HEKTOEN ENTERIC (HE) AGAR

Cat. no. G63 Hektoen Enteric (HE) Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. J139 Hektoen Enteric (HE) Agar / SS Agar, 15x100mm Biplate,
10ml/10ml
10 plates/bag

INTENDED USE

Hardy Diagnostics Hektoen Enteric (HE) Agar is a selective and differential medium used for the isolation and differentiation of gram-negative enteric pathogens.

SUMMARY

King and Metzger developed HE Agar in an effort to increase the recovery of Salmonella and Shigella species over the previously formulated Salmonella-Shigella (SS) Agar.(7) This medium is particularly useful in the isolation of Shigella species.

The present formulation of HE Agar incorporates larger amounts of peptone in order to offset the inhibitory effect of bile salts. Also, sodium deoxycholate has been eliminated and the amount of bile salts reduced. Bile salts allow for the selective nature of HE Agar by inhibiting gram-positive organisms. Bile salts can also be toxic for some gram-negative strains. Salicin, sucrose, and lactose are the fermentable carbohydrates present. They provide optimal differentiation of enteric pathogens. Lactose and sucrose, in increased concentration, aid in the differentiation of enteric pathogens from slow lactose fermenters. Bromothymol blue and acid fuchsin (Andrade's) are added as acid-base indicators. The addition of ferric ammonium citrate and sodium thiosulfate enable the detection of H2S, noted by the production of black centered colonies. Sodium thiosulfate serves as the sulfur source while ferric ammonium citrate serves as the indicator.

HE Agar is currently recommended as one of several plating media for the culture of Enterobacteriaceae from stool specimens. This is due to its moderately selective nature as well as for its differentiation property.

FORMULA

Ingredients per liter of deionized water:*

Peptic Digest of Animal Tissue 12.0gm
Lactose 12.0gm
Sucrose 12.0gm
Bile Salts 9.0gm
Sodium Chloride 5.0gm
Sodium Thiosulfate 5.0gm
Yeast Extract 3.0gm
Salicin 2.0gm
Ferric Ammonium Citrate 1.5gm
Acid Fuchsin 0.1gm
Bromothymol Blue 0.064gm
Agar 13.5gm

Final pH 7.7 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. Products should not be used if there are any signs of contamination, deterioration, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation. Consult listed references for information on specimen collection.(1-6)

Method of Use: Medium should be brought to room temperature prior to inoculation. If material is being cultured directly from a swab, roll the swab over a small area of the surface edge. Streak the inoculum to obtain isolated colonies. A nonselective medium should also be inoculated. This increases the chance of recovery when the population of gram-negative organisms is low. It also provides indication of other organisms present in the specimen. Incubate plates for 18-24 hours at 35ºC. protected from light. If negative after 24 hours, reincubate for an additional 24 hours.

INTERPRETATION OF RESULTS

HE Agar is examined for typical colonial morphology after incubation. Fermentation of lactose, sucrose or salicin results in the production of acid which give rise to yellow-orange to salmon colored colonies. Colonies of Salmonella and Shigella spp. are green to bluish-green in color. Salmonella spp. that produce H2S appear as blue-green colonies with black centers. H2S producers form black-centered colonies in the presence of ferric ammonium citrate and sodium thiosulfate.

Consult listed references for further interpretation of growth and other identification tests to identify growth of organisms in this medium.(1-6)

LIMITATIONS

Cultural growth may be delayed or inhibited by the presence of antimicrobial agents in the specimen. Additionally, antimicrobics may alter the characteristic appearance of the organism on the medium.

It is recommended that selective enrichment broths (GN or Selenite Cystine) be used in conjunction with selective plating media for optimal isolation of enteric pathogens.

Bile salts in the medium may crystallize over time. They appear as small spider-like puff-balls within the medium and do not affect performance.

The color of HE Agar may shift from green to brownish during shipment. This is normal and should not affect the performance of the medium. Placing plates in refrigerated conditions (2-8°C) upon receipt overnight will result in the medium returning to a normal green appearance.

Colonies of Proteus, which may or may not be inhibited, may resemble Salmonella or Shigella.

The recovery of most Shigella and many Salmonella spp. from unpreserved stool specimens may be jeopardized if processing delays exceed 2-3 hours.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC® 14028
A 24hr 35°C Aerobic Growth; blue to blue-green colonies with black centers
Shigella flexneri
ATCC® 12022
A 24hr 35°C Aerobic Growth; green to blue-green colonies
Enterococcus faecalis
ATCC® 29212
B 24hr 35°C Aerobic Inhibition; may be slight growth of yellow colonies
Escherichia coli
ATCC® 25922
B 24hr 35°C Aerobic Partial inhibition; may be slight growth of yellow to salmon colored colonies

User Quality Control

PHYSICAL APPEARANCE

Hektoen Enteric (HE) Agar should appear clear, and green in color.

S. flexneri growing on HE Agar

Shigella flexneri (ATCC® 12022) colonies growing on Hektoen Enteric Agar (Cat. no. G63). Incubated aerobically for 24 hours at 35ºC.

S. typhimurium growing on HE Agar

Salmonella enterica (ATCC® 14028) colonies growing on Hektoen Enteric Agar (Cat no. G63). Incubated aerobically for 24 hours at 35ºC.

E. faecalis inhibited on HE Agar

Enterococcus faecalis (ATCC® 29212) growth inhibited on Hektoen Enteric Agar (Cat no. G63). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

3. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.

6. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

7. King, S., and Metzger, W.I. 1968. Applied Microbiology ; 16:577-579.

8. Centers for Medicare & Medicaid Services (CMS). Individualized Quality Control Plan (IQCP).


ATCC is a registered trademark of the American Type Culture Collection.

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