Coagulase test is an analytical method that demarcates the staphylococci species into coagulase-positive and coagulase-negative species. Coagulase is an enzyme, which can either bound to the cell surface of bacteria as “Bound coagulase” or reside within the extracellular space as “Free coagulase”.
- Staphylococcus aureus, S. intermedius, S. hyicus, are the coagulase-positive organisms.
- Staphylococcus epidermis, S. saprophyticus, S. haemolyticus, S. lugdonensis etc. are coagulase-negative organisms.
Slide and tube coagulation method come into use to confer the presence of bound and free coagulase, respectively. Coagulase binds with prothrombin, which results in the proteolysis of fibrinogen into fibrin.
In this context, we will discuss the definition, principle, purpose and the two popular methods of the coagulase test. You would also get to know the meaning of some important terms related to this context.
Content: Coagulase Test
Definition of Coagulase Test
Coagulase test is a biochemical test, prevalently employed to differentiate coagulase-positive Staphylococcus aureus from the other Staphylococcus isolates, depending upon the production of coagulase enzyme. Coagulases break the peptide bonds of fibrinogen to convert it into fibrin clots. Coagulase test procedure involves two methods, namely slide and tube coagulase test.
- Slide coagulase method determines the formation of bound coagulase by the bacterial isolates.
- Tube coagulase method determines the presence of free coagulase.
Let us discuss some important terms, which we must keep in mind while studying this article.
It refers to a protein enzyme, which associates with thrombin and forms Staphylothrombin complex. It results in activation of the proteases, which eventually cause proteolysis of fibrinogen (glycoprotein). Thus, the peptide bonds of the fribrinogen breaks to produce visible strands of fibrin clots.
Coagulase positive organisms produce coagulase enzyme, which causes conversion of soluble fibrinogen into the solid fibrin clots. In contrast, the coagulase-negative microorganisms cannot convert fibrinogen, as they lack coagulase exoenzyme. Staphylococcus aureus and other coagulase-positive organisms produce two kinds of coagulase exoenzyme, namely bound and free coagulases.
- Bound coagulase: It refers to a “Clumping factor”, whose presence becomes evident after performing the slide coagulase method. Bound coagulase is stable against heat and it serves as a non-virulent factor. It is a screening method to detect the existence of coagulase in a test organism.
- Free coagulase: It refers to an extracellular coagulase, whose presence can be spotted after performing the tube coagulase method. Free coagulase enzyme is unstable in action to heat and it acts as a virulent factor. It is a confirmatory method to identify whether a test organism is coagulase-positive or negative.
It refers to the fibrinogenase, which primary function is to form fibrin clots by causing proteolysis of internal fibrinopeptide in the fibrinogen’s plasma protein.
Thrombin works as a clotting enzyme, which comes into action after the splitting of prothrombin by the enzymatic activity of prothrombinase. Then, thrombin acts upon fibrinogen glycoprotein that further splits it into individual strands of fibrin.
It is a kind of glycoprotein, which functions as a substrate of thrombin. Fibrinogen is an extracellular protein in the plasma that acts as a “Determiner of platelet aggregation”. It has a molecular weight of 340kDa, and an average concentration of 150-400 mg/dl in the blood plasma.
Thrombin acts upon the substrate (fibrinogen). It breaks the fibrinopeptide bonds of α and β chains towards the amino-terminal. The hepatocytic cells of the liver produce fribrinogen glycoprotein.
It is an insoluble protein, which appears as the long fibrous threads. Fibrin is an active protein that participates in the blood clotting. It helps in aggregating the platelets to form a spongy mass that stiffens to form a blood clot.
Principle of Coagulase Test
The mechanism of the coagulase test is based on the agglutination reaction. An extracellular coagulase enzyme produced by the coagulase-positive microorganisms break the plasma protein (fibrinogen). Coagulase test differentiates organisms into two categories (coagulase-positive and coagulase-negative bacteria).
Coagulase positive microorganisms produce bound and free coagulase enzyme. The bound coagulase combines with the fibrinogen protein of blood plasma and ultimately leads to the precipitation or clumping of fibrinogen.
Free coagulase enzyme combines with the coagulase reacting factor, which results in the clotting of fibrinogen to fibrin strands. Coagulase enzyme is also responsible for the pathogenicity of the microorganisms.
The cell membrane of some bacteria possess coagulases, which provide resistance against phagocytosis and other host defences. Therefore, the coagulase test also differentiates between the coagulase-positive and potentially virulent organism (Staphylococcus aureus) from the other isolates of Staphylococci.
The coagulase test aims the following:
- It is used to detect the production of exocellular coagulase by the microorganisms.
- Coagulase test discriminates the two specific groups of bacteria, namely coagulase-positive species (CPS) and coagulase-negative species (CNS), based on the coagulase production.
- It also checks the organism’s ability to coagulate the plasma protein fibrinogen into fibrin clots.
Slide Coagulase Method
It detects the cell-bound coagulase, which directly agglutinates the plasma into fibrin clots. It involves the mixing of bacterial suspension with a drop of plasma. The agglutination of plasma indicates positive coagulase test.
In contrast, the slides with a negative result are confirmed by following tube coagulase test. Therefore, the slide coagulase test is a screening method, which only checks the presence or absence of coagulase.
Slide coagulase test involves the following steps:
- Take a clean, grease-free glass slide.
- Pour one drop of undiluted blood plasma towards the centre of the glass slide.
- Sterilize an inoculating loop, and carefully take the bacterial suspension.
- After that, mix the bacterial suspension with the drop of blood plasma by moving the sterilized inoculating loop in a clockwise and anticlockwise direction, until the formation of milky suspension.
- Allow the glass slide to stand for a few seconds, and then note down the results based on agglutination reaction.
Tube Coagulase Method
It detects the presence of free-coagulase (Staphylocoagulase), which indirectly reacts with fibrinogen. Free coagulase reacts with the coagulase reacting factor (thrombin) and converts it into fibrin. It involves incubation of the media containing bacterial suspension added with plasma clotting within 4 hours. It is a confirmatory test that confirms the presence of coagulase.
Tube coagulase test involves the following steps:
- Perform serial dilution of the blood plasma in a series of sterile test tubes.
- Pour 1 ml of diluted plasma into the sterile tube.
- Sterilize an inoculating loop under the flame, and carefully take the bacterial suspension.
- After that, emulsify the bacterial suspension with plasma until the formation of a milky suspension.
- Allow the test tube to stand for 4 hours in a hot water bath.
- At last, note down the results based on the clot formation by slightly tilting the tube.
Positive result: The formation of visible clots develop within 4 hours.
Negative result: No formation of fibrin clots.