Microdase (Modified Oxidase) Test- Principle, Procedure, Results

The microdase test is a simple and rapid method to differentiate gram-positive, catalase-positive cocci (micrococci from staphylococci). Gram-positive cocci are spherical bacteria that stain purple with the Gram stain and produce bubbles when exposed to hydrogen peroxide (catalase test). However, these characteristics are not enough to distinguish between the genera Micrococcus and Staphylococcus, which have different clinical and epidemiological significance. Micrococci are generally harmless commensals of the skin and mucous membranes, while staphylococci are opportunistic pathogens that can cause a variety of infections, such as skin infections, food poisoning, endocarditis, and toxic shock syndrome. Therefore, it is important to identify these bacteria accurately in the laboratory.

The microdase test is based on the presence or absence of oxidase enzyme in the bacterial cells. Oxidase is an enzyme that transfers electrons from a donor molecule to molecular oxygen, forming water or hydrogen peroxide. Oxidase is part of the electron transport chain in some bacteria, such as micrococci, that use oxygen as the final electron acceptor in aerobic respiration. Staphylococci, on the other hand, do not have oxidase enzyme and use other molecules as electron acceptors.

The microdase test uses a filter paper disk impregnated with an oxidase reagent, which is a chemical compound that changes color when oxidized by the oxidase enzyme. The most commonly used oxidase reagent is tetramethyl-p-phenylenediamine dihydrochloride (TMPD), which turns blue or purple when oxidized. By rubbing a small amount of bacterial colonies onto the disk and observing for color change within 2 minutes, one can determine whether the bacteria are oxidase-positive (micrococci) or oxidase-negative (staphylococci).

The microdase test is also known as modified oxidase test because it uses dimethyl sulfoxide (DMSO) as a solvent for the oxidase reagent. DMSO is a polar organic compound that can penetrate the cell membrane and enhance the permeability of the bacterial cells to the reagent. This increases the sensitivity and specificity of the test and reduces the false-negative results that may occur with some strains of micrococci that have low levels of oxidase enzyme.

The media used in the microdase test are filter paper disks that contain the oxidase reagent. The oxidase reagent is a chemical compound called tetramethyl-p-phenylenediamine dihydrochloride (TMPD), which is dissolved in dimethyl sulfoxide (DMSO). DMSO is a solvent that helps the reagent penetrate the bacterial cells.

The filter paper disks are also known as oxidase discs or microdase discs. They are commercially available and come in a stock bottle. The discs should be stored in a dark and cool place to prevent oxidation of the reagent.

The oxidase reagent acts as an electron donor for the oxidase enzyme, which is present in some bacteria. The oxidase enzyme transfers electrons from the reagent to cytochrome C, a protein involved in cellular respiration. This process produces indophenol, a colored compound that turns blue or purple blue on the disc.

The color change indicates a positive microdase test, which means that the bacteria have oxidase enzyme and belong to the genus Micrococcus. A negative microdase test shows no color change on the disc, which means that the bacteria do not have oxidase enzyme and belong to the genus Staphylococcus. However, some exceptions exist, such as S. sciuri, S. lentus, and S. vitulus, which are oxidase-positive staphylococci.

The microdase test is a simple and quick method to differentiate Staphylococcus from Micrococcus based on the presence or absence of oxidase enzyme. The procedure involves the following steps:

• Using sterile forceps, transfer a microdase disk from the stock bottle to a clean and dry petri dish. The microdase disk is a filter paper disk impregnated with tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) in dimethyl sulfoxide (DMSO). The oxidase reagent is colorless in its reduced form and turns blue or purple blue in its oxidized form.
• Using a wooden applicator stick, rub a small amount of several colonies of an 18-24 hour pure culture grown on blood agar onto the top of the microdase disk. Do not use a metal loop or needle as they may interfere with the reaction. Avoid touching the disk with your fingers as they may contain oxidase-positive bacteria from the skin flora.
• Incubate the disk at room temperature for 2 minutes. Do not incubate longer than 2 minutes as false-positive results may occur due to atmospheric oxidation of the reagent.
• Observe the color change on the disk. A positive test is indicated by the development of a blue or purple blue color on the disk within 2 minutes. A negative test is indicated by no color change on the disk.

The following image shows an example of a positive microdase test (left) and a negative microdase test (right).

The microdase test is based on the color change of the oxidase reagent when it reacts with the oxidase enzyme present in some bacteria. The oxidase reagent is a colorless compound that turns blue or purple blue when it is oxidized by the enzyme. The color change indicates a positive test result, meaning that the bacteria have the oxidase enzyme and belong to the genus Micrococcus. A negative test result means that the bacteria do not have the oxidase enzyme and belong to the genus Staphylococcus (with some exceptions).

To interpret the results of the microdase test, observe the color of the microdase disk after 2 minutes of incubation at room temperature. If the disk turns blue or purple blue within 2 minutes, the test is positive and the bacteria are micrococci. If the disk remains colorless or shows a faint pink color, the test is negative and the bacteria are staphylococci (except for S. sciuri, S. lentus, and S. vitulus, which are oxidase-positive staphylococci).

The microdase test is a simple and rapid method to differentiate between micrococci and staphylococci, which are both gram-positive, catalase-positive cocci. However, it is important to use a pure culture of bacteria and a fresh microdase disk to avoid false results. The microdase test should also be performed along with other biochemical tests to confirm the identification of bacteria.

• The microdase test is not suitable for differentiating all staphylococci from micrococci, as some staphylococcal species may produce a positive reaction. These include S. sciuri, S. lentus, and S. vitulus. These species are usually associated with animals and are rarely isolated from human infections.
• The microdase test should be performed on pure cultures only, as mixed cultures may give false results due to the presence of oxidase-positive bacteria other than micrococci, such as Neisseria, Pseudomonas, Aeromonas, etc.
• The microdase test should be performed within 2 minutes of inoculation, as prolonged exposure to air may cause oxidation of the reagent and lead to false-positive results.
• The microdase test should be performed with fresh reagent disks, as the reagent may deteriorate over time and lose its sensitivity.
• The microdase test should not be performed on colonies grown on media containing glucose or other fermentable sugars, as these may interfere with the oxidation reaction and inhibit color development.

To ensure the validity and accuracy of the microdase test, quality control strains should be tested along with the unknown isolates. A positive control strain that produces oxidase enzyme and a negative control strain that does not produce oxidase enzyme should be used. The recommended quality control strains are:

• Positive: Micrococcus luteus (ATCC10240)
• Negative: Staphylococcus aureus (ATCC25923)

Micrococcus luteus is a gram-positive, catalase-positive, oxidase-positive coccus that forms yellow colonies on blood agar. Staphylococcus aureus is a gram-positive, catalase-positive, oxidase-negative coccus that forms golden colonies on blood agar and produces coagulase enzyme.

The quality control strains should be tested in the same manner as the unknown isolates. A small amount of each strain should be rubbed onto a microdase disk and incubated at room temperature for 2 minutes. The color change on the disk should be observed and recorded.

The expected results are:

• Positive: Micrococcus luteus - blue or purple blue color on the disk
• Negative: Staphylococcus aureus - no color change on the disk

If the quality control results are as expected, then the test is valid and the results of the unknown isolates can be interpreted. If the quality control results are not as expected, then the test is invalid and the results of the unknown isolates cannot be interpreted. Possible reasons for invalid results include:

• Contamination of the culture or the disk
• Improper storage or expiration of the disk
• Inadequate amount or age of the culture
• Incorrect incubation time or temperature
• Interference from other substances in the culture

In case of invalid results, the test should be repeated with fresh culture and disk, following the proper procedure and precautions. If the problem persists, then a different method of oxidase detection should be used.

The microdase test is a simple and rapid method to differentiate micrococci from staphylococci, which are both gram-positive, catalase-positive cocci. This differentiation is important for clinical and epidemiological purposes, as micrococci and staphylococci have different pathogenic potentials and antibiotic susceptibilities.

Micrococci are generally considered as commensals or opportunistic pathogens that can cause infections in immunocompromised hosts or in patients with indwelling medical devices. Micrococci are usually susceptible to penicillin and resistant to lysozyme. Some species of micrococci, such as Micrococcus luteus and Micrococcus roseus, can produce yellow or pink pigments on agar media.

Staphylococci are more common and virulent pathogens that can cause a variety of infections, such as skin and soft tissue infections, abscesses, endocarditis, osteomyelitis, septicemia, and toxic shock syndrome. Staphylococci are usually resistant to penicillin and susceptible to lysozyme. Some species of staphylococci, such as Staphylococcus aureus and Staphylococcus epidermidis, can produce coagulase or biofilms.

The microdase test can help to distinguish micrococci from staphylococci based on their oxidase activity. Micrococci are oxidase-positive, meaning that they have the enzyme oxidase that can oxidize the tetramethyl-p-phenylenediamine reagent to produce a blue or purple color on the filter paper disk. Staphylococci are oxidase-negative, meaning that they lack the enzyme oxidase and do not change the color of the disk.

The microdase test can also be used to identify some species of micrococci based on their degree of oxidase activity. For example, Micrococcus luteus has a strong oxidase activity and produces a dark blue color on the disk within 10 seconds. Micrococcus varians has a weak oxidase activity and produces a light blue color on the disk within 2 minutes. Micrococcus sedentarius has a delayed oxidase activity and produces a blue color on the disk after 2 minutes.

The microdase test is a useful tool for the identification and characterization of gram-positive, catalase-positive cocci in clinical microbiology laboratories. However, it should be used in conjunction with other biochemical tests and molecular methods to confirm the results and avoid misidentification.

The microdase test is a simple and rapid method to differentiate between micrococci and staphylococci, which are both gram-positive, catalase-positive cocci. This differentiation is important for the identification and classification of these bacteria, as well as for the diagnosis and treatment of infections caused by them.

Micrococci are generally harmless commensals of the skin and mucous membranes, but they can occasionally cause opportunistic infections such as endocarditis, meningitis, septicemia, and wound infections. Micrococci are resistant to penicillin and other beta-lactam antibiotics, but they are susceptible to erythromycin, tetracycline, and vancomycin.

Staphylococci are more common and more pathogenic than micrococci. They can cause a variety of infections such as boils, abscesses, impetigo, cellulitis, mastitis, osteomyelitis, pneumonia, toxic shock syndrome, and food poisoning. Staphylococci are also notorious for their ability to develop resistance to multiple antibiotics, especially methicillin-resistant Staphylococcus aureus (MRSA), which poses a serious threat to public health.

The microdase test can help to distinguish between these two groups of bacteria based on their oxidase activity. Micrococci produce the enzyme oxidase, which catalyzes the oxidation of the oxidase reagent to form a blue or purple color on the disc. Staphylococci do not produce oxidase, and thus do not change the color of the disc.

The microdase test can also be used to identify some other bacteria that produce oxidase, such as Neisseria, Pseudomonas, Aeromonas, Campylobacter, and Vibrio. However, these bacteria are gram-negative and can be easily differentiated from micrococci by their morphology and staining properties.

The microdase test is a useful tool for the preliminary identification of gram-positive cocci in clinical microbiology laboratories. However, it should be confirmed by other biochemical tests and molecular methods for accurate identification and characterization of bacterial isolates.

The microdase test is a useful tool for the identification and differentiation of gram-positive, catalase-positive cocci, especially micrococci and staphylococci. Micrococci are widely distributed in nature and can be found in soil, water, dust, air, and various surfaces. They are also part of the normal flora of the skin, mucous membranes, and oropharynx of humans and animals. Some species of micrococci, such as Micrococcus luteus and Micrococcus roseus, can cause opportunistic infections in immunocompromised hosts or those with prosthetic devices.

Staphylococci are also common inhabitants of the skin and mucous membranes of humans and animals. They are responsible for a variety of infections ranging from minor skin infections to life-threatening diseases such as endocarditis, septicemia, pneumonia, and toxic shock syndrome. The most important pathogen among staphylococci is Staphylococcus aureus, which can produce several virulence factors such as coagulase, hemolysins, leukocidins, enterotoxins, exfoliative toxins, and toxic shock syndrome toxin.

The microdase test can help to distinguish micrococci from staphylococci based on their ability to produce oxidase enzyme. Oxidase enzyme catalyzes the transfer of electrons from a reduced donor to molecular oxygen, forming water or hydrogen peroxide. The oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride) acts as an artificial electron donor that changes color from colorless to blue or purple-blue when oxidized by the enzyme.

Micrococci possess cytochrome c oxidase, which reacts with the oxidase reagent and produces a positive result. Most staphylococci lack this type of cytochrome and give a negative result. However, there are some exceptions among staphylococci that can produce a positive microdase test. These include Staphylococcus sciuri, Staphylococcus lentus, and Staphylococcus vitulus . Therefore, the microdase test should be used in conjunction with other biochemical tests to confirm the identification of gram-positive cocci.

The microdase test is a simple, rapid, and inexpensive method that can be performed in any microbiology laboratory. It requires only a filter paper disk impregnated with the oxidase reagent and a pure culture of the organism to be tested. The test can be completed within 2 minutes and does not require any special equipment or incubation conditions. The microdase test is a valuable addition to the battery of tests used for the identification of gram-positive cocci.

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