required pracs Flashcards
Give examples of variables that could affect the rate of an enzyme controlled reaction:
Enzyme conc
Substrate conc
Temp of solution
pH of solution
Inhibitor concentration
Describe how temperature can be controlled:
Use a thermostatically controlled water bath
Monitor using a thermometer at regular intervals and add hot/cold water if temperature fluctuates
Describe how pH can be controlled:
Use a buffer solution
Monitor using a pH meter at regular intervals
Why were the enzyme and substrate solutions left in the water bath for 10 mins before mixing?
So solutions equilibrate/reach temp of water bath
Describe a control experiment:
Use denatured enzymes (e.g by boiling)
Everything else same as experiment
Describe how rate of an enzyme-controlled reaction can be measured:
Measure time taken for reaction to reach a set point (e.g conc./volume/mass/colour of substrate/product)
- rate of reaction = 1/time
Measure conc/volume/mass/colour of substrate at regular intervals throughout reaction
- plot graph with time (x) and whatever is being measured (y)
- draw tangent at particular time
- initial rate = change in y/change in x
Suggest a safety risk to dealing with enzymes and how to reduce this risk:
Handling enzymes may cause an allergic reaction
Avoid contact with skin by wearing gloves/eye protection
Explain why using a colorimeter to measure colour change is better than comparison to colour standards:
Not subjective so more accurate
Explain a procedure to stop an enzyme controlled reaction:
Boil/add strong acid/alkali: denature enzymes
Put in ice- lower kinetic energy so E-S complexes can’t form
Add high conc. of inhibitor- no E-S complexes form
Explain why the rate of reaction decreases over time throughout each experiment:
Initial rate is highest as substrate conc. not limiting/many E-S complexes form
Reaction slows as substrate used up, and often stops as there is no substrate left
Describe how to prepare squashes of cells from plant root tips:
Cut a thin slice of root tip using scalpel and mount to slide
Soak root tip in HCl and then rinse
Stain for DNA
Lower coverslip using a mounted needle at 45º without trapping air bubbles
Squash by firmly pressing down on glass slip but do not push sideways
Why are root tips used?
Where dividing cells are found/mitosis occurs
Why is a stain used?
To distinguish chromosomes
Chromosomes not visible without stain
Why squash/press down on cover slip?
To create a single layer of cells
So light passes through to make chromosomes visible
Why not push cover slip sideways?
Avoid rolling cells together/breaking chromosomes
Why soak roots in acid?
Separate cells/cell walls
To allow stain to diffuse into cells
To allow cells to be more easily squashed
To stop mitosis
Describe how to set-up and use an optical microscope:
Clip slide onto stage and turn on light
Select lowest power objective lens
Use coarse focusing dial to move stage close to lens, and then turn to move stage away from lens until image comes into focus
Adjust fine focusing dial to get clear image
Swap to higher power objective lens, then refocus
What are the rules of scientific drawing?
Look similar to specimen
No sketching/shading- only clear continuous lines
Include a magnification scale
Label with straight uncrossed lines
What is a mitotic index?
Proportion of cells undergoing mitosis
Mitotic index = number of cells undergoing mitosis / total number of cells
Explain how to determine a reliable MI from observed squashes:
Count cells in mitosis in field of view
Count only whole cells - standardise counting
Divide this by total number of cells in field of view
Repeat with many/at least 5 fields of view selected randomly so representative
Calculate a reliable mean
Suggest how to calculate the time cells are in a certain phase of mitosis:
Identify proportion of cells in named phase at any one time
Multiply length of cell cycle
What two equations can be used to calculate dilutions?
C1 x V1 = C2 x V2
V2 = V1 + volume of distilled water to dilute with
Describe a method to produce a calibration curve with which to identify the water potential of plant tissue:
Create a series of dilutions using a 1 mol/dm^3 sucrose solution
Use scalpel/cork borer to cut potato into identical cylinders
Blot dry with a paper towel and record initial mass of each piece
Immerse one chip in each solution and leave for a set time in a water bath at 30ºC
Blot dry with paper towel and measure/record final mass of each piece
Calculate % change in mass
Plot a graph with conc. on x axis and % change in mass on y axis
Identify conc. where line of best fit intercepts x axis
Use a table in a textbook to find water potential of that solution
Why calculate % change in mass?
Enables comparison.shows proportional change
As plant tissue samples had different initial masses
Why blot dry before weighing?
Solution on surface will add to mass
Amount of solution on cube varies
Explain the changes in plant tissue mass when placed in different concentrations of solute:
Increase in mass:
- water moved into cell by osmosis
- as water potential of solution higher than inside cells
Decrease in mass:
- water moved out of cells by osmosis
- as water potential of solution lower than inside cells
No change:
- no net gain/loss of water by osmosis
- as water potential of solution same as water potential of cells
Describe a method to investigate the effect of a named variable on the permeability of cell-surface membrane:
Cut equal sized/identical cubes of plant tissue of same age/type using a scalpel
Rinse to remove pigment released during cutting or blot on paper towel
Add same number of cubes to 5 different test tubes containing same volume of water
Place each test tube in a water bath at a different temperature
leave for same amount of time
Remove beetroot and measure intensity of colour of surrounding solution:
- semi-quantitatively: use a known conc of extract and distilled water to prepare a dilution series, and compare results with colour standards to estimate conc.
- quantitatively: measure absorbance of known conc. using a colorimeter and draw calibration curve. Read off calibration curve to find conc. of unknown sample
What are the issues with comparing to a colour standard?
Matching colour is subjective
Colour obtained might not match any of colour standards
Why wash the beetroot before placing it in water?
Wash any pigment off surface to show that release is only due to named variable
Why regularly shake each test tube containing cubes?
To ensure all surfaces of cubes remain in contact with liquid
Maintain a conc. gradient for diffusion
Why control the volume of water?
Too much water would dilute the pigment so solution will appear lighter
So results are comparable
How could you ensure beetroot cylinders were kept at the same temperature throughout experiment?
Take readings in intervals throughout experiment of temperature in tube using a digital thermometer/temp sensor
Use corrective measure if temp has fluctuated
What does a high absorbance suggest about cell surface membranes?
More permeable/damaged
As more pigment leaks out making solution more conc.
Explain how temperature affects the permeability of cell-surface membranes
As temp increases, permeability increases
- phospholipids gain kinetic energy and fluidity increases
- transport proteins denature at high temps as H bonds break, changing tertiary structure
At very low temperatures permeability increases
- ice crystals pierce the cell membrane and increase permeability
Explain how pH affects the permeability of cell surface membrane:
High or low pH increases permeability
- transport proteins denature as H/ionic bonds break, changing tertiary structure
Explain how lipid soluble solvents (alcohol) affect permeability of cell-surface membranes:
As conc. increases, permeability increases
Ethanol may dissolve phospholipid bilayer
Describe precautions that should be followed when performing a dissection:
Cover any cuts with a waterproof dressing
When using a scalpel, cut away from body onto a hard surface
When using a scalpel, use a sharp blade and carry with blade protected/pointing down
Wear disposable gloves and disinfect hands/surfaces/equipment
Safe disposal into bin
If poisonous chemicals, work in well ventilated area
Suggest an ethical consideration when dissecting animals:
Morally wrong to kill animals just for dissection
So use animals for dissection that have already been killed for meat
Describe how you could prepare a temporary mount of a piece of plant tissue for observation:
Add drop of water to glass slide
Obtain a thin section of specimen and place on slide
Stain
Lower coverslip at an angle using mounted needle without trapping air bubbles
Explain examples of aseptic techniques:
Wash hands with soap/disinfect surfaces- kill microbes/prevent contamination
Sterilise pipette/spreader/boil agar growth medium- kill microbes/prevent contamination
Flame neck of bottle of bacteria- kill microbes/prevent contamination
Bunsen burner close- upward current of air draws airborne microbes away to prevent contamination
Lift lid of petri dish slightly/minimise opening- prevent entry of microbes/contamination
Describe a method to investigate the effect of antimicrobial substances on microbial growth:
Prepare area using aseptic techniques
Use sterile pipette to transfer bacteria from broth to agar plate using aseptic techniques
Use a sterile spreader to evenly spread bacteria over plate
Use sterile forceps to place same size discs that have been soaked in different conc./types of antimicrobials fro same length of time onto agar plate
Lightly tape lid onto plate, invert and incubate for 25ºC for 48 hrs
Measure diameter of inhibition zone around each disc and calculate area using π r^2
Why is it important to maintain a pure culture of bacteria?
Bacteria may outcompete bacteria being investigated
Or could be harmful to humans
Why hold lid with 2 pieces of tape instead of sealing completely?
Allows O2 in, preventing growth of anaerobic bacteria which are more likely to be pathogenic/harmful to humans
Why use paper disc with water/no antimicrobial agent?
Acts as a control, ensuring antimicrobial prevented growth, not paper disc
Why incubate upside down?
Condensation drips onto lid rather than agar surface
What if inhibition zones are irregular?
Repeat readings in different positions and calculate mean
Why not use a higher antimicrobial conc?
More bacteria killed. so clear zones may overlap
Why incubate below 25ºC?
Below human body temp so prevent growth of pathogens
Explain the presence and absence of clear zones?
Clear zones: antimicrobial diffuses out of disc into agar, killing/inhibiting growth of bacteria
- larger clear zones so more bacteria killed therefore more effective antimicrobial
No clear zones- if antibiotic is used, bacteria may be resistant/ineffective against that specific bacteria
