Standard Operating Procedure 

 

ETHIDIUM BROMIDE


#1 PROCESS

Use of ethidium bromide for visualization of nucleic acids.


#2 HAZARDOUS CHEMICALS/CLASS OF HAZARDOUS CHEMICALS

Potent mutagen. Moderately toxic.


#3 PERSONAL PROTECTIVE EQUIPMENT

Wear chemical safety goggles when using ethidium bromide. Avoid skin contact, ethidium
bromide may be absorbed through the skin. Use NATURAL rubber gloves.


#4 ENGINEERING/VENTILATION CONTROLS

Use only inside a chemical fume hood with adequate ventilation. Safety shower and eye wash should be easily accessible where ethidium bromide is used.


#5 SPECIAL HANDLING PROCEDURES AND STORAGE REQUIREMENTS

Store in a cool, dry place away from strong oxidizing agents. Keep containers tightly
closed. Use with adequate ventilation.


#6 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. Call the UCSD EHS-42823
If spill is on equipment, use ultraviolet light to locate spill, then use the decontamination 
procedure outlined below.

Wear protective clothing. 

Small spill: If in solution, absorb freestanding liquid. Use ultraviolet light to locate spill,
then use the decontamination procedure outlined below.

Large Spill: Notify others in room of spill. Evacuate room/immediate area. Call EHS for
cleanup. Post room with warning notifying others of spill. Prevent unnecessary entry into
area until arrival of EHS response team. Provide assistance and information to spill
responders.


#7 WASTE 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,  the following disposal procedures for ethidium bromide are recommended:

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 that if the concentration is less than 0.1% ethidium bromide, gels may be placed in laboratory trash.  If concentration is equal to or greater than 0.1%, gels must be placed in Biohazard Burn Box for incineration.

     Less than 0.1% ethidium bromide:  place in laboratory trash 
     More than or equal to 0.1%:  place in biohazard box for incineration. 

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 /deactivated using one of these methods: 

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 two 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.  Kit is available through Schleicher and Schuell or VWR. 

  • 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 BagŪ" 

Another simple charcoal filtration method is the Green Bag, manufactured by BIO 101.  The Green BagŪ 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 back in the biohazardous waste box for incineration.

Green Bags are available through VWR (www.vwr.com )- this is a non-catalog item -
 

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 two 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.

EHS strongly recommends charcoal filtration over chemical deactivation. 

Solutions containing heavy metals, organics, cyanides or sulfides should be disposed as hazardous waste. 

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.
 

Contact UCSD EHS-42823 for further disposal instructions.


#8 SPECIAL APPROVAL REQUIRED

PI approval before initial use of this material.


#9 DECONTAMINATION

Wash the area with a paper towel soaked in a decontamination solution consisting of 4.2g
of sodium nitrite and 20 ml of hypophosphorous acid (50%) in 300 ml of water. Then wash
five times with wet paper towels using a fresh towel each time. Soak all the towels in
decontamination solution for 1 hour. Using a U- V light, check for completeness of
decontamination, and if satisfied, then call EHS for disposal of the decontamination
solution.

The decontamination solution must be prepared just prior to use.

If the decontamination solution (pH 1.8) is felt to be too corrosive for the surface to be
decontaminated, then use six rather than five washes following the decontamination
solution wipe. Again, soak all the towels in decontamination solution for at least 1 hour
before disposal.


#10 DESIGNATED AREA

Recommended.


#11 LABELING

Whenever ethidium bromide is mixed and stored outside its original container, it must be clearly marked
with its concentration and a description of its hazards. 


#12 REFERENCES