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10.13: Citrate Test

HGTC BIO 225: Microbiology Lab Manual · LibreTexts

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    Learning Objectives
    • Understand the principle of the citrate agar test as a differential medium used to differentiate bacteria based on their ability to utilize citrate as the sole carbon source.
    • Interpret test results by observing color changes in the citrate agar medium, distinguishing between positive (blue color development) and negative (no color change) reactions.
    • Correlate citrate utilization with the presence or absence of the enzyme citrate permease.
    • Analyze and interpret experimental data obtained from the citrate agar test to draw conclusions about the metabolic capabilities of tested bacterial species.

    Citrate Test

    All living things need carbon to survive. The carbon-containing molecules that bacteria can utilize as a carbon source differ based on the bacterial species and is dependent on their genes. Their genes dictate what enzymes the bacterial species can produce. Since enzymes are necessary for a cell's metabolic reactions, the genes of a bacterial species dictate their ability to use different carbon sources.

    Some bacterial species can utilize citrate as a source of carbon. Organisms that can survive using citrate as the sole source of carbon have a citrate permease enzyme that can transport citrate molecules into the cell. The citrate is then made into pyruvate, which can be converted into a variety of different products in the cell.

     

    bacteria with citrate permease

    Figure 1: Bacteria with the citrate permease gene can produce the enzyme citrate permease by gene expression (left). Citrate permease is a membrane protein that enables the transport of citrate into the cell. (Right) When a bacterial cell produces citrate permease, citrate molecules are transported into the bacterial cell. This enables the bacteria to utilize citrate as a source of carbon.

     

    bacteria without citrate permease

    Figure 2: Bacteria without the citrate permease gene cannot produce the protein citrate permease (left). Without the citrate permease protein, citrate is unable to enter the bacterial cell (right). As a result, the bacteria cannot use citrate as a source of carbon.

     

    Simmons' citrate is a chemically defined microbiological medium that contains sodium citrate as the sole carbon source. A pH indicator, bromothymol blue, is also included in Simmons' citrate medium. Bacteria that can grow on this medium (i.e., that can use citrate as the sole source of carbon) produce alkaline byproducts that will change the medium color from green (neutral pH) to blue (alkaline pH).

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    Figure 3: Results of the citrate test. When the medium stays green (no color change), the test indicates the bacterial species is citrate negative (left). When the medium changes color to blue, the test indicates the bacterial species is citrate positive (right).

    Bacterial species that are capable of metabolizing citrate are considered citrate-positive and will cause Simmons' citrate agar to change from green to blue. Bacterial species that are incapable of metabolizing citrate are considered citrate-negative and will result in Simmons' citrate agar remaining green (no color change). Since only some bacterial species can metabolize citrate, the citrate test is useful for bacterial identification and characterization.

    Note

    A small inoculum and/or incomplete incubation can prevent color change. Therefore, growth in the absence of color change is also considered a positive result. 

     

     

    Real World Connections

    Shigella dysenteriae

    Simmons citrate agar can be used to differentiate fecal coli, bacteria that target the gut, based on carbon usage. Simmon citrate can be used to determine if a gut infection is caused by bacteria belonging to the genus Shigella. Shigella bacteria cause shigellosis, a severe form of diarrhea. There are about 450,000 Shigella infections each year across America. (CDC, 2024). Most of these infections are resistant to antibiotics and cost the healthcare industry $93 million in treatment efforts. (CDC, 2024). One specific Shigella strain, Shigella dysenteriae type 1, causes life-threatening dysentery and is highly resistant.

    S. dysenteriae infections develop when a person ingests the bacteria. The bacteria is present in stool and can be transmitted from mouth to GI tract by direct contact with fecal matter or by consuming contaminated food or water. Young children are especially vulnerable to these infections as they are more likely to put unwashed hands in their mouth. Travelers are also more likely to develop infections as they may encounter untreated water and contaminated food more often. Infections can easily be reduced by washing your hands with soap and water, not drinking water from stagnant sources, and checking food and beverage preparation when traveling. (CDC, 2024)

    Patients experiencing diarrhea will need to submit a stool sample to determine if the cause is S. dysenteriae or another fecal coli bacterium. S. dysenteriae growth is inhibited on Simmons citrate media and the media will stay green in color. Positive S. dysenteriae tests may need to be followed up with antibiotic resistance testing to determine what antibiotic would be effective for that patient. Severe cases of S. dysenteriae can develop into chronic arthritis, bloodstream infections, and seizures. (CDC, 2024)

    Laboratory Instructions

    Citrate Test

    1. Obtain two test tubes with Simmons' citrate agar slants.
    2. Use tape to label the test tubes with your group name, bacteria name, section, and date.
    3. Using a sterile inoculation needle, aseptically obtain a sample of bacteria and streak the entire slant of the citrate agar using a fishtail pattern. Repeat for both species.
      • A small sample must be used because the presence of growth may be considered a positive citrate test. Bacteria from a heavy inoculum could be misconstrued as a positive test result. 
    4. Incubate the inoculated tube at 37 °C until the next lab session.
    5. Observe the color of the Simmons’s citrate slant.
      • bright blue is positive
      • no color change and no observable growth is negative
        • *Please note, if the agar is green but growth has occurred, the test is positive. 

    Resources

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