Hello everyone.

I'll be doing a yeast uptake experiment with the use of the fluorescent sugar esculin, and the yeast strain Saccharomyces cerevisiae W303, as detailed in this article :

Patzke, K., Prananingrum, P., Klemens, P. A., Trentmann, O., Rodrigues, C. M., Keller, I., Fernie, A. R., Geigenberger, P., Bölter, B., Lehmann, M., Schmitz-Esser, S., Pommerrenig, B., Haferkamp, I., & Neuhaus, H. E. (2018). The Plastidic Sugar Transporter pSuT Influences Flowering and Affects Cold Responses. Plant Physiology, 179(2), 569‑587. https://doi.org/10.1104/pp.18.01036

Usually, i do a liquid culture of W303 during night, with YP(D)AU (yeast extract peptone dextrose - Adenine Uracile), where I add the antibiotics Geneticin/G418 for selection purpose. Usually, I plan to do an observation after the night.

However, I need to fix the culture and conserve it during one day before observing the fluorescence with a confocal microscope.

I found the bisaria fixation protocol as detailed here : https://openwetware.org/wiki/McClean:_Fixation_of_Yeast_(Bisaria_Protocol)

• Prepare eppendorf tubes with 50μL of 37% formaldehyde, one per sample.
• Add 450μL of culture to 50μL of formaldehyde in an eppendorf tube and mix by inversion. (The goal is to have a final concentration of formaldehyde around 3.7%. So you can adjust the cell and formaldehyde volumes accordingly, as long as you end up with 3.7% formaldehyde).
• Incubate the tube at room temperature for 15-20 minutes.
• Spin down gently for 5 minutes at 8000rpm in the small eppendorf centrifuge.
• Resuspend cells in 75-100μL of 0.1M potassium phosphate
• Store samples at 4°C until you are ready to image.

Has anyone already done this protocol with this yeast strain in the purpose of fluorescence observation and had success with it ?

I was wondering whether or not this would work by replacing the 0.1M potassium phosphate by a 25 mM phosphate buffer pH 6 (made with sodium phosphate monobasic anhydrous and sodium phosphate dibasic dihydrate).

I'm sorry if something is unclear or if this was not the right way to do it, that is my first time doing this kind of thing.

Thank you very much.