyrimidine” and “thiazole” portions are active in the yeast growth method as proposed by Williams, McMahan, and Eakin (5) ; in addition, this organism is not sensitive to cocarboxylase. The microbiological method with a strain of Staphylococcus aureus as the test organism, as reported by West and Wilson (6), is also subject to error, since this organism is able to synthesize thiamine from the two cleavage products of the vitamin (7). The purpose of this communication is to introduce a thiamine assay procedure which takes advantage of an organism for which neither cleavage product, alone or in combination, is active. As little as 0.01 millimicrogram of thiamine per ml. of medium can be quantitatively detected. Under the conditions tested, cocarboxylase is approximately 40 per cent more active than thiamine, calculated on a molecular basis, thus requiring an enzymatic hydrolysis for precise determinations in some food products.