1. Laboratory techniques for biologists

Question 1

How are risks minimised in laboratories?

Answer 1

• Control of substances hazardous to health (COSHH) regulations cover substances that are hazardous to health.
• A COSHH assessment form allows a risk assessment to be carried out for any substance which is potentially hazardous.
• In this form, factors such as exposure, disposal procedures and handling procedures are considered.

Question 2

What are serial dilutions?

Answer 2

• Repeated dilution from a stock solution.
• Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3
• Dilutions in a log dilution series differ by a constant proportion, for example 10‑1, 10‑2, 10‑3

Question 3

What is a colorimeter?

Answer 3

• A colorimeter measures the absorbance of specific wavelengths of light by a solution.
• A colorimeter works by passing a light beam, at a specific wavelength, through a cuvette containing a sample solution.

Colorimeters can give two types of readings:
* absorbance - how much light has been absorbed by the sample;
* transmission - how much light passed through the sample without being absorbed.

Question 4

What is a standard curve?

Answer 4

A series of ‘standards’ of known concentration are measured and graphed. This graph can then be used to determine the concentration of an unknown sample.

Question 5

What is a pH buffer?

Answer 5

• A pH buffer is a solution whose pH changes very little when a small amount of acid or base is added to it.
• Buffer solutions are used as a means of keeping pH at a nearly constant value.

Question 6

What is centrifugation?

Answer 6

• Centrifugation allows substances to be separated according to their density.
• The densest materials separate out first and form a pellet at the bottom of the tube.
• The liquid which remains above the pellet is called the supernatant.

Question 7

What is gel electrophoresis?

Answer 7

• Proteins and nucleic acids can be separated by gel electrophoresis.
• This process separates macromolecules based upon their charge and/or size/shape.
• Small proteins travel further through the gel than large proteins.
• SDS–PAGE separates proteins by size alone
• It is also possible to run native gels where the protein is not denatured before the gel electrophoresis. This allows the scientist to analyse the proteins in their folded state.

Question 8

What is iso-electric point?

Answer 8

• Proteins can be separated from a mixture using their IEPs.
• IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
• If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate.

Question 9

What is chromatography?

Answer 9

• Chromatography can be used to separate mixtures of amino acids and sugars.
• The mixture is dissolved in a fluid known as the mobile phase.
• The components of the mixture are separated as they pass through a stationary phase.

Question 10

What is paper chromatography?

Answer 10

• In paper chromatography, the stationary phase is a strip of chromatography paper.
• A sample of the mixture being separated is placed in a dot or line, near the bottom of a strip of chromatography paper which is then placed in a solvent.
• The solvent moves up through the chromatography paper and carries the components of the mixture with it, which will travel at different rates depending on their properties.

Question 11

What is thin layer chromatography?

Answer 11

• Rather than using a stationary phase of chromatography paper, TLC uses a strip of absorbent material, such as silica gel, on a non-reactive backing.
• A solvent moves up through the stationary phase and carries the components of the mixture with it.
• The components will travel different distances depending on how soluble they are in the solvent and how much they bind to the stationary phase.

Question 12

What is affinity chromatography?

Answer 12

• Affinity chromatography relies on the binding interactions between a protein and specific molecules bound to a matrix or gel. A specific molecule is immobilised on an inert support in a column and a protein mixture is passed through the column.
• The target protein, which is complementary to the specific molecule in the column, will bind to it and remain in the column when the other components are washed away.
• The target protein can then be stripped from the support, resulting in its separation and purification from the original sample.

Question 13

What is immunoassay?

Answer 13

• Immunoassay techniques are used to detect and identify specific proteins.
• These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies
• An antibody specific to the protein antigen is linked to a chemical ‘label’.

Question 14

What is western blotting?

Answer 14

• Western blotting is a technique, 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. This can be in the form of a colour change.

Question 15

What is bright field microscopy?

Answer 15

Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells

Question 16

What is fluorescence microscopy

Answer 16

Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.

Question 17

What is aseptic technique?

Answer 17

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

Question 18

What is microbial culture?

Answer 18

A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients

Question 19

How are animal cells cultured?

Answer 19

Animal cells are grown in medium containing growth factors from serum.

Question 20

What are primay and tumor cell lines?

Answer 20

In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions.

Question 21

How is the number of cells in a culture counted?

Answer 21

• Plating out of a liquid microbial culture on solid media allows the number of colony-forming units to be counted and the density of cells in the culture estimated.
• Serial dilution is often needed to achieve a suitable colony count

Question 22

What is a haemocytometer?

Answer 22

• A haemocytometer is a specialised slide that has a counting chamber with a known volume of liquid
• It allows an estimation of the concentration of cells in a culture to be made. It has a grid made up of perpendicular lines etched into the glass.
• The number of cells in more than one square should be counted and an average calculated.

Question 23

How are viable cells identified?

Answer 23

• The haemocytometer is set up using cells stained with trypan blue dye, which is taken up by dead cells but not by living cells.
• A live cell count can then be performed where only unstained cells are counted.