1.1 - Cells And Proteins Flashcards
What are buffers?
Buffers are solutions that resist PH changes
Why are buffers used in experiments?
They are used to maintain pH level in experiments in which pH is a possible confounding variable
The solubility and affinity of an amino acid is dependent on what?
It’s r group
What is centrifugation used to?
Separate components of a suspension that have a different density.
How do you separate components of a suspension that have a different density?
Centrifugation
What does homogenisation mean?
Using a motar and pestle, sieve or liquidiser to break open all cells.
Describe the process of centrifugation
A suspension is placed in a centrifuge tube.
The tube is then supn in a centrifuge machine.
After a time,
The denser components are separated into a pellet.
While less dense components remain suspended in the supernatant.
What are toxic chemicals?
Substances that are harmful when inhaled, ingested, injected or absorbed.
What are corrosive chemicals?
A reactive substance that can damage living tissue
What is a reactive substance that can damage living tissue?
A corrosive chemical
How do corrosive chemicals act?
Either by chemically destroying part of the body or indirectly by causing inflammation
What corrosive substances are commonly found in biology labs?
Acids and bases
why should lab workers be aware of sources of heat?
To prevent burns and scalds
What sources of heat should lab workers be aware of?
Lighted Bunsen burners
Electric ovens
Hot plates
Steam baths
What are pathogenic organisms?
Organisms that can cause disease
Are pathogenic organisms a biohazard?
Yes
What pieces of mechanical equipment are hazards that lab workers should be aware of?
Machines that have; Moving parts Vibrating parts Hot surfaces Sharp components Heavy components
What are the main physical risks from mechanical equipment?
Noise
Hand/arm/whole body vibration
Heat stress
What are the main mechanical risks from mechanical equipment?
Cuts
Lacerations
Needle punctures
Crushing
What is risk?
Risk is the likelihood of harm arising from exposure to a hazard
What does a risk assessment involve?
Identifying risk levels
Their likely severity
And the control measures that can be used to minimise risks.
Some control measures include
Using appropriate handling and disposal techniques
Using appropriate masses,volumes and concentrations of substances
Use of protective clothing such as lab coats and gloves
Use of protective equipment such as googles and masks
Use of aseptic technique in microbiology
What are liquid volumes expressed in?
Ml or CM3
When are linear dilutions used?
When the substance being diluted is the idependent variable in an experiment
How do liner dilutions differ from each other?
By an equal interval
How do you make a linear dilution of a substance?
Start with a stock solution with a known concentration
Add increasing, stepped volumes of the stock solution to separate tubes
Then add pure distilled water to each tube to make the volume up to the same for each tube
When are log dilutions often used?
In microbiology,
To estimate the concentration or density of cells in a stock culture.
How do log dilutions differ?
Dilutions in a log dilution differ, by a constant proportion eg. 10-1, 10-2, 10-3
How are log dilutions created?
By diluting a stock solution by a factor and then further diluting the dilution produced by the same factor and so on.
How can log dilutions be used to estimate the concentration or density of cells in a stock culture?
The serial dilution produces low concentrations of cells that can be cultured on an agar plate.
Producing a number of easily countable colonies.
From this result , the estimated number of cells in the original stock can be calculated.
What can colorimeters be used to determine?
The concentration of solutions that have been coloured by an indicator
Or
The turbidity of a cell culture
How does a colorimeter determine the concentration of solutions that have been coloured by an indicator?
Light is passed though a sample of the solution
The sample is contained in a small tube called a curvette
An electronic sensor detects how much light has been absorbed as it is passed through the solution or culture suspension.
What us Turbidity proportional to?
The density of cells in the culture
What is the density of cells in the culture proportional to?
The turbidity
How is the turbidity of a cell culture determined with a colourimeter?
Light is passed through a sample of the culture in a cuvette
An electronic sensor detects how much light has been transferred through the culture suspension
What must be done before measuring concentration or turbidity with a colorimeter?
The instrument must be calibrated using a blank as a baseline
What is a blank?
A curvette containing only the solvent used when making dilutions or a sample of the mediums used in the cell culture.
Most ——————— reactions are dependent on PH
Biological
Why are buffer solutions used in in-vitro experiments?
So that changes in PH during the reaction dont act as confounding variables and cause a mistaken association between independent and dependent variables
A linear dilution of a substance , such as glucose, can be used to produce a ——————- curve
A standard curve
What does homogenisation mean?
Splitting open of cells
What are some homogenation methods?
Pestle and mortar
Sieve
Liquidiser
What does homogenisation mean?
Splitting open of cells
What is centrifugation used to separate?
Components of a suspension that have a different density
How does centrifugiation work?
The cell homgenate is placed in a centrifuge tube
Which us then spun a centrifuge machine at between 200 and 120,000 revolutions per minute
After a time the denser components of the cells are separated into a pellet
While less dense components remain suspended in the supernatant
What is chromatography used to?
Separate different solutes such as amino acids and sugars
How does paper and thin layer chromatography work?
Mixtures of these substances dissolved in a solvent cab be added to a paper strip or to a metal foil strip with a thin layer of silica or cellulose bonded to it.
The speed that each solute travels along the strop depends on its differing solubility in the chromatography solvent used, and its differing affinity for the paper or thin layer.
If the substances being separated are colourless, Luke amino acids, they must be made visible on the paper or thin layer using a developing agent.
The solubility and affinity of an amino acid is dependent on its R group.
What is affinity?
The degree to which a substance is attracted to and tends to bind to another.
What is affinity chromatography?
A technique used to separate and purify proteins based on a specific binding interaction between an immobilised ligand and its binding partner.
What is an antigen?
A specific protein with which antibodies can bind an immune response.
What is aseptic technique?
Procedures in place to prevent contamination, including sterilising equipment and work surfaces.
What is bright-field microscopy?
Microscopy commonly used to observe whole organisms , parts of organisms, thin sections of dissected tissue or individual cells.
What is a buffer?
A solution used to set and maintain a particular pH
What is centrifugation?
A process that used centrifugal forces to separate components of different densities in a mixture.
List 5 hazards found in biology laboratories
Toxic chemicals Corrosive chemicals Flammable chemicals Pathogens Mechanical hazards
Describe what is meant by a risk assessment
Identifying risks
Identifying control measures
Describe how you would make a linear dilution series of a 1M glucose solution
Mix 9ml of stock with 1 ml solvent to give 0.9M
8ml stock with 2ml solvent to give 0.8M
And so on
Describe how you would make estimate the bacterial cell density in a sample of Escherichia Coli in a culture of stock solution
Carry out serial log dilutions to give 10-5/very dilute suspension
Plate out dilute suspension and incubate
Count colonies
Calculate original cell density
Describe how you could use colourimetry and a standard curve to identify the glucose concentration of an unknown solution
Make up glucose solutions of known concentration
Put each through colorimeter to determine absorbance
Draw standard curve
Put unknown through colorimeter
And determine absorbance
Read concentration from standard curve
Explain what buffers are and why they are used in experiments
Solutions that resist pH changes
Used to maintain pH level in experiments in which pH is a possible confounding variable
Explain how centrifugation achieves separation of a suspension into a pellet and a supernatant
Centrifugation separates by density
The most dense material is in the pellet
The least is left in the supernatant
Describe how a solution with three different amino acids can be separated
Add spot of solution to origin line of paper/thin layer chromatography strip
Dip in (appropriate) solvent and allow it to run in
Develop with amino acid stain
Describe how proteins in a mixture can be separated by affinity chromatography
Add mixture to column
Target protein binds to ligand/antibody in column
Other proteins run through
Target protein then collected by washing out column
Explain the difference between native and SDS-PAGE techniques
In native PAGE, proteins are separated in electric field on the basis of mass and charge
In SDS-PAGE proteins are denatured/all given negative change and separated in electric field on the basis of mass
Explain how iso electric point can be used to separate proteins
At its iso electric point, a protein has not net charge, it precipitates
If a mixture is run through a pH gradient gel each protein precipitates at its iso electric point.
Explain what is meant by a monoclonal antibody
Supply of antibodies all with the same specificity/which bind to the same antigen
Describe how a reporter enzyme works
Bound to an antibody that binds to an antigen in immunoassay well
Substrate added, which is converted to coloured protein
Describe western blotting
Proteins separated by gel electrophoresis
Separated protein blotted onto membrane
Treated with reporter
Explain why aseptic technique is used when working with cell cultures
To prevent contamination
Prevent competition with target microbe
