EMB is a selective, differential agar medium used for isolation of gram negative rods in a variety of specimen types. It is used frequently in clinical laboratories.
The selective/ inhibitory agents of EMB are the dyes eosin Y and methylene blue. Methylene blue inhibits the gram + bacteria (eosin to a lesser extent), while eosin changes color, to a dark purple, when the medium around the colony becomes acidic.
EMB contains lactose and sucrose sugars, but it is the lactose that is the key to the medium. Lactose-fermenting bacteria (E. coli and other coliforms) produce acid from lactose use, and the combination of the dyes (which serve as pH indicators in this medium) produces color variations in the colonies because of the acidity. Strong acidity produces a deep purple colony with a green metallic sheen, whereas less acidity may produce a brown-pink coloration of colony. Nonlactose fermenters appear as translucent or pink.
SOME bacteria gram + bacteria may grow–although not well–particularly if you let cultures sit for more than a couple of days. Usually those species will show as pinpoint colonies.
It is a blend of two stains, eosin and methylene blue in the ratio of 6:1. EMB is a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E.
Preparation of EMB agar
Weigh and suspend 35.96 grams of dehydrated media in 1000 ml distilled water.
Mix until the suspension is uniform and heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Cool to 45-50°C, and with frequent gentle swirling, pour the media into sterile Petri plates. Note: frequent swirling is recommended to restore the blue color o methylene blue and to suspend the flocculent precipitate if any.
Label with initials of the name of the medium, and the date of preparation.
Store the plates upside down (lids below) in the refrigerator until use.
Colony Morphology EMB agar inoculated with Escherichia coli demonstrating growth with green-metallic sheen colonies. (Image source:Carmen Moreno González)
Organism
Colonial appearance on EMB agar
Escherichia coli
Colonies are 2-3 mm in diameter and have a greenish metallic sheen in reflected light, dark or even black center in transmitted light.
Enterobacter aerogenes
Colonies are 4-6mm in diameter, raised and mucoid, tending to become confluent. No metallic sheen, grey-brown centers by transmitted light
Salmonella and Shigella spp
Translucent and colorless colonies
Pseudomonas spp
Colorless irregular colonies
Proteus spp
Colorless colonies
Gram-positive cocci
Partially inhibited or no growth
Coagulase-positive staphylococci
Colorless, “pin-point” colonies on modified EMB
Principle of EMB Agar
EMB agar is characterized by the presence of a combination of the two dyes eosin and methylene blue in the ratio of 6:1.
Gram-negative bacteria that ferment the lactose produce acid which lowers the pH. This encourages dye absorption by the colonies and turns the colonies dark purple as the acid acts upon the dyes.
In addition, certain lactose-fermenting bacteria produce flat, dark colonies with a green metallic sheen.
Other lactose fermenters produce larger, mucoid colonies, often purple only in their center.
In EMB agar, most of the strains of E. coli colonies have a characteristic green sheen. Rapid fermentation of lactose & production of strong acids, thus a rapid reduction in the pH of the EMB agar the critical factor in the formation of the green metallic sheen observed with E. coli, rapid fermentation of lactose and formation of strong acids.
Lactose non-fermenters, on the other hand, may increase the pH by deamination of proteins. This ensures that the dye is not absorbed. The colonies will be colorless.
Lactose non-fermenters are thus either colorless or light lavender.
Peptic digest of animal tissue serves as a source of carbon, nitrogen, and other essential growth nutrients.
Lactose and sucrose are the sources of energy by being fermentable carbohydrates.
Eosin-Y and methylene blue serve as differential indicators. Phosphate buffers the medium.
FAQ
What ingredients make EMB plates differential?
It is a selective medium due to inhibition of gram-positive bacteria by methylene blue. EMB agar’s differential property comes from the eosin and methylene blue complex formation under acidic pH.
What is the differential agent in the EMB test?
Eosin–Methylene Blue (EMB) Agar is a differential medium for the detection of Gram negative enteric bacteria. The medium contains peptone, lactose, sucrose, dipotassium phosphate, eosin and methylene blue dyes.
