Butyrate Disk Test: Principle, Procedure, Results, Uses

Last updated on June 23rd, 2021

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.


  • Butyrate disk test helps to identify Moraxella catarrhalis.
  • Butyrate disk test helps to differentiate Neisseria gonorrhoeae (negative) and Moraxella catarrhalis (positive), both are oxidase-positive, Gram-negative diplococci.

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:

  • Discard disks if they do not appear white with no visible color.
  • Perform quality control on each new lot and shipment of disks or MUB reagents prior to using them.
  • Organism:
    • Moraxella catarrhalis ATCC 25240: butyrate positive
    • Neisseria gonorrhoeae ATCC 43069: butyrate negative

References and further readings

  1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814
About Acharya Tankeshwar 474 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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