CHAPTER IV
THE YEAST TEST FOR VITAMINE "B"
As far back as the days of Pasteur a controversy arose over the power of yeast cells to grow on a synthetic medium composed solely of known constituents. This controversy hinged on a discussion as to whether these media were efficient unless reinforced with something derived from a living organism. In 1901 Wildier in France published an article in which he showed that extracts of organic matter when added to synthetic media had the power to markedly stimulate the growth of yeast organisms. He did not attempt at the time to identify the nature of this stimulatory substance, but since it was derived from living organisms, he called it "Bios." Soon after the discovery of vitamines the bacteriologists began to discover that they or an analogous factor apparently played a part in the growth of certain strains of bacteria, especially the meningococcus. In 1919 Roger Williams working in Chicago University was struck with the bearing of Wildier's work on the vitamine hypothesis and formed the theory that Wildier's "bios" might be the water-soluble vitamine "B." He proceeded to test out this theory and demonstrated that extracts of substances rich in the "B" vitamine had a marked effect on the stimulation of yeast growth. He developed these experiments and devised a method of comparing the growth of yeast cells when stimulated by such extracts. The results were so striking as to appear to justify his view and he then suggested that his method might be used as a test for the measure of "B" vitamine in a given source. William's method consisted essentially in adding the extract of an unknown substance to hanging drops in which were suspended single yeast cells and observing the rate of growth under the microscope. Soon after, Miss Freda Bachman reinvestigated the problem with various types of yeast and found that practically all types of yeast respond to the stimulation of these "bios" extracts. Her method consisted in the use of fermentation tubes and the stimulatory effect was measured by the amount of CO_2 produced in a given time. By this method she confirmed Williams' view that the "bios" of Wildier was apparently identical with vitamine "B" and that most yeasts require this vitamine for their growth. She also suggested that her method might be made the basis of a test for vitamine content. In 1919 Eddy and Stevenson made extended experiments with these two methods in the attempt to improve the technique and make it serve as a quantitative measure. Their experiments served two purposes, first to bring out certain difficulties in the methods of the two authors from the quantitative viewpoint and the development of a technique to correct these difficulties and secondly to add more data bearing on the specificity of the test. Soon after their publication Funk became interested and coming to the same conclusions as to specificity devised a centrifugating method for measuring the yeast growth. Williams also improved his original method and devised a gravimetric method for the same purpose. From the viewpoint of methodology we now have methods which are suitable as quantitive procedures for determining the effect of extracts of unknown substances on yeast growth and hence if the stimulatory substance is vitamine "B," a means of determining within a space of twenty-four hours the approximate content of stimulatory material in a given source. Since the Funk method is the simplest of these and illustrates the principles involved it will suffice to describe that.
Funk method of yeast test with Eddy and Stevenson modification
1. To a basal diet of 9 cc. of sterile culture medium such as a von Nageli solution [Footnote: von Nageli's solution consists of the following ingredients NH_4NO_3, 1 gram; Ca_3(PO_4)_2, 0.005 gram; MgSO_4, 0.25 gram dextrose 10.0 grams made up to 100 cc. with distilled water. Other culture media may be used and such combinations will be found in any text on yeasts. They all permit a certain amount of growth but all are apparently stimulated by the addition of vitamine extracts.] in a sterile test tube is added 1 cc. of the sterile, neutral, watery extract of the source of the vitamine. A pure culture of Fleischman's yeast (Funk prefers brewer's yeast) is maintained on an agar slant and twenty-four hours before the test is to be made, a transplant is made to a fresh agar slant. One standardized platinum loopful of the twenty-four hour yeast growth is then used to inoculate the contents of the tube, the tube stoppered with cotton and incubated for from twenty-four to seventy-two hours at a temperature of 31°C. The seventy-two hour incubation period yields nearly optimum growth for this purpose.
2. At the end of this time the yeasts are killed by plunging the tube in water heated to 80°C. and maintained at this temperature for fifteen minutes. The contents of the tubes are then poured into a Hopkins centrifuge tube which has a capillary tip graduated in hundredths of a cubic centimeter. After twenty minutes centrifugating at a speed of about 2400 revolutions per minute the yeasts in the solution have all been packed into the tip and the volume can then be read accurately to thousandths of a cubic centimeter (with the aid of a scale and magnifier). With a control tube containing 9 cc. of the sterile media and 1 cc. of distilled water in place of the 1 cc. of extract a comparison can be obtained which is an accurate measure of the stimulatory effect of the extract. If this stimulus is due purely to vitamine it is obvious that this procedure would enable us to compare extracts of known weights of and arrive at comparisons which would be measures of their vitamine content. In other words the procedure is now in a satisfactory form for testing and its value depends merely upon our ability to show that the stimulus given the yeast is due solely to vitamine "B."
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.