Methods and reagents: Disposal of ethidium bromide Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses the various methods used to dispose of ethidium bromide waste. For details on how to partake in the newsgroup, see the accompanying box. Everyone is a little nervous about sending a potentially mutagenic substance through the waste-water treatment plant. One topic that comes up often on the newsgroup is how to dispose of ethidium bromide solutions that are used to visualize DNA trapped within agarose and polyacrylamide gels. According to a small sample survey of 12 netters, performed by Kieron Walsh of Schleicher and Schuell (kierondes@aol.com), despite many published warnings and protocols for detoxifying, many people are still unsure of how to dispose of ethidium bromide waste properly. Surprisingly, most of those responding said that, after considering the alternatives, they eventually end up pouring it down the sink. Taken on a global scale, this widespread practice may result in huge amounts of untreated ethidium bromide leaving research laboratories each year. Mutagenicity ************ The latest discussion over this issue drew some heated debate over whether or not ethidium bromide is as dangerous to work with as previously thought, or even if it is a mutagenic substance at all. Some feel that it is very dangerous and that dumping it in the sink should be avoided at all cost. One person wrote that ethidium bromide is certainly a mutagenic substance, causing DNA damage to bacteria, and that this is enough evidence to treat it with the utmost care and respect. By contrast, as demonstrated by a negative SOS chromotest (a colorimetric assay that measures the expression of genes induced by genotoxic agents in Escherichia coli), ethidium bromide has not been confirmed to be a genotoxic compound.[1] Others are not convinced of its danger and think that, even if it were to get into the water system in low quantities, it would not cause serious harm, since it probably cannot cause substantial damage to mammalian cells. The skeptics propose that the manner in which it had been tested for mutagenicity, namely the Ames test for detecting reversion mutations in Salmonella typhimurium [2], leaves some doubt and is open to interpretation. They claim that it has not yet been adequately proved to be mutagenic to humans in small quantity. Disposal practices ****************** Some scientists justify the dumping of ethidium bromide into the sink as reasonable because it will be absorbed by many substances within the sewage-treatment process, and will eventually be degraded or detoxified by the natural flora of microorganisms at the waste-water management plant. However, there still seems to be some doubt as to whether this is the best approach. One option for reducing the waste is to lower the amount of ethidium bromide used for each experiment. This may be accomplished by adding 0.5 ug/ml directly to the gel instead of soaking it afterwards, eliminating the need for larger volumes of staining solution. Problems do exist with this method, however, because you are still left with the problem of disposing of the stained gel and any contaminated electrophoresis buffer. Some people argue that post-electrophoresis staining instead produces less contaminated waste, since the staining bath can be used repeatedly before decontaminating or discarding it. A common practice to dispose of the waste is to mix dilute ethidium bromide solutions with hypochlorite (bleach) in order to stifle its mutagenicity. Some netters strongly disagree with this practice, stating that this converts it into an even more mutagenic substance [3]. One the other hand, no evidence exists for this product being more dangerous to humans than ethidium bromide [4]. Another way is to extract the ethidium bromide with an organic solvent such as isoamyl alcohol or butanol, and pour the aqueous phase down the sink. However, the contaminated organic solvent must now be disposed of. Two other methods have been published for the conversion of ethidium bromide to less hazardous chemicals. One involves oxidation by potassium permanganate and the other involves destruction by deamination with sodium nitrite and hypophosphorous acid [5]. Problems associated with these methods include the time spent for detoxification, the cost of extra materials and the risk of exposure to other hazardous substances. To avoid dealing with these issues, many scientists choose to send the nasties off elsewhere or simply tip them down the sink. As implied earlier [3], methods employing complex steps and additional hazardous chemicals are unlikely to survive in the lab. An incentive for most scientists to dispose of ethidium bromide properly would be a simple, safe and cost-efficient means of handling of the waste. One easier method that can be used for dilute solutions is to add a nonionic, polymeric, adsorbent resin, such as Amberlite XAD-16, or activated charcoal (charcoal that has been pulverized to create a larger surface-to-volume ratio for adsorption), at 0.5 g per 100 ml (Ref. 6). The slurry is then allowed to sit for at least 1 h before the solid particles are filtered out onto Whatman [R] paper. The liquid can be poured down the sink and the paper destroyed by incineration. Charcoal filter traps or retaining cartridges, which may cost substantially less than would other decontamination materials, are also becoming available from some biotechnology companies. A closed container of charcoal can also be used as a depository, with an absorbent cloth material, kitty litter or vermiculite added to soak up the excess liquid. When saturated, the water can be allowed to evaporate for some time to reduce the volume before incineration. Although there is no consensus among the netters for how to dispose of ethidium bromide, it is clear that there is a need for awareness of the problem and for a simple method of safe disposal. Alternatives ************ Nucleic acid stains other than ethidium bromide may be more useful for some purposes. For example, one netter wrote that the DNA-binding dye SYBR [TM] Green I, available from Molecular Probes Inc., gives 100-1000-fold more sensitive detection of low quantities of 200-400 base-pair DNA probes or polymerase chain reaction (PCR) products when using a 254 nm transilluminator. In addition, larger DNA fragments gave 20-100-fold stronger signal. A phone call to Molecular Probes revealed that tests performed by the company showed that the dye can be used to detect less than 20 pg of DNA per band in an agarose gel and gives a 20-35-fold increase in detection over ethidium-bromide-stained DNA visualized on a 300 nm transilluminator. Since SYBR [TM] Green I has a large fluorescence enhancement when bound to DNA, it may be used as a more sensitive replacement in the standard ethidium bromide spot test for quantitating very dilute samples of DNA, discussed in a previous Methods and reagents article (TIBS 19:93-94), or for detection of PCR products of low abundance. The down side is that the DNA-binding properties of the dye have not yet been fully investigated, and no tests for mutagenicity and/or genotoxicity have been performed. In addition, the increased sensitivity may not necessarily be an advantage in all experiments, because it costs nearly 2000 times more per gel than ethidium bromide. Nontoxic alternatives to ethidium bromide for staining DNA in agarose gels include basic dyes such as methylene blue, toluidine blue, azure A and brilliant cresyl blue [7]. One netter even reported that using methylene blue to visualize a DNA fragment resulted in a tenfold increase in the efficiency of a subsequent subcloning experiment as compared with using ethidium bromide. References: [1] Quillardet, P. and Hofnung, M. (1993) Mutation Res. 297,235-279 [2] Ames, B. N., McCann, J. and Yamasaki, E. (1975) Mutation Res. 31,347-364 [3] Cohen, B. L. (1987) Trends Genet. 3,308 [4] Quillardet, P. and Hofnung, M. (1988) Trends Genet. 4,89 [5] Lunn, G. and Sansone, E. B. (1987) Anal. Biochem. 162,453-458 [6] Bensaude, O. (1988) Trends Genet. 4,89-90 [7] Santillan-Torres, J. L. and Ponce-Noyola, P. (1993) Trends Genet. 9,40 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jun94.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1994. Methods and reagents - Disposal of ethidium bromide. Trends in Biochemical Sciences 19(6):257-258. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************