Coagulase Test: Principle, Procedure, Results

Two blood cultures, the same Gram stain, oppositive meaning

Two patients, two positive blood cultures, and under the microscope the smears look identical: Gram-positive cocci in clusters. Both are catalase positive, so both are staphylococci.

The first patient has a central line and a low-grade fever. The second has a new heart murmur, a fever, and no obvious source.

For both, the next test is the same, and it costs almost nothing: coagulase.

If the organism is coagulase negative, the first result is probably a skin contaminant picked up when the line was drawn. One bottle, no fever spike, coagulase-negative staphylococci: the lab reports it, the clinician watches, and often nothing more is needed. But if that same patient had a prosthetic valve or an indwelling device, coagulase-negative Staphylococcus epidermidis becomes a real pathogen colonizing the hardware, and the test result reads the same.

If the organism is coagulase positive, it is Staphylococcus aureus until proven otherwise, and the second patient's new murmur is now infective endocarditis until an echocardiogram says it is not. S. aureus in the blood is never dismissed. It seeds heart valves, bone, and joints, and it kills.

Coagulase is the enzyme that separates these two paths. It is not a perfect test, and the ways it fails, a slide-negative S. aureus, a citrate false positive, a dog-associated species that mimics aureus, are the reason this article is longer than a three-second test seems to deserve.

Principle of the coagulase test

Coagulase is an enzyme or more precisely, a protein with enzyme-like activity produced by Staphylococcus aureus that causes plasma to clot. Its detection is the cornerstone of S. aureus identification in clinical microbiology, because among clinically significant staphylococci, only S. aureus (and a few rarely encountered animal-associated species) produces coagulase in quantities sufficient to give a positive test.

Why does S. aureus produce coagulase?

Coagulase functions as a virulence factor of S. aureus. By converting soluble fibrinogen in plasma into insoluble fibrin, coagulase coats the bacterial cell surface with a layer of fibrin. This fibrin coat serves two protective purposes:

1. It masks bacterial surface antigens from recognition by the host immune system
2. It physically protects the organism from opsonization and subsequent phagocytosis by neutrophils and macrophages

In essence, S. aureus uses coagulase to hide inside a clot of the host's own plasma, evading the very immune mechanisms designed to destroy it.

Bound coagulase vs. free coagulase

S. aureus produces two distinct forms of coagulase, and the coagulase test has a separate method for detecting each:

Because not all S. aureus strains express bound coagulase (particularly MRSA strains), a negative slide test must always be confirmed by a tube coagulase test. The tube test is the definitive, gold-standard method for S. aureus identification.

Slide Coagulase Test

Slide coagulase test is done to detect “bound coagulase” or “clumping factor”. The clumping factor is a fibrinogen-binding cell surface receptor present in the cell walls of most, but not all, S. aureus strains. The clumping factor acts directly on the fibrinogen in the plasma, resulting in clumping. Slide coagulase test is rapid, but it requires several colonies and lacks sensitivity.

As the clumping factors are not present in all S. aureus, some strains will give a negative slide coagulase test. In addition, clumping factors can be masked by cell surface capsular polysaccharides. So, the negative slide tests must be confirmed by tube coagulase test. Other species of Staphylococcus, namely, S. lugdunensis and S. schleiferi may give positive slide coagulase test results.

Note: Colonies from a mannitol salt agar (MSA) culture are not suitable for coagulase testing. The organism must first be cultured on nutrient agar or blood agar.

Slide Coagulase Test Procedure

1. Emulsify a staphylococcal colony in a drop of water on a clean and grease-free glass slide with a minimum of spreading (If the isolate does not form a smooth, milky suspension, do not proceed with the test).
2. Make similar suspensions of control positive and negative strains to confirm the proper reactivity of the plasma.
3. Dip a flamed and cooled straight inoculating wire into the undiluted plasma at room temperature, withdraw, and stir the adhering traces of plasma (not a loopful) into the staphylococcal suspension on the slide. Flame the wire and repeat for the control suspensions.
4. Read as positive a coarse clumping of cocci visible to the naked eye within 10 seconds. Read as negative the absence of clumping or any reaction taking more than 10 seconds to develop, but re-examine any slow reacting strains by the tube coagulase test.

Observation

1. Coagulase positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop.

2. Coagulase-negative: No clumping in either drop

Interpretation

Slide coagulase test is the main method used to identify S. aureus in clinical laboratories but it has some limitations.

1. About 15% of ordinary strains of S. aureus and many more of MRSA give negative reactions.
2. Few species of coagulase-negative staphylococci give positive reactions.

Note: All coagulase-negative slides must be confirmed using a tube coagulase test as the definitive test for S. aureus.

Tube Coagulase Test

Tube coagulase test detects free coagulase (staphylocoagulase) which reacts with coagulase-reacting factor (CRF). CRF is a plasma protein, often described as a thrombin-like molecule, that serves as an activator. Staphylocoagulase forms a complex with CRF, and this complex then indirectly converts soluble fibrinogen in plasma to insoluble fibrin, resulting in a clot.

When a suspension of the organism is incubated with plasma at 35°C, clot formation within 4 hours indicates a positive test.

The coagulase clot thus formed can be destroyed by S. aureus fibrinolysin or staphylokinase. Staphylokinase is more active at 35°C than at room temperature (around 25°C). Therefore, if a clot forms early and is then lysed by staphylokinase at incubation temperature, you might miss it. This is why tubes must be examined periodically, and negative tubes are held overnight at room temperature, where staphylokinase activity is reduced, allowing any delayed or transient clot to persist and be observed.

Tube Coagulase Test Procedure

1. Prepare a 1-in-6 dilution of the plasma in saline (0.85% NaCl) and place 1 ml volumes of the diluted plasma in small tubes.
2. Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma to give a milky suspension.
3. Incubate the tube at 35°C in ambient air or in a water bath for 4 hours.
4. Examine at 1, 2, and 4 hours for clot formation by tilting the tube through 90°.
5. Leave negative tubes at room temperature overnight and re-examine. This step is essential for some strains of S. aureus, including many MRSA, which produce a delayed clot that is rapidly lysed at incubation temperature by the organism's staphylokinase.

Observation

Read as positive any degree of clot formation. Often the plasma is converted into a stiff gel that remains in place when the tube is tilted or inverted, but sometimes clots are seen floating in the fluid.

1. Coagulase Positive: Clot of any size eg. Staphylococcus aureus
2. Coagulase Negative: No clot (plasma remains wholly liquid or shows only a flocculent or ropy precipitate). eg. Staphylococcus epidermidis

Note: Rabbit plasma is preferable, as it gives better clotting, is free from inhibitors, and is safe. Human plasma contains sodium citrate as an anticoagulant, and some citrate utilizing bacteria such as Enterococcus faecalis can destroy the anticoagulant and cause clotting. False-positive or false-negative results can occur if the plasma is not sterile.

Slide vs. tube coagulase test

Reporting

• Report as Staphylococcus aureus if the tube coagulase test is positive and the organism is catalase-positive and a Gram-positive coccus in clusters.
• For a negative tube coagulase test from catalase-positive, Gram-positive cocci in clusters that have creamy white colonies, report as “coagulase-negative staphylococci.”
• A positive slide coagulase test can be reported as S. aureus; however, the test should be confirmed with a tube test from nonhemolytic or only slightly hemolytic colonies from sterile sites, such as blood, to separate S. aureus from S. lugdunensis and S. schleiferi.
• A negative slide coagulase test is not valid and should be followed with a tube test for confirmation.

Quality Control

• Do not use plasma that has not been stored refrigerated or frozen or that appears turbid.
• Perform quality control of coagulase plasma on new lots prior to using them.
• Organisms
  • S. aureus ATCC 25923: coagulase positive
  • Staphylococcus epidermidis ATCC 12228 or ATCC 14990: coagulase-negative

Where I can get the plasma?

Rabbit plasma may be obtained by centrifuging blood to which 0.1% EDTA has been added as anticoagulant. Alternatively, it may be obtained lyophilized from a commercial supplier and reconstituted by the addition of water.

Citrated human plasma may be obtained from a blood bank, but the blood must have been screened and found free from viral (Hepatitis B, HIV) antigens and antibodies. It must be handled with the precautions appropriate for all human body fluids.

Store the plasma in small portions at −20°C, and keep a stock of in-use plasma at 4°C, bringing it to room temperature before use.

Pitfalls and Limitations of the Coagulase Test

1. Do not use citrated plasma, as false-positive results can occur. Sodium citrate anticoagulates by chelating calcium: it locks up the free calcium ions that the clotting cascade needs, keeping the plasma liquid. Some organisms, notably Enterococcus faecalis, can consume citrate as a carbon source. When they do, they remove the chelator. The calcium that was bound is now free again, the clotting cascade proceeds normally, and the plasma clots, producing a false-positive result that has nothing to do with staphylocoagulase. This is a property of the plasma being used, not of the test organism, which is why rabbit plasma (anticoagulated with EDTA rather than citrate) is preferred.
2. Do not perform coagulase testing from colonies grown on mannitol salt agar. High salt concentrations in MSA can interfere with the enzyme activity or cause autoagglutination of some Staphylococci, leading to false-positive or unclear results.
3. Methicillin-resistant S. aureus (MRSA) can be deficient in bound coagulase, which results in a negative slide test.
4. S. intermedius and S. hyicus may be positive in the tube test; these species are usually associated with animals (dogs and pigs, respectively) and reach humans through animal contact. They can cause clinically significant, sometimes serious human infections and are easily mistaken for S. aureus on standard tests. Both form nonhemolytic colonies on fresh blood agar plates and are Voges-Proskauer negative, which separates them from S. aureus. S. intermedius is also pyrrolidonyl-β-naphthylamide (PYR) positive
5. S. lugdunensis and S. schleiferi give positive slide test but negative tube test. They can be separated from S. aureus by their strongly positive PYR reaction and from S. intermedius by a negative tube coagulase test.
6. Staphylococcus argenteus is a recently described species that is tube coagulase-positive and, like S. aureus, PYR negative. Because it shares those results with S. aureus, biochemical tests cannot separate the two. One clue at the bench: S. argenteus lacks the golden carotenoid pigment of S. aureus and forms whiter or greyish colonies (hence argenteus, meaning silvery). Definitive identification is by molecular methods.

How to Remember

• Bound = Body of the cell = Brief. Bound coagulase sits on the cell body and clumps in seconds on the slide. Free coagulase is floating (secreted) and needs the full incubation in the tube. If you remember that "bound is on the cell and fast," the whole slide-versus-tube split follows.
• Slide screens, tube confirms, and MRSA breaks the slide. The one clinical rule that prevents the most errors: a negative slide is never final, because MRSA is the organism that most often reads slide-negative while tube-positive.

Where students get confused

"A positive slide test is enough to call it S. aureus." For most routine isolates it is, but the slide test detects bound coagulase (clumping factor), and S. lugdunensis and S. schleiferi also carry it. From a sterile site such as blood, a slide-positive result should be confirmed by the tube test before you commit to S. aureus, because those two species are tube-negative. The slide test screens; the tube test confirms.

Reading the two tests as detecting the same thing. They do not. The slide test detects bound coagulase, a receptor fixed to the cell wall that grabs fibrinogen directly and clumps in seconds. The tube test detects free coagulase, secreted into the plasma, which works indirectly through coagulase-reacting factor to build a clot over hours. A negative slide with a positive tube is not a contradiction. It is one form of coagulase absent and the other present, which is exactly why a negative slide is never the final answer.

Forgetting which way MRSA fails. Many MRSA strains lack bound coagulase, so they are the classic slide-negative, tube-positive organism. A student who stops at a negative slide will miscall an MRSA as coagulase-negative staphylococci, the opposite of the truth. If the slide is negative, go to the tube every time.

The citrate false positive. This trips people because the clot has nothing to do with Staphylococcus. Human plasma is anticoagulated with citrate, which works by locking up calcium. Some organisms, notably Enterococcus faecalis, consume citrate, release the calcium, and let the plasma clot on its own. The result is a clot from an organism that makes no coagulase at all. The fix is the plasma, not the reading: use rabbit plasma anticoagulated with EDTA.

Testing straight from mannitol salt agar. MSA is where you often first see a staphylococcus, so it is tempting to test from it directly. The high salt causes autoagglutination and interferes with the reaction, giving false or uninterpretable results. Subculture to blood agar or nutrient agar first, then test.

Assuming a tube-positive result is definitively S. aureus. The tube test is the gold standard, but it is not perfectly specific. S. intermedius and S. hyicus (animal-associated) and the newer S. argenteus are all tube coagulase-positive. In routine human clinical work these are uncommon, but a tube-positive isolate that behaves oddly, nonhemolytic, from an animal-contact history, or PYR positive, deserves a second look before it is reported as S. aureus.

References and further readings

• Tille, P. M. (2022). Bailey and Scott's Diagnostic Microbiology (15th ed.). St. Louis: Elsevier.
• Procop, G. W., Church, D. L., Hall, G. S., Janda, W. M., Koneman, E. W., Schreckenberger, P. C., & Woods, G. L. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Philadelphia: Wolters Kluwer.
• Leber, A. L. (Ed.). (2016). Clinical Microbiology Procedures Handbook (4th ed.). Washington, DC: ASM Press. https://doi.org/10.1128/9781555818814
• Katz, D. S. (2010). Coagulase test protocol. American Society for Microbiology.
• Staphylococcus argenteus: another coagulase-positive Staphylococcus. (2021). American Society for Microbiology, Editors in Conversation podcast.