Hektoen Enteric Agar, a selective and differential medium, is used to distinguish Salmonella and Shigella species from other Enterobacteriaceae. Sylvia King, William I. Metzger introduced the medium in 1968. They developed HE Agar medium during their time at the Hektoen Institute, Chicago, in order to improve the recovery of Salmonella and Shigella from clinical specimens.
Principle of Hektoen Enteric Agar
Hektoen Enteric (HE) is a medium that uses bile salts to selectively inhibit and two indicators systems: acid fuchsin and bromothymolblue as indicators of carbohydrate digestion and ferric iron, which are used as indicators for the formation of hydrogen sulfurate from thiosulfate. Because bile salts inhibit these organisms, HE agar allows for good growth of Shigella.
The medium provides good colonial differentiation and inhibits some coliforms and other nonlactose-fermenting bacteria, thereby facilitating the identification of Salmonella and Shigella from food products. HE agar contains proteose peptone and yeast extract as well as sucrose, lactose. Salmonella can produce transparent green or blue-green colonies on HE agar with or without black centers. They also appear almost entirely black.
Shigella produces green, transparent colonies. Biochemical and serological confirmation tests are required for other organisms that form colonies like Salmonella or Shigella. Salmon-colored colonies are formed by lactose, sucrose or salicin fermentation of gram-negative bacteria.
Composition of Hektoen Enteric Agar
| Ingredients | Gms/liter |
| Protease peptone | 12.00 |
| Yeast extract | 3.000 |
| Lactose | 12.00 |
| Sucrose | 2.000 |
| Salicin | 9.000 |
| Bile Salts mixture | 9.000 |
| Sodium chloride | 5.000 |
| Sodium thiosulfate | 5.000 |
| Ferric ammonium citrate | 1.500 |
| Acid fuchsin | 0.100 |
| Bromothymol blue | 0.065 |
| Agar | 14.00 |
Final pH (at 25°C): 7.5±0.2
Preparation and Method of Use of Hektoen Enteric Agar
- Take 72.66 grams and 1000 ml purified/distilled water.
- To dissolve the medium completely, heat to boiling
- Keep it at 45-50 degrees Celsius
- Mix everything together and pour onto sterilized Petri dishes.
- Inoculate the medium with fresh faeces in Ringers solution, or with rectal swabs.
- Spread the inoculum for well-separated colonies.
- At 37°C, incubate for 18-24 Hours
- Additional incubation can improve differentiation between Salmonella et Shigella.
Result Interpretation on Hektoen Enteric Agar
- Rapid lactose fermenters, such as E. coli, are moderately inhibited. They produce bright-orange colonies to salmon pink colonies.
- Salmonella colonies are typically blue-green with black centers due to hydrogen sulfide.
- Shigella appears greener than Salmonella and the color fades to the edges of the colony.
- Proteus strains can be somewhat inhibited. Colonies that grow are smaller transparent, more glistening or less watery than species like Salmonella and Shigella.
| Organisms | Growth |
| Salmonella Typhimurium | Blue-green with or without black centers |
| Salmonella Abony | Blue-green with or without black centers |
| Salmonella Enteritidis | Blue-green with or without black centers |
| Salmonella Typhi | Blue-green with or without black centers |
| Escherichia coli | Orange (may have bile precipitate) |
| Shigella flexneri | Greenish blue |
| Shigella sonnei | Growth good to excellent; colonies light green |
| Proteus | Variable, blue-green to blue or salmon, most strains with black center or completely black |
| Enterobacter/Klebsiella | Large, yellow to salmon color |
Uses of Hektoen Enteric Agar
- Hektoen Enteric Agar Medium can be used to isolate Salmonella and Shigella species from enteric pathological specimens, in accordance with the United States Pharmacopoeia.
- The United States Pharmacopoeia recommends this medium for testing the presence Salmonella in dietary supplements.
- This medium is used to test for Salmonella in food samples according to various standards.
- HE agar can be used to plat fecal specimens in a direct or indirect manner. This helps increase the recovery of Salmonella and Shigella species from high levels of normal fecal flora.
- It can be used to extract gastrointestinal pathogens such as Salmonella or Shigella from food, water and fecal samples that may contain these organisms.
Limitations of Hektoen Enteric Agar
- One medium will rarely be able to capture all pathogens in a specimen. Additional media must be used to isolate Salmonella, Shigella, and other enteric pathogens from the specimen.
- This medium may make Proteus mirabilis colonies look like Salmonella.
- Some Shigella strains may require a 42-48 h incubation.
- While some diagnostic tests can be performed directly on the medium, it is not possible to identify all organisms.
- It is necessary to confirm and identify biochemically as well as serologically any colonies suspected to be Salmonella or Shigella.
