Hello! For previous experiments, I have been culturing P. tricornutum non-axenically. However, I am in the process of preparing another experiment which requires an axenic culture of the P. tricornutum strain I am working with. There are various protocols that I have found but none specific for P.tricornutum.
Do any of you have any advice on how to proceed? What sort of lab supplies would I need to have in order to ensure the successful preparation of an axenic culture?
Thank you for you time!
Hi Katrin!
Thank you VERY much for getting back to me!!! I really appreciate it!!
Just wondering - how much of the culture, and does it have to be at a specific point of growth cycle, do you use in Step 1? Also, how much do you resuspend the pellet in in Step 2?
How crucial is the light intensity for the incubation steps?
Thank you again!
Fiona
Standard bacteriology works for us( plates, antibiotics, colony picking). If I remember correctly, 100microg/mL Chloram is what is needed for most bacteria and the treatment time is overnight as Chloram is toxic to Phaeo. You could also try plating on plates that have gradients of antibiootic concentrations. Streak along the gradient in the agar. Pick colonies of Pt that are alone and not growing with bacteria along side them. You may want to use marine medium with a small concentration of sugar on which the bacteria will grow. Many antibiotics only work if the cells are graowing. More dicrete colonies of Pt can be obtained if the Ca concentration in the medium is about 0.25mM. Try Robert Anderson's book on handling algae. All this is covered there.
Thanks Keith for your response! I hope you had a lovely festive season and a wonderful start to the New Year!
I will be attempting to create axenic cultures of Pt in the coming weeks. I will let you know what protocol was implemented and how it went.
Thank you for your time!
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