PHENYLALANINE DEAMINASE TEST

PHENYLALANINE DEAMINASE TEST

AIM

      To differentiate the Proteus sp., Morganella sp. and Providencia sp. based on their ability to produce Phenylpyruvic/ Pyruvic acid by Oxidative deamination form other members of Enterobacteriaceae family.

PRINCIPLE       

     In 1950, Hendriksen demonstrated that Proteus spp. were able to convert the amino acid phenylalanine to phenylpyruvic acid. Later, Buttiaux in 1954 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 further modified by Ewing in 1957 by omitting Proteose peptone. 

        Phenylalanine Deaminase Test is used to differentiate members of the Gram negative bacterial genera Proteus, Morganella (which were originally classified under the genus Proteus), and Providencia from other Enterobacteriaceae. Phenylalanine agar, also known as Phenylalanine deaminase medium, contains nutrients and DL-Phenylalanine. The Phenylalanine serves as the substrate for enzymes, which are able to deaminate it to form Phenylpyruvic acid. Yeast extract in the medium supports the growth of the organisms. Sodium chloride maintains osmotic equilibrium.

         Microorganisms that produce Phenylalanine deaminase remove the Amine (NH₂) from the amino acid Phenylalanine. The reaction results in the production of Ammonia (NH₃) and Phenylpyruvic acid. The Phenylpyruvic acid is detected by adding a few drops of 10 % Ferric chloride which acts as a chelating agent. A Green colored complex is formed between these two compounds indicating a positive test. If the medium remains a straw color, the organism is negative for Phenylalanine deaminase production.

MATERIALS REQUIRED

1. Test bacteria
2. Phenylalanine agar slant
3. 10 % Ferric chloride
4. Inoculation loop
5. Incubator

PROCEDURE

a)   Inoculate an inoculum of test bacteria (18 to 24 hours old) by streaking on the Phenylalanine agar slant.

b)     Incubate the inoculated Phenylalanine agar slant at 37 °C for 18 – 24 hours.

c)     Following incubation, apply 4 - 5 drops of a 10 % Ferric Chloride solution directly to the slant.

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

OBSERVATION AND RESULTS

·   Positive Result - Green color develops on the Phenylalanine agar slant within 1 -5 minutes after applying 10 % Ferric chloride reagent. 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.

·       Negative Result - Absence of a green color reaction. Negative results will take on a yellow color due to the color of the 10 % Ferric chloride.

(Left – Positive reaction; Right – Negative reaction)

Figure – 1: Phenylalanine Deaminase Test (Source: pinterest.es)