We can use our knowledge in bacterial enzymes to identify the bacteria and distinguish between bacterial species Enzyme Substrate Product Enzymes Types of Enzymes ID: 528291
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Slide1Slide2
In the microbiology lab, biochemical test relays on enzymes which is glycoprotein or protein that act as catalyst by lowering the activation energy of certain biological reaction.
We can use our knowledge in bacterial enzymes to identify the bacteria and distinguish between bacterial species.
EnzymeSubstrate Product
EnzymesSlide3
Types of Enzymes
According to site of the reactionEndoenzymes : where substrate and enzyme react inside the cell.(ex: oxidase, catalase, urease, nitrate reductase).Exoenzymes: where substrate and enzyme react outside the cell. (ex: free coagulase, gelatenase, amylase, lipase, casienase).
According to enzyme productioninducible : produced only when needed or induced.constitutive : produced continuously Slide4
Notes:
Every genus of bacteria has it’s unique set of enzymes, so we can identify it.Endoenzymes may act outside the cell in case of the presence of high concentration of it’s substrate.
The same enzyme could be inducible and constitutive in different generaSlide5
Kinds of bacterial enzymatic reactions
The breakdown of toxic wastes such as hydrogen peroxide or urea (ex: catalase)The reduction of nitrate or oxygen (ex: nitrate
reductase).The degradation of specific amino acids (ex:
treptophanase).The utilization of noncarbohydrate
carbon sources for growth (ex: urease).Slide6
Catalase Test
Enzyme name \ catalaseSubstrate name \ hydrogen peroxide
Enzyme action \ breakdown the toxic H2O2 producing oxygen gas and water
Hydrogen peroxide produce due to the aerobic respiration of the cells and have to be breakdown to prevent it’s toxic action on DNA and cell membrane
Catalase
2H2O2
2H2O
+ O2Slide7
By catalase test we can distinguish between:
G (+ve
) cocci : staphylococcus is
catalase positive where streptococcus is catalase negative
G (+ve
) bacilli :
Bacillus
is
catalase
positive where
Clostridium
is catalase negative
All
Enterobactreacae
(a gram negative bacilli) are
catalse
positive
Lesteria monocytogenes
( a gram positive bacilli) are catalase positive
When hydrogen peroxide is added to a colony
of
catalase-producing bacteria, it is broken down
and
the oxygen that is produced can be seen as
bubbles. Slide8
How to do the Test
Slide methodAdd
one drop of 3% Hydrogen peroxide on a clean glass slide.Aseptically take a loopful of the test organism and emulsify in the H2
O2 drop.
Capillary tube methodInoculate
the test organism on agar slant and incubate for 24
hours
.
Allow 1 mL of 3% hydrogen peroxide to flow over the slant.
Adding hydrogen peroxide directly to a pure slant culture.Slide9Slide10
Be careful when using bacteria from blood agar culture and avoid touching the agar by the loop because blood cell in agar also had catalase enzyme.
false positiveAlso don’t use bacteria from old culture because the enzymes activity drops by time. false negative
Notes:Slide11Slide12
Coagulase Test
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.Slide13
Significance
The coagulase test is used to differentiate the potentially pathogenic species Staphylococcus aureus
from other Gram-positive cocci, the usually non-pathogenic species.The
S. aureus (potentially pathogenic in humans and animals, but S. epidermidis
(is not pathogenic)Slide14
Types Of Coagulase
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
.Slide15
Test methods
There are
2 methods:
Tube Method (detects the presence of either bound or
free).
Slide Method
(
detects only bound
coagulase).Slide16
Procedure of Slide Method
Place a drop of coagulase plasma on a clean, dry glass slide
.Place a drop of distilled water or saline near the drop of plasma as a control
.With
a sterile loop or wooden stick, emulsify an amount of the isolated colony being tested into each drop.
Inoculating
the water or saline first
.
Try
to create a smooth suspension.Slide17
Observe
for clumping in the coagulase plasma and a homogenous suspension in the control.Clumps
that will not mix uniformly into coagulase plasma indicate a positive test whereas a uniform suspension is indicative of a negative test.
Clumping in both tests indicate that the organism autoagglutinates
and is unsuitable for the slide coagulase test. When
autoagglutination is observed.
the tube coagulase test should be employed as an alternative to the slide agglutination test.Slide18
Procedure of Tube Method
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.
Incubate the inoculated tube at 35-37 degrees C. for
1 to 4 hours.
Negative
tests at 4 hours should be held at room temperature for a total of 24 hours before reporting results
.
Read
by gently tilting the tube while observing for clotting of plasma
.Slide19
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.
Results should be read at 4 hours.
A positive test for coagulase production results in a clotting of the rabbit plasma.Any degree of clotting is a positive test.
All
"0"
results after 4 hours should be held at room temperature for a total of 24 hours incubationSlide20
When
the slide test is employed, all negative slide reactions must be confirmed by the tube
test .
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.
spontaneous agglutination may occur when rough cultures are used.
The tube test is more reliable than the slide test
.
NotesSlide21
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.Slide22Slide23
END OF LECTURE