Transcript Foundations in Microbiology Sixth Edition

Lecture PowerPoint to accompany
Foundations in
Microbiology
Sixth Edition
Talaro
Chapter 17
Diagnosing Infections
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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; antibodyantigen 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