• Applicable Regulations
• Overview of Procedure
• Waste Minimization Procedure
• Known Limitations
• Safety & Health Precautions/Personal Protective Equipment
• Benefits
• Disadvantages
• Project Related Costs

Overview of Procedure
Researchers and students must immobilize Drosophila for examination and manipulation. Traditionally, immobilization has been accomplished using ethyl ether or triethylamine. Ethyl ether is extremely flammable, potentially explosive, and can cause depression of the central nervous system and other health effects. Triethylamine is flammable, can cause severe respiratory and skin irritation, and burns. As an alternative to ethyl ether and triethylamine, CO₂,_, a nonflammable gas, may be used to immobilize the Drosophila. CO₂ may also be used for short term immobilization of other insects, replacing traditional "kill jars" containing toxic compounds such as cyanide.

Waste Minimization Procedure
I. Procedure for examining Drosophila

A. Set-up of system

1. A cylinder of CO₂ is attached (through a gas regulator) by plastic tubing to an aquarium gangvalve or a series of valves.

2. The valves are connected to a specially constructed diffusing pad of porous polyethylene sheeting, which allows the passage of the CO₂. The pad should fit on the stage of a dissecting microscope.

3. One valve from each gangvalve should have only a free piece of tubing.

B. Use of CO₂ system

1. Following proper operating procedure for gas cylinders, first open the gangvalves to the diffusing pads that will be used; second, open the regulator valves; third, open the tank valve. Opening the valves in the incorrect order can lead to a pressure build up in the lines.

2. Place the end of the free piece of tubing into an anesthetizing chamber (any small wide-mouth glass bottle will suffice). Use a foam stopper or short, wide stem plastic powder funnel to cap the bottle. Open the gangvalve for 5-20 seconds to fill the chamber with gas.

3. Quickly shake the flies into the chamber. Tap it on the bench to settle the flies to the bottom; CO₂ is heavier than air and will sit on the bottom of the bottle. The flies should stop moving immediately. If they do not, use the free tubing to add a little more CO₂ into the bottle. It is important to open the valve just a little or the flies will be blown around quite violently.

4. Shake or brush the flies onto the diffusing pad. The flies will remain immobile as long as CO₂ is delivered through the pad. If starting a new culture, brush the flies directly from the pad to the new food bottle or vial. The flies will awaken within five to ten minutes, depending on the length of time anesthetized.

5. When finished, close the valves in the reverse order that they were opened: first, close the tank valve; second, close the regulator valve; and third, close the gangvalve.

II. Procedure for short term immobilization of insects, which can be used in the field

Note: this works best for insects with high metabolic requirements, such as flying insects.

1. Two vials or bottles with tightly sealed lids or stoppers are connected with a piece of tubing (aquarium tubing works well).

2. Place the insect in one of the vials.

3. The other vial is the CO₂ generating chamber. Alka-seltzer tablets may be used or a mixture of sodium bicarbonate and citric acid (or use vinegar as the activating agent instead of water). Add water to the vial to generate the CO₂ and quickly reseal the lid. If too much water is added, the liquid will flow into the vial and drown the insect.

4. The CO₂ should immobilize the insect, depending on the type, while it remains in the chamber. If longer examination is required, the insect may be placed on a freezer block. Otherwise, the insect will quickly awaken when removed from the CO₂ environment.

Known Limitations
Some mutant strains of Drosophila may be less tolerant of long term exposure to CO₂ (greater than 20-30 minutes).

The CO₂ vial method may not be effective for insects with low metabolic requirements.

Safety & Health Precautions/Personal Protective Equipment
Follow all applicable safety and health protocols and regulations as established by your institution.

Benefits
CO₂ is much less likely to kill or sterilize Drosophila than ethyl ether or triethylamine.

The diffusion pad permits continuous application of the immobilizing agent, whereas the effects of ethyl ether and triethylamine will wear off in less than 10 minutes and 20-40 minutes, respectively.

CO₂ is not flammable nor reactive, is odorless, and a natural component of air. Conversely, ethyl ether is extremely flammable, may react with air to produce explosive peroxides, has a strong chemical odor, and potential negative health effects. Triethylamine, normally used in very small quantities, is nevertheless potentially flammable and corrosive.

Elimination of the purchase of ethyl ether is advantageous for the ignitable characteristic waste property and being banned from land disposal in accordance with the Resource Conservation and Recovery Act. In addition, ethyl ether is not stable and should be stored in explosion-proof facilities. Empty containers retain product residue and may be dangerous.

Finally, the CO₂ vial method may be advantageous for use with young students or in cases where the insects are to be released unharmed after a short examination.

Disadvantages
Initial time and monetary investments are required for assembling the CO₂ cylinder system. Gas cylinders are cumbersome and under very high pressure. Large laboratories may require a large number of cylinders.

Project Related Costs
Initial cost for the CO₂ cylinder-method equipment and materials is a cost consideration as well as the small rental fee for the cylinder itself. Unused CO₂ can be returned to the vendor along with the cylinder.