EMB Agar, Levine

Intended Use

Recommended for isolation, enumeration and differentiation of members of Enterobacteriaceae. It is recommended by BIS committee under the specifications IS:5887 (Part I)-1976, Reaffirmed 2005, IS:5401(Part 1)-2012.

Composition**

Final pH (at 25°C): 7.1±0.1

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 37.5 grams 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. AVOID OVERHEATING. Cool to 45-50°C and shake the medium in order to oxidize the methylene blue (i.e. restore its blue colour) and to suspend the precipitate which is an essential part of the medium. Mix well and pour into sterile Petri plates.

Precaution: Store the medium away from light to avoid photooxidation.

Principle And Interpretation

Levine EMB Agar was developed by Levine (1,2) and is used for the differentiation of Escherichia coli and Enterobacter aerogenes and also for the rapid identification of Candida albicans. This medium is recommended by BIS committee for isolation, identification and enumeration ofEscherichia coli from foods (3) and estimation of coliform bacteria in food and animal feeding stuffs (4). It is also recommended for the detection, enumeration and differentiation of members of the coliform group by American Public Health Association (3,5,6).

Eosin-Y and methylene blue make the medium slightly selective and inhibit certain gram-positive bacteria. These dyes differentiate between lactose fermenters and nonfermenters. Some gram-positive bacteria such as faecal Streptococci, yeasts grow on this medium and form pinpoint colonies. Weld (7,8) proposed the use of Levine EMB Agar, with added Chlortetracycline hydrochloride, for the rapid identification of Candida albicans in clinical specimens. A positive identification of Candida albicans can be made after 24-48 hours incubation at 35-37°C in 10% carbon dioxide atmosphere, from specimens such as faeces, oral and vaginal secretions and nail or skin scraping etc. However, the typical appearance is variable.

Type of specimen

Foodstuffs

Specimen Collection and Handling

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (5).

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. A non-selective medium should be inoculated in conjunction with EMB Agar.
2. Confirmatory tests should be further carried out for identification of isolated colonies.
3. Some strains of Salmonella and Shigella species do not grow in the presence of eosin and methylene blue.

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: Light pink to purple coloured homogeneous free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium: Reddish purple coloured slightly opalescent gel with greenish cast and finely dispersed precipitate, forms in Petri plates.

Reaction: Reaction of 3.75% w/v aqueous solution at 25°C. pH: 7.1±0.1

Cultural Response

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

Key: (*) Corresponding WDCM numbers (#) Formerly known as Enterobacter aerogenes

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°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 clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (9,10).

Reference

1. Levine M., 1918, J. Infect. Dis., 23:43.
2. Levine M., 1921, Bull. 62, Iowa State College Engr. Exp. Station.
3. Bureau of Indian Standards, IS : 5401, 1969 (Second reprint - June 1990).
4. Bureau of Indian Standards, IS: 5887 (Part - I) 1976, reaffirmed 2005.
5. 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.
6. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
7. Weld J. T., 1952, Arch. Dermat. Syph., 66:691.
8. Weld J. T., 1953, Arch. Dermat. Syph., 67(5):433.
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.