Lab Techniques

Question 1

Define ‘hazard’

Answer 1

A hazard is anything with the potential of causing harm or injury to a person or the environment.

Question 2

Define ‘risk’

Answer 2

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

Question 3

What are the three types of hazards?

Answer 3

Substances, organisms and equipment

Question 4

Describe the process of an SSERC protocol.

Answer 4

Identify the hazard, decide who may be harmed and how, evaluate risks and decide on precautions, record your findings and implement them, review and update your assessment.

Question 5

What is a risk assessment?

Answer 5

A risk assessment is a process where you identify and implement control measures to minimise risks.

Question 6

Give examples of hazards and explain how they cause harm.

Answer 6

SUBSTANCE - ionic chemicals can enter the body and bioaccumulate, corrosive chemicals can cause severe burns and skin irritation, flammable substances.

ORGANISMS - some plants and animals excrete substances that can cause harm to people, parthenogenesis organisms can cause virulent disease.

Question 7

Give examples of control measures to minimise harm.

Answer 7

Appropriate handling techniques, protective clothing and equipment, aseptic technique.

Question 8

What are the benefits of using dilutions in an experiment?

Answer 8

Dilutions can be used to control potential confounding variables in an experimental procedure.

They can be used to generate a suitable range for an independent variable.

They can be used to modify the dependent variable so a measurable value can be obtained.

Question 9

Define ‘confounding variable’

Answer 9

A confounding variable is a variable other than the independent variable that may affect the dependent variable.

Question 10

Describe the properties of a linear dilution

Answer 10

Linear dilutions differ by equal intervals. This means that each concentration is made individually and any measurement errors will only affect one dilution.

Question 11

Describe the properties of a log dilution.

Answer 11

Log dilutions differ by a constant proportion. This means that all concentrations after the standard dilution will be affected if a measurement error were to occur.

Question 12

What is ‘colorimetry’ and what is it used for?

Answer 12

Colorimetry is a technique used to determine the concentration of a pigment in solution and or the turbidity of liquid or density of cells in a culture.

Question 13

Define ‘turbidity’

Answer 13

Turbidity refers to a solutions degree of cloudiness.

Question 14

Comment on the use of a standard curve.

Answer 14

A standard curve is made by plotting the absorbance of a solution against the known concentrations of the culture. A standard curve can be used to estimate the concentration of a solution of unknown concentration of the same substance. This is done by interpolation.

Question 15

Define ‘interpolation’

Answer 15

Interpolation is a type of estimate used to find data points based on a range of known data points.

Question 16

What are buffers and how are they used?

Answer 16

Buffers are aqueous solutions that show very little change in pH upon the addition of an acid or alkali. A buffer is used to ensure the pH of a solution stays constant.

Question 17

Define ‘extrapolation’

Answer 17

Extrapolation refers to predicting a data point based on its last known value

Question 18

Define ‘centrifugation’ and its uses.

Answer 18

Centrifugation is a method used for separating materials in suspension according to their differing densities.

Question 19

State the speed at which materials are spun in a centrifuge.

Answer 19

The material is rotated at between 2000 and 120000 resolutions per minute.

Question 20

State the names given to the differing components of a centrifuge tube.

Answer 20

The dense component formed at the bottom of the tube after spinning is referred to as the ‘pellet’
The liquid sitting on top of the pellet is known as the ‘supernatant’

Question 21

What is the use of ‘thin-layer chromatography’?

Answer 21

Thin layer chromatography allows amino acids and sugars to be separated according to their relative solubilities.

Question 22

Of what material is the paper in thin-layer chromatography made of?

Answer 22

The paper in thin-layer is made from silica gel or cellulose.

Question 23

Define ‘affinity chromatography’

Answer 23

Affinity chromatography is used to separate a specific protein from a mixture.

Question 24

Describe in detail the process of affinity chromatography.

Answer 24

STEP 1 - an antibody / ligand specific to binding with the selected protein is immobilised in gel or a solid matrix and packed into a column.

STEP 2 - A mixture of proteins is then poured through the column and only the protein specific to the ligand or antibody will bind to the column. The other proteins will not bond and are washed out.

STEP 3 - The column is then washed again with a buffer of a different pH which lowers the target proteins affinity to the column, allowing it to be collected.

Question 25

Explain simply, the purpose of gel electrophoresis.

Answer 25

Gel electrophoresis uses current flowing through a buffer to separate proteins/nucleic acids

Question 26

Describe in detail, the process of gel electrophoresis.

Answer 26

STEP 1 - a sample is first loaded into a gel matrix and an electric field is applied. STEP 2 - negatively charged molecules then migrate toward the positively charged electrode, with smaller molecules moving faster through the gel pores. STEP 3 - the separated molecules are visualised by applying stains.

Question 27

What factors affect the rate at which macromolecules migrate through gel?

Answer 27

Size and charge are factors that affect macromolecules rate of migration through gel.

Question 28

How are SDS-PAGE proteins separated?

Answer 28

SDS-PAGE proteins are separated by size alone in an unfolded state. The protein is unfolded by denaturing bonds with heat, which results in a linear protein with a uniform negative charge across its length.

Question 29

Define ‘iso-electric point’

Answer 29

The isoelectric point of a protein is the pH at which it has no net charge.

Question 30

How does water interact with proteins?

Answer 30

Above and below its pH there will either be a majority of positive or negative charges at the surface of the protein allowing it to become soluble. Water molecules interact with these charges to keep it suspended in solution.

Question 31

What is the benefit of iso-electric point?

Answer 31

The overall neutral charge at IEP allows the protein to form a solid and precipitate out of solution. This can be useful in separating proteins. By slowly altering the pH of a solution using buffers, proteins with IEP at those specific pH’s will precipitate out of solution and therefore be collected.

Question 32

What is the purpose of immunoassay techniques?

Answer 32

Immunoassay techniques are useful in the detection and identification of specific proteins.

Question 33

What are antibodies? Where are they produced? How do they work?

Answer 33

- Antibodies are Y-shaped globular proteins produced by B-lymphocytes as part of an immune response by a vertebrate. - B-lymphocytes are produced by the spleen and bone marrow. - each lymphocyte produces a specific antibody for a specific antigen. The antibody will then bind to its antigen rendering it useless.

Question 34

What term is used to describe a serum made with many different antibodies against an antigen?

Answer 34

A serum made with many different antibodies against an antigen is referred to as polyclonal.

Question 35

What is an essential stage in all immunoassay techniques?

Answer 35

An essential stage in all immunoassay techniques is manufacturing antibodies that are identical and will bind to exactly that antibody.

Question 36

How are monoclonal antibodies produced?

Answer 36

Monoclonal antibodies are produced by growing a line of lymphocytes secreting the same specific antibody.

Question 37

What are monoclonal antibodies used for?

Answer 37

Monoclonal antibodies are used to diagnose and detect disease.

Question 38

What are reporter enzymes?

Answer 38

Reporter enzymes are enzymes attached to an antibody. They catalyse a reaction in the presence of a specific enzyme.

Question 39

Define and describe the process of ‘western blotting’

Answer 39

- western blotting is a technique used to identify specific proteins separated by SDS-PAGE electrophoresis. STEP 1 - proteins are blotted from the gel onto a solid medium STEP 2 - the filter is then flooded with fluorescent labelled monoclonal antibodies. Several antibodies can be used as long as each has a different coloured fluorescent label. STEP 3 - once antibodies have bound to their target proteins, unbound ones are flushed out. STEP 4 - the medium is then exposed to light of different wavelengths, the antibodies allow the precise location of the proteins to be identified.

Question 40

Define ‘histochemistry’

Answer 40

Histochemistry is the study of tissues using stains and microscopy.

Question 41

Give an example of a use for fluorescent immunohistochemical staining.

Answer 41

Fluorescent immunohistochemical staining is used to visualise the distribution of specific cellular components in live cells

Question 42

What is the benefit of fluorescent microscopy?

Answer 42

It allows specific protein structures to be visualised in a particular way that was not previously possible.

Question 43

Define ‘aseptic technique’

Answer 43

Aseptic technique is a set of precautions taken to eliminate unwanted microbial contamination.

Question 44

What does aseptic technique involve?

Answer 44

The sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contamination.

Question 45

Why is aseptic technique important?

Answer 45

Aseptic technique is crucial in preventing bacterial or fungal growth from rapidly outcompeting and spoiling a culture of slow growing animal and plant cells

Question 46

Give examples of mediums which microbes grow on.

Answer 46

Microbes can be grown in suspension in a liquid medium (broth) with suitable nutrients or on or within a solid medium such as agar jelly (substrate)

Question 47

How can rapid growth of cells be ensured?

Answer 47

Rapid growth of chosen cells can be ensured by providing the media with optimum conditions in terms of pH, nutrients and gases.

Question 48

How can anaerobic organisms be cultured?

Answer 48

Anaerobic organisms can be cultured in non-aerated suspension or in agar.

Question 49

How can a microbial culture be cultivated?

Answer 49

A microbial culture can be cultivated with an inoculum where cells are added in a volume of liquid medium from a previous culture. The inoculum is gathered by touched a sterile hoop to the broth on the culture plate.

Question 50

How are mammalian cells cultured?

Answer 50

Mammalian cells are cultured in liquid in a flask.

Question 51

How can you ensure rapid growth in a mammalian culture?

Answer 51

To ensure rapid growth and cell division in a mammalian cell culture, most mammalian cells require the addition of a complex medium containing chemical growth factors from serum.

Question 52

Give an example of an animal serum and explain what it is.

Answer 52

An example of an animal serum is foetal bovine serum otherwise known as ‘FBS’ FBS is a mixture containing proteins, salts, vitamins and glucose other than growth factors.

Question 53

Why are antibiotics added to the culture?

Answer 53

Antibodies are added to minimise the chances of spoiling by microorganisms.

Question 54

Define ‘growth factors’

Answer 54

Growth factors are proteins that promote growth and proliferation (rapid increase).

Question 55

Explain what a ‘cell line’ is.

Answer 55

A cell line is a genetically uniform cell culture developed from a single cell.

Question 56

Explain the difference between a primary and a tumour cell line.

Answer 56

PRIMARY cell lines tend to die after a certain amount of divisions. This means they have a shorter lifespan. TUMOUR cell lines however are immortal and have an infinite amount of divisions. They will not die.

Question 57

How are colony forming units counted?

Answer 57

Colony forming units are counted by plating out a liquid microbial culture on solid media. This also allows the cell density to be estimated. Serial dilutions are often used to determine suitable cell count.

Question 58

What is a haemocytometer and how is it used?

Answer 58

A haemocytometer is a graduated microscope slide. The overslip of the haemocytometer sits a known distance from the slide allowing the viewer to look through a known volume of medium. Cell density can be calculated by counting the NUMBER OF CELLS in a particular part of the grid. Haemocytometers also make total cell counts. If vital staining is used, viable cell counts as well.