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Diagnostic Testing in the
Microbiology Laboratory
Jane Wong
Public Health Microbiologist
September 30, 2003
[email protected]
Topics
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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
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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% CO2
– 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
– 272 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 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
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
Other Bacillus
species are
motile.
B. anthracis
is non-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:
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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
800
700
600
500
Total
Pass
400
300
200
100
0
CA
US
not US