Citrate Agar (Hydrolysis test)

What is Hydrolysis

The use of water to split complex molecules

What is the enzyme used in hydrolytic reactions?

hydrolytic enzymes

What is intracellular enzyme?

enzyme inside the cell

What is extracellular enzyme (or exoenzyme)?

enzyme that is secreted and act on substrates in the environment

What is tested for in citrate agar?

citrate metabolism

Why is citrate agar a low nutrient medium?

Because it only has citrate as the source of carbon and only ammonium dihydrogen phosphate as nitrogen source

What are components in citrate agar?

• sodium chloride

• sodium citrate

• ammonium dihydrogen phosphate

• dipotassium phosphate

• magnesium sulfate

• bromothymol blue

• agar

What is purpose of sodium citrate in the citrate agar media?

carbon source

What is purpose of ammonium dihydrogen phosphate in the citrate agar media?

nitrogen source

What is indicator of the citrate agar media?

bromothymol blue

What is the color of citrate agar in acidic condition?

bromothymol blue turns yellow

What is the color of citrate agar in basic condition?

bromothymol blue turns blue

What are 2 enzymes required for citrate fermentation?

• citrate permease

• citrate lyase

What is the function of citrate permease in citrate fermentation?

To bring citrate into the cell

What happened if the organism that does not have citrate permease?

Cannot grow on citrate media

What is the function of citrate lyase in citrate fermentation?

To convert citrate to oxaloacetate and acetate

Oxaloacetate is converted to pyruvate to generate energy for the cell

What leads to the increase in pH?

Accumulation of ammonia via dipotassium ammonium phosphate usage

What is the positive result of citrate agar?

Growth with and without color change to blue

Why is the color change to blue does not matter in positive result?

Because the blue of bromothymol indicates the basic media, result of nitrogen source ultilization not citrate ultilization. 

What is the negative result of citrate agar media?

No growth at all
That means the organism lack enzyme that breaks down citrate to survive

How to avoid a false positive result?

Use needle instead of inoculation loop

Brief protocol for Citrate Agar

1. Obtain 3 citrate slants, label (E.coli & Enterobacter aerogenes). Have 1 control tube

2. Sterilize the inoculate needle

3. Aseptically transfer each species: stab the slant and squiggle up the surface.

4. Incubate for 24-48 hours

5. Read result. Remove tapes in liquid rank to be autoclaved