Coagulase Test- Principle, Procedure, Types, Result, Uses

Staphylococci are Gram-positive bacteria that form clusters and are commonly found on the skin and mucous membranes of humans and animals. Some species of Staphylococci are harmless commensals, while others can cause infections ranging from minor skin infections to life-threatening diseases such as endocarditis, septicemia, and toxic shock syndrome. One of the most important and pathogenic species of Staphylococci is Staphylococcus aureus, which is responsible for many hospital-acquired and community-acquired infections.

To identify and diagnose S. aureus infections, it is essential to differentiate it from other Staphylococcal species that may have similar morphology and biochemical characteristics. One of the most reliable and widely used methods for this purpose is the coagulase test. The coagulase test is based on the ability of S. aureus to produce an enzyme called coagulase, which can cause the clotting or clumping of blood plasma. Coagulase production is considered a virulence factor of S. aureus, as it helps the bacteria evade the host immune system by forming a protective layer of fibrin around them.

The coagulase test can be performed in two ways: the slide test and the tube test. The slide test is a rapid and simple method that detects bound coagulase or clumping factor on the surface of the bacterial cells. The tube test is a more sensitive and specific method that detects free coagulase or extracellular coagulase in the culture supernatant. Both methods use plasma as a source of fibrinogen, which is converted to fibrin by coagulase. The presence or absence of clotting or clumping indicates whether the organism is coagulase positive or negative.

The coagulase test is useful for differentiating S. aureus from other Staphylococcal species, as S. aureus is usually coagulase positive, while most other Staphylococci are coagulase negative. However, there are some exceptions and limitations to this rule, which will be discussed later in this article. Therefore, the coagulase test should always be performed along with other tests to confirm the identity of S. aureus.

In this article, we will explain the principle, procedure, types, result, uses, and limitations of the coagulase test in detail. We will also provide some examples and images to help you understand this test better.

The coagulase test is based on the ability of some bacteria to produce an enzyme called coagulase, which can cause the clotting of blood plasma. Coagulase is a virulence factor that helps the bacteria evade the host immune system by forming a protective layer of fibrin around them.

There are two types of coagulase: bound and free. Bound coagulase, also known as clumping factor, is attached to the bacterial cell wall and can directly agglutinate plasma or latex particles coated with fibrinogen. Free coagulase is secreted by the bacteria and requires a coagulase-reacting factor (CRF) in the plasma to form a complex that can convert fibrinogen to fibrin.

The coagulase test can be performed by two methods: slide and tube. The slide method is a rapid screening test that detects bound coagulase by mixing a bacterial suspension with a drop of plasma on a glass slide and observing for clumping within 10 seconds. The tube method is more sensitive and specific and detects both bound and free coagulase by incubating a bacterial culture with diluted plasma in a test tube and observing for clot formation within 24 hours.

The coagulase test is mainly used to differentiate Staphylococcus aureus from other Staphylococci species, as S. aureus is usually coagulase-positive while most other Staphylococci are coagulase-negative. However, some exceptions exist, such as S. lugdunensis and S. schleiferi, which are coagulase-positive but not S. aureus, and some methicillin-resistant S. aureus (MRSA), which are coagulase-negative but still pathogenic.

Coagulase is an enzyme that can cause blood clotting by converting fibrinogen into fibrin. S. aureus produces two types of coagulase: bound coagulase and free coagulase.

Bound coagulase, also known as clumping factor, is attached to the bacterial cell wall and can directly act on fibrinogen in the plasma to form clumps. This can be detected by the slide test, which is a rapid but less sensitive method. Bound coagulase can also interact with another substance in the cell wall of S. aureus, called protein A, which binds to the Fc portion of human immunoglobulin G (IgG). This enhances the clumping reaction when latex or erythrocyte particles coated with IgG and fibrinogen are used.

Free coagulase, on the other hand, is secreted by the bacteria into the surrounding medium and requires a coagulase-reacting factor (CRF) in the plasma to form a complex. This complex then acts on fibrinogen to form a clot. This can be detected by the tube test, which is a more sensitive but slower method. Free coagulase is usually measured using rabbit plasma, which has a higher CRF activity than human plasma.

Both bound and free coagulase are important virulence factors of S. aureus, as they help the bacteria evade phagocytosis and immune clearance by forming a protective layer of fibrin around them. They also facilitate the invasion of tissues and organs by breaking down blood vessels and causing inflammation. However, not all strains of S. aureus produce both types of coagulase, and some may even be coagulase-negative. Therefore, other tests are needed to confirm the identification of S. aureus.

The coagulase test is mainly performed on Gram-positive cocci in clusters that are catalase-positive, as part of the identification of S. aureus. These bacteria are commonly isolated from skin, wound, and blood infections. The coagulase test can also be used for rapid detection of S. aureus in positive blood cultures containing Gram-positive cocci in clusters.

The main reagent used in the coagulase test is frozen plasma with EDTA. EDTA is an anticoagulant that prevents the plasma from clotting by itself. The plasma can be obtained from rabbit or human sources, but rabbit plasma is preferred as it is more sensitive and less infectious than human plasma. The plasma should be stored at -20°C and thawed at 37°C before use.

The other reagent that may be used in the coagulase test is 5% CaCl2 (optional). This is added to the tube test if no clot formation is observed after 24 hours. CaCl2 can enhance the coagulation process by providing calcium ions that are required for fibrin formation.

The supplies needed for the coagulase test are:

• Loops or sterile sticks for inoculating the bacteria
• Glass or plastic tubes for the tube test
• Glass slides for the slide test

The coagulase test requires a fresh culture of the bacteria to be tested, preferably grown on a non-selective medium such as blood agar or tryptic soy agar. The culture should not be older than 24 hours, as older cultures may lose their coagulase activity or produce autoagglutinins that interfere with the test results.

The coagulase test can be performed by two different methods: the slide test and the tube test. The slide test is a rapid and simple method to detect bound coagulase or clumping factor on the surface of the bacterial cells. The tube test is a more sensitive and reliable method to detect free coagulase that is released by the bacteria into the medium. The following are the steps for each method:

Slide test

1. Add about 10 µl of deionized water or physiological saline to a clean glass slide.
2. Pick several colonies from a fresh culture of the test organism with an inoculating loop or a sterile stick and emulsify them in the water to obtain a smooth milk-colored suspension.
3. Add a drop of rabbit or human plasma to the slide and mix it with the bacterial suspension.
4. Observe the slide immediately for clumping or agglutination of the bacterial cells, not exceeding 10 seconds.
5. Record the result as positive or negative.

Tube test

1. Dilute rabbit plasma with physiological saline in a ratio of 1:10 (add 0.2 ml plasma to 1.8 ml saline) in a small tube or vial.
2. Transfer 0.5 ml of the diluted plasma to a clean glass or plastic tube.
3. Add about 5 drops of the test organism culture (preferably from an overnight broth culture) to the tube containing the diluted plasma.
4. Mix the tube gently and incubate it at 37°C for an hour.
5. Examine the tube for clot formation by tilting it slightly. If no clotting is observed, continue to incubate the tube and examine it at 30 minutes intervals for up to 6 hours.
6. Record the result as positive or negative.

The coagulase test results can be interpreted by observing the presence or absence of clumping or clotting in the slide or tube methods. The clumping or clotting indicates that the organism produces coagulase, which reacts with the fibrinogen in the plasma and forms fibrin. The fibrin then binds the bacterial cells together and forms a visible aggregate.

The slide test is a rapid and simple method to detect the bound coagulase or clumping factor on the surface of the bacterial cells. A positive slide test is indicated by the agglutination of the bacterial cells within 10 seconds after adding a drop of plasma to the bacterial suspension. A negative slide test is indicated by no agglutination within 10 seconds.

The tube test is a more sensitive and reliable method to detect the free coagulase that is secreted by the bacterial cells. A positive tube test is indicated by the formation of a clot in the plasma within 24 hours of incubation at 37°C. The clot may be complete or partial, and it may be firm or friable. A negative tube test is indicated by no clot formation within 24 hours of incubation at 37°C.

Some organisms may produce weak or delayed coagulase reactions, which can be enhanced by adding a few drops of 5% calcium chloride (CaCl2) solution to the tube. Calcium ions act as cofactors for the coagulase enzyme and facilitate its activity. If a clot forms after adding CaCl2, the test is considered positive. If no clot forms even after adding CaCl2, the test is considered negative.

The coagulase test can be used to differentiate Staphylococcus aureus from other Staphylococcal species, as S. aureus is usually coagulase positive, while most other Staphylococci are coagulase negative. However, some exceptions exist, such as S. intermedius and S. hyicus, which are coagulase positive but are usually found in animals rather than humans. Some other Staphylococcal species, such as S. lugdunensis and S. schleiferi, may produce slide coagulase but not tube coagulase, and they may require human plasma rather than rabbit plasma for the reaction.

Therefore, it is important to confirm the identity of S. aureus by performing other tests, such as mannitol fermentation test, catalase test, DNAse test, and antibiotic susceptibility test, along with the coagulase test.

• The coagulase test is used to determine the production of coagulase by different microorganisms .
• This test can also be used to differentiate S. aureus from other Staphylococcal species .
• Staphylococcal species are differentiated into coagulase-negative and coagulase-positive species on the basis of the production of the coagulase enzyme.
• Coagulase-positive Staphylococci are usually more pathogenic and virulent than coagulase-negative Staphylococci.
• S. aureus is the most common coagulase-positive Staphylococcus that causes various infections in humans and animals.
• The coagulase test can help in the rapid identification and confirmation of S. aureus from clinical specimens and positive blood cultures .
• The coagulase test can also help in the detection of methicillin-resistant S. aureus (MRSA), which is a major cause of hospital-acquired infections.
• The coagulase test can also be used to identify some rare strains of S. aureus that do not produce coagulase by using thermonuclease test.

The coagulase test is a simple and rapid method to identify S. aureus and differentiate it from other Staphylococcal species. However, the test has some limitations and potential sources of error or false results that should be considered.

• Some strains of methicillin-resistant S. aureus (MRSA) may be deficient in bound coagulase, which results in a negative slide test. These strains can be detected by the tube test or by other methods such as latex agglutination or PCR.
• Some animal-associated Staphylococcal species such as S. intermedius, S. hyicus, S. delphini, and S. schleiferi may also produce coagulase and cause false-positive results in the coagulase test. These species are usually found in dogs, pigs, dolphins, and cats, respectively, but they can also infect humans and cause diseases similar to S. aureus. Therefore, it is important to confirm the identity of coagulase-positive isolates by other biochemical or molecular tests.
• Some coagulase-negative Staphylococcal species such as S. lugdunensis and S. schleiferi may produce slide coagulase, but not tube coagulase. These species can cause false-positive results in the slide test, but they can be distinguished by using human plasma instead of rabbit plasma, which is more sensitive to their clumping factor. These species are also more virulent than other coagulase-negative Staphylococci and can cause serious infections such as endocarditis and osteomyelitis.
• The quality and source of plasma used in the coagulase test can affect the accuracy of the results. Human plasma is commonly not used for the test, as it is less sensitive and potentially infectious with human pathogenic viruses. Rabbit plasma is preferred, but it should be stored at -20°C and thawed only once before use. Repeated freezing and thawing can reduce the activity of fibrinogen and cause false-negative results. Citrated blood should not be used as it can cause false-positive results due to the presence of anticoagulant.
• The incubation time and temperature of the tube test can also influence the results. The optimal incubation time is 4 hours at 37°C, but some strains may require up to 24 hours to produce a visible clot. Longer incubation times can increase the risk of contamination or nonspecific clotting due to bacterial growth or plasma degradation. The optimal incubation temperature is 37°C, but some strains may produce coagulase at lower or higher temperatures. Therefore, it is recommended to perform the tube test at both 25°C and 35°C to detect temperature-dependent coagulase production.

The coagulase test is a useful tool for identifying S. aureus and differentiating it from other Staphylococcal species. However, it should be performed with caution and interpreted with other tests to avoid false results and misidentification of coagulase-producing organisms.

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