Coagulase Test Principle, Procedure, Application, Result.
Coagulase Test Principle, Procedure, Application, Result.

Microbiology Techniques

Coagulase Test Principle, Procedure, Application, Result.

It is a type of biochemical test, which is used to differentiate Staphylococcus aureus from coagulase-negative staphylococci. S.aureus produces two forms of...

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This article writter by MN Editors on August 19, 2021

Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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Coagulase test Definition

It is a type of biochemical test, which is used to differentiate Staphylococcus aureus from coagulase-negative staphylococci. S.aureus produces two forms of coagulase (i.e., bound coagulase and free coagulase).

  • Coagulase is a type of protein enzyme produced by many microorganisms which helps in the conversion of fibrinogen to fibrin. This protein enzyme helps to distinguish between different types of Staphylococcus isolates. 
  • Most importantly, S. aureus is commonly coagulase-positive, which means that a positive coagulase test confirms the presence of S. aureus or any of the other 11 coagulase-positive Staphylococci. A negative coagulase test would instead show the presence of coagulase-negative organisms for example S. epidermidis or S. saprophyticus. 
  • In some organisms, the coagulase enzyme functions as a virulence factor,  as it interacts with the fibrinogen present on the host’s cell surface.
  • The coagulase-containing microorganisms usually contain a protective barrier around themselves which helps to increase their pathogenicity and resistance against the immune system.
  • There are present two types of Coagulase such as free coagulase and bound coagulase.
  • The bound coagulase is known as the “clumping factor”, which can be easily detected by using slide coagulase test, while a free coagulase can be detected by using a tube coagulase test.

Aim of Coagulase test

  • To describe how pathogenic species of Staphylococci can be differentiated from nonpathogenic species.
  • To understand the biochemistry of the enzyme coagulase.
  • To explain how coagulase confers a survival advantage to bacteria that produce this enzyme.

Principle of Coagulase test

The enzymatic protein Coagulase is a thermostable thrombin-like substance, it helps to converts the fibrinogen into fibrin which results in clotting or clumping. S. aureus carries two different types of coagulase such as free coagulase and bound coagulase.

Bound coagulase

The slide test techniques are used to detect this type of clumping factor, but the disadvantage is this test requires several colonies and lacks sensitivity. The protein A  produced from the cell wall of S. aureus is combined with the FC moiety of human immunoglobulin G (IgG) and thus functions as a coagulase-reacting factor.

When latex or erythrocyte particles are coated with IgG and with human fibrinogen, a staphylococcus will agglutinate if either clumping factor or protein A is present in the bacterial cell wall. The appearance of the clumping factor is shown by the strength of the organism to act immediately on the fibrinogen in the plasma to clump it in a slide assay.

The analysis for the clumping factor is a speedy process but needs several colonies, and also the factor might not be present in all S. aureus organisms.

Free coagulase

The tube method is used for the detection of Free coagulase. The free coagulases are different as compared to bound coagulase, for the clotting mechanism they required the activation of a plasma coagulase-reacting factor (CRF), which is a transformed or derived thrombin molecule, to form a coagulase-CRF complex. 

The rabbit plasma is added to the tube which acts as a binding factor. After that, the complex reacts with fibrinogen and forms the fibrin clot in a test tube.

Coagulase Test reagent

  • Loops or sterile sticks
  • Glass or plastic tubes
  • Glass slides
  • 5% CaCl2 
  • Frozen plasma (preferably rabbit plasma) with EDTA. Human plasma can not be used for this test, because it is less sensitive and potentially infectious with human pathogenic viruses.

Coagulase Test Procedure

The Coagulase test can be done by two methods;

  1. Tube Method
  2. Slide Method

Coagulase Slide Test

This method measures bound coagulase. The bound coagulase is also known as the clumping factor. It cross-links the α and β chain of fibrinogen in plasma to form fibrin clot that deposits on the cell wall. As a result, individual coccus stick to each other and clumping is observed. 

  1. Divide the slide into two sections with grease pencil. One should be labeled as “test” and the other as “control
  2. 2Place a small drop of distilled water on each area
  3. Emulsify one or two colonies of Staphylococcus on blood agar plate on each drop to make a smooth suspension
  4. The test suspension is treated with a drop of citrated plasma and mixed well with a needle
  5. Do not put anything in the other drop that serves as control. The control suspension serves to rule out false positivity due to auto agglutination
  6. Clumping of cocci within 5-10 seconds is taken as positive. 
  7. Some strains of S.aureus may not produce bound coagulase, and such strains must be identified by tube coagulase test

Coagulase Tube Test

This method helps to measure free coagulase. The free coagulase secreted by S.aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.

  1. Three test tubes are taken and labeled “test”, “negative control” and “positive control”.
  2. Each tube is filled with 1 ml of 1 in 10 diluted rabbit plasma.
  3. To the tube labeled test, 0.2 ml of overnight broth culture of test
  4. bacteria is added.
  5. To the tube labeled positive control, 0.2 ml of overnight broth culture of known S.aureus is added
  6. To the tube labeled negative control, 0.2ml of sterile broth is added.
  7. All the tubes are incubated at 37oC and observe the suspensions at half hourly intervals for a period of four hours.
  8. Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube.
  9. If the test remains negative until four hours at 37oC, the tube is kept at room temperature for overnight incubation.

Coagulase Test Results and observation

Slide Test

  • Positive test: Agglutination formation of the bacterial cells after the plasma is added.
  • Negative test: The lack of agglutination.

Tube Test

Positive coagulase test

  • Complete clot formation or any degree of clot formation before 24 hours.
  • No clot formation after the addition of 1 or 2 drops of 5% CaCl2 to a tube without a clot at 24 hours.

Negative coagulase test

  • A lack of clot formation at 24 h at 25°C.
  • No clot after 24 hours at 35°C, but after the addition of 1 or 2 drops of 5% CaCl2 to the tube, a clot forms.
Coagulase Test Principle, Procedure, Application, Result.
Image Source: http://laboratorytests.org/coagulase-test/

List of Coagulase Test Positive and Negative Organisms

SpeciesTube Coagulase TestSlide Coagulase Test
Staphylococcus aureus++
Staphylococcus schleiferi+
Staphylococcus lugdunesis+
Staphylococcus delphini+
Staphylococcus intermedius+d
Staphylococcus hyicusd
Staphylococcus epidermidis
Staphylococcus saprophyticus

(+) — Delayed reaction
d — 11- 89% of strains are positive

Coagulase Test
Image source: https://commons.wikimedia.org/wiki/File:Coagulase_test_in_S._aureus_and_S._epidermidis.jpg

Other organisms which shows negative coagulase test are; Staphylococcus warneri, Staphylococcus hominis, Staphylococcus caprae

Coagulase Test Purpose

  • This test is used to differentiate between the pathogenic and nonpathogenic strains of the Staphylococcus. The pathogenic strains of S. aureus show positive results in the Coagulase test, while the nonpathogenic species(S. epidermidis) show negative results in the coagulase test.
  • The slide coagulase test is useful for screening.
  • The tube coagulase test is used for the confirmation of coagulase test.

Limitations of Coagulase test

  • The slide test should be read very quickly, as false positives can occur.
  • Auto agglutination can occur.
  • Make sure saline is used instead of water during the mixing.
  • The organism is used in the slide test method, should not be taken from the high-salt media  for example Mannitol Salt Agar, as the salt content can leads to the false positives result.
  • Over-mixing can lead to the breakdown of the clot.
  • The tube test method is more reliable as compared to the slide test method.
  • The coagulase test can not be used when we are identifying unknowns.
  • Samples must be observed for clotting within 24 hours. This is because some strains that produce coagulase also produce an enzyme called fibrinolysin, which can dissolve the clot. Therefore, the absence of a clot after 24 hours is no guarantee that a clot never formed. The formation of a clot by 12 hours and the subsequent disappearance of the clot by 24 hours could produce a so-called false negative if the test were only observed at the 24-hour time.
  • A false-positive result can occur if Citrated blood is used.

References

  • https://en.wikipedia.org/wiki/Coagulase
  • https://microbiologyinfo.com/coagulase-test-principal-procedure-types-interpretation-and-examples/
  • https://microbeonline.com/diagnostic-tests-biochemical-tests-coagulase-test/
  • https://microbenotes.com/coagulase-test-principle-procedure-and-result-interpretation/
  • https://vlab.amrita.edu/?sub=3&brch=73&sim=703&cnt=1
  • https://www.microrao.com/micronotes/coagulase.pdf
  • http://laboratorytests.org/coagulase-test/
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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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