Lecture15: Overview of the Clinical Microbiology Laboratory Flashcards Preview

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Flashcards in Lecture15: Overview of the Clinical Microbiology Laboratory Deck (37):
1

When working with specimen 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. 

2

Standard Microbial Practices

  • Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory
  • Eating, drinking, smoking, handling of contact lenses, apply cosmetics, and storing food for human consumption must not be permitted in laboratory
  • Mouth pipetting is prohibited; mechanical pipetting devices must be used
  • Policies for safe handling of sharps (such as needles, scalpels, pipettes, and broken glassware) must be developed and implemented
  • Procedures must be developed and implemented to minimize the creation of splashes and/or aerosols 
  • Work surfaces must be decontaminated with appropriate disinfectant after completion of work and after any spill or splash of potentially infectious material 
  • All cultures, stocks, and toher potentially infectious materials must be decontaminated using an effective method before disposal 
  • A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present
  • Persons must report all injuries incurred in laboratory to the laboratory supervisor as soon as possible 

3

Microbiology Risk Groups 

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4

Specimen collection

  • Numerous methods used; choice of methods depends on specimen
    • Specimen should represent the diseased area
    • quantity should be adequate
    • collection to avoid contamination
    • proper container, promptly sent to laboratory
    • obtain specimen before antimicrobial treatment

5

  • Identification of Microorganisms from Specimen
    • Direct vs. Indiret indentification methods

6

  • Direct identification methods
    • growth and biochemical characteristics 
    • microscopy 
    • molecular methods
    • bacteriophage typing
    • immunologic tests
  • Indirect identification methods
    • serology 
    • immunogluorescence 

7

Example of some bacteria that are not routinely cultured

  • Rickettsias, chlamydiae, and mycoplasmas
    • these are identified with special stains, immunologic tests, or molecular methods such as PCR 

8

Rapid Methods of Identification 

9

Rapid Methods of Identification

  • Manual biochemical systems 
    • e.g., API 20 E system
  • Mechanized/automated systems
  • Immunologic systems 

10

how are speciment fixed for microscopy

  • wet-mounts
  • heat-fixed
  • chemically fixed

11

  • Choice of microscopy depends on possible pathogen
    • ex.?

  • Dark-field microscopy
    • Detection of spirochetes in skin lesions associated with syphilis
  • Fluorescence microscopy
    • Direct microscopic of specimens to detect fungi
  • (note stains that are often used)
    • Gram stain and acid fast fast stain

12

Monoclonal Antibodies (mAB)

  • Produced by hybridoma cells
    • "Immortal" cancerous B cell fused with an antibody-producing normal B cell 
  • Recognize a single epitope 

13

How are monoclonal antibodies (Mabs) produced 

  1. a mouse is injected with a specific antigen that will induce antibodies against that antigen 
  2. The spleen of the mouse is removed and homogenized into a cell suspension. The suspension includes B cells that produce antibodies against the injected antigen
  3. The spleen cells are then mixed with myeloma cells that are capable of continuous growth in culture but have lost the ability to produce antibodies. some of the antibody-producing spleen cells and myeloma cells fuse to form hybrid cells. These hybrid cells are now capable of growing continuously in culture while producing antibodies 
  4. The mixture of cells is placed in a selective medium that allows only hybrid cells to grow
  5. hybrid cells proliferate into clones called hybridomas. The hybridomas are screened for production of the desired antibody
  6. The selected hybridomas are then cultured to produce large quantities of monoclonal antibodies 

14

Chimeric Mabs

  • Genetically modified mice that produce Ab with a human constant region

15

Humanized Mabs

Mabs that are mostly human, except for mouse antigen-binding

16

Fully human antibodies

  • Mabs produced from a human gene on a mouse 

17

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 without changing antibody's ability to bind a specific antigen
  • Can be used as direct fluorescent-antibidoy (FA) technique or indirect fluorescent antibody (IFA) technique assay 

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18

Identification of parasites

  • Identification by microscopic examination of clinical specimens
    • definitive diagnosis obtained by identification and characterization of ova, trophozoites, and cysts in the specimen
  • Histological staining of blood, negative staining of other body fluids, and immunofluorescence staining are routinely used in identifiation of parasites

19

Molecular Genetic Methods of testing

  • Accurate, routine, used in clinical microbiology labs 
    • comparison of proteins
    • nucleic acid-based detection methods
  • Molecular methods widely used
    • Nucleic acid probes; DNA hybridization 
    • PCR and real time PCR
    • Ribotyping (16S rRNA analysis)
    • Multilocus sequence typing (MLST)
    • Genomic fingerprinting
    • Plasmid fingerprinting 

20

Ribotyping

  • Used to identify bacterial genera
  • based on high level of 16S rRNA gene conservation among bacteria
  • rRNA encoding genes or fragments are amplified by PCR
  • The nucleotide sequence of the amplified DNA is determined and compared with those in the National center for Biotechnology (NCBI)

21

Plasmid Fingerprinting

  • Characterizes bacteria based on number of plasmids and their molecular weight

22

Immunolgocial Techniques

  • Detection of antigens or antibodies in specimens 
    • especially useful when cultural methods are unavailable or impractical or antimicrobial therapy has been started
  • Use of immunological systems has many advantages 

23

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24

Clinical Immunology

  • Number, sensitivity, and sepcificity of serological tests increased due to better understading of 
    • immune cell surface antigens (CDs)
    • Lympocyte biology
    • production of monoclonal antibodies
    • development of sensitive antibody-binding reporter systems
  • Test selection and timing of specimen colleciton are essential to the proper interpretation of immunologic tests

25

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
    • e.g. since virulence factor genes often occur in same clone wiht genes for antigenic cell wall material, it is possible to serologically identify pathogen by testing for cell wall antigens 

26

What is a Quellung reaction

Swelling of capsular material following addition of antisera specific to a capsular type 

27

Serological tests

 

  • Direct
    • detect antigens (from patient sample) 
  • Indirect
    • detect antibodies (in patient s serum)
  • Agglutination
    • Particulate antigens
  • Hemagglutination
    • Agglutination of RBCs
  • Comlement fixation
    • Binding of complement to an antigen-antibody complex
    • RBCs are indicator
  • ELISA
    • (enzyme-linked immunosorbent Assay)
    • Peroxidase enzyme is the indicator 
  • Precipitation
    • Soluble Antigens 
  • Fluorescent-antibody technique
    • Antibodies linked to fluorescent dye
  • Neutralization
    • Inactivates toxin or virus 

28

Visible clumps or aggregates of cells or particles 

Agglutinates

29

examples of Agglutination tests

  • Widal test
    • diagnostic for typhoid fever
  • Latex agglutination tests
    • pregnancy test
    • viral hemagglutination
  • Antibody titer measurments

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31

What is an antibody titer

  • Concentrtion of antibodies against a particular antigen 
    • (note that the wider the dot means there is more agglutination and thus more antibodies)

32

Viral Hemagglutination-inhibition

  • Some viruses agglutinate RBCs in viro
  • Abs prevent hemagglutination by binding to viruses 

33

Complement fixation

  • Binding of complement to an antigen-antibody complex
  • Basis of diagnostic tests that determine if antibodies to an antigen are present in patient's serum
  • Very sensitive, measure extremely small amounts of antibody
  • When complement binds to an antigen-antibody complex, it become "fixed" and "used up." 
    • thus a known antigen is mixed with test serum lacking complement. when immune complexes have had time form, complement is added to the mixture. if immune complexes are present, they will fix and consume complement. Afterward, sensitized indicator cells (usually sheep RBCs previously coated with antibodies) are added. If complement is present then indicator cells will be lysed (negative test because antibodies where not for test antigen) 

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34

ELISA (enzyme-linked immunosorbent Assay) 

  • One of the most widely used serological tests
    • direct test can be used to detect antigens in a sample
      • antibody placed in wells of plate (or attached to membrane). Test antigen added. Antibody-enzyme complex added thus you get (Ab-Ag-Ab) or sandwich 
    • indirect test
      • can be used to detect antibodies in a sample
      • antigen in buffer is incubated and absorbed into walls of plate. Free antigen washed away and serum added, and if antibody is present, it binds to the antigen. (can also be done with beads). Then antibody that has been covalently added to enzyme is added. conjugates to antibody on antigen. and visualized with chromogen (which acts on the enzyme portion) 
  • Reaction visualized by addition of chromogen 
  •  

35

Immunoprecipitation

  • Detects soluble Ag reacting with Ab (precipitins) 
  • Binding of Ab to Ag forms lattice that precipitates
  • Lattice formation occurs only in optimal ration of Ag to Ab

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36

Flow cytometry

  • Detection based on cytometric parameters or by use of fluorochromes 
    • fluorochromes often bind to antibodies or oligonucleotides
  • Flow cytometer
    • Forces suspension of cells through laser beam and measures amount of light scattering of fluorescence
    • can detect heterogeneous microbial populations with different responses to antimicrobial treatments

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