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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
