Introduction
Ethidium bromide (EtBr), 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide, is commonly used as a non-radioactive marker for identifying and visualizing nucleic acid bands in electrophoresis and other methods of gel-based nucleic acid separation. EtBr is a dark red crystalline that readily fluoresces reddish-brown when exposed to ultraviolet (UV) light. EtBr is a potent mutagen and toxic after an acute exposure. It is an irritant to the skin, eyes, mouth, and upper respiratory tract. Direct contact with EtBr should be avoided.
Handling Procedures
The best practice for handling EtBr, as with handling any chemical, is to consult the MSDS prior to usage. Pure EtBr should always be handled in a chemical fume hood. A laboratory that uses EtBr can easily become contaminated if appropriate precautions are not taken. The following best management practices are recommended for all EtBr laboratories:
- Designate an area where EtBr work is going to occur and only handle it in this area (i.e. chemical fume hood, bench top). The area within a laboratory where EtBr is used should be limited.
- It is recommended that the surfaces of the designated area be covered with an appropriate liner (i.e. plastic, chemical resistant liner). If the liner becomes contaminated, it can simply be replaced and disposed of as EtBr contaminated debris.
- If a liner cannot be used, then the surfaces of the work area should be decontaminated routinely (refer to decontamination procedures below).
- Good industrial hygiene should be practiced at all times. All spills or evidence of contamination should be cleaned immediately using the decontamination procedures below.
Personal Protective Equipment
Ensure all personal protective equipment (PPE) is in good condition and worn at all times when using EtBr. At a minimum, PPE should include a lab coat, closed-toe shoes, chemically-resistant gloves, and chemical safety goggles. Latex gloves give very little protection against EtBr! Nitrile gloves are an effective short-term barrier. Upon removal of the gloves, wash hands thoroughly. If using EtBr for extended periods, double-gloving gives better protection. Remember to change outer gloves frequently to minimize contamination.
For accidental exposures to:
- Eyes - immediately flush with water for at least 15 minutes. Seek medical attention.
- Skin - immediately wash the area with soap and water. Seek medical attention.
- Clothing - remove immediately and place in a sealed plastic bag or container. Seek medical attention.
Waste Disposal Procedures
EtBr waste must be collected and managed as hazardous waste, not biological waste. Items that have come into contact with EtBr such as PPE, absorbent materials, liners or other debris must also be collected as hazardous waste. All solid EtBr waste, including debris and dry agarose gels, should be placed in an appropriate container and labeled accordingly. Please double-bag your solid waste in two black trash bags. Do Not use orange biohazard waste bags unless the waste used to be infectious. All EtBr waste containers must be suitable for transportation and must not be leaking. For removal of EtBr waste from your laboratory, submit a Hazardous Materials Pickup Request Form.
The buffers and solutions you use with ethidium bromide gels can be submitted to REM as waste or put down the drain only if you use a filter especially designed for removal of EtBr. An ethidium bromide extractor such as the Whatman Extractor™ System or similar device can be purchased from several bioscience, laboratory, or chemical supply companies.
Decontamination Procedures
Decontamination should be performed using the following solution:
- 4.2 grams of sodium nitrite (NaNO2)
- 20 milliliters of 50% hypophosphorous acid solution (H3PO2)
- 300 milliliters of water
To perform decontamination:
- Soak a paper towel in the decontamination solution and thoroughly wash the contaminated area.
- Once the contaminated area has been thoroughly washed with the decontamination solution, rinse the area 5 times with tap water using a clean paper towel for each rinse.
- Soak all spent paper towels in the decontamination solution for one hour. Gently wring out excess solution and dispose of as hazardous waste with contaminated gloves, pipette tips or any other solid EtBr debris.
- Using a UV light, check to ensure all EtBr has been removed.
- The decontamination solution should be collected in an appropriate container and labeled as hazardous waste (i.e. Water 95%, Hypophosphorus Acid 3%, Sodium Nitrite 1%, Ethidium Bromide 1%).
- Include both the debris and the waste decontamination solution on a Hazardous Materials Pickup Request form
Note: Hypophosphorous acid is a DEA List chemical. When purchasing it, you and your PI will have to fill out and sign an “Authorized Purchaser” form. This is simply a company’s method of ensuring you will not misuse the material.
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