Name: assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface
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The authors gratefully acknowledge financial support from the Flanders Research Foundation to Marta Calatayud Arroyo (FWO postdoctoral fellowship-12N2815N). Emma Hernandez-Sanabria is a postdoctoral fellow supported by Flanders Innovation and Entrepreneurship (Agentschap voor Innovatie door Wetenschap en Technologie, IWT).

1. Strains and Culture Conditions
NOTE: The synthetic oral community was composed by strains commonly present in the oral microbiome3.
<ol>
	<li>Obtain the following strains from the American Type Culture Collection (ATCC): Aggregatibacter actinomycetemcomitans (ATCC 43718), Fusobacterium nucleatum (ATCC 10953), Porphyromonas gingivalis (ATCC 33277), Prevotella intermedia (ATCC 25611), Streptococcus</e...

<ol>
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U., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+interpretation+of+bacterial+viability+by+using+the+LIVE/DEAD+BacLight+kit+in+combination+with+flow+cytometry.">Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry.</a> Applied and Environmental Microbiology. 73, 3283-3290 (2007).</li><li>Props, R., Monsieurs, P., Mysara, M., Clement, L., Boon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+biodiversity+of+microbial+communities+by+flow+cytometry.">Measuring the biodiversity of microbial communities by flow cytometry.</a> Methods in Ecology and Evolution. 7, 1376-1385 (2016).</li><li>Yamashita, Y., Takeshita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oral+microbiome+and+human+health.">The oral microbiome and human health.</a> Journal of Oral Science. 59, 201-206 (2017).</li><li>Wade, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oral+microbiome+in+health+and+disease.">The oral microbiome in health and disease.</a> Pharmacological Research. 69, 137-143 (2013).</li><li>Yu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+resource+for+cell+line+authentication,+annotation+and+quality+control.">A resource for cell line authentication, annotation and quality control.</a> Nature. 520, 307 (2015).</li><li>Pelaseyed, 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=The+mucus+and+mucins+of+the+goblet+cells+and+enterocytes+provide+the+first+defense+line+of+the+gastrointestinal+tract+and+interact+with+the+immune+system.">The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.</a> Immunological Reviews. 260, 8-20 (2014).</li><li>Bengoa, A. A., 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=Simulated+gastrointestinal+conditions+increase+adhesion+ability+of+Lactobacillus+paracasei+strains+isolated+from+kefir+to+Caco-2+cells+and+mucin.">Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin.</a> Food Research International. 103, 462-467 (2018).</li><li>Kleiveland, C. R., et al., Verhoeckx, K., 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=.">.</a> The Impact of Food Bioactives on Health: in vitro and ex vivo models. , 197-205 (2015).</li><li>Laparra, J. 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The oral microbiome is a key element in human health as recently reported by several authors20,21. Previous findings suggest that the ingestion of saliva containing large loads of bacteria can influence the microbial ecosystem of the small intestine, which is one of the main sites for immune priming. The combination of a static upper gastrointestinal digestion model with the host interface represented by intestinal epithelial and mucus-secreting cells, served to ...

Humans cohabit with bacteria, which are present at the same number as human cells1. Hence, it is of crucial important to obtain a comprehensive understanding of the human microbiome. The oral cavity is a unique environment in that it is divided into several smaller habitats, thus containing a large variety of bacteria and biofilms in those different locations. Being an open ecosystem, some species in the mouth may be transient visitors. However, certain microorganisms colonize soon after birth and form organized biofilms2. These are found in the teeth surface above the gingival crevice, the subgingival crevice, tongue, mucosal surfaces and dental prosthetics and fillings3. Bacteria can also be present as flocs and planktonic cells in the lumen of the tooth canal, either intermixed with necrotic pulp tissue or suspended in a fluid phase.
There is active, continuous cross-talk between host cells and the resident microbiota4. Bacteria communicate within and between species, and only a small proportion of the natural colonizers can adhere to tissues, while other bacteria attach to these primary colonizers. For instance, cell-cell binding between microorganisms is key for integrating secondary colonizers into oral biofilms, and building complex networks of interacting microbial cells4. Around 70% of bacterial aggregates in a saliva sample are formed by Porphyromonas sp., Streptococcus sp., Prevotella sp., Veillonella sp., and unidentified Bacteroidetes. F. nucleatum is an intermediate colonizer in the subgingival biofilm and aggregates with the late colonizers P. gingivalis, T. denticola, and Tannerella forsythia, which are implicated in periodontitis5. In addition, Streptococcus mitis occupies both mucosal and dental habitats, while S. sanguinis and S. gordonii prefer to colonize teeth3. Thus, S. sanguinis is present in lower incisors and canines, while Actinomyces naeslundii has been found in upper anteriors6.
In addition, the indigenous microbiome plays a role in maintaining human health2. Resident microbiota participates in immune education and in preventing pathogen expansion. This colonisation resistance occurs because the native bacteria may be better adapted at attaching to surfaces, and more efficient at metabolising the available nutrients for growth. Although probiotic strains survive the gastrointestinal passage and remain active, the persistence of autochthonous bacteria swallowed from an upper location of the gastrointestinal tract has not been fully described. Thus, we subjected an artificial community, representative of the oral ecosystem, to simulated gastrointestinal transit conditions. Viability of bacterial cells was assessed using a multicompartment model resembling the gut epithelium. Current gut simulators offer suitable reproducibility in terms of analysis of the luminal microbial community7. However, bacterial adhesion and host-microbe interaction are separately addressed, as combining cell lines with microbial communities is challenging8. In contrast, we present a framework that provides potential mechanistic explanation of successful colonization events reported on the gut interface. Indeed, this model can be jointly used with a static gut model to evaluate the impact of microbial communities on host surface signalling.

<table>
	<tbody>
		<tr>
			<td>STRAINS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aggregatibacter actinomycetemcomitans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 43718</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusobacterium nucleatum </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 10953</td>
			<td></td>
		</tr>
		<tr>
			<td>Porphyromonas gingivalis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33277</td>
			<td></td>
		</tr>
		<tr>
			<td>Prevotella intermedia </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25611</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mutans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25175</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sobrinus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33478</td>
			<td></td>
		</tr>
		<tr>
			<td>Actinomyces viscosus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15987</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus salivarius&#160; TOVE-R</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mitis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49456</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sanguinis </td>
			<td>BCCM/LMG Bacteria Collection</td>
			<td>LMG 14657</td>
			<td></td>
		</tr>
		<tr>
			<td>Veillonella parvula </td>
			<td>Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures</td>
			<td>DSM 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus gordonii </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49818</td>
			<td></td>
		</tr>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caco-2 cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>86010202</td>
			<td></td>
		</tr>
		<tr>
			<td>HT29-MTX cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>12040401</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS AND CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI) broth</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar 2</td>
			<td>Oxoid</td>
			<td>CM0055</td>
			<td>Blood Agar medium</td>
		</tr>
		<tr>
			<td>Menadione</td>
			<td>Sigma</td>
			<td>M9429</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma</td>
			<td>H9039</td>
			<td></td>
		</tr>
		<tr>
			<td>5% sterile defibrinated horse blood</td>
			<td>E&#38;O Laboratories Ltd,</td>
			<td>P030</td>
			<td></td>
		</tr>
		<tr>
			<td>InnuPREP PCRpure Kit</td>
			<td>Analytik Jena</td>
			<td>845-KS-5010250</td>
			<td>PCR purification kit</td>
		</tr>
		<tr>
			<td>Big Dye</td>
			<td>Applied Biosystems</td>
			<td>4337454</td>
			<td>Dye for sequencing</td>
		</tr>
		<tr>
			<td>ABI Prism BigDye Terminator v3.1 cycle sequencing kit</td>
			<td>Applied Biosystems</td>
			<td>4337456</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR&#160;Green I</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 culture flasks uncoated, cell-culture treated, vented, sterile</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 
			0.4%, liquid, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM cell culture media, with GlutaMAX and Pyruvate</td>
			<td>Life technologies</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polyester membrane cell culture inserts</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3450-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin from porcine stomach Type II&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactivated fetal bovine serum</td>
			<td>Greiner Bio One</td>
			<td>758093</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X 100 for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without calcium, magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce LDH Cytotoxicity Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>88953</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning HTS Transwell-24 well, pore size 0.4 &#181;m</td>
			<td>Corning Costar Corp</td>
			<td>3450</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Serva Electrophoresis</td>
			<td>28539010</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber improved</td>
			<td>Carl Roth</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>653118</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer 24 Homogenizer</td>
			<td>MoBio</td>
			<td>13155</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>186-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Flush system</td>
			<td>Custom made</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>InnOva 4080 Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>8261-30-1007</td>
			<td>Shaker for 2.10</td>
		</tr>
		<tr>
			<td>Memmert CO2 incubator</td>
			<td>Memmert GmbH &#38; Co.</td>
			<td>ICO150med</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS (Electrical Resistance System)</td>
			<td>EMD Millipore, Merck KGaA</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore Milli-Q academic, ultra pure water system</td>
			<td>Millipore, Merck KGaA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker (ROCKER 3D basic)</td>
			<td>IKA</td>
			<td>4000000</td>
			<td>Shaker for 6.10</td>
		</tr>
	</tbody>
</table>

assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

This protocol leads to the generation of a model suitable for elucidating the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. The counts of intact cells from individual strains is approximately 108 cells mL-1 prior the creation of the synthetic community, while the multispecies microcosm contained above 90% of viable cells during the establishment of the community (Figure 1A </s...

Watch this Scientific Journal Video about Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface at JoVE.com

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

This model is the next step in setting host microdirections for the upper digestive tracts. It is allowing addressing mechanistic research questions and it offers many opportunities for the food, the pharma, and nutraceutical industry and also toxicology. The main advantage of this technique is that it combines a model of the gastrointestinal system with the host micro interface including a complex microbe community.It is very applicable and it is very flexible as well. This methodology can be adopted using other cell types to assess pathogen viability and subsequent information. Changes can also be used to assist colonization capacity of probiotic mixtures and ultimately the potential effect of bacterias on the per systemic circulation.To begin, autoclave the medium, and then add five microgram per milliliter of hemen and one microgram per milliliter of menadione. Dispense nine milliliters of the medium into Hungate tubes, and close the tubes with a rubber stopper and an aluminum cap. Then flush the tubes with a mixture of 90%nitrogen and 10%carbon dioxide.Use the syringe to retrieve one milliliter of anaerobic medium, and transfer it to a 1.5 milliliter microcentrifuge tube. Pick single colonies of each bacterial species and resuspend them in the medium. Then transfer the colonies into the anaerobic modified BHI, and incubate them at 37 degrees celsius for 48 hours.Following the incubation, use filtered sterilized PBS to dilute the cultures to 10 to the fifth cells per milliliter. Then use flow cytometry with the cyber green propidium iodide stain to measure the cell number. Evaluating the microbiome community composition and viability is essential for avoiding bias in our experiment.Remove the cell culture medium from the flasks, then use five milliliters of PBS without calcium or magnesium to wash the cells once. Add two milliliters of trypsin EDTA solution to the cells. Distribute the solution by gently moving the flask.Then remove 1.5 milliliters of the trypsin solution, and incubate the flask at 37 degrees celsius for 10 minutes. After checking under the microscope if the cells have become detached, add five milliliters of supplemented cell culture medium to the flask to inactivate the trypsin, and pipette up and down to obtain a homogenous single cell suspension. Immediately mix a 50 microliter aliquot of the cell suspension with sterile 0.4%trypan blue solution in a 1:1 volume to volume proportion for KCO2 cells for a 1:5 volume to volume for HT29MTX.Mix by pipetting, and load the suspension into a counting chamber before counting the cells under the microscope. After preparing KCO2 and HT29MTX cell suspensions according to the text protocol, homogenize the suspensions, and add the corresponding volume of KCO2 and HT29MTX cells to a pre-filled sterile container with supplemented cell culture medium. Aspirate the pre-incubated medium from the apical side of the previously prepared chamber.Gently mix the cell suspension by pipetting, and transfer 1.5 milliliters to the apical compartment of the chamber inserts mixing the cell suspension every one to three wells to ensure constant homogenization. Gently move the plates back and forth and then right to left to evenly spread the cells in the well. Transfer the plates to the incubator, and repeat the movement of the plates.The sitting of the cells need to be done with constant homogenization of the cell suspension to ensure an even spread in the well. To measure the epithelial barrier integrity before starting the ASA, ensure that tier equipment is fully charged before starting the measurements. Disconnect the tier equipment from the power supply, and cover with a plastic autoclave bag.Make a hole in the bag to introduce the electrode into the input port on the meter. Then spray the bag with 70%ethanol before transferring the tier equipment into the flow cabinet. To disinfect the electrode, immerse the electrode tips in 70%ethanol for 15 minutes.Allow the tips to air dry for 15 seconds, then use cell culture medium to rinse the electrode. Allow the cells to come to room temperature inside the flow cabinet. Make sure that the meter is disconnected from the charger, then set the mode switch to Ohms, and turn on the power switch.Measure the cell resistance by immersing the electrode with the shorter tip in the insert and the longer tip out of the well. Keep the electrode at a 90 degree angle to the plate insert. To simulate oral digestion, transfer three milliliters of the bacterial community in a 50 milliliter sterile tube, and mix with three milliliters of simulated saliva fluid or SSF.Add 75 units per milliliter of alpha-amylase from human saliva type 9A and two grams per liter of mucin from porcine stomach type 2. Incubate the mixture at 37 degrees celsius and 100 RPM for two minutes. Collect a two milliliter sample for DNA extraction and flow cytometry quantification.To simulate gastric digestion, add four milliliters of simulated gastric fluid or SGF to three milliliters of the bacteria. Use one molar HCl to adjust the pH to three if necessary. Incubate the sample at 37 degrees celsius and 100 RPM for two minutes.Then collect a two milliliter sample. For small intestine digestions, add four milliliters simulated intestinal fluid or SIF to the bacteria. Use sodium hydroxide to adjust the pH if necessary.Then incubate the sample at 37 degrees celsius and 100 RPM for two minutes. And collect a two milliliter sample. After washing and adjusting the cell density of the small intestine digestion according to the text protocol, remove the apical medium from the transwell system, and add 1.5 milliliters of the bacterial suspension.Co-culture the system under general cell-culture conditions for two hours. Following incubation, measure the tier values to evaluate the integrity of the epithelial barrier as demonstrated earlier in the video. Add 0.5 milliliters of 10 millimolar n-acetylcysteine and HPSS to solubilize the mucus produced by the HT29MTX and to recover the adhered bacteria.Incubate the plates at 37 degrees celsius for one hour under agitation. Recover the NAC HPSS in a microcentrifuge tube, and use one milliliter of DPBS to wash the cells twice. Add 0.5 milliliters of 0.5%triton-x100 on top of the monolayers, and disrupt the cells by pipetting.Then quickly recover the liquid in vortex for one minute. Speed is crucial to avoid damaging the bacterial cells with the detergent. Analyze the samples according to the text protocol.As reported here, the counts of intact cells from individual strains were approximately 10 to the eighth cells per milliliter prior to the creation of the synthetic community while the multi-species microcosm contained above 90%viable cells during the establishment of the community. Based on the live-dead quantification, bacterial viability decreases after each digestion step as the result of acid pH during gastric passage and of the bio-salts contained in the small intestine digestion fluids. The harsh conditions of stomach and small intestine digestion can switch bacteria to viable but not in cultureable cells which are live bacteria that do not either grow or divide.As demonstrated in this figure, viable cells recovered from the mucosal interface are colored blue in the flow-cytometry plot. However, no growth was observed when these mucus samples were plating. The fraction in which bacteria show higher viability rates is the cellular debris as revealed by plate counting.The number of colonies may be variable, but the presence of metabolically active bacteria is found in the less diluted samples. After this development, this technique can be used by researches from the fields of nutrigenomics or pharmacomicrobiomics to study the host microcosmically. Following this protocol, other techniques like transcriptome or metabolic analysis can be performed to obtain mechanistic understanding in the successful colonization process accordingly.After watching this video, you should have a good understanding in how to establish complex in vitro model representative of the digestive processes including the cross talk between the host cells and the rest of the microbiome. Don't forget that working with pathogenic bacteria can be extremely hazardous, and it's important to consider bio-safety precautions.

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