Butyrate Disk Test – Principle, Procedure, Result

Overview of Butyrate Test

• Butyrate Disk is a reagent-coated disc that can be used to find the enzyme butyrate esterase. This can help identify Moraxella catarrhalis that has been isolated from clinical samples.
• Moraxella (Branhamella) catarrhalis is now known to be a major pathogen that causes otitis media, sinusitis, conjunctivitis, bronchitis, and pneumonia.
• Also, a large number of M. catarrhalis strains make an enzyme called P-lactamase.
• This organism is a lot like the saprophytic strains of Neisseria spp. in a few ways. Both live in the same place, and they both break down sugar.
• Conventional tests can take anywhere from 24 to 48 hours to finish. We need other ways to identify people that are quick and easy to use.
• Members of the Neisseriaceae family have different esterase activities. This is something that has been known for a long time. Since Berger’s early research, tributyrin (glycerol tributyrate) hydrolysis has been used as a key test to tell M. catarrhalis apart and find it.
• Several substrates and ways to find butyrate esterase have been suggested. Recently, Vaneechoutte et al. wrote about a fluorogenic method that gave accurate results after 5 minutes of incubation. The goal of this study was to find out how useful a spot fluorescence technique that makes this method easier and faster is.
• In the lab, substrates like bromochloro-indolyl butyrate or 4-methylumbelliferyl butyrate are broken down by water to show that butyrate esterase is present.
• Either the disc test or the tube method can be used to do the test. Both of these tests are for quick identification, but the disc test is easier to do because you don’t have to prepare reagents like you do for the tube test.
• In the disc test, the fluorescence from the products of the hydrolysis can show that the substrate is being broken down by water.

Butyrate Disk Test Purpose

• This is a quick way to find out if Moraxella catarrhalis has the enzyme butyrate esterase.

Principle of Butyrate Disk Test 

• The butyrate test is a quick qualitative test that looks for the enzyme butyrate esterase. It can also be used to identify M. catarrhalis.
• The butyrate test can be used to confirm that Moraxella catarrhalis is present when it is combined with the way the organism looks on blood agar plates, a typical Gram stain, and a positive oxidase test.
• Butyrate esterase breaks apart the ester bond between the butyrate group and the substrate, 5-bromo-4-chloro-3-indolyl butyrate, by adding water.
• When the butyryl part breaks off, it releases indolyl, which reacts with another indolyl molecule in the presence of oxygen to make indigo, a blue pigment that doesn’t dissolve.
• When 4-methylumbelliferyl substrate is broken down by water under UV light, a molecule that gives off light is made.
• Because of this, the hydrolysis can be seen by how the products change colour or glow.

Requirements

• Bromochloro-indolyl butyrate and 4-methylumbelliferyl impregnated discs
• Bacteriologic loops or sterile wooden applicator sticks
• Petri dish, slide, or tube filled with distilled water
• UV radiation with a wavelength of 360 nanometers

Test Organism

Gram-negative, oxidase-positive diplococci developing as white colonies on blood agar that do not separate when pulled with a loop or wire.

Quality control

Throw away discs that don’t look white and have no colour. Before you use a new batch or shipment of discs or MUB reagents, you should check their quality.

Organism:

• Moraxella catarrhalis ATCC 25240: butyrate positive
• Neisseria gonorrhoeae ATCC 43069: butyrate negative

Procedure of Butyrate Disk Test

1. Take out a disc and set it down on a glass microscope slide.
2. Add a drop of reagent-grade water. This should leave a small amount of water on the disc.
3. A pure culture of oxidase-positive, Gram-negative diplococci should be grown for 18 to 24 hours, and then numerous colonies should be rubbed onto the disc using a wooden applicator stick.
4. Leave to incubate for up to 5 minutes at room temperature.

Expected results

• Positive: During a 5-minute incubation, a blue colour or fluorescence can form if MUB is used as a substrate.
• Negative: No color change

Important Notes on Butyrate Disk Test

• Moraxella catarrhalis is a gram-negative, oxidase-positive diplococcus that meets the following conditions. a. Grows in colonies on blood agar that stay together when a sample is taken and b. Is butyrate a good thing?
• Check for DNase production in butyrate-negative colonies that look like M.oraxella catarrhalis based on the above criteria. Report colonies with DNase as M. catarrhalis.
• You can use 4methylumbelliferyl butyrate (MUB) instead of bromochloro-indolyl butyrate (IB), but you must meet the following requirements:
  • Long-wave UV light (360 nm; Wood’s lamp)
  • Instead of blue to blue-violet colour (IB substrate) or fluorescence, i.e. hydrolysis of the MUB substrate makes a fluorescent compound that can be seen under UV light.

Uses of Butyrate Disk Test

• The Butyrate Disk Test is a quick, likely identification test that is used to find M. catarrhalis.
• The butyrate test is also used to see how well an organism can break down butyrate substrate with the help of the enzyme butyrate esterase.
• The test is also used as a biochemical test to tell the difference between oxidase-positive, Gram-negative diplococci Neisseria gonorrhoeae (negative) and Moraxella catarrhalis (positive).

Limitation

• This is just one part of the whole plan for identifying people. For confirmation, we need to do more tests.
• Butyrate esterase is found in many strains of Moraxella and some other organisms, but it is not found in most Neisseria spp.
• If the incubation time is longer than 5 minutes, the false-positive reaction will happen.
• If the amount of inoculum is too small, false-negative reactions may happen. If tests for suspected organisms come back negative, try again with a bigger sample and then use other methods.
• Some Moraxella spp. bacillary strains may give a positive or weakly positive reaction.
• Unrelated organisms such as staphylococci and pseudomonads may also give a positive reaction.

References

• Pérez, J & Pulido, A & Pantozzi, Florencia & Martin, Roshan. (1990). Butyrate esterase (4-methylumbelliferyl butyrate) spot test, a simple method for immediate identification of Moraxella (Branhamella) catarrhalis [corrected]. Journal of clinical microbiology. 28. 2347-8. 10.1128/JCM.28.10.2347-2348.1990. 
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