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MUG Test :Objective, Principle, Procedure, Result, Uses, Limitations

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What is MUG Test?

MUG Test: MUG stands for 4-Methylumbelliferyl-β-D Glucuronide and it acts as a substrate for the organisms those having enzyme β-glucuronidase. This enzyme is present in 97% strains of E. coli .Organisms other than E. coli of the Enterobacteriaceae family Salmonella, Shigella and Yersinia also possess the enzyme β-glucuronidase.

Objectives of MUG test 

To identify a variety of Enterobacteriaceae genera and Escherichia coli strains that produce verotoxin.

Principle of MUG test

β-D-Glucuronidase, an enzyme generated by most strains of E. coli and other Enterobacteriaceae, hydrolyzes β-d-glucopyranosid-uronic derivatives to aglycons and d-glucuronic acid. The disc is impregnated with the substrate 4-methylumbelliferyl-beta-D-glucuronide. A fluorogenic substrate of β -glucuronidase is 4 -methylumbelliferyl-beta-D-glucuronide which is abbreviated as MUG. The organism that generates the – D- Glucuronidase enzyme may break the substrate 4- Methylumbelliferyl— D- Glucuronide, resulting in the fluorescent moiety 4-methylumbelliferyl (4-MU). Under long wavelength UV light, the final product of hydrolysis, 4-methylumbelliferyl (4-MU), fluoresces blue. MUG is not produced by verotoxin-producing E. coli strains. Hence a negative test result could suggest the presence of a clinically significant strain. At low (1.97-6.72) and high (7.12-10.3) pH, 4MU fluorescence has excitation maxima of 320 and 360 nm, respectively.

Procedure of MUG test

Direct Disk Test

  1. Add a drop of demineralized water to a MUG disc which is kept in an empty sterile petri dish.
  2. On the disc, smear 2-3 isolated colonies from an 18-24 hour old culture.
  3. To create a humid environment, place a piece of filter paper saturated with water in the lid of the petri dish.
  4. At 35°-37°C, incubate aerobically for up to 30 minutes.
  5. After incubation, inspect the disc for fluorescence in a darkened area using longwave UV light (360nm).

Tube Test

  1. In a clean glass tube, pour 0.25ml of demineralized water.
  2. In the tube, make a heavy suspension containing 3- 4 colonies of test isolate.
  3. Place a MUG disc in the tube with forceps and shake vigorously to ensure enough substrate elution in the surrounding liquid.
  4. Incubate aerobically for up to 1 hour at 35°-37°C.
  5. After incubation, inspect the disc for fluorescence in a darkened area using longwave UV light (360nm).

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MUG Test :Objective, Principle, Procedure, Result, Uses, Limitations

Result of MUG Test

Positive test: blue fluorescence

Negative test: no fluorescence

Uses of MUG Test 

  1. The test is used to presumptively identify the Enterobacteriaceae genera.
  2. To identify Escherichia coli strains that produce verotoxin. (Strains of E. coli that produce verotoxin do not produce MUG. Hence a negative test result could indicate the presence of a clinically significant strain.)
  3. Escherichia coli can be detected in water and food samples with this tool.

Limitations of MUG Test 

  1. This test is merely one element of a larger method for detecting E. coli. For conclusive identification, additional biochemical and serological testing is required.
  2. The majority of E. coli 0157:H7 strains are MUG negative. For quick identification of isolates during outbreaks, an E. coli 0157:H7 latex agglutination test has to be used in combination with the MUG test.
  3. MUG-positive Shigella strains exist. To distinguish Shigella and E. coli, serological tests may be required.
  4. When test colonies isolated from media-containing dyes (EMB, MAC) are utilized, false-negative results may be reported, making interpretation problematic.
  5. Salmonella, Shigella, Staphylococcus, and Streptococcus are examples of organisms that have the enzyme -Glucuronidase and are MUG positive. Only lactose-positive, gramme negative rods are tested for – Glucuronidase activity, which helps prevent other organisms from being misidentified as E. coli.

Quality control of MUG test

Positive: Escherichia coli (ATCC25922)

Negative: Klebsiella pneumoniae (ATC13883)


Mug Test Citations

  1. Remel MUG disk. 2010. 12076 Santa Fe Drive, Lenexa, KS 66215, USA

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