To estimate the amount of reducing sugars in a grape

Transcription

1 生物實驗室 Experiment 1: To estimate the amount of reducing sugars in a grape Explanation of method: To estimate the amount of reducing sugars in a grape, the method of test for reducing sugars must be known first. To test the presence of reducing sugars in a solution, Benedict's test can be carried out. In Benedict's test, equal volume of Benedict's solution should be added into the unknown solution. The mixture is then boiled. If reducing sugars are present, there should be some brick red precipitates. This method can be done quantitatively. The amount of precipitates can indicate the amount of reducing sugars. The more the precipitates are formed, the more the reducing sugars are present in the solution. So by comparing the amount of precipitates formed in the grape juice with the amount of precipitates formed in a standard sugar solution, one can estimate the amount of reducing sugars in a grape. In this experiment, a standard glucose solution of concentration 0.01M was used. It was diluted into different concentration. The amount of reducing sugars was determined by Benedict's test and the results were plotted in a standard curve. Procedure: (1) Prepare 1ml of glucose solutions of different concentrations from a stock 0.01M glucose solution by adding suitable amounts of distilled water. The dilution table was shown: Molarity of glucose solution (M) Volume of stock glucose solution added (ml) Volume of distilled water added (ml) (2) Peel off the epidermis of the grape, put the grape into a beaker, grind the grape tissue with a glass rod to get as much juice as possible, pour the juice into a measuring cylinder and add distilled water into it up to 25ml.

2 (3) Pipette 1ml of the prepared diluted juice into a test tube G. Pipette 1ml of the sugar solutions into test tube A to F. (4) Place 2ml of Benedict's solution into each of the test tube A-G. Boil all tubes in a water bath for 5 minutes. Allow the precipitates to settle for 10 minutes. Estimate the amount of precipitates in each tube.

3 Experiment 2: To study the action and activity of amylase on starch digestion Principles of design of the experiment: In order to find out the action of amylase on starch, amylase solution can be added to starch solution. After some times, the presence of starch in the mixture can be tested by iodine solution. The positive test for the starch is dark blue colouration. If there is no starch, iodine solution remains brown. If there is less starch or no starch in the mixture, it can be concluded that amylase can change starch into another chemicals. This can show amylase can have some action on starch. To the study the activity of amylase on starch, the amount of starch in the mixture can be estimated by observing the colour change in the iodine solution at a regular time interval. The colour changes from dark blue to brwon indicates the disappearance of starch in the mixture. If the colour changes very rapidly, it shows that the activity of amylase is high. If the change is slow, it shows the acitivity of amylase is slow Procedure: (1) Two drops of iodine solution were put into each depression in a spotting tile. (2) 5 ml of 1% starch solution was added into test tube A with a 5ml pipette. (3) 0.5 ml of 1% amylase solution was added into the same tube with a 1ml pipette. (4) The solution was mixed thoroughly. (5) At a half-minute interval, two drops of mixture were taken out from test tube A and transferred to each depression of the spotting tile. (6) The iodine solution and the mixture was mixed into the depression by a glass rod. (7) The colour of the iodine solution was observed and recorded. (8) Step 5 to 7 were repeated until the colour of the iodine solution remained brown. Explanation of results: Starch molecules can combine with the active sites of the amylase. Amylase can then speed up the digestion of starch into maltose. When amylase was mixed with starch, starch is

4 digested. Since less starch is present in the mixture, if we test the presence of the starch by iodine solution, the colour of the iodine is less dark blue. When there is a complete digestion of starch, the colour of the iodine solution will remain brown. The reaction rate is fast at first since the amount of starch in the mixture is large. However, the reaction rate is slower after 1.5 minutes, since the concentration of substrate decreases. Precautions : 1. The experiment should be carried out at the same temperature. 2. The solutions must be well mixed since the starch and amylase are not completely dissolved. 3. Adding too much iodine solution and mixture into the depressions must be avoided. This may cause the over-flow of the solution from the depressions. Mixing of the solutions in the depressions is not easily done. Sources of errors: 1. The volume of each drop of solutions may not be the same. 2. The amylase or starch solution left on the inside surface of the test tube may cause an inaccuracy in the amount of solutions added. 3. The amylase and starch are not completely soluble. Improvement: 1. The colour change is not easy to observe. If a colorimeter and data-logging device are used. The results can be quantitized and the results can be easily compared. 2. A shorter time-interval can be used. This makes the result more clearly observed. Of course, this may have difficulty when the process is done by one person.

5 Experiment 3: To find out the water potential of potato tuber cells by weighing method Principles of design of the experiment: When the water potential of potato tuber cells is lower than that of outside solution, there is a net movement of water into the cells. The cells increase in weight. When the water potential of the cells is higher than that of outside solution, there is a net movement of water out of the cells. The cells decrease in weight. When the water potential of the cells is the same as the water potential outside, there is no net movement of water in or out of the cells. The weight of the cells will not change. We can measure the change in weight of the cells. If there is no change in weight, the water potential should be the same as that of outside. In the experiment, we can use potato cylinder to do the experiment. However, since there are many cells in the cylinder and they may have different water potential. The water potential found in this experiment should be the average of the water potential of all the cells in the cylinder. In the experiment, we will measure the % change in weight because all the cylinders have no identical weight. We plot a graph showing % change in weight of various cylinder against different concentration of sucrose solution. We can, from the graph, find out the zero % change in weight. Procedure: (1) Peel off the skin of potato tuber. (2) Prepare 10 potato cylinders by means of a cork borer. (3) From a 20% stock solution of sucrose solution, 10 different concentrations of sucrose solution are prepared. (4) Label test tube Each test tube should contain sucrose solution of different concentrations. (5) Weigh each tuber. After 30 minutes, take off the tubers and blot them with a tissue paper. (6) Immediately weigh the tuber again. (7) Calculate the percentage change in weight in each tuber.

6 (8) Plot a graph showing % change in weight of all the cylinders against different sucrose solution. (9) Find the concentration of sucrose solution at which zero % change in weight is found. Precautions : 1. The skin must be peeled off. The skin is impermeable to water. 2. The cylinders must be immersed in the solutions completely so that all cells are being tested. 3. The cylinders must be placed in the solutions for at least 30 minutes because osmosis takes time to occur. 4. The water on the surface of the cylinders must be blotted away by a tissue paper. The amount of water on the surface affects the result. Sources of errors: 1. The evaporation of water in the test tubes may increase the concentration slightly. 2. When the water on the surface of the cylinders is blotted away, some of the water entered the cells may be drawn out by the tissue paper. 3. The size of the cylinder is not so great. The percentage change in weight is not very great. Improvement: 1. Larger piece of cylinder should be used. 2. The mouth of each test tube can be sealed by a plastic film. 3. More cylinders should be placed in each of the sucrose solution. The % change should be calculated as the average of the % change.

7 Experiment 4: To find out the water potential of Zebrina leaf epidermis by 50% plasmolysis Theory: When the water potential of leaf epidermal cells is lower than that of outside solution, there is a net movement of water into the cells. The cells become turgid. When the water potential of the cells is higher than that of outside solution, there is a net movement of water out of the cells. Plasmolysis occurs. Plasmolysis is a phenomenon that the cell membrane detaches from the cell wall. Plasmolysed cells can be observed in the microscope. When the water potential of the cells is the same as the water potential outside, there is no net movement of water in or out of the cells. The cells are in the state of incipient plasmolysis. However, the cell in this state cannot be distinguished from the turgid cells. Since different cells have different water potential, when the whole piece of tissue is submerged in a solution in which 50% cells show plasmolysis, we can conclude that the water potential of the solution is equal to the average water potential of the cells. Principles of design of the experiment: The epidermal peel is observed under microscope to observe the plasmolysed cells. To find out which solution produces a 50% plasmolysis result, sucrose solutions of different concentration were produced. The peels were put inside the solution for some times to allow osmosis to take place. We select several views to obtain a more accurate results. A graph plotting % of plasmolysis against concentration of sucrose solution was made. From the graph, 50% plasmolysed cells are found and the water potential of the sucrose solution was obtained. Procedure: (1) Peel off a lower epidermis of a Zebrina leaf. (2) Put a small piece of epidermal peel onto a slide. (3) Observe the peel under a microscope.

8 (4) Randomly select a view to observe. Count the total number of cells observed and number of plasmolysed cells. (5) Calculate the percentage of plasmolysis. (6) Repeat step (4) and (5) for 2 more views. Precautions : 1. Allow time for osmosis to occur. 2. Observe several views to get a more accurate results. Sources of errors: 1. Some cells are broken off when the epidermal peels were made.

9 Experiment 5: To demonstrate the presence of catalase in pig's liver Background information: Catalase is an enzyme found in many animal tissues such as meat, liver, and many plant tissues such as stems of seedlings. Catalase is found in the organelle peroxisomes and microbodies in a cell. In industry, it is used to generate oxygen to convert latex to foam rubber. The presence of catalase is important because it can decompose hydrogen peroxide which is a by-product of certain cell oxidations and is also very toxic. Catalase can eliminate the hydrogen peroxide immediately. It is a fastest-acting enzyme know. Its activity can be demonstrated by dropping a piece of fresh liver into hydrogen peoxide, when rapid evolution of oxygen is observed. Principles of design of the experiment: The evolution of gas should be observed when catalase is placed into hydrogen peroxide. The hydrogen peroxide is decomposed into water and oxygen. As a result, when gas evolves, catalase should be present. The amount of gas is related with the amount of catalase. Procedure: (1) Grind 10g of fresh pig's liver in 10cm3 of distilled water in a mortar with a pestle. (2) Filter the liver extract. (3) Dilute the liver filtrate by 100% with distilled water. (4) Add a drop of the filtrate to 5cm3 of hydrogen peroxide. (5) Observe the evolution of gas. (6) Repeat step (4) with distilled water instead of hydrogen peroxide. Then repeat again with distilled water instead of liver filtrate. These are the controls. (7) If you want to test any other tissues, repeat the procedure with any other tissues.

10 Precautions : 1. Hydrogen peroxide solution is corrosive. It may hurt your skin. Wash your fingers if you can get contact with the solution. 2. The extract must be filtrated so that the residues will not affect the result.

11 Experiment 6: To find out the water potential of potato tuber tissues by density method Background information: When potato tuber tissues are placed into sucrose solution of different concentrations, there may be a difference in water potential between the potato tuber tissues and that of the surrounding sucrose solution. As a result, there may be a net movement of water molecules into or out of the cells. When the amount of water surrounding the potato tuber tissues are not so large. The outward movement of water from the cells or the inward movement of water from the sucrose solution will lead to a change in density of the sucrose solution. In other words, if there is a net movement of water molecules into the cells, the density of sucrose molecules in the solution becomes higher and the density of the solution will be higher. If there is a net movement of water molecules out of the cells, the density of sucrose molecules in the solution becomes lower and the density of the solution will be lower. Principles of design of the experiment: When potato tube tissues are submerged into sucrose solution of different concentrations, and if there is a difference in water potential, there will be a net outward or inward movement of water molecules. The density of the surrounding solutions will be changed. If these solutions are coloured and one drop of these solutions is released into the middle level of the solutions of original concentration, the drop of coloured liquid may move due to the difference of the density. If the density is lowered after osmosis, the drop of coloured liquid will rise. If the density is raised after osmosis, the drop of coloured liquid will sink. If no osmosis takes place, the density remains the same. Therefore, the coloured drop will be stationary. Procedure: (1) Prepare two sets of solution with 5 sucrose solutions of different concentrations: 1%, 2%, 3%, 4% and 5%. Label the 5 test tubes A-E and 1-5. Test tubes A and 1 contain 1% sucrose solution and so forth.

12 (2) Prepare 5 potato tuber cylinders by a cork borer. They must be of the same length. (3) Put one cylinder into each of the test tube A-E. (4) Wait for 15 minutes for osmosis to take place, if any. (5) Drop one drop of methylene blue dye into the test tube A-E. Mix the dye with the sucrose solution. (6) Carefully take out the potato tuber cylinders. (7) Withdraw one drop of the coloured sucrose solution in A-E. Slowly insert the dropper into the test tube 1-5. (8) Slowly release the drop of sucrose solution into the colourless sucrose solutions. (9) Observe the movement of the coloured drop inside the solutions. Precautions : 1. The skin of the potato tuber cylinders should be peeled off because the skin is impermeable to water. 2. The potato tuber cylinders should be the same length as far as possible. This is to make the total number of cells in each of the cylinders nearly the same. 3. The cylinders should be placed in the solutions for 15 minutes or longer to allow osmosis to take place. 4. The methylene blue should be mixed thoroughly in the sucrose solution. 5. The drop of coloured solution must be carefully released into the sucrose solution. No extra force should be applied onto the drops.

13 Experiment 7: To determine the optimum ph for trypsin Background information: Trypsin is a kind of protease. This enzyme is present in small intestine and can break down protein into amino acid. Different enzymes may have different optimum ph. In the optimum ph, the enzymes work best. The activity is the highest. In lower ph or higher ph, the excessive hydrogen ions or hydroxide ions may break the ionic bonds. This changes the shape of the enzyme. The shape of active site also alters. This lowers the catalytic activity. Photographic film has a protein called gelatin which coats on the surface of it. If it is removed, a whitish stain will appear. Principles of design of the experiment: In this experiment, we can put a photographic film strips into a trypsin solution. The protease will digest the gelatin coating and make it whitish. Different buffer solutions will be added to the solutions as well to change the surrounding ph. The degree of digestion of the gelatin reflects the activity of the enzyme. Therefore, the optimum ph can be estimated. Procedure: (1) Set up the water bath and adjust the temperature to 37 degrees Celsius. (2) Pipette 1mL buffer solution of ph 1.0 into a test tube. (3) Add a strip of photographic film into the solution and put it into the water bath for 5 minutes. (4) Pipette 1mL of trypsin solution into another test tube. Put it into the water bath for 5 minutes. (5) Pour the 1mL of trypsin solution into test tube with film. (6) Record the time taken for complete disappearance of gelatin on the film. (7) Repeat the experiment with buffer solution of ph around 2.0, 4.0, 6.0, 8.0, 10.0.

14 Precautions : 1. The temperature must be kept constant since temperature is a factor affecting the enzymatic activity. 2. The gelatin coating is easily scratched and damaged and it should be handled with care. 3. The enzyme, and the film have to be put into the water bath for 5 minutes before mixing. This allows both substrates and enzymes reach the optimum temperature before mixing. 4. The tubes must be mixed from time to time. 5. If the gelatin coat remains for over 1 hour. We can assume the time to be infinity.

15 Experiment 8: To separate chlorophyll extract of tomato leaves into its constituent pigments Background information: Chlorophyll is a mixture of pigments found in the grana of the chloroplasts in plants. There are chlorophyll a, chlorophyll b, carotene and xanthophyll. Their absorption spectra vary. Paper chromatography is a method to separate a mixture of pigments. The pigments can then be identified by their colours and their Rf values in a particular solvent. The colour of chlorophyll b is green, that of chlorophyll a is blue green and that of xanthophyll and carotene is orange yellow and yellow respectively. Principles of design of the experiment: The chlorophyll is generally soluble in non-polar solvent. The leaves are ground and the chlorophyll is extracted by a extracting solvent. The chlorophyll extract is then placed onto a filter paper for chromatography. The paper is placed inside a test tube containing developing solvent. The solvent will go up due to capillary action. The pigments in the chlorophyll are soluble in the developing solvent and they have a tendency to adsorb onto the paper. Since different pigment molecules have different solubilities and degree of adsorption. They will be carried up by the movement of the solvent. If the solubility is not very large and the adsorpiton tendency is great, that pigment will not move up too much. The distance of movment is less. Different pigments move up to a different distance from the starting point. The number of pigments can thus be found. Different pigments can be identified by their colours as well. Procedure: (1) A tomato leaf was ground in a mortar with small amount of extracting solvent (propanone).

16 (2) A pencil line was drawn across the filter paper strip at 1.5 cm from the base. A spot was made at the centre of the line. (3) Using a micro-capillary tube, a drop of chlorophyll extract was transferred to the spot. (4) Step 3 was repeated after the extract was dried, until a concentrated deep green spot of chlorophyll was observed on the spot. (5) A development solvent was poured into a boiling tube. (6) The strip of filter paper was fitted into a split cork and placed into the boiling tube in such a position that the base of the paper just touch the solvent. (7) The rise of solvent and the pigments were observed. (8) Different pigments were identified by their colours. (9) After the solvent front almost reached the top, the paper was taken out. The locations of the colour spots were marked by a pencil. The Rf value of each pigment was calculated. [Rf=(distance moved by the pigment)/(distance moved by the solvent front)] Precautions : 1. The amount of extraction solvent must not be too much, otherwise it takes a lot of time to get a concentrated dark green spot. 2. The paper strip should be long enough to touch the developing solvent. It should not be too long, otherwise, it will curve and bend in the boiling tube. 3. The spot should not touch the developing solvent. The developing solvent can soak the pigments out. 4. The base line on the filter paper must be drawn by pencil. The colour of ball pen may also be moved up by the developing solvent. 5. We should wait until the pigment spot to dry before we apply the next spot of chlorophyll. The paper may be punctured by the capillary tube if it is wet. 6. The boiling tube should not be shaken and moved when the pigments were being developed.

17 Experiment 9: To find out the relationship between transpiration rate and stomatal density Background information: On the surface of the leaves, there are stomata which are holes for gaseous exchange. Water vapour can be lost through the stomata. This is a process called transpiration. Transpiration occurs through the cuticle and stomata on the leaves. When water on the wall of the mesophyll evaporates and diffuses out of the leaves, water is lost. Principles of design of the experiment: If most of the water is lost through stomata, the transpiration rate is related to the stomatal density. If most of the water is lost through cuticle of the leaves, the transpiration rate is not closely dependent on the stomatal density. We can measure the rate of water lost by timing the change in colour of the dry cobalt chloride paper. If the cobalt chloride paper changes to pink in a short time, it means that the transpiration rate is relatively higher. Then, we can mesure the number of stomata on the leaves surface by observation under a microscope. The relative transpiration rate can then be related with the stomatal density. Procedure: (1) Place a dry cobalt chloride paper onto a adhesive tape. (2) Put it onto the leaf surface. Make sure that the cobalt chloride paper is enclosed by the adhesive tape to prevent water vapour in the air reaching the cobalt chloride paper (3) Record the time for the cobalt chloride paper to change from blue to pink. (4) Repeat the Step 1 to 3 by placing another testing paper on different regions of the leaf. (5) Then, peel the epidermis of the leaf with forceps. Mount the epidermis on a glass slide. (6) Observe under microscope. Count a total 5 random fields of view and calculate the average number of stomata per field.

18 (7) In order to relate relative transpiration rate with stomatal density, two or more different types of leaves should be tried out. Step 1 to 6 will be repeated. Precautions : 1. The cobalt chloride paper must be very dry before the experiment. 2. The cobalt chloride paper must be covered by the adhesive paper to prevent effect caused by moisture in air. 3. More areas on the leaf should be done. After taking an average, the relative rate of transpiration will be more accurate.

19 Experiment 10: To study the enzyme activity of the gut of grasshopper Background information: Grasshopper is a herbivore. It usually feeds on grass. In order to digest the grass well, its gut should have certain adaptation for plant digestion. As we know there are startch and sucrose in the grass, there should also be amylase and sucrase in the gut. In this experiment, we are trying to find out which part of its gut contain amylase and sucrase. Principles of design of the experiment: First of all, we should cut out different regions of the gut. By definition, the part starting from the mouth to the gizzard is the foregut, the part starting from the malpighian tubules to the anus is the hindgut. In between is the midgut. After the identification, we should cut the different regions out and cut open the guts. We extract enzymes out of the gut and add starch or sucrose. If the starch is digested, it means the extract contains amylase. If the sucrose is digested, it means the extract contains sucrase.. Procedure: (1) Dissect the freshly killed grasshopper. (2) Take out its gut and cut it into three corresponding portions. (3) Cut open the guts and rinse with distilled water to remove any residue food inside the gut. (4) Place the gut into a beaker and macerate it with a glass rod. (5) Then, add 5ml of distlled water to get an extract. (6) Pipette 1ml of gut extract into a test tube. Add 1ml of starch solution into it. (7) Incubate the tube for at least 30 minutes. (8) Pipette 3ml of Benedict's solution into the tube and boil the mixture in a water bath for 5 minutes. (9) Observe for red precipitates.

20 (10) Repeat the experiment (Step 6-9) using sucrose solution instead of starch solution. (11) Do the control experiment using boiled extracts. Precautions : 1. The different portions of gut should be clearly separated. 2. The contents inside the gut should be removed by running distilled water. 3. Don't add more than 5ml of distilled water into the gut. It will dilute the enzyme solution.

21 Experiment 11: To find out which one, ripe banana or non-ripe banana, releases ethane Background information: Ethene is a colourless gas. Ethene, also known as ethylene, is a plant growth substance. It can be produced by almost all parts of higher plants, though the rate of production is highest where cell division occurs. Its production is increased during leaf fall, flower senescence and fruit ripening. In addition, stress factors such as wounding, flooding, chilling, disease, high temperatures and drought seem to induce ethene synthesis. Ethene has a simple structure and is lighter than air, so it diffuses rapidly amongst the air spaces of a plant, and between plants. Certainly plants are sensitive to it, and responses occur at only 1 part ethene per million of air. Yet the concentration of ethene in the tissues of a ripening apple is 2500 times greater! Ethene is an important plant growth substance, and plays an essential role in the triggering of ripening in many fruits such as bananas, avocados, figs, mangoes, peaches, pears, persimmons, plums and tomatoes. This has been exploited by commercial suppliers in management of their fruit stores - so that the fruit is ripened at just the right time for sale. Principles of design of the experiment: Ethene is an important plant growth substance, and plays an essential role in the triggering of ripening in many fruits. The production of ethene occurs during a short part of the ripening process. Presence of ethene can be detected by a relatively simple biological assay, using germinating seedlings. Since the ethene can trigger certain responses made by young seedlings, grown in the dark, then exposed to ethene. The morphology of the seedlings treated with ethene changes noticeably, compared to controls. For example, the ethene-treated seedlings are shorter, with decreased length of hypocotyl and root are fatter, with increased girth of hypocotyl. We can place the ripe banana and non-ripe one together with the young seedlings in a closed light-proof container. The ethene produced will trigger the changes in the young seedlings.

22 Procedure: (1) Prepare two identical light-proof cupboard. (2) Place 10 young seedlings into the cupboard. (3) Measure the length of the hypocotyl and the girth of root. (4) Put one ripe banana into a cupboard and put a non-ripe banana into another cupboard. (5) Measure the length of hypocotyl and the girth of root in a one-day time interval for one week. (6) Plot a graph to show the changes. Precautions : 1. The cupboards should be light-proof. 2. A large number of seedlings should be used to eliminate the individual differences. An average increase or decrease is more accurate. 3. The cupboard should be a closed one. If not, the ethene will diffuse out and cannot be kept inside the container.

23 Experiment 12: To find out which one, glucose or sucrose is a better respiratory substrate in yeast respiration Background information: Yeast belongs to unicellular fungi. It can carry out aerobic respiration when a food supply of sugar is provided. By taking in the sugar into the cell, the yeast cell can break it down to carbon dioxide and water with capture of some of the energy in the form of ATP. Principles of design of the experiment: A certain amount of sugar is added into the yeast suspension. Evolution of carbon dioxide can be observed. The rate of gas evolution can be measured in the syringe or a pipette with liquid index. In the case of syringe, the reading can show directly the amount of carbon dioxide produced. In the case of pipette, a liquid index is firstly introduced in the tube, the carbon dioxide will move the index outwards. The distance moved by the index can show the rate of gas evolution. Procedure: (1) Obtaine a yeast culture. Activate the culture by some glucose and warm temperature for 15 minutes. (2) Put 2 ml of yeast suspension into test tube A. (3) Place 2 ml of glucose into the test tube and stopper the tube. (4) The tube is connected with a syringe or a pipette with liquid index. (5) Measure the reading of the syringe before the start of experiment. Measure the reading at a 1 minute time interval. (6) Plot a graph to show the changes. (7) Repeat the experiment with sucrose. Precautions :

24 1. The activated yeast suspension should be kept in a warm water bath. The mixture of yeast and sugar should be kept in the same temperature. 2. Mix the yeast suspension well to ensure uniformity. 3. Always agitate the yeast and sugar mixture. 4. Time should be allowed for the yeast to adapt to the new environment. 5. The piston of the syringe should be made smooth for measurement of amount of gas produced. 6. Seal all joints with vaseline to prevent leakage of gases.