CORE PRACTICAL ONE Describe how the effect of caffeine on heart rate in Daphnia can be investigated practically, and

discuss whether there are ethical issues in the use of invertebrates. Daphnia, the water flea, is a small freshwater crusta cean which lacks physiological methods of maintaining a constant body temperatur e. This means that if the environmental temperature changes, its body temperatur e does so too and its metabolic rate will be expected to rise or fall accordingl y. 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 o f caffeine changes, the heartbeat rate (cardiac frequency) of Daphnia also chang es. Fortunately Daphnia is relatively transparent and its heart can be seen quit e easily under the low Power of the microscope. Setting up the experiment 1. Select a large specimen and, with a pipette, transf er 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 seek er 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 wate r 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 temper ature probe of a digital thermometer, into position. Estimating the cardiac frequency A convenient way of doing this is to time how l ong 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 solution s of concentration 1mol dm-3 at 30C. Estimate the cardiac frequency. Switch on th e heater so that the water gradually warms up. If the temperature of the water r ises too rapidly, switch off the heater and, if necessary, add a few ice chippin gs. Estimate the cardiac frequency at caffeine concentrations of 2 mol dm-3, 3 m ol dm-3, 4 mol dm-3, 5 mol dm-3 and 6 mol dm-3, noting the temperature each time . Compiled by Stafford Valentine Redden 1

Present your results in a table and. if you have sufficient readings, draw a gra ph 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 enhan ce scientific knowledge. Monkeys are commonly used for brain research, dogs are used in behavioural experiments, rabbits and mice are often dissected in laborat ories, mice and fruit flies are used in genetic research, etc. Is it cruel and u nfair to utilise these organisms for research? The issue is very controversial a nd 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 scienti fic studies, for the following reasons: Daphnia has reduced awareness of pain be cause 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 r eproduce asexually and may be clones, therefore there is no loss of genetic vari ation. Factors to be controlled - Size of daphnia - Habitat from which daphnia i s obtained - Temperature of the surrounding - Oxygen concentration of the water surrounding the daphnia If daphnia is treated with a chemical, - The volume and concentration of the chemical should be controlled - The duration of exposure to the chemical should be controlled - Time should be allowed for acclimatisation SAQ1. Sam studied the effect of varying the concentration of the stimulant drug caffeine on heart rate. She chose to use the water flea, Daphnia, for ethical re asons. In her study, Sam attempted to keep the temperature of the various caffei ne solutions constant. As an extension of this work, she decided to investigate the effect of temperature on heart rate in more detail. In this new investigatio n Sam used a small glass chamber which could hold the Daphnia and water at a set temperature. The whole apparatus could be placed under a microscope so that the Daphnia heart could be seen. She videoed four Daphnia at each of five different temperatures for 30 seconds. She used a slow motion replay of the video to coun t the number of heart beats in 30 seconds for each Daphnia at each temperature. Her data are summarised in the table below: (a) (i) State and explain one ethical reason why Sam chose to use Daphnia for th is investigation. (ii) Suggest one reason for her choice of maximum temperature (30 C) and one reason for her choice of minimum temperature (5 C) used. (2) (iii) In her investigation, 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 t he Daphnia? (1) (b) (i) Calculate the mean heart rate in beats per minute for ea ch temperature. Write your answers in the spaces provided in the table. Show you r working in the space below. (3) Compiled by Stafford Valentine Redden 2

(ii) Use these data to plot a fully-labelled graph to show the effect of tempera ture on the mean heart rate of Daphnia. On your graph, show the variability of t he data. (c) In order to get some idea of the validity of her data, Sam searched the Internet for similar studies. She could not find any studies that had used her method exactly, especially the video technique, but she did find data from s tudies in which direct observation had been used to count heart rates in Daphnia . She compared the results from one such study, shown in the following table, wi th her own. (i) State one similarity and one difference in the conclusions Sam could make ab out the effect of temperature on Daphnia heart rate, based on these two sets of data. (2) (ii) Suggest one explanation for the similarity and one explanation fo r the difference you have given above. (4) SNAB SAM 2008 SAQ2. Daphnia (water fl eas) can be used to determine the effect of chemicals on heart rate. (a) (i) Explain one reason why Daphnia is a suitable organism for this experimen t. (1) (ii) An experiment was carried out to investigate the effect of caffeine on the heart rate of Daphnia. State two variables that you would need to control to produce reliable results. (2) (iii) Suggest why Daphnia needs a heart and ci rculatory system. (2) (b) Caffeine increases human heart rate. Suggest and expla in why high caffeine consumption could increase a person's risk of developing card iovascular disease. (2) SNAB Unit 1 Jun 2005 SAQ3. (a) The photograph below show s Daphnia (a water flea). It is a small animal that lives in freshwater Compiled by Stafford Valentine Redden 3

Daphnia has a heart which pumps fluid around its body. This fluid has a higher s olute concentration than the freshwater that Daphnia lives in. The table below g ives four statements concerning transport in Daphnia. If a statement is correct, place a tick (J) in the box to the right of that statement and if a statement i s incorrect, place a cross ( x ) in the box. Statements about transport in Daphn ia (i) The movement of fluid through the heart is an example of mass transport ( ii) Daphnia uses diffusion to transport oxygen into muscle cells (iii) Daphnia t ends to lose water to the freshwater by osmosis (iv) Daphnia can use active tran sport to move ions from the freshwater into its body Tick or cross (b) A student investigated the effect of caffeine on the heart rate of Daphnia. Three different Daphnia were used, A, B and C. The table below shows her results at the end of the investigation. Daphnia Caffeine Duration of Number of concent ration/ observation heart beats counted / seconds arbitrary units A A A B B B C C C 5 5 5 10 10 10 15 15 15 10 10 10 10 10 10 10 10 10 50 53 47 73 76 76 101 99 100 (i) Calculate the mean number of heart beats per 10 seconds for each Daphnia Dap hnia A .............................. heart beats per 10 seconds Daphnia B ..... ......................... heart beats per 10 seconds Daphnia C ................. ............. 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. (1) Compiled by Stafford Val entine Redden 4

(iii) Suggest three reasons why the prediction you made for (ii) above may not b e very reliable. (3) (Total 9 marks) SNAB Unit 1 Jan 2008 SAQ4. (a) Daphnia heart rate increases when Daphnia are given the stimulant, caf feine. A student used this knowledge to estimate the caffeine content of three d rinks. To do this she set up a calibration curve. Initially, she placed one Daph nia in pond water with no caffeine and counted the number of its heart beats in one minute. She found this to be 180. She then placed the Daphnia in pond water with 10 mg of caffeine per 100 cm3 of pond water and recorded the time taken for 180 heart beats. This was repeated for several different caffeine concentration s and the results are shown in the calibration curve below. All measurements wer e taken at 15 0C. The student now repeated the study, using the same Daphnia and keeping the tempe rature at 15 0C throughout, but with instant coffee as the source of caffeine. S he recorded the time taken for 180 heart beats to occur. She then repeated this using the two other drinks. The results are shown in the table below. Use the ca libration curve to complete the third column of the table. (b) By using the same Daphnia throughout the investigation, the student was able to control certain variables that could have affected her results. Give three v ariables that the student controlled by using the same Daphnia. (3) (c) At the e nd of the investigation the student removed the Daphnia from the tea and placed it in pond water. She then recorded its heart rate and found it to be 190 beats per minute. 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. (2) (To tal 8 marks) SNAB Unit one Compiled by Stafford Valentine Redden 5

CORE PRACTICAL TWO Describe how to investigate the vitamin C content of food and drink. PROCEDURE Add Vitamin C solution of a known concentration (CONCA), drop by drop, with a pipette, to 2 cm3 of the DCPIP (blue) solution in a test tube. S hake the tube gently after the addition of each drop and continue to add drops u ntil the DCPIP solution is decolourised. Record the exact volume of vitamin C (V OLA) you added. Repeat the procedure and calculate the mean volume. Repeat the procedure with the fruit juice, containing vitamin C at unknown conce ntration (CONCB). Record the volume of juice (VOLB) required to decolourise 2 cm 3 of the same concentration of DCPIP solution. Note: If only one or two drops of fruit juice are required to decolourise DCPIP, dilute the juice five times and try again. Using the same technique, compare the vitamin C contents of different food and drinks. Use the equation below to estimate the concentration of vitami n C in the fruit juices. CONCB = (VOLA X CONCA) / VOLB VOLA = Volume of vitamin C solution in ml CONCA = Concentration of vitamin C solution in mg ml-1 VOLB = V olume 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 pla nt origin. It is unstable and easily destroyed. An investigation was carried out into the effects of boiling on the vitamin C content of some vegetables. The vi tamin C content of each of the vegetables was determined in their raw state and after boiling. The results are shown in the table below. (a) Complete the table below to make a reliable comparison of the effect of boil ing on the vitamin C content on each vegetable. (b)(i) Describe how the vitamin C content of the fresh vegetables could be determined. (ii) State three precauti ons which would need to be taken when carrying out the investigation your descri bed in (b) (i) (c) Suggest two ways, other than boiling, in which vitamin C may be lost from fresh fruit and vegetables. (2) [Total 11 marks] SAQ6. Give an acco unt of an experiment you could carry out to investigate the effect of storage ti me on the ascorbic acid (vitamin C) content of fresh orange juice. [Total 10 mar ks] Compiled by Stafford Valentine Redden (2) (4) (3) 6

SAQ 7 Some students carried out an investigation into the effect or temperature on the vitamin C content of orange juice. They extracted 600 cm3 of orange juice from a batch of fresh or anges and divided it into 100 cm3 samples. Three samples were kept at 4 C and thr ee samples were kept at 30 C. The vitamin C content of each sample was measured i mmediately after extraction and then each day for the next five days. A copy of the entries in their laboratory notebook is shown below. (a) (i) Prepare a table and organise the data in a suitable way so that the effe ct or temperature on the vitamin C content of orange juice can be displayed. (4 marks) (ii) Use the information in your table to present the information in a su itable graphical form. (4 marks) (b) What conclusions can be drawn from the resu lts of this investigation? (2 marks) (Total 10 marks) Factors to be controlled Volume and concentration of DCPIP - Concentration of Vitamin C solution - Exten t of shaking of the test tube with DCPIP must be standardised During extraction of Vitamin C juice from fruits or vegetables, - The mass of fruit or vegetable t issue and the volume of distilled water used for making a pulp must be standardi sed. - The conditions of storage of the fruits and vegetables must be standardis ed. - The duration of storage (age) must also be standardised. Compiled by Staff ord Valentine Redden 7

Note: A less reliable method would be to count the number of drops of the vitami n C solution and juice needed to decolourise the DCPIP. This method is less reli able because the size of drops could be highly variable in volume. CORE PRACTICA L THREE Describe how membrane structure can be investigated practically, eg by t he effect of alcohol concentration or temperature on membrane permeability. The colour of beetroot is due to the presence of a red pigment, anthocyanin, in the cell sap. You are going to investigate the effect of temperature on the selectiv ely permeable membranes of beetroot. Safety TAKE CARE WITH CORK BORER AND MOUNTE D NEEDLE MIND YOUR FINGERS WEAR GOGGLES ONCE THE BUNSEN BURNER IS LIT 1. Use a c ork borer to cut cylinders of fresh beetroot tissue. Place on a tile and cut int o 3 mm wide discs. 2. Place all the discs in a small beaker and wash under a run ning tap for at least 5 minutes. 3. Meanwhile, label the test tubes - 30 C, 40 C, 50 C , 60 C, 70 C, 80 C and 100 C and use a graduated pipette to add 6 cm3 cold water to each . 4. Prepare a water bath using a large beaker, tripod and gauze and Bunsen burn er. 5. Heat gently to 30 C and remove the Bunsen burner. 6. Gently pick 6 beetroot discs with a forcep, one by one. 7. Place the discs in the water bath for exact ly 1 minute. Then drop them into the test tube labelled 30 C. 8. Leave the discs i n the test tubes for at least 20 minutes. 9. Repeat the procedure for the other tubes. 10. Shake the tubes, hold to the li ght and compare the colour of the liquids in each. 11. If possible, use a colori meter to compare the colours of each liquid. SAQ8 Amir decided to investigate th e permeability of beetroot cell membranes. Beetroots are root vegetables. They a ppear red because their cells contain a water soluble red pigment in their vacuo les, which cannot pass through membranes. He carried out an experiment to invest igate the effect of temperature on the permeability of beetroot membrane. High t emperature disrupts the structure of the membranes. Several beetroot discs were cut of equal dimensions. Each disc was rinsed in distilled water and dried using absorbent tissue. One beetroot discs was then placed in a tube containing 25 cm of distilled water and left for 30 minutes at 20 C. The procedure was repeated for temperatures of 30 C, 40 C, 50 C, 60 C and 70 C. After 30 minutes, each beet root disc was removed from the water. The water had become red and the discs wer e slightly pink. Each coloured solution was stirred and a sample removed and pla ced in a colorimeter. The intensity of red coloration (absorbance) was determine d by the colorimeter. The experiment was repeated three times. The results of Am ir's investigation are shown in the table below. Temperature / oC Intensity of red colouration (absorbance) / Arbitrary units Trial one Trial two Trial 3 Mean 20 0.2 0 0.1 30 0.4 0.3 0.3 40 0.5 0.6 0.6 50 0.9 1.0 1.1 60 1.6 1.6 1.7 70 1.8 1.6 1.8 Compiled by Stafford Valentine Redden 8 0 0 0 0 0 0 3

a) i) Suggest why it was necessary to rinse the beetroot discs before they were added to the distilled water. (1) ii) Suggest how the choice of temperatures cou ld reduce the sensitivity of the results. (2) iii) Suggest a reason for the appe arance of red colour in the distilled water at 20 0C. (1) iv) State how Amir ens ured the reliability of his data. (2) v) Give one other way in which the reliabi lity of the data can be increased. (1) b) Calculate the mean Absorbance in arbit rary units after 30 minutes for each temperature. Write your answers in the spac es provided in the table. Show your working in the space below. (3) c) Use these data to plot a fully-labelled graph to show the effect of temperature on the pe rmeability of the membrane. (4) d) In order to get some idea of the validity of his data, Amir searched the Internet for similar studies. He could found similar studies that had used his method. He compared the results from one such study, shown in the following table, with his own. Temperature (C) Mean Absorbance (arbi trary units) 20 0.0 25 0.2 30 0.3 35 0.4 40 0.7 45 1.2 50 1.8 55 1.8 60 1.8 i) S tate one similarity and one difference in the conclusions Amir could make about the effect of temperature on permeability of the membrane, based on these two se ts of data. (2) ii) Suggest one explanation for the similarity and one explanati on for the difference you have given above. (4) CHSE First Semester 2008 SAQ9 Th e diagram below shows the fluid mosaic model of the cell membrane. (2) (a) Name the structures labeled A. B, C and D. (b) Describe two ways in which hy drophilic molecules, such as glucose, are able to pass through intact membranes. (3) (c) The graph below shows the results or an experiment to investigate the e ffect of temperature n the permeability of beetroot cell membranes. The intensit y of the color in the water surrounding the beetroot was measured at the tempera tures indicated oil the graph. Compiled by Stafford Valentine Redden 9

(i) Outline a procedure that you could have used to produce these results. (4) ( ii) Explain the effect or the increase in temperature on the permeability of the membranes of beetroot cells. (3) Total 12 marks SNAB Jan 2005 SAQ10 Beetroots a re root vegetables. They appear red because their cells contain a water soluble red pigment in their vacuoles, which cannot pass through membranes. An experimen t was carried out to investigate the effect of alcohol on the permeability of be etroot membrane. Alcohol disrupt the structure of the membranes. Several beetroo t discs were cut of equal dimensions. Each disc was rinsed in distilled water an d dried using absorbent tissue. Five beetroot discs were then placed in a tube c ontaining 25 cm 0 3 of 0.2% ethanol and left for 30 minutes at 20 C. The procedure was repeated for different concentrations of ethanol and one set of discs was left in distilled w ater. After 30 minutes, each set of beetroot discs was removed from the solution s and from the water. The ethanol in each tube had become red and the discs were slightly pink. There was no change in the colour of the discs in the water and the water remained colourless. The ethanol in each tube was stirred and a sample removed and placed in a colorimeter. The intensity of red coloration (absorbanc e) was determined by the colorimeter. The results of the investigation are shown in the graph below. a) Explain why the cell membrane is described as having a fluid mosaic structure . (2) b) Suggest why it was necessary to rinse the beetroot discs before they we re added to the bile salt solution. (1) Compiled by Stafford Valentine Redden 10

c) Describe the effect of increasing ethanol concentration on the intensity of t he red colour (absorbance) of the solution. (3) d) Suggest an explanation for th ese results. (4) e) The experiment was repeated using a second beetroot. Suggest why the readings obtained might be slightly different from those for the first beetroot. (1) SVR CHSE 2009 Working of the colorimeter Cuvette contains pigment solution Factors to be controlled - The storage conditions and age of beetroot must be ta ken into account - The diameter and thickness of the discs must be standardised - The volume of water or ethanol in the test tube must be controlled - The numbe r of discs in the test tube must be the same for all trials - The temperature of the water bath must be controlled - The duration of temperature or ethanol trea tment must also be standardised - The volume of red pigment in the cuvette must be standardised - A blue filter must be used in the colorimeter during measureme nt of absorbance CORE PRACTICAL FOUR Describe how enzyme concentrations can affect the rates of r eactions and how this can be investigated practically by measuring the initial r ate of reaction. Investigating an enzyme controlled reaction: catalase and hydro gen peroxide concentration Procedure SAFETY: Wear eye protection and protect clo thing from hydrogen peroxide. Rinse splashes of peroxide and pureed potato off t he skin as quickly as possible as it is an oxidising agent. Investigation a Use the large syringe to measure 20 cm3 pureed potato (pulp) int o the conical flask. b Put the bung securely in the flask twist and push careful ly. c Half-fill the trough, bowl or sink with water. d Fill the 50 cm3 measuring cylinder with water. Invert it over the trough of water, with the open end unde r the surface of the water in the bowl and with the end of the rubber tubing in the measuring cylinder. Clamp in place. Compiled by Stafford Valentine Redden 11

e f g h i j Measure 2 cm3 of hydrogen peroxide into the 2 cm3 syringe. Put the syringe in pl ace in the bung of the flask but do not push the plunger straight away. Check th e rubber tube is safely in the measuring cylinder. Push the plunger on the syrin ge and immediately start the stop clock. After 30 seconds, note the volume of ox ygen in the measuring cylinder in a suitable table of results. Empty and rinse t he conical flask and measure another 20 cm3 pureed potato into it. Reassemble th e apparatus, refill the measuring cylinder, and repeat from d to g with another concentration of hydrogen peroxide. Use a 100 cm3 measuring cylinder for concent rations of hydrogen peroxide over 20 vol. Calculate the rate of oxygen productio n in cm3/ s. Plot a graph of rate of oxygen production against concentration of hydrogen peroxide. Enzyme Kinetics This means measuring the rate of enzyme reactions. 1. Firstly yo u need a signal to measure that shows the progress of the reaction. The signal s hould change with either substrate (S) or product (P) concentration, and it shou ld preferably be something that can be measured continuously. Typical signals in clude colour changes, pH changes, mass changes, gas production, volume changes o r turbidity changes. If the reaction has none of these properties, it can someti mes be linked to a second reaction, which does generate one of these changes. 2. If you mix your substrate with enzyme and measure your signal, you will obtai n a time-course. If the signal is proportional to substrate concentration it wil l start high and decrease, while if the signal is proportional to product it wil l start low and increase. In both cases the time-course will be curved (actually an exponential curve). 3. How do you obtain a rate from this time-course? One t hing that is not a good idea is to measure the time taken for the reaction, for as the time-course shows it is very difficult to say when the reaction ends: it just gradually approaches the end-point. A better method is to measure the initi al rate - that is the initial slope of the time-course. This also means you don' t need to record the whole time-course, but simply take one measurement a short time after mixing. 4. Repeat this initial rate measurement under different conditions (such as diff erent substrate concentrations) and then plot a graph of rate vs. the factor. Ea ch point on this second graph is taken from a separate initial rate measurement (or better still is an average of several initial rate measurements under the sa me conditions). Draw a smooth curve through the points. Be careful not to confus e the two kinds of graph (the time-course and rate graphs) when interpreting you r data. An example of time course measurement is shown in the SAQ below. SAQ11 The total amount of product formed in an enzyme - controlled reaction was investigated at two different temperatures, 55 C and 65 C. The results are shown in the graph. Compiled by Stafford Valentine Redden 12

a) (i) Explain how you would calculate the rate of the reaction at 55C over the f irst 2 hours of the investigation. (1) (ii) Explain why the initial rate of this reaction was faster at 65 C than it was at 55 C. (3) b) Use your knowledge of enz ymes to explain the difference in the two curves between 4 and 6 hours. (1) c) I n this investigation, the enzyme and its substrate were mixed in a buffer soluti on. What was the purpose of the buffer solution? (1) (Total 6 marks) An example of initial rate measurement reaction is shown in the SAQ below. SAQ 12 The graph below shows the results of an investigation into the effect of substrate concen tration on the initial rate of enzyme controlled reaction. 0.20 B Initial 0.15 rate of reaction / mg of products s 1 0.10 C 0.05 A 0 0 10 20 30 40 Substrate concentration / mg cm 3 50 60 a) Suggest two conditions apart from temperature that should be kept constant in this investigation. (2) b) Explain why changes in the substrate concentration c ause an increase in the rate of reaction between points A and B on the graph. (2 ) c) Suggest why the curve levels off between points B and C. (2) Compiled by St afford Valentine Redden 13

d) On the graph, sketch a curve to show how the results for the investigation wo uld change if it were repeated at a lower temperature. Explain any difference be tween the two curves. (3) (Total 9 marks) January 2002 Unit 1, Edexcel SAQ13 A s tudent carried out an experiment to test the hypothesis that an increased concen tration of the substrate increases the initial rate of an enzyme-controlled reac tion. For this investigation, hydrogen peroxide was used as the substrate, and l iver tissue was used as the 3 source of the enzyme catalase. The enzyme was extr acted by breaking up 3.0g or liver in 250cm of water 3 -3 in a blender for 45 se conds. At the start. 20cm of 31.7 mmol dm hydrogen peroxide was placed in a beak er. One 6mm diameter disc of filter paper. previously soaked in Iiver extract. w as then put at the bottom of the beaker and a stop watch as started. Oxygen bubb les, produced as a result of the reaction, caused the disc to rise to the surfac e of the liquid in the beaker. The time in seconds for the disc to reach the sur face was recorded. This procedure vas repeated for two more discs and the mean t ime was recorded. The experiment was then continued using increasing concentrati ons of hydrogen peroxide. A copy of the student's laboratory notes showing the m ean times for the discs to rise to the surface at each concentration is given be low. (a) The initial rate or reaction can be calculated using the formula below. Init ial rate = 1 / Mean time Prepare a table and present the data to show the initia l rate of reaction at each concentration of hydrogen peroxide. (4) (b) Use the d ata in your table to present the information in a suitable graphical form. (4) ( c) Describe the trends and patterns shown by these data. (2) (d) What conclusion s regarding the hypothesis can be drawn from this investigation? (2) (e) Describ e two limitations of this technique that could give rise to unreliable results. (2) (f) Compare the reliability of this method with the method describes in the core practical described above and give reasons for your answer. (g) Describe ho w a solution containing 160 mmol of hydrogen peroxide per dm3 would be diluted t o prepare a solution containing 80 mmol of hydrogen peroxide per dm3. (2 ) SVR Compiled by Stafford Valentine Redden 14

CORE PRACTICAL FIVE Describe the stages of mitosis and how to prepare and stain a root tip squash in order to observe them practically. Method PROCEDURE: 1. Pla ce some 1 molar Hydrochloric acid in the watch glass. Be careful not to get the acid on your skin or clothing. 2. In to this acid place the terminal 3 or 4 mm o f the 1 cm long onion root. 3. In a short time (a few minutes) the root tip will feel soft when touched with a dissecting needle. 4. Now, using forceps or a nee dle, pick up the softened root tip and transfer it in to a drop of acetocarmine stain on a clean slide. 5. Using a razor blade or a sharp scalpel, shop the root tip in to tiny pieces. Note: Iron in the scalpel or dissecting needle reacts wi th the acetocarmine stain to give a better staining reaction. 6. Once this proce dure is complete, apply a clean cover slip to the slide and heat it gently over an alcohol lamp or slide warmer. Do not boil. Then invert the slide on a blottin g paper and push down ward firmly, applying pressure with your thumb over the co ver glass. This should flatten the cells and disperse them so they can be observ ed under the microscope. SAQ14 (a) The table below describes some of the key eve nts that occur during mitosis Complete the table by writing the name of the stag e of mitosis next to its description. Key events Chromatids separate and move to opposite poles of the dividing cell. Chromosomes shorten and thicken. The nucle ar envelope breaks down and the spindle forms. The spindle fibres break down, th e nuclear membrane re-forms and the chromosomes elongate. Chromosomes line up on the equator of the cell, attached to spindle fibres by their centromeres. (4) ( b) The graph below illustrates the change in DNA content during the cell cycle S tage Mass of DNA / arbitrary units 6 5 4 3 A 2 1 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time / hours 26 G1 B C D (i) Calculate the percentage of the cell cycle time spent in G1. (3) (ii) At whi ch point, A, B, C or D, does chromosome replication (the S phase) begin? Explain your answer. (Total 9 marks) SAQ 15 The diagram below shows cells from a root t ip, prepared by the root tip squash method. Compiled by Stafford Valentine Redden 15

(a) Describe how you would prepare a root tip squash so that mitosis can be stud ied. (b) State which of the cells labelled AE is in: (i) metaphase .............. ..... (ii) anaphase ..................... (c) State two events that take place d uring interphase. (2) (Total 8 marks) SAQ 16 The photograph shows a cell in mito sis as viewed using a transmission electron microscope. (4) (2) (a) The actual diameter of the cell between points X and Y is 50 m. Calculate the magnification of this photograph. Show your working. (3) (b) In the space below make an accurate drawing of the cell. Label the chromosomes, spindle fibres and centrioles. (5) (c) State the stage of mitosis that this cell is in. (1) (d) St ate one function of each of the following. Spindle fibres Centrioles (2) (Total 11 marks) SAQ 17 The diagrams below show four stages of mitosis. Compiled by Stafford Valentine Redden 16

A B C D (a) (i) Write the letters of the stages in the sequence in which they occur duri ng mitosis. (ii) Name stage D. (b) The graph below shows how the quantity of DNA varies with time in a cell cycle. 4 Quantity of DNA / arbitrary units 3 (1) (1) 2 1 0 0 5 10 15 20 25 Time/hours (i) Explain the changes in the quantity of DNA that take place: between 10 to 15 hours at 20 hours (2) (1) (1) (Total 6 marks) (ii) What is happening - in the cell between 15 and 20 hours? (iii) What is the minimum length of time that interphase would occupy in this cell cycle? SAQ 18 The flow diagram below shows a method for preparing and staining cells in order to study stages of mitosis. Compiled by Stafford Valentine Redden 17

Treat plant material with hydrochloric acid Place in stain and warm Break open plant material Mount on slide Squash gently (a) Name a suitable part of a plant to use, giving a reason for your choice. (b) (i) Explain why staining is necessary in this preparation. (ii) Name a suitable stain for this technique. (c) Explain why it is necessary to squash the prepara tion. (2) (1) (1) (1) (Total 5 marks) SAQ19 An investigation was performed to determine the length of time that a cell in an onion root tip spends in each stage of mitosis. A growing root from an on ion was selected and a root tip squash was made. This was examined under a light microscope and the percentage of cells in each stage of mitosis was determined. The results are shown in the table below. Stage of mitosis Prophase Metaphase A naphase Telophase Percentage of cells in this stage 2.43 1.40 0.70 2.78 (4) (a) Describe how you would prepare a root tip squash so that mitosis could be st udied. (b) The percentage of cells in a stage of mitosis is proportional to the duratio n of that stage. Use this information to compare the duration of each stage of m itosis in these root tip cells by drawing a suitable graph. (3) (c) The duration of each stage of mitosis can be calculated using the equation b elow. Duration of a stage = Percentageof cells in thatstage 18 cell cycle time 100

Compiled by Stafford Valentine Redden

The cell cycle time for these cells is 1200 minutes. Determine the duration of e ach stage of mitosis and present the data in a suitable table. SAQ 20 The diagra ms below show some cells in different stages of mitosis. A B (2) (Total 9 marks) C D (a) Name the stages of mitosis shown by the cells labelled A, B and C. (b) Descr ibe the events that occur in the stage of mitosis shown by cell D. (c) Explain t he significance of the stage shown by cell D. SAQ21 The diagram below shows the stages in the cell cycle of a plant root cell. (1) (3) (2) (Total 6 marks) Cytokinesis Telophase Anaphase Metaphase Prophase Interphase (a) The cell had 2 arbitrary units of DNA at the start of interphase. State the number of arbitrary units of DNA in this cell in each of the following stages. ( i) at the end of prophase (1) (ii) during anaphase (1) (b) Describe how you woul d prepare a root tip squash to observe the stages of mitosis. (5) Compiled by Stafford Valentine Redden 19

(c) Two onion bulbs were grown to obtain some roots. One onion bulb was grown in water, the other in a solution of a drug called vincristine, as shown below. Onion bulb Roots Water Solution of vincristine Vincristine is a drug used in the treatment of cancer. It prevents spindle forma tion during mitosis. The result of a root tip squash on the roots grown in a sol ution of vincristine showed an increase in the percentage of cells found in one of the phases compared with roots grown in water. (i) Suggest in which phase of mitosis this increase occurs. (1) (ii) Give an explanation for your answer. (2) (iii) State the precautions that must be taken for the results of the comparison to be reliable. (4) iv) Use the information in the pie chart to draw a bar grap h, showing the relative length of each stage in the cell cycle. (4) SVR (Total 1 8 marks) CORE PRACTICAL SIX Describe how totipotency can be demonstrated practic ally using plant tissue culture techniques. Tissue culture techniques for the ve getative propagation of plants (micropropagation) have a number of advantages ov er other methods. Growth is very rapid. Growth is independent of seasons. The te chnique can be applied to a number of species that are otherwise difficult to pr opagate. Apical meristems in infected plants often remain virus-free. Their use for tissue culture has, therefore, permitted the elimination of viruses from inf ected stocks of a range of species. Certain types of callus culture give rise to clones that have inheritable characteristics different from those of the parent plant. Improved varieties arising in this way can be propagated and used commer cially. In the future, it is likely that new varieties and hybrids produced usin g other modern techniques will be commercially propagated by tissue culture meth ods. A recent development has been the commercial use of callus cultures of the shikon plant to produce large quantities of shikonin, a medically valuable produ ct found, in a lower concentration, in the shikon root. Plants are valuable sour ces of many such products, and the application of tissue culture techniques in t his way may, therefore, become more economically important. Procedure Collect al l the apparatus that you will need and prepare any solutions that are required. Set up and sterilize your work-bench as suggested in the notes on aseptic handli ng techniques. 1. Select a clean floret from a fresh cauliflower head. Place it on a tile and. holding it with forceps. Carefully trim off mini-fIorets' from th e curd to produce 20 cuboids' of curd tissue, approximately 3 mm x 5mm x 5 mm. The se will be your explants. Compiled by Stafford Valentine Redden 20

The next stage of the procedure is to steriiize the surface of the explants with a chlorate (I) solution (Bleach). Caution: You will be ushig a fairly strong bl each solution so take extreme care. From now on you must use aseptic handling te chniques (p. 17), so return your forceps to the ethanol beaker. 2. Quickly trans fer your explants to a clean (preferably sterile) screw-top jar and add chlorate (l) solution to leave a small head space. Reseal the jar and shake the contents for five seconds. 3. Shake the jar for five seconds every minute for exactly 10 minutes. 4. After exactly 10 minutes. pour off the hypochlorite solution into t he 'waste' beaker, using the Jar lid to trap the sterilized explants. 3 5. wash th e explants four times as follows. Add approximately 100cm sterile water to the j ar. Reseal it, shake for five seconds and pour off the liquid into the 'waste' b eaker as in Step 4. The explants may be left in the last wash until they are req uired. 6. Using sterile forceps (cooled in the wash water) transfer six explants to each of the three petri dishes containing growth medium. The explants should be widely spaced and pressed gently onto the agar. Flame sterilize and cool you r forceps when each dish is complete. 7. Seal each dish with Parafilm or insulat ing tape to reduce dehydration. 8. Label each dish clearly on its base and incub ate them in the light at 200C to 280C. 9. Examine each culture weekly. record an d sketch any changes you observe, If some of the explants show signs of contamin ation, the remainder should be aseptically transferred to fresh medium (Steps 6 8 ). Aseptic techniques the systematic precautions taken to keep cultures pure and fr ee from contamination. All apparatus (Petri dishes, pipettes, flasks, etc) must be sterilised by autocl aving at 121 0C for 15 minutes at 103 kPa. Or by irradiating with ultraviolet ra ys (Wavelength - 254nm). Sterile the culture medium by autoclaving. Wipe the ben ch / work surfaces with 70% ethanol before and after work. Flame the necks of bo ttles to prevent air-borne contamination. Flame wire loops and forceps before an d after use or sterilise by dipping in ethanol. Lids of containers should not le ft on benches. Petri dishes opened slightly during operation to avoid contaminat ion. Cultures after study and contaminated equipment must be autoclaved / steril ised before disposal or reuse. The composition of the medium used for growth is shown in the table below. Growth substances used in tissue culture experiments Phytochromes Photosensitive pigments.. Caution: Some phytohormones are toxic, so care must be taken when pr eparing stock solutions, e.g. protective gloves should be worn. Students should not, of course, come into direct physical contact with media. Auxins (Stimulate root development) e.g Indol 3-yl acetic acid (IAA) 1-Naphthyl acetic acid (NAA) 2,4-dichlorophenoxy acetic acid (2,4-D) Cytokinins (Stimulate cell division) 6-f urfurylaminopurine (Kinetin) 6-benzylaminopurine (BAP) Compiled by Stafford Valentine Redden 21

Outline of plant tissue culture Compiled by Stafford Valentine Redden 22

SAQ 22 Plant tissue culture is a method used to propagate plants. The flow diagr am shows one method of plant tissue culture. (a) Name the type of reproduction involved in plant tissue culture. (1 mark) (b) Give two advantages of producing plants using this method rather than from seed s. (2 marks) (c) Why is a viral infection more likely to destroy a complete batc h of plants grown by plant tissue culture than a batch of plants grown from seed s? (1 mark) (d) Callus tissue develops into either shoots or roots depending on the relative concentration of the plant growth regulators used. Use your knowled ge of genes to suggest how these plant growth regulators determine the type of p lant tissue formed. (1 mark) Compiled by Stafford Valentine Redden 23

(e) The data below shows the growth of five callus' grown at different concentrati ons of sucrose in the culture medium. Sucrose concentration 0.1 mmol dm Initial mass of callus 2.1 g Final mass of callus 2.6g (After one week) -3 0.2 mmol dm 0.3 mmol dm 0.4 mmol dm 0.5 mmol dm 2.6g 3.8g 2.3g 4.6g 2.1g 4.2g 2. 7g 5.4g -3 -3 -3 -3 i) Prepare a table and organise the data in a suitable way, so that the growth o f the callus at different sucrose concentrations can be compared. (4) ii) Presen t your data in a suitable graphical form. (4) iii) Suggest why the reliability o f the data may be low. (3) iv) List the factors that must be controlled to get r eliable results. (4) SVR SAQ 23 `Take-all' is a disease of wheat caused by a fungus. It can cause serious damage to the crop. There is no gene for resistance to thi s fungus in wheat. There is, however, a gene for resistance to this fungus prese nt in oats. The diagram shows how this gene might be transferred to wheat. (a) (i) The wheat plant with the resistance gene contains recombinant DNA. What is recombinant DNA?(1) (ii) The plasmids act as vectors for the resistance gene. What is a vector? (1) (iii) Suggest how cells with the resistance gene might be selected. (2) b)i) Suggest which tissue would be most suitable for the explants . (1) ii) Explain why explants containing large quantities of xylem do not usual ly grow into a callus. (2) c) State what is meant by the term totipotency. (2) d ) Distinguish between differentiation and dedifferentiation. (2) e) Suggest why adult animal cells usually do not develop into complete organisms, but plant tis sues do.(2) SVR Compiled by Stafford Valentine Redden 24

CORE PRACTICAL SEVEN Describe how to determine the tensile strength of plant fib res practically. Extracting fibres from plants The objectives of this procedure are: to extract fibres with possible commercial use from plants to develop inves tigative and experimental skills: devise a hypothesis, plan an experiment to pro duce appropriate results to test that hypothesis, use apparatus and a procedure that is suitable to produce valid results. Introduction Fibres have been extract ed from plant stems for centuries and used in the commercial manufacture of a wi de range of textiles and paper. The term `fibres' is used to describe a range of `fibr e-like' structures, not just the sclerenchyma. The use of different fibres depends on their properties. Strength can be defined as the maximum stress a material c an withstand without failing (breaking). Tensile strength is the maximum stress caused by a pulling force that a material can stand without failing. Concrete ha s a tensile strength of 2 x 106 N m-2. Compression strength is the maximum stren gth caused by a pushing force that a material can stand without crushing. Plant stems must not only be strong. Often they must be able to bend in the wind and r eturn to their original shape without any permanent distortion. They must not be too stiff. Useful part of the plant Applications Stem of flax plant Linen for c lothing Hairs on the seeds of a plant belonging to the Cotton for clothing mallo w family Hemp Fibres from stem/ leaves of the hemp plant Used for ropes and carp et-backing Coir Fibre from the husks of the fruit of the coconut Floor coverings , ropes Jute Fibre from the stem of the jute plant Hessian sacking and carpets M anila Hard fibres from the leaves of a type of Marine cables and other ropes, ne ts banana and matting Pulp Softwood trunks Paper, cardboard Fibres can be remove d from the plant stems by retting. In field retting, the plant stems are cut or pulled up and left to rot in the field where microbial action breaks down the st alks. In water retting, stems are immersed in water. During soaking, bacteria an d fungi break down the soft tissues of the stems leaving the cellulose intact. T his produces more uniform, higher quality fibres, but is more expensive and prod uces nitrogen-rich waste water that must be treated before discharge. It is rela tively easy to remove the cellulose-rich fibres, as described below. Procedure S AFETY: Wear eye protection and gloves when handling the plant material. Wash you r hands after handling the soaked material. When testing fibres to breaking poin t, make sure the loads on the material can fall without causing injury. Preparat ion of plant fibres Remove the stems from the water. Wash the stems to remove th e softened tissue and then dry the remaining fibres on a paper towel. The outsid e cuticle and epidermal layer will rub away and the central pith will be left wh en you peel away the fibres. These fibres are made up of vascular tissue; they c ontain both the xylem vessels and sclerenchyma fibres. Fibre Flax Cotton Compiled by Stafford Valentine Redden 25

SAQ24 Sisal is a material used to make rope. It is made from the sclerenchyma fi bres found in the leaves of the plant, Agave sisalana. After extraction of the f ibres, the waste leaf material can be used in the production of organic fertilis er. The four countries that produce most of the world's sisal are Brazil, Kenya, T anzania and Madagascar. The table below shows the annual harvest of freshly-cut Agave sisalana leaves together with the total annual production of sisal. (a) (i) Complete the table to show the total annual production of sisal. (1) (ii ) Calculate the total percentage of sisal produced from freshly-cut leaves. Show your working. (2) (b) Nylon is a synthetic (man-made) fibre which can be used t o make ropes. Nylon ropes are lighter and stronger than those made using sisal. Suggest two advantages of using sisal rather than nylon to make ropes. (2) (c) ( i) Explain what is meant by the term tensile strength of a fibre. (1) (ii) Sugge st how you could carry out a practical investigation to compare the tensile stre ngth of sisal and nylon fibres. (4) 1. idea of suspending {fibres / bundles of f ibres} with weights on / pulling them with a forcemeter/eq; 2. { fibres / bundle s of fibres} of same diameter used / different diameters accounted for; 3. { fib res / bundles of fibres} of same initial length used; 4. detail of how {weights added / forcemeter pulled}; 5. description of measurable endpoint eg {breaking p oint / stretched to standard length}; 6. repeated readings taken at each {weight / forcemeter reading} (using different fibre NOT just checking the reading with same fibre); 7. reference to a safety procedure eg {goggles in case fibre snaps / precaution against falling weights}; 8. ref to control of {temperature / humi dity / other relevant factor}; (d) Describe two ways in which the structure of x ylem vessels is similar to that of sclerenchyma fibres. (2) Compiled by Stafford Valentine Redden 26

CORE PRACTICAL EIGHT Describe how to investigate plant mineral deficiencies prac tically. The aim of this practical is to investigate the effects of mineral defi ciency on the growth of plants. Seedlings are grown in solutions containing a ra nge of mineral salts, including those which lack phosphate, nitrate, calcium, po tassium, magnesium, iron and sulphate, plus the complete medium. Suitable seedli ngs which can be used for this experiment include: maize (Zea mays), castor bean s (Ricinus communis), tomato (Lycopersicon esculentum) and cabbage (Brassica ole racea). Seeds are germinated in moist vermiculite (a nutrient free medium that r etains moisture and anchors roots at the same time), then transferred to the nut rient solution, as shown in the Figure. At weekly intervals, the plants are meas ured and any deficiency symptoms noted. Method 1. Make up the culture media and set up eight tubes containing the following: Complete (normal) medium Medium lac king phosphate Medium lacking nitrate Medium lacking calcium Medium lacking pota ssium Medium lacking magnesium Medium lacking iron Medium lacking sulphate 2. Wr ap each tube in aluminium foil. 3. Select eight seedlings and set up the culture s as shown in Figure. 4. Leave the experiment at room temperature and each week record the following: Shoot and root length Leaf number and size Internode numbe r and length Deficiency symptoms, such as changes in shape or yellowing of leave s. SAQ 25 The apparatus shown below can be used to study mineral nutrition in fl owering plants. (a) Suggest the function of each of the following. (i) The stream of air (ii) Th e black paper cover (2) (2) (b) The nutrient solution contains various mineral ions including magnesium and phosphate. Give one reason why each of these ions is essential to the plant. Mag nesium Phosphate (2) (Total 6 marks) Compiled by Stafford Valentine Redden 27

SAQ26 An investigation was carried out into the absorption of mineral ions by be ech tree seedlings. The absorption of phosphate ions by beech roots was measured in moist air, and in an atmosphere of moist nitrogen. The results are shown in the graph below. 10 9 8 7 Absorption of 6 phosphate ions / arbitrary units 5 4 3 2 1 0 0 5 10 15 20 Time / hours 25 30 Roots in air Roots in nitrogen (a) Calculate the rate of absorption of phosphate ions by beech roots in air bet ween 10 hours and 20 hours. Show your working. (3) (b) (i) Compare the rates of absorption of phosphate ions by roots in air and roots in nitrogen. (2) (ii) Sug gest an explanation of the difference in rates of absorption. (2) (c)(i) Suggest two reasons why the atmosphere in which the roots are kept has to be moist. (2) (ii) Suggest two factors that should be kept constant in this experiment. (2) ( Total 11 marks) Compiled by Stafford Valentine Redden 28

SAQ 27 An experiment was carried out to investigate the uptake of different mine ral ions by barley plants. A large number of barley seedlings was grown in a nut rient solution containing a range of mineral ions including potassium (K+), calc ium (Ca2+), magnesium (Mg2+) and nitrate (NO3). The experiment was set up as show n in the diagram below. The concentration of these ions in the solution were measured at the beginning a nd at the end of the experiment. The results are shown in the table below. Concentration of ions in nutrient solution / arbitrary units Mineral ion At star t of experiment Nitrate Potassium Magnesium Calcium 7.0 3.0 2.0 5.0 At end of ex periment 1.8 0.0 2.1 5.6 (a) Calculate the percentage difference between the concentration of nitrate ion s at the beginning and at the end of the experiment. Show your working. (3) (b) What do the results suggest about the mechanism of absorption of potassium ions? Explain your answer. (3) (c) Suggest an explanation for the changes in concentr ations of magnesium and calcium ions during the experiment. (2) (d) State two pr ecautions which should have been taken to ensure that results for all the barley seedlings were comparable. (2) (e) Describe the pathway taken by mineral ions a s they pass from the nutrient solution to the xylem in the roots of the seedling s. (3) (Total 13 marks) Compiled by Stafford Valentine Redden 29

SAQ 28 Duckweeds are small, green plants that float on the surface of freshwater ponds. Experiments were carried out on the growth of two species of duckweed, L emna gibba and Lemna polyrrhiza. L. gibba plants contain non-photosynthetic air sacs which enable them to float on the surface of the water. In these experiment s the two species were grown in complete mineral nutrient solutions as follows. Experiment A Lemna gibba and Lemna polyrrhiza together Experiment B Lemna polyrr hiza only Experiment C Lemna gibba only The dry mass of the plants was measured each week for 8 weeks. The results for experiment A are shown in the table below . Dry mass / mg Week Lemna polyrrhiza 0 1 2 3 4 5 6 7 8 25 69 87 106 87 100 87 81 37 Lemna gibba 22 84 169 197 289 344 344 347 345 (5) (4) (a) Plot the data in suitable graphical form on graph paper. (b) Describe the gr owth of L. polyrrhiza and L. gibba in experiment A. (c) The results for experime nts B and C are shown in the table below. Week Experiment B Experiment C Lemna polyrrhiza Lemna polyrrhiza 0 63 50 1 100 119 2 188 200 3 306 259 4 381 291 5 448 356 6 562 350 7 594 369 8 641 400 (i) Compare the growth of L. polyrrhiza in experiments A and B (2) (ii) Suggest a reason for the difference in growth in these two experiments. (1) (d) (i) How does the gro wth of L. polyrrhiza in experiment B differ from L. gibba in experiment C? (ii) Suggest an explanation for this difference. (2) (Total 15 marks) Compiled by Stafford Valentine Redden 30

CORE PRACTICAL NINE Describe how to investigate the antimicrobial properties of plants. Preparation of media and plate pouring Introduction There are many diffe rent types of media which are used for the culture of micro-organisms. These may be obtained in ready-formulated preparations, or can be made using separate ing redients. In this practical, we look at the method for preparing and sterilizing nutrient agar medium. Nutrient agar medium can be used for the culture of bacte ria. If ready-formulated media are used, then it is made up using distilled wate r. Materials Ready-formulated Nutrient agar medium. Distilled water 50 ml Univer sal bottle Petri dishes Autoclave Incubator Gas burner Glass rods Electronic wei ghing balance Measuring cylinders Conical flasks, sterilized cotton Method Nutri ent agar medium Add 28 g of formulated ingredients in 1 litre of distilled water and stir it until it dissolve. Dispense into a suitable containers and autoclav e at 121 C for 15 minutes. Procedure for pouring a plate Allow liquid agar to coo l to about 50 C (just cool enough for comfortable handling) after removal from au toclave. Unscrew cap of bottle with little finger as shown in Figure (a). It can be held by little finger during the procedure to avoid placing it on the bench where it could become contaminated. `Flame' mouth of bottle for a few seconds. This is done to produce an upward flow of air from the bottle so that any organisms i n the area will not fall into the bottle. (It is not done to kill microorganisms ; the bottle would have to be heated for too long to achieve this effect). Refer Figure (b). Pour the molten agar (about15-20 cm3) slowly into the base of the s terile Petri dish, lifting the lid only as much as necessary. Do not spill agar on the edges of the dish (if so, start again). Replace the lid and allow the aga r to set. Refer Figure (c). If the plate is to be used for `streaking' or `spreading' th e surface of the agar and lid can be dried once the agar is set. They are best d ried upside down in an incubator at about 37 C, and arranged as shown in Figure ( d) to minimize the risk of contamination (slight in these circumstances). Prepar ation of streak plate of bacteria Introduction In this practical, an agar plate will be poured using sterile nutrient agar and inoculated using a culture of a s uitable bacterium, such as Bacillus subtilis. Streak plates are useful to isolat e pure culture as, individual colonies will have grown from a single cell. Singl e colonies can be used to subculture another agar plate to obtain a pure isolate Boiling water bath Compil . Materials Sterile Petri dishes Sterile nutrient agar ed by Stafford Valentine Redden 31

Bacteriological loop Slope culture of Bacillus subtilis, or other suitable bacte rium. Chinagraph pencil or spirit marker pen Method Pouring a sterile agar plate 1. Before starting, wipe the bench surface u sing a suitable disinfectant solution 2. Melt the agar in a boiling water bath, remove carefully using tongs allow to cool to about 45 C. At this temperature, th e agar will be cool enough to handle safely, but will remain molten. Agar starts to set below about 42 C. 3. On the base of sterile Petri dish, write your name, the date and the name of the organism with which the plate will be inoculated. P etri dishes should be always be labeled on the base, as it is possible for lids to be transposed. 4. Working near a Bunsen burner with a blue flame, hold the mo lten, but cooled, nutrient agar in one hand and using little finger of the other hand, remove the lid of the bottle. Do not place the lid on the bench. 5. Pass the neck of the bottle through the Bunsen flame using the hand in which you are holding the lid of the bottle, raise the lid of the Petri dish to an angle of ab out 45 and carefully pour in the agar until the dish is nearly half full. Replac e the Petri dish lid, flame the neck of the bottle again and replace the lid. 6. Leave the agar plate to set. Method Preparing a streak plate 1. Have ready your sterile agar plate and slope culture of the bacterium to be used. 2. Sterilize the bacteriological loop by holding it in a blue Bunsen flame until red hot. 3. Allow the loop to cool and whilst still holding the loop, remove the lid from th e slope culture using the little finger of the hand in which you are holding the loop. Do not place the lid on the bench. 4. Pass the neck of the culture bottle through the Bunsen flame, then use the loop to remove a small portion of the cu lture. Replace the lid on the culture bottle. 5. Now lift the lid of the Petri d ish and use the loop to streak out the culture as shown in Figure. Be careful no t to `Plough up' the surface of the agar. When you have finished, flame the loop aga in before placing it on the bench. 6. Fasten the lid of the Petri dish using two pieces of adhesive tape. Invert the dish, and incubate at 30 C for 24 hours. Res ults and discussion 1. Record the appearance of your streak after incubation. We re you successful in obtaining single colonies of the bacterium? 2. List the dif ferent methods of sterilization, which have been used in this practical. 3. Expl ain why it is important: (a) to avoid ploughing up the surface of the agar when inoculating (b) not to seal the dishes all around with adhesive tape (c) to inve rt the dishes when they are incubated. SAQ 29 Mutation in bacteria can be invest igated by inoculating agar plates containing concentrations of antibiotic suffic iently high to prevent bacterial growth. Only bacteria that have mutated and bec ome resistant to the antibiotic will grow and form colonies. An experiment was c arried out to investigate the mutagenic potential of three compounds, A, B and C . Bacteria of a strain known to be sensitive to penicillin were exposed for two minutes to varying concentrations of compounds A or B or C. Plates containing pe nicillin were then inoculated with 107 bacteria and incubated for three days. Compiled by Stafford Valentine Redden 32

The results are shown in the table below. Concentration of compound / g cm3 0 10 20 30 40 50 100 200

Number of colonies Compound A 2 3 6 7 11 14 24 40 Compound B 1 4 9 14 23 34 7 0 Compound C 2 4 3 5 2 1 3 6 (a) Describe the results obtained with each compound and in each case suggest a reason for the result. Compound A Compound B Compound C (6) (b) Mutation rate is defined as the chance that a mutation would occur in one generation. Calculate the mutation rate for the bacteria in compound A at 200 g cm -3 concentration. Sh ow your working. (2) (c) (d) Suggest two reasons why bacteria are suitable organ isms for studies of mutation. (2) Suggest why antibiotics used to treat human di sease should not be used to treat disease in animals reared for food. (2) (e) Dr aw a graph to compare the effects of the concentration of the compound A,B and C on the growth of the colonies. (4) (Total 16 marks) SAQ 30 An experiment was ca rried out to test the sensitivity of the bacterium Bacillus subtilis to differen t antibiotics. The surface of a nutrient agar plate was flooded with a broth cul ture of the bacterium. The excess broth was discarded. Five filter paper discs, (A, B, C, D and E), each containing a different antibiotic, were placed on the n utrient agar surface. The plate was allowed to stand for 30 minutes at room temp erature before being incubated at 30 C for 24 hours. The appearance of the plate after the incubation is shown in the diagram. A Bacteria growing on agar Clear zones E B Antibiotics discs D C (a) (b) State two ways in which antibiotics may inhibit the growth of bacteria such as B acillus subtilis. (2) Suggest why the surface of the nutrient agar was flooded w ith the broth culture rather than being inoculated by a streaking technique. (1) 33 Compiled by Stafford Valentine Redden

(c) (d) Suggest why the plate was allowed to stand for 30 minutes at room temperature be fore being incubated. (1) Suggest what the results indicate about the sensitivit y of B. subtilis to the antibiotics. (1) (Total 5 marks) SAQ 31. In the culturing of microorganisms in the laboratory it is necessary to use aseptic techniques to prevent contamination. The diagram below shows how a s imple autoclave is used to sterilise laboratory equipment and culture media. Val ve Valve open closed Mass added Steam Aluminium foil Cotton wool plug Water Left to cool Heat Heat (a) (b) (c) The pressure of steam builds up to 103 kPa. This raises the boiling point of wat er to 121C. The contents of the autoclave are maintained at this temperature for 15-20 minutes. Explain why these conditions are necessary for effective sterilis ation. (2) Suggest why the cotton wool plugs of the flasks are covered with alum inium foil. (1) Describe and explain two precautions, other than the use of ster ile equipment, you would need to take to prevent contamination of cultures of mi croorganisms. (4) (Total 7 marks) SAQ 31 A suspension of bacteria was spread evenly over solid medium in a petri d ish. The culture was incubated at 25 C for 12 hours. After 12 hours two discs wer e placed on the surface of the medium. Each disc had been soaked in a different antibiotic (antibiotic A and antibiotic B). The culture was then incubated for 4 8 hours to allow the antibiotics to diffuse into the medium. The appearance of t he culture before and after incubating with the antibiotic-soaked discs is shown below. Antibioticsoaked disc B A Bacterial colonies Appearence of culture after 12 hour s, on adding the antibiotic-soaked discs Compiled by Stafford Valentine Redden B A Appearence of culture 48 hours after adding the discs 34

(a) (b) Describe the effect of each antibiotic on the growth of bacteria. Suggest a reas on for the effect of each antibiotic. Antibiotic A Antibiotic B (4) Suggest why antibiotics such as penicillin affect bacterial cells but not the cells of the p atient they are used to treat. (3) (Total 7 marks) Reliability: When the variability in replicated results is very high, the reliab ility is relatively low. This may be due to certain variables not being controll ed of faulty experimental procedures. Range bars or error bars on the graph will give a fair indication of the reliability. If there is considerable overlap bet ween error bars, then variability is very high and reliability is low. Students should be able to identify factors that may decrease the reliability of the resu lts. Accuracy: accuracy can be defined as the difference between the actual valu es and the measured values. If the difference is high, then accuracy is low and vice versa. Accuracy can be improved by using appropriate apparatus and methods for making measurements. Question one (Core practical) Data organisation. a. Tabulation The Independent variable comes in the first column. Arrange values in ascending order. The independent variable is controlled by the experimenter. The dependent variable is measured to give the results. The dependent factor de pends on the independent variable. Label all columns and rows appropriately and accurately. Include SI units in the headings of the columns and rows. Be consist ent with significant figures / decimal places. You are generally asked to find t he mean or the percentage difference. Mean = sum of readings / number of reading s. ( e.g. Mean = 12 + 11.2 + 13.1 / 3 = 12.1) Percentage Difference = (Differenc e / Initial reading) x 100 b. Graph Axes: Independent variable on X - axis. Labe l appropriately with units. Dependent variable on Y - axis. Label appropriately with units. Scale: The curve should cover more than 50 % of the graph paper. Plo t all points accurately. Mark the plotted point with a dot and a circle or with a cross. Line: Join each point with a neat straight line, passing exactly throug h the point. Do not extrapolate. c. Describe the trends in the graph. Do NOT mak e theoretical assumptions. Make conclusions based on the experimental data. Refe r to the hypothesis being investigated, while making conclusions. d. Limitations are genuine sources of error. Look out for variables like temperature, pH, etc. not being controlled. Also check if the experiment has been replicated. Every e xperiment has two main components: 1. Control of variable / factors. Ensure that you control all Variables and factors that can affect your results. State sensi ble values, with SI units and describe sensible ways of controlling these variab les. Describe the type of apparatus that could be used. Controlling factors effectively will improve the reliability of your results. 2. Making quantifiable measurements. The results of your experiment should have numerical values, so that the data can be analysed and results can be drawn. Using an appropriate method and suitable apparatus can improve the accuracy of y our results. Note: Examiners usually check whether you know the factors that need to be contr olled for the core practical. They also expect you to devise or be aware of suit able methods of making accurate measurements. Compiled by Stafford Valentine Red den 35