Catalase Test Principle If organisms have catalase enzyme they will hydrolyze hydrogen peroxide substrate leading to production of oxygen end product 2H 2 ID: 569888
Download Presentation The PPT/PDF document "Biochemical Reactions" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Biochemical ReactionsSlide2
Catalase Test:
Principle:
If organisms have catalase enzyme they will hydrolyze hydrogen peroxide
(substrate)
leading to production of oxygen
(end product)
2H
2
O
2
>> 2H
2
O + O
2
End point: air bubbles >> catalase positive
No air bubbles >> catalase negative
Used to differentiate staphylococci from streptococciSlide3Slide4
Coagulase test:
Principle:
if organisms have coagulase enzyme it will converts fibrinogen (plasma) to fibrin (clot)
This test is done by slide method or tube method
Used to differentiate between
S.
aureus
(CPS) and other Staphylococcus species (CNS)Slide5Slide6Slide7
Oxidase Test:
Used to identify bacteria that produce cytochrome c oxidase enzyme
Principle:
cytochrome c oxidase enzyme oxidizes the
(reagent)
T
etra
M
ethyl
P
henylene
D
iamine
(TMPD) to indophenol -purple color-
(end product)
in the presence of atmospheric oxygen
End point: purple color>> oxidase positive
No purple color>> oxidase negativeSlide8Slide9
DNAse
test:
Principle:
if organisms have
deoxyribonuclease
(
DNAse
) enzyme it will
hydrolyse
DNA in the media and give clear area around the colony after adding HCL
End point: clear halo around the colony>>
DNAse
positive
No clear halo>>
DNAse
negativeSlide10Slide11
Bile solubility test:
Used to differentiate
Streptococcus
pneumoniae
(bile soluble) from other alpha- hemolytic Streptococcus like
Streptococcus
viridans
(bile insoluble)
Add bile salt to the bacterial colony>> colony disappears>> bile soluble bacteria
Add bile salt to the broth:
Clear>> bile soluble
Turbid>> bile insolubleSlide12Slide13
Nitrate test:
Principle:
if organisms have nitrate
reductase
enzyme it will reduce nitrate (NO
3
) to nitrite (NO
2
) which give red color after adding the
reagents
Nitrate broth medium is used in
this test. It contains nutrients and potassium nitrate as a source of nitrate.
End point: red color broth>> nitrate positive yellow color broth>> nitrate negativeSlide14Slide15
Indol
test:
Used to identify bacteria that produce
tryptophanase
enzyme
Principle:
tryptophanase
enzyme breakdown tryptophan (substrate) to
indol
(end product) and give red color ring after adding the reagent
Medium used: is tryptophan broth which contains the
a.a
trptophan
End point: red ring>>
indol
positive
no red ring>>
indol
negativeSlide16Slide17
Amino Acid Decarboxylation Test:
Principle:
determines the ability of organisms to produce amino acid decarboxylase enzyme that removes carboxyl groups from amino acids
Medium used is decarboxylase base containing nutrients, dextrose and pH indicators with added amino acids (either arginine, ornithine or lysine). The
a.a
content gives the broth an alkaline
pH.
Un inoculated broth is purple in colorSlide18
Bacteria first ferment dextrose to produce acids>> PH changes from alkaline (purple) to acidic (colorless-light yellow).
Following dextrose fermentation:
If org. produces the decarboxylase enzyme specific to the
a.a
in the tube, it will yield alkaline products>> pH reverts to alkaline (purple).
If not, the broth remains acidic. Color stays colorless-light yellow.
End point: if broth is yellow, the organism is decarboxylase -
ve
for that
a.a
.
If the medium is purple, the organism is decarboxylase +
ve
for that
a.a
.Slide19Slide20
A.A Decarboxylation Results of some EnterobacteriaceaeSlide21
Methyl Red- Vogues
Proskaur
(MR-VP):
Bacteria metabolize sugars by different pathways to produce either stable acidic end products or non- stable acids that quickly convert to neutral end products.
MR-VP broth is the medium in which both the Methyl Red and
Voges-Prosakuer
tests can be performed. It contains peptone, buffers, and either the sugar dextrose or glucose.
Organisms that metabolize sugars using the mixed acid pathway produce stable acids such as lactic acid, acetic acid and formic acid (remain acidic).
Organisms using the butylene glycol pathway produce unstable acids that are further metabolized to yield neutral end products such as
acetoin
and
butanediol
.Slide22
Methyl Red Test Principle and Results:
Tests for orgs. that use mixed acid fermentation of sugars
MR detects PH change and is +
ve
when the PH is acidic.
If broth turns red after adding MR reagent (methyl red), the result is positive.
If, after the reagent has been added, a copper-yellow color is present, the result is -
veSlide23
Vogues
Proskaur
Principle and Results:
Tests for bacteria that metabolize sugars by the
bytylene
glycol pathway
VP detects alcohols and is +
ve
when PH is neutral.
When VP reagents (VP1,VP2) are added to MR-VP broth that has been inoculated with an org. that uses the butylene glycol pathway, a red color is produced indicating a +
ve
result.
If, after the VP reagents have been added, a copper-yellow color is present, the result is -
ve
.Slide24
Note: MR & VP can’t BOTH be positive; they test for different fermentation pathways, the bacteria will only be using oneSlide25
Ureas
test:
Light orange media, contain urea
Indicator: phenol red
Principle: If organism produce urea's enzyme, it will break down urea to ammonia and carbon dioxide, the PH will change from acidic to alkaline and the color of media will change from light orange to deep pink color (+
ve
)
If organism do not produce urea's enzyme, the media remain light orange(no color change) -
veSlide26Slide27
Citrate test:
Test the ability of organism to utilize citrate as a carbon source for the production of energy.
Citrate agar contains sodium citrate as the sole source of carbon, ammonium phosphate as the sole source of nitrogen , other nutrients, and the pH indicator
bromothymol
blue.
Bacteria that can utilize citrate as their sole carbon source produce an enzyme, citrate
permease
to transport the citrate into the cell for the production of energy
The pH change turns the
bromthymol
blue indicator from green to blue
End point: green color>> citrate –
ve
.
blue color>> citrate +
ve
Slide28Slide29
Triple Iron Sugar Test (TSI):
Composition of TSI:
Lactose, Sucrose and Glucose (10:10:1)
Iron: Ferrous sulfate: Indicator of H2S formation
Phenol red: Indicator of acidification (It is yellow in acidic condition and red under alkaline conditions). Slide30
Interpretation of Triple Sugar Iron Agar Test:
Lactose and sucrose are both present in very large amounts (1%). If either one is fermented, a large amounts of acid are produced and the whole tube turns from red to yellow. Some species generate gases, which producing bubbles/cracks on the medium
Glucose is present in a very small amount (0.1%) and if it is the only sugar fermented only a very small amount of acid can be produced from it. Therefore, the butt only become (yellow). Some species may also produce gas from glucose. Organisms that produce H2S react with the ferric sulfate to form black ferrous sulfide.
IF neither lactose/sucrose nor glucose is fermented, both the butt and the slant will be red. Slide31
K/A= red/yellow= glucose fermentation only = NLFSlide32
A/A= yellow/yellow= glucose and lactose and/or sucrose fermentation= LFSlide33
K/K= red/red= no
fermentationSlide34