Exercise 14: Phenylalanine Deamination

What is deamination?

The removal of an amino group from an organic molecule.

What is the purpose of the phenylalanine deamination test?

To determine whether an organism produces phenylalanine deaminase, which removes the amino group from phenylalanine.

• The main products include phenylpyruvic acid and ammonia

How can bacteria use phenylalanine to support growth and reproduction?

They can incorporate phenylalanine into proteins. They can also deaminate it and use the resulting carbon skeleton in further metabolism or biosynthesis.

What test preparation is used for phenylalanine deamination in your course?

A commercial KEY phenylalanine tablet suspended in sterile water and inoculated with a heavy amount of bacteria.

• Phenylalanine is the essential substrate supplied by the tablet

How is each phenylalanine test tube prepared in your course?

Add one KEY phenylalanine tablet to a small tube, then add 1 mL of sterile water. Gently tap to partially dissolve the tablet, add a heavy loopful of bacteria, mix gently, cap, and incubate at 35°C until the next lab period.

Why is a heavy inoculum essential in this test?

Bacteria do not grow in this test preparation. Enough cells must be added initially to supply sufficient enzyme for a detectable reaction.

• A false-negative result can occur from uses to little bacteria

  • Not enough cells to supply phenylalanine deaminase

Is the KEY tablet preparation a selective growth medium?

No. It is a substrate preparation used in a differential enzyme test, rather than a medium that selects organisms through growth.

What reagent is added after incubation, and what does it detect?

Acidified ferric chloride, FeCl₃. It reacts with phenylpyruvic acid to form a green compound.

• The green color results from a chemical reaction; it’s not a pH indicator

How is the phenylalanine deamination test developed after incubation?

Add 3–5 drops of acidified ferric chloride to each tube. Gently tap to mix and observe for green color within 5 minutes.

What indicates a positive phenylalanine deamination result? Negative?

• Positive: a definite light-to-dark green color within 5 minutes after ferric chloride is added

• Negative: the solution remains yellow

Does ferric chloride directly detect phenylalanine deaminase or ammonia?

No. It detects phenylpyruvic acid. Enzyme activity is inferred from production of this reaction product.

Which organisms are the positive and negative controls used in your course’s procedure?

• Positive: Proteus vulgaris

• Negative: Enterococcus faecalis