MUG (Beta-Glucuronidase) Test for E. coli

MUG is an acronym for 4-methylumbelliferyl-β-D-glucuronide. Since 97% of Escherichia coli strains produce the enzyme β-D-glucuronidase, MUG test can be used for rapid identification of E. coli. Verotoxin-producing E. coli (E. coli O157:H7) strains do not produce MUG. Please remember that some rare strains of Salmonella, Yersinia, and Shigella also produce the enzyme β-glucuronidase.

Beta-D-glucuronidase is an enzyme that 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 are best suited for confirmatory testing of the presence of E. coli after a presumptive positive result for fecal coliform bacteria.

MUG test can be used to separate potential verotoxin-producing E. coli from other E. coli strains (usually MUG positive) in gastrointestinal specimens, once the isolate has been identified as E. coli.

Microorganisms Tested

  • Fresh colonies on blood agar plate of possible E. coli that are indole-positive, oxidase-negative, Gram-negative rods, whether they are lactose positive or negative.
  • Do not use this test as part of an algorithm to rapidly identify E. coli in abdominal sources, since occasional isolates of both Salmonella and Shigella can be MUG positive.

Procedure for MUG test

MUG tube method

  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 a MUG disk 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 2 hours.
  5. Observe fluorescence using long-wave UV light in a dark room.

MUG disk method

  1. Place MUG disk in a sterile empty petri dish and wet with 1 drop of water. If excess water is used, test may be falsely negative.
  2. Using a wooden stick or bacteriological loop, roll a colony of suspected E.coli from a blood agar plate onto the disk.
  3. Incubate at 35C for a minimum of 2 hours.
  4. Observe the disk using long-wave UV light in a darkened room.

Expected results of MUG Test

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

Quality control of MUG Test

  1. Perform quality control on each new lot or shipment of disks or liquid reagent prior to use.
  2. Organisms
    1. E. coli ATCC 25922—blue fluorescence (positive)
    2. Klebsiella pneumoniae ATCC 13883 or ATCC 27736—no fluorescence (negative)

Limitations

  • Not all E. coli are MUG positive. A Negative MUG test does not mean that the organism is not E. coli.
  • Do not use media that contain dyes (e.g., EMB agar or MacConkey agar) for the disk test. Dyes do not interfere with the tube test.
  • As some strains of Salmonella, Shigella, and Yersinia also MUG positive. Do not test lactose-negative organisms from abdominal sources or from blood with this method to avoid misidentification.
  • Do not perform MUG test for oxidase-positive organisms. Some fluorescing organisms, such as Pseudomonas aeruginosa, may resemble a positive MUG result.

References and further readings

  1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

3 thoughts on “MUG (Beta-Glucuronidase) Test for E. coli

  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.

    Regards

    1. 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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