Key Area 1

Question 1

What are examples of hazards in a lab?

Answer 1

Toxic or corrosive chemicals.
Heat or flammable substances.
Pathogenic organisms.
Mechanical equipment.

Question 2

What is a risk?

Answer 2

Risk is the likelihood of harm arising from exposure to a hazard.

Question 3

What does a risk assessment do?

Answer 3

Identifies control measures to minimise the risk.

Question 4

What are examples of control measures?

Answer 4

Using appropriate handling techniques.
Protective clothing and equipment.
Aseptic technique.

Question 5

Linear Dilutions

Answer 5

Differ by an equal interval, e.g. 0·1, 0·2, 0·3 …

Question 6

Log Dilutions

Answer 6

Differ by a constant proportion, e.g. 10-1, 10-2, 10-3 …

Question 7

What does a standard curve tell us?

Answer 7

Plotting values for known concentrations to produce a line/curve allows the concentration of an unknown to be determined.

Question 8

What do buffers do?

Answer 8

Allows the pH of a reaction mixture to be kept constant.
Addition of acid or alkali has very small effects on the pH of a buffer.

Question 9

Use of colorimeter to measure concentration:

Answer 9

Calibration with suitable blank as a baseline.
Use of absorbance to determine conc. of a solution using correct wavelength filter.

Question 10

Use of colorimeter to measure turbidity:

Answer 10

Use of percentage transmission to determine turbidity.
e.g cells in suspension.

Question 11

How do solutions separate during centrifugation?

Answer 11

More dense components settle in the pellet (bottom).
Less dense components remain in the supernatant (above pellet).

Question 12

Paper and thin layer chromatography:

Answer 12

Separate different substances such as amino acids and sugars.
Speed of each solute travels depends on its solubility in the solvent used.

Question 13

How does affinity chromatography separate proteins?

Answer 13

1. Solid matrix/gel column have specific
   molecules bound to the matrix/gel.
2. Soluble target proteins in a mixture with a high
   affinity for molecules in matrix/gel attach to
   them as the mixture passes them.
3. Molecules with a weaker affinity are washed
   out.

Question 14

How does gel electrophoresis separate proteins?

Answer 14

2 types:
- Native gels
- SDS-PAGE
Charged macromolecules move through an electric field applied to a gel matrix.

Question 15

Native Gels

Answer 15

Separate proteins by their shape, size and charge.
Do not denature the molecule.

Question 16

SDS–PAGE

Answer 16

Separates proteins by size alone.
Gives all the molecules an equally negative charge.
Denatures molecules.

Question 17

Isoelectric Points (IEPs)

Answer 17

pH at which a soluble protein has no net charge and will precipitate out of solution.

Question 18

IEPs and buffers:

Answer 18

Only proteins that have an IEP of that specific pH will precipitate.

Question 19

IEP electrophoresis:

Answer 19

Soluble proteins separated using an electric field and a pH gradient.
Protein stops migrating through the gel at its IEP because it has no net charge.

Question 20

What are immunoassay techniques used for?

Answer 20

To detect and identify specific proteins using monoclonal antibodies.

Question 21

Monoclonal antibodies:

Answer 21

Stocks of antibodies with the same specificity.

Question 22

Chemical labels

Answer 22

Shown when:
- An antibody is specific to the protein antigen.
- A specific antigen detects the presence of
antibodies.
Usually it’s a reporter enzyme producing a colour change.
Other chemical labels include:
- Chemiluminescence
- Fluorescence

Question 23

Western blotting:

Answer 23

Used after SDS–PAGE electrophoresis.
Separated proteins from the gel are transferred (blotted) onto a solid medium.

Question 24

How are proteins identified in western blotting?

Answer 24

Using specific antibodies that have reporter enzymes attached.

Question 25

Bright-field microscopy:

Answer 25

Used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.

Question 26

Fluorescence microscopy:

Answer 26

Uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.

Question 27

Aseptic technique:

Answer 27

Sterilisation of equipment and culture media by heat or chemical means to eliminate unwanted contaminants when culturing microorganisms or cells.

Question 28

How is a microbial culture started:

Answer 28

Using microbial cells on an agar medium, or in a broth with suitable nutrients.
There are different culture media for specific types of microbes.

Question 29

How are animal cells grown?

Answer 29

Grown in a medium containing growth factors from a serum.

Question 30

What are growth factors?

Answer 30

Proteins that promote cell growth and proliferation.
Essential for the culture of most animal cells.