It has been reported that the enzyme beta-glucuronidase is generally present in many strains o the E. coli and also in other organisms like Salmonella, Shigella, Staphylococcus, Streptococcus along with E. coli which contains the enzyme Beta-glucuronidase.
Thus, the detection of this enzyme, Beta-glucuronidase is determined in the biochemical laboratories by using this method which helps us to identify and differentiate the certain organisms.
The substance known as MUG (4-methylumbellferyl-Beta-D-glucuronide is considered as a sensitive and a selective substrate for detecting the activity of Beta-glucuronidase.
Thus, MUG test is considered as a conjunction with the substrate like oxidase, indole and the fermentation of lactose, which is generally performed in an effective manner in order to identify the species of E. coli and the other such related organisms.
MUG is an acronym of 4-methylumberlliferyl Beta-D-glucuronide. Shortly, MUG test is used for rapid identification of the E. coli which has the capability to produce Verodoxin from the strain that do not produce it.
MUG Test Objective
The main aim of the test is to identify the activity of glucuronidase in the organisms by fluorescence, when it is observed under the UV light source having a long wave of about 365nm.
MUG Test Principle
E. coli and the other species of Enterobacteriaceae produces an enzyme known as Beta-D-glucuronidase, that hydrolyses the Beta-D-glucopyranoside-uronic derivatives into the aglycons and the d-glucuronic acid.
Here, the substrate 4-methylumbelliferyl-β-d-glucuronide which is impregnated into the disk and it is further hydrolyzed by the enzyme in order to yield methyl umbelliferyl moiety, that fluoresces blue under a long wavelength in the UV light.
Hence, if the test organism produces the enzyme known as glucuronidase which helps in breaking the substrate thus resulting in the formation of a fluorescence, which is noted as the positive test.
On the other hand, if there is no desired enzymatic activity, then the substrate will not be broken down into fluorescence on test, which is notes as a result for negative test.
MUG Test Reagents
Media: MUG disks are generally prepared by using the impregnating the MUG i.e., 4-mrthylumbelliferyl Beta-glucuronide onto a high-quality filter paper disk.
2 to 3 isolated colonies of organisms
Long wave UV light
MUG Test Procedure
Generally, two methods are used for performing the MUG test;
i. Direct Disk MUG Test
Initially, the petri dish is made wet using a drop of water. It should not be saturated.
With the help of a wooden applicator small portion of a colony is rubbed from an 18 to 24-hour culture.
Then the culture is incubated in a closed container for about 35 to 37º C for about 2 hours.
After incubating, the disk is observed under a ultra violet light, a long wave of about 360nm in a darkened room, fluorescence can be observed.
ii. Tube MUG Test
Initially 0.25 ml of deionized water is taken in a clean glass tube.
Then a heavy suspension is created using up to 3 to 4 colonies of the isolated test in the glass tube.
With the help of the force, a MUG disk is placed in the tube and it is shaken strenuously to make sure that the adequate elution of the substrate in the surrounding medium.
Further the medium is incubated aerobically for about one hour at a temperature of 35 to 37ºC.
After incubation, the tubes are taken out and they are detected for the presence of fluorescence by using a longwave of ultra violet for about 360nm in a darkened room.
MUG Test Results
Positive MUG Test Results: In case of positive result, there will be fluorescence, which resembles an electric blue.
Negative MUG Test Results: Whereas in case of negative results there will be absence of blue fluorescence.
MUG Test Uses
This test is widely used for identifying the different genera which belongs to the family of Enterobacteriaceae in a presumptive manner.
This test also helps us to characterize the Verotoxin producing E. coli. As the Verotoxin producing strains of E. coli does not have the capability to produce a MUG, and a negative test indicates the presence of a clinically important strain.
This test also aids in detecting the Escherichia coli in the water and in food samples.
MUG Test Limitations
In MUG test, the colonies that are isolated from the media containing dyes, are not suggested for test as it makes the interpretation difficult.
This test is reliable only for oxidase-positive organisms as some of the oxidative negative organisms has its fluorescence naturally.
It is also suggested that the tests such as biochemical, immunological, molecular or a mass spectrometry are generally performed in the colonies that is present in the pure culture for the complete identification.
Where as some strains of the Shigella results as MUG positive. Serological testing is usually required to differentiate the species of Shigella and E. coli.
MUG Test Citations