PSEUDOMONAS ISOLATION AGAR

Cat. no. G145 Pseudomonas Isolation Agar, 15x60mm Plate, 11ml 10 plates/bag
Cat. no. G219 Pseudomonas Isolation Agar, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Pseudomonas Isolation Agar is recommended for the selective isolation and differentiation of Pseudomonas aeruginosa .

SUMMARY

Non-fermentative gram-negative bacteria (NFB) can exploit the wet environments in hospitals and are often associated with nosocomial infections. For instance, non-fermentative bacteria have been implicated in the contamination of faucet aerators, respiratory therapy equipment, as well as sterile solutions and medications. Among the NFB, Pseudomonas aeruginosa is the most clinically significant bacteria. (8)

Hardy Diagnostics Pseudomonas Isolation Agar is a modification of the formula developed by King, et al. (1) The medium is designed to enhance pyocyanin production, thereby improving the differentiation of pseudomonads. The modified formula contains a low phosphorous content, glycerol, magnesium chloride and potassium sulfate, all of which promote pyocyanin production. (1) Pyocyanin production is unique to Pseudomonas aeruginosa and is noted as a blue-green water soluble pigment that imparts a greenish color into the media. (8)

Pseudomonas Isolation Agar also includes glycerol as an energy source, while peptone provides nutrients necessary for bacterial growth. Irgasan ® is incorporated into the medium to inhibit the growth of many gram-positive and gram-negative microorganisms other than Pseudomonas spp. (2)

FORMULA

Ingredients per 980ml of deionized water:*

Pancreatic Digest of Gelatin 20.0gm
Potassium Sulfate 10.0gm
Magnesium Chloride 1.4gm
Irgasan ® 25.0mg
Glycerol 20.0ml
Agar 13.6gm

Final pH 7.0 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection. (4,5) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold.

Method of Use:

1. Allow the plates to warm to room temperature and dry the agar surface before inoculating.

2. Inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over one third of the agar surface.

3. Streak for isolation with a sterile loop.

4. Incubate plates aerobically at 35ºC. for 18-48 hours. Some pseudomonads do not grow well at 35ºC. If after 24 hours no growth appears, reincubate the plate(s) at 25ºC. for an additional 24 hours.

5. Examine plates daily for colony morphology and growth characteristics.

INTERPRETATION OF RESULTS

Pseudomonas aeruginosa will produce blue to blue-green pigmented colonies.

Pseudomonas fluorescens and other non-fermenting bacteria will produce colonies that are not blue-green in color.

LIMITATIONS

Some pseudomonads do not grow well at 35ºC. If no growth appears after 24 hours of incubation at 35ºC., the plates should be reincubated at 25ºC. for 24 hours.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, slides, staining supplies, other culture media, microscopes, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Pseudomonas aeruginosa
ATCC ® 27853
A 18-48hr 35°C Aerobic Growth; blue-green colonies
Pseudomonas aeruginosa
ATCC ® 9027
A 18-48hr 35°C Aerobic Growth; blue-green colonies
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
A 18-48hr 35°C Aerobic Growth; no pigment
Escherichia coli
ATCC ® 25922
B 18-48hr 35°C Aerobic Inhibited
Staphylococcus aureus
ATCC ® 25923
B 18-48hr 35°C Aerobic Inhibited

User Quality Control

PHYSICAL APPEARANCE

Pseudomonas Isolation Agar should appear slightly opalescent, and light amber in color.

P. aeruginosa growing on Pseudomonas Isolation Agar

Pseudomonas aeruginosa (ATCC ® 27853) growing on Pseudomonas Isolation Agar (Cat. no. G145). Incubated aerobically for 24 hours at 35ºC.

B. cepacia growing on Pseudomonas Isolation Agar

Burkholderia ( Pseudomonas ) cepacia (ATCC ® 25416) growing on Pseudomonas Isolation Agar (Cat. no. G145). Incubated aerobically for 24 hours at 35ºC.

Pseudomonas Isolation Agar

Uninoculated plate of Pesudomonas Isolation Agar (Cat. no. G145).

REFERENCES

1. King, E.O., et al. 1954. J. Lab. Clin. Med.; 44:301.

2. Soap and Chemical Specialties, January 1968.

3. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

4. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. Bodey, G.D., et al. 1989. "Infections caused by P. aeruginosa". Rev. Infect. Dis.; 5:279-313.


ATCC is a registered trademark of the American Type Culture Collection.
Irgasan is a registered trademark of Geigy Chemical Corp.

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