Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate,
Thane(W)-400604, MH, India.
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Intended Use
This medium is prepared by completely replacing animal based peptones with vegetable peptones. Recommended for detection of Escherichia coli and coliforms in water samples.
Composition**
ISO 9308-1:2014 Specification -Chromogenic Coliform agar (CCA Agar)
| Ingredients | g/L |
|---|---|
| Enzymatic digest of casein | 1.000 |
| Yeast extract | 2.000 |
| Sodium chloride | 5.000 |
| Sodium dihydrogen phosphate, 2H2O | 2.200 |
| Disodium hydrogen phosphate | 2.700 |
| Sodium pyruvate | 1.000 |
| Sorbitol | 1.000 |
| Tryptophan | 1.000 |
| Tergitol-7 | 0.150 |
| 6-chloro-3-indoxyl β-D-galactopyranoside | 0.200 |
| 5-bromo-4-chloro-3-indoxyl- β-D-glucuronic acid cyclohexyl ammonium salt, monohydrate (X-beta-G-glucuronide CHX salt) | 0.100 |
| IPTG (Isopropyl-β-D-thiogalactopyranoside) | 0.100 |
| Agar | 9.0 to 18.00 |
| Final pH (at 25°C) | 6.8±0.2 |
MV1991I - HiCrome® Chromogenic Coliform HiVeg® agar
| Ingredients | g/L |
|---|---|
| HiVeg® hydrolysate # | 1.000 |
| Yeast extract | 2.000 |
| Sodium chloride | 5.000 |
| Sodium dihydrogen phosphate, 2H2O | 2.200 |
| Disodium hydrogen phosphate | 2.700 |
| Sodium pyruvate | 1.000 |
| Sorbitol | 1.000 |
| Tryptophan | 1.000 |
| Tergitol-7 | 0.150 |
| 6-chloro-3-indoxyl β-D-galactopyranoside | 0.200 |
| 5-bromo-4-chloro-3-indoxyl- β-D-glucuronic acid cyclohexyl ammonium salt, monohydrate (X-beta-G-glucuronide CHX salt) | 0.100 |
| IPTG (Isopropyl-β-D-thiogalactopyranoside) | 0.100 |
| Agar | 15.000 |
| Final pH (at 25°C) | 6.8±0.2 |
**Formula adjusted, standardized to suit performance parameters
# Enzymatic digest of casein
Directions
Suspend 30.92 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). This medium is the modification of the same, replacing animal peptones with vegetable peptones to avoid BSE/TSE risks associated with animal peptones. The medium contains three chromogenic substrates. The enzyme ß-D-galactosidase produced by coliforms cleaves 6-chloro-3-indoxy 1-ß-D-galactopyranoside to form pink to red coloured colonies (1). The enzyme ß-D glucuronidase produced by E.coli, cleaves 5-bromo-4chloro-3-indoxy 1-ß-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.
HiVeg® hydrolysate, 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 Kovacs 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 undereath, 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
- Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
- 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.
- 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 yellow coloured opalescent gel forms in Petri plates
Reaction
Reaction of 3.09% w/v aqueous solution at 25°C. pH: 6.8±0.2
pH
6.60-7.00
Cultural Response
Productivity:
Cultural response observed after an incubation at 36°C ± 2 for 21 ± 3 hours. Recovery rate is considered as 100% for bacteria growth on Reference medium - Soyabean Casein Digest Agar (Tryptone Soya Agar).
Selectivity:
Cultural response observed after an incubation at 36°C ± 2 for 21 ± 3 hours.
Specificity:
Cultural response observed after an incubation at 36°C ± 2 for 21 ± 3 hours.
| Organism | Inoculum (CFU) | Growth | Recovery | Colour of Colony# |
|---|---|---|---|---|
| Productivity | ||||
| Escherichia coli ATCC 25922 (00013)* | 50-100 | luxuriant | >=70% | dark blue to violet |
| Escherichia coli ATCC 8739 (00012)* | 50-100 | luxuriant | >=70% | dark blue to violet |
| Citrobacter freundii ATCC 43864 (00006)* | 50-100 | luxuriant | >=70% | pink to red |
| Selectivity | ||||
| ##Klebsiella aerogenes ATCC 13048 (00175)* | 50-100 | luxuriant | >=70% | pink to red |
| Enterococcus faecalis ATCC 19433 (00009)* | >=104 | inhibited | ||
| Specificity | ||||
| Pseudomonas aeruginosa ATCC 10145 (00024)* | 103-104 | growth | - | colourless |
Key *: Corresponding WDCM numbers
#: either on plate or membrane
## Formerly knownas 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).
| Product Name | HiCrome™ Chromogenic Coliform HiVeg™ Agar |
|---|---|
| SKU | MV1991I |
| Customized Product Available | No |
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