HiCrome™ EC O157 : H7 Selective Agar Base, Modified

Intended Use

Recommended for selective isolation and easy detection of Escherichia coli O157:H7 from food samples.

Composition**

Final pH (at 25°C): 6.8±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 31.85 gram in 990 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Add rehydrated contents of 1 vial of NP Selective Supplement (FD187) aseptically. Mix well and pour into sterile Petri plates.

Principle And Interpretation

Enterohaemorragic E.coli strains are also termed as verocytotoxin-producing E.coli (VTEC/ EHEC). Although many different serotypes of Escherichia coli are known to produce verocytotoxin (1) those of Escherichia coli O157:H7 and O157:H are so far the common types causing human infections. O157 VTEC strains have several unusual biochemical characters that are exploited in methods for their laboratory identification. They belong to the minority of E.coli that are ß-glucuronidase negative and do not ferment sorbitol or rhamnose within 24 hours. These can be isolated from faecal specimens by plating on media containing D-sorbitol instead of lactose.

HiCrome™ EC O157:H7 Agar is based on the formulation described by Rappaport and Henigh (2). The medium contains sorbitol as fermentable carbohydrate and chromogenic mixture instead of lactose and indicator dyes respectively. The chromogenic substrate is specifically and selectively cleaved by Escherichia coli O157:H7 resulting in a dark purple to magenta coloured moiety. E.coli forms bluish green coloured colonies.

Tryptone and yeast extract provides carbonaceous and nitrogenous compounds, long chain amino acids, vitamins and growth nutrients. Sodium chloride maintains osmotic equilibrium. Addition of NP Selective Supplement (FD187) makes the medium selective (3). Potassium tellurite selectively inhibits Aeromonas and Providencia species. Novobiocin inhibits gram-positive bacteria. Sodium lauryl sulphate helps to inhibit the accompanying gram-positive flora.

Type of specimen

Food samples.

Specimen Collection and Handling

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (4). After use, contaminated materials must be sterilized by autoclaving before discarding.

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. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user’s unique requirement.
3. Further biochemical and serological test are necessary for 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 homogeneous free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

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

Reaction: Reaction of 3.18% w/v aqueous solution at 25°C. pH : 6.8±0.2

pH: 6.60-7.00

Cultural Response

Cultural characteristics observed with added NP Selective Supplement (FD187) after an incubation at 35-37°C for 18-24 hours.

Key : (*) Corresponding WDCM number and **Formerly known as Bacillus subtilis subsp. spizizenii

Storage and Shelf Life

Store between 15-25°C in a tightly closed container and the prepared medium at 2-8°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 sample must be decontaminated and disposed of in accordance with current laboratory techniques (5,6).

Reference

1. Smith and Scottland, 1988, J. Med. Microbiol., 26:77-85.
2. Rappaport F. and Henigh E., 1952, J. Clin. Pathol. 5:361.
3. Zadik P. M., Cahpman P. A. and Siddons C. A., 1993, J.Med. Microbiol., 39, 155-158.
4. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
6. 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.