chap 35 lec micro

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  • 1. Chapter 35 Clinical Microbiology

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. Figure 35.1 4. Working with specimens

  • safety concerns
    • Standard Microbiological Practices have been established bythe 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

5. Collection

  • numerous methods used
  • choice of method depends on specimen

6. Skin and mucous membranes

  • sterile swab
    • greater risk of contamination
    • limited volume can be collected

Figure 35.2 (a) 7. Body fluids, etc.

  • needle aspiration
    • blood and cerebrospinal fluid
  • intubation
    • insertion of tube into body canal or hollow organ
    • stomach specimens
  • catheter
    • tubular instrument used to withdraw or introduce fluids into body cavity
    • urine

8. Figure 35.2 (d) 9. Body fluids

  • clean-catch method
    • collection of midstream portion of urine
  • sputum cup
    • sputum
      • mucous secretion expectorated from lungs, bronchi, and/or trachea

10. Figure 35.2 (e) 11. Handling

  • proper labeling
    • patient information
    • possible diagnosis
    • current antimicrobial therapy
    • physician information
    • type of specimen

12. Transport

  • should be timely
  • may involve use of special media that preserve microbes in specimen
  • special treatment may be needed if anaerobe is to be identified in specimen
  • temperature control may be needed

13. Transport Media

  • may require supplementation to support microbial survival or inhibit normal flora
    • e.g., use of antibacterial antibiotics such as penicillin to ensure recovery of fungi
    • e.g., use of polyvinyl alcohol-based preservatives for fixation of ova and parasites in clinical specimens

14. Figure 35.3 used to collect specimen specimen transferred from syringe to vial medium in vial retards oxygen diffusion 15. Select Agents Legislation

  • governs policy for possession, use and transport (beyond collection point) of potential biothreat agents
  • requires specific packaging and approvals for transport of specimens containing these organisms

16. Standard Microbiological Practices

  • areminimumguidelines that should be supplemented with other precautions based on exposure risks and lab biosafety level regulations
  • goal is to protect workers from contact with agents by their taking precautions and by their working in a safe laboratory environment

17. More on Standard Microbiological Practices

  • e.g., workers can limit their contact with microbes by not eating or smoking in lab and by preventing injuries caused by sharp objects
  • e.g., coverings such as lab coats and bandages should be used
  • e.g., workers should know how to use emergency eye wash and shower stations
  • e.g., work space should be disinfected
  • e.g., hands should be washed throughly afer any exposure and before leaving lab

18. Biosaftety Levels

  • recommended guidelines for additional precautions reflect the laboratorys biosafety level (BSL)
    • BSL 1 not known to cause disease in healthy adults
    • BSL 2 associated with human disease
    • BSL 3 disease may have serious or lethal consequences
    • BSL 4 agent poses high risk of life-threatening disease

19. 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

20. Microscopy

  • wet-mount, heat-fixed, or chemically fixed specimens can be examined
  • choice of microscopy depends on possible pathogen
    • e.g., dark-field microscopy
      • detection of spirochetes in skin lesions associated with syphilis
    • e.g., fluorescence microscopy
      • direct microscopic of specimens to detect fungi
  • stains often used
    • Gram stain and acid fast stain

21. Monoclonal Antibodies (MAB)

  • produced by hybridoma cells
  • recognize a single epitope
    • fluorescently-labeled MABs used diagnostically
      • technique has replaced use of polyclonal antisera for culture confirmation

22. 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 antibodys ability to bind a specific antigen
  • can be used as direct fluorescent-antibody (FA) technique or indirect fluorescent-antibody (IFA) technique assay

23. Figure 35.4 (a) FA technique 24. Figure 35.4 (b) IFA technique 25. Growth and Biochemical Characteristics

  • techniques used depend on nature of pathogen
  • for some pathogens, culture-based techniques have limited use

26. Viruses

  • 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

27. Fungi

  • cultures used to recover fungus from patient specimens
  • identification
    • direct microscopic (fluorescence) examination
    • immunofluorescence
    • serological tests (for some)
    • rapid identification methods (most yeasts)

28. Parasites

  • culture-based techniques not commonly used
  • identification by microscopic examination of clinical specimens
    • definitive diagnosis obtained by identification and characterization of ova, trophozoites and cysts in the specimen
  • histological staining of blood, negative staining of other body fluids and immunofluorescence staining are routinely used in identification of parasites

29. Bacteria

  • most bacteria:
    • culturing involves use of numerous kinds of growth media
      • can provide preliminary information about biochemical nature of bacterium
    • additional biochemical tests 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

30. Table 35.1 31. Table 35.2 32. Figure 35.5 (a) 33. Figure 35.5 (b) 34. Rapid Methods of Identification

  • manual biochemical systems
    • e.g., API 20 E system
  • mechanized/automated systems
  • immunologic systems

35. API