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It is generally accepted that to create an environment conducive to cell growth, a CO2 incubator is used to provide a controlled atmosphere with a temperature of 37°C, 5% CO2 concentration and 95% relative humidity (RH)


For many applications, these parameter values are an accurate representation of the conditions in which cultures will grow with the expected characteristics and at the expected rates. However, many applications have different requirements. For example, when studying a stressed cellular system, the ability to manipulate relative humidity is advantageous. When studying the effects of V. cholerae on human gastrointestinal cells, the manipulation of all three environmental variables, including RH, is required, because the stress on the cellular system can be more accurately defined by mimicking in the in vivo response to infection (i.e., dehydration).

As another example, psychrotrophic and thermophilic bacteria grow best at temperatures below and above (respectively) 37°C (e.g., psychrotrophs Vibrio marinus, Thiobacillus novellus, and Vibrio cholerae; and thermophiles Bacillus flavothermusand Thermus aquaticus), making the manipulation of the temperature variable critical for the growth of such cultures.

Technologies to provide precise, user-defined control of temperature and CO2 are widely available, so these two variables are commonly manipulated for experimental purposes; however, in most incubators, RH is not a parameter that is actively monitored and controlled, even though the manipulation of RH is a critical factor in cell growth. This directly impacts research validity and reliability by negatively impacting culture growth and making it less predictable.

Microbes Sensitive to Relative Humidity


Many incubator manufacturers claim to provide “optimal” conditions for healthy cell growth, but how are they defining “optimal?” Our opinion is that the definition of “optimal” depends on the specific needs of your cells. In other words, you should be defining the parameters that contribute to an optimal growth environment, not the incubator.

Below are several examples of fungal and bacterial species that require precise, user-defined control of relative humidity in a CO2 incubator in order to provide a truly optimal environment for culturing.

Peltaster fructicola Johnson et al., anamorph (ATCC® MYA-686™)

Temperature: 37.0°C
Humidity: <85% structure affected without fruiting body; >88% fruiting body present

Sp. Bacillus cereus, strain ip5832

Temperature: 37.0°C
Humidity: <65.0% morphological and elastic properties leading to cell wall structure change. Change to accommodate lack of moisture.

Sp. E. coli, strain K12

Temperature: 37.0°C
Humidity: <84.0% morphological and elastic properties leading to cell wall structure change and failure. Change to accommodate lack of moisture.

Sp. Burkholderia cepacia

Temperature: 37.0°C
Humidity: 73.0% survivability and growth

Sp. Cyanobacteria (class of bacteria)

Temperature: 25.0°C (in an incubator; lower in its natural environment of the Arctic Desert)
Humidity: 75.0% or less

Various Acidobacteria and Proteobacteria spp.

Temperature: avg. 16.5°C
Humidity: <65% morphological and elastic properties leading to cell wall structure change. Change to accommodate lack of moisture.

Does Your CO2 Incubator Really Offer Active Humidity Control?


Some CO2 incubators provide just one humidity setting without allowing the user to adjust it (i.e., there is no humidity “control”). Other systems allow users to dial in to a specific humidity set point (e.g., 90%), which the system then purports to maintain. However, not all of these latter types of systems give the user true control over RH. To find out if your CO2incubator provides user-defined, active control over RH, download our RH paper.

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