作者感谢教授保罗COS和LMPH，安特卫普大学，比利时期间，在他的实验室拍摄过程中他的支持。这项工作是由芬兰工程学院（项目272266和282981）和瑞典技术科学院芬兰资助。硕士Janni Kujala的技术贡献得到承认。

1.成长的细菌<ol><li>预培养物在37℃下在胰蛋白酶大豆肉汤（TSB）中的细菌过夜以220rpm振荡（16-18小时）。 </li><li>稀释预培养100-1,000倍（取决于细菌的生长速率;在这里，1000倍，用于金黄色葡萄球菌 ）的新鲜TSB，让它生长在37℃和200rpm下达到指数生长（光密度在0.2和0.6）之间的595纳米（OD 595）。 注:此步骤需要特定应变优化。 </li></ol> 2.生物膜的形成:前和暴露后<ol><li>稀释成倍增长文化的100倍（这相当于每毫升约10 6个菌落形成单位（CFU /毫升））。 </li><li> 预曝光协议 <ol><li>对于未经处理的对照样品，加入200微升每孔稀释细菌培养无菌96微孔板的。 </li><li>添加测试化合物或对照的4微升抗生素（50X原液）和196微升每孔稀释细菌培养。 </li><li>在37℃下以200rpm搅拌18小时孵育它在板振荡器上。 注意:在这里我们使用2毫米的轨道振动筛。 </li></ol></li><li> 暴露后协议 <ol><li>对于所有的样品，加入200微升每孔稀释细菌培养无菌96微孔板的。 </li><li>在37℃下以200rpm搅拌18小时孵育它在板振荡器上。 注意:在这里我们使用2毫米的轨道振动筛。 </li><li>小心地取出整个浮游的解决方案，而不触及生物膜，使用多道移液器。 </li><li>添加4微升测试化合物或控制抗生素（50X原液），并每孔196微升TSB的。 </li><li>在37℃下孵育其上以200rpm板振荡器另外24小时。 注意:在这里我们使用2毫米的轨道振动筛。 </li></ol></li></ol> 3.刃天青染色Protocol对于生物膜的可行性分析<ol><li>制备的在无菌PBS 0.1毫克/毫升刃天青（0.4毫摩尔）的储备溶液。保持这个股票不育，从曝光的保护，并在4℃。 </li><li>稀释刃天青库存1:50在无菌磷酸盐缓冲盐水（PBS），以实现20微米的最终浓度。 </li><li>传送整个浮游液（离开生物膜在孔中）小心，不接触生物膜或产生气泡，要使用多道移液器一个单独的，干净的96孔板中。 </li><li>通过加入每孔200μl的用无菌PBS洗涤生物膜一次，并小心地用多道移液器将其删除。 </li><li>加入200μl每使用多道移液器生物膜板以及稀释刃天青。 </li><li>孵育它在黑暗中，在室温（RT），并以200rpm，约20分钟，振摇直到未处理的生物膜控制是均匀的粉红色。 </li><li>测量在荧光λEXC = 560纳米和λEM =用酶标仪（顶端读数）590纳米。 </li></ol> 4.结晶紫染色协议生物膜生物量定量使用同一板块如步骤3 <ol><li>使用多道移液器井小心取出刃天青染色。 </li><li>固定用200μl甲醇的生物膜15分钟。 </li><li>让板空气干燥10分钟。 </li><li>加入190微升0.02％（体积/体积，在去离子水稀释）龙胆紫液，小心使用多道移液器。避免与同时移液污渍接触孔的侧面和防止空气气泡的形成不按移液器完成吹出。 </li><li>孵育它在RT 5分钟。 </li><li>小心取出色斑，使用多道移液器。 </li><li>用去离子水（200每次微升）洗两次。 </li><li>让各孔在RT干燥5分钟，溶解剩余的污点在96％乙醇或33％乙酸。 </li><li>孵育它在RT 1小时，并在595nm处读取吸光度。 </li></ol> 5.浮游菌评价杀菌效果使用相同的样品板作为生物膜<ol><li>测量在含有从3.3浮游溶液板的595nm处的浊度。 </li><li>加入10微升刃天青股票，每孔用染色的刃天青浮游细菌。吹打拌匀。 </li><li>它孵育在黑暗中在室温约23分钟，直到被处理控件是均匀的粉红色。 </li><li>在测量EXCλ= 560nm的荧光，λ= EM 590纳米。 </li></ol> 6.麦胚凝集素染色矩阵量化和生物膜的荧光显微成像<ol><li> 量化矩阵 <ol><li>制备在无菌PBS WGA的探针的5微克/毫升的溶液。 </li><li>使用并行样品解放军忒这个染色。除去从孔浮游溶液并用无菌PBS（每孔200微升）洗一次，小心用多道移液器而不触及生物膜。 </li><li>加入200μl的WGA解决每个被染色良好。 </li><li>它孵育在黑暗中在4℃下2小时。 </li><li>通过用200μl的PBS清洗3次洗涤各孔除去未结合的污点。 </li><li>让板干燥在室温15分钟。 </li><li>溶解在33％乙酸结合的染色，使用每孔200μl的。 </li><li>密封该孔与带状帽和使用水浴超声波仪在室温30秒和40千赫超声处理它们。 </li><li>将培养板在RT 1小时。 </li><li>重复超声步骤。井可以在超声处理步骤之间保持密封。 </li><li>测量在λEX = 495nm处的荧光，λEM =用酶标仪（顶端读数）520纳米。 </li></ol></li><li> 可视化与基质荧光显微镜<ol><li>使用相同的协议，上面直到步骤6.1.6。 </li><li>在干燥步骤之后，可视化的样品用荧光显微镜，使用FITC标记的过滤器（或其它合适的绿色激发过滤器）。 </li></ol></li></ol>在生物膜内7.染色活细胞和死细胞与荧光显微镜和信号的定量绿色到红色比（G / R）成像<ol><li> 成像与荧光显微镜 <ol><li>制备各探针（一个染色的活细胞，另一种染色死细胞）的溶液中，根据制造商的指导方针。 </li><li>除去从孔浮游溶液，并用无菌PBS洗涤一次（每孔200微升）用多道移液器。 </li><li>加入6微升每孔的染色溶液中。 </li><li>孵育在黑暗中板15分钟。 </li><li>在显微镜之前，除去多余的液态间全线采用多道移液器。 </li><li>捕获使用荧光显微镜图像，使用例如FITC过滤器（绿色荧光，活细胞）或TRITC滤波器（红色荧光，死细胞）。 </li></ol></li><li> 信号作为绿色到红色荧光比的定量（G / R） <ol><li>按照相同的协议，在步骤7.1.1和7.1.2。 </li><li>加入200μl每孔的染色溶液的孵育它们在黑暗中15分钟。 </li><li>测量与激发/发射波长为绿色和红色荧光535分之485nm和635分之485，分别为（顶端读数）读板器的荧光。 </li></ol></li></ol>

<ol>
	<li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Dufour, D., Leung, V., L&#233;vesque, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilm:+structure,+fuction,+and+antimicrobial+resistance.">Bacterial biofilm: structure, fuction, and antimicrobial resistance.</a> Endodontic Topics. 22, 2-16 (2012).</li><li>Donne, J., Dewilde, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenging+World+of+Biofilm+Physiology.">The Challenging World of Biofilm Physiology.</a> Adv. Microb. Physiol. 67, 235-292 (2015).</li><li>Otto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+infections:+mechanisms+of+biofilm+maturation+and+detachment+as+critical+determinants+of+pathogenicity.">Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity.</a> Annu. Rev. Med. 64, 175-188 (2013).</li><li>Cos, P., Tote, K., Horemans, T., Maes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+an+extra+hurdle+for+effective+antimicrobial.">Biofilms: an extra hurdle for effective antimicrobial.</a> Curr. Pharm. Des. 16, 2279-2295 (2010).</li><li>Bjarnsholt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+biofilm.">The in vivo biofilm.</a> Trends Microbiol. 21, 466-474 (2013).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persister+cells.">Persister cells.</a> Annu. Rev. Microbiol. 64, 357-372 (2010).</li><li>Mulcahy, L. R., Burns, J. L., Lory, S., Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+Pseudomonas+aeruginosa+strains+producing+high+levels+of+persister+cells+in+patients+with+cystic+fibrosis.">Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis.</a> J. Bacteriol. 192, 6191-6199 (2010).</li><li>Buckingham-Meyer, K., Goeres, D. M., Hamilton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+biofilm+disinfectant+efficacy+tests.">Comparative evaluation of biofilm disinfectant efficacy tests.</a> J. Microbiol. Methods. 70, 236-244 (2007).</li><li>Goeres, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+growing+a+biofilm+under+low+shear+at+the+air-liquid+interface+using+the+drip+flow+biofilm+reactor.">A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.</a> Nat. Protoc. 4, 783-788 (2009).</li><li>Goeres, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+assessment+of+a+laboratory+method+for+growing+biofilms.">Statistical assessment of a laboratory method for growing biofilms.</a> Microbiology. 151, 757-762 (2005).</li><li>Parker, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruggedness+and+reproducibility+of+the+MBEC+biofilm+disinfectant+efficacy+test.">Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test.</a> J. Microbiol. Methods. 102, 55-64 (2014).</li><li>Ceri, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Calgary+Biofilm+Device:+new+technology+for+rapid+determination+of+antibiotic+susceptibilities+of+bacterial+biofilms.">The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.</a> J. Clin. Microbiol. 37, 1771-1776 (1999).</li><li>Harrison, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+susceptibility+testing+of+microbes+growing+on+peg+lids:+a+miniaturized+biofilm+model+for+high-throughput+screening.">Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening.</a> Nat. Protoc. 5, 1236-1254 (2010).</li><li>Konrat, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bead+Assay+for+Biofilms:+A+Quick,+Easy+and+Robust+Method+for+Testing+Disinfectants.">The Bead Assay for Biofilms: A Quick, Easy and Robust Method for Testing Disinfectants.</a> PLoS One. 11, e0157663 (2016).</li><li>Cragg, G. M., Newman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+products:+A+continuing+source+of+novel+drug+leads.">Natural products: A continuing source of novel drug leads.</a> Biochim Biophys Acta. 1830, 3670-3695 (2013).</li><li>Sandberg, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pros+and+cons+of+using+resazurin+staining+for+quantification+of+viable+Staphylococcus+aureus+biofilms+in+a+screening+assay.">Pros and cons of using resazurin staining for quantification of viable Staphylococcus aureus biofilms in a screening assay.</a> J. Microbiol. Methods. 78, 104-106 (2009).</li><li>Mariscal, A., Lopez-Gigosos, R., Carnero-Varo, M., Fernandez-Crehuet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+assay+based+on+resazurin+for+detection+of+activity+of+disinfectants+against+bacterial+biofilm.">Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm.</a> Appl. Microbiol. Biotechnol. 82, 773-783 (2009).</li><li>Peeters, E., Nelis, H., Coenye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+multiple+methods+for+quantification+of+microbial+biofilms+grown+in+microtiter+plates.">Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.</a> J. Microbiol. Methods. 72, 157-165 (2008).</li><li>Pettit, R., Weber, C., Pettit, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+high+throughput+Alamar+blue+biofilm+susceptibility+assay+to+Staphylococcus+aureus+biofilms.">Application of a high throughput Alamar blue biofilm susceptibility assay to Staphylococcus aureus biofilms.</a> Ann. Clin. Microbiol. Antimicrob. 8 (28), (2009).</li><li>Stepanovic, S., Vukovic, D., Dakic, I., Savic, B., Svabic-Vlahovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+microtiter-plate+test+for+quantification+of+staphylococcal+biofilm+formation.">A modified microtiter-plate test for quantification of staphylococcal biofilm formation.</a> J. Microbiol. Methods. 40, 175-179 (2000).</li><li>Stiefel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+biofilm+removal+properly+assessed?+Comparison+of+different+quantification+methods+in+a+96-well+plate+system.">Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.</a> Appl. Microbiol. Biotechnol. , (2016).</li><li>Sandberg, M., M&#228;&#228;tt&#228;nen, A., Peltonen, J., Vuorela, P. M., Fallarero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automating+a+96-well+microtitre+plate+model+for+Staphylococcus+aureus+biofilms:+an+approach+to+screening+of+natural+antimicrobial+compounds.">Automating a 96-well microtitre plate model for Staphylococcus aureus biofilms: an approach to screening of natural antimicrobial compounds.</a> Int. J. Antimicrob. Agents. 32, 233-240 (2008).</li><li>Thomas, V. L., Sanford, B. A., Moreno, R., Ramsay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+lectinsorbent+assay+measures+N-acetyl-D-glucosamine+in+matrix+of+biofilm+produced+by+Staphylococcus+epidermidis.">Enzyme-linked lectinsorbent assay measures N-acetyl-D-glucosamine in matrix of biofilm produced by Staphylococcus epidermidis.</a> Curr. Microbiol. 35, 249-254 (1997).</li><li>Burton, E., Yakandawala, N., LoVetri, K., Madhyastha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microplate+spectrofluorometric+assay+for+bacterial+biofilms.">A microplate spectrofluorometric assay for bacterial biofilms.</a> J. Ind. Microbiol. Biotechnol. 34, 1-4 (2007).</li><li>Skogman, M. E., Vuorela, P. M., Fallarero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+biofilm+matrix+measurements+with+biomass+and+viability+assays+in+susceptibility+assessments+of+antimicrobials+against+Staphylococcus+aureus+biofilms.">Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms.</a> J. Antibiot. Tokyo. 65, 453-459 (2012).</li><li>Skogman, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antibacterial+and+anti-biofilm+activities+of+cinchona+alkaloid+derivatives+against+Staphylococcus+aureus).">Evaluation of antibacterial and anti-biofilm activities of cinchona alkaloid derivatives against Staphylococcus aureus).</a> Nat. Prod. Commun. 7, 1173-1176 (2012).</li><li>Fallarero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(+)-+Dehydroabietic+Acid,+an+Abietane-Type+Diterpene,+Inhibits+Staphylococcus+aureus+Biofilms+in+Vitro.">(+)- Dehydroabietic Acid, an Abietane-Type Diterpene, Inhibits Staphylococcus aureus Biofilms in Vitro.</a> Int J Mol Sci. 14, 12054-12072 (2013).</li><li>Manner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+derivatives+of+dehydroabietic+acid+target+planktonic+and+biofilm+bacteria+in+Staphylococcusaureus+and+effectively+disrupt+bacterial+membrane+integrity.">New derivatives of dehydroabietic acid target planktonic and biofilm bacteria in Staphylococcusaureus and effectively disrupt bacterial membrane integrity.</a> Eur. J. Med. Chem. 102, 68-79 (2015).</li><li>Muller, G., Kramer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+index+of+antiseptic+agents+by+parallel+assessment+of+antimicrobial+activity+and+cellular+cytotoxicity.">Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity.</a> J. Antimicrob. Chemother. 61, 1281-1287 (2008).</li><li>Tot&#233;, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+efficacy+of+various+antibiotics+on+matrix+and+viable+mass+of+Staphylococcus+aureus+and+Pseudomonas+aeruginosa+biofilms.">Inhibitory efficacy of various antibiotics on matrix and viable mass of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.</a> Int. J. Antimicrob. Agents. 33, 525-531 (2009).</li><li>Djordjevic, D., Wiedmann, M., McLandsborough, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+plate+assay+for+assessment+of+Listeria+monocytogenes+biofilm+formation.">Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation.</a> Appl. Environ. Microbiol. 68, 2950-2958 (2002).</li><li>Li, X., Yan, Z., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+variation+of+biofilms+among+strains+in+natural+populations+of+Candida+albicans.">Quantitative variation of biofilms among strains in natural populations of Candida albicans.</a> Microbiolog. 149, 353-362 (2003).</li><li>Barbosa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+and+simple+micro+assay+for+sulfated+glycosaminoglycans+quantification+in+biological+extracts+and+its+use+in+skin+and+muscle+tissue+studies.">Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies.</a> Glycobiology. 13, 647-653 (2003).</li><li>Tot&#233;, K., Vanden Berghe, D., Maes, L., Cos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+colorimetric+microtitre+model+for+the+detection+of+Staphylococcus+aureus+biofilms.">A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms.</a> Lett. Appl. Microbiol. 46, 249-254 (2008).</li><li>Arciola, C. R., Campoccia, D., Ravaioli, S., Montanaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+intercellular+adhesin+in+biofilm:+structural+and+regulatory+aspects.">Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects.</a> Front Cell Infect Microbiol. 5 (7), (2015).</li><li>Ausbacher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+biofilm+susceptibility+to+small+and+potent+beta(2,2)-amino+acid+derivatives.">Staphylococcus aureus biofilm susceptibility to small and potent beta(2,2)-amino acid derivatives.</a> Biofouling. 30, 81-93 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

没有可以同时测量上的可行性，生物质，和生物膜基质的化合物的效果单一方法。因此，有必要在为了检测在三个端点的效果，优选在初次筛选阶段结合测定。 刃天青是仅由加入氧化还原探针的一个非常简单的染色方案。然而，建立与刃天青生物膜的最佳温育时间是该测定的成功是至关重要的。在某些细菌菌株，刃天青探针的粉红色，荧光试卤灵的减少发生得非常快，而在?...

细菌可以两种非常不同的生活方式，浮游和固着，其中生物膜是最常见的例子之间进行切换。在生物膜，细菌形成嵌入在自产矩阵1结构化的社区。此自产矩阵是细菌，它们的外部环境之间的屏障，它保护了的微生物细胞，使它们在接近。生物膜基质的组合物之间，甚至在物种而异，但它主要是由脂多糖，胞外DNA和蛋白质的紧密网。基质用作物理屏障抑制有害剂的入口，但它也可以防止脱水生物膜并防止营养物质逸出的小区2。 生物膜被视为现代生物医学的最具挑战性的课题之一，它们是对抗生素耐受感染3超过80％的据称负责。他们disp已铺设针对外部威胁的固有的高耐受性:湿度，渗透压，机械应力4，热，紫外线辐射5，消毒剂，抗菌剂，和宿主免疫系统1。例如，为了杀死生物膜所需的所需的抗微生物剂的浓度已显示相比较，以杀死浮游细菌所需的要高的1000倍。此更高公差的解释似乎是多因素的。基质提供了降低抗生素渗透到生物膜的物理屏障。另外，生物膜内的细胞表型多样;他们由于氧，营养物，和生物膜6的内和外部分之间的代谢物的现有梯度不同的代谢状态之间转变。因此，在一些生物膜地区，如核心，细菌被剥夺氧气和营养，生活在一个代谢活性较低或完全休眠状态<suP&gt; 7。完全休眠细胞被称为持留细胞，它们是不易于常规抗微生物治疗8。因此，它很可能是生物膜的韧性源自的当前建议等，未知，机制的组合。 葡萄球菌属。仍然是最有问题的革兰氏阳性菌中，引起严重的，通常生物膜相关的，感染4。有人建议，所有的细菌高达99％的生物膜内的关联，使得它的主要细菌生活方式3。但是，所有的现有的抗生素已经开发针对单小区（浮游）细菌。迄今，分子的一个非常有限的剧目存在可在成熟生物膜选择性作用。这种情况已经在推动药物发现一个渐进的转变，其中的抗生物膜搜索已敦促占据了更加突出的位置。 从methodologiCal中的角度来看，额外的挑战存在，因为只有生物膜的方法有限数目已经由标准制定组织，特别是那些适用于化学文库的高通量筛选开发的。所有的标准化测定（只有一个例外）都基于生物膜反应器中，并且这些方法需要大量的工作容积和大量的化合物进行测试，这通常是在早期研究性阶段9-12不可用。现有的唯一标准筛选适用的测定法是所谓的卡尔加里生物膜的装置，从该商购的最小生物膜消除浓度（MBEC）系统的开发13-15。然而，该测定的限制是该生物膜生长在钉，并且不是所有的细菌物种或同一物种内甚至菌株能够形成这个装置上生物膜。而且，可以特别适用于天然化合物的探索方法必要的。天然产品已经在抗菌药物发现在过去的16世纪创新的主要来源。它们可提供具有独特的作用机制也可以是针对持留细胞有效新颖抗生物化合物。因此，自然和自然的启发库的探索有前途的生产和独特的抗生物引线的高机会。 这里，我们提出测定的一个平台，是为使用三个测定法来测量上的可行性，总生物量，和金黄色葡萄球菌生物膜基质的影响抗生物化合物的化学筛选开发的实验细节。第一测定测量生物膜的可行性，并且它是基于刃天青染色。刃天青是氧化还原染色是蓝色和非荧光在其氧化态和由细菌的代谢活性降低时变为粉红色，高荧光试卤灵。这是一个非常简单和快速米ethod适合初级筛查17-20。第二个实验中，根据结晶紫染色，测量总质量的生物膜。结晶紫为在生物膜19,21-23研究细菌和细菌广泛使用的污点。该测定法是基于廉价的试剂和具有简单的吸光度端点读数。最后，第三个测定目标通过小麦胚凝集素生物膜（WGA），其特异性结合聚-N-乙酰葡糖胺残基（PNAG）存在于葡萄球菌生物膜24的基体的胞外聚合物物质（EPS） -矩阵。 WGA的结合有可使用荧光强度读者25被检测的荧光团。我们在座的基本原理和我们开发，包括示例应用程序的平台的详细信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Resazurin</td>
			<td>Sigma Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>HT90132&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>W11261</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD BacLight&#160;</td>
			<td>Molecular Probes</td>
			<td>L7012</td>
			<td>SYTO 9 for staining viable cells green and propidium iodide for staining dead cells red</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone soy agar</td>
			<td>Lab M, Neogen</td>
			<td>LAB011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptine soy broth</td>
			<td>Lab M, Neogen</td>
			<td>LAB004</td>
			<td></td>
		</tr>
		<tr>
			<td>F96 Well Plate Polystyrene Sterile Clear Flat bottom</td>
			<td>Thermo Fisher Scientific</td>
			<td>161093</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND caps, strips of 8</td>
			<td>Sigma Aldrich</td>
			<td>BR781413-300EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson CPX series ultrasonic bath</td>
			<td>Sigma Aldrich</td>
			<td>Z769428-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipipette</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multidrop dispenser</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biomek 3000</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan Flash Multiplate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC&#160;</td>
			<td>25923</td>
			<td></td>
		</tr>
	</tbody>
</table>

a platform of anti-biofilm assays suited to the exploration of natural compound libraries

生物膜被视为现代生物医学的最具挑战性的课题之一，它们是潜在的负责抗生素耐受性感染的80％以上。生物膜已显示化疗极高的耐受性，这被认为是多因素的。例如，基体提供了降低抗生素渗透到生物膜的物理屏障。另外，生物膜内的细胞的表型多样性。可能的是，生物膜韧性源于这些和其他，未知，机制的组合。所有的现有的抗生素已经开发针对单细胞（浮游）细菌。因此，到目前为止，分子的一个非常有限的剧目存在可在成熟生物膜选择性作用。这种情况已经在推动药物发现一个渐进的转变，其中的抗生物膜搜索已敦促占据了更加突出的位置。一个额外的挑战是，有一非常有限的生物膜研究标准化方法，尤其是那些可用于化学文库的大通量筛选号码。在这里，药剂筛选试验的抗生物平台提出。在聚-N-乙酰氨基葡萄糖，PNAG的它采用三个实验使用小麦胚芽凝集素测量生物膜活力（带刃天青染色），总生物量（用结晶紫染色），和生物膜矩阵（，WGA-荧光染色基础， 分数）。所有的测定用金黄色葡萄球菌作为模型细菌开发的。的平台如何可以用于初步筛选以及用于鉴定抗生物命中功能表征实例。这个实验序列还允许根据测量的终点命中的分类。它还提供了对他们的行动模式的信息，特别是在长期和短期化疗的效果。因此，它是非常ADVAntageous用于快速识别，可以作为起点关于各种生物医学应用的高质量的命中化合物。

在所提出的平台上的可行性，生物质，和生物膜基质的影响量化。在工作序列（ 图1），一个样品板沾有刃天青，随后用结晶紫同时评价对细菌生物膜存活和总生物膜的生物量的影响。这两个实验可以连续在同一个板块，因为它被证明早些时候与刃天青第一染色对结晶紫染色结果（P = 0.4149无统计学显著影响水晶紫激光印版之间最大的信号吸光度单位的?...

Watch this Scientific Journal Video about A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries at JoVE.com

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

a-platform-anti-biofilm-assays-suited-to-exploration-natural-compound

Research

JoVE Journal

Immunology and Infection

17.5K 次观看.  University of Helsinki. Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects. 

视频: A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

科研

免疫与感染

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...