Lecture15: Overview of the Clinical Microbiology Laboratory Flashcards
1
Q
When working with specimen specimen should
A
- 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
Q
Standard Microbial Practices
A
- 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
Q
Microbiology Risk Groups
A
4
Q
Specimen collection
A
- 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
Q
- Identification of Microorganisms from Specimen
- Direct vs. Indiret indentification methods
A
6
Q
- Direct identification methods
- growth and biochemical characteristics
- microscopy
- molecular methods
- bacteriophage typing
- immunologic tests
- Indirect identification methods
- serology
- immunogluorescence
A
7
Q
Example of some bacteria that are not routinely cultured
A
- Rickettsias, chlamydiae, and mycoplasmas
- these are identified with special stains, immunologic tests, or molecular methods such as PCR
8
Q
Rapid Methods of Identification
A
9
Q
Rapid Methods of Identification
A
- Manual biochemical systems
- e.g., API 20 E system
- Mechanized/automated systems
- Immunologic systems
10
Q
how are speciment fixed for microscopy
A
- wet-mounts
- heat-fixed
- chemically fixed
11
Q
- Choice of microscopy depends on possible pathogen
- ex.?
A
- 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
Q
Monoclonal Antibodies (mAB)
A
- Produced by hybridoma cells
- “Immortal” cancerous B cell fused with an antibody-producing normal B cell
- Recognize a single epitope
13
Q
How are monoclonal antibodies (Mabs) produced
A
- a mouse is injected with a specific antigen that will induce antibodies against that antigen
- 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
- 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
- The mixture of cells is placed in a selective medium that allows only hybrid cells to grow
- hybrid cells proliferate into clones called hybridomas. The hybridomas are screened for production of the desired antibody
- The selected hybridomas are then cultured to produce large quantities of monoclonal antibodies
14
Q
Chimeric Mabs
A
- Genetically modified mice that produce Ab with a human constant region
15
Q
Humanized Mabs
A
Mabs that are mostly human, except for mouse antigen-binding
17
Q
Immunofluorescence
A
- 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
23
Q
A
30
Q
A
33
Q
Complement fixation
A
- 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)
35
Q
Immunoprecipitation
A
- 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
37
Q
A
