Virus cultivation and purification Flashcards
Most viruses can be grown in
- cultured cells
- embryonated hen’s egg
- laboratory animals
Cell culture
involves the growth of dispersed animal cells in vitro, either as cells in suspension or as a monolayer on a solid surface
primary cell cultures
- cultures established from tissue taken directly from humans or animals
- contain several differentiated cell types, which is why they are the best cell culture systems
- used in producing viral vaccines
Secondary cultures
subcultures from primary cultures
serially propagated cell cultures (cell lines)
- diploid cell lines
- consist of a homogenous population of a single cell type derived from human embryos or subcultures of a primary culture
continuous (Heteroploid) cell lines
- cells are single type that are capable of indefinite propagation in vitro
- derived from cancer cells
- their use is prohibited in vaccine production
Trypsinization
the organ/tissue is minced and treated with a protease( trypsin or collagenase) to separate the cells
Growth medium
- a chemically defined medium containing all the nutrients required for cell growth, including amino acids, vitamins, inorganic salts and other growth factors such as glucose
- serum 5-10%
Maintenance medium
- contains 1-2% serum
- used when cell growth is complete
pH indicator
- phenol red
- most media are buffered to pH 7.4
antimicrobial agents
penicillin, streptomycin, gentamicin, mycostatin, and kanamycin
Shell vial (1-dram) cell culture
- a small borosilicate glass vial containing a coverslip. the monolayer is grown on the coverslip. it is ideal for centrifuge- enhanced virus inoculation of the monolayer
Co-cultivated cells
-this technique involves combinations of different cell types grown together as a single monolayer
yolk sac
- 5-7 day old embryo
- 22 gauge, 1 1/ in length needle
Chorioallantoic membrane
- 9-12 day old embryo
- 26-28 gauge needle
Amniotic route and Allantoic route
- 7-13 day old embryo
Intravenous route
- 10- 12 day old embryos
Some responses which may be noticed following inoculation of the embryonating egg are:
- stunted growth
- urate deposits in mesonephros
- hemorrhage and congestion
- sluggish movement
- death of embryo
- lesions of the extracellular membranes
- hemaggulutinins in embryonic fluids
high-speed equilibrium centrifugation
-used to obtain highly purified and con concentrated preparations of viruses
buoyant density
density at which a virus neither sinks nor floats when suspended in a density gradient
