Coagulase Test

Key points to remember

• Coagulase = Virulence enzyme of S. aureus.
• Slide test → detects bound coagulase (rapid).
• Tube test → detects free coagulase (confirmatory).
• Positive = Pathogenic (S. aureus)
• Negative = Usually harmless staph species

Keywords

Coagulase, Bound coagulase, Free coagulase, Fibrinogen, Staphylococcus aureus, S. epidermidis, Rabbit plasma, EDTA plasma, Tube test, Slide test.

Introduction

The coagulase test is one of the most important biochemical tests in microbiology, mainly used to differentiate Staphylococcus aureus (a major human pathogen) from other Staphylococcus species. It detects the enzyme coagulase, which helps bacteria convert fibrinogen into fibrin, causing plasma to clot. This reaction is a key virulence factor that allows pathogens to evade host defenses.

Principle

Certain bacteria, especially Staphylococcus aureus, produce coagulase that reacts with plasma components to produce a visible clot.

• Bound coagulase (clumping factor): Found on the bacterial cell wall; causes direct agglutination on a slide.
• Free coagulase: Secreted enzyme that reacts with plasma coagulase-reacting factor (CRF) to form a clot in liquid plasma.

Because coagulase helps bacteria form protective barriers (fibrin layers), this enzyme plays a major role in pathogenicity. Therefore, detecting its presence is vital for diagnostic microbiology.

Reagents and Materials

Reagent / Material	Description
Rabbit plasma (EDTA-treated)	Used as the coagulase reagent. Prevents false positives caused by citrate metabolism.
Sterile saline or distilled water	For preparing bacterial suspensions in slide test.
Calcium chloride (optional)	Sometimes added to enhance clot formation in weak reactions.
Control strains	S. aureus (positive) and S. epidermidis (negative).
Equipment	Slides, tubes, inoculating loops, pipettes, incubator (37 °C), marker, and safety gear.

Methods

1. Slide Coagulase Test (Bound Coagulase)

Used for rapid screening.

Procedure

1.     Draw two circles on a clean slide.

2.     Add a drop of saline in one circle and rabbit plasma in the other.

3.     Emulsify a small amount of bacterial colony in each drop (use separate sterile loops).

4.     Mix gently and observe for 10–15 seconds.

Interpretation

• Positive: Clumping in plasma drop only → Bound coagulase present (S. aureus).
• Negative: No clumping → Perform tube test for free coagulase.
• Autoagglutination: Clumping in saline as well → Invalid; use tube test.

2. Tube Coagulase Test (Free Coagulase)

Detects secreted coagulase.

Procedure

1.     Add 0.5 mL rabbit plasma to a sterile test tube.

2.     Inoculate with 2–3 colonies of the test organism.

3.     Mix gently and incubate at 35–37 °C.

4.     Observe for clot formation every hour up to 4 hours; if negative, check again at 24 hours.

Interpretation

• Positive: Visible clot or solidification (even partial) — S. aureus.

·         Negative: No clot after 24 hours — Coagulase-negative Staphylococcus.

Note: Avoid shaking the tube, as it can break the clot and give false negatives.

Results Overview

Test	Positive Result	Negative Result	Example Organism
Slide Test	Clumping in plasma drop	No clumping	S. aureus (+), S. epidermidis (–)
Tube Test	Plasma clot forms	No clot at 24 h	S. aureus (+), S. saprophyticus (–)

Common Results by Species

Species	Slide Test	Tube Test	Interpretation
Staphylococcus aureus	+	+	Pathogenic
S. epidermidis	–	–	Skin commensal
S. lugdunensis	+	–	Opportunistic
S. intermedius	+	+	Animal-associated
S. saprophyticus	–	–	UTI pathogen (non-coagulase type)

Clinical Relevance

• A positive coagulase test confirms Staphylococcus aureus, a common cause of:
• Skin and wound infections
• Pneumonia
• Osteomyelitis
• Sepsis and endocarditis
• Coagulase-negative staphylococci (CONS), such as S. epidermidis, are usually non-pathogenic but can cause device-related infections (catheters, prosthetic valves).

Because S. aureus is more virulent, rapid detection using the coagulase test is crucial for timely treatment.

Precautions

• Always use EDTA-treated rabbit plasma, not human plasma.
• Avoid prolonged incubation beyond 24 hours (clot may dissolve).
• Include positive and negative controls with every batch.
• Use fresh, pure cultures (24-hour growth).
• Dispose of biohazard waste safely after testing.

Conclusion

The coagulase test remains one of the most reliable and cost-effective diagnostic tools for distinguishing Staphylococcus aureus from other staphylococci. Its rapid results guide early clinical decisions and infection control. Even in modern molecular labs, this classic test continues to serve as a cornerstone of bacterial identification.