How to Perform a Hugh-Leifson (OF) Test: Oxidative-Fermentative Metabolism

The Hugh-Leifson (OF) test is a microbiological method used to determine whether a bacterium metabolizes a specific carbohydrate through oxidative pathways (requiring oxygen), fermentative pathways (occurring without oxygen), or is unable to metabolize the carbohydrate at all. This test is particularly useful for differentiating gram-negative bacilli, especially nonfermentative species such as Pseudomonas from fermentative Enterobacteriaceae, and for characterizing environmental and clinical isolates in teaching and research laboratories. The test relies on a semisolid OF medium containing the carbohydrate of interest and a pH indicator (bromothymol blue), with two tubes per carbohydrate: one open to air (oxidative) and one sealed with mineral oil (fermentative). Color changes from green to yellow indicate acid production, and the pattern of acid production in the open versus sealed tube reveals the metabolic pathway.

At a Glance

Scientific Principle

The Hugh-Leifson OF test is based on the differential ability of bacteria to metabolize carbohydrates via two distinct biochemical pathways: oxidation (aerobic respiration) and fermentation (anaerobic metabolism). The test medium contains a low concentration of peptone (to minimize alkaline byproducts from protein degradation), a high concentration of the test carbohydrate (typically 1%), and bromothymol blue as a pH indicator. Bromothymol blue is green at neutral pH (approximately 7.0–7.2), turns yellow under acidic conditions (pH below 6.0), and becomes blue under alkaline conditions (pH above 7.6).

When a bacterium oxidizes a carbohydrate, it uses oxygen as the terminal electron acceptor, producing acidic end products (such as organic acids) only in the presence of oxygen. In the open tube, oxygen diffuses into the medium, allowing oxidative metabolism to occur, and acid production turns the medium yellow near the surface. In the sealed tube, oxygen is excluded by the mineral oil overlay, preventing oxidative metabolism; therefore, no acid is produced, and the medium remains green or may turn blue due to alkaline byproducts from peptone utilization.

When a bacterium ferments a carbohydrate, it uses an organic molecule as the terminal electron acceptor, producing acidic end products (such as lactic acid, acetic acid, or formic acid) regardless of oxygen presence. In both the open and sealed tubes, acid production occurs, turning the entire medium yellow. Some fermentative organisms may also produce gas, visible as bubbles or cracks in the agar.

If a bacterium cannot metabolize the test carbohydrate, it will use peptone as an energy source, producing alkaline byproducts (such as ammonia) that turn the medium blue. This occurs in both tubes, though the sealed tube may show a more pronounced alkaline reaction due to limited oxygen.

The test was originally developed by Hugh and Leifson in 1953 to differentiate gram-negative bacilli, particularly to distinguish oxidative genera like Pseudomonas from fermentative genera like Escherichia and Enterobacter. The method remains a cornerstone in bacterial identification schemes, especially for nonfermentative gram-negative rods, as described in the rapid identification system by Otto and Pickett (1976) [1].

Materials and Instrumentation

OF Medium Preparation

The Hugh-Leifson OF medium is commercially available as a dehydrated powder or can be prepared from individual components. The base medium contains:

• Peptone (2 g/L)
• Sodium chloride (5 g/L)
• Dipotassium phosphate (0.3 g/L)
• Bromothymol blue (0.08 g/L)
• Agar (2–3 g/L for semisolid consistency)

The carbohydrate of interest is added separately at a final concentration of 1% (w/v). Common carbohydrates include glucose, lactose, sucrose, maltose, mannitol, xylose, and arabinose. The choice of carbohydrate depends on the bacterial species being tested and the identification scheme being used.

Preparation steps:

1. Suspend the dehydrated OF base in distilled water according to manufacturer instructions.
2. Heat to boiling to dissolve completely.
3. Adjust pH to 7.1–7.2 if necessary (the medium should be green).
4. Dispense into flasks or tubes and sterilize by autoclaving at 121°C for 15 minutes.
5. After cooling to 50–55°C, add the filter-sterilized carbohydrate solution (10% stock) to achieve a 1% final concentration.
6. Aseptically dispense 4–5 mL into sterile 13 × 100 mm screw-cap tubes.
7. Allow the medium to solidify in an upright position.

Important considerations:

• The agar concentration should be low (0.2–0.3%) to create a semisolid consistency that allows motility observation and oxygen diffusion. Higher agar concentrations inhibit oxygen diffusion and may give false-negative oxidative results.
• The carbohydrate must be added aseptically after autoclaving because heat sterilization can degrade some sugars (e.g., lactose, sucrose) and alter the pH.
• The medium should be green at neutral pH. If it appears yellow or blue, the pH is incorrect and must be adjusted before use.

Mineral Oil

Sterile mineral oil (light paraffin oil) is used to create an anaerobic environment in the sealed tube. The oil must be sterile and free of contaminants that could affect the test. Autoclave the oil at 121°C for 15 minutes in a heat-resistant container, or use commercially available sterile mineral oil.

Inoculation Equipment

• Sterile inoculating needle (straight wire) for stab inoculation
• Bunsen burner or microincinerator for sterilization
• Sterile pipettes or syringes for adding oil overlay

Controls

Appropriate control strains are essential for validating the test system. Recommended controls include:

• Oxidative positive control: Pseudomonas aeruginosa (ATCC 27853 or equivalent) – produces acid from glucose only in the open tube
• Fermentative positive control: Escherichia coli (ATCC 25922 or equivalent) – produces acid from glucose in both open and sealed tubes
• Negative control (non-saccharolytic): Alcaligenes faecalis (ATCC 8750 or equivalent) – produces no acid from glucose; medium turns blue in both tubes
• Uninoculated medium control: One tube of each carbohydrate medium left uninoculated and incubated alongside tests

Controls and Quality Assurance

Internal Controls

Each batch of OF medium must be tested with known control strains before use in diagnostic or research work. The control strains should produce expected results:

• P. aeruginosa: Open tube turns yellow within 24–48 hours; sealed tube remains green or turns blue
• E. coli: Both open and sealed tubes turn yellow within 24 hours; gas production may be visible
• A. faecalis: Both tubes remain green or turn blue; no yellow color develops

If control strains do not produce expected results, the medium is faulty and must be discarded. Common causes of failure include incorrect pH, degraded carbohydrate, contaminated oil, or improper incubation conditions.

Uninoculated Controls

An uninoculated tube of each carbohydrate medium should be incubated alongside test tubes to monitor for spontaneous color changes or contamination. The uninoculated tube should remain green throughout the incubation period. If it turns yellow, the medium is contaminated or the carbohydrate has degraded.

Replicate Testing

For critical identifications, perform the OF test in duplicate or triplicate to ensure reproducibility. Discrepant results between replicates indicate technical error or mixed cultures and require repeat testing from a pure culture.

Documentation

Record the following information for each test:

• Organism identification (strain number or source)
• Carbohydrate(s) tested
• Date and time of inoculation
• Date and time of oil overlay addition
• Incubation temperature
• Observations at each reading interval (24, 48, 72 hours, 7 days)
• Final interpretation (oxidative, fermentative, or non-saccharolytic)
• Control strain results
• Any anomalies or deviations from protocol

Conceptual Workflow

Step 1: Prepare the OF Medium

Prepare Hugh-Leifson OF medium with the desired carbohydrate(s) as described in the Materials section. Allow the medium to solidify in upright tubes. The medium should be green and semisolid. If the medium appears yellow or blue, do not use it.

Step 2: Inoculate the Tubes

For each bacterial isolate and each carbohydrate to be tested, inoculate two tubes of OF medium:

1. Using a sterile inoculating needle, pick a single colony from a pure culture (18–24 hours old).
2. Stab the needle straight down through the center of the medium to approximately 1 cm from the bottom of the tube.
3. Withdraw the needle along the same line to minimize disruption of the medium.
4. Repeat for the second tube.

Important: Do not stab multiple times or swirl the needle, as this introduces excess oxygen and may damage the semisolid consistency. Use a light inoculum; heavy inoculation can cause false-positive results due to preformed enzymes or carryover of nutrients.

Step 3: Add Oil Overlay

Within 15–30 minutes of inoculation, add sterile mineral oil to one of the two tubes:

1. Using a sterile pipette or syringe, carefully layer sterile mineral oil over the surface of the medium.
2. Add enough oil to create a layer 1–2 cm deep (approximately 1–2 mL for a standard tube).
3. Ensure the oil completely covers the agar surface without trapping air bubbles.
4. Leave the second tube without oil (open to air).

The oil overlay creates an anaerobic environment by preventing oxygen diffusion into the medium. This is critical for distinguishing oxidative from fermentative metabolism.

Step 4: Incubate the Tubes

Incubate both tubes (open and sealed) at 30–35°C for up to 7 days. The standard incubation temperature is 35°C for clinical isolates, but 30°C may be used for environmental strains or when testing temperature-sensitive organisms.

Reading schedule:

• Read at 24 hours
• Read at 48 hours
• Read at 72 hours (if negative at 48 hours)
• Read at 7 days (if negative at 72 hours)

Some slow-growing or weakly oxidative organisms may require extended incubation. Do not discard tubes as negative before 7 days.

Step 5: Interpret the Results

Observe the color of the medium in both tubes. Record the color at each reading interval. The interpretation is based on the pattern of color change:

Oxidative result: Acid production occurs only in the open tube, where oxygen is available. The sealed tube remains green or may turn blue due to alkaline byproducts. The yellow color typically appears first at the surface of the open tube and may spread downward over time.

Fermentative result: Acid production occurs in both tubes, regardless of oxygen availability. Both tubes turn yellow, often throughout the entire medium. Gas production (bubbles, cracks, or displacement of the agar) may be visible in fermentative organisms.

Non-saccharolytic result: No acid production in either tube. The medium may remain green or turn blue. This indicates the organism cannot metabolize the test carbohydrate and relies on peptone for energy.

Alkaline reaction: The medium turns blue in both tubes, indicating strong alkaline byproducts from peptone utilization. This is common in non-saccharolytic organisms and some oxidative organisms that produce alkaline end products.

Step 6: Record and Report Results

Document the results for each carbohydrate tested. Include the color observations at each reading interval, the final interpretation, and any notes on gas production or motility (visible as turbidity spreading from the stab line).

Quality Checks and Troubleshooting

Common Problems and Solutions

Troubleshooting Guide

Problem: All tubes turn yellow, including uninoculated controls

• The medium pH is too low (acidic). Prepare fresh medium and verify pH is 7.1–7.2.
• The carbohydrate has degraded during storage. Use freshly prepared carbohydrate solutions.
• The bromothymol blue indicator is contaminated. Use a fresh batch of indicator.

Problem: No color change in any tube, including controls

• The medium pH is too high (alkaline). Adjust pH to 7.1–7.2 before autoclaving.
• The carbohydrate concentration is too low. Verify that 1% carbohydrate was added.
• The incubation temperature is incorrect. Verify incubator temperature with a calibrated thermometer.
• The organism is dead or nonviable. Check viability on nutrient agar.

Problem: Inconsistent results between replicates

• Mixed culture. Streak the original culture for isolation and repeat the test from a single colony.
• Inoculum size varies. Standardize the inoculum by using a single colony and consistent stabbing technique.
• Oil overlay thickness varies. Use a consistent volume of oil (1–2 mL) for all sealed tubes.

Problem: Sealed tube turns yellow but open tube remains green

• This is unusual and may indicate a contaminant that grows anaerobically. Gram stain the sealed tube and repeat the test.
• The oil overlay may have been added before the medium solidified, creating an anaerobic environment from the start. Allow medium to solidify completely before adding oil.

Limitations and Considerations

Organism-Specific Limitations

• Fastidious organisms: Some bacteria require enriched media or specific growth factors and may not grow in the minimal OF medium. For these organisms, alternative methods (e.g., API strips, commercial identification systems) may be more appropriate.
• Slow-growing organisms: Some oxidative organisms (e.g., Burkholderia, Ralstonia) may require 7–14 days for visible acid production. Extended incubation may be necessary.
• Alkaline-producing organisms: Some bacteria produce strong alkaline byproducts that mask weak acid production. In such cases, the medium may turn blue before any yellow color develops. Reading at early time points (24 hours) may help detect transient acid production.
• Motile organisms: Motile bacteria may spread throughout the semisolid medium, causing uniform turbidity. This does not affect the color change interpretation but should be noted.

Medium-Specific Limitations

• Carbohydrate concentration: The standard 1% carbohydrate concentration is suitable for most organisms, but some may require higher (2%) or lower (0.5%) concentrations for optimal results. Refer to specific identification schemes for guidance.
• Agar concentration: The semisolid consistency is critical for oxygen diffusion. If the agar concentration is too high (e.g., >0.4%), oxygen diffusion is inhibited, and oxidative organisms may appear non-saccharolytic. If the agar concentration is too low, the medium may not remain intact after stabbing.
• pH indicator: Bromothymol blue is the standard indicator, but it may be toxic to some organisms at high concentrations. Use the recommended concentration (0.08 g/L) and avoid overheating during preparation.

Interpretation Limitations

• Weak oxidative reactions: Some organisms produce only small amounts of acid, resulting in a faint yellow color that may be difficult to distinguish from green. Use a white background and good lighting for reading. Compare with uninoculated controls.
• Delayed reactions: Some organisms may show acid production only after 5–7 days. Do not discard tubes prematurely.
• Gas production: Gas production is not always visible in semisolid medium. If gas is suspected but not seen, use a Durham tube in a liquid fermentation medium for confirmation.
• Mixed metabolic pathways: Some organisms can switch between oxidative and fermentative metabolism depending on conditions. The OF test provides a snapshot of the predominant pathway under the test conditions.

Biosafety Considerations

The OF test is typically performed with BSL-1 organisms in teaching and research laboratories. However, when testing clinical isolates or unknown environmental samples, follow BSL-2 practices as outlined in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL) guidelines [2]. Key biosafety measures include:

• Perform all work in a biological safety cabinet (BSC) when handling BSL-2 organisms.
• Use proper personal protective equipment (PPE): lab coat, gloves, and eye protection.
• Decontaminate all waste (tubes, pipettes, oil) by autoclaving before disposal.
• Do not use the OF test for select agents or highly pathogenic organisms without appropriate containment and authorization.
• Follow institutional biosafety committee (IBC) guidelines for recombinant or synthetic nucleic acid work, as described in the NIH Guidelines [3].

Documentation and Reporting

Laboratory Record

Maintain a detailed laboratory record for each OF test, including:

• Date and time of inoculation
• Organism identification (strain number, source, Gram stain result)
• Carbohydrate(s) tested
• Tube labeling (open/sealed)
• Incubation temperature
• Observations at each reading interval (24, 48, 72 hours, 7 days)
• Color of medium in each tube (green, yellow, blue)
• Any gas production or motility
• Control strain results
• Final interpretation
• Technician initials

Reporting Results

Report the OF test result as one of the following:

• Oxidative: "The organism oxidizes [carbohydrate] (OF test positive in open tube only)."
• Fermentative: "The organism ferments [carbohydrate] (OF test positive in both open and sealed tubes)."
• Non-saccharolytic: "The organism does not metabolize [carbohydrate] (OF test negative in both tubes)."
• Alkaline reaction: "The organism produces an alkaline reaction in OF medium with [carbohydrate]."

Include the reading time (e.g., "positive at 48 hours") and any relevant notes (e.g., "gas production observed").

Integration with Other Tests

The OF test is often used in conjunction with other biochemical tests for bacterial identification. Common companion tests include:

• Oxidase test
• Catalase test
• Motility test (semisolid agar method)
• Nitrate reduction test
• Indole test
• Methyl red and Voges-Proskauer tests
• Citrate utilization test

For gram-negative nonfermentative bacilli, the OF test is particularly useful when combined with the oxidase test and growth on selective media, as described by Otto and Pickett (1976) [1].

Frequently Asked Questions

1. Why do I need two tubes for the OF test? Can I use just one tube with oil?

No, you must use two tubes per carbohydrate. The open tube allows oxygen diffusion and detects oxidative metabolism, while the sealed tube (with oil overlay) creates an anaerobic environment to detect fermentative metabolism. Using only one tube would not distinguish between oxidative and fermentative pathways. The comparison between the two tubes is essential for correct interpretation.

2. What should I do if the oil overlay turns yellow during incubation?

If the oil overlay itself turns yellow, this indicates contamination or a chemical reaction, not a valid test result. The oil should remain colorless. Discard the test and repeat with fresh sterile oil. Check your aseptic technique and ensure the oil is properly sterilized before use.

3. Can I use the OF test for gram-positive bacteria?

The OF test was originally developed for gram-negative bacilli, but it can be used for some gram-positive organisms with appropriate modifications. However, many gram-positive bacteria require enriched media and may not grow well in the minimal OF medium. For gram-positive organisms, alternative carbohydrate utilization tests (e.g., phenol red broth base with Durham tubes) are often more reliable.

4. How long should I incubate the OF test before calling it negative?

Incubate for at least 7 days before calling a test negative. Some oxidative organisms, particularly environmental isolates and slow-growing species, may require 5–7 days or longer to produce detectable acid. Read the tubes at 24, 48, and 72 hours, and again at 7 days. If the medium remains green or turns blue after 7 days, the organism is considered non-saccharolytic for that carbohydrate.

References and Further Reading

1. Rapid method for identification of gram-negative, nonfermentative bacilli. Otto LA, Pickett MJ. (1976). PubMed. https://pubmed.ncbi.nlm.nih.gov/780371/ – Describes a rapid identification system for nonfermentative gram-negative bacilli using oxidative attack of substrates, including comparison with the OF method.

2. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. CDC and NIH. (2020). https://www.cdc.gov/labs/bmbl/index.html – Authoritative guidelines for biosafety practices in microbiological laboratories, including risk assessment and containment for BSL-1 and BSL-2 organisms.

3. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. National Institutes of Health. https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules/ – Framework for biosafety and biosecurity in research involving recombinant or synthetic nucleic acids.

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

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