FUNGI AS TEST ORGANISMS 209 



assayed, in a water-soluble condition; (4) a standard sample of the sub- 

 stance to be analyzed ; (5) preparation of a range of concentrations of the 

 known and unknown substances in the basal medium ; (6) uniform inocula- 

 tion; (7) incubation under uniform conditions; (8) measuring the response 

 of the test organism; (9) construction of the standard curve from the 

 response of the test organism to kno\\Ti amounts of the substance under 

 test; (10) calculating the content of the substance contained in the sample. 

 The above discussion assumes the use of pure compounds in obtaining 

 standard curves. The utility of microbiological assay methods is not 

 confined to the assay of known compounds. They are of great utihty 

 in studies of methods of isolation of new growth factors and other active 

 compounds. These occur in complex natural products and, before they 

 are isolated, are known only by the physiological effects they produce in 

 living organisms. Given a deficient fungus, or other organism, it is pos- 

 sible to follow the efficiency of the various steps in an isolation procedure. 

 The isolation of many of the water-soluble vitamins has been facilitated 

 by the use of test fungi. The use of a biotin-deficient yeast enabled 

 Kogl and Tonnis (1936) to isolate biotin for the first time as a pure 

 compound. 



GENERAL PROCEDURES 



The following discussion of the steps involved in microbiological assay 

 may serve also as a guide to the quantitative study of the physiology of 

 fungi. Such studies are the surest way to gain knowledge and under- 

 standing of the physiology of the fungi. 



Selection of test organisms. The first requirement of a test organism 

 is specificity for the compound under assay. A fungus which responds 

 to either or both moieties of thiamine is less suitable than one which 

 requires the intact thiamine molecule. Other considerations may out- 

 weigh the advantages of strict specificity, but the response of the test 

 organism to moieties, vitamers, and related compounds must be known. 



Other considerations besides specificity enter into the selection of test 

 organisms. Test organisms should be easily maintained in culture, 

 easily handled in the laboratory, and have stable biochemical character- 

 istics. Rapid and uniform growth is desirable. The habit of growth is 

 important. A fungus which forms mucilaginous colonies which adhere 

 to the walls of the flasks is difficult to harvest, and yeasts which clump 

 are difficult to determine by turbidimetric methods. 



The basal medium. Except for the compound or element under 

 investigation the basal medium should be complete and balanced. If a 

 test organism is deficient for more than one factor, all the factors except 

 the one under investigation should be present in optimum amounts. 

 Other requirements are easily available sources of carbon and nitrogen 

 and a medium which is adequately buffered in the optimum pH range. 



