Hektoen Enteric Agar

Intended Use

Hektoen Enteric (HE) Agar is a modrately selective medium used in qualitative procedures for the isolation and cultivation of gram-negative enteric microorganisms, especially Shigella, from a variety of clinical and nonclinical specimens.

Summary and Explanation

Through the years many media have been devised for the isolation of enteric pathogens. These various formulations have differed in their degree of selectivity for the pathogenic species. Some were designed to isolate and differentiate Shigella species whereas others were formulated for the selective isolation of the salmonellae. Media that isolated a broader spectrum of enteric pathogens were less inhibitory to members of the nonpathogenic intestinal flora.
Hektoen Enteric Agar was developed in 1967 by King and Metzger of the Hektoen Institute in order to increase the frequencies of isolation of Shigella and Salmonella organisms when compared with their recovery on other media frequently utilized in clinical laboratories at that time.^1-3 This medium is considered to be moderately selective, and is particularly useful in the isolation of Shigella species. The present formulation differs from that of the original in that sodium desoxycholate has been eliminated and the concentration of bile salts is reduced. Additionally, the peptone concentrations have been increased in order to offset the inhibitory effects of the bile salts.⁴
HE Agar is currently recommended as one of several plating media for the culture of Enterobacteriaceae from stool specimens.⁵ Foods containing poultry, eggs or dairy products are the most frequent vehicles for foodborne salmonellosis, and a variety of procedures have been developed using Hektoen Enteric Agar as part of the multi-step procedure to isolate Salmonella.^6-9

Principles of the Procedure

The selective nature of Hektoen Enteric Agar is due to the incorporation of bile salts in the formulation. These substances inhibit gram-positive organisms but also can be toxic for some gram-negative strains.
This medium contains three carbohydrates, lactose, sucrose (saccharose) and salicin, for optimal differentiation of enteric pathogens by the color of the colonies and of the medium adjacent to the colonies. The lactose concentration is higher than in many other media used for enterics in order to aid in the visualization of enteric pathogens and minimize the problem of delayed lactose fermentation. Ferric ammonium citrate and sodium thiosulfate in the medium enable the detection of hydrogen sulfide production, thereby aiding in the differentiation process due to the production of blackcentered colonies. The indicator system, consisting of acid fuchsin and bromthymol blue, has a lower toxicity than that of many other enteric media, resulting in improved recovery of enteric pathogens.

User Quality Control

Identity Specifications
Hektoen Enteric Agar
Dehydrated Appearance: Light beige, may have a slight green cast, freeflowing,
                                    homogeneous.
Solution:                       7.6% solution, soluble in purified water upon
                                    boiling. Solution is brown with greenish cast,
                                    slightly opalescent.
Prepared Appearance:     Green with yellowish cast, slightly opalescent.
Reaction of 7.6%
Solution at 25°C:           pH 7.5 ± 0.2
Cultural Response
Hektoen Enteric Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-24 hours.

Formula

Difco™ Hektoen Enteric Agar
Approximate Formula* Per Liter
Proteose Peptone....................................................... 12.0 g
Yeast Extract................................................................ 3.0 g
Bile Salts No. 3............................................................. 9.0 g
Lactose...................................................................... 12.0 g
Saccharose................................................................. 12.0 g
Salicin......................................................................... 2.0 g
Sodium Chloride........................................................... 5.0 g
Sodium Thiosulfate....................................................... 5.0 g
Ferric Ammonium Citrate............................................... 1.5 g
Agar.......................................................................... 14.0 g
Bromthymol Blue....................................................... 65.0 mg
Acid Fuchsin................................................................. 0.1 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 76 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat to boiling with frequent agitation to dissolve completely. Do not overheat. DO NOT AUTOCLAVE.
3. Cool to 45-50°C and use immediately.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens. A nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen.
Incubate plates, protected from light, at 35 ± 2°C for 18-24 hours.

Expected Results

After incubation most plates will show an area of confluent growth. Because the streaking procedure is, in effect, a “dilution” technique, diminishing numbers of microorganisms are deposited on the streaked areas. Consequently, one or more of these areas should exhibit isolated colonies of the organisms contained in the specimen. Better isolation is obtained due to the inhibitory action of the medium.

Limitations of the Procedure

Proteus species may resemble salmonellae or shigellae. Further testing should be conducted to confirm the presumptive identification of organisms isolated on this medium.

*Store at 2-8°C.

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