Core Practical 3 Biology

Describe what information the cell counts give about each stage of mitosis. CP6

The cell counts show the realtive duration of each stage in the cell cycle. The longer a phase, the more cells are likely to be going through that phase at any point in time.

The cellulose cell walls of plant cells are permeable whereas the cell membranes of plant cells are partially permeable. Explain the meaning of the terms permeable and partially permeable. CP3

Permeable allows any type/size of molecule to pass through. Partially permeable: Only allows some molecules of a certain size/type to pass through

CP1 Procedure (Benedicts Reagent)

Plan how you will use the stock 2% glucose solution to make the following five concentrations of glucose solution: 2%, 1%, 0.5%, 1.5% and 0.25%. Use the waterproof pen to label six test tubes and five small beakers with the different glucose concentrations they will contain. Label the sixth test tube 'fruit juice'. Use the syringes, the distilled water and the 2% glucose solution to create 5 cm3 of each solution in the corresponding labelled beaker. Use a clean syringe to add 2 cm3 of Benedict's reagent to each of the six labelled test tubes. Using a clean syringe each time, add 1 cm3 of each glucose solution to the corresponding labelled test tubes. Add 1 cm3 of fruit juice to the last labelled test tube. Give each tube a gentle shake to ensure the contents are mixed. Place all six test tubes in the water bath and start the timer. After 2 minutes, use tongs to remove the test tubes from the water bath and place them in the test tube rack. Observe the tubes and record the results in a suitable table.

CP1 Procedure (Iodine Solution)

Plan how you will use the stock 2% starch solution to make the following five concentrations of starch solution: 2%, 1%, 0.5%, 0.2% and 0.1% Use the waterproof pen to label five test tubes and five small beakers with the different starch concentrations they will contain. Label the sixth test tube 'unknown'. Use the syringes, the distilled water and the 2% starch solution to create 5 cm 3 of each solution in the corresponding labelled beaker. Use a clean syringe to add 0.5 cm 3 of iodine to each of the six labelled test tubes. Add a further 10 cm 3 of distilled water to each tube. Using a clean syringe each time, add 5 cm 3 of each starch solution to the corresponding labelled test tube. Add 5 cm 3 of the 'unknown' starch solution to the sixth test tube. Compare the colour produced in this tube with the colour of the diluted test tubes. Record your results in a suitable table.

Core Practical 3 - Effect on temperature

Prepare eight water baths pre-set to a range of temperatures between 0 and 70 °C. Take eight test tubes and label each one with the temperature of one of the water baths. Use a syringe to add 10 cm 3 of distilled water to each test tube. Place each tube in the water bath set to the corresponding temperature and leave for 5 minutes. Check the temperature of each bath using a thermometer. The temperatures are unlikely to be exactly right, so record the actual temperatures. Use a cork borer to cut eight beetroot cylinders (see figure A). Use a knife, ruler and white tile to trim them all to the same length (1 cm is sufficient). Wash the cylinders thoroughly with water until the water runs clear, then gently pat dry with a paper towel. Add one beetroot cylinder to each test tube and leave in the water bath for 15 minutes. Shake the tubes once. Then, working quickly and carefully, use forceps to remove the cylinder from each tube. Discard the cylinders but keep the supernatant liquid (the clear liquid above the solid). It may be easier to decant this liquid into clean test tubes. Set the colorimeter to a blue/green filter and percentage transmission. Zero the colorimeter using a blank cuvette filled with distilled water. Transfer liquid from each test tube in turn into a colorimeter cuvette, place in the colorimeter and take the percentage transmission reading. Record your results in a suitable table.

Describe how the area of a zone of inhibition could be determined

Measure {diameter / radius} of zone Apply area formula (HAVE TO INCLUDE FORMULA)

Describe the role of mitosis in the life of an organism. CP6

Mitosis produces identical daughter cells for growth, replacement and repair.

What type of scale would you use when plotting these concentrations on a graph? CP4

A logarithmic scale

Explain why the plates should be incubated at a temperature lower than 30 °C. CP9

Temperatures above 30C are closer to human body temperature, so there is a risk of incubating human pthogens which could infect you.

Suggest why the tubes were placed in the water baths for 5 minutes before the cylinders were added. CP3

The temperature must be equilibrated to ensure the tubes contain water at the correct temperature before the experiment begins. This allows confidence that the effect of the correct temperature is being assessed.

Describe how the companion cells in phloem contribute to translocation. CP7

Translocation in the phloem happens through tubes connected in chains called the seive tube element. Seive tube element is next to the companion cell. The companion cells are connected to the seive tube element with plasmodesmata, which allows materials to flow between cells. The seive tube elements are alive, yet they lack organelles such as nuclei/ribosomes. Organelles of the companion cells carry out all of the metabolic functions of the sieve-tube element. Without the companion cells, translocation would not be possible because the sieve tube element would die.

Explain briefly how cells can be measured using a microscope. CP5

Use a stage micrometer and eyepiece graticule. Calibrate the eyepiece graticule with the objective lens that will be used. Do this by measuring the objective eyepiece scale against the scale of the stage micrometer. Measure the cell using the eyepiece scale and convert into length units (um)

Describe how the procedure shown in the flow chart could be used to investigate the effect of pH on the mitotic index.

Use range of pH solutions (in which plants) grown (several) root tips (from each pH) Take cells from same part of root tips Count number of cells undergoing mitosis and total number of cells In several fields of view

Suggest one way in which this procedure could be modified to provide more accurate data. CP8

Use smaller masses, so the mass at which the string breaks is closer to the true value.

Suggest one reason why syringes were used in this investigation rather than burettes. CP2

Using very small very volumes of solution requires a more precise piece of apparatus than a burette. Syringes are easier to control, allowing smaller volumes to be added. Ease of use, and relatively low cost.

`Explain why vitamin C is described as an antioxidant. CP2

Vitamin C/absorbic acid readily loses electrons/becomes oxidised and this prevents other cellular components/chemicals becoming oxidised.

Formula for concentration of vitamin C in fruit juice. CP2

Volume of standard solution/volume of fruit juice x concentration of standardised solution

State the dependent variable in the investigation of testing antimicrobial properties of substances

AREA of zone of inhibition

To calculate the tensile strength of the fibre, the cross-sectional area has to be determined. Devise a method to determine the cross-sectional area of a fibre, using the following equipment: a sharp blade a microscope a microscope slide and coverslip an eyepiece graticule a stage micrometer

(transverse) section/layer/slice of the fibre is cut Ensure section is flat Graticule calibrated (with stage micrometer) Diameter measured/found and converted to measurement with calibration data Area calculated using circle equation

Explain how you could use serial dilution to make a range of concentrations from a stock solution of 10% fructose.

1 ml from stock solution into 9 ml of distilled water makes a 1 in 10/x10 dilution of 1% so 1 ml of that in 9 ml of water gives 0.1%. 5 ml of stock solution and 5 ml of distilled water = 5% solution . 5 ml of that and 5 ml of water = 2.5% solution. (5 in 10 x 5) 9 ml stock sol and 1 ml water = 9% solution 8 ml stock and 2 ml water = 8% solution

A cell from epithelial tissue in the trachea was found to be approximately cylindrical in shape. The cell had a length of 20 µm and a diameter of 4 µm. The volume (v) of a cylinder may be calculated from v = πr 2 h. Calculate the volume of the cell. Show your working. CP5

2x2 = 4 V = 3.14 x 4 x 20 = 251.36 um3

Under a high-power objective lens, 300 µm on a stage micrometer was found to be equivalent to 87 eyepiece units. What is the size of each eyepiece unit in µm? Using the same objective, the diameter of a red blood cell was measured as 2.5 eyepiece units. What is the actual diameter of the red blood cell? CP5

300/87 = 3.45/3.5 2.5 x 3.4 = 8.5

Explain why a scratched cuvette should not be used. CP1

A scratched surfacce would absorb some light/interfere with absorption.

Explain the term 'serial dilution'. CP4

A stepwise dilution of a solution. The dilution factor at each step is constant/the concentration decreases by the same factor with each step.

Explain what is meant by a 'reducing sugar'. CP1

A sugar that has a free aldehyde or ketone group in its molecule that can reduce (donate electrons to) Benedicts reagent, changing the copper (II) ions.

If the surface of the cuvette is scratched, this can result in a greater absorbance of light. If the cuvette used for the reaction was scratched (but the reference cuvette was not), would this give a random or a systematic error? Explain your answer. CP4

A systematic error, because it would cause absorbance readings to be higher than the true value for every measurement.

____________ fruit juices will not completely decolorise the DCPIP; instead, the solution will turn pink. CP2

Acidic

How should data be recorded in a table?

All numbers should be recorded to the same degree of precision. They should all be to the same number of decimal places. The data from the replicates should be recorded and a mean values calculated and shown.

Sterile (aseptic) technique is important in microbiology. Explain why this is the case even in this activity, where the bacterial species have been selected because they are harmless to humans. CP9

Aseptic techniques are vital in microbiology to ensure that there is no contamination of cultures by microorganisms from the environment, and that people and the environment are not contaminated by the microorganisms being handled. Even cultures thought to be low risk should be treated with caution because: Bacteria may mutate to form pathogenic strains Our knowledge of hazards may be incomplete The culture may have become contaminated.

Core Practical 4 Procedure - Enzyme concentration

Check your dilution calculations, then make up the solutions as planned. Place 2 cm 3 of trypsin solution and 2 cm 3 of distilled water in a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero. Measure 2 cm 3 of milk suspension into a second cuvette. Add 2 cm 3 of your first trypsin solution to the milk in the cuvette. Working quickly, mix the trypsin solution and the milk, then place the cuvette into the colorimeter and start the datalogger. Measure absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance. Rinse the cuvette with distilled water and repeat steps 3-5 for each of the other four concentrations. Remember to use the reference cuvette to zero the colorimeter before each new set of readings.

Explain your results in detail in terms of what is happening to the beetroot membrane. CP3

At lower temperatures, the tonoplast and plasmalemma are intact. Betalain molecules are too large to pass through these membranes easily, so light transmission remains high. (If cells freeze, damage to the membranes may cause pigment release) The higher temperature, the greater the kinetic energy and the faster the movement and diffusion of pigment molecules. Greater kinetic energy also causes phospholipids of the membrane to become more fluid and bonds between the fatty acid tails can begin to separate, allowing some pigment molecules to pass through. As more pigment passes through the membrane, less light can pass through the liquid (percentage transmission decreases).The point of sudden increase in percentage transmission occurs when proteins in the membrane begin to lose their tertiary structure. At higher temperatures, the protein molecules in the membrane become completely denatured and the membrane developed gaps through which the pigment can flood out, so the change in transmission levels out as the concentration of pigment is the same inside and outside the cells. Change in alcohol concentration affects integrity of the phospholipid bilayer. Because phospholipids are soluble in alcohol, the membrane loses the ability to orientate towards and away from water. The loss of hydrophobic and hydrophilic interactions results in holes forming in the membrane which allow the pigment out of the cells. The increase in the pigment outside of the cells reduces the ability of light to penetrate the solution, therefore decreasing transmission.

In an investigation, the tensile strength of fibres from five plant species was determined. The breaking force was measured by finding the mass in grams that would break the fibre. Describe a valid method that could be used to compare the breaking force of these fibres.

Attached to a retort stand/hanging from a clamp Gradual addition of masses Constant variable: Relative humidity, temperature, length of fiber Replicate the experiment/measurements Calculate the mean/SD

Tea tree oil is poisonous if taken internally. It contains many compounds, including terpenes, which produce the characteristic odour. These are unsaturated hydrocarbons containing a cyclic molecule with the formula C 10 H 16 .The antibacterial ingredient in garlic is allicin, produced when alliin in chopped garlic is acted upon by the enzyme alliinase. Its formula is C 6 H 10 OS 2 . Discuss the problems of using data from the disc diffusion method to promote these products as effective antibacterials. CP9

Because a substance that works in a Petri disc does not mean it will work in the same way on the skin. Garlic is not poisonous but when ingested may be digested by enzymes and the active ingredient may not remain intact/active. Tea tree oil is poisonous so cannot be used internally.

Ideally, you would repeat the procedure for each factor several times. Explain why it is important to measure the initial rate of the reaction rather than an average rate over a longer time period. CP4

Because the reaction is rapid and the milk (substrate) concentration quickly declines. The rate slows as the subtrate is used up. Therefore, it is only possible to make valid comparisons at the start of the reaction, when controlled variables such as substrate concentration are the same for all levels of the independent variable.

Plant fibres consist of the polymer cellulose. Name the monomers that make up cellulose.

Beta glucose

Describe a method for comparing the size of the xylem tissue in the root and stem samples. CP7

By using a micrometer slide and an eyepiece graticule. The graticule is calibrated using the micrometer slide. The size of the xylem tissue can then be calculated and compared.

Suggest improvements to the method that was used to describe the size of cells in a tissue. CP5

Calculate a mean. Measure more cells to estimate a more reliable mean. The volume of the cells would be a better descriptor of size than the linear dimensions.

Core Practical 8 Procedure

Carefully remove nine fibrous strings from the celery stalks, using the knife. Use a white tile as a cutting surface. Carefully inspect the strings to ensure there are no breaks or cuts. Each string should have a constant diameter along its whole length. Cut the strings so you have three that are 10 cm long, three 15 cm long and three 20 cm long. Clamp one of the 10 cm strings between two retort stands. Ensure it is held securely. Place the cushioning underneath the string. Ensure the tray is kept beneath the masses, so they do not strike the bench when the string breaks. Add masses 10 g at a time until the string breaks (see figure A). Record the mass in the results table. Repeat steps 4-6 with each of the other strings.

Explain how high temperatures and ethanol damage cell membranes. Make reference to the fluid mosaic model in your answer. CP3

Cell membranes consist of proteins floating in a phospholipid bilayer. High temperatures can denature proteins by disrupting the bonds that hold their tertiary structure in place. High temperatures increase the fluidity of the phospholipid bilayer and may melt the lipids, causing gaps in the bilayer. Ethanol at high enough concentrations can denature some proteins by altering hydrophobic interactions. Alcohol disrupts the phospholipid bilayer and this is more severe in plant cell membranes as they lack cholesterol which helps stabilise the membrane.

Core Practical 4 Procedure - Substrate concentration

Check your dilution calculations, then make up the solutions as planned. Place 2 cm 3 of 1% trypsin solution and 2 cm 3 of distilled water in a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero. Measure 2 cm 3 of the 0.2% milk suspension into a second cuvette. Add 2 cm 3 of trypsin solution to the milk in the cuvette. Working quickly, mix the trypsin solution and the milk, place the solution into the colorimeter and start the datalogger. Measure absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance. Rinse the cuvette with distilled water and repeat steps 3-5 for each of the other four milk concentrations. Remember to use the reference cuvette to zero the colorimeter before each new set of readings.

Core Practical 5 - Calibration

First, you will need to calibrate the eyepiece graticule (see figure A). Place a micrometer slide on the stage of the microscope and focus on the micrometer scale, using the low-power objective. The smallest division of the micrometer scale is usually 100 µm. Move the slide and rotate the eyepiece to align the scales of the eyepiece graticule and the stage micrometer in the field of view. Count the number of divisions (eyepiece units or epu) on the eyepiece graticule that are equivalent to a known length on the micrometer slide and work out the length of one eyepiece unit. (For example, if 100 µm is equivalent to 2 epu, then 1 epu 100/ 50 2=μm at this magnification.) Repeat steps 1-3 with the medium- and high-power objectives.

Core Practical 7 Procedure

Collect a piece of plant stem. Add a few drops of water to the centre of the white tile and wet the razor to reduce friction. Hold the plant stem firmly, keeping your fingers away from the edge of the razor. Cut several transverse sections (across the stem), keeping them as thin as possible. Incomplete thin sections may sometimes be better than thicker complete ones. Use a brush to transfer the sections to water in a watch glass. Select the thinnest section and place it on a slide. Add a drop of water. Remove excess water by carefully touching the edge with absorbent paper. Wearing gloves and eye protection, add two drops of toluidine blue O stain and leave for 2-4 minutes. Then add a coverslip and gently remove excess stain with a paper towel. Turn the objective lens to low power. Examine the slide under the microscope. To do this, bring the lens as close to the slide as possible while watching it from the side. Then, looking through the eyepiece, focus using the coarse focusing knob, moving the lens away from the stage. This avoids damage to the slide and lens. Use the fine focus until a clear view of the section is established. Still on low or medium power, draw and annotate a simple outline plan of the section. Show the arrangement of tissues within the stem but do not include any cell details. Use figure A to help you identify the different tissues and label them. Repeat steps 1-5 using a piece of root. To make a leaf cross section, place the leaf between two thin pieces of polystyrene. Gently stroke the razor blade across the top of the leaf and polystyrene to shave off four or five thin sections.

The vitamin C content of food and drink can be measured using DCPIP. The concentration of vitamin C in a solution can be determined by finding the volume of the solution that causes DCPIP to change colour. The higher the concentration of vitamin C in the solution, the smaller the volume needed to cause the colour change. Explain what happens to DCPIP in the presence of vitamin C.

Color change from blue to colorless because DCPIP is reduced

Explain how the companion cells are adapted to their function. CP7

Companion cells are able to carry out translocation because they usually have a large number of ribosomes and mitochondria than a typical plant cell. This makes the companion cells more metabolically active than a 'typical' plant cell and it is therefore able to carry out translocation. The cytoplasm of the companion cell is connected to the sieve tube element by plasmodesmata, allowing materials to flow between the companion cell and the sieve tube element.

How could you evaluate the accuracy of your experimental value for the concentration of glucose in the fruit juice? CP1

Compare with the reference values on the nutrition labels which show the glucose content Calculate the concentration of glucose in the juice and then compare with the estimate generated experimentally

Name a variable that you controlled in the iodine investigation, explain how you controlled it and describe the possible effect on the result if it had not been controlled. CP1

Concentration of Iodine was controlled by using the same solution and diluting by the same amount each time. Failure to control iodone concentration would result in over/underestimation of starch concentration.

Starch is a polymer of glucose. Name the type of reaction involved when two glucose molecules join together by a 1,4 glycosidic bond.

Condensation

Devise an experiment, which will produce valid data, to investigate the deficiency of a names inorganic ion on the growth of a plant.

Control of {species/ type/ age} of plant Control of temperature / soil pH /light intensity/ time of growth Complete solution described and solution without named ion Measurement of dependent variable described (Height, Mass, Number, Size of leaves) Replication to measure variability/find SD

Describe the direction of movement of electrons during the reaction between DCPIP and vitamin C. CP2

DCPIP gains electrons from asorbic acid.

Core Practical 9 Procedure

Day 1 Wash your hands with soap and water and disinfect your bench area, leaving it to soak for 10 minutes before wiping it down. Place a piece of garlic in the mortar and grind it into a paste with the pestle. Add 10 cm 3 of alcohol to the garlic paste. Use the forceps to pick up a paper disc and place it in the mortar to soak up the garlic and alcohol solution. Use the forceps to place the disc on the sterile agar plate to dry. Use a marker pen to mark four quarters on the bottom of the agar plate that has been seeded with bacteria; label one garlic, one mint and two control. Open the lid of the Petri dish containing the bacteria-seeded agar. Open it away from yourself and only open it slightly. Use the forceps to place the dry disc in the centre of the quarter labelled garlic. Close the Petri dish. Clean the pestle and mortar, then repeat steps 2-7 with the mint. Add a small amount of alcohol to the small beaker. Using clean forceps, add two paper discs to the beaker. Remove them and place them on the sterile agar plate dish to dry. Open the lid of the seeded Petri dish, making sure you open it away from yourself and only open it slightly. Add one disc to each of the quarters labelled control. Place one small piece of tape on each side of the Petri dish lid to hold it down. Do not tape it all the way around. Invert the plate and incubate at a temperature no higher than 30 °C for 24 hours; alternatively, leave at room temperature for 48 hours. Day 2 Use a ruler to measure the diameter of the clear zone around each disc. (It is also possible to measure the area of the clear zone using squared paper.) Record your results in a suitable table. Clear away all the equipment you have used. Petri dishes should be returned for sterilisation. Wash your hands and disinfect surfaces before leaving the laboratory.

Explain why the mitotic index is affected by pH as shown by the graph (Optimum in graph is 6.2)

Enzymes are involved that are protein. 6.2 is the optimum pH (for these enzymes). At pH {below optimum/ above optimum} the active site is not the right shape/ enzyme denatures/ bonds holding shape change Enzyme substrate complexes do not form

Describe the control disc used in the investigation

Filter paper discs qualified (Controlled variable, same size, thickness, paper type) Soaked in solvent/water

Describe how you would carry out a serial dilution of the 1% trypsin stock solution to make concentrations of 0.1%, 0.01% and 0.001%. You may use a labelled diagram to help with your explanation. CP4

Mix I cm of the 1% stock with 9 cm of distilled water. This produces a 10-fold dilution/0.1% solution. Then mix 1 cm of the 0.1% solution with 9 cm of distilled water to make the 0.01% solution. Then mix 1 cm of 0.01% solution with 9 cm of distilled water to make the 0.001% solution. Suitable equipment for measuring volumes - pipette

Mitotic index

Number of cells containing visible chromosomes/total cells in field view

Suggest one variable that would normally be controlled in an enzyme-catalysed reaction but which has not specifically been controlled in your investigation. Explain why this variable would usually be controlled carefully and suggest how this could be done. CP4

For a pH investigation, the rate of reaction of enzymes varies with pH due to changes in the shape of the active site. An enzyme has the highest rate of reaction at its optimum pH. A buffer might be used to maintain pH at a suitable level. For temperature, substrate concentration and enzyme concentration investigations. Temperature because the rate if reaction of enzymes varies with temperature. As temperature increases, particles gain more energy and more collisions take place between enzyme and substrate particles. Enzymes have an optimum temperature at which the rate of reaction is at its peak. Above that temperature, enzymes will begin to denatue, changing the shapes of the active site and preventing further analysis. A water bath and thermometer could be used to maintain a suitable temperature.

Explain why the Benedict's test is not a suitable test for measuring the total sugar content of fruit juice. CP1

Fruit juices contain sucrose, which is a non-reducing sugar and does not react with Benedict's reagent.

Beetroot cells contain a red pigment called betalain. This pigment does not leak out of the cells, unless their membranes are damaged. A student investigated the effect of detergent on beetroot cell membranes. Discs of tissue were cut from a beetroot. Any betalain on the outside of the discs was removed. One disc was placed into each of five test tubes, containing 0.2% detergent solution. The five tubes were left for 30 minutes at 20°C.The discs were then removed carefully. Betalain had leaked from the discs and formed a red solution in each test tube. Light was shone through this solution. The percentage of the light transmitted through the solution was recorded.The same procedure was repeated using detergent concentrations of 0.0%, 0.5%, 1.0% and 2.0%. Explain why the discs were removed carefully.

In order to avoid (physical/more) damage to the discs/membranes/cells which would cause more pigment release causing the results to be invalid

What were the independent and dependent variables in this investigation? CP2

Independent variable was type of fruit juice used. Dependent variable was volume of juice added to decolorise DCPIP.

What were the independent and dependent variables in this investigation? CP4

Independent: Temperature, pH, trypsin concentration, substrate concentration Dependent: Rate of reaction in absorbance s-1

Suggest why, when adding ascorbic acid to DCPIP, the tube should be shaken gently and not vigorously with the addition of each drop of DCPIP. CP2

It becomes colorless when reduced and that is the end point. Shaking too vigorously would introduce oxygen to the DCPIP/reoxidise it and the blue color would return/would make it difficult to find the true end point.

Core Practical 2 Procedure

It is possible to determine the concentration of vitamin C in a solution by using dichlorophenolindophenol (DCPIP). Vitamin C is an antioxidant, so it reduces the DCPIP causing a colour change. By using a solution of vitamin C with a known concentration, it is possible to calculate the concentration of vitamin C in other solutions, for example, in fruit juices (see figure A). Use the 5 cm3 syringe to draw up 5 cm3 of 1% DCPIP. Shake the syringe to expel any air bubbles. Add 1 cm3 of DCPIP to a test tube. Use a clean 5 cm3 syringe to draw up 5 cm3 of the 1% vitamin C solution. Add the vitamin C solution to the test tube containing the DCPIP, one drop at a time. After each drop, shake the test tube slightly to ensure the solutions have mixed. Continue to add vitamin C solution until the blue colour of the DCPIP disappears. Record the volume of vitamin C solution added. You can find this volume by subtracting the value on the syringe from the original 5 cm3 in the syringe. Repeat steps 2-6 twice more and calculate a mean value for the volume of 1% vitamin C solution needed to decolorise 1 cm3 of DCPIP. Add 1 cm3 of DCPIP to a clean test tube. Use a clean syringe to draw up 5 cm3 of a fruit juice. Add the fruit juice to the DCPIP one drop at a time. Record the volume of juice needed to cause the blue colour to disappear. Repeat steps 8 and 9 twice more, then calculate the volume of fruit juice needed to decolorise 1 cm3 of DCPIP. Repeat steps 8-10 with the other fruit juices.

Explain why it was important not to use too much stain when preparing the slide. CP7

It will be difficult to see anything using the microscope if too much stain is used. Any differences between scells will be hidden as the stain will absorb all the light.

Vitamin C is water soluble and cannot be stored within the body. Many mammals, including dogs, can make vitamin C in certain cells. Humans cannot synthesise vitamin C and have to obtain it from their diet. However, if they consume more than needed, the excess is filtered from the blood in the kidneys and passes out in the urine. Imagine that you are a scientist in a physiology research lab. Outline the design of an investigation to determine whether taking vitamin C supplements benefits people. CP2

Large sample of volunteers Matched age/gender/mass/lifestyle/diet Collect a day's urine and test for vitamin C to scertain base levels - are they already taking plenty of vitamin C or not enough? Divide into groups - one given no vitamin C supplement, one group given small supplment and one given a larger one. Could make it blind/double blind and give the first group a placebo. Make sure they stick to normal diet/stay in a lab so scientists control their diet. Collect urine again and test for vitamin C concentration (Extra being passed out?)

Suggest one way in which the results of the Benedict's reagent investigation could have been made more reliable. CP1

Larger quantities of each sample concentration could have been made so repeat readings could be taken (Results from the whole class could be collated to give an average estimate)

Suggest why light of wavelength 650 nm was used in the colorimeter. CP1

Light of 650 nm is red and will be absorbed by the blue/black solution

State the effect of maceration and pressing the slide preparation on the dividing cells. CP6

Maceration and pressing the preparation separates the cells in the meristem tissue into individual cells in a single layer. This makes it easier to see the chromosomes and to identify the stages of division.

Why was each titration completed three times to calculate a mean? CP2

Makes it easier to spot anamolies. Repeated readings also reduce the effect of errors.

By what process does water pass across cell surface membranes?

Osmosis

Core Practical 4 Procedure - Temperature

Prepare water baths for your chosen range of temperatures. Use thermometers to ensure each water bath stays at the correct temperature. Take five test tubes and add 2 cm 3 of 1% trypsin solution to each one. Label the tubes with the temperatures and place each one in the corresponding water bath. Take five clean test tubes and add 2 cm 3 of milk to each one. Label the tubes with the temperatures and place each one in the corresponding water bath. Leave the test tubes for 5 minutes so they reach the required temperature. Place 2 cm 3 of trypsin solution and 2 cm 3 of distilled water in a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero. Pour 2 cm 3 of milk suspension at the first temperature being tested into the second cuvette. Add 2 cm 3 of trypsin solution from the same water bath to the milk in the cuvette. Working quickly, mix the trypsin solution and the milk by shaking gently, then place the solution into the colorimeter and start the datalogger. Measure the absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance. Rinse the cuvette with distilled water and repeat steps 6-8 for each of the other four temperatures. Remember to use the reference cuvette to zero the colorimeter before each new set of readings.

Silk and wool are fibres obtained from animals. What is the nature of the polymers in these animal fibres?

Protein

Explain the difference between qualitative, semi-quantitative and quantitative tests. CP1

Qualitative tests tells you whether something is present or absent. A quantitative test enables you to determine exactly how much of a substance is present. A semi-quanitative test enables you to estimate roughly how much of a substance is present.

Explain how xylem is adapted to its function. CP7

Ring of lignin - Very strong and provides supportive structure for the plant, prevents the walls of xylem from collapsing under the hydrostatic pressure of transpiration pull, also waterproof, preventing any water from leaking out of the xylem Dead cells - Enables it to carry water through it, when it dies, it shrivels, causing the cell inside the cell wall to die, meaning it is hollow within the cell wall Pits - Helps the transfer of water from one tracheid to another and allows water to move from side to side even though there is waterproofing lignin Preforation plates - Cells can form a long, open, hollow tube. This allows water to move up in a continuous column: the water does not have to diffuse by osmosis from one cell to another.

Describe how you would investigate the effect of acetic acid on beetroot cell membranes. CP3

Same equipment and protocol as CP3 but use different concentrations of acetic acid. (Marks for: Hypothesis, euipment, procedure, IV and DV, control variables, how to deal with the data)

Core Practical 5 Procedure - pH

Select the buffer solutions for the pH values being investigated. Place 1 cm 3 of trypsin solution, 1 cm 3 of buffer solution and 2 cm 3 of distilled water into a cuvette. Use this as a reference cuvette to set the colorimeter absorbance to zero. Add 1 cm 3 of trypsin solution and 1 cm 3 of your first buffer solution to a cuvette. Measure 2 cm 3 of milk suspension into a second cuvette. Add the mixture made in step 2 to the milk in the cuvette. Working quickly, mix the solution and the milk by shaking gently, then place the solution into the colorimeter and start the datalogger. Measure absorbance immediately and then at 15-second intervals (more frequently if recording electronically) for 5 minutes, or until there is little change in absorbance. Rinse the cuvette with distilled water and repeat steps 2-5 for each of the other four pH values. Remember to use the reference cuvette to zero the colorimeter before each new set of readings.

Core Practical 3 - Effect on Alcohol

Take five test tubes and add 10 cm 3 of ethanol to each one. Use a different concentration of ethanol in each tube (distilled water can be used for a 0% concentration). Use a cork borer to cut five beetroot cylinders. Use a knife, ruler and white tile to trim them all to the same length (1 cm is sufficient). Wash the cylinders thoroughly with water until the water runs clear, then gently pat dry with a paper towel. Add one beetroot cylinder to each of the five tubes and leave for 15 minutes. Shake the tubes once. Then, working quickly and carefully, use forceps to remove the cylinder from each tube. Discard the cylinders but keep the supernatant liquid (the clear liquid above the solid). It may be easier to decant this liquid into clean test tubes. Practical activities have been safety checked but not trialled. Users may need to adapt the risk assessment information to local circumstances. This document may have been altered from the original. Set the colorimeter to a blue/green filter and percentage transmission. Zero the colorimeter using a blank cuvette filled with distilled water. Transfer liquid from each test tube in turn into a colorimeter cuvette, place in the colorimeter and take the percentage transmission reading. Record your results in a suitable table.

A length of fibre from a pumpkin stem broke when a mass of 300 g was suspended from it. The diameter of the fibre was 2 mm. Calculate the tensile strength of this fibre in N m −2 CP8

Tensile strength = Breaking force/cross sectional area Breaking force = 300 g = .3 kg = 2.94 N Diameter = 2mm = 0.002m so radius = .001 Cross sectional area = 3.142 x (.001)squared = 0.000003142 m2 (x) Tensile strength = 2.94/x = 935709 m-2

Suggest one further investigation that could be undertaken to determine the validity of the conclusions drawn from this investigation. CP8

Test fibers from other types of plant to see if the pattern shown by the celery strings is replicated. Use a greater range of lengths of celery string, to determine if the pattern continues.

Write a plan to investigate the tensile strength of banana skin. CP8

Testable hypothesis/prediction stated Mark lengths of 0.25/0.5/0.75/1.0/1.25/1.5 cm along an intact banana Cut the banana into sections, cutting at these points. Push an edible part out of the banana and discard. Hang a loop of banana skin on a clamp and secure it Hand slotted mass from the lower end of the loop until breaking point is reached Repeat three times to find mean Repeat with different lengths of banana skin loops Tabulate and graph data

How could these investigations have been adapted to produce quantitative results? CP1

The absorbance of the samples could have been measrued using a colorimeter.

Explain why the root tip is heated with acid. CP6

To break up the tissues into individual cells. The cellulose walls of the plant cells are held together by pectins that can be broken down by HCl treatments.

Tea tree oil is poisonous if taken internally. It contains many compounds, including terpenes, which produce the characteristic odour. These are unsaturated hydrocarbons containing a cyclic molecule with the formula C 10 H 16 .The antibacterial ingredient in garlic is allicin, produced when alliin in chopped garlic is acted upon by the enzyme alliinase. Its formula is C 6 H 10 OS 2 . Discuss the problems with using the disc diffusion method to compare the antibacterial properties of different compounds. CP9

The active ingredients have molecular sizes and so this affects their diffusion into the agar gel Tea tree oil may have many different ingredients Some of the chemicals may remain bound to the paper disc

Explain why a blank - a cuvette containing distilled water and dilute iodine/KI solution - is used to set the absorbance of the colorimeter to zero. CP1

The blank will be pale brown. If we set the absorbance to 0, this eliminated any absorbance due to just the iodine solution so we are measuring the absorbance due to the iodine/starch complex.

Explain the advantages of using serial dilutions. CP4

The dilutions cover a wide range (and are therefore good for pilot studies)

Explain why the blackcurrant juice was first decolorised.(by charcoal) CP2

The juice was decolorised to make it easy to spot the endpoint. (The point when the DCPIP just becomes decolorised). This point would be difficult to judge in colored juices.

Use the trend line of one of your graphs to describe the effect of temperature or alcohol concentration on the percentage transmission. CP3

The percentage transmission decreases as temperature rises. Initially there is a little increase, but at around 40-60C the percentage transmission decreases sharply. At higher temperatures, the rate decrease levels out. The percentage transmission decreases as the alcohol concentration increases. At low concentrations of alcohol (0-10%) there may be little noticable effect but as the concentration is increased the decrease in transmission should be proportional until leveling off between 60%-80%.

Explain why measuring the area of the clear zone around each disc is more accurate than measuring the diameter of the clear zone. CP9

The shape of the clear zone may be irregular, so the diameter may not represent an accurate measurement. Measuring the area of the clear zone enables a fair comparison to be made between discs.

Identify one variable in this investigation that was not controlled and describe how this may have affected the data obtained. CP8

The strings may have come from different parts of the stalk. Some parts of the stalk may have denser fibers, so the data may have provided higher or lower values. Storage conditions or age of the celery. The tissues may have been degraded or weaker, so they snapped at a lower value. The time between cutting the strings and using them may have allowed some strings to dry out. This may have caused them to be more brittle and to snap at a lower value.

List the variables that were controlled during the experiment and state how they were controlled. This could be done using a table. CP3

The volume of bathing or ethanol in each tube The surface area and volume of beetroot cylinders The equilibrium time in the temperature experiment Soaking time for cylinders The volume of coloured liquid in the cuvettes The colorimeter filter/wavelength used (blue/green)

Dietary vitamin C is an antioxidant that may be linked to the risk of cardiovascular disease (CVD). Explain what is meant by this statement.

There is a correlation (between vitamin C in the diet and CVD) (1) Effect of vitamin C on an aspect of CVD (Affects plaque formation/LDLs/ cholesterol / damage to endothelium/ atherosclerosis)

Suggest why a stain was used on the stem and root cells. CP7

Thin sections of stem and root do not absorb much light, so they are difficult to see using a light microscope. The stain absorbs more light, making it easier to see the structures. Not all of the cells are stained so differences can be seen.

Core Practical 6 Procedure

This first step may have been done for you. Fill a small bottle with 1 mol dm −3 hydrochloric acid and place it in a thermostatically controlled water bath set at 55 °C. Leave the bottle for 15 minutes to allow the acid to warm to the temperature of the water bath. Place a garlic clove in the top of the bottle so that the roots are submerged in the hydrochloric acid at 55 °C. Leave the roots in the acid for 5 minutes. After 5 minutes, take out the garlic clove and rinse the roots thoroughly in tap water. Use a pair of sharp scissors to cut off several root tips of 5-10 mm in length. Let them fall into a small vial of acetic orcein standing on a white tile. Use the scissors to make sure the root tips are immersed in the stain. Place a lid or laboratory stretch film over the vial. Lids should have a pin-prick hole, or be slightly loose if they are screw caps, to prevent the ejection of liquid during heating. Place the vial containing the root tips in acetic orcein in the 55 °C water bath for 5 minutes to intensify the staining. After 5 minutes, use forceps to remove the root tips from the vial and place them on a microscope slide. Add a drop of water to the root tips on the slide. Tease each root tip apart with needles (maceration), to spread out the cells a little. Cover with a coverslip. Replace the lid on the vial of stain and return it to the teacher as instructed. Wrap the slide in several layers of paper towel and press gently on the paper to squash the tissues. Take care not to twist the slide as you press down, or the coverslip will break. Examine under the microscope on low power to identify the area of dividing cells or meristem (see figure A). Position the cells in the centre of the field of view. Meristem cells are small and square, have no obvious vacuoles and are usually found in rows. Move to high power (×400). Identify as many stages of the cell cycle as you can in your field of view. Count the number of cells in each of the stages of mitosis, plus interphase, in the field of view. Record your results in a table. Draw and annotate one cell from each of the stages you have identified. Your drawings will be simple outlines of the cells and the groups of chromosomes in them; few other structures will be visible. Aim to show the relative sizes and positions of the chromosomes and the cell accurately. Annotate your drawings to describe what is happening.

State the purpose of staining specimens in light microscopy. CP7

To add contrast to the image To identify the prescence of chemical compounds of interest To locate/see the position of particular types of chemical component To differentiate between different types of chemicals/compounds/tissues

State the purpose of staining specimens in light microscopy. CP5

To add contrast to the image. To identify the prescence of chemical compounds of interest. To locatesee the position of particular types of chemical components. To differentiate between different types of chemicals/components/tissues.

Suggest why plates (in bacteria CP) were kept at 5C before they were incubated

To allow (chemicals in the extract)to diffuse into agar / to stop growth of bacteria

Suggest a reason for including the control discs in this investigation. CP9

To allow comparison and to check that the paper discs themselves were not responsible for the inhibition of growth. Control discs enable us to be sure that the only factor that was different was the independent variable.

Beetroot cells contain a red pigment called betalain. This pigment does not leak out of the cells, unless their membranes are damaged. A student investigated the effect of detergent on beetroot cell membranes. Discs of tissue were cut from a beetroot. Any betalain on the outside of the discs was removed. One disc was placed into each of five test tubes, containing 0.2% detergent solution. The five tubes were left for 30 minutes at 20°C.The discs were then removed carefully. Betalain had leaked from the discs and formed a red solution in each test tube. Light was shone through this solution. The percentage of the light transmitted through the solution was recorded.The same procedure was repeated using detergent concentrations of 0.0%, 0.5%, 1.0% and 2.0%. Explain why five discs were used for each concentration of detergent.

To calculate standard deviation / measure reliability / variability to identify anomalies

Give one reason why a coverslip was places over the root tips and squashed gently

To locate and then magnify the cells

Why were the beetroot cylinders washed with distilled water and dried before the experiment began? CP3

To remove excess surface pigment from the cut cells at the edge. This excess pigment would distort the transmission readings, giving inaccurate results.

Give one reason why root tips were places on a microscope slide and broken apart using mounted needles

To spread/separate the cells out

Give one reason why root tips would be placed in acetic orcein

To stain the CHROMOSOMES (Cells rejected on ms)

Core Practical 5 - Making observations

Wash your hands with soap and water. Take a cotton bud and gently rub it on the inside of your cheek, then rub the cotton bud in a small circle in the centre of a glass slide. Immediately place the cotton bud in a beaker of disinfectant solution. Add a few drops of methylene blue to the sample, then cover with a coverslip. Turn the objective lens to low power and examine the stained slide under the microscope. To do this, bring the lens as close to the slide as possible while watching it from the side of the instrument. Then, looking through the eyepiece, use the coarse focusing knob to focus, moving the lens away from the stage. This avoids damage to the slide or lens. Finally, use the fine focus until a clear view of the cells is established. Carefully sketch a few of the cells. Use figure B to help you identify the parts of the cell. Use the eyepiece graticule to measure a cell's diameter. Add a scale bar to your diagram. Add a title and include the magnification at which you made your observations. For example, with an eyepiece lens magnification of ×10 and an objective of ×10, the total magnification will be ×100. Remember, this is not the same as the magnification of the drawing. Now, turn the objective disc to the medium-power lens, and focus until the cells are clear and distinct. Identify as many details of the cells as you can. Finally, turn the objective disc to the high-power lens and focus using the fine-focusing knob only. Draw and label the detail of the cells as accurately as you can. Measure the length and breadth of two cells. Record these measurements in your diagram. Place the glass slide in the beaker of disinfectant solution.

Explain one safety precaution that must be taken when carrying out this procedure (Staining and measuring mitotic index in cells)

Wear gloves because HCl (or Acetic orcein) is corrosive

This method can be used to suggest that an extract has an antimicrobial effect. Other factors can affect the size of the zone of inhibition. State two factors, other than the antimicrobial effect, that could affect the area of the zone of inhibition in this investigation.

{solubility/ concentration/ volume} of extract/ solvent used Size of molecules in the extract Rate of diffusion of extract into agar