Butyrate Disk Test- Principle, Procedure, Results, Uses, Limitations

Butyrate disk test is a simple and rapid method for the identification of Moraxella catarrhalis, a Gram-negative, oxidase-positive diplococcus that causes various respiratory infections in humans. M. catarrhalis is also known for its ability to produce β-lactamase, an enzyme that confers resistance to penicillin and ampicillin. Therefore, it is important to distinguish M. catarrhalis from other similar organisms, such as Neisseria gonorrhoeae and Neisseria lactamica, which are butyrate-negative.

The butyrate disk test is based on the detection of butyrate esterase, an enzyme that hydrolyzes butyrate substrates such as bromochloro-indolyl butyrate or 4-methylumbelliferyl butyrate. The hydrolysis of these substrates results in the formation of color or fluorescence that can be easily observed within 5 minutes. The test can be performed on a glass slide, a petri dish, or a tube, using disks impregnated with the substrates. The test is highly specific and sensitive for M. catarrhalis and can be used as a presumptive identification tool along with other tests such as Gram stain, oxidase test, and morphology on blood agar plates.

The butyrate disk test is a convenient and inexpensive alternative to conventional tests that may require longer incubation times or more complex reagents. The test is also useful for the differentiation of M. catarrhalis from other Moraxella species, as well as Eikenella and Acinetobacter, which may also produce butyrate esterase but have different characteristics. However, the test has some limitations that need to be considered, such as the possibility of false-positive or false-negative results due to various factors such as inoculum size, incubation time, or substrate quality.

In this article, we will discuss the objectives, principle, procedure, results, uses, and limitations of the butyrate disk test in detail.

The main objectives of the butyrate disk test are:

• To detect and identify M. catarrhalis from clinical samples as a presumptive identification method. M. catarrhalis is a major pathogen that causes various respiratory infections and is often resistant to penicillin and ampicillin. Therefore, it is important to identify it quickly and accurately for appropriate treatment and infection control.
• To detect the ability of an organism to hydrolyze butyrate substrate by the production of butyrate esterase. Butyrate esterase is an enzyme that catalyzes the breakdown of butyrate into butyric acid and alcohol. This enzyme is present in M. catarrhalis and some other Moraxella species, but not in Neisseria species or other Gram-negative diplococci. Therefore, the butyrate disk test can help differentiate M. catarrhalis from other similar organisms.

The butyrate disk test is a simple, rapid, and inexpensive test that can be performed in any laboratory setting with minimal equipment and reagents. It can provide a presumptive identification of M. catarrhalis within 5 minutes, which can be confirmed by other tests such as DNase production or molecular methods. The butyrate disk test can also be used as a screening test for the presence of butyrate esterase in other organisms that may have clinical significance or research interest.

The butyrate disk test is based on the ability of M. catarrhalis to produce an enzyme called butyrate esterase, which can hydrolyze (break down) certain substrates that contain butyrate. The substrates used in this test are either bromochloro-indolyl butyrate or 4-methylumbelliferyl butyrate, which are impregnated on disks.

When the organism is inoculated onto the disk, the butyrate esterase enzyme reacts with the substrate and releases a product that can be detected by a color change or fluorescence. The product of bromochloro-indolyl butyrate hydrolysis is indoxyl, which forms a blue to blue-violet color in the presence of oxygen. The product of 4-methylumbelliferyl butyrate hydrolysis is 4-methylumbelliferone, which emits fluorescence under UV light.

The color change or fluorescence indicates a positive test result, meaning that the organism has butyrate esterase activity and is presumptively identified as M. catarrhalis. A negative test result means that the organism does not have butyrate esterase activity and is not M. catarrhalis.

The butyrate disk test is a rapid and simple method that can provide a presumptive identification of M. catarrhalis within 5 minutes. However, it should be used in conjunction with other tests, such as Gram stain, oxidase test, and morphology on blood agar plates, to confirm the identification of M. catarrhalis.

The butyrate disk test is mainly used to identify Moraxella catarrhalis, a Gram-negative, oxidase-positive diplococcus that causes respiratory tract infections such as otitis media, sinusitis, bronchitis, and pneumonia. M. catarrhalis is also known to produce β-lactamase, which confers resistance to penicillin and ampicillin.

M. catarrhalis can be distinguished from other Gram-negative diplococci by its characteristic morphology on blood agar plates. It forms white, smooth, convex colonies that remain together when lifted with a loop or wire. The colonies are non-hemolytic and do not ferment carbohydrates.

The butyrate disk test can also differentiate M. catarrhalis from Neisseria gonorrhoeae and Neisseria lactamica, which are also Gram-negative, oxidase-positive diplococci that grow on blood agar plates. However, these organisms are butyrate-negative and have different biochemical properties.

N. gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted infection that affects the mucous membranes of the genital, urinary, and reproductive tracts. N. gonorrhoeae can be identified by its ability to ferment glucose but not maltose or lactose.

N. lactamica is a commensal organism that colonizes the nasopharynx of humans. It is usually harmless but can occasionally cause opportunistic infections in immunocompromised individuals. N. lactamica can be identified by its ability to ferment glucose, maltose, and lactose.

Other organisms that may give a positive or weakly positive butyrate disk test include some strains of Moraxella nonliquefaciens, Moraxella osloensis, Eikenella corrodens, and Acinetobacter spp. However, these organisms are usually not clinically significant and can be differentiated from M. catarrhalis by their different morphological and biochemical characteristics.

Therefore, the butyrate disk test is a useful tool for the rapid presumptive identification of M. catarrhalis from clinical samples when used in conjunction with other tests such as Gram stain, oxidase test, and DNase test.

The reagents and supplies used for the butyrate disk test are as follows:

• Reagent disks: These are commercially available disks impregnated with either bromochloro-indolyl butyrate or 4-methylumbelliferyl butyrate. The disks should be stored in a refrigerator (2 to 8°C) in a tightly closed vial and protected from light. The disks should be discarded if they do not appear white with no visible color.
• Sterile wooden applicator sticks or bacteriologic loops: These are used to collect and transfer the inoculum to the disk. The sticks or loops should be sterile to avoid contamination and false results.
• Distilled water: This is used to moisten the disk before inoculation. The water should be free of any impurities or contaminants that might interfere with the test.
• Petri dish, slide, or tube: These are used to hold the disk during the test. The petri dish, slide, or tube should be clean and dry before use.
• Long-wave (360-nm) UV light: This is used to observe the fluorescence produced by the hydrolysis of 4-methylumbelliferyl butyrate. The UV light should be in good working condition and have a suitable wavelength for the detection of fluorescence.

The butyrate disk test can be performed either by using disks impregnated with bromochloro-indolyl butyrate or 4-methylumbelliferyl butyrate. Both substrates can be hydrolyzed by the enzyme butyrate esterase, which is produced by M. catarrhalis. The following steps describe the procedure of the butyrate disk test:

1. Remove a disk from the vial and place it on a clean glass slide or petri dish. Do not touch the disk with your fingers or any other object.
2. Add a drop of distilled or deionized water to the disk to moisten it. Do not use tap water or saline as they may interfere with the test.
3. Collect a heavy, visible inoculum of the organism with a sterile wooden applicator stick or a bacteriologic loop from a 24 to 72 hours culture on blood agar plate. The culture should be an oxidase-positive, Gram-negative diplococcus with typical morphology.
4. Smear the inoculum onto the disk and spread it evenly over the entire surface. Do not rub or press too hard as this may damage the disk or cause false-positive results.
5. Incubate the inoculated disk at room temperature (15 to 30°C) for up to 5 minutes. Do not incubate longer than 5 minutes as this may lead to false-positive results.
6. Observe the disk for any color change or fluorescence under UV light. A positive test is indicated by a blue to blue-violet color (bromochloro-indolyl substrate) or fluorescence (4-methylumbelliferyl substrate) within 5 minutes, indicating the hydrolysis of the substrate by butyrate esterase. A negative test is indicated by the absence of any color change or fluorescence.

The result of the butyrate disk test should be interpreted along with other tests and criteria for the identification of M. catarrhalis, such as Gram stain, oxidase test, and DNase test.

Quality control is an essential step in any laboratory test to ensure the accuracy and reliability of the results. For the butyrate disk test, the following quality control measures should be followed:

• The disks should be stored in a cool and dry place, away from direct sunlight and moisture. The disks should be discarded if they do not appear white with no visible color.
• The disks should be used before the expiration date indicated on the label. The disks should not be used if they are damaged or contaminated.
• The inoculum should be taken from a pure culture of the organism, preferably from a 24 to 72 hours growth on blood agar plates. The inoculum should be heavy and visible, but not too thick to obscure the disk.
• The inoculated disk should be incubated at room temperature (15 to 30°C) for up to 5 minutes. Incubation for longer periods might yield false-positive results due to nonspecific hydrolysis of the substrate by other enzymes.
• The result should be read within 5 minutes of incubation. A positive test is indicated by a blue to blue-violet color (bromochloro-indolyl substrate) or fluorescence (4-methylumbelliferyl substrate) within 5 minutes, indicating the hydrolysis of the substrate by butyrate esterase. A negative test is indicated by the absence of a color change or fluorescence.
• As a form of quality control, known positive and negative control organisms should be tested along with the unknown samples. The recommended control organisms are M. catarrhalis ATCC (positive) and Neisseria gonorrhoeae or Neisseria lactamica (negative).
• The results should be interpreted in conjunction with other tests, such as Gram stain, oxidase test, and DNase test, to confirm the identification of M. catarrhalis. The organism must be an oxidase-positive, Gram-negative diplococcus with typical morphology to be accurately identified as M. catarrhalis.

The result of the butyrate disk test depends on the type of substrate used and the presence or absence of butyrate esterase in the tested organism. The test can be read within 5 minutes of inoculation, and any color change or fluorescence after that time should be disregarded.

If the substrate is bromochloro-indolyl butyrate, a positive test is indicated by a blue to blue-violet color on the disk, which means that the organism has hydrolyzed the substrate and released indigo. A negative test is indicated by no color change on the disk, which means that the organism does not have butyrate esterase or cannot hydrolyze the substrate.

If the substrate is 4-methylumbelliferyl butyrate, a positive test is indicated by fluorescence on the disk under UV light, which means that the organism has hydrolyzed the substrate and released 4-methylumbelliferone. A negative test is indicated by no fluorescence on the disk under UV light, which means that the organism does not have butyrate esterase or cannot hydrolyze the substrate.

The image below shows an example of a positive and a negative result using bromochloro-indolyl butyrate disks.

A positive result for butyrate disk test is presumptive evidence for the identification of Moraxella catarrhalis, which is an oxidase-positive, Gram-negative diplococcus that grows on blood agar plates as white colonies that remain together when lifted with a loop or wire. A negative result for butyrate disk test excludes M. catarrhalis from identification.

However, some other organisms may also give a positive or weakly positive result for butyrate disk test, such as some strains of other Moraxella species, Eikenella corrodens, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa. Therefore, additional tests are required to confirm the identity of M. catarrhalis, such as DNase production, carbohydrate fermentation, and antibiotic susceptibility.

The results of the butyrate disk test should be reported along with the other characteristics of the organism, such as morphology, Gram stain, and oxidase test. The butyrate disk test is not a definitive identification test by itself, but rather a presumptive test that can help to narrow down the possible candidates.

The result is reported as Moraxella catarrhalis if an oxidase-positive, Gram-negative diplococcus meets the following criteria:

• Grows on BAP as colonies that remain together when sampled and
• Is butyrate positive

The butyrate-negative colonies that are suggestive of M. catarrhalis can be tested by the above-listed criteria for DNase production. DNase-positive colonies are reported as Moraxella catarrhalis.

The result is reported as Neisseria gonorrhoeae or Neisseria lactamica if an oxidase-positive, Gram-negative diplococcus meets the following criteria:

• Grows on BAP as colonies that separate when sampled and
• Is butyrate negative

The result is reported as other Moraxella species, Eikenella, or Acinetobacter if an oxidase-positive, Gram-negative coccobacillus or rod meets the following criteria:

• Grows on BAP as colonies that may or may not remain together when sampled and
• Is butyrate positive or weakly positive

The result is reported as Staphylococcus or Pseudomonas if an oxidase-negative or variable, Gram-positive or Gram-negative coccus or rod meets the following criteria:

• Grows on BAP as colonies that may or may not remain together when sampled and
• Is butyrate positive

The result is reported as negative or no growth if no colonies are observed on BAP after incubation.

The result is reported as inconclusive or needs further testing if the organism does not fit any of the above criteria or if there is any doubt about the accuracy of the test. In such cases, additional tests such as DNA sequencing, MALDI-TOF MS, or PCR may be required to confirm the identity of the organism.

Butyrate disk test is used for the following purposes:

• It is a rapid and simple presumptive identification test for the detection of Moraxella catarrhalis, a major pathogen that causes various respiratory infections and is often resistant to penicillin and ampicillin. The test can be performed within 5 minutes and does not require any special equipment or reagents.
• It is a biochemical test that measures the ability of an organism to hydrolyze butyrate substrate by the production of butyrate esterase, an enzyme that is present in M. catarrhalis but not in most other bacteria. The test can differentiate between M. catarrhalis and other oxidase-positive, Gram-negative diplococci such as Neisseria gonorrhoeae and Neisseria lactamica, which are butyrate negative.
• It is a confirmatory test that can be used along with other criteria such as morphology, Gram stain, oxidase test, and DNase test to provide a definitive identification of M. catarrhalis from clinical samples. The test can also be used to monitor the quality of culture media and reagents.

The butyrate disk test is a rapid and simple test for the presumptive identification of M. catarrhalis, but it has some limitations that should be considered before reporting the results.

• The test relies on the detection of butyrate esterase, an enzyme that hydrolyzes butyrate substrates to produce color or fluorescence. However, not all strains of M. catarrhalis produce this enzyme, and some strains may produce it in low amounts or only under certain conditions. Therefore, a negative test does not rule out the presence of M. catarrhalis, and further confirmatory tests may be needed.
• Conversely, a positive test does not necessarily indicate the presence of M. catarrhalis, as some other organisms may also produce butyrate esterase and give a false-positive reaction. These include some strains of other Moraxella species, as well as Eikenella and Acinetobacter. Therefore, a positive test should be interpreted in conjunction with other criteria, such as Gram stain, oxidase test, and colony morphology on blood agar plates.
• Another limitation of the test is that it requires careful timing and observation of the reaction. The disks should not be incubated beyond 5 minutes, as prolonged incubation may lead to false-positive results due to non-specific hydrolysis or oxidation of the substrates. The color or fluorescence change should also be observed under appropriate lighting conditions, as ambient light or UV light may interfere with the detection of the reaction.
• Additionally, the test may be affected by the quality and storage of the disks, as well as the inoculum size and age of the culture. The disks should be stored in a dark and dry place at room temperature, and discarded if they appear discolored or contaminated. The inoculum should be heavy and visible, and taken from a fresh culture (24 to 72 hours old) to ensure optimal enzyme activity. If the inoculum is too small or the culture is too old, the test may give a false-negative result.

In summary, the butyrate disk test is a useful tool for the rapid identification of M. catarrhalis, but it has some limitations that should be taken into account before reporting the results. The test should be performed and interpreted carefully, and confirmed by other methods if necessary.

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