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beefzoologistBiotechnology

Feb 21, 2013 (4 years and 8 months ago)

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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

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

0
100
200
300
400
500
600
700
800
CA
US
not US
Total
Pass