ducation m bridge A YEARS ss es al E Ca 25 on W king for ove or r i sm WITH rnat e n t In t e Cambridge International AS & A Level Biology Practical Skills Salma Siddiqui 9781510482869.indb 1 31/08/21 1:55 PM Cambridge International copyright material in this publication is reproduced under licence and remains the intellectual property of Cambridge Assessment International Education. Cambridge Assessment International Education bears no responsibility for the example answers to questions taken from its past question papers which are contained in this publication. These have been written by the authors. Some Practice questions in Chapters 4 and 7 have been written by the authors. In examinations, the way marks are awarded may be different. References to assessment and assessment preparation are the publisher’s interpretation of the syllabus requirements and may not fully reflect the approach of Cambridge Assessment International Education. Cambridge International recommends that teachers consider using a range of teaching and learning resources in preparing learners for assessment, based on their own professional judgement of their students’ needs. Third-party websites and resources referred to in this publication have not been endorsed by Cambridge Assessment International Education. The Publishers and author would like to thank Michael Smyth for his valuable contributions to this book. The Publishers would like to thank the following for permission to reproduce copyright material. Photo credits p18 left © Dr Keith Wheeler/Science Photo Library; p19 © Ed Reschke/Stone/Getty Images; p23 © Victor Shahin, Prof. Dr. H.Oberleithner, University Hospital Of Muenster/Science Photo Library; p25 © CNRI/Science Photo Library; p42 © Pfeiffer/Shutterstock.com; p54 © Ed Reschke/Stone/ Getty Images; p60 (and p10 of answers) © Steve Gschmeissner/Science Photo Library; p73 (and p12 of answers) © Kevin Mackenzie, University of Aberdeen Every effort has been made to trace all copyright holders, but if any have been inadvertently overlooked, the Publishers will be pleased to make the necessary arrangements at the first opportunity. Although every effort has been made to ensure that website addresses are correct at time of going to press, Hodder Education cannot be held responsible for the content of any website mentioned in this book. It is sometimes possible to find a relocated web page by typing in the address of the home page for a website in the URL window of your browser. Hachette UK’s policy is to use papers that are natural, renewable and recyclable products and made from wood grown in well-managed forests and other controlled sources. The logging and manufacturing processes are expected to conform to the environmental regulations of the country of origin. Orders: please contact Hachette UK Distribution, Hely Hutchinson Centre, Milton Road, Didcot, Oxfordshire, OX11 7HH. Telephone: +44 (0)1235 827827. Email: education@hachette.co.uk Lines are open from 9 a.m. to 5 p.m., Monday to Friday. You can also order through our website: www.hoddereducation.co.uk ISBN: 978 1 5104 8286 9 © Salma Siddiqui 2021 First published in 2021 by Hodder Education, An Hachette UK Company Carmelite House 50 Victoria Embankment London EC4Y 0DZ www.hoddereducation.co.uk Impression number 10 9 8 7 6 5 4 3 2 1 Year 2025 2024 2023 2022 2021 All rights reserved. Apart from any use permitted under UK copyright law, no part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying and recording, or held within any information storage and retrieval system, without permission in writing from the publisher or under licence from the Copyright Licensing Agency Limited. Further details of such licences (for reprographic reproduction) may be obtained from the Copyright Licensing Agency Limited, www.cla.co.uk Cover photo © Tatiana Zorina - stock.adobe.com Illustrations by Integra Software Services Pvt. Ltd., Pondicherry, India Typeset in 11/14pt Minion Pro Regular by Integra Software Services Pvt. Ltd., Pondicherry, India Printed in the UK A catalogue record for this title is available from the British Library. 9781510482869.indb 2 31/08/21 1:55 PM Contents Introduction 4 1 Safety 5 AS Level Practical Skills 7 2 Manipulation, measurement and observation 7 2.1 Carrying out investigations 7 2.2 Measurements used in biology 7 2.3 Setting up and using the light microscope 2.4 Using apparatus 27 2.5 Solutions 29 2.6 Tests you need to know 40 8 3 Presentation of data and observations 47 3.1 Presenting data in tables 47 3.2 Presenting data in graphs 47 3.3 Writing practical procedures 53 4 Practice questions 54 A Level Practical Skills 66 5 Planning an investigation 66 5.1 Developing your own procedure 66 6 Analysis and conclusions 69 6.1 Analysing data 69 6.2 Drawing conclusions 70 6.3 Evaluation 70 7 Practice questions 73 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 3 Photocopying prohibited 3 31/08/21 1:55 PM Introduction This workbook aims to provide you with the practical skills and knowledge required for the Cambridge International AS & A Level Biology syllabus (9700). Whatever the approach and whatever the techniques, the ultimate purpose of practical work is to explore and investigate the world of living things and not merely to pass an examination. This Practical Skills Workbook is designed to enable you to carry out essential biology investigations and to gain expertise in particular techniques. The first chapter covers safety in the laboratory. The rest of the book is divided into two halves: Chapters 2 to 4 cover the skills and knowledge required by the AS Level qualification; Chapters 5 to 7 cover the additional skills and knowledge you will need if you are studying the full A Level. The first two chapters in each half of the book relate to skills specified by the practical assessment section of the syllabus: ‘Manipulation, measurement and observation’ and ‘Presentation of data and observations’ for AS Level; ‘Planning an investigation’ and ‘Analysis, conclusions and evaluation’ for the full A Level. These chapters contain guidance, examples and exercises that you can work through alongside your study of the main syllabus content and refer back to when required. Chapters 4 and 7 feature practice questions to give you an opportunity to check your understanding and put the skills you have learned into practice. Some are taken from previous Cambridge International AS & A Level Biology (9700) examination papers and have information about the source at the end of the question; others have been written by the author in the style of exam questions. There are spaces for you to write your calculations and answers in the book. Answers to these questions have been written by the author and can be found online at www.hoddereducation.com/cambridgeextras. Assessment overview There are two assessment components that test your ability to understand, plan, carry out and evaluate practical investigations. l Paper 3 Advanced Practical Skills – a laboratory-based paper that requires you to carry out an investigation or investigations and to answer related questions. All students take this paper, which tests the skills covered in Chapters 2 and 3. l Paper 5 Planning, Analysis and Evaluation – a written paper testing the practical skills of planning, analysis, conclusions and evaluation. Only students studying for the full A Level qualification take this paper, which tests the skills covered in Chapters 5 and 6. The information in the above section is taken from the Cambridge International syllabus for examination from 2022. You should always refer to the appropriate syllabus document for the year of your examination to confirm the details and for more information. The syllabus document is available on the Cambridge International website at www.cambridgeinternational.org. 4 9781510482869.indb 4 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 1 Safety A science laboratory is potentially a dangerous place. Although any potential dangers are pointed out in each investigation, it is your responsibility to be vigilant, so take heed of instructions and warnings. Your teachers will have carried out a risk assessment for any practical you carry out and will point out hazards verbally and in any written instructions provided, or may expect you to work these out as part of an investigation. You should know the difference between a hazard and a risk as you will be penalised if you use the terms interchangeably. l A hazard is something that may cause harm if safety precautions are not observed, for example Bunsen burners, chemicals, electricity, spillage of liquids and glassware. l Risk is the chance that a hazard will cause someone harm. Risk is assessed as low, medium or high. For example, a spillage of water in the lab would be classed as low risk, while working with corrosive substances, such as acids and alkalis, would be high risk. You may be required to identify hazards and assess the risks of a given practical investigation or in an investigation you plan as low, medium and high. Some general rules that you should follow in the laboratory include the following. Make sure you know what to do in case of fire, including how to raise the alarm and the location of fire extinguishers. Remember, the most important consideration at all times is human life. l Know the location of the first aid kit. l Treat all chemicals as hazardous. Observe, understand and follow all warning labels for specific hazards. You should be familiar with the hazard symbols and codes in Table 1.1. l i Explosive Corrosive l l l l l l l l l l l l Toxic Oxidising Irritant Flammable n Harmful Radioactive C corrosive MH moderate hazard HH health hazard T acutely toxic F flammable O oxidising N hazardous to the aquatic environment Table 1.1 Hazard symbols and codes Know what you are doing before you start work. Never eat, drink or smoke in the laboratory. Wear protective clothing, for example laboratory coat/apron, while carrying out an investigation. Wear eye protection when working with chemicals or heating liquids in glassware. Wear gloves when working with harmful chemicals or plant and animal tissue. Take care not to cut yourself when using scalpels and glassware. Using heating equipment is a hazard that is classified as low risk if safety precautions are followed. You should be familiar with the safety precautions for the equipment you use. Liquids should be heated in a water bath at a temperature suited to the investigation. Keep flammable materials well away from flames. Hold test tubes so that they point away from you and everyone else. Do not allow electrical equipment to come into contact with water. Work in a logical, tidy manner, keeping your working area orderly and tidy. Clean up afterwards. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 5 Photocopying prohibited 5 31/08/21 1:55 PM SAFETY l l l l l Keep hands away from your face, eyes and mouth when working with biological material, chemicals, specimens and microorganisms. If any chemicals or other agents splash into your eyes, wash them immediately with distilled water or the saline solution for eye washing in your first aid kit. Dispose of chemicals and specimens safely and ethically, as instructed. Report or clean up spillages and breakages immediately. Report accidents to your teacher, however minor. Always wash your hands before leaving the laboratory. 6 9781510482869.indb 6 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS 2 Manipulation, measurement and observation 2.1 Carrying out investigations For many of the investigations you are asked to carry out throughout the course, you will be provided with a handout with clearly defined objectives and instructions. Sometimes, however, you will be expected to plan and describe how you would carry out certain procedures that you may have learned as part of your course, or adapt them for a new scenario — for example, to change an investigation into the effect of temperature on an enzyme into one to investigate the effect of pH. Following a given procedure Here are some tips that you should consider and keep in mind before you begin an investigation for which you have been given the procedure. l Read the whole procedure carefully before you start. l Before starting any investigation, make sure you know the aim of the investigation, the hypothesis being tested and what you are expected to do. l If the procedure requires you to wait for any length of time, think about whether you can work on another part of the investigation – drawing tables, heading graphs, etc. – while waiting for the reaction to occur. l Be prepared to accurately record all observations, measurements, timings and any special precautions and alterations. Tabulate your results neatly (scraps of paper have a habit of getting lost) so that you do not have to copy them later. These records will be useful when writing your reports. l If you make a mistake, you will need to repeat the investigation – if possible. If not, you will need to note any mistakes/alterations you have made. These will help explain your results later. l You should write up your practical reports as soon as possible. If you do not, you are likely to forget important details, and it is much easier to write up one report than many at a time. 2.2 Measurements used in biology The international conventions for measurements are governed by the Système Internationale d’Unités, and are usually referred to as SI units. This is an internationally recognised form of measurement and represents the accepted scientific convention for measurement of physical quantities. Units and symbols that you are likely to encounter in this course are listed in Table 2.1. Measured quantity Name of SI unit Symbol Other common units for this quantity Length metre m millimeter (mm) micrometer (μm) nanometer (nm) Ångstrom (Å) (10−10 m) Mass kilogram kg gram (g) milligram (mg) microgram (μg) Volumes (solids) cubic metre m3 cubic centimetre (cm3) cubic millimetre (mm3) cubic micrometre (μm3) Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 7 Photocopying prohibited 7 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Measured quantity Name of SI unit Symbol Volumes (fluids) decimetre dm3 Time second s Frequency hertz Hz Energy joule J Pressure pascal Pa Force newton N Temperature Kelvin K Celsius °C Sedimentation rate of a particle when centrifuged Svedberg unit S Acidity pH - Other common units for this quantity Table 2.1 Common units of measurement Most measurements consist of a number and a unit. The number expresses the ratio of the fixed quantity to a fixed standard, and the unit is the name for the measure or the dimension. Certain measurements can be expressed as dimensionless ratios or logarithms (e.g. pH), and thus do not require a qualifying unit. 2.3 Setting up and using the light microscope The compound microscope is an expensive, precision instrument. Handle it with care. l When carrying the instrument always use both hands, holding it by the limb and under the base. (Never carry a microscope by the microscope tube or stage.) l Do not remove eyepieces, objectives, etc. without permission from your teacher and unless you know what you are doing. If there is dust inside your instrument, ask your teacher to help you because unnecessarily opening these parts introduces more dust into the instrument. l Keep the lenses and working surface (the stage) clean and dry. Take care to avoid touching the lenses and make sure the solvents, stains and mounting fluids do not come into contact with the lenses. l Lenses and the mirror should be wiped only with a lens tissue or lint-free soft duster kept solely for this purpose. Setting up a microscope at low power l l l Before using the microscope, familiarise yourself with its components. Time spent now in learning to set your instrument to give the best possible image will pay dividends later. The microscope will be your companion in many investigations throughout your course. If possible, try to use the same microscope each time, so that you can familiarise yourself with it. Follow these instructions carefully. Failure to do so could result in breaking the slide and may damage the microscope. Referring to Figure 2.1, proceed as follows. 1 Place the microscope on the bench in front of you, so that you can sit comfortably and look down the eyepiece lens without straining yourself. 8 9781510482869.indb 8 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation using the simple microscope (hand lens) You should bring the thing you are looking at nearer to the lens and not the other way round. eyepiece lens using the compound microscope turret– as it is turned the objectives click into place, first the mediumpower, then the high-power objective lenses – ×4 (low); ×10 (medium); ×40 (high power) coarse focus – used to focus the low- and medium-power objectives stage – microscope slide placed here fine focus – used to focus the high-power objective condenser – focuses light on to the object with iris diaphragm – used to vary the intensity of light reaching the object built-in light source Figure 2.1 Light microscope 2 Plug in and adjust the lamp setting to the minimum and switch on. Adjust the lamp setting to about twothirds of the maximum. 3 Familiarise yourself with the various objective lenses (×4, ×10, ×40, ×100) mounted on a rotating turret called the nosepiece, which clicks into position. Rotate the turret to click the low-power (×4) objective into position. 4 Find the coarse and fine adjustment knobs, movement of which usually moves the microscope tube up and down (in some microscopes the stage moves) and focuses the image. 5 Do not touch the condenser and iris diaphragm below the stage. Fiddling with these will require trained staff to readjust them. 6 When you are fully familiar with the controls, find a suitable stained slide, place it on the stage with the coverslip (thin glass covering the section) uppermost, and clip it into position so that the specimen is illuminated from the light below. 7 Now focus the image of the specimen first with the coarse-adjustment knob, then with the fineadjustment knob until the image is in sharp focus. 8 Reduce the illumination if too bright. 9 Your microscope is now set to give you the best possible image. If you are having trouble, ask your teacher or technician for help. 10 Observe a selection of prepared slides, for example of Protoctista (single-celled eukaryotic organisms), blood and other suitable tissues. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 9 Photocopying prohibited 9 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Setting up a microscope at high power 1 For high-power magnification, first focus the microscope at low power for the specimen to be examined as described previously using the coarse-adjustment knob only. 2 Then carefully turn the objective nosepiece around until the medium- or high-power objective (×10, ×40) is clicked into place. (The ×100 objective is generally used in a special technique – oil-immersion – which you are not required to learn.) If the microscope was in focus at low/medium power, then the high-power objective lens will be close to the prepared slide, but not touching it. 3 Look down the microscope and fine-focus the image by slowly racking up with the fine-adjustment knob only. Using the coarse-adjustment knob will break the slide and may damage the microscope. 4 Do not remove the slide with the high-power objective lens in position. Move all objectives out of line and move the stage down using the coarse-adjustment knob. 5 Examine prepared slides at low and high magnification, until you are sure you can do so with ease. 6 When you have finished your observations, reset the microscope objectives to low power. 7 Remove the prepared slide and store it in the correct slide tray. See that the microscope is clean and dry before you put it away. Preparation of biological material for optical microscopy Preparation techniques are crucial to successful microscopic investigations. Although living or preserved biological specimens can be observed with the compound microscope, dead, fixed and stained specimens are usually used. Staining Cells and tissues to be examined under a microscope must be sufficiently transparent for light to pass through them. As specimens of biological material tend to be colourless and transparent, stains are often used to make different parts stand out or show contrast. ‘Contrast’ involves highlighting tiny differences in the structure of the specimen. In biological material, contrast is normally achieved by staining the material. These stains usually colour specific parts of a cell so can be extremely useful. As long as the specimen is very small, there is no limit as to what you can view under the light microscope. Living, moving cells and ‘fixed’ stained sections can be examined. Much of what is known about the structure of cells was first learnt by observation of thin sections of tissues that had been preserved, sectioned, stained and permanently mounted on microscope slides. In light microscopy, staining is used to add contrast to the image as well as to highlight components of interest and to locate particular cells, tissues or organs. In ultraviolet (UV) microscopy, contrast is obtained using fluorescent stains. The physicochemical properties of the stain cause it to attach to specific structures preferentially, or be taken up across cell membranes. Cell fractionation Cell fractionation is the technique by which the tiny structures within the cells, known as organelles, are isolated so that structure and function can be investigated outside the organism. The steps in cell fractionation are summarised in Figure 2.2. 10 9781510482869.indb 10 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation 1. The chilled tissue is cut up in isotonic buffer solution 2. The tissue fragments are homogenised in a blender 3. The suspension is filtered through several layers of muslin 4. The filtrate is centrifuged at low speed to remove large cell debris 5. Supernatant is pooled chopped tissue in buffer solutions iced water 6. The supernatant containing 7. Ultra centrifugation at organelles is pipetted on 100 000g overnight continuous sucrose density gradient sucrose density gradient supernatant containing organelles filtrate iced water 8. Fraction of the content removed containing various cell organelles at different densities ribosomes mitrochondria chloroplasts nuclei cell debris 9. Different cell organelles separated density of organelles The organelles are identified by chemical analyses Figure 2.2 Separating cell parts using cell fractionation Drawing observations from a microscope As part of your practical work, you are required to use a light microscope to study an extensive range of biological specimens, make accurate visual observations and then record your observations by drawing. Drawing what you see under the microscope can be daunting, but the technique can be learnt – it requires no artistic ability, just attention to detail, accurate observations and practice. You will have plenty of opportunities to practise these skills throughout the course. Your drawings should prove useful as a permanent record for future reference, helping you to remember what you have observed and recorded. In Paper 3, one question requires you to use the microscope and record your observations by drawing. You will need: l plain paper l a good selection of pencils (B and HB are most useful) l a good eraser l a ruler l a sharpener. Pretend that you are drawing for someone who has never seen this specimen. They should be able to see exactly what it looks like and what size it is from your drawing without seeing the specimen. Learn the difference between plan, high-power and annotated drawings, and follow instructions. For example, if asked to draw two cells, draw two cells. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 11 Photocopying prohibited 11 31/08/21 1:55 PM (a) AS LEVEL PRACTICAL SKILLS 1 Your drawing should fill at least half the provided space, with ample space on either side for labels. 2 The drawing should be drawn at a defined magnification. Often you need to draw larger than the original specimen, and then have to specify the exact magnification. If you have difficulty in making a start, measure the specimen: say it is 2 cm, then decide how large you want your drawing to be, for example 2 cm 5 × the specimen so, 5 × 2 cm = 10 cm (Figure 2.3). (b) (a) 10 cm Figure 2.3 Drawing an object: (a) determine linear dimensions; (b) construct a frame and outline 2 cm (b) Take a ruler and mark out 10 cm in the centre of the space, now start by drawing very faint ‘construction lines’ using a sharp, soft pencil, to allow you to get the basic proportions correct before progressing. These can be erased once the drawing is complete. 3 Mark out the main structures in the correct position and proportion. Again, if you have trouble with drawing the correct proportions, use the same technique of measuring and multiplying. If the drawing needs to be smaller than the specimen, use the same procedure, but divide instead of multiplying. After some practice, you will not need to follow this method. 4 Using a soft pencil draw faint, clear, smooth lines. Avoid hesitant fuzzy or broken lines. If you do draw in such a manner, make sure to rub these out and replace them with smooth, continuous lines. 5 Now add the main structures. 10 cm 6 Observation and attention to detail are key to a good drawing. Look carefully at how each structure is linked to the main plan of construction (Figure 2.4). Make sure that junctions between lines are properly drawn. For example, if you are drawing a plant, observe exactly how the leaves are attached to the main stem. Is there a little stalk joining the main leaf blade to the stem or does the blade join directly on to the stem? Look at the exact arrangements of the leaves. Are the two leaves arranged exactly opposite each other or are they alternating? 7 Draw exactly what you see and not what you expect to see, and do not copy from a textbook. 8 Shading or use of coloured pencils is not normally allowed. Once you are satisfied with your drawing, go over with fine, firm lines, rubbing out any mistakes and fuzzy, broken lines. 9 Drawings should be clearly labelled. Annotations are strongly recommended; however, do read the question carefully and follow any specific instructions. 10 Work out and record the magnification at which you observed the image. This magnification is: eyepiece lens × the objective lens. 12 9781510482869.indb 12 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation Incorrect Correct avoid fuzzy broken lines avoid careless drawings pay attention to detail avoid unnecessary detail make sure labels point exactly Figure 2.4 Common errors in biological drawings Annotating drawings Annotated drawing includes labels and additional brief comments to describe structures or functions, or both, of the labelled part. Annotations are most helpful when using your drawings for revision. An annotated diagram can be used to answer questions about the structure and function of the heart, for example. l Always keep labels well away from the drawing. l Print labels using a sharp pencil on either side of the drawing, with lines drawn with a ruler, pointing precisely to the structure and without arrowheads. l Labels lines should never cross each other. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 13 Photocopying prohibited 13 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS EXERCISE 2A Annotate the main structures of the heart (Figure 2.5) to describe its structures and functions. [16] Figure 2.5 Main structures of the heart EXERCISE 2B This exercise requires you to dissect a mammalian heart (the video at the following location shows you how to do this: https://youtu.be/WBwPhWAP394). Record your observations by drawing and then answer the following questions. External features 1 You should be able to identify the: ventral (front) and dorsal (back) sides of the heart (the ventral side is more rounded); the four chambers; and all the blood vessels entering and leaving the heart. Note, the main veins enter the heart via the dorsal side. 2 Identify the inferior and superior vena cava; pulmonary artery and vein; and the aorta. Note also, the coronary vessels that supply the ventricle muscle; the aorta is a thick-walled rubbery tube. The outer surface of the heart often shows an accumulation of fat, largely around the main blood vessels and over the upper part of the ventricles. 14 9781510482869.indb 14 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation 3 Make annotated diagrams of the external appearance of the ventral and dorsal sides of the heart. Internal features 4 Dissect the heart and make labelled drawings to show all the structures you are asked to identify. Locate the valves at the base of the aorta, at its junction with the ventricle wall. Note the semilunar valves, tricuspid valves and tendineae chordae (heartstrings). 5 Note the difference in the thickness of the walls of the atrium and the ventricles. 6 Make labelled diagrams to show all the major internal structures of the heart. The drawing should have clean, unbroken lines and no shading anywhere. Label lines should just touch structures and not pass into them. Label lines should not have arrowheads. The labels should include: l left and right atria l left and right ventricles l atrio-ventricular valves. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 15 Photocopying prohibited 15 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Other structures, such as major blood vessels, if visible should be correctly labelled. a Describe the functions of the following structures: aorta, pulmonary artery and vein, left and right atrium, left and right ventricles, bicuspid valve, tricuspid valves, semilunar valves, vena cava. [11] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 16 9781510482869.indb 16 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� b The heart is described as myogenic. What does ‘myogenic’ mean? [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� c Why is the right ventricle thicker than the left ventricle? [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� d Which two nerves of the autonomic system regulate the heartbeat? [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� e Where is the pacemaker (sino-atrial node) and what is its function? [3] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� f Where is the atrioventricular node and what is its function? [3] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� g What other body system plays a direct role in regulating the heartbeat? [1] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 17 Photocopying prohibited 17 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Plan drawings A plan drawing indicates the distribution of the main tissues within an organ. it requires the following. l Only draw the main structures (best observed at low power). l Draw each structure in exact position and proportion (structures should be the right size relative to each other and in the correct position). l Draw each tissue completely enclosed by sharp lines. l Be precise about detail. l Do not draw individual cells. l Avoid fuzzy or broken lines. l Do not shade. l Label and/or annotate the main structures as shown in Figure 2.6. l Give the drawing its main label – usually what is written on the slide, for example transverse section (TS) of a dicotyledon stem – and make a habit of always indicating at what magnification the observations were made. vascular bundle epidermis sclerenchyma of the vascular bundle cortex (parenchyma) phloem Figure 2.6 Dicotyledon stem: (a) TS and (b) plan drawing TIP Use the ×4 and ×10 objectives so that you can see the whole specimen. Pay special attention to the structures and proportions. High-power drawings This is usually of a specific structure, or of one or two cells only. 1 Observe and draw the number of cells you are asked to draw – no more and no less. 2 Detail of only one cell may be needed, with outlines of adjacent cells to show their relative positions. 3 Observe the shape of each cell and how it attaches to other cells. 4 Remember that each cell has a separate cell wall/plasma membrane. 5 Label/annotate as required. 6 Only label what you can see, not what you expect should be present. 18 9781510482869.indb 18 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation In the image shown in Figure 2.7, individual cells and their cell walls are visible. The shape of each cell is different. The middle lamella is clearly visible between the xylem vessels and should be drawn with three lines where the boundaries of the two xylem vessels meet. Using different pencils, such as HB and B, would help to differentiate between xylem and surrounding parenchyma cells. Figure 2.7 High power image of TS dicotyledon root EXERCISE 2C Follow the method below to prepare a temporary slide to observe diffusion and osmosis in onion epidermis cells, then record your observations as guided below. Method 1 Cut a 0.5 cm long piece from an onion. 2 Separate two of the thin layers of the onion. 3 Using forceps, peel off the inner translucent membrane between the layers. This is the epidermal layer. 4 Place two drops of iodine solution onto the centre of a microscope slide. 5 Place the epidermal tissue on top of the iodine solution, making sure it is flat and not folded. Avoid trapping air bubbles. 6 Carefully lower a coverslip onto the slide. Do this by placing one edge of the coverslip on the slide and using a mounted needle to lower the other edge down onto the slide. Avoid trapping air bubbles. 7 Leave for five minutes while the iodine solution penetrates the cell membrane. 8 Soak up any liquid from around the edge of the coverslip using some filter paper. 9 Observe some cells under higher-power objective lens. 10 Make a clear, labelled drawing of two epidermal cells and their parts. 11 Place a few drops of sugar solution on the slide, to one side of the coverslip. 12 Use the blotting paper on the other side of the coverslip to draw the solution across. 13 Observe under the microscope and record your observations. 14 Draw and label a diagram of two cells. 15 Observe plasmolysis in as many cells as you can and calculate the percentage of cells showing plasmolysis. 16 Wash your hands thoroughly. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 19 Photocopying prohibited 19 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Observations a Use the space below to draw your observation of four cells mounted in iodine solution. Label the parts of one cell. [5] b Use the space below to record and draw your observations of two cells mounted in sugar solution. Label these cells. [4] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 20 9781510482869.indb 20 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation c Explain the effects of immersing plant cells in iodine solution and sugar solutions by using ideas about diffusion, water potential, osmosis and plasmolysis. [5] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� d Calculate the percentage of cells showing plasmolysis for each concentration. Show your working. [2] e Suggest why onion tissue was chosen for this practical. [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� TIP When drawing cells, look carefully at the shape of each cell and how the cells join with other cells. Draw a clear, continuous outline for each cell with no shading. The drawings should include a title that describes the specimen, and labels for each visible structure. Magnification of a microscopic image The magnification of a microscopic image is calculated by multiplying the objective magnification (×4, ×10, ×40) by the eyepiece magnification (usually ×10 or ×16). However, you may be asked to calculate the magnification of your drawing or of a given image. Magnification is simply how much bigger or smaller the drawing is compared with the actual specimen. For example, if you are provided with a leaf and asked to draw it, you may draw it much bigger than the actual size of the leaf, or you may draw it smaller. Magnification is worked out using the following: M (magnification) = I (drawing size) A (specimen size) Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 21 Photocopying prohibited 21 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS This equation can be rearranged to give the actual size of an object where the size of the image and magnification are known. A= I M For example, if the actual width of the leaf is 25 mm and on your drawing this measures 100 mm across, the magnification would be greater than 1. M= 100 =4 25 You would write your magnification as ×4. If your drawing is smaller than the specimen, for example 10 mm, the magnification would be less than 1. M= 10 = 0.4 25 The magnification is written as ×0.4. TIP l l Magnification does not have units. To avoid mistakes in calculations always measure in the smallest unit, for example use mm rather than cm and mm. Remember: l 1 metre (m) = 1000 millimetres (mm) l 1 millimetre (mm) = 1000 micrometres (µm) l 1 micrometre (µm) = 1000 nanometres (nm) You may need to work out the magnification or size of a cell or cell organelle. It helps to have an idea of the approximate size of plant and animal cells and some organelles, in case you make a mistake in your calculations (Table 2.2). Cell/organelle Average size Prokaryotic cell (bacteria) ~ 1–5 μm Plant cell ~ 100 μm Animal cell ~ 10–50 μm Nucleus ~ 5–7 μm Mitochondrion ~ 0.5–1 μm Chloroplast ~ 4–10 μm Ribosome ~ 15–25 nm Cell wall ~ 2 μm Plasma membrane ~ 7.5 nm Table 2.2 Typical sizes of plant and animal cells, and some organelles A rough estimate of the magnification can be made by comparing the size of the image to the diameter of the field of view. The field of view can be roughly estimated by focusing on a transparent millimetre ruler under low power. For example, if the field of view at an overall magnification of ×40 is 4 mm, then at a total magnification of ×100 it will be (40/100) × 4 mm = 1.6 mm (1600 µm). 22 9781510482869.indb 22 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation EXAMPLE 2.1 Calculate the magnification of the image of the nuclear pore shown in Figure 2.8. 10 nm scale bar Figure 2.8 Scanning electron micrograph (SEM) of nuclear pore Method To calculate the magnification, we first convert all figures to the smallest unit, in this case nm. 24 millimetres = (24 × 1000 × 1000) = 24 000 000 nanometres magnification = size of image/actual size of object = 24 000 000/120 = ×200 000 A better method is to use an eyepiece graticule and a stage micrometer slide. See the following section for more information on measuring microscopic objects. EXERCISE 2D Figure 2.9 shows a plan drawing of tissues in a transverse section of a dicotyledonous leaf. A B Figure 2.9 Dicotyledonous leaf Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 23 Photocopying prohibited 23 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS The actual thickness of the leaf along the line AB is 0.6 mm. Calculate the magnification of the diagram. Show your working. [2] Measuring microscopic objects It is important to be able to measure the dimensions of biological material. The size of an object observed under the microscope can be measured by using an eyepiece graticule and a stage micrometer slide. l An eyepiece graticule is a plastic or photographic film with a printed grid, or a tiny circular piece of glass with a grid etched on it (in arbitrary units). l A stage micrometer slide is a microscope slide with a scale, which is used like a tiny ruler to measure the distance between each division on the eyepiece graticule. Familiarise yourself with both and observe the grid on them by holding up to the light and using a hand lens (Figure 2.10). 0 1 2 3 4 5 6 7 8 910 Figure 2.10 A stage micrometer slide Ask your teacher to demonstrate how to install the eyepiece graticule into the microscope before you attempt it yourself. This is done by carefully removing the eyepiece from the microscope and unscrewing the top lens (Figure 2.11). One way to fit the eyepiece graticule is to place it (right side up) on the ‘shelf’ halfway down in the microscope eyepiece. The upper eyepiece lens is screwed back and returned to the microscope. Eyepiece graticule The scale (arbitry units) usually 1 cm long and divided into 100 divisions 0 1 2 3 4 5 6 7 8 9 10 3. Replace top lens lack 1. Unscrew top lens 2. Place eyepiece in the microscope graticule inside the from microscope 4. Look down the microscope, superimpose the eyepiece microscope eyepiece eyepiece image of the specimen on the scale of the graticule, and record the specimen size in arbitrary units 1 0 0 0.1 2 0.2 0.3 0.4 1.5 (15 units) 0.24 mm 5. Replace the specimen slide with the stage micrometer slide and superimpose the scale of the eyepiece graticule with the scale on the stage micrometer; calibrate the scale of the graticule at the magnification used Figure 2.11 Using an eyepiece graticule with a stage micrometer slide 24 9781510482869.indb 24 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation The grid on the eyepiece graticule is in arbitrary units. It needs to be calibrated (that is, you need to work out the distance between each division), using the stage micrometer slide. Once you have calibrated the eyepiece graticule for a particular magnification, you can measure the size of any specimen viewed at that magnification under the microscope. EXERCISE 2E Figure 2.12 shows a mitochondrion from a cell as seen with an electron microscope. B A Figure 2.12 Transmission electron micrograph (TEM) of a mitochondrion magnification × 65 000 Calculate the actual length of the organelle as shown by the line AB in the diagram. Show your working and express your answer to the nearest micrometre (µm). [3] Calibration of the eyepiece graticule 1 Hold up the stage micrometer slide in the light and observe the scale using a hand lens, for example 1 mm with 100 divisions. Each stage micrometer slide division is therefore equal to 0.01 mm = 10 µm. Remember that the scale on the micrometer slide can vary. 2 Place the micrometer slide on the stage of the microscope. 3 Look down the eyepiece of the microscope at low power. 4 Focus on the divisions on the micrometer slide under the microscope. 5 Rotate the microscope eyepiece until the graticule scale lies superimposed on the stage micrometer slide. The graticule scale is divided in arbitrary units, that is, not sub-divisions of a millimetre. 6 Use the stage micrometer slide like a mini ruler to measure the distance between divisions on the graticule (Figure 2.13). stage scale: 1 division = 10 µm 0 0 10 2 20 4 6 8 eyepiece scale Figure 2.13 Eyepiece graticule scale Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 25 Photocopying prohibited 25 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS For example, if 2 eyepiece graticule divisions are equivalent to 6 stage micrometer slide divisions, then one eyepiece graticule division = 6 = 3 stage micrometer divisions 2 Since each stage micrometer division = 10 µm Each eyepiece graticule division = 3 × 10 = 30 µm So, if you replace the stage micrometer slide with a prepared slide and measure the length of a cell on the slide to be 1.5 eyepiece graticule divisions or units, then the cell is 1.5 × 30 = 45 µm. 7 Observe and record how many eyepiece units (epu) (eyepiece graticule divisions) are equivalent to how many micrometer slide divisions at each objective lens. (As the magnification is increased the eyepiece scale will cover a smaller area of the object and each division measures a smaller length.) 8 Once you have calibrated the divisions on the eyepiece graticule, you can remove the stage micrometer slide and use the eyepiece graticule to measure accurately any structure/cell in a slide. EXERCISE 2F a Repeat the technique described previously by taking measurements of : l the diameter of a parenchyma cell from the central pith of the stem l the diameter of a nucleus (stained preparation) l the width of a capillary l the diameter of the largest xylem vessel in the stem (TS) l the diameter of a red blood cell. Measure several cells and calculate the average size of the cells. Decide how many cells you are going to measure. Choose cells you can see clearly and record their diameter in epu. If you measure five cells, for example, add the five epus and divide by 5 to give the mean diameter. Add your measurements to the table. Complete the table. Cells [16] Diameter of cell or structure/epu Diameter/μm Mean size of cell/ structure/μm Parenchyma cell Nucleus Capillary Xylem vessel Red blood cell b Compare and contrast plant cells with animal cells with respect to size and structural organisation. Tabulate your observations. [4] Plant cell 26 9781510482869.indb 26 Photocopying prohibited Animal cell Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation 2.4 Using apparatus You will be provided with appropriate equipment for each investigation, such as syringes, pipettes, measuring cylinders, rulers and micrometer slides – according to the task at hand. It is important to learn to use equipment to measure consistently and accurately and to obtain the best possible results. You will also be required to make decisions about the suitability of apparatus for a particular task. The suitability of apparatus relates to whether it measures to the appropriate accuracy. For example, whether to use the wall clock or a stopwatch when recording time for a chemical reaction to change colour, or whether to use a 10 cm3 pipette or a 1 cm3 pipette to dispense a 1 cm3 sample. The 1 cm3 pipette will dispense more accurately. Accuracy relates to closeness to the standard measure and precision refers to the closeness of two or more measurements to each other. Accuracy will depend on the equipment used, and precision will depend on your ability to use the equipment properly. For example, if you need to dispense 0.1 cm3, you will get a more accurate result using a 1 cm3 graduated pipette, which has the smallest measuring unit of 0.01 cm3, than using a 10 cm3 pipette, with a smallest measuring unit of 0.1 cm3. The precision (pipetting exactly the same volume each time) will depend how carefully you pipette. Precision refers to how small the units of measurements are, i.e. the number of decimal places any measurement can be recorded to. It is determined by the apparatus used – for example, a 1 cm3 graduated pipette has the smallest measuring unit of 0.01 cm3, so the precision is limited to 0.005 cm3, which is half of this smallest unit. Using glassware Choose the right glassware to measure liquids, keeping in mind the accuracy required, the volumes being dispensed and the number of times the measurement must be taken. You need to take care when using glassware. l Do not use chipped or cracked glassware – it may break on heating or under the slightest strain. l If possible, use heat-resistant glassware, especially if heating and cooling are involved. l Do not force stoppers in glassware; they are very hard to take out, and you may cause breakage and injury. l Dispose of broken glass in appropriate bins, so that the cleaning staff do not get injured. Certain liquids may cause problems. Never mouth-pipette any chemicals such as acids or poisonous solutions. Use a syringe or a pipette filler. Check with your teacher if you are uncertain. l Viscous liquids, such as glycerol, may be difficult to dispense; allow time for all the liquid to transfer. It may be easier and more accurate to weigh than pipetting. l Organic solvents evaporate quickly, making measurements inaccurate and difficult; work rapidly and seal containers quickly. Work in a fume cupboard or in a well-ventilated room. l Certain solutions, such as proteins and detergents, are prone to frothing; try to avoid forming bubbles by dispensing gently. Take the sample from within the liquid, below the froth. l Suspensions such as cell cultures and soil samples tend to sediment. Mix thoroughly before dispensing. l Measuring cylinders and volumetric flasks To accurately measure liquids, follow the following steps. 1 Stand the measuring cylinder or volumetric flask on a level surface. 2 Fill to just below the required volume. 3 With your eye level with the meniscus, fill carefully to the final mark using a Pasteur pipette until the meniscus is level with the final mark. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 27 Photocopying prohibited 27 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS 4 Always take the measurement from the bottom level of the meniscus. 5 If you need to measure solutions of differing concentration, it may not be possible to use a fresh syringe/ pipette for each one. Start at the lowest concentration and rinse the syringe/pipette between each measurement. 6 When working with different samples, rinse well with distilled water each time to avoid contamination. Graduated pipettes 25 ml TD 20ºC 10 ml Pipettes are precision glassware used for accurately measuring volumes to within one drop, which is 0.05 cm3. Pipettes are available in various sizes. Choose the right size for the volume to be dispensed, i.e. do not use a 10 cm3 pipette to dispense 0.1 cm3; use a 1.0 cm3 pipette. Bulb pipettes are of fixed volume – for example, 5 cm3, 10 cm3, 50 cm3 – and are very accurate. Take care to check the volume scale before use. Most pipettes empty from full volume to zero, others fill from zero to full, and some refer to the shoulder of the tip (Figure 2.14). 10 0 9 1 8 2 7 3 6 4 5 5 4 6 3 7 2 8 1 9 0 10 Figure 2.14 Graduated pipettes 1 Using a pipette filler, draw the solution until the meniscus is just above the required gradation mark. 2 Keep the pipette vertical and your line of sight horizontal with the gradation, and allow the solution to drip slowly out of the pipette until the bottom of the meniscus is on the gradation (Figure 2.15). 50 meniscus 40 eye level 30 20 Figure 2.15 Keep your line of sight horizontal until the bottom of the meniscus is on the gradation 3 Dispense the liquid into the test tube/beaker as required. 28 9781510482869.indb 28 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation 4 Touch the tip of the pipette to the side of the glassware to allow the last drop to trickle out. Do not blow out the last drop. 5 The pipette needs to be cleaned by rinsing with a little deionised water and then rinsed with a little of the solution to be used, which is then discarded. Syringes Syringes without an attached hypodermic needle are often used instead of pipettes. Needles should not be used. 1 Place the tip into the solution and gently pull the plunger to a little more than the required volume on the scale. Stiff syringes may need lubricating by drawing up water before using. 2 Expel any air bubbles by inverting and gently tapping the syringe. Then expel the air and the excess liquid. Top pan balance Make sure you know how to use the balance accurately. l Never weigh anything directly onto a balance pan – there may be contamination from previous users. l Use a clean spatula and new piece of aluminium foil or filter paper. Otherwise, weigh directly into the vessel in which you are mixing the solution. l Balances can also be used to weigh liquids that you have dispensed accurately. This is especially useful for viscous liquids such as glycerol. l Convert mass to volume using the following equation: volume = mass density The density of most liquids is written on the bottle, or can be looked up in textbooks or on the internet. Tips for obtaining good, consistent results Avoid: l reading instruments incorrectly – for example, temperatures of a liquid should be taken after stirring the liquid and while the bulb of the thermometer is still in the liquid l not following instructions – carefully read and understand exactly what you are supposed to be doing, and why l sample/solution mix-ups – avoided by careful planning and labelling l careless measurements – avoided by attention to detail l using different batches of solutions – avoided by planning and making excess solution l not stirring fully – always mix solutions prior to use, especially those that form a sediment l gas escaping before bung insertion – unavoidable at times, but ensure you have the right bung, etc., beforehand l blocked tubes/airlocks – unavoidable at times l instrument malfunction – test equipment before use If you encounter problems during your investigation, mention them in your discussion, suggesting how you would overcome and remedy them in further studies. 2.5 Solutions A solution is formed by adding solute to a solvent (usually distilled water). The solute molecules become evenly distributed between the solvent molecules to give a homogeneous solution. Most investigations require solutions of a particular concentration. Before preparing any such solutions, ensure you understand what is required for the particular task. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 29 Photocopying prohibited 29 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Concentration In SI units, the concentration of a solute is expressed in mol m−3, which is convenient for most biological purposes. The concentration of a solute is represented by square brackets, for example [KCl]. However, there are several alternative ways of expressing concentration, which you may come across in your practical work, or in textbooks. Molarity Molarity is the term used to denote concentration C, expressed as moles of solute per litre volume of solution (mol dm−3 = molar). The symbols previously in common use for molar (M) and millimolar (mM) solutions are at odds with the SI system, and now mol dm−3 and mmol dm−3 are preferred. A 1.0 molar (1.0 mol dm−3) NaCl solution would contain 58.44 g in a decimetre (dm3) of solution. This is based on the molecular mass, or the sum of the atomic masses, which can usually be found on the side of the container (Figure 2.16): Na = 23, Cl = 35.44; 23 + 35.44 = 58.44. A 1.0 molar (1.0 mol l−1 or 1.0 mol dm−3) KCl solution would contain 74.55 g (molecular mass) in a litre of solution (K = 39.10, Cl = 35.45; 39.10 + 35.45 = 74.55 g) or you can work it out from the molecular formula using a periodic table or the internet. Relative molecular mass 58.08 Highly flammable Keep container in a well ventilated place Keep away from sources of ignition – no smoking Propanone Minimum assay 99% (CH3)2CO Density (g cm–3) 0.789–0.782 at 20 oC (acetone) Molecular mass Boiling range 98% minimum distils between 55.5 and 56.5 oC Do not breath vapour Maximum limits of impurities Free acid (CH3CCCH) 0.0005% Take precautionary measures against static discharge Non volatile residue 0.005% Highly flammable Figure 2.16 Molecular mass usually appears on the label of a chemical container Percentage concentration (% w/v) Percentage concentration can be based on w/v (weight/volume), where a mass of the solid is added to make up a specific volume of solution. This is the number of grams of solute per 100 cm3 solution. The solution can be accurately prepared by weighing out the required amount of solute and then making this up to a known volume using a volumetric flask or a measuring cylinder. For example, a 10% w/v sucrose solution contains 10 g sucrose made up to 100 cm3 with water. TIP When water is the solvent, this is not usually specified. However, if another solvent is used it has to be specified, for example an 80% w/v ethanol solution would contain 80% ethanol and 20% water. 30 9781510482869.indb 30 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation l l l l l l l l l If the mass required is too small to be weighed accurately, make up a stock solution of a higher concentration, which can be diluted further. Use the right glassware for the job – for example, do not use a litre measuring cylinder to measure 50 cm3; use a 50 cm3 or 100 cm3 measuring cylinder. Make sure the glassware is clean (sterilised if required). Always use distilled or deionised water to make solutions. Always make sure all the solute is dissolved before adjusting the final volume. Adjust the pH, if required, before adjusting the final volume. Use a funnel when pouring the solution to avoid spillage. Make up the volume using the meniscus at eye level. Transfer the solution to a labelled reagent bottle. Making aqueous solutions of known concentration In most cases, solutions are made for you by the laboratory staff, but you are required to know how to prepare one should the need arise. Follow this simple procedure. 1 Work out or decide on the concentration of the chemical required for your investigation. 2 Work out the total volume of solution required (always make a little extra as you may spill some or make a mistake in dispensing). 3 Find the molecular mass of the chemical. (This is the sum of the atomic elements and can usually be found on the side of the container. See Figure 2.16.) 4 Work out the mass of the chemical required that would give the desired concentration in the required volume, which can be calculated using the following equation: C= mass of solute/molecular mass volume of solution Suppose you need to make 200 cm3 (0.2 dm3) of 0.5 mol dm−3 NaCl solution. (Molecular mass of NaCl = 58.44) Using the above equation: 0.5 = mass of solute/58.44 0.2 Therefore mass of solute = 0.5 × 0.2 × 58.44 = 5.844 g 5 Add the weighed chemical to a clean beaker. 6 Then add distilled water – a little less than the final volume required. 7 Stir until all the chemical is fully dissolved. (You may need to heat gently to dissolve some chemicals. Make sure the chemicals do not alter with heating.) 8 Check and adjust the pH, if required. 9 Pour the solution into a measuring cylinder or volumetric flask, as required. 10 Adjust the volume accurately using a Pasteur pipette. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 31 Photocopying prohibited 31 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS EXAMPLE 2.2 To prepare 100 cm3 of 0.1 mol dm−3 NaCl solution: The molecular mass of NaCl is 58.44. To make 1.0 dm3 (litre) of 1.0 mol dm−3 solution, you would need 58.44 g of NaCl. You are only making 100 cm3 of 0.1 mol dm−3. Therefore: 58.44 = 0.5844 100 Round off to two decimal places. You require 0.58 g of NaCl in 100 cm3 of water. Proceed as follows. 1 Label a 100 cm3 beaker 0.1 mol dm−3 NaCl. 2 Weigh out 0.58 g of NaCl, either straight into the beaker or on a piece of filter paper, depending on the balance available to you. 3 Mix the NaCl with distilled water in the beaker until all the salt dissolves. 4 Transfer the solution to a volumetric flask or a 100 cm3 measuring cylinder (volumetric flasks are more accurate) and adjust the final volume, adding the last few cm3 with a Pasteur pipette, and holding the vessel up to your eye level. The bottom of the meniscus should touch the final volume mark. 5 Transfer the solution to a labelled container. EXERCISE 2G The molecular mass of glucose (C6H12O6) is 180. a Describe how to make 250 cm3 of a 1.5% (w/v) solution of sucrose in water. [2] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� The density of glycerol is 1.26 g m−3 b Describe how a top pan balance and a dry beaker could be used to measure 35 cm3 of glycerol accurately using the density information. [4] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 32 9781510482869.indb 32 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation c Describe how you would make 500 cm3 of 0.5 mol dm−3 stock solution. [4] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� Labelling and storing solutions Label all stored solutions/chemicals carefully with the following information: the chemical name, the concentration, the pH if measured and the date when made. Make sure solutions are stored appropriately, either in the refrigerator or a freezer, and allow time for them to come to room temperature before use. Stock solutions Stock solutions are convenient if you are making a range of solutions of various concentrations or for serial dilutions. A stock solution is always made at a higher concentration than the final concentration required and is diluted appropriately. Standard dilutions 1 Decide on the concentration of solutions you need to prepare. 2 Calculate the volume of stock solution required, using the following equation: volume of stock solution required = required concentration × final volume required concentration of stock solution 3 To calculate the volume of distilled water to add, you subtract the volume calculated in step 2 from the final volume required. EXAMPLE 2.3 You wish to make 200 cm3 of 0.1 mol dm−3 NaCl solution using a stock solution of 0.5 mol dm−3 NaCl. Method Using the above instructions, calculate the volume of stock solution required: volume of stock solution required = = required concentration × final volume required concentration of stock solution 0.1 × 200 = 40 cm3 0.5 volume of distilled water required = 200 − 40 = 160 cm3 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 33 Photocopying prohibited 33 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Dilution of stock solution To dilute 10% stock solution to 100 cm3 of each of 5%, 2.5% and 1.25% solutions: 1 Label three beakers 5%, 2.5% and 1.25%. 2 50 cm3 of 10% stock solution made up to 100 cm3 with distilled water will give you 100 cm3 of 5% solution. 3 50 cm3 of the 5% solution made up to 100 cm3 will give you 100 cm3 of 2.5% solution, and so on. If you need to make a whole set of samples, it is best to prepare a table. Label beakers and arrange them in order to avoid confusion. For example, if you wish to prepare 100 cm3 of each of 0.1, 0.2, 0.3, 0.4 mol dm−3 NaCl solution using a 0.5 mol dm−3 solution you can use the volumes given in Table 2.3. Final concentration/mol dm−3 0.1 0.2 0.3 0.4 Volume of stock solution/cm3 20 40 60 80 Volume of distilled water/cm3 80 60 40 20 Table 2.3 EXERCISE 2H Using a 0.5 mol dm−3 solution, how would you make 10 cm3 of the following concentrations: 0.1, 0.2, 0.3, 0.4 mol dm−3? Complete the table. Concentration/mol dm−3 0.1 0.2 0.3 [8] 0.4 Volume of stock solution/cm3 Volume of distilled water/cm3 Serial dilutions l l l l Clearly label the vessel containing each dilution – it is easy to get confused. Solutions must be thoroughly mixed before measuring out volumes for the next dilution. When deciding on the final volume required, allow for the volume removed when making up the subsequent dilutions in the series. Remember to discard the excess from the last vessel in the series if volumes are critical. EXAMPLE 2.4 Prepare 200 cm3 of 10% stock solution. Use this to make up 100 cm3 of each of the following sucrose solutions: 10%, 5%, 2.5%, 1.25%. Method 1 Weigh out 20 g of sucrose in a beaker. 2 Add about 150 cm3 distilled water and dissolve the sucrose. 3 Transfer the solution to a 250 cm3 measuring cylinder. Adjust the final volume using a Pasteur pipette to add the last few cm3. 4 Transfer to a labelled reagent bottle. 34 9781510482869.indb 34 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation Use the stock solution to prepare 100 cm3 of the 5%, 2.5% and 1.25% solutions as follows. 1 Take 100 cm3 of 10% solution, add 100 cm3 distilled water to make a 5% solution. 2 Take 100 cm3 of the 5% solution, add 100 cm3 distilled water to make a 2.5% solution. 3 Take 100 cm3 of 2.5% solution, add 100 cm3 distilled water to make a 1.25% solution. 4 Discard 100 cm3 of the 1.25% solution so that 100 cm3 is left. EXAMPLE 2.5 Use a 10% stock solution to prepare 5 cm3 of the following dilutions: 1%, 0.1%, 0.01% and 0.001%. (It is better to make a little more solution than required.) Method 1 Label four test tubes: 1, 0.1, 0.01 and 0.001. 2 Add 1 cm3 of 10% solution to the test tube labelled 1. Add 9 cm3 of distilled water, mix well. 3 Transfer 1 cm3 from this test tube to the test tube labelled 0.1. Add 9 cm3 of distilled water, mix well. 4 Transfer 1 cm3 from the 0.1 test tube to the test tube labelled 0.01. Add 9 cm3 of distilled water, mix well. 5 Transfer 1 cm3 from the 0.01 test tube to the test tube labelled 0.001. Add 9 cm3 of distilled water, mix well. EXERCISE 2I a Describe how you would prepare a 200 cm3 10% w/v NaCl solution. [2] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� b Complete the table to show how you would prepare the following serial dilutions using the 10% NaCl solution: 1.0%. 0.1%, 0.01%, 0.001%. Concentration/% 1.0 0.1 0.01 [8] 0.001 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 35 ▶ ▶ Volume of stock solution/cm3 ▶ Volume of distilled water/cm3 Photocopying prohibited 35 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS c Explain why the concentrations of the stock solutions in part b will be more accurate if a clean, dry pipette is used each time. [3] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� pH pH is a measure of the concentration of hydrogen ions [H+] in a solution – a measure of the solution’s acidity (from the French pouvoir hydrogène, ‘hydrogen power’). The pH scale runs from 0 to 14 (Figure 2.17). Pure distilled water is neutral, at pH 7. The concentrations of free OH− and H+ ions in pure water are equal. If the concentration of hydrogen ions [H+] exceeds that of [OH−] then the pH is lower than 7, indicating acidity. If [OH−] exceeds [H+] then the pH is higher than 7, indicating a base. l pH affects the solubility of many substances and the activity of most biological systems. The rate of chemical reactions alters with slight changes in pH because the change in pH will alter the shape of the active site of the enzyme, and thus the functioning of the enzymes. l Many compounds act as acids, bases or alkalis. Those that are almost completely ionised in solution are usually called strong acids or strong bases, while weak acids or bases ionise only slightly in solution. sodium hydroxide lime water washing soda 14 very alkaline 13 12 11 10 baking soda blood (7.3–7.5) pure water milk urine (5.0–7.0) black coffee orange juice lemon juice gastric fluid hydrochloric acid 9 8 7 neutral 6 5 4 3 2 1 0 very acidic Figure 2.17 pH values of some common solutions l The pH scale is not SI; nevertheless, it is still widely used in biological sciences. 36 9781510482869.indb 36 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation l pH is determined experimentally using either coloured indicators or an electronic pH meter, which is equipped with a probe sensitive to hydrogen ions (Figure 2.18). 250 200 150 100 Figure 2.18 A portable combined pH meter TIP Remember the following points. l An acid is a compound that acts as a proton donor. l A base is a compound that acts as a proton acceptor. l An alkali is a compound that liberates hydroxyl ions when it dissociates. Since hydroxyl ions are strongly basic, this will reduce the proton concentration. Buffer solutions A solution that resists change in its pH when small amounts of acid or alkali are added is called a buffer solution. Buffers are usually made by mixing weak acid and the soluble salt of the same acid. A buffer of any pH can be made by selecting a suitable acid and the appropriate acid-to-salt ratio. Buffers are common in biological systems because the complex physiological reactions in living organisms are susceptible to changes in pH. Even a very slight change in pH can result in death. For example, the pH of human blood is 7.35; a change of more than 0.3 of a pH unit could be fatal. Controlled variables In scientific investigations there are three types of variable: l independent variable – the variable you can change l dependent variable(s) – the variable you are measuring l variables that you need to control – variables that must be kept the same; if they are not, then the investigation will not be valid. For example, if you wish to investigate the effect of temperature on the rate of an enzyme-controlled reaction, perhaps an investigation to see the effect of temperature on the hydrolysis of starch using amylase. You would perform the investigation at different temperatures, keeping all other variables the same. Therefore, you would: l use the same concentration of substrate (starch) in the same volume of liquid l use the same concentration of enzyme (amylase) in the same volume of liquid l allow them to react with each other for the same length of time l repeat the investigation at different temperatures. In this investigation, the independent variable is the temperature (you can control at which temperature you will perform the investigation). Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 37 Photocopying prohibited 37 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS The dependent variable would be the rate of the reaction, i.e. how quickly the substrate (starch) turns to product (glucose). You could observe this by taking samples at regular intervals and then l testing for starch (disappearance of starch, using the starch test), or l performing Benedict’s test (appearance of product). Controls Many investigations require a control to prove that any reactions occurring in the investigation are, in fact, due to the stated cause and not due to other factors. For example, if you wished to see if what you observed was due to the presence of the enzyme you could substitute the enzyme in your control test tube with water or buffer. All other variables, such as substrate concentration, temperature, volume, pH, timing, should remain constant. The control will then provide a reference point of comparison for the main investigation. EXERCISE 2J In this exercise you will compare the action of immobilised and free enzymes using yeast. Yeast can be immobilised in alginate beads or suspended in a solution. Yeast contains an enzyme called sucrase that hydrolyses sucrose to glucose and fructose. The presence of glucose can be tested for using commercially available glucose test-strips. You will need: l 30 cm3 yeast that has been freshly immobilised in alginate beads* l 30 cm3 of freshly prepared yeast suspension (7 g of baker’s yeast made up to 100 cm3 in warm distilled water) l 12 glucose test strips l 150 cm3 of 5% (w/v) sucrose solution in distilled water l one 25 cm3 measuring cylinder l two 100 cm3 beakers l two 250 cm3 conical flasks l two filter funnels l two discs of filter paper l permanent marker or waterproof labels l stopwatch or access to a wall clock with a second hand * Add 4 g of sodium alginate to 50 cm3 distilled water and mix well. Add 7 g yeast and make up to 100 cm3 with distilled water. In a different beaker, make a 1.5% (w/v) solution of calcium chloride. Take up the yeast-alginate mix into a syringe. From a height of about 20–30 cm, release individual drops from the syringe into the calcium chloride solution. Beads will form. Leave for at least 20 minutes for the beads to solidify. Method 1 Label one beaker ‘free enzyme’ and the other ‘immobilised enzyme’. 2 Add 25 cm3 sucrose solution to each beaker. 3 Add the yeast suspension to the beaker labelled ‘free enzyme’ and the alginate beads to the beaker labelled ‘immobilised enzyme’. 4 Test the contents of both beakers immediately using a glucose test strip. Leave each strip in the liquid for 30 seconds. 5 Repeat this test procedure every 2 minutes until 12 minutes have passed since adding the yeast. 6 Record your results for the presence or absence of glucose in the observations part a. 38 9781510482869.indb 38 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation Observations a Draw one table that shows time and presence or absence of glucose in each beaker. Record your results in this table. [3] b The yeast was present at the same concentration in both beakers. i Describe what your results show about the activity of free and immobilised enzymes. [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ii Explain any difference in the activities. [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� c In a different investigation, the immobilised enzyme can be tested for activity at different temperatures. i State the independent and dependent variable in this investigation. [2] Independent�������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� Dependent����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ii List three variables that must remain the same in this investigation. [3] 1������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 2������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ 3����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 39 Photocopying prohibited 39 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS 2.6 Tests you need to know Starch test 1 Place a small amount of crushed food to be tested into a test tube (or on a tile); add water. 2 Add a few drops of iodine solution (I2/KI). 3 Observe a blue-black colour where starch is present. TIP Do not refer to iodine solution as iodine. Testing for fat and oil 1 Place a small amount of the food to be tested into a test tube and add about 5 cm3 of ethanol/alcohol. 2 Shake the tube to dissolve as much of the food as possible and allow to stand for a few minutes. 3 Decant off the top 2 cm3 of ethanol into a second test tube containing 2 cm3 of water. 4 Look for signs of opacity in the water; the more opaque the water is, the more fat or oil is present. 5 When left to stand it forms a cloudy emulsion. Smear or grease spot test Rub a small amount of sample on filter paper; if the paper becomes translucent when held up to the light, the sample contains fat. Testing for protein (biuret test) Biuret solution is copper(ii) sulfate solution mixed with sodium hydroxide. These should only be mixed immediately prior to use, as mixing them earlier causes precipitation. 1 Place a small amount of the crushed food to be tested into a test tube and add about 2 cm3 of water. Shake to allow as much of the food as possible to dissolve. 2 Add 1 cm3 of biuret solution and shake gently. Or Add 2 cm3 of sodium hydroxide solution and 1 cm3 of copper(ii) sulfate solution and shake gently. 3 Presence of a pink/purple or lilac colour indicates presence of protein. Testing for reducing sugars Benedict’s reagent is used to test for the presence of reducing sugars. It contains a mildly alkaline solution of copper(ii) sulfate, which is blue in colour. The reducing sugar reduces the blue copper(ii) ion to a reddish copper(i) oxide precipitate, the intensity of which is proportional to the amount of sugar present. 1 Mix a small sample of crushed food with about 1 cm3 of water in a test tube and add 2 cm3 Benedict’s solution (Benedict’s solution should be in excess). 2 Place the test tube into the boiling water bath (+80 °C) and leave it there for 5 minutes, agitating gently. 3 Carefully observe and make a note of the sequence of colour changes. Any change from the turquoise blue is indicative of the presence of reducing sugar. The colour changes to light green, yellow, orange or brick red. 40 9781510482869.indb 40 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation Testing for non-reducing sugars TIP Only carry out this test on samples that test negative for reducing sugars. 1 Mix a small sample of the food to be tested with about 1 cm3 of 0.1 M HCl and heat it in the boiling water bath for 2 minutes. (This breaks the disaccharide non-reducing sugars, if present, into monosaccharides or reducing sugars that produce a colour change with Benedict’s solution.) 2 Remove the tube and cool it under a tap until it reaches room temperature. 3 Add small amounts of sodium hydrogen carbonate to the mixture until further additions do not cause any effervescence. 4 Test the pH. This must be neutral. 5 Add about 2 cm3 of Benedict’s solution and test as for a reducing sugar. 6 Place the test tube into the boiling water bath and leave it there for 5 minutes, agitating gently. 7 Carefully observe and make a note of the sequence of colour changes. Any change from the turquoise blue is indicative of presence of reducing sugar. The colour changes to light green, yellow, orange or brick red. A semi-quantitative Benedict’s test Set up a thermostatically controlled water bath to 100 °C or heat a large beaker half-filled with tap water. Bring the water in the water bath or beaker to the boil. Prepare standards using five solutions of known glucose concentration, one of which should be distilled water, 0% glucose and proceed as follows. 1 Label seven clean test tubes 0–6 and a place them in a rack. 2 Add 0.5 cm3 of distilled water to the test tube labelled 0. 3 Add 0.5 cm3 of appropriate glucose solution to each of the labelled test tubes 1–5. 4 Add 0.5 cm3 of solution X into test tube 6, which has an unknown concentration of glucose. 5 Add 5 cm3 Benedict’s solution to each test tube. Mix well. 6 Place all the test tubes into the boiling water bath for 5 minutes, remove from water bath. 7 Use a colorimeter to record the optical density of each sample, reading against the sample 0. 8 Prepare a table and record the optical density of each tube against its concentration. 9 Plot a graph of % glucose concentration against optical density/arbitrary units. Read the concentration of glucose in solution X from your graph. You can also filter the precipitate onto a weighed filter paper and weigh the precipitate formed. The precipitate is proportional to the concentration of the reducing sugar. Qualitative vs quantitative tests Qualitative analysis is primarily exploratory research. Qualitative results are observations with no numerical value – for example, colour changes, which are effectively subjective. If you were asked to carry out qualitative analysis on some food samples you would need to find out if the food tests were positive or negative – for example, whether or not each sample was a carbohydrate, protein or fat. Quantitative analysis (also known as numerical data) would involve figures – for example, sample X has more carbohydrate than sample Y; you may need to work out exactly how much more. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 41 Photocopying prohibited 41 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS EXERCISE 2K a Describe how you would carry out a qualitative analysis of an apple to find out if it contains l reducing sugars l proteins l fat. [10] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� b Gum acacia (Senegalia senegal) is a small thorny tree (Figure 2.19) that grows in semi-desert regions of Sudan, India, Oman and Pakistan. It is considered to be a very useful plant as cattle can graze on its leaves, seed pods and seeds; humans can consume the seeds. Figure 2.19 Gum acacia (Senegalia senegal) A student wants to find out if the seeds contained reducing sugar, protein or fat. i Name the tests the student needs to carry out. [3] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 42 9781510482869.indb 42 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation ii Describe in detail how you would estimate which part of the plant (leaves, seed pods or seeds) have the highest content of reducing sugar. Your method should give all the necessary details for another student to be able to reproduce your results. [10] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� TIP l l A qualitative test can tell you whether the test is positive or negative. A quantitative test tells you by how much. The colorimeter A colorimeter is a piece of equipment that measures the quantity of light that is transmitted by (passes through) a coloured solution or that is absorbed by a coloured solution. The measurement of either transmittance or absorbance is made in comparison to a ‘blank’ which consists of the solvent only and none of the coloured solute. Transmittance or absorbance are usually given as a decimal or percentage. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 43 Photocopying prohibited 43 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS The ‘blank’ solution is placed into a small square-section tube called a cuvette, which is made from glass or transparent plastic. The cuvette is then placed inside the colorimeter, usually under a light-tight lid. Care must be taken to ensure that the cuvette is inserted the correct way. If the sides of the cuvette are not all the same, then those sides that are most transparent should be facing the light source and the detector. Some colorimeters need time to warm up, so that the reading is stable and comparable over time. Whether or not this is the case, it is better not to switch the colorimeter off between readings. With the ‘blank’ solution in place, the colorimeter is set to zero. After this, the test solution can be placed in a different, clean cuvette and inserted into the colorimeter, in place of the ‘blank’. The ‘blank’ should be inserted again between every sample reading to ensure the colorimeter reading has not drifted away from zero. If it has, then it can be adjusted again back to zero. EXERCISE 2L In this activity, you will use a colorimeter to follow the course of an enzyme-catalysed reaction. You will use a solution of milk protein, which is white and opaque. The milk protein is broken down by protease, which turns the solution clear and transparent. Therefore, we would expect the absorbance of the enzyme-substrate mixture to decrease with time. The colorimeter is a useful way to measure this change. Later, you have the option to use enzyme concentration, substrate concentration, temperature or pH as the independent variable for the rate of the enzyme-catalysed reaction. You will need: l 100 cm3 of a 2% (w/v) solution of skimmed milk powder in distilled water l 10 cm3 of a 1% (w/v) solution of trypsin (a protease) in distilled water l 10 cm3 of a 1% (w/v) solution of boiled trypsin in distilled water l access to distilled water l test tubes and rack l graduated pipettes or 10 cm3 syringes l colorimeter and 2 cuvettes l stopwatch or access to a wall clock with a second hand l water baths at different temperatures and thermometer (optional) l buffer solutions at various pH values (optional) Preliminary experiment procedure The aim of the preliminary experiment is to find a set of conditions that give a suitable rate of reaction. The reaction must not proceed too quickly for us to measure. Nor must it proceed too slowly. Start by bringing the 20 cm3 of the substate and 20 cm3 of the enzyme solution to a temperature of 25–35 °C. Add 1 cm3 of the enzyme to the substrate solution and mix well. Keep this mixture at your chosen temperature and check the intensity of the colour of the solution just by looking. Ideally, you should be able to see a decrease in the intensity of the white colour within about 5 minutes. Your reaction mixture should have a volume of about 40 cm3. 44 9781510482869.indb 44 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 2 Manipulation, measurement and observation Main experiment procedure 1 Prepare a ‘blank’ by mixing the same volumes of substrate solution and boiled enzyme as you have chosen for your experiment. The volume of boiled enzyme in the ‘blank’ should be the same as that of fresh enzyme in the experiment. 2 Place a sample of this ‘blank’ mixture into a cuvette and use this to set the absorbance of the colorimeter to a maximum, or a suitable high starting value advised by your teacher. 3 Start your enzyme-substrate reaction as you planned from the preliminary experiment. 4 Immediately remove a sample of the mixture and record its absorbance in the colorimeter. 5 Discard this sample and wash the cuvette. 6 Check the colorimeter reading again using the ‘blank’ to make sure it has not changed. 7 Remove another sample after 1 minute and record its absorbance. 8 Repeat steps 5, 6 and 7 until you think the reaction has stopped. Observations a Draw a table to show your results. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 45 [3] Photocopying prohibited 45 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS b Plot a graph of your results on the grid below. Put time on the x-axis and absorbance on the y-axis.[4] c Explain the trend in your results. [4] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� If you have time, you could plan and carry out an investigation to determine how the rate of reaction between trypsin and the milk protein depends on any of these: l enzyme concentration l substrate concentration l temperature l pH 46 9781510482869.indb 46 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM Presentation of data and observations 3 Once you have performed your investigation, you will be expected to present and analyse the results and your conclusions in a specified manner. Your data should be presented in a clear and concise form. There are multiple ways of presenting data, such as tables, various forms of graphs and drawings. It is good practice to record results as they are determined. Raw data (data you have collected during the investigation) presented in a simple table make the results easier to understand. There are strict rules to follow when presenting data in tables and graphs. 3.1 Presenting data in tables Height of seedlings for germinated seeds Water Trial De-ionised Tap water Trial average seedling Overall average seedling height/mm ± 0.5 mm height/mm ± 0.5 mm 1 13.0 2 11.8 1 57.8 2 61.1 13.4 59.5 Table 3.1 A table provides a neat and accurate record of your data values l l l l l l l l l l Use a sharp pencil to draw a large, clear table. Draw an appropriate numbers of rows and columns. Include separate columns for averages, standard deviation, etc. The rows and columns must have appropriate and precise headings, and units where required. Include uncertainty, if appropriate. The seedling heights in Table 3.1 are shown as ± 0.5 mm. This means that they could be up to 0.5 mm taller or shorter. The absolute uncertainty here is 1.0 mm as this is the range over which we are uncertain. Never put units in the body of the table (only in headings). The independent variable is usually in the left-hand column. In some cases results may be presented horizontally, in which case the independent variable is the first row. Each column must be arranged in a logical and meaningful way. Avoid giving unnecessary information. Quote values to a sensible number of significant figures. For example, in your average column, if the number of bubbles produced comes to 2.93, round it to 3.0 – you cannot have part of a bubble. If several tables are used they should be numbered so they can be quoted in the text. If several repeated readings were taken, the mean value should be calculated and presented in the table. 3.2 Presenting data in graphs Unless specified, choose carefully which type of graph would be best for your data. In many cases you will generate continuous data, so your graph will be a line graph. Line graphs l l l Line graphs should provide an immediate visual summary of your tabulated data, which should be relatively easy to understand. Draw a large graph, using a sharp pencil, with appropriate scales on both axes. Choose divisions that are easy to read, for example each one small square = 2 units rather than 3. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 47 Photocopying prohibited 47 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS l l l l l l l l l l l l Graphs should be self-contained – they should have all the information necessary to convey the appropriate message without having to refer to the text. Consider the layout and scale of the axes carefully, so that the plotted points cover at least half of the available grid in both x and y directions. In some cases, it may be inappropriate to start the scale at zero. In such cases show the break as —//—, just beyond zero. Most graphs illustrate the relationship between two variables (x and y) and have two axes at right angles. Make sure you have the axes the right way around. There is no actual rule, but it is accepted practice to put the independent variable along the x-axis. The dependent variable, the one measured, goes along the y-axis. Each axis must have a descriptive label showing what it represents, together with the appropriate unit measurements. Each point on the graph should be visible either as a small cross or a small dot with a circle around it, plotted exactly on the intended spot. Symbols must be exact and must not exceed half a small square in size. The points marked on the graph are a record of the actual observations made and should be joined by straight lines using a ruler, by a smooth curve or in some cases by a ‘line of best fit’ (Figure 3.1). Plot anomalous results, but ignore them when you are joining the dots. If you are required to read a value or values from the graph, carefully draw dashed straight lines to the axes to indicate the coordinates. If there is more than one set of data on the graph, make sure to distinguish each line clearly, for example you could plot one set of data using circled dots and the other with crosses, and use a key to label them. If there are repeats you could add range bars, to show the spread of data (draw a line above and below your data point to show highest and lowest values, then join with a ruler through the point), which will indicate the variability of the data. Range bars typically extend the same distance either side of a mean point. 120 Time/days 0 2 4 6 8 10 12 14 16 18 20 22 24 26 E Mean height in mm 100 80 60 I 40 20 0 10 20 30 Time in days Mean height/mm 0 1 2 10 22 38 60 80 90 100 108 110 110 110 Key: I = interpolation E = extrapolation (See text below) Figure 3.1 Graph showing mean height of oat seedlings l l If several graphs are plotted, they should be numbered so that they can be referred to in your analysis. Only the points on the graph represent actual data. Estimations of other values can be obtained from reading off coordinates at any point on the line; this is called interpolation. Similar coordinates outside the range of the graph can also be estimated by extending the lines on the graph, a technique known as extrapolation. No extrapolation should be carried out unless this can be assumed from the data. Extrapolation and interpolation should only be carried out if you are asked to do so, and should be shown with broken lines. Figure 3.1 shows an extrapolated point (E). 48 9781510482869.indb 48 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 3 Presentation of data and observations TIP Key things to remember when plotting a line graph l Identify and label the independent (x-axis) and dependent (y-axis) variables correctly. l Label both axes with appropriate units. l Choose an appropriate scale for each axis. l Plot data points accurately and visibly. l Join the points with a ruled line unless instructed otherwise. Some other types of graph that may be useful are described in this chapter. EXERCISE 3A A student carried out an investigation into the rate of diffusion from a Visking tubing bag into surrounding distilled water. The student took samples from the Visking tubing bag at regular intervals and measured the glucose concentration. The results are shown in Table 3.2. Time/mins Glucose concentration/arbitrary units (au) 4 0.27 8 0.13 12 0.07 16 0.03 20 0.02 Plot a graph of the data in Table 3.2 on the grid provided. Use a sharp pencil for drawing your graph. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 49 [4] Photocopying prohibited 49 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Histograms The histogram (Figure 3.2) is the most suitable form of data presentation when the number of data points is too few to allow a trend line to be drawn. The x-axis represents continuous values of independent variables grouped into small classes of equal size, for example the leaf size for a particular plant. The columns are adjacent to each other. Histograms can also be used to represent frequency data or distributions. 16 Number of leaves 14 Leaf length/mm Numbers 0–50 51–100 101–150 151–200 201–250 251–300 301–350 351–400 401–450 451–500 2 5 6 12 16 14 8 5 3 2 12 10 8 6 4 2 0 50 100 150 200 250 300 350 400 450 500 Leaf length/mm Figure 3.2 Histogram Bar graphs/charts 350 Banana Bar graphs or charts (Figure 3.3) are drawn when the data are discontinuous or categoric, that is, one of the two variables is not numerical, for example the number of flowers per plant or the energy values of different fruits. They should be made up of narrow blocks of equal width, which do not touch. The blocks can be arranged in any order. 300 Fruit Melon Grapefruit 100 Orange Plum 150 Peach Cherry 200 Apple kJ / 100 g fresh fruit 250 50 0 kJ/100 g fresh fruit Banana 318 Apple 193 Cherry 193 Peach Plum 155 134 Orange 96 Grapefruit 92 Melon 29 Fruits Figure 3.3 Bar graph 50 9781510482869.indb 50 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 3 Presentation of data and observations EXERCISE 3B A student carried out an investigation to determine the percentage by mass of lipid in the seeds of five species of plant, A–E. The results are shown in Table 3.3. Species from which seed was taken Lipid content of seed/% A 39 B 25 C 13 D 31 E 6 Table 3.3 Draw a bar chart of the data in Table 3.3 on the grid provided. Each bar should be separated for each species of plant. Use a sharp pencil for drawing bar charts. [4] Frequency diagrams Frequency diagrams are drawn when plotting the frequency of distribution with continuous data (e.g. frequency of leaves of different length). The blocks should be drawn in decreasing order or in increasing magnitude, and they should be touching. Pie charts You will not be expected to draw a pie chart in a practical paper, but you may be presented with one as part of a question and required to answer questions about the data it represents. Pie charts are generally drawn with sectors in descending order, beginning at noon and proceeding clockwise (Figure 3.4). If a comparison is to be made between two pie charts, the second chart should keep the same sequence of segments as the first, whatever their relative sizes. Comparison between pie charts can also be made by making the area of the circle proportional to the size of each sample. Preferably, pie charts should contain no more than six segments. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 51 Photocopying prohibited 51 31/08/21 1:55 PM Flies 4 Wasps 2 AS LEVEL PRACTICAL SKILLS Arthropods on a Eucalyptus tree Bugs 6 Bark lice 8 Ants 36 Spiders 15 Ants 36 Bark lice 8 Bugs 6 Flies 4 Spiders 15 Wasps 2 Figure 3.4 Pie chart EXERCISE 3C A student investigated the effect of changing surface area to volume ratio on diffusion rate. The student was given a large block of agar that had been soaked in Universal Indicator solution. The student cut cube-shaped blocks from the agar of different sizes. The blocks were placed in different beakers and 2.0 mol dm−3 hydrochloric acid was poured over each block. The student timed how long it took for the Universal Indicator in each block to change colour completely from green to red. The student’s results are shown in Table 3.4, which is not complete. Complete Table 3.4 by calculating: a the volume of each cube [1] b the surface area of each cube [1] c the surface area to volume ratio of each cube [1] d the rate of diffusion using the equation: rate of diffusion = Side length of agar cube/mm distance from face to centre of cube in mm time taken to change colour Volume of agar cube/mm3 Surface area of agar cube/mm2 Surface area to volume ratio [1] Time taken for whole cube to change colour/s 3 49 5 117 7 162 9 583 11 930 Rate of diffusion/mm s−1 Table 3.4 52 9781510482869.indb 52 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 3 Presentation of data and observations e Plot a graph to show how the rate of diffusion depends on the surface area to volume ratio of these cubes. Include a line or curve of best fit. [4] f Hydra is a small multicellular animal that lives in water. Hydra has a hollow, tube-shaped body and a maximum length of 10 mm. Hydra has no transport system. Use the results to suggest why Hydra does not need a transport system, whereas larger multicellular animals, such as fish, that live in water do need a transport system. [3] ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 3.3 Writing practical procedures The procedure should be written in a logical sequence of events under appropriate headings. The headings are as follows. Title The title should be a clear statement outlining the aim, for example ‘Investigation to determine the water potential of potato tuber’. The title should briefly and concisely outline what you intend to find out or prove. Procedure The procedure should be written in a logical manner so that another scientist using this information should be able to carry out the same investigation without having to ask questions such as: How much? How long? At what temperature? For example: 1 Label five test tubes 1, 2, 3, 4 and 5. 2 To each, add 5 cm3 Benedict’s solution. 3 … Numbering each stage is helpful, especially for those carrying out the investigation. It is easy to follow, and it is helpful to tick each point as it is completed. It also allows you to say, for example, ‘repeat stage 2’ and avoid duplicating detail. If you have forgotten a stage, you can insert at the correct point in the procedure and renumber the stages that follow. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 53 Photocopying prohibited 53 31/08/21 1:55 PM 4 Practice questions 1 a Figure 4.1 is a slide of a stained transverse section through a plant stem. Figure 4.1 TS dicotyledon stem You are not expected to be familiar with this specimen. An eyepiece graticule scale can be used to measure the layers of tissues and to help draw a plan diagram with the correct shape and proportions of the tissues, without needing to calibrate the eyepiece graticule scale. You are required to use a sharp pencil for drawings. i Select one vascular bundle from one of the corners of the specimen on K1. The grid and your eyepiece graticule should help you draw the vascular bundle with the correct shape and proportions of the tissues. Draw on the grid opposite a large plan diagram of the vascular bundle you have selected. 54 9781510482869.indb 54 Photocopying prohibited [5] Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 4 Practice questions Use one ruled label line and label to identify the xylem. ii Observe the tissue (cortex) between the epidermis and the vascular bundle. Select one group of four cells made up of: l two cells from the epidermis l two cortex cells which touch each other and at least one of the epidermis cells. Make a large drawing of this group of four cells. Use one ruled label line and label to identify the cell wall of one cell. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 55 [5] Photocopying prohibited 55 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS b Figure 4.2 is a photomicrograph of a stained transverse section of a stem of a different plant species. Figure 4.2 TS of a stem i magnification ×125 Calculate the actual length of the line A shown on Figure 4.2. You may lose marks if you do not show your working. actual length …………. µm 56 9781510482869.indb 56 Photocopying prohibited [2] Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 4 Practice questions ii A student observed a different plant of the same species shown in Figure 4.2. The student determined the ratio of the radius of the stem to the length of one air space as 266:82. However, a ratio may be simplified to the smallest possible whole number on each side. In this example, both sides of the ratio 266:82 are divisible by 2, so the simplest ratio for the measurements taken by the student is 133:41. The actual radius of the stem in Figure 4.2 is 825 µm. Determine the simplest ratio of the radius of the stem in Figure 4.2 to the length of an air space (line A). ratio ................................ [1] iii Suggest a habitat where this plant might grow and one observable feature shown in Figure 4.2, which enables it to live in this habitat. habitat: ……………………………………………………………. feature: ……………………………………………………………. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 57 [1] Photocopying prohibited 57 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS c Prepare the space below so that it is suitable for you to record observable differences between the specimen on Figure 4.1 and Figure 4.2. Record your observations in the space you have prepared. [4] [Total: 18] Cambridge International AS & A Level Biology (9700) Paper 33 Q2, October/November 2015 58 9781510482869.indb 58 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 4 Practice questions 2 All new cells come from previously existing cells. New cells are formed by the process of cell division known as mitosis. To study the stages of mitosis, you need to look for tissues where there are many cells dividing. a State precisely where in a plant you would observe mitosis taking place. [2] ....................................................................................................................................................................................................... b Describe the procedure to observe mitotic cell division in plants. [8] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... c Give three reasons why this particular tissue was chosen to observe mitosis. [3] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... d List hazards and safety precautions for this procedure. [2] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 59 Photocopying prohibited 59 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS e Mitosis is divided into the following stages: l prophase l metaphase l anaphase l telophase. i Identify the stages of mitosis in Figure 4.3. [4] Figure 4.3 Mitosis ii Find a cell at metaphase and make a clear labelled diagram. [4] iii Mitotic index is defined as the ratio between the number of cells in a population undergoing mitosis and the total number of cells in a population. Calculate the mitotic index using Figure 4.3. Show your working. [4] [Total: 27] 60 9781510482869.indb 60 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 4 Practice questions 3 Enzyme E hydrolyses (breaks down) starch to reducing sugar. You are required to investigate the effect of temperature (independent variable) on the activity of E by recording the time taken for the starch to be hydrolysed (dependent variable). a You will investigate the hydrolysis of starch: l at room temperature l at other temperatures, starting at 40 °C to a maximum of 80 °C. i State the temperatures, other than room temperature, that you will investigate. [2] ................................................................................................................................................................................................. You are provided with: contents hazard volume/cm3 E 2.0% enzyme E solution harmful 20 S starch solution none 50 iodine iodine solution none 15 labelled You are advised to wear suitable eye protection. Iodine solution is a stain. If E or iodine comes into contact with your skin, wash it off immediately under cold water. The enzyme and starch solutions are mixed together and then a sample of the mixture is removed every 15 seconds and tested for starch. The end-point of the hydrolysis of the starch is when a sample does not change the colour of iodine solution, showing that all of the starch has been hydrolysed. Figure 4.4 shows a spotting tile with drops of iodine solution that have been labelled with the time, in seconds, that they will be used to test a sample. 2 drops of iodine solution 15 30 45 60 75 90 105 120 135 150 165 180 Figure 4.4 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 61 Photocopying prohibited 61 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS Proceed as follows: 1 Set up a water-bath at room temperature. 2 Set up a spotting tile, as shown in Figure 4.4. 3 Measure the temperature of the water-bath and record its temperature in Table 4.1, in (a)(ii). 4 Label one test-tube E and label another test-tube S. 5 Put 1 cm3 of E into the test-tube labelled E. 6 Put 3 cm3 of S into the test-tube labelled S. 7 Put both of these test-tubes into the water-bath. Leave for 1 minute. 8 After 1 minute, remove both of the test-tubes and put them into a test-tube rack. 9 Heat the water-bath to 40 °C, which is the next temperature that you will test. Continue with step 10 while the water is heating. Read step 10 to step 13 and (a)(ii) before proceeding. Note: as soon as the starch is added to the enzyme the reaction will start. 10 Put the starch solution from test-tube S into the enzyme solution in test-tube E and mix. Start timing. 11 After 15 seconds, use the glass rod to remove a sample of the mixture and stir it into the iodine solution labelled ‘15’ on the spotting tile, as shown in Figure 4.4. Wipe the end of the glass rod with a paper towel. Continue removing and testing samples every 15 seconds until the colour of the iodine solution does not change, up to a maximum of 180 seconds. Each time, use the next labelled iodine solution on the spotting tile and wipe the end of the glass rod clean between tests. 12 Record the colour of the iodine solution for each test in Table 4.1, in (a)(ii). These are the raw results. 13 Use a paper towel to wipe the spotting tile clean. 14 Repeat step 2 to step 13 for all of the temperatures you stated in (a)(i), each time heating the waterbath in step 9 to the next temperature that you are going to use. 62 9781510482869.indb 62 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 4 Practice questions ii Record your raw results by completing Table 4.1, using the letters stated below for the colours. You will observe a range of colours depending on the concentration of starch in the sample. You will see some of the following colours and should record the colours by using these letters. BB blue/black DB dark brown DP dark purple B brown P purple PB pale brown PP pale purple Y yellow/orange (colour of iodine solution at start) [3] colour of iodine solution time/s 15 30 45 60 75 90 105 120 135 150 165 180 temperature/°C Table 4.1 iii Prepare the space below and, using your raw results from Table 4.1 in (a)(ii), record the time taken for enzyme E to hydrolyse all of the starch at each temperature. Record ‘more than 180’ if there is still starch present at 180 seconds. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 63 [4] Photocopying prohibited 63 31/08/21 1:55 PM AS LEVEL PRACTICAL SKILLS . iv Describe two significant sources of error in this investigation. [2] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. v A student using the same enzyme concluded that the source of enzyme E was not from humans. [2] Explain how your results support the student’s conclusion. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. vi This procedure investigated the effect of temperature on the activity of enzyme E, using the time taken to hydrolyse starch. To modify this procedure for investigating another variable, temperature (the previous independent variable) would need to be standardised. Describe how the temperature could be standardised. ................................................................................................................................................................................................. ................................................................................................................................................................................................. Think about how you could modify this procedure to investigate the effect of enzyme concentration (the new independent variable) on the time take to hydrolyse the starch. Describe the modifications needed to investigate the effect of enzyme concentration.[3] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. 64 9781510482869.indb 64 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 4 Practice questions b A student investigated the effect of temperature on the activity of an enzyme extracted from an organism living in very low temperatures. All the variables were standardised at each temperature. The student’s results are shown in Table 4.2. temperature/°C activity of enzyme/arbitrary units 4.0 19.00 7.0 17.50 10.5 14.75 12.0 3.25 19.5 0.75 Table 4.2 Use a sharp pencil for graphs. i Plot a graph of the data shown in Table 4.2. [4] ii Describe the effect of temperature on the activity of this enzyme. [1] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. [Total: 21] Cambridge International AS & A Level Biology (9700) Paper 33 Q1, February/March 2017 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 65 Photocopying prohibited 65 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS 5 Planning an investigation 5.1 Developing your own procedure If you have been tasked with developing your own procedure, you may be required to do the following. l Define the problem, using information provided about the aims of the investigation. l Provide a hypothesis to test and suggest a workable method in both familiar and unfamiliar contexts. l Use knowledge and understanding of the topic under consideration to make a quantitative and testable prediction of the likely outcome of the investigation. l Provide a description, including diagrams, of how the investigation would be performed. l Provide a list of apparatus and equipment of appropriate precision suitable for the task. l Identify the controlled and uncontrolled variables. l Describe how the variables will be controlled. l Describe suitable controls. l Evaluate risks and hazards, suggesting suitable safety precautions to be taken. l State how the data will be collected and presented. l Explain how the data will be analysed; propose appropriate statistical analysis (see Chapter 6). If you are planning an investigation to conduct in class, here are some steps to follow: 1 Identify the question you need to explore. For example, you know that some seeds only germinate in the absence of light, so usually need to be covered in a thin layer of soil, while others require the presence of light, so have to be on top of the soil. Write out a clear statement. This could be: To see if light affects germination of lettuce seeds. 2 Discuss the question with your teacher; carry out research in textbooks and on the internet. 3 Put forward a hypothesis. A hypothesis is a clear statement that specifies what you think will happen, based on your existing knowledge. For example, your hypothesis could be: Lettuce seed germination requires absence of light. 4 For statistical analysis of your data, you could write a null hypothesis. A null hypothesis usually negates your hypothesis and you go on to disprove it. Your null hypothesis could be: Exposure to light will not affect lettuce seed germination. Your investigation will prove or disprove your null hypothesis. 5 From your research, you would need to decide: l the species of seeds to use l the number of seeds you will grow in light and in dark l the suitable number of repeats you will carry out l the medium you will grow them in – for example, on moist filter paper, in Petri dishes, in soil l how you will grow the seeds – on top of the soil or x mm below the surface l the temperature at which you will keep the seedlings l how you will record your observations, for example will the appearance of the first pair of leaves or the emergence of the shoot be recorded as germination l how you will analyse and evaluate your data. 6 Make a list of your requirements: l quantities of chemicals (if any); water (most seeds germinate in tap water) l glassware, for example pipettes, beakers, measuring cylinders, Petri dishes l any other equipment, for example a camera to record results visually. 66 9781510482869.indb 66 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 5 Planning an investigation 7 Outline a risk assessment and how you would mitigate any hazards. 8 You should also be able to point out the limitations of your procedure. This will depend upon: l the time available – you may not be able to do as many repeats as you would like l the equipment available – you may not have the most suitable equipment l the resources available – you may not have sufficient funds l variables beyond your control, for example weather, time of year in an ecological study, age or weight of individuals in a study, lighting, location. 9 Write out a procedure. Show your teacher your proposed plan. They will be able to help you identify any flaws in your plan and how to overcome them. Once you are satisfied with your plan, you will need to give the list of requirements to your teacher or lab technicians to ensure you have everything you need. Variables In any investigation there will be variables, some of which you can control and others that you cannot. Note which variables you will need to keep constant – for example, species of seeds, medium for growth, temperature, location – and which variable will change, such as the height of the shoot over time. When you plan an investigation you have to define and establish some of these parameters from the start. The parameter that you can change and manipulate is referred to as the independent variable (for the height of seedlings over time that would be the time interval). You have control as to how often you will take the measurements, for example daily, every other day, weekly. The parameter that you will be measuring, which is changing – in this example, shoot height – is the dependent variable. EXERCISE 5A The effect of light intensity on the rate of photosynthesis can be measured using a photosynthometer by the evolution of oxygen from an aquatic plant such as Canadian pondweed, Elodea, or Hydrilla at different levels of illumination. A simple approach to doing this is to illuminate the plant using a lamp set at various distance (10, 20, 30,40, 50, 60, 70 and 80 cm) from the plant. The volume of gas evolved per unit time can be measured by collecting the gas evolved. plastic tube syringe heat trap light stand bubble of gas being measured bubble of gas collecting end of capillary tube Elodea capillary tube stop clock dilute hydrogencarbonate solution Figure 5.1 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 67 Photocopying prohibited 67 31/08/21 1:55 PM Rate of photosynthesis/ bubbles of oxygen released A LEVEL PRACTICAL SKILLS 2 4 6 8 10 12 Light intensity/arbitrary units 14 16 Figure 5.2 The rate of photosynthesis can be calculated by plotting a graph of the volume of oxygen produced against light intensity. a State the dependent variables in this investigation. [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� b Explain why sodium hydrogen carbonate was added to the beaker of water. [1] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� c In any investigation, some variables must be kept constant, except the one you are investigating. Suggest two variables that may be difficult to control in this investigation. [2] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� d Observe the graph of the results obtained by some students, Figure 5.2. Describe how light intensity affected the rate of photosynthesis. Explain your answer. [4] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 68 9781510482869.indb 68 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 6 Analysis and conclusions 6.1 Analysing data Dealing with data l l l l l l l You may be asked to calculate the standard deviation of the data; if so, you will be given the formula, but you need to know how to use it and what it means. l High standard deviation → low reliability (values deviate a lot from the mean – they are spread out). l Low standard deviation → high reliability (values do not deviate much from the mean – they are clustered). You may be asked to calculate the standard error and/or 95% confidence intervals (95% CI). You may also be asked to use the following statistical analyses: l chi-squared l t-test l Pearson’s linear correlation l Spearman’s rank correlation. You are expected to select the appropriate statistical tests for your analysis. Although you will usually be told to use a specific test and provided with the appropriate formulae, you do need to know how to use the formulae. You must be able to identify anomalies (outliers, unexpected or odd readings) and exclude them from the calculation. You must think if: l the suspected anomaly was recorded in error l the suspected anomaly was recorded in different conditions to the other values. If the anomaly is spotted at the time of the experiment, time permitting, you should repeat the reading. If that is not possible, the anomaly should be omitted from the data set and the mean should be calculated without this value. You should, however, show it on your graph and explain in your write up what may have caused the anomalous reading. If the expected results are not obtained (as is quite often the case) do not get discouraged. It is quite normal. Investigations must be repeated to get good results. However, you must try to explain what went wrong and how you could overcome the problem to prove or demonstrate the aim of your investigation. You may be asked to explain how you could make your results more accurate and reliable (see Section 6.3), and how you could improve your investigation further. Interpreting graphs Interpretation is important. Describe the trends, i.e. the overall pattern of your results, using vocabulary such as linear relationship, rises/falls sharply, steady increase, plateaus, exponential increase. Consider your responses carefully and ensure that you express yourself clearly, using appropriate scientific terminology. Using suitable terms and vocabulary can turn a vague answer into a much more focused response. l Know the difference between describe and explain. Describe means to say what you see, for example, ‘it rises sharply between 10 s and 50 s, after which the curve plateaus’. Explain means to explain why you think it is happening. l Use a ruler to read off graphs precisely, quoting the units. l If the graph has several phases, it is easier to describe if you label each phase (Figure 6.1). Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 69 Photocopying prohibited 69 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS 120 Mean height in mm 100 80 60 40 20 0 Phase A 10 20 30 Phase B Phase C Time in days Figure 6.1 Graph showing different phases l If asked to compare/contrast, always mention both things in each sentence – for example, ‘X is much bigger, whereas Y is smaller’ – or, better still, make a table to ensure comparative points. Consider both comparable differences and similarities between them. 6.2 Drawing conclusions l l l l l Describe and explain what happens to the dependent variable as you change the independent variable. Describe and explain the trends in your graph line as it changes gradient. Make sure to include the appropriate units when discussing data. Explain the results using your relevant biological knowledge. What do your results show and why do you think this is so? Determine whether the evidence supports what you started out to investigate, or whether it supports the hypothesis you set out to test. Evaluate validity by examining flaws and irregularities in your method. Suggest explanations for observations and trends, and make predictions from the patterns and trends in data. Suggest improvements to and explanations for flaws in the procedure, and how to move forward with the investigation. 6.3 Evaluation Reliability l l l l Reliability considers the spread of the data from the mean; this can be shown on the graph as error/range bars. Reliability can be improved by carrying out more repeats of your measurements, as this will reduce the effect on the mean of any anomalous results. You should aim to repeat a minimum of three times – preferably five times. A reliable method is one that produces reproducible results. Calculate the standard deviation of the data or look at how closely grouped the repeated measurements are. Accuracy and precision l l l Accuracy is an assessment of how close the obtained value is to the true value. Accuracy can also be assessed by commenting on how calculated values compare to known values or published data, and how the trend line compares to the theoretical trend line/predicted line, or how close the values are to a line of best fit. The accuracy of a set of data is influenced by the choice of apparatus (see ‘Limitations’ section) and how it is used, as explained later. 70 9781510482869.indb 70 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 6 Analysis and conclusions l l l Accuracy incorporates precision, which is to do with the size of the gradations on the scale of the measuring equipment used. Precision measures how closely two or more measurements agree with each other. Precision is sometimes referred to as ‘repeatability’ or ‘reproducibility’. A highly reproducible measurement tends to give values that are very close to each other. Validity l l Validity is the confidence that can be placed on the conclusion, given the level of accuracy and reliability as well as the sources of error and limitations within the strategy. The outcome of a statistical test can be used to assess the confidence that can be placed in a conclusion. Limitations The limitations of an investigation are those factors of your methodology that impacted or influenced the interpretation of your results. These may have been design faults due to accuracy of the equipment available, bias, human reaction times, insufficient sample size for statistical measurement or beyond your control. For example, in an investigation to measure the volume of oxygen evolved during photosynthesis, it is not possible to consider the volume of oxygen being used by the plant for respiration or control the changes in temperature of the water by altering the distance of lamp from the plant, or inability to repeat due to time constraints. The bubbles may appear so fast that you miss counting some of them. These can be described as design faults and because they are inherent, they will affect each run and replicate equally throughout the investigation. You can suggest how to address these limitations, instead of counting the number of bubbles, use a data logger, use a gas syringe, and allow the experiment to proceed for longer and repeat several times. To maintain temperature in the test tube, arrange to place the test tube in a cooling jacket with circulating water bath. In ecological studies there are many conditions beyond your control. For example, you may have planned an investigation to count to number of insects in a particular field, and the farmer next door sprays his field with insecticides, or it is a particularly windy day. You should mention the limitations in the discussion of your results but don’t simply list the limitations, justify the choices you made and focus on techniques to minimize their impact, and suggest how they can be addressed in further studies. A good phrase to use is: ‘The findings of this investigation have to be seen in light of some limitations.’ EXERCISE 6A In 2019, a new type of coronavirus called COVID-19 infected many people throughout most countries. All affected countries reported the number of cases of COVID-19 to the World Health Organization. Table 6.1 shows the total numbers of reported cases from some countries by the end of April 2021. Country Total number of COVID-19 cases up to end of April 2021 USA 33 476 781 Brazil 15 184 790 Indonesia 1 713 684 Iraq 1 112 785 Sweden 1 007 792 Barbados 3 942 Tanzania 509 Table 6.1 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 71 Photocopying prohibited 71 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS Explain the limitations of comparing the total numbers of COVID-19 cases from different countries using data such as those in Table 6.1. [6] �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� Errors It is important to identify main sources of error in your investigation. These can be systematic or random errors. Systematic errors These are errors that are the same throughout the investigation – for example, they could arise due to a faulty measuring device, imperfect observation methods, incorrect calibration or always reading liquid measurements from the top of the meniscus. Such an error is usually constant, or yields results proportional to the measurement’s true value, so systematic errors may not affect the trend in results. Random errors Random errors are ‘one-off’ events that can affect some, but not all, of your results – for example, not equilibrating mixtures to the correct temperature before use, not rinsing the pipette each time, or reading liquid measurements from the top of the meniscus in some cases and from the bottom of the meniscus in others. Random errors will affect the trend. For measurement with an instrument, the reading error is ± one-half of the smallest unit. Percentage error A percentage error calculation indicates how close a measured value is to the true value. It is calculated by working out the difference between the experimental and theoretical values, divided by the theoretical value, multiplied by 100. 72 9781510482869.indb 72 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions 1 Liver cells were homogenised (broken up) to release the cell organelles. The homogenate was separated by cell fractionation into: complete homogenate, cytosol, mitochondria and nuclei. Each sample was incubated with glucose or pyruvate and tested for the production of carbon dioxide. a Put a tick or cross in the boxes that would test positive for liberation of carbon dioxide. Complete homogenate Cytosol Mitochondria [4] Nuclei Glucose Pyruvate b Name the stage(s) in cellular respiration where carbon dioxide is produced. [2] ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ c In the first step in this procedure, the liver was homogenised by grinding in cold buffer solution containing sucrose. Explain why. [3] ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ d Using the samples obtained, how would you observe the structure of the mitochondria? [2] ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ e Identify the cell structures in the electron micrograph in Figure 7.1. [9] Figure 7.1 TEM of liver cell [Total: 26] Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 73 Photocopying prohibited 73 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS 2 Resistance to antibiotics within a population of bacteria is due to selection pressure. This can be linked to the use of antibiotics by patients. A study was carried out into the link between antibiotic use and the presence of resistant Escherichia coli (E. coli) populations in human communities. l Over 30 000 patients were involved in the study. l Only patients attending large medical clinics took part in the study. l The number of prescriptions issued by each clinic was used as an estimate of antibiotic use. l Urine from patients attending the clinics was used as a possible source of antibiotic resistant E. coli. l Antibiotic resistance of E. coli in the urine samples was measured using the disc diffusion method. a The number of prescriptions issued for antibiotics varied considerably between clinics. The researchers wanted to find out whether there was a correlation between the number of prescriptions for each of the five antibiotics issued by a clinic and the percentage of urine samples containing resistant E. coli. Spearman’s rank correlation test was used for this analysis. The results of this analysis are shown in Table 7.1. antibiotic Spearman’s rank correlation coefficient (rs) cephalosporin 0.30 trimethoprim 0.62 co-amoxiclav 0.23 ampicillin 0.71 quinolone 0.44 Table 7.1 Table 7.2 shows the critical values for rs at five levels of significance for the data collected in this study. level of significance (p) 0.20 0.10 0.05 0.02 0.01 critical value of rs 0.240 0.306 0.362 0.425 0.467 Table 7.2 i Suggest why the Spearman’s rank correlation test was used in this study. [1] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ii State a null hypothesis for the Spearman’s rank correlation test for this study. [1] ................................................................................................................................................................................................. ................................................................................................................................................................................................. 74 9781510482869.indb 74 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions iii Using Table 7.1 and Table 7.2, identify which antibiotics showed a statistically significant correlation between the number of prescriptions and the presence of resistant strains of E. coli in urine samples. Give a reason for your answer. [2] antibiotics............................................................................................................................................................................. reason.................................................................................................................................................................................... ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. b The percentage of patients with ampicillin-resistant E. coli infections varies with age and gender, as shown in Figure 7.2. percentage ampicillin resistance 80 male 70 60 female 50 40 0 20 40 60 age / years 80 100 Figure 7.2 Describe and explain the trends shown by these data. [3] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... [Total: 7] Cambridge International AS & A Level Biology (9700) Paper 53 Q2 c & d, October/November 2018 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 75 Photocopying prohibited 75 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS 3 A student investigated the rate of respiration in two different tissues, A and B, using the redox dye methylene blue as an indicator. The student made a suspension of each tissue using the following procedure: l a sample of each tissue was homogenised in a blender with ice-cold osmotic buffer l osmotic buffer was added to each homogenate and stirred to make a suspension l the two suspensions were incubated at 20 °C before testing. The results of this investigation are shown in Table 7.3. time for methylene blue to become colourless / s−1 test 1 test 2 test 3 test 4 test 5 test 6 test 7 test 8 test 9 test 10 mean time ± s rate / s−1 Tissue A 70 56 59 54 52 56 55 75 59 50 55 ± 3.14 18 × 10−3 Tissue B 124 126 136 126 122 125 121 123 124 125 124 ± 1.73 8 × 10−3 Table 7.3 Outline the procedure the student could use to obtain these results. Your method should be detailed enough for another person to use. [8] ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... 76 9781510482869.indb 76 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... ................................................................................................................................................................................................................... [Total: 8] Cambridge International AS & A Level Biology (9700) Specimen paper 5 Q2 a, 2016 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 77 Photocopying prohibited 77 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS 4 Cancer of the blood, including leukaemia and lymphoma, can be caused by mutations of stem cells in the bone marrow. A long-term study into the effects of radiation on the frequency of blood cancer was carried out on two groups of people: group 1 and group 2. These people were all born to mothers exposed to nuclear radiation during pregnancy. Table 7.4 summarises information about the two groups of people included in this study. group 1 group 2 when born between 1948 and 1988 between 1950 and 1961 how the mothers were exposed to radiation working in a nuclear power plant and living in the town next to the nuclear power plant living next to a river contaminated by nuclear wastes from an accident at the same nuclear power plant time when mothers were exposed to radiation any time between January 1948 and December 1982 any time between January 1950 and December 1960 method of determining radiation exposure of mothers using badges worn by workers at the nuclear power plant to record their exposure to radiation from external radiation levels measured in the area individuals for whom blood cancer data were collected people who continued to live in the same town as the nuclear power plant people who continued to live in the area where they were born when blood cancer data were collected January 1948 until December 2009 January 1953 until December 2009 Table 7.4 Until 2005, the data sources used for all of this information were paper based and obtained from hospitals, clinics and medical records. After 2005, data were collected electronically from databases at cancer clinics and from online death certificates. a Table 7.5 shows some of the results from this study. group 1 group 2 number of people in the group studied 8 466 male female group 1 and group 2 combined 11 070 19 536 4 361 5 588 9 949 4 105 5 482 9 587 number of people not developing any cancer who were still alive 4 053 5 648 9 701 number of people not developing any cancer who had died 898 1 864 2 762 number of people developing any cancer 220 288 508 number of deaths from any cancer 103 145 248 number of people developing blood cancer 32 26 58 number of deaths due to blood cancer 21 15 36 number of people where outcome not known 3 295 3 270 6 565 outcomes up to December 31 2009 Table 7.5 78 9781510482869.indb 78 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions i In group 1, the proportion of people who were known to develop blood cancer out of all the people who were known to develop any cancer was 0.145. Calculate for group 2 the proportion of people who were known to develop blood cancer out of all the people who were known to develop any cancer. Give your answer to three decimal places. [1] ii It is possible to carry out a chi-squared test on the data in Table 7.5 to test whether there is a difference in the probability of individuals in group 1 and group 2 developing blood cancer. State one reason why the chi-squared test can be used with these data. [1] ................................................................................................................................................................................................. ................................................................................................................................................................................................. b The data were analysed to assess how the number of people who developed blood cancer was affected by their mothers’ exposure to radiation during pregnancy. Figure 7.3 shows the results of this analysis for the combined data from group 1 and group 2. Each plotted number includes all those people whose mothers’ exposure to radiation during pregnancy was below, or up to, the exposure to radiation shown. number of people in group 1 and group 2 who developed blood cancer 60 50 40 30 20 10 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 exposure of mothers to radiation during pregnancy/arbitrary units Figure 7.3 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 79 Photocopying prohibited 79 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS i Use Fig. 7.3 to describe the relationship between the number of people who developed blood cancer [3] and their mothers’ exposure to radiation during pregnancy. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ii Suggest an explanation for the relationship shown between the number of people who developed blood cancer and their mothers’ exposure to radiation during pregnancy. [1] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. 80 9781510482869.indb 80 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions c Evaluate the validity of the results of this study with reference to all the information provided. [3] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... d Plant scientists were interested in the effect of radiation on the germination of seeds. [3] They exposed seeds to the same intensity of radiation for different lengths of time and measured the proportion of seeds that germinated. Suggest three variables, other than intensity of radiation, that would need to be standardised in an investigation of this type. ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... [Total: 12] Cambridge International AS & A Level Biology (9700) Paper 52 Q2, March 2019 Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 81 Photocopying prohibited 81 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS 5 Below is a diagram of a respirometer. A respirometer can be used to measure the oxygen uptake of living organisms. graduated syringe A clips A and B are closed B hypodermic needle respirometer tube water bath germinating seeds soda lime pellets soda lime pellets U-tube manometer A B Figure 7.4 a Describe how a respirometer could be set up to determine the rate of respiration of germinating seeds at different temperatures. [9] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... 82 9781510482869.indb 82 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... b Suggest a suitable control to add to tube A. [2] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... c Explain the use of soda lime. Suggest what would happen if soda lime was not used. [2] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... e State what is meant by the term respiratory substrate. [1] ....................................................................................................................................................................................................... Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 83 Photocopying prohibited 83 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS The respiratory quotient (RQ), can be calculated using the formula: RQ = volume of carbon dioxide exhaled per minute volume of oxygen inhaled per minute The basic equation for the respiration of glucose is: C6H12O6 + 6O2 → 6CO2 + 6H2O f Calculate RQ for the reaction. [2] RQ = g When fatty acids are being respired it is 0.7. Explain. [2] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... h Sunflower seeds contain lipids. State the advantages of storing lipid for use as a respiratory substrate in seeds.[2] ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... ....................................................................................................................................................................................................... [Total: 22] 84 9781510482869.indb 84 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions 6 Figure 7.5 shows some of the plants growing in a pond and on the land around the pond. Some students decided to investigate the changes in the distribution and abundance of species of land plants at different distances from the edge of the pond. They started their investigation at the plants growing next to the water, as shown in Figure 7.5. land plants water plants start of investigation Figure 7.5 a i State the independent and dependent variables in this investigation. [2] independent variable........................................................................................................................................... dependent variable.............................................................................................................................................. ii Describe a systematic sampling method the students could use to find out how the distribution and abundance of the plant species changed as the distance from the edge of the pond increased. Your description of the sampling method should be detailed enough for another person to use. [8] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. b The students also collected samples of soil at different distances from the pond edge and estimated the water content. The students wanted to find out if the water content of the soil at the different distances sampled was related to the number of different plant species found at the same distances. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 85 Photocopying prohibited 85 31/08/21 1:55 PM A LEVEL PRACTICAL SKILLS To do this, a Spearman’s rank correlation (rs) was carried out using the data in Table 7.6. sample rank difference (D) D2 water content/arbitrary units rank number of species rank 1 28 1 3 10 –9 81.00 2 26 2 4 9 –7 49.00 3 21 3 5 8 –5 25.00 4 18 4 6 7 –3 9.00 5 15 5.5 8 6 –0.5 0.25 6 14 7.5 9 4.5 3 9.00 7 15 5.5 10 3 2.5 6.25 8 14 7.5 9 4.5 3 9.00 9 13 9.5 11 2 7.5 56.25 10 13 9.5 12 1 8.5 72.25 ΣD2 = Table 7.6 The formula for Spearman’s rank correlation is: 2 rs = 1 – 6 ×3 ΣD n –n When: rs = Spearman’s rank correlation n = number of pairs of observations D = difference between each pair of ranked measurements Σ = sum of i Complete Table 7.6 to show ΣD2.[1] ii Use the information in Table 7.6 to calculate the value for rs. Show the values for: l 6 × ΣD 2 l n3 – n[2] rs = iii State what the value for rs shows about the relationship between soil water content and the number of species present. [1] ................................................................................................................................................................................................. ................................................................................................................................................................................................. 86 9781510482869.indb 86 Photocopying prohibited Cambridge International AS & A Level Biology Practical Skills Workbook 31/08/21 1:55 PM 7 Practice questions c i The group of students then investigated the relationship between soil air content and the number of different plant species at the same sampling points. The students calculated the rs value as +0.86. Table 7.7 shows part of a Spearman’s rank probability table. n (number of pairs) 8 9 10 11 12 significance level 5% 0.738 0.700 0.648 0.618 0.618 significance level 1% 0.881 0.883 0.794 0.755 0.727 Table 7.7 The students concluded that their rs value of +0.86 for the relationship between soil air content and the number of species present was significant at both the 5% level and 1% level. Explain how the students reached this conclusion. [2] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ii Based on the result of their Spearman’s rank test and the significance of the rs value, the students concluded that: Soil air content caused the difference in the number of plant species that could grow at different distances from the edge of the pond. Suggest why this conclusion may not be valid. [2] ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. ................................................................................................................................................................................................. Cambridge International AS & A Level Biology Practical Skills Workbook 9781510482869.indb 87 [Total: 18] Photocopying prohibited 87 31/08/21 1:55 PM Reinforce learning and deepen understanding of the key practical skills required by Cambridge International AS & A Level Biology syllabus (9700); an ideal course companion or homework book for use when carrying out and analysing practical work throughout the course. » Support students’ learning and provide guidance on practical skills with extra practice questions and activities, tailored to topics in the Student Book » Keep track of students’ work with readyto-go write-in exercises which once completed can also be used to recap learning for revision » Offer extra support for the mathematical and statistical parts of the course » Answers can be found at www.hoddereducation.com/ cambridgeextras Ca ducation W For over 25 years we have been king for ove or r trusted by Cambridge schools 25 around the world to provide YEARS quality support for teaching and i es at sm ent Intern learning. For this reason we have been selected by Cambridge Assessment International Education as an official publisher of endorsed material for their syllabuses. ss on al E m bridge A WITH Use with Cambridge International AS & A Level Biology Student’s Book Second Edition 9781510482876 This resource is endorsed by Cambridge Assessment International Education ✓ Provides learner support for the syllabus for examination from 2022 ✓ Has passed Cambridge International’s rigorous quality-assurance process ✓ Developed by subject experts ✓ For Cambridge schools worldwide 9781510482869.indb 88 31/08/21 1:55 PM