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AH Biology > Lab Techniques > Flashcards

Lab Techniques Flashcards

1
Q

What is a hazard?

A

Anything which could cause harm to people or ecological damage if it’s released from lab

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2
Q

3 types of hazard

A

Substances e.g. toxic/corrosive chemicals and flammable substances
Organisms e.g. pathogenic organisms
Equipment e.g. mechanical equipment if not used correctly

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3
Q

What is risk?

A

Likelihood of harm arising from exposure to a hazard

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4
Q

What does a risk assessment involve?

A

Identifying control measures to minimise the risk

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5
Q

Control measures include using…

A

Appropriate handling techniques
Protective clothing
Protective equipment
Aseptic technique

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6
Q

Control measures do not remove hazard, they…

A

Bring it down to an acceptable level (if not possible lab work cannot proceed)

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7
Q

Why are dilutions used in many experimental procedures?

A

To control confounding variables, generate a suitable range in independent variable or as a way of modifying the dependent variable so a measurable value can be obtained

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8
Q

Solutions allow…

A

Transfer of parts for sampling and are diluted for better/easier analysis

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9
Q

What does a linear dilution series consist of?

A

Range of values that differ by equal intervals

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10
Q

What does a log dilution series consist of?

A

Range of dilutions that differ by a constant proportion e.g. 10-1 10-2 10-3…

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11
Q

To make a linear dilution series…

A

Add different volumes of stock solution to different volumes of solvent, each concentration is made individually so any errors only affect one concentration

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12
Q

To make log dilution series…

A

Each dilution acts as stock for subsequent dilution, each concentration depends on previous ones so any errors are compounded in later dilutions

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13
Q

A colorimeter is used to…

A

Measure concentration of pigment in a coloured solution, the turbidity (cloudiness) of a liquid or density of cells in a culture

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14
Q

A colorimeter works by…

A

Passing a light beam, at a specific wavelength, through a cuvette containing sample solution and recording how much of the light is absorbed by the sample

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15
Q

A colorimeter can display…

A

Absorbance- used to determine concentration of coloured solution
Percentage transmission- used to determine turbidity

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16
Q

What must happen between each colorimetry experiment?

A

Machine is calibrated using ‘blank’ cuvette (contains solution only) which acts as a baseline for comparison

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17
Q

What must be used in colorimetry depending on the solution being measured?

A

Suitable wavelength filters

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18
Q

A standard curve is used to determine…

A

Concentration of an unknown

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19
Q

How is a standard curve produced?

A

A series of known concentrations (‘standards’) are measured and results are plotted to produce a line/curve which is used as a reference for samples of unknown concentration

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20
Q

What are most biological processes dependent on?

A

pH therefore cells and their secretions tend to contain pH buffers

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21
Q

What are buffers?

A

Aqueous solutions that show very little variation in their pH despite addition of acids or alkalis, allow the pH of reaction mixtures to be kept constant

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22
Q

Centrifugation allows substances to be separated according to their…

A

Density (for substances in suspension)

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23
Q

The centrifuge must be…

A

Balanced with samples of similar volumes placed on opposite sides

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24
Q

What does centrifugation separate mixture into?

A

Supernatant- less dense, remains in liquid fraction
Pellet- most dense components at the bottom of the tube

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25
Q

Paper and thin-layer chromatography separates components of mixture according to…

A

Characteristics of solubility, used for amino acids ad sugars

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26
Q

Equation for Rf value…

A

distance travelled by dot
distance travelled by solvent

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27
Q

Steps in paper/TLC

A
  1. Sample being separated placed in dot near bottom of chromatography paper
  2. Paper placed in solvent
  3. Solvent moves through chromatography paper and carries components of mixture
    (TLC is the same process but thin layer is silica gel or cellulose)
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28
Q

Speed that solute travels along chromatogram depends on…

A

Differing solubility in the solvent used
(most soluble=travels furthest)

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29
Q

Affinity chromatography is used to separate…

A

Target proteins from mixture

30
Q

Steps for affinity chromatography

A
  1. Solid matrix or gel column created with specific molecules bound to the matrix or gel
  2. Soluble target proteins in mixture with high affinity for these molecules become attached to them as the mixture passes down the column
  3. Other non-target molecules with weaker affinity are washed out
  4. Column washed with specific molecules that target protein has high affinity for so target molecule binds and is removed from column
31
Q

Gel electrophoresis separates…

A

Proteins and nucleic acids based on charge, shape or size

32
Q

Process of gel electrophoresis

A

Charged macromolecules macromolecules move through an electric field applied to a gel matrix (proteins loads onto gel and then a current is applied to separate them)

33
Q

2 types of electrophoresis…

A

Native gels- separate proteins by shape, size and charge by ensuring that they do not denature the molecule
SDS-PAGE- separate proteins by size alone by giving all molecules an equally negative charge and denaturing them

34
Q

What is the isoelectric point (IEP) ?

A

pH at which a soluble protein has no net charge and therefore protein can form a solid and will precipitate out of solution

35
Q

Proteins can be separated from mixtures using their…

A

IEP’s as if a solution is buffered to a specific pH, only proteins with an IEP of that pH will precipitate

36
Q

How are soluble proteins separated using their IEP’s in electrophoresis?

A

Each protein in sample is loaded onto gel and will move until it reaches the pH corresponding to its IEP
At this pH the protein will migrate no further as it has no net charge so precipitates and forms a band (can be seen after staining)

37
Q

What are antibodies?

A

Y-shaped, globular proteins produced by B lymphocytes as part of the immune response

38
Q

Immunoassay is a technique used to…

A

Detect and identify specific proteins

39
Q

Immunoassay uses…

A

Stocks of antibodies with the same specificity (identical and bind to same feature of antigen, known as monoclonal antibodies)

40
Q

Process of immunoassay…

A
  1. Antibody specific to target antigen is bound to the surface of a multi-well plate
  2. Sample is added and if target antigen is present it binds to antibody
  3. To see if the antigen has bound, another antibody specific to the antigen added is added (is linked to a chemical label)
41
Q

What is a chemical label?

A

Often a reporter enzyme producing a colour change but other reporters can also be used e.g. chemiluminescence, fluorescence

42
Q

Immunoassay can also be used to…

A

Detect the presence of antibodies using a specific antigen

43
Q

Western blotting is a technique used to …

A

Identify specific proteins after they have been separated e.g. by SDS-PAGE electrophoresis

44
Q

Process of western blotting…

A

Separated proteins from the gel are transferred/blotted onto a solid medium (allows final positions to be recorded on a more convenient material
Proteins are then identified using specific antibodies with reporter enzymes attached

45
Q

What are the 2 types of microscopy?

A

Bright field microscopy
Fluorescence microscopy

46
Q

What is bright field microscopy used to observe?

A

Whole organisms (unicellular)
Parts of organisms
Thin sections of dissected tissue
Individual cells

47
Q

What does fluorescence microscopy use?

A

Specific fluorescence labels to bind to and visualise certain molecules or structures within cells or tissues

48
Q

What is the purpose of aseptic technique?

A

To eliminate unwanted microbial contaminants when culturing micro-organisms or cells

49
Q

What does aseptic technique involve?

A

Sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants

50
Q

Examples of aseptic technique

A

Wearing gloves
Washing hands
Cleaning work surfaces
Wearing facemasks
Bunsen burner

51
Q

What is a cell culture?

A

Cells artificially grown in a laboratory environment e.g. for safety

52
Q

How can a microbial culture be started?

A

Using an inoculum of microbial cells on agar jelly or in a broth (liquid medium) with suitable nutrients

53
Q

Many culture media exist which…

A

Promote the growth of specific types of cells and microbes

54
Q

What are growth factors?

A

Proteins that promote cell growth and proliferation, essential for the culture of most animal cells
e.g. foetal bovine serum (FBS)

55
Q

What are animal cells cultured in?

A

Medium containing growth factors from serum which allow rapid growth and cell division

56
Q

What do plant and animal cell cultures require?

A

Complex culture media which contain all the requirements of the cell (water, salts, amino acids, vitamins, glucose)

57
Q

Why are antibodies added to cell cultures?

A

To minimise the chances of spoilage by microorganisms

58
Q

What are cell lines?

A

Genetically uniform cell cultures developed from a single cell

59
Q

In culture primary cell lines can…

A

Divide a limited number of times reducing the length of time a primary cell line can be maintained

60
Q

In culture tumour cell lines can…

A

Perform unlimited divisions (subcultured indefinitely)

61
Q

Why must some cells be subcultured into a fresh culture flask?

A

To keep the cloned cell line alive as cells quickly use up nutrients in the medium

62
Q

What does plating out of liquid microbial culture on solid media allow for?

A

Number of colony-forming units to be counted and density of cells in culture estimated

63
Q

What is often needed to achieve a suitable colony count?

A

Serial dilution

64
Q

What is a haemocytometer?

A

Graduated microscope slide (grid is etched into glass)

65
Q

What can a haemocytometer be used for?

A

Estimate cell numbers in a liquid culture
Make total cell counts
Make viable cell counts (if vital staining is used as this can distinguish living cells)

66
Q

Example of a vital stain

A

Methylene blue

67
Q

What is put over a haemocytometer to allow density to be calculated?

A

Cover slide, creates a chamber with known depth of 0.1mm

68
Q

How can reliability be improved when using a haemocytometer?

A

Counting number of cells in more than one square and calculating average

69
Q

What is the rule for counting using a haemocytometer when cells are half in/out?

A

Top and right border counted
Left and bottom border not counted

70
Q

What is a viable cell count?

A

Number of live, actively growing/dividing cells in a sample

71
Q

What is the total cell count?

A

Number of live and dead cells

72
Q

How are dead and live cells distinguished?

A

Vital stain such as trypan blue dye which is only taken up by dead cells

AH Biology (13 decks)

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