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Advanced Higher Biology > Laboratory Techniques For Biologists > Flashcards

Laboratory Techniques For Biologists Flashcards

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1
Q

What can present a hazard?

A

Substances, organisms and equipment in a laboratory.

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2
Q

What is a hazard?

A
  • A toxic or corrosive chemical
  • Heat or flammable substances
  • Pathogenic organisms
  • Mechanical equipment
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3
Q

What is a risk?

A

The likelihood of harm arising from exposure to a hazard

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4
Q

How can Biologists identify and control measures to minimise risks and reduce hazards?

A

Creating a risk assessment

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5
Q

What is a control measure?

A
  • Using appropriate handling techniques
  • protective clothing and equipment
  • Aseptic techniques
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6
Q

What are linear dilutions?

A

When dilutions differ by an equal amount e.g. 0.1, 0.2, 0.3

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7
Q

What are log/ serial dilutions?

A

Dilutions that differ by a constant proportion e.g. 0.1, 0.01, 0.001

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8
Q

What is a standard curve?

A

Plotting known measurements onto a graph to determine unknown measurements

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9
Q

How does a buffer control pH?

A

A buffer is a solution where adding acids or alkalis has very little effect on the pH. This allows pH in a reaction mixture to be kept constant.

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10
Q

What can a colorimeter be used for?

A

A colorimeter can be used to quantify concentration and turbidity (the cloudiness)

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11
Q

How do colorimeters work?

A
  • Before use, the colorimeters need to be calibrated with an appropriate blank sample to provide a baseline reading.
  • Light is split into its component colours and filtered so there is one wavelength of light.
  • This is then passed through the sample solution where detector picks up how much light has been absorbed by the sample or transmitted (passed through)
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12
Q

What can absorbance be used for?

A

To determine the concentration of a coloured solution using a suitable wavelength filter.

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13
Q

What can percentage transmission be used for?

A

Percentage transmission can be used to determine turbidity, such as cells in suspension

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14
Q

How does centrifuge separate substances?

A

Samples are spun at incredibly fast speeds. More dense components settle to form the pellet whilst less dense components remain in the supernatant.

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15
Q

How does paper and thin layer chromatography separate different amino acids and sugars?

A

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

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16
Q

How does affinity chromatography separate proteins?

A

A solid matrix or gel is created with specific molecules, usually receptors, bound to it. Soluble, target proteins in a mixture, 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.

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17
Q

How does Gel electrophoresis separate proteins and nucleic acids?

A

Samples are loaded into wells in a gel which will have an electric current running through it. As they are usually charged molecules they will move towards the opposing charge. Smaller molecules will travel faster than larger molecules so they will travel further.

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18
Q

How do native gels separate proteins?

A

They separate proteins by their shape, size, and charge. They do this by ensuring they do not denature the molecule.

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19
Q

How do SDS-PAGEs separate proteins?

A

They separate by size alone. They do this by giving all molecules an equally negative charge and denature them.

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20
Q

How do IEPs separate proteins from a mixture?

A

Proteins with different IEPs are loaded into a gel matrix. The proteins migrate towards the charges until they reach an area with the pH or their IEP. Proteins stop migrating as they have no net charge and precipitate out.

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21
Q

What happens if the solution is buffered (IEP) ?

A

Only the proteins that have an IEP of that pH will precipitate out.

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22
Q

Explain the relationship between electrophoresis and IEPs

A

Proteins can be separated using their IEPs in electrophoresis

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23
Q

What is immunoassay?

A

Its a technique which is used to detect and identify specific proteins.

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24
Q

What do immunoassay techniques use?

A

Stocks of antibodies with the same specificity, known as monoclonal antibodies.

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25
Q

What is linked to a chemical label?

A

An antigen that is specific to an antibody

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26
Q

What is a chemical label?

A

Often a reporter enzyme producing a colour change.

27
Q

What is immunoassay used for?

A

Using a specific antigen to detect the presence of antibodies to test for diseases.

28
Q

What is western blotting?

A

A technique used after SDS-PAGE electrophoresis.

29
Q

How does western blotting work?

A

Once the proteins have been separated in the gel they are transferred or blotted onto a solid medium. The proteins can then be identified using specific antibodies with reporter enzymes attached.

30
Q

What is bright field microscopy?

A

Form of microscopy commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.

31
Q

What is Fluorescence Microscopy?

A

Form of microscopy which uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.

32
Q

What does an aseptic technique do?

A

Eliminates unwanted microbial contaminants when culturing micro-organisms or cells

33
Q

How can microbial culture be started?

A

Using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients.

34
Q

What are animal cells grown in?

A

A medium containing growth factors from serum. The growth factors promote growth and proliferation.

35
Q

What is the difference between primary and tumour cells?

A

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

36
Q

How can the number of colony-forming units be counted and the density of the cells in the culture be estimaed?

A

Plating out of a liquid microbial culture on solid media.

37
Q

Why is a serial dilution often used?

A

To achieve a suitable colony count.

38
Q

What is a haemocytometer and what does it do?

A

Specialised microscope cells which are used to estimate cell numbers in a liquid culture.

39
Q

What is needed in order to identify and count living cells?

A

Vial staining is required, any living cells will absorb the stain whilst non-living cells do not.

40
Q

List 5 hazards found in biology laboratories

A 
Toxic chemicals
corrosive chemicals
flammable chemicals 
pathogens 
mechanical hazard
41
Q

describe what is meant by a risk assessment

A

Identifying risks; identifying control measures

42
Q

Describe how you could use colorimetry and a standard curve to identify the glucose concentration of an unknown solution

A

make up glucose solutions of known concentration; put each through a colorimeter to determine absorbance; draw standard curve; put unknown concentration through colorimeter and determine absorbance; read concentration from standard curve

43
Q

Explain how centrifugation achieve separation of a suspension into a pellet and a supernatant

A

Centrifugation separates by density; the most dense material is in the pellet, the least dense is left in the supernatant

44
Q

Describe how a solution with three different amino acids can be separated

A

Add a spot of solution to the origin line of a thin layer chromatography strip; dip in appropriate solvent and allow it to run; develop with amino acid stain

45
Q

Describe how proteins in a mixture can be separated by affinity chromatography

A

Add mixture to the column; target proteins bind to ligand/ antibody in the column; other proteins run through; target proteins add then collected by washing out the column

46
Q

What are monoclonal antibodies?

A

antibodies with the same specificity

47
Q

What is the ELISA technique?

A

Enzyme-linked immunosorbent assay is an analytical technique which uses antibodies to detect the presence of an antibody within a solution

48
Q

What are forms of ELISA

A

direct
indirect
sandwich

49
Q

What happens during direct ELISA

A

The antigen is allowed to bind to the surface of a multiwell plate, a primary antibody linked to a reporter enzyme is added to the antigen

50
Q

What happens during indirect ELISA

A

The antigen is allowed to bind to the surface of a multiwell plate, a primary antibody is added to the well and is allowed to bind to the antigen. A second antibody linked to a reporter enzyme, is then added, which binds to the primary antibody

51
Q

What happens during sandwich ELISA

A

A capture antibody is bound to the surface of a multiwell plate, the antigen is added and allowed to bind to the capture antibody. A primary antibody, which binds to the antigen is added to the well a secondary antibody, linked to a receptor enzyme is then added which binds to the primary antibody

52
Q

What is required to identify and count viable cells?

A

Vital staining. Any living cells will absorb the stain whilst non-living cells do not

53
Q

How to use a haemocytometer

A

Calculate out the volume of each of the ‘large’ squares in the grid by multiplying the area by the depth (usually 0.1mm) count the number of cells in each corner grid, including those that are touching the top and left sides, and calculate an average cell density=average cells per square/volume of the small square

54
Q

How is an antibody extracted and purified

A

Centrifugation

55
Q

What are monoclonal antibodies used for

A

Diagnosis and detection of diseases

56
Q

Give some examples of aseptic techniques

A

Pass the neck of a bottle through a Bunsen, pass the loop through a Bunsen

57
Q

How are risks minimised

A

By identifying and controlling measures

58
Q

Why do we often have to dilute substances

A

in order to change their concentration

59
Q

What is gel electrophoresis used for

A

To separate proteins and nucleic acids

60
Q

Explain how isoelectric point can be used to separate proteins.

A

At it’s isoelectric point, a protein has no net charge, it precipitates; if a mixture is run through pH gradient gel each protein precipitates at its isoelectric point

61
Q

why does washing occur in immunoassay, and what is the implication of its not done properly

A

washing between steps ensures that only specific (high affinity) binding events are maintained

62
Q

Explain what is meant by monoclonal antibody

A

supply of antibodies all with the same specificity/ which bind to the same antigen

63
Q

Describe how a reporter enzyme works

A

an enzyme linked to an antibody specific to a protein antigen; they are used in immunoassay techniques

64
Q

Explain why aseptic technique is used when working with cell cultures

A

To prevent contamination; prevent competition with target microbe

Advanced Higher Biology (4 decks)

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