Diagnostic Testing in the Microbiology Laboratory

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Diagnostic Testing in the Microbiology Laboratory

• Jane Wong
• Public Health Microbiologist

September 30, 2003 jwong_at_ucbcidp.org
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Topics

• Some basic principles of microbiology testing
• A crash course in microbiology
• Follow a specimen through the lab
• Laboratory staffing issues

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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

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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

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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

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Specimens from Sterile Sites

• Any organism growing in a normally sterile site
  is significant
• Identify it

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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

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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

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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

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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

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Specimen

• Appropriateness
• Collection
• Transport to lab
• Inoculation of media
• Culture and isolation
• Confirmation
• Report

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Appropriate Specimen

• From relevant body site
• Adequate amount
• Quality

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Collection

• No contamination
• Appropriate equipment
• Good instructions to patient

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Transport to Laboratory

• Safe packaging
• Good labeling
• Temperature

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Inoculation of Media

• Use appropriate culture media
• What kind of specimen is it?
• What test did the physician request?

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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

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Microbiological Culture Media
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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 CO2
• Usually first examined after 24 hours

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Streaking a Plate
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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
• 272 organisms in one colony after 24 hours!

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• Classical bacterial identification can only be
  performed on pure cultures of bacteria (ideally,
  all descendants from one bacterial cell)

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Mixed Culture of Soil Organisms Containing
Bacillus anthracis
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Colony Picking

• Sterile needle or loop is touched to surface of
  colony and transferred to fresh, sterile media
• Incubation for another 24 hours

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Colonies of Bacteria in Pure Culture
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Pure Culture of Francisella tularensisColonies
After 72 hours Growth
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Pure Culture of Yersinia pestisColonies on Blood
Agar After 48 hours of Growth
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Yersinia pestis Colonial Morphology Viewed With
Transmitted Light
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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)

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Gram Stain of Streptococcus sp.
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Yersinia pestisGram stain
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Gram stain of Brucella sp.
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B. anthracis Gram stainshowing spores
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Gram stain of B. anthracis from broth culture
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Examples of Biochemical Tests
Left API 50 Test Above Antimicrobial
Sensitivity Test
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Yersinia pestis E-Test (Antimicrobial Sensitivity
Test)
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Nitrate and Urea Reactions
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Reactions on MacConkey Agar
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Triple Sugar Iron (TSI) Test
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Case Study

• Patient arrives in emergency room with fever
  (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

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Patient Admitted to Hospital

• Blood cultures ordered
• Blood drawn and immediately placed in blood
  culture bottles

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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.

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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

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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

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Bacillus anthracisIndia Ink Preparation
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Growth on a Blood Agar Plate (Petri Dish) After
18-24 Hours
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Gram stain of B. anthracis from broth culture
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Motility
B. anthracis is non-motile.
Other Bacillus species are motile.
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Laboratory Cannot Rule Out Bacillus anthracis

• Refers the culture to a reference laboratory that
  is part of the Laboratory Response Network (LRN)

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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

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Molecular Tests

• Biotechnology has given diagnostic laboratories
  very powerful tools
• for rapid detection and identification of human
  pathogens
• for strain typing for epidemiological
  investigations

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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.

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Other Factors in Personnel Shortage

• Training opportunities have been drastically
  reduced
• Pay is not competitive
• Much of the work force is approaching retirement
  age

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Licensing Applications/Year For Clinical
Laboratory Scientist Certification
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