Cetrimide Agar: Composition, Preparation, Uses

By Nisha Rijal •  Updated: 04/11/22 •  4 min read

Cetrimide agar also known as pseudosel agar is used for the selective isolation and presumptive identification of Pseudomonas aeruginosa.

The medium was first developed by Lowburry and is a modification of Tech Agar (developed by King et al.) with the addition of 0.1% cetrimide (cetyl trimethyl ammonium bromide) for the selective inhibition of organisms other than Pseudomonas aeruginosa. Because of the increased purity of the inhibitory agent, the concentration was later reduced to 0.03%, as reported by Lowbury and Collins in1955.


Cetrimide is a quaternary ammonium salt, which acts as a cationic detergent when comes in contact with the bacterial cell, causes the release of nitrogen and phosphorous which in turn has denaturing effects on membrane proteins of a bacterial cell. It exhibits inhibitory actions on a wide variety of microorganisms including Pseudomonas species other than Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a number of water-soluble pigments, including the yellow-green or yellow-brown fluorescent pigment pyoverdin (fluorescein). When pyoverdin combines with the blue water-soluble pigment pyocyanin, the bright green color characteristic of Pseudomonas aeruginosa is created.

Cetrimide enhances the production of both pyocyanin and fluorescein pigment. Besides cetrimide, the media also contains gelatin pancreatic digest which provides nitrogen, vitamins, minerals, and amino acids essential for growth.

Composition of Cetrimide Agar

Ingredients Gram/liter
Enzymatic digest of gelatin20 g
Magnesium chloride1.4 g
Potassium chloride10 g
Cetrimide (cetyltrimethylammonium bromide)0.3 g
Glycerol10 mL
Agar13.6 g

Final pH: 7.2 ± 0.2 at 25°C

Quality Control:

Perform QC on each new lot or shipment of media prior to using them. Inspect cetrimide agar for evidence of contamination, cracks, dehydration, and bubbles prior to storage and before use. Perform performance testing of the media with following organisms;

  1. P. aeruginosa ATCC 27853—growth; yellow-green to blue pigment
  2. Escherichia coli ATCC 25922—inhibited

Procedure for media preparation

Cetrimide Agar A. Positive, B. Negative
Cetrimide Agar
A. Positive, B. Negative
  1. Suspend 45.3 g of the medium and 10 ml of glycerol in one liter of purified water.
  2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
  3. Autoclave at 121°C for 15 minutes.
  4. Mix well and pour into sterile Petri plates.

Typical colony characteristics

After 18 – 48 hr of incubation, the plates should show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation. Colonies may be presumptively identified as Pseudomonas aeruginosa when it exhibits a blue-green to green pigment and fluoresces under short wavelength (254 nm) ultraviolet light.

Note:  Certain strains of P. aeruginosa may not produce pyocyanin. Other species of Pseudomonas do not produce pyocyanin, but fluoresce under UV light. Some non-fermenters and some aerobic spore formers may exhibit a water-soluble tan to brown pigmentation on this medium. Serratia may exhibit pink pigmentation


Cetrimide test

To perform cetrimide test, take isolated colonies of non-glucose-fermentative, Gram-negative rods that are suggestive of P. aeruginosa and inoculate on cetrimide agar slant.


  1. Streak the cetrimide agar slant back and forth with inoculum picked from the center of a well-isolated colony.
  2. Place cap loosely on tube.
  3. Incubate aerobically at 35 to 37°C for up to 7 days.
  4. Observe for growth and pigment.
  5. If no pigment is visible, examine growth under UV light for the presence of fluorescein.

Expected Results

  1. Positive: Growth of organism is seen in the slant. Optionally a yellow-green (fluorescein) to dark blue-green (pyocyanin) color may be observed.
  2. Negative: No growth


References and further readings

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

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