PHENYLALANINE AGAR

Cat. no. L21 Phenylalanine Agar, 16x100mm Tube, 5.5ml Slant 20 tubes/box

INTENDED USE

Hardy Diagnostics Phenylalanine Agar is recommended for use in the differentiation of gram-negative enteric bacilli based on the ability of the microorganisms to produce phenylpyruvic acid by oxidative deamination.

SUMMARY

In 1950, Hendriksen demonstrated that Proteus spp. were able to convert the amino acid phenylalanine to phenylpyruvic acid. Later, Buttiaux et al. developed a culture medium for detecting the formation of phenylpyruvic acid from phenylalanine by members of the Proteus , Providencia , and Morganella groups. (1) This medium was modified by Bynae and further modified by Ewing et al. who simplified Bynae's formula by omitting proteose peptone. (2,3) Hardy Diagnostics Phenylalanine Agar follows the formulation established by Ewing.

The deamination of phenylalanine by oxidative enzymes results in the formation of phenylpyruvic acid. After incubation, an aqueous solution of ferric chloride is added. If phenylpyruvic acid is present, a light to deep green color is produced. Of the Enterobacteriaceae , only Proteus , Providencia , and Morganella species possess enzymes capable of deaminating phenylalanine. (4)

FORMULA

Ingredients per liter of deionized water:*

Sodium Chloride 5.0gm
Yeast Extract 3.0gm
Phenylalanine 2.0gm
Dipotassium Phosphate 1.0gm
Agar 12.0gm

Final pH of 7.3 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 15-30ºC. Media should not be used if there are any signs of contamination, deterioration or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: This medium is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organisms.

Method of Use:

1. Prior to inoculation, allow the medium to equilibrate to room temperature.

2. Using a loopful of inoculum from an 18-24 hour pure culture, streak the slant surface using a fishtail motion.

3. Incubate the inoculated slant aerobically at 35ºC. for 18-24 hours.

4. Following incubation, apply 4-5 drops of a 10% Ferric Chloride solution (Cat. no. Z63) directly to the slant.

5. Gently agitate the tube and observe for the development of a green color within 1-5 minutes.

INTERPRETATION OF RESULTS

A positive phenylalanine deamination reaction is indicated by the development of a light to dark green color within 1-5 minutes after applying ferric chloride reagent.

A negative test is indicated by the absence of a green color reaction. Negative results will take on a yellow color due to the color of the ferric chloride.

LIMITATIONS

The green color reaction of a positive test fades rapidly. Test results must be interpreted within 5 minutes following the application of ferric chloride or false-negative results may occur.

Slight agitation of the tube containing ferric chloride will dislodge surface colonies and produce a faster more pronounced color reaction.

Refer to the keyword "Limitations", in the Hardy Diagnostics' software program HUGO™, for more information regarding general limitations on culture media.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, slides, staining supplies, ferric chloride reagent (Cat. no. Z63), 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
Proteus mirabilis
ATCC ® 12453**
E 18-24hr 35°C Aerobic Growth; turns green after the addition of 4-5 drops of ferric chloride with agitation, may take 1-5 minutes
Escherichia coli
ATCC ® 25922**
E 18-24hr 35°C Aerobic Growth; ferric chloride remains yellow

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

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

P. mirabilis, positive phenylalanine reaction

Showing positive phenylalanine deaminase reaction.
Ferric Chloride Reagent, 10% (Cat. no. Z63) added to phenylalanine slant (Cat. no. L21) with heavy growth of Proteus mirabilis (ATCC ® 12453). P. mirabilis was incubated aerobically for 24 hours at 35ºC.

E. coli, negative phenylalanine reaction

Showing negative phenylalanine deaminase reaction.
Ferric Chloride Reagent, 10% (Cat. no. Z63) added to phenylalanine slant (Cat. no. L21) with heavy growth of Escherichia coli (ATCC ® 25922). E. coli was incubated aerobically for 24 hours at 35ºC.

Phenylalanine Agar

Uninoculated tube of Phenylalanine Agar (Cat. no. L21).

REFERENCES

1. Buttiaux, et al. 1954. Ann. Inst. Pasteur; 87:375-386.

2. Ewing, et al. 1957. Pub Hlth Lab.; 15:153.

3. Hendriksen. 1950. J. Bacteriol.; 60:225.

4. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

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

6. 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.

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

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


ATCC is a registered trademark of the American Type Culture Collection.

040416gr