Microbiology: A Systems
Approach, 2nd ed.
Chapter 17: Diagnosing Infections
17.1 Preparation for the Survey of
Microbial Diseases
• Methods used to identify bacteria to the level
of genus and species
– Phenotypic methods
• Morphology
• Physiology or biochemistry
– Immunologic method
• Serological analysis
– Genotypic techniques
• More and more often used as a sole resource for
identifying bacteria
Phenotypic Methods
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Microscopic morphology
Macroscopic morphology
Physiological/Biochemical characteristics
Chemical analysis
Microscopic Morphology
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Cell shape and size
Gram stain reaction
Acid fast reaction
Special structures
Macroscopic Morphology
• Colony appearance
• Speed of growth
• Patterns of growth
Physiological/Biochemical
Characteristics
• Traditional mainstay of bacterial identification
• Diagnostic tests for determining the presence of
specific enzymes and assessing nutritional and
metabolic activities
• Examples
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Fermentation of sugars
Capacity to metabolize complex polymers
Production of gas
Presence of enzymes
Sensitivity to antimicrobic drugs
Chemical Analysis
• Analyzing the types of specific structural
substances that the microorganism contains
• Examples
– Chemical composition of peptides in the cell wall
– Lipids in membranes
Genotypic Methods
• Primary advantage over phenotypic methods:
actually culturing the microorganisms is not
always necessary
• Also are increasingly automated with results
obtained very quickly
Immunologic Methods
• Antibody response to antigens
• Blood testing- often easier than testing for the
microbe itself
• Laboratory kits available for immediate
identification of a number of pathogens
17.2 On the Track of the Infectious
Agent: Specimen Collection
• The success of identification and treatment
depends on how specimens are collected,
handled, and stored
• General aseptic procedures must be used
Figure 17.1
Overview of Laboratory Techniques
• Direct tests using microscopic, immunologic,
or genetic methods
• Cultivation, isolation, and identification of
pathogens using a wide variety of general and
specific tests
• Results of specimen analysis entered in a
summary patient chart
Figure 17.2
Figure 17.3
17.3 Phenotypic Methods
• Immediate Direct Examination of Specimen
– Gram stain
– Acid-fast stain
– Direct fluorescence antibody (DFA) tests
– Direct antigen testing
Figure 17.4
Cultivation of Specimen
• Isolation media
• Biochemical testing
– Carbohydrate fermentation (acid and/or gas)
– Hydrolysis of gelatin, startch, and other polymers
– Enzyme actions such as catalase, oxidase, and
coagulase
– By-products of metabolism
Figure 5
Figure 17.6
Miscellaneous Tests
• Phage typing
• Animal inoculation
• Antimicrobial sensitivity
Determining Clinical Significance of
Cultures
• Is an isolate clinically important?
• How do you decide whether it is a
contaminant or part of the normal biota?
• Possible criteria
– Number
– Repeated isolation of a relatively pure culture of
any microorganism
17.4 Genotypic Methods
• DNA Analysis Using Genetic Probes
– Hybridization- can identify a bacterial species by
analyzing segments of its DNA
– Small fragments of single-stranded DNA or RNA
called probes
• Known to be complementary to the specific sequences
of DNA from a particular microbe
• Unknown test DNA from cells is bound to blotter paper
• Add probes to blotter
• Observe for signs that the probes have become fixed to
the test DNA
Figure 17.7
Nucleic Acid Sequencing and rRNA
Analysis
• Comparison of the sequence of nitrogen bases
in rRNA
• Effective for differentiating general group
differences
• Can be fine-tuned to identify at the species
level
Polymerase Chain Reaction
• Rapid identification of pathogens
• Developed for a wide variety of bacteria,
viruses, protozoa, and fungi
• Biosensor
17.5 Immunologic Methods
• Characteristics of antibodies can reveal the
history of a patient’s contact with
microorganisms or other antigens
• Serological testing
• Serology: the branch of immunology that
traditionally deals with in vitro diagnostic
testing of the serum
Figure 17.8
General Features of Immune Testing
• Strategies
– Agglutination
– Precipitation
– Immunodiffusion
– Complement fixation
– Fluorescent antibody tests
– Immunoassay tests
• Specificity and sensitivity
Figure 17.9
Visualizing Antigen-Antibody
Interactions
Figure 17.10
Agglutination and Precipitation
Reactions
• Agglutination: antigens are whole cells such
as red blood cells or bacteria with
determinant groups on the surface
• Precipitation: the antigen is a soluble
molecule
Agglutination Testing
• Antibodies cross-link the antigens to form visible
clumps
• Performed routinely to determine ABO and Rh blood
types
• Widal test: tube agglutination test for diagnosing
salmonelloses and undulant fever
• Rapid plasma regain (RPR) test: tests for antibodies to
syph8ilis
• Weil-Felix reaction: diagnoses ricketsial infections
• Latex agglutination tests: tiny latex beads with
antigens affixed
Precipitation Tests
• The soluble antigen is precipitated by an
antibody
• Reaction is observable as a cloudy or opaque
zone at the point of contact
• VDRL (Veneral Disease Research Lab) test
• Double diffusion (Ouchterlony) method
• Immunoelectrophoresis
Figure 17.11
Figure 17.12
The Western Blot for Detecting
Proteins
• Involves electrophoretic separation of proteins
followed by an immunoassay to detect those
proteins
• Counterpart of the Southern blot test
• Test material is electrophoresed in a gel to
separate out particular bands
• Gel transferred to a special blotter that binds the
reactants in place
• Blot developed by incubating it with a solution of
antigen or antibody labeled with radioactive,
fluorescent, or luminescent labels
Figure 17.13
Complement Fixation
• Lysin or cytolysin: an antibody that requires
complement to complete the lysis of its
antigenic target cell
Figure 17.14
Miscellaneous Serological Tests
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Treponema pallidum immobilization (TPI) test
Toxin neutralization tests
Serotyping
Quellung test
Flurorescent Antibodies and
Immunofluorescence Testing
• Direct testing: an unknown test specimen or
antigen is fixed to a slide and exposed to a
fluorescent antibody solution of known
composition
• Indirect testing: the fluorescent antibodies
are antibodies made to react with the Fc
region of another antibody
Figure 17.15
Immunoassays
• Extremely sensitive methods that permit rapid
and accurate measurement of trace antigen or
antibody
• Radioactive isotope labels
• Enzyme labels
• Sensitive electronic sensors
Radioimmunoassay (RIA)
• Antibodies or antigens labeled with a radioactive
isotope used to pinpoint minute amounts of a
corresponding antigen or antibody
• Compare the amount of radioactivity present in a
sample before and after incubation with a
known, labeled antigen or antibody
• Large amounts of a bound radioactive component
indicate that the unknown test substance was not
present
• Radioimmunosorbent test (RIST)
• Radioallergosorbent test (RAST)
Enzyme-Linked Immunosorbent Assay
(ELISA)
• Enzyme-antibody complex that can be used as
a color tracer for antigen-antibody reactions
• Indirect
• Direct
Figure 17.16
Tests that differentiate T Cells and B
Cells
• Mix with untreated sheep red blood cells
– T cells bind RBCs into a rosette formation
Figure 17.17
In Vivo Testing
• Tuberculin test
• Other diagnostic skin tests
A Viral Example
Figure 17.18