UNIVERSITY OF CALIFORNIA PUBLICATIONS IN AGRICULTURAL SCIENCES Vol. 3, No. 5, pp. 63-102 November 30, 1917 TOXIC AND ANTAGONISTIC EFFECTS OF SALTS ON WINE YEAST (SACCHAROMYCES ELLIPSOIDEUS) BY S. K. MITBA CONTENTS PAGE Introduction 64 Acknowledgments 65 Method of experimentation 65 Choice of solution 66 Method of determining the activity of yeast 67 Salts used 68 Single salts — Toxic effects 68 Combinations — Antagonistic effects 68 Binary combinations 68 Ternary combinations 68 Experimentation with single salts — Toxic effects 68 Series I. Potassium chloride 69 Series II. Magnesium chloride 71 Series III. Calcium chloride 72 Series IV. Sodium chloride 74 Series V. Effect of the toxicity of salts on microscopical appearance of yeast cells 75 Experimentation with combinations of salts — Antagonistic Effects 80 Series VI. Antagonism between Magnesium Chloride and Calcium Chloride 81 (a) Plants 83 (ft) Animals 83 (c) Bacteria 84 Series VII. Antagonism between Potassium Chloride and Calcium Chloride 84 (a) Plants 86 (&) Animals 87 (c) Bacteria 87 Series VIII. Antagonism between Magnesium Chloride and Sodium Chloride 87 (a) Plants 89 (6) Animals 89 (c) Bacteria 90 64 University of California Publications in Agricultural Sciences [Vol. 3 PAGE Series IX. Antagonism between Potassium Chloride and Sodium Chloride 90 (a) Plants 90 (&) Animals 90 (c) Bacteria 92 Series X. Antagonism between Potassium Chloride and Magnesium Chloride 92 (a) Plants 94 (6) Animals 94 (c) Bacteria 94 Series XL Antagonism between Calcium Chloride and Sodium Chloride .... 94 (a) Plants 95 (&) Animals 96 (c) Bacteria 96 Eelative antagonisms of various combinations 97 Summary 99 Part A. Toxic effects of single salts 99 Part B. Antagonistic effects of combinations of salts 99 Literature cited 101 Part A. Toxic effects of single salts 101 Part B. Antagonistic effects of combinations of salts 101 Introduction Most of the published studies of wine yeast deal with its activities as related to wine making. Its botanical characteristics and still less its fundamental physiological reactions have apparently received little attention. ' Among the most important and interesting investigations of higher plants, bacteria and animals in recent years have been studies of the effects of various single salts and various combinations of salts on the physiology of these organisms. For example, it has been found by Osterhout 6 ' 7 that practically all of the simple salts, such as sodium chloride, potassium chloride, calcium chloride, etc., have a decided toxic action upon the plant when it is subjected to the action of a single pure salt. Further, if certain combinations of two or more salts were used in certain ratios the toxicity was reduced. This reduction of toxicity is commonly termed antagonism between the salts used. It was found also that a combination of all the salts in the ratios in which they occur in the soil solutions or in other solutions to which the plant is accustomed afford the best conditions for growth. Such a combination is spoken of as a physiologically balanced solution. Loeb working with marine animals and C. B. Lipman with soil and other bacteria obtained results similar to those obtained by Oster- hout with the higher plants. As was to be expected, the reaction of animals to the salts was not identical with that of bacteria, nor does either reaction follow the behavior of the higher plants closely. In 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 65 general, however, the results with all three classes were of the same kind; that is, single salts, hitherto considered non-toxic, were found to be toxic to organisms, while various combinations of these salts showed antagonism or reduction of toxicity in the presence of each other. In accordance with these facts, a physiologically balanced solution can be made by using proper concentrations and proportions of the various salts found in the solutions to which the organism is accustomed. So far as the writer could ascertain, no one has investigated the behavior of yeast in this respect. Results of investigations of the effects of the heavy metallic salts, such as mercuric chloride, silver nitrate, etc., on yeast have been published (Bokarny 12 ), but nothing has appeared upon the toxic and antagonistic effects of such salts as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. This field therefore seemed especially inviting, and it was with the idea of studying the fundamental relations existing between yeast and the chlorides that the writer undertook the work summarized in the following pages. Acknowledgments The experiments on which this paper is based were carried out under the general supervision of Professor W. V. Cruess, and I am indebted to Professor P. T. Bioletti for suggestions and critical read- ing of the manuscript. Method of Experimentation In the selection of a yeast for my investigation I was led to use the wine yeast, Saccharomyces ellipsoideus, by the fact that it is one of the most useful of all yeasts and is universally used in wine making. It is also one of the most vigorous, is easy to grow, and gives definite results in a few days. The particular yeast, no. 66, used in this experiment, was isolated by William V. Cruess from one of the wineries in northern California. It has been found by repeated trials that specimens of Saccharomyces ellipsoideus collected from different sources are not identical as to their physiological characters in every respect, and so do not respond in different salt solutions in the same way. An experiment showing this will be described later. Although work on this line may not have immediate practical 66 University of California Publications in Agricultural Sciences [Vol. 3 value to the zymologist, it is of considerable scientific interest. It is with this thought that these experiments have been carried out in the Laboratory of Zymology.* The salts tested in these experiments were the chlorides of potas- sium, magnesium, calcium and sodium, each being taken up separately. The reason for choosing these chlorides was that their metallic ions (cations) are those most abundant in the ash of grape juice. Besides, Loeb 1 and Lipman 11 have shown that the positive ions of these salts have the most effect, while their negative ions (anions) have the least. Owing to the fact that the effect of the chlorine ion is uniform in all cases, the metallic ions show their characteristic effects on the yeast culture very clearly. Choice of Solution. — It has been shown by Loeb with marine ani- mals (Fundulus) , by Osterhout with higher plants (wheat), and by Lipman with soil bacteria (Bacillus subtilis) that, for the growth of living organisms, a nutrient solution must be physiologically balanced. In order to grow the yeast in a medium whose constituents were known both in quality and quantity, it was necessary to prepare a nutrient solution from pure materials. Such a solution must con- tain an adequate amount of nitrogen and phosphorus in order that the yeast may grow rapidly. For this purpose a number of sub- stances were tried, such as Witte's peptone, asparagin, urea, and ammonium phosphate, in different concentrations, with pure cane sugar or pure dextrose. Witte's peptone proved to be impure, being very high in ash, and the others did not give satisfactory results. As dextrose is not easily available in the market at present, pure cane sugar had to be used as a carbohydrate food and as a source of fer- mentable material. Later a synthetic solution was made with hydrolizecl pure cane sugar, phosphoric acid, and ammonia, which was suitable for the growth of yeast for experimental purposes. Although this synthetic solution produces a slower rate of growth than grape juice, which is a perfect physiologically balanced solution for yeast fermentation, it gives sufficient growth for experimental work. To make it, a 50 per cent pure cane sugar syrup was made with distilled water and a measured amount (1 gram per 100 c.c. of syrup) of phosphoric acid added. This syrup was hydrolized by boiling for one-half hour on a slow fire, and was then neutralized with dilute ammonium hydroxide. Litmus solution was used to test the neutrality of the syrup. The * These experiments were earried out under the general supervision of William Y. Cruess. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast f>7 ammonium phosphate that eventually formed furnished both the nitrogen and phosphorus needed by the yeast. As yeast eells grow easily in moderately acid, but not in alkaline solutions, the syrup was left slightly acid, being tested by titration with N/10 solution of sodium hydroxide. To facilitate the work, the syrup was boiled down to 65°Balling and put into a corked bottle. From this concentrated synthetic solution a measured quantity was drawn off and diluted with distilled water to 5°Bal. for use in the cultures. Method of Determining the Activity of the Yeast. — The experi- ments were carried on in a series of 200 c.c. Erlenmyer flasks. To each flask the weighed amount of salt was added and 100 c.c. of the diluted synthetic solution placed in the flask by means of a 100 c.c. pipette. The salts were weighed according to their respective molecular concentrations. The flasks as soon as filled were plugged with cotton and sterilized. After they had cooled down to the room temperature they were inoculated with the yeast from a new culture. For this purpose the new culture was prepared in a 200 c.c. Erlenmyer flask containing 100 c.c. of the synthetic solution. The yeast thus became habituated to this solution and therefore grew rapidly and uniformly in the flasks. The new culture was transferred from a mother culture in grape juice and put into the incubator for forty-eight hours at 28° C. At the end of this period the flasks containing the salts were inoculated with one cubic centimeter of the new culture. After inoculation, the flasks were put into the incubator, which was kept at an approximately even temperature of 28° C. during the entire experiment. As the alcoholic fermentation in the synthetic solution was not rapid enough to serve as a criterion, the multiplication of the cells was taken as the measure of the activity of the yeast. Accordingly a microscopical count was made every forty-eight hours with a cali- brated microscope. Five counts were made in each experiment, during a period of about twelve days. In every case two blanks, with no added salts, were made up, and the tables given represent the average of two sets of duplicate experiments, except in the cases of potassium chloride and magnesium chloride, where the results were so close that only the first set of duplicates was used. It may be added that the incubator did not keep exactly the same temperature throughout the experiments, but ranged from 27 °C. to 29 °C. This difference of 2°C., however, did not interfere appreciably with the uniformity of growth 68 University of California Publications in Agricultural Sciences [Vol. 3 and the check flasks controlled any slight variation it may have caused. Salts Used. — During February, 1916, a series of experiments in two parts was planned. The first concerned the toxicity of the single salts and the second the antagonistic effects of all their binary and ternary combinations, as follows: I. Toxicity of the Single Salts 1. KC1 3. CaCl 2 2. MgCL 4. NaCl II. Antagonistic Effects of Combinations A. Binary Combinations 1. MgCl 2 + CaCL 4. KC1 + NaCl 2. KC1 + CaCL 5. KC1 + MgCL 3. MgCl 2 + NaCl 6. CaCL + NaCl B. Ternary Combinations 1. NaCl + KC1 + CaCL 3. NaCl + MgCL + CaCL 2. NaCl + KC1 + MgCL 4. KC1 + CaCL + MgCL A. Experiments With Single Salts — Toxic Effects In all cases chemically pure salts (Baker's analyzed) were used. Each amount of the single salts was carefully weighed and put into the flasks, except .001M and .01M, which were added as solutions of known strength according to molecular weights. The following pro- portions were taken: 1. KCl — .001M to 2.2M* Molecular 2. MgCL — .001M to 1.2M 3. CaCl 2 — .001M to .7M 4. NaCl — .001M to .2M All of these solutions were clear except the calcium chloride, which gave an appreciable amount of coagulated precipitate of calcium phos- phate with the phosphorus of the synthetic solution. This, however, did not interfere with the experiment, as the precipitate disappeared with the growth of the yeast and the solution finally became almost clear. The tables and curves given in each case show the growth of yeast at every forty-eight hours in the different molecular concen- * M represents the degree of concentration in a solution which contains one gram molecule of the substance in one litre of solution. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 69 trations of each salt as indicated above. In the microscopical count one million yeast cells per cubic centimeter was taken as an appreci- able number ; below that it was not considered that any appreciable growth had taken place. SERIES I— POTASSIUM CHLORIDE Thirty 200 c.c. Erlenmyer flasks were arranged in duplicate, in- cluding two blanks. The first pair had no salt added and was used as a check. The second pair had .001M KC1 and the third .01M KC1, and so on to the last pair, which had 2.2M KC1, as shown in the table below. Then, with a 100 c.c. pipette one hundred cubic centimeters of the diluted synthetic solution (5°Bal.) were put into each flask. The flasks were plugged with cotton, sterilized, and inoculated with yeast, as stated before, and put into the incubator, and counted every forty-eight hours. The results are shown in table 1 and the curves in figure 1. The curves have been plotted from the results of every forty-eight hours' growth, taking the various concentrations of potassium chloride as abscissae and the number of yeast cells, counted in millions, as ordinates (fig. 1). Following the table and the curves, it is evident at a glance that potassium chloride up to the concentration of .2M accelerates the multiplication of the yeast. Bej^ond this it becomes gradually more and more toxic until at 2.2M the yeast cells entirely cease to multiply. Both Magowan 10 and Lipman, 11 especially the former, found a strong resemblance between potassium chloride and sodium chloride in their action on wheat and on Bacillus subtilis. The yeast shows physiological characteristics differing from those of either the bacteria or the wheat. Lipman 11 found sodium chloride the least toxic to Bacillus subtilis and potassium chloride second. Magowan, 10 with wheat, found this position reversed, the potassium chloride being less toxic. Both ob- servers found that these salts were very similar in their degree of toxicity. To yeast sodium chloride is the most toxic of the four salts and potassium chloride the least. Ostwald 4 in experimenting with animals (Grammarus) found potassium chloride the most toxic, and Loeb 's work 2 ' 3 with Fundulus corroborates this to a certain extent. The reaction of yeast, therefore, differs from that of bacteria of the higher plants or of animals. 70 University of California Publications in Agricultural Sciences [Vol. 3 Table 1 — Toxic Effect of KCl on Saccharomyces ellipsoideus M.KCl 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. .00 1,695,000 7,237,000 11,636,000 12,892,000 15,835,000 .001 3,325,000 10,786,000 15,400,000 16,217,000 19,670,000 .01 7,797,000 15,600,000 18,905,000 21,479,000 23,568,000 .1 7,262,000 20,558,000 24,208,000 24,608,000 27,915,000 .2 5,650,000 18,493,000 29,689,000 28,783,000 30,541,000 .4 5,425,000 20,227,000 25,006,000 27,560,000 28,914,000 .6 1,130,000 8,535,000 20,928,000 22,802,000 23,802,000 .8 809,000 5,298,000 17,670,000 19,508,000 20,982,000 1.0 226,000 6,787,000 15,205,000 17,986,000 18,453,000 1.2 1,102,000 8,986,000 16,207,000 16,951,000 1.4 452,000 8,765,000 11,584,000 12,882,000 1.6 3,204,000 5,794,000 8,232,000 1.8 904,000 1,243,000 6,252,000 2.0 904,000 2,051,000 2.2 226,000 A .6 .8 1.0 1.2 Concentration of salt Fig. 1. — The ordinates represent millions of yeast cells and the abscissae, the various concentrations of KCl. The ordinates at represent the number of yeast cells in the check cultures. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 71 SERIES II— MAGNESIUM CHLORIDE The same method of procedure was used with MgCl 2 as with KC1. The results are shown in Table 2. Table 2- -Toxic Effect of MgCl 2 on S. ellipsoideus M. MgClo 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. .00 2,412,000 8,108,000 12,205,000 16,837,000 17,202,000 .001 4,972,000 10,753,000 13,673,000 18,871,000 19,775,000 .01 8,751,000 15,798,000 18,703,000 19,588,000 20,374,000 .1 3,482,000 12,227,000 20,521,000 25,013,000 26,501,000 .2 2,356,000 7,204,000 11,342,000 15,530,000 18,617,000 .4 1,695,000 5,400,000 9,504,000 12,837,000 17,413,000 .6 989,000 2,157,000 7,332,000 11,253,000 11,905,000 .8 226,000 1,130,000 4,294,000 6,943,000 8,436,000 1.0 226,000 1,875,000 2,712,000 2,904,000 1.2 226,000 .01 .1 .2 .4 Concentration of salt Fig. 2. — The ordinates represent the number of yeast cells in millions and the abscissae, the concentration of magnesium chloride. The ordinate at represents the number of yeast cells in the check cultures. 72 University of California Publications in Agricultural Sciences [Vol. 3 From both table 2 and the curves in figure 2, it is evident that magnesium chloride is more toxic than potassium chloride. Up to the concentration of .1M, it is favorable to the growth of yeast, but beyond this it becomes more and more toxic until at 1.2M concen- tration there is little or no growth at all. In the case of yeast, mag- nesium chloride and calcium chloride show less similarity than that found by Lipman with soil bacteria, and the toxic effect of magnesium chloride is nearer to that of calcium chloride than to that of potassium chloride. Magnesium chloride is not so toxic to yeast as Lipman found it with Bacillus subtilis. In the case of yeast, .7M concen- tration of calcium chloride altogether inhibits its growth. The same concentration, however, of magnesium chloride allows an appreciable number of yeast cells to grow, and the same toxic effect as that of .7M CaCL is not attained until a concentration of 1.2M MgCl 2 is reached. In fact, magnesium chloride stands midway between the two extremes of toxicity of these four salts, namely, the more toxic NaCl and CaCl 2 and the less toxic, KC1. Loeb, 1, 2 with marine organisms, found that a .5M solution of magnesium chloride inhibits the development of embryos in the eggs of Fundulus, and that even .125M Ca(N0 3 ) 2 is toxic. In his experi- ment with soil bacteria Lipman 11 has met with about the same result. He found that .4M MgCl 2 inhibits the growth of Bacillus subtilis, while for the same effect on yeast a concentration of 1.0M MgCl 2 is needed. But Magowan 10 has shown that with wheat magnesium chlor- ide is the most toxic of all the four salts ; in this respect yeast resembles neither the animals, nor bacteria, nor the higher plants. It must be noted that the magnesium chloride used in all the experiments was MgCl 2 .6H 2 0, as this is less hygroscopic than the same salt having two molecules less of water (MgCl 2 .4H 2 0), which is difficult to weigh accurately. However, magnesium chloride was found more toxic than potassium chloride and more favorable than calcium chloride, which is directly opposite to the result obtained with higher plants. SERIES III— CALCIUM CHLORIDE The experiment with calcium chloride was carried on in the same Avay. From both table 3 and the curves in figure 3, it is evident that .01M concentration of CaCl 2 gives the highest growth, while beyond this favorable concentration CaCL is more and more toxic. In its 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 7:i toxicity to yeast, CaCl 2 stands second, NaCl being the most toxic of the four salts. A .5M concentration of CaCL allows an appreciable growth of yeast, while even ,2M NaCl stops all growth. Table 3 — Toxic Effect of CaCl 2 on S. ellipsoideus M. CaCI 2 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. .00 2,599,000 8,136,000 11,752,000 13,108,000 16,336,000 .001 3,051,000 9,989,000 • 12,656,000 13,965,000 17,751,000 .01 3,503,000 11,050,000 13,926,000 15,885,000 19,251,000 .1 226,000 6,034,000 8,468,000 10,904,000 16,674,000 .2 '. 1,695,000 3,256,000 3,821,000 15,778,000 .3 904,000 1,130,000 3,090,000 14,561,000 .4 226,000 904,000 1,130,000 9,771,000 .5 226,000 987,000 2,935,000 .6 226,000 904,000 .7 226,000 19 18 16 ( 8 14 & 12 / y i \ tvJ •> \ 3 s - w o c 10 8 8< 6 4 \ v\ / i Vs \ W \ l<5> V* \ ° \ *- \ ^ \ ^ s* < 2 \ c » .001 .1 .2 .3 .4 .5 .6 .7 Concentration of salt Fig. 3. — The ordinates represent the number of yeast cells in millions and the abscissae the concentration of CaCL. The ordinate at represents the number of yeast cells in the check cultures. The work of other investigators with regard to the toxicity of CaCl 2 shows a general agreement with the results obtained by Loeb with Fundulus, 3 Ostwald 4 with fresh water, Grammarus, and Lip- man 11 with soil bacteria, all of which show CaCL to be extremely toxic. An exception to this general statement is found in the work of 74 University of California Publications in Agricultural Sciences [Vol. 3 Magowan, who showed in her experiments that, in the case of wheat, CaCl 2 is the least toxic of the four salts. Here also we find that yeast exhibits a peculiar physiological character which does not agree with either of the above divisions, the animals or the plants. Perhaps this may throw some light on the relation of yeast to these two groups. SEKIES IV— SODIUM CHLORIDE As NaCl is the most toxic of all the salts, only a few pairs of flasks were taken, from .001M to .2M concentration, together with a pair of blanks. The experiment was carried on in the same way as the others. From table 4 and the curves in figure 4, it is evident that NaCl is the most toxic to the yeast. Even the concentration of .01M NaCl did not stimulate the growth of yeast, as it did with the other salts. The highest growth in this case was in .001M, and beyond that it was toxic. Table 4- -Toxic Effect of NaCl on 8. ellipsoideus M.NaCl 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. .00 2,424,000 8,059,000 9,267,000 11,978,000 15,142,000 .001 3,819,000 9,605,000 12,430,000 12,995,000 17,967,000 .01 3,164,000 7,458,000 10,283,000 10,504,000 13,060,000 .1 226,000 452,000 452,000 452,000 1,130,000 .2 226.000 18 16 « 14 ■S 12 10 tf V ^>7 if* $^ .001 .01 .1 .2 Concentration of salt Fig. 4. — The ordinates represent the number of yeast cells in millions and the abscissae, the concentration of NaCl. The ordinate at represents the number of yeast cells in blank cultures. 1917] Miira: Toxic and Antagonistic Effects of Salts on Wine Yeast 75 NaCl shows a directly opposite reaction witli yeast from that found by Lipman 11 with soil bacteria. Both Loeb and Ostwald found NaCl to be toxic for animals, but less so than we have found with yeast. The toxicity of NaCl to animals may be compared with the toxicity of NaCl 2 to yeast. Loeb 3 found it impossible to develop embryos in the egg of Fundulus at .625M NaCl. Osterhout 8 ' 9 found that a .375M solution of sodium chloride is fairly toxic to marine plants. Young plants of a fresh-water alga, Vaucheria sessilis, could not live at a .094M concentration of sodium chloride, and even a concentration of .0001M NaCl was found to be toxic. Magowan has shown that sodium chloride is very toxic to wheat seedlings and down to .02M the root hairs did not grow at all. The relation of yeast to plants is thus to a certain extent shown by similar physiological behavior. It may be noted here that the experiments with yeasts have been conducted on the same general principle followed by previous investi- gators with animals, plants, and bacteria. The number of yeast cells was taken as the measure of multiplication or activity and was de- termined by a microscopical count of each flask every forty-eight hours. It must be admitted that experimental errors may occur in counting, but as the numbers were taken from the average results of two sets of duplicates it does not interfere with the validity of the final result, as the range of variation between the results of these two sets of duplicates was only between and 10 per cent calculated from the mean variation. SERIES V— EFFECT OF THE TOXICITY OF SALTS ON THE MICROSCOPICAL APPEARANCE OF YEAST CELLS It is generally known that all salts at certain concentrations are more or less toxic to living organisms. Yeast shows its physiological condition in relation to various salts in characteristic ways. It is evident from the above experiments that in this respect it occupies a place between the animal and the plant kingdoms. Although yeast grows normally in a physiologically balanced solution, for which grape juice answers in every way, the addition of a small amount of a favorable salt, as potassium chloride, may stimulate the growth a great deal. This is of some practical zalue to zymologists. Yeast is affected very remarkably by the toxicity of salts at dif- ferent concentrations. In the extreme concentrations it apparently 76 University of California Publications in Agricultural Sciences [Vol. 3 dissolves. This occurs in the cultures having 2.2M KC1, 1.2M, MgCl 2 , .7M CaClo, and .2M NaCl respectively. At lower concentrations there is a degenerated condition, various shapes occurring, as shown in figure C. Such diseased cells show a heavy black membrane, especially in the case of CaCl 2 and NaCl, with transparent cell-illusions or black spots within the cells. Moreover, they vary in size. This variation in size occurs also with KC1 and MgCl 2 , but in these cases the yeast cells are larger than with CaCl 2 and NaCl. In all instances, as the concentration of salt increases beyond the favorable degree of con- centration the cells become smaller and smaller until finally, in the extreme concentrations, they dissolve. Table 5 (a. b, c, d) and the curves in figure 5 {a, b) show the effect on the size of yeast cells in different salt solutions. Table 5a — Effect of KCl on Size of Yeast Cells (S. ellipsoideus) Concentration of salt (M.KC1) Av. length and breadth of yeast cells in Mu. Av. volume yeast cells calculated from length and breadth .00 4.7 X 4.6 77 .001 5.4x5.4 122 .01 6.7x6.7 232 .1 6.7x6.7 232 .2 6.7x6.7 232 .4 6.6 x 6.6 223 .6 6.6 x 6.6 223 .8 5.8x5.8 151 1.0 5.8x5.8 151 1.2 5.8x5.8 151 1.4 5.8x5.8 151 1.6 4.9x4.9 91 1.8 4.9 x 4.9 91 2.0 3.3x3.3 28 2.2 3.3x3.3 28 •Effect of MgCl 2 on Size of Yeast Cells (S. elli\ Concentration of salt (M.MgClo) Av. length and breadth of yeast cells in Mu. Av. volume yeast cells calculated from length and breadth .00 4.5x4.5 71 .001 5.1x5.1 103 .01 6.2 x 6.2 185 • .1 6.2x6.2 185 .2 5.0x5.0 98 .4 5.1x5.1 103 .6 5.0x5.0 98 .8 4.9x4.9 91 1.0 4.4 x 4.4 66 1.2 3.3 x 3.3 28 1917 J Ultra: Toxic and Antagonistic Effects of Salts on Wine Yeast 77 Table 5c— Effect of CaCl 2 on Size of Yeast Cells (S. ellipsoideus) Concentration of salt (M.CaClo) .00 .001 .01 .1 .2 .3 .4 .5 .6 .7 Av. length and breadth of yeast cells in Mu. 4.7x4.4 Av. volume of yeast cells calculated from length and breadth 70 4.6x4.6 75 4.1x4.1 53 3.3x3.3 28 3.3x3.3 28 2.8x2.8 17 2.8x2.8 17 2.8x2.8 17 2.6x2.6 14 2.6x2.6 14 Table 5d — Effect of NaCl on Size of Yeast Cells (S. eUipsoides) Concentration of salt (M.NaCl) .00 .001 .01 .1 .2 Av. length ind breadth of of yeast cells in Mu. Av. volume of yeast cells calculated from length and breadth 5.1x4.8 91 5.0x4.4 75 4.4x3.3 37 4.1x3.3 34 2.6x2.6 14 .001 .01 .8 1.0 1.2 1.4 Concentration of salt 1.6 1.8 2.0 Fig. 5a. — Curves showing the average relative volumes of yeast cells in various concentrations of CaCL and MgCl 2 . The ordinates represent the average volume of the yeast cells and the abscissae, the concentrations of KC1 and MgCL used. The ordinate at represents the volume in blank cultures. 78 University of California Publications in Agricultural Sciences [Vol. 3 200 1 9 5 100 ^ L* )^ \ ,N ) c .001 .01 .1 .2 .3 .4 .6 .7 Concentration of salt Fig. 5b. — Curves showing the average relative sizes in volumes of yeast cells in various concentrations of KC1 and MgCL. The orclinates represent the volume of the yeast cells and the abscissae, the concentrations of the salts used. The ordinate at represents the volume in blank cultures. Fig. 5c. — Appearance of yeast cells in extreme concentrations of salts. Normal yeast cells in 1, 2 and 3, diseased yeast cells from extreme concen- trations of KC1 and MgCl 2 in 4 and 5; (white) and diseased yeast cells from extreme concentrations of CaCl 2 and NaCl in 6, 7 and 8 (black or shadowy) (X5000). 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 79 The measurements given are the average of five counts in each case. The volumes from which the curves have been drawn in figure 5 (a, b) have been calculated, for purposes of comparison, as though the cells were cylindrical. Both from table 5 (a, b, c, d) and the curves in figure 5 (a, b) it is evident that KC1 and MgCl 2 favor growth in size up to the most favorable concentration, beyond which the cells decrease in size until the extreme concentration is reached, where they dissolve. Both NaCl and CaCl 2 limit the growth even in minute concentrations, thus show- ing their extreme toxicity to yeast cells. Yeast cells seem to have remarkable resistant power. Many of them with cell wall thickened to a heavy membrane have been found in extreme concentrations. Perhaps this heavy membrane is formed to resist the osmotic pressure outside the cell. Besides some of the cells in these extreme concentrations are in norma] condition and are even budding, thus showing the power of adaptability of yeast cells to new conditions. After they have become habituated to the presence of toxic salts, they grow normally and reproduce. It is probably owing to fhe adaptability of yeast to different conditions that the same yeast, S. ellipsoideus, collected from various sources, shows dis- similar physiological characters. Besides in many cases I have observed that the diseased yeast cells in extreme toxicity of KC1 and MgCl 2 form a white membrane with normal cell contents, while those of CaCl 2 and NaCl form a rather dark cell membrane with shadowy cell contents. A similar case to that of Loeb 5 ' 13 may be cited here. In his experiments with sea urchin eggs he found two distinct phases of cytolysis which he terms "black cytolysis" and "white cytolysis." With regard to the effect of the salts on the size of the yeast cells, NaCl is the most and KC1 the least toxic, while CaCl 2 and MgCL stand midway. The effect is parallel with that of the multiplication of cells. An experiment was carried on with a second culture of 8. ellip- soideus collected from another source by Cruess and named no. 60. This experiment was also made in duplicate. With this yeast potas- sium chloride and magnesium chloride gave the same results as with no. 66, but NaCl and CaCl 2 showed a marked difference and CaCl 2 was the most toxic of all. 4M NaCl gave an appreciable number of yeast cells, while even .3M CaCl 2 stopped the growth altogether. Further, the number of yeast cells was much lower than that of yeast no. 66. Evidently yeast no. 60 was less vigorous than the other, though other- wise there was no fundamental difference between them. 80 University of California Publications in Agricultural Sciences [Vol. 3 B. Experiments With Combinations of Salts — Antagonistic Effects The toxic effects of the single salts KC1, MgCl 2 , CaCL and NaCl upon a wine yeast, 8. ellipsoideus, have been shown in the first part of this paper. The results of the study indicate that the reactions of yeast differ from those of plants, animals or bacteria. This second part of the paper gives the results of an investigation to ascertain the effects of various binary combinations of the salts named upon the same yeast. From the four salts, six combinations of two salts each are possible. All of these were tested. Judging from analogous work of other investigators with animals, plants and bacteria, it was expected that these salts would exhibit mutually antagonistic action, i.e., that the toxicity of one salt would be reduced by the presence of another and that the total effect of two salts together would be less than the sum of their individual effects. In some cases definite antagonistic effects were found. In others antagonism was not so well defined. In a few instances there was no antagonism shown. In the discussion of results, considerable space has been given to the findings of other investigators because it was considered important to point out how the effects on other organisms compare with those on yeast. A few words on the development of the idea of antagonism in binary combinations of salts will be of value as an introduction to the data in this paper. Considerable work on the antagonistic effects of salts has been done by Einger, Locke, Howell, Loeb, Osterhout, Overton, Ostwald, Loew, Lipman and others. That the poisonous effect of one salt is reduced by the addition of another salt has been known for a long time, especially among animal physiologists. In this matter we owe a great deal of our knowledge to Loeb, whose investigations brought forth a large number of unexpected results. It was he who first developed the theory that the valences of metallic ions have consid- erable influence on their toxic and antagonistic effects, and that mono- valent cations may be antagonized ; by bivalent, trivalent or tetravalent but not by monovalent cations. His results show some parallelism to the work of Linder and Picton.* This general statement does not apply in all cases to plants, animals and bacteria, experimented upon by various other investigators. Neither does it apply always to yeast. * Hober and Gordon, Beitr. zur chem. physiol., vol. 5, p. 432, 1904, cited by Osterhout. 22 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 81 The experiments with binary salts were made in the same general way as those with simple salts, but with slight modifications of tech- nique. The flasks were arranged as before in duplicate, but in com- bining the salts in different molecular concentrations the method followed differed from those of previous investigators. Of the two salts to be tested for antagonism, one was weighed from the minimum concentration to that of extreme toxicity according to the molecular concentration, and the other was weighed and added to the former in the reverse way in the corresponding flasks. The flasks containing the extreme concentration of each salt did not receive any addition of the other salt. Aside from this, the methods of in- oculation, incubation, and microscopical counting were the same as those described for the single salts. Duplicates were made in all cases and two blanks were used in each series, as checks on the growth of the yeast in the treated flasks. The same yeast, S. dlipsoideus, no. 66, was employed in these experiments as in the ones with simple salts. The results given are therefore the average of duplicate experi- ments. SEEIES VI— ANTAGONISM BETWEEN MAGNESIUM CHLOKIDE AND CALCIUM CHLORIDE In this series MgCl 2 and CaCl 2 were combined in various mole- cular concentrations. A series of 16 Erlenmyer flasks was arranged in duplicate with two blank cultures. First, amounts of MgCl 2 cor- responding to from OM to 2.2M were weighed and put in the flasks, as was done with the single salts. The CaCl 2 also was weighed ac- cording to its molecular concentration and put in the same flasks in reverse order, leaving the extreme concentrations of each salt free from the addition of the other. Thus the first two flasks received .72M CaCl 2 without any addition of MgCl 2 ; the second received .66M CaCl 2 and .001 MgCl 2 ; the third .60M CaCl 2 and .01M MgCl 2 , and so on to the last couple, which contained only 1.2M MgCl 2 and no addition of CaCl 2 . The remaining flasks were combined in different molecular concentrations, as shown in table 1. Two blanks were taken to which no salt was added. In order to facilitate the plotting of the curves, the different combinations of salts have been indicated by letters A, B, C, D, etc. A represents the blank cultures, while the other letters represent the different molecular combinations shown in the table below: 82 University of California Publications in Agricultural Sciences [Vol. 3 Table 6 — Antagonistic Effect Between MgCl, and CaCl No. MgClo vs. CaCl 2 M. Cone. 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. A .00 x.0.0 1,954,000 6,290,000 10,556,000 14,108,000 16,944,000 B .00 .00] x.72 x.66 n 226,000 4,520,000 452,000 D .01 x.60 452,000 2,034.000 3,842,000 5,650,000 E .1 x.48 8,362,000 20,860,000 23,120,000 24,730,000 26,842,000 F .2 x.36 10,848,000 26,024,000 29,706,000 30,856,000 31,960,000 G .4 x.18 9,718,000 28,996,000 32,284,000 33,974,000 36,120,000 H .6 x.06 8,804,000 25,286,000 30,256,000 31,865,000 32,556,000 I .8 x.01 452,000 4,972,000 8,289,000 10,298,000 12,684,000 J 1.0 x .001 226,000 1,130,000 2,260,000 3,129 000 T\ 1.2 x.00 A B MgCl 2 D E F G H Concentration of salts K CaCl 2 Fig. 6. — Curves of yeast growth showing antagonism between MgCL and Cacl 2 . The ordinates represent the number of yeast cells in millions and the abscissae, the concentration of the salts in combination. The ordinates at A represent the number of yeast cells in blank cultures. 1917] Ultra: Toxic and Antagonistic Effects of Salts on Wine Yeast Hi', From both table 6 and the curves in figure 6 it is evident that there is a distinct antagonism between these two salts. For example, in the experiments with simple salts MgCl 2 alone at .8M concentration allowed the growth of yeast cells up to only 8% millions, but in com- bination with .01M CaCl 2 the growth was increased up to 12% millions, i.e., 50 per cent increase. Similarly, .6M CaCL alone allowed an increase to about one millions, and, with the addition of .01 MgCl 2 , an increase to 5% millions, showing 5% times more growth. The highest number in MgCl 2 alone was 26% millions at .1M, and in CaCl 2 alone 19 millions at .01M concentration. In this binary combination the highest number was obtained at G, the point where AM. MgCl 2 and .18M CaCL were combined with a ratio of about 2 :1. For purposes of comparison let us now consider the results obtained in similar experiments with these four salts on plants, animals, and bacteria. (a) Plants. — Kearney and Cameron 8 found a distinct antagonism between Mg and Ca ions for higher plants. In their experiments with leguminous plants Lupinus albus and Medicago sativa they found that, for a combination of these two salts, the plants show about five times as much tolerance as for the salts separately. The plants also dis- played a remarkable degree of tolerance when MgS0 4 was used in- stead of MgCL, thus showing in addition the relative difference be- tween different anions of the same salt. Loew and his pupils, 10 ' 18 in their experiments with lower plants (Spirogyra), have found a strong antagonism between Mg and Ca ions. (6) Animals. — Loeb 2 with sea urchins (blastulae and gastrulae) found that a mixture of MgCl 2 (10/8n) and CaCL (10/Sn) will allow them to swim for about forty-eight hours, while each of the salts singly at the same concentration is extremely poisonous and kills the animals. The same investigator 15 working with a jellyfish (Poly or- chis) has shown that the addition of a small quantity of CaCL to a mixture of NaCl and MgCl 2 favors the normal, rhythmical contrac- tions, while MgCL alone stops them altogether. Contrary to the above results, Loeb 12 in his experiments with frogs has found that a combination of Mg and Ca ions completely inhibits the rhythmical muscular contractions. This has been corroborated by Anne Moore, 7 in her experiments with the contraction of the lymph hearts of frogs. Lillie 6 has found that the ciliary movement of the larvae of 84 University of California Publications in Agricultural Sciences [Vol. 3 Arenicola goes on normally for a time in a mixture of MgCl 2 and CaCl 2 with the ratio of 4:1 at 10/8n concentration, though either of the two salts used alone would stop it entirely. Matthews, 11 in his work with the development of embryos in the eggs of Fundulus, found a distinct antagonism between Mg and Ca. Meltzer and Auer 21 have shown with rabbits and a monkey that the poisonous action of MgCl 2 in subcutaneous injection is similarly diminished by the injection of CaCl 2 . They found also a strong antagonism between the nitrates, acetates and sulfates of these two salts respectively. (c) Bacteria. — Lipman, 23 ' 24 with a soil bacterium, Bacillus siib- tilis, found little or no antagonism between the two salts, but, on the contrary, the addition of one salt to the other was found to be more toxic than either of the two salts used alone. All of the above mentioned experiments, except those of the three cases of Lipman, Loeb, and Anne Moore, are in agreement with the antagonistic effects between Mg and Ca ions that occur with yeast. In addition, it may be noted here that the antagonistic effect between MgCl 2 and CaCl 2 with yeast has been found to be the strongest of all the combinations. This corroborates the opinion advanced by Loew that there is a strong antagonism between calcium and magnesium both with plants and animals. 10 SERIES VII— ANTAGONISM BETWEEN POTASSIUM CHLORIDE AND CALCIUM CHLORIDE In this series the experiments were carried on in the same way as with MgCl 2 and CaCl 2 . Table 7 and the curves in figure 7 show there is a distinct antagonism between the two salts. In this case marked antagonism was found on the side of CaCl 2 , but little or none on the side of KC1. For example, the combination of .001M KC1 with .66M CaCl 2 allowed the yeast to grow up to 6% millions, while in CaCl 2 at .6 alone the yeast was found to increase only up to about one million. Thus there was 6!/2 times as much growth where the KC1 was present. But, on the other hand, the combination of .001M CaCl 2 to 2.0M KC1 did not accelerate the growth. This unexpected result may be accounted for by the fact that the higher concentrations of KC1 being very high in comparison to the small concentrations of CaCl 2 the latter was not sufficient to reduce the toxicity of the KC1 at such a high concentration. It is also very probable that a concen- 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 85 tration of 2.0M KC1 exerts a strong osmotic effect and that the toxicity is due to osmotic influences rather than to the usual toxicity of the ion itself. If this were true we would expect little antagonism from other salts. Loeb 12 in his experiments with a jellyfish (Gonionemus) met with a similar difficulty. In this case the KC1 concentration was so high that a small concentration of NaCl did not remove the toxicity, and so the combination inhibited the contraction of the animal, while the same concentrations used in the case of another kind of fish, Fundulus, allowed the development of embryos in the eggs. He has pointed out the fact that in the embryos of Fundulus the solutions in which cleav- age proceeds normally interferes seriously with the heartbeat of Coni- onemus, if the proportion of KC1 exceeds a certain limit. In this instance we find proof of the fact that in the same organism cell- division and muscular contractility are influenced by entirely different combinations of ions, and therefore these vital activities must depend on widely different chemical constitutions. However, the highest growth in the case of yeast was obtained at H, where .6M KC1 and .36M CaCl 2 have been combined, a ratio of about 2:1. In the case of KCl alone the highest growth was obtained at .2M concentration, allowing growth up to 3OV2 millions per c.c. CaCl 2 allowed growth up to 19 millions at .01M concentration. Table 7 — Antagonistic Effects Between KCl and CaCl, No. KCl vs. CaCl 2 M. Cone. 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. A .00 x.00 2,101,000 8,589,000 13,908,000 16,896,000 17,520,000 B .00 .00] x.72 x.66 <^ 226,000 2,034,000 3,985,000 6,722,000 D .01 x.60 452,000 4,972,000 13,315,000 ] 8,604,000 22,720,000 E .1 x.54 4,020,000 10,328,000 20,245,000 23,266,000 25,120,000 F .2 x.48 5,558,000 12,840,000 22,190,000 26,880,000 29,380,000 G .4 x.42 3,034,000 12,840,000 26,852,000 19,126,000 32,285,000 H .6 x.36 1,017,000 8,398,000 13,645,000 28,904,000 34,500,000 I .8 x.30 226,000 4,256,000 10,250,000 14,966,000 18,732,000 ,T 1.0 x.24 2,965,000 6,126,000 3,986,000 9,551,000 16,159,000 K 1.2 x.18 1,130,000 5,410,000 7,119,000 T, 1.4 x.12 452,000 2,550,000 2,712,000 4,438,000 M 1.6 x.06 904,000 1,130,000 3,906,000 TsJ 1.8 2.0 x.01 x.001 452,000 904,000 226,000 2,652,000 O 1,130,000 P 2.2 x.00 86 University of California Publications in Agricultural Sciences [Vol. 3 C D KC1 F G H I J K Concentration of salts M N O CaClo Fig. 7. — Curves of yeast growth showing antagonism between KC1 and CaCL. The ordinates represent the number of yeast cells in millions and the- abscissae, the concentration of salts in combination. The ordinate at A represents the number of yeast cells in blank cultures. For comparison with these results, a number of cases dealing with plants, animals and bacteria may be cited below : (a) Plants. — Osterhout 22 has shown that for higher plants a com- bination of 100 c.c. KC1 and 5 c.c. CaCL at the concentration of .12M is best suited for the highest development of roots. Benecke 19 has shown that for lower plants (Spirogyra) the harmful effect of the K ion is very distinctly antagonized by the addition of the Ca ion at a certain definite concentration. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 87 (b) Animals. — In regard to the development of embryos in the eggs of Fundiilus, Loeb 1 has met with a marked antagonism between the two salts, using 75 c.c. of KC1 (5/8n) and 25 c.c. of CaCl 2 (10/8n). This combination allowed the development of a number of embryos, while in the same concentration of KC1 alone no development was shown. He also obtained a similar result with the muscular contrac- tion of a jellyfish (Poly orchis), 15 thus showing an antagonistic effect between the two salts. The same investigator 3 in his experiments with the hydromedusa Gonionemus has shown that the combination of K ion (5/8n) and Ca ion (10/8n) is poisonous to the animals. Anne Moore 7 obtained a similar result in her experiments on the contraction of the lymph heart of frogs. Meltzer and Auer 21 have shown that with rabbits and a monkey in subcutaneous injection there is a limited antagonism between the two salts. Matthews 11 with Fundulus met with a similar result. He found that at the dilution of M/1600 CaCL to 6/8n KC1 the develop- ment of embryos in the eggs was found to be the best. Lillie 6 found that with the larvae of Arenicola the ciliary activity went on when he used 97.5 c.c. CaCL (10/8n) and 2.5 c.c. KC1 (5/8n), showing an antagonism between the two salts. (c) Bacteria. — Lipman 23 has shown that for Bacillus subtilis the highest production of ammonia is found at the point where 100 c.c. KC1 and 5 c.c. CaCL at the concentration of .35M is used, thus showing a distinct antagonism between the two salts. His work has a striking similarity to that of Osterhout on wheat. Summarizing the antagonism between K and Ca, it may be said that the toxicity of high concentrations of Ca is greatly reduced by the presence of K, but that the toxicity of high concentrations of K is not appreciably reduced by small amounts of Ca. The optimum ratio of KC1 to CaCL was about 2 :1 for yeast. SERIES VIII— ANTAGONISM BETWEEN MAGNESIUM CHLOEIDE AND SODIUM CHLOEIDE The experiments in this series were carried on in the same way as the others. Both table 8 and the curves in figure 8 show that there is a distinct antagonism between these two salts. The highest growth in this case was found at G, the point where .4M MgCL and .06M NaCl were combined, a ratio of about 6 :1. As already shown, when used singly MgCL allows the highest growth at .1M, i.e., 26y 2 millions, 88 University of California Publications in Agricultural Sciences [Vol. 3 Table 8 — Antagonistic Effects Between MgClo and NaCl No. A B C D E F G H I J K MgClo vs. NaCl M. Cone .00 X.00 .00 x.208 .001 x .180 .01 .1 .2 .4 .6 .8 1.0 1.2 x.160 x.128 x.096 x.064 x.032 x.01 x.001 x.00 48 hrs. 3,100,000 226,000 3,250,000 5,424,000 2,260,000 1,582,000 904,000 96 hrs. 7,644,000 452,000 6,212,000 10,396,000 12,656,000 8,684,000 3,102,000 904,000 144 hrs. 13,686,000 452,000 11,201,000 16,368,000 20,696,000 14,956,000 6,358,000 1,872,000 192 hrs. 15,203,000 226,000 678,000 14,258,000 21,690,000 25,882,000 17,009,000 10,605,000 3,896,000 240 hrs. 17,009,000 226,000 1,130,000 17,255,000 25,793,000 28,890,000 21,583,000 15,430,000 4,276,000 A MgCl 2 D ■ E F G H Concentration of salts NaCl Fig. 8. — Curves of yeast growth showing antagonism between MgCL and NaCl. The ordinates represent the number of yeast cells in millions and the abscissae, the concentration of salts in combination. The ordinate at A repre- sents the number of yeast cells in blank cultures. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 89 and NaCl at .001M, i.e., 18 millions. But in combination the two salts permit the highest growth of 29 millions per c.c. at .4M and .06M respectively. The antagonism between these two salts in the case of yeast is found very distinctly at both ends of the curves. For example, .1M NaCl alone shows a growth of scarcely more than one million, while in combination with .1M MgCl 2 it shows over 17 millions, or 17 times as much. On the other hand, .8M MgCl 2 alone allowed a growth of about 8y 2 millions, while in combination with .01M NaCl the growth was increased to about 15% millions, or about twice as much. In comparison with these results, a number of cases dealing with the effects of combinations of MgCl 2 and NaCl on plants, animals and bacteria are cited below. (a) Plants. — Osterhout 5 found a distinct antagonism between the two salts with the growth of a fungus (Botrytis cinerea). He found that 15.M NaCl alone was very toxic, but that when this concentration of NaCl was combined with .4 M MgCl 2 the toxicity was much re- duced. He also found with wheat that neither NaCl nor MgCl 2 at .12M alone allowed root development, but in a combination in the proportion of 100 c.c. NaCl to 75 c.c. MgCl 2 the root developed very well. The same investigator obtained a negative result with green algae. 20 Kearney and Cameron 8 with Lupinus albus and Medicago sativa have shown that the addition of MgCl 2 to NaCl raised the tolerance of these plants to the latter 3-10 times. (b) Animals. — Loeb 12 with Fundulus has found that in a mixture of 98 c.c. 5/8n NaCl and 2 c.c. 10/8n MgCl 2 all the eggs develop embryos, while the same salts alone at the same concentration are extremely toxic. Even an equal proportion of the two salts in the same concentration allowed about 75 per cent of the embryos to de- velop. He also found a similar antagonism with a sea urchin (Ar- bacia) and a jellyfish {Poly orchis). Lillie 6 found that with the larvae of Arenicola the ciliary move- ment continued for a time when he added 10 c.c. MgCl 2 (10/8n) to 90 c.c. NaCl (5/8n), while the same concentrations of NaCl alone would stop it immediately. Matthews with Fundulus found an an- tagonism between the two salts. Ostwald, 13 however, with fresh-water Grammarus obtained con- trary results. In this case a combination of the two salts was found 90 University of California Publications in Agricultural Sciences [Vol. 3 to be more toxic than NaCl alone, isotonic with sea water (2.7 per cent NaCl in sea water or about .4M NaCl). (c) Bacteria. — Lipman 23 with Bacillus subtilis obtained a result similar to that of Osterhout. A mixture of the same concentration of MgCl 2 and NaCl (.35M) in the ratios of 10:1 gave the maximum production of ammonia. To summarize the results of these experiments, it may be said that there is a distinct antagonism between MgCl 2 and NaCl, which is evident on both ends of the curves in figure 8. In this case the yeast agrees with the observations on plants, animals and bacteria except in the two instances cited above in regard to fresh-water Grammar us and green algae. SERIES IX— ANTAGONISM BETWEEN POTASSIUM CHLORIDE AND SODIUM CHLORIDE In this series the flasks were arranged as before. It has been pointed out by Loeb that two salts with ions of like valence, especially in the case of monovalent ions, do not antagonize the toxicity of each other, but rather show a moderately increased toxicity when com- bined. This is evident with yeast, as is shown by table 9 and the curves in figure 9. The highest growth in this case was found at F, where .2M KC1 and .12M NaCl have been combined, having a ratio of about 2 :1. KC1 alone at .2M concentration allows the growth about iy 2 times that found in this combination. Thus the antag- onism of NaCl for KC1 is found to be negative. But, on the other hand, there is a distinct antagonism of KC1 for NaCl. For example, NaCl alone at .17M concentration hardly allowed any growth, but in combination with .01M KC1 the growth was accelerated up to about 15 millions, thus showing a distinct antagonism. The reason of this unexpected negative result on the side of KC1 is perhaps the same that I have suggested in the case of KC1 and CaCh in Series II. For comparison with these results, a number of cases dealing with plants, animals and bacteria are cited below: (a) Plants. — Osterhout 22 with wheat (Early Genesee) has found a slight antagonism between K and Na ions. But in his work 17 on a green alga he obtained a negative result using 3/8M concentration of two salts in combination. (~b) Animals. — Loeb 1 with Fundulus found a slight antagonism between the K and the Na ion in relation to the development of em- 1917] Alitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 91 Table 9 — Antagonistic Effects Between KCl and NaCl No. KCl vs. NaCl M. Cone 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. A B .00 .00 .00] .01 x.00 x.208 L x .192 x.176 1,954,000 6,290,000 10,556,000 14,108,000 16,944,000 f! 226,000 7,184,000 1,808,000 9,256,000 6,780,000 12,176,000 7,888,000 14,952,000 D 2,678,000 E .1 x.160 5,424,000 10,786,000 14,690,000 16,922,000 18,566,000 F G H # 2 .4 .6 x.144 x.128 x.112 2,938,000 2,260,000 678,000 12,339,000 7,838,000 6,780,000 15,942,000 11,526,000 8,678,000 18,206,000 15,830,000 12,687,000 21,250,000 17,248,000 13,266,000 I J .8 1.0 x.096 x.080 226,000 5,650,000 4,156,000 2,906,000 7,205,000 5,882,000 10,256,000 8,120,000 7,750,000 12,984,000 9,886,000 8,^05,000 K 1.2 x.064 4,900,000 Ti 1.4 x.048 1,130,000 3,390,000 1,130,000 226,000 4 968 000 6 983 000 M 1.6 1.8 x.032 x.010 452,000 2,260,000 1,130 000 4,452,000 2,960,000 904 000 N O 2.0 2.2 x .001 x.00 452,000 P a s B C KCl F G H I J Concentration of salts M N O NaCl Fig. 9. — Curves of yeast growth showing antagonism between KCl and Nacl. The ordinates represent the number of yeast cells in millions and the abscissae, the concentration of salts in combination. The ordinate at A repre- sents the number of cells in blank cultures. 92 University of California Publications in Agricultural Sciences [Vol. 3 bryos in the eggs. He also found a similar result with sea-urchins, Hydromedusa gonionemus, and a jellyfish, Polyorchis. Lillie 6 found that with the larvae of Arenicola the ciliary move- ment goes on in a solution containing 20 parts of NaCl (5/8n) and 8 parts of KC1 (5/8n), while each salt used alone stops the movement altogether. Ostwald 13 with fresh-water Gammarus has shown that there is a distinct antagonism between K and Na ions in regard to the duration of life of that animal. Matthews 11 has found that it takes twice as much KC1 to neutralize the toxicity of NaCl in the case of the devel- opment of embryos in the eggs of Fundulus. This is rather similar to the case of yeast, where it takes .2M KC1 to neutralize the toxicity of .14M NaCl to allow the highest growth. (c) Bacteria. — Lipman 23 with Bacillus subtilis has found that none of the combinations of these two salts gives as favorable conditions for growth as is found with each salt alone at the same concentration, thus showing non-antagonism between the two salts. To summarize the results in this experiment, it may be said that with yeast, like valences prevent the antagonistic effects, contrary to what was found by Lipman with soil bacteria, but in accordance with the results of Osterhout with wheat, Loeb with Fundulus, and other investigators with other organisms. The yeast agrees in this case with all the above-mentioned cases except with that of green algae tested by Osterhout and that of Bacillus subtilis by Lipman. SERIES X— ANTAGONISM BETWEEN POTASSIUM CHLORIDE AND MAGNESIUM CHLORIDE The experiments in this series were conducted like the others. The highest growth in this case was found at H, the point where .6M KC1 and .5M MgCl 2 were combined in a ratio of about 1:1. In the case of simple salts KC1 alone at .2M concentration allowed the highest growth up to about 30i/ 2 millions and MgCl 2 at .1M about 26y 2 millions. KC1 alone at .6M and MgCL at .5M permitted the growth of yeast more than is found in this combination at H. But this indicates a mild antagonism, because the toxic effect was less than the sum of the separate toxic effects of the two salts used alone. Distinct antag- onism to the effects of MgCl 2 is shown by KC1, but not the converse. For example, .8M MgCl 2 alone allows the yeast to grow only to 8 millions, while in the combination with .1M KC1 the growth has 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 93 been increased to HV2 millions. On the other hand, the smaller con- centrations of MgClo with higher concentrations of KC1 did not show any antagonism. The reason for this unexpected result is perhaps that previously mentioned in the case of KC1 vs. CaCL, in Series VII. Table 10 — Antagonistic Effects Between KCl and MgCl., KCl vs. No. MgCl 2 M. Cone. 48 hrs. 96 hrs. 144 hrs. 192 hrs. 240 hrs. A .00 X .00 2,356,000 9,701,000 12,170,000 14,890,000 17,526,000 B .00 .00] X y 1.2 1.0 n 226,000 1,130,000 2,260,000 3,845,000 11,560,000 D .01 X .9 226,000 1,356,000 6,780,000 10,070,000 E .1 X .8 1,130,000 6,780,000 7,408,000 10,975,000 12,180,000 F .2 X .7 1,356,000 7,458,000 11,578,000 13,449,000 14,328,000 G .4 X .6 2,486,000 4,838,000 7,006,000 14,690,000 15,500,000 H .6 X .5 904,000 3,816,000 5,296,000 12,850,000 16,280,000 I .8 X .4 678,000 2,612,000 4,852,000 8,286,000 9,856,000 J 1.0 X .3 452,000 2,260,000 3,706,000 5,463,000 5,902,000 K 1.2 X .2 452,000 2,040,000 3,295,000 4,895,000 5,240,000 L 1.4 X .1 678,000 1,926,000 2,940,000 3,656,000 4,864,000 M 1.6 X .05 226,000 904,000 3,050,000 3,006,000 4,628,000 N 1.8 X 01 226,000 2,260,000 2,990,000 3,862,000 O 2.0 2.2 X X .001 .00 2,226,000 1,130,000 P B C KCl F G H I J Concentration of salts M N MgCl 2 Fig. 10. — Curves of yeast growth showing antagonism between KCl and MgCl 2 . The ordinates represent the number of yeast cells in millions and the abscissae, the concentration of salts in combination. The ordinate at A repre- sents the number of cells in blank cultures. 94 University of California Publications in Agricultural Sciences [Vol. 3 For comparison with these results a few cases may be cited as follows : (a) Plants. — Osterhout 20 with wheat (Early Genesee) has shown that the root develops better in a solution having 100 c.c. KC1 and 7.5 c.c. MgCL at .12M concentration than in KC1 alone. He also found with a marine alga, 20 Enteromorpha hopkirkii, that both salts are poisonous when used alone, but a combination in the proportion of 100 c.c. MgCL and 40 c.c. KC1 allows considerable growth. He found a similar antagonism with liverworts. 20 (b) Animals. — Matthews 11 found with Fundulus that in order to permit development of the embryos in the eggs at the concentration of 33/48n KC1 at least about M/160 MgCL is needed. He also found that a solution of 6/8n KC1 requires M/80 MgCL to give the best result. (c) Bacteria. — Lillie 6 has shown that a combination of 10/8n MgCL and 5/8n KC1 allows the ciliary activity of the larvae of Arenicola, which is stopped when one salt is used alone. To summarize, it may be said that a distinct antagonism was found by Osterhout with higher and lower plants and by Matthews and Lillie with animals. With yeast a slight antagonism is found, which is shown on the curves in figure 10 on the side of MgCL. SERIES XI— ANTAGONISM BETWEEN CALCIUM CHLORIDE AND SODIUM CHLORIDE The plan of this series of experiments was the same of that of the others. In the case of simple salts both CaCL and NaCl were found to be very toxic, and it may be owing to this extreme toxicity that the combinations of the two salts showed increased toxicity. Both from table 11 and the curves in figure 11 it is evident that this toxicity is very marked. The highest growth was found at E, where .1M CaCL and .12M NaCl have been combined in the ratio of 1:1. But even here the number of yeast cells went up only to 8 millions, which is far below the highest growth obtained when the salts were used alone. However, CaCL shows slight antagonism to the toxicity of NaCl, for example, .1M NaCL alone allows the growth only to one million, while in combination with .1M CaCl it reached more than 8 millions. On the whole, however, both from the table and the curves it is evident that the combinations of the two salts are more toxic than the single salts. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 95 Table 11 — Antagonistic Effects Between CaCl 2 and NaCl No. A B C D E F G H I J K CaCl 2 vs. NaCl M. Cone .00 x.00 .00 x.208 .001 x. 18 .01 .1 .2 .3 .4 .5 .6 .7 x.16 x.12 x.09 x.06 x.03 x.01 x.001 x.00 48 hrs. 2,356,000 226,000 226,000 226,000 96 hrs. 9,381,000 226,000 1,582,000 1,808,000 1,356,000 226,000 144 hrs. 12,172,000 226,000 3,390,000 5,650,000 3,482,000 1,130,000 . 226,000 192 hrs. 14,890,000 904,000 4,682,000 7,910,000 4,520,000 5,650,000 3,390,000 240 hrs. 17,108,000 1,130,000 5,842,000 8,290,000 7,042,000 6,820,000 4,526,000 452,000 A B CaCl 2 D E F G H Concentration of salts J K NaCl Fig. 11. — Curves of yeast growth showing effects of NaCl on CaCL. The ordinates represent the number of yeast cells in millions and the abscissae, the concentration of salts in combination. The ordinate at A represents the number of yeast cells in blank cultures. For comparison with other organisms the following cases are cited : (a) Plants. — Osterhout 5 with wheat found a distinct antagonism between the two salts. He obtained a similar result with green algae in which he used 100 c.c. NaCl and 10 c.c. CaCL at the concentration of 3/8M. Kearney and Cameron, 8 with leguminous plants, found that a combination of the two salts increased the tolerance of the plants for CaCl 2 three times. 96 University of Calif ornia Publications in Agricultural Sciences [Vol. 3 (b) Animals. — Loeb 1 with Hydromedusa Gonionemus has shown that a combination of 10/8n CaCl 2 and 5/8n NaCl is harmless to animals. He 15 also found a distinct antagonism with a jellyfish, Polyorchis, using 50 c.c. NaCl and 1 c.c. CaCl 2 , which allowed the animal to swim, while NaCl alone was poisonous. The same investi- gator found a distinct antagonism between these two salts working with the development of embryos in the eggs of the Fundulus. Anne Moore 7 with the contraction of the lymph heart of frogs and Lingle 4 with that of the turtle's heart noted similar phenomena, thus corrob- orating the work of Loeb. Lillie 6 working with the larvae of Arenicola has found a distinct antagonism between Ca and Na ions. MacCallum 14 found the same with his experiments on cathartics. Meltzer and Auer 21 found a distinctly antagonistic effect with animals in subcutaneous injections. Ostwald 13 wth fresh-water Gram- marus found a strong antagonism between NaCl and CaCL in regard to the duration of life of that animal. Finally, Matthews 11 has shown that there is a slight antagonism between the two salts in the develop- ment of embryos in the eggs of Fundulus. (c) Bacteria. — Lipman 24 with Bacillus subtilis found a marked lack of antagonism between the two salts. In his case any combi- nation of the two salts at .35M concentration was found to be more poisonous than a single salt. All these experiments except that of Lipman show that there is antagonism between CaCl 2 and NaCl. The yeast agrees very mark- edly with Bacillus subtilis in showing little or no antagonism between the two salts, CaCl 2 and NaCl 2 . 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 97 Relative Antagonisms of Various Combinations Table 12 is intended to show the relative antagonisms of the various combinations. The data used in constructing the table are the final counts in each flask. The average of the counts in all the check flasks is taken as the basis from which to estimate the influence of the various salts and of their combinations. The calculation is made as follows : Yeast growth in check flasks = 17 (millions). Yeast growth with single salt no. 1 = a. Yeast growth with single salt no. 2 = o. Yeast growth with combination no. 1 + 2 = c. Toxicity — expected = (17 — a) + (17 — &). Toxicity — observed = 17 — c. Antagonism of combinations* ■= (17 — a) + (17 — o) — .(17 — c). m ' . Antagonism == 17 + c — a — o. Table 12 — Range of Antagonism of the Binary Combinations Calculated From the Last Microscopical Count* No. MgCl 2 X CaCl 2 KC1 X CaCl 2 MgCl 2 X NaCl KC1 X NaCl KOI X MgCl 2 CaCl 2 X NaCl At 17,000,000 17,000,000 17,000,000 17,000,000 17,000,000 17,000,000 B C 4,000,000 5,000,000 4,000,000 D 4,000,000 17,000,000 8,000,000 10,000,000 4,000,000 E 27,000,000 17,000,000 8,000,000 9,000,000 9,000,000 7,000,000 F 24,000,000 18,000,000 25,000,000 8,000,000 7,000,000 7,000,000 G 20,000,000 30,000,000 27,000,000 7,000,000 16,000,000 6,000,000 H 21,000,000 31,000,000 14,000,000 7,000,000 9,000,000 I 7,000,000 8,000,000 11,000,000 10,000,000 J 1,000,000 1,000,000 2,000,000 10,000,000 K 1,000,000 L M N O P f Millions on average. * These results are shown graphically by the curves in figure 12. * This defines ' antagonism' as the difference between the expected and the observed toxicity. The curves have been drawn to show the antagonism of the com- binations and not the actual growth of the yeast as has been shown in the previous curves. 98 University of California Publications in Agricultural Sciences [Vol. 3 | 32 SO — ( / Fni v fi„m i MgCl 2 X CaCl 26 /,< \ ■ ' - MgUIo a JNaUJ KC1 X MgClo \ < \\ \ """""'" ' ■■ CaCl 2 x NaCl \ / \ / \ i / \ \ \ i c in , / , \ / \ \\ / i \ • % i / i \ 1 ' c r> fv, i < \ \ ! 7 \ K ) — < A \\ \\\ \ \ i ' \ w \ i i /I \ 4t \ 1 4 ; < \ // 1 ( ~\ ^ > v F G H I J K L Combinations of Salts M N Fig. 12. — Curves showing range of antagonism of binary combinations of salts. The ordinates represent the average number of yeast cells in millions and the abscissae, the concentration of salts in combinations. The ordinate at A represents the average number of cells in blank cultures. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 99 Summary PART A— TOXIC EFFECTS OF SINGLE SALTS 1. Each of the four single salts— KC1, MgCl 2 , CaCl 2 , and Nad- is more or less toxic to the yeast, Saccharomyces ellipsoideus, at certain concentration. KC1 is the least toxic and NaCl the most for the yeast (no. 66) used. 2. The lower concentrations of each salt stimulate the growth of yeast. The highest number of yeast cells in microscopical count was found at .2M KC1, .1M MgCL, .01M CaCL, and .001M NaCl, KC1 being the most favorable and NaCl the least. Beyond the favorable concentrations the various salts are toxic to yeast. 3. The concentrations of salts that inhibited the growth of yeast cells were found at 2.2M KC1, 1.2M MgCL, .7M CaCl 2 , and .2M NaCl. 4. The results of the experiments are quite different from those found with either bacteria, the higher plants or animals. The yeast stands in this respect midway between plants and animals and swings to either direction according to the environment. 5. The salts used had a marked effect on the size and appearance of the yeast. Taking decrease in size as a criterion, the salts affected the yeast toxically in the same relative ways as indicated by the rate of multiplication of the cells. PART B— ANTAGONISTIC EFFECTS OF COMBINATIONS OF SALTS 1. As shown by growth of yeast, the variation in antagonism be- tween the four single salts in all possible combinations may be ar- ranged in order as follows : 1. MgCL vs. CaCL (most) 2. KC1 vs. CaCL 3. MgCL vs. NaCl 4. KClvs. NaCl 5. KClvs. MgCL 6. CaCL vs. NaCl (least) 2. The effect of binary salts with yeast, whether positively or nega- tively antagonistic in comparison to animals, plants and soil bacteria, may be tabulated as follows: Yeast Animals Plants Soil bacteria + + and - + — + and - + and — + + + + and — + and — + + and- + •4- and — + and - + and — + + + + + and — — : mild antago nism. — — strong increase of toxicity. — — slight 100 University of California Publications in Agricultural Sciences [Vol. 3 Binary salts 1. MgCL vs. CaCL 2. KC1 vs. CaCL 3. MgCL vs. NaCl 4. KClvs. NaCl 5. KC1 vs. MgCL 6. CaCL vs. NaCl -f- — strong antagonism, increase of toxicity. 3. In regard to the effects of valences of ions the following results have been obtained with yeast : (a) That divalent ions may antagonize monovalent ions is evident from the combinations of MgCl 2 vs. NaCl and CaCL vs. NaCl. Ngative results were ob- tained from the combinations of KC1 vs. CaCL and KC1 vs. MgCl 2 . (b) That a divalent ion may be antagonized by a divalent ion is evident from the combination of MgCL vs. CaCl 2 . (c) That monovalent ions may antagonize divalent ions is shown in the combinations of KC1 vs. CaCl 2 ; MgCL vs. NaCl and KC1 vs. MgCL. (d) That a monovalent ion may antagonize a monovalent ion, though not very markedly, has been found in the combination of KC1 vs. NaCl. 1917] Mitra: Toxic and Antagonistic Effects of Salts on Wine Yeast 101 Literature Cited PAET A— TOXIC EFFECTS OF SINGLE SALTS 1 1900. Loeb, J. On the different effects of ions upon myogenic and neuro- genic rhythmical contractions and upon embryonic and muscular tissues, Am. Jour. Physiol., vol. 3, pp. 383-396. 2 1900. — On the ion proteid compounds and their role in the mechanics of life phenomena, Am. Jour. Phyiol., vol. 3, pp. 327-338. 3 1902. — Studies on the physiological effects of the valency and possibly the electrical charges on ions, Am. Jour. Physiol., vol. 6, pp. 411-433. 4 1905. Ostwald, W. Studies on the toxicity of sea-water for fresh-water animals (Grammarus Pulex De Geer), Publ. Univ. Calif. Physiol., vol. 2, pp. 163-191. s 1905. Loeb, J. On fertilization, artificial parthenogenesis and Cytolysis of the sea urchin egg, Univ. Calif. Publ. Physiol., vol. 2, no. 8, pp. 73-81. 6 1906. Osterhout, W. J. V. On the importance of physiologically balanced solutions for plants, Bot. Gaz., vol. 42, pp. 127-134. 7 1907. — On the importance of physiologically balanced solutions for plants, Bot. Gaz., vol. 44, pp. 259-272. 8 1909. — On the similarity of behavior of sodium and potassium, Bot. Gaz., vol. 48, pp. 98-106. 9 1906. — Extreme toxicity of NaCl and its prevention by other salts, Jour. Biol. Chem., vol. 1, pp. 363-369. io 1908. Magowan, F. W. The Toxic Effects of certain common salts of the soil on plants, Bot. Gaz., vol. 45, pp. 45-49. ____ ii 1909. Lipman, C. B. Toxic and antagonistic effects of salts as related to ammonification by Bacillus subtilis, Bot. Gaz., vol. 48, pp. 105-125. 12 1912. Bokurny, T. The effects produced by metallic salts on yeasts and other fungi, Centrbl. Bakt., 2. Abt. Bd. 35, no. 6-10, pp. 118-197. is 1913. Loeb, J. Artificial Parthenogenesis and Fertilization, pp. 173-190. PART B— ANTAGONISTIC EFFECTS OF COMBINATIONS OF SALTS i 1900. Loeb, J. On the different effects of ions, Am. Jour. Physiol., vol. 3, p. 383. Complete entry to be found in Part A. 2 1900. Loeb, J. On the ion proteid compounds, Am. Jour. Physiol., vol. 3, p. 327. Complete title in Part A. 3 1900. Loeb, J. Ueber die Bedeutung der Ca and K ionen fur die Herzthat- igkeit, Pnuger's Archiv, vol. 80, pp. 229-232. * 1900. Lingle, D. J. In action of certain ions, Am. Jour. Physiol., vol. 4, p. 265-282." 5 1900. Osterhout, W. J. V. Die Schiitzwirkung des Natriums fiir Pflanzen, Jahrb. Wiss. Bot., vol. 46, p. 121-136. 6 1901. Lillie, R. On the differences in the effects of, etc., Am. Jour. Physiol., vol. 5, pp. 56-85. 7 1901. Moore, A. Effects of ions on the contraction of, etc., Am. Jour. Physiol., vol. 5, pp. 86-94. s 1902. Kearney & Cameron. Some mutual relations between alkali soils and vegetation, U. S. Dept. Agr. Report, no. 71. 9 1902. Loeb, J. Studies on the physiological effects, Am. Jour. Physiol., vol. 6, p. 411. Complete title in Part A. io 1903. Loew, O. The physiological role of mineral nutrients in plants, U. S. Dept. Agr. B. P. I., Bull. 45. ii 1904. Matthews, A. P. Toxic and antagonistic effects of salts, Jour. Am. Physiol., vol. 12, pp. 419-443. 12 1905. Loeb, J. Studies in general physiology, vol. 2, pp. 518, 572, 584. 13 1905. Ostwald, W. Studies on the toxicity of sea-water, Publ. Univ. Calif. Physiol., vol. 2, pp. 163-191. 102 University of California Publications in Agricultural Sciences [Vol.3 1 4 1905. MacCallum, J. B. The action of purgatives in a crustacean, Bull. Univ. Calif. Physiol., vol. 2, no. 6, pp. 65-70. is 1906. Loeb, J. The stimulating and inhibitory effects of Mg and Ca., Jour. Biol. Chem., vol. 1, p. 427-436. i 6 1906. Osterhout, W. J. V. On the importance of physiologically balanced solutions for plants, Bot. Gaz., vol. 42, pp. 127-134. - — - 17 1906. Extreme toxicity of NaCl and its prevention by other salts, Jour. Biol. Chem., vol. 1, pp. 363-369. 18 1907. Loew & Azo. On the physiologically balanced solutions, Bull. Agric. Univ. Tokyo, vol. 7, no. 3. is 1907. Benecke, W. Uber die Giftwirkung verscheidener Salze auf Spy- rogyra und ihre Entgiftung durch Calciumsalze, Ber. deutsch. botanisch. Ges., vol. 25, pp. 322-337. 20 1908. Osterhout, W. J. V. Antagonism of Mg and K on plants, Bot. Gaz., vol. 45, pp. 117-129. 2i Meltzer & Auer. The antagonistic action of calcium, Am. Jour. Physiol., vol. 21, pp. 400-419. 22 1909. Osterhout, W. J. V. On the similarity of behavior of Na and K, Bot. Gaz., vol. 48, pp. 98-104. 23 1909. Lipman, C. B. Toxic and antagonistic effects of salts, Bot. Gaz., vol. 48, p. 105. Complete title in Part A. 24 1910. Lipman, C. B. On the lack of antagonism, Bot. Gaz., vol. 49, pp. 41-50. 25 1910. Lipman, C. B. On physiologically balanced solutions for bacteria, Bot. Gaz., vol. 49, pp. 207-215.