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Chapter 10:
Methods for Classifying and
Identifying Microorganisms
Phylogenetic
Relationships
• Taxonomy
─ The science of classifying organisms
─ Provides universal names for organisms
─ Based on phylogenetic studies
◦ Phylogeny: the study of the evolutionary
relatedness among organisms
The Three-Domain System:
Classification Based on Cell Type
• Bacteria
• Archaea
• Eukarya
• Cell types differ in properties including rRNA
sequences, membrane lipid structure, tRNA molecules,
sensitivity to antibiotics, cell wall structure
─ rRNA sequences: primary means of determining
phylogenetic relationships (especially for
microorganisms)
Table 10.1
Applying Classification Strategies:
Cladograms
• Reflect evolutionary relatedness
─ Determined by rRNA sequences
• The length of a shared line corresponds to the degree of
similarity
• Each branch point (fork) indicates the evolution of a novel
property used to separate one group/organism from another
Figure 10.18.2
Species Definition
• Eukaryotic species:
─ A group of closely related organisms that breed among
themselves
• Prokaryotic species:
─ A population of cells with similar characteristics
─ Clone: Population of cells derived from a single cell
─ Strain: Group of cells within the same species that
have a distinct characteristic from others in the species
Classification vs. Identification
• Classification: based on phylogenetic/evolutionary
relationships
─ Groups similar organisms together
─ Largely based on rRNA sequences (microorganisms)
• Identification: utilizing observations and tests to
identify an unknown organism
─ Clinical settings—for diagnosis, disease treatment
scheme, etc.
◦ Needs to be efficient
Microorganism Identification
Methods
• Bergey’s Manual of
Determinative Bacteriology:
identification scheme reference
• Morphological characteristics:
Shape/structure; useful for
identifying eukaryotes
• Differential staining: Gram
staining, acid-fast staining
• Serology: Relies on pathogenspecific antibodies for
identification
• DNA-based Strategies
• Biochemical tests: Determines
presence of bacterial enzymes
Figure 10.8
Microorganism Identification Methods:
Serology
• Serology utilizes
pathogen-specific
antibodies as a tool
for identification
─ Example:
Slide agglutination
Known antiserum + unknown bacteriumreaction?
Positive test
Negative test
Figure 10.10
Serology:
ELISA
• Enzyme-linked Immunosorbent Assay (ELISA)
─ Antibody detection of specific pathogenic antigens
Pathogenspecific
antibody
Patient sample
http://www.komabiotech.com
Microorganism Identification Methods:
Phage Typing
• Phage typing: tests
which bacteriophages a
bacterium is susceptible to
─ Phages are specific to a
particular species or
strain of bacteria
─ Many phages cause lysis
of the cells they infect
◦ Areas of clearing/
plaques
─ Can be used for
comparisons (i.e. of two
individuals’ infections)
Figure 10.13
Microorganism Identification Methods:
DNA Strategies
• DNA fingerprinting
─ DNA of known microbe or
unknown sample is
digested with restriction
enzymes
Patient
sample
Known bacteria:
A
B
C
D
E
F
G
─ DNA fragments separated
and patterns are
compared
• Polymerase Chain
Reaction (PCR)
─ Amplification of a
microbe-specific DNA
fragment to
detectable levels 
identification
Figure 10.14, modified
Microorganism Identification Methods:
DNA Strategies
• Nucleic Acid Hybridization
─ Strands may be separated with heat
─ Complimentary sequences hybridize
(bind) to one another
Figure 10.15
Microorganism Identification Methods:
Biochemical Tests
• Test for the presence of specific bacterial enzymes
─ Develop a biochemical/enzymatic profile for an
unknown organism  identify the organism based on
this profile
Microorganism Identification Methods:
Numerical Identification (Biochemical)
• Rapid identification of
medically important
bacteria
─ Enterics (Enterotube)
• Several biochemical
tests simultaneously
Figure 10.9
Applying Identification Method Results:
Biochemical Tests & Dichotomous Keys