Go CO2 free

In our recent article, we describe how investigators can grow the bacteria causing gonorrhoea without the need for atmospheric CO2.
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Improved Neisseria gonorrhoeae culture media without atmospheric CO2 - Applied Microbiology and Biotechnology

Abstract Bacterial culture on solid media is the crucial step in diagnosing Neisseria gonorrhoeae infections and is the gold standard for determining their antimicrobial resistance profile. However, culture of Neisseria spp. can be challenging in resource poor areas, relying on specialist incubators or other methods of supplying 5% CO2 for growth of the bacteria. Even when such incubators are available, the CO2 to run them may be scarce; there were CO2 shortages during the COVID-19 pandemic, for example. Although culture jars with gas packs or candles can be used, these are inefficient in terms of use of incubator space and researcher time. To achieve simplicity in culturing of N. gonorrhoeae, the standard Oxoid GC agar base medium, made with the Kellogg’s glucose and iron supplements was improved with the addition of 0.75 g/l sodium bicarbonate (NaHCO3), which is inexpensive and readily available. This improved media in a standard incubator performed as well as standard Oxoid GC agar media with supplements in a 5% CO2 incubator. Chocolate agar and Thayer-Martin agar with sodium bicarbonate were also developed, with all showing good growth of N. gonorrhoeae without the need for atmospheric CO2. Key points • Neisseria spp. (N. gonorrhoeae, N. meningitidis) require atmospheric CO2 to grow. • Sources of CO2 may be scarce depending on geography and lab supply availability. • We have developed GC, Chocolate, and Thayer-Martin media that does not need CO2.

The growth of all bacteria in a laboratory requires specific environmental conditions, including the nutrients in the media, the temperature, and the atmosphere. Much of the media that we use today was created decades ago to provide the needed nutrients to bacteria of interest.

Temperatures for bacterial growth cultures are generally set by the temperature of the natural environment in which the bacteria are found; for this reason, most pathogenic bacteria are grown at human body temperature, 37°C.

Lastly, bacteria need to have the right atmospheric environment to be able to grow in a lab; this also often mimics the conditions where the bacteria is found in nature. Some bacteria grow fine in the lab in a normal atmosphere. Some bacteria require there to be no oxygen, an anaerobic atmospheric environment like would be found in the human gut. Other bacteria need something different, such as the bacteria that cause gonorrhoea, Neisseria gonorrhoeae; these need enhanced levels of CO2 to be able to grow in the lab.

 
Petri dishes in a lab 

Due to this requirement for CO2, special incubators are often used to grow N. gonorrhoeae that have regulators to add CO2 from gas tanks into the atmosphere within the incubator. Growth like this requires there to be a readily available supply of CO2 gas and for there to be the special incubators able to use this gas.

There are some alternatives, such as putting the agar plates with the bacteria into sealed jars with a small candle to deplete oxygen and thus raise CO2 levels in the jar. But, these jars then take up additional space in an incubator, where space can be precious.

 
Glass jar with candle and petri dishes. 

It would be so much easier, and more readily accessible to remote and resource limited areas, to be able to grow Neisseria gonorrhoeae and bacteria like it on media that eliminates the need for CO2.

This is what we have done in our paper, ‘Improved Neisseria gonorrhoeae culture media without atmospheric CO2.’ In Applied Microbiology and Biotechnology (10.1007/s00253-025-13449-7).
 
During the pandemic, there were shortages in available CO2 gas cylinders. Even outside of the pandemic, CO2 cylinders run out of gas, ruining experiments and analysis of samples. In remote areas and resource limited areas, there can be issues trying to grow bacteria such as Neisseria gonorrhoeae that have non-standard atmospheric requirements.

Our modified media solves this problem. By simply adding sodium bicarbonate to the three commonly used growth media for these bacteria, we have demonstrated growth of Neisseria gonorrhoeae in normal, standard atmosphere incubators. The GC agar, Chocolate agar, and Thayer Martin agar growth media with sodium bicarbonate are as effective as growth of the bacteria in a CO2 incubator (shown below, A in CO2, B on our media) . This opens up a world of possibilities about where and when these bacteria can be grown in laboratory conditions.

 
Petri dishes showing equal bacterial growth on each.

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