Lab Techniques

Question 1

What 3 things might present a hazard in the lab?

Answer 1

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

Question 2

What is risk?

Answer 2

The likelihood of harm arising from exposure to a hazard.

Question 3

What is risk assessment?

Answer 3

Identification of significant hazards, quantifying the level of risk and implementation of sufficient control measures to mitigate these risks.

Question 4

What are the 5 main types of control measures used to mitigate risk from most preffered to least preffered?

Answer 4

Elimination, substitution, equipment, education, appropriate PPE.

Question 5

Define how to create log dilution series and a practical application.

Answer 5

Log series’ are made from diluting a solution by a proportion of its concentration, then diluting the diluted solution by the same proportion again, it can be used to count cell colonies otherwise too dense to count.

Question 6

Define linear dilution series and what it might be used for.

Answer 6

Each test tube in the series is made separately from the stock solution, they differ by an equal amount. e.g. 0.9, 0.8, 0.7… They can be used to find an appropriate concentration for a chemical reaction or to use to plot a standard curve.

Question 7

What is a standard curve, how is it made and what is it used for?

Answer 7

Concentration against absorbance, it is made from many different colorimetry (absorbance) readings
(y-axis) plotted against the corresponding concentration for each of those solutions, used to determine the concentration of of a solution for which it is unkown based off a colourimetry (absorbance) reading.

Question 8

What is a pH buffer used for / what does it do?

Answer 8

pH buffers keep the pH of the solution relatively constant even when strong acid or base is added as they can be proton acceptors or donors e.g. pH buffers needed in blood.

Question 9

How is a colorimeter used to determine concentration and turbidity of a solution?

Answer 9

A cuvette of the solute is inserted clear sides facing forwards and back as a control, then the unknown solution is inserted in the same way into the colorimeter and an absorbance reading is taken, this is compared to a reading on a standard curve where concentration can be found based on the absorbance of the solution.

Question 10

What is a centrifuge, how does it work and what are the products called?

Answer 10

A centrifuge spins capsules of solution at incredibly fast speeds and separates solutions by their densities into the pellet (bottom) and the supernatant (top).

Question 11

What might paper and thin layer chromatography be used for?

Answer 11

Separating different substances such

as amino acids and sugars.

Question 12

How does affinity chromatography work and what is it used for.

Answer 12

A gel or matrix containing immobilised antibodies/receptors spicific to the antigen/ligand is used to separate the antigens/ligands from a mixture. The antigens/ligands will bind to the antibodies/receptors, other mollecules will pass through.

Question 13

What is native gel electrophoresis and how is it used to separate proteins and nucleic acids?

Answer 13

A charged electric field is used to separate proteins based on charge, shape and size.

Question 14

What is SDS-PAGE gel electrophoresis?

Answer 14

All proteins are denatured and given a uniform negative charge, so the distance they migrate through the gel is based on size alone.

Question 15

What is an isoelectric point (IEP) and how can it be used to separate proteins?

Answer 15

The IEP of a protein is the pH at which it has no net charge (different for every protein), as it is neutral it will precipitate out of solution because water is a polar mollecule so proteins can be separated this way if their IEP is known.

Question 16

How can IEPs be used to separate proteins in gel electrophoresis?

Answer 16

A pH gradient is used so mollecules will stop moving at their IEPs since they have no net charge.

Question 17

How are antibodies used to detect specific proteins or antigens (proteins produced by pathogens)?

Answer 17

Stocks of antibodies with the same specificity in binding, known as monoclonal antibodies are used to bind to the protein. These antibodies are linked to a chemical “label” which could be a flourescent marker, making the protein detectable.

Question 18

What is western blotting?

Answer 18

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 19

What is bright field microscopy used for and how does it work?

Answer 19

Bright-field microscopy is commonly used to observe whole organisms, parts of organisms,
thin sections of dissected tissue or individual cells and uses a lamp under the smaple to illuminate it creating a “bright field” around it.

Question 20

What is flourescence microscopy used for and how does it work?

Answer 20

Fluorescence microscopy uses specific fluorescent labels (e.g. antibodies with flourescent markers) to bind to and visualise certain molecules or structures within cells or tissues.

Question 21

Why is asceptic technique used?

Answer 21

To eliminate unwanted microbial contaminants when culturing micro-organisms or cells.

Question 22

Name 3 different kinds of asceptic technique and give examples of each.

Answer 22

Sterile work environment (bench wiped down with 60% ethanol), sterile equipment (sterilised in an autoclave at high temperature and pressure) and personal (PPE such as gloves and goggles worn to reduce contamination risk).

Question 23

How is a microbial cell culture started?

Answer 23

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

Question 24

What is required for animal cell culture specifically?

Answer 24

Growth factors from serum (the clear part of blood when separated).

Question 25

In culture what is different about the number of times that primary cell lines and tumour cell lines can divide?

Answer 25

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

Question 26

Why might a liquid microbial culture be plated out on solid media?

Answer 26

This allows the number of colony-forming units to be counted and the density of cells in the culture estimated.

Question 27

What may need to be done in order to count a small number of cells in order to estimate density?

Answer 27

Serial dilution (probably log dilution).

Question 28

What is a haemocytometer and how might it be used to estimate cell count in a liquid culture?

Answer 28

It is a glass slide with a counting chamber with a grid on it. The counting chamber has known volume so the cell number can be scaled up to estimate the size of the entire colony.

Question 29

What is used to identify and count viable cells?

Answer 29

Vital staining with a dye such as methylene blue is used, the dye penetrates the membrane of dead cells staining them blue and living cells reamin as they were before.