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