PRACTICAL SKILLS Flashcards
What 5 things can you are 5 variables you can change on rate of enzyme controlled reaction
enzyme conc
substrate conc
temperature
pH
inhibitors
What can you use as a control when testing pH with enzyme/
use water instead of trypsin solution (buffer + trypsin)
proves that temp or buffer itself is not causing the digestion of the substate (only the enzyme is)
What does it mean if no change has occurred beyond around 25mins/
the enzyme has denatured
What must you do before you find means between trials
eliminate the anomalies
what is important when you are plotting data on a table
all figures go to 3sf
how do you find rate of reaction
1 / time taken
what do you plot on y and x axis when finding effect of variable on rate in enzyme controlled reaction
mean reaction rate (yaxis)
variable e.g pH (x-axis)
When do you go dot to dot or find best line of fit line?
when not enough data points to predict a patter, go dot to dot
if you can easily predict the patter then draw line of best fit
You must always critique the method and not the experimenter, hence what are some method limitations when testing variable against rate on enzyme controlled reaction?
- end point is subjective, e.g the point at which the film turned clear is subjective leading to ‘inaccurate’ time taken measurements
- water bath isnt thermostatically controlled so temperature is deacuresing throughout duration of practical
- not enough intermediate variables being tested to accurately identify the optimum values
- range of variable is not wide enough hence unable to find the point at which the enzyme denatures
You must always critique the method and not the experimenter, hence what are some method limitations when testing variable against rate on enzyme controlled reaction?
- end point is subjective, e.g the point at which the film turned clear is subjective leading to ‘inaccurate’ time taken measurements
- water bath isnt thermostatically controlled so temperature is deacuresing throughout duration of practical
- not enough intermediate variables being tested to accurately identify the optimum values
- range of variable is not wide enough hence unable to find the point at which the enzyme denatures
Why is HCL, acetic orcein stain, mounted needle and root tip of plant used?
HCL - softens and loosens the root tissues so when it comes to squashing the root its easier
acetic orecien stain - stains chromosomes in the nucleus so they are visible under the microscope
mounted needle - lowers the cover slip at 45 degree angle to prevent air bubbles under the slide
root top- growing region where mitosis is occurring
describe process of producing mounted sample to see mitosis
heat 5mm root top with HCL in a 60 degree water bath for 10 mins
pour out acid and place root tip on watch glass, rinse with distilled water
place root tip on microscope slid and soak any excess water with filter paper
add 2 drops of acetic orcein stain to root tip
use mounted needle and lower coverslip and leave for 10 mins (provides time to stain chromosomes)
place filter paper on top of slide and gently press down (dont press to hard and break glass or slide glass to side)
why must you squash the root tip
speeds out the cells to get a single layer 1 cell thick so light can pass thru when viewing through microscope
what might happen if u slide the cover slip?
end up sliding a layer of cells ontop of another layer and the light won’t pass thru or damage the chromosomes so it ruins image
What are hazards, risk and precaution in mitotic index rp
scalpel , cut urself, cut ontop of white tile to prevent slipping and cut away from body
HCL, irritant, wear goggles and wash hands
how do you calculate mitotic index
number of cells in mitosis / total number of cells (x100)
why are
why is root tip stained
so chromosomes are visible
How can you ensure accuracy counting of cells to find mitotic index
examine for large number of fields of view then find average value
this ensures its more representative of entire root tip
Why is temp controlled when experimenting on osmosis
kinetic energy will affect the rate of osmosis so it must be kept the same for all experiments
Why must all test subjects of osmosis be the same length/have the same surface area
it affects rate of diffusion
why must you blot the test subject dry before weight
so that the excess water on the outside of the cells dont effect the initial mass
why do you measure the initial and final mass of test subject
to find change in mass to see if there was gain or loss due to osmosis
Why do you find percentage change in mass
fair comparisons as it takes into account that not all test subjects will have had the same initial mass
What are the axis of a calibration curve when testing osmosis
y-axis = percentage change in mass
x-axis = sucrose concentration
How do you find the water potential of test subject from the calibration curve
find the point at which the curve crosses the x-axis
use a conversion table to look up the conversion of mol dm=3 into water potential
how do you make a serial dilution
What should you undergo if asked to find either the qualitative or quantitative results on membrane permeability
qualitative = observations of colour
quantitative = use colorimeter and compare absorbance
How is a colour standard made when comparing the pigments release from eating cell membrane permeability?
What are the axis on the colour standard calibration curve?
y-axis = absorbance at 520nm (or any wavelength depending on the colour of the pigment)
x-axis + percentage extract (of pigment extract in solution)
when testing permeability of cell membranes, what must be controlled and why
temperature as it affects the permeability of cell membranes
should be around 30 degrees as anything above may begin to denature membrane proteins causing more pigment to be released
Why must a bung be placed in each test tube when testing cell permeability
to ensure the alcohol does not evaporate and change the concentration of the solutions that the “beetroot” is in
Why must 2 discs of beet root be put into each test tube?
so theres same surface area and amount so theres same potential maximum amount of vettelin that could move out
Why is beetroot blotted dry before placed in test tube
remove excess water and remove excess betalin that is caused by the cutting of the beetroot
Why should you shake the tubes with alcohol and beetroot every min
ensures it mixes fully with the alcohol and ensures the betalin can be released from the vacuole
How do we find the percentage extract of beetroot from our results when testing beetroot pigment with different concentrations of alcohol
make calibration curve of percentages conc of ethanol (x-axs) with absorbance (y-acxis)
extrapolate the chosen conc of alcohol back to the line and find the absornace
use this absorbance on the colour standard calibration curve and extrapolate back to find percentage extract
Why would these conditions cause betalin to be released : higher temp, alcohol, acid
high temp = membrane protein denature so more betalin released
alcohol = phospholipid soluble in alcohol so alcohol will start to dissolve the phospholipid bilayer causing damage and pigment to be released
acid = causes membrane proteins to denature and pigment can be released
Whats the use of dehydrogenase enzyme + where is it found
naturally occurs in chloroplasts and catalyses reactions involved in NADP accepting electrons (from photoionisation of chlorophyll and photolysis)
What is DCPIP
redox indicator that turns from blue to colourless when it gets reduced/gains electrons
(picks up electrons from LDR instead of NADPH)
Why is ammonium hydroxide used in finding effect of a named factor on the rate of dehydrogenase activity in extracts of chloroplasts.
its an alkaline which can denature the dehydrogenase enzyme
it can also accept electrons instead of DCPIP and NADP
what is the hypothesis on effect of a ammonium hydroxide on the rate of dehydrogenase activity in extracts of chloroplasts.
rate of reaction decrease with addition of ammonium hydroxide
explain the 5 different test tubes you have when testing the effect of a ammonium hydroxide on the rate of dehydrogenase activity in extracts of chloroplasts.
How do you form the chloroplast suspension and explain
homogenise/blend spinach leaves and isolation medium which is ice cold with salts to make it isotonic
filter through muslin cloth
What do you do with ur 5 test tubes to test effect of a named factor on the rate of dehydrogenase activity in extracts of chloroplasts.
place at a set distance from light (as its the light dependant reactant)
wait for the colours of test tubes to turn the same colour as the testable with only with chloroplast solution (proved that DCPIP has decolourised)
time how long that took
limitations in effect of testing ammonium hydroxide on the rate of dehydrogenase activity in extracts of chloroplasts.
end point is subjective
unequal distribution of light
foil isnt blocking out light fro all directions
limitations in effect of testing ammonium hydroxide on the rate of dehydrogenase activity in extracts of chloroplasts.
end point is subjective
unequal distribution of light
foil isnt blocking out light fro all directions
How does a respirometer work and what is it used for
used to estimate rate of respiration
works due to volume and pressure changes caused by gas ABSORPTION
What are the 2 different test tubes in a repirometer and why are they sealed with bungs and what are they connected by
control tube and experimental tube
bung make test tubes air tight as we are looking at changes in volume and pressure so have to ensure no gases enter or leave
thin capillary tube known as manometer contains a red liquid and is connected to the 2 tubes
Why is there a scale/rule behind the manometer?
so you can measure how far the liquid moves over a period of time
Why is there a string on the control tube
if you wish to do repeats, you can reset your equipment so you can move the res liquid back to the starting point
Why is there a string on the control tube
if you wish to do repeats, you can reset your equipment so you can move the res liquid back to the starting point
What is in the experimental tube?
respiring organism e.g maggots separated from the soda lime (which absorbs carbon dioxide) by a metal mesh
What is in the control tube?
glass beads and soda line
Why is there a metal mesh separating the respiring organism from soda lime
just incase the soda lime irritates the organism
Why is soda lime used
to absorb carbon dioxide and ensure any changes in volume and pressure are purely due to oxygen being absorbed
Why are gas beads used
same mass of glass beads used as respiring organism as they won’t be retiring and can act as a control
explain how the red liquid moves
in the experimental tube, the respiring organism is using up oxygen and the co2 it produces is absorbed by the soda lime so the overall pressure in the experimental tube decreases and volume of gas in experimental tube decreases
no respiration is occurring in control tube volume of gas and pressure in control tub is the same BUT it has a higher pressure compared to experimental tube
so force exerted on liquid in manometer and pushes it towards the experimental tube
How do you find rate of respiration from respirometer
rate = volume / ( time x mass)
units= cm3 min-1 g-1
volume of manometer (cross section are of manometer x change in length of red liquid)
mass of respiring organisms
What is the use of dehydrogenase enzyme in yeast
catalyses reactions involving removing H from coenzymes and carbon compounds
(involved in all stages of aerobic respiration when NADH is made but also is oxidative phosphorylation where NADH is re-oxidised to NAD.
What is TTC
redox indicator which is colourless when oxidised and red when reduced
it can pick up a hydrogen removed by dehydrogenase in respiration
What happens as you increase them temp to the yeast and TTC mixture
itll turn red faster indicating a faster rate of reaction as the temp increases
describe the process of varying temp on the enzyme reaction of sugar and yeast with TTC and determining rate of reaction
place TTC and yeast sugar mixture in separate test tubes and place in thermostatic water bath, leave for 5 mins to equilibrate the temps of both solutions
when both solutions are same temp, combine the solutions and start a timer, wait for the solution to turn a certain standard colour of pink (not red because the yeast mixture is beige).
ensure to mix whilst waiting as the yeast can fall to the bottom of the tube
when the colour of solution has been reached, record the time taken
repeat experiment at varying temperatures
plot a table of the temps, time taken to turn pink in seconds, rate of reaction
plot a spearman’s rank graph with data
What are the limitations of the TTC yeast experiment
end-point is subjective (different eyes have different views of pink end point in mind)
difficulties in seeing colour change in water bath
Why did the TTC turn red
dehydrogenase enzyme which is naturally occurring in yeast removed H from NADH and TTC picked up this H.
TTC got reduced and formed red precipitate
At what stages of respiration will dehydrogenase be removing hydrogen
NADH is made in glycolysis, links and Krebs as it removed H from carbon compounds.
iT ALSO REMOVED h FROM REDUCED COENZYMES IN OXIDATIVE PHOSPHORYLATION
How do you make a control chamber when testing kinesis and taxis of organisms
leave all areas of the chamber uncovered so it can be assumed that there will be an even spilt of invertabrae across the chamber
What are some risk assessments when using invertebrates to test kinesis and taxis
may have pathogens on them which cause infection so use equipment to transfer them instead of ur hands and wash hands after practical
How can you test whether there was a significant difference in invertaebrea kinesis and taxis?
use chi squared statistic
if theres less than 5% probability that theres difference in distribution due to chance, it can be concluded that environmental variables cause a signig=ficant difference in distribution
How can you test whether there was a significant difference in invertaebrea kinesis and taxis?
use chi squared statistic
if theres less than 5% probability that theres difference in distribution due to chance, it can be concluded that environmental variables cause a signig=ficant difference in distribution
After invertabrae has moved along maze/chamber you need to wipe it down with cotton, why?
the invertabrae can leave a trail of chemicals where they move which can influence the choices other invertabrae make
what is the purpose of the control maze
to show any differences in movement within maze are due to independent variables
why is it important to repeat with at least 10 invertebrates
so that a mean and statistic can be calculated to see if the difference in turning direction is due to chance or significance
How would you make the following conditions in a chamber: dry, humid, dark
dry - use silica beads (absorbs moisture)
humid - dampened filter paper
dark - cover with black paper and sellotape it
Why is a nylon fabric placed between lid and base
easier for organisms to crawl over sheet rather than over the sections of the chamber
ensures they dont touch the silica gel beads
Why is a nylon fabric placed between lid and base
easier for organisms to crawl over sheet rather than over the sections of the chamber
ensures they dont touch the silica gel beads
How do you insert the bugs into the chamber
through a hole in the middle of the chamber lid so they all start in the middle
why should you wait 5 mins after adding dampened filter paper to chamber
provides time for the water from the filter paper to evaporate to create a humid environment and reach the nylon mesh
whats a more accurate way to count the invertabrea after removing the lids
remove lid and take instant photo and count from the picture
this ensures that you are able to count them before they move along to a different chamber once lid is removed
why do invertebrate move to dark and humid chambers
humidity is advantageous to prevent desiccation (drying out0
darkness advantageous as its harder for predators to locate and may prevent desiccation
What is the serial dilution equation
C1 V1 = C2 V2
c1= starting conc
v1 = volume you are trying to transfer
c2 = concentration your trying o make
v2 = final volume
What is the serial dilution equation
C1 V1 = C2 V2
c1= starting conc
v1 = volume you are trying to transfer
c2 = concentration your trying o make
v2 = final volume
How do you test a urine same for its glucose concentration?
make a series dilution of glucose concentration
add Benedictus reagent
place it in a boiling water bath for 5 mins and colour should change
use a colorimeter to measure the absorbance of each glucose concentration
plot a calibration curve of glucose concentration (x-axis) against absorbance (y-axis)
add Benedictus eacgent to urine samples and boil them in water bath
place the solution into colorimeter and find the rabsorbance
compare the absorbance of urine samples to calibration curve (extrapolate the absorbance back to the line and find the glucose conc in the urine)
Why is sampling used and how do you ensure its accurate
to accurately represent population
- random to eliminate bias
- line transects to examine a change over distance
-large number of samples 30+
How do you know to use a capture release method or quadrate
if population is mobile then use capture method
if population is slow moving or non-mobile, use quadrate
what are the 2 method for using quadrate depending on if theres uniform or non uniform distribution
uniform = random sampling
non-uniform = line transect
how do you do random sampling
place 2 tape measures act right angle to form gridded area
use random number generator to make 2 coordinates
place quadrate and collect data
repeat at least 30 times and find mead
what is belt and interrupted belt transect
belt = quadrate placed at every position alone tape measure
interrupted = quadrate placed at uniform intervals along tape measure
when would you choose interrupted belt transect over normal belt?
if there isnt much variation at every position alone the tape measurement
or to make things faster/more efficient
when would you choose interrupted belt transect over normal belt?
if there isnt much variation at every position alone the tape measurement
or to make things faster/more efficient
how do you line transects
- place tape measure at right angle TO SHORE LINE
- PLACE QUADRATE EVERY 5M OR POSITION
- collect data
- REPEAT at RIGHT ANGLES to shoreline about 30 times
what are the different ways to collect data in a quadrate and how
- local frequency
- find the percentage of squares in the quadrate occupied by the species - Density
- number of species in given area
( area of quadrate is 0.25m2 so
(area of entire field/0.25m2 ) x number of species in quadrate = total number of species in field - Percentage cover
- proportion of ground occupied by spe died (how many squares do all the species in the quadrate take up)
pros and cons of local frequency
quick way to sample large area
useful if too hard to identify individual organism
poor accuracy as doesnt consider overlapping plants or its size
pros and cons of density
more accurate if species is easily distinguishable and not too many to count
can be used to estimate species richness
more time consuming
pros and cons of percentage cover
faster than density
useful if too hard to identify individual organism or if too many to count
subjective so limits accuracy
doesnt consider overlapping or size of plants
pros and cons of percentage cover
faster than density
useful if too hard to identify individual organism or if too many to count
subjective so limits accuracy
doesnt consider overlapping or size of plants
Describe the process of mark-release-recapture
- initial sample of population captured
- marked and released back into wild. capture number is recorded
- after period of time to allow animals to randomly disperse throughout habitat
- second sample is captured
- total number captured in 2nd sample and unumber recaptured with marking is recorded
- size of population is estimated on principle that proportion marked in 2nd sample = proportion of marked individuals in population as a whole
ow to increase reliability of mark-release-recapture
repeat several times
the more repeated the more reliable
whats the equation for mark-release-recapture
estimated total population = ( no organisms initially caught x no organisms in 2nd sample) / no of marked organisms recaptured
what are some considerations about marking animals
non toxic
must increase chance of predetaion
musnt reduce chances of reproduction
what assumptions are made with mark-release-recapture
population size is constant (no birth, no death, no migration )
animals always redistribute evenly (can huddle unreality)
what assumptions are made with mark-release-recapture
population size is constant (no birth, no death, no migration )
animals always redistribute evenly (can huddle unreality)
What does a potometer measure
rate of transpiration
why must plant be cut underwater for potometer
xylem has negative pressure so tis constantly pulling water up from lower down
so if cut in air, it will pull air into xylem (breaks column of water and transpiration dont work efficiently)
so you need to cut in water to maintain the water column in order for transpiration
why is entire potometer assembled under water
to remove all air bubbles
why is position where plant is attached to potometer smothered in petroleum jelly and have rubber seal
make equipment air tight
prevent any water leaking out
ensure water can only leave by evaporation out of stomata on plant
how is air bubble introduced into the capillary section of potomenter
life the potometer out of the beaker of water for 5-20 seconds and replace
how is air bubble introduced into the capillary section of potomenter
life the potometer out of the beaker of water for 5-20 seconds and replace
how do you find rate of transpiration from potometer
wait for introduced air bubble to reach zero on capillary tube scale
have ur variable on
start the timer and measure the time taken for the air bubble to travel a distance
find the volume of water that the air bubble travels ( cross sectional area of capillary tube x distance travelled by air bubble) / time taken for air bubble to move
how do you remove the air bubble to reset th epotometer
open the tap to the reservoir and flush the air bubble out
what variable would be controlled between 2 different plant species testing for rate fo transpiration (not including light, humidity etc)
surface area of leaves / number and size of leaves
what variable would be controlled between 2 different plant species testing for rate fo transpiration (not including light, humidity etc)
surface area of leaves / number and size of leaves
