Problem |
Possible Cause
|
Suggested Solution |
Rapid pH shift
in medium |
Incorrect carbon dioxide (CO2)
tension |
Increase or decrease percentage of CO2 in the incubator based on concentration of sodium bicarbonate in medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use CO2 amounts of 5% to 10%, respectively.
Switch to CO2-Independent Medium.
|
|
|
Overly tight caps on tissue culture flasks |
Loosen caps one-quarter turn.
|
|
|
Insufficient bicarbonate buffering
|
Add HEPES buffer to a final concentration of 10 to 25 mM.
|
|
|
Incorrect salts in medium
|
Use an Earle’s salts-based medium in a CO2
environment and a Hanks’ salts-based medium in atmospheric conditions.
|
|
|
Bacterial, yeast, or fungal contamination
|
Discard culture and medium.
Try to decontaminate culture.
|
Precipitate in medium, no change
in pH
|
Residual phosphate left over from detergent
washing, which may precipitate powdered medium components
|
Rinse glassware in deionized, distilled water
several times, then sterilize.
|
|
|
Frozen medium
|
Warm medium to 37°C and swirl to dissolve. If precipitate remains, discard medium.
|
Precipitate in medium, change in
pH
|
Bacterial or fungal contamination
|
Discard medium.
Try to decontaminate culture.
|
| Cells not adhering to culture vessel
|
Overly trypsinized cells
|
Trypsinize for a shorter time, or use less
trypsin.
|
|
|
Mycoplasma contamination
|
Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.
|
|
|
No attachment factors in medium
|
For serum-free formulations, be sure they contain attachment factors.
|
| Decreased growth of culture
|
Change in medium or serum
|
Compare media formulations for differences in glucose, amino acids, and other components.
Compare the old lot of serum with the new lot in a growth experiment.
Increase initial cell inoculum.
Adapt cells sequentially to new medium.
|
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