Lecture 7 Flashcards
Why are bacteria used in the lab?
- Useful for studying/using phage
Are bacteria easy to culture?
- Bacteria are not fussy eaters: Will grow easily, to high density, in flasks or on agar plates containing ill-defined rich broth, or minimal media w/ maybe no more than inorganic salts & glucose.
Are mammalian cells easy to culture?
- Mammalian cells are fussy eaters.
Covered in chemically-defined nutrient-rich medium + blood serum - At 37 ºC
- In defined atmosphere
- Bacteria must be kept out !! (aseptic technique) (bacteria grow faster and would take over)
monolayer
cells attached to the lower surface of a dish or
flask, as a layer that is one cell thick
Can liquid cells be stored indefinitely?
Living cells can be stored indefinitely, frozen in liquid nitrogen if necessary (suspended animation!)
What do mammalian cell cultures need?
Glucose, salt, and blood serum
What is in blood serum?
Serum contains (~~ mystery components ~~ ..):
– Binding/transport proteins (Transferrin/Fe, albumin) ?
– Growth factors ? Insulin ?
– Lipids/fatty acids ?
What else is blood serum used for?
- Viscosity of medium (shear protection of cells)
– Detoxification
What are some potential problems with adding serum?
- Composition is poorly defined
– Often, batch-to-batch variation (in growth factors, exosomes, etc).
– Contaminated with infectious agents? (viruses, mycoplasma, prions)
– Expensive
What are the differences in natural versus artificial blood serums?
Chemically-defined serum substitutes generally proprietary, but probably contain:
- Purified proteins (serum albumin, transferrin, insulin, hormones)
- Lipids, salts, amino acids, trace elements
Can you see a cell monolayer under a microscope?
yes!
What do you do if the cells are confluent?
They stopped dividing (contact inhibition of growth). Therefore, “passage”
them (add trypsin protease to detach them from plastic surface -> dilute & re-
attach just a portion of them).
How do you purify a virus?
Centrifugation!
What is differential centrifugation?
Based on the low mass of the virion (in relation to
nuclei, mitochondria, microsomes etc.)
Basically doing centrifugation a ton of times at different speeds
Density gradient centrifugation
The semi-pure virus is layered on top of a gradient of continuously increasing density (eg. of
sucrose), -> the gradient is centrifuged. Everything will travel at a rate dictated by its mass.
Isopycnic centrifugation
If gradient density encompasses density of virus, then virus will stop moving when it reaches its OWN buoyant density, even if force continues to be applied. It may take a while to reach this equilibrium, and may require gradient of high density, toxic material (CsCl) instead of sucrose.
Rate zonal centrifugation
The whole gradient is less dense than the virus but different-density particles will still move at different rates and separate. NOTE: To recover pure virus, the centrifuge must be stopped before the virus eventually reaches the bottom/piles up with everything that arrived before it.
What do they have in common?
During either process, the virus particles are initially layered on the top, of the gradient. During centrifugation they move downwards as a flat layer (“band”), arriving somewhere in the gradient.
What do you do after centrifugation?
Virus harvested from band
How do you harvest the virus from the band?
puncturing side of tube or puncturing bottom and letting garbage beneath the virus drip through first (discard) followed by the virus band which can be isolated in just one collection tube.
How do you harvest the virus from the band?
puncturing side of tube or puncturing bottom and letting garbage beneath the virus drip through first (discard) followed by the virus band which can be isolated in just one collection tube.
How to measure the virus titer
count virus particles by EM
What are potential risks with counting virus particles?
- Viruses may be delicate - may become inactivated during laboratory purification.
- Can be solved by assays that can count infectious particles
Gross intracellular changes
- Gross morphological changes (actin filament breakdown; apoptosis: A->B, left picture, below)
- “Inclusion bodies” (solid bodies inside the cell containing virus) e.g. virus assembly factories
- Cell surface changes (cell-cell fusion into giant syncytia – right picture, below)
Molecular changes
- Shutdown of host cell mRNA synthesis/protein synthesis, so that only virus proteins made
- Bypass natural pauses in cell cycle (-> proliferation of virus-infected cells)
- Fight with host defenses (interferon response, MHC, etc.).
What happens if a single LYTIC virion lands on a monolayer?
A single lytic virus particle landing will infect a single cell and infect outward in almost a ring
plaque
large hole in sheet
Non-lytic viruses can form plaques through ________
cell migration
“Plaquing” a virus
Add low number of
infectious virus particles (< 100) to a cell
monoloyer (‘cell sheet’, containing thousands
of cells) then wash sheet after 1 hr to
remove non-attached virus.
Over subsequent 2-3 days plaquing a virus:
Every one initialvirion that entered a cell during the 1 hr incubation -> one necrotic patch (‘plaque’)(assuming progeny virus from first cell did not float away, but instead infected neighboring cells only).»_space;»>
Count of plaques =
count of infectious virus particles that were in the initial inoculum, referred to as “pfu” (plaque-forming units) of virus.
if a virus is able to form plaques
you can use plaque Assay to determine virus titer
Steps to make titer:
- Make serial dilutions of the virus
- Infect a different well of cells from each dilution
- Incubate wells for 2-3 days
- Stain cell sheets and count plaques
- Back-calculate pfu/ml of original inoc.
Key points:
- Bacteria are not fussy eaters (salt & sugar may be OK).
- Mammalian cells tend to be fussy eaters: Require more complex media, serum, defined atmosphere.
- Mammalian cells grown in lab as “monolayer” attached to lower surface of plastic dish
- Stop growing upon confluency
- Mammalian cells in lab come from animals (initially called “primary” cells): Digest from animal tissue and seed the lab culture. Many viruses can only be grown successfully on primary cells
- BUT primaries soon die out unless you are lucky and they undergo “immortalization” (ie get cancer = become tumor cells)
- However, the best immortal cells used in culture may have come originally from a tumor in the original animal
- The most aggressively immortal cells in lab culture are easy to grow, but really messed up inside (“de-differentiated”), and may not support virus replication, and may not attach to culture dish.
