UNIT 1 - lab techniques Flashcards
what is a hazard?
a hazard is anything which could cause harm
what are some examples of hazards in labs?
toxic or corrosive chemicals
heat or flammable substances
pathogenic organisms
mechanical equipment
what is a risk?
a risk is the likelihood of harm being caused by exposure to a hazard
risk assessment meaning?
risk assessment involves identifying control measures to minimise the risk
examples of hazardous substances?
corrosive chemicals - sulfuric acid
toxic chemicals - lead ethanoate (can bioaccumulate in the human body and can non competitively inhibit enzymes)
flammable substances - ethanol
examples of hazardous organisms?
animals - new zealand flatworm
plants - euphorbia plants release latex when cut that causes inflammation to the skin
microorganisms - e. coli is a common lab bacterium
examples of hazardous equipment?
sharp scalpels
glassware
centrifuge loaded unevenly
gas taps
electrical sockets
what are control measures used for?
control measures are used to minimise risk
what are the three main categories of control measures?
appropriate handling techniques - e.g clamping test tube before heating
protective clothing and equipment - lab goggles
aseptic technique - sterilise with ethanol to prevent exposure to or escape of a pathogen
how are dilutions helpful?
dilutions can help with
- controlling confounding variables
- setting up a suitable range for the independent value
- making sure the dependent variable can be measured evenly
what is a pipette?
used for accurately measuring out set volumes
what is an autopipette?
used for very small volumes and has a disposable tip meaning that you can cut down contamination risks when taking samples
burettes
used to add a small volume in a controlled manner
these are used when calculating an unknown concentration.
what is a linear dilution?
a range of dilutions which differ by equal intervals e.g 0.1m 0.2m 0.3m 0.4m
what is good about a linear dilution?
allows for less error in measurement since each dilution is made independently of the others
what is a log dilution?
a range of dilutions that differ by a constant proportion in this, each dilution acts as a stock for the next dilution
what happens if you make an early error in a log dilution?
if you make an early error it is then compounded in later dilutions
what is a buffer?
they are aqueous solutions that are used in experiments to keep the pH of a reaction constant.
what is a standard curve used for?
determining the concentration of an unknown solution
how is a standard curve produced?
by plotting measured values for known concentrations
what is a colourimeter used for?
determining the turbidity of a solution
how does a colourimeter work?
an appropriate blank is used as a baseline.
the measurement of the absorbance is used to calculate the concentration of the coloured solution using suitable wave length filters.
the measurement of percentage transmission is is used to determine turbidity
what is centrafugation?
a technique used to separate substances of differing density. more dense components settle in a pellet; less dense components remain in the supernatant
what is affinity chromatography?
a separation technique used to separate target proteins from a mixture of proteins. this is done as:
- the mixture is put down a gel column and the target protein within the mixture becomes attached to the ligands/antibodies.
- non-target molecules with a weaker affinity pass through the column.
- the target molecules inside the column are washed out separately and then collected
what is gel electrophoresis?
a technique that is used to separate proteins and nucleic acids where charged macromolecules move through an electric field applied to a gel matrix
what is native gel electrophoresis?
this technique separates proteins and nucleic acid based by their shape and size and charge
what are toxic chemicals?
toxic chemicals are substances that are harmful when inhaled, ingested, injected or absorbed. the degree of toxicity is determined by the concentration of the substance
what is a corrosive chemical?
a corrosive chemical is a reactive substance that can damage living tissue. they either act directly by chemically destroying part of the body or indirectly by causing inflammation.
what do native gels do?
native gels do not denature the molecules so the separation is by size, shape and charge
what is SDS-PAGE?
gives all the molecules an equally negative charge and denatures them, separating the molecules by size alone.
what is a proteins isoelectric points?
A protein IEP is the pH value which a protein is electrically neutral with surface charges that are balanced. proteins can be separated from a mixture using their IEPs. this is the pH at which a soluble protein has no net charge and will precipitate out of the solution.
This works as proteins that have a pH above the IEP are positive and proteins with a pH lower than the IEP are negative. This allows them to stay suspended in the solution
at IEP the protein loses solubility in water and precipitates out of the solution.
what happens if the solution is buffered to a specific pH (IEPs)?
if the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
what is a gradient gel used for?
soluable proteins can be separated using an electric field and a pH gradient gel
when does a protein stop moving through the gel in a gel gradient?
when the protein has reached its IEP in the pH gradient because it has no net charge
what is an immunoassy?
they are techniques that are used to detect and identify specific proteins using stocks of antibodies with the same specificity
what are proteins detected by?
proteins can be detected using antibodies
what are monoclonal antibodies?
stocks of antibodies that are specific to a particular antigen
what is a chemical label?
the label is often a reporter enzyme producing a colour change but chemiluminescence, fluorescence radioactivity and other reporters can be used.
what is an antibody specific to the protein antigen linked to?
an antibody specific to the protein antigen is linked to the chemical label
what can be used to detect the presence of antibodies?
in some cases, the assay uses a specific antigen to detect the presence of antibodies
what is western blotting?
a technique used after SDS-PAGE electrophoresis. this is where separated proteins from the gel are transferred (blotted) onto a solid medium.
what are proteins identified by?
specific antibodies with reporter enzymes attached
what is bright field microscopy used for?
this is commonly used to observe the whole organism, parts of an organism, thin sections of dissected tissue or individual cells
what is the aseptic technique?
procedures in place to prevent contamination including sterilising equipment and working surfaces. this is done by heat or chemical means. it eliminates unwanted microbial contaminants..
what is a fluorescence microscopy?
it uses UV light to detect specific florescent stains which bind and visualise certain molecules and structures within cell walls/tissues
what does the aseptic technique eliminate?
contamination and competition
how is a microbial culture started?
using an inoculum of microbial cells on an agar medium or in a broth with suitable nutrients
what is culture media used for?
many culture media exist to promote the growth of specific cells and microbes
how are animal cells grown?
animal cells are grown in medium containing serum with growth factors
what are growth factors?
growth factors are proteins that promote cell growth proliferation. growth factors are essential for the culture of most animal cells.
what are serial dilutions usually used for?
serial dilutions are usually used to achieve a suitable colony count
what is vital staining required for?
vital staining is used to identify and count viable cells. this uses harmless dye to strain either living or dead cells for microscopical observation to allow a viable cell count to be made.
what is a haemocytometer used for?
they are used to estimate cell numbers in a liquid culture
what does plating out a liquid of microbial culture on solid media allow?
the number of colony forming units to be counted and the density of cells in culture to be estimated
what does v1 c1 = v2 c2 mean?
v1 - volume of stock /starting solution
c1 = concentration of stock solution
v2 = volume of diluted solution
c2 = concentration of diluted solution
what is western blotting?
it is used to identify and locate specific proteins in a sample of tissue homogenate or extract based on their ability to bind to specific antibodies.
what is serum?
vitally important as a source of growth factors, hormones, lipids and minerals for the culture of cells
what is the difference between primary and tumour lines
in culture primary cell lines can divide a limited number of times whereas tumour cell lines can preform unlimited devisions.
what is paper chromatography?
- it is used to separate amino acids and sugars.
- it works by adding a spot of the solution to an origin line of a piece of paper or a metal foil strip that has a thin layer of silica/cellulose bound to it.
- the solute is allowed to run.
- the speed that each solute runs along the strip/paper depends on the solubility of the solute used and the differing affinity for the paper or thin layer used.
what are the steps of western blotting?
1) protein is transferred to a membrane by blotting
2) antibodies bind to a protein as the membrane is treated with antibodies specific to a target protein
3) excess is washed away but some antibodies bind specifically to target
4) the second antibody with the radioactive label is added and binds to antibodies present
5) membrane placed against a photographic film with a mark showing where the radioactive label was
