Cultivation of Viruses Flashcards
Can you streak a small amount of virus on growth medium and see growth after a few hours?
NO -> bacteria easier to grow than viruses
Can a virus multiply outside of the host cell?
No they are inert particles outside of the host cell and don’t have the genetic capability to replicate.
3 ways to cultivate viruses
- cell/tissue culture
- inoculation of embryonated eggs
- lab animals
organ culture
parts of an organ or a whole organ cultured in vitro; new technique with technical difficulties
cell culture
removal of cells from an animal or plant and subsequent growth in a favorable artificial environment; grown in suspension or as monolayer
suspension culture
cells which do not require attachment for growth or do not attach to the surface of culture vessels
monolayer culture
the bottom of the culture vessel is covered by a continuous layer of cells, usually one cell in thickness
primary cell culture
growth of cells dissociated directly from parental tissue; cells are morphologically similar to cells of parent tissue
advantages of primary cell culture
- best system for propagation and isolation of viruses
- heterogenous -> many cell types
- closest to animal
- used in producing viral vaccines
disadvantages of primary cell culture
- difficult to obtain
- short lifespan in culture (5-20 subcultures)
- susceptible to contamination
- may not fully act like parent tissue
secondary culture
transfer culture; when a primary culture is sub-cultured, it is known as a secondary culture or cell line
subculture
aka passage; transfer of cells from one culture vessel to another
What are the 2 types of cell lines?
- finite/diploid
2. continuous
finite cell line
- limited life span and # of cell generations
- population of a single cell type
- derived mainly from embryos or secondary cell cultures
- have contact inhibition, anchorage and density limitation
- slow growth rate
- easy to use than continuous cell lines ‘
ex. WI-38
continuous cell lines
aka immortal/heteroploid
- most homogeneous
- derived from cancer cells
- abnormal morphology and chromosome #
- absence of contact inhibition and anchorage dependence
- rapid growth rate
- hassle free to use
- ex. HeLa cells
T/F continuous cell lines can be used for vaccine production
F: FDA prohibits
morphology of cells in culture (3)
- fibroblastic
- epithelial-like
- lymphoblast-like
fibroblastic cells
bipolar or multipolar, elongated shapes, grow attached to a substrate
epithelial-like cells
polygonal in shape with more regular dimensions, grow attached to a substrate in discrete patches
lymphoblast like cells
spherical in shape and usually grown in suspension without attaching to a surface
culture medium
provides all necessary nutrients (amino acids, inorganic salts, vitamins and glucose) required for growth of cells
What is the most widely used animal serum supplement?
FBS (fetal bovine serum)
What is added to culture media as a source of adhesion factors, attachment and spreading factors, low molecular weight nutrients, and hormones and growth factors?
serum
Commonly used pH indicator?
phenol red
T/F It is necessary to use exogenous CO2 when using media buffered with a CO2-bicarbonate based buffer
T: 4-10% CO2 common for most cell culture experiments
Why would you use antimicrobial agents in cell culture?
To prevent contamination with bacteria, mycoplasma, yeast, molds
Trypsin and collagenase are types of ______.
proteases
*EDTA binds Ca so integrin proteins won’t function properly and cells won’t adhere properly -> released
What are ways to dispense cells?
- polystyrene flasks
- polystyrene dishes
- microwell plates
- roller bottles
- leighton tube
cytopathic effect (CPE)
damage or morphological changes to host cells during virus invasion
Shell Vial technique
- inoculate vial with specimen
- coverslip with tissue culture monolayer in shell vial
- centrifuge to enhance infection of monolayer
- incubate at 35C for 1-5 days
- stain with anti-viral fluorescent monoclonal antibodies
- mount coverslip on slide and read with fluorescent microscop or check cytopathic effect on coverslip
co-cultivated cells
single monolayer consisting of multiple different cell types
ideal for isolation of multiple viruses
viral antigens detected using a pool of fluorescein-labeled monoclonal antibodies
*R-mix
egg candling
tests if eggs can be used for propagating viruses (shows cracks)
routes of inoculation in eggs
yolk sac
allantoic cavity
amniotic cavity
chorioallantoic membrane(CAM)
evidence of viral growth in eggs
- death of embryo
- paralysis (sluggish movement)
- stunted growth
- urea deposits in mesonephros
- hemorrhage and congestion
- hemagglutins in embryonic fluids
- extracellular membrane lesions
lab animal inoculation
- intracerebral
- intraperitoneal
