THE NATURE OF VITAMIN REQUIREMENTS 325 



Especially with negative results, i.e., the finding that an organism 

 does not need a vitamin, still further precautions may be mentioned. 

 First, the basal medium must be free of the vitamin; even cotton plugs 

 may contribute growth factors (248, 251). Second, it is essential that 

 the organism be serially subcultured in the vitamin-free medium for 

 at least three transfers. In some investigations (131) the inoculum has 

 no effect, but this cannot be assumed. The criterion of serial subcul- 

 ture is the one most frequently disregarded, and in consequence most 

 reports of a lack of need for a vitamin — especially those, e.g., biotin, for 

 which the requirement is always small — are not wholly reliable. 



The use of crude extracts of natural products in the preliminary 

 search for vitamin requirements is, of course, helpful and, within lim- 

 its, valuable. However, the simple observation that such an extract 

 increases growth has no necessary bearing on vitamin nutrition, since 

 so many other materials, known and unknown, are present in these ex- 

 tracts. Thus, the effect of agar on growth of several fungi (132) and the 

 effect of yeast extract on cytochrome synthesis by Ustilago sphaerogena 

 (89) are both attributable to the zinc content of the natural product, not 

 at all to organic materials. 



The vitamin content of many common constituents of culture media 

 is known (34, 44, 95, 206, 217, 224, 264); occurrence in other natural 

 products is reviewed in general works on the vitamins (225, 249). 



2. THE NATURE OF VITAMIN REQUIREMENTS 



It is generally assumed that a fungus or other organism which is in- 

 dependent of an externally supplied vitamin is so by virtue of its ability 

 to synthesize the compound: a thiamine-independent fungus synthe- 

 sizes thiamine, etc. The validity of this assumption is borne out by 

 experimental evidence in all cases so far investigated, and is logically 

 deduced from the premise that the known vitamins have essential 

 functions in all cells. This premise, it should be noted, extends only 

 to the water-soluble or B vitamins. 



Further evidence, if such is needed, for the essentiality of vitamins 

 in those fungi which do not require an external supply is found in the 

 fact that deficient mutants can usually be obtained by appropriate tech- 

 niques; examples, in addition to the classical Neurospora crassa, in- 

 clude species of Penicillium (17) and Aspergillus (115, 195, 199), gen- 

 era in which naturally occurring vitamin deficiencies are uncommon. 



The simplest situation in vitamin studies is that of complete inabil- 

 ity of a given fungus to synthesize one or more vitamins. In this cir- 

 cumstance, the cell depends absolutely on an external supply and, at 



