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PSEUDOMONAS CETRIMIDE AGAR (USP, EP)

Code: CM0579

Pseudomonas Cetrimide Agar is used for the selective isolation and identification of Pseudomonas aeruginosa.

Typical Formula*

gm/litre

Gelatin peptone
20.0
Magnesium Chloride
1.4
Potassium Sulphate
10.0
Cetrimide
0.3

Agar

13.6

Final pH 7.2 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

Directions
Suspend 45.3g of Pseudomonas Cetrimide Agar in 1 litre of distilled water. Add 10ml of glycerol and boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes. Cool the medium to approximately 50°C and pour into sterile Petri dishes.

Description
A modification of the medium described by Brown and Lowbury3 for the selective isolation and differentiation of Pseudomonas aeruginosa from a range of samples.

Cetrimide is a quarternary ammonium compound with bactericidal activity against a broad range of Gram-positive organisms and some Gram-negative organisms.

Pseudomonas aeruginosa produces a number of water soluble iron chelators, including the yellow-green or yellow-brown fluorescent pyoverdin. When pyoverdin combines with the blue water-soluble pyocyanin, the bright green colour characteristic of Pseudomonas aeruginosa is created. The addition of magnesium chloride and potassium sulphate enhances the production of these chelators.

Cetrimide Agar is recommended in the United States Pharmacopoeia XXVI1 and European Pharmacopoeia IV2 for use in Microbial Limit Tests. The formulation is also in the A.O.A.C. guidelines4 for isolation of Pseudomonas aeruginosa from cosmetics and in the A.O.A.C. method5 for testing disinfectants on hard surfaces.

Microbial limit tests
The US and EU Pharmacopoeia state that pharmaceutical articles ranging from raw materials to finished products must be monitored for the total number of viable aerobic organisms present (Total Aerobic Microbial Count) and also that they must be completely free from the following organisms; Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella species and Escherichia coli.

For the examination of materials for the absence of specific organisms the sample is first dissolved or suspended in a recovery medium such as Tryptone Soya Broth (CM0129, CM0876 or CM1065). The sample should be incubated in this medium at 35-37°C for long enough to allow sub-lethally injured organisms to be revived, but not to multiply (usually 2-5 hours). If antimicrobials are present in the material to be examined, they must be adequately neutralised. This may be achieved by the addition of 0.1-1% of a neutralising agent such as Polysorbate and/or lecithin to the recovery broth. The level of neutralising agent required for each type of sample will need to have been predetermined using recommended control strains.

After the appropriate incubation time a portion of any positive broth is inoculated onto a range of recommended selective media which are incubated appropriately and examined for bacterial growth. Further tests are carried out on any colonies present on the selective media to confirm identification.

Technique
Follow the methods and procedures stated in the appropriate standard method. Plates are usually inoculated by streak or spread method from non-selective medium or directly from the specimen. Incubate the plates at 35-37°C for up to 48 hours.

Pseudomonas aeruginosa colonies are yellow-green or yellow brown in colour and fluoresce under UV light. Presumptive identification by colonial morphology should be confirmed using further tests such as oxidase and inoculation onto media for the detection of pyoverdin and pyocyanin.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Translucent, light straw coloured gel

Quality control

Positive control:

Expected results

Pseudomonas aeruginosa ATCC14207 *Fluorescent, green colonies
Negative control:  
Escherichia coli ATCC® 8739 *

No growth

* This organism is available as a Culti-Loop®

Precautions
Cetrimide Agar should be used for in vitro diagnostic purposes only.
Do not use Cetrimide Agar beyond the stated expiry date, or if the product shows any signs of deterioration

References
1.
United States Pharmacopeia 2002. Microbiological Limit Tests, United States Pharmacopeia, 26th Ed. United States Pharmacopial Convention, Rockville, M.D.
2. European Pharmacopeia 2002. Microbial Examination of Non-Sterile Products, (Test for Specified Microorganisms). European Pharmacopoeia, 4th Ed.
3. Brown V. I. and Lowbury, R. J. L. Use of an Improved Cetrimide Agar Medium and other Culture Methods for Pseudomonas aeruginosa. J. Clin. Pathol. 18; 752-756 (1965)
4.Official Analytical Chemists. 1995. Bacteriological Analytical Manual, 8th Ed. AOAC International, Gaithersburg, M.D.
5. Official Methods of Analysis of A.O.A.C. International. 17th Edition, Revision 1, 2002.

 
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