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

What is a Cetrimide Agar?

• Cetrimide agar, also known as pseudomonas agar Cetrimide, is a specialized selective and differential medium used for the isolation and identification of Pseudomonas aeruginosa from various sources, including water and clinical specimens. It was first developed by Lowburry as a modification of Tech Agar, which was developed by King et al. Cetrimide agar contains 0.1% cetrimide (cetyl trimethyl ammonium bromide), which acts as a selective agent to inhibit the growth of organisms other than Pseudomonas aeruginosa.
• Cetrimide is a quaternary ammonium salt and functions as a cationic detergent. It reduces surface tension upon contact and exerts precipitant, complexing, and denaturing effects on bacterial membrane proteins. This compound exhibits inhibitory actions on a wide range of microorganisms, including other Pseudomonas species, but it allows Pseudomonas aeruginosa to develop typical colonies.
• Pseudomonas aeruginosa is a bacterium that can grow well on standard laboratory media, but for specific isolation and identification purposes, a medium with selective agents and constituents to enhance pigment production is preferred. Cetrimide agar fulfills these requirements by inhibiting the growth of many microorganisms while allowing Pseudomonas aeruginosa to thrive and develop its characteristic colonies.
• One of the distinguishing features of Pseudomonas aeruginosa is its production of pyocyanin, a blue, water-soluble, non-fluorescent phenazine pigment. This pigment, along with the colonial morphology and grape-like odor of aminoacetophenone, helps identify Pseudomonas aeruginosa. Cetrimide agar, therefore, plays an essential role in the identification of this species. It is used in various applications, including the examination of cosmetics and clinical specimens for the presence of Pseudomonas aeruginosa, as well as the evaluation of disinfectants against this organism.
• The composition of cetrimide agar includes gelatin peptone, which provides necessary nutrients for Pseudomonas aeruginosa, and sodium chloride, which maintains osmotic equilibrium in the medium. Additionally, magnesium chloride and potassium sulfate are added to stimulate pyocyanin production.
• To isolate Pseudomonas aeruginosa, cetrimide agar plates are typically inoculated from a non-selective medium such as Brain Heart Infusion Broth or Soybean Casein Digest Medium. If the bacterial count is high, the sample can be directly inoculated onto cetrimide agar. Pseudomonas aeruginosa colonies on cetrimide agar may appear pigmented in shades of blue, blue-green, or non-pigmented. Presumptive positive identification is often determined by colonies exhibiting fluorescence at 250nm and a blue-green pigmentation.
• It’s important to note that certain strains of Pseudomonas aeruginosa may not produce pyocyanin, while other Pseudomonas species may fluoresce under UV light without producing pyocyanin. Some non-Pseudomonas species may exhibit water-soluble tan to brown pigmentation on cetrimide agar, and Serratia may show pink pigmentation. Therefore, biochemical tests and serological procedures should be performed to confirm the findings obtained from cetrimide agar.
• In summary, cetrimide agar is a specialized medium that selectively inhibits the growth of microorganisms other than Pseudomonas aeruginosa. It aids in the isolation and presumptive identification of Pseudomonas aeruginosa based on its characteristic pigment production and colony morphology.

Principle of Cetrimide Agar

The principle of Cetrimide Agar lies in its ability to selectively inhibit the growth of microorganisms other than Pseudomonas aeruginosa. This is achieved through the inclusion of cetrimide, a quaternary ammonium salt that acts as a cationic detergent. When cetrimide comes into contact with bacterial cells, it reduces surface tension and exerts precipitating, complexing, and denaturing effects on the bacterial membrane proteins.

Most bacteria are unable to withstand the germicidal activity of cetrimide, as it causes the release of nitrogen and phosphorous, which slows down or kills the organisms. However, Pseudomonas aeruginosa has developed resistance to cetrimide and can grow in its presence. Therefore, cetrimide agar acts as a selective medium for the isolation of Pseudomonas aeruginosa from various clinical specimens.

Pseudomonas aeruginosa is distinguished by its characteristic production of pyocyanin, a blue, water-soluble, non-fluorescent phenazine pigment. In addition to its colonial morphology and the characteristic grape-like odor of aminoacetophenone, the presence of pyocyanin helps identify Pseudomonas aeruginosa. This species is the only known Pseudomonas or gram-negative rod that excretes pyocyanin.

To enhance the production of pigments, cetrimide agar includes magnesium chloride and dipotassium sulfate. These components stimulate the production of pyoverdin, a yellow-green or yellow-brown fluorescent pigment (also known as fluorescein), and pyocyanin. The combination of these pigments creates the bright green color characteristic of Pseudomonas aeruginosa colonies.

The gelatin peptone present in the medium provides the necessary nutrients for the growth of Pseudomonas aeruginosa. Sodium chloride is included to maintain osmotic equilibrium, while agar serves as the solidifying agent. Overall, cetrimide agar creates a selective environment that supports the growth of Pseudomonas aeruginosa while inhibiting the growth of other microorganisms.

Composition of Cetrimide Agar

Ingredients	Grams/liter
Gelatin peptone	20.0gm
Magnesium chloride	1.4gm
Potassium sulfate	10.0gm
Cetrimide	0.3gm
Glycerol	10.0ml
Agar	13.6gm

Final pH (at 25°C) 7.2±0.2

Preparation of Cetrimide Agar

To prepare Cetrimide Agar, follow these steps:

1. Suspend 46.7 grams of Cetrimide Agar powder in 1000 ml of purified or distilled water.
2. Add 10 ml of glycerol to the suspension.
3. Heat the mixture while stirring until it reaches boiling point, ensuring complete dissolution of the medium.
4. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
5. Allow the medium to cool down to a temperature of 45-50°C.
6. If desired, aseptically add the rehydrated contents of 1 vial of Nalidixic Selective Supplement (FD130) to the 1000 ml of medium.
7. Mix the medium well to ensure proper distribution of all components.
8. Pour the prepared medium into sterile Petri plates.
9. Allow the medium to solidify before use.

By following these steps, you can prepare Cetrimide Agar for selective isolation and identification of Pseudomonas aeruginosa from various specimens.

Procedure or Method of Use of Cetrimide Agar

The following is a general procedure for the use of Cetrimide Agar, which can be adjusted based on the specific organism or product being tested. It is important for laboratories to verify and validate the correct testing procedures according to their regulatory guidelines or licensure.

Procedure for General Sample Preparation:

1. Take a 10.0g sample and place it in 100.0mL of sterile deionized water or appropriate buffer. Use a paddle blender (such as a Stomacher, WhirlPak, or Blender bags) to mix the sample and liberate microorganisms from it.
2. Take a 1.0mL aliquot of the sample suspension and transfer it to a separate dilution flask.
3. Prepare three subsequent 10X dilutions by adding fresh sterile deionized water or buffer (e.g., Butterfield’s Buffer) to the dilution flask. Make dilutions of 1/10, 1/100, and 1/1000 from the original sample suspension. If the colony counts are too high to be counted accurately, further dilutions may be necessary.
4. Plate the diluted samples onto three separate plates of Cetrimide Agar using the spread plate technique or streaking to achieve isolated colonies. Ensure that each plate corresponds to the appropriate dilution. Incubate the plates at the recommended temperature and duration suitable for the target organism.

Procedure for Inducing Sporulation:

1. Take a 10.0g sample and place it in 100.0mL of deionized water.
2. Transfer a 10.0mL aliquot of this dilution to a sterile, screw-cap, 20x150mm tube.
3. Heat-shock the tube in a water bath at 95°C to 100°C for fifteen minutes to induce sporulation.
4. Remove the tube from the water bath and rapidly cool it in an ice bath (0°C to 4°C).
5. Vortex the heat-shocked tube for at least ten seconds to resuspend the sporulated cells.
6. Transfer two 1.0mL aliquots from the heat-shocked tube to two primary dilution tubes containing 9.0mL of sterile water.
7. Vortex the primary dilution tubes for at least ten seconds to ensure proper mixing.
8. Transfer 1.0mL from the primary dilution tubes to a second dilution tube containing 9.0mL of sterile water. Repeat this step once more if necessary for the desired population count.
9. Use the spread plate technique or the pour plate method to plate 1.0mL aliquots from the dilution tubes onto Tryptic Soy Agar. This is done to confirm colony counts.
10. Incubate the plates at the specified temperature (55-60°C) for 48 hours.
11. After incubation, count the number of colony-forming units (CFUs) on each plate. The plates should ideally have counts between 30-300 CFUs, but not less than 6 CFUs per USP guidelines.

The procedures mentioned for the use of Cetrimide Agar have certain limitations that should be taken into consideration:

1. Nutritional Variation: Due to variations in nutritional requirements among different strains of microorganisms, some strains may grow poorly or fail to grow on Cetrimide Agar. This can potentially lead to false-negative results or difficulty in isolating certain organisms.
2. Yellowing of the Medium: Occasionally, enteric bacteria may cause a slight yellowing of the medium. However, this yellow coloration does not fluoresce and can be easily distinguished from true fluorescein production. Care must be taken to differentiate between these non-fluorescent yellow colonies and the desired characteristics of Pseudomonas aeruginosa.
3. Pigmentation of Non-Fermenters and Aerobic Spore Formers: Some non-fermenting bacteria and certain aerobic spore-forming organisms may exhibit a water-soluble tan to brown pigmentation on Cetrimide Agar. This can create challenges in accurately identifying and differentiating these organisms from Pseudomonas aeruginosa. Additionally, Serratia strains may exhibit a pink pigmentation, further complicating interpretation.
4. Loss of Fluorescence: Studies have shown that Pseudomonas aeruginosa cultures on Cetrimide Agar can lose their fluorescence under UV light if the cultures are left at room temperature for a short period. However, fluorescence can reappear when the plates are re-incubated. This temporary loss of fluorescence can lead to potential misinterpretation of results if not considered.
5. Confirmatory Testing: Although Cetrimide Agar provides presumptive identification of Pseudomonas aeruginosa, further tests are necessary to confirm the presence of this organism. Additional biochemical tests and serological procedures should be performed to validate the findings and ensure accurate identification.

Result Interpretation of Cetrimide Agar

The interpretation of results on Cetrimide Agar is as follows:

• Positive Result: If there is the presence of pigmented blue or blue-green colonies on the Cetrimide Agar, it is considered a positive result. These colonies typically exhibit fluorescence at 250nm and have a blue-green pigmentation. The presence of these characteristics is indicative of the growth of Pseudomonas aeruginosa. However, it is important to note that this result is presumptive and should be confirmed through further biochemical tests and serological procedures.
• Negative Result: If there is no growth or the absence of colonies on the Cetrimide Agar, it is interpreted as a negative result. This indicates that there is no growth of Pseudomonas aeruginosa or other organisms that can withstand the selective properties of cetrimide. It is important to consider that a negative result does not necessarily rule out the presence of Pseudomonas aeruginosa in the original sample, and further testing may be required for confirmation.

The interpretation of results on Cetrimide Agar also can be as follows:

1. Pigment Production: Cetrimide Agar is used to isolate and detect pigment production by Pseudomonas aeruginosa. Pseudomonas aeruginosa produces a variety of water-soluble pigments. The presence of pigmentation is determined by visual examination of the colonies on the plates.
2. Pyocyanin: Pseudomonas aeruginosa produces pyocyanin, a blue phenazine pigment. Typically, colonies of P. aeruginosa on Cetrimide Agar exhibit a blue-green coloration due to the presence of pyocyanin. However, in some cases, the colonies may appear light aqua or dark blue.
3. Fluorescein: Another pigment produced by Pseudomonas aeruginosa is fluorescein. It appears as a bright yellow-green halo around the colonies, which can be observed under short wavelength UV light (254 nm). The presence of fluorescein is indicative of P. aeruginosa.
4. Pyurubin: Pseudomonas aeruginosa can also produce the pigment pyurubin, which is pink to red in color. It is often formed simultaneously with pyocyanin and/or fluorescein.
5. Non-Pigmented Strains: It should be noted that occasionally, non-pigmented strains of P. aeruginosa may be encountered. In such cases, the presence of growth on Cetrimide Agar alone is considered a positive result for the identification of P. aeruginosa.
6. Additional Tests for Complete Identification: To complete the identification process, further biochemical and/or serological tests should be performed on isolated colonies from pure culture.

Quality Control

Quality control of Cetrimide Agar involves several parameters to ensure its performance and reliability:

1. Appearance: The Cetrimide Agar should have a cream to yellow homogeneous free-flowing powder appearance. Any deviation from this appearance may indicate a quality issue.
2. Gelling: The gelled medium should be firm and comparable to a 1.5% Agar gel. This ensures proper solidification and consistency of the medium.
3. Colour and Clarity of Prepared Medium: The prepared medium should have a light amber color with opalescence. A slight precipitate may form in the Petri plates, which is considered normal.
4. Reaction: A 4.67% w/v aqueous solution of the medium containing 1% glycerol should have a pH of 7.2±0.2 at 25°C. This pH range ensures the optimal growth conditions for the targeted organisms.
5. pH: The pH of the medium should fall within the range of 7.00-7.40. This pH range is suitable for the growth of the desired microorganisms.
6. Cultural Response: The cultural response of the medium is observed after incubation at 30-35°C for a specified time. The recovery rate, indicated as a percentage, should be 100% for bacteria growth on Soyabean Casein Digest Agar.
7. Organism Inoculum and Growth: Various organisms, including Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophila, Staphylococcus aureus, Salmonella Typhimurium, and Proteus mirabilis, are inoculated onto the medium. The growth or inhibition of these organisms is observed after incubation at 30-35°C for a specific period of time.
8. Recovery Rate: The recovery rate of the organisms on the medium should be 100% for desired bacterial growth.

Uses of Cetrimide Agar

Cetrimide Agar has several important uses in microbiology:

• Selective Isolation of Pseudomonas aeruginosa: Cetrimide Agar is primarily used for the selective isolation of Pseudomonas aeruginosa from clinical and nonclinical specimens. It inhibits the growth of many microorganisms, allowing for the specific isolation of Pseudomonas aeruginosa colonies on the agar. This selective property helps in the identification and study of this particular species.
• Presumptive Identification of Pseudomonas aeruginosa: The growth of Pseudomonas aeruginosa on Cetrimide Agar is presumptively identified based on its characteristic colonial morphology and the production of pyocyanin, a blue, water-soluble, non-fluorescent phenazine pigment. The presence of these traits helps in distinguishing Pseudomonas aeruginosa from other bacteria.
• Detection of Fluorescein and Pyocyanin Production: Cetrimide Agar is also used to determine the ability of an organism to produce fluorescein and pyocyanin. Pseudomonas aeruginosa produces these pigments, which can be observed as fluorescence under specific light conditions. This feature aids in the preliminary identification of Pseudomonas aeruginosa colonies on the agar.
• Isolation and Purification of Pseudomonas aeruginosa: Cetrimide Agar is utilized for the isolation and purification of Pseudomonas aeruginosa from contaminated specimens. It provides a selective environment that inhibits the growth of other microorganisms while supporting the growth of Pseudomonas aeruginosa. This allows for the isolation and purification of pure cultures of this bacterium for further study and research.

Limitations of Cetrimide Agar

Cetrimide Agar, despite its usefulness, has certain limitations that should be taken into account:

1. Poor Growth of Some Strains: Cetrimide is highly toxic, and as a result, some strains of bacteria, including certain strains of Pseudomonas aeruginosa, may show poor growth on the medium. This can lead to difficulties in the isolation and identification of these strains.
2. Presence of Yellowing and Other Pigmentations: Occasionally, enteric bacteria may cause a slight yellowing of the medium. However, this yellow coloration does not fluoresce and can be distinguished from true fluorescein production. Additionally, some non-fermenters and aerobic spore formers may exhibit water-soluble tan to brown pigmentation on the medium, and Serratia strains may exhibit pink pigmentation. These pigmentations can interfere with the accurate interpretation of results.
3. Loss of Fluorescence: Pseudomonas aeruginosa cultures on Cetrimide Agar may lose their fluorescence under UV light if the plates are left at room temperature for a short period. However, fluorescence can reappear when the plates are re-incubated. This temporary loss of fluorescence can affect the reliability of the results.
4. Need for Confirmatory Testing: While Cetrimide Agar provides presumptive identification of Pseudomonas aeruginosa based on its selective properties and pigment production, further biochemical tests and serological procedures are required to confirm the findings and establish a definitive diagnosis of Pseudomonas aeruginosa.

FAQ

References

• https://microbiologie-clinique.com/cetrimide-agar.html
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• https://hardydiagnostics.com/media/assets/product/documents/Cannabis%20Testing%20Guide.pdf
• https://hardydiagnostics.com/media/assets/product/documents/CetrimideSelAgar.pdf
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