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

Abstract

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host'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'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's GIT.

Introduction

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 studies^1,2,3. 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 isolates^2,3. 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 studies^6,7,8,9.

Considered a semi-essential substance, myo-inositol (MI) has been reported to play a pivotal role in diverse metabolic, physiological, and regulatory processes^10,11. 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 poultry^10,11. 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 animal^13,14. 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 ileum^16,17,18,19.

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.

Protocol

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 Regierungspräsidium Tübingen, Germany with the approval numbers HOH50/17 TE and HOH67-21TE.

1. Obtaining samples for the cultivation of anaerobic bacteria

1. Maintenance of animals
   1. 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.
2. Preparation of transport solution
   1. 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.
   2. Adjust the pH of the medium to 6.0 ± 0.2 at 25 °C and autoclave at 121 °C for 15 min at 15 psi pressure.
   3. After the medium has cooled to approximately 50 °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 µm) 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).
   4. Attach the 100% nitrogen (N₂) tube to the syringe filter, and sparge N₂ gas into the bottle for 10 min at 1 psi.
   5. 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.
3. Slaughtering and sample collection
   1. 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.
   2. 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.
   3. 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.
   4. 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
4. Transportation of sample
   1. 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 °C, maintain the temperature of the sample by using warm packs. Ensure the time of transportation does not exceed 4 h.
   2. Transfer the tubes carefully inside the anaerobic station for further process.

2. Isolation of anaerobic bacteria

1. 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.
   1. 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.
   2. Prepare the carbohydrate solution in 1/4^th 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.
   3. Prepare the protein solution in 1/4^th 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.
   4. 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.
   5. 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.
   6. Autoclave the protein solution, agar solution, and mineral solutions at 121 °C for 15 min at 21 psi pressure. Autoclave the carbohydrate solution at 110 °C for 30 min at 5 psi.
   7. Once all solutions are sterile, cool them down to 50 °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.
   8. Once the media has been mixed and degassed, pour into sterile Petri dishes or tubes inside the anaerobic station.
2. 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.
   1. 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).
   2. Inside the anaerobic station, transfer the section from the hungate tube to the sterile Petri dish using autoclaved forceps.
   3. 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.
   4. Perform a 10-fold serial dilution using a sterile physiological solution (0.85% NaCl).
   5. Dispense 0.1 mL of the sample from dilutions 10−4 to 10−7 into sterile and properly labeled Petri dishes media plates and spread with a sterile Drigalski spatel.
   6. Incubate the plates inside the anaerobic station for 24–48 h at 39 °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.
3. Targeted isolation of a specific bacterial group.
   NOTE: The following methodology explains an isolation strategy for anaerobic bacteria that can potentially assimilate MI.
   1. 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.
   2. 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 °C inside the anaerobic station.
   3. 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 °C.
   4. 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.
   5. 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 °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.
   6. Upon agar solidification, incubate the Petri dishes for 24 h at 39 °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.
   7. Using a sterile inoculating loop, carefully pick individual bacterial colony and transfer it into separate tubes of minimal broth medium.
   8. After transferring the colonies to individual tubes, anaerobically incubate the inoculated tubes at 39 °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. Steps 2-8 should be carried out inside the anaerobic station.

3. Identification of anaerobic bacteria

1. Extract DNA from cultures of 24 h to 48 h incubation time, following an enzymatic lysis protocol²².
2. Centrifuge the culture at 3000 x g for 10 min at 4 °C and discard the supernatant using a pipette.
3. 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 °C and discard the supernatant carefully.
4. Suspend the cell pellet in 0.2 mL of PBS pH 7.4 and incubate at 50 °C with 1 U of lysozyme and 1 U of recombinant mutanolysin for 30 min, followed by an incubation of 30 min at 56 °C with 5 µL of proteinase K.
5. Add 4 µL 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.
6. 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 °C.
7. 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.
8. Purify the PCR product using a commercial PCR purification kit to get rid of primers, nucleotides, and other contaminants.
9. 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 °C or at -20°C.
10. 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
11. 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.

4. Preservation of pure bacterial cultures

1. 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.
2. 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 °C.
3. 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.
4. 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.
5. 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 °C (Figure 3).
   NOTE: All steps before transferring the cryovials to -80 °C should be done inside the anaerobic station.

Representative Results

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 of sequencing results
DNA extraction of isolates having chromatogram data with excessive background noise or disruption in baseline, peak with low signals intensity, presence of multiple or overlapping peaks at single position, and inconsistent peak heights were repeated using fresh and pure culture as they indicate contamination of the sample, uneven amplification, or problem during sequencing process. On performing BLAST search in NCBI, the strain showing highest percent identity and high query cover was the related strain to our isolate leading to the identification of the isolate.

Cultivation of a broad spectrum of anaerobic bacteria
The isolation of anaerobic bacteria resulted in more than 600 isolates obtained in five different media prepared and sterilized in separate bottles. The identification using the 16S rRNA gene resulted in 15 different genera of bacteria (Figure 4). Among the total number of isolates, more than half represent strict anaerobic bacteria and the rest are facultative anaerobic.

The diversity of isolates includes eight strains, demonstrating novel taxonomic species related to members of the families Clostridiaceae, Lactobacillaceae and Oscillospiraceae (Table 5)²⁵. These results highlight the significance of adhering to good practices in culture media preparation and use of diverse range of media for the success of cultivation studies.

Targeted isolation
The observation of colonies on minimal agar medium suggests the potential presence of anaerobic bacteria capable of utilizing MI as a carbon source (Figure 5). To conclusively confirm these findings at genetic level, comprehensive analysis through whole genome sequencing can be conducted. This sequencing approach enables the identification of specific genes associated with or contributing to MI metabolism, including iolA, iolB, iolC, iolD, iolE, iolF, iolG, iolH, iolI, iolJ, iolT, and iolW.

Figure 1: Process of sampling and transporting GIT sections for anaerobic bacteria cultivation. 1) Sections are clamped closed before cutting. The dotted lines indicate the recommended cut position. 2) Transfer of the sampled section into a Hungate tube filled with transportation solution. 3) Replacement of the air atmosphere by injecting transportation solution to maintain anaerobic conditions. Please click here to view a larger version of this figure.

Figure 2: Illustration of Hungate tubes containing samples obtained from ileum section of chicken. The image demonstrates proper filling of the Hungate tubes with transport solution, ensuring the absence of air space and maintenance of consistent color compared to the control having no sample. Noticeable turbidity in sample tubes is attributed to the dissolution of digesta. Please click here to view a larger version of this figure.

Figure 3: Sequential steps in the preservation of anaerobic bacteria. 1) Biomass harvest from 24 h to 48 h axenic culture in an isotonic solution. 2) Washing of bacterial cells by the process of centrifugation and supernatant removal to get rid of leftover media. 3) Repetition of washing process. 4) Pellet dissolution in culture media and 50% glycerol solution followed by storage at -80 °C in sterile cryovials. Please click here to view a larger version of this figure.

Figure 4: Taxonomic distribution of isolated bacteria at genus level. The stacked bar chart shows the diversity of bacteria isolated from five distinct media formulations, emphasizing the beneficial impact of using diverse media formulations in this study. The media formulation is listed in Table 1. Please click here to view a larger version of this figure.

Figure 5: Anaerobic bacterial colonies on minimal agar medium. Anaerobic bacterial colonies thriving on minimal agar media after 24 h of incubation at 39 °C. The development of colonies suggests the potential assimilation of myo-inositol as a primary carbon source, among other carbon sources. Two dilutions are presented, (A) 10^-1, revealing a colony count surpassing a practical enumeration due to their abundance; and (B) 10^-2, displaying distinguishable, countable colonies, facilitating selective analysis. Please click here to view a larger version of this figure.

Media	1	2	3	4	5
	Peptones media	Polysaccharides media	Short fatty acids media	Carbohydrates media	Sulphato-reducing bacteria (SRB) media
Source of C	Dextrose 0.2%	Poultry feed (2%)	Acetic acid 0.17%, Isovaleric acid 0.01%, Propionic acid 0.2%, Butyric acid 0.2%	Dextrose 5%	Na-DL-lactate, Ascorbic acid
	Starch 0.05%
Source of N	Amino acids from soybean (1%)	-	Amino acids from Tryptone and meat extract (0.1%)	Amino acids from soy peptones (0.1%), (NH4)3 citrate (0.1%)	NH4Cl (0.1%)
Source of minerals	K2HPO4 (0.05%)	-	K2HPO4 (0.05%)	K2HPO4 (0.05%)	K2HPO4 (0.05%)
Agar	1.50%	1.50%	1.50%	1.50%	1.50%
Cofactor (Added after sterilization)	Yeast extract (0.1%)	-	Vitamin mix (0.01%)	Vitamin mix (0.01%)	Yeast extract (0.01%)
Inhibitors	none	none	Tween 80 (1%)	Menadione (0.05%)	-

Table 1: Composition of the culture media for broad range of bacterial isolation. Composition of five agar media, each uniquely formulated based on variations in carbon source, nitrogen source, source of mineral, and the incorporation of co-factors and inhibitors.

Composition	Amount (g/L)
Casein peptone	17
Soya peptone	3
Sodium chloride	5
Dipotassium hydrogen phosphate	2.5
Myo-inositol	2.5

Table 2: Enrichment medium composition. Formulation of medium designed with a blend of complex ingredients to support the growth of a broad spectrum of bacteria. The quantity of each ingredient is specified in g/L.

Composition	Minimal Agar Media  (g/mL)/ L	Minimal Broth Media  (g/mL)/ L
L-cystine	0.5	0.5
Sodium chloride	0.3	0.3
Sodium thioglycolate	0.5	0.5
Sodium hydrogen phosphate dihydrate	0.53	0.53
Potassium dihydrogen phosphate	0.41	0.41
Ammonium chloride	0.3	0.3
Calcium chloride	0.11	0.11
Magnesium chloride	0.1	0.1
Tryptone	10	10
EDTA	0.00773	0.00773
Trace element solution SL7	1	1
Vitamin mix	0.2	0.2
Myo-inositol (20 mM)	3.6	3.6
Agar	15	--
pH (using 1 mM HCl)	6 ± 0.2	6 ± 0.2

Table 3: Minimal medium composition. The medium is formulated with myo-inositol as the primary carbon source.

Initial Denaturation	95 °C	4 min	1x
Denaturation	95 °C	30 s
Annealing	52 °C	30 s	30x
Extension	68 °C	90 s
Final Extension	68 °C	10 min	1x
	4 °C	∞

Table 4: Optimal conditions for 16S rRNA gene amplification. The ideal temperature, duration, and cycling parameters to be employed in the thermocycler for efficient amplification of the target gene.

Novel taxonomy assignation	Genome size	GC content	Gut region	16S accession
Limosilactobacillus galli	1861581	50.43	crop	OM760982
Limosilactobacillus avium	2158112	48.49	ileum	OM760983
Limosilactobacillus pulli	1924125	45.87	ileum	OQ831033
Clostridium butanoliproducens	3507657	28.34	crop	OQ831034
Faecalispora anaeroviscerum	2921858	50.36	ileum	OM760984
Limosilactobacillus viscerum	1993658	44.9	ileum	OM760986
Ligilactobacillus hohenheimensis	1381484	50.47	jejunum	OM760988
Limosilactobacillus difficilis	1998683	47.38	ileum	OQ832131

Table 5. Taxonomic identification of novel bacterial species. The novel bacterial species isolated and identified from the gastrointestinal tract of chickens in this study, are detailed alongside their genome size, GC content, section, and 16S rRNA gene accession. This table has been modified from²⁵.

Discussion

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 mice^1,26,27 report on selection of media for chicken is rather narrow and possibly underestimated even for sections of high interest such as feces and caeca^2,28. In this sense, the formulation and preparation of media used in this work offered 80 different conditions that intend to cover the metabolic demands of microorganisms reported by sequencing approaches in other works^29,30,31.

Along with the selection of media and supplementation, the formulation of culture media and interaction of the ingredients during preparation and sterilization is observed. The combination of phosphates and agar, reported to generate H₂O₂, was avoided by preparing the phosphate and agar solution separately. This modification improved the spectrum of isolates diversity by enhancing the viability of catalase negative bacteria. To avoid the formation of Maillard reaction products (MRP) during sterilization, carbohydrates were prepared in a separate solution and sterilized under different conditions. These two modifications increase the demand of material and worktime but provide the advantage to mix and formulate an exponential number of different media combinations.

The preparation of separate solutions in smaller volumes might represent a challenge during dissolution of the ingredients and finding adequate sterilization conditions to avoid precipitations. But culture media formulations normally do not reach saturation. We prefer filtered-sterilized mineral solutions dissolved at 60 °C to prevent precipitations. For solutions sterilized by heat and pressure in the autoclave, special attention was paid to carbohydrate solutions' oxidation at high temperatures and pressure. To avoid any chemical modification, all carbohydrate solutions must be sterilized at 110 °C for 30 min at 5 psi. These precautions improve the availability of nutrients such as essential amino acids, cofactors like copper, zinc, or iron, as well as the reduction on the hydrolytic activity of some proteins modified by the generated carbonyl compounds³².

Across studies, very few authors discuss the direct influence of culture media on the cultivation results. Common treatments applied in human cultivation studies (pre-incubation in blood culture, rumen fluid, or mucin) have been repeated indistinctively along gut culturomics studies on other species with different intestinal conditions^2,28,33. In this work, the plant-based diet given to chicken supported the modification on the formulation of media by using major components of plant-based nutrients (peptides and carbohydrates) that mimic the GIT environment found in chicken and considered nutrient availability.

Most of the studies have a cultivation approach on the lower digestive regions of chicken (caeca and feces)^2,3,28,34. Few cultivation approaches have isolated members of the family Lactobacillaceae, Clostridiaceae and Enterobacteriaceae^35,36 from the upper digestive tract. However, none of them used the modern protocols for culturomics. In 2021, Zenner et al., reported the sole isolation of Ligilactobacillus aviarius obtained from ileum of chicken while the rest of the cultures originated from caeca. Contrastingly, the diversity recovered in this work reported a redundant isolation of L. aviarius, together with other species from the genera Lactobacillus, Ligilactobacillus and Limosilactobacillus²⁵. This difference illustrates the advantage of using separately prepared media over commercially available ready-to-use formulations.

Maintaining anaerobic conditions throughout the isolation and cultivation process is a very crucial step, as even a small amount of oxygen can adversely affect the growth of anaerobes. To monitor and sustain anaerobic conditions during sample transportation, sodium resazurin is added to the transport solution. This chemical compound undergoes reduction when exposed to oxygen, resulting in a color transition from blue to pink. At low concentration, it does not impact bacterial viability. However, it is essential to avoid excessive concentrations, as they may lead to unintended impact on bacteria. Careful control of sodium resazurin levels ensures accurate monitoring of anaerobic conditions without compromising the integrity of the bacterial samples. For the targeted isolation, the pour plate method was employed using the spread plate method due to the advantage of maintaining a homogeneous distribution of bacterial colonies, facilitating the precise selection of individual bacteria. Furthermore, this method exhibits reduced susceptibility to airborne or external contamination. Additionally, it offers a consistent growth environment for diverse bacterial types, both fast- and slow-growing species allowing each bacterial strain to multiply at its own individual pace without interference from other microbial species.

For preservation, pellet-based glycerol stock was chosen over broth-based glycerol stock as it facilitates a higher bacterial concentration and lower susceptibility of contamination. This method proves to be an efficient method for preserving the bacterial culture at -80 °C, ensuring long-term storage stability.

The initial part of this methodology outlines an approach for isolating novel anaerobic cultures, with the primary goal to explore the diversity of previously uncultured strains within the chicken intestine. Cultivating and identifying anaerobic bacteria serves as a valuable tool for better understanding of poultry health, animal welfare, microbe-host interaction, and the potential isolation of probiotic strains. Additionally, it can also provide a deeper understanding of genetic makeup and metabolite capabilities, paving the way for advancements in manipulating the gut microbiota to achieve desired outcomes in poultry production. The subsequent part of the methodology focuses on the potential isolation of MI metabolizing anaerobes. This endeavor holds significant promise for elucidating the MI metabolization pathway, a process that has remained unclear to date. Notably, the versatility of these methodologies extends beyond chickens, offering an isolation technique applicable to various sample types. Therefore, the method presented in this study is one of the efficient approaches for isolation of anerobic cultures.

Materials

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