4-Methylumbelliferyl-β-D Glucuronide Test: Introduction, Principle, Procedure, Result Interpretation, Uses and Keynotes

4-Methylumbelliferyl-β-D Glucuronide Test: Introduction, Principle, Procedure, Result Interpretation, Uses and Keynotes

Introduction of 4-Methylumbelliferyl-β-D Glucuronide Test

4-Methylumbelliferyl-β-D Glucuronide Test short form is MUG test and it acts as a substrate for the organisms those having the enzyme β-glucuronidase. This enzyme is present in 97% of strains of E. coli. Organisms other than E. coli of the Enterobacteriaceae family  Salmonella, Shigella, and  Yersinia also possess the enzyme β-glucuronidase. This test uses for rapid identification of E. coli, the most common gram-negative rod seen in clinical specimens. Since verotoxin-producing E. coli strains are among the few E. coli strains that do not produce MUG, this test can also be used to detect the absence of the enzyme in a fecal isolate of E. coli to alert the microbiologist to the possible presence of a verotoxin-producing strain. Therefore, the MUG test is useful in the laboratory to identify and differentiate such organisms.

Principle of MUG Test

Escherichia coli and only a few other members of the family Enterobacteriaceae (Salmonella, Yersinia, and Shigella) produce the enzyme b-glucuronidase. This enzyme hydrolyzes the MUG, releasing 4-methylumbelliferone, which fluoresces blue under long-wave UV light.

Requirements for 4-Methylumbelliferyl-β-D Glucuronide Test

  • MUG disk
  • Test organisms-Fresh colonies on blood agar plate of possible E. coli organisms that are indole-positive, oxidase-negative, gram-negative rods, whether they are lactose positive or negative.
  • Long-wave UV light (366 nm; e.g., Wood’s lamp)
  •  Inoculating loops
  • Incubator
  • Empty petri dish ( test tube if necessary)
  • Control strains

Positive Control (PC): E. coli ATCC 25922

Negative Control (NC): Klebsiella pneumoniae ATCC 13883 or ATCC 27736

Procedure of 4-Methylumbelliferyl-β-D Glucuronide Test

  1. Place the disk in a sterile empty petri dish and wet with 1 drop of water. ( Excess water in the test may cause falsely negatives.)
  2. 2. Using inoculating loop roll colony from a blood agar plate onto the disk.
  3. Incubate at 35°C for a minimum of 2 hours (test can be read up to 24 hours, but 2 hours is usually sufficient.)
  4. Observe the disk using long-wave UV light in a darkened room.
  5. When interpreting weak reactions with the disk test, after incubation, place the disk in 2 ml of sterile saline. Observe as for tube test after 10 minutes.

Result and Interpretation of MUG Test

MUG test positive: Presence of blue  fluorescence

MUG test negative: Absence  of blue fluorescence

Escherichia coli ATCC 25922: positive

Klebsiella pneumoniae ATCC 13883 or ATCC 27736:negative

Keynotes on 4-Methylumbelliferyl-β-D Glucuronide Test

  • Escherichia coli is definitively identified if a gram-negative rod is indole positive, oxidase negative, and MUG positive.
  • Escherichia coli O157:H7 is indole positive and MUG test negative.
  • The test is also to use other than E. coli for presumptive identification of various members of the family Enterobacteriaceae like Salmonella, Shigella, and Yesenia.
  • To characterize verotoxin-producing Escherichia coli. (Vero toxin-producing strains of E. coli do not produce MUG, and a negative test result may indicate the presence of a clinically important strain.)
  • It is also helpful in the detection of Escherichia coli from water and food samples.

Limitations of the 4-Methylumbelliferyl-β-D Glucuronide Test

  1. All strains of  E. coli organisms are not  MUG positive and therefore a negative test does not mean that the organism is not E. coli.
  2. Culture media that contain dyes like EMB, MAC can not use for the disk test, although the dyes do not interfere with the tube test.
  3. Shigella species that are indole positive, approximately 8% are also MUG positive. Rare isolates of Salmonella and Yersinia are also MUG positive. However, they are rarely indole positive. Thus, to avoid a misidentification, lactose-negative organisms from abdominal sources or from blood should not be tested using this method.
  4. Some fluorescing organisms, such as Pseudomonas aeruginosa, may resemble a positive MUG result. Therefore, the test should not be performed on oxidase-positive bacteria.
  5. Some bacteria fluoresce orange, which is not considered a positive reaction.
  6. Rare MUG test positive E. coli O157 strains have also been reported.

Further Readings on 4-Methylumbelliferyl-β-D Glucuronide Test

  1. Cowan and Steel’s, manual for the identification of medical bacteria
  2. Lynne S. Garcia, Clinical Microbiology Procedures Handbook
  3. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
  4. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MUGDisks.html
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