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

Finansiering från P30 DK034854 stödde VY, LB och studier i Massachusetts Host-Microbiome Center och finansiering från NIH / NEI R01 EY022054 stödde MG.

Alla procedurer som involverar möss följer riktlinjerna från kommittén för institutionell djurvård och användning. Följ laboratoriesäkerhetsriktlinjerna (enligt din institutionella miljöhälso- och säkerhetsavdelning) när du arbetar med mikroorganismer och potentiellt förorenade material. Använd lämpliga avfallsbehållare och saneringsförfaranden innan potentiellt biologiskt förorenade material bortskaffas.
1. Förberedelse av ögonsvabb, arbetsfältsuppsättning, ögonsvabbning...

<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. 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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. 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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. 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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? 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På grund av den okulära ytans paucibacterial tillstånd har många laboratorier haft svårt att isolera okulär commensals7,20, vilket resulterar i lågt antal prover med tillväxt, lågt överflöd och låg mångfald8. Denna metod förbättrar avsevärt standardkulturpraxis4,21 genom att lägga till ett anrikningssteg, samt en omdesignad ögonvabb och identifiering av MALDI-TOF ...

Syftet med detta protokoll är att förbättra specifik isolering av livskraftiga och sällsynta aeroba och facultative anaeroba mikrober från okulär bindhinnan att karakterisera okulär microbiome. Omfattande studier har profilerat commensal slemhinnor på huden, tarmen, luftvägarna och könsorganen och visar att dessa samhällen påverkar utvecklingen av immunsystemet ochsvar 1,2,3. Okulär commensal samhällen har visat sig förändras under vissa sjukdom patologier, såsom Torra ögonsjukdom 4, Sjögrens syndrom5 och diabetes6. Ändå hämmas förmågan att definiera en typisk okulär yta commensal gemenskap av deras relativt låga överflöd jämfört med de andra slemhinnor6,7,8. Detta leder till en kontrovers om huruvida det finns ett resident okulärt mikrobiom och om det finns, om det skiljer sig från hudens mikrobiom och följaktligen dess lokala effekt på det medfödda immunsystemets utveckling och svar. Det här protokollet kan hjälpa till att lösa den här frågan.
I allmänhet är metoder för att definiera den okulära commensal nischen baserade på sekvensering och kulturbaserade tekniker4,7,9. 16 S rDNA-sekvensering och BRISK-analys7 visar en bredare mångfald än kulturbaserade tekniker, men kan inte skilja mellan levande och döda mikrober. Eftersom den okulära ytan är fientlig mot många mikrober på grund av att tårfilmens antimikrobiellaegenskaper 4 genererar ett stort antal DNA-fragment, kommer DNA-baserade metoder att upptäcka dessa artefakter som kan skeva data mot identifiering av döda bakterier som bosatta commensals snarare än föroreningar. Detta resulterar i avvikande kommensal identifiering och karakterisering av det okulära utrymmet som högre i mikrob överflöd och mångfald10. Detta gör det svårt att definiera det bosatta okulära mikrobiomet via DNA-baserade metoder. Standardkulturbaserade tekniker kan inte upptäcka commensals eftersom belastningen är för låg11. Vår metod förbättrar standardpraxis genom att använda en tunn svabb som kan rikta in sig på bindhinnan, vilket undviker förorening från närliggande hud, liksom konceptet att livskraftiga organismer kan berikas av kort kultur i näringsrika täta medier med målet att återuppliva livskraftiga men icke-odlingsbara, liksom berikande för sällsynta livskraftiga mikrober.
Resultaten, relativa överflöd av okulär commensals per öga svabb, karakterisera bindhinnan bosatta mikrobiom och är viktiga för jämförande ändamål. Våra data visar att det finns en skillnad mellan hud och konjunktival mikrobiota, liksom större mångfald med ökad ålder och en könsspecifik skillnad i överflöd. Dessutom har detta tillvägagångssätt reproducerbart funnit kommensala skillnader i knockout möss12. Detta protokoll kan tillämpas för att beskriva okulär mikrobiom som kan variera på grund av lokaliseringsmetoder, geografi eller sjukdomstillstånd, liksom de lokala effekterna av kommensala metaboliter och produkter på immunsystemets utveckling och svar.

<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

Okulär ytan ansågs en gång immun privilegierad och abiotisk, men nyligen verkar det som om det finns en liten, men ihållande commensal närvaro. Identifiering och övervakning av bakteriearter vid okulär slemhinnan har varit utmanande på grund av deras låga överflöd och begränsade tillgänglighet av lämplig metodik för kommensal tillväxt och identifiering. Det finns två standardmetoder: kulturbaserade eller DNA-sekvenseringsmetoder. Den första metoden är problematisk på grund av de begränsade återvinningsbara bakterierna och det andra tillvägagångssättet identifierar både levande och döda bakterier som leder till en avvikande representation av det okulära utrymmet. Vi utvecklade en robust och känslig metod för bakteriell isolering genom att bygga vidare på standard mikrobiologiska odlingstekniker. Detta är en svabbbaserad teknik, med hjälp av en "in-lab" gjord tunn svabb som riktar sig mot den nedre bindhinnan, följt av ett förstärkningssteg för aeroba och fakultativa anaeroba släkten. Detta protokoll har gjort det möjligt för oss att isolera och identifiera konjunktival arter såsom Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. Tillvägagångssättet är lämpligt för att definiera kommensal mångfald hos möss under olika sjukdomsförhållanden.

Representativa resultat för en ögonvabbplatta som visar olika metoder för plätering visas i figur 3A som visar morfologiskt olika isolat från C57BL/6 mus. För varje distinkt isolate räknades kolonierna i remsan och det relativa överflöd, unika koloniformningsenheter (CFUs) per ögonvabb, beräknade och plottade för jämförelseändamål. För mikrobiologisk karakterisering plockades bakterier från enskilda mus öga svabbplattor för att producera en master TSA platta (skapas genom...

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

Conjunctival Commensal Isolation and Identification in Mice

Presenteras här är ett protokoll för isolering och förstärkning av aeroba och facultative anaeroba mus konjunktival kommensala bakterier med hjälp av en unik öga svabba och kultur-baserade anrikning steg med efterföljande identifiering av mikrobiologiska baserade metoder och MALDI-TOF massa spektrometri.

Konjunktivval kommensal isolering och identifiering hos möss

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

Denna metod identifierar livskraftiga okulär konjunktival mikroorganismer, vilket är utmanande på grund av ögonen fientlig miljö. Det kan hjälpa till att svara på om det finns ett okulärt mikrobiom och hur bakteriell närvaro förändras vid ögonsjukdom. I motsats till DNA-sekvensering, som identifierar både livskraftiga och icke-livskraftiga mikroorganismer, identifierar denna metod endast livskraftiga mikroorganismer, vilket resulterar i en tydligare förståelse för det okulära kommensala samhället.Studier tyder på att svårt att diagnostisera ögonsjukdomar som icke autoimmuna och autoimmuna torra ögon har unik okulär bakteriell närvaro. Denna metod kan användas för att diagnostisera ögonsjukdomar med distinkta mikrobiella signaturer. Börja med att autoklavera lämplig mängd bomullsslag och tandpetare för antalet möss som ska swabbed, nyp sedan av 1/2 en centimeter lång bit bomullsslag och reta ut det genom att dra på kanterna för att bilda ett platt enda poröst lager som stannar precis innan slagningen faller isär.Snurra slagningen runt en av tandpetarens vassa ändar genom att lätt hålla den utsträckta biten på tandpetarspetsen när den är vriden. Den färdiga ögonpinnen kommer att ha ett mycket tunt lager bomull sträckt över spetsen som sträcker sig ungefär 1/2 till en centimeter från spetsen. Sätt in svabbproverna i en liten bägare, svabba sidan nedåt och täck sedan över och autoklavera dem.Rengör arbetsområdet med ett desinfektionsmedel för att minimera kontaminering och alikvot 0,5 milliliter steril hjärnhjärtainfusion, eller BHI-media till märkta 1,5 milliliter sterila mikrocentrifugrör. Kapa rören i ett rack och ställ racket på is. Ställ in arbetsflöde från vänster till höger från vänster till höger från och med musbedövande station, som innehåller buret med de experimentella mössen, tom steril bur, rumstemperaturbedövning, 25 gauge nål och en en milliliter spruta.Förbered sedan en ögonsvabbstation som innehåller den alikvoterade BHI på is, steriliserade ögonvabbar, smörjande ögondroppar, biohazardbehållare, rena pappershanddukar och 70% isopropanolspray. Slutligen, ställ in pläteringsstationen med blodagarplattor i rumstemperatur, en 10 mikroliterpipett, 10 mikroliter sterila engångsspetsar och en biohazard avfallsbehållare. Se till att musen är ordentligt sövd genom att klämma på en bakfotsplatta.Fortsätt bara om det inte finns någon rörelse. Tilldela en hand för att hantera bedövade möss och de andra händerna för att hantera ögonvabben och kulturen. Ta bort musen från buret och placera den ovanpå arbetsytan placerad på sidan med vänster öga exponerat.Spraya handskhänder med isopropanol och torka dem med en pappershandduk. Packa upp det märkta BHI-mikrocentrifugröret med den dedikerade mediehanteringshanden och placera de två tillbaka i racket. Doppa ögonpinnen i BHI:s bomullsbelagda spets och dra sedan ut svabbpinnen ur röret medan du virvlar spetsen två gånger mot det inre röret för att ta bort överflödig vätska och ta bort den.Håll försiktigt musen i nacken med mushanteringshanden. Med den andra handen, placera ögonpinnens spets mot den mediala konjunktivalregionen på vänster öga. Tryck lätt in ögongloben och flytta svabbpinnen i en fönstertvättrörelse mellan det nedre ögonlocket och ögat 10 gånger, vilket upprätthåller konstant tryck.Utan att vidröra pälsen, ta försiktigt bort toppen av svabbpinnen vinkelrätt mot där den sattes in. Placera bomullssidan i bomull, direkt i ett märkt mikrocentrifugrör med BHI-media. Applicera ett ögonfall på det svabbade ögat.Om så önskas, skaffa hud- eller pälsprover för kontrollprover, sterilisera handskar på lämpligt sätt mellan varje svabbprov. När du är klar, sätt tillbaka musen i buret. Låt svabbpinnen stå i 10 till 15 minuter på is, sterilisera sedan handske och ta bort svabbpinnen medan du blandar spetsen i media i 10 rotationer.Dra ut svabbpinnen genom att virvla spetsen mot rörets innervägg i fem rotationer och kassera den i en biohazard behållare. Upprepa processen för varje mus. Berika provet genom att inkubera röret statiskt i en timme vid 37 grader Celsius.Under inkubationen, märk en TSA-platta i rumstemperatur per musögon eller kontrollsvabb och dela den i hälften. Ta bort de berikade proverna från inkubatorn och lägg dem på is. Virvel kort proverna för att blanda, sedan alikvot 10 mikroliter av provet på TSA-plattan och luta plattan för att bilda en remsa.Upprepa detta två gånger. På andra sidan av plattornas delningslinje, skapa 10 punkter med 10 mikroliter prov vardera. Inkubera plattorna vid 37 grader Celsius i 18 timmar, två dagar och fyra dagar i en ren kammare som förhindrar att agarplattor torkar.Räkna kolonierna i remsorna. Notera morfologi och beräkna kolonibildande enheter per svabbprov för morfologiskt liknande isolat. Titta på prickarna där unika organismer inte fångas i remsorna.För att underlätta visualiseringen pläterade vi bakterier tätt i mitten och rätt plattor. Vanligtvis observerade vi mycket färre bakterier i ögonvabbplattor. Observera att ögonvabbarna ibland inte kan ge återvinningsbara bakterier.En representativ ögonpinneplatta med morfologiskt olika isolat från en C57 svart sex mus visas här. För varje distinkt isolate räknades kolonierna i remsan och det relativa överflöd beräknades och plottades. För mikrobiologisk karakterisering plockades bakterier från enskilda mus ögonvabbplattor för att producera en master TSA-platta.När tillväxt uppstod, gjordes ytterligare tester för att karakterisera eller identifiera mikroberna. Huvudplattan användes för att ge tillräckligt med inokulat för att expandera respektive isolat. För att identifiera isolat, en TSA platta var streaked och inkuberade över natten, och MALD-TOF MS utfördes.Exempel på isolat som visas här identifierades som Streptococcus acidominimus och Aerococcus viridans. Betydligt olika nivåer av kommensala organismer återfanns från manliga och kvinnliga C57 svarta sex möss. Streptococcus acidominimus, Aerococcus viridans koagulerar negativa Staphylococci och E.coli isolerades från C57 svarta sex N möss.När du försöker detta protokoll, kom ihåg att det bästa resultatet kommer att uppnås genom att täcka svabben tunt och ta tid under ögonvabbsteget. Om tillgången till MALDI-TOF MS är begränsad kan isolaten identifieras genom mikrobiologisk och biokemisk testning.

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.

Isolering och analys av Brain-binds Leukocyter från<em&gt; Plasmodium berghei</em&gt; ANKA-infekterade möss

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

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.

Snabb identifiering av gramnegativa bakterier från Blood Culture Broth Använda MALDI-TOF masspektrometri

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.

Isolering, identifiering och rening av murina Thymic epitelceller

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.

Neutrofil Isolering och analys för att fastställa deras roll i lymfom Cell Känslighet för terapeutiska medel

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.

Identifiering av Plasmodesmal lokalisering sekvenser i proteiner i Planta

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.

Isolering av Salmonella typhimurium-innehållande Phagosomes från makrofager

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.

Identifiering och isolering av Oligopotent och härstamning-begått myeloida från mus benmärg

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.

Isolering och identifiering av extravaskulära immunceller i hjärtat

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.

En Galleria mellonella Oral Administration modell till Study Commensal-Induced medfödda immunsvar

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.