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

Coagulase Test – Introduction

The coagulase test is used to detect the production of coagulase enzyme by Staphylococcus aureus. Coagulase is an enzyme that causes plasma to clot by reacting with fibrinogen and converting it into fibrin. Staphylococcus aureus can be classified into coagulase-positive and coagulase-negative strains based on their ability to produce this enzyme.

1. Classification of Staphylococci:
   • Coagulase-positive Staphylococci, mainly Staphylococcus aureus, produce coagulase enzyme.
   • Coagulase-negative Staphylococci, such as S. epidermidis and S. saprophyticus, lack or produce minimal coagulase.
2. Virulence Factor:
   • Coagulase acts as a virulence factor by interacting with fibrinogen, promoting the formation of protective fibrin clots.
   • The ability to form clots enhances the pathogenicity of organisms, contributing to their evasion of the host immune system.
3. Protective Barrier:
   • Organisms producing coagulase can create a protective barrier, which aids in their survival within the host.
   • The barrier formed by fibrin clots can protect bacteria from the host’s immune defenses, facilitating the establishment of infections.
4. Types of Coagulase:
   • Bound Coagulase (Clumping Factor): This form is associated with the surface of bacterial cells and can cause clumping or agglutination.
   • Free Coagulase: This form is secreted into the surrounding environment, leading to the formation of clots in the medium.
5. Detection Methods:
   • Slide Test (Detection of Bound Coagulase): Rapid method where clumping on a slide indicates the presence of bound coagulase.
   • Tube Test (Detection of Free Coagulase): Detects clot formation in the medium, indicating the presence of free coagulase.

Understanding the coagulase status of Staphylococci is crucial in clinical microbiology for accurate identification and assessment of their pathogenic potential.

Objectives of Coagulase Test

1. Detection of Coagulase Enzymes:
   • Identify the presence of both free coagulase and bound coagulase produced by various organisms.
2. Differentiation of Staphylococcal Species:
   • Distinguish coagulase-positive Staphylococci from coagulase-negative Staphylococci.
3. Identification of S. aureus:
   • Specifically identify and differentiate Staphylococcus aureus from other Staphylococcal species.

Principle of Coagulase Test

Coagulase Enzyme:

• Coagulase is an enzymatic protein that exhibits thermostable thrombin-like properties.
• Its function involves converting fibrinogen into fibrin, leading to the formation of clots or clumping.

Forms of Coagulase in S. aureus:

1. Bound Coagulase (Clumping Factor):
   • Detected rapidly in the slide test but requires several colonies.
   • Involves the clumping factor, a substance present in the cell wall of S. aureus.
   • Protein A, also produced by S. aureus, contributes to agglutination by binding to immunoglobulin G (IgG).
   • Clumping factor may not be present in all S. aureus organisms.
2. Free Coagulase:
   • Released by the organism.
   • Requires the activation of a plasma Coagulase-Reacting Factor (CRF), forming a coagulase-CRF complex.
   • Rabbit plasma, acting as a binding factor, is often preferred in the test tube.
   • The complex reacts with fibrinogen to form a fibrin clot in the test tube.

Microorganisms Tested

• Colonies: Gram-positive cocci in clusters that are catalase-positive, aiding in the identification of S. aureus.
• Blood Cultures: Positive blood cultures containing Gram-positive cocci in clusters for the rapid detection of S. aureus.

The Coagulase Test is crucial in clinical microbiology for its role in distinguishing pathogenic S. aureus from other less virulent Staphylococci.

Reagents and Supplies Used

Reagents

1. Frozen Plasma:
   • Preferably rabbit plasma with EDTA.
   • Human plasma is not commonly used due to lower sensitivity and potential infection risk with human pathogenic viruses.
2. 5% CaCl2 (optional):
   • Calcium chloride, an optional additive.

Supplies

1. Loops or Sterile Sticks:
   • Used for collecting bacterial colonies.
2. Glass or Plastic Tubes:
   • For performing the tube test.
3. Glass Slides:
   • Utilized in the slide test.

Procedure of Coagulase Test

Coagulase can be detected by two different methods: the tube test and the slide test.

Slide Test

1. Add about 10 µl of deionized water or physiological saline to a slide.
2. Collect several colonies from a fresh culture using an inoculating loop.
3. Emulsify the colonies into the water, creating a smooth, milk-colored suspension.
4. Add a drop of rabbit or human plasma to the slide.
5. Observe clumping immediately, not exceeding 10 seconds.

Tube Test

1. Dilute the plasma with physiological saline (Add 0.2 ml plasma in 1.8 ml saline).
2. Add 5 ml of the diluted plasma to a test tube.
3. Add about 5 drops of the test organism culture to the test tube.
4. Mix the contents and incubate at 37°C for an hour.
5. Observe the tube for clot formation. If no clotting is observed, examine the tube at 30-minute intervals for up to 6 hours.

The Coagulase Test, whether performed by tube or slide method, is a vital tool for differentiating Staphylococcus aureus from other Staphylococci based on coagulase production.

Result Interpretation of Coagulase Test

Slide Test:

• Positive Test: Agglutination of bacterial cells after adding plasma.
• Negative Test: Lack of agglutination.

Tube Test:

• Positive Test Criteria:
  • Complete clot formation or any degree of clot formation before 24 hours.
  • No clot formation after 24 hours, with the addition of 1 or 2 drops of 5% CaCl2 to a tube without a clot.
• Negative Test Criteria:
  • No clot formation at 24 h at 25°C.
  • No clot after 24 hours at 35°C, but clot forms after the addition of 1 or 2 drops of 5% CaCl2 to the tube.

Coagulase Positive Organisms:

• Staphylococcus aureus and other animal host bacteria like S. pseudintermedius, S. intermedius, S. schleiferi, S. delphini, S. hyicus, S. lutrae, S. hyicus.

Coagulase Negative Organisms:

• Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae, etc.

Uses of Coagulase Test

• Determine the production of coagulase by different microorganisms.
• Differentiate S. aureus from other Staphylococcal species.
• Distinguish Staphylococcal species into coagulase-negative and coagulase-positive based on coagulase enzyme production.

Limitations of Coagulase Test

• Methicillin-resistant S. aureus can be deficient in bound coagulase, leading to a negative slide test.
• S. intermedius and S. hyicus may show positive results in the tube test; these species are generally found in dogs and pigs, respectively, but can be as infectious as S. aureus when they infect humans.
• S. lugdunensis and S. schleiferi produce slide coagulase, but the reaction is more efficient if human plasma is used rather than rabbit plasma.
• Citrated blood should not be used, as false-positive results can occur.
• Coagulase testing cannot be performed from growth on mannitol salt agar.