chapter 32- clinical microbiology
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Chapter 32Chapter 32
Clinical MicrobiologyClinical Microbiology andand ImmunologyImmunology
SpecimensSpecimensClinical microbiologistClinical microbiologist
major function is to isolate and identify microbes major function is to isolate and identify microbes from clinical specimens rapidlyfrom clinical specimens rapidly
Clinical specimenClinical specimen portion or quantity of human material that is portion or quantity of human material that is
tested, examined, or studied to determine the tested, examined, or studied to determine the presence or absence of specific microbespresence or absence of specific microbes
Working with SpecimensWorking with SpecimensSafety concernsSafety concerns
Standard Microbiological Practices have Standard Microbiological Practices have been established by the Centers for been established by the Centers for Disease Control and Prevention (CDC)Disease Control and Prevention (CDC)
Specimen should:Specimen should: represent diseased area and other represent diseased area and other
appropriate sitesappropriate sites be large enough for carrying out a variety of be large enough for carrying out a variety of
diagnostic testsdiagnostic tests be collected in a manner that avoids be collected in a manner that avoids
contaminationcontamination be forwarded promptly to clinical labbe forwarded promptly to clinical lab be obtained prior to administration of be obtained prior to administration of
antimicrobial agents, if possibleantimicrobial agents, if possible
Identification of Identification of Microorganisms from Microorganisms from
SpecimensSpecimensPreliminary or definitive identification Preliminary or definitive identification
of microbe based on numerous types of microbe based on numerous types of diagnostic proceduresof diagnostic proceduresmicroscopymicroscopygrowth and biochemical characteristicsgrowth and biochemical characteristicsimmunologic testsimmunologic testsbacteriophage typingbacteriophage typingmolecular methodsmolecular methods
CollectionCollection
numerous methods usednumerous methods usedchoice of method depends on choice of method depends on
specimenspecimen
ImmunofluorescenceImmunofluorescence
process in which fluorescent dyes are exposed to process in which fluorescent dyes are exposed to UV, violet, or blue light to make them fluoresce UV, violet, or blue light to make them fluoresce
dyes can be coupled to antibody molecules with dyes can be coupled to antibody molecules with changing antibody’s ability to bind a specific changing antibody’s ability to bind a specific antigenantigen
can be used as can be used as direct direct fluorescent-antibody (FA) fluorescent-antibody (FA) technique or technique or indirectindirect fluorescent-antibody (IFA) fluorescent-antibody (IFA) technique assaytechnique assay
Figure 32.2a
FA technique
Figure 32.2b
IFA technique
Growth and Biochemical Growth and Biochemical CharacteristicsCharacteristics
techniques used depend on nature of techniques used depend on nature of pathogenpathogen
for some pathogens, culture-based for some pathogens, culture-based techniques have limited usetechniques have limited use
VirusesVirusesIdentified by:Identified by:
isolation in living isolation in living cellscells
immunodiagnostic immunodiagnostic teststests
molecular molecular methodsmethods
replication in replication in culture detected culture detected by:by: cytopathic effectscytopathic effects
morphological morphological changes in host changes in host cellscells
hemadsorptionhemadsorptionbinding of red binding of red
blood cells to blood cells to surface of infected surface of infected cellscells
FungiFungi
Cultures used to recover fungus from patient Cultures used to recover fungus from patient specimensspecimens growth medium depends on type(s) of fungus being growth medium depends on type(s) of fungus being
isolatedisolated
IdentificationIdentification direct microscopic (fluorescence) examinationdirect microscopic (fluorescence) examination immunofluorescenceimmunofluorescence serological tests (for some)serological tests (for some) rapid identification methods (most yeasts)rapid identification methods (most yeasts)
BacteriaBacteria
Most bacteria:Most bacteria: culturing involves use of numerous kinds of culturing involves use of numerous kinds of
growth mediagrowth mediacan provide preliminary information about can provide preliminary information about
biochemical nature of bacteriumbiochemical nature of bacterium additional biochemical tests and staining additional biochemical tests and staining
used following isolationused following isolation some bacteria are not routinely culturedsome bacteria are not routinely cultured
rickettsias, chlamydiae, and mycoplasmasrickettsias, chlamydiae, and mycoplasmas identified with special stains, immunologic identified with special stains, immunologic
tests, or molecular methods such as PCRtests, or molecular methods such as PCR
Rapid Methods of Rapid Methods of IdentificationIdentification
manual biochemical systemsmanual biochemical systemsmechanized/automated systemsmechanized/automated systems immunologic systemsimmunologic systems
BiosensorsBiosensors
based on the linkage of traditional based on the linkage of traditional antibody-based detection systems to antibody-based detection systems to sophisticated reporting systems sophisticated reporting systems
can be based oncan be based on microfluidic antigen sensorsmicrofluidic antigen sensors real time PCRreal time PCR highly sensitive spectroscopy systemshighly sensitive spectroscopy systems liquid crystal amplification of microbial immune liquid crystal amplification of microbial immune
complexes complexes
Molecular Methods and Molecular Methods and Analysis of Metabolic Analysis of Metabolic
ProductsProductsseveral methods widely usedseveral methods widely used
examples includeexamples includenucleic acid probes nucleic acid probes ribotypingribotypinggenomic fingerprintinggenomic fingerprinting
Genomic FingerprintingGenomic Fingerprinting characterizes characterizes
bacteria based on bacteria based on restriction restriction endonuclease endonuclease digestion of DNAdigestion of DNA
plasmid plasmid fingerprintingfingerprinting uses uses number of plasmids, number of plasmids, their molecular their molecular weight, and weight, and restriction digestion restriction digestion patternpattern
Figure 32.5
Immunological TechniquesImmunological Techniques
Detection of antigens or antibodies in Detection of antigens or antibodies in specimensspecimens especially useful when cultural methods are especially useful when cultural methods are
unavailable or impractical or antimicrobial unavailable or impractical or antimicrobial therapy has been started therapy has been started
Clinical Immunology & Clinical Immunology & SerotypingSerotyping
Clinical Immunology:Clinical Immunology: many antibody-antigen interactions that many antibody-antigen interactions that
occur in vivo can also be used under occur in vivo can also be used under controlled laboratory conditions for (in controlled laboratory conditions for (in vitro) diagnostic testingvitro) diagnostic testing
Serotyping :Serotyping : use of serum antibodies to detect and use of serum antibodies to detect and
identify other moleculesidentify other molecules can be used to differentiate serovars or can be used to differentiate serovars or
serotypes of microbes that differ in serotypes of microbes that differ in antigenic composition of a structure or antigenic composition of a structure or productproduct
AgglutinationAgglutinationAgglutinatesAgglutinates
visible clumps or aggregates of cells or particlesvisible clumps or aggregates of cells or particles e.g., e.g., Widal testWidal test
diagnostic for typhoid feverdiagnostic for typhoid fever e.g., latex agglutination testse.g., latex agglutination tests
pregnancy testpregnancy test e.g., e.g., viral hemagglutinationviral hemagglutination
can be used to indicate the presence of virus-specific antibodiescan be used to indicate the presence of virus-specific antibodies
Figure 32.8
Agglutination TestsAgglutination Tests
titer = reciprocal of highestdilution positive for agglutination
Enzyme-Linked Enzyme-Linked Immunosorbent Assay Immunosorbent Assay
(ELISA)(ELISA) can be used to detect antigens or can be used to detect antigens or antibodies in a sampleantibodies in a sample
test involves the linking of various “label” test involves the linking of various “label” enzymes to either antigens or antibodiesenzymes to either antigens or antibodies
two basic methods usedtwo basic methods used direct immunoabsorbant assaydirect immunoabsorbant assay indirect immunoabsorbant assayindirect immunoabsorbant assay
Immunoblotting (Western Immunoblotting (Western Blot)Blot)procedureprocedure
proteins separated by electrophoresisproteins separated by electrophoresisproteins transferred to nitrocellulose proteins transferred to nitrocellulose
sheetssheetsprotein bands visualized with enzyme-protein bands visualized with enzyme-
tagged antibodiestagged antibodiessample usessample uses
distinguish microbesdistinguish microbesdiagnostic testsdiagnostic testsdetermine prognosis for infectious diseasedetermine prognosis for infectious disease
Radioimmunoassay (RIA)Radioimmunoassay (RIA)
purified antigen labeled with purified antigen labeled with radioisotope competes with radioisotope competes with unlabeled standard for antibody unlabeled standard for antibody bindingbinding
amount of radioactivity associated amount of radioactivity associated with antibody is measuredwith antibody is measured
BibliographyBibliography
Lecture PowerPoints Prescott’s Lecture PowerPoints Prescott’s Principles of Microbiology-Mc Graw Principles of Microbiology-Mc Graw Hill Co.Hill Co.
http://en.wikipedia.org/wiki/Scientific_method
https://files.kennesaw.edu/faculty/jhendrix/bio3340/home.html
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