10%3% 0%

6% 0%

1%

26%

4%

0%

0%

0%

40%

10%

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10%3%

0%

5% 0%

3%

21%

3%

0%

0%

0%

40%

15%Dino.Crypto.ChrysoPrasino.ChloroBolido.Trausto.-likeRhodo.Prymnesio.Dictyochia.Chlorarchnio.CiliatesOthers

Dino

56%

1%4%6%

1%

4%

3%

0%

0%

15%

0% 5%5%

Dino.Crypto.ChrysoPrasino.ChloroBolido.Trausto.-likeRhodo.Prymnesio.Dictyochia.Chlorarchnio.CiliatesOthers

42%

2%3%8%2%10%

8%

1%

1%

2%

1% 11%

9%Dino.Crypto.ChrysoPrasino.ChloroBolido.Trausto.-likeRhodo.Prymnesio.Dictyochia.Chlorarchnio.CiliatesOthers

Ciliates

Others:AcantheraCercozoaDevelopayellaAmoebidiniumRhinosporidium

Crypto PrasinoBolido

Thrausto

Rhodo?

He00 04-12Individuums Species

Orkney 4/00

Cultures from Helgoland• Take fresh sample• Filter twice through 3 µm• Prepare serial dilutions from

10-1 - 10-4 in IMR and Drebes• For each sample 12 x 2 ml

10-1, 12 x 10-2 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

Site Date Precultures StatusHelgoland 27.04.00 96 50ml flasksHelgoland 03.08.00 96 50ml flasksHelgoland 05.10.00 96 50ml flasksHelgoland 06.12.00 96 50ml flasksHelgoland 17.01.01 96 24 well platesHelgoland 18.02.01 96 24 well platesHelgoland 22.03.01 96 24 well platesHelgoland 18.04.01 96 24 well plates

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 conformationpolymorphism)

Do PCR with one phophorylated andone dephosphorylated primer. Digestphosphorylated strand and separate single strands on non-denaturingSDS 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