School Science Lessons
Plant physiology, respiration, transpiration
2014-08-19
Please send comments to: J.Elfick@uq.edu.au

Table of contents
9.10.0 Respiration in organisms
9.18.0 Transpiration, conduction of water, stomates, potometer, root pressure

9.10.0 Respiration in organisms
6.6.18 Alcoholic fermentation, yeast, Saccharomyces cerevisiae
6.6.1.1 ATP, adenosine triphosphate
6.6.19 Butyric acid fermentation
4.38 Calorific value of fuel
3.37 Carbon dioxide and respiration
6.6.20 Glucose fermentation
22.5.7 Heat of combustion, bomb calorimeter, energy values of food
6.6.17.2 Energy from breakfast cereal
6.6.17.1 Energy from peanuts
9.160 Food used in plant respiration
9.156 Heat energy from respiration of peas
9.158 Heat of respiration, bakers' yeast, Saccharomyces cerevisiae
9.157 Production of carbon dioxide during plant respiration
3.37 Respiration, carbon dioxide and respiration
5.05 Respiration, carbon dioxide gas is produced during respiration
9.4.0 Respiration, aerobic respiration
8.6.5 Respiration is a form of combustion
6.6.1 Respiration, limewater tests for carbon dioxide
6.6.2 Respiration of flower heads over mercury
6.6.11 Respiration of small animals and temperature
6.6.5 Respiration of soaked peas over mercury
9.5.8 Respiration of soaked peas, grasshopper
6.6.9 Respiratory quotient of Compositae flowers, mung bean seedlings
6.6.13 Respiratory quotient using an alternative design respirometer
9.159 Rotting banana and rotting grass
9.154
Study respiration with a respiration apparatus
6.6.7 Tests for oxygen absorption during plant respiration
6.6.12 Tests for gases collected in a respirometer
9.155 Tests for respiration of soaked peas with limewater, respiration apparatus, mouse

9.18.0 Transpiration, conduction of water, stomates, potometer, root pressure
9.185 Conduction of water and salts through the stems
9.183 Conduction of water in plants, cut flowers in coloured water
9.188 Control of evaporation by potato skin, apple peel
9.196
Guttation
6.2.12 Path of the transpiration stream, cut flower stems in ink
5.06 Transpiration, plastic bag over leaves (Primary)
9.190 Transpiration and water transport in plants
9.191 Transpiration and weight of plants
9.189 Transpiration and temperature
6.2.13 Transpiration by leaves exerts suction
9.195 Transpiration causes a drop in water level
9.194.1 Transpiration into a plastic bag, plastic bag over leaves
9.192 Transpiration rates using a potometer
9.186 Transpiration sites
9.193 Transpiration through stomates
9.184 Water transport in plants, root pressure

5.05 Respiration, carbon dioxide gas is produced during respiration
See diagram 3.34.1: Limewater test for carbon dioxide
Demonstrating respiration in plants is not easy. However, you can say that plants breathe out the same gases as humans and then do the limewater test for carbon dioxide. This demonstration always interests students because it lets them see the effect of the carbon dioxide they breathe out. You need fresh limewater for this demonstration so do your preparation the day before the lesson.
Experiment
Heat calcium carbonate, (coral, shells), strongly to change it into quicklime, (calcium oxide). Add this to water, shake and stand for a few days until the top of the liquid is clear. To show the presence of carbon dioxide gas, breathe down a tube or a drinking straw to make bubbles in the clear liquid. The carbon dioxide gas will turn the clear liquid a white milky colour.
Plants (and animals) breathe in oxygen gas from the air to breakdown food into carbon dioxide gas and water.
The energy stored in the food is then let out and can be used for growth, movement and to keep the plant (or animals) alive.
This process is called respiration.
Respiration equation: oxygen gas + simple sugar (food) --> carbon dioxide gas + water + energy
The carbon dioxide gas produced by respiration goes out into the air.
Photosynthesis and respiration are reverse processes.
Photosynthesis equation: carbon dioxide gas + water + energy --> food + oxygen gas
During photosynthesis, energy from the sun is taken in and stored in food. During respiration, that energy taken in is let out and used.
5.06 Transpiration
See diagram 9.194.1: Plastic bag over leaves
Two hours before the lesson, place a dry clear plastic bag over a small branch of a plant growing in the sun. Tie the mouth of the bag tightly around the stem with string. Also find a similar branch, pull all the leaves off and tic another dry clear plastic bag over the bare branch. Be prepared to show what happens when a newly picked leaf is placed in water that is nearly boiling. Bubbles come out from the leaf.
Show the students the leafy branch and the bare branch, each covered with a plastic bag.
Look carefully inside the bags. What do you see? [Water]
What is the difference between the two branches? [One has leaves and the other has no leaves.]
What is the difference between the two plastic bags? [The bag over the leafy branch has water on the inside. The bag over the bare branch has no water in it. Both bags were dry when put on the branches.]
Where did the water come from? [The leaves.]
The loss of water by leaves is called transpiration.
Leaves give out water when they are growing in the sunlight. The sunlight heats the water and turns it into water vapour. This is called evaporation. When a liquid is heated, it forms a gas. This gas is called a vapour. The water vapour comes out through holes in the lower side of the leaf.
The loss of water by evaporation cools the leaf. (Special note: This is a difficult idea. The sun heats the water in the leaf but the water evaporating cools the leaf.)

6.2.12 Path of the transpiration stream, cut flower stems in ink
Herbaceous plants lose several hundred times their own weight of water in a day, mostly through stomata on the leaves. Within the mesophyll of the leaf a large wet surface is exposed to enable the adsorption of carbon dioxide for photosynthesis. When the air outside the leaf is drier than the air within, water vapour can diffuse out through the stomates causing more water to evaporate from the leaf cells. Then the suction pressure of the leaf cells rises, draws water from the veins and creating negative hydrostatic pressure in them to draw water up the xylem vessels. The xylem vessels are merely pipes and the water flows passively so water can be made to flow in the reverse direction by sealing the normal base end, cutting off the apex of the shoot and dipping the cut stem into water.
1. To show that the leaves are mainly responsible for causing the flow of water, you can remove leaves and compare the rates of water movement. Use two leafy shoots of balsam to study of water conduction in stems. Cut a fresh surface on the lower end of each shoot with a sharp razor blade, keeping the end wet under water as you cut. Leave in water for at least two minutes. Place one shoots in a tube of dye provided and watch continuously until you see colour rise in the vascular bundles in the stem and has reached the leaves. Remove the other shoot from the water, dry the cut end and seal with petroleum jelly. Cut the stem under water near the terminal rosette and leave inverted in water for two minutes. Transfer the shoot to the dye provided and watch continuously until you see colour flow. Note that the direction of flow is the reverse of that previously noted.
2. Stand the cut stems of white flowers and some cut shoots in dilute red ink or eosin dye. Note the stain reaching the petals and leaves. Cut across the stems to see the die in the xylem of the vascular bundles in the veins.

6.2.13 Transpiration by leaves exerts suction
Do NOT use elemental mercury for school experiments!
See diagram 9.192.1: Suction pressure
Make all joints air tight and do not include any air bubbles. The mercury will rise in the narrow glass tube.

6.5.1 Carbon dioxide in the air is necessary for photosynthesis
See diagram 9.151: Split rubber stopper
1. Insert a split rubber stopper firmly. Smear petroleum jelly over the surface of the stopper to prevent any air entering the vessel either between the stopper and the neck or between the stopper and the plant stem. Place the apparatus in sunlight. Leave for two days, then do the iodine tests for starch on a leaf from that part of the twig inside the vessel and also a leaf outside the vessel. The potassium hydroxide has absorbed the carbon dioxide in the enclosed air.

See diagram 9.146: Nasturtium
2. Put a mature nasturtium plant in a plant pot in a dark place in the evening. On the following morning, fill a glass container one third full with 20% potassium hydroxide solution. Place the nasturtium plant beside the glass container. Bend a leaf stem without breaking it and insert one leaf into the upper air filled section of the glass container. Cover the jar with a slotted cover to let the leaf stem to pass through the slot. Seal the slot around the stem with adhesive tape. Completely seal the glass container with adhesive tape Potassium hydroxide absorbs carbon dioxide so the leaf sealed inside the glass container is in an atmosphere without carbon dioxide. Put the plant and culture jar in direct sunlight so that the leaf inside the glass container is exposed to the light source. After three hours, detach the leaf inside the glass container from the plant. Also detach some other leaves, which have had equal exposure to light. Kill the cells in the leaves by dropping them into boiling water. After one minute, remove them with tweezers and place them in 96% ethanol. After 2 hours the leaves become almost colourless. Rinse the leaves in water and pour potassium iodide solution over them. Note which leaf is coloured blue violet by the iodine potassium iodide solution.

6.5.4 Chloroplasts, Spirogyra, Zygnema, Closterium
See diagram 9.39.1: Filamentous algae, Spirogyra, Zygnema, Closterium
The green leaf pigment chlorophyll is not usually present in plant cells in solution, but in chloroplasts. To study the shape of the chloroplasts in various plant species, use forceps to detach a few leaves from a stem of moss that has large leaves. Put them in a drop of water on a microscope slide. Mount a coverslip. Examine the slide under low power. Use forceps to place a few cell filaments of Spirogyra in a drop of water on a slide. Mount a coverslip. Examine the slide under high power. Study cell filaments of a stellate algae, Zygnema. Use a pipette to put cells of a desmid, Closterium, on a microscope slide. Mount a coverslip. Examine the preparation under high power. Note the shapes of the chloroplasts and their position inside the cell. Study other species of plants including seed bearing plants, ferns, mosses, and algae to determine the shape of their chloroplasts.

6.6.1 Respiration, limewater tests for carbon dioxide
See diagram 3.34.1: Limewater tests for carbon dioxide
1. Calcium hydroxide is only slightly soluble in water. Prepare the weak alkali calcium hydroxide solution, limewater, by adding solid calcium hydroxide, slaked lime, to demineralized water. Shake vigorously and leave to stand. Calcium hydroxide solid is only slightly soluble in water. When the white solid has settled as a fine white sediment, decant the clear limewater above the sediment. To replenish the limewater, add more demineralized to the sediment in the stock bottle, shake and leave to settle. The settling process may take several days.
2. Pass carbon dioxide through the clear limewater. The solution becomes a milky because of a fine precipitate of calcium carbonate.
Ca(OH)2 (aq) + CO2 (g) --> Ca(CO3)2 (s) + 2HCl (l)
Pass more carbon dioxide through the limewater. The solution becomes clear again because of the formation of soluble calcium hydrogen carbonate.
CaCO3 (s) + CO2 (g) + H2O (l) --> Ca(HCO3)2 (aq)
Pass air through freshly made limewater. After a long time you may see a faint cloudy precipitate.
3. The air contains about 0.4% carbon dioxide. Use a drinking straw to exhale into the limewater. A cloudy precipitate soon forms because you exhaled breath contains about 4% carbon dioxide.
4. Carbon dioxide gas is produced during respiration. Demonstrating respiration in plants is not easy. However, you can say that plants breathe out the same gases as humans and then do the limewater tests for carbon dioxide. limewater tests for carbon dioxide. This demonstration always interests students because it lets them see the effect of the carbon dioxide they breathe out. You need fresh limewater for this demonstration so do your preparation the day before the lesson. Heat calcium carbonate (coral) strongly to change it into quicklime (calcium oxide). Add this to water, shake and stand for a few days until the top of the liquid is clear. To show carbon dioxide gas, breathe down a tube or a straw to make bubbles in the clear liquid. The carbon dioxide gas will turn the clear liquid a white milky colour. Plants (and animals) breathe in oxygen gas from the air to breakdown food into carbon dioxide gas and water. The energy stored in the food is then let out and can be used for growth, movement and to keep the plant (or animals) alive. This process is called respiration. Respiration equation: oxygen gas + simple sugar (food) --> carbon dioxide gas + water + energy. The carbon dioxide gas produced by respiration goes out into the air. Photosynthesis and respiration are reverse processes. Photosynthesis equation: carbon dioxide gas + water + energy --> food + oxygen gas. During photosynthesis energy from the sun is taken in and stored in food. During respiration that energy taken in is let out and used.

6.6.1.1 ATP, adenosine triphosphate
See diagram 6.6.1.1: ATP, adenosine triphosphate
ATP, adenosine triphosphate, is the molecule that allows extraction of energy from food by cycling between ADP, adenosine diphosphate.
1. The RNA nucleotide adenosine monophosphate contains the purine base adenine has one phosphate group, PO4. Adenosine triphosphate, ATP, has three phosphate groups. The terminal third phosphate of ATP can be transferred to other molecules to make them more reactive, the stable glucose molecule can receive a phosphate from ATP, phosphorylation, become glucose-phosphate and be quickly broken down. ATP is made in mitochondria from the oxidation of glucose, cellular respiration when glucose combines with oxygen, oxidation, to form carbon dioxide, water and 38 molecules of ATP. During oxidation, electrons from glucose pass step by step through the cytochrome enzyme system, that contains iron, in the mitochondria.
2. DNA (deoxyribonucleic acid), RNA (ribonucleic acid): 1. messenger RNA, M-RNA 2. transfer RNA, T-RNA 3. ribosomal RNA.
During protein synthesis M-RNA molecules are made from sections of DNA in the nucleus, by transcription, then travel to ribosomes. T-RNA molecules attach to amino acids in the cytoplasm and bring them to ribosomes where they join with base triplets, codons, along the M-RNA strand, translation. A complementary base triplet on each T-RNA, anticodon, joins with the codon of M-RNA, the anticodon AUG joins with the codon UA3. Different T-RNA molecules carry different amino acids, depending on their anticodons. With 64 codons (4 × 4 × 4) and 20 different amino acids in human protein, the same codons of M-RNA can stand for the same amino acid, codons UUA, UUG, CUU, CUC, CUA and CUG stand for the amino acid leucine. As each amino acid is linked to the growing polypeptide, a molecule of water is released until an enzyme, antibody or structural protein forms in animals or plants.

6.6.2 Respiration of flower heads over mercury
See diagram 9.156.2: Flowers in a flask
Do NOT use elemental mercury for school experiments!
Collect living flowers and push them down into a flask. Invert the flask over mercury, so that the mouth of the flask is below the surface. Use a bent tube to introduce strong caustic potash solution to the surface of the mercury inside the neck of the flask. After a few hours, the mercury will rise inside the flask because of the respiratory activity of the flowers. During the process, oxygen is absorbed from the enclosed atmosphere of the flask, and an almost equal volume of carbon dioxide is given off. The reduced volume of gases in the flask is owing to the absorption of carbon dioxide by the caustic potash.

6.6.5 Respiration of soaked peas over mercury
See diagram 9.156.4: Soaked peas over mercury
Do NOT use elemental mercury for school experiments!
1. Fill a test-tube with mercury and, sealing the end with the finger, invert it over a dish of mercury, with the mouth of the tube below the surface. Insert soaked peas, one at a time, into the mouth of the tube. These peas will rise to the top of the tube. Support the tube in this position with a clamp. After 24 hours, the peas will have given off a gas that has forced the mercury some distance down the tube. Show this gas to be carbon dioxide by allowing a strong caustic potash solution to rise into the test-tube from a bent tube inserted at the mouth. The potash, now in contact with the carbon dioxide, absorbs it, and the mercury rises again in the tube.
2. To measure the respiration rate of soaked peas over mercury, insert the short end of an L-shaped tube into a flask containing soaked peas. Insert the long end of the tube into a reservoir of mercury and note the level of mercury in the tube. In time the soaked peas will use all the oxygen in the flask and tube and change to anaerobic respiration with breakdown of starch reserves. Note the level of mercury in the tube at equal time intervals, e.g. every hour.

6.6.7 Tests oxygen absorption during plant respiration
See diagram 9.157
Plants take oxygen in and give carbon dioxide out during respiration. Green plants also take in carbon dioxide and give out oxygen during photosynthesis. Plant respiration can only be noted when there is no photosynthetic activity . So this experiment uses plants or parts of plants that have no chlorophyll necessary for photosynthesis, e.g. mushrooms or flowers of Compositae. Use the burning time of a candle in an enclosed known volume of air to prove the presence of oxygen. Smear the bottom edge of a 5 litre polystyrene bell jar with glycerine to provide a good seal, and is then put on a glass plate. Put a lighted candle in the bell jar using the candle holder. Close the neck of the bell jar immediately with the rubber stopper of the candle holder. Record the burning time of the candle. The candle burns for 90 to 120 seconds enclosed in the bell jar.

6.6.9 Respiratory quotient, Compositae flowers, mung bean seedlings
See diagram 9.156.2: Flower heads
The ratio of the quantity of carbon dioxide given out during respiration to the quantity of oxygen taken in CO2:O2, is called the respiratory quotient. If sugars are consumed in respiration, 6 moles of carbon dioxide and 6 moles of water are produced by the complete combustion of one mole of glucose by 6 moles of oxygen.
C6H12O6 + 6O2 ---> 6CO2 + 6H2O
The carbon dioxide given out and the oxygen taken in are in the ratio 1.1, the respiratory quotient = 1. If during respiration substances low in oxygen are consumed, oil, more oxygen must be used for the complete combustion to carbon dioxide and water. So the respiratory quotient is less than 1. If substances rich in oxygen are consumed, organic acids, the respiratory ratio rises above 1.
1. Respiratory quotient of Compositae flowers
Attach a double burette clamp to a Bunsen burner stand and clamp a respiration vessel to each side. Fill both two thirds filled with small flowers of Compositae with the green leaflets of the calyx removed to prevent photosynthesis. In one respiration vessel is placed a specimen tube containing 6 pellets of potassium hydroxide. Smear the ground glass stoppers with glycerine to provide a good seal and insert in the respiration vessels. Half fill two 100 mL beakers with deionized water and place them under the two respiration vessels. Adjust the latter such that their capillary ends are immersed to a depth of 2 cm in the water. Attach a scale to each capillary. Warm both respiration vessels by placing a hand around them so that a few air bubbles escape from the capillaries. On cooling, the deionized water rises into the region of the scale. After 15 minutes record the level of the water in the two capillaries. Repeat this every quarter of an hour. The meniscus in the capillary of the respiration vessel containing potassium hydroxide rises uniformly. The reduction in volume indicates the uptake of oxygen owing to the respiration of the flowers. Only a small rise in the water level is noted in the other respiration vessel so the amount of CO2 given out must be almost as much as the oxygen taken in. The respiratory ratio of the flowers is thus 1. In general this applies for all higher plants. It clearly indicates that sugar is the predominant substance consumed in the respiration process.

2. Respiratory quotient calculation for mung bean seedlings:
See diagram 9.72.1: Mung bean
Table 6.6.10
Mung bean seedlings 1. with foliage leaves developed 2. with radicle just emerged
A 0.72 cm3 per hour 3.24 cm3 per hour
B 0. 06 cm3 per hour 1.20 cm3 per hour
(A - B) 0.66 cm3 per hour 2. 04 cm3 per hour
RQ = (A - B) / A 0.92 0.63

6.6.11 Respiration of small animals and temperature
Use a U-tube to connect the syringe to the pipette and put the apparatus in a water bath. At each new temperature wait 10 minutes before taking readings. Do not use temperatures that cause discomfort to small animals. See graph 4.9.12.2.1 of respiration rates of ten worms measured at 20oC, 25oC, 30oC and 35o3.

6.6.12 Tests for gases collected in a respirometer
1. Invert the apparatus so that the collected gas is near the open end of the syringe. Push the plunger to introduce a column of gas into the pipette.
2. Seal the end of the gas column with the solution in the syringe to trap a column of 0.8 cm3 gas inside the pipette. Fix the apparatus in a vertical position.
3. Record the volume of gas in the pipette, V1.
4. Expel most of the solution at the lower end of the gas column. Draw in some 2 M sodium hydroxide solution. Keep the tip of the pipette in the sodium hydroxide solution and move the gas column up and down to assist the absorption of carbon dioxide.
5. Record the volume of the gas column every five minutes until the reading, V2, becomes steady. V2 measures the volume of the gas column without carbon dioxide. % carbon dioxide =, V 1 - V2, / V1 × 100
Example result for pond Selaginella:
Initial volume of gas column, v = 0.75 cm3
Volume of gas column after absorbing carbon dioxide, v2, = 0.74 cm2
Percentage of carbon dioxide = V1 - V2 × 100 = 1.33%
6. The percentage of oxygen in the gas collected in the syringe can be estimated using alkaline pyrogallol solution. However, this chemical is too dangerous for use in school science experiments.

6.6.13 Respiratory quotient using an alternative design respirometer
Make sure that the joints are airtight, e.g. between the rubber stopper and the boiling tube. If the rubber stopper loses its elasticity after a long period of storage, ve an airtight condition is difficult. The respirometer is sensitive to volume changes owing to slight fluctuations in air temperature so errors in measurement may arise if you do not set up a control for comparison. When measuring the rate of photosynthesis by collecting the oxygen evolved from a pond weed, Figure I c, errors may occur if the gas bubbles generated from the cut ends of the aquatic stem are released at an erratic rate or in variable size, or when the gas bubbles are trapped on the leaf surface or wall of the apparatus. These errors make counting the number of gas bubbles evolved or measuring the volume of gas collected a less than reliable method for assessing the rate of photosynthesis.

6.6.17.1 Energy from peanuts
Put 20 mL of water in a test-tube or 100 mL of water in an aluminium can. Weigh a peanut (about 1 gram). Push the blunt end of a needle into a cork then stick the sharp end into the peanut or make a peanut holder by winding a 15 cm length of 10 gauge nichrome into a conical spiral by winding over the conical end of a cork borer. Record the temperature of the water in the test-tube. Set alight the peanut with a match or Bunsen burner then immediately hold it under the test-tube or aluminium can. The peanut burns with a sooty flame and may leave some blackened ash in the shape of the peanut. Record the temperature of the water in the test-tube. Weigh the burnt remains of the peanut. Heat in calories = weight of water multiplied by the specific heat of water multiplied by the rise in temperature of the water. One calorie = 4.186 joules, J. Nutrition information often uses the kilocalorie, food Calorie = 1000 calories. Most school experimenters get values of about 12 kJ per gram but nutrition information on food labels usually quote peanuts at 25 kJ per gram and peanut butter at 27 kJ per gram. Half a peanut from a commercial package weighs about 0.5 g and can be burned to heat 100 mL of water by about 15oC. Repeat the experiment with cashew, marshmallow and popcorn.
6.6.17.2 Energy from breakfast cereal
Repeat the previous experiment with breakfast cereal. Compare the result of the experiment with the information on the packet. The packet information is more accurate than this experiment because in a science laboratory, a "bomb calorimeter" is used to burn food samples and calculate the energy stored from the increase in temperature of water around the bomb calorimeter. No heat is lost or unaccounted for during this procedure so may find that the calorific value on the packet may be three times the result of this experiment.

6.6.18 Alcoholic fermentation, yeast, Saccharomyces cerevisiae
See diagram 9.156.1: Yeast with sucrose solution | See diagram 9.204: Yeast cell forming bud | See diagram 11.209.3: Wine and spirits hydrometer
Many organisms can breakdown organic substances without atmospheric oxygen, anaerobic degradation. The process is called fermentation. The amount of energy produced is less than in an aerobic reaction since further substances of varying energy content are formed. Instead of atmospheric oxygen the intermediate products of the decomposition are used here as hydrogen acceptors. Because the decomposition results from the splitting of molecules, the fermentation can also be defined as a dissimulation. Fission respiration, the most familiar example, is alcoholic fermentation.
In the Bible is caution against putting new wine into old wine-skin containers. This action would burst the containers by the gases released by further fermentation: "Neither do men put new wine into old bottles [wine-skins]; else the bottles break, and the wine runneth out, and the bottles perish [are spoilt]" Matthew, ix, 17.
6.6.18.1 Alcoholic fermentation, yeast, Saccharomyces cerevisiae
1. Half fill a test-tube with water, add a piece of yeast the size of a pea, and stir the mixture to produce a uniform suspension of the yeast cells. Three quarters fill two test-tubes with 10%t sucrose solution, and fill a third test-tube with the same amount of water. Add 10 drops of water to one of the test-tubes containing sucrose solution, and add 10 drops of the yeast suspension to the other two test-tubes. The contents of each of the test-tubes are poured into fermentation tubes, taking care that the upright limbs of the tubes are completely filled with liquid and contain no air bubbles. During the next few days a gas collects in the upright limb of the fermentation tube containing sugar and yeast suspension. No gas collects in the two control tubes, containing sucrose and water, or water and yeast suspension.
2. Insert two pellets of potassium hydroxide in the fermentation tube in which gas was produced. The gas soon disappears, indicating the presence of carbon dioxide. Pour the contents of this fermentation tube into a flat glass dish and note the distinct smell of alcohol. Yeasts ferment sugar to produce alcohol and carbon dioxide.
3. Half fill a test-tube with water, add a piece of yeast the size of a pea, and stir the mixture to produce a uniform suspension of the yeast cells. Three quarters fill two test-tubes with 10% sucrose solution, and fill a third test-tube with the same amount of water. Add 10 drops of water to one of the test-tubes containing sucrose solution, and add 10 drops of the yeast suspension to the other two test-tubes. The contents of each of the test-tubes are poured into fermentation tubes, taking care that the upright limbs of the tubes are completely filled with liquid and contain no air bubbles In the course of the next few days a gas collects in the upright limb of the fermentation tube containing sugar and yeast suspension. This does not happen in the control tubes, containing sucrose and water, or water and yeast suspension. If 1 - 2 pellets of potassium hydroxide are inserted in the fermentation tube in which gas was produced, the gas disappears within a few minutes, indicating the presence of carbon dioxide. On pouring out the contents of this fermentation tube into a flat glass dish the smell of alcohol is very distinct. Yeasts ferment sugar to give alcohol and carbon dioxide.

6.6.19 Butyric acid fermentation
See diagram 9.161: Cut potato
The soil contains micro-organisms, among which are spores and vegetative rods of Bacillus amylobacter. This bacillus is able to breakdown the middle lamella of plant cells forming, among other things, butyric acid CH3-CH2-CH2-COOH. From this breakdown it obtains the energy necessary for the maintaining of its metabolic processes. Butyric acid fermentation is of practical importance in flax and hemp production since it loosens the bundles of fibres from the structural matter of the stalks. Bacillus amylobacter is the most important butyric acid producer in the soil. The following experiment shows its activity: Make a cut in a medium size potato, infected by rubbing soil into it. It is well covered with water, contained in a 600 mL beaker, and left to stand at room temperature After 5 days a vigorous fermentation process can be seen to be taking place. Bubbles of gas rise from the cut made in the potato. It smells of butyric acid. The spores and vegetative rods of Bacillus amylobacter present in the garden or arable soil can develop and propagate under the above experimental conditions. The bacillus breaks down the middle lamella of the potato cells, wet rot, thus forming butyric acid. Bacillus amylobacter is anaerobic, dislikes oxygen, so potatoes must be covered with water to keep them away from atmospheric oxygen.
6.6.20 Glucose fermentation
See diagram 6.6.20: Gas produced from fermentation
Wear safety glasses and rubber gloves to complete all tasks
1. Prepare 180 mL of glucose solution at 10% (w/v). Dissolve 18 g of glucose powder in distilled water and make up to 180 mL. Add the glucose solution to a 250 mL conical flask and warm to the ideal temperature for yeast activity using the thermometer, a hot plate, incubator, water bath or bunsen burner.  Weigh 1.5 g  of dried brewer’s yeast onto filter paper. and add the yeast to the glucose solution in the conical flask. Gently stir the yeast and glucose solution. To allow gases to escape, reduce loss of alcohol by evaporation,  and avoid bacteria or fungi contamination, fix cotton balls firmly into the neck of the flask and put 5 cm square of aluminium foil to sit on top of the flask.
2. Weigh the flask, solution, cotton wool balls and aluminium foil square and record the initial mass, M1. Maintain the solution at 20-25oC, if not specified differently on the yeast packet. The time required for fermentation may be 24 hours at ideal temperature and gently stirred, or aerated,  or up to 5-6 days, if just left on a window sill. Fermentation is completed when no more bubbles are released. Reweigh the flask, fermented solution, cotton wool balls and aluminium foil square, M2. Calculate any changes in mass, M1- M2.
3.  Secure the rubber stopper into the top of the flask with the tubing fitted snugly through the stopper. Maintain the solution at the ideal temperature for fermentation. When bubbles form to show that  fermentation is taking place, measure 25 mL limewater into a test-tube and immerse the end of the tubing into the limewater. Observe and record any changes in the appearance of the limewater.
4. See diagram 6.6.20
Insert the stopper in the top of the flask with the tubing passing through the stopper. Fill the beaker and the measuring cylinder with water and arrange the equipment to allow measurement of the volume of gas produced from the fermentation. Maintain the solution at the ideal temperature for fermentation. When fermentation is completed , observe and record the amount of gas produced in the upturned measuring cylinder.
5. Teacher demonstration, wear safety glasses, rubber gloves and a lab coat. Concentrated sulphuric acid is dangerous.
Use a pipette to collect 5 mL of the fermented solution in a test-tube, secured safely in a test tube rack. Use a control using 5mL of distilled water to allow comparison of the results. Use a clean pipette to add 2 drops of concentrated sulphuric acid to the fermented solution in the test-tube. Put a small amount of potassium dichromate on the wooden end of a matchstick and put it in the solution in the test-tube. If the colour change is not immediately obvious, gently heat the solution by sitting it in a beaker of hot water for a few minutes. Observe and note any colour changes.

9.154 Study respiration with a respiration apparatus
See diagram 9.3.42: Respiration of a mouse | See diagram 9.159: Respiration experiments
The respiratory activity of organisms can be shown with an apparatus that moves air over leaves, insects or a small animal and bubbles it through a weak solution of limewater, Ca(OH)2. The system must be isolated from atmospheric carbon dioxide. Set up the apparatus as shown in the diagram but leave the third bottle empty. Run the apparatus by siphoning the large container, a carboy, until it is empty. Note the results. Replace the solutions in all bottles, and this time put loosely packed leaves or an animal into the third bottle. Compare the results of the first run, the control, with the second run. limewater turns from clear to cloudy in the presence of carbon dioxide. This can be shown by blowing through a drinking straw into a container of clear limewater. Plant leaves produce oxygen and carbon dioxide in the light and produce carbon dioxide in the dark.
Do of the experiments suggested in 6.6.1 in the light and in a dark room, and compare the results.

9.155 Tests for respiration of soaked peas with limewater, respiration apparatus
See diagram 9.155: Respiration of soaked peas | See diagram 9.160: Mouse in respiration apparatus
1. The respiratory activity of organisms can be shown with an apparatus that moves air over leaves, insects or a small animal and bubbles it through a weak solution of limewater, Ca(OH)2. The system must be isolated from atmospheric carbon dioxide. Set up the apparatus as shown in the diagram but leave the fourth container empty. Note the results. Replace the solutions in all bottles. Put peas in the fourth container. Draw air slowly through the apparatus with a filter pump. When the air current bubbles through limewater before passing the soaked peas the limewater remains clear. When the air current bubbles through limewater after passing the soaked peas the limewater becomes milky. As limewater turns from clear to cloudy in the presence of carbon dioxide, the peas must have been respiring.
2. Draw air slowly through the apparatus with a filter pump. The air current bubbles through limewater before passing the soaked peas. That limewater remains clear. The air current bubbles through limewater after passing the soaked peas. That limewater becomes milky.

9.156 Heat energy from respiration of peas
See diagram 9.156.3: Heat of respiration
Prepare two thermos flasks fitted with one-hole stoppers for the insertion of thermometers. Put dry pea seeds and water to the first thermos flask. Boil the same number of seeds and put them and water in the second thermos flask. Adjust the thermometers in both thermos flasks so that the bulb of each thermometer touches the peas. Note the rising temperature in the first thermos flask. Actively respiring plants generate heat. Note the steady temperature in the second thermos flask until the activity of micro-organisms causes a sharp rise in temperature. Examine the contents of the two thermos flasks.

9.157 Production of carbon dioxide during plant respiration
See diagram 9.157: Production of carbon dioxide during plant respiration
1. Plant respiration can only be observed where no photosynthetic activity occurs. So use fungi or parts of plants that have no chlorophyll necessary for photosynthesis, e.g. mushrooms or the white flowers of the Compositae family, e.g. daisy. Remove the green leaflets of the calyx to prevent photosynthesis. Use the burning time of a candle in an enclosed known volume of air to prove the presence of oxygen. Smear the bottom edge of a big jar with petroleum jelly then put it on a glass plate. Open the neck of the jar then put a lighted candle down the neck on to the glass plate. Be careful! Melting wax from a burning candle can cause severe skin burns so use safety glasses and insulated heat-proof gloves. Close the neck of the jar immediately. Record the burning time of the candle. Put mushrooms or white flowers in the jar. Close the neck of the jar. Two hours later, put the lighted candle into the jar. Record the burning time of the candle. The candle burns a shorter time because plants extract oxygen from the air during respiration.
2. Repeat the experiment by pumping air from the jar through limewater. Continue pumping until the limewater becomes milky to show the presence of carbon dioxide.

9.158 Heat of respiration, bakers' yeast, Saccharomyces cerevisiae
See diagram 9.156.1: Heat of respiration of yeast
Heat 450 mL 10% sucrose solution to 35oC then add 25 g of baker's yeast, Saccharomyces cerevisiae. Stir the suspension then pour it into a thermos flask. Fit a two-hole stopper with a thermometer inserted through one hole. As a control, heat 450 mL 10% sucrose solution to 35oC then pour the solution into an identical thermos flask. Fit a two-holes stopper with a thermometer inserted through one hole. Record the temperatures every 15 minutes. The temperature in the thermos flask containing the sugar solution and yeast rises but the temperature in the control decreases. Energy is liberated during respiration. Part of it is given off to the outside as heat.

9.159 Rotting banana and rotting grass
1. Squeeze very ripe fruit into a watery mash, banana. Put the mash in a small bottle then fill the bottle with water. Attach a balloon to the mouth of the bottle. Put the bottle and balloon in a warm place. Measure the size of the balloon at the same time each day. The balloon inflates because of the carbon dioxide gas produced by the action of bacteria on the sugars in the rotting fruit.
2. Sterilize rotting grass by boiling in water. Prepare sterile agar containing beef broth in test-tubes sealed with cotton wool. Remove the cotton wool and pour into 2 sterilized dishes, Dish 1 and Dish 2. After pouring the agar immediately replace the dish lids. When the agar is set, use sterilized forceps to rub the sterilized rotten grass over the agar in Dish 1, the control. Replace the lid, and seal with adhesive tape. Rub rotting grass over the agar in Dish 2 and seal with adhesive tape. Leave the dishes upside down and undisturbed in a warm dark place for 4 days. Examine the dishes each day for bacteria and fungi growing in small colonies like dots. The bacterial colonies are usually shiny and smooth. The fungal colonies are usually fuzzy or furry. Note whether bacterial or fungal colonies appeared first and whether they appeared in Dish 1 or Dish 2.

9.160 Food used in plant respiration
See diagram 9.113.2d: Bean seed germination
Put absorbent paper in two aluminium foil trays and add 50 g of wheat grains to each tray. To one tray add water shaken with thymol or chloroform prevent mould growth. Put the trays under jars raised to admit air. Put both trays in the dark. When the yellow seedlings are more than 5 cm long, dry both dishes in an oven until the weight is constant. Record the results as the weight of 1. dry seeds 2. dry seeds after heating 3. germinated seeds after heating.

9.183 Conduction of water in plants, cut flowers in coloured water
Water is distributed in a plant through the xylem vessels in the vascular bundles. The water absorbed by the roots of plants goes to all cells and to replace water lost through transpiration. Water travels up cut stems by capillary action.
1. Cut the stem of a white flower, e.g. carnation or lily, under water. Keep the cut end wet then put it into a red dye, e.g. red ink diluted with water. After some hours the petal will change colour.
2. Examine the arrangement of vascular bundles in the stalk. Use a one-sided razor blade to cut thin wedge-shaped slices, e.g. maize, buttercup, tulip, iris. Cut off a small piece at the end of the stalk with a cut perpendicular to the axis and cut away from you or cut plant material down on a bench top and wear protective gloves. Drop the sections into a beaker of water. Examine the sections under low power. Identify the vascular bundles and note how their distribution is different in monocotyledons and dicotyledons.
3. Put ink or food colouring or 0.5% of the magenta dye acid fuchsine in test-tubes. Put a small flowering sprig in the test-tubes, e.g. camomile. The white florets of camomile become pale red within 10 minutes. The solution of dye rises with the transpiration stream into the flowers.
4. Use a white carnation flower with a long stem. Cut the stem in half along its length and put the half stems in test-tubes containing different water colours to create a flower with two colours.
5. Repeat the above experiments with a celery stem. Cut a length of a stem under water and add red ink to the water. Watch the red ink moving up the celery stem. Calculate the speed of movement of the ink up the stem.

9.184 Water transport in plants, root pressure
1. Use two white flowers with stalks cut with a slanting cut. Fill a test-tube two-thirds full with 1% of the magenta dye fuchsine acid solution and put in the two white flowers. Note any changes in the colour of the petals.
2. Fill a test-tube two-thirds full with 1% fuchsine acid solution. Cut off a side shoot from a pot plant and put it in the coloured solution. After 15 minutes cut through the side shoot and note any change in colour.
9. Make a horizontal cut 5 cm above the soil level of a pot plant. Fix a glass tube to the cut end. Note any water moving up the glass tube. The process of osmosis takes up water from the soil through the roots, even if the foliage of a plant has been removed. Root pressure raises the water level in the glass tube.

9.185 Conduction of water and salts through the stems
Water is speedily distributed over a plant along special pathways, vascular bundles.
1. To examine the arrangement of vascular bundles in the stalk, use a razor blade to cut thin wedge-shaped slices, e.g. maize, buttercup, tulip, iris. Cut off a small piece at the end of the stalk with a cut perpendicular to the axis. If right-handed, take the stalk in the left hand and hold it with the thumb and first two fingers. Hold a razor blade between the thumb and index finger of the right hand. Pull the blade along its whole length from left to right and towards the body. Drop the sections into a beaker of water. Examine the sections under low power. Identify the vascular bundles and note how their distribution is different in monocotyledons and dicotyledons.
2. Put a young plant, e.g. balsam, in a beaker with its roots in water containing red ink or eosin or 5% of the magenta dye acid fuchsine. After two hours, cut thin sections of the stem above the level of the solution and examine them under low power. Note the xylem vessels are coloured but the phloem is not coloured. The xylem vessels, not the phloem, conduct water and other dissolved salts up the stem.

9.186 Transpiration sites
See diagram 9.186: Sites of transpiration
1. Smear the rim of a large jar with petroleum jelly. Put a potted plant and a dish containing a known weight of calcium chloride on a piece of glass. Invert the large jar and place it over the potted plant and dish of calcium chloride. Leave to stand for six hours then again weigh the calcium chloride.
2. Cover the leaves of a potted plant with a thin layer of petroleum jelly then put the plant under a large jar. Also, put a dish containing a known weight of calcium chloride in a large jar. Leave to stand for 6 hours then again weigh the calcium chloride. Note an increase in the weight of calcium chloride in 1.1 whereas in 1.2 there is no change in weight of calcium chloride. The calcium chloride becomes heavier because it absorbed water from transpiration. The calcium chloride in 1.2 did not absorb any water and remained the same weight because the leaves were covered by the petroleum jelly that prevented the loss of any water vapour by transpiration.
3. Find the surface of the leaf through which transpiration takes place more rapidly. Fix one piece of dry cobalt (II) chloride paper on the upper surface of one leaf of the potted plant with adhesive tape. Fix another piece of dry cobalt (II) chloride paper on the lower side of another leaf of the same potted plant. Put the pot plant on a piece of glass and cover it with a glass jar. Apply petroleum jelly to where the jar touches the glass to ensure that the apparatus is airtight. Leave the experiment for two hours then note how fast any change in colour takes place in the cobalt (II) chloride papers. The piece of dry cobalt (II) chloride paper attached to the lower surface of the leaf changed from blue to pink much more rapidly than that fixed to the upper surface of a leaf of the same potted plant. So the lower surface of leaves gives off more water than the upper surface.

9.188 Control of evaporation by potato skin, apple peel
1. Plants continually lose water from their surfaces through evaporation, transpiration. The water lost by transpiration must be replaced. Many plants have protective devices to control transpiration. Compare the water lost by an unpeeled and a peeled potato. Use two potatoes of different size. Peel the larger potato and weigh it. After peeling it should still be heavier than the unpeeled potato. Cutting small slices from the peeled potato until the peeled and unpeeled potato have the same weight. Put each potato in an open flat glass dish and leave them at room temperature. Weigh both potatoes at the same time each day and record the results.
2. Apple peel provides protection against loss of water by evaporation. Select two apples of equal size. Peel one apple as thinly as possible and leave the other apple as a control. Put each apple in a beaker and weigh the apple + beaker. Leave the apples to stand for some days. During the next few days, the peeled apple shrinks and becomes increasingly wrinkled. However, the unpeeled apple shows no noticeable changes. Weigh each apple + beaker again. Calculate the percentage loss in weight of the two apples. The peel of an apple is only very slightly permeable to water so water can only evaporate only slowly from the fruit pulp inside it. If the skin is not removed or damaged, fruit can be stored for a long time.

9.189 Transpiration and temperature
See diagram 9.189: Effect of temperature on transpiration
1. Set up a vertical metal heating sheet with its lower edge 20 cm above the surface of the bench. Place a Bunsen burner below the heating sheet. Be careful! Use safety glasses and insulated heat-proof gloves. Do not get too close to the flame. Prepare 4 fresh cuttings, e.g. poplar, with about the same leaf area and the bottom 5 cm of stem cut off diagonally to promote good suction. Do the cutting by cutting down on the stem under water. Put each cutting in a graduated cylinder containing water. Fill the graduated cylinders to the 100 mL mark with water and cover the surface of the water with paraffin oil or hot wax to prevent evaporation. Place one cutting near the heating sheet and the rest farther distances away from the heating sheet. Compare the levels of water in the measuring cylinders after two hours. The losses of water are caused only by the transpiration of the cuttings. The cuttings placed at a higher temperature transpire more.

9.190 Transpiration and water transport in plants
1. To show transpiration under a variety of conditions use a plaster of Paris pot, with a rubber stopper and glass tube, held upside
down. The glass tube sits in coloured water. A fan heater is blown over the pot, water evaporates, drawing up water from the beaker, as shown by a bubble moving up.
(in diagram below, although it appears to be open at the 'top', this is the base of the pot, upside down)
2. Make dry cobalt (II) chloride paper by putting 1 cm squares of absorbent paper or white newspaper into a 5% solution of cobalt (II) chloride then dry in an oven. The dry cobalt (II) chloride paper is blue when dry but turns pink when exposed to a humid atmosphere or dipped in water. Store the dry cobalt (II) chloride paper in a sealed test-tube or in a desiccator or over anhydrous calcium chloride. Put a piece of dry cobalt (II) chloride paper in contact with the upper and lower surface of a leaf and quickly apply a coverslip. Note the time required for the blue colour to fade for each piece of dry cobalt (II) chloride paper.
3. Do a similar experiment with dried copper (II) sulfate paper. This liquid turns white anhydrous copper (II) sulfate to blue. This liquid turns blue paper soaked in cobalt (II) chloride solution pink. So the liquid is water. This water could not have come from any other source except the plant.
4. Plants continuously evaporate water and absorb water. Fill a 100 mL measuring cylinder with tap water to 2 cm below the 100 mL mark. Insert rooted shoots of Tradescantia with well-developed leaves. Pour paraffin oil on the water so that no water can evaporate from the surface. The leaves of the Tradescantia shoots, inside the measuring cylinder above the layer of paraffin, and outside the measuring cylinder must all be completely dry. Wipe any drops of water off them with absorbent paper. Record the level of the water surface in the measuring cylinder, e.g. 98.5 mL. Put the measuring cylinder on a sheet of glass and place a large jar over the measuring cylinder. Where the rim of the large jar touches the glass make a seal with petroleum jelly so no air or water can enter or leave the large jar. By the following day, drops of water have appeared on the inside surface of the large jar and the level of the water in the measuring cylinder has fallen. The Tradescantia shoots have absorbed water then lost water through their leaves as water vapour that has condensed on the inside surface of the large jar. Absorbing water through the roots must compensate for loss of transpired water from the leaves.
4. Pick a green leaf then immediately put it in cold water. Nothing happens. Put a green leaf in hot water. Bubbles of air come from holes in the lower side of the leaf where the stomates are situated.

9.191 Transpiration and weight of plants
1. Use a well-watered plant in a flowerpot, e.g. Pelargonium or fuchsia. Cover the surface of the soil and the sides and base of the flowerpot with plastic sheet. Cover the soil surface in the flowerpot with a plastic sheet with a slit for the stem. Seal around the stem with rubber solution. Fix the ends of the plastic sheet to the flowerpot so no moisture can be lost to the air. Weight the whole apparatus then stand in sunlight for several hours. Weigh the whole apparatus again and note any loss in weight because of transpiration.

9.192 Transpiration rates using a potometer
See diagram 9.3.43: Potometer to measure transpiration
1. A potometer measures the rate of water intake rather than transpiration. However, assume that water intake equals water loss. Cut the end of the shoot of the potted plant under water. Pass a cork borer of slightly bigger diameter than the shoot through the hole in a 3-hole cork stopper from below. Still working under water, slide the shoot into the cork borer from above, and then remove the cork borer by pulling downward. Flood the apparatus to eliminate any air bubbles. The end of the capillary tube must not stick out below the cork stopper because air may become trapped there. When all the air is eliminated from the apparatus, close the reservoir tap. Leave the apparatus for four hours.
2. Introduce a small air bubble in the end of the bent capillary glass tube. Note the time taken by the bubble to move towards the cut shoot. This movement is a measure of the rate of transpiration. Measure the rate of transpiration under different conditions, e.g. wind velocity and temperature.
3. Put a leaf on graph paper and draw a line around the leaf margin to measure the leaf area then calculate the rate of transpiration per cm2 of leaf.

9.193 Transpiration through stomates
See diagram 9.193: Transpiration through stomates
1. Half fill with water four identical containers, A, B, C, D. Prepare two similar leafy shoots A1 and B1, each with two similar leaves. Smear petroleum jelly over both sides of the leaves of B1. A1 is the control. Cut the ends of both stems under water. Pick up each stem carefully so that a drop of water sticks to the cut end. Put each stem in its container. Adjust the level of water in the four containers to the same height. Add 0.5 cm of cooking oil to containers A, B and C. Use a grease pencil to mark the original water level on each container. Leave the stems in the sunlight for hours then the next day record the water levels in each container. Repeat the experiment using an electric fan. Container, B is the experiment and containers A, C and, D are controls.
2. Choose four similar leaves of a plant with thin leaves and stomates only on the lower sides of the leaves
2.1 Smear both surfaces of leaf 1 with petroleum jelly.
2.2 Smear only the upper surface of leaf 2 with petroleum jelly.
2.3 Smear only the lower surface of leaf 3 with petroleum jelly.
2.4 Let leaf 4 hang freely in the air as a control without smearing it with petroleum jelly.
The next day, examine the leaves. Leaf 1 and leaf 3 show signs of wilting. Leaf 2 and leaf 4 show no signs of wilting.

9.194.1 Transpiration into a plastic bag, plastic bag over leaves
See diagram 9.194.1: Transpiration in plastic bag
1. Tie a dry plastic bag over the leaves of a small tree. Wind wire or string around the mouth of the bag to make it air tight. Examine the bag after a few hours. Note the drops of water inside the plastic bag. Tie the plastic bag around the stem with the leaves pulled off. No water forms in the bag. The water comes from the leaves.
2. Tie a piece of polythene around a pot containing a young growing plant. Put the pot under a large jar or plastic container on a sheet of glass. Set up a similar large empty large jar as a control. Apply petroleum jelly to where the large jar touches the glass to ensure that the apparatus is airtight. Droplets of liquid appear on the walls of the large jar after two hours.

9.195 Transpiration causes a drop in water level
See diagram 9.195: Water level drops
1. Remove a nasturtium plant from a pot or the soil. Wash the plant and put it into a jar of water. Pour oil on the surface of the water. Note the level of the oil in the jar. Hours later, note the level of the oil again. Water has been lost from the jar by transpiration through the plant.
2. Put water in a jar. Mark the water level. Fix plastic cling film over the top of the jar. Cut a slit in the cling film just long enough to insert through it the stem of a plant. Use some glue to seal the slit. The water level will drop as the plant stem absorbs water.

9.196 Guttation
See 14: Diseases of taro plants
If you observe a heavy dew on grass early in the morning some of the moisture may not have come from the air but from guttation by the grass. Guttation occurs on cool clear nights when stomates are closed to reduce water lost by transpiration but water lost occurs by xylem through modified stomates called hydathodes along the edges of leaves or at leaf blade tips. Although observed usually in grasses guttation occurs in hundreds of genera. It may occur in other genera during the day in very humid tropical regions with high soil temperature to assist in loss of water by transpiration and cause negative osmotic pressure in the xylem compare to the osmotic pressure of the soil water.