(From the ,,Centraalbureau voor Schimmelcultures", Baarn).

C U L T U R I N G OF M Y X O M Y G E T E S by

H. A. D I D D E N S

1)

(F.cccived December 24, 1940).

Most of tile studies on myxomycetes have been made with contaminated plasmodia and under uncontrolled conditions. Therefore it seems important to discuss tile methods of COHEN (2) who succeeded in the attaining of absolutely pure cultures of myxomycetes under controlled conditions. In his investigations he aims at a better understanding of the nutritional physiology of these organisms; the chief problems he attacks are: a. are myxomycetes able to feed saprophytically; b. which is the nutritional relation - - if an3' - - between the myxomycetes and their accompanying organisms. I. P U R I F I C A T I O N (ISOLATION). Most of his cultures were obtained as plasmodia or sclerotia in the field, as the germination of spores may offer difficulties; moreover not all myxomycetes which are suitable for cultivation by their ability of forming readily plasmodia in culture, develop spores. For the purification of the field material from the ever present contaminants (moulds, bacteria) two methods were used: 1. T h e m i g r a t i o n method. 2. T h e e n r i c h m e n t method. Before applying these methods tile generally heavily contaminated collected plasmodia were cleansed of contaminants by allowing them to crawl over some plates of non-nutrient agar (2% agar). Thereupon a part of the plasmodium-bearing substrate was placed on moist filter ') Presented at the meeting of the ,,Nedertandsche Vereeniging voor Microbiologie", Utrecht, October 19, 1940; on that occasion COHEN'S cultures, kept at the ,,Centraalbureau voor Schimmelcultures", were demonstrated. This paper gives a review of an article by A. I. COVlEN(2).

8

H.A.

l)iddens,

p a p e r in a p e t r i d i s h . As t h e p l a s m o d i u m c r e p t over t h e p a p e r , o a t m e a l was s p r i n k l e d on or n e a r it ( m o d i f i c a t i o n of HowAI~D's (3) c u l t u r e m e t h o d ) , care b e i n g t a k e n t h a t not too m a n y g r a i n s were a d d e d lest t h e c o n t a m i n a n t s get t h e u p p e r h a n d . A piece of this filter p a p e r b e a r i n g t h e p l a s m o d i u m was t r a n s f e r r e d to a sterile a g a r plate. 1. In t h e m i g r a t i o n method small pieces of p l a s m o d i u m from this l a t t e r a g a r p l a t e were placed in a p l a t e with w a s h e d a g a r 1) n e a r t h e t)eriphery. As soon as t h e c r a w l i n g p l a s m o d i u m h a d r e a c h e d t h e o p p o s i t e side of the dish w i t h o u t crossing its own t r a c k , it was t r a n s f e r r e d to a n o t h e r p l a t e of w a s h e d a g a r ; t h i s process was r e p e a t e d u n t i l t h e p u r i t y of t h e p l a s m o d i u m was b e y o n d all d o u b t . G e n e r a l l y t h r e e t r a n s f e r s sufficed for this purpose. The m i g r a t i o n m e t h o d is effective b y t h e s t a r v i n g of the c o n t a m i n a n t s on tile v e r y spot where t h e y were p l a c e d on the e x t r e m e l y p o o r m e d i u m (washed agar), the p l a s m o d i u m c r a w l i n g a w a y from them. 2. The second, t h e e n r i c h m e n t m e t h o d , allows t h e m y x o m y c e t e to feed a n d g r o w a l ) u n d a n t l y , w h e r e a s t h e c o n t a m i n a n t s are not s u p p l i e d with sufficient n o u r i s h m e n t . W a s h e d a g a r p l a t e s were s t r e a k e d with a suspension of w a s h e d living cells of Saccharomvces dlipsoideus H a n s e n '-'-)(streaks 5 cm long); a piece of t)lasnaodium (from the a g a r p l a t e used p r e l i m i n a r y to p u r i f i c a t i o n ) was t r a n s f e r r e d at one end of t h e s t r e a k a n d a f t e r r e a c h i n g the o t h e r end, i n o c u l a t e d a g a i n in a second s i m i l a r p l a t e , a n d so o11. The living y e a s t cells serve as food onh" t h e m y x o m y c e t e which can engulf t h e m b y m e a n s of its p l a s m o d i a l s t r a n d s , w h e r e a s t h e y do not s a t i s f y tile needs of t h e u n d e s i r e d c o n t a m i n a t i n g organislns. Tile p u r i t y of t h e p l a s m O d i u m in b o t h m e t h o d s was t e s t e d at each ~) 100 g g r a n u l a t e d Difco Bacto a g a r are washed for 5 clays with freq u e n t changes of distilled water. The a g a r is squeezed o u t in fine cheese cloth untill all free w a t e r is driven out. F r o m this a g a r 2~, (dry weight basis) is p r e p a r e d and buffered a t p H 6.0 (KH.~POdNa~HPO, in a molar ratio of 8/2 : 1.45 g KH2POd, 0.96 g Na,HPOd (molar q u a n t i t y being M/75 t o t a l POd). "-) The v e a s t is cultured in 1 1 flasks c o n t a i n i n g y e a s t - e x t r a c t dextrose b r o t h (cI. note 2, page 9), w i t h o u t p h o s p h a t e ; from time to time the flasks are shaken for aeration. After 2 d a y s the flasks are inclined until the y e a s t settles and the excess m e d i u m is d e c a n t e d off t h r o u g h a flame. The y e a s t is washed several times with sterile distilled water. The final suspension is centrifuged and only a volume equal to t h a t of the yeast layer is left.

Culturing of Myxomycctcs.

9

transfer by inoculating in yeast-extract broth 1) and yeast-extract dextrose broth 2). When these broths remained clear, it was considered as a proof of tile t)urity of tile plasmodium. In case of the second method the yeast did not grow appreciably in the broth without dextrose. Most contaminants (for the greater part bacteria) form nondescript spreading colonies in streak plates on the opaque, colourtess media (washed agar, oatmeal agar) ; obviously in cultures along with the plasmodia they may be only - - with difficulty - - detected by careful microscopical and cultural examination. So the testing for purity by inoculating at regular intervals in yeast-extract broth is needed. An agar-medium remaining clear is no sufficient proof of purity of a culture (cf. SKt:PtExsKf (4)). The migration method is simpler, gives on the whole better results than the enrichment method, but it may happen that a plasmodium is so completely devitalized by continued inanition that it can no longer be transferred successfully ; then it may cross its own track a~ld pick up contaminants again ; moreover for want of food it may start fructification or sclerotization, (ither of them undesirable phenomena, as conditions influencing the germination of spores, a.s.o, are generally unknown. The success of the enrichment method largely depends on the feeding rate of the plasmodium which must be rather fast. The too stow-feeding plasmodia may arrive at the end of tile streak more heavily contaminated than at the moment of inoculation ; moreover there is the danger of sclerotization. Fast-inoving plasmodia are easily purified with both methods. II. C U L T U R E . Plasmodia purified by either methods described above, may be cultured in two ways: 1. in t w o - m e m b e r e d cultures (,,zweigliederige Kultur") 2. in p u r e c u l t u r e s . 1. In t w o - m e m b e r e d cultures the plasmodia are feeding upon a second organism which develops on the supplied medium. ') Yeast-extract (Difco, powdered) 2.5 g, tapwater 1 1. This broth is dispensed in tubes and sterilized by autoclaving 20 minutes at 15 pounds' pressure. 5) Similar to the preceding, with addition t~f 10 g dextrose and 1.4 g Na,HPO~ . 12H~O.

10

H.A.

I)iddens,

I n m o s t of his e x p e r i m e n t s Com~:x used Saccharomwes ellipsoidcus H a n s e n as s e c o n d o r g a n i s m , b e c a u s e t h i s y e a s t is f a s t - g r o w i n g a n d n e i t h e r t o o small, nor g l u t i n o u s . Some o t h e r y e a s t s a n d m o u l d s t e s t e d lack these advantages. T h e p u r e p l a s m o d i u m was t r a n s f e r r e d into o a t m e a l a g a r 1) flasks w i t h S. ellipsoideus. This m e d i u m allows t h e y e a s t to grow m o d e r a t e l y , w h e r e a s richer m e d i a yield too h e a v y a g r o w t h of y e a s t . W h e n the i s o l a t i o n was m a d e b y e n r i c h m e n t m e t h o d it s e l d o m was n e c e s s a r y to i n o c u l a t e the y e a s t , as t h e p l a s m o d i u m c a r r i e d sufficient y e a s t w i t h it. 2. F o r p u r e culture t h e l)urified p l a s m o d i u m was s t r e a k e d with t h e a u t o c l a v e d b a k e r s ' y e a s t ") - - in the s a m e w a y as t h e living y e a s t was s t r e a k e d in t h e e n r i c h m e n t m e t h o d - - on w a s h e d a g a r slants. O t h e r m e d i a t r i e d (plugs of p o t a t o , c a r r o t , b a n a n a , a p p l e , o a t m e a l agar, y e a s t - e x t r a c t agar, pea agar, etc.) g a v e n e g a t i v e results. The good results on t h e w a s h e d a g a r m e d i u n l are in e n t i r e a g r e e m e n t with t h e good r e s u l t s of t h e t w o - m e m b e r e d cultures. P l a s m o d i a i s o l a t e d b y e n r i c h m e n t m e t h o d were freed from t h e y e a s t b y some m i g r a t i o n s o v e r w a s h e d agar. Beside t h e claim of p t t r i t y ( ' o n r x insisted on t h e need t h a t t h e cultures s h o u l d not decline in v i g o u r t h r o u g h m a n y transfers, or - - b e t t e r still - - s h o u l d show good g r o w t h i n d e f i n i t e l y . H e was successful in c u l t u r i n g : Badhamia /oliicola Lister, B. utricularis (Bull.) Berk., Didymium squamu.losmn Fr., Fuligo septica (L.) W e b e r , Physarum polycephalum Schw. a n d Slemonilis a.~i/cra M a c b r i d e in p u r e culture. III. STORAGE OF CULTURES. M y x o m y c e t c - c u l t u r e s ask for a good d e a l of t r a n s f e r r i n g , as v a r i o u s p r o d u c t s of Wastage soon i n j u r e t h e v i t a l i t y of t h e p l a s m o d i a . P u r e c u p t u r e s were k e p t at 25 ° C. a n d t h e gross a n d t w o - m e m b e r e d c u l t u r e s at room temperature. ') Quaker rolled Oats is dispensed into 125 ml E r l e m n e y e r flasks to an even l a y e r one grain in t h i c k n e s s ; 20 ml unwashed a g a r (pH 6.0, cf. washed agar) is pot, red into each flask. Sterilization by a u t o c l a v i n g 20 m i n u t e s a t 15 p o u n d s ' pressure. ~) E q u a l q u a n t i t i e s by' weight of bakers' y e a s t and distilled w a t e r a r e well mixed in a m o r t a r and dispensed into t u b e s in 5 ml q u a n t i t i e s for a u t o c l a v i n g (20 m i n u t e s a t 15 poun(ls' pressure). Before use the tubes are to be shaken.

Culturing of Myxomycetes.

II

In order to cut down the large number of transfers, cultures of¢en were sclerotized by the method of HOWARD (3), a slow drying process, and kept in envelopes in the refrigerator. Not all these experiments proved to be successful. A sclerotium, however, on moistening may change within a few hours into a fast-crawling pla.smodium. From COHEN'S experiments it is evident that the plasmodium and yeast are not in competition for the nutritive mediunl (a view brought forward by Cm~zAszcz (1)); neither seem metabolic products of the yeast or the secreted slime to serve as nutrients, as 3"east cells are seen to be engulfed and destroyed by the plasmodium. Moreover his studies show that the growth of myxomycetes is greatly influenced by temperature, size of inocutum, pH, etc., factors which may change tile balance between plasmodium and its co-organism in two-membered cultures. The foregoing may have given an impression of the useful piece of work done by COHEN in this rather neglected field of botany; he succeeded in cultivating in pure culture several myxomycetes which offer difficulties in handling. In view of the results arrived at already by COHEN, one hopes that this author m a y continue his experiments. Literature. 1. T. CHRZASZCZ,Centralbl. f. Bakt. II, 8, 431, 1902. - - 2. A. I. COHEN, Nutrition of the Myxomycetes. 1. Pure culture and two membered culture of myxomycete plasrnodia, Bot. Gazette 101,243, 1939. -- 3. F.L. HOWARD, Amer. Journ. Bot. 18, 116and 624, 1 9 3 1 . - 4. F. X. SKUPIENSKI, Recherches sur le cycle ~volutif des certaius myxomyc~tes. Paris, 1920; Acto Soc. bot, Poloniae 5, 255, 1928.