to test the anti-microbial activity and efficacy

Standard protocol for an anti-microbial susceptibility test against Propionibacterium acnes:

Materials Needed:
- Propionibacterium acnes strain (e.g., ATCC 6919)
- Reinforced Clostridial broth or Brucella broth with hemin and vitamin K1
- Reinforced Clostridial agar or Brucella agar with hemin, vitamin K1, and 5% defibrinated sheep blood
- Antimicrobial agent(s) to be tested
- Sterile petri dishes
- Sterile swabs or spreaders
- Anaerobic incubator or anaerobic jar system
- Microbial loop or inoculating needle

Procedure:

1. Preparation of Propionibacterium acnes Culture:
   a. Inoculate Propionibacterium acnes into Reinforced Clostridial broth or Brucella broth with hemin and vitamin K1.
   b. Incubate the broth culture at 35-37°C for 48-72 hours under anaerobic conditions to obtain a fresh, actively growing culture.

2. Standardization of Inoculum:
   a. Adjust the turbidity of the broth culture to match the 0.5 McFarland turbidity standard (approximately 1-2 x 10^8 CFU/mL).
   b. This can be done by diluting the broth culture with sterile saline or broth.

3. Preparation of Agar Plates:
   a. Prepare Reinforced Clostridial agar or Brucella agar supplemented with hemin, vitamin K1, and 5% defibrinated sheep blood according to the manufacturer's instructions.
   b. Allow the agar to solidify and dry before use.

4. Inoculation of Agar Plates:
   a. Dip a sterile swab or spreader into the standardized inoculum and streak it evenly onto the agar plate to obtain a uniform lawn of bacterial growth.
   b. Allow the inoculum to dry for a few minutes before proceeding.

5. Application of Antimicrobial Agent(s):
   a. Depending on the type of antimicrobial agent (e.g., discs, wells, or strips), follow the appropriate method for applying the agent(s) to the inoculated agar plate.
   b. For disc diffusion method, place antimicrobial discs onto the inoculated agar surface using sterile forceps.
   c. For well diffusion method, cut wells into the agar using a sterile cork borer or similar tool, and pipette the antimicrobial solution into the wells.

6. Incubation:
   a. Incubate the inoculated agar plates at 35-37°C for 48-72 hours under anaerobic conditions (anaerobic incubator or anaerobic jar system).

7. Interpretation:
   a. After incubation, examine the plates for zones of inhibition around the antimicrobial agent(s).
   b. Measure the diameter of the zones of inhibition in millimeters (mm) using a caliper or ruler.
   c. Refer to standard guidelines or interpretive criteria (e.g., CLSI or EUCAST) to determine the susceptibility or resistance of Propionibacterium acnes to the tested antimicrobial agent(s).