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microbiological diagnosis microbiological diagnosis Presentation Transcript

  • Diagnostic Testing in the Microbiology Laboratory Jane Wong Public Health Microbiologist September 30, 2003 [email_address]
  • Topics
    • Some basic principles of microbiology testing
    • A crash course in microbiology
    • Follow a specimen through the lab
    • Laboratory staffing issues
  • Media and Culture
    • Media: Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats
    • Culture: The propagation of microorganisms using various media
  • Direct and Indirect Testing
    • Direct: Demonstration of the presence of an infectious agent
      • Culture
      • Microscopy
      • Molecular methods such as PCR
    • Indirect: Demonstration of presence of antibodies to a particular infectious agent
      • Serology
  • Sterile versus Non-sterile Body Sites
    • Sterile body sites:
      • These sites normally do not contain any bacteria, so any bacteria found there are significant
        • Blood
        • Spinal fluid
    • Non-sterile body sites:
      • These sites are open to the external environment and normally contain bacteria
        • Throat
        • Feces
  • Specimens from Sterile Sites
    • Any organism growing in a normally sterile site is significant
    • Identify it
  • Specimens from Non-Sterile Sites
    • Only look for specific pathogens
    • Physician will order test for a specific organism, or group of organisms
    • Other “normal flora” bacteria will be present, but are not be identified
  • Sensitivity
    • The fraction of those with the disease correctly identified as positive by the test.
    • Isolation and identification of a known pathogenic organism may not be a very sensitive test
      • If the organism is present, it may not be found 100% of the time
        • There can be false negatives
  • Specificity
    • The fraction of those without the disease correctly identified as negative by the test.
    • Isolation and identification of a known pathogenic organism is a very specific test
      • If the organism is not present in the specimen it will not be found
  • Documentation
    • Specimen is logged in upon arrival in laboratory
    • All tests and results are recorded and initialed by microbiologist
    • All media and reagents are batch tested with positive and negative controls
    • All equipment is checked at least once a day to be sure it is operating within predetermined parameters
  • Specimen
    • Appropriateness
    • Collection
    • Transport to lab
    • Inoculation of media
    • Culture and isolation
    • Confirmation
    • Report
  • Appropriate Specimen
    • From relevant body site
    • Adequate amount
    • Quality
  • Collection
    • No contamination
    • Appropriate equipment
    • Good instructions to patient
  • Transport to Laboratory
    • Safe packaging
    • Good labeling
    • Temperature
  • Inoculation of Media
    • Use appropriate culture media
      • What kind of specimen is it?
      • What test did the physician request?
  • Culture media
    • Used to grow bacteria
    • Can be used to:
      • Enrich the numbers of bacteria
      • Select for certain bacteria and suppress others
      • Differentiate among different kinds of bacteria
  • Microbiological Culture Media
  • Isolation of Individual Bacteria
    • Specimen is “streaked”, using a sterile loop, onto solid media.
    • The agar plates (media) are incubated at appropriate temperature and atmosphere
      • Often at 35º C.
      • Often at 5% CO 2
      • Usually first examined after 24 hours
  • “Streaking a Plate”
  • Growth of Colonies
    • Bacterial Colony
      • Result of one bacterium being isolated from others during “streaking procedure”
      • That bacterium grows in numbers exponentially
      • Many bacteria have a generation time of 20 minutes
      • 2 72 organisms in one colony after 24 hours!
    • Classical bacterial identification can only be performed on pure cultures of bacteria (ideally, all descendants from one bacterial cell)
  • Mixed Culture of Soil Organisms Containing Bacillus anthracis
  • Colony “Picking”
    • Sterile needle or loop is touched to surface of colony and transferred to fresh, sterile media
    • Incubation for another 24 hours
  • Colonies of Bacteria in Pure Culture
  • Pure Culture of Francisella tularensis Colonies After 72 hours Growth
  • Pure Culture of Yersinia pestis Colonies on Blood Agar After 48 hours of Growth
  • Yersinia pestis Colonial Morphology Viewed With Transmitted Light
  • Confirmation
    • Now we have a pure culture of bacteria
    • Testing is now done to confirm the identification of the bacteria culture
      • Stains
      • Biochemical tests
      • Serological tests (using known antibodies)
      • Molecular tests (nucleic acid probes)
  • Gram Stain of Streptococcus sp.
  • Yersinia pestis Gram stain
  • Gram stain of Brucella sp.
  • B. anthracis Gram stain showing spores
  • Gram stain of B. anthracis from broth culture
  • Examples of Biochemical Tests Left: API 50 Test Above: Antimicrobial Sensitivity Test
  • Yersinia pestis E-Test (Antimicrobial Sensitivity Test)
  • Nitrate and Urea Reactions
  • Reactions on MacConkey Agar
  • Triple Sugar Iron (TSI) Test
  • Case Study
    • Patient arrives in emergency room with f ever (temperature greater than 100 degrees F). The fever is accompanied by chills or night sweats.
    • Flu-like symptoms.
    • Non-productive cough, chest discomfort, shortness of breath, fatigue, muscle aches
  • Patient Admitted to Hospital
    • Blood cultures ordered
    • Blood drawn and immediately placed in blood culture bottles
  • Blood Bottles Incubated
    • Bottles are automatically tested every 10 minutes.
    • Positive results are tagged for quick processing.
    • Negative bottles can be batch-scanned out of the system and unloaded at the end of protocol.
  • 18 Hours of Incubation
    • Blood culture incubator signals that there is growth in one of the bottles.
    • It is removed and a Gram stain is performed
  •  
  • Microbiologist Suspects Bacillus anthracis
    • Reports results so far to supervisor
    • Streaks a fresh blood agar plate and incubates it
    • May perform wet mount test with India Ink to see “capsule” around individual bacteria
    • Inoculates media to observe motility
  • Bacillus anthracis India Ink Preparation
  • Growth on a Blood Agar Plate (Petri Dish) After 18-24 Hours
  • Gram stain of B. anthracis from broth culture
  • Motility B. anthracis is non-motile. Other Bacillus species are motile.
  • Laboratory Cannot Rule Out Bacillus anthracis
    • Refers the culture to a reference laboratory that is part of the Laboratory Response Network (LRN)
  • Report
    • Final report goes to physician
    • The validity of this report is dependent upon:
      • Appropriateness of specimen
      • Proper collection and adequacy of specimen
      • Appropriate transport to lab
      • Use of media of known quality
      • Culture and isolation by knowledgeable personnel using equipment known to be operating correctly
      • Confirmation by tests of known quality
      • Results interpreted and reported by professional staff
      • No transcription or computer errors
  • Molecular Tests
    • Biotechnology has given diagnostic laboratories very powerful tools
      • for rapid detection and identification of human pathogens
      • for strain typing for epidemiological investigations
  • The Flip Side!
    • Biotechnology companies attract recent college graduates
      • Majors in biology and allied fields
      • Salaries usually higher than clinical or government public health labs offer
      • Appeal to public service only goes so far!
    • Result: public health and clinical laboratories have trouble recruiting and retaining laboratory personnel.
  • Other Factors in Personnel Shortage
    • Training opportunities have been drastically reduced
    • Pay is not competitive
    • Much of the work force is approaching retirement age
  • Licensing Applications/Year For Clinical Laboratory Scientist Certification
  •