1.1 Lab Techniques for Biologists Flashcards Preview

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Flashcards in 1.1 Lab Techniques for Biologists Deck (73)
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1
Q

what can present a hazard in a lab

A

substances, organisms and equipment

2
Q

name hazards in a lab

A

toxic or corrosive chemicals
heat or flammable substances
pathogenic organisms
chemical equipment

3
Q

how are risks minimised

A

by identifying and controlling measures

4
Q

how are hazards reduced

A

by creating a risk assessment

5
Q

what is a risk

A

the likelihood of harm arising from exposure to a hazard

6
Q

what is a risk assessment

A

involves identifying control measures to minimise risk

7
Q

what do control measures include

A

appropriate handling techniques
protective clothing and equipment
aseptic techniques

8
Q

why do we often have to dilute substances

A

in order to change their concentration

9
Q

which methods are used for dilutions

A

linear dilutions

log (serial) dilutions

10
Q

what are linear dilutions

A

dilutions differ by an equal interval e.g. 0.1, 0.2, 0.3 and so on

11
Q

what are log (serial) dilutions

A

dilutions differ by a constant proportion e.g. 10-1 (0.1), 10-2 (0.01), 10-3 (0.001) and so on

12
Q

what is a standard curve

A

a graph which can be used to determine the concentration of an unknown solution
plotting measured values for known concentrations to produce a line of an unknown to be determined from the standard curve

13
Q

what is a buffer

A

a solution where adding acids or alkalis have very small effects on the pH
this allow pH in a reaction mixture to be constant

14
Q

why are buffers extremely useful for biological reactions

A

as pH can affect proteins and thus the overall reaction

15
Q

what is a colorimeter

A

a device that is used to measure the absorbance of a specific wavelength of light by a solution

16
Q

what are colorimeters used for

A

quantify concentrations and turbidity (cloudiness)

17
Q

how do colorimeters work

A

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 a detector picks up how much light has been absorbed by the sample or transmitted (passed through)

18
Q

what has to be done first when using a colorimeter

A

colorimeter needs to be calibrated with an appropriate blank sample to provide a baseline reading

19
Q

what is absorbance used for (colorimetery)

A

to determine concentration of a coloured solution using suitable wavelength filters

20
Q

what is percentage transmission used for (colorimetery)

A

to determine turbidity such as cells in suspension

21
Q

how are substances separated

A

centrifuge
paper chromatography
thin layer chromatography
affinity chromatography

22
Q

what is centrifugation

A

a process which uses centrifugal forces to separate components of a mixture

23
Q

what is the process of centrifugation

A

samples are spun at incredibly high speeds, sometimes up to 18000 rpm

24
Q

how does centrifugation separate substances

A

according to density

more dense components settle to form the pellet whilst less dense components remain in the supernatant

25
Q

what is chromatography

A

a set of techniques which separates the components of a mixture

26
Q

what is paper and thin-layer chromatography used for

A

to separate different substances such as amino acids and sugars

27
Q

what does the speed of the solute travelling along the chromatogram depend on

A

its differing solubility in the solvent used

28
Q

what is affinity chromatography used for

A

to separate proteins

29
Q

what is the process of affinity chromatography

A

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 molecule with a weaker affinity are washed out.

30
Q

what are the staged of affinity chromatography in order

A

loading
separation
elution

31
Q

what is loading in affinity chromatography

A

the protein mixture is poured into teh gel column

the column is lined with receptors specific to protein we are interested in

32
Q

what is separation in affinity chromatography

A

the target proteins bind to the receptors in the gel

non-target molecules are unable to bind so are washed out of the column and disposed off

33
Q

what is elution in affinity chromatography

A

the target proteins are removed from the receptors and washed out of the column where they can then be retrieved

34
Q

what is gel electrophoresis

A

a process which applies an electric current across a gel to separate components of a mixture

35
Q

what is gel electrophoresis used for

A

to separate proteins and nucleic acids

36
Q

how does gel electrophoresis work

A

the 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 will travel further

37
Q

what separates proteins by their shape, size and charge

A

native cells

they do this by ensuring they do not denature the molecule

38
Q

what separate the proteins by size only

A

SDS-PAGE

they do this by giving all the molecules an equal negative charge and denatures them

39
Q

what is the isoelectric point (IEP) used for

A

to separate proteins from a mixture

40
Q

what is the isoelectric point

A

the IEP is the specific pH at which a soluble protein has no net charge and will participate out of a solution
if the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
this can be used along with electrophoresis to separate out proteins

41
Q

process of isolelectric point

A

two different proteins with differing IEPs loaded into a gel matrix
proteins migrate towards the charges intol they reach an area with the pH of their IEP
proteins stop migrating through the gel at its IEP in the pH gradient because it has no net charge

42
Q

what are immunoassay techniques used for

A

to detect and identify specific proteins

43
Q

what is immunoassay

A

technique which uses antibodies to reporter enzymes to cause a colour change in the presence of a specific antigen

44
Q

what are monoclonal antibodies

A

antibodies with the same specificity

45
Q

what is the antibody specific to the protein antigen linked to in immunoassay

A

a chemical label

46
Q

what is a label in immunoassay

A

it is often a reporter enzyme producing a colour change

47
Q

which reporters other than the label can be used

A

chemiluminescence, fluorescence

in some cases, the assay uses a specific antigen to detect the presence of antibodies

48
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

49
Q

what are the forms of ELISA

A

direct
indirect
sandwhich

50
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 well and binds to the antigen

51
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 allowed to bind to the antigen
a second antibody linked to a reporter enzyme, is then added, which binds to the primary antibody

52
Q

what happens in 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

53
Q

what is western blotting

A

used after SDS-PAGE electrophoresis
the separated proteins form the gel are transferred (blotted) onto a solid medium
the proteins can be identified using specific antibodies that have reporter enzymes attached (ELISA)

54
Q

what are the types of microscopy

A

bright field

fluorescence

55
Q

what is bright field microscopy used for

A

to observe whole organisms, parts of organisms thin sections of dissected tissue or individual cells

56
Q

what is fluorescence microscopy used for

A

specific labels to bind to and visualise certain molecules or structures within cells or tissues

57
Q

what is aseptic technique

A

procedures used in laboratories to reduce contamination as well as the unwanted growth or spread of micro-organisms

58
Q

what does aseptic technique eliminate

A

unwanted microbial contaminants when culturing micro-organisms or cells

59
Q

what does aseptic technique involve

A

the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants

60
Q

what is microbial culture

A

a method of multiplying microorganisms by letting them reproduce in a culture medium under controlled laboratory conditions

61
Q

how is a microbial culture started

A

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

62
Q

what do many culture medias promote

A

the growth of specific types of cells and microbes

63
Q

animal cells are grown in medium containing

A

growth factors from serum

64
Q

what are growth factors

A

proteins that promote cell growth and proliferation

growth factors are essential for the culture of most animal cells

65
Q

what are primary cell lines

A

cell lines which have a limited number of cell divisions and are sourced directly from normal animal tissue

66
Q

what are tumour cell lines

A

cell lines which have an indefinite number of cell divisions and are sourced from tumours

67
Q

the immortal life of Henrietta Lacks

A

in January 1951 Henrietta Lacks had a tumour biopsied during treatment for cervical cancer
the cells were taken without her permission and were cultured into a cell line called HeLa
these cell, which are still widely used to this day, is one of the most important cell lines in medical research and have been a source of invaluable medical data to the present day
they are used to study the effects of toxins, drugs, hormones and viruses on the growth of cancer cells without experimenting on humans
they have been used to test the effects of radiation and poisons, to study the human genome, to learn more about how viruses work, and played a crucial role in the development of the polio vaccine

68
Q

how are cells in cultured estimated

A

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

69
Q

what is often needed to achieve a colony count

A

serial dilutions

70
Q

what is hemocytometer used for

A

to estimate cell numbers in a liquid cell culture

71
Q

what are heamocytometers

A

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

72
Q

how to use a haemocytometer

A

caclulate 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

73
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