I. Introduction: Dictyostelium discoideum has long been used as a model organism for studying differentiation, since the vegetative amoebae aggregate and form multicellular "slugs" after the food supply (bacteria) has been depleted. Once the amoebae have aggregated, they begin differentiating into prespore and prestalk cells. After the slug has formed, the anterior half of the slug is destined to become stalk cells, while the posterior half will become spores. If some of the amoebae are fed Serratia marcescens which contains the reddish pigment prodigiosin, the amoebae will become pink (the pigment becomes deposited in vacuoles as the amoebae eat the bacteria. Another population of amoebae is fed Escherichia coli, and will remain white to cream-colored. If the anterior of a pink slug is grafted onto the posterior of a white slug, the fruiting body subsequently formed will consist of white spores on a pink stalk. If the graft is white anterior to pink posterior, pink spores will be found on a white stalk.

II. Materials:

• Dictyostelium discoideum
• E. coli (a non-mucoid strain like K-12 or B/r)
• Serratia marcescens
• 100 ml Petri dishes containing sterile Hay Infusion (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 1.0N KOH. Add 7.5 g agar, autoclave and dispense in plastic petri dishes
• 100 ml Petri dishes containing sterile NTGY agar: 1.25 g tryptone
  1.25 g yeast extract
  0.25 g glucose
  0.25 g K₂HPO₄
  5 g agar
  (bring to 250 ml final volume with distilled water, autoclave and dispense into Petri dishes
  
• sterile Pasteur pipets and bulbs
• transfer loop
• bunsen burner to sterilize loop and transfer needles
• transfer needle (scalpel blade, etc.)
• compound and dissecting microscopes
• bent glass rod to spread inocula on plates
• sterile distilled water (SDW)
• 75% alcohol in wide-mouth container for dipping utensils to be flamed

• A. Streak one NTGY plate with E. coli and another with Serratia marcescens with with transfer loop. Grow for several days at room temperature.
• B. Take a large loopful of E. coli and add to 3 drops of sterile distilled water in the center of an HI Petri dish.
• C. On another HI plate do the same with the Serratia marcescens culture.
• D. Use a transfer loop to collect a large number of D. discoideum fruiting bodies. Transfer them to the drops of water containing bacteria on each plate.
• E. Sterilize the bent glass rod by dipping it into alcohol and briefly flaming it before use (do not flame and then dip it into the alcohol or it will fracture). Use the flamed rod to spread the bacterium/D. discodeum material all over the surface of the plate.
• F. Within 24 hours, numerous amoebae will be observed mixed with the food bacteria on each plate. Use a sterile loop to collect some of them and place them in a drop of distilled water on a microscope slide. Add a glass coverslip and observe with a compound microscope to determine the mode of locomotion (amoeboid motion with extension of filose pseudopodia).
• G. After 48 hours, turn the plate upside down and look through the bottom of the plate with a 4 X or 10 X objective on a compound microscope to see aggregation of amoebae in streams to a forming center.
• H. Within 5 or 6 hours of streaming, slugs (elongated, multicellular masses of cells) can be seen migrating over the agar surface. Note that the slugs grown on E. coli as a food source are pale white while the slugs grown on S. marcescens are pale pink in color.
• I. Place a dish of cells grown with E. coli under a dissecting microscope and sever a slug at the mid line (anterior/posterior). Transfer the anterior part of the slug to one fresh HI plate and the posterior part of the slug to another HI plate.
• J. Place a dish of cells grown with S. marcescens under a dissecting microscope and sever a slug at the mid line (anterior/posterior). Attach the posterior part to the anterior part of the slug grown on E. coli. Attach the anterior part to the posterior part of the slug grown on E. coli.
• K. Repeat steps I and K above until you have 4 or 5 grafted slugs on the fresh HI plates.

IV. Results Expected: After 24 hours, fruiting bodies should have formed from these grafted slugs. Careful examination should show white spores on pink stalks (from grafted slugs with pink anteriors and white posteriors) and slugs with pink spores on white stalks (from grafted slugs with white anteriors and pink posteriors).

Observation of fruiting bodies on the plates from which the slugs were removed should show all white fruiting bodies on the E. coli plates and all pink fruiting bodies on the S. marcescens plates.

V. Cautionary Notes: If the grafted slugs get significantly disorganized during transfer or the grafting process itself, they may go back to a vegetative mode and reorganize such that the differentiation of the two cell types by color will be difficult (they can become randomly reorganized within a newly-formed slug).

VI. Reference:

Bonner, J. T. 1967. The Cellular Slime Molds. Princeton University Press, New Jersey.

Raper, K.B. 1984. The Dictyostelids. p. 146-149. Princeton University Press, New Jersey.