Subculturing Sulfite-Reducing Anaerobic Bacteria from an Agar Culture

Begin with a differential agar plate pre-inoculated with a food sample and containing grown bacterial colonies.

Aerobic bacteria grow on the surface, while anaerobic bacteria develop deeper in the agar, where sulfite-reducing bacteria reduce sulfite to hydrogen sulfide.

The hydrogen sulfide then reacts with ferric ammonium citrate in the medium, forming a black precipitate.

Observe the agar plate for black spots in the deeper layers, indicating sulfite-reducing bacteria.

To subculture these bacteria, use a sterile cell scraper to gently remove the unwanted layer of aerobic bacteria, creating a clean entry point.

Insert a sterile eye dropper into the medium without introducing air to preserve anaerobic conditions.

Carefully extract the embedded black colony.

Transfer the colony into a sterile tube containing fresh growth medium.

Finally, seal the tube tightly and incubate it under anaerobic conditions to promote further growth of the sulfite-reducing bacteria.

After the incubation, examine plates for bacterial growth.

Aerobic bacteria may be present at the surface of the Agar, while anaerobic bacteria will be embedded in the Agar. Sulfite reducing bacteria will turn the surrounding Agar black, and possible C perfringens colonies will be black and embedded in the Agar. If there is a dense amount of aerobic bacterial growth on the surface of the plate, use a cell scraper to remove colonies from selected areas.

Use a sterile single-use eye dropper to pluck the suspected C perfringens colonies from the Agar, making sure to expel the air prior to piercing the Agar. Transfer the colonies to 10 milliliters of fresh RPM in a 15 milliliter conical tube. Multiple colonies could be sampled from the same plate into separate RPM cultures.

Tightly secure the conical tube lids, wrap them in paraffin film, and incubate them overnight at 37 degrees Celsius.