Hello everyone,
I am encountering some trouble maintaining the pH balance in our media solution. Originally I thought it was a problem with the reagents, but I have narrowed it down to the following:
Once the pH was recorded at 7.20, I took a couple of samples and put into glass vials and left it at room temperature. I also put one sample in the refrigerator where the media is stored between uses (for up to 3 weeks).
After 5 days, the media left at RT and in the glass vials is still recording a pH of 7.20, but the media in the flask is recording a pH of a whopping 7.65. The only thing I can think of is contamination, but I filtered the media using a Steritop 0.22µm filter into an autoclaved glass media flask. I've also observed this pH drift in previous instances where I filtered the media into a Stericup which is sterilized by radiation.
It never seems to drift higher than 7.7 but that's too high to maintain viable cultures.
Possible contaminants or contributing factors:
- air in the refrigerator (perhaps the containers or lids are not sealed appropriately)
- contaminants in the autoclaved flask
- contaminants in the air while recording the pH and osmolality
- pipette aid is contaminated (however, using only sterilized serological pipettes)
- temperature of the refrigerator is unstable (seems to fluctuate between 0 and 7 degrees Celsius)
In the meantime, I am going to take samples of the media at every step in the process of making it until I can see a clear indicator of where a contaminant or unsuitable material is evidently contributing to this problem.
Thank you for your help!
Hi Taylor,
This doesn't sound like contamination but rather have you considered the CO2 levels in the atmosphere? Tissue culture media is made to be used at 5% CO2 and not atmospheric CO2. If you want to really prove it to yourself, put some media in a flask and put it at 37 degrees C and 5% CO2 then test the pH
Bacterial or yeast contamination usually makes the media go turbid, acidic and yellow.
Hi Colin,
Thank you for your response. I am testing the following: CO2 is filtered out of the media during sterilization which is increasing the pH prior to incubation. The pH calculations we have for the media only considered temperature as a variable and not the amount of CO2 lost in filtration. The CO2 buffers in the incubator (5% CO2 at 37 degrees) but at a variable rate depending on the surface area of the media and the ventilation of the container. I will likely have to make changes to the tubes we incubate them in (switch to vented caps and a greater surface area to increase rate of buffering) or gas the flask which the media is stored in.
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