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BIOLO

An Introductory
iaboralory Manual

V\/ALD • ALBERSHEIM • DOVS/LIN

ass^^^^s^^s^E

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Marine Biological Laboratory Library

Woods Hole, Mass.

Presented by

Dr. George Wald
Sept. 18, 1962

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TWENTY-SIX AFTERNOONS OF

BIOLOGY

AN INTRODUCTORY LABORATORY MANUAL

TWENTY-SIX AFTERNOONS OF

BIOLOGY

GEORGE WALD
PETER ALBERSHEIM
JOHN DOWLING
JOHNS HOPKINS III
SANFORD LACKS

Harvard University

ADDISON-WESLEY PUBLISHING COMPANY, INC.

READING, MASSACHUSETTS • PALO ALTO • LONDON

ADDISON-WESLEY PUBLISHING COMPANY, INC.
Printed in the United States of America

All rights reserved. This book, or ports thereof, may
not be reproduced in any form without written per-
mission of the publisher.

PREFACE

The introductory biology course for which this
book is the laboratory manual comes in a period
of extraordinary changes. On the one hand, we
are undergoing a revolution in biology, which for
the first time is approaching its problems sys-
tematically at the molecular level. With the
emergence of biology at this level, already occu-
pied by chemistry and physics, science as a whole
has achieved a new unity.

At the same time we are undergoing a funda-
mental revolution in American education. Its
main seat is not in the colleges, but the high
schools. Exhortations, threats, normal internal
developments, improved economic conditions,
federal programs for retraining teachers, science
fairs and competitions, and probably most im-
portant of all, the advanced placement program-
all these have had their effect, and students
now enter college in a very different condition
from what obtained just a few years ago. Many
of them know much more science and mathe-
matics than they ever did before. Indeed, many
of them have gone far past what we taught
juniors and seniors in the colleges only a few
years ago.

What is much more important is that high-
school students quite generally have developed a
new eagerness to learn and understand science.
The glamor that used to go with athletic achieve-
ment seems largely now to be accorded scientific
achievement. The elation that students used to
derive from working their muscles, many now
seem to achieve also by working their heads.
Surprisingly learned, eager, responsive, deeply
interested — this is the new college freshman.
This book is dedicated to him.

The course for which this book is the labora-
tory guide has been given on a reasonably large
scale to approximately 350 students, mainly
freshmen and sophomores, about evenly divided

between general education students and those
intending to concentrate in the sciences, mainly
premedical students. No distinction whatever is
made in handling these two groups together; and
it is noteworthy that after a short initial lag, the
general education students keep up thoroughly
with the others.

Each student has one three-hour laboratory
session weekly throughout two semesters. At
Harvard this comes out to thirteen sessions per
semester. Each laboratory section contains
about twenty-five students, supervised by two
graduate student assistants, and under the gen-
eral 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 labora-
tory work with the lectures; each attempts to
develop its own logic. Nevertheless numerous
points of correspondence and overlap develop
between these two aspects of the course, and by
its end lectures and laboratory tend to form a
reasonably unified whole. Some idea of the con-
tent and sequence of the lectures can be gained
from the outline of lecture topics that follows.

I. Origin of life (2 lectures)
II. Ultimate particles

1. Interconversion of matter and energy

2. Structure of the atomic nucleus

3. Nuclear transformations: origin of
sunlight

vi PREFACE

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. Hydrogen bonds

5. Van der Waals forces

6. Polar molecules: surface forces, the
association of water

V. Organic molecules

1. Special position of C, H, N, O

2. The major groups (hydroxyl, car-
bonyl, carboxyl, amino, sulfhydryl,
etc.)

VI. Biomolecules

1. Sugars, disaccharides, polysaccha-
rides

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 con-
struction of three-dimensional models of the
molecules in the laboratory is an important ele-
ment 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 36 key organic molecules:
glucose, ribose, deoxyribose, neutral
fat, phospholipid, 20 amino acids, 5
nucleotides

VIII. Macromolecules

1. Proteins

2. Nucleic acids

3. Nucleoproteins; viruses; bacterio-
phage

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

5. Chemoautotrophy: the nitrogen cycle

XII. Organization of the cell, microscopic and
ultramicroscopic

XIII. Mitosis and meiosis

XIV. Classical genetics

1. Mendel's laws; linkage and crossing-
over; chromosome mapping

2. Sex determination

3. Heteroploidy and polyploidy; chro-
mosomal 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

PREFACE vi

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, spon-
taneous activity

b. Receptors: generator potentials

c. Nervous integration: synapses, re-
flex 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 transfu-
sion 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. Our introductory labora-
tories formerly had been furnished only with very
low tables, bearing only microscope lamps, sup-
posed to facilitate long hours of microscopy, and
incidentally to keep the students fixed in posi-
tion. This kind of thing has been more or less
standard laboratory furniture for biology courses
in the past.

In the present course 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 in-
clude 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 an-
other, 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 ap-
pended lists of materials and apparatus at the
end of each exercise. We reserve one afternoon
per week, on which no laboratory sessions are
held, for setting up and going over the week's
work with the graduate assistants. It will prob-
ably not surprise the readers of this manual to
learn that a number of the exercises involve
procedures that were new to most of our grad-
uate students, and indeed to most of the staff
including the professor in charge. This is a
symptom of what it means to be teaching the new
biology.

PREFACE

The exercises likely to present special prob-
lems— notably those in microbiology and elec-
trophysiology — have detailed appendices that
include information on sources of materials,
apparatus, and prices. The prices are as of
1961-62 and are of course subject to change.

When we first began to prepare this course,
we asked advice of many persons, and ex-
amined 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 ap-
preciation to the National Science Foundation,
which through a generous grant of funds gave us
the opportunity to explore the possibilities in
this type of instruction far beyond what would
otherwise have been possible. We should like
particularly to acknowledge our indebtedness
to Dr. Bowen C. Dees, Assistant Director of the
Division of Scientific Personnel and Education
of the N.S.F., and to Dr. Charles A. Whitmer,
Head of the Course Content Improvement Sec-

tion. The help we have received from the
National Science Foundation implies a public
obligation which we gladly accept and are
anxious to fulfill. We shall be glad to help in
any way we can with the use of this manual
and the institution of this type of instruction in
biology.

It hardly needs saying, however, 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 im-
proving the present experiments, and suggestions
of new experiments.

Cambridge, 1962

G. W.
P. A.
J. E. D.
J. H.
S. L.

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 sub-
stitute for reality. They always fall short of
saying what needs to be said. Even after one
has learned to talk easily about nature in cer-
tain ways, after the words and phrases and con-
cepts 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 whatever 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. Scien-
tists of all kinds — physicists, chemists, geolo-
gists, 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 intui-
tions 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 ex-
clusively of carbon, hydrogen, nitrogen, and
oxygen — so-called organic molecules. All of
them are interesting, and all have special proper-
ties; 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

X A FOREWORD TO THE STUDENT

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 mean-
ing to our concepts.

This is our aim in the laboratory, therefore —
to make direct contacts with living organisms
and with the molecules that compose them. A
great Harvard biologist, Louis Agassiz, the
founder of the Museum of Comparative Zool-
ogy, 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 hap-
pen 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 direct
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 note-
book 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 informa-
tive, 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. Draw-
ings, 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 labora-
tory. 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 com-
ing to the laboratory, the more you will get out
of it.

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

A FOREWORD TO THE STUDENT xi

The Science of Biology, by Paul B. Weisz,
McGraw-Hill Book Co., 1959 (referred to here-
after as "Weisz").

Bioliogy. by Claude A. Villee. W. B. Saunders
Co.. 1962 (referred to hereafter as "Villee").

Life, by G. G. Simpson, C. S. Pittendrigh,
and L. H. Tiffany, Harcourt, Brace and Co.,
1957 (referred to hereafter as "S.P.T.").

Numerous other books are referred to through-
out the manual, where they are fully identified, as
are others listed in the Bibliography at the back
of the manual.

Scientific American articles are identified both
by date of issue, and for the benefit of those who
have access to the reprints issued by W. H.
Freeman and Company, by reprint number.

Equipment. You will need dissecting tools:
1 scalpel, I pair of scissors, 1 pair of forceps,
I dissecting needle, represent a minimum set.
Students going on in biology may wish to pur-
chase 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 what-
ever is left. Carelessness in rinsing may spoil a
later experiment.

I. Living Cells (1) 1

A note on the compound microscope 3

II. Living Cells (2); Cell Models 7

III. Chemical Components of Cells: Macromolecules of
Yeast and Their Subunits (1) 12

IV. Chemical Components of Cells: Macromolecules of
Yeast and Their Subunits (2) 21

V. Enzymes 25

VI. Studies in Microbiology (1). Bacterial Growth; A Bac-
terial Enzyme; Comparative Biochemistry .... 33
VII. Studies in Microbiology (2). Bacterial Mutation; Resist-
ance to Antibiotics; Radiation Effects; Action of Lyso-
zyme; Bacterial Anatomy 38

VIII. Studies in Microbiology (3). Genetic Transformation

of Bacteria 43

IX. Studies in Microbiology (4). Viruses: Their Identifica-
tion, Mode of Reproduction, and Filterability ... 47

X. Photosynthesis 50

XI. Fermentation and Respiration 54

XII. and XIII. The Array of Living Organisms .... 57

A short guide to plant and animal classification . . 65

XIV. Vertebrate Anatomy 69

XV. Organization of Higher Plants; The Transport of Sap . 74

XVI. Blood and Circulation 80

XVII. Permeability and Active Transport: The Hamster Gut . 86

XVIII. The Nerve Impulse 91

XIX. Muscle 99

XX. Electrical Activity of a Sense Organ: The Limulus Eye . 1 04
XXI. Plant Grov/th and Tropisms; Carbon Dioxide Fixation

and Translocation of Plant Substances 1 09

XXII. Introduction to the Genetics of Man and the Fruit Fly;

Regeneration of Planaria 114

XXIII. Fertilization and Early Development; Continuation of

the Genetics Experiment 1 20

XXIV. Development of the Chick; Continuation of the Genetics
Experiment 123

XXV. Completion of the Genetics Experiment .... 1 26

XXVI. Sensory Receptors 1 29

Appendix A. Outline for the Instructor on the Preparation for

Microbiological Experiments (Exercises VHX) . 135

Appendix B. Notes to the Instructor on the Electronic Equip-
ment Used in Exercise XVIII 141

Appendix C. Supplementary Experiments on Chemical Com-
ponents of Cells: The Biochemistry of Milk . . 143

Appendix D. Exponents and Logarithms 1 49

Appendix E. The Periodic System of the Elements . . . 151

Appendix F. Table of Atomic Weights 152

BIBLIOGRAPHY 154

xiii

CONTENTS

80218

LIVING CELLS (I)

(Readings: Weisz, pp. 55-67. S.P.T., pp. 39-58 and 488^98. Villee, pp. 35-^2.
J. Brachet, "The Living Cell," Sci. Am. 205, No. 3, pp. 50-62, Sept. 1961,
Reprint No. 90.)

The cell is the minimum organization that
displays the properties and processes that we
refer to collectively as "life." We know life only
in the form of living cells. They are called
"cells" because each is enclosed in a continuous
boundary, the cell membrane, and sometimes
also a cell wall.

Some living organisms are composed of a
single cell; others are multicellular. A multi-
cellular organism may be composed of many
different types of cell, each type playing a dif-
ferent role. A group of similar cells, specialized
for a single type of function, is called a tissue.
Between the unicellular and the multitissued
organisms, we find a few multicellular forms
that, because all the cells are of much the same
type, we speak of as "colonial."

Most living organisms can be characterized
clearly as plant or animal, though one tends
now to recognize a third great kingdom, that
of the protists, which includes all unicellular
and colonial forms. Typical plant cells are
characterized by a rigid cell wall, made largely
of cellulose, and may contain organs of photo-
synthesis, the chloroplasts. Both plant and ani-
mal organisms may be uni- or multicellular, or
colonial. Among the unicellular or colonial

forms, in addition to those that are clearly
plant (algae) and clearly animal (protozoa), there
is an interesting group that does not fit easily
into either category — the green flagellates.

We shall devote the first two laboratories to
examining a variety of living cells and what
they do. You will see that though they look
very different from one another, they share
many properties in common. Toward the end
of the second laboratory, we will try to mimic
some of their activities with simple inorganic
models. The cells are made of molecules, much
the same types of molecules in all living cells.
Immediately after this work with living cells, we
shall go to work with the molecules. It is a
striking fact that the chemistry of living organ-
isms varies much less than their anatomy.

CELLS OF MULTITISSUED ORGANISMS
Onion epidermis

Remove a fresh inner scale from an onion.
With a scalpel and forceps strip off a layer of
the epidermis from the inner side of the scale.
Mount a piece in tap water on a slide, and with

2 LIVING CELLS (1)

Exercise I

forceps place a cover glass over it, putting one
edge of the cover glass down first and then
letting the other end down slowly so as to drive
out all bubbles of air. Examine under the low
power of the microscope. The epidermal cells
of the onion are typical plant cells in that they
consist of (1) a cellulose cell wall, (2) a thin
layer of cytoplasm which lines the cell wall,
(3) a nucleus, and (4) a large central vacuole.
Observe as many features of the living cell as
you can. Stain a piece of this tissue with aceto-
carmine. This is a dye which stains basic pro-
teins red, made up in 45% acetic acid which
coagulates protoplasm (like cooking an egg).
Sketch one cell, showing cell wall, cytoplasm,
vacuole, and nucleus.

Human epidermis

Having seen some onion skin under a micro-
scope, you may enjoy seeing your own. With a
reasonably clean fingernail, or the blunt end of
your scalpel, scrape the inside of your cheek
lightly. Stir the scrapings into a drop of tap
water on a slide, cover with a cover slip, and
find the cells under low power. They will
appear as small masses of colorless, granular
material. Under high power, study their struc-
ture. This is a large, flat type of cell (squamous
epithelium) that, as in the onion, forms tissue
surfaces. Individual cells are best seen at the
margins of a group. Note the cell membrane,
junctures with neighboring cells, the granular
cytoplasm, and the small, rounded, highly re-
fractile nucleus, itself surrounded by a mem-
brane. Sketch one cell. Compare the cell mem-
brane with that of the onion cell; this is one of
the features by which one distinguishes plant
and animal cells.

Highly specialized cells: the Elodea leaf

Elodea is a flowering plant that grows in
fresh water. Pluck a young leaf and mount it
whole in tap water, top side up, under a cover
slip. Under low power find a group of elongated
cells near the midrib and toward the base of the

leaf. The green structures are chloroplasts.
They are the organs of photosynthesis, and the
green chlorophyll and other pigments they con-
tain absorb the light used in this process. In
some cells you can observe a circulation of the
protoplasm (cyclosis). If you find this, try the
effect on it of changing the brightness of the
light. Make a sketch of a single cell, showing
the relation to its neighbors, and as much of
the internal structure as you have seen.

You have already seen an example of the
cellulose wall that typically encloses a plant cell,
and of the delicate surface membrane that sur-
rounds the cytoplasm of all living cells, the
so-called plasma membrane. We shall take this
opportunity to demonstrate the relationship be-
tween these structures in Elodea cells. When
such a plant cell is laid in a salt solution more
concentrated than its own cytoplasm, the salt
solution draws water out of the cell, causing it
to shrink away from the rigid cell wall, so ex-
posing the plasma membrane.

Mount a whole Elodea leaf as above, and
when you have a good field of cells in focus
under the microscope, replace the tap water by
a concentrated solution of sodium chloride
(2 M). This is done by using a medicine dropper
to place a single drop of the salt solution on the
slide beside the cover slip, just making contact
with its edge. Then touch the margin of liquid
at the opposite side of the cover slip with a
small piece of lens paper, so that the lens paper
draws up the liquid, sucking the salt solution
under the cover slip to replace the liquid you
have withdrawn. When you have done this
two or three times, the liquid under the cover
slip will have been completely replaced by the
salt solution. This is the general method used
for changing solutions under a cover slip, so
that their effects can be observed as the change
progresses.

Potato

Cut a thin slice from a freshly cut surface of
a potato tuber. Lay the section in a drop of
water on a slide and examine under low power.

Exercise I

LIVING CELLS (1) 3

Stain by adding a drop of iodine-potassium
iodide solution (I2 + KI, 0.01 A/ each). Cover
with a cover slip and look for the deep purple
color that indicates the presence of starch.

COLONIAL ORGANISMS

Spirogyra, a green alga

Spirogyra is commonly referred to as pond
scum. It tends to float on the surface in many
fresh-water streams and ponds and is recognized
by its bright green color and slippery feeling.
Place a few filaments of Spirogyra on a slide in
a drop of water. Cover with a cover slip. Under
low power select a group of cells with regular,
spiral, green chloroplasts. Examine under high
power. Note the pyrenoids in the chloroplast.
These are associated with starch formation.
Where is the nucleus and how is it held in place?
Sketch a cell showing the various structures
you see.

Test for starch by adding to a strand of
Spirogyra on a slide a drop of iodine-potassium
iodide solution as above. Note on your sketch
the structures that stain most deeply with
iodine.

Volvox, a "colonial" green flagellate

A Volvox colony may contain several thou-
sand cells, embedded at the surface of a gela-
tinous sphere, with the flagella — two per cell —
directed outward. The cells are interconnected
by delicate strands of protoplasm. (You will see
the arrangements better if you stain by drawing
a drop of methylene blue under the cover slip.)
Moreover the cells vary in size, shape, and func-
tion. For both reasons this is more than a
simple collection of cells; it represents a genuine
approach to a differentiated, multicellular or-
ganism. Sketch a colony. Do not try at this
time, however, to see very much of the structure
of individual cells. You will be able to do that
better with closely related unicellular green
flagellates.

OTHER ALGAE

Your instructor may also have other algae in
the laboratory for optional study, perhaps an
example of such a stonewort as Nitella; perhaps
a diatom, one of the golden algae; or a desmid;
or such a unicellular green flagellate as Chlamy-
domonas.

What is the simplest cell you have seen today?
What would you say of the simplicity of the
organism of which it is a part? The complexity
of a multitissued organism is achieved through
the specialization, and concomitant simplifica-
tion, of its individual cells. Does specialization
always imply simplification?

A NOTE ON THE
COMPOUND MICROSCOPE

Realms of dimension

In a development that stretched over nearly
three centuries, the compound microscope
brought biologists into a world of new dimen-
sions. Their dissections had previously been
concerned with the gross anatomy of tissues and
organs. Now they could penetrate to cellular
anatomy. This involved a leap in dimensions
of about 1,000 times, roughly from the level of
millimeters to that of microns (10~^ cm). A
cell in a multicellular plant or animal is usually
one to several microns in diameter, though some
algae are enormously larger, and bacteria in
general very much smaller. The limit of resolu-
tion in visible light, that is, the separation at
which two points in the object are seen as two
rather than as a single blob, is about 0.2 micron.
No details finer than this can ordinarily be dis-
tinguished, no matter how fine the instrument.

Recently the electron microscope has per-
mitted a further leap in dimensions of approxi-
mately another 1000 times, from microns to
millimicrons [1 mju = 10~' cm = 10 angstrom
units (A)]. This has brought us from micro-
scopic to ultramicroscopic anatomy, from the

4 LIVING CELLS (1)

Exercise I

anatomy of cells to that of subcellular particles.
It has also made the larger molecules visible,
for 10 angstrom units corresponds to the diam-
eter of a rather small protein. The larger pro-
teins and the nucleic acids and viruses can readily
be distinguished under the electron microscope,
and at times even identified by their characteris-
tic shapes.

To go further requires radiations of still
shorter wavelengths (x-rays). Methods of x-ray
diffraction, simple in principle though compli-
cated and laborious in practice, permit us to
determine the positions of the individual atoms
in molecules. Here the limit of resolution is a
fraction of an angstrom unit. With such meth-
ods the characteristic distances and angles be-
tween the atoms in molecules can be determined.

This is the last reach of anatomy. It is not
only that biological interest does not penetrate
further; that might change with time. It is
rather that beyond this point, anatomy becomes
indeterminable. Particles smaller than atoms are
subject to the limitations of physical indeter-
minacy; it is impossible to assign definite
meaning to their individual spatial relations.

So the domain of biological anatomy stretches
over an enormous range, from gross anatomy
to molecules, from centimeters to angstrom
units, from the anatomy of the dissecting pan
to that of x-ray crystallography. There are no
sharp boundaries; and nowadays biologists
must be concerned with the entire continuum.

Use of the microscope

A compound microscope is a delicate and
expensive instrument. Treat it tenderly. Don't
begin by twiddling knobs, and then trying to
find out what you twiddled. Find out what to
do before you do it. Your instructor, charts,
booklets, this outline — all will help.

The following summary of practical directions
will get you started :

(1) Pick the microscope up by its arm. Don't
let it knock against anything, and set it down
gently to avoid jarring its parts out of line.

(2) Place the microscope on your desk with
the arm toward you. Identify the ocular (eye-
piece), nosepiece and objectives, the body tube,
the coarse and fine adjustments for focusing,
the stage, iris diaphragm, condenser (if present),
and the mirror — one face of which is plane,
the other concave — which reflects light through
the opening in the stage into the objective.

(3) Revolve the nosepiece to bring the low-
power (the shorter) objective into line with the
body tube. A spring catch engages the nose-
piece to hold each objective in its correct posi-
tion.

(4) Adjust the mirror so that the concave side
reflects light from your microscope lamp through
the opening in the stage into the objective. If
the light is too intense, close down the iris
diaphragm until the brightness suits you.

(5) Always look at the object first under low
power. Place a slide on the stage with its ends
held by the spring clips (or in a mechanical
stage), and position it so that the part you want
to study lies at the center of the opening. Lower
the tube until the bottom lens of the objective
is about a quarter-inch above the slide. Now,
while looking through the ocular, focus slowly
upward with the coarse adjustment, until the
image is visible. Continue to focus up and down
with the fine adjustment until the image is
sharpest.

(6) To use the high-power objective, first
focus the object under low power and move the
slide until the detail you wish to examine under
higher magnification is almost exactly centered
in the field. Turn the nosepiece slowly to swing
the high-power objective into line. Watch
meanwhile from the side, to see that this longer
objective does not strike the slide or cover glass.
Now focus slowly upward with the fine adjust-
ment (the coarse adjustment is never used with
the high-power objective). If no image is seen,
carefully lower the objective until it almost
touches the slide, then focus upward again.
The point is to avoid any possible damage to
the objective by permitting it to touch the slide.
Finally, bring the image into sharpest focus by
playing back and forth with the fine adjustment.

Exercise I

LIVING CELLS (1) 5

THE COMPOUND MICROSCOPE

Ocular:

{5X or 10X)

Coarse adjustment knob

Fine adjustment
knob

Objectives: high
power (4 mm
40 X, and low
power (16 mm), 10 X

Condenser with
iris diaphragm

Mirror: one
side concave,—
the other flat

Base

(7) When examining permanent slides, you
may find it more comfortable to tilt the micro-
scope toward you, at an angle with its base.
For work with fresh preparations, always keep
the stage of the microscope horizontal.

(8) It is good practice to keep both eyes open
when looking through the microscope. You
will soon learn to disregard the image from the
"off" eye, and so will avoid the strain of holding
that eye closed.

(9) Dirty lenses give poor results. Clean them
only with the special lens paper which is pro-
vided, not with your handkerchief, Kleenex, or
anything else. If after it is gently wiped with

lens paper your microscope still does not yield
a clear image, ask your instructor to help.

(10) The magnification of any combination of
objectives and oculars is the product of the
magnifications of the separate components.
Thus a lOX ocular combined with the low-
power, lOX objective yields a total magnifica-
tion of 100 diameters. Each of the objectives
is marked with its magnification and the dis-
tance from the object at which it yields an
approximate focus. The low-power objective is
in focus at about 16 millimeters above the object,
the high-power (44 X) objective at about 4 milli-
meters above the object.

6 LIVING CELLS {!)

Exercise I

(11) By moving your slide about gently while
it is in focus, try to get used to the fact that the
microscope reverses as well as magnifies every
motion. In a little while you should have this
under control. Also, in fresh preparations sus-
pended in a liquid medium, do not be surprised
to find all very small particles engaged in a con-

tinuous, random motion. This is called Brown-
ian movement. It is caused by the fact that
all objects suspended in a fluid medium are
continuously bombarded by the molecules all
about them; and sufficiently small particles
are continuously knocked about by this bom-
bardment.

EQUIPMENT

Throughout the manual these lists and the instructions which often
accompany them are included for the use of the instructor.

Per student

compound microscope

Per 8 students

slides and cover slips

onion

potato

Elodea

cultures of Spirogyra

Volvox

other algae

iodine and potassium iodide in water (0.01 M each)

methylene blue solution

sodium chloride solution (2 M)

Per laboratory

charts on the compound microscope

LIVING CELLS (2); CELL MODELS

UNICELLULAR ORGANISMS
Paramecium, a ciliate

This is one of the commonest fresh-water
protozoa. It is found in many pools, where it
feeds on bacteria which, in turn, feed on decay-
ing vegetation. The ciliates are the most com-
plexly organized protists, at the opposite ex-
treme from the Rhizopods. Indeed, because
they present such a remarkable differentiation
of structures and activities, it is hard to remem-
ber that these organisms are single-celled. In
deference to their unicellularity, we speak of
their organs as "organelles."

Put a small drop of Paramecium culture on a
slide, and add about an equal drop of 4%
methyl cellulose. Making the medium viscous
will slow down the Paramecia so that you can
observe them more easily. Carefully cover with
a cover slip, supported on small broken pieces
of another cover slip, and study under low and
high power. Note the minute, whiplike cilia,
whose regular, synchronized beat propels the
animal through the water. Note the differences
in length of cilia in different regions of the body;
where are they longest?

Observe the two clear pulsating structures,
the contractile vacuoles, near each end of the
body. What functions do they serve? What
human organ performs analogous functions?

Note that the Paramecium is asymmetrical.
Note its "mouth," a groove or depression lead-

ing to a funnel-shaped gullet, at the end of
which food vacuoles form.

Unicellular organisms ordinarily reproduce by
fission, whereby a mature cell divides to form
two equivalent daughter cells. Each individual
could in this way become the origin of an
immortal line, perpetually renewing itself by
repeated division. If reproduction were perfect,
that would do well enough, but as in any com-
plicated form of life, aging processes occur. The
genetic material of all cells is subject to random
changes, called mutations. Aging is in part the
result of the accumulation of mutations, which
are usually deleterious.

For this reason there is great advantage in
some arrangement that permits the individuals
of any stock of organisms to mix their genetic
material from time to time, so that out of all
possible combinations, individuals emerge that
possess particularly advantageous constellations
of genetic characters. Sexual reproduction is
such a device for regularly mixing genetic ma-
terial. Such ciliates as Paramecium, though
usually reproducing by fission, at times inter-
polate another process, a form of sexual repro-
duction, called conjugation. Two mature Para-
mecia']o\n together side-to-side, exchange genetic
material, separate, and then resume asexual
reproduction by fission (see S.P.T., pages 491-
492).

Identical Paramecia ordinarily do not con-
jugate. Conjugants, even though they may have

8 LIVING CELLS (2)

Exercise

developed in the same culture, are heritably
different from each other. We speak of such
different strains as different mating types. At
least 28 mating types are now known. These
occur as 14 complementary pairs. Fertile con-
jugations occur only between individuals of
complementary mating types.

We shall examine conjugation in two comple-
mentary mating types of Paramecium aurelia.
Types XIII and XIV. Place 3 drops of Type
XIII culture in the left depression and 3 in the
center depression of a 3-depression glass slide.
Place 3 drops of Type XIV culture in the right
and center depressions. The right and left
depressions will serve as controls for what hap-
pens in the center depression, where both types
have been mixed.

When two mating types are compatible, as
are these, the individuals first clump, their cilia
sticking together. At this stage a narrow space
can still be seen between them. After a short
time, the Paramecia pair off, the mating indi-
viduals uniting side by side, and the pellicles
fusing. It is at this stage that exchange of
genetic material (haploid gametic micronuclei)
takes place. Conjugation goes on for several
hours. Then the mating individuals separate,
and each resumes reproduction by fission.

After you have observed the original clump-
ing, set the slide aside and go on with the rest
of the exercise. After about an hour, a few
pairs should have separated. Find such pairs
and make a quick sketch of what you can see.

Euglena, a green flagellate

Place a drop of Euglena culture on a clean
slide. Add a drop of 4*^ methyl cellulose.
Cover with a cover slip, and observe, first under
low, then under high power. Note the whiplike
flagellum (often better seen by dimming the
light), the chloroplasts, and the eye-spot, which
seems to be a genuine light-receptor, guiding
the motion of the organism toward or away
from the light. Sketch one cell and its parts.

The green flagellates are hard to classify.
Zoologists include them among the one-celled

animals, the protozoa; botanists among the
algae; or both avoid the issue by calling them
"protists." What are their plant, and what
their animal characteristics? One characteristic
of typical plants is that they can incorporate
inorganic nitrogen (nitrates, ammonium salts),
whereas animals require their nitrogen in or-
ganic form (e.g., amino acids). On the basis
of this criterion, how would you set up an
experiment to classify Euglena ?

Ameba, a Rhizopod (Sarcodine)

This is the famous protozoan whose name
has become a household word for the simple
and listless among living organisms. Put a drop
of ameba culture on a clean slide, and observe
with the naked eye against a dark background.
The amebas can be seen as whitish dots about
as large as a pinpoint. Look at them under low
power, without a cover slip. See how they move
by means of outward bulgings of the cytoplasm
(pseudopodia = "false feet"). Watch carefully
the formation of a pseudopod, and the asso-
ciated flow of cytoplasm. Distinguish in the
cytoplasm a clear outer layer, an inner, granular
mass, and the single, ovoid nucleus. Make a
series of 6 outline sketches of a progressing
ameba, recording the time of each sketch.

For study under high power, it is preferable
to use a cover slip. So that this will not crush
the ameba, place four bits of broken cover slip
around the drop of culture, and set the cover
slip on these. Under high power observe the
granules, food vacuoles, nucleus, contractile
vacuoles, and pseudopodia.

It must be plain to you now that the ameba
has been maligned. It is not as simple as it
looks. It packs more into a small space than
anything yet designed by man. It can move,
react to stimuli, reproduce, adapt to the environ-
ment (how?), ingest solid food, excrete waste,
and regulate its water content. How do you
manage to do more? With all that churning up
of its contents, how does the ameba keep its
functions sorted out and balanced ?

Exercise

LIVING CELLS (2) 9

Most amebas are free-living, but one notori-
ous human parasite in this group is Endameba
histolytica, which causes amebic dysentery. It
is easy to think of such Khizopods as the ameba
as primitive and as ancestral to other types of
protozoa; but an interesting argument considers
the flagellates to be the most primitive protists,
and derives the ameba from them (cf. Weisz,
pp. 664-667).

Reactions of Paramecium to its environment

To another drop of Paramecium culture on a
fresh slide, add a small drop of a suspension of
powdered carmine before adding the methyl
cellulose. Observe how the granules of carmine
accumulate in a food vacuole at the end of the
gullet, which is pinched off and then pursues a
definite course around the cell. Follow one
such vacuole through its circuit. Unlike the
ameba, Paramecium has a special area which
serves for the egestion of solid wastes. This
anal spot is on the surface, about level with the
posterior end of the gullet. It can be detected
only during the act of egestion. You may be
able to see the elimination of carmine particles
through the anal spot. {Note: If the methyl
cellulose does not slow the organism sufficiently
for these observations, use in addition or instead
a bit of lens paper laid in your preparation.)

Paramecia propel barbed, harpoonlike tricho-
cysts when disturbed. Place a large drop of
Paramecium culture on a slide, and put a very
small drop of ink next to it but not touching it.
Now bring the two drops into contact, put a
cover slip over them, and quickly examine
under low power. Note what happens when a
Paramecium swims into a blue zone.

In a fresh preparation made up with lens
paper and not containing methyl cellulose, note
how Paramecium reacts to obstacles. Do you
see what is meant by its "trial-and-error" be-
havior? Can a Paramecium back up? How do
you suppose it does so? How does it synchronize
and integrate the beating of its cilia? Has it a
nervous system? Of what could a subcellular
nervous system be composed ?

Other protozoa and algae

Your instructor will also have available some
pond water containing protozoa and algae other
than those already studied. We are not inter-
ested in identifying them except in the roughest
way; but they are interesting to find and watch,
and to assign to the major groups. How many
different kinds do you see? Are organisms pres-
ent that are not unicellular? Of what types?
(Your instructor and reference books will help
you answer these questions.)

You have now seen a wide range of living
cells, from the comparatively simple ones to the
exceedingly complex. With simplicity and com-
plexity we often associate such terms as "primi-
tive" and "advanced," or "lower" and "higher."
Would you say that Paramecium is a "lower"
organism? Does it seem "primitive" to you?
Does it seem more "primitive" perhaps than an
onion or a man, of which you have seen epider-
mal cells? Which is "higher" or more "ad-
vanced," Euglena or the ameba which may be
derived from similar flagellates?

MODELS OF LIFE

A discouraging thing about defining life is
that once one has made a definition, it is easy
to construct a model that satisfies the definition,
yet clearly is not alive. Such models are them-
selves instructive, because they sometimes pre-
sent much simpler systems that display proper-
ties exhibited by living organisms in ways that
permit closer analysis, and suggest physical and
chemical bases for these phenomena in the living
organisms themselves. Such an application of
models can be misleading; one needs to judge
carefully how far to pursue a model, and when
to leave it.

In any case, the model we are about to
examine should be thought of in two ways : as
a demonstration that some phenomena of living
organisms are easily reproduced in inorganic

10 LIVING CELLS (2)

Exercise

systems; and to raise the question whether the
model and the organism behave similarly for
the same reasons.

An artificial "ameba"

Into a clean Syracuse watch glass laid on a
piece of white paper pour dilute nitric acid to a
depth of about ^ inch. Into this introduce a
drop of mercury about ^ inch in diameter; the
mercury is best introduced by putting the tip of
the pipet that contains it under the surface of
the nitric acid. Be careful not to spill any mer-
cury. Drop a crystal of potassium dichromate
about ^ inch in diameter or somewhat larger
into the nitric acid about f inch from the mer-
cury drop. You will immediately see the potas-
sium dichromate beginning to dissolve in the
nitric acid and diffusing from the crystal in all
directions. As the boundary of this yellow dif-
fusion zone reaches the mercury drop, things
begin to happen. Watch this for a time and
describe the phenomena you see. Are the mo-
tions you observe comparable in any way with
the mode of locomotion of an ameba? Have
you observed anything resembling cell division?

The physico-chemical basis of this behavior is
as follows. Mercury has an exceedingly high
surface tension, the highest of any known liquid,
and for this reason assumes an approximately
spherical form on a surface, though flattened
by its own weight. The potassium dichromate
in nitric acid oxidizes the surface of the mer-
cury, lessening momentarily the surface tension
at this point, causing a local outflow of mercury.
Such points of oxidation, distributed asym-
metrically over the surface from moment to
moment, lead to the motions and cleavages you
have observed.

(Go on watching this experiment as long as
you like. When you decide to clean up, be sure
not to drop any mercury on the floor or to let
any run into the sink. There will be a container
available into which to pour it. Mercury blocks
and rots plumbing; but much more serious is
mercury spilled around the room or on the
floors. There it enters the dust and may be

inhaled or otherwise absorbed by the body in
this form. Since it is not readily excreted, the
body tends to accumulate it, and in larger
amounts it can produce very serious disturb-
ances. Make certain, therefore, that mercury is
not spilled, and that if any is spilled by accident,
it is immediately picked up. Any of it that is on
the floor can be brushed into a dust pan by
using a wet brush.)

Ingestion, digestion, excretion

In feeding, a protozoan exhibits some de-
gree of choice. Ordinarily a protozoan takes in
some objects and not others. Having taken in
a particle of potential food, the cell digests it in
part and excretes what remains. The process of
digestion is well understood, but the mechanisms
by which the organism ingests some objects and
excretes others are only partly understood. In
performing the following experiment we should
like you to note what analogies to these processes
it presents, and to ask yourselves to what degree
the simple mechanisms it involves are related to
the comparable phenomena in living cells.

Put about 2 inches of distilled water into a
6-inch test tube, and drop into this 6 or 7 drops
of chloroform. Swirl the water in the test tube
and wait a minute for the chloroform to coalesce
at the bottom to form a single more-or-less
spherical drop about ^ inch across. Draw a
clean piece of glass rod, about ^ inch thick, to
a fine tip. Now attempt to insert the tip of the
rod into the drop of chloroform. Does the drop
accept it? Now wipe the rod dry, and dip the
very end into a solution of shellac. Blow on it
until it is dry. Now try again to make it enter
the drop of chloroform, watching closely what
happens. Does the drop accept it in the first
moment? later?

Such a drop of chloroform under water, like
that of mercury, approximates a spherical shape.
It does so not so much because of its own sur-
face tension but because of the surface tension
of the water that surrounds it. Surface tension
is a force well described by its name: the mole-
cules of a fluid attract one another more or less

Exercise

LIVING CELLS (2) 11

Strongly depending upon the substance of which
it is composed, and at the surface, where this
attraction is all directed inwardly, it produces a
tension which tends constantly to contract the
surface to a minimum. This is why such fluids
tend when possible to assume the spherical form
which presents the smallest possible surface
for a given volume. Any distortion from
the spherical form is resisted by the surface
tension.

Glass is, of course, insoluble in chloroform,
and the introduction of a glass rod would in-
crease the surface of the drop. Its resistance to
this increase of surface is the force that tends to
expel the rod from the drop, or vice versa. The
coating of the rod with a substance soluble in
chloroform (e.g., shellac) entirely changes these
relationships, since a coated rod no longer offers
an incompatible surface to the chloroform, but
instead a substance ready to enter the same
phase with it. As a result, the drop now accepts
the glass rod. In the happy event that the size

of the chloroform drop and the amount of
shellac on the rod come out about right, one
might observe that after accepting the shellac-
coated rod for a while, the drop spontaneously
moves apart from it again. The explanation is
that the drop has finished dissolving the shellac
off the rod and now rejects the rod itself, as
originally. One can think of this as a model of
a cell taking in an object which is partly food
and partly indigestible, digesting off the food,
and excreting the remainder. One sees also in
the behavior of this drop that through simple
forces of surface tension, the surface of separa-
tion between two immiscible phases (water and
chloroform in this case, but equally water and
air, or any others) forms a kind of skin with
special properties, an approach to a surface
membrane. This resists penetration by sub-
stances which it cannot dissolve or with which
it cannot react, and on the other hand it is
readily penetrated by substances that it can dis-
solve or with which it can react.

EQUIPMENT

Per student

compound microscope

slides and cover slips

Syracuse watch glass

piece of glass rod, 6 to 8" long, about J" thick

6" test tube

medicine dropper

Per 4 students

dropping bottle of 4% methyl cellulose
package of lens paper

Per 8 students

dropping bottle cultures of Paramecium (including
Paramecium aurelia Types XIII and XIV), Euglena,
ameba, and pond water

dropping bottle of blue ink

dropping bottle of carmine suspension

dilute nitric acid (about 1 M; dilute about 60 ml of

concentrated acid to 1 liter)

clean mercury (10 cc)

potassium dichromatic crystals (10 gm)

chloroform (25 cc)

white shellac (10 cc)

Per laboratory

reference books containing pictures of various

microorganisms

pure-line cultures of opposite mating types suitable

for demonstrating conjugation (Paramecium bur-

saria) can be obtained from the General Biological

Supply House, 8200 So. Hoyne Ave., Chicago 20, 111.

CHEMICAL COMPONENTS OF CELLS:
MACROMOLECULES OF YEAST AND
THEIR SUBUNITS (I)*

(Readings: Weisz, pp. 17-38 and 149-156; Villee, pp. 26-31. F. H. C. Crick,
"TTie Structure of the Hereditary Material," Sci. Am. 191, No. 4, Oct. 1954,
Reprint No. 5. P. Doty, "Proteins," Sci. Am. 197, No. 3, Sept. 1957, Reprint
No. 7.)

A cell lives by virtue of its composition and
organization. Both are unique: the composition
in large part because of the universal presence
of certain classes of very large molecules, so-
called macromolecules, the largest and most
complex in all chemistry; and these are respon-
sible also for many of the most distinctive fea-
tures of cellular organization and behavior.

Our approach to the macromolecules is enor-
mously simplified by three circumstances : (a) All
of them fall into three great classes — poly-
saccharides, nucleic acids, and proteins — com-
mon to all cells, and sharing common properties
within each class, (b) Each type of macromole-
cule is composed of a limited number of repeat-
ing subunits, bound together to form long
chains. The subunits of the polysaccharides
are sugars; those of nucleic acids, nucleotides;
and those of proteins, amino acids. Rather
than dealing with the individual atoms of which

*An alternative or supplementary pair of exer-
cises on the biochemistry of milk will be found in
Appendix C.

these molecules are composed, which may run
into many hundred thousands, we deal with the
much smaller numbers of subunits. (c) In all
types of macromolecule, the subunits are bound
to one another through the same device, the
elimination of a molecule of water between
each pair. Conversely every macromolecule
may be broken down into its subunits by the
reverse process, the insertion of a molecule of
water between each pair. The latter process is
called hydrolysis. Digestion is a series of such
hydrolyses, catalyzed by enzymes in the diges-
tive system, which cleave all the macromolecules
of the food into their constituent subunits :

Sugars

Nucleo-
tides

Synthesis
-(n-l) H2O

+(n-l)H20
Hydrolysis,
Digestion

Polysaccharides
(glycogen, starch)

Nucleic acids

Proteins

Amino
acids

In this and the next laboratory session we
will separate the major types of macromolecules

Exercise III

CHoOH

C-
H/H
C

-O

CHEMICAL COMPONENTS OF CELLS (1]
CH2OH

-0

C-
H/H
C

\H H/H \H

c c c

|\OH H/l |\OH H/l

-0 — I C C I — o — I c — c I — o-

H OH T H OH

HO— H

from a cell, and learn something of their proper-
ties and the units of which they are composed.
For convenience we shall work with yeast cells,
but the results would be much the same if we
used any others.

The simplest of the macromolecules are the
polysaccharides. They include the starches and
glycogens, forms in which the cell stores sugar
for future use; and such inert structural poly-
saccharides as cellulose, the principal component
of plant cell walls. Each of these molecules,
though very large, is made of a single repeating
unit, glucose.

Glucose contains 6 carbon, 12 hydrogen, and
6 oxygen atoms, so that its empirical formula is
C6H12O6. What is much more important is
the arrangement of these atoms in the molecule,
the so-called structural formula:

H— C=0

I CH2OH

H— C— OH \

I C O

HO— C— H H/l \ H

I 1/ H \|

H— C— OH ^ C C

I |\ OH H /I

H— C— OH OH\| 1/ OH

I c — c

H— C— OH I I

I H OH

These are two forms in which glucose exists at
all times, in equilibrium with each other. The
straight-chain form at the left is present in
minor amounts, but exposes the aldehyde
(HC=0) reducing group upon which the test

you will perform next week depends. The ring
structure at the right is by far the more prevalent.

A starch or glycogen is formed by stringing
hundreds or thousands of glucose molecules
together by eliminating a molecule of water
between each pair, as shown above. That is, «

(C6Hi206)^[(C6H,o05)„-H20] + (/7-l)H20,

in which n is the number of glucose units in-
volved. [Why is the number of water mole-
cules eliminated {n — 1)?]

Such a chain, several hundred glucose units
in length but unbranched, is the component of
starch that yields a blue color when treated
with iodine. A second component of starch is
formed of similar chains, but highly branched;
and glycogen, the characteristic storage poly-
saccharide of animal tissues and yeast, consists
entirely of such highly branched chains. The
highly branched polysaccharides yield brownish
or reddish colors when treated with iodine.

The nucleic acids are composed of units
called nucleotides, tied together to form long
unbranched chains, thousands of nucleotide
units long. Each nucleic acid contains four
different nucleotides; and since so many of
these units are involved, and they can be ar-
ranged in any sequence along the chain, it is
possible to construct in this way an enormous
number of different nucleic acids. Such variety
is needed, for among other things nucleic acids
form the functional components of the genes,
and it takes a lot of genes to account for the
heredity of all living things.

Any cell, even one that looks as simple as
yeast, contains a large number of different
nucleic acids, representatives of the two great
families, ribonucleic (RNA) and deoxyribonu-
cleic acid (DNA). Each, as already said, is
made of four different nucleotides; and each

14 CHEMICAL COMPONENTS OF CELLS (1)

Exercise III

nucleotide is itself composite, being made of a
nitrogenous purine or pyrimidine base, a
5-carbon sugar (ribose in RNA and deoxyribose
in DNA), and phosphoric acid, united to one
another by the same principle of elimination of
water between them. The hydrolysis of nucleic
acid not only may cleave the nucleotides, but
may sever all these linkages, leaving us with a
mixture of the free nitrogenous bases, 5-carbon
sugars, and phosphoric acid. The acid hydroly-
sis that we will perform releases nearly all the
purine bases, but only a small fraction of the
pyrimidines.

The ultimate components of the two families
of nucleic acids are:

RNA

adenine!
guanine]
cytosine
uracil
ribose
phosphoric
acid

purmes

DNA

(adenine
{guanine

. ... (cytosine
pynmidmes { ,

(thymme

deoxyribose

phosphoric

acid

The first four substances named in each column
are the nitrogenous bases. A nucleotide can be
written: base-ribose-phosphoric acid, the nu-
cleotides of each nucleic acid differing only in
their bases. The fundamental arrangement of
nucleotides in nucleic acid is:

base-sugar-phosphoric acid

where each bond represents a point at which
molecules have been united by elimination of
water, and conversely can be hydrolyzed by the
insertion of water.

The third class of macromolecules, the pro-
teins, is composed of up to 20 different amino
acids, joined together to form chains hundreds
to thousands of amino acids in length. Since

proteins are of many sizes, and their amino
acids can be united in any proportions and in
any sequences, almost an infinite variety of dif-
ferent proteins can exist. Living organisms
take full advantage of this possibility, for as far
as we know every living species, animal and
plant, contains specific proteins different from
those of all other living species. Proteins account
for much of the internal structure of cells, and
all known enzymes are proteins.
An amino acid has the general formula:

I
H— C— NH2

c=o

in which — NHo is the amino, — COOH the
carboxyl (acid) group, and R may be any one
of 20 different groups (— H, — CH3, — CH2OH,
etc.). Amino acids are joined to one another
by taking out a water molecule between the
— NH2 group of one and the — COOH group
of its neighbor. The joint that results,

II H
— C— N—

is called a peptide bond. Groups of amino acids
linked together in this way are called polypep-
tides until they get big enough to be called
proteins.

The general arrangement of amino acids in a
segment of polypeptide or protein follows. The
insertion of molecules of water at the places
indicated by arrows hydrolyzes the structure
into its constituent amino acids:

H C

1/ \
C P

C N H

/l\ / \l.
4 H C C

HO— H

Exercise

CHEMICAL COMPONENTS OF CELLS (]

A WORD ON MOLECULAR
STRUCTURE

Molecular structure is anatomy carried to the
level of small dimensions. We hope that by
now you would have no difficulty recognizing
an ameba or a Paramecium when seeing one
under the microscope. In exactly the same
sense you should learn to know a sugar, fat, or
a section of a protein or nucleic acid molecule
from its molecular appearance. Molecules are
three-dimensional structures, with characteristic
anatomies upon which many of their properties
depend. Some violence is done by the habit of
portraying them on the plane surfaces of paper
and blackboards; yet even such two-dimensional
representations are useful and recognizable.
After all, this is no greater violence than is in-
volved in pictures of animals and plants.

Fortunately, however, we can do something
much better, and we hope you will take full
advantage of it. You will find in the laboratory
sets of molecular models, from which you can
construct sugars, fats, representative sections of
proteins and nucleic acids, and many other types
of molecule that we encounter in this course.
With these models you can also inquire into
such interesting and important matters as optical
activity, associated with the right- or left-handed-
ness characteristic of many of the organic mole-
cules found in cells.

It would be altogether wrong to deal with
these molecules simply as words, the names of
abstractions. Use this opportunity in the labora-
tory to handle them and look at them as things,
which is what they are. Make yourselves models
of glucose, and join them together by taking
out molecules of water between them, as in
polysaccharide formation; then split them apart
again by inserting water molecules, as in hy-
drolysis. Similarly construct a polypeptide
chain from a few generalized amino acids, and
see what it means to hydrolyze such a chain,
the process catalyzed by such protein-hydrolyz-
ing enzymes as are found in pancreatic extracts.

From now on whenever you have a little free
time in the laboratory, one good thing to do

with it is to construct molecular models, and
carry out reactions with them. This is fun to
do, it will help you greatly, and it is as close to
synthetic organic chemistry as many of you will
ever come.

One last word about these models. They are
probably of a relatively inexpensive type, that
represents fairly correctly interatomic distances
and bond angles. The little balls that represent
the atoms, however, show only the relative
positions of the centers of those atoms, not the
space they occupy. In a more correct and much
more expensive type of molecular model, which
tries in addition to represent the space-filling
properties of the atoms, one sees that molecules
are much more solid structures. In such a more
correct model, for example, the six-membered
ring of glucose is seen to have almost no hole
in the middle.

EXPERIMENTAL PROCEDURE

Yeasts are a unicellular type of fungus which
reproduces by budding. The species of yeast we
shall use, Saccharomyces cereviseae, serves many
human uses. Different strains of it have been
developed as baker's yeast, for raising dough;
brewer's yeast, for fermenting malt to make
beer; and various types of wine yeast. We shall
be working with baker's yeast, which ordinarily
comes in cakes with starch as a binding mate-
rial. We have carefully washed the starch away,
leaving a clean suspension of yeast cells with
which to work.

Stir a pinch of yeast into 1 ml of glucose
medium, and set it aside. Toward the end of
the laboratory period, when you have time,
make a slide of a drop of this, and examine
the budding cells under the high power of the
microscope. During this interval the yeast will
have begun to ferment the glucose, and you
will see the bubbles of carbon dioxide which is
one of the products.

Our work in the laboratory will involve a
number of processes that are new to many of
you: centrifuging, neutralization of acids with

16 CHEMICAL COMPONENTS OF CELLS (1)

Exercise

bases, and dialysis. After describing the pro-
cedure, we shall discuss each of these processes.
That discussion is an integral part of the pro-
cedure, so be sure to read it before you begin
work. We will begin with a general account of
the procedure, and then give explicit directions
in the form of a flow sheet.

Yeast cells are enclosed in a tough, cellulose-
like outer wall. The wall is made of a poly-
saccharide called glucan, which contains only
glucose units, but bound to one another differ-
ently than in cellulose, starch, or glycogen. The
first operation is to break the cell walls by
grinding the yeast with sand in a mortar, re-
leasing the contents of the cells.

Extraction and hydrolysis of glycogen

The cell contents are stirred into trichloracetic
acid solution (TCA) which dissolves the glyco-
gen, leaving the nucleic acids and proteins as
solid particles in suspension. This suspension
is decanted from the sand, and the solid material
separated off by centrifuging.

The glycogen is precipitated from the solution
with ethyl alcohol, and this precipitate separated
off by centrifuging. It is redissolved in 1 N
hydrochloric acid (HCl), and this solution is
divided in halves. One half is immediately
neutralized with 1 yV sodium hydroxide (NaOH),
to prevent hydrolysis. The other half is heated
for 30 to 60 minutes in a bath of boiling water
(i.e., at approximately 100°C), and then is neu-
tralized in the same way. The heating in acid
solution hydrolyzes the glycogen completely.
Each half is now placed in a dialysis sac, and
the sacs are suspended in test tubes containing
distilled water, and stored in a refrigerator until
next week, when we will test for the presence of
glycogen and glucose inside and outside the sacs.

(NaCl), by stirring and heating in a boiling
water bath, leaving an insoluble residue of
coagulated proteins. The proteins are separated
off by centrifuging, and the nucleic acids in the
supernatant are precipitated with ethyl alcohol.
This precipitate is collected by centrifuging, and
dissolved in 1 A' sulfuric acid (H2SO4). The
solution is divided into halves, and one half
heated for 30 to 60 minutes at 100°C to hy-
drolyze. Then both solutions are neutralized
with barium hydroxide (Ba{OH)2). The salt
that results from the neutralization, barium
sulphate (BaS04), precipitates out. The reason
for acidifying with H2SO4 in this case, and
neutralizing with Ba(OH)2, is to get rid of
this salt on neutralization because the paper
chromatography of these solutions that you will
carry out next week goes much better in the
absence of salt.

Hydrolysis of protein

We are now ready to deal with the coagulated
proteins. It would take many hours of boiling
in strong acid or alkali to hydrolyze them. In-
stead, we perform this hydrolysis rapidly and
at room temperature by using enzymes, as do
living systems. We shall use a mixture of pro-
tein-digesting enzymes from a mammalian pan-
creas, which in life would have delivered this
mixture of enzymes to the small intestine.

Small portions of the solid protein residue are
transferred into each of two test tubes. One is
stirred into a buffered solution of pancreatic
enzymes, the other into a solution containing
the buffer alone, to serve as control. Both will
be stored by your instructor until next week,
when their contents will be analyzed by paper
chromatography.

Extraction and hydrolysis of nucleic acids

The solid residue of the yeast cell contents
after the removal of glycogen contains nucleic
acids and proteins. The nucleic acids are ex-
tracted into strong sodium chloride solution

DOING AN EXPERIMENT

The way to go at a job such as this, whether
it is simple or complicated, is to read through
the instructions and then make a plan of attack,
in which you try to see yourself going through

Exercise Itl

CHEMICAL COMPONENTS OF CELLS (1) 17

the whole business. That is a very important
part of getting ready to do an experiment. If
you can see in your mind's eye just what you'll
be doing and how you'll be doing it halfway
through — whether, for example, you'll be hold-
ing the test tube in your right or your left hand
at that moment — then you are ready to go to
work. So your first job after reading through
the above procedure and the further discussion
of the manipulations below is to make yourself
a schedule of just what you expect to do and
when. It might come out somewhat as follows:

(1) Grind cells, extract with TCA, centrifuge.

(2) Precipitate glycogen, extract and precipi-
tate nucleic acid.

(3) Centrifuge both preparations.

(4) Get both nucleic acid and glycogen sam-
ples ready to hydrolyze. Put into boiling water
bath together.

(5) Prepare the protein hydrolysis.

(6) Look at the yeast.

(7) Neutralize the acid hydrolysates and start
the dialysis of glycogen.

THE MANIPULATIONS
Centrlfuging

In its most primitive form a centrifuge might
be a boy whirling a bucket of water in circles
around his head, which as you know can be
done without spilling any water. If the water
had small particles of sand suspended in it, this
motion would make them settle faster to the
bottom of the bucket. In its most complex
form, an ultracentrifuge spins quartz tubes in
an evacuated chamber at tens of thousands of
revolutions per minute, developing forces well
over 100,000 times gravity. Under these circum-
stances macromolecules, being somewhat denser
than water, are sedimented. Your centrifuge
operates in between these two extremes. Its
maximum rate is about 3000 revolutions per
minute (rpm), and you will always use it at its
top speed.

A centrifuge is a potentially dangerous instru-
ment, and certain precautions must be observed

even with such relatively slow types as you are
using:

(1) Use only plastic tubes, and do not fill
higher than about i inch from the top.

(2) Each tube in the centrifuge must be bal-
anced against another of the same weight just
across from it. In your experiment it will be
enough to have both these tubes contain the
same volume of solution, gauged by eye. In
faster centrifuges it is necessary to balance the
pairs of tubes, preferably with their cups, against
each other on a sensitive balance.

(3) Place the centrifuge well away from the
edge of the work table, and be sure that it is
level. Otherwise it may creep off the table while
running.

(4) Close the lid before starting the centrifuge
and leave it closed until the centrifuge has
stopped spinning. Let it stop by itself; do not
brake it by hand.

(5) If the centrifuge begins to vibrate strongly
and clatter while running, stop it at once and
check the balance of the tubes.

(6) Bring it to top speed gradually.

(7) Since others will be sharing the instrument,
do your centrlfuging efficiently. Get everything
ready before occupying the instrument, and get
your tubes out of it immediately when the job
is finished.

Handling reagents

We have designed the fractionation scheme
to keep the use of strong acids and bases to a
minimum. You will nevertheless be using nor-
mal sulfuric and hydrochloric acids, and barium
and sodium hydroxides. Try not to spill, but
if you should spill any on the table or floor,
clean up immediately, using a fair amount of
water to dilute the acid or base. If any spills on
you, rinse at once with water, and tell the in-
structor. Any large spill of acid can be neutral-
ized by sprinkling it with sodium bicarbonate
(baking soda).

The alcohol that we use to precipitate glyco-
gen and nucleic acid is flammable, and should
not be used close to any flame. If you or your

18 CHEMICAL COMPONENTS OF CELLS (1)

Exercise

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Exercise

CHEMICAL COMPONENTS OF CELLS (1) 19

neighbors need to have a flame at the bench,
either delay the alcohol precipitation or do it at
a distance. Locate the fire extinguisher and fire
blanket early in the period, in case they may be
needed.

Neutralization of acids and bases

You have four neutralizations of acids to per-
form, two on the hydrolysates of glycogen and
nucleic acid, the other two on the controls for
these reactions. The glycogen had been taken
up in normal (i.e., 1 A') HCl, and is neutralized
with 1 N NaOH. A normal solution contains
1 gram molecular weight of hydrogen or hy-
droxy! ions per liter. The molecular weight of
HCl is 36.5 grams, so that a normal solution
contains this amount of HCl per liter; similarly
a normal solution of NaOH contains 40 grams
per liter. (How do we obtain these numbers?)
A given volume of 1 A'^ HCl should be very
nearly neutralized by the addition of the same
volume of 1 N NaOH. What are the products
of this neutralization?

To neutralize the glycogen solution in HCl,
add about 30 drops of NaOH solution (about
1.5 ml), stir with a glass rod, and then touch the
end of the wet rod to a piece of red litmus paper.
Such paper is red when acid, but turns blue on
being made alkaline. You will find that the wet
spot you have made remains red, as it should at
the beginning of such a titration. Now add
more NaOH drop by drop, stirring well each
time with the glass rod, and touching a fresh bit
of the litmus paper each time with the wet rod.
Do this until the addition of a final drop just
turns the paper blue. That marks the end of
your titration. The faint blueness of the paper
shows that your solution is now slightly alkaline,
which is how we want it.

The same essential procedure is used to neu-
tralize the H2SO4 in which nucleic acid was
hydrolyzed. The sulfuric acid solution also is

1 N. Since each molecule of H2SO4 contains

2 atoms of hydrogen, to make a normal solution
we dissolve half the gram molecular weight of
H2SO4 (i.e., ^ of 98 = 49 grams) in one liter.

In this case we use barium hydroxide to neu-
tralize the acid. The reason is that the salt
formed by the neutralization, BaS04, is insolu-
ble and precipitates, leaving a salt-free solution
in which the paper chromatography to be done
next week will go better than if the salt were
present.

Fractionation and isolation of molecules

One of the principal tasks in biochemistry is
to divide a complicated mixture of molecules
into its components, ultimately separating out
single molecular species. This is the enterprise
in which you are now engaged, and we should
like to say a little more about the procedures
you are using.

The crudest of them involves separating a
soluble from an insoluble fraction. That could
be done by filtration, the filter paper holding
back the insoluble material and permitting the
clear solution to run through. We accomplish
the same thing more rapidly and cleanly by
centrifugation, which among other things avoids
losing any material such as inevitably would
have stuck to filter paper. This is one of the
commonest procedures in biochemistry and can
be used to throw down any particles which are
denser than the liquid in which they are sus-
pended.

A second method of fractionation in these
experiments is dialysis. This is a refined kind
of filtration, in which the dialysis sac is the
filter. It is composed of a cellulose membrane
that contains tiny pores which allow water and
small molecules to pass through but block the
passage of macromolecules. In other words,
this process divides small from large molecules.
In your dialysis setup, the large molecules
should stay inside the sac, and the small mole-
cules distribute themselves evenly inside and
outside. If, however, you had chosen to replace
the outside solution repeatedly with fresh dis-
tilled water, you would eventually have removed
all the small molecules, and only the large mole-
cules would have remained inside the sac. In
this way you could have washed the macro-

20 CHEMICAL COMPONENTS OF CELLS (1)

Exercise III

molecules inside the sac, freeing them from all
contamination with small molecules. Dialysis
therefore is one of the most useful procedures,
not only for distinguishing large from small
molecules, but for purifying large molecules.

Next week we shall work with an ultimate
fractionation method, paper chromatography.

This can separate individual molecular species,
one from another, even when they differ only
slightly in structure. It requires also extremely
small amounts of material. Its sensitivity and
accuracy of resolution make it one of the most
useful procedures now available for biochemical
analysis.

EQUIPMENT

Per student

4 small test tubes with stoppers

8 test tubes (16 X 150 mm)

4 50-ml plastic centrifuge tubes

3 stirring rods

400-ml (or 250-ml) beaker

bunsen burner

test-tube rack

6" medicine dropper

2 ft of f" dialysis tubing

Per 2 students

mortar and pestle
wax marking pencil

Per 8 students

matches

5% trichloracetic acid (250 ml)

2 vials of indicator paper

2 dropping bottles of distilled water (with glass

stopper)

2 dropping bottles of 1-A' HCl (with glass stopper)

2 dropping bottles of I-TV H2SO4 (with glass stopper)

2 dropping bottles of l-A' NaOH (with rubber

stopper)

2 dropping bottles of 1-A' saturated Ba(OH)2 (with

rubber stopper)

dropping bottle of 0.5-M glucose (with rubber

stopper)

10% NaCl (200 ml)

95% ethanol (500 ml)

Per 30 students

2 clinical centrifuges

pair of scissors

2 ink marking pencils ("Magic Markers")

1 lb of purified sea sand

phosphate butfer (0.1 M, pH 7.0) (200 ml)

0.1% pangestin in phosphate buffer (0.1 M, pH 7.0)

(200 ml)

thymol (^ lb)

Per laboratory

molecular models (can be purchased from E. H.

Sargent & Co., Chicago, III.)

water baths at 100°C

refrigeration space for 2 test tubes per student

yeast preparations

Commercial brewer's yeast contains very little gly-
cogen and is therefore unsatisfactory for this experi-
ment. Baker's yeast does contain glycogen, but the
cakes in which it is supplied are held together with
starch binder. The starch is in the form of grains
which are larger and denser than the yeast cells, so
that they are easily removed by sedimentation.
Suspend the yeast in water or dilute salt solution,
and centrifuge very briefly, for about 15 sec, at 500
to 1000 rpm; or let the suspension stand until the
starch grains settle. The upper layer of the suspen-
sion can then be decanted and centrifuged to pack
the yeast cells. A few repetitions of this procedure
should be enough to remove all starch. This point
is easily demonstrated by staining one drop on a
slide with iodine-KI (Lugol's) solution (see page 24).
Starch, of course, stains blue or purple, whereas the
yeast glycogen stains reddish brown, entirely within
the cells.

Test tubes to be put into the water bath can be
labeled with masking tape.

CHEMICAL COMPONENTS OF CELLS:
MACROMOLECULES OF YEAST AND
THEIR SUBUNITS (2)

^4S'*

(Reading: W. H. Stein and S. Moore, "The Structure of Proteins," Sci. Am.
192, No. 5, 36-^1, May 1955.)

Paper chromatography separates compounds
on the basis of their different rates of migration
on filter paper (cellulose). The rates of migra-
tion depend upon the solvent which is flowing
up or down the paper, and on the relative
strengths of adsorption which hold the mole-
cules more or less tightly to the paper. Some
substances can be separated fairly well in dis-
tilled water, but mixtures of water with various
organic solvents are usually more effective.
Depending upon whether the solvent flows down
or up the paper, one speaks of descending and
ascending chromatograms. We will use ascend-
ing chromatograms.

Chromatography of amino acids and protein
hydrolysates

In this experiment you will chromatograph on
a single sheet of filter paper your unhydrolyzed
protein, the protein hydrolysate you have pre-
pared, an unknown amino acid, and five known
amino acids (alanine, aspartic acid, histidine.

lysine, and methionine). All these things will be
lined up as though getting ready for a race.
The measurement will consist in permitting
them to run for a time, and then finding how
far each has gone. The ratio of the distance a
given substance has moved from the starting
point to the distance traveled by the solvent
front is called the Rp. Two substances having
the same Rp, particularly when this has been
measured in a variety of solvents, are probably
identical; and the Rp of any known substance
under particular conditions is an important
identifying property.

Lay a square piece of filter paper, 12-cm
across, on wax paper, and draw a fine line, with
a lead pencil, parallel to and 1.5 cm from one
edge. This will be the bottom of your chromato-
gram. (Note: Avoid excess handling of the
filter paper, since your hands may contaminate
it with amino acids. Touch it only at the edges.)
On this line mark pencil dots, 13 mm apart,
starting 20 mm from one edge. These are to
indicate the positions for placing your samples.

22 CHEMICAL COMPONENTS OF CELLS (2)

Exercise IV

a
o

Unknown
amino acid

Protein
hydrolysate

Unhydrolyzed
protein

Label each sample directly on the paper below
the line, as shown on the diagram.

The samples are applied to the paper with a
fine glass capillary. Draw a little solution into
a capillary, and touch it to the paper at a pencil
dot. Let this dry, and repeat. Each spot should
be not more than 3 mm in diameter. Two such
superimposed applications should be enough
with the amino acid solutions, and four with
the protein and protein hydrolysate solutions.
It will be advantageous to place your unknown
amino acid in the middle, between the third
and fourth known amino acids. Now roll the
sheet into a cylinder, and tie the edges together
with needle and thread, leaving a gap, as shown
in the diagram and as demonstrated by your
instructor.

Pour 30 ml of solvent (formic acid : isopropa-
nol:water = 10:70:20) into a quart jar. Line
the walls of the jar with a piece of filter paper

dipping into the solvent in order to help saturate
the atmosphere. Splash the solvent about. Now
stand your filter paper cylinder in the jar, keep-
ing it away from the walls, close the jar, and let
it stand quietly. Wait until the solvent has risen
within 0.5 cm from the top of the paper before
removing the cylinder and letting it dry. Cut
the threads, dip the paper into the ninhydrin-
acetone reagent, and set it aside to dry. After
the acetone has evaporated, place the paper in
the warm oven (80°) for a few minutes. Do not
overheat! Remove the paper (you may now
handle it), and immediately outline with pencil
the spots that you see. (The reason for this is
that they fade in the light.)

Make a table showing the Rp values of the
known amino acids. Also enter in this table the
Rp value of your unknown amino acid. On
this basis, what do you think it is? Compare
the chromatograms yielded by the unhydrolyzed

Exercise IV

CHEMICAL COMPONENTS OF CELLS (2) 23

protein and the protein hydrolysate. Interpret
your results.

Chromatography of nucleic acid components

Your extract of yeast nucleic acids contained
both RNA and DNA (see the discussion at the
beginning of Exercise III). The hydrolysis that
you have performed not only broke the nucleic
acid into its component nucleotides, but went
on to hydrolyze the nucleotides into their unit
components. What we are looking for now,
therefore, are the isolated nitrogenous bases.
The RNA brought in four such bases: adenine,
cytosine, uracil, and guanine; but since guanine
is relatively insoluble and difficult to detect on
chromatograms, we disregard it in this experi-
ment. Also, since DNA is present in yeast in
much smaller amounts than RNA, we shall dis-
regard its distinctive base, thymine.

Prepare a sheet of filter paper, just as before,
to run on a single chromatogram your unhy-
drolyzed and hydrolyzed nucleic acid solutions,
a series of three known nitrogenous bases —
adenine, cytosine and uracil — and a mixture of
the bases. When the paper is ready, put five
superimposed applications of each of these solu-
tions at each of the labeled starting positions.

Prepare a second quart jar just as you did the
other, but using as solvent acetic acid :butanol:
water = 15:60:25. Set this chromatogram up
just as you did the other, stopping it when the
solvent has reached 0.5 cm from the top of the
paper. Then remove the paper, let it dry, and
cut the threads.

Instead of staining the paper this time, as we
did to find amino acids, we shall take advantage
of the fact that nucleic acids, because of the
nitrogenous bases which they contain, strongly
absorb ultraviolet light of wavelengths about
260 m/u. After drying your chromatogram, hold
it under a source of ultraviolet light. The
organic bases will appear as dark spots against
the light background. {Caution: Do not look
into the ultraviolet light. It is harmful to the
eyes. Do not expose your skin for more than
a few seconds.)

Dialysis of glycogen

Get the test tubes containing the dialyzed
samples of your unhydrolyzed and hydrolyzed
glycogen from the refrigerator. The point is
now to test for glycogen and its subunit, glucose,
both inside and outside each of the sacs.

Pour the contents of each bag into a separate
clearly labeled test tube so that you now have
four solutions: unhydrolyzed inside, unhydro-
lyzed outside, hydrolyzed inside, hydrolyzed
outside. Pour about 1 ml of each of these solu-
tions into a labeled test tube. Into a fifth test
tube measure 1 ml of water as a blank. {Note:
1 ml = 20 drops.) To each tube now add 2 ml
of iodine reagent (iodine-potassium iodide solu-
tion), which stains glycogen red-brown. (Do
you remember the purple staining of the starch
grains in potato slices and Spirogyral That
color resulted from the fact that starch contains
a straight-chain blue-staining component, in
addition to a branched red-staining component.
See page 3.)

After you have determined and recorded the
fractions in which glycogen is located, wash out
the 5 test tubes in which you did the iodine
test. Pour a fresh 1-ml sample of each of the
4 fractions and 1 ml of water into the 5 test
tubes. To each add 3 ml of Benedict solution,
swirl to mix, and place all 5 test tubes in the
boiling water bath for 3 min. Now compare
the colors.

The Benedict test is given by all sugars that
contain reducing groups (aldehyde or ketone)
that can reduce blue cupric (Cu++) ions to red,
insoluble cuprous (Cu+) ions. The Benedict
test is negative for glycogen or starch, because
in them the repeating glucose subunits, each of
which has a potential aldehyde group (see dis-
cussion in III), use up these groups in the
glucose-glucose linkages. However, when the
glucose units are freed by hydrolysis, the alde-
hyde groups become available for reaction.

What are your results and what do they mean ?
The Benedict test is not given by the sugar most
familiar to you, cane sugar or sucrose. Why
not? Make a model of sucrose.

24 CHEMICAL COMPONENTS OF CELLS (2)

Exercise IV

EQUIPMENT

Per student

test-tube rack

400-ml (or 250-ml) beaker

bunsen burner

ring

wire gauze

2 stirring rods

7 test tubes (16 X 150 mm)

2 quart jars with tops

dropping pipet

Per 8 students

matches

capillary tubing

2 needles and white thread

2 small test tubes of 10~'^ Af solutions of each of the
following: alanine, aspartic acid, histidine, lysine,
and methionine

bottle of Benedict solution (250 ml) prepared as

follows:

Dissolve 173.0 of sodium citrate and 100 gm of

sodium carbonate in 800 ml of water by heating.

Filter if necessary. Dissolve 17.3 gm of copper sul-
fate in 100 ml of water. Add it slowly to the citrate-
carbonate solution, with constant stirring. Make up
to 1 liter with water.

2 small test tubes of 1.0 mg/ml solutions of each of
the following: adenine, cytosine, uracil, and 1 of a
mixture of the three

bottle of Lugol's solution (250 ml) prepared as fol-
lows:

Dissolve 1 gm of iodine (I2) and 2 gm of potassium
iodide (KI) in 20 ml of water. Add this to 980 ml
of aqueous solution containing 25% KCI (w/v).

Per 30 students

0.5% ninhydrin in acetone (1 liter)
formic acid, isopropanol, water (10:70:20) (2 liters)
acetic acid, butanol, water (15:60:25) (2 liters)
12-cm- Whatman No. 1 filter paper (300 sheets)
wax paper (2 rolls)

Per laboratory

ultraviolet germicidal lamp and safety glasses

warm oven

water baths at 100°C

ENZYMES

(Readings: Weisz, pp. 135-141 and 271-273. S.P.T., pp. 93-96. Villee,
pp. 57-65, 306.)

Living cells have the remarkable capacity to
perform rapidly and under mild conditions
chemical reactions which under the same cir-
cumstances would proceed extremely slowly
outside the organism. A homely example: sugar
exposed to oxygen burns to carbon dioxide and
water, generating considerable heat in the proc-
ess. If you touch a match to the sugar, thus pro-
viding energy of activation, this reaction goes
very rapidly, as you know. Without the match,
i.e., at room temperature, the same reaction goes
in exactly the same way, yielding just as much
carbon dioxide, water, and heat, but so slowly
as to be negligible. In a frog at room tempera-
ture, however, or in yourself at a slightly higher
temperature, the same reaction occurs rapidly,
yielding exactly the same products, and exactly
the same amount of energy, though the latter,
before being degraded finally to heat, is used
for all the multiple activities of the organism.

The enzymes of living cells greatly accelerate
such chemical reactions, and by governing their
relative rates, regulate the overall directions of
metabolic change. Enzymes are catalysts: they
greatly speed a chemical reaction, without them-
selves being used up in the process. It is not
that they don't take part in the reaction. They
do, by combining for a moment with the react-

ant, the substrate; but at the end of the reaction
the enzyme is returned and can be used again:

enzyme -f substrate ^

enzyme-substrate complex ;^

enzyme -|- products.

This is what we mean by a catalyst; and for
this reason a little enzyme goes a long way.

Since the enzyme is returned unchanged at
the end of the reaction, it can contribute nothing
to the final result. If the reaction is reversible,
the presence of the enzyme hastens, but does
not change, the final equilibrium. That is, in
any reversible system, the enzyme speeds up
equally the forward and the back reaction. This
behavior also is typical of all catalysts. Thus
the pancreatic enzymes you have already used
catalyze equally well the hydrolysis and the
synthesis of peptide linkages; yet because the
equilibrium of this pair of opposed reactions
lies far over toward hydrolysis, and because the
reaction usually occurs in the presence of over-
whelming concentrations of water, an almost
irreversible hydrolysis is the end result.

All known enzymes are proteins, and many
of their properties depend upon this fact. Their
activity depends, as do many other protein

26 ENZYMES

Exercise V

properties, on the hydrogen ion concentration
of the medium. Each enzyme tends to be most
active over a narrow range of hydrogen ion
concentration, the "pH optimum." Enzymes
are rapidly destroyed by boihng, as are proteins
generally.

Another general property of enzymes, as of
other proteins, is specificity. Each enzyme cata-
lyzes only one or a narrow class of chemical
reactions. Hence thousands of different enzymes
are needed to catalyze the multitude of chemical
reactions carried out by living cells.

It is one of the triumphs of modern bio-
chemistry to extract enzymes and enzyme sys-
tems from cells and have them catalyze in the
test tube the same reactions and reaction se-
quences that we find in living organisms.
Indeed a great number of enzymes have been
prepared pure and crystalline, and many are
now bought and sold commercially like other
organic substances.

In this period we shall work with three enzyme
systems, each of which has something special to
tell us. Succinic dehydrogenase is an oxidation-
reduction (hydrogen-transferring) enzyme, of
central importance in cellular respiration. With
it we can demonstrate hydrogen transfer, and the
mechanism of action of a powerful respir-
atory poison. Amylase is a digestive enzyme,
which catalyzes an almost irreversible hy-
drolysis; with this system we can readily measure
the effects of changing enzyme concentration,
pH, and temperature on the rate of reaction.
Phospliorylase catalyzes the coming to equilib-
rium of a reversible system, and so permits us to
study synthesis as well as degradation, depending
upon how the system is constituted.

SUCCINIC DEHYDROGENASE

The citric acid or Krebs cycle is central among
the enzyme systems concerned with cellular
respiration, the process by which organic mole-
cules are burned with molecular oxygen to
carbon dioxide, water, and energy in forms use-
ful for cellular work. One of the steps of this

cycle is the oxidation of succinic acid to fumaric
acid. This reaction is catalyzed by the enzyme
succinic dehydrogenase:

COOH

CH, ^

1 - Succinic

dehydrogenase

1
CH

II + 2H

CHa

COOH

Succinic acid

Fumaric acid

This reaction can be followed by observing
the loss of color of the dye methylene blue
(MB) as it is reduced to the colorless form
"methylene white" (MB-H2) by accepting the
two hydrogen atoms removed from succinic acid.

We have already spoken of the specificity of
enzymes for their substrates. Succinic dehydro-
genase, so far as we know, catalyzes only the
dehydrogenation of succinic acid, in part be-
cause the catalytically active site on the enzyme
molecule combines readily with succinic acid to
form the enzyme-substrate complex. Sometimes,
however, it is possible to fool an enzyme by
offering it a molecule that so greatly resembles its
normal substrate that the enzyme combines with
the impostor instead. Such a molecule in the
present instance is malonic acid :

COOH

I
CH2

I
COOH

Succinic dehydrogenase, having combined with
malonic acid rather than succinic acid, can
neither dehydrogenate it nor lose it again.
Thus its active site is blocked, and the enzyme
is inhibited or poisoned. The inhibition is as
specific as the enzyme action and for the same
reason. It can be reversed by adding an excess
of succinic acid, which competes with malonic
acid for the catalytic site. We call malonic acid
for this reason a competitive inhibitor. Its action
on succinic dehydrogenase makes it about as
powerful a poison of cellular respiration as
cyanide.

Exercise V

ENZYMES 27

Try to tind time after doing the experiments to
make molecular models of succinic and malonic
acids. What resemblance between these mole-
cules do you suppose fools the enzyme?

Experiment

Succinic dehydrogenase occurs in the cell
particles known as mitochondria. It can be ob-
tained directly from beef heart. A piece of meat
about the size of a small marble should be used
for each assay. The meat should be cut up
further and washed a few times by vigorous
shaking with water in a test tube, followed by
decantation of the wash water in order to re-
move any substrates already present. Add re-
agents to the labeled tubes as follows, agitating
so that added substances are evenly distributed
throughout the muscle suspension:

Tube 1: no meat; add an equivalent volume of
water plus 3 drops succinic acid (0.5 M)
and 7 drops methylene blue (MB) solu-
tion (0.01%).

Tube 2: meat plus 3 drops succinic acid.

Tube 3: meat plus 7 drops MB.

Tube 4: meat plus 3 drops succinic acid plus 7
drops MB.

Tube 5: meat plus 3 drops succinic acid plus 3
drops malonic acid (1 M) plus 7 drops
MB.

Tube 6: meat plus 9 drops succinic acid plus 3
drops malonic acid plus 7 drops MB.

Tube 7: boiled (2 min) meat plus 3 drops suc-
cinic acid plus 7 drops MB.

Bring the solutions in all tubes to the same
total volume (19 drops, as in Tube 6) by adding
distilled water.

Pour mineral oil down the side of each tube
so as to form a surface layer not more than 1 cm
thick. The oil keeps oxygen from diffusing in,
and so prevents reoxidation of MB-H2. Place
the tubes in the water bath at 37°C, and watch
for color changes while you go on with other
experiments.

What changes have you observed? Why was
the experiment set up in seven test tubes as
above? What does each mixture contribute?
Could you have learned as much from fewer
mixtures? At the end of the experiment, you
can demonstrate the rapid oxidation of re-
duced methylene blue by air by stoppering
Tube 4 with your finger and shaking it vio-
lently.

SALIVARY AMYLASE

Starches are very large carbohydrate mole-
cules, made by stringing hundreds to thousands
of glucose molecules together in long straight
and branched chains (Review Exercise III).
Saliva, which is secreted by the salivary glands in
amounts of the order of 1 liter daily, contains an
enzyme that catalyzes the hydrolysis (digestion)
of starch through a series of smaller and smaller
intermediates (so-called dextrins) to the final
product maltose, which consists of two glucose
molecules joined together as in starch. To hy-
drolyze maltose to glucose requires another
enzyme, maltase, not present in saliva, but
secreted by both the pancreas and the small
intestine.

The salivary enzyme that hydrolyzes starch to
maltose is called salivary amylase. It has been
prepared in crystalline condition. Older names
for it are salivary diastase and ptyalin.

Its action can be followed readily with the
iodine test. Iodine yields a deep blue color with
starch (actually only with the straight-chain,
amylose fraction of starch). As the starch is hy-
drolyzed, repeated tests with iodine go from the
initial blue color to red or reddish brown (dex-
trins), and eventually to colorlessness (smaller
dextrins, maltose).

A word about the shapes of molecules and
specificity of enzymes. Like starch, cellulose is
made of glucose molecules tied together to form
very long chains. The only essential difference
between starch and cellulose — one of the most
biochemically reactive, and one of the most
inert molecules — is that in starch the glucose

28 ENZYMES

Exercise V

molecules are bound in so-called alpha-linkage,
in cellulose in beta-linkage:

CHoOH

c— o

CHoOH
C— O

c
c— c

-0-

-'\

c— c

CH20H

c— 0

c— c

Maltose, with
two glucose
molecules linked
as in starch.

-0-

V Cellobiose. with

r^ two glucose

^ molecules linked

^ as in cellulose.

C— C

C— O

I
CHoOH

(Write all the missing — H and — OH groups
into the above structures.)

The essential difference between starch and
cellulose is therefore one of molecular shape; but
that is reason enough for the amylases, which
digest starch, to have no effect on cellulose, and
for the rare group of enzymes that digest cellu-
lose (cellulases) to have no effect on starch.

When you have time, make the molecular
models of maltose and cellobiose, starting in
each case with two molecules of glucose.

Experiments

Reaction rate vs. enzyme concentration. Stimu-
late your flow of saliva by chewing a piece of
gum, and collect about 5 ml in a test tube.
Working with your partner, make a series of
dilutions in tap water as follows:

Dilution Concentration of saliva
(ml saliva: ml tap water) C^)

1:9 10

1:19 5

1:49 2

1:99 1

These are conveniently made by taking 1 ml of
saliva for each dilution (using a pipet) and
making it up to the indicated total by adding

tap water from a 50-ml graduate. (Do not use
the same pipet for the starch solutions.) Meas-
ure the activity of these four dilutions of saliva
as follows:

(1) Pipet 1 ml of each concentration of saliva
into a test tube and label.

(2) Into each of a second series of four test
tubes pipet 2 ml of 0.59^, starch suspension,
made up in 0.259( NaCl. (Salt is added because
chloride ions specifically activate salivary amyl-
ase.) Add 2 ml of buffer solution, pH 6.8. to
each tube (this is the optimal pH for the en-
zyme).

(3) Place the two sets of tubes (eight in all)
in the water bath at 37°C. Leave for several
minutes until they reach that temperature. (Note:
This experiment can also be performed at room
temperature but will go more slowly (see next
exercise).

(4) At a recorded time, pour the contents of
one tube containing starch mixture into the tube
with the highest concentration of saliva. Swirl
to mix, and return it quickly to the bath.

(5) Working with your partner, test for starch
by removing a drop of the reaction mixture
with a medicine dropper and adding it to a drop
of an aqueous solution of lo in KI (each 0.01
M) on a test plate. {Note: The lo-KI solution
should not be allowed to stand in the test plate
depressions for more than a few minutes. Dis-
pense it one drop at a time as needed.) These
tests should be started at a time as near zero as
possible, and continued at 10-second intervals
thereafter. The initial color should be blue;
continue the tests until they yield no color
change at all. Note the colors you see, and
record the time required to reach the endpoint,
the point at which the mixture has the same
color as the iodine test solution.

(6) Repeat the procedure of steps 4 and 5
with the 1:19 dilution. Depending on the rate
of reaction, the time intervals between tests can
be lengthened.

Exercise V

ENZYMES 29

(7) If the activity of the saliva is not too great
(if, for example, it takes more than a minute
for the 1:19 dilution to reach the endpoint),
the remaining two dilutions (1 :49 and 1 :99) can
be run simultaneously to save time, and the test
intervals can be increased again to keep pace
with the rates of reaction.

Plot a graph showing the reciprocal of the
time (1/min) required to reach the endpoint vs.
the concentration of saliva. The reciprocal of
the time is a measure of the rate of reaction.

Compare the activity of the saliva used in
your tests with that used by other students in
terms of the times required to reach the achro-
matic endpoint in the tubes to which 2% saliva
was added. Compare the result in your own
experiment with the minimum, maximum, and
mean values of the class as a whole. What
would you conclude?

Reaction rate vs. temperature. (Note: Half
the students in the class should do this experi-
ment, the other half the experiment on acidity
below, in each case working in pairs.) Using
the same techniques and the same pH as in the
previous experiment, and selecting a saliva con-
centration that yields an endpoint in 3 to 4
minutes, determine the rate of the reaction at
0°C (ice in water), room temperature, 37°C
(water bath), and 100°C (boiling water). At 0°
and 100°, tests can be made at intervals of 1, 5,
or 10 minutes, after it has become clear that
the reaction is going slowly.

Plot a graph of 1/min to endpoint vs. tem-
perature.

Chemical reactions in general go 2 to 3 times
faster for every 10° rise in temperature. The
same tends to be true of enzyme-catalyzed reac-
tions, with a special twist: as the temperature
rises, it reaches a point at which it begins to
destroy the enzyme, as it does other proteins,
and thereafter the reaction rate falls instead of
rising further. The result is that as the tem-
perature is raised from some low initial value,
the rate of the catalyzed reaction first rises, then
falls. At a certain temperature, just before it
begins to fall, the rate is at its highest, the
so-called temperature optimum.

From your observations, about where do you
estimate the temperature optimum for salivary
amylase to lie? How is it related to your body
temperature? If you now brought both the 0°
and the 100° samples to 37°, what reaction rates
would result? Why?

Reaction rate vs. pH. Determine the time to
reach the endpoint at 37° in reaction mixtures
buffered at pH 3.4, 5.0, 6.8, and 8.0. Mix 2 ml
of the starch-NaCl solution, 2 ml of the appro-
priate buffer, and 1 ml of a dilution of saliva
deemed suitable on the basis of your previous
measurements.

What do you conclude to be the approximate
pH optimum of salivary amylase? On what
side of neutrality does it lie? How is it related
to the pH of your saliva? (Measure this by
touching the end of a piece of pHydrion paper
to your tongue and comparing with the color
scale.)

Whichever of the last two experiments you
did, find out what results were obtained in the
other experiment, and note them in your labora-
tory notebook. In general we want you to know
everything that goes on in your laboratory,
whether you do it yourself or not.

PHOSPHORYLASE

For a long time it was thought that such
amylases as you have just examined are responsi-
ble for degrading glycogen in animal tissues. Yet
liver and muscle degrade glycogen very much
more quickly than any known amylases can
accomplish. In 1935 a new class of polysac-
charide-splitting and -synthesizing enzymes was
discovered, called phosphorylases. The splitting
of glycogen by a phosphorylase requires the
presence of inorganic phosphate, and the prod-
uct is not glucose, but glucose- 1 -phosphate.
Whereas amylases break glycolytic linkages by
introducing water (hydrolysis), phosphorylases
do the same job by introducing phosphoric acid
(phosphorolysis), as shown on the next page.

30 ENZYMES

Action of amylase and phosphorylase on polysaccharides:

Exercise V

CH2OH

-K)-

HO-^H

I— o-

CH20H

J-O.

-0—

HO-f-H

polysaccharide + water

amylase

maltose

CHoOH

<-°>

^o— ^

phosphorylase

(0H)20P0— H (0H)20P0— H

polysaccharide + phosphoric acid

CH2OH

-O

'— op=o +

CHoOH

-0

-0—

glucose-1-phosphate + shorter chain

These two ways of degrading polysaccharides
differ in fundamental ways. The difference in
rate has already been mentioned; the phos-
phorylases are among the most active of known
enzymes. Second, amylases end by cleaving
polysaccharides into maltose units, which require
a second enzyme, maltase, to yield glucose. The
phosphorylases yield instead glucose phosphate
units, which are hydrolyzed further to glucose
and phosphoric acid by the enzyme phosphatase.

The most interesting and important difference
in the action of these enzymes, however, involves
the reversibility of the phosphorylase reaction.
Whereas the hydrolysis of polysaccharides by
amylases is virtually irreversible, their phos-
phorolysis goes readily in either direction. It is
important that you understand the reason for
this difference.

Hydrolyses in general tend to be virtually ir-
reversible for two reasons: (1) in polysaccharides,
for example, the glucose-glucose bond has an
energy of about 3 kcal/mole. The hydrolysis of
the polysaccharide results in a loss of this
energy; and conversely one should have to add
this amount of energy per glucose-glucose link
from outside in order to resynthesize a poly-

saccharide. In the absence of such added energy,
only the hydrolysis can occur. (2) Such enzy-
matic reactions, in and out of the cell, ordinarily
occur in the presence of an overwhelming con-
centration of one of the reactants, water. The
molar concentration of pure water is 55.6 M
(why so?), and most aqueous solutions approach
this concentration of water. If we write a
reversible equation for a hydrolysis, this enor-
mous concentration of water on one side of the
equation pushes the equilibrium very far in the
other direction ("mass action effect").

A phosphorolysis presents a very different
situation in both regards. On the one hand, the
energies of the reactants and products are fairly
evenly balanced : a glucose-phosphoric acid bond
has very nearly the same energy as a glucose-
glucose bond. Hence little energy is lost or need
be added in going in either direction. Further-
more the concentrations of reactants and prod-
ucts are more evenly balanced, since here phos-
phoric acid in relatively low concentration takes
the place that water occupies in a hydrolysis.
The phosphorylase reaction therefore is freely
reversible. In neutral solution and at room
temperature the equilibrium lies somewhat over

Exercise V

ENZYMES 31

toward polysaccharide synthesis: the system
tends to synthesize rather than degrade poly-
saccharides.

This synthesis, however, can occur only if two
conditions are realized: (1) It proceeds, not from
glucose, but from glucose- 1 -phosphate. The
organism must begin by spending considerable
energy in forming the initial glucose-phosphoric
acid bonds. (2) The synthesis requires the
presence of some polysaccharide on which to
build ("primer"). The reaction adds glucose
units to the end of already existing polysac-
charide chains. The phosphorylase reaction
builds up and degrades only straight polysac-
charide chains. The branching of such chains,
or the attack upon branched chains, requires
another type of enzyme, which exchanges glu-
cose-1,6-glucose links at branch points for the
glucose- 1,4-glucose links of straight chains.
Unaided phosphorylase, therefore, synthesizes
only straight-chain polysaccharides, or straight-
chain projections from highly branched polysac-
charides. In such reactions the molar concentra-
tion of polysaccharide does not change. One
starts with the concentration of the primer, and
all that happens during the synthesis is that the
primer grows bigger as glucose units are added
to it:

phosphorylase

glucose- 1 -phosphate + primer =^

primer-glucose + H3PO4

Phosphorylases occur in many animal and
higher plant tissues and in yeast. Today we will
extract phosphorylase from the potato tuber.
In Exercise I (p. 3) you examined starch grains
in potato cells, staining them with the lo-KI
reagent. Try this again today if you like.

Experiment

Prepare the following test tubes:

(1) 3 ml of 0.01 M glucose

(2) 3 ml of 0.01 M glucose- 1 -phosphate

(3) 3 ml of 0.01 M glucose- 1 -phosphate

(4) 3 ml of 0.01 A/ glucose- 1 -phosphate

(5) 3 ml of 0.01 M glucose- 1 -phosphate +
1 ml of0.2MKH2PO4

(6) 3 ml of 0.2% soluble starch +
1 mlof0.2MKH2PO4

(7) 3 ml of 0.2% soluble starch +
1 ml of 0.2 A/ KH2PO4

Add a very small drop of the 0.2% starch
solution to Tubes 1, 2, 4, and 5 to act as primer.
There should be so little starch present in these
tubes that the I2-KI test is negative — check it.

With a paring knife, peel a small potato and
cut it into small cubes. Place these in a Waring
blendor, add 40 ml of 0.01 N sodium fluoride,
and grind for 30 sec. {Note: FLUORIDE IS A
POISON! We use it here to inhibit potato
phosphatase, which would otherwise hydrolyze
glucose- 1 -phosphate to glucose and phosphoric
acid.)

Filter the homogenate through a double layer
of cheesecloth into a beaker. Squeeze out as
much of the liquid as you can. Centrifuge the
suspension for 3 min, then decant and keep the
supernatant. Test this extract to see that it is
negative to the I2-KI reagent. Transfer approxi-
mately 10 ml of the extract to a test tube, and
heat for 5 min in a boiling water bath.

Now add 3 ml of the enzyme preparation to
Tubes 1, 2, 3, 5 and 6; and 3 ml of the boiled
enzyme preparation to Tubes 4 and 7. {Note: Use
the enzyme as soon as you have finished prepar-
ing it, since it deteriorates rapidly.)

Test each of these mixtures at once and at
3-min intervals thereafter with lo-KI. The re-
action should be completed within about 30
min. Record and explain your results.

What was the purpose of each component in
the mixtures you prepared ? What was the point
of each mixture? What might have happened
had you left the fluoride out of the enzyme
preparation? If you have time, try doing that.
How do you account for the fact that though the
number of polysaccharide molecules has not
changed (see above), you now obtain a test
with I2-KI whereas initially you didn't?

32 ENZYMES

Exercise V

EQUIPMENT

Per 2 students

24 test tubes (small or medium); 8 more if possible

8 dropping bottles

1-ml pipet

2 5-ml pipets

50-ml graduate

6 6" medicine droppers

2 beakers suitable for 0°C and 100°C water baths

potato

porcelain spot plate

cheese cloth (10" X 20")

Per 8 students

fresh beef heart

mineral oil

50-ml dropping bottle succinate solution (0.5 M, adj.

to pH 7.5)

50-ml dropping bottle malonate solution (1 M, adj.

to pH 7.5)

50-ml dropping bottle 0.01 % methylene blue solution

50-ml dropping bottle iodine and potassium iodide

solution (0.01 M)

50-ml dropping bottle 0.5% starch (boiled) in 25%

NaCl

50-ml dropping bottle glucose (0.01 M)

50-ml dropping bottle glucose-1-phosphate (0.01 M)

50-ml dropping bottle KH2PO4 (0.2 M)

50-ml dropping bottle 0.2% soluble starch

50-ml dropping bottle sodium fluoride (0.01 A')

50-ml dropping bottle Mcllvaine buffers (0.1 M) at

pH 3.4, 5.0, 6.8, and 8.0

vial of pHydrion paper

Per laboratory

water baths at 0°C, 37°C, and 100°C
chewing gum (1 stick per student)
Waring blendor
clinical centrifuge

STUDIES IN MICROBIOLOGY (ly

Bacterial Growth; A Bacterial Enzymc;
Comparative Biochemistry

(Readings: R. Y. Stanier, M. Doudoroff, and E. A. Adelberg, The Microbial
World, Prentice-Hall, 1957, pp. 26-37, 225-239, and 255-256. K. V. Thimann,
Life of Bacteria, Macmillan, 1955, pp. 3-31 and 550-560. Weisz, pp. 173-175.
S.P.T., pp. 484^88. Villee, pp. 131-138.)

Bacteria are single-celled organisms, much
smaller than the yeasts, algae, and protozoa,
examples of which we have already seen. Many
kinds of bacteria can be distinguished on the
basis of such characteristics as shape, color,
nutritional requirements, and biochemical con-
stitution. We shall first study the bacterium
Serratia marcescens. It is rodlike in shape, red
in color, and requires a source of animal or
vegetable material for growth.

As the bacterium takes in nutrient substances
from its surroundings and converts them into
its own proteins, it grows up to a point at which
it splits into two cells, each of which continues
to grow in the same manner. That is, it repro-
duces by fission. We shall follow the growth of
Serratia by counting the number of cells present
before and after letting the bacteria grow for
two hours.

*A detailed discussion of preparations for the
microbiological experiments in Exercises VI through
IX will be found in Appendix A (pp. 135-140).

In order that growth occur rapidly, the bac-
teria will be suspended in broth, aerated in order
to allow the cells to respire freely, and incubated
at 37°C. Furthermore, our source of bacteria
will be a young culture, that is, one in which
the cells are growing rapidly. In an old culture
the cells have used up all the nutrients and are
no longer growing. To start growing again
when transferred to fresh medium, the cells
have to reorganize their machinery, and this
results in a delay — a lag period — before growth
begins.

The number of cells present in a bacterial
suspension can be counted by spreading a
dilute sample on the surface of agar (a jelly-
like material) to which nutrients are added.
Each cell grows into a colony, and one counts
the colonies.

With the help of the enzymes they contain,
microorganisms can carry out numerous chemi-
cal reactions. Serratia marcescens contains the
enzyme catalase which breaks down hydrogen
peroxide to water and oxygen (2H2O2 — * 2H2O

34 STUDIES IN MICROBIOLOGY (1]

Exercise Vi

+ O2). Catalase is a red iron-porphyrin-protein
closely related chemically to the blood pigment
hemoglobin. The oxygen which is produced can
be measured with a volumeter; thus the en-
zyme action is easily followed. In particular, we
shall study the behavior of the enzyme when
different amounts of the substrate (hydrogen
peroxide) are added. We shall also investigate
the inhibition of catalase activity by hydroxyl-
amine (NH2OH). This chemical interferes with
the enzyme by attaching to and hence block-
ing the iron atoms upon which its action
depends.

Catalase is found in many organisms. We
shall study it in bacteria, the horse, and potato
plants. In all of them it possesses the same type
of enzyme activity, and, as can be judged from
inactivation by hydroxylamine, this activity is
based on the same active group. (Actually these
catalases do vary somewhat in their properties,
owing to differences in the amino acid composi-
tion of their protein components.) How do you
suppose organisms so different from one another
come to possess such similar enzymes?

EXPERIMENTS

A note on sterile procedure. In working with
bacteria it is necessary to minimize the possi-
bility of stray microorganisms from the air
entering the cultures and agar plates. Sterilized
glassware and pipets should be used whenever
possible. Containers should be opened for as
brief a time as possible when material is being
transferred. The wire loop which is used to
sample a suspension and spread it on agar must
be heated in a flame before use. If necessary,
dropper pipets and tubes may be sterilized by
heating them in a boiling water bath for five
minutes. However, always allow an instrument
to cool before using it, or else the heat will kill
the bacteria with which you are working. Avoid
touching pipet tips or the sterile part of cotton
plugs with your fingers; also do not place them
on the bench.

Bacterial growth

Into a sterile 4-inch test tube pour with sterile
precautions 2 ml (^ inch) of the young Serratia
culture, and into a wide 6-inch tube pour 20 ml
of nutrient broth (about 3 inches). Also obtain
nine sterile dropper pipets; wrap these in a clean
paper towel before placing them on your bench.
With a sterile 5-ml pipet, transfer 5 ml of broth
to a wide tube, and without putting down the
pipet, deliver 0.9-ml portions of broth to eight
4-inch tubes which will be used for dilutions.
With a dropper pipet, inoculate the tube con-
taining 5 ml of broth with 2 drops of Serratia
culture. Swirl to suspend the bacteria evenly.
Label the tube with your name; this will be
your culture tube.

Set up a dilution series A with four small
tubes, labeled Al, A2, A3, A4. Transfer 2 drops
from your culture tube into tube No. 1. Swirl
contents to mix, and with a fresh pipet add 2
drops of No. 1 to No. 2. Continue in this man-
ner so that you have a series of four tubes each
of which has only -^ as many cells as the one
before it. (A drop contains 0.05 ml.) Be careful
to add just the right number of drops, since
your calculation of the number of cells present
depends on the accuracy of the dilutions.

Now, insert a sterile aerator into your culture
tube in place of the cotton plug. Attach the
long arm of the aerator to the air hose adjacent
to the water bath. Adjust the flow of air so
that it bubbles gently through your culture.
Incubate the tube in the bath at 37°C.

Take an agar plate. Turn the petri dish up-
side down, and with a wax pencil divide the
bottom into four quadrants, labeled Al, A2,
etc. Pass a wire loop through the flame, wait
a moment for it to cool, then dip it into the
No. 4 tube. Make sure you have a loopful of
liquid. (A loopful contains just about 0.001 ml.)
Gently spread the contents of the loop on the
agar in the appropriate quadrant. Repeat with
the other tubes. It is not necessary to flame the
loop each time when going from lower to higher
concentrations. Why?

After your culture has grown for two hours,
repeat this procedure to determine the number

Exercise VI

STUDIES IN MICROBIOLOGY (1) 35

of bacteria now present. Label this series B.
Take the agar plates home with you. Keep
them in a fairly warm spot. Within a few days
colonies should appear. Count them. Calculate
the number of bacteria in your culture before
and after the two hours of growth. (Remember
that a drop contains 0.05 and a loop contains
O.OOI ml.) How many divisions did each cell
undergo in this period? How much time was
needed for a cell to duplicate itself?

Bacterial calalase

While your culture is growing, continue with
other parts of the experiment. Rinse out the
small tubes and dropper pipets which were used
for dilution series A. These will now be used
in the study of catalase. It is not necessary to
use sterile procedure in this part of the experi-
ment.

Obtain 5 ml (1 inch) of the old Serratia cul-
ture in a 6-inch test tube. The old culture is

more convenient for enzyme studies since it con-
tains more cells and hence more enzyme than
the young culture. Add 10 drops of this culture
to a small tube. Add 10 drops of hydrogen
peroxide. Wait a few minutes. Observe. What
happens when you dip a glowing splinter into
an empty test tube? into the reaction tube?
Why?

We shall now measure catalase activity with
a "volumeter," a device that measures gas ex-
changes in terms of changes in the total volume
of gas at constant pressure (see illustration).
One of the volumeter test tubes will contain the
experimental material. The other is left empty,
to act as a thermobarometer — a means of cor-
recting for changes of gas volume owing to
trivial changes of temperature or barometric
pressure in the course of the experiment. Such
changes should be equal in both tubes if both
contain about the same volumes of gas.

Working in pairs, place 3 drops of the bac-
terial suspension in one of the volumeter test

36 STUDIES IN MICROBIOLOGY (1)

Exercise VI

tubes. Replace the stopper, making sure that
the side-arm, as also that of the thermobarom-
eter, is horizontal. Place a drop of kerosene in
the end of each side-arm. Unclamp the escape
tube, and with the aid of a medicine dropper in-
serted into the rubber tubing withdraw enough
air to pull this indicator drop back to the
proximal end of the scale on the side-arm.
When all is in order, add one drop of 3% hydro-
gen peroxide solution to the test tube containing
bacteria through the escape tube. Immediately
clamp it shut, and read the positions of the in-
dicator drops in both tubes. From now on re-
read both tubes every minute, agitating the
whole volumeter back and forth for 15 seconds
before each reading to hasten the escape of
oxygen. When the indicator drop has ceased to
move, add a second drop of hydrogen peroxide
to the bacterial suspension, and repeat the entire
performance. {Note: The position of the in-
dicator drop can be moved back to the proximal
end of the scale just before adding the second
drop of hydrogen peroxide.)

Subtract all changes recorded in the thermo-
barometer from those measured in the experi-
mental tube. Plot a graph showing the volume
of oxygen emitted vs. time. (To turn your
measurements of the distance the indicator drop
has traveled into gas volumes you must of course
measure the internal diameter of the side-arm.
Better still, calibrate the volume of the side-arm
by injecting known volumes of air through the
escape tube with a 1-cc syringe.) Does the
evolved gas account for all the oxygen you would

expect to obtain from one drop (0.05 ml) of 3%
hydrogen peroxide? [The equation for this
change is: H2O2 "'^'"") H2O + ^Oj. Starting
with the fraction of a mole of H2O2 added,
knowing from this the fraction of a mole of
oxygen that should be evolved, turn this into a
gas volume through the relationship: 1 mole of
gas at 273°K (0°C) and 1 atmosphere pressure
(as here) occupies 22,400 ml. At room tem-
perature on the absolute scale (r°K), this vol-
ume is increased by the factor 7"°/273.]

Add two drops of hydroxylamine solution to
the test tube containing the bacteria. After one
minute, add two more drops of hydrogen
peroxide. Take readings until sure of the result.
Now add 10 more drops of hydrogen peroxide,
and again follow the reaction. Describe your
observations. How does the "noncompetitive"
inhibition of catalase by hydroxylamine differ
from the competitive inhibition of succinic
dehydrogenase by malonate studied in Exercise
V?

An experiment in comparative biochemistry

Cut off a 5 inch cube of potato. Mash it up
with a glass rod in 1 ml of water in a 4-inch
test tube. Set up three 4-inch test tubes con-
taining (1) two drops of Serratia culture, (2) two
drops of horse blood, (3) two drops of potato
extract. Add a drop of hydrogen peroxide to
each. Observe. After foaming has subsided add
a drop of hydroxylamine to each. Swirl to mix.
Add a drop of hydrogen peroxide. Is there a
reaction in any of the tubes?

EQUIPMENT

Per student

5-ml pipet, sterile

10 dropper pipets, sterile

test tube, sterile

2 test tubes, nonsterile

2 wide tubes, sterile

aerator assembly, sterile

bacteriological loop
2 nutrient agar plates
bunsen burner
wood splint
marking pencil

Per 8 students

nutrient broth in 250-ml Erlenmeyer flask (20 ml)

Exercise VI STUDIES IN MICROBIOLOGY (1) 37

young culture of 5. marcescens in wide tube (20 ml) small potato

old culture of S. marcescens in 250-ml Erlenmeyer paper towels

flask (50 ml) 4 volumeters

4 dropping bottles of 3% hydrogen peroxide

2 dropping bottles of 10% hydroxylamine, neu- Per laboratory

tralized water baths at 37°C with rack accommodating wide

dropping bottle of horse blood diluted 1;20 in tube

0.85% sodium chloride provision for aeration at water baths

STUDIES IN MICROBIOLOGY (2)

Bacterial Mutation; Resistance to Antibiotics;
Radiation Effects; Action of Lysozyme;
Bacterial Anatomy*

(Reading: H. J. Muller, "Radiation and Human Mutation," Sci. Am. 193, No. 5,
Nov. 1955, Reprint No. 29. Further readings are suggested at the end of this
exercise.)

A bacterial population, even though it may
have descended from a single cell, contains many
cells which differ from the original bacterium
and from most of the cells about them. These
variants, or mutants, arise spontaneously as the
result of aberrations in the molecules concerned
with transmitting inheritance from parent to
daughter cells, the deoxyribose nucleic acids
(DNA). The aberrations responsible for muta-
tion are believed to involve the substitution
of one or more nucleotides for others originally
present in the DNA sequences. Such errors in
the replication of DNA probably occur while
these molecules are being multiplied prior to
cell division; but whenever they occur, they are
propagated thereafter from generation to gen-
eration. In this way what begins as a small
molecular change can end in forming a new
population, a new strain of bacteria.

*Directions for setting up these experiments will
be found in Appendix A.

Even though such changes are rare, there are
many of them in a large population. If one in
ten thousand bacteria is a mutant, a bacterial
population of ten million is likely to have one
thousand such mutants. This therefore consti-
tutes a tremendous potentiality for variation,
present in all bacterial cultures.

Most mutations are disadvantageous, and
thus most mutant strains tend to die out rather
than to propagate and expand. A change in
environmental conditions, however, may favor
a previously unsuccessful mutant. Indeed, a
drastic change may kill all the other bacteria
and allow one mutant form alone to survive,
and — since it is now relieved from competition —
to flourish. This is exactly what happens when,
after you have taken heavy doses of an anti-
biotic, you may find that the antibiotic no longer
works.

The experiment to be performed today should
show you that it is not difficult to develop
strains of bacteria that are resistant to penicillin

Exercise VII

STUDIES IN MICROBIOLOGY (2) 39

or streptomycin. Some of you may find strains
that are resistant to both antibiotics. Such
strains do not develop by the reorganization of
cells of the original parent stock. On the con-
trary, the antibiotic eliminates these, and per-
mits only one or a few antibiotic-resistant mutant
forms that happen to be present to multiply to
form a new antibiotic-resistant population.
What we observe, therefore, is not the inherit-
ance of an acquired character, but, as always,
the selection of those individuals which through
mutation already possess that character.

As you must know very well by now from
discussions of atom bombs and fallout, it is
possible to increase the rate of mutation far
above that which occurs naturally. One way is
by exposing cells to high-energy radiation.
Ultraviolet light of wavelengths near 260 m/i
has this effect. It is high in energy, correspond-
ing with its short wavelength (£ = hcj\, in
which E is the energy per quantum, /; is Planck's
constant, c is the velocity of light, and X is
the wavelength). The organic bases in the nucleic
acid chains strongly absorb these wavelengths
of ultraviolet light. Indeed, such ultraviolet
light in large enough doses kills all living cells
through its destructive effects upon their nucleic
acids. If one subjects a population of cells to a
large enough dose of ultraviolet light to kill
many of them but not all, the survivors usually
display an extraordinarily high incidence of
mutation.

Today you will perform such an experiment
upon Serratia marcescens and look for induced
mutants among the surviving cells. The brilliant
red pigmentation of this bacterium makes it
particularly suitable for such studies, since
mutants that lack the normal pigmentation and

hence look pink, white, or speckled are easily
recognized.

Isolation of antibiotic-resistant strains

Prepare your own agar plate containing an
antibiotic concentration gradient by the follow-
ing procedure (see diagram): Pour enough
melted nutrient agar (about 15 ml) into a slanted
petri dish (place a stirring rod under one end)
so that you have just covered the bottom of the
dish. Let the agar harden. Place the plate in a
horizontal position and add enough additional
agar containing penicillin (P) or streptomycin
(S) (whichever one you choose, your neighbor
should use the other) to just cover the already
solidified agar. (Don't fill the dish to the top.)
The antibiotic will establish a linear concentra-
tion gradient during subsequent incubation by
diffusing into the nutrient agar below it. Before
inoculation, dry the surface of the agar by open-
ing the dish slightly by propping up one edge of
the lid and incubating the dish for one hour at
37°C. Mark with an arrow on the bottom of
the dish the direction of the gradient of anti-
biotic concentration.

The plate is inoculated with either S. mar-
cescens or Escherichia coli bacterial suspension,
parallel to the gradient. Give the labeled plates
to your instructor to store until next week.

Radiation effects

With sterile technique pour about 1 ml of the
diluted saline suspension of S. marcescens (10*
cells/ml) into a sterile test tube. Obtain an agar
plate containing synthetic medium and divide it
into four quadrants marked on the bottom of
the glass with wax pencil. Label the quadrants
150, 120, 90 (standing for seconds of radiation),

40 STUDIES IN MICROBIOLOGY (2)

Exercise VII

and control. Spread a loop of the bacterial sus-
pension on the quadrant marked 150 and expose
the uncovered plate to ultraviolet light at a dis-
tance of 25 inches for 30 seconds. (Caution: Do
not look into the ultraviolet light. Do not
expose your skin for more than a few seconds.)
Next spread a loop of the suspension on the
quadrant marked 120 and expose the plate to
ultraviolet light for an additional 30 seconds.
Now spread a third loop on the quadrant
marked 90 seconds and expose the plate for
90 seconds. Finally spread a loop of the sus-
pension on the control quadrant, cover, and give
the labeled plates to your instructor. He will
incubate them for two days at room tempera-
ture and will then store them in the refrigerator
until next week.

Microscopic examination of
Bacillus megatherium

Bacillus megatherium is a giant among bac-
teria even though it is only 1 micron wide by
4 micra long. (A micron is 1/1000 mm, or
1/25,000 inch.) The other strains which we have
been using are much smaller, and in order to
see them more elaborate microscopy is needed.

Prepare a wet mount of B. megatherium as
follows. Place a droplet of water on a clean
glass slide. This may be done conveniently with
a glass rod. Stir a loopful of B. megatherium
culture into the drop. Gently place a cover glass
over the drop; try to avoid leaving air bubbles
under the glass. Focus on the bacteria under
the high-power objective of your microscope.
To do this, watching from the side, bring the
objective down until it Just fails to touch the
cover glass. Now, looking through the ocular,
slowly raise the objective by means of the fine
adjustment until the field is in focus. To see
the bacteria well it will be necessary to close
down the diaphragm (with the lever under the
stage) so that the field is only dimly illuminated.

The bacteria may be seen more easily after
staining them with a dye. Remove the cover
glass. Let the suspension dry. Pass the slide,
face up, through a bunsen flame three times.

The heat will coagulate bacterial proteins and
fix the bacteria to the slide. When it is cool,
flood the area with a drop of methylene blue.
Wait one minute, then rinse the slide with water.
Gently blot it dry with a paper towel. Examine
the slide once again under the high power.
Draw what you see.

Action of lysozyme: bacterial protoplasts

The enzyme lysozyme breaks down the com-
plex polysaccharides of which the cell walls of
many bacteria are composed, leaving the cell
covered only by its delicate plasma membrane.
Whereas the intact bacterium may have been
rod-shaped, it becomes spherical on losing its
rigid cell wall. Such naked, spherical cells are
called protoplasts. We shall watch cells of B.
megatherium being lysed by lysozyme, and form-
ing protoplasts. This experiment does not de-
mand sterile conditions.

Quite frequently, as in the present instance,
the contents of a cell are considerably more con-
centrated than the surrounding medium. As a
result, water tends to flow from the medium
into the cell, making it swell. In B. megatherium,
as in many other bacteria, this tendency to swell
is resisted by the rigid cell wall. (Recall the
opposite effect of suspending Elodea in strong
salt solution, in Exercise I, which made the cell
shrink away from the cell wall.) When bacterial
cells have lost their cell walls through the action
of lysozyme, this restraint is removed. The
entrance of water from the medium, swelling
the cell, subjects its plasma membrane to great
strain. Eventually it ruptures, and the cell con-
tents pour out into the medium.

This is easily seen by adding lysozyme to a
turbid suspension of bacteria. The suspension
rapidly clears as the cell walls are hydrolyzed
away, and the bacteria burst or lyse. If sucrose
is added to the medium, so that its osmotic
concentration is equal to that of the cell interior,
the cell no longer swells, and the result is a
stable, spherical protoplast.

Two suspensions of B. megatherium are pro-
vided, identical except that one is suspended in

Exercise VII

STUDIES IN MICROBIOLOGY (2) 41

dilute phosphate buffer alone (pH 7.0) and the
other in phosphate buffer to which sucrose has
been added to a concentration of 0.15 M, making
the medium isosmotic with the cell contents.

Pour about 2 ml of the suspension of cells in
buffer alone into a small test tube. Note the
turbidity of the suspension. Add 4 to 5 drops
of lysozyme solution and swirl, watching the
tube as you do so. You should soon see the
suspension clarify, as the cells lyse. Examine
the end result under the microscope.

Repeat this experiment, using the suspension
of cells containing sucrose. Do you still note
changes in turbidity? Again look at the result
under the microscope. The spherical protoplasts
should be visible.

It will be worth preparing a wet mount of
B. megatherium in phosphate buffer containing
sucrose, and adding one drop of lysozyme on
the slide while looking at the cells. The dissolu-
tion of the cell wall can be seen, and all the
stages in the formation of protoplasts.

Microorganisms in the air

Label a plate of nutrient agar with your
name, and leave it open, exposed to the air, for
30 minutes. Don't place it too close to where
anyone is working, lest he spill bacteria near
your plate. These plates should be incubated
at home for two days at room temperature
(about 20° to 25°C), then placed in the refrigera-
tor so that you can examine them next week.

Further microscopy of bacteria

The microscopes you have been using do not
have sufficient magnification to make most bac-
teria visible. A few higher-power microscopes
may be available, possessing an oil immersion
objective lens. Since in this case the light is not
required to pass from glass to air and back,
greater magnification can be achieved. In addi-
tion, this microscope may provide phase con-
trast, which enhances the contrast wherever
there is a difference in refraction of light within
the object or between it and its surroundings.

Examine wet mounts of Serratia marcescens
and Pneumococcus under the highest power avail-
able to you, using phase contrast if you have it.
After placing the slide on the stage, put a drop
of immersion oil on the center of the cover
glass. Watching from the side, bring the high-
power objective down until it dips into the oil
and almost touches the cover glass. With the
fine adjustment slowly raise the objective until
the bacteria come into focus.

Bacteria are often divided into two groups on
the basis of shape: bacilli (rods) and cocci
(spheroids). B. megatherium is clearly a rod.
Serratia is more difficult to classify; it is con-
sidered to be a short rod. Pneumococcus is, of
course, considered to be a coccus. Do you find
its shape to be perfectly round ? Cocci that are
strung along in chains are called streptococci
(strepto, Gr. = chain); those which occur in
pairs are diplococci. What would you call Pneu-
mococcus 7

Further reading

C. A. Knight and D. Fraser, "The Mutation of

Viruses," Sci. Am. 193, No. 1, pp. 74-78, July

1955, Reprint No. 59.

S. E. LuRiA, "The T2 Mystery," Sci. Am. 192, No.

4, pp. 92-98, April 1955, Reprint No. 24.

H. Fraenkel-Conrat, "Rebuilding a Virus," Sci.

Am. 194, No. 6, 42-47, June 1956, Reprint No. 9.

R. Y. Stanier, M. Doudoroff, and E. A. Adel-

BERG, The Microbial World, Prentice-Hall, 1957,

pp. 365-371.

K. V. Thimann, Life of Bacteria, Macmillan, 1955,

pp. 85-94.

EQUIPMENT

Per student

5-ml pipet, sterile

8 dropper pipets, sterile

4 nutrient agar plates
2 wide tubes, sterile
aerator assembly, sterile
bacteriological loop
bunsen burner

14 small test tubes with 1 ml soft agar

5 ml nutrient broth

Per 8 students

suspension of phage T4 in broth at lO'* particles/ml
(for phage reproduction experiment) (5 ml)

suspension of T2, T4, T4r, and unlabeled phage, in
broth at 10^ particles/ml (for phage morphology
experiment) (5 ml each)

dropping bottle of E. coli culture in logarithm phase
of growth (10-20 ml)

2 porcelain filter cylinders assembled in suction
flasks, sterile

Per laboratory (30 students)

water bath at 37°C, with aeration assembly

2 water baths at 45°C containing small tubes with
soft agar

PHOTOSYNTHESIS

mmsmsm

(Readings: Weisz, pp. 241-263. S.P.T., pp. 95-100. Vill^e, pp. 94-103. E. I.
Rabinowitch, "Photosynthesis," Sci. Am. 179, No. 2, pp. 24-34, Aug. 1948,
Reprint No. 34. D. I. Arnon, "The Role of Light in Photosynthesis," Sci.
Am. 203, No. 5, 104-118, Nov. 1960, Reprint No. 75.)

The energy that supplies all life on the earth
comes ultimately from sunlight, through the
process of photosynthesis. Each year plants on
the earth reduce about 550 billion tons of carbon
dioxide, using about 25 billion tons of hydrogen,
and releasing about 400 billion tons of oxygen
into the atmosphere. About nine-tenths of this
activity goes on in the surface layers of the
oceans.

No industrial process yet invented converts
light economically into useful forms on a large
scale. For this reason our economy still depends
largely upon the combustion of fossil fuels,
themselves the products of photosynthesis in
past ages. We have only recently begun to
understand how plants accomplish this feat.

For light to be used, it must be absorbed;
and substances which absorb visible light are
by that token pigments. The pigments which
absorb the light used in photosynthesis are
found in the chloroplasts of green plants, and
in similar particles called chromatophores in
photosynthetic bacteria. The principal pigment
of chloroplasts is chlorophyll a. Chlorophyll b
and the yellow carotenoids play secondary roles,
transferring the energy they absorb as light to

chlorophyll a for use in photosynthesis. Photo-
synthetic bacteria possess a special bacterio-
chlorophyll, and also a number of specific
carotenoids.

The net action of light in photosynthesis is to
split water, thus providing hydrogen for reduc-
tions and eliminating oxygen as a by-product:

I2H2O

light

chloroplasts

^ 24H + 6O2.

The H atoms supplied in this way are used to
reduce CO2 to carbohydrate and water:

6CO2 + 24H

18 ATP

CeHiaOe + 6H2O.

Thus the overall reaction is

light

6CO2 + I2H2O

chloroplasts

C6H12O6 + 6H2O + 6O2.

To fix one molecule of CO2 in the form of
carbohydrate requires not only 4 H atoms but
also 3 "high-energy" phosphate bonds of
adenosine triphosphate (ATP). The structure
of ATP and some of its sources are discussed
in Exercise XI. It is now recognized that the

Exercise X

PHOTOSYNTHESIS 51

energy absorbed as light by chloroplasts gen-
erates not only hydrogen, but also ATP. Indeed,
isolated chloroplasts can carry out the whole
process of photosynthesis.

Carbohydrate, having been prepared by
photosynthesis, is in turn degraded to provide
all the cell's energetic needs. The two principal
processes for deriving energy by the degradation
of sugars are fermentation and respiration. We
shall examine both processes in the next labora-
tory session. Fermentation is the process by
which cells derive energy anaerobically, by rear-
ranging the atoms of sugar to yield products of
lower energy. Respiration is a combustion, in
which sugar is burned with molecular oxygen to
yield carbon dioxide, water, and energy in the
form of ATP.

Photosynthesis and respiration are opposed
reactions. The overall equation of the former
is just the reverse that of the latter. Green
plants respire in the dark; they simultaneously
respire and photosynthesize in the light. The
consumption of oxygen is a measure of their
respiration; the evolution of oxygen measures
their photosynthesis. In the light, with both
processes going on simultaneously, the oxygen
exchange represents a balance between these
opposed reactions. If the light is sufficiently
bright, however, photosynthesis may go so much
faster than respiration as to dominate the
oxygen exchange.

With a fine capillary, apply this mixture to
the longer side of a 4.5 x 4.5-inch filter paper in a
narrow line, 3 inches long, 1 cm from the bot-
tom. Develop the chromatogram with a mix-
ture of 9 petroleum ether : 1 acetone.

In this solvent the carotenes (C4nH,5fi) move
the fastest, followed by the xanthophylls
(C4oH54(OH)2) and then the chlorophylls a
and b. Outline the visible pigment spots lightly
with a pencil. Then examine the paper under
ultraviolet light, noting the fluorescence of the
various pigments and the presence of any addi-
tional spots which were not apparent in visible
light. (Caution: Recall our earlier warnings not
to look into the light.)

Determine where the petroleum ether extract
of plant pigments has absorption maxima by
looking at this solution through the hand spec-
troscope. Chlorophyll a has a major absorption
band at about 680 m// and chlorophyll b at
about 665 myu. Those of you who have time
may cut out the two chlorophyll bands on your
chromatogram and elute the pigments by leach-
ing out the paper strips in a small test tube with
a few ml of acetone. Remove the filter paper
with forceps and observe the absorption of these
two solutions at the specified wavelengths in the
hand spectroscope, or measure it in a spectro-
photometer. Which pigment migrated faster on
your chromatogram? What is the chemical dif-
ference between chlorophylls a and A? Why is
chlorophyll green?

EXPERIMENTS

Analysis of chloroplast pigments

The chlorophylls and carotenoids (xantho-
phylls and carotenes) are the major pigments of
the chloroplasts. These pigments can be ex-
tracted from green plant tissues with lipid sol-
vents, and separated by chromatographic ad-
sorption.

Such an extract has been prepared before the
laboratory session by homogenizing spinach
leaves with 95*^^, ethanol in a Waring blendor.
The extract has been filtered, evaporated to
dryness, and redissolved in petroleum ether.

The Hill reaction

The photolytic cleavage of water in the pres-
ence of chloroplasts is known as the Hill reac-
tion. It can be represented by the following
equation :

A + H2O

light

chloroplasts

H2A + i02.

In this reaction "A" represents an electron (or
hydrogen) acceptor. In plants this is usually
the coenzyme TPN. In our experiment we shall
use an artificial electron acceptor, the dye 2,6
dichlorophenolindophenol, which is reduced

52 PHOTOSYNTHESIS

Exercise X

with the concomitant evolution of oxygen.
You will be able to follow the course of the
reaction by observing the loss of blue color as
the dye is reduced:

Dye (blue) + H2O —T^, — *

■' ^ ' chloroplasts

Dye — H2 (colorless) + JO2.

Spinach chloroplasts have been prepared as
follows: Leaves were homogenized with 0.5- M
sucrose solution at 0°C for 30 sec in a Waring
blendor. The suspension was then filtered
through two layers of cheese cloth. The filtrate
was centrifuged at 50 times the force of gravity
(50 G) for 10 minutes. The supernatant was
then decanted and recentrifuged for 10 minutes
at 600 G. The supernatant was decanted and
discarded. The pellet at the bottom, containing
the chloroplasts, was resuspended in 0.5-M
sucrose. It is important to keep the chloroplasts
at 0°C; they deteriorate rapidly at higher tem-
peratures.

In each of two test tubes, mix:

2 ml of phosphate buffer, 0.1 M, pH 6.5;
2 ml of dye solution

(2,6 dichlorophenolindophenol,

2.5 X 10-4 M).
0.1 ml of chloroplast suspension (2 drops);
6 ml of distilled water.

Swirl to stir, wrap one tube immediately in
aluminum foil to protect it from light, and
expose the other to bright light for 10 minutes.
Compare. (Protect the chloroplasts from heat
radiation by placing a glass tumbler filled with
water between the light source and the reaction
tubes.)

Devise experiments to show (a) that the
chloroplasts and dye must be illuminated
together to obtain this result; (b) that the reac-
tion depends upon catalysis by enzymes. In-
clude the results of these experiments in your
notes.

Oxygen evolution in photosynthesis

Place three leafy sprays of Elodea in one test
tube of the volumeter described in Exercise VI

(pp. 35-36), and fill this tube with 1% sodium
bicarbonate solution. Add sufficient solution so
that when the rubber stopper is inserted, an air
space of about 3 to 6 mm is left between the
liquid surface and the stopper. The bicarbonate
solution will provide the carbon dioxide used
in photosynthesis. Fill the second tube, which
as before will serve as thermobarometer,
with the same volume of sodium bicarbonate
solution.

A 60-100 watt lamp mounted upright in a
standard receptacle will be used as light source.
Between this lamp and the volumeter place ajar
or glass filled with water, to serve as a heat filter,
which, by absorbing the infrared (heat) radia-
tion from the lamp, will prevent large tempera-
ture changes in the volumeter from distorting the
readings. Place the lamp as close to the volu-
meter as it will go with the heat filter in between.
Draw a drop of kerosene into the proximal end
of each side-arm, as described in Exercise VI,
and close the pinch clamps. Allow the system
to equilibrate for 5 to 10 minutes. This equilibra-
tion time is needed for the oxygen evolved to
saturate the water; thereafter all the gas pro-
duced is given off".

Take readings in both side-arms at 2-minute
intervals, each time subtracting the reading in
the thermobarometer from that in the experi-
mental tube. Go on with the readings until the
rate of change remains constant through three
consecutive readings.

After the rate has stabilized, move the light
source to twice the distance from the plant,
and after taking readings at this distance double
the light distance once again. Assuming that
the light intensity is inversely proportional to
the distance, plot the rate of oxygen evolution
(change of volume in units/min) vs. light inten-
sity (relative). {Note: The intensity of light
coming from a point source falls off" as the square
of the distance; i.e., at twice the distance the
intensity has fallen to one-quarter. Your lamp,
however, is not a point source, particularly if
frosted, or equipped with a reffector; and the
light intensity declines more nearly in propor-
tion to the distance.)

Exercise X

PHOTOSYNTHESIS 53

Optional experiments

(1) In the above experiment the hght inten-
sity limits the rate of photosynthesis. If several
independent steps are involved in an overall
reaction, the rate at which the process goes is
determined by the step which has the lowest
rate, i.e., the limiting step. Determine at what
concentration the bicarbonate becomes limiting
at maximum light intensity.

(2) Shake a few pieces of leaf for 3 to 4
minutes in a test tube with petroleum ether.
Pour off the petroleum ether into another test
tube, replace with methanol, and shake the
leaves as before in this solvent, heating mean-
while under the hot-water tap. Pour off the
methanol extract into a clean test tube, and
compare with the petroleum ether extract.
Which contains more pigment? Which is

therefore the better solvent for leaf pigments?
Now mix both together, shake once vigorously,
and let the two layers separate. Which contains
more pigment? How do you explain these
observations?

(3) Determine in a spectrophotometer the
absorption spectra of the mixture of chloro-
plast pigments and of the separate pigments
isolated on your chromatograms. By dividing
this job among several students, you may be
able to do all of it. To obtain enough of the
isolated pigments it will probably be necessary
to combine several chromatographed samples in
as small a volume of solvent (acetone or ethanol)
as the spectrophotometer will handle. The mix-
ture of pigments and the chlorophylls should be
measured between 380 and 720 mix; the caro-
tenoids between 380 and 550 m/x. The instructor
will show you how to use the spectrophotometer.

EQUIPMENT

Per student

6 large test tubes
2 small test tubes
quart jar

Per 2 students

volumeter

electric light source

glass tumbler

Per 30 students

95% ethanol

100 ml sucrose (0.5 M)

500 ml 2,6-dichlorophenolindophenol (2.5

500 ml phosphate buffer (0.1 M, pH 6.5)

2 liters of 1% sodium bicarbonate

1 liter absolute methanol

X 10-

0.5 liter petroleum ether

2 liters acetone

50 filter paper sheets, 4.5" X 4.5"

fresh spinach leaves

crushed ice

100 fresh Elodea stems

fine capillaries for applying pigments to filter paper

roll of aluminum foil

Per laboratory

6 hand spectroscopes

spectrophotometer (the Bausch and Lomb "Spec-

tronic 20" instrument will perform adequately)

cheese cloth \

Waring blendor > for instructor before lab

centrifuge j

0.5 M sucrose

ultraviolet lamp and safety glasses

FERMENTATION AND RESPIRATION

ijjsm

(Readings: Weisz, pp. 319-351. S. P. T., pp. 129-132. A. L. Lehninger, "How
Cells Transform Energy," Sci. Am. 205, No. 3, 62-73, Sept. 1961. See also
R. Y. Stanier, M. DoudorofF, and E. A. Adelberg, The Microbial World, Prentice-
Hall, 1957, pp. 147-150, 577-583, and K. V. Thimann, Life of Bacteria, Mac-
millan, 1955, pp. 376-383.)

The great metabolic processes by which cells
obtain energy are fermentation and respiration.
Fermentation is Pasteur's "life without air"; it
provides energy in the absence of oxygen. The
essence of this process is the rearrangement of
the atoms of a sugar to yield a compound of
lower energy, making the difference in energy
available to the cell. Respiration is a cold com-
bustion: molecular oxygen is used to burn or-
ganic molecules — frequently sugars — to yield
carbon dioxide, water, and exactly the same
total energy as if the same molecules had been
burned in a flame.

In both respiration and fermentation part of
the energy is liberated as heat. The organism

N=C— NH2

I I
HC C— N

cannot use this, however, except to warm itself;
for living organisms are chemical machines, not
heat engines. The energy the cell needs to
maintain itself, to make new molecules, grow,
move, and reproduce, must be provided in
chemical form. Usually this is in the form of
adenosine triphosphate, ATP. It takes about
8 kcal of energy per mole to attach the terminal
phosphate group to adenosine diphosphate to
make ATP (ADP + P ^^ ATP), and this energy
is made available again when the terminal phos-
phate is transferred to other molecules. Such a
high-energy phosphate group is frequently desig-
nated by the symbol ~P; ATP can be written
AP ~ P ~ P. The terminal ~P of ATP is the
energy currency with which the cell pays for its
varied activities.

N— C— N-

-C C— CH2— O— P— O— P— O— P— OH

l\H H/H I I I

H C— C OH OH OH

OH OH

ribose, a 5-C sugar

3 phosphoric acids

adenosine

triphosphate

Exercise XI

FERMENTATION AND RESPIRATION 55

The main business of fermentation and
respiration is to supply cells with ~P; and the
yield of such groups is a measure of the efficiency
of these processes.

Yeast, a unicellular organism, can live nor-
mally either by fermentation, when no oxygen
is available, or by respiration, when oxygen is
present:

Yeast fermentation:

CnHiL'Oo -^ 2C2H5OH + 2CO2 + 2~P.

elhylalcohol

Yeast respiration:

CgHi20g + 6O2 -^

6CO2 + 6H2O + (approx.) 38~P.

Various other microorganisms ferment sugar
to different products, namely butyric acid, ace-
tone, etc., but the principle is always the same.
Animal cells also ferment sugar. Muscle cells,
for example, are often required to work more
rapidly than they can be supplied with oxygen,
and do so by fermenting sugar to lactic acid:

Muscle fermentation :

CgHioOb -> 2C3H6O3 + 2~P.

lactic acid

It will be noted that the chemical changes of
respiration just reverse those of photosynthesis;
similarly the energy of sunlight stored in sugars
by photosynthesis is released in respiration to
make high-energy phosphate bonds. Green
plants carry out both processes, photosynthesis
in the light, and respiration at ail times.

You have already measured photosynthesis in
Elodea by the rate of oxygen evolution in the
light. Now we will measure respiration in a
higher plant by the rate of oxygen consumption.
As you see by the above equation for respira-
tion of sugars, one molecule of CO2 is produced
for each molecule of O2 consumed, so that,
according to Avogadro's law, one would expect
no change in gas volume. We shall absorb CO2
as fast as it is formed, however, with soda lime
(a mixture of solid sodium hydroxide and cal-
cium hydroxide). Write the equation for this
process.

The rate of respiration varies greatly over the
life span of many organisms, being most rapid
during growth and development and slowing
down with maturity. Pea seedlings that are 3
to 4 days old have very rapid rates of respira-
tion, and thus were chosen for this experiment.
Since the products of respiration are also the
reactants of photosynthesis, it is advisable to
hold the latter process to a minimum during
your measurements. For that reason, the pea
seedlings were germinated in the dark, and so
lack chlorophyll. They green rapidly, however,
when exposed to light, so keep them shaded.

EXPERIMENTS
Respiration

We shall be working again with the volumeter
described originally in Exercise VI (pp. 35-36).
Fill one of the test tubes to within 2 inches of
the top with pea seedlings, tapping the test tube
against your hand to pack the seedlings. Insert a
cotton plug over the seedlings, and layer about 1
inch of soda lime over the cotton plug, to absorb
all carbon dioxide. Be sure that no soda
lime touches the seedlings. The second test tube,
which again will act as thermobarometer, should
be filled to 2 to 3 inches from the top with water,
to approximate the volume occupied by solid
material in the experimental tube.

Insert the rubber stoppers and adjust indicator
drops in the side-arms, this time placing the drop
in the experimental tube near the distal end of
the scale. Clamp the escape tubes and wait
about 5 minutes for equilibration. Take readings
in both side-arms at 3-minute intervals, each
time subtracting the reading in the thermo-
barometer from that in the experimental tube,
until the rate of change in the experimental tube
becomes constant. This measures the rate of
oxygen consumption.

If you now knew the rate of oxygen consump-
tion minus carbon dioxide production, you could
calculate the rate of evolution of carbon dioxide.
Figure out how to do this yourself; then do the
experiment.

56 FERMENTATION AND RESPIRATION

Exercise XI

The rate of carbon dioxide production di-
vided by the rate of oxygen consumption is the
so-called respiratory quotient (R. Q.). What do
you find it to be? What does the equation for
the respiration of sugar, shown above, predict?
What would the R. Q. be if an organic acid
(e.g., palmitic acid, CisH.nCOOH) or an amino
acid were being respired rather than a sugar?
Can you conclude from your R. Q. what types
of metabolites are being respired by these pea
seedlings?

Fermentation

Working in pairs, stir about Ysofa yeast cake
into 20 drops (1 ml) of glucose solution in one of
the volumeter test tubes. The other tube, to
serve as thermobarometer, should contain 3 ml
of water. Adjust kerosene drops at the proximal
ends of the side-arms in the volumeter, and take
readings in both side-arms every minute, each
time subtracting the volume changes in the
thermobarometer from those in the experimental
tube. It may take a few minutes for a constant
rate of change to be established, since the sugar
solution must first be saturated with carbon
dioxide.

Repeat this experiment, this time stirring the
yeast into 1 ml of galactose solution, to test the
ability of yeast to ferment this sugar.

To verify that the gas produced in fermenta-
tion is carbon dioxide, make use of the fact that
carbon dioxide reacts with calcium hydroxide to
yield an insoluble precipitate of calcium carbon-
ate. Pour the yeast suspension in glucose solu-
tion from the volumeter test tube into a small test
tube, connect a gas-delivery tube, and let the gas
bubble through limewater (calcium hydroxide
solution; this is prepared by stirring powdered
calcium hydroxide in water for a few minutes,
and filtering). If the gas production is not rapid
enough, add more yeast and glucose to your
fermentation mixture. It may also take a little
time for the carbon dioxide produced to break
through the foam.

Yeast ferments glucose, fructose, and mannose
indiscriminately. Why? Does it ferment galac-
tose? Why? It will help you to construct and
compare the molecular models of these sub-
stances.

The total energy change in fermenting a mole
of glucose (how many grams?) to alcohol and
carbon dioxide is about 20 kcal. If this change
makes 2 moles of ~P available, what is its
efficiency in producing useful energy?

Calculate the weights of carbon dioxide and
ethyl alcohol produced in fermenting one mole
of glucose. How do they compare?

The total energy change in respiring one mole
of glucose is 672 kcal. If this produces 38 ~P,
what is its efficiency ?

EQUIPMENT

Per student

2 wide test tubes

microscope

slide and cover slip

Per 8 students

4 test tubes, with assembly consisting of No. 0

rubber stopper with 5-mm diameter hole; 2" piece

of 6-mm diameter glass tubing

4 volumeters

4 gas-delivery tubes: 6" piece of rubber tubing Yg"

in diameter and a 6"-long dropper tube

2 dropping bottles of 10% glucose

2 dropping bottles of 10% galactose

2 250-ml beakers

funnel

filter paper

cotton (nonabsorbent)

soda lime

calcium hydroxide

cake of yeast

pea seedlings

XII, XIII

THE ARRAY OF LIVING ORGANISMS

(Readings: Weisz, Chapter 29. S.P.T., Chapter 19. Villee, pp. 83-84. R. Y.
Stanier, M. Doudoroff, and E. A. Adelberg, The Microbial World, Prentice-
Hall, 1957, Chapters 3-6. Other readings are listed at appropriate places within
the exercise.)

So far this semester we have studied general
properties and processes in living organisms,
emphasizing components and reactions rather
than the specific organisms in which they were
examined; and so we shall go on doing. Yet
we have already encountered a fair variety of
organisms, and next semester we shall deal with
many more. Also next term we shall study more
highly integrated phenomena as they appear in
more complex organisms.

Up to now, whenever we have encountered a
new organism, something has been said of its
biological position. The time has come to go
beyond such a piecemeal approach and to gain
a view of the entire array of living things.

Biological order as history and experiment

At first glance nature appears to abound with
an enormous diversity of living organisms.
Careful examination of the different forms, how-
ever, enables us to group them on the basis of
similarities in anatomical organization, embryo-
logical development, chemical constitution, and
other criteria. The catalogue of types that
results is highly useful in itself, both in reducing
the diversity to manageable proportions and in
enabling us to ascertain readily the general na-

ture of an organism once we are aware of its
name or of enough of its properties to place it
in the classification scheme.

But emerging from this classification, and
indeed woven inseparably into its fabric, we
find two tremendous concepts: that of origin
and descent; and that of progressive adaptation,
of ceaseless problem-solving, accompanying the
ceaseless expansion of organisms into every
environment that can support life, working out
in all environments their universal problems of
nutrition and reproduction. That is, we come
out not only with a history, but one that in-
volves direction, indeed many simultaneous di-
rections; in essence, the history of the explora-
tion of this planet as an abode for life. It is
this that transforms what might otherwise be a
tedious catalogue into a profound intellectual
adventure.

The dominant view that guides the construc-
tion of a scheme of classification — once one has
simply made order, grouping similar organisms
together and separating the groups in proportion
to their differences — is that all living organisms,
plant and animal alike, are linked together by
descent from common ancestors, from which
they evolved along separate paths to their pres-
ent state of divergence. Although each group

58 THE ARRAY OF LIVING ORGANISMS

Exercises XII and Xll!

TABLE 1

Plants

Animals

Perform photosynthesis

Live on organic materials, ultimately supplied by plants

Possess functional chlorophyll

Chlorophyll is not present, except rarely as functionless

pigment, retained from plant diet

Use starch as the principal food

Use glycogen or fat as principal food reserve

reserve

Have rigid cell walls

No cell walls

Display no active movement

Usually display active movements

(sessile)

Grow indefinitely to various

Usually grow to fixed size and shape

sizes and shapes

now alive has as long a history of evolution as
any other — no contemporary organism is the
ancestor of any other contemporary organism —
certain groups have changed relatively little over
long periods of time. From these, and from
genuine ancestors preserved as fossils, it is pos-
sible to construct a genealogy of living things,
a tree of life, that shows the lines of ancestry
and divergence among living forms. On such a
tree, all present-day organisms have equivalent
status, at the tips of branches. It is the stems
and branch points that express their evolution.

A first approach: the three kingdoms

All living things may be divided into three
great kingdoms: plants, animals, and protists:

Plants
Seed plants
Ferns
Mosses, liverworts

Animals
Vertebrates
Invertebrates
Sponges, jellyfish

Protists

Plant-like

Animal-like

Blue-green Algae Slime molds Protozoa

algae
Bacteria Fungi

The protists are mainly single-celled organ-
isms, sometimes containing many nuclei within

a single cell membrane (multinucleate). Some
are multicellular, but then display little or no
differentiation of tissues to perform specific
functions. We regard such aggregated protists
as colonial, to distinguish them from the multi-
tissued organisms. Usually the cells of such
colonial forms can also live independently, and
can give rise by division to new colonies. Most
protists are small; but red or brown seaweeds
may achieve great size and very complicated
shapes — giant kelps may be 150 feet long —
yet with little differentiation of tissues. (Vari-
ous authors include different groups within
the protists. Simpson, et al., include the proto-
zoa among them, but place the algae among the
plants. Weisz does not use the category protist
at all, and puts the animal-like protists among
the animals and the plant-like protists among
the plants. Stanier, et al., in The Microbial
World group them as here, and provide an ex-
cellent discussion of the relations among them.)
Fortunately most of you have good, working
notions of animals and plants, and could prob-
ably decide fairly accurately into which group
to place even unfamiliar forms. The character-
istics that divide these two kingdoms are sum-
marized in Table 1.

Orders of classification

The members of each kingdom are arranged
in a hierarchy of groupings. The major groups

Exercises XII and XIII

THE ARRAY OF LIVING ORGANISMS 59

TABLE 2

Paramecium

Corn

Lobster

Man

Phylum

Ciliophora

Tracheophyta

Arthropoda

Chordata

(Subphyium)

Pteropsida

Vertebrata

Class

Ciliata

Angiospermae

Crustacea

Mammalia

Order

Holotricha

Monocotyledonae

Eucarida

Primates

Family

Hymenostomata

Gramineae

Decapoda

Hominidae

Genus

Paramecium

Zea

Homarus

Homo

Species

caudatum

mays

americanus

sapiens

are called phyla; from these one works down
through smaller and smaller divisions, finally to
the double name, genus and species, by which
any single type of organism is called.

Some idea of the task involved in classifying
and naming living organisms may be gained
from the realization that there are about 300,000
living plant and over a million animal species.
It is a little like assigning a meaningful status
and relationships to everyone in Los Angeles.

The main taxonomic divisions can best be
illustrated by classifying a few familiar organ-
isms from the three kingdoms, as we have done
in Table 2. How much of this kind of thing do
we want you to know? We want you to know
the really important things that are involved in
and lie behind such classification schemes. We
will try to point them out to you as we go along,
and they are summarized in the diagrams which
follow.

A depressing thing about much of the tech-
nical terminology used in classification is that
it keeps changing. Even at any given time, we
find great disagreement involving even the main
categories. For example, the terms "Trache-
ophyta" and "Pteropsida" used in the classifica-
tion of corn in Table 2 are characterized as
"abandoned" by a recent authority (H. C. Bold,
The Plant Kingdom, Prentice-Hall, 1960). In-
deed, what are eight plant phyla in Simpson,
et ai, have now been reclassified by Bold into
24 "divisions."

What saves this situation from its zealots are
two things: common names, which do stay in
use; and the possibility of expressing most of

the fundamental relationships in plain English.
Thus in place of the above technical classifica-
tion of corn, already obsolete according to some
authorities, we can describe it safely as a vascu-
lar, flowering, seed plant, one of the grasses —
indeed, Indian corn or maize.

Another point important for us is that we
classify only to the extent that serves our needs.
For example, most biologists would say of
Paramecium that it is a protozoan and a ciliate,
and let it go at that. As for the lobster, most of
us are content to know that among the Arthro-
pods it is a decapod crustacean. For the most
part, the remaining terms used in Table 2 would
be used only by specialists.

So relax, use common names and ordinary
English as much as you like, but do learn to
recognize the important groups of protists, ani-
mals, and plants, and learn as much as you can
of the relationships among them. Read your
text, and use the following pages as a guide to
what we most want you to learn.

THE PROTISTS
Guidelines

(1) Close relations between bacteria and blue-
green algae as structurally simplest pro-
tists.

(2) The "flagellate line."

(3) Colonial algae as first approaches to dif-
ferentiated multicellular organisms.

60 THE ARRAY OF LIVING ORGANISMS

Exercises XII and XIII

Blue-green
algae

Bacteria

HIGHER PLANTS

Stoneworts
Plant- like

flagellates
Green )
Brown i ,
Red °'9°«-

GoldenI

PROTISTS

Lichens

Fungi

Slime

molds

HIGHER ANIMALS

PROTOZOA
Animal-like flagellates
Ciliates

(Paramecium)
Rhizopods

(ameba)
Sporozoa

(parasitic:

Plasmodium

of malaria)

(Ancestral flagellates?)

Each of these groups is classified at present os a distinct phylum, except that the term
"Protozoa" now designates o subkingdom within either the protist or animal kingdom; and
the flagellates as a whole constitute the Phylum Mastigophoro (whip-bearers), which includes
the plant- and animal-like flagellates and the dinoflagellates.

Among the protists the bacteria and blue-
green algae stand somewhat apart. They are
highly successful groups, particularly the bac-
teria; yet structurally they represent the simplest
of living cells, and so are sometimes spoken of
as "lower" protists, though not ordinarily to
imply an ancestral position. The blue-greens,
like the true algae, are photosynthetic, evolving
oxygen in this process. The bacteria may be
photosynthetic or not, but never evolve oxygen
in photosynthesis. These two groups share the
following further properties:

(1) Small size, including the smallest living
cells.

(2) No separate nucleus, surrounded by a
membrane.

(3) No chloroplasts.

(4) Motionless cytoplasm.

(5) A chemical distinction, the exclusive pos-
session of diaminopimelic acid.*

The flagellates may provide the thread that
binds the protists together, and leads off" in the
directions of the multitissued plants and animals.
It is this that has prompted the thought of a

flagellate ancestor, probably photosynthetic,
which may have given rise on the one hand,
with some structural retrogression, to the bac-
teria and blue-greens, and on the other to the
more highly developed protists, both plant-like
and animal-like. It is as though, starting with
an ancestral green flagellate, the photosynthetic
capacity had been exploited in developing the
algae, and the motility exploited in developing
such protozoa as the ciliates.

Modern flagellates include both green, photo-
synthetic types (e.g., Euglena and Chlamydo-
nionas, both of which you have studied), and
colorless animal-like forms, such as the trypan-
osomes responsible for African sleeping sickness.
Flagellated cells form one stage in the develop-
ment of slime molds. We have also the colonial
green flagellates, some of which display a first
differentiation of function, certain cells being
specialized for reproduction (recall Volvox).
These may represent first approaches to the
formation of the multitissued plants. On the
other hand the sponges, the first of the animal
phyla, possess characteristic flagellated collar
cells that greatly resemble free-living flagellated
protozoa. The cells of sponges also display an

'■ HOOC— CHNHa— (CH2):i— CHNH2— COOH, a carboxylated lysine.

Exercises XII and XIII

THE ARRAY OF LIVING ORGANISMS 61

THE PLANT KINGDOM

Thallophytes _
(nonvascular)

Bryophyfes (mosses:
first lond forms;
thickened epidermis)

Plant-like
protists

Mostly extinct

Sphenopsids

(horsetails)

Lycopsids

(club mosses)

Psilopsids

_Tracheophytes_
(vascular)

Monocotyledons Dicotyledons

(grasses) (others)

Angiosperms
(flowering plants:
enclosed seeds and fruits)

Filicinae
(ferns:

roots, stems,
leaves)

Gymnosperms

(conifers: dry
fertilization, seeds)

Pteropsids
(leafy plants)

extraordinary independence on occasion. A
sponge can be pressed through cheese cloth so
that all the cells are separated. Left to them-
selves, they reaggregate to form a new sponge.

Stoneworts, though a small group, are the
most complex algae. You already know one of
them, Nitella.

Lichens are composite associations of algae
with fungi, living together to their mutual benefit
(symbiosis), the alga photosynthesizing and the
fungus providing water and a source of nitrogen
for both partners.

The slime molds display both plant-like and
animal-like characteristics, passing through
stages of free-living, unicellular flagellates and
amebae. Then the ameboid individuals mi-
grate together and form a great multinucleate
slug, bounded by a single membrane. This may
then differentiate into a beautiful fruiting body,
carrying a bulb containing spores at the end of a
long stalk, very plant-like in appearance.

THE PLANT KINGDOM

(Readings: S. P. T., Chapter 21. Weisz, Chapter 30.
Villee, Chapters 11 and 12. Optional readings are
C. P. Swanson, The Cell, Prentice-Hall, 1960, and
H. C. Bold, The Plant Kingdom, Prentice-Hall, 1960.)

Principal groups and numbers of species

Flowering plants (250,000)

Conifers (600)

Gingko (maidenhair tree) (1)

Cycads (100)

Ferns (9500)

Horsetails (25)

Club mosses (1000)

Mosses (14,000)

Liverworts (9000)

Total : approximately 300,000 species

62 THE ARRAY OF LIVING ORGANISMS

Exercises XII and XIII

Guidelines

(1) Alternation of haploid gametophyte and
diploid sporophyte generations.

(2) Progression from gametophyte-dominance
(mosses, liverworts) to sporophyte-dom-
inance.

(3) Emergence from water to land : vasculari-
zation.

(4) Mosses, liverworts, and ferns as the
amphibia among plants.

(5) Development of dry fertilization in con-
ifers and flowering plants.

A major difference between plants and ani-
mals, one which runs throughout the entire
plant kingdom and extends back among the
colonial algae, involves their reproductive habits.
Animals are almost invariably diploid (the
nuclei of their cells contain /?fl/>.y of chromosomes,
forming a double set) except for the mature germ
cells (gametes: eggs and sperm), which are
haploid (i.e., each contains a single set of
chromosomes).

In contrast with this, plants alternate a diploid,
spore-bearing (sporophyte) generation with a
haploid, gamete-bearing (gametophyte) genera-
tion. It is true that in higher plants the gameto-
phytes, male and female, are very small, and live
upon the diploid sporophyte, which forms the
main body of the plant. Yet in lower plants these
relations are reversed, and all plants display the
basic pattern of alternation of generations. This
is how it works:

(1) Diploid sporophyte by meiosis (cell divi-
sion with reduction from double to
single chromosome number) yields hap-
loid spores.

(2) Haploid spore by ordinary cell division
(mitosis) yields a haploid gametophyte,
bearing haploid gametes.

(3) Fertilization of an egg by a sperm restores
the diploid number of chromosomes, and
by mitosis yields a new diploid sporophyte.

The forerunners of modern plants were un-
doubtedly aquatic. They had to face neither the
problem of conveying food and water over rela-

tively long distances, as must be done in the
larger land plants, nor providing devices by
which the germ cells could find one another,
which is no problem in water.

Mosses and liverworts, most of which made
the transition from water to land, have compro-
mised with both these problems. They are non-
vascular (i.e., lack conducting vessels), and hence
are restricted to a small size, which keeps them
close to the ground. Also, at the time of sexual
fertilization they must manage to collect enough
water for the sperms to swim to the eggs. In
these plants the gametophyte is the dominant,
free-living generation; it is what we mainly see
as the plant. The sporophyte is a relatively small
structure that remains permanently attached to
the gametophyte.

With the evolution of specialized tissues to
overcome the difliculties of terrestrial living,
plants came to cover almost all the land masses.
The primary step in this development was the
evolution of vessels to conduct water and dis-
solved materials throughout the organism. This
step is so important that the plant kingdom has
traditionally been divided into two subking-
doms: the nonvascular thallophytes (algae,
fungi, bryophytes) and the vascular tracheo-
phytes, the higher land plants. The tracheo-
phytes have also developed further specialized
tissues, leaves, roots and stems, which have aided
in the colonization of the land. The simplest
tracheophytes, the psilopsids, club mosses, and
horsetails, most of which are now extinct, dis-
play the beginnings of all these developments.

In all the tracheophytes the sporophyte is the
dominant generation; it is what we see as the
plant. In ferns, the gametophyte is still free-
living, though reduced to a very small size. In
the more advanced groups the gametophytes are
represented by only a few cells.

The leafy plants (Pteropsids) have diverged
in the course of their evolution to form three
large groups: the ferns, conifers, and flowering
plants. All ofthem have well-differentiated roots,
stems, and leaves. The ferns, however, have still
not won freedom from one condition of aquatic
life; they still need water in which the sperms,

Exercises XII and XIII

THE ARRAY OF LIVING ORGANISMS 63

which develop in one part of the tiny ganieto-
phyte, can swim to the eggs, which develop in
another part of the gametophyte, at the bottom
of a cleft.

The conifers have made further steps in
adaptation to land life. They have developed
two types of spores, which give rise respectively
to male and female gametophytes. The male
gametophyte, now a pollen grain, is dispersed by
the wind or by insects, so eliminating the need
for water. The female gametophyte is entirely
parasitic, living always within the tissues of the
sporophyte. On fertilization of the egg by the
sperm delivered by a pollen grain, it yields a
sporophyte embryo, which, provided with food
and a protective coat, is the seed. On being
planted, this develops into the mature sporo-
phyte.

In the flowering plants (angiosperms) the re-
productive systems achieve further refinement.
Stems and leaves are modified to form flowers,
which contain the gametophyte generation. (We
shall study flower structures in detail next
semester, so we need not go into them deeply
now.) The gaily colored flowers with their
perfumes and nectars attract insects and birds,
which willy-nilly transport pollen from one
flower to another, ensuring efficient fertilization.
Fertilization of the egg within the ovary of a
flower leads, as in conifers, to the growth of an
embryo sporophyte, which, with its surrounding
tissues and protective coat, constitutes the seed.
The angiosperms, however, go one step further
than the conifers, enclosing the seed in a fruit,
which develops from tissues of the flower. The
fruits may be eaten by animals, which dissemin-
ate the seeds over the countryside.

The angiosperms are the most complex and
successful land plants. They include about | of
all living plant species. They divide into the so-
called monocots and dicots on a rather trivial
basis, whether the cotyledons, the food-con-
taining, leaflike structures within the seeds, are
single (as in a corn seed) or double (as in a
peanut). The monocots include the grasses, and
several minor groups, palms, lilies, onions, and
orchids. The dicots are almost everything else.

THE ANIMAL KINGDOM

(Readings: S.P.T., Chapters 22 and 23. Villee, pp.
195-207; Chapters 14 and 15. Weisz, pp. 667-702.
An excellent additional source that does almost the
whole job in a couple of hours of pleasant reading is
the Golden Science Guide, Zoology, by H. S. Zim,
H. I. Fisher, and R. W. Burnett. Also, see an excel-
lent discussion in Weisz, Chapters 29 and 31, and
the fine pictures in Ralph Buchsbaum's Animals
Without Backbones, University of Chicago Press,
rev. ed., 1948.)

Principal groups and numbers of species

PHYLUM CHORDATA (50,000)

Subphylum: vertebrates
Classes: mammals
birds
reptiles
amphibia
bony fishes
cartilaginous fishes
placoderms (extinct, armored,

jawed fishes)
jawless fishes (Cyclostomes)
Subphyla (3) of protochordates (Amphioxus,
acorn worms, tunicates)
PHYLUM ECHiNODERMATA, "spiny-skinncd"

(6000)
PHYLUM ARTHROPODA, "jointed-legs" (1,000,000)
Classes: insects

arachnids (spiders, horseshoe

crab)
Crustacea (crabs, lobsters, bar-
nacles)

PHYLUM MOLLUSC A (100,000)

Classes: gastropods (snails, slugs, whelks)
pelecypods (clams, mussels)
cephalopods (squid, octopus,
nautilus)
PHYLUM ANNELIDA, segmented worms (10,000)
PHYLUM NEMATODA, roundworms (10,000)

PHYLUM PLATYHELMINTHES, flatworms (10,000)

PHYLUM COELENTERATA, corals, jcllyfishes, hydro-
zoa (10,000)

PHYLUM PORiFERA, sponges (15,000)

64 THE ARRAY OF LIVING ORGANISMS

Exercises XII and Xlli

THE ANIMAL KINGDOM

Crustacea
(crabs, lobster)

Insects

Birds

Mammals

Arachnids (spiders,
horseshoe crab)

Arthropods
("jointed legs"

Molluscs —^
(snails, clams,
squid)

Annelids — ^—
(segmented worms)

Platyhelminthes-
(flatworms)

- Reptiles

- Amphibia

-Bony fishes
-Cartilaginous fishes

- Jawless fishes

Vertebrates

■ Roundworms,
rotifers

Chordctes

Protochordates
(Amphioxus, acorn
worms, sea squirts)

Echinoderms-

PROTOSTOMIA

DEUTEROSTOMIA

Three cell layers

Coelenterates
(corals, jellyfish)

Porifera ■
(sponges)

Ctenophores
(comb jellies)

Two cell layers

PROTOZOA

Of the approximately 1,200,000 species of liv-
ing animals, about 97% are invertebrates and
about 75% are insects.

Guidelines

(1) Two cell layers (sponges, coelenterates) to
three cell layers.

(2) Development of a true body cavity
(coelom) lined with mesoderm.

(3) Adaptations for emergence from water to
land and air.

(4) Grouping of annelids, arthropods, and
molluscs as protostomes ("annelid super-
phylum") and of echinoderms and chor-
dates as deuterostomes ("echinoderm
superphylum").

The coelenterates and comb jellies are built
of two layers of cells, the internal endoderm

lining the digestive cavity, and the external ecto-
derm. All further phyla add a third layer be-
tween these two, the mesoderm.

The coelenterates and flatworms have only a
single opening into the digestive cavity, which
therefore serves as both mouth and anus. The
higher phyla possess tubular digestive systems,
open at both ends. In arriving at this condition,
the annelids, arthropods, and molluscs (proto-
stomes) convert the primitive opening to a
mouth, and break through a new opening for the
anus. In the echinoderms and chordates (deuter-
ostomes), the primitive single opening becomes
the anus, and a new opening is broken through
to form the mouth.

One of the most important developments is
the formation of a body cavity (coelom) lined
with mesoderm, in which the internal organs lie.
The sponges and coelenterates have no meso-
derm, and no such cavity. The flatworms have
a solid mesoderm, and no cavity. The round
worms have a restricted mesoderm, and apart

Exercises XII and XIII

PLANT AND ANIMAL CLASSIFICATION 65

from it an internal space lined by ectoderm and
endoderm. The higher phyla all possess a true
coelom.

In the protostomes, the mesoderm is formed
by cells which wander in from the ectoderm and
bud off the endoderm to form a solid layer, in
which the cavity later develops that becomes the
coelom. In the deuterostomes, the mesoderm is
formed by outpocketings of the endoderm to
form hollow pouches, the cavity of which is the
coelom.

WORK ASSIGNMENT

These two laboratory sessions will be devoted
to a study of the diversity of organisms, animal
and plant. Representative plants and animals
of all the major groups will be on display, and
the student should study each of them carefully.
There will also be a group of unlabeled plants
and animals to examine and to compare with the
labeled specimens.

Working independently, place the unlabeled
organisms within their major categories: phyla
for the most part; but also subphyla among the
vascular plants, and classes among the Pterop-
sids; similarly, classes among two of the animal
phyla, the Arthropods and Chordates. In a few
sentences and perhaps a sketch, defend each of
your identifications. Hand in your results at
the end of the period.

To prepare for these laboratory sessions, and
to supplement them, students should, if possible,
spend two or three hours in the nearest accessi-
ble museum that has a display of representative
organisms of the major plant and animal phyla.
For such an excursion to be meaningful, the
student should first abstract the pertinent ma-
terial from our list of readings, and probably
have with him the charts that appear in this
manual, as well as the outline that follows.
Notes should be taken about the phyla observed,
their distinctive characteristics, the range of
organisms they include, and the relationships
among them.

A Short Guide to
Plant and Animal Classification

During the year a variety of organisms will be
used in the laboratory. It will be worth while to
attempt to classify them. As an aid in doing this,
an abbreviated guide is included here. Various
authors disagree on minor points of classifica-
tion, but practically any textbook of botany or
zoology can be consulted for more details.

PLANT KINGDOM*

Plants are usually considered as organisms
with stiff cell walls and with chlorophyll.

* Adapted from C. A. Villee, Jr., Biology, 4th ed.,
Saunders, 1962.

Subkingdom Thallophyta (Gr. thallos, young
shoot; phyton, plant). Plants not forming em-
bryos. These are the simplest plants without true
roots, stems, or leaves; there is little differentia-
tion of tissues.

1. PHYLUM CYANOPHYTA (or Myxophyta) (Gr.
myxa, mucus; phyton, plant). The blue-green
algae. Chloroplasts and nuclei not distinct.

2. PHYLUM EUGLENOPHYTA (Gr. eu, Well, truc;
glene, pupil of the eye or socket of a joint). The
Euglenoids.

3. PHYLUM CHLOROPHYTA (Gr. chloros, green;
phyton, plant). The green algae. Contain dis-
tinct nuclei and chloroplasts. Spirogyra and
Oedogonium.

66 PLANT AND ANIMAL CLASSIFICATION

Exercises XII and XIII

4. PHYLUM CHRYSOPHYTA (Gr. chrysos, gold;
phyton, plant). The yellow-green algae, the
golden-brown algae, and the diatoms.

5. PHYLUM PYRROPHYTA (Gr. pyrrho, red; phy-
ton, plant). The Cryptomonads and dino-
flagellates.

6. PHYLUM PHAEOPHYTA (Gr. pfiaios, dun-
colored; phyton, plant). The brown algae;
multicellular, often large bodies; large seaweeds
such as Fucus.

1. PHYLUM RHODOPHYTA (Gr. rhodon, rose;
phyton, plant). The red algae; multicellular;
usually marine; sometimes impregnated with
calcium carbonate.

8. PHYLUM SCHIZOMYCOPHYTA (Gr. scluzein,
to cleave; mykes, fungus; phyton, plant). The
bacteria.

9. PHYLUM MYXOMYCOPHYTA (Gr. myxa,
mucus; mykes, fungus; phyton, plant). The
slime molds. Made up of protoplasm contain-
ing many nuclei but without division into dis-
tinct cells. Movement is ameboid.

10. PHYLUM EUMYCOPHYTA (Gr. eu, well, true;
mykes, fungus; phyton, plant). The true fungi.
This phylum contains the Phycomycetes (bread
and leaf molds), the Ascomycetes (yeasts,
mildews, cheese molds), the Basidiomycetes
(mushrooms, toadstools, and rusts), and the
Fungi Imperfecti (fungi which are difficult to
classify, such as that causing athlete's foot).

Subkingdom Embryophyta (Gr. embryon, to
swell; phyton, plant). Plants forming embryos.

11. PHYLUM BRYOPHYTA (Gr. bryon, moss;
phyton, plant). No conducting tissue; multi-
cellular; terrestrial; alternation of sexual and
asexual generations (the prominent plant is the
sexual generation, the gametophyte). Mosses,
liverworts, and hornworts.

12. PHYLUM TRACHEOPHYTA (Gr. tracheia, artery:
phyton, plant). Vascular plants.

Subphylum Psilopsida: rootless and leafless
vascular plants.

Subphylum Lycopsida: clubmosses; small
green leaves and a simple conducting system.

Subphylum Sphenopsida: horsetails; jointed
stems and scalelike leaves.

Subphylum Pteropsida: complex conducting
systems and large, conspicuous leaves. This
subphylum is divided into three major classes:
FiUcinae (ferns), Gymnospermae (conifers,
cycads, most evergreens and shrubs — no
true flowers or ovules present — the seeds are
born naked on the surface of the cone scales),
and Angiospermae (flowering plants with
seeds enclosed in an ovary). The Angio-
sperms may be subdivided into the Dicotyle-
dons and Monocotyledons. The dicots have
embryos with two cotyledons (seed leaves);
most flowering plants belong to this sub-
class. The grasses, lilies, and orchids, how-
ever, are monocots, their embryos having only
one seed leaf.

ANIMAL KINGDOM*

Ten questions are particularly useful in dis-
tinguishing phyla of the animal kingdom:

(1) Unicellular or multicellular?

(2) Diploblastic or triploblastic? (Is the
body composed of two layers, the ecto-

* Adapted from M. F. Guyer, Animal Biology, 3rd
ed.. Harper Brothers, 1941.

derm and endoderm, or are there three
layers, ectoderm, endoderm, and meso-
derm?)

(3) Body saclike or built on tube-within-a-
tube plan?

(4) True digestive cavity present or absent?

(5) Segmented or nonsegmented?

(6) Asymmetry, bilateral symmetry, or radial
symmetry?

Exercises XII and XIII

PLANT AND ANIMAL CLASSIFICATION 67

(7) Appendages present or absent? If pres-
ent, jointed or not?

(8) What is the nature and position of the
skeleton (exoskeleton or endoskeleton)?

(9) Notochord or vertebral chord present or
absent?

(10) What is the structure and position of vari-
ous organ systems?

I. Protozoa (unicellular animals)

1. PHYLUM PROTOZOA (Gr. protos, first;
zoon, animal). Single cells or loosely ag-
gregated colonies of single cells. Amoeba,
Euglena, Paramecium, Stentor, Volvox, etc.

II. Metazoa (multicellular animals containing
specialized tissues)

A. Parazoa (no true digestive cavity)

2. PHYLUM PORIFERA (L. pours, porc ; ferre,
to bear). Sponges. Sessile; aquatic; diplo-
blastic; radially symmetrical body consist-
ing of cylinder closed at one end: budding
and folding of body often present; digestion
does not occur in central cavity but rather
in individual cells; generally a skeleton
present.

B. Enterozoa (true digestive cavity present)
(a) Enterocoela (only cavity in the body is
the digestive cavity)

3. PHYLUM COELENTERATA (Gr. koUoS, hol-

low; enteron, intestine). Hydroids, jelly-
fishes, sea anemones, and corals. Diplo-
blastic; body may be tubular (polyp) or
bell- or umbrella-shaped (medusa); in some
organisms these forms alternate during life
cycle; budding to form colonies is common;
body is a double- walled sac; body cavity
is not separate from digestive tract; radiate
symmetry.

4. PHYLUM CTENOPHORA (Gr. kterjos, comb;
p/wros, bearing). Sea walnuts, comb jellies.
Triploblastic (ectoderm, endoderm, and
mesoderm); radial combined with bilateral
symmetry; body with eight meridionally
arranged rows of swimming plates; a few
species are ribbon-shaped.

(b) Coelomocoela (coelom present, tube-
within-tube structure)
(i) Nonsegmented

5. PHYLUM PLATYHELMINTHES (Gr. platys,

broad; helmintlws, worm). Flatworms.
Bilaterally symmetrical; no true segmenta-
tion, flattened dorsoventrally, no blood
vascular system; no anus; mostly parasitic.
The flatworms are triploblastic but don't
have a definite coelom. They do, however,
have gonocoels which represent a primitive
form of coelom.

6. PHYLUM NEMATHELMINTHES (Gr. nematos,
thread; hehninthos, worm). Roundworms.
Bilaterally symmetrical; unsegmented; usu-
ally long and thin; most often contain an
alimentary tract with mouth and anus;
body cavity present; papillae or spines at
anterior extremity of body; both parasitic
and free-living forms.

7. PHYLUM ROTIFERA (L. rota, v^\itt\; ferre,
to bear). Common small aquatic forms;
usually found in fresh water but also may be
marine and parasitic; ciliary movements on
anterior end suggest a rotating wheel; ner-
vous system present; body enclosed in flexi-
ble cuticle; body usually roughly cylindrical
tapering at posterior end to form a foot;
well-developed digestive system with mouth,
pharynx, glandular stomach, intestine, and
anus.

8. PI YLUM BRYOZOA (Gr. bruon, moss;
zoon, animal). Moss animals and sea mats.
Small; aquatic, sessile, unsegmented; usu-
ally colonial; ciliated tentacles surround
mouth; U-shaped intestine with anus near
mouth; colonies often look superficially
like hydroid colonies.

9. PHYLUM BRACHIOPODA (L. bracluum, arm;
Gv. podos,foo\). Lamp shells. Unsegmented
body covered with calcareous bivalve shell;
mouth is between two spiral, ciliated arms
which lie within shell; many fossil forms.

10. PHYLUM ECHINODERMATA (Gr. ecllinOS,

hedgehog; derma, skin). Sea lilies, starfish,
sea cucumbers, sea urchins. Marine; radi-

68 PLANT AND ANIMAL CLASSIFICATION

Exercises XII and XIII

ate symmetry in adults; most forms with
spiny skin; triploblastic with large coelom
and distinct ahmentary canal; calcareous
plates provide protective exoskeleton; tube
feet for locomotion.

11. PHYLUM MOLLUSCA (L. moUuSCUS, Soft).

Chitons, snails, slugs, whelks, clams, scal-
lops, oysters, ship worms, squids, octopuses.
Unsegmented; appendages not jointed; usu-
ally a shell and mantle; "foot" usually
present.

(ii) Segmented

12. PHYLUM ANNELIDA (L. annelus, a little
ring). Segmented worms such as earth-
worms, marine worms, leeches. Blood ves-
sels, excretory organs, and nervous system
segmentally arranged; distinct coelom; ven-
tral double nerve cord of "ladder type";
appendages not jointed.

13. PHYLUM ARTHROPODA (Gr. arthron,]omi\
podos, foot). Crustaceans such as crayfish,
water fleas, crabs, barnacles; centipedes,
millipedes; insects; spiders, mites, ticks,

scorpions. Segmented (but body cavity is
continuous and without transverse septa;
segmentation shown internally in arrange-
ment of organs); groups of segments tend
to fuse into larger regions (head, thorax and
abdomen); paired, jointed appendages; exo-
skeleton; nervous system of "ladder type";
main longitudinal blood vessel dorsal to
alimentary canal.

14. PHYLUM CHORDATA (Gr. chorde, cord).
Internal skeleton with a notochord sometime
during life history; gill clefts in throat some-
time during life history; central nervous
system is tubular and dorsal. The phylum
Chordata consists of several subphyla, the
most important of which is the Craniata (or
Vertebrata), the backboned animals. Among
the members of the subphylum are the
Cyclostomata (lampreys and hagfishes),
the Elasmobranchii (sharks and rays), Pisces
(bony fish). Amphibia (salamanders, frogs
and toads), Reptiha (lizards, snakes, turtles,
crocodiles), Aves (birds), and Mammaha
(mammals).

EQUIPMENT

Laboratory exhibit of plants and animals representing all major groups, labeled and unlabeled

XIV

VERTEBRATE ANATOMY

(Readings: Weisz, pp. 201-223; also browse through Chapters 12, 13, 14, 20
and 21, looking especially at the diagrams and photographs. S. P. T., pp. 1 17-
158, and browse through to p. 220. Villee, pp. 234-242, and browse through
Chapters 17-25.)

Our first semester in the laboratory was de-
voted primarily to the forms and activities of in-
dividual cells. We stressed mainly widespread or
universal similarities among cells, whether ani-
mal, plant, or protist.

This semester we will study the aggregations of
cells which compose the tissues and organs of the
higher organisms. A tissue is a group of cells
which display common functional and/or mor-
phological properties. Usually in a tissue the
cells are bound together to form sheets, layers, or
more-or-less solid structures; but this need not
always be so. The circulating blood cells in
vertebrates, for example, constitute a tissue. The
higher animals are composed mainly of four
types of tissue: epithelial, connective, muscular,
and nervous. Higher plants also possess four
primary types of tissue (protective, meristematic,
"fundamental," and conductive or vascular). An
organ is a structure composed of two or more
tissues that performs a specialized function.

Just as we started last semester by surveying
types of cells, the first two laboratory sessions
this semester will be devoted to studying a few
typical higher organisms. In this first period we
shall study vertebrate anatomy; next week we
will examine the anatomy of higher plants.

The work this week may be done in pairs, one
partner dissecting a rat, the other a frog. As you
dissect, compare the similarities and differences
between these vertebrates. Learn the names,
locations, appearance, and functions of the
various organs you encounter. Your textbook
will help, as also will wall charts posted in the
laboratory. You and your partner should try
to dissect in the same body area at the same
time.

A systematic procedure, outlined below, will
direct you to the major structures. Strike out on
your own, if you like; but if you do, plan ahead
how you are going to go about it.

When you have finished examining an organ,
it is often helpful to remove it. If you choose to
do this as you go along, slice into each structure
you remove. Note its internal appearance,
whether it is solid or possesses a cavity, and
whether it appears homogeneous or differenti-
ated into distinct regions. Keep your animal and
the organs you have removed from it moist, and
they will retain their natural shape and appear-
ance throughout the period.

Stained sections of the major organs will
probably be available from your instructor.
Although this will be a good time to look at them.

70 VERTEBRATE ANATOMY

Exercise XIV

we will examine such sections in greater detail
later, so don't spend too much time with them
now.

The animals will be given to you alive but
anesthetized. One of the problems that biologists
face, and that you are facing now, is how to
examine what goes on inside animals without
causing them pain. "Anesthesia" literally means
lack of feeling or sensation. We try to achieve
this either by rendering the higher centers of the
brain functionless with the use of a narcotic
("anesthetic") or by destroying those centers.
Whatever one does in this regard should be done
skillfully and quickly. If you pith your own frog,
know exactly what you are going to do, and be
ready to do it before you make the first move.
All the biologists we know take a lot of trouble
with such procedures. Speed and skill are of the
essence. If an animal needs to be killed in the
course of a laboratory procedure, one takes
similar precautions, trying whenever possible to
kill in one stroke an animal that has been handled
gently up to that point.

The particular point of the procedures we have
used here with the rat and frog is to abolish pain,
yet permit you to examine the organs in a func-
tional state. Take full advantage of this op-
portunity, and be prepared to begin as soon as
the animal is ready. Work as fast as you can,
consistent with care and thoroughness.

The rats will be given an overdose of bar-
biturate by the instructor at the start of the
period. Watch the anesthesia take hold. This in
itself is instructive. The barbiturates inhibit the
higher centers of the brain first — the cortical
centers — lessening the animal's coordination.
The first effect is a staggering gait. (This is what
ethyl alcohol does to us.) Gradually the animal
becomes immobile, though its reflexes still re-
spond to external stimuli. The reflexes are con-
trolled through lower centers of the brain or
through the spinal cord, both of which are more
resistant to narcosis. Eventually the reflexes also
cease to respond, yet the animal continues to
breathe. The respiratory center in the brain
stem is extremely resistant to narcosis. Why do
you think this is so?

At this point, when the animal no longer
responds to stimuli, but is still breathing, begin
the dissection.

The frogs are "anesthetized" by destroying
(pithing) their brains. One of the instructors will
demonstrate this procedure, and will pith your
own frog if you wish.

Pithing is done by quickly inserting a dissecting
needle, directed forward into the skull, at the
point where it joins the backbone, meanwhile
moving the point of the needle from side to side
as far as it will go, to cut as many as possible of
the nervous connections. If you hold a frog
gently but securely, and bend its head a little
downward, you can find a little depression at the
back of the head that marks its joint with the
backbone. This is where the needle should be
inserted, with one swift motion that gets inside
the brain cavity, stirring as it advances.

THE RAT

First look carefully at the external appearance
of your animal, and compare it with the frog.
Note diff'erences in texture of the skin. Where
does the head end ? What do you see in the way
of ears? How many digits are on the feet? Feel
such major body landmarks as the rib cage, back-
bone, and the connection of the backbone with
the skull. Where is the heartbeat strongest?
Note the breathing movements.

Lay the animal on its back, and slit open the
skin from the jaw to the genital openings. Be
careful not to cut through the underlying tissues.
Separate the skin laterally, using your fingers
or the blunt end of the scalpel. (If you have
difficulty here, the instructor will demonstrate.)

Note: The popular image of a biologist has
him dissecting with a scalpel. The truth is that
the cutting edge of the scalpel is used relatively
little. Most cutting is done with scissors, and
most dissecting is done by prying, pushing, and
lifting things apart rather than cutting. You will
probably find the spade-shaped, blunt end of the
scalpel more generally useful than the blade.
When you really want to slice, as, for example,

Exercise XIV

VERTEBRATE ANATOMY 71

an organ that you have removed, a single-edged
razor blade ordinarily does better than the
scalpel.

Open the abdomen about midway down from
the rib cage. Extend a longitudinal slit forward
to the ribs (be careful not to go beyond) and back
to the genital openings. Make lateral slits down
the side of the body wall parallel to the rib cage.
Observe the packing of the abdominal contents;
poke about, noting connections and relative
positions. Don't be afraid to lift the organs
away from one another. Watch the slow peristal-
tic movements of the intestine. Pull the liver
down and observe the lung through the trans-
parent part of the diaphragm.

Cut through one side of the diaphragm and the
rib cage on that side, staying close to the midline.
Note the collapse of the lung. (Why does it
collapse?) Pull open the rib cage and observe
the heartbeat. Describe the motions. Do all the
parts beat simultaneously? Collapse the lung
on the other side and remove the ribs and ster-
num, over the heart. This will allow you to
inspect the heart more closely. Try to trace the
major vessels to and from the heart (the vena cava
and aorta). Can you distinguish arteries and
veins? It might help to remove the white thymus
gland, which is found anterior to the heart and
may obscure its atria (auricles). (You may
keep your animal's heart beating longer and ob-
serve the expansion of the lungs by inserting a
dropper pipet attached to a rubber tube into the
trachea and breathing for the rat. The instruc-
tor will demonstrate this for you.)

By this time, anoxia is probably overcoming
the heart and other tissues. The muscles may
twitch, but these are "automatic" responses; the
animals feels nothing. After you have observed
the heart, cut a slit into the end of the ventricle
with your scalpel to bleed the animal. Take it
over to the sink, and wash away the blood. Now
start dissecting the abdominal contents.

First, examine the liver. The veins draining the
intestines pass through the liver on their way
to the heart. (Of what importance do you think
this might be in the light of liver function?)
Trace these veins as best you can to and from the

liver. Remove the liver carefully, freeing it as far
as possible from its attachments to surrounding
tissues. Examine the excised liver, noting its
consistency, lobes, etc. (Make such a cursory
examination of every organ you remove.)

The stomach is found just below the liver on
the animal's left. Find the esophagus, following
it to where it penetrates the diaphragm. (We will
dissect it completely later.) Examine the in-
testinal tract. Pull it out, noting the mesentery
that attaches the intestine to the body wall (all
the organs in the body cavity are surrounded and
supported by mesenteries). Note the fanlike
arrangement of blood vessels in the mesentery.
Note the large caecum of the rat. (Do we have a
caecum?) Below the caecum, the digestive tract
is called the colon ; above, the intestine. Next find
the pancreas, buried in the mesenteries just below
the stomach. Starting at either end, free and re-
move the intestinal tract in one long string. How
long is the intestine? Comparing your height
and the length of the rat, how long would you
estimate your own intestinal tract to be?

Look next at the spleen, located below and to
the right of the stomach. Why is the speen so
dark in color? What is known of its function?

Study the kidneys. Try to trace the ureters to
the bladder. Find the adrenal glands in the fat just
above the kidney. These are round, brownish
bodies, easy to miss. Note the prominent blood
vessels leading from the kidney to enter or leave
the heart through the vena cava and the aorta.
Remove the kidneys and adrenal glands. (Be
sure to slice open these organs and look at their
cross sections.)

Now move to the thorax. If a model of the
human thorax is available in your laboratory,
study it before going on with your dissection.
Look at the lungs, heart, esophagus, and major
vessels that run forward from the heart and then
back along the ridge of the spinal column. Fol-
low the aorta and vena cava backward as far
as you can. Try to reach the place where they
fork before entering the legs. Remove the heart;
identify the ventricles and atria. Slice it to look
at the internal structure. (We will study the
mammalian heart in some detail later in the

72 VERTEBRATE ANATOMY

Exercise XIV

course, so don't linger with it here.) Next find
the trachea; look for the thyroid g\&nds clinging
to its side about two-thirds of the way to the
mouth. Free the trachea from the esophagus and
carefully dissect the bronchi and lungs free.

Next look at the internal reproductive organs.
If your rat is a male, push the testis up into the
abdomen. Note the tubular epididymis attached
to the testis. Follow the ductus deferens to the
seminal vesicles. Slit open the epididymis and
squeeze some of its contents onto a microscope
slide. Add a drop of saline and cover with a
cover slip. Observe the sperm under the micro-
scope. If your preparation is reasonably
fresh, the sperm should still be motile. Describe
their motions. In the female, find the ovaries.
Follow Xht fallopian tubes to the uterus.

Now turn to the muscular system. First skin
the animal. This can be done easily with blunt
dissection and the fingers. The pelt can be re-
moved in one piece. Dissect one foreleg and hind
leg. Identify as many muscles as you can. (The
best procedure here is to peel off the overlying
connective tissue, cut across the muscle or its
tendon at one end, and strip the whole muscle
back.) Note the large nerves entering the limbs;
these are seen best by excising the muscles on
the under side of the forelimbs or on the rear
of the hind limbs.

Next cut through one side of the jaw. Look
at the tongue and remove it. Find the opening
of the esophagus. How do the teeth differ from
ours? How many different types of teeth do you
see?

Rongeurs will be available for exposing the
brain. First remove the eyes, and then cut across
the skull between the eyes. Making small snips,
cut away the brain case overlying the brain. (Be
careful; the brain is very fragile!) When you
have exposed the brain, lift it from the anterior
end. Note the cranial nerves (especially the optic
nerves) on the underside. Free the brain, and
either remove it entirely or continue the dissec-
tion down the spinal cord. Note the nerves com-
ing from the spinal column. Note also the way
the cord bulges where the nerves to the fore limbs
come off. Where does the spinal cord terminate?

If you continue to dissect down the spinal
cord, it may be helpful to turn the animal on its
back. Follow an arm or leg nerve out into a
limb. Eventually remove both cord and brain.
Cut the brain through longitudinally. Note the
grey and white matter (especially in the cere-
bellum). What do they represent? Cut across
the cerebral hemispheres, and note the tracts of
white matter. Cut other cross sections, and at-
tempt to follow some tracts.

THE FROG

Follow the directions given for the rat as far as
they are applicable. You should be able to find
almost all the organs mentioned in the rat dis-
section. On opening the abdomen, however,
note immediately that the frog has no diaphragm.
How does it breathe?

Look at the beating heart under the dissecting
microscope. Follow the major vessels. Can you
see the blood flowing? Look at all the body con-
tents of the frog under the dissecting microscope.
When you find interesting things, show them to
your partner.

Follow the dissection guide given for the rat.
(If you have a female frog, the abdominal con-
tents may be filled with two masses of dark
spherical bodies. These are the ovaries filled
with growing eggs. Remove them immediately
on beginning the abdominal dissection.)

You will have an easier time dissecting the
visceral organs than your partner. You may
have trouble identifying the kidneys. They are
long, narrow organs found close to the midline
on the dorsal body wall. The adrenal glands
are the long yellow structures applied to the
kidney's surface. The testes are small white
bodies suspended from the kidney. Compare
your dissection with that of your partner and
note the differences in the two animals.

The frog nerves and muscles will remain ex-
citable for most of the day. Stimulators will be
available (your instructor will demonstrate them
for you). Try stimulating various nerves and
muscles; learn their names, and show them to
your partner.

Exercise XIV

VERTEBRATE ANATOMY 73

You will not be able, of course, to dissect the
brain, but the frog eye is large and can be ex-
amined. Identify its parts. Excise the eye, and
carefully cut around its equator. Lift offthe

cornea, iris, and lens (which will come away
together). Then with a thin blunt instrument,
lift the retina and pigment layers from the back
of the eye cup.

EQUIPMENT

Per student

dissecting tools

microscope (dissecting and compound)

slides and cover slips

dropper and 12" rubber tubing

dissecting pan

frog or rat

Per 8 students

stimulator and electrodes

box of pins

25 cc saline (0.9%)

Per 30 students

4 rongeurs

bottle of nembutal (50 mg/ml) (50 cc)
1-cc syringe and No. 27 needle

5 pithing needles

1 pr. gloves for handling rats

stained sections of major frog and rat organs

wall charts of frog and rat dissections

skeletons of frogs and rats

model of human thorax

ORGANIZATION OF HIGHER
PLANTS; THE TRANSPORT OF SAP

(Readings: Weisz, pp. 171-199; 231-239. S. P. T., pp. 55-63; 137-141; 368-378.
Villee, pp. 104-106; 117-129. Review the discussion of the plant kingdom in
Exercises XII and XIII. V. Grant, "The Fertilization of Flowers," Sci. Am.
184, No. 6, 52-56, June 1951, Reprint No. 12. M. H. Zimmerman, "The Move-
ment of Organic Substances in Trees," Science, 133, Jan. 13, 1961, pp. 73-79.)

This week we shall examine the organization
of flowering plants (angiosperms), which repre-
sent the peak of plant evolution, just as the
vertebrates studied last week represent the peak
of animal evolution. We shall also inquire into
an important aspect of their function, the trans-
port of sap, which plays a role in vascular plants
comparable with the circulation of blood in ani-
mals; and into the osmotic relations of plant
cells, upon which the transport of sap largely
depends.

Surely you are already familiar with the gross
division of higher plants into stems, roots, leaves,
and flowers. In the course of this period we shall
examine these organs more closely, dissect a
flower and a fruit, and examine under the micro-
scope the tissues of which such organs are com-
posed.

We shall begin, however, by setting up experi-
ments on the rise of sap, and on plasmolysis.
Once these have been started, they need only
occasional attention; and while they are going
on, you can examine the anatomy of plants and
plant structures.

WATER MOVEMENT IN PLANTS

One of the major problems in the life of vas-
cular plants is the transport of sap. This flows
in two streams, one generally downward, carry-
ing organic molecules prepared by photo-
synthesis in the leaves; the other upward, carry-
ing water and dissolved ions absorbed from the
soil by the roots. For the plant to survive and
grow, both streams must penetrate to all its
tissues.

Of these two streams, the upward stream of
water and salts from the roots is the larger and
more continuous. A fraction of it supplies the
downward stream, and to this degree we may
speak of the flow of sap as a "circulation." A
further fraction contributes to the growth of the
plant, and is retained in new tissues. Much of the
ascending water, however, is lost by evaporation
from the leaves.

The upward stream presents the major prob-
lem. To bring sap from the roots to the top of a
high tree demands a very large force. The high-
est trees — California redwood, for example, and

Exercise XV

ORGANIZATION OF HIGHER PLANTS 75

eucalyptus — may be over 300 feet high. How sap
is raised to such heights is a problem that has
plagued plant physiologists for generations. A
prevalent type of theory, entertained for a time,
was that as water evaporated from the leaves, it
left a vacuum in the ascending vessels (xylem),
which drew water upward. Even if one could
establish a perfect vacuum in the upper vessels
of a tree — a very unlikely possibility — this
would provide a pressure of only 1 atmosphere
to raise the sap. One atmosphere pressure
raises water about 34 feet. To bring sap to
the top of a 300-foot tree would require about
9 times this force, that is, about 9 atmospheres
pressure.

OSMOTIC PRESSURE AND
PLASMOLYSIS

There is little doubt that the major force for
the ascent of sap in plants is osmotic pressure.
Whenever two solutions are separated by a semi-
permeable membrane — a membrane that readily
passes water and small molecules, but blocks the
passage of larger molecules and some ions — water
tends to flow through the membrane from the
more dilute to the more concentrated solution.
It is easy enough to understand why. The more
concentrated side in terms of dissolved molecules
is the more dilute side in terms of water. Sup-
pose that there were pure water on one side of
such a semipermeable membrane, and a 10%
solution of molecules that could not go through
the membrane on the other side. At every instant
large numbers of molecules collide with the
membrane from both sides. On the side contain-
ing pure water, of every 100 molecules that hit
the membrane, 100 would go through. On the
other side, of every 100 molecules that hit the
membrane, only 90 would go through, that is,
only the molecules of water. The result is a tide
of water into the solution, exercising a water
pressure (the osmotic pressure) that tends to raise
its level to such a point that the added weight of
water pressing downward counterbalances the
further entrance of water. The height to which

the level of the solution rises on the more con-
centrated side is a measure of its osmotic pres-
sure.

A simple formula makes this relationship
quantitative. You know from last semester that
1 mole of any gas in a volume of 22.4 liters has
a pressure at 0°C of 1 atmosphere. In exactly
the same way, 1 mole of solute that cannot get
through a semipermeable membrane, distributed
in a volume of 22.4 liters of water, has an osmotic
pressure at 0°C of 1 atmosphere. That is, 1 mole
of such nondiffusing material dissolved in 22.4
liters of solution (a 0.045 M solution) exerts an
osmotic pressure that can raise water 34 feet.
To raise water 300 feet by this means would
require only about a 0.4 M solution.

It is important to note that what one is con-
cerned with in accounting for osmotic pressure is
the total concentration of particles that do not
penetrate the membrane, whatever their nature.
The "particles" may be all alike, or greatly
mixed, small or macromolecules, or even molec-
ular aggregates, indeed, anything dispersed in
water that does not go through the membrane.
The essential factor is the degree to which such
particles dilute the water on both sides of the
membrane.

The protoplasm of plant cells contains dis-
solved substances which do not readily diffuse
through the semipermeable plasma membrane.
Hence when a cell is immersed in water, more
water molecules diffuse into the cell than diffuse
out. The net tide of water into the cell inflates it,
producing a pressure against the cell wall. This
turgor pressure keeps the cell plump and relatively
rigid. Conversely, drying the cell or placing it in
a more concentrated solution, by withdrawing
water, decreases the turgor pressure, causing the
cell to soften or wilt.

When a plant cell is placed in water, water
enters until the turgor pressure is large enough to
counteract its further (net) entrance. At this
point the turgor pressure, driving water out of
the cell, equals the osmotic pressure, drawing
water in. In this state of equilibrium, water has
not stopped moving in and out of the cell, but it
is moving in and out at equal rates.

76 ORGANIZATION OF HIGHER PLANTS

Exercise XV

THE ASCENT OF SAP

The cells in the root have a higher osmotic
pressure than the salt solution in the soil. This
so-called root pressure draws water in from the
soil and pushes it upward through the vessels of
the xylem. A second source of osmotic pressure
originates in the leaves. Here water is con-
tinuously evaporated from mesophyll cells, and
water vapor finds its way to the exterior through
the small openings in the leaves called stomata.
This loss of water from the leaves (transpiration),
since it concentrates continuously the contents of
their cells, creates an osmotic pressure that tends
to draw water into them from adjoining cells,
and eventually from the sap-filled conducting
vessels of the xylem.

As already noted, if this removal of water
from the vessels of the xylem acted by creating a
vacuum at the top of the sap column, that could
at most develop a pressure of 1 atmosphere and
could raise sap at most 34 feet. It is now realized,
however, that owing to the great cohesion of
water (why has it so great a cohesion?), a con-
tinuous column of water can support a tension
of at least 20 to 30 atmospheres and perhaps
much more before breaking. Added to this, the
cellulose walls of the xylem and its conducting
vessels not only imbibe much water (up to 30
to 40% of the plant's dry weight) but also bind
the water of the sap columns by powerful elec-
trostatic forces and hydrogen bonding to the
— OH groups of cellulose. Very long columns of
sap can be lifted by this combination of forces:
transpiration of water pulling from the top, the
internal cohesion of the sap column, soaking
up of water through imbibition by the cell walls,
and the adhesion of the sap columns to the walls
of the vessels.

This entire view of the process is spoken of as
the transpiration-cohesion-tension theory of sap
rise. It appears principally to account for the rise
of sap in trees in full leaf, with root pressure as a
secondary force. Transpiration pulls and root
pressure pushes the sap upward. In the early
spring, of course, before the leaves appear, there
is little if any transpiration, and the sap must
ascend mainly by root pressure.

EXPERIMENTS
Plasmolysis

Sucrose, though a small molecule, enters cells
only very slowly. If a plant cell is placed in a
sucrose solution whose concentration is greater
than that of the total dissolved contents of the
cell (i.e., a hypertonic solution), water leaves the
cell. When enough water has left, the protoplasm
of the cell within its plasma membrane contracts
away from the cell wall. This process is called
plasmolysis. The concentration of sucrose at
which plasmolysis just becomes detectable is
equivalent to the osmotic concentration of the
cell contents.

We shall determine in this way the osmotic
concentration of epidermal cells of the red onion.
With scalpel and tweezers remove strips of
epidermis. The color of these cells is due to a
red, water-soluble anthocyanin pigment, and
will help you to detect the first withdrawal of the
cytoplasm from the cell wall.

Place strips of epidermis in a graded series of
sucrose solutions ranging in concentration from
0.1 to 0.6 M. Leave them for 30 to 60 minutes,
and then determine the degree of plasmolysis by
observing them under low power in the com-
pound microscope. From your observations
estimate the approximate osmotic concentration
of the cells. What osmotic pressure (in atmos-
pheres) should they develop when placed in
water? How high could this osmotic pressure
raise a column of water?

Transpiration

Water absorbed by the roots travels through
the vessels of the xylem, which form a con-
tinuous conducting system from the young roots
to the mesophyll tissue of the leaves. Most of
the water absorbed by a plant in leaf is lost by
evaporation from the surfaces of the mesophyll
cells. The water vapor finds its way through
intercellular spaces in the mesophyll to the ex-
ternal air via the stomatal openings.

A geranium plant is available for each two
students. The device for measuring transpira-

Exercise XV

ORGANIZATION OF HIGHER PLANTS 77

Planf

Funnel

Open capillary
with scale

A=»4;«

tion (called a potometer) should be set up as
shown in the diagram. Keep the stem of the cut
plant in water until the rest of the setup is ready
to receive it. Wire all joints for a tight fit.

Before attaching the plant, be sure all other
joints are tight, and fill the entire apparatus with
water. Shut off" the funnel, which serves as a
water reservoir, from the rest of the apparatus
with the pinchcock.

When the potometer is ready, slice off" the
bottom one-fourth to one-half inch of the stem
Vyhile it is still under water; then quickly push it
into the open end of the rubber tubing which is
filled with water. Be sure no bubble of air is
caught in the joint. Use the tubing stretchers to
aid in inserting the plant; then coat the outside of
the joint with a little vaseline. (Be sure not to get
vaseline on the cut surface!) Clamp the plant so
that the cut end is at about the same level as the
top of the capillary. Add water if needed to the
funnel, so that its level is higher than the top of
the capillary. Then open the pinchcock so as to
fill the capillary with water, and tightly close it
again.

As water is lost from the leaves by transpira-
tion, the water level in the capillary drops. Let
this go on for a convenient interval, and note the

time and change of water level. By reopening the
pinchcock, bring the water level in the capillary
back to near the starting point, and repeat the
measurement.

After a few consecutive readings are in good
agreement with one another, calculate the rate
of transpiration, per hour, day, and year. Direct
a light on the geranium plant; does the rate of
transpiration increase? What is the eff"ect of a
stream of air blowing on the leaves ?

PLANT STRUCTURE

We have already noted that the tissues of
higher plants, like those of higher animals, can
be divided into four types. Meristematic tissue
is responsible for the production of new cells,
and the growth of the plant. The new cells
formed from the meristems may diff"erentiate
into any one of the other three types of plant
tissue. The protective tissues (epidermis and
cork) comprise the outermost layers of the plant.
The "fundamental" tissues are more variable in
function and type. Some of them provide sup-
port (fiber tissue : sclerenchyma and collenchyma);
others are concerned with photosynthesis (paren-
chyma). There are two kinds of conductive

78 ORGANIZATION OF HIGHER PLANTS

Exercise XV

tissues, the xylem which transports water and
dissolved minerals upward from the roots, and
phloem which transports food materials from the
leaves to all parts of the plant. Phloem cells
usually do not have as thick walls as xylem, and
may be distinguished from them on this basis.

Stem anatomy

Begin by looking at the slides of the herbaceous
(green) angiosperm stems, alfalfa (Medicago), a
dicot, and corn {Zea mays), a monocot. Both
contain easily recognized vascular bundles of
both xylem and phloem. In dicots, however, the
vascular bundles are arranged in a ring, while in
monocots the vascular bundles are scattered
throughout the stem. This is one of the major
differences between mono- and dicots.

A second significant difference is that dicots
may retain some meristematic tissue in the stem
(called cambium) which lies between the xylem
and phloem, and which may lay down new xylem
and phloem. Most monocot stems, on the other
hand, lack a cambium.

Identify the following cell types in the stems:
epidermis, parenchyma, phloem, xylem, fiber
tissue, and cambium.

Next study the cross sections of the 2- to 4-
year-old woody stem of the tulip tree (Lirio-
dendron). Here the cambium has produced new
(secondary) xylem and phloem. The earlier
(primary) xylem has been left behind as orderly
rows of cells (wood), but the primary phloem
has been crushed to a thin layer which lies just
beneath the epidermis. The xylem cells that
form in the spring of the year are bigger than
those that form in the summer and fall. The
latter also have thicker walls. These differences
account for the annual rings visible in a tree
trunk.

You will find also some tangential and radial
sections of stems of Liriodendron and Thuja
(arborvitae).

There will also be pieces of various woods
available, cut in different planes. Examine these,
correlating their grains with the microscopic
sections.

Leaf anatomy

Examine the fixed and stained cross section of
a leaf of privet (Ligustrum). Note the following
layers:

1. Cuticle and epidermis of the upper surface
of the leaf.

2. Mesophyll. This is made up of two layers,
the palisade parenchyma and the spongy
parenchyma. (Of what advantage are the inter-
cellular spaces in the spongy parenchyma?)
The veins (vascular bundles) are distributed
through the mesophyll. The xylem here again
has thicker walls than the phloem and is stained
pink.

3. Epidermis and cuticle of the under surface
of the leaf. Note the specialized epidermal
cells called guard cells. They occur in pairs
with a pore or stoma (plural: stomata) be-
tween them. Gas exchange and water loss
occur through the stomata. The guard cells
are capable of swelling or shrinking, through
changes in osmotic pressure, depending on the
light and other conditions. When they are
turgid, the stomata are open; when they wilt,
the stomata close. This is an important regu-
latory mechanism in the leaf.

Root anatomy

Study the prepared slides of median longitu-
dinal sections of root tips of corn {Zea mays).
Identify the root cap, the meristematic zone, the
zone of cell elongation, and the root hairs. The
meristematic zone provides all the new cells for
the growth of the root.

Also examine the slides of a mature root of the
dicot, buttercup {Ranunculus). Three layers
should be distinguished: the outer epidermis,
from which the root hairs arise; the cortex, made
up primarily of cortical parenchymal cells, which
may contain starch grains (stained violet); and
the stele, a cylindrical area enclosing the con-
ducting elements. Note that the xylem (thick-
ened walls, stained red), in the center, is in the
form of a star. The phloem cells are located be-
tween the arms of the xylem.

Exercise XV

ORGANIZATION OF HIGHER PLANTS 79

Flower and fruit

Each pair of students will be provided with a
flower (probably a tulip). Study the following
structures, progressing from the outside inward.
The pericanth consists of an outer series of green
leaves (sepals), not always present, and colored,
modified leaves (petals). The inner sex organs
are called stamens (male) and pistils (female). A
"perfect" flower has both stamens and pistils.
Each stamen consists of a slender stalk at the
end of which is the anther, which produces the
pollen grains. Examine some pollen under the
microscope. Recall that this represents the male
gametophyte.

The pistil consists of an enlarged basal portion,
the ovary, which supports a slender tube, the

style. The tip of the style is slightly flattened,
forming the stigma. When the flower is receptive
to fertilization, the stigma becomes sticky, help-
ing it to retain the pollen grains. Inside the ovary
are several ovules, which produce the female
gametophytes. (Read carefully the discussion of
the formation of the egg and pollen, and fertiliza-
tion in Weisz, pp. 556-559; S. P. T., pp. 368-
378; Villee, pp. 182-184.)

After fertilization and formation of the em-
bryo, the ovule tissues harden, forming a seed
coat. In some plants, the seeds are retained in
the ovary, which develops into a fruit. Examine
either an apple or pear, cut in cross sections.
Identify as many structures as you can. After
that, it's yours!

EQUIPMENT

Per student

compound microscope

porcelain spot plate

slides and cover slips

The following prepared slides:

stem of Medicage (alfalfa)

stem of Zea mays (corn)

2- to 4-year old woody stem of Liriodendron (tulip

tree)

tangential section of Liriodendron

radial section of Thuja (arborvitae)

leaf of Liqustrum (privet)

root tip of Zea mays

root of Ranunculus (buttercup)

Per 2 students

medium-size funnel

capillary tubing (about 3 ft) or 0. 1 ml calibrated pipet

T-tube

punch cock and screw clamp

2 ring stands with 4 clamps

rubber tubing

400-ml beaker

geranium plant

tulip or other flower

apple or pear

Per 8 students

graded sucrose solutions (0.1 to 0.5 M) (20 ml)

vaseline

flexible wire for joints

red onion

Per laboratory

assorted pieces of polished wood

wall charts of stem, leaf, and root anatomy

BLOOD AND CIRCULATION

(Readings: Weisz, pp. 433-457. S.P.T., pp. 141-153; 163-168. Villee, Chapters
16 and 17. Also B. W. Zweifach, "The Microcirculation of the Blood," Sci.
Am. 200, No. 1, 54-60, Jan. 1959, Reprint No. 64. C. J. Wiggers, "The Heart,"
Sci. Am. 196, No. 5, May 1957, Reprint No. 62. W. B. Wood, Jr., "White
Blood Cells vs. Bacteria," Sci. Am. 184, No. 2, 48-52, Feb. 1951, Reprint
No. 51. M. B. Zucker, "Blood Platelets," Sci. Am. 204, No. 2, 58-64, Feb.
1961.

One of the principal problems facing a cell as
part of a multicellular organism is that it no
longer has free access to the external environ-
ment. To obtain water, salts, and organic
nutrients, to get rid of wastes, and for gas ex-
change, it must depend on some sort of circula-
tory system. The importance of the circulation
in maintaining an animal can hardly be over-
estimated. By far the largest cause of death in
man is failure of the circulation.

Beyond its nutritive and excretory roles, the
circulatory system in vertebrates performs an
essential function in defending the animal from
invasions of foreign organisms and foreign
molecules. A failure of these defense mechan-
isms can lead to death as surely as the failure
in the nutritive and excretory functions of the
blood.

The blood of higher animals is a complex
tissue. It may be separated by centrifugation
into a fraction composed of cells, and a cell-free
liquid fraction called the plasma. The plasma is
a complex solution of proteins, sugars, salts, and
other substances. One of the plasma proteins,
fibrinogen, is the precursor of the insoluble _/zin>j

of the blood clot. The remainder of the plasma
after the clot has been removed is called serum.
For both the nutritive and defensive roles of the
circulatory system, both cells and plasma are
needed.

Let us first consider the nutritive function of
the blood. Many substances are carried in water
solution in the plasma and are transported to the
cells in this fashion. Other substances are ad-
sorbed on proteins in the blood and are carried
in this way. Gas exchange presents further
problems. A little oxygen and somewhat more
carbon dioxide can be dissolved in the plasma;
but the major transport of both these gases in
vertebrates depends upon the red pigment,
hemoglobin, an iron-porphyrin-protein. The
hemoglobin is carried in specialized cells, the red
blood cells or erythrocytes. About as much
hemoglobin is packed into these cells as they
can possibly hold. Some 30% of the red blood
cell or 95% of its dry weight is hemoglobin.
The red blood cells are nonmotile, and do little
more than carry hemoglobin. In mammals these
cells lose their nuclei before maturing; and as you
would expect, from that point on they run down

Exercise XVI

BLOOD AND CIRCULATION 81

metabolically, dying after an average life of
about 120 days.

Human red blood cells are about 7.5 microns
in diameter and have a biconcave disc shape
which facilitates gas exchange. They are present
in great numbers in the blood; a normal young
man may have nearly six million erythrocytes
per cubic millimeter of blood. (If the human
blood volume is 6 liters, how many new red
blood cells must be produced per day to keep the
total number constant?)

For defense, the body depends on both plasma
proteins and cells. The plasma contains a special
group of proteins, called antibodies, which com-
bine with and hence inactivate foreign proteins,
viruses, or polysaccharides, and also cause in-
vading bacteria to clump together. Each anti-
body is specific for the substance or type of cell
with which it reacts. Somehow our defense
machinery knows the shapes of our own pro-
teins and leaves them alone. When foreign pro-
teins or polysaccharides called antigens are intro-
duced into the circulation, antibodies against
them are quickly synthesized.

The cells of the defense system, the white
blood cells or leucocytes, in marked contrast to
the red blood cells, are motile and highly active.
They can travel about in the blood stream, or by
going through the wall of a blood vessel can
wander out into the tissues and tissue spaces.
They move more or less as does an ameba, by
flowing in one direction or another. When in-
fection strikes, they quickly travel to the inva-
sion site in great numbers. There they destroy
large numbers of mvading organisms by in-
gesting them, a process called phagocytosis, and
also release special substances which help or-
ganize the defense. The pus formed in and
around an infection consists of dead white
blood cells.

A specialized group of white blood cells, the
plasma cells (plasmocytes), produce antibodies.
White blood cells can be divided into two groups :
the round, smooth-nucleated lymphocytes and
the granulocytes, which have irregularly lobed
nuclei. White blood cells are slightly larger than
red blood cells, and are present in considerably

smaller numbers (about 8000 per cubic milli-
meter of blood). During infection, however,
their number increases enormously, and this in-
crease provides a sensitive warning that an infec-
tion is present.

A third group of elements in the blood, the
platelets (thrombocytes), is involved in clotting.
When a blood vessel is cut open, an interlacing
network of fibrin forms a clot which eventually
closes the wound. This process is complicated,
involving the platelets, calcium ions, and the
plasma proteins thrombin and fibrinogen (throm-
bin is a proteinase which activates fibrinogen by
hydrolyzing off part of it, turning it into fibrin).

In addition to its nutritive and defensive
activities, the blood provides a constant internal
environment for the cells and tissues of the body.
In a mammal the pH, temperature, and sugar
concentration of the blood are held within very
narrow limits. This relative stability of the in-
ternal environment makes it possible for a mam-
mal to experience enormous changes in the ex-
ternal environment without damage. The great
nineteenth century physiologist, Claude Bernard,
was thinking of this when he said, "The con-
stancy of the internal environment is the condi-
tion of a free life."

During this period you will prepare and ex-
amine a stained smear of your own blood, and
will determine your blood type. We shall ex-
amine also the anatomy of the circulatory sys-
tem and the heart, and will observe the absorp-
tion spectrum of hemoglobin and its changes on
combination with oxygen and carbon monoxide.

BLOOD CELLS

Swab the ball of your middle or forefinger with
70% alcohol. Using a new, sterile lancet, punc-
ture the skin lightly, so that you can squeeze out
a drop of blood. Touch this to a microscope
slide about 1 cm from the end.

[At the same time suspend another drop or
two of blood in about 5 drops of isotonic saline
solution (0.9% sodium chloride in water) in a
small test tube. This concentration of salt solu-

82 BLOOD AND CIRCULATION

Exercise XVI

tion has the same osmotic pressure as the blood
and will keep the blood cells in good condition.
This suspension of blood cells will be used for
blood typing.]

The drop of blood that has been placed on the
slide should be spread evenly and very thinly
by drawing it along the slide with the end of a
second slide. When the film of blood has dried,
cover it with a few drops of methyl alcohol, and
let it stand for 2 or 3 minutes. Drain off the
alcohol, and immerse the slide for 6 seconds in
the red stain which has been provided. Then
rinse in a gentle stream of tap water for a few
seconds. Allow the slide to drain again, and
immerse for 6 seconds in the blue stain. Rinse
again in tap water, drain, and examine under
the microscope.

Identify the various types of blood cells. Most
common of course will be the erythrocytes,
which appear red. The nuclei of the leucocytes
stain blue; it should be possible to tell the
difference between the lymphocytes and granulo-
cytes by the shapes of their nuclei.

BLOOD GROUP TYPING:

AN ANTIGEN-ANTIBODY REACTION

The entire human race can be divided into
four categories on the basis of their blood types
(A, B, AB, O). Erythrocytes may contain anti-
genic proteins on their surfaces, designated A
or B, or AB, if both are present. If neither anti-
gen is present, the letter O is used. Persons of
the A type have in their serum an antibody
known as anti-B, which specifically reacts with

erythrocytes containing the antigen B. Similarly,
persons of the B type have an antibody, anti-A,
which reacts with A erythrocytes. Blood of
type O contains both anti-A and anti-B anti-
bodies, and AB blood contains neither of these
antibodies.

If the blood from an A person is transferred
into a B person the antibodies of the host serum
react with the antigens of the donor's red blood
cells and cause them to clump together or
agglutinate. This blocks the blood vessels and
may kill the person. It is the antigen more than
the antibody of the donor which causes severe
damage when injected into an incompatible
person, since the bulk of the blood cells ag-
glutinated are those of the donor. What happens
when B blood is transfused into an A recipient ?

Although blood-group typing is an example
of an antibody-antigen reaction, it is unusual in
that these antibodies are present in the cir-
culatory system without having been stimulated
by an invasion of foreign material. Usually anti-
bodies are made only in response to the presence
of a foreign antigen. Yet no blood group antigen
need ever have been present in man for the blood
group antibodies to develop.

Blood groups are determined genetically.
They are distributed differently in the various
human races. The distribution among white
Americans is as shown in the table, where the
plus sign indicates agglutination or clumping of
cells.

To determine your blood grouping, use the
suspension of cells that you prepared in normal
saline solution. Draw a line across the middle of
a microscope slide with a wax crayon. Place 1

Blood group

Blood cells agglutinated by:

%in

White

Americans

Anti-A serum from
Group B donors

Anti-B serum from
Group A donors

—

-f

Exercise XVI

BLOOD AND CIRCULATION 83

drop of the cell suspension on each half of the
slide. To the drop on one side, add a drop of
anti-A serum, to which a blue dye has been
added; and to the drop on the other side, add a
drop of anti-B serum, which has been dyed
yellow. (Be sure to mark which is which!)
Mix the drops by jittering the slide gently for
15 to 20 seconds, being careful not to let the
drops run into each other. Can you observe
any agglutination? Place cover slips on the slide
and examine both drops under the microscope.
Compare with a drop of blood suspension to
which no serum has been added. What is your
blood group?

Recently a number of blood factors in addi-
tion to the A, B, AB and O groups have been
discovered. Probably the most important of
these is the Rh factor. About 85% of the white
race and 99 to 100% of certain other groups
(Chinese, Japanese, African Negroes, and
North American Indians) have an antigen called
the Rh factor in their red blood cells. (The ab-
breviation "Rh" refers to the Rhesus monkey in
whose erythrocytes, as part of the cell mem-
brane, this antigen was first discovered.) Blood
containing the Rh factor is known as Rh-
positive; that lacking it, Rh-negative. If blood
from an Rh-positive person is transfused into
an Rh-negative recipient, an antibody (anti-Rh
factor) is produced. This in itself is not harmful,
but if a second transfusion is given, the anti-Rh
antibody which has accumulated in the recipi-
ent's blood reacts with the Rh antigen introduced
with the new red blood cells, and the result is
often fatal.

The anti-Rh antibody may also be produced
in an Rh-negative woman who, having an Rh-
positive husband, bears Rh-positive children.
During pregnancy some fetal red blood cells
containing the Rh antigen may leak into the
mother's circulation and cause the formation of
anti-Rh antibody. This has no ill consequences
for the mother, unless she later receives a trans-
fusion of Rh-positive blood. Usually also the
first child is not seriously harmed, since the
mother's antibody titer is still low. During
later pregnancies, however, the mother's anti-Rh

antibody may enter the fetal circulation and
destroy the fetal blood cells. This condition,
known as erythroblastosis fetalis^ causes the
death of the child, unless its blood can be re-
placed in a massive transfusion by Rh-positive
blood free from the antibody.

The test to determine the presence of Rh factor
in blood is tricky, and inaccurate results are often
obtained unless great care is taken to standardize
the procedure. For this reason we will not at-
tempt it in this laboratory.

THE HEART

(Do not fail to read Wiggers's fine paper listed
in the Readings. Bring it to the laboratory, if
possible.)

The heart, which is a single, two-chambered
organ in fishes, is a double organ in the birds and
mammals, with a lung heart on the right and a
body heart on the left. The right atrium (auri-
cle) receives blood drained by the veins from the
tissues throughout the body and passes it into
the right ventricle, which pumps it to the lungs
for gas exchange. The oxygenated blood is
brought back to the left atrium, which passes it
to the left ventricle, which sends it out through
the arteries to the body tissues.

The heartbeat has an associated pattern. First
the atria, their walls relaxed, distend with blood.
Then the atria contract while the ventricles re-
lax, transferring the blood to the ventricles.
Then the ventricles contract, driving the blood
to the lungs and tissues, the bicuspid and tri-
cuspid valves preventing it from re-entering the
atria. Similarly the semilunar valves prevent any
suck-back of blood from the aorta and pul-
monary artery during the next relaxation of the
ventricle.

Body tissues — > venae cavae — > right atrium

tricuspid semilunar

* right ventricle — > pulmonary

valve

artery
atrium

valve

body tissues

-^ lungs

bicuspid

valve

pulmonary vein

left

semilunar

* left ventricle — ^ aorta

valve

84 BLOOD AND CIRCULATION

Exercise XVI

Beef or sheep hearts will be available for ex-
amination. Identify the four chambers of the
heart: the two large ventricles forming the tip,
and the two smaller atria which lie above. Find
the openings from the veins into the atria, and
from the ventricles into the arteries. Examine
the valves of the heart. The tricuspid valve is
between the right atrium and ventricle, the bi-
cuspid (or mitral) between the left atrium and
ventricle. Between each ventricle and its artery,
a semilunar valve is found. Try to imagine the
action of each chamber and valve while the heart
is beating and blood is flowing through it.

CIRCULATION OF BLOOD

After you understand the organization of the
beef or sheep heart, two of you working together
should obtain a pithed frog. Draw out the
tongue, and pin it across the hole in the frog
board, or pin out the webbing of one foot across
this opening. (The tongue is usually more satis-
factory because it has less pigmentation to ob-
scure the capillaries.) Position the whole assem-
bly on the stage of a compound microscope. You
should be able to see clearly the circulating
blood, and identify arteries (or arterioles), veins,
and the interconnecting capillaries. Note the
elasticity of the red blood cells as they course
through the blood vessels. Can you find a
leucocyte pushing through a capillary wall?

Slit open the abdomen of the frog to expose
the beating heart. Trace the circulation through
the lungs (pulmonary circulation). Find the
aorta, the venae cavae, and the other major
blood vessels. Take this opportunity to review
the organs of the viscera.

Make a smear preparation of frog's blood on
a microscope slide and stain it as you did your
own blood. What striking difTerence do you see
between human and frog blood cells?

Examine the prepared slides of an artery and a
vein in cross section and of a piece of lung in
cross section. Arterial walls are thicker and more
rigid than those of veins, though both are com-
posed of three layers. You will probably see
red blood cells within the blood vessels.

HEMOGLOBIN

Most of the oxygen is transported in the blood
stream in loose combination with hemoglobin.
In vertebrates, the hemoglobin is entirely con-
tained within the red blood cells. It is composed
of a protein, globin, to which heme is attached
as prosthetic group. Heme is a complex of
ferrous iron (Fe++) with protoporphyrin.

The function of hemoglobin depends upon its
capacity to combine reversibly with oxygen:
Hb + 02^ Hb02. Hemoglobin absorbs oxy-
gen in the lungs, where the oxygen concentration
is high, and gives it up again in the tissues, where
the oxygen concentration is low.

Hemoglobin possesses a characteristic absorp-
tion spectrum, and each of its derivatives has a
different spectrum. Much of the chemistry of
hemoglobin has been learned by observing these
spectra. We shall examine the spectra of hemo-
globin and several typical derivatives with a hand
spectroscope.

Begin by orienting yourself in the visible spec-
trum. See that it stretches from a wavelength of
about 400 TUfi, in the far violet, to 700 m/x, in the
far red. The absorption spectra of hemoglobin
and its derivatives are visible as shadows in the
green and yellow regions. These are called ab-
sorption bands, and each is characterized by the
wavelength at which the shadow is deepest, the
absorption maximum.

Oxyhemoglobin

Examine a few milliliters of a diluted prepara-
tion of blood (1 ;100) in the hand spectroscope.
Note two absorption bands in the green, at
about 577 m/z and 539 mju. These are the bands
of oxyhemoglobin, Hb02. Any hemoglobin ex-
posed to air, as this is, is oxygenated.

Reduced hemoglobin

To the preparation you have just examined,
add a minute amount of the reducing agent,
sodium hydrosulfite (sodium dithionite,
Na2S204), and re-examine the spectrum. You

Exercise XVI

BLOOD AND CIRCULATION 85

will now see the two former bands replaced by a
single broad absorption band centering at about
565 m/i. This is the absorption band of reduced
hemoglobin (Hb). The hemoglobin can be re-
oxygenated by shaking vigorously; the former
bands re-appear. This cycle can be repeated over
and over again; indeed, this is how hemoglobin
functions in the body.

Carboxyhemoglobin

A preparation of diluted blood through which
carbon monoxide has been bubbled will be
available. Examine its spectrum. Add sodium
dithionite. What happens?

Spectrophotometers are available with which
these absorption spectra can be accurately
measured. Working in groups of 2 to 4, measure

the absorption of each of the above solutions
at 5 m^u intervals throughout the visible range
(400 to 700 myu). Plot the optical density against
the wavelength.

Optical density or extinction is the most useful
measure of absorption of light. If the intensity of
light of a given wavelength entering a solution is
/o, and the intensity of light that emerges is /,
then ///o is the fraction transmitted, or trans-
mittance. The fraction absorbed is (I — ///o).
The optical density or extinction is logio/n//, i.e.,
the logarithm of 1/transmittance. It has the
special virtue of being proportional to the con-
centration of pigment, and to the depth of layer.
For example, on doubling either the concentra-
tion of hemoglobin or the depth of layer meas-
ured, one doubles its extinction at all wave-
lengths.

EQUIPMENT

Per student

lancet, sterile, disposable
microscope slides
cover slips
2 small test tubes

Per 2 students

frog

cork board

box of pins

compound microscope

dissecting microscope

slide of mammalian lung

slide of mammalian artery and vein

Per 8 students

bottle 70% ethanol
absorbent cotton

dropping bottle 0.9% sodium chloride solution
dropping bottle methyl alcohol
differential staining solutions for blood cells*
wax crayon

blood typing sera: anti-A and anti-Bf
mammalian blood J

carboxyhemoglobin solution: the carbon monoxide
complex of hemoglobin is prepared easily by bub-
bling a gentle stream of carbon monoxide through
defibrinated blood diluted 1:100 with water. This
must be done in a hood!

Per laboratory

beef hearts

bottle sodium hydrosulfite (dithionite) Na2S204

spectrophotometer (e.g., Bausch and Lomb "Spec-

tronic 20")

hand spectroscope

*Red and blue stains as provided by Scientific Products Co. (Division of American Hospital Supply Corp.,
1210 Leon Place, Evanston, III.; branch offices in many cities. Wright's stain may be used instead.

tAnti-A and anti-B serum can be obtained from most medical supply houses or from Hyland Labora-
tories, Los Angeles, Cal.

jFresh mammalian blood, most easily obtained from a slaughter house, can be defibrinated by shaking
vigorously in a bottle with glass beads. Clotting can also be prevented by the addition of 0.1-0.2 ml 10%
potassium oxalate for every 10 ml of blood.

— ',«y-vK55- fir<-""Sis"!?»5^^r-

'B^li^^^R'

XVII

PERMEABILITY AND ACTIVE
TRANSPORT: THE HAMSTER GUT

(Readings: Weisz, pp. 277-285. Villee, pp. 44-^6; 299-303; 330-334. See also
H. W. Smith, "The Kidney," Sci. Am. 188, No. 1, 40-48, Jan. 1953, Reprint
No. 37; and further discussion of the kidney in S.P.T., pp. 156-158 and in
Weisz, pp. 459^62.)

Living organisms, plants and animals alike,
are to a degree divided into compartments,
separated from one another and from the ex-
ternal environment by membranes. The com-
partments may be cells, cell organelles, tissues,
organs, or indeed entire multicellular organisms;
but they have in common the fact that they are
divided off from other compartments by mem-
branes.

Each cell has its membrane. Each of such
intracellular structures as the nucleus and mito-
chondrion has its own membrane. An entire
tissue or group of tissues stretched between two
spaces or bounding the surface of an organ may
also function as a membrane. So, for example,
the multi-tissued animals may be thought of as
essentially saclike or tubular in construction,
with an outer surface facing the external environ-
ment, and an inner surface surrounding the di-
gestive cavity, both lined by membranes. Food-
stuffs, waste products, salts, water, oxygen, and
carbon dioxide — the continuous flow of mate-
rial into and out of the organism that is a large
part of its life — must all be transported through
membranes.

This transfer takes place in various ways.
Even the simplest biological membranes are
semipermeable. They allow certain substances to
pass through the membrane, while blocking the
passage of others. In general, for water-soluble
substances, this choice depends mainly on
molecular size. The membrane acts as though it
possessed pores of a certain effective size, which
permit small enough molecules to go through
and block the passage of larger molecules.

A second factor, added to semipermeability, is
selective permeability. So, for example, cell mem-
branes tend to pass fat-soluble molecules, almost
regardless of size. So also many cell membranes
tend to pass uncharged molecules much more
readily than charged molecules; and many exer-
cise a further selection by passing, for example,
negative ions more readily than positive ions.

In the types of permeability so far mentioned,
the driving force is the difference in concentra-
tion of the permeating ion or molecule on both
sides of the membrane. Granted that the mem-
brane permits a molecule to pass through it, the
net diffusion is always from the more con-
centrated to the less concentrated side, and the

Exercise XVII

PERMEABILITY AND ACTIVE TRANSPORT 87

rate of diffusion is directly proportional to the
difference in concentration on the two sides of
the membrane.

A third factor regulating the penetration of
substances through biological membranes is ac-
tive transport. This is of the highest importance,
and is the special subject of this week's experi-
ment. In active transport, a specific mechanism
exists, and work is done, to carry a substance
through a biological membrane. Specificity and
the expenditure of energy are the earmarks of
this process. Diffusion is an energy-yielding
process that can do work. Active transport is
an energy-demanding process; work must be
done upon it. Such active transport may take
substances from a higher to a lower concen-
tration, through a membrane that would other-
wise block their passage. What is much more
remarkable, active transport can take sub-
stances from a lower to a higher concentration,
that is, against the concentration gradient, bring-
ing them to many times the concentration they
possess in the medium from which they are being
absorbed.

Active transport can be thought of as a process
of pumping. Little is know of the mechanism by
which it occurs. On the other hand, it is clear
that energy is required, and this is usually sup-
plied as ATP. The specificity of the process is
also apparent. Certain molecules may be passed
by the membrane and concentrated, while very
similar molecules are blocked. So, for example,
many cells are able to concentrate L-amino
acids, but not their "unnatural" D-amino acid
isomers. These distinctions are frequently rela-
tive rather than absolute; the specificity fre-
quently takes the form of a difference in rate of
transport. So, for example, galactose, glucose,
and fructose are all isomeric 6-carbon sugars
(C(5Hi206), all of about the same size and shape.
Yet a mammalian intestine absorbs galactose
more rapidly than glucose, and glucose more
rapidly than fructose. Similarly glucose pene-
trates the wall of the intestine much faster than
such a 5-carbon sugar as xylose or arabinose,
though the latter, being smaller, would diffuse
faster through a semipermeable membrane. On

the death of the cells lining the lumen of the in-
testine, all such distinctions are lost. Now all the
hexoses penetrate at the same speed, and 5-
carbon sugars faster than 6-carbon sugars.

We are going to study an example of the active
transport of glucose between two compartments
in the hamster: the inside or lumen of the small
intestine, and the "outside," normally filled by
the blood and lymph.

The absorption of foodstuffs in mammals
takes place almost entirely in the small intestine.
The mucosa lining the intestine is thrown into
folds and ridges. Its surface is velvety with
numerous tiny, fingerlike projections, the villi;
and the individual cells lining the lumen have so-
called brush borders, tiny projections of cyto-
plasm upon their outer surface, so that this also
is velvety at another level of dimensions. All
these devices increase enormously the absorbing
surface of the intestine. In man it has been
estimated that the total intestinal area effective
for absorption is about 10 m^. Compare this
with the total area of skin, which is less than
2m2.

After passing through the intestine, food sub-
stances are absorbed into the blood vessels or
lymph channels, and are transported first to the
liver and then to other tissues throughout the
body. The cells which cover the villi take an
active part in transferring some of these sub-
stances from the lumen of the intestine (the
mucosal side) to the outer space (the serosal side).
If the transfer of such a substance as glucose
were a matter of simple diffusion, the concentra-
tion on the serosal side would never become
greater than that on the mucosal side. In fact,
however, glucose is actively transported through
the intestine, so that it can become several times
more concentrated on the serosal than on the
mucosal side.

The preparation and procedure we shall use
for studying the active transport of molecules
across the wall of the small intestine was devised
by Dr. T. H. Wilson of the Harvard Medical
School. The small intestine of the rat or ham-
ster is removed and cut into sections a few centi-
meters long. These sections are everted (turned

88 PERMEABILITY AND ACTIVE TRANSPORT

Exercise XVII

inside out), filled with the solution to be studied,
and tied off at both ends to form sausage-like
sacs. The eversion places the serosal side toward
the inside of the sac, and the mucosal side out-
ward, so that the cells engaged in active transport
can be kept supplied with oxygen. The sac is laid
in the same solution it contains, and incubated
with continuous aeration for some time. After
incubation, the solutions inside and outside the
sac are analyzed to reveal any change of concen-
tration that may have occurred.

The hamster gut should be set up as described
below as soon as you enter the laboratory.
There are, however, several things that should
be done during the 60 to 90 minutes of incuba-
tion. During this time the materials for the
glucose analysis should be prepared. It would be
wise to run through the analysis of the standard
solution to make sure that everything is working
well.

The hamster from which you removed the gut
should also be used to review vertebrate anat-
omy. Trace the alimentary canal. Examine the
prepared slides of intestinal tissues. Review also
the circulatory system. Compare the hamster
heart with that of the frog. Note the cheek
pouches. What do you notice about the hamster
stomach ?

Try to find the major places in the body where
substances pass from one "compartment" to the
other. In which of these is one "compartment"
the outside environment, or open to it? In this
connection, do you regard the inner cavity of the
gastrointestinal system as inside or outside the
animal? How about the body cavity (coelom)?
Does it possess an opening to the outside en-
vironment, not blocked by a membrane? in
males? in females?

One of the principal organs for the exchange of
dissolved substances is the kidney. Portions of
the kidney provide prime examples both of diffu-
sion through a semipermeable membrane, and
active transport (cf. Homer Smith's article listed
in the Readings). Examine the hamster kidney.
Examine also the prepared slides, and identify
Bowman's capsule, glomerulus, and tubules.
Which substances are excreted by the kidney?

Which retained? Where do these things occur,
and what kinds of permeability are involved?

PROCEDURE

Students, working in pairs, will be given a
freshly killed hamster. Slit open the belly so as
to expose the viscera, being careful not to dam-
age the intestine. Find the stomach. Snip off
the upper end of the duodenum just below the
stomach, and carefully uncoil the small in-
testine, using scissors to cut away the mesentary
when necessary. When the lower end of the small
intestine is reached, snip it loose, and place the
entire intestine in a petri dish half filled with
Krebs phosphate Ringer solution containing 20
millimolar (0.36%) glucose. Do not allow the
intestine to dry!

All the remaining mesentery and fat should be
stripped by hand from the gut. Cut the intestine
into at least two (preferably three or four if
your animal is large) 5- or 6-cm sections, begin-
ning at the upper end. Using a dropper, gently
force a little solution through the sections to
wash out the contents.

The sections of gut are now ready to be
everted and tied off.

The mucosal surface is extremely delicate and
the success of your experiment depends upon it.
Handle it as little and as gently as possible,
taking special pains not to scrape or bruise it.
Using the glass rods provided, push one end of
the gut into the lumen until it appears at the
opposite end. Complete the eversion by rolling
the gut along the rod. Slip the gut off the rod,
and immerse in fresh glucose-Ringer solution.

Tie a thread ligature tightly around one end.
Fill a dropper with the glucose-Ringer solution
and insert the dropper into the gut. Loosely
knot a thread around the open end of the gut so
that it can be tightened quickly. Force the solu-
tion from the dropper into the intestinal sac until
the sac is completely filled but not grossly dis-
tended. Tighten the ligature quickly as the
dropper is withdrawn.

Place the sac in a test tube containing 5.0 ml of
the fresh glucose-Ringer solution, and leave it at

Exercise XVII

PERMEABILITY AND ACTIVE TRANSPORT 89

room temperature for 60 to 90 min (the longer,
the better). Arrange for air to bubble through
the solution during this time. This should be
done gently so as not to mar the intestinal sac.
Note the exact length of time that each sac is
incubated.

Prepare the other sections of intestine exactly
as above. One (or two) sacs should be incubated
with aeration, as above, to allow metabolic
processes to continue normally. The other sec-
tions of intestine are to have their metabolism
stopped by adding an inhibitor of the respiratory
production of ATP, dinitrophenol (DNP), and/
or by stopping the aeration. To the tubes con-
taining the sacs whose metabolism is to be in-
hibited, either add 1 drop of DNP solution, or
stop aerating, or do both.

After the incubation is complete (60 to 90
min), remove the sac from the tube, blot it dry
on a paper towel, and carefully cut open the end
so as to collect the liquid inside in a small test
tube or in the depression of a spot plate. This
is a tricky operation, so use extreme care or you
may lose the results of the experiment ! Save the
solution also in which the sac has been incubated.

Glucose analysis

Three drops of the solution to be tested are
placed in a test tube, and 2 ml of Benedict's solu-

tion are added. Mix, and place in a boiling water
bath for 5 min. The approximate concentration
of glucose can be determined by comparison
with known glucose standards run similarly. The
following analyses should be carried out:

(1) solutions inside each of the intestinal sacs
(4 tubes),

(2) solutions outside each of the intestinal sacs
(4 tubes),

(3) the original Krebs phosphate Ringer solu-
tion with 20 mM glucose (1 tube),

(4) tubes containing 0, 1,2, and 3 drops of the
standard 50 mM glucose solution (4 tubes).

Be sure each tube is carefully labeled in such a
way that the boiling water will not obliterate the
label! Masking tape high up on the tube is
convenient for this.

Benedict's reaction depends upon the reduc-
tion of blue cupric (Cu++) to red cuprous (Cu+)
ions by the aldehyde group of the sugar. CU2O
is the red product formed. The variation in color
is a measure of the amount of glucose originally
present. A clear blue solution indicates none; a
deep red indicates a high concentration. (Re-
call a previous use of the Benedict test in Exer-
cise IV, p. 23.)

Report your results as the approximate ratio
of glucose inside the sac to that outside.

EQUIPMENT

Per 2 students

hamster (killed immediately before use, preferably
without anesthesia, or by injecting a fatal dose of
nembutal)

dissecting instruments
2 petri dishes

6 or 8 droppers (some or all should be long)
sewing cotton

glass rod (1.5 mm X 15 to 20 cm)
2 pipets (2 ml and 5 ml of solution have to be de-
livered)
18 test tubes
spot plate

paper towels

400-ml (or 250-ml) beaker for boiling water bath

bunsen burner

ring stand

metal gauze

Per laboratory

Benedict solution; prepare as directed in Exercise IV
Krebs phosphate Ringer solution
Stock solutions :

(1) 0.9% NaCl (0.154 M)

(2) 1.15% KCl (0.154 M)

90 PERMEABILITY AND ACTIVE TRANSPORT

Exercise XVII

(3) 1.22% CaCb (0.11 M)

(4) 3.82% MgS04 • 7H2O (0.154 M)

(5) phosphate buffer (0.1 M, pH 7.4) (17.8 gm of
Na2HP04 • 2H2O + 20 ml NHCl diluted to 1
liter)

Use 100 parts of solution 1 ; 4 parts of solution 2;
3 parts of solution 3; 1 part of solution 4; 20 parts
of solution 5. Add glucose to make 20-mA/ (0.36%)
and 50-mM (0.90%) solutions just before using.

The solution should be stirred while adding the
phosphate to prevent precipitation.

dinitrophenol, saturated solution

prepared slides of intestine

prepared slides of kidney

large water baths at 100°C may be used instead of
individual ones

XVIII

THE NERVE IMPULSE

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»ll)iiiijjjjMji,4.j|jiniSi

'■^,. !'-!'< :W"

(Readings: B. Katz, "The Nerve Impulse," Sci. Am. 187, No. 5, 55-64, Nov.
1952, Reprint No. 20. This excellent article contains what you really need to
know. Read also R. D. Keynes, "The Nerve Impulse and the Squid," Sci. Am.
199, No. 6, Dec. 1958, Reprint No. 58; Weisz, pp. 475^80; Villee, pp. 354-358.)

One of the most important aspects of animal
evolution is the development of systems of rapid
intercommunication through nerve cells. Even
some of the protists, notably the ciliates, possess
intercommunicating systems of fibrillae that
seem to help to integrate the cell's motions, and
may be thought of as a sub-cellular nervous
system. The coelenterates possess a diffuse nerve
net. All the higher animals possess nervous
systems made up of discrete, intercommunicating
nerve cells.

One nerve cell meets another at a boundary
called a synapse. Though a nerve cell conducts
impulses equally well in both directions, a
synapse transmits impulses only in one direction.
It is this characteristic that limits nervous trans-
mission to particular pathways and particular
lines of flow. In general we distinguish excita-
tions flowing into the central nervous system
from the receptors (afferent) from excitations
flowing out of the central nervous system
(efferent) toward the effectors (the muscles and
glands).

* The instructor should consult Appendix B for
information about the electronic equipment.

A nerve cell consists of a cell body containing
the nucleus and its cytoplasm, from which
springs a specialized, threadlike, conducting ele-
ment, the nerve fiber or axon. In a higher animal
the nerve cell bodies are all inside or just beside
the central nervous system — the brain and cen-
tral nerve cord. The nerves that one finds roam-
ing about the body are bundles of axons.

The business of a nerve axon is to conduct an
excitation. If we dissect a nerve out of the body
together with the muscle that it innervates (for
example, the frog sciatic nerve and the gastro-
cnemius muscle), we can stimulate one end of the
nerve and know that it has transmitted an ex-
citation by the fact that a moment later the
muscle contracts. As the nerve conducts its
excitation, sufficiently delicate instruments can
measure the passage of an electrical change.
This electrical change, which invariably ac-
companies the nerve response, is the nerve im-
pulse, action potential, or action current.

An electric current is a flow of electrons from
a region in which they are more concentrated to
a region of lower concentration: i.e., from a
more negatively to a more positively charged
region. By an odd historical convention, the

92 THE NERVE IMPULSE

Exercise XVIII

electric current is usually stated to be flowing
just the other way, from positive to negative.
You will have to remember, while using this con-
vention, that the positive pole is where there are
fewest electrons, and that when a current flows,
the electrons flow toward the positive pole.

In any flow of current we must take account of
three factors:

(1) An intensity factor, the potential, measured
in volts. This is the pressure of electrons to
flow from the region of higher to that of
lower concentration of electrons. It is just
like the pressure of water to flow from a
higher to a lower level. Just as with water,
one could measure this pressure without
allowing any flow. It is this pressure of
electrons that we measure as the electrical
potential or voltage.

(2) A quantity factor, the current or amount of
flow, measured in amperes. There is a
certain pressure in the water mains,
whether or not you use any water. You
can then turn on a tap and allow the water
to flow gently or strongly. The same is true
of an electric current.

(3) The third factor is the resistance off'ered
by the conductor, measured in ohms. To
follow out the water analogy, one might
have a narrow pipe, which even when
entirely open allows the water to flow
through it only slowly, or a wide pipe,
which can conduct it very rapidly. Simi-
larly a thin copper wire off"ers considerable
resistance to the flow of current compared
with a thick wire. In these cases the po-
tential may be identical; but the flow of
current is very different.

These three quantities are bound together in
the simple relation expressed in Ohm's Law:
E = IR, in which E is the potential (volts), / is
the current (amperes), and R is the resistance
(ohms).

If a very fine electrode is inserted into the in-
terior of a nerve fiber, and another electrode
touches its surface, one finds a more-or-less con-

stant electrical potential between these two elec-
trodes, called the resting potential. Such an ex-
periment is best done on the giant nerve fibers of
the squid (see Keynes's article). This resting
potential is about 75 to 90 millivolts (mv), with
the outside of the nerve fiber positive to the in-
side, the nerve membrane forming the interven-
ing boundary. The source of the potential is a
differential distribution of ions: more K+ ions
inside, and more Na+ and Cl~ ions outside.
The selective permeability of the nerve mem-
brane, which is largely responsible for this
diff"erential distribution of ions, is spoken of as
its polarization.

A nerve impulse results from a local depolariz-
ation of the membrane, permitting ions to flow
through it more freely. The active point on the
nerve fiber has momentarily ceased to maintain
the diff"erential distribution of ions just described.
As a result, the surface of the cell at this point
has lost its special positivity; it is therefore nega-
tive relative to the remaining cell surface. This
change is self-propagating, each such active
point stimulating the adjacent region of the fiber.
For this reason the nerve impulse appears as a
wave of negativity that sweeps down the length
of the nerve fiber. It is important to realize that
when a nerve fiber carries an impulse, all that it
conducts is this excitation. There is no actual
flow of either energy or material from one end
of the fiber to the other; there is only the passage
of excitation.

This point can be made clear by a simple ex-
ample. The conduction of a nerve impulse is not
like the conduction of water through a pipe, or
of electricity through a wire. It is like the flash
that passes down a train of gunpowder if we
ignite one end. If you will think of such a train
of gunpowder, you will understand readily many
important properties of the nerve impulse. So,
for example, it is obvious that at each point the
gunpowder generates its own response, inde-
pendently of all the other points. You would get
the same response whether you lit one end with a
spark or a bonfire. Where there is a lot of gun
powder, there will be a big response; where the
gunpowder is thinly sprinkled, there will be a

Exercise XVIII

THE NERVE IMPULSE 93

Analysis of a diphasic
action potential

-»- Time

0 ^

Voltage-measuring

Active
region

Nerve
fiber

Wm>M/M'^y/^y,.ymmm.

weak response. At each point, however, the gun-
powder will give everything it has. That is, the
response will vary in strength from point to
point with the amount of gunpowder, but at
each point it will be all or nothing. In the same
way, and for much the same reasons, the re-
sponse of a nerve fiber is independent of the
strength of the stimulus, provided it is strong
enough to excite at all; and is also all or nothing.
We measure nerve response by placing two
electrodes on the nerve and connecting them
through a sufficiently sensitive recording device.
The quantity measured is the potential. (The
nerve membrane has a very high resistance, so
that the current flow is very small.) The two elec-
trodes, touching the outside of the fiber, nor-
mally have zero potential between them. If we
now excite the fiber, as the nerve impulse comes
under the first electrode that electrode becomes

zm.

negative to the other electrode. The instrument
records that negativity. As the impulse sweeps
on, it may come to involve equally both elec-
trodes. In that case there is momentarily again
no difference of potential between the two elec-
trodes. Then the nerve impulse has passed the
first electrode and involves only the second,
which now momentarily becomes negative to the
first; the recorded potential is now just opposite
to what it was before. Then the impulse is past,
and again, as at rest, there is zero potential be-
tween the electrodes.

The result is a so-called diphasic change of po-
tential : the potential starts at zero, sweeps up to
a maximum in one polarity, comes down again
through zero to a maximum in the opposite
polarity, and returns to zero. These changes
should be obvious from the accompanying dia-
gram.

94 THE NERVE IMPULSE

Exercise XVIII

All this discussion has been in terms of a
single nerve fiber. Nowadays physiologists often
work with such single nerve fibers, either the
giant nerve fiber of the squid or with the very
much smaller nerve fibers of higher animals in-
cluding mammals. For the latter one needs to
use microelectrodes, which may be only 1/x in
diameter.

You must remember that the nerves that you
see in animals and with which you will be work-
ing are bundles of many such nerve fibers.
Though each individual nerve fiber exhibits all-
or-nothing behavior, the nerve bundle does not,
since a strong stimulus may excite many nerve
fibers whereas a weaker stimulus may excite few.
Many other characteristics of the nerve impulse,
however, can be studied in such nerve bundles.

In order to measure nerve impulses, very sensi-
tive devices must be used. At the present time
all such work is done with electronic amplifiers
and recorded with oscilloscopes. The expense
and complexity of such equipment has in the past
restricted neurophysiological experimentation to
relatively few places and relatively advanced
courses. This is a great pity, for the phenomena
are of the highest importance, generality, and
interest. The experiments we are about to de-
scribe involve the use of the equipment described
in Appendix B. It is the finest equipment of its
kind that is available, quite adequate for ad-
vanced research in neurophysiology, yet de-
signed for maximum dependability and simplic-
ity of operation. Fundamentally it is not very
much harder to use than a television set. We
hope that it will be a relief to you to see what
electronics can do when it is not producing soap
operas.

In this first period, working in groups of four,
everyone should learn to operate the electronic
equipment and set up and record the impulses
from a frog sciatic nerve. The best arrangement
is for two of you to begin at once to dissect out
the frog sciatic nerve, while the other two learn
how to operate the equipment. Then, after the
preparation is set up, the students who know
how to work the apparatus can teach the others
how to do it while recording from the nerve.

THE ELECTRONIC EQUIPMENT

This equipment may at first glance seem com-
plex and forbidding. It is, in fact, simpler than it
looks. Many of the dials are simply multipliers
of the same adjustment, or of no concern in the
normal operation of the instrument. There are
only five or six controls you must learn to
operate.

The general principle for study of such small
electrical changes as found in nerve cells is first
to amplify the potentials and then to observe
their magnitude, duration, and shape by means
of a recording device. From the nerve chamber,
which will enclose the preparation, note the
leads into the preamplifier. We shall speak of the
nerve potential and its changes as the signal. The
preamplifier amplifies this signal 100 or 1000
times. The other dials on the amplifier allow
one to select certain frequencies of signal while
eliminating others. These need not concern us
today, but next week we will have opportunity
to use these controls.

To observe and measure the response, the
magnified signal is led into the indicator, an
oscilloscope with its amplifier. In the indicator,
the impact of a narrow beam of electrons on a
fluorescent screen makes a spot of light, as in a
TV set. The beam of electrons on its way to the
screen passes between two metal plates, charged
with the amplified biological potential. The beam
of electrons, themselves negatively charged, is de-
flected toward the positively charged plate and
away from the negatively charged plate, so dis-
placing the spot of light on the screen from the
zero position which it occupies when both plates
are equally charged. In this way the polarity
and magnitude of the signal are reflected in the
direction and extent of the deflection of the spot
of light on the screen.

The controls on the indicator {intensity, focus,
position, etc.) are familiar to anyone who has
ever adjusted a TV set. The calibrated amplifier
in the indicator allows further magnification or
attenuation of the input signal.

Besides polarity and amplitude, we are in-
terested also in the change of biological potential

Exercise XVIII

THE NERVE IMPULSE 95

Recording
leads

with time. To study this, the signals are applied
to the electron beam while it is moving hori-
zontally across the screen at some prescribed
rate. The horizontal sweeping of the electron
beam is controlled by the waveform generator,
which sets high voltage potentials across a pair
of plates that deflect the beam in the horizontal
direction. We control the rate of the horizontal
sweep by the waveform duration dial. The
operating mode dial lets us select how the sweep
is to be controlled, whether automatically or
manually. (Ignore the "gated" and "triggered"
selections on the equipment; these are for special
applications.)

These instruments and the power supply com-
plete the recording setup. However, to study
nerve impulses, we must stimulate the nerve to
fire. This can be done artificially by applying a
brief electrical shock. (Next time we shall study

the biological initiation of nerve impulses by
light striking a photoreceptor.)

The pulse generator provides controlled elec-
trical stimuli. It, like the indicator sweep, is
activated by the waveform generator. Thus for
every sweep of the indicator beam, one stimulus
pulse is delivered. The pulse generator has
three controls: the pulse delay knob sets the
point during the horizontal sweep at which the
stimulus pulse is delivered; the pulse width
knobs control the duration of the stimulus pulse;
and, finally, the pulse amplitude controls its
voltage.

That is all there is to the equipment. To
familiarize yourself with the controls, connect
the lead from the "pulse out" terminal of the
pulse generator to the "vertical input" of the
indicator. (The pulse generator provides enough
voltage so that the preamplification can be

96 THE NERVE IMPULSE

Exercise XVIII

Gray

Stimulating leads

omitted.) Convenient settings to begin with
are:

Indicator:

"5 volts/division" amplification;
Waveform generator:

"recurrent operating mode,"

100 msec "waveform duration";
Pulse generator:

0.3 "pulse delay,"

10 msec "pulse width,"

0 "pulse amplitude."

Turn the instruments on and allow a few min-
utes for them to warm up. Focus, center, and
brighten the beam if necessary. With the "wave-
form duration" dial of the waveform generator,
alter the rate of horizontal sweeping of the
electron beam. A setting of 100 msec means that
it takes 100 msec (0.1 sec) for the electron beam
to complete a sweep. Then, with the wave dura-
tion set at 100 msec, very slowly turn up the
pulse amplitude of the pulse generator, and ob-
serve the square wave deflection on the oscillo-
scope screen. (Check that the pulse ampHtude
you are putting in is giving a deflection that
agrees with the indicator amplifier.) Now vary
all the controls mentioned above, and become

' familiar with them. The only caution is not to
put so much pulse amplitude voltage into the
indicator that the beam is deflected off the
screen; this might cause damage.

NERVE PREPARATION

After pithing the frog, strip the skin off one
leg. Lay the animal on the dissecting pan, belly
down, and expose the sciatic nerve. With
glass needles, free as much of the nerve as you
can (about 4 cm) from the surrounding tissues,
being careful not to stretch or injure it. The more
nerve you expose, the better for your experiment.
Keep the nerve wet with Ringer solution
throughout the dissection. To expose the upper
part of the nerve, you will have to remove the
overlying pelvic girdle. When the nerve is freed
from the surrounding tissues, tie a short length
of thread to each end; measure the distance be-
tween the threads. Cut beyond the threads, and
remove the nerve. Keep track of which end is
proximal and which distal.

Mount the nerve in the nerve chamber so
that each end passes underneath an electrode,
while the middle section weaves over and under
the intervening electrodes. (See diagram.) The

Exercise XVIII

THE NERVE IMPULSE 97

proximal end of the nerve should be in contact
with the three closely spaced electrodes used for
stimulation. Extend the nerve to its in situ
length in the chamber; do not overstretch it. Fix
the ends of the threads to the edge of the cham-
ber with plasticene. Pour mineral oil into the
chamber to cover the nerve. The oil insulates
the nerve and prevents it from drying out.

EXPERIMENTAL PROCEDURE

Note: Before connecting any leads to the nerve
chamber, make sure that the pulse amplitude
dial on the pulse generator is turned to 0. If you
accidentally put high voltages through the nerve,
it will be ruined very quickly.

Two leads (gray and red) from the pulse gen-
erator should be connected to the first two,
closely spaced, stimulating electrodes; and two
leads (red and black) from the first two recording
electrodes to the input plug of the amplifier. The
gray ground lead should be connected to the
third, closely spaced electrode. (See diagram.)
Use the following settings :

Preamplifier amplification = 100

Indicator amplifier = 0.5 volts/division

Waveform duration = 10 msec

Pulse width =0.1 msec

Pulse delay = 0.3

Pulse amplitude = 0

Pulse = negative

Now very slowly increase the pulse amplitude.
Note first the stimulus artifact, and then the
appearance of the nerve action potential. In-
crease the pulse amplitude until the nerve im-
pulse is maximal in height.

The nerve impulse should appear as a di-
phasic wave. Why? What is its maximal volt-
age? What is its duration? Make a graph of
voltage vs. time. Calculate the latency of the
nerve impulse by measuring the distance from
the start of the stimulus artifact to the beginning
of the response (the stimulus artifact is con-
ducted down the outside of the nerve and is re-
corded instantaneously). Most of the latency is
occupied by the time it takes for the nerve im-

pulse to travel from the stimulating electrode to
the first recording electrode. Estimate the speed
of conduction of the main group of sciatic nerve
fibers from the latency and the distance between
these electrodes. Express this velocity in meters
per second.

Determine the minimal stimulus voltage
needed to evoke minimal (threshold) and maxi-
mal responses. Why can't you increase the
amplitude of the nerve potential indefinitely by
increasing the stimulus? How does the observed
grading of the response fit in with the all-or-none
law? Decrease the duration of the stimulus
(pulse width) and redetermine the voltages that
produce minimal and maximal responses. Try
several different pulse widths. Make a plot of
pulse duration vs. threshold voltage. What do
you conclude?

Next, crush the distal end of the nerve with a
fine pair of metal forceps just before it passes
underneath the most distal electrode, and recon-
nect the recording leads to the two most distal
electrodes (one on each side of the crushed por-
tion). Observe that the nerve impulse is now
monophasic. Why?

There are several classes of fibers in the frog
sciatic nerve which conduct impulses at different
rates. With monophasic recording you may
notice humps on the down sweep of the nerve im-
pulse. These represent the slower conducting
a2, ;S, or 7 fibers separating from the prominent
and most rapidly conducting ai fibers. To see
these humps clearly, set the "operating mode" to
manual, and trigger the impulses by hand at a
rate of about 1 per second. Try to estimate the
rates of conduction of each of the fiber classes.
See if the groups of fibers differ in threshold, by
determining the pulse amplitude needed to pro-
duce a maximal response in each group.

Finally, determine the maximum frequency
with which the nerve can respond. This is done
by progressively decreasing the "waveform dura-
tion," so decreasing the intervals between
successive stimuli. At what frequency of stimu-
lation do the responses begin to decline in am-
plitude? What can you conclude concerning the
refractory period (see Katz's article) ?

98 THE NERVE IMPULSE

Exercise XVIII

ACTION POTENTIALS OF HUMAN
NERVE AND MUSCLE

It is possible to record indirectly the nerve and
muscle action potentials in your own hand by
means of remote electrodes when the hand is in
a nonconducting medium such as distilled water.
For this experiment, fill a porcelain pan with dis-
tilled water and suspend about 6 inches of the
two heavy copper wire electrodes in the water at
the two ends of the pan with clamps. Make sure
the exposed ends of the electrodes are entirely
under the surface of the water. Connect the
leads from the electrodes to the binding post
terminals on the side of the copper cage, and
connect the input leads from the amplifier to
these terminals. Use the following settings:

Amplification = 1000

Indicator amplifier = 0.05 volts/division

Waveform duration = 1000 msec

Hold your hand relaxed in the water. When
the baseline has settled down to a steady trace,
clench your fist. Individual action potentials of
200 to 300 ^v should be seen. Remember that in
the frog nerve, you were stimulating all the
nerve fibers simultaneously, so that it appeared
as if you were eliciting just one large action

potential. Here the action potentials in the vari-
ous fibers (both nerve and muscle) are staggered
in time, and consequently appear as smaller
single spikes. To see the single action potentials
more clearly, increase the sweep speed by de-
creasing the waveform duration to 100 msec.
Note that the action potentials continue as long
as the fist is clenched. One nerve impulse serves
only to twitch a muscle. For a muscle to remain
contracted, it must receive a continuous train of
impulses. Alternately clench and relax your fist
as quickly as you can ; note the bursts of impulses
that excite each tightening of the fist.

From this preparation, you can also learn
something about the anatomy of the hand.
Many of the movements of the hand, such as
flexion and extension of the fingers are performed
by muscles in the forearm which connect to the
fingers by tendons. Flex your fingers under the
water and note that you see no action potentials.
Try spreading your fingers sideways, and bringing
them together again. What do you conclude?
Move your thumb and hand in as many ways as
possible and try to decide which movements are
performed by the muscles in the hand and which
by the muscles in the forearm. Check your con-
clusions in an anatomy atlas.

EQUIPMENT

Per 2-4 students

electronic recording and stimulating setups

frog

dissecting pan

goose-neck lamp

thread (1 spool)

plasticene

beaker (400 ml)

medicine dropper

plastic ruler

nerve chamber

2 glass dissecting needles

mineral oil (to fill nerve chamber)

frog ringers (25 ml)

porcelain pan (approx. 12" X 8" X 8")

2 heavy copper electrodes (heavy copper wire
flattened about 6" at one end and otherwise insu-
lated works very well)

2 electrode clamps
distilled water

MUSCLE

(Readings: Weisz, pp. 356-362; 446-457. Villee, pp. 48-50; 344-351. H. E.
Huxley, "The Contraction of Muscle," Sci. Am. 199, No. 5, 66-82, Nov. 1958,
Reprint No. 19. C. J. Wiggers, "The Heart," Sci. Am. 196, No. 5, 74-87, May
1957, Reprint No. 62.)

The ability to move rapidly is one of the major
characteristics of animal life. In all but the
lowest animals, such motions are accomplished
by muscles. Throughout the animal kingdom,
muscle tissue is built upon a common plan, and
is remarkably uniform.

During the past few years we have begun to
learn how muscle works. Upon excitation of a
muscle, through a nerve or by the same artificial
devices one uses to excite nerves, a wave of de-
polarization much like the nerve impulse passes
down the muscle membrane. Somehow this
releases ATP, which reacts with the proteins,
actin and myosin, of which the muscle fibrils are
mainly composed, causing the muscle to con-
tract. In this process, ATP is broken down to
ADP, yielding with the release of its high-energy
phosphate bond the chemical energy that is con-
verted in the muscle contraction to mechanical
work. Actin and myosin are long, fiber proteins,
arranged in alternate, overlapping sequences
along the muscle fiber. During contraction, the
actin and myosin filaments slide over one an-
other so that they overlap further, causing
shortening (see Huxley).

How ATP causes these changes is not known;
nor do we know how the depolarization of the

muscle cell membrane excites this reaction. We
do know, however, that the wave of excitation
that passes over a muscle fiber on stimulation is
very much like the nerve impulse.

Three types of muscle tissue are found in
vertebrates: striated, cardiac, and smooth mus-
cle. The rapidly contracting "voluntary" mus-
cles of our arms, legs, and trunk are striated.
The cardiac muscle that forms the wall of the
heart, is also striated, but is otherwise inter-
mediate in structure and speed of contraction.
Smooth muscle, found in the gut and blood
vessels, undergoes slow, sustained contractions,
as for example the slow peristaltic motions of the
intestine. All three types of muscle, though
histologically and functionally distinct, owe their
contractility to actin and myosin.

Since they do contain the same contractile pro-
teins, one might wonder why these different
types of muscle possess such different properties.
One reason is large differences in structural
arrangement, apparent in part under the micro-
scope, and persisting down to the molecular
level. Another factor is that the cell membranes
of the various types of muscle, as also of nerve,
have very different excitatory characteristics.
So, for example, the larger frog nerves conduct

100 MUSCLE

Exercise XIX

impulses at about 30 m/sec. Striated muscle
conducts at only 2 to 5 m/sec; heart muscle at
less than I m/sec, and smooth muscle at only
5 to 20 cm/sec. The more slowly the excitatory
impulse is conducted over a muscle fiber, the
more slowly it responds.

The excitability of such cell membranes can be
drastically altered by chemical reagents. Nerve
cells transmit their excitation across a synapse
with another nerve or with a muscle by releasing
at the synapse excitatory or inhibitory substances
(neurohumors, "nerve hormones"). The usual
excitatory neurohumor is acetylcholine, though
at the first line of synapses in the sympathetic
nervous system it is epinephrine (adrenaline).
In such cases the neurohumor released by an
excited nerve cell at the synapse locally de-
polarizes the membrane of the succeeding nerve
or muscle cell, so exciting it in turn.

In addition to such substances which de-
polarize nerve and muscle cell membranes, ex-
citing them or lowering their thresholds to other
stimuli, there are also hyperpolarizing substances,
which increase the polarization of the membrane,
raising the threshold of the cell and making it
more difficult to excite. These are therefore not
excitatory, but inhibitory substances, and may
be released at nerve endings which are concerned
with inhibition, rather than with excitation.
7-amino butyric acid,

(CH2NH2 • CH2 • CH2 • COOH),

acts in this way; but, as we shall see, acetyl-
choline also may at times, as in the heart, inhibit
rather than excite.

The characteristics of the excitable membranes
of nerves and muscles can be altered and con-
trolled by the application of such substances,
released naturally at nerve endings, or applied
artificially. The rate of the heart beat, for ex-
ample, as well as its amplitude, are controlled in
this way. The heart muscle beats automatically,
as a result of an intrinsic cycle of excitation and
recovery. The heart rate is regulated through the
activity of two nerves, which are opposed in their
effects: a nerve from the sympathetic system,
which releases epinephrine at its synapse with the

heart muscle and excites the heart to beat more
quickly; and the vagus nerve from the parasym-
pathetic nervous system, which inhibits the heart
by releasing acetylcholine, slowing the beat.

During this period we shall examine the struc-
ture of the various types of muscle, the con-
tractile effects of ATP upon the muscle proteins,
and the effects of excitatory and inhibitory sub-
stances on the frog heart.

EXPERIMENTAL PROCEDURE
Types of muscle

First examine the prepared sections of striated,
smooth, and cardiac muscle. Striated and car-
diac muscle may be recognized by their cross-
banding or striations. Smooth muscle has no
visible banding. Each of the long, spindle-
shaped cells of smooth muscle contains a promi-
nent, elongated nucleus. The fibers of striated
muscle seen under the microscope are distinct
cells, lying parallel to one another. The similarly
striated fibers of cardiac muscle, however,
branch widely with one another, forming an
intercommunicating network that contains many
nuclei, but no apparent cell boundaries. Until
recently the entire ventricle of the heart was
thought to constitute a single, multinucleate
cell (a so-called syncitium). Recently, however,
membranes that divide cardiac muscle cells have
been found with the electron microscope.

Using Huxley's article as guide, study the
banding of a microscopic section of striated
muscle. Identify the A and / bands and the Z
and H lines. What arrangement of molecules
accounts for the A and / bands?

Contraction of glycerinated muscle

Next, we shall study the contraction of muscle
fibers on addition of ATP, using the famous and
extraordinarily important preparation devised
by Albert Szent-Gyorgyi. The psoas muscle of
a rabbit contains exceedingly little connective
tissue, being composed almost entirely of long,
parallel muscle fibers. It is the lack of connec-
tive tissue that makes this the "tenderloin." A

Exercise XIX

MUSCLE 101

rabbit had been killed, hastily eviscerated, and
the body wall chilled. The psoas muscles lie at
both sides over the backbone. They are readily
divided into narrow strips, and each such strip
was tied at its ends to an applicator stick at the
slightly stretched length at which it was found in
the body. Then the muscle strips, tied to their
sticks, were cut away, and soaked in a half-and-
half mixture of glycerol and water at 0°C for two
weeks or longer.

This procedure removes almost all the con-
tents of the muscle fibers except for the contrac-
tile proteins, actin and myosin. The muscle still
retains its striated appearance under the micro-
scope, and can still contract when supplied with
ATP. Indeed it retains these properties for many
months in the cold glycerol solution.

(The following experiment should be com-
pleted during the first hour of the laboratory.)

One such strip of glycerinated muscle will be
given to each group of four students. With
scissors cut the muscle just inside the sutures
(knotted threads) that hold it to the stick, so as
to get as long a piece as possible, and then cut
this in half, so that each pair of students gets half
the strip. Drop each piece into a small petri dish
containing cold glycerol-water mixture.

With fine forceps and dissecting needles,
tease out narrow threads of muscle about 15 mm
long and about as thick as silk thread. Place one
of these in a drop of cold glycerol-water mixture
on a microscope slide, and examine its structure
in the microscope. Can you see the striations?
(To see them clearly, you may have to separate
out a single fiber.) Transfer the slide to the stage
of a dissecting microscope, and measure the
length of the muscle with a ruler. Add a drop of
0.25% ATP solution containing 0.05M KCl and
0.001 M MgCl2. Remeasure the length of the
muscle, and re-examine its striations in the com-
pound microscope. (If the strip curls during con-
traction, it is too thick.) Can you distinguish any
diff"erences?

Try this several times, each time measuring
the original and final length of the fibers. Record
your results. Does a second drop of the ATP-
salt solution have any further effect? For con-

traction to occur, certain ions must be present in
specific proportions, in addition to ATP. To a
degree, other ions can be substituted for the
K+ and Mg++ that we use here, but no other
substance seems to substitute for ATP, which
seems specific for this process. Convince your-
self of the need for the ions and the ATP by add-
ing drops of ATP alone, and KCl-MgClo mix-
ture alone, to narrow threads of muscle.

Hormonal control of the frog heart

By the end of the hour, turn to the next experi-
ment, on the effects of acetylcholine and
epinephrine (adrenaline) on the frog heart. Work
in pairs. While one partner is preparing the ani-
mal, the other should become familiar with the
operation of the kymograph. This will be
demonstrated by the instructor. A revolving
drum has a piece of smoked paper wrapped
around it. (Be careful not to touch or otherwise
smudge the paper!) The speed of the drum is
controlled with a knob on the base.

Expose the heart of a pithed frog, freeing it
from the pericardial membranes, and cutting
away the body wall over it. Grasp the tip of the
ventricle with a pair of fine forceps, and pass a
thread through it with a needle and thread. Tic it
securely (this is called a suture in surgery), but
not so tightly as to cut the tissue. Keep the heart
continuously wet with fresh Ringer solution.
Place the frog on the frog board, with the heart
directly beneath the kymograph lever, and at-
tach the other end of the suture to the lever with
a bit of modeling clay. The heart should extend
well out of the chest cavity and the lever should
be about parallel with the table. If it seems to
strain the heart unduly to lift the lever, help to
balance the lever with a little lump of clay at
the end attached to the heart. The lever should
now be moving up and down rhythmically with
the heartbeat. Move the kymograph so that the
tip of the lever just touches the smoked paper,
and the lever is tangential to the drum. (See
diagram.) Make sure you start your record high
up on the drum, so that you can get another
record below it.

102 MUSCLE

Exercise XIX

Kymograph

Smoked
paper

Plasticene
counterbalance

Frog board

Keep dripping Ringer solution on the heart;
if you let it dry, the amplitude of contraction will
decline. When the heart is beating evenly,
rotate the drum a little way so as to draw a
control record of 6 to 8 beats. Rinse the heart
with 2 to 3 drops of Ringer, wait 30 seconds, and
repeat. There should be no difference in the two
records. (Writing on the smoked paper with your
dissecting needle, indicate on the record when
you add anything to the heart.)

Now rinse the heart with 2 (only 2!) drops of
acetylcholine solution in Ringer. Watch the
heart, and when you see a perceptible change of
beat, run a short record. (If nothing happens
within 2 minutes, add 2 more drops of the acetyl-
choline solution, but no more. Too much
acetylcholine will cause the heart to stop com-
pletely. If your heart does stop from too much
acetylcholine, rinse it thoroughly with Ringer
solution, and it should recover within about 5
minutes.)

What has happened to the heart rate and the
amplitude of contraction? After waiting another

minute or so, run another record. Has the heart
begun to recover? Now rinse the heart with
fresh Ringer. The beat should return to normal
in a few minutes.

Run another control record. Now rinse with
2 drops of epinephrine solution in Ringer. What
does it do to the heart beat? Now rinse with 2
drops of acetylcholine solution in Ringer, and
again record the result.

Again rinse the heart with Ringer, and wait for
about 5 minutes. The heart may still show some
effects of the epinephrine, which wear off much
more slowly than those of acetylcholine.- Run
another control record. Add 5 drops of atropine
solution in Ringer to the heart, wait for 30
seconds, and run a record. Has anything hap-
pened? Now add 2 to 4 drops of acetylcholine
solution, and record again. What has happened ?
What did the atropine do?

The heart is self-excitatory. Its beat originates
in a small specialized area in the wall of the sinus
venosus, the SV node. In this area, the mem-
brane allows a continuous, small leakage of Na+

Exercise XIX

MUSCLE 103

into the cells, so depolarizing them and causing
them to fire impulses regularly. Acetylcholine
exerts its effect only on this area, apparently by
increasing the permeability of the cells to K+, so
allowing some K+ to leak out. This hyper-
polarizes the cells, and lengthens the time re-
quired for the sodium leak to depolarize them
to the point of firing.

Epinephrine exerts its effect both at the SV
node and over the entire heart, by increasing the
cell permeability to Na+, thereby tending to de-
polarize the membranes, and facilitating the
conduction of impulses. Epinephrine also seems
to have some effect on the contractile process
itself, since it increases the amplitude of con-
traction of the heart fibers. Did you see this
effect?

Atropine is thought to exert its effect by com-
bining with the same sites on the membrane that

acetylcholine would otherwise combine with,
much as an inhibitor, by combining with an
enzyme, blocks the substrate.

Finally, show that the ventricular beat origin-
ates in the atria by tying a suture (a thread)
around the heart between the atria and ventri-
cle. This pinches the specialized connecting
cells which transmit the excitation from the atria
to the ventricle. These cells normally delay the
impulse long enough for the atria to complete
their contractions before the ventricle begins to
contract. When pinched by the thread, they may
not conduct at all, or may conduct so slowly
that they transmit only one impulse for every
two or three contractions of the atria. This is
called a heart block.

After you have caused a heart block, stimulate
the ventricle electrically with a stimulator. Does
the ventricle contract? What do you conclude?

EQUIPMENT

Per student

dissecting microscope

compound microscope

microscope slides

prepared slide of striated, cardiac, and smooth

muscle

petri dish

fine forceps

plastic ruler

Per 4 students

solution of 0.25% ATP in 0.05-M KCl + 0.001-M

MgCb

solution of 25% ATP

solution of 0.05-M KCI + 0.001 -M MgCb (above

three solutions in 10-ml dropping bottles)

15% glycerol-water mixture (25 ml)

strip of glycerinated rabbit psoas muscle (soaked in

50% glycerol and water mixture at 0° for 2 weeks;

changed to cold 15% glycerol and water mixture

1 hour before using) (preparation of muscle fibers
described in text; see also A. Szent-Gyorgyi's book:
Chemistry of Muscular Contraction, Academic
Press, New York, 1951)

Per 2 students

kymograph and smoked paper
frog board
kymograph lever
ring stand
needle (small)
thread

medicine droppers

acetylcholine solution (2 X lO""* M)
epinephrine solution (2 X 10"'* M)
atropine solution (5 X 10~* M) (above three solu-
tions in 10-ml dropping bottles)
frog ringer (50 ml, in dropping bottle)
plasticene
frogs (pith one hour before using)

ELECTRICAL ACTIVITY OF
A SENSE ORGAN: THE LIMULUS EYE

(Readings: Weisz, pp. 485-490. S. P. T., pp. 188-201. L. J. Milne, "Electrical
Events in Vision," Sci. Am. 195, No. 6, 113-122, Dec. 1956. W. R. Loewenstein,
"Biological Transducers," Sci. Am. 203, No. 2, 98-108, Aug. 1960, Reprint
No. 70. W. H. Miller, F. RatlifF, and H. K. Hartline, "How Cells Receive
Stimuli," Sci. Am. 205, No. 3, 222-238, Sept. 1961, Reprint No. 99.)

During the past two weeks we have studied the
electrical responses in peripheral nerve axons
and the spontaneous activity of the heart. Today
we will examine the electrical activity of a re-
ceptor and its attached nerve. The receptor we
have chosen is the eye of the horseshoe crab or
king crab, Limulus polyphemus. Though this
animal is called a crab, it is not a crustacean, but
an arachnid, closely related to the spiders. Many
of the characteristics of its eye are shared by all
types of eye, and indeed by all other types of
sensory receptor.

The sensory receptors are the outposts of the
nervous system. Their business is to translate
various types of stimuli into meaningful patterns
of nerve impulses. The stimulus is always some
exchange of energy or material with the environ-
ment. This may be light (photoreceptors), heat
(hot and cold receptors), mechanical (touch,
pressure, sound), or chemical substances (taste,
smell, common chemical sense). The receptors
translate all such stimuli into relatively slow,
local electrical potentials, that depolarize the

associated nerve fibers, causing them to fire
trains of all-or-nothing impulses. These are con-
ducted to other portions of the nervous system,
and sometimes eventually out again to excite
muscles and glands. The more intense the stim-
ulus, the larger the depolarization of the re-
ceptor, and the higher the frequency and greater
the number of impulses in the associated nerve.

That is, the response of the receptor cell to the
external stimulus is not all or nothing, but small
or large, depending upon the intensity of the
stimulus; and this graded potential in the re-
ceptor is then translated into frequency and
number of all-or-nothing discharges in the asso-
ciated nerve fibers. Such slow receptor poten-
tials, called in general generator potentials, have
special names in the different receptors. In an
eye such as that of Limulus they are called retinal
potentials, and the records of them are called
electroretinograms (ERG's).

How a receptor transduces ("leads over")
stimuli of all kinds into electrical activity is not
known; but in photoreceptors we do know

104

Exercise XX

ELECTRICAL ACTIVITY OF A SENSE ORGAN 105

CH3 H

CH3

1 c

1
^ /

C C /"\

/'\8/'\l0/ I2CH

/^\ /

c 1

H2C4 6C CH3 H

1 H3C-C13

1 1/

H \

H2C3 iC

uCH

\2/ \

C CH.-,

HC15

W-cis retinene, the chromophore of visual pigments

CH3

I
C

H
C

H2C

CH3 H

light

CH3

H
C

CH3

H
C

\ / \ / \ / \

C
H

C
H2

CH3

all-?ra/u retinene, C19H27CHO

something of the action of hght on the receptor.
All photoreceptors so far examined contain light-
sensitive pigments, substances which are changed
on absorbing visible light so as somehow to in-
duce a nervous excitation. Each such visual pig-
ment is composed of a colorless protein, called
an opsin, to which is attached as chromophore
or color group the yellow, fat-soluble substance,
retinene (vitamin A aldehyde, C19H27CHO). It
is for this reason that vision depends upon vita-
min A: the first symptom of vitamin A deficiency
in man and other animals is the failure of vision
called night-blindness.

Retinene itself is very light yellow in color.
The visual pigment of the Limulus eye that is
formed by the attachment of retinene to opsin is
red in color. It is this red pigment that absorbs
the light which is effective in vision. Molecules
of retinene come in a variety of shapes, cis-trans
isomers of one another. A special, unstable, bent
and twisted shape of retinene (the l\-cis isomer)
is the only one that can join with opsin to form a

visual pigment. When a quantum of light is ab-
sorbed by the visual pigment, the effect is to
straighten out the retinene to the aW-trans
isomer. Somehow, perhaps by exposing an ac-
tive site on the opsin which had been covered
before, this leads to the depolarization and
nervous excitation. (1 1-m retinene is both bent,
as are all cis molecules, and twisted, owing to
the — H on Cio running into the — CH3 on C13,
which keeps the molecule from lying flat.)

Before a molecule that has responded in this
way can participate again in excitation, the
retinene must be rebent and retwisted back into
the active shape that can recombine with opsin
to regenerate the visual pigment. That is, the
effect of light on the visual pigment is both to
excite vision and to inactivate temporarily the
pigment. The result is a temporary decline of
visual sensitivity, called light adaptation. Then
some time must elapse during which the visual
pigment is regenerated, and the eye regains its
maximal sensitivity. This is dark-adaptation.

106 ELECTRICAL ACTIVITY OF A SENSE ORGAN

Exercise XX

You must understand that though light is
needed to inactivate the pigment, it regenerates
equally well in light or darkness. In a steady
light the pigment is continuously inactivated and
continuously regenerated, so that a balance is
struck between these processes, in which some
pigment is constantly present, permitting vision
to continue. In the dark, only regeneration
occurs, bringing the visual pigment back up to
its maximum concentration and returning the
eye to its maximum sensitivity.

In today's experiment you will examine the
retinal generator potential (ERG), dark-adapta-
tion as measured by the ERG, the relation of the
ERG to action potentials in the optic nerve, and
the patterns of nerve impulses in the optic nerve.

Equipment

The light stimulus is provided by a micro-
scope lamp, and its intensity is controlled with
neutral filters inserted in the beam. The light
will be focused on the eye with a condensing lens,
and the duration of the stimulus controlled by
raising and lowering a piece of cardboard that
shuts off the beam. Before beginning the experi-
ment, look over this setup and try it out.

Since exposed wick electrodes will be used, the
preparation must be shielded. For this it is set
up inside the copper cage. Make sure that the
wick electrodes are connected with the binding-
post terminals on the side of the copper cage.
The input cable from the preamplifier is then
connected to these binding posts.

EXPERIMENTAL PROCEDURE

Note. In this first part of the experiment, use
as little light as possible so as not to light-adapt
your preparation strongly. The Limulus eye is
not stimulated by deep red light, since its visual
pigment does not absorb the long wavelengths of
the spectrum. Red lamps will be available, and
can be used freely without affecting the prepara-
tion.

Plan to work in groups of two to four. Place
the horseshoe crab on the block of wood in the

shielded cage, and fasten it down with nails
through the edges of the shell. Putting nails
through the shell causes no more pain than cut-
ting your fingernails.

Identify the prominent faceted eyes, so-called
compound eyes. With a fresh razor blade, gently
scrape the horny surface of the eye (again a pain-
less operation). This removes the highly water-
resistant waxy substance that helps to make the
eye waterpoof. Don't scrape too long or too
hard; it is better to do too little than too much.
Then with the tip of a sharp scalpel, dig a tiny
hole through the shell directly back of the eye,
just large enough to admit the tip of a wick elec-
trode.

Set the animal in position in the shielded cage,
and focus the light beam on its eye, using very
dim light, and exposing it only for short inter-
vals. The cotton wicks used as electrodes will
have been soaked with sea water, so as to con-
duct the electric current. Place one such wick on
the cornea of the eye, and insert the tip of the
other through the small hole behind the eye.

Convenient settings on the recording instru-
ment are:

Preamplifier magnification = 1000

Indicator amplification = 0.05 volts/division

Waveform duration = 1000 msec (1 sec)

The trace should be steady and should not ex-
hibit waves due to interference from the 60-cycle
power lines. If you do have hash on the screen,
readjust the electrodes to make a better contact,
If the hash persists, you may have to scrape the
surface of the eye a little more, but consult the
instructor first.

Stimulate the eye with a dim, brief flash of
light (through density 2.0 filter), and observe
the response. How long does the response last
compared with the stimulus? How does it com-
pare in duration with a nerve action potential?
Let the animal dark-adapt for a few minutes, and
stimulate the eye again. If the response has
grown, let the animal continue to dark-adapt
until the responses have become constant. This
may take up to 15 minutes or longer.

Exercise XX

ELECTRICAL ACTIVITY OF A SENSE ORGAN 107

Compare the responses elicited with three
different durations of the stimulating flash, of
about ^ sec (as fast as you can move the card-
board), 1 sec, and 3 sec. What is the relationship
between the ERG and duration of stimulus at
constant intensity?

Now investigate the way in which the ERG
varies with light intensity. The intensity is con-
trolled with a series of neutral filters. A neutral
filter is one that absorbs light more-or-less
equally throughout the spectrum, and hence
looks colorless (gray). Such filters are graded on
a density scale, in which density equals log
1/transmission. That is, a filter that transmits
1/10 of the light has density log 10 = 1.0; a
filter that transmits 1/100 of the light has density
log 100 = 2.0; and so on.

Starting with the dimmest light (neutral filter,
density 3.5), and a stimulus of 1 sec, measure the
height of the ERG. Now, keeping the duration
of the stimulus constant, progressively increase
the light intensity by steps of 0.5 log unit; i.e.,
use progressively lighter filters in which the
density falls by steps of 0.5. Make at least two
measurements at each intensity that agree with
each other. Wait at least one minute between
exposures to allow the animal to recover. At the
higher intensities you will probably have to re-
adjust the amplification setting on the indicator
to keep the response on the screen. Plot the
magnitude of response (in millivolts) against the
light intensity in log units. How big is the range
of light intensities over which you find the re-
sponse to vary? How big is it in ordinary arith-
metic units? Describe the relationship between
intensity of stimulus and the ERG in words, and
draw what conclusions you can concerning the
animal's capacity to respond to and distinguish
various brightnesses of light.

Using a moderate intensity of light (density
1.0), and a 1-sec exposure, remeasure the magni-
tude of response to a flash. Light-adapt the ani-
mal for 5 minutes with the brightest light avail-
able, and remeasure the response at density 1.0.
Let the animal remain in the dark, and periodi-
cally remeasure the response to a flash of this
intensity and duration of light. Start by making

a measurement every minute, and as the change
slows down lengthen this interval, eventually to
every 3 to 5 minutes. (Don't go on with this
longer than 30 minutes.) How long does the
horseshoe crab take to dark-adapt? Make a
graph of the relation between the height of the
ERG and time in the dark.

Remove the animal from the cage and kill it
by turning it on its back and slitting it up and
down the middle with a scalpel. Prepare to ex-
pose the optic nerve by first cutting a square,
about 1 inch on a side, through the carapace
around the unused eye of the animal, using a
sharp scalpel or one-edged razor blade. Then
carefully raise this piece of carapace at its upper
edge, and begin to free it from the underlying
tissue with the blunt end of a scalpel. Work very
slowly, watching carefully for the optic nerve. It
is a very fine, glassy structure that runs forward
from the eye. If you have trouble, call an in-
structor.

When you find the nerve, free it from the bulk
of the surrounding connective tissue and tie a
suture around its distal end. Now remove the
square of carapace containing the eye with its
attached nerve from the animal, and continue to
clean away the connective tissue from its back.
Go as far with this as you can, but be very care-
ful not to damage the eye itself. The cleaner the
nerve, the better your experiment will go. Keep
the nerve moist with sea water.

Mount the eye upside down, as shown in the
diagram, on a block of plasticene. Position the
block of plasticene in the shielded cage, and re-

108 ELECTRICAL ACTIVITY OF A SENSE ORGAN

Exercise XX

focus the light on the eye. Touch one wick elec-
trode to the front of the eye, and sling the optic
nerve over the other wick electrode. Be sure that
the wick touches nothing but the nerve. On
stimulating the eye, you should see small nerve
impulses superimposed on the ERG. Remember
that the eye has probably been light-adapted
during your manipulations, so if the responses
seem small, wait a few minutes for them to grow
larger. (Getting good reponses from a prepara-
tion like this may take some fussing. If your
responses are small, or none is visible, try re-
adjusting the nerve on the electrode. It is usually
advantageous to have the electrode close to,
though not touching, the eye. If the nerve is too
wet, the responses may be shorted out by the sea
water; and the nerve should be dried with a bit
of cotton. On the other hand, if the nerve is too
dry, it will not make suitable contact, and should
be moistened. So if your responses are not ideal,
keep fiddling and don't get discouraged. Your
instructor may also have suggestions.)

Examine the relationship between the in-
tensity of the light, the height of the ERG, and
the relative number of impulses in the optic
nerve. Examine also the responses to short and
long flashes at one intensity. Describe your
observations and draw conclusions.

If you wish to study the nerve impulses alone,
you can filter out the ERG by turning the low-

frequency dial on the amplifier to the 80-cycle
setting. This makes the amplifier unresponsive
to signals that have a time course longer than
1/80 sec. Examine the eff'ect of a long flash of
light on the train of impulses. What changes in
frequency of impulses do you see? At what
point, relative to the onset of stimulus and the
shape of the ERG, does the nerve response reach
the highest frequency? Does the response stop
completely after the stimulus has been on for a
time? What would you conclude of the animal's
sensations?

It seems to be a general rule that receptors re-
spond most strongly to change, rather than to
steady stimulation. Demonstrate this for your-
self by flickering the light to the Limulus eye by
rapidly moving the cardboard back and forth
through the light beam.

It is possible to separate out single fibers from
the optic nerve of Limulus. If you have time at
the end of the experiment, try teasing out small
bundles of fibers with glass needles and fine
forceps. Move both electrodes to the back
of the eye, and sling one such nerve bundle
across both wicks. If you are lucky, you may
be able to separate out a bundle that contains
only one or a few active fibers. This is not an
easy thing to do, and several tries may be
necessary.

EQUIPMENT

Per 2-4 students

electronic recording equipment

copper cage (2 ft square)

pair of wick electrodes (see notes on electronic

equipment)

wooden block, 3" X 3"

2-3 nails

3 neutral density filters (0.5, 1.0, 2.0) (partly exposed

films will do)

condensing lens

microscope lamp, wired for d-c

piece of cardboard, 8" X 10"

2 blocks of plasticene, 2" X 2"

2 glass needles

flashlight with red cellophane filter

limulus, 2" to 4" across carapace

thread

razor blades

^.^■Mff'r:'^*''^'' ■■ ^v\

:SSBSSaKSOS!lBS£.

PLANT GROWTH AND TROPISMS;
CARBON DIOXIDE FIXATION
AND TRANSLOCATION OF
PLANT SUBSTANCES

(Readings: Weisz, pp. 253-263. S.P.T., pp. 183-185; 57-63. Villee, pp. 107-
113; 126-127. Review Exercise X on "Photosynthesis." G. Wald, "Life and
Light," Sci. Am. 201, No. 4, 92-108, Sept. 1959, Reprint No. 61.)

All organisms respond to stimuli, though not
all of them with as swift integration and motions
as provided by the neuromuscular systems of
higher animals. Plants, for example, from uni-
cellular molds to flowering plants, respond to a
variety of stimuli with appropriate motions.

When we plant seeds in the ground, for ex-
ample, we pay no attention to how they are
oriented, yet the stems always grow upward and
the roots downward. Similarly, in any situation
in which light comes regularly from one side,
plants tend to bend toward the light.

These responses are obviously highly ad-
vantageous, directing the organs of the plant
where they can do the most good. Such directed
motions in response to directional stimuli are
called tropisms. (If the entire organism, rather
than one of its parts, moves toward or away from
the stimulus, this is sometimes called a taxis.) In
the case of growing upward or downward, the
force is gravity, and the direction is the center of

the earth. We speak of such responses as geo-
tropisms, and distinguish the directions toward
and away from the center of the earth as positive
and negative. So one describes the growing
downward of roots as positive geotropism, the
growing upward of shoots as negative geotrop-
ism. Similarly, bending toward the light is posi-
tive phototropism, whereas bending away from
the light would have been called negative photo-
tropism.

Since they lack contractile tissues, plants per-
form these motions by differential growth. Light,
for example, inhibits the axial growth of shoots.
Hence the side toward the light grows more
slowly than the shaded side, with the result that
the shoot bends toward the light. Some of the
lower invertebrates that are attached as are
plants exhibit similar tropisms. The hydroid
Eudendrium, for example, a coelenterate, bends
toward light by differential growth, just as does
a plant.

109

no PLANT GROWTH AND TROPISMS

Exercise XXI

As you know, plants of all sizes and ages al-
ways retain meristematic tissue that is capable of
new growth. In a young shoot, growth in length
is confined to a rather narrow zone toward the
tip. This growth is controlled by a hormone
called auxin. The most prominent auxin is in-
dole-3-acetic acid (lAA), which has the following
formula :

C O

/- \ ^

HC C C— CH2 • C

N
H

Indole

Auxin acts by promoting cell elongation,
rather than cell division. It is synthesized in the
tip of the shoot, though small amounts of auxin
are also produced in roots, leaves, and fruit. The
auxins are distributed throughout the plant from
the apical buds via the phloem. The highest con-
centration of auxin is found nearest the apical
bud, and the concentration falls off rapidly
toward the basal portions of the plant. Auxin is
inactivated or destroyed during growth, and must
be continuously supplied from the apical bud.
Within the range of low auxin concentrations, if
one portion of a plant has more auxin than an-
other, it grows faster. The differential distribu-
tion of auxin accounts for much of the differen-
tial growth, and hence the tropisms of plants.

Among today's experiments, you will have the
opportunity to examine the effects of auxin on
growth, the responses of plants to light and
gravity, and other aspects of the physiology of
plant growth. We shall use for these experiments
the classic oat shoot (Avena). The young shoot
consists of a colorless tubular sheath, the
coleoptile, which surrounds the yellow or green
primary leaf. It is the coleoptile that is princi-
pally responsible for the bending reactions. We
shall also take the opportunity to examine under
the microscope the tissues of a higher plant con-
cerned with growth and translocation.

One of the most useful techniques developed
for investigating cellular metabolism depends
upon isotope-labeled molecules. Such molecules
have exactly the same chemical properties as
those lacking the label, and can be used to follow
the pathways and ultimate fates of metabolites
in the organism. Today, we shall offer CO2
labeled with the radioactive isotope of carbon,
C'^ (therefore C'"'02), to a bean leaf, and inves-
tigate its uptake in light and darkness, and the
subsequent translocation of the carbon com-
pounds newly synthesized from it.

Radioactive compounds emit radiations that,
like light, affect a photographic film, producing a
latent image which darkens on development.
We will measure both the uptake and distribu-
tion of the radioactive carbon in the leaf by
exposing a film to it.

Carbon-I4 is a relatively stable radioisotope
that emits /3-rays (electrons). This is not a very
penetrating radiation; one thickness of paper can
usually block it. For this reason €'■* compounds
are relatively safe to use; yet take care with them.
Be careful not to spill any radioactive materials.
Also place any contaminated materials as soon
as you are through with them into the special
containers which are provided. Wash your
hands thoroughly before leaving the laboratory.

PLANT GROWTH AND TROPISMS

First test the effects of auxin on the growth of
the stem. You will be supplied with 4 oat seed-
lings that are 3 days old (the first leaf should
not as yet have pushed through the coleoptile).
With a razor blade cut off the terminal 3 mm
of each tip, and discard it (why?). Then, using
a sharp razor blade, cut a segment exactly 10 mm
long from each plant. Place 2 such segments in
each of 2 small petri dishes. Fill one dish with a
2% sucrose solution, the other with 2% sucrose
containing 2 mg per liter of indole acetic acid,
brought to a slightly acid pH with KH2PO4.
Place the dishes in the dark (your desk drawer).
After at least 2 hours have passed, measure the
length of the seedlings to the nearest quarter

Exercise XXI

PLANT GROWTH AND TROPISMS 111

millimeter, under the dissecting microscope. If
you leave the experiment until the following
morning, your results will be much plainer.
Calculate the percentage increase of length per
hour in each of the solutions.

Next, working in groups of four or eight, test
the effects of light and gravity on Avena seed-
lings. Each group should obtain a dish contain-
ing young seedlings. Weed out any seedlings
that are not straight. Place the dish under the
wooden box that is provided, at the end away
from the aperture that holds a light filter. At
the various tables in the room, the boxes contain
different light filters, red, yellow, blue, and green.
Move a microscope lamp close to the aperture,
so that light penetrating the filter reaches the
plants. Irradiate the plants in this way for 90
minutes. Then note whether or not they have
bent toward the light, what proportions have
responded, and about what angle the tip of the
plant assumes with the vertical. To make this
measurement more quantitative, lay the plant
on a piece of graph paper and trace the bend.
You should get a sufliciently accurate measure-
ment of the degree of bending to compare with
your neighbors' results.

By comparing your results with those obtained
at other tables, grade the effectiveness of the
different colored lights in stimulating bending.
Draw a graph of this effectiveness against wave-
length in the spectrum (representative wave-
lengths: violet, 410 m/x; blue, 470 mp; green,
520 mn; yellow, 580 m/x; orange, 600 m/x; red,
650 m/i). Such a graph, when corrected for the
energy content of the various colored lights, is
called an action spectrum.

Phototropic bending in plants, like vision in
animals, is mediated by light-sensitive pigments.
This is necessarily true; for light in order to have
any effect, chemical or physical, must be ab-
sorbed; and substances that absorb visible light
are pigments. The effectiveness of the various
wavelengths of light in stimulating vision or
phototropism depends in the first instance on the
capacity of the photoreceptor pigments to ab-
sorb those wavelengths. Hence an action spec-
trum tells us not only the region of the spectrum

most effective in stimulating the response, but
by the same token the region of the spectrum
most strongly absorbed by the photoreceptor
pigment. This tells us the color of the pigment,
and sometimes provides a clue to its chemical
nature.

Of the various pigments present in Avena seed-
lings, the chlorophylls a and b absorb light in
the blue and red, and hence are green in color;
whereas the carotenoids, xanthophyll and caro-
tene, as also riboflavin, absorb light only in the
blue, and hence look yellow. Judging by your
observations, which of these pigments might
possibly mediate the phototropic response?

Test tube

Bean plant

CO2 FIXATION AND
TRANSLOCATION

In this experiment, work in pairs. Obtain a
bean plant and a small test tube which contains
1 to 2 /xgm of radioactive barium carbonate from
your instructor. (Be careful not to spill any of
the carbonate; if you do, tell your instructor so
that he can get rid of it.) Tape the test tube to
one of the sticks provided, and place the stick
upright in the earth surrounding your bean
plant, directly underneath one of the bean
leaves. Adjust the height of the stick so that the
leaf rests firmly against the mouth of the test
tube, as shown in the diagram. Gently push the

112 PLANT GROWTH AND TROPISMS

Exercise XXI

leaf aside, and ring the top of the test tube with
vaseline. Carefully introduce 2 drops of H2SO4
into the bottom of the test tube with the capillary
tube that is provided. (Your instructor will
demonstrate this for you.) Make sure the acid
reaches the bottom of the tube, and that there is
not an air bubble holding back the second drop
of acid. If there is, hit the tube sharply with a
snap of your finger, until the acid falls to the
bottom of the tube. Also, be careful not to allow
any acid to wet the top of the test tube.

As soon as the acid is in the test tube, quickly
replace the leaf, very gently pushing it down until
coming in contact with the vaseline, it is sealed
over the mouth of the test tube. The leaf should
now be left undisturbed for 10 minutes, while
the plant is brightly illuminated with the lamp
that is provided.

You should be able to see small bubbles of
C^*02 rising through the acid. This is formed by
the reaction :

BaCi^Os + H2SO4 -^ BaS04 + H2O + C'^Oa.

After exactly 10 minutes, cut off the leaf at the
base of its stem with a pair of scissors. Carefully
wipe off the vaseline, and lay the leaf flat within
a folded piece of paper that is marked with your
name and the letter "A." Place the paper in the
refrigerator.

Now obtain another test tube with radio-
active carbonate, and repeat the above experi-
ment on another leaf, except that immediately
after the leaf has been placed over the test tube
generating C'*02, cover the whole plant with
the black hood that is provided. Again, expose
the plant for exactly 10 minutes.

At this point, remove the black hood and as
quickly as possible place the test tube generating
C'^Oo under a third leaf. Cut off the second leaf
as you did the first, remove the excess vaseline,
lay it within a folded piece of paper marked with
your name and "B," and also place it in the
refrigerator.

To prepare the third leaf, again brightly il-
luminate the plant, and leave it undisturbed for
25 to 30 minutes. Now remove the leaf, and

prepare as before, marked with your name
and "C."

After the third leaf has been in the refrigerator
for at least 10 minutes, remove all three leaves
from the refrigerator and take them into a room
which has been outfitted as a darkroom, illumi-
nated only with dim red light.* There take two
pieces of x-ray film and cut off one corner of each
piece to identify an end. (Try to perform all
manipulations with the x-ray film in the dark, or
as nearly so as you can manage. The film rapidly
fogs when exposed to light, even to red light.)
Place the three leaves in the order A, B, C from
top to bottom on one piece of film, with the cut
corner at the top. Cover this with the second
piece of film, also with the cut corner at the top,
so that the leaves lie between the two emulsions.
Sandwich the films between two pieces of card-
board, holding everything together with rubber
bands around each end. Place the sandwich in
a black envelope, seal with rubber bands, mark
with your name, and put the package into the
freezer of a refrigerator.

Sometime during the next laboratory period
develop your film in the dark room. Again try
to work in as little light as possible. Remove
the film from the sandwich, and throw the radio-
active leaves in the can which is provided. Attach
a clothespin to one side of each piece of film and
immerse both films in developer for 3 minutes,
then rinse in water, and immerse both in fixer
for 5 minutes. Then wash the films in running
water for at least 10 minutes before looking at
them.

{Caution: Photographic developer stains, and
fixer eats at clothing. Keep both from dripping
around; and be particularly careful to keep any
trace of fixer out of the developer.)

Wherever a (3-particle from the radioactive
carbon hits the film emulsion, a silver ion is
reduced to metallic silver, which in the de-
veloper catalyses the reduction of a whole grain
of the emulsion, resulting in a black spot. Com-
pare the patterns and intensity of radioactivity

*If a Geiger counter is available, count the radio-
activity incorporated into each leaf at this point and
record the results in your notes.

Exercise XXI

PLANT GROWTH AND TROPISMS 113

in the three leaves as displayed by your film.
What do you conclude about the effects of light
on the incorporation of CO2? What evidence do
you observe of the translocation of recently
synthesized organic molecules?

In the intervals of waiting for things to hap-
pen, study the prepared slides of the apex of the
flowering plant Coleus. The small, darkly stained
cells that form a small mound at the apex of the
stem represent the apical meristem. Remember
that these cells are responsible for the further
growth of all the remaining plant structures that
are above ground. Note the young leaves de-

veloping around the apex. Careful observation
will reveal much of the differentiation of the
tissues that compose the lateral stems and leaves.
Note the area that will become vascular tissue.
Follow bundles of vascular tissue back to the
apex from the largest leaves and the base of the
stem.

Tissue differentiation is much easier to study
in plants than animals, because all stages of
differentiation appear in a linear sequence start-
ing at the apical meristem and working toward
whatever type of tissue interests you.

EQUIPMENT

Per student

8 Avena seedlings, grown 3-5 days

2 petri dishes

2% sucrose solution (5 ml)

2% sucrose solution with 2 mg/1 indole acetic acid,

brought to pH 6.5 with KH2PO4 (5 ml)

razor blade

graph paper

0.5-ml centrifuge tube containing BaCos

wooden sticks (12" long)

piece of 5" X 7" (no screen) x-ray film

piece of 5" X 7" cardboard

2 rubber bands

black paper envelope (8" X 10")
prepared slide of apex of coleus

Per 2 students

bean plant {2-A weeks old)

black wooden box with colored light (red, green,

blue, and yellow) filter

Per 8 students

roll of Scotch tape

H2SO4, 1 M (5 ml) and capillary pipet

jar of vaseline

Per laboratory

space outfitted as a darkroom

developer and fixer for processing x-ray film

w^sm

XXII

INTRODUCTION TO THE
GENETICS OF MAN AND THE FRUIT
FLY; REGENERATION OF PLANARIA

(Readings: C. M. Williams, "The Metamorphosis of Insects," Sci. Am. 182,
No. 4, 24-28, April 1950, Reprint No. 49. Weisz, Chapter 27. S.P.T., pp. 159-
161; 240. Villee, pp. 496-508. R. Buchsbaum, Animals Without Backbones,
Univ. of Chicago Press, rev. ed., 1948, Chapters 10 and 12.)

In this laboratory section, beyond considering
a few simple examples of human genetics, we
will begin a four-week program of genetics ex-
periments on the fruit fly, Drosophila. While
these experiments develop, we shall have ample
time to do other things. During the present
period you will begin experimenting with the
regeneration of a planarian. There will also be
slides on display demonstrating meiosis and
mitosis in a variety of animals and plants; and
also stained preparations of the giant chromo-
somes of the salivary glands of Drosophila
larvae.

ASPECTS OF HUMAN GENETICS
PTC tasting

The substance phenlythiocarbamide (PTC;
phenylthiourea) tastes very bitter to some per-
sons ("tasters") but is tasteless to others ("non-

tasters"). The abiUty to taste it is inherited as a
dominant characteristic. About 70% of the
American population taste PTC, the other 30%,
who are homozygous for the recessive allele, do
not taste it.

Pieces of paper which have been impregnated
with PTC will be provided. Hold a piece in your
mouth for about 30 seconds to determine
whether or not you are a taster. How does the
class come out as a whole?

Excretion of methyl mercaptan

Asparagus contains the organic sulfur com-
pound dimethylthetin ((CH3)2S"^-CH2COOH).
About 60% of the American population possess
an enzyme which catalyzes the conversion of
dimethylthetin to methyl mercaptan (CH3SH).
It is the latter substance that gives urine its
characteristic odor after asparagus is eaten. The
presence of the enzyme is a dominant trait.

114

Exercise XXII

INTRODUCTION TO GENETICS OF MAN AND FRUIT FLY 115

Sex-linked genes in man

The most common sex-linked human trait is
red-green color blindness. This occurs in about
8% of the male and 0.5% of the female popula-
tion. The recessive gene responsible for color
blindness is in the X chromosome, and since
men have only one X chromosome, while women
have two, a father transmits his X chromosomes
to all his daughters but never to his sons, where-
as a mother gives one X chromosome to each of
her children regardless of sex. It follows that
the sons of a color-blind mother are all color
blind, but daughters have normal vision if the
father has normal vision. The daughters, how-
ever, carry the color-blindness trait; if married
to men with normal vision, their daughters are
normal, but half their sons are color blind. How
is a color-blind woman produced?

Hemophilia, the failure of the blood to clot, is
another sex-linked recessive trait, also therefore
almost entirely restricted to males. One of the
troubles with European royalty is that Queen
Victoria, a carrier of the hemophilia gene, tended
to have royal descendants who bled for the
wrong reasons.

Attached or free ear lobes
Full lips, thin lips
Freckles

DROSOPHILA GENETICS

The common fruit fly, Drosophila melano-
gaster (i.e., "black-belly"), has been highly im-
portant in genetics since introduced half a
century ago by T. H. Morgan. Its short genera-
tion time, ease of handling, large number of off-
spring, and convenient size all tended to make
this the most widely used organism in genetics.
Only lately has it been superseded by micro-
organisms, which offer still further conveniences
and potentialities for experiment, once one has
learned to handle them.

A further advantage of Drosophila is that it
possesses as the diploid number only four pairs of
easily identified chromosomes. Also the salivary
glands of the larvae contain giant chromosomes,
the structures of which have provided important
anatomical correlations with genetic linkage
maps, and which have furthered the analysis of
chromosome functions and rearrangements.

Other human genetic traits

You have already typed your own blood. (See
page 327 of S. P. T., or pp. 471-472 in ViUee, for
a description of genetic aspects of blood types.)

You may be interested in the following ex-
amples of other human Mendelian traits:
Blood types Rh+, Rh"
Tongue rolling
Tongue folding
Widow's peak
Dimpled cheeks
Mongolian eyefold
Hyperextension of distal thumb joint
Albinism

Straight hair, curly hair
Mid-digital hair on fingers
Far-sightedness
Near-sightedness
Astigmatism

Overall plan of the experiment

We have planned an experiment that demon-
strates Mendel's laws of segregation and inde-
pendent assortment. It involves two recessive
mutants, the genes for which are located in
separate chromosomes: dumpy {dp) and ebony
(e). Flies homozygous for dumpy have truncated
wings, only about two-thirds as large as wild
type. Those homozygoous for ebony have shiny
black bodies, much darker than wild type.

A week before this laboratory session initial
crosses were made between males and virgin
females, the flies of one sex taken from a stock
homozygous for dumpy body, the other from one
homozygous for ebony. The parent flies remain
in the vials that you have been given, and will
shortly be removed. The eggs already laid by
these females will hatch to form the Fi genera-
tion with which the experiment will be continued.

116 INTRODUCTION TO GENETICS OF MAN AND FRUIT FLY

Exercise XXII

The schedule for the entire experiment is as

follows:

This week: Remove the parent flies, following

the directions below.
Week 2: Cross the F\ flies, and record their

phenotypes.
Week 3: Remove the Fi parents.
Week 4: Score the results of this cross and of

a more complex cross which will be

given you.

Each week you will find detailed instructions
for proceeding with the experiment.

This week's work

The main job this week, apart from removing
the adults from your vials, is to get to know the
flies and learn to handle them.

Begin by removing the adults from the vials,
etherizing them as described below. Have the
vials ready beforehand to be stored for incuba-
tion. Be sure each vial is labeled with your name
and a description of the cross and the date it was
made. Each mutant gene has a special symbol,
dp for dumpy, e for ebony, and + for wild type.
We will use diploid formulas that represent the
somatic cells of the parents, rather than haploid
formulas that would represent mature germ cells.
The formula for the female should be written
on the left, followed by X and the formula for
the male. The pair of gene symbols for each
chromosome pair is written one above the other,
like a fraction; so for example, homozygous
dumpy is represented by dpjdp. The initial cross,
therefore, in which homozygous dumpy females
were mated with homozygous ebony males, can
be written dpjdp X eje.

As soon as the adults have been taken out of
the vials, return the vials to the boxes so that
they can be incubated until next week.

The etherized adults should be examined care-
fully under the dissecting microscope. You
should be able to distinguish males from females
and the mutant types from wild-type flies, several
of which will be provided for comparison. You
will find descriptions to guide you below and in
your reading.

Examine also Drosophila eggs, larvae, and
pupae under the dissecting microscope. These
stages will be given you. Do not take any eggs
from your experiment.

In any free time, examine the prepared slides
which are set up under the demonstration
microscopes.

Life cycle

At 25°C the entire life cycle of Drosophila
is usually completed within 10 days. It includes
four stages: egg, larva, pupa, and imago
(adult), as in all Diptera (true flies). The eggs,
about 0.5 mm in length, are sausage-shaped
white structures bearing a pair of filaments at
the end, which help to keep them from sinking
into the soft food on which the eggs are always
laid. The larvae are little white maggots which
burrow in the food at the bottom of the vials.
Drosophila larvae undergo two molts after
emerging from the egg; the larval period thus
consists of three stages (instars). Larvae may
be up to 4.5 mm long in the final stage; it is
from them that the giant salivary gland chromo-
somes are obtained. At the end of the third
instar, the larvae crawl up to a dry spot on the
wall of the container, where they pupate in
small dark cocoons. Pupation lasts about four
days at 25°C, after which the adult fruit fly
emerges. The adult is at first light in color, and
its wings are crumpled; but within a few hours
the wings expand and the adult takes on its
familiar appearance.

Determining the sex of adults

Males can be told from females with the
naked eye, using several dilTerent criteria.
Though the external genitalia are more com-
plex in males, this difference is difficult to see.
The abdomen of the female is long and pointed
at the end, whereas that of the male is consider-
ably shorter and somewhat stubby. Further-
more, the entire rear portion of the male
abdomen is black, whereas in the female dark
and light bands alternate to the tip. One of the

Exercise XXII

INTRODUCTION TO GENETICS OF MAN AND FRUIT FLY 117

most helpful signs of maleness is the possession
of "sex combs," consisting of a series of about
10 stout, black bristles on the basal (upper)
tarsal joint of the first legs; these can be seen
with the naked eye.

Your initial matings were made with virgin
females. These are obtained by emptying all
the adults out of an active culture of Drosophila.
One or two hours later, one finds a few new
adults that have emerged in the interim, and
have surely not yet mated. If one now segre-
gates such virgin females, they can be kept until
wanted for mating. The males of course don't
require such precautions; they can be taken
from the culture at any time.

Drosophila culture

Drosophila can be raised in the laboratory in
3-inch glass vials closed with cotton plugs and
held at a constant temperature of 25°C. The
food consists of a cooked-up mixture of corn
meal, agar, molasses, water, and a mold-preven-
tive. The hot food is poured into vials, and
allowed to cool, the mass being stiffened by the
setting of the agar into a gel as it cools. A thin
suspension of yeast sown on the surface of the
food grows rapidly on this medium, providing
food for the flies. If the food is too soft or the
vial too wet, the adult flies readily stick to the
walls or drown. Precautions must be taken to
prevent these things from happening. It is im-
portant also that flies not be allowed to escape
into the laboratory, since adventitious matings
could invalidate your results. Containers are
provided for the disposal of used vials, and flies
with which you have finished, first killed
by overetherizing, should be placed in the
"morgues" (jars containing kerosene oil).

Handling Drosophila

Flies are anesthetized with ether to keep
them quiet during examination or transfer.
Care must be taken not to overetherize them;
there is only a narrow gap between anesthetizing
and killing them with ether. Flies killed in this
way can be recognized, since their wings are

drawn up away from the abdomen, the pro-
boscis is everted, the legs are stiffly extended
and bunched together, and the body is curled
and has stopped twitching.

There are two types of etherizer available. To
use the plastic Burco model, put not more than
two drops of ether into the chamber through the
spout. The ether should last an hour or more.
Shake the flies to the bottom of your vial;
remove the vial top and place the funnel top
over the open vial. Now invert the vial and
etherizer together and tap gently to shake flies
into the chamber. Immediately after the last
fly becomes still, remove the cap at the bottom
of the chamber and pour the flies out.

The other type of etherizer is made of a glass
bottle with a tightly fitting cork holding a piece
of cotton. To use it, drop a few drops of ether
on the cotton, quickly shake the flies from the
culture bottle into the etherizer bottle, and
quickly close it with the cork, with its ether-
wetted cotton inside. (Be sure that the cotton
on your etherizer plug is just moistened, not
soaked with ether. Any liquid ether that touches
the flies is instantly fatal.) Some practice may
be needed to do this smoothly. It is helpful
first to tap the culture vial sharply against the
palm of the hand, so as to shake the flies away
from the cotton plug, yet not so violently that
they become stuck in the food. Immediately
pull out the plug, and set the mouth of the
culture vial into the mouth of the etherizer
bottle, holding the latter down. Tapping lightly
on the upturned bottom of the culture vial, and
holding the etherizer bottle toward the light,
help to get the flies into the etherizer bottle.
Don't tap so hard as to knock pieces of food
in on top of them.

Caution: Since ether is dangerously explosive,
there must be no flames or lighted cigarettes in
the room.

At most 10 seconds after the flies in the
etherizer have stopped moving, empty them out
for examination. If the anesthetization wears
off" before you have finished examining them,
they can be re-etherized. A re-etherizer is made
from one section of a petri dish, with a piece

118 INTRODUCTION TO GENETICS OF MAN AND FRUIT FLY

Exercise XXII

of absorbent cotton or paper taped on the in-
side. A drop or two of ether is put on the
cotton or paper, and the dish is placed over the
flies for a few seconds. Alternatively, the flies
can be covered with the open chamber of the
plastic etherizer. Flies are more easily killed by
a second exposure to ether than by the first, so
be particularly careful not to overdo it.

The anesthetized flies should be dumped out
of the etherizer onto a white paper card and
examined under the dissecting microscope; use
whatever magnifications are convenient. The
flies are moved around on the card with a
camel's hair brush or a dissecting needle. When
dividing the sexes, it is convenient to line up
all the flies at the center of the card and then
to run down the line pushing males to one side
and females to the other.

REGENERATION OF PLANARIA

Planarians have a remarkable capacity to re-
generate parts of their bodies which have been
removed. Regeneration occurs in all animals,
yet to diff"erent degrees, tending to diminish as
one ascends the evolutionary scale, until in
mammals it is restricted to wound-healing.

Planarians are members of the phylum
Platyhelminthes, the flatworms. (For their sys-
tematic position, see Weisz, pp. 731-732;
S. P. T., pp. 528-531 ; Villee, pp. 201-203.) They
are small animals, less than an inch long, and
have a primitive brain, eyes, digestive organs,
muscles, and an excretory system. They repro-
duce either sexually or by fragmentation.

Our planarian is Dugesia dorotocephala, a
relatively large species that is uniformly darkly
pigmented. It is found in the middle-western
states in wells or spring-fed streams. In the
laboratory, planarians are kept in spring water
or dechlorinated water, and are fed occasionally
on bits of beef liver. During the course of
regeneration, however, they should be starved.

Each student will be given two or three
animals. It will be up to you to design your
own experiments to demonstrate regeneration.

This laboratory guide is only to suggest possi-
bilities. Everyone should read the chapters in
Buchsbaum's book before coming to the labora-
tory in order to see the variety of simple experi-
ments that can be done. After the initial opera-
tions have been performed, the animals must be
disturbed as little as possible. If you would
rather carry out this experiment at home, per-
form the surgery there also.

Before operating on the animals, you might
determine their sensitivity to light. They have
well-defined eyes, which can discriminate bright-
nesses and the direction from which light comes,
but which probably do not resolve images. Use
the lamp from the dissecting microscope as a
light source. Simpson, Pittendrigh, and Tiffany
(p. 240) and Buchsbaum (pp. 118-120) describe
several experiments on the behavior of these
animals, even one experiment that suggests
learning. You might want to repeat these or to
devise experiments of your own.

Planarians are best observed under low powers
of the dissecting microscope, in either a small
petri dish or on a slide. Be sure to use a prepara-
tion of bicarbonate-versene-tap water (BVT) and
never the untreated tap water, which may kill
them. They are best transferred from one con-
tainer to another with a small camel's hair
brush, or with a bit of tissue paper grasped in
forceps so as to serve as a brush. To make a
cut, wait until the animal has flattened out, and
then make a quick slash, perpendicular to the
plane of its body, with a clean, sharp razor
blade. After the operation, transfer the animal
or its parts to the containers and label them
carefully. Keep them cool, though not cold,
and in little or no light. The water should be
replaced two or three times a week with fresh
BVT, and dead animals must be removed at
once. Do not feed them during the month or so
it will take to complete regeneration. The ani-
mals should be left in the laboratory and dis-
turbed as little as possible. They are quite
fragile after the operation and will disintegrate
if shaken.

Experiments of this kind have disclosed a
number of principles which govern regenera-

Exercise XXII

INTRODUCTION TO GENETICS OF MAN AND FRUIT FLY 119

tion. Two of these are: (1) The pieces of the
animal retain the polarity they had in the whole
animal; a new head grows from the end origi-
nally nearest the head, and a new tail from the
end originally nearest the tail. (2) Pieces cut
from near the head regenerate better than those
from near the tail. Your own experiments can
demonstrate both points. It has been suggested
that differences in the rate of metabolism, graded
downward from the anterior end, may explain
the polar nature of regeneration. The regenerat-

ing parts at first lack the pigmentation of the
original tissue and are thus easily recognized.

Planarian monsters possessing two heads or
two tails can be made by slicing the animal
parallel to the long axis of the body, the cut
extending about a third of the body length,
either through the head or through the tail.
Since there is a great tendency for the divided
parts to rejoin and heal together, the slit should
be reopened every day if necessary.

EQUIPMENT'

Per laboratory

prepared slides demonstrating meiosis and mitosis
stained preparations of salivary glands of Drosophila
larvae

ether in dropping bottles

PTC paper

white cards

dissecting microscope for each student

etherizers t and re-etherizers

"morgues" (jars containing kerosene oil)

razor blades

camel's hair brushes

Dugesia dorotocephala (2 or 3 per student)

petri dishes (2 or 3 per student)

solution of bicarbonate, versene, and tap water

(BVT), prepared as follows:

(1) NaHCOs (2 gm/100 ml); sodium (di)ethylene-

diamine tetracetate (sodium versenate) 1 gm/100

ml
(2)CaCl2(1.5gm/100ml)

To prepare 1 liter of BVT, put 5.0 ml of solution
(1) and 5.0 ml of solution (2) into some hot tap
water, and then make up to 1 liter with hot tap
water. (Using hot water gets rid of the chlorine
faster.) The solution can be used after standing
overnight.

*Detailed information about designing experiments with Drosophila and obtaining stocks can be found in
the Drosophila Guide, by M. Demerec and B. P. Kaufmann, which can be obtained for 25^ from the Car-
negie Institution, 1530 P St. N. W., Washington 5, D. C.

fEtherizers can be made from small, wide-mouthed bottles to the corks of which have been tacked bits of
cotton. Polyethylene anesthetizers can be bought from Burdick Drosophila Supply Co., 250 Lincoln Street,
West Lafayette, Ind.

XXIII

FERTILIZATION AND EARLY
DEVELOPMENT; CONTINUATION OF
THE GENETICS EXPERIMENT

(Readings: Weisz, pp. 532-536; 594-601.
420-421; 430-432.)

S.P.T., pp. 335-340. Villee, pp.

During fertilization, a haploid sperm nucleus
fuses with a haploid egg nucleus to form the
diploid nucleus of a new cell, which by repeated
mitoses and differentiation develops into an
adult organism. All the somatic cells of the
adult organism, including the precursors of the
mature germ cells (spermatocytes and oocytes)
have the double chromosome number (2/2). As
part of the process of maturation of the germ
cells, this is halved in the reduction division of
meiosis. The sperms that engage in fertilization
are wholly mature and haploid. In most ani-
mals, however, the egg does not complete its
maturation until after the sperm head has
entered it. The egg nucleus then completes its
meiosis, throwing off the supernumerary nuclei
in one or two polar bodies and achieving the
haploid condition just before fusing with the
sperm nucleus. In many coelenterates and
echinoderms, the egg has finished its matura-
tion divisions before the sperm enters, so that
fusion of nuclei and cleavage can proceed
immediately.

THE SEA URCHIN

During this laboratory period we shall ob-
serve fertilization and the first stages of develop-
ment in an echinoderm, the sea urchin. The
sea urchin can be induced to shed its eggs and
sperm by passing a weak electric current through
it or by injecting a small quantity of potassium
chloride solution; or the ovaries and testes can
be removed by dissection. It has been estimated
that one sea urchin contains about 10'^ sperm,
or about 8 million eggs.

Procedure

Obtain a petri dish containing a suspension of
eggs in sea water, and examine them under the
dissecting microscope at convenient magnifica-
tions. Note the thick jelly coat that holds the
eggs apart. Add one drop of dilute sperm sus-
pension to the eggs, swirl gently to mix, and
record the time and room temperature.

The schedule of sea urchin development varies
slightly in different batches of eggs and varies

120

Exercise XXIII

FERTILIZATION AND EARLY DEVELOPMENT 121

greatly with the temperature. An approximate
schedule of development at 23°C for an East
Coast sea urchin, Arbacia punctulata, is as
follows:

Sperm comes into contact with egg

0 min

Completion of fertilization membrane

Union of pronuclei

Completion of hyaline layer

Streak stage

20-35

Nuclear membrane breaks

Prophase

Metaphase

Anaphase

Telophase

1st cleavage

2nd cleavage

3rd cleavage

103

4th cleavage

130

5th cleavage

157

Blastula (about 1000 cells)

7-8 hours

Gastrula

12-15 hours

Skeleton begins

19 hours

Pluteus (larva)

1 day

Other species of sea urchin have different
schedules of development, usually slower.

After penetrating the jelly coat, a sper-
matozoon touches the surface of the egg. At
this point an entrance or fertilization cone forms,
within about 20 seconds, which engulfs the
sperm head. The cone is very difficult to see,
so don't be disappointed if you miss it. A
fertilization membrane also begins immediately
to form around the egg, and to lift off, leaving
a space between it and the egg surface. This
takes about 2 minutes. The changes that take
place in the egg during the next 40 minutes or
so are difficult to see in v/vo, but slides are
available showing sections of eggs in various
stages of mitosis.

Depending upon the species and the tempera-
ture, about 45 to 90 minutes after fertilization,
sea urchin eggs begin to undergo their first
cleavage. Prepare a sample containing 30 to 50
eggs, and record the time at which the first

eggs have cleaved, and then at intervals of 2-3
minutes, record how many eggs have cleaved
until all that are going to have done so. Draw
a graph showing the percentage of cells that
have undergone first cleavage (ordinate) against
time in minutes (abscissa). Draw another graph
showing the percentage of cells that have cleaved
per one- or two-minute interval, i.e., the rate
of cleavage (ordinate) against time in minutes
(abscissa).

The latter curve usually has the typical bell-
shaped form of a "population curve," the dis-
tribution of any measured property in a popula-
tion of independent individuals. The former
curve (usually S-shaped or sigmoid) is the
typical summed-over or integral form of a
population curve. If in this class, for example,
you measured everyone's height, and then
plotted two curves — one of the number of per-
sons in each height range (66-68 inches, 68-70
inches, and so on) as ordinate against the height
as abscissa, the other curve recording the total
number of persons under each height as ordinate,
against the height as abscissa — you would prob-
ably obtain a similar pair of curves. (To say
this in the language of calculus: the bell-shaped
distribution curve is the differential form; the
sigmoid curve is its integral.)

In order to determine the schedule of develop-
ment through the first four cleavages, you will
want to examine fertilized eggs during the first
five or six hours after fertilization. Your
instructor will provide you with two batches of
eggs, fertilized 3 hours and 1.25 hours before
the laboratory period begins. With these two
batches and the eggs you have fertilized at the
beginning of the period, you will have samples
of fertilized eggs at all stages during the first
six hours of development. Working in pairs, set
up a sampling schedule so that you can follow
the progress of development at half-hour inter-
vals, using the time of fertilization of the three
batches as starting times. Thus, using the egg
fertilized 3 hours before the laboratory session
began, you may get time intervals up to 6 hours
after fertilization. A sample should be taken
by placing a few drops of eggs selected randomly

122 FERTILIZATION AND EARLY DEVELOPMENT

Exercise XXIII

from the large batch onto a depression slide.
Examine under low power and count 30 to 50
eggs, classifying them according to cleavage
stage (the larger your sample, the more reliable
your results will be). For each succeeding count,
withdraw a fresh sample from the appropriate
batch of eggs. Tabulate your results when you
finish according to time (time from fertilization
to sampling) and cleavage stage (expressed as
per cent of total sample counted). Make a
graph showing the percentage of eggs uncleaved
and in each stage of cleavage as ordinate, against
the time as abscissa (use different colors for the
different cleavage stages). In such a graph, the
uncleaved eggs should form an S-shaped curve,
the various cleavage stages bell-shaped curves.
The peak of each of the latter curves represents
the characteristic time for that stage of develop-
ment. With these characteristic times, and
noting the temperature, prepare a schedule of
development for the species you have worked
with.

MAMMALIAN SPERM

Recently methods have been devised for
freezing and storing bull sperm for long periods
of time. Currently, artificial insemination, using

sperm from a few superior bulls, is common
practice in the dairy industry. A suspension of
bull sperm will be available for examination.
Put a drop or two on a microscope slide, cover
with a cover slip, and observe immediately
under the compound microscope. Do not allow
the preparation to dry out.

GENETICS EXPERIMENT
(continued: second week)

During a lull, make the new matings in your
Drosophila experiment. The flies now in the
vials are the Fi generation. Etherize them care-
fully and examine them under the dissecting
microscope. Record the phenotype of every
fly. If they aren't as expected, consult an in-
structor; a mistake may have been made in the
original mating.

Prepare two new sets of matings, placing 3
males and 3 females in each of two vials. Label
the vials, and store them in the boxes until next
week, when we will remove the parents. Two
weeks from today, the F2 generation will have
emerged, and the results of the experiment will
be analyzed.

Why have no precautions been taken to ob-
tain virgin females for today's matings?

EQUIPMENT

Per student

small petri dish

2 droppers

microscope slides and cover slips

depression slide

dissecting microscope

compound microscope

sea water

prepared slides of sea urchin development

diluted suspensions of sea urchin eggs and sperm*

frozen bull spermf

equipment for handling Drosophila is the same as

for Exercise XXII

2 vials for Drosopliila

*Detailed instructions for setting up the sea urchin experiment will be found in E. B. Harvey's excellent
book, Tlie American Arbacia and Otlier Sea Urchins (Princeton University Press, 1956). Arbacia punctulata
is common on the East Coast, and Sirongylocentrotiis purpuratiis or S. franciscanus on the West Coast. The
latter forms, containing mature eggs and sperm, can be obtained during the fall and winter from the Pacific
Bio-Marine Supply Co., P. O. Box 285, Venice, Cal.

fThis must be kept at dry-ice or liquid-nitrogen temperature. Deep freezers are not cold enough to main-
tain such preparations. Sources of bull sperm will be found in the yellow pages of telephone directories,
listed under "Livestock Breeders," or by contacting the county agent in agricultural communities, or any
agricultural college.

XXIV

DEVELOPMENT OF THE CHICK,
CONTINUATION OF THE GENETICS
EXPERIMENT

(Readings: J. D. Ebert, "The First Heartbeats," Sci. Am. 200, No. 3, 87-96,
March 1959, Reprint No. 56. C. H. Waddington, "How Do Cells Differentiate?"
Sci. Am. 189, No. 3, 108-116, Sept. 1953, Reprint No. 45. See also the handsome
photographs showing the progressive stages of chick development in the little
book by E. Bosiger and J. M. Guilcher, A Bird Is Born, Sterling Pub. Co., 1959.)

1...

The chick egg has been a classic object for
the study of embryonic development for the last
three-hundred years. It achieved this position
in the great work of Wilham Harvey (whom
you already know as the discoverer of the cir-
culation of the blood) on The Generation of
Animals. This work contains on the title page
the aphorism, Ex ova omnia, "all life from the
egg." The chick egg provides fine material for
the analysis of development beyond the earliest
stages, which have already passed before the
egg is laid.

The egg is fertilized immediately after ovula-
tion, as soon as it enters the oviduct. Usually
five or six sperm enter, a common condition in
the large eggs of certain amphibia, reptiles, and
birds, though abnormal in most other animals.
One sperm head eventually fuses with the egg
nucleus; the others disintegrate.

At the time of sperm entry the egg nucleus is
just entering its first maturation division, and
must go on to complete its meiosis before the

egg and sperm pronuclei fuse. Then cleavage
begins, and goes through to early gastrulation
within the hen, before the egg is laid. Also the
walls of the oviduct secrete a layer of albumen
around the egg, which serves later to float the
embryo within the shell and provides it with an
aqueous environment. The shell membranes
and the porous limestone (calcium carbonate)
shell are subsequently laid down by the shell
gland. Only the yolk with its small disc of
protoplasm represents the true ovarian egg. All
the rest is accessory structure. All vertebrate
embryos develop in an aqueous environment;
and such eggs as this represent a device for
bringing and maintaining an aqueous environ-
ment ashore — in a sense, an enclosed pond.
How do you think the size of the yolk is corre-
lated with the time it takes various types of egg
to develop?

In today's experiment each student can
examine an early stage in the development of
the chick embryo. Record your observations

123

124 DEVELOPMENT OF THE CHICKS

Exercise XXIV

in a labeled sketch. Your partner will at the
same time be examining an embryo at another
stage, so that each pair of students will have a
more-or-less complete picture of early develop-
ment. Each student also will perform a test for
cytochrome oxidase on his chick embryo.

Prepared slides of chick embryos will be avail-
able for examination under the microscope,
representing stages of development both earlier
and later than your live embryos. Examine
them carefully, tracing the development of
various parts of the embryo from stage to
stage: the heart, brain, eye, limbs, musculature,
and so on.

EARLY STAGES OF THE
CHICK EMBRYO

Two students will work together on this
experiment. Each pair will be given two eggs,
one of which has been incubated at 38°C for 3
days and one for 5 days. Each egg is marked
with the number of days of incubation.

In an egg left resting in one position for any
length of time, the embryo has rotated so that
the blastodisc is at the top, owing to the yolk
being heavier. You cannot rely on this in the
eggs given to you, and you should "candle"
your egg to find where the embryo lies. This is
conveniently done by holding the egg in front
of a microscope lamp. Mark with a pencil the
place on the egg where a shadow shows the
embryo to lie, and keep this uppermost. Lay
the egg in a petri dish, partly filled with warm
Ringer solution, and carefully cut around the
middle of the shell with scissors. Pick the shell
off carefully. The unbroken yolk and embryo
then will lie free in the Ringer solution. If a
living embryo is not present, get another egg
from the instructor. Use a dissecting microscope
for observing the embryo.

Up to gastrulation, the chick follows much
the same pattern of development as does the
echinoderm egg. The differences are due mainly
to the large amount of yolk in the chick egg,
which crowds the protoplasm of the egg into a

flat disc. Development proceeds primarily in
this disc, rather than in the whole sphere of the
egg as in a sea urchin. The embryonic heart
begins to beat after 2 days, and some circula-
tion of blood may be detected then. Anterior
to the heart is the head, with its bulging, partly
formed eyes. After 2 days the optic vesicles
reach the optic cup stage and lenses form. Also
the somites, precursors of the muscles, appear
at about the same time, as blocks of tissue lined
up in two rows along the trunk of the embryo.
The vitelline blood vessels which carry food to
the embryo emerge from the middle of the trunk
and branch out over the yolk.

Three-day embryo. The embryo is bent back
on itself, and lying on its side. Note the size of
the head and the development of the heart.
Two limb buds should be visible on each side
of the embryo, as projecting lumps of tissue.
The anterior limb buds will give rise to the
wings, the posterior limb buds to the legs.
Count the number of somites and note the
development of the blood vessels surrounding
the embryo. Record your observations in a
labeled sketch.

Five-day embryo. The increased size and
vascularity at this stage are obvious. It may
help make fine details visible to rinse the embryo
with several changes of warm Ringer solution.
Blood vessels can be seen pressed close against
the shell. What is their function? Toward the
tail end of the embryo you should be able to see
a fluid-filled sac, the allantois, which functions
as a urinary bladder, and is one of the extra-
embryonic membranes. As the embryo metabo-
lizes the food material of the yolk, waste prod-
ucts accumulate in this sac. The embryo is sur-
rounded by the amnion, another extraembryonic
membrane, but this is difficult to see after the
egg has been opened.

CYTOCHROME OXIDASE IN THE
CHICK EMBRYO

One can detect the presence of cytochrome
oxidase in tissues with the so-called NADI

Exercise XXIV

reagent. This is a mixture of alpha-/;ophthol
and ^/methyl-para-phenylenediamine (hence
"NADI"), which is oxidized to the blue pig-
ment, indophenol blue, by cytochrome oxidase
in the presence of oxygen.

Remove an embryo from the yolk and rinse
it in warm saline solution. Draw off the saline,
and replace it immediately with warm NADI
reagent. Record the time. Now record the
time for the first trace of blue color to appear,
and continue to record its location and extent
at three-minute intervals. Compare the results
of your experiment with those of your partner
on an older or younger embryo.

DEVELOPMENT OF THE CHICKS 125

GENETICS EXPERIMENT
(continued: third week)

At some time during this period, go the next
step in your Drosophila experiment. Last week,
flies of the Fi generation were mated. Today,
these parent flies should be removed from the
vials and disposed of in the morgues. Then
replace the vials in the boxes for further incuba-
tion. Next week this experiment will be com-
pleted. The F2 generation of adults will have
emerged, and the results of the experiment can
be analyzed.

EQUIPMENT

equipment for handling Drosophila as in Exercise

XXII

fertile eggs incubated for 3 and 5 days (I per student)

prepared slides of chick development

dissecting scope

petri dish

ringer solution, kept at 37°C (0.9% NaCl may be
substituted for ringer solution if there is no desire
to keep the embryo alive for an extensive period)

NADI reagent

Preparation of NADI reagent. This should be pre-
pared just prior to use. Combine equal parts of
0.01-M alpha-naphthol, 0.01-M dimethyl-/7-phenyI-
enediamine (PPD), and phosphate buffer, pH 5.8.
The alpha-naphthol is made by dissolving 1.44 gm
in 1 liter of physiological saline (0.9% NaCl solu-
tion). Heat to dissolve. The PPD solution contains
1.36 gm in 1 liter of physiological saline. The phos-
phate buffer is a mixture of Na2HP04 (9.5 gm/1 dis-
tilled water), and KH2PO4 (9.07 gm/l distilled
water), mixed in the proportions 7.8 mm Na2HP04
solution; 92.2 mm KH2PO4 solution.

XXV

COMPLETION OF THE
GENETICS EXPERIMENT

The adult Fg flies in the dumpy-ebony experi-
ment have now emerged, and this laboratory
period will be devoted to examining them and
evaluating the results. Since the flies will not be
needed again, they may be overetherized before
counting. The more flies counted, the more
reliable the results will be.

Eight possible classes of flies can be dis-
tinguished from the crosses you have made:
wild-type males, ebony males, dumpy males,
ebony-dumpy males, and the same four classes
of females. Determine the number of flies in
each of the eight categories. From this you can
tell whether the mutant genes are dominant or
recessive, linked or not, and sex-linked or not.

After all the students have finished their
counts, all the results will be summed up to give
class totals, which can be treated as one large
experiment.

Consider the dumpy : wild-type and ebony:
wild-type ratios separately. What are they?

Make a diagram showing the genotypes of
members of the parental generation, the Fi
generation, and the Fa generation.

CHROMOSOME MAPPING:
A THREE-GENE EXPERIMENT

The dumpy-ebony cross has illustrated simple
segregation and independent assortment. To
demonstrate a more complex situation in

Drosophlla genetics, you will work with a hatch-
ing generation in which three genes are segre-
gated: apricot, cut, and bar. Apricot refers to
the eye color which is much lighter than the
wild-type red; cut to a marginal cleft in the
wing; and bar to the eye shape: in the male the
eye is restricted to a narrow vertical bar and
in the female to a kidney-shaped slit. These
characteristics are easily spotted. The symbol
for apricot is w" (since apricot is an allele of
white); for cut it is ct, and B stands for bar.

Examine your flies (you may overetherize
them) and record the various combinations of
these genes and wild-type genes separately for
the sexes.

There are eight possible phenotypes:

The parental types :

+ B red eye, normal wing, bar eye
ct + apricot, cut wing, normal eye

Single crossovers in region I.

M'" ct B apricot, cut wing, bar eye
+ + 4- comnletelv wilH-tvnp

completely wild-type

Single crossovers in region II:

H" + B apricot, normal wing, bar eye
-\- ct + red eye, cut wing, normal eye

Possible double crossovers:

w" -f + apricot, normal wing, normal eye
-\- ct B red eye, cut wing, bar eye

126

Exercise XXV

COMPLETION OF THE GENETICS EXPERIMENT 127

Are all genes on the same chromosome? How
do you know? If they do appear linked, calcu-
late the percentage of crossing over between
them. Prepare a map indicating relative posi-
tions of these genes on the chromosome(s). Dia-
gram two generations of crosses giving rise to
these offspring.

Interference of crossing over in one region
with crossing over in another region can be tested
in the following way :

Coincidence =

% double crossovers
(% crossovers in region I)
X {% crossovers in region 1 1)

(The denominator is the percentage of double
crossovers that is expected.)

If the coincidence is less than 1.0, crossing
over in one region interferes with that in an-
other. One sometimes expresses what is called
the "interference" as (1 — coincidence). Is
there interference in this test cross?

PROBABILITY IN GENETICS

The interpretation of breeding experiments in
genetics often requires statistical analysis; with-
out the use of statistics it is sometimes impos-
sible to decide whether the results of an experi-
ment agree with those predicted by theory. A
thorough treatment of the mathematics of
genetics is beyond the scope of this course, but
it will be helpful to consider a few elementary
principles of probability in interpreting the
Drosophila experiment and in understanding
many aspects of segregation of genes.

The probability (P) that some event (.v) will
occur can be represented by a fraction between
0 and 1. This fraction is the proportion of
times the event occurs {m) in a very large num-
ber of trials («), or

nix
riz

When in a very large number of trials, every
trial yields the event, then m = n and P = \\
the event is inevitable. When the event does not

occur at all in a very large number of trials,
P = 0; the event is impossible. Everything
that happens has a probability that lies between
these limits. The nearer Pj is to 1, the more
probable the event.

Probability values are theoretical; they are
merely mathematical expressions of expecta-
tions. It is necessary to perform a very large
number of trials, and for an event to occur
many times, for the observed frequency of suc-
cesses to equal the probability. That is, the
more trials and more often an event takes place,
the more closely the proportion of successes
will approach P^.

To illustrate this, perform the following tests:

(a) Flip a coin four times and record the
number of heads and of tails; repeat this four
times. Note the variation in results.

(b) Flip the coin 10 times and again record
the number of heads and of tails.

(d) If you have time, extend this to 100 or
more flips.

(e) Sum up the totals for heads and tails
from (a), (b), (c), and (d) above.

(f) Calculate the ratio of heads (or tails) to
the total number of tosses in each of (a), (b),
(c), (d), and (e) above.

Just from the shape of a coin we expect the
probability of a head (or tail) coming up on
any flip to be about 0.5. Which coin-flipping
test above provides the most reliable agreement
with the theoretical value of P?

Simultaneous occurrence of independent
events

The probability that several independent
events will occur together is equal to the product
of their separate probabilities, or

Px.y.z... —P X X P ,j X Pz...

For instance, when two dice are tossed the

128 COMPLETION OF THE GENETICS EXPERIMENT

Exercise XXV

probability of "box cars" is the product of the
probabilities of turning up a six on each die
separately (1/6), or/' = 1/6 X 1/6 = 1/36.

Genetics makes frequent use of this principle.
At fertilization an egg and a sperm combine
randomly. For any genetic trait, the egg may
contain either a dominant or a recessive gene,
and likewise the sperm may contain either a
dominant or a recessive gene. If one considers
that the probability of an egg containing a
dominant (or recessive) gene is 0.5, the same is
true for the sperm. The probability, then, that
the fertilized egg (zygote) will contain two
dominant genes is the product 0.5 X 0.5, or
0.25. That is, in a suitably large population of
offspring, 25% will carry two dominant genes
(homozygous dominant). Likewise, 25% can
be expected to carry two recessive genes (homo-
zygous recessive). Another 25% of the offspring
will receive a dominant gene from the father
and a recessive from the mother; in the final
25% this is reversed, and a recessive gene will
come from the father and a dominant from the
mother. Thus 50% of the offspring should
possess one dominant and one recessive gene
(heterozygous).

Further coin-tossing tests help to illustrate
the probability of occurrence of joint inde-
pendent events such as these.

(a) Toss two coins at a time 12 times, and
record the results: (h, h); (h, t); (t, t). Now

using the principle that P^^y = Px X Py, calcu-
late the probability of each paired outcome (2
heads, 2 tails, or a head and a tail). How
closely do the results agree with the theoretical
prediction? You might try tossing the two
coins 100 times to see if the agreement is better.

(b) Repeat the above test tossing three coins
16 times. Calculate the probability of each com-
bination: (h, h, h); (h, h, t); (h, t, t); and (t, t, t).

(c) Can you derive a general relationship
that could be used to predict the results when
n coins are tossed together a large number of
times?

In a family with five children what is the
probability that all will be daughters? that all
will be of the same sex? (This is a problem in
either-or probability. Whereas the probability
that several events will all happen together is
the product of their several probabilities, the
probability that any one of several possible
events will occur is the sum of their separate
probabilities.)

For a more complete treatment of the use of
statistical methods in heredity, consult any
modern textbook of genetics. The chapter on
"Statistical Inference in Genetics" in Principles
of Genetics, by Sinnott, Dunn, and Dobzhansky,
5th ed., McGraw-Hill, 1958, is particularly
recommended.

EQUIPMENT

equipment for handling Drosophila as in Exercise
XXII

a hatching generation of Drosophila to illustrate
sex-linkage. Although apricot, cut, and bar have

been used as markers in the exercise here, many
others will serve. Details can be found in any
genetics text or in the Drosophila Guide mentioned
in Exercise XXII.

msm

XXVI

SENSORY RECEPTORS

(Readings: G. von Bekesy, "The Ear," Sci. Am. 197, No. 2, 66-78, Aug. 1957,
Reprint No. 44. G. Wald, "Eye and Camera," Sci. Am. 183, No. 2, 32^0,
Aug. 1950, Reprint No. 46. W. Loewenstein, "Biological Transducers," Sci.
Am. 203, No. 2, 98-108, Aug. 1960, Reprint No. 70. S. P. T., pp. 195-208.
Weisz, pp. 480-495. Villee, pp. 373-386.)

All that we know we lean] through our sense
organs. They are our ultimate instruments for
exploring the environment. It is of the highest
importance that we understand what kind of
instruments they are, what they can do, and
where they fail.

The anatomical unit of every receptor system
is the single receptor cell or end-organ, particu-
larly sensitive to one kind of stimulus, and
giving rise to one quality of sensation in the
brain. The effect of the stimulus upon such an
end-organ is a depolarization ("generator poten-
tial"), in most cases long-lasting compared with
the depolarizations that stimuli excite in nerve
or muscle fibers — which in turn causes the firing
of the attached nerve fiber. In some instances,
as for example in touch spots, there may be
only one or two all-or-nothing discharges in the
nerve fiber in response to each stimulus. In
most receptor systems, however, the depolariza-
tion of the end-organ lasts relatively long, and
results in a long burst of all-or-nothing responses
in the attached nerve fiber, which may cease
after a time though the stimulus continues (e.g..

smell), or may go on as long as the stimulus
lasts, as in vision.

TOUCH

The skin contains a wide variety of end-
organs, specific for pain, heat, cold, pressure,
and touch. Touch receptors are of two kinds :
bulbous arrangements of cells enclosing the
naked terminal twigs of a sensory nerve fiber
(Pacinian and Meissner corpuscles), or the
widely branching terminal arborization of such
a nerve fiber around the basal bulb or "root"
of a hair ("hair crown"). With very small
stimuli one can map out the locations of the
sharply localized points at which stimulation
conveys any one of the skin sensations.

We shall map out the touch spots in various
areas of skin in this way, using a bristle as
stimulus. This will demonstrate a general con-
dition of all receptor systems — that the receptors
form a discontinuous mosaic of isolated sensitive
points, relatively coarse in the case of touch, and

129

130 SENSORY RECEPTORS

Exercise XXVI

varying greatly from one area of skin to another.
Our sense of spatial continuity — of the smooth-
ness of a surface — as well as of pattern is con-
veyed by such discontinuous mosaics of recep-
tors.

The capacity of a sensory surface for evaluat-
ing pattern is measured by determining the
"two-point threshold," which is the smallest
separation at which two point stimuli are per-
ceived as two. This measures the density of
receptors, since for two stimuli to be appreciated
as two, they must excite two touch spots having
at least one unexcited touch spot between them.

Perform the following experiments in pairs,
one student, with eyes closed during each test,
serving as subject, the other as experimenter
and recorder.

Distribution of touch spots

With a pen outline a square hairless or shaved
area about 3 cm on a side on the inner forearm.
Explore this area by touching it lightly with the
tip of a bristle, noting the points from which a
distinct sensation of touch is felt. Mark each of
them with a spot of ink. Draw a diagram of the
area, showing the locations of the touch spots.
Estimate the number per square centimeter.

Two-point thresholds

To test for these, the experimenter touches
various points in a region of skin very lightly
with one or both of the blunted points of a pair
of dividers, in haphazard order. At each touch
the subject reports the sensation as either "one"
or "two."

At the start of each test adjust the separation
of the dividers so that all double stimuli are
reported as "two" and all single stimuli as
"one." Then gradually lessen the separation
until only about 8 in 10 reports are correct.
The separation of the points in centimeters is
then the approximate minimum perceptible sepa-
ration, or two-point threshold. In some areas
of skin this is much the same in all orientations
of the dividers; in others it differs greatly.

Determine and record in a table the two-point
thresholds for the upper arm (longitudinal),
upper arm (transverse), forearm (longitudinal),
forearm (transverse), back of hand, palm of
hand, fingertip, and lips.

Calculate the number of receptors per square
centimeter in each area tested, and enter this in
the table. In those areas in which the two-point
threshold is about the same in all orientations
of the dividers, use the formula : N/cm~ = 4/L^,
in which A'^ is the number of touch spots and L
is the two-point threshold in centimeters. This
formula is based on the assumption that the
two-point threshold represents twice the dis-
tance between neighboring touch spots. Why
twice the distance?

For areas of skin in which the longitudinal
two-point threshold (Li) differs from the trans-
verse (Lo), the density is TV/cm^ = 4/(Li X /.2)-

How does the density of touch spots on the
forearm, calculated from the two-point thresh-
old, compare with the density you found by
direct mapping?

TASTE

The special senses (sight, hearing, smell, taste)
are associated with dense aggregates of recep-
tors, concentrated in limited areas, some of them
supplied with highly adapted accessory struc-
tures, such as in the eye and ear. The sense of
taste is limited to the mucosa of the tongue and
mouth. The receptors are clustered in "taste
buds" and are of several types, each type mediat-
ing a primary taste. Since the receptors for the
several primary tastes are not uniformly dis-
tributed over the sensory surface, their nature
and distribution can be determined by applying
various solutions to different regions of the
tongue.

A solution may stimulate more than one kind
of taste cell, resulting in a wide variety of taste
sensations. Other senses frequently enter: a
solution that is both bitter and hot may give
rise simultaneously to sensations of bitterness,
warmth, and perhaps pain. Very often also, the

Exercise XXVI

SENSORY RECEPTORS 131

same solutions stimulate the sense of smell, and
taste and smell together give us composite sen-
sations of flavor.

Experiment

Again, work in pairs, one student serving as
subject, the other as experimenter. The experi-
menter should moisten small rolls of filter paper
in each of the following solutions:

quinine sulphate,
5% sugar (sucrose),
10% sodium chloride,
1% acetic acid,

and, after shaking off excess liquid, apply each
in turn with forceps to different regions of the
tongue of the subject, for about 10 seconds.
At each application the subject should report the
sensation as "bitter," "sweet," "salt," "sour,"
or "none," and should rinse the mouth with
water after each test.

On a diagram of the tongue mark each region
from which sensation is reported, using a dif-
ferent symbol (circle, square, triangle, cross) for
each primary taste.

To demonstrate the role of smell in deciding
flavor, alternately place a bit of apple and a bit
of onion on the subject's tongue, while he keeps
his eyes closed and holds his nose shut. Can the
subject distinguish them by taste alone?

Demonstration

Taste and smell are confined to mucous sur-
faces. The stimuli for these chemo-receptors
are always substances in aqueous solution. For
substances to reach our olfactory areas and
stimulate smell sensations they must obviously
be in the gaseous state, so that they can be
inhaled; but in this case also these substances
must dissolve in the layer of mucous that covers
the olfactory patch before they can stimulate
the smell receptors.

Animals which have wet skins apparently
have such chemo-receptors distributed over
large areas or the entire surface. This is true
in general of fishes and amphibia.

Destroy the brain of a frog by pithing or by
cutting off the head just at the angle of the jaws.
Allow the frog to lie undisturbed for a time to
recover from the shock. Now lay a bit of filter
paper soaked in dilute acid on its flank, and
observe what happens. Touch a similar piece
of filter paper soaked in the same acid to your
tongue. Note that to you this is a stimulus for
taste, not for pain. Presumably it is the same
kind of thing for the frog, and unlike us, the
frog apparently can taste all over.

This response in the absence of the brain is
an extraordinary demonstration of a complex
spinal reflex. Here a mild stimulus, no more
than "distasteful," evokes a reasonable and
accurate response, all handled at the level of
the spinal cord.

SENSORY JUDGMENTS OF INTENSITY:
THE ESTIMATION OF WEIGHT

All measurements are ultimately sensory
judgments of quantity. Yet the response of a
receptor to a stimulus varies with duration and
state of adaptation, so that sensory reports of
intensity of stimulation are at best relative. In
general, as regards intensity, our receptors per-
mit only three kinds of measurement: (a) the
absolute threshold: the strength of stimulus that
just excites the sensation; (b) the intensities at
which two stimuli seem just equal; (c) the
intensities at which two stimuli are just per-
ceptibly unequal. This last is the "difference
threshold." It plays the same role in our estima-
tion of intensity as the two-point threshold does
in our estimation of space.

Beyond these judgments, all measurement
ceases. So, for example, I can say accurately
the intensity of light that is just visible, its
absolute threshold; or how much I need to
increase the intensity of a light to make it just
perceptibly brighter, the difference threshold;

132 SENSORY RECEPTORS

Exercise XXVI

or that two lights are equal in brightness. What
would it mean, however, for me to say that one
light is 2.3 times as bright as another?

Weber (1834), experimenting initially with
weights, discovered that the difference threshold
varies with the intensity of the stimulus in a
peculiar way that became known as Weber's
law: the difference threshold (A/) is a constant
fraction of the intensity of stimulus (/), that is,
A/// = constant. The ratio A/// is called the
Weber fraction. It is an inverse measure of the
capacity to discriminate intensities; the larger
this ratio, the poorer is the capacity for making
such discriminations. Weber's law holds only
approximately and over a limited range. The
ratio A/// remains approximately constant for
many senses over the middle ranges of intensity,
but rises at both the low and high extremes of
intensity.

Estimation of weight

A weight held in the hand is supported by
muscle tensions in the hand and arm. These
stimulate tension receptors in the muscles and
tendons, the reports of which help to guide the
limb and also excite sensations. If two weights
are successively lifted, there exists a minimal
difference in weight such that one is judged
just heavier than the other, the difference thresh-
old. This may be determined for various weights,
and the constancy of the Weber ratio tested.

Working in pairs, perform the following ex-
periment. You have two 125-ml Erlenmeyer
flasks. Mark one of them with a crayon, to be
the test flask. Add water to both so as to bring
them to equal weight at about 50 grams. The
experimenter now hands the flasks to the sub-
ject, whose eyes are closed. The subject holds
the flasks either cupped in his palms or with his
fingers by the necks, but whichever way he
chooses should be maintained throughout the
experiment. For this first experiment the flasks
should be held steady, and the subject says
whether they feel equal or unequal in weight.
Presumably they feel equal. Now the experi-
menter takes the flasks again, and adds water

to the test flask in 2-ml portions, each time
handing the flasks back to the subject, randomly
mixing right and left, each time giving the sub-
ject all the time he needs to decide the relative
weights. When the test flask feels just per-
ceptibly heavier than the other, record the vol-
ume of water that was added to it. This is also
the added weight, since 1 ml of water weighs
1 gram. The difference in weight is then the
difference threshold for a weight of 50 grams.

Now repeat this procedure with the flasks
initially made equal in weight at about 100,
200, and 500 grams (the latter two in 500-ml
flasks) and tabulate the results.

Prepare a graph plotting the Weber ratio
(A weight/lower weight) on the vertical axis
against the lower weight on the horizontal axis.

Repeat this experiment for at least one
weight, or all the way through if you have time,
with the subject wagging the flasks up and down
as he estimates their weights. Do you find a
difference in the Weber ratio? Motion in gen-
eral produces a much stronger and more per-
sistent excitation than a stationary stimulus.
Why? (Recall the effectiveness of flickered as
compared with steady light, in Exercise XX,
p. 108.)

Has the Weber ratio remained approximately
constant in your experiments? What do you
conclude of the accuracy with which weights
can be estimated? How do the Weber ratios of
other subjects compare with yours? This last
question illustrates one example of the "per-
sonal equation" that is involved in every type
of sensory judgment.

VISION

The blind spot

The point at which the optic nerve leaves the
retina is blind, since this area contains no visual
receptors. Lay a sheet of blank white paper on
the desk, and draw a small cross to the left of
center. The subject, holding his left eye closed,
should stare fixedly at the cross with his right
eye 30 cm from it. (Staring fixedly at anything
means holding its image within the central fovea

Exercise XXVI

SENSORY RECEPTORS 133

Blind spot

of the retina, so fixing its position on the retinal
surface.) The experimenter, without jogging the
paper, slowly advances a small target (a pencil
point will do) into the subject's field of vision,
starting about 2 to 4 inches to the right of the
cross. There is a point at which the target dis-
appears. The experimenter marks this point on
the paper, and starts again from another angle.
By repeating this performance, advancing the
target from various angles around the cross, one
can plot the entire boundary of the blind spot.

If there is time, repeat this for the left eye;
this time, however, the target should be intro-
duced at the left of the cross.

The accompanying figure shows diagram-
matically the optics of this experiment. Study
it carefully, and see that you understand every-
thing in it, for it contains the essential elements
of image formation in the eye. From the results
of your experiment calculate the diameter of
the blind spot in the eye, and also the distance
of its center from the fixation point within the
central fovea. This is done very easily, since
the projected dimensions on the paper are to
the dimensions on the retinal surface as the
distance from the paper to the eye (300 mm)
is to 17 mm, the focal length of the human eye.

Note that, like any other simple lens system,
the eye inverts the images of all objects at which
one looks, and equally inverts all spatial rela-
tions. Why then do we not see upside down,
and wrong end to?

Retinal blood vessels

As a consequence of the way it develops
embryologically, the vertebrate retina points
away from the light. Light must pass through
the entire thickness of the retina, including the
retinal blood vessels, before reaching the visual
receptors. The blood vessels therefore cast a
continuous shadow upon the visual field; and
the only reason we are not aware of this at all
times is that one cannot continue to see any
image that is fixed in position on the retinal
surface. To make the blood vessels visible, all
that is needed is to make their shadows move.

Make a small hole, about 1 mm across, in a
card, and look through it at a brightly illumi-
nated white surface, meanwhile giving the hole
a rapid side-to-side or rotary motion. Shortly
you should become aware of a delicate, lacy
network, with a central open space, as though
a hole were torn in it. The hole will move
wherever the eye is fixated. The network repre-
sents the shadows of the retinal capillaries, the
hole the central fovea, from which blood vessels
are lacking.

Those of you who see this plainly might like
to estimate the diameter of the fovea. This can
be done by estimating the width of the image of
the capillary-free area as projected on the white
surface; from this and the distance of the sur-
face from the eye, you can complete the calcu-
lation as you did above for the blind spot.

134 SENSORY RECEPTORS

Exercise XXVI

EQUIPMENT

Per laboratory

safety razor

filter paper

1% quinine sulfate

5% sucrose

10% sodium chloride

1% acetic acid

onions and apples

frog

pan balance

Per pair of students

bristle

dividers

2 125-ml Erlenmeyer flasks

marking pencil

pipet

2 500-ml flasks

sheet of white paper

3" X 5" white card

OUTLINE FOR THE INSTRUCTOR ON THE PREPARATION
FOR MICROBIOLOGICAL EXPERIMENTS (EXERCISES VI
THROUGH IX)

APPENDIX A

A. MATERIALS

1. Glassware

Pyrex culture tubes without lips are preferable.
They will be needed in three sizes : 1 3 X 100 mm,
16 X 150 mm, and 20 X 150 mm. Along with
the 5-ml serological pipets and Erlenmeyer
flasks, they may be obtained from any scientific
supply house.

Dropper pipets of sufficient length for transfers
from tubes (about six inches) are difficult to
obtain commercially and probably will have to
be made up. Alternatively, 1-ml serological
pipets or Pasteur pipets may be used.

Sterile disposable petri dishes, 15 X 100 mm,
available from Falcon Plastics, 5500 West 83rd
St., Los Angeles 45, or from Scientific Products,
1210 Leon Place, Evanston, Illinois, at a cost of
about five cents apiece, are recommended since
their use obviates the need for much tedious
cleaning, washing, and sterilizing, and also
allows the students to take plates home with
them to observe growth.

2. Media and Chemicals

Ready-mixed media such as nutrient broth,
nutrient agar, and tryptose blood agar base can
be obtained from Difco Laboratories, Detroit 1,
Michigan. They should be made up according
to the directions on the bottles.

Of the constituents for the Pneumococcal
media, Casamino acids, tryptone, yeast extract,
and brain-heart infusion are obtained from

Difco. All other organic materials including
vitamins, amino acids, sugars, streptomycin,
deoxycholic acid, methylene blue, serum albu-
min, and sterile horse blood may be obtained
from Nutritional Biochemicals, Cleveland, Ohio.
Fresh yeast can be procured from Standard
Brands, Inc.

Inorganic chemicals, reagent grade, are avail-
able from any chemical supply house. Antifoam
may be obtained from the Dow Chemical Com-
pany.

3. Miscellaneous

a. Constant-temperature equipment

Water baths may be rigged up from parts
which can be obtained at relatively low cost
from an aquarium supply house. A tank
16" X 10" X 12" deep, fitted with a 100-watt
thermostat aquarium heater and a 100-watt
constant heating element and either an air line
or aquarium bubbler for stirring, will provide
room for eight students. However, if at all pos-
sible, it is recommended that water baths or
their components be obtained from scientific
research supply houses in order to achieve more
reliable temperature regulation.

Two neoprene-coated test-tube racks with
holes large enough to accommodate tubes 20
mm in diameter (obtainable from Emil Greiner
Co., New York City) may be supported in the
baths by means of platforms made of i" mesh-
wire screening.

135

136 APPENDIX A

Air-

Cotton

E=3 EiS

Qr*-Screw clamp

■Air

No. 2 rubber
stopper with

,-^

Rubber hose
and clamps

two 5-mm holes

^""^b-mm pyrex
y^ tubing

20 X 150

culture tu

be
■

Aerator sto

■1.

j^-«^Cotton
,^,.,. ~ZJtr plugs

^C.^^1>

2-liter flask

. l-llter
culture

An incubator cabinet of moderate size, avail-
able from any scientific supply house, is useful
for the preparation of large quantities of culture
and is essential for the incubation of plates con-
taining Pneumococcus.

b. Aeration

An aerator assembly, leading from either a
compressed air line or an aquarium bubbler
(alternatively, suction may be used to drive the
aerator), composed as shown in the diagram,
provides for aeration of four student cultures.

For aeration of large volumes of culture
(100 ml to 1000 ml), an aerator stone (available
from Fisher Scientific Co.) attached by gum
rubber tubing to a pyrex tube plugged at the
opposite end with cotton should be used.

c. Ultraviolet irradiation

A satisfactory source of ultraviolet light is a
General Electric 15-watt germicidal lamp in-
stalled in an ordinary fluorescent desk fixture.

To avoid injury to the eyes, students should
wear safety glasses of either glass or plastic.

d. Filtration

Seitz filters and filter assembles for prepara-
tive sterilization of solutions containing heat-

labile material may be purchased from any
scientific supply house.

Porcelain candle filters, made by Coors, size
10 mm X 55 mm, porosity No. 5, for use in
the virus filterability experiment may be ob-
tained from Arthur H. Thomas Co., Philadel-
phia, Pa. The filters should be inserted into
rubber stoppers to fit into 500-ml suction flasks
and permanently marked for use in phage or
bacterial filtration. The filtration assembly
should look as shown in the diagram.

Cotton
padding

g^ 500-ml suction
"'' flask

PREPARATION FOR MICROBIOLOGICAL EXPERIMENTS 137

e. Bacteriological loops

These should be made from nichrome wire,
No. 23 gauge. The loop should be about \" in
diameter; it is convenient to form such loops by
bending the wire around two nails of appropriate
size imbedded in a wood block, as shown in the
diagram.

The wire is either inserted into a commercially
available loop holder or fused into the end of a
4" length of thick-walled capillary tubing to
serve as a handle. Alternatively loops may be
purchased already assembled.

B. WASHING OF GLASSWARE

All glassware should be thoroughly washed
with detergent and rinsed at least five times in
order to remove traces of detergent which might
be toxic for the bacteria. It is not necessary to
rinse this glassware with distilled water, though
the latter should be used for making up all
media and solutions. It is advisable that the
students be taught to do as much of the washing
and plugging of pipets and aeration tubes as
possible.

C. STERILIZATION

In general, vessels containing liquids, or
assemblies containing rubber parts, should be
autoclaved at 120°C (15-lb pressure) for 15 min.
Large volumes (greater than 200 ml), particu-
larly of viscous liquids such as agar, should be
autoclaved for longer periods (30 min to 1 hr).

Empty glassware should be dry-sterilized in
an oven at 160°C for at least 90 min.

Serological pipets, culture tubes, flasks, and
aerators must be plugged with nonabsorbent
cotton before sterilization. If proper cans are
not available, serological pipets, dropper pipets,
and aerators may be wrapped in bunches with
aluminum foil so that sterility is preserved on
removal.

Sugar solutions are best sterilized by heating
for 20 min in a boiling water bath or for one
hour in an inspissator (steam box). However,
concentrated glucose solutions (10%) may be
wet-autoclaved the same as media and then
measured out after cooling.

Solutions of labile materials must be sterilized
by filtration.

D. SOURCES OF CULTURES

Cultures of Bacillus megatherium, Serratia
marcescens, and Escherichia coli B may be ob-
tained from the American Type Culture Collec-
tion, 212 M Street, N.W., Washington, D.C.
They should be propagated every two months
by streaking a sample of the old culture onto
the surface of a fresh nutrient agar slant and
incubating until growth is completed, after
which the cuhures should be conserved in the
cold. Agar slants are prepared by adding about
5 ml of liquid nutrient agar to a sterile screw-
cap vial, about 16 mm X 150 mm in dimen-
sions. Tilting the tube before solidification
results in a slant surface of considerable area,
in a tube of small cross section, and hence rela-
tively little risk of contamination.

Strains of Pneumococcus which are nonencap-
sulated or rough, and therefore nonpathogenic,
must be used. A normal strain sensitive to
streptomycin, as well as a streptomycin-resistant
strain, can be obtained from universities or insti-
tutes carrying on research on the transformation
of Pneumococcus. Among such institutions are
The Rockefeller Institute, New York City;
University of Colorado Medical Center, Denver,
Colorado; Laboratoire de Ge'netique Physiolo-
gique, C.N.R.S., Gif-sur-Yvette (Seine-et-Oise),
France; and Brookhaven National Laboratories,
Upton, New York.

138 APPENDIX A

Pneumococcal strains are best propagated by
growing an inoculum in the medium described
in part E-3 of this outline, supplemented with
^ volume of fresh yeast extract. (Fresh yeast
extract is prepared by crumbling 1 pound of
fresh yeast in 1 liter of water, bring to a boil,
cooling, centrifuging, and sterile-filtering the
supernatant.) The culture is grown at 37°C
until visibly turbid; ^ volume of sterile glycerol
is added, and the culture is frozen at — 20°C.
Such frozen cultures retain their viability for
three months to a year. Competent cultures of
streptomycin-sensitive cells to be transformed
are grown in the same fashion; they retain
optimal transformability for a week or two.

Bacteriophage must be procured from labora-
tories doing research on bacteriophage. Most
universities, medical schools, or research insti-
tutes could either supply the virus or else suggest
where it could be obtained. The virus is propa-
gated by addition of a sample to a culture of
E. coli B in logarithmic growth at a density of
about 10^ cells/ml. Incubation is continued
until the culture lyses. The resultant phage sus-
pension may be kept sterile by addition of a
drop of chloroform. (All chloroform must be
removed by aeration before using the virus.)
The concentration of particles may be deter-
mined by the method described in Exercise IX
or, more accurately, by the agar layer technique.
For details of this technique and other useful
information on bacteriophage properties and
handling, see M. H. Adams, Bacteriophages,
New York, Interscience Publishers, Inc., 1959.

E. DETAILS OF PREPARATION*
1. Exercise VI

Agar plates. Disperse 350 gm of nutrient
agar in 10.5 liters HoO in six 2-liter Erlenmeyer
flasks. Plug. Autoclave 40 min. Let flasks
cool to about 60°C. Pour layer equivalent to
30 ml into each of 300 petri dishes. Store
plates at room temperature.

Broth. Dissolve 40 gm of nutrient broth and
24 gm NaCl in 5 liters HjO. Add a squirt of
antifoam. Distribute: 200 ml in each of thirteen
500-ml flasks, 800 ml in a 2-liter flask containing
a stone aerator, 300 ml in a 500-ml flask. Plug.
Autoclave 20 min.

3% H2O2. Dilute 30 ml of 30% H2O2
(Superoxal) with 270 ml H2O.

10% hydroxylamine. Dissolve 10 gm hy-
droxylamine hydrochloride in 50 ml H2O. Add
sufficient 10% NaOH to give pH 7. Add water
to give a total volume of 100 ml.

Blood. Dilute 5 ml defibrinated horse blood
with 95 ml NaCl, 0.85%. Refrigerate.

Cultures. Inoculate loopful of Senatia
marcescens from agar slant into 800 ml of
broth. Incubate at 37°C with gentle aeration
for 10-20 hours. Culture should be heavy.

Transfer 3 ml with aerator to 300-ml broth 4
to 6 hours before class begins. Aerate at 37°C.
Divide up the rest of the culture into flasks to
be used as the "old culture." Refrigerate these
until class begins. Just before class, distribute
the "young culture" (transfer made 4-6 hours
earlier) in sterile fashion.

2. Exercise VII

Agar plates.

plates.

See E-1 above. Prepare 500

For a class of 100 students.

Broth. See E-1 above. Two 200-ml portions
with aerators for growing cultures of Serratia
marcescens and Bacillus megatherium are needed.

Cultures. Inoculate broth with a loopful from
agar slants of Serratia marcescens and Bacillus
megatherium and grow the two cultures over-
night at 37°C with aeration. {Note: When
grown at temperatures over 30°C, Serratia
marcescens may not develop its characteristic
red pigment.)

Alkaline methylene blue. Dissolve 1 gm
methylene blue in 100 ml of 95% alcohol. Add
300 ml 0.01% KOH.

PREPARATION FOR MICROBIOLOGICAL EXPERIMENTS 139

3. Exercise VIII

Blood agar streptomycin plates. Prepare four

l-liter batches in 2-liter flasks. Disperse 35 gm

tryptose blood agar in 1 liter H2O. Autoclave

30 min at 120°C. Cool to SOX. Add, per flask:

5 ml sucrose, 20Tc, sterilized by heating

15 min in boiling water,
5 ml streptomycin sulfate, 10 mg/ml,
sterilized by filtration,
20 ml sterile horse blood.

Pour 120 plates, each containing about 30 ml.

Growth medium for streptomycin-resistant
cells. Prepare three 1.5-liter batches in 2-liter
flasks. To 1.5 hters H2O add

10 gm brain-heart infusion,
10 gm Difco yeast extract,
10 gm Casamino acids,
10 gm tryptone,
3 gm glucose.

Adjust pH to about 7.5 with 10% NaOH.
Autoclave.

Pneumococcal medium for competent strepto-
mycin-sensitive cells — basal. Dissolve :

36 mg tryptophan,
200 mg cycteine-HCl,
12 gm sodium acetate,
30 gm Casamino acids,
51 gm K2HPO4

in 6 liters H2O. Distribute: 200 ml in each of
sixteen 500-ml flasks, and 900 ml in each of
three 2-liter flasks. Plug. Autoclave 15 min.
Store at room temperature.

Addition mix. Dissolve in 200 ml H2O the
following substances (this will make sufficient
mix for 4 liters of basal medium):
2gmMgCl2-6H20,
10 mg CaClo,

100MgMnsd4-4H2O,

0.8 ng biotin,

0.8 mg nicotinic acid,

0.8 mg pyridoxine-HCl

0.8 mg thiamine- HCl,

0.4 mg riboflavin.

2.4 mg calcium pantothenate,

2MgFeS04-7H20,

2MgCuS04-5H20,

2/igZnS04-7H20,
20 mg choline,
40 mg glutamine,
200 mg asparagine,
20 mg adenine,

2 gm serum albumin (Armour Fraction V).

Adjust pH to 7. Sterilize by filtration.

Citrate-saline. Dissolve 6 gm NaCl and 20
gm sodium citrate in 700 ml H2O.

Deoxycholate solution. Dissolve 5 gm deoxy-
cholic acid in 80 ml H2O by addition of 10%
NaOH to bring pH to about 7.5. Add water to
bring to 100 ml.

Growth of streptomycin-resistant cells. Inocu-
late 2 drops of thawed culture of SR into each
of three flasks. Incubate without aeration for
15 hours at 37°C or until growth is maximal.
Refrigerate until 1-2 hours before class. Cen-
trifuge cells. Resuspend in 700 ml citrate-saline.
(If growth has been poor, resuspend in less
volume.)

(Note: All cultures should be examined micro-
scopically in order to determine that they are
not grossly contaminated. It may be advisable
to grow cultures in duplicate to allow for those
discarded because of contamination.)

Growth of competent streptomycin-resistant
cells. Add 5 ml addition mix and 1 ml auto-
claved 20% glucose to every 100 ml of basal
medium to form the complete medium. Inocu-
late with a drop or two of the frozen culture of
streptomycin-resistant cells and incubate as
above. Dispense convenient volumes into
sterile tubes for use in class. These must be
kept in ice.

4. Exercise IX

E. co// culture. Inoculate loopful of E. coli B
from agar slant into 20 ml nutrient broth.
Aerate at 37°C overnight. Inoculate 10 ml of

140 APPENDIX A

this culture into 1.3 liters of nutrient broth.
Aerate at 37°C for 4 hours or until culture con-
tains from 5 X 108 to 1 X iqs cells per ml.
Use this young culture.

Phage suspension. Grow 20 ml of culture of
E. coli B with aeration at 37°C to a density of
about 2 X 10* cells/ml. Infect with a drop of
phage suspension containing 10^ to 10* infective
units. Continue incubation until visible lysis

occurs. Determine infective titer. Dilute as
called for.

Soft agar.

bacto-tryptone, 10 gm,
Difco agar, 7 gm,
sodium chloride, 5 gm,
water to 1 liter.

Nutrient agar plates. 2.3% nutrient agar in
distilled water.

NOTES TO THE INSTRUCTOR ON THE ELECTRONIC
EQUIPMENT USED IN EXERCISE XVIII

APPENDIX B

The most satisfactory equipment now avail-
able for teaching electrophysiology to students
at any level is the Tektronix "160 series" of
instruments, manufactured by Tektronix, Inc.,
Beaverton, Oregon. This equipment, designed
originally for teaching medical school neuro-
physiology, has proved to be extremely depend-
able, easy to operate, and of high research
quality. For the experiments chosen here, we
purchased the following pieces of equipment
(prices as of early 1961):

Type 360 Tektronix Indicator Unit at $250.00

at $125.00

Type 762 Tektronix Waveform
Generator

Type 161 Tektronix Pulse

Generator at $125.00

Type 160A Tektronix Power

Supply at $175.00

Type 122 Tektronix Preamplifier at $130.00

Type 125 Tektronix Amplifier

Power Supply at $250.00*

The waveform generator provides the proper
voltage to drive the horizontal sweep (100 ^lsec
to 10 sec) of the electron beam of the indicator,
and also to trigger the pulse generator. Pulse
stimuli of 0 to 50 volts amplitude and 10 /isec
to 0.1 sec duration from the pulse generator are
therefore always synchronized with the sweep

* One 125 preamplifier power supply serves for
four setups.

of the horizontal beam of the indicator, thereby
facilitating the observation of electrically evoked
responses.

The 122 preamplifier (a-c, 0.16 cycles to 40
kilocycles) provides amplification of 100 or
1000, and together with the 360 indicator unit
provides a sensitivity of 50 /xv/cm.

The 160A power supply provides the required
voltages and currents for the 360 indicator unit,
the 162 waveform generator, and the 161 pulse
generator; while the 125 preamplifier power
supply powers up to four 122 preamplifiers.
The need for batteries with this equipment is
therefore eliminated; all the current needed is
provided by a 110- or 220-volt a-c wall outlet.

The instruments may be attractively and con-
veniently mounted in frames of standard rack-
mount dimensions, and either placed in open
racks or cabinets. We have frame-mounted the
360 indicator, 161 pulse generator, 162 wave-
form generator, and 160A power supply to-
gether; and with a rack-mounting model 122
preamplifier housed our instruments in a Bud
cabinet (model CR 1736, Bud Radio Co.,
Cleveland, Ohio). Each 125 amplifier power
supply is placed conveniently to four such
setups, and connected via cables with the
amplifiers.

The nerve chambers which we have found
satisfactory were purchased from the Harvard
Apparatus Company, Dover, Mass. ($13.75 a-
piece). The wick electrodes used for recording
the electrical activity of the Limulus eye were
designed for this experiment, but undoubtedly
would be satisfactory for any experiment in
which wick electrodes were required.

141

142 APPENDIX B

Wick electrode mount

Bind

";^

^^Bi

Front view

End view

•i

As shown on the diagram, heavy cotton
thread extending through a tapering glass pipet
(a) filled with sea water provides a convenient
wick. The pipet is fastened with small fuse clips
to a small piece of lucite (b) (2" X 1" X i"),
which is mounted on the large lucite base (c)
(3" X 6" X f") with heavy stopcock grease.
The pipet is thereby readily movable with regard
to the base and can be positioned in any desired
direction. A silver-silver chloride wire runs
through the pipet and attaches to a binding post
screwed into the top of the base. The rear end

of the pipet is sealed off with a rubber cap, pre-
venting the pipet from drying out rapidly.
When not in use, the wick end of the pipet
should be immersed in sea water.

We have not found it necessary to shield the
input cables for these experiments; but when
using the exposed wick electrodes, a copper
shielding cage is necessary to enclose the prepa-
ration. We have attached standard banana
plugs on all our leads for convenience in con-
necting the instruments both to nerve chambers
and electrodes.

SUPPLEMENTARY EXPERIMENTS ON THE CHEMICAL
COMPONENTS OF CELLS: THE BIOCHEMISTRY
OF MILK*

APPENDIX C

PART 1

(Reading: S. T. P., pp., 70-85; 121-123.)

Milk, the cellular product of a mother,
contains everything needed for the cellular
growth of a baby: proteins, fats, sugars, mineral
salts, and vitamins.

In this exercise we will work with skimmed
milk, since the isolation and properties of the
fatty components are already familiar to you in
the form of butter. Incidentally, one of the
most general properties of a fat is that it makes
a nonvolatile grease stain on paper; but you
certainly don't need to do that in the laboratory.

Proteins are the large molecules of which the
cell principally builds its structure and ma-
chinery. They are composed of chains of amino
acids. We shall isolate a protein called casein
by neutralizing the negative charges on the
casein which cause the molecules to repel each
other. This will be done by adding acid, that
is, a source of positively charged hydrogen ions
(protons). When the hydrogen ions bind to the
negatively charged casein molecules, the latter
no longer repel one another, and they begin to
aggregate, causing precipitation of the protein,
and permitting its filtration.

After the casein is removed, the other proteins
will be coagulated by heating and evaporating
the solution. Heat causes proteins to lose their
delicate normal structure; the normally coiled
chains of amino acids unwind. If there are
enough molecules in close contact, chains of

* This material covers two laboratory sessions.

different molecules intertwine, giving rise to an
insoluble coagulum. How does evaporation of
the solution enhance this process? (When a
protein is thrown out of solution relatively un-
changed, so that it can be redissolved, we call
that precipitation. In coagulation its structure
has been unraveled irreversibly, and it cannot be
redissolved.)

Two important mineral ions of milk are cal-
cium (Ca++) and phosphate (P04°). Both of
them are essential in the formation of bone, and
calcium ions are necessary also for such diverse
biological processes as blood clotting and
muscle action. On continued evaporation of the
milk, calcium and phosphate ions come into
closer and closer contact until they reach such
a concentration that attractive forces between
them cause them to precipitate as the salt, cal-
cium phosphate.

Sugars serve living cells as sources of fuel and
material, performing as middlemen which en-
able chemical reactions carried out in one cell
or organism to support activity in another cell
or organism. They are small molecules com-
posed of carbon, hydrogen, and oxygen atoms,
in the proportion (CH20)n. The sugar present
in milk is lactose. It will be isolated by adding
the residue from the milk to acetone. Lactose
is insoluble in acetone; that is, the lactose mole-
cules tend to stick together in an orderly array
rather than float about singly in the acetone.
Keeping the solution cold hastens crystallization

143

144 APPENDIX C

by reducing the tendency of the molecules to
move around.

Specific tests enable us to characterize the iso-
lated components. The reagent in the biuret
test gives a color with substances composed of
linked amino acids. Similarly, Benedict solution
reacts with aldehyde groups ( — HC=0) found
in sugars.

Differentiation between large and small mole-
cules can be accomplished by placing the milk
in a sac which has pores so fine that only small
molecules may pass through. The wall of such
a sac is thus a semipermeable membrane, and
the process of ultrafiltering mixtures of large
and small molecules through such a semiperme-
able membrane is called dialysis.

The amino acid composition of casein will be
examined by allowing a proteolytic enzyme to
break it down. The proteolytic enzyme is a
protein isolated from the pancreas gland, which
forms important enzymes for the digestive sys-
tem. It is capable of splitting the links between
the amino acids in a protein. The breakdown
products of casein will be analyzed by paper
chromatography.

ON THE SEPARATION OF
COMPOUNDS

Organic chemistry began as the chemistry of
carbon compounds that occur in, or are pro-
duced by, living organisms. So it remained for
a time until it was discovered that it was pos-
sible to synthesize innumerable unnatural com-
pounds of carbon, and organic chemistry went
its own way to leave the naturally occurring
compounds in the realm of biological chemistry.

The major effort in biochemistry was devoted
for many years to its medical aspects, and
methods were developed for the qualitative and
quantitative determination of constituents of
milk, saliva, blood, urine, feces, and gastric
juices. These are materials easily obtainable for
analysis, and valuable for the clinical diagnosis
of human diseases.

Many of the methods developed in this con-
nection, however, are not exact, since chemical

tests which may be satisfactory for the detection
of a substance in one biological fluid or extract
are unsatisfactory in other preparations. The
difficulty is that such complicated mixtures vary
greatly in chemical composition (even within
the different tissues of the same organism), and
interfering compounds occur in some prepara-
tions though not in others. For this reason it
has been necessary to devise procedures to
separate mixtures into simpler fractions, in the
best case containing pure or nearly pure com-
pounds.

One of the most rapid and convenient methods
for doing this is paper chromatography. This
method in combination with the use of relatively
specific chemical tests provides a general scheme
for evaluating the chemical composition of all
kinds of complex mixtures of biological origin.

You will use this technique, not only to reveal
the complexity of a large natural molecule, the
protein casein, but also to identify a single sub-
stance among a variety of possibilities.

The movement of substances on filter paper
depends on their solubility in the developing
solvent, adsorption on the paper, and often on
partition between two solvents. Some sub-
stances can be separated fairly well in distilled
water, but mixtures of water with various organic
solvents are usually more satisfactory. The
aqueous portion usually contains acid or base
to minimize the existence of more than one
ionized form of any dissociable substance in the
sample. This is not always necessary, and neu-
tral or buffered solvents are frequently used.
The volatile alcohols (methanol, propanol,
butanol) and acids (formic, acetic, hydrochloric)
are convenient, and ammonia is the most
generally satisfactory base.

EXPERIMENTAL PROCEDURES

Measure 200 ml of skimmed milk into a
400-ml beaker. Add hydrochloric acid drop
by drop until a precipitate of casein appears.
About 50 drops will be needed. Stir with a
glass rod while adding the acid. Add 40 drops,

THE BIOCHEMISTRY OF MILK 145

then titrate drop by drop, touching the glass rod
to the indicator paper. Let the precipitate settle
for 5 minutes. Filter it through cloth as follows:
place a piece of cloth over a beaker; depress
the middle. Pour in the suspension, slowly.
When the flow slows down, shape the cloth into
a bag, and squeeze as much of the excess liquid
into the beaker as possible.

Add one marble chip to the filtrate in the
beaker, mark the level of the liquid with a wax
pencil, and boil gently over the flame of a bunsen
burner until the volume of liquid is reduced to
a little less than half. (The marble chip will pre-
vent bumping during the boiling.)

Meanwhile, continue working with the casein.
With the precipitate still in the cloth, press out
excess moisture with paper towels. When this is
done, transfer the precipitate to a beaker, and
add sufficient alcohol to cover it. (Caution:
Highly inflammable.) Break up the particles of
casein with a glass rod. Let the casein settle
and pour off the liquid. Casein does not dissolve
in alcohol, but washing it in this way removes
impurities which do. Press out the excess alco-
hol from the precipitate in the beaker with a
paper towel. Repeat the alcohol washing. Press
out again and set it aside to dry. At the end
of the afternoon transfer the casein to a card-
board container.

Filter out the coagulated proteins using the
suction funnel. Put a paper filter into the funnel,
wet it down with a little water, turn on the
aspirator, and connect the suction hose to the
flask. Pour in the solution containing the coagu-
lated protein. When the liquid has all passed
through, disconnect the suction hose or release
pressure by letting air enter. (Do not release
suction by turning off" the aspirator, since this
will cause water from the line to back up into
the flask.) Pour the filtrate into a beaker.
Wash the coagulated protein on the filter pad
by pouring alcohol over it and sucking it dry.
Do this twice; then remove the material from
the filter into a cardboard container with a glass
rod or spatula. Label.

Rinse out the beakers and suction flask before
using them in subsequent steps.

Evaporate the solution further. Use a very
gentle flame and stir constantly with a glass rod,
lest the liquid boil out of the beaker. (In heating
liquids avoid at any time having your face near
the mouth of the beaker or tube so that acci-
dental spurting will not result in injury. And
don't forget also to be your neighbor's keeper.)
Continue evaporation until a white precipitate
of calcium phosphate appears. (The volume of
the liquid will probably be about 20 ml.) Cool
to room temperature. Filter with suction. Set
aside the filtrate. Wash the precipitate three
times with small portions (about 5 ml) of cold
water. Suck dry. Transfer to another card-
board container.

Evaporate the filtrate over a very gentle
flame until it becomes syrupy, or to a volume
of about 10 ml. Stir constantly and remove
the flame if necessary so that neither excessive
foaming nor charring of the sugar occurs. Add
this syrup to about 6 ml (about two inches) of
acetone in a test tube. (Caution: Highly in-
flammable.) Stopper, and shake to disperse
contents. Label the test tube with your name
and place it in the refrigerator until next
week.

Make a dialysis sac out of cellophane tubing.
Obtain about 9 inches of tubing. Don't bend it
sharply while it is dry or it will develop leaks.
Wet it thoroughly with water, and open it by
running water through it. Tie a sturdy knot to
seal off' one end, and knot it again for good
measure. With a pipet introduce 5 ml of milk
into the sac. (In using the pipet, hold it between
the thumb and middle finger; the index finger
over the end acts as a valve.) Carefully tie a
knot in the top part of the bag. Put it in a large
test tube. Add water to the level of the milk.
Cover with a stopper, label, and leave it in the
refrigerator.

Digestion of casein

It requires many hours of boiling in strong
acid or alkali to hydrolyze a protein molecule.
Living systems accomplish the same thing in
relatively neutral solution, and at relatively low

146 APPENDIX C

temperatures. The difference is that in living
organisms such reactions are catalyzed by en-
zymes. All known enzymes are proteins, and
hence possess typical protein properties.

In vertebrates generally, the pancreas secretes
into the digestive system a number of enzymes
(trypsin, chymotrypsin, etc.) that catalyze the
breakdown of proteins into smaller units, called
peptides, and individual amino acids. You will
be supplied with a dilute pancreatic extract in
order to carry out at room temperature the
digestion of the casein you have prepared.

Measure about 0.2 gm of casein into a test
tube. Add approximately 2 ml of O.^T; pan-
creatic extract, dissolved in 0.1 M phosphate
buffer at pH 7.0. Swirl the contents of the test
tube to dissolve or suspend the casein. Add a

small crystal of thymol to prevent the growth of
microorganisms. Label and initial your tube,
stopper it, and give it to your instructor to store
until the next laboratory period.

A >vord on molecular structure

Read the material on this topic in Exercise III
and make yourselves models of a representative
fat, and the sugars glucose and galactose. See
what it means to join glucose and galactose to-
gether, taking out a molecule of water, to yield
lactose. Similarly construct a polypeptide chain
from a few generalized amino acids, and see what
it means to insert a molecule of water so as to
break (hydrolyze) such a chain, the process
catalyzed by such protein-hydrolyzing enzymes
as are found in pancreatic extracts.

PART 2

(Readings: G. Wald, "The Origin of Life," Sci. Am. 191, No. 2, 45-53, Aug.
1954, Reprint No. 47. P. Doty, "Proteins," Sci. Am. 197, No. 3, 173-184,
Sept. 1957, Reprint No. 7. E. O. P. Thompson, "The Insulin Molecule,"
Sci. Am. 192, No. 5, 36^1, May 1955, Reprint No. 42.)

Shake the tube containing the lactose crystals,
and filter with suction. Wash them with two
5-ml portions of acetone. (Acetone is a fire
hazard. When you are through, flush it down
the drain with plenty of water.) Suck the crys-
tals dry, and transfer them to a cardboard
container.

We will now examine some of the specific
properties of the substances isolated from milk.
Record all observations immediately. First note
the colors and textures of these substances.
Then carry out the following general tests for
protein and sugar on each of the four com-
ponents.

size of a BB) of casein in one, of coagulated
protein in another, of lactose in a third and Ca
phosphate (or oxalate) in the last.

Biuret test for proteins

Add 3 ml of sodium hydroxide to each of one
series of test tubes. Gentle warming of a tube
in a water bath will hasten the solution of
whatever material it contains, but run the rest
at room temperature. Add a few drops of
copper sulfate solution. Note any appearance
of color. The biuret test is given not only by
proteins, but by any substance that contains
so-called peptide bonds ( — CO — NH — ).

EXPERIMENTAL PROCEDURE

Prepare two groups of test tubes, with five in
each series. Leave one tube of each series
empty to serve as a blank. In the other four
tubes of each series place a pinch (about the

Benedict test for sugars

Add 5 ml of Benedict solution to each test
tube in the second series. Hold them in the
boiling water bath for 2 minutes. Compare the
colors. The Benedict test is given by all sugars
that contain groups (aldehyde or ketone) that

THE BIOCHEMISTRY OF MILK 147

can reduce blue cupric (Cu++) to red cuprous
(Cu+) ions. It is not given by the sugar most
familiar to you, sucrose (cane sugar). Why not?
Run the Benedict test with a graded series of
sugar concentrations prepared as follows: Label
5 small test tubes, #1 to #5. To a pinch of
lactose in test tube #1, add 20 drops of water,
and swirl to dissolve. Add 10 drops of water to
each of the other 4 test tubes. Now transfer 10
drops of lactose solution from test tube #1 to
#2 and mix; then transfer 10 drops from #2 to
#3, and so on. Discard 10 drops of solution
from tube #5, after mixing. You now have a
series of test tubes, each containing half as much
sugar as the one before it. Add 3 ml of Benedict
solution to each, and heat in the boiling water
bath for 2 minutes. Note the colors, and set
aside.

Completion of the dialysis experiment

The biuret and Benedict tests can now be used
to determine whether protein, sugar, or both
have passed out of the dialysis sac prepared last
week. Remove the sac from the surrounding
solution in the test tube. The latter is called
the dialysate. Empty the contents of the sac
into a beaker. Test 5 drops of each solution
for both sugar and protein. Compare the colors
with those of the blanks, prepared above, and
with the graded sugar series. Does the dialysate
contain sugar? How much sugar, compared
with the sac contents? Does it contain protein?
How do you explain these observations?

Paper chromatography of amino acids and
casein hydrolysate

In this experiment, six known amino acids
[alanine, aspartic acid, lysine, proline (an imino
acid), histidine and methionine], one unknown
amino acid, and your casein hydrolysate are
chromatographed on a single sheet of filter
paper.

Place a piece of filter paper, 4" X 5", on wax
paper, and draw a fine line with lead pencil
parallel to and 1.5 cm from one long edge, which
will be the bottom of your chromatogram. On
this line mark pencil dots about 1 cm apart.

starting about 2 cm from one edge. These are
to indicate the positions for placing your sam-
ples; you can label each sample directly on the
paper below the line.

The samples are applied to the paper with a
fine glass capillary. (The instructor will show
you how to make capillaries.) Draw a little
solution into a capillary, touch it to the paper
at a pencil dot, let dry, and repeat. Each spot
should not be more than 3 mm in diameter.
Two such superimposed applications should be
sufficient with the amino acid solutions, and
four with the casein hydrolysate. It will be
advantageous to place your unknown amino
acid in the middle, between the third and fourth
known amino acid. (Note: Avoid excess han-
dling of the filter paper, since your hands might
contaminate it with amino acids. Touch it only
at the edges.) Now roll the sheet into a cylinder,
with the short dimension vertical, and tie the
edges together with staples so that they do not
touch each other, as shown in the diagram in
Exercise IV.

Pour about 30 ml of solvent (formic acid:
isopropanol : water = 10:70:20) into a quart
jar. Line the walls of the jar with a piece of
filter paper dipping into the solvent, to act as a
wick and help to keep the atmosphere in the jar
saturated with solvent. Splash the solvent
about. Now insert your cylinder, keeping it
away from the walls, close the jar, and let it
stand quietly. Wait until the solvent has risen
within 0.5 cm from the top of the paper before
removing the cylinder and letting it dry. Then
dip it into the ninhydrin-acetone reagent, and
after the acetone has evaporated, place the
paper in the warm oven (80°) for a few minutes.
Do not leave it too long or let it overheat! Look
each minute, and take it out as soon as you can
clearly see the spots. Then immediately outline
the spots that you see with pencil. (You can
now handle the paper freely but the spots fade
in the light.) Which of the amino acids is your
unknown?

The ninhydrin test yields purple colors with
amino acids and some related substances, and
a yellow spot with the imino acid proline.

148 APPENDIX C

EQUIPMENT

First Milk Experiment
Per student

^ pint of milk

1 ft- of cloth

9" of dialysis tubing, ^" in diameter
large test tube (20 X 150 mm)
test tube (16 X 150 mm)

2 stirring rods
wax pencil
box of matches

4 cardboard containers

No. 0 rubber stopper or cork
No. 4 rubber stopper or cork
bunsen burner
2 400-ml beakers

dropping bottle of 4 A'^ hydrochloric acid
spatula

5-ml serological pipet
test-tube rack, with holes 1 in-
tripod or ring
wire gauze
250-ml beaker

Per 2 students

Buchner funnel, 56 mm in diameter

500-ml suction flask

aspirator

Per 8 students

small bottle of glass beads

2 boxes of paper towels

1 gal 95% alcohol

1 pint of acetone

box of Whatman No. 1 filter paper, 56 mm in

diameter

buffer solution (200 ml)

dropping bottle of enzyme solution

Second Milk Experiment
Per student

14 test tubes (16 X 150 mm)

5 small test tubes (13 X 100 mm)

2 6" medicine droppers
5-ml serological pipet
400-ml beaker
250-ml beaker
spatula
test-tube rack

Per 2 students

Buchner funnel
500-ml suction flask

Per 8 students

acetone

filter paper

10% sodium hydroxide (500 ml)

dropping bottle of 0.5% copper sulfate solution

Benedict solution.
Exercise IV

1 liter prepared as directed in

Paper Chromatography Experiment

Per student

soft glass tubing

quart jar

filter paper

stapler

oven

wax paper

beaker (water bath)

2 test tubes, with corks

formic acid

isopropanol

acetone

ninhydrin

alanine (10~^ M)

aspartic (10~^ M)

lysine (IQ-^ M)

proline (10-^ M)

histidine (10"^ M)

methionine (10"'^ M)

pangestin

phosphate buffer (0.1 M, pH 7.0)

thymol

EXPONENTS AND LOGARITHMS

APPENDIX D

*rfiJSiS«^

Number

As power of 10

log

0.001

10-3

-3 or 3

0.01

10-2

-2 or 2

0.1

10-1

-1 orT

100

101

100

102

1000

103

Multiplication

103 X 103 ^ 106 (1000 X 1000 = 1 million),

.-. log 103 + log 103 (2 X log 103) = 3 _|. 3 = 6
(i.e., multiplying numbers = adding exponents
or logs).

Division

/1, 000,000

106 ^ 102 = 104 ^ : — = 10,000

V 100

)•

.". in logs: 6 — 2 = 4 (i.e., dividing numbers
subtracting exponents or logs).

Number

log

Number

log

— infinity

0.7782

0.0000

0.8451

0.3010

0.9031

0.4771

0.9542

0.6021

1.0000

0.6990

Make a graph, plotting these numbers against
their logs. From this you can interpolate inter-
mediate numbers. From your graph read the
logs of 2.35, 9.76, 3.87.

The log of

5.76 = 0.7604,

576 = 2.7604,

576 million = 8.7604,
0.576 = T.7604,

0.00576 = 3.7604.

The number before the decimal point is called
the characteristic:

characteristic 43210 T2345
number 00000.00000

To multiply decimals:

0.003 X 0.090 = 0.00027

logs: 3.48 + 2.95 = 4.43

Check with your graph.

An exercise in logarithms: the pH scale

In pure water or in any aqueous solution,
whatever its alkalinity or acidity, the product of
the concentrations of hydrogen and hydroxyl
ions in moles per liter is lO-i*:

(H+)(OH-) = 10-14

.-. log (H+) + log (0H-) = - 14

or, changing signs,

-log(H+) - log(OH-) = 14

— log (H+) is called the pH (Sorensen). In a
neutral solution, (H+) = (OH-) = IQ-^ moles
per liter, i.e.,

log(H+) = -7

.-. pH = 7

149

150 APPENDIX D

In an acid solution, pH is less than 7; in an
alkaline solution it is more than 7.

pH 0: the solution contains 1 mole (H+) per
liter. Explain.

pH 14: the solution contains I mole (OH")
per liter. Explain.

What is the concentration of (H+) in a solu-
tion of pH 7.35? 9.73?

What is the approximate pH of a solution
containing: 0.03 moles per liter of hydrochloric
acid? 0.0007 moles per liter of potassium
hydroxide?

THE PERIODIC SYSTEM OF THE ELEMENTS
(Niels Bohr's Arrangement)

APPENDIX E

K^^

Electronic Configurations of the Inert Gases

Ca
20

Atom and
atomic number

Helium (2)
Neon (10)
Argon (18)
Krypton (36)
Xenon (54)
Radon (86)

Electrons in quantum groups
1st 2nd 3rd 4th 5th 6th

2 +

+ 8

+ 18+8

+ 18 + 18 + 8

+ 18 + 32 + 18 +

Mn Fe Co Ni

25 26 27 28

-Transitional Elements ■

Cu Zn Ga Ge

29 _J

Y Zr Cb Mo Ma Ru Rh Pd

9 ^^40^ 41--^ 42^ 43-^ 44^ 45^ 46

30 31

Agi Cd
47. 1 48,

Kr
36

Ba
56

Ce Pr Yb

58 59 * 70

Lu Hf Ta W Re Os Ir Ft

■ 1\ Jl^ll^l^ 75 76 77 78

In Sn Sb te I X

49 50 51 52 53 54

\ \ 1

Aui Hg Tl Pb Bi Po - Rn

79 ; 80 81 82 83 84 85 86

Ac
89

Th
90

U
92

Np Pu Am Cm Bk
93 94 95 96 97

E
99

Fm Mv No
100 101 102

'Artificial" Elements

*Rare earths: Nd Pm Sm Eu Gd Tb Dy Ho Er Tm
60 61 62 63 64 65 66 67 68 69

151

TABLE OF ATOMIC WEIGHTS

APPENDIX F

MiliJJlA.J^MMi

Element

Sym-
bol

At.
no.
Z

At. wt.
(chem.
scale)

Element

Sym-
bol

At.
no.
Z

At. wt.
(chem.
scale)

Actinium

(227)

Gadolinium

157.26

Aluminum

26.98

Gallium

69.72

Americium

(243)

Germanium

72.60

Antimony

121.76

Gold

197.0

Argon

Arsenic

Astatine

Ar
As
At

18
33
84

39.944
74.91
(210)

Hafnium

Helium

Holmium

Hf
He
Ho

178.50
4.003
164.94

Barium

137.36

Hydrogen

1.0080

Berkeiium

Beryllium

Bismuth

Boron

Bromine

97
4

83
5

(247)
9.013

(209)
10.82
79.916

Indium
Iodine
Iridium
Iron

In
I

Ir
Fe

49
53

77
26

114.82
126.91
192.2
55.85

Krypton

83.80

Cadmium

Calcium

Californium

Carbon

Cerium

48
20
98
6
58

112.41
40.08
(251)
12.011

140.13

Lanthanum
Lead
Lithium
Lutetium

La
Pb
Li
Lu

138.92
207.21

6.940
174.99

Cesium

132.91

Magnesium

24.32

Chlorine

35.457

Manganese

54.94

Chromium

52.01

Mendelevium

101

(256)

Cobalt

58.94

Mercury

200.61

Copper

63.54

Molybdenum

95.95

Curium

(247)

Neodymium

144.27

Dysprosium

162.51

Neon
Neptunium

Ne
Np

10
93

20.183
(237)

Einsteinium
Emanation
Erbium
Europium

Es
Em
Er
Eu

99
86
68
63

(254)
(222)(Rn)

167.27

152.0

Nickel
Niobium or

(Columbium)(Cb)
Nitrogen
Nobelium

102

58.71

92.91
14.008
(253)

Fermium

100

(253)

Osmium

190.2

Fluorine

19.00

Oxygen

16.0000

Francium

(223)

(standard)

(cont.)

152

TABLE OF ATOMIC WEIGHTS 153
Table of Atomic Weights (cont.)

Element

Sym-
bol

At.

no.

At. wt.
(chem.
scale)

Element

Sym-
bol

At.
no.
Z

At. wt.
(chem.
scale)

Palladium

106.4

Tantalum

180.95

Phosphorus

30.975

Technetium

(98)

Platinum

195.09

Tellurium

127.61

Plutonium

(244)

Terbium

158.93

Polonium

(210)

Thallium

204.39

Potassium

39.100

Thorium

(232)

Praseodymium

140.92

Thulium

168.94

Promethium

(145)

Tin

118.70

Protactinium

(231)

Titanium

47.90

Tungsten (Wolfram)

183.86

Radium

(226)

Rhenium
Rhodium

Re
Rh

75
45

186.22
102.91

Uranium

(238)

Rubidium
Ruthenium

Rb
Ru

37
44

85.48
101.10

Vanadium

50.95

Samarium
Scandium

Sm
Sc

62
21

150.35
44.96

Xenon

131.30

Selenium

Silicon

Silver

Se
Si

34
14

78.96

28.09

107.88

Ytterbium
Yttrium

Yb
Y

70
39

173.04
88.92

Sodium

22.991

Strontium

87.63

Zinc

65.38

Sulfur

32.066

Zirconium

91.22

BIBLIOGRAPHY

ARTICLES FROM SCIENTIFIC AMERICAN

INTRODUCTION

Wald, G., Innovation in Biology, 199, No. 3, 100-113, Sept. 1958.
Wald, G., Tlie Origin of Life, 191, No. 2, 45-53, Aug. 1954.

THE COSMOLOGICAL SETTING

Gamow, G., The Evolutionary Universe, 195, No. 3, 136-154, Sept. 1956.
HOYLE, P., The Steady-State Universe, 195, No. 3, 157-166, Sept. 1956.
Sandage, a. R., The Red-Shift, 195, No. 3, 170-182, Sept. 1956.

ELEMENTARY PARTICLES

BuRBiDGE, G., and Hoyle, P., Anti-Matter, 198, No. 4, 34-39, April 1958.

Gamow, G., The Principle of Uncertainty, 198, No. 1, 51-57, Jan. 1958.

Gell-Mann, M., and Rosenbaum, E. P., Elementary Particles, 197, No. 1, 72-88, July 1957.

Morrison, P., and Morrison, E., The Neutron, 185, No. 4, 44-53, Oct. 1951.

ATOMS

Brown, H., The Age of the Solar System, 196, No. 4, 80-94, April 1957.

Darrow, K. K., The Quantum Theory, 186, No. 3, 47-54, March 1952.

Fowler, W. A., The Origin of the Elements, 195, No. 3, 82-91, Sept. 1956.

Gamow, G., The Exclusion Principle, 201, No. 1, 74-86, July 1959.

Peierls, R. E., The Atomic Nucleus, 200, No. 1, 75-82, Jan. 1959.

Perlman, I., and Seaborg, G. T,, The Synthetic Elements, I, 182, No. 4, 38-47, April 1950.

MOLECULES

BuswELL, A. H., and Rodebush, W. H., IVater, 194, No. 4, 76-89, April 1956.
Roberts, J. D., Organic Chemical Reactions, 197, No. 5, 117-126, Nov. 1957.
Wannier, G. H., The Nature of Solids, 187, No. 6, 39^8, Dec. 1952.

PHYSICAL METHODS POR THE STUDY OP MOLECULES
Gray, G. W., Electrophoresis, 185, No. 6, 45-53, Dec. 1951.
Gray, G. W., The Ultracentrifuge, 184, No. 6, 42-51, June 1951.
Kamen, M. D., Tracers, 180, No. 2, 30-41, Peb. 1949.

Stein, W. H., and Moore, S., Chromatography, 184, No. 3, 35^1, March 1951.

154

BIBLIOGRAPHY 155

MOLECULES OF LIVING ORGANISMS

Abelson, p. H., Paleobiochemistry (10), 195, No. 1, 83-92, July 1956.

Crick, F. H. C, The Structure of the Hereditary Material, 191, No. 4, 54-61, Oct. 1954.

Doty, P., Proteins, 197, No. 3, 173-184, Sept. 1957.

FiESER, L. F., Steroids, 192, No. 1, 52-60, Jan. 1955.

Pauling, L., Corey, R. B., and Hayward, R., The Structure of Protein Molecules, 191, No. 1, 51-59,

July 1954.
Stein, W. H., and Moore, S., The Structure of Proteins, 204, No. 2, 81-92, Feb. 1961.
Thompson, E. O. P., The Insulin Molecule, 192, No. 5, 36^1, May 1955.

ENERGY TRANSFORMATIONS IN CELLS

Arnon, D. I., The Role of Light in Photosynthesis, 203, No. 5, 104-1 18, Nov. 1960.

Bassham, J. A., The Path of Carbon in Photosynthesis, 206, No. 6, 88-100, June 1962.

Lehninger, a.. Energy Transformation in the Cell, 202, No. 5, 102-114, May 1960. How Cells Transform

Energy, 205, No. 3, 62-73, Sept. 1961.
Rabinowitch, E. I., Photosynthesis, 179, No. 2, 24-34, Aug. 1948.
Wald, G., Life and Light, 201, No. 4, 92-108, Sept. 1959.

GENES, VIRUSES, GENE ACTION
Allfrey, V. G., and Mirsky, A. E., How Cells Make Molecules, 205, No. 3, 74-82, Sept. 1961.
Beadle, G., The Genes of Men and Molds, 179, No. 3, 30-38, Sept. 1948.
Benzer, S., The Fine Structure of the Gene, 206, No. 1, 70-84, Jan. 1962.
Burnet, F. M., Viruses, 184, No. 5, 43-51, May 1951.
Fraenkel-Conrat, H., Rebuilding a Virus, 194, No. 6, 42^7, June 1956.
Hoagland, M. B., Nucleic Acids and Proteins, 201, No. 6, 55-61, Dec. 1959.
Horowitz, N. H., The Gene, 195, No. 4, 78-90, Oct. 1956.

HoTCHKiss, R. D., and Weiss, E., Transformed Bacteria, 195, No. 5, 48-53, Nov. 1956.
Hurwitz, J., and Furth, J. J., Messenger R.N.A., 206, No. 2, 41-49, Feb. 1962.
Ingram, V. M., How Do Genes Act?, 198, No. 1, 68-74, Jan. 1958.
Mirsky, A. E., The Chemistry of Heredity, 188, No. 2, 47-57, Feb. 1953.
Stent, G. S., The Multiplication of Bacterial Viruses, 188, No. 5, 36-39, May 1953.
Wollman, E. L., and Jacob, F., Sexuality in Bacteria, 195, No. 1, 109-118, July 1956.
Zinder, N. D., Transduction in Bacteria, 199, No. 5, 38^2, Nov. 1958.

THE CELL AND HEREDITY

Bracket, J., The Living Cell, 205, No. 3, 50-62, Sept. 1961.

DOBZHANSKY, T., The Genetic Basis of Evolution, 182, No. 1, 32^0, Jan. 1950.

HOLTER, H., How Things Get into Cells, 205, No. 3, 167-180, Sept. 1961.

Mazia, D., Cell Division, 189, No. 2, 53-63, Aug. 1953. How Cells Divide, 205, No. 3, 100-120, Sept. 1961.

MoROWiTZ, H. J., and Tourtellotte, M. E., 206, No. 3, 117-126, March 1962.

Muller, H. J., Radiation and Human Mutation, 193, No. 5, 58-68, Nov. 1955.

156 BIBLIOGRAPHY

Robertson, J. D., The Membrane of the Living Cell, 206, No. 4, 65-72, April 1962.
SONNEBORN, T. M., Partner of the Genes, 183, No. 5, 30-39, Nov. 1950.
Taylor, J. H., The Duplication of Chromosomes, 198, No. 6, 36-42, June 1958.

EMBRYONIC DEVELOPMENT
Butler, W. L., and Downs, R. J., Light and Plant Development, 203, No. 6, 56-63, Dec. 1960.
Ebert, J. D., The First Heartbeats, 200, No. 3, 87-96, March 1959.

FiscHBERG, M., and Blackler, A. W., How Cells Specialize, 205, No. 4, 124-140, Sept. 1961.
Grant, V., The Fertilization of Flowers, 184, No. 6, 52-56, June 1951.
Gray, G. W., ''The Organizer;' 197, No. 5, 79-88, Nov. 1957.
MoscoNA, A. A., How Cells Associate, 205, No. 4, 142-162, Sept. 1961.
Singer, M., Regeneration of Body Parts, 199, No. 4, 79-88, Oct. 1958.
Tyler, A., Fertilization and Antibodies, 190, No. 6, 70-75, June 1954.
Waddington, C. H., How Do Cells Differentiate?, 189, No. 3, 108-116, Sept. 1953.
Wigglesworth, V. S., Metamorphosis and Differentiation, 200, No. 2, 100-110, Feb. 1959.
Williams, C. M., The Metamorphosis of Insects, 182, No. 4, 24-28, April 1950.

ORGANS: STRUCTURE AND FUNCTION

Hayashi, T., How Cells Move, 205, No. 3, 184-202, Sept. 1961.

Huxley, H. E., The Contraction of Muscle, 199, No. 5, 66-82, Nov. 1958.

Katz, B., The Nerve impulse, 187, No. 5, 55-64, Nov. 1952. How Cells Communicate, 205, No. 3, 209-220,

Sept. 1961.
Keynes, R. D., The Nerve Impulse and the Squid, 199, No. 6, 83-90, Dec. 1958.
LoEWENSTEiN, W. R., Biological Transducers, 203, No. 2, 98-108, Aug. 1960.
Miller, W. H., Ratliff, F., and Hartline, H. K., How Cells Receive Stimuli, 205, No. 3, 222-238, Sept.

1961.
Smith, H. W., The Kidney, 188, No. 1, 40-48, Jan. 1953.
VON Bekesy, G., The Ear, 197, No. 2, 66-78, Aug. 1957.
Wald, G., Eye and Camera, 183, No. 2, 32^0, Aug. 1950.
Wiggers, C. J., The Heart, 196, No. 5, 74-87, May 1957.

NERVOUS INTEGRATION

EccLES, J. C, The Physiology of the Imagination, 199, No. 3, 135-142, Sept. 1958.

French, J. D., The Reticular Formation, 196, No. 5, 54-60, May 1957.

Gray, G. W., The Great Ravelled Knot, 179, No. 4, 26-39, Oct. 1948.

Olds, J., Pleasure Centers in the Brain, 195, No. 4, 105-116, Oct. 1956.

Snider, R. S., The Cerebellum, 199, No. 2, 84-90, Aug. 1958.

Sperry, R. W., The Growth of Nerve Circuits, 201, No. 5, 68-75, Nov. 1959.

Walter, W. G., The Electrical Activity of the Brain, 190, No. 6, 54-63, June 1954.

BEHAVIOR AND SOCIAL INTEGRATION

Tinbergen, N., The Evolution of Behavior in Gulls, 203, No. 6, 118-130, Dec. 1960.

BIBLIOGRAPHY 157

BOOKS

PHYSICS

Bonner, F. T., and Phillips, M., Principles of Physical Science. Addison-Wesley, 1957.

Feather, N., Mass, Length and Time. Edinburgh Univ. Press, 1959.

Hecht, Selig, Explaining the Atom. Viking Press, 2nd ed., 1954.

HOLTON, G., and Roller, D. H. D., Foundations of Modern Physical Science. Addison-Wesley, 1958.

McCuE, J. J. G., The World of Atoms. Ronald Press, 1956.

Rogers, E. M., Physics for the Inquiring Mind, Princeton Univ. Press, 1960.

CHEMISTRY
Brown, G. I., Simple Guide to Modern Valency Theory. Longmans, Green, 1954.
Brown, G. I., Electronic Theories of Organic Chemistry. Longmans, Green, 1958.
Cram, D. J., and Hammond, G. S., Organic Chemistry. McGraw-Hill, 1959.
Handbook of Chemistry and Physics. Chemical Rubber Pub. Co., Cleveland, Ohio.
Hitchcock, D. L, Physical Chemistry. Little, Brown, 4th ed., 1953.
Lessing, L. P., Understanding Chemistry. Interscience Pub. Co., 1959.
Pauling, L., College Chemistry. Freeman, 1957.
Pauling, L., General Chemistry. Freeman, 1953.

Seaborg, G. T., and Valens, E. G., Elements of the Universe. Button, 1958.
Speakman, J. C, Introduction to the Electronic Theory of Valency. London: Arnold, 1955.
Stott, R. W., Electronic Theory and Chemical Reactions. Longmans, Green, 1958.

BIOLOGY

Adrian, E. D., Physical Background of Perception. Oxford Univ. Press, 1947.

Adrian, E. D., Mechanism of Nervous Action. Univ. of Penn. Press, 1932.

Best, C. H., and Taylor, N. B., The Livig Body. Henry Holt, 4th ed., 1958.

Bonner, J. T., and Galston, A. W., Principles of Plant Physiology. Freeman, 1952.

Brazier, M. A. B., Electrical Activity of the Nervous System. Macmillan, 1951.

Buchsbaum, Ralph M., Animals without Backbones. Univ. of Chicago Press, rev. ed., 1948.

Cannon, W. B., Wisdom of the Body. Norton, 1939.

Carter, G. S., General Zoology of the Invertebrates. Sidgwick and Jackson, 1951.

Conant, J. B., ed.. Case Histories in Science. No. 7, "Pasteur's and Tyndall's Spontaneous Generation."

Harvard Univ. Press, 1953.
Elliott, A. M., and Ray, C, Jr., Biology. Appleton-Century-Crofts, 1960.
Evlenberg-Wiener, Fearfully and Wonderfully Made. Macmillan, 1938.
Gabriel, M., and Fogel, S., eds., Great Experiments in Biology. Prentice-Hall, 1955.
Garrod, a. E., Inborn Errors of Metabolism. London: Froude, Hodder and Stoughton, 2nd ed., 1923.
Gray, James, How Animals Move. Cambridge Univ. Press, 1960.
Griffin, D. R., Echoes of Bats and Men. Anchor Books, 1959.
Griffin, D. R., Listening in the Dark. Yale Univ. Press, 1958.
Harvey, W., Motion of the Heart and Blood. Translated by R. Willis. Everyman.
LocY, W. A., The Story of Biology. Garden City, 1925.

158 BIBLIOGRAPHY

LuRiA, S. E., General Virology. Wiley, 1953.

Marsland, Douglas, Principles of Modern Biology. Henry Holt, 3rd ed., 1957.

Medawar, p., Uniqueness of the Individual. Methuen, 1957.

Oginsky, E. L., and Umbreit, W. W., Introduction to Bacterial Physiology. Freeman, 1959.

PiRENNE, M. H., Vision and the Eye. Chapman and Hall, 1948.

Prosser, C. L., ed.. Comparative Animal Physiology. Saunders, 1950.

ROMER, A. S., The Vertebrate Story. Univ. of Chicago Press, 1959.

RoMER, A. S., Man and the Vertebrates. Pelican Books.

Schmidt-Nielsen, K., Animal Physiology. Prentice-Hall, 1960.

Sherrington, C. S., Integrative Action of the Nervous System. Yale Univ. Press, 1952.

SiNNOTT, E. W., Dunn, L. C, and Dobzhansky, Th., Principles of Genetics. McGraw-Hill, 5th ed., 1958.

Srb, A. M., and Owen, R. D., Genetics. Freeman, 1952.

Stanier, R. Y., Douderoff, M., and Adelberg, E. A., The Microbial World. Prentice-Hall, 1957.

Sussman, Maurice, Animal Growth and Development. Prentice-Hall, 1960.

SwANSON, C. P., The Cell. Prentice-Hall, 1960.

Thimann, K. v.. The Life of Bacteria. Macmillan, 1955.

TiNBERGEN, N., Social Behavior in Animals. Wiley, 1953.

ViLLEE, C. E., Biology. Saunders, 3rd ed., 1957.

VON Frisch, Karl, The Dancing Bees. Harcourt, 1955.

Waddington, C. H., How Animals Develop. Norton, 1936.

Walter, W. G., The Living Brain. Norton, 1953.

Winchester, A. M., Genetics. Houghton Mifflin, 1951.

Young, J. Z., The Life of Mammals. Oxford Univ. Press, 1957.

Young, J. Z., The Life of Vertebrates. Oxford Univ. Press, 1950.

GENERAL PERSPECTIVES
Anfinsen, C. B., The Molecular Basis of Evolution. Wiley, 1959.
Blum, H. P., Time's Arrow and Evolution. Princeton Univ. Press, 1951.
Henderson, L. J., Fitness of the Environment. Beacon Press, 1958.
Oparin, a. I., The Origin of Life on the Earth. Oliver and Boyd, 1957.
Rush, J. H., The Dawn of Life. Hanover House, 1957.
Schrodinger, E., What is Life? Cambridge Univ. Press, 1944.
Simpson, G. G., The Meaning of Evolution. Yale Univ. Press, 1950.
Smith, H. W., From Fish to Philosopher. Little, Brown, 1953.
Snow, C. P., Two Cultures (Oxford).

BIOCHEMISTRY

Baldwin, Ernest, Dynamic Aspects of Biochemistry. Cambridge Univ. Press, 3rd ed., 1957.

Baldwin, Ernest, Introduction to Comparative Biochemistry. Cambridge Univ. Press, 3rd ed., 1948.

BoREK, E., Man, The Chemical Machine. Columbia Univ. Press, 1953.

Butler, J. A. V., Inside the Living Cell. Basic Books, 1959.

Harrison, K., A Guide-Book to Biochemistry. Cambridge Univ. Press, 1959.

Harrow, B., and Mazur, A., Textbook of Biochemistry. Saunders, 1958.

BIBLIOGRAPHY 159

SUGGESTIONS FOR A "50-CENT"
PERSONAL SCIENCE LIBRARY

NON FICTION
Adler, Irving, How Life Began. Signet.

Carson, Rachel L., Tlie Edge of the Sea (Mentor); The Sea Around Us (Mentor); Under the Sea Wind
(Mentor).

Crompton, John, The Life of lite Spider. Mentor.

Darwin. Charles, The Origin of Species (Mentor); The Voyage of the Beagle (Bantam).

Davis, Helen Miles, The Chemica/ Elements. Ballantine Books.

DE Kruif, Paul, Microbe Hunters. Cardinal.

Dunn, I. C, and Dobzhansky, Th., Heredity, Race and Society. Mentor.

Gamow, George, The Birth and Death of the Sun (Mentor); One, Two, Three . . . Infinity (Mentor); The

Creation of the Universe (Mentor).
Haggard, H. W., Devils, Drugs and Doctors. Cardinal.

Hoyle, Fred, Frontiers of Astronomy (Mentor); The Nature of the Universe (Mentor).
Mutton, Kenneth, Chemistry, The Conquest of Materials. Penguin Books.
Huxley, Julian, Evolution in Action. Mentor.
Jeans, James, The Growth of Physical Science. Premier.
Jones, H. Spencer, Life on Other Worlds. Mentor.
Lessing, Lawrence P., Understanding Chemistry. Mentor.

Malthus, Thomas, Julian Huxley, and Frederick Osborn, Three Essays on Population. Mentor.
Opik, Ernst J., The Oscillating Universe. Mentor.
Payne-Gaposchkin, Cecilia, Stars in the Making. Cardinal.
Rapport, Samuel, and Helen Wright, editors. The Crust of the Earth. Mentor.
Sanderson, Ivan T., How to Know the American Mammals. Signet.

Shapley, Harlow, Of Stars and Men (Washington Square Press); Stars in the Making (Pocket Books).
Simpson, George Gaylord, The Meaning of Evolution. Mentor.
Storer, John H., The Web of Life. Signet.
Whitehead, Alfred North, Science and the Modern World. Mentor.

FICTION

Capek, Karel, War with the Newts. Bantam.

Hoyle, Fred, The Black Cloud. Signet.

Lewis, Sinclair, Arrowsmith. Harcourt.

Snow, C. P., The Affair (Signet); The Search (Signet).

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