Name: conjunctival commensal isolation and identification in mice
Uploaded: 2021-05-01T13:00:00
Description: Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

来自P30 DK034854的资金支持VY，LB和马萨诸塞州主机微生物中心的研究，以及NIH/NEI R01 EY022054支持MG的资金。

所有涉及小鼠的程序均遵循机构动物护理和使用委员会的准则。在与微生物和潜在受污染材料合作时，请遵循实验室安全指南（由您的机构环境健康和安全部门指导）。在处置可能受到生物危害的受污染材料之前，使用适当的废物贮器和净化程序。
1. 眼拭子准备、工作现场设置、鼠标眼拭子和样品浓缩
<ol><li>准备无菌眼拭子 注：无菌眼拭子是薄涂层的木制牙...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+diversity+of+ocular+microbial+communities+in+diabetic+patients+compared+with+healthy+subjects.">Distribution and diversity of ocular microbial communities in diabetic patients compared with healthy subjects.</a> Current Eye Research. 43 (3), 314-324 (2018).</li><li>Doan, 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=Paucibacterial+microbiome+and+resident+DNA+virome+of+the+healthy+conjunctiva.">Paucibacterial microbiome and resident DNA virome of the healthy conjunctiva.</a> Investigative Ophthalmology and Visual Science. 57 (13), 5116-5126 (2016).</li><li>Kugadas, A., Gadjeva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+microbiome+on+ocular+health.">Impact of microbiome on ocular health.</a> Ocular Surface. 14 (3), 342-349 (2016).</li><li>Dong, Q., 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=Diversity+of+bacteria+at+healthy+human+conjunctiva.">Diversity of bacteria at healthy human conjunctiva.</a> Investigative Ophthalmology and Visual Science. 53 (8), 5408-5413 (2011).</li><li>Zegans, M. E., Van Gelder, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+understanding+the+ocular+surface+microbiome.">Considerations in understanding the ocular surface microbiome.</a> American Journal of Opthalmology. 158 (3), 420-422 (2014).</li><li>Fleiszig, S. M., Efron, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+flora+in+eyes+of+current+and+former+contact+lens+wearers.">Microbial flora in eyes of current and former contact lens wearers.</a> Journal of Clinical Microbiology. 30 (5), 1156-1161 (1992).</li><li>Lu, X., 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=Neutrophil+L-Plastin+Controls+Ocular+Paucibacteriality+and+Susceptibility+to+Keratitis.">Neutrophil L-Plastin Controls Ocular Paucibacteriality and Susceptibility to Keratitis.</a> Frontiers in Immunology. 11, 547 (2020).</li><li>Johnson, T. R., Case, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Experiments in Microbiology. , (2010).</li><li>Reiner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalase+Test+Protocol.">Catalase Test Protocol.</a> American Society for Microbiology. , (2010).</li><li>UK SMI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standards for Microbiology Investigation. UK SMI. , (2014).</li><li>Sharp, S. E., Searcy, C. <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+mannitol+salt+agar+and+blood+agar+plates+for+identification+and+susceptibility+testing+of+Staphylococcus+aureus+in+specimens+from+cystic+fibrosis+patients.">Comparison of mannitol salt agar and blood agar plates for identification and susceptibility testing of Staphylococcus aureus in specimens from cystic fibrosis patients.</a> Journal of Clinical Microbiology. 44 (12), 4545-4546 (2006).</li><li>Siegman-Igra, Y., Azmon, Y., Schwartz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milleri+group+streptococcus--a+stepchild+in+the+viridans+family.">Milleri group streptococcus--a stepchild in the viridans family.</a> European Journal of Clinical Microbiology and Infectious Diseases. 31 (9), 2453-2459 (2012).</li><li>Mohan, B., Zaman, K., Anand, N., Taneja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerococcus+Viridans:+A+Rare+Pathogen+Causing+Urinary+Tract+Infection.">Aerococcus Viridans: A Rare Pathogen Causing Urinary Tract Infection.</a> Journal of Clinical and Diagnostic Research. 11 (1), 1-3 (2017).</li><li>Senneby, E., Nilson, B., Petersson, A. C., Rasmussen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+is+a+sensitive+and+specific+method+for+identification+of+aerococci.">Matrix-assisted laser desorption ionization-time of flight mass spectrometry is a sensitive and specific method for identification of aerococci.</a> Journal of Clinical Microbiology. 51 (4), 1303-1304 (2013).</li><li>Wan, S. 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=IL-1R+and+MyD88+contribute+to+the+absence+of+a+bacterial+microbiome+on+the+healthy+murine+cornea.">IL-1R and MyD88 contribute to the absence of a bacterial microbiome on the healthy murine cornea.</a> Frontiers in Microbiology. 9, 1117 (2018).</li><li>Ozkan, 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=Temporal+Stability+and+Composition+of+the+Ocular+Surface+Microbiome.">Temporal Stability and Composition of the Ocular Surface Microbiome.</a> Scientific Reports. 7 (1), 9880 (2017).</li><li>Oliver, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viable+but+non-culturable+state+in+bacteria.">The viable but non-culturable state in bacteria.</a> Journal of Microbiology. 43 (1), 93-100 (2005).</li><li>Epstein, S. 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+phenomenon+of+microbial+uncultivability.">The phenomenon of microbial uncultivability.</a> Current Opinion in Microbiology. 16 (5), 636-642 (2013).</li><li>Whelan, F. 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=Culture-enriched+metagenomic+sequencing+enables+in-depth+profiling+of+the+cystic+fibrosis+lung+microbiota.">Culture-enriched metagenomic sequencing enables in-depth profiling of the cystic fibrosis lung microbiota.</a> Nature Microbiology. 5 (2), 379-390 (2020).</li><li>Raymond, F., 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=Culture-enriched+human+gut+microbiomes+reveal+core+and+accessory+resistance+genes.">Culture-enriched human gut microbiomes reveal core and accessory resistance genes.</a> Microbiome. 7, 56 (2019).</li><li>Peto, L., 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=Selective+culture+enrichment+and+sequencing+of+feces+to+enhance+detection+of+antimicrobial+resistance+genes+in+third-generation+cephalosporin+resistant+Enterobacteriaceae.">Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.</a> PLoS One. 14 (11), 0222831 (2019).</li><li>Lauer, B. A., Masters, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+effect+of+calcium+alginate+swabs+on+Neisseria+gonorrhoeae.">Toxic effect of calcium alginate swabs on Neisseria gonorrhoeae.</a> Journal of Clinical Microbiology. 26 (1), 54-56 (1988).</li><li>Dubois, 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=Performances+of+the+Vitek+MS+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+system+for+rapid+identification+of+bacteria+in+routine+clinical+microbiology.">Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid identification of bacteria in routine clinical microbiology.</a> Journal of Clinical Microbiology. 50 (8), 2568-2576 (2012).</li><li>Kawakita, 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=double-blind+study+of+the+safety+and+Efficacy+of+1%D-3-Hydroxybutyrate+eye+drops+for+Dry+Eye+Disease.">double-blind study of the safety and Efficacy of 1%D-3-Hydroxybutyrate eye drops for Dry Eye Disease.</a> Scientific Reports. 6, 20855 (2016).</li><li>Lee, H. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+response+to+lipopolysaccharide+on+the+ocular+surface+in+a+murine+dry+eye+model.">Inflammatory response to lipopolysaccharide on the ocular surface in a murine dry eye model.</a> Investigative Ophthalmology and Visual Science. 57 (6), 2444-2450 (2016).</li><li>Miller, D., Ioviano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microbial+flora+on+the+ocular+surface.">The role of microbial flora on the ocular surface.</a> Current Opinion in Allergy and Immunology. 9 (5), 466-470 (2009).</li><li>Nayyar, A., Gindina, S., Barron, A., Hu, Y., Danias, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+epigenetic+changes+caused+by+commensal+microbiota+contribute+to+development+of+ocular+disease?+A+review+of+evidence.">Do epigenetic changes caused by commensal microbiota contribute to development of ocular disease? A review of evidence.</a> Human Genomics. 14 (1), 11 (2020).</li><li>Stevenson, W., 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=Dry+eye+disease:+an+immune-mediated+ocular+surface+disorder.">Dry eye disease: an immune-mediated ocular surface disorder.</a> Archives of Ophthalmology. 130 (1), 90-100 (2012).</li></ol>

由于眼表面的低菌态，许多实验室难以分离眼科共生体7、20，导致生长、丰度低、多样性低的样品数量少。这种方法通过增加充实步骤以及MALDI-TOF MS重新设计的眼拭子和识别，显著改进了标准文化实践4，21。浓缩步骤通过放大细菌负荷来解决低可回收性问题。可回收细菌从不到100个CFU显著增...

本议定书的目的是加强从眼结膜中分离出可行和罕见的有氧和足性厌氧微生物的具体隔离，以特性眼微生物群。广泛的研究已经剖析了皮肤、肠道、呼吸道和生殖器上的粘膜群落，并表明这些社区影响免疫系统的发展和反应1，2，3。眼科共生社区已被证明在某些疾病病理学中发生改变，如干眼病4、Sjogren综合征5和糖尿病6。然而，与其他粘膜部位6、7、8相比，其丰度相对较低，妨碍了定义典型眼表面共体群落的能力。这引发了一场关于是否有常驻眼微生物群以及是否存在、它是否与皮肤微生物群不同以及其对与生俱来的免疫系统发育和反应的局部影响的争议。此协议可以帮助解决此问题。
一般来说，定义眼部共融利基的方法是基于测序和基于文化的技术4，7，9。16 S rDNA测序和BRISK分析7显示，与基于文化的技术相比，其多样性更为广泛，但无法区分活微生物和死微生物。由于眼部表面由于撕裂膜的抗微生物特性而对许多微生物怀有敌意，因此基于DNA的方法将检测这些文物，这些文物可能会使数据偏向于将死细菌识别为常驻共产物而不是污染物。这导致异常的共体识别和描述的眼部空间是较高的微生物丰度和多样性10。这使得很难通过基于DNA的方法定义常驻眼微生物群。然而，基于文化的标准技术无法检测共体，因为负载太低11。我们的方法改进了标准做法，使用薄拭子，可以瞄准结膜，从而避免来自邻近皮肤的污染，以及可行的生物体可以通过营养密集介质的短暂培养来丰富的概念，目的是恢复可行但不可耕种的微生物，以及丰富稀有可行的微生物。
结果，每个眼拭子的眼部共生体相对丰度，是结膜常驻微生物群的特征，对比较目的很重要。我们的数据显示，皮肤和结膜微生物之间有差异，随着年龄的增长和性别的丰度差异，多样性也越来越大。此外，这种方法在淘汰小鼠12中重新发现了共性差异。此协议可用于描述可能因细胞实践、地理或疾病状态而变化的眼微生物群，以及共生代谢物和产品对免疫系统发育和反应的局部影响。

<table>
	<tbody>
		<tr>
			<td>0.1 to 10 &#181;l pipet tip</td>
			<td>USA Scientific</td>
			<td>1110-300</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>0.5 to 10 &#181;l Eppendorf pipet</td>
			<td>Fisher Scientific</td>
			<td>13-690-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Fisher Scientific</td>
			<td>BD309623</td>
			<td>1 syringe for each eye swab group</td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>1000 &#181; ml pipet tip</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>200 to 1000&#181;l Gilson pipetman (P1000)</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>1 needle per eye swab group</td>
		</tr>
		<tr>
			<td>3 % Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>S25359</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176; C Incubator</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 % Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>PX1840-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ana-Sed Injection (Xylazine 100 mg/ml)</td>
			<td>Santa Cruz Animal Health</td>
			<td>SC-362949Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BBL Gram Stain kit</td>
			<td>Fisher Scientific</td>
			<td>B12539</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean paper towels</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Batting/Sterile rolled cotton</td>
			<td>CVS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>E.coli</td>
			<td>ATTC</td>
			<td>ATCC 8739</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>Ketamine (100mg/ml)</td>
			<td>Henry Schein</td>
			<td>9950001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mac Conkey Agar Plates</td>
			<td>Fisher Scientific</td>
			<td>4321270</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Carolina Biological Supply</td>
			<td>784641</td>
			<td>Prepare plates according to mfr&#39;s instructions, store at 4 &#176;C for 1 week</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Labs</td>
			<td>C57/BL6J</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
			<td>for Mannitol Salt agar plates</td>
		</tr>
		<tr>
			<td>RPI Brain Heart Infusion Media</td>
			<td>Fisher Scientific</td>
			<td>50-488525</td>
			<td>prepare according to directions and autoclave</td>
		</tr>
		<tr>
			<td>SteriFlip (0.22 &#181;m pore size polyester sulfone)</td>
			<td>EMD/Millipore, Fisher Scientifc</td>
			<td>SCGP00525</td>
			<td>to sterilize anesthesia</td>
		</tr>
		<tr>
			<td>Sterile Corning Centrifuge Tube</td>
			<td>Fisher Scientific</td>
			<td>430829</td>
			<td>anesthesia preparation</td>
		</tr>
		<tr>
			<td>Sterile mouse cage</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth picks (round bamboo)</td>
			<td>Kitchen Essentials</td>
			<td></td>
			<td>autoclave before use and swab preparation</td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar II with 5% Sheep&#39;s Blood Plates</td>
			<td>Fisher Scientific</td>
			<td>4321261</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Vitek target slide</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS CHCA matrix solution</td>
			<td>BioMerieux Inc. Durham, NC</td>
			<td>411071</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use eye drops</td>
			<td>CVS Pharmacy</td>
			<td></td>
			<td>Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each</td>
		</tr>
	</tbody>
</table>

conjunctival commensal isolation and identification in mice

眼表面曾经被认为是免疫特权和非生物，但最近看来，有一个小的，但持久的共生存在。眼粘膜细菌物种的鉴定和监测一直具有挑战性，因为其丰度低，而且缺乏适当的共生和识别方法。有两种标准方法：基于文化或DNA测序方法。第一种方法是有问题的，由于有限的可回收细菌和第二种方法识别活细菌和死细菌导致异常的眼部空间表示。我们利用标准的微生物培养技术，开发出一种坚固灵敏的细菌分离方法。这是一种基于拭子的技术，使用"实验室内"制成的薄拭子，瞄准下结膜，然后是有氧和足部厌氧基因的放大步骤。该协议使我们能够分离和识别结膜物种，如 科琳细菌、凝固负葡萄球菌、链球菌等。该方法适用于定义不同疾病条件下小鼠的同体多样性。

显示不同电镀方法的眼拭板的代表性结果见 图 3A， 显示与 C57BL/6 鼠标的形态多样性隔离。对于每个不同的隔离，殖民地被计算在条和相对丰度，独特的殖民地形成单位（CFU）每个眼拭子，计算和绘制的比较目的。对于微生物特征，细菌从单个鼠标眼拭子板中采摘，以产生主 TSA 板（通过从每个鼠眼拭子板中采摘三元形的形态分明分离物创建）。从主板块，在第二天或当增长...

Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

Conjunctival Commensal Isolation and Identification in Mice

这里介绍的是一个协议，用于分离和放大有氧和体性厌氧小鼠结膜共体细菌使用独特的眼拭子和基于培养的丰富步骤，随后通过微生物为基础的方法和MALDI-TOF质谱识别。

小鼠的共性共性隔离和识别

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

这种方法识别可行的眼结膜微生物，这是具有挑战性的，由于眼睛敌对的环境。它可以帮助回答眼微生物群是否存在，以及眼部疾病的细菌存在如何变化。与DNA测序（可识别可行和不可行微生物）不同，该方法只识别可行的微生物，从而更清楚地了解眼共体群落。研究表明，难以诊断的眼疾，如非自身免疫和自身免疫性干眼有独特的眼部细菌存在。这种方法可用于诊断具有独特微生物特征的眼部疾病。首先自动切割适量的棉击和牙签，让老鼠数量被擦拭，然后捏掉一厘米长的棉击1/2块，然后拉到边缘，形成一个扁平的单孔层，在击球分崩离析之前停止。在牙签尖端扭曲时轻轻握住伸出的一块，围绕牙签的一端旋转击球。成品眼拭子将有一个非常薄的棉层伸展在尖端延伸约1/2至1厘米远的尖端。将棉签插入一个小烧嘴中，向下拭子侧，然后盖住并高压灭菌。使用消毒剂清洁工作区域，以尽量减少污染，并引用 0.5 毫升无菌脑心脏输液，或 BHI 介质放入标有 1.5 毫升无菌微离心管。将管子盖在架子上，并将机架放在冰上。从左到右设置工作流程，从小鼠麻醉站开始，该站包含带实验鼠的笼子、空无菌笼、室温麻醉、25号针头和1毫升注射器。接下来，准备一个眼拭子站，其中含有冰上引用的 BHI、消毒眼拭子、润滑眼药水、生物危害容器、干净的纸巾和 70% 异丙酚喷雾剂。最后，设置了带室温血脂板的电镀站、10微升移液器、10微升无菌一次性提示和生物危害废物容器。通过挤压后脚垫，确保鼠标正确麻醉。只有在没有移动的情况下才能继续。分配一只手处理麻醉小鼠，另一只手处理眼拭子和文化。将鼠标从笼子中取出，放在工作面的顶部，使其侧面露出左眼。用异丙酚喷洒戴手套的手，用纸巾擦干。用专用介质处理手解开标记的 BHI 微型离心机管，并将两者放回机架中。将棉质涂层的眼拭子尖浸入 BHI 中，然后从管中取出拭子，同时将尖端两次旋转到内管上，以去除多余的液体并将其取出。用鼠标处理手，轻轻地按住鼠标的脖子擦伤。另一方面，将眼睑尖端贴在左眼的中间结合区域。轻轻压低眼球，在下眼睑和眼睛之间移动拭子10次，保持恒定的压力。无需触摸毛皮，请轻轻取出垂直于插入位置的拭子尖端。将棉签侧向下放置，直接放入带有 BHI 介质的标记微型离心机管中。将眼滴涂在擦拭的眼睛上。如有需要，获取皮肤或毛皮拭子作为控制样本，在每个拭子之间适当消毒手套。完成后，将鼠标返回笼子。让拭子在冰上站立10至15分钟，然后消毒戴手套的手，取出拭子，同时在介质中混合尖端10次旋转。将尖端旋转到管内壁上进行五次旋转，将其丢弃在生物危害容器中，从而提取拭子。重复每个鼠标的过程。通过在37摄氏度下静态地孵化管一小时来丰富样品。在孵化过程中，给每个鼠标眼拭子或控制拭子贴上一个室温 TSA 板的标签，并将其分成两半。从孵化器中取出浓缩样品，放在冰上。短暂地将样品漩涡混合，然后将样品的 10 微升引到 TSA 板上，并将板倾斜以形成条状。重复两次。在板分界线的另一边，创建 10 个点，每个点有 10 微升样品。在37摄氏度的高温下将盘子孵化18小时，两天四天，在一个干净的室内，防止阿加板干燥。数一数条带中的殖民地。注意形态，并计算每个拭子的菌落形成单位，以形成形态相似的分离物。看看在条带中没有捕获独特生物的点。为了便于可视化，我们在中间和右板密集地镀细菌。通常，我们观察到的眼拭子板中的细菌要少得多。请注意，有时，眼拭子可能不会产生可恢复的细菌。此处显示了具有代表性的眼拭板，其形态多样化的分离物与 C57 黑色六只鼠标分离。对于每个不同的隔离区，殖民地被计算在条中，相对丰度被计算和绘制。对于微生物特征，细菌是从单个鼠标眼拭子板中挑选出来的，以产生主TSA板。当生长出现时，会进行其他测试来描述或识别微生物。主板用于提供足够的接种，以扩大各自的隔离。为了识别隔离物，TSA 板在一夜之间被条纹和孵育，并进行了 MALDI-TOF MS。此处显示的分离物示例被确定为链球菌酸性氨基尼穆斯和气管病毒。从雄性C57黑六只小鼠身上发现了明显不同水平的共生生物。链球菌酸性氨基尼穆斯、气可可病毒凝固阴性葡萄球菌和大肠杆菌从C57黑六N小鼠中分离。在尝试此协议时，请记住，最佳结果将通过在眼拭子步骤中涂上拭子并花时间来实现。如果获得 MALDI-TOF MS 的机会有限，则可以通过微生物和生化测试识别分离物。

conjunctival-commensal-isolation-and-identification-in-mice

Research

JoVE Journal

Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

科研

免疫与感染

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

心脏外免疫细胞的分离与鉴定

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.