Cetrimide Agar: Composition, Principle, Preparation and Uses

Last updated on May 7th, 2021

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.

Principle

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.

  • Glycerol acts as the carbon source.
  • Magnesium chloride and potassium chloride enhance the production of pyocyanin and fluorescein.
  • Agar is the solidifying agent.
  • Cetrimide is the selective agent. It is a toxic substance that inhibits the growth of many bacteria.

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

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.

Expected Results

  1. Positive: Growth of organism is seen in the slant (see the image above)
  2. Negative: No growth

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

Quality Control:

  • Positive: Pseudomonas aeruginosa
  • Negative: Escherichia coli

Limitations

  • Growth on cetrimide agar alone is not sufficient for identification of P. aeruginosa to the species level, since other non-glucose-fermenting species such as Achromobacter xylosoxidans subsp. xylosoxidans and Alcaligenes faecalis may grow. Pigment must also be present.
  • Lack of growth on cetrimide agar does not rule out the identification of P. aeruginosa.
About Nisha Rijal 41 Articles
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.

1 Comment

  1. When we preserve any strain of Salmenella Can it be Preserved with out mutatic change ? After long term preservation will it change its biochemical test result or it will be same.?

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