《黑胶虫污染》ppt课件

Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant,abstract,Cell culture model systems are utilized for their ease of use, relative inexpensiveness, and potentially limitless sample size. Reliable results cannot be obtained, however, when cultures contain contamination. This report discusses the observation and identification of mobile black specks移动的黑点observed in multiple cell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies分离菌. The 16S rDNA gene was PCR amplified using primers that will amplify the gene from many genera, and then sequenced. Sequencing results matched the members of the genus Achromobacter, bacteria common in the environment. Achromobacter species have been shown to be resistant to multiple antibiotics.Attempts to decontaminate the eukaryotic真核cell culture used multiple antibiotics at different concentrations. The contaminating chromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics ciprofloxacin and piperacillin.环丙沙星、哌拉西林,In the case of non-commercially经济 available lines, all attempts are usually taken to clean up the culture by selectively killing the contamination without harming the cell line. Often the culprits首因of culture contamination is mycoplasma支原体, intracellular bacteria that can be almost undetectable in culture. Approximately 28% of 460 human cell lines surveyed contained mycoplasma 1. Numerous commercial kits试剂盒are available to identify mycoplasma contamina- tion 2,3 and antibiotic solutions are commercially available to rid cultures of them. Additional culture contamination includes other bacteria, mold, and fungi,Unknown bacterial contamination can be transient. Contami- nation is common with poor aseptic technique and can be devas- tating in a research setting 3. While many undesired organisms may steal nutrients from cell lines in culture, they may also prey on the cells themselves. Predatory bacteria have been shown to feed on other bacteria 5,6, particularly in a limited nutrient environ- ment7. Experimental results mayalso be altered due to unwanted activation of cells. Different cellular functions, including those triggered by Toll-like receptors, can be activated by a variety of bacterial components. Several online science blogs discuss a cell culture contaminate that looks like black specks in the cell culture (http:/www. http:/www.proto col-online.org/forums/index.php?sbc4fecd655ee6898191ab6ed 43f5a5a7&showtopic9373&pid30730&st0). The researchers on these blogs describe black dots that fidget or swim and look more like rods than dots. Our laboratory has experienced sporadic cell culture contamination fitting this description. Here we report the identification of a bacterial cell culture contaminate, a member of the Achromobacter genus, matching anecdotal descriptions of cell culture contaminants of multiple eukaryotic cell lines. In addition, we provide information regarding antibiotic resistance and treatment effectiveness.,Antibiotics and doses tested to treat Achromobacter contaminated cells. Antibiotic Dose ( m g/ml) Gentamicin庆大5002000 Amikacin 502500 Tobramycin妥布霉素50600 Imipenem 264 Erythromycin 25500 Piperacillin哌拉西林0.51000 Choramphenicol氯霉素50500 Ciprofloxacin环丙沙星10750 Plasmocin 2562.5清除支原体污染,Antibiotics were added to the complete media which was replaced every 35 days with fresh media and antibiotics.,Achromobacter can survive and multiply in water and moist soil. Sources have been identified as untreated well water, swimming pools, dialysis fluids, distilled water, deionized water, tap water, disinfectants, water systems, ventilators, humidifiers, IV fluids, and infant formula,未经处理的水，游泳池、透析液、蒸馏水、去离子水、自来水，消毒剂、水系统、风机、加湿器、静脉输液，和婴儿配方奶粉,Initial attempts to lyse裂解the culture contamination were done in the eukaryotic cell culture supernatant. Boiling (510 min at 95 .C)and sonicating声波降解标本(1530 s at output power 4) did not result in available DNA for PCR. It is possible that the large amount of protein present in eukaryotic真核culture media, primarily from the FBS, could have protected the bacterial cells. DNA was finally isolated using a commercial kit from Promega from isolated colonies grown on blood agar琼脂.,The PCR products, about1400 base pairs in size, were isolated by ethanol乙醇precipitation沉淀and sent for sequencing. After the sequencing, the resulting sequences were checked for similarity to other known sequences using NCBIs BLAST and the Ribosomal Database Project (RDP). Sequence 1 shared 99% sequence identity with Alcaligenes and Achromobacter 16S rDNA gene sequences, as,Members of the Achromobacter and Alcaligenes genera have reclassified to and from these two genera, including Achromobacter (Alcaligenes) xylosoxidans and Achromobacter (Alca- ligenes) denitrificans 22,23. It is unclear whether the similarity to both members of the Achromobacter and Alcaligenes genera is due to uncertainty in the nomenclature of previously submitted sequences or due to the actual sequence of the contaminating bacteria. RDP, a web-based program containing only 16S rDNA sequences, was utilized to confirm the contaminating bacterias genus. Results using SeqMatch from the RDP identified the sequences as belonging to the genus Achromobacter. Phylogenetic trees based on the BLASTand RDP results grouped the bacteriawith predominantly Achromobacter 16S rDNA sequences (data not shown). Sequences 2, 3, and 4 share 99% sequence identity with sequence 1,Achromobacter 革兰氏阴性 ，氧化酶阳性，有鞭毛，在3742 C，pH为6.58.5生长良好 In our This contaminant was cultured from FBS and NCS, tap water, distilled water, the water bath (in the water and colony material on the metal), phospho-buffered saline, and tris-buffered saline. FBS and NCS were suspected as the primary, but not only, sources of contamination for several reasons. First, cells thawed from cryo- storage (stored in 95% FBS or NCS and 5% dimethyl sulfoxide), which had been previously uncontaminated, contained the bacteria once thawed解冻. Second, multiple sources of FBS and NCS appeared to be contaminated with the rod-like棒状motile structures, presumed to be bacteria, when samples were examined under a microscope. Third, this contaminant morphology and motility has been observed in cells obtained from other labs using NCS from the same source.,Cultures of Achromobacter were visualized using microscopy. Cultures were mixed with an equal volume of agarose and placed on a slide. Fig. 1A shows many bacterial cells while 1B shows,IMCE大肠腺瘤, YAMC小鼠结肠上皮, MC38小鼠结肠癌,小鼠胚胎成纤维3T3-L1,individual cells. Cells were approximately 45 m m and were rod-Shaped.One member of the genus, Achromobacter xylosoxidans木糖has been found to be the causative agent病原体in multiple cases of septicemia败血症, mainly in immunocompromised individuals, and sometimes due to contaminated medical supplies 2530. Achromobacter is becoming a serious threat to the health of individuals with cystic fibrosis囊性纤维化.,Eukaryotic cells were killed at various concentrations of antibi- otics, depending on cell type. For example, a ciprofloxacin concentration greater than 60 m g/ml killed both MC38 and YAMC cells. A combination of two antibiotics, piperacillin and cipro- floxacin哌拉西林、环丙沙星, at a concentration of 10 m g/ml each, was best at selec-tively killing the Achromobacter contaminant without appearing to harm the eukaryotic cells. Achromobacter have been previously been shown to be susceptible to antibiotics administered together. A. xylosoxidans is resistant to ciprofloxacin, but susceptible to a combination of piperacillin and tazobactam,Inthis study, isolation of an unknown contaminantwas revealed as member of the genus Achromobacter. The bacteria in this genus can survive in a wide range of environments including tap water, the water bath, FBS, NCS, and many common laboratory solutions. In addition, commercial cells were found to contain this contami- nant. It is important that researchers utilizing cell culture for experiments become aware of this multi-drug resistant bacterial contaminant. Selective killing of these bacteria in eukaryotic cell culture was successful only with a combination of two antibiotics, piperacillin and ciprofloxacin, both at a concentration of 10 m g/ml each. This report presents the novel identification of this cell culture contaminant and a possible way to remove the swimming black dots from cell culture.,