Dino Ciliates Others: Acanthera Cercozoa Developayella Amoebidinium Rhinosporidium Crypto Prasino Bolido Thrausto Rhodo? He Individuums Species Orkney 4/00

Cultures from Helgoland Take fresh sample Filter twice through 3 µm Prepare serial dilutions from in IMR and Drebes For each sample 12 x 2 ml 10 -1, 12 x etc. in both media (4x12x2 = 96) Incubate at approx. the same temperature as sea water at sampling time Wait for 1-4 months for growth Transfer to 50 ml tubes, allow to grow for 1-2 months Check with microscope and flow Sort „unique populations“ by flow

Generate stable cultures Check by microscopy Analyse by flow cytometry Sort homogenous populations Prepare serial dilutions ?

Check by microscopy and flow. Check by HPLC ??? (In Bremerhaven or Barcelona) Check by DGGE or SSCP, reamplify and sequence fragment? Amplify and (partially?) sequence 18S. Send cultures to Wenche. Classify stable cultures

Single cloneTotal DNAs SSCP (Single strand conformation polymorphism) Do PCR with one phophorylated and one dephosphorylated primer. Digest phosphorylated strand and separate single strands on non-denaturing SDS gel.

SSCP versus DGGE/TGGE Generates ssDNA by nuclease digestion Uses non-denaturing „ordinary“ PAGE Mostly one band per species/clone Not widely accepted In my hands works with 528F Generates dsDNA with GC clamp Uses denaturing gradient PAGE Frequently >1 band per species Well established in many labs Works with 528F?

Generate SSCP products for many of the clones from 18S libraries Run them separately on PAGE and combine appropriate ones to create a „species ladder“ Use this ladder to identify bands in SSCP profiles from natural samples and from cultures Possible application of SSCP for monthly monitoring and culture identification

Colaborations on PICODIV cultures Prof. Kirst, Univ. Bremen, will study DMSP production Prof. Heinz, Univ. Hamburg, will study PUFA production