Foundations in Microbiology

Sixth Edition

Chapter 17Diagnosing Infections

Lecture PowerPoint to accompany

Talaro

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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 up3. 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 17.1Sampling 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 17.10Cellular\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