9.5: Eosin Methylene Blue Media, EMB
- Demonstrate proper aseptic techniques and safety protocols while handling microbial cultures and laboratory equipment.
- Explain how Eosin Methylene Blue (EMB) is both a selective and differential medium.
- Understand the principle of the EMB test in differentiating between lactose fermenters and non-fermenters.
- Interpret the results of the EMB test accurately, distinguishing between colonies exhibiting various degrees of lactose fermentation based on color changes and colony morphology.
- Discuss the clinical significance of identifying lactose-fermenting and non-fermenting bacteria in the context of diagnosing and treating infectious diseases.
Eosin-Methylene Blue (EMB) Medium
Eosin-methylene blue (EMB) medium is a selective medium and a differential medium used for the isolation of gram-negative bacteria and differentiation of members of the Enterobacteriaceae family, particularly coliforms such as Escherichia coli and other lactose-fermenting bacteria, from non-fermenting bacteria. EMB is a selective medium because it inhibits gram-positive bacterial growth. The two dyes in this medium, eosin , and methylene blue act as selective agents to inhibit gram-positive bacteria but allow for the growth of gram-negative bacteria.
EMB enables the differentiation of bacterial species that are coliforms due to dyes and lactose content. EMB contains two types of sugars: lactose and sucrose. It is the lactose that is the key to this medium's ability to differentiate coliform bacteria. Lactose-fermenting bacteria ( Escherichia coli and other coliforms) produce acid as lactose is fermented. When acids are produced by bacteria, the combination of the dyes (which serve as pH indicators in this medium) produces color variations in the colonies. Strong acidity produces a deep purple/black colony with a green metallic sheen (Fig.1.), whereas less acidity may produce a brown-pink coloration. Non-lactose fermenters appear as translucent.
Coliform: A classification of bacterial species that is typically associated with animal digestive tracts and fecal contamination in the environment. These bacterial species are gram-negative bacilli that do not produce endospores and have the ß-galactosidase gene, and can therefore break down lactose. Coliform bacteria break down lactose to produce acid and gas.
EMB Test: Checking the Purity of Drinking Water
To ensure our water sources are not contaminated with fecal-borne diseases, for example cholera, we test for the presence of coliforms. Coliforms are organisms commonly found in the intestinal tracts of warm-blooded organisms. Therefore, the presences of these organisms in a water sample could indicate fecal contamination. Coliforms are often gram-negative bacteria that have the ability to ferment the carbohydrate lactose. Eosin Methylene Blue, or EMB, media is a selective and differential agar that can be used to isolate and identify fecal coliforms. The EMB agar will only allow gram negative bacteria due to the presence of gram-positive inhibiting dyes. If bacteria grown on the EMB media can ferment lactose, such as coliforms, their colonies will appear purple/black or brown/pink. Therefore, if purple/black or brown/pink colonies grow on EMB agar when inoculated from a water sample, there is a high probability that water is contaminated with fecal coliforms.
Laboratory Instructions
- Use a sharpie to divide the EMB plate into 4 sections. Label the bottom of the plate appropriately, making sure to mark each section with the name of the inoculum. Label the outer edge of the plate with your group name, course section, and date.
-
To observe the effects of EMB medium as selective and differential, aseptically spread a sample of each bacterial species (see below) into each quadrant. Make sure to use a new sterile cotton swab with each sample and take caution not to cross-contaminate adjacent quadrants.
- Escherichia coli
- Enterobacter aerogenes
- Proteus mirabilis
- Staphylococcus epidermidis
- Parafilm the plates.
- Invert and incubate the plates for 24-48 hours at 37 °C.
- Observe, record, and interpret the results.
Attributions
- "Microbiology Laboratory Manual: Labs, 1.28 EMB Agar" by Dr. Rosanna Hartline , West Hills College Lemoore, LibreTexts: Biology is licensed under CC BY-NC 4.0
- Cover Image: Escherichia coli EMB.jpg by Gene Drendel is licensed under CC BY 4.0
- MB352 General Microbiology Laboratory 2021 (Lee) by Alice_Lee@ncsu.edu has an undeclared license
- Microbiology Labs I by Delmar Larsen is licensed under an undeclared license
- "Isolation and Identification of Coliform Bacteria and Multidrug-Resistant Escherichia coli from Water Intended for Drug Compounding in Community Pharmacies in Jordan" by Abu-Sini et al. , Healthcare 2023, 11(3), 299; https://doi.org/10.3390/healthcare11030299 , Multidisciplinary Digital Publishing Institute is licensed under CC BY 4.0
-
"Cholera: About Cholera" by U.S. Center for Disease Control and Prevention , CDC Use of Materials Statement