MICROBIOLOGICAL ASSAY 



Phycomyces Assay Method 



Although yeast is the micro-organism most commonly used for the 

 estimation of aneurine, other micro-organisms have been used. Next 

 in importance to yeast is the. mould, Phycomyces Blakesleeanus, which 

 was employed by W. H. Schopfer/^ A. P. Meiklejohn/^H. M. Sinclair ^' 

 and T. Morell.^^ A suitable aneurine-free medium to which graded 

 amounts of the test solution have been added is inoculated with 

 mould spores, incubated for seven days and the mycelium then re- 

 moved, dried and weighed. The results are compared with those 

 obtained using the same basal medium to which have been added 

 known amounts of aneurine, and the aneurine content of the unknown 

 solution is calculated from the dose-response curve obtained with the 

 standard. The method has been used to estimate the aneurine and 

 cocarboxylase contents of blood plasma and cerebrospinal fluid ^^ and 

 for the routine assay of vegetable extracts. ^^ A. P. Meiklejohn 21 

 used the Phycomyces method to estimate the aneurine content of 

 potatoes and found that the green sprouts contained a factor toxic for 

 the mould, whilst the centre of the tubers from April to August, and 

 the skin layer always, contained an adjuvant factor that stimulated 

 the growth of the mould only in presence of aneurine. 



Assays with Other Organisms 



Lactobacillus fermenti was suggested by H. P. Sarett and V. H. 

 Cheldelin,^^ growth being measured turbidimetrically sixteen to 

 eighteen hours after inoculation. Cocarboxylase was 30 % more- 

 active than aneurine. Staphylococcus aureus was used by P. M. West 

 and P. W. Wilson, ^^ Glaucoma piriforme by L. Emerique-Blum and 

 A. Lwoff,2* Streptococcus salivarius by C. F. Niven and K. L. Smiley, ^^ 

 and a yeast, Saccharomyces macedoniensis , by Emery et al.^^ 



Probably the most satisfactoiy of these methods is that of Sarett 

 and Cheldelin. Under the conditions prescribed by the authors, the 

 organism, Lactobacillus fermenti 36, does not respond to the pyrimidine 

 and thiazole components of aneurine, alone, together or in presence of 

 aneurine. A quantitative response is obtained in presence of 0-005 

 to 0-04 jLtg. of aneurine per 10 ml. of mediimi. Unfortunately, the 

 basal medium is rather complicated, consisting of alkali- treated 

 peptone, acid-hydrolysed casein, glucose, sodium acetate, cystine, 

 adenine, guanine and uracil, with the usual inorganic salts and the 

 following members of the vitamin B complex : riboflavine, calcium 

 pantothenate, ^-aminobenzoic acid, nicotinic acid, pyridoxine, biotin 

 and folic acid. The growth-response is measured turbidimetrically. 



The original method of Sarett and Cheldelin gave unsatisfactory 

 results with some materials containing inhibitory or stimulatory 



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