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