Lab Techniques

Question 1

What are 4 examples of hazards in the lab?

Answer 1

Toxic chemicals, corrosive chemicals, heat or flammable substances, pathogenic organisms, mechanical equipment

Question 2

What is the difference between a risk and a hazard?

Answer 2

Hazard - source of potential harm; risk - likelihood of harm occurring from a hazard

Question 3

What are some control measures that may be put in place to reduce risks?

Answer 3

Clear the area of combustible materials. Oxidising agents and volatile flammable substances (like meths!) should be covered and kept away from the flame. Hand sanitiser is flammable - make sure hands are dry and sanitiser is away. Sources of draught should be identified. The investigator should have hair tied back, safety glasses on and should be standing. Flame should not be left unattended.

Question 4

Define: accuracy, validity, reliability.

Answer 4

Accuracy is the closeness of a measured value to the true value. Reliability is the overall consistency of a measure. In a valid investigation only one factor will change. All other variables will be controlled.

Question 5

What is the difference between a log dilution and a linear dilution and give an example of each?

Answer 5

Dilutions in a linear dilution series differ by an equal interval, for example 0·1, 0·2, 0·3 and so on. Dilutions in a log dilution series differ by a constant proportion, for example 10-1 , 10-2 , 10-3 and so on.

Question 6

What formula do you use for linear dilutions?

Answer 6

V1C1 = V2C2

Question 7

How can a standard curve be produced and what is an example of a practical application of 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

Why would a buffer be used?

Answer 8

A buffer is a system that resists change in pH. For example – blood pH must be kept between 7.35 to 7.45.

Question 9

Why must a colorimeter be calibrated between uses and what should a calibration ‘blank’ contain?

Answer 9

Sometimes your ‘blank’ will be a solution otherwise it is distilled water. Your ‘blank’ is used to calibrate your colorimeter to provide a baseline.

Question 10

When is it appropriate to use absorbance when using a colorimeter?

Answer 10

Use absorbance with reagents

Question 11

When would transmission be more appropriate?

Answer 11

Cells suspension

Question 12

How can a centrifuge be used to separate molecules?

Answer 12

More dense components settle in the pellet; less dense components remain in the supernatant

Question 13

How can chromatography be used to separate molecules?

Answer 13

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

Question 14

How can affinity chromatography be used to separate molecules?

Answer 14

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 15

How can gel electrophoresis be used to separate molecules. Describe the differences between Native Gel Electrophoresis and SDS-PAGE.

Answer 15

Charged macromolecules move through an electric field applied to a gel matrix. SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone. Native gels do not denature the molecule so that separation is by shape, size and charge.

Question 16

How can molecules be separated using their isoelectric points?

Answer 16

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) that have an IEP of that pH will precipitate. Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.

Question 17

What are immunoassay techniques and how do they work?

Answer 17

There are a number of antibody techniques used in Biology for the detection and identification of proteins. These include: Use of monoclonal antibodies, Use of reporter enzymes, Western Blotting, Use of the ELISA technique

Question 18

What are monoclonal antibodies?

Answer 18

Monoclonal just means all one type. So each MAB is a lot of copies of one type of antibody. MABs are made in a laboratory. These antibodies can be used to detect specific proteins so aid in the detection of certain conditions such as liver damage. MABs are also used as a therapy for treating some cancers.

Question 19

What are reporter enzymes? What do they do? Explain how they are used.

Answer 19

When MABs are used in the detection of disease, reporter enzymes are often used. An antibody specific to the protein antigen is linked to a chemical ‘label’. The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used. In some cases the assay uses a specific antigen to detect the presence of antibodies. This colour change is used to determine a positive test for the protein (an indicator of the disease). The change in colour can also be analysed to determine the quantity of protein in the sample.

Question 20

How and why does fluorescence microscopy work?

Answer 20

Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues. A fluorescence microscope is similar to a bright field microscope but instead of using visible light (400-700 nm) it uses a higher intensity light source that is capable of exciting a fluorescent molecule in the sample on the slide. This molecule then emits a lower energy light of a longer wavelength to produce the magnified image. Fluorescence microscopy allows specific protein structures to be visualised.

Question 21

What can a haemocytometer be used for?

Answer 21

Blood counts, Sperm counts, cell culture, Beer brewing, Cell processing for downstream analysis, Measurement of cell size.

Question 22

How and why is vital staining used?

Answer 22

Vital staining involves adding a stain - usually trypan blue to a cell culture and then observing which cells have taken up the stain. You can then count the number of stained cells (dead) and the number of live cells so that you can estimate a viable cell count.

Question 23

Why do we use the aseptic technique when handling microorganisms?

Answer 23

To avoid contamination

Question 24

What are the 2 main media for culturing cells?

Answer 24

Agar medium or in a broth with suitable nutrients

Question 25

What do we need to include in the medium when growing animal cells?

Answer 25

Serum

Question 26

What is the difference between a primary cell line and a tumour cell line?

Answer 26

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

Question 27

What benefit is there in plating out cultures on solid media?

Answer 27

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 28

Why do we use serial dilutions and plate out all dilutions?

Answer 28

Serial dilution is often needed to achieve a suitable colony count

Question 29

What is vital staining?

Answer 29

Vital staining is required to identify and count viable cells.