Diagnostic Testing in the Microbiology Laboratory

Diagnostic Testing in the Microbiology Laboratory
Presentation by: Jane Wong
Public Health Microbiologist

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
o Culture
o Microscopy
o Molecular methods such as PCR
* Indirect: Demonstration of presence of antibodies to a particular infectious agent
o Serology

Sterile versus Non-sterile Body Sites

* Sterile body sites:
o These sites normally do not contain any bacteria, so any bacteria found there are significant
+ Blood
+ Spinal fluid
* Non-sterile body sites:
o These sites are open to the external environment and normally contain bacteria
+ Throat
+ Feces

Specimens from Sterile Sites
Specimens from Non-Sterile Sites
Sensitivity
Specificity
Documentation
Specimen

* Appropriateness
* Collection
* Transport to lab
* Inoculation of media
* Culture and isolation
* Confirmation
* Report
Appropriate Specimen
Collection
Transport to Laboratory
Inoculation of Media
Culture media

* Used to grow bacteria
* Can be used to:
o Enrich the numbers of bacteria
o Select for certain bacteria and suppress others
o Differentiate among different kinds of bacteria

Microbiological Culture Media
Isolation of Individual Bacteria
Growth of Colonies
Mixed Culture of Soil Organisms Containing Bacillus anthracis
Colony “Picking”
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
o Stains
o Biochemical tests
o Serological tests (using known antibodies)
o 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
Blood Bottles Incubated
18 Hours of Incubation
Microbiologist Suspects 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
Laboratory Cannot Rule Out Bacillus anthracis
* Refers the culture to a reference laboratory that is part of the Laboratory Response Network (LRN)
Report
Molecular Tests
The Flip Side!
Other Factors in Personnel Shortage
Licensing Applications/Year For Clinical Laboratory Scientist Certification

Diagnostic Testing in the Microbiology Laboratory