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