Cetrimide Agar: Composition, Principle, and the Confirmatory Test Non-Fermenters Fail

The colony that didn't look like much, until it was given the right stage

A sputum sample from a cystic fibrosis patient grows a non-fermenting, Gram-negative rod on the primary plate. No strong odor, no obvious pigment, nothing that immediately says "Pseudomonas aeruginosa," the single most consequential chronic colonizer in CF lung disease, one whose presence changes antibiotic strategy, prognosis, and infection control practice.

Subcultured onto cetrimide agar, the same organism looks different. Cetrimide, a cationic detergent, doesn't just suppress most competing organisms, it specifically enhances P. aeruginosa's own production of pyocyanin and fluorescein, the pigments responsible for its signature blue-green color. A strain that looked unremarkable on the primary plate can reveal its true colors once it's given the right medium to do so.

That's the entire reason this medium exists: not every P. aeruginosa strain announces itself, and cetrimide agar is built specifically to coax out the signature even when a strain doesn't show it readily elsewhere.

Principle

Cetrimide agar, also called pseudosel agar, is used for the selective isolation and presumptive identification of Pseudomonas aeruginosa. Cetrimide is a quaternary ammonium salt acting as a cationic detergent; on contact with a bacterial cell, it releases nitrogen and phosphorus, denaturing membrane proteins and inhibiting a wide range of organisms, including Pseudomonas species other than P. aeruginosa. Cetrimide also enhances P. aeruginosa's production of pyocyanin (blue pigment) and fluorescein/pyoverdin (yellow-green fluorescent pigment); the combination produces the organism's characteristic bright green appearance.

Composition of Cetrimide Agar

Final pH: 7.2 ± 0.2 at 25°C

Cetrimide 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.

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 the following organisms;

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

Media Preparation

Figure: 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.

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

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.

How to Remember?

Anchor for the two pigments: "one glows, one just is." Fluorescein needs short-wavelength UV light to reveal its yellow-green glow clearly. Pyocyanin doesn't need any special light at all, it's visible as a blue-green color under ordinary conditions. A strain can show either, both, or, occasionally, neither.

Anchor for the definitive confirmatory step: "same glow, different comfort zone." P. fluorescens and P. putida can mimic P. aeruginosa's fluorescent glow on cetrimide agar, but only true P. aeruginosa tolerates growth at 42°C. When the pigment picture is ambiguous, this temperature test, not another look at the color, is what actually settles it.

Pulling the Real Bench Traps Together

• A non-pigmented colony doesn't rule out P. aeruginosa. Some strains genuinely don't produce pyocyanin. The absence of visible pigment is a limitation of this medium, not proof the organism isn't there.
• Growth alone, without pigment, isn't sufficient for species-level identification. Other non-glucose-fermenting genera, Achromobacter xylosoxidans and Alcaligenes faecalis among them, can also grow on cetrimide agar. Pigment has to be present for a presumptive call based on this medium alone.
• Fluorescence under UV light doesn't confirm P. aeruginosa on its own. Pseudomonas fluorescens and Pseudomonas putida can also grow and fluoresce on cetrimide agar. The feature that actually separates them is growth at 42°C: true P. aeruginosa tolerates it, its fluorescent look-alikes don't.
• A pink-pigmented colony on cetrimide agar isn't automatically ruled out as contamination. Serratia species can produce a pink pigment on this medium, worth remembering before dismissing an unexpected color as an artifact.

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.

Procedure

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

Reporting

• Gram-negative oxidase-positive bacilli that grow on cetrimide agar and produce a blue-green (pyocyanin) pigment can be definitively identified as P. aeruginosa.
• Pseudomonas fluorescens and Pseudomonas putida may also grow on cetrimide agar and may produce a fluorescent pigment but are separated from P. aeruginosa because they do not grow at 42°C.

Key exam facts in one table

References and further readings

1. Forbes, B. A., Sahm, D. F., & Weissfeld, A. S. Bailey & Scott's Diagnostic Microbiology (11th ed.).
2. Linscott, A. J. (2016). Clinical Microbiology Procedures Handbook (4th ed.). ASM Press. https://doi.org/10.1128/9781683670438

3. Lowbury, E. J., & Collins, A. G. (1955). The use of a new cetrimide product in a selective medium for Pseudomonas pyocyanea. Journal of Clinical Pathology.