Hey Iago,

I am purifying proteins from pichia with the pPICZ system too. I have experienced similar problems in the cultivation with bacterial contamination. We don't have BSCs for microorganisms here so I have to do all work at the banch.

For me it turned out (as mentioned previously) to be an autoclaving issue. My yeast extract/peptone medium (which you use for BMGY and so on) was contaminated. Ever since I store the medium only short time after autoclaving in the cold room and filter the media right before usage. That worked well in my case.

Good luck!

If possible, try doing your manipulations (taking samples, adding methanol, etc..) in a biological safety cabinet or laminar flow hood. I always did it in BSC and never had problems with contamination of my Pichia cultures (growth or induction).

Also, make sure all of your media/solutions are sterile (autoclaved or filtered), sterilze them aliquoted so there is less manipulations and less chance of contamination. I even filter sterilized the methanol. Probably not necessary but like I said I never had any contamination doing it this way.

Good Luck!

This is clearly a sterilization issue. Autoclaving can be a problem over time since the settings can vary.  Make sure the media alone is not getting contaminated.

I used to work with recombinant methylotrophic yeast Hansenula polymorpha in the past and could confirm that work with such kind of organisms require the same approaches as with all other microorganisms, i.e. sterility at the first term. Two previous answers are absolutely correct. I would also recommend to use sterile water and tips for molecular biology work to be sure that you do not have contamination in inoculum. 

Hey Iago,

I am purifying proteins from pichia with the pPICZ system too. I have experienced similar problems in the cultivation with bacterial contamination. We don't have BSCs for microorganisms here so I have to do all work at the banch.

For me it turned out (as mentioned previously) to be an autoclaving issue. My yeast extract/peptone medium (which you use for BMGY and so on) was contaminated. Ever since I store the medium only short time after autoclaving in the cold room and filter the media right before usage. That worked well in my case.

Good luck!

Hi Iago

I also use Pichia pastoris with pPICZb.  I also, unfortunately, get contamination on occasions.  I ensure everything is autoclaved and filter sterilised, but it seems every so often, contamination sneaks in and ruins my growth.  When not contaminated, I get a clean pellet when collecting the cells and can pour off clean media.  When I have contamination my pellet is loose and sloppy, and the media remains cloudy.  So now, as well as adding zeocin, I have started adding ampicillin as well.  This seems to be working for me so far.

Recently, we got some contaminant that caused the foam of the cultures to curdle like cottage cheese. We were hoping it was because our protein of interest was expressed and secreted at a high level, but no such luck. Sometimes we can tell contamination from the smell of the culture. Instead of smelling like beer or bread, the smell is like dirty socks or E. coli.

We finally have ordered a BSC and probably will turn to filtration of our media after autoclaving.

Directly related to my above post, it turns out our lab building had a severe mold contamination problem due to water intrusion after a large rain and high humidity when our air conditioning system went down for a week after the rain. We had to bring in an outside company to test the air, decontaminate several pieces of equipment with hydrogen peroxide, and whole building air scrubbing. We finally got the mold out of the air and have 8 small air purifiers that run 24-7 to keep the air clean.

That was why all of our Pichia cultures were getting contaminated. We also have our Biological Safety Cabinet and open flasks only while in that cabinet.