Lab

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Lab 23, 24, 25

• Micro lab

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Starch hydrolysis

• Starch molecules are too large to enter the
  bacterial cell, so some bacteria secrete
  exoenzymes to degrade starch into subunits that
  can then be utilized by the organism. 
• Starch agar is a simple nutritive medium with
  starch added.  Since no color change occurs in
  the medium when organisms hydrolyze starch, we
  add iodine to the plate after incubation.
• Iodine turns blue, purple, or black (depending on
  the concentration of iodine) in the presence of
  starch.
• A clearing around the bacterial growth indicates
  that the organism has hydrolyzed starch.

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Starch hydrolysis

• Iodine has been added to this starch agar plate.
  The zone of clearing surrounding
• Bacillus megaterium and Bacillus subtilis
  indicate that both were able to hydrolyze starch.

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Lipase test

• In our lab, the Lipase test we perform is with
  tributyrin agar .
• Tributyrin agar is a differential medium that
  tests the ability of an organism to produce an
  exoenzyme, called lipase, that hydrolyzes
  tributyrin oil.  Lipases break down lipids
  (fats). Tributyrin oil is a type of lipid called
  a triglyceride. Other lipase tests use different
  fat sources such as corn oil, olive oil, peanut
  oil, egg yolk, and soybean oil.
• Lipase allows the organisms that produce it to
  break down lipids into smaller fragments.
  Triglycerides are composed of glycerol and three
  fatty acids.  These get broken apart and may be
  converted into a variety of end-products that can
  be used by the cell in energy production or other
  processes.
• Tributyrin oil forms an opaque suspension in the
  agar. When an organism produces lipase and breaks
  down the tributyrin, a clear halo surrounds the
  areas where the lipase-producing organism has
  grown.

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Lipase test

• The Salmonella typhimurium on the left is
  negative for lipase production.  The Pseudomonas
  aeruginosa on the right is positive for lipase
  production, as evidenced by the clearing
  surrounding the growth.

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Casease test

• Casein forms an opaque suspension in milk that
• makes the skim milk agar appear white.

• Skim milk agar is a differential medium that
  tests the ability of an organism to produce an
  exoenzyme, called casease, that hydrolyzes
  casein. Casease allows the organisms that produce
  it to break down casein  into smaller
  polypeptides, peptides, and amino acids that can
  cross the cell membrane and be utilized by the
  organism. 
• When casein is broken down into these component
  molecules, it is no longer white.  If an organism
  can break down casein, a clear halo will appear
  around the areas where the organism has grown.

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Casease test

• The Bacillus megaterium on the left is negative
  for casease production.  The Bacillus subtilis on
  the right is positive

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Urease test

• This test is used to detect the enzyme urease,
  which breaks down urea into ammonia. Ammonia is a
  base and thus will raise the pH of the media if
  it is present. This change in pH is indicated by
  a pH indicator called phenol red which is present
  in the media.
• A color change from yellow to bright pinkish-red
  is positive lack of color change is a negative
  result. Inoculate the liquid media with a
  transfer loop.
• Members of genus Proteus are known to produce
  urease.

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Urea
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Nitrate test

• There are various ways that a bacterium can
  utilize nitrate as the final electron acceptor in
  anaerobic respiration. 
• The first obvious product of reduction to look
  for is reduction to N2 gas, called
  denitrification
• If there is no nitrogen gas, there are still a
  couple of possible interpretations---nitrate
  reduction to nitrite (NO2), reduction to ammonia,
  or no reduction of nitrate at all.    

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Nitrate reduction

• Nitrate reduction test is used to test for the
  reduction of nitrate to nitrite, ammonia or
  nitrogen gas.
• A deep red color produced after adding
  alpha-naphtylamine reagent is a positive result
  for nitrate reduction by nitrate reductase. NO3
  ------?NO2
• If there is no color change, the nitrate might
  not be reduced at all or the nitrate has been
  reduced to ammonia or N2.
• A pinch of zinc dust is added to colorless
  solution. If the addition of zinc does not
  produce a color change, the nitrates has been
  reduced all the way into ammonia or N2, showing a
  positive result. (red color is a neg result at
  this point)

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CATALASE TEST

• Some bacteria and macrophages can reduce diatomic
  oxygen to hydrogen peroxide or superoxide. Both
  of these molecules are toxic to bacteria.
• Some bacteria, however, possess a defense
  mechanism which can minimize the harm done by the
  two compounds. These resistant bacteria use two
  enzymes to catalyze the conversion of hydrogen
  peroxide and superoxide back into diatomic oxygen
  and water.
• One of these enzymes is catalase and its presence
  can be detected by a simple test.
• The catalase test involves adding hydrogen
  peroxide to a culture sample or agar slant. If
  the bacteria in question produce catalase, they
  will convert the hydrogen peroxide and oxygen gas
  will be evolved.
• The evolution of gas causes bubbles to form and
  is indicative of a positive test.

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catalase

• Catalase test is used to test the ability of
  bacteria to degrade hydrogen peroxide by using
  the enzyme catalase.
• The production of bubbles after addition of
  hydrogen peroxide (H202) shows that the catalase
  is present

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Oxidase

• Cytochrome oxidase is an enzyme found in some
  bacteria that transfers electrons to oxygen, the
  final electron acceptor in some electron
  transport chains. Thus, the enzyme oxidizes
  reduced cytochrome c to make this transfer of
  energy.
• Presence of cytochrome oxidase can be detected
  through the use of Chromogenic reducing agent
  which produces color when oxidized.
• No cytochrome oxidase---No color change indicates
  a negative test.
• Cytochrome oxidase present ---purple color
  indicates a positive test

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oxidase

• Oxidase test is used to determine the ability of
  certain bacterial species to produce cytochrome
  oxidase.
• If the cytochrome oxidase is produced, the
  colonies will turn into blue or black coloration
  10-15 seconds after the addition of chromogenic
  reducing reagent