How to Perform a Citrate Utilization Test: Simmons Citrate Agar Protocol

The citrate utilization test is a biochemical method used to determine whether a bacterium can use citrate as its sole carbon source. This test is performed by inoculating a Simmons citrate agar slant, which contains citrate as the only carbon source and ammonium phosphate as the sole nitrogen source. When bacteria metabolize citrate, they produce alkaline byproducts that raise the pH of the medium, causing the bromothymol blue pH indicator to change from green to blue. This test is particularly useful for differentiating members of the Enterobacteriaceae family, distinguishing citrate-positive organisms such as Klebsiella pneumoniae and Enterobacter species from citrate-negative organisms such as Escherichia coli. The Simmons citrate agar test is a standard component of the IMViC (Indole, Methyl Red, Voges-Proskauer, Citrate) series used in clinical and environmental microbiology.

At a Glance

Scientific Principle

The citrate utilization test relies on the ability of bacteria to transport citrate into the cell and metabolize it via the tricarboxylic acid (TCA) cycle. Simmons citrate agar contains sodium citrate as the sole carbon source and ammonium dihydrogen phosphate as the sole nitrogen source. Bacteria that possess the enzyme citrate permease can transport citrate across the cell membrane. Once inside the cell, citrate is converted to oxaloacetate and acetate by citrate lyase, entering the TCA cycle for energy production.

The critical biochemical event for detection is the metabolism of ammonium phosphate. When bacteria utilize citrate, they also consume ammonium ions from the medium. This consumption releases ammonia, which reacts with water to form ammonium hydroxide. The accumulation of hydroxide ions raises the pH of the medium from approximately 6.9 to 8.0 or higher. The pH indicator bromothymol blue is green at neutral pH (6.9) and turns blue at alkaline pH (7.6 or above). Therefore, a color change from green to blue indicates successful citrate utilization.

Some bacteria may produce visible growth on the slant without an immediate color change. In such cases, extended incubation (up to 48 hours) may be necessary for sufficient alkalinity to develop. The test is considered positive only when both growth and a distinct blue color are observed.

Materials and Instrumentation

Simmons Citrate Agar

Simmons citrate agar is commercially available as dehydrated powder or prepared slants. The medium composition per liter of deionized water includes:

• Sodium citrate: 2.0 g (sole carbon source)
• Ammonium dihydrogen phosphate: 1.0 g (nitrogen source)
• Dipotassium phosphate: 1.0 g (buffer)
• Sodium chloride: 5.0 g (osmotic balance)
• Magnesium sulfate: 0.2 g (cofactor for enzymatic reactions)
• Bromothymol blue: 0.08 g (pH indicator)
• Agar: 15.0 g (solidifying agent)

The final pH is adjusted to 6.9 ± 0.2 before autoclaving. Prepared slants should be stored at 2–8°C and used within the manufacturer's expiration date. Do not use slants that appear dehydrated, cracked, or contaminated.

Inoculation Equipment

• Sterile inoculating loop (10 µL loop for liquid cultures or single-use sterile loops)
• Sterile needle (for colony picking from solid media)
• Bunsen burner or microincinerator for loop sterilization
• Biosafety cabinet (for clinical or environmental isolates)

Controls

• Positive control: Klebsiella pneumoniae ATCC 13883 or known citrate-positive strain
• Negative control: Escherichia coli ATCC 25922 or known citrate-negative strain
• Uninoculated control: An uninoculated slant incubated alongside tests to verify medium sterility and color baseline

Incubation

• Standard microbiological incubator set to 35–37°C
• Timer or log for recording incubation start and end times
• Aerobic conditions (loose caps or vented tubes)

Quality Checks Before Starting

Before performing the citrate test, verify the following:

1. Medium integrity: Simmons citrate agar slants should be green, with no cracks, dehydration, or visible contamination. The slant surface should be moist but not wet.
2. Control strains viability: Subculture positive and negative control strains onto non-selective media (e.g., tryptic soy agar) 18–24 hours before testing to ensure active growth.
3. Incubator temperature: Confirm incubator temperature is within 35–37°C using a calibrated thermometer.
4. Sterility check: Incubate one uninoculated slant from each batch at 35–37°C for 48 hours to confirm no contamination.
5. Purity of test isolate: Ensure the test organism is a pure culture. Gram stain or colony morphology check on a non-selective plate is recommended before biochemical testing.

Conceptual Workflow

Step 1: Prepare the Medium

If using dehydrated powder, prepare Simmons citrate agar according to manufacturer instructions. Dispense into screw-cap tubes (approximately 5 mL per tube) and autoclave at 121°C for 15 minutes. After autoclaving, tilt tubes at an angle to create slants with a 1-inch butt. Allow to solidify completely. Prepared slants can be stored at 2–8°C for up to 4 weeks in sealed containers to prevent dehydration.

Step 2: Inoculate the Slant

Using a sterile inoculating loop or needle, pick a single well-isolated colony from an 18–24 hour pure culture. Lightly streak the surface of the Simmons citrate agar slant in a zigzag pattern from bottom to top. Do not stab the butt—the test requires aerobic conditions for citrate metabolism. The butt serves as a color reference for the uninoculated medium.

For liquid cultures, dip a sterile loop into the culture and streak the slant surface. Avoid transferring excessive liquid, which may dilute the medium.

Step 3: Incubate Aerobically

Loosen the tube cap to allow air exchange (aerobic conditions). Place tubes in an incubator at 35–37°C for 18–24 hours. Some slow-growing citrate-positive organisms may require up to 48 hours. Examine tubes at 24 hours; if negative, re-incubate and read again at 48 hours.

Step 4: Read and Interpret Results

Examine the slant for two criteria:

• Growth: Visible bacterial growth along the streak line
• Color change: Medium turns from green to blue (alkaline)

Record results as:

• Positive: Growth with blue color (partial or complete)
• Negative: No growth or minimal growth; medium remains green
• Weak positive: Growth present but only slight blue tint at the top of the slant

Step 5: Document Results

Record the following in your laboratory notebook or electronic system:

• Test organism identification and source
• Date and time of inoculation
• Incubation temperature and duration
• Positive and negative control results
• Test result (positive/negative) with description of color change
• Any observations (e.g., heavy growth, delayed reaction)
• Technician initials

Result Interpretation

Positive Result

A positive citrate test shows visible bacterial growth on the slant surface accompanied by a color change from green to blue. The blue color may appear first at the top of the slant (where oxygen is highest) and gradually extend downward. The intensity of blue can range from pale blue to deep Prussian blue depending on the extent of citrate utilization.

Examples of citrate-positive bacteria:

• Klebsiella pneumoniae
• Enterobacter cloacae
• Serratia marcescens
• Proteus mirabilis (variable)
• Salmonella enterica (most serovars)
• Citrobacter freundii

Negative Result

A negative citrate test shows no visible growth on the slant surface, and the medium remains green. Some organisms may produce slight growth without a color change; this should be recorded as negative because citrate is not being utilized as the sole carbon source.

Examples of citrate-negative bacteria:

• Escherichia coli
• Shigella species
• Yersinia enterocolitica
• Morganella morganii
• Providencia stuartii (variable)

Weak or Delayed Positive

Some organisms may show growth after 24 hours but only a faint blue color at the top of the slant. Re-incubate for an additional 24 hours. If the blue color intensifies, record as positive. If no color change occurs after 48 hours, record as negative.

Common Pitfalls in Interpretation

• False positive: Contamination of the medium with organic carbon sources (e.g., from dirty loops or excessive inoculum) can support growth without true citrate utilization. Always use a light inoculum.
• False negative: Insufficient incubation time, particularly for slow-growing organisms. Always incubate for 48 hours before finalizing a negative result.
• Dehydrated medium: Slants that have lost moisture may concentrate the pH indicator, giving a false blue appearance. Compare with uninoculated control.
• Mixed culture: If two colony types appear, the test is invalid. Repeat with a pure culture.

Troubleshooting

Limitations

The citrate utilization test has several important limitations that users must understand:

1. Not all citrate-positive organisms produce rapid color change: Some bacteria metabolize citrate slowly and may require 48–72 hours for a visible reaction. Always incubate negative results for the full 48 hours.

2. Inoculum size matters: A heavy inoculum can introduce trace nutrients from the original culture medium, supporting initial growth that is not due to citrate utilization. Use a light inoculum from a pure culture.

3. Medium quality varies by manufacturer: Different commercial formulations may have slight variations in buffer capacity or indicator concentration. Always follow manufacturer instructions and validate with known controls.

4. Cannot differentiate all Enterobacteriaceae: While useful in the IMViC panel, the citrate test alone cannot identify organisms to species level. It must be used in conjunction with other biochemical tests (e.g., urease, indole, oxidase).

5. Some organisms give variable results: Strains within the same species may differ in their ability to utilize citrate. For example, Proteus mirabilis can be citrate-positive or negative depending on the strain.

6. Not suitable for fastidious organisms: Bacteria that require growth factors (e.g., Haemophilus, Neisseria) cannot be tested on Simmons citrate agar because it lacks complex nutrients.

7. Environmental isolates may behave differently: Bacteria isolated from soil, water, or other environmental sources may show atypical reactions compared to clinical reference strains. Always include appropriate controls for your specific application.

Documentation

Proper documentation of citrate utilization test results is essential for quality assurance, reproducibility, and regulatory compliance. Record the following information in your laboratory records:

Essential Documentation Elements

• Test identification: Unique sample ID, organism name or code
• Date and time: Inoculation date and time, reading date and time
• Medium details: Manufacturer, lot number, expiration date, preparation date (if prepared in-house)
• Control results: Positive control (organism, result), negative control (organism, result), uninoculated control (color)
• Incubation conditions: Temperature, duration, aerobic/anaerobic
• Result: Positive, negative, or weak positive; description of color change
• Technician: Name or initials of person performing the test
• Any deviations: Notes on delayed reading, unusual observations, or repeat testing

Quality Control Records

Maintain a log of quality control results for each new lot of Simmons citrate agar. Test each new lot with positive and negative control strains before using for diagnostic purposes. Document:

• Lot number and date received
• Date of QC testing
• Positive control result (should be positive within 24–48 hours)
• Negative control result (should remain negative at 48 hours)
• Sterility check result (uninoculated slant should remain sterile and green)
• Technician signature
• Supervisor review (if required by laboratory policy)

Reporting Results

In clinical or research reports, present citrate test results clearly. For example:

• "Citrate utilization: Positive (blue color at 24 hours)"
• "Citrate utilization: Negative (no color change at 48 hours)"
• "Citrate utilization: Weak positive (faint blue at 48 hours)"

Include the incubation time in your report because delayed reactions may have different diagnostic significance.

Biosafety Considerations

The citrate utilization test is routinely performed at Biosafety Level 1 (BSL-1) when using non-pathogenic teaching strains such as Escherichia coli K-12 or Klebsiella pneumoniae ATCC 13883. However, when testing clinical isolates or environmental samples that may contain pathogens, follow BSL-2 practices as outlined in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition [6].

BSL-1 Practices (Teaching Strains)

• Standard microbiological practices: no eating, drinking, or applying cosmetics in the laboratory
• Hand washing after handling cultures and before leaving the laboratory
• Decontamination of work surfaces daily and after spills
• Use of personal protective equipment (lab coat, gloves, safety glasses)
• Mechanical pipetting only (no mouth pipetting)
• Proper waste disposal: autoclave all contaminated materials before disposal

BSL-2 Practices (Clinical or Environmental Isolates)

All BSL-1 practices, plus:

• Work performed in a Class II biosafety cabinet for procedures that may generate aerosols
• Restricted access to the laboratory during work
• Biohazard signage on laboratory doors
• Sharps disposal in puncture-resistant containers
• Spill kit available and staff trained in spill cleanup
• Medical surveillance as required by institutional policy

Decontamination

All Simmons citrate agar slants, whether positive or negative, must be decontaminated before disposal. Autoclave at 121°C for 30 minutes in validated biohazard bags. Alternatively, incinerate or use chemical disinfection according to institutional biosafety guidelines.

Recombinant or Synthetic Nucleic Acids

If the test organism contains recombinant or synthetic nucleic acid molecules, follow the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules [7]. This may require Institutional Biosafety Committee (IBC) approval and additional containment measures.

Frequently Asked Questions

1. Why must I avoid stabbing the butt of the Simmons citrate agar slant?

Stabbing the butt creates anaerobic conditions in the deeper portion of the medium. Citrate utilization requires aerobic metabolism through the tricarboxylic acid cycle. Under anaerobic conditions, bacteria cannot efficiently metabolize citrate, leading to false-negative results. Additionally, the butt serves as a color reference for the uninoculated medium, allowing you to compare the original green color with any color change on the slant surface.

2. Can I use Simmons citrate agar in Petri plates instead of slants?

Yes, Simmons citrate agar can be poured into Petri plates for streak plate inoculation. However, slants are preferred because the slanted surface provides a larger aerobic area for growth, and the butt serves as a built-in color control. If using plates, ensure they are incubated in a humidified chamber to prevent dehydration, and include an uninoculated plate as a color reference.

3. What should I do if my positive control turns blue but my test organism shows growth without color change?

This situation can occur with organisms that utilize citrate slowly or produce insufficient alkaline byproducts. Re-incubate the test slant for an additional 24 hours (total 48 hours). If the medium remains green after 48 hours, record the result as negative. Some environmental isolates may show growth on Simmons citrate agar without true citrate utilization if they can use trace contaminants in the medium. Always confirm with a pure culture and appropriate controls.

4. How does the citrate test fit into the IMViC series for identifying Enterobacteriaceae?

The citrate test is the "C" in IMViC (Indole, Methyl Red, Voges-Proskauer, Citrate). The four tests together help differentiate key genera:

• Escherichia coli: Indole (+), MR (+), VP (-), Citrate (-)
• Klebsiella pneumoniae: Indole (-), MR (-), VP (+), Citrate (+)
• Enterobacter aerogenes: Indole (-), MR (-), VP (+), Citrate (+)
• Citrobacter freundii: Indole (-), MR (+), VP (-), Citrate (+)

The citrate test alone cannot identify an organism; it must be interpreted alongside the other IMViC tests and additional biochemical reactions for definitive identification.

References and Further Reading

1. Sedláček I, Holochová P, Sedlář K, et al. Two new psychrotolerant Massilia species inhibit plant pathogens Clavibacter and Curtobacterium. (2025). https://pubmed.ncbi.nlm.nih.gov/40681538/ — Describes citrate utilization as part of biotyping for novel Massilia species isolated from Antarctic environments.

2. Safari M, Ghahroodian S, Abyarazimi Mv, et al. Comprehensive genomic analysis of a novel Bacillus cereus decomposing toluene potentially applicable in bioremediation. (2026). https://pubmed.ncbi.nlm.nih.gov/41313188/ — Demonstrates use of biochemical tests including citrate utilization for characterizing environmental bacterial isolates.

3. León MJ, Vera-Gargallo B, de la Haba RR, et al. Integrating genomic evidence for an updated taxonomy of the bacterial genus Spiribacter. (2024). https://pubmed.ncbi.nlm.nih.gov/39627276/ — Discusses metabolic diversity and carbon source utilization in halophilic bacteria, relevant to understanding citrate metabolism.

4. Arbab S, Ullah H, Wang W, et al. Prevalence and antimicrobial drug resistance of gram-negative bacteria in dairy feed and water: a One Health concern. (2025). https://pubmed.ncbi.nlm.nih.gov/41070378/ — Uses IMViC biochemical tests including citrate utilization for identifying E. coli and Salmonella from environmental samples.

5. Kayange NM, Malande OO, Scialaba S, et al. Non-malarial etiology of acute febrile episodes in children attending five healthcare facilities in Mwanza, Tanzania years 2020-2021. (2025). https://pubmed.ncbi.nlm.nih.gov/41038868/ — References use of biochemical identification methods for bacterial isolates from clinical specimens.

6. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services (2020). https://www.cdc.gov/labs/bmbl/index.html — Authoritative principles for risk assessment, containment, and microbiological laboratory practice.

7. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules/ — Framework for biosafety when working with genetically modified organisms.

8. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. https://www.ncbi.nlm.nih.gov/books/ — Searchable collection of authoritative biomedical books and methods references.

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