Principle of Phenylalanine Deaminase (PDA) Test
The phenylalanine deaminase test is also known as the phenyl pyruvic acid (PPA) test used to differentiate among the urea positive gram-negative rods based on the ability of the microorganisms to produce phenyl pyruvic acid by oxidative deamination. Phenylalanine is an amino acid that, upon deamination by oxidase enzymes, results in the formation of phenyl pyruvic acid. The deamination of phenylalanine to phenyl pyruvic acid is detected by the addition of a ferric chloride solution that acts as a chelating agent with the keto acid by-product to produce a light to deep green cyclic compound. The genus of Enterobacteriaceae like Proteus, Providencia and Morganella are capable of deaminating phenylalanine and they are also urease positive and gram-negative rods.
Requirements of Phenylalanine Deaminase Test
A. Test organisms
For phenylalanine deaminase test take gram-negative rods that grow well on MacConkey agar, are oxidase negative, and are usually urea positive.
Phenylalanine agar slants
Composition of the medium
- DL-Phenylalanine: 2 gm
- Yeast extract: 3 gm
- Sodium chloride: 5 gm
- Disodium phosphate: 1 gm
- Agar: 12 gm
- Distilled water: 1000 ml
- pH: 7.3
10% Ferric chloride, acidiﬁed
- Ferric chloride: 12 gm
- Concentrated HCl: 2.5 mL
- Distilled water: 100
- Dissolve 12 g of ferric chloride in 97.5 ml of water.
- Slowly add 2.5 ml of concentrated HCl to a fume hood.
- Store in a brown bottle at 4°C.
D. Other accessories
- Sterile wooden sticks or inoculating loops
Quality control strains
Organisms for positive control (PC) Proteus mirabilis ATCC 12453 and negative control (NC) Escherichia coli ATCC 25922
Procedure of Phenylalanine Deaminase Test
- Before inoculation, allow the medium to equilibrate at room temperature.
- Using a heavy inoculum from an 18 to 24 hours pure culture, streak the slant surface using a ﬁshtail motion.
- Incubate the inoculated slant aerobically at 37°C for 18 to 24 h. If you are using a heavy inoculum, incubate only for 4 to 6 hours.
- After incubation, apply 4 or 5 drops of ferric chloride directly to the slant.
- Gently roll the reagent over the slant to dislodge surface colonies.
After application of ferric chloride, observe for the development of a green color within 1 to 5 minutes.
Result and Interpretation of Phenylalanine Deaminase Test
Phenylalanine deaminase Test positive: phenylalanine deamination reaction is indicated by the development of a light to dark green color within 1 to 5 min after applying ferric chloride reagent.
Test Negative: It is indicated by the absence of a green color reaction i.e. negative results will take on a yellow color due to the color of the ferric chloride
Positive Control: Proteus mirabilis turns green after the addition of ferric chloride
Negative Control: Escherichia coli remains yellow after the addition of ferric chloride
Note: Test result is positive as shown above image.
Keynotes on Phenylalanine Deaminase Test
- The test can also use to identify other Enterobacteriaceae, Buttiauxella, Rahnella, and Tatumella, which are PDA positive but are urea negative.
- Tryptophan are also alternative for phenylalanine; tryptophan deamination releases indole-pyruvic acid, which results in a purple to black color with the addition of ferric chloride.
- Either tryptophan or phenylalanine can be used to differentiate among the Proteus group of gram-negative rods.
- The rapid test is available using the disk.
Limitations of Phenylalanine Deaminase Test
Following are the limitations of the phenylalanine deaminase test:
- The green color reaction of a positive test fades rapidly and therefore, it must interpret within 5 minutes after the application of ferric chloride, or false-negative results may occur.
- Slight agitation of the tube containing ferric chloride will produce a faster, more expressed color reaction.
- Tryptophan deaminase is the substitute of PDA with the same interpretation of results.
- It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
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