INORGANIC SOURCES OF NITROGEN. 209 



yeast elements are required to induce fermentation." The limita- 

 tion implied by the words italicised was not determined until 

 twenty-two years later. 



WILDIERS (I.), in 1901, was the first to show, by the use of 

 pure cultures of top-fermentation beer yeast of the Sacch. cere- 

 visice 1., Hansen type, that neither fermentation nor yeast repro- 

 duction took place in 125 c.c. of a saccharified nutrient solution 

 of mineral salts, when only a very small number of yeast cells 

 were used for inoculation, e.g., about as many as are contained in 

 two drops of a culture grown in beer wort, or in 0.25-1.0 c.c. of 

 a mixture of pressed yeast with ten parts of water. On the other 

 hand, both fermentation and reproduction took place when the in- 

 oculation was accompanied by the addition of a few c.c. of a decoction 

 of yeast, or of Liebig's meat extract, peptone or wort. From these 

 observations Wildiers concluded that nitrogen in inorganic com- 

 bination is insufficient for the needs of theyeast cell, the growth and 

 fermentation also requiring a certain quantity of a special unknown 

 substance, absent from inorganic foodstuffs, and which he pro- 

 posed to term " Bios " (Gr. = Life). This substance is not an ash 

 constituent; it is destroyed (rendered inactive) by boiling in 

 20 per cent, sulphuric acid, can be dialysed, is soluble in water, 

 and can be extracted with this solvent from yeast (especially on 

 boiling). Yeast, though containing bios, is incapable of elaborat- 

 ing it ; so that when a small amount of yeast is taken for 

 inoculation, the quantity of bios introduced into the mineral 

 nutrient solution is insufficient for reproduction, whereas with a 

 more plentiful inoculation enough is introduced to allow new cells 

 to be formed at the expense of such as are moribund. 



Owing to their highly important bearing on the study of the 

 nutrition of yeast, these observations deserve a thorough experi- 

 mental investigation ; but at the outset they were yearly sub- 

 jected to deprecatory criticism, as being opposed to the ruling 

 dogma. Some asserted that the dependence of the result of the 

 experiments on the amount of the inoculation was due to the 

 presence, in Wildiers' nutrient solutions, of poisons, such as 

 copper, derived (in traces) from the distilled water or present in 

 the air of the laboratory, or ultramarine contained in the 

 commercial saccharose used in the experiments, although 

 Wildiers expressly stated that no difference in the results was 

 obtained by working with invert sugar. To bios was ascribed 

 the task of rendering these poisons innocuous, becoming thereby 

 itself inactive and unsuitable as a yeast food, the further quan- 

 tities, present only in larger sowings, being required for the needs 

 of the cells. This opinion was tested, in a series of exhaustive 

 experiments, by A. AMAND (I.), who showed that bios does not 

 play the part of an antidote. 



One of the next measures was to obtain quantitative data 

 on the new problem. Wildiers mainly judged the results of his 



