Foundations in Microbiology Sixth Edition

Foundations in Microbiology Sixth Edition

Lecture PowerPoint to accompany Foundations in Microbiology Seventh Edition Talaro

Chapter 17 Diagnosing Infections Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 17.1 Survey of Microbial Disease Methods of identifying unknown microbes

fall into three categories: 1. Phenotypic observable microscopic and macroscopic characteristics 2. Genotypic genetic make up 3. Immunological serology; antibodyantigen reactions Phenotypic Methods

Microscopic morphology fresh or stained microorganisms from specimen; shape, size, stain reaction, cell structures Macroscopic morphology colony appearance; texture, size, shape, pigment, growth requirements Physiological/biochemical characteristics

detection of presence or absence of particular enzymes or metabolic pathways Chemical analysis analyze specific chemical composition; cell wall peptides, cell membrane lipids Genotypic Methods

Assess genetic make-up Culture is not necessary Precise, automated methods, quick results Immunological Methods Specific antibodies are used to detect antigens

Easier than testing for the microbe itself 17.2 Specimen Collection and Laboratory Methods Sampling body sites or fluids for suspected infectious agent Results depend on specimen collection,

handling, transport, and storage Aseptic procedures should be used Figure 17.1 Sampling sites Overview of Laboratory Techniques

Routes taken in specimen analysis Direct tests (microscopic, immunologic, or genetic) Cultivation, isolation, and identification (general and specific tests) Two categories of results

Presumptive Confirmatory Figure 17.2 Scheme of specimen isolation and identification 17.3 Phenotypic Methods

Immediate direct examination Microscopic differential and special stains Gram, DFA, direct antigen testing Phenotypic Methods Cultivation of Specimen Colony appearance, growth requirements,

appropriate media Biochemical testing Physiological reactions to nutrients as evidence of the absence or presence of enzymes

Figure 17.4 Rapid tests Figure 17.5 Phenotypic Methods Miscellaneous tests Phage typing

Animal inoculation Antimicrobial sensitivity Important to consider whether microbe recovered and identified is actually causing the disease or simply normal flora

17.4 Genotypic Methods DNA analysis Hybridization Probes complementary to the specific sequences of a particular microbe PCR

DNA and RNA analysis Ribosomal RNA Comparison of the sequence of nitrogen bases in ribosomal RNA 17.5 Immunological Methods Serology in vitro diagnostic testing of

serum Antibodies have extreme specificity for antigens Visible reactions include precipitates, color changes, or the release of radioactivity Tests can be used to identify and to determine the amount of antibody in serum

titer Figure 17.7 Basic principles of serological testing using antibodies and antigens Figure 17.8 Specificity and sensitivity in immune testing

Agglutination and Precipitation Reactions Agglutination testing antibody cross links whole-cell antigens, forming complexes that settle out and form visible clumps Blood typing, some bacterial and viral diseases

Precipitation tests soluble antigen is made insoluble by an antibody VDRL Most precipitation reactions are carried out in agar gels Figure 17.9 Agglutination and precipitation reactions

Insert figure 17.10 Cellular\molecular view Figure 17.10 Precipitation reactions Figure 17.11 Immunoelectrophoresis of normal human serum

The Western Blot for Detecting Proteins Electrophoretic separation of proteins, followed by an immunoassay to detect these proteins Highly specific and sensitive Sites of specific antibody binding will appear as

a pattern of bands Second test used to verify HIV status Figure 17.12 The Western blot procedure Complement Fixation Lysin mediated hemolysis

Test uses four components Antigen, antibody, complement, and sensitized sheep RBCs Steps of test 1. Test antigen reacts with test antibody 2. Contents of tube from (1.) are mixed with sheep

RBCs Complement used up in first stage, no hemolysis Unfixed complement, hemolysis

Figure 17.13 Complement fixation test Fluorescent Antibody and Immunofluorescent Testing Fluorescent antibody Monoclonal antibody labeled by a fluorescent dye

Two ways FABs are used Direct testing Indirect testing Figure 17.14 (a, b)

Figure 17.14 (c) Immunoassays Extremely sensitive to detect trace antigens and antibodies Radioimmunoassay (RIA) antigens or antibodies labeled with radioactive isotopes

Enzyme-linked immunosorbent assay (ELISA) enzyme-antibody complex produces a colored product when an enzyme-substrate reaction occurs Indirect Capture

Figure 17.15 Methods of ELISA testing Tests that Differentiate T Cells and B Cells Rosette formation Mix T cells with sheep red blood cells

Fluorescent techniques Differentiates T and B cells and subgroups them In vivo Testing Antigens are introduced directly into the body to determine the presence or

absence of antibodies Tuberculin skin test, allergy testing A Viral Example Viruses present special difficulties because they are not cells Viruses are labor intensive to culture in the

laboratory Figure 17.17 Diagnosing viral infections

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