HEKTOEN ENTERIC AGAR

INTENDED USE

Hektoen Enteric Agar is a selective medium for the isolation and differentiation of pathogenic enterobacteria from biological samples of animal origin, water samples, dairy products and other food products. The medium is particularly suited for the culture of Shigella, and prevents invasion by Proteus.

HISTORY

Hektoen Enteric Agar was formulated in 1967 by King and Metzger at the Hektoen Institute in order to increase the isolation frequency of Shigella and Salmonella species in comparison to that obtained with other selective isolation media. Although this medium enabled a wide variety of pathogenic enterobacteria to be isolated, it was less inhibitory towards non-pathogenic enteric bacteria. The current formulation differs from the original by the suppression of desoxycholate and the reduced concentration of bile salts. In parallel, the concentration of peptones was increased to offset the inhibitory effect of bile salts.

PRINCIPLES

- Gram-positive flora are inhibited by bile salts which can also slightly inhibit the growth of a few Gram-negative microorganisms. - The medium contains three carbohydrates: lactose, sucrose and salicin. The high lactose concentration favors the visualization of enterobacteria by avoiding the problem of late fermentations. The other carbohydrates were added to insure a higher degree of differentiation and to reduce the toxicity caused by colored indicators so as to obtain excellent recovery of Shigella.

- In the presence of sodium thiosulfate, H2S producing bacteria reduce ferric ammonium citrate and are detected by darkening due to the production of iron sulfide at the center of the colonies. - The color indicator system, composed of bromthymol blue and acid fuchsin, yields orange yellow colonies for lactose-positive enterobacteria and blue-green colonies in the case of lactose-negative strains.

PREPARATION

- Suspend 75.1 g of dehydrated medium (BK067) in 1 liter of distilled or deionized water. - Slowly bring to boiling, stirring with constant agitation until complete dissolution. - Continue boiling for 2 minutes. - Do not autoclave.

NOTE : Incomplete agar melting during preparation will invariably lead to significant inconsistency in the gel strength of the solidified agar, after sterilization and cooling.

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INSTRUCTIONS FOR USE

- Cool and maintain the medium at 44-47°C. - Pour into sterile Petri dishes. - Let solidify on a cold surface. - Dry in an incubator with the covers partially removed. - Inoculate onto plates prepared as above by streaking enrichment media used for the detection of Salmonella. - Simultaneously inoculate another selective medium such as COMPASS Salmonella agar (BM066). - Incubate at 37°C for 24 and 48 hours.

RESULTS

The principle for reading the results is based on the possible fermentation of the three carbohydrates in the medium (lactose, sucrose, salicin). Strains fermenting at least one of them form salmon colored colonies, others forming blue or green colonies. In the presence of sodium thiosulfate, hydrogen sulfide-producing bacteria give colonies with a black center after reacting with ferric citrate.

The colonies have the following appearance :

Characteristics Microorganisms

Escherichia coli, Citrobacter, Klebsiella, Salmon-yellow colonies Enterobacter, Arizona, Serratia

Salmon-yellow colonies with a black center Proteus vulgarism

Green colonies with a black center Proteus mirabilis, Salmonella

Shigella, Salmonella, Providencia, Green or bluish colonies Proteus morganii, Proteus rettgeri

Small blue or brownish colonies Pseudomonas (oxidase positive)

TYPICAL COMPOSITION (can be adjusted to obtain optimal performance)

For 1 liter of medium : - Peptic digest of meat ...... 12.0 g - Yeast extract ...... 3.0 g - Lactose ...... 12.0 g - Sucrose...... 12.0 g - Salicin ...... 2.0 g - Bile salts...... 9.0 g - Sodium chloride ...... 5.0 g - Sodium thiosulfate ...... 5.0 g - Ferric ammonium citrate ...... 1.5 g - Bromthymol blue ...... 65.0 mg - Acid fuchsin...... 40.0 mg - Bacteriological agar ...... 13.5 g pH of the ready-to-use medium at 25°C : 7.6 ± 0.2.

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QUALITY CONTROL

- Dehydrated medium : beige powder, free-flowing and homogeneous. - Prepared medium : dark green agar. - Typical culture response after 48 hours of incubation at 37°C (qualitative method of inoculation):

Microorganisms Growth Characteristics

Salmonella Typhimurium ATCC® 14028 good, score 2 green colonies with a black center Salmonella Enteritidis CIP 82.97 good, score 2 green colonies with a black center Shigella flexneri ATCC 29903 good, score 2 green colonies Escherichia coli ATCC 25922 partially inhibited, salmon-orange colonies score 0-1 Enterococcus faecalis ATCC 29212 inhibited, score 0 Staphylococcus aureus ATCC 25923 inhibited, score 0

STORAGE / SHELF LIFE

Dehydrated medium : 2-30°C. - The expiration date is indicated on the label. Prepared medium (benchmark value*) : - Media in plates : 8 days at 2-8°C.

PACKAGING Code

Dehydrated medium : - 500 g bottle BK067HA

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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com

PHOTO SUPPORT :

Product reference : BK067HA

Media used for : Isolation and differentiation of pathogenic enterobacteria.

Salmonella Typhimurium

Typical aspect

Green colonies with black center, becoming nearly all black after sufficient incubation.

Hektoen Enteric Agar Ref : BK067HA

Incubation 24-48 hours at 37°C (plating)

Salmonella Typhimurium : Green colonies with black center, becoming nearly all black after sufficient incubation. (Reduction of Ferric ammonium citrate, absence of lactose fermentation)

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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com

BIBLIOGRAPHY

KING, S., and METZGER, W.I.. 1968. A New Plating Medium for the Isolation of Enteric Pathogens: I. Hektoen Enteric Agar. Applied and Environmental Microbiology, 16 : 577-578.

KING, S., and METZGER, W.I.. 1968. A New Plating Medium for the Isolation of Enteric Pathogens: II.Comparison of Hektoen Enteric Agar with SS and EMB Agar. Appl. Microbiol., 16 : 579-581.

TAYLOR, W.I., and SCHELHART, D.. 1971. Isolation of Shigellae. VIII. Comparison of Xylose Lysine Deoxycholate Agar, Hektoen Enteric Agar, Salmonella-Shigella Agar and Eosin Methylene Blue Agar with stool specimens. Applied Microbiology, 21 : 32-37.

BISCIELLO, N.B., and SCHRADE, T.P.. 1974. Evaluation of Hektoen enteric Agar for the detection of Salmonella in foods and feeds. Journal of A.O.A.C., 57 : 992-996.

XP CEN ISO/TS 11133-2 (V 08-104-2). Janvier 2004. Microbiologie des aliments. Guide pour la préparation et la production des milieux de culture. Partie 2 : Guide général pour les essais de performance des milieux de culture.

NF EN ISO 21567 (V 08-411). Mars 2005. Microbiologie des aliments. Méthode horizontale pour la recherche de Shigella spp.

NF EN ISO 6579 (V 08-013). Mars 2007. Microbiologie des aliments. Méthode horizontale pour la recherche des Salmonella spp.

NF U 47-102. Janvier 2008. Méthodes d’analyse en santé animale. Isolement et identification de tout sérovar ou de sérovar(s) spécifié(s) de salmonelles chez les mammifères.

*Benchmark value refers to the expected shelf life when prepared under standard laboratory conditions following manufacturer’s instructions. It is provided as a guide only and no warranty, implied or otherwise is associated with this information.

The information provided on the package take precedence over the formulations or instructions described in this document. The information and specifications contained in this technical data sheet date from 2010-01-11. They are susceptible to modification at any time, without warning. Code document : BK067/A/2003-01 : 7. 5/5

Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com