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
What is the time duration needed for incubation for tube test?
4 hrs
What is the principle (and purpose) of microdase test?
The microdase test is a rapid method to differentiate Staphylococcus from Micrococcus by detection of the enzyme oxidase. In the presence of atmospheric oxygen, the oxidase enzyme reacts w/ the oxidase rgnt and cytochrome C to form the colored compound, indophenol
What is the procedure of microdase test?
1) Using a wooden applicator stick, rub a small amt of several colonies of an 18 - 24 hr pure culture grown on blood agar onto a small area of the microdase disk; note: do not rehydrate the disk before use
2) Incubate at room temp for 2 mins
What are the results (or expected results) for microdase test?
1) (+): development of purple-blue color
2) (-): no color change
What is used to rub small amt of colonies from a 18 - 24 hr pure culture grown on blood agar onto a small area of the microdase disk?
Wooden applicator stick
What is the temperature used for incubation in microdase test?
Room temp
What is the time duration of incubation in microdase test?
2 mins
What is the enzyme detected in microdase test?
Oxidase
What is the principle of latex test for the identification of Staph aureus?
Latex particles have been coated w/ Ab that can recognize bound coagulase as well as Ig that will bind to protein A present on the surface of most strains of Staph aureus
What coats the latex particles?
Ab
What is the protein that is present on the surface of most strains of Staph aureus?
Protein A
What is the purpose of MSA?
It is a selective and differential medium used for the isolation and presumptive identification of Staph aureus
What is the action that occurs in MSA?
The high salt concentration inhibits growth of many organisms that inhabit skin and mucosal membranes. The phenol red indicator incorporated into the medium detects acid production (yellow) resulting to the fermentation of mannitol
What is the indicator incorporated in MSA?
Phenol red
Is the salt concentration in MSA high or low?
High
What is the color of acid production in MSA?
Yellow
What is the procedure (or steps) of novobiocin susceptibility?
1) Inoculate MH agar or BA w/ suspensions equivalent to a 0.5 McFarland std
2) Place Novobiocin disks (5 ug) on the agar surface and incubate for 24 hrs at 35 DC
3) ZOI measuring < or equal to 16 mm indicate novobiocin resistance, while > or equal to 16 indicates susceptible (or susceptibility)
What are the media that can be used for Novobiocin susceptibility test?
1) MH agar
2) BA
What is the time duration needed for incubation in Novobiocin susceptibility test?
24 hrs
What is the temp needed for Novobiocin susceptibility test?
35 DC
