Introduction to Biotechnology
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Transcript Introduction to Biotechnology
Chapter 32
Clinical Microbiology and
Immunology
Specimens
Clinical microbiologist
major function is to isolate and identify microbes from
clinical specimens rapidly
Clinical specimen
portion or quantity of human material that is tested,
examined, or studied to determine the presence or
absence of specific microbes
Working with Specimens
Safety concerns
Standard Microbiological Practices have been
established by the Centers for Disease Control
and Prevention (CDC)
Specimen should:
represent diseased area and other appropriate
sites
be large enough for carrying out a variety of
diagnostic tests
be collected in a manner that avoids
contamination
be forwarded promptly to clinical lab
be obtained prior to administration of antimicrobial
agents, if possible
Identification of Microorganisms
from Specimens
Preliminary or definitive identification of
microbe based on numerous types of
diagnostic procedures
microscopy
growth
and biochemical characteristics
immunologic tests
bacteriophage typing
molecular methods
Collection
numerous
methods used
choice of method depends on
specimen
Immunofluorescence
process in which fluorescent dyes are exposed to UV,
violet, or blue light to make them fluoresce
dyes can be coupled to antibody molecules with changing
antibody’s ability to bind a specific antigen
can be used as direct fluorescent-antibody (FA) technique
or indirect fluorescent-antibody (IFA) technique assay
FA technique
Figure 32.2a
IFA technique
Figure 32.2b
Growth and Biochemical
Characteristics
techniques used depend on nature of
pathogen
for some pathogens, culture-based
techniques have limited use
Viruses
Identified by:
isolation in living
cells
immunodiagnostic
tests
molecular methods
replication in culture
detected by:
cytopathic effects
morphological
changes in host cells
hemadsorption
binding of red blood
cells to surface of
infected cells
Fungi
Cultures used to recover fungus from patient specimens
growth medium depends on type(s) of fungus being isolated
Identification
direct microscopic (fluorescence) examination
immunofluorescence
serological tests (for some)
rapid identification methods (most yeasts)
Bacteria
Most bacteria:
culturing involves use of numerous kinds of
growth media
can provide preliminary information about biochemical
nature of bacterium
additional biochemical tests and staining used
following isolation
some bacteria are not routinely cultured
rickettsias, chlamydiae, and mycoplasmas
identified with special stains, immunologic tests,
or molecular methods such as PCR
Rapid Methods of Identification
manual biochemical systems
mechanized/automated systems
immunologic systems
Biosensors
based on the linkage of traditional antibodybased detection systems to sophisticated
reporting systems
can be based on
microfluidic antigen sensors
real time PCR
highly sensitive spectroscopy systems
liquid crystal amplification of microbial immune
complexes
Molecular Methods and Analysis
of Metabolic Products
several methods widely used
examples
nucleic
include
acid probes
ribotyping
genomic fingerprinting
Genomic Fingerprinting
characterizes bacteria
based on restriction
endonuclease digestion
of DNA
plasmid fingerprinting
uses number of
plasmids, their
molecular weight, and
restriction digestion
pattern
Figure 32.5
Immunological Techniques
Detection of antigens or antibodies in specimens
especially useful when cultural methods are
unavailable or impractical or antimicrobial therapy has
been started
Clinical Immunology & Serotyping
Clinical Immunology:
many antibody-antigen interactions that occur in
vivo can also be used under controlled
laboratory conditions for (in vitro) diagnostic
testing
Serotyping :
use of serum antibodies to detect and identify
other molecules
can be used to differentiate serovars or
serotypes of microbes that differ in antigenic
composition of a structure or product
Agglutination
Agglutinates
visible clumps or aggregates of cells or particles
e.g., Widal test
e.g., latex agglutination tests
diagnostic for typhoid fever
pregnancy test
e.g., viral hemagglutination
can be used to indicate the presence of virus-specific
antibodies
Agglutination Tests
titer = reciprocal of highest
dilution positive for agglutination
Figure 32.8
Enzyme-Linked Immunosorbent
Assay (ELISA)
can be used to detect antigens or antibodies in a
sample
test involves the linking of various “label”
enzymes to either antigens or antibodies
two basic methods used
direct immunoabsorbant assay
indirect immunoabsorbant assay
Immunoblotting (Western Blot)
procedure
proteins
separated by electrophoresis
proteins transferred to nitrocellulose sheets
protein bands visualized with enzyme-tagged
antibodies
sample uses
distinguish
microbes
diagnostic tests
determine prognosis for infectious disease
Radioimmunoassay (RIA)
purified antigen labeled with radioisotope
competes with unlabeled standard for
antibody binding
amount of radioactivity associated with
antibody is measured
Bibliography
Lecture PowerPoints Prescott’s Principles
of Microbiology-Mc Graw Hill Co.
http://en.wikipedia.org/wiki/Scientific_
method
https://files.kennesaw.edu/faculty/jhend
rix/bio3340/home.html