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

P30 DK034854からの資金は、マサチューセッツ州ホストマイクロバイオームセンターでのVY、LBおよび研究を支援し、NIH/ NEI R01 EY022054からの資金提供はMGをサポートしました。

マウスを含むすべての手順は、制度的動物ケアと使用委員会のガイドラインに従います。微生物や汚染される可能性のある材料を扱う際には、(お客様の機関環境安全衛生部門が指示する)実験室の安全ガイドラインに従ってください。バイオハザード汚染材料の廃棄前に、適切な廃棄物容器と除染手順を使用してください。
1.アイスワブの準備、作業場のセットアップ、マ...

<ol>
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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=Ocular+pathogen+or+commensal:+a+PCR+based+study+of+surface+bacterial+flora+in+normal+and+dry+eyes.">Ocular pathogen or commensal: a PCR based study of surface bacterial flora in normal and dry eyes.</a> Investigative Ophthalmology and Visual Science. 48 (12), 5616-5623 (2007).</li><li>Wang, C., 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=Sj&#246;gren-Like+Lacrimal+Keratoconjunctivitis+in+Germ-Free+Mice.">Sj&#246;gren-Like Lacrimal Keratoconjunctivitis in Germ-Free Mice.</a> International Journal of Molecular Sciences. 19 (2), 565-584 (2018).</li><li>Ham, B., Hwang, H. B., Jung, S. H., Chang, S., Kang, K. D., Kwon, M. 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. S., Hattori, T., Stevenson, W., Cahuhan, S. K., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+toll-like+receptor+4+contributes+to+corneal+inflammation+in+experimental+dry+eye+disease.">Expression of toll-like receptor 4 contributes to corneal inflammation in experimental dry eye disease.</a> Investigative Ophthalmology and Visual Science. 53 (9), 5632-5640 (2012).</li><li>Simmons, K. T., Xiao, Y., Pflugfelder, S. C., de Paiva, C. 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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>

眼表面の小児性細菌状態のために、多くの研究室は、7、20の眼部の分離が困難であり、その結果、成長、低存在量、低多様性8の低いサンプル数を生じる。この方法は、濃縮工程の追加、およびMALDI-TOF MSによるアイスワブおよび同定を加えることによって、標準的な培養プラクティス4,21?...

このプロトコルの目的は、眼のマイクロバイオームを特徴付けるために、眼結膜から実行可能でまれな有酸素性および気性嫌気性微生物の特異的な分離を強化することである。広範な研究は、皮膚、腸、呼吸器および生殖管上の経経膜粘膜群集をプロファイリングし、これらのコミュニティが免疫系および応答1、2、3の発達に影響を及ぼすことを示している。眼部の男性群は、ドライアイ病4、シェーグレン症候群5および糖尿病6などの特定の疾患病態の間に変化することが示されている。しかし、典型的な眼表面の連動性共同体を定義する能力は、他の粘膜部位6、7、8と比較して比較的少ない存在によって妨げられている。これは、常駐眼マイクロバイオームがあるかどうか、それが存在する場合、それが皮膚マイクロバイオームと異なるかどうか、そして結果的に、自然免疫系の発達と応答に対する局所的な影響があるかどうかについての論争を促す。このプロトコルは、この問題を解決するのに役立ちます。
一般に、連視ニッチを定義するアプローチは、シーケンシングと文化ベースの技術4、7、9に基づいています。16 S rDNAシーケンシングおよびBRISK分析7は、培養ベースの技術よりも広い多様性を示すが、生きた微生物と死んだ微生物を区別することができない。眼表面は、大量のDNA断片を生成する涙膜の抗菌特性4のために多くの微生物に敵対しているため、DNAベースのアプローチは、汚染物質ではなく常駐的な汚染物質として死んだ細菌の同定に向けてデータを歪める可能性のあるこれらのアーティファクトを検出します。これは、微生物の存在量および多様性10においてより高いとして眼空間の異常な分別識別および特徴付けをもたらす。これにより、DNAベースの方法を介して常駐眼マイクロバイオームを定義することが困難になります。一方、標準的な文化ベースの技術は、負荷が低すぎるため、コメンサルを検出することができません11.我々の方法は、結膜を標的とすることができる薄い綿棒を使用して、近隣の皮膚からの汚染を避けることによって、ならびに実行可能な生物が生存可能であるが非カルト的な、ならびに希少生存微生物を豊かにすることを目的に、栄養密度の高い培地の短い培養によって豊かにすることができるという概念を使用することによって、標準的な慣行を改善する。
結果は、眼綿棒あたりの眼のコメンの相対的な豊富さ、結膜の常駐微生物叢を特徴付け、比較目的のために重要である。我々のデータは、皮膚と結膜微生物叢の間に違いがあり、年齢の増加と性別固有の豊富な違いと共に多様性が高いことを示している。さらに、このアプローチは、ノックアウトマウス12における因果的な違いを再現的に発見した。このプロトコルは、ケージの慣行、地理、または疾患状態、および免疫系の発達および応答に対するコメンサル代謝産物および産物の局所的影響によって異なる可能性のある眼マイクロバイオームを記述するために適用することができる。

<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

眼表面はかつて免疫特権と非生物性と考えられていたが、最近では小さいが持続的な経常存在があるように見える。眼粘膜における細菌種の同定とモニタリングは、その存在量が少なく、産経的成長と同定のための適切な方法論の利用可能性が限られているため、困難であった。培養法とDNAシーケンシング法の2つの標準的なアプローチがあります。第1の方法は、限られた回収可能な細菌のために問題であり、第2のアプローチは、眼空間の異常な表現につながる生きている細菌と死んだ細菌の両方を識別する。標準的な微生物培養技術を基に、細菌の分離のための堅牢で高感度な方法を開発しました。これは、下の結膜を標的とする「ラボ内」製の薄い綿棒を利用した綿棒ベースの技術であり、続いて有酸素性および好気性嫌気性性の生殖器の増幅ステップを利用する。このプロトコルは、 コリネバクテリウムspp.、コアグラーゼ陰性ブドウ球菌spp.、ストレプトコッカス属などの結膜種を分離し、同定することを可能にしました。このアプローチは、異なる疾患条件下でのマウスの男性の多様性を定義するのに適している。

めっきのための異なる方法を示す目の綿棒板の代表的な結果は、C57BL/6マウスからの形態学的に多様な分離株を示す 図3A に示されている。各別個の分離物について、コロニーはストリップに数え、相対的な豊富さ、目の綿棒あたりのユニークなコロニー形成単位(CFUs)を計算し、比較目的でプロットした。微生物学的特徴付けのために、細菌を個々のマウスアイスワブプ?...

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

Conjunctival Commensal Isolation and Identification in Mice

ここでは、微生物学的手法とMALDI-TOF質量分析によるその後の同定を用いたユニークな眼綿棒および培養ベースの濃縮ステップを用いた有酸素性および気性嫌気性マウスの結合性のコンメンサルト菌の分離と増幅のためのプロトコルを提示する。

マウスにおける結膜経月の分離と同定

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

この方法は、目の敵対的な環境のために挑戦している実行可能な眼結膜微生物を識別します。これは、眼マイクロバイオームが存在するかどうか、および細菌の存在が眼疾患でどのように変化するかを答えるのに役立ちます。生存可能な微生物と生存不可能な微生物の両方を識別するDNAシーケンシングとは対照的に、この方法は生存可能な微生物のみを識別し、眼部のコメンザルコミュニティを明確に理解する結果となる。研究は、非自己免疫および自己免疫ドライアイのような眼疾患を診断することは困難であり、独特の眼細菌存在を有することを示唆している。この方法は、特徴的な微生物シグネチャを持つ眼疾患を診断するために使用することができます。スワッピングするマウスの数に合わせて綿のバッティングと爪楊枝を適切な量に詰め込み、長さ1/2センチメートルの長い綿のバッティングをつまみ、端を引っ張ってバッティングがばらばらになる直前に平らな単一の多孔質層を形成していじめます。爪楊枝の鋭い端の1つの周りにバッティングを軽く持って、爪楊枝の先端に伸ばした部分をねじって回します。完成した目の綿棒は、先端から約1/2〜1センチメートル離れた先端の上に伸ばされた綿の非常に薄い層を持つことになります。小さなビーカーに綿棒を挿入し、綿棒の側面を下に置き、それらをカバーしてオートクレーブします。消毒剤で作業領域をきれいにして、0.5ミリリットルの無菌脳心注入、またはBHI培地を標識された1.5ミリリットルの無菌マイクロ遠心チューブに最小限に抑えます。チューブをラックにキャップし、ラックを氷の上に置く。実験マウス、空の滅菌ケージ、室温麻酔、25ゲージ針、1ミリリットルの注射器を備えたケージを含むマウス麻酔ステーションから左から右にワークフローを設定します。次に、氷の上に引用されたBHI、滅菌された目綿棒、潤滑点眼薬、バイオハザードコンテナ、きれいなペーパータオル、および70%イソプロパノールスプレーを含む目スワッピングステーションを準備します。最後に、常温の血液寒天プレート、10マイクロリットルピペット、10マイクロリットルの滅菌使い捨てチップ、およびバイオハザード廃棄物容器を備えためっきステーションを設置します。後ろ足パッドを絞ってマウスが正しく麻酔されていることを確認します。動きがない場合にのみ進みます。麻酔付きマウスを扱う片手と、目の綿棒と培養を処理するもう一方の手を割り当てます。ケージからマウスを取り出し、左目を露出させた状態で側面に配置された作業面の上に置きます。手袋をした手にイソプロパノールをスプレーし、ペーパータオルで乾かします。専用のメディアハンドリングハンドでラベル付きのBHIマイクロ遠心分離管をアンキャップし、ラックに戻します。BHIに目の綿棒の綿でコーティングされた先端を浸し、その後、余分な液体を取り除くために内側のチューブに対して2回渦巻きながら、チューブから綿棒を引き出します。マウスハンドで、首の擦り傷でマウスをそっと握りしめる。一方、左目の内側結膜領域に対して、目の先端をスワブに置きます。眼球を軽く押し下げ、下眼瞼と眼の間の洗浄運動で綿棒を10回動かし、一定の圧力を維持する。毛皮に触れることなく、綿棒の先端を挿入した場所に垂直にそっと取り除きます。綿棒綿を下に下に置き、BHI培地付きのラベル付きマイクロ遠心管に直接入れる。スワッピングアイに目のドロップを適用します。必要に応じて、コントロールサンプル用の皮膚または毛皮綿棒を取得し、各綿棒の間に適切に手袋を殺菌する。終了したら、マウスをケージに戻します。綿棒を氷の上で10〜15分間放置し、手袋をした手を殺菌し、10回転のためにメディアの先端を混合しながら綿棒を取り除きます。5回転のためにチューブの内壁に先端を旋回させることによって綿棒を引き出し、バイオハザード容器に処分します。各マウスに対してこの手順を繰り返します。37°Cで1時間静かにチューブをインキュベートしてサンプルを濃縮します。インキュベーション中に、マウスの目の綿棒またはコントロール綿棒ごとに1つの室温TSAプレートにラベルを付け、半分に分割します。濃縮されたサンプルをインキュベーターから取り出し、氷の上に置きます。サンプルを簡単に渦を混ぜて混合し、次にサンプルの10マイクロリットルをTSAプレートにアリコートし、プレートを傾けてストリップを形成する。これを 2 回繰り返します。プレート分割線の反対側に、それぞれ10マイクロリットルのサンプルを含む10ドットを作成します。寒天プレートの乾燥を防ぐクリーンチャンバーで、18時間、2日、4日間、摂氏37度でプレートをインキュベートします。ストリップ内のコロニーを数えます。形態に注意し、形態学的に類似した分離物のための綿棒ごとのコロニー形成単位を計算する。ユニークな生物がストリップに捕獲されていない点を見てください。可視化を容易にするために、真ん中と右のプレートに細菌を密にメッキしました。典型的には、目の綿棒プレートの細菌がはるかに少ない観察を受けた。時折、目の綿棒は回復可能な細菌を得ることができないことに注意してください。C57ブラックシックスマウスからの形態学的に多様な分離株を有する代表的な眼綿棒板をここに示す。それぞれの異なる分離について、コロニーをストリップで数え、相対的な存在量を計算してプロットした。微生物学的特徴化のために、細菌は、マスターTSAプレートを生成するために、個々のマウス目綿棒プレートから選ばれた。成長が現れたとき、微生物を特徴付けるか、または識別するために追加のテストが行われました。マスタープレートは、それぞれの分離株を拡大するのに十分な接種を提供するために使用された。分離株を同定するために、TSAプレートをストリークして一晩インキュベートし、MALDI-TOF MSを実施した。ここに示す分離株の例は、レンサ球菌アシドミニムスおよびアエロコッカスビリダンとして同定された。男性および女性C57黒6匹のマウスから有意に異なるレベルのコメンザル生物が回収された。連鎖球菌酸菌、アエロコッカス・ビリダンスは、負のブドウ球菌および大腸菌をC57黒6Nマウスから単離した。このプロトコルを試みるとき、綿棒を薄くコーティングし、目の綿棒のステップの間に時間を取ることによって最良の結果が達成されることを覚えておいてください。MALDI-TOF MSへのアクセスが制限されている場合、分離株は微生物学的および生化学的試験によって同定され得る。

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.

脳から隔離された白血球の単離と解析<em&gt;マラリアベルゲイ</em&gt; ANKA感染マウス

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

免疫・感染学

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.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

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.

単離、同定、およびマウス胸腺上皮細胞の精製

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.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

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.

タンパク質Planta Plasmodesmal ローカリゼーション シーケンスの同定

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.

サルモネラの分離-大食細胞のファゴゾームを含む

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.

寡能性と系列拘束されたマウスの骨髄から骨髄前駆細胞の分離と同定

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.

心の血管外免疫細胞の分離と同定

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.

ハチミツガ口腔管理モデル Study Commensal-Induced 自然免疫応答

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.