Exercise 13.6 - Suspension cultures of D. discoideum
Axenic culture selected for suspension growth (e.g. D.
discoideum Strain AX-3 24)
Cultures supplemented with 10 M cAMP25
Fresh flasks of growth media, with and without cAMP
MES-PDF containing 10 mM EDTA (Optional)
- 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).
- Select amoeba from the cAMP treated culture and compare the
amoeba to those grown in the absence of the nucleotide.
- 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.
- Prepare a growth curve for each of the amoeba subcultures
(i.e. amoeba grown in the presence and absence of cAMP).
- Note on the growth curve the times for any observed
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
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.
Harvest the cells by centrifugation and analyze for size of the
aggregates as well as formation of the multicellular structures.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org