Coagulase Test Introduction

Many types of biochemical and antimicrobial tests are being performed in a laboratory in order to detect the specific characteristic of a micro-organism especially a pathogen and its reactions to the certain enzymes.

One such biochemical test is Coagulase test. Coagulase test is generally used to differentiate the bacterium, Staphylococcus aureus, positive from the coagulase negative Staphylococci.

Staphylococcus aureus synthesis an enzyme named Coagulase which helps in converting the soluble from of fibrinogen present in the plasma into the insoluble form.

What is Coagulase Test?

Coagulase test is one the biochemical test which is used to differentiate Staphylococcus aureus from other species of Streptococci such as S. epidermidi and S. saprophyticus on the basis on their ability to synthesis the enzyme, coagulase.

The coagulase test is very important to differentiate the species of the gene Staphylococci, and it is divided into two types namely Positive Staphylococci and the Coagulase Negative Staphylococci.

The enzyme, coagulase acts as a essential factor in many of the organisms and it interacts with the factor fibrinogen that is present on the surface of the host cell.

Organisms which contain coagulase usually contains a protective barrier around themselves, that increases their pathogenicity and their resistance against the hosts immune system.

Coagulase is categorized into two ways; free coagulase and as bound coagulase, each of which is determined by different methods.

The bound coagulase is known as clumping factor and it is usually detected by using a slide test.

The free coagulase is usually detected by a test tube which results in the formation of a clot.

Coagulase Test Objective

• The aim of the main of the test is to detect free coagulase and bound coagulase produces by different organisms.

• To differ coagulase positive coagulase Staphylococci from the coagulase negative staphylococci.

• To identify and differentiate the species of Staphylococcus aureus from other Staphylococcal species.

Coagulase Test Principle

Coagulase is one of the enzymatic proteins, which is thermostable and it is a thrombin-like substance, that contains fibrinogen into fibrin and results in the clot or clumping.

In Staphylococcus aureus, two different forms of coagulase are found. Free coagulase and the bound coagulase.

I. Bound Coagulase Test

The clumping factor, known as bound coagulase is detected rapidly by slide test, however, this test requires several colonies and it lacks sensitivity.

Staphylococcus aureus, produces a substance in its cell wall protein, which binds to a Fc moiety of a human immunoglobin G and it behaves like a coagulase-reacting factor.

When the latex or the erythrocyte particles are coated with Immunoglobin G and the human fibrinogen, a staphylococcus agglutinates, either of the clumping factor that is present in the cell wall of bacteria.

Thus, the presence of the clotting factor is demonstrated by the ability of the organism to act upon the factor fibrinogen, which is present in the plasma to form a clot during the process of slide assay.

However, the test for clumping factor is rapid and it also requires several colonies, and the factor may not present in all the organisms of S. aureus.

II. Free Coagulase Test

This is one of the other types of coagulase which is released by the organism.

Free coagulase is different from the bound coagulase in the mechanisms of clotting as free coagulase requires the activation of a plasma-coagulase reacting factor known as CRF.

It is known as the modified or derived form of Pro-thrombin, to form a coagulase complex, CRF.

The plasma added to the tube is preferably rapid plasma that acts as a binding factor.

The complex formed then reacts with the fibrinogen to form fibrin clot inside a test tube.

Coagulase Test Reagent

Supplies:

 Loops or sterile sticks

 Glass or plastic tubes

 Glass slides

Reagents:

 Frozen Plasma with EDTA

 Here, most preferably rabbit plasma is used and human plasma is not commonly used for this test, as it is less sensitive and it causes potential infections with the human pathogenic viruses

 5% of calcium chloride

Coagulase Test Procedure

Usually, coagulase test is experimented using two methods such as Tube test and Slide test.

I. Coagulase Slide Test

For slide test, about 10µl of physiological saline or deionized water is added to a slide.

Then using an inoculating loop several colonies from a fresh culture are collected and are emulsified into the water, in order to get a smooth milk-colored suspension.

Further, a drop of rabbit plasma is placed on the slide and clumping can be observed immediately.

Mostly clumping does not exceeds more than 10 seconds.

II. Coagulase Tube Test

For tube test, Initially the plasma is diluted with the physiological saline by adding, 0.2ml of plasma in 1.8 ml of saline.

Then about 5 ml of diluted plasma is then added into a test-tube along with 5 drops of test organism culture.

Then the test tube is mixed thoroughly and it is incubating at a temperature of about 37ºC for about one hour.

Then the tube is finally observed for formation of a clot.

In case, if there is no formation of any clumps, then the is tube is examined for 30 minutes for a regular interval of 6 hours.

Coagulase Test Result

Slide Test:

o Usually, a positive test is demonstrated by the agglutination of the bacterial cells after addition of plasma.

o Whereas, the negative test is demonstrating when is no formation of clumps.

Test Tube:

o In tube test, few criteria are followed for determining the positive and negative results.

Positive Results:

o In positive result, Complete clot is formed or there will be any degrees of clot formation within 24 hours.

o If there is no clot formation, after adding 1 to 2 drops of calcium chloride, after 24 hours.

Negative Results:

o In case of negative results, there will be no formation of clumps or clots.

o If there is no clot observed after 24 hours in a temperature of 35ºC, then 2 to 3 drops of calcium chloride is added and it results in formation of a clot.