I have tried several times now to make competent Pichia KM71H PDI cells now. Everytime I seem to get a bacterial contamination in my main culture.

The set up: I use 5 ml sterile YPD media (autoclaved and checked visually before use) then I take a bit of the glycerol stock and use my pipette tip (shortly flamed before use) to distribute the cells from the stock in the media. Everthing is done next to a flame. I put the 50 ml greiner tube at 30 °C overnight.

The next day I have a turbid solution. I take 1 ml ONC to inoculate the main culture (50 ml YPD + 1 ml ONC, in a sterile 200 ml flask). After 2 hours of incubation at 30 °C my OD600 is at 1.4 and when microscoping the culture you see a bacterial contamination. Under the microscope the glycerol stock looks fine.

Does anyone have experience with that type of contamination ?