4-Methylumbelliferyl-β-D-Glucuronide (MUG) Test Principle, Procedure, Results

  • MUG Disk is a reagent-coated disc that can be used to quickly and probably identify Escherichia coli in qualitative procedures.
  • MUG stands for 4-Methylumbelliferyl-β-D Glucuronide and it acts as a substrate for the organisms that have the enzyme β-glucuronidase.
  • This enzyme is found in 97% of E. coli strains. Salmonella, Shigella, and Yersinia are also in the Enterobacteriaceae family with E. coli, and they all have the enzyme -glucuronidase.
  • This test is used to quickly find E. coli, which is the most common type of gram-negative rod found in clinical samples.
  • Since verotoxin-producing E. coli strains are some of the few E. coli strains that don’t make MUG, this test can also be used to find the absence of the enzyme in a faecal isolate of E. coli to alert the microbiologist to the possible presence of a verotoxin-producing strain.
  • So, the MUG test can be used in the lab to identify and tell these organisms apart.
  • In 1976, Kilian and Bulow found that 97% of E. coli strains have the enzyme -glucuronidase.
  • In an evaluation of methods based on microbial enzyme activity profiles, Godsey et al. showed that their findings were correct.
  • They found that 4-methylumbelliferyl—D-glucuronide (MUG) was both sensitive and specific for detecting -gluc-uronidase activity.
  • Trepeta and Edberg combined the MUG test with oxidase, indole, and lactose fermentation to find E. coli quickly and cheaply.


  • This test is used to make a best guess about the type of Enterobacteriaceae and Escherichia coli that makes verotoxin.

Principle of MUG Test

  • Most strains of E. coli make an enzyme called β-D-glucuronidase. This enzyme cuts the substrate, 4-methylumbelliferyl—D-glucuronide (MUG), and makes methylumbelliferone, which is a fluorescent product.
  • A source of long-wave ultraviolet light can be used to find this compound.
  • In the indole test, the enzyme tryptophanase attacks the side chain of the tryptophan molecule, leaving behind the indole form of the aromatic ring.
  • The indole is then found by adding -dimethylaminobenzaldehyde (Kovacs’ Reagent), which changes the colour of the solution to red.


  • Reactive Ingredients: 4-methylumbelliferyl-β-D-glucuronide, Tryptophan
  • Loop sterilization device
  • Inoculating loop, swabs, collection containers
  • Incubators, alternative environmental systems
  • Supplemental media
  • Quality control organisms
  • Forceps
  • Longwave ultraviolet light
  • Kovacs’ Indole Reagent (REF R21227)
  • Petri dish, filter paper, test tube,
  • Demineralized water.

Quality control of MUG Test

Using the following quality control organisms, all lot numbers of MUG Disk have been tested and found to be good. Control organisms should be tested using quality control methods that have already been set up in the lab. If there are problems with quality control, patient results shouldn’t be sent out.

  • E. coli ATCC 25922 Incubate at Aerobic, 30-60 min @ 35-37°C—blue fluorescence (positive), Indole (-)
  • Klebsiella pneumoniae ATCC 13883 or ATCC 27736 Incubate at Aerobic, 30-60 min @ 35-37°C—no fluorescence (negative), Indole (-)
  • Proteus mirabilis ATCC® 12453 incubate at Aerobic, 30-60 min @ 35-37°C –
  • MUG (-), Indole (-)

Procedure of MUG Test

Method A (Direct Disk Test)

  1. Put a MUG Disk in an empty, sterile Petri dish and add one drop of demineralized water. You can also place the disc directly on the agar surface. If you do this, you won’t need to add water because the moisture in the medium will rehydrate the disc.
  2. Spread two or three separate colonies on the disc.
  3. Put a piece of wet filter paper in the lid of the petri dish to make the environment humid.
  4. At 35–37°C, incubate for up to 30 minutes with air.
  5. After incubation, look for fluorescence on the disc in a dark room with a longwave ultraviolet light (360 nm). Note: You have to figure out what the MUG reaction means before you add Kovacs’ Indole Reagent.
  6. Once the disc has been checked for fluorescence, add one drop of Kovacs’ Indole Reagent to it. Watch right away for a red colour to appear.

Method B (Tube Test)

  1. Add 0.25 ml of water that has no minerals to a clean tube made of plastic or glass.
  2. Put a lot of the test isolate (3-5 colonies from a blood agar plate left out for 18–24 hours) in the tube. Note: A known MUG-negative organism can be put into a second tube to act as a negative control and help figure out what the test means.
  3. Using forceps, put a MUG Disk in the tube and shake it hard to make sure the substrate is eluted into the liquid around it.
  4. Incubate at 35–37°C for 1 hour with air.
  5. After incubation, check the tube for fluorescence in a dark room with a longwave ultraviolet light (360 nm). Note: You have to figure out what the MUG reaction means before you add Kovacs’ Indole Reagent.
  6. After looking at the tube to see if it fluoresces, add 2 to 3 drops of Kovacs’ Indole Reagent and mix. Watch right away for a red colour to appear.

Results of MUG Disk Test (Direct or Tube Test)

  • Positive Test: Blue fluorescence
  • Negative Test: No fluorescence
Results of MUG Disk Test (Direct or Tube Test)
4-Methylumbelliferyl-b-d-Glucuronide (MUG) test.
A, Positive. B, Negative.

Limitation of MUG Disk Test

  • This test is just one part of a bigger plan to find out what kind of bacteria E. coli is. For sure identification, more biochemical and/or serological tests are needed. Refer to the right sources for more information.
  • MUG-negative strains of E. coli O157:H7 are the most common. For quick identification of isolates during outbreaks, it is recommended to use both the MUG test and an E. coli 0157:H7 latex agglutination test.
  • Some types of Shigella test positive for MUG. Serology may be needed to tell the difference between Shigella and E. coli.
  • Inoculum taken out of MacConkey agar and triple sugar iron agar has been said to cause false-negative reactions.
  • Salmonella, Shigella, Staphylococcus, Streptococcus, and other organisms have the enzyme -glucuronidase and are MUGpositive. Testing for -glucuronidase activity on only lactose-positive, gram-negative rods can help make sure that other organisms aren’t mistaken for E. coli.

Keynotes on MUG Test

  • Escherichia coli can be ruled out if a gram-negative rod is positive for indole, negative for oxidase, and positive for MUG.
  • Escherichia coli O157:H7 tests positive for indole but fails the MUG test.
  • The test will also use bacteria other than E. coli to try to identify Salmonella, Shigella, and Yesenia, which are all in the same family as E. coli.
  • To learn about Escherichia coli that makes verotoxin. (E. coli strains that make Vero toxin don’t make MUG, and a negative test result may mean that a clinically important strain is present.)
  • It can also be used to find Escherichia coli in samples of water and food.

Uses of MUG Test

  • The test is used to guess which genera of Enterobacteriaceae are present.
  • To learn about Escherichia coli that makes verotoxin. (E. coli strains that make verotoxin don’t make MUG, and a negative test result may mean that a clinically important strain is present.)
  • It helps to find Escherichia coli in samples of water and food.


  • Kilian, M. and P. Bulow. 1976. Acta Pathol. Microbiol. Scand. Sect. B. 84:245-251.
  • Godsey, J.H., M.R. Matteo, D. Shen, G. Tolman, and J.R.Gohlke. 1981. J. Clin. Microbiol. 13:483-490.
  • Trepeta, R.W. and S.C. Edberg. 1984. J. Clin. Microbiol. 19:172-174.
  • Raj, P. 1993. Clin. Microbiol. Newsl. 15:89-93.
  • Murray, P.R., E.J. Baron, J.H. Jorgensen, M.L. Landry, and M.A. Pfaller. 2007. Manual of Clinical Microbiology. 9th ed. ASM Press, Washington, D.C.
  • Forbes, B.A., D.F. Sahm, and A.S. Weissfeld. 2007. Bailey and Scott’s Diagnostic Microbiology. 12th ed. Mosby Elsevier, St. Louis, MO.
  • Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology.

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