1.1 Lab Techniques

Question 1

Potential hazards in the lab

Answer 1

Toxic/corrosive chemicals
Heat/flammable substances
Pathogenic organisms
Mechanical equipment

Question 2

Risk

Answer 2

The likelihood of harm arising from exposure to a hazard

Question 3

What does risk assessment involve

Answer 3

identifying control measures to minimise the risk

Question 4

Hazard

Answer 4

A source of potential harm

Question 5

Control measures include

Answer 5

using appropriate handling techniques
protective clothing/equipment
aseptic techniques

Question 6

Dilutions in a LINEAR dilution series differ by…

Answer 6

equal interval e.g. 0.1, 0.2, 0.3 and so on

Question 7

Dilutions in a LOG dilution series differ by…

Answer 7

a constant proportion e.g. 10^-1, 10^-2, 10^-3 and so on

Question 8

Plotting measured values for known concentrations to produce a line or curve allows

Answer 8

the concentration of an unknown to be determined by the standard curve

Question 9

buffers

Answer 9

can be used to set and maintain a particular pH

Question 10

Does the addition of an acid/alkali have a small/large effect on the pH of a buffer?

Answer 10

It has very small effects on the pH of a buffer, allowing the pH of a reaction mixture to be kept constant.

Question 11

What is a colorimeter used for?

Answer 11

measuring the quantity of light that a sample absorbs or transmits

Question 12

Key elements of colorimeter (3)

Answer 12

• calibration with an appropriate blank as a baseline
• use of absorbance to determine concentration of coloured solution using suitable wavelength filters
• use of percentage transmission to determine turbidity (such as cells in suspension)

Question 13

4 separation techniques

Answer 13

• centrifuge
• chromatography
• gel electrophoresis
• iso-electric points

Question 14

5 ways molecules are separated on

Answer 14

• density
• size
• solubility
• charge
• affinity

Question 15

(centrifugation)
The solid found at the base of the tube is called…
the liquid is called the

Answer 15

pellet

supernatent

Question 16

The purpose of centrifugation

Answer 16

to separate pellet and supernatent of differing density

Question 17

Paper and thin layer chromatography can be used for separating different substances such as

Answer 17

amino acids and sugar

Question 18

(chromatograpy)

The speed that each solute travels along the chromatogram depends on

Answer 18

its differing solubility in the solvent used

Question 19

Explain affinity chromatography

Answer 19

A solid matrix/gel column (stationary phase) is created with specific molecules bound to the gel.
The sample mixture is poured over the stationary phase. Soluble, target proteins in the mix, with a high affinity become attached to the molecules as they pass down.
Non-target molecules with a lower affinity are washed out.

Question 20

Protein electrophoresis

Answer 20

uses a current flowing through a buffer to separate proteins and DNA
The charged macromolecules move through the electric field applied to a gel matrix (usually agarose jelly)

Question 21

Native gels (2)

Answer 21

• native gels separate proteins by their shape, size, and charge
• native gels do not denature the molecules (they are in their natural folded state)

Question 22

SDS-PAGE separates proteins by

Answer 22

size alone

Question 23

How does SDS-PAGE work?

Answer 23

SDS-PAGE gives all the molecules an equally negative charge and denatures them, separating by size alone

Question 24

Proteins can be separated from a mixture using their

Answer 24

isoelectric points (IEPs)

Question 25

IEP

Answer 25

IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution If a solution is buffered to a specific pH, only the proteins that have an IEP of that pH will precipitate

Question 26

Proteins can also be separated using their IEPs in

Answer 26

electrophoresis Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.

Question 27

What does ELISA stand for?

Answer 27

Enzyme Linked Immunosorbent Assay

Question 28

What can an ELISA be used to detect?

Answer 28

Determine the presence of antibodies or antigens in a sample and can be used diagnostically

Question 29

An antibody specific to the protein antigen is linked to a chemical 'label'. What is the 'label'?

Answer 29

The 'label' is often a reporter enzyme producing a colour change, other things like fluorescence can also be used.

Question 30

Advantages of monoclonal antibodies

Answer 30

Provides a stable source of antibodies of known, uniform, specificity

Question 31

What is Western Blotting Technique?

Answer 31

It is used after SDS-PAGE electrophoresis. The separated proteins from the gel are transferred (blotted) onto a solid medium. The proteins can be identified using specific antibodies that have reporter enzymes attached

Question 32

Fluorescence microscopy

Answer 32

uses specific fluorescent labels to bind to and visualise certain molecules/structures within cells or tissues

Question 33

Bright field microscopy

Answer 33

allows you to look at whole organisms, parts of organisms, thin sections of dissected tissue or individual cells

Question 34

What does aseptic technique eliminate?

Answer 34

Eliminates unwanted microbial contaminants when culturing microorganisms or cells

Question 35

What does aseptic technique involve?

Answer 35

Involves the sterilisation of equipment and culture media by heat/chemicals. And the exclusion of microbial contaminants

Question 36

What can microbial culture be started with?

Answer 36

an inoculum of microbial cells on an agar medium/ in a broth with suitable nutrients

Question 37

What are animal cells grown in?

Answer 37

a medium containing growth factors from serum

Question 38

What are growth factors?

Answer 38

proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.

Question 39

In a culture, what are PRIMARY cell lines?

Answer 39

Primary cell lines can divide a limited number of times

Question 40

In a culture, what are TUMOUR cell lines?

Answer 40

can perform unlimited divisions

Question 41

What is the inoculum?

Answer 41

The starting material you use to start growing a culture from.

Question 42

What does planting out of a liquid microbial culture on solid media allow?

Answer 42

It allows the number of colony forming units to be counted and the density of cells in the culture estimated.