1. Microbiology: A Systems Approach, 2nd ed.
Chapter 17: Diagnosing Infections

2. 17.1 Preparation for the Survey of Microbial Diseases
Methods used to identify bacteria to the level of genus and species
Phenotypic methods
Morphology
Physiology or biochemistry
Immunologic method
Serological analysis
Genotypic techniques
More and more often used as a sole resource for identifying bacteria

3. Phenotypic Methods Microscopic morphology Macroscopic morphology
Physiological/Biochemical characteristics
Chemical analysis

4. Microscopic Morphology
Cell shape and size
Gram stain reaction
Acid fast reaction
Special structures

5. Macroscopic Morphology
Colony appearance
Speed of growth
Patterns of growth

6. Physiological/Biochemical Characteristics
Traditional mainstay of bacterial identification
Diagnostic tests for determining the presence of specific enzymes and assessing nutritional and metabolic activities
Examples
Fermentation of sugars
Capacity to metabolize complex polymers
Production of gas
Presence of enzymes
Sensitivity to antimicrobic drugs

7. Chemical Analysis
Analyzing the types of specific structural substances that the microorganism contains
Examples
Chemical composition of peptides in the cell wall
Lipids in membranes

8. Genotypic Methods
Primary advantage over phenotypic methods: actually culturing the microorganisms is not always necessary
Also are increasingly automated with results obtained very quickly

9. Immunologic Methods Antibody response to antigens
Blood testing- often easier than testing for the microbe itself
Laboratory kits available for immediate identification of a number of pathogens

10. 17.2 On the Track of the Infectious Agent: Specimen Collection
The success of identification and treatment depends on how specimens are collected, handled, and stored
General aseptic procedures must be used

11. Figure 17.1

12. Overview of Laboratory Techniques
Direct tests using microscopic, immunologic, or genetic methods
Cultivation, isolation, and identification of pathogens using a wide variety of general and specific tests
Results of specimen analysis entered in a summary patient chart

13. Figure 17.2

14. Figure 17.3

15. 17.3 Phenotypic Methods Immediate Direct Examination of Specimen
Gram stain
Acid-fast stain
Direct fluorescence antibody (DFA) tests
Direct antigen testing

16. Figure 17.4

17. Cultivation of Specimen
Isolation media
Biochemical testing
Carbohydrate fermentation (acid and/or gas)
Hydrolysis of gelatin, startch, and other polymers
Enzyme actions such as catalase, oxidase, and coagulase
By-products of metabolism

19. Figure 5

20. Figure 17.6

21. Miscellaneous Tests Phage typing Animal inoculation
Antimicrobial sensitivity

22. Determining Clinical Significance of Cultures
Is an isolate clinically important?
How do you decide whether it is a contaminant or part of the normal biota?
Possible criteria
Number
Repeated isolation of a relatively pure culture of any microorganism

23. 17.4 Genotypic Methods DNA Analysis Using Genetic Probes
Hybridization- can identify a bacterial species by analyzing segments of its DNA
Small fragments of single-stranded DNA or RNA called probes
Known to be complementary to the specific sequences of DNA from a particular microbe
Unknown test DNA from cells is bound to blotter paper
Add probes to blotter
Observe for signs that the probes have become fixed to the test DNA

24. Figure 17.7

25. Nucleic Acid Sequencing and rRNA Analysis
Comparison of the sequence of nitrogen bases in rRNA
Effective for differentiating general group differences
Can be fine-tuned to identify at the species level

26. Polymerase Chain Reaction
Rapid identification of pathogens
Developed for a wide variety of bacteria, viruses, protozoa, and fungi
Biosensor

27. 17.5 Immunologic Methods
Characteristics of antibodies can reveal the history of a patient’s contact with microorganisms or other antigens
Serological testing
Serology: the branch of immunology that traditionally deals with in vitro diagnostic testing of the serum

28. Figure 17.8

29. General Features of Immune Testing
Strategies
Agglutination
Precipitation
Immunodiffusion
Complement fixation
Fluorescent antibody tests
Immunoassay tests
Specificity and sensitivity

30. Figure 17.9

31. Visualizing Antigen-Antibody Interactions
Figure 17.10

32. Agglutination and Precipitation Reactions
Agglutination: antigens are whole cells such as red blood cells or bacteria with determinant groups on the surface
Precipitation: the antigen is a soluble molecule

33. Agglutination Testing
Antibodies cross-link the antigens to form visible clumps
Performed routinely to determine ABO and Rh blood types
Widal test: tube agglutination test for diagnosing salmonelloses and undulant fever
Rapid plasma regain (RPR) test: tests for antibodies to syph8ilis
Weil-Felix reaction: diagnoses ricketsial infections
Latex agglutination tests: tiny latex beads with antigens affixed

34. Precipitation Tests The soluble antigen is precipitated by an antibody
Reaction is observable as a cloudy or opaque zone at the point of contact
VDRL (Veneral Disease Research Lab) test
Double diffusion (Ouchterlony) method
Immunoelectrophoresis

35. Figure 17.11

36. Figure 17.12

37. The Western Blot for Detecting Proteins
Involves electrophoretic separation of proteins followed by an immunoassay to detect those proteins
Counterpart of the Southern blot test
Test material is electrophoresed in a gel to separate out particular bands
Gel transferred to a special blotter that binds the reactants in place
Blot developed by incubating it with a solution of antigen or antibody labeled with radioactive, fluorescent, or luminescent labels

38. Figure 17.13

39. Complement Fixation
Lysin or cytolysin: an antibody that requires complement to complete the lysis of its antigenic target cell

40. Figure 17.14

41. Miscellaneous Serological Tests
Treponema pallidum immobilization (TPI) test
Toxin neutralization tests
Serotyping
Quellung test

42. Flurorescent Antibodies and Immunofluorescence Testing
Direct testing: an unknown test specimen or antigen is fixed to a slide and exposed to a fluorescent antibody solution of known composition
Indirect testing: the fluorescent antibodies are antibodies made to react with the Fc region of another antibody

43. Figure 17.15

44. Immunoassays
Extremely sensitive methods that permit rapid and accurate measurement of trace antigen or antibody
Radioactive isotope labels
Enzyme labels
Sensitive electronic sensors

45. Radioimmunoassay (RIA)
Antibodies or antigens labeled with a radioactive isotope used to pinpoint minute amounts of a corresponding antigen or antibody
Compare the amount of radioactivity present in a sample before and after incubation with a known, labeled antigen or antibody
Large amounts of a bound radioactive component indicate that the unknown test substance was not present
Radioimmunosorbent test (RIST)
Radioallergosorbent test (RAST)

46. Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme-antibody complex that can be used as a color tracer for antigen-antibody reactions
Indirect
Direct

47. Figure 17.16

48. Tests that differentiate T Cells and B Cells
Mix with untreated sheep red blood cells
T cells bind RBCs into a rosette formation
Figure 17.17

49. In Vivo Testing
Tuberculin test
Other diagnostic skin tests

50. A Viral Example
Figure 17.18