Starting Axenic Cultures from Bacterial Plates
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- Harvest cells from the edge of a growing clone (at least 1 cm diameter) of D. discoideum from a bacterial plate
by scraping
with a loop. Preferably use SM plates, which yield 5 times as many cells as SM/5 plates. Try to minimize the amount of
contaminating bacteria, but don't be overly concerned about it. Use fresh plates; the viability rapidly decreases after a
few days at 4°C.
- Suspend the cells in 1 ml of HL5 in 13x100 mm sterile tubes or in wells of a 24-well plate. The HL5 should contain 300-500 µg/ml
of dihydrostreptomycin-sulfate. Start at least 4 tubes for each strain.
- Incubate a couple of days at 21-22°C without shaking. This allows the cells to consume the remaining bacteria and
become axenic.
- Change the medium.
- After two more days the contents of the tubes are transferred to a flask with regular HL5 (10 ml in 50 ml flask);
shake at 21-22°C. You'll have a growing culture in a week. Cells that have been cultured for several transfers on bacterial
plates seem to take longer to get established in axenic medium.
J. Franke, 1983 (rev. 12/92; 05/2002; 11/2005)
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