Ethidium Bromide

Ethidium Bromide (EtBr), a dark red, crystalline, non-volatile powder that is moderately soluble in water, is widely used in research laboratories as a visualizing agent for nucleic acids.  The powder is used to make a stock solution of 10 mg/mL, and the working concentration for gel-staining is 1 µg/mL.  Because EtBr can bind with DNA, it is considered highly toxic as a mutagen.  It may potentially cause carcinogenic or teratogenic effects, although no scientific evidence showing either health effect has been found.  Exposure routes of EtBr are inhalation, ingestion, and skin absorption.  An acute exposure to EtBr causes irritation of the mouth, upper respiratory tract, skin, and eyes.

Ethidium bromide is not regulated as hazardous waste; however, St. Olaf College policy requires that the following precautions and procedures are taken while using or handling this chemical.

Personal Protective Equipment
  • Chemical splash-resistant safety goggles and gloves MUST be used when handling pure EtBr, preparing ethidium bromide solutions, staining gels, or visualizing nucleic acids in gels.


Special Handling Procedures
  • All work with EtBr is confined to RNS 395 to the fume hood, the gel running and pouring benches, and the gel doc areas (which are labeled).  Gels are occasionally brought to a designated bench in RNS 375 for slicing.
  • Making an EtBr solution from powder must be done in the RNS 395 fume hood to avoid the potential for an acute inhalation hazard (i.e., open the container of pure EtBr only inside the RNS 395 fume hood). 
  • Before you leave the EtBr work area you MUST remove your gloves to prevent EtBr being transferred to other parts of the lab, or to outside of RNS 395.



Primary and Secondary Containment; Labeling
  • The EtBr powder is kept tightly closed and stored in the RNS 395B refrigerator.
  • The 10 mg/mL stock solution is to be kept in a tightly closed and labeled container, in a labeled secondary containment tray.
  • Containers for the 1 µg/mL working solutions must be clearly labeled, have lids, and placed in a labeled secondary containment tray.
  • All primary and secondary containers must be labeled “Designated for ethidium bromide use only.”
  • Make sure to include the EtBr concentration on all labels.


Procedure for Gel Staining
  • Wearing all PPE as stated above, place the gel in one of the staining trays containing EtBr in a 1 µg/mL concentration.
  • Close the lid of the tray; you may rinse and remove gloves and leave them at the staining station until gel is ready for viewing (about 10-15 minutes).
  • Put gloves and PPE back on, use the designated spatula to remove the gel from the staining tray.
  • Place the gel in to a tray of distilled water (may leave for a few minutes if destaining is required).
  • Use the designated spatula to place the gel on to the light box, and return the spatula to the hood.
  • You must remove gloves to use the computer keyboard when imaging; the keyboard must remain a non-EtBr space for all users.
  • When you are finished viewing and/or photographing the gel, put it in the designated yellow metal waste container labeled “ethidium bromide waste.”
  • Rinse the Li-Cor EtBr imaging tray with deionized water and then wash with 70% Ethanol and dry with a Kimwipe; the wipes and your gloves should be safe for disposal in the regular trash (turn gloves inside-out).


Small Spills; Decontamination of Counters or Equipment
  • Before decontamination, de-energize any electrical equipment that might need cleaning, and wear the appropriate PPE.
  • The decontamination solution is prepared by adding 20mL of 50% hypophosphorous acid to a solution of 4.2g of sodium nitrite in 300mL of water.  This solution will be acidic; wear gloves.
  • Wash the area with a paper towel soaked in the decontamination solution. Then wash another five times with soaked paper towels using a fresh towel each time.
  • Place all the used towels in a large container and soak them in fresh decontamination solution for at least one hour.  Using a U-V light, check for completeness of decontamination.
  • Adjust the used (and acidic) decontamination solution and towels to a pH of 5-9 with sodium bicarbonate.  The towels can then be discarded in the trash, and the solution may be rinsed down the sanitary sewer with a strong stream of running water.


Disposal/Destruction Methods: Ethidium Bromide Solutions
  • Aqueous solutions of EtBr that contain ≤ 10 µg/mL.
    • These working/staining solutions can be poured down the laboratory sink with a strong stream of water.
  • Aqueous solutions of EtBr that contain > 10 µg/mL.  These Stock Solutions must either be poured into a suitable screw-top container and disposed of as hazardous waste, or these solutions can be rendered harmless (and poured down the sink) using one of the three bench-top treatment procedures:
    • AMRESCO Destaining Bag.  
      • Place a Destaining Bag into the solution.  Allow the bag to remain in the solution for the allotted time (read the directions).
      • Remove the bag and place it into the yellow metal EtBr waste container.
      • Pour the remaining filtrate down the laboratory sink with a strong stream of running water.
    • Lunn and Sansone Ethidium Bromide Destruction Method (Lunn, George, and Sansone, Eric B. 1994. Destruction of Hazardous Chemicals in the Laboratory. John Wiley and Sons, Inc. pp. 185.)
      • Carry out the following steps in a fume hood and follow all laboratory safety precautions, including proper protective clothing.
      • Dilute solutions containing EtBr to concentration <0.05% w/v (50mg/100mL).
      • For each 100mL of EtBr solution add 20mL of fresh 5% hypophosphorous acid and 12mL of fresh 0.5M sodium nitrite solution.  Check that the pH of the solution is <3.0.  Stir briefly.
      • After reacting for at least 20 hours, adjust the pH to 5-9 with sodium bicarbonate, then pour the solution down the laboratory sink with a strong stream of running water.
    • Armour Ethidium Bromide Destruction Method (Kaufman, James A. ed. 1990. Waste Disposal in Academic Institutions. Lewis Publishers. pp. 127-8)
      • Carry out the following steps in a fume hood and follow all laboratory safety precautions, including proper protective clothing.
      • Dilute solutions containing EtBr to concentration <0.034% w/v (34mg/100mL).
      • Add 10mL of fresh bleach for every 1mg EtBr.
      • Stir at room temperature for at least 4 hours.
      • The EtBr is converted to the physiologically inactive product 2-carboxybenzophenone.  The solution can then be poured down the laboratory sink with a strong stream of running water.


Disposal of Sharps, Gels, Gloves, Glassware, Destaining Bags, etc.
  • Solid waste including contaminated pipet tips, gloves, destaining bags, and all used gels are to be deposited in the designated yellow metal waste container in RNS 395.  Follow the instructions on the waste container.
  • Needles, scalpels, Pasteur pipettes and other sharps contaminated with EtBr should be disposed of directly into a sharps container.
  • Volumetric or transfer pipettes, and other disposable glassware contaminated with EtBr should be disposed of in the designated glass disposal container.  Grossly contaminated (visibly contaminated) glassware should be washed with bleach before disposal in the designated 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 tubes can then be disposed of in the trash.  Grossly contaminated (visibly contaminated) tubes should be washed with bleach prior to disposal.
  • Most other disposable labware (e.g. sample vials, disposable beakers, etc.) contaminated with EtBr may be disposed of in the normal trash.  Grossly contaminated (visibly contaminated) disposable labware should be washed with bleach prior to disposal.


Spill and Accident Procedures

In case of contact with eyes, immediately flush eyes with copious amounts of water for at least 15 minutes.  In case of contact with skin, immediately wash skin with soap and copious amounts of water.  If inhaled, remove to fresh air.  Contact the Chemical Hygiene Officer (Pat Ceas, X-3560; 507-321-0379).



Acknowledgments

Much of the above information was modified from the James Cooke University “Draft Protocols for handling and disposal of ethidium bromide and similar compounds.”  Other information was obtained from:

http://dehs.umn.edu/hazwaste_chemwaste_umn_cwmgbk_sec5.htm#eb

https://ehs.research.uiowa.edu/ethidium-bromide