Handling Issues with Your Cell Culture
There are several common problems encountered when doing cell culture. Problems in primary cell cultures may have different causes than the same problem in established cell lines. The examples below address some of the common problems encountered when doing cell culture along with their possible causes and suggested actions to resolve them.
Problem: Rapid pH shift in medium
Possible Causes:
1. Incorrect carbon dioxide (CO2) tension
Action: 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.
2. Overly tight caps on tissue culture flasks
Action: Loosen caps one-quarter turn.
3. Insufficient bicarbonate buffering
Action: Add HEPES buffer to a final concentration of 10 to 25 mM.
4. Incorrect salts in medium
Action: Use an Earle’s salts-based medium in a CO2 environment and a Hanks’ salts-based medium in atmospheric conditions.
5. Bacterial, yeast, or fungal contamination
Action: Discard culture and medium or try to decontaminate culture.
Problem: Precipitate in medium, no change in pH
Possible Causes:
1. Residual phosphate left over from detergent washing, which may precipitate powdered medium components
Action: Rinse glassware in deionized, distilled water several times, then sterilize.
2. Frozen medium
Action: Warm medium to 37°C and swirl to dissolve. If a visible precipitate remains discard remaining media and start with fresh reagents.
Problem: Precipitate in medium, change in pH
Possible Causes:
1. Bacterial or fungal contamination
Action: Discard medium or try to decontaminate culture.
Problem: Cells not adhering to culture vessel
Possible Causes:
1. Overly trypsinized cells
Action: Trypsinize for a shorter time, or use less trypsin.
2. Mycoplasma contamination
Action: Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.
3. No attachment factors in medium
Action: For serum-free formulations, be sure they contain attachment factors.
Problem: Decreased growth of culture
Possible Causes:
1. Change in your current cell culture medium, fetal bovine serum or other sera
Action: Compare media formulations for differences in glucose, amino acids, and other components.
Action: Perform growth studies to compare the old lot of sera vs the new lot.
Action: Increase plating density.
Action: Gradually adapt your cells to the new cell culture media.
Invitrogen™ provides solutions for your animal cell, stem cell and primary cell culture needs with GIBCO® cell culture media, fetal bovine serum and more.
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