HiCrome™ ECC Selective Agar Base

Intended Use:

Recommended for detection of Escherichia coli and coliforms in water and food samples.

Composition**

Final pH (at 25°C): 6.8±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 26.48 gram in 1000 ml purified/ distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15lbs pressure (121°C) for 15 minutes. If desired, selective medium can be prepared by aseptically adding the rehydrated contents of 1 vial of ECC Selective Supplement (FD190) to previously cooled to 45-50°C sterile medium. Mix well and pour into sterile Petri plates. Medium may show haziness, but it does not affect the performance of the medium.

Principle And Interpretation

HiCrome™ ECC Selective Agar is a selective medium recommended for the simultaneous detection of Escherichia coli and total coliforms in water and food samples (1,2). The chromogenic mixture contains two chromogenic substrates. The enzyme B-D-galactosidase produced by coliforms cleaves one of the chromogen to form salmon to red coloured colonies (3). The enzyme ẞ-D-glucuronidase produced by E.coli, cleaves X-glucuronide, the other chromogen (4). Colonies of E.coli gives dark blue to violet coloured colonies due to cleavage of both the chromogens. Addition of L-Tryptophan improves the indole reaction, thereby increasing the detection reliability. Peptone special, sodium pyruvate and sorbitol provide nitrogenous substances, fermentable carbohydrate and other essential growth nutrients for the organisms. Phosphates buffer the medium. The media formulation helps even sublethally injured coliforms to recover and grow rapidly. Tergitol inhibits gram-positive as well as some gram-negative bacteria other than coliforms (5). Addition of ECC Selective Supplement (FD190) helps to inhibit the accompanying heterogeneous microflora.

The medium is inoculated either by pour plate technique or by spreading the sample on the surface of plated medium. Membrane filter technique can also be used. To confirm E. coli, add a drop of Kovac's reagent on the dark blue to violet colony. Formation of cherry red colour indicates a positive reaction.

Type of specimen

Food samples; Water samples

Specimen Collection and Handling

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (5).

For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (6).

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. B-glucuronidase is present in 97% of E.coli strains, however few E.coli may be negative. Some species may show poor growth due to nutritional variations.
2. 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

Light yellow to pink homogeneous free flowing powder

Gelling

Firm, comparable with 1.0% Agar gel

Colour and Clarity of prepared medium

Light pink coloured clear to slightly opalescent gel forms in Petri plates

Reaction

Reaction of 2.65% w/v aqueous solution at 25°C. pH: 7.4±0.2

pH

6.60-7.00

Cultural Response

Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.

Key : *Corresponding WDCM numbers

#- Formerly known as Enterobacter aerogenes.

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 (7,8).

Reference

1. Frampton E.W., Restaino L. and Blaszko N., 1988, J.Food Prof., 51:402.
2. M. and Bülow P., 1976, Acta. Pathol. Microbiol. Scand Sect. B, 84:245.
3. LeMinor L. and Hamida F., 1962, Ann. Inst. Pasteur > 102:267.
4. Manafi M. and Kneifel W., 1989, Zentralbl. Hyg., 189:225.
5. 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.
6. Lipps WC, Braun-Howland EB, Baxter TE, eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
7. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
8. 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.