Cetrimide Agar- Composition, Principle, Preparation, Results, Uses

Cetrimide agar is a selective and differential medium that is used for the isolation and identification of Pseudomonas aeruginosa, a gram-negative, rod-shaped, opportunistic pathogen that can cause various infections in humans and animals. Pseudomonas aeruginosa is known for its ability to produce pigments, such as pyocyanin and fluorescein, that give it a characteristic green color on cetrimide agar. Cetrimide agar contains cetrimide, a quaternary ammonium salt that inhibits the growth of most bacteria except Pseudomonas aeruginosa, which is resistant to it. Cetrimide agar also contains other ingredients that enhance the production of pigments by Pseudomonas aeruginosa, such as magnesium chloride, potassium sulfate, and glycerol. Cetrimide agar was first developed by Lowburry in 1948 as a modification of Tech Agar, which was originally devised by King et al. in 1939. Cetrimide agar is widely used in clinical and environmental microbiology for the detection and enumeration of Pseudomonas aeruginosa from various specimens. Cetrimide agar can also be used to determine the ability of an organism to produce pyocyanin and fluorescein, which are important virulence factors of Pseudomonas aeruginosa. Cetrimide agar is easy to prepare and interpret and has a high specificity and sensitivity for Pseudomonas aeruginosa. However, cetrimide agar also has some limitations, such as the toxicity of cetrimide, the variability of pigment production by different strains of Pseudomonas aeruginosa, and the possibility of false positive or negative results due to other bacteria that may grow or produce pigments on cetrimide agar. Therefore, cetrimide agar should be used in conjunction with other tests and procedures to confirm the identity of Pseudomonas aeruginosa.

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Pseudomonas aeruginosa is a gram-negative, rod-shaped, motile bacterium that belongs to the family Pseudomonadaceae. It is an opportunistic pathogen that can cause a variety of infections in humans and animals, especially in immunocompromised or hospitalized patients. It is also a common cause of nosocomial infections and biofilm formation on medical devices. Some of the infections caused by P. aeruginosa include pneumonia, urinary tract infections, wound infections, septicemia, endocarditis, meningitis, otitis externa, and eye infections.

Pseudomonas aeruginosa can be identified by several methods based on its morphological, biochemical, and molecular characteristics. Some of the common methods are:

• Gram staining: P. aeruginosa appears as pink rods with rounded ends under the microscope after gram staining.

• Oxidase test: P. aeruginosa is oxidase positive, meaning that it produces a dark purple color when exposed to an oxidizing agent such as tetramethyl-p-phenylenediamine dihydrochloride (TMPD).

• Catalase test: P. aeruginosa is catalase positive, meaning that it produces bubbles of oxygen when exposed to hydrogen peroxide.

• Growth on cetrimide agar: P. aeruginosa grows well on cetrimide agar and produces characteristic green colonies with a metallic sheen due to the production of pyocyanin and fluorescein pigments.

• Fluorescence under UV light: P. aeruginosa produces a yellow-green fluorescent pigment called pyoverdin or fluorescein that can be detected under UV light at 250 nm wavelength.

• Pigment production: P. aeruginosa produces various pigments such as pyocyanin (blue-green), capsorubin (red-brown), and pheomelanin (brown-black) that can be observed on different media or in liquid cultures.

• Odor production: P. aeruginosa produces a distinctive grape-like or fruity odor due to the production of aminoacetophenone.

• Molecular methods: P. aeruginosa can be identified by various molecular methods such as polymerase chain reaction (PCR), DNA hybridization, DNA sequencing, or MALDI-TOF mass spectrometry-based on its specific genetic markers.

These methods can help in the presumptive identification of P. aeruginosa from clinical and nonclinical specimens. However, further confirmation and characterization may be required by performing additional tests such as serotyping, antibiotic susceptibility testing, or genotyping.

Cetrimide agar is a selective medium that contains the following ingredients:

• Cetrimide: 0.1 g

• Gelatin peptone: 20 g

• Sodium chloride: 5 g

• Magnesium chloride: 1.4 g

• Potassium sulfate: 10 g

• Glycerol: 10 ml

• Agar: 15 g

• Distilled water: 1000 ml

The final pH of the medium is 7.2 ± 0.2 at 25°C.

Cetrimide is the key ingredient that makes this medium selective for Pseudomonas aeruginosa. It is a quaternary ammonium salt that acts as a cationic detergent and has bactericidal effects on many microorganisms by disrupting their cell membranes and releasing nitrogen and phosphorus. Pseudomonas aeruginosa is resistant to cetrimide and can grow on this medium, while other bacteria are inhibited.

Gelatin peptone provides the necessary nutrients for the growth of Pseudomonas aeruginosa. Sodium chloride maintains the osmotic balance of the medium. Magnesium chloride and potassium sulfate stimulate the production of pyocyanin, a blue-green pigment that diffuses into the medium and helps to identify Pseudomonas aeruginosa. Glycerol serves as the carbon source for the bacteria. Agar is the solidifying agent that gives the medium a firm consistency.

Cetrimide agar is based on the principle that cetrimide, a quaternary ammonium salt, acts as a selective agent that inhibits most bacteria by affecting their membranes. Cetrimide causes the release of nitrogen and phosphorus from the bacterial cells, which slows or kills the organisms. However, Pseudomonas aeruginosa is resistant to cetrimide and can grow on this medium.

Cetrimide agar also contains gelatin and glycerol as sources of nitrogen and carbon for the growth of P. aeruginosa. The medium also has magnesium chloride and potassium sulfate that stimulate the production of pyocyanin and fluorescein, two water-soluble pigments that are characteristic of P. aeruginosa. Pyocyanin is a blue-green pigment that diffuses into the medium, while fluorescein is a yellow-green or yellow-brown fluorescent pigment that can be detected under ultraviolet light. When these two pigments combine, they create a bright green color that is typical of P. aeruginosa.

Therefore, cetrimide agar is used to determine the ability of an organism to grow in the presence of cetrimide, a toxic substance that inhibits the growth of many bacteria. It also allows for the identification of P. aeruginosa due to its characteristic production of pyocyanin and fluorescein, which give a distinctive color and fluorescence to the colonies. Cetrimide agar is a selective and differential medium for P. aeruginosa.

To prepare cetrimide agar, follow these steps:

• Add 45.3 grams of cetrimide agar powder to 1 liter of distilled water in a flask or beaker.

• Add 10 milliliters of glycerol and mix well.

• Heat the mixture to boiling until the agar dissolves completely.

• Sterilize the medium by autoclaving at 121°C for 15 minutes.

• Cool the medium to approximately 50°C and pour it into sterile Petri dishes.

• Allow the medium to solidify and store in a refrigerator until use.

Alternatively, you can use ready-made cetrimide agar plates or tubes from a commercial supplier.

To inoculate cetrimide agar, follow these steps:

• Using a sterile loop or swab, streak a pure culture of the test organism on the surface of the agar plate or tube.

• Incubate the plate or tube at 35-37°C for 18-24 hours in an aerobic atmosphere.

• Examine the growth and pigmentation of the colonies under normal and UV light.

The result of cetrimide agar is based on the observation of the colony morphology, pigmentation, and fluorescence of the bacteria grown on the medium. The following table summarizes the expected results for Pseudomonas aeruginosa and other bacteria:

| Bacteria | Colony Morphology | Pigmentation | Fluorescence |

| -------- | ----------------- | ------------ | ------------ |

| Pseudomonas aeruginosa | Smooth, round, convex, translucent to opaque | Blue-green due to pyocyanin and fluorescein production | Bright green under UV light at 250 nm |

| Other Pseudomonas species | Variable, may be similar to P. aeruginosa | Variable, may produce yellow-green or brown pigments | Variable, may or may not fluoresce |

| Enterobacteriaceae | Inhibited or very poor growth | None or slight yellowing of the medium | None |

| Other gram-negative rods | Inhibited or very poor growth | None or tan to brown pigmentation | None |

| Serratia species | Inhibited or very poor growth | Pink pigmentation | None |

Colonies exhibiting fluorescence at 250 nm and a blue-green pigmentation are considered presumptive positive for Pseudomonas aeruginosa. However, some strains of P. aeruginosa may lose their fluorescence under UV light if the cultures are left at room temperature for a short time. Fluorescence reappears when plates are re-incubated. Therefore, it is recommended to observe the fluorescence under UV light after incubation and before exposure to room temperature.

To confirm the identification of P. aeruginosa, additional tests such as oxidase test, motility test, growth at 42°C, and biochemical tests should be performed. Serological tests can also be used to differentiate P. aeruginosa from other Pseudomonas species.

Cetrimide agar has several uses in microbiology, especially for the detection and identification of Pseudomonas aeruginosa, a gram-negative bacterium that can cause various infections and diseases in humans and animals. Some of the uses of cetrimide agar are:

• It is primarily used for the selective isolation and presumptive identification of Pseudomonas aeruginosa from clinical and nonclinical specimens, such as pus, sputum, urine, blood, wounds, burns, eye infections, ear infections, etc. Cetrimide agar inhibits most bacteria other than P. aeruginosa by acting as a detergent that disrupts their cell membranes. P. aeruginosa can be recognized by its characteristic production of fluorescein and pyocyanin, two water-soluble pigments that give the colonies a yellow-green to blue-green color under ultraviolet light or natural light.

• It is also used for determining the ability of an organism to produce fluorescein and pyocyanin, which are also known as antibiotics because they have antibacterial properties against other microorganisms. Fluorescein and pyocyanin are produced by P. aeruginosa as iron chelators that help the bacterium scavenge iron from the environment. The production of these pigments is enhanced by cetrimide agar, which contains magnesium chloride and potassium sulfate as stimulants.

• It is used to isolate and purify Pseudomonas aeruginosa from contaminated specimens, such as water, soil, cosmetics, pharmaceuticals, etc. Cetrimide agar can help eliminate other microorganisms that may interfere with the identification or analysis of P. aeruginosa. Cetrimide agar can also be used to test the purity and quality of products that may contain or be exposed to P. aeruginosa.

Cetrimide agar is a useful medium for the study of Pseudomonas aeruginosa, a versatile and opportunistic pathogen that can cause serious infections in immunocompromised or hospitalized patients. Cetrimide agar can help isolate and identify this bacterium from various sources and determine its pigment production ability.

• Cetrimide agar is a selective medium that may inhibit the growth of some strains of P. aeruginosa or other non-fermentative gram-negative bacilli. Therefore, it should not be used as the sole medium for isolation and identification of these organisms.

• Some enteric bacteria may produce a slight yellowing of the medium, which can be confused with fluorescein production by P. aeruginosa. However, this yellowing does not fluoresce under UV light and can be distinguished from the true positive reaction.

• Some non-fermenters and some aerobic spore-formers may produce a water-soluble tan to brown pigmentation on cetrimide agar. Serratia species may produce pink pigmentation. These pigments are different from the blue-green pyocyanin and the yellow-green fluorescein produced by P. aeruginosa.

• P. aeruginosa may lose its fluorescence under UV light if the cultures are left at room temperature for a short time. Fluorescence reappears when plates are re-incubated.

• Cetrimide agar alone is not sufficient for the 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 and produce pigments on this medium. Additional biochemical tests and serological procedures should be performed to confirm the findings and to differentiate P. aeruginosa from other similar organisms.

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