HEKTOEN 54386 63894 64284 SELECTIVE MEDIUM for the ISOLATION of SALMONELLA Spp

HEKTOEN 54386 63894 64284 SELECTIVE MEDIUM FOR THE ISOLATION OF SALMONELLA spp. AND SHIGELLA spp.

2014/04

1- INTENDED USE Hektoen agar is a selective medium for the isolation and differentiation of enteropathogenic Gram (-) bacilli, particularly Salmonella spp. and Shigella spp.

2- PRINCIPLE The selectivity of this medium is based on the presence of bile salts, bromothymol blue and acid fuchsin (which inhibit the growth of Gram (+) bacteria, E. coli and, to a lesser degree, Proteus and Citrobacter). The nutrient properties of peptones and sugars neutralize the inhibitory effect of bile salts on certain fastidious bacteria and allow good growth of Shigella. Differentiation of Enterobacteriaceae is based on their ability to ferment various sugars: lactose, sucrose, salicin. The reaction is visualized by two indicators: bromothymol blue, which turns yellow in the presence of acid, and fuchsin which turns red in the presence of aldehyde. An additional differentiation based on the production of hydrogen sulphide is also possible due to the presence of sodium thiosulphate and iron citrate. A positive test consists of colonies with a black centre due to the formation of iron sulphide.

3- HOW SUPPLIED  Ready to use medium: - box of 20 Petri dishes (90 mm) (HEKT) code 63894  Ready to use medium (to be dispensed) - 6 x 100 ml bottles (HEKT) code 54386  Dehydrated medium - Bottle of 500 g code 64284

4- THEORETICAL COMPOSITION (g/l of distilled water) Hektoen medium is prepared according to the formula described by S. Kings et al. (1). Proteose peptone 12 Yeast extract 3 Sodium chloride 5 Sodium thiosulphate 5 Bile salts 9 Ammoniacal iron citrate 1.5 Salicin 2 Lactose 12 Sucrose 12 Acid fuchsin 0.1 Bromothymol blue 0.065 Agar 14 Final pH 7.5  0.2

Preparation of the medium: Homogenize the powder contained in the bottle. Add 75 grams of dehydrated medium to one litre of freshly distilled water. Heat gently, shaking frequently, then heat to boiling until complete dissolution. Allow to cool to 50°C. Precaution for use: DO NOT AUTOCLAVE.

5- STORAGE  Ready to use medium: at +2-8°C.  Ready to use medium (to be dispensed): at +2-8°C.  Dehydrated medium: tightly sealed bottle in a dry place at +15-25°C. The expiry date and batch number are indicated on the packaging.

6- INSTRUCTIONS Material:  Material provided: Hektoen medium.

Inoculation: Inoculate by streaking directly from the specimen to be examined (stool suspension or swab). Refer to current recommendations for storage of biological specimens (2).

Incubation: Incubate for 24-48 hours at 37°C.

Reading: Fermentation of at least one of the sugars results in a salmon-pink colour of the colonies. The absence of fermentation results in a blue or green colour of the colonies. The production of hydrogen sulphide (H2S) is characterized by colonies with a black centre.  Salmon-pink colonies: Escherichia, Levinea, Citrobacter, Enterobacter, Serratia, Yersinia  Salmon-pink colonies with a black centre: Proteus vulgaris.  Blue-green or green colonies: suspicion of Shigella or Salmonella to be differentiated from Proteus morganii or Proteus rettgeri of Providencia, Hafnia, Levinea, Plesiomonas, Pseudomonas (Pseudomonas can be differentiated by the oxidase test).  Blue-green or green colonies with a black centre: suspicion of Salmonella or Proteus mirabilis (P. mirabilis can be differentiated by the urease test).

7- PERFORMANCE/QUALITY CONTROL OF THE TEST  Appearance of the ready to use medium: clear green agar.  Appearance of the dehydrated medium: pink powder.  The growth performances of Hektoen medium are verified with the following strains:

STRAINS CULTURE RESULT AFTER 24 to 48 hours at 37°C Escherichia coli ATCC 25922 Salmon-pink colonies, partial inhibition Enterobacter aerogens ATCC 13048 Salmon-pink colonies Shigella flexneri ATCC 12022 Green colonies Shigella sonnei ATCC 25931 Green colonies Proteus mirabilis ATCC 25933 Blue-green colonies with a black centre Staphylococcus aureus ATCC 25923 Total inhibition

8- QUALITY CONTROL OF THE MANUFACTURER All manufactured reagents are prepared according to our Quality System, starting from reception of raw material to the final commercialization of the product. Each lot is submitted to quality control assessments and is only released to the market, after conforming to pre-defined acceptance criteria. The records relating to production and control of each single lot are kept within Bio-Rad.

9- LIMITS OF USE  Some strains of Salmonella arizonae, Shigella sonnei and Vibrio cholerae may form pink-yellow colonies.  Pseudomonas strains can form small brown and bluish colonies.  Complementary tests must be performed to identify the species of the strain isolated.

10- REFERENCES 1. Kings S. et al, 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: p.577-578. 2. Basic Laboratory Procedures in Clinical Bacteriology. World Health Organization. Geneva.1991. 1st edition. 3. TAYLOR W.I et al, 1971. Appl Microbiol. 21: p.32-37.

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