Ethidium Bromide

General Information

Ethidium bromide (EtBr) or 3,8-Diamino-5-ethyl-6-phenylphenanthridinium is commonly used as a non-radioactive marker to stain DNA in order to identify and visualize nucleic acid bands in electrophoresis and other gel-based methods of nucleic acid separation. EtBr is a dark red, crystalline, non-volatile, odorless powder that is moderately soluble in water. This material fluoresces a red-orange color under ultraviolet (UV) light and with increased fluorescence when bound to double-stranded DNA. While it is an effective tool for genomic research, its hazardous properties require special safe handling and disposal.

Health and Safety

EtBr is a potent mutagen (can cause genetic damage), and moderately toxic after an acute exposure. EtBr can be absorbed through skin, so it is important to avoid any direct contact with the chemical. The powder form is considered an irritant to the upper respiratory tract, eyes, and skin. Even though there is no evidence at this time of human carcinogenicity or teratogenicity, this material should be considered a possible carcinogen or teratogen. Some alternative stains have been found to be less mutagenic and less toxic than EtBr. If the toxicological data is lacking or unclear, the stain should be handled in the same way as EtBr. Some alternative stains are suspended in dimethyl sulfoxide (DMSO), which has health implications of its own, including increased skin absorption of organic compounds.

Good Lab Practices

Maintaining overall good laboratory work practices helps reduce the risks of hazardous exposures.

Work in a fume hood when handling powders, crystals, or solutions, in order to prevent inhalation exposure.

Wear a lab coat, long pants, closed-toe shoes, eye protection (Safety Glasses or Goggles), and nitrile or chloroprene gloves when working with EtBr. Leave lab coats, gloves, and other PPE in the lab, when your work is complete, to prevent the spread of this or other chemicals outside of the lab.

Provide safety training to the researchers/users on the hazards, use, and proper cleanup procedures for EtBr. Review of the relevant standard operating procedures (SOPs) for the work, Safety Data Sheet (SDS). Please contact EH&S if your lab does not currently have an approved SOP for the use of ethidium bromide.

Transport in secondary containment and work with small quantities of EtBr to minimize the risk of accidents.

Careful housekeeping is necessary when working with DNA stains. Delineate and restrict the area in which DNA stains may be used. Check other areas with UV light in a darkened room and follow the decontamination procedures below for contaminated surfaces.

When an ultraviolet light source is used with EtBr procedures, added caution is required. As a general rule, avoid exposing unprotected skin and eyes to intense UV sources. If the UV light is aimed upwards, wear a UV protective face shield when you are standing near the source. For prolonged work close to UV light boxes or other intense sources, it may be useful to wrap the end of the lab coat sleeves loosely with masking tape to prevent gaps where the wrist could be exposed.

Ethidium bromide should be stored away from strong oxidizing agents in a cool, dry place, and the container must be kept undamaged and tightly closed.

Safer Alternatives

Several alternatives to EtBr exist which manufacturers claim are less toxic than EtBr and may not require UV light sources. However, the SDSs and manufacturers’ guidelines need to be reviewed prior to use. The following represent a few of the available alternatives to EtBr:

MaestroSafe®, produced by Maestrogen, is a safe class of nucleic acid stains for visualization of double-stranded DNA, single-stranded DNA, and RNA in agarose gels. MaestroSafe products are non-carcinogenic by the Ames-test. The results are negative in both the mouse marrow chromophilous erythrocyte micronucleus and mouse spermary spermatocyte chromosomal aberration tests.

SYBR® Safe DNA Gel Stain is a highly sensitive stain for visualization of DNA in agarose or acrylamide gels. Specifically formulated to be a less hazardous alternative to ethidium bromide that can be used with either blue-light or UV excitation. Supplied as either a concentrate or a ready-to-use solution that can be used like an ethidium bromide solution. The stain is also suitable for staining RNA in gels.

GelRed and GelGreen
New generation fluorescent nucleic acid gel stains designed to replace the ethidium bromide (EtBr). GelRed and GelGreen from Biotium offer sensitivity, cell membrane impermeability for true safety, extreme heat and light stability as well compatibility with all downstream manipulations.

EZ-Vision® DNA Dye is an all-in-one solution containing a fluorescent visualization dyed in a loading buffer.

Emergency Response

Eye care: If EtBr comes in contact with the eyes, immediately flush them with copious amounts of cold or cool water for at least 15 minutes, preferably in an emergency eyewash.

Skin care: In the event of skin exposure, remove contaminated clothing and immediately wash the affected area with soap and copious amounts of cold or cool water for 15 minutes.

If swallowed or inhaled: In the case of ingestion obtain medical attention immediately. If EtBr dust is inhaled move to a source of fresh air.

Spills: All labs should have a Spill Kit available and anyone who uses it must first be trained.

Spills of ethidium bromide solutions should be absorbed and the area decontaminated with soap and water. Avoid raising dust when cleaning up solid spills by mixing with water and then absorbing the solution. All spill cleanup materials and absorbents should be bagged or placed in a sealed container with a hazardous waste label. Request a hazardous waste pickup from EH&S.

Some facilities use a handheld UV lamp to check for residual ethidium bromide contamination following spill cleanup. A reddish-orange fluorescence can be detected under both "long" and "short" UV wavelengths. Users of the handheld lamps should be aware that their ability to detect small spills is not guaranteed. The ease of detection depends upon a variety of factors including the chemical composition of the sample, the wavelength of the UV lamp, and the intensity of the lamp. Use of a handheld UV lamp to detect traces of ethidium bromide may serve as an occasional check of laboratory practices, but it cannot substitute for good cleanliness and careful contamination control.