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

Introduction

Coagulase is a protein enzyme produced by several microorganisms that enables the conversion of fibrinogen to fibrin. In the laboratory, it is used to distinguish between different types of Staphylococcus isolates.

Staphylococcus aureus is known to produce coagulase, which can clot plama into gel in tube or agglutinate cocci in slide. This test is useful in differentiating S.aureus from other coagulase-negative staphylococci. Most strains of S.aureus produce two types of coagulase, free coagulase and bound coagulase. While free coagulase is an enzyme that is secreted extracellularly, bound coagulase is a cell wall associated protein. Free coagulase is detected in tube coagulase test and bound coagulase is detected in slide coagulase test. Slide coagulase test may be used to screen isolates of S.aureus and tube coagulase may be used for confirmation. While there are seven antigenic types of free coagulase, only one antigenic type of bound coagulase exists. Free coagulase is heat labile while bound coagulase is heat stable.

Principle

Coagulase reacts with prothrombin in the blood. The resulting complex is called staphylothrombin, which enables the enzyme to act as a protease to convert fibrinogen, a plasma protein produced by the liver, to fibrin. This results in clotting of the blood. Coagulase is tightly bound to the surface of the bacterium S. aureus and can coat its surface with fibrin upon contact with blood. The fibrin clot may protect the bacterium from phagocytosis and isolate it from other defenses of the host.

The coagulase test identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot. Organisms that produce catalase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents.

The coagulase slide test is used to identify the presence of bound coagulase or clumping factor, which is attached to the cell walls of the bacteria. Bound coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate. This causes the cells to agglutinate, or clump together, which creates the “lumpy” look of a positive coagulase slide test.  You may need to place the slide over a light box to observe the clumping of cells in the plasma.

The coagulase tube test has been set up as a demo for you to observe in class. This version of the coagulase test is used to identify the presence of either bound coagulase or free coagulase, which is an extracellular enzyme. Free coagulase reacts with a component of plasma called coagulase-reacting factor. The result is to cause the plasma to coagulate.  In the demo, the coagulase plasma has been inoculated with Staphylococcus aureus and Staphylococcus epidermidis and allowed to incubate at 37˚C for 24 hours.  Staphylococcus aureus produces free coagulase; Staphylococcus epidermidis does not.

The coagulase test is useful for differentiating potentially pathogenic Staphylococci such as Staphylococcus aureus from other Gram positive, catalase-positive cocci.

Procedure

The enzyme coagulase is demonstrated invitro by two methods:

• The Slide coagulase test
• The Tube coagulase test

The Slide coagulase test

Principle

This method measures bound coagulase. The bound coagulase is also known as 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.

Procedure Of Slide Coagulase Test

1. Divide the slide into two sections with grease pencil. One should be labeled as “test” and the other as “control
2. Place 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.

The Tube Coagulase Test

Principle

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.

Procedure Tube Coagulase Test

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 Interpretation

Clumping in both drops of slides indicates that the organism auto agglutinates and is unsuitable for the slide coagulase test. All the negative slide test must be confirmed using the tube test. 

During slide test, there may be chance to false positive results in case of citrate utilizing bacteria ( Enterococcus and Pseudomonas). In this case also, tube test should be performed and confirmed.

Coagulase Positive Organisms: Staphylococcus aureus and other animal host bacteria likeS. 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

1. The coagulase test is used to distinguish between pathogenic and nonpathogenic members of the genus Staphylococcus. All pathogenic strains of S. aureus are coagulase positive whereas the nonpathogenic species(S. epidermidis) are coagulase negative.
2. While slide coagulase test is useful in screening, tube coagulase test is useful in confirmation of coagulase test.
3. Not all S.aureus strains produce coagulase; such rare strains are identified by thermonuclease test.

Limitations Of Coagulase Test

1. The slide test should be read very quickly, as false positives can occur.
2. Auto agglutination may occur.
3. Use water instead of saline for mixing.
4. The slide test should not be performed with organisms taken from high-salt media such as Mannitol Salt Agar, as the salt content can create false positives.
5. Over mixing may cause the clot to break down.
6. The tube test is more reliable than the slide test.
7. We generally don’t use the coagulase test when identifying unknowns.
8. 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.

Further Reference

1. Zahlane K, Haouach K, Zouhdi M. Staphylococci: actual state of epidemiology and antibioresistance in University Hospital Center of Rabat. Maroc Médical 29 (2007): 279-285.
2. Zaineb El Houssaini, Nadia Harrar, Khalid Zerouali, Houria Belabbes, Naima Elmdaghri. Prevalence of coagulase-negative staphylococci in blood cultures at the IbnRochd University Hospital in Casablanca. The Pan African Medical Journal 33 (2019): 193.
3. The United States Pharmacopeia (USP 31)-NF 26. Microbial Limit Tests. United States Pharmacopeia Convention Inc. Rockville, MD. USA (2008).
4. Gilles Berrut Laure de Decker. Immunosenescence: a review. Geriatr Psychol Neuropsychiatr Vieil 13 (2015): 7-14.
5. Agon SIA, Olowo PP. Determination of species and antibiotic resistance profiles of strains of Staphylococci isolated from pathological samples at CNHU-HKM. End of training memory for obtaining a professional degree. Polytechnic school of Abomey-Calavi (EPAC), University of Abomey-Calavi (UAC). Bénin (2012): 53.
6. Faye-Kette H, Kouassi MY, Akoua-Koffi G, et al. Microbial Epidemiology of Surgical Site Infections (ISO) in Abidjan Trauma Department and Sensitivity of Germs to Antibiotics. Revue Bio-Africa 6 (2008): 25-31.
7. European Pharmacopoeia. Control of microbial contamination in non sterile products – Solution and recommended culture media. Council of Europe (2007).