Preparation of Freezer Stocks of Physarum Myxamoebae

Tim Burland   (Last Update May 10, 1993)

1. Grow Amoebae

• Inoculate an SM plate with a 0.1ml spot of SBS, then inoculate the spot of SBS with amoebae - either using a toothpick to transfer from another plate, or 20uL of a freezer stock.
• Spread amoebae and bacteria over SM plate with a glass spreader, making sure the suspension does NOT dry into the plate while spreading.
• Incubate plates at 26^o (except for  selfing strains, which should be grown at 30^o, and temperature-sensitive mutants, which should be grown at permissive temperature).
• Incubate plates until cells become confluent —typically 5-6 days—and continue incubating plates for another 2 days to allow plenty of time for encystment.

2. Harvest and Freeze Cells

• Check  visually that the plate is not contaminated
• Flood plate with 5mL sterile 10%  w/v glycerol (Sigma cell culture grade, Cat# G-2025)
• Scrape off the myxamoebae with a glass spreader, and recover the suspension from the plate with a pipette.
• Transfer 2mL of suspension to each of two vials (e.g. BioRad #223-9842 cryovials) on ice.
• Transfer the vials to a freezer (-70 to -80^oC).
• Check viability every 5-10 years.

Viability also appears to be OK for at least five years at -20^o—handy for a separate space for backup stocks in labs with only one deep freeze.