Phenylalanine Deaminase Test – Procedure, Principle and Uses

Phenylalanine Deaminase Test is a procedure used to find out the capability of Proteus species to deaminate phenylalanine to phenylpyruvic acid with the aid of an enzyme; a vital and useful reaction in distinguishing the members of Enterobacteriaceae family.

Specifically, it is used to differentiate Proteus and Providencia organisms from Enterobacteriaceae family.

Using the Phenylalanine Deaminase Test, the organism’s ability to produce deaminase enzyme will be identified. What this enzyme does is it removes the amine group from the amino acid phenylalanine and ending up producing phenylpyruvic acid and ammonia.

The medium used for this test is phenylalanine agar/phenylalanine deaminase and it contains DL-phenylalanine and nutrient is used for testing the medium. ^{(1, 2, 3, and 4)}

Image 1: The test tube of negative phenylalanine deaminase test remains the same in color while the one that turned out positive has green discoloration.
Picture Source: slideplayer.com

Image 2: Reagents used for phenylalanine deaminase tets.
Picture Source: orioner.com

Purpose of Phenylalanine deaminase test

The main purpose of phenylalanine deaminase test is to determine if the microbe produces phenylalanine deaminase enzyme, which is vital for it to use amino acid phenylalanine as a source of carbon and energy in order to grow. ^{(4, 5)}

Compositions of phenylalanine agar slant

• Sodium chloride
• Yeast extract
• Disodium phosphate
• Agar
• DL-Phenylalanine
• Final pH at 25 C temperature ⁽⁵⁾

Preparing the agar slant

• Suspend 26 grams in 1 liter of distilled water.
• Bring to boil until the medium is dissolved completely.
• Dispense in the tube and autoclave for 15 minutes at 121 degree Celsius.
• Allow the medium to cool by placing the tube in a slanting position.

Note: after autoclaving, the medium should be light amber in color. You will also notice a slightly opalescent gel formation in the tube. ^{(4, 5, 6, and 7)}

How is the procedure performed?

1. Inoculum from a pure culture is transferred to a tube containing phenylalanine agar to streak the slant.
2. The tube is incubated for 24 hours at a temperature of 35-37 C.
3. A reagent is added to determine the result. ^{(6, 7)}

What type of reagent is added?

The reagents added are 10% ferric chloride and 0.1 N HCl five drops each. The tube is shaken and the result is interpreted within five minutes. ^{(7, 8)}

Steps to follow when inoculating the medium

• Choose the phenylalanine agar slant medium.
• Light up the Bunsen burner.
• Choose the inoculating loop tool and sterilize it by placing it on the fire in the Bunsen burner.
• Remove the test tube cap and flame the mouth of the test tube.
• Using the sterile inoculating tool, pick up the inoculum from the culture tube and transfer the inoculum into the sterile medium.
• Flame the test tube mouth and replace the cap.
• Re-flame the inoculating tool. ^{(4, 7, and 8)}

Incubating the inoculated medium

• Put the inoculated tube inside the incubator at a temperature of 35 to 37 C.
• Leave it in the incubator for at least 24 hours. ^{(9, 10)}

Adding the reagents

• Choose the appropriate dropper tool and reagents.
• Take the cap off from the tube and put the end of the dropper into the test tube.
• Add 10% ferric chloride reagent to the sample.
• Place the dropper’s end into the test tube and add the next reagent 0.1N HCl reagent to the sample.  ^{(1, 4, and 7)}

Image 3: The image explains the green discoloration of the test tube slant indicating a positive result.
Picture Source: slideplayer.com

Result Interpretation

Positive Result

The test is positive if the medium turns dark green. The green color is a result of degradation product produced from phenylalanine. It will eventually combine with iron compound in the acidic environment leading to dark green color.  Examples of organisms that test positive for phenylalanine deaminase test are:

• Proteus sp
• Morganella sp
• Providenica sp
• Proteus vulgaris
• Proteus mirabilis
• Providencia alcalifaciens
• Providencia alcalifaciens ^{(3, 6, and 10)}

Negative Result

The result is negative if the color of the medium didn’t change. It remains straw to yellow in color. Examples of organisms that test negative for phenylalanine deaminase test are:

• Escherichia coli
• Enterobacter aerogenes

Note: The result should be interpreted within five minutes after adding the reagent. The changes in color fade quickly and so it is a must to immediately interpret the result. ^{(2, 4, and 8)}

References

1. https://microbeonline.com/phenylalanine-deaminase-test-principle-procedure-results-uses/
2. http://www.austincc.edu/microbugz/phenylalanine_deaminase_test.php
3. http://www.vumicro.com/vumie/help/VUMICRO/Phenylalanine_deaminase_Test.htm
4. https://quizlet.com/71417469/phenylalanine-deaminase-test-micro-flash-cards/
5. https://www.coursehero.com/file/p1giid/The-phenylalanine-deaminase-test-was-done-to-determine-if-the-bacteria-has/
6. https://www.studyblue.com/notes/note/n/phenylalanine-deaminase-test/deck/6258000
7. https://en.wikipedia.org/wiki/Phenylalanine_ammonia-lyase
8. http://comenius.aeprosa.pt/wp.php/?p=13331859.html
9. https://mltgeeks.com/phenylalanine-deamination-test-procedure-and-result-interpretation/
10. https://microbenotes.com/phenylalanine-deaminase-test/