Cell Culturing Flashcards
Why is propagating viruses essential
To apply Koch’s postulates to include viruses. Viruses require a host to replicate in. Often use of chicken egg. Now rare due to welfare and costs
Benefits of propagating viruses in chicken eggs
Specific pathogen free, can be used to support numerous mammalian and avian viruses, able to inoculate the membrane which best supports the specific virus and can grow high titre stocks of virus
What are primary cell lines
- cells directly isolated from organism tissue
- normal chromosome number, morphology and differentiation
- require higher concentration of growth factors
- includes non-diving cells (neurons)
- limited number of cell division
- similar to the in vivo state
What are continuous cell lines
- divide indefinitely
- require fewer growth factors for viability
- can be derived from cancers or induced from primary cell lines by viral, oncogene transformation, mutagenesis
- stores in liquid nitrogen with cryoprotectants
- HeLa cell line most used
What is required for culturing cells in tissue culture medium
- isotonic salts
- sodium bicarbonate buffer
- PH indicator (phenol red)
Essential amino acids
Vitamins
Glucose (energy source)
Foetal bovine serum
Non essential:
- feeder layer and antibiotics
What are the Incubator, sterile and environmental requirements for culturing cells
Incubator: controllled 37º temp, humidified, 5% Co2 to maintain correct pH with buffer
Sterile: disposable equipment, UV irradiation, 70% ethanol swab, sterile hood
Safe working environment: gloves, biological safety cabinet, negative air flow
What is passaging cells and why is passaging cells essential
Passaging cells is taking some cells from a cell culture and diluting them in a fresh medium.
It is important to prevent overcrowding and keep cells healthy and viable
Describe quality control and regulation of cells and what are risk groups
Quality: cell lines can be authenticated and obtained from reliable sources, must be mycoplasma free (parasitic bacteria lacking a cell wall which changes growth rate, morphology, chromosome number)
Control: Physical containment according to PC2, PC3 and PC4 guidelines, SSBA and most exotic animal viruses can only be handled in approved facilities
What is a cytopathic effect
The morphological changes as a result of viral infections. Can indicate virus classification. Also dependent on how cells are imaged, and stained, cell type, the virus and mutations
How to detect infected cells with viruses that are non-cytopathic
Immunofluorescence, immunohistochemistry, PCR (DNA virus) or RT-PCR (RNA or DNA virus)
Electron microscopy
What are plaque assays and how are they formed
Used to quantify infectious virus in a stock, measured as PFU/mL
- dishes with confluent layer of cells
- overlay to stop virus infecting all cells on dish
- plaque formed by many rounds of replication
- based on dilution, estimate concentration of OG stock
- Only works on viruses with cytopathic effects
Stain the cells
What is TCID50
Endpoint dilution assay referring to volume of virus that will infect 50% of cells in wells
