PREFACE

The introductory biology course for which this book is the laboratory manual is now completing its sixth year. It was begun in a period of extraordinary changes, which have since developed further, and show no signs as yet of settling down. Just as the opening years of this century ushered in a revolution in physics, so we are now undergoing a revolution in biology; and that is transforming the entire scientific outlook, for biology no longer develops apart from chemistry and physics, but in intimate association with them. All three sciences come together in the attempt to understand the molecular structure and function of living organisms; and though this is by no means all of biology, or will ever be, it is the great new development. Morphology begins with molecular structure, physiology with chemical reactions; and these initial impulses, pursued to higher levels of organization, suffuse all biology. In the hierarchy molecule-cell-organism, if at times the molecule seems to be given special consideration, that is not because it takes any final precedence, but because its contribution to that trinity is the special task and contribution of our time, and because the molecule lies at the heart of the new unity of science.

In parallel with these great developments, we have been undergoing a fundamental revolution in American education. Six years ago we could say that its main seat was in the high schools; but by now the effects of that revolution have begun to permeate the colleges and graduate

schools. Indeed, by now the high schools are entering a period of consolidation in this regard; the center of experimentation and change has moved into the colleges. A great many colleges have basically revised their introductory courses, and during the last few years an entirely new literature has arisen to cater to this enterprise. As fast as introductory courses are revised, the more advanced curriculum has had to respond. We are witnessing a nationwide upgrading of instruction in biology, a usual feature of which is an integration in depth with chemistry and physics.

An important element in this process is that a new generation of biologists is emerging from the graduate schools, who have grown up in the new biology, and are much more familiar with its concepts and procedures than we ourselves were when we began. We dedicated the first edition of this book to the "new freshman" ; but now that "new freshman" is helping to teach our courses and indeed has helped to revise this manual.

Over these years our course has been given on a reasonably large scale, each year to 350-400 students, mainly freshmen and sophomores. One special feature of the course from the start has been that we mix indiscriminately students taking biology as a General Education course and science majors, and this remains one of its most interesting and rewarding aspects. A science course for General Education students alone labors under a formidable difficulty: there is not one committed person in the room, and that realization governs the students' attitudes. When one mixes General Education with science students, all students take on the commitment of the latter group.

The presence of the General Education students also does important things for the science students. Everything one teaches has to be justified there and then on its merits and not as preparation for future courses. One has to tell a story that hangs together and is reasonably complete on its level, and continually has to develop its relevance to other aspects of the culture. I have no doubt that this makes the best kind of introductory instruction for science majors also. If through some mischance we had to go back again to handling General Education and science students separately, I would change nothing in our present procedure, whichever group we ended with.

There was initially a pleasant surprise in this relationship that by now we count upon. At the beginning of the course the General Education students fear having to compete with the science majors, who are mainly premedical students. In the first hour quiz, given after about a month, the General Education students tend to come out a few points behind, so few as to encourage them. By the second quiz, given early in December, the General Education students have caught up, and thereafter do at least as well as the others. This induces in them a curious state of pride and elation that we can all enjoy.

We should in this connection say something of the preparation of our students. Our Department offers another one-semester introductory biology course, intended for students who have had a reasonably good biology course in school. Since we turn away about 100 students each year for lack of space, we try to send away those who seem best prepared to take the one-semester course. As a result we tend to draw our class, not from the top but from the bottom of the pile, as regards preparation in the sciences. This year their preparation has come out as follows: 2 percent of our students had no science in school; 24 percent had one year of science preparation; 38 percent, two years; 31 percent, three years; and 5 percent, four years. Sixty-five percent have had a course in biology; 85 percent have had chemistry; and 62 percent, physics. Forty-four percent have had a year of calculus, and 88 percent have had three years or more of mathematics.

Each student has one three-hour laboratory session weekly throughout two semesters. At Harvard this comes out to thirteen sessions each semester. Each laboratory section contains about twenty-five students, supervised by two graduate student assistants, and under the general supervision of one of the senior staff who is continuously available. In twenty-six laboratory sessions we do everything described in this book. It makes a keyed-up, busy laboratory, yet not a harrassed one. If at any point we thought the work of the laboratory was becoming too pressed for time, we would cut down on its content.

It may help place the laboratory work in perspective to know something of its relation to the lectures in our course. We have three one-hour lectures per week through two semesters. No attempt is made to synchronize the laboratory work with the lectures; each attempts to develop its own coherence. Nevertheless numerous points of correspondence and overlap develop between these two aspects of the course, and by the end lectures and laboratory tend to form a reasonably unified whole. Some idea of the content and sequence of the lectures can be gained from the outline of lecture topics that follows.

I. Origins of life (2 lectures): the cosmological setting, limiting conditions, evolution of metabolism (sets themes for first semester)

II. Elementary particles

1. Interconversions of matter and energy
2. Structure of the atomic nucleus
3. Nuclear transformations: origin of sunlight

III. Structure of the atom

1. Atomic orbitals; inert gases
2. Periodic system of the elements

IV. Chemical combination

1. Ion formation
2. Molecule formation: the covalent bond
3. Coordinate valence (the dative bond)
4. Properties of special elements: H, C, N, O
5. Properties of special molecules: H₂0, NH₃, C0₂, 0₂
6. Acids and bases
7. Weak binding forces: electrostatic, hydrogen bonds, van der Waals (hydrophobic) forces

V. Organic molecules

1. The major groups (hydroxyl, carbonyl, carboxyl, amino, sulfhydryl, etc.), developed with 2-carbon and 3-carbon examples
2. Isomerism; optical activity
3. Polar and nonpolar molecules; interfaces

VI. Biomolecules

1. Sugars, disaccharides, polysaccharides
2. Neutral fats; phospholipids
3. Amino acids
4. Nucleotides

(This entire treatment of molecules, beginning with the discussion of chemical combination, is "morphological." It is conducted entirely in terms of structural formulas. There is rarely an empirical formula in our discussions. The construction of three-dimensional models of the molecules in the laboratory is an important element in this instruction.)

VII. "The alphabet of organisms"

1. Four ultimate particles: protons, neutrons, electrons, photons
2. Seventeen to twenty bioelements: C, H, N, O; S and P; Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, Cl^-; the trace elements, Mn, Fe, Zn, Cu, Co (I, Mo, B, Al, V)
3. About 30 key organic molecules: glucose, ribose, deoxyribose, neutral fat, phospholipid, 20 amino acids, 5 nitrogenous bases for forming nucleotides
4. About 30 key organic molecules: glucose, ribose, deoxyribose, neutral fat, phospholipid, 20 amino acids, 5 nitrogenous bases for forming nucleotides

VIII. Macromolecules

1. Proteins
2. Nucleic acids
3. Nucleoproteins; viruses; bacteriophage

IX. Energetics of chemical reaction

1. Thermodynamics: free energy, heat of reaction, entropy
2. Temperature, molecular activation, and reaction rate

X. Enzymes and catalysis

XI. Cellular energetics

1. Fermentation
2. Respiration
3. Hexosemonophosphate (HMP) cycle
4. Photosynthesis

XII. Organization of the cell, microscopic and ultramicroscopic

XIII. Mitosis and meiosis

XIV. Classical genetics

1. Mendel's laws; linkage and crossingover; chromosome mapping
2. Sex determination
3. Heteroploidy and polyploidy; chromosomal balance

XV. Fine structure genetics

1. Recombination in bacteriophage
2. Protein and nucleic acid synthesis and coding
3. The molecular basis of mutation

XVI. Embryonic development

1. Fertilization and cleavage
2. The early embryo : vertebrate, in vertebrate, higher plants to seed formation
3. Differentiation a. induction b. nuclear changes c. nuclear-cytoplasmic relations

XVII. Endocrine control and hormones

1. General nature of hormonal action
2. Hormonal control of the sexual cycle in animals
3. Hormonal control of plant growth and development

XVIII. Physiological mechanisms: structure and function

1. The nervous system a. Nerve: structure, membrane po tentials, the nerve impulse, spontaneous activity b. Receptors: generator potentials c. Nervous integration: synapses, reflex arcs, organization of the spinal cord, autonomic nervous system, brain
2. Muscle a. Muscle structure and function b. The chemistry of muscular activity
3. Digestion a. The course of digestion; enzymes b. Absorption, transport of food c. Role of the liver
4. Osmotic and ionic balance a. Kidney structure and function b. Ionic composition of blood and tissues c. Regulation of pH
5. The blood a. Blood cells and plasma b. Immunological reactions c. Individuality: problems of transfusion and organ transplantation d. Respiratory pigments: transport of oxygen and carbon dioxide

XIX. Evolution and its mechanisms

1. Time scale of evolution; major events in animal and plant evolution
2. Mechanisms of evolution: natural selection, artificial selection, sexual selection
3. Biochemical evolution (For traditional discussion of mech anisms of evolution and phylogeny we rely principally upon the reading.)
4. The evolution of man

It may be helpful also to say something of our laboratory facilities. We have stand-up benches, with adjustable stools for when the student must sit. The benches are in double rows, back to back, with facilities and a drain running down the middle. The facilities at each place include water outlets (one equipped with an aspirator), electricity, and gas. At the end of each double pair of benches is a large sink, for washing up and other uses.

The stand-up benches are important. They do not prevent microscopy, which seems to go as well on high stools and benches as nearer the floor. On the other hand, our students are not fastened down. They move about a great deal during a laboratory session, talking with one another, seeing what other students are doing, frequently going to the blackboard to argue a point. This is of course just what we want. If one of our laboratory sessions seems inordinately quiet and orderly, we know that something is wrong and try to stir it up.

To assist instructors in setting up, we have appended lists of materials and apparatus at the end of each exercise. We reserve Monday afternoons on which no laboratory sessions are held, for setting up and going over the week's work with the graduate assistants. The exercises likely to present special problems-notably those in microbiology and electrophysiology - have detailed appendices that include information on sources of materials, apparatus, and prices. The prices are as of 1966 and are of course subject to change.

When we first began to prepare this course, we asked advice of many persons, and examined many other laboratory manuals. We should like to thank all those who generously contributed their advice and information.

We should like also to express our deep appreciation to the National Science Foundation, which through a generous grant of funds gave us the initial opportunity to explore the possibilities in this type of instruction far beyond what would otherwise have been possible.

We want to acknowledge also our debt to some of our Teaching Fellows who in the course of conducting laboratories have made important suggestions for improving them. They are too numerous to list; yet we should particularly mention Thomas E. Powell III (chromosome staining and frog development), John Menninger (transformation of B. subtilis), and Al Ruesink (action spectrum of plant phototropism).

It hardly needs saying that we need more help than we can provide. The present contents of this manual represent little more than work in progress. We are anxious to improve it, and would be most grateful to hear from any of our readers their criticisms, suggestions for improving the present experiments, and suggestions of new experiments.

Cambridge, 1966
G. W.

WHY A BIOLOGY LABORATORY?

A Foreword to the Student

Science is an attempt to understand reality. The questions we ask, and the answers, are put into words, and we try to give the words the clearest meanings we can. But they are no substitute for reality. They always fall short of saying what needs to be said. Even after one has learned to talk easily about nature in certain ways, after the words and phrases and concepts have grown familiar, the contact with the thing itself is always surprising. It has a quality of newness and freshness; one feels that for the first time one really understands - or, what is at least as good, that one has never understood at all - that the familiar words had been concealing mysteries. Often it looks as though something were being explained, when in fact it is only being named. A lot of scientific terminology is of this kind. It does well enough in a world of words, but fails immediately in a world of things.

Nowhere is this as true as in biology. The word "life" itself balks all attempts to define it. The trouble is that whateyer definitions of life we make are easily fulfilled with models that clearly are not alive. What we do about life is not define it, but recognize it. It would be an interesting experiment to see whether you could be fooled now; whether if we showed you a lot of different things, alive and dead, you would have trouble telling the one from the other.

In any case we hope you will do better after your experiences in this laboratory; better, not only in telling what is alive from what is dead, but in knowing what to expect of living things, what they do, how they behave, what they can tolerate, and what is likely to kill them. This is what biologists sometimes talk about as "the feel" of living organisms, something one gets only by living with them - by observing, playing with, and experimenting with them in their great variety, until one has developed intuitions of what kinds of things they do and don't do, and what one can do and not do with them. Scientists of all kinds-physicists, chemists, geologists, astronomers - are turning their attention to biology as never before; and this is a fine thing. Many biologists think, however, that what some of these visitors lack is just this "feel" for organisms. Sometimes they know the words, but make obvious mistakes or miss the point entirely, because they do not know living organisms and do not have useful intuitions about them.

Living organisms are made of molecules, and it is important not only to develop a'"feel" for the organisms, but equally for the molecules that compose them. They are for the most part a special group of molecules, made almost exclusively of carbon, hydrogen, nitrogen, and oxygen - so-called organic molecules. All of them are interesting, and all have special properties; but particularly the big ones, the proteins and nucleic acids, have qualities of their own that set them apart to a degree from all other molecules. They are at once the largest and most complicated molecules we know. Here again the words fail. It is only by preparing and handling them, by learning what they will tolerate by way of handling, and what destroys them, that we gradually acquire a "feel" for proteins and nucleic acids, just as one does for organisms. Indeed, the one greatly helps the other, for many of the basic properties of living organisms derive from their proteins and nucleic acids. Here again it is only long experience with these molecules in their great variety that develops the intuitions that give point and meaning to our concepts.

This is our aim in the laboratory, therefore - to make direct contact with living organisms and with the molecules that compose them. A great biologist, Louis Agassiz, the founder of the Harvard Museum of Comparative Zoology, is often quoted as having said, "Study nature, not books." The statement is a little foolish if taken literally; for one thing, you have just read it in a book. I think he really meant that we should do both, but wished to remind us that studying nature is a very different thing from studying books, and at times more reliable. In any case, our job in the laboratory is the study of nature itself.

We will pursue it there for its own sake, not merely to illustrate and amplify the content of the lectures. Indeed, laboratory work develops on its own, independently of the lectures; and you should approach it with this in mind. If something comes up in the laboratory that has not been mentioned at the lectures, as will happen regularly, master it then and there. We will try to help you in every way we can, but much of it is up to you. Know what you are doing in the laboratory at all times. No mistake would be as great as to go through a laboratory session in a state of confusion, hoping that some later lecture will clear it up. We hope that later lectures will make things clearer. In fact, we hope the whole course hangs together in that regard. But each laboratory experience must be met on its own terms, then and there.

One last word: your business in the laboratory is with living organisms and the molecules that compose them. This laboratory guide, your instructors, the instructors' questions, are all to

help deepen and enrich that experience. They are not objectives in themselves. Come to the laboratory as a scientist, to put questions directly to nature. Experiment and observe generously, not just what we suggest, but whatever interests you. Try to raise your own questions; we will appreciate them more than the ones we ask you. This is your opportunity to have a meaningful experience with a lot of things you may never have in your hands again. Make the most of it.

A few technical matters

Notebooks. Get a three-ring loose-leaf notebook for the laboratory and a block of unlined paper on which you can take notes. Note down whatever is essential in your experiments, in good English and in good order, so as to give a clear and connected account of what you have done, your observations, and the results of your experiments. Whenever a drawing helps, make one. The point is for it to be clear and informative, not necessarily beautiful.

Don't copy out sections of this laboratory guide into your notes. Whatever you need to describe, put into your own words. Answer all questions.

The notes may be in pencil or in ink. Drawings, of course, are better done in pencil. Do not use a soft pencil for either notes or drawings, since it smudges. A No. 3 pencil is of about the right hardness.

Preparatory reading. At the beginning of each exercise you will find references to textbooks and often also to Scientific American articles. These should be read before you come to the laboratory. Often it would be useful for you to have a textbook in the laboratory with you, but only for reference, not for extensive reading. Read the directions beforehand on the experiment you are about to undertake, and try to get a good idea of what you will be doing and in what sequence. The better prepared you are on coming to the laboratory, the more you will get out of it.

The books most commonly referred to in the preparatory reading are:

C. A. VILLEE, Biology, 4th Edition, W. B. Saunders Co., Philadelphia, 1962 (referred to hereafter as "Villee").

P. B. WEISZ, The Science of Biology, 2nd Edition, McGraw-Hill Book Company, New York, 1963 (referred to hereafter as "Weisz").

G. G. SIMPSON and W. S. BECK, Life, 2nd Edition, Harcourt Brace and World, Inc., New York, 1965 (referred to hereafter as "SimpsonBeck").

J. W. KIMBALL, Biology, Addison-Wesley Publishing Company, Reading, Mass., 1965 (referred to hereafter as "Kimball").

Numerous other books are referred to throughout the manual, where they are fully identified.

Scientific American articles are identified both by date of issue, and by the reprint numbers under which they can be purchased separately from W. H. Freeman and Company, 660 Market Street, San Francisco, Cal. 94104. A selected list of these articles, grouped by topic, is included in the Bibliography (pp. 171-175).

Equipment. You will need dissecting tools: 1 scalpel, 1 pair of scissors, 1 pair of forceps, 1 dissecting needle, represent a minimum set. Students going on in biology may wish to purchase high-quality instruments and more of them; a large and a small pair of forceps, for example, and a large and small pair of scissors. You may also want a laboratory apron.

Cleaning up. Leave the laboratory as you find it, or better still, as you wish you had found it. Wash any dirty glassware and other equipment with detergent or other cleanser, using brushes when needed. Then rinse each article at least five times, so that no soap whatever is left. Carelessness in rinsing may spoil a later experiment.

 

CONTENTS

1. Living Cells (1)A note on the compound microscope
2. Living Cells (2); Cell Models
3. Chemical Components of Cells: Macromolecules of Yeast and Their Subunits (1).
4. Chemical Components of Cells: Macromolecules of Yeast and Their Subunits (2)
5. Enzymes
6. Studies in Microbiology (1). Bacterial Growth; A Bacterial Enzyme; Comparative Biochemistry
7. Studies in Microbiology (2).Bacterial Mutation; Resistance to Antibiotics; Radiation Effects; Action of Lysozyme; Bacterial Anatomy
8. Studies in Microbiology (3).Genetic Transformation of Bacteria
9. Studies in Microbiology (4).Viruses: Their Identification, Mode of Reproduction,
10. Photosynthesis
11. Fermentation and Respiration
12. The Array of Living Organisms
13. A short guide to plant and animal
14. Vertebrate Anatomy
15. Organization of Higher Plants; The Transport of Sap
16. Blood and Circulation
17. Permeability and Active Transport: The Hamster
18. The Nerve Impulse
19. Muscle
20. Electrical Activity of a Sense Organ: The Limulus Eye
21. Plant Growth and Tropisms; Carbon Dioxide Fixation and Translocation of Plant Substances
22. Introduction to the Genetics of Man and the Fruit Fly; Regeneration of Planaria
23. Fertilization and Early Development; Continuation of the Genetics Experiment
24. Development of the Chick; Giant Chromosomes of Drosophila; Continuation of the Genetics Experiment
25. Completion of the Genetic Experiment
26. Sensory receptors

Appendix A. Outline for the Instructor on the Preparation of Microbiological Experiments (Exercises VI-IX)

Appendix B. Notes to the Instructor on the Electronic Equipment used in Exercise XVII

Appendix C: Alternative Experiment on Chemical Components of Cells: The Biochemistry of Milk.

Appendix D: Film Loops

Appendix E: Exponents and Logarithms

Appendix F: The Periodic System of the Elements

Appendix G: Table of Atomic Weights

Bibliography