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Lab Manual SB015 & SB025

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MATRICULATION DIVISION BIOLOGY LABORATORY MANUAL SEMESTER I & II SB015 & SB025 TWELFTH EDITION MATRICULATION DIVISION MINISTRY OF EDUCATION MALAYSIA BIOLOGY LABORATORY MANUAL SEMESTER I & II SB015 & SB025 MINISTRY OF EDUCATION MALAYSIA MATRICULATION PROGRAMME TWELFTH EDITION First Printing, 2003 Second Printing, 2004 Third Printing, 2005 (Sixth Edition) Fourth Printing, 2006 (Seventh Edition) Fifth Printing, 2007 (Eighth Edition) Sixth Printing, 2011 (Ninth Edition) Seventh Printing, 2013 (Tenth Edition) Eighth Printing, 2018 (Eleventh Edition) Ninth Printing, 2020 (Twelfth Edition) Copyright © 2020 Matriculation Division Ministry of Education Malaysia ALL RIGHTS RESERVED. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system, without the prior written permission from the Director of Matriculation Division, Ministry of Education Malaysia. Published in Malaysia by Matriculation Division Ministry of Education Malaysia, Level 6 – 7, Block E15, Government Complex Parcel E, Federal Government Administrative Centre, 62604 Putrajaya, MALAYSIA. Tel : 603-88844083 Fax : 603-88844028 Website : http://www.moe.gov.my/v/BM Malaysia National Library Biology Laboratory Manual Semester I & II SB015 & SB025 Twelfth Edition eISBN 978-983-2604-48-8 NATIONAL EDUCATION PHILOSOPHY Education in Malaysia is an on-going effort towards further developing the potential of individuals in a holistic and integrated manner, so as to produce individuals who are intellectually, spiritually and physically balanced and harmonious based on a firm belief in and devotion to God. Such an effort is designed to produce Malaysia citizens who are knowledgeable and competent, who posses high moral standards and who are responsible and capable of achieving a high level of personal well-being as well as being able to contribute to the betterment of the family, society and the nation at large. NATIONAL SCIENCE EDUCATION PHILOSOPHY In consonance with the National Education Philosophy, science education on Malaysia nurtures a science and technology culture by focusing on the development of individuals who are competitive, dynamic, robust and resilient and able to master scientific knowledge and technological competency. iii FOREWORD I am delighted to write the foreword for the Laboratory Manual, which aimed to equip students with knowledge, skills, and the ability to be competitive undergraduates. This Laboratory Manual is written in such a way to emphasise students’ practical skills and their ability to read and understand instructions, making assumptions, apply learnt skills and react effectively in a safe environment. Science process skills such as making accurate observations, taking measurement in correct manner, using appropriate measuring apparatus, inferring, hypothesizing, predicting, interpreting data, and controlling variables are further developed during practical session. The processes are incorporated to help students to enhance their Higher Order Thinking Skills such as analytical, critical and creative thinking skills. These skills are crucial to prepare students to face upcoming challenges in the 21st century era. The manipulative skills such as handling the instruments, setting up the apparatus correctly and drawing the diagrams can be advanced through practical session. The laboratory experiments are designed to encourage students to have enquiry mind. It requires students to participate actively in the science process skills before, during and after the experiment by preparing the pre- report, making observations, analysing the results and in the science process skills before, during, after the experiment by preparing the pre-report, making observations, analysing the results and drawing conclusions. It is my hope and expectation that this manual will provide an effective learning experience and referenced resource for all students to equip themselves with the skills needed to fulfil the prerequisite requirements in the first-year undergraduate studies. Dr. HAJAH ROSNARIZAH BINTI ABDUL HALIM Director Matriculation Division iv CONTENTS Page Foreword iv Content v Learning Outcomes vii Introduction x SEMESTER I 1 7 Experiment Title 14 1 Basic Techniques In Microscopy 17 2 Plant Tissues 21 3 Transport Across Membrane 29 4 Cell Division – Mitosis 5 Inheritance 6 Basic Techniques in Isolating DNA v SEMESTER II Experiment Title 7 Diversity of Bacteria 32 8 Plant Diversity – Bryophytes and Pteridophytes 38 9 Biocatalysis 48 10 Cellular Respiration 11 Photosynthesis 53 12 Dissection 56 61 References 76 Acknowledgements 77 vi 1.0 Learning Outcomes 1.1 Matriculation Science Programme Educational Objectives Upon a year of graduation from the programme, graduates are: 1. Knowledgeable and technically competent in science disciplines in-line with higher educational institution requirement. 2. Communicate competently and collaborate effectively in group work to compete in higher education environment. 3. Solve scientific and mathematical problems innovatively and creatively. 4. Engage in life-long learning with strong commitment to continue the acquisition of new knowledge and skills. 1.2 Matriculation Science Programme Learning Outcomes At the end of the programme, students should be able to: 1. Acquire knowledge of science and mathematics fundamental in higher level education. (PEO 1, MQF LOD 1) 2. Demonstrate manipulative skills in laboratory work. (PEO 1, MQF LOD 2) 3. Communicate competently and collaborate effectively in group work with skills needed for admission in higher education institutions. (PEO 2, MQF LOD 5) 4. Apply logical, analytical and critical thinking in scientific studies and problem solving. (PEO 3, MQF LOD 6) 5. Independently seek and share information related to science and mathematics. (PEO 4, MQF LOD 7) vii 1.3 Biology 1 Course Learning Outcome At the end of the course, student should be able to: 1. Explain the basic concepts and theories in cells, biomolecules, inheritance, genetics and biological development. (C2, PLO 1, MQF LOD 1) 2. Conduct biology laboratory work on microscopy, biological molecules, histology and genetics information by applying manipulative skills. (P3, PLO 2, MQF LOD 2) 3. Solve problems related to cells, biomolecules, inheritance, genetics and biological development. (C4, PLO 4, CTPS3, MQF LOD 6) 1.4 Biology 2 Course Learning Outcome At the end of the course, student should be able to: 1. Explain the basic concepts and theories in transport system processes, mechanisms for adaptations in living things, ecological and environmental issues in biology. (C2, PLO 1, MQF LOD 1) 2. Conduct biology laboratory work on diversity of bacteria and plant, biocatalysis, cellular respiration, chromatography and dissecting technics by applying manipulative skills. (P3, PLO 2, MQF LOD 2) 3. Solve problems related to transport system processes, mechanisms for adaptations in living things, ecological and environmental issues in biology. (C4, PLO 4, CTPS 3, MQF LOD 6) viii 1.5 Biology Practical Learning Outcomes Biology experiment is to give the students a better understanding of the concepts of Biology through experiments. The aims of the experiments in this course are to be able to: • know and practice the necessary safety precautions to be taken. • use the correct techniques of handling apparatus. • plan, understand and carry out the experiment as instructed. • observe, measure and record data consistency, accuracy and units of the physical quantities. • define, analyse data and information in order to evaluate and deduce conclusions from the experiments. • discuss data and information logically and critically. • analyse and draw conclusions from biological data. • develop solution to biological problems. • acquire scientific skills in measuring, recording and analysing data as well as to determine the uncertainties (error) in various physical quantities obtained in the experiments. • understand the limitations to the accuracy of observations and measurements. ix INTRODUCTION A. General Guidelines Laboratory Regulation 1. Always wear laboratory coats and covered shoes in the lab. 2. Do not eat or drink in the laboratory. 3. Use the apparatus and materials wisely. 4. Do not throw rubbish and residues into the sink. Wrapped and throw them into the dustbin provided. 5. At the end of the experiment, students must a) clean the apparatus using the detergent provided. b) soak the apparatus in acidic solution containing mild hydrochloric acid. c) wash the sink and work station. d) make sure that all the tables are clean and neat. e) place the materials and apparatus in their respective places. Sectioning and Staining Plant Tissues 1. Sectioning of plant tissues or parts must be made and stained before they are examined under the microscope. 2. Use sharp blade or microtome to make a thin slice of the specimen. 3. Clean the blades with water and dry them using tissue paper after being used. Preparation for Experiment 1. You are advised to read the manual before carrying out the experiment. You are also advised to make additional references about the topic. 2. Prepare a rough layout of the experiment that consists of tables, graphs and space for drawing. 3. Identify the equipments and materials that are going to be used in the experiment. This will maximise the time used for experiment. 4. Follow strictly the instructions in the manual. 5. Record only what you observe in the experiment. x Laboratory Report and Evaluation 1. Report should contain the following:- Title Objective(s) Introduction (hypothesis/variable/problem statement) Procedures (in passive voice, past tense, in reporting style) Observation (tables, graphs, data, drawing) Analysis / Discussion regarding tables, graphs, data or drawings Conclusion Questions References 2. Reports must be handwritten or typed. 3. Diagrams should be drawn on the blank sheet using a 2B pencil. All diagrams must be labelled. 4. Metric system must be used in writing numerical data. 5. Data can be presented in the form of graphs, tables, flow charts or diagrams. Give suitable titles to the graph, table, flow charts and diagrams. 6. Record the following on the front page of the report. College’s name: Student’s name: Matriculation number: Practicum group: Title: Date: Tutor’s / lecturer’s name: 7. Submit your report to your lecturer at the end of the practical session. The report and the attendance for each lab will be evaluated and included in the assessment. Scientific Drawing 1. Diagrams drawn must be based on the observation of the specimen and not copied from books. 2. All parts of the specimens observed must be drawn using the right scale. 3. An overall drawing or plan drawing must be made to show the parts where the drawings are made. 4. Show clear orientation of the specimen so that the position and the relationship with other organs can be determined. 5. Use a sharp 2B pencil to draw thin, clear and continuous lines. xi 6. Drawing must not be coloured or shaded to differentiate the systems from tissues. For this purpose, students are allowed to use various patterns to differentiate systems. 7. Label all your drawings. All labels must be written on the right and left side of the diagram. Do not write the labels on or in the diagram. Labels must be written horizontally. Straight line must be used to connect the structure. 8. Magnification used in the drawing from observation under the microscope must be mentioned; e.g.: 40x or 100x actual magnification. Caring for Plants and Animals 1. Water the plants every day. Make sure the soil is damp and wet. 2. Clean the animal cages every day. Make sure the cages are in good condition. 3. Feed the animal daily. B. Introduction to Microscopy The discovery of microscope started a new era in biology since for the first time man was able to observe cells, the basic units of life. The optical properties of lenses have been known for the last 300 years B.C. , but these knowledge were not used to the fullest until the seventeenth century when Antonio Van Leeuwenhoek (1632-1732), a Dutch, and his colleagues discovered a simple workable microscope. With the discovery of the simple microscope, many people were able to observe minute living organisms in great details. One of them was Robert Hooke who in 1665 gave the first extensive description of his experience in observing cork tissue using the simple microscope. This marked to the beginning of the study of cells. Below is the excerpt from the journal Micrographia by Hooke of what he observed from the cork tissue under the microscope: “ I could exceedingly plainly perceive it to be all perforated and porous….. these pores, or cells, ….. were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any writer or person, who has made any mention of them before this”. Although the description by Hooke about the cork tissue might sound hilarious, you may have described them in the same way had you lived in the seventeen century when the concept of cell as the fundamental unit of life was something unknown. xii 1. What is a Microscope? Microscopes are precision instruments, and therefore need to be handled carefully. Many people think that microscopes can only be used to observe objects in higher magnification. If a microscope can only be used to observe a magnified image, then its usage is limited. In fact, microscope can be used to magnify an object, determining the size of an object and observing fine details of an object, all of which are not discernible to our naked eyes. Therefore, before one can properly use a microscope, first he has to be familiar with the microscope and be able to identify the components of the microscope and their functions. With the advancement of technology in microscopy, many high-quality microscopes have been designed for many specific uses. Nowadays, many microscopes are of the compound types which use two sets of lenses. The first set of lens constitutes the objective lens which supplies the initial real magnified image. The second set of lens constitutes the ocular lens which magnifies further the image formed by the first set of lens and converts the real image into virtual image which is in turn viewed by the user’s eyes. In compound microscopes, the actual magnification is calculated as the magnification of objective lens multiplied by the magnification power of the ocular lens. Today there are many types of light microscope, for example the phase- contrast microscope that allows user to view living cells or specimens without the use of stains to increase the contrast. Contrast is based on the differential absorption of light by parts of the specimen. There are compound microscopes, which employ ultraviolet light as the source of light, making it possible to view specimens that emit fluorescence. Such microscopes are now commonly used in diagnostics laboratories and research. There are also other compound microscopes which use either dark field or light field. Another type of microscope is compound microscope with inverted objective, called inverted microscope, which is used to observe living cell cultures. A microscope is not only capable of producing the image of an object but also capable of distinguishing between two adjacent points on the object. This capacity is termed as the resolving power of the lenses or the resolving power of the microscope. The higher the resolution of the microscope, the higher is the ability to distinguish details of the object. Microscope quality depends upon the capacity to resolve, not magnify, objects. Magnification without resolving power, however, is not worthless in the field of microscopy. xiii The resolving power of a light microscope depends upon the wavelength of light (colour) being used, and not on a value called the numerical aperture (N. A) of the lens system used. The numerical aperture is derived from a mathematical expression that relates the light transferred to the specimen by the condenser to the light received by the objective lens. This relationship is given by the following expression: Resolving power = Shortest diameter of the observed structure = Wavelength () Numerical Aperture (N.A) Thus, the resolving power is increased by reducing the wavelength of the light used. The shorter the wavelength used, the shorter will the diameter of the structure being observed, or in other words, the resolving power is increased. The resolving power cannot be increased substantially because the light spectrum is narrow (500 nm). However, we can increase the resolving power by increasing the numerical aperture in the lens system of the microscope. When the specimen is illuminated with light from direct or oblique direction, the relationship is given as follows: Resolving power = Wavelength () 2 x N.A where λ - wavelength of light N.A - Numerical Aperture The condenser located below the mechanical stage or slide holder can transfer oblique and direct light sources to the specimen and this can approximately double the numerical aperture (N.A). Thus, the resolving power can be increased. Therefore, the condenser has to be properly focused to achieve high resolving power. Light enters the specimen, and some of it will be refracted as it goes through the air. This light will not enter the objective lens. By placing oil of immersion in the space between specimen and objective lens, we can reduce the light refraction and increase the amount of light entering the objective lens resulting in a brighter and clearer image. The oil of immersion used should have the same refractive index (R.I) as the glass to reduce refraction. xiv After understanding some principles of microscopy, we need to identify the components of the microscope and know their functions. The microscope to be used in the laboratory is the bright field light compound. The diagram of the microscope is shown in Figure 1. Familiarise yourself with a microscope and its functions before using it (refer Table 1). Figure 1: Compound light microscope Each objective lens will generate an image in a specific field of view. The size (diameter) of the field of view depends on the type of objective lens used. As the magnifying power of the objective lens increases, the size of the field of view decreases, and the working distance, the distance between the slide and the objective lens, also decreases. When the specimen field of view is wide, more light will enter the objective lens, so it is important to regulate the amount of light. Figure 2 shows the relationship between objective lens, fields of view and working distance for each of the objective lens. xv Figure 2 Comparison of working distance at three different objective magnification Table 1 Components of microscope and their functions Component Function 1a. Ocular lens or eyepiece lens: 1a. Magnifies the real image and This lens is found at the top of the microscope. It normally has a converts it to a virtual image to be magnification power of 10x or 15x. viewed by user’s eyes. 1b. Ocular control knob 1b. To adjust and compensate the differences in binocular image of the eyes. 2. Body tube Body tube is the hollow housing which supports the ocular lenses at the top and connects them with the objective lenses below it. 3. Rotating nosepiece: The structure to which the objective You will hear clicking sounds when the lenses are mounted. By gently rotating objective lens is in its correct position the nosepiece, you may choose the above the specimen. You may practise objective lens you want and correctly this by rotating and changing the place it over the specimen. objective lenses. 4. Objective lenses: Normally there are 3-4 objective lenses mounted on the nosepiece, and these can be rotated and changed as you require. a. Scanning objective lens (4x): a. Used to scan the specimen before Coarse specimen field = 5 mm identifying the specific part to be viewed further. xvi b. Low-power objective lens (10x): b. Used to view major part of the Small specimen field = 2 mm specimen. c. High-power objective lens (40x): c. Used to view specific part of the Very small specimen field = 0.5 mm specimen. d. Oil immersion objective lens (100x): d. Used to view microorganisms such as Immersion oil is placed in the space bacteria and microstructures in the cells. between the specimen and objective lens to reduce the light refraction from the specimen. Very small specimen field = 0.2 mm 5a. Stage: a. The horizontal surface on which a The horizontal surface which has a hole specimen is placed. in the centre to allow light from below to focus on the specimen. 5b. Slide clips b. The stage is usually equipped with slide clips to hold the slide in place. 5c. Slide adjustment knob c. Two knobs are used to move the slide to the left, right, forward or backward. Move these knobs to learn how the slide is moved into position. 6a. Condenser: a. Used to focus and deliver light to the Condenser is located immediately specimen. under the stage b. Used to adjust the condenser. 6b. Condenser control knob c. Used to focus the light. 6c. Condenser lens knob 7. Iris diaphragm: Controls the amount of light entering An adjustable light barrier of iris type and leaving the condenser. built into condenser. The size of the diaphragm is controlled by rotating the knob either to the left or right. Rotate the knob to the left and to the right and observe what happens. xvii 8a. Off/on switch. The source of light is a tungsten bulb located at the base of a microscope. 8b. Light control knob. Please ensure that either of the switches is OFF or MINIMUM, respectively before you use the microscope. 9. Body arm The metal part used to carry a 10. Base microscope. The heavy cast metal part used as the base and for support. 11. Coarse adjustment knob: Used to bring specimen into focus by Use this knob only when using low- moving the stage to the specimen. power objective lens. Rotate this knob carefully, and observe what happens. Does the stage or the body tube move? 12. Fine focus adjustment knob Used to bring specimen into focus while using high-power or oil immersion objective lenses. Now that you have become familiar with the component parts of the microscope, you can proceed to use the microscope. Check the microscope to ensure that it is in good working conditions. 2. Setting Up of a Light Microscope a) Plug the microscope to a power source. Before switching on the plug, check that the light switch is OFF or the light control knob is set at MINIMUM b) Switch on the power. Turn on the light control knob or adjust the light diaphragm to deliver the light to the specimen field (but not too much light). To focus the condenser, do the following: i) Take a prepared slide and place it on the stage. ii) Rotate the nosepiece and put the coarse objective lens into position above the specimen. iii) Move the stage upwards by rotating the coarse adjustment knob until it stops completely. xviii iv) While looking through the oculars, move the stage downwards using the fine adjustment knob until the specimen is in focus. v) To focus the condenser, you need to bring the specimen and the condenser into focus in the same plane. Close down the iris diaphragm and reduce the amount of light. 3. Focusing a Specimen a) Place a prepared slide on the stage (for this exercise you may use any of the prepared slides available in the lab). Move the slide so that the specimen is placed in the centre and under the objective lens. b) First you need to ensure that either the scanning objective lens (4x) or low-power objective lens (10x) is placed above the specimen. c) While looking at the slide from the side, move the stage upwards until it stops completely. Use the coarse adjustment knob to do this. d) Now observe the specimen through the ocular lens. The specimen will appear blur because it is still not focused. To focus the specimen, gently move the stage downwards until the specimen comes into sharp focus and clear. Use the fine adjustment knob to do this. Look through the ocular lens with both eyes. You may see the image differently between your right and left eyes. Do the following to adjust the ocular lenses for the differences between your eyes. Determine which ocular lens is adjustable. Close the eye over that lens and bring the specimen into sharp focus for the open eye (right eye). Open the other eye (left eye) and close the first eye (right eye). If the specimen is still not in sharp focus, turn the adjustable ocular control knob (1b) until the specimen is in focus. You may now look with your eyes through both ocular lenses. e) After the specimen has been focused by the low-power objective lens, rotate the nosepiece to change to the high-power objective lens (40x). You will hear a clicking sound when the objective lens comes into its correct position right above the specimen. The microscope used should be of PARFOCAL type, that is once a specimen has been focused using a particular objective lens, it will stay focused for the other objective lenses. Using this microscope, you do not need to refocus the specimen when you change the objective lens. You just need to adjust the fine focus adjustment knob. f) If the field of view is dark or too bright, adjust the amount of light by using the light control or diaphragm knob. g) When you have finished using the microscope, rotate the nosepiece to place the coarse objective lens (4x) back in position over the centre of the stage. Remove the last slide and clean the stage if necessary. xix 4. Using Oil Immersion Objective Lenses The oil immersion objective lens is used when you want to observe a specimen at the highest resolution with the light microscope or when the resolution of other objective lens is not sharp and clear enough. The objective lens is usually used to observe microorganisms such as bacteria and protozoa or to observe microorganelles in the cell. Before using the objective lens, the specimen has to be fixed and stained to increase its contrast. a) Follow steps (a) to (d) in procedure (3). b) Rotate the nosepiece to bring the high-power objective lens (40x) half way as shown in Figure 3. c) While holding the nosepiece in this position, apply a single small droplet of immersion oil to the illuminated spot on the slide. Figure 3 Using oil immersion objective lenses d) Rotate the nosepiece again to move the high-power objective lens into position until you hear a clicking sound. The objective lens is now right above the specimen and will be immersed in the oil. e) Open up the iris diaphragm to increase the amount of light. f) While looking at the specimen through the ocular lens, use the fine adjustment knob until the specimen comes into sharp focus and become clear. If you have any problems, consult the instructor / tutor. g) When you have finished using the oil immersion objective lens, do the following steps: i) Carefully move the stage downwards. ii) Clean the oil immersion objective lens by gently wiping it with clean lens tissue. If the objective lens is still dirty, clean it with a little amount of xylene and rub it gently with clean, dry lens tissue. xx iii) Remove the slide off the stage. iv) Gently rotate the nosepiece again to place the low-power objective lens back in position over the centre of the stage. v) If oil is found on the stage, wipe the oil off with lens tissue and with some alcohol. 5. Storage of Microscopes When you have finished using the microscope, do the following to store the microscope. a) Check that you have not left a slide on the stage. b) Check that the stage is clean without any trace of water or dust on it. If there is any water on the stage, wipe it off with dry tissue. If it is oil, wipe it off with dry tissue with some alcohol. c) If you use oil immersion objective lens, gently wipe it with clean lens tissue. d) Check that the scanning objective lens (4x) is placed back in position over the centre of the stage. e) Turn off the light switch or close down the iris diaphragm to reduce the amount of light to a minimum and then switch off the power. f) Tie up the power chord below the body arm. g) Ensure the slide clips are placed on the stage and they are not protruding. h) HOLD THE MICROSCOPE WITH BOTH HANDS, that is hold the body arm of the microscope with one hand and the base of the microscope with the other hand. 6. The Dissecting Microscope (Stereoscopic Microscope) The dissecting microscope (Figure 4) is used for observations at low magnification in binocular view (involving 2 ocular lenses) or in three dimensions. Specimens are often viewed in a fresh state and need not be placed on a slide. The microscope is ideal for dissection of a small specimen. The procedures of using a dissecting microscope are basically similar to the procedures for a light microscope; however, it is simpler to use than a light microscope. 1. Place a specimen on the specimen plate at the base (5). 2. Illuminate the specimen, by switching on the light source. a) The eyepiece lenses need to be adjusted to suit your eyes and to ensure that the image remains clear at different magnifications. Turn the adjustment knob (2) to position ‘O’ b) Adjust ocular 1 so that the two oculars fit well with both eyes. xxi c) Turn the magnification control knob (4) to select the magnification to 4x. d) Look through both ocular lens and focus the specimen by turning the focus knob (8). e) Change the magnification to 0.8x by turning the control knob (4). f) Observe the image with your right eye and focus using the adjustment knob located on the right ocular until the image becomes clear. The microscope has now been adjusted to suit your eyes so that you can take advantage of the stereoscopic effect. 3. Look through the oculars with both eyes. Focus the image by turning the focus knob (8). Specimen as high as 20 mm may be focused using the adjustment knob. 4. For specimen higher than 20 mm, the microscope may be focused by moving its body (3) upwards. This is done by turning the body screw (7) loose and moving the body upwards or downwards along the stand (6) as far as the stop screw (9). Tighten the body screw (7) when the body is at the right position. 5. The stop screw (9) prevents the body of the microscope from crashing on to the specimen plate at the base. Figure 4 A dissecting microscope xxii 7. Electron Microscope This microscope makes use of the electron beams instead of light source. Electron beams have very short wavelength of approximately 0.005 mm, and therefore theoretically, the microscope can resolve objects as small as 0.0025 nm in diameter. The resolution of an electron microscope is usually 1 to 1.2 nm. With electron microscope, magnifications up to 250,000 are commonly obtained with biological materials. The shorter wavelengths of electrons are said to have greater resolving power than those of light microscope. There are two types of electron microscope, namely the transmission electron microscope and the scanning electron microscope. In transmission electron microscope, the electron beams are used instead of light source. An image will be formed on a photographic film screen. The microscope uses an electromagnetic lens as a condenser and the electron source is focused by the condenser lens through the specimen. The image is then magnified by the objective lens and the projector lens. An image taken from the electron microscope is called a transmission electron micrograph. In transmission electron microscope (TEM), only very thin sections of specimen of < 30 nm are used for microscopic observation. They are placed on a copper grid used for support. Electrons cannot be seen with the human eye, so the image is made visible by shinning the electrons on to a fluorescent screen. This will only produce black-and-white pictures. The electron microscope can be used only for dead tissue materials because they are viewed in vacuum. In scanning electron microscope, specimens are coated with a heavy metal such as gold. Electron beams will not be focused through the specimen, and when the electron beams collide with the specimen, some electrons will be absorbed while some are deflected or scattered. Those parts of the specimen which are denser will absorb more electrons and will appear darker in the final pictures. Density differences are due to differences in the contour of the coated surfaces of the specimen. The image produced will be in three dimensions, and the pictures are called scanning electron micrograph (SEM). xxiii BIOLOGY 1 SB015 SB015 Lab Manual EXPERIMENT 1: BASIC TECHNIQUES IN MICROSCOPY Course Learning Objective: Conduct biology laboratory work on microscopy, biological molecules, histology and genetics information by applying manipulative skills. (P3, CLO 2, PLO 2, MQF LOD 2) Learning Outcomes: At the end of this lesson, students should be able to: i. To obtain accurate images ii. To determine the depth of field iii. To determine the field of view iv. To calculate the actual magnification v. To apply the use of oil immersion with high magnification (oil immersion lens) Student Learning Time (SLT): Face-to-face Non face-to-face 2 hour 0 Before doing the following exercises, you must read and understand the basic techniques of using a microscope. Exercise 1.1: Images, Depth of Fields and Field of View of the Microscope Apparatus Compound light microscope Materials ‘e’ prepared slide Crossthreads prepared slide (3 colours eg: yellow, red and blue) Transparent ruler (10 mm size) or graph paper prepared slide Updated: 13/09/2017 1 SB015 Lab Manual Procedures and Observation 1.1.1 Images under the microscope 1. Observe the ‘e’ prepared slide using the 4x objective lens. 2. What do you observe using the 4x objective lens? Draw what you have observed. 3. Determine the position of ‘e’ (inverted/original position) (Figure 1.1). Figure 1.1: Letter ‘e’ to be observed under microscope 1.1.2 The depth of field The depth of field refers to the thickness of the plane of focus. With a large depth of field, all of the threads can be in focused at the same time. With a smaller or narrower depth of field, only one thread or a part of one thread can be focused, everything else will be out of focus. In order to view the other threads, you must focus downward to view the ones underneath and upward view the ones that are above. Do the following exercises to determine the depth of field of microscope. 1. Observe the position of the thread on the slide with your naked eyes. Identify the colour of thread a) at the top b) in the middle c) at the bottom 2. Observe the crossthreads under the microscope using 4x and 10x objective lens. 3. Determine what happens to the depth of field when the power of objective lens increases. Updated: 13/09/2017 2 SB015 Lab Manual Bottom: Yellow Middle: Red Top: Blue Figure 1.2 :Cross thread prepared slide 1.1.3 The field of view The simplest method of estimating linear dimension is to compare the size of the image to the diameter of the field of view. You can make a rough estimate of the field diameter by focusing on the millimetre scale of a transparent ruler using the lowest power objective. To calculate the field of view, use this formula: Diameter of field of view under low High magnification power magnification power Low magnification power = Diameter of field of view under high magnification power Do the following exercises to determine the diameter of the field of view for each of the objective lens on your microscope. 1. Place a transparent ruler on the stage. 2. Observe the transparent rulerusing the 4x, 10x and 40x objective lenses. (Increase the amount of light by adjusting the control knob to the maximum). 3. What do you observe using the 4x objective lens? Draw what you have observed. Updated: 13/09/2017 3 SB015 Lab Manual Figure 1.3 : Diameter field of view The diameter of field of view for the 4x objective lens is mm or m. Determine the diameter of field of view for the 10x and 40x objective lens in mm or m. The diameter of field of view for the 10x = ____ mm = ____ m. The diameter of field of view for the 40x = ____ mm = ____ m. 4. Using 40x objective lens, determine the size of a cell from a piece of cork tissue with approximately 20 cells in horizontal position and 10 cells in vertical position. Exercise 1.2: Magnification Procedures and Observation 1. Determine the actual magnification of a specimen by using the formula below. Magnification power x Magnification Actual magnification = of objective lens a power of ocular lens 2. Calculate the actual magnification in Table 1.1. Table 1.1 Actual magnification of a specimen Magnification Actual magnification power of ocular Magnification power of objective lens lens 4x 10x 40x 100x 10x Updated: 13/09/2017 4 SB015 Lab Manual Exercise 1.3: Oil Immersion Objective Lens Apparatus Compound microscope Materials Prepared slide of bacteria Lens tissue papers Immersion oil Methylated spirit (only for specific use) Procedures and Observation 1. Observe the prepared slide under the microscope. (Caution: Use immersion oil only for 100x objective lens). 2. Draw your observation. (Caution: Draw only the bacteria and not artifacts such as air bubbles, dust, fibre, etc.) (Refer to the method in Introduction to Microscopy) Questions A) For questions 1 to 7, choose the correct answer from the following list: A Scanning objective lens (4x) B Low-power objective lens (10x) C High-power objective lens (40x) D Oil immersion objective lens (100x) 1. Which is the shortest objective lens? 2. Which objective lens should you use when you begin to focus a specimen? 3. Which objective lens should be in position before you store a microscope? 4. Which objective lens will deliver the highest amount of light? 5. Which objective lens requires immersion oil to fill up the space between the specimen and the lens? Updated: 13/09/2017 5 SB015 Lab Manual 6. Which objective lens will still remain in focus when placed at the longest working distance from the specimen? 7. When using an ocular lens with 10x magnification power, which objective lens should be used to obtain the following actual magnification? (a) 100 times of its diameter (b) 1000 times of its diameter B) Answer the following questions. 1. Based on laboratory practices, what do you use to clean the microscope lenses? 2. While observing a moving microorganism under a microscope, you found that the organism has moved out of the field of view to the right. In order to keep observing the microorganism, which direction do you move your slide (right/left)? 3. How do you adjust the slide when the specimen is out of the field of view to the top? C) Complete the following sentences. 1. A microscope is called a compound microscope when it consists of more than one set of ………………………………… 2. Condenser and iris diaphragm are useful to coordinate…………………………………………... Updated: 13/09/2017 6 SB015 Lab Manual EXPERIMENT 2: PLANT TISSUES Course Learning Objective: Conduct biology laboratory work on microscopy, biological molecules, histology and genetics information by applying manipulative skills. (P3, CLO 2, PLO 2, MQF LOD 2) Learning Outcomes: At the end of this lesson, students should be able to: i. To identify different types of plant tissues ii. To compare the structure and distribution of tissues in monocots and dicots Student Learning Time (SLT): Face-to-face Non face-to-face 2 hour 0 Introduction Plant tissues are divided into two types: meristematic tissues and permanent tissues. Meristematic tissues are located in the apical meristem, or zone of active cell division. The division of cells on apical meristems, located at the tips of the roots and shoots, cause elongation of the root or the shoot, known as primary growth. The cells are smaller in size, with large nucleus, thin walls, large amount of cytoplasm and without inter- cellular space. Permanent tissues consist of mature cells that have specialized structure and functions. Permanent tissues can be divided into three: dermal tissues (epidermal and peridermal), ground tissues (parenchyma, sclerenchyma, collenchyma and endodermis) and vascular tissues (xylem and phloem). Updated: 13/09/2017 7 SB015 Lab Manual Apparatus Compound light microscope Materials Prepared slides of cross sections of monocot stem and root Prepared slides of cross sections of dicot stem and root Procedures and Observation 1. Examine the prepared slides of cross sections of dicot and monocotstems and roots using the 4x objective lens. 2. Identify the distribution of the tissues (epidermis, parenchyma, collenchyma, sclerenchyma, phloem, xylem and cambium). 3. Draw the arrangement of the tissues. Note the differences in the following characteristics: cell size, shape, wall thickness and stained parts. 4. Select a section and observe it under 40x objective lens. Draw and label the section showing the different tissues (epidermis, parenchyma, collenchyma, sclerenchyma, phloem, xylem and cambium). Use the figures provided to assist you in your tissue investigation. Updated: 13/09/2017 8 SB015 Lab Manual starch granule primary cell wall intercellular space Figure 2.1 Parenchyma cells (c.s) primary cell wall Figure 2.2 Collenchyma cells (c.s) primary cell wall secondary cell wall lumen Figure 2.3 Sclerenchyma cells (c.s) (Source: http://en.wikipedia.org/wiki/File:Plant_cell_type_sclerenchyma_fibers .png) Updated: 13/09/2017 9 SB015 Lab Manual tracheid vessel element Figure 2.4 Structure of xylem vessel element and tracheid (l.s) sieve tube companion cell sieve plate Figure 2.5 Structure of phloem sieve tube and companion cell (l.s) epidermis phloem xylem parenchyma Figure 2.6 Cross section of monocot stem (Source:http://www.phschool.com/science/biology_place/biocoach/pla nts/images/monstmlb.gif) Updated: 13/09/2017 10 SB015 Lab Manual Figure 2.7 Illustrated diagram of cross section of monocot stem (Source : https://farm9.staticflickr.com/8504/8426623226_56e9e1f488_o.jpg) Figure 2.8 Structure and distribution of vascular bundles in cross section of dicotyledon stem (Source:http://3.bp.blogspot.com/Pr_pAO5SlOo/UVKowiOM9II/AAAA AAAAFVs/9OctyttLZy8/w1200-h630-p-k-no-nu/Dicot+stem.jpg Updated: 13/09/2017 11 SB015 Lab Manual Figure 2.9 Cross section of dicotyledon stem (Source : http://botanystudies.com/wp-content/uploads/2017/03/Cell- Types-Tissues.jpg) Figure 2.10 Structure and distribution of vascular bundles in cross section of monocotyledon root (Source:http:// http://cssmith.co/monocot-root-cross-section-diagram/) Updated: 13/09/2017 12 SB015 Lab Manual Figure 2.11 Structure and distribution of vascular bundles in cross section of dicotyledon root Questions 1. You are given two slides of cross sections of Angiosperms stems and roots. How would you differentiate between the following? (a) the stems of monocot and dicot plants (b) the roots of monocot and dicot plants 2. Explain the structures and functions of parenchyma, collenchyma and sclerenchyma cells. Updated: 13/09/2017 13 SB015 Lab Manual EXPERIMENT 3: TRANSPORT ACROSS MEMBRANE Course Learning Objective: Conduct biology laboratory work on microscopy, biological molecules, histology and genetics information by applying manipulative skills. (P3, CLO 2, PLO 2, MQF LOD 2) Learning Outcomes: At the end of this lesson, students should be able to: i. To determine the sucrose concentration which is isotonic to potato cells ii. To determine the osmotic pressure of potato cells in atmospheric unit Student Learning Time (SLT): Face-to-face Non face-to-face 2 hour 0 Introduction The cell membrane is a selective permeable structure because only selected materials can pass through it. Water molecules can easily pass through the membrane and the movement of water is called osmosis. The direction of movement of water molecules is determined by the concentration of the solutes of both sides of the membrane. The water potential inside and outside of the cell is said to be isotonic, that is the movement of water molecules in both direction is at the same rate. The vacuolar membrane is also a selective structure and the condition in the vacuole is isotonic to the cell environment. In a hypertonic environment, water molecules will move out of the cell and the cell shrinks. The shrinking of cell is due to the hypertonic environment outside the plant and animal cells. The shrinking of plant cell is called plasmolysis while the shrinking of animal cell is called crenation. When a plant cell is in a hypotonic environment, it will expand but the increase in size is restricted by the cell wall (turgid). On the other hand, animal cells which are in the hypotonic environment will expand and burst and this is called lysis or haemolysis. Updated: 13/09/2017 14 SB015 Lab Manual Apparatus Boiling tube Beaker Cork borer Electronic balance Forceps Measuring cylinder (25 ml) Petri dish Pipette (10 ml) Materials Distilled water Filter paper Fresh potato tuber Graph paper Labelling paper Razor blade Ruler Sucrose solutions 1.0 M (40 ml per student) Tile Procedures and Observation Exercise: Osmotic pressure of potato cells 1. Prepare 20 ml of sucrose solution with different molarities in boiling tubes using the dilution method. The molarities required are 0.1M, 0.2M, 0.3M, 0.4M and 0.5M. Record in Table 3.1 the volumes of sucrose solution (1M) and the distilled water used in preparing the sucrose solutions. Table 3.1 Determination of molarities of sucrose solutions using dilution method Final molarity of the sucrose solutions 0.1 M 0.2 M 0.3 M 0.4 M 0.5 M Volume of 1.0 M sucrose (ml) Volume of distilled water (ml) Updated: 13/09/2017 15 SB015 Lab Manual 2. Prepare 15 pieces of potato strips using cork borer to have 3 replicates for each concentration. The length of each strip is 4 cm. 3. For every concentration, take 3 potato strips, record their average weight in a table. 4. Put all potato strips into the boiling tubes containing different sucrose concentrations. 5. After 30 minutes, remove the three strips from the boiling tube, wipe and immediately record their average weight. 6. Based on your results, draw a graph to show the changes in weight of the potato strips against the molarities of the sucrose solutions. 7. From the graph obtained in step 6, determine the sucrose concentration which is isotonic to potato cells. 8. Based on the values given in Table 3.2, draw a standard graph of osmotic pressure against the molarity of sucrose solution. Table 3.2 Values for constructing a standard graph Molarity 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 (M) Osmotic 1.3 2.6 4.0 5.3 6.7 8.1 9.6 11.1 12.6 14.3 16.0 pressure (atm) 9. From the graph obtained in step 8, determine the osmotic pressure of potato cells in atmospheric unit (atm). Updated: 13/09/2017 16 SB015 Lab Manual EXPERIMENT 4: CELL DIVISION - MITOSIS Course Learning Objective: Conduct biology laboratory work on microscopy, biological molecules, histology and genetics information by applying manipulative skills. (P3, CLO 2, PLO 2, MQF LOD 2) Learning Outcomes: At the end of this lesson, students should be able to: i. To prepare onion root tip slides ii. To identify stages in mitosis Student Learning Time (SLT): Face-to-face Non face-to-face 2 hour 0 Introduction In most tissues, new cells are formed as a result of mitosis. If the chromosomes of such cells are selectively stained with a dye such as aceto-orcein, stages in mitosis can be observed. An example of a tissue that undergoes mitosis is the meristematic tissue. This tissue is located in the cell division zone of the apical meristem at the root tip and shoot apex. Apparatus Compound light microscope Beakers Blades Needle Prepared slides of various stages of mitosis Slides and cover slips Watch glass Water bath Updated: 13/09/2017 17 SB015 Lab Manual Materials Filter paper Tissue paper 1M hydrochloric acid (HCl) Onion root tips (3-4 days old) Acetic alcohol (3 parts of absolute alcohol:1 part of acetic acid, freshly prepared) Aceto–orcein (Freshly prepared. Heat 45 mL 70% acetic acid and when the acid is hot, add 2g of orcein. Allow the solution to cool and dilute with 55 mL of distilled water. Filter the solution prior to use.) Procedures and Observation Exercise 4.1: Prepared slides of mitosis 1. Examine the prepared slides of various stages of mitosis. 2. Draw and label the stages of mitosis observed. Exercise 4.2: Preparation of onion root tips slide 1. Rest onion bulb on the rim of a container of water. Leave until the roots develop for 3 to 4 days. 2. Cut off the root tips 1 - 2 cm long. Put them in a small volume of acetic alcohol for 10 minutes. 3. Wash root tips in ice cold water for 4 - 5 minutes, then dry them on filter paper. 4. Transfer root tips to pre-heated 1M HCl at 60°C for 5 minutes. Repeat step 3. Caution - they will be very fragile. 5. Transfer two root tips onto a clean microscope slide. Cut each root tip about 1 mm from the growing tip. Keep the tips, discard the rest. 6. Tease the root tips with a mounted needle. Add one small drop of aceto–orcein stain for 2 minutes. 7. Cover with a cover slip, and blot firmly with several layers of tissue or filter paper and press gently to spread root tips. 8. View under the microscope using 40x objective lens and observe the chromosome behaviour in the mitotic stages. Updated: 13/09/2017 18 SB015 Lab Manual 9. Identify, draw and label cells showing the stages of mitosis: prophase, metaphase, anaphase and telophase. Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Updated: 13/09/2017 19 SB015 Lab Manual Figure 4.2 : Stages of mitosis seen under light compound microscope (Source:https://fthmb.tqn.com/uWS8dstnohIso29NAme1H0iugYg=/768 x0/filters:no_upscale()/139812087-56a2b3cd3df78cf77278f2cb.jpg) Questions 1. Why is the root tip placed in acetic alcohol? 2. What is the purpose of using HCl in this experiment? 3. What is the stage in the mitosis that is frequently observed? Why? 4. Explain the chromosome behavior at each stage in mitosis. 5. Where does mitosis actively take place in plants? Updated: 13/09/2017 20 SB015 Lab Manual EXPERIMENT 5: INHERITANCE Course Learning Objective: Conduct biology laboratory work on microscopy, biological molecules, histology and genetics information by applying manipulative skills (P3, PLO 2, MQF LOD 2) Learning Outcomes: At the end of this lesson, students should be able to: i. To determine the inheritance of genetic traits controlled by single genes in human ii. To determine the inheritance of ABO blood groups Student Learning Time (SLT): Face-to-face Non face-to-face 2 hour 0 Introduction A number of human characteristics are determined by single genes. These characteristics include the shape of nose, earlobe, the ability of tongue rolling, the presence of dimple and left-handed (Figure 5.1). A single gene also determines pigmentation of iris and the ability to taste phenylthiocarbamide (PTC). Procedures and Observation Exercise 5.1: Inheritance of genetic traits in human 1. The exercise will require information to be gathered from every student in the class. 2. Below are six inherited characteristics in human: (i) Shape of nose : Straight nose (E_) is dominant to curved nose (ee). (ii) Earlobe : Free earlobe (P_) is dominant to attached earlobe (pp). (iii) Tongue Rolling : Ability of tongue rolling into “U” shape (C_) is dominant to inability of tongue rolling into “U” shape (cc). Updated: 13/09/2017 21 SB015 Lab Manual (iv) Dimple : Individual with are genotypically(D_) dominant compared to those without dimple (dd). (v) Left-handed : The right-handed characteristic (H_) is dominant to left- handed (hh). (vi) Hitch hiker thumb: The ability to bend thumb at 60 angle or more are genotypically(tt) recessive compared to normal thumb- bending (T_). 3. Based on the above characteristics, fill in the information below in Table 5.1. (i) Determine your genotype for each of the six characteristics. (ii) Calculate the observed and expected frequencies for each of the six characteristics. Updated: 13/09/2017 22 SB015 Lab Manual Shape of nose Curved nose Straight nose Earlobe Free earlobe(detached) Attached earlobe https://www.users.rowan.edu https://www.users.rowan.edu Tongue rolling Ability to roll tongue Inability to roll tongue https://askabiologist.asu.edu https://askabiologist.asu.edu Updated: 13/09/2017 23 SB015 Lab Manual Dimple With dimple Without dimple https://www.news.makemeheal.com https://www.sharewhy.com Left-handed Left handed Right handed http://www.edquest.ca http://www.edquest.ca Hitch hiker thumb Normal thumb Hitch hiker thumb https://askabiologist.asu.edu https://askabiologist.asu.edu Figure 5.1 The six inherited characteristics in human Updated: 13/09/2017 24 SB015 Lab Manual Results : Table 5.1 Observed and expected frequencies of each genotype for six characteristics in the class Characteristic Phenotype Genotype Tick (√) Observed Expected your frequency frequency Shape of nose Straight nose E_ own Earlobe Curved nose ee of each of each Free earlobe P_ genotype genotype in genotype in Tongue Attached pp Rolling earlobe the class the class Ability of C_ Dimple tongue rolling Left-handed into “U” shape cc Inability of D_ Hitch hiker tongue rolling dd thumb into “U” shape H_ Have dimple hh Without T_ dimple Right-handed tt Left-handed Normal thumb- bending Ability to bend thumb at 60 angle or more Updated: 13/09/2017 25

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EXPERIMENT 1 

DIVERSITY OF BACTERIA

EXPERIMENT 2

PLANT DIVERSITY: BRYOPHYTES AND PTERYDOPHYTES

EXPERIMENT 3

BIOCATALYSIS

EXPERIMENT 4

CELLULAR RESPIRATION

EXPERIMENT 5

PHOTOSYNTHESIS

EXPERIMENT 6

MAMMAL ORGAN SYSTEM

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PRE LAB MODULE

Experiment 7: Diversity of Bacteria

Experiment 8: Plant Diversity- Bryophytes & Pteridophytes

Experiment 9: Biocatalysis

Experiment 10: Cellular Respiration

Experiment 11: Photosynthesis

Experiment 12: Mammal Organ System