Butyrate Disk test - Tri-butyryl esterase test, Principle, Purpose, Procedure, Result, Interpretation

Introduction to Butyrate Disk test / Tri-butyryl esterase test

The butyrate disk test (Tri-butyryl esterase test) is a rapid test used to detect if the microorganism produces the enzyme butyrate esterase.

Due to its rapid results and qualitative procedures, this test is significant in the identification of etiological agents and treatments in cases of infections. Conventional methods of identification may take up to 48 hours to perform which is costly in both time and economy.

Principle of Butyrate Disk test / Tri-butyryl esterase test

Both 4- methylumbelliferyl butyrate and bromochloro-indolyl butyrate can be used as substrates in this test.

The substrate bromochloro-indolyl butyrate is hydrolyzed by microorganisms capable of producing the enzyme butyrate esterase, which releases Indoxyl. In the presence of oxygen, Indoxyl gives an indigo color (blue to blue-violet color) which is positive for the butyrate disk test.

Principle of Butyrate Disk test / Tri-butyryl esterase test:

Bromochlorindoyl butyrate==^{(butyrate esterase)}==>Indoxyl ==^{(presence of O2)} ==>Indigo (blue to blue-violet colour)

If substrate 4- methylumbelliferyl butyrate is used, it produces a fluorescent compound visible under UV light (long wave: 360 nm). The change in color is detected by fluorescence from the products.

Purpose of Butyrate Disk test / Tri-butyryl esterase test

The purpose of the Butyrate Disk test / Tri-butyryl esterase test is as follows:

• This test is important in the presumptive identification Moraxella catarrhalis (formerly named Branhamella catarrhalis).

• It also differentiates Neisseria gonorrhea (negative) and Moraxella catarrhalis (positive) as both pathogens are oxidase-positive gram-negative diplococci.

Procedure of Butyrate Disk test / Tri-butyryl esterase test

Disks impregnated with bromo-chloro-indolyl butyrate or 4- methylumbelliferyl butyrate are used in this test.

The procedure of the test are:

1. Place a disc impregnated with bromo-chloro-indolyl butyrate on a surface (usually a microscope glass slide)

2. Add 1 drop of reagent-grade water to the disc

3. Pick 3-5 freshly cultured test colonies using a wooden applicator stick and smear them on the disc

4. Inbuate for 5 minutes at room temperate

5. Observe for color change

Result, Interpretation of Butyrate Disk test / Tri-butyryl esterase test

Result, Interpretation of Butyrate Disk test / Tri-butyryl esterase test is as follows.

Positive

Blue coloration is developed on the disc within a 5-minute incubation period. (if bromochloro-indolyl substrate is used)

Fluorescence under UV light is observed within a 5-minute incubation period. (if 4-methylumbelliferyl substrate is used)

Negative

No color change within a 5-minute incubation period.