-Olivia Heath 03/29/19

This is a lab made hypoxic chamber to study the effects of hypoxia or anoxia in c. elegans. This set up was originally derived from this Jove article.

• Glass tupperware container with airtight lid. I recommend a lasagna pan to max out the n of the experiment.
• Nitrogen tank- purity depends on the %O2 that you are trying to achieve.
• Regulator for Nitrogen tank
• Flow meter for Nitrogen tank
• Lab tubing approved for gas
• Barbed fittings- at least three
• Bulkhead fittings- at least two
• Exacto knife
• Oxygen meter
• Teflon tape
• Liquid silicon
• 2L flask
• 2L filter flask
• 1 hole cork to fit filter flask
• 25ML stripette
• Barrier hose

1. To begin, you will need to make holes in your tupperware lid that fit the bulkhead fittings. I recommend putting the bulkheads on the outer edges of the lid so you are using the most real estate of the chamber.You want the flat end of your bulkhead to be inside the chamber while the male end is outside of the lid.The best way to measure your hole is to trace the outer thread of your bulkhead on your lid and use a hot exacto knife to cut the hole. You will know you did it right if your bulkhead does not slip in but rather require you to screw it in. Remember, this must be air tight to prevent Oxygen from entering the chamber.
2. Once your bulkheads are in place, you will want to tighten them. DO NOT OVER-TIGHTEN!! You will know if you over tighten if the rubber washers escape from under the fitting. (insert pic here) Next you will add the barbed fittings by first adding teflon tape to the thread of the bulkhead. Teflon tape should go with the thread so that when you tighten the barbed fitting, the teflon tape tightens as well.
3. Now you are ready to start attaching tubes. One of your barbed fittings is going to be the input, going from the nitrogen tank to the bubbler and then to your chamber. The other is the output that goes straight into a 2L flask with water.
4. To make your bubbler, you will grab your 2L filter flask. You will find a cork that fits snugly into the flask, and using a cork borer you will make a hole to fit a stripette. You want about two inches of stripette to be outside of the cork and the rest to be going down into the flask. The goal here is to force all of the incoming Nitrogen gas to go through water so we don't desiccate our worms. If the Stripette does not reach all the way to an inch above the bottom of the flask, you may need to elongate it with a small piece of tubing.
5. You will connect a piece of tubing from the output of your filter flask to the input (barbed fitting) of your chamber. If you have problems getting the tube on, you may need to use some lubricant. 80% glycerol usually does the trick.

Additional important or sometimes important information that is not part of the flow of steps in the protocol.

list the full manufacturer name and manufacturer part number for all reagents, and then for all equipment. If these already have wiki pages you should be able to get the numbers there. PART NUMBERS will always have two sections, first Reagents and then Equipment. Please place these items in 2 tables.