Phenylalanine Deaminase Test Introduction

Biochemical tests are usually performed to detect the ability of the microorganism to distinguish their enzymatic activity and to define their characteristics.

Each of the organism acts accordingly based on their surrounding enzymatic reactions and their host cell they live upon.

These biochemical tests are performed with the help of inoculating cultures and specific chemical reagents and the indicators used.

Phenylalanine deaminase test is one such test which is used to differentiate the species belonging to the group of urea-positive gram-negative bacilli on the basis of their ability to produce phenyl.

What is Phenylalanine Deaminase Test?

Phenylalanine deaminase test is commonly known as Phenyl pyruvic acid test, which is commonly called as PPA test.

Phenylalanine deaminase test is due to ability of the organism to produce deaminase.

Phenylalanine deaminase enzyme helps in removing the amino group from the amino acid phenylalanine which thus produces and phenyl pyruvic acid and ammonia by the process of oxidative deamination of phenylalanine.

Phenyl pyruvic acid reacts along with the ferric iron and produces a visible green color.

Phenylalanine agar, commonly known as phenylalanine deaminase medium, as it contains the DL- phenylalanine and the nutrients in their medium.

Phenylalanine Deaminase Test

Phenylalanine deaminase tests is usually used to determine the ability of the organisms to produce the enzyme deaminase.

Micro organisms which are capable of producing phenylalanine deaminase.

Micro organisms that are capable of producing the enzyme phenylalanine deaminase removes the amide group from the phenylalanine thus results in release of the free molecules of ammonia.

Thus, the deamination of phenylalanine results in the formation of phenylalanine pyruvic acid with the helps of the oxidative enzymes.

Hendriksen in the year 1950 demonstrated the species of proteus which helps us in converting the amino acid phenylalanine into the acid form known as phenylpyruvicacid.

But in later times, Buttiaux et al. developed a medium of culture for detecting the formation of phenyl pyruvic acid from the members of the phenylalanine namely Proteus, Providencia and other groups such as Morganella.

This medium is modified again by Ewing et al. and further by Bynae. Bynae simplified this medium by discarding the process of protease peptone in the medium.

Usually, the micro-organisms which detects the phenylamino acid by the enzyme phenylalanine deaminase is identified by adding 4 to 5 drops of ferric chloride.

Whereas addition of ferric chloride results in the production of green colored complex between the compounds of the slant test and also in the tube which indicates the positive results.

On the other hand, the absence of green colored complex determines the negative result of the test.

Phenylalanine Deaminase Test

• The main aim of the test is to detect the ability of the organism to deaminate the phenylalanine oxidatively and to convert it into phenyl pyruvic acid.

• To differentiate the certain species of gram-negative bacilli from the Enterobacteriaceae family.

Phenylalanine Deaminase Test Reagents

Materials required for preparing a medium:

Ingredients	Gram/Liter
DL – Phenylalanine	2.0
Yeast extract	3.0
Sodium chloride	5.0
Disodium phosphate	1.0
Agar	12.0
Distilled water	1 liter
pH	7.3

Here, yeast extract plays an important role in acting as a source of carbon and nitrogen. On the other hand, meat extract and protein hydrolysates cannot be used as the natural varying agent of the phenylalanine.

Phenylalanine Deaminase Test Procedure

Initially a loop full of inoculum is collected from an 18 to 24-hour pure culture, and it is streaked against the slanted surface with the help o a fishtail motion or by using a Phenylalanine inoculate of about 1 drop from the 24-hour brain-heart infusion medium.

The prepared medium is inoculated at a temperature of about 35 degree Celsius for about 18 to 24 hours.

After completing the process of incubation, about 4 to 5 drops of 10% ferric chloride solution is applied directly upon the slant.

Then the tube containing a slant is agitated gently and if the results are positive then the development of green color can be observed within a minimum of 1 minute and a maximum of 5 minutes.

Phenylalanine Deaminase Test Result

Positive Result: In case of positive result, the development of green color can be observed along the slant after adding few drops of ferric chloride. This reaction is usually seen within 5 minutes of addition of ferric chloride.

Negative Result: Here the negative results are determined by absence of green color in the medium after 6 to 7 minutes of addition of a ferric chloride. But negative results are determined by appearance of yellow color which is due to the presence of the ferric chloride.

Phenylalanine Deaminase Test Uses

This test is usually recommended for differentiating the species of gram-negative enteric bacilli on the basis of the ability of the micro-organism to produce the phenyl pyruvic acid by the process of oxidative deamination.

Generally, the genera like Morganella, Proteus and Providencia are differentiated by following these tests to differentiate them from Enterobacteriaceae family.