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28 Cards in this Set
- Front
- Back
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What are the 3 ways to identify a disease?
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How to Identify the Disease
-Direct testing (microscopic, immunological genetic methods) -Time required: from few minutes… -Cultivation of microorganisms -Time required: few days to few weeks (mycobacterium tuberculosis) -Based on symptoms (athlete’s foot) |
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What are the 3 ways to identify unknown bacteria?
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Phenotypic Methods:
-Macroscopic morphology -Microscopic Morphology -Physiological/ Biochemical Characteristics Genotypic Methods: -Culturing not necessary -Results obtained quickly Immunological Methods: -Looking for the specific antibody in patient’s blood |
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Describe specimen collection.
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-Can be collected by a member of a clinical team or b a patient
-Aseptic procedures should be followed -Avoid taking samples of normal biota (e.g. saliva when taking throat swab *can lead to misleading results and conclusions*) -Samples should be promptly transported to the lab |
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What is immediate direct examination of specimen? What type of method is it?
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Phenotypic Method
-Direct microscopic observation: -Fresh -Stained (Gram staining, acid fast staining) -Direct fluorescence antibody test: -Antibody labeled with a fluorescent dye |
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How is a specimen cultivated?
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-A dominant organism has to be isolated into a pure culture
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When a specimen is cultivated how is the isolated microorganism characterized?
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The isolated microorganism is characterized by
-Microscopic morphology -Staining reaction (G+ or G-) -Cultural appearance -Motility -Oxygen requirements -Biochemical characteristics |
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Describe biochemical tests.
(Phenotypic method) |
Biochemical Tests
-Enzymatic activity (fermentation of glucose, lactose…, production H@S, presence of catalase, use of citric acid as sole carbon source...) -Differentiate among the bacterial species -Rapid biochemical systems for identification of medically important bacteria have developed -Disadvantage of the method: mutation can result in strains with different characteristics |
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What is the disadvantage of biochemical tests?
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mutation can result in strains with different characteristics
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What is phage typing?
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Phage Typing:
-Identifies with phage affects specific bacteria strain -Can distinguish strains within one species -Enables tracking the source of the outbreak |
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Hybridization with a probe is what type of method?
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Genotypic method
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What is hybridization with a probe?
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Hybridization with a probe
-The method used to detect specific nucleotide sequence in an unknown sample by using a gene probe -Gene probe is a short segments of DNA of a known sequence -A probe carries a radioactive label |
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Describe DNA typing for restriction fragment length polymorphism (RFLP)
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DNA typing for restriction fragment length polymorphism (RFLP)
-DNA from M. Tuberculosis from 17 patients is cut with a restriction enzyme -By comparing the pattern of DNA fragments it is possible to determine which patients were infected with the same bacterial strain |
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What is Diagnostic immunology?
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Diagnostic Immunology
-Use of immunological methods to identify pathogenic microbes -Main characteristics of this method – Specificity – Specific antibody will react only with a specific antigen -Either antigen or antibody can be used -Reactions are visible with a naked eye |
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What are the types of immunologic tests?
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-Precipitation tests
-Agglutination tests -Immunodiffusion -Complement fixation -Fluorescence- antibody technique -Immune assay |
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What is an Agglutination Reaction?
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-Particulate antigens (microbial cells) react with antibodies forming visible aggregates
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What happens during a precipitation reaction?
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Precipitation Reaction
-Soluble antigens react with antibodies forming large molecular aggregates – visible precipitate |
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What are the two techniques for precipitation tests?
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-There are two techniques:
-Precipitation in liquid -Precipitation in agar |
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Describe precipitation in agar.
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-Precipitation in agar “double diffusion test”
-The wells in the agar are filled with test antigens and various antibodies -They diffuse in a gar -The positive reaction is the line- they precipitate between two wells -Used for the detection of antibodies in syphilis |
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How is Quantification of antibodies in the serum done?
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Quantification of antibodies in the serum
-Determining the titer -Patient’s serum is serially diluted -Mixed with antigen -Incubate and centrifuge -Tubes inspected for clumps -Titer is the dilution of the last tube that shows agglutination -A change of the titer over time indicates whether the disease is subsiding or advancing |
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What are the complements needed for the Complement fixation test?
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-Complement needed for the test:
-Antibody -Antigen -Complement -Sensitized sheep red blood cells |
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Describe the 1st stage of Complement fixation test
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1st Stage
-test antigen allowed to react with the known antibody in the absence of complement -Complement added -If antibody and antigen made a complex, they will fix the complement to them |
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Describe the 2nd stage of Complement fixation test
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2nd Stage of testing
-Sheep RBC containing cytolysins are mixed with the content of the stage 1 tube -Hemolysis (clearing of the solution is observed -If the complement is fixed to antigen –antibody complex – no hemolysis will occur – the reaction is positive |
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Describe the procedure for neutralization reaction test.
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-Procedure:
-Patient’s serum + erythrocytes + a virus suspension -If the serum contains the antibodies against a specific virus hemagglutination will not occur |
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What is neutralization reaction?
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-Clumping of red blood cells
-Some viruses (measles and influenza) can clump the red blood cells -Not an antigen-antibody reaction -Clumping of red blood cells -Some viruses (measles and influenza) can clump the red blood cells -Not an antigen-antibody reaction Antibodies can block exotoxins or a virus -This is used for diagnosis of influenza, measles, mumps, etc. by viral hemagglutination inhibition test |
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Describe Immunofluorescence Testing
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Direct Fluorescent-antibody test
-Antibodies are tagged with the fluorescent antibody -Sample is flooded with fluorescently labeled antibody -If the tested sample has receptors for the particular antibody they will conjugate -Those cells will fluroresce when illuminated with UV light (Fluorescent microscopy). |
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Describe Enzyme-linked immunosorbent assay testing.
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Enzyme-linked immunosorbent assay (ELISA)
-A known antigen is absorbed onto a surface of the well -An unknown serum is added -Well is rinsed -An antibody with an attached enzyme that can react with the unknown test antibody is added -Well is rinsed -The substrate for the enzyme is placed in the well -If the enzyme-linked antibody recognized the unknown antibody – the color will develop -The intensity of the color is proportional to the amount of the unknown antibody |
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What does ELISA detect?
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-Detects antibodies
-Detects antigens (drugs) |
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What are the uses for ELISA?
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Indirect ELISA is used for screening for the antibodies to:
-HIV -Hepatitis A and C -Cholera -Heliobacter (gastric ulcers) Sandwich ELIS is used for detection of: -Hantavirus -Rubella Virus -Toxoplasma (protozoan) |
