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Gram Negative BacilliEnterobacteriacae

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Gram Negative Bacilli-Enterobacteriacae. Oxidase test. Used to distinguish ... the four Biochemical tests performed on the Gram Negative Bacilli being studied ... – PowerPoint PPT presentation

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Title: Gram Negative BacilliEnterobacteriacae


1
Gram Negative Bacilli-Enterobacteriacae
  • Oxidase test
  • Used to distinguish fermenters (Oxidase negative)
    from non-fermenters(Oxidase positive).
  • Testing for the presence of the enzyme indophenol
    oxidase.
  • The reagent (Tetramethyl-para-phenylenediamine)
    will be oxidized in the presence of oxygen by the
    enzyme indophenol oxidase producing a dark-purple
    product called indophenol.
  • Production of a dark purple product (within 10
    30 seconds) is a positive
  • ( ) test for oxidase.

2
  • Urease test
  • Testing for the presence of the enzyme urease.
  • This enzyme will break down urea to ammonia and
    carbon dioxide.
  • CO(NH2)2 NH3 CO2
  • The ammonia produced increases the pH of the
    media (becomes more alkaline) and causes the
    indicator present in the media (Phenol red) to
    change from a yellow-orange color to a hot pink
    color.
  • A hot-pink color is an indication of a positive
    test for Urease.

3
  • Triple Sugar Iron (TSI) Test
  • Test conditions
  • Provides information about sugar fermentation and
    the possible by-products produced by the
    organisms tested.
  • Three sugars, glucose, lactose, and sucrose are
    present in the media at different concentrations
    (Glucose at 0.1, Lactose at 1.0, and Sucrose at
    1.0).
  • Fermentation is an anaerobic process.
  • When sugar is fermented there may be an acid
    end-product produced.
  • Phenol red is again used in the media as a pH
    indicator and will change from red to yellow
    under acidic conditions.
  • Some organisms also may produce hydrogen gas (H2)
    as a by-product of fermentation.
  • Also present in the media is ferrous sulfate,
    which will react with any hydrogen sulfide (H2S)
    produced as a another possible by-product to form
    a black precipitate (ferrous sulfide). H2S
    production is an anaerobic process so the black
    precipitate produced will only appear in the butt
    of the tube.

4
  • Procedure
  • Work in pairs
  • You will be testing three organisms
  • Pseudomonas aeruginosa
  • Escherichia coli
  • Proteus vulgaris
  • You will need one TSI agar slant for each
    organism/group (3 tubes).
  • The tests require anaerobic conditions, therefore
    the organisms must be injected into the agar
    media. For this reason you will use sterile
    inoculating needles (not loops) for inoculating
    the tubes.
  • The best method is to obtain one of the
    organisms, preferably on a nutrient agar plate,
    and roll the tip of a sterile inoculating needle
    in the organism.
  • Next stab the agar slant nearly to the bottom of
    the agar tube and when removing the inoculating
    needle, lightly streak the surface of the slant.
  • Follow the same procedure for each organism
    tested.
  • The tubes are placed in the incubator for 18 - 24
    hours at 37 C.

5
  • Interpretation of Results
  • Many of the enteric organisms will ferment
    glucose with the production of acids which will
    change the color of the medium in the butt and
    along the slant from red to yellow because of a
    reduction in the pH (within the first few hours).
  • However, since the glucose is present in small
    amounts (0.1), the supply is soon exhausted and
    the organisms growing on the surface of the slant
    in the presence of oxygen are forced to
    catabolize peptones and amino acids in the media
    for their energy supply. Alkaline end-products (
    NH4OH) are produced from these substances which
    revert the pH of the slant to an alkaline pH and
    thus change the color of the agar slant back to
    red (after 18-24 hours).
  • Organisms such as Salmonella spp. or Shigella
    spp. and other organisms which attack glucose but
    do not ferment lactose or sucrose will produce an
    alkaline slant and acid butt in TSI slants in 18
    to 24 hours. Since metabolism is progressing at a
    slower rate in the butt, this reversion does not
    usually take place in the butt until 48 hours or
    longer.
  • If the glucose is metabolized to CO2, the gas
    will be seen as bubbles or cracks in the agar
    butt. If hydrogen sulfide is formed during
    growth, a gray or black streak of iron sulfide is
    seen originating where the inoculating needle
    entered and throughout the agar butt.
  • Organisms which attack lactose and/or sucrose,
    such as Escherichia, will produce acid slants and
    acid butts usually with the formation of gas. In
    these cases, the acid slants do not revert to an
    alkaline status because lactose (1) and sucrose
    (1) are being fermented and are present in
    concentrations ten times that of glucose.
  • Some organisms (e.g., Pseudomonas, Acinetobacter)
    fail to ferment even glucose, and because they
    are strictly aerobic, they fail to grow in the
    butt of the tube. In these cases, the butt will
    be unchanged in color, and the slant either
    alkaline or unchanged.

6
TSI Test Results
Control
Eschericihia Klebsiella Enterobacter
Pseudomonas aeruginosa
Proteus vulgaris
Shigella
Salmonella enteritidis
7
Recording the Results of the TSI Tests
  • K alkaline Red  A acid Yellow  NC No
    change  G gas produced  H2S hydrogen
    sulfide produced
  • Acid or alkaline results in the slant are
    reported first, followed by the butt results
    (e.g., K/A would be read as "K over A" or
    "alkaline over acid" and refers to an alkaline
    slant and acid butt).
  • Slant result / Butt result
  • K/A Only Glucose fermented
    peptone utilized
  • A/A Glucose and Lactose/Sucrose fermented
  • K/K No sugars fermented, only peptone
    utilized
  • K/NC No sugars fermented, Peptone used
    aerobically only
  • NC/NC No growth, neither sugars or peptone
    used
  • A/AG Glucose and Lactose/Sucrose fermented,
    gas produced
  • A/A H2S Glucose and Lactose/Sucrose
    fermented, H2S produced
  • A/AG H2S All sugars fermented and both H2S
    and gas produced
  • K/AG Only Glucose fermented peptone utilized,
    gas produced
  • K/A H2S Only Glucose fermented peptone
    utilized, H2S produced
  • K/AG H2S Only glucose fermented Peptone
    utilized, both gas and H2S
  • produced

8
Motility Test
  • To test for bacterial motility a diluted agar is
    used. Normally the agar concentration is 1.5 g of
    agar per mL of nutrient media broth. Motility
    agar is usually 0.7 g agar per mL of nutrient
    broth.
  • When the lower concentration agar solidifies it
    is less gel-like and more fluid allowing bacteria
    with flagella to move into the surrounding media.
    Additionally, a special reagent,
    Triphenyltetrazonium chloride, or TTC, is used to
    make the motility more visual in the tube. TTC is
    colorless and soluble in its oxidized form, but
    becomes insoluble and dark red in color when in
    its reduced state. Any metabolic process, such
    as the expenditure of energy for flagellar
    movement, will reduce the TTC and cause the
    appearance of the red, reduced TTC in areas where
    motile bacteria are present.

9
Identification of GNB IMViC Tests
  • Mnemonic for the four Biochemical tests performed
    on the Gram Negative Bacilli being studied
  • Indole test
  • Methyl Red test
  • Voges-Proskauer test
  • Citrate utilization test
  • These tests divide the Enterobacteriaceae into
    two major groups
  • Escherichia coli group
  • Enterobacter-Klebsiella group

10
  • Indole test
  • Tests for the production and secretion of the
    enzyme tryptophanase.
  • If the organism can produce tryptophanase and
    break down tryptophan to Indole the Indole will
    react with Kovacs reagent (p-dimethylaminobenzald
    ehyde) forming a pink-colored chemical complex.
  • We use commercially prepared slides impregnated
    with Kovacs reagent

11
Methyl Red Test
  • Some organisms produce acid from the metabolism
    of glucose (fermentation) in a sufficient
    quantity to alter the pH of the media to about
    4.4. These are stable acids and are not further
    metabolized.
  • Methyl red indicator is used to detect the
    presence of these acids in the MRVP broth medium.
    Methyl red indicator at this pH ( 4.4) changes
    to a bright cherry red color.

12
Voges-Proskauer Test
  • Some microorganisms produce organic acids from
    glucose metabolism, but further metabolize the
    acid produced to various neutral end products,
    like acetoin, and 2,3-butanediol.
  • There is an initial pH drop in the MRVP broth,
    but the neutral end product raise the pH so that
    the methyl red test will be negative.
  • The presence of acetoin, and 2,3-butanediol is
    tested for using a-naphthol which reacts with
    these two neutral products to produce a mahogany
    red color.

13
Citrate Test
  • Citrate may be used as a carbon source by some
    microorganisms.
  • The Citrate test uses an agar medium with citrate
    and the pH indicator Bromothymol blue present. At
    the pH of the un-inoculated medium the color is
    Blue-green.
  • If the organism can utilize Citrate as a carbon
    source the breakdown of citrate releases
    bicarbonate ions (HCO3-) into the medium. The
    bicarbonate ions raise the pH of the medium above
    7.4 pH. This causes the Bromothymol blue
    indicator to turn dark blue in color.
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