Cell Culture Troubleshooting Guide

There are several common problems encountered when culturing cells. Problems in primary cultures may have different causes than the same problem in established cell lines.

 The table below addresses some of the common problems encountered when culturing cells, along with their possible causes and suggested solutions.

Problem Possible Cause Suggested Solution
Rapid pH shift in mediumIncorrect carbon dioxide (CO2) tensionIncrease 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 flasksLoosen caps one-quarter turn.

Insufficient bicarbonate bufferingAdd HEPES buffer to a final concentration of 10 to 25 mM.
 Incorrect salts in mediumUse an Earle’s salts-based medium in a CO2 environment and a Hanks’ salts-based medium in atmospheric conditions.
 Bacterial, yeast, or fungal contaminationDiscard culture and medium.

Try to decontaminate culture.  (See Decontaminating Cultures with Antibiotics and Antimycotics.)
Precipitate in medium, no change in pHResidual phosphate left over from detergent washing, which may precipitate powdered medium componentsRinse glassware in deionized, distilled water several times, then sterilize.



Frozen mediumWarm 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.  (See Decontaminating Cultures with
Antibiotics and Antimycotics.)
Cells not adhering to culture vessel
Overly trypsinized cellsTrypsinize for a shorter time, or use less trypsin.  (See Dissociation of Cells from
Culture Vessels.)

Mycoplasma contaminationSegregate culture and test for mycoplasma infection.  Clean hood and incubator. If culture is contaminated, discard.

No attachment factors in mediumFor 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.