PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College C H A P T E R 10 Classification of Microorganisms
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.
Table 10.1 Some Characteristics of Archaea, Bacteria, and Eukarya
Table 10.2 Prokaryotic Cells and Eukaryotic Organelles Compared
The Three Domains Eukaryotes originated from infoldings of prokaryotic plasma membranes Endosymbiotic bacteria developed into organelles
Figure 10.2 A model of the origin of eukaryotes. Early cell Bacteria Chloroplast Archaea Mitochondrion DNA Eukarya
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
Scientific Nomenclature Common names vary with languages and geography Binomial nomenclature is used worldwide to consistently and accurately name organisms Genus Specific epithet (species)
Table 1.1 Making Scientific Names Familiar
Figure 10.5 The taxonomic hierarchy.
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
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
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
Glucose Gas Lysine Ornithine H 2 S Indole Adonitol Lactose Arabinose Sorbitol V P Dulcitol Phenylalanine Urease Citrate 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. 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. Comparing the resultant code number with a computerized listing shows that the organism in the tube is Citrobacter freundii. Code Number Microorganism Atypical Test Results 62352 Citrobacter freundii Citrate 62353 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
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.
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 Larger Polyacrylamide gel Proteins 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 Gel Sponge 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.
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). A mixture of cells is treated to label cells that have certain antigens with fluorescent-antibody markers. Fluorescently labeled cells Cell mixture leaves nozzle in droplets. Laser Laser beam Laser beam strikes each droplet. Detector of scattered light Fluorescence detector Electrically charged metal plates Collection tubes Electrode 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.
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
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. The DNA is separated into single strands. DNA probes are added to the DNA from the unknown bacteria. DNA probes hybridize with Salmonella DNA from sample. Then excess probe is washed off. Fluorescence indicates presence of Salmonella. Fluorescent probe Salmonella DNA 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
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.
Dichotomous Keys: Overview PLAY Animation: Dichotomous Keys: Overview
Dichotomous Keys: Sample with Flowchart PLAY Animation: Dichotomous Keys: Sample with Flowchart
Dichotomous Keys: Practice PLAY Animation: Dichotomous Keys: Practice