Core practical one :Effect of Caffeine on Daphnia heart rate:

Method: 1. Select a large specimen and transfer it to the centre of a small, dry Petri dish with a pipette. Use a filter paper to remove excess water from around the specimen so that it is completely stranded. 2. Use a seeker to 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. 3. Fill the petri dish with water at 30C 4. Allowed to acclimatize 4. Place the Petri dish on the stage of a microscope and observe the animal under low Power. 5. Time how long it takes for the heart to beat 50 times using stopper 6. Repeat the experiment using Daphnia with similar size and from same habitat 6. Replace the distilled water in the Petri dish with caffeine solutions of different concentration. Independent variables Relative caffeine concentration Dependent variables Heartbeat rate of Daphnia Controlled variable in order to produce reliable results 1. Size of daphnia ( Same age, same growth condition) as size or surface area to volume ratio could influence the rate of absorption of caffeine into the body. It is not possible to measure the size or S.A. of Daphnia, but Daphnia of similar size can be selected for the experiment by simple observation and judgment 2. Habitat from which Daphnia is obtained 3. Temperature of the surrounding Metabolic activity is slowed down at low temperatures due to lack energy as respiratory enzymes have low kinetic energy 4. Oxygen concentration of the water surrounding the Daphnia This is to ensure metabolic activity is not limited by lack of oxygen for respiration This can be controlled by aerating the solutions by passing air through it to saturate the solution with oxygen 5.Time in solution prior to counting heart rate

Explain why Daphnia is a suitable organism for this experiment. 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 and can be seen quite easily under the low power of the microscope

Daphnia is abundant in nature and there is no threat to it or its dependent species ( food chain) Daphnia can reproduce asexually and may be clones, therefore there is no loss of genetic variation

Explain why Daphnia is not a suitable organism for this experiment Use of any animal is wrong How can we be sure what the Daphnia can feel Possibility that the Daphnia could die

Why better not to keep the Daphnia on the cavity slide for too long after removing it from the water: As the temperature could change and thereby influence the heartbeat rate

Why better not to use tap water: Tap water contains chlorine, which could be toxic to daphnia. Solution: A beaker containing tap water can be left open overnight to allow chlorine to evaporate.

Why a value lies away from the trend This is a random error. All other readings lies close to the expected values and follow a trend. Only this value lies away from the trend and is unexpected. A systematic error will lead to the entire set of data deviating from the actual trend

Plot graph: Single neat line drawn either with a ruler from point to point or freehand curve No extrapolation Range bars plotted accurately

Counting of heart beat can be inaccurate -- Method to reduce counting error: Use a microscope fitted with a video camera and integrate timer to record the heartbeat rate. Then replay the video in slow motion and count the number of heartbeats in one minute as shown on the integrated timer

Possible evaluation issues 1. If left too long under microscope, temp increase due to lamp -- > increased heart rate 2. Too high concentration of caffeine kills Daphnia

How to ensure the reliability of the data? For each temperature, use four different Daphnia to carry out the experiment. Then calculate the mean heart rate per minute in 30 seconds.

Suggest one reason for choosing 30C as the maximum temperature and one reason for her choice of minimum temperature used. Metabolic activity is slowed down at low temperatures due to lack energy as respiratory enzymes have low kinetic energy

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 Genotype Age Physiological state Absorption rate Size Gender

Caffeine increases human heart rate. Suggest and explain why high caffeine consumption could increase a persons risk of developing cardiovascular diseases. 1. 2. 3. 4. 5. Increase heart rate increase heart pressure Risk of damage to arteries increases Increase risk of heart attack/ stroke/ Aneurysm More chance of atherosclerosis Vasocontriction

Suggest why Daphnia needs a heart and circulatory system 1. Very active and high metabolic rate 2. Requires heart and circulatory system to supply O2 and remove CO2 3. Slow diffusion through body surface because of low SA/V ratio 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. Variation due to chance Still have some caffeine present within the organism

Need time to recover from the effect of caffeine Inaccuracy of measurement

Purpose of control in this experiment To find out heart rate under normal conditions to compare treated animal with the untreated

Suggest why the experiment was repeated at each concentration of caffeine Can calculate a mean increase the reliability of the experiment To reduce the effect of anomalies

Explain why the specimens were kept at a constant temperature during the experiment Reduce variable so that factors other than ____ are kept constant Daphnia lacks physiological methods of maintaining a constant body temperature. If the environmental temperature changes, its body temperature does so too and its metabolic rate will be expected to rise or fall accordingly. Moreover, change in temperature will affect heart rate

Core practical two: Investigate the vitamin C content of food and drink Procedure: 1. Pipette 2 cm3 blue DCPIP into test tube 2. Add vitamin C solution of a known concentration, drop by drop, to 2 cm3 of the DCPIP solution in a test tube using burette. 3. Add drops until the DCPIP is decolourised 4. Shake the tube gently after the addition of each drop 5. Record the exact volume of vitamin C you added. 6. Repeat the procedure and calculate the mean volume 7. Repeat above procedure with the fruit juice, containing vitamin C at unknown concentration. 8. Record the volume of juice required to decolorize 2 cm3 of the same concentration of DCPIP solution 9. Use the equation below the estimate the concentration of vitamin C in the fruit juice. Conc B X Vol B ( known) = Vol A ( known) X conc A ( unknown) Or, use calibration curve to determine vitamin C concentration Method to investigate the effect of storage time on vitamin C content Procedure being repeated at regular time interval everyday

How to prepare a known concentration of vitamin C solution? Weigh 6 mg of vitamin C powder by using an electronic balance. Add distilled water into a volumetric flask of 100cm3 and dissolve the power into the distilled water to prepare 100 cm3 of the vitamin C solution If only one o two drops of fruit juice are required to decolorize DCPIP: Dilute the juice five times and try again

What is the objective of this experiment? To compare the vitamin C content in different fruits and vegetables

What is the control of this experiment?

Distilled water

How can you obtain faster result? Use less DCPIP solution or a more dilute DCPIP solution

What precaution should be taken when carrying out the investigation Use freshly prepared juices in the experiment. Otherwise, vitamin C content will be decreased because it is oxidized if the juice are allowed to stand in the air for a long time Do not use hot water to extract the juices from the fruits and vegetables. Otherwise, the high temperature will lower the reducing power of any vitamin C in the samples. Because vitamin C is unstable at high temperature When mixing the samples and the DCPIP solution, do not shake the test tube too much. Otherwise, oxygen in the air bubbles produced by shaking will lower the reducing power of any vitamin C in the sample because oxygen will slightly restore the DCPIP to blue. Therefore, extent of shaking of the test tube with DCPIP must be standardized. Rinse the dropper thoroughly for each sample

What are the possible sources of errors in this experiment? Suggest ways for improvement Oxidation of vitamin C in the juices. Work quickly with the freshly prepared juice The observation of complete decolorization of DCPIP solution is subjective, especially when the juice are colored. Therefore, it is difficult to accurately observe the point at which the DCPIP becomes colorless. Moreover, there are other reducing substances, e.g. glucose and fructose in the juice

What is the limitation of using DCPIP solution to compare the vitamin C content in different fruits or vegetables? Fruits or vegetables that have juices very dark in colour cannot be used. The dark colour of the juices masks the decolourization of DCPIP solution, therefore difficult to observe the exact point at which the DCPIP changes colour. So the accuracy of the readings is likely to be low and hence the validity of the results will be low

What is the conclusion of this experiment? Vitamin C is present in the fruits and vegetables tested. Among them, the green pepper has the highest vitamin C content, followed by the lemon and the orange.

How can you make sure that the results are reliable? Take any dilution factor of the juices into consideration in the comparison of vitamin C content.

Repeat the experiment with more samples from the same types of fruits and vegetables.

Explain the difference in effects of vitamin C solution and boiled vitamin C solution on DCPIP solution. Boiling destroys the reducing property of vitamin C. Thus, only the vitamin C solution that has not been boiled can reduce the blue DCPIP solution and decolourize it.

Do the amounts of the samples used in the food tests need to be measured very accurately? No. This is because these food tests are qualitative tests for showing the presence of certain food substances. They are not quantitative tests.

Do you need to measure the volume of DCPIP solution accurately in this experiment? Explain. Yes. Accurate measurement is necessary because a comparison of vitamin C content in different fruits and vegetables is needed.

What are the controlled variables in this experiment? Amount of each type of fruits / vegetables used to extract the juices, volume and concentration of DCPIP solution used with each sample, temperature of sample and DCPIP solution, etc.

Why this method is not reliable? This method is less reliable because the size of drops could be highly variable in volume

What is the risk of this experiment? The DCPIP solution could stain clothes and skin This could be minimized by using rubber gloves or wearing a laboratory coat/ using a pipette to transfer DCPIP

Core Practical Three --- Investigate the membrane structure --- temperature Procedure: 1. Use a cork borer to cut cylinders of fresh beetroot tissue. 2. Place on a tile and cut into 3 mm wide discs which therefore have equal dimensions 3. Place all the discs in a small beaker and wash under a running tap for at least 5 minutes and dried using absorbent tissue 4. Mean while, label the test tubes and use a graduated pipette to add 6 cm3 cold water to each. 5. Prepare a water bath using a large beaker, tripod and gauze and Bunsen burner 6. Heat gently to 30C and remove the Bunsen burner 7. Gently pick 6 beetroot discs with a forcep, one by one 8. Place the discs in the water bath for exactly 1 minutes. Then drop them into the test tube labeled 30C. 9. Leave the discs in the test tube for at least 20 minutes 10. Repeat the procedure for the other tubes 11. Shake the tubes. 12. Use a colorimeter to compare the colours of each liquid. Why we better use discs cut from the same beetroot. So that the genetic variation, storage conditions and growth conditions will not influence the result. If we use different beetroots, they may have different concentration of pigment, and the stability of cell membrane may vary due to different storage conditions or age of the samples Suggest why it was necessary to rinse the beetroot discs before they were added to the distilled water. To remove cell debris and red pigment from the disc surface.

If we dont rinse the beetroot discs before adding, why there are some red coloration in the tube containing only water/ Cells damaged by cutting up of pieces As a result pigment could leak out of vacuoles and outside the cells

Why beetroots is used Because their cells contain a water soluble red pigment in their vacuoles, which cannot pass through membranes.

Core Practical Three --- Investigate the membrane structure --- Alcohol Suggest an explanation for why increasing ethanol concentration will increase the intensity of the absorbance of the solution. Ethanol is an organic solvent, thus lipids can dissolve in alcohol the pigment is contained within the vacuoles of the cell and therefore would only be able to leak out of the beetroot into the water if the integrity of the vacuole membrane and the cell membrane had been disrupted. Ethanol dissolved the phospholipids in the cell membrane and disrupts the phospholipid bilayer. This makes the membrane freely permeable to the red pigment which can rapidly diffuse out of the cell. Moreover, the orientation of phospholipids depends on water around it, Increase in ethanol cause the solution to be less polar

Core Practical four --- Investigate enzyme activities --- catalase and H2O2

Controlled variables: SA/V ratio of the meat: cut meat into cubes of equal length breath and height by using a very sharp scalpel and rule Protein content: use Meat from the same source or animal Temperature: by using a thermostatic water bath. pH of the solution should be stable: using a buffer solution

Why initial rate of reaction falls off sharply after some time Concentration higher than __%, the graph reach plateau It is because the concentration of the substrate in the reaction mixture has fallen. As less substrate is made available, the rate of reaction decreases. Substrate concentration become limiting factor

Core practical five --- investigate cell division How to prepare a root tip squash so that mitosis can be studied 1. 2. 3. 4. 5. 6. 7. 8. The tip of a root (5mm only) cut off and retained Tip transferred to a watch glass which has 3 drops of conc. HCl and 30 drops of acetic orcein stain Gently heated for 3-5 minutes using a steam bath. If excess evaporation occurs, more stain added Tissues transferred to a microscope slide and root tip cells gently teased apart with mounted needle. Additional drops of stain added followed by placing a cover slip slowly to the slide Squash the tissue by applying gentle pressure by thumb pressure on the top of the cover slip Observe under high power magnification to observe the chromosomes and cells in different stages of the cell cycle.

Name a suitable part of a plant to use. Give a reason for your answer Root tip, shoot tip or meristematic tissue Actively Dividing cells are present It is site of cell division Suitable stain for this experiment Acetocarmine, Schiffs reagent, lactoproprionic acid

Why we need to treat the root tissue with dil HCL for 4-5 minutes: This softens the tissue and break middle lamella and allows easy squashing

There are various risks associated with the production of a root tip squash. Suggest two risks and the precaution you would take to minimize each risk Cut Acid Heat Stain Coverslip How to avoid contact with the acid: Use a teat pipette to transfer the acid into a watch glass

Why we need to place a cover slop slowly To ensure no bubbles are produced

Why do we need to squash the tissue at the end of the process? Squashing opens up cells so that chromosomes become visible Flatten the cells and disperse them and get a single layer of cell so they can be observed under the microscope.

Why do we need to heat the slide gently? Heating makes the stain darker so that chromosomes are more visible.

Core practical six --- demonstrate totipotency using plant tissue culture techniques e.g. Explain how you could use a plant tissue culture technique to show tissue A or tissue B is totipotent

Procedure ( checked)

1. 2. 3. 4. 5. 6. 7. 8.

All apparatus must be sterilized by autoclaving at 121C for 15 minutes at 103kPa Wipe the bench with 70% ethanol before and after work Small piece of tissue is removed from a plant Place on a sterile agar which has certain growth hormones added and covered by a clean plastic film Leave for some time Cells divide by mitosis to form a cluster of cells which is a mass of unspecialized cell called callus tissue Divide cluster and then transfer these callus tissue to different culture medium containing nutrients Add different growth regulators to above the agar which stimulate plant cells to differentiate into roots, stem and leaves 9. Ref to control variable What is the advantages of tissue culture techniques for the vegetative propagation of plants over other methods Growth is very rapid Growth is independent of seasons The technique can be applied to a number f species that are otherwise difficult to propagate Apical meristems in infected plants often remain virus-free. Their use for tissue culture thus permit the elimination of viruses from infected 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 commercially

Give two advantages of producing plants using this method rather than from seeds Grow more rapidly No genetic variation, so desirable characters can be preserved

Why is a viral infection more likely to destroy a complete batch of plants grown by plant tissue culture than a batch of plants grown from seeds No genetic variation in plants produced by tissue culture so if virus destroy one plant, all other will likely to be destroyed In contrast, in plants produced by seed there will be genetic variation so some plants may have features that help to resist the viral attack

Core practical seven determine the tensile strength of plant fibres practically

What is tensile strength? The maximum stress caused by a pulling force that a material can stand without failing

Suggest how you could carry out a practical investigation to compare the tensile strength of sisal and nylon fibres 1. 2. 3. 4. 5. 6. 7. 8. 9. Fibres of same diameter and same initial length are used Connect fibres between 2 clamp stands Gradually add weight in the middle Increase the weight in suitable increments. Initially add larger increments and as the fibre begins to stretch then add smaller weights to get a more accurate measurement Keep adding weights till fibres breaks Repeated readings taken at each weight using same fibre Repeat above procedures using different fibre Reference to a safety procedure: goggles in case fibre snaps, precaution against falling weights Reference to control of temperature, humidity.

Precaution: Take care to add the weights gently and do not drop the weights onto the holder

Core practical seven investigate plant mineral deficiency practically

Core practical nine --- investigate effect of garlic on bacterial growth Procedure: Filter paper soaked in extract placed on agar plate With bacterial lawn Reference to sterile or aseptic approach e.g. appropriate reference to sealing Incubate at 25C but not 37C An appropriate time for incubation e.g. 24hours Clear area shows no bacteria Reference to suitable control

The effectiveness of the extract can be estimated by measuring the size of the clear zone. Suggest an accurate method for finding the size of the clear zone. Use graph paper tracing Count the number of squares covered by the clear zone and then subtract area of well / paper The number of squares is equal to the area of the clear zone in mm2

Count only squares that are covered more than half

The clear zone around some disc is not a circle. Suggest how you would calculate the mean diameter of this clear zone Record several measurements Divide by number of measurements to obtain mean

How would you determine the minimum strength of tea tree oil that would be as effective as the 100% tea tree oil 3 or more dilutions of tea tree oil from 50% downwards Look for min strength when diameter is same as original strength One other named variable kept constant

Suggest one reason why it was good safety practice to incubate the Petri dish at 25C rather than at 37C 37C is human temperature This temp allows growth of pathogenic bacteria because this temp encourages more rapid growth and reproduction

Suggest what is meant by the phrase suitably-prepared Petri dish in this experiment Agar Bacteria need to be distributed Single bacterial strain Appropriate microbiological technique employed , e.g. aseptic or sterile plates

What is control in this experiment and what is its function To allow a comparison with the other discs to show that any difference between the discs is due to the treatment given to those discs

Explain why clear zones are found around disc with teatree oil Tea tree oil diffused out of the disc Killed the bacteria

Core practical scientific reading

Discuss one economic implication of modern drug trialing, compared with Witherings method More expensive Long time Legal costs Equipment qualified

Identify one similarity and one difference, other than economic factors, between Witherings drug trial and a modern drug trial. In each case, give an explanation for your answer. Similarities: Both isolated a possible drug or treatment testing on people small to start with and scale up later Careful recording of results and publication Finding effective dosage Similar response to safety issues Monitoring patients Need for replication

Difference: Animal tests Large scale synthesis Placebo, double blind trials Sample size

Write a risk assessment for the practical work carried out. This should include suggestions of the risks and how to minimize them. Breathing problems --- wearing mask Possible skin reaction to enzyme: wear gloves, wash with copious water Getting enzyme into blood through cut: wear gloves High level of alkali damages skin or eyes: glove and goggles

Comment on the reliability

Wide range / high variability of data. Greater reliability for data at 1% 4% and 5% Outliers / anomalies

Suggest conclusions that the student could make about efficiency and cost effectiveness when using this enzyme in a biological washing powder. Efficiency: Best concentration to use is at__. No improvement above this concentration Cost effectiveness: Enzyme costs money (1M), reduced cost effectiveness at higher concentration

Suggest how you would investigate the validity of these sources of information Check for any bias of sponsor, e.g. funding source Check for credentials of contributor Cross check with another source