I. Objectives: To observe the germination of a sclerotium of Physarum polycephalum, its migration across an agar surface, cytoplasmic streaming of organelles through its "veins", the formation of a new sclerotium, and formation of fruiting bodies.

Il. Materials:

• 100 mm plastic Petri dishes
• sclerotia of Physarum polycephalum
• forceps
• scalpel
• 100 ml Petri dishes containing sterile HI agar: steep 1.25 g hay in 500 ml steaming distilled water for 30 min; filter supernate through several layers of Kimwipes; bring volume to 500 ml with distilled water and adjust pH to 7.0 with 1.0 N KOH; add 7.5 g agar, autoclave and dispense in 100 ml plastic petri dishes 100 mm diameter Whatman #1 filter paper distilled water in squirt bottles sterile Quaker oat flakes

III. Procedure:

• Tear off piece of filter paper with sclerotium (several mm in diameter) with sterilized forceps and place face down on a fresh dish of HI agar. Sprinkle a half dozen sterile oat flakes near the filter paper.
• Put one piece of Whatman #1 filter paper in the top of a plastic Petri dish, flood with distilled water and then pour off excess water. Add a piece of sclerotium about 1 cm in diameter to the wetted filter paper and then sprinkle about 6 oat flakes onto the filter paper. Cover with the bottom of the Petri dish.
• After the plasmodium on the HI plate has migrated away from the oats (probably after 2-3 days of feeding), cut out a piece of agar without any oat flakes near the plasmodium (about 2 cm in diameter) and place face down on top of a Petri dish containing two freshly wetted pieces of Whatman filter paper. Cover the dish. After 24 hours, crack the dish by lifting the bottom slightly and propping on the top, leaving a small air space. Observe over the next 5-7 days.

IV. Results: After 24 hours, the sclerotium will have germinated to produce a moving plasmodium. The plasmodium will move toward the oats and will enlarge after several days of feeding on the oats. Subsequently, the plasmodium will move away from the oat flakes. At that time, if the Petri dish bottom over the wetted filter paper set-up is lifted and set slightly askew on the Petri dish top, the filter paper will dry rapidly overnight, causing the plasmodium to sclerotize. The resulting sclerotium may be stored for several months at room temperature, with the expectation that it will germinate if set up as in PROCEDURE "B" above. The sclerotium placed on the HI plate will also germinate within 24 hours, at which time the plate can be placed upside down on a compound microscope stage. Using a 4 X or 10 X lens, the "veins" of the plasmodium can be observed. The cytoplasmic contents will be seen to stream rapidly in one direction from 15-40 sec, slow down, stop, and then reverse direction for about the same amount of time. The granular material rushing by in the veins consists of nuclei, mitochondria, and vacuoles. The two-piece of filter paper set-up should allow slow enough drying that the plasmodium will fruit to form black, pleomorphic fruiting bodies. These generally will not yield viable spores with this technique, but give a fairly good example of the fruiting process nonetheless.

V.Cautionary Notes: Since sterile techniques are not rigidly adhered to in these procedures, some yeast or bacterial grown can be expected on agar/filter paper surfaces. This should normally not be a problem. If producing sclerotia for long-term storage, allow them to migrate over agar surfaces repeatedly until they are not leaving behind trails of bacteria or yeasts before transferring to sterile filter paper wetted with sterile distilled water for the sclerotizing process described above.

VI. Reference: