MGY277H1 Study Guide - Final Guide: Single-Access Key, Microorganism, Prokaryotic Small Ribosomal Subunit
8 Jun 2018
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Unit 7 – Microbial Identification and
Classification
Learning Objectives:
1. Describe how prokaryotes are identified, classified, and assigned names.
2. Describe how phenotypic characteristics—including microscopic morphology, culture characteristics,
metabolic capabilities, serology, and fatty acid analysis—can be used to identify prokaryotes.
3. Describe five distinct methods to distinguish different strains.
4. Describe how 16S rRNA sequences are used to classify and identify prokaryotes.
Determining phylogeny of prokaryotes
• Difficult to apply same classification criteria
• Do not undergo sexual reproduction
• Relied heavily on phenotypic characteristics for classification
Strategies to classify prokaryotes
• Goal: group by evolutionary relatedness
• Historically: based on phenotypic traits
o Size, shape, staining, metabolic capabilities
o Drawbacks:
▪ Phenotypic differences may be due to a few gene products or single mutation
▪ Phenotypically similar organisms may be distantly related
▪ Closely related organisms may appear dissimilar
• New molecular techniques are more accurate and less prone to human bias
o Evolutionary chronometers = DNA sequences
o Can use DNA sequences to construct phylogenetic tree!
Systems to classify prokaryotes
• Taxonomic based
o Species – group of closely related isolate or strains
o permits identification
• Informal groupings
o Can be genetically unrelated
o Examples: lactic acid bacteria, anoxygenic phototrophs, endospore-formers, sulfate reducers.
• Horizontal (lateral) gene transfer complicates classification.
o Gene content between two strains of E. coli can differ more than humans and fish.
What are STRAINS?
• Stains are levels of categorization underneath species.
• Typically identify specific isolates of a given species.
• Bergey’s Manual of Systemic Bacteriology
o Describes all known species, including those not cultivated yet.
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Strategies to identify prokaryotes
• Procedures: microscopy, culture characteristics, biochemical test, nucleic acid analysis
• Clinical setting – patient’s symptoms can be either misleading or limited in informational value.
o Rule out presence of disease-causing organism rather than conclusively identifying it.
o Patients’ fecal material can be tested for organisms that cause diarrhea or fever.
Phenotypic characteristics to identify cellular pathogens
1. Microscopic morphology
2. Culture characteristics
3. Metabolic capabilities
4. Serology
5. Fatty acid analysis
6. Mass spectrometry
Microscopic Morphology
• Initial step to quickly determine: Size, shape, staining characteristics.
o Can diagnose eukaryotic infections and start treatment.
• Gram stain and special stains (acid-fast, endospore) info → start appropriate therapy.
Culture Characteristics
• Colony morphologies:
o Streptococci = small
o Serratia marcescens = red @ 22°C
o Pseudomonas aeruginosa = green pigment + fruity smell
• Differential media:
o Steptococcus pyogenes (strep throat ) = B-hemolytic colonies on blood agar
o E. coli (UTI) = fermets lactose, pink colonies on MacConkey agar
Metabolic Capabilities
• Biochemical tests provides certainty of identification.
o Catalase test
▪ Catalase + = bacteria that can grow in the presence of oxygen.
▪ Except lactic acid bacteria.
o pH indicator tests
▪ Sugar fermentation, urease production.
o Enterotube II, API test strip
▪ Less labor intensive, more consistent, rapid identification.
• Relies on dichotomous key – a flow chart of tests with + or – results.
• Diagnosis can be done without culturing for some tests.
o Breathe test or urease to identify H. pylori.
Serology
• Antibodies isolated from animals to diagnose pathogen.
• Can identify markers of pathogens:
o LPS O-antigen, capsule, flagella, pili, proteins, polysaccharides
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find more resources at oneclass.com
Fatty Acid Analysis
• Prokaryote produce different types, and quantities of FA.
• FA composition is determined by gas chromagraphy.
o Procedure:
▪ Cells cultured under standard conditions
▪ Treated to convert FA to FA methyl ester (FAME)
▪ Separated and measured by gas chromagraphy
▪ Compare chromatogram to known profiles
Methods to characterize strain differences
1. Biochemical typing
2. Serological typing
3. Molecular typing (MLST, RFLP)
4. Phage typing
5. Antibiograms
Biochemical Typing
• Group of strains with characteristic pattern of growth are called a biovar or biotype.
o Different biovars can grow under different conditions.
Serological Typing
• Markers: proteins and carbohydrates.
o Group of strains based on characteristic antigens are called serovar or serotype.
o E. coli can be distinguished by type of:
▪ Flagella (H antigen)
▪ Capsule (K antigen)
▪ Lipopolysaccharide (O antigen)
Molecular Typing
• Restriction Fragment Length Polymorphisms (RFLPs)
o Different DNA fragment patterns indicate different strains.
• Multi-Locus Sequence Typing (MLST)
o Compare 8-10 housekeeping genes.
o Perform PCR and sequence.
o Align sequence to database of known species isolates.
▪ Not every species has a MLST database
o Similar sequences = related microbes.
Phage Typing
• Different bacterial strains have different susceptibility to bacteriophages.
o Patterns can be seen with different bacteriophage suspensions.
• Replaced by molecular methods.
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Determining phylogeny of prokaryotes: difficult to apply same classification criteria, do not undergo sexual reproduction, relied heavily on phenotypic characteristics for classification. Systems to classify prokaryotes: taxonomic based, species group of closely related isolate or strains, permits identification. Informal groupings: can be genetically unrelated, examples: lactic acid bacteria, anoxygenic phototrophs, endospore-formers, sulfate reducers, horizontal (lateral) gene transfer complicates classification, gene content between two strains of e. coli can differ more than humans and fish. What are strains: stains are levels of categorization underneath species, typically identify specific isolates of a given species, bergey"s manual of systemic bacteriology, describes all known species, including those not cultivated yet. Phenotypic characteristics to identify cellular pathogens: microscopic morphology, culture characteristics, metabolic capabilities, serology, fatty acid analysis, mass spectrometry. Initial step to quickly determine: size, shape, staining characteristics: can diagnose eukaryotic infections and start treatment, gram stain and special stains (acid-fast, endospore) info start appropriate therapy.
