Ethyl Violet Azide Broth (E.V.A. Broth)

Intended use

Recommended for selective and confirmatory detection of Enterococci as an indicator of faecal pollution in water and other specimens. It is recommended by BIS Committee under the specifications IS 5887(Part II): 1976, Reaffirmed 2005.

Composition**

Final pH (at 25°C)
7.0±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 35.8 grams in 1000 ml purified/distilled water. Heat if necessary to dissolve the medium completely. Dispense in tubes in 10 ml amounts and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Principle And Interpretation

EVA broth is based on the formulation of Litsky et al (1) and present medium is a modification (2) with the reduced amount of dextrose and increased dye concentration which is highly specific for Enterococci. The presence of Enterococci acts as a valuable index of faecal or sewage pollution in water (3). It is recommended by BIS for detection of faecal Streptococci (4). BIS has recommended EVA Broth for enumeration of Enterococci using MPN technique. EVA Agar can be prepared by adding 1.5% agar to EVA Broth before autoclaving. EVA Agar plates are used for isolation of Enterococci.

EVA Broth is used in conjunction with Azide Dextrose Broth (M345). Larkin et al (5) used Azide Dextrose Broth as a presumptive medium and EVA Broth for the confirmation of the presence of Streptococci in frozen foods. They found that generally faecal Streptococci were recovered more consistently and in greater number than the coliforms and could be used in preference to coliforms as an indicator bacteria in frozen foods.

Tryptone provides carbon, nitrogen compounds, long chain amino acids and dextrose provides the necessary nutrients for the growth of Enterococci. Sodium azide and ethyl violet inhibit gram-negative bacilli and gram-positive spore formers.

Type of specimen

Food samples; Water sample

Specimen Collection and Handling:

For food samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (6). For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (7).

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 :

• Further biochemical and serological tests must be carried out for complete identification.
• Some strains may show poor growth due to nutritional variations.

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 coloured homogeneous free flowing powder

Colour and Clarity of prepared medium
Light amber coloured clear solution without any precipitate.

Reaction
Reaction of 3.58% w/v aqueous solution at 25°C. pH: 7.0±0.2

pH
6.80-7.20

Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 24-48 hours

Key: (*) Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 15-30°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 (3,8).

Reference

1. Litsky W., Mallmann W.L. and Fifield C.W., 1953, Am. J. Publ. Health, 43:873.
2. Litsky W., Mallmann W.L. and Fifield C.W., 1955, Am. J. Publ. Health, 45:104.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
4. Bureau of Indian Standards IS: 5887 (Part II) 1976, reaffirmed 2005.
5. Larkin, Litsky and Fuller, 1955, Appl. Microbiol., 3:98, 102, 104, 107.
6. 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.
7. Lipps WC, Braun-Howland EB, Baxter TE, eds. Standard methods for the Examination of Water and Wastewater, 24th ed. Washington DC:APHA Press; 2023.
8. 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.