FACT SHEET # 23
March 2004
REVISION: SECOND

NORTHEASTERN UNIVERSITY PROCEDURE FOR MANAGEMENT AND DISPOSAL OF ETHIDIUM BROMIDE

General Information:
Ethidium Bromide (EtBr) is a commonly used stain for the visualization of nucleic acids in agarose gels. It is widely used by scientists due to its high sensitivity, rapid staining and very inexpensive price. While it is not specifically regulated as a hazardous waste, the mutagenic properties may present a hazard if it is not managed properly in the laboratory.

Safer Alternative:
There is now a safer, more convenient and sensitive alternative to EtBr. Please go to the Molecular Probes website for additional information.

Personal Protection:
When handling EtBr always wear a lab coat, nitrile gloves, and chemical splash goggles. Proper skin and eye protection are needed when a ultraviolet (UV) light source is used while working with EtBr. Avoid exposing unprotected skin and eyes to intense UV sources. Wear a face shield if UV source is pointing upwards. When working with a UV source for a long time, wrap up lab coat sleeves with tape or other means where the wrist could be exposed.

DISPOSAL OF ETHIDIUM BROMIDE: 

Electrophoresis Gels:
Trace amounts of EtBr (less than 0.1%) in electrophoresis gels do not pose a serious hazard so they can be discarded in the trash if properly bagged and secured. If the gels contain more than 0.1% EtBr they should be placed in an appropriate container for hazardous waste disposal. Environmental Health and Safety (EH&S) has a variety of containers that are available to collect and dispose of gels.
Aqueous Solutions:
These can be decontaminated or deactivated using the following methods.

Charcoal Filtration:
Filtering the aqueous EtBr waste solutions (free of other contaminants) through a bed of activated charcoal is a relatively simple and effective method for removal of EtBr. The filtrate may be poured down the drain. There are two kits available for charcoal filtration.

Funnel Kit:
Commercial filter funnel kits are available that use a packaged charcoal disk that is graduated for easily tracking the amount of aqueous solution that can be run through it. This is particularly useful for labs that generate large amounts of solutions at a time. The kit (Extractor ETBR Waste Reduction System) is available through Sigma-Aldrich or Thomas Scientific.

 

  • Filter the EtBr solution through the charcoal filter.
  • Pour filtrate down the drain.
  • Place charcoal filter in a sealed container (mayonnaise jar) and label as a hazardous waste.

Greenbag Kit:

Another simple charcoal filtration method is the Green Bag, manufactured by BIO 101; The Green Bag® Kit allows rapid and trouble-free concentration of EtBr from large volumes of solutions into a small “tea” bag containing activated carbon, which is then conveniently disposed along with other solid (contaminated debris) hazardous wastes. One kit has the capacity to remove 500 mg of ethidium bromide from solutions (10 mg EtBr/bag).

 

  • Place the Green Bag into the EtBr solution.
  • Allow to sit for the allotted time.
  • Pour filtrate down the drain.
  • Dispose of the Green Bag in a sealed container (mayonnaise jar) and label as a hazardous waste.

CHEMICAL DEACTIVATION:
Solutions containing EtBr can be deactivated and rendered nonhazardous by using one of three methods. Deactivation may be confirmed using UV light to detect fluorescence. The three methods are as follows:

Armour Method:
This is the simplest method, but is somewhat controversial. One study found traces of mutagenic reaction mixtures using this method. (Lunn, G.and E. Sansone, Analytical Biochemistry, vol. 162, pp. 453-458, 1987)

 

  • Combine equal amounts of EtBr solution and household bleach.
  • Stir constantly for four hours or let sit for 2-3 days.
  • Adjust pH to 4-9 with sodium hydroxide.
  • Pour down drain with copious amounts of water.

Lunn and Sansone Method: For each 100 ml of EtBr solution:

 

  • Add 5% hypophosphorus acid.
  • Add 12 ml of 0.5 M sodium nitrite.
  • Stir briefly and let stand for 20 hours.
  • Adjust pH to 4-9 using sodium hydroxide.
  • Pour down drain with copious amounts of water.

Quillardet and Hoffnung Method:
This method uses 0.5 M potassium permanganate and 2.5 M hydrochloric acid. Since chlorine gas may be released in significant concentration, EH&S does not recommend using this method.

Crystals and Powders:
Label containers containing crystals and powders as hazardous waste. EH&S should be contacted when the material is ready for a pickup.

Contaminated Debris:
Contaminated debris includes gloves, bench paper and other non-labware items. Place contaminated debris into an appropriate containers for hazardous waste disposal. All containers used for collection must have a top or cover that can be secured. Hazardous waste labels must be used and EH&S contacted when the material is ready for disposal. Bags must not be used unless they are doubled up and kept sealed.

Contaminated Labware:
Contaminated labware includes needles, pipettes, test tubes, etc. that are contaminated with EtBr. Depending on the waste you generate, follow the procedures below.

Needles, scalpels, Pasteur pipettes, and other sharps contaminated with EtBr should be disposed of directly into a sharps container. These will be disposed of like other sharps waste.

Volumetric or transfer pipettes, and other disposable glassware incidentally contaminated with EtBr should be disposed of in a sharps container. Grossly contaminated (visibly contaminated) glassware should be washed with bleach before disposal in a sharps container.

Test tubes and centrifuge tubes contaminated with EtBr should first be emptied, with the liquid disposed of according to the procedures given above. Empty incidentally contaminated tubes can be disposed of in the trash. Grossly contaminated (visibly contaminated) tubes should be collected with other laboratory debris, labeled and disposed of as a hazardous waste.

Most other disposable labware (e.g. sample vials, disposable beakers, etc.) incidentally contaminated with EtBr may be disposed of in the normal trash. Grossly contaminated (visibly contaminated) disposable labware should be labeled and managed as a hazardous waste.

Accident Procedures:
Be very careful when using EtBr. Call EH&S at 373-2769 for assistance with spills. Below are procedures to follow in case of contact:
In the case of eye contact, flush eye(s) with water for at least 15 minutes lifting upper and lower eyelids occasionally.

If skin is exposed, remove contaminated clothing and wash skin with soap and water immediately.

If EtBr vapors are inhaled, remove to fresh air.

If swallowed, get medical attention immediately.

Spill Clean-up:
In case of a small spill, absorb freestanding liquid. Use ultraviolet light to locate the spill. Then prepare decontamination solution by mixing of 4.2 grams of sodium nitrite and 20 mL of hypophosphorous acid (50%) in 300 mL of water. To decontaminate the spill area, wash it with a paper towel soaked in the decontamination solution. Wash the spill area with wet paper towels (soaked in the decontamination solution) an additional 5 times (using fresh paper towel each time). After cleaning up the area put all the used towels in the decontamination solution for 1 hour. Check the completeness of decontamination using the ultraviolet light. If satisfied, put all the decontamination solution in a bottle, label it with a hazardous waste label and call EH&S for waste pick-up.

In case of large spill notify all the people in the laboratory. Evacuate the laboratory immediately. Call EH&S for assistance with a clean up. Post warning signs on all laboratory doors notifying others of the spill. Prevent unnecessary entry into the room until the EH&S personnel arrive. Explain all the details to the EH&S personnel. Contaminated Equipment:
Laboratory equipment (e.g. transilluminators, laboratory floors and countertops, etc.) contaminated with aqueous solutions of more than 10 mg/L (0.01 %) EtBr should be decontaminated using the spill clean-up procedures listed above.

Waste Minimization:
The U.S. Congress has made waste minimization a national policy and goal of each waste generator. You as a generator of EtBr have the responsibility to minimize the waste you generate. Waste minimization has benefits such as decreasing your exposure to hazardous substances, protection of the environment, and decreasing the cost of disposal which can exceed the initial cost of the material by many times. EtBr waste minimization should be considered at all times. The following are suggestions for minimizing waste.

Substituting less or non-hazardous materials for hazardous materials.

Using dilute rather than concentrated solutions.

Using micro or semi-micro techniques.

Using computer modeling/simulation, films, videotapes or demonstrations instead of performing individual experiments.

Use commercially available filtrating vacuums or EtBr extractors to remove EtBr from buffers and other dilute aqueous solutions.

If you have any questions or would like assistance with waste minimization, please contact the Office of Environmental Health and Safety at x2769.