Citrate Utilization Test – Principle, Media, Procedure And Result

The natural world is filled with living creatures, from animals and plants to microscopic bacteria.

Citrate Utilization Test - Principle, Media, Procedure And Result

You may have wondered how scientists are able to classify different species of bacterium, when all of them are too small to be perceived by the naked eye.

One way that scientists can differentiate species of microorganisms is to find out what they feed and eat on.

Certain bacteria will be able to eat different kinds of sugar, depending on their metabolism, among other numerous factors.

The citrate utilization test, is one of the four IMViC tests.

These are a collection of scientific experiments that can be used to determine if a bacterium is capable of utilizing specific substances as a source of energy.

As such, they are a useful tool when classifying different species of bacteria.

In this article we will be telling you what the citrate utilization test is, how to perform it, and how to read the results.

What Is The Citrate Utilization Test?

As its name suggests, the citrate utilization test is a scientific experiment used to determine if a bacterial culture can use citrate as a source of energy.

It takes place in a medium where citrate is the only source of carbon, with inorganic ammonium salts used as the sole source of nitrogen.

When carbon-based microorganisms are introduced to this solution, they will have no choice but to feed upon the citrate or perish.

Bacteria that are capable of feeding on citrate will produce a special enzyme called citrate permease. This enzyme converts citrate into another substance called pyruvate.

As the bacteria utilize the citrate to produce energy and growth, the ammonium salts will also be broken down.

This reaction produces ammonia, which will raise the PH of the medium significantly.

When the test is positive, the increase in PH will cause a bromothymol blue indicator to change color from green to blue once the solution reaches a PH of 7.6

What Media Is Required For This Test?

In microbiology, a medium is a chemical solution that promotes the growth of a bacterial culture.

For the citrate utilization test, a special medium must be prepared where citrate is the only available source of food.

This medium is known as Simmons Citrate Agar, named after the scientist who first developed it.

Below you will find a list of ingredients for Simmons citrate agar, along with the precise quantities required for each one.

Ingredients For Simmons Citrate Agar

• Sodium Chloride: 5.0gm
• (Dehydrated) Sodium Citrate: 2.0gm
• Ammonium Dihydrogen Phosphate: 1.0gm
• Dipotassium Phosphate: 1.0gm
• Magnesium Sulfate (heptahydrate)
• Bromothymol Blue
• Agar
• Deionized water: 1,000ml

How To Prepare Simmons Citrate Agar

Now that you know the ingredients required to make Simmons Citrate Agar, here is a step-by-step guide on how to prepare them.

  1. Dissolve all of the salts (the first five ingredients listed above) in the deionized water.
  2. Monitor the PH to make sure it is resting at 6.9.
  3. Add the agar and bromothymol blue.
  4. Stir the mixture while gently heating it until it has boiled and all of the agar has dissolved.
  5. Add 5ml of this solution into two 16 mm test tubes.
  6. Autoclave the test tubes for 15 minutes at 121 °C and a pressure of 15psi
  7. Let the tubes cool in a slanted position.
  8. You can store the tubes in the fridge until you are ready to add your bacterial sample. They will keep for up to 8 weeks.
  9. Your medium should be a deep forest green before any bacteria have been added.

Procedure For The Citrate Utilization Test

Procedure For The Citrate Utilization Test

Take one of your test tubes, setting the other one to the side to act as the control sample.

Streak the slant in a zigzagging motion with a light inoculum taken from your chosen bacterial culture.

Once this is done, you need to incubate the sample in an aerobic environment at a temperature between 35 and 37 °C for as long as 4 to 7 days.

If the test is positive, you will notice a color change taking place along the slant as your media turns from green to blue.

A negative result will be indicated by a lack of color change.

Common examples of bacteria that will yield a positive result from the citrate utilization test include salmonella, Serratia, and Klebsiella pneumoniae.

The last example is commonly used as a quality control to test if the Simmons Citrate Agar used for the test is behaving as it should.

Other species that can’t feed off citrate include Shigella, Yersinia and Escherichia coli, all of which will produce a negative result when subjected to this experiment.

One of the most common uses for the citrate utilization test is to differentiate between members of the Enterobacteriaceae family.

Are There Any Limitations To The Citrate Utilization Test?

As with any scientific procedure, there are limits to what we can learn from the citrate utilization test.

For instance, some microorganisms can grow on citrate without producing a color change.

As such, make sure to check for growth as well as color change when interpreting your results.

Furthermore, the results from this test won’t help you identify the exact species of bacteria present in a given culture.

It can help you narrow down the possibilities, but other tests will be required to know exactly which species you are working with.


The citrate utilization test is a useful tool for microbiologists to be able to classify the different species of bacteria.

There are other tests that can be used to test if bacteria will eat citrate, however, they don’t use citrate as the sole food source.

As such, this is the best test available for knowing if a given culture can survive on citrate alone when there are no other sources of food.

Jennifer Dawkins