Transcript Biochemical Reactions

Biochemical Reactions
Catalase Test:
• Principle: If organisms have catalase enzyme they
will hydrolyze hydrogen peroxide (substrate) leading
to production of oxygen (end product)
2H2O2 >> 2H2O + O2
• End point: air bubbles >> catalase positive
No air bubbles >> catalase negative
• Used to differentiate staphylococci from streptococci
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)
Oxidase Test:
• Used to identify bacteria that produce cytochrome c
oxidase enzyme
• Principle: cytochrome c oxidase enzyme oxidizes the
(reagent) TetraMethylPhenyleneDiamine (TMPD) to
indophenol -purple color- (end product) in the
presence of atmospheric oxygen
• End point: purple color>> oxidase positive
No purple color>> oxidase negative
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 negative
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 insoluble
Nitrate test:
• Principle: if organisms have nitrate reductase
enzyme it will reduce nitrate (NO3) to nitrite (NO2)
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 negative
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 negative
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 color
• 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 colorlesslight 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.
A.A Decarboxylation Results of some
Enterobacteriaceae
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.
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 -ve
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 copperyellow color is present, the result is -ve.
Note: MR & VP can’t BOTH be positive; they test for
different fermentation pathways, the bacteria will
only be using one
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) -ve
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
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).
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.
K/A= red/yellow= glucose fermentation only
= NLF
A/A= yellow/yellow= glucose and lactose and/or
sucrose fermentation= LF
K/K= red/red= no fermentation