1.1 lab techniques Flashcards
Hazard definition
Anything which can cause harm to a person
Hazards examples
Corrosive chemicals
Heat or flammable substances
pathogenic organisms
Mechanical equipment
COSSH
Control of substances hazardous to health
CLEAPSS
consortium of local educational authority for the provision of science services
Risk definition
The likelihood of a hazard occurring
What is in COSHH
chemicals
Nanotechnology
Biological agents
Standard PPE
Safety goggles
Lab coat
Gloves
Long trousers
Closed toe shoes
Linear dilutions
A series of solutions which differ in concentration by an equal interval
Log Dilutions
A series of solutions which differin concentration by a constant proportion
Making up linear dilutions
Add equal intervals of solution into each test tube and make up to the same volume with water
making up log dilutions
add 1cm of solution from sample into test tube and make up to 10cm3, then use 1cm3 of test tube 1 and make up to 10cm3 in test tube 2 and repeat
Semi log graphs
A graph with a linear axis and a log axis
Advantages of linear dilution
Each concentration is made up individually so errors only affect one solution.
Application of log dilution
Log dilution down the concentration of a bacteria solution
Then count her number of colonies on an agar plate.
Multiply up and you can find the concentration of the original solution
Buffer solution definition
A solution which remains at a constant pH when small quantities of acid or base are added
colourimetry uses
percentage transmission used to measure turbidity
absorbtion to measure colour and concentration
What is a centrifuge used for
Separation of substances of different density
more dense substance in centrifuge
will form a pellet at the bottom of the tube.
What happens to the less dense substance in a centrifuge
It remains in the supernatant
Supernatant
The solvent in a centrifuge
Types of chromatography
Paper
Thin layer
Affinity
Paper chromatography
Uses a special type of paper as the chromatogram, since paper contains cellulose which is polar it will allow for non polar molecules to travel further up the paper.
Thin layer chromatography
Uses silica gel, cellulose or an absorbent material.
Affinity chromatography
Used to separate one specific protein from a mixture by sending the mixture through a column with specific antibodies which will bind to the specific protein. This will mean the specific protein remains in the column whilst other proteins pass through.
Chromatography purpose
To be able to separate and purify the components of a mixture
Chromatography basic principle
Draw a line on your chromatogram a the bottom and apply substance in a dried dot on the line
Place the bottom of the chromatogram in the solvent and put into a chromatography chamber, allow for the solvent front to travel up the chromatogram in the mobile phase.
What affects the RF value of a substance in chromatography
The solubility of the substance in the solution and the ability of the substance to bind to the stationary phase
Rf value
Distance traveled by the dot/distance traveled by solvent front
gel electrophoresis
Where a protein is separated from a mixture/solution using electricity.
Factors affecting SDS page electrophoresis
Based on size
Factors affecting native gel electrophoresis
Size shape or charge
Types of gel electrophoresis
Native gel and SDS page
Native gel electrophoresis
Native gels analyse the proteins in their folded state without denaturing them
SDS page electrophoresis
Gives each protein a negative charge and denatures them to separate the proteins out based on size.
Separation by size alone rule
Small proteins travel further along the electrophoresis
ELIZA definition
A technique which uses antibodies to bind to a specific antigen, which allows a reporter enzyme to produce a colour change and indicate the antigens presence.
Isoelectric point definition
The pH that a protein’s overall charge becomes neutral and it falls out of solution
Immunoassay
Where a specific antibody is used to bind to an antigen on a protein, which allows a reporter enzyme to produce a colour change. This is done to identify a specific protein in a mixture.
ELISA stands for
Enzyme linked immunosorbent assay
Direct ELISA
antigen binds to a multi well plate
Antibody binds to the antigen
A reporter enzyme is added to then well and a colour change occurs.
Types of ELISA
direct ELISA
Indirect ELISA
sandwich ELISA
Indirect ELISA
An antigen will bind to a multi well plate
The primary antibody will bind to the antigen
The secondary antibody binds to the primary antibody
The reporter enzyme is added and causes a colour change
Sandwich ELISA
A primary antibody binds to a multi well plate
An antigen binds to the primary antibody
A secondary antibody binds to the antibody
The reporter enzyme linked to the second antibody causes colour change.
When is western blotting used
After SDS page electrophoresis
What is western blotting
Where after an electrophoresis proteins are transferred to a solid medium
Protein blotting
Western blotting using proteins
Fluorescent microscopy
Where fluorescent light emitting chemicals are used to bind to proteins so they can be identified under a microscope.
Bright field microscopy
A where a sample is put under a microscope and a white light is shined through the sample
Bright light microscopy samples
Whole organisms, parts of organisms, sections of tissues and individual cells
Immunofluorescence
Where antibodies are used to fluorescent tag a protein.
Cell culture
Growing cells in a lab
Culture media contents
Water
Salts
Amino acids
Vitamins
Glucose
Animal cell culture requirements
Growth factors
Aseptic techniques
Eliminate and kill all unwanted microorganism contamination when culturing microorganisms
Precautions taken when working with cultures
Sterilise work area with cleaning products before
Good personal hygiene at all times
Sterile reagents and media must be used
Sterile handling must occur such as wiping down containers with ethanol, flaming containers when opened and exposing the all reagents to air for as little time as possible.
Haemocytometer
A piece of apparatus which create a 0.1 mm chamber with a grid on the side.
Total cell count
Count both viable and dead cells
Dye for dead cells
Trypan blue
Live cell count
Stain dead cells with trypan blue then count the colourless cells.
Percentage viability
Live cells/ total cells x100
