HiCrome™ Coliform Agar (CCA) w/1% Agar

Intended Use

Recommended for detection of Escherichia coli and coliforms in water samples. The composition and performance criteria of this medium are as per the specifications laid down in ISO 9308-1:2014.

Composition

M1991AI - HiCrome® Chromogenic Coliform Agar (CCA) w/1% Agar

Final pH (at 25°C): 6.8±0.2

**Formula adjusted, standardized to suit performance parameters

# Enzymatic digest of casein

Directions

Suspend 25.92 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified/ distilled water. Heat to boiling to dissolve the medium completely. DO NOT AUTOCLAVE. DO NOT OVERHEAT. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

HiCrome® Chromogenic Agar is a selective medium recommended by ISO for enumeration of Escherichia coli and coliform bacteria (1). The medium contains three chromogenic substrates. The enzyme β-D-galactosidase produced by coliforms cleaves 6-chloro-3-indoxy l-β-D-galactopyranoside to form pink to red coloured colonies (1). The enzyme β-D glucuronidase produced by E.coli, cleaves 5-bromo-4chloro-3-indoxy l-β-D-glucuronic acid (1) Colonies of E.coli give dark blue to violet coloured colonies due to cleavage of both the chromogens. The presence of the third chromogen IPTG enhances the colour reaction. Addition of L-Tryptophan improves the indole reaction thereby increasing the detection reliability.

Tryptone, 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 sub-lethally injured coliforms to recover and grow rapidly. Tergitol-7 inhibits gram-positive as well as some gram-negative bacteria other than coliforms (1). 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

Water samples

Specimen Collection and Handling

Processing (1)

Filtration: Filter 100ml of the sample using membrane filter. The minimum volume for filtration should be 10ml (or dilution) so that to ensure even distribution of the bacteria on the membrane filter.

Incubation and differentiation: After filtration place the membrane filter on HiCrome® Chromogenic Coliform agar (CCA Agar), ensuring that no air is trapped underneath, invert petri dish and incubate at 36°C ± 2 for 21 ± 3 hours. Examine the colony on membrane filters for color change.

Confirmation: Biochemical and serological tests are performed for confirmation.

Warning and Precautions

Read the label before opening the container. The media should be handled by trained personnel only. 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.

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 1.0% Agar gel.

Colour and Clarity of prepared medium: Light yellow coloured opalescent gel forms in Petri plates

Reaction: Reaction of 2.59% w/v aqueous solution at 25°C. pH : 6.8±0.2

pH: 6.60-7.00

Cultural Response: Cultural characteristics observed after an incubation at 36°C ± 2 for 21 ± 3 hours. Recovery is considered on TSA.

Key * : Corresponding WDCM numbers # : either on plate or membrane ## Formerly known as Enterobacter aerogens

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 (2,3).

Reference

1. International Organization for Standardization. Water quality: Enumeration of E.coli and coliform bacteria. Part I- Membrane filtration methods for bacteria with low bacterial background flora. ISO 9308-1:2014.
2. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
3. 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.