1. Microbiology: A Systems Approach
PowerPoint to accompany
Microbiology: A Systems Approach
Cowan/Talaro
Chapter 17
Diagnosing Infections
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2. Chapter 17 Topics - Sample Collections - Phenotypic Method
- Genotypic Method
- Immunological Method

3. Sample Collection Typical samples Analysis scheme Saliva Sputum Blood
Urine
Fecal
Skin
Cutaneous membrane
Analysis scheme

4. Typical sites and methods of collection.
Fig Sampling sites and methods of collection

5. A general scheme used to isolate and identify a specimen.
Fig A scheme of specimen isolation and identification

6. An example of a clinical data form.
Fig Example of a clinical form used to report data

7. Phenotype Microscopic Cultivation Biochemical Miscellaneous
Clinical significance

8. Microscopic Common methods Gram stain Acid-fast stain
Direct fluorescence
Direct antigen testing

9. Stains Determine cell shape Determine cell wall property
Gram positive (thick peptidoglycan layer)
Gram negative (thin peptidoglycan layer)
Mycolic acid on cell surface

10. A direct fluorescence antibody test enables specific identification of microbes, and is visualized under the microscope.
Fig Direct fluorescence antigen test

11. Cultivation Enable growth of specific microbe
Specialized media
Selective media
Defined media
Differential media
Biochemical media
Enable pure culture isolation

12. Biochemical
Indirect method
Common tests
Rapid test
Schemes

13. Indirect method Indirect method of identification
Identify enzyme and product
Ideal for bacterial identification
Haploid
Express genes related to nutrient use

14. Common tests Carbohydrate fermentation Hydrolysis of gelatin, starch
Catalase
Oxidase
coagulase

15. An example of a rapid test, which includes 23 biochemical tests.
Fig Rapid tests

16. Flow charts, which contain data generated from Gram stain and biochemical tests, are helpful in species identification.
Fig Flowchart to separate primary genera of gram-pos

17. Miscellaneous Bacteriophages - Staphylococcus
Animal – Mycobacterium leprae
Avian embryos - rickettsias
Antisensitivity test - Clostridium

18. Clinical significance
Number of microbes present
Presence of microbes in a sterile site (eg. Cerebrospinal fluid)
Repeated isolation
Isolation of a true pathogen (eg. Mycobacterium tuberculosis)

19. Genotypic Method Genetic probes rRNA sequencing
Polymerase Chain Reaction (PCR)
G + C base composition

20. Genetic probes
Specific DNA probes for genes that identify a particular microbe
Probes bind to complimentary sample DNA (hybridization), enabling positive identification

21. Restriction digests, followed by hybridization with different probes generates specific patterns that enables identification of a particular microbe.
Fig DNA typing of restriction fragment length
Polymorphisms.

22. rRNA sequencing rRNA and protein make-up a ribosome
rRNA sequence is stable
Identification or signature for a particular microbe
Distinguishes the three Domains
Evolutionary relationships

23. PCR Amplify specific fragment DNA or gene
Sensitive – amplify small amounts of sample DNA
Quick identification – 1 day

24. G + C base composition General indicator of relatedness
Very small rate of change
Taxonomic determination
Non-specific – cannot be used to identify specific microbe

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

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

27. 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.

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

29. 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)

30. 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.

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

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

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

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

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

36. 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

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

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

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

40. Common assays Radioimmunoassay (RIA)
Enzyme-Linked Immunosorbent Assay (ELISA)
T and B cell differentiation
Virus testing

41. RIA Antibody or antigen is labeled
Detect corresponding antibody or antigen
Competition between unlabeled and labeled molecule

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

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

44. The rosette formation method and the fluorescent technique are used to identify lymphocytes (T an B cells), in order to evaluate immune dysfunctions.
Fig Tests for characterizing T cells and B cells.

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