1.1 Lab Techniques

Question 1

Define a Hazard

Answer 1

A source of potential harm

Question 2

Define a Risk

Answer 2

The likelihood that a hazard would cause significant harm.

Question 3

What is the porpose of a risk assessment?

Answer 3

Identify control measures to minimise risk
e.g. clothing, handling rechniques and aseptic technique

Question 4

What are the two types of dilutions

Answer 4

• Linear Dilutions
• Log (Serial) dilutions

Question 5

Describe Linear Dilutions

Answer 5

This consists of a range of dilutions that differ by an equal interval. For example, solutions of concentrations 0.1, 0.2, 0.3, 0.4, 0.5

Question 6

Describe Serial Dilutions

Answer 6

A log dilution consists of a range of different dilutions that differ by a constant proportion. For example, solutions of concentrations 10-1, 10-2, 10-3, 10-4 and 10-5

Question 7

Describe the problem with serial dilutions

Answer 7

Each concentration depends on those made before and any earlier measurement errors will be carried over into later dilutions.

Question 8

What is a Standard curve

Answer 8

A standard curve is made by plotting measured values for known concentrations to produce a line or curve

Question 9

What is the purpose of a standard curve?

Answer 9

Once the line (the standard curve) has been produced, it can be used as a reference for any samples of unknown concentration. Through interpolation of the standard curve, we can estimate the concentration of our unknown sample

Question 10

What are Buffers?

Answer 10

Buffers are aqueous solutions that show very little variation in their pH despite addition of acids or alkalis.

Question 11

What is the Role of a Buffer?

Answer 11

Buffers can be selected so that the pH of solutions can be controlled

Question 12

What is the purpose of a colorimetre?

Answer 12

A colorimeter is used to measure the concentration of a pigment in a solution or the turbidity of liquid.

Question 13

How does a colorimetre work?

Answer 13

The machine works by passing particular wavelengths of light through the sample.

Question 14

What does is absorbance determined from a colorimetre used for?

Answer 14

Absorbance is used to determine the concentration of a coloured solution (using a suitable filter)

Question 15

what is a colourimetric blank

Answer 15

a baseline or control value for comparison.

Question 16

What does percentage transmittion determine?

Answer 16

turbidity (such as cells in suspension)

Question 17

What are 4 Separation Techniques?

Answer 17

• Centrifugation
• Electrophoresis
• Isoelectric point
• Chromatography

Question 18

Describe Centrifuging

Answer 18

A centrifuge is a machine used to separate substances by density.

The machine spins samples very fast and the more dense components settle in the pellet (a solid lump found at the bottom of the tube) while less dense components remain in the supernatant (the liquid).

Question 19

Describe Paper and Thin Layer Chromatography

Answer 19

These techniques can be used to separate different substances such as amino acids and sugars by their solubility.

The Substances being tested are spotted towards the base of the chromatography medium and a solvent mix pulls the different constituents up the chromatogram.

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

Question 20

Describe Affinity Chromatography

Answer 20

This technique is used to separate a specific protein from a mixture.

A solid matrix or gel column is created with specific molecules bound to the matrix/gel.

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 21

What is the purpose of Gel electrophoresis

Answer 21

Gel electrophoresis is used to separate proteins and nucleic acids.

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

Question 22

How does Gel electrophoresis work?

Answer 22

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

Question 23

What are the two types of Gel Electrophoresis

Answer 23

Native gels and SDS-PAGE.

Question 24

Describe Native Gels Electrophoresis

Answer 24

Native gels separate molecules by their shape, size and charge. Native gels do not denature the molecule so that separation is by all three of these characteristics

Question 25

Describe SDS-PAGE Electrophoresis

Answer 25

SDS-PAGE separates proteins by size alone. The proteins are denatured by heat in the presence of a detergent which causes them to unfold. This results in a linear protein with an equally negative charge. When these gels are run only size of the molecule becomes a consideration.

Question 26

What is the Isoelectric Point?

Answer 26

Isoelectric point (IEP) is the pH at which a soluble protein has no net charge and will precipitate out of solution

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

Question 27

How can IPE’s be used in Electrophoresis

Answer 27

Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its isoelectric point in the pH gradient because it has no net charge.

Question 28

What are antibodies?

Answer 28

Antibodies are Y-shaped globular proteins produced by B lymphocytes as part of an immune response.

Question 29

What is another name for Antibody techniques

Answer 29

Immunoassay techniques

Question 30

What us the purpose of Immunoassay techniques

Answer 30

Used in the detection and identification of specific proteins

Question 31

What is a Monoclonal antibodies

Answer 31

Each B lymphocyte produces one specific antibody that binds to one specific antigen. This binding helps to render an antigen harmless.

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

Question 32

How do Immunoassay techniques work?

Answer 32

Immunoassay techniques (such as ELISA) involve the use of monoclonal antibodies that have an enzyme attached.

This reporter enzyme catalyses a colour change reaction that is used to detect and quantify the presence of a specific antigen.

If the antigen is present, the antibody binds allowing the reporter enzyme to produce a coloured product; providing visual confirmation of the antigen’s presence. If the antigen is not present then the antibody can not bind and the enzyme is washed away before a reaction can take place.

Blood samples can be screened for either antigens or antibodies themselves using this technique. Chemical labels other than reporter enzymes include chemiluminescence and fluorescence.

Question 33

What is Western Blotting?

Answer 33

Western blotting is a technique for identifying specific proteins that have been separated using SDS-PAGE electrophoresis

Question 34

Describe Western Blotting?

Answer 34

The proteins are blotted from a gel onto a solid medium. It is then flooded with fluorescent labelled monoclonal antibodies (several different ones can be used simultaneously if they have different coloured markers). Once the antibodies have bound to their target proteins, the excess is washed away.

When exposed to particular wavelengths of light the antibodies fluoresce allowing the precise location of target proteins to be identified.

Question 35

What is Bright-field microscopy Used for?

What is Fluorescence microscopy?

Answer 35

Bright-field microscopy is commonly used to observe whole organisms, parts of organisms or 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 36

What is Aseptic technique

Answer 36

Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells. It involves the sterilisation of equipment and culture media by heat or chemical means and therefor the subsequent exclusion of contaminants.

Question 37

Describe A microbial culture

Answer 37

A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients. Many different types of culture media exist that promote the growth of specific types of cells and microbes.

The type of media required should be investigated prior to culturing. Animal cells are grown in medium containing growth factors from serum.

Question 38

Growth factors regarding microbial cultures are….

Answer 38

proteins that promote cell growth and proliferation

Question 39

What is a Cell Line

Answer 39

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

Question 40

Why are cell lines required?

Answer 40

In culture the cells soon use up the nutrients in the medium so to keep the cloned cell line alive some must be sub cultured into a fresh culture flask.

Newly created animal cell lines tend to die after a finite number of divisions (about 60 – the Hayflick limit). This reduces the length of time a primary cell line cultured can be maintained.

However, this limit does not exist in cancer cell lines, which are immortal and can be sub cultured indefinitely

Question 41

How do you count and controll the numbers of cell colonys in cell lines?

Answer 41

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. Even then serial dilution is often needed to achieve a suitable colony count

Question 42

What are Haemocytometers used for

Answer 42

To estimate cell numbers in a liquid culture

Question 43

What is A haemocytometer

Answer 43

A haemocytometer is a graduated microscope slide used to count cell density.

Question 44

Describe the design of A haemocytometer coverslip and its purpose

Answer 44

The coverslip of a haemocytometer slide is designed to sit a known distance from the slide. This means that the viewer is looking through a known volume of medium. By counting the number of cells in a particular area of the grid, a cell density can be calculated

Question 45

What is Viable cell count?

Answer 45

Using a haemocytometer to make total cell counts or if a vital stain is used then the living cells can be distinguished.

Question 46

Give an example of a vital stain

Answer 46

An example of a vital stain is methylene blue; when yeast is viewed under the microscope at high power using this stain the vital or living cells are colourless, whereas dead cells are blue.