The cetrimide agar or cetrimide is a selective solid culture medium designed for the isolation of Pseudomonas aeruginosa. It is based on showing the production of characteristic pigments of this species and was made from the modification of Tech agar, created by King, Ward and Raney.
The original formula contained salts of magnesium chloride, potassium sulfate, pancreatic digestion of gelatin, and agar-agar. The modification of the formula consisted of the addition of cetrimide (cetyl trimethyl ammonium bromide) and glycerol.
Cetrimide agar seeded with Pseudomonas aeruginosa Source: BiotechMichael, from Wikimedia Commons
Cetrimide agar is useful for the microbiological study of samples where the presence of Pseudomonas aeruginosa is suspected. It should be noted that this bacterium is extremely important, because although it is part of the normal environmental microbiota, it frequently behaves as an opportunistic pathogen.
For this reason, one of the most common problems caused by this germ is nosocomial infections, that is, those that occur within the hospital environment, attacking patients who have a depressed immune system.
On the other hand, due to the affinity that this microorganism has with humidity, the most vulnerable contamination targets are: assisted breathing equipment, medicines, nebulizers, water sources, air conditioners, disinfectants, soapy solutions, injectable solutions, open wounds, catheters, urinary tubes, among others.
In this sense, cetrimide agar is useful to carry out microbiological controls and cultures to the previously mentioned elements.
Basis
Cetrimide agar is based on the ability of the medium to promote the growth of P. aeruginosa, stimulate the production of its pigments and in turn inhibit the growth of other microorganisms.
These properties are due to the function of each of its components. The gelatin peptone present serves as a source of nitrogen, vitamins and minerals. Glycerol or glycerin works as a carbon source.
For its part, cetrimide (cetyl trimethyl ammonium bromide) is the substance that inhibits the growth of bacteria other than P. aeruginosa, including other species belonging to the same genus.
Inhibition occurs because cetramide acts as a cationic detergent, managing to destabilize the plasma membrane of most bacteria, except for P. aeruginosa and some others that manage to survive.
On the other hand, it contains magnesium chloride and potassium sulfate. These compounds stimulate phenotypic expression related to the ability of Pseudomonas aeruginosa to produce various pigments, including: pyocyanin, pyoverdin, pyorrubin, pyomelanin, and fluorescein. Finally, it contains agar-agar, which gives it a solid consistency.
Interpretation
The interpretation of the growth obtained in this agar is carried out as follows:
The observation of round, smooth colonies with regular edges, with the production of blue-greenish, green, brown or reddish pigments, plus the emission of fruity odor (aminoacetophenone), is a presumptive result of the presence of this bacterium in said sample.
On the other hand, the observation of a bright greenish-yellow pigment on the colonies is indicative of P. aeruginosa when the plate is exposed to ultraviolet light.
Fluorescence of P. aeruginosa colonies on cetrimide agar under ultraviolet light.
It should be noted that each color observed is due to the production of a specific pigment. The blue-green pigment corresponds to the production of pyocyanin, the green to pyoverdin, the reddish to pyorubin, the brown to pyomelanine, and the bright yellow-green fluorescence under UV light to fluorescein.
Preparation
Weigh out 43 g of the dehydrated medium and dissolve in distilled water. Add 10 ml of glycerol. Take the mixture to a heat source. Let it boil for a few minutes until complete dissolution.
Autoclave at 121 ° C for 15 minutes. Let stand and serve in sterile Petri dishes when the temperature is around 50 ° C.
Allow to solidify, invert, order in plaqueros and store in the refrigerator until use. Cetrimide agar plates should be removed from the refrigerator in advance to seeding and allowed to come to room temperature.
The final pH of the medium should be 7.2 ± 0.2.
The color of the dehydrated medium is beige and the preparation is opaque white.
Applications
All kinds of samples in which the presence of Pseudomonas aeruginosa is suspected can be sown on cetrimide agar. Therefore, it is useful in all areas of microbiology (environmental, industrial, clinical, water and food).
It is of great importance to analyze hospital environments and thus be able to apply corrective measures, since this microorganism reaches patients through contaminated equipment, medicines, solutions and supplies that are used by the patient.
In this way, the microorganism can infect the lower respiratory tract, urinary tract and wounds of immunosuppressed patients.
Colony counts of P. aeruginosa can also be done in microbial limit tests.
Sown
Cetrimide agar can be used as a primary culture. The plate is inoculated on one of its edges and from there it is distributed by exhaustion to the rest of the plate. Liquid samples can be surface seeded with a drigalski spatula.
The plates are incubated aerobically at 37 ° C for 24 hours of incubation.
Limitations
-A small percentage of Pseudomonas aeruginosas strains do not produce pyocyanin, so a false negative can be interpreted.
-Some Pseudomonas species of clinical importance are inhibited in this medium.
-Despite observing the characteristics described for Pseudomonas aeruginosa, they must be corroborated with additional identification tests. A test that should not be missed is the oxidase test, it must give positive.
-Some Enterobacteriaceae can grow in this medium and develop a yellow pigment, but it differs from Pseudomonas aeruginosa in that when the plate is subjected to ultraviolet light there is no fluorescence.
- Serratia marcescens manages to develop and produces a pink pigment.
-If the plates seeded with cetrimide agar are exposed for a time at room temperature, the P. aeruginosa strains can lose the fluorescence observed under ultraviolet light, however the property is recovered if they are incubated again at 37 ° C.
QA
To analyze the good performance of cetrimide agar, control strains can be used, such as: Pseudomonas aeruginosa ATCC 9027, Stenotrophomonas maltophilia ATCC 13637, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.
The expected results are:
- Good growth for P. aeruginosa, with blue-green pigment and positive fluorescein.
- S. maltophilia and S. aureus will be partially to completely inhibited.
- Escherichia coli is expected to be completely inhibited.
References
- Callicó A, Cedré B, Sifontes S, Torres V, Pino Y, Callís A, Esnard S. Phenotypic and serological characterization of clinical isolates of Pseudomonas aeruginosa. VacciMonitor. 2004; 13 (3): 1-9.
- Conda Pronadisa Laboratories. Cetrimide agar base. 2014.Available at: condalab.com
- Britannia Laboratories. Cetrimide agar. 2015.Available at: britanialab.com
- BD Laboratories. BD Pseudosel agar (Cetrimide agar). 2013.Available at: bd.com
- Francisco Soria Melguizo Laboratory, CA Cetrimide agar. 2009.Available at: