Diagnostic Testing in the
Microbiology Laboratory
Jane Wong
Public Health Microbiologist
September 30, 2003 jwong@ucbcidp.org

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

Other Bacillus species are motile.
Motility
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:
– 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
400
300
200
100
0
Total
Pass
CA US not US