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How to recognize bacterial contamination in your cell culture

Lab Academy

Macroscopic detection

  • Increased turbidity of culture medium, medium appears cloudy.
  • If medium contains phenol red as pH indicator, a rapid color change to yellow indicates a sudden decrease in pH
Culture media contaminated with bacteria (left) appear turbid and yellow whereas non-contaminated media (right) appear clear and red.

Microscopic detection

Bacteria are much smaller than eukaryotic cells. They appear as dark rod-like structures, spheres or spiral structures under the microscope, and they may exist as single cells, in pairs, chains, or clusters.

Bacteria can be visualized using phase contrast and 100x - 400x magnification. Phase contrast facilitates detection, especially at low contamination levels.
Common shapes of bacteria: rod (bacillus), spherical (coccus), and spiral (spirilla).

Moving bacteria in cell culture

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Some bacteria show active movement. You are not sure whether what you see in your culture are cell debris or bacterial contamination? Try to focus on one of the potential contaminants and follow it with your eyes. Cell debris or any precipitates from the medium will only fidget but mostly stay in the same place. Some bacterial strains will actively move away from their current position.

Low levels of non-moving bacteria with round shapes are especially difficult to distinguish from any harmless particles fidgeting between the cells. In this case, only time can bring certainty: without antibiotics the number of particles will increase overnight if a contamination is present. If no increase occurs without antibiotics, most likely harmless debris or precipitates are at fault.

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