Exercise 13.5 - D.discoideum Growth on Agar

LEVEL II

Materials

• Dictyosteleum discoideum grown axenically on agar plates
  
• Petri plates with SM agar media
  
• Broth culture of Klebsiella aerogenes
  or E. coli (non-mucoid variety)
  
• Wire loop for transfers
  
• Microscope, slides, coverslips

Procedure

1. Select a petri plate with SM agar media, a broth culture of and a culture of D. discoideum containing fruiting bodies.

2. Inoculate an agar plate with a thin layer of the bacteria. Select a fruiting body from the fungal culture and place it in the center of the petri plate containing the bacterial suspension.

3. Continue to monitor the growth of the fungus for the next week (make daily observations - new fruiting bodies will appear in about 4 days).
   Note the initial germination of the spores from the fruiting bodies, the subsequent growth and the increased number of amoeba in the culture. As the culture begins to dry, the amoeba will aggregate and form the migrating slug. The slugs will migrate around the plate, consuming the bacteria as food.
   Eventually, the slug will anchor itself to the agar and undergo a process known as culmination. During this stage, the cells in the anterior of the slug will begin differentiating into pre-stalk cells, while those in the rear will develop into pre-spore cells.

4. Sketch each stage in the development of the fungus and note the time of each stage appearance within the culture.

Optional

Periodically remove a sample of the cells or slug and fix with Carnoy fixative. Place the cells on a slide and allow to air dry. Stain with a a basophilic dye and note any alterations in the nuclear material during each phase. In particular, note the presence and size of the nucleoli, and the degree of basophilia demonstrated by the cytoplasm.

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