Phenylalanine deaminase test also known as phenylpyruvic acid (PPA) test is used to test the ability of an organism to produce enzyme deaminase. This enzyme removes the amine group from the amino acid phenylalanine and produces phenylpyruvic acid (PPA) and ammonia i.e. oxidative deamination of phenylalanine. Phenylpyruvic acid reacts with ferric iron (10% ferric chloride is added to the medium) producing a visible green color.
Phenylalanine agar, also known as phenylalanine deaminase medium which contains DL-phenylalanine and nutrients is used as a test medium.
Media and Reagents
Phenylalanine agar medium is prepared and poured as a slant into a tube. The composition of the medium is as follows:
- DL-Phenylalanine: 2 gm
- Yeast extract: 3 gm
- Sodium chloride: 5 gm
- Disodium phosphate: 1 gm
- Agar: 12 gm
- Distilled water: 1 L
- pH: 7.3
Yeast extract serves as carbon and nitrogen source. Meat extracts or protein hydrolysates cannot be used because of the varying natural content of phenylalanine.
- Ferric chloride: 12 gm
- Concentrated HCL: 2.5 mL
- Distilled water: 100 mL
- Take or prepare a phenylalanine deaminase agar medium
- Inoculate the phenylalanine slant (with a loop on the surface) with a test organism.
Note: If you are using the test medium i.e. phenylalanine agar for the first time use positive (Proteus vulgaris) and negative control (Escherichia coli) to check the efficacy of the test medium.
- Incubate the test medium at 37°C for overnight.
- Add 4-5 drops of 10% aqueous ferric chloride (FeCl3) solution to the slant. After addition of the reagent, rotate the tube to dislodge the surface colonies.
- Positive test: Production of green color (Phenylpyruvic acid thus formed reacts with ferric chloride producing a green colored compound thus turning the medium dark green). Proteus sp., Morganella sp., Providenica sp give positive PPA test.
- Negative: No color change (medium remains straw/yellow color; no PPA to react with ferric chloride).
Uses of Phenylalaine Deaminase Test
Phenylalaine deaminase test is used to differentiate members of the genera Proteus, Morganella, and Providencia (+ve) from other members of Enterobacteriaceae which give negative results.
Each new batch of medium or reagent must be tested with positive and negative control.
- Positive control: Proteus species
- Negative control: Escherichia coli