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PSEUDOMONAS AGAR BASE

Code: CM0559

For the selective isolation of Pseudomonas species when supplemented with SR0102 or SR0103.

* Adjusted as required to meet performance standards

PSEUDOMONAS CN SELECTIVE SUPPLEMENT

Code: SR0102

PSEUDOMONAS CFC SELECTIVE AGAR SUPPLEMENT

Code: SR0103

Directions
To Prepare the Agar Base
Suspend 24.2g of the agar base, in 500ml of distilled water. Add 5ml of glycerol. Bring to the boil to dissolve completely, sterilise by autoclaving at 121°C for 15 minutes. Allow the medium to cool to 50°C.
To Prepare Pseudomonas CN Agar
To 500ml of agar base cooled to 50°C add the contents of 1 vial of Pseudomonas CN Supplement (SR0102) rehydrated as directed. Mix well and pour into sterile Petri dishes.
To Prepare Pseudomonas CFC Agar
To 500ml of agar base cooled to 50°C add the contents of 1 vial of Pseudomonas CFC Supplement (SR0103) rehydrated as directed. Mix well and pour into sterile Petri dishes.

Description
Pseudomonas Agar Base is designed so that by the addition of the appropriate supplement (SR0102 or SR0103) the medium becomes selective for Pseudomonas aeruginosa or Pseudomonas spp. generally. The base medium is a modification of King’s A Medium¹ in which magnesium chloride and potassium sulphate are present to enhance pigment production.

Pseudomonas CN Supplement (SR0102) is recommended for the selective isolation of Pseudomonas aeruginosa. The formula of the supplement was described by Goto and Enomoto² following the demonstration of cetrimide as a selective agent by Lowbury and Collins³. Goto and Enomoto showed that addition of nalidixic acid at 15µg/ml, while at the same time reducing the cetrimide content to 200mg, improved performance. The medium gave better recovery of Pseudomonas aeruginosa with enhanced pigment formation whilst strongly suppressing Klebsiella, Proteus and Providencia spp., the latter organisms being the troublesome contaminants of conventional cetrimide media.

Pseudomonas CFC Supplement (SR0103) is recommended for the selective isolation of Pseudomonas spp. generally. Mead and Adams⁴ showed that reducing the cetrimide content to 10mg/ml allowed the growth of all pigmented and non- pigmented psychrophilic pseudomonads. To improve its selective action they added cephaloridine 50µg/ml) and fucidin (10µg/ml). This combination of agents gave a new and more specific medium for isolating pseudomonads from chilled foods and processing plants. Incubation should be carried out at 25-30°C for 48 hours.

Considerable importance is given to detection of Burkholderia cepacia (formerly Pseudomonas cepacia) in water systems, particularly where the water is to be used for the preparation of pharmaceuticals and cosmetics⁵. The organism is resistant to many commonly-used disinfectants. Burkholderia cepacia has emerged as an important opportunistic pathogen in urinary, abdominal, respiratory and other infections.

Growth characteristics of Pseudomonas species on Oxoid Pseudomonas Agar Base with Supplements.

Incubation carried out at 30°C and plates read after 18 hours incubation.

Technique
Clinical Specimens for Pseudomonas aeruginosa Investigations
1. Prepare Pseudomonas CN Medium as directed.
2. Pour plates and dry the surface.
3. Swab a large inoculum over half the area of the plate.
4. Using a sterile loop, streak out the inoculum over the remainder of the plate to obtain isolated colonies.
5. Incubate at 35°C and examine after 24 and 48 hours, using both white and ultraviolet light.

Food, Water and Environmental Samples for Pseudomonads
1. Prepare Pseudomonas CFC Medium as directed.
2. Pour plates and dry the surface.
3. Prepare food samples by diluting 1 in 5 or 1 in 10 with 1% (w/v) sterile Peptone Water (CM0009) and homogenise in a `Stomacher’ or a laboratory blender.
4. Pipette 0.5 or 1ml of the homogenate on to the plate and spread over the surface with a sterile glass spreader. Inoculate water and swab samples directly on the surface of the medium.
5. Incubate at 25°C and examine after 24 and 48 hours, using both white and ultraviolet light.

Colonial Appearance
Growth on CN or CFC Medium is usually limited to Pseudomonas spp. but some members of the family Enterobacteriaceae may also be present. The presence of blue-green or brown pigmentation, or fluorescence may be taken as presumptive evidence of Pseudomonas spp. but further tests must be carried out to confirm the identity of the organism.
Stanbridge and Board⁶ modified CFC Medium to differentiate pseudomonads from Enterobacteriaceae developing on beef steaks packaged in modified atmospheres. Arginine 1% w/v and phenol red 0.002% w/v were added to the medium.
Pseudomonads produce ammonia from the arginine and colonies may be distinguished by a pink coloration.

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 medium at 2-8°C.

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

Quality control

* This organism is available as a Culti-Loop®

Precautions
Fresh media should be prepared as required. Molten agar should not be kept longer than 4 hours. Medium should not be stored and remelted. If swarming colonies of Proteus species are a problem in food samples then the incubation temperature can be lowered to 20°C for a period of 3-5 days. Chilled foods may carry a wide range of pseudomonads and the colonies on CFC Medium, incubated at lower temperatures, may be Pseudomonas fluorescens or Pseudomonas putida as well as Pseudomonas aeruginosa. Aeromonas species will also appear as pink/brown colonies, particularly from fish products.

References
1. King E.O., Ward M.K. and Raney D.E. (1954) J. Lab. & Clin. Med. 44. 301-307.
2. Goto S. and Enomoto S. (1970) Jap. J. Microbiol. 14. 65-72.
3. Lowbury E.J. and Collins A.G. (1955) J. Clin. Path. 8. 47-48.
4. Mead G.C. and Adams B.W. (1977) Br. Poult. Sci. 18. 661-667.
5. Geftic S.G., Heymann H. and Adair F.W. (1970) App. & Environmental Microbiol. 37. 505-510.
6. Stanbridge L.H. and Board R.G. (1994) Lett. Appl. Microbiol. 18. 327-328.