Practical work and student investigations 1 Tests for glucose and starch, lipid and protein 2 Controlled experiments to illustrate how enzyme activity can be affected by changes in temperature and pH 3 Simple experiments on diffusion and osmosis using living and non-living systems 4 Controlled experiments to investigate photosynthesis, showing the evolution of oxygen from a water plant, the production of starch and the requirements of light, carbon dioxide and chlorophyll 5 A simple experiment to determine the energy content of a food sample 6 Controlled experiments to demonstrate the evolution of carbon dioxide and heat from respiring seeds or other suitable living organisms 7 Simple controlled experiments to investigate the effect of light on net gas exchange from a leaf, using hydrogen-carbonate indicator 8 A simple experiment to investigate the effect of exercise on breathing in humans 9 Experiments to investigate the role of environmental factors in determining the rate of transpiration from a leafy shoot 10 A simple experiment to investigate the effect of exercise on heart rate in humans 11 A simple experiment to show how the sensitivity of the skin differs on finger tips, back of hand, wrist and forearm 12 A practical exercise comparing floral structure in insect-pollinated and wind-pollinated flowers 13 Controlled experiments to demonstrate phototropic and geotropic plant growth responses 14 The use of quadrats to estimate the population size of an organism in two different areas 15 A simple experiment to investigate carbon dioxide production by yeast in different conditions. 1 1 Tests for glucose, starch, lipid and protein The tests that are expected: • Benedict’s test for glucose Testing for sugars Benedict’s solution is blue. Add Benedict’s solution and heat in a water bath. Solution should turn from blue to green to yellow and finally red if glucose is present. If solution remains blue, then glucose is not present. • iodine test for starch (test not required by specification) Testing for starch Iodine is orange/brown Add iodine solution, if it turns blue black it means starch is present. If it stays yellow/orange means there is no starch present • the emulsion test for lipid Testing for lipid (fat) Dissolve the lipid in ethanol then add water and shake vigorously, if it goes cloudy white lipid is present. • biuret reagent for protein(test not required by specification). Testing for protein Biuret reagent is pale blue. Add Biuret reagent to a solution of food, if it turns purple protein is present. If it remains pale blue no protein is present These tests could be carried out: • on prepared samples of pure substances • on foods that contain the substances • in a context. Example To demonstrate health applications, a fake ‘urine sample’ can be made by colouring water with iodine solution. A similar solution, with glucose added, could mimic the urine of an untreated diabetic. Students can use Benedict’s test to identify the diabetic – if glucose is present in urine the patient is diabetic. The iodine test could be introduced when teaching that plants manufacture starch by photosynthesis, or when investigating the effect of amylase on starch digestion. 2 Controlled experiments to illustrate how enzyme activity can be affected by changes in temperature 1. The effect of amylase on starch digestion At each temperature selected, from 0°C to 100°C, samples of amylase solution and of starch solution are brought to temperature before being added together. The mixture is then kept at the same temperature. To measure the rate of reaction, drops of the mixture can be collected at intervals of one minute and added to individual iodine drops on a white tile. Iodine tests for the presence of starch – iodine goes black when starch is present. The time taken for the starch to disappear is recorded for each temperature – indicated when the iodine no longer changes colour (remains orange/brown). The temperature can be controlled using water baths. The digestive enzyme amylase breaks down starch into the sugar maltose. If the speed at which the starch disappears is recorded, this is a measure of the activity of the amylase. 2. The effect of catalase on the breakdown of hydrogen peroxide. Catalase is found on the surface of potato and liver. Catalase converts hydrogen peroxide into water and oxygen. The rate of oxygen production can be measured as an indication of enzyme activity. An upturned boiling tube previously filled with water can be used to collect and measure the volume of oxygen evolved. Or the time take for ‘bubbling’ to stop can be measured. The liver/potato can be exposed to different temperature to demonstrate the effect of temperature. Enzymes are proteins that speed up chemical reactions in our cells. Enzymes work best at their optimum temperature. This is why homeostasis is important - to keep our body temperature at a constant 37°C. As the temperature increases, so does the rate of chemical reaction. This is because heat energy causes more collisions, with more energy, between the enzyme molecules active sites and other molecules. However, if the temperature gets too high, the enzyme is denatured and stops working. A common error in exams is to write that enzymes are killed at high temperatures. Since enzymes are not living things, they cannot be killed. Graph showing the effect of temperature on enzyme reactions 3 3. Effect of changing pH on enzyme’s rate of reaction. 1 cm3 sodium carbonate solution 0.5 cm3 sodium carbonate solution 2 cm3 acetic acid 4 cm3 acetic acid Place 5 cm3 starch solution in each tube. Note the time and add 1 cm3 amylase solution to each Then test for starch using iodine – take a sample every 30 seconds and time how long it takes for the iodine to no longer turn blue/black. The most rapid breakdown of starch will be in the region of pH 6-7. When the pH in the mouth was tested, the result was probably about pH 7. Logically you would expect the pH in the mouth to be the one which favoured a rapid breakdown of starch. The other pH values seem to slow/inhibit the action of the enzyme – pH can denature the enzyme so that the enzyme’s active site changes shape and the substrate can no longer bind to it.. 4 Simple experiments on diffusion and osmosis using living and non-living systems a Cubes of agar jelly placed into solutions of methylene blue or potassium permanganate will absorb the pigment by diffusion. The cubes are left in the pigmented solution for different measured periods of time and are then sliced open. The distance between the edge of each cube and the edge of the coloured agar may be used as a measure of the distance the pigment molecules have moved by diffusion. Demonstration of diffusion in a jelly Agar jelly has a consistency similar to the cytoplasm of a cell. Like cytoplasm, it has a high water content. Agar can be used to show how substances diffuse through a cell. This demonstration uses the reaction between hydrochloric acid and potassium permanganate solution. When hydrochloric acid comes into contact with potassium permanganate, the purple colour of the permanganate disappears. A Petri dish is prepared which contains a 2 cm deep layer of agar jelly, dyed purple with potassium permanganate. Three cubes of different sizes are cut out of the jelly, with side lengths 2 cm, 1 cm and 0.5 cm. The cubes are carefully dropped, at the same time, into a beaker of dilute hydrochloric acid (Figure 1.10). The time is taken for each cube to turn colourless. The smaller the cube the larger its surface area to volume ratio, so the quicker the rate of diffusion (the shorter the time for the cube to go colourless). b A crystal of potassium permanganate can be dropped into a beaker of water and the appearance of the water noted over time. 5 6 c To demonstrate osmosis, Visking tubing (dialysis tubing) can be tied at one end and filled with 20 per cent sucrose solution. The other end is attached to a capillary tube. The level of the sucrose can be noted before and after the tubing has been placed in a beaker of water for about 30 minutes. Visking tubing has microscopic holes in it, which let small molecules like water pass through (it is permeable to them) but is not permeable to some larger molecules, such as the sugar sucrose. This is why it is called ‘partially’ permeable. The sucrose molecules are too big to pass through the holes in the partially permeable membrane. The water molecules can pass through the membrane in either direction, but those on the right are attracted to the sugar molecules. This slows them down and means that they are less free to move – they have less kinetic energy. As a result of this, more water molecules diffuse from left to right than from right to left. In other words, there is a greater diffusion of water molecules from the more dilute solution (in this case pure water) to the more concentrated solution. 7 d Onion epidermis can be peeled away, cut into squares and mounted on slides in different concentrations of sucrose solution. Observation under a microscope will show the effects of osmosis. Plasmolysis - The state of a plant cell in a hypertonic solution, the cell shrinks. Turgid - Plant cells in a hypotonic solution, the cell swells against the cell wall and the cell is rigid and firm Flaccid - Plant cells in a hypertonic solution, the cell shrinks away from the cell wall and the cell is limp. 8 e Red blood cells in blood obtained from a butcher may be mounted on slides in hypotonic, isotonic and hypertonic saline, and observed under a microscope to show the effects of osmosis. Blood plasma has a concentration equivalent to a 0.85% salt solution. If fresh blood is placed into solutions with different concentrations, the blood cells will gain or lose water by osmosis. This can be demonstrated using sterile animal blood (available from suppliers of biological materials). Three test tubes are set up, containing these solutions: A 10 cmᶟ of distilled water (hypotonic) B 10 cmᶟ of 0.85% salt solution (isotonic) C 10 cmᶟ of 3% salt solution (hypertonic) 1 cmᶟ of blood is added to each tube, and the tubes are shaken. A sample from each tube is examined under the microscope. The sample from tube A is found to contain no intact cells The blood cells on the right (tube C) were placed in a 3% salt solution and the normal blood cells on the left(tube B) were in a 0.85% salt solution. The cells from tube B look normal, but those from tube C are shrunken, with crinkly edges It is important that animal cells are surrounded by a solution containing the correct concentration of dissolved solutes. If the surrounding solution does not have the right concentration, cells can be damaged by the effects of osmosis. The red blood cells placed in water absorb the water by osmosis, swell up and burst, leaving a red solution of haemoglobin in the test tube. When placed in 3% salt solution, the red blood cells lose water by osmosis and shrink. The three tubes are now placed in a centrifuge and spun around at high speed to separate any solid particles from solution. Tube A contains a clear red solution and no solid material at the bottom of the tube = the cells have burst due to water entering via osmosis and haemoglobin is released into the water Tubes B and C both contain a colourless liquid and a red precipitate at the bottom = the cells have remained intact so haemoglobin remains in the cells. 9 f Osmosis can be demonstrated by using strips of potato, and this basic experimental method provides a good opportunity for students to carry out individual whole investigations. Because of the difficulty of the osmosis concept, it is better to keep this investigation until the latter part of the course so that students will have had previous experience of carrying out investigations on simpler topics. Students enjoy the reference to ‘chips’, but should quickly realise that it can be difficult to keep the size constant – to achieve consistency lengths of potato tissue can be drilled from a potato using a cork borer. The ‘chips’ are measured by mass or by length and are placed into sucrose solutions of different concentrations for at least one hour. The percentage change in mass or length is a measure of the degree of osmosis that has occurred. A potato tuber is a plant storage organ. It is a convenient tissue to use to investigate the effects of osmosis on the mass of the tissue. A boiling tube is half-filled with tap water and a second with concentrated (Molar) sucrose solution. A third tube is left empty. A potato is ‘chipped’ – cut into chips 5cm x 1cm x 1cm, making these measurements as accurate as possible to ensure the chips have the same surface area. No skin is left on the potato (it would provide a waterproof layer). Each chip is gently blotted to remove excess moisture and weighed to find the starting mass of each. One chip is placed into each of the three tubes, and after a fixed time the chips are removed, gently blotted to remove excess moisture and reweighed. The chips can be felt to see and change in texture and re-measured to see if there is any changed in dimensions (length/width/breadth). The change in mass (+/-) is calculated for each chip and the % change is found. (% change in mass takes into account the difference in starting masses of the chip so they can be compared). % change = change in mass x 100 Starting mass Boiling tube with water - water moves into the cells The water in the tube is more concentrated than the water in the cells, so water molecules will move from an area of higher concentration of water (in the tube) to an area of lower concentration of water (in the potato cells). The cell membrane is semi-permeable so the water molecules can get through and each cell will swell as the water flows in. As all the cells swell, the potato chip will increase in mass, and length/width/breadth and become more rigid. Boiling tube with concentrated sugar solution - water moves out of the cells The water in the tube is a lower concentration than the water in the cells, so water molecules will move from an area of higher concentration of water (in the potato cells) to an area of lower concentration of water (in the tube). The cell membrane is semi-permeable so the water molecules can get through and each cell will shrink as the water flows out. As all the cells shrink, the potato chip will decrease in mass, and length/width/breadth and become more flexible/floppy. Boiling tube with nothing - some water moves out of the cells Water molecules will move from an area of higher concentration of water (in the potato cells) to an area of lower concentration of water (in the tube). The cell membrane is semi-permeable so the water molecules can get through and each cell will shrink as the water flows out. As all the cells shrink, the potato chip will decrease in mass, and length/width/breadth and become more flexible/floppy. The effect will be less than in the tube with concentrated sugar solution. 10 g A variation on this theme is to cut potato cubes of different sizes, which have different surface area to volume ratios. After measuring and recording the masses of the cubes, they are immersed in water. After one hour, the cubes are blotted dry and their masses measured and recorded again. The percentage increase in mass for cubes of different surface area to mass ratio can be compared, to explore the concept of how surface area to volume ratio influences water uptake. The surface area over which the osmosis occurs can change the speed at which it happens dramatically. The larger the surface area between the two solutions the faster osmosis will happen. This is due to more of each solution will be in contact with the membrane at one moment. This means that a larger amount of the free water molecules in the solution can move across the membrane at the same time therefore making the process happen much faster. Eight 1cm³ cubes One 8cm³ cube 11 Controlled experiments to investigate photosynthesis, showing the evolution of oxygen from a water plant, the production of starch by leaves and the requirements for light, carbon dioxide and chlorophyll a The evolution of oxygen from a water plant can be seen if a water plant (typically elodea or a similar species) is placed in a beaker of water and covered with a glass funnel that has a water-filled test tube placed over its opening. After 24 hours, a colourless gas will have displaced water from the test tube. A test for oxygen is then carried out. Re-light a glowing splint 12 b To measure the rate of oxygen production, the stem of a water plant is cut under water, and the plant kept immersed in water in a beaker or boiling tube. The number of bubbles of gas given off over a measured time period can be counted. This simple experimental set up can enable students to carry out individual investigations into the effect of different factors on the rate of bubble production. Suitable variables include: light intensity (the plant is exposed to a light source and the rate of bubble production measured at different light intensities by changing the distance between the light source and the water plant); colour/wavelength of light (coloured filters are placed between the plant and the light source); and carbon dioxide availability (the plant is immersed in solutions of different concentration of sodium hydrogen carbonate). It is vital to have a stabilising period of a minimum 5 minutes before any measurements are taken. An increase in light intensity should bring about an increase in gas production and, until a limiting factor comes into operation, the two should be proportional. Increasing the light intensity makes available a greater amount of energy to drive the photosynthetic reaction. Since oxygen is a product of photosynthesis, a higher rate of photosynthesis should result in a higher rate of oxygen production. Unless the gas produced by Elodea is pure oxygen it would be possible for the plant to double its oxygen output without doubling the volume of gas if the proportion of oxygen in the gas mixture were increased. One would need to know that the percentage of oxygen in the bubbles remains constant, before it could be assumed that doubling the volume of gas meant doubling the production of oxygen. It is quite likely that the gas production will increase in direct proportion to the light intensity over the whole range of this experiment. Limiting factors may operate, however, at the higher light intensities. It is unlikely that gas production would continue to increase indefinitely with increasing light intensity since carbon dioxide would become limiting or internal factors such as the available chloroplasts or the diffusion rate of carbon dioxide would impose a limit on the rate of photosynthesis. As the bench lamp comes closer to the beaker it might be expected that there would be a rise in temperature of the water in the beaker. Since a rise in temperature speeds up many chemical reactions it might also accelerate some stages of photosynthesis. (In practice, the temperature is unlikely to rise more than 1° C.) 13 c Starch production can be investigated by placing a plant in the dark for 24 hours to de-starch the leaves. A starch test on a leaf from a plant that has been kept in the dark will not give a blue-black colour, whereas a similar test on a control leaf from a plant kept in the light will give a blue-black colour. Remove a green leaf from a plant that has been exposed to sunlight for a few hours Half-fill a beaker with water. Heat the water until it boils. Keep the water at boiling point. Use the forceps to place the leaf in the boiling water. Boil for 2 minutes. Boiling the leaf in water: Removes the waxy cuticle which prevents entry of iodine/potassium iodide solution. Ruptures cell membranes to make starch granules in cytoplasm and chloroplasts accessible to iodine/potassium iodide solution. Cell membranes are selectively permeable and do not readily allow the penetration of iodine. Denatures enzymes, particularly those which convert starch to glucose e.g. diastase. Boiling arrests all chemical reactions, since enzymes which catalyse the reactions are denatured. Denatured enzymes have altered or destroyed active sites due to heat, pH, ionic concentration Place the boiled leaf in a boiling tube containing 90% ethanol. Place the boiling tube in hot water and boil for 10 minutes or until the leaf decolourizes. Boiling the leaf in ethanol: Removes chlorophyll which is a green pigment and so masks the colour change of the iodine test for starch, so the leaf needs to be ' decolourized' for changes to be observed. A decolourized leaf is pale yellow or green. Ethanol is an organic solvent and so extracts chlorophyll from the leaf. Gently remove the leaf and wash with a fine trickle of cold tap water. Spread the leaf evenly on a white tile. Add a few drops of iodine/potassium iodide solution to the leaf and note any observations. The iodine solution will turn from brown to blue - black if starch is present. 14 d A starch test on a variegated leaf can be used to demonstrate that chlorophyll is needed for photosynthesis. Place a plant with variegated leaves in the dark for at least 24 hours to make sure that there is no starch in the leaves i.e. ‘de-starching the leaves’. Variegated Leaves: have green and white areas – the green areas have chlorophyll but chlorophyll is absent in the white areas. Leave the plant in the light with de-starched leaves in good light for 6 hours at room temperature (20˚C). Then test the leaf for the presence of starch using iodine. Compare the stain pattern with the colour map. The green chlorophyll rich parts are blue-black with starch. The white parts without chlorophyll are yellow-brown without starch. Since starch is only produced if chlorophyll is present, then chlorophyll is needed for photosynthesis e To show that carbon dioxide is needed for photosynthesis, a leaf on a plant may be surrounded by air with no carbon dioxide by inserting it into a conical flask containing a small amount of potassium or sodium hydroxide. The plant is left in good light for 24 hours. NaOH removes CO₂ The test leaf and a control leaf from the plant are then tested for starch. To test leaves for starch: • drop into very hot/boiling water for one minute (to destroy the cell membranes so that chlorophyll molecules can pass through) • drop into hot ethanol (to remove/dissolve the green chlorophyll) • place leaf into water (to rehydrate and soften the leaf so that it can be spread out) • drop iodine solution onto the leaf (test for starch) – blue-black colour will show the presence of starch. 15 A simple experiment to determine the energy content of a food sample Fat-containing foods such as dried crisps work very well. • A known mass of the food sample is weighed and the mass noted. • A boiling tube is prepared, containing a known volume of water. The water temperature is recorded. • The food sample is put in a crucible or burning spoon and ignited (for example in the flame of a Bunsen burner). • The food sample is quickly placed under the boiling tube. As soon as the food sample has completely burnt the water temperature in the boiling tube is re-measured. • The equation used to calculate the energy content of the food is: Energy content of food sample (joules per gram) = mass of water heated (g) x temperature rise (oC) x 4.2 mass of food sample (g) To obtain an accurate result, all the energy in the food sample needs to be transferred to the water, but unless you use a bomb calorimeter this won’t happen. Bomb calorimeter NB: The energy gets lost to the surroundings as not all energy is direct to heat the water, also there is a need to heat the glass apparatus first. 16 Controlled experiments to demonstrate the evolution of carbon dioxide and heat from respiring seeds or other suitable living organisms Vacuum flasks are needed for this activity. Surface sterilised seeds are put into a flask, which is sealed with a bung. To show heat production the flask needs a cotton wool bung with a thermometer going through the bung into the flask. Students could demonstrate that they produce carbon dioxide by breathing out through a tube into lime water or hydrogen carbonate indicator. thermometer cotton wool Living seeds Dead seeds A B The temperature will increase significantly in the flask containing the live seeds. Respiration produces the energy the seeds need to germinate and the by-product of the use of the energy is heat (energy). 17 A glass tube runs from inside the flask, through the bung and into an indicator solution of either limewater or hydrogen carbonate. The carbon dioxide produces changes the colour of the indicator solution. The lime water associated with living organisms should be milky. The lime water receiving air from nonliving organisms should be clear. Carbon dioxide is the gas which turns lime water milky. The results suggest that carbon dioxide is given out by the organisms. The air from non-living organisms failed to turn lime water milky. Thus the carbon dioxide which was in sufficient concentration from living organisms to turn lime water milky so must have come from the living organisms rather than the air around them NB: The experiment provides evidence for only the organisms in the test-tubes. The failure of dead organisms to turn lime water milky shows that the production of carbon dioxide results from a process that occurs in living organisms but not in dead ones. Draw gas off from respiring seeds Bubble gas through limewater – if it goes cloudy then CO₂ is present 18 Simple controlled experiments to investigate the effect of light on net gas exchange from a leaf, using hydrogen carbonate indicator A water plant can be placed in a sealed tube of air-equilibrated hydrogen carbonate solution (red in colour) and placed in the light or in the dark. The solution will turn purple if kept in the light and will turn yellow if kept in the dark. A variation could involve the use of water snails or, if not available, small land insects placed on a gauze platform above the indicator, with and without the water plant. This variation allows students to think about the balance between carbon dioxide used by photosynthesis and carbon dioxide produced by respiration. Tube A B C Colour of indicator yellow purple red Level of CO₂ in the water high low normal Cause Respiration Photosynthesis Respiration and Photosynthesis A useful demonstration uses four tubes containing hydrogen carbonate solution: one with water plant only, one with animals only, one with both water plant and animals and one with no living organisms. One set of the tubes is exposed to light and left for 12 to 24 hours and another set is placed in the dark for the same length of time. 19 A simple experiment to investigate the effect of exercise on breathing in humans The breathing rate can be measured at rest and after a period of exercise by counting the number of inhalations per minute. Exercise also influences the rate of breathing by increasing the volume of each breath, this can be measured by taking the volume of one exhalation before and after exercise. This can be done by breathing through a tube into a plastic container filled with water. The volume of displaced water can be measured. The breathing rate at rest and after exercise can be calculated as number of breaths per minute x volume of each breath. Volume of each breath before exercise Volume of each breath during exercise 20 Experiments to investigate the role of environmental factors in determining the rate of transpiration from a leafy shoot A bubble potometer can be used to illustrate the effects of light, wind, temperature and air humidity. Plants covered with dark polythene bags simulate darkness and can be compared with plants covered with transparent polythene bags. Hairdryers can simulate wind. The use of potted plants is also acceptable, where the pot and the soil is sealed with polythene and the mass of the potted plant is measured before and after a period of exposure to the environmental factor. Effect of different factors on transpiration rate: An increase in rate will result from transferring the shoot from darkness to light, moving from shade to sunlight, from still to moving air and removing the plastic bag. A decrease will occur when the shoot is enclosed in a plastic bag and on any reduction of light intensity. The stomata probably respond to increased light intensity by opening more widely so allowing more rapid diffusion. Direct sunlight will also warm up the leaves and increase the rate of evaporation. Inside the plastic bag, the humidity will rise to 100% saturation and so prevent the establishment of a diffusion gradient for water vapour from the leaf to the atmosphere. In moving air, the water vapour is carried away from the leaf so maintaining a steep diffusion gradient. Changes in light intensity and diffusion gradients may affect the rate of photosynthesis. Changes in photosynthetic rate are, however, unlikely to produce such large changes in water uptake. The most dramatic increase in transpiration will be seen when the shoot is moved from shade to sunlight. The rate may increase to 10 times that in the shade of the laboratory. It is difficult to separate the effects of temperature and light in these circumstances. On moving from the laboratory to outside there will be changes in light intensity, temperature, air movement and perhaps humidity. The inside of the plastic bag may become misted with condensed water. The significance is: (a) that the water must have come from the plant (b) that the air in the bag must be saturated with water vapour. An increase in light intensity will increase the rate of photosynthesis if other factors are not limiting. An increased rate of photosynthesis will need more water, which may be reflected in the rate of uptake. The distinction between uptake and loss is made because not all the water taken up is necessarily transpired or, alternatively, evaporation may be taking place faster than uptake in some circumstances. It is assumed that the bulk of the water taken up is evaporated and that the changing conditions affect evaporation far more than any other process in the leaf. 21 A simple experiment to investigate the effect of exercise on heart rate in humans Students should be shown how to measure and record their pulse. They can measure this at rest. A short period of exercise can follow, stepping on and off a low stool, or running up and down a flight of stairs for five minutes. (Safety note: careful supervision is required.) The pulse rate should now be recorded for five minutes or until it returns to its rest level. This experiment can provide a great deal of interesting data on the variation in resting heart rate, the effects of exercise and how quickly the heart recovers. Discussion can include measures of fitness, heart disease, cardiac output and the effects of long-term exercise on stroke volume. The muscles start to produce more CO2 as they work harder To get rid of the extra CO2 you start to breathe deeper and faster Your heart rate increases to pump more oxygen around the body Your arterioles widen to stop your blood pressure increasing Blood is diverted away from inactive organs (stomach / liver) and towards the working muscles through vasoconstriction and vasodilation When the muscles contract they squeeze blood back towards your heart The heart contracts even stronger to pump more blood with each beat 22 A simple experiment to show how the sensitivity of the skin differs on finger tips, back of hand, wrist and forearm Students should work in pairs. A piece of hard cardboard or cork can be used to fix the two prongs of a hairpin or two pins 5 mm apart. This is then used by one student to lightly touch the fingertips of another who is looking away. The first student can use both points or one point as a stimulus. The second student then has to judge whether one or two points were used and their response recorded as correct or incorrect. This can be repeated 10 times for each area of the hand. It is then repeated using pins 1 cm apart and 2 cm apart. Students can then identify the most sensitive area as this should have the most correct responses with the smaller distance. Conclusions can be made about the number of sensory nerve endings, receptive field size and the thickness of skin. This practical also provides opportunities to discuss data analysis, experiment design and anomalous results, and the benefits of grouping class results. Finger-tips will probably recognize the two points even when they are only about 2 mm apart, i.e. the lower limit of this instrument. The back of the hand will probably need a separation of l0 mm to achieve a score of 8-10. Other areas will probably be even less sensitive. Possibilities: (a) One point does not touch a receptor. It seems unlikely that, on the back of the hand, a pressure actually indenting the skin will not affect at least one receptor. Moreover, recognition of two stimuli when they are separated by half a second suggests that it is not shortage of receptors alone which causes poor discrimination. (b) Both points touch receptors which feed impulses into one nerve fibre. Since a single sensory fibre may receive branches from sensory endings covering a region of several square millimetres this explanation is quite plausible. The recognition of the non-simultaneous double stimulus could be explained by the additional burst of impulses initiated by a second, stimulus. It has been claimed that single hair follicle receptors are sensitive over an area several centimetres in diameter but the 65 mm separation claimed for the back could hardly be explained in this way. (c) There are two receptors and two sensory fibres but unless the impulses are separated in time, they cannot be discriminated by the brain. (a) Only one receptor affected (b) Both receptors feed impulses into the same fibre 23 (c) Only one fibre but stimuli separated in time A practical exercise comparing floral structure in insect-pollinated and wind-pollinated flowers Most areas should have access to suitable specimens. Insect-pollinated flowers can be examined and the various structures observed. Wind-pollinated grasses should be available but have fewer structures to see. Insect Pollinated Wind Pollinated large, brightly coloured petals - to attract insects small petals, often brown or dull green - no need to attract insects often sweetly scented - to attract insects no scent - no need to attract insects usually contain nectar - to attract insects no nectar - no need to attract insects moderate quantity of pollen - less wastage than with wind pollination pollen produced in great quantities - because most does not reach another flower pollen often sticky or spiky - to stick to insects pollen very light and smooth - so it can be blown in the wind and stops it clumping together anthers firm and inside flower - to brush against insects anthers loosely attached and dangle out - to release pollen into the wind stigma inside the flower - so that the insect brushes against it stigma hangs outside the flower - to catch the drifting pollen stigma has sticky coating - pollen sticks to it stigma feathery or net like - to catch the drifting pollen Insect pollinated flowers rose sweet pea Wind pollinated flowers ragweed 24 Controlled experiments to demonstrate phototropic and geotropic plant growth responses Plant material such as wheat, maize, oat or cress seedlings can be used to demonstrate phototropism. Petri dishes containing moist cotton wool and the plant material can be put into light-proof boxes such as shoe boxes. To create unilateral light a small slit can be cut in one side of the box and light can be shone into the box. Control seedlings can either have aluminium caps put on their tips or can be kept in a shoe box without a slit for light. A clinostat needs to be used to demonstrate geotropism. To test the response to or effect of light on a plant shoot. A growing herbaceous stem is placed in a box that has a small opening on one side only, where light enters. Auxins are plant hormones that make some parts of a plant stem grow faster than others. The result is that the plant stem bends towards the light. You may have noticed that a houseplant grows towards the window and turns its leaves towards the light. It does this because light coming from the window side of the plant destroys the auxin in that side of the stem. So growth on that side slows down. On the shaded side of the plant there is more auxin. So growth on this side speeds up. The result is that the shoots and leaves are turned towards the light for photosynthesis. Use of a clinostat to show geotropism in seedlings 25 A few beans are soaked in water overnight then placed on wet cotton wool for a few days until the first root of each seed (the radicle) grows to a length of about 2cm. Wet cotton wool is attached to the cork disc of two clinostats. Three or four of the germinating bean seeds are pinned onto each of the discs with their radicles pointing outwards. Covers are placed on them to keep the air moist. The clinostats are turned on their sides. One is left switched on, the other is switched off to act as the control (to allow you to compare your results) Both clinostats are left set up for a few days. The cotton wool is kept damp. After this time the radicles of the beans on the control clinostat will have grown downwards, whereas those on the beans on the rotating clinostat will be growing straight out horizontally. Explanation: Gravity would have been 'pulling' consistently at 90° to the horizontal radicles in the stationary box. In the clinostat the gravitational force also acts at 90° but on all sides of the radicle in turn. 26 The use of quadrats to estimate the population size of an organism in two different areas Quadrats can be used to sample part of each area. Calculation will be needed to work out the estimated population size. For example, if 10 quadrats have been used and the total area amounts to 100 quadrats, the estimated population size will be the number of organisms counted in the 10 sample quadrats multiplied by 10. It is important to place the sample quadrats randomly – this avoids bias and improves the reliability of the data. An interesting way to practise the technique is to throw plastic beads on the floor of the classroom and ask students to guess how many beads there are. The quadrat sampling procedure can be used in front of students to get an estimate. The beads can then be collected and counted. The actual number can be compared to the estimated number and used to see how accurate the estimation was. A quadrat is a square (of either metal, wood, or plastic) used in ecology and geography to isolate a sample, usually about 1m2 or 0.25m2. The quadrat is suitable for sampling plants, slowmoving animals (such as millipedes and insects), and some aquatic organisms. When an ecologist wants to know how many organisms there are in a particular habitat, it would not be feasible to count them all. Instead, he or she would be forced to count a smaller representative part of the population, called a sample. Sampling of plants or animals that do not move much (such as snails), can be done using a sampling square called a quadrat. A suitable size of a quadrat depends on the size of the organisms being sampled. For example, to count plants growing on a school field, one could use a quadrat with sides 0.5 or 1 metre in length. It is important that sampling in an area is carried out at random, to avoid bias. For example, if one were sampling from a school field, but for convenience only placed quadrats next to a path, this might not give a sample that was representative of the whole field. It would be an unrepresentative, or biased, sample. One way one can sample randomly is to place the quadrats at coordinates on a numbered grid. 27 A simple experiment to investigate carbon dioxide production by yeast in different conditions This allows students to see that carbon dioxide is released during anaerobic respiration (fermentation). Students add yeast to glucose solution in a side-arm test tube. Anaerobic conditions are achieved by putting a drop of oil (cooking oil will do) onto the yeast and glucose mixture. A rubber tube is attached to the side arm of the test tube and a glass pipette is inserted at the other end of the rubber tube. The pipette is placed under water to allow the bubbles of carbon dioxide gas to be counted. Temperature is the easiest condition to investigate. Glucose concentration and pH could also be investigated. tap open 3-way tap (open) capillary tube water bath tap closed thin layer of oil lime water yeast and glucose A dead yeast and glucose B The lime water associated with tube A should go milky. Lime water connected to tube B should remain clear. Tube A will show signs of fermentation, bubbles of gas and frothing. Tube B should show little or no change. Lime water going milky is evidence of the presence of carbon dioxide. Respiration involves the breaking down of carbohydrates to carbon dioxide. The production of carbon dioxide thus supports the contention that respiration is taking place. If a simple chemical reaction between the yeast and glucose was producing carbon dioxide it should be produced in tube B as well as in tube A, assuming that the chemicals in yeast had not been decomposed by temperatures as low as 100 °C. Air should be bubbled through lime water, using a pump or filter pump, at the same rate (counting the bubbles) as in the experiment to discount the idea that CO₂ in the air is causing the change. If after the same time interval as in the experiment the lime water remains clear, the argument is refuted. The experiment was designed to show that it was due to anaerobic respiration by boiling the water to expel the dissolved gases, including oxygen. The layer of oil prevented air re-dissolving in the solution at the start of the experiment. The glucose is the substrate for respiration. The yeast obtains energy from the glucose by breaking it down to alcohol and carbon dioxide. 28