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

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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><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&amp;nbsp;</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 &amp;amp; Co. KG, D&amp;uuml;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% N&lt;sub&gt;2&lt;/sub&gt; (quality level 5.0), 15% CO&lt;sub&gt;2&lt;/sub&gt; (quality level 3.0) and 5% H&lt;sub&gt;2&lt;/sub&gt; (quality level 5.0))</td><td>Westfalen Gase GmbH, Germany</td><td>-</td><td></td></tr><tr><td>Mutanolysin, recombinant (lyophilisate)</td><td>A&amp;amp;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&amp;nbsp;</td><td>QIAGEN Ribonuclease A (RNase A)&amp;nbsp;</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>&amp;beta;-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.

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

Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry

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.

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, ...

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Biology

642 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. 

Video: Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Immunology and Infection

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Biochemistry

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Environment

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

This study establishes a set of standard operating procedures (SOPs) for efficiently screening and isolating intestinal bacteria capable of cleaving C-glycosides.

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, ...

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines.&#160;The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry.&#160;Eggs were quickly collected after laying at a broiler breeder farm.&#160;They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator.&#160;The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Here we describe a method for the generation of germ-free chicks from eggs of a commercial broiler line, the Ross PM3. This method can be adapted for the generation of germ-free animals from other poultry species.

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed &#34;extremophiles&#34;, these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.
Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.
Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.
Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).
Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.
One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.
This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Enrichment Cultures, Selective and Differential Media

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, ...

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Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias