Foundations in

Microbiology
Sixth Edition
Chapter 17
Diagnosing Infections
Lecture PowerPoint to accompany
Talaro
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Survey of Microbial Disease
Methods of identifying unknown microbes
fall into three categories:
1. Phenotypic - observable microscopic and
macroscopic characteristics
2. Genotypic genetic make up
3. Immunological serology; antibody-
antigen reactions

Phenotypic Methods
Microscopic morphology fresh or stained
microorganisms from specimen; shape, size, stain
reaction, cell structures
Macroscopic morphology colony appearance;
texture, size, shape, pigment, growth
requirements
Physiological/biochemical characteristics
detection of presence or absence of particular
enzymes or metabolic pathways
Chemical analysis analyze specific chemical
composition; cell wall peptides, cell membrane
lipids

Genotypic Methods
Assess genetic make-up.
Culture is not necessary.
Precise, automated methods, quick results
Immunological Methods
Specific antibodies are used to detect
antigens.
Specimen Collection and
Laboratory Methods
Sampling body sites or fluids for
suspected infectious agent
Results depend on specimen collection,
handling, transport and storage.
Aseptic procedures should be used.


Insert figure 17.1
Sampling sites
Phenotypic Methods
Observation
macroscopic - cultivation colony
appearance, growth requirements,
appropriate media
microscopic - differential and special stains
Gram, AFB, fluorescent antibody stains
Direct antigen/antibody testing
Biochemical testing physiological
reactions to nutrients as evidence of the
absence or presence of enzymes
Important to consider whether microbe
recovered and identified is actually
causing the disease or simply normal flora
Genotypic Methods
DNA analysis
Assess the proportion of G + C nucleotides
relative to A + T content.
Determine DNA or ribosomal RNA sequences
using probes and polymerase chain reactions.
Immunological Methods
Serology attempts to detect signs of
infection in a patient by identifying specific
antibodies in vitro
Visible reactions include precipitates, color
changes, or the release of radioactivity.
Tests can be used to identify and to
determine the amount of antibody in
serum titer.
Agglutination and Precipitation
Reactions
Agglutination testing antibody cross links
whole-cell antigens, forming complexes that
settle out and form visible clumps
blood typing, some bacterial and viral diseases
Precipitation tests soluble antigen is made
insoluble by an antibody
syphilis,
Western blot immunoelectrophoresis;
separates antigens into bands
HIV

Insert figure 17.10
Cellular\molecular view

Complement Fixation
Detect antibodies that fix complement and
lyse target cells
antigen, antibody, complement, and sensitized
sheep RBCs
If complement is fixed by the Ag-Ab, the RBCs
will not be lysed.
Immunoassays
Extremely sensitive to detect trace antigens
and antibodies
Radioimmunoassay (RIA) antigens and
antibodies labeled with radioactive isotopes
Enzyme-linked immunosorbent assay
(ELISA) enzyme-antibody complex
produces a colored product when an
enzyme-substrate reaction occurs
In vivo Testing
Antigens are introduced directly into the
body to determine the presence or
absence of antibodies.
tuberculin skin test, allergy testing