Practicals

Cards (18)

  • Investigate the effect of caffeine on heart rate in Daphnia.
    In humans, caffeine is a stimulant which increases heart rate by increasing release of excitatory neurotransmitters. Daphnia are water fleas, used in this case because they have a translucent body through which the heartbeat can be observed, and hence the effect of caffeine on heart rate can be measured.

    Equipment
    ● Daphnia
    ● Cavity slides
    ● Dropping pipettes
    ● Distilled water
    ● Caffeine solution
    ● Cotton wool
    ● Test tubes
    ● Stop clock
    ● Filter paper
    Optical microscope

    Method
    1. Dilute the caffeine solution with distilled water to produce several different concentrations.
    2. Place some cotton wool (to restrict movement) on a cavity slide. Add one large water flea.
    3. Use filter paper to absorb the water around the flea. 4. Then use a dropping pipette to add a few drops of distilled water to the slide.
    Do not use a coverslip to prevent conditions from becoming anoxic.
    1. Use a stop clock to time a minute and record the number of heartbeats.
    2. Repeat the experiment, replacing the distilled water with a caffeine solution.

    Hazards
    1. Biohazard; Contamination; Use disinfectant, wash hands with soap after handling Daphnia.
    2. Broken glass; Cuts from sharp object; Take care when handling glassware, keep away from edge of desk

    Graph
    ● Plot a graph of heart rate against caffeine concentration.

    Conclusion
    ● Caffeine increases the heart rate of Daphnia by increasing the release of stimulatory neurotransmitters. As concentration of caffeine increases, heart rate also increases.

    NB: it is important to treat the Daphnia ethically during the experiment and release them back into a stream/pond afterwards.
  • Investigate the vitamin C content of food and drink.
    DCPIP is a redox indicator dye (which turns from blue to colourless when it is reduced and accepts electrons). Vitamin C is an antioxidant and therefore DCPIP is decolourised in the presence of Vitamin C. This can be used to measure the Vitamin C content of fruit juice.

    Equipment
    ● DCPIP solution
    ● Vitamin C solution
    ● Fruit juices
    ● Test tubes
    ● Pipette

    Method
    1. Transfer 1cm of DCPIP solution into a test tube with a pipette.
    2. Add Vitamin C solution dropwise to the DCPIP solution. Shake after each drop.
    3. Record the volume of Vitamin C that is required to change the colour of the DCPIP.
    4. Repeat the experiment and replace the Vitamin C solution with the fruit juices.

    Risk Assessment
    1. DCPIP; Irritant to skin and eyes, may cause staining; Wear eye protection, keep away from edge of desk
    2. Broken glass; Cuts from sharp object; Take care when handling glassware, keep away from edge of desk
    3. Bags/stools; Tripping; Keep under desks and away from workspace

    Graph
    ● Plot a graph of absorbance against ethanol concentration/temperature.
  • Investigate membrane structure, including the effect of alcohol concentration or temperature on membrane permeability.
    Cell-surface membranes are made up of a phospholipid bilayer which makes them selectively permeable. This permeability can be changed by different variables, such as temperature and concentration of solvents, like ethanol.

    The permeability of a membrane can be measured by using beetroot cells, which contain a purple pigment called betalain. When the cell-surface membrane has a higher permeability, more pigment leaks out of cells. The permeability can therefore be measured by the amount of pigment leaked from beetroot cells into an aqueous solution using a colorimeter.

    Equipment
    ● Water baths
    ● Thermometer
    ● Distilled water
    ● Syringe
    ● Beetroot
    ● Cork borer
    ● White tile
    ● Knife
    ● Syringe
    Pipette
    ● Test tubes
    ● Colorimeter
    Cuvettes
    ● Forceps

    Method
    1. Cut beetroot into 8 identical cylinders using a cork borer and wipe/rinse to clean off any pigment released as a result.
    2. Place each of the cylinders of beetroot in 10 ml of distilled water. Place each test tube in a water bath at a range of temperatures between 0 and 70°C.
    3. Leave the samples for 15 minutes- pigment will leak out of the beetroot.
    4. Record the exact temperature of the water bath using the thermometer.
    5. Remove the test tubes from the water baths and remove the cylinders of beetroot from them. Decant the liquid into clean test tubes.
    6. Set the colorimeter to a blue filter and zero using a cuvette with distilled water. Filter each sample into a cuvette using filter paper.
    7. Measure the absorbance for each solution. A higher absorbance indicates higher pigment concentration, and hence a more permeable membrane.

    Risk Assessment
    1. Scalpel; Cuts from sharp object; Cut away from fingers, use forceps to hold sample whilst cutting, keep away from edge of desk
    2. Broken glass; Cuts from sharp object; Take care when handling glassware, keep away from edge of desk
    3. Hot liquids; Scalding; Handle with care, use tongs to remove boiling tubes from water bath, wear eye protection, keep away from edge of desk
    4. Ethanol; Irritant/ flammable; Wear eye protection, keep away from naked flames

    Graph
    ● Plot a graph of absorbance against ethanol concentration/temperature.

    Conclusion
    ● As the temperature increases, the permeability of the cell-surface membrane also increases. This is because the proteins in the membrane denature as the heat damages the bonds in their tertiary structure. This creates gaps in the membrane so it is easier for molecules to pass through it.
    ● At low temperatures, phospholipids have little energy and are packed closely together to make the membrane rigid. This causes a decrease in permeability and restricts molecules from crossing the membrane.

    NB: At very low temperatures, ice crystals can form which pierce the cell membrane and increase the permeability.

    Modification
    The method can be modified to investigate the effect of ethanol on membrane permeability by having concentration of ethanol as the independent variable. Ethanol causes the cell-surface membrane to rupture, releasing the betalain pigment from the cell. Higher concentrations of ethanol will cause more disruption to the membrane and more gaps will form, thus as concentration of ethanol increases, so does the permeability of the cell-surface membrane.
  • Investigate the effect of enzyme and substrate concentrations on the initial rates of reactions.
    The rate of reaction of an enzyme-controlled reaction is influenced by different factors: the temperature, pH, concentration of the substrate, and the concentration of the enzyme. 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.

    Initial rate of reaction is measured because the rate of an enzyme-controlled reaction is high, this is because enzymes act as biological catalysts, so the concentration of reactants changes rapidly. The initial rate is the only point during the reaction when concentration of reactants and products is known.

    Equipment
    ● Milk powder solution
    ● Trypsin solution (1%)
    ● Test tubes
    ● Test tube holder
    ● Stopclock
    ● 5cm3 pipettes
    ● Goggles
    ● Colorimeter
    ● Cuvettes
    ● Distilled water

    Method
    1. Dilute stock solution of trypsin with distilled water to produce solutions with concentrations of 0.2%, 0.4%, 0.6% and 0.8%.
    2. Make a control by adding 2cm3 of trypsin solution and 2cm3 of distilled water. Use this to set the colorimeter absorbance to zero.
    3. To another cuvette, add 2cm3 of milk suspension and 2cm3 of the stock trypsin solution. Mix, place in the colorimeter and measure absorbance at 15 second intervals for 5 minutes.
    4. Rinse the cuvette with distilled water.
    5. Repeat step 3 at all trypsin concentrations

    rate of reaction = 1/mean time

    Risk Assessment
    1. Broken glass; Cuts from sharp object; Take care when handling glass objects, keep away from edge of desk
    2. Hot liquids; Scalding; Handle with care, use tongs to remove boiling tubes from water bath, wear eye protection, keep away from edge of desk
    3. Enzymes; Allergies; Avoid contact with skin/eyes, wear eye protection

    Graph
    ● Plot a graph of rate of reaction against temperature.

    Conclusion
    ● Milk contains a white protein called casein which, when broken down, causes the milk to turn colourless. Trypsin is a protease enzyme which hydrolyses the casein.
    ● As concentration of trypsin increases, the number of enzyme-substrate complexes forming also increases because enzymes and substrates are more likely to collide. This means that the rate of reaction increases up to the optimum enzyme concentration.
    ● The rate plateaus at the point where all substrates occupy an active site. Increasing the enzyme concentration won't increase rate as substrate concentration is limiting the rate.

    Modification
    The procedure can be modified to measure the effect of substrate concentration on initial rate of reaction by diluting the milk suspension to produce different concentrations and controlling concentration of trypsin.
  • Prepare and stain a root tip squash to observe the stages of mitosis.
    Plant cells undergo mitosis at shoot and root tips in areas called meristems. Cells in the meristems are totipotent and retain the ability to differentiate.

    The mitotic index of a sample is the ratio of cells undergoing mitosis to the total number of cells in a sample. To find the mitotic index, cells from the meristem must be viewed under an optical microscope.

    Equipment
    Garlic root tip
    1M hydrochloric acid
    ● Toluidine blue stain
    ● Distilled water
    ● Watch glasses
    ● Sample tube
    ● Pipettes
    ● Microscope slides and coverslips
    ● Forceps
    ● Filter paper
    ● Scissors
    ● Optical microscope

    Method
    1. Cut a 5mm sample of the root tip using a scalpel.
    2. Transfer root tip to sample tubes containing HCl and leave for 5 minutes.
    3. Transfer to watch glass containing cold distilled water. Leave for 5 minutes.
    4. Dry root tips on filter paper.
    5. Place tip on a microscope slide. Macerate with a needle to spread the cells out. This makes the chromosomes visible and will therefore show which cells are undergoing mitosis.
    6. Add a drop of toluidine blue to the slide and leave to stain for 2 minutes.
    7. 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. 8. Wrap in a paper towel and gently 'squash' the slide.
    8. Place under a microscope and set the objective lens on the lowest magnification, then 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 (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.

    mitotic index = no. of cells w/ visible chromosomes/ total no. cells in sample

    Risk Assessment
    1. Hydrochloric acid; May cause harm/irritation to eyes or in cuts; Wear eye protection, avoid contact with skin, keep away from edge of desk
    2. Toluidine blue O stain; May cause harm/irritation to eyes or in cuts; Wear eye protection, avoid contact with skin
    3. Scalpel; Cuts from sharp object; Cut away from fingers, use forceps to hold sample whilst cutting, keep away from edge of desk
    4. Broken glass; Cuts from sharp object; Take care when handling slides and coverslips, keep glassware away from edge of desk
  • Identify sclerenchyma fibres, phloem sieve tubes and xylem vessels and their location within stems through a light microscope.
    Microscopy is used to increase magnification and resolution of an object. Microscopes can be optical or electron, and electron microscopes can be transmission or scanning. Magnification can be calculated by dividing the size of the image by the size of the actual object.

    Equipment
    ● Plant stem
    ● Acidified phloroglucinol in ethanol
    ● Optical microscope
    ● Microscope slides and coverslips
    Scalpel
    ● Razor blade
    ● Paintbrush
    ● White tile
    ● Watch glass
    Pipette
    ● Wax crayon

    Method
    1. Calibrate the eyepiece graticule by placing both on the stage and lining up the divisions of the stage micrometer (which have a known length) with the divisions of the eyepiece graticule (for which the length is unknown) to calculate the length of one division of the graticule.
    2. Cut transverse sections of the plant stem (on the white tile using a razor, wet to reduce friction) as thinly as possible. Select the thinnest sections.
    3. Place one section on a microscope slide. Draw a line in wax crayon from top to bottom of the slide either side of the specimen to prevent the dye from spreading. Add a few drops of concentrated phloroglucinol and lower the cover slip down carefully onto the slide. Make sure there are no air bubbles in the slide which may distort the image.
    4. Place under a microscope and set the objective lens on the lowest magnification, then use the coarse adjustment knob to move the lens down to just above the slide.
    5. Use the fine adjustment knob to carefully re-adjust the focus until the image is clear (can use a higher magnification if needed).
    6. Observe and draw the stem.
    7. Measure the size of the stem diameter and vascular bundle using the calibrated eyepiece graticule.

    Risk Assessment
    1. Acidified phloroglucinol; Corrosive and highly flammable; Wear eye protection, avoid contact with skin
    2. Razor; Cuts from sharp object; Cut away from fingers, use forceps to hold sample whilst cutting, keep away from edge of desk
    3. Broken glass; Cuts from sharp object; Take care when handling slides and coverslips, keep glassware away from edge of desk
    4. Ethanol; Irritant/ flammable; Wear eye protection, keep away from naked flames, keep away from edge of desk
  • Investigate plant mineral deficiencies.
    Plants require a range of nutrients to grow, survive and reproduce.
    These minerals include:
    Nitrate, which is used to form DNA and amino acids.
    Calcium, which is used to form calcium pectate for the middle lamella and in membrane permeability.
    Magnesium, which is used in the production of chlorophyll.

    The effects of deficiencies of any of these minerals can be investigated specifically via Bryophyllum plants. Bryophyllum reproduce asexually via budding, which produces genetically identical 'daughter' plantlets.

    Equipment
    ● Bryophyllum plantlets
    ● Nutrient solutions:
    ○ All minerals present
    ○ Without nitrogen
    ○ Without magnesium
    ○ Without calcium
    ○ Without any nutrients
    ● Measuring cylinder
    Test tube
    ● Test tube rack
    ● Tinfoil

    Method
    1. Use the measuring cylinder to fill test tubes with each of the nutrient solutions.
    2. Cover the top of the test tube with tinfoil. Poke a hole through the tinfoil.
    3. Push the roots of the Bryophyllum plantlets through the hole in the tinfoil into the solution.
    4. Wrap the test tubes in tinfoil (to prevent light getting in) and place them under a sunny window.

    Risk Assessment
    1. Biohazard; Contamination; Use disinfectant, wash hands with soap after handling.
    2. Broken glass; Cuts from sharp object; Take care when handling glassware, keep away from edge of desk
    3. Potassium nitrate; Flammable, reducing agent; Keep away from naked flame, avoid contact with metals and other flammable substances
    4. Iron chloride; Irritant when solid; Wear eye protection, keep away from naked flames.
    5. Potassium sulphate; Low hazard; Wear eye protection.

    Conclusion
    ● Magnesium deficiency: stunted growth, yellowed leaves (because chlorophyll cannot be synthesised).
    ● Nitrate deficiency: yellowed leaves with red-brown cast (because chlorophyll cannot be synthesised as protein synthesis is restricted).
    ● Calcium deficiency: stunted growth, weakened stem (because the support from the cell wall is reduced and metabolism is restricted due to decreased membrane permeability).
  • Determine the tensile strength of plant fibres.
    Plant fibres can be used to refer to any fibre-like structure within a plant but is generally used to describe the vascular bundle - the xylem (hollow tube of cells which water is transported through) and phloem (cells with sieve plates which transports assimilates like sucrose) - and the sclerenchyma fibres (lignified support tissues).

    Tensile strength describes the maximum pulling force that can be applied to a tissue before the tissue breaks.

    Equipment
    Forceps
    White tile
    Sample of New Zealand flax plant
    Scalpel
    Suspended masses

    Method
    1. Use the forceps to separate the fibres.
    2. Test the tensile strength of the fibres by using suspended masses to compare
    e.g. types of fibres, internal vs external fibres etc.

    Risk Assessment
    1. Scalpel; Cuts from sharp object; Cut away from fingers, use forceps to hold sample whilst cutting, keep away from edge of desk
    2. Biohazard; Contamination; Use disinfectant, wash hands with soap after handling

    Modification
    The plant used could also be stinging nettles instead of the New Zealand flax plant. The practical works the same way, but the nettles must be soaked for a week to soften and rubber gloves must be worn to avoid being stung
  • Investigate the antimicrobial properties of plants, including aseptic techniques for the safe handling of bacteria.
    Aseptic technique is used to avoid contamination of the sample from outside substances such as microorganisms. This is important to get reliable and repeatable data.

    Aseptic Technique
    ● Wipe down surfaces with antibacterial cleaner both before and after experiment.
    ● Use a Bunsen burner in the work space so that convection currents draw microbes away from the culture.
    ● Flame the wire hoop before using to transfer bacteria. ● Flame the neck of any bottles before use to prevent any bacteria entering the vessel (air moves out so unwanted organisms don't move in).
    ● Keep all vessels containing bacteria open for the minimum amount of time.
    ● Close windows and doors to limit air currents.

    Equipment
    ● Agar plate seeded with bacteria
    ● Sample of garlic
    ● Sample of mint
    ● Mortar and pestle
    ● Methylated spirit
    ● Pipette
    ● Paper discs
    ● Petri dish
    ● Forceps
    ● Tape

    Method
    1. Carry out aseptic techniques detailed above.
    2. Crush 3g of the garlic and mint (separately) with methylated spirit. Shake occasionally.
    3. Use a sterile pipette to transfer plant extract to paper disc.
    4. Leave paper discs to dry for 10 minutes.
    5. Use sterile forceps to place the paper disc onto a petri dish.
    6. Lightly tape a lid on, invert and incubate at 25°C for 24 hours. DO NOT tape around the entire dish as this prevents oxygen entering and so promotes the growth of more harmful anaerobic bacteria.
    7. Sterilise equipment used to handle bacteria and disinfect work surfaces.
    8. Measure the diameter of the inhibition zone (clear circle) for each plant. DO NOT remove lid from agar plate.
    9. Work out the area of the inhibition zone using the formula: A = πd/4
    where d is the diameter.

    NB: Bacteria sample is incubated at 25°C as incubating at 37°C (human body temperature) could enable pathogens to grow that are harmful to humans.

    Risk Assessment
    1. Disinfectant; Flammable; Keep away from naked flame
    2. Biohazard; Contamination, infection; Use disinfectant, wash hands with soap after dissection, do not incubate at human body temperature, do not open agar plate post incubation
    3. Naked flame; Fire hazard, burns; Keep away from flammable materials, tie up long hair, keep away from edge of desk
    4. Methylated Spirit; Flammable and toxic; Keep away from naked flame. Do not ingest. Wear gloves and goggles.

    Graph
    ● Plot a bar chart of the area of the inhibition zone against plant.
    ● Graph could include range bars to show the uncertainty from the ruler in measuring the diameter.

    Conclusion
    ● If there is a larger inhibition zone around the plant, it has killed more bacteria. Therefore, the larger the inhibition zone, the better the antimicrobial properties of the plant.
  • Carry out a study on the ecology of a habitat, such as using quadrats and transects to determine distribution and abundance of organisms, and measuring abiotic factors appropriate to the habitat.
    The distribution of a species is determined by a range of different variables. These can be grouped into abiotic (non-living) and biotic (living) factors. Abiotic factors include light intensity, amount of water and nutrients, and temperature. Biotic variables include competition for resources, the number of predators and disease.

    Note: there are other ways of completing this practical than the method listed below. You might also have used quadrats and random sampling or measured other abiotic variables.

    Equipment
    ● Quadrat
    ● Transect (20m rope marked at 1m intervals)
    ● Clipboard
    ● Appropriate equipment to measure variable

    Method
    1. Choose a site where there is an obvious gradient in an abiotic variable. Place the transect down. Select a species that changes in abundance along the gradient. 2. Place the quadrat at each of the marks on the transect, placing the bottom left corner on the mark every time.
    2. Record the percentage cover for the chosen species. This can be done by recording how many of the quadrat's 100 squares contain the chosen species. A square should only be counted if half or more of it is covered.
    3. At each coordinate, a measure of the independent variable should be taken. For example, if investigating light intensity, a photometer can be used to take a reading for the light intensity at each coordinate.

    Risk Assessment
    1. Biohazard; Allergies, soil bacteria, contamination; Wash hands after practical
    2. Slippery surfaces; Slip hazard; Wear appropriate footwear; don't run

    Graph/Data Analysis
    ● Plot a graph of the percentage cover against the chosen independent variable.
    ● Various statistical tests, including Spearman's Rank, T-test and Chi Squared, can be carried out on the collected data.

    Conclusion
    ● You should be able to see a correlation from the graph which will indicate the effect of the chosen variable on the distribution of the species.
    ● Be aware that correlation is not necessarily causation: there could be a range of factors that influence the results.
  • Investigate photosynthesis using isolated chloroplasts (the Hill reaction).
    Dehydrogenase is an enzyme found in plant chloroplasts that is crucial to the light dependent stage of photosynthesis. In the light dependent stage, electrons are accepted by NADP. This reaction was discovered in 1938 by Robin Hill and thus is often called the Hill reaction. Dehydrogenase catalyses this reaction. When a redox indicator dye is present, such as DCPIP (which turns from blue to colourless when it is reduced), electrons are accepted by this instead.

    Equipment
    ● Leaf sample
    ● Scissors
    ● Mortar and pestle (cold)
    ● Nylon mesh
    ● Filter funnel
    ● Centrifuge
    ● Centrifuge tubes
    ● Ice-water-salt bath
    ● Glass rod
    ● Measuring cylinder
    ● Beaker
    ● Pipettes
    ● Bench lamp
    Buffer
    ● Isolation medium
    ● DCPIP solution

    Method
    1. Remove stalks from leaf samples. Cut into small sections. Grind sample using a pestle and mortar and place into a chilled isolation solution.
    2. Place several layers of muslin cloth into funnel and wet with isolation medium to filter sample into a beaker. 3. Suspend the beaker in an ice water bath to keep sample chilled.
    3. Transfer to centrifuge tubes and centrifuge at high speed for 10 minutes. This will separate chloroplasts into the pellet.
    4. Remove supernatant and add pellet to fresh isolation medium.
    5. Store isolation solution on ice.
    6. Set the colorimeter to the red filter. Zero using a cuvette containing chloroplast extract and distilled water.
    7. Place test tube in rack 30cm from light source and add DCPIP. Immediately take a sample and add to cuvette.
    8. Measure the absorbance of the sample using the colorimeter
    9. Take a sample and measure its absorbance every 2 minutes for 10 minutes.
    11.Repeat for different distances from lamp up to 100 cm. This will vary the light intensity.

    NB: This experiment should be done in a darkened room to make results more reliable. The sample should not be put too close to the lamp as temperature may affect results.

    Risk Assessment
    1. DCPIP; Irritant to skin and eyes, may cause staining; Wear eye protection
    2. Biohazard; Allergies, soil bacteria, contamination; Wash hands after use
    3. Lamps; Temporary damage to eyes; Do not look directly at lamp
    4. Electrical appliances; Liquids near electrical appliances; Do not touch lamp/wires with wet hands, keep liquids away from lamp/wires

    Graph
    ● Plot a graph of absorbance against time for each distance from the light.

    Conclusion
    ● As the light intensity decreases, the rate of photosynthesis also decreases. This is because the lowered light intensity will slow the rate of photoionisation of the chlorophyll pigment, so the overall rate of the light dependent reaction will be slower.
    ● This means that less electrons are released by the chlorophyll, hence the DCPIP accepts less electrons. This means that it will take longer to turn from blue to colourless.
    ● When the DCPIP is blue, the absorbance is higher. The rate at which the absorbance decreases can therefore be used to determine the activity of the dehydrogenase enzyme. A higher rate of decrease, shown by a steep gradient on the graph, indicates that the dehydrogenase is highly active.
  • Investigate the effect of temperature on the rate of an enzyme-catalysed reaction, to include Q10.
    The rate of reaction of an enzyme-controlled reaction is influenced by different factors: the temperature, pH, concentration of the substrate, and the concentration of the enzyme. 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. Initial rate of reaction is measured because rate of an enzyme-controlled reaction is high, because enzymes act as biological catalysts, so concentration of reactants changes rapidly. The initial rate is the only point during the reaction when concentration of reactants and products is known.

    The effect of changing temperature on rate can be quantified up to optimum temperature via calculating the temperature coefficient (Q10) for the reaction. This indicates the change in rate of reaction caused by a 10 degree increase in temperature, and is calculated via dividing rate of reaction at temperature T + 10 degrees by rate of reaction at temperature T.

    Note: there are other ways to measure rate of reaction than the one outlined below. This method works because the enzyme catalase breaks down hydrogen peroxide into water and oxygen so rate can be calculated by measuring the volume of oxygen gas produced.

    Equipment
    ● Water bath
    ● Boiling tube
    ● Bung
    ● Soaked peas
    ● Hydrogen peroxide solution
    ● Delivery tube
    Gas syringe
    Stop clock
    ● Mortar and pestle

    Method
    1. Grind a known mass of peas in distilled water and place in a boiling tube.
    2. Add 5cm of hydrogen peroxide solution to the peas. 3. Fit the syringe into a delivery tube and the delivery tube into the boiling tube with a bung.
    3. Place the boiling tube into a water bath at a known temperature.
    4. Time for a set length of time e.g. 5 minutes. Measure the volume of gas produced at regular intervals e.g. 30 seconds.
    5. Repeat the experiment at different temperatures.

    Risk Assessment
    1. Biohazard; Contamination; Use disinfectant, wash hands with soap after handling
    2. Broken glass; Cuts from sharp object; Take care when handling glassware, keep away from edge of desk
    3. Hot liquids; Scalding; Handle with care, use tongs to remove boiling tubes from water bath, wear eye protection, keep away from edge of desk
    4. Hydrogen peroxide; May cause harm/irritation to eyes or in cuts; Wear eye protection, avoid contact with skin

    Graph
    ● Plot a graph of temperature against ethanol concentration/temperature.

    Conclusion
    ● Rate can be calculated by dividing volume of gas produced by time.
    ● Q10 can be calculated by dividing rate at T+10 degrees by rate at T degrees.
    ● Q10 for catalase is about 2; the rate doubles for every 10 degree increase.

    Note: Q10 can be only be used up to optimum temperature.
  • Investigate the effects of temperature on the development of organisms (such as seedling growth rate, brine shrimp hatch rates).
    Changes in average temperature have been recorded via use of ice cores, peat bogs, dendrochronology etc. This type of climate change is caused by the greenhouse effect - the increase in greenhouse gases such as methane, water vapour etc. in the atmosphere causing increased absorption of infrared light and increasing average temperature. Climate change has many effects - melting of the polar ice caps, increased incidence of natural disasters etc. - but one of those could be changes to morphology, number and distribution of organisms.

    Equipment
    ● Brine shrimp egg cysts
    ● Sea salt
    ● De-chlorinated water
    ● Salt water
    Beakers
    ● Water bath
    ● Stirring rod
    ● Magnifying glass
    Forceps
    ● Pipette
    ● Bright light
    Refrigerator
    ● Graph paper

    Method
    1. Place 2g of sea salt into a beaker containing 100 cm of dechlorinated water. Stir with the stirring rod until the salt completely dissolves.
    2. Put some eggs onto a sheet of paper.
    3. Wet a piece of graph paper in salt water. Place it face-down onto the sheet of paper so it picks up some eggs.
    4. Observe the graph paper under a microscope and count out 40 eggs.
    5. Remove the rest of the eggs/the paper so there are only 40 eggs there.
    6. Place the graph paper upside-down into the beaker and leave for 3 minutes/until all eggs have detached into the water.
    7. Incubate the beaker at a set temperature (between about 5 and 35 degrees Celsius - this mimics the conditions in the wild) for 24 hours.
    8. Remove the beaker from the incubator.
    9. Shine a bright light on the beaker. Any hatched larvae will swim towards the light and can then be removed with a pipette.
    10. Return the beaker to the incubator and repeatedly remove and count hatched larvae.
    11. Repeat all steps at a range of temperatures.

    NB: treat the shrimps, eggs and larvae responsibly and ethically for the duration of the experiment and release them into salt water when it is completed.

    Risk Assessment
    1. Broken glass; Cuts from sharp object; Take care when handling glassware, keep away from edge of desk
    2. Hot liquids; Scalding; Handle with care, use tongs to remove boiling tubes from water bath, wear eye protection
    3. Biohazard; Contamination; Use disinfectant, wash hands with soap after handling

    Graph
    ● Plot a graph of temperature against hatched larvae.

    Conclusion
    ● As the temperature increases, the number of shrimp hatched increases, up to an optimum of about 25 degrees Celsius.
    ● After the optimum temperature, as temperature increases, number of shrimp hatched will decrease.
    ● This indicates that as climate change increases temperature of sea water, the development of brine shrimps will decrease.

    Modification
    The method can be modified to measure the effect of climate change on seedling growth rate. Optimum temperature for seedlings depends on the species, but they show a similar pattern of increase in growth up to an optimum temperature (as rate of photosynthesis increases) and then decrease (as water loss will increase and crenation may occur). This indicates that plants are similarly vulnerable to climate change.
  • Use gel electrophoresis to separate DNA fragments of different length.
    Gel electrophoresis is used to separate DNA fragments of different sizes. In gel electrophoresis, DNA fragments (cut with restriction endonuclease enzymes) are loaded into wells in agarose gel and a current is applied. DNA has a negative charge, so it moves towards the anode, but different DNA fragments of different sizes will move at different rates and therefore create bands at different heights. This is used as part of DNA profiling.

    Equipment
    ● amplified DNA samples for suspects A-D (prepared in last lesson)
    crime scene allele ladder - 20µl
    ● orange G loading dye
    micropipette and tips(x20)
    ● waste tip beaker
    ● gel electrophoresis chamber
    power supply
    ● 3% agarose gel
    TAE running buffer
    fast blast DNA stain
    ● gel staining tray

    Method
    1. Practise using the micropipette to put samples into wells in the practise gel.
    2. Collect your 5 PCR tubes (from previous lesson) and place in a capless tube adaptor in a rack at your work station.
    3. Use a micropipette and a fresh tip, to pipette 10µl of Orange G loading dye (tube labelled LD) into your PCR reaction tube. Mix the contents well by gently pipetting up and down. Do this for each PCR tube using a fresh tip each time.
    4. Centrifuge each PCR tube briefly in order that all DNA is collected at the bottom, and not stuck up the tube sides.
    5. Remove the tape from around the edges of the agarose gel to be used in the electrophoresis, and set up in the chamber. The wells will be at the cathode (-) end which is black. The anode (+) is at the other end of the gel (red electrode).
    6. Fill the chamber with enough TAE buffer to cover the gel, but no more.
    7. Using the table below as a guide, load 20µl of each DNA sample in the order indicated.

    Lane Sample Load Volume (µl)
    1 Allele ladder 20
    2 Crime scene 20
    3 Suspect A 20
    4 Suspect B 20
    5 Suspect C 20
    6 Suspect D 20

    1. Run the gel at 100V for approximately 30 minutes. Do not let the orange dye front migrate to the end of the gel.
    2. The gel is now ready to be stained. The Biology Technician or Teacher will switch off the electrophoresis chamber and place the gels into a staining tray.
    3. Pour the fast blast stain into the staining tray to immerse the gel. Stain the gel for 5 minutes with gentle agitation. The Biology Technician will now remove the stain from the gels and return them to the lab.
    4. At this point the gels are placed on white light box and results are recorded by drawing or photography.

    Risk Assessment
    1. Electrical equipment can provide lethal shock;
    Ensure that all power cords are undamaged, and that all switches are in proper working condition.
    1. Pipette tips could potentially pierce skin;
    Immediately dispose of all used tips in a sharps container
    1. Fast Blast DNA stain could potentially act as an irritant; Wear gloves and goggles, rinse immediately if stain comes into contact with skin.
    2. Using an unbalanced centrifuge could cause injury;
    Always ensure to load all centrifuge tubes in a balanced manner.
    1. TAE buffer solution is an irritant; Avoid inhalation. Wear goggles and gloves to avoid skin contact.

    Validity (control variables)
    1. Voltage at which the electrophoresis chamber is operated; Operate the chamber at 100V for the duration of the procedure.
    2. Temperature at which the experiment is carried out;
    Perform all experiments in the same, draft-free laboratory, Do not move for the duration of the experiment.
    1. pH of the running buffer; Use the same running buffer for all repeats.

    Conclusion
    an exact match heavily implicates a suspect
  • Investigate the effect of different antibiotics on bacteria.
    Aseptic technique is used to avoid contamination of the sample from outside substances, such as microorganisms. This is important to get reliable and repeatable data. Inoculating an antibiotic agar plate (to provide nutrients), growing bacteria on it and then determining zone of inhibition can be used to measure the effectiveness of different antibiotics against particular species of bacteria.

    Equipment
    ● Agar plate seeded with bacteria
    ● Pipette
    ● Bunsen burner
    ● Disinfectant
    ● Soap
    ● Paper towels
    ● Forceps
    ● Antibiotic impregnated paper discs
    ● Sellotape
    Incubator

    Aseptic Technique
    ● Wipe down surfaces with antibacterial cleaner both before and after experiment.
    ● Use a Bunsen burner in the work space so that convection currents draw microbes away from the culture.
    ● Flame the wire hoop before using to transfer bacteria. ● Flame the neck of any bottles before use to prevent any bacteria entering the vessel (air moves out so unwanted organisms don't move in).
    ● Keep all vessels containing bacteria open for the minimum amount of time.
    ● Close windows and doors to limit air currents.

    Method
    1. Carry out the whole experiment using aseptic technique.
    2. Flame the forceps and pick up a paper disc.
    3. Slightly lift the lid of the petri dish and place the paper disc onto the agar.
    4. Tape the dish with two pieces of sellotape (don't tape all the way around to avoid conditions becoming anoxic).
    5. Wash your hands and disinfect the bench.
    6. Incubate for 24 hours at approximately 30 degrees.
    7. Measure the radius of the clear zone on the agar plate. Calculate the area

    Risk Assessment
    1. Disinfectant; Flammable; Keep away from naked flame
    2. Naked flame; Fire hazard, burns; Keep away from flammable materials, tie up long hair, keep away from edge of desk
    3. Biohazard; Contamination; Use aseptic technique, wash hands, wear eye protection
    4. Broken glass; Cuts from sharp object; Take care when handling glass objects, keep away from edge of desk

    Graph
    ● Plot a bar graph of type of antibiotics against area of clear zone.

    Conclusion
    ● The area of the zone of inhibition/ 'clear zone' will be more effective when the antibiotics are more effective against the type of bacteria being used.
    ● How effective an antibiotic is against a certain type of bacteria is dependent on whether the bacteria are gram-positive or gram-negative and what type of antibiotics are used.

    NB: gram-negative bacteria do not retain crystal violet stain, and appear pink due to secondary dye.
  • Investigate rate of respiration practically.
    A respirometer is a piece of equipment which measures the rate of respiration. It works by the addition of a drop of coloured liquid to a length of tubing. As the organism respires and the volume of oxygen in the tube decreases, the pressure also decreases, and the liquid moves down the pressure gradient towards the respirometer.

    Equipment
    ● Respirometer
    ● Actively respiring organisms
    ● Soda lime
    ● Coloured liquid
    Pipette
    Solvent
    ● Cotton wool
    ● Stop clock

    Method
    1. Assemble the respirometer.
    2. Add 5g of one organism to the boiling tube and replace the bung.
    3. Place a drop of coloured manometer fluid in the open end of the respirometer.
    4. Use a syringe to draw the fluid as far from the respirometer as possible and record its starting position.
    5. Close the tap. Start the stop clock.
    6. After five minutes, open the tap. Record the end position of the coloured liquid.
    7. Repeat the process for the other organism.

    Risk Assessment
    1. Broken glass; Cuts from sharp object; Take care when handling glass objects, keep away from edge of desk
    2. Soda lime; Corrosive; Wear eye protection, avoid contact with skin, keep away from edge of desk
    3. Biohazard; Contamination; Use disinfectant, wash hands with soap after handling organisms

    Analysis
    ● Convert distance moved by the liquid in the time into volume of gas by using the πD formula with the diameter of the respirometers tube to produce a cross-section and then multiplying by distance moved.
    ● Convert volume into rate by dividing by five minutes. ● Convert rate into rate per gram of organism by dividing by five grams.

    Conclusion
    ● Soda lime absorbs carbon dioxide that is given out during respiration, so any changes in volume are assumed to be only due to differences in oxygen uptake.
    ● Gas exchange due to photosynthesis is ignored and all of the gas is assumed to be oxygen.
    ● Different organisms have different rates of respiration - the animals have a higher rate of respiration per gram than the plants, as they have a higher metabolic rate and require much more energy to be released for movement/reproduction/etc.
  • Investigate the effects of exercise on tidal volume, breathing rate, respiratory minute ventilation and oxygen consumption using data from spirometer traces.
    A spirometer is a device which measures breathing and respiration. It can measure breathing rate (number of breaths per minute), tidal volume (volume of gas of one normal breath), respiratory minute ventilation (volume of gas inhaled per minute), and vital capacity (volume of gas of one forced deep breath).

    A spirometer consists of a chamber filled with air, connected by tubes and suspended in a tank of water. When the volume of gas changes, the lid of the chamber moves up and down. The chamber contains soda lime to absorb the exhaled carbon dioxide, so changes in the position of the lid are due to changes in the volume of oxygen inhaled/exhaled by the person breathing into the tubes. The person wears a nose clip so that all breathing occurs through the mouth. The graph produced by a spirometer is called a spirograph or spirometer trace.

    Equipment
    ● Spirometer
    ● Datalogger/computer/kymograph
    ● Soda lime
    ● Disinfectant
    ● Nose clip

    Method
    1. Find the vertical scale by emptying the chamber, starting the kymograph and then forcing a known volume of air into the chamber. This measures the volume of gas in the chamber, and by reading the trace, the movement of the pen on the kymograph can be calibrated to the actual volume of air.
    2. Find the horizontal scale by setting it to 1 mm per second, using the switch (or as close to 1 mm per second as possible). This is the speed at which the drum turns.
    3. A trained member of staff can fill the spirometer with medical grade oxygen.
    4. Disinfect the mouthpiece and attach it to the tube. Turn the tap so the tube is not attached to the spirometer.
    5. Subject attaches the nose clip and breathes into the tube for a while to practice. When they are comfortable, start the datalogger/kymograph and turn the tap to attach the tube to the spirometer.
    6. Subject takes one forced deep breath and then breathes normally into the spirometer for a duration of 5 minutes maximum.

    Risk Assessment
    1. Disinfectant; Flammable; Keep away from naked flame
    2. Soda Lime; Corrosive; Wear eye protection, avoid contact with skin, keep away from edge of desk
    3. Spirometer; Breathing/circulatory problems; Read manufacturer's notes before using, only use spirometer supervised, don't use with breathing/circulatory issues

    Conclusion
    ● From the calibration of the kymograph, volume of air can be linked to the movement (e.g. in number of squares) of the pen on the kymograph. Therefore, distance can be linked to volume.
    ● Interpreting the spirometer trace:
    ○ The tidal volume is the distance from peak to trough, when the subject is breathing normally.
    ○ The vital capacity is the distance from peak to trough, when the subject takes a forced deep breath.
    ○ Breathing rate is the number of peaks in a time corresponding to a minute (e.g. total peaks divided by 5).
    ○ Respiratory minute ventilation is calculated by multiplying breathing rate by tidal volume.
    ● If the experiment is repeated after exercise (although not during exercise, because a spirometer can create resistance to breathing and therefore isn't safe for use during exercise):
    ○Tidal volume increases.
    ○ Vital capacity remains the same (although can be impacted long-term by regular aerobic exercise).
    ○ Breathing rate increases.
    ○ Respiratory minute ventilation increases.
    ● This is because respiration increases during exercise because of increased muscle contraction. Therefore, more oxygen is required and more carbon dioxide is produced, so breathing rate, tidal volume and therefore respiratory minute ventilation increases to cope with this demand up to the maximum aerobic rate. After this point, minute ventilation will plateau to a maximum and further respiration will be anaerobic.

    NB: informed consent should be obtained before anyone uses the spirometer, as they are a participant in scientific research. Anyone should be allowed to refrain from participating or contributing their data, and can stop at any time during the practical.
  • Investigate habituation to a stimulus.
    When snails are touched between the eye buds they retract into their shells very quickly and take some time to re-emerge. This is an innate response to what is perceived as a threat. Habituation is the decreasing of an innate response, such as this one over time, as the stimulus is repeated and is learned to be benign.

    Equipment
    ● Snail
    ● Cotton wool bud
    ● Distilled water
    ● Chopping board
    ● Stopwatch

    Method
    1. Dampen the cotton wool bud in distilled water.
    2. Touch the snail between the eye buds with the cotton wool bud
    3. Time the length of time it takes for the snail to emerge fully from its shell again.
    4. Repeat for a total of ten touches.

    Risk Assessment
    1. Biohazard; Contamination; Use disinfectant, wash hands with soap after handling
    2. Bags/stools; Tripping; Keep under desks and away from workspace

    Graph
    ● Plot a graph of number of touches against time to re-emerge from shell.

    Conclusion
    ● Results can be tested using Spearman's Rank Correlation Coefficient to determine the correlation between the two variables and whether it is statistically significant.
    ● As number of touches increases, the time taken for the snail to re-emerge decreases because the snail becomes habituated to the stimuli. This is because calcium channels in the presynaptic membrane become less responsive to stimuli (the threshold increases) over time, so less neurotransmitter is released into the synaptic cleft.
    ● This is because it is evolutionarily advantageous not to use resources on responding to a benign stimulus.

    Note: it is important to treat the snails ethically during the experiment and to re-release them afterwards.