Pseudomonas aeruginosa Counting Service

Pseudomonas aeruginosa Counting Procedure

Materials and Equipment

• Sample: e.g., water, environmental, or clinical sample.
• Sterile containers and pipettes
• Sterile dilution blanks (e.g., 0.1% peptone water or sterile saline)
• Membrane filtration apparatus
• 0.45 µm pore-size sterile membrane filters
• Selective Agar Plates: Typically, cetrimide agar (or another selective medium like Pseudomonas CN agar) is used.
• Incubator: Set at 35–37°C.
• Forceps: Sterile, for handling membrane filters.
• Disposable gloves and lab coat: For personal protection.
• Colony counter (manual or automated), if available

Procedure

1. Sample Preparation and Dilution

   • a. Homogenize the Sample (if necessary):
     If you are dealing with a complex matrix (e.g., food or soil), homogenize the sample in sterile saline or buffer.
   • b. Prepare Serial Dilutions:
     To ensure countable colony numbers, prepare serial dilutions (e.g., 10^–1, 10^–2, 10^–3, etc.) using sterile dilution blanks. For water samples with expected low bacterial counts, you might filter the entire volume (e.g., 100 mL) directly without dilution.

2. Membrane Filtration

   • a. Assemble the Filtration Apparatus:
     Set up the sterile membrane filtration system in a clean area (ideally in a biosafety cabinet if working with clinical or environmental pathogens).
   • b. Filter the Sample:
     Filter a measured volume (e.g., 100 mL or an aliquot from your dilutions) through a 0.45 µm sterile membrane filter. If you are using dilutions, record the volume filtered for each dilution.
   • c. Transfer the Filter:
     Using sterile forceps, carefully transfer the filter onto the surface of a cetrimide agar plate. Ensure the filter lies flat on the agar surface.

3. Incubation

   • a. Incubate the Plates:
     Place the agar plates in an incubator set at 35–37°C.
   • b. Incubation Time:
     Incubate for 24 to 48 hours. P. aeruginosa typically produces characteristic colonies (often with a bluish-green pigment due to pyocyanin production) within this timeframe.

4. Colony Identification and Counting

   • a. Identify Colonies:
     After incubation, inspect the plates for colonies that have the typical appearance of P. aeruginosa (rough, pigmented, sometimes with a fruity odor). Because cetrimide agar is selective, most colonies should be P. aeruginosa, but confirmatory tests (e.g., oxidase test, pigment production, Gram stain) may be warranted for regulatory purposes.
   • b. Count Colonies:
     Choose the dilution plate that yields 30–300 colonies (if possible) for accurate counting. Count each colony (either manually or with an automated colony counter).