Ethidium Bromide Laboratory Spill and Waste Handling Procedures (FDWSRU)

Ethidium Bromide Laboratory Spill and Waste Handling Procedures

Introduction

Ethidium bromide intercalates (inserts itself) between nucleic acid base pairs and exhibits fluorescence under ultraviolet light, providing molecular biologists with an excellent stain for visualizing and imaging nucleic acids. While the ultimate goal of scientists working with ethidium bromide stains is to intercalate and visualize plant nucleic acids, all laboratory workers should be aware that ethidium bromide will intercalate human nucleic acids to cause mutations which may be potentially harmful to their health. Thus, all employees are expected to use adequate safety precautions in the handling and disposal of ethidium bromide stain solutions to prevent exposing themselves and their coworkers to the powerful mutagenic and moderately toxic properties of this chemical.

The purpose of this standard operating procedure is to provide laboratory workers with guidelines for the proper clean up of ethidium bromide spills and disposal of ethidium bromide wastes. NEVER DISPOSE OF UNTREATED ETHIDIUM BROMIDE SOLUTIONS DOWN THE DRAIN OR IN REGULAR TRASH. The mutagenic and toxic nature of "untreated" ethidium bromide stock and staining waste solutions require capturing, handling and disposal through our location's hazardous waste program. Activated charcoal filtration or chemical neutralization methods are presented as two pre-treatment options currently available that provide ethidium bromide waste volume reduction or reduce the mutagenic activity of waste solutions. Please contact Bonnie DiSalvo, NAA ASHM or Glen Davis, NAA CEPS, for additional handling and disposal guidance.

Gloves should always be worn when working with solutions containing ethidium bromide. Evaluate the size of the spill and determine whether spill control pillows or absorbent towels are required. Attempt to minimize the area of the spill by absorbing the solution carefully. Ethidium bromide contaminated spill control pillows or towels should be placed in a plastic bag, sealed and labeled with the contents and the words "Hazardous Waste." After absorption, decontaminate the spill site by cleaning with a laboratory detergent solution. Wearing UV eye protection, scan the decontaminated spill area using a hand-held UV light to locate any remaining traces of ethidium bromide.

Toxic fumes will be generated from fires involving ethidium bromide and thus will require immediate evacuation of the laboratory and building following our emergency evacuation plan. If you are working with ethidium bromide in a laboratory fumehood and a small fire occurs in that fumehood, extinguish the fire if that can be safely accomplished, close the sash and leave the exhaust fan running. Evacuate the area and immediately notify the authorities.

Treatment may be accomplished by extracting ethidium bromide from staining solutions using activated charcoal or by chemical neutralization to reduce the mutagenicity. Activated charcoal extraction of ethidium bromide is a hazardous waste volume reduction method, whereas a chemical neutralization method may not result in waste volume reduction. A selection of the following treatment methods will depend upon ethidium bromide concentration.

A. Activated Charcoal Absorption Procedure (using noncommercial equipment)

Add 100 mg of powdered activated charcoal to each 100 ml of stain waste solution

Store solution for one hour at room temperature, shaking it intermittently

Filter solution through a Whatman #1 filter and discard the nonhazardous filtrate (drain disposal will be subject to approval by the Ft. Detrick Hazardous Waste Manager)

Seal the filter containing the activated charcoal/ethidium bromide in a plastic bag and dispose of filter as hazardous waste

B. Activated Charcoal Absorption Procedure (using commercially available extraction units)

Disposable activated charcoal filter units designed for the single pour through filtration of ethidium bromide solutions are available. These filters are designed to remove ethidium bromide from up to 10 liters of stain solution with > 99% efficiency. This is a low cost, extraction efficient, no mess method for treatment of ethidium bromide stain solutions from laboratories generating small volumes of ethidium bromide stain waste. (Source: Schleicher & Scheill, Inc. at 800-245-4024, Supelco at 800-247-6628)

For those laboratories generating large volumes of ethidium bromide stain waste, cartridge systems are available. These systems utilize replacement cartridges, each capable of serving up to 100 liters of stain solution. (Source: American Bioanalytical at 800-443-0600). Filters and cartridges must be disposed of through our hazardous waste program.

C. Amberlite XAD-16 Absorption Procedure

Add 2.9 grams of Amberlite XAD-16 to each 100 ml of solution. This material is a nonionic, polymeric absorbent, available from Rohm and Haas

Incubate for 12 hr at room temperature, shaking intermittently

Filter solution through a Whatman #1 filter and discard the nonhazardous filtrate (drain disposal will be subject to approval by the Ft. Detrick Hazardous Waste Manager)

Seal the filter containing the Amberlite resin/ethidium bromide in a plastic bag and dispose of as hazardous waste

A. Hypophosphorus acid / Sodium nitrate/ Sodium bicarbonate neutralization

Dilute the stain solution with water to reduce the concentration to <0.5 mg/ml

Add 0.2 volume of fresh 5% hypophosphorus acid and 0.12 volume of fresh 0.5 M sodium nitrate. Mix carefully and check that the solution pH is <3.0.

(Hypophosphorus acid is usually supplied as a 50% solution, which is corrosive and should be handled with care. Sodium nitrate solution (0.5 M) may be prepared by dissolving 34.5 g of sodium nitrate in water to a final volume of 500 ml)

After incubation for 24 hr at room temperature, add a large excess of 1 M sodium bicarbonate. Mix carefully and discard per chemical transport officer instruction.

B. Potassium permanganate/ Hydrochloric acid / Sodium hydroxide neutralization

Dilute the stain solution with water to reduce the concentration to <0.5 mg/ml

Add 1 volume of 0.5 M KmnO₄ , mix carefully and add 1 volume of 2.5 N HCl. Mix carefully, and allow the solution to stand at room temperature for several hours.

Add 1 volume of 2.5 N NaOH. Mix carefully and discard per chemical transport officer instruction.

Detailed instruction and references to these procedures are found in "Molecular Cloning, A Laboratory Manual", Volume 3, 2nd edition by Sambrook, Fritsch and Maniatis, Appendix E (Commonly Used Techniques in Molecular Cloning), pages 8-9, 1989.