Phenylalanine Deaminase Test Principle, Procedure, Result

• The Phenylalanine Deaminase (PDA) test can be utilized to identify Gram-negative bacilli that are urea-positive due to the capacity of microorganisms to generate phenyl-pyruvic acids through the process of oxidative deamination.
• The test is also referred to as the phenyl-pyruvic acids (PPA) test. Phenylalanine is an amino acid that when deaminated through oxidase enzymes, produces ammonia and phenylpyruvic acid. released.
• The deamination is confirmed by the addition of a ferric-chloride solution, which is a chelating agent that interacts the -keto acid byproduct that produces a light deep green chemical compound that is cyclic.
• Hendriksen in 1950 showed the fact that Proteus spp. were capable of converting phenyl- Alanine into phenyl-pyruvic acids.
• This discovery was incorporated into an environment by Ewing and al. and then into a disk with Urea by Ederer and Ederer. . Of the Enterobacteriaceae which are urea positive and belong to the Proteus, Providencia, and Morganella group can deaminate the phenylalanine.
• This test could also be used to determine different Enterobacteriaceae, Buttiauxella, Rahnella and Tatumella that are PDA positive, but not urea positive.

Objective of Phenylalanine Deaminase Test

• To assess the capacity of an organism’s ability to produce the enzyme Deaminase.
• To distinguish between the Gram-negative urea-positive bacteria by the capacity of microorganisms to make phenyl-pyruvic acids through the process of oxidative deamination.
• Learn the biochemical basis of the phenylalanine test
• Make sure you are performing the phenylalanine desaminase test correctly;
• Differentiate between negative and positive reactions as well as
• Learn how this information can be used to discriminate and differentiate Microbial species.

Principle of Phenylalanine Deaminase Test

• Phenylalanine Agar, also referred to as phenylalanine-deaminase medium, has nutrient as well as DL which is phenylalanine.
• The phenylalanine is the base of the enzyme Phenylalanine deaminase (PAD) that is capable of de-amining it into phenyl pyruvic acid.
• This enzyme is employed by some bacteria to make use of this amino acid to create an energy source and carbon source.
• Enteric bacteria do not make this enzyme, however species like Morganella, Providencia, and Proteus do.
• Microorganisms that make phenylalanine deaminase eliminate the amino (NH2) from the phenylalanine.
• This reaction causes the creation of ammonia (NH3) and Phenyl pyruvic acid. Phenyl pyruvic acid can be identified by adding some drops of ferric chloride at 10% that acts as an agent for chelation.
• A green-colored complex forms between the two compounds, indicating that the test is positive.

Requirements For the test

• Test organisms: For phenylalanine deaminase test take gram-negative rods that grow well on MacConkey agar, are oxidase negative, and are usually urea positive.
• Media: Phenylalanine agar
• Reagents: 10% Acidified Ferric chloride
• Apparatus: Inoculating loops, Incubator at 35 to 37°C, Distilled water

Composition of Phenylalanine agar

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

Preparation of Phenylalanine agar slant

1. Submerge 26 grams of the substance in 1 Liter of water that has been distilled.
2. Bring to a boil, stirring until the medium dissolves completely.
3. Dispense in a tube and then place it within the autoclave for 15 mins at 121 degrees Celsius.
4. Let the medium get cool before placing it in a slanting manner.

Note: Following autoclaving, the medium should appear lighter in color. Also, you will notice the formation of a slight opalescent gel within the tube.

Composition of 10% Acidified Ferric chloride

Ferric chloride:	12 gm
Concentrated HCl:	2.5 mL
Distilled water:	100

Preparation of 10% Acidified Ferric chloride

1. Dissolve 12 g ferric chloride using 97.5 milliliters of water.
2. Slowly slowly add 2.5 Ml of concentrated HCl to an exhaust hood.
3. Keep in a brown container at 4°C.

Procedure of Phenylalanine Deaminase Test

Step 1: Inoculation of Medium

1. Choose the phenylalanine agar medium that has a slant.
2. Start your Bunsen burner.
3. Choose the inoculating loop for inoculation. Then, use a flame in order to make it sterilized.
4. Take off the caps of the test tube. Burn the insides of your test tubes’ mouths.
5. Utilize the sterile inoculating tool to collect an inoculum that was taken from the culture tube from the unidentified bacteria. The inoculum should be immediately transferred into the fresh sterilized, safe medium.
6. Again Flame the mouths of your tubes.   
7. Replace the caps that are on test tubes.
8. Re-flame the tool used to inoculate.  

Step 2: Incubation of the Inoculated Medium

1. Place the tube with the inoculation into the 35 to 37 C incubator.
2. Hit the New Day button to move forward for 24 hours. Allow to incubate for the proper amount of time. The test can be properly read within 24 hours.
3. Remove desired incubated cultures from the incubator. 

Step 3: Adding the reagents

1. Select the right dropper instrument and Reagents.
2. Remove the cap from the tube and place the bottom of the dropper in the test tube.
3. Add 10 percent ferric chloride reagent into the sample.
4. The dropper’s end should be placed in the test tube. Add the next ingredient 0.1N HCl reagent to the sample. 

Result Interpretation

• Positive Result: The test is considered positive If the medium becomes dark green. This color change is the consequence of degradation products created by Phenylalanine. It will eventually mix the iron compounds in an acidic environment, resulting in a the dark color of green.
• Negative Result: The outcome is negative if the hue of the medium doesn’t change. It is still straw-yellow in the color (No PPA to react with ferric chloride).

Phenylalanine Deaminase Positive Test organisms

The organisms that can test positive for the test for phenylalanine aminase include:

• Proteus sp
• Morganella sp
• Providenica sp
• Proteus vulgaris
• Proteus mirabilis

Phenylalanine Deaminase Negative Test organisms

Some examples of organisms that are negative for the test phenylalanine desaminase include:

• Escherichia coli
• Enterobacter aerogenes

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).

Use of Phenylalanine Deaminase Test

• Phenylalaine deaminase test can be used to distinguish members belonging to three genera Proteus, Morganella, and Providencia (+ve) from other members of Enterobacteriaceae which yield negative results.

Keynotes on Phenylalanine Deaminase Test

• Tryptophan can also be a substitute for phenylalanine. tryptophan deamination causes the release of indole-pyruvic acid which causes an ethereal black or purple color by adding ferric chloride.
• Phenylalanine or tryptophan can be used to distinguish between those belonging to the Proteus group of rods with gram-negative chemistry.
• The test can be performed quickly through the disk.

Limitations of Phenylalanine Deaminase Test

Here are the drawbacks of the test phenylalanine deaminase:

• The green color of a positive test will fade rapidly and, therefore, the test must interpret within 5 minutes following an application of ferric chloride or false-negative results could occur.
• A gentle stirring of the tube containing ferric chloride can cause an enhanced, quicker color reaction.
• Tryptophan deaminase is the replacement in the absence of PDA that has the exact interpretation of the results.
• It is suggested that biochemical, immunological mass spectrometry, or molecular testing be carried out on pure-cultured colonies to ensure complete identification.

References

• Deirdre L. Church, 2016. 3.17 Biochemical Tests for the Identification of Aerobic Bacteria, Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.CMPH.ch3.17
• ://dspace.vpmthane.org:8080/jspui/bitstream/123456789/7584/1/PHENYL%20ALANINE%20DEAMINASE%20TEST.pdf
• https://www.onlinebiologynotes.com/phenylalanine-deaminase-test-principle-requirements-procedure-and-results-interpretations/
• https://microbeonline.com/phenylalanine-deaminase-test-principle-procedure-results-uses/
• https://universe84a.com/phenylalanine-deaminase-test/
• https://laboratoryinfo.com/phenylalanine-deaminase-test/
• https://www.vumicro.com/vumie/help/VUMICRO/Phenylalanine_deaminase_Test.htm
• http://crcooper01.people.ysu.edu/microlab/PDA-test.pdf