Butyrate Disk Test
Many of the biochemical tests are performed to identify the characteristics of a specific micro-organism and to find about its pathogenic activities, especially during some of the specific enzymes.
These tests help us to find the nature of the pathogen and its disease-causing activity. Which helps us to define the organism and its characteristics in the hosts body especially in humans.
One such biochemical test is Butyrate disk test which helps to detect the morphology and identification, of the specific organism Moraxella Catarrhalis, commonly called as Branhamella.
What is Butyrate Disk Test?
Butyrate disk test is one of the rapid identification tests for detecting the Moraxella catarrhalis based on their ability to utilize bromochloro-indolyl butyrate or 4-methylumbelliferyl butyrate.
In recent days Moracella catarrhalis is recognized as the major pathogen which causes sinusitis, conjunctivitis, bronchitis, ottis media and pneumonia. In addition to large number of Moraxella catarrhalis strains produces an enzyme known as Beta-lactamase which are resistant to penicillin and ampicillin.
For the completion of conventional test, it requires up to 24 to 48 hours, However, the alternative tests are easy to perform, whereas for identification methods butyrate disk tests can be used.
Usually in biochemical tests, butyrate esterase is demonstrated by hydrolyzing the substrates like bromochloro-indolyl butyrate or four-methyl umbelliferyl butyrate.
The test can be performed either by using disk test or using the tube method. Both of these tests are included as the rapid identification methods, But the disk tests are considered as a most feasible one as it does not require any preparations like reagents in the test tubes.
Butyrate Disk Test Objective
• The main aim of this test is to identify and detect the species known as Moracella catarrhalis in the clinical samples, as a presumptive identification method.
• To detect the capacity of the organism to hydrolyze butyrate substrate by producing butyrate.
Butyrate Disk Test Principle
Butyrate test is considered as one of the rapid qualitative tests used for detecting the enzyme known as butyrate esterase, which further provides a judgment for identifying the organism, Moracella catarrhalis.
While performing this test, it is important to know the characteristic morphology of a specific organism which is placed on the plate of blood agar, typical gram stain and the positive oxidase tests are very much useful in identification of this species.
Both the reagents, Bromochloro-indolyl butyrate and the 4-methylumbelliferyl butyrate serves as a substrate.
During hydrolyzing the bromochloro-indolyl substrate with butyrate esterase it releases indoxyl.
Thus, indoxyl in the presence of oxygen changes its form into indigo. Indigo is one of the chromogenic compounds, which appears in the blue color or in a shade of blue-violet.
On hydrolyzing 4-methylumbelliferyl substrate it produces a fluorescent compound, which is visible only under a UV light.
However, Hydrolysis is detected by change in color or by the detecting the fluorescence from the products.
Butyrate Disk Test and Types of Micro Organism
Here, the Gram-negative species of oxidase positive diplococci that is growing on the blood agar plates in the form of white colonies which remain together when lifted using a loop or a wire is detected.
Butyrate Disk Test Reagent
• Disks impregnated with Bromochloro-indolyl butyrate and 4-methylumbelliferyl
• Bacteriologic loops
• Sterile wooden applicators
• Petri dish
• Distilled water
• Long wave of UV light, about 360nm.
Butyrate Disk Test Procedure
Initially the disks are removed from the vial and they are placed on the clean glass slide or in a petri dish.
Then a drop of deionized or distilled water is placed on the disk, in order to provide a moisture.
A heave inoculum, that is visible is collected from the 24 to 72-hour culture using a sterile wooden applicator stick and it is placed on the disk.
Further the inoculated disks are incubated by maintaining a room temperature of about 15 to 30ºC for about 5 minutes.
Incubation for a slightly longer period is required for yielding a false-positive result.
Butyrate Disk Test Quality Control
• The disks are discarded in case if they are not appearing in white color or without any visible colors.
• According to quality control, the organisms like Catarrhalis ATCC gives a butyrate positive result. Neisseria gonorrhea or Neisseria lactamica gives butyrate negative results.
Butyrate Disk Test Result
• A positive result is determined in the butyrate disk test when there is a change in color from blue to violet or appearance of fluorescence under UV light. This reaction is seen within 5 minutes, which indicates the hydrolysis of bromochloro-indoyl and the 4-methylumbelliferyl, by the enzyme butyrate esterase.
• Whereas the negative test is indicated by no change in the color
Butyrate Disk Test Uses
• This test helps us to identify the Moraxella catarrhalis.
• This test also helps us to differentiate Neisseria gonorrhea and Moraxella catarrhalis; where both are oxidase positive, gram negative diplococci.
Butyrate Disk Test Limitations
• Incubation should not be maintained more than 5 minutes, as it results in false positive results.
• False-negative reactions also occurs of the inoculum is smaller than usual.
• Tests are often performed in conjunction with the chromogenic substrate tests or in carbohydrate fermentations, oxidase reaction, gram stain and morphology for the complete identifation of the organism.
• Non-human species of Branhmella subgenus, Moraxella are butyrate esterase-positive. Some strains of the subgenus Moraxella five a positive weak reaction in few cases.
• IN some cases, the unrelated organisms like Staphylococcus and pseudomonads gives a positive result,
Butyrate Disk Test Citations