HiCrome™ M-Coliconfirm Agar Base

Intended use

Recommended for the selective isolation and identification of E.coli and coliforms from water samples using membrane filtration technique.

Composition**

Final pH ( at 25°C): 7.2±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 33.97 gram in 1000 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. Aseptically add the rehydrated contents of one vial of TTC Solution, 1% (FD057). Mix well and pour into sterile Petri plates.

Principle And Interpretation

It is possible to remove bacteria from fluids by passing them through filters with such small pore size that bacteria are arrested. This filtration technique enables fairly large volumes of water to pass rapidly under pressure, but prevents the passage of any bacteria present. These nutrients are retained on the surface of the membrane which is then brought into contact with suitable liquid nutrients. These diffuse upwards through the pores thereby inducing the organisms to grow as surface colonies which can be counted (1).

Escherichia coli, a member of the family Enterobacteriaceae is a normal flora of the intestinal tract of humans and a variety of animals. Although most of the E.coli does not cause gastrointestinal illness, certain groups of E.coli can cause life threatening diarrhoea and severe disability. For water testing, detection of E.coli and total coliforms is very important. There are various media available for the detection of E.coli and total coliforms.

Tryptone, growth factors provide nitrogenous and carbonaceous compound, long chain amino acids, vitamins and other essential nutrients. Phosphates buffers the medium well. Selective agents inhibit gram positive bacteria and other accompanying bacteria. The chromogenic mix incorporated in the medium aids in the detection of β-glucuronidase positive organisms which gives blue coloured organisms. Coliforms produce red coloured colonies other than Escherichia coli as they reduce TTC (2,3,5-triphenyl tetrazolium chloride). Thus, the resulting colour distinction allows simple interpretation of test without further confirmation.

Type of specimen

Water samples.

Specimen Collection and Handling

For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (2). 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. This medium is based on the principle of detection of β-glucuronidase enzyme. 97% of the E.coli are β-glucuronidase positive and will result in blue colour.
2. Certain other Salmonella and Shigella species may also exhibit β glucuronidase activity.
3. Final identification must be carried out by biochemical tests.

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

Reaction: Reaction of 3.4% w/v aqueous solution at 25°C. pH : 7.2±0.2

pH: 7.00-7.40

Cultural Response

Cultural characteristics observed on membrane filter or plate after an incubation at 35-37°C for 18-24 hours.

Key: (#) Formerly 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 (3,4).

Reference

1. Cruickshank R., Duguid J. P., Marmion B. P., Swain R. H. A., (Eds.), Medical Microbiology, 1975, 12th Ed. Vol. II, Churchill Livingstone
2. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
4. 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.