Transcript Coagulase

THE ISLAMIC UNIVERSITY OF GAZA
FACULTY OF SCINCE
DEPARTMENT OF MEDICAL TECHNOLOGY
COAGULASE TEST
Kholoud Younes Shaheen
Student ID: 220051113
Supervisor :
Dr. Abdelraouf Elmanaama
1
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.
2
Why microorganism produce coagulase?
It has been proposed that fibrincoated staphylococci resist
phagocytosis making the bacteria
more virulent.
3
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.
4
Coagulase +ve
Coagulase -ve
5
signifigance
The coagulase test is used to
differentiate the potentially
pathogenic species Staphylococcus
aureus from the usually nonpathogenic species Staphylococcus
epidermidis.
6
The S. aureus (potentially
pathogenic in humans and animals,
but S. epidermidis (is not
pathogenic)
7
Scanning Electron Microscope
Staphylococcus
8
Staphylococcus aureus
9
Manitol salt agar
10
Types of coagulase
There are 2 types of cagulase
ensyme:
1- bound coagulase.
2-free coagulase.
11
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.
12
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.
13
Test methods
There are 2methods:
1-slide method.
2- tube method.
14
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.
15
Procedurer
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.
16
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.
17
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.
18
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.
19
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.
20
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.
21
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.
22
All "0" results after 4 hours should be
held at room temperature for a total
of 24 hours incubation.
23
24
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.
25