Citrate utilization test objective, principle, procedure and result

Citrate utilization test: objective, principle, procedure and result

What is Citrate utilization test ?

The Citrate Utilization Test is a biochemical test used to assess whether bacteria can use citrate as their sole carbon source for energy production. This method is particularly useful for identifying different bacterial species, including those in the Enterobacteriaceae family such as E. coli, Klebsiella, and Salmonella.

When bacteria can metabolize citrate, they produce alkaline byproducts that cause a color change in the medium, helping to distinguish citrate-positive from citrate-negative organisms

.Objective

The key purpose of the Citrate Utilization Test is to determine whether bacteria:

Are capable of using citrate as the only carbon source for their metabolism.

Produce the enzyme citrate permease, which enables the bacteria to take in and metabolize citrate.

This test is widely used to differentiate bacterial species based on their ability to metabolize citrate, especially when identifying members of the Enterobacteriaceae family.

Principle of the Test

In the Citrate Utilization Test, bacteria are grown on a medium where sodium citrate is the only carbon source available. While most bacteria prefer glucose or other sugars as a carbon source, some can switch to citrate when necessary.

Bacteria that can use citrate produce an enzyme called citrate permease, which allows citrate to enter the bacterial cell. Inside the cell, citrate is broken down into oxaloacetate and acetate, and these are further converted into pyruvate, which provides energy through cellular respiration. This metabolic process results in the production of ammonia (NH₃), which raises the pH of the medium, making it more alkaline.

The test medium contains bromothymol blue, a pH indicator that is:

Green at neutral pH (approximately 6.9).

Blue when the pH becomes alkaline (above 7.6).

If the bacterium can metabolize citrate, the medium turns blue, indicating a positive result. If the bacterium cannot use citrate, the medium remains green, showing a negative result.

Requirements

To conduct the Citrate Utilization Test, the following items are necessary:

Simmons citrate agar slants, which provide sodium citrate as the sole carbon source and bromothymol blue as the pH indicator.

Sterile inoculating loop for transferring the bacterial sample.

Bacterial cultures (such as E. coli or Klebsiella) for testing.

Incubator set to 35-37°C.

Procedure

Prepare the Medium: Simmons citrate agar, containing sodium citrate and bromothymol blue, is poured into test tubes and allowed to solidify in a slanted position.

Inoculate the Agar: Use a sterile loop to pick a small amount of the bacterial sample and streak it gently over the surface of the agar slant. Avoid adding too much bacteria, as it can lead to false results.

Incubation: Place the inoculated slant in an incubator set at 35-37°C for 24 to 48 hours. Make sure the test tube cap is not tightly closed, allowing oxygen exchange, which is necessary for citrate metabolism.

Observation: After incubation, examine the agar slant for color changes. Compare it to an uninoculated control tube, which should remain green.

Result Interpretation

Positive Result

A color change to blue indicates that the bacteria can use citrate as their carbon source. This happens because ammonia is produced during citrate metabolism, raising the pH and turning the bromothymol blue indicator blue. Organisms like Klebsiella pneumoniae or Salmonella often yield positive results, as they can utilize citrate.

Negative Result

If the medium remains green, it means the bacteria cannot use citrate, and there has been no change in the pH. This indicates a negative result. An example of a citrate-negative bacterium is E. coli, which cannot metabolize citrate as its only carbon source.

Applications of the Citrate Utilization Test

Identifying Bacteria: The Citrate Utilization Test is one of the tests in the IMViC series (Indole, Methyl Red, Voges-Proskauer, and Citrate tests), commonly used to differentiate bacteria within the Enterobacteriaceae family.

Distinguishing Between Species: The test is particularly useful for separating citrate-positive organisms, like Klebsiella and Enterobacter, from citrate-negative ones, such as E. coli.

Limitations

False Results: Adding too much bacteria to the medium may cause a false positive result by increasing the likelihood of growth without true citrate utilization.

Incubation Time: Some bacteria may require more than 48 hours to show a result, so extending the incubation period can improve the accuracy of the test.

Aerobic Process: Citrate metabolism requires oxygen, so the test tubes should not be tightly closed during incubation to allow for proper air exchange.

Frequently Asked Questions (FAQ)

What is use of Citrate utilization test ?

The citrate utilization test helps identify if a bacterium can use citrate as its only carbon source, assisting in distinguishing and identifying bacterial species, especially in the Enterobacteriaceae family.

Explain the applications of the Citrate Utilization Test ?

The citrate utilization test is used in microbiology to distinguish and identify bacterial species based on their capacity to use citrate as their only carbon source, which is especially useful for identifying members of the Enterobacteriaceae family and evaluating bacterial metabolic processes.

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