12 Eng Biology Lab Manual
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Laboratory Manual
Biology
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Class XII
FOREWORD
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The National Council of Educational Research and Training (NCERT) is the apex body concerning all aspects of refinement of School Education. It has recently developed textual material in Biology for Higher Secondary stage which is based on the National Curriculum Framework (NCF)–2005. The NCF recommends that children’s experience in school education must be linked to the life outside school so that learning experience is joyful and fills the gap between the experience at home and in community. It recommends to diffuse the sharp boundaries between different subjects and discourages rote learning. The recent development of syllabi and textual material is an attempt to implement this basic idea. The present Laboratory Manual will be complementary to the textbook of Biology for Class XII. It is in continuation to the NCERT’s efforts to improve upon comprehension of concepts and practical skills among students. The purpose of this manual is not only to convey the approach and philosophy of the practical course to students and teachers but to provide them appropriate guidance for carrying out experiments in the laboratory. The manual is supposed to encourage children to reflect on their own learning and to pursue further activities and questions. Of course the success of this effort also depends on the initiatives to be taken by the principals and teachers to encourage children to carry out experiments in the laboratory and develop their thinking and nurture creativity. The methods adopted for performing the practicals and their evaluation will determine how effective this practical book will prove to make the children’s life at school a happy experience, rather than a source of stress and boredom. The practical book attempts to provide space to opportunities for contemplation and wondering, discussion in small groups, and activities requiring hands-on experience. It is hoped that the material provided in this manual will help students in carrying out laboratory work effectively and will encourage teachers to introduce some open-ended experiments at the school level.
New Delhi 21 May 2008
YASH PAL Professor and Chairperson National Steering Committee National Council of Educational Research and Training
PREFACE
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The development of the present laboratory manual is in continuation to the NCERT’s efforts to improve upon comprehension of concepts and practical skills among the students. The present laboratory manual will be complementary to the textbook of Biology for Class XII. The expansion of scientific knowledge and consequently the change in the system of education has led to the development of new methods of instructions. Today the stress is laid on the enquiry approach and discussion method instead of lecture method of teaching. Biology is now something more than observation of living organisms. Study of Biology includes microscopic observations to reveal minute internal details of the organism, biochemical testing to understand complex reactions taking place inside the organisms, experiments with live organism to understand various physiological processes and even much more. In other words experiments in Biology truly represents an interdisciplinary approach of learning. The new syllabus of Biology has been designed to cater to the needs of pupil who are desirous of pursuing science further. The fundamental objective of this course is to develop scientific attitude and desired laboratory skills required for pursuing Biology as a discipline at this level. A similar approach has been taken while formulating the practical syllabus of Biology for higher secondary stage. The practical syllabus includes content based experiments, which help in comprehension of the concepts. There are altogether twenty-five exercises in the present manual which are based on Biology curriculum for Class XII. For each practical work, principle, requirements, procedure, precautions, observations, discussion and the questions are given in the book. The methodology of preparation of any reagent, if required, has been given alongwith the requirements, for the convenience of students and teachers. The questions are aimed to develop learner’s understanding of the related problems. However, teacher may provide help in case the problem is found to be beyond the capability of the learner. Precautions must be well understood by the learners before proceeding with the experiments and projects. In addition to the core experiments enlisted in the syllabus for Class XII emphasis has also been given for pursuing Investigation Project Work. It is expected that these informations will motivate the students to take up independent project work on topics of their interest. Appropriate appendices related to the observation and study of organisms are given along with the experiment. International symbols for units, hazards and hazard warnings are given at appropriate places in the book. It is expected that this will make the learners more careful about the environment and make them careful while dealing with the equipments and chemicals in the laboratory.
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It gives me a pleasure to express my thanks to all those who have been associated at various stages of development of this laboratory manual. It is hoped that this practical book will improve teaching-learning process in Biology to a great extent. The learners will be able to understand the subject well and will be able to apply the acquired knowledge in new situations. I acknowledge with thanks the dedicated efforts and valuable contribution of Dr Dinesh Kumar, coordinator of this programme and other team members who contributed and finalised the manuscript. I especially thank Professor G. Ravindra, Director (Incharge), NCERT for his administrative support and keen interest in the development of this laboratory manual. I am also grateful to the participating teachers and subject experts who participated in the review workshop and provided their comments and suggestions which helped in the refinement of this manual. We warmly welcome comments and suggestions from our readers for further improvement of this manual.
H UKUM SINGH
Professor and Head Department of Education in
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Science and Mathematics
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LABORATORY MANUAL DEVELOPMENT TEAM MEMBERS Animesh K. Mohapatra, Associate Professor, Regional Institute of Education, NCERT, Ajmer
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B.K. Tripathi, Professor, DESM, NCERT, New Delhi
C.V. Shimray, Assistant Professor, DESM, NCERT, New Delhi
N.V.S.R.K. Prasad, Associate Professor in Botany, Sri Venkateshwara College, New Delhi P.K. Durani, Professor (Retired), DESM, NCERT, New Delhi
Sunita L. Varte, Assistant Professor, DESM, NCERT, New Delhi
S.P. Sinha, Professor of Zoology (Retired), TM Bhagalpur University, Bhagalpur V.V. Anand, Associate Professor, Regional Institute of Education, Mysore
MEMBER-COORDINATOR
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Dinesh Kumar, Associate Professor, DESM, NCERT, New Delhi
ACKNOWLEDGEMENT
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The National Council of Educational Research and Training (NCERT) acknowledges the valuable contribution of the individuals and organisations involved in the development of this laboratory manual. The council also acknowledges the valuable contribution of the following academics for reviewing and refining the manuscript of the laboratory manual: A.K. Sharma, Reader in Zoology, University of Lucknow, Lucknow; Iswant Kaur, D.M. School, RIE, Bhopal; K. Muralidhar, Professor, Department of Zoology, University of Delhi, Delhi; K.K. Sharma, Professor Department of Zoology, M.D.S. University, Ajmer; M.M. Chaturvedi, Professor Department of Zoology, University of Delhi, Delhi; Nazir Ahmad Kakpori, Department of Education, Govt of Jammu & Kashmir, Srinagar; Reena Mohapatra, St. Stephen’s Senior Secondary School, Ajmer; Savita Sharma, Mount Carmel School, Dwarka, New Delhi; Savithri Singh, Professor and Principal, Acharya Narendra Dev College, New Delhi; Shalu Dhawan, Amity International School, Saket, New Delhi; Shivani Goswami, Mother’s International School, New Delhi; V.K. Srivastava, Reader in Zoology, J.N. College, Pasighat; Vijay Kumar, Delhi State Science Teacher Forum, New Delhi. We also acknowledge the contributions of Anil Kumar and Binita Kumari, Junior Project Fellows, DESM, NCERT, New Delhi. Special thanks are also due to Hukum Singh, Professor and Head, DESM, NCERT for his interest in the work and administrative support. The Council also acknowledges the efforts of Surender Kumar, Narender Kumar Verma, Monika Rajput and Girish Goyal, DTP Operators, for helping in shaping this laboratory manual. The contributions of Publication Department of NCERT in printing out this laboratory manual are also duly acknowledged.
CONTENTS FOREWORD
iii v
Introduction
1
Exercise 1
:
Exercise 2
:
Exercise 3
:
Exercise 4
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Exercise 5
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Exercise 6
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Exercise 7
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Exercise 8
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Exercise 9
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PREFACE
To study the reproductive parts of commonly available flowers
5
12
To study pollen tube growth on stigma
14
To study the discrete stages of gametogenesis in mammalian testis and ovary
16
To study and identify various stages of female gametophyte development in the ovary of a flower
18
Preparation and study of mitosis in onion root tips
20
Study of stages of meiosis using permanent slides
25
To study the blastula stage of embryonic development in mammals, with the help of permanent slide, chart, model or photograph
30
To verify Mendel's Law of Segregation
32
Exercise 10 :
To verify the Mendel’s Law of Independent Assortment
35
Exercise 11 :
Preparation and analysis of Pedigree Charts
39
Exercise 12 :
To perform emasculation, bagging and tagging for controlled pollination
45
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To calculate percentage of pollen germination
Staining of nucleic acid by acetocarmine
47
Exercise 14 :
To identify common disease-causing organisms and the symptoms of the diseases
49
Exercise 15 :
To study the texture of soil samples
53
Exercise 16 :
To determine the water-holding capacity of soils
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Exercise 13 :
To study the ecological adaptations in plants living in xeric and hydric conditions
62
Exercise 18 :
To study the adaptations in animals living in xeric and hydric conditions
65
Exercise 19 :
To determine the pH of different water and soil samples
68
Exercise 20 :
To study turbidity of water samples
71
Exercise 21 :
To analyse living organisms in water samples
75
Exercise 22 :
To determine the amount of Suspended Particulate Matter (SPM) in air at different sites in a city
83
Exercise 23 :
To study plant population density by quadrat method
85
Exercise 24 :
To study plant population frequency by quadrat method
87
Exercise 25 :
Study of homologous and analogous organs in plants and animals
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Exercise 17 :
Investigatory Project Work
92–104
Project 1
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To study the effect of pH on seed germination
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Project 2
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Quantitative analysis of phytoplankton in a water body
102
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Introduction
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Laboratory is a place where ideas and concepts can be tested through experiments. Biology, like any other discipline of science, is based on experimental work and therefore practical forms an integral part of learning. Biology laboratory provides a unique learning environment where learners inculcate scientific temper, develop relevant skills and get exposed to realms of techniques and methodologies of scientific investigations. Laboratory investigations in Biology increase the reasoning abilities, bring scientific attitude in a learner and also help in acquisition of skills of scientific processes. Also, observation of nature and the living organisms found in it is no less important for the understanding of many aspects of the subject especially the diversity of the living organisms, their systematic study, their relationships among themselves and with the environment. Knowledge in the field of Biology can be acquired or constructed only on the basis of correct observations and experimentally verifiable processes. Biology laboratory thus provides the learners an environment where the process of learning is facilitated by hands-on experiments. Biology is a unique discipline in the sense that it does not merely deal with the study of morphology, anatomy, physiology and reproduction of the living organisms, rather, understanding of the subject requires understanding of a number of interdisciplinary areas and approaches. On one hand, a biologist needs to be sufficiently skilled in handling the enormous diversity of the living organisms, be it plants, animals, fungi or even microscopic bacteria, while on the other hand, a biologist should be able to understand the biochemical, molecular, physiological, behavioural, genetic and many other phenomena pertaining to the living organisms. The study of intricate relationship of different types of organisms among themselves and also with its environment is an important concern of a biologist. Thus, experiments and exercises in Biology train a learner about skills of observations, manipulation of the organisms for the study of internal details, biochemical as well as molecular composition and processes, investigation of the abiotic environment and even analysis of phenomena like inheritance and evolution. As far as the study of the living organism is concerned, correctness of the method is very important. Such a study may be very simple, e.g., study of habit, habitat and external features of the plants or animals, or, it may involve certain manipulations like dissection and section cutting of the parts of the organisms to study the minute details. Very often observation and study of the magnified image of the minute parts under a microscope provides a better insight about the features of the organisms. However, microscopic study involves certain specific skills depending on type of the organisms/ tissues/cells to be studied. It involves specific preparations (peeling, section
L ABORATORY MANUAL: BIOLOGY
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cutting, fixation, staining, dehydration, mounting, etc.) so that microscopic examination reveals the expected details. As histological and cytological observations give us only static pictures of the continuous processes, analysis of biochemical, physiological and ecological aspects need certain other kinds of skills such as preparation of chemicals and reagents, designing and performing an experiment, observation and recording of data and ultimately interpretation and drawing conclusions. While performing experiments, honesty in recording of data and its correct presentation is very important as it is not only useful in the logical interpretation but also helps in the identification of errors. In order to perform experiments successfully, a learner needs to go to the Biology laboratory well prepared. This includes the following: 1.
Laboratory Record Book: For maintaining all the information including recording of data and its interpretation.
2.
Dissection Box: A dissection box is required in the Biology laboratory for various purposes like handling and manipulation of living materials, performing experiments, preparation of slide, etc. A dissection box should contain scissors (two pairs, one small with fine tip and one larger), scalpels (one small and one medium sized), forceps (two, one small with sharp fine tips and the other medium sized with blunt tips), dissecting needles (two), razor, hand lens, dropper, fine brush, etc.
3.
Laboratory Manual
4.
A Laboratory Coat or Apron
5.
A Hand Towel
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While in the laboratory a student should be very careful and methodical. One should listen carefully to the instructions given by the teacher/ instructor before performing an experiment. In the biology laboratory a student has to handle a number of sharp objects and hence necessary precaution and care should always be taken while handling objects like scissors, forceps, needles, scalpel, razor, etc. It is also very important to follow the safety instructions mentioned on the instruments and/or on the label of the reagent/chemical. Students should also be aware about the use of the First-aid Box so that in case of any accident or injury the preliminary aid can be provided to the affected person. While describing the experiment students are expected to follow a pattern in which the aim of the experiment, its principle, list of the materials to be used, procedure, observation table (if required), inference and discussion should be given. Necessary precautions to be taken should also be mentioned appropriately in the procedure or at the end. There are a few experiments in which field visit is essentially required. For this all the necessary preparations (materials, equipments, reagents and chemicals)
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IENTRODUCTION XERCISE 1
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should be made in advance. Drawing of illustrations is also an important component of the practical in Biology. Students are expected to follow certain fundamental rules while drawing the illustrations so that it reflects your observations correctly. • Make your illustrations using pencil only and always use white drawing sheet. Illustration should be in the centre of the page. • Drawing of an object (plant, animal or experimental set-up) should be proportionate in size. • Draw your illustrations keeping the object before you. • Drawing must be clear with simple outlines. • Appropriately label your drawing. Parts of the drawing should be indicated by straight horizontal line or arrow. Two lines or arrow should never cross each other. As far as possible, labelling should be done on the right side of the drawing. An appropriate legend or heading of the drawing should also be given below it. About the Manual
The main objective of the manual is to introduce the students of higher secondary stage to the fascinating world of plants, animals and microbes and their complex biological phenomena. The manual covers a complete description of the experiments and exercises. The suggested experiments cover almost all the units/topics including those on diversity in living world, plant, animal and human physiology, genetics, bio-technology and human welfare and environment. A standard format has been used to describe each experiment which includes
• •
Aim: It gives a brief title of the experiment under investigation.
•
Materials required: This includes the names of plants/animals to be used as 'samples', the type of apparatus, the type and quantity of glasswares required, reagents, chemicals and solutions needed, their concentration and other specifications, method of preparations of solutions and reagents. If a particular material/chemical/glassware is not available, sufficient alternatives have been suggested.
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•
Principle: It is a very brief introduction of the experiment under investigation and explains the biological phenomenon involved. It gives brief but comprehensive ideas about the design of the experiment and explains the significance of the phenomenon being studied.
Procedure: This section includes full details of experimental procedure explained stepwise, including special precautions necessary to be taken while the experiment is being conducted. Drawings of the samples, apparatus and the experimental setup, wherever found necessary, have been included to facilitate the students to perform the experiment as accurately as possible.
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L ABORATORY MANUAL: BIOLOGY
Observation and Results: This section deals with the recording of all observations made during the experiment. Students are advised to consider the entire data. Data can be represented in the form of tables, graphs and histograms wherever possible. Use of units in which various quantities are measured has been indicated in the manual.
•
Discussion: Included in this heading is a statement of the conclusions drawn from the experimental results and compared thesis (wherever possible) with any comparable data from other sources. The relevance of the conclusions drawn from the experimental results to the various processes under investigation and to the life of plant, animal and microbes has been prompted out.
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•
•
Precaution: This section contains all the necessary precautions to be taken during experimentation to obtain results free of errors. However, attempts have been made to mention required precautions along with the procedure also.
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A great emphasis has been laid on a student getting valid results and interpreting them. It is essential that the teacher should properly explain each experiment so that inexperienced students will be able to obtain accurate results within a reasonable time. Teachers are also expected to help students in identifying errors and mistakes committed during experiments and ways for correcting them. It is possible that some of the students may undoubtedly be capable of doing more sophisticated work than that represented in the manual. But introductory course of this sort has been designed to help all students for some useful and joyful experience by conducting the experiments of their own. The manual also aims that students and teachers not be discouraged by either incomplete experiments or experiments which yield apparently meaningless results. With the objectives of inculcating scientific temper among learners and providing them an opportunity to undertake independent scientific investigation, Investigatory Project Work has been included as an integral part of the practical curriculum of Class XII. Such investigatory projects are expected to provide thrill in the learning process. It is also expected to serve the real purpose of practicals, i.e., developing an ability to hypothesise and design experiments to address certain problems, to make observations methodically and to draw conclusions out of the experimental data. A comprehensive idea about undertaking investigatory project has been given in the book with a list of a few problems on which investigatory project work can be undertaken. However, the list is only suggestive and considering the wider scope students can undertake any kind of investigatory project work depending on their region, its climatic condition, availability of resources, etc.
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Exercise 1 Aim: To study the reproductive parts of commonly available flowers
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Principle: The male reproductive parts of a flower are the stamens collectively called androecium and the female reproductive parts are the carpels/pistils collectively called gynoecium. The individual units of stamen consist of a filament, which supports the anther lobes. Gynoecium consists of stigma, style and ovary. Many variations are found in different characteristics of both the stamens and carpels. We shall try to study these variations in the reproductive parts of flowers in the exercise. Requirement: Commonly available flowers, needles, forceps, razor/scalpel blade, brush, slides, cover slip, watch glass, magnifying lens, dissecting microscope, compound microscope, etc.
Procedure
(i) Familiarise with the terms to describe the reproductive parts of flowers given in annexures of Exercise No. 11 of Laboratory Manual: Biology (Class XI) and at the end of this experiment. (ii) Observe the flower with the naked eye, hand lens or under a dissecting microscope. Study their reproductive parts and count the number of stamens and record their cohesive and adhesive features. (iii) Cut L.S. of the flower and place it on a slide to observe the following characters: (a) Placement of anthers
(b) Position of the ovary: epigynous/perigynous/hypogynous.
(iv) Mount one stamen on a slide and study the following characters: (a) Attachment of filament to anther
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(b) Dehiscence pattern of the anther lobes for discharge of pollen. (v) Cut T.S. of anther lobe to observe the number of pollen sacs. (vi) Mount the pistil on a slide and study style, stigma and ovary. Record the number of stigma and nature of pistil. (vii) Cut T.S. of ovary, mount it on a slide and observe (a) Number of locules in the ovary
L ABORATORY MANUAL: BIOLOGY
(b) Type of placentation (c) Number of ovules per locule (viii) Draw labelled figures of your preparation and observations.
Questions 1. Name the most common type of placentation observed.
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2. What is the most common type of dehisence pattern in anthers? 3. Name a few unisexual flower-bearing plants studied by you.
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4. “Flower is a modified shoot.” Justify the statement based on your observation.
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EXERCISE 1
Annexure 1 Description of reproductive parts of flowers Androecium Number of stamens
Stamens may be free or united. If united they can be of the following type:
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Cohesion (Fig. 1.1 a–e)
The number of stamens may vary from a few to many in different flowers
(i)
(ii)
(iii)
Adhesion (Fig. 1.2)
no t Dehiscence pattern
Synandrous : Stamens fused all through their length, e.g., Cucurbita
Adelphous: Anthers remain free and filaments are united. Adelphous condition can be (a)
Monoadelphous—United to form 1 bundle, e.g., China rose
(b)
Diadelphous—United to form 2 bundles, e.g., Pea
(c)
Polyadelphous—United into more than two bundles, e.g., Lemon
Fusion of stamens with other parts of the flower (i)
(ii)
Attachment of filament to anther (Fig.1.3 a–d)
Syngenesious : Filaments free and anthers united, e.g., Sunflower.
(i)
Epipetalous : Stamens fused with petals, e.g., Sunflower, Datura
Epiphyllous : Stamens fused with perianth, e.g., Lily
Basifixed: Filament attached to the base of anther, e.g., Mustard
(ii)
Adnate : Filament attached along the whole length of anther, e.g., Michelia, Magnolia
(iii)
Dorsifixed : Filament attached to the back of anther, e.g., Passion flower
(iv)
Versatile : Anther lobes attached with filament in the middle portion with both ends free, e.g., Gramineae family
(i)
Porous : Pollens released through pores, e.g., Brinjal, Potato
(Fig. 1.4 a,b)
(ii)
Longitudinal: Pollens released through the longitudinal slit of another lobes, e.g., China rose, Cotton
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L ABORATORY MANUAL: BIOLOGY
Gynoecium Number of stamens
(i)
Epigynous: Position of ovary inferior to other floral parts, e.g., Mustard, China rose
(ii)
Perigynous : Other floral parts are attached around the ovary, e.g., Apple, Guava
(iii)
Hypogynous: Position of ovary superior to other floral parts, e.g., Sunflower
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Position of ovary (Fig. 1.5 a–d)
The number of stamens may vary from a few to many in different flowers
Cohesion (Fig. 1.6 a–c)
Number of locules in ovary
If number of carpels is more than one, they may be (i)
Apocarpous : Carpels are free. Each carpel has its own style and stigma, e.g., Rose
(ii)
Syncarpous: Carpels are united, e.g., Lady’s finger, Tomato
Vary from one to many (i)
(ii)
Placentation (Fig. 1.7 a–e)
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Bilocular: Two locules, e.g., Datura
(iii)
Multilocular : Many locules, e.g., Lady’s finger, China rose
(i)
Marginal : The placenta forms a ridge along the ventral suture of the ovary and the ovules are borne on this ridge, e.g., Pea
(ii)
Axile: The ovary is partitioned into several chambers or locules and the placentae are borne along the septa of the ovary, e.g., Tomato, China rose
(iii)
Parietal: The ovules develop on the inner wall of the ovary or on peripheral part. Ovary unilocular but in some cases becomes two chambered due to formation of a false septum, e.g., Mustard
(iv)
Free central : Ovules are borne on the central axis and septa are absent, e.g., Carnation, Chilly
(v)
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Unilocular : One locule, e.g., Rose, Pea
Basal: Placenta develops at the base of the ovary, e.g. ,Sunflower.
EXERCISE 1
(b)
(c)
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(a)
(d)
(e)
Fig.1.1 Cohesion of stamens: (a) Syngenesious (b) Synandrous (c) Monoadelphous (d) diadelphous (e) Polyadelphous
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Fig.1.2 Adhesion of Stamens: Epipetalous/Epiphyllous
(a)
(b)
(c)
(d)
Fig.1.3 Attachment of filament to anther: (a) Basifixed (b) Adnate (c) Dorsifixed (d) Versatile
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L ABORATORY MANUAL: BIOLOGY
(a)
(b)
Fig.1.4 Dehiscence pattern of anther: (a) Porous (b) Longitudinal
(a)
(b)
(c)
(d)
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Fig.1.5 Position of ovary: (a) Epigynous (b–c) Perigynous (d) Hypogynous
(a)
(b)
(c)
Fig.1.6 Cohesion of carpels: (a) Apocarpous (b–c) Syncarpous
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EXERCISE 1
(a)
(b)
(d)
(c)
(e)
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Fig.1.7 Placentation: (a) Marginal (b) Axile (c) Parietal (d) Free central (e) Basal
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Exercise 2 Aim: To calculate percentage of pollen germination
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Principle: In nature, pollen grains germinate on the compatible stigmas of the carpel. Pollen grains can also be induced to germinate in a synthetic medium. During germination, intine (inner wall) of pollen grain emerges out as pollen tube through one of the germ pores in exine (outer wall).
Requirement: Calcium nitrate, boric acid, sucrose, distilled water, petridish, slides, coverslips, brush, needle, microscope, and mature pollen grains of Tradescantia/balsam/Jasmine/lily/ pomegranate/grass/Vinca/China rose/Petunia.
Procedure
(i) Prepare the pollen germination medium by dissolving 10g sucrose, 30mg calcium nitrate and 10mg boric acid in 100ml of distilled water. Alternatively 10% sucrose solution can also be used.
(ii) Take a drop of medium or 10% sucrose solution on a cover slip and sprinkle mature pollen grains on the drop. (iii) Invert the cover glass on to a slide
(iv) After 10 minutes, observe the slide under microscope.
(v) Count (a) total number of pollen grains seen in the microscope field, and (b) the number of pollen grains that have germinated.
Observation
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Several pollen grains germinate and put forth pollen tubes. Count the total number of pollen grains and the number of germinated pollen grains in 3-5 different microscope fields. Tabulate your observations and calculate the percentage of pollen germination. Nameoftheplantusedassourceofpollen…………………………… Number of pollen grains in a field of microscope = N Number of germinated pollen grains in a field of microscope = n Percent pollen germination =
n 100n × 100 or N N
EXERCISE 2 Number of
Total number of pollen (N) Total number of pollen germinated (n) % pollen germination
n × 100 N
observation
1. 2. 3.
5. Average
Discussion
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4.
Although pollen grains of many species germinate in this medium, the percentage of germinations and the time taken for germination varies in different species. Draw a germinating pollen grain and label.
Questions
1. How many pollen tubes emerge from a pollen grain? 2. What does the pollen tube carry?
3. Can you explain as to why some pollen grains fail to germinate?
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4. Why do we use sucrose as the medium for pollen germination?
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Exercise 3 Aim: To study pollen tube growth on stigma
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Principle: Pollen grains germinate and form pollen tubes after they get deposited by the process of pollination on compatible stigma. Pollen tube, made up of cellulose, is an extension of the inner wall of pollen grain (intine). It emerges through one of the germ pore and passes through tissues of stigma and style to reach the ovule. The growing pollen tube is observed by staining with cotton blue. Requirement: 5–6 excised styles with stigma of Petunia/grass/maize/sunflower/Abelmoschus (Lady's finger), beaker, water, slides, cover slips, cotton blue stain, microscope, brush, needle.
Procedure
(i) Place the stigmas in boiling water in a beaker for softening the tissues for 5–10 minutes.
Pollen grains
(ii) Stain with cotton blue for 3–5 minutes and wash with water to remove excess stain.
(iii) Mount one stigma in a drop of glycerine on a slide. Place a cover slip on the stigma and gently press the cover slip on the material. Observe the slide under a microscope.
Pollen tube Style
(iv) If you fail to observe pollen tubes mount another stigma.
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Observation
Look for long blue-coloured tubular structures traversing through the tissues of stigma and style (Fig. 3.1).
Fig.3.1 Growth of pollen tube in the style of a carpel
EXERCISE 3
Discussion Pollen tubes are seen amidst the stylar tissue. Many pollen tubes may be seen. Trace the origin of pollen tubes to the pollen grains present around the surface of the stigma.
Questions
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1. Can pollen grains of one plant species germinate on stigma of other species? Give reasons. 2. Do all pollen tubes reach the ovules?
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3. Are all the pollen tubes of equal length? If not, why?
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Exercise 4 Aim: To study the discrete stages of gametogenesis in mammalian testis and ovary
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Principle: In all male and female organisms gamete formation takes place in their gonads, i.e., testis and ovary respectively. The process of gamete formation, called gametogenesis involves meiotic cell division. The gametogenic development in testis is called spermatogenesis and in ovary it is oogenesis. They exhibit marked differences and can be examined in transverse section (T.S.) of these organs. Requirement: Permanent slides of T.S. of testis and ovary, compound microscope, lens-cleaning paper and cleaning fluid
Procedure
(i) Clean the slide and microscope’s eye and objective lenses with the help of lens cleaning paper using any cleaning fluid. (ii) Place the slide on the stage of the microscope and observe first under lower magnification and then in higher magnification. Observe various stages of gamete development. (iii) Record your observations in the notebook and draw labelled diagrams.
Observation
Seminiferous tubule
Spermatozoa
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T.S. of testis (i) You will observe a large number of seminiferous tubules under lower magnification. Observe a complete tubule in higher magnification and view various stages of gamete development from periphery towards lumen (Fig. 4.1) and identify the following types of cells namely, Germinal epithelium, Spermatogonial cells, Primary spermatocytes, Secondary spermatocytes, Spermatids and Spermatozoa.
Germinal Epithelium Spermatogonia
Fig. 4.1 T.S. of mammalian testis
EXERCISE 4
(ii) In T.S. of testis the space between tubules are filled with blood vessels and a specific cell type called Leydig's cell or Interstitial cells. T.S. of Ovary (i) In the section of ovary, there is a mass of tissue lined with germinal epithelium. Inside that you will observe an ovum, which is a cell surrounded by one to several layers of follicular cells. As the ovum matures, the number of surrounding follicular cell layer increases (Fig. 4.2).
Graafian Follicle
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Antrum
(ii) In the later stage of follicular development a cavity called antrum appears.
Corpus luteum
Corpus albicans
Fig. 4.2 Section of mammalian ovary
(iii) The cavity gets further enlarged and the follicle grows bigger. This is the stage of Graafian follicle ready to release the ovum (ovulation).
(iv) In the next stage, you may notice a Corpus luteum, and/or Corpus albicans, which differ from each other and also from Graafian follicle in their features.
Discussion
Spermatogenesis is a continuous process after attainment of puberty, and that is why gamete development and spermatozoa are observed in a single seminiferous tubule. In case of ovary, the follicular development stages are observed.
Questions
1. What would happen if meiosis fails to occur in gametocyte? 2. At which stage of follicular development, is ovum released?
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3. Spermatogenesis is a continuous process. Justify the statement. 4. Draw a labelled diagram of T.S. of testis. 5. Draw a labelled diagram of T.S. of ovary. 6. What would happen if sperms are devoid of their tail? 7. What are the consequences of failure of ovulation?
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Exercise 5 Aim: To study and identify various stages of female gametophyte development in the ovary of a flower
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Principle: In flowering plants, female gametophyte (embryo sac) is a microscopic structure situated deep inside the ovule. An ovule generally has one female gametophyte. Development of female gametophyte begins with megaspore mother cell. Most common type of female gametophyte is the monosporic, 8-nucleate, 7-celled type. Requirements: Permanent slides of V.S. of ovary, photographs/chart or models showing stages of female gametophyte development and microscope
Procedure
(i) In a V.S. of ovary we generally find several ovules. Carefully observe each ovule and locate as many stages of female gametophyte development as possible. (ii) Draw the diagrams as observed under microscope.
Observation
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(i) Record the features of ovule like number of integuments, nucellus and micropylar and chalazal poles. Fig 5.1 shows the female gametophyte (embryo sac) as seen in a V.S. of an ovule. Different stages of development of female gametophyte are shown in Fig. 5.2.
(ii) Observe the placement of embryo sac close to the micropylar pole.
Chalaza
Embryo sac
Outer integument Inner integument Micropyle
Funiculus
Fig. 5.1 V.S. of an ovule
EXERCISE 5
Egg Synergids
(a)
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Central cell Secondary nucleus
(b)
(c)
(d)
(e)
Antipodals
Fig. 5.2 Stages of gametophyte development: (a) megaspore with 2 nucleus (b) 4-nucleate stage (c) 8- nucleate stage (d) 8- nucleate stage showing 3+2+3 distribution of nuclei (e) mature embryo sac.
(iii) Note the contents of embryo sac, namely, an egg apparatus (2 synergids and egg) at micropylar end, secondary nucleus in the center and three antipodal cells at the chalazal end (Fig. 5.2).
Questions
1. Explain the difference between gamete and a gametophyte. 2. Name two differences between synergids and egg.
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3. What is the function of polar nuclei?
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Exercise 6 Aim: Preparation and study of mitosis in onion root tips
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Principle: Somatic growth in plants and animals takes place by the increase in the number of cells. A cell divides mitotically to form two daughter cells wherein the number of chromosomes remains the same (i.e., unchanged) as in the mother cell. In plants, such divisions rapidly take place in meristematic tissues of root and shoot apices, where the stages of mitosis can be easily observed. In animals, mitotically dividing cells can be easily viewed in the bone marrow tissue of a vertebrate, epithelial cells from gills in fishes and the tail of growing tadpole larvae of frog. Requirement: Onion bulbs, wide mouth glass tubes/jar/bottle, glacial acetic acid, ethanol 2-4% acetocarmine/acetoorcein stain, N/10 HCl, spirit lamp/hot plate, slide, cover slips, blotting paper, molten wax/nail polish and compound microscope
Procedure
Growing of root tips
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Select a few medium-sized onion bulbs. Carefully remove the dry roots present. Grow root tips by placing the bulbs on glass tubes (of about 3–4 cm. diameter) filled with water. Care should be taken so that the stem portion of the bulb (basal part) just touches the water. A few drops of water may be added periodically to compensate evaporation losses. New roots may take 3–6 days to grow. Cut 2–3 cm long freshly grown roots and transfer them to freshly prepared fixative, i.e., aceto-alcohol (1:3:: glacial acetic acid : ethanol). Keep the root tips in the fixative for 24 hours and then transfer them to 70% ethanol (for preservation and use in future). Onion root-tip cells have a cell cycle of approximately 24-hour duration, i.e., they divide once in 24 hours, and this division usually takes place about two hours after sunrise. Therefore, roots grown on water should be cut only at that time to score maximum number of dividing cells. Preparation of slide
Take one or two preserved roots, wash them in water on a clean and greasefree slide. Place one drop of N/10 HCl on the root tip followed by 2–3 drops of aceto-carmine or aceto-orcein stain on it. Leave the slide for 5–10 minutes
EXERCISE 6
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on a hot plate (or warm it slightly on spirit lamp). Care should be taken that the stain is not dried up. Carefully blot the excess stain using blotting paper. Now cut the comparatively more stained (2–3 mm) tip portion of the root and retain it on the slide and discard the remaining portion. After (10–20 seconds) put one or two drops of water and blot them carefully using blotting paper. Again put a drop of water on the root tip and mount a cover slip on it avoiding air bubbles. Place the slide in between the folds of blotting paper using the fingers in such a way that the cover slip mounted on the slide is properly held. Now slowly tap the cover slip using the blunt end of a pencil so that the meristematic tissue of the root tip below the cover slip is properly squashed and spread as a thin layer of cells. Carefully seal the margins of the cover slip using molten paraffin wax or nail polish. This preparation of onion root tips cells is now ready for the study of mitosis. Study of slide
Place the slide on the stage of a good quality compound microscope. First observe it under the lower magnification (10 X objective) to search for the area having a few dividing cells. Examine the dividing cells under higher magnification of the microscope to observe the detailed features of mitosis.
Observation
The stages of mitosis can be broadly categorised into two parts: karyokinesis (division of nucleus) followed by cytokinesis (division of cytoplasm, and ultimately of the cell). Those cells, which are not in the phases of cell division are considered to be in interphase. You may observe that most of the cells in a microscope field are in interphase
Interphase
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The cells are mostly rectangular, oval or even circular in shape, with almost centrally situated densely stained nucleus. The chromatic (coloured) material of the nucleus is homogeneous and looks granular. The boundary of the nucleus is distinct. One or few nucleoli (sing: nucleolus) can also be observed inside the nucleus (Fig. 6.1a).
Stages of Mitosis (a)
Prophase
Intact nuclear outline is seen. The chromatin (seen as a homogeneous material in the nucleus at interphase) appears as a network of fine threads (chromosomes). Nucleoli may or may not be visible (Fig. 6.1b).
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L ABORATORY MANUAL: BIOLOGY
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a. Interphase
b. Prophase
c. Metaphase
d. Anaphase
e. Telophase
Fig.6.1 Interphase (a) and stages of mitosis (b - e) – actual microscopic view on left side and its diagrammatic representation on the right hand side
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If the cell under observation is in the early stage of prophase then the chromatin fibres (chromosomes) are very thin. However, in the cells at late prophase, comparatively thicker chromatin fibres would be visible. Besides this, in the late prophase the nuclear membrane may not be noticed.
(b) Metaphase The nuclear membrane disappears. Chromosomes are thick and are seen arranged at the equatorial plane of the cell (Fig. 6.1c). Each chromosome at
22
EXERCISE 6
this stage has two chromatids joined together at the centromere, which can be seen by changing the resolution of the microscope. Nucleolus is not observed during metaphase. (c) Anaphase
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This stage shows the separation of the chromatids of each chromosome. The chromatids separate due to the splitting of the centromere. Each chromatid now represents a separate chromosome as it has its own centromere. The chromosomes are found as if they have moved towards the two poles of the cell. The chromosomes at this stage may look like the shape of alphabets 'V', 'J' or 'I' depending upon the position of centromere in them. Different anaphase cells show different stages of movement of chromosomes to opposite poles, and they are designated to represent early, mid and late anaphase (Fig. 6.1d). (d) Telophase
Chromosomes reach the opposite poles, lose their individuality, and look like a mass of chromatin (Fig. 6.1e). Nuclear membrane appears to form the nuclei of the two future daughter cells.
Cytokinesis
In plants, a cell plate is formed in the middle after telophase. The plate can be seen to extend outwards to ultimately reach the margin of the cell and divide the cell into two. Such cell plates are characteristic of plant cells (Fig. 6.2). However, in an animal cell, the two sides of the cell show inpushings or constrictions formed from the peripheral region in the middle of the cell, which grow inward and meet to divide the cell into two daughter cells. Draw labelled diagrams of all the phases of mitosis.
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Fig. 6.2 Cytokinesis
Discussion
Mitotic index (MI) is defined as a ratio of the total number of dividing cells (n) and the total number of cells (N) in a particular focus chosen randomly under n the microscope and is calculated as MI = N × 100 . By randomly selecting 5 to 10 such foci, one can estimate the mitotic index for a given type.
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L ABORATORY MANUAL: BIOLOGY
The effect of different samples of water (polluted or contaminated) can be assayed on the mitotic-index (an indicative feature of somatic growth rate in them). Further, the impact of different types of pollutants on different phases of mitosis can also be assayed. Tabulate your observations in the tabular form given below Interphase
Karyokinesis
Cytokinesis
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Features
Prophase
Metaphase
Anaphase Telophase
1. Cell morphology
2. Nuclear morphology
3. Chromosomes/chromatids
Questions
1. Suggest names of a few tissues, which are suitable for the study of mitosis. 2. Why is mitosis also known as equational division?
3. What shape would a metacentric and a sub-metacentric chromosome exhibit during the anaphase stage?
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4. How does cytokynesis differ in plant and animal cells?
24
Exercise 7 Aim: Study of stages of meiosis using permanent slides
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Principle: Meiosis is a type of cell division in which the number of chromosomes is halved (from diploid to haploid) in the daughter cells, i.e., the gametes. The division is completed in two phases, meiosis I and meiosis II. Meiosis I is a reductional division in which the chromosomes of homologous pairs separate from each other. Meiosis II is equational division resulting in the formation of four daughter cells. Stages of meiosis can be observed in a cytological preparation of the cells of testis tubules or in the pollen mother cells of the anthers of flower buds. Requirement: Permanent slides of meiosis and compound microscope
Procedure
Place the slide on the stage of the microscope and search for the dividing cells using lower magnification. When dividing cells are located observe them under higher magnification.
Observation
Observe various stages of meiosis and identify them on the basis of the specific features given in the table 7.1. A significant number of cells will be in the Interphase. These cells have a centrally positioned densely stained nucleus. In case of slide of animal tissue a few mitotically dividing spermatogonial cells may also be seen.
Table 7.1 Different stages of meiosis and their features
Meiosis I
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1. Prophase I
Unlike the prophase of mitosis, it is a comparatively complex phase characterised by a number of events. Five sub-phases can be identified in it. (a) Leptotene (leptos = slender tene = band or thread) (i)
The nuclear membrane and nucleolus are not distinctly observable (Fig. 7.1 a).
(ii)
Fine network of thin threads are seen uniformly distributed in the nucleus.These are chromatin threads, which may be observed as more prominent structures in the later stages.
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L ABORATORY MANUAL: BIOLOGY
(a) Leptotene
(b) Zygotene
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(c) Pachytene
(d) Diplotene-Diakinesis
Fig. 7 . 1 Sub-phase of Prophase I (a-d) – actual microscopic view on left side and its diagrammatic representation on the right hand side
26
EXERCISE 7
(b) Zygotene ( Zygon = paired) This stage is characterised by the pairing of the homologous chromosomes, which can be seen as paired chromatin threads (bivalents) (Fig. 7.1b). (c) Pachytene (pachy = thick)
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The chromatin threads get condensed and appear shortened and thick. Pairs of homologous chromosomes can be seen. Each chromosome has two chromatids and thus each bivalent consists of four chromatids. This configuration is called tetrad (Fig. 7.1c). (d) Diplotene (diplos = double)
The homologous chromosomes (each made up of two chromatids) show distinct separation from each other except at few regions where attachments are seen (Fig. 7.1d). These are chiasmata (sing. chiasma) representing the site of exchange of the parts between two homologous chromosomes (i.e. crossing over). (e) Diakinesis (Dia = opposite; kinesis= separation or movement)
(i) The homologous pair of chromosomes appear more shortened, thick and prominent (Fig. 7.1d).
(ii) Chiasmata can be still observed.
(iii) All the homologous pairs appear scattered in the cell.
2. Metaphase I
Homologous chromosomes are still in pairs, and are arranged along the equatorial plane of the cell (Fig. 7.2a). At this stage, the number of bivalents can be counted. Chiasmata may still be seen in a few bivalents.
3. Anaphase I
The chromosome pairs appear to have moved towards the two opposite poles of the cell. At the later stage, the anaphase - I may show the assembly of chromosomes at two poles (Fig. 7.2b). This results into the reduction of number of chromosomes to half. This stage can be identified by the presence of two chromatids in each chromosome.
4. Telophase I
The chromosomes present at the two poles appear decondensed and form two distinct nuclei (Fig. 7.2c).
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Note: After the telophase I stage there may or may not be cytokinesis. Thereafter the cell enters into the second meiotic division.
Meiosis II
1. Prophase II
(i) Distinct thread- like chromatin fibres or rod- shaped chromosome are seen.
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L ABORATORY MANUAL: BIOLOGY
(b) Anaphase I
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(a) Metaphase I
(c) Telophase I
Fig. 7.2 Phases of Meosis I (a-c) – actual microscopic view on left side and its diagrammatic representation on the right hand side.
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(a) Metaphase II
(b) Anaphase II
Fig.7.3 Phases of Meosis II (a,b) – actual microscopic view on left side and its diagrammatic representation on the right hand side.
28
EXERCISE 7
2. Metaphase II
This phase is similar to that of mitotic division (i) The chromosomes having two chromatids attached at the centromere are observed arranged at the equatorial plane of the cell. Note: Metaphase II of meiosis can be differentiated from metaphase-I on the basis of the following features: Each chromosome of metaphase II has two chromatid (Fig. 7.3a) whereas in metaphase I these are paired homologous chromosomes each having two chromatids thus forming tetrad.
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(ii)
(iii)
3. Anaphase II
In the metaphase I of meiosis, a few chiasmata are observed, where as no chiasmata are observed during metaphase II.
The two chromatids of each chromosome after separation appear to lie at the two poles of the cell (Fig. 7.3b).
Note: Anaphase II can also be distinguished from the anaphase I of meiotic division on the basis of chromatids: In anaphase I, each chromosome has two distinct chromatids, but in anaphase II, each chromosome is represented by one chromatid only.
4. Telophase II
The separated chromosomes appear decondensed and form nuclei (Fig. 7.3c).
Questions
1. What is the significance of meiosis?
2. What is synapsis and crossing over?
3. How can anaphase I and anaphase II be distinguished from each other?
4. Indicate distinguishing feature of metaphase I of meiosis and metaphase of mitosis. 5. How many daughter cells are produced at the end of meiosis?
6. The daughter cells produced at the end of meiosis are genetically different. Explain.
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7. What is the significance of synapsis?
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Exercise 8 Aim: To study the blastula stage of embryonic development in mammals, with the help of permanent slide, chart, model or photograph
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Principle: The zygote undergoes a few cycles of mitotic divisions to form a solid ball of cells called morula. The cells continue to divide and at a later stage a cavity is formed within it. This stage is blastula. The internal structural details of blastula can be observed in its transverse section. Requirement: Permanent slide, chart/model of T.S. of blastula, compound microscope, lens cleaning fluid and paper
Procedure
Observe the slide under lower magnification of the microscope. In case of chart/models/photographs, note the feature of blastula in your practical record and draw labelled diagram.
Observation
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In transverse section, the blastula appears as a sphere with a cavity, called blastocoel within it (Fig. 8.1). Notice an outer layer of blastomeres called trophoblasts. A cellular mass, adhered to the trophoblast is present on one end of the blastula. It is called inner cell mass.
Fig.8.1 Blastula stage of a mammal
EXERCISE 8
Questions 1. What are the differences between blastula and morula? 2. What are the main structures you observe in T.S. blastula? 3. Match the stages in column I with features in column II Column I (i) (ii) (iii) (iv)
Dividing cells of the morula Outer layer of blastula Solid ball of cells Cavity
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(a) Trophoblast (b) Morula (c) Blastocoel
Column II
31
Exercise 9 Aim: To verify Mendel's Law of Segregation
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Principle: When two pure lines with contrasting forms of a particular character (phenotypes) are crossed to produce the next generation (F1 generation), all the members of the progeny are of only one phenotype i.e. of one of the two parents. The phenotype that appears is called dominant, and the one that does not appear is called recessive. When the F1 plants are selfed, the progeny i.e. the F2 generation is in the ratio of 3 dominant: 1 recessive (¾: ¼ or 75%: 25%). This reappearance of the recessive phenotype in F2 generation verifies law of segregation. Requirement: 64 yellow and 64 green plastic beads, all of exactly same shape and size, (when beads are not available, pea seeds may be coloured using paint, these beads represent the gametes of a specific trait), plastic beakers/petri dishes and a napkin/hand towel
Procedure
Students have to work in pairs to perform the experiment. The following steps are to be strictly followed in the sequence mentioned below.
(i) Put 64 yellow beads in one beaker/petridish and 64 green beads in the other to represent respectively male and female gametes. Let the yellow bead be indicated by ‘Y’ and green bead by ‘y’. (ii) Take a bead from each container and place them together (it represents fertilisation) on the napkin spread before you on the table. (One student to take out beads and to put in the hands of the other student who will put them on the table). (iii) Just like the previous step, continue to pick beads and arrange them in pairs. Thus 64 pairs of beads are obtained representing the 64 heterozygous F1 progeny.
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Note that all the F1 individuals are represented by one yellow and one green bead.
(iv) Put 32 F1 progeny in one petridish and the remaining 32 in another petridish (representing the F1 males and females). (v) Stir the beads of each petridish with a pencil/pen for about 10 times taking care that no bead falls off.
EXERCISE 9
(vi) To obtain the F2 generation, one student would withdraw one bead from one beaker labelled male and one from the other beaker labelled female keeping his/her eyes closed (to ensure randomness), and put them together in the stretched palm of the partner, who will put them together on the napkin spread over the table. Continue this process till all the beads are paired. Thus 64 offsprings of F2 are obtained. (vii) Note the genotype (YY or Yy or yy) of each pair, and their possible phenotype.
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(viii) Have six repeats of the experiment (steps i to vii) with partners changing their roles. Pool all the data from the six repeats together.
(ix) Calculate the genotypic and phenotypic ratios of your pooled data. Note that larger the sample size, more accurate is the result.
Observation
Record the result in the following table: Generation
F1
Repeat No.
Total no. of
Genotype (s)
individuals
YY Yy yy
Phenotype (s)
1. 2. 3. 4. 5. 6.
Total
F2
1. 2. 3. 4. 5.
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6.
Phenotypic Ratio:
Total
in F1…….
in F2…….
Genotypic Ratio:
in F1…….
in F2…….
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L ABORATORY MANUAL: BIOLOGY
Discussion
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The results are so because each diploid individual contains two copies of every gene - one copy on each of the two homologous chromosomes. These two copies of the gene may be of similar type (YY or yy) or are dissimilar Yy. The former (YY or yy) are called homozygous for that particular character, and the Yy are called heterozygous ones. The pure lines in the above cross are homozygous ones, which contributed only one copy of their gene (as a result of meiosis) to their F1 progeny to restore its diploid nature with genotype Yy (heterozygous) where only one form (allele) is expressed (dominant) and the other form (allele) is not expressed (recessive). This is the phenomenon of Dominance.
When the F1 individuals are crossed together to raise the F2 generation, each F1 individual produces two types of gametes: 50% having dominant allele, and the remaining 50% having recessive allele. These gametes undergo random fusion during fertilisation to produce the F2 generation. According to simple probability of mixing of opposite sex gametes (sperms and ova), offsprings of three genotypes are likely to appear as follows: [(half of gametes of Y type + half of remaining gamete y type) X (half gametes of Y type + half of remaining gamete of y type)] = One-fourth of F2 individuals of YY phenotype + half of F2 individual Yy type + one-fourth of F2 individul of yy type. Among these proportion of dominant phenotype would be YY+ Yy = yellow and recessive phenotype yy i.e. green phenotypes in 3:1 or 75%:25% ratio.
This ratio of 3:1 in the F2 suggests that in the F1 heterozygotes, the recessive allele does not get destroyed and remains only in the recessive (dormant) state to get an opportunity to express itself when it has separated from the influence of the dominant allele (Y). This is called Law of Segregation of the alleles.
Questions
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1. Do you expect the same results in terms of 3:1 ratio in F2 if you had started with smaller number of beads (say 10 beads)?
34
Exercise 10 Aim: To verify the Mendel’s Law of Independent Assortment Principle: In a dihybrid cross, the segregation of one gene pair is independent of the segregation of the other pair. It means that genes of two different traits assort independently to give a probability ratio equal to segregration probability ratio of one allele pair X segregation probability ratio of other allele pair, which comes to, (3:1) X (3:1) = 9:3:3:1 Requirement: Plastic beakers; 64 plastic beads each of yellow, green, red and white to represent, yellow and green colour of seed coat and red and white flowers respectively and napkin/hand towel
Procedure Students are to work in pair. The following steps are to be followed sequentially: (i) Place 64 beads of each colour in four separate beakers. (ii) Put the beakers containing the yellow and red beads on your left side, and those containing the green and white beads on your right side. The beakers on your left side represent plants bearing yellow seed and red flower (dominant character YY, RR). Beakers on the right side represent plants bearing green seeds and white flowers (recessive character yy, rr). These are the two parental types having contrasting forms of two different characters. (iii) Stir the beads in each beaker with a pencil/pen. Each bead now represents alleles in the male and female gametes. (iv) Pick up one yellow, one green, one red and one white bead, and put them together on the napkin spread on the table. (v) Continue picking up and putting together of the beads of all colours as mentioned in the previous step, till all the beads are utilised. (vi) Note that in all, 64 such 4-bead clusters are obtained representing the F1 individuals. Ascertain their genotype and phenotype. (vii) Next step is to cross these F1 individuals to raise the F2 generation. Let us suppose half of the 4-bead clusters (32 clusters) represent the male parents and the remaining half (32 clusters) the female parents. Now put the 32 red and 32 white beads together in one beaker (numberedI), and similarly put 32 yellow and 32 green beads together in other
L ABORATORY MANUAL: BIOLOGY
beaker (numbered-II). These two beakers represent F1 female. Similarly put remaining 32 red + 32 white beads in beaker numbered-III, and 32 yellow and 32 green one in beaker numbered-IV to represent the F1 male. The arrangement can be presented as below Female F1
Male F1
32 red + 32 white (Beaker I)
32 red+32 white (Beaker III)
32 yellow + 32 green (Beaker II)
32 yellow + 32 green (Beaker IV)
(viii) Stir the beads in each beaker with a pencil. In order to raise the F2 generation, pick up (with eyes closed) one bead from the beaker-I of female and one bead from the beaker-III of the male, and put into the palm of the partner student. Similarly, pick up one bead each from the beaker-II of female and beaker IV of male to put in the palm of the partner. This partner would now keep all the four beads together (to represent the F2 individual). Continue this process till all beads are utilised. At the end, 64 F2 individuals (each represented by a 4-bead cluster) are obtained. (ix) Determine the genotype and phenotype of each of the 64 F2 individuals and write down the number of individuals of different genotypes and phenotypes in the tabular form (given below), remembering that Y (yellow seed colour) is dominant over y (green seed) and R (red flower) is dominant over r (white flower). (x) Repeat the whole procedure (steps i to ix) six times, and tabulate your results.
Observation Tabulate the results as follow: Symbol (-) indicates the presence of corresponding dominant or recessive allele e.g. Y or y and R or r. Summarise your results (adding together the data of all the six repeats)
F1 Generation
36
(a)
Total number of individuals:
_________________________
(b)
Phenotype (s)
_________________________
(c)
Genotype (s)
_________________________
EXERCISE 10 Generation & repeat
Total No. of offsprings Y-R-
Genotype Y-rr
yyR-
Phenotype yyrr
No.
Yellow
Yellow
Green
Green
Red
White
Red
white
F1 1. 2. 3. 4. 5. 6. Total F2 1. 2. 3. 4. 5. 6. Total
F2 Generation (a)
Total number of individuals
_________________________
(b)
Phenotypes
_________________________
(c)
Number of individuals in each phenotypic class: Number
Phenotype
__________________
_____________________
__________________
_____________________
__________________
_____________________
__________________
_____________________
(d)
Phenotypic ratio
_____________________
(e)
Genotypic ratio
_____________________
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(f)
(g)
Number of individuals of each genotypic class: Number
Genotype
__________________
_____________________
__________________
_____________________
__________________
_____________________
__________________
_____________________
__________________
_____________________
Genotypic Ratio ______________________________________
Discussion The four phenotypic classes in the F2 generation are in ratio of 9:3:3:1 as expected from the Law of Independent Assortment. The genotypic ratio would be (1:2:4:2): (2:1):(2:1):1. Note 1.
In case six repeats of the experimental procedure are not feasible due to time limitations, either the number of repeats be slashed down to three or the data from single repeat of six different pair of students may be pooled together to make the final calculations.
2.
This Law of Independent Assortment was later found to be true only for traits present on two different homologous pair of chromosomes, that is, the two are not linked together. The linked traits do not assort independently, rather they are inherited together (linked) except when crossingover separates them.
3.
It is quiet likely that you may not find your data exactly in the expected ratio, instead almost approximate to it. The statistical significance of this deviation from the exact expected ratio due to probality can be checked using chi-square (χ 2) test, about which you will study in higher classes.
Questions 1. Linked traits fail to assort independently. Explain. 2. How is independent assortment of alleles important from the point of view of variation?
38
Exercise 11 Aim: Preparation and analysis of Pedigree Charts Principle: The Mendelian concept of dominance and segregation can also be studied in humans by preparing and then analysing the pedigree charts. The internationally approved symbols for indicating males and females, marriages, various generations (I, II, III), etc., are given below.
Requirement: Information about characters/traits in a family for more than one generation
Procedure Select a family in which any one of the monogenic traits such as tongue rolling, widow's peak, blood groups’, red-green colour blindness, dimple in
L ABORATORY MANUAL: BIOLOGY
the cheek, hypertrichosis of ear, hitch-hiker's thumb, etc., is found. Ask the person exhibiting the trait to tell in which of his/her parents, grand parents (both maternal and paternal), their children and grand children the trait in question is present. Among surviving individuals the trait may also be examined. The information made available is the basis for the preparation of pedigree chart using the appropriate symbols. A careful examination of the pedigree chart would suggest whether the gene for the character is autosomelinked dominant or recessive, X - chromosome linked dominant or recessive, Y- chromosome linked or not. Explanation 1.
Autosome Linked Dominant traits: These are the traits whose encoding gene is present on any one of the autosomes, and the wildtype allele is recessive to its mutant allele, i.e., the mutant allele is dominant. The pedigree-chart can be of the undernoted pattern (Fig. 11.2), where the female being interviewed is exhibiting the trait, and is indicated by an arrow-mark in the chart.
The characteristic features of inheritance of such type of traits are: (a) Transmission of traits occurs from parents of either sex. (b) Males and females are equally affected. (c) The pedigree is vertical, i.e., the trait is marked to be present in each of the generations. (d) Multiple generations are characteristically affected. Brachydactyly, polydactyly, dimple in the cheek are some of the common traits of this type.
40
EXERCISE 11
2.
Autosomal Recessive trait: These are the traits whose mutant allele is recessive to its wild type allele. The pedigree chart can be more or less of the pattern given below (Fig. 11.3), where the lady (marked by the arrow) is showing the trait. The bar
in the example represents the presence of corresponding dominant or recessive allele for the specific trait. Suppose the given trait is albinism. Denote its dominant allele as ‘A’ that produces pigments, and the recessive allele as ‘a’ that fails to synthesise the pigment, melanin. The female (our subject in generation III) is therefore of genotype aa. She must have received each of her ‘a’ allele from both the parents (generation-II), who are therefore themselves normal but are definitely of genotype Aa, and are carriers of the trait. The allele a must also have been present in her grand parents too, of course in heterozygous condition also to make them carriers (generation-I) Albinism in the subject’s children (generation-IV) suggests her husband too to be of genotype Aa, a carrier. Marriage of her albino daughter to an albino man is bound to produce all her grand-children albino (gen-V). The following are the salient features of the inheritance of such type of traits. (a) Occur in equal proportions in multiple male and female siblings, whose parents are normal but carriers; (b) The siblings are homozygous for the defective allele, but their parents, though some may appear normal, are obviously heterozygous, i.e., are merely carriers of the trait. (c) Consanguinity (marriage between man and woman genetically related to each other, such as cousins) occasionally results in the appearance of such traits.
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3.
X-Linked Dominant traits: These are the traits whose encoding gene is present on the X- chromosome, and the mutant allele of which is dominant over its wild-type allele. Such traits are very rare, and are almost difficult to find in the population. One example is oral-facial-digital syndrome (Duchene Muscular Dystrophy), which results in absence of teeth, cleft (bifid) tongue associated with mental retardation. The pedigree chart may appear as follows (Fig. 11.4):
The possible genotypes of the above pedigree can be written as follows (Fig 11.5):
Fig. 11.5 Genotypes of individuals shown in Fig. 11.4
42
EXERCISE 11
Here, the dominant mutant allele is denoted by ‘D’, and its recessive wild type allele is denoted by ‘d’. Remember that human females have two X-chromosomes (XX), and the males have only one X and one Y chromosome. Males receive their lone X-chromosome from their mother, and the Y-chromosomes from their father, whereas females receives one of her X-chromosome from her mother, and the other X from her father. The characteristics of such inheritance are: (a) The trait appears in almost all the generations, and the inheritance is vertical. (b) If the female is affected, then about half of her sons are affected. (c) If the male is affected then all of his daughters would be affected, but none of his sons are affected. (d) In short, the pedigree resembles the pattern of inheritance of autosomal dominants, except that there is no male-to-male transmission. 4.
X-linked Recessive traits: These are the traits whose encoding gene is present on the X-chromosome and its mutant allele is recessive to its wild-type allele. Red-green colour blindness and hemophilia, are some of its well known examples. The characteristic features of such inheritance are:
(a) Females express the trait only when they are homozygous for the mutant allele, whereas the males do so even when they are hemizygous for it. The pedigree chart would appear as the following one (Fig. 11.6):
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L ABORATORY MANUAL: BIOLOGY
(b) About half of the sons of the carrier (heterozygous for the trait) females are affected. In case of homozygous females showing the trait, fifty percent of her daughters and all of her sons are likely to be affected. Therefore, the males are most affected in the population. (c) Affected persons are related to one another through the maternal side of their family. (d) Any evidence of male-to-male transmission of the trait rules out the X- linked inheritance. 5.
Y-chromosome linked traits: These are the traits whose gene is present on the Y-chromosome. The females do not have any Y-chromosome, whereas all the males must have a Y-chromosome to be a male, and this Y-chromosome they get from their father. Therefore, any trait linked to the Y- chromosome must be present only in males, and certainly not in any of the females. This is why these traits are also called male-sex limited traits. All the sons of the affected male would express the trait whereas none of his daughters would do so. The pattern of the pedigree chart would be as follows (Fig 11.7):
Hypertrichosis of the ear (presence of hairs on pinna) is one most common example of such traits. Note: Students may be asked to prepare the pedigree-chart from given data and analyse the pattern of inheritance. The work may be done as a project.
Questions 1. How will you differentiate between autosome linked dominant and sex chromosome linked dominant pedigree chart? Explain. 2. Discuss the differences in the patterns of autosome linked recessive and sexchromosome linked pedigree.
44
Exercise 12 Aim: To perform emasculation, bagging and tagging for controlled pollination
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Principle: Conventional plant breeding programmes involve bringing under human control reproductive processes that lead to seed and fruit formation. For this controlled pollination is desirable using male and female parent having desired traits. One of the process that can be easily brought under human control is emasculation. For this the knowledge of flower structure, mechanism of pollination, fertilisation and physiology of flowering is essential for this. In emasculation technique the stamens are removed before anthesis to obtain female parent and pollen from the desired male parent is transferred on to its stigma. Requirement: Ornamental plants/ wild plants bearing large bisexual flower, magnifying lens, tweezers, small sharp scissors, brush, alcohol, rubber bands, paper bags, paper clips and tags
Procedure
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(i) Select a flower in bud condition where antheses has not occurred. Open the bud carefully and remove the stamens (Fig. 12.1). Mark this as female parent plant.
Fig. 12.1 Showing process of Emasculation
L ABORATORY MANUAL: BIOLOGY
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(ii) Cover the emasculated flower with a plastic bag to protect it from undesired pollen (Bagging) (Fig. 12.2). The bag should be held securely in place with a paper clip/ string/thread. Select the size of bag in accordance with the flower size. Bags must be transparent with minute pores.
Fig. 12.2 Bagging of an emasculated flower
(iii) Bring into physical contact anthers of a desired male plant containing mature pollen grains with the stigmatic surface of emasculated female flower (Fig. 12.3). Use tweezers/brush if necessary to dust the stigmatic surface with pollen.
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Fig. 12.3 Showing cross pollination on an emasculated flower
(iv) Cover the pollinated flower again with the bag immediately. For identification, label the female parent (Tagging). Each pollinated flower should bear a label containing the name of the seed parent, the letter X (to signify a cross), the name of the pollen parent, and the date on which the cross was effected.
Questions
1. Why is emasculation performed before anthesis? 2. What are the advantages of using a bag containing minute pores?
46
Exercise 13 Aim: Staining of nucleic acid by acetocarmine
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Principle: Acetocarmine combines with nucleic acid present in the nuclei of cells to form a deep red conjugate. Requirements: Onion bulb, onion root tips, 2 to 4% acetocarmine/acetoorcein stains, slide and coverslips, brush/needle, pair of fine scissors, filter paper and microscope
Procedure
(i) Peel off epidermis from the fleshy leaf of onion and put it on a slide. Add a few drops of water over it to avoid desication. (ii) Cut out a small piece (about 0.5 cm size) of the epidermal peel and discard the remaining portion. (iii) Wipe out the water with a filter paper.
(iv) Put 2 drops of acetocarmine on the epidermal peel and heat gently on a spirit lamp.
(v) Apply a coverslip over the peel avoiding air bubbles and wrinkles of the material. (vi) Wipe out the excess stain with help of blotting paper.
(vii) Examine the material under low magnification of a microscope.
Observation
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Record your observations with regard to shape of cell, the number of nuclei and their position in the cell. Draw a diagram based on your preparation and label its parts.
Discussion
Nuclei in cells are extremely rich in nucleic acid which exist in a conjugated form with protein to form nucleoproteinous structures, called chromatin fibres/chromosomes.
L ABORATORY MANUAL: BIOLOGY
Questions 1. What are the building blocks of the nucleic acid? 2. What is DNA and how is it different from RNA?
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3. Name different nitrogenous bases present in the nucleic acid.
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Exercise 14 Aim: To identify common disease-causing organisms and the symptoms of the diseases
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Principle: There are quite a large number of organisms that are parasitic/pathogenic to humans. These organisms substantialy damage the human body and cause diseases, which may even be fatal sometimes. These organisms exhibit characteristic features in their external morphology. Symptoms of the diseases caused by them are also specific. Requirement: Preserved specimens/permanent slides/photographs of Ascaris, Entamoeba, Plasmodium, Ring-worm fungus and compound microscope
Procedure
Observe the preserved specimens/slides/photographs and note down the features in the practical record book. Take care to observe all the minute details and draw labelled diagrams of the pathogens.
Observation
A. Entamoeba
Observe the following features of the parasite in the slide or photograph: (i) It is unicellular.
(ii) Shape of the cell is irregular due to pseudopodia. (iii) A single nucleus is present eccentrically in the cell.
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(iv) *In the nucleus a peripheral ring of granule of nucleoprotein and central karyosome are observed. Rest of the space in the nucleus looks empty (Fig. 14.1). (v) A few food vacuoles may be seen in the cytoplasm. Contractile vacuoles are absent.
(vi) *Mature quadrinucleated cysts may be present.
Fig.14.1 An Entamoeba
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Note: Entamoeba is an intestinal parasite in humans and causes amoebic dysentery. The symptoms of the disease are frequent loose, mucus filled watery stools, abdominal pain and spasms.
Systematic position –
Protozoa
Class
–
Rhizopoda
Type
–
Entamoeba histolytica
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Phylum
* Distinctive feature of the pathogen
B. Plasmodium vivax
(i) It is an intracellular endoparasite seen easily within the RBC of the infected person.
(ii) It is unicellular.
(iii) The most diagnostic stage of the parasite is "signet ring" stage in the erythrocytes, within which it appears as a rounded body (Fig. 14.2). (iv) It has a big vacuole inside, and the cytoplasm is accumulated at one place containing the nucleus. Because of the above mentioned features, the parasite appears as a ring.
Search the stage in the blood film slide, find the signet-ring stage, and draw its labeled diagram. Note: It is a protozoan parasite causing malaria in humans. When an infected female anopheles mosquito bites a healthy person, it injects the infective stage, sporozoite, into the peripheral blood vessels. The infective stage undergoes several rounds of multiplication in liver and erythrocytes. Symptoms: Intermittent high fever with chills followed by profuse sweating at an interval of alternate days.
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Systematic position
50
Phylum
–
Protozoa
Class
–
Sporozoa
Type
–
Plasmodium vivax
EXERCISE 14
C.
Ascaris
The external features of round worm are as follows:
Mouth
(i) Body long (20 to 40 cm), cylindrical (5 to 6 mm diameter) with no segmentation (Fig. 14.3). (ii) Sexes are separate; the females are longer than the males.
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(iii) Both the ends are pointed; posterior end of male is ventrally curved.
(iv) Mouth is situated at the anterior end, and is surrounded by three lips, one present middorsally and rest two lips are situated ventrolaterally (for viewing these lips a magnifying lens is needed).
Female genital aperture
Penial spicule
(v) Single longitudinal lines are present on the dorsal, ventral and on the two lateral sides, all along the length of the body. Out of these the lateral lines are comparatively more distinct than the others lines.
(b)
(vi) Excretory pore is present on the ventral surface slightly behind the anterior end.
(vii) In addition to the ventrally curved posterior tip, the male worm has a pair of penial spicules very close to the cloacal opening.
(a)
Fig.14.3 Ascaris (a) Female (b) Male
(viii) In case of female specimen a female genital aperture is present mid-ventrally about one third distance from the anterior end.
Systematic position –
Class
–
Type
–
Aschelminthes Nematoda
Ascaris lumbricoides
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Phylum
Note: Round worm or Ascaris is one of the common parasite found in the intestine of human beings. Symptoms: (a) Irregular bowel, (b) Occasional vomiting, (c) Anaemia
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L ABORATORY MANUAL: BIOLOGY
Trichophyton (Ringworm fungus) It is a fungus that feeds on keratin of the skin of human beings. The features as observed under the microscope are: 1. Texture of hyphae is waxy, glabrous to cotton like. 2. Unstained hyphae are white, yellowish brown to reddish brown in colour.
Systematic position –
Fungi
Class
–
Deuteromycetes
Type
–
Trichophyton rubrum
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Kingdom
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Symptoms Ringworm is a contagious fungal infection of the skin. Infected area of skin is itchy, red, raised, scaly patches (with sharply defined edges). It is more red on the periphery than in the center creating a ring like appearance.
52
Exercise 15 Aim: To study the texture of soil samples
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Principle: Texture is one of the most important physical properties of soil. The soil texture is based upon division of the size of soil particles into three size fractions viz., Sand (2–0.05mm average particle diameter), Silt (0.05–0.002mm) and Clay ( less then 0.002mm). If one of these fractions dominates the properties of a soil, the name of that fraction is included in the name of the texture. A soil which has all of these fractions in nearly equal proportion is called a loam soil. The four terms—sand, silt, clay and loam— are combined in various ways to name 12 different textural classes. The 12 textural classes and the percentages of sand, silt and clay fractions that are included in each are shown in textural triangle (Fig. 15.1). Texture affects several physicochemical properties of soil like density, capillary and non-capillary pore spaces, water holding capacity, aeration, temperature and also the root penetration. Requirement: Oven/stove dried soil samples, balance, weights, mechanical sieve set and blotting sheets/old newspapers
Procedure
Fig.15.1 Soil Textural Triangle
Three methods are suggested here. Any one of these may be followed.
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Method I
(i) Collect about 300–500g of soil from two different locations. Label them as sample A and B. (ii) Dry the samples in an oven, or stove or in sun to remove the soil moisture (capillary and bound water).
(iii) Select the 3 sieves of different mesh sizes (2mm, 0.05mm and 0.002mm). Arrange them in a collecting chamber as shown in Fig. 15.2. (iv) Place 200g of the soil in the Ist sieve (sieve of 2mm mesh) and close the lid. To sieve the soil, shake the set manually for 5–10 minutes and collect the three soil fractions.
L ABORATORY MANUAL: BIOLOGY
(v) Weigh the soil fractions viz-sand, silt and clay collected in the 3 compartments
Lid
– .........g
Wt of sand fraction
– .........g
Wt of silt fraction
– .........g
Wt of clay fraction
– .........g
The weight of three fractions should be equal to the total weight of sample taken for analysis.
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2mm mesh
Wt of soil sample taken
0.05mm mesh
0.002mm mesh
Observations
Calculate the percentages of the various soil fractions and tabulate: Calculate the percentages of sand, silt and clay fractions.
Collecting chamber
Fig.15.2 Sieve set
Use the textural triangle now. Note that the three sides of the textural triangle represent 0 to 100% of sand, silt and clay respectively. Note that (i) the percentage lines for clay run paralled to the base line of sand, (ii)
the precentage lines of silt run parallel to the clay side of the triangle and, (iii) perentage lines of sand run parallel to the silt silde of the triangle. In reading the textural triangle, any two particle fractions will locate the textural class at the point where these two intersect.
Soil sample
Percentage (%)
Sand
A
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B
Silt
Texture class
Clay
Note for Teachers: The sieve sets contain a number and an abbreviation BSS/ASTM/ ISS on each sieve. In the given table (Table No. 15.1) the corresponding aperture size of the sieves is listed. For example, BSS 30 sieve aperture size will be 500 microns.
54
EXERCISE 15
Appendix 1
Table 15.1 Mesh No. and the corresponding Aperture size Sl. No.
BSS Mesh
ASTM Mesh
ISS Mesh
Aperture
4
5
480
4.75 mm
2
5
6
340
3.35 mm
3
6
7
280
2.80 mm
7
8
240
2.36 mm
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
8
10
200
2.00 mm
10
12
170
1.70 mm
12
14
140
1.40 mm
14
16
120
1.18 mm
16
18
100
1.00 mm
18
20
85
850 micron
22
25
70
710 micron
25
30
60
600 micron
30
35
50
500 micron
36
40
40
425 micron
44
45
35
355 micron
52
50
30
300 micron
60
60
25
250 micron
72
70
20
212 micron
85
80
18
180 micron
100
100
15
150 micron
120
120
12
125 micron
150
140
10
106 micron
170
170
9
90 micron
200
200
8
75 micron
240
230
6
63 micron
300
270
5
53 micron
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26
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1
27
350
325
4
45 micron
28
400
380
3
38 micron
29
25 micron
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L ABORATORY MANUAL: BIOLOGY
Method II Texture by Feel
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The texture of the soil sample can also be estimated by feeling it in the dry, moist and wet states. Sand is coarse and gritty, silt feels smooth like flour and clay is sticky and plastic. The smallest soil particles that one can see are coarse silt. Feel the known texture samples first, then feel the unknown ones and decide their textures.
Procedure
(i) Feel the dry soil first. Does it crumble easily or is it hard to break? Hard soil samples contain a moderate amount of clay.
(ii) Take in your palm a lump of soil sample about the size of a one-rupee coin and wet it to the consistency of modeling clay. Try to press it into a ribbon between the thumb and forefinger. An alternate test is to form a wire by rolling the wet soil until it is about 1/8" in diameter.
(iii) If a long wire or ribbon can be formed readily the soil is plastic and probably contains over 40% of clay. Its texture must therefore be clay/ silty clay/or sandy clay. If a ribbon or wire can be formed easily but also breaks easily, the soil sample is probably a clay loam/silty clay loam/or sandy clay loam. A heavy loam/silt loam/or sandy loam sample may form ribbon or a wire if the moisture content is just right but these will be still weaker than the ribbons and wires formed by the clay loam samples. (iv) Next determine whether sand or silt is dominant. If there is a gritty feel without the smooth floury touch of silt, choose a texture-name that includes the word ‘sandy’. If the smooth floury feel predominates and there is not much gritty feel, choose one of the ‘silty’ texture names. Use the name without a prefix if neither smoothness nor grittiness predominates (simply clay or sand or silt). Often this can best be determined by adding more water until the soil is in a wet state.
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If the soil is very sandy, you must choose between sandy loam, loamy sand and sand. In the moist state, sandy loam samples will have some tendency to stick together but loam sand and sand samples will not do so. Use the wet state to determine whether a sample is sand or loamy sand. After handling wet sand, your hands will be moist but clean loamy sand will make the hands slightly soiled.
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EXERCISE 15
Method III Requirements: Soil samples, balance, weights, glass rod, standard sieves of 2mm and 0.5mm mesh size, blotting sheets/old newspapers, evaporating dish and water
Procedure
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(i) Collect 200-300g of soil samples from different sites, and dry them as suggested previously to remove the moisture. (ii) Sieve the sample through a 2mm sieve to remove stones, pebbles, roots etc. (iii) Take 100-150 g of the sieved soil sample and further sieve it through a 0.05 mm sieve to separate the sand fraction (collected in the sieve) from silt and clay (collected on a blotting sheet). Weigh the amount of sand fraction and silt + clay fraction. (iv) Take a large evaporating dish (a shallow clay plate, glass trough or a shallow iron plate) and record its weight. (v) Add the clay and silt fraction to the dish and note the weight. (vi) Add water to the dish leaving half an inch space empty at the top and stir the liquid thoroughly with a glass rod taking care that the contents do not spill out. Allow it to stand for several hours. Decant off the cloudy supernatant liquid (clay fraction). Repeat the process three to four times until the decanted liquid is quite clear. (vii) Dry the silt left in the evaporating dish to dryness. Cool the dish and weigh it.
Observation
Record your observation in the following table:
A
B
Weight of the soil sample taken Weight of sand fraction
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Weight of silt & clay fraction Weight of silt fraction
Subtract the weight of silt fraction from the weight of silt + clay fraction. The difference will be the weight of clay decanted. Calculate the % of sand, silt and clay fraction of the soil and express the texture.
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L ABORATORY MANUAL: BIOLOGY
Discussion
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Correlate the texture with the plants growing in the area from which the soil sample has been collected. Discuss how the texture of soil can affect the root penetration, tillage, soil aeration, moisture content, water holding capacity and other aspects related to plant growth. In sandy soil the non-capillary pore spaces will be more and the capillary pore spaces will be less. The condition will be reverse in case of clay soil. The pore space in turn determines water holding capacity, percolation rate, aeration, root penetration and soil flora and fauna. Clay particles are anionic colloides and adsorb mineral nutrients and minimise their leaching.
Questions
1. Which type of soil is better for root-penetration and better aeration?
2. Among sandy and clay soil which one has higher water holding capacity? Explain. 3. If the clay content is high, will it affect soil fertility? Explain.
4. Which type soil has poor nutrient status and high leaching?
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5. What kind of plants grow in smooth texture soil? Name two plants that grow in heavy-textured soil.
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Exercise 16 Aim: To determine the water-holding capacity of soils
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Principle: Water holding capacity of the soil is the amount of water retained in the capillary spaces of the soil after the percolation of gravitational water into the deeper layers. Water holding capacity depends upon the capillary pore spaces in the soil. Sandy soil has very low water holding capacity, whereas clayey soils have very high water holding capacity. Requirement: Soil samples from different sites (garden, road side, bank of river, paddy field etc.), Gooch crucible (china clay crucible with perforated bottom), filter-paper, pestle and mortar, petridish, beaker, glass rod, balance and blotting paper
Procedure
(i) Dig a small pit about 10cm x 10cm x 10cm, Scoop 100–300 g of soil from the pit and collect it in a small polythene bag. (ii) Remove the pebbles and large lumps from the soil sample.
(iii) Pass the soil through a coarse sieve to remove small lumps and dead decaying leaves and twigs.
(iv) Spread the soil into a thin layer on a sheet of blotting paper or old newspaper and sun dry it for 2–3 hours or dry it in a pan kept on stove. Alternatively dry the soil sample in oven at 1080C for 1 hour. (v) With the help of pestle and mortar grind the sample into fine powder.
(vi) Put a small disc of blotting paper at the base of the Gooch crucible. Weigh the crucible along with the blotting paper and note its weight.
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(vii) Transfer the soil sample into the crucible. Tap the rim of the crucible gently several times with the help of glass rod so that soil is compactly filled and forms a uniform layer at the top. Add more soil if necessary. (viii) Weigh the crucible along with soil sample and note its weight. (ix) Fill the petridish with water and place two small glass rods in it parallel to and at a small distance from each other. (x) Place the crucible on the two glass rods in such a manner that its bottom is in contact with water.
(xi) Leave the set up undisturbed till water appears at the upper surface of the soil. Wait till entire soil surface is wet.
L ABORATORY MANUAL: BIOLOGY
(xii) Remove the crucible and allow all the gravitational water to flow out from the bottom. When no more water percolates, wipe the bottom dry with the blotting paper. (xiii) Weigh the crucible and note its weight.
Observation
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Record your observation in the following table. Calculate the % water holding capacity of the soil as follows. Weight of crucible + blotting paper:
Ag
Weight of crucible + blotting paper + soil sample before experiment:
Bg
Weight of dry soil:
B - A= Cg
Weight of crucible + blotting paper + wet soil sample after experiment:
Dg
Weight of wet soil after the experiment:
D - A= Eg
Mass of water absorbed by soil:
E - C= Ng
% Water holding capacity:
Tabulate your results as shown below
Sample No. Wt. of Crucible Wt. of Crucible Wt. of soil Wt. of crucible Wt. of wet Amount of % water + blotting + blotting paper sample + blotting paper soil (D-A) water holding paper (A) + soil sample (B) (B-A) = (C) + wet soil (D) = E absorbed capacity (E-C) = N
A Garden soil
B Road side soil
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C……...
D……...
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EXERCISE 16
Discussion
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Compare % water holding capacity of soil collected from different habitat conditions. The variation in water holding capacity is due to varying proportion of sand, silt and clay in the soil of different habitats. Soil with very high proportion of sand have very low water holding capacity due to large pore spaces between the particles which enables the water to percolate freely into deeper layers leaving upper layers practically dry. In clay soil, due to very small size of the pore spaces (fine capillaries) the water is retained in the capillary spaces as capillary water. In these soil the water does not percolate freely. Soil with more or less equal proportion of sand, silt and clay (loam soil) combines the properties of sand and clay and therefore has optimum water holding capacity and optimum soil-air for root growth.
Questions
1. What are heavy soil and light soil?
2. Give examples of a plant seen in heavy soil and light soil.
3. How does pore space determine the % water holding capacity of soil? 4. Why is clay soil often referred to as physiologically dry soil? 5. Which type of soil is suitable for cultivation of crop plants? 6. How can water-holding capacity of soil be improved?
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7. Dead decomposed organic matter is usually added in the fields before the cultivation of crops. Apart from providing the mineral nutrients, what additional role does organic matter play in the cultivation of crop plants?
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Exercise 17 Aim: To study the ecological adaptations in plants living in xeric and hydric conditions
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Principle: Successful adjustment of plants and animals under prevailing environmental conditions is known as adaptation. For terrestrial plants, the habitats vary from extremely dry conditions as in deserts to extremely wet conditions as in marsh lands. For aquatic plants the habitats may vary from deep water bodies like oceans and lakes to shallow ponds and pools. The plants are adapted to diurnal, seasonal or annual fluctuations of the habitat conditions. For land plants the main limiting factor is the availability of soil water whereas, for aquatic plants the main limiting factors are the fluctuations in water level, availability of gases like CO2 and O2 and the light intensity. Adaptation of land plants are primarily for conservation of available soil water, avoidance of bright sunlight and intense heat and for aquatic plants, adaptation are for conservation of gases and efficient utilization of available sunlight. On the basis of availability of water, plants are classified as: (a) Xerophytes: These are plants growing in extreme dry conditions throughout the year. For example, plants growing in deserts (psammophytes), on rock (lithophytes) or alpine plants growing above 14000 feet altitude. (b) Mesophytes: These are plants growing in soils with optimum soil water conditions prevailing for major part of the year. (c) Hydrophytes: These are aquatic plants growing in fresh to marine water.
The morphological, anatomical and physiological attributes of terrestrial plants are different from the aquatic plants.
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Requirement: Plant specimens from xeric and hydric habitat conditions. The specimens from xeric condition may include a few cacti, succulents (Euphorbia, Bryophyllum, Kelancho) cycas leaves, pine needles, twigs of Acacia, Nerium, Parkinsonia, Casuarina etc. The aquatic plants: Salvinia, Eichornia, Pistia, Hydrilla, Vallisnaria, Utricularia, Lymnophila; some reeds like Typha, Phragmites, amphibious plants like Marsilea and halophyte like Rhizophora. Beakers, glassjars, microscope, slide, coverslips and rajor blades
Procedure
Prepare temporary stained transverse sections of leaf, stem and root of the specimens. Study the morphological and anatomical features of the plants
EXERCISE 17
collected and look for the following adaptations. Write the name of the plant in which a particular adaptation is observed.
Observations
Xerophytes Adaptations
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Record your observation in the given tables:
Modifications (Morphological/Anatomical)
Examples (from the specimen collected)
Conservation of Water
a. Leaves few or absent or represented by spines only b. Petiole modified into leaf like structure c. Stem reduced, branching sparse d. In some cases stem flattened, leaf like, green, photosynthetic in nature
------
2.
Storage of Water
Thick, fleshy and succulent leaves as well as stem
--------
3.
Prevention of loss of water by transpiration
a. Intercellular spaces reduced b. Spongy parenchyma/ palisade parenchyma present c. Stomata on lower surface, sunken in stomatal pits d. Leaves needle like e. Thick cuticle on leaf surface
---------
4.
Prevention of excessive heat
a. Leaves covered with dense hairs; b. Leaf surfaces shiny or glaborous c. Leaf blade remains rolled during the day
-------------
5.
Efficient mechanism of water absorption
a. Long and profusely branched roots b. Dense root hairs c. Well developed xylem
-----------------
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1.
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L ABORATORY MANUAL: BIOLOGY Hydrophytes Adaptations
Modifications (Morphological/Anatomical)
1.
a. Leaves long and cylindrical b. Petioles flexible to withstand currents of water and to carry the leaf blade on the surface of water c. Petioles are modified into air pockets d. Leaf blade pale green in colour, finely dissected e. Leaf blade waxy with thin cuticle
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Buoyancy and resistance to currents of water
Examples
2. Transpiration
a. Stomata absent
b. Stomata present on upper surface of leaves
3. Absorption of water
4. Gaseous circulation and storage of air
a. Poorly developed roots b. Root hairs absent c. Roots with air pocket to help in buoyancy
Parenchymatous tissue of stem, roots, petioles and leaves modified into aeranchyma in the form of air channels in a. b. c. d.
5. Mechanical tissues
Root Stem Petiole Leaf
a. Poorly developed xylem b. Poorly developed sclerenchyma c. Sclerides present
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Questions
1. Give three adaptive features of water hyacinth suitable to aquatic life. 2. What are the features present in plants of xeric habitat for the prevention of loss of water? 3. What is the importance of succulent leaves and stem for a xerophytic plant? 4. Why is air stored between tissues in aquatic plants?
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Exercise 18 Aim: To study the adaptations in animals living in xeric and hydric conditions
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Principle: The aquatic ecosystem exhibits a different pattern of abiotic factors as compared to those in terrestrial ecosystem. The temperature of the water, penetration of sun light, the physicochemical characteristics of water body affect the growth and survival of the biotic community. In order to overcome the cumulatory effects of these factors, certain morphological and anatomical features, as well as physiological processes develop in the organisms. These modifications in animals are called adaptive features. We will study adaptations in selected animals living in aquatic and xeric condition. Requirement: Animal specimen/models of xeric (rat, camel, squirrel) and hydric (fish, frog, prawn, etc.) conditions
Procedure
Observe the animals provided and note down their adaptive features in the observation table with example.
Observations
Hydric adaptations Features
Body colour
(a) On dorsal surface (b) On ventral surface (a) (b) (c) (d) (e) (f) (g)
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Body contour
Adaptations
Locomotory
Streamlined Disappearance of neck constriction Tail enlargement Position of external nostrils Loss of external ears Position of eyes Presence of eye protecting membrane
(a) Fins or fin-like expansions of the body wall (b) Loss of limbs (c) Webbed feet
Example (For students)
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Integument
Presence of dermal/epidermal derivatives (a) Scales (b) Hairs (c) Mucous glands (d) Oil glands (a) Position (b) Presence of teeth a. Upper jaw b. Lower jaw
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Mouth
Respiratory organs
(a) Gills/lungs (b) Cutaneous
Xeric adaptations Features
Adaptations
Moisture getting
(a) Preference for juices as food (b) Hygroscopic skin
Moisture Conservations
(a) Storage of water in body (b) Avoidance of evaporation (non-perspiring)
Body colour
(a) Protective mimicry (b) Predating mimicry
Body contour
(a) Position of a. Nostrils directly upward b. Reduction to pin-head size (b) Position of eyes a. Covering of eyes b. Size
Skin
(a) Hard (b) Spiny (c) Poison glands
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Limbs
Scrotum
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(a) Speed (b) Long slender (c) Padded feet Present or Absent
EXERCISE 18
Discussion You may have noticed many features in the body of aquatic animals which support their life. As the different aquatic bodies vary to a great extent, there are many other adaptive features you may notice. For example the aquatic organism in ponds, lakes, river and sea.
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Questions 1. Name the features that helps a frog for aquatic life.
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2. What are the adaptations present in xeric animals for conservation of water?
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Exercise 19 Aim: To determine the pH of different water and soil samples
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Principle: The pH value of a water/soil sample can be determined by (i) indicator dye method, (ii) electrometric method using a pH meter and (iii) colorimetric method. For routine purposes the indicator dye method using universal pH indicator solution (containing a wide range of pH indicator dyes) or paper strips containing the pH indicators are preferred though it is not as accurate as the electrometric method.
Requirement: Soil or water samples A, B and C collected from different sites (for example soils from road side, garden, humus rich sites; water samples from borewell, handpump, pond, sewage), balance, weights, filter paper, distilled water, measuring cylinder (50 mL), droppers, cavity tile, funnel, beakers (100 mL), funnel stand, universal pH indicator solution and pH indicator paper (narrow range and broad range)
Procedure
(i) Weigh 10 g of the soil sample A. Add 50 mL of distilled water to soil sample to make a soil solution. (ii) Filter the soil solution through a filter paper and collect the filtrate in a beaker. Label it as soil solution -A.
(iii) Take a clean dry porcelain cavity tile. Place 5 drops of soil solution A in three cavities of the tile as shown in Fig. 19.1. Soil - A Soil - B Soil - C
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Universal pH indicator solution pH indicator paper (Broad range) pH indicator paper (Narrow range)
Fig. 19.1 Porcelain cavity tile
EXERCISE 19
(iv) To the 5 drops of soil solution present in one cavity add 5 drops of universal pH indicator solution. Note the colour developed and compare it with the colour chart given on the universal pH indicator solution bottle. (v) To the soil solution present in the second cavity, dip a small strip of broad range pH indicator paper (pH 2-11). Note the colour and compare with the colour chart given on the broad range indicator paper and get a rough estimate of pH of the sample solution.
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(vi) Choose a suitable narrow range pH indicator paper (for e.g. If the pH of soil is determined by you as 8.0, choose a narrow range 7.0 to 9.0) and dip a small strip of it in the soil solution present in the third cavity. Note the colour developed and determine the pH to the nearest possible value with the help of the colour chart.
Repeat the same steps for determining the pH of sample B and C. Follow the same procedure for water samples collected from different sites.
Observation
Record your observations in the given table.
Table: Measurement of pH of soil samples A, B and C pH value as determined by
Soil Samples
A
B
C
Universal indicator solution
Broad range indicator paper
Narrow range indicator paper
Discussion
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Based on the pH values obtained, categorise the samples into acidic, basic, neutral type. Record the plant species present in the site from which the samples are collected. Note for teachers: The colour developed should be noted against direct sun light. Also, sometimes the soil solution colour may interfere with the readings. Thus one has to be careful while making the observations.
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Questions 1. What will be the pH of chalk (calcareous) soil? 2. pH measurement with indicator paper is not very accurate. Comment. 3. Water logged soils are acidic. Comment.
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4. Why are soil around mineral mining areas acidic?
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Exercise 20 Aim: To study turbidity of water samples
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Principle: Various characters that control the quality of water are taste, smell, colour, amount of dissolved nutrients, dissolved O2 and CO2, pH and different types of plants and animals and their density. Turbidity of the water body determines the depth upto which light can penetrate and thus affects the distribution and photosynthesis of phytoplanktons and macrophytes. More turbid the water body less is the thickness of its photic zone. In polluted water bodies turbidity is due to:
1. Effluents: A water body which receives domestic sewage, run off from adjacent agricultural fields and liquid wastes from nearby small and large industries remains turbid. 2. Planktons: A water body may be turbid due to very high density of phytoplanktons and zooplanktons, especially when the water body is rich in nutrients. I. Secchi’s Disc method
Requirement: Secchi’s Disc, rope of moderate thickness, meter rod, black and white paints; paint br ush. Prepare a Secchi's disc by taking an iron disc of about 6 inches diameter, to which a weight is attached in the centre on one side and an iron hook on the other side. Tie a plastic rope of sufficient length to the hook. Divide the upper surface of the disc into 4 equal segments and paint two of these white and the other two segments black in such a way that black and white segments alternate with each other (Fig. 20.1).
Procedure
(i) Visit a nearby pond.
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(ii) Reach to the center of the pond in a small boat.
(iii) Slowly immerse the Secchi disc into water vertically holding the rope tightly in the hand till the black and white segments of the disc just begin to disappear. On reaching to a particular depth, the disc becomes completely invisible. Mark the length of the rope when the disc just disappears (say A cm).
Fig . 20.1 A S ecchi’s Disc
L ABORATORY MANUAL: BIOLOGY
(iv) Slowly pull up the disc and find out the length of the rope where the black and white segments of the disc just reappear (say B cm). (v) Find out the mean length (X) of the rope by the following method.
(vi) Repeat the process at different sites of the pond.
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Tabulate the results in the given table Water body
Depth at which Disc
Depth at which
Depth of Photic
disappears (A cm)
disc reappears (B cm)
zone
Pond Site 1
Site 2 Site 3 -
-
Observations
The value X represents the depth of the photic zone upto which sunlight penetrates in the water body and photosynthesis takes place.
Discussion
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Greater the value of 'X' less turbid is the water. In crystal clear deep lakes, the value of 'X' will be very high indicating, thereby, that the water body does not have large quantities of flocculating silt or organic matter residues. This may be due to no discharge of effluents or domestic sewage into the water body. The high clarity of water is also an indication of very less density of phyto and zooplanktons. These water bodies are called as non-productive or oligotrophic, while highly turbid water bodies are eutrophic in nature.
Precautions Students are advised to perform this experiment under the strict supervision of teacher to prevent incidents due to drowning.
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EXERCISE 20
II. Measurement of turbidity using measuring cylinder Requirements: Water samples from different sources, three measuring cylinders (500mL) of the same height.
Procedure (i) Collect about 2 liters each water samples from different sources.
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(ii) Transfer 500ml of water sample in the measuring cylinders of same volume and height.
(iii) Mark the three cylinders A, B and C and leave them undisturbed overnight.
Observations
Observe the amount of sediment settled at the bottom of each cylinder and also note whether the water above the sediment is still turbid. Record your observations in the following table: Water sample
Thickness of sediment
Clarity of water—turbid/ semiturbid/clear
'A' 'B' 'C'
Discussion
Do all the samples show same amount of sediments?
z
Which sample shows maximum sedimentation and correlate it with the source of the sample?
z
Find out whether in all the cylinders, water above the sediment is clear or turbid. Explain with reasons.
z
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z
Draw conclusions on the basis of the observations.
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L ABORATORY MANUAL: BIOLOGY
Questions 1. Is turbid water fit for drinking? Explain. 2. Why is the penetration of sunlight in any water body important? 3. Green plants are seen only in photic zone. Comment. 4. It is a common practice to use alum for clearing turbid waters. Explain.
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5. Turbidity of water body varies with season. Comment.
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Exercise 21 Aim: To analyse living organisms in water samples
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Principle: The productivity and the trophic status of a water body is determined by assessing the number and type of organisms (micro as well as macro) present in the water body. Water body with very high density of phytoplankton per unit area is a productive water body. Such water bodies are usually turbid and have high amounts of nutrients and dissolved oxygen. These water bodies support fairly large number of organisms of different trophic levels. This is in contrast to non-productive water bodies, which have very low density of organisms per unit area, fairly transparent waters with low mineral concentration and dissolved oxygen and also fewer trophic levels. The status of health of a water body can be determined by analysing water samples for the number and type of organisms present in it at a given time. Such assays also help us to find out whether a water body is polluted as some of the organisms are strong indicators of water pollution. Requirement: Water samples from different water bodies (lake, pond, river etc.), beakers, a few vials or small test tubes, slides and cover slips, watch glasses, dropper, compound microscope and 5% FAA (Formalin Aceto Alcohol 5:5:90:Formalin: Acetic acid: Ethanol) as preservative
Procedure
(i) Collect about a liter of water sample from nearby water body (pond lake, reservoir, river etc). (ii) Add about 5 ml of FAA to fix and preserve the living organisms present in each sample at the place of collection.
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(iii) In the laboratory, transfer the water sample into a measuring cylinder of one litre capacity. Label each water sample to indicate the site from which the water sample has been collected. (iv) Leave the water samples undisturbed for 48-72 hours. (v) Decant off the clear water, leaving concentrated sediment at the bottom.
(vi) Transfer the sediment into a vial or a small test tube. Cork and label each vial for future use. (vii) With the help of a dropper, transfer a few drops of sediment liquid from a vial into a watch glass. Dilute the sediment with water if the sediment is highly concentrated.
L ABORATORY MANUAL: BIOLOGY
(viii) With the help of a dropper transfer a drop of water from the watch glass on the center of a slide and mount it. Blot the excess water using blotting paper. (ix) Prepare a few more slides of each water sample in the same way. (x) Observe each slide, first under lower magnification and then under higher magnification.
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Observations 1.
Record the different types of organisms present.
2.
Count the number of organisms under each field of microscope.
3.
Some of the commonly found organisms of water bodies are given in Annexure 2.
Discussion
Prepare a list of organisms observed in each water sample and make an assessment of type and density of different organisms in each water sample. Polluted waters may contain very few types of organisms but in very high density. The non-polluted waters will have large variety of organisms in low density.
Questions
1. Why do you find few organisms in polluted water? Explain.
2. Why is FAA (Formaline Aceto Alcohol) added after collecting the water sample?
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3. Name at least one phytoplankton and zooplankton commonly found in polluted water.
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EXERCISE 21
Annexure 2
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Cosmarium
Desmickum
Stigeoelomium
Spirogyra
Draparnaldiopsis
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Nitella
Dinobryon
Euglena
Moubeotia
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L ABORATORY MANUAL: BIOLOGY
Ceratium
Gymnodinum
Peridinium
Calothrix
Rivularia
Cyanomonas
Gleotrichia
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Anabaena
Cylindespermum
Scytonema
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Fischerella
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EXERCISE 21
Chlamydomonas
Volvox
Coelastrum
Closterium
Golenkinia
Pedrastrum
Chlorella
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Hydrodictyon
Ankistrodesmus
Scenedesmus
Staurastrum
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L ABORATORY MANUAL: BIOLOGY
Synechococcus
Gloeocapsa
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Microcystis
Phormidium
Oscillatoria
Synechocystis
Spirulina
Lyngbya
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Nostoc
Melosira Chaetoceros
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EXERCISE 21
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Rhizoselina
Amphora
Biddulphia
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Bacillaria
Synedra
Centronella Nitzschia
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L ABORATORY MANUAL: BIOLOGY
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Navicula
Somphonema
Fragilaria
Pleurosigma
Coscinodiseus
Skeletonema
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Lauderia
Cocconeis Asterionella
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Exercise 22 Aim: To determine the amount of Suspended Particulate Matter (SPM) in air at different sites in a city
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Principle: Environmental pollution is the unfavorable alteration of our surroundings wholly or largely as a by-product of man's action through direct or indirect effects of changes in energy patterns, radiation levels, chemical and physical constitutions of environment and abundance of organisms. Substances that cause pollution to the environment are called pollutants. They are the residues of things that man makes, uses and throws away. These residues pollute soil, water and air. The atmosphere in highly populated area is very rich in dust, smoke and SPM all due to vehicular exhausts and industrial emission. Requirement: A few freshly cut broad leaves, Vaseline, laboratory balance, weights, brush, paper clips and twine thread
Procedure
This experiment is an outdoor activity and may be conducted by assigning 2–3 students into a group. (i) Collect a few locally available broad leaves from a nearby tree plant (Canna, Peepal, etc.). (ii) Wash the leaves gently in running water to remove any dust settled on their surfaces.
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(iii) Blot dry the surface area of the leaves. To calculate the area of the leaf, trace the outline of the leaf on graph paper (Fig 22.1). Within the traced area calculate the total number of full squares, 1/2, 1/3 and 2/3 squares and individual small squares. Add all the squares to get the total leaf area. Multiply their value with two to obtain total area of both the surfaces.
(iv) Take 8–10 feet long twine thread and tie five leaves leaving a foot distance in between. Apply an extremely thin layer of vaseline on both surfaces of each leaf. Make a bundle of these leaves and pack
Fig. 22.1 Calculating the area of a leaf on a graph paper
L ABORATORY MANUAL: BIOLOGY
them in polythene bags. Ensure that the outer surface of polythene bag does not have any vaseline sticking on it. (v) Make three such bundles of smeared leaves, each bundle containing 5 leaves. (vi) Mark bundles as A, B and C and carefully weigh each bundle of leaves along with the polythene bags.
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(vii) Select three spots (X, Y and Z) near by your school. Spots selected should be in a manner that spot 'X' has very heavy vehicular traffic, the spot Y has moderate traffic and spot 'Z' has little or no vehicular traffic. At spot 'X' expose each leaf of bundle 'A' by stretching the attached thread and tie the two ends to two poles or branches of trees preferably at 10 feet height above ground. Keep leaves exposed for about two hours.
(viii) After exposure at spot 'X', collect the leaves and carefully re-bundle exposed leaves and place them along with the string in the polythene cover 'A'. Record your findings in the following table:
Site
Leaf bundle sample
Weight of leaves (g)
Before exposure (W1)
X Y Z
Weight of suspended particle (W2 -W1 )
Total leaf area (cm2) of five leaves
After exposure (W2)
'A'
'B' 'C'
(ix) Repeat the same process at spot 'Y' and 'Z' exposing leaves of 'B' and 'C' bundles respectively.
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(x) At the end of the experiment, return back to the laboratory. Reweigh each bundle of exposed leaves along with their respective polythene cover. - Calculate the amount of suspended particles deposited in mg cm2 of leaf at each spot.
- Compare the results of three different spots and interpret.
Since the weight of suspended particles will be in milligrams or even less it is advised to use a very sensitive laboratory balance.
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Exercise 23 Aim: To study plant population density by quadrat method
Density=
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Principle: Density represents the numerical strength of a certain plant species in the community per unit area. The number of individuals of the species in any unit area is its density. The unit area may be as small as 5 square cm to as large as 10 square metre depending on the size and nature of the plant community under study. For herbaceous vegetation a metre square quadrat is normally used. Density which gives an idea of degree of competition is calculated as follows. Total number of individual(s) of the species in all the sampling unit (S) Total number of sampling units studied (Q)
The value thus obtained is then expressed as number of individuals per unit area. When the measured unit area is divided by the number of individuals the average area occupied by each individual is obtained. Requirement: Cotton/nylon thread (five meters), 4 nails and a hammer
Procedure
(i) In the selected site of study, make a 1 m X 1 m quadrat with the help of nails and thread. Hammer the nails firmly and make sure that the vegetation is not damaged while laying the quadrat.
(ii) List the names of the plant species seen in the quadrat (if the name is not known mark these as species A or B etc., and the same species if seen in other quadrats assign the same alphabet). (iii) Count the number of individuals of each species present in the quadrat and record the data as shown in the table.
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(iv) Similarly make nine more quadrats randomly in the site of study and record the names and number of individuals of each species.
Observations
Record the total number of species seen in the ten quadrats. This will give an idea about the composition of the vegetation. There will be difference in the species composition in the quadrats made in shady areas, exposed areas with bright sunlight, dry or wet areas etc.
L ABORATORY MANUAL: BIOLOGY
Table 23.1: Density studies of the given vegetation Plant Species
Quadrats employed in study & no. of individuals in each quadrat I 2
Z
1
III
IV
V
5 2
4
8
VI
VII VIII
7
10
3
IX
Total no. Density (D) of Quadrats studied (Q)
X 3
2
27
10
27/10 = 2.7
20
10
20/10 = 2.0
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A
II
Total No. of individuals (S)
Discussion
Plants growing together exhibit mutual relationships among themselves and also with the environment. Such a group of plants in an area represent a community. The number of individuals of a species varies from place to place, making it necessary to take many random sample areas for reliable results. Density values are significant because they show relative importance of each species. With increasing density the competition stress increases and the same is reflected in poor growth and lower reproductive capacity of the species. Data on population density are often very essential in measuring the effects of reseeding, burning, spraying and successional changes. Discuss the vegetation composition of the area (herbs/shrubs) and comment on the dominant component species.
Questions
1. What factors influence the population density? 2. What is the significance of quadrat method?
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3. What conclusion can be drawn if density of a plant species is low?
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Exercise 24 Aim: To study plant population frequency by quadrat method
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Principle: Frequency is concerned with the degree of uniformity of the occurrence of individuals of a species within a plant community. It is measured by noting the presence of a species in random sample areas (quadrats) which are distributed as widely as possible throughout the area of study. Frequency is the number of sampling units (as %) in which a particular species (A) occurs. The frequency of each species (sps. A or sps. B or sps. X etc) is expressed in percentage and is calculated as follows. % Frequency or
Frequency Index
=
Number of sampling units (quadrats) in which the species occurs Total number of sampling units (quadrats) employed for the study
Requirements: Cotton/nylon thread of 5 metres, 4 nails and a hammer
Procedure
(i) In the selected site of study, make a 1 m X 1 m quadrat with the help of nails and thread. Hammer the nails firmly and make sure that the vegetation is not damaged while laying the quadrat.
(ii) List the names of the plant species seen in the quadrat (if the name is not known mark these as species A or B etc. and if the same species is seen in other quadrats assign the same alphabet) (iii) Similarly lay nine more quadrats randomly in the site of study and record the names of individuals of each species.
(iv) Calculate the percentage frequency of occurrence using the formula given.
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Observations
Record the total number of species seen in the ten quadrats. This will give an idea about the composition of the vegetation. There will be difference in the species composition in the quadrats made in shady areas, exposed areas with bright sunlight, dry or wet areas etc. Observe that the frequency of occurrence is not the same for all species.
L ABORATORY MANUAL: BIOLOGY
Table 24.1: Frequency studies for the given vegetation Plant Species
Number of quadrats employed in the study (Q) I
B C
√
III
IV
√
√
V
VI
VII VIII
IX
√
X √
√
Percentage of frequency F=N/Q X 100
5
5/10
100 = 50%
1
1/10
100 = 10%
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A
II
No. of quadrats in which the species is present (N)
√
√
√
√
4
4/10
100 = 40%
Discussion
Variation in distribution of a species is caused by factors like soil conditions, quantity and dispersal of gemmules, vegetative propagation, grazing, predation, diseases and other biotic activities. Also frequency values differ in different communities. They are influenced by micro-habitat conditions, topography, soil and many other environmental characteristics. Thus unless frequency is not correlated with other characters such as density, frequency alone does not give correct idea of the distribution of a species. Frequency determinations by means of sample areas are often needed in order to check general impressions about the relative values of species. Many species having low cover or population density also rate low in frequency, but some may have high frequency because of their uniform distribution. Usually if the cover and population density are high, the frequency will be high. The plants with high frequency are wide in distribution.
Questions
1. If frequency of a plant is high, what will be your interpretation?
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2. Can many micro-habitat in an area affect frequency of a species? Comment.
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Exercise 25 Aim: Study of homologous and analogous organs in plants and animals
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Principle: In plants and animals there are several organs or parts thereof, apparently alike in their function and appearance, but markedly different from each other in their origin and anatomical structure. These organs are called analogous organs, and the seeming similarity among them is the result of convergence, that is, adaptation to similar habitat and identical ecological niche. On the other hand, there are organs or parts thereof, which apparently are quite dissimilar to each other in appearance and perform different functions, but have the same origin and anatomy. The differences in their function and also in their appearances are the result of divergence, due to adaptive radiation to different habit, habitat and ecological niche. These organs are called homologous organs.
Requirement: Plant specimens showing tendrils, thorns, etc., as given in the text or any other locally available plants, a plant with normal stem, potato and onion bulb, prickly pear, specimens of phylloclade, cladode, wings of bird, cockroach and bat, and cervical, thoracic and lumbar vertebrae of a mammal/lizard
Observations
1. Homologous Organs in Plants
(i) Tendrils of passion flower and thorns of pomegranate Tendrils of passion fruit and thorns of pomegranate are structurally and functionally different but they have similar origin i.e. they arise from axillary bud (Fig. 25.1a & b).
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Tendril
Thorn (a)
(b)
Fig. 25.1 (a) Tendrils of passion fruit (b) Thorns of pomegranate
L ABORATORY MANUAL: BIOLOGY
(ii) Tendrils of Vitis and thorns of Carissa
Thorns
Tendril
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Tendrils of Vitis and thorns of Carissa originate from the terminal bud, but they are functionally different (Fig. 25.2 a & b).
(a)
(b)
Fig. 25.2 (a) Tendrils of Vitis (b) Thorns of Carissa Tendril
(a)
Both are modifications of floral bud, but they perform different functions. Tendrils help in climbing but bulbils are meant for reproduction (Fig. 25.3 a & b).
(b)
Fig. 25.3 (a) Tendrils of baloon vine (b) Bulbils of Agave
Spines
(a)
(iii) Tendrils of baloon vine (Cardiospermum) and bulbils of Agave.
(b)
Fig. 25.4 (a) Scale leaves of onion (b) Spines of cactus
(iv) Scale leaves of onion and spines of prickly pear (Opuntia) Both the scale leaves and spines are modifications of leaves but are structurally and functionally different. Scale leaves of onion are thick and fleshy and store food. On the other hand spines of cactus are defensive organs (Fig. 25.4 a & b).
2. Analogous Organs in Plants
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Tendril
Tendril (b) (a) Fig. 25.5 (a) Tendrils of pea (b) Tendrils of Vitis
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(i) Stem tendrils and leaf tendrils
All tendrils are analogous with one another, being structurally and functionally similar, irrespective of their origin. Example: Tendrils of pea and tendrils of Vitis. Tendrils of pea are modification of leaf and in Vitis it is the modification of terminal bud (Fig. 25.5 a & b).
EXERCISE 25
(ii) Thorns and spines Thorns and spines are analogous structures being defensive in function. Thorns are modifications of axillary or terminal buds, and spines are (a) (b) modifications of leaves. Fig. 25.6 (a) Modified root of carrot (b) Rhizome of ginger e.g: Thorns of pomegranate and spines of prickly pear. (iii) Modified underground stems and modified roots
Spine
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Modified stems (rhizome, corm, tuber) are analogous to modified roots (carrot, radish) as they perform similar function of storage of food but their origin is different. Rhizome of ginger, potato tuber, (a) (b) Colocasia are stems and beetroot, radish etc. are Fig. 25.7 (a) Phylloclade (b) Cladode of roots. (Fig. 25.6 a & b) ruscus
(iv) Phylloclade, cladode and leaves
They perform the same function i.e. they photosynthesise but phylloclade and cladode are modifications of stem. Phylloclade of Opuntia, Parkinsonia, Asparagus and leaves of any local plant like mango are analogous organs. (Fig. 25.7 a & b)
3. Homologous Organs in Animals
(i) Wings of birds, and forelimb of mammals/reptiles/ frog: All have the same bony elements (humerus radioulna, carpals, metacarpals and phalanges), but perform different (flying in birds, for holding or walking etc. in other) functions. (Fig. 25.8 a & b)
4. Analogous Organs in Animals
(a)
(b)
Fig. 25.8 Fore limb of (a) human (b) bat
(a)
(b)
Fig. 25.9 Wing of (a) dragonfly (b) bird
(i) Wings of dragonfly/cockroach/butterfly and of birds. (Fig. 25.9 a & b) (ii) Mandible of cockroach and mandible (lower jaw) of a vertebrate. (Fig. 25.10 c & d)
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Note: Students and teachers are suggested to discuss more examples.
(a)
(b)
Fig. 25.10 Mandible of (a) cockroach (b) rabbit
Questions
1. Suggest examples of homologous and analogous organs other than what are given in the manual. 2. Why are stem and leaf tendrils considered as analogous organs?
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Investigatory Project Work
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Investigations are more open-ended than practical exercises involving a search to understand the unknown and begins with a question or a hypothesis. You are not instructed exactly what to do, but are given only general guidance. These give you more opportunity to plan your work. For example, you might investigate what traits you and your classmates inherit from your parents and forefathers (both maternal and paternal).
Projects are even more open-ended than investigations. These are practical investigations carried out by an individual or a group of students. Projects are largely your own initiative. It also requires evaluation of your findings, redefining ideas and designing further investigations. This may lead to evidence that enables answering the question posed at the outset. Some of these projects would take about few hours to complete. Other may take few weeks. Some are laboratory based, others involve fieldwork. Many could be carried out at home.
Investigatory projects are part of obligatory assignment involving purely experimental procedures so that you report on, duplicate, or adapt something that someone else has already discovered. It may involve some other form of investigation also. For example, you may undertake to investigate the richness and patterns of biodiversity (flora and fauna) in your school campus and prepare a mural of it or to investigate the effects of physical fitness on your pulse rate.
Choosing an Investigatory Project
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You may be guided by your teacher for your choice of topic. The more original or new the project is, the better it would be. But it must be realistic in terms of the time available and at a level attained in the higher secondary biology. You must review the available literature to find out what type of work has been done. This will help you to reject some of the alternatives, and possibly cause you to modify others. It may also be the source of new ideas. By doing these investigatory projects you will gain experience of research besides providing opportunity for learning skills such as photography, electronics, etc.
Identifying the Objectives of the Project Having identified a possible project, you should be able to identify and list the tentative objectives you hope to attain by completing that investigation. For example,
INVESTIGATORY PROJECT WORK
¨
Suppose your project involves studying the biodiversity of birds in your district/state, examine the data in the light of some questions (say, how do the birds in Rajasthan differ from those in Assam or Bihar?) your investigation might attempt to answer.
¨
Suppose your project involves investigating leaf mosaics revealing the complexity of the growth correlations that lead to efficient light interception, suggest also the factors that might affect this type of study.
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Keep the aim of your project simple. Investigate only one factor at a time and never allow yourself to be side-tracked. Remember that time may be too short for follow-up and any fascinating secondary aspects that you may come across.
Designing Projects
Having established the objectives of your chosen project, you must have an experimental design. This will allow you to collect the data you need in a scientific way to test the hypothesis. For example, if your project involves investigating the hypothesis that stale milk contains more bacteria than fresh milk, devise the procedure you would adopt to carry out your investigation.
Planning Investigations
Having decided your topic for scientific investigation, you should give careful thought to the plan of your investigation in some detail. These may include What hypothesis can you make?
¨
How can you ensure that the experimental tests and measurement you carry out are accurate and reliable?
¨
What controls do you need?
•
How many variables are you investigating? Correctly identify key variables as independent and dependent.
¨
Are your variables discrete or continuous?
¨
Identify appropriate control variable for fair test.
¨
How many repeat observations or samples will you require?
¨ ¨ ¨ ¨
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What instruments/equipment or techniques will you use to obtain relevant information? Identify suitable materials and equipment to be used. If your investigation requires the use of a questionnaire, design and standardise before implementation. Is your intended procedure safe and ethically permitted, i.e., taking care of the distress or suffering of living organisms and damage to the environment? How will you collect your data?
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¨
How do you plan to analyse your results? Would you employ statistical or other methods? Are scale range, interval, number of values chosen are adequate and reasonable ?
Executing the Project
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Following planning, a brief description of the expected procedures has to be approved in advance by the teacher. Having decided what controls you need to use, list the components of your experiment and decide what quantities of substances to use, how to set the experiment. You should also decide what type of readings or measurements you are going to make, how often and how many. Note the source of error, if any, that you come across.
¨ ¨
Handle instruments and equipments appropriately to give accuracy.
¨
Keep proper controls and the variables constant.
Repeat measurement.
Reporting/Writing of Project
A format, such as given below, can be followed.
(i) Title of the investigatory project: Write the title of the project, for example, ‘Inheritance pattern of eye colour’.
(ii) Objectives: Express as clearly as possible the effect of one variable that the experiment is designed to investigate.
(iii) Materials needed: This might be just a list, or a diagram if a particular piece of apparatus was used. (iv) Method: Describe the procedure stepwise including the precautions taken, if any. (v) Result: A suitable chart or table for recording and organising your readings or measurements should be made out before you start the experiment.
(vi) Analysis and interpretation: Observation data are factual, and may not be as expected by you.
(vii) Discussion: Discuss briefly the implication of your results and suggest extensions of any kind that can be undertaken.
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(viii) Conclusion: In view of the results obtained and related work done on the topic of the project, write conclusion briefly. (ix) References: Any work related to the project which you have come across through books/articles or any other source should be written as reference, for example: Michael Michalco (2001), Cracking Creativity, Berkeley, Ten Speed Press.
This write up is meant to train the students in scientific methods. In other words, it accentuates the spirit of enquiry and investigation in young minds.
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INVESTIGATORY PROJECT WORK
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The operational aspects of doing a project include choosing a hypothesis or problem to be investigated, collecting data in a designed manner, analysing the data in a scientific way, drawing conclusions which are justified and discussing the results in the light of known knowledge and bringing out its importance. Finally it includes the scientific way of communicating the findings. While your discovery during the investigatory project may not merit a Nobel Prize it may help you discover something, a fact or relationship that was unknown to you and that was not recorded in any book available to you. Scientists refer to this as an independent discovery. Your investigation will certainly give a sample of the thrill of discovery. Following are pages on procedural guideline about a few suggestive investigatory project work.
1. Investigating the pH of a water sample Background information
Monitoring the physico-chemical properties of water is of vital importance. Normal maximum permissible limit of pH for our life and health is 6.5–8.5.
Abnormal levels of pH and their consequences are given below: pH 3 to 5 is too acidic for most organisms to survive, when the pH of water falls below 4.5 most of the fishes die, leaving only a small number of acid-tolerant insects such as water boatman and whirligig. These insects (beetles) can survive and multiply even at pH 3.5. Similarly, pH>8.5 is too basic for most organisms to survive. Materials needed ¨
Universal indicator test paper (broad range, narrow range PH 2–11)
¨
Water sample
2. Investigating the biochemical (also called biological oxygen demand [BOD]) of a water sample as pollution indicator. Background information
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A dissolved oxygen (DO) test measures the current status of oxygen in a water body. This is a useful starting point. However, DO content can vary considerably from day-to-day as affected by many factors like temperature, wind velocity, eutrophication, pollution, etc. The unpolluted water is characteristically rich in DO and low in BOD. Higher the BOD, lower would be DO. Conversely, the polluted water has high values of BOD. Water for drinking should have a BOD less than 1. Typical BOD value for raw sewage run from 200–400 mg of oxygen/litre. The maximum
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L ABORATORY MANUAL: BIOLOGY
permissible limit of BOD followed by Central Pollution Control Board (India) for national water quality monitoring purposes is less than or equal to 3mg/L. For example, in a study the prevalence of some organisms were done at two different sites in a water body. The result can be tabulated on the basis of following facts: A = Abundant; R = Rare; X = Very few Concentration
Site 1
Site 2
Red sledge worm (Tubifex worm)
A
A
Larvae of midge (Chironomus)
R
A
Blood Worm
X
X
Leech (Hirudinea)
X
X
Water louse, water skaters (Asellus)
X
X
X
X
Fresh water shrimp (Gammarus)
X
X
Water boatman
X
X
Diving beetle (Dytiscus)
X
X
Caddisfly larva (Ochrotricha)
X
X
Damselfly larva (Lestes)
X
X
Stonyfly nymph (Isoperla)
X
X
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Organisms in order of tendency to disappear as degree of pollution increases
Indicator organism
Mayfly nymph (Stenonema)
X
X
Snail (Lymnaea)
none
X
Clams (Corbicula)
X
X
X
Bacteria (anaerobic)
X
X
Utricularia
X
X
Chara
X
X
Water fern (Salvinia)
X
X
Water velvet (Azolla)
X
X
Water meal (Wolffia)
X
X
Lesser duckweed (Lemna minor)
X
X
Greater duckweed (Spirodela)
X
X
Diatom
X
X
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none
Fungi
3. Population density of plants (i) Identify any 5 weeds from your locality.
(ii) Collect information about them from various sources. Focus on their economic importance especially their medicinal importance (collect samples for herbarium preparation).
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INVESTIGATORY PROJECT WORK
(iii) Study their distribution in different localities by quadrat method. (iv) Register their data and draw comparisons of their distribution by histograms. (v) Try to analyse the differences in distribution and density. (vi) Correlate their presence with their habitat/adaptability.
4. To make an inventory of local tree, shrub and herb
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Informations can be listed in following categories: (i) Avenue trees
(ii) Wind Breakers (iii) Road dividers
(iv) Sound barriers
(v) Medicinal and other uses
5. Agrochemicals and their effects
The project may be carried out in a survey mode with a questionnaire prepared with the help of the teacher to cover the following aspects.
(i) List of pesticides used, amount used/hectare or acre, periodicity of spray, name of the crop plant grown, recommended dose and the dose employed, known effects on pest, whether the chemical pesticide is biodegradable or not, alternate ecofriendly biocides.
(ii) List of fertilisers used, cost incurred/acre/year, recommended dosage/ time of use and the dosage used, known effects on fertility of soil, any decrease in crop productivity, use of ecofriendly biofertilisers (VAM fungi, leaf mold, green manure, dung, etc.).
6. Ecological role of some animals observed in a local area
Record the various plant species growing in the area under study—trees, shrubs, annual/perennial herbs, etc.
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Note the season when flower/seed is formed.
Note the various types of insects, birds, reptiles, amphibians, mammals, etc., and record their role as a/an (i) Herbivore
(ii) Pollinator
(iii) Agent in seed dispersal (iv) Prey
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L ABORATORY MANUAL: BIOLOGY
(v) Predator (vi) Vectors for transmission of diseases (vii) Any other
07. Study the effect of a local industry on environment (i) Select an industry of your choice.
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(ii) Note the source of energy used, product formed, raw materials used (locally available or imported) mode of transport used to move the final product.
(iii) Possible types of pollutants released by the industry (air/water/ soil).
(iv) Measures undertaken by the management to comply with the standard set by Central Pollution Control Board (CPCB), PCBs, etc. (v) Awareness about ISO 2000.
(vi) Impact assessment carried out or not.
08. Study of the effect of chemicals and pollutants on the Mitotic Index of the mitotically dividing onion root tip cells This study may include
(i) Growing of onion root tip cells in the solution of pollutant/chemical and also in normal water as control.
(ii) Preparation and observation of slide for the study of mitotic index both in experimental and control set-ups.
(iii) Analysis of the effect of pollutant/chemical by comparing the data of mitotic index between experimental and control variable.
09. Study of the genetic markers in the human population
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In this investigation a few selected inherited traits can be investigated in the family members of a small population in the locality. The compilation and analysis of data will provide an about prevalence of trait in the said population.
10. Inventory of weeds in aquatic bodies/agricultural fields 11. Inventory of birds in your locality, their ecological role as scavangers, pollinators, etc.
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INVESTIGATORY PROJECT WORK
12. Impact of local industry on the environment and the remedial measures taken by the industry
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The guidelines and a brief outline of a few projects have been given with a purpose to design and perform such investigation. Students and teachers can think and design investigatory projects based on almost all concepts about which experimental protocol have been given in the manual. However, a small list of suggestive projects are also given below. Please note that these are only suggestive and it is expected that students and teachers will take up many more types of investigatory projects depending on the specificity of their area, need and problems.
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Investigatory Project
1
Aim: To study the effect of pH on seed germination
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Principle: pH is one of the most important factors that controls the composition of flora and fauna in different terrestrial and aquatic ecosystems. pH of soils is essentially controlled by the amount and type of various minerals and also by the quality and quantity of humus (dead, decaying organic matter) present in it. Seed germination is controlled by pH of the germinating medium. Seeds of different species prefer a specific range of pH for maximum germination. pH not only controls the germination of seeds but also growth and development, reproduction and various other metabolic activities, of the plant. Objectives: After completing the project, the students will be able to 1. Plan out an experiment and understand the use of appropriate chemicals, apparatus and equipment, and learn the preparation of solutions. 2. Understand research methodology. 3. Generate, analyse and interpret the data and draw conclusions. 4. Conceive and choose other different themes related to pH and plant growth.
Materials required: 1. 125 seeds each of sunflower, mustard, green moong, alfa alfa, fenugreek and barley (selection of seeds of different species may be made as per their availability) 2. Phosphate buffers 3. Distilled water 4. Petridishes of 7.5 cm diameter (15 pairs) 5. Blotting papers cut into circular discs to the size of petridishes
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Procedure
(i) Prepare a range of pH buffers using Na2HPO 4 and KH2PO4.
(ii) Wash the seeds with water and blot them dry.
(iii) Select an appropriate place in the laboratory where there is sufficient light. Arrange petridishes in three horizontal lines, with 5 dishes in each line. Arrange petridishes horizontally in three rows A, B and C with seven dishes in each row.
INVESTIGATORY PROJECT 1
(iv) Place one blotting paper disc in each petridish. (v) Wet the blotting papers with small quantities of buffer solution of 4.0 pH for petridish No. 1, 5.0 pH for petridish No. 2 and so on till all the blotting papers in petridishes No. 1–5 in row 'A' are wet with appropriate buffer solution. (vi) Repeat the process for rows B and C also. (vii) Spread selected seeds in each of the five petridishes in such a way that each petridish contains 25 seeds in row 'A'.
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(viii) Repeat the process for rows B and C using two other types of seeds.
(ix) Cover the petridishes and record your observation after every 24 hours for 7 days. Tabulate your results as given.
Observation
Observe the emergence of radicle as an indicator of germination and record in the table. Calculate the percentage of germination every day. Table: Percentage of ................ seed germination No and % of seed germination
pH 4 5 6 7 8
1
2
3
4
5
Total % of seed germination
6
7
A
B
C
Use the data of the table for graphic presentation.
Inferences and conclusion
• • • •
•
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The inferences and conclusion may be drawn on the basis of observations and the points given below
Find out at what pH range seeds of different species had maximum % of germination. Find out at what pH seeds failed to germinate or showed minimum % of germination. Is there any general pattern of seed germination with regard to pH ranges? What are the common features exhibited by the various types of seeds under varied pH ranges? For example, did all the type of seeds show maximum germination in acidic range or alkaline range or did pH preference varied between acidic and alkaline ranges? Did you observe any relationship among time period, seed germination and pH range?
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Investigatory Project
2
Aim: Quantitative analysis of phytoplankton in a water body
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Principle: The species composition and the density of the phytoplanktons determine the productivity status of a water body. Phytoplanktons are the principal producers in a water body. Based on the density of phytoplanktons, the water bodies are classified into nonproductive or oligotrophic and highly productive or eutrophic. Oligotrophic water bodies support only a few species, whereas eutrophic water bodies support large number of species. Further, the species composition of the phytoplanktons indicates the status of health of the water body. Through phytoplankton assays, limnologists make an estimate of degree of pollution in the water body. High density of cyanophycean algae, diatoms, volvocales, etc., are the indicators of high degree of pollution. It should be noted that density and the species composition of phytoplanktons exhibit diurnal, seasonal and annual fluctuations. It therefore becomes important to monitor water bodies at regular intervals for drawing specific conclusions related to their ecology. It is in this context, the procedure for phytoplankton analysis on qualitative (species composition) and quantitative estimation (density/unit area) is suggested for students who want to enter into fascinating realms of aquatic ecology. Objectives: After completing the project, the students will be able to 1. Plan out an experiment. 2. Identify and quantify phytoplanktonic forms present in an aquatic ecosystem. 3. Interpret the data and draw conclusions. 4. Recognise the indicator species of pollution.
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Requirements: Plankton net with plankton bucket, graduated plastic bucket (15 L), slides, cover slips, compound microscope, watch glasses, dropper, and 5% F.A.A (Formaldehyde Acetic acid, Alcohol)
Procedure (i)
Plankton net resembles the butterfly net in several aspects. Plankton net, however, is prepared from bolting silk cloth which is readily available at shops dealing in scientific equipments and chemicals. Procure about one metre of bolting silk cloth of 40 mesh size and stich out of it a 40 cm
INVESTIGATORY PROJECT 2
long cone with a diameter of about 20 cm at the mouth and a diameter of 3–4 cm at the other end (both the sides open). Fasten to the mouth of the cone a circular iron ring (with handle) of about 20 cm diameter with the help of twine thread. Fasten a small steel bucket (plankton bucket) or glass specimen tube of 50 ml capacity at the lower end. Visit a nearby pond, pool or river bank carrying along with you the plankton net fitted with the plankton bucket, graduated plastic bucket and 5–10 ml of F.A.A.
(iii)
Since this is a group activity, ask your friend to hold the plankton net firmly a few centimetres above the water surface.
(iv)
Immerse and fill the plastic bucket with water completely upto 15–litre – graduated mark and filter the water through the plankton net. Repeat the process several times (say10 times).
(v)
At the end calculate the quantity of water in litres (X) filtered by multiplying the amount of water in one bucket and number of buckets of water filtered.
(vii)
During this process of filtering, the planktons are collected in plankton bucket. Only the water free of planktons escapes through the mesh of the net.
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(ii)
Splash a few buckets of water against the net from outside taking care that no water enters into the cone from the mouth. This will wash all the planktons sticking against the inside wall of the net into the plankton bucket.
Detach the plankton bucket from the net and add a few drops (1–2 ml) of 5% F.A.A. to the plankton concentrate. Transfer the concentrate collected into a suitable specimen tube and cork it. Note the volume of the concentrate (Y).
In the laboratory
1. With the help of 1 ml pipette, draw 1 ml of concentrate and transfer it dropwise into the watch glass. Count the total number of drops that make 1 ml of concentrate (A).
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2. Transfer one drop of plankton concentrate from the watch glass on a clean slide. Cover it with square shaped cover slip. (For convenience divide the area of the cover slip into parts with the help of lines drawn by Indian ink).
Observation
Observe the slide under microscope and count the number of total organisms (B) by moving the slide from one corner of the cover slip to another horizontally as well as vertically till the entire sample under the cover slip is completed. With the help of following calculations find out the total number of different organisms per litre of water.
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L ABORATORY MANUAL: BIOLOGY
Unit cells/L = Unit/L =
1000×(A BY) x
Where A = number of drops in 1ml concentrate B = number of organisms counted in 1 drop of concentrate X = total amount of water filtered Y = total volume of concentrate after filtration
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Note: Another alternate method is the use of haemocytometer to calculate the density of organisms under the guidance of teacher.
Inferences and conclusion
Find out the density and composition of organisms in different water samples (polluted/non-polluted).
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Note the common organisms in both the water samples and those specific to each sample.
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© to N be C E re R pu T bl is he d Notes
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