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.
Common blood culture contaminants
Coagulase-negative staph, Bacillus, corynebacteria, propionibacteria
What is the significance of Mycoplasma in the nasal cavity of a cat?
Uncertain - can be isolated from healthy cats and cats with rhinosinusitis
What level of bacterial growth from a cystocentesis sample is significant?
Any level significant, most >10^3 CFU
When is anaerobic urine culture indicated?
If there is evidence of emphysematous cystitis
In an animal with a urinary catheter and suspected UTI; what should be cultured?
Urine (obtained by cysto or freshly placed catheter) - NOT catheter tip (likely contaminated with urethral commensals
What is the structure of mold?
Molds exist as filaments - hyphae
Mass of filaments = mycelium
Granule formed by compacted mat of fungal filaments = actinomycotic mycetoma
What is the structure of yeast?
Single-celled organisms, reproduce by budding
Sometimes buds remain attached and form a chain - pseudohyphae
What is a dimorphic fungi?
Exist as mold in the environment and convert to yeast form in the body
How to fungi reproduce?
By forming spores - sexual or asexual reproduction possible
What are the terms for the a) asexual and b ) sexual stage of a fungus?
a) Anamorph
b) Teleomorph
What are the two types of nucleic detection assays - give an example of each
Probe-based assays - ISH
DNA amplification - PCR
Describe DNA/RNA probes
Short single-stranded nucleic acids, complementary to a specific segment of DNA. Labelled with fluorescent or chemical tag
Describe how PCR works
2 short DNA primers used in conjunction with a thermocycler to amplify DNA segment
What is the CT number of a PCR and how does it relate to the quantity of target DNA present?
The number of cycles that elapse in a RT-PCR before fluorescence appears
Lower the CT number = more target DNA
What is RT-PCR used for?
RNA detection
What is nested PCR? What is an advantage and disadvantage of the technique?
Small amount of amplified PCR product subjected to another set of primers
Increases specificity and greatly increases sensitivity
More affected by contamination
