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 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