Laboratory Procedures (Staphylococci | F) Flashcards
What is the principle of catalase test?
The enzyme catalase mediates the breakdown of hydrogen peroxide (H2O2) into oxygen and H2O. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into H2O2 (30% for the slide test), and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production
What is the procedure (or steps) of catalase test?
1) Use a loop or sterile wooden stick to transfer a small amt of colony growth to the surface of a clean, dry glass slide
2) Place a drop of 30% H2O2 onto the medium
3) Observe for the evolution of oxygen bubbles
What are the expected results (or results) of catalase test?
1) (+): copious bubbles produced
2) (-): no or few bubbles produced
* note: some organisms (enterococci) produce a peroxidase that slowly and the test may appear weakly (+). This rxn is not a truly (+) test
What is the purpose of coagulase test?
This test is used to differentiate Staph aureus (+) from CONS (-)
What is the principle of coagulase test?
Staph aureus produces 2 forms of coagulase: bound and free. Bound coagulase, or “clumping factor”, is bound to the bacterial cell wall and reacts directly w/ fibrinogen. This results in an alteration of fibrinogen so that it precipitates on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed w/ plasma.
The presence of bound coagulase correlates well w/ free coagulase, an extracellular protein enzyme that causes the formation of a clot when Staph aureus colonies are incubated w/ plasma. The clotting mechanism involves activation of a plasma coagulase-reacting factor (CRF), w/c is a modified or derived thrombin molecule, to form a coagulase-CRF complex. This complex in turn reacts w/ fibrinogen to produce the fibrin clot
What are the 2 methods of coagulase test?
1) Slide test
2) Tube test
What is the procedure (or steps) of slide test (coagulase test)?
1) Place a drop of coagulase plasma (preferably rabbit plasma w/ EDTA) on a clean, dry glass slide
2) Place a drop of distilled H2O or saline next to the drop of plasma as a control
3) W/ a loop, straight wire, or wooden stick, emulsify a portion of the isolated colony being tested in each drop, inoculating the H2O or saline first. Try to create a smooth suspension
4) Mix well w/ a wooden applicator stick
5) Rock the slide gently for 5 - 10 secs
What are the results (or expected results) for slide test (coagulase test)?
1) (+): macroscopic clumping in 10 secs or less in coagulated plasma drop and no clumping in saline or H2O drop
2) (-): no clumping in either drop; note: all (-) slide tests must be confirmed using the tube test
3) Equivocal: clumping in both drops indicates that the organism autoagglutinates and is unsuitable for the slide coagulase test
What should be done if the slide test resulted to (-)?
All (-) slide tests must be confirmed using the tube test
What is the preferred coagulase plasma to be used in slide test?
Rabbit plasma w/ EDTA
What acts as a control in slide test?
Distilled H2O or saline
What is the time duration needed for rocking the slide gently in slide test?
5 - 10 secs
What is the procedure (or steps) of tube test (coagulase test)?
1) Emulsify several colonies in 0.5 mL of rabbit plasma (w/ EDTA) to give a milky suspension
2) Incubate tube at 35 DC in ambient air for 4 hrs
3) Check for clot formation; note: tests can be (+) at 4 hrs and then revert to (-) after 24 hrs
4) If (-) at 4 hrs, incubate at room temp overnight and check again for clot formation
What are the results (or expected results) for tube test (coagulase test)?
1) (+): clot of any size
2) (-): no clot
What is the temp for incubation in tube test?
35 DC
