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Study Outline
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  1. Specimens
    1. Clinical microbiologists are microbiologists whose main function is to isolate and identify microorganisms from clinical specimens, and to do so as rapidly as possible
    2. Specimen-human material that is tested, examined, or studied to determine the presence or absence of specific microorganisms
      1. Because of safety concerns, specimens must be handled carefully; universal safety precautions have been recommended by the CDC to address safety issues in specimen handling
      2. Specimens should be:
        1. Representative of the diseased area
        2. Adequate in quantity for a variety of diagnostic tests
        3. Devoid of contamination, particularly by microorganisms indigenous to the skin and mucous membranes
        4. Forwarded promptly to the clinical laboratory
        5. Obtained prior to the administration of any antimicrobials
    3. Collection
      1. Sterile swabs-used to collect specimens from skin and mucous membranes; associated with greater risk of contamination and have limited volume capacity, and so their use is generally discouraged
      2. Needle aspiration-used to collect blood and cerebrospinal fluid; skin surface microorganisms must be excluded by the use of stringent antiseptic techniques; anticoagulants are used to prevent blood clotting
      3. Intubation-used to collect specimens from stomach
      4. Catheterization-used to collect urine
      5. Clean-catch midstream urine-first urine voided is not collected, because it is likely to be contaminated with surface organisms
      6. Sputum-mucous secretion expectorated from the lungs, bronchi, and/or trachea
    4. Handling-includes any special additives (e.g., anticoagulants) and proper labeling
    5. Transport-should be timely; temperature control may be needed; special treatment may be needed for anaerobes
  2. Identification of Microorganisms from Specimens
    1. Microscopy-direct examination of specimen, or examination of specimen after various staining procedures
    2. Growth and biochemical characteristics
      1. Viruses-identified by isolation in cell (tissue) culture, by immunodiagnosis, and by molecular detection
        1. Viral cultivation 1) Cell cultures-viruses are detected by cytopathic effects (observable morphological changes in host cells) or by hemadsorption (binding of red blood cells to infected cells) 2) Embryonated eggs-virus can be inoculated into allantoic cavity, amniotic cavity, or the chorioallantoic cavity; virus is detected by development of pocks on the chorioallantoic membrane, by development of hemagglutinins in the allantoic and amniotic fluid, and by death of the embryo 3) Laboratory animals (e.g., suckling mice)-observed for signs of disease or death
        2. Serological tests (e.g., monoclonal antibody-based immunofluorescence) can be used to detect virus in tissue-vial cultures
      2. Fungi
        1. Direct microscopic examination with fluorescent dyes
        2. Examination of cultures
        3. Serological tests for antifungal antibodies
        4. Yeast can be identified by the use of rapid ID methods
      3. Parasites-identified by examining specimens for eggs, cysts, larvae, or vegetative cells; some serological tests are available
      4. Bacteria (other than rickettsias, chlamydiae, and mycoplasmas)
        1. Isolation and growth of bacteria are required before many diagnostic tests can be used
        2. Initial identity may be suggested by source of specimen; microscopic appearance and Gram reaction; pattern of growth on selective, differential, and other media; and by hemolytic, metabolic, and fermentative properties
        3. After pure cultures are obtained, specific biochemical tests can be done
      5. Rickettsias-identified by immunoassays or by isolation (the later can be hazardous)
      6. Chlamydiae-identified by Giemsa staining, immunofluorescent staining of tissues with anti-chlamydia monoclonal antibodies, DNA probes, and PCR
      7. Mycoplasmas-identified immunologically or by the use of DNA probes
    3. Rapid methods of identification
      1. Manual biochemical systems such as the API 20E system for enterobacteria
        1. Consists of 20 microtube inoculation tests
        2. Results are converted to a seven- or nine-digit profile number
        3. The number is compared to the API Profile Index to determine the name of the bacterium
      2. Mechanized/automated systems
      3. Immunological systems
    4. Immunological techniques-detection of antigens or serum antibodies in specimens by the various procedures discussed in chapter 33
    5. Bacteriophage typing-the host range specificities of bacteriophages are dependent upon surface receptors on the particular bacteria; therefore, this can be a reliable method of identification
    6. Molecular methods and analysis of metabolic products
      1. Some of these procedures have been discussed (e.g., protein comparisons, enzyme characterizations, nucleic acid-base composition, nucleic acid hybridization, and nucleic acid sequencing)
      2. Nucleic Acid-based detection methods-ssDNA molecules that have been cloned from organism or prepared by PCR technology can be used in hybridization procedures; rRNA genes can be used to identify bacterial strains (ribotyping)
      3. Gas-liquid chromatography (GLC)-used to identify specific microbial metabolites, cellular fatty acids, and products of pyrolysis; usually for nonpolar substances that are extractable in ether
      4. Plasmid fingerprinting-separation and detection of the number and molecular weight of different plasmids, which are often consistently present in a strain of bacteria
  3. Susceptibility Testing
    1. Thought by many to be the most important testing done
    2. Used to help physician decide which drug(s) and which dosage(s) to use
  4. Computers in Clinical Microbiology
    1. Test ordering-specific requests, patient data, and accession number
    2. Result entry
    3. Report printing-flexible format to meet the needs of physician
    4. Laboratory management
    5. Interfaced with automated instruments

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