I have noticed several times that there are hard particles in the Pichia expression medium that block pipette tips when I try to wash the cells with buffer. I looked at these particles under the microscope and these seem to be salt crystals. What may cause salt precipitation? I use classical BMGY/BMMY media at 20 degrees Celsius. When a yeast strain is cultured in identical conditions in four different flasks, there may be particles in one of the flasks and not in the others. I added the picture of the crystals. The appearance of crystals is not specific to the protein being currently expressed.
Hi Kaia
Are you sure these crystals are salts and not proteins or others.
Do you prepare medium yourself or purchase it? Maybe overdosage in salt mother solution?
According to shape of crystals in picture looks like it is a salt.
Hi Patrick
I prepare the medium myself (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 x 10-5% biotin, 1% glycerol or 0.5% methanol). YNB contains all sorts of salts. If there was a problem with the stock, I would assume crystals in all flasks.
Hi Kaia
If these are salts, you may possibly obtain similar cristals by plating medium alone. Another hypothesis is concentration of the medium by evaporation not likely at 20°C; depending if incubation time is long ?..
You could also collect the medium instead of discarding it and shortly centrifuge to separate the cristals and apply protein dosage on the pellet..?
These are just some simple suggestions. Hope the problem in not more complicated..;-)
Good luck
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