EMB agar | Principle | Preparation | Interpretation
☰ Content :
Ⅰ. Overview
Eosin Methylene Blue Agar (EMB) is a selective and differential medium used for the isolation of gram-negative bacilli in foods, dairy products and clinical samples.
EMB Agar was originally formulated in 1916 by Holt-Harris and Teague, then the agar was modified by Levine in 1918 which removed the sucrose from the formula and increased the lactose content.
Today, EMB Agar is a combination of the formulation described by Holt-Harris and Teague and Levine.
E.coli in EMB Agar
Ⅱ. Preparation / composition
Suspend the components, dehydrated powder, in water (36 grams in 1000 ml of purified/distilled water). The medium is boiled for a few seconds until the ingredients are completely dissolved. Sterilize in an autoclave at 15 lbs (121°C) pressure for 15 minutes. avoid overheating.
Cool to 45-50°C and stir the medium to oxidize the methylene blue (i.e. restore its blue color) and suspending the flocculant precipitate. Pour into Petri-dishes.
Composition EMB Agar |
|||
|---|---|---|---|
| Ingredients | Gram/liter | ||
| Animal Tissue Peptic Digest | 10,000 | ||
| Dipotassium Phosphate | 2,000 | ||
| Lactose | 5,000 | ||
| Sucrose | 5,000 | ||
| Eosin – Y | 0.400 | ||
| Methylene blue | 0.065 | ||
| Agar | 13.500 | ||
Ⅲ. Principle
EMB Agar contains two dyes, eosin and methylene blue which inhibit the growth of many microorganisms Gram-positives that accompany it. These dyes serve as differential indicators in response to carbohydrate fermentation.
- Lactose-fermenting gram-negative bacteria acidify the medium, and under acidic conditions the dyes produce a deep purple complex that is usually associated with a green metallic sheen. This metallic green sheen is an indicator of the vigorous lactose and/or sucrose fermentation capacity typical of fecal coliforms.
- A smaller amount of acid production, which is the result of slow fermentation (by slow-fermenting lactose organisms), results in a brown-pink coloration of growth.
- Lactose non-fermenters, such as Shigella and Salmonella, form clear, colorless or amber colonies that are easily distinguished from coliforms.
Lactose and sucrose are sources of energy by being fermentable carbohydrates.
The phosphate buffers the medium.
Note:
The distinctive metallic sheen produced by E. coli on this medium is due to acid production resulting in an amide bond between eosin and methylene blue, other coliforms not producing enough acid to cause this reaction.
Klebsiella in EMB Agar
Ⅳ. Results
◈ After 24 hours at 37°C aerobically, in general:
| Organisms | Growth |
|---|---|
| E. coli | Blue-black colonies with green metallic sheen |
| Klebsiella | Pink and mucoid colonies |
| Enterobacter aerogenes | Good growth; pink, dull |
| Proteus | Variable in size, blue-green to blue or salmon in color, most stumps being black in the center or over their entire area |
| Salmonella | Colorless Colonies |
| Shigella | Colorless Colonies |
| Pseudomonas | Colorless Colonies |
| Gram positive bacteria | Inhibition (partial to complete) |
