Cetrimide Agar Base

Intended Use

Cetrimide (Pseudosel) Agar is used for the selective isolation and identification of Pseudomonas aeruginosa. Meets United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)^1-3 performance specifications, where applicable.

Summary and Explanation

King et al. developed Medium A (Tech Agar) for the enhancement of pyocyanin production by Pseudomonas.⁴ Cetrimide (Pseudosel) Agar has the formula for Tech Agar but is modified by the addition of cetrimide (cetyl trimethy¹ ammonium bromide) for the selective inhibition of organisms other than P. aeruginosa.⁵

In 1951, Lowbury described the use of 0.1% cetrimide in a selective medium for P. aeruginosa.⁵ Because of the increased purity of the inhibitory agent, the concentration was later reduced, as reported by Lowbury and Collins in 1955.⁶ Brown and Lowbury employed incubation at 37°C with examination after 18 and 42 hours of incubation.⁷
Strains of P. aeruginosa are identified from specimens by their production of pyocyanin, a blue, water-soluble, nonfluorescent, phenazine pigment in addition to their colonial morphology⁸ and the characteristic grapelike odor of aminoacetophenone.⁹ P. aeruginosa is the only species of Pseudomonas or gramnegative rod known to excrete pyocyanin. Cetrimide (Pseudosel) Agar, therefore, is a valuable culture medium in the identification of this organism.

Cetrimide (Pseudosel) Agar is widely recommended for use in the examination of cosmetics,¹⁰ clinical specimens^8,11 for the presence of P. aeruginosa, as well as for evaluating the efficacy of disinfectants against this organism.¹² It is also used in the microbiological examination of nonsterile pharmaceutical products for Pseudomonas aeruginosa.¹

Principles of the Procedure

Gelatin peptone supplies the nutrients necessary to support growth. The production of pyocyanin is stimulated by the magnesium chloride and potassium sulfate in the medium.¹³
Cetrimide is a quaternary ammonium, cationic detergent compound, which is inhibitory to a wide variety of bacterial species including Pseudomonas species other than P. aeruginosa. Agar is a solidifying agent. Cetrimide Agar Base is supplemented with 1% glycerol as a source of carbon.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.
Identity Specifications
Cetrimide Agar Base
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                       4.53% solution with 1% glycerol, soluble in
                                   purified water upon boiling. Solution is light
                                   amber, opalescent, with a precipitate.
Prepared Appearance:    Light amber, opalescent, with precipitate.
Reaction of 4.53%
Solution with 1%
glycerol at 25°C:            pH 7.2 ± 0.2
Cultural Response
Cetrimide Agar Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-48 hours. Incubate plates with E. coli ATCC 8739 and P. aeruginosa ATCC 9027 at 30-35°C for 18-72 hours.

Formula

Cetrimide Agar Base
Approximate Formula* Per Liter
Pancreatic Digest of Gelatin......................................... 20.0 g
Magnesium Chloride..................................................... 1.4 g
Potassium Sulfate...................................................... 10.0 g
Cetrimide (Tetradecyltrimethylammonium Bromide)......... 0.3 g
Agar......................................................................... 13.6 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 45.3 g of the powder in 1 L of purified water containing 10 mL of glycerol. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens. Incubate plates in an inverted position (agar side up) at 35 ± 2°C for 18-48 hours. Inoculate tubes with either pure cultures or with specimen material.
Incubate tubes at 35 ± 2°C for 18-24 hours in an aerobic atmosphere. Refer to USP General Chapters <61> and <62> for details on the examination of nonsterile products and tests for isolating Pseudomonas aeruginosa using Cetrimide Agar.¹

Expected Results

Colonies that are surrounded by a blue-green pigment and fluoresce under short wavelength (254 nm) ultraviolet light may be presumptively identified as Pseudomonas aeruginosa. Note, however, that certain strains of P. aeruginosa may not produce pyocyanin. Other species of Pseudomonas do not produce pyocyanin, but fluoresce under UV light. Most non-Pseudomonas species are inhibited, and some species of Pseudomonas may also be inhibited. Gram staining, biochemical tests and serological procedures should be performed to confirm findings.

Limitations of the Procedure

1. The type of peptone used in the base may affect pigment production.^7,14
2. No single medium can be depended upon to exhibit all pigment-producing P. aeruginosa strains.
3. Occasionally some enterics will exhibit a slight yellowing of the medium; however, this coloration is easily distinguished from fluorescin production since this yellowing does not
fluoresce.⁷
4. Some nonfermenters and some aerobic sporeformers may exhibit a water-soluble tan to brown pigmentation on this medium. Serratia strains may exhibit a pink pigmentation.⁷
5. Studies of Lowbury and Collins6 showed 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 reincubated.

* Store at 2-8°C.
† QC testing performed according to USP/EP/JP performance specifications.

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