Unit 1 - Lab Techniques

Question 1

Hazard

Answer 1

Anything that can cause harm or injury to people, or can cause ecological damage.

There are 3 types - substances, organisms and equipment.

Question 2

Toxic chemicals

Answer 2

Chemicals that poison cells by acting as non-competitive enzyme inhibitors.

They can bioaccumulate over long periods (lead ethanoate), or can be acute, fast acting poisons (potassium cyanide).

Question 3

Corrosive chemicals

Answer 3

Cause severe burns to exposed skin. eg. sulfuric acid.

Question 4

Flammable substances

Answer 4

Catch fire easily at room temperature. eg. ethanol.

Question 5

Hazardous organisms

Answer 5

Produce substances harmful to health eg. New Zealand flatworm mucus and Euphorbia latex both cause severe skin irritation.

Question 6

Pathogenic organisms

Answer 6

Organisms that cause disease (usually micro-organisms) eg. virulent strains of E.coli.

Question 7

Hazardous equipment

Answer 7

Laboratory equipment with the potential to cause harm eg. sharp scalpels, centrifuge rotors (if the lid is opened early).

Question 8

COSHH

Answer 8

Control of substances hazardous to health. Likelihood of harm resulting from exposure to a hazard must be considered before starting lab work, by carrying out a risk assessment.

Question 9

Risk assessment

Answer 9

Identify control measures that minimise the risk posed by a particular hazard. The hazard is not removed, but risk is reduced to an acceptable level.

Question 10

Control measures

Answer 10

Reduce the risk from a hazard.

3 categories : appropriate handling techniques (eg. tongs); protective clothing and equipment (eg. gloves, goggles) and aseptic technique.

Question 11

Dilution series

Answer 11

Used to generate a suitable range for an independent variable, or to modify a dependent variable so that it can be measured.

Question 12

Linear dilution series

Answer 12

A range of dilutions that differ by an equal interval eg. 0.1M, 0.2M, 0.3M etc.

To make a 0.1M solution, add 1ml of 1M stock solution to 9ml water.
To make a 0.2M solution add 2ml of 1M stock solution to 8ml water etc.

Question 13

Log dilution series

Answer 13

A range of dilutions that differ by a constant proportion. eg. by a factor of 10.

Each dilution acts as the stock solution for subsequent dilutions, so any measurement errors are compounded. Often used to estimate microbial cell density.

Question 14

Colorimeter

Answer 14

Used to measure the concentration of a pigment in solution, the turbidity of a liquid or the density of cells in a culture.

A small sample in a transparent cuvette is illuminated and the light absorbed by the sample is measured electronically. A suitable wavelength filter is used, and the machine is calibrated using a blank cuvette containing solvent only.

Question 15

Standard curve

Answer 15

Absorbance readings for a series of known concentrations are plotted, and are used as a reference for unknown samples, which are estimated by interpolation.

Question 16

Buffer

Answer 16

Aqueous solution showing little variation in pH, despite addition of acids or alkalis.

Used to keep the pH of reaction mixtures constant.
Found in all cells and their secretions eg. blood, as most biological processes are pH dependent.
Used to prevent pH changes in cell culture, which may occur due to build up of waste.

Question 17

Centrifugation

Answer 17

Used to separate materials in suspension according to their differing density. eg. cell constituents or enzymes from homogenised (blended) tissue.

Dense components form a pellet, while the less dense fractions remain in the liquid supernatant.

Question 18

Chromatography

Answer 18

Separation of molecules by their solubility. The material to be tested is spotted on to the base of the chromatography strip, and a solvent mixture pulls them up the chromatogram. Different solubility results in different speeds, so the constituents travel different distances.

Paper chromatography uses cellulose paper, thin layer chromatography uses a layer of silica gel or cellulose. Both require a hydrophobic solvent.

Question 19

Affinity chromatography

Answer 19

A technique for separating one specific soluble protein from a mixture.

An antibody or ligand specific to the required protein is immobilised in gel and packed into a column. The mixture is poured through, and only the target protein will bind.

The target protein is recovered by washing with either a buffer of different pH (to lower its affinity for the antibody/ligand) or a solution containing a free ligand.

Question 20

Gel electrophoresis

Answer 20

A current flowing through a buffer is used to separate proteins or nucleic acids by size and charge in their native folded state, by dragging them through a gel.

Question 21

SDS-PAGE Electrophoresis

Answer 21

Proteins are separated using gel electrophoresis in their denatured (unfolded) state by size alone.

Proteins are denatured by heat and detergent, resulting in an unfolded linear protein with a uniform negative charge along its length.

Question 22

Isoelectric point (IEP)

Answer 22

The pH at which a protein has no net charge, which allows it to precipitate out of a solution.
Used to separate a mixture of proteins from one another by slowly changing the pH of a solution.

Question 23

Antibody

Answer 23

Y-shaped globular proteins produced by B lymphocytes, as part of the immune response in vertebrates.

Each B lymphocyte produces a specific antibody that binds to one specific antigen.

Question 24

Polyclonal serum

Answer 24

Obtained from the blood of animals that have been exposed to material containing a particular antigen.
The blood is centrifuged to separate the different antibodies that have formed in response to different parts of the antigen. Each different antibody is made by a different B lymphocyte, so is described as polyclonal.

Question 25

Monoclonal antibodies

Answer 25

Antibodies that are identical and will bind to exactly the same feature of an antigen.

Produced from a single line of identical B lymphocytes, producing the same specific antibody. Essential for immunoassays.

Question 26

Immunoassay

Answer 26

The use of monoclonal antibodies for the detection and diagnosis of disease or pregnancy.

ELISA uses monoclonal antibodies with a reporter enzyme attached.
The enzyme catalyses a colour change, which is used to detect and quantify the presence of a particular antigen.

Question 27

Western blotting

Answer 27

Used to identify specific proteins that have been separated using SDS-PAGE gel electrophoresis.

The proteins are blotted from the gel, which is then flooded with fluorescent labelled monoclonal antibodies. Allows the precise location of a target protein to be identified.

Question 28

Fluorescence microscopy

Answer 28

Used to visualise the distribution of specific cellular components in live cells, once they have been stained with fluorescent-immunohistochemical staining.

eg. a specific fluorescent labelled antibody for tubulin will show spindle fibres stained in a particular colour.

Question 29

Histochemistry

Answer 29

The study of tissues using stains and microscopy.

Question 30

Bright field microscopy

Answer 30

Used to examine whole organisms, parts of organisms such as tissue sections or whole cells.

Specimens must be small and thin enough to fit on a slide under a coverslip, or on a cavity slide. Stains can be used to improve definition. Specimens can be magnified to 400x (or 1000x with oil immersion - a drop of oil is placed between the objective and the slide)

Question 31

Aseptic technique

Answer 31

A set of precautions taken to eliminate microbial contamination.

It involves the sterilisation of equipment and culture media using heat or chemicals, followed by exclusion of unwanted microbes.

Question 32

Microbial culture

Answer 32

Growing microbes in suspension in liquid broth, or on solid agar jelly containing nutrients.

Optimum growing conditions are provided by manipulating pH, nutrients and gases.

Question 33

Inoculum

Answer 33

Used to start a microbial culture.

Growing cells are added by collecting a small volume of liquid from an existing culture, or by touching a culture plate with a sterile loop.

Question 34

Animal cell culture

Answer 34

Animal cells are cultured in a flask in liquid containing a complex medium.

An animal serum (such as FBS - foetal bovine serum) must be added, as it contains growth factors + proteins, salts, vitamins and glucose. Growth factors promote cell growth and proliferation.

Antibiotics are added to prevent spoilage by micro-organisms.

Question 35

Subculture

Answer 35

Some cells are transferred to a fresh culture flask, in order to keep a culture alive once the nutrients in a flask have been used up.

Question 36

Primary cell line

Answer 36

A newly created animal cell line. They tend to die after around 60 divisions (Hayflick limit) which reduces the time that a primary cell line can be maintained.

Question 37

Tumour cell line

Answer 37

Immortal cell line which can be subcultured indefinitely, as they perform unlimited divisions. eg HELA cells.

Question 38

Plating out

Answer 38

A liquid microbial culture is spread onto an agar plate, so that the number of colony forming units can be counted. This allows the density of cells in the culture to be estimated.

Question 39

Haemocytometer

Answer 39

A graduated microscope slide used to count cell density.

It contains a known volume, and by counting the number of cells in a particular area of the grid, it allows the cell density to be calculated.

Counting rules are essential.

Question 40

Vital stain

Answer 40

Used to estimate what proportion of a cell culture is living (viable).

eg. methylene blue stains dead cells blue, while living (viable) cells remain colourless.