EMB Agar, Levine, without Lactose

Intended Use

Eosin Methylene Blue Agar, Levine is a slightly selective and differential plating medium for the isolation of gram-negative enteric bacteria. EMB Agar, Levine, without Lactose is provided for convenience in genetic studies of enteric bacilli.

Summary and Explanation

Shortly following the publication by Holt-Harris and Teague of a paper describing a new culture medium for the differentiation of enteric microorganisms through the use of eosin and methylene blue dyes,1 Levine described a modification of their formulation which he claimed gave better differentiation between what are now referred to as Escherichia and Enterobacter
species.2 The two formulations differ in that Levine EMB Agar does not contain sucrose. Both of these formulations were developed to improve upon the differentiating properties of Endo

Agar,3 which was developed previously. Levine EMB Agar has become the predominant enteric plating medium that utilizes dyes as selective agents. It is recommended for use in the microbiological examination of dairy products and foods by the American Public Health Association.4,5 EMB Agar, Levine, without Lactose can be supplemented with a carbohydrate as the sole carbon source for the study of hybrid enteric bacilli.6

Principles of the Procedure

The eosin Y and methylene blue dyes in Levine EMB Agar render the medium slightly selective in that they inhibit gram-User positive bacteria to a limited degree. These dyes also play a role in differentiating between lactose fermenters and lactose nonfermenters due to the presence or absence of dye uptake in the bacterial colonies. Coliforms, as lactose-fermenting organisms, are visualized as blue-black colonies, whereas colonies of Salmonella and Shigella, as lactose nonfermenters, appear colorless, transparent or amber. Some gram-positive bacteria, such as fecal streptococci, staphylococci and yeasts, will grow on this medium and usually form pinpoint colonies. A number of nonpathogenic, lactose-nonfermenting gram-negative bacteria will grow on this medium and must be distinguished from the pathogenic strains by additional biochemical tests.

Quality Control
Identity Specifications
Eosin Methylene Blue Agar, Levine
Dehydrated Appearance: Fine, homogeneous, may contain up to a large
                                   amount of minute to small dark red purple particles.
Solution:                      3.74% solution, soluble in purified water upon
                                   boiling. Solution is medium to dark, green
                                   orange brown, hazy.
Prepared Appearance:    Medium to dark, green orange brown, hazy.
Reaction of 3.74%
Solution at 25°C:          pH 7.1 ± 0.2
EMB Agar, Levine, without Lactose
Dehydrated Appearance: Fine, homogeneous, may contain up to a large
                                   amount of minute to small dark red purple particles.
Solution:                       2.74% solution, soluble in purified water upon
                                   boiling. Solution is medium to dark, green
                                   orange brown, hazy.
Prepared Appearance:    Medium to dark, green orange brown, hazy.
Reaction of 2.74%
Solution at 25°C:           pH 7.1 ± 0.2
Cultural Response
Eosin Methylene Blue Agar, Levine or EMB Agar, Levine, without Lactose
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 24 hours.

Formula

Eosin Methylene Blue Agar, Levine
Approximate Formula* Per Liter
Pancreatic Digest of Gelatin....................................... 10.0 g
Lactose................................................................... 10.0 g
Dipotassium Phosphate.............................................. 2.0 g
Eosin Y..................................................................... 0.4 g
Methylene Blue........................................................ 65.0 mg
Agar....................................................................... 15.0 g
EMB Agar, Levine, without Lactose
Consists of the same ingredients without the lactose.
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water:
BBL™ Eosin Methylene Blue Agar, Levine – 37.4 g;
BBL™ EMB Agar, Levine, without Lactose – 27.4 g.
Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens. A nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen. Incubate plates, protected from light, at 35 ± 2°C for 18-24 hours. If negative after 24 hours, reincubate an additional 24 hours. Follow established procedures when using the medium without lactose.

Expected Results

Typical colonial morphology on Eosin Methylene Blue Agar, Levine is as follows:
Escherichia coli......................... Large, blue-black, green metallic sheen
Enterobacter/Klebsiella...............Large, mucoid, blue-black
Proteus....................................Large, colorless
Salmonella...............................Large, colorless
Shigella....................................Large, colorless
Pseudomonas...........................Irregular, colorless
Gram-positive bacteria...............No growth to slight growth
Results obtained with Levine EMB Agar without Lactose are dependent upon the substituted carbohydrate.

*Store at 2-8°C. 

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