Sporulation by Hansenula Mrakii on vegetable media by O. B. WILLIAMS and ARTHUR NEWTON
(Department o] Bacteriology, University o/ Texas, Austin)
(23.9.195o) Methods and media for inducing the formation of ascospores by yeasts have long been a subject of investigation. This has been due chiefly to the diagnostic importance of the number, mode of formation and morphology of the spore, but also to some extent because of the significance of ascospores in connection with studies in the developing field of microbial genetics. Two main lines of attack on the problem have been followed. One has been to grow the yeast on a pre-sporulation medium, transfer the vegetative cells to some non-nutrient support as plaster of Paris blocks, wood or clay blocks, filter paper, silica gel or washed agar which is kept moist with water or with some dilute solution of a test material, prepare smears at intervals and examine for the development of spores. The other procedure has been to grow the yeast directly on sporulation media, which in some instances supported luxuriant growth, and in others allowed only a limited growth. The general concensus is that no one medium or method is adequate for all types, and that there is a tack of uniformity in behavior among species, and even among strains of the same species. PHAFF and MRAK (9) have recently reviewed the literature dealing with sporutation in yeasts, hence in the present instance reference need be made only to especially pertinent publications. (See also HENRICI (2) and LINDEGREVr and LINDEGREN (4)). A mixed vegetable medium with an especially high spore inducing potential was described by MRAI~, PILAFF and DOUGLAS (6) in 1942. This medium, which was prepared from carrots, beets, cucumbers and potatoes, came nearer being a universal sporulation medium than any previously described. However, LINDEGREN and LINDEGREN (4) found it to be ineffective with some types, and following studies to develop a more nearly optimal medium they described one containing extracts of both beet leaves and beet roots, apricot juice, grape juice, dried yeast and glycerin, and neutralized with calcium carbonate. The medium m a y be used either as a presporulation nutrient or directly as a sporulation medium. They did not include carrot in any of their experiments, stating that in earlier work this vegetable, which has long been used as a sporulation medium, had not been found useful. WlCI~ERHAM, FLICKIVIGER and BURTON (13) described a medium, which they said was first described to them by HENRICI in 1943, prepared with V--8, a commercial mixture of juices of tomato, celery, carrot, parsley, lettuce, beet, spinach and water cress, and containing salt and "seasoning." The medium has yeast added in the proportion of one half of a small yeast cake (or 10 grams of yeast) to each liter of medium. Because of ease of preparation and high spore productivity the V - - 8 medium has been extensively used. Most sporulating yeasts will form spores fairly readily on the medium (ETcHELLS (1)).
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O.B.
WILLIAMS AND ARTHUR NEWTON
Little is known about the causal relationship to sporulation of the various components of the medium of MRAK et al or of the V - - 8 medium. The present report gives the results of an effort to evaluate the various constituents of these media as spore inducing nutrients.
Assay organism In a study of the comparative spore inducing potential of different nutrients the choice of an assay Organism is of importance. The test organism should show on the reference or control medium a substantial percentage of the cells as a~.ci, preferably between 35 % and 65 %, in order that significant increases or decreases in sporulation may be readily detected. It is desirable that sporulation proceed to a reproducible maximum fairly rapidly, and it is helpful in ready recognition if the organism has distinctive ascospores. Tests on the organisms shown in the tabulation gave the percentage of asci indicated after I days incubation on tomato juice agar. Saccharomyces cerevisiae . . . . . . . . . . . . S a c c h a r o m y c e s cerevisiae var. d l i p s o i d e u s . . . . P i c h i a / e r m e n t a n s vat. rugosa . . . . . . . . . Z y g o s a c c h a r o m y c e s gIobi/ormis . . . . . . . . . Hansenula mrakii . . . . . . . . . . . . . . .
1 i
15 25 32
The requirements listed were fairly well met b y H a n s e n u l a m r a k i i , consequently this organis m was the one Selected for study. This organism has not been officially described but a description is now in process of preparation b y WlCKERHAN (11) and will appear in due course in a Technical Bulletin of the U.S. Department of Agriculture. The organism has been referred to b y WlCKERHAM and BURTON (12) and was one of the species discussed b y WlCKERHAM, GILL and BURTON (14) at the 1950 meeting of the Society of American Bacteriologists. The strain used was obtained from Dr. E. M. MRAK, of the University of California, and was maintained on nutrient agar.
Materials and methods Vegetable base media were prepared b y grinding washed unpeeled vegetables in a Waring blendor, mixing with an equal volume of distilled water, adjusting to the desired p H with a glass electrode potentiometer, adding 2 % agar and sterilizing at 15 pounds steam pressure for 15 minutes. V - 8 and tomato juice were diluted with an equal volume of distilled water and handled in a similar manner. Petri dishes were poured and inoculated b y streaking to give isolated colonies. Incubation was at 30C. Ratio counts of sporulating cells were made on smears prepared from the surface of colonies and stained b y the method used b y WYNNE (15) for bacterial spores. MRAK (5) has noticed for H . m r a k i i a tendency to shift from conjugation preceding sporulation to sporulation without conjugation. However, in the work here reported no tendency for sporulation without conjugation was noted. At least 300 cells were counted on each smear from fields selected at random. At first counts were made at daily intervals but later when experience showed that maximum sporulation had occurred b y the fourth day the counts were made at this interval only. The results reported are the average of percentage of asci from three determinations in close agreement.
SPORULATION BY HANSENULA MRAKII ON VEGETABLE MEDIA
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Results One of the conditions early recognized as of importance for ascospore formation was a free access to atmospheric oxygen, and apparently all later observations are in accord on this point. It would appear t h a t the difference in aeration between the surface and the depths of a colony is significant since LINDEGREN and HA~II.TO~ (3) found spores only in the surface layer of prepared cross sections of colonies of Saccharomyces cerevisiae. One of the preliminary experiments done as a part of the present work was an examination of smears from the surface and from the b o t t o m levels of colonies grown on tomato juice medium for 4 days. The surface layer showed 24 % of the cells as asci, the b o t t o m layer only 16 %. In all subsequent experiments care was used to obtain the cells for examination from the surface layer of the colony, and since results have been readily reproducible in replicate experiments it is believed t h a t there are no errors in the results due to faulty sampling. Other preliminary experiments included a test of the significance of the p H of the medium and of yeast extract as a component of the medium. The medium of MRAK et al, and V - 8 medium with 0 . 1 % yeast extract substituted for yeast cake, were prepared with p H values of 4.3 and of 6.8. The 4 day ascus count on the medium of MRAI~ et al was 11 and 10, and on the V - - 8 medium 4.0 and 36 at these two p H values. It was clear that p H did not exercise a significant effect on sporulation b y H. mrakii, and in all later experiments the media were adjusted to p H 6.8. The importance of yeast extract as a component of the medium was tested with several media, each with 0 . 1 % yeast extract and with no yeast extract. The 4 day ascus count on each of the media tested was as follows: V--8 Plain 42
+ ye 40
Carrot + Plain y e 16 12
Tomato+ Plain y e 32 32
Spinach+ Plain ye 14 13
Celery + Plain y e 34 34
Lettuce+ Plain y e 7 8
F r o m these data it was concluded that yeast extract in the amount used was not significant for sporulation b y H. rarakii, and consequently yeast extract was not added to the media used in later experiments. On the basis of observations b y a number of workers it might be expected t h a t a greater amount of yeast extract would be stimulatory to sporutation, but in the interest of keeping the media as simple as possible no further tests were made. The d a t a show very clearly t h a t striking differences in spore productivity m a y be encountered among different vegetable base media. The next experiment was designed to establish the spore productivity of the vegetable components of the ~/~RAK et gl and the V - 8 media. The 4 day ascus counts which were obtained are as follows: Celery . . . . . . . . . . . . . . . Commercial tomato juice--salted . Commercial tomato j u i c e - - n o added Fresh tomato . . . . . . . . . . . Carrot . . . . . . . . . . . . . . . Spinach . . . . . . . . . . . . . . Cucumber . . . . . . . . . . . . . Water Cress . . . . . . . . . . . . Beet (root) . . . . . . . . . . . . Parsley . . . . . . . . . . . . . . Potato . . : . . . . . . . . . . . Lettuce . . . . . . . . . . . . . .
- ..... . . . . . . salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
34 32 22 21 16 14 12 12 10 10 9 7
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WILLIAMS AND A R T H U R NEW'TON
Tables of the proximate composition of the vegetables tested were consulted to see if there were either qualitative or quantitative differences which would suggest an explanation of the results observed. However, no explanation based on tl~s idea seemed probable. The results of tests made to determine whether the difference in count on the two lots of commercial tomato juice might be explainable on the basis of the amount of salt present did not support such an idea. Salt at a level of 2 % was inhibitory to sporulation; lesser amounts were only slightly or not at all stimulatory. The differences in spore productivity among the various vegetable media made it seem worthwhile to test combinations to see whether the productivity of one could be altered by the addition of another. The mixtures were in equal proportions except as noted. The combinations tested and the counts recorded for each were as follows: Carrot and celery . . . . . . . . . . . Carrot and spinach . . . . . . . . . . Carrot and potato . . . . . . . . . . Carrot and tomato . . . . . . . . . . Celery and spinach . . . . . . . . . . Celery and potato . . . . . . . . . . Cucumber and tomato . . . . . . . . Beet and tomato . . . . . . . . . . . 25 % b e e t - 75 % tomato . . . . . . 50 % beet - - 50 % tomato . . . . . . 75 % beet - - 25 % tomato . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
. . . . . . . . . . .
21 23 I8 14 18 10 10 15 14 14 15
It will be noted that in the instance of the cucumber-tomato mixture the count is essentially the same as that obtained with cucumber Mone-the less productive member of the combination. The beet-tomato and carrot-celery combinations gave counts intermediate between the counts for the components alone, while the carrot-potato combination gave a count essentially the same as that of the more productive member of the pair. Such data scarcely permit of a generalization regarding the effects of one vegetable on the productivity of another, but rather speak for the necessity of testing each combination of nutrients. The fact that growth of essentially equal luxuriance was obtained on the various vegetable base media but with significant differences in spore yield suggested either that certain of the media carried a factor(s) stimulatory to spore formation, or others a sporulation inhibitor. Several workers have noted the stimulatory effect of one or another substance (Oct~MANN (8) STANTIAL (10) NICKERSON and THIMANN (7)) and a few experiments were conducted to determine whether the superiority of tomato juice might be due to the presence of a factor(s) stimutatory to spore production. Salt free juice was separated into liquid and insoluble solids fractions by centrifuging, and media prepared from each fraction. The liquid fraction supported 24 % sporulation, the solid fraction only 6 %. Further testing was done with the liquid fraction of the juice. Control observations were made on cultures from media consisting of half strength tomato juice and gave asci counts within the range 23 to 32 %. The effect of each of the various factors tested was evaluated by incorporating treated juice, or material removed from juice, in medium in a concentration which corresponded to the amount of untreated juice in the controls. The heat stability of the factor(s) at acid, neutral and alkaline reactions was tested by mixing two parts of juice with 1 part of 4 N hydrochloric acid or sodium hydroxide, and by adjusting a third lot to pH 6.8. These were auto-
SPORULATION BY HANSENULA MRAKII ON VEGETABLE MEDIA
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claved at 15 pounds steam pressure for 30 minutes. Each was then adjusted to pH 6.8 and incorporated in medium. Asci counts from these media gave values of 0, 13 and 23 % for the acid, neutral and alkali treated juice, respectively. Attempts to remove the factor(s) by ether extraction at pH values of 4.4, 6.8 and 9.1 were not successful. Cultures on media containing ether extracted juice gave asci counts of 25 % while cultures from media containing an aqueous solution of the material removed by ether extraction and remaining after evaporation of the ether were negative for asci. The active material(s) was removed by wecipitation with both silver nitrate and basic lead acetate. Precipitation with silver nitrate was effected by adding a 4 % aqueous solution until no further precipitate formed, filtering and removing tile excess silver with 6N hydrochloric acid. The silver chloride was filtered off and the filtrate evaporated on a steam bath to near dryness to remove the excess hydrochloric acid. The volume was restored with distilled water, and the filtrate incorporated in medium in the usual manner. Lead acetate precipitation was effected with 2 % basic lead acetate, filtering and removing the excess lead from the filtrate with hydrogen sulfide. The lead sulfide was removed by filtration and the excess hydrogen sulfide by boiling. The filtrate was restored to volume with distilled water and incorporated in medium. Good growth was obtained on each of the media. No asci were noted on the medium containing the lead acetate precipitated juice, and less than 1 % on the silver nitrate precipitated juice. Adsorption of juice with norite in the ratio of 1 part norite to 2 parts of soluble solids at pH 3.2, 6.8 and 9.0 showed little or no effect at pH 3.2, essentially complete removal of the factor(s) at pH 6.8 and some removal at pH 9.0. The factor(s) could not be eluted by refluxing the norite used for adsorption with either 5 % ammonia in ethanol or by a mixture of 5 % pyridine, 45 % ethanol and 50 % distilled water. These various data are inadequate to characterize the factor(s), but do suggest that there is present in tomato juice a factor(s) stimulatory to spore production by H. mrakii, and that the factor(s) is probably a neutral substance(s). Summary A determination of the ascospore producing potential for Hansenula mrakii of several vegetable media, showed celery and tomato media to be much superior to all others tested, and with celery slightly better than tomato medium. The stimulatory effect on ascospore production, at least in the instance of tomato, appears to be due to a neutral substance which is not extractable with ether, is destroyed by heating under pressure in the presence of acid, and which can be removed by norite adsorption at pH 6.8 and by precipitation with silver nitrate or basic lead acetate. References
J. L. 1949. Personal communication. 2. HENRICI, A. T. ]941. The yeasts, Bact. Reviews, 5, 97--179. 3. LINDEGREN, CARL C. and HAMILTON, ELIZABETH. 1944. Autolysis and sporulation in the yeast colony. Bot. Gazelte 105, 316--321. 4. LINDEGREN, CARL C. and LINDEGREN, GERTRUDE. 1944. Sporulation in Saccharomyces cerevisiae. Bot. Gazette, 105, 304---316. .
ETCHELLS,
6 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
O. B. WILLIAMS AND ARTHUR NEWTON MRAK, E. 1V[. 1950. Personal communication. MRAK,~E. M., PHAFF, H. J. and DOUGLAS, H. C. 1942. A sporulation stock medium for yeasts and other fungi. Science 96, 432. NICKERSON, W. J. and THII~ANN, K. V. 1941. The chemical control of conjugation in Zygosaccharomyces. Amer. four. Bot. 28, 617--621; 1943. 30, 94--101. 0CHMANN,W. 1932. ~(3ber den Einfluss der Stickstoffnahrung auf die sporenbildung yon verschiedenen Hefen. Centrbl. Bakt. Parasitenk. II. 86, 458--465. PHArr, H. J. and MRAK, E. M. 1949. Sporulation in yeasts, Part II. Wallerstein Lab. Comm. 12, 29--44. STANTIAL,H. 1935. The sporulation of yeast. Second paper. Trans. Roy. Soc. Cann. III 29, 175--188. WICKERHAM, LYNFERD J. 1950. Personal communication. WICKERHAM,LYNFERD J. and BURTON, KERMIT A. 1948. Carbon assimilation tests for the classification of yeasts, four. Bact. 56, 3 6 3 3 7 1 . WICKERHAM,LYNFERD J., FLICKINGER, M. H. and BURTON, Z. A. 1946. A modification of HENRICI'S vegetable juice sporulation medium for yeasts. four. Bact. 52, 611--612. WICKERHAM,L. J., GILL, R. J. and BURTON, K. A. ]950. New concepts in the phylogeny of yeasts as revealed by a study of the genus HansenuIa Hansen. Bact. Proc. 36--37. WYNNE,E. S. 1948. Physiological studies on spore formation in Clostridium botulinum, four. In/. Dis. 83, 243--249.
