Methods and reagents: Floaties in the gene pool 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 a strange contaminant thought to be lurking in a waterbath, as well as some other items of unfinished business. For details on how to partake in the newsgroup, see the accompanying box. One researcher, Stephen J. Palmieri (rocco@mail.utexas.edu) had a very strange result when transformation experiments were performed with either XL1-Blue [TM] (Stratagene) or SURE [TM] (Life Technologies) strains of frozen competent Escherichia coli cells. Bacteria transformed with DNA, then plated onto LB media containing 50 ug/ml ampicillin appeared as odd-looking colonies after just a few hours of incubation at 37 degrees C. Since these colonies did not look like the cream colored run-of-the-mill E. coli type, and because some more normal looking colonies began to appear on the plates several hours later, it became quite obvious that what he had on his hands were contaminants. Oddly, the faster-growing colonies did not appear on control plates spread with bacteria that had no DNA added, but did appear when cells were mixed with DNA that had been ligated. This led him to suspect that perhaps the strange bacteria were transformants, or that the ligation buffer or T4 DNA ligase enzyme stock was contaminated. Not surprisingly, when mini-preps were performed, and isolated DNA was run on an agarose gel to determine if these colonies contained plasmid DNA, none of the samples had the expected plasmid, even though the bacteria grew on media with antibiotic added. When competent E. coli cells were mixed with purified DNA of a covalently closed plasmid, and this was used for transformation instead of the ligation mixture, 80-90% of the colonies proved to contain the expected plasmid by mini-prep analysis. Because he had sterilized all the solutions used in the experiment, even going as far as changing to a different source of distilled water, this netter was distraught to find that the contaminants continued to appear on his plates in further experiments and would not go away. Battle of the bulb ****************** Since all tubes and media used had been autoclaved, some netters immediately thought that the contamination must be coming from bacteria within the pipettes or the pipette bulb used for the aseptic transfer of the media, or that the micropipette tips had not been sterilized properly. It was also thought that, perhaps, DNA from a commonly used plasmid vector such as pBR322 could be present throughout the lab and could possibly be coating much of the laboratory glassware or benchtop, allowing another strain of bacteria to acquire it through transformation, and then this, somehow, made its way into one or more of the solutions. Initiated graduate students have heard the story about one PhD student who thought he had discovered a unique antibiotic-resistant plasmid within a Gram-negative bacterium that was not E. coli. This student went to tremendous efforts to sequence much of it, only to find that by running a BLAST comparison search, the sequence matched that of pBR322. It was only later that the newly acquired penicillin resistance factor was thought to have arisen from contaminating plasmid DNA left behind by previous students. It was pointed out by one person that the general methods used for microbiological sterilization would not destroy replicative plasmid DNA, which could be spread throughout molecular biology labs, possibly being harbored within pipettes or other equipment. Therefore, it was recommended that to rid the lab of contaminating DNA, it would be necessary to bake the pipettes at 180 degrees C for more than four hours, or to wash them for several minutes with 2 M HCl. Someone else suggested that all plastic micropipettes be thoroughly washed and autoclaved, and that aerosol-resistant pipette tips should be used to help keep the contamination to a minimum. Others added that the contaminating substance could not only be another species of bacterium, plasmid DNA or the like, but could even be a lysogenic bacteriophage that could alter the growth characteristics of E. coli. A waft of familiarity ********************* Some people describe having similar problems, saying that the smell of the bacterial colonies could be indicative of where the contaminant was coming from. They suggested that if the colonies smelled like old socks or dirty laundry they could be a species of Pseudomonas. Someone else mentioned that they once had contaminating colonies that grew extremely fast, were resistant to just about every antibiotic in the laboratory and smelled like a cheap detergent. This netter, who apparently was very accustomed to the smell of dirty laundry, also suspected Pseudomonas based on the growth rate. It was also thought that if the colonies were innoculated into broth, and grew as a bright green colored culture, then it is most likely P. fluorescens. If it smells like grape juice, however, it is probably P. aeruginosa. Both species are well known for their ability to grow in moist areas with minimal amounts of organic matter. The general consensus was that, on one hand, this netter had not skimmed his waterbath for a long time, and it had become overrun with bacteria. On the other hand, it was not clear how the bacteria could have gotten into the microfuge tubes while they were in the waterbath, unless the water seeped through the lip contacts, or somehow adhered to the sides of the tube and got into the plated material through handling. One person who had exactly the same problem took on the task of determining what the contaminating bacterium was by sending a sample out to a diagnostic microbiology lab. The bacteria were biochemically identified as P. fluorescens, and this netter could trace the source back to a single waterbath. After thorough cleaning and the addition of generous amounts of sodium azide, the problem went away. Other ways to keep your waterbath free from bacteria and fungi are to include antimicrobials such as chlorinating agents as used in swimming pools, or copper sulfate or cationic detergents. Although the exact source is still unknown, Mr Palmieri managed to rid himself of the contaminants and complete his cloning experiments. After washing out his pipettor with soap and water followed by rinsing with ethanol, and then extracting the ligation reactions with 1:1 phenol/chloroform and precipitating the DNA prior to transformation, the odd colonies disappeared. SYBR Green I passes the Ames test ********************************* In a previous Methods and reagents column (TiBS 19, 257-258), I described a relatively new dye, SYBR Green I (SGI), that can be used instead of ethidium bromide for staining nucleic acids within agarose or polyacrylamide gels. The choice between these two dyes continues to be debated in methods-reagnts. Although some netters are not convinced that ethidium bromide is as bad as some have made it out to be, an independent study supported by Molecular Probes (MP), the company that sells SYBR Green I, indicates that this more sensitive DNA stain might not be as harmful as ethidium bromide. MP now advertises SGI as being less mutagenic than ethidium bromide according to the Ames test for chemical mutagenicity. While the laboratory that conducted the study remains unknown, results are expected to be published soon showing that SGI produced an approximately twofold increase in histidine (+) revertants in Salmonella typhimurium strains TA98 and TA102. In the same assay, ethidium bromide produced an approximately 70-fold increase in strain TA98, 15-fold in TA1537, 4-fold in TA97a, 80-fold in TA 1538, and 2-fold in TA102 (the same as with SGI). No mutagenic activity was observed for SGI in strains TA100, TA1535, TA1537, TA1538, and TA97a, and no activity was found for ethidium bromide in strains TA100 or TA1535. It is still a matter of debate whether that reversion frequency in bacteria is enough to give some peace of mind, and whether this will outweigh all the added expense, as SGI has not dropped in price over the past year, still tipping the scales at US$200 per ml. Membrane SWAP ************* Molecular biology kit components are frequently improved upon, replaced or altogether discontinued. One problem occurs when biotechnology companies switch kit components and do little or nothing to inform researchers that some of the components have been changed. Not only does this cause the researcher to face an unexpected change in results, but often they have to re-adjust to new conditions only after some very precious materials have been lost. Scientists using kits are all too familiar with this type of switch, because it often happens with the undesirable side effect that previously published experimental results might not be reproducible under the conditions described in their publications. (for a more detailed discussion on the pitfalls of using molecular biology kits, see TiBS 19, 46-47). This month it was learned that within the past year some companies switched manufacturers of nylon-based transfer membranes, without effectively informing researchers, and leaving a lot of people with the sheets pulled out from under them. Recently, as part of a dispute over patent rights, companies that sell membranes as kit components were forced to seek an alternative supplier. For example, the Tropilon-Plus [TM] positively charged membrane by Tropix, supplied with their Southern-Light [TM] chemiluminescent detection kit, was switched and the newer membrane is still being marketed under the same name as the older one, causing some confusion. In addition to the switch, this company inadvertently continued their advertisements with literature developed with the older membrane, showing side-by-side comparisons of blots done with several brands of membranes. Netters call this kind of action a SWAP (switching without appropriate publicity). Upon hearing through bionet.molbio.methds-reagnts that their membrane SWAP was adversely affecting researchers and that their product was possibly misrepresented by comparisons made using the wrong membrane, Tropix quickly removed their product flyer and changed their catalog pages. Their swift response to this problem is to be commended. ******************************************************************************* 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/dec95.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1995. Methods and reagents - Floaties in the gene pool. Trends in Biochemical Sciences 20(12):522-523. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************