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Introduction of Pseudomonas agar

Pseudomonas agar is clear from its name and thus it is recommended selective isolation of Pseudomonas species.

Principle of Pseudomonas agar

The incorporation of tryptone and gelatin peptone provides nitrogenous and carbonaceous compounds, long chain amino acids, and other essential growth nutrients for the organisms. Magnesium chloride and potassium sulphate of medium enhance pigment production. Cetrimide and Sodium nalidixate used as a supplement makes  the medium specific for isolation of Pseudomonas from clinical specimens. Observe  inoculated plates after 24 hours and 48 hours using both white and UV light. The presence of blue-green or brown pigmentation may be considered as presumptive evidence of P.  aeruginosa . Alteromonas spp. may form brown or pink colonies on the medium.

Composition of Pseudomonas agar

Ingredients  Gms / Litre
Tryptone: 10.0
Gelatin peptone: 16.0
Potassium sulphate: 10.0
Magnesium chloride, anhydrous: 1.4
Agar: 11.0
Final pH ( at 25°C) 7.1±0.2

Extra-

Glycerol- 5.0 ml(sufficient for 500ml of medium)

One CN Selective Supplement vial  for 500 ml medium)-
Cetrimide: 100.0mg
Sodium nalidixate: 7.5mg

 Preparation of Pseudomonas agar

1. Suspend 24.2 grams in 500 ml purified / distilled or deionized water.
2. Add 5 ml glycerol.
3. Heat to boiling to dissolve the medium completely.
4. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
5. After autoclaving,  leave for cooling to 45-50°C.
6. Add  CN Selective Supplement one vial and mix well.
7. Pour medium into each plate and leave plates on the sterile surface until the agar has solidified.
8. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Pseudomonas agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
• Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( clinical samples-pus, urine, body fluids, or growth of bacteria)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Proteus vulgaris ATCC 13315 and Pseudomonas aeruginosa
  ATCC 27853)

Procedure of Pseudomonas agar

1. Allow the plates to warm at 37°C or  to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37ºC. for 24-48hours.
6. Examine colonial characteristics.

Result-Interpretation of Pseudomonas Agar

Proteus vulgaris ATCC 13315: No growth

Pseudomonas aeruginosa ATCC 27853: Good-luxuriant growth with blue-green

Pseudomonas aeruginosa: Good-luxuriant growth with blue-green/ greenish yellow/ reddish brown/ brown to black pigments

Colony Morphology of Pseudomonas agar

• Proteus vulgaris ATCC 13315: No growth
• Pseudomonas aeruginosa ATCC 27853 (00025*): Good-luxuriant growth with blue-green
• P. aeruginosa ATCC 9027 (00026*): Good to luxuriant growth with blue-green pigmentation
• Pseudomonas  aeruginosa ATCC 10145 (00024*): Good-luxuriant growth having blue-green pigmentation
• P. cepacia ATCC 10661: No growth
• P. fluorescens ATCC 13525 (00115*): No growth
• Enterococcus faecalis ATCC 29212: No growth
• Escherichia coli ATCC 25922: No growth
• Burkholderia cepacia ATCC® 25416: Good growth; straw coloured colonies with brown pigmentation

Uses of Pseudomonas agar

• Pseudomonas agar having CN selective supplement is used for selective isolation Pseudomonas species from clinical specimens.
• Pseudomonas agar having CFC (cetrimide, fucidin and cephalosporin) selective supplement is used for selective isolation Pseudomonas species from chilled foods and processing plants, environmental samples and water.

Keynotes on Pseudomonas agar

1. CN supplement suppresses Klebsiella, Proteus and Providencia species
2. Pseudomonas Agar Base is a Kings A medium modification.
3. Lowbury and Collins studied cetrimide as a selective agent.
4. CFC selective Supplement was Mead and Adams formulation.
5. Molten agar should not be kept longer than 4 hours.
6. Types of specimens may be used according to selective supplements. Nature of specimens are clinical samples – pus, urine, body fluids, Food samples, and water samples.
7. One vial contents are (each vial is sufficient for 500ml of medium)-cetrimide (5mg), fucidin (5mg), and cephalosporin (25mg).

Further Readings 

• Goto S. and Entomoto S., 1970, Jap. J. Microbiol., 14:65.
• Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
• http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0559&c=UK&lang=EN
• https://himedialabs.com/TD/M085.pdf
• King E.O., Ward M.K. and Raney D.E., 1954, J.Lab and Clin. Med., 44:301.
• Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
• Lowbury E.J. and Collins A.G., 1955, Clin. Path., 8:47.
• Baird R.B., Eaton A.D., and Rice E.W., (Eds.), 2015, Standard Methods for the Examination of Water and Wastewater, 23rd ed., APHA, Washington, D.C.
• Mead G.C. and Adams B.W., 1977, Br. Poult. Sci., 18:661.
• Salfinger Y., and Tortorello M.L. , 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.