Cetrimide Agar Test- Principle, Procedures, Results, Uses
Cetrimide agar test is a biochemical test that is used to identify and differentiate Pseudomonas aeruginosa from other microorganisms. Pseudomonas aeruginosa is a gram-negative rod that can cause various infections in humans, such as urinary tract infections, wound infections, and septicemia. It is also known for producing pigments, such as pyocyanin (blue-green) and fluorescein (yellow-green), that give it a characteristic appearance on culture media.
The test is based on the ability of Pseudomonas aeruginosa to grow in the presence of cetrimide, a quaternary ammonium salt that acts as a cationic detergent. Cetrimide inhibits most other bacteria by affecting their cell membranes and causing the release of nitrogen and phosphorus from the cells. Pseudomonas aeruginosa, however, is resistant to cetrimide and can use it as a source of nitrogen.
Cetrimide agar is a selective medium that contains cetrimide as well as other ingredients that enhance the growth and pigment production of Pseudomonas aeruginosa. These include gelatin peptone, glycerol, sodium chloride, magnesium chloride, and potassium sulfate. The medium has a pH of 7.2 and is solidified with agar.
Cetrimide agar test is used for the isolation and identification of Pseudomonas aeruginosa from clinical and non-clinical specimens, such as water, pharmaceutical products, and environmental samples. It can also be used to detect the ability of an organism to produce pyocyanin and fluorescein, which are indicative of Pseudomonas aeruginosa. The test is simple, rapid, and inexpensive compared to other methods of identification.
The main objective of cetrimide agar test is to identify and differentiate Pseudomonas aeruginosa from other microorganisms. Pseudomonas aeruginosa is a common pathogen that can cause various infections in humans, such as urinary tract infections, wound infections, pneumonia, and septicemia. It is also resistant to many antibiotics and disinfectants, making it difficult to treat and control. Therefore, it is important to have a reliable and specific method to detect and isolate this organism from clinical and environmental samples.
Another objective of cetrimide agar test is to detect the ability of different organisms to grow on cetrimide agar. Cetrimide agar is a selective medium that contains cetrimide, a quaternary ammonium salt that inhibits the growth of most bacteria except for Pseudomonas aeruginosa and a few other Pseudomonas species. Cetrimide agar also enhances the production of pyocyanin, a blue-green pigment that is characteristic of Pseudomonas aeruginosa. By observing the growth and pigmentation of the organisms on cetrimide agar, one can determine whether they belong to the genus Pseudomonas or not.
Cetrimide agar test is based on the principle that some bacteria can grow in the presence of cetrimide, a quaternary ammonium salt that acts as a cationic detergent. Cetrimide is toxic to most bacteria by causing the release of nitrogen and phosphorus from the bacterial cell, which slows or kills the organism. However, some bacteria, such as Pseudomonas aeruginosa, are resistant to cetrimide and can grow on cetrimide agar.
Pseudomonas aeruginosa is a gram-negative rod that produces two distinctive pigments: pyocyanin and pyoverdin. Pyocyanin is a blue-green, water-soluble, non-fluorescent phenazine pigment that gives P. aeruginosa its characteristic color. Pyoverdin is a yellow-green, water-soluble, fluorescent siderophore that chelates iron from the environment. When pyoverdin combines with pyocyanin, the bright green color of P. aeruginosa is enhanced.
Cetrimide agar contains gelatin peptone that provides the necessary nutrients for P. aeruginosa growth. Sodium chloride maintains the osmotic equilibrium of the medium and prevents the change of pH during growth. Magnesium chloride and potassium sulfate in the medium stimulate the production of pyocyanin and pyoverdin by P. aeruginosa. The medium also contains glycerol as a source of carbon.
The principle of cetrimide agar test is to isolate and identify P. aeruginosa from other microorganisms by observing its growth and pigmentation on cetrimide agar. P. aeruginosa is the only Pseudomonas species or gram-negative rod known to excrete pyocyanin, which can be detected visually or under UV light. Other bacteria that may grow on cetrimide agar, such as Pseudomonas fluorescens and Pseudomonas putida, do not produce pyocyanin and can be differentiated from P. aeruginosa by other tests, such as growth at 42°C.
The microorganism tested by the cetrimide agar test is Pseudomonas aeruginosa, a common gram-negative, rod-shaped bacterium that can cause infections in humans and animals. P. aeruginosa is found widely in the environment, such as in soil and water, and can also contaminate medical devices and surfaces in health care settings. P. aeruginosa is an opportunistic pathogen that mainly affects people with compromised immune systems or underlying diseases, such as cystic fibrosis or burns. P. aeruginosa can cause infections in various parts of the body, such as the blood, lungs, urinary tract, and skin. P. aeruginosa is also known for its intrinsic and acquired resistance to many antibiotics, making it difficult to treat.
P. aeruginosa is identified by the cetrimide agar test based on its ability to grow in the presence of cetrimide, a quaternary ammonium salt that inhibits most other bacteria. P. aeruginosa can also produce distinctive pigments and odors that help in its identification. The most characteristic pigment is pyocyanin, a blue-green, water-soluble phenazine that gives the colonies a greenish hue. Pyocyanin is unique to P. aeruginosa among pseudomonads and gram-negative rods. Another pigment that may be produced by P. aeruginosa is fluorescein, a yellow-green fluorescent compound that can be detected under UV light. P. aeruginosa also produces a grape-like odor due to the production of aminoacetophenone.
The cetrimide agar test is performed on isolated colonies of non-glucose-fermenting, gram-negative rods that are suggestive of P. aeruginosa. However, growth on cetrimide agar alone is not sufficient for identification of P. aeruginosa to the species level, as some other non-glucose-fermenting bacteria (e.g., Achromobacter xylosoxidans subsp. xylosoxidans and Alcaligenes faecalis) may also grow on this medium. Therefore, additional tests, such as oxidase test and growth at 42°C, are required to confirm the identity of P. aeruginosa.
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:
|Pancreatic Digest of Gelatin||20.0 gm|
|Potassium Sulfate||10.0 gm|
|Magnesium Chloride||1.4 gm|
|Cetyltrimethylammonium Bromide||0.3 gm|
The final pH of the medium is 7.2 +/- 0.2 at 25 degrees C.
- Sterile inoculating loops or sticks
- Wood’s or UV light (360 nm) or short-wavelength (254-nm) UV light
- Sterile Petri dishes
The procedure of cetrimide agar test involves the following steps:
- Preparation of media: Cetrimide agar can be prepared in the lab by adding 46.7 grams of the dehydrated powder or lab-prepared media to 1000 milliliters of distilled or deionized water containing 10 ml glycerol. The medium is heated up to boiling to dissolve it completely. Optionally, a selective supplement containing nalidixic acid can be added aseptically to the medium. The medium is then distributed into tubes and sterilized in an autoclave at 121°C for 15 minutes. The tubes are cooled at a slanted position to obtain butts of 1.5 – 2.0 cm depth.
- Inoculation of media: A well-isolated colony of a non-glucose-fermentative, Gram-negative rod that is suggestive of P. aeruginosa is collected from an 18-24 hour old pure culture or directly from the specimen with a sterile inoculating needle or loop. The cetrimide agar tubes are inoculated by streaking the surface of the slant back and forth to ensure proper inoculation with the loop or the inoculating stick. The cap of the test tubes is left loosened to ensure adequate aeration.
- Incubation of media: The tubes are incubated aerobically at 35-37°C for up to 7 days. The test tubes are examined daily for 4 days and again at 7 days before discarding the result as negative.
- Observation of results: The tubes are observed for bacterial growth, color change, pigment production, and odor. A positive result is indicated by growth on the slant, which may vary in color from yellow-green (fluorescein) to dark blue-green (pyocyanin). A negative result is indicated by no growth on the slant. Optionally, a UV light can be used to detect the fluorescence of fluorescein.
Quality control is the process of ensuring that the laboratory results are accurate, reliable, and reproducible. Quality control in microbiology involves monitoring the performance of the media, reagents, instruments, and personnel involved in the test. Quality control also helps to identify and correct any errors or problems that may affect the test outcome.
Some of the quality control measures for the cetrimide agar test are:
- Media: The cetrimide agar should be checked for sterility, pH, color, and appearance before use. The media should also be stored and handled according to the manufacturer`s instructions. The media should be discarded if there is any sign of contamination, deterioration, or expiration.
- Reagents: The reagents used for the test, such as cetrimide and nalidixic acid supplements, should be checked for purity, potency, and expiration date. The reagents should also be stored and handled according to the manufacturer`s instructions. The reagents should be discarded if there is any sign of contamination, deterioration, or expiration.
- Instruments: The instruments used for the test, such as inoculating loops, incubators, and UV lights, should be calibrated, cleaned, and maintained regularly. The instruments should also be checked for proper functioning and accuracy before use.
- Personnel: The personnel performing the test should be trained, qualified, and competent in the test procedure. The personnel should also follow the standard operating procedures (SOPs), wear appropriate personal protective equipment (PPE), and adhere to the biosafety guidelines.
- Positive and negative controls: As a form of quality control for the cetrimide agar test, two different organisms can be taken as a positive and negative control. Pseudomonas aeruginosa can be used as a positive control, as it grows well on cetrimide agar and produces a blue-green pigment. Escherichia coli can be used as a negative control, as it does not grow on cetrimide agar. The controls should be inoculated and incubated along with the test samples. The results of the controls should match the expected outcomes. If not, the test should be repeated or investigated for possible sources of error.
- Quality assessment: Quality assessment is the process of evaluating the quality of the laboratory results and identifying areas for improvement. Quality assessment in microbiology involves internal audits, external quality assessment (EQA) or proficiency testing (PT), and accreditation. Internal audits are periodic reviews of the laboratory processes and records by internal staff or experts. EQA or PT are external evaluations of the laboratory performance by comparing the results with those of other laboratories or reference standards. Accreditation is a formal recognition of the laboratory`s competence and quality by an external agency or authority.
Quality control in microbiology is essential for ensuring the validity and reliability of the cetrimide agar test and other tests performed in the laboratory. Quality control also helps to improve patient care and public health by providing accurate and timely diagnosis of infections caused by Pseudomonas aeruginosa and other microorganisms.
The result of the cetrimide agar test is based on the observation of growth and pigment production on the medium. The growth indicates the resistance of the organism to cetrimide, whereas the pigment production indicates the presence of P. aeruginosa.
A positive result is demonstrated by growth on the medium. Optionally, a yellow-green (fluorescein) to dark blue-green (pyocyanin) color may be observed on the colonies or around the growth. The color change is due to the production of water-soluble pigments by P. aeruginosa that can be detected under ultraviolet light or by visual examination. A positive result indicates the presence of P. aeruginosa or other cetrimide-resistant organisms.
A negative result is indicated by no growth or poor growth on the medium. A negative result indicates the susceptibility of the organism to cetrimide or the absence of P. aeruginosa.
The result should be confirmed by other biochemical tests and morphological characteristics before reporting. P. aeruginosa is definitively identified if an oxidase-positive, Gram-negative rod grows on cetrimide agar and produces a blue-green (pyocyanin) pigment. Some other Pseudomonas species may also grow and produce a fluorescent pigment on this medium but can be differentiated from P. aeruginosa by their inability to grow at 42°C. Some enteric organisms may also grow and exhibit a weak yellow color in the medium, which can be distinguished from fluorescein production.
Therefore, the cetrimide agar test is a useful tool for the selective isolation and presumptive identification of P. aeruginosa from various specimens. However, it should not be used as a sole criterion for identification, as some other organisms may also grow or produce pigments on this medium. Further tests are required to confirm the identity of P. aeruginosa and rule out other cetrimide-resistant or pigment-producing organisms.
The results of the cetrimide agar test should be reported based on the growth and pigmentation of the colonies on the medium. The following criteria can be used to report the results:
- Pseudomonas aeruginosa is definitively identified if an oxidase-positive, Gram-negative rod grows on cetrimide agar and produces a blue-green (pyocyanin) pigment. Optionally, a yellow-green (fluorescein) fluorescence can be observed under ultraviolet light (254 nm).
- 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.
- Other non-glucose-fermenting species (e.g., Achromobacter xylosoxidans subsp. xylosoxidans and Alcaligenes faecalis) may grow but do not produce any pigment.
- No growth or poor growth indicates that the organism is not P. aeruginosa or other Pseudomonas species.
The results should be confirmed by additional biochemical tests if necessary. The results should also be interpreted in conjunction with the clinical history and other laboratory findings of the patient.
Cetrimide agar test has several uses in microbiology, such as:
- It is primarily used for the selective isolation and presumptive identification of Pseudomonas aeruginosa from clinical and non-clinical specimens. Pseudomonas aeruginosa is a common pathogen that can cause infections like urinary tract infections, wound infections, pneumonia, and septicemia in humans. It is also resistant to many antibiotics and disinfectants. Therefore, it is important to isolate and identify this organism from various sources.
- It is also used for determining the ability of an organism to produce fluorescein and pyocyanin. Fluorescein and pyocyanin are water-soluble pigments that are produced by Pseudomonas aeruginosa and give it a characteristic yellow-green or blue-green color. These pigments can be detected by visual examination or by using ultraviolet light. The production of these pigments is enhanced by the presence of magnesium chloride and potassium sulfate in the medium.
- It is used to isolate and purify Pseudomonas aeruginosa from contaminated specimens. Cetrimide agar test can be used for microbial limit testing for non-sterile products, such as cosmetics, pharmaceuticals, food, and water. Cetrimide agar test can help to detect the presence of Pseudomonas aeruginosa in these products and prevent potential health hazards.
- 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.
- Some strains may develop poorly or not at all on this medium as a result of nutritional variation.
- On this medium, several non-fermenters and aerobic spore formers may exhibit a water-soluble tan to brown coloring. Strains of Serratia may have a pink hue.
- Cetrimide agar is a selective medium, and thus, some strains may show poor growth as cetrimide is highly toxic.
Keep in mind that the cetrimide agar test should be interpreted in conjunction with other tests and clinical findings to ensure accurate identification of Pseudomonas aeruginosa and to rule out other organisms.
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