Laboratory Techniques for Biologists

Question 1

What is a hazard ?

Answer 1

A hazard is anything which could harm

Question 2

What can present a hazard ?

Answer 2

Substances, organisms, and equipment in a laboratory can present a hazard, including;

-Toxic or corrosive chemicals
-Heat or flammable substances
-Pathogenic organisms
-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 a risk assessment ?

Answer 4

Risk assessment involves identifying control measures to minimise the risk.

Question 5

What are control measures of a risk ?

Answer 5

Control measures are any measures taken to eliminate or reduce the risk of injury or bodily harm.

Control measures include using;
-appropriate handling techniques
-protective clothing and equipment (Safety goggles, lab coats, gloves etc)
-aseptic technique

Question 6

What is and what are types of dilution ?

Answer 6

-A dilution is taking an original solution and making the concentration weaker.

-Includes linear and log dilutions.

Question 7

What is a linear dilution ?

Answer 7

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

Question 8

What is a log dilution ?

Answer 8

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

Question 9

How can the production of a standard curve be used to determine an unknown ?

Answer 9

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 10

How are buffers used to control pH

Answer 10

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 11

Describe the method and uses of a colorimeter to quantify concentration and turbidity

Answer 11

Calibration with appropriate blank as a baseline;
-Use of absorbance to determine concentration of a coloured solution using suitable wavelength filters, such as haemoglobin in blood.
-Use of percentage transmission of light to determine turbidity, such as cells in suspension.

Turbidity = the quality of being cloudy, opaque, or thick with suspended

Question 12

What are the separation techniques ?

Answer 12

1) Centrifugation
2) Paper, thin-layer, and affinity chromatography
3) Gel electrophoresis

5) Iso-electric points

4) Separation of proteins

Question 13

What is a centrifuge used for ?

Answer 13

-Separates substances of different densities
-More dense components settle in the pellet; less dense components remain in the supernatant

Question 14

What is paper and thin layer chromatography used for ?

Answer 14

-Can be used for separating different substances such as amino acids and sugars

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

Question 15

What is affinity chromatography used for ?

Answer 15

-Separating proteins

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

Question 16

What is gel electrophoresis ?

Answer 16

-Used to separate proteins and nucleic acids.

-Charged macromolecules move through an electric field applied to a gel matrix (will move towards positive electrode)

-Native gel and SDS-PAGE

Question 17

What is native gel electrophoresis ?

Answer 17

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

Native gels separate proteins by their shape, size and charge

Question 18

What is SDS-PAGE

Answer 18

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

Question 19

What are Isoelectric points ?

Answer 19

-IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
-Can be used to separate proteins in a mixture, If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
-Can also be used in electrophoresis

Question 20

Describe isoelectric points in electrophoresis

Answer 20

-Proteins can also be separated using their IEPs in 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 21

What are immunoassay techniques ?

Answer 21

-Immunoassay techniques use antibodies to detect and identify specific proteins.

-Antibodies can be used to detect both the presence and concentration of a protein within a solution.

-They rely on the specificity of antibodies; their ability to recognise and bind with only one protein.

(The thing we are trying to detect is called the “antigen”)

Question 22

How do immunoassay techniques work ?

Answer 22

-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’. The ‘label’ is often a reporter enzyme
producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.

-In some cases the assay uses a specific antigen to detect the presence of antibodies.

Question 23

What is Western Blotting ?

Answer 23

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.

Question 24

What is microscopy ?

Answer 24

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

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

Question 25

What is aseptic technique ?

Answer 25

Aseptic technique eliminates unwanted microbial contaminants when culturing microorganisms or cells

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

Prevents the contamination of people/cell culture/environment by microorganisms.

Examples: Cleaning with ethanol, using bunsen burner to prevent airborne microrganims landing in workspace (convection), glass autoclaving…

Bacterial or fungal contaminants will rapidly out compete and spoil a culture of slower-growing plant or animal cells.

Question 26

How can a microbial culture be started ?

Answer 26

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

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

(Inoculum = cells from a source)

Question 27

How are animal cells cultured ?

Answer 27

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 28

Describe cell division in lines of different cell types, in culture.

Answer 28

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

Question 29

How can cells in culture be counted ?

Answer 29

-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.

-Method and use of haemocytometer to estimate cell numbers in a liquid culture

-Vital staining is required to identify and count viable (living) cells.

(Haemocytometer allows estimate of concentration of cells in a sample “cell count”)
(Vital staining allows distinguishment between living and non-living cells, so can complete a live/total cell count)

Question 30

What are disadvantages of using a haemocytometer to obtain a cell count ?

Answer 30

-Dead cells are not distinguished from live cells (unless stained)

-Small cells difficult to locate

-Number obtained are only an estimate

-Time consuming

-Clumping of cells