Pseudomonas Agar F and P (Tech Agar)

Cat. no. G198 Pseudomonas Agar F, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. G201 Pseudomonas Agar P, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. L96 Tech Agar, 16x100mm Tube, 5.5ml Slant 20 tubes/box

INTENDED USE

Hardy Diagnostics Pseudomonas Agar F is used for the detection and differentiation of Pseudomonas aeruginosa by enhancement of fluorescein production. Pseudomonas Agar P, also known as Tech Agar, is recommended for enhancement of pyocyanin production by Pseudomonas aeruginosa.

SUMMARY

Pseudomonas aeruginosa is an environmental isolate commonly found in soil, water, food, and in many man-made products. The organism can utilize an array of nutrients and will thrive in a variety of environments, including medical equipment such as catheters, infusion fluids, disinfectants, and cosmetics. P. aeruginosa can also readily produce a biofilm, an aggregate of cells that can adhere to many surfaces. Because its cells can grow on most surfaces, P. aeruginosa is considered an opportunistic pathogen, particularly in immunocompromised persons, and can cause ocular, burn wound, and respiratory tract infections; it is the most common non-fermentative, gram-negative rod isolated by clinical microbiologists. (5,6)

Most strains of P. aeruginosa secrete a variety of pigments, such as pyoverdin, a yellow, water-soluble, fluorescent pigment, and pyocyanin, a blue-green, water- and chloroform-soluble, non-fluorescent pigment. Pyoverdin is most often produced by P. aeruginosa and other pseudomonads commonly isolated from humans. (2) However, when present, pyoverdin can suppress pyocyanin production. In contrast, P. aeruginosa is the only Pseudomonas spp. known to produce pyocyanin, a pigment that diffuses into the surrounding medium: yet, some strains are apyocyanogenic. (2)

The detection of pigment generating strains was first described by King et al. who developed two different media to detect pigment producing pseudomonads. (4) King et al. described Medium A, also known as Pseudomonas Agar P or Tech Agar, for the detection of pyocyanin and Medium B, also known as Pseudomonas Agar F or Flo Agar, for the detection of pyoverdin. The media were later modified according to recommendations made by the U.S. Pharmacopoeia and are also listed in the FDA Bacteriological Analytical Manual (BAM) for the differentiation of Pseudomonas aeruginosa . (8, 9)

Pseudomonas Agar F incorporates equal concentrations of peptones, which contain phosphorous to stimulate fluorescein production. Dipotassium phosphate added to the medium increases the phosphorous concentration and further enhances pigment production. Essential ions are provided by magnesium sulfate. Pseudomonas Agar P contains digest of gelatin to provide essential amino acids and other nitrogenous compounds to promote bacterial growth. Digest of gelatin is low in phosphorous, which helps inhibit the production of pyoverdin, a pigment that suppresses pyocyanin production. (2) Moreover, magnesium, potassium and sulfate ions promote the production of pyocyanin. Both media contain agar as the solidifying agent and glycerol as the energy source.

FORMULA

Ingredients per liter of deionized water:*

Pseudomonas Agar F:
Pancreatic Digest of Casein 10.0gm
Proteose Peptone No. 3 10.0gm
Glycerol 10.0gm
Dipotassium Phosphate 1.5gm
Magnesium Sulfate 1.5gm
Agar 15.0gm

Final pH 7.0 +/- 0.2 at 25ºC.

Pseudomonas Agar P and Tech Agar:
Pancreatic Digest of Gelatin 20.0gm
Potassium Sulfate 10.0gm
Glycerol 10.0gm
Magnesium Chloride 1.4gm
Agar 15.0gm

Final pH 7.0 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store Cat. no. G198 and G201 at 2-8ºC. and Cat. no. L42 and L96 at 2-30ºC. 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, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Consult listed reference for more information on specimen collection. (2,3,5,9) Specimens must be isolated from a pure culture on a suitable medium. Colony morphology and Gram stain characteristics should be confirmed as appropriate for Pseudomonas spp. prior to use.

Method of Use:

1. Using a sterile inoculating loop, streak the sample over the surface of the agar.

2. Incubate the specimen at 35 to 37ºC. for 18 to 24 hours. If the isolate fails to grow, reincubate the sample at 25 to 30ºC. for 1 to 2 days.

3. Observe cultures for growth and pigment production. If examining Pseudomonas Agar F, a long wavelength UV light (Cat. no. UVL56) is needed.

INTERPRETATION OF RESULTS

Colonies growing on Pseudomonas Agar F should be observed using a long wavelength UV lamp (Cat. no. UVL56) for fluorescein pigmentation. Fluorescein production should appear as a greenish-yellow fluorescent pigment in the colonies and surrounding medium.

Colonies growing on Pseudomonas Agar P and Tech Agar should display a blue to blue-green pigment in the colony as well as the surrounding medium, indicating pyocyanin production. (2) Confirm the presence of pyocyanin by performing a chloroform (CHCl 3 ) extraction and observe for a blue pigment solubilized in the chloroform solution. (9)

LIMITATIONS

Some strains of Pseudomonas will yield only small amounts of pigment in the medium. If this occurs, a yellow-green color will be evident on Pseudomonas Agar F or a blue-green pigment will be visible on Pseudomonas Agar P and Tech Agar. If the blue-green coloration is observed on Pseudomonas Agar P or Tech Agar, confirm the presence of pyocyanin by performing extraction with chloroform.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, UV Lamp (Cat. no. UVL56), other culture media, 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 Agar F:
Pseudomonas aeruginosa
ATCC ® 27853
A 18-24hr 35°C Aerobic Growth; greenish-yellow fluorescent pigment
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
A 18-24hr 35°C Aerobic Growth; no pigment, no fluorescence

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Pseudomonas Agar P and Tech Agar**:
Pseudomonas aeruginosa
ATCC ® 27853**
E 18-24hr 35°C Aerobic Growth; blue to blue-green pigment
Burkholderia ( Pseudomonas ) cepacia
ATCC ® 25416
E 18-24hr 35°C Aerobic Growth; no pigment

USER QUALITY CONTROL

Physical Appearance

Pseudomonas Agar F and Pseudomonas Agar P should appear clear, slightly opalescent, and light to medium amber in color.

REFERENCES

1. 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.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

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

4. King, E.O., M.K. Ward and D.E. Raney. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med.; 44: 301.

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

6. Reyes, E.A.P., M.J. Bales, W.H. Cannon, and J.M. Matsen. 1981. Identification of Pseudomonas aeruginosa by Pyocyanin Production on Tech Agar. J. Clin. Microbio.; 13(3):456-458.

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

8. Wentworth, B. 1987. Diagnostic Procedures for Bacterial Infection, 7th ed. APHA, Washington, D.C.

9. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm


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