The authors acknowledge the Rehovot-Hohenheim partnership program and Deutsche Forschungsgemeinschaft (DFG) SE 2059/7-2. This project was developed as part of the research unit P-FOWL (FOR 2601).

The protocol is divided into four parts: sampling, bacterial isolation, identification, and preservation of the obtained microorganisms. Approved permissions on the use of animals were issued by the ethical commission of Regierungspr&#228;sidium T&#252;bingen, Germany with the approval numbers HOH50/17 TE and HOH67-21TE.
1. Obtaining samples for the cultivation of anaerobic bacteria
<ol>
	<li>Maintenance of animals
	<ol>
		<li>Maintain animals on an ad libitum commercial corn-based diet (Legehennen/ Junghennenfutter; see Table of Materials) and house at the experimental station of the University of Hohenheim.</li>
	</ol>
	</li>
	<li>Preparation of transport solution
	<ol>
		<li>At 1-2 days before sample collection, prepare the transport solution containing 1.00 g/L sodium thioglycolate, 0.10 g/L calcium chloride dihydrate (CaCl2.2H2O), and 0.5% of cysteine21 and 0.1% sodium resazurin solution.</li>
		<li>Adjust the pH of the medium to 6.0 &#177; 0.2 at 25 &#176;C and autoclave at 121 &#176;C for 15 min at 15&#160;psi pressure.</li>
		<li>After the medium has cooled to approximately 50 &#176;C, replace the cap of the medium bottle with sterile screw cap with a bore and a rubber stopper. Insert two sterile syringe needles into the rubber stopper at different positions and affix a hydrophobic syringe filter (0.22 &#181;m) onto one of the needles. 
		NOTE: Replacement of caps and insertion of needles and filter should be carried out inside a laminar air flow (LAF).</li>
		<li>Attach the 100% nitrogen (N2) tube to the syringe filter, and sparge N2 gas into the bottle for 10 min at 1 psi.</li>
		<li>Subsequently, transfer the medium bottle and sterile Hungate tubes into the anaerobic station and aseptically transfer the transport medium into the Hungate tubes. Completely fill the Hungate tubes to the top, ensuring absence of any air inside.</li>
	</ol>
	</li>
	<li>Slaughtering and sample collection
	<ol>
		<li>During the slaughtering process, maintain axenic conditions to avoid contamination by using gloves, mask, plastic lab apron, and sterile surgical instruments throughout the entire procedure. Moreover, properly sanitize the working bench using ethanol and tissues to uphold a sterile environment.</li>
		<li>In the experimental station, anesthetize 10, 50-week-old Lohmann laying hens using a bottled gas mixture consisting of 35% N2, 35% CO2, and 30% O2 and immediately decapitate them. 
		NOTE: The anesthetization and sacrificing should be conducted by a laboratory animal technician well-versed in animal welfare and scientific procedures. This ensures that the sacrificing procedure adheres to the ethical standards, legal regulations, and safety guidelines.</li>
		<li>Before proceeding with the dissection of the GIT section, secure the required intestinal segment, namely the crop, ileum, and jejunum, using sterile Kelly hemostat (also known as a Kelly clamp; Figure 1). 
		NOTE: The dissection of the animal should be performed by either a veterinarian or a well-trained person who is familiar with the anatomy of chickens.</li>
		<li>Transfer each individual section into a separate Hungate tube filled with transport medium and close the lid properly. If required, add extra transport medium to the tube to displace any remaining air. 
		NOTE: The transfer of the section is a crucial step, and it is essential to minimize any delay or opening of the section to prevent prolonged exposure to oxygen</li>
	</ol>
	</li>
	<li>Transportation of sample
	<ol>
		<li>Handle the transportation of the Hungate tubes with care, placing them inside a Styrofoam box to prevent any risk of breakage. In case the outside temperature is lower than 15 &#176;C, maintain the temperature of the sample by using warm packs. Ensure the time of transportation does not exceed 4 h.</li>
		<li>Transfer the tubes carefully inside the anaerobic station for further process.</li>
	</ol>
	</li>
</ol>
2. Isolation of anaerobic bacteria
<ol>
	<li>Culture media preparation 
	NOTE: The outlined method presents various considerations for culture media preparation. Table 1 illustrates an example of how solutions are divided for the diverse isolation of microorganisms.
	<ol>
		<li>Divide the ingredients of the media formulation to be used in four solution-groups: carbon source, nitrogen source, agar, and mineral source. For rich media like Brain-Heart infusion (BHI), separation is not always possible (see example of medium 2 in Table 1). In such cases, ensure separate preparation and autoclaving of agar or additional solutions.</li>
		<li>Prepare the carbohydrate solution in 1/4th of the final volume of media required, e.g., in 250 mL for a total volume of 1 L of media. Following the example of medium 1 in Table 1, dissolve 2 g of dextrose and 0.5 g of starch in 250 mL of distilled water.</li>
		<li>Prepare the protein solution in 1/4th of the final volume of media required, e.g., in 250 mL for a total volume of 1 L of media. Following the example of medium 1 in Table 1, dissolve 10 g of peptone in 250 mL of distilled water.</li>
		<li>Prepare agar solution in 45% to 48% of the final volume of media, e.g., in 450 to 480 mL for a total volume of 1 L of media. Following the example of mediu 1 in Table 1, dissolve 15 g of agar in 450 to 480 mL of distilled water.</li>
		<li>Prepare the mineral solution in the left 20 to 50 mL volume of media when the final volume of media is 1 L. Following the example of medium 1 in Table 1, dissolve 0.5 g of K2HPO4 in 20 to 50 mL of distilled water.</li>
		<li>Autoclave the protein solution, agar solution, and mineral solutions at 121 &#176;C for 15 min at 21 psi pressure. Autoclave the carbohydrate solution at 110 &#176;C for 30 min at 5 psi.</li>
		<li>Once all solutions are sterile, cool them down to 50 &#176;C and mix under sterile conditions into an appropriate autoclaved glass bottle with a cap having a bore and a rubber stopper. After this, follow steps 1.2.3 and 1.2.4. 
		NOTE: The mixing of solutions should be done inside a LAF.</li>
		<li>Once the media has been mixed and degassed, pour into sterile Petri dishes or tubes inside the anaerobic station.</li>
	</ol>
	</li>
	<li>Isolation of anaerobic bacteria 
	NOTE: The following methodology explains an isolation strategy for cultivating a broad range of anaerobic bacteria that inhabit the digestive tract of chicken.
	<ol>
		<li>Transfer the samples to an anaerobic station containing a bottled gas mixture composed of 80% N2 (quality level 5.0), 15% CO2 (quality level 3.0) and 5% H2 (quality level 5.0) provided by a commercial supplier (see Table of Materials).</li>
		<li>Inside the anaerobic station, transfer the section from the hungate tube to the sterile Petri dish using autoclaved forceps.</li>
		<li>Carefully cut open the section using a sterile pair of scissors and extract approximately 1 g of digesta content from the intestinal segment using a sterile spatula. 
		NOTE: After taking 1 g of digesta content, scrap and transfer the remaining digesta to its respective transport media tube for the targeted isolation.</li>
		<li>Perform a 10-fold serial dilution using a sterile physiological solution (0.85% NaCl).</li>
		<li>Dispense 0.1 mL of the sample from dilutions 10&#8722;4 to 10&#8722;7 into sterile and properly labeled Petri dishes media plates and spread with a sterile Drigalski spatel.</li>
		<li>Incubate the plates inside the anaerobic station for 24&#8211;48 h at 39 &#176;C. 
		NOTE: When incubating inside an incubator, transfer the Petri dishes into an airtight box before taking the Petri dishes out of the anaerobic station.</li>
	</ol>
	</li>
	<li>Targeted isolation of a specific bacterial group. 
	NOTE: The following methodology explains an isolation strategy for anaerobic bacteria that can potentially assimilate MI.
	<ol>
		<li>For the preparation of enrichment medium, minimal agar medium, and minimal broth medium, follow the composition specified in Table 2 and Table 3, adhering to the guidelines outlined in step 2.1. Add 0.2 mL/L of filter-sterile vitamin mixture (as outlined in the composition) into minimal agar media and minimal broth media after the autoclaving process.</li>
		<li>Homogenize the initial sample using a vortex mixer for 10-15 s. Subsequently, transfer the homogenized sample into the tube containing enrichment medium at a rate of 5% (e.g. if the volume of enrichment media is 10 mL, inoculate 0.5 mL of homogenized sample) using a pipette. Incubate the freshly inoculated sample for 24 h at 39 &#176;C inside the anaerobic station.</li>
		<li>Following incubation, mix the enriched tube thoroughly by either using a vortex mixer for 10-15 s or by pipetting. Subsequently, transfer the mixed sample to sterile minimal broth medium at a rate of 5% and then incubate for 24 h at 39 &#176;C.</li>
		<li>Serially dilute the samples in sterile saline solution (i.e., 0.85% NaCl), starting with a 1:10 dilution and continuing till 1:1000 dilution. Following each dilution, homogenize the sample thoroughly at room temperature by using a vortex mixer for 10-15 s or by pipetting.</li>
		<li>Under sterile and anaerobic conditions, transfer 1 mL of diluted sample to a Petri dish. Pour approximately 15-20 mL of melted minimal agar medium into the Petri dish and gently swirl horizontally for uniform mixing. 
		NOTE: The temperature of agar should be around 45 &#176;C so that no lumps are present and to prevent the sample from being killed by heat. Medium pouring should be done under anaerobic conditions.</li>
		<li>Upon agar solidification, incubate the Petri dishes for 24 h at 39 &#176;C in inverted position inside an anaerobic station or in incubator. After 24 h of incubation, bacterial colonies appear as distinct, small dots embedded in the media. 
		NOTE: When incubating the Petri dish inside an incubator, transfer the plates to an airtight box before taking them out of anaerobic station.</li>
		<li>Using a sterile inoculating loop, carefully pick individual bacterial colony and transfer it into separate tubes of minimal broth medium.</li>
		<li>After transferring the colonies to individual tubes, anaerobically incubate the inoculated tubes at 39 &#176;C for 24 h. After incubation, increased turbidity in the media will indicate bacterial growth. 
		NOTE: Every time before transferring the media bottles inside the anaerobic station, they should be degassed as mentioned earlier in step 2.1.7.&#160;Steps 2-8 should be carried out inside the anaerobic station.</li>
	</ol>
	</li>
</ol>
3. Identification of anaerobic bacteria
<ol>
	<li>Extract DNA from cultures of 24 h to 48 h incubation time, following an enzymatic lysis protocol22.</li>
	<li>Centrifuge the culture at 3000 x g for 10 min at 4 &#176;C and discard the supernatant using a pipette.</li>
	<li>Wash the pellet 2x with phosphate buffered saline (PBS) by suspending in 2 mL of PBS. Homogenize the sample by using a vortex mixer for 10-15 s. Repeat the centrifugation step at 3000 x g for 10 min at 4 &#176;C and discard the supernatant carefully.</li>
	<li>Suspend the cell pellet in 0.2 mL of PBS pH 7.4 and incubate at 50 &#176;C with 1 U of lysozyme and 1 U of recombinant mutanolysin for 30 min, followed by an incubation of 30 min at 56 &#176;C with 5 &#181;L of proteinase K.</li>
	<li>Add 4 &#181;L unit of RNAseA to each tube and incubate at room temperature for 10 min. Centrifuge tubes at 21168 x g for 1 min. Recover the supernatant and purify using magnetic beads.</li>
	<li>Perform quantification of DNA with a fluorometric method according to manufacturer instructions for dsDNA quantification kit (mentioned in Table of Materials). 
	NOTE: Extracted DNA can be stored at -20 &#176;C.</li>
	<li>Use DNA samples as templates for PCR amplification using the primers 27f and 1492r23 following the conditions shown in Table 4. To ensure that the PCR reaction was successful, perform agarose gel electrophoresis according to standard agarose gel electrophoresis method using 1X TAE buffer and 1% agarose.</li>
	<li>Purify the PCR product using a commercial PCR purification kit to get rid of primers, nucleotides, and other contaminants.</li>
	<li>Send the PCR product for Sanger sequencing to the suitable sequencing service provider. 
	NOTE: Along with sequencing, service providers can also provide services including purification of the PCR product with extra fees. PCR products can be stored at 4 &#176;C or at -20&#176;C.</li>
	<li>After getting the sequencing results in FASTA format along with chromatogram file (commonly in ABI or SCF format), analyze sequences using bioinformatics tools. Use sequence analysis software to open and visualize the chromatogram file. 
	NOTE: The overall quality of chromatogram was checked based on clear and distinct peaks without excessive noise</li>
	<li>Compare amplicons and align to the closely related species at the non-redundant GenBank 16S ribosomal RNA database from the National Center for Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) tool24 and the Refseq genome database. 
	NOTE: When using NCBI BLAST, copy the whole FASTA sequence in the search bar.</li>
</ol>
4. Preservation of pure bacterial cultures
<ol>
	<li>Harvest bacterial cells from a well-grown culture of 24 h to 48 h either by centrifugation (3000 x g for 10 min) or biomass collection from an axenic culture on a plate.</li>
	<li>Suspend the bacterial culture in a suitable isotonic sterile solution (NaCl 0.85%) and wash by centrifuging the bacterial solution at 3000 x g for 10 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the bacterial pellet in a small volume of sterile isotonic solution (0.5 mL). To ensure the removal of any growth medium residue, repeat 1x.</li>
	<li>Resuspend the pellet in 0.8 mL to 1 mL of sterile culture medium concentrate and homogenize either with a vortex mixer for 5-10 s or by pipetting gently.</li>
	<li>Transfer 0.5 mL of cell suspension to a sterile and properly labelled 1 mL cryovial and add 0.5 mL of autoclaved 50% glycerol solution. Homogenize the cryovial using a vortex mixer, and store in a freezer rack at -80 &#176;C (Figure 3). 
	NOTE: All steps before transferring the cryovials to -80 &#176;C should be done inside the anaerobic station.</li>
</ol>

Cultivation Strategies for Uncultured Microbial Diversity in the Chicken GI Tract

<ol>
	<li>Wylensek, 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=A+collection+of+bacterial+isolates+from+the+pig+intestine+reveals+functional+and+taxonomic+diversity.">A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.</a> Nat Comm. 11 (1), 6389 (2020).</li><li>Medvecky, 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=Whole+genome+sequencing+and+function+prediction+of+133+gut+anaerobes+isolated+from+chicken+caecum+in+pure+cultures.">Whole genome sequencing and function prediction of 133 gut anaerobes isolated from chicken caecum in pure cultures.</a> BMC Geno. 19 (1), 561 (2018).</li><li>Zenner, 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=Early-life+immune+system+maturation+in+chickens+using+a+synthetic+community+of+cultured+gut+bacteria.">Early-life immune system maturation in chickens using a synthetic community of cultured gut bacteria.</a> mSystems. 6 (3), 1110-1128 (2021).</li><li>Borda-Molina, 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=Effects+on+the+ileal+microbiota+of+phosphorus+and+calcium+utilization,+bird+performance,+and+gender+in+japanese+quail.">Effects on the ileal microbiota of phosphorus and calcium utilization, bird performance, and gender in japanese quail.</a> Animals. 10 (5), 885 (2020).</li><li>Bonnet, M., Lagier, J. C., Raoult, D., Khelaifia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+culture+through+selective+and+non-selective+conditions:+The+evolution+of+culture+media+in+clinical+microbiology.">Bacterial culture through selective and non-selective conditions: The evolution of culture media in clinical microbiology.</a> New Micro New Infect. 34, 100622 (2020).</li><li>Tanaka, 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=A+hidden+pitfall+in+the+preparation+of+agar+media+undermines+microorganism+cultivability.">A hidden pitfall in the preparation of agar media undermines microorganism cultivability.</a> Appl Environ Microbiol. 80 (24), 7659-7666 (2014).</li><li>Kawasaki, K., Kamagata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate-catalyzed+hydrogen+peroxide+formation+from+agar,+gellan,+and+&#954;-carrageenan+and+recovery+of+microbial+cultivability+via+catalase+and+pyruvate.">Phosphate-catalyzed hydrogen peroxide formation from agar, gellan, and &#954;-carrageenan and recovery of microbial cultivability via catalase and pyruvate.</a> Appl Environ Microbiol. 83 (21), e01366-e01417 (2017).</li><li>Kato, S., 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=Isolation+of+previously+uncultured+slow-growing+bacteria+by+using+a+simple+modification+in+the+preparation+of+agar+media.">Isolation of previously uncultured slow-growing bacteria by using a simple modification in the preparation of agar media.</a> Appl Environ Microbiol. 84 (19), e00807-e00818 (2018).</li><li>Lewis, W. H., Tahon, G., Geesink, P., Sousa, D. Z., Ettema, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovations+to+culturing+the+uncultured+microbial+majority.">Innovations to culturing the uncultured microbial majority.</a> Nat Rev Microbiol. 19 (4), 225-240 (2021).</li><li>Su, X. B., Ko, A. L. A., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulations+of+myo-inositol+homeostasis:+Mechanisms,+implications,+and+perspectives.">Regulations of myo-inositol homeostasis: Mechanisms, implications, and perspectives.</a> Adv Biol Regul. 87, 2212-4926 (2023).</li><li>Monastra, G., Dinicola, S., Unfer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physiological and pathophysiological roles of inositols in A clinical guide to inositols. , (2023).</li><li>Kanehisa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+understanding+the+origin+and+evolution+of+cellular+organisms.">Toward understanding the origin and evolution of cellular organisms.</a> Prot Sci. 28 (11), 1947-1951 (2019).</li><li>Lee, S. A., Nagalakshmi, D., Raju, M. V., Rao, S. V. R., Bedford, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+phytase+superdosing,+myo-inositol+and+available+phosphorus+concentrations+on+performance+and+bone+mineralisation+in+broilers.">Effect of phytase superdosing, myo-inositol and available phosphorus concentrations on performance and bone mineralisation in broilers.</a> Animal Nutri. 3 (3), 247-251 (2017).</li><li>Farhadi, D., Karimi, A., Sadeghi, G., Rostamzadeh, J., Bedford, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+high+dose+of+microbial+phytase+and+myo-inositol+supplementation+on+growth+performance,+tibia+mineralization,+nutrient+digestibility,+litter+moisture+content,+and+foot+problems+in+broiler+chickens+fed+phosphorus-deficient+diets.">Effects of a high dose of microbial phytase and myo-inositol supplementation on growth performance, tibia mineralization, nutrient digestibility, litter moisture content, and foot problems in broiler chickens fed phosphorus-deficient diets.</a> Poultry Sci. 96 (10), 3664-3675 (2017).</li><li>Weber, M., Fuchs, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+in+the+niche:+A+large-scale+genome-based+survey+reveals+inositol+utilization+to+be+widespread+among+soil,+commensal,+and+pathogenic+bacteria.">Metabolism in the niche: A large-scale genome-based survey reveals inositol utilization to be widespread among soil, commensal, and pathogenic bacteria.</a> Microbio Spect. 10 (4), e02013-e02022 (2022).</li><li>Kawsar, H. I., Ohtani, K., Okumura, K., Hayashi, H., Shimizu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+transcriptional+regulation+of+myo-inositol+operon+in+Clostridium+perfringens.">Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens.</a> FEMS Microbiol Lett. 235 (2), 289-295 (2004).</li><li>Hellinckx, J., Heermann, R., Felsl, A., Fuchs, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+binding+affinity+of+repressor+IolR+avoids+costs+of+untimely+induction+of+myo-inositol+utilization+by+Salmonella+Typhimurium.">High binding affinity of repressor IolR avoids costs of untimely induction of myo-inositol utilization by Salmonella Typhimurium.</a> Sci Rep. 7 (1), 44362 (2017).</li><li>Yebra, M. 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=Identification+of+a+gene+cluster+enabling+Lactobacillus+casei+BL23+to+utilize+myo-inositol.">Identification of a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol.</a> Appl Environ Microbiol. 73 (12), 3850-3858 (2007).</li><li>Zhang, W. Y., 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=Comparative+analysis+of+iol+clusters+in+Lactobacillus+casei+strains.">Comparative analysis of iol clusters in Lactobacillus casei strains.</a> World J Microbiol Biotechnol. 26, 1949-1955 (2010).</li><li>Labeda, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bergey's manual of systematic bacteriology. , (2001).</li><li>Peterson, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+media+on+transport+and+recovery+of+anaerobic+bacteria.">Effect of media on transport and recovery of anaerobic bacteria.</a> Clin Infect Dis. 25, 134-136 (1997).</li><li>Ulrich, R. L., Hughes, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+procedure+for+isolating+chromosomal+DNA+from+Lactobacillus+species+and+other+Gram-positive+bacteria.">A rapid procedure for isolating chromosomal DNA from Lactobacillus species and other Gram-positive bacteria.</a> Lett Appl Microbiol. 32 (1), 52-56 (2008).</li><li>Relman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+bacterial+16s+rRNA+amplification+and+sequencing.">Universal bacterial 16s rRNA amplification and sequencing.</a> Diag Mol Microbiol. , 489-495 (1993).</li><li>Altschul, S. 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=Gapped+BLAST+and+PSI-BLAST:+A+new+generation+of+protein+database+search+programs.">Gapped BLAST and PSI-BLAST: A new generation of protein database search programs.</a> Nuc Acid Res. 25 (17), 3389-3402 (1997).</li><li>Rios-Galicia, B., 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=Novel+taxonomic+and+functional+diversity+of+eight+bacteria+from+the+upper+digestive+tract+of+chicken.">Novel taxonomic and functional diversity of eight bacteria from the upper digestive tract of chicken.</a> Int J Syst Evol Microbiol. 74 (1), 006210 (2024).</li><li>Lagier, J. 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=Microbial+culturomics:+Paradigm+shift+in+the+human+gut+microbiome+study.">Microbial culturomics: Paradigm shift in the human gut microbiome study.</a> Clin Microbiol Infect. 18 (12), 1185-1193 (2012).</li><li>Lagkouvardos, I., 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+mouse+intestinal+bacterial+collection+(mibc)+provides+host-specific+insight+into+cultured+diversity+and+functional+potential+of+the+gut+microbiota.">The mouse intestinal bacterial collection (mibc) provides host-specific insight into cultured diversity and functional potential of the gut microbiota.</a> Nat Microbiol. 1 (10), 1-15 (2016).</li><li>Gilroy, 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=Extensive+microbial+diversity+within+the+chicken+gut+microbiome+revealed+by+metagenomics+and+culture.">Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture.</a> PeerJ. 9, e10941 (2021).</li><li>Huang, P., 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+chicken+gut+metagenome+and+the+modulatory+effects+of+plant-derived+benzylisoquinoline+alkaloids.">The chicken gut metagenome and the modulatory effects of plant-derived benzylisoquinoline alkaloids.</a> Microbiome. 6 (1), 1-17 (2018).</li><li>Zhang, Y., 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=Improved+microbial+genomes+and+gene+catalog+of+the+chicken+gut+from+metagenomic+sequencing+of+high-fidelity+long+reads.">Improved microbial genomes and gene catalog of the chicken gut from metagenomic sequencing of high-fidelity long reads.</a> GigaScience. 11, 116 (2022).</li><li>Segura-Wang, M., Grabner, N., Koestelbauer, A., Klose, V., Ghanbari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-resolved+metagenomics+of+the+chicken+gut+microbiome.">Genome-resolved metagenomics of the chicken gut microbiome.</a> Front Microbiol. 12, 726923 (2021).</li><li>Borrelli, R. C., Fogliano, V., Monti, S. M., Ames, 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=Characterization+of+melanoidins+from+a+glucose-glycine+model+system.">Characterization of melanoidins from a glucose-glycine model system.</a> Euro Food Res Technol. 215, 210-215 (2002).</li><li>Ferrario, 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=Untangling+the+cecal+microbiota+of+feral+chickens+by+culturomic+and+metagenomic+analyses.">Untangling the cecal microbiota of feral chickens by culturomic and metagenomic analyses.</a> Environ Microbiol. 19 (11), 4771-4783 (2017).</li><li>Crhanova, 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=Systematic+culturomics+shows+that+half+of+chicken+caecal+microbiota+members+can+be+grown+in+vitro+except+for+two+lineages+of+clostridiales+and+a+single+lineage+of+bacteroidetes.">Systematic culturomics shows that half of chicken caecal microbiota members can be grown in vitro except for two lineages of clostridiales and a single lineage of bacteroidetes.</a> Microorganisms. 7 (11), 496 (2019).</li><li>Rubio, 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=Lactobacilli+counts+in+crop,+ileum+and+caecum+of+growing+broiler+chickens+fed+on+practical+diets+containing+whole+or+dehulled+sweet+lupin+(lupinus+angustifolius)+seed+meal.">Lactobacilli counts in crop, ileum and caecum of growing broiler chickens fed on practical diets containing whole or dehulled sweet lupin (lupinus angustifolius) seed meal.</a> British Poult Sci. 39 (3), 354-359 (1998).</li><li>Bjerrum, 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=Microbial+community+composition+of+the+ileum+and+cecum+of+broiler+chickens+as+revealed+by+molecular+and+culture-based+techniques.">Microbial community composition of the ileum and cecum of broiler chickens as revealed by molecular and culture-based techniques.</a> Poult Sci. 85 (7), 1151-1164 (2006).</li></ol>

The authors declare that they do not have any competing financial or personal interests related to the work reported in this script.

The purpose of this methodology is to enhance the cultivation of anaerobic intestinal bacteria by improving the quality of sampling conditions, sample processing, and media formulation and preparation. The physicochemical conditions of samples (pH, the availability of carbon, nitrogen, and cofactors) must be taken into consideration when formulating the culture media. Compared to bacterial culture collections obtained from pigs, humans, or mice1,26,<su...

The resurgence of cultivation in studying microorganisms has complemented insights from metagenomic studies by providing material to test metabolic hypotheses that were previously only partially described and quantified. Cultivation of intestinal bacteria provides material to sustain future research on microbial-host interactions, facilitate targeted colonization studies, and improve molecular interaction studies1,2,3. The knowledge gained about gastrointestinal microorganisms has improved animal nutrition and welfare by influencing diet formulations and enhancing nutrient availability4. This understanding has contributed to performance improvements in utilizing prebiotic and probiotic interaction. However, in-depth research is required to gain a complete understanding of how biochemical and physicochemical conditions interact and impact the microbial profile and its structure. To achieve this objective, cultivation remains imperative, serving as a crucial tool to delve into the intricate dynamics of microbial communities within the gastrointestinal environment.
In contrast to the extensive research on microbes associated with the human gut and clinical cultivation studies5, reports on microorganisms from livestock have predominantly utilized a limited range of media for isolation, potentially constraining the diversity of isolates2,3. Furthermore, improvements in the formulation of media and studies on the interaction of phosphate and salts with agar, as elucidated by Tanaka et al. and Kawasaki et al., have not yet been implemented for gut-microbiome studies6,7,8,9.
Considered a semi-essential substance, myo-inositol (MI) has been reported to play a pivotal role in diverse metabolic, physiological, and regulatory processes10,11. These include involvement in bone mineralization, breast muscle development, cellular signaling, promotion of ovulation and fertility, modulation of neuronal signaling, and acting as a regulator of glucose homeostasis and insulin regulation in poultry10,11. MI plays a role as a precursor through its interconversion within pivotal biochemical processes, including the glycolysis/gluconeogenesis process, the citric acid cycle, and the pentose phosphate pathway. Additionally, it also serves as a precursor of phosphatidylinositol (PI), which is further involved in glycerophospholipid metabolism12. Few investigations have reported that the metabolization of MI leads to alterations in bone stability and animal performance. This includes enhancements in feed conversion rate and body weight gain, demonstrating its impact after absorption and utilization within the animal13,14. However, the pathway for MI metabolization and its impact on poultry metabolism remains elusive15. Furthermore, few studies propose a potential role of bacteria in MI utilization, particularly in regions of high metabolic activity such as the ileum16,17,18,19.
Efforts on cultivating bacteria from the GIT of animals aim to enhance genomic databases and expand research, verify genome-based hypothesis, and understand the ecological importance of these resources20. The objective of this work is to improve strategies for bacterial cultivation from the GIT of chicken to enhance the isolation diversity and the targeted isolation of an ecological group of interest that assimilate and metabolize myo-inositol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>VWR</td>
			<td>20104.334</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>VWR</td>
			<td>97064-332</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Carl Roth</td>
			<td>P726.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerobic station</td>
			<td>Don Whitley Scientific</td>
			<td>A35 HEPA</td>
			<td></td>
		</tr>
		<tr>
			<td>Butyric acid</td>
			<td>Merck</td>
			<td>8.0045.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate&#160;</td>
			<td>VWR</td>
			<td>97061-904</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken lysozyme (Muramidase)</td>
			<td>VWR</td>
			<td>1.05281.0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine</td>
			<td>VWR</td>
			<td>97061-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>VWR</td>
			<td>90000-908</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-potassium hydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>P749.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Legehennen/ Junghennenfutter</td>
			<td>Deutsche Tiernahrung Cremer GmbH &#38; Co. KG, D&#252;sseldorf, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>MagAttract HMW DNA Kit</td>
			<td>Qiagen</td>
			<td>67563</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Carl Roth</td>
			<td>2189.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed gas (80% N2 (quality level 5.0), 15% CO2 (quality level 3.0) and 5% H2 (quality level 5.0))</td>
			<td>Westfalen Gase GmbH, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Mutanolysin, recombinant (lyophilisate)</td>
			<td>A&#38;A Biotechnology</td>
			<td>1017-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Myo-inositol</td>
			<td>Carl Roth</td>
			<td>4191.2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>ChemSolute</td>
			<td>8418.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Carl Roth</td>
			<td>6026.1</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiFluor dsDNA System</td>
			<td>Promega</td>
			<td>E2671</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A&#160;</td>
			<td>QIAGEN Ribonuclease A (RNase A)&#160;</td>
			<td>19101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>27800.291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium resazurin</td>
			<td>VWR</td>
			<td>85019-296</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thioglycolate</td>
			<td>Sigma-Aldrich</td>
			<td>102933</td>
			<td></td>
		</tr>
		<tr>
			<td>Soy Peptone, GMO-Free, Animal-Free</td>
			<td>VWR</td>
			<td>97064-186</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth</td>
			<td>8952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween80</td>
			<td>Carl Roth</td>
			<td>9139.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin mix (supplement)</td>
			<td>VWR</td>
			<td>968290NL</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Star Lab</td>
			<td>07127/92930</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Carl Roth</td>
			<td>9257.05</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-D-Fructose</td>
			<td>VWR</td>
			<td>53188-23-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

strategies to enhance cultivation of anaerobic bacteria from gastrointestinal tract of chicken

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken&#39;s GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken&#39;s GIT.

Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

We aim to isolate and cultivate bacteria from the intestine of chickens to improve our knowledge of microbial networks and interactions with the host. The characterization of these anaerobic bacteria supports the knowledge of intestinal processes and enrich bacteria databases for future research. One of the main challenges is the prevention of oxygen exposition and oxygen-reactive species along the sampling and cultivation process to prevent bacterial inactivation.To our experience, this has improved the diversity recovery of anaerobic bacteria. Our protocol addresses the gap in understanding myo-inositol metabolism in poultry. By isolating potential myo-inositol-metabolizing bacteria from chicken intestine, our goal is to shed light on the specific bacterial community and metabolic pathways involved, advancing knowledge in this area.Our primary goal is understanding how microbial communities interact with a host, especially in the gut. This is where diet is broken down, nutrients are absorbed, and significant interactions occurs with these microorganisms.

Monitoring of anaerobic conditions during transportation 
Due to addition of sodium resazurin, the change in color of transport solution to pink before the transfer of sample into the tube indicates a disruption or failure in maintenance of anaerobic conditions. Hence, the tube showing color change were refrained from being used during transport and only the tubes showing no color change were used, as can be seen in Figure 2.
Analysis...

Read this Scientific Article Protocol about Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry at JoVE.com

The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance.

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...

strategies-to-enhance-cultivation-anaerobic-bacteria-from

Research

JoVE Journal

Biology

Anaerobic Bacterial Isolation from Chicken Digesta as a Cultivation Improvement

To begin, combine 250 milliliters of each carbohydrate, protein, agar and mineral solutions in an autoclave glass bottle. Seal the bottle with a cap having a bore and a rubber stopper. After degassing the mixture with nitrogen, pour it into sterile Petri dishes inside an anaerobic station.Next, transfer the chicken intestinal samples to the anaerobic station containing a bottled gas mixture of nitrogen, carbon dioxide, and hydrogen. With a pair of sterile forceps, transfer the intestinal samples from the Hungate tube to the sterile Petri dish. Use sterile scissors to carefully cut the open section.Then use a sterile spatula to extract one gram of the digested content. Transfer the digested content into a tube containing a sterile physiological solution, and perform tenfold serial dilution. Pipette 0.1 milliliter of the sample from dilutions 10 to the negative fourth to 10 to the negative seven into sterile and properly-labeled media plates.Spread the sample with a sterile Drigalski spatula. Finally incubate the Petri dishes inside the anaerobic station in an inverted position for 24 to 48 hours at 39 degrees Celsius. 15 different genera of bacteria were isolated.The diversity of isolates includes eight strains, demonstrating novel taxonomic species related to members of the families Clostridiaceae, Lactobacillaceae, and Oscillospiraceae.

Targeted Isolation of Specific Myo-Inositol Utilizing Bacterial Groups from Chicken Digesta

To begin, homogenized chicken digest are harvested from an extracted chicken intestine on a vortex for 10 to 15 seconds. Transfer the homogenized sample into the tube containing enrichment medium. After incubation, use a vortex mixer to mix the enriched tube thoroughly for 10 to 15 seconds.Then transfer the mixed sample to sterile minimal broth medium before incubating as before. Next, serially dilute the samples in sterile saline solution, starting with a one to 10 dilution and continuing until one to 1000 dilution. Homogenize the sample thoroughly on a vortex mixer after each dilution.Transfer one milliliter of the diluted sample to a Petri dish under sterile and anaerobic conditions. Then pour 15 to 20 milliliters of melted minimal agar medium into the Petri dish. Swirl the dish gently to ensure uniform mixing.When the agar has solidified, incubate the dishes in an inverted position in an anaerobic station for 24 hours. at 39 degrees Celsius. The colonies should appear as distinct, small embedded dots at the end of incubation.Now use a sterile inoculation loop to carefully pick each individual bacterial colony. Transfer the colonies into separate tubes containing five milliliters of minimal broth medium. The increased turbidity of the end of incubation is indicative of bacterial growth.Bacterial colonies capable of utilizing myo-inositol were observed on minimal agar medium.

481 Views.  University of Hohenheim.  The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance. 

Cultivation Strategies for Uncultured Microbial Diversity in the Chicken GI Tract

Podsumowanie