THE ACTION OF METHYLENE-BLUE AND CERTAIN OTHER DYES ON LIVING AND DEAD YEAST BY CHAS. G. FRASER
In the experiments of Scheurlen, Beckmann,2 Paul and KrOr~ig,~ and Laird,5 the number of cells remaining alive after immersion for a measured time in a poisonous liquid was determined by mixing a known volume of the cell suspension with melted agar jelly, pouring on a petri dish, incubating, and counting the number of colonies that appeared. This procedure involves a wait of three or four days after the completion of the poisoning experiment before its result i s known. Other criteria of death have been employed in similar investigations. Bokorny6 for instance, who compared the toxicities of some go different chemicals towards infusoria, algae, etc., relied on the mobility of the test objects or of their cilia, etc., or on the persistance of protoplasmic streaming in the cells as signs of life; Kahlenberg and True7 and Healds relied on the cessation of growth of the rootlets used as test-objects and on their general appearance (flabbiness) as evidence of death; Galeotti, used this last characteristic in his experiments on the poisoning of iris petals. According to Mol1,'O the fact that plasmolysis occurs only with living cells has been employed to distinguish living from dead cells by Langel' and by Strecke.12
* la
le
Arch. exp. Path. Pharm., 37, 74 (1895). Centralbl. Bakteriologie, 20, Abt. I , 577 (1896). Zeit. phys. Chem., 21, 4 1 4 (1896). Jour. Phys. Chem., 24, 570 (1920). Ibid., 24, 664 (1920). Pfliiger's Archiv Physiol., 110, 174 (1905); 111, 341 (1906). Bot. Gazette, 22, 8 1 (1896). Ibid., 22, 1 2 5 (1896). Zeit. wiss. Mikroskopie, 11, 1 7 2 (1894). Progressus rei botanicae, 2, 297 (1908). Flora, 1891,5 2 ; see Moll: loc. cit. Tnaug. Diss. Amsterdam ( I 904) ; see Moll: loc. cit.
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With the possible exception of the last mentioned, none of these seems adapted to the case of yeast. There seemed more to hope from the use of dyes. According to Klocker,’ “The distinguishing of dead cells from living ones has been assiduously carried on in most brewery laboratories since the microscope came into general use. But the value of the indications given by the reagents employed for this purpose has been very much overestimated. The question seems to deserve proper investigation. According to Wehmer, a half percent methylene-blue solution will stain the dead cells indigo blue, while the living cells remain colorless.” MOSSO~ has published a paper with the title “Anwendung des Methylgriin ziir Erkennung der chemischen Reaktion und des Todes der Zellen;” on reading it, however, it appears that methylgreen acted as a poison towards the cells studied (leucocytes, ciliated epithelia, etc.) and that.by the time enough to color them anyway deeply had been absorbed,.they died. Similar observations were made by Bokorny ; in discussing experi~ ~ for instance, ments with infusoria in 0 . 0 1 Victoria-blue,3 he says, “one can see quite distinctly that the body of the infusorian becomes colored while it is still alive (recognized by its movements) and that death occurs only when the coloration has reached a certain intensity.” The name “vital dyes” has been given to a group of substances, thionine derivatives prominent among them, which have the power of staining the nerves of certain animals while they are still alive. Ehrlich, to whom this discovery is due, was of opinion that the staining could occur only during the lifetime of the tissue affected ; subsequent experiments, however, have shown that this conclusion was too broad; indeed, Galeottij who besides contributing work of his own on salamanders and on iris petals has provided a most convenient summary of Fermentation Organisms, tr. by Allan aud Millar, Longmans-Green (1903). Pfliiger’s Arch. Physiol., 113, 397 (1888). Ibid., 110, 178 (1905). 4 Bid. Centralbl., 6, 2 2 1 (1897). 6 Zeit. wiss. Mikroskopie, 11, 172 (1894).
Action of Methylene-Blue on Living and Dead Yeast 743 his predecessors’ work, comes to the exactly opposite conclusion, viz., that only cells whose vitality has been impaired are capable of taking on the stain. The Action of a Number of Dyes on Yeast Cells To be of use for the purpose in hand, a dye must be able to distinguish sharply between living and dead yeast cells, more particularly when death has been brought about by the action of phenol; it must have no toxic action of its own on the yeast, a t least in the time necessary for the test, and a t the concentration used; if in addition it should stain quickly, so much the better. A number of the dyes commonly used in microscopic work were tested from this point of view, including Grubler’s methylene-blue, fuchsin, Congo-red, erythrosin, and safranin (water soluble) ; Merck’s methylene-blue, gentian violet, and methyl-green, and Kahlbaum’s methylene-blue 6 B extra. For most of these I am indebted to the kindness of Prof. Thomson, of the department of Botany and of Prof. Piersol, of the department of Biology. Aqueous solutions of each were made containing 0.5 g per IOO cc (gentian violet 0.4 g); these were suitably diluted and added in turn to a suspension of yeast cells. To test their action on living yeast, a suspension was made from 0.6 g Fleischmann’s yeast cake and IOO cc distilled water; the reactions of this suspension towards dyes remained unaltered for twenty-four hours a t room temperature. The action on dead cells was tested with a portion of this suspension which had been heated to boiling and cooled again (“boiled yeast”) ; and a third suspension (“mixed yeast”), made by mixing equal volumes of “live” and “boiled,” was used to compare the colors in the same microscopic field. A magnification of 430 diameters was employed; the fraction given as stained is a rough estimate only; the percentage of dye is the number of grams dye per IOO cc of the liquid containing dye and yeast. Congo-red: (liviutg) 0 . 2 5 % ~ I min. o std.; 60 min. I or 2 cells std.; (boiled) 0.25Oj0, all light orange after standing an hour; (mixed) 0.2570, I min. o std.; 60 min. about half.
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Eosin: (living) 0 . 2 5 7 0 , none stained in an hour; (boiled) 0.25% min. all stained, not so dark as erythrosin; 0 . 0 5 7 ~stained ~ pink slowly; (mixed) distinction poor. Erythrosin: (living) 0 . 2 5 7 ~none stained in IOO min.; (boiled) 0.2570 all stained pink in I rnin., dark red in about 3 rnin.; (mixed) 0 . 2 5 % half std., sharp distinction after 3 min. Fuchsin: (living) 0.25% I min. nearly all std.; 0.05% I min. onefifth std. 13 min. nearly all std.; 0.005% IOO min. none std.; (mixed) 0.05%) 2 min. 5% std.; 15 rnin.’ less than half std.; 60 min. about half faintly stained. Gentian-violet: (living) 0.20%~ 0 . 0 2 % ~ I min. all std. deep violet; 0.002% 60 min. o std.; (boiled) 0 . 0 0 2 % ~ all stain slowly; (mixed) 0.002010, 3 min. about half std. light purple. *Methylene-blue 6B extra: (living) 0.05%, I min. o std., 45 min. about one quarter of the cells dark blue; 0.01% 60 min. o std.; (boiled) 0.005% I min. all std. distinctly; (mixed) 0.005% I min. half std., good distinction. Methyl-green: (living) 0.2570, I min. few std., 15 min. nearly all ~ min. o std.; (mixed) 0.05Y0,half std., good distincstd.; 0 . 0 5 7 ~60 tion. Neutral-red: (living) 0.01%, I min. three-quarters std. ; 0 . 0 0 2 % ~ I O min. o std., 75 min. about half faintly std. (boiled) 0.02% I min. all pink-violet; 0 . 0 0 2 7 ~I min. o std., IOO min. all faint orange. Safranin: (living) 0 . 2 5 % , 0.05%) I min. all std.; 0.005% I min. o std., 2 0 min. one-quarter std., 45 min. half std.; (mixed) 0.005% I min. half std. a dirty pink. I
a
Phenol (about one percent) was then added to a portion of the yeast suspension, and left a t room temperature for three hours, which is longer than is necessary to kill all the cells. This “phenolized yeast” was then mixed with an equal volume of the various dye solutions, giving a solution containing one-half percent of phenol, and a dye concentration chosen in the light of the preceding results. Eosin: 0.05%~ I min. all std. faintly; 80 min. much darker. Erythrosin: 0.2570 needs a minute to stain; color as deep as with methylene-blue 0.01Yo. Fuchsin: 0.005% very light stain; 0.01% not as dark as with methylene-blue 0.01%. Gentian-violet: 0.0027~stain light, comes slowly; 0.00470 not SO dark as with methylene-blue 0.0170. Methylene-blue 6B extra: 0.005% needs a minute to stain light blue; 0.01% much darker ana quicker stain. Methyl-green: 0.05% stained about as dark as methylene-blue
0.017~.
* T h e other brands of methylene blue were somewhat more toxic.
Action of Methylene-Blue on L i v i n g and Dead Yeast 745 Neutral-red:
0.002%
absence of phenol. Safranin:
0.005%
none stained; 0.01% all stained as in the
all stained light orange-pink.
From these results it appears that gentian-violet, neutralred and safranin have too much action on living yeast to be convenient indicators; Congo-red on the other hand has too little action on the dead cells; fuchsin, neutral-red and safranin are too faint ; while eosin, erythrosin, metKyl-green and methylene-blue 6 B extra would serve. The question whether the presence of these dyes in small amounts would prevent reproduction of the yeast in culture media was then taken up, and for this purpose a “malt” solution was prepared by dissolving 30 g of malt extract (a brown sticky stuff) in water, making up to 2 0 0 cc, boiling and filtering. To 4 cc of this “malt” was added 0.5 cc or 1.0cc of the solution of a dye, and 2 cc of yeast suspension; the mixtures were left in test tubes plugged with cotton wool in an incubator at 25’ C. The concentration of dye in the mixture was in each case less than would completely stain unboiled yeast, viz., Congo-red 0.04%~ eosin 0.04%~ erythrosin 0.047~, fuchsin 0.014%, gentian-violet 0.006%~ methylene-blue 0.0770, neutral-red 0.00370, safranin 0.014%. After a dozen hours the occurrence of fermentation could be recognized in every case, by a stream of small bubbles rising through the tube; and in every case reproduction of the yeast was taking place, under the microscope an increase in the number of cells could be observed, and numerous double cells and chains of three and four were to be seen. This result made it easy in certain cases to answer the next question, viz., when a dye has acted on yeast long enough to stain most of the cells, have those remaining unstained lost their power of reproduction? Mixtures were made up of the same composition as before, but instead of mixing in the order: malt, dye, yeast, the yeast was added directly t o the dye, which was thus present in concentration two or three times greater than in the former instance; and before putting in the malt, the mixture was let stand until most of
746
Chas. G. Fraser
the cells were stained. Even when only a few percent of the cells remained colorless, fermentation and reproduction took place after adding the malt; this was not quite what was expected, as in a number of cases where wort-agar had been seeded with a mixture of stained and unstained cells, the number of colonies formed was much less than corresponded to the number of unstained cells.. When the dye had acted for a long time, and no unstained cells could be found under the microscope, no fermentation took place after adding the malt. Methyl-green seemed to behave quite differently from the other dyes, though not enough time was spent on the matter to make sure that the results obtained with it could not be duplicated with others. When 0 . 2 5 cc or less, of 0 . 5 0 7 ~ methyl-green solution was added to 4 cc malt, and then 2 cc yeast suspension was put in, fermentation took place in from 7 to 30 hours; but if 0.5 cc or 1.0 cc of the dye were used, no fermentation was observed, no increase in the number of cells, and no chains of cells. Examined under the microscope, the cells were turgid, but after some days in the solution they became granular; they remained unstained by the methyl-green, and were not stained when methylene-blue was added to a concentration of o.oog%. When the dyemalt-yeast mixture was heated to 100' C, the cells promptly stained green; the same when phenol was added; thus the dye had evidently not lost its staining power by mixing with the malt. An hour after adding 2 cc yeast suspension (count 150) to the solution made from 1 . 0 cc methyl-green 0.507~ and 4 cc malt, 0.25 cc of the mixture was pipetted into 4 cc sterile malt and put away at 25' C ; similar tubes were prepared after 2 , 4, 17 and 2 9 hours; neither fermentation nor reproduction took place in these tubes in 5 days, although the concentration of the dye was much less than that at which yeast grows readily. When some more of the original yeast (kept in water in the meantime) was put in, fermentation took place about as quickly in a parallel experiment with malt; this shows that the cell, not the medium, was at fault.
Actiow of Methylene-Blue on Living and Dead Yeast 747 Two percent agar-agar was dissolved in malt, and a drop of the original malt-dye-yeast mixture added 7 hours after it had been made up; a second plate was seeded from the same solution 98 hours after it had been made up. Yeast colonies grew plentifully on both plates; where the dye had acted 7 hours about 2 0 to 25% of the cells grew, after 98 hours about one percent. This work with methyl-green is being continued; it is only mentioned here because of the evidence it affords that yeast cells may be alive, unstainable, and capable of reproduction in some media, and yet incapable of reproduction in other media in which normal yeast cells grow readily. In case of contradiction arising between the evidence of stain and plates this should be borne in mind. Of the four dyes named on page 745 as suitable for indicators, erythrosin gives better distinction than eosin, while the behavior of methyl-green in hindering reproduction in the culture medium makes it for some work less safe than methylene-blue ; the choice thus lies between erythrosin and methylene-blue. For use with the camera, blue is not the best, as it requires a long exposure during which the light and heat may increase the number of cells stained. With a Wratten screen B (green), or with an “isolar” screen, using 4 mm objective, no ocular, IOO watt tungsten lamp, and Ilford rapid plates, about thirty seconds exposure were needed to give a good negative. For direct observation with the microscope, however, blue is much less fatiguing to the eyes than red; I, therefore, decided to adopt methylene-blue in my further work, and studied its behavior with a number of reagents apt to be used in quantitative toxicological work with yeast. In the presence of carbinol 2070, alcohol 2 0 7 ~ or ~ acetone 2 0 % ~ and methylene-blue 0.00570, boiled cells stain green. With M. B. 0 . 0 0 5 ~and ~ acetic acid 0 . 0 5 7 ~they also stain green, in shade about as dark as the blue obtained in the absence of the acid; with 0.1570 acetic acid the staiq is a very faint green, addition
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of ammonia restores the blue. No great pains need be taken to avoid excess of ammonia, immersion in normal ammonia for a quarter of an hour does not cause living cells to stain. Mercuric chloride 0.1% gives a purplish precipitate and the cells do not stain. Sodium chloride 1 2 y 0 gives a purplish precipitate, and under the microscope long hair-like crystals (Pelet-Jolivetl found that the amount of methylene-blue absorbed by wool is influenced by the addition of sodium sulphate, and Ross2 that the action of the same dye on leucocytes is interfered with by neutral salts). If silver nitrate, 0 . 1 2 % ~ be added to an equal volume of the yeast suspension, boiled a moment to kill the cells, (“silver-yeast”) and then an equal volume of 0.03yomethylene-blue be added, the cells are barely stained a greenish tint; if the concentration of the silver be decreased or that of the dye be increased, the cells stain bluer. With this silver-yeast, neutral-red behaves much as methylene-blue; eosin stains the cells but forms a pink film visible under the microscope ; erythrosin, gentianviolet, and methyl-green act as with ordinary boiled cells. My thanks are due to Prof. W. Lash Miller under whose direction this work was carried out. Summary The behavior of nine dyes with’living yeast and with yeast killed by boiling or by the action of phenol, was studied with the object of finding a convenient criterion of death in quantitative toxicological investigations with microbes. Erythrosin and methylene-blue 6 B extra proved the best. In a solution of extract of malt to which methyl-green has been added, yeast cells may lose their power of reproduction without becoming stained. The University of Toronto J u l y 1920 1
2
Arch. Sci. phys. nat. Geneve, (4)27, 27; from Chem. Centr., 1909,I, 879. Jour. Physiol., 37, 327; Chem. Centralbl., 1908,11, 1693.
