MUG Test- Objective, Principle, Procedure, Result, Limitations

Objective of MUG Test

To identify various genera of Enterobacteriaceae and verotoxin-producing Escherichia coli.

Principle of MUG Test

MUG is an acronym for 4-methylumbelliferyl-beta-D-glucuronide, a fluorogenic substrate of β-glucuronidase. β-D-Glucuronidase is an enzyme produced by most strains of E. coli and other Enterobacteriaceae which hydrolyzes β-d-glucopyranosid-uronic derivatives to aglycons and d-glucuronic acid. The substrate 4-methylumbelliferyl-beta-D-glucuronide is impregnated in the disc. The organism capable of producing the β- D- Glucuronidase enzyme has the ability to cleave the substrate 4- Methylumbelliferyl- β- D- Glucuronide and thereby produces a  fluorescent moiety 4-methylumbelliferyl (4-MU). The end product of hydrolysis, 4-methylumbelliferyl (4-MU), fluoresces blue under long-wavelength ultraviolet light. However, verotoxin-producing strains of E. coli do not produce MUG, and a negative test result may indicate the presence of a clinically important strain. 4-MU fluorescence is pH-dependent with excitation maxima of 320 and 360 nm at low (1.97-6.72) and high (7.12-10.3) pH, respectively.

Procedure of MUG Test

Direct disk test

1. Place a MUG disk in an empty sterile petri dish and add a drop of demineralized water.
2. Smear 2-3 isolated colonies from 18-24 hour old culture on the disk.
3. Place a piece of filter paper saturated with water in the lid of the petri dish to provide humid environment.
4. Incubate aerobically at 35°-37°C for up to 30 minutes.
5. Following incubation, examine the disk for fluorescence using longwave ultraviolet light (360nm) in a darkened room.

Tube test

1. Add 0.25ml of demineralized water to a clean glass tube.
2. Make a heavy suspension with 3- 4 colonies of test isolate in the tube.
3. Using forcep, place a MUG disk in the tube and shake vigorously to ensure adequate elution of substrate in the surrounding liquid.
4. Incubate aerobically at 35°-37°C for up to 1 hour.
5. Following incubation, examine the disk for fluorescence using longwave ultraviolet light (360nm) in a darkened room.

Result Interpretation of MUG Test

Positive test:  blue fluorescence

Negative test: no fluorescence

Limitations of MUG Test

1. This test is only part of the overall scheme for identification of E. coli. Further biochemical and serological testing is required for definitive identification.
2. Most strains of E. coli 0157:H7 are MUG negative. The use of an E. coli 0157:H7 latex agglutination test in conjunction with the MUG test is recommended for rapid identification of isolates during outbreaks.
3. Some strains of Shigella are MUG positive. Serological testing may be required to differentiate Shigella and E. coli.
4. False negative reactions may be reported when test colonies isolated from medias containing dyes (EMB, MAC) are used and hence it may make the interpretation difficult.
5. Organisms other than E. coli like Salmonella, Shigella, Staphylococcus, Streptococcus possess the enzyme β-Glucuronidase and are MUG positive. Testing only lactose positive, gram negative rods for β – Glucuronidase activity helps preclude misidentification of other organisms as E. coli.

Quality Control of MUG Test

Positive: Escherichia coli (ATCC25922)

Negative: Klebsiella pneumoniae (ATC13883)

References

1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
2. Remel MUG disk. 2010. 12076 Santa Fe Drive, Lenexa, KS 66215, USA https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU21135.pdf