| Subcultivation |
Protocol: Scrape the bottom of the flask, agitate quickly, and aseptically withdraw 0.2 ml of cell suspension. Inoculate in a T25 flask containing 10 ml of ATCC medium 1525 bacterized with Klebsiella pneumoniae subsp. pneumoniae ATCC 700831. Note: The amoebae settle very quickly so the transfer must be done rapidly. |
| Cryopreservation |
Harvest cells 2 to 3 days post-peak density. Decant the culture fluid leaving approximately 1 ml to cover the attached cells. Prepare a 20% DMSO solution using the decanted fluid. Detach cells using a sterile cell scraper and determine the volume of the concentrated cell suspension. Add an equal volume of the 20% DMSO solution, yielding a final concentration of 10%. Aliquot the suspension in 0.5-ml amounts to screw-capped 1.2-ml Nalgene cryovials and transfer them to a Nalgene 5100 Cryo 1 C Freezing Container ("Mr. Frosty"). After 3 hours at -80 C, rapidly plunge the vials into liquid nitrogen.
|
| References |
carapace of an American lobster, Homarus americanus, Connecticut
Thomas A Nerad, personal communication
Smirnov AV, Kudryavtsev AA. Pellitidae n. Fam. (Lobosea, Gymnamoebia) - a new family, accommodating two amoebae with an unusual cell coat and an original mode of locomotion, Pellita catalonica n.g., n.sp. and Pellita digitata comb. nov. Eur. J. Protistol. 41: 257-267, 2005.
|
