Name: evaluation of the efficacy of organic peroxyacids for eradicating dairy biofilms using an approach combining static and dynamic methods
Uploaded: 2022-12-09T13:50:00
Description: Watch this Scientific Journal Video about Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods at JoVE.com

这项研究得到了魁北克生物工业研究与创新联盟（CRIBIQ）（2016-049-C22），Agropur，Groupe Sani Marc和加拿大自然科学与工程研究委员会（NSERC）（RDCPJ516460-17）的支持。我们感谢Teresa Paniconi对手稿的批判性审查。

本文所载的工作需要一个生物安全2级实验室，并已获得拉瓦尔大学机构生物安全委员会的批准（项目编号119689）。
注意：图1中的流程 图 总结了结合静态和动态方法的方法，该方法用于评估有机过氧酸根除生物膜的功效。
1. 材料准备
<ol><li>微生物分离物<ol><li>使用前15分钟打开生物安全柜（BSC），并用70%（v / v）酒精?...

<ol>
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O., 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=Efficiency+of+different+disinfectants+on+Bacillus+cereus+sensu+stricto+biofilms+on+stainless-steel+surfaces+in+contact+with+milk.">Efficiency of different disinfectants on Bacillus cereus sensu stricto biofilms on stainless-steel surfaces in contact with milk.</a> Frontiers in Microbiology. 9, 2934 (2018).</li><li>Sedlak, D. L., von Gunten, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry.+The+chlorine+dilemma.">Chemistry. The chlorine dilemma.</a> Science. 331 (6013), 42-43 (2011).</li><li>vander Veen, S., Abee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+species+biofilms+of+Listeria+monocytogenes+and+Lactobacillus+plantarum+show+enhanced+resistance+to+benzalkonium+chloride+and+peracetic+acid.">Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid.</a> International Journal of Food Microbiology. 144 (3), 421-431 (2011).</li><li>Saa Ibusquiza, P., Herrera, J. J., Cabo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+benzalkonium+chloride,+peracetic+acid+and+nisin+during+formation+of+mature+biofilms+by+Listeria+monocytogenes.">Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes.</a> Food Microbiology. 28 (3), 418-425 (2011).</li><li>Goetz, C., Larouche, J., Velez Aristizabal, M., Niboucha, N., Jean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+organic+peroxyacids+for+eliminating+biofilm+preformed+by+microorganisms+isolated+from+dairy+processing+plants.">Efficacy of organic peroxyacids for eliminating biofilm preformed by microorganisms isolated from dairy processing plants.</a> Applied and Environmental Microbiology. 88 (4), 0188921 (2022).</li><li>Vimont, A., Fliss, I., Jean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+virucidal+potential+of+organic+peroxyacids+against+norovirus+on+food-contact+surfaces.">Study of the virucidal potential of organic peroxyacids against norovirus on food-contact surfaces.</a> Food and Environmental Virology. 7 (1), 49-57 (2015).</li><li>ASTM E2562-17. Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor. ASTM International Available from: <a target="_blank" href="https://www.astm.org/e2562-17.html">https://www.astm.org/e2562-17.html</a> (2017)</li><li>ASTM E2871-19. Standard Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method. ASTM International Available from: <a target="_blank" href="https://www.astm.org/e2871-19.html">https://www.astm.org/e2871-19.html</a> (2019)</li><li>Niboucha, N., 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=Comparative+study+of+different+sampling+methods+of+biofilm+formed+on+stainless-steel+surfaces+in+a+CDC+biofilm+reactor.">Comparative study of different sampling methods of biofilm formed on stainless-steel surfaces in a CDC biofilm reactor.</a> Frontiers in Microbiology. 13, 892181 (2022).</li><li>ASTM E2799-17. Standard Test Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay. ASTM International Available from: <a target="_blank" href="https://www.astm.org/e2799-17.html">https://www.astm.org/e2799-17.html</a> (2022)</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> Journal of Microbiological Methods. 102, 55-64 (2014).</li><li>Stewart, P. S., Parker, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+antimicrobial+efficacy+against+biofilms:+A+meta-analysis.">Measuring antimicrobial efficacy against biofilms: A meta-analysis.</a> Antimicrobial Agents and Chemotherapy. 63 (5), 00020 (2019).</li><li>Lindsay, D. K., Fouhy, K., Loh, M., Malakar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CDC+biofilm+bioreactor+is+a+suitable+method+to+grow+biofilms,+and+test+their+sanitiser+susceptibilities,+in+the+dairy+context.">The CDC biofilm bioreactor is a suitable method to grow biofilms, and test their sanitiser susceptibilities, in the dairy context.</a> International Dairy Journal. 126, 105264 (2022).</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> Journal of Microbiological Methods. 70 (2), 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=Statistical+assessment+of+a+laboratory+method+for+growing+biofilms.">Statistical assessment of a laboratory method for growing biofilms.</a> Microbiology (Reading). 151, 757-762 (2005).</li><li>Williams, D. L., Woodbury, K. L., Haymond, B. S., Parker, A. E., Bloebaum, R. D. <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+CDC+biofilm+reactor+to+produce+mature+biofilms+on+the+surface+of+peek+membranes+for+an+in+vivo+animal+model+application.">A modified CDC biofilm reactor to produce mature biofilms on the surface of peek membranes for an in vivo animal model application.</a> Current Microbiology. 62 (6), 1657-1663 (2011).</li><li>Pieranski, M. K., Rychlowski, M., Grinholc, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Streptococcus+agalactiae+biofilm+culture+in+a+continuous+flow+system+for+photoinactivation+studies.">Optimization of Streptococcus agalactiae biofilm culture in a continuous flow system for photoinactivation studies.</a> Pathogens. 10 (9), 1212 (2021).</li><li>Mendez, E., Walker, D. K., Vipham, J., Trinetta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+a+CDC+biofilm+reactor+to+grow+multi-strain+Listeria+monocytogenes+biofilm.">The use of a CDC biofilm reactor to grow multi-strain Listeria monocytogenes biofilm.</a> Food Microbiology. 92, 103592 (2020).</li><li>Salgar-Chaparro, S. J., Lepkova, K., Pojtanabuntoeng, T., Darwin, A., Machuca, L. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+during+hexadecane+degradation+and+the+effects+of+flow+field+and+shear+stresses.">Biofilm formation during hexadecane degradation and the effects of flow field and shear stresses.</a> Annual Transactions - The Nordic Rheology Society. 21, 341-346 (2013).</li><li>Gilmore, B. F., Hamill, T. M., Jones, D. S., Gorman, S. 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MBEC测定（生物膜微孔板测定）是第一种被ASTM17认可为标准生物膜根除测试的方法。我们的研究和其他研究表明，使用该测定时有两个关键步骤：超声处理步骤（时间和功率）和消毒剂处理时间18。斯图尔特和帕克还提出了可能影响测定结果的其他参数，例如微生物种类，生物膜年龄，表面积/体积比等。19. 最后，重要的是要注意，微孔板盖上?...

乳制品行业是全球主要工业部门，包括在加拿大，那里有超过 10，500 个奶牛场，每年生产近 9000 万升牛奶1。尽管乳制品行业（包括加工厂）有严格的卫生要求，但牛奶是微生物的重要培养基，因此，乳制品可能含有微生物，包括腐败或病原微生物。这些病原体可引起各种疾病;例如，沙门氏菌属和单核细胞增生李斯特菌可分别引起胃肠炎和脑膜炎2。腐败微生物可以通过产生气体、细胞外酶或酸来影响乳制品的质量和感官特性3.牛奶的外观和颜色也可能改变，例如通过假单胞菌spp.4。
其中一些微生物可以在不同的表面上形成生物膜，包括不锈钢。这种生物膜使微生物能够在设备表面持续存在和繁殖，从而污染乳制品5。生物膜也存在问题，因为它们能够阻碍传热并加速设备的腐蚀，导致设备过早更换，从而导致经济损失6。
原位清洗（CIP）程序使食品工业能够控制微生物的生长。这些程序涉及依次使用氢氧化钠、硝酸，有时还包括含有次氯酸和过氧乙酸7，8 的消毒剂。次氯酸虽然对微生物非常有效，但它也会与天然有机物发生反应，导致有毒副产物的形成9。过氧乙酸不产生有害副产物10;然而，它在食品工业中对生物膜的有效性变化很大10，11。最近，已经研究了其他过氧酸，包括过丙酸和过乳酸的抗菌活性，它们似乎是控制生物膜中微生物生长的良好替代品12，13。
因此，本研究旨在评估有机过氧酸（过氧乙酸、过丙酸和过乳酸以及基于过氧乙酸的消毒剂）根除乳品生物膜的功效，该方法结合了最小生物膜根除浓度 （MBEC） 测定、静态高通量筛选方法和美国疾病控制中心 （CDC） 生物膜反应器（一种模拟原 位 的动态方法 条件。MBEC测定在协议中以下称为“生物膜微量滴定板”。这里介绍的协议和代表性结果证明了有机过氧酸的功效及其在控制乳制品行业中微生物生物膜的潜在应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >0.2 &#181;m filters&#160;</td>
			<td >Corning</td>
			<td >09-754-28</td>
			<td >diameter: 50 mm, PTFE- Membrane</td>
		</tr>
		<tr >
			<td >316 stainless-steel disc coupon</td>
			<td >Biosurface Technologies Corporation</td>
			<td >RD128-316</td>
			<td></td>
		</tr>
		<tr >
			<td >316 stainless-steel slide coupon</td>
			<td >Biosurface Technologies Corporation</td>
			<td >CBR 2128-316</td>
			<td></td>
		</tr>
		<tr >
			<td >96-microtiter plate</td>
			<td >Corning</td>
			<td >07-200-89</td>
			<td>cell Culture-Treated, flat-Bottom Microplate&#160;</td>
		</tr>
		<tr >
			<td >Acetic acid</td>
			<td >Sigma Aldrich</td>
			<td >27225</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Aluminium stubs</td>
			<td >Electron Microscopy Science</td>
			<td >75830-10</td>
			<td>32x5mm</td>
		</tr>
		<tr >
			<td >Aqueous glutaraldehyde EM Grade 25%</td>
			<td >Electron Microscopy Sciences</td>
			<td >16220</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr >
			<td >AB204-S/FACT Analytical balance</td>
			<td >Mettler Toledo</td>
			<td >AB204-S</td>
			<td></td>
		</tr>
		<tr >
			<td >Bacterial Vent Filter (0.45 &#181;m)</td>
			<td >Biosurface Technologies Corporation</td>
			<td >BST 02915</td>
			<td></td>
		</tr>
		<tr >
			<td >BioDestroy</td>
			<td >Groupe Sani Marc</td>
			<td >09-10215</td>
			<td>commercial peracetic acid-based disinfectant, store at RT</td>
		</tr>
		<tr >
			<td >Carboy LDPE 20 L</td>
			<td >Cole Parmer</td>
			<td >06031-52</td>
			<td></td>
		</tr>
		<tr >
			<td >CDC biofilm reactor</td>
			<td >Biosurface Technologies Corporation</td>
			<td >CRB 90</td>
			<td>bioreactor</td>
		</tr>
		<tr >
			<td >Cerium (IV) sulphate</td>
			<td >Thermo Scientific</td>
			<td >35650-K2</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Confocal laser scanning microscope&#160; LSM 700</td>
			<td >Zeiss</td>
			<td >LSM 700</td>
			<td></td>
		</tr>
		<tr >
			<td >Dey-Engley neutralizing broth</td>
			<td >Millipore</td>
			<td >D3435-500G</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >EMS950x + 350s gold sputter&#160;</td>
			<td >Electron Microscopy Sciences</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Epoxy resin</td>
			<td >Electron Microscopy Sciences</td>
			<td >14121</td>
			<td>with BDMA</td>
		</tr>
		<tr >
			<td >Ethyl alcohol 95%, USP</td>
			<td >Greenfield global</td>
			<td >P016EA95</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Ferroin indicator solution</td>
			<td >Sigma Aldrich</td>
			<td >318922-100ML</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Filling/venting cap</td>
			<td >Cole Parmer</td>
			<td >RK-06258-00</td>
			<td></td>
		</tr>
		<tr >
			<td >FilmTracer LIVE/DEAD Biofilm Viability Kit</td>
			<td >Invitrogen</td>
			<td >L10316</td>
			<td >fluorescent cell viability kit (SYTO 9: green fluorescent stain, Propidium iodide: red fluorescent stain), store at - 20 &#176;C</td>
		</tr>
		<tr >
			<td >Glass flow break</td>
			<td >Biosurface Technologies Corporation</td>
			<td >FB 50</td>
			<td></td>
		</tr>
		<tr >
			<td >Gold with silver paint&#160;</td>
			<td >Electron Microscopy Sciences</td>
			<td >12684-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Heating plate set</td>
			<td >Biosurface Technologies Corporation</td>
			<td >110V Stir Plate</td>
			<td></td>
		</tr>
		<tr >
			<td >Hex screwdriver</td>
			<td >Biosurface Technologies Corporation</td>
			<td >CBR 5497</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrogen peroxide</td>
			<td >Sigma</td>
			<td >216763</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >Inoculating loops</td>
			<td >VWR</td>
			<td >12000-812</td>
			<td>sterile, 10 &#181;l</td>
		</tr>
		<tr >
			<td >Lactic acid</td>
			<td >Laboratoire MAT</td>
			<td >LU-0200</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >MASTERFLEX L/S 7557-04 W/ 7557-02 with EASY-LOAD II peristaltic pump and 77200-50 Head</td>
			<td >Cole Parmer</td>
			<td >77200-60</td>
			<td></td>
		</tr>
		<tr >
			<td >MBEC (Minimum Biofilm Eradication Concentration) assay biofilm inoculator with a 96-well base</td>
			<td >Innovotech</td>
			<td >19111</td>
			<td>Biofilm microtiter plate</td>
		</tr>
		<tr >
			<td >Oxford agar base</td>
			<td >Thermo Scientific</td>
			<td >OXCM0856B</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr >
			<td >Plastic coupon holder</td>
			<td >Biosurface Technologies Corporation</td>
			<td >CBR 2203</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic slide holder rod</td>
			<td >Biosurface Technologies Corporation</td>
			<td >CBR 2203-GL</td>
			<td></td>
		</tr>
		<tr >
			<td >Potassium iodide</td>
			<td >Fisher Chemical</td>
			<td >P410-500</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Precision slotted screwdriver (1.5 mm x 40 mm)</td>
			<td >Wiha</td>
			<td >26015</td>
			<td></td>
		</tr>
		<tr >
			<td >Propionic acid</td>
			<td >Laboratoire MAT</td>
			<td >PF-0221</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Sartorius BCE822-1S Entris&#174; II Basic Essential Toploading Balance</td>
			<td >Cole Parmer</td>
			<td>&#160;UZ-11976-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Scanning electron microscope JSM-6360LV model</td>
			<td >JEOL</td>
			<td >JSM-6360LV</td>
			<td>SEM and user control interface</td>
		</tr>
		<tr >
			<td >Screw cap tube, 15 mL</td>
			<td >Sarstedt</td>
			<td >62.554.205</td>
			<td>(Lx&#216;): 120 x 17 mm, material: PP, conical base, transparent, HD-PE</td>
		</tr>
		<tr >
			<td >Screw cap tube, 50 mL</td>
			<td >Sarstedt</td>
			<td >62.547.205</td>
			<td>(Lx&#216;): 114 x 28 mm, material: PP, conical base, transparent, HD-PE</td>
		</tr>
		<tr >
			<td >Sodium Cacodylate Trihydrate</td>
			<td >Electron Microscopy Sciences</td>
			<td>&#160;12300</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr >
			<td >Sodium thiosulfate</td>
			<td >Thermo Scientific</td>
			<td >AC124270010</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Sonication bath</td>
			<td >Fisher</td>
			<td >15-336-122</td>
			<td>5,7 L</td>
		</tr>
		<tr >
			<td >Starch solution</td>
			<td >Anachemia</td>
			<td >AC8615</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Sulfuric acid</td>
			<td >Sigma Aldrich</td>
			<td >258105-500ML</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Tryptic soy agar</td>
			<td >BD Bacto</td>
			<td >DF0369-17-6</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Tryptic soy broth</td>
			<td >BD Bacto</td>
			<td >DF0370-17-3</td>
			<td>store at RT</td>
		</tr>
		<tr >
			<td >Tubing Masterflex L/S 16 25&#39;</td>
			<td >Cole Parmer</td>
			<td >MFX0642416</td>
			<td></td>
		</tr>
		<tr >
			<td >Tubing Masterflex L/S 18 25&#39;</td>
			<td >Cole Parmer</td>
			<td >MFX0642418</td>
			<td></td>
		</tr>
		<tr >
			<td >Tygon SPT-3350 silicon tubing&#160;</td>
			<td >Saint-Gobain</td>
			<td >ABW18NSF</td>
			<td>IDx OD: 1/4 in.x 7/16 in.</td>
		</tr>
		<tr >
			<td >Vortex</td>
			<td >Cole Palmer</td>
			<td >UZ-04724-00</td>
			<td></td>
		</tr>
		<tr >
			<td >Water bath&#160;</td>
			<td >VWR</td>
			<td >89202-970</td>
			<td></td>
		</tr>
		<tr >
			<td >Zen software</td>
			<td >Zeiss</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of the efficacy of organic peroxyacids for eradicating dairy biofilms using an approach combining static and dynamic methods

乳制品行业中生物膜的存在是一个主要问题，因为它们对加工厂中经常使用的大多数原位清洗（CIP）程序具有很高的抵抗力，因此可能导致生产不安全和改变的乳制品。因此，为乳品行业开发新的生物膜控制策略势在必行。该协议旨在评估有机过氧酸（过氧乙酸，过丙酸和过乳酸以及基于过氧乙酸的商业消毒剂）使用静态和动态方法的组合根除乳制品生物膜的功效。使用最小生物膜根除浓度（MBEC）测定（一种静态高通量筛选方法）对所有生物膜或混合生物膜中最强的产生生物膜的细菌进行测试。与推荐浓度的消毒剂接触时间为5分钟，成功消除了单个和混合生物膜。目前正在进行研究，以使用疾病控制中心（CDC）生物膜反应器来证实这些观察结果，这是一种模拟 原位 条件的动态方法。这种类型的生物反应器可以使用不锈钢表面，该表面构成了大多数工业设备和表面。反应器的初步结果似乎证实了有机过氧酸对生物膜的功效。本研究中描述的组合方法可用于开发和测试用于控制生物膜和根除微生物的新生物或化学制剂。

SEM分析显示生物膜微孔板钉上存在由偶氮假单胞菌PFl1A产生的生物膜（图2A）。可以观察到三维生物膜结构。P. azotoformans PFl1A先前被确定为使用96孔微量滴定板12的强大生物膜生产者（A570>1.5）。
此外，使用生物反应器在不锈钢载玻片上形成的偶氮福曼斯PFl1A生物膜似乎非常致密，并显示出具有三维结构的成熟...

Watch this Scientific Journal Video about Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods at JoVE.com

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

该协议描述了一种结合静态和动态方法的方法，以评估有机过氧酸在乳制品行业中根除生物膜的功效。这种方法也可用于测试新的生物或化学配方控制生物膜的有效性。

使用静态和动态方法相结合的方法评估有机过氧酸根除乳品生物膜的功效

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their ...

生物膜形成微生物是食品工业（包括乳制品工业）的主要问题。由于它们对产品质量的负面影响，因此制定生物膜控制策略非常重要。这种方法结合了静态高通量筛选方法和模拟原位条件的动态方法。这两种方法对于研究消毒剂对生物膜的功效是互补的。这种方法可用于测试新的生物或化学配方，以控制食品、医疗或环境领域的生物膜。首先涡旋偶氮假单胞菌细菌培养管。无菌将100微升细菌培养物转移到10毫升无菌胰蛋白酶大豆肉汤或TSB培养基中，以达到每毫升约10至第七CFU的两倍的终浓度。涡旋培养管。然后使用多通道移液器，将150微升稀释的细菌培养物一式三份转移到生物膜微量滴定板孔中。将150微升TSB培养基装入三个新孔中作为对照。接下来，将生物膜微量滴定板在30摄氏度下孵育24小时而不搅拌。清洁并风干生物反应器的部件，然后将扁平刀片放入通过磁棒连接到支架的一升玻璃烧杯内。使用连接到生物反应器盖内侧的塑料条保持设置直立。使用螺丝刀将不锈钢试样或滑块放在聚丙烯棒上。将试样或滑轨插入盖子上的孔中，不要将其定位销放在凹口中，以免蒸汽在灭菌过程中逸出。用铝箔覆盖所有生物反应器通风口，并用铝箔包裹其余设备，即 18 号管和 16 号管、玻璃流制动器、容器盖、螺丝刀、镊子和 0.2 微米过滤器。将生物反应器在121摄氏度的干循环中高压灭菌20分钟。为了以间歇模式在生物反应器中执行生物膜形成的第一步，在生物安全柜内，将 18 号管道的一端连接到生物反应器的出口，并保持另一端包裹在铝箔中。从生物反应器盖上取下一个试样或载玻片支架，并将其放入无菌50毫升管中。然后使用50毫升血清移液器，通过棒占据的孔向生物反应器烧杯填充340毫升每毫升300毫克TSB培养基。为了在生物反应器中接种培养基，使用五毫升移液管通过孔口向每毫升偶氮假单胞菌细菌溶液中的第八个CFU中加入一毫升10，并将杆放回其原始位置。将已放置在盖孔中的杆定位，以使销钉适合相应的凹口。然后将0.2微米的细菌空气吹扫过滤器放在位于生物反应器盖子上的最小直径管的末端。另一根相同直径的管子仍然用金属螺帽或紧密关闭的硅胶塞永久堵塞。将生物反应器放在设定在30摄氏度的加热板上24小时，并以130 RPM搅拌。24小时后，在连续流动模式下在生物反应器中执行生物膜形成的第二步。将装有18升无菌蒸馏水的大瓶放入生物安全柜中。然后加入两升每升1000毫克的TSB培养基，以获得每升100毫克的终浓度。用连接两根管子的无菌盖盖住容器。第一个是固定在盖子接口上的硅胶管，用于泵送介质。第二个16号管连接到外部端口，允许液体流向生物反应器。将 16 号管连接到玻璃流动制动器的末端后，将后者的另一端插入生物反应器盖上最大的管中，然后将 16 号管安装在蠕动泵中。使用另一个 20 升大瓶收集生物反应器的流出物。将连接到生物反应器出口出水口的管端连接到废物容器的盖子上。将0.2微米过滤器插入该容器盖上的管中。完成后，以每分钟 11.3 毫升的流速启动蠕动泵，并使系统运行 24 小时。24小时后，关闭蠕动泵，停止搅拌和加热生物反应器。要恢复细菌生物膜，请在40毫升PBS中冲洗优惠券或载玻片以消除浮游细菌。然后将它们释放到含有40毫升PBS的无菌50毫升锥形管中。涡旋 30 秒后，以 40 千赫兹的速度对试管进行超声处理 30 秒。重复此操作三次。完成后，将40毫升生物膜悬浮液收集在无菌的50毫升锥形管中，并用两种无菌PBS溶液冲洗试样或载玻片。回收这种漂洗液并将其添加到已经收集的生物膜悬浮液中。从30摄氏度下取出微量滴定板。将 200 微升 PBS 加入 96 孔微孔板的三个孔中。为了清洗生物膜并消除浮游细菌，将带有在钉子上形成的生物膜的生物膜微孔板的盖子转移到含有PBS的96孔微孔板上10秒。准备所需浓度的消毒剂，并将每种消毒剂浓度的200微升一式三份加入新的96孔微量滴定板的孔中。将生物膜微量滴定板的盖子转移到含有消毒剂的96孔微量滴定板上，并在室温下孵育板所需的曝光时间。接下来，将 200 微升 Dey-Engley 中和肉汤添加到新的 96 孔微量滴定板的孔中。将生物膜微量滴定板的盖子转移到含有中和肉汤的96孔微量滴定板上。用封口膜密封微量滴定板，并将其放入40千赫兹的浴超声仪中30分钟。30分钟后，从超声板的第一列，将100微升分离的生物膜转移到新的96孔微量滴定板的第一排。然后将 180 微升无菌 PBS 加入 96 孔板中，第一排除外。接下来，将20微升生物膜溶液从第一行转移到含有180微升PBS的第二排孔中，以达到稀释10至负1。然后将20微升从第二排转移到下一行的孔中，以达到10到负2的稀释度。重复此操作以获得 10 至负 5 和 10 至负 7 之间的稀释度。在TSA上接种100微升所需的稀释液，并根据细菌的生长参数孵育板。在生物反应器的试样上形成偶氮假单胞菌PFLA 1生物膜，取出装有试样的杆，并在含有30毫升PBS的锥形管内冲洗棒以去除浮游细胞。使用螺丝刀将每张优惠券放入无菌的 50 毫升锥形管中。然后加入四毫升适当的有机过氧酸溶液或PBS用于对照。孵育五分钟后，加入 36 毫升 Dey-Engley 中和肉汤。将锥形管涡旋 30 秒后，以 40 千赫兹的速度对其进行超声处理 30 秒。重复该过程三次以获得生物膜悬浮液。用其他棒重复相同的步骤，制备生物膜悬浮液的连续妄想，并如前所述将悬浮液铺在TSA培养基上。SCM分析显示偶氮假单胞菌PFL1A在生物膜微孔板钉上存在生物膜。使用生物反应器在不锈钢载玻片上形成的偶氮假单胞菌PFL1A生物膜看起来非常致密，并显示出成熟三维生物膜的形态特征。该分离株在动态系统中产生的生物膜的细菌计数结果显示，TSB培养基中显着的细胞密度对应于每平方厘米8.74 log CFU，无菌脱脂牛奶中对应于7.86 log CFU平方厘米。用B.vesicularis分离物获得的结果表明，营养浓度从每升100毫克增加到900毫克导致生物膜的细菌计数从6.11 log CFU平方厘米增加到8.71log CFU平方厘米。此外，当流速降低到每分钟6.0毫升时，观察到显着的生物膜生长，表明某些因素可能会影响生物反应器中生物膜的形成。在五分钟的接触时间内，没有一个生物膜含有可检测的活细胞，其中有机过氧酸为百万分之500，百万分之一，过氧化氢浓度的消毒剂通常应用于乳制品厂。SCM表现出未处理的最小生物膜根除浓度或MBEC生物膜的三维结构，而处理后的MBEC生物膜则失去了三维构象。在执行此协议时必须保持无菌状态。此外，用户必须记住，生成的结果与独特的分离物和消毒剂相关联。可以评估将消毒剂与机械振动、超声或酶处理相结合，以提高生物膜根除的功效。这种方法使用静态和动态方法形成生物膜，为研究人员筛选和研究新的抗生物膜配方铺平了道路，以更好地控制各个领域的生物膜。

Research

JoVE Journal

Biology

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

科研

生物学

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

三维白色脂肪组织结构的全装染色可视化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Development of an Individual-Tree Basal Area Increment Model using a Linear Mixed-Effects Approach

使用线性混合效应方法开发单树基数增量模型

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots ...