EMB Agar: Composition, Principle, Preparation, Uses And colony characteristics

Introduction

Eosin-methylene blue (EMB) agar was initially formulated in 1916 by Holt-Harris and Teague, to visibly differentiate between the lactose fermenting and non-fermenting microorganisms through the use of eosin and methylene blue dyes. The medium also included sucrose to differentiate between coliforms that were able to ferment sucrose more rapidly than lactose and those that were unable to ferment sucrose.

Later in 1918, EMB agar was modified by Levine to enable the medium to differentiate between faecal and nonfecal types of the coliform bacteria group. However, this medium differs from the one formulated by Holt-Harris and Teague in that it includes a single peptone as a base and supplementing it with dipotassium phosphate as a buffer and increased concentration of lactose and absence of sucrose. Salmonellae and other non-lactose- fermenters from the coliforms could also be differentiated.

In recent times, Bacto EMB agar which is a combination of the EMB agar described by Holt-Harris and Teague and Levine is being used. This medium contains lactose and sucrose (as formulated by Holt-Harris and Teague) and also contains peptone (peptic digest of animal tissue) and phosphate (as modified by Levine). The two indicator dyes, eosin and methylene blue, are used in a ratio to impart minimum toxicity while providing best differentiation.

EMB Agar Principle

EMB agar is selective and differential medium for gram-negative bacteria. The dye methylene blue in the medium inhibits the growth of gram-positive bacteria; small amounts of this dye effectively inhibit the growth of most gram-positive bacteria. Eosin is a dye that responds to changes in pH, going from colorless to black under acidic conditions. EMB agar medium contains lactose and sucrose, but not glucose, as energy sources. The sugars found in the medium are fermentable substrates which encourage growth of some gram-negative bacteria, especially fecal and nonfecal coliforms.

Differentiation of enteric bacteria is possible due to the presence of the sugars lactose and sucrose in the EMB agar and the ability of certain bacteria to ferment lactose in the medium. Lactose-fermenting gram-negative bacteria (generally enteric) acidify the medium, and under acidic conditions the dyes produce a dark purple complex which is usually associated with a green metallic sheen. This metallic green sheen is an indicator of vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms (E.coli)A smaller amount of acid production, which is a result of slow fermentation (by slow lactose-fermenting organisms), gives a brown-pink coloration of growth with no green metallic sheen. (Enterobacter, klebseilla and other coliforms)Colonies of nonlactose fermenters appear as translucent or faint pink (Salamonella & Shigella).

Note: If you are maintaining pH at 7.2 means you will get good metallic sheen colonies.

Preparation Of EMB Agar

Composition Of EMB Agar

Reagents And Materials

• Glass beaker
• Conical Flask / Erlenmeyer Flask
• Spatula
• Measuring Cylinder
• pH meter
• Weighing balance
• Distilled Water
• Butter Paper
• Magnetic stirrer and pellet
• Pipettes and tips
• Petri plates and/or test tubes
• 1N NaOH
• 1N HCl

Preparation Procedure

1. Weigh the ingredients separately with respect to the volume of the media. (Here, we are considering 1L of the media).
2. Suspend the ingredients (except agar) in a glass beaker containing about 900mL of distilled water.
3. Dissolve the components in the beaker using a magnetic stirrer. (Heat may be applied to dissolve the medium completely).
4. Adjust the pH of the medium to the desired value.
5. Adjust the broth to a final volume of 1L using distilled water.
6. Transfer the broth to conical flask or aliquot into smaller volumes.
7. Now add agar accordingly with respect to the volume of the media (i.e., 13.5 gms agar for 1L of the media).
8. Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
9. Autoclave for 20 min at 15 psi (1.05kg/cm²) on liquid cycle.
10. Mix well and pour into sterile Petri plates.

User Quality Control

Inoculate representative samples with the following strains. Incubate plates aerobically at 35 ± 2°C for 18 to 24 hours.

Storage

• Store the media plates at 4^◦C until they are utilized. The test sample can be directly streaked on the medium plates and then the plates should be incubated, protected from light.

• However, in order to obtain isolated colonies, standard procedures should be followed. A non-selective medium should be inoculated along with conjunction with EMB Agar. Identification of isolates should be done with further confirmatory tests.

Result Interpretation/Colony Characteristics on EMB Agar

Typical colonial morphology is as follows:

Uses of EMB Agar

• EMB Agar (Eosin Methylene Blue Agar) is recommended for the isolation and differentiation of gram negative enteric bacteria from clinical and nonclinical specimens.
• It is useful in differentiating gram positive and gram-negative bacteria.
• It helps in the isolation and differentiation of enteric bacilli and gram-negative bacilli.
• It is used in testing the quality of water, especially in determining if the water is contaminated by harmful microorganisms.
• It differentiates microorganisms in the colon-typhoid-dysentery group.
• EMB media assists in visual distinction Escherichia coli, other nonpathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera.
• It also helps in the isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli.

Limitations of EMB Agar

• A non-selective medium should be inoculated in conjunction with EMB Agar.
• It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
• Some strains of Salmonella and Shigella may fail to grow on EMB Agar.
• Some gram-positive bacteria, such as enterococci, staphylococci, and yeast will grow on this medium and usually form pinpoint colonies.
• Non-pathogenic, non-lactose-fermenting organisms will also grow on this medium. Additional biochemical tests must be performed in order to distinguish these organisms from pathogenic strains.
• Serial inoculation may be required to ensure adequate isolation of mixed flora samples.
• Some strains of E. coli may fail to produce a characteristic green metallic sheen; consequently, the green metallic sheen is not diagnostic for E. coli.

Further References

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.
2.  Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS), Wayne, PA.
3. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.
4. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS), Wayne, PA.
5. MacConkey, A.T. 1905. Lactose-fermenting bacteria in feces. J. Hyg.; 5:333-379.
6. Holt-Harris, J.E. and O. Teague. 1916. A new culture medium for the isolation of Bacillus typhosa from stools. J. Infect. Dis.; 18:596.
7. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
8. Colthup, N. B., L. H. Daly, and S. E. Wiberley 1964 Introduction to infrared and raman spectroscopy, p. 263-265, 332, 382-384. Academic Press Inc., New York.
9. Bopp, C.A., Brenner, F.W., Fields, P.I., Wells, J.G., and N.A. Strockbine. 2003. Escherichia,Shigella, and Salmonella. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C.