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Ethidium Bromide Disposal
Ethidium Bromide (3,8 diamino-5-ethyl-6-phenyl phenanthridinium bromide, dromilac, CAS #1239-45-8) , is a compound used in many of the Health Sciences Campus Laboratories. While it is not currently regulated as a hazardous waste, the mutagenic and moderately toxic properties may present a hazard if handle or disposed of improperly.
Based on these considerations, Environmental Health & Safety (EH&S) recommends the following disposal or destruction procedures for ethidium bromide(EtBr).
Surface Decontamination of Ethidium Bromide (Bensaude, 1988)
- Wipe up excess liquid with paper towels. Wet surface with ethanol and sprinkle activated charcoal on the surface.
- Wipe up the charcoal/ethanol mixture with paper towels. Seal the wastes in a plastic bag, label, and dispose of the bag through EH&S.
- PPE and materials mildly contaminated with Ethidium Bromide may be disposed of as red bag waste.
Electrophoresis Gels
All gels containing ethidium bromide should be collected and disposed through EH&S as hazardous chemical waste. The container, which can be provided by EH&S, should be labeled as “Hazardous Waste – Ethidium Bromide Gels.” When the container is approximately 85% full, call EH&S at (212)305-6780 for hazardous chemical waste disposal or facsimile the Chemical Waste Disposal sheet at 795-5847.
Ethidium Bromide Solutions
Buffer solutions containing greater than 0.5ug/ml ethidium bromide should be filtered, decontaminated, or destroyed using one of the methods below or collected as hazardous chemical waste.
While aqueous buffer solutions containing less than 0.5ug/ml ethidium bromide can be released down the drain, EH&S recommends using one of the following filter methods before release.
Any solution containing heavy metals, organic solvents, cyanides, sulfides, acids (pH<=2), or bases (pH=>12) must be disposed of as hazardous chemical waste.
As with all hazardous chemical waste, waste solutions can not be diluted for release.
Methods of Decontamination/Destruction
- Commercially Available Filtration Kits
Filtering ethidium bromide buffer solutions through bed of activated carbon is a simple and effective method of removing ethidium bromide. The filtrate can be drain disposed. One such kit is available from BIO101 (www.bio101.com), who offers the EtBr Green Bag Kit, for ethidium bromide filtering. The Green Bag is essentially a charcoal “teabag” which is place in the ethidium bromide solution and each bag treats 10 mg EtBr/bag. One kit (50 bags) has the capacity to remove 500 mg of ethidium bromide from solutions. After the solution has been treated, it may be poured down the drain and the bag can be disposed of as hazardous chemical waste.
- Decontamination of Dilute Solutions (Up to 100 ug/ml)
Method 1 (Lunn and Sansone 1987)
1. Add 2.9 g of Amberlite XAD-16 for each 100 ml of solution up to 100 ug/ml ethidium bromide. Amberlite XAD-16, a nonionic, polymeric absorbant, is available from Rohm and Haas and sold by Sigma Chemical Company.
2. Store the solution for 12 hours at room temperate, shaking it intermittently.
3. Filter the solution through a Whatman No. 1 filter. Discard the filtrate to the drain.
4. Seal the filter and Amberlite in a plastic bag, label, and dispose of the bag through EH&S
Method 2 (Bensaude 1988)
1. Add 300mg of powdered activated charcoal for each 100 ml of up to 10 ug/ml solution of ethidium bromide.
2. Store the solution for 1 hour at room temperature, shaking it intermittenly.
3. Filter the solution through a Whatman No. 1 filter. Discard the filtrate to the drain.
4. Seal the filter and activated charcoal in a plastic bag, label, and dispose of the bag through EH&S.
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Destruction of Concentrated Solutions (0.5 mg/ml) (Lunn and Sansone 1987)
1. Add sufficient water to reduce the concentration of ethidium bromide to 0.5mg/ml or less. Work in a chemical fume hood.
2. For each 100 ml of ethidium bromide solution, add 20 ml of fresh 5% hypophosphorous acid and 12 ml of fresh 0.5 molar sodium nitrite. Mix carefully. Important: Check that the pH of the solution is less than Hypophosphorous acid is usually supplied as a 50% solution, which is corrosive and should be handled with care. It should be freshly diluted before use: 10 ml of hypophosphorous acid into 90 ml of water, stirred briefly. Use a chemical fume hood to prepare this solution.
3. Sodium nitrate solution, 0.5 molar, should be freshly prepared by dissolving 3.45 grams of sodium nitrite in water to a final volume of 100 ml. Use a chemical fume hood to prepare this solution.
4. After incubation for 24 hours at room temperature, bring the pH to between 5 and 9 with sodium bicarbonate. Discard the solution to the drain. As per RCNY Title 15, Chapter 19 Section 3 , no solution with a pH lower than 5 or greater than 11 can be drain disposed.
*Do Not Use hypochlorite (bleach) to treat ethidium bromide. Bleach treatment can produce mutagenic products and leave behind up to 20% of the original ethidium bromide.*
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