Cultivation of Viruses

Question 1

Can you streak a small amount of virus on growth medium and see growth after a few hours?

Answer 1

NO -> bacteria easier to grow than viruses

Question 2

Can a virus multiply outside of the host cell?

Answer 2

No they are inert particles outside of the host cell and don’t have the genetic capability to replicate.

Question 3

3 ways to cultivate viruses

Answer 3

1. cell/tissue culture
2. inoculation of embryonated eggs
3. lab animals

Question 4

organ culture

Answer 4

parts of an organ or a whole organ cultured in vitro; new technique with technical difficulties

Question 5

cell culture

Answer 5

removal of cells from an animal or plant and subsequent growth in a favorable artificial environment; grown in suspension or as monolayer

Question 6

suspension culture

Answer 6

cells which do not require attachment for growth or do not attach to the surface of culture vessels

Question 7

monolayer culture

Answer 7

the bottom of the culture vessel is covered by a continuous layer of cells, usually one cell in thickness

Question 8

primary cell culture

Answer 8

growth of cells dissociated directly from parental tissue; cells are morphologically similar to cells of parent tissue

Question 9

advantages of primary cell culture

Answer 9

• best system for propagation and isolation of viruses
• heterogenous -> many cell types
• closest to animal
• used in producing viral vaccines

Question 10

disadvantages of primary cell culture

Answer 10

• difficult to obtain
• short lifespan in culture (5-20 subcultures)
• susceptible to contamination
• may not fully act like parent tissue

Question 11

secondary culture

Answer 11

transfer culture; when a primary culture is sub-cultured, it is known as a secondary culture or cell line

Question 12

subculture

Answer 12

aka passage; transfer of cells from one culture vessel to another

Question 13

What are the 2 types of cell lines?

Answer 13

1. finite/diploid

2. continuous

Question 14

finite cell line

Answer 14

• 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

Question 15

continuous cell lines

Answer 15

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

Question 16

T/F continuous cell lines can be used for vaccine production

Answer 16

F: FDA prohibits

Question 17

morphology of cells in culture (3)

Answer 17

1. fibroblastic
2. epithelial-like
3. lymphoblast-like

Question 18

fibroblastic cells

Answer 18

bipolar or multipolar, elongated shapes, grow attached to a substrate

Question 19

epithelial-like cells

Answer 19

polygonal in shape with more regular dimensions, grow attached to a substrate in discrete patches

Question 20

lymphoblast like cells

Answer 20

spherical in shape and usually grown in suspension without attaching to a surface

Question 21

culture medium

Answer 21

provides all necessary nutrients (amino acids, inorganic salts, vitamins and glucose) required for growth of cells

Question 22

What is the most widely used animal serum supplement?

Answer 22

FBS (fetal bovine serum)

Question 23

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?

Answer 23

serum

Question 24

Commonly used pH indicator?

Answer 24

phenol red

Question 25

T/F It is necessary to use exogenous CO2 when using media buffered with a CO2-bicarbonate based buffer

Answer 25

T: 4-10% CO2 common for most cell culture experiments

Question 26

Why would you use antimicrobial agents in cell culture?

Answer 26

To prevent contamination with bacteria, mycoplasma, yeast, molds

Question 27

Trypsin and collagenase are types of ______.

Answer 27

proteases

*EDTA binds Ca so integrin proteins won’t function properly and cells won’t adhere properly -> released

Question 28

What are ways to dispense cells?

Answer 28

• polystyrene flasks
• polystyrene dishes
• microwell plates
• roller bottles
• leighton tube

Question 29

cytopathic effect (CPE)

Answer 29

damage or morphological changes to host cells during virus invasion

Question 30

Shell Vial technique

Answer 30

• 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

Question 31

co-cultivated cells

Answer 31

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

Question 32

egg candling

Answer 32

tests if eggs can be used for propagating viruses (shows cracks)

Question 33

routes of inoculation in eggs

Answer 33

yolk sac
allantoic cavity
amniotic cavity
chorioallantoic membrane(CAM)

Question 34

evidence of viral growth in eggs

Answer 34

• death of embryo
• paralysis (sluggish movement)
• stunted growth
• urea deposits in mesonephros
• hemorrhage and congestion
• hemagglutins in embryonic fluids
• extracellular membrane lesions

Question 35

lab animal inoculation

Answer 35

• intracerebral

- intraperitoneal