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Ethidium Bromide Disposal

Ethidium bromide is commonly used in molecular biology laboratories.  While it is not regulated as hazardous waste, the mutagenic properties of this substance may present a hazard if it is poured down the drain untreated or placed in the trash.

Based on these considerations, EHS recommends the following disposal procedures for ethidium bromide.

Electrophoresis Gels

Trace amounts of ethidium bromide in gels should not pose a hazard.  Higher concentrations, e.g., when the color of the gel is dark pink or red, should not be placed in laboratory trash.  EHS recommends the following:
  • Less than 0.1% ethidium bromide:  place in laboratory trash
  • More than or equal to 0.1%:  place in biohazard box for incineration.

Consider substituting with a less hazardous material, such as GelRad Nucleic Acid Gel Stain.

Ethidium Bromide Solutions

  • Aqueous solutions containing <10ug/ml ethidium bromide can be released to the drain.
  • Aqueous solutions containing >10ug/ml ethidium bromide should be filtered or deactivated using one of the methods described below.  EHS strongly recommends charcoal filtration over chemical deactivation.
  • Solutions containing heavy metals, organics, cyanides or sulfides should be disposed as hazardous waste.

Charcoal Filtration

Filtering the aqueous ethidium bromide waste solutions, free of other contaminants, through a bed of activated charcoal is a relatively simple and effective method for removal of ethidium bromide.  The  filtrate may be poured down the drain.

There are three simple kits available for charcoal filtration:

Funnel Kit
Schleicher and Schuell supply a commercial  filter funnel kit that uses a packaged charcoal disk that is graduated for easily tracking the amount of aqueous solution calculated for a fixed quantities of ethidium bromide residue.  This is particularly useful for labs that generate large amounts of solutions at a time.  The kit is available through VWR and other suppliers.


 
 

  • Filter the ethidium bromide solution through the charcoal filter.
  • Pour filtrate down the drain.
  • Place charcoal filter in a sealed bag (e.g., zip-lock) and place in biohazardous waste box for incineration.

The Green Bag

Another simple charcoal filtration method is the Green Bag, manufactured by BIO 101.  The Green Bag® Kit allows rapid and trouble-free concentration of ethidium bromide from large volumes of solutions into a small "tea" bag containing activated carbon which is then conveniently disposed along with other solid hazardous wastes. One kit has the capacity to remove 500 mg of ethidium bromide from solutions (10mg EtBr/bag).

  • Place the Green Bag into the ethidium bromide solution.
  • Allow to sit for the allotted time.
  • Pour filtrate down the drain.
  • Dispose of the Green bag in the biohazardous waste box for incineration.

Green Bags are available through BIO101 (www.bio101.com) or through VWR (www.vwr.com - this is a non-catalog item - use #BIO101 22350-200).

Destaining Bags

Amresco Destaining Bags are similar to the Green Bag Kits and remove up to 5 mg of ethidium bromide and other biological stains, including Coomassie Blue. Follow the same procedures as for the Green Bag kits.

Chemical Neutralization

Solutions containing ethidium bromide can be deactivated, neutralized and poured down the drain with copious amounts of water.  Deactivation may be
confirmed using UV light to detect fluorescence.  There are three recognized methods for deactivation:

Armour Method

This is the simplest method, but is somewhat controversial.  One study found traces of mutagenic reaction mixtures using this method. (Lunn, G. and E. Sansone, Analytical Biochemistry, vol. 162, pp. 453-458, 1987)
  • Combine equal amounts of ethidium bromide solution and household bleach.
  • Stir constantly for four hours or let sit for 2-3 days.
  • Adjust pH to 4-9 with sodium hydroxide.
  • Pour down drain with copious amounts of water.

    • Lunn and Sansone Method

    For each 100 ml of ethidium bromide solution:
  • Add 5% hypophosphorus acid.
  • Add 12 ml of 0.5 M sodium nitrate.
  • Stir briefly and let stand for 20 hours.
  • Adjust pH to 4-9 using sodium hydroxide.
  • Pour down drain with copious amounts of water.

    • Quillardet and Hoffnung Method

    This method uses 0.5 M potassium permanganate and 2.5 M hydrochloric acid.  Since chlorine gas may be released in significant concentration, EHS does not recommend using this method.

    Gloves, Equipment and Debris

    Gloves, test tubes, paper towels, etc., that are grossly contaminated with ethidium bromide should be placed in medical waste for incineration.  Consider deactivating in bleach before disposal if the items are significantly contaminated.

    If you have any questions about disposal of ethidium bromide or any other hazardous material, contact Kyle Angjelo or Robin Izzo at 258-5294.


           
           
         

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