Unit 1: KA1 - Lab Techniques

Question 1

Define the term Hazard.

Answer 1

A source of potential harm

Question 2

Define the term Risk.

Answer 2

The likelihood of a hazard causing significant harm.

Question 3

What 3 things must a risk assessment include?

Answer 3

• Identification of risk
• Likelihood it occurs
• control measures to minimise risk

Question 4

Name 3 appropriate measures to minimise risk

Answer 4

• appropriate handling techniques
• PPE
• Aseptic techniques to culture cells

Question 5

Describe linear dilution and what it’s used for.

Answer 5

• Equal intervals

- Used for plotting standard curves

Question 6

Describe log dilution and what it’s used for.

Answer 6

• Constant proportion (eg. 1x10^-1, 1x10^-2)

- Used for counting cell colonies

Question 7

What is the formula for calculating dilutions?

Answer 7

V₁C₁ = V₂C₂

Question 8

What is the purpose of adding a buffer to a reaction?

Answer 8

To allow the pH to remain constant within the reaction mixture.

Question 9

What is the use of a colorimeter?

Answer 9

To quantify colour variation or turbidity of solutions.

Question 10

What does absorbance measure?

Answer 10

Concentration of pigment?

Question 11

What does Transmission determine?

Answer 11

Turbidity (cloudiness)

Question 12

How does a Centrifuge separate components?

Answer 12

It spins at a high speed to separate components of differing densities into a Pellet (solid) and a supernatant (liquid).

Question 13

How does paper and thin layer chromatography separate components and what is an example?

Answer 13

Distance travelled is dependent on a substance’s solubility in the solvent.

Amino acids and Sugars

Question 14

Describe Affinity Chromatography.

Answer 14

• A solid matrix or Gel column is created with specific molecules.
• Soluble target proteins in a mixture with high affinity for these molecules become attached when passed through.
• Other non-target molecules are washed out.

Question 15

Describe gel electrophoresis

Answer 15

Charged macromolecules move through an electric field applied to the gel matrix.
Size and charge are factors that affect rate of which any macromolecules migrate through the gel.

Question 16

How do native gels separate proteins?

Answer 16

By shape size and charge.

Fully folded proteins

Question 17

How does SDS-Page electrophoresis separate proteins?

Answer 17

By size alone.

Denatured proteins

Question 18

Describe Western blotting.

Answer 18

• Used after SDS Page electrophoresis.
• Separated proteins are transferred to a solid medium.
• The proteins can be identified using specific antibodies with reporter enzymes.

Question 19

Define the term isoelectric point

Answer 19

The pH at which a particular molecule carries no net electrical charge.

Question 20

Give two ways proteins can be separated by isoelectric point.

Answer 20

1- Buffer to specific pH- only proteins of that IEP will precipitate out
2- (electrophoresis) soluble proteins can be separated using an electric field and a pH gradient.
A protein stops moving through the gel at its IEP in the pH gradient because it has no net charge.

Question 21

What are the two types of microscopy?

Answer 21

Bright field

Fluorescence

Question 22

What 4 things is bright field microscopy used to observe?

Answer 22

• Whole organisms
• parts of organisms
• thin sections of dissected tissue
• individual cells

Question 23

How does Fluorescent microscopy work?

Answer 23

Uses specific fluorescent labels to bind to and visualise:

• certain molecules
• structures within cells or tissues

Question 24

What is the purpose of immunoassay techniques?

Answer 24

to identify and detect specific proteins

Question 25

What are monoclonal antibodies?

Answer 25

Identical antibodies with the same specificity.

Question 26

Describe immunoassay.

Answer 26

• Monoclonal antibodies are bound to plate
• substance being tested for specific protein is washed through and binds to antibodies
• plate is washed and any unbound proteins are washed away.
• enzyme substrate for protein is added creating detectable change if specific protein is present.

Question 27

What is the advantage of aseptic technique?

Answer 27

Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells

Question 28

What does aseptic technique include?

Answer 28

• sterilisation of equipment using heat or chemicals

- exclusion of microbial contaminants.

Question 29

Define the term inoculum.

Answer 29

An original stock of cells.

Question 30

How can a microbial culture be started?

Answer 30

using an inoculum of microbial cells on an agar medium or in a broth with suitable nutrients.

Question 31

What is serum?

Answer 31

It is added to animal cell culturing media as it contains growth factors.

Question 32

What is the difference between primary cell lines and tumour cell lines?

Answer 32

Primary cell lines can divide a limited number of times, whereas tumour cell lines can divide an unlimited number of times.

Question 33

Describe plating out.

Answer 33

• Liquid microbial culture is plated onto a solid media, allowing a number of colonies to form
• The number of these colonies can be counted, giving an indication of cell density.

Question 34

What is serial dilution’s use?

Answer 34

to allow a countable number of colonies after plating out.

Question 35

What is a haemocytometer?

Answer 35

A special slide used to estimate total and viable cell counts in a sample

Question 36

What are the rules in counting viable cell count in a haemocytometer?

Answer 36

• Include cells touching middle top and left line

- Exclude cells touching middle bottom and right line

Question 37

What does vital staining achieve?

Answer 37

Vital staining is required to identify and count viable cells.

Question 38

How does vital staining work?

Answer 38

Dye is only taken up by non-viable cells, so viable cells are easily counted.