Cetrimide Agar (CA): Principle, Composition, Preparation, and Uses in Microbiology

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Cetrimide Agar (CA) is a selective and differential medium specifically designed for the isolation and identification of Pseudomonas aeruginosa from clinical, pharmaceutical, cosmetic, and water samples. The medium was originally formulated by King et al. and has become a gold standard in microbiology laboratories for detecting the characteristic pigment-producing bacteria.

🧪 Principle of Cetrimide Agar

Cetrimide (cetyltrimethylammonium bromide) is a quaternary ammonium compound that acts as a selective agent by inhibiting the growth of most microorganisms other than Pseudomonas aeruginosa. This bacterium has a unique resistance mechanism to cetrimide, allowing it to survive and grow while other bacteria are suppressed.

In addition, P. aeruginosa produces characteristic pigments such as pyocyanin (blue-green pigment), pyoverdin (fluorescent yellow-green pigment), and pyorubin (red-brown pigment). These pigments diffuse into the medium and can be easily visualized under normal or UV light, providing a simple differentiation method.

⚗️ Composition of Cetrimide Agar (per liter)

IngredientQuantity
Peptone20.0 g
Magnesium chloride1.4 g
Potassium sulfate10.0 g
Cetrimide0.3 g
Agar13.6 – 15.0 g
Distilled water1000 mL

Final pH: 7.2 ± 0.2 at 25°C

⚙️ Preparation Procedure

  1. Weigh and suspend 45.3 g of the dehydrated Cetrimide Agar medium in 1 liter of distilled water.
  2. Heat the mixture with gentle agitation until the medium dissolves completely.
  3. Autoclave at 121°C for 15 minutes.
  4. After sterilization, cool to 45–50°C and pour into sterile Petri plates.
  5. Allow the plates to solidify and store them in a refrigerator (2–8°C) if not used immediately.

🧫 Inoculation and Incubation

  1. Using a sterile inoculating loop, streak the sample onto the surface of solidified Cetrimide Agar plates.
  2. Incubate at 35–37°C for 24–48 hours.
  3. Observe the plates for typical growth and pigment production.

Note: For water or pharmaceutical samples, membrane filtration can be performed before inoculation to concentrate the microorganisms.

🌈 Interpretation of Results

OrganismGrowthPigment Production
Pseudomonas aeruginosaLuxuriant growthBlue-green or fluorescent yellow-green pigment
Other Pseudomonas speciesGood growthMay or may not produce pigment
Other bacteriaInhibited or no growth—

🧬 Quality Control (QC)

Before use, each new batch of Cetrimide Agar should be tested with known control strains to ensure selectivity and differential capability:

  • Pseudomonas aeruginosa ATCC 27853: Luxuriant growth with blue-green pigment
  • Escherichia coli ATCC 25922: Inhibited growth
  • Staphylococcus aureus ATCC 25923: Inhibited growth

🧯 Storage and Shelf Life

  • Store the dehydrated medium at 10–30°C in a tightly closed container.
  • Prepared plates should be stored at 2–8°C and used within 2 weeks.
  • Do not use the medium if it becomes discolored or shows signs of contamination.

📌 Applications of Cetrimide Agar

  • Isolation and identification of Pseudomonas aeruginosa from clinical samples (wounds, sputum, urine, etc.).
  • Microbial limit testing in pharmaceutical and cosmetic products (as per IP, BP, USP guidelines).
  • Water testing for the detection of P. aeruginosa in purified and potable water.
  • Environmental monitoring and sterility assurance in cleanrooms and microbiology labs.

🔬 Advantages of Cetrimide Agar

  • Highly selective for P. aeruginosa.
  • Enables pigment-based identification under UV light.
  • Easy to prepare and interpret results.
  • Recommended by major pharmacopeias (IP, BP, USP, EP).

⚠️ Limitations

  • Some Pseudomonas strains may lose pigment-producing ability.
  • Other fluorescent bacteria like Pseudomonas fluorescens may produce similar pigment.
  • Confirmatory biochemical tests (oxidase test, growth at 42°C) are required for final identification.

📚 References

  • Indian Pharmacopoeia (IP), Current Edition
  • United States Pharmacopeia (USP), Microbiological Examination of Non-sterile Products
  • British Pharmacopoeia (BP), Microbial Contamination Tests
  • King, E.O. et al. (1954). Journal of Laboratory and Clinical Medicine

💡 Summary

Cetrimide Agar is an essential medium in microbiology for isolating and identifying Pseudomonas aeruginosa. Its selective property and pigment-based differentiation make it invaluable in pharmaceutical, clinical, and environmental testing laboratories. By following proper preparation and incubation techniques, accurate identification of this pathogen can be achieved efficiently.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
Mobile: 09505626106

📱 Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.

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