Coagulase test procedure complete guide 2023

The Coagulase test is a biochemical assessment that checks for the ability of bacteria to produce coagulase enzyme. Microbiologists primarily use the test for differentiating coagulase-positive Staphylococcus aureus from other coagulase-negative Staphylococci.

Bacteria produce coagulase enzyme, a virulent factor that interacts with fibrinogen attached to the host’s cell—interacting with fibrinogen results in the formation of a clot around it, protecting itself from phagocytosis by immune cells. These bacteria usually have a layer protective against the enzyme’s activity.

Coagulase test principle and pathogenicity

Bacteria produce two forms of coagulase: bound coagulase and free coagulase.

Bound coagulase is a protein found on the surface of the cell wall of coagulase-producing bacteria. The protein is a fibrinogen receptor since its structure complements fibrinogen.

Laboratory Technicians assess Bound coagulase using a suspension of test bacteria on a slide emulsified in rabbit plasma. If present, the bacteria clump together aided by adhesin, a compound that adheres fibrinogen to plasma.

On the other hand, free coagulase requires the activation of a plasma coagulase-reacting factor (CRF) to form a coagulase-CRF compound. The coagulase-CRF complex reacts with fibrinogen in rabbit plasma to form a fibrin clot. Technologists assess free coagulase using the tube method.

Human plasma vs. rabbit plasma

Microbiologists prefer rabbits over mice due to their diverse natural repertoire and larger spleen which exhibit a higher affinity for antigens. Rabbit plasma is preferred over human plasma for coagulase tests for safety, better clotting ability and is free from inhibitors. Other technologists widely use their plasma in ELISA assays, immunochemistry, immunohistochemistry, and other antibody-based applications as blocking agents.

Antibiotic circulation and antibody production must be considered when you have to use human plasma for coagulase testing, as they can give false-negative results. You should pool the plasma from at least 5 – 10 people to ensure sufficient fibrinogen levels in the test plasma.

Some bacteria like Enterococcus faecalis, Serratia marcescens, and pseudomonas species can contaminate citrate in anticoagulants used in plasma, resulting in false-positive results. EDTA is the preferred anticoagulant because bacteria do not use it.

Test guidelines and limitations

Commercially provided rabbit plasma is recommended for the test as it is standardized to meet the required specifications.

You should conduct the test in a sterile environment and check for signs of contamination like cloudiness in the plasma.

Test colonies are highly infectious; always wear protective personal equipment when conducting the test.

DO NOT use plasma with citrate anticoagulant. If human plasma, it should be pooled from 5 to 10 people and distributed into smaller aliquots. 

Some species of staphylococci, like Staphylococcus intermedius and Staphylococcus schleiferi, give false positive bond coagulase results.

DO NOT agitate the tube, as it may disintegrate the clot.

Using fresh cultures in the Coagulase test is essential because they are more prime for producing coagulase enzymes than older cultures.

Coagulase tube test requirements include a test colony, Staphylococcus aureus (positive control), rabbit plasma, pipette, normal saline, test tube, and inoculating wire.

Coagulase tube test requirements include a test colony, normal saline, Staphylococcus aureus (positive control), rabbit plasma, slide, incubator, sterile broth (negative control), and inoculating wire.

Coagulase tube test procedure 

1. Dilute rabbit plasma in normal saline at a ratio of 1: 10. (0.2 ml of plasma in 1.8 ml normal saline).
2. Take 3 test tubes and label them as; Test, Positive control, and Negative control.
3. Pipette 0.5 ml of diluted plasma into each test tube.
4. In the test tube marked ‘test,’ inoculate your test colony, ‘Positive control’ inoculate Staphylococcus aureus colony, and ‘Negative control’ innoculate sterile broth.
5. Mix and incubate the test tubes at 37^oC for 4 hours, and check for clotting every 1 hour. If no clot is formed, reincubate at room temperature and check for clot formation after 20 hours.

Note: Incubating for longer than 4 hours risks lysing of the clot by Staphylococcus aureus fibrinolysin.

Coagulase slide test procedure 

1. Pick two slides and label them as ‘Test’ and ‘Control.’
2. Place a drop of normal saline on each of the slides.
3. Using a sterile loop, emulsify the test colony in both slides. 
4. Add a drop of rabbit plasma on the slide labeled ‘Test’ and mix gently.
5. Check for agglutination/ clumping within 10 seconds.
6. Compare the two slides. The slide marked ‘Control’ helps compare with the normal granular appearance of the bacteria.

NOTE: If clumping appears on the two slides, the slide test is unsuitable for assessing the test bacteria. Confirm all negative results with the tube method.

Coagulase positive bacteria

Examples of bacteria that produce coagulase enzymes include Staphylococcus aureus in humans, Staphylococcus pseudintermedius, Staphylococcus hyicus, Staphylococcus lutrae, S. intermedius, Staphylococcus delphini, and Staphylococcus schleiferi.

Coagulase-negative bacteria

Examples of coagulase-negative bacteria include Staphylococcus epidermidis, S. warneri, S. hominis, S. saprophyticus, and S. caprae.