from Scientific American, January 1966, pages 116-121

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Conducted by C. L. Stong

THE AMATEUR SCIENTIST

How to cultivate the slime molds and perform experiments on them

You can usually stump players in guessing games such as Twenty Questions by introducing into play one of the 450 species of amoeboid slime molds. These are organisms that do not fit neatly into the conventional categories of animal, vegetable or mineral. Strictly speaking they are none of these. Some player may argue that the zoologist classifies the organisms as animals -- protozoa in the group of animals called Mycetozoa -- on the basis that they cannot manufacture food and that during much of their life cycle they move freely in response to light, heat and chemical stimuli. To this argument the botanist can respond just as stoutly that slime molds are plants, which he calls Myxomycophyta, because at maturity they develop cell walls of cellulose, become securely attached to some feature of the environment, produce fruiting bodies and release spores. Some biologists take the position that the concept of plant or animal simply cannot he applied to these organisms. At some point in the course of evolution the slime molds ran up a blind alley, where they are now trapped -- relatively harmless and apparently good for nothing except as specimens for some of the most engaging experiments in biology. A. C. Lonert, who is director of research at the General Biological Supply House in Chicago, explains how to culture and experiment with two of the most interesting species. "I shall discuss two kinds of slime mold belonging to different orders," he writes, "because of the astonishing diversity of their life cycles and the ease with which they can be cultured. Both organisms offer a rich field for experimentation.

A slime mold growing in a natural environment
A slime mold growing in a natural environment

"The first, Physarum polycephalum, is a member of an extremely interesting order, the widely distributed Myxogastrales (also called Myxomycetes) of the subclass Mycetozoa. Viewed from the perspective of an imaginative microscopist, the sporangia, or fruiting bodies, of the many species appear as enchanted forests in which the fruits become pulsating, devouring monsters.

"When found in the early stage of growth, the myxomycete is most like an animal and generally consists of an unattractive patch of naked protoplasm, either colorless or pigmented, that can attain a diameter of several feet. This hungry protoplasmic mass, called a plasmodium, creeps about in a moist and well-shaded environment, seeking food in rotting vegetation and decomposing wood. In the process of feeding, the organism aids the economy of nature by processing waste into compounds that can be utilized by other organisms.

"At a propitious time, governed by the availability of food and such factors as temperature und moisture, the oozy mass leaves its dank haunts and, as it reaches toward the light, transforms itself into upright plantlike structures. These are the sporangia. The shape, color and size of those bodies vary considerably among the many genera of Myxogastrales, but the bodies usually have two features in common; a delicate network of noncellular threads and strands, called the capillitium, in which the spores are enmeshed, and a fragile, sometimes beautifully formed and tinted spore case, called the peridium.

"After the drying and rupture of the spore case, expansion of the threads of the capillitium exposes the spores to the action of wind and rain. At the same time the expansion controls the rate at which the spores are released. The life cycle is repeated when spores fall in a suitable environment. Free-swimming swarm cells, or myxamoebae, are then released, They feed for a time and multiply by splitting in two. Eventually two of the cells fuse to form a zygote, which is equivalent to fertilized egg. This fusion constitutes the beginning of a new plasmodium, which can grow by nuclear division or by fusion with other zygotes. Unfortunately for those who enjoy the exotic and beautiful in nature, these fantastic groves are usually at ground level. Because they are underfoot they usually remain unobserved and unappreciated except by those who know where to look.

"Most of the slime molds, when taken from their natural environment, are difficult to culture. This difficulty need not stop anyone from enjoying the real-life adventure of observing the strange organism at home or in the laboratory. A culture can be started easily from a fragment of Physarum polycephalum plasmodium in the sclerotial stage, a dried condition. This stage is sometimes found in nature and can be induced in the laboratory by the method to be described.

"Sclerotia that will remain viable for a long time and have the ability to transform themselves into active plasmodium within several hours are available commercially. One can also buy the necessary supplies for experimenting with the organisms. All the items can he obtained from distributors such as General Biological Supply House, Incorporated, 8200 South Hoyne Avenue, Chicago, Ill. 60620.

Arrangement of an improvised chamber for cultivating slime mold
Arrangement of an improvised chamber for cultivating slime mold

Details of the culture chamber
Details of the culture chamber

"To grow a plasmodium of Physarum polycephalum from the sclerotial stage, first improvise a culture chamber by placing a dish -- for example an inverted saucer or a Petri dish -- within a larger, covered container, such as a casserole or a pair of facing Pyrex pie plates, so that the smaller dish serves as a platform [see illustration right]. For this experiment omit the rubber separators shown in the illustration. Lay a sheet of filter paper or paper toweling across the platform. The paper should droop to the bottom of the dish. Wet the paper thoroughly with distilled water, pouring off the excess. From time to time add enough water to prevent drying. Incidentally, distilled water should be used in all experiments to be described.

"Place a small fragment of selerotium on the platform and wet it with a drop of water. Within a few hours the organism will awaken from its deathlike torpor. Keep the unused portion of sclerotium in the refrigerator. When the plasmodium has emerged and has begun to seek food, put a flake of uncooked oats in contact with the rapidly spreading growth. Use old-fashioned rolled oats, not the 'quick' variety.

"Keep the covered dish at room temperature in an area that does not get direct sunlight. As the plasmodium increases in size, place more oat flakes along the growth front. A rapidly moving plasmodium will consume a larger number of oat flakes than a sluggish one. In either case the feeding organism will show a decided preference for fresh oat flakes and will abandon partially digested ones. Flakes that have first been moistened with a drop of water are accepted more readily than dry oats. To maintain a clean culture, transfer the organisms to a fresh sheet of filter paper or paper toweling weekly, avoid overfeeding and remove abandoned food that shows signs of becoming moldy or slimy. Occupied oat flakes (those covered with yellow plasmodium) or sections of paper bearing plasmodium can he transferred repeatedly to clean sheets of paper to perpetuate the plasmodial stage.

"The organism at this stage has been compared to a giant amoeba: a thin, irregularly shaped mass of creeping jelly that is threaded by a network of veins. To observe the complex circulation in these vessels, transfer several occupied oat flakes to a Petri dish containing a thin layer of nonsterile, nonnutrient 1.5 percent agar. The agar culture is maintained in the same way as the paper culture, but there is no need to add water, The protoplasmic streaming and its periodic reversal can be observed with any microscope capable of magnifying to 50 diameters or more. Observation should be made with light transmitted through the specimen from the substage. Cut one of the vessels when the circulation is active and observe the effect. Make a transfer of scraped plasmodium and watch it reconstitute itself. Place a drop of vinegar near the plasmodium and note the reaction.

"Usually cultures can be kept in the plasmodial stage for some weeks by maintaining ample food and water and periodically transferring specimens as described. Occasionally, however, specimens will for some unknown reason enter the sporangial, or fruiting, stage spontaneously. This stage can bc induced at any time by removing most of the food amd allowing the plasmodium to roam while simultaneously keeping the organism moist. The transformation will occur suddenly, usually at night, within a week or so. If the observer is fortunate enough to witness the actual transformation, he will see the entire plasmodium, now more orange than yellow, appear to separate into uniformly rounded masses that ascend from the surface on stalks and then develop into weird, multilobed bodies [see illustration below].

Sporangia of the genus Physarum polycephalum
Sporangia of the genus Physarum polycephalum

"Under the higher powers of the microscope, spores obtained from crushed sporangia can be seen to germinate with the emergence of an amoeba-like protoplast, which afterward gives rise to an amoeboid and then to flagellum-bearing swarm cells-cells equipped with whiplike tails for swimming. The series of transformations usually requires about three days. Eventually pairs of swarm cells fuse to become zygotes. From these a new plasmodium originates.

"The germination and subsequent transformations up to, but usually not including, the plasmodial stage can be observed by setting up a hanging-drop preparation. This consists of a drop of water, containing specimens, that clings to the underside of a thin sheet of glass. The glass is usually a cover slip of the type used to protect specimens on a microscope slide. Evaporation is prevented by enclosing the drop in a small, airtight vessel. The vessel may consist of a microscope slide into which a depression has been ground. The other items needed for doing the experiment are a sterilized medicine dropper, a quarter-inch loop of fine wire supported at the end of a pencil-sized piece of wood, a pair of tweezers, Vaseline, a 70 percent solution of isopropyl alcohol and a gas burner or an alcohol lamp.

"Sterilize a microscope slide and cover slip in the flame. A few sporangia are then placed in isopropyl alcohol for one minute and dried on the sterilized slide. Next the dried specimens are placed between another pair of sterilized slides with a drop or two of water and crushed. The sterilized cover slip is held horizontally, by an edge, with the tweezers. A drop of water is applied to the underside of the cover slip at the center.

"This suspended drop is inoculated with spores by first stirring the crushed specimen with the wire loop and touching the loop to the bottom of the drop. The rim of the depression in the microscope slide is now lightly coated with Vaseline. Finally, the cover slip is placed on the coated slide with the suspended drop centered over the depression. The cover slip is pressed gently into place to seal with Vaseline the space between the cover slip and the slide. The combination is placed on the stage of the microscope, which is focused on the contents of the suspended drop. The recommended sterile technique is not absolutely necessary but it increases the probability that more of the early stages will develop. It is difficult, however, to generate a fresh plasmodium by this technique.

"To preserve Physarum polycephalum for possible future use, return the organism to the dormant, sclerotial stage, from which it can be conveniently aroused again when it is needed. A method that I developed is quite simple and produces a good yield of very durable sclerotia. Set up a culture chamber with 10½-inch Pyrex pie plates, 10-inch disks of paper and either the top or the bottom of a six-inch Petri dish to serve as an elevated platform. Lay the paper across the platform and center it on the lower pie plate. Moisten the paper with distilled water and push down the portion of the paper that extends beyond the platform. The depressed paper forms a circular moat. Pour off excess water. Transfer oat flakes occupied by plasmodium from a desired culture to the center of the paper. Cover the pie plate with a similar but inverted dish.

"When the plasmodium begins to move about, place fresh oat flakes along the growth front -- wetting each flake to facilitate rapid occupation. By the following day plasmodial fronts, moving toward the edge of the culture chainber, will have developed. Put fresh oat flakes along these more distant fronts and, when they are occupied, lift them from the paper with a knife, replacing them promptly with fresh flakes, and reposition the occupied flakes toward the center ot the platform. Place the flakes next to each other. Continue this procedure until a circular area of occupied flakes about three inchcs in diameter has been concentrated ceutrally on the platform. As much specimen material as desired can usually be gathered after 48 hours of consecutive feeding. Occupied flakes can be taken from more than one culture; if several cultures are maintained simultaneously, a good supply can he accumulated in a much shorter period of time.

"To begin the induction of sclerotization, take the plasmodium-bearing paper out of the culture chamber. Remove surplus moisture by superposing the paper gently, with occupied oat flakes uppemrost, on a piece of clean, dry paper of the same size. Allow the papers to remain in in contact. Wash and dry the platform and bottom culture plate, then promptly reassemble the chamber and restore the partly drained culture paper to its former position, reforming the moat.

"Circle the paper moat with dry oat flakes. The flakes form an entangling barrier that prevents wastage of active Plasmodium by premature drying at the edge of the plate. Next insert one or two thicknesses of rubber tubing, which have been slit lengthwise, between the upper and the lower culture plates at four equidistant points [see illustration]. If the relative humidity is high (from 50 to 60 percent at temperatures ranging from 80 to 90 degrees Fahrenheit), use two thicknesses of rubber tubing and increase the spacing of occupied flakes; if the relative humidity is low (from 15 to 25 percent at 75 to 80 degrees F.), use one thickness of rubber tubing. These are, of course, rough approximations. The object is to dry the culture paper in not less than 24 hours and not more than 48 hours. (One should avoid, however, the use of an artificially created air current to promote drying.) The crustlike sclerotia produced by this method will retain viability for years if they are stored in closed jars under refrigeration at 42 degrees F.

"The second slime mold, Dictyostelium discoideum, a member of the order Acrasiales, differs in a number of remarkable ways from Physarum polycephalum and other members of that order. It is a bacterial feeder and also exhibits both animal and plant characteristics. The events of its life cycle can be ascertained rather easily, but several of its other properties have not yet yielded to the assaults of the investigator. Apparently similar cells play at least three distinctive roles on a simple level -- first, as free-living myxamoebae; second, as massed structures of cells exhibiting interdependence and coordination, and third, as cells differentiating into several kinds of structure.

"About 18 to 24 hours after inoculation of a bacterial culture with spores of Dictyostelium discoideum there will appear within the bacterial growth numerous minute, refractive lumps that have bright centers when they are viewed slightly above focus at a magnification of 100 diameters. These lumps are actually the minute myxamoebae busily eating their way through what, on the scale of comparative sizes, must be described as jungles of bacteria. Simultaneously the organisms multiply at a prodigious rate through binary fission, or splitting. Even a hand magnifier will show the digestion of circular patches of bacterial growth at this stage. Incidentally, if the experimenter is working with an inexpensive microscope objective of .25 or .65 numerical aperture (corrected for use with a cover slip) and a 160-millimeter tube length, a fairly good compensation for the aber- ration caused by the absence of the cover slip can be made by increasing the tube length by 30 mm.

Wandering myxamoebae, enlarged 525 diameters
Wandering myxamoebae, enlarged 525 diameters

"At the end of 24 hours the culture will begin to teem with hordes of the voracious organisms, which are new clearly visible as they sweep the agar surface clean of bacteria. The disorganized, searching myxamoebae as they appear with a fairly oblique illumination are shown in the accompanying photomicrograph [see picture right]. The organisms are difficult to see except under such oblique lighting or with phase illumination.

"By the end of 48 hours, with the food supply nearing depletion in some portions of the plate, a remarkable change will begin to take place. The organisms stop feeding and spontaneously converge on centers of aggregation. According to John Tyler Bonner of Princeton University, the release of acrasin, a chemotactic substance, is responsible for the strange phenomenon. As the myxamoebae continue to press themselves into these centers of aggregation several moundlike structures are built up, each with a characteristic tip. The myxamoebae pack themselves together but persist in maintaining their individuality. There is no fusing of cells into a mass that contains a large number of nuclei as there is in the case of Physarum polycephalum; the pseudo-plasmodium remains associational in character. The individual aggregates grow rapidly through the continued accretion of myxamoebae, elevating at first and then, after a period of elongation, tipping horizontally to become migrating, sluglike creatures.

"The slug soon assumes the characteristics of a creature equipped with special organs. For example, it has a front-to-back orientation, with the tip, observed to form early in the mound stage, exhibiting sensitivity to light and heat. In the presence of such a stimulus the top appears to head the parade with the rest of the body following, leaving a trail of slime behind it. The motion of the slug is apparently produced by the concerted action of the individual myxamoebae, but it is not yet known what coordinates their remarkable performance. It is possible, by exerting gentle pressure, to dissociate amoebae from the mass. Subsequently they recombine to reconstitute the slug! Even a stained section of the slug fails to demonstrate the mechanism of communication. It is at this stage of develop- ment that the slime mold lends itself particularly well to experimentation.

"Boner has shown that when the tip of one slug is cut and joined to the tail end of another slug, or replaces the tail of another slug, it becomes evident that differentiation has occurred, that certain groups of the amoebae have acquired unique traits [see "Differentiation in Social Amoebae," by John Tyler Bonner; Scientific American, December, 1959]. Myxamoebae of the tip that have been transplanted, for example, will begin to migrate to their own social level and join the tip cells of the host. On the other hand, a graft of the hind part of one slug to replace the hind part of another slug produces no appreciable migration. To tag the myxamoebae for these and other experiments Bonner stained the specimens with a harmless dye. The stains must be used in highly diluted form; they include Janus green B, methylene blue, neutral red and brilliant vital red. Pink slugs can also be developed from colorless slugs for experimentation by starting a culture with the red Serratia marcescens bacterium as thc food organism. The myxamoebae are unable to eliminate the bacterial pigment.


Stages in the fruiting of Dictyostelium discoidium
Stages in the fruiting of Dictyostelium discoidium


"The final stage should develop in about 60 hours. By that time a number of slugs will have begun to elevate and change into fruiting bodies. In the process the myxamoebae located toward the front end and along the central axis of the slug undergo a drastic change, literally giving up their lives to become a firm supporting core of cellulose-lined dead cells constituting the sporophore, or stalk. The rest of the myxamoebae begin to be raised upward. Those amoebae that do not become stalk cells are eventually lifted in a body to the top of the stalk, where they are transformed into capsule-shaped spores encased in a globular mass of slime. This body constitutes the sorus. The complete fruiting sequence is shown in the accompanying illustration [see illustration right]. If spores are sown on a clean agar surface without food organisms, freshly hatched myxamoebae, the beginning of a new cycle, may be observed in about 18 hours.

"A suitable culture medium can be made by first cooking 35 grams of hay for 10 minutes in one liter of tap water. Filter this solution and add enough water to restore the original volume. Add the filtered infusion to a flask containing 15 grams of agar. Boil to dissolve the agar. Sterilize in steam at 15 pounds of pressure for 20 minutes.

"Another satisfactory medium is described in Plants in Perspective, by Eldon H. Newcomb, Gerald C. Cerloff and William F. Whittingham, published by W. H. Freeman and Company in 1964. It consists of .1 percent lactose and .1 percent peptone in 2 percent agar sterilized as above. Melt the agar first and then add the other ingredients.

"To start the culture pour the medium into sterile Petri dishes and test tubes. Avoid aerial contaminants. Test-tube media are usually sterilized after pouring. After preparing fresh stock cultures inoculate the selected medium with a nonmucoid strain of Escherichia coli or Serratia marcescens and then inoculate the same plate with Dictyostelium discoideum spores. By restricting to limited areas the spore inoculation of the Petri-dish culture (the entire plate, however, can be inoculated with bacteria) almost any stage of developent can be found in other portions of the culture for several days after formation of the first organisms. The bacteria that serve as the food organism can be transferred in the form of a suspension or they can be streaked over the agar surface. Incubate the culture at room temperature.

"To prepare stock cultures transfer the food organism to two or more agar plates and add Dictyostelium discoideum spores to one of them. When the stock culture has developed, stopper it with the separate culture of bacteria and store under refrigeration. Transfer the stock cultures every three months."


Bibliograhy

Growth and Development of Dictyostelium discoideum with different bacterial associates.
Kenneth B. Raper in Journal of Agricultural Research, Vol. 55, No. 4, pages 289-316; August 15, 1937.

An Introduction to the Plant Kingdom.
Norman H. Russell. The C. V. Mosby Company, 1958.

Morphology and Taxonomy of Fungi.
Ernst Athearn Bessey. Hafner Publishing Company, 1961.