Rapid HiColiform™ Broth w/ Tryptophan

Intended Use

For detection and confirmation of Escherichia coli and total coliforms from water and food samples, using a combination of chromogenic and fluorogenic substrates.

Composition**

Final pH ( at 25°C): 6.8±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 17.03 gram in 1000 ml purified/distilled water. For double strength broth use 34.06 grams of M1453A in 1000 ml purified/distilled water. Heat if necessary to dissolve the medium completely. Dispense in tubes or flasks as desired. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Principle And Interpretation

Rapid HiColiform™ Broth w/Tryptophan is a modification of LMX Broth described by Manafi and Kneifel (1). This medium is useful for the detection and confirmation of Escherichia coli and total coliforms in water samples on the basis of chromogenic and fluorognic substrates (1,2,3,4,5,6).

The fluorogenic substrate, 4-methylumbelliferyl-β-D-glucuronide (MUG) is split by enzyme ß-D-glucuronidase specifically found in Escherichia coli. The reaction is indicated by the development of a blue fluorescence under UV light. The presence of total coliforms is indicated by blue-green colourations due to the cleavage of the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. IPTG amplifies enzyme synthesis and increases the activity of ß-D-galactosidase. To confirm presence of E.coli overlay the medium with Kovac's reagent. The layer turns red within 2 minutes in case of positive reaction. Tryptose serves as a source of carbon and nitrogen compounds, long chain amino acids, vitamins and other essential growth nutrients. Sorbitol is the fermentable carbohydrate. The phosphate salts provide buffering action for rapid growth of coliforms. Sodium lauryl sulphate makes the medium selective by inhibiting accompanying microflora, especially the gram-positive organisms. L-Tryptophan aids in improving the indole 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 (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. 97% of the E.coli strains are ß-D-glucuronidase positive, however few strains may show negative fluorescence.
2. Further biochemical and serological test needs to be carried out 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

Colour and Clarity of prepared medium: Light yellow coloured, clear solution having slight precipitate in tubes

Reaction: Reaction of 1.70% 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 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 inorder 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 clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (9,10).

Reference

1. Manafi. M. and Kneifel W., (1989), Zbl. Hygiene and Umweltmedizin 189:225-234.
2. Hahn G. and Wittrock E., (1991), Acta Microbiologica Hungarica 38(3-4):265-271.
3. Manafi M., (1990), Forum Stadte-Hygiene 41:181-184.
4. Manafi M., (1991), Ernahrung / Nutrition, 15, Nr. 10.
5. Manafi M. and Kneifel W., (1991), Acta Microbiologica Hungarica 33(3-4):293-304.
6. Manafi M., Kneifel B. and Bascon S., (1991), Microbiol. Rev., 55:335-348.
7. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, 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.