1.1 Lab techniques for biologists

Question 1

What presents a hazard in a lab?

Answer 1

Substances, organisms, and equipment in a laboratory can present a hazard.

Question 2

Give four examples of hazards in a lab

Answer 2

Hazards in the lab include:

1. Toxic or corrosive chemicals
2. Heat or flammable substances
3. Pathogenic organisms
4. Mechanical equipment.

Question 3

What is risk?

Answer 3

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

Question 4

What is risk assessment?

Answer 4

Risk assessment involves identifying control measures to minimise the risk.

Question 5

Give three examples of control measures used to minimise risk

Answer 5

Control measures include:

1. Using appropriate handling techniques
2. Protective clothing and equipment
3. Aseptic technique.

Question 6

Linear dilutions

Answer 6

Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3 and so on.

Question 7

Log dilutions

Answer 7

Dilutions in a log dilution series differ by a constant proportion, for example 10-1, 10-2, 10-3 and so on.

Question 8

How does a standard curve work?

Answer 8

Plotting measured values for known concentrations to produce a line or curve allows the concentration of an unknown to be determined from the standard curve.

Question 9

How do pH buffers control pH?

Answer 9

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

Question 10

What are colorimeters used for?

Answer 10

Colorimeters can be used to quantify concentration and turbidity (cloudiness).

Question 11

Before use, colorimeters must be…

Answer 11

Calibrated with an appropriate blank sample as a base line.

Question 12

In a colorimeter, what is absorbance used to determine?

Answer 12

Absorbance can be used to determine concentration of a coloured solution using suitable wavelength filters.

Question 13

In a colorimeter, what is percentage transmission used to determine?

Answer 13

Percentage transmission can be used to determine turbidity, such as cells in suspension.

Question 14

What is a centrifuge used for?

Answer 14

A centrifuge is used to separate substances of differing density.

Question 15

How does a centrifuge work?

Answer 15

Samples are spun so that more dense components settle in the pellet (solid) and less dense components remain in the supernatant (liquid).

Question 16

What substances do paper and thin layer chromatography separate?

Answer 16

Paper and thin layer chromatography can be used for separating different substances such as amino acids and sugars.

Question 17

How do paper and thin layer chromatography work?

Answer 17

The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.

Question 18

What is affinity chromatography used for?

Answer 18

Affinity chromatography is used to separate proteins.

Question 19

How does affinity chromatography work?

Answer 19

A solid matrix or gel column is created with specific molecules (usually receptors) bound to it.
Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column.
Other non-target molecules with a weaker affinity are washed out.

Question 20

What is gel electrophoresis used for?

Answer 20

Gel electrophoresis is used to separate proteins and nucleic acids.

Question 21

How does gel electrophoresis work?

Answer 21

Charged macromolecules move through an electric field applied to a gel matrix.

Question 22

Native gels

Answer 22

Native gels do not denature the molecule so that separation is by shape, size and charge.

Question 23

SDS–PAGE

Answer 23

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

Question 24

What is isoelectric point?

Answer 24

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

Question 25

What is IEP used for?

Answer 25

Proteins can be separated from a mixture using their isoelectric points. Proteins can also be separated using their IEPs in electrophoresis.

Question 26

pH buffers and IEP

Answer 26

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

Question 27

How does separation using IEPs work?

Answer 27

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 28

What are immunoassay techniques used for?

Answer 28

Immunoassay techniques are used to detect and identify specific proteins.

Question 29

What do immunoassay techniques use?

Answer 29

Immunoassay techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies

Question 30

Chemical 'labels'

Answer 30

An antibody specific to the protein antigen is linked to a chemical ‘label’. The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.

Question 31

What is sometimes used with immunoassay techniques?

Answer 31

In some cases the assay uses a specific antigen to detect the presence of antibodies to test for diseases.

Question 32

When is Western blotting used?

Answer 32

Western blotting is a technique used after SDS–PAGE electrophoresis.

Question 33

How does Western blotting work?

Answer 33

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 34

Bright-field microscopy

Answer 34

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

Question 35

Fluorescence microscopy

Answer 35

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

Question 36

What is aseptic technique?

Answer 36

Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.

Question 37

What does aseptic technique do?

Answer 37

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

Question 38

How can a microbial culture be started?

Answer 38

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

Question 39

Growth of specific cells or microbes

Answer 39

Many culture media exist that promote the growth of specific types of cells and microbes.

Question 40

Culture of animal cells

Answer 40

Animal cells are grown in medium containing growth factors from serum. Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.

Question 41

Primary cell lines

Answer 41

In culture, primary cell lines can divide a limited number of times.

Question 42

Tumour cell lines

Answer 42

Tumour cells lines can perform unlimited divisions.

Question 43

Colony counts and estimating the density of cells

Answer 43

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 44

What are haemocytometers used for?

Answer 44

Haemocytometers are used to estimate cell numbers in a liquid culture.

Question 45

How do you use a haemocytometer?

Answer 45

1. Calculate the volume of the 'large' squares in the grid. 2. Count the number of cells in each 'large' square and calculate an average. 3. Cell density = average no. of cells per square / volume of the square.

Question 46

What is vital staining needed for?

Answer 46

Vital staining is required to identify and count viable cells.