Butyrate Disk Test: Introduction, Principle,Test Requirements, Procedure, Result-Interpretation, Keynotes, and Limitations

Introduction of Butyrate Disk Test 

Butyrate disk test is useful to differentiate Moraxella catarrhalis from Neisseria species like Neisseria gonorrhoeae, Neisseria lactamica.

Neisseria gonorrhoeae in Urethral Diacharge Gram Staining Showing Gram Negative Diplococci
Fig. Neisseria gonorrhoeae in Urethral Diacharge Gram Staining Showing Gram Negative Diplococci
Neisseria gonorrhoeae growth on blood agar
Fig. Neisseria gonorrhoeae growth on blood agar
Neisseria gonorrhoeae Colony Morpholohy on Chocolate Agar
Fig. Neisseria gonorrhoeae Colony Morpholohy on Chocolate Agar
Neisseria gonorrhoeae OXidase Test -Positive
Fig. Neisseria gonorrhoeae OXidase Test -Positive

Principle of Butyrate Disk Test 

The butyrate disk test is a rapid test for the detection of the enzyme butyrate esterase. Bromochloro-indolyl butyrate (IB)  is the substrate. Hydrolysis of the IB substrate by the butyrate esterase of the test organism(Moraxella catarrhalis) releases indoxyl, which in the presence of oxygen spontaneously forms indigo, a chromogenic compound that appears blue to blue-violet.

Morxalella growth on blood agar
Fig. Morxalella growth on blood agar

Requirements for Butyrate Disk Test

  • Test organism-Gram-negative, oxidase-positive diplococci growing on BAP as white colonies that remain together when lifted with a loop or wire
  • Disks impregnated with IB(Store at 2 to 8°C and protect from light.)
  • Sterile wooden applicator sticks or bacteriologic loops
  • Distilled water or deionized water
  • slide
  • Control Organisms

Positive control(PC): Moraxella catarrhalis ATCC 25240

 Negative Control(NC): Neisseria gonorrhoeae  ATCC43069 or Neisseria lactamica ATCC23970

Note: Discard disks if they appear visible color and perform quality control(QC)on each new lot and shipment of disks.

Procedure of Butyrate Disk Test

  1. Remove butyrate disk from vial and place on a clean glass slide.
  2. Add 1 drop of distilled water to moisten the disk.
  3. Obtain a heavy, visible inoculum with a sterile wooden applicator stick or loop from a 24 to 72 hours culture and rub it onto the disk.
  4. Incubate at room temperature for up to 5 min.
  5. Incubation for slightly longer periods may yield false-positive results. Do not read after 5 minutes.

Result and Interpretation of Butyrate Disk Test

  • Butyrate esterase test positive:  Development of a blue to violet color
  • Test Negative: No color change
  • Moraxella catarrhalis ATCC 25240 : butyrate test positive
  • Neisseria gonorrhoeae  ATCC43069 or Neisseria lactamica ATCC23970: butyrate  test negative
Butyrate Disk Test-Positive
Fig. Butyrate Disk Test-Positive

Keynotes on Butyrate Disk Test

  1. Report as Moraxella catarrhalis is an oxidase-positive, gram-negative diplococcus meets the following criteria. a. Grows on blood agar as colonies that remain together when sampled and b. Is butyrate positive
  2. Test butyrate-negative colonies that are suggestive of Moraxella  catarrhalis by the above-listed criteria for DNase production. Report DNase-positive colonies as M. catarrhalis.
  3. In place of bromochloro-indolyl butyrate (IB), another substrate i.e. 4methylumbelliferyl butyrate (MUB) can be used but the following requirements are necessary-

Long-wave (360 nm) UV light (Wood’s lamp)

In place of blue to blue-violet color (IB substrate)or fluorescence, i.e. hydrolysis of the MUB substrate produces a fluorescent compound visible under UV light.

Limitations of the Test

Do not incubate test beyond 5 minutes, to avoid false-positive reactions.

  1. Many strains of other Moraxella species, as well as Eikenella and Acinetobacter, may give a positive or weakly positive reaction. The organism must be an oxidase-positive, gram-negative diplococcus with typical morphology to be accurately identified as Moraxella catarrhalis.
  2. Unrelated organisms such as staphylococci and pseudomonads may also give positive results.
  3. False-negative tests may result from using too small an inoculum. If the organism is suspected but the test is negative, repeat with a large inoculum or test for DNase production.

Further Readings

  1. https://assets.thermofisher.com/TFS-Assets/MBD/Instructions/IFU901424.pdf
  2. https://www.keyscientific.com/files/productfiles/K9131.PDF
  3. Cowan and Steel’s, manual for the identification of medical bacteria
  4. Lynne S. Garcia, Clinical Microbiology Procedures Handbook

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