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 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 thermometer, 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.

Factors to be controlled

- Size of daphnia

- Habitat from which daphnia is 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 reasons. In her study, Sam attempted to keep

the temperature of the various caffeine 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 investigation 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 count the number of heart beats in 30 seconds for each Daphnia at each

temperature. 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. (ii) Suggest one reason for her choice of maximum temperature (30 °C) and one reason for her choice of m i n i m u m t e m p e r a t u r e ( 5 °C ) u s e d . (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 the Daphnia? (1) (b) (i) Calculate the mean heart rate in beats per minute for each temperature. Write your answers in the spaces provided in the table. Show your working in the space below. (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. On your graph, show the variability of the 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 studies 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, 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, based on these two sets of data. (2) (ii) Suggest one explanation for the similarity and one explanation for the difference you have given above. (4) SNAB SAM 2008

SAQ2. Daphnia (water fleas) can be used to determine the effect of chemicals on heart rate.

Pic 06

a) (i) Explain one reason why Daphnia is a suitable organism for this experiment. (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 circulatory system. (2) (b) Caffeine increases human heart rate. Suggest and explain why high caffeine consumption could increase a person’s risk of developing cardiovascular disease. (2) SNAB Unit 1 Jun 2005 SAQ3. (a) The photograph below shows Daphnia (a water flea). It is a small animal that lives in freshwater

Pic 07

Daphnia has a heart which pumps fluid around its body. This fluid has a higher solute concentration than the freshwater that Daphnia lives in.

The table below gives 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 is incorrect, place a cross ( x ) in

the box.

Pic08(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

pic 09

(i) Calculate the mean number of heart beats per 10 seconds for each Daphnia

Daphnia 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.

(iii) Suggest three reasons why the prediction you made for (ii) above may not be very reliable. (3) (Total 9 marks) SNAB Unit 1 Jan 2008 SAQ4. (a) Daphnia heart rate increases when Daphnia are given the stimulant, caffeine. A student used

this knowledge to estimate the caffeine content of three drinks. To do this she set up a calibration

curve. Initially, she placed one Daphnia 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 concentrations and the results are shown in the calibration

curve below. All measurements were taken at 15 0C.

The student now repeated the study, using the same Daphnia and keeping the temperature at 15 0C

throughout, but with instant coffee as the source of caffeine. She 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 calibration 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 variables that the student controlled by

using the same Daphnia. (3) (c) At the end 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) (Total 8

marks) SNAB Unit one

pic 04 ,05, 06

pic 07

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. Shake the tube gently after the addition of each drop and

continue to

add drops until the DCPIP solution is decolourised. Record the exact volume of vitamin C (VOLA) you

added. Repeat the procedure and calculate the mean volume.

Pic11 pic12

Repeat the procedure with the fruit juice, containing vitamin C at unknown concentration (CONCB).

Record the volume of juice (VOLB) required to decolourise 2 cm3 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 vitamin 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 = Volume of fruit juice in ml

CONCB = Concentration of vitamin C in fruit juice in mg ml-1SAQ5 Vitamin C is a water-soluble vitamin which is found in foods of plant 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 vitamin C content of each of the vegetables was determined in their raw state and

after boiling. The results are shown in the table below.

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. (2)

(b)(i) Describe how the vitamin C content of the fresh vegetables could be determined. (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, other than boiling, in which vitamin C may be lost from fresh fruit and vegetables. (2) [Total 11 marks] SAQ6. 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. [Total 10 marks]

pic 11 , 12, 13

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 oranges and divided it into 1 0 0 c m3 s a m p l e s . T h r e e s a

m p l e s w e r e k e p t a t 4 °C a n d t h r e e s a m p l e s w e r e k e p t a t 3 0 °C . T h e v i t a m i n C content of each

sample was measured immediately after extraction and then each day for the next five days. 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. (4 marks) (ii) Use the information in your table to present the information in a suitable graphical form. (4 marks) (b) What conclusions can be drawn from the results of this investigation? (2 marks) (Total 10 marks)

Factors to be controlled

- Volume and concentration of DCPIP

- Concentration of Vitamin C solution

- Extent of shaking of the test tube with DCPIP must be standardisedDuring extraction of Vitamin C juice from fruits or vegetables, - The mass of fruit or vegetable tissue and the volume of distilled water used for making a pulp

must be standardised.

- The conditions of storage of the fruits and vegetables must be standardised.

- 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. This method is less reliable because the size of drops could be highly variable in volume. CORE PRACTICAL THREE Describe how membrane structure can be investigated practically, eg by the 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 selectively permeable membranes of beetroot. Safety TAKE CARE WITH CORK BORER AND MOUNTED NEEDLE – MIND YOUR FINGERS WEAR GOGGLES ONCE THE BUNSEN BURNER IS LIT

1. Use a cork borer to cut cylinders of fresh beetroot tissue. Place on a tile and cut into 3 mm wide

discs.

2. Place all the discs in a small beaker and wash under a running tap for at least 5 minutes.

3. Meanwhile, label the test tubes - 30C, 40C, 50C, 60C, 70C, 80C and 100C and use a

graduatedpipette to add 6 cm3 cold water to each. 4. Prepare a water bath using a large beaker, tripod and gauze and Bunsen burner. 5. Heat gently to 30C 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 exactly 1 minute. Then drop them into the test tube labelled 30C. 8. Leave the discs in the test tubes for at least 20 minutes.

Pic 15

9. Repeat the procedure for the other tubes.

10. Shake the tubes, hold to the light and compare the colour of the liquids in each.

11. If possible, use a colorimeter to compare the colours of each liquid.

SAQ8 Amir decided to investigate the permeability of beetroot cell membranes. Beetroots are root

vegetables. They appear red because their cells contain a water soluble red pigment in their vacuoles,

which cannot pass through membranes. He carried out an experiment to investigate the effect of

temperature on the permeability of beetroot membrane. High temperature 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 3

of distilled water and left for 30 minutes at 20 0C. The procedure was repeated for temperatures of 30 0C, 400C, 50 0C, 600C and 70 0C. After 30 minutes, each beetroot disc was removed from the water. The water had become red and the discs were slightly pink. Each coloured solution was stirred and a sample removed and placed in a colorimeter. The intensity of red coloration (absorbance) was determined by the colorimeter. The experiment was repeated three times. The results of Amir’s investigation are shown in the table below.

Pic16

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 could reduce the sensitivity of the results. (2) iii) Suggest a reason for the appearance of red colour in the distilled water at 20 0C. (1) iv) State how Amir ensured the reliability of his data. (2) v) Give one other way in which the reliability of the data can be increased. (1) b) Calculate the mean Absorbance in arbitrary units after 30 minutes for each temperature. Write your answers in the spaces 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 permeability 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.

Pic 17i) State 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 sets of data. (2) ii) Suggest one explanation for the similarity and one explanation for the difference you have given above. (4) CHSE First Semester 2008 SAQ9 The 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 hydrophilic 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 effect of temperature n the permeability of beetroot cell membranes. The intensity of the color in the water surrounding the beetroot was measured at the temperatures indicated oil the graph.

(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 are root vegetables. They appear red because their cells contain a water soluble red

pigment in their vacuoles, which cannot pass through membranes. An experiment was carried out to

investigate the effect of alcohol on the permeability of beetroot membrane. Alcohol disrupt 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. Five beetroot discs were then placed in a tube containing 25 cm 3

of 0.2% ethanol and left for 30 minutes at 20 0C. The procedure was repeated for different concentrations

of ethanol and one set of discs was left in distilled water. After 30 minutes, each set of beetroot discs

was removed from the solutions 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 (absorbance) 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 were added to the bile salt solution. (1) c) Describe the effect of increasing ethanol concentration on the intensity of the red colour (absorbance) of the solution. (3) d) Suggest an explanation for these 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 taken 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 number 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 treatment 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 measurement of absorbance