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Title: Carbohydrate Fermentation: 24-48 hour read (check for


1
Microbiology Laboratory
  • Oct 30 Oct 31

2
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

5
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.

6
Carbohydrate Fermentation 24-48 hour read (check
for growth)
7
Carbohydrate Results
Negative orange or red
Positive yellow, or yellow with gas
8
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).

9
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
10
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!

11
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.

12
Nitrate Reduction Procedure
13
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.

14
Motility Test Results
Positive Cloudiness in whole tube
Negative Cloudiness only around stab mark
15
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

16
Simmons Citrate Results
Positive color changes from green to blue
Negative - No color change
17
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

18
Urea Results
  • Positive - Cerise (a hot pink-cherry color)
  • Negative Yellow, Orange, or light pink

19
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.

20
Kligler's Iron Agar Results
Negative - no precipitate, any yellow, red, or
orange color without a precipiate
Positive dark precipitate
21
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

22
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.

23
Gelatinase Test Results
Positive Gelatin remains liquid after chilled
Negative Gelatin is firm after chilled
24
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

25
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.

26
Starch Hydrolysis Results
Positive
Negative
27
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

28
Casein Results
Positive zone of clearing around organism
Negative no zone of clearing
29
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

30
Lipid Hydrolysis Results
Positive zone of clearing around organism
Negative no zone of clearing around organism
31
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.

32
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

33
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

34
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

35
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

36
Example of page from Bergeys Manual
37
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

38
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

39
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

40
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

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