BIOCHEMICAL REACTIONS

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BIOCHEMICAL REACTIONS
By
Dr. Emad AbdElhameed Morad
Lecturer of Medical Microbiology and Immunology
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Biochemical reactions

• Sugar fermentation
• Composed of
• Six tubes.
• Each tube contains peptone water specific sugar
  in a concentration of 1.
• The sugars are glucose, lactose, maltose,
  mannite, sucrose, salicin.
• Each tube contains also Andrades indicator which
  changes to pink if acid is produced.
• An inverted tube (Durhams tube) is placed in the
  medium. Gas production is revealed by collection
  of bubbles at its apex.

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• Used to
• Differentiate between bacteria according to
  their fermentative effect on different sugars and
  whether the fermentation is associated with gas
  production or not.

Fermentation of glucose, maltose, mannite with
acid only
Fermentation of all sugars with acid gas
production
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• Indole test
• Principle
• Demonstrates the ability of certain bacteria to
  decompose the amino acid tryptophan present in
  peptone water to indole.
• Indole is then tested for by adding few drops of
  Kovacs reagent which gives a pink ring in the
  presence of indole.
• Procedure
• The organism is inoculated in peptone water and
  after incubation at 37 degree for 24 hours,
  Kovacs reagent is added.
• Interpretation
• If a pink ring is produced, the organism is
  indole Ve (E. coli).
• If a yellow ring is produced, the organsim is
  indole Ve (Klebsiella).

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• Voges-Proskauers reaction (VP)
• Principle
• Some bacteria ferment glucose with production of
  acetyl methyl carbinol.
• Procedure
• Bacteria is grown in glucose phosphate peptone
  water for 48 hours.
• Then KOH is added to test for acetyl methyl
  carbinol formation.
• Interpretation
• If an eosin pink color is produced, VP reaction
  is Ve.

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• Methyl red test (MR)
• Principle
• Detects the ability of some bacteria to produce
  large amounts of acid on fermentation of glucose,
  thus lowering the pH of the medium below 4.
• Procedure
• The bacteria is grown in glucose phosphate
  peptone water.
• After incubation at 37 degree for 48 hours, few
  drops of methyl red indicator are added.
• Interpretation
• A positive test gives bright red color.
• A negative test gives a yellow color.

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• Urease test
• Principle
• Some organisms produce urease enzyme.
• This enzyme splits urea with the release of
  ammonia.
• Ammonia causes alkalinity and increases pH of
  the medium.
• The phenol red indicator turns deep pink.
• Procedure
• The bacteria is grown on a medium containing urea
  and phenol red indicator for 24 hours.
• Interpretation
• Urease positive organisms such as proteus will
  turn the medium deep pink.

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Ve
-Ve
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• Oxidase test
• Principle
• Some bacteria produce oxidase enzyme which
  reduces the oxidase reagent (tetramethyl-p-phenyle
  ne-diamine hydrochloride) to a deep purple color.
• Procedure
• Done by picking up a portion of the colony tested
  and smearing it on a filter paper impregnated
  with oxidase reagent.
• Interpretation
• The immediate development of a deep purple color
  indicates positive test.
• Examples neisseria pseudomonas vibrios.

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• H2S test
• Principle
• Production of H2S from proteins.
• Procedure
• Inoculate the organism on peptone water.
• After incubation, put lead acetate paper at the
  mouth of the tube.
• Interpretation
• If the organism produces H2S, a black color is
  formed.
• Examples proteus Salmonella typhi.

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• Catalase test
• Principle
• Tests the ability of some bacteria to produce
  catalase enzyme.
• Procedure
• Pick up the test colony on a platinum loop and
  immerse it in few drops of 3 H2O2 (hydrogen
  peroxide).
• Interpretation
• Rapid effervescence indicates oxygen production
  and a positive test.
• This test is used to differentiate between
  staphylococci (Ve) and streptococci (-Ve).

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Catalase Ve
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• Coagulase test
• Detects the coagulase bound to the organism.
• Homogenous suspension of the test organism is
  made in a drop of saline on a slide then mixed
  with a drop of undiluted human or rabbit plasma.
• Staphylococcus aureus clumps within 15 sec.
  because coagulase enzyme precipitates the fibrin
  in the plasma on the cell surface.

Slide method
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• Detects the free coagulase.
• It is done by adding 5 drops of an overnight
  broth culture of the test organism to 0.5 ml of
  human or rabbit plasma diluted 1/10 in sterile
  saline.
• The tubes are incubated for 6-12 hours and
  inspected hourly for coagulation.
• Staphylococcus aureus is coagulase positive.

Tube method
Positive
Negative
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• DNAase test
• Principle
• This test is used to identify Staph. aureus as it
  produces DNAase enzyme.
• Procedure
• The tested organism is cultured on a medium which
  contains DNA.
• After overnight incubation, the colonies are
  tested for DNAase production by flooding the
  plate with weak HCL.
• The acid precipitates unhydrolyzed DNA.
• Interpretation
• DNAase producing colonies (such as Staph. aureus)
  are surrounded by clear areas due to DNA
  hydrolysis.

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DNAase Ve
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• Analytical profile index (API)
• Principle
• It is composed of a plastic strip with cupules
  containing dehydrated substances. Each cupule has
  a small hole at the top.
• Procedure
• A saline suspension of the test organism is
  dropped in the cupules.
• The strip is covered with a lid and placed in a
  humidified plastic chamber and incubated at 37
  degree for 24-48 hours.
• Interpretation
• Biochemical profiles are determined by reading
  the color change and interpret according to the
  available charts.
• These are then converted to numerical codes which
  will be read from a profile index to identify the
  bacteria.

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API
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• Automated bacterial identification systems
• Principle
• Examples Vitek system, microscan, phoenix.
• These systems identify the organism and its
  antibiotic sensitivity by detecting color changes
  or turbidity in special plastic cards inoculated
  with the organism.
• Such cards are composed of tiny wells that
  contain substrates for detection of biochemical
  reactions and antibiotic sensitivity.
• Once the card has been inoculated and placed in
  the instrument, it will automatically perform all
  readings.
• Results are available within 4-6 hours.

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Vitek card
Vitek system
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GOOD LUCK