Staphylococcus Aureus Counting Service

Materials and Equipment

• Sample (e.g., food, swab, or clinical specimen)
• Sterile diluent (e.g., buffered peptone water or saline solution)
• Stomacher or homogenizer (for solid samples)
• Sterile stomacher bags or tubes
• Pipettes and sterile pipette tips
• Sterile dilution tubes or containers
• Selective agar medium:
  • Baird–Parker Agar supplemented with egg yolk tellurite (commonly used for S. aureus enumeration)
    (Other selective media may be used depending on the protocol.)
• Sterile spreaders or disposable loops
• Incubator set at 35–37°C
• Colony counter or manual counting aids
• Personal Protective Equipment (PPE) (lab coat, gloves, eye protection)

Procedure

1. Sample Preparation

• Weigh or Measure the Sample:
  For solid samples (e.g., food), aseptically weigh 25 g and place it in a sterile stomacher bag. For liquid samples, measure an appropriate volume.

• Dilute the Sample:
  Add 225 mL of sterile diluent to a 25 g sample to obtain an initial 1:10 dilution. Mix thoroughly using a stomacher or by vigorous shaking to ensure a homogeneous suspension.

2. Serial Dilution

• Prepare Serial Dilutions:
  • Transfer 1 mL of the homogenized sample into 9 mL of sterile diluent to achieve a 1:100 dilution.
  • Continue making further tenfold (1:10) serial dilutions as needed to obtain dilutions that yield countable colony numbers on the plates (ideally between 30 and 300 colonies per plate).

3. Plating

• Plating Technique:
  • For each selected dilution, pipette 0.1 mL onto the surface of a pre-dried Baird–Parker agar plate.
  • Use a sterile spreader (or disposable spreader) to evenly distribute the inoculum over the agar surface.
  • Work carefully to avoid cross-contamination and to maintain aseptic conditions.

4. Incubation

• Incubate the Plates:
  • Place the inoculated plates in an incubator set to 35–37°C.
  • Incubate for 24 to 48 hours (check your specific protocol; some guidelines suggest 48 hours to allow for the typical colony morphology to develop).

5. Colony Identification and Counting

• Examine Plates:
  • After incubation, inspect the plates for colonies. On Baird–Parker agar, S. aureus typically produces characteristic colonies:
    • Appearance: Grey to black, shiny, convex colonies
    • Halo: Often surrounded by a clear zone (due to lecithinase activity)
• Count Colonies:
  • Select plates that have between 30 and 300 colonies for reliable enumeration.
  • Count all typical colonies on the plate. If counts vary between dilutions, choose the dilution that provides the most statistically reliable count.