Unit 1 - section 1 Flashcards
Name 2 things that can be intrinsically harmful when working in a lab.
Chemicals
Organisms
Who or what is at risk in a lab?
People
Other organisms
Environment
Name the control measures
Elimination Substitution Isolation Education Personal protective equipment
Describe elimination
The hazardous chemical/ organism is replaced with a less harmful equivalent or the step is removed from the protocol
Describe substitution
If the hazard is very particular to a chemical/ organism involved it may be possible to use alternatives which do not produce the same level of risk
Describe isolation
The procedure would be carried out in a contained environment
Describe education (control measure)
People would be trained to follow standard methods of practice that reduce the risk
Describe personal protective equipment
Can include wearing a lab coat, dust mask, gloves etc
What is the purpose of risk assessments?
To enable any hazards to be identified and ensure appropriate safety is in place to minimise the risk. Ensures working conditions are safe.
What are burettes used for?
For titration. Can make accurate measurements of small volumes
What are pipettes used for?
Less accurate than other methods. Bulb is depressed before insertion into the liquid and then gently (almost fully) released
What are syringes used for?
Very accurate method of measurement
What are autopipettes used for?
Allow w small volumes of liquid to measures very accurately. A disposable tip is used to prevent contamination
What are measuring cylinders used for?
Measuring volumes between 5 and 2000cm3. Scale is read from the bottom of the meniscus
Define the term dilution
To make a solution less concentrated by adding another solution or water
Formula for dilution calculations
V1C1=V2C2
Describe log dilution
Performed using a variety of chemicals or microorganisms.
Involves dilution by 1/10 each time
Useful when culturing microbes
Describe linear dilution
Involves even quantity separations
Useful for standard curves
What is a standard curve and what is it used for?
A graph of known concentrations that allows you to determine unknown concentrations
How can pH be controlled in an experiment?
By adding a buffer solution
2 ways pH can be tested
PH meter
Universal indicator and a colour chart
Describe the process of centrifugation
Used to separate substances by their density.
Define the terms pellet and supernatant
Pellet- solid that’s forms at the bottom of the vessel as the machine spins
Supernatant- the remaining liquid
How are the pellet and supernatant formed during centrifugation
As the material is rotated in the centrifuge tube the resulting g-force causes the constituents to separate
Name the 3 types of chromatography
Paper
Thin-layer
Affinity
Describe paper chromatography
Amino acids with different solubilities travel different distances up the paper support
Describe thin-layer chromatography
Amino acids with differing solubilities travel different distances up the solid support eg glass/ plastic coated in gel
Describe affinity chromatography
Separating biochemical mixtures based on a highly specific interaction. The molecules that are to be separated will bind to the equipment, which will be designed to only attract that substance.
Example interactions- antigen and antibody, enzyme and substrate, receptor and ligand
2 types of molecules that can be separated using chromatography
Amino acids
Proteins
What is responsible for the separation of molecules on chromatography
Paper and thin-layer chromatography- solubility
Affinity chromatography- affinity for specific antibody or ligand
Define electrophoresis
Proteins are put in a gel with opposite charges at either end. The charges cause the proteins to move different distances depending on their charge and polypeptide chain length
2 factors that affect the migration of molecules in the gel (electrophoresis)
By charge or size
What molecules can electrophoresis be used to separate?
Proteins
How can electrophoresis be used to separate molecules in solution?
?
How can pH be used to separate proteins/amino acids
Through iso-electric focusing
Gel set up with pH gradient. Protein/amino acid will migrate until it reaches the pH where it has an overall neutral charge. Since proteins/amino acids carry a charge but have no charge at specific pH they stop at specific points in the gel allowing them to be identified
What happens to amino acids when the overall charge is neutral? (iso-electric focusing)
Amino acids precipitate out of solution
Define iso-electric point
pH at which the amino acid precipitates out of solution
What are antibody techniques used to detect?
Proteins
Feature of antibodies that make them suitable to use to detect proteins
Antibodies bind to specific antigens so specific proteins can be targeted
Define immunoassay
Based on antibody and antigen binding principles. ????
Describe the process of direct ELISA to detect specific antigens
Protein sample added to multi-well plate. Proteins adhere to sides of well
Well washed out to remove non-adhered proteins
Specific antibody for the protein of interest is labelled with a reporter enzyme
Antibody attaches to protein of interest
Well washed out to remove non-adhered antibodies
Substrate added and enzyme causes colour change which identifies presence of specific protein
The deeper the colour, the higher the concentration of specific protein
Describe the process of indirect ELISA to detect specific antigens
Protein sample added to multi-well. Proteins adhere to sides of well
Well washed out to remove no-adhered proteins
Specific primary antibody added and attached to specific protein
Secondary antibody which has a reporter enzyme attached, is added to plate
Labelled antibody attaches to the primary antibody
Well washed out to remove non-adhered antibodies
Substrate added and enzyme causes colour change which identifies presence if specific protein
The deeper the colour,the higher the concentration of the specific protein
Describe the process of capture ELISA to detect specific antigens
Antibody that binds to target protein is added to multi-well plate
Well is washed to remove excess antibody
Protein sample added to multi-well plate
Well washed out to remove non-adhered proteins
Second antibody is added which attaches to target protein
Another antibody that is labelled with a reporter enzyme is added
Labelled antibody attaches to second antibody
Well washed out to remove non-adhered antibodies
Substrate added and enzyme causes colour change which identifies presence of specific protein
The deeper the colour, the higher the concentration of specific protein
What is the role of reporter enzymes in immunoassays?
To catalyse a colour change reaction that is used to detect and quantify the presence of a specific antigen.
If the antigen is present the antibody binds allowing the reporter enzyme to produce a coloured product. (Visual confirmation of the antigens presence)
What is a way antibodies can be labelled for detection
With fluorophore or a reporter enzyme
Describe the process of protein blotting
Protein samples are added to well on electrophoresis gel
Electric current used to separate proteins based on charge or size
Separated proteins are blotted onto a membrane
Membrane treated with fluorescently labelled antibodies that bind to a specific target protein
Membrane passed under UV light. Bound antibodies containing fluorophore fluorescence identifying if target protein is present in sample
Describe the use of immunohistochemical staining
Tissue sample is taken usually from a biopsy
Tissue is stained using a solution that contains labelled antibodies that are specific to a target protein. The label can be a fluorophore or a reporter enzyme
Tissue is washed to remove and unbound antibodies
Substrate added for enzyme that causes colour change or cell sample assessed under UV light. Colour change or fluorescence can be used to identify the presence of target protein
What are monoclonal antibodies and what are they used for
Antibodies that are identical and will bind to exactly the same feature of the antigen
Used to allow a specific antigen to be targeted
Can be used in the treatment of diseases
2 types of cell that are fused when producing monoclonal antibodies
B lymphocytes
Myeloma (cancer) cells
Why are B lymphocytes used to produce monoclonal antibodies?
They allow a specific antigen to be targeted
Why are myeloma cells used to produce monoclonal antibodies?
B lymphocytes do not divide in culture so myeloma cells allow duplicate cells to be produced
What is the name of the cell that results from the fusion of B lymphocytes and myeloma cell?
Hybridomas
Name the technique used for the fusion of B lymphocytes and myeloma cells?
PEG (polyethylene glycol)
What are the 2 types of microscope?
Bright field
Fluorescence
What are bright field microscopes used for?
To examine whole organisms,parts of organisms or thin sections of tissue
What are fluorescence microscopes used for?
To detect specific proteins that have been bound to fluorescently labelled antibodies
Define aseptic technique
Procedure carried out to ensure sterile conditions
State and justify the use of the stages of aseptic technique
Sterilisation of containers, equipment and materials - kills off bacteria/contaminants to avoid cross contamination in the experiment
Disinfection of working area - gets rid of bacteria on surface to prevent contamination interfering with experiment
Wire loop flames before use - gets rid of previous contaminants on the wire loop, keeping it sterile
McCartney bottle lid flamed - sterilises the bottle and remove contaminants
Inoculating loop flames after use - ensures the sample does not infer the future cultures/experiments
Petri dish lid only partly opened - makes sure no outside contaminants interfere
Culture dish sealed - ensure that the materials inside the dish stay in the dish/don’t fall out
Define the term inoculum
Original stock of cells
What are explants and how are they created?
Small cuttings of whole tissue
What is the name of the price of equipment that can be used to calculate cell counts?
A haemocvtometer
What is the purpose of staining when making cells counts?
So you can see the cells
What is vital staining and how does it work?
A method of staining where only the dead cells change colour. Allows you to distinguish between dead and living cells. Both types of cells would take up the dye but the living cells would pump it back out preventing them from changing colour
Define viable cell count
Total number of living cells in the sample
Define non-viable cell counts
Total number of dead cells in the sample
Define total cell count
The total number of living and dead cells in the sample
How would you perform a cell count using a haemocytometer?
Work out volume under grid
Divide 1 by volume to find the number of times the volume goes into 1cm3
Count the cells under the grid
Multiply number of cells by the times the volume goes into 1cm3 to find the cells concentration
What are the contents and purpose of a simple culture medium?
Allows conditions for gas exchange, has a suitable pH and temperature and has a suitable growing surface
Purpose is to provide the basic requirements for cell growth
What are the contents of a complex culture medium?
Contains different nutrients depending on what you are growing
Examples
Growth factors - stimulate proliferation of cells
Serum - source of growth factors
Food source eg glucose - provides energy for proliferation
pH buffer - prevents change in pH
Auxins - plant growth regulator
Why is serum added to media when culturing animal cells?
It is a source of growth factors meaning it stimulates proliferation of cells
What is a monolayer? (when culturing mammalian cells)
Single cell thick confluent layer of cells
Compare the lifetime of primary and cancer cell lines
Primary cells have a shorter lifetime than cancer cells and cancer cells divide infinitely as they don’t have normal controls
What is added to media when culturing plant cells? Why is this required?
Auxins because they regulate plant growth
This allows plants to grow in the most energy absorbing way
