Unit 1- Part 1 Flashcards
What should happen before any activity is carried out in the Laboratory?
The protocol should first be considered in terms of hazards and risk.
What is a hazard?
Anything that poses a potential threat to an individual or the environment.
Describe different types of hazards in the Laboratory.
Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms and mechanical equipment.
What is a risk?
Risk is the likelihood of harm arising from exposure to a hazard.
What is a risk assessment?
A risk assessment needs to be carried out before any practical work in the lab.
What do risk assessments involve?
Identifying hazards, risks and control measures to minimise the risk.
Describe some control measures that can be put in place to reduce the risk.
Using appropriate handling techniques
Protective clothing and equipment (PPE)
Aseptic technique
What is PPE?
Personal Protective Equipment
Why is PPE worn?
PPE is worn to reduce the risk of harmful substances coming into contact with the body.
Why are biological systems put into solution?
They are frequently put into solution for analysis.
What do solution allow you to do?
Solutions allow you to transfer parts for sampling and can be diluted so you can better analyse.
When working with liquids and solutions what should be considered when choosing apparatus?
Accuracy
Precision
The volumes used
What would you use to make a really large dilution?
Log dilution series
What would you use to make a small dilution?
Linear dilution series
Describe a linear dilution series?
Dilutions in a linear dilution series differ by an equal interval, e.g. 0.1, 0.2, 0.3, and so on.
How is a linear dilution series achieved?
This is usually achieved by using different volumes of the same stock solution and combining with different volumes of a suitable solvent, e.g. water.
Describe the formula C1V1=C2V2
Use the formula C1V1 = C2V2
C1 = stock concentration
V1 = volume of stock required
C2 = concentration required for working solution
V2 = volume required of working solution
Describe a Log dilution series.
Dilutions in a log dilution series differ by a constant proportion, e.g. 10-1, 10-2, 10-3, and so on.
How is a Log dilution series is achieved?
This is usually achieved by using successive dilutions as the new stock solution.
When are log dilution series useful?
Are particularly useful when diluting bacterial suspensions.
A large scale dilution is usually required to obtain countable numbers of colonies.
Often a range of dilutions are made and then plated out onto nutrient agar plates until a countable number of colonies is achieved.
What is a standard curve used to determine?
The concentration of a solution.
Describe a standard curve.
For a standard curve, a series of “standards” of known concentration are measured and graphed.
This graph can then be used to determine the concentration of an unknown solution.
What is a titration used to determine?
Titrations can be used to determine the concentration of an unknown solution.
Describe how to do a titration.
Titrations are carried out using a burette to deliver a solution of known volume and concentration to a solution of unknown concentration underneath.
The volume and concentration of the known solution taken to reach the endpoint of the reaction are used to calculate the concentration of the unknown solution.
Describe a Buffer.
Buffers are an important addition to an in vitro experiment, e.g. during an enzyme assay. It allows the pH of the reaction mixture to be kept constant (minimising pH as a confounding variable) because the addition of acid or alkali has very small effects on the pH of a buffer
What experiment is a Buffer useful in?
In Vitro
Describe how a colorimeter works.
It works by passing a light beam, at a specific wavelength, through a cuvette containing a sample.
Some of the light is absorbed by the sample, light transmitted is detected and displayed as an absorbance value.
What does a colorimeter measure?
A colorimeter measures the absorbance or transmission of light through a solution.
What can we determine by measuring the absorbance of light by a sample?
By measuring absorbance of light by a sample, we can determine the concentration of a coloured substance using suitable wavelength filters.
What is the relationship is there between absorbance and concentration?
A linear relationship between absorbance and concentration.
By measuring percentage transmission of light through a sample, what can we measure?
We can measure turbidity of a sample.
A colorimeter is used when calculating the cells in a suspension in microbiology work. What is the relationship between transmission and turbidity?
The lower the transmission, the higher the turbidity.
Tell me how to set up a colorimeter
To use a colorimeter the correct wavelength of light must be selected
An appropriate reference blank is required to calibrate the instrument and provide a baseline reading.
This is usually distilled water but depends on the reaction being investigated. The references should show you the absorbance in the absence of your coloured product.
The blank is placed in the colorimeter and the calibration button pressed (usually the R button). This will give a reading of 0.
Then sample cuvettes can be used by pressing the test button (T)
Biological systems often involve mixtures, name a few types of mixtures
Cells, DNA fragments, proteins and other compounds.
What can biological mixtures be separated based on?
These can be separated based on differences between the component parts. This can be differences in; solubility, size, shape or charge, or indeed any combination of these attributes.
What is a centrifuge?
A centrifuge is a piece of equipment that spins a sample at high speed.
What does centrifugation separate materials based on?
Density
Describe what happens in a centrifuge once it stops, where do the different densities of material go?
The largest and densest materials separate out first and form a pellet at the bottom of the tube. The less dense components remain in the supernatant.
What are paper and thin layer chromatography used for?
Are used for separating different substances such as amino acids and sugars. The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.
What is paper chromatography?
Uses paper as the stationary phase and the solvent as mobile phase.
What is thin layer chromatography?
Uses cellulose or silica gel on a glass plate as stationary phase
Describe Affinity chromatography
Is used to separate proteins.
A solid matrix or gel column is created with specific molecules bound to the matrix or gel.
Soluble, target proteins in a mixture, with a high affinity for these molecules.
The protein mixture is passed through the column.
The target proteins become attached to the molecules.
Other non-target molecules with a weaker affinity are washed out.
What is Gel electrophoresis used to do?
Is used to separate proteins and nucleic acids.
What does Gel Electrophoresis separate molecules based on?
This process separates proteins based upon their charge and/or size/shape.
Describe Gel Electrophoresis
Protein electrophoresis uses current flowing through a buffer to separate proteins. The gel used in protein electrophoresis acts as a sieve, separating the proteins.
Describe SPD page electrophoresis
SDS-PAGE electrophoresis, this procedure denatures proteins and they are given a uniform negative charge.
This means that the proteins can be separated based on their size alone.
Small proteins travel further through the gel than large proteins.
Describe Native Gel electrophoresis.
Native gel electrophoresis is when the protein is not denatured before the gel electrophoresis. This allows the scientist to analyse the proteins in their folded state. In this case, separation is by shape, size and charge.
What is the Isoelectric point?
The IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
Describe the use of the Isoelectric point in separating proteins?
If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate.
Proteins can also be separated using their IEPs in electrophoresis.
Soluble proteins can be separated using an electric field and a pH gradient.
A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What are Immunoassay techniques used to detect and identify?
Immunoassay techniques are used to detect and identify specific proteins.
Immunoassay techniques use to stocks antibodies with what to recognizes one antigen?
These techniques use stocks of antibodies with the same specificity, recognises one antigen.
hese techniques use stocks of antibodies with the same specificity, recognises one antigen.
What are these antibodies known as?
Monoclonal antibodies.
Describe Immunoassay techniques.
Immunoassay techniques are used to detect and identify specific proteins.
These techniques use stocks of antibodies with the same specificity, recognises one antigen.
These antibodies are known as monoclonal antibodies. An antibody specific to the protein antigen is linked to a chemical ‘label’ to allow scientists to detect when binding has occurred.
The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used. In some cases the assay uses a specific antigen to detect the presence of the antibodies.
Bright-field microscopy is commonly used for what?
Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.
What does Fluorescence microscopy use?
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
What does aseptic techique do?
Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells.
What does aseptic technique involve?
This involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
Sterilising benches with virkon
When opening bottles – the neck must be immediately passed through a hot flame
Complete work close to a Bunsen burner flame where air currents are drawn upwards.
When using petri dishes, limit exposure of the sterile inner surface to contamination from air.
A microbial culture can be started using what?
A microbial culture can be started using an inoculum (a small volume) of microbial cells on an agar medium, or in a broth with suitable nutrients.
What exists to promote the growth of specific types of cells and microbes.
Culture media
