KA1.1 - Laboratory Techniques for Biologists

Question 1

Define a hazard in a laboratory setting, and give examples.

Answer 1

• Anything that can cause harm.
• Examples: Toxic/corrosive chemicals, heat/flammable substances, pathogenic organisms, mechanical equipment.

Question 2

Define risk in a laboratory context.

Answer 2

The likelihood of harm arising from exposure to a hazard.

Question 3

What is the purpose of carrying out a risk assessment in a laboratory?

Answer 3

To determine the level of risk from a hazard and identify control measures to minimise harm to individuals.

Question 4

Name a key control measure related to how substances and equipment are managed.

Answer 4

Using appropriate handling techniques.

Question 5

Name a common control measure involving personal protection.

Answer 5

Protective clothing and equipment

Question 6

What is a crucial control measure for preventing contamination, especially with biological materials?

Answer 6

Aseptic technique

Question 7

State the two main methods of creating appropriate dilutions of a chemical.

Answer 7

• Linear dilution
• Log dilution

Question 8

Describe a linear dilution series.

Answer 8

Dilutions differ by an equal interval (e.g., 0.1, 0.2, 0.3).

Question 9

Describe a log dilution series.

Answer 9

Dilutions differ by a constant proportion (e.g., 10⁻¹, 10⁻², 10⁻³)

Question 10

What formula is used to calculate dilutions?

Answer 10

C₁V₁ = C₂V₂

Question 11

What is the purpose of making a standard curve?

Answer 11

To determine the unknown concentration of a solution.

Question 12

Briefly describe how a standard curve is made and used.

Answer 12

A series of “standards” (known concentrations) are measured and graphed. This graph is then used to determine the concentration of an unknown solution.

Question 13

Why are buffers used during experiments?

Answer 13

To control pH; they keep the pH of a reaction mixture constant despite small additions of acid or alkali.

Question 14

What two properties can a colorimeter be used to measure?

Answer 14

• Concentration
• Turbidity

Question 15

What is necessary for accurate measurements when using a colorimeter?

Answer 15

Calibration with an appropriate blank as a baseline.

Question 16

How is the absorbance value from a colorimeter used?

Answer 16

It can be used to determine the concentration of a coloured solution, using suitable wavelength filters.

Question 17

How is the percentage transmission value from a colorimeter used?

Answer 17

It can be used to determine turbidity, such as the concentration of cells in suspension.

Question 18

How is the best wavelength of light for measuring absorbance determined?

Answer 18

Choosing the wavelength that provides the highest absorbanace

Question 19

Name four common laboratory separation techniques.

Answer 19

1. Centrifugation
2. Chromatography
3. Gel Electrophoresis
4. Isoelectric Points (IEP)

Question 20

What is centrifugation, and how does it separate substances?

Answer 20

A separation technique that separates substances of differing density. Denser components settle as a pellet; less dense remain in the supernatant.

Question 21

What is the general principle by which chromatography separates mixtures?

Answer 21

Components of a mixture are separated based on their differing solubility in the solvent used, which affects their speed of travel along the chromatogram.

Chromatogram = TLC plate / paper

Question 22

Name three types of chromatography.

Answer 22

1. Paper chromatography
2. Thin layer chromatography
3. Affinity chromatography

Question 23

Explain how Affinity Chromatography separates a mixture of proteins.

Answer 23

• A solid matrix/gel with specific molecules bound is created.
• Soluble target proteins with high affinity for these molecules attach as the mixture passes.
• Non-target molecules are washed out.

Question 24

What is Gel Electrophoresis used to separate, and on what principle?

Answer 24

Separates mixtures of proteins and nucleic acids. Charged macromolecules move through an electric field applied to a gel matrix.

Negatively charged DNA is attracted to postitive electrode of tank

Question 25

In gel electrophoresis, how does the size of a protein affect its migration speed?

Answer 25

Smaller proteins migrate faster due to less resistance from the gel matrix.

Question 26

What are the key features of Native Gel Electrophoresis?

Answer 26

It does **NOT** denature proteins and separates them based on their **shape, size, and charge**.

Question 27

What are the key features of SDS-PAGE denaturing electrophoresis?

Answer 27

Proteins are given an equally negative charge and denatured, separating them by **size only**.

Question 28

Define a protein's Isoelectric Point (IEP).

Answer 28

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

Question 29

How can proteins be separated from a mixture by precipitation using their IEPs?

Answer 29

If the solution is buffered to a specific pH, only the protein(s) that have an IEP matching that pH will precipitate out.

Question 30

How can proteins be separated using their IEPs in electrophoresis?

Answer 30

In an electric field with a pH gradient, a protein moves through the gel and stops at its IEP because it has no net charge there. | The gel has a pH gradient, e.g. low to high from one end to the other.

Question 31

What is the primary purpose of immunoassay (antibody) techniques?

Answer 31

To detect and identify specific proteins.

Question 32

What are monoclonal antibodies?

Answer 32

antibodies with the same specificity

Question 33

What is a chemical "label" in immunoassay, and what are common types?

Answer 33

A chemical linked to a specific antibody. Often a reporter enzyme (producing a colour change), but can also be chemiluminescence, fluorescence, etc

Question 34

What is the general principle of how immunoassay techniques work to detect proteins?

Answer 34

An antibody specific to the protein of interest's antigen is linked to a chemical "label," allowing its detection (or a specific antigen can be used to detect antibodies)

Question 35

What is Western blotting used for, and what common lab technique precedes it?

Answer 35

An immunoassay technique used to identify specific proteins **after** SDS-PAGE electrophoresis.

Question 36

Briefly describe the key steps of the Western blotting process.

Answer 36

Separated proteins from the SDS-PAGE gel are transferred (blotted) onto a solid medium, then identified using specific antibodies with reporter enzymes attached.

Question 37

State the **two** types of microscopy

Answer 37

1. Bright-field 2. Fluorescence

Question 38

What is bright-field microscopy commonly used to observe?

Answer 38

Whole organisms, parts of organisms, thin sections of dissected tissue, or individual cells.

Question 39

How does fluorescence microscopy work?

Answer 39

Uses specific fluorescent labels that bind to and visualize certain molecules or structures within cells or tissues

Question 40

What is the primary purpose of **aseptic technique** in culturing?

Answer 40

Eliminates unwanted microbial contaminants when culturing micro-organisms or cells.

Question 41

What does aseptic technique involve?

Answer 41

Sterilisation of equipment and culture media (by heat or chemical means) and subsequent exclusion of microbial contaminants.

Question 42

How can a microbial culture be started?

Answer 42

Using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients. | inoculum = sample

Question 43

What is the purpose of "plating out" a liquid microbial culture on solid media?

Answer 43

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

Question 44

Why is serial dilution often needed when working with microbial cultures?

Answer 44

To achieve a suitable colony count for accurate estimation of cell density. | High concentration = too many colonies to count

Question 45

Define 'primary cell lines'

Answer 45

Cells which can divide a limited number of times.

Question 46

Define 'tumour cell lines'

Answer 46

Can perform unlimited divisions

Question 47

What essential components are found in the medium for culturing animal cells?

Answer 47

Medium containing growth factors from serum

Question 48

What are growth factors in cell culture?

Answer 48

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

Question 49

How are animal or plant cell numbers estimated?

Answer 49

Using a haemocytometer.

Question 50

What is the purpose of a vital stain when counting cells, and how does it work?

Answer 50

It's required to identify and count viable (living) cells. It colours dead cells, but living cells remain uncoloured as they remove or metabolise the stain.