Download Unknown Report: Outline Introduction o Goal: determine genus of an

Unknown Report: Outline
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Introduction
o Goal: determine genus of an unknown organism
o Methodology: use numerous specific microbiological tests to analyze various properties of the
organism
o Temporary name: Insert unknown number here.
Body
o Basic characteristics
 Shape
 Discerned using simple staining (provides information about shape, size, and
arrangement of organism)
o Slide is a heat-fixed (process by which bacterial samples are more permanently
fixed to a slide by slightly heating them) bacterial smear (process of “smearing”
bacteria on a slide in preparation for viewing)
 Kills cells and allows stains to penetrate better
 Makes cells stick to the slide better (by denaturing the proteins)
 Preserves the slide before staining it
o Basic dyes (dyes with alkaline pH levels) are used to color bacterial smears
 Three main cell morphologies (fancy word for “shapes”): rod, sphere, spiral
 Cells of organism may be arranged differently – clumped in groups, branched, linear, etc.
 Gram stain – differential staining process used to discern different organisms with cell walls
 Process
o Primary stain (first stain used in a staining process): crystal violet (violet-colored
basic dye)
o Rinse
o Decolorize with ethanol
o Rinse
o Counterstain with safranin (pink-colored basic dye)
o Rinse
 Results
o Gram-positive: bacteria that retain crystal violet; Gram-positive bacteria have a
thick peptidoglycan (layered sugar-protein structure) layer in their outer membrane
that retains basic dye due to electric charges; only Gram-positive bacteria can form
endospores (see “Structures” section)
o Gram-negative: bacteria that lose crystal violet and pick up safranin; Gramnegative bacteria have a lipopolysaccharide (LPS) (layered sugar-lipid structure)
layer in their outer membrane with large pores that allow basic dyes to “leak” into
and out of the cell; ethanol shrinks pores of LPS, enabling Gram-negative organisms
to retain safrinin
 Colony characteristics
 Colony: well-isolated group of bacterial cells on a solid medium
 [See pg. 96, figure 15.1 in lab notebook]
 Streak plate: method for producing colonies in a plate
 Growth characteristics
 Agar deep
o Growth near point of inoculation near the surface of agar  bacterium is an obligate
aerobe (see “Environment” section)
o Growth all over stab line  bacterium is a facultative anaerobe (see “Environment”
section)
o Cracks in growth  bacterium produces gas
 Broth
o Color of growth
o
o
o Change of color in broth  bacterium utilized something in broth to create products
Structures
 Structures for motility – flagella
 Wet mount
o Organism is suspended in water and viewed under a microscope
o Gives some bacteria their figure back, by adding water to the organism
o Can be used to analyze movement, cell mechanics (e.g. cytoplasmic streaming), etc.
o Ways to move
 Flagella
 Brownian movement: movement caused by vibrations in an environment that
cause organisms to move; characterized by uncoordinated, erratic movement in
water
 Soft deeps
o Agar deep with less agar (hence, it is called “soft”), making flagellar movement
possible
o Created using stab inoculation
o Growth only on stab line or near point of inoculation  nonmotile; growth
throughout tube  motile
 SIM deeps: growth only on stab line or near point of inoculation  nonmotile; growth
throughout tube  motile
 Endospores - NSM media
 Endospore: small, water-tight, heat-resistant structure found in Gram-positive bacteria
that contains genetic information from its parent cell that has the ability to grow in good
living conditions
 Nutrient sporulation medium (NSM): medium used to increase rate of endospore
growth in endospore formers
 Can be seen with a microscope using endospore staining
o Create bacterial smear using growth from NSM, heat and soak in malachite green
(green dye), dry, view under microscope
o Endospores found in one of three positions
 Central: in the center of an organism
 Subterminal: between center and one end of an organism
 Terminal: at one end of an organism
Metabolism
 Fermentation: energy-producing process in which organic molecules act as electron donors
and acceptors
 Different from oxidation because oxidation utilizes inorganic terminal electron acceptors
(e.g. O2)
 Acids and gases can be produced during fermentation
o Acids lower pH of environment and can be detected using phenol red (pH indicator
that turns red at pH > 8.4 and yellow at pH < 6.4)
o Gases can be detected using Durham tube (smaller test tube inverted within a
larger test tube, which will show a bubble in the presence of gas production)
 Substances tested for fermentation
o Glucose
o Lactose – presence of β-galactosidase (enzyme responsible for hydrolysis of
lactose into glucose and galactose) indicates ability to utilize lactose as a carbon
source
o Sucrose
o Mannitol salt: a selective and differential medium that contains mannitol (sixcarbon sugar alcohol) and 7.5% NaCl
 NaCl concentration selects for staphylococci
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Tests
 Methyl red test: test that utilizes methyl red (an indicator) to indicate the
fermentative products created by bacteria; turns red at a pH of 4 and yellow at a pH of 6
o Mixed acid fermenters: bacteria that produce acids in fermentation; positive for
methyl red test
o Butanediol fermenters: bacteria that produce butanediol (a four-carbon
molecule with two hydroxyl groups attached to it) in fermentation; negative for
methyl red test
 Voges-Proskauer test: test that utilizes Barritts’ reagent (an indicator) to indicate
the presence of acetoin (a precursor molecule to the production of butanediol)
 In the presence of acetoin, Barritts’ reagent produces a cherry red color, a positive
Voges-Proskauer test
 The absence of acetoin produces no color change, a negative Voges-Proskauer test
Catabolism and catabolic enzymes
 Starch: polysaccharide made up of glucose monomers
 Ability to hydrolyze starch = ability to use starch as a source of glucose
 α-amylase: enzyme responsible for hydrolysis of starch into dextrins, maltose, and
glucose
 Starch hydrolysis can be detected by using iodine and a starch agar – brown area around
bacterial growth means that starch was not hydrolyzed (negative test for starch
hydrolysis), zone of hydrolysis (clear area around bacterial growth) means that starch
was hydrolyzed (positive test for starch hydrolysis)
 Lipid: organic compound that is only slightly soluble or insoluble in water
 Can be hydrolyzed using lipases (enzymes responsible for hydrolysis of lipids) to a
variety of products, mainly fatty acids (non-polar, uncharged monomeric units of lipids),
to be used for a variety of purposes
 Phospholipids (lipids containing phosphate) produce a pearl-like precipitate upon
hydrolysis
 Lipid hydrolysis can be detected by using egg yolk agar, which contains phospholipids
 Casein: protein found in milk that gives milk its white color
 Can be broken down by proteases (enzymes responsible for breaking down proteins),
which produces a zone of proteolysis (clear area around a bacterium after the bacteria
has secreted active proteases) in a medium
 Presence of casein proteases can be determined by using a skim milk agar
 Hydrogen sulfide (H2S)
 H2S gas production
o Result of breaking down proteins rich in sulfur-containing amino acids (e.g. cysteine)
o Reduction of inorganic sulfur-containing compounds
 Can be detected using SIM medium (medium that checks for the presence of sulfide,
indole [by-product of the breakdown of tryptophan], and motility; semi-soft agar
containing peptones, thiosulfate, and ferrous ammonium sulfate)
o Uses ferrous ammonium sulfate as the H2S indicator
o Presence of black ferrous sulfide precipitate in medium after inoculation with
organism and incubation  positive test for H2S; absence of black ferrous sulfide
precipitate in medium after inoculation with organism and incubation  negative test
for H2S
 Tryptophan
 Tryptophanase: enzyme responsible for breaking down tryptophan; breaks tryptophan
down into indole, pyruvic acid, and ammonia
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o
Mannitol and phenol red differentiate between staphylococci; Staphylococcus
aureus ferments mannitol and turns the medium yellow (due to fermentative
acids) while Staphylococcus epidermidis does not ferment mannitol or change
the color of the medium
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o
Can be detected using SIM medium and checking for indole production; Kovacs’
reagent is used to detect the presence of indole in a medium
o Red color (rosindole dye) after addition of Kovacs’ reagent  positive test for
indole
o No change in color after addition of Kovacs’ reagent  negative test for indole
 Citrate: six-carbon, tricarboxylic organic compound (you don’t need to know that)
 Organisms with citrate permease (enzyme responsible for facilitating the transport of
citrate into the cell) can use citrate as a carbon source
 Citrate permease-producing organisms can be detected using Simmons agar (agar
containing sodium citrate and bromothymol blue [a pH indicator]; citrate is the only
carbon source in this agar)
o Use of citrate permease will eventually create CO2, which combines with sodium from
the sodium citrate and water to form sodium carbonate, a basic compound
o An increase in pH produces a blue color, a positive test for citrate catabolism; no
growth  no presence of citrate permease, a negative test for citrate catabolism
 Urea: nitrogen-containing compound; condensed structure formula: H2N-CO-NH2
 Can be catabolized using urease (enzyme responsible for catalyzing the breakdown of
urea) into CO2, H2O, and ammonia (a basic molecule; NH3)
 Organisms that can breakdown urea can be detected in a medium containing urea, the
organism, and phenol red (an indicator); a change in color from orange-red to cerise
(deep pink or purple) indicates a positive test for urease, and an absence of change in
color indicates a negative test for urease
 Nitrate: nitrogen-containing compound NO3 Nitrate can be used as a terminal electron acceptor in chemolithoautotrophs (via
reduction)
o Can be reduced by organisms that can produce nitrate reductase (enzyme
responsible for reducing nitrate)
o Reduced to nitrite (NO2-) in the reaction
NO3- + 2 H+ + 2 e - − [nitrate reductase]  NO2- + H2O
and can be further reduced by other enzymes
NO2- − [other enzymes]  NH3+ − [other enzymes]  ½ N2
 Testing – nitrate reduction test
o Inoculate broth containing 0.5% potassium nitrate (KNO3) with organism and
incubate
o Production of gas can be checked using Durham tube
o Reduction of nitrate to nitrite can be checked by adding sulfanilic acid and N,Ndimethyl-1-naphthylamine to the medium
 Results:
 Presence of nitrite will cause the medium to turn pink or red (positive test for
reduction of nitrate to nitrite)
 Lack of color change is a negative test for reduction of nitrate to only nitrite;
lack of color change may be due to reduction of nitrite to ammonia
Pathogenic activity
 Gelatin: a soluble mixture of polypeptides
 Gelatinases: enzymes responsible for breaking down gelatin
 Ability to utilize gelatin correlates to pathogenicity – collagen can be broken down by
gelatinase  pathogens with gelatinase can break down host tissue
 Coagulase: enzyme responsible for causing coagulation in a medium
 Pathogens can use it to form a protective barrier, preventing the host’s defensive
systems from attacking the pathogen
 Pathogens do not need to be able to produce coagulase for them to be pathogenic
 Presence of coagulase can be detected by analyzing a mixture of plasma and an
organism; a cloudy and solidified mixture after four hours indicates that coagulation has
o
taken place (positive test for coagulase), and no coagulation after four hours indicates
that no coagulation has taken place (duh) (negative test for coagulase)
 Hemolysis: degradation of hemoglobin (molecular substance in a red blood cell
responsible for carrying oxygen)
 Pathogens that produce hemolysin (protein that degrades red blood cells) can break
down red blood cells in the host
 Hemolytic activity can be detected by inoculating blood agar with an organism; after
incubation, a partial or complete clearing may be visible (positive test for hemolysin) or
nonexistent (negative test for hemolysin)
 Two types of hemolysis
o α-hemolysis: partial degradation of hemoglobin; appearance of a green color and
indistinct margins in blood agar inoculated with an organism
o β-hemolysis: complete degradation of hemoglobin; appearance of a clear zone in
blood agar inoculated with an organism
 Antibiotic resistance
 Can be detected using the Kirby-Bauer method
o Create bacterial lawn
o Inoculate lawn with antibiotic disk and incubate
o Measure diameter of zone of inhibition (area of clearing around antibiotic disk, due
to interactions between antibiotic and organism) and comparing it to an
antibiogram (pattern of antibiotic susceptibility in which certain diameters of zones
of inhibition are known to have particular effects on organisms)
 Possible results
o Resistant strains: bacterial strains that are completely unaffected by an antibiotic;
no clearing whatsoever around antibiotic disk
o Intermediate strains: bacterial strains that are partially affected by an antibiotic;
some clearing around antibiotic disk
o Susceptible strains: bacterial strains that are greatly affected by an antibiotic;
significant clearing around antibiotic disk
Environment
 Oxygen requirement
 Oxygen
o Generally used as an electron acceptor, to produce energy
 Oxidase: enzyme that helps a cell to use O 2 as an electron acceptor in the
electron transport chain
 Testing
 KEY oxidase test strip: strip containing tetramethyl-p-phenylenediamine
dihydrochloride that, on contact with oxidized cytochrome c (molecule
involved in the energy production of the electron transport chain), reduces
cytochrome c
o Positive test for oxidase is a blue or purple color on the test strip
o Negative test for oxidase is a light pink color or no color on the test strip
o Toxicity
 Some forms of oxygen are toxic to cells because they can destroy cellular
components
 Hydrogen peroxide: H2O2
 Superoxide: O2o Enzymes needed to convert toxic forms to non-toxic forms
 Superoxide dismutase (SOD): enzyme that converts superoxide to oxygen
and hydrogen peroxide
2O2- + 2H+ – [SOD]  O2 + H2O2
 Catalase: enzyme that converts hydrogen peroxide to water and oxygen
2H2O2 – [catalase]  2H2O + O2
 Strict anaerobes lack these enzymes  they cannot tolerate oxygen
Testing for the presence of oxygen-related enzymes done by adding some drops of
H2O2 to a medium containing the organism in question
 Classification of organisms based on oxygen requirement
o Obligate aerobes: organisms that require oxygen; in media, growth is strictly near
open surface
o Microaerophiles: organisms that require oxygen at less-than-atmospheric levels; in
media, growth is slightly below open surface
o Facultative anaerobes: organisms that do not require oxygen but grow better in
its presence; in media, growth is both near open surface and throughout the medium
o Aerotolerant anaerobes: organisms that do not require oxygen but are not
harmed by it; in media, growth is evenly distributed throughout medium
o Obligate anaerobes: organisms that cannot tolerate oxygen; in media, growth is
generally far away from the open surface
 Testing for oxygen requirements
o Eugon deeps
 Heat agar, inoculate agar with organism, cool and incubate
 During cooling and incubation, organism will orient itself in agar to maximize
growth (e.g. if the organism is microaerophilic, it might inflate or deflate gas
vesicles to position itself in the appropriate place in the medium)
 Cracking in agar indicates production of CO2 by organism
o Thioglycollate broth: broth that contains sulfhydryl groups, which are oxidized by
O2 to create an anaerobic environment; growth in tube  organism is a strict
anaerobe, aerotolerant anaerobe, or a facultative anaerobe
o GasPak Anaerobic System: jar that utilizes H2 and palladium to create an
anaerobic environment
 Agar plates of organism are placed inside jar
 Process
 Add water to “gas generator envelope” to produce H 2 and CO2
 H2 combines with O2 to create an anaerobic environment; palladium pellets
act as a catalyst for the reaction 2 H2 + O2  2 H2O
 CO2 helps organism to grow quicker
 Organisms that grow in jar are either strict anaerobes, aerotolerant anaerobes,
or facultative anaerobes
 Can be compared to plates grown in an aerobic environment to check for
facultative anaerobes (which will grow better in aerobic than anaerobic
environment)
Temperature
 Temperature can affect the enzymes of an organism, so it is important that organisms
maintain a temperature at which their enzymes can function without denaturing
 Classification of organisms based on temperature requirements
o Psychrophiles: organisms that grow between 0º C and 15º C
o Mesophiles: organisms that grow between 20º C and 45º C; most bacteria are
mesophiles
o Thermophiles: organisms that grow at temperatures above 55º C
 Thermoduric: adjective used to describe organisms that can endure long periods of
boiling temperature, even though they do not grow; many spore formers are thermoduric
 Organisms can be tested for temperature growth by inoculating plates and incubating
them at different temperatures
pH
 pH can affect the balance of electrical charges, so it is important that organisms maintain
a pH at which their electrical charges will stay balanced
 Classification of organisms based on optimal pH growth
o
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o
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Acidophiles: organisms that grow at a pH between 0.0 and 5.5; yeasts, molds, and
algae seem to favor acidic conditions
o Neutrophiles: organisms that grow at a pH between 5.5 and 8.0; most bacteria are
neutrophiles
o Alkalophiles: organisms that grow at a pH between 8.5 and 11.5
 Organisms can be tested for optimal pH growth by inoculating solutions of particular pHs
and measuring their turbidity (using a spectrophotometer)
 Salt concentration
 Salt concentration can affect the balance of water in a cell
o Isotonic: amount of solute and solvent is equal inside and outside the cell
o Hypertonic: low solute and high solvent concentrations on the outside of a cell
relative to the inside of the cell; solvent rushes into the cell and may cause it to
undergo lysis if its cell wall is not strong enough
o Hypotonic: high solute and low solvent concentrations on the outside of a cell
relative to the inside of the cell; solvent leaves the cell and may cause it to undergo
plasmolysis (plasma membrane “shrinks away” from cell wall)
 Organisms can be tested for salt concentration growth by inoculating plates containing
different amounts of sodium chloride (NaCl) and checking for growth
Conclusion
o Tentative genus identification: Insert tentative genus here.
o Reasoning for genus identification
 Factors that promote correct genus identification
 Factors that disprove incorrect genus identification
o Possible errors in genus identification
 Errors in testing
 Differential identification (i.e. two genii, A and B, are very similar to your unknown, and you
identify your unknown as part of genus A when your unknown actually belongs to genus B)
o Retesting
 Necessity
 Tests that would provide more conclusive results