Identifying Cell Culture Contamination: Bacteria, Fungi and Mycoplasma

A morning in the cell culture room: you put the flask under the microscope and see extra specks, some of them moving. The two worst reactions now are throwing the flask away on instinct, or deciding it looks fine and carrying on. Contamination comes in several forms, each looking different through the eyepieces, and each calling for a different response.

1. Contamination is not the same as poor condition

Slow growth, rounded morphology and weak attachment are not automatically contamination. A new serum lot, over-trypsinisation, expired medium or wrong CO2 will produce the same picture. The first check is the medium itself: clear or cloudy. Clear medium with unhappy cells means look at culture conditions; cloudiness or an abnormal colour shift justifies thinking about contamination.

2. Bacterial contamination: fastest and clearest

Bacteria are quick. Within hours the medium turns visibly cloudy, and phenol red shifts from orange-red to yellow through acid production, or to purple through alkali production. Under the microscope the background fills with fine particles around one micrometre across, most in irregular Brownian motion, some swimming actively.

The decisive sign is that bacteria fill the whole liquid layer, not just the area around the cells. Focus up and down: if the fluid away from the cells is also full of specks, the diagnosis is settled. Gram staining then helps identify the type and likely source.

There is no partial fix: seal the flask and discard it. Antibiotics in cell culture only hide the problem while it spreads through the incubator.

3. Fungi and yeast: two different things

Fungal contamination is recognised by hyphae, long filamentous structures usually radiating from a single point. It grows slowly but once established is very hard to clear. Spores are round or oval bodies, larger than bacteria at 3 to 5 micrometres, often in chains.

Yeast is different. It is single-celled, appearing as smooth round bodies with visible budding, like a small bunch of grapes. Larger than bacteria, smaller than cells, with little motility, it clouds the medium more slowly.

Both require discarding the batch and decontaminating the incubator thoroughly: change the water pan, UV the interior, and wipe with hydrogen peroxide or a dedicated disinfectant. Spores survive for a long time, so discarding flasks alone means the problem returns within days.

4. Mycoplasma: the invisible one

Mycoplasma is the most troublesome contaminant in cell culture because it lacks a cell wall and measures only 0.2 to 0.3 micrometres, invisible in an ordinary light microscope. The medium stays clear and the colour normal. The only hints are slower growth, deteriorating morphology, lower transfection efficiency and higher rates of chromosomal abnormality.

Confirmation needs a dedicated assay: PCR is the usual choice, or Hoechst 33258 staining viewed under fluorescence to look for fine fluorescent specks on and around the cells. Routine screening is worth more than retrospective diagnosis, and well-run facilities test quarterly.

Confirmed contamination means clearing the batch, since mycoplasma does not disappear on its own and can spread by aerosol to other cells in the same incubator.

5. Debris that is not contamination

Cell debris: clumps left from incomplete trituration, over-digestion or dead cells after thawing. Irregular fragments with soft edges, non-motile, reducing after a medium change or the next passage.

Serum precipitate: lipoproteins and fibrin from heat inactivation or freeze-thaw cycles form fine, evenly distributed particles that do not travel with the cells and persist after a medium change. This is normal; buy serum from a consistent lot.

Scratches or plastic reflections on the flask: they look like a field full of specks at one focus position and vanish as you turn the knob.

The simple test is whether it disappears when you change the medium or drops after passage. If so, it is not contamination.

6. Two more that are easy to miss

Cross-contamination: two lines mixed together show inconsistent morphology, and the faster grower gradually takes over. The microscope will not settle this; STR profiling will. Journals now routinely require cell line authentication.

Protozoan contamination: rare but real, appearing as organisms smaller than cells, larger than bacteria and moving quickly across the field. Water and handling are the usual sources.

7. A diagnostic order to follow

When something looks wrong: check medium clarity and colour, then focus above and below the cells to inspect the fluid layer, then passage and see whether it clears, and finally run Gram staining and mycoplasma PCR. Do not skip steps, and do not throw away an irreplaceable batch on a hunch.