Phenylalanine Deaminase Test- Principle, Procedure, Results

Phenylalanine deaminase test, also known as phenylpyruvic acid (PPA) test, is a biochemical test used to determine the ability of bacteria to produce an enzyme called phenylalanine deaminase. This enzyme catalyzes the oxidative deamination of the amino acid phenylalanine, which means it removes the amine group (NH2) from phenylalanine and produces phenylpyruvic acid and ammonia as products .

Phenylpyruvic acid is a key intermediate in the metabolism of phenylalanine and tyrosine, two essential amino acids that are involved in the synthesis of various proteins, hormones, neurotransmitters and pigments. Phenylpyruvic acid can react with a chelating agent called ferric chloride, which forms a green-colored complex that can be detected visually . This color change is the basis of the phenylalanine deaminase test.

The phenylalanine deaminase test was first developed by Hendriksen in 1950 to differentiate Proteus spp., which are bacteria that can produce phenylalanine deaminase, from other members of Enterobacteriaceae, which are bacteria that cannot produce this enzyme. Later, Ewing and his team formulated a medium containing phenylalanine to test the bacterial capacity to deaminate phenylalanine oxidatively. In 1971, Ederer and his associates developed a disc method to rapidly detect the bacteria`s ability to produce both urease and phenylalanine deaminase enzymes. These methods have been modified over time and are now widely used in microbiology laboratories for identification and differentiation of various bacterial species.

The phenylalanine deaminase test is especially useful for distinguishing Proteus spp., Morganella spp. and Providencia spp., which are positive for this test, from other Enterobacteriaceae, which are negative for this test . These bacteria are commonly found in soil, water, plants and animals, and some of them can cause infections in humans, such as urinary tract infections, wound infections, septicemia and meningitis. Therefore, the phenylalanine deaminase test can help in the diagnosis and treatment of these infections by providing information about the bacterial identity and susceptibility to antibiotics.

The phenylalanine deaminase test (PDA) is a biochemical test that can be used for two main purposes:

• To determine the ability of bacteria to synthesize the phenylalanine deaminase enzyme, which oxidatively deaminates (removes NH2) from the amino acid phenylalanine. This enzyme is present in some bacteria but not in others, and it can be detected by a colorimetric reaction with ferric chloride.
• To differentiate Enterobacterales and identify the Proteus spp., which are a group of bacteria that can cause urinary tract infections, wound infections, and septicemia. Proteus spp. are among the few Enterobacterales that produce phenylalanine deaminase enzyme, and they can be distinguished from other members of this family by the PDA test.

The PDA test is a simple, inexpensive, and rapid test that can be performed in a laboratory setting with minimal equipment and materials. It can provide useful information for the identification and characterization of bacterial isolates.

Some bacteria have the ability to produce an enzyme called phenylalanine deaminase, which catalyzes the removal of an amino group (NH2) from the amino acid phenylalanine. This reaction results in the formation of phenylpyruvic acid and ammonia as by-products. The phenylpyruvic acid can be detected by adding a chemical reagent called ferric chloride, which forms a green-colored complex with phenylpyruvic acid. The intensity of the green color is proportional to the amount of phenylpyruvic acid present in the medium. Therefore, the presence or absence of phenylalanine deaminase enzyme in bacteria can be determined by observing the color change of the medium after adding ferric chloride.

The phenylalanine deaminase test is based on this principle and uses a special medium called phenylalanine agar, which contains phenylalanine as the sole source of carbon and nitrogen for bacterial growth. The medium also contains yeast extract, sodium chloride, disodium hydrogen phosphate, and agar. The medium is inoculated with a heavy load of bacteria and incubated for 18 to 24 hours. After incubation, ferric chloride solution is added to the slant and the color change is observed. A positive test is indicated by the development of a green color on the slant, while a negative test is indicated by no color change or a yellow color due to ferric chloride.

The phenylalanine deaminase test is useful for differentiating some members of the family Enterobacterales, especially the genus Proteus, which are known to produce phenylalanine deaminase enzyme. The test can also help in identifying some other bacteria that produce this enzyme, such as Morganella and Providencia. However, the test is not a confirmatory test and should be used along with other biochemical tests for accurate identification of unknown bacteria.

The phenylalanine deaminase test requires the following components:

• Culture Media: Phenylalanine agar medium (also called the phenylalanine deaminase medium) is used for the PDA test. It is a differential medium that contains phenylalanine as the sole source of carbon and nitrogen for the bacteria. The medium also contains yeast extract, sodium chloride, disodium hydrogen phosphate and agar. The pH of the medium is adjusted to 7.3±0.2 at 25°C.

• Reagents: Acidic 10% ferric chloride solution is used as the indicator reagent for the PDA test. It reacts with the phenyl pyruvic acid produced by the deamination of phenylalanine and forms a green complex. Alternatively, 10% aqueous ferric chloride solution can be used in place of acidic ferric chloride solution. For the rapid method, 1 N HCl and urea-PDA disks are also required.

• Equipment and materials: The equipment and materials needed for the PDA test include:

  • PPE and other general laboratory materials such as gloves, goggles, lab coat, etc.
  • Test organism (sample bacteria) from a well-isolated colony or pure culture
  • Positive Control: Proteus mirabilis ATCC 12453
  • Negative Control: Escherichia coli ATCC 25922
  • Sterile loop or needle
  • Test tubes with screw caps or cotton plugs
  • Incubator
  • Dropper or pipette
  • Plastic test tubes (for rapid method)
  • Sterile saline (for rapid method)

Phenylalanine medium is a differential medium that contains phenylalanine as the sole source of carbon and nitrogen for the bacteria. It also contains yeast extract, sodium chloride, disodium hydrogen phosphate and agar to provide essential nutrients and maintain the pH and consistency of the medium. The medium is prepared as follows:

• Measure the appropriate amount of phenylalanine powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company.
• Stir well using a magnetic stirrer or manually and heat to boiling so that all the components dissolve completely in water.
• Dispense the media in a test tube (about 5 mL in each or appropriate volume as per requirement) and loosely put on the screw cap (or use a cotton plug to cover the opening).
• Autoclave the tubes with the medium at 121°C and 15 lbs pressure for 15 minutes.
• Let it cool and solidify in a slanting position at room temperature.

The prepared medium should be clear and light amber in color. It should be stored in a refrigerator at 2-8°C and used within one month of preparation. Before use, check the medium for any signs of contamination, discoloration or deterioration. If any of these are observed, discard the medium and prepare a fresh batch.

The main reagent used in the phenylalanine deaminase test is acidic 10% ferric chloride solution. This reagent reacts with the phenyl pyruvic acid produced by the deamination of phenylalanine and forms a green-colored complex. The intensity of the green color indicates the amount of phenyl pyruvic acid present in the medium.

To prepare acidic 10% ferric chloride solution, follow these steps:

• Dissolve 12 grams of ferric chloride in 97.5 mL of distilled water
• Slowly add 2.5 mL of concentrated hydrochloric acid (HCl) and mix well
• Store the solution in a dark bottle and away from light

Alternatively, you can use 10% aqueous ferric chloride solution (10 grams of ferric chloride in 100 mL distilled water) instead of acidic ferric chloride solution.

For the rapid method, you will also need 1 N HCl and urea-PDA disks. The 1 N HCl is used to lower the pH of the bacterial suspension before adding the ferric chloride solution. The urea-PDA disks contain both urea and phenylalanine and can be used to detect both urease and phenylalanine deaminase enzymes in one test.

To prepare 1 N HCl, follow these steps:

• Measure 83 mL of concentrated HCl and transfer it to a 1 L volumetric flask
• Add distilled water to make up the volume to 1 L and mix well
• Store the solution in a glass bottle with a tight cap

The urea-PDA disks can be purchased from commercial suppliers or prepared in-house by impregnating filter paper disks with urea and phenylalanine solutions.

Some additional sentences to conclude the point are:

• Make sure to label the reagents and store them properly to avoid contamination and deterioration.
• Always check the reagents for discoloration or precipitation before use and discard them if they are expired or damaged.
• Follow the safety precautions when handling the reagents as they are corrosive and toxic.

To perform the phenylalanine deaminase test, you will need the following equipment and materials:

• Test tubes with screw caps or cotton plugs to hold the phenylalanine agar medium and the bacterial suspension.
• Conical flask or glass bottle to prepare the phenylalanine agar medium.
• Magnetic stirrer or manual stirring device to mix the medium components.
• Autoclave to sterilize the medium and the test tubes.
• Bunsen burner or other flame source to sterilize the inoculating loop and create aseptic conditions.
• Laminar flow chamber or other clean work area to prevent contamination of the medium and the bacteria.
• Dispose jar or other waste container to discard the used test tubes and loops safely.
• Incubator to provide optimal temperature and aerobic conditions for bacterial growth and enzyme production.
• Phenylalanine agar medium or phenylalanine deaminase medium, which contains DL-phenylalanine and nutrients for bacterial growth and enzyme detection.
• Ferric chloride solution (10% acidic or aqueous), which acts as a chelating agent with the phenyl pyruvic acid produced by deamination and forms a green complex.
• 1 N HCl and urea-PDA disks (optional), which are used for a rapid method of phenylalanine deaminase test that also detects urease activity.
• Inoculating loop to transfer bacteria from pure cultures or isolated colonies to the phenylalanine agar slant or saline suspension.
• Test organism (sample bacteria) that you want to test for phenylalanine deaminase enzyme production. You should use fresh, actively growing bacterial cultures (18 to 24 hours old) for optimal results.
• Positive control and negative control bacteria to check the validity of the medium and the reagent. For example, you can use Proteus mirabilis ATCC 12453 as a positive control and Escherichia coli ATCC 25922 as a negative control.

There are two methods to perform the phenylalanine deaminase test: the agar method and the rapid method. The agar method is more sensitive and reliable, but it takes longer time and requires more materials. The rapid method is less sensitive and reliable, but it takes less time and requires fewer materials. Both methods use the same principle of detecting the phenyl pyruvic acid produced by the deamination of phenylalanine.

Agar Method

• Using a sterile loop, pick up a heavy load of test bacteria from a well-isolated colony (or pure culture) or fresh culture (18 to 24 hours old culture) and heavily inoculate the slant by streaking method.
• Incubate the inoculated tube aerobically at 35±2°C for 18 to 24 hours. (If heavy inoculum is used for streaking, incubation for about 6 hours can be enough to detect the enzyme production.)
• After incubation, drop 4 to 5 drops of acidic 10% ferric chloride solution directly over the slant.
• Roll the tube gently so that the reagent reaches all over the slant surface.
• Observe for color change and development of green color within 5 minutes.

Rapid Method

• In a test tube (preferably a plastic test tube), add 0.25 mL of sterile saline and make a heavy suspension of the test bacteria from a fresh, actively growing bacterial culture.
• Add a urea-PDA disk.
• Incubate the tube aerobically at 37°C for up to 2 hours.
• Observe for the development of pink color over the disk, indicating a positive urease test.
• Add 2 drops of 1 N HCl.
• Add 2 drops of acidic 10% ferric chloride solution and shake well and observe for the development of green color.

The result of the phenylalanine deaminase test is based on the color change of the medium or the suspension after the addition of acidic ferric chloride solution. The color change is due to the reaction of phenyl pyruvic acid with ferric ions, forming a green complex. The result should be read within 5 minutes of adding the reagent, as the color may fade over time.

• Positive test: development of a light to dark green color within 1-5 minutes after applying ferric chloride reagent. This indicates that the bacteria can produce phenylalanine deaminase enzyme and deaminate phenylalanine to phenyl pyruvic acid. Examples of bacteria that give a positive PDA test are Proteus spp., Morganella spp., and Providencia spp.
• Negative test: absence of a green color reaction or yellow coloration due to the color of the ferric chloride. This indicates that the bacteria cannot produce phenylalanine deaminase enzyme and do not deaminate phenylalanine. Examples of bacteria that give a negative PDA test are Escherichia coli, Klebsiella spp., and most other Enterobacteriaceae .

Quality control measures are essential to ensure the accuracy and reliability of the phenylalanine deaminase test results. Some of the quality control measures are:

• Use pure and fresh cultures of the test bacteria for inoculation. Contaminated or old cultures may give false results.
• Use appropriate media and reagents for the test. Check the expiry date and storage conditions of the media and reagents before use. Discard any media or reagents that show signs of deterioration or contamination.
• Use positive and negative control strains along with the test bacteria to verify the performance of the media and reagents. The positive control strain should produce a positive reaction (green color) and the negative control strain should produce a negative reaction (no color change) after the addition of ferric chloride solution.
• Follow the standard procedure and incubation conditions for the test. Do not over-inoculate or under-inoculate the media. Do not incubate for longer or shorter than the recommended time.
• Read and interpret the results within 5 minutes of adding ferric chloride solution. The green color may fade away after some time, leading to false-negative results.
• Record and report the results carefully. Use a standardized format and terminology to describe the results. Include any relevant information such as culture source, incubation time, and control results.

By following these quality control measures, one can ensure the validity and reproducibility of the phenylalanine deaminase test results.

• The phenylalanine agar medium should be checked for sterility and contamination before use. If the medium is turbid or discolored, it should be discarded and replaced with a fresh one.
• The inoculation of the test bacteria should be done with a sterile loop or needle and care should be taken to avoid cross-contamination or over-inoculation of the slant.
• The incubation of the inoculated tubes should be done at the optimal temperature and time for the growth of the test bacteria. If the incubation is too short or too long, it may affect the enzyme production and the test results.
• The acidic ferric chloride solution should be added carefully and evenly over the slant or the suspension. Excess or insufficient amount of the reagent may alter the color reaction and lead to false results.
• The color change should be observed within 5 minutes of adding the reagent. If the observation is delayed, the green color may fade or disappear due to oxidation or reduction of phenyl pyruvic acid.
• The test results should be interpreted in conjunction with other biochemical tests and morphological characteristics of the test bacteria. A positive PDA test alone is not sufficient to identify a bacterial species.

The phenylalanine deaminase test has several applications in microbiology, especially for the differentiation and identification of bacteria. Some of the applications are:

• To differentiate Proteus spp., Morganella spp., and Providencia spp. among other Enterobacterales . These bacteria are known to produce phenylalanine deaminase enzyme and give a positive test result, while most other members of Enterobacterales do not .
• To identify Proteus spp. in clinical isolates . Proteus spp. are opportunistic pathogens that can cause urinary tract infections, wound infections, septicemia, and other infections in humans. They are also known for their ability to produce urease enzyme and form kidney stones. The phenylalanine deaminase test can help to confirm the presence of Proteus spp. among other urease-positive bacteria.
• To use in biochemical identification of unknown bacteria . The phenylalanine deaminase test can be used as one of the biochemical tests to determine the metabolic characteristics of an unknown bacterium. It can help to narrow down the possible genera or species of the bacterium based on its ability to produce phenylalanine deaminase enzyme.

The phenylalanine deaminase test is a simple, inexpensive, and reliable method to detect the presence of phenylalanine deaminase enzyme in bacteria. It can be used as a supplementary test along with other tests to differentiate and identify bacteria of clinical and environmental importance.

The phenylalanine deaminase test is a useful biochemical test to differentiate some members of the Enterobacterales and identify the Proteus spp. However, it has some limitations that should be considered when interpreting the results. Some of the limitations are:

• The test is not a confirmatory test; hence, it needs other tests for the complete identification of unknown bacteria. For example, the PDA test cannot distinguish between different species of Proteus, Morganella, or Providencia. Other tests such as indole, methyl red, Voges-Proskauer, citrate, urease, or motility tests may be required to further differentiate these bacteria.
• The developed green color fades rapidly, so the result must be read within 5 minutes of the addition of ferric chloride solution. If the result is read after 5 minutes, it may give a false-negative result due to the loss of color intensity. Therefore, it is important to observe the color change immediately after adding the reagent and record the result accordingly.
• The test may give false-positive results if the phenylalanine medium is contaminated with other amino acids that can also be deaminated by some bacteria. For example, tyrosine can also be deaminated by some bacteria such as Pseudomonas aeruginosa or Alcaligenes faecalis and produce a green color on the slant. Therefore, it is essential to use a pure and fresh phenylalanine medium for the test and avoid cross-contamination with other media or reagents.
• The test may give false-negative results if the bacterial culture is too old or too diluted. The phenylalanine deaminase enzyme requires an active and heavy growth of bacteria to produce enough phenyl pyruvic acid for the reaction. If the bacterial culture is too old (more than 24 hours) or too diluted (less than 10^8 CFU/mL), it may not produce enough enzyme or substrate for the reaction and result in a negative test. Therefore, it is recommended to use a fresh and heavy inoculum of bacteria for the test and incubate it for an optimal time (18 to 24 hours) at an optimal temperature (35±2°C).
• The test may give variable results depending on the strain of bacteria and the environmental conditions. Some strains of bacteria may produce more or less phenylalanine deaminase enzyme than others depending on their genetic makeup and metabolic activity. Similarly, some environmental factors such as pH, oxygen level, temperature, or nutrient availability may affect the enzyme production and activity. Therefore, it is important to standardize the test conditions and use appropriate controls to ensure reliable and consistent results.

These are some of the limitations of the phenylalanine deaminase test that should be taken into account when performing and interpreting the test. Despite these limitations, the test is still a valuable tool for identifying some bacteria that can cause infections in humans and animals.

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