Pipettes and Tissue Culture

Question 1

What are the 3 types of pipettes?

Answer 1

Pipettes
Pasteur pipettes
Micropipettes

Question 2

Describe a pipette

Answer 2

A plastic of glass tube
Graduated to deliver any amount from 1-25mls
Suction of liquid via bub or pipette aid

Question 3

Describe a Pasteur pipette

Answer 3

Small, tapered glass tube
Used with a bulb
Used to dispense liquid if volume not critical

Question 4

Describe a micropipette

Answer 4

Use to dispense small volumes from 1-1000 microlitres

Used with disposable tips

Question 5

How do you use a micropipette?

Answer 5

Depress the plunger to the first stop
Insert the tip into the liquid
Slowly release the plunger to pull liquid into the tip
Dispense by pushing the plunger to the second stop

Question 6

How should a micropipette be held?

Answer 6

Hold vertically

Question 7

What is molarity?

Answer 7

The measure of the concentration of a solute in solution

Question 8

What is molarity measured in?

Answer 8

Moles

Question 9

What are moles?

Answer 9

The molecular weight of a substance expressed in grams?

Question 10

What is one mole of a substance?

Answer 10

Avogadro’s number of molecules of that substance

Question 11

What does the weight/volume of a solution refer to?

Answer 11

The weight of a solute as a percentage of the volume

Question 12

For aqueous solutions weight/weight is the same as weight/volume
T/F

Answer 12

T

Question 13

When is volume/volume used?

Answer 13

To make a solution of two or more liquids

Question 14

What does a “10X Buffer” mean?

Answer 14

You would need to dilute by 10 to get the 1x working concentration

(ie a final volume of 10ml needs 1ml of 10X buffer)

Question 15

What is the formula for calculating dilution?

Answer 15

C1V1=C2V2

Question 16

What is the goal of a lowry protein assay?

Answer 16

To use known concentrations to generate a standard curve

In order to calculate unknown protein concentrations

Question 17

What is the Lowry protein assay?

Answer 17

Method involving copper bonding to measure protein concentrations through colorimetric techniques

Question 18

Describe what happens in the lowry assay

Answer 18

Divalent copper ions form complexes with peptide bonds in the protein under alkaline condition
It is reduced then to a monovalent ion which reacts with Folin’s reagent along with radical groups of tyrosine, tryptophan and cysteine
This forms an unstable product reduced to molybdenum blue
Spectrophotometry used

Question 19

How should a spectrophotometer be used?

Answer 19

Calibrate machine to appropriate wavelength
Set to zero with a control
Transfer liquid to cuvette (avoid touching the sides)
Place in spec and shut the lid
Read absorbance

Question 20

What is the purpose of tissue culture?

Answer 20

To grow cells and passage them to allow propagation of the cell line

Question 21

How can you continue growth of the cell line in tissue culture?

Answer 21

Provide cells with nutrients and a medium to grow in in vitro
This allows division by mitosis by the cells and the population can continue to grow
Until limited by nutrient depletion at which point they can be passaged

Question 22

How many cell types do tissue culture dishes normally contain?

Answer 22

One

Question 23

What is a “clone” population of cells?

Answer 23

A homogenous population of cells derived from a single parental cell

Question 24

Give examples of applications for cell culture

Answer 24

Chemo-resistance testing
Genetic tests
Receptor type recordings

Question 25

What is passaging?

Answer 25

Removal of medium and transfer of a proportion of the cells to a fresh growth medium

Question 26

Why does passaging allow more growth?

Answer 26

Gives cells more space and fresh medium

Question 27

In which phase do the cells not divide?

Answer 27

Lag phase

Question 28

In which phase do the cells grow exponentially?

Answer 28

Log phase

Question 29

What does the length of the lag phase rely on?

Answer 29

Growth phase of the cell line at the time of subculture and the seeding density

Question 30

What is the role of PBS in tissue culture?

Answer 30

To buffer the solution | Reduce amount of Mg2+ and Ca2+ ions

Question 31

What is trypsin?

Answer 31

A protease which interferes which adhesion proteins

Question 32

What is the role of EDTA in tissue culture?

Answer 32

Reduces Ca2+ and Mg2+ as these normally enhance cell adhesion

Question 33

Why do we want to detach the cells in tissue culture?

Answer 33

They cannot be passaged when stuck to bottom of dish

Question 34

What was in the DMEM?

Answer 34

``` 10% foetal bovine serum Glutamine 1% sodium pyruvate 1% pen/strep Phenyl red dye ```

Question 35

What is the purpose of 10% foetal bovine serum in the DMEM?

Answer 35

Growth factors and attachment proteins

Question 36

What is the purpose of the pen/strep in the DMEM?

Answer 36

To stop contamination

Question 37

What is the purpose of the phenyl red in the DMEM?

Answer 37

Indicator

Question 38

Why is PBS used to wash off the media?

Answer 38

Trypsin is inhibited by Mg, Ca and FBS in the media

Question 39

Which component of the FBS affects the trypsin binding?

Answer 39

Alpha-1-anti-trypsin

Question 40

What is trypsin?

Answer 40

A proteolytic enzyme serine protease

Question 41

What is the role of trypsin?

Answer 41

Breaking bonds to detach cells from surface

Question 42

What was the %CO2 of the incubator ?

Answer 42

5% Diff per cell type - you get a guide with the cells

Question 43

Why should you always passage your cells a couple of times before you do anything with them?

Answer 43

To establish they are healthy and growing ok Establish no issues with freezing Bulk up the cell number (not many are frozen)