Methods and reagents: Emergency sterilization using microwaves 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 use of a home microwave oven for sterilizing laboratory equipment. For details on how to partake in the newsgroup, see the accompanying box. Ever have one of those days when you desperately need two sterile bottles and there's only one left on the shelf? This happens often enough for some to consider quickly sterilizing a bottle using a microwave oven. But is this really a good idea? Recently, netters discussed the pros and cons of sterilizing laboratory plastic- and glassware by microwaves as a last resort. One person discovered that 5 mins of full power (600W) is enough to bend large polystyrene dishes without any water present and wondered if the technique could be used to sterilize culture flasks and other equipment. Someone else mentioned that a large commercial microwave sterilizer is currently on the market, but that rather than directly killing microorganisms by microwaves, it also uses a microwave generator to produce steam for the sterilization process. By contrast, common household kitchen microwaves are not equipped with such a device and it is not known what effect it would have on bacteria microwaved on a dry surface. Cooking times for contaminants ****************************** In order to test if his household microwave oven could kill dried bacteria, Sebastian Bunka (Sebastian.Bunka@vu-wien.ac.at) did an experiment with a plastic tube filled with a suspension of Staphylococcus aureus at approximately 10^5 colony forming units (CFU) ml-1. The culture was completely dried into the tube by vacuum and then placed in the microwave for 10 mins at full power (600W). After the tube had cooled, it was rinsed with sterile broth and serial dilutions plated onto blood agar to determine the number of survivors. To his surprise, the experiment showed almost no reduction in the cell viability by dry microwaving. However, Michael Prigge (prigge@darkwing.uoregon.edu) wrote that he has some experience with surface-sterilizing hundreds of Arabidopsis seeds at a time in order to germinate them on agar plates. He feels that steam is not necessary as his experiments show contaminanting bacteria and fungi can be eliminated from dry seeds by microwaving them in a 15 ml polypropylene tube twice for 5 mins at full power, along with a beaker of 100 ml water and boiling chips in the oven to absorb any unquenched microwaves. After comparing plates of microwaved vs non-microwaved seeds, he is convinced that contaminating fungi that are normally overrun the plates are killed by the microwaves. Although this report is encouraging, netters feel that the risk of contamination is too great for glassware to be treated this way for important experiments. Most still believe that steam and pressure are necessary for eliminating unwanted bacteria, particularly because spore-forming bacteria require high pressure for destruction. One netter suggested that an alternative is UV light. A quick sterilization can be accomplished by placing glassware in a UV cross-linking oven and subjecting it to 5000-8000 uJ cm-2. As this kind of sterilizing has been standard practice for some time, this person feels it is far more reliable. In addition, the UV treatment would be more useful for equipment involved in amplification of minute amounts of DNA, and as the PCR can give false positives from any leftover bacterial cell debris, this would be a better solution for a quickie fix. Others thought that use of a microwave is okay depending on the experiment for which the glassware is used. Liquid media that is made fresh from powder and then boiled for several minutes could be used for growing bacterial cells without getting into too much trouble, given that most Gram-negative bacteria would not survive the boiling treatment. Alternatively, preparation of a large mass of protein or plasmid DNA could even be done without sterilizing the media if it were to be used right away for growing Escherichia coli. The media could be made in a non-sterile flash from powder, mixed with water, and immediately inoculated with a log-phase primary bacterial culture. Because the Gram-negative bacteria generally used for isolating vector DNA have such a short doubling time (15-20 mins), a bacterial culture can be inoculated with an astronomical amount of cells, and a bloom of the culture should occur within 2-3 hrs. This trick can be used for creating a rapidly growing culture with minimal risk of obtaining a contaminating vector from any single cell contaminants lurking in the dry media or a dry flask. Pick of the day *************** Toothpick or not toothpick? That is the question. In a previous Methods and reagents column (TiBS 20, 160-161) the use of wooden toothpicks for transferring DNA from a gel into polymerase chain reaction (PCR) tubes for reamplification of the banded fragment was discussed. Despite the warnings about the possible dangers of introducing substances inherent to the wood or impregnated disinfectants that could interfere with the PCR, netters have continued to use the wooden toothpicks for the purpose of screening bacterial colonies for cloned DNA by the PCR without worries. Netters typically use the flat-style of Kaybee brand toothpicks manufactured by Keenan Industries Limited, Owen Sound, Canada, for colony screening and they say the initial report [1] of a contaminant inhibiting the PCR from the picks is not as worrisome as previously thought. They think that the article might have been misleading because they have never experienced adverse results when using the picks for colony screening. That is not to say that a very low amount of DNA transferred using a wooden toothpick would not be inhibited when added to the PCR, as might be the case when stabbing a DNA band in a gel. Nevertheless, netters are quite inflexible these days when it comes to choosing between paper and plastic. The woodmongers prefer the flat toothpicks over the larger pointed cocktail sticks available at most supermarkets because they say the ends are too sharp. Scooping a portion of the colony is more trouble with these, and the points always end up stabbing holes in the agar or transferring more bits of agar than need be. In addition, the cost of the flat wooden toothpicks, in comparison to the plastic sticks, swords, pipet tips or plastic loops recommended by others, is much less, and the wooden ones are bio-degradable. References ********** [1] Lee, A. B. and Cooper, T. A. (1995) BioTechniques 18, 225-226 ******************************************************************************* 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/feb97.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1997. Methods and reagents: Emergency sterilization using microwaves. Trends in Biochemical Sciences 22(2):68-69. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************