This study investigates the interaction between Corynebacterium propinquum and pathogenic Staphylococcus in a bioassay setup. The focus is on how C. propinquum inhibits Staphylococcus growth through the secretion of siderophores.
Begin with bioassay plates, one pre-colonized on the left side of the nutrient agar with Corynebacterium propinquum and the other containing nutrient agar alone.
Next, take a multi-well plate containing pathogenic Staphylococcus suspension.
Lower an inoculation stamp into the wells, with its tips dipping into the Staphylococcus culture.
Swirl the stamp, then lift it and verify that each tip carries a uniform droplet to ensure consistent inoculation.
Align the stamp with the right side of the bioassay plate, avoiding contact with the existing colonies.
Remove the stamp, leaving Staphylococcus inoculum spots on the agar.
Repeat the stamping on the agar alone plate to generate a monoculture. Incubate the plates upside down.
In the coculture, C. propinquum secretes siderophores, small molecules that scavenge iron from the agar, thereby inhibiting Staphylococcus growth.
Post-incubation, compare Staphylococcus growth in the co-culture and the monoculture.
Significant inhibition in the co-culture suggests interaction between the two species.
After the six-day incubation of the bioassay plate, prepare overnight cultures of the target organism as previously described. Prepare the target plate by filling each well of an empty 12-well plate with 1.8 milliliters of BHI and 200 microliters of the target overnight culture. Place a sterile inoculation stamp into the target plate.
Gently swirl the cultures around the wells, ensuring that they do not cross-contaminate neighboring wells. Lift the inoculation stamp, and ensure that there is a droplet of diluted target culture on each stamp tip. Place the inoculation stamp on an uninoculated bioassay plate, and gently rock the stamp so that the culture droplet inoculates each well.
Once the stamp is removed, a droplet of culture should be visible in the wells. If any wells are not inoculated with the inoculation stamp, spot 3 microliters of diluted overnight culture onto the wells using a pipette. Then, inoculate the bioassay plates as previously described, but align the stamp tips with the right side of the 12-well plate, ensuring that the stamp does not contact the existing bacterial colony. Carefully remove the stamp and place it back into the target plate.
If the agar has solidified in the wells unevenly, gently rock the stamp back and forth, but be careful not to shift the stamp into the test organism growth.
Repeat the inoculation for each bioassay plate until all the plates are inoculated, then incubate the plates upside down at the appropriate temperature for seven days.
After co-culturing the test and target organism for one week, score the interactions based on visual assessment. Score the wells with the target organism growth that exhibits indistinguishable growth from the monoculture as zero. Score the wells with diminished growth as one, and no growth as two.
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