Pseudomonas Isolation Agar Base

Intended Use

For selective isolation and identification of Pseudomonas aeruginosa from clinical and nonclinical specimens.

Composition**

Final pH (at 25°C) 7.0±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 45.03 grams in 1000 ml purified/distilled water containing 20 ml glycerol. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

Pseudomonas aeruginosa is an important human pathogen commonly found in nosocomial infections. It successfully combines adaptability to a variety of moist environments with a collection of potent virulence factors (1). Pseudomonas infections usually occur at any site where moisture tends to accumulate e. g. tracheostomies, in-dwelling catheters, burns, the external ear and weeping cutaneous wounds (2). Pseudomonas Isolation Agar Base, used for the selective isolation and identification of P. aeruginosa, is a modification of Medium A, originally formulated by King, Ward and Raney (3). The medium contains pigment-enhancing components and the selective agents, triclosan (4) which selectively inhibits non-pseudomonads. The pigment-enhancers i.e. potassium sulphate and magnesium chloride enhance the blue or blue-green pigment production by P. aeruginosa, thus aiding in its identification.

Peptone provides nitrogenous compounds and other essential growth nutrients. Glycerol is a source of energy and promotes pyocyanin i.e. pigment production which is characteristic of Pseudomonas (5,6). Potassium sulphate and magnesium chloride enhance pyocyanin production. Triclosan (7) selectively inhibits gram-positive and gram-negative bacteria but Pseudomonas species are resistant to it. Some pyocyanin producing strains may also produce small amounts of fluorescein, resulting in the production of a blue-green to green pigment. Presumptive Pseudomonas should be further confirmed by performing biochemical tests, as some strains of Pseudomonas do not produce pyocyanin (8).

Type of specimen

Clinical samples - pus, urine, wounds, etc; Food samples; Water samples

Specimen Collection and Handling:

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (9,10).

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (11).

For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (12).

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use. For professional use only. Read the label before opening the container. The media should be handled by trained personnel only. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture.

Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitation

1. Presumptive Pseudomonas should be further confirmed by performing biochemical tests, as some strains of Pseudomonas do not produce pyocyanin (8).

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance

Cream to yellow homogeneous free flowing powder

Gelling

Firm, comparable with 1.36% Agar gel.

Colour and Clarity of prepared medium

Yellow coloured clear to slightly opalescent gel forms in Petri plates.

Reaction

Reaction of 4.5% w/v aqueous solution at 25°C. pH: 7.0±0.2

pH

6.80-7.20

Cultural Response

Cultural characteristics observed after an incubation at 35-37°C for 18-48 hours.

Key: (*) - Corresponding WDCM numbers

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (6,7).

Reference

1. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
2. Koneman E. W., Allen S. D., Janda W. M., Schreckenberger P. C., Winn W. C. Jr., 1992, Colour Atlas and Textbook of Diagnostic Microbiology, 4th Ed., J. B. Lippinccott Company.
3. King F. O., Ward M. K. and Raney D. E., 1954, J. Lab. Clin. Med., 44 :301.
4. Collee J. G., Fraser A. G., Marmion B. P., Simmons A., (Eds.), Mackie and McCartney, Practical Medical Microbiology, 1996, 14th Edition, Churchill Livingstone
5. Finegold S. M. and Baron E. J., 1986, Bailey and Scotts Diagnostic Microbiology, 7th Ed., The C. V. Mosby Co., St. Louis.
6. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I, Williams and Wilkins, Baltimore.
7. Furia T. E. and Schenkel A. G., 1968, Soap and Chemical Specialties 44:47
8. Gaby W. L. and Free E., 1958, J. Bacteriol., 76:442
9. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
10. 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.
11. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I, Williams and Wilkins, Baltimore.
12. Lipps WC, Braun-Howland EB, Baxter TE, eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.