HiCrome™ ECC Selective Agar Base, Modified

Intended use

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

Composition**

Final pH ( at 25°C): 7.0±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 39.30 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. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

HiCrome® ECC Selective Agar, Modified 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 ß-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 give 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 provide nitrogenous and carbonaceous compounds, long chain amino acids and other essential growth nutrients for the organisms. Sodium pyruvate serves as a growth factor and sorbitol is the fermentable carbohydrate. Phosphates buffer the medium. The media formulation helps even sub lethally injured coliforms to recover and grow rapidly. Sodium lauryl sulphate inhibits gram-positive bacteria.

Type of specimen

Food and dairy samples; Water samples.

Specimen Collection and Handling

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (5,6,7). For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (8). 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. Certain strains of E.coli may exhibit salmon colour if they lack the ß-glucuronidase enzyme.
2. The intensity of the colour produced by E.coli and coliforms depends on the amount of enzyme produced by the organism.
3. Certain species of Shigella and Salmonella are ß-glucuronidase positive which may appear as light blue.

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 yellow coloured, clear to slightly opalescent gel forms in Petri plates

Reaction: Reaction of 3.93% w/v aqueous solution at 25°C. pH : 7.00±0.2

pH: 6.80-7.20

Cultural Response

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

Key : (#) Formely known as Enterobacter aerogenes, (*) Corresponding WDCM numbers

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 (9,10).

Reference

1. Kilian M. and Bülow P., 1976, Acta. Pathol. Microbiol. Scand Sect. B, 84:245.
2. LeMinor L. and Hamida F., 1962, Ann. Inst. Pasteur 102:267.
3. Manafi M. and Kneifel W., 1989, Zentralbl. Hyg., 189:225.
4. Frampton E.W., Restaino L. and Blaszko N., 1988, J.Food Prof., 51:402.
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. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
7. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
8. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
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.