Cetrimide Agar Test- Principle, Procedures, Results, Uses

Last Updated on October 10, 2020 by Sagar Aryal

What is Cetrimide Agar Test?

Cetrimide agar test is a biochemical test performed to identify or differentiate Pseudomonas aeruginosa from other microorganisms.

  • The test works on the principle of the ability of an organism to grow in the presence of cetrimide.
  • Cetrimide is a toxic quaternary ammonium detergent that is toxic to most bacteria except for few organisms like P. aeruginosa.
  • Pseudomonas aeruginosa is a common pathogen commonly associated with infections like urinary tract infections and bacteremia in humans.
  • The ability of the organism to survive in the presence of cetrimide enables the test to be used for the differentiation of P. aeruginosa from other microorganisms.
  • P. aeruginosa is identified on the basis of their characteristic production of pyocyanin, a blue, water-soluble, non-fluorescent phenazine pigment, the characteristic grape-like odor of aminoacetophenone, and their colony morphology.
  • P. aeruginosa is the only Pseudomonas species or gram-negative rod known to excrete pyocyanin, which is detected in this test.

Objectives of Cetrimide Agar Test

  • To identify and differentiate P. aeruginosa from other microorganisms.
  • To detect the ability of different organisms to grow on cetrimide agar.

Principle of Cetrimide Agar Test

  • Cetrimide is a quaternary ammonium salt, which acts as a cationic detergent that is toxic to most bacterial cells.
  • Cetrimide reduces surface tension in the point of contact and results in precipitant, complexing and denaturing effects on bacterial membrane proteins.
  • Inhibition of growth is observed in a wide variety of microorganisms including Pseudomonas species other than Pseudomonas aeruginosa.
  • When cetrimide is in contact with bacteria, nitrogen and phosphorus are released from the bacterial cell.
  • Organisms other than P. aeruginosa and a few other Pseudomonas are unable to withstand this germicidal activity.
  • The media further contains gelatin peptone that provides the necessary nutrients for P. aeruginosa.
  • Sodium chloride maintains the osmotic equilibrium of the medium and prevents the change of pH of the medium during growth. Magnesium chloride and potassium sulfate in the medium enhance the production of pyocyanin and pyoverdin (fluorescein) by  P. aeruginosa.

Microorganism tested

Isolated colonies of non-glucose-fermentative, Gram-negative rods that are suggestive of P. aeruginosa.

Media, Reagents, and Supplies Used

Medium Used

  • Cetrimide Agar can be bought commercially in the form of dehydrated powder. It can also be prepared in the lab if the necessary constituents of the media are available.
  • The following is the composition of the cetrimide agar:
S.N Ingredients Gram/liter
1. Gelatin peptone 20.0
2. Magnesium chloride 1.4
3. Potassium sulfate 10.0
4. Cetrimide 0.3
5. Agar 15.0
Final pH at 25°C: 7.2 ±0.2

Supplies Used

  • Sterile inoculating loops or sticks
  • Wood’s or UV light (360 nm) or short-wavelength (254-nm) UV light

Procedure of Cetrimide Agar Test

Preparation of media

  • In a beaker, 46.7 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water containing 10 ml glycerol.
  • The medium is heated up to boiling in order to dissolve the medium completely.
  • The boiled medium is then distributed into tubes and sterilized in an autoclave at 15 lbs pressure (121°C) for 15 minutes.
  • If necessary, rehydrated contents of 1 vial of Nalidixic Selective Supplement (FD130) can be added aseptically to the 1000 ml medium.
  • The tubes are taken out after autoclaving and cooled at a slanted position to a temperature of about 40-45°C. The position should be maintained at an appropriate angle to obtain butts of 1.5 – 2.0 cm depth.

Cetrimide Agar Test

  • A well-isolated colony is collected from an 18-24 hour culture with a sterile inoculating needle or loop.
  • The cetrimide agar tubes are inoculated by streaking the surface of the slant. The slant should be streaked back and forth to ensure proper inoculation with the loop or the inoculating stick.
  • The cap of the test tubes should be left loosened to ensure adequate aeration.
  • The tubes are then incubated aerobically at 35-37°C for up to 7 days.
  • The test tubes should be examined daily for 4 days and again at 7 days before discarding the result as a negative.

Quality Control

  • As a form of quality control for the cetrimide agar test, two different organisms can be taken as a positive and negative control.
Control Incubation Results
P. aeruginosa Aerobic incubation at 33-37°C for 24-48 hours. Cetrimide agar positive (growth; yellow-green to blue pigment)
Escherichia coli Aerobic incubation at 33-37°C for 24-48 hours. Cetrimide agar negative (no growth)

Result and Interpretation of Cetrimide Agar Test

Cetrimide Agar Test- Principle, Procedure and Result Interpretation

Figure: Cetrimide Agar Test. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

  • A positive result is demonstrated by growth. Optionally a yellow-green (fluorescein) to dark blue-green (pyocyanin) color may be observed.
  • The inhibition of growth indicates a negative result.

Reporting results

  • aeruginosa is definitively identified if an oxidase-positive, Gram-negative rod grows on cetrimide agar and produces a blue-green (pyocyanin) pigment.
  • Pseudomonas fluorescens and Pseudomonas putida may also grow and may produce a fluorescent pigment on this medium but are separated from P. aeruginosa because they do not grow at 42°C.

Uses of Cetrimide Agar Test

  • Cetrimide agar test is used for the selective isolation of Pseudomonas aeruginosa from water and clinical specimens.
  • It is also used to detect the ability of an organism to tolerate cetrimide and exhibit growth on cetrimide agar.

Limitation of Cetrimide Agar Test

  • Growth on this medium alone is not sufficient for identification of P. aeruginosa to the species level, since other non-glucose-fermenting species (e.g., 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.
  • Cetrimide agar is a selective medium, and thus, some strains may show poor growth as cetrimide is highly toxic.

References and Sources

  • Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,–
  • 7% – http://www.himedialabs.com/TD/M024.pdf
  • 2% – https://microbiologyinfo.com/cetrimide-test/
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  • 1% – https://www.sciencedirect.com/topics/medicine-and-dentistry/achromobacter-xylosoxidans
  • 1% – https://orbitbiotech.com/pseudomonas-aeruginosa-isolation-and-identification/
  • 1% – https://microbiologynotes.com/cetrimide-test-principle-procedure-result-interpretation-and-limitation/
  • 1% – https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU1292.pdf
  • <1% – https://www.who.int/water_sanitation_health/resourcesquality/wqmchap10.pdf
  • <1% – https://www.techylib.com/en/view/mexicorubber/pathogenic_microbiology_college_of_computer_mathematical
  • <1% – https://www.cram.com/flashcards/non-fermentative-gram-negative-rods-1568966
  • <1% – https://biologicalindicators.mesalabs.com/wp-content/uploads/sites/31/2014/02/Unique-Cycles-Sterilizing-Liquid-Loads.pdf

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