COAGULASE AND AMYLASE TEST

INTRODUCTION Coagulase test is one of the biochemical tests. It is very important test in the microbiology. The coagulase test identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot. Organisms that produce Coagulase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents.

Why microorganism produce coagulase? It has been proposed that fibrin-coated staphylococci resist phagocytosis making the bacteria more virulent.

principle Coagulase reacts with prothrombin in the blood.it causes blood to clot by converting fibrinogen to fibrin. The presence of coagulase can be detected by heavily inoculating the test organism into rabbit plasma and incubating the mixture for 4 to 24 hours. Any degree of clotting during this time, from a loose clot suspended in the plasma to a solid, immovable clot is a positive result.

Result Coagulase +ve Coagulase -ve

signifigance The coagulase test is used to differentiate the potentially pathogenic species Staphylococcus aureus from the usually non-pathogenic species Staphylococcus epidermidis.

The S. aureus (potentially pathogenic in humans and animals, but S The S. aureus (potentially pathogenic in humans and animals, but S. epidermidis (is not pathogenic)

Staphylococcus

Types of coagulase There are 2 types of cagulase ensyme: 1- bound coagulase. 2-free coagulase.

bound coagulase. It is tightly bound to the surface of the bacteria S. aureus and can coat its surface with fibrin upon contact with blood. Bound coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate. This causes the cells to agglutinate, or clump together, which creates the “lumpy” look of a positive coagulase slide test. You may need to place the slide over a light box to observe the clumping of cells in the plasma.

free coagulase. It is an extracellular enzyme. Free coagulase reacts with a component of plasma called coagulase-reacting factor. The result is to cause the plasma to coagulate.

Test methods There are 2 methods: 1-slide method. 2- tube method.

coagulase slide test is used to identify the presence of bound coagulase or clumping factor. For this method we use: 1-lyophilized rabbit plasma. 2-slide. 3-loop. 4- Staphylococcus plate.

Procedure 1.Place a drop of coagulase plasma on a clean, dry glass slide. 2.Place a drop of distilled water or saline near the drop of plasma as a control. 3.With a sterile loop or wooden stick, emulsify an amount of the isolated colony being tested into each drop. 4.inoculating the water or saline first. 5.Try to create a smooth suspension.

6.Observe for clumping in the coagulase plasma and a homogenous suspension in the control. 7.Clumps that will not mix uniformly into coagulase plasma indicate a positive test whereas a uniform suspension is indicative of a negative test

8. Clumping in both tests indicate that the organism autoagglutinates and is unsuitable for the slide coagulase test. 9.When autoagglutination is observed. the tube coagulase test should be employed as an alternative to the slide agglutination test.

When the slide test is employed, all negative slide reactions must be confirmed by the tube test because of : 1-The slide agglutination technique may lead to false-positives: since some strains produce clumping factor resulting in a positive slide test and a negative tube coagulase test. 2- spontaneous agglutination may occur when rough cultures are used.

Tube method is used to identify the presence of 2 types of coagulase ( bound and free). For this method we use: 1-lyophilized rabbit plasma. 2-2tubes. 3-loop. 4- Staphylococcus plate.

Procedurer 1.Using a culture that is less than 24 hours old, inoculate the CoaguStaph™ by emulsifying one loopful (2-4 colonies) of bacteria from a non-inhibitory agar plate into the tube of plasma. 2.Incubate the inoculated tube at 35-37 degrees C. for 1 to 4 hours. 3.Negative tests at 4 hours should be held at room temperature for a total of 24 hours before reporting results. 4. Read by gently tilting the tube while observing for clotting of plasma.

5. Results should be read at 4 hours. 6 5. Results should be read at 4 hours. 6. A positive test for coagulase production results in a clotting of the rabbit plasma. 7.Any degree of clotting is a positive test. 8.Results can be reported across a range 0 to 4+, 0 meaning the plasma remained liquid (no coagulase activity) and 4+ meaning the plasma completely hardened (the consistency of an agar) due to strong coagulase activity.

All "0" results after 4 hours should be held at room temperature for a total of 24 hours incubation

Notes The slide test should be read very quickly, as false positives can occur. The slide test should not performed with organisms taken from high-salt media such as Mannitol Salt Agar, as the salt content can create false positives. The tube test is more reliable than the slide test. We generally don’t use the coagulase test when identifying unknowns.

Amylase Production (Starch Hydrolysis Test) Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism.  Starch agar is a simple nutritive medium with starch added.  Since no color change occurs in the medium when organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch. A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.

Procedure Streak the test organism across a small portion of the agar surface. Incubate at 37 oC for 48 hours. Cover the surface with iodine. Rotate to distribute the iodine into a thin layer. Do not flood the plate. Iodine will turn blue when it reacts with starch. A clear zone will be seen where starch has been digested.

Starch Hydrolysis Test