1.1 Lab Techniques for Biologists Flashcards
what can present a hazard in a lab
substances, organisms and equipment
name hazards in a lab
toxic or corrosive chemicals
heat or flammable substances
pathogenic organisms
chemical equipment
how are risks minimised
by identifying and controlling measures
how are hazards reduced
by creating a risk assessment
what is a risk
the likelihood of harm arising from exposure to a hazard
what is a risk assessment
involves identifying control measures to minimise risk
what do control measures include
appropriate handling techniques
protective clothing and equipment
aseptic techniques
why do we often have to dilute substances
in order to change their concentration
which methods are used for dilutions
linear dilutions
log (serial) dilutions
what are linear dilutions
dilutions differ by an equal interval e.g. 0.1, 0.2, 0.3 and so on
what are log (serial) dilutions
dilutions differ by a constant proportion e.g. 10-1 (0.1), 10-2 (0.01), 10-3 (0.001) and so on
what is a standard curve
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
what is a buffer
a solution where adding acids or alkalis have very small effects on the pH
this allow pH in a reaction mixture to be constant
why are buffers extremely useful for biological reactions
as pH can affect proteins and thus the overall reaction
what is a colorimeter
a device that is used to measure the absorbance of a specific wavelength of light by a solution
what are colorimeters used for
quantify concentrations and turbidity (cloudiness)
how do colorimeters work
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)
what has to be done first when using a colorimeter
colorimeter needs to be calibrated with an appropriate blank sample to provide a baseline reading
what is absorbance used for (colorimetery)
to determine concentration of a coloured solution using suitable wavelength filters
what is percentage transmission used for (colorimetery)
to determine turbidity such as cells in suspension
how are substances separated
centrifuge
paper chromatography
thin layer chromatography
affinity chromatography
what is centrifugation
a process which uses centrifugal forces to separate components of a mixture
what is the process of centrifugation
samples are spun at incredibly high speeds, sometimes up to 18000 rpm
how does centrifugation separate substances
according to density
more dense components settle to form the pellet whilst less dense components remain in the supernatant
what is chromatography
a set of techniques which separates the components of a mixture
what is paper and thin-layer chromatography used for
to separate different substances such as amino acids and sugars
what does the speed of the solute travelling along the chromatogram depend on
its differing solubility in the solvent used
what is affinity chromatography used for
to separate proteins
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.
what are the staged of affinity chromatography in order
loading
separation
elution
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
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
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
what is gel electrophoresis
a process which applies an electric current across a gel to separate components of a mixture
what is gel electrophoresis used for
to separate proteins and nucleic acids
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
what separates proteins by their shape, size and charge
native cells
they do this by ensuring they do not denature the molecule
what separate the proteins by size only
SDS-PAGE
they do this by giving all the molecules an equal negative charge and denatures them
what is the isoelectric point (IEP) used for
to separate proteins from a mixture
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
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
what are immunoassay techniques used for
to detect and identify specific proteins
what is immunoassay
technique which uses antibodies to reporter enzymes to cause a colour change in the presence of a specific antigen
what are monoclonal antibodies
antibodies with the same specificity
what is the antibody specific to the protein antigen linked to in immunoassay
a chemical label
what is a label in immunoassay
it is often a reporter enzyme producing a colour change
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
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
what are the forms of ELISA
direct
indirect
sandwhich
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
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
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
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)
what are the types of microscopy
bright field
fluorescence
what is bright field microscopy used for
to observe whole organisms, parts of organisms thin sections of dissected tissue or individual cells
what is fluorescence microscopy used for
specific labels to bind to and visualise certain molecules or structures within cells or tissues
what is aseptic technique
procedures used in laboratories to reduce contamination as well as the unwanted growth or spread of micro-organisms
what does aseptic technique eliminate
unwanted microbial contaminants when culturing micro-organisms or cells
what does aseptic technique involve
the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants
what is microbial culture
a method of multiplying microorganisms by letting them reproduce in a culture medium under controlled laboratory conditions
how is a microbial culture started
using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients
what do many culture medias promote
the growth of specific types of cells and microbes
animal cells are grown in medium containing
growth factors from serum
what are growth factors
proteins that promote cell growth and proliferation
growth factors are essential for the culture of most animal cells
what are primary cell lines
cell lines which have a limited number of cell divisions and are sourced directly from normal animal tissue
what are tumour cell lines
cell lines which have an indefinite number of cell divisions and are sourced from tumours
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
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
what is often needed to achieve a colony count
serial dilutions
what is hemocytometer used for
to estimate cell numbers in a liquid cell culture
what are heamocytometers
specialised microscope cells which are used to estimate cell numbers in a liquid culture
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
what is required to identify and count viable cells
vital staining
any living cells will absorb the stain whilst non-living cells do not
