Understanding Clinical Microbiology: Isolation and Identification of Microbes from Specimens

1. Chapter 32 Clinical MicrobiologyandImmunology

2. Specimens 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

3. Working with Specimens Safety concerns • Standard Microbiological Practices have been established by the Centers for Disease Control and Prevention (CDC) Specimen should: • represent diseased area and other appropriate sites • be large enough for carrying out a variety of diagnostic tests • be collected in a manner that avoids contamination • be forwarded promptly to clinical lab • be obtained prior to administration of antimicrobial agents, if possible

4. Identification of Microorganisms from Specimens Preliminary or definitive identification of microbe based on numerous types of diagnostic procedures • microscopy • growth and biochemical characteristics • immunologic tests • bacteriophage typing • molecular methods

5. Collection • numerous methods used • choice of method depends on specimen

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 with 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

7. FA technique Figure 32.2a

8. IFA technique Figure 32.2b

9. Growth and Biochemical Characteristics • techniques used depend on nature of pathogen • for some pathogens, culture-based techniques have limited use

10. Identified by: isolation in living cells immunodiagnostic tests molecular methods replication in culture detected by: cytopathic effects morphological changes in host cells hemadsorption binding of red blood cells to surface of infected cells Viruses

11. Fungi Cultures used to recover fungus from patient specimens • growth medium depends on type(s) of fungus being isolated Identification • direct microscopic (fluorescence) examination • immunofluorescence • serological tests (for some) • rapid identification methods (most yeasts)

12. Bacteria Most bacteria: • culturing involves use of numerous kinds of growth media • can provide preliminary information about biochemical nature of bacterium • additional biochemical tests and staining used following isolation • some bacteria are not routinely cultured • rickettsias, chlamydiae, and mycoplasmas • identified with special stains, immunologic tests, or molecular methods such as PCR

13. Rapid Methods of Identification • manual biochemical systems • mechanized/automated systems • immunologic systems

14. Biosensors • based on the linkage of traditional antibody-based detection systems to sophisticated reporting systems • can be based on • microfluidic antigen sensors • real time PCR • highly sensitive spectroscopy systems • liquid crystal amplification of microbial immune complexes

15. Molecular Methods and Analysis of Metabolic Products • several methods widely used • examples include • nucleic acid probes • ribotyping • genomic fingerprinting

16. Genomic Fingerprinting • characterizes bacteria based on restriction endonuclease digestion of DNA • plasmid fingerprinting uses number of plasmids, their molecular weight, and restriction digestion pattern Figure 32.5

17. Immunological Techniques Detection of antigens or antibodies in specimens • especially useful when cultural methods are unavailable or impractical or antimicrobial therapy has been started

18. Clinical Immunology & Serotyping Clinical Immunology: • many antibody-antigen interactions that occur in vivo can also be used under controlled laboratory conditions for (in vitro) diagnostic testing Serotyping : • use of serum antibodies to detect and identify other molecules • can be used to differentiate serovars or serotypes of microbes that differ in antigenic composition of a structure or product

19. 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 • e.g., viral hemagglutination • can be used to indicate the presence of virus-specific antibodies

20. Agglutination Tests titer = reciprocal of highest dilution positive for agglutination Figure 32.8

21. Enzyme-Linked Immunosorbent Assay (ELISA) • can be used to detect antigens or antibodies in a sample • test involves the linking of various “label” enzymes to either antigens or antibodies • two basic methods used • direct immunoabsorbant assay • indirect immunoabsorbant assay

22. Immunoblotting (Western Blot) • procedure • proteins separated by electrophoresis • proteins transferred to nitrocellulose sheets • protein bands visualized with enzyme-tagged antibodies • sample uses • distinguish microbes • diagnostic tests • determine prognosis for infectious disease

23. Radioimmunoassay (RIA) • purified antigen labeled with radioisotope competes with unlabeled standard for antibody binding • amount of radioactivity associated with antibody is measured

24. Bibliography • Lecture PowerPoints Prescott’s Principles of Microbiology-Mc Graw Hill Co. • http://en.wikipedia.org/wiki/Scientific_method • https://files.kennesaw.edu/faculty/jhendrix/bio3340/home.html