Exercise 13.6 - Suspension cultures of D. discoideum

Procedure

1. Collect amoeba from the normal media cultures. Examine the culture for the presence of the amoeba and note the stage of their development (i.e. individual amoeba, aggregates, slugs).

2. Select amoeba from the cAMP treated culture and compare the amoeba to those grown in the absence of the nucleotide.

3. Using a pipette, transfer an aliquot of the amoeba previously grown in the absence of cAMP to fresh media without cAMP. Establish a second culture by transferring the same amoeba to media containing cAMP. Follow the techniques outlined for the transfer of eukaryotic cells in Chapter Twelve.

4. Prepare a growth curve for each of the amoeba subcultures (i.e. amoeba grown in the presence and absence of cAMP).

5. Note on the growth curve the times for any observed differentiation.

Optional

When the amoeba aggregate, the position the cells have within the slug determines its ultimate fate. If a cell is in the anterior of the slug, it will become part of the subsequent stalk. If in the posterior, the same cell would become part of the spore forming body.
The pseudoplasmodia are attracted toward a strong light, and this taxis has been used to orient slugs and consequently separate cells on the basis of their position.
Plate Ax-3 cells onto Millipore filters for development (5 x 10 cells/cm). At the finger stage, shake the cells from the filters and dissociate them into single cells by vigorous pipetting in cold MES-PDF containing 10 mM EDTA. Shake the suspension for 3 hours under one of the following conditions:

No added cAMP @230 RPM
1 mM cAMP, with hourly additions to 100 microM, 230 rpm
No added cAMP @ 70 RPM

The fast shaking cultures will remain as predominantly single cells while those in the slowly shaking culture will aggregate. 26
Harvest the cells by centrifugation and analyze for size of the aggregates as well as formation of the multicellular structures.

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