Hektoen Enteric Agar: Selective and Differential Properties for Enteric Pathogens

Hektoen Enteric (HE) agar is a selective and differential culture medium designed for the isolation and preliminary differentiation of enteric pathogens, particularly Salmonella and Shigella species, from mixed microbial populations. This medium is most useful when processing fecal specimens, food samples, or environmental swabs where the target pathogens may be present alongside a background of normal enteric flora. HE agar achieves selectivity through bile salts that inhibit Gram-positive bacteria and many Gram-negative commensals, while differential properties rely on carbohydrate fermentation patterns and hydrogen sulfide (H₂S) production visualized through pH indicators and ferric ammonium citrate. Unlike MacConkey agar, which primarily differentiates lactose fermenters, HE agar incorporates multiple carbohydrates and an H₂S detection system that provides enhanced discrimination among enteric pathogens.

At a Glance

Scientific Principle

HE agar operates on two fundamental microbiological principles: selective inhibition and differential visualization. The selective properties derive from bile salts incorporated into the medium at concentrations that inhibit Gram-positive bacteria and many Gram-negative commensals while permitting the growth of enteric pathogens. Bile salts disrupt bacterial cell membranes, and organisms that have evolved to tolerate bile—such as members of the Enterobacteriaceae family—can grow in its presence. However, the concentration used in HE agar is calibrated to suppress even some bile-tolerant commensals, favoring pathogens like Salmonella and Shigella.

The differential properties of HE agar are more complex than those of simpler media like MacConkey agar. The medium contains three carbohydrates—lactose, sucrose, and salicin—along with a dual pH indicator system (bromothymol blue and acid fuchsin). Organisms that ferment any of these carbohydrates produce acid, which lowers the pH and causes the indicators to change color. Non-fermenters produce alkaline conditions or neutral pH, resulting in a different color. This multi-carbohydrate approach is particularly valuable because some enteric pathogens that appear as non-lactose fermenters on MacConkey agar may ferment sucrose or salicin, revealing their metabolic capabilities.

The hydrogen sulfide detection system is a critical differential feature. Ferric ammonium citrate reacts with hydrogen sulfide gas produced by certain bacteria during the reduction of sulfur-containing compounds (typically sodium thiosulfate in the medium). The reaction produces ferric sulfide, an insoluble black precipitate that accumulates in the center of colonies. This black center is a hallmark of H₂S-producing Salmonella species and helps distinguish them from Shigella species, which do not produce H₂S.

Materials and Instrumentation Choices

Medium Preparation Options

HE agar is available in dehydrated powder form from multiple manufacturers, including BD Difco, Oxoid, and Hardy Diagnostics. Each manufacturer's formulation may have slight variations in the concentration of selective agents or indicators, which can affect performance characteristics. When selecting a commercial source, review the manufacturer's quality control data and ensure the medium meets your laboratory's performance specifications. Dehydrated medium should be stored in a cool, dry environment and used before the expiration date indicated on the container.

For preparation, suspend the dehydrated powder in distilled or deionized water according to the manufacturer's instructions. Heat the suspension with frequent agitation and boil for one minute to dissolve completely. Do not autoclave HE agar, as excessive heat can degrade the selective agents and indicators. After boiling, cool the medium to approximately 45–50°C in a water bath before pouring plates. Pour plates to a depth of approximately 4–5 mm (about 20–25 mL per standard 100 mm Petri dish). Allow plates to solidify on a level surface, then store them inverted in sealed plastic bags at 2–8°C. Properly prepared plates are stable for approximately two weeks when stored under refrigeration.

Pre-Poured Commercial Plates

Many laboratories choose to purchase pre-poured HE agar plates from commercial suppliers. This option eliminates variability in preparation and provides documented quality control. When using commercial plates, verify the lot number, expiration date, and storage conditions upon receipt. Commercial plates should be stored refrigerated and allowed to warm to room temperature before inoculation to prevent condensation that could interfere with colony isolation.

Inoculation Equipment

Standard microbiological loops (calibrated 10 μL loops for quantitative work or 1 μL loops for qualitative streaking) are appropriate for specimen inoculation. For environmental or food samples, sterile swabs or spreaders may be necessary. When processing liquid samples, sterile pipettes with appropriate volume ranges should be used. All equipment must be sterile before use, and aseptic technique must be maintained throughout the procedure.

Incubation Equipment

A standard microbiological incubator capable of maintaining 35–37°C is required. The incubator should have temperature monitoring and recording capabilities to ensure consistent conditions. While HE agar plates are typically incubated aerobically, some protocols recommend incubation in ambient air without added CO₂, as elevated CO₂ can alter pH indicator responses.

Controls

Positive Controls

Positive controls verify that the medium supports the growth of target organisms and produces expected reactions. For HE agar, the following controls are recommended:

• Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028 or equivalent): Expected growth with blue-green colonies and black centers (H₂S positive)
• Shigella flexneri (ATCC 12022 or equivalent): Expected growth with green to blue-green colonies, no black centers (H₂S negative)
• Enterococcus faecalis (ATCC 29212 or equivalent): Expected complete inhibition (no growth)

Negative Controls

Negative controls confirm that the medium does not support the growth of organisms that should be inhibited. A sterile swab streaked across the plate surface serves as a negative control for contamination. Additionally, Escherichia coli (ATCC 25922) can serve as a differential control, producing yellow-orange colonies due to lactose fermentation.

Control Frequency

Quality control testing should be performed with each new lot of medium, after any preparation method change, and periodically (e.g., monthly) during routine use. Document all control results, including lot numbers, dates, and technician initials. If control organisms fail to produce expected results, investigate the medium preparation, storage conditions, and inoculum viability before using the medium for diagnostic purposes.

Conceptual Workflow

Step 1: Specimen Collection and Preparation

Collect specimens using appropriate techniques for the sample type. For fecal specimens, collect a small amount (approximately 1–2 grams) in a sterile container. For food or environmental samples, follow established protocols for sample collection and transport. Transport specimens to the laboratory promptly and process within two hours of collection, or store at 2–8°C for up to 24 hours if processing is delayed.

Step 2: Inoculation

Allow HE agar plates to reach room temperature before inoculation to prevent condensation. Using a sterile loop, pick a small amount of the specimen and streak the plate using the quadrant streak method to obtain isolated colonies. For liquid samples, use a sterile pipette to transfer 0.1 mL to the plate surface and spread evenly with a sterile spreader. For swab samples, roll the swab over a small area of the plate, then streak for isolation.

Step 3: Incubation

Place inoculated plates in an incubator set to 35–37°C with the lids slightly ajar for the first 15–30 minutes to allow excess moisture to evaporate, then close the lids fully. Incubate for 18–24 hours. Do not exceed 24 hours of incubation, as prolonged incubation can lead to overgrowth of background flora and deterioration of differential reactions.

Step 4: Examination

After incubation, examine plates for the presence of isolated colonies. Record the following characteristics for each colony type:

• Color: Note whether colonies are yellow-orange (fermenter), green or blue-green (non-fermenter), or colorless
• Black centers: Presence or absence of black precipitate in colony centers
• Size: Measure colony diameter in millimeters
• Transparency: Note whether colonies are transparent, translucent, or opaque
• Elevation: Describe colony elevation (raised, flat, umbonate)

Step 5: Interpretation

Interpret colony characteristics according to the differential reactions described in the Result Interpretation section. Record preliminary identification based on colony morphology and HE agar reactions. Confirm presumptive identifications with biochemical tests or serological methods as appropriate for the laboratory's scope.

Quality Checks

Medium Appearance

Before use, inspect HE agar plates for signs of deterioration. The medium should be a clear, green to blue-green color. Cracking, discoloration, or excessive condensation indicates improper preparation or storage. Discard plates that show any signs of contamination, such as visible colonies or turbidity.

pH Verification

The pH of properly prepared HE agar should be 7.5 ± 0.2 at 25°C. Verify pH using a calibrated pH meter or pH indicator strips. Incorrect pH can alter the selectivity and differential properties of the medium.

Sterility Testing

Incubate one plate from each prepared batch at 35–37°C for 24–48 hours to verify sterility. If any growth appears, investigate the preparation process and discard the entire batch.

Performance Testing

As described in the Controls section, test each new lot of medium with control organisms to verify that selective and differential properties are functioning correctly. Document all performance test results.

Result Interpretation

Colony Color and Fermentation Patterns

The dual pH indicator system in HE agar produces distinct colors based on carbohydrate fermentation:

• Yellow-orange colonies: Indicate fermentation of one or more carbohydrates (lactose, sucrose, or salicin). This reaction is typical of Escherichia coli, Klebsiella pneumoniae, and Enterobacter species. These organisms are generally considered non-pathogenic enteric flora in the context of HE agar screening.

• Green to blue-green colonies: Indicate no fermentation of the carbohydrates present. This reaction is typical of Salmonella and Shigella species, as well as some strains of Proteus, Providencia, and Edwardsiella. These organisms are potential pathogens and warrant further investigation.

• Colorless or transparent colonies: May indicate organisms that do not utilize the carbohydrates or that produce alkaline reactions. Some Pseudomonas species may appear colorless on HE agar.

Hydrogen Sulfide Production

The presence of black centers in colonies indicates H₂S production. The black precipitate (ferric sulfide) forms when hydrogen sulfide gas reacts with ferric ammonium citrate in the medium. Key interpretations include:

• Blue-green colonies with black centers: Strongly suggestive of Salmonella species, particularly Salmonella enterica subsp. enterica serovar Typhi and many non-typhoidal serovars. Some strains of Proteus mirabilis and Edwardsiella tarda may also produce black centers.

• Green colonies without black centers: Typical of Shigella species, which do not produce H₂S. However, some Salmonella strains (particularly Salmonella serovar Paratyphi A) may also lack H₂S production.

• Yellow-orange colonies with black centers: Uncommon but can occur with some H₂S-producing organisms that also ferment carbohydrates, such as Citrobacter freundii.

Colony Size and Morphology

• Salmonella species typically produce colonies 2–4 mm in diameter after 18–24 hours of incubation.
• Shigella species often produce smaller colonies (1–2 mm) that may be more translucent.
• Rapidly growing organisms like Proteus species may produce larger colonies or show swarming motility.

Limitations in Interpretation

HE agar provides presumptive identification only. Colony morphology and color reactions are screening tools that guide further testing. Confirmatory tests, including biochemical panels (e.g., triple sugar iron agar, lysine iron agar, urea agar) and serological typing, are necessary for definitive identification. Additionally, some enteric pathogens may produce atypical reactions on HE agar. For example, Salmonella serovar Paratyphi A is typically H₂S negative, and some Shigella strains may show weak fermentation reactions.

Troubleshooting

Limitations

HE agar has several important limitations that users must understand to avoid misinterpretation of results. First, the medium is not inhibitory to all Gram-negative organisms. Some non-pathogenic enteric bacteria, including Escherichia coli and Klebsiella species, grow readily on HE agar and may obscure the presence of pathogens if present in high numbers. This limitation is particularly relevant when processing specimens from patients with disrupted gut flora, such as those receiving antibiotic therapy.

Second, HE agar does not differentiate between all enteric pathogens. For example, Yersinia enterocolitica may produce variable reactions on HE agar, and some strains may be inhibited entirely. Similarly, Campylobacter species, which are important enteric pathogens, do not grow on HE agar and require specialized media and incubation conditions.

Third, the H₂S detection system can produce false-positive results. Some Proteus species and Citrobacter freundii produce black-centered colonies that can be mistaken for Salmonella. Conversely, H₂S-negative Salmonella strains (e.g., Salmonella serovar Paratyphi A) may be missed if H₂S production is the primary screening criterion.

Fourth, the medium's selectivity can be affected by the specimen type and the presence of inhibitory substances. Fecal specimens from patients with gastrointestinal bleeding may contain blood that interferes with the pH indicator system. Highly mucoid specimens may trap bacteria and prevent proper colony isolation.

Fifth, HE agar is not suitable for quantitative enumeration of enteric pathogens. The selective agents may inhibit a proportion of viable target organisms, leading to underestimation of bacterial numbers. For quantitative work, use non-selective media in parallel.

Finally, the medium has a limited shelf life once prepared. Dehydrated medium stored properly may last for years, but prepared plates should be used within two weeks. Expired or improperly stored plates may lose selectivity or differential properties.

Documentation

Record Keeping Requirements

Maintain comprehensive records for all HE agar use, including:

• Medium lot number and manufacturer
• Date of preparation or receipt of pre-poured plates
• Storage conditions and expiration date
• Quality control test results with dates and technician initials
• Specimen source and collection date
• Inoculation date and incubation conditions
• Colony morphology observations
• Preliminary interpretation
• Confirmatory test results (if performed)
• Final identification and disposition

Standard Operating Procedures

Each laboratory should maintain a written standard operating procedure (SOP) for HE agar use. The SOP should include:

• Purpose and scope of the procedure
• Principle of the method
• Materials and equipment required
• Step-by-step protocol
• Quality control procedures
• Result interpretation criteria
• Troubleshooting guidance
• Safety considerations
• References

Review and update the SOP annually or whenever procedures change. Ensure all personnel are trained on the current SOP and document training completion.

Reporting Results

Report HE agar results in a standardized format that includes:

• Specimen identification
• Colony morphology description
• Presumptive identification based on HE agar reactions
• Recommendation for confirmatory testing (if applicable)
• Date and time of report
• Technician signature or initials

For teaching and research laboratories, results may be reported in laboratory notebooks or electronic data management systems. Ensure all entries are legible, dated, and signed.

Biosafety

Risk Assessment

HE agar is used to culture enteric bacteria, some of which are human pathogens. Even when working with known non-pathogenic strains, there is always a risk of exposure to unexpected pathogens in clinical or environmental specimens. Conduct a risk assessment before beginning any work with HE agar, considering the specimen source, the organisms likely to be present, and the laboratory's containment capabilities.

Biosafety Level Considerations

According to the CDC and NIH guidelines [6], work with clinical specimens that may contain enteric pathogens should be conducted at Biosafety Level 2 (BSL-2). BSL-2 requires:

• Restricted access to the laboratory during work
• Personal protective equipment (lab coat, gloves, eye protection)
• Biological safety cabinet for procedures that may generate aerosols
• Autoclave for waste decontamination
• Sharps disposal containers
• Handwashing facilities

For teaching laboratories using known non-pathogenic strains (e.g., Escherichia coli K-12, non-pathogenic Salmonella mutants), BSL-1 practices may be appropriate. However, consult institutional biosafety committees and follow local regulations.

Personal Protective Equipment

Minimum personal protective equipment for HE agar work includes:

• Laboratory coat or gown
• Disposable gloves (nitrile or latex)
• Safety glasses or goggles (if splash risk exists)

Change gloves between handling different specimens and after any suspected contamination. Remove gloves before leaving the laboratory and wash hands thoroughly.

Waste Disposal

All materials that come into contact with bacterial cultures must be decontaminated before disposal. Autoclave all plates, loops, pipettes, and other contaminated items at 121°C for at least 30 minutes. Alternatively, use chemical disinfection with appropriate disinfectants (e.g., 10% bleach solution, 70% ethanol) for surfaces and non-autoclavable items. Follow institutional waste disposal policies and local regulations.

Spill Response

In the event of a culture spill:

1. Alert others in the area and restrict access
2. Cover the spill with absorbent material (paper towels)
3. Apply disinfectant (10% bleach or appropriate alternative) around the spill perimeter
4. Allow contact time as specified by the disinfectant manufacturer (typically 10–20 minutes)
5. Clean up using absorbent materials and dispose of as biohazardous waste
6. Decontaminate the area again with disinfectant
7. Report the spill to the laboratory supervisor

Training Requirements

All personnel working with HE agar must receive training on:

• Safe handling of microbiological cultures
• Proper use of personal protective equipment
• Biological safety cabinet operation (if applicable)
• Emergency procedures (spills, exposures)
• Waste disposal protocols
• Institutional biosafety policies

Document all training and maintain records according to institutional requirements.

Frequently Asked Questions

1. Can HE agar be used to isolate Vibrio cholerae from stool specimens?

No, HE agar is not suitable for isolating Vibrio cholerae. This organism requires alkaline conditions and specialized media such as thiosulfate-citrate-bile salts-sucrose (TCBS) agar. The bile salts and pH of HE agar may inhibit V. cholerae growth. For suspected cholera cases, use TCBS agar or other Vibrio-selective media.

2. Why do some Salmonella colonies appear green without black centers on HE agar?

Some Salmonella serovars, particularly Salmonella Paratyphi A, do not produce hydrogen sulfide and therefore lack the characteristic black centers. Additionally, H₂S production can be weak or delayed in some strains, and colonies examined before 18 hours of incubation may not show visible black precipitate. Always incubate plates for the full 18–24 hours before interpreting H₂S reactions.

3. How does HE agar compare to xylose-lysine-deoxycholate (XLD) agar for Salmonella isolation?

Both HE agar and XLD agar are selective and differential media for enteric pathogens, but they use different indicator systems. XLD agar relies on xylose fermentation, lysine decarboxylation, and H₂S production, while HE agar uses lactose, sucrose, and salicin fermentation. XLD agar may be more sensitive for some Salmonella serovars, particularly those that ferment xylose. However, HE agar provides better differentiation of Shigella from other non-fermenters. Many laboratories use both media in parallel for comprehensive screening.

4. Can HE agar plates be stored at room temperature instead of refrigeration?

Refrigeration (2–8°C) is strongly recommended for storing prepared HE agar plates. Room temperature storage accelerates medium deterioration, including degradation of selective agents and pH indicators. Plates stored at room temperature may lose selectivity within days and produce unreliable results. If refrigeration is unavailable, prepare fresh plates for each use and discard any unused plates after 24–48 hours.

References and Further Reading

1. De Chiara I, Marasco R, Della Gala M, Fusco A, Donnarumma G, Muscariello L. Probiotic Properties of Lactococcus lactis Strains Isolated from Natural Whey Starter Cultures. 2024. https://pubmed.ncbi.nlm.nih.gov/38540945/

2. Wysok B, Dymkowski A, Sołtysiuk M, Kobuszewska A. Assessment of Microbial and Heavy Metal Contamination of Natural Sheep Casings from Different Geographic Regions. 2025. https://pubmed.ncbi.nlm.nih.gov/40361603/

3. Janda JM, Duman M. Expanding the Spectrum of Diseases and Disease Associations Caused by Edwardsiella tarda and Related Species. 2024. https://pubmed.ncbi.nlm.nih.gov/38792860/

4. Kengne MF, Tsobeng OD, Dadjo BST, Kuete V, Mbaveng AT. Multidrug Resistant Enteric Bacteria from Cancer Patients Admitted in Douala Laquintinie Hospital, Littoral Region of Cameroon. 2024. https://pubmed.ncbi.nlm.nih.gov/39036470/

5. Kuan S, Chin NL, Tee TP, Hasnan NZN. Microbiome Diversity in Seafood Factories via Next-Generation Sequencing for Food Safety Management System (FSMS) Certifications in Malaysia. 2025. https://pubmed.ncbi.nlm.nih.gov/40361602/

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

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/

8. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. https://www.ncbi.nlm.nih.gov/books/

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