Coagulase test is an analytical method that demarcates the staphylococci species into coagulase-positive and coagulase-negative species. Staphylococcus aureus, S. intermedius, S. hyicus, are the coagulase-positive organisms, whereas Staphylococcus epidermis, S. saprophyticus, S. haemolyticus, S. lugdonensis etc. are coagulase-negative organisms. Coagulase is an enzyme that can either bound to the cell surface of bacteria as “Bound coagulase” or present in the extracellular space as “Free coagulase”.
Slide and tube coagulation method come into use for the detection of bound and free coagulase, respectively. Coagulase binds with prothrombin that results in proteolysis of fibrinogen into fibrin.
Content: Coagulase Test
Definition of Coagulase Test
Coagulase test is a biochemical test, which is employed to primarily differentiate coagulase-positive Staphylococcus aureus from the other Staphylococcus isolates. It detects the production of coagulase enzyme that breaks the peptide bonds of fibrinogen to convert it into fibrin clot. Coagulase test includes two methods, namely slide and tube coagulase test. Slide coagulase method is generally used to check the formation of bound coagulase by the bacterial isolates. Tube coagulase method is used to check the presence of free coagulase.
Let us discuss some important terms that we must keep in mind while reading this article.
It can define as a “Protein enzyme” that combines with thrombin and forms Staphylothrombin complex. This complex then activates the protease enzyme that breaks down the peptide bonds of the “Fibrinogen” glycoprotein and converts it into visible strands of “Fibrin clots”.
Coagulase positive organisms produce coagulase enzyme that causes conversion of soluble fibrinogen into the solid fibrin clots. In contrast, the coagulase-negative microorganisms cannot convert fibrinogen due to lack of coagulase exoenzyme. Staphylococcus aureus and other coagulase-positive organisms produce two kinds of coagulase exoenzyme, namely bound and free coagulase.
- Bound coagulase: It also refers as “Clumping factor”, whose presence is observed after performing the slide coagulase method. Bound coagulase is stable against heat and non-virulent. It is a screening method to detect the existence of coagulase in a test organism.
- Free coagulase: It also refers as “Extracellular coagulase”, whose presence is spotted after performing the tube coagulase method. Free coagulase enzyme is unstable in action to heat and acts as a virulent factor. It is a confirmatory method to identify whether the organism is coagulase-positive or negative, in a test organism.
It also refers as “Fibrinogenase”, and its primary function is to form fibrin clots by the proteolysis of internal fibrinopeptide in fibrinogen plasma protein. Thrombin works as a clotting enzyme that forms 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 which acts as a “Determiner of platelet aggregation”. Thrombin acts upon the substrate (fibrinogen) and breaks the fibrinopeptide bonds of α and β chains towards the amino-terminal. It is glycoprotein that produces from the hepatocytic cells of the liver. It has a molecular weight of 340kDa, and its average concentration is 150-400 mg/dl in the blood plasma.
It is an insoluble protein, which appears as long fibrous threads. Fibrin is an active protein that participates in the blood clotting, as it helps in aggregation of 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 of plasma protein “Fibrinogen” by the action of extracellular coagulase enzyme produced by coagulase-positive microorganisms. 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 results in precipitation or clumping of fibrinogen. Free coagulase enzyme combines with the coagulase reacting factor, which results in the clotting of fibrinogen, as fibrin strands.
Coagulase enzyme is also responsible for the pathogenicity of the microorganisms, as it attaches to the cell membrane of bacteria and provides 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 Staphylococci.
The purposes of the coagulase test are as follows:
- It is used to detect the production of exocellular “Coagulase” by the microorganisms.
- Coagulase test is used to distinguish among the two specific groups of bacteria, namely coagulase-positive species (CPS) and coagulase-negative species (CNS) based on coagulase production.
- It is used to check the organism’s ability to coagulate plasma protein fibrinogen into fibrin clots.
Slide Coagulase Method
It detects the cell-bound coagulase, which directly agglutinates the plasma to the fibrin clot. It involves the mixing of bacterial suspension with a drop of plasma. The agglutination of plasma indicates positive coagulase test, while the slides with a negative result are confirmed by performing tube coagulase test. Therefore, the slide coagulase test is a screening method to check whether coagulase is present or absent, while the tube coagulase test confirms the coagulase presence or absence.
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 by reacting with coagulase reacting factor (Thrombin), and converts it into fibrin. It involves incubation of the media containing bacteria suspension added with plasma clotting within 4 hours, 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 1ml 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 clot formation the slightly tilting the tube.
Positive result: Confirms by the formation of visible clots within 4 hours.
Negative result: Here, the fibrin clots will not form.