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Individual colonies on agar are round, convex, and 1 to 4 mm in diameter with a sharp border.
Coagulase test is used to differentiate Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (CONS). Emulsify a staphylococcal colony in a drop of water on a clean and grease free glass slide with a minimum of spreading.
Make similar suspensions of control positive and negative strains to confirm the proper reactivity of the plasma.
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. Coagulase Positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop. Note: All coagulase negative slides must be confirmed using tube coagulase test as the definitive test for S. 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.

Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma* to give a milky suspension.
Coagulase Negative: No clot (plasma remains wholly liquid or shows only a flocculent or ropy precipitate).
Note: Rabbit plasma is preferable, as it gives better clotting, is free from inhibitors and is safe. Rabbit plasma may be obtained by centrifuging blood to which 0.1% EDTA has been added as anticoagulant. 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. 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. Quality Control: With each batch of the tests include tubes with known coagulase-positive and coagulase-negative cultures as controls, and a tub of unseeded diluted plasma to confirm that it does not clot spontaneously.
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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. 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. Human plasma contains sodium citrate as anticoagulant, and some citrate utilizing bacteria such as Enterococcus faecalis can destroy the anticoagulant and cause clotting. Alternatively, it may be obtained lypophilized from a commercial supplier and reconstituted by the addition of water. I am teaching Microbiology to Medical Students in Patan Academy of Health Sciences, School of Medicine, Lalitpur, Nepal.

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