Section 1 Flashcards
When is cell culture indicated
Detection of organisms dependent on living host cells for replication
What are the disadvantages of cell culture?
Slow
Costly
Technically difficult to perform and interpret results
What are the main applications of cell culture?
Discovery of new pathogens
Pathogen identification when results of molecular testing/serology equivocal
Research
Vaccination
Establishment of efficacy of novel antimicrobial drugs
What is the significance of timing of collection of samples for cell culture?
Must be collected early (within first week), when viral shedding occurring
What are the different cell lines for cell culture? When might one be preferred?
Primary - directly derived from animal
Immortalised/continuous cell line - derived from cancer cells or from introduction of cellular mutations
Primary are needed for isolation of some viruses
List examples of immunoassays
IFA (direct/indirect), IHC, ELISA, Western immunoblotting, agglutination, gel immunodiffusion, serum neutralisation
Describe direct IFA
Used to detect antigen
Uses antibody tagged with fluorescent label, or an enzyme
What is IHC/ICC?
ICC - antibody vs antigen tagged with enzyme, substrate added, enzyme causes substrate to change into insoluble precipitate
IHC - same method applied to tissue
How does indirect IFA differ from direct IFA
Normally (not always) used to detect antibody
When detecting antigen more sensitive than direct
Patient serum diluted and reacted with known antigen on glass slide. Second antibody designed to react to dog/cat antibody added, conjugated with marker
Describe ELISA testing
Can detect antigen or antibody
Antigen detection - antigen immobilised in wells of plate (direct binding or bound using capture antibody - sandwich ELISA). Antigen detected using antibody bound to enzyme.
Describe Western immunoblotting
Used for antibody detection
After physical/chemical disruption of an organism, proteins separated based on molecular mass using gel electrophoresis. Protein bands then blotted onto a membrane using an electrical current.
Membrane cut into vertical strips, if antibodies in serum to antigens in organism, antibodies bind to antigens in the strip. Visualised using immunoperoxidase reaction
Describe agglutination testing
When might a false negative be seen
Detects antibody or antigen
Serial dilutions of serum tested for ability to cause or inhibit agglutination
When excess antibody present (prozone effect)
How should anaerobes be sample for culture and how should samples be stored?
Aspirates/tissue preferred, not refrigerated
Which organisms are detected with acid-fast stains?
Give an example of a acid fast stain
Nocardia, Mycobacterium
Ziehl-Neelsen, Fite’s, Kinyoun
What are the advantages of dark field and phase contact microscopy?
Allow visualisation of organism without fixation or staining, allows motility to be assessed.