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What is a CO2 Incubator?

biological incubator

CO2 incubators are sealed, climate-controlled boxes used in life science laboratories to grow biological cell cultures. They are required to maintain the same conditions as inside the human body.

  • 7.4 pH neutral
  • 98.6°F (37°C)
  • > 90% relative humidity

These three numbers create the optimal conditions for biological cell growth.

Why is additional CO2 needed in an incubator?

Humans are most comfortable at CO2 levels at or slightly above 400 ppm (0.04%) which raises the question, why would a CO2 incubator that is used to grow tissue cultures need CO2 levels of 5 - 10%? 

In order to culture cells under optimum conditions, the media they grow in needs to stay at neutral pH (around pH 7). The H2O in the cells can be turned into a carbonic acid (H2CO3) buffer by adding additional CO2. The combination of H2O and CO2 results in bicarbonate (HCO3-) and H2CO3 which keeps the pH neutral, and therefore has been found to affect the growth of biological cells the least. Here's a short video that explains the chemical reaction.

In other words, by adding additional CO2 at the right level you prevent the pH inside the cells from becoming either alkaline or acidic, which both inhibit cell growth.

Maintaining CO2 Levels

CO2 levels inside a CO2 incubator are measured with accurate optical nondispersive infra-red (NDIR) sensors. There are two concerns when using an NDIR CO2 sensor:

  1. In high-altitude environments, the sensor readings must be adjusted to the change in barometric pressure.
  2. The sensor be guarded against high humidity. Too much water in the air can result in condensation inside the CO2 sensor which will render the sensor inoperative.

Maintaining Humidity Levels

While a water pan is often used to improve humidity, it creates potential problems with contamination and irregular humidity levels as the door is opened and closed. For that reason, an atomizer may be used to boost the humidity level along with a %RH sensor to control the humidity level.


Risks of contamination inside a CO2 incubator are primarily around cross contamination from fungi, viruses and bacteria. This can be controlled by sterilization between samples. It is important however, that sterilization with heat or superheated water not come into direct contact with the CO2 sensor. For this reason, the CO2 sensor should be integrated into the incubator in such a way that it is not impacted by the sterilization cycle. This can be solved by

  • Gas sampling CO2 sensor (fixed or handheld) outside the incubator
  • CO2 sensor with heat and moisture-resistant stainless-steel hood

Photo by Shinryuu - Own work, Public Domain

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