State of Michigan Act 451 Part 111.
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.
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
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
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.
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.
& Health Precautions/Personal Protective Equipment
Follow all applicable
safety and health protocols and regulations as established by your institution.
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.
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
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.