Funding from&#160;P30 DK034854&#160;supported VY, LB and studies in the Massachusetts Host-Microbiome Center and funding from NIH/NEI&#160;R01 EY022054 supported MG.

All procedures involving mice follow the Institutional Animal Care and Use Committee guidelines. Follow laboratory safety guidelines (as directed by your Institutional Environmental Health and Safety department) when working with microorganisms and potentially contaminated materials. Use appropriate waste receptacles and decontamination procedures prior to disposal of potentially biohazard contaminated materials.
1. Eye swab preparation, work field set-up, mouse eye swabbing and sample enrichment
<ol>
	<li>Prepare sterile eye swabs 
	NOTE: Sterile eye swabs are thinly coated wooden toothpicks with a fiber thin layer of cotton batting and made in house.
	<ol>
		<li>Autoclave appropriate amount of cotton batting and toothpicks for number of mice to be swabbed.</li>
		<li>Pinch off a half a centimeter-long piece of cotton batting with thumb and forefinger. Tease out batting by pulling on the edges with thumbs and forefingers to form a flat single porous layer, stopping just before the batting falls apart (small bits of cotton dispersed throughout the stretched batting are acceptable).</li>
		<li>Swirl the batting around one of the sharp ends of the toothpick by lightly holding the stretched-out piece on the toothpick tip as it is twisted, see Figure 1. The &#8220;completed&#8221; eye swab will have a very thin layer of cotton stretched over the tip, extending approximately one half to one centimeter away from the tip.</li>
		<li>Insert &#8220;completed&#8221; swabs into small beaker, swab side down and cover. Autoclave.</li>
	</ol>
	</li>
	<li>Set up the workspace
	<ol>
		<li>Prepare anesthesia containing 2 mL of ketamine (100 mg/mL), 400 &#181;L of xylazine (100 mg/ml) and 27.6 mL of sterile saline (0.9% NaCl in dH2O) in a sterile 50 mL sterile centrifuge tube. Filter sterilize and aliquot anesthesia for immediate use (may be stored at 4 &#176;C for 1 month; always allow to equilibrate to room temperature prior to use).</li>
		<li>Clear and clean the work area with disinfectant to minimize contamination.</li>
		<li>Aliquot 0.5 mL of sterile Brain Heart Infusion media (BHI) into labeled 1.5 mL sterile microcentrifuge tubes (1 tube per mouse and control); arrange in microcentrifuge rack. Set the rack on ice.</li>
		<li>Set up work flow as follows (from left to right): mouse anesthetizing station (cage containing experimental mice, empty sterile cage, room temperature anesthesia, 25 G needle and 1 mL syringe), eye swabbing station (aliquoted BHI on ice, sterilized eye swabs,&#160;clean paper towels, 70% isopropanol spray) and plating station (room temperature blood agar plates, 10 &#181;L pipette, 10 &#181;L sterile disposable tips, biohazard waste container).</li>
	</ol>
	</li>
	<li>Eye swabbing
	<ol>
		<li>Administer 10 &#181;L of anesthesia per 1 g of mouse weight dosage of ketamine and xylazine respectively intraperitoneally to each adult mouse and place into an autoclaved cage. Test anesthetization by squeezing&#160;hindfoot pad; no movement indicates appropriate anesthetization.</li>
		<li>Assign one hand to only handle anesthetized mice and the other hand to only handle the eye swab and the culture. For swabbing, remove the fully anesthetized mouse from the cage; place on top of clean work surface&#160;positioned on its&#8217; side with left eye exposed. Spray gloved hands with isopropanol, dry with clean paper towel.</li>
		<li>Uncap specifically labeled BHI micro-centrifuge tube with dedicated media handling hand. Place the tube back into the micro-centrifuge rack on ice.&#160; Dip the cotton coated tip of the eye swab in BHI, withdraw the eye swab from the tube swirling the tip 2 times against the inner tube to remove excess liquid.&#160;</li>
		<li>With the mouse handling hand, gently hold the mouse by the scruff of the neck. With the other hand, place the tip of eye swab against the medial conjunctival region of the left eye, see Figure 2A. Lightly depress the eyeball and move the swab in a window washing motion (Figure 2B) back and forth, between the lower eyelid and eye, 10 times. Maintain constant pressure.</li>
		<li>Without touching the fur, gently remove the tip of swab, perpendicular to where it was inserted, and place the swab cotton side down directly into a labeled micro-centrifuge tube containing BHI media.&#160;Apply a lubricating eye drop to the swabbed eye.</li>
		<li>Return the mouse to the cage.</li>
		<li>Let the swab stand for 10 to 15 min on ice and remove eye swab with the sterilized gloved hand by mixing the tip in the media for 10 rotations. Withdraw the swab by swirling tip against the inner wall of the micro-centrifuge tube for 5 rotations. Dispose of the swab in biohazard container.</li>
		<li>Repeat steps 1.3.2 to 1.3.7 for each mouse and for control samples (skin or fur swab). Sterilize gloves appropriately between each swab.</li>
	</ol>
	</li>
	<li>Enrichment
	<ol>
		<li>Enrich the sample by incubating the tube statically for 1 h at 37 &#176;C</li>
		<li>During incubation, label one room temperature Trypticase Soy with 5% sheep&#8217;s blood agar plate (TSA plate) per mouse and divide in half.</li>
		<li>After eye swab culture incubation, remove enriched samples from the incubator and place on ice.</li>
		<li>Briefly vortex samples to mix and plate according to the schematic in Figure 3A. Aliquot 10 &#181;L of the sample onto the TSA plate and tilt the plate to form a strip, repeat 2 times.</li>
		<li>On the other side of the plate&#8217;s dividing line, dot 10 &#181;L of sample on agar, repeat 9 times.</li>
		<li>Incubate plates at 37 &#176;C for 18 h, 2 days, and 4 days (in a clean chamber that prevents agar plates from drying). Count colonies in strips, note morphology and calculate colony forming units (CFUs) per swab for morphologically similar isolates. Look at dots for unique organisms not captured in strips.</li>
	</ol>
	</li>
</ol>
2. Master plate, characterization, and identification of ocular microbes
<ol>
	<li>Master plate
	<ol>
		<li>Draw grids on the back of a TSA plate (Figure 3B). Pick a colony with a sterile toothpick from each mouse eye swab plate and streak the master plate square (grids), label grid with colony number and mouse information. Pick and plate three different colonies for each morphologically distinct colony.</li>
		<li>Incubate the plate overnight at 37 &#176;C. Continue incubation for 96 hours, checking daily for the appearance of new isolates. 
		NOTE: The Commensal population will vary based on mouse age, nutrition, disease state and caging practices. Isolate characterization is based on the predominant aerobic and facultative anaerobic bacteria found in our laboratory.</li>
	</ol>
	</li>
	<li>Characterization
	<ol>
		<li>Duplicate13 plate microbes on Mannitol Salt Agar (MSA) and MacConkey (MAC) plates, incubate overnight at 37 &#176;C. Note growth for each isolate and presence of waxy colonies or yellow colonies with halo on MSA.</li>
		<li>For Gram positive cocci, test with the catalase test13,14. Place a drop of 3% hydrogen peroxide on a clean glass slide. Using a sterile toothpick, pick the corresponding microbe from the eye swab plate. No bubble formation indicates Streptococcus spp., slight bubble formation indicates Corynebacterium spp. and perhaps Aerococcus spp. and rapid bubble formation indicates Staphylococcus spp.</li>
	</ol>
	</li>
	<li>Identification: MALDI-TOF MS analysis 
	NOTE: Bacterial identifications are carried out using the MALDI-TOF and the Software Database. This system employs matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for developing spectra profiles which are compared to known bacterial spectra for identification. E.coli, ATCC 8739, is used for system calibration.
	<ol>
		<li>Plate bacterial isolates onto TSA plates containing 5% sheep blood and incubate overnight with 5% carbon dioxide at 37 &#730;C.</li>
		<li>Using a 1 &#181;L loop, apply a thin layer of the pure bacteria to the MALDI-TOF target slide; 1 &#181;L of MALDI-TOF MS CHCA matrix solution (alpha-cyano-4-hydroxycinnamic acid) is overlaid and allowed to air dry completely before loading the slide into the MALDI-TOF instrument.</li>
		<li>Each isolate is spotted in duplicate.</li>
		<li>Reports containing probability scores greater than 80%, which indicate a high discrimination value, are accepted as reliable results and the bacterial identification accepted.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Arpaia, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolites+produced+by+commensal+bacteria+promote+peripheral+regulatory+T+cell+generation.">Metabolites produced by commensal bacteria promote peripheral regulatory T cell generation.</a> Nature. 504 (7480), 451-455 (2013).</li><li>Hooper, L. V., Littman, D. R., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+the+microbiota+and+the+immune+system.">Interactions between the microbiota and the immune system.</a> Science. 336 (6086), 1268-1273 (2010).</li><li>Nagpal, R., 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=Human-origin+probiotic+cocktail+increases+short+chain+fatty+acid+production+via+modulation+of+mice+and+human+gut+microbiome.">Human-origin probiotic cocktail increases short chain fatty acid production via modulation of mice and human gut microbiome.</a> Scientific Reports. 8 (1), 12649 (2018).</li><li>Graham, J. 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. 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>

No conflict of interest to disclose.

Due to the paucibacterial state of the ocular surface, many laboratories have had difficulty isolating ocular commensals7,20, resulting in low number of samples with growth, low abundance and low diversity8. This method significantly improves upon standard culture practices4,21 by the addition of an enrichment step, as well as a redesigned eye swab and identification by MALDI-TOF M...

The aim of this protocol is to enhance specific isolation of viable and rare aerobic and facultative anaerobic microbes from the ocular conjunctiva to characterize the ocular microbiome. Extensive studies have profiled commensal mucosal communities on the skin, gut, respiratory and genital tracts and show that these communities influence the development of the immune system and response1,2,3. Ocular commensal communities have been shown to change during certain disease pathologies, such as Dry eye disease4, Sjogren&#8217;s syndrome5 and diabetes6. Yet, the ability to define a typical ocular surface commensal community is hampered by their relatively low abundance compared to the other mucosal sites6,7,8. This prompts a controversy over whether there is a resident ocular microbiome and if it exists, whether it differs from the skin microbiome and consequently, its local effect on the innate immune system development and response. This protocol can help resolve this question.
Generally, approaches to define the ocular commensal niche are based on sequencing and culture-based techniques4,7,9. 16 S rDNA sequencing and BRISK analysis7 show a broader diversity than culture-based techniques, but are unable to differentiate between live and dead microbes. Since the ocular surface is hostile to many microbes due to tear film&#8217;s anti-microbial properties4 generating a large array of DNA fragments, DNA based approaches will detect these artifacts which may skew the data toward identification of dead bacteria as resident commensals rather than contaminants. This results in aberrant commensal identification and characterization of the ocular space as being higher in microbe abundance and diversity10. This makes it difficult to define the resident ocular microbiome via DNA based methods. Whereas, standard culture-based techniques are unable to detect commensals because the load is too low11. Our method improves upon standard practices by using a thin swab that can target the conjunctiva, thus avoiding contamination from neighboring skin, as well as the concept that viable organisms can be enriched by brief culture in nutrient dense media with the goal of resuscitating viable but non-culturable, as well as, enriching for rare viable microbes.
The results, relative abundance of ocular commensals per eye swab, characterize the conjunctiva resident microbiome and are important for comparative purposes. Our data shows that there is a difference between skin and conjunctival microbiota, as well as greater diversity with increased age and a sex specific difference in abundance. Furthermore, this approach has reproducibly found commensal differences in knock-out mice12. This protocol can be applied to describe the ocular microbiome which may vary due to caging practices, geography, or disease state, as well as the local effects of commensal metabolites and products on immune system development and response.

<table><tbody><tr><td>0.1 to 10 &amp;micro;l pipet tip</td><td>USA Scientific</td><td>1110-300</td><td>autoclave before use</td></tr><tr><td>0.5 to 10 &amp;micro;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 &amp;micro; ml pipet tip</td><td>USA Scientific</td><td>1111-2021</td><td>autoclave before use</td></tr><tr><td>200 to 1000&amp;micro;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 &amp;deg; 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>&lt;em&gt;E.coli&lt;/em&gt;</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 &amp;deg;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&#039;s instructions, store at 4 &amp;deg;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 &amp;micro;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&#039;s Blood Plates</td><td>Fisher Scientific</td><td>4321261</td><td>store at 4 &amp;deg;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

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Representative results for an eye swab plate demonstrating different methods for plating are pictured in Figure 3A showing morphologically diverse isolates from C57BL/6 mouse. For each distinct isolate, the colonies were counted in the strip and the relative abundance, unique Colony Forming Units (CFUs) per eye swab, calculated and plotted for comparison purposes. For microbiological characterization, bacteria were picked from individual mouse eye swab plates to produce a master TSA plate (c...

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

Conjunctival Commensal Isolation and Identification in Mice

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

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

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

Research

JoVE Journal

Immunology and Infection

4.3K Views.  Harvard Medical School. Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

Video: Conjunctival Commensal Isolation and Identification in Mice

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Bioengineering

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 &#181;L. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 &#181;L of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 &#181;L sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 &#176;C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

We describe here a method of intravitreal injection and subsequent bacterial quantitation in mouse model of bacterial endophthalmitis. This protocol can be extended for measuring host immune responses and bacterial and host gene expression.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Neuroscience

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

Biology

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, M&#252;ller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse M&#252;ller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, M&#252;ller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for M&#252;ller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn&#39;t be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

This manuscript describes a detailed protocol for isolating retinal glial M&#252;ller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of M&#252;ller cells.

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...

Swab-based Conjunctival Commensal Bacteria Isolation: A Technique to Isolate Commensal Bacteria from Conjunctiva of Murine Model

Source: Smith-Page, K. et al. <a href="https://www.jove.com/video/61672" target="_blank">Conjunctival Commensal Isolation and Identification in Mice</a>. J. Vis. Exp. (2021)
This video describes a swab-based technique to isolate viable commensal bacteria from the ocular conjunctiva. The technique can be used to profile the commensal bacterial microbiome of the conjunctiva, which plays a defensive role against pathogens.

Source: Smith-Page, K. et al. Conjunctival Commensal Isolation and Identification in Mice. J. Vis. Exp. (2021)
This video describes a swab-based technique ...

JoVE Encyclopedia of Experiments

Rodent Models

Adult

Bacterial Endophthalmitis Mouse Model: A Method for Generating Bacterial Endophthalmitis via Intravitreal Injection of Bacterial Suspension into Eye of Mouse

Source: Mursalin, M. H. et al., <a href="https://www.jove.com/v/61749">Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis.</a> J. Vis. Exp. (2021).
This video describes a method for generating a mouse model that mimics bacterial endophthalmitis &#8211; an intraocular infection resulting from a bacterial invasion into the eye&#8217;s vitreous cavity &#8211; via intravitreal injection of a bacterial suspension into the eye. This mouse model helps study the disease mechanism and evaluate therapeutics for it.

Source: Mursalin, M. H. et al., Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis. J. Vis. ...