Principle
Some bacteria have the ability to deaminate the amino acid phenylalanine, as they can produce the enzyme ‘phenylalanine deaminase’.This enzyme deaminates phenylalanine by removing its amino group, thereby producing phenylpyruvic acid (a keto acid) and ammonia (NH3). Phenylpyruvic acid forms green colour with ferric chloride solution.
In the phenylalanine deaminase test, the test bacterium is grown on agar slants containing phenylalanine. If the bacteria have the ability to deaminate phenylalanine, the color of the slants changes from yellow to green, upon dropping of ferric chloride solution onto it.
Experiment
Reagents And Materials Required
- Test tubes
- Conical flask
- Cotton plugs
- Inoculating loop
- Autoclave
- Bunsen burner
- Laminar flow chamber
- Dispose jar
- Incubator
- Phenylalanine agar
- Ferric chloride solution (10%)
- Isolated colonies or pure cultures of bacteria.
Procedure
- The ingredients of phenylalanine agar medium (containing phenylalanine as the main components) or its ready-made powder required for 100 ml of the medium is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
- Its pH is determined using a pH paper or pH meter and adjusted to 7.2 using 0.1N HCI if it is more or using 0.1N NaOH if it is less.
- The flask is heated to dissolve the agar in the medium completely.
- Before it solidifies, the medium in warm molten condition is distributed into 5 test tubes (approximately 20 ml each).
- The test tubes are cotton-plugged, covered with craft paper and tied with thread or rubber band.
- They are sterilized at 121°C (15 psi pressure) for 15 minutes in an autoclave.
- After sterilization, they are removed from the autoclave and kept in a slanting position to cool and solidify the medium, so as to get phenylalanine agar slants.
- The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, by streaking on surface of the slants with the help of a flame-sterilized loop. The loop is sterilized after each inoculation.
- The inoculated slants are incubated at 37°C for 24 to 48 hours in an incubator.
- 4-5 drops of ferric chloride solution (10%) is added to each slant.
Observations
1. Color of slant changes to green. Phenylalanine deaminase positive.
(Phenylpyruvic acid thus formed reacts with ferric chloride producing a green colored compound thus turning the medium dark green). Proteus sp., Morganella sp., Providenica sp give positive PPA test.
2. Color of slant remains yellow: Phenylalanine deaminase negative.(medium remains straw/yellow color; no PPA to react with ferric chloride).
Uses of Phenylalaine Deaminase Test
Phenylalaine deaminase test is used to differentiate members of the genera Proteus, Morganella, and Providencia (+ve) from other members of Enterobacteriaceae which give negative results.