1.1 lab techniques for biologists Flashcards
what do risk assessments involve?
identifying hazards and implementing control measures to minimise the risk
what can control measures include?
handling techniques
PPE
aseptic techniques
what is the use of a burette?
to dispense precise volumes of liquid reagents
what is dilation?
reducing the concentration of a substance in a solution
what is serial dilution?
repeated dilution from a stock solution
how do linear dilutions work?
Dilutions differ by an equal interval
how do logarithmic dilutions work?
Dilutions differ by a
constant proportion
what is a colorimeter used for?
to determine the concentration of a pigmented compound
how does a colorimeter work?
it passes a light beam at a specific wavelength through a cuvette containing the sample solution
it measures the absorbance of this wavelength by the solution
how does a standard curve work to determine unknown concentrations?
a series of standards of known concentrations are measured and graphed
this graph is used to determine the concentration of an unknown sample
what is a pH buffer solution?
a solution whose pH changes very little when a small quantity of acid or base is added to it
how do buffers work?
they allow the addition of hydrogen/hydroxide ions without affecting the pH of the solution
according to which factors can substances be separated?
according to their
solubility
size
shape
charge
how does centrifugation separate substances?
according to their size
how do the largest and densest substances behave in a centrifuge?
they separate first and form a pellet at the bottom of the centrifuge
what is the name given to the liquid remaining at the top of the centrifuge?
the supernatant
describe proteins at their iso-electric point (a specific pH)
overall neutral charge and precipitate out of solution
describe the charges of proteins above and below their isoelectric point
positive charge at pH below their IEP
negative charge at pH above their IEP
what happens during isoelectric focussing?
a solution is buffered to a specific pH and only proteins with that IEP will precipitate
how does electrophoresis separate molecules?
based on their charge, size and shape
how does protein electrophoresis work?
current flows through a buffer
the gel acts as a sieve, separating the proteins
which factors does SDS-PAGE separate proteins based upon?
size only - proteins are denatured and given a uniform charge
which factors affect protein migration in gel electrophoresis?
size and charge (shape if proteins are not denatured beforehand)
what does gel electrophoresis separate?
proteins and nucleic acids
what does chromatography separate?
mixtures of amino acids and sugars
the speed that each solute travels along the chromatogram depends on…
its solubility in the solute
how does affinity chromatography work?
a ligand is immobilised and bound to a matrix of a gel column
a target protein which is complementary to the ligand (strong affinity) will bind to it and remain in the column while the others are washed away (weak affinity)
target protein can then be separated and purified
what is the purpose of an immunoassay?
to produce antibodies and use them to identify proteins
which antibodies are used in an immunoassay?
stocks of antibodies with the same specificity (monoclonal)
how do immunoassays work?
the antibody specific to the antigen is linked to a chemical label
what is usually used as a chemical label?
a reporter enzyme that produces a colour change
how can the immunoassay process be used in an alternative way?
by using the antigen to detect the antibody
what is the purpose of protein/western blotting?
to detect proteins from a tissue or cell extract
how does protein/western blotting work?
after SDS-PAGE, the separated proteins from the gel are transferred onto a solid medium and can be identified using specific antibodies with reporter enzymes attached
how does bright-field microscopy work?
a sample is mounted on a slide and illuminated from below
the sample will often be stained beforehand to increase contrast
how does fluorescence microscopy work?
uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
what is the purpose of a haemocytometer?
to estimate the concentration of cells in a sample
describe the process of using a haemocytometer
- cell culture counted and gently mixed
- some pipetted under a coverslip and haemocytometer placed under a microscope
- cells within one of the 1mm x 1mm areas and counted
what does the count of cells in the 1mm x 1mm square provide an estimate of?
the number of cells per 0.1 microlitres of culture medium
how do you get the number of cells per microlitre?
multiply by 10
how do you get the number of cells per cubic centimetre?
multiply by 10000
how can the reliability of the results be affected when using a glass haemocytometer and why?
by incorrect placement of the coverslip - this provides a chamber with unknown volume
how can viable cell counts be estimated?
using vital staining
what is serial dilution sometimes needed for?
to achieve a suitable colony count
what is cell culture?
the ability to grow cells in an artificial laboratory environment
which process require cell culture?
growing bacterial cells
culturing mammalian cells
cancer studies
what does typical cell culture media contain?
water, salts, amino acids, vitamins and glucose
what does animal cell culture contain that plant does not?
growth factors from serum
what is the purpose of growth factors?
to promote cell growth and proliferation
how can a microbial culture be started?
using an inoculum of microbial cells on an agar medium/ broth with suitable nutrients
how many times can primary cell lines divide?
a limited number
how many times can tumour cell lines divide?
unlimited times
what is the aim of aseptic techniques?
the keep the culture free from microorganisms and bacteria
what are the essential aspects of aseptic techniques?
sterile work area
sterile reagents and media
sterile handling
how can equipment and culture media be sterilised?
by heat or chemicals
