[principle]
Pathogenic staphylococcus can produce plasma coagulase, so that the fibrinogen in plasma into insoluble fibrin, attached to the surface of bacteria, the formation of coagulation, thus has the role of anti-phagocytosis. Coagulase test is very helpful to determine whether the strain has pathogenicity. Staphylococcus coagulase test has been widely used in routine identification of Staphylococcus aureus and other staphylococcus.
There are two kinds of coagulase produced by staphylococcus: binding coagulase and free coagulase.
Binding coagulase (coagulation factor) : an enzyme binding to the cell wall of bacteria, it directly acts on the fibrinogen in plasma, making it become fibrin, and causing staphylococcus agglutinate into a block, glass method positive result is caused by this enzyme (coagulation factor).
Free coagulase the original thrombin substance, which does not act directly on plasma fibrinogen, but becomes a heat-resistant thrombin like substance when activated by plasma activator (coagulase activator). This substance can change liquid fibrinogen in plasma into solid fibrin, thus making plasma coagulate. The positive result of test tube was caused by this enzyme.
【 method 】
Method: (1) glass in the middle of the 1 a clean glass add 1 drop of 9 g/L sodium chloride (nacl) solution, by inoculating loop influenza virus cultures and their hybrid (positive and negative control) made of bacteria suspension, if after 10 ~ 20 s and no the phenomenon of self-curing, belong to rabbit fresh plasma 1 ring, mixed with bacteria suspension, observations.
(2) Test tube method: dilute the fresh rabbit plasma 4 times with normal saline, take 0.5ml in test tube and add 0.5ml of bacterial concentration suspension to be tested (positive control and negative control are required). , the mixture was placed in a 37℃ water bath, and the results were observed every 30 minutes. If the test tube and the positive care appear solidification within 3 hours, the negative care (that is, the concentrated bacteria liquid tube) does not solidification, it is positive; If negative, continue to observe 24 hours, do not coagulate for negative.
[Result judgment]
(1) Slide method: agglutination within 5 ~ 10s is positive.
(2) Test tube method: if there is coagulation or whole tube agglutination, it is positive. If the above phenomenon does not appear after 4 hours, it should be placed overnight and observed again.
【 application 】
This test is only used for identification of pathogenic staphylococcus.
[Precautions]
(1) Glass slide method: observation results within 10 seconds, if more than 10 seconds may appear false positive, so test tube method must be used to verify the condensation factor test.
(2) Optional slide agglutination test also includes latex agglutination test to detect agglutination factor and protein A. However, the results of latex agglutination test and slide coagulase test are not completely consistent. Although the reliability of latex agglutination test for identification of Staphylococcus lugdunensis is poor, the specificity and sensitivity of identification of Staphylococcus aureus are higher than that of traditional slide coagulase test. Some saprophytic staphylococcus and Staphylococcus squirrel and some micrococci species may be positive for latex agglutination test, but glass coagulase test is usually negative.
(3) Test tube method: thick and uniform bacterial suspension must be prepared to observe the results.
A. Those who are positive for staphylococcal coagulase test by test tube should see obvious fibrin gel clumps, wooly or fibrous precipitants that are not really coagulated and should be judged negative. Intermediate staphylococcus, staphylococcus suis need a longer time (more than 4H) incubation, can appear positive.
B. If the incubation time exceeds 4H, the following points should be considered:
ⅰ. After prolonged culture, some strains produced staphylococcal kinase (fibrinolytic protease), which cleaved clots and produced false negative results;
Ii. Both false positive and false negative results may occur if the plasma used is not sterile (or partially sterile);
ⅲ. The bacteria to be tested are not pure. After prolonged culture time, contaminated bacteria may lead to false positive results. Edta-containing plasma is superior to citrate plasma in this regard because citrate utilizing bacteria (such as some streptococcus) may promote agglutination by consuming citrate.
ⅳ. Plasma used lacks fibrinogen (defibrated plasma).
(4) It is better to use EDTA anticoagulant rabbit plasma, do not advocate human anticoagulant blood, unless the test proves that it does not contain infectious factors, no coagulation ability and no inhibitory factors. Different animal plasma factors are different, coagulation state is also different, rabbit plasma clot firm, coagulation speed is faster, better than human plasma.
(5) Colonies should not be collected on high salt medium because self-coagulation or false positive may occur.
(6) If the detected bacteria are old broth culture (more than 18 ~ 24H), or the growth is poor, the strain with low coagulase activity may not be detected.
(7) In order to prevent false positive reaction of slide test, the operation must be carried out in strict accordance with the National Clinical Test Operation Procedures. When the bacteria to be tested is suspected to be Staphylococcus aureus, the negative result of coagulase on the glass slide should be confirmed by coagulase test in vitro. The plasma criteria for the coagulase test by slide and test tube methods are: it must contain sufficient concentration of coagulase reaction factor and fibrinogen, and it must be free of fibrinolytic activity and inhibitor.