1 - laboratory techniques for biologists

Question 1

How do you identify and control hazards and assess the risk in a lab environment ?

Answer 1

Hazard, risk and control of risk in the lab by risk assessment.

Question 2

What three things can present a hazard in a laboratory?

Answer 2

Substances, organisms and equipment in a lab can present a hazard

Question 3

Give examples of hazards in a lab

Answer 3

Hazards in the lab include toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms, and mechanical equipment.

Question 4

Define the terms risk and risk assessment in relation to health and safety

Answer 4

Risk is the likelihood of harm arising from exposure to a hazard.
Risk assessment involves identifying control measures to minimise the risk.

Question 5

Describe the use of control measures include using appropriate handling techniques, protective clothing and equipment and aseptic technique

Answer 5

Control measures include using appropriate handling techniques, protective clothing and equipment, and aseptic technique.

Question 6

Describe how to carry out linear and log dilution series

Answer 6

Linear dilation series defer by an equal interval. Log dilutions differ by a constant proportion.

Question 7

Describe how unknown concentrations can be determined using a standard curve

Answer 7

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 8

Describe the role of buffers in maintaining and controlling pH

Answer 8

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 9

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

Answer 9

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

Question 10

Describe the use of centrifugation to separate substances of different density

Answer 10

More dense components settle in the pellet and less dense components remain in the supernatant.

Question 11

Describe the use of paper and thin layer chromatography for separating different substances such as amino acids and sugars

Answer 11

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

Question 12

Describe the principle of affinity chromatography and its use in separating proteins

Answer 12

A solid matrix or gel column is created with specific molecules bound to the matrix or 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 13

Describe the principle of gel electrophoresis and its use in separating proteins and nucleic acids

Answer 13

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

Question 14

What is the difference between native gels and SDS PAGE ?

Answer 14

Native gels do not denature the molecule so that separation is by shape, size and charge. SDS PAGE gives all molecules an equally negative charge and denatures them, separating proteins by size alone.

Question 15

What can proteins be separated from a mixture using?

Answer 15

Proteins can be separated from a mixture using their isoelectric points (IEPs)

Question 16

What is an isoelectric point?

Answer 16

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

Question 17

What proteins will precipitate if a solution is buffered to a specific pH?

Answer 17

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

Question 18

How can proteins be separated using their IEPs in electrophoresis?

Answer 18

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

Question 19

What are immunoassay techniques used to detect and identify?

Answer 19

Immunoassay techniques are used to detect and identify specific proteins

Question 20

What are monoclonal antibodies used for?

Answer 20

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

Question 21

What are the types of labels used to identify antibody binding?

Answer 21

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

Question 22

What is the use of Western blotting?

Answer 22

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 23

What can bright field microscopy be used to examine?

Answer 23

Bright field microscopy can be used to examine whole organisms, pats of organisms, thin sections of dissected tissue or individual cells

Question 24

What does fluorescence microscopy use?

Answer 24

Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues fluorescence microscopy allows particular protein structures to be visualised

Question 25

What is aseptic technique and give examples?

Answer 25

Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent excursion of microbial contaminants.
Aseptic technique eliminates unwanted microbial contaminants when culturing microorganisms or cells.

Question 26

How can microbial culture be started?

Answer 26

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

Question 27

What is the role of growth factors from serum in culturing animal cells?

Answer 27

Animal cells are grown in a 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

In culture what is the difference between the amount of divisions primary cell lines can do compared to tumour cell lines?

Answer 28

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

Question 29

What does plating out of a liquid microbial culture on a solid media allow?

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.

Question 30

What is often needed to achieve a suitable colony count?

Answer 30

Serial dilution is often needed to achieve a suitable colony count.

Question 31

Describe the method and use of hemocytometer to estimate cell numbers in a liquid culture

Answer 31

By looking at the grid you can count the number of cells or particles in a specific volume of fluid and thereby calculate the concentration of the cells in the fluid overall.

Question 32

Describe the use of vital staining to identity and count viable cells

Answer 32

Vital staining is required to identify and count viable cells.