MUG (beta-Glucuronidase) test for rapid identification of E. coli

E. coli use MUG as a substrate, releasing a fluorogenic compound

MUG is an acronym for 4-methylumbelliferyl-beta-D-glucuronide. Escherichia coli produces the enzyme beta-D-glucuronidase. Beta-D-glucuronidase is a enzyme which hydrolyzes the beta-D-glucopyranosid-uronic derivatives to aglycons and D-glucuronic acid.

E. coli use MUG as a substrate, releasing a fluorogenic compound
E. coli use MUG as a substrate, releasing a fluorogenic compound

The substrate 4-methylumbelliferyl-beta-D-glucuronide is impregnated in the disc and is hydrolyzed by the enzyme (beta-D-glucuronidase) to yield the 4-methylumbelliferyl moiety, which fluoresces blue under long wavelength ultraviolet light.

Significance of MUG Test: 

U.S. Environmental Protection Agency has approved 4-methylumbelliferyl-β-D-glucuronide Escherichia coli broth medium (EC-MUG) is an effective and rapid method for detection and verification of E. coli in food, water, and environmental samples.

Standard analysis of water includes the most probable number (MPN) for the presumptive and quantitative detection of coliform and fecal coliform bacteria  in water samples. According to ASM EC broth and agar media with MUG is best suited for confirmatory testing of the presence of E. coli after a presumptive positive result for fecal coliform bacteria.

Procedure for MUG Test (b-Glucuronidase test)

  1. Prepare a dense milky suspension of the organism to be tested in a small tube containing 0.25 ml of saline. The suspension should be prepared from colonies growing on MacConkey agar.
  2. Add one tablet of 4-nitrophenyl-β-D-glucopyranosiduronic acid (PGUA) for detection of β-glucuronidase activity.
  3. Place a stopper in the tube and agitate vigorously for a few seconds.
  4. Incubate the tube at 35-37 °C for 4 hours.
  5. The development of a yellow colour in the supernatant indicates a positive test for Ecoli.

Expected results of MUG Test

  1. Positive: Electric blue fluorescence
  2. Negative: Lack of fluorescence

Quality control of MUG Test

  1. Positive: Escherichia coli
  2. Negative: Pseudomonas aeruginosa
About Acharya Tankeshwar 458 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. Hello Tankeshwar,

    Nice to read your article. I was looking into EC MUG issues as we are using it in our lab for E. coli testing. I prepared a new batch and used E. coli as a positive control, but my results do not appear to be positive. First of all the media is cloudy its not clear, is that common?

    Secondly, any idea why our E. coli control is not MUG positive? Wrong strain may be?
    Anyways, I appreciate your input.


    • Dear Muhammad Ali, sorry to know that you are having trouble with MUG test. We used to perform MUG test earlier during water quality analysis, and now we are again reverting back to plating in EMB and conventional biochemical tests (when required). In any enzyme based assay, multiple factors affect the result outcome. Main components to look is substrate quality and enzyme producing ability of the organism, and an environment which facilitate or prevent enzyme production. As you do not know where is the problem in your case in substrate or in organism or the environment, its better to collaborate with nearby lab which performs MUG test.

  2. Your blog is very helpful for me as am venturing into food quality and safety analysis. how do I process foods like cooked beef stew, cooked rice, coleslaw, bacon soups.

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