Complete Cell Culture Protocol: Revival, Passaging, Cryopreservation

critical

Mycoplasma Contamination - Silent Growth Inhibitor

Slowed cell growth, altered cellular metabolism, chromosomal aberrations, and interference with cell attachment. No visible turbidity in medium. Difficult to detect by light microscopy due to small size (0.15-0.3 µm).

💡 6 · ✓ 7

critical

No/Few Viable Cells After Thawing Cryopreserved Stock

Upon thawing cryopreserved cell stocks, researchers observe minimal or no viable cells via trypan blue exclusion or hemocytometer counting. Cells appear non-adherent, crenated, or lysed.

💡 5 · ✓ 5

critical

Cell Death from Bacterial or Fungal Contamination

Media becomes rapidly turbid with visible particles or cloudiness. Cells show signs of toxicity, detachment, and death. Microscopy reveals motile bacteria or fungal filaments. Note: mycoplasma rarely causes acute cell death.

💡 4 · ✓ 6

severe

Cell Death from CO₂/pH Imbalance in Bicarbonate Buffer

Cells show progressive death over 24-48 hours with media color shift (phenol red turning yellow or purple). Cells exhibit membrane damage and eventual lysis despite sterile conditions.

💡 5 · ✓ 5

severe

Cell Death from Poor Quality or Toxic Reagents

Sudden cell death occurs immediately after introducing new lot of media, serum, trypsin, or buffer. Previously healthy cultures show rapid necrosis or apoptosis within 12-24 hours.

💡 5 · ✓ 6

severe

Bacterial Contamination with Rapid Turbidity

Rapid-onset turbidity and color change of culture medium (when phenol red is present). Medium becomes cloudy and pH indicator changes color quickly.

💡 7 · ✓ 9

severe

Fungal and Yeast Contamination

Visible turbidity and color change of medium. Fungal structures visible under standard light microscopy. Rapid onset similar to bacterial contamination. May include visible mold growth.

💡 6 · ✓ 8

moderate

Incubator-Associated Mold Contamination

Recurring fungal contamination in cultures after incubation. Multiple cultures affected over time. Visible mold growth may appear on incubator surfaces. Persistent contamination despite replacing affected cultures.

💡 6 · ✓ 8

moderate

Chemical Contamination from Residues

Poor cell growth, cell death, or altered cell behavior without visible microbial contamination. May include effects from free radicals, metal ions, disinfectant/detergent residues, or endotoxins persisting after bacterial contamination is cleared.

💡 6 · ✓ 6