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discuss whether there are ethical issues in the use of invertebrates. Daphnia, the water flea, is a small freshwater crustacean which lacks physiological methods of maintaining a constant body temperature. This means that if the environmental temperature changes, its body temperature does so too and its metabolic rate will be expected to rise or fall accordingly. So the temperature of the organism must be kept constant during the procedure. In this investigation we shall test the hypothesis that as the concentration of caffeine changes, the heartbeat rate (cardiac frequency) of Daphnia also changes. Fortunately Daphnia is relatively transparent and its heart can be seen quite easily under the low Power of the microscope. Pic 01
Setting up the experiment 1. Select a large specimen and, with a pipette, transfer it to the centre of a small, dry Petri dish. With filter paper remove excess water from around the specimen so that it is completely stranded. 2. With a seeker place a small blob of silicone grease onto the floor of the Petri dish. Then wipe the needle clean and use it to gently push the posterior end of the animal into the grease so that it is firmly anchored. Now fill the Petri dish with water at 300C. 3. Place the Petri dish on the stage of a microscope and observe the animal under low Power. The figure above shows the position of the heart, watch it beating. Don't confuse the beating of the heart with the flapping of the legs. 4. Surround the animal with a circular heating coil and fix it in position as shown in the figure below. Also clamp a small mercury thermometer, or the temperature probe of a digital ther mometer, into position. Pic 02
Estimating the cardiac frequency A convenient way of doing this is to time how long it takes for the heart to beat 50 times. If it is beating too frequently for every beat to be counted, make a mark on a piece of paper every tenth beat. Do several practice runs to get used to the technique when you feel ready, proceed as follows: Replace the distilled water in the Petri dish with caffeine solutions of concentration 1mol dm-3 at 30°C. Estimate the cardiac frequency. Switch on the heater so that the water gradually warms up. If the temperature of the water rises too rapidly, switch off the heater and, if necessary, add a few ice chippings. Estimate the cardiac frequency at caffeine concentrations of 2 mol dm -3, 3 mol dm-3, 4 mol dm -3, 5 mol dm -3 and 6 mol dm -3, noting the temperature each time. Present your results in a table and. if you have sufficient readings, draw a graph of the cardiac frequency as a function of the caffeine concentration. Ethical issues in the use of invertebrates Animals are often used for research to enhance scientific knowledge. Monkeys are commonly used for brain research, dogs are used in behavioural experiments, rabbits and mice are often dissected in laboratories, mice and fruit flies are used in genetic research, etc. Is it cruel and unfair to utilise these organisms for research? The issue is very controversial and the ethical guidelines vary from country to country and person to person. In the UK it is considered ethical to use invertebrates, such as Daphnia in scientific studies, for the following reasons: Daphnia has reduced awareness of pain because of the lack of a well developed nervous system. It is transparent and its heart is visible without the need for dissection. Daphnia is abundant in nature and there is no threat to it or its dependent species (food chains). Some people also feel that it is bred for fish food and will thus die anyway. Daphnia can reproduce asexually and may be clones, therefore there is no loss of genetic variation.
for ethical reasons.The duration of exposure to the chemical should be controlled . On your graph. Sam searched the Internet for similar studies. shown in the following table. Daphnia. In her study. She compared the results from one such study. Sam studied the effect of varying the concentration of the stimulant drug caffeine on heart rate. As an extension of this work.The volume and concentration of the chemical should be controlled .Time should be allowed for acclimatisation SAQ1.Oxygen concentration of the water surrounding the daphnia If daphnia is treated with a chemical. She videoed four Daphnia at each of five different temperatures for 30 seconds. Daphnia (water fleas) can be used to determine the effect of chemicals on heart rate.Factors to be controlled . she decided to investigate the effect of temperature on heart rate in more detail.Habitat from which daphnia is obtained . (ii) Suggest one reason for her choice of maximum temperature (30 °C) and one reason for her choice of minimu memperature 5C) sed.Size of daphnia . based on these two sets of data. but she did find data from studies in which direct observation had been used to count heart rates in Daphnia. how did Sam try to ensure the reliability of her data? (1) (iv) Which aspect of her investigation was improved when Sam decided to video the Daphnia? (1) (b) (i) Calculate the mean heart rate in beats per minute for each temperature. In this new investigation Sam used a small glass chamber which could hold the Daphnia and water at a set temperature. (c) In order to get some idea of the validity of her data. (3 Pic 04 (ii) Use these data to plot a fully-labelled graph to show the effect of temperature on the mean heart rate of Daphnia. especially the video technique. The whole apparatus could be placed under a microscope so that the Daphnia heart could be seen. with her own Pic 05 (i) State one similarity and one difference in the conclusions Sam could make about the effect of temperature on Daphnia heart rate. . Sam attempted to keep the temperature of the various caffeine solutions constant. Write your answers in the spaces provided in the table.Temperature of the surrounding . show the variability of the data. Show your working in the space below. She used a slow motion replay of the video to count the number of heart beats in 30 seconds for each Daphnia at each temperature. (4) SNAB SAM 2008 SAQ2. . t (° u (2) (iii) In her investigation. (2) (ii) Suggest one explanation for the similarity and one explanation for the difference you have given above. She chose to use the water flea. She could not find any studies that had used her method exactly. Her data are summarised in the table below Pic 03 (a) (i) State and explain one ethical reason why Sam chose to use Daphnia for this investigation.
(3) (c) At the end of the investigation the student removed the Daphnia from the tea and placed it in pond water.. place a cross ( x ) in the box.. using the same Daphnia and keeping the temperature at 15 0C throughout... All measurements were taken at 15 0C. She then repeated this using the two other drinks... (2) (iii) Suggest why Daphnia needs a heart and circulatory system. but with instant coffee as the source of caffeine.. Three different Daphnia were used... (b) By using the same Daphnia throughout the investigation.... (2) SNAB Unit 1 Jun 2005 SAQ3. Initially. she placed one Daphnia in pond water with no caffeine and counted the number of its heart beats in one minute. Suggest and explain why high caffeine consumption could increase a person¶s risk of developing cardiovascular disease. The table below gives four statements concerning transport in Daphnia....... (3) (Total 9 marks) SNAB Unit 1 Jan 2008 SAQ4. (2) (Total 8 marks) SNAB Unit one ...... (1) (ii) An experiment was carried out to investigate the effect of caffeine on the heart rate of Daphnia.... (iii) Suggest three reasons why the prediction you made for (ii) above may not be very reliable........ State two variables that you would need to control to produce reliable results..... heart beats per 10 seconds Daphnia C .Pic 06 a) (i) Explain one reason why Daphnia is a suitable organism for this experiment. Suggest two reasons why the Daphnia heart rate was higher at the end of the investigation compared to the 180 beats per minute at the start...... The table below shows her results at the end of the investigation pic 09 (i) Calculate the mean number of heart beats per 10 seconds for each Daphnia Daphnia A .... caffeine... heart beats per 10 seconds Daphnia B ........ A student used this knowledge to estimate the caffeine content of three drinks.. heart beats per 10 seconds (ii) Use your answers from (i) above to predict the mean number of heart beats in 10 seconds for another Daphnia placed in a caffeine concentration of 35 arbitrary units.. Use the calibration curve to complete the third column of the table.. To do this she set up a calibration curve..... A. Give three variables that the student controlled by using the same Daphnia. Pic08 (b) A student investigated the effect of caffeine on the heart rate of Daphnia. the student was able to control certain variables that could have affected her results.. She found this to be 180. This fluid has a higher solute concentration than the freshwater that Daphnia lives in. She then recorded its heart rate and found it to be 190 beats per minute... It is a small animal that lives in freshwater Pic 07 Daphnia has a heart which pumps fluid around its body.. (2) (b) Caffeine increases human heart rate.... (a) The photograph below shows Daphnia (a water flea)... This was repeated for several different caffeine concentrations and the results are shown in the calibration curve below.... If a statement is correct. place a tick (J) in the box to the right of that statement and if a statement is incorrect.... She recorded the time taken for 180 heart beats to occur.. The student now repeated the study... She then placed the Daphnia in pond water with 10 mg of caffeine per 100 cm 3 of pond water and recorded the time taken for 180 heart beats. The results are shown in the table below... (a) Daphnia heart rate increases when Daphnia are given the stimulant...... B and C..
06 .pic 04 .05.
(2) (b)(i) Describe how the vitamin C content of the fresh vegetables could be determined. with a pipette. Record the volume of juice (VOLB) required to decolourise 2 cm 3 of the same concentration of DCPIP solution. containing vitamin C at unknown concentration (CONC B). drop by drop. Note: If only one or two drops of fruit juice are required to decolourise DCPIP. other than boiling. Use the equation below to estimate the concentration of vitamin C in the fruit juices. in which vitamin C may be lost from fresh fruit and vegetables. Repeat the procedure and calculate the mean volume. Pic11 pic12 Repeat the procedure with the fruit juice. compare the vitamin C contents of different food and drinks. Shake the tube gently after the addition of each drop and continue to add drops until the DCPIP solution is decolourised. The vitamin C content of each of the vegetables was determined in their raw state and after boiling. Pic 13 (a) Complete the table below to make a reliable comparison of the effect of boiling on the vitamin C content on each vegetable. CONCB = (VOLA X CONCA) / VOL B VOLA = Volume of vitamin C solution in ml CONCA = Concentration of vitamin C solution in mg ml-1 VOLB = Volume of fruit juice in ml CONCB = Concentration of vitamin C in fruit juice in mg ml-1 SAQ5 Vitamin C is a water-soluble vitamin which is found in foods of plant origin. An investigation was carried out into the effects of boiling on the vitamin C content of some vegetables.pic 07 CORE PRACTICAL TWO Describe how to investigate the vitamin C content of food and drink. dilute the juice five times and try again. (4) (ii) State three precautions which would need to be taken when carrying out the investigation your described in (b) (i) (3) (c) Suggest two ways. (2) [Total 11 marks] . The results are shown in the table below. PROCEDURE Add Vitamin C solution of a known concentration (CONCA). to 2 cm 3 of the DCPIP (blue) solution in a test tube. Record the exact volume of vitamin C (VOLA) you added. Using the same technique. It is unstable and easily destroyed.
12. t t a f ji f t f ii itt . Give an account of an experiment you could carry out to investigate the effect of storage time on the ascorbic acid (vitamin C) content of fresh orange juice. (4 marks) (b) What conclusions can be drawn from the results of this investigation? (2 marks) (Total 10 marks) . s w k t t° a t s w k tt ° . (4 marks) (ii) Use the information in your table to present the information in a suitable graphical form.SAQ6. v Coeoaha pws t tf l mauem eayexcnnhnahaoeevdays. esrd mdear r oadeecdyr nxe i itl tetait t f fh if t t A copy of the entries in their laboratory notebook is shown below. Pic 14 (a) (i) Prepare a table and organise the data in a suitable way so that the effect or temperature on the vitamin C content of orange juice can be displayed. 13 SAQ 7 Some students carried out an investigation into the effect or temperature on the vitamin C content ornecTeercd0c 3ornecrmbcoehrneadven100 m sampl esThr ee ampl es ere epa4C ndhree ampl es ere epa30CThe i tami n cnnecsme a o guehyx e60m oagueo aahrsoagsndddo c 3 fa j i . [Total 10 marks] pic 11 .
Each coloured solution was stirred and a sample removed and placed in a colorimeter. 400C. Pic16 . hold to the light and compare the colour of the liquids in each. SAQ8 Amir decided to investigate the permeability of beetroot cell membranes.The duration of storage (age) must also be standardised Note: A less reliable method would be to count the number of drops of the vitamin C solution and juice needed to decolourise the DCPIP. He carried out an experiment to investigate the effect of temperature on the permeability of beetroot membrane. Pic 15 9. The colour of beetroot is due to the presence of a red pigment. You are going to investigate the effect of temperature on the selectively permeable membranes of beetroot. 5.Extent of shaking of the test tube with DCPIP must be standardised During extraction of Vitamin C juice from fruits or vegetables. Safety TAKE CARE WITH CORK BORER AND MOUNTED NEEDLE ± MIND YOUR FINGERS WEAR GOGGLES ONCE THE BUNSEN BURNER IS LIT 1. Shake the tubes. Gently pick 6 beetroot discs with a forcep. Each disc was rinsed in distilled water and dried using absorbent tissue. After 30 minutes. Place on a tile and cut into 3 mm wide discs. 70 C. . 2. . label the test tubes . 600C and 70 0C. Then drop them into the test tube labelled 30 C. .Factors to be controlled . anthocyanin. 50 C. 6. Meanwhile.The conditions of storage of the fruits and vegetables must be standardised. which cannot pass through membranes. Heat gently to 30 C and remove the Bunsen burner. 7.The mass of fruit or vegetable tissue and the volume of distilled water used for making a pulp must be standardised. 50 0C. 8. Leave the discs in the test tubes for at least 20 minutes. The results of Amir¶s investigation are shown in the table below. If possible. This method is less reliable because the size of drops could be highly variable in volume. The procedure was repeated for temperatures of 30 0C. The experiment was repeated three times. Prepare a water bath using a large beaker. High temperature disrupts the structure of the membranes. They appear red because their cells contain a water soluble red pigment in their vacuoles. One beetroot discs was then placed in a tube containing 25 cm 3 of distilled water and left for 30 minutes at 20 0C. Beetroots are root vegetables. 40 C.30 C.Volume and concentration of DCPIP . The intensity of red coloration (absorbance) was determined by the colorimeter. 60 C. The water had become red and the discs were slightly pink.Concentration of Vitamin C solution . one by one. in the cell sap. Several beetroot discs were cut of equal dimensions. 11. 10. Use a cork borer to cut cylinders of fresh beetroot tissue. tripod and gauze and Bunsen burner. eg by the effect of alcohol concentration or temperature on membrane permeability. 80 C and 100 C and use a graduated pipette to add 6 cm3 cold water to each. Place the discs in the water bath for exactly 1 minute. 3. each beetroot disc was removed from the water. 4. Place all the discs in a small beaker and wash under a running tap for at least 5 minutes. CORE PRACTICAL THREE Describe how membrane structure can be investigated practically. use a colorimeter to compare the colours of each liquid. Repeat the procedure for the other tubes.
are able to pass through intact membranes. (3) c) Use these data to plot a fully-labelled graph to show the effect of temperature on the permeability of the membrane. (1) ii) Suggest how the choice of temperatures could reduce the sensitivity of the results. shown in the following table. (1) iv) State how Amir ensured the reliability of his data. 3) (c) The graph below shows the results or an experiment to investigate the effect of temperature n the permeability of beetroot cell membranes. (1) b) Calculate the mean Absorbance in arbitrary units after 30 minutes for each temperature.a) i) Suggest why it was necessary to rinse the beetroot discs before they were added to the distilled water. (b) Describe two ways in which hydrophilic molecules. such as glucose. . based on these two sets of data. (2) iii) Suggest a reason for the appearance of red colour in the distilled water at 20 0C. B. The intensity of the color in the water surrounding the beetroot was measured at the temperatures indicated oil the graph. He compared the results from one such study. Show your working in the space below. He could found similar studies that had used his method. (2) v) Give one other way in which the reliability of the data can be increased. Amir searched the Internet for similar studies. C and D. with his own. Write your answers in the spaces provided in the table. (4) CHSE First Semester 2008 SAQ9 The diagram below shows the fluid mosaic model of the cell membrane. (4) d) In order to get some idea of the validity of his data. Pic 17 i) State one similarity and one difference in the conclusions Amir could make about the effect of temperature on permeability of the membrane. (2) (a) Name the structures labeled A. (2) ii) Suggest one explanation for the similarity and one explanation for the difference you have given above.
(i) Outline a procedure that you could have used to produce these results. each set of beetroot discs was removed from the solutions and from the water. (3) Total 12 marks SNAB Jan 2005 SAQ10 Beetroots are root vegetables. (1) SVR CHSE 2009 Working of the colorimeter Cuvette contains pigment solution ± Factors to be controlled . a) Explain why the cell membrane is described as having a fluid mosaic structure. (1) c) Describe the effect of increasing ethanol concentration on the intensity of the red colour (absorbance) of the solution.The volume of water or ethanol in the test tube must be controlle d .The diameter and thickness of the discs must be standardised . (4) (ii) Explain the effect or the increase in temperature on the permeability of the membranes of beetroot cells. Several beetroot discs were cut of equal dimensions.The duration of temperature or ethanol treatment must also be standardised .A blue filter must be used in the colorimeter during measurement of absorbance .The temperature of the water bath must be controlled . The results of the investigation are shown in the graph below.The storage conditions and age of beetroot must be taken into account . Five beetroot discs were then placed in a tube containing 25 cm 3 of 0. The intensity of red coloration (absorbance) was determined by the colorimeter. The ethanol in each tube had become red and the discs were slightly pink. The procedure was repeated for different concentrations of ethanol and one set of discs was left in distilled water. Each disc was rinsed in distilled water and dried using absorbent tissue. After 30 minutes. (4) e) The experiment was repeated using a second beetroot. Alcohol disrupt the structure of the membranes. The ethanol in each tube was stirred and a sample removed and placed in a colorimeter. (2) b) Suggest why it was necessary to rinse the beetroot discs before they were added to the bile salt solution.2% ethanol and left for 30 minutes at 20 0C.The number of discs in the test tube must be the same for all trials .The volume of red pigment in the cuvette must be standardised . They appear red because their cells contain a water soluble red pigment in their vacuoles. An experiment was carried out to investigate the effect of alcohol on the permeability of beetroot membrane. (3) d) Suggest an explanation for these results. There was no change in the colour of the discs in the water and the water remained colourless. which cannot pass through membranes. Suggest why the readings obtained might be slightly different from those for the first beetroot.
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