Decontamination of Dilute Solution of Ethidium Bromide

e.g. electroforesis buffer containing 0.5 ug/ml of ethidium bromide (EtBr)

Method 1 (Lunn and Sansone 1987)

1. Add 2.9 g of amberlite XAD-16 for each 100 mL of solution. Amberlite XAD-16, a nonionic, polymeric absorbent, is available from Rohm and Haas.
2. Store the solution for 12 hours at room temperature, shaking it intermitently.
3. Filter the solution through Whatman No.1 filter, and discard the filtrate.
4. Seal the filter and Amberlite resin in a plastic bag, and dispose of the bag in the hazardous waste.

Method 2 (Bensaude 1988)

1. Add 100 mg of powdered activated charcoal for each 100 mL of solution.
2. Store the solution for 1 hour at room temperature, shaking it intermitently.
3. Filter the solution through a whatman No.1 filter and discard the filtrate.
4. Seal the filter and activated charcoal in a plastic bag and dispose of the bag in the hazardous waste.

Notes:

1. Treatment of dilute solutions of EtBr with Hypochlorite (bleach) is not recommended as a method of decontamination. Such treatment reduces the mutagenic activity af EtBr in the Salmonella/microsome assay by about 1000-fold, but it converts the dye into a compound that is mutagenic in the absence of microsomes (Quillardet and Hofnung 1988)
2. EtBr decomposed at 262 Celcius and is unlikely to be hazardous after incineration under standar condition.
3. Slurries of amberlite XAD-16 or activated charcoal can be used to decontaminate surfaces that become contaminated by EtBr.

Decontamination of Ethidium Bromide Solution (Solutions containing >0.5mg/ml)

Ethidium Bromide (EtBr) is a powerfull mutagen and is moderatelly toxic. Gloves should be worn when working with solutions that contains this dye. After use, these solution should be decontaminated by one of the methods described below.

Decontamination of Concentrated Solutions of EtBr (Solutions containing >0.5 mg/ml)
Method 1
This method (Lunn and Sansone 1987) reduces the mutagenetic activity of EtBr in the Salmonella/ microsome assay by approximately 200-fold.

1. Add Sufficient water to reduce the concentration of EtBr to <0.5>
2. To the resulting solution, add 0.2 volume of fresh 5% hypophosphorous acid and 0.12 volume of fresh 0.5M sodium nitrite. Mix carefully. Check that the pH of the solution is <3.0.
   
3. After incubation for 24 hours at room temperature, add a large excess of 1M sodium bicarbonate. The solution may now be discarded.

note: Hypophosphorous acid is usually supplied as 50% solution, which is corrosive and should be handled with care. It should be freshly diluted immediatelly before use.
Sodium nitrite solution (0.5M) should be freshly prepared by dissloving 34.5 g of sodium nitrite in water to a final volume of 500 ml.

Method 2
This method (Quillardet and Hofnung 1988) reduces the mutagenenic activity of EtBr in Salmonella/microsome assay by approximately 300-fold. However, there are reports (Lunn and Sansone, 1987) of mutagenic activity in occasional batches of "blanks" treated with decontamination solutions.

1. Add sufficient water to reduce the concentration of ETBr to<0.5>
2. Add 1 volume of 0.5 M KMnO4. Mix carefully, and then add 1 volume of 2.5 N HCl. Mix carefully, and allow the solution to stand at room temperature for several hours. Caution:KMnO4 is irritant and is explosive. Solutions containing KMnO4 should be handled in a chemical hood.
3. Add 1 volume 0f 2.5 N NaOH. Mix carefully, and then discard the solution.