The butyrate disk test is a rapid test detecting 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 helpful for definitively identifying Moraxella catarrhalis.
Butyrate Test Overview
|Method||Butyrate Disk Test (Catarrhalis Test)|
|Detection of||Enzyme butyrate esterase|
|Use||Identification of Moraxella catarrhalis|
|Time Required||5-7 minutes|
|Expected result||Development of blue color|
|Limitations||False positive reactions if incubation is longer than 5 minutes.|
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. In the presence of oxygen, indoxyl spontaneously forms indigo, a chromogenic compound that appears blue to blue-violet. If the filter paper disk is blue, 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.
Gram-negative, oxidase-positive diplococci growing on blood agar as white colonies that remain together when lifted with a loop or wire.
- 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 before using them.
- Moraxella catarrhalis ATCC 25240: butyrate positive
- Neisseria gonorrhoeae ATCC 43069: butyrate negative
- 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
- Remove a disk from the vial and place it on a glass microscope slide
- Add 1 drop of reagent-grade water. This should leave a slight excess of water on the disk.
- 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 onto the disk.
- Incubate at room temperature for up to 5 minutes.
- Positive: Development of blue color or fluorescence (if MUB is used as a substrate) during 5 minute incubation period.
- Negative: No color change
- The false-positive reaction is seen if the incubation period is longer than 5 minutes.
- False-negative reactions may occur if the inoculum is too small. If suspected organisms give negative test results, repeat with a larger inoculum and follow up with additional methods.
References and further readings
- Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814
- Michael A Noble, Bailey and Scott’s Diagnostic Microbiology, Fourteenth Edition (2017). Betty Forbes, Daniel F. Sahm, and Alice S. Weissfeld. St. Louis, MO: Mosby.