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College
CHAPTER
10
Classification of
Microorganisms
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The Study of Phylogenetic Relationships
Learning Objectives
10-1 Define taxonomy, taxon, and phylogeny.
10-2 Discuss the limitations of a two-kingdom
classification system.
10-3 Identify the contributions of Linnaeus,
Whittaker, and Woese.
10-4 Discuss the advantages of the three-domain
system.
10-5 List the characteristics of the Bacteria,
Archaea, and Eukarya domains.
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The Study of Phylogenetic Relationships
• Taxonomy is the science of classifying organisms
• Shows degree of similarity among organisms
• Systematics, or phylogeny, is the study of the
evolutionary history of organisms
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The Study of Phylogenetic Relationships
• 1735: Linnaeus—kingdoms Plantae and Animalia
• 1800s: Bacteria and fungi put in kingdom Plantae
(Nägeli); Kingdom Protista proposed for bacteria,
protozoa, algae, and fungi (Haeckel)
• 1937: Prokaryote introduced to distinguish cells
without a nucleus
• 1968: Murray—Kingdom Prokaryotae
• 1969: Whittaker—five-kingdom system
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Check Your Understanding
 Of what value is taxonomy and systematics?
10-1
 Why shouldn't bacteria be placed in the plant
kingdom?
10-2, 10-3
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The Three Domains
• Developed by Woese in 1978; based on
sequences of nucleotides in rRNA
• Eukarya
• Animals, plants, fungi
• Bacteria
• Archaea
• Methanogens
• Extreme halophiles
• Hyperthermophiles
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Figure 10.1 Three-Domain System.
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Table 10.1 Some Characteristics of Archaea, Bacteria, and Eukarya
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Table 10.2 Prokaryotic Cells and Eukaryotic Organelles Compared
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The Three Domains
• Eukaryotes originated from infoldings of
prokaryotic plasma membranes
• Endosymbiotic bacteria developed into organelles
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Figure 10.2 A model of the origin of eukaryotes.
Early cell
Bacteria
Chloroplast
Archaea
Mitochondrion
DNA
Eukarya
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Figure 10.3 Cyanophora paradoxa.
Bacterium
Eukaryotic host cell
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A Phylogenetic Tree
• Grouping organisms according to common
properties
• Fossils
• Genomes
• Groups of organisms evolved from a common
ancestor
• Each species retains some characteristics of
its ancestor
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Figure 10.4a Fossilized prokaryotes.
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Figure 10.4b Fossilized prokaryotes.
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Figure 10.4c Fossilized prokaryotes.
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Check Your Understanding
 What evidence supports classifying organisms into
three domains?
10-4
 Compare archaea and bacteria; bacteria and
eukarya; and archaea and eukarya.
10-5
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Classification of Organisms
Learning Objectives
10-6 Explain why scientific names are used.
10-7 List the major taxa.
10-8 Differentiate culture, clone, and strain.
10-9 List the major characteristics used to
differentiate the three kingdoms of
multicellular Eukarya.
10-10 Define protist.
10-11 Differentiate eukaryotic, prokaryotic, and viral
species.
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Scientific Nomenclature
• Common names vary with languages and
geography
• Binomial nomenclature is used worldwide to
consistently and accurately name organisms
• Genus
• Specific epithet (species)
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Table 1.1 Making Scientific Names Familiar
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The Taxonomic Hierarchy
• A series of subdivisions developed by Linnaeus to
classify plants and animals
• Eukaryotic species: a group of closely related
organisms that breed among themselves
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Figure 10.5 The taxonomic hierarchy.
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Check Your Understanding
 Using Escherichia coli and Entamoeba coli as
examples, explain why the genus name must
always be written out on first use. Why is binomial
nomenclature preferable to common names?
10-6
 Find the gram-positive bacteria Staphylococcus in
Appendix F. To which bacteria is this genus more
closely related: Bacillus or Streptococcus?
10-7
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Classification of Prokaryotes
• Prokaryotic species: a population of cells with
similar characteristics
• Culture: bacteria grown in laboratory media
• Clone: population of cells derived from a single parent
cell
• Strain: genetically different cells within a clone
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Figure 10.6 Phylogenetic relationships of prokaryotes.
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Classification of Eukaryotes
• Protista: a catchall kingdom for a variety of
organisms; autotrophic and heterotrophic
• Grouped into clades based on rRNA
• Fungi: chemoheterotrophic; unicellular or
multicellular; cell walls of chitin; develop from
spores or hyphal fragments
• Plantae: multicellular; cellulose cell walls; undergo
photosynthesis
• Animalia: multicellular; no cell walls;
chemoheterotrophic
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Classification of Viruses
• Not a part of any domain; not composed of cells;
require a host cell
• Viral species: population of viruses with similar
characteristics that occupies a particular
ecological niche
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Check Your Understanding
 Use the terms species, culture, clone, and strain
in one sentence to describe growing methicillin-resistant
Staphylococcus aureus (MRSA).
10-8
 You discover a new multicellular, nucleated, heterotrophic,
organism with cell walls. To what kingdom does it belong?
10-9
 Write your own definition of protist.
10-10
 Why doesn't the definition of a viral species work for a
bacteria?
10-11
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Methods of Classifying and Identifying
Microorganisms
Learning Objectives
10-12 Compare and contrast classification and
identification.
10-13 Explain the purpose of Bergey's Manual.
10-14 Describe how staining and biochemical tests
are used to identify bacteria.
10-15 Differentiate Western blotting from Southern
blotting.
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Methods of Classifying and Identifying
Microorganisms
Learning Objectives
10-16 Explain how serological tests and phage typing
can be used to identify an unknown bacterium.
10-17 Describe how a newly discovered microbe can
be classified by DNA base composition, DNA
fingerprinting, and PCR.
10-18 Describe how microorganisms can be identified
by nucleic acid hybridization, Southern blotting,
DNA chips, ribotyping, and FISH.
10-19 Differentiate a dichotomous key from a
cladogram.
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Methods of Classifying and Identifying
Microorganisms
• Classification: placing organisms in groups of
related species
• Lists of characteristics of known organisms
• Identification: matching characteristics of an
"unknown" organism to lists of known organisms
• Clinical lab identification
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Methods of Classifying and Identifying
Microorganisms
• Bergey's Manual of Determinative Bacteriology
provides identification schemes for identifying
bacteria and archaea
• Approved Lists of Bacterial Names lists species of
known classification
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Methods of Classifying and Identifying
Microorganisms
• In clinical microbiology, lab requisition forms are
used to note types of specimens collected and
tests to be conducted
• Transport media is used to collect and transport
pathogens to a laboratory
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Figure 10.7 A clinical microbiology lab report form.
Filled out by one person
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Filled out by different person
Methods of Classifying and Identifying
Microorganisms
• Morphological characteristics: useful for identifying
eukaryotes; tell little about phylogenetic
relationships
• Differential staining: Gram staining, acid-fast
staining; not useful for bacteria without cell walls
• Biochemical tests: determine presence of bacterial
enzymes
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Figure 10.8 The use of metabolic characteristics to identify selected genera of enteric bacteria.
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Applications of Microbiology 10.1
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Biochemical Tests
• Rapid identification methods perform several
biochemical tests simultaneously
• Results of each test are assigned a number
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Figure 10.9 One type of rapid identification method for bacteria: EnteroPluri test from BD Diagnostics.
One tube containing media for 15 biochemical tests
is inoculated with an unknown enteric bacterium.
Citrate
Urease
Dulcitol
Phenylalanine
V–P
Sorbitol
Arabinose
Lactose
Adonitol
Indole
H2S
Ornithine
Lysine
Gas
Glucose
After incubation, the tube is observed for results.
The value for each positive test is circled, and
the numbers from each group of tests are
added to give the code number.
4
Comparing the resultant code number with a
computerized listing shows that the organism in
the tube is Citrobacter freundii.
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Code Number
Microorganism
Atypical Test Results
62342
Citrobacter freundii
Citrate
62343
Citrobacter freundii
None
Serology
• The science that studies serum and immune
responses in serum
• Microorganisms are antigenic—they stimulate the
body to form antibodies in the serum
• In an antiserum, a solution of antibodies is tested
against an unknown bacterium
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Serology
• In the slide agglutination test, bacteria
agglutinate when mixed with antibodies produced
in response to the bacteria
• Serological testing can differentiate between
species and strains within species
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Figure 10.10 A slide agglutination test.
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Serology
• Enzyme-linked immunosorbent assay (ELISA)
• Known antibodies and an unknown type of bacterium
are added to a well; a reaction identifies the bacteria
• Western blotting
• Identifies antibodies in a patient's serum; confirms HIV
infection
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Figure 10.11 An ELISA test.
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Figure 18.14a The ELISA method.
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Figure 10.12 The Western blot.
If Lyme disease is suspected in a patient:
Electrophoresis is used to separate Borrelia
burgdorferi proteins. Proteins move at
different rates based on their charge and size
when the gel is exposed to an electric current.
Lysed
bacteria
Polyacrylamide
gel
Proteins
Larger
Smaller
The bands are transferred to a nitrocellulose
filter by blotting. Each band consists of many
molecules of a particular protein (antigen). The
bands are not visible at this point.
Paper towels
Salt
solution
Sponge
Gel
Nitrocellulose
filter
The proteins (antigens) are positioned on the filter
exactly as they were on the gel. The filter is then
washed with patient's serum followed by antihuman
antibodies tagged with an enzyme. The patient
antibodies that combine with their specific antigen
are visible (shown here in red) when the enzyme's
substrate is added.
The test is read. If the tagged antibodies stick to
the filter, evidence of the presence of the
microorganism in question—in this case, B.
burgdorferi—has been found in the patient's
serum.
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Phage Typing
• Test for determining which phages a bacterium is
susceptible to
• On a plate, clearings called plaques appear where
phages infect and lyse bacterial cells
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Figure 10.13 Phage typing of a strain of Salmonella enterica.
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Fatty Acid Profiles
• FAME: Fatty acid methyl esters provide profiles
that are constant for a particular species
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Flow Cytometry
• Uses differences in electrical conductivity between
species or fluorescence
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Figure 18.12 The fluorescence-activated cell sorter (FACS).
A mixture of cells is
treated to label cells
that have certain
antigens with
fluorescent-antibody
markers.
Cell mixture leaves
nozzle in droplets.
Fluorescently
labeled cells
Laser beam strikes
each droplet.
Laser beam
Detector of
scattered light
Laser
Electrode
Fluorescence
detector
Electrically
charged
metal plates
Fluorescence detector
identifies fluorescent
cells by fluorescent
light emitted by cell.
Electrode gives
positive charge to
identified cells.
As cells drop between
electrically charged
plates, the cells with
a positive charge
move closer to the
negative plate.
The separated cells
fall into different
collection tubes.
Collection
tubes
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DNA Base Composition
• DNA base composition
• Guanine + cytosine %
• Two organisms that are closely related have similar
amounts of various bases
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DNA Fingerprinting
• DNA fingerprint
• Electrophoresis of restriction enzyme digests of an
organism's DNA
• Comparing fragments from different organisms provides
information on genetic similarities and differences
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Figure 10.14 DNA fingerprints.
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Nucleic Acid Amplification Tests (NAATs)
• Use of PCR to amplify DNA of an unknown
microorganism that cannot be cultured
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Nucleic Acid Hybridization
• Nucleic acid hybridization measures the ability
of DNA strands from one organism to hybridize
with DNA strands of another organism
• Greater degree of hybridization, greater degree of
relatedness
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Figure 10.15 DNA-DNA hybridization.
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Nucleic Acid Hybridization
• Southern blotting uses nucleic acid hybridization
to identify unknown microorganisms using DNA
probes
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Figure 10.16 A DNA probe used to identify bacteria.
Plasmid
Salmonella
DNA
fragment
A Salmonella DNA
fragment is cloned in
E. coli.
Unknown bacteria
are collected
on a filter.
The cells are lysed,
and the DNA
is released.
Cloned DNA fragments are marked
with fluorescent dye and separated
into single strands, forming
DNA probes.
DNA probes are added
to the DNA from the
unknown bacteria.
The DNA is separated into
single strands.
Fluorescent probe
Salmonella DNA
DNA probes hybridize with
Salmonella DNA from sample.
Then excess probe is washed
off. Fluorescence indicates
presence of Salmonella.
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DNA from
other bacteria
DNA Chips
• A DNA chip (also known as a microarray)
contains DNA probes and detects pathogens by
hybridization between the probe and DNA in the
sample
• Detected by fluorescence
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Figure 10.17a-b DNA chip.
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Figure 10.17c-d DNA chip.
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DNA Chips
• Ribotyping
• rRNA sequencing
• Fluorescent in situ hybridization (FISH)
• Fluorescent DNA or RNA probes stain the
microorganisms being targeted
• Determines the identity, abundance, and relative activity
of microorganisms in an environment
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Figure 10.18 FISH, or fluorescent in situ hybridization.
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Putting Classification Methods Together
• Dichotomous keys
• Identification keys based on successive questions
• Cladograms
• Maps that show evolutionary relationships among
organisms; based on rRNA sequences
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Figure 10.19 Building a cladogram.
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Dichotomous Keys: Overview
PLAY
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Animation: Dichotomous Keys: Overview
Dichotomous Keys: Sample with Flowchart
PLAY
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Animation: Dichotomous Keys: Sample
with Flowchart
Dichotomous Keys: Practice
PLAY
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Animation: Dichotomous Keys: Practice
Check Your Understanding
 What is in Bergey's Manual?
10-13
 Design a rapid test for a Staphylococcus aureus.
(Hint: See Figure 6.10, page 162.)
10-14
 What is tested in Western blotting and Southern
blotting?
10-15
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Check Your Understanding
 What is identified by phage typing?
10-16
 Why does PCR identify a microbe?
10-17
 Which techniques involve nucleic acid
hybridization?
10-18
 Is a cladogram used for identification or
classification?
10-12, 10-19
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