Introduction and Background

Eosin Methylene Blue (EMB) agar is a selective and differential medium used to support the growth of Gram-negative bacteria (selective) and determine whether these bacteria can metabolize the sugar lactose (differential).  It is a growth medium commonly used in characterizing gastrointestinal bacteria, which can live in the gastrointestinal tract as part of the normal flora of the gastrointestinal tract.  EMB agar is designed to support the growth of these organisms and their differentiation in the following manner:

  1. Eosin Y and Methylene Blue – Inhibit the growth of Gram-positive bacteria (selective)
  2. Lactose – Allows to differentiate between species of Gram-negative bacteria
  3. Eosin Y and Methylene Blue –pH indicators that changes color if lactose is fermented.

If you obtain bacterial growth on your EMB agar plate, it is most likely a Gram-negative bacterium.  Then you can determine if it is a lactose fermenter by viewing the color of the medium (uninoculated MacConkey agar is light purple in color):

  1. Positive for lactose fermentation – The bacterial colonies turn red to purple
  2. Negative for lactose fermentation – The bacterial colonies remain off-white or their natural color

Examples of bacteria in both categories of lactose fermentation include Escherichia coli (positive) and Proteus vulgaris (negative).  Of note is the fact that Escherichia coli ferment lactose so vigorously that their colonies do not turn a red-purple color, but a metallic green-brown color.  For this reason, EMB agar is an essential test to run if you suspect that you have E. coli as an unknown bacterium. 

Laboratory Procedure

  1. Obtain the following materials:
    • One plate of EMB agar
    • Inoculation loop
    • Bacterial culture
  2. Take a loopful of the bacterial culture and inoculate the EMB agar plate in a zig-zag pattern. If using multiple bacteria, split the plate into the appropriate number of sections and inoculate each section in the same manner.
  3. Label the bottom of the plate and incubate the plate with the lid down for 2-3 days in the designated incubation area.
  4. Examine the plate for bacterial growth and lactose fermentation.

Perform the following data analysis and submit the following  answers to all questions:

1. Organism Name

2. Sketch or Describe Appearance of Bacteria on Plate

3. Color Change in Bacteria?

4. Lactose Reaction



2. Growth present

3. Red




2. Growth present

3. Off-white


1.What is the difference in lactose fermentation that separates coli from other Gram-negative lactose fermenters?

2. K. pneumoniae has a metabolic profile that is very similar to E. coli. It will sometimes even produce the metallic green-brown color that E. coli produces on EMB plates.  Using the EMB plate, how can you tell if you have K. pneumoniae instead of E. coli, even if the lactose usage is similar?

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