Phenylalanine Deaminase Test

Phenylalanine Deaminase Test

Phenylalanine Deaminase Test

Objectives of Phenylalanine Deaminase Test

  • To determine the ability of an organism to oxidatively deaminate phenylalanine to phenylpyruvic acid.
  • To differentiate enteric Gram-nega­tive bacilli on the basis of their ability to produce phenylpyruvic acid from phenylalanine.

Principle of Phenylalanine Deaminase Test

Phenylalanine deaminase medium tests the ability of an organism to produce the enzyme deaminase. Microorganisms capable of producing  phenylalanine deaminase remove the amine (NH2) from phenylalanine and releases the amine group as free ammonia. The deamination of phenylalanine by oxidative enzymes results in the formation of phenylpyruvic acid.  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. This medium was modified by Bynae and later on further modified by Ewing et al. who simplified Bynae’s formula by omitting proteose peptone in the medium. The phenylpyruvic acid produced by the action of phenylalanine deaminase produced by certain microorganism is detected by adding a few drops of 10% ferric chloride which result in the formation of a greencolored complex  between these two compounds in the slant of the test tube indicating the positive test result. The absence of this green coloured complex is an indicative of absence of deaminase enzyme implying negative test result.

Media Used in Phenylalanine Deaminase Test

Phenylalanine agar

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

Final pH of 7.3 +/- 0.2 at 25ºC.

Procedure of Phenylalanine Deaminase Test

  1. Using a loopful of inoculum from an 18-24 hour pure culture, streak the slant surface with sterile inoculating wire.
  2. Incubate the inoculated slant for 18-24 hours at 35°-37°C in ambient air with cap loose.
  3. Following incubation, add 4-5 drops of a 10% Ferric Chloride solution directly to the slant. Gently agitate the tube and observe for the development of a green color within 1-5 minutes.

Result Interpretation of Phenylalanine Deaminase Test

Result Interpretation of Phenylalanine Deaminase Test

Positive test: development of a light to dark green color within 1-5 minutes after applying ferric chloride reagent.

Negative test: absence of a green color reaction or yellow coloration due to the color of the ferric chloride.

Limitations of Phenylalanine Deaminase Test

  • Biochemical, immunological, molecular, or mass spectrometry testing must be performed on colonies from pure culture for complete identification.
  • The green color reaction of a positive test fades rapidly. Hence 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.

Quality Control of Phenylalanine Deaminase Test

Positive: Proteus mirabilis (ATCC12453)

Negative: Escherichia coli (ATCC25922)

References

  1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
  2. Snyder JW, Atlas RM. Handbook of Media for Clinical Microbi­ology
  3. Ederer, G. M., Chu, J. H., & Blazevic, D. J. (1971). Rapid Test for Urease and Phenylalanine Deaminase Production. Applied Microbiology21(3), 545.

Phenylalanine Deaminase Test

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