|Rabbit plasma after centrifugation and separation
from red cells
|Coagulase test (Stahpylococcus aureus)
both tubes are positive
|Coagulase Test & Clumping factor
Coagulase is an enzyme produced by some bacteria that reacts with prothrombin in the blood, forming a complex named
staphylothrombin, which causes blood to clot by converting fibrinogen to fibrin. This mechanism protect bacteria from phagocytosis.
Frequently coagulase test is used to distinguish between different types of Staphylococcus isolates. S.aureus produces two forms of
coagulase: bound coagulase and free coagulase. Bound coagulase (clumping factor), can be detected by slide coagulase test, and
free coagulase can be detected tube coagulase test.
* Rabbit plasma
Harvest blood from a rabbit using EDTA to prevent coagulation. Separate plasma
from blood cells deposit by centrifugation. This should be carried out for a minimum
of 10 minutes at 1500-2000 rpm at room temperature. Do not use brake to stop
centrifuge. It will result two layers: plasma (up), erythrocytes (bottom).
* Slide test
First test the strain for autoagglutination in a saline drop. If negattive, then proceed to
Emulsify another saline drop with the test organism using a wire loop. A drop of rabbit
plasma is placed on the inoculated saline drop corresponding, and mixed well, then
the slide is rocked gently for about 10 seconds.
Positive result: macroscopic clumping would be observed in the plasma within 10
seconds, with no clumping in the saline drop.
Negative result: no clumping.
If the slide coagulase test is negative, a tube test should follow as a confirmation.
* Tube test
Inocculate rabbit plasma with bacteria culture and incubate the tube in a 37 ºC
incubator for 3-4 hours. Examine hourly the clot formation. If negative, then continue
incubation up to 24 hours.
Result is positive if the serum coagulates resulting a clot (or a solid block when the
reaction is very intense). Handle the tube with care. The clot may be fragile and may
be disrupted before reading the reaction.
Result is negative if the plasma remains liquid.
Utilization of 2% Na citrate for coagulase tesr is no longer recommended. Some
bacteria can utilize citrate from plasma and false positive reactions may appear. In
order to avoid false positive coagulase reactions, tests should be performed with
plasmas which contain anticoagulants other than citrate, e.g. oxalate or heparin.
Coagulase-positive staphylococci: Staphylococcus aureus subsp. aureus and
anaerobius, S. intermedius, S. pseudintermedius, S. delphini, S. hyicus (variable), S.
lutrae, S. schleiferi subsp. coagulans and S. agnetis (usually negative).
Clumping factor-positive species: Staphylococcus aureus subsp. aureus, S.
lugdunensis and S. schleiferi subsp. schleiferi. Variable results for Staphylococcus
intermedius, S. muscae, S. vitulinus, Macrococcus bovicus and M. carouselicus.
S. argenteus and S. schweitzeri are biochemically very similar to S. aureus, and
1. Helgomar Raducanescu, Valeria Bica-Popii,1986. Bacteriologie veterinara, Editura Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.
3. Mushin, R. and Kerr, V. J. (1954). Clotting of Citrated Plasma and Citrate Utilization by Intestinal Gram-negative Bacilli. J. Gen.
Microbiol. 10, 445-451.
4. Karl-Heinz Schleifer and Julia A. Bell, 2009. Family VIII. Staphylococcaceae fam. nov.. In: (Eds.) P.D. Vos, G. Garrity, D. Jones, N.R.
Krieg, W. Ludwig, F.A. Rainey, K.-H. Schleifer, W.B. Whitman. Bergey’s Manual of Systematic Bacteriology, Volume 3: The Firmicutes,
5. Costin Stoica. ABIS online Staphylococcus database version 5.4.7-052018, www.tgw1916.net/bacteria_logare_desktop.html,
accessed Nov. 2019.
(c) Costin Stoica