COAGULASE TEST: RATIONALE, PROCEDURE, AND USES - BIOLOGY - 2025

The coagulase test is a laboratory technique used to reveal the presence of the enzyme coagulase. This enzyme has the property of coagulating plasma. Loeb in 1903 was the first to describe this enzyme.

This test is performed on Gram-positive, catalase-positive cocci, allowing to distinguish the Staphylococcus aureus strains from the rest of the staphylococci, since it is the only microorganism of clinical importance that produces it.

Both images reveal a positive Coagulase Test. Source: Left image: Philippinjl. Right image: Photo taken by the author MSc. Marielsa Gil.

In this sense, members of the Staphylococaceae Family that test negative are often referred to as coagulase negative Staphylococcus.

There are some strains other than S. aureus that can produce coagulase, such as Staphylococcus schleiferi spp coagulans, S. hyicus, S. intermedius and S. delphini.

However, the first three are of clinical importance at the veterinary level and could very rarely be found as a causative agent of infections in humans, while S. delphini is only found in marine environments.

Furthermore, they are easily differentiated because S. hyicus and S. intermedius do not ferment mannitol and S. schleiferi spp coagulans does not ferment maltose or trehalose, while S. aureus does ferment these carbohydrates.

The presence of the coagulase enzyme has been linked to the virulence of the strains. However, this theory has been falling apart, as other virulent coagulase-negative species capable of producing significant infections are observed.

Basis

Interpretation

Agglutination within 5-20 sec (strong positive test).

Variable agglutination occurring between 20 seconds and one minute (delayed positive test).

Some degree of agglutination after one minute (doubtful evidence). It is recommended to repeat the test or confirm by the tube method.

There is no agglutination (negative test).

Result with SSF. It must always give negative, if it gives positive automatically the test result is invalidated.

-Tube coagulase test

materials

-Sterile test tube

-Plasma

-Bath of Mary at 37 ° C.

Technique

Pipette 0.5 ml of plasma into a 12 x 75 test tube with a sterile pipette. Load the platinum loop with 2 to 4 pure colonies to study from a solid culture for 18 to 24 hours and dissolve in the plasma carefully mix and incubate at 37 ° C for 4 hours.

Examine the tube in the first hour without shaking it, just gently tilt it. If a clot is still not seen, it can continue to be observed every 30 minutes until the 4 hours are completed. If after 4 hours it is still negative, it can be left for up to 24 hours but at room temperature. Observe and report the result.

Based on experience, some microbiologists recommend using 500 µl of a bacterial suspension from an 18-hour culture in liquid medium to perform the test.

It appears to offer faster and more reliable results than when emulsifying colonies from solid media, especially if human plasma obtained from the blood bank has been used.

The use of strains from a broth helps to dilute the possible presence of human anti-staphylococcal antibodies in the plasma that can inhibit the action of coagulase.

Interpretation

If a clot is seen that encompasses all of the fluid (complete clotting) or clot with nothing in the remaining fluid (partial clotting) it should be considered a positive test.

If no clot is formed, that is, the suspension remains homogeneous, the test is negative.

-Coagulase test using fibrinogen

Fibrinogen is used the same as plasma and is used for both the slide and tube tests. Proceed as described for plasma and interpret in the same way.

Use

It is used to differentiate Staphylococcus aureus from coagulase negative staphylococci.

QA

Have fresh cultures of a S. aureus strain available to be used as a positive control. A S. epidermidis strain is also available as a negative control.

Limitations

-A positive test should not be left in incubation for 24 hours, since S. aureus produces a fibrinolysin that dissolves the clot.

-For a reliable test, fresh or newly reconstituted plasma should be used, as well as it is important to use fresh bacterial cultures (18 to 24 h). This avoids false negatives.

-The test must be performed together with a negative and a positive control.

-Some solid media can interfere with the coagulase test. It is not recommended to use colonies from salty mannitol agar.

-If citrated plasma is used, it is recommended to place 5 units of heparin per ml of plasma to avoid false positives. This is because some microorganisms other than S. aureus can break down citrate and cause the plasma to clot. In this case, it is advisable to do a Gram and a catalase test.

-It is important, in the tube test, to monitor the reaction every 30 minutes, as there are strains of S. aureus that produce high concentrations of fibrinolysin and will dilute the newly formed clot quickly. Avoid false negatives.

-When monitoring the test, avoid shaking the tube abruptly, this can destroy the initiation of clot formation that will not be restored later, causing false negatives.

References

1. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
2. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
3. Mac Faddin J. (2003). Biochemical tests for the identification of bacteria of clinical importance. 3rd ed. Editorial Panamericana. Buenos Aires. Argentina.
4. Pro-Lab Laboratories. Rabbit coagulates plasma. Available at: pro-lab.com
5. "Coagulase." Wikipedia, The Free Encyclopedia. 12 Feb 2019, 04:23 UTC. 22 Apr 2019, 15:50 wikipedia.org.