Chapter 10:

Identification and
Classification of
Prokaryotic
Microorganisms
Principles of Taxonomy
 Taxonomy is the science that studies organisms to
order and arrange them
 Taxonomy can be viewed in three areas
• Identification
• Process of characterizing in order to group them
• Classification
• Arranging organisms into similar or related groups
• Nomenclature
• System of assigning names
Principles of Taxonomy
 Phylogeny considers evolutionary relatedness
 Species is basic unit: in prokaryotes, a group of
closely related isolates or strains
• May be difficult to determine separate species or strains
within a species
 Informal groupings also used based on phenotype,
physiology
• Lactic acid bacteria
• Endospore-formers
• Sulfate reducers
• Anoxygenic
phototrophs
Principles of Taxonomy
 Three domain system has replaced five-kingdom
system proposed by Robert Whittaker (1969)
• Animalia
• Plantae
• Fungi
• Protista
• Monera

Carl Woese and others based on comparisons

of small subunit ribosomal RNA
Principles of Taxonomy
 Another view of three domain system
• Based on ribosomal protein sequence data
• Includes bacteria not
grown in culture
Principles of Taxonomy
 Bergey’s Manual of Systematic Bacteriology
• Describes all known prokaryotic species
• Newest edition in five volumes
• Classifies by genetic relatedness (phylogeny)
• Provides information on ecology, methods of
enrichment, culture, and isolation, and methods for
maintenance
 International Code of Nomenclature of Bacteria
• Provides rules for naming bacteria and archaea
Identification Methods
 Strategies used to identify microorganisms
• Microscopic examination
• Culture characteristics
• Biochemical tests
• Nucleic acid analysis
• Patient symptoms (for pathogens)
 Identification may be more important than
determining evolutionary relationships
Identification Methods
 Methods used to identify microorganisms
Identification Methods Based on Phenotype
 Microscopic morphology important initial step
• Size, shape, staining characteristics
• Sometimes enough to diagnose eukaryotic infections
• Gram stain: Gram positive or Gram negative
• Results may be enough to start appropriate therapy
• Special stains may help
• E.g., acid-fast stain on Mycobacterium tuberculosis
Identification Methods Based on Phenotype
 Culture characteristics can give clues
• Serratia marcescens colonies often red at 22℃
• Pseudomonas aeruginosa often produces green pigment
• Cultures also have distinct fruity odor
• Differential media aids in identification
• Streptococcus pyogenes (strep throat) yields β-
hemolytic colonies on blood agar
• E. coli (urinary tract infections) ferments lactose, forms
pink colonies on MacConkey agar
Identification Methods Based on Phenotype
 Metabolic capabilities revealed by biochemical tests
• Catalase test: positive if O2 bubbles are seen after H2O2 is
added to culture
• Many tests rely on pH indicators
• Sugar fermentation lowers pH and may trap gas in
inverted tube
• Urease raises pH
Identification Methods Based on Phenotype
 Metabolic capabilities revealed by biochemical tests
Identification Methods Based on Phenotype
 Basic strategy for identification using biochemical
tests relies on dichotomous key: series of choices
• Simultaneous tests speed process, more thorough
• Some tests accomplished without culturing (e.g., breath
test to assay urease and identify Helicobacter pylori)
Identification Methods Based on Phenotype
 Commercial kits allow rapid identification via
biochemical tests
• Require incubation
• Pattern of results scored, leads to identification
Identification Methods Based on Phenotype
 Serological testing uses antibodies to detect
specific molecules
• Proteins, polysaccharides of prokaryotic cells can serve
as identifying markers
• Most useful include surface structures of cell wall,
capsule, flagella, pili
• Some Streptococcus species contain unique
carbohydrates in cell wall
Identification Methods Based on Phenotype
 Protein profile can be determined by MALDI-TOF
• Matrix-assisted laser desorption ionization time of flight
mass spectrometry
Identification Methods Based on Genotype
 Detecting specific nucleotide sequences
• Can identify sequences unique to species or group
• Nucleic acid probes
• Nucleic acid amplification tests (NAATs) (e.g., PCR)
• Limitation is each detects only single possibility
• Need to run multiple probes if organism being tested
could be one of multiple different species or groups
Identification Methods Based on Genotype
 Sequencing ribosomal RNAs or their DNA genes
• Sequences relatively stable
• 16S rRNA most useful because of moderate size
• Approximately 1,500 nucleotides
• Sequence compared with databases
• Can identify organisms that
cannot be grown in culture
Characterizing Strain Differences
 Characterizing different strains is important
• Foodborne illnesses
• Forensic investigations of bioterrorism, biocrimes
• Diagnosing certain diseases
Characterizing Strain Differences
 Biochemical Typing
• Group with characteristic pattern: biovar or biotype
 Serological Typing
• E. coli distinguished by antigenic type of flagella, capsule,
lipopolysaccharide molecules
Characterizing Strain Differences
 Molecular Typing
• Differences in DNA sequences
distinguish strains
• Cut DNA with restriction enzyme
• Separate via gel electrophoresis
• Yields RFLPs (restriction
fragment length polymorphisms)
• Different RFLPs indicate
different strains
Characterizing Strain Differences
 Phage typing
• Relies on differences in susceptibility to
bacteriophages
• Clearing zone forms as phage lyses
cells
• Largely replaced by molecular
methods, but still useful in labs lacking
equipment for molecular typing
Characterizing Strain Differences
 Antibiograms
• Reveal differences in susceptibility to antimicrobials
• Discs containing different antimicrobials
Classifying Microorganisms
 Classification historically based on phenotypic traits
• Size, shape, staining, metabolic capabilities
• But phenotypically similar organisms may be only
distantly related; conversely, closely related organisms
may appear dissimilar
 New molecular techniques more accurate
• Provide relative measure of time elapsed since
divergence from common ancestor
• Random mutations accumulate over time
• DNA sequencing allows construction of phylogenetic tree
Classifying Microorganisms
Classifying Microorganisms
 Sequence analysis of ribosomal components
• Reliable indicators because ribosomes perform crucial
tasks in all organisms
• Only a limited number of mutations can be tolerated
• Some sequences will be similar even if organisms
diverged long ago
• Ribosomal genes not commonly horizontally transferred
Classifying Microorganisms
 DNA Hybridization: relatedness of organisms
determined by similarity of nucleotide sequences
• Measured by how completely single strands of their DNA
hybridize with each other
• Bacterial strains with over 70% similarity are considered
members of same species
Classifying Microorganisms
 G+C Content: percent of G-C pairs in DNA
• If different by more than a few %, organisms unrelated
• Similarity does not guarantee relatedness
• DNA with higher G+C content melts at higher temperature