Cultivation of Viruses Flashcards
is it easier to grow bacteria or viruses
bacteria
ways to grow/cultivate viruses
cell/tissue culture
inoculation in embryonated eggs
laboratory animals
organ culture
parts of an organ or a whole organ are cultured in-vitro
cell culture
growth of cells in artificial media outside host
suspension culture
cells which do not require attachment for growth or do not attach to the surface of the culture vessels
monolayer culture
bottom of the culture vessel is covered with a continuous layer of cells, usually one cell thick.
primary cell culture
growth of cells dissociated directly from the parental tissue of animal/human origin. cells share the same chromosomes as the original tissue. Heterogeneous. short life span. closest to animal cells, used in producing viral vaccines
secondary cell culture & two types
when a primary culture is sub-cultured (transferred to another culture vessel). Grow quickly. Two types- finite and continuous
finite/diploid cell lines
limited life span and a limited number of cell generations. can sub-culture up to 100x before cells die. Growth is slow (doubling time 24-96hrs)
continuous/immortal/heteroploid cell lines
divide indefinitively. immortal cells lines. subcultures after subcultures and still maintained. Growth is rapid (doubling time is 12-24hrs). Cannot use for vaccination.
fibroblastic morphology
cells are bipolar or multipolar, have elongated shapes, and growth attached to a substrate
epithelial-like morphology
cells are polygonal in shape with more regular dimensions, and growth attached to a substrate in discrete patches. clumps
lymphoblast-like morphology
cells are spherical in shape and usually grown in suspension without attaching to a surface. detached living in suspension
cell culture media
provides all the necessary nutrients (AAs, inorganic salts, vitamins, and glucose) required for the growth of cells
serum in culture media
vital source of adhesion factors, attachment and spreading factors. Most used- fetal bovine serum (FBS)
phenol red
pH indicator, detects contamination via a pH change. metabolic waste can also lead to change in pH. Changes color when pH decreases (acidity)
antimicrobial agents in media
to prevent contamination wit bacteria, mycoplasma, yeast, molds, etc.
human and mammals optimal cell line growth temperature
36-37ºC
insect optimal cell line growth temperature
27ºC
avian optimal cell line growth temperature
38.5ºC
cold-blooded animals optimal cell line growth temperature
15-26ºC
shell vial technique
inoculate with specimen, centrifuge to enhance infection monolayer, incubate at 35ºC for 1-5d, stain with anti-viral fluorescent monoclonal antibodies, mount coverslip on slide, read with fluorescent microscope
cultivation of viruses in eggs
specific pathogen free eggs, egg candler, betadine, sterile swab, drill, microdrill bits
infertile egg
cannot be used in viral growth
fertile egg
can be used in viral growth
yolk sac inoculation
place inoculum below the embryo and within the yolk material
allantoic cavity inoculation
inoculate specimen into the allantoic cavity
amniotic cavity inoculation
into amniotic cavity
chorioallantoic membrane inoculation (CAM)
drill 2 holes, move air sac to the side of the egg, inoculate on the dropped CAM
evidence of virus growth inside egg
embryo death, paralysis, stunted growth, urate deposits in the mesonephros, hemorrhage, hemagglutins in embryonic fluids, extracellular membrane lesions
pocks
necrotic lesions due to viral replication
pocks assay determines…
number of pocks determine viral infection
ultracentrifugation
gravitational force of 200,000 x 150.000 RPM. efficiently sediment even the smallest viruses
rate-zonal centrifugation
sample is layered. density gradient. bands are formed depending of substances masses. light particles settle slow, heavy particles settle quick
isopychic centrifugation/buoyant or equillibrium separation
particles will never sediment to the bottom of the tube, state with a uniform mixture. use cesium chloride as a suitable density gradient medium
buoyant density
if the object has exactly the same density as the fluid, then its buoyancy equals its weight. it will remain submerged in the fluid, but it will neither sink nor float
isopycnic point
the point where the buoyant density of a particle equals that of the surrounding density gradient medium
Binding of the viruses on ion exchange membranes depends on……….
the charge distribution on the virus
