Lab 23: Biochemical Assays: Indole production, urea hydrolysis, and deamination of phenylalanine

What ability is measured to differentiate enteric bacteria?

Ability to break down tryptophan amino acid

What enzyme breaks down tryptophan?

Tryptophanase

What is chemical reaction break down of tryptophan?

½ O2 + tryptophan → indole + pyruvic acid + ammonia

What happens to the 3 substances that are produced after tryptophan is broken down?

Pyruvic acid enters Krebs cycle and is used by cells to produce energy. Indole and ammonia are excreted. 

What is measured to test for the break down of tryptophan?

Presence of indole

What substances are added to test for indole?

Kovacs reagent, 5% DMAB in 75% amyl alcohol, 25% concentrated HCl

What occurs when Kovacs reagent reacts with indole?

Produces rosindole dye which gives red color

How does the amyl alcohol affect indole?

Extracts the indole from the medium, making it rise to the top

What indicates a positive indole test?

Bright red layer

Which organism provides a positive control for the tryptophan broth test?

E. coli

Which enzyme breaks down urea?

Urease

What does the break down of urea produce?

Ammonia and carbon dioxide

The ability to produce urease is an indication of which type of bacteria?

Gram-negative and distinguishes from enterics

What effect does adding antibiotics to animal feed have on growth and why?

Ammonia reduces animal cells so adding antibiotics would remove gut microbes that produce urea, increasing growth rate

What is used to test the ability of organisms to break down urea?

Urea agar, yeast extract, buffer, phenol red

What is indicated when phenol red shows yellow?

pH 6.6; urease is not present

What is indicated when phenol red shows bright pink?

pH>8.1, urease is present 

Which organism provides a positive control for the urea agar test?

P. vulgaris

What is oxidative deamination?

Process an organism does to remove the amine group from an amino acid

What is produced from the oxidative deamination process?

Keto acid and ammonia

What type of bacteria is associated with the production of enzyme phenylalanine deaminase?

Gram-negative

What is the purpose of phenylalanine deaminase?

Remove amine group from amino acid phenylalanine 

What is produced from the break down of phenylalanine?

Alpha-keto acid, phenylpyruvic acid, ammonia

What is indicated to show presence of alpha-keto acid on phenylalanine medium?

Dark green color

What is indicated to show presence of alpha-keto acid on tryptophan medium?

Brown color

What solution must be added to the culture to test for alpha-keto acid presence?

10% ferric chloride

Which organism is the positive control for the phenylalanine agar test?

P. vulgaris