Hektoen Enteric Agar, w/ 1. Agar

Intended Use

Recommended for isolation of Shigella and Salmonella species from food samples. The composition and performance criteria of this medium is in accordance with FDA BAM, ISO 21567:2004, APHA, ISO 6579-1:2017(E).

Composition**

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 75.67 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE OR OVERHEAT. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

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 (1, 2, 3, 4). Hektoen Enteric Agar w/ 1.4% agar is used for the differential isolation of Salmonella and Shigella from food specimens in accordance with FDA BAM, Chapter 5 Revision 2021(1). This medium is recommended by APHA, ISO 21567, ISO 6579-1 (2,5,6).

The increased concentration of carbohydrates and peptone helps to reduce the inhibitory effect of bile salts and indicators thus allowing the good growth of Salmonella and Shigella species while inhibiting the normal intestinal flora. The medium contains three carbohydrates i.e lactose, sucrose and salicin for differentiation of enteric pathogens. The higher lactose concentration aids in the visualization of enteric pathogens and minimizes the problem of delayed lactose fermentation. Salicin is fermented by many coliforms including those that do not ferment lactose and sucrose. Combination of ferric ammonium citrate and sodium thiosulphate in the medium enables the detection of hydrogen sulfide production indicated by the black coloured colonies. The indicator system, consisting of acid fuchsin and bromothymol blue, has lower toxicity as compared to other enteric media, resulting in improved recovery of enteric pathogens. Low concentration of bile salts allows the growth of Shigella and Salmonellae. Higher concentration of peptone minimizes the inhibitory effects of the bile salts (7,8).

Type of specimen

Foods samples

Specimen Collection and Handling

FDA BAM:
Grow suspected sample in TetraThionate broth (M032F) overnight. Mix (vortex, if tube) and streak 3 mm loopful (10 µl) incubated TT broth on Hektoen Enteric Agar w/ 1.4% agar. Appearance of blue-green to blue colonies with or without black centers indicates the presence of Salmonella. Many cultures of Salmonella may produce colonies with large, glossy black centers or may appear as almost completely black colonies.

Processing: ISO 21567:2004 (5), APHA (2)

Enrichment:

Add x g or x ml of test portion to 9x ml of Shigella broth containing 0.5mcg of novobiocin to make a 1 in 10 dilution of the test sample. Incubate Shigella broth under anaerobic conditions with at 41.5°1°C for 16h to 20h.

Plating out:

Using the cultures obtained from enrichment, inoculate the surface of Hektoen enteric (HE) agar. Incubate at 37±1°C for 20-24h. Examine typical Shigella colonies and carry out biochemical tests for confirmation.

Processing: ISO 6579-1:2017(6)

Pre-enrichment:

Samples (25 grams in 225 ml) are pre-enriched in Buffered Peptone Water (M14941) and incubated at 34°C to 38°C for 18 h ± 2 hours.

Selective enrichment:

0.1 ml of pre-enriched sample is inoculated in 10 ml RVS Broth (M1448I) or MSRV Agar (M1428) and incubated at 41.5 ± 1°C for 24 ± 3 hours and 1 ml of culture is inoculated in MKTTn broth (M14961) and incubated at 36±2°C for 24 ± 3 hours.

Isolation:

The culture thus obtained is then plated on XLD Agar, Modified (M0311) and incubated at 36° 2°C for 24 ± 3 hours. Simultaneously plating on second isolation agar is carried out like Hektoen Enteric Agar, Bismuth Sulphite Agar, Brilliant green Agar

Confirmation:

Biochemical and serological tests are performed for confirmation.

Warning and Precautions

Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Further incubation will improve differentiation between Salmonella and Shigella. Proteus species may resemble Salmonella or Shigella; hence further testing must be carried out for confirmation.
2. Since the medium is selective it must be used in conjunction with other media.
3. Certain Salmonella species are lactose positive or H2S negative which are not identified on this medium. Further biochemical and serological tests or chromogenic medium (M2116) may be used for complete identification and confirmation.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance
Cream to yellow with tancast homogeneous free flowing powder

Gelling
Firm, comparable with 1.4% Agar gel

Colour and Clarity of prepared medium
Green coloured, clear to slightly opalescent gel forms in Petri plates

Reaction
Reaction of 7.57% w/v aqueous solution at 25°C. pH : 7.5±0.2

pH
7.30-7.70

Cultural Response
Cultural characteristics observed after an incubation at 35°C for 22-26 hours.

Key: (*) Corresponding WDCM numbers. (#) Formerly known as Enterobacter aerogenes

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (9,10).

Reference

1. FDA Bacteriological Analytical Manual, 2005, 18th Ed., AOAC, Washington, DC.
2. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
3. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
4. Williams, (Ed.), 2005, Official Methods of Analysis of the Association of Official Analytical Chemists, 19th Ed.AOAC, Washington, D.C.
5. ISO 21567:2004, Microbiology of food and animal feeding stuffs-Horizontal method for the detection of Shigella spp.
6. ISO 6579-1:2017(E), Microbiolgy of the food chain-Horizontal method for the detection, enumeration and serotyping of Salmonella.
7. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I. Williams & Wilkins, Baltimore, Md.
8. Murray P. R., Baron E. J., Jorgensen J. H., Pfaller M. A., Yolken R.H., (Eds.), 8th Ed., 2003, Manual of Clinical Microbiology, ASM, Washington, D.C.
9. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
10. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.