classification of microorganisms - · pdf filewhen the gel is exposed to an electric current....

PowerPoint® Lecture Presentations prepared byBradley W. Christian, McLennan Community College

C H A P T E R

© 2016 Pearson Education, Inc.

Classification of Microorganisms

10


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


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


Figure 10.1 Three-Domain System.


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


Figure 10.4c Fossilized prokaryotes.


Scientific Nomenclature

• Common names vary with languages and geography

• Binomial nomenclature is used worldwide to consistently and accurately name organisms• Genus • Specific epithet (species)


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


Figure 10.5 The taxonomic hierarchy.


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?


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


Figure 10.6 Phylogenetic relationships of prokaryotes.


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


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


Use the terms species, culture, clone, and strainin one sentence to describe growing methicillin-resistant Staphylococcus aureus (MRSA).


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


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


Filled out by one person Filled out by different person

Figure 10.7 A clinical microbiology lab report form.


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


Figure 10.8 The use of metabolic characteristics to identify selected genera of enteric bacteria.


Applications of Microbiology 10.1


Biochemical Tests

• Rapid identification methods perform several biochemical tests simultaneously• Results of each test are assigned a number


One tube containing media for 15 biochemical testsis inoculated with an unknown enteric bacterium.

After incubation, the tube is observed for results.

The value for each positive test is circled, andthe numbers from each group of tests areadded to give the code number.

Comparing the resultant code number with acomputerized listing shows that the organism inthe tube is Citrobacter freundii.

Glu

cose

Gas

Lysi

ne

Orn

ithin

e

H2S

Indo

le

Ado

nito

l

Lact

ose

Ara

bino

se

Sorb

itol

V–P

Dul

cito

lPh

enyl

alan

ine

Ure

ase

Citr

ate

Code Number Microorganism Atypical Test Results

62352

62353

Citrobacter freundii

Citrobacter freundii

Citrate

None

Figure 10.9 One type of rapid identification method for bacteria: EnteroPluri test from BD Diagnostics.


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


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


Figure 10.10 A slide agglutination test.


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


Figure 10.11 An ELISA test.


Figure 18.14a The ELISA method.


If Lyme disease is suspected in a patient: Electrophoresis is used to separate Borreliaburgdorferi proteins. Proteins move atdifferent rates based on their charge and sizewhen the gel is exposed to an electric current.

The bands are transferred to a nitrocellulosefilter by blotting. Each band consists of manymolecules of a particular protein (antigen). Thebands are not visible at this point.

The proteins (antigens) are positioned on the filterexactly as they were on the gel. The filter is thenwashed with patient's serum followed by antihumanantibodies tagged with an enzyme. The patientantibodies that combine with their specific antigenare visible (shown here in red) when the enzyme'ssubstrate is added.

The test is read. If the tagged antibodies stick tothe filter, evidence of the presence of themicroorganism in question—in this case, B.burgdorferi—has been found in the patient'sserum.

Lysedbacteria

Larger

PolyacrylamidegelProteins

Smaller

Paper towels

Salt solution

Gel

Sponge

Nitrocellulosefilter

Figure 10.12 The Western blot.


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


Figure 10.13 Phage typing of a strain of Salmonella enterica.


Fatty Acid Profiles

• FAME: Fatty acid methyl esters provide profiles that are constant for a particular species


Flow Cytometry

• Uses differences in electrical conductivity between species or fluorescence


Figure 18.12 The fluorescence-activated cell sorter (FACS).

Fluorescentlylabeled cells

Laser beamLaser

Fluorescencedetector

Electricallychargedmetal plates

Collectiontubes

The separated cellsfall into differentcollection tubes.

As cells drop betweenelectrically chargedplates, the cells witha positive chargemove closer to thenegative plate.

Electrode givespositive charge toidentified cells.

Detector ofscattered light

Laser beam strikeseach droplet.

Cell mixture leavesnozzle in droplets.

A mixture of cells istreated to label cellsthat have certainantigens withfluorescent-antibodymarkers.

Electrode Fluorescence detectoridentifies fluorescentcells by fluorescentlight emitted by cell.


DNA Base Composition

• DNA base composition• Guanine + cytosine %• Two organisms that are closely related have similar

amounts of various bases


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


Figure 10.14 DNA fingerprints.


Nucleic Acid Amplification Tests (NAATs)

• Use of PCR to amplify DNA of an unknown microorganism that cannot be cultured


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


Figure 10.15 DNA-DNA hybridization.


Nucleic Acid Hybridization

• Southern blotting uses nucleic acid hybridization to identify unknown microorganisms using DNA probes


Plasmid

SalmonellaDNAfragment

Unknown bacteriaare collectedon a filter.

A Salmonella DNAfragment is cloned inE. coli.

The cells are lysed,and the DNAis released.

Cloned DNA fragments are markedwith fluorescent dye and separatedinto single strands, formingDNA probes. The DNA is separated into

single strands.

DNA probes are addedto the DNA from theunknown bacteria.

Fluorescent probe

Salmonella DNA

DNA fromother bacteria

DNA probes hybridize withSalmonella DNA from sample.Then excess probe is washedoff. Fluorescence indicatespresence of Salmonella.

Figure 10.16 A DNA probe used to identify 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


Figure 10.17a-b DNA chip.


Figure 10.17c-d DNA chip.


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


Figure 10.18 FISH, or fluorescent in situ hybridization.


Putting Classification Methods Together

• Dichotomous keys• Identification keys based on successive questions

• Cladograms• Maps that show evolutionary relationships among

organisms; based on rRNA sequences


Figure 10.19 Building a cladogram.

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