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  • Writer's pictureKaun Lab

How do I humidify air and CO2?

Flies can sense humidity in their environment, and change their behavior when the air is too wet or dry (more on this on ‘Does humidity affect behavior?’). Essentially, the humidity during rearing, collection, storage and while performing behavior is important for consistency of responses in behavior experiments. Below are some tips for ensuring your flies don’t get dried out:

Use a humidified incubator to rear your flies. You can do this with an expensive incubator with humidity controls or by placing an open tub of water at the bottom of the incubator. The incubator with humidity controls is typically a bit more consistent, but the tub on the bottom of the incubator can be surprisingly reliable (depending on how often the incubator is opened or how long it is left open). 

Check the food quality of the food your flies are being reared in. Make sure it isn’t too dry or too wet. You can take a look at examples of food problems on ‘Which fly food recipe should I use to raise flies for behavior?’. If your food is too dry, you can add a couple of drops of water along the side of the rearing vial (not enough to drown the eggs or larvae). If your food is too wet, you can add a piece of filter paper or folded Kimwipe into the middle of the food (poke it down a bit using a probe or the end of your brush/feather).

Humidify your CO2 while fly pushing. Humidifying your CO2 will ensure your flies don’t get dried out or dessicate while you are sorting them. You can tell they are getting dried out or have been on the CO2 too long when their wings raise up in a heldup phenotype. An added benefit is that it reduces static, making your flies easier to push. You can also use the sound and sight of the CO2 bubbling to gauge how much CO2 you are giving your flies. 

You can buy a pre-built CO2 bubbler system that will humidify your CO2, or you can build one yourself relatively inexpensively. You add a fish tank air stone to the end of a tube, submerge it in a clean water bottle and then through another tube inserted just into the lid of the jar, push the humidified CO2 towards your CO2 pad. 

Use humidified air when performing behavior experiments: Humidify your behavior environment either by performing your experiments in a humidified room, or humidifying your environment chamber using a small household humidifier plugged into a humidity sensor that controls the on/off of the humidifier. 

An alternative to performing your experiment in a humidified room or behavior environment is to run humidified air through the behavior arena itself. You can do this using a glass bubbler and depending on the type of experiment you are performing you can use a larger one (pictured here) or you might want to use a smaller one such as glass air stone set or smaller glass bottles with added luer lock connections to the cap. You can also build your own system similar to the CO2 system mentioned above. Tip: depending on the humidity and flow rate you need, sometimes you don’t need to bubble the air directly through the water, but just pass it over the surface of the water.

Once you have the water bubbler set up, you want to push air through to your behavior chamber. You can generate air using a room air valve (if you are very lucky your institute has this) or, like us, you can use compressed air (ie delivered via a large tank like the CO2 tank). If you are using compressed air, beware that the air has a distinct, unpleasant odor near the end of the tank, and you need to switch out your tank as soon as you smell this. An inexpensive alternative we have also used is to generate air using a fish-tank or fish-pond pump. The downside to this method is they use a rubber compressor and when these are run for a while the air starts to smell like burning rubber. 

Instead of actively pumping air through your arena, you can also set up a vacuum system that sucks air from the room, through (or across the surface of) the water in your bubbler and through the behavior arena. Most labs come equipped with vacuums so this can be a bit easier to set up. Make sure to have a way to control and measure the flow rate of your vacuum though since we have found the room vacuum can fluctuate in flow rate a lot.

Tip: make sure to switch out your water each day before you start your experiment since the water can retain odors from the air.

Tip: You will want to use a low flow rate when running humidified air through your behavior chambers because high flow rates will affect activity of the flies, and can potentially cause them to gather near the odor port or vacuum port depending on the flow rate.

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