Lecture 7

Question 1

Why are bacteria used in the lab?

Answer 1

• Useful for studying/using phage

Question 2

Are bacteria easy to culture?

Answer 2

• 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.

Question 3

Are mammalian cells easy to culture?

Answer 3

• 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)

Question 4

monolayer

Answer 4

cells attached to the lower surface of a dish or
flask, as a layer that is one cell thick

Question 5

Can liquid cells be stored indefinitely?

Answer 5

Living cells can be stored indefinitely, frozen in liquid nitrogen if necessary (suspended animation!)

Question 6

What do mammalian cell cultures need?

Answer 6

Glucose, salt, and blood serum

Question 7

What is in blood serum?

Answer 7

Serum contains (~~ mystery components ~~ ..):
– Binding/transport proteins (Transferrin/Fe, albumin) ?
– Growth factors ? Insulin ?
– Lipids/fatty acids ?

Question 8

What else is blood serum used for?

Answer 8

• Viscosity of medium (shear protection of cells)
  – Detoxification

Question 9

What are some potential problems with adding serum?

Answer 9

• Composition is poorly defined
  – Often, batch-to-batch variation (in growth factors, exosomes, etc).
  – Contaminated with infectious agents? (viruses, mycoplasma, prions)
  – Expensive

Question 10

What are the differences in natural versus artificial blood serums?

Answer 10

Chemically-defined serum substitutes generally proprietary, but probably contain:
- Purified proteins (serum albumin, transferrin, insulin, hormones)
- Lipids, salts, amino acids, trace elements

Question 11

Can you see a cell monolayer under a microscope?

Answer 11

yes!

Question 12

What do you do if the cells are confluent?

Answer 12

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).

Question 13

How do you purify a virus?

Answer 13

Centrifugation!

Question 14

What is differential centrifugation?

Answer 14

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

Question 15

Density gradient centrifugation

Answer 15

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.

Question 16

Isopycnic centrifugation

Answer 16

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.

Question 17

Rate zonal centrifugation

Answer 17

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.

Question 18

What do they have in common?

Answer 18

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.

Question 19

What do you do after centrifugation?

Answer 19

Virus harvested from band

Question 20

How do you harvest the virus from the band?

Answer 20

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.

Question 21

How do you harvest the virus from the band?

Answer 21

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.

Question 22

How to measure the virus titer

Answer 22

count virus particles by EM

Question 23

What are potential risks with counting virus particles?

Answer 23

• Viruses may be delicate - may become inactivated during laboratory purification.
• Can be solved by assays that can count infectious particles

Question 24

Gross intracellular changes

Answer 24

• 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)

Question 25

Molecular changes

Answer 25

- 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.).

Question 26

What happens if a single LYTIC virion lands on a monolayer?

Answer 26

A single lytic virus particle landing will infect a single cell and infect outward in almost a ring

Question 27

plaque

Answer 27

large hole in sheet

Question 28

Non-lytic viruses can form plaques through ________

Answer 28

cell migration

Question 29

“Plaquing” a virus

Answer 29

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.

Question 30

Over subsequent 2-3 days plaquing a virus:

Answer 30

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). >>>>>

Question 31

Count of plaques =

Answer 31

count of infectious virus particles that were in the initial inoculum, referred to as “pfu” (plaque-forming units) of virus.

Question 32

if a virus is able to form plaques

Answer 32

you can use plaque Assay to determine virus titer

Question 33

Steps to make titer:

Answer 33

1. Make serial dilutions of the virus 2. Infect a different well of cells from each dilution 3. Incubate wells for 2-3 days 4. Stain cell sheets and count plaques 5. Back-calculate pfu/ml of original inoc.

Question 34

Key points:

Answer 34

- 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.