AS practicals Flashcards
RP1 - measuring enzyme activity*
how can the progress of enzyme-catalysed reactions can be investigated by?
1) by measuring the rate of formation of a product using a catalyst
2) by measuring the rate of disappearance of a substrate using a catalyst
common factors to investigate and how to investigate them:
-temperature
-pH
-substrate concentration
-enzyme concentration
-inhibitors
(the effect of each of these can be determined by changing a single variable and measuring its effect on the rate of reaction. it is important to keep all other variables constant so that they do not influence the results)
common enzymes to investigate:
-amylase
-catalase
-protease
-trypsin
trypsin practical
trypsin practical equipment:
-powdered milk suspension
-trypsin solution (0.5%)
-distilled water
-hydrochloric acid
-5cm³ syringe
-flat bottomed tubes
-timer
what is the independent variable in the trypsin practical?
temperature
method of trypsin practical:
1) take three test tubes and measure 5cm³ milk into each. place in water bath at
10°C for 5 minutes to equilibrate
2) add 5cm³ trypsin to each test tube simultaneously and start the timer immediately
3) record how long it takes for the milk samples to completely hydrolyse and become colourless
4)) repeat steps 2-3 at temperatures of 20°C, 30°C, 40°C and 50°C
5) find the mean time for the milk to be hydrolysed at each temperature and use this to work out the rate of reaction
rate of reaction formula:
1/ mean time
risk assessment: hazards
-broken glass
-hydrochloric acid
-hot liquids
-enzymes
broken glass
(risk, safety precaution, in emergency)
risk: cuts from sharp object
safety precaution: take care when
objects; keep away from edge of desk
in emergency: elevate cuts; apply pressure; do not remove glass from wound; seek medical assistance
hydrochloric acid
(risk, safety precaution, in emergency)
risk: may cause harm/irritation to eyes or in cuts
safety precaution: wear eye protection; avoid contact with skin, tie up long hair
in emergency: wash off skin immediately;
flood eye/cuts with cold water
hot liquids
(risk, safety precaution, in emergency)
risk: scalding
safety precaution: handle with care; use tongs to remove boiling tubes from water bath; wear eye protection, keep away from the edge of the desk
in emergency: run burn under cold water; seek medical assistance
enzymes
(risk, safety precaution, in emergency)
risk: allergies
safety precaution: avoid contact with skin/eyes; wear eye protection
in emergency: seek assistance
milk & colourlessness
-milk contains a protein called casein which, when broken down, causes the milk to turn colourless
-trypsin is a protease enzyme which hydrolyses the casein protein
method limitations:
-the end-point was subjective which leads to inaccurate ‘time taken’ measurements
-the water bath was not thermostatically controlled and therefore the temperature decreased during the practical
-the range of tested pH values was not wide enough as the point at which trypsin denatures was not identified (e.g. test beyond pH 11)
RP2 - root tips
plant mitosis
plant cells undergo mitosis at shoot and root tips in areas called meristems
equipment of root tip practical:
-100ml beaker
-hydrochloric acid
-microscope slide & cover slip
-acetic orcein stain
-filter paper
-mounted needle
-scalpel
-distilled water
-watch glass
-forceps
-onion
-paper towel
-light microscope
method of root tip practical:
(part 1)
1) heat 1 mol dm HCl at 60°C in a water bath
2) cut a small sample of the root tip using a scalpel
3) transfer root tip to HCl and incubate for 5 minutes
4) remove from HCI and wash sample in cold distilled water, remove the tip using a scalpel
5) place tip on a microscope slide and add a few drops of stain
method of root tip practical:
(part 2)
6) lower the cover slip down carefully onto the slide. make sure there are no air bubbles in the slide which may distort the image, and that the coverslip doesn’t slide sideways which could damage the chromosomes
7) place under a microscope and set the objective lens on the lowest magnification
8) use the coarse adjustment knob to move the lens down to just above the slide
9) use the fine adjustment knob to carefully re-adjust the focus until the image is clear (you can use a higher magnification if needed)
10) to calculate mitotic index, cells undergoing mitosis must be counted (cells with chromosomes visible), as well as the total number of cells.
why is hydrochloric acid used?
it softens and loosens the root tissues
why is acetic orcein stain used?
it makes the chromosomes visible and will show which cells are undergoing mitosis
why is a mounted needle used?
to lower the cover slip and prevent air bubbles under the cover slip
why was motion tip used?
the tip is the growing region, therefore mitosis should be occurring here
risk assessment: hazards
-hydrochloric acid
-stain
-scalpel
-broken glass
hydrochloric acid
(risk, safety precaution, in emergency)
risk: may cause harm/irritation to eyes or in cuts
safety precaution: wear eye protection; avoid contact with skin, tie up long hair
in emergency: wash off skin immediately; flood eye/cuts with cold water
stain
(risk, safety precaution, in emergency)
risk: may cause harm/irritation to eyes or in cuts
safety precaution: wear eye protection; avoid contact with skin
in emergency: wash off skin immediately; flood eye/cuts with cold water
scalpel
(risk, safety precaution, in emergency)
risk: cuts from sharp object
precaution: cut away from fingers; use forceps to hold sample whilst cutting, keep away from the edge of the desk
in emergency: elevate cuts; apply pressure; seek medical assistance
broken glass
(risk, safety precaution, in emergency)
risk: cuts from sharp object
safety precaution: take care when handling slides and coverslips; keep glassware away from edge of desk
in emergency: elevate cuts; apply pressure; do not remove glass from wound; seek medical assistance
what is mitotic index?
the proportion of cells (in a group of cells or a sample of tissue) that are undergoing mitosis
mitotic index: formula
number of cells with visible chromosomes ÷ total number of cells
(x100 if percentage is needed)
a student who wanted to observe mitosis prepared a sample of cells. they counted a total of 42 cells in their sample, 32 of which had visible chromosomes. calculate the mitotic index for this sample of cells (give your answer to 2 decimal places)
mitotic index = 32 ÷ 42
mitotic index = 0.76
how can root tip slides be prepared?
squash technique
(root tips are stained and then gently squashed, spreading the cells out into a thin sheet and allowing individual cells undergoing mitosis to be clearly seen)
mm → µm
x1000
RP3 - water potential
what are calibration curves?
graphs used to determine an unknown concentration of a sample by comparing the unknown to a set of standard samples with known concentrations
hypotonic
-lower concentration
-higher water potential
(water moves out of cell)
isotonic
-same concentration
-same water potential
hypertonic
-higher concentration
-lower water potential
(water moves in)
equipment of water potential practical:
-large potato
-cork borer
-1 mol dm-3 sucrose solution
-distilled water
-boiling tube rack
-six boiling tubes
-marker pen
-thermometer
-10cm measuring cylinder
-white tile
-scalpel
-ruler
-paper towels
-stop clock
-digital balance
-forceps
method of water potential practical:
1) make a series of dilutions of 1M sucrose solution
↳ these should be at 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0M sucrose
↳ dilute using distilled water
2) measure 10cm³ of each dilution into separate test tubes and label them
3) use a cork borer to cut out six potato chips and cut down the sections into identically lengthed chips (with ruler)
↳ dry each chip using a paper towel to remove excess water but do not squeeze
4) weigh each before the start of the experiment and put into graph
5) place a potato chip in each test tube (one per sucrose concentration) and leave for 20 minutes
6) remove each potato chip, dry gently using paper towel, and weigh them in turn, put this into a table
7) calculate the percentage change in mass for each sucrose solution
risk assessment: hazards
-scalpel
-broken glass
scalpel
(risk, safety precaution, in emergency)
risk: cuts from sharp object
safety precaution: cut away from fingers; use forceps to hold sample whilst cutting, keep away from the edge of the desk
in emergency: elevate cuts; apply pressure; seek medical assistance
broken glass
(risk, safety precaution, in emergency)
risk: cuts from sharp glass
safety precautions: take care when handling glass objects; keep glassware away from edge of desk
in emergency: elevate cuts; apply pressure; do not remove glass from wound; seek medical assistance
osmosis analysis:
calculate the percentage change in mass for each potato cylinder
percentage change in mass
(formula)
change in mass/initial mass
x100
what does a positive percentage change in mass entail?
the potato has gained water by osmosis
↳ the solution had a higher water potential than the potato
↳ this would make the potato cells turgid
↳ the water exerts turgor pressure (or hydrostatic pressure) on the cell walls
what does negative percentage change in mass entail?
the solution had a lower water potential than the potato
↳ the potato cylinder in the strongest sucrose concentration will have decreased in mass the most
↳ more water molecules will move out of the potato cells by osmosis, making them flaccid
what does no change in mass entail?
-no overall net movement of water into or out of the potato cells
-the solution that this particular potato cylinder was in had the same water potential as the solution found in the cytoplasm of the potato cells
the format of the calibration curve graph:
-plot a graph of change in mass against concentration of sucrose solution
-the point at which the line of best fit crosses the x axis indicates the point at which the solution is isotonic
RP4 - permeability
what are cell membranes comprised of?
-a phospholipid bilayer which makes them selectively permeable
-this permeability can be changed by different variables
examples of variables that permeability is affected by:
-temperature
-concentrations of solvents (ethanol)
beetroot components:
-beetroot cells contain a purple pigment called betalain (in the vacuole)
↳ betalain can’t cross the plasma membrane unless the membrane is damaged
-when the cell-surface membrane has a higher permeability, more pigment leaks out of cells
why is beetroot used in the practical?
permeability can be measured by the amount of pigment leaked from beetroot cells into an aqueous solution using a colorimeter
equipment of permeability practical:
-beetroot
-scalpel
-forceps
-ruler
-tongs
-distilled water
-boiling tube & boiling tube rack
-colorimeter
-cuvettes
-filter paper
-timer
-water bath
-thermometer
-ethanol
method of permeability practical:
(temperature)
1) coloured agar is made up and cut into equal-sized cubes (a cork borer can also be used, as long as the cores are cut to the same length)
2) rinse the beetroot pieces
3) place each of the cubes of beetroot in an equal volume of distilled water (eg: 5ml)
4) place each test tube in a water bath at a range of temperatures (20, 30, …)
5) leave the samples for 20 minutes - the pigment will leak out of the beetroot
6) remove the beetroot pieces, leaving just the coloured liquid in the five test tubes
7) set the colorimeter to a blue filter and zero using a cuvette with distilled water
8) filter each sample of water into a cuvette using filter paper
9) use a colourimeter to measure how much light is absorbed as it passes through each of the five samples of coloured liquid
10) measure the absorbance for each solution. a higher absorbance indicates higher pigment concentration, and hence a more permeable membrane
the method of the permeability practical:
(ethanol)
1) coloured agar is made up and cut into equal-sized cubes (a cork borer can also be used, as long as the cores are cut to the same length)
2) rinse the beetroot pieces
3) create a dilution series of ethanol using distilled water. ethanol concentrations should range from 0-100% ethanol.
5) leave the samples for 20 minutes - the pigment will leak out of the beetroot
6) remove the beetroot pieces, leaving just the coloured liquid in the five test tubes
7) set the colorimeter to a blue filter and zero using a cuvette with distilled water
8) filter each sample of water into a cuvette using filter paper
9) use a colourimeter to measure how much light is absorbed as it passes through each of the five samples of coloured liquid
10) measure the absorbance for each solution. a higher absorbance indicates higher pigment concentration, and hence a more permeable membrane
ethanol
(risk, safety precaution, in emergency)
risk: irritant/ flammable
safety precaution: wear eye protection; keep away from naked flames
in emergency: wash eyes and skin with cold water
what to plot for permeability practical:
absorbance (y) against ethanol concentration/temperature (x)
why are equal sized beetroot cubes used?
the pieces must have the same dimensions so that they all have equal surface areas and volumes, as these factors could affect the rate at which the pigment leaks out
why are the beetroot cubes rinsed?
to remove excess pigment
conclusions of the practical:
(permeability)
as temperature increases, membrane permeability also increases
↳ phospholipids in cell membrane move more because they have more energy
↳ phospholipids are not as tightly packed together → permability increases
permeability at very high temperatures:
-the phospholipid bilayer may even to melt and breakdown, further increasing the permeability of the membrane
-the volume of water inside the cells expands, putting pressure on the membrane, causing channel and carrier proteins to deform → they can’t control what enters and leaves the cell
↳ increased permeability
how high temperatures affect proteins
-affects the conformation (3D shape) of proteins
-at high temperatures the intermolecular forces between amino acids are broken which affects the protein’s specificity and function
cell membranes at very low temperatures (below 0):
membrane permeability may also be increased (once the cells have thawed again)
-channel or carrier proteins deform at these low temperatures
-ice crystals that form can also pierce the cell membrane, making it highly permeable
how does ethanol affect permeability?
-ethanol causes the cell-surface membrane to rupture, releasing the betalain pigment from the cell
-higher concs of ethanol cause more disruption to the membrane → more gaps form
-as concentration of ethanol increases, the permeability of the cell-surface membrane also increases
limitations of the permeability practical:
-cuvette
-beetroot pieces
limitations: cuvettes
(+ how to rectify)
cuvettes may differ in thickness (slightly):
↳ a thicker (or scratched) cuvette will absorb slightly more light than a thinner unscratched cuvette
↳ this can be overcome by using the same cuvette for every reading, or repeating the investigation many times and finding a mean
limitations: beetroot
(+ how to rectify)
the beetroot pieces may not be identical in size and shape, meaning some test tubes could contain slightly more beetroot tissue than others
↳ this can be overcome by cutting the discs as accurately as possible using a scalpel and ruler, and by repeating each investigation several times to find a mean
RP5 - dissection
the importance of dissections:
-essential for understanding the function of internal biological functions
what are the issues with dissections?
ethical issues
examples of the ethical issues of dissections:
-people worry about how the animals for dissections are raised and killed
-it goes against the religious beliefs of some individuals
equipment for the dissections:
-scalpel
-scissors
-tweezers
-pins & labels
-dissection tray
-newspaper/paper towels
-specimen’s heart
-camera
-plastic gloves
-disinfectant spray
the method of the dissection:
1) take a picture of the outside, identify the coronary arteries
2) place the specimen on the dissecting board
3) make a cut along each side of the heart to see inside of the four chambers
4) look for the atrioventricular valve, there will be tendinous cords attached to these
↳ use a dissection needle to pull on these cords to feel how strong they are. lift the valves to see how thin they are.
5) examine the thickness of the walls of the chambers.
6) observe how much thinner the muscular walls of the atria are than the ventricles
what can scissors be used for?
cutting large sections of tissue (cuts do not need to be precise)
what do scalpels enable?
finer, more precise cutting
(it needs to be sharpe to ensure this)
how to clean up after the dissection:
-place all dissection equipment in a beaker of disinfectant.
-place the heart, gloves and any paper towels in disposal bags
-clean your work surface with a disinfectant spray and clean and disinfect the dissecting
what are the hazards of the practical?
-biohazard
-disinfectant
-sharp tools
biohazard
(risk, safety precaution, in emergency)
risk: contamination
precautions: use disinfectant; keep sample on dissection board; wash hands with soap after dissection
in emergency: seek assistance
disinfectant
(risk, safety precaution, in emergency)
risk: flammable
safety precaution: keep away from naked flame
in emergency: out of fire
what should be worn during the dissection?
-lab coat
-eye protection
-gloves
tips for drawing a labelled diagram:
-use a sharp HB pencil
-add labels to all structures (lines should be drawn with a ruler)
-no shading
-use single and continuous lines (no sketching
-no colour
-include a magnification/scale
limitations of dissection practical:
-it can be hard to see some of the smaller, finer structures within organs
-the specimens do not reflect how the tissue would look in a living organism
-if only a single specimen is dissected then anomalies found within that specimen may be ignored or glossed over
RP6 - aseptic techniques
what are aspetic techniques?
they ensure that the microbes being investigated don’t escape or become contaminated with another unwanted, and possibly pathogenic, microbe
examples of aseptic techniques:
-washing hands thoroughly to disinfect them
-no food or drink allowed in the lab
-disinfecting work surfaces with disinfectant or alcohol
aseptic techniques before the practical:
-sterilise all equipment
-sterilise work surfaces with disinfectant
-wash hands with soap
-close windows and doors to reduce draughts
(to kill microbes)
equipment of the aseptic practical:
-petri fish with agar
-diluted bacterial broth
-pipettes
-plastic spreader
-bunsen burner
-gloves
-goggles
-virkon disinfectant
-heatproof mat
-loop
-antibiotic disc
steps of aseptic practical:
1) sterilise the equipment (spreader, work surface, loop)
2) turn a bunsen burner on (keep it on the whole time)
3) open a bottle (containing bacteria) & flame the bottleneck
4) use an inoculating loop to transfer broth onto the agar plate (flame the wire hoop before using it to transfer bacteria)
4) spread a sample of the diluted bacterial broth onto the surface of the sterile agar plate (only open the petri dish quickly & slightly, when necessary)
5) use sterile forceps to put an antibacterial disc onto the agar plate
6) lightly tape a lid on, invert and incubate at 25°C for 48 hours
7) put all of the used equipment into virkon & disinfect the work surface
steps of aseptic practical:
(after incubation)
1) measure the diameter of the inhibition zone (clear circle) for each antibiotic.
↳ don’t remove the lid from the agar plate
2) work out the area of the inhibition zone using the formula
why is a bunsen burner used?
so that convection currents draw microbes away from the culture
why are bottles flamed?
air moves out so unwanted organisms don’t move in
clear zones:
(extra)
-the clear area will end when the conc of antibiotic reaches a level that the bacteria are no longer susceptible to
-more effective antibiotics need a lower concentration to kill bacteria and so they will produce larger clear zones
-if a bacteria is completely resistant to an antibiotic then there will be no clear zone around that particular paper disk
hazards:
-disinfectant
-biohazard
-naked flame
disinfectant
(risk, safety precaution, in emergency)
risk: flammable
safety precaution: keep away from naked flame
in emergency: put out fire; seek assistance
biohazard
(risk, safety precaution, in emergency)
risk: contamination; infection
safety precaution: use disinfectant; wash hands with soap; do not incubate at human body temperature
in emergency: seek assistance
naked flame
(risk, safety precaution, in emergency)
risk: fire hazard, burns
safety precaution: keep away from flammable materials; tie up long hair, wear goggles
in emergency: put out fire; seek assistance; run burns under cold water immediately
how do you tape an agar plate?
don’t tape around the entire dish, this prevents oxygen from entering and so promotes the growth of more harmful anaerobic bacteria
