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AH biology > U1 - KA - ALL Lab Techniques For Biologists In AH > Flashcards

U1 - KA - ALL Lab Techniques For Biologists In AH Flashcards

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1
Q

What is a hazard in a laboratory

A

A hazard is anything that can cause harm or injury to people, that can cause ecological damage if it is released from the laboratory however not all hazards are created equal.

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2
Q

What are the types of hazards

A

1) toxic chemicals : substances which are toxic when inhaled or ingested , injected or absorbed (essentially are poisonous) some are inorganic some are produced by living organisms. Degree of toxicity depends on conc
2) corrosive chemicals : reactive substance that can damage living tissue. Act either directly by chemically destroying part of the body or indirectly by causing inflammation (acids and bases or often corrosive ) (eg sulfuric acid can cause severe burns to skin)
3) flammable substance : lighted Bunsen burners , electro governs , hot plates, steam baths. Flammable substances are this whuch can be easily ignited at room temperature.
4) organisms :some animals and plants can present hazard. Organism which need to be considered in the laboratory are micro organisms. Biggest hazard are presented by pathogenic organisms - they are organism which can cause disease and are a bio hazard
5) mechanical equipment: machines may have moving /vibrating parts.hot surfaces or sharp / heavy components

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3
Q

What is risk in the laboratory

A

Risk is defined as the likelihood of harm rising from exposure to a hazard. A crucial aspect of laboratory work is the control of risks and this is achieved by carrying out a risk assessment

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4
Q

Risk assessment - what does it involve

A
  • identifying risk levels, their severity and the control measures that can be used to minimise these risks
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5
Q

Control measures include…

A
  • using appropriate handling and disposal techniques
  • using appropriate masses , volumes and concentrations of substances
  • use of protective clothing (lab coat or gloves )
  • use of protective equipment such as googles / masks
  • use of aseptic technique in microbiology
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6
Q

Liquids and solutions :
dilution series
- what are they used for

A
  • many substances are found in cells at extremely low concentrations.
  • to estimate the concentration (number of colonies, organisms, bacteria, or viruses) of an unknown sample
  • they can be used to control potential conforming variables in an experiment - to generate a suitable range in an independent variable or as a way of modifying the dependent variable so that a measurable value can be obtained
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7
Q

Linear dilutions series

  • when is it often used
  • what does it consist Of
  • how do you carry out a linear dilution series
A
  • Linear dilution is often used when the substance being diluted is the independent variable
  • linear dilution series consists of a range of dilutions which differ by an equal interval eg solutions of concentration 0.1, 0.2,0.3,0.4,0.5,0.6 would represent a linear dilution series
  • to make a linear dilution series: add different volumes of stock solution to different volumes of solvent (add stock volume then fill up with solvent so to make the same volume as rest )
    In this way each concentration is made up individually and any measurement errors effect only the one concentration
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8
Q

Log dilution series

  • what is it
  • how is it carried out
A
  • a log dilution series consists of a range of different dilutions that differ by a constant proportion eg solutions of concentrations 10-1, 10-2, 10-3 , 10-4 etc would represent a log dilution series.
  • to make a log dilution series , it is normal for each dilution to act as the stock for the subsequent solution. In this way , each concentration depends on those made before and any earlier measurement errors are compounded in later dilutions
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9
Q

Colorimeter :

What is it used to determine

A

colorimeter can be used to determine the concentration of solutions that have been coloured by an indicator Regent by measuring how much light they absorb.
- they can also be used to determine the turbidity of cell culture

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10
Q

How is a colorimeter used

A
  • light is passed through a sample of the solution contained in a small tube called a cuvette ; an electronic sensor detects how much light has been absorbed as it passed through the solution or culture suspension
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11
Q

Turbidity is proportional to the ______ of cells in the culture
- how is colorimter used to measure turbidity

A

Density
- light is passed through a sample of the culture in a cuvette , an electromic sensor detects how much light has been transmitted through the culture suspension

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12
Q

How are standard curve used

A

Plotting measured values for known
concentrations to produce a line or curve
allows the concentration of an unknown to be
determined from the standard curve.

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13
Q

What is a buffer

A

Buffers are solutions that can resist changes of pH even although acid or alkali is added.
This allows the pH of a reaction mixture to be kept constant in spite of the production of acidic or alkaline products

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14
Q

Why are buffers used in vitro experiments

A
  • most biological reactions are dependent on pH, so buffer solutions are often used in in vitro experiments on these reactions so that changes in pH during the reaction don’t act as confounding variables and cause a mistaken association between the independent and dependent variables
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15
Q

What are some separation techniques

A
  • centrifugation
  • paper and thin layer chromatography
  • affinity chromatography
  • gel electrophoresis
  • isoelectric point
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16
Q

What is centrifugation used for

A

Centrifugation is used to separate components of a suspension that have a different density

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17
Q

Eg - diffrent components of cells can be seperated by homogenisation of tissue, followed by centrifugation- explain

A
  • the cell homogenate is placed in a centrifuge tube , which is them spun in a cebtrifuge machime berween 200 and 120000 revolutions per minute
  • after a time , denser components of the cell are seperated into a pellet while less dense components remain susoended in the supernatant
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18
Q

The pellet and supernatant

A
  • denser conponents of the cells seperate into a pellet (the bottom of tube) while less dense components remain suspended in the supernatant
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19
Q

What is paper and thin layer chromatography used for

A
  • can be used to seperate different solutes such as amino acids and sugars
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20
Q

Explaim how paper and think layer chromatography is carried out

A
  • mixtures of these susbstances (eg amino acid mixture) dissolved in a solvent can be added to a paper strip or metal foil strip , which has a thin layer of silica gel bonded to it.
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21
Q

The speed that each solute travels along the strip depends on…..

A

Depends on its differing solubility in the chromatography solvent used , and its differing affinity for the paper or thin layer

22
Q

Solubility and affinity of an amino acid is dependent on its __ ______

A

R group

23
Q

If the susbtances being seperated are colourless (like amino acids) what must be done

A

Susbtances must be made visible on the paper or thin layer using a developing agent

24
Q

What is affinity chromatography used for

A
  • can be used to seperate target proteins from a mixture of proteins
25
Q

How is affinity chromatography carried out

A
  • a solid gel column in a glass tube is produced with specific molecules such as antibodies or ligands bound to the gel.
  • soluble target proteims with a high affinity for these specific molecules become attached to them as a mixture of proteins passes down the column.
  • non target proteins with a weaker affinity or no affinity are washed out , the target protein can then be washed out seperatley and collected
26
Q

what is gel e

What happens in gel electrophoresis

A
  • separation of macromolecules based on size and charge
    charged macromolecules such as proteins or nucleuic acids move through an electric field applied to a buffered gel matrix (polyacrylamide gel electrophoresis or PAGE)
27
Q

What happens when Native PAGE gel is used

A
  • native PAGE gels do not denature the molecules being seperated- they preserve their structure and function , so the seperation is by shape , size and charge, but it is tricky to carry out
28
Q

What happens when SDS PAGE gels are used

A
  • SDS-PAGE gels contain sodium dodecyl sulfate (SDS), which denatures molecules passing through. It gives molecules present an equally negative charge , seperating proteins by size alone.
  • it is simple to carry out , but the structure and function of any seperated protein is lost
29
Q

Proteins have net charges caused by….

A

Caused by the R groups of their hydrophilic surface amino acids

30
Q

What is the isoelectric point of a protein

A
  • the isoelectric point is the pH value at which a protein is electrically neutral , with surface charges that are balanced
31
Q

At pHs below the isoelectric point, the net charges are _______
At pHs above the isoelectric point, the net chargees are _______
-what does this allow

A
  • positive
  • negative
  • allowing the protein to remain in solution or suspension
32
Q

What happens at isoelectric point of a protein

A

At IEP, the protein looses solubility in water and starts to solidify and precipitate out of solution

33
Q

How can IEP be used to seperate a mixture of proteins

A
  • IEP can be used to seperate a mixture of proteins by a type of electrophoresis in which the mixture is passed through a gel with a built in pH gradient. The individual proteins reach their IEP one by one and are collected as precipitate
34
Q

What are immunosassay techniques , and how do they work

A
  • immunoassy techniqes are used to identify specific proteins
  • these techniques use stocks of antibodies with the same specifity known as “monoclonial antibodies” - antibodies that are identical and will bind to exactly the same feature of antigen
35
Q

The antibodies can be linked to a chemical label - explain this

A

Can be linked to a chemical label , often an enzyme will be attached to the antibodies

  • the reporter enzyme produces a colour change identifying a specific antigen
  • reporters that show chemiluminescence , fluorescence or radioactivity can also be used
36
Q

If the antigen is present the antibody will do what

A
  • the antibody binds to it, allowing the reporter enzyme to produce a coloured product
  • if antigen is not present , it wont bind and the enzyme is washed away before any reactiok takes place
37
Q

Explain how a sample would be assayed

A
  • sample to be assayed is added to container, antigenic material sticks to surface (bottom if container)
  • to identify if specific antigen is present , antibody(specific to antigen) with reporter enzyme is added , then washed out
  • reporter enzyme is colourless before added , and will change to coloured product if antigen is present
38
Q

Other immunoassay techniques involve..

A
  • Involve the antibody being pre attached to container
  • sample to be analysed is then added
  • if antigen is present in sample it will bind to antibody , and antibody cant wash away
  • a second antibody with a reporter that binds to the first can be added
39
Q

What is western blotting used for

A
  • is a technique for identifying specific proteins that have been seperated using SDS page gel electrophoresis
40
Q

Explain western blotting

A
  • the proteins are blotted from the gel onto a solid medium and dried
  • proteins can be labelled by soaking blot with specific antibodies, the antibodies bind to specific proteins
  • second antibody with reporter enzymes / fluorescent markers is attached
  • the presence of protein cam be seen , as reporter emzymes substrate will change colour , or seen by fluorescece in UV light
41
Q

What is bright field microscopy used for

A
  • to examine whole organisms, parts of organisms , thin sections of dissected tissue or individual cells
42
Q

What is fluorescence microscopy used for

A
  • uses UV light to detec specific fluorescent staiks , which bind to certain molecules or structures within cells
  • technique is used to visualise the distribution of specifif cellular components in live cells
  • in fluorescense microscopy particular protein structures can be visulised in a way that previously was not possible
43
Q

What does aseptic technique involve

A
  • steralistaion of equipment and culture media by heat or chemical means
44
Q

Why is aseptic technique used

A
  • to eliminate unwanted microbial contaminents when culturing micro organisms or cells
45
Q

How can microbial culture be started

A
  • using an inoculum of microbial cells on an agar medium or in a broth with suitable nutrients
46
Q

What are growth factors

A
  • proteind that promote cell growth and proliferation and are essential for the culture of most animal cells
47
Q

______ dilution is often needed to achieve a suitable colony count

A

Serial

48
Q

What is a haemocytometer

A
  • a microscopic grid used to estimate cell numbers in a liquid culture
49
Q

What is required to count viable cells and why

A
  • Vital staining is required to identify and count viable cells , because the stain can distingusih between cells that are alive or dead.
  • the stain only colours the dead cells
49
Q

What is required to count viable cells and why

A
  • Vital staining is required to identify and count viable cells , because the stain can distingusih between cells that are alive or dead.
  • the stain only colours the dead cells
50
Q

in culture
primary cell lines can divide….
tumour cell lines can perform…

A
  • primary cell lines can divide a limited number of times

- tumour cell lines can perform unlimited divisions

51
Q

plating out of a liquid microbial culture allows…

A

the number of colony forming units to be counted and the density of cells in the culture estimated

AH biology (23 decks)

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