1. Chapter 17
Topics
Phenotypic
Genotypic
Immunological Method

2. Phenotyic Methods
Phenotype – appearance of a gene (expression of a genotype)
Subtypes:
Microscopic observations (gram stain)
Macroscopic observations (colonies)
Physiological/Biochemical (SIM, carbohydrate fermentation)
Chemical Composition (chemical analysis of cell wall or the cell structures)

3. Genotypic Methods
Genotype – the combination of two alleles (one from mother, one from father) that creates a trait. (With microbes, analysis of the Genes)
Polymerase Chain Reaction (PCR)
Fast and more precise
Restriction Fragment Length Polymorphism (RFLP)
Long but more specific
G + C composition
Percentage of Guanine and Cytosine

4. Immunological Methods
Basic principle
Agglutination
Precipitation
Western blot
Complement fixation
Immunofluorescence
Common assays

5. Basic principle Serology In vitro method
Antibodies have specificity for antigens
Detect
Identify
Quantify
Sensitive

6. Serological identification of disease and serotyping are two methods that can be used in serology testing.
Fig Basic principles of serological testing using antibodies
and antigens.

7. Serological tests are specific and sensitive.
Fig Specificity and sensitivity in immune testing

8. Agglutination Antigens are whole cells (eg. RBC or bacteria)
Antibodies cross-link the antigens
Determine serum titer
Types of tests
Rapid plasma test (syphilis)
Cold agglutinin test (mycoplasma)
Weil-Felix test (rickettsial)
Latex agglutination test (pregnancy)

9. Agglutination reactions involve whole cell antigens, while precipitation reactions involve soluble antigens.
Fig Cellular/molecular view of agglutination and
Precipitation reactions that produce visible Ag-Ab complexes.

10. Precipitation Soluble antigens
Binding of antibody to antigen, makes the complex insoluble or visible
Types of test
Tube precipitation
Ouchterlony (double diffusion)
Immunoelectrophoresis

11. An example of the tube precipitation test and the Ouchterlony method.
Fig Precipitation reactions.

12. Immunoelectrophoresis enables the identification of specific antibody classes that bind antigen from a serum sample.
Fig Immunoelectrophoresis of normal human serum.

13. Western blot Electrophoretic separation of protein (antigen) sample
Specific antibodies bind to protein sample
Specific and sensitive
Counterpart of the Southern blot (DNA test)

14. An example of human serum being tested for antibodies against HIV antigens using the Western blot method.
Fig The Western blot procedure.

15. Complement fixation
Antibody (lysin), antigen, complement, and sensitized sheep rbc are required
Complement is fixed to a Ab-Ag-complex
Fixed complement cannot participate in rbc lysis = positive reaction or identification

16. An example of the complement fixation test.
Fig Complement fixation test.

17. Immunofluorescence
A monoclonal antibody labeled with a fluorescent dye
Direct testing
Indirect testing

18. An example of direct and indirect immunofluorescence testing.
Fig Immunofluorescence testing

19. Common assays
Enzyme-Linked Immunosorbent Assay (ELISA)

20. ELISA Horseradish peroxidase or alkaline phosphatase
Enzyme releases dye (chromogen)
Types of test
Indirect
Capture or Sandwich

21. An example of the indirect and capture ELISA methods.
Fig Methods of ELISA testing.

22. Different methods commonly used to detect and identify viral infections.
Fig Summary of methods used to diagnose
Viral infections.