How to Perform a Hektoen Enteric (HE) Agar Test: Selective and Differential Properties

Hektoen Enteric (HE) agar is a selective and differential medium designed for the isolation and preliminary identification of Salmonella and Shigella species from fecal specimens and other clinical or environmental samples. This method exploits the ability of HE agar to inhibit Gram-positive bacteria and many coliforms while allowing pathogenic enteric bacteria to grow, and it differentiates organisms based on lactose fermentation and hydrogen sulfide (H₂S) production. The test is useful when a laboratory needs to screen for enteric pathogens in mixed microbial populations, particularly in stool cultures or food microbiology investigations, where the presence of Salmonella or Shigella must be confirmed or ruled out.

At a Glance

Scientific Principle of Hektoen Enteric Agar

HE agar operates on two fundamental microbiological principles: selectivity and differentiality. The selectivity is achieved through the incorporation of bile salts, which inhibit the growth of Gram-positive bacteria and many Gram-negative commensals. The differential properties rely on two distinct biochemical systems: carbohydrate fermentation and hydrogen sulfide production.

Selective Mechanism

The bile salts in HE agar (a mixture of bile salts and bromothymol blue) disrupt the cell membranes of Gram-positive bacteria, preventing their growth. The acid fuchsin dye further contributes to selectivity by inhibiting many non-pathogenic Gram-negative organisms, particularly those that are rapid lactose fermenters. This selective pressure allows Salmonella and Shigella species, which are more resistant to these agents, to grow while suppressing the background flora.

Differential Mechanism: Carbohydrate Fermentation

HE agar contains three carbohydrates: lactose, sucrose, and salicin. The inclusion of multiple sugars increases the medium's ability to differentiate between enteric bacteria. Organisms that ferment any of these carbohydrates produce acid, which lowers the pH of the medium. The pH indicator, bromothymol blue, changes color in response to this acid production. At neutral pH (approximately 7.0), the medium appears green. When acid is produced, the pH drops, and the indicator turns yellow or orange-yellow.

• Lactose fermenters (e.g., Escherichia coli, Klebsiella pneumoniae): Produce yellow or orange-yellow colonies, often with a yellow halo in the surrounding medium.
• Non-lactose fermenters (e.g., Salmonella, Shigella, Proteus): Remain green, blue-green, or transparent because they do not produce acid from the carbohydrates.

The inclusion of sucrose and salicin helps differentiate Salmonella and Shigella from other non-lactose fermenters that might ferment these alternative sugars. For example, some Proteus species can ferment sucrose, producing yellow colonies that could be confused with lactose fermenters.

Differential Mechanism: Hydrogen Sulfide Production

The second differential system detects hydrogen sulfide (H₂S) production. HE agar contains sodium thiosulfate as a sulfur source and ferric ammonium citrate as an indicator. Bacteria that produce H₂S (e.g., Salmonella species) reduce thiosulfate to hydrogen sulfide gas. The H₂S then reacts with ferric ions to form ferric sulfide, an insoluble black precipitate. This precipitate accumulates in the colony center, producing colonies with black centers. The blackening can be so intense that the entire colony appears black, or it may be limited to a central black dot.

• H₂S-positive organisms: Salmonella species typically produce colonies with black centers. Proteus species may also produce H₂S but often appear as swarming colonies with black centers.
• H₂S-negative organisms: Shigella species, Escherichia coli, and most other enteric bacteria do not produce H₂S and therefore lack black precipitate.

The combination of carbohydrate fermentation and H₂S production allows for presumptive identification. For example, Salmonella typically appears as green or blue-green colonies with black centers (non-lactose fermenter, H₂S-positive), while Shigella appears as green or blue-green colonies without black centers (non-lactose fermenter, H₂S-negative).

Materials and Instrumentation Choices

HE Agar Preparation

HE agar is available commercially as dehydrated powder or as pre-poured plates. The choice between these formats depends on laboratory volume, quality control requirements, and budget.

Dehydrated powder: Requires careful weighing, suspension in distilled water, heating to boiling to dissolve completely, and autoclaving at 121°C for 15 minutes. After autoclaving, the medium should be cooled to 45–50°C before pouring into sterile Petri dishes. The pH should be checked and adjusted to 7.0 ± 0.2 before autoclaving. This format is economical for high-volume laboratories but requires precise preparation and quality control.

Pre-poured plates: Ready-to-use plates are convenient for low-volume laboratories or when consistency is critical. They come with a manufacturer's quality control certificate, but the user should still perform in-house quality control testing upon receipt and before use.

Critical considerations:

• Overheating during preparation can degrade the selective agents and pH indicators, reducing the medium's performance.
• The agar should be poured to a depth of approximately 4–5 mm in standard 100 mm Petri dishes. Thinner agar may not provide sufficient nutrients or may dry out during incubation.
• Plates should be stored at 2–8°C, protected from light, and used within the manufacturer's expiration date. Light exposure can degrade the pH indicators.

Inoculation Equipment

• Sterile inoculating loops: Calibrated loops (1 µL or 10 µL) are recommended for quantitative work, but standard 4 mm loops are acceptable for qualitative isolation.
• Bunsen burner or microincinerator: For sterilizing loops between inoculations.
• Sterile saline or phosphate-buffered saline (PBS): For preparing inoculum suspensions if direct plating is not possible.
• Vortex mixer: For homogenizing liquid samples before inoculation.

Incubation Equipment

• Incubator: Set to 35–37°C, with temperature monitoring and logging. The incubator should have a thermometer calibrated annually.
• Timer: For tracking incubation duration (18–24 hours).

Quality Control Strains

• Positive control for H₂S and non-lactose fermentation: Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028 or equivalent)
• Negative control for H₂S and lactose fermentation: Shigella flexneri (ATCC 12022 or equivalent) or Shigella sonnei (ATCC 25931)
• Inhibited organism control: Escherichia coli (ATCC 25922) – should show weak growth or be inhibited
• Sterility control: Uninoculated plate incubated alongside samples

Controls and Their Importance

Controls are essential for validating that the HE agar is performing correctly and that the results are interpretable. Without proper controls, a negative result could be due to medium failure rather than absence of target organisms.

Positive Controls

A positive control using Salmonella enterica should produce:

• Green or blue-green colonies (non-lactose fermenter)
• Black centers (H₂S-positive)
• Moderate to good growth

If the positive control fails to grow or does not produce the expected reactions, the medium may be expired, improperly prepared, or contaminated with inhibitory substances.

Negative Controls

A negative control using Shigella species should produce:

• Green or blue-green colonies (non-lactose fermenter)
• No black precipitate (H₂S-negative)
• Moderate growth

If Shigella fails to grow, the medium may be too selective, possibly due to excessive bile salts or overheating during preparation.

Inhibited Organism Control

Escherichia coli should show either no growth or very weak growth with small, yellow colonies. If E. coli grows vigorously, the selective agents may be degraded or insufficient.

Sterility Control

An uninoculated plate incubated under the same conditions should show no growth. Any growth indicates contamination of the medium or the plate.

Frequency of Controls

• Each new lot number: Test all controls before using the medium for diagnostic work.
• Each day of use: At minimum, run a positive and negative control.
• When troubleshooting: Run full controls if unexpected results occur.

Conceptual Workflow

Step 1: Sample Preparation

For fecal specimens, a small amount (approximately 1–2 mm diameter) of stool is emulsified in sterile saline or PBS. For rectal swabs, the swab is rolled directly onto the agar surface. For food or environmental samples, appropriate enrichment and dilution steps should be performed according to standard methods.

Important: Do not use excessive inoculum, as this can overwhelm the selective agents and allow background flora to grow, obscuring target colonies.

Step 2: Inoculation

Using a sterile loop, streak the inoculum onto the HE agar plate using a quadrant streak method to obtain isolated colonies. The goal is to dilute the sample sufficiently so that individual colonies can be observed after incubation.

Streaking pattern:

1. First quadrant: Spread the inoculum over approximately one-quarter of the plate.
2. Second quadrant: Streak from the first quadrant into a fresh area, overlapping slightly.
3. Third quadrant: Streak from the second quadrant into a fresh area.
4. Fourth quadrant: Streak from the third quadrant into the remaining area.

Flame the loop between quadrants to ensure proper dilution.

Step 3: Incubation

Place the inoculated plates in an incubator at 35–37°C, with the lids facing upward (agar side down). Incubate aerobically for 18–24 hours. Do not incubate longer than 24 hours, as prolonged incubation can cause:

• Overgrowth of slow-growing organisms
• Diffusion of acid from fermenters into the medium, causing false-positive color changes
• Deterioration of the H₂S precipitate

Step 4: Examination

After incubation, examine the plates for:

• Growth: Presence or absence of colonies
• Colony color: Green/blue-green (non-fermenter) vs. yellow/orange (fermenter)
• Black precipitate: Presence or absence of black centers
• Colony morphology: Size, shape, edge characteristics

Record observations for each colony type present.

Quality Checks

Pre-Use Quality Checks

1. Visual inspection: The agar should be green to blue-green, with no cracks, bubbles, or contamination. The surface should be smooth and moist but not wet.
2. pH verification: If preparing from powder, verify the pH is 7.0 ± 0.2. Use a calibrated pH meter or pH indicator strips.
3. Expiration date: Do not use expired medium.
4. Storage conditions: Ensure plates have been stored at 2–8°C and protected from light.

During-Use Quality Checks

1. Incubator temperature: Verify the incubator temperature is 35–37°C at the start and end of incubation.
2. Incubation time: Record the exact time of inoculation and removal.
3. Control performance: Document the results of all controls.

Post-Use Quality Checks

1. Photographic documentation: If possible, photograph plates with typical reactions for reference.
2. Result recording: Record all observations in a laboratory notebook or electronic system.
3. Discrepancy investigation: If controls fail, investigate the cause before reporting results.

Result Interpretation

Colony Appearance and Presumptive Identification

Confirmatory Testing

HE agar provides only presumptive identification. All suspect colonies should be confirmed using:

• Biochemical tests: Triple Sugar Iron (TSI) agar, urea hydrolysis, citrate utilization, indole production, lysine decarboxylase
• Serological testing: Agglutination with specific antisera for Salmonella (O and H antigens) or Shigella (group-specific antisera)
• Commercial identification systems: API 20E, VITEK, or MALDI-TOF MS

Reporting Results

Results should be reported as "Presumptive Salmonella" or "Presumptive Shigella" until confirmatory testing is complete. The report should include:

• Colony morphology on HE agar
• Biochemical reactions observed
• Any serological results
• Final identification after confirmation

Troubleshooting

Limitations

Specificity Limitations

HE agar is not specific for Salmonella and Shigella. Other organisms can produce similar colony appearances:

• H₂S-positive, non-lactose fermenters: Proteus mirabilis, Proteus vulgaris, Edwardsiella tarda, Citrobacter freundii (some strains), and Arizona species can all produce green colonies with black centers, mimicking Salmonella.
• H₂S-negative, non-lactose fermenters: Yersinia enterocolitica, Providencia species, Morganella morganii, and some Serratia species can appear similar to Shigella.
• Lactose-fermenting Salmonella: Rare strains of Salmonella can ferment lactose, producing yellow colonies that may be mistaken for coliforms.

Sensitivity Limitations

• Low bacterial load: If the sample contains very few target organisms (e.g., early infection or carrier state), HE agar may not detect them without prior enrichment. As noted by Gal-Mor (2019), persistent Salmonella infections can involve low-level shedding, making detection challenging without enrichment steps [1].
• Inhibited strains: Some strains of Shigella are sensitive to the selective agents in HE agar and may not grow, leading to false-negative results.

Interpretation Limitations

• Mixed cultures: When multiple colony types are present, it can be difficult to distinguish target organisms from background flora. Subculture to fresh HE agar or other selective media may be necessary.
• Overlapping reactions: Some organisms produce weak or delayed reactions that may be misinterpreted. For example, Shigella sonnei may appear as pale green colonies that could be confused with non-pathogenic non-fermenters.

Medium Limitations

• Batch variation: Different lots of HE agar can vary in selectivity and performance. Always perform lot-to-lot quality control.
• Storage sensitivity: HE agar is sensitive to light and temperature. Improper storage can degrade the selective agents and indicators.
• Expiration: The medium has a limited shelf life, typically 4–8 weeks when stored properly.

Documentation Requirements

Proper documentation is essential for traceability, quality assurance, and regulatory compliance. The following should be recorded:

Pre-Analytical Documentation

• Medium information: Lot number, expiration date, manufacturer, date received, date opened
• Quality control results: Results of positive, negative, and sterility controls for each lot
• Sample information: Patient or sample identifier, collection date and time, sample type, requesting physician

Analytical Documentation

• Inoculation date and time
• Incubation conditions: Temperature, duration, atmosphere
• Observations: Colony morphology, color, presence of black precipitate, quantity of growth
• Control results: Performance of controls on the day of testing

Post-Analytical Documentation

• Interpretation: Presumptive identification based on HE agar reactions
• Confirmatory testing: Results of biochemical and serological tests
• Final report: Final identification and any relevant comments
• Discrepancy reports: Any issues encountered and corrective actions taken

Record Retention

Laboratory records should be retained according to institutional policy and regulatory requirements, typically for a minimum of 2–5 years.

Biosafety Considerations

Risk Assessment

HE agar is used to culture potentially pathogenic organisms, including Salmonella and Shigella species, which are classified as BSL-2 agents. The CDC and NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL) guidelines recommend BSL-2 practices for work with these organisms [2]. However, when using known non-pathogenic strains for teaching or quality control, BSL-1 practices may be sufficient.

BSL-2 Practices for Clinical Samples

When processing clinical specimens that may contain Salmonella or Shigella:

1. Personal protective equipment (PPE): Wear a laboratory coat, gloves, and eye protection. Use a face shield if there is risk of splashing.
2. Biological safety cabinet (BSC): Perform all manipulations of potentially infectious materials in a Class II BSC.
3. Sharps disposal: Dispose of all sharps in puncture-resistant containers.
4. Decontamination: Decontaminate all work surfaces before and after use with an appropriate disinfectant (e.g., 10% bleach or 70% ethanol).
5. Waste disposal: Autoclave all contaminated materials before disposal.

BSL-1 Practices for Teaching Laboratories

For teaching laboratories using known non-pathogenic strains:

1. PPE: Wear a laboratory coat and gloves.
2. Work surface: Use a designated area with a non-porous surface that can be disinfected.
3. Hand washing: Wash hands thoroughly after handling cultures.
4. No eating or drinking: Prohibit food and drink in the laboratory.
5. Decontamination: Disinfect work surfaces before and after use.

Spill Procedures

In case of a spill:

1. Alert others in the area.
2. Cover the spill with absorbent material.
3. Apply disinfectant (10% bleach) and allow 20–30 minutes contact time.
4. Clean up the spill using absorbent material and dispose of in biohazard waste.
5. Decontaminate the area again.

Transportation and Storage

• Transport: Use secondary containment (e.g., a leak-proof container) when moving cultures within the facility.
• Storage: Store cultures in a designated refrigerator or incubator, clearly labeled with biohazard symbols.

Frequently Asked Questions

1. Can HE agar distinguish between Salmonella and Shigella?

No, HE agar cannot definitively distinguish between these two genera. Both Salmonella and Shigella appear as non-lactose fermenters (green/blue-green colonies). The key difference is that most Salmonella species produce hydrogen sulfide, resulting in black-centered colonies, while Shigella species do not produce H₂S and lack black centers. However, some Salmonella strains (e.g., Salmonella serovar Typhi) may produce weak or no H₂S, and some Shigella strains may produce atypical reactions. Therefore, HE agar provides only presumptive identification, and confirmatory biochemical and serological testing is required.

2. Why does my HE agar plate show all yellow colonies even though I inoculated with a known non-fermenter?

This can occur for several reasons. First, the medium may be too old or improperly stored, causing the pH indicator to degrade and produce a false yellow color. Second, if the inoculum is too heavy, acid produced by even weak fermenters in the sample can overwhelm the buffer system and turn the entire plate yellow. Third, contamination of the medium with fermenting organisms during preparation can cause uniform yellowing. Finally, some non-fermenters can produce alkaline byproducts that interact with the indicator. Always run a positive control (non-fermenter) to verify the medium is performing correctly.

3. How long can I incubate HE agar plates before reading results?

The standard incubation time is 18–24 hours at 35–37°C. Plates should not be incubated longer than 24 hours. Prolonged incubation can lead to several problems: the H₂S precipitate may diffuse into the surrounding medium, making interpretation difficult; slow-growing lactose fermenters may eventually produce enough acid to change colony color; and the selective agents may break down, allowing background flora to overgrow. If no growth is observed after 24 hours, re-incubate for an additional 24 hours only if the sample is known to contain slow-growing organisms (e.g., Yersinia).

4. Can I use HE agar for environmental or food samples?

Yes, HE agar is suitable for environmental and food microbiology applications, but with some modifications. For food samples, an enrichment step (e.g., using buffered peptone water or Rappaport-Vassiliadis broth) is typically required to recover sublethally injured Salmonella cells. Direct plating of food samples onto HE agar may not detect low levels of contamination. For environmental samples (e.g., water, soil), the sample should be concentrated or filtered before plating. Always follow standard methods (e.g., FDA Bacteriological Analytical Manual, ISO 6579) for specific sample types.

References and Further Reading

1. Persistent Infection and Long-Term Carriage of Typhoidal and Nontyphoidal Salmonellae – Gal-Mor O. (2019). This review discusses the epidemiology, pathogenesis, and laboratory diagnosis of persistent Salmonella infections, including the challenges of detecting low-level shedding in carriers. PubMed

2. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition – CDC and NIH (2020). The authoritative guide for risk assessment and containment practices in microbiological laboratories, including BSL-2 recommendations for work with Salmonella and Shigella. CDC

3. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules – National Institutes of Health. Provides the institutional framework for biosafety and biosecurity in research settings, relevant when working with genetically modified organisms on HE agar. NIH

4. NCBI Bookshelf: Molecular Biology and Laboratory Methods – National Center for Biotechnology Information. A searchable collection of authoritative biomedical books and methods references, including detailed protocols for enteric pathogen isolation and identification. NCBI

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