Hektoen Enteric Agar- Composition, Principle, Preparation, Results, Uses

Hektoen enteric agar (HE agar) is a selective and differential medium that is used to isolate and identify Salmonella and Shigella species from fecal and other specimens. HE agar contains the following ingredients and their quantities in grams per liter:

• Proteose peptone: 12.0
• Yeast extract: 3.0
• Sodium chloride: 5.0
• Lactose: 12.0
• Sucrose: 12.0
• Salicin: 2.0
• Bromothymol blue: 0.065
• Acid fuchsin: 0.1
• Sodium thiosulfate: 5.0
• Ferric ammonium citrate: 1.5
• Bile salts mixture: 20.0
• Agar: 13.5
• Distilled or deionized water: 1000 ml

The final pH of the medium is adjusted to 7.5 ± 0.2 at 25°C.

The following table lists the ingredients and their quantities in grams per liter for preparing Hektoen Enteric Agar:

The final pH of the medium should be adjusted to 7.5±0.2 at 25°C.

Hektoen enteric agar (HEA) is a selective and differential medium that is designed to isolate and differentiate Salmonella and Shigella species from other enteric bacteria. HEA has several components that contribute to its principle:

• Bile salts: These are added to inhibit the growth of most gram-positive bacteria and some gram-negative bacteria that are not part of the normal intestinal flora. Bile salts also enhance the lactose fermentation by some coliforms, resulting in acid production and color change of the indicators.
• Lactose, sucrose, and salicin: These are the fermentable carbohydrates that serve as substrates for differentiating bacteria based on their ability to produce acid from them. Lactose and sucrose are more readily fermented by most coliforms, while salicin is more specific for Shigella species. The acid production lowers the pH of the medium and changes the color of the indicators from green to yellow or salmon pink.
• Bromothymol blue and acid fuchsin: These are the pH indicators that detect the acid production from carbohydrate fermentation. Bromothymol blue turns yellow at pH below 6.0, while acid fuchsin turns salmon pink at pH below 6.8. The combination of these two indicators produces a range of colors from green (neutral or alkaline) to yellow or salmon pink (acidic).
• Sodium thiosulfate and ferric ammonium citrate: These are the sources of sulfur and iron, respectively, that allow the detection of hydrogen sulfide (H2S) production by some bacteria. H2S is formed when bacteria reduce sulfur to sulfide, which then reacts with iron to form a black precipitate of ferrous sulfide (FeS). The blackening of the medium or the colonies indicates H2S production.
• Peptone and yeast extract: These are the sources of nitrogen, carbon, vitamins, and minerals that support the growth of bacteria on the medium.

The principle of HEA is based on the following characteristics of Salmonella, Shigella, and other enteric bacteria:

• Salmonella species are non-lactose fermenters that produce H2S from thiosulfate. They grow as blue-green colonies with or without black centers on HEA.
• Shigella species are non-lactose fermenters that do not produce H2S. They grow as green colonies on HEA.
• Coliforms are lactose fermenters that do not produce H2S. They grow as yellow or salmon pink colonies on HEA.
• Proteus species are non-lactose fermenters that produce H2S and also deaminate phenylalanine to produce phenylpyruvic acid, which alkalinizes the medium. They grow as small, transparent, glistening colonies with a brown halo on HEA.

HEA allows a good growth of Shigella because the inhibition of these organisms by bile salts is reduced by the addition of relatively large amounts of peptone and carbohydrates. The medium provides good colonial differentiation and inhibits some coliforms and other non-lactose-fermenting bacteria, thereby facilitating the identification of Salmonella and Shigella from food products.

However, HEA is not a definitive medium for identifying Salmonella and Shigella, as some other organisms may produce similar colonies on this medium. Therefore, biochemical and serological confirmatory tests are necessary for complete identification.

Hektoen enteric agar is a ready-to-use medium that can be purchased from commercial suppliers or prepared in the laboratory. The preparation and method of use of hektoen enteric agar are as follows:

• If preparing the medium in the laboratory, suspend 72.66 grams of the dehydrated powder in 1000 ml of purified or distilled water. Heat to boiling to dissolve the medium completely. Do not autoclave the medium as it may affect its performance. Cool the medium to 45-50°C and mix well before pouring into sterile Petri plates.
• If using the commercially available medium, follow the manufacturer`s instructions for reconstitution and storage. The medium should be clear and greenish in color before use.
• Inoculate the medium with fresh fecal specimens suspended in Ringer`s solution or directly with rectal swabs. Use a sterile loop or swab to streak the inoculum over the surface of the agar, spreading it to obtain well-separated colonies. Alternatively, use a sterile pipette to deliver 0.1 ml of the inoculum onto the center of the plate and spread it evenly with a sterile spreader.
• Incubate the plates for 18-24 hours at 37°C in an aerobic atmosphere. Further incubation up to 48 hours may improve the differentiation between Salmonella and Shigella species.
• Examine the plates for typical colony morphology and color changes. Refer to the result interpretation section for details.

Hektoen enteric agar (HEA) is a differential and selective medium that allows the identification of Salmonella and Shigella species based on their colony morphology and color. The medium contains two indicators of carbohydrate fermentation: bromothymol blue and acid fuchsin, which turn yellow in acidic conditions. The medium also contains sodium thiosulfate and ferric ammonium citrate, which react with hydrogen sulfide (H2S) produced by some bacteria, forming a black precipitate. The medium also contains bile salts, which inhibit the growth of most gram-positive and some gram-negative bacteria.

The interpretation of the results on HEA is as follows:

• Rapid lactose fermenters (such as E. coli) are moderately inhibited and produce bright-orange to salmon pink colonies. These colonies have a yellow halo around them due to the acidification of the medium.
• Salmonella colonies are blue-green typically with black centers from hydrogen sulfide gas. These colonies do not ferment lactose, sucrose, or salicin, and produce H2S from thiosulfate.
• Shigella appear greener than Salmonella, with the color fading to the periphery of the colony. These colonies do not ferment lactose, sucrose, or salicin, and do not produce H2S from thiosulfate.
• Proteus strains are somewhat inhibited; colonies that develop are small transparent and more glistening or watery in appearance than species of Salmonella or Shigella. These colonies may produce H2S from thiosulfate, but they also swarm on the surface of the medium, forming concentric rings.
• Other non-lactose fermenting gram-negative bacteria may grow on HEA, but they usually produce colorless or pale green colonies that do not produce H2S from thiosulfate.

It is important to note that although HEA can provide useful clues for the identification of Salmonella and Shigella species, it is not sufficient for a definitive diagnosis. Biochemical and serological tests are necessary to confirm the presence and identity of these pathogens. Some strains of Shigella may require longer incubation periods to show visible growth on HEA. Some strains of Salmonella may not produce H2S from thiosulfate. Some strains of Proteus may resemble Salmonella on this medium. Therefore, careful observation and interpretation of the results on HEA are essential for accurate diagnosis.

Hektoen Enteric Agar allows the differentiation of enteric pathogens based on their ability to ferment lactose, sucrose, and salicin, and to produce hydrogen sulfide gas from sodium thiosulfate. The medium contains two pH indicators, bromothymol blue and acid fuchsin, that change color in response to acid production from carbohydrate fermentation. The medium also contains ferric ammonium citrate, which reacts with hydrogen sulfide gas to form a black precipitate. The following table summarizes the growth and color characteristics of different organisms on Hektoen Enteric Agar:

Hektoen Enteric Agar (HEA) is a medium that has various uses in microbiology, especially for the isolation and differentiation of Salmonella and Shigella species from different types of specimens. Some of the common uses of HEA are:

• HEA is recommended for differential and selective isolation of Salmonella and Shigella species from enteric pathological specimens in accordance with United States Pharmacopoeia.
• HEA is recommended by United States Pharmacopoeia, 2009 for testing the presence of Salmonella in dietary supplements.
• HEA is recommended in the testing of Salmonella in food samples by various standards.
• HEA is used as a plating medium to recover gastrointestinal pathogens, such as Salmonella and Shigella, from food, water, and fecal samples suspected of containing these organisms.
• HEA is used as both a direct and indirect plating medium for fecal specimens to enhance the recovery of species of Salmonella and Shigella from heavy numbers of mixed normal fecal flora.
• HEA may also be used to subculture the overnight growth from enrichment broths (such as gram-negative broth or selenite broth) inoculated with fecal specimens suspected of containing low numbers of Salmonella.

Hektoen Enteric Agar is a useful medium for the isolation and differentiation of Salmonella and Shigella species from enteric specimens, but it also has some limitations that should be considered:

• A single medium is only rarely able to recover all pathogens contained in a specimen. Therefore, additional media for the isolation of Salmonella and/or Shigella and possibly for other enteric pathogens must be inoculated with the specimen.
• Proteus mirabilis colonies may resemble Salmonella on this medium, as they may produce blue-green colonies with black centers due to hydrogen sulfide production. Biochemical and serological confirmation is necessary to differentiate them.
• Some Shigella strains may require a 42-48 h incubation to produce visible colonies on this medium. The color of Shigella colonies may also fade to the periphery, making them less distinct from the background.
• While some diagnostic tests can be performed directly on this medium, it is not possible to identify all organisms based on their colony morphology alone. Biochemical, immunological, molecular, or mass spectrometry testing should be performed on colonies from pure culture for complete identification.
• Colony morphology is only presumptive identification and hence biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

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