Biochemical tests of Gram positive bacteria L. Samira

Coagulase Test Purpose Principle The Coagulase Test is typically used to differentiate Staphylococcus aureus from other Gram-positive cocci Principle Staphylococcus aureus is an opportunistic pathogen that can be highly resistant to both the normal immune response and antimicrobial agents. Its resistance is due, in part, to the production of a coagulase enzyme. Coagulase works in conjunction with normal plasma components to form protective fibrin barriers around individual bacterial cells or groups of cells.

Coagulase enzymes occur in two forms—bound coagulase and free coagulase. Bound coagulase, also called clumping factor, is attached to the bacterial cell wall and reacts directly with fibrinogen in plasma. The fibrinogen then precipitates causing the cells to clump together in a visible mass. Free coagulase is an extracellular enzyme (released from the cell) that reacts with a plasma component called coagulase-reacting factor (CRF). The resulting reaction is similar to the conversion of prothrombin and fibrinogen in the normal clotting mechanism.

There are Two forms of the Coagulase test Tube Test and the Slide Test There are Two forms of the Coagulase test Tube Test and the Slide Test. The Tube Test detects the presence of either bound or free coagulase, while the Slide Test detects only bound coagulase. to interrupt normal clotting mechanisms Both tests utilize rabbit plasma treated with anticoagulantto interrupt normal clotting mechanisms to interrupt normal clotting mechanisms.

Procedures The Tube Test is performed by adding the test organismto rabbit plasma in a test tube. Coagulation of the plasma(including any thickening or formation of fibrin threads)within 24 hours indicates a positive reaction The plasma is typically examined for clotting (without shaking) after about 4 hours because it is possible for coagulation to take place early and revert to liquid within 24 hours.In the slide test, bacteria are transferred to a slide containing a small amount of plasma. .

Agglutination of the cells on the slide within one to two minutes indicates thepresence of bound coagulase . Equivocal or negative Slide Test results are typically confirmed using the Tube Test

Coagulase Tube Test

Coaguliase Slide Test

Catalase Test Purpose The Catalase Test is used to identify organisms that produce the enzyme catalase. It is most commonly used to differentiate members of the catalase-positive Micro coccaceae from the catalase-negative Streptococcaceae Variations on this test may also be used in identification of Mycobacterium

Principle The electron transport chains of aerobic and facultatively anaerobic bacteria are composed of molecules capable of accepting and donating electrons as con ditions dictate. As such, these molecules alternate between the oxidized and reduced form, passing electrons down the chain to the final Aerobic and facultatively anaerobic bacteria produce enzymes capable of detoxifying these compounds. Super - oxide dis mutase catalyzes conversion of superoxide radicals (the more lethal of the two compounds) to hydrogen peroxide Catalase converts hydrogen peroxide into water and gaseous oxygenelectron acceptor

Catalase Slide Test

Hemolysis test Blood agar is used to differentiate bacteria based on their hemolytic characteristics, especially within the genera Streptococcus, Enterococcus, and Aerococcus . there are three major types of hemolysis are Beta-hemolysis gama-hemolysis. alpha-hemolysis

Principle Several species of Gram-positive cocci produce exotoxins called hemolysins, which are able to destroy red blood cells (RBCs) and hemoglobin. Blood Agar, sometimes called Sheep Blood Agar because it includes 5% sheep blood in a Tryptic Soy Agar base, allows differentiation of bacteria based on their ability to hemolyze RBCs

􀀀 Streak plate of Streptococcus pneumoniae demonstrating alpha-hemolysis. The greenish zone around the colonies results from incomplete lysis of red blood cells

Beta-HEMOLYSIS Streptococcus pyogenes demonstrates beta-hemolysis Beta-HEMOLYSIS Streptococcus pyogenes demonstrates beta-hemolysis.The clearing around the growth is a result of complete lysis of red bloodcells.

Gama-Hemolysis This streak plate of Staphylococcus epidermidison a Sheep Blood Agar illustrates no hemolysis.

Mannitol Salt Agar Purpose Mannitol Salt Agar (MSA) is used for isolation and differentiation of pathogenic staphylococci, principally Staphylococcus aureus Principle Mannitol Salt Agar contains the pH indicator phenol red. This medium selective for staphylococci The pathogenic species of Staphylococcus ferment mannitol produce acid, which turns the pH indicator yellow. Nonpathogenic staphylococcal species grow, but produce no color change.

MANNITOL SALT AGAR

MANNITOL SALT AGAR MSA inoculated with Staphylococcus aureus (top) and S. epidermidis (bottom). (Note: Some strains of S. epidermidis are inhibited by this medium). The yellow halo around S. aureus is due to mannitol fermentation with acid end products