Phenylalanine Deaminase Test – Principle, Procedure, Result

The phenylalanine deaminase test is a biochemical test used to identify bacteria that can deaminate the amino acid phenylalanine. It is the process where certain organisms convert phenylalanine into phenylpyruvic acid by removing the amine group. This is referred to as oxidative deamination and it is one of the important reactions for separating the PPM group (Proteus, Providencia, and Morganella) from other Enterobacteriaceae where this enzyme is absent.

In this test, the bacteria is grown on a medium containing phenylalanine. If the organism produces phenylalanine deaminase, the enzyme acts on phenylalanine forming phenylpyruvic acid and ammonia. The phenylpyruvic acid formed is colorless and cannot be detected directly. It is the addition of ferric chloride (FeCl₃) reagent after incubation that helps in detecting the reaction. The reaction is as follows– phenylpyruvic acid reacts with ferric ions forming a green colored complex.

In this step, the appearance of a distinct green color indicates a positive test. The color is unstable and fades quickly, so the result is read within 1 to 5 minutes. If the slant remains yellow without any color change, it is considered as negative. This process occurs when the organism has the enzyme phenylalanine deaminase and thus this test is used as an important differential test in microbiology for identifying the members of the PPM group.

Objectives of Phenylalanine Deaminase Test

• To detect the ability of bacteria to produce the enzyme phenylalanine deaminase which acts on phenylalanine.
• To find out if oxidative deamination of phenylalanine is taking place forming phenylpyruvic acid and ammonia.
• To differentiate the important Proteus, Providencia, and Morganella groups from other Enterobacteriaceae.
• To help in identifying some non-enteric Gram-negative species which show positive or negative reaction.
• To check if the organism can use phenylalanine as a source of carbon and energy.

Principle of Phenylalanine Deaminase Test

The Phenylalanine Deaminase test is based on the capacity of certain bacteria to produce the enzyme phenylalanine deaminase. It is the process in which the amino acid phenylalanine is oxidatively deaminated, and the amine group (NH₂) is removed in presence of oxygen. In this reaction phenylalanine is changed into ammonia and a keto acid called phenylpyruvic acid. The phenylpyruvic acid formed is colourless, so it cannot be detected directly in the medium. For this reason a reagent of 10% ferric chloride (FeCl₃) is added, and the ferric ions combine with phenylpyruvic acid forming a green coloured complex. This is referred to as the positive reaction. The appearance of the green colour indicates that the reaction is completed. The test is used mainly to differentiate Proteus, Morganella, and Providencia species because these organisms can form the enzyme, while most other Enterobacteriaceae cannot.

Requirements of Phenylalanine Deaminase Test

1. Culture Media (The Substrate)
   • Phenylalanine agar is used as the main medium. It is generally prepared as a slant.
   • The medium contains DL-phenylalanine, yeast extract, sodium chloride, disodium phosphate, and agar.
   • Yeast extract is the major source of vitamins and growth factors. It is used instead of meat extract because meat extract may contain phenylalanine naturally.
   • DL-phenylalanine act as the substrate on which the enzyme phenylalanine deaminase acts.
   • Commercial PDA tablets can also be used. These are dissolved in distilled water before inoculation.
2. Reagents (The Developer)
   • 10% Ferric chloride solution is the main reagent for detection.
   • It is the reagent that reacts with phenylpyruvic acid to form green color.
   • Some ferric chloride solutions are acidified using concentrated HCl to prevent precipitation of iron salts.
   • The reagent must be stored in dark bottles because it is light sensitive.
3. Equipment and Supplies
   • Inoculating loop or needle is required for transferring culture. A heavy inoculum is important.
   • Incubator maintained at 35°C–37°C.
   • Sterile test tubes with loose caps are needed because the process occur in aerobic condition.
   • For rapid test methods, sterile saline and 1N HCl is used along with ferric chloride.
4. Quality Control Organisms
   • Proteus mirabilis or Proteus vulgaris is used as positive control because the slant turns green.
   • Escherichia coli is used as negative control where the slant remains yellow or straw-colored.

Procedure of Phenylalanine Deaminase Test

Standard Agar Slant Method

1. A sterile Phenylalanine agar slant is taken for the test.
2. The medium contains yeast extract sodium chloride disodium phosphate and DL-phenylalanine.
3. A heavy inoculum is taken from a pure bacterial culture (18–24 hours old) with the help of sterile inoculating loop.
4. The surface of the agar slant is streaked by fishtail manner so that the growth spreads over the slant.
5. The butt of the medium is not stabbed as this reaction is aerobic in nature.
6. The inoculated tube is incubated at 35–37°C for 18–24 hours.
7. The cap of the tube is kept loosely closed to maintain aerobic condition.
8. In case of very heavy inoculation result may be obtained within 4–6 hours.
9. After incubation the tube is removed from incubator.
10. About 4–5 drops (approximately 0.2 ml) of 10% Ferric chloride (FeCl₃) reagent is added directly on the surface of the slant.
11. In some procedure acidified ferric chloride containing dilute HCl is used.
12. The tube is gently rotated so that the reagent covers the entire bacterial growth on the slant.
13. The colour change is observed immediately or within 1–5 minutes. Development of light to dark green colour indicates positive test. No colour change and medium remains yellow or straw colour indicates negative test. The green colour is unstable therefore result should be recorded within 5 minutes.

Alternative Rapid Methods

Tablet Method

1. A Phenylalanine deaminase tablet is dissolved in 1 ml of distilled water.
2. The tube is heavily inoculated with a loopful of bacterial growth.
3. It is incubated for 24 hours or for 4–6 hours in case of heavy inoculation.
4. One to two drops of ferric chloride reagent is added and green colour is observed.

Disk Method (Urea-PDA)

1. A heavy suspension of test organism is prepared in 0.25 ml sterile saline.
2. A urea-PDA disk is added and incubated at 37°C for up to 2 hours.
3. After observing urease reaction 0.1N HCl is added followed by 10% ferric chloride.
4. The tube is shaken gently and observed for green colour development.

Quality Control

1. Positive control – Proteus mirabilis or Proteus vulgaris gives green colour.
2. Negative control – Escherichia coli shows no colour change or yellow colour.

Result of Phenylalanine Deaminase Test

Positive Result (+)

• Green colour is developed on the agar slant or in the test solution within 1–5 minutes after addition of ferric chloride.
• It indicates the presence of phenylalanine deaminase enzyme in the organism.
  This enzyme removes the amine group (NH₂) from phenylalanine forming phenylpyruvic acid and ammonia.
  The phenylpyruvic acid reacts with ferric chloride (FeCl₃) producing green coloured complex.
• The result should be read immediately as the green colour is unstable and fades rapidly on exposure to air and light.
• This result is commonly shown by Proteus group organisms such as Proteus Providencia and Morganella.

Negative Result (−)

• No green colour is produced after addition of ferric chloride.
  The medium remains yellow or straw colour of the reagent.
• It indicates absence of phenylalanine deaminase enzyme.
  Phenylalanine is not converted to phenylpyruvic acid therefore ferric chloride does not react.
• Most members of Enterobacteriaceae such as Escherichia coli and Klebsiella gives negative reaction.

Invalid or Indeterminate Result

• If the tube is not observed within 5 minutes the green colour may fade and appear yellow or brownish giving false negative result.
• Improper or deteriorated ferric chloride reagent and dried medium may also give incorrect result. Hence quality control should be done using known positive and negative organisms.

Quality control strains

• Organisms for positive control (PC) Proteus mirabilis ATCC 12453 (Proteus mirabilis changes color after adding ferric chloride).
• Organisms for negative control (NC) Escherichia coli ATCC 25922 (Escherichia coli remains yellow after the addition of ferric chloride).

Uses of Phenylalanine Deaminase Test

• It is used for differentiation of Proteus Providencia and Morganella group from other members of Enterobacteriaceae.
• It helps in identification of Proteus mirabilis which is commonly associated with urinary tract infection and kidney stone formation.
• It is useful in routine identification of clinical isolates showing swarming growth or non lactose fermenting colonies.
• It helps in separating PPM group from organisms like Escherichia coli Klebsiella Enterobacter Salmonella and Shigella.
• It is used to identify some uncommon gram negative bacilli which possess phenylalanine deaminase enzyme.
• It is used to demonstrate the ability of organism to deaminate phenylalanine forming phenylpyruvic acid.
• The test is also useful in biochemical characterization and classification of enteric bacteria in laboratory diagnosis.

Advantages of Phenylalanine Deaminase Test

• It is simple and easy biochemical test to perform in routine laboratory work.
• It helps in clear differentiation of Proteus Providencia and Morganella group from other Enterobacteriaceae.
• The test gives distinct colour reaction which is easy to observe and interpret.
• It provides direct evidence of phenylalanine deaminase enzyme production by organism.
• Rapid methods are available which reduce time required for identification.
• It does not require complex equipment or special conditions for performance.
• It is useful as a confirmatory test in identification of enteric and non fermenting gram negative bacteria.
• The reagents used are easily available and inexpensive.

Limitations of Phenylalanine Deaminase Test

• The green colour formed after addition of ferric chloride is unstable and fades rapidly if not observed within few minutes.
• Delay in reading the result may lead to false negative reaction.
• The test is not confirmatory and should be used along with other biochemical tests for final identification.
• Some organisms other than Proteus Providencia and Morganella may also give positive reaction.
• Ferric chloride reagent is sensitive to light and deterioration of reagent can affect the result.
• A heavy inoculum is required otherwise weak enzyme production may give false negative result.
• Poor growth of organism on the medium may interfere with proper colour development.
• The test cannot differentiate species within the PPM group.