Introduction to Biotechnology

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20 Φεβ 2013 (πριν από 4 χρόνια και 7 μήνες)

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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





Figure 32.2a

FA technique

Figure 32.2b

IFA technique

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 antibody
-
based 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 include


nucleic 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


diagnostic for typhoid fever


e.g., latex agglutination tests


pregnancy test


e.g.,
viral hemagglutination


can be used to indicate the presence of virus
-
specific
antibodies


Figure 32.8

Agglutination Tests

titer = reciprocal of highest

dilution positive for agglutination

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