1. Clinical Microbiology and Immunology 1 36 Copyright © McGraw-Hill Global Education Holdings, LLC. Permission required for reproduction or display.

2. The Clinical Microbiology Laboratory Clinical microbiologist –major function is to isolate and identify microbes from clinical specimens rapidly Clinical specimen –portion or quantity of human material that is tested, examined, or studied to determine the presence or absence of specific microbes 2

3. Microbiology Risk Groups 3

4. Biosafety Levels Recommended guidelines for additional precautions reflect the laboratory’s biosafety level (BSL) 4

5. Immunological Techniques Detection of antigens or antibodies in specimens –especially useful when cultural methods are unavailable or impractical or antimicrobial therapy has been started –serology is the study of blood, namely serum, to identify antibodies and other blood components –often referred to as a titer – a measurement of the antibodies or antigen based on a method that utilizes serial dilution 5

6. Immunofluorescence Process in which fluorescent dyes are exposed to UV, violet, or blue light to make them fluoresce Dyes can be coupled to antibody molecules without changing antibody’s ability to bind a specific antigen Can be used as direct fluorescent-antibody (FA) technique or indirect fluorescent- antibody (IFA) technique assay 6

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8. Agglutination Agglutinates –visible clumps or aggregates of cells or particles e.g., Widal test –diagnostic for typhoid fever e.g., latex agglutination tests –pregnancy test –viral hemagglutination e.g., antibody titer measurements 8

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11. Complement Fixation Binding of complement to an antigen-antibody complex Basis of diagnostic tests that determine if antibodies to an antigen are present in patient’s serum Very sensitive, measure extremely small amounts of antibody 11

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13. Enzyme-Linked Immunosorbent Assay (ELISA) One of the most widely used serological tests –direct test can be used to detect antigens in a sample –indirect test can be used to detect antibodies in a sample Reaction visualized by addition of chromogen 13

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16. Immunoblotting (Western Blotting) Procedure –proteins separated by electrophoresis transferred to nitrocellulose sheets –protein bands visualized with enzyme-tagged antibodies Sample uses include –distinguish microbes –diagnostic tests –prognosis of infections 16

17. Immunoprecipitation Detects soluble Ag reacting with Ab (precipitins) Binding of Ab to Ag forms lattice that precipitates Lattice formation occurs only in optimal ratio of Ag to Ab 17

18. Immunodiffusion Precipitation reaction that occurs in agar gel medium Two commonly used techniques –single radial immunodiffusion (RID) assay (antibody in agar, antigen diffuses) quantitates antigen –double diffusion agar assay (Ouchterlony technique) antibody and antigen both diffuse identifies antigens 18

19. Immuno- electrophoresis Antigens first separated by electrophoresis according to charge Antigens visualized by precipitation reaction Has greater resolution than immunodiffusion assays 19

20. Flow Cytometry Detects organisms in clinical samples Detection based on cytometric parameters or by use of fluorochromes –fluorochromes often bound to antibodies or oligonucleotides Flow cytometer –forces suspension of cells through laser beam and measures amount of light scattering of fluorescence –can detect heterogeneous microbial populations with different responses to antimicrobial treatments 20

21. Radioimmunoassay (RIA) Purified antigen labeled with radioisotope competes with unlabeled standard for antibody binding Amount of radioactivity associated with antibody is measured 21