BIOLOGY 5th EDITION LABORATORY MANUAL DB014 & DB024 MINISTRY OF EDUCATION MATRICULATION DIVISION
MATRICULATION DIVISION MINISTRY OF EDUCATION MALAYSIA BIOLOGY LABORATORY MANUAL SEMESTER I & II DB014 & DB024 MINISTRY OF EDUCATION MALAYSIA MATRICULATION PROGRAMME FIFTH EDITION
First Printing, 2011 (First Edition) Second Printing, 2015 (Second Edition) Third Printing, 2018 (Third Edition) Fourth Printing, 2020 (Fourth Edition) Fifth Printing, 2022 (Fifth Edition) Copyright © 2022 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/ Printed in Malaysia by Malaysia National Library Biology Laboratory Manual Semester I & II DB014 & DB024 Fifth Edition e ISBN 978-983-2604-75-4
ii NATIONAL SCIENCE EDUCATION PHILOSOPHY In consonance with the National Education Philosophy, science education in 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. 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 Malaysian citizens who are knowledgeable and competent, who possess 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.
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 21st century skills are crucial to prepare students to succeed in Industrial Revolution (I.R.) 4.0. 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 prereport, 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 1.0 Student Learning Time (SLT) v 2.0 Learning Outcomes v 3.0 Guidance for Students viii 4.0 Introduction to Microscopy x Semester I Experiment Title Page Flowering Plants 16 6 Plant Growth 18 Semester II Experiment Title Page 7 Diversity of Bacteria 22 24 29 10 Chloroplast in Aquatic Plant (Elodea sp.) 33 11 Homeostasis 36 12 Coordination 43 References 48 Acknowledgements 49 9 Animal Diversity – Invertebrates and Vertebrates 8 Plant Diversity – Gymnosperms and Angiosperms 1 Basic Techniques in Microscopy 1 2 Animal Tissue 4 3 Plant Tissue 9 4 Cell Division – Mitosis 15 5 Reproductive Structures of
v 1.0 Student Learning Time (SLT) Students will be performing the experiment within the time allocated for each practical work. Face-to-face Non face-to-face 2 hours 0 2.0 Learning Outcomes 2.1 Matriculation Science Programme Educational Objectives Upon a year of graduation from the programme, graduates are: i. Knowledgeable and technically competent in science disciplines study in-line with higher educational institution requirement. ii. Able to apply information and use data to solve problems in science disciplines. iii. Able to communicate competently and collaborate effectively in group work to compete in higher education environment. iv. Able to use basic information technologies and engage in life-long learning to continue the acquisition of new knowledge and skills. v. Able to demonstrate leadership skills and practice good values and ethics in managing organisations. 2.2 Matriculation Science Programme Learning Outcomes At the end of the programme, students should be able to: i. Acquire knowledge of science and mathematics as a fundamental of higher level education. (MQF LOC i – Knowledge and understanding) ii. Apply logical, analytical and critical thinking in scientific studies and problem solving. (MQF LOC ii – Cognitive skills) iii. Demonstrate manipulative skills in laboratory works. (MQF LOC iii a – Practical skills)
vi iv. Collaborate in group work with skills required for higher education. (MQF LOC iii b – Interpersonal skills) v. Deliver ideas, information, problems and solution in verbal and written communication. (MQF LOC iii c – Communication skills) vi. Use basic digital technology to seek and analyse data for management of information. (MQF LOC iii d – Digital skills) vii. Interpret familiar and uncomplicated numerical data to solve problems. (MQF LOC iii e – Numeracy skills) viii.Demonstrate leadership, autonomy and responsibility in managing organization. (MQF LOC iii f – Leadership, autonomy and responsibility) ix. Initiate self-improvement through independent learning. (MQF LOC iv – Personal and entrepreneurial skills) x. Practice good values attitude, ethics and accountability in STEM and professionalism. (MQF LOC v – Ethics and professionalism) 2.3 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 LOC i) 2. Solve problems related to cells, biomolecules, inheritance, genetics and biological development. (C3, PLO 2, MQF LOC ii) 3. Conduct biology laboratory work on microscopy, biological molecules, histology and genetics information by applying manipulative skills. (P3, PLO 3, MQF LOC iii a)
vii 2.4 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 LOC i) 2. Solve problems related to transport system processes, mechanisms for adaptations in living things, ecological and environmental issues in biology. (C3, PLO 2, MQF LOC ii) 3. Conduct biology laboratory work on diversity of bacteria and plant, photosynthesis, homeostasis and coordination by applying manipulative skills. (P3, PLO 3, MQF LOC iii a) 2.5 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: 1. know and practice the necessary safety precautions to be taken. 2. use the correct techniques of handling apparatus. 3. plan, understand and carry out the experiment as instructed. 4. observe, measure and record data consistency, accuracy and units of the physical quantities. 5. define, analyse data and information in order to evaluate and deduce conclusions from the experiments. 6. discuss data and information logically and critically. 7. analyse and draw conclusions from biological data. 8. develop solution to biological problems. 9. 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. 10. understand the limitations to the accuracy of observations and measurements.
viii 3.0 Guidance for Students 3.1 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. 3.2 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. 3.3 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.
ix 3.4 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 for each lab will be evaluated and included in the assessment.
x 3.5 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. 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. Reminder: NO PLAGIARISM IS ALLOWED. 3.6 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. 4.0 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
xi 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. 4.1 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 highquality 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 phasecontrast 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
xii 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. 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 structure = Shortest diameter of the observed = Wavelength (λ) 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 (λ) where 2 x N.A λ - wavelength of light N.A - Numerical Aperture
xiii 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. 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
xiv 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. 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: This lens is found at the top of the microscope. It normally has a magnification power of 10x or 15x. 1b. Ocular control knob 1a. Magnifies the real image and converts it to a virtual image to be viewed by user’s eyes. 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.
xv 3. Rotating nosepiece: You will hear clicking sounds when the objective lens is in its correct position above the specimen. You may practise this by rotating and changing the objective lenses. The structure to which the objective lenses are mounted. By gently rotating the nosepiece, you may choose the objective lens you want and correctly place it over the specimen. 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): Coarse specimen field = 5 mm a. Used to scan the specimen before identifying the specific part to be viewed further. b. Low-power objective lens (10x): Small specimen field = 2 mm c. High-power objective lens (40x): Very small specimen field = 0.5 mm d. Oil immersion objective lens (100x): Immersion oil is placed in the space between the specimen and objective lens to reduce the light refraction from the specimen. Very small specimen field = 0.2 mm b. Used to view major part of the specimen. c. Used to view specific part of the specimen. Used to view microorganisms such as bacteria and microstructures in the cells. 5a. Stage: The horizontal surface which has a hole in the centre to allow light from below to focus on the specimen. 5b. Slide clips 5c. Slide adjustment knob a. The horizontal surface on which a specimen is placed. b. The stage is usually equipped with slide clips to hold the slide in place. c. Two knobs are used to move the slide to the left, right, forward or backward. Move these knobs to
xvi learn how the slide is moved into position. 6a. Condenser: Condenser is located immediately under the stage 6b. Condenser control knob 6c. Condenser lens knob a. Used to focus and deliver light to the specimen. b. Used to adjust the condenser. c. Used to focus the light. 7. Iris diaphragm: An adjustable light barrier of iris type 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. Controls the amount of light entering and leaving the condenser. 8a. Off/on switch. 8b. Light control knob. Please ensure that either of the switches is OFF or MINIMUM, respectively before you use the microscope. The source of light is a tungsten bulb located at the base of a microscope. 9. Body arm The metal part used to carry a microscope. 10. Base The heavy cast metal part used as the base and for support. 11. Coarse adjustment knob: Use this knob only when using lowpower objective lens. Rotate this knob carefully, and observe what happens. Does the stage or the body tube move? Used to bring specimen into focus by moving the stage to the specimen.
xvii 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. 4.2 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. 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. 4.3 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.
xviii 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 highpower 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 4.4 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
xx is still dirty, clean it with a little amount of xylene and rub it gently with clean, dry lens tissue. iii) Remove the slide off the stage. iv) Gently rotate the nosepiece again to place the lowpower 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. 4.5 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.
xxi 4.6. 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. 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.
xxii Figure 4: A dissecting microscope 4.7. 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-andwhite pictures. The electron microscope can be used only for dead tissue materials because they are viewed in vacuum.
xxiii 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).
BIOLOGY 1 DB014
DB014 Lab Manual Updated: 26/04/2022 1 EXPERIMENT 1: BASIC TECHNIQUES IN MICROSCOPY Objectives: At the end of this lesson, students should be able to: i. handle microscope properly; ii. obtain accurate images; and iii. calculate the actual magnification. Experiment 1.1: Handling Microscope Properly Before doing the following experiments, you must read and understand the basic techniques of using a microscope. Refer to Introduction: to Microscopy. Apparatus: Compound light microscope Experiment 1.2: To obtain accurate images Apparatus: Compound light microscope Materials: ‘e’ prepared slide Prepared slides of cross sections of monocot or dicot root Procedures and Observation: Images under the microscope 1. Observe the prepared slides of ‘e’ using the 4x and 10x objective lens. Determine the position of ‘e’ and draw your observation. 2. Observe the prepared slides of monocot or dicot root using the 4x and 10x objective lens. Draw your observation under 10x objective lens.
DB014 Lab Manual Updated: 26/04/2022 2 Experiment 1.3: Magnification Procedures and Observation: 1. Determine the actual magnification of a specimen by using the formula below. Actual Magnification = Magnification power of objective lens x Magnification power of ocular lens 2. Calculate the actual magnification in Table 1.1. Table 1.1 Actual magnification of a specimen Actual magnification Magnification power of ocular lens Magnification power of objective lens 4x 10x 40x 100x 10x
DB014 Lab Manual Updated: 26/04/2022 3 Questions: Part A: Handling microscope properly. 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 …………………. 3. Based on laboratory practices, what do you use to clean the microscope lenses? 4. 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)? 5. How do you adjust the slide when the specimen is out of the field of view to the top? Part B: To obtain accurate image 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 still remain in focus when placed at the longest working distance from the specimen? 5. 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
DB014 Lab Manual Updated: 26/04/2022 4 EXPERIMENT 2: ANIMAL TISSUES Objectives: At the end of this lesson, students should be able to: i. identify the animal tissues under the compound light microscope; and ii. identify the structure of epithelial tissues, nerve tissues, muscle tissues and connective tissues. Introduction: Four main types of animal tissues are epithelial tissues, muscle tissues, connective tissues and nerve tissues. Epithelial tissues form the surface lining of other tissues either inside or outside the body. Epithelial tissues consist of epithelial cells which are closely arranged between the cells. Epithelial tissues have specialized functions for growth, protection, excretion and absorption. The main function of muscle tissues is for contraction. Based on structures, muscle tissues can be divided into three types: striated muscle, smooth muscle and cardiac muscle. Physiologically, muscle tissues can be divided into voluntary muscle and involuntary muscle. Bone tissue, cartilage tissue and blood tissue are grouped into connective tissues. The functions of the connective tissues are for support, connecting tissues and organ, transport and defence. Connective tissues consist of three components: cell, ground substance (substance which is deposited in between the connective tissues and serves to hold the cells together) and fibre. The fibre together with the connective tissue is embedded in the matrix. The main function of nerve cells or neuron is to conduct nerve impulses. Apart from neuron, the nerve tissue consists of neuroglia which gives internal support. Apparatus: Compound light microscope Materials: Prepared slides of epithelial tissues Simple squamous epithelium Simple cuboidal epithelium Simple columnar epithelium
DB014 Lab Manual Updated: 26/04/2022 5 Prepared slides of nerve tissues Motor neuron Prepared slides of muscle tissues Smooth muscle tissue Prepared slides of connective tissues Blood Procedures and Observation: 1. Examine the slides given using either 10x or 40x objective lens. 2. Draw and label the structure of each of the following tissues. Use the figures provided to assist you in your cell investigation. a. Epithelial tissues i. Simple squamous epithelium ii. Simple cuboidal epithelium iii. Simple columnar epithelium b. Nerve tissue i. Motor neuron c. Muscle tissues i. Smooth muscle tissue d. Connective tissues i. Blood Figure 2.1 Simple squamous epithelium w.m. (Source: Simple squamous epithelium (Solomon et.al, Biology 11th Edition, Page: 824))
DB014 Lab Manual Updated: 26/04/2022 6 Figure 2.2 Simple cuboidal epithelium (c.s.) Figure 2.3 Simple columnar epithelium (l.s.) (Source: Simple columnar epithelium (Solomon et.al, Biology 11th Edition, Page: 824))
DB014 Lab Manual Updated: 26/04/2022 7 Figure 2.4 Blood smear Figure 2.5 Smooth muscle (l.s.)
DB014 Lab Manual Updated: 26/04/2022 8 Figure 2.6 Motor neuron Source: Nervous tissue (Solomon et.al, Biology 11th Edition, Page: 830) Questions: 1. Describe the structure, functions and distributions of tissues below: a. Epithelial tissues i. Simple squamous epithelium ii. Simple cuboidal epithelium iii. Simple columnar epithelium b. Nerve tissue i. Motor neuron c. Muscle tissues i. Smooth muscle tissue d. Connective tissues i. Blood
DB014 Lab Manual Updated: 26/04/2022 9 EXPERIMENT 3: PLANT TISSUES Objectives: At the end of this lesson, students should be able to: i. identify the plant tissue under the compound light microscope; and ii. identify different types of ground and vascular tissues. Introduction: Permanent tissues consist of mature cells that have specialized structure and functions. Permanent tissues can be divided into three: dermal tissues, ground tissues (parenchyma, sclerenchyma, and collenchyma) and vascular tissues (xylem and phloem). Apparatus: Compound light microscope Materials: Prepared slides of cross sections of monocot/ dicot stem and root Structures of plant tissues 1. Examine the prepared slides of any types of plant tissues (root/ stem) 2. Identify the distribution of the tissues (parenchyma, collenchyma, sclerenchyma, phloem and xylem). 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 (parenchyma, collenchyma, sclerenchyma, phloem, and xylem). Use the figures provided to assist you in your tissue investigation.
DB014 Lab Manual Updated: 26/04/2022 10 Figure 3.1 Structure and distribution of vascular bundles in cross section of monocot root. Source: Cross section of dicot stem (Reece et.al, Biology 12th Edition, Page:769) Figure 3.2 Cross section of monocot root Source: Monocot root C.S (Jaime K., 2022)
DB014 Lab Manual Updated: 26/04/2022 11 Figure 3.3 Structure and distribution of vascular bundles in cross section of dicot root Source: Dicot root (Solomon et. al, Biology 11th Edition, Page:764)
DB014 Lab Manual Updated: 26/04/2022 12 Figure 3.4 Cross section of dicot root Source: Stele of dicot root (Solomon et. al, Biology 11th Edition, Page:764) Figure 3.5 Structure and distribution of vascular bundles in cross section of monocot stem. Source: Cross section of monocot stem (Reece et. al, Biology 12th Edition, Page:770)
DB014 Lab Manual Updated: 26/04/2022 13 Figure 3.6 Structure and distribution of vascular bundles in cross section of dicot stem. Source: Cross section of dicot stem (Reece et. al, Biology 12th Edition, Page:770) Figure 3.7 Structure of different plant tissues in cross section of monocot and dicot stem Source: Dicot stem vs monocot stem (Harold, G. 2018)
DB014 Lab Manual Updated: 26/04/2022 14 Questions: 1. Describe the structure, functions, and distributions of tissues below: a. Ground tissues i. Parenchyma cells ii. Collenchyma cells iii. Sclerenchyma cells b. Vascular tissues i. Xylem tissue ii. Phloem tissue
DB014 Lab Manual Updated: 26/04/2022 15 EXPERIMENT 4: CELL DIVISION - MITOSIS Objective: At the end of this lesson, students should be able to identify different stages of mitosis. 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. Apparatus: Compound light microscope Prepared slides of various stages of mitosis Procedures and Observation: 1. Examine the prepared slides of various stages of mitosis. 2. Draw and label the stages of mitosis observed. Questions: 1. Describe the four stages of mitosis. 2. Where does mitosis actively take place in plants?
DB014 Lab Manual Updated: 26/04/2022 16 EXPERIMENT 5: REPRODUCTIVE STRUCTURES OF FLOWERING PLANTS Objectives: At the end of this lesson, students should be able to: i. observe the reproductive structure of flowering plants; and ii. differentiate between germinated and non-germinated pollen grains. Introduction: The growth of a pollen tube is a fascinating phenomenon. Pollen grains are morphologically simple, small structures which contain two nuclei when released from the anther at anthesis. When a viable pollen grain lands on the stigma of a compatible flower, it produces a tube several hundred to several thousand micrometres long in which the pollen nuclei travel to the ovary of the flower. The process of tube formation is a relatively uncomplicated example of growth and development. Pollen germination represents a short, yet very critical event in a series of steps leading to the double fertilization of the ovule. Apparatus: Compound light microscope Cavity well slides and cover slips Dropper Dissecting microscope Dissecting needle Scalpel/ Razor blade/ lancet Petri dish Watch glass Labelling stickers Material: Hibiscus flower Hibiscus or Spider Lily pollen grains Hibiscus or Spider Lily pollen grains in germinating solution *Germinating solution (10g sucrose, 0.01g boric acid and 0.03g calcium nitrate in 100 ml distilled water)
DB014 Lab Manual Updated: 26/04/2022 17 Experiment 5.1: Reproductive structure of flowering plants Procedures and Observation: 1. Remove the sepals and petals from hibiscus flower by gently pulling them off the receptacle. 2. Locate the stamen, each of which consists of a thin filament with a pollenfilled anther of the tips. Note the number of stamen. 3. Locate the pistil. The stigma at the top of the pistil is often sticky. The style is a long, narrow structure that leads from the stigma to the ovary. 4. Observe the stamen and pistils of the hibiscus flower with dissecting microscope. 5. Draw and label the male and female reproductive system of hibiscus flower. Experiment 5.2: Pollen germination Procedures and Observation: 1. Add 1-3 drops of water on a cavity well slide and transfer pollen grains onto it. Cover with a cover slip. 2. Observe the pollen grains under a compound light microscope using 40x objective lens. Draw and label your observation. 3. Repeat step 1 and 2 with pollen grains in germinating solutions. Questions: 1. Why stigma at the top of the pistil is often sticky? 2. Why pollen is important in fertilization?
DB014 Lab Manual Updated: 26/04/2022 18 EXPERIMENT 6: PLANT GROWTH Objectives: At the end of this lesson, students should be able to: i. observe and record the growth of a bean sprout; and ii. measure growth parameter (height of plant and length of leaf). Introduction: Growth is a fundamental characteristic of all living organism. Growth is always associated with an increase in size. However, increase in size is not the sole factor to define growth. There are stages in organism development where there is an increase in cell numbers but not body size. Growth and development goes together in tandem. Development can also be described as an increase in complexity. For metazoan, the growth can be divided into three phases; cell division, cell enlargement, and cell differentiation. Growth can be measured by plotting parameters such as length, height, mass, surface area, volume and number against time. Sigmoid curve is a typical growth pattern for many organisms. Apparatus: Cotton wool/ filter paper Graph paper Plastic container (e.g. Mineral bottle, plastic cup etc.) Ruler Thread Materials: Mung beans Water Procedures and Observation: 1. Place the cotton wool/ filter paper into the plastic container. 2. Pour adequate water into the container. 3. Place the 15 mung bean seeds on the cotton wool. 4. Observe what happens to the mung beans for 8 days. Water them daily. 5. The observation includes the height of plant and the length of the leaf.
DB014 Lab Manual Updated: 26/04/2022 19 6. Record your observation in Table 6.1. Table 6.1 Mean height of plant and length of leaf Days 0 1 2 3 4 5 6 7 Mean height of plant (mm) Mean length of leaves (mm) 7. Plot the growth curves of the bean sprouts in a single graph. Questions: 1. Based on the graph drawn, what is the shape of the growth curve? Explain why. 2. Which of the parameter shows rapid growth? Explain your answer.
BIOLOGY 2 DB024
DB024 Lab Manual Updated: 26/04/2022 22 EXPERIMENT 7: DIVERSITY OF BACTERIA Objectives: At the end of this lesson, students should be able to: i. identify different shapes of bacteria; and ii. apply the use of oil immersion with high magnification (oil immersion lens). Introduction: Bacteria are unicellular prokaryotes belong to the Domain Bacteria. Bacteria can be identified based on cell shapes, Gram staining and position of flagella. The four common shapes of bacteria are coccus, bacillus, spirillum and vibrio. Apparatus: Compound light microscope Materials: Prepared slides of bacteria Escherichia coli Staphylococcus aureus Immersion oil Lens tissue papers Methylated spirit/ Xylene (only for specific use) Procedures and Observation: 1. Observe the prepared slide under the microscope using 100x objective lens. (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.) REMINDER: Make sure the oil immersion lens is properly cleaned at the end of experiment (Refer to section INTRODUCTION: TO MICROSCOPY)
DB024 Lab Manual Updated: 26/04/2022 23 Figure 7.1 Escherichia coli (under 100x objective lens) Source: S. M. Lutful Kabir (2016) Gram negative, pink coloured, small rod shape E. coli under light microscope. Figure 7.2 Staphylococcus aureus (under 100x objective lens) Source: J. Scott (2022). Gram stain of Staphylococcus aureus. https://www.ncbi.nlm.nih.gov/books/NBK470553/figure/article-22388.image.f1/ Questions: 1. Describe the diversity of bacteria based on cell shapes. 2. What are the precautions taken when handling immersion oil?
DB024 Lab Manual Updated: 26/04/2022 24 EXPERIMENT 8: PLANT DIVERSITY - GYMNOSPERMS AND ANGIOSPERMS Objective: At the end of this lesson, students should be able to identify morphological reproductive structure of gymnosperms and angiosperms. Introduction: Gymnosperms Gymnosperms are plants that produce naked seeds on scaly structure, named strobilus or cones. Its sporophyte generation produces 2 types of spores: microspores that will develop into male gametophytes and megaspores that develop into female gametophytes which is nutritionally dependent on living sporophyte. Angiosperms Angiosperms are the most abundant, diverse and geographically widespread flowering plants. The success of angiosperms in dominating the terrestrial habitats is due to some factors: structural diversity, efficient vascular system, mutualism and short generation period. Their sizes vary from 1mm up to 100m in height. The sporophyte generation is dominant while the gametophyte is microscopic and fully dependent on the sporophytes. Experiment 8.1: Diversity of Gymnosperms Apparatus: Compound light microscope Materials: Fresh specimens: Pinus sp. (Phylum Coniferophyta) Male and female cones of Pinus sp. Prepared slides: Megaspores of Pinus sp. cones Microspores of Pinus sp. cones
DB024 Lab Manual Updated: 26/04/2022 25 Procedures and Observation: 1. Observe the given specimens. 2. Draw and label the specimens to show their morphological differences. 3. Examine the prepared slides of megaspores and microspores. Draw and label the megasporophyll, megasporangium, microsporophyll and microsporangium. Figure 8.1 Male cone of Pinus sp. Figure 8.2 Female cone of Pinus sp. microsporophyll megasporophyll