Dictyostelium amphisporum Cavender et al.
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1. Prepare media by streaking center of agar surface with a large X of Escherichia coli (ATCC 23437) and incubating at 25°-30°C one day before inoculating organism. Several replicates are suggested for optimum results.
2. To thaw frozen ampoule, place in a 37°C water bath, immerse ampoule to depth of one millimeter above the level of frozen material in the ampoule. Keep ampoule immersed until material is thawed but no more than 3 minutes. Do not agitate or vortex the ampoule.
3. Immediately after thawing, wipe down ampoule with 70% ethanol and aseptically transfer one loop full of contents onto center of X grown out with Escherichia coli.
4. Incubate the plates at the temperature recommended.
5. Allow culture to incubate for 2-5 days. Visually inspect by inverting plate under 10X objective. Look for swarms of amoebae feeding on bacteria and the initials of fruiting bodies and/or fruiting bodies rising from the surface of the agar.
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Cavender JC, et al. New species of small Dictyostelids from the great Smoky Mountains National Park. Mycologia 97: 493-512, 2005.