1.1 Lab Techniques for Biologists Flashcards

1
Q

what can present a hazard in a lab

A

substances, organisms and equipment

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2
Q

name hazards in a lab

A

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

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3
Q

how are risks minimised

A

by identifying and controlling measures

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4
Q

how are hazards reduced

A

by creating a risk assessment

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5
Q

what is a risk

A

the likelihood of harm arising from exposure to a hazard

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6
Q

what is a risk assessment

A

involves identifying control measures to minimise risk

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7
Q

what do control measures include

A

appropriate handling techniques
protective clothing and equipment
aseptic techniques

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8
Q

why do we often have to dilute substances

A

in order to change their concentration

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9
Q

which methods are used for dilutions

A

linear dilutions

log (serial) dilutions

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10
Q

what are linear dilutions

A

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

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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

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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

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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

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14
Q

why are buffers extremely useful for biological reactions

A

as pH can affect proteins and thus the overall reaction

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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

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16
Q

what are colorimeters used for

A

quantify concentrations and turbidity (cloudiness)

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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)

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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

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19
Q

what is absorbance used for (colorimetery)

A

to determine concentration of a coloured solution using suitable wavelength filters

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20
Q

what is percentage transmission used for (colorimetery)

A

to determine turbidity such as cells in suspension

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21
Q

how are substances separated

A

centrifuge
paper chromatography
thin layer chromatography
affinity chromatography

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22
Q

what is centrifugation

A

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

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23
Q

what is the process of centrifugation

A

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

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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

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25
what is chromatography
a set of techniques which separates the components of a mixture
26
what is paper and thin-layer chromatography used for
to separate different substances such as amino acids and sugars
27
what does the speed of the solute travelling along the chromatogram depend on
its differing solubility in the solvent used
28
what is affinity chromatography used for
to separate proteins
29
what is the process of affinity chromatography
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
what are the staged of affinity chromatography in order
loading separation elution
31
what is loading in affinity chromatography
the protein mixture is poured into teh gel column | the column is lined with receptors specific to protein we are interested in
32
what is separation in affinity chromatography
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
what is elution in affinity chromatography
the target proteins are removed from the receptors and washed out of the column where they can then be retrieved
34
what is gel electrophoresis
a process which applies an electric current across a gel to separate components of a mixture
35
what is gel electrophoresis used for
to separate proteins and nucleic acids
36
how does gel electrophoresis work
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
what separates proteins by their shape, size and charge
native cells | they do this by ensuring they do not denature the molecule
38
what separate the proteins by size only
SDS-PAGE | they do this by giving all the molecules an equal negative charge and denatures them
39
what is the isoelectric point (IEP) used for
to separate proteins from a mixture
40
what is the isoelectric point
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
process of isolelectric point
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
what are immunoassay techniques used for
to detect and identify specific proteins
43
what is immunoassay
technique which uses antibodies to reporter enzymes to cause a colour change in the presence of a specific antigen
44
what are monoclonal antibodies
antibodies with the same specificity
45
what is the antibody specific to the protein antigen linked to in immunoassay
a chemical label
46
what is a label in immunoassay
it is often a reporter enzyme producing a colour change
47
which reporters other than the label can be used
chemiluminescence, fluorescence | in some cases, the assay uses a specific antigen to detect the presence of antibodies
48
what is the ELISA technique
enzyme-linked immunosorbent assay is an analytical technique which uses antibodies to detect the presence of an antibody within a solution
49
what are the forms of ELISA
direct indirect sandwhich
50
what happens during direct ELISA
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
what happens during indirect ELISA
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
what happens in sandwich ELISA
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
what is western blotting
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
what are the types of microscopy
bright field | fluorescence
55
what is bright field microscopy used for
to observe whole organisms, parts of organisms thin sections of dissected tissue or individual cells
56
what is fluorescence microscopy used for
specific labels to bind to and visualise certain molecules or structures within cells or tissues
57
what is aseptic technique
procedures used in laboratories to reduce contamination as well as the unwanted growth or spread of micro-organisms
58
what does aseptic technique eliminate
unwanted microbial contaminants when culturing micro-organisms or cells
59
what does aseptic technique involve
the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants
60
what is microbial culture
a method of multiplying microorganisms by letting them reproduce in a culture medium under controlled laboratory conditions
61
how is a microbial culture started
using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients
62
what do many culture medias promote
the growth of specific types of cells and microbes
63
animal cells are grown in medium containing
growth factors from serum
64
what are growth factors
proteins that promote cell growth and proliferation | growth factors are essential for the culture of most animal cells
65
what are primary cell lines
cell lines which have a limited number of cell divisions and are sourced directly from normal animal tissue
66
what are tumour cell lines
cell lines which have an indefinite number of cell divisions and are sourced from tumours
67
the immortal life of Henrietta Lacks
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
how are cells in cultured estimated
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
what is often needed to achieve a colony count
serial dilutions
70
what is hemocytometer used for
to estimate cell numbers in a liquid cell culture
71
what are heamocytometers
specialised microscope cells which are used to estimate cell numbers in a liquid culture
72
how to use a haemocytometer
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
what is required to identify and count viable cells
vital staining | any living cells will absorb the stain whilst non-living cells do not