RIBOFLAVINE 



present. Again, starch contains material that stimulates the growth 

 of L. helveticus, leading to high results ; to eliminate errors from 

 this cause, preliminary treatment with takadiastase is recommended. ^^ 

 E. C. Bart on -Wright, ^^ however, stated that digestion with acid or 

 ptyalin was preferable to digestion with takadiastase. Another 

 stimulatory substance encountered in extracts of rice bran, wheat 

 bran and whole wheat flour,^* was not destroyed by takadiastase or 

 papain, and no satisfactory method of eliminating it was discovered. 

 Yet other growth-stimulating substances were reported by Bauernfeind 

 et al}^ in certain foodstuffs ; to eliminate these, the use of clarified 

 aqueous extracts of the solvent -extracted material was recommended. 

 It has also been suggested that a photolysed extract of the product 

 to be assayed might be added to the assay medium, in order to com- 

 pensate for the additional growth produced by the presence of such 

 impurities. Another stimulatory substance was obtained when certain 

 types of foodstuff were autoclaved at 15 lb. pressure with o-i N 

 hydrochloric acid for 15 minutes ; it was eliminated by adjusting to 

 pH 4*5 and filtering off the precipitate that formed.^ ^ 



The amount of acid produced by L. helveticus was said to be altered 

 by variations in the concentrations of metal ions.^^ Small amounts 

 of certain fatty acids also exerted an inhibitory effect on the organ- 

 ism,i8» ^^ whilst other fatty acids had a stimulatory effect. 2* To 

 prevent interference from this source, all materials should be extracted 

 with ether, chloroform or other suitable solvent after hydrolysis. 

 Solvent extraction was also employed in assaying buttermilk. ^^ In 

 addition, the casein in the basal medium was replaced by 4 % gelatine 

 plus 0-2 % of cystine. 



An important practical point in carrying out riboflavine assays is 

 that great care must be taken to maintain the temperature of incuba- 

 tion constant ; 21 a variation of 4 to 5° C. may cause marked differences 

 in the growth response. The temperature inside some types of bac- 

 teriological incubators varies considerably at different points, and 

 these variations may be sufficiently great to give erroneous results 

 with L. helveticus and, indeed, with other micro-organisms. 



Although the customary method of measuring the growth response 

 of L. helveticus is to titrate the lactic acid produced, a method has 

 been proposed in which changes in the pH of the culture were measured 

 and compared with those produced by the addition of known amounts 

 of riboflavine. 22 Another modification of the method utilises the 

 Heatley technique for assaying antibiotics ; the test solution is put in 

 holes cut in an agar plate seeded with L. helveticus, and the diameters 

 of the zones of stimulation are measured after incubation of the plates.^^ 



In the face of this multiplicity of methods it is difficult to select, 

 with any degree of confidence, the most reliable. E. C. Barton- 



158 



