Tube coagulase Test

Tube coagulase Test

1. Detects staphylocoagulase which reacts with coagulase-reacting factor (CRF)
2. CRF is a thrombin-like molecule
3. Staphylocoagulase and CRF combine to indirectly convert fibrinogen to fibrin
4. A suspension of organism is suspended and incubated with plasma at 37°C
5. Clot formation within 4 hours indicates a positive test
   • ƒ Positive test indicates Staphylococcus aureus
   • ƒ Some species of Coagulase negative staphylococcus can be positive
6. Negative tubes should be held overnight at room temp.
   •  Some species possess enzyme that can cause dissolution of clot after prolonged
     incubation

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 tube at 35^oC in ambient air or in water bath for 4 hours.
4. Examine at 1, 2 and 4 hour for clot formation by tilting the tube through 90°. (Clots may liquefy after their formation)
5. Leave negative tubes at room temperature overnight and re-examine.
   (This step is essential, for some strains of S. aureus, including many MRSA, produce a delayed clot which is rapidly lysed at 37°C by the organism’s staphylokinase.

Observation

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

Coagulase Test

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 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.
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 lypophilized 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 vial (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-used plasma at 4°C bringing it to room temperature before use.

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