Butyrate Disk Test: Principle, Procedure, Results, Uses

By Acharya Tankeshwar •  Updated: 06/23/21 •  2 min read

The butyrate disk test is a rapid test for the detection of the enzyme butyrate esterase. When used in conjunction with characteristic morphology on a blood agar plate, typical Gram stain, and a positive oxidase test, the butyrate test is useful for the definitive identification of Moraxella (Branhamella) catarrhalis.

Suspected bacterial colonies are applied on the filter paper disk impregnated with bromo-chloro-indolyl butyrate (butyrate esterase substrate) and are examined for the presence of a blue color.


Hydrolysis of bromo-chloro-indolyl butyrate impregnated in the disks by the enzyme, butyrate esterase releases indoxyl, which in the presence of oxygen spontaneously forms indigo, a chromogenic compound that appears blue to blue-violet. If the filter paper disk is blue in color, the organism is identified as M. catarrhalis If the disk is colorless (white), the butyrate esterase reaction is recorded as negative.

4-methylumbelliferyl butyrate (MUB) can also be used as a substrate. Hydrolysis of the MUB produces a fluorescent compound visible under UV light.

Test Organism

Gram-negative, oxidase-positive diplococci growing on blood agar as white colonies that remain together when lifted with a loop or wire.


Procedure of Butyrate Disk Test

  1. Remove a disk from the vial and place it on a glass microscope slide
  2. Add 1 drop of reagent-grade water. This should leave a slight excess of water on the disk.
  3. Using a wooden applicator stick, rub a small amount of several colonies of oxidase-positive, Gram-negative diplococci from an 18 to 24 hour pure culture on to the disk.
  4. Incubate at room temperature for up to 5 minutes.
Butyrate disk test
Butyrate disk test A, Positive test. B. Negative test

Expected results:

  1. Positive: Development of blue color or fluorescence (if MUB is used as a substrate) during 5 minute incubation period.
  2. Negative: No color change

Quality control:

References and further readings

  1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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