The interest of the vitamine student in this test will be easily understood for it is so simple of manipulation and so rapid in producing results that it is the nearest approach to a chemical test of satisfactory nature yet proposed but unfortunately evidence soon began to accumulate to show that the stimulation produced by extracts of various sources is not a matter of pure vitamine. If we plot a curve of stimulation for various dilutions of a given extract we find that the stimulation is not directly proportional to the concentration of vitamine present but is a composite of several factors. The chart derived from experiments by Eddy and Stevenson shows the general nature of this curve. Other experimenters have reached similar results and some have gone so far as to maintain that the stimulation is not due to vitamine "B" at all. It is therefore evident that until this controversy is settled the yeast test cannot be used for the purpose proposed. Our own experiments at present make us still firm in our belief that _one_ of the factors and perhaps the most important factor in the stimulation effect is the vitamine but until we can devise a basal medium that is comparable to that used in rat feeding experiments, i.e., one that contains all the elements for optimum growth of yeasts except vitamine "B" it will be unsafe to draw conclusions from the test as to vitamine content. It may be possible to so treat our extracts as to eliminate from them all other stimuli except the vitamine or to destroy the vitamine in them and thus permit the comparison of an extract with the vitamine destroyed against one in which it is present and thus arrive at the result desired. At any rate all we can say at present is that the yeast test is unreliable as a measure of vitamine content but that if it can be made quantitative its advantages are so great that it is very much worth while to continue work upon it until it is certain that it cannot be made to produce the desired result. [Illustration: FIG. 7. GROWTH RATE OF YEAST UNDER ALFALFA EXTRACT STIMULATION This chart shows the effect of varying concentrations of an alfalfa extract on the growth rate of the yeast cell. The rate of growth was determined after the Funk method by centrifuging the cells after seventy- two hours incubation and measuring the volume in cubic centimeters. The shape of the curve shows that this method will not give comparative results unless the extracts tested are dilute enough for the determinations to fall in the steep part of the curve.] Another reason for our attention to this test is that if it can be made to show vitamine effect it provides an excellent medium for investigation of vitamine "B" reactions, and a method for studying the effect of the vitamine upon the protoplasm of a single cell. CHAPTER V THE SOURCES OF THE VITAMINE