Acytostelium singulare Cavender et al.
ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
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
4. immersed until material is thawed but no more than 3 minutes. Do not agitate or vortex the ampoule.
5. 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.
6. Incubate the plates at the temperature recommended.
7. 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.
Cavender JC, et al. New species of small Dictyostelids from the great Smoky Mountains National Park. Mycologia 97: 493-512, 2005.