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. 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 digestive content. Transfer the digestive 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 4 to 10 to the 7 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. To begin, homogenize chicken digesta 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 1, 000 dilution. Homogenize the sample thoroughly on a vortex mixture after each dilution. Transfer one milliliter of the diluted sample to a Petri dish under sterile and anaerobic conditions.

Then bore 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 at the end of incubation is indicative of bacterial growth. Bacterial colonies capable of utilizing myo-inositol were observed on minimal agar medium.