Citrate Utilization Test- Principle, Procedure, Results, Uses

Citrate utilization test is a biochemical test that determines whether a bacterium can use citrate as its sole source of carbon and energy. Citrate is a tricarboxylic acid that is an intermediate in the Krebs cycle, a metabolic pathway that generates energy from carbohydrates, fats, and proteins. Some bacteria have the ability to ferment citrate in the presence of an enzyme called citrase, which breaks down citrate into oxaloacetate and acetate. These bacteria can grow on a medium that contains citrate as the only carbon source and ammonium salts as the only nitrogen source.

Citrate utilization test is one of the four tests that make up the IMViC test, which stands for Indole, Methyl Red, Voges-Proskauer, and Citrate test. The IMViC test is a set of biochemical tests that are used to differentiate Gram-negative bacilli of the Enterobacteriaceae family, which includes many medically important bacteria such as Escherichia coli, Salmonella typhi, Shigella dysenteriae, and Klebsiella pneumoniae. The IMViC test is based on the different metabolic reactions of these bacteria to various substrates and indicators.

The principle of the citrate utilization test is based on the ability of some bacteria to use citrate as their sole source of carbon and energy. Citrate is an intermediate metabolite in the Krebs cycle, which is a series of chemical reactions that generate energy from carbohydrates, fats, and proteins.

The citrate utilization test uses a medium called Simmon`s citrate agar, which contains sodium citrate as the only carbon source and ammonium dihydrogen phosphate as the only nitrogen source. The medium also contains bromthymol blue as a pH indicator, which is green at neutral pH and turns blue at alkaline pH.

Bacteria that can utilize citrate have an enzyme called citrase, which breaks down citrate into oxaloacetate and acetate. Oxaloacetate is further converted into pyruvate and carbon dioxide. The carbon dioxide reacts with water and sodium to form sodium carbonate, which raises the pH of the medium. The increase in pH causes the bromthymol blue to change color from green to blue, indicating a positive result.

Bacteria that cannot utilize citrate do not grow on the medium and do not change its color, indicating a negative result.

The citrate utilization test is useful for differentiating between members of the Enterobacteriaceae family, which are Gram-negative bacilli that inhabit the intestinal tract of humans and animals. Some genera, such as Klebsiella, Enterobacter, and Citrobacter, are usually citrate positive, while others, such as Escherichia, Shigella, and Yersinia, are usually citrate negative. Proteus is a variable genus that may or may not utilize citrate depending on the species.

The citrate utilization test is performed along with other biochemical tests, such as indole, methyl red, and Voges-Proskauer tests, to form the IMViC test battery. The IMViC test helps to identify and differentiate between various species of Enterobacteriaceae based on their metabolic characteristics.

The citrate utilization test is mainly used to differentiate Gram-negative bacilli of the Enterobacteriaceae family. These are a large group of bacteria that inhabit various environments, including soil, water, plants, and animals. They are also common in the human intestinal tract and can cause various infections, such as urinary tract infections, septicemia, and gastroenteritis.

The Enterobacteriaceae family consists of many genera and species that have different biochemical characteristics and pathogenic potentials. One of the methods to identify and classify these bacteria is the IMViC test, which consists of four tests: indole production, methyl red test, Voges-Proskauer test, and citrate utilization test. Each test gives a positive or negative result that can be used to form a four-digit code for each organism.

The citrate utilization test is particularly useful for distinguishing between Escherichia coli and other members of the Enterobacteriaceae family. Escherichia coli is one of the most common causes of urinary tract infections and diarrheal diseases in humans. It is usually citrate negative, meaning that it cannot use citrate as a sole source of energy. On the other hand, most other members of the Enterobacteriaceae family are citrate positive, meaning that they can use citrate as a sole source of energy.

Some examples of citrate positive bacteria are:

• Salmonella: a genus of bacteria that causes typhoid fever, paratyphoid fever, and foodborne illnesses. Salmonella can be further divided into serotypes based on their surface antigens.
• Edwardsiella: a genus of bacteria that causes edwardsiellosis, a disease that affects fish and reptiles. Edwardsiella can also cause opportunistic infections in humans with compromised immune systems.
• Citrobacter: a genus of bacteria that can cause urinary tract infections, septicemia, meningitis, and brain abscesses in humans. Citrobacter can also cause diarrhea in infants and young children.
• Klebsiella: a genus of bacteria that can cause pneumonia, urinary tract infections, wound infections, and septicemia in humans. Klebsiella can also cause nosocomial infections in hospitals and health care facilities.
• Enterobacter: a genus of bacteria that can cause urinary tract infections, respiratory tract infections, wound infections, and septicemia in humans. Enterobacter can also cause nosocomial infections in hospitals and health care facilities.
• Serratia: a genus of bacteria that can cause urinary tract infections, respiratory tract infections, wound infections, endocarditis, and septicemia in humans. Serratia can also cause nosocomial infections in hospitals and health care facilities.
• Providencia: a genus of bacteria that can cause urinary tract infections, wound infections, and septicemia in humans. Providencia can also cause traveler`s diarrhea.

Some examples of citrate negative bacteria are:

• Escherichia: a genus of bacteria that includes Escherichia coli and other strains that can cause urinary tract infections, diarrheal diseases, septicemia, and meningitis in humans. Escherichia can also be part of the normal flora of the human intestinal tract.
• Shigella: a genus of bacteria that causes shigellosis or bacillary dysentery, a severe form of diarrhea that can lead to dehydration and death. Shigella can also cause hemolytic uremic syndrome (HUS), a condition that affects the kidneys and blood cells.
• Morganella: a genus of bacteria that can cause urinary tract infections, wound infections, and septicemia in humans. Morganella can also cause gas gangrene or necrotizing fasciitis (flesh-eating disease).
• Yersinia: a genus of bacteria that includes Yersinia pestis (the causative agent of plague), Yersinia enterocolitica (the causative agent of yersiniosis or enteric fever), and Yersinia pseudotuberculosis (the causative agent of pseudotuberculosis or Far East scarlet-like fever). Yersinia can also cause lymphadenitis (swelling of the lymph nodes) and reactive arthritis.

Some examples of citrate variable bacteria are:

• Proteus: a genus of bacteria that can cause urinary tract infections, wound infections, septicemia, and kidney stones in humans. Proteus can also produce urease (an enzyme that breaks down urea into ammonia) and swarming motility (a rapid movement on solid surfaces).

Media Used

Simmon’s Citrate agar is used as the medium to test the ability of an organism to utilize citrate as a sole source of energy.

Simmon’s Citrate agar is sold commercially by different vendors in the form of dehydrated powder. However, it can also be prepared in the laboratory if the necessary ingredients are available.

The following is the composition of Simmon’s Citrate Agar:

The pH of the medium is adjusted to 6.9 ± 0.2 at 25°C.

Reagents Used

No additional reagents are required for the citrate utilization test.

Supplies Used

• Inoculating loops or needles
• Incubator at 35-37°C

The citrate utilization test is performed by inoculating Simmon`s citrate agar with a pure culture of the organism to be tested. The following are the steps involved in the test:

1. Prepare the Simmon`s citrate agar tubes by heating and sterilizing them in an autoclave. Cool them to 40-45°C and slant them to obtain a butt of 1.5-2.0 cm depth. The agar should appear as forest green colored slightly opalescent gel slants.
2. Take a well-isolated colony from an 18-24 hour culture of the organism with a sterile inoculating needle. Streak the surface of the slant with the needle, back and forth, to cover the entire slant. Do not stab the agar, as the test requires an aerobic environment.
3. Loosen the cap of the tube and incubate it aerobically at 35-37°C for up to 4 days. Examine the tube daily for any change in color or growth.
4. Observe the results and interpret them according to the following criteria:
   • A positive test is indicated by growth with a color change from green to intense blue along the slant. This means that the organism can utilize citrate as a sole source of energy and produce alkaline products that raise the pH of the medium.
   • A negative test is indicated by no growth and no color change, and the slant remains green. This means that the organism cannot utilize citrate as a sole source of energy and does not produce alkaline products that affect the pH of the medium.

The following image shows an example of a positive and a negative citrate utilization test:

Quality control measures are essential to ensure the validity and reliability of the citrate utilization test results. The following are some of the quality control measures that should be followed while performing the test:

• The Simmon`s citrate agar should be prepared according to the manufacturer`s instructions or the standard protocol. The pH of the medium should be checked and adjusted to 6.9 ± 0.2 before sterilization.
• The medium should be sterilized properly and cooled to 40-45°C before dispensing into tubes. The tubes should be slanted to obtain a butt of 1.5-2 cm depth and a slant of about 3 cm in length.
• The medium should be stored in a dark and cool place to prevent deterioration. The medium should not be used if it shows signs of contamination, cracking, dehydration, bubbles, or discoloration.
• The inoculum should be taken from a pure and fresh culture of 18-24 hours old. The inoculum size should be small to avoid carryover of substances from previous media that might interfere with the test results.
• The inoculation should be done by streaking the surface of the slant with a sterile inoculating needle or loop. The slant should not be stabbed as the test requires an aerobic environment.
• The inoculated tubes should be incubated aerobically at 35-37°C for up to 4 days. The tubes should be examined daily for growth and color change. A positive result is indicated by growth with a blue color along the slant, while a negative result is indicated by no growth and no color change.
• Positive and negative controls should be included in each batch of tests to verify the performance of the medium and the inoculation technique. Klebsiella pneumoniae is used as a positive control, while Escherichia coli is used as a negative control for the citrate utilization test.
• The test results should be interpreted carefully and in conjunction with other biochemical tests for identification to the species level. Some organisms may show weak or delayed reactions, while others may show variable reactions depending on the strain or environmental conditions.

By following these quality control measures, one can ensure the accuracy and consistency of the citrate utilization test results.

The citrate utilization test is based on the observation of growth and color change on the Simmon`s citrate agar medium. The growth indicates the ability of the organism to use citrate as a sole source of carbon and energy, while the color change indicates the production of alkaline metabolites due to citrate breakdown.

A positive test is indicated by growth with a color change from green to intense blue along the slant. This means that the organism has citrase enzyme and can utilize citrate as a sole carbon source. The blue color is due to the increase in pH above 7.6 as a result of ammonia or sodium carbonate formation.

A negative test is indicated by no growth and no color change, and the slant remains green. This means that the organism does not have citrase enzyme and cannot utilize citrate as a sole carbon source. The green color is due to the neutral pH of the medium.

Some examples of positive and negative results are given below:

• Klebsiella pneumoniae is a Gram-negative bacillus that belongs to the Enterobacteriaceae family. It is a common cause of pneumonia, urinary tract infections, and septicemia. It is also a part of the normal flora of the mouth, skin, and intestines. Klebsiella pneumoniae is citrate positive, meaning that it can use citrate as a sole source of carbon and energy. It produces a blue color on Simmon`s citrate agar medium.
• Escherichia coli is another Gram-negative bacillus that belongs to the Enterobacteriaceae family. It is one of the most common bacteria in the human gut and can cause various infections such as diarrhea, urinary tract infections, meningitis, and septicemia. Some strains of E. coli are beneficial, while others are pathogenic. Escherichia coli is citrate negative, meaning that it cannot use citrate as a sole source of carbon and energy. It does not produce any growth or color change on Simmon`s citrate agar medium.
• Proteus mirabilis is a Gram-negative bacillus that belongs to the Enterobacteriaceae family. It is a common cause of urinary tract infections, especially in patients with catheters or stones. It can also cause wound infections, septicemia, and otitis media. Proteus mirabilis is citrate variable, meaning that some strains can use citrate as a sole source of carbon and energy, while others cannot. It may produce either a positive or a negative result on Simmon`s citrate agar medium depending on the strain.

The following table summarizes the results of some common bacteria on Simmon`s citrate agar medium:

The results of the citrate utilization test should be interpreted along with other biochemical tests such as indole, methyl red, Voges-Proskauer, and urease tests for the identification of Gram-negative bacilli to the species level.

The citrate utilization test is a useful tool for the identification and differentiation of Gram-negative bacilli of the Enterobacteriaceae family. The test can help to distinguish between citrate-positive and citrate-negative organisms based on their ability to use citrate as a sole source of carbon and energy. The test can also help to differentiate between fecal coli and members of the aerogenes group, which are usually citrate-positive.

However, the citrate utilization test also has some limitations that should be considered before interpreting the results. Some of these limitations are:

• The test requires an aerobic environment, so the slant should not be stabbed or inoculated from broth cultures.
• The test may give false-positive results if a heavy inoculum is used, as it may carry over substances from previous media that can affect the pH of the citrate agar.
• The test may give false-negative results if the incubation period is too short or the temperature is too low, as some organisms may take longer to utilize citrate or grow slowly at lower temperatures.
• The test may give equivocal results if there is growth on the slant without a color change, or if there is no growth but a slight color change. In such cases, the test should be repeated with a smaller inoculum or a longer incubation period.
• The test alone is not sufficient for the identification of an organism to the species level, as some organisms may show variable reactions or share similar reactions with other organisms. Therefore, additional tests should be performed to confirm the identity of an organism.

Therefore, the citrate utilization test is a simple and reliable method for testing the ability of an organism to use citrate as a sole source of energy, but it should be performed carefully and interpreted along with other tests for accurate identification.

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