Carbohydrate Fermentation: 24-48 hour read (check for

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Microbiology Laboratory

• Oct 30 Oct 31

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Review of Biochemical Tests

• Why do we need to focus on the metabolic
  characteristics of organisms?
• Recording your results remember to write all
  this down on your biochemical result sheet in the
  lab manual

3
Catalase Test Immediate Read(p. 50 photographic
atlas)

• Test identifies bacteria that produce the enzyme
  catalase
• This enzyme enables obligate aerobes to breakdown
  toxic byproducts of respiration (ex hydrogen
  peroxide of superoxide radicals)
• A positive result was indicated by the appearance
  of bubbles

4
Oxidase Test Immediate Read (p. 72 photographic
atlas)

• The oxidase test involved substituting an
  artificial substrate p-phenylenediamine for the
  reduced cytochrome
• A positive result was indicated by a purple color
  formed on the slide

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Carbohydrate Fermentation 24-48 Hour Read
(check for growth)

• The ability to ferment following carbohydrates
  glucose, sucrose, lactose and mannose.
• Tests for gas production color change due to pH
  change.
• Phenol red changes to yellow when conditions are
  acidic-when acidic, sugar is being fermented.

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Carbohydrate Fermentation 24-48 hour read (check
for growth)
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Carbohydrate Results
Negative orange or red
Positive yellow, or yellow with gas
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Nitrate Reduction Test(p. 68-70 photographic
atlas)

• Nitrate can be reduced to nitrite (NO2-) and some
  microorganisms can reduce the nitrite further to
  ammonia (NH3) or even to nitrogen gas (N2).

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Nitrate Reduction 24/48 hour check for growth,
refrigerate until next lab session

• Check for the presence of gas in the Durham tube.
  
• If there is gas in the Durham tube, it is
  nitrogen and this observation alone is a positive
  test for nitrate reduction.

Positive ? Stop
Negative ? Go to next step
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Step 2 Gas Not Present ? Do Following

• Add 10 to 15 drops of Nitrite A reagent to your
  tube
• Nitrite A 0.8 sulfanilic acid in 5 N acetic
  acid
• To same tube, add same amount Nitrite B reagent
• Nitrite B 0.6 dimethyl-alpha-naphthylamine in
  5 N acetic acid
• Note that dimethyl-alpha-naphthylamine is closely
  related to compounds that are carcinogenic.
• If any of this reagent contacts your hands, wash
  them immediately
• Keep gloves on for nitrate test
• Positive - If the culture turns red within 15
  min. it is positive for the presence of nitrite
  and positive for nitrate reduction.
• Negative - If after 15 min. there is no color
  change, then one of two events have occurred
  either the nitrate has not been reduced or
  nitrate has been reduced beyond nitrite to
  ammonia or nitrogen gas. ? Perform next test!

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Step 3 No change after Nitrate A and B were
added

• Add small amount of zinc powder to test tube
• Let me do this step as
• Zinc is explosive and
• We need to keep track of our zinc waste
• Red color within 15 min test is positive for
  presence of nitrate, but negative for nitrate
  reduction.
• No color change nitrate has been reduced to
  either ammonia or nitrogen gas and is positive
  for nitrate reduction.

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Nitrate Reduction Procedure
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Motility Test 24-48 hour read (check for
growth)(p. 67-68 photographic atlas)

• True motility (directed movement) is different
  than Brownian movement.
• Motility can be observed in a wet mount or
  hanging drop preparation of the organism.
  However, wet mounts tend to dry out quickly
  rendering the organisms immotile.

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Motility Test Results
Positive Cloudiness in whole tube
Negative Cloudiness only around stab mark
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Simmons Citrate 24-48 hour read (check for
growth)(p. 51-52 photographicatlas)

• This test determines if an organism can transport
  citrate and use it as the sole carbon source
• Organisms that live in this medium can also use
  ammonium ions (instead of amino acids) as the
  sole nitrogen source
• The pH indicator is brom thymol blue. This
  indicator is green at neutral pH but turns blue
  above pH 7.6
• When citrate is being utilized, bacteria increase
  the alkalinity of the solution
• As the alkalinity increases, a characteristic
  blue color will appear

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Simmons Citrate Results
Positive color changes from green to blue
Negative - No color change
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Urea Hydrolysis 24-48 hour read (check for
growth)(p. 79 photographic atlas)

• This test differentiates organisms by their
  ability to hydrolyze urea
• If urease is present, ammonia will be released
  and the pH will rise
• Similar to the Simmons citrate test, as the pH
  rises, the indicator will change color

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Urea Results

• Positive - Cerise (a hot pink-cherry color)

• Negative Yellow, Orange, or light pink

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Kligler's Iron Agar 24-48 hour read (check for
growth) (p.61-62 photographic atlas)

• Kligler's iron agar is used to test for the
  production of H2S often results from the
  deamination of the sulfur containing amino acid
  cysteine.
• A positive test shows a dark precipitate that has
  formed in the tube. The absence of a precipitate
  is a negative test.
• Since this medium also contains glucose, lactose
  and phenol red, the medium might also turn yellow
  due to the fermentation of these carbohydrates.
• Note that a yellow color in the tube without a
  dark precipitate is still a negative test for H2S
  production.

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Kligler's Iron Agar Results
Negative - no precipitate, any yellow, red, or
orange color without a precipiate
Positive dark precipitate
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Gelatinase Test 1 week read (check for growth)
(p. 59 photographic atlas)

• Gelatin is a heterogeneous mixture of very large,
  water-soluble proteins and is prepared from
  collagen by boiling skin, tendons, ligaments,
  bones etc., with water.
• Many microorganisms produce an enzyme called
  gelatinase that can degrade or breakdown the
  gelatin into smaller polypeptides and amino acids
  that can be taken up and used by the cell.
• Gelatin liquefies at temperatures above 30?C but
  solidifies at 4?C. When hydrolyzed by the enzyme
  gelatinase, however, gelatin does not gel when
  placed at 4? or 5?C.
• Essentially, when the enzyme has broken the
  gelatin molecule by adding water, it is no
  longer able to solidify

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Gelatinase Test Results

• After one week incubation, chill the tubes in an
  ice-water bath.
• Do not shake the tubes when transferring them to
  the ice bath as this medium is already a bit
  "loose."
• Negative - Gelatin tube should "firm up" when
  chilled.
• Positive - If your unknown organism produced
  gelatinase and hydrolyzed the gelatin, the
  gelatin will remain liquid after chilling.
• If your unknown organism did not hydrolyze the
  gelatin after one week incubation, continue
  incubating your unknown for another week.

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Gelatinase Test Results
Positive Gelatin remains liquid after chilled
Negative Gelatin is firm after chilled
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Starch Hydrolysis 24-48 hour growth check,
refrigerate until next lab session(p. 75-76
photographic atlas)

• Starch is a complex polysaccharide that can be
  hydrolyzed by a variety of microorganisms via
  extracellular enzymes called a-amylases.
• Again-these enzymes are adding water to the
  molecules to break the bonds
• Starch molecules are much too large to be taken
  into the cell, and must be broken down to
  transport
• similar to the process many large proteins
  undergo

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Starch Hydrolysis Results

• Add a few drops of Gram's iodine to your
  refrigerated plate
• use just enough to cover the surface of the plate
• Areas on the plate that contain starch will form
  a dark blue or purple complex.
• Areas around colonies in which the starch has
  been hydrolyzed will appear as clear zones.
• Positive - A clear zone around your test organism
  after treatment with Gram's Iodine.

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Starch Hydrolysis Results
Positive
Negative
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Casein (milk protein) Hydrolysis24-48 hours,
check for growth and read

• Proteins often need to be broken down into
  individual amino acids or small peptides (chains
  of a few amino acids) in preparation for
  transport into the cell

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Casein Results
Positive zone of clearing around organism
Negative no zone of clearing
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Lipid Hydrolysis 1 week growth check, continue
to incubate if necessary(p. 62-63 photographic
atlas)

• Lipases (or esterases) are enzymes which
  hydrolyze the ester linkages that hold fatty
  acids to glycerol
• Positive zone of clearing around organism.
• Negative no zone of clearing (lightening of the
  medium is also a negative result)
• Be careful when interpreting this result
• If you do not have a definite positive, check
  with a TA
• You may let your plate incubate for another week
  if you do not have a positive result

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Lipid Hydrolysis Results
Positive zone of clearing around organism
Negative no zone of clearing around organism
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Facultative Anaerobes 1 week read time (check
for growth)

• Many bacteria can grow both aerobically and
  anaerobically
• Organisms that can grow in the presence or
  absence of oxygen are call "facultative
  anaerobes"
• E. coli is an example
• Check to see if your culture grew in an
  anaerobic chamber. If so, you have a facultative
  anaerobe.

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Bergeys Manual of Systematic Bacteriology

• The goal of this entire environmental unknown
  project has been to obtain an organism from the
  environment, isolate it from other organisms,
  carry out a series of tests and use the results
  of those tests to identify the organism
• So far weve done all but the last part

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Bergeys Manual of Systematic Bacteriology

• Bergeys Manual of Systematic Bacteriology is
  going to help us with the last part
• This book is based upon years of research with
  specific organisms
• The volumes we have rely mainly on the results of
  biochemical tests while newer volumes focus on
  rRNA

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Bergeys Manual of Systematic Bacteriology

• This manual will help you determine the identity
  of your organism
• You will use the information presented and the
  process of elimination
• You need to identify it down to the genus level
• Do your best to identify to the species level
• It is separated into two volumes
• Volume I
• Volume II

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Bergeys Manual of Systematic Bacteriology

• Start with your morphology and your Gram reaction
• Ex Gram positive rods, Gram negative rods, Gram
  positive cocci
• Then use the results of your other tests to
  eliminate certain groups of organisms (genus
  level) or certain organisms (species level)
• Ex. A Gram positive rod that is catalase
  positive, gelatinase negative and ferments
  glucose will eliminate several other types of
  organisms

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Example of page from Bergeys Manual
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Bergeys Manual of Systematic Bacteriology

• In addition to the biochemical tests results you
  should pay attention to the location from which
  you isolated your organism
• Was it from the soil? Was it from a tree?
• If you are working to narrow down two groups or
  organisms or are deciding between two organisms
  and one is isolate from fish intestines and the
  other from soil which do you think is the more
  likely possibility?
• The point is-you have to read the descriptions of
  the organisms not just base your conclusions off
  the tables

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Bergeys Manual of Systematic Bacteriology

• Common abbreviations
• d 11-69 of strains are positive
• ND no data available
• D different in different taxa
• or - 90 or more of the strains are positive
  or negative

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Environmental Isolate Staining

• In the time remaining continue microscopic
  examination of your environmental unknown(s),
  including simple, capsule stain, acid-fast and
  endospore stain
• Should have time next week
• Record your results on the back of your
  biochemical sheet
• Remember to use controls (look at your handout if
  you forget my directions)
• Endospore stain should be done with organisms
  from a stress plate

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Next Week

• Turn in The bacterial growth graphs
• Exercise 8
• You will get back your Hamburger Report
• Should have time to work on staining of your
  environmental unknown