ISE Biology Laboratory Manual

(Thirteenth Edition) - eBook PDF

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 ISE Biology Laboratory Manual
(Thirteenth Edition) - eBook PDF
Visit to download the full and correct content document:
https://ebooksecure.com/download/ise-biology-laboratory-manual-thirteenth-edition-e
book-pdf/
Biology
Laboratory Manual

Thirteenth Edition

Darrell S. Vodopich
Baylor University

Randy Moore
University of Minnesota
BIOLOGY LABORATORY MANUAL

Published by McGraw Hill LLC, 1325 Avenue of the Americas, New York, NY 10019. Copyright ©2023 by
McGraw Hill LLC. All rights reserved. Printed in the United States of America. No part of this publication
may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system,
without the prior written consent of McGraw Hill LLC, including, but not limited to, in any network or other
electronic storage or transmission, or broadcast for distance learning.

Some ancillaries, including electronic and print components, may not be available to customers outside the
United States.

This book is printed on acid-free paper.

1 2 3 4 5 6 7 8 9 LMN 27 26 25 24 23 22

ISBN 978-1-265-13673-4
MHID 1-265-13673-4

Cover Image: ©Darrell S. Vodopich

All credits appearing on page or at the end of the book are considered to be an extension of the copyright page.

The Internet addresses listed in the text were accurate at the time of publication. The inclusion of a website
does not indicate an endorsement by the authors or McGraw Hill LLC, and McGraw Hill LLC does not
guarantee the accuracy of the information presented at these sites..

mheducation.com/highered
Contents

Preface ​ ​v Exercise 16
Teaching and Learning Tools ​  ix Molecular Biology and Biotechnology: DNA Isolation and Genetic
Transformation ​ 175
Welcome to the Biology Laboratory ​xii

Exercise 1 Exercise 17
Genetics: The Principles of Mendel ​183
Scientific Method: The Process of Science ​1

Exercise 2 Exercise 18
Evolution: Natural Selection and Morphological Change in
Measurements in Biology: The Metric System and Data Analysis ​11
Green Algae ​199

Exercise 3 Exercise 19
The Microscope: Basic Skills of Light Microscopy ​21
Human Evolution: Skull Examination ​211

Exercise 4 Exercise 20
The Cell: Structure and Function ​33
Ecology: Diversity and Interaction in Plant Communities ​223

Exercise 5 Exercise 21
Solutions, Acids, and Bases: The pH Scale ​51
Community Succession ​233

Exercise 6 Exercise 22
Biologically Important Molecules: Carbohydrates, Proteins, Lipids, and
Population Growth: Limitations of the Environment ​241
Nucleic Acids ​59

Exercise 7 Exercise 23
Pollution: The Effects of Chemical, Thermal, and Acidic Pollution ​249
Separating Organic Compounds: Column Chromatography, Paper
Chromatography, and Gel Electrophoresis ​73
Exercise 24
Survey of Prokaryotes: Domains Archaea and Bacteria ​259
Exercise 8
Spectrophotometry: Identifying Solutes and Determining Their
Concentration ​ 83 Exercise 25
Survey of Protists: Algal Autotrophs ​275
Exercise 9
Diffusion and Osmosis: Passive Movement of Molecules in Biological Exercise 26
Systems ​ 95 Survey of Protists: Protozoan Heterotrophs ​289

Exercise 10 Exercise 27
Cellular Membranes: Effects of Physical and Chemical Stress ​109 Survey of the Kingdom Fungi: Molds, Sac Fungi, Mushrooms, and
Lichens ​ 299
Exercise 11
Enzymes: Factors Affecting the Rate of Activity ​117 Exercise 28
Survey of the Plant Kingdom: Liverworts, Mosses, and Hornworts of
Phyla Hepatophyta, Bryophyta, and Anthocerophyta ​315
Exercise 12
Respiration: Aerobic and Anaerobic Oxidation of Organic
Molecules ​ 129 Exercise 29
Survey of the Plant Kingdom: Seedless Vascular Plants of Phyla
Pterophyta and Lycophyta ​325
Exercise 13
Photosynthesis: Pigment Separation, Starch Production, and CO2
Uptake ​ 141 Exercise 30
Survey of the Plant Kingdom: Gymnosperms of Phyla Cycadophyta,
Ginkgophyta, Coniferophyta, and Gnetophyta ​337
Exercise 14
Mitosis: Replication of Eukaryotic Cells ​153
Exercise 31
Survey of the Plant Kingdom: Angiosperms ​347
Exercise 15
Meiosis: Reduction Division and Gametogenesis ​163

TOC–1 iii
Exercise 32 Exercise 43
Plant Anatomy: Vegetative Structure of Vascular Plants ​363 Human Biology: Muscles and Muscle Contraction ​507

Exercise 33 Exercise 44
Plant Physiology: Transpiration ​377 Human Biology: Breathing ​515

Exercise 34 Exercise 45
Plant Physiology: Tropisms, Nutrition, and Growth Regulators ​385 Human Biology: Circulation and Blood Pressure ​525

Exercise 35 Exercise 46
Bioassay: Measuring Physiologically Active Substances ​397 Human Biology: Sensory Perception ​539

Exercise 36 Exercise 47
Survey of the Animal Kingdom: Phyla Porifera and Cnidaria ​403 Vertebrate Anatomy: External Features and Skeletal
System of the Rat ​549
Exercise 37
Survey of the Animal Kingdom: Phyla Platyhelminthes and Exercise 48
Mollusca ​ 419 Vertebrate Anatomy: Muscles and Internal Organs of the Rat ​557

Exercise 38 Exercise 49
Survey of the Animal Kingdom: Phyla Annelida and Nematoda ​435 Vertebrate Anatomy: Urogenital and Circulatory Systems of the Rat ​567

Exercise 39 Exercise 50
Survey of the Animal Kingdom: Phylum Arthropoda ​449 Embryology: Comparative Morphologies and Strategies
of Development ​579
Exercise 40
Survey of the Animal Kingdom: Phyla Echinodermata and Exercise 51
Chordata ​ 463 Animal Behavior: Taxis, Kinesis, and Agonistic Behavior ​589

Exercise 41 Appendix I
Vertebrate Animal Tissues: Epithelial, Connective, Muscular, and Nervous Dissection of a Fetal Pig ​595
Tissues ​ 483
Appendix II
Exercise 42 Conversion of Metric Units to English Units ​602
Human Biology: The Human Skeletal System ​499

iv TOC–2
Preface
Contents
W e have designed this laboratory manual for an intro-
ductory biology course with a broad survey of basic
laboratory techniques. The experiments and procedures are
simple, safe, easy to perform, and especially appropriate for
large classes. Few experiments require more than one class
meeting to complete the procedure. Each exercise includes
many photographs and illustrations, traditional topics, and
experiments that help students do biology as they learn about
life. Procedures within each exercise are numerous and dis-
crete so that an exercise can be tailored to the needs of the stu-
dents, the style of the instructor, and the facilities available.
TO THE STUDENT
We hope this manual is an interesting guide to many areas
of biology. As you read about these areas, you’ll probably
spend equal amounts of time observing and experimenting.
Don’t hesitate to go beyond the observations that we’ve
outlined—your future success as a scientist and an informed
citizen depends on your ability to seek and notice things that
others may overlook. Now is the time to develop this ability
with a mixture of hard work and relaxed observation. Have
fun, and learning will come easily. Also, remember that this
manual is designed with your instructors in mind as well. Go
to them often with questions—their experience is a valuable
tool that you should use as you work.
TO THE INSTRUCTOR
This manual’s simple, straightforward approach emphasizes
experiments and activities that optimize students’ investment
of time and your investment of supplies, equipment, and
preparation. Simple, safe, and straightforward experiments
are most effective if you interpret the work in depth. Most
experiments can be done easily by a student in 2 to 3 hours.
Terminology, structures, photographs, and concepts are lim-
ited to those that the student can readily observe and under-
stand. In each exercise we have included a few activities
requiring a greater investment of effort if resources are avail-
able, but omitting them will not detract from the objectives.
This manual functions best with an instructor’s guid-
ance and is not an autotutorial system. We've provided back-
ground information for context and understanding, but the
focus of each exercise remains on students doing interesting
and meaningful activities to learn basic information about
P–1
biology. We’ve tried to guide students from observations to
conclusions, to help students make their own discoveries,
and to make the transition from observation to understand-
ing biological principles. But discussions and interactions
between student and instructor are major components of a
successful laboratory experience. Be sure to examine the
“Questions for Further Study and Inquiry” in each exercise.
We hope they will help you expand students’ perceptions
that each exercise has broad application to their world.
DIGITAL INTEGRATION
Today’s students are digital learners, and this lab manual
integrates that learning with interesting activities that help
students learn about biology. Virtually every exercise of this
manual is accompanied by tailor-made digital resources,
including assignable questions and a variety of high-definition
videos, PowerPoint images, and other resources that demon-
strate basic techniques, emphasize biological principles, test
for understanding, and engage students as they learn biology
in the laboratory.
Digital resources are available to instructors at connect
.mheducation.com. Instructors will want to assign these
resources to help students know what they’ll be doing, what
principles they’ll be investigating, and what concepts they’ll
need to understand before coming to lab.
WHAT’S NEW IN THIS EDITION
Throughout the manual, we have expanded and improved
several of the most popular and effective features of
previous editions, including
∙ Learning Objectives have been updated to provide an
overview of what students will do and learn in the exercise.
∙ Procedures and Doing Biology Yourself require stu-
dents to do biology as they apply skills they’ve learned to
develop and study hypotheses they formulate about biology.
∙ Questions throughout each exercise encourage students to
pause and think about their data and what they’ve learned.
∙ Questions for Further Study and Inquiry at the
end of each exercise help students apply what they’ve
learned to broader topics and issues in biology.
v
∙ Writing to Learn Biology encourages students to use writ-
ing to develop their ideas about what they learned in lab.
∙ Caution and Safety First icons make students aware of
safety issues associated with the procedures they’ll use
in lab.
∙ Boxed readings titled Inquiry-Based Learning encour-
age students to apply what they’ve learned to indepen-
dently answer questions about intriguing biological topics.
∙ Updated health-related exercises help students better
understand how topics such as genetics, cell biology,
blood pressure, atherosclerosis, and their risk of cardio-
vascular disease relate to our health.
∙ Several illustrations have been replaced with photographs
to provide more realistic images to support the Exercise
content.
∙ Approximately 90 illustrations and photos have been
revised.
∙ Questions within procedures now include lines on which
students can write their answers.
∙ An assignable, updated library of videos and Connect
questions helps students prepare for lab and understand
the instruments and techniques that will be important
for their investigations. Instructors may assign these
videos before class time to help ensure that students
arrive prepared for lab.
Exercise-Specific Changes
∙ Exercise 1—Edited text for improved readability and
relevance (e.g., climate change, COVID-19); Improved
questions to help students better understand what sci-
ence is and how science is done
∙ Exercise 2—Improved the readability of the text and the
presentation of metric units; Specified the differences
in using a triple-beam balance and an electronic scale;
Emphasized the importance of significant figures in
measurements; Emphasized that in biology, the mean is
usually preferred to the median when reporting descrip-
tive statistics; Added a question about measurements of
COVID-19
∙ Exercise 3—Improved the instructions for how to use a
compound light microscope
∙ Exercise 4—Added an objective for understanding the
relative sizes of cells and organelles; Added a boxed
insert about surface-area-to-volume ratios in cells; Added
a boxed insert about cellular structure and human disease
∙ Exercise 5—Reorganized and edited the text for
increased understanding and readability
vi
∙ Exercise 6—Replaced figure 6.9 with a better, more
informative image; Added a table for students to sum-
marize the biochemical tests they performed in the lab;
Added a question to emphasize the significance of acid
precipitation; Added a boxed insert about using the
iodine test to detect counterfeit money; Added a boxed
insert about dietary fats
∙ Exercise 7—Reorganized the procedures for better use
of time in the lab
∙ Exercise 9—Revised the Introduction and Diffusion
sections to emphasize the relevance of osmosis and dif-
fusion to general physiology; Enhanced the safety notice
to use appropriate PPE; Added question for problem-
solving based on experimental data; Revised captions for
figures 9.7 and 9.9 to emphasize the flow of water into
and out of cells
∙ Exercise 10—Revised the Introduction to reinforce
understanding of how membranes regulate the move-
ment of materials into and out of cells
∙ Exercise 12—Replaced figure 12.1 (i.e., rising bread
dough) to show the production of carbon dioxide; Edited
questions for improved understanding; Updated the ter-
minology for the citric acid cycle
∙ Exercise 13—Replaced figure 13.1 to emphasize the
production of oxygen by photosynthesis; Edited the text
for improved readability and understanding; Corrected
figure 13.10 for improved entry of data by students
∙ Exercise 14—Enhanced the readability of the Introduc-
tion; Expanded the description of chromatids versus
chromosomes; Added new figure 14.6 showing the
metaphase plate and chromosomal alignment
∙ Exercise 15—Revised the Introduction to emphasize
the value of genetic recombination for adaptation to
changing environments; Revised labels of figure 15.1 to
better distinguish maternal homologues from paternal
homologues; Revised figure 15.2 to emphasize (1) the
replication of chromosomes and (2) the formation of
chromatids; Added new figure 15.6 of spermatogenesis
to emphasize the steps of maturation from spermatogo-
nium to spermatozoa
∙ Exercise 16—Updated the information about the use
and yield of genetically modified crops; Edited questions
to emphasize critical thinking about genetically modi-
fied crops
∙ Exercise 17—Edited the text for improved readability
and understanding; Added updates about phenylketon-
uria, Huntington’s disease, and familial hypercholester-
emia; Added information and a new image to improve
students’ understanding of transposons
P–2
∙ Exercise 18—Added an example of calculating Hardy-
Weinberg frequencies
∙ Exercise 19—Revised figure 19.2 to reflect recent
discoveries about human evolution; Revised Procedure
19.2 to compare the sizes of brain cases in apes versus
humans; Added new figure 19.10 comparing skeletons
of humans and chimpanzees
∙ Exercise 20—Clarified the definitions of soil types;
Revised Procedure 20.3 to clarify calculations
∙ Exercise 21—Edited the objectives for improved
understanding
∙ Exercise 22—Plagues; Added a boxed insert about
Population Growth and Our Carbon Footprint; Updated
information in the text about population and population
growth; Expanded table 22.1 to include 10 generations
of bacterial growth; Emphasized and added a question
about how population growth affects public health, eco-
nomic stability, social structure, and the well-being of our
environment
∙ Exercise 23—Edited text to improve readability and
accuracy
∙ Exercise 24—Relabeled figure 24.6 to help students
better understand the structure of bacterial cell walls;
Replaced figure 24.7 to better show steps of the Gram
stain procedure; Revised the description and interpreta-
tion of antibiotic effectiveness apparent on bacterial
sensitivity plates
∙ Exercise 25—Enhanced explanations of autotrophic
versus heterotrophic protistans; Added new figure 25.1
to distinguish between algae and protozoans; Replaced
figure 25.5 to better explain Chlamydomonas life cycle;
Expanded the explanation of asexual versus sexual
reproduction in unicellular algae; Rearranged the descrip-
tions of brown algae and red algae to adhere to current
phylogeny based on molecular taxonomic techniques
∙ Exercise 26—Moved the coverage and procedures about
slime molds forward to better reflect current phylogeny;
Added new figure 26.8 showing a scanning electron
micrograph that emphasizes the cell surface of a ciliate
∙ Exercise 27—Multiple clarifications of the structures and
processes of asexual versus sexual reproduction in fungi;
Revised figure 27.1 to highlight aseptate hyphae; Revised
figure 27.2 to distinguish between sporangia and sporan-
giophores; Expanded the coverage of the major phyla of
fungi to include phylum Glomeromycota; Added new
figure 27.3b to show infection by chytrid fungi; Revised
table 27.1 to include description and artwork of key repro-
ductive features of Glomeromycota; Updated figure 27.4
to better illustrate stolons, spores, and sporangiophores
P–3
of Zygomycota; Expanded explanation of asexual versus
sexual reproduction in Zygomycota; Revised figure 27.6b
to emphasize distinctions between sexual reproduction
and asexual reproduction in bread molds; Expanded
descriptions in Procedure 27.3 to help students better
interpret conjugation plates of Rhizopus; Revised figure
27.9 to better distinguish between a sporangium and
conidiophore; Revised figure 27.13 to better distinguish
asexual from sexual reproductive structures and processes;
Revised figure 27.15 to emphasize sexual reproduction in
mushrooms; Included coverage and new procedures for
examining Glomeromycota and other mycorrhizae; Added
descriptions and illustrations of mycorrhizae, including
arbuscular and ectomycorrhizae forms; Added new figure
27.18e illustrating the structure of a lichen cross section
∙ Exercise 28—Updated classification information;
Replaced figures 28.6 and 28.11 to help students better
understand the information
∙ Exercise 29—Enhanced figures 29.1 and 29.11 for bet-
ter understanding
∙ Exercise 30—Edited text for better readability and
understanding; Added a question about the distinguish-
ing features of the groups of plants that students exam-
ined in this lab
∙ Exercise 31—Improved table 31.1 and figure 31.5 for
better understanding; Improved “Dichotomous Key to
Major Types of Fruit”; Replaced figure 31.18 with bet-
ter, more informative images and information; Added a
question to emphasize the differences between mono-
cots and eudicots
∙ Exercise 32—Edited text for improved readability and
understanding; Improved the description of the endoder-
mis and its function; Replaced figure 32.1 to better show
the differences in tap versus fibrous root systems; Added
scale-markers to figures; Edited the text to better empha-
size the differences between gymnosperms and angio-
sperms; Enhanced figure 32.16 for better understanding;
Added a question to emphasize the differences between
stomata and lenticels
∙ Exercise 33—Edited the Introduction for improved
understanding; Removed the redundant instruction in
Procedure 33.2; Added an alternate procedure for making
a leaf-impression for counting and visualizing stomata
∙ Exercise 34—Emphasized and added a question about
how plants, unlike animals, have a small number of growth
regulators that influence many traits; Added scale-markers
to figures; Added information about the use of 2,4-D;
Added information about how gibberellic acid is important
for increasing yields and profits for grape growers
vii
∙ Exercise 35—Added text to improve understanding
about bioassays and standard curves; Added a more spe-
cific question to the “Inquiry-Based Learning” assign-
ment; Added graph paper for reporting students’ results
∙ Exercise 36—Clarified functional relationships among
spicules, spongin fibers, porocytes, and amoebocytes;
Expanded the description of water flow through a wall
of a sponge as depicted in figure 36.4; Revised figure
36.12 to show the relative size of cnidarian medusae;
Revised figure 36.16 to show the relative size of ephy-
rae; Expanded the description of corals to include infor-
mation about coral bleaching and coral symbioses with
algae
∙ Exercise 37—Significantly revised the sequence of cover-
age of invertebrate phyla to adhere to current phylogeny
based on molecular taxonomic techniques; Included
taxonomic classifications of lophophorazoa and ecdy­
sozoa; Positioned coverage of nematodes to immediately
precede coverage of arthropods, as both are now consid-
ered ecdysozoans; Mollusk coverage now immediately
follows that of flatworms, as they are both considered
lophophorazoans; Added new figure 37.3 to illustrate a
trochophore larva; Revised table 37.1 to replace nematode
descriptions with mollusk descriptions; Replaced figure
37.3 with new art illustrating flatworm anatomy; Replaced
figure 38.5 with new art illustrating molluscan radula
∙ Exercise 38—Coverage of nematodes now follows that
of annelids
∙ Exercise 39—Revised figure 39.16 to clarify position of
retinula cells
∙ Exercise 40—Revised legend of figure 40.18 to better
describe the evolution of jaws among fish ancestors;
Changed common name of chordate class Actinopteriy-
gii from boney fish to ray-finned fish; Added new table
40.3 to provide space for students to organize classes of
vertebrates and their major characteristics
∙ Exercise 41—Revised Procedure 41.1 to emphasize
safety when using stains; Revised figure 41.5 to clearly
label nuclei of simple columnar epithelial cells; Clari-
fied the varied functions of connective tissues; Expanded
Procedure 41.3 to describe the appearance of red blood
cells and leukocytes on prepared slides; Included new
terminology of central canals in place of Haversian sys-
tems of bones
∙ Exercise 42—Clarified the differences between ten-
dons and ligaments; Added new figure 42.1 to illustrate
the parts of the human skeleton; Revised figure 42.2
to include labels of the ileum, ischium, and pubis;
Expanded the Questions for Further Study and Inquiry
viii
∙ Exercise 43—Modified labels of figure 43.2 to show the
origin and insertion of triceps brachii
∙ Exercise 44—Revised figure 44.4 to emphasize how
changes of internal air pressure affect the mechanics
of breathing; Emphasized the value of measuring lung
capacity to understanding respiratory disease; Clarified
Procedure 44.2 to better describe the use of a spirometer
∙ Exercise 45—Expanded the procedure for examining a
cow heart to include the use of a heart model; Added a
new question to describe heartbeat sounds heard with
a stethoscope; Revised figure 45.2 to better show dif-
ferences in the walls of arteries versus veins; Revised
Procedure 45.2 to better describe the steps to measure
blood pressure; Added new figure 45.7 to illustrate the
anatomy of venous valves; Updated the table for scoring
risk factors of cardiovascular disease; Questions for Fur-
ther Thought and Inquiry now include library research
to understand diseases of the heart and circulatory
system
∙ Exercise 46—Quantified differences in retinal resolu-
tions among humans and other animals; Described and
distinguished sensorineural versus nerve deafness;
Clarified the steps of Procedure 46.8 to better determine
nerve deafness; Updated figure 46.6 to show the size of
the ear drum; Modified Procedure 46.1 to include safety
procedures
∙ Exercise 47—Expanded Questions for Further Study
and Inquiry include an analysis of bipedalism
∙ Exercise 48—Added new figure 48.7 to include art and
a photograph showing the structure of microvilli; Rela-
beled figure 48.6 to show the common bile duct
∙ Exercise 49—Added new figure 49.4 to illustrate kidney
anatomy with sagittal section
∙ Exercise 50—Clarified the distinction between an
embryo and a zygote; Expanded the description of gray
crescent formation; Added new figure 50.5 to illustrate
the formation of a gray crescent; Added new figure 50.8
to illustrate differences between the vegetal pole and
animal pole; Relabeled figure 50.9 to clearly distinguish
the endoderm and mesoderm; Quantified the egg sizes
among birds to emphasize variety in egg anatomy; Rela-
beled figure 50.12 to show albumin
∙ Exercise 51—Added questions to encourage students to
think about agonistic behaviors in humans and why it is
important to try to integrate all aspects of an organism’s
behavior
∙ Appendix II Updated information about the metric
system
P–4
Teaching and Learning Tools
Contents

McGraw Hill Connect® McGraw Hill CreateTM

McGraw Hill Connect provides online presentation, assign- With McGraw Hill Create, you can easily rearrange exer-
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Resources tab.
While the biological sciences are hands-on disciplines,
instructors are now often being asked to deliver some of
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These simulations help each student learn the practical
and conceptual skills needed, then check for understanding and
provide feedback. With adaptive pre-lab and post-lab assess-
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From the instructor’s perspective, these simulations
may be used in the lecture environment to help students visu-
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or Gram staining, while at the same time providing a valu-
able connection between the lecture and lab environments.

T–1 ix
Instructors: Student Success Starts with You
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Welcome
Contents
to the Biology Laboratory

W elcome to the biology laboratory! Although reading

your textbook and attending lectures are important
ways of learning about biology, nothing can replace the
importance of the laboratory. In lab you’ll get hands-on
experience with what you’ve heard and read about biology—
for example, you’ll observe and manipulate organisms, do
experiments, test ideas, collect and organize data, and make
conclusions about what you’ve learned. You’ll do biology.
You’ll enjoy the exercises in this manual—they’re
interesting and informative and can be completed within
the time limits of your laboratory period. We’ve provided
questions to test your understanding of what you’ve done; in
some of the exercises, we’ve also asked you to devise your
own experiments to test hypotheses and answer questions
that you’ve posed. To make these exercises most useful and
enjoyable, follow these guidelines noted in the next sections.
THE IMPORTANCE OF COMING TO CLASS
Biology labs are designed to help you experience biology
firsthand. To do well in your biology course, you’ll need to
attend class and pay attention. Remember this: Attending
and being prepared for class are critical for learning
about biology and earning a good grade in this course.
To appreciate this, examine figure 1, which is a graph

100
A

B
80
C

D
60
Grade (%)

40

20

0
0 20 40 60 80 100
Attendance (% of classes attended)

Figure 1 Relationship of students’ grades in an introductory biology course to their rates of class attendance.

xii W–1
showing how students’ grades in an introductory biology
course correlate to their rates of class attendance. Data are
from a general biology class at the University of Minnesota.
On page xv, write an analysis of the data shown in figure 1.
What do these data mean?
BEFORE COMING TO LAB
Watch the lab video. Videos are provided for several of the
labs in this manual. Be sure to watch any assigned video
associated with the lab you will be completing. These videos
will help you know more about what you will be doing, what
principles you will be investigating, and what concepts you
need to understand before coming to lab.
Read the exercise before coming to lab. This will give
you a general idea about what you’re going to do, as well as
why you’re going to do it. Knowing this will not only save
time, it will also help you finish the experiments and make
you aware of any safety-related issues associated with the lab.
Review any of the lab safety concerns. Before doing
any procedures, you’ll encounter a section of each exercise
titled “SAFETY FIRST” that is marked with its icon:
This icon will warn you of safety concerns (e.g., solvents,
acids, bases, hotplates) associated with the work. If you have
questions about these safety issues, contact your lab instructor
before starting the lab work.
Notify your instructor if you are pregnant, are color-
blind, are taking immunosuppressive drugs, have allergies,
or have any other conditions that may require precautionary
measures. Also, before coming to lab, cover any cuts or
scrapes with a sterile, waterproof bandage.
WHEN IN LAB
1. Know what you are going to do. Read and understand
the lab before coming to lab.
2. Don’t start the exercise until you’ve discussed the
exercise with your laboratory instructor. She or he will
give you specific instructions about the lab and tell
you how the exercise may have been modified.
3. Work carefully and thoughtfully, and stay focused as
you work. You’ll be able to finish each exercise within
the allotted time if you are prepared and stay on task.
W–2
4. Discuss your observations, results, and conclusions
with your instructor and lab partners. Perhaps their
comments and ideas will help you better understand
what you’ve observed.
5. Always follow instructions and safety guidelines pre-
sented by your instructor. Speak up!
6. If you have questions, ask your instructor.
SAFETY IN THE LABORATORY
Laboratory accidents can affect individuals, classes, or the
entire campus. To avoid such accidents, the exercises in this
manual were designed with safety as a top priority. You’ll
be warned about any potentially hazardous situations or
chemicals with this image:
When you see this image, pay special attention to the
instructions.
The laboratory safety rules listed in table 1 will help
make lab a safe place for everyone to learn biology. Remem-
ber, it is much easier to prevent an accident than to deal with
its consequences.
Read the laboratory safety rules listed in table 1. If
you do not understand them, or if you have questions, ask
your instructor for an explanation. Then complete table 1
and sign the statement at the bottom of page xv.
BEFORE YOU LEAVE LAB
Put away all equipment and glassware, and wipe clean your
work area.
AFTER EACH LABORATORY
Soon after each lab, review what you did. What questions
did you answer? What data did you gather? What conclu-
sions did you make?
Also note any questions that remain. Try to answer
these questions by using your textbook or visiting the
library. If you can’t answer the questions, discuss them with
your instructor.
Welcome to the biology laboratory!
xiii
 Table 1
Laboratory Safety Rules
Why is this rule important?
Rule What could happen if this rule is not followed?
Behave responsibly. No horseplay or fooling around while in lab.
Do not bring any food or beverages into lab, and do not eat, drink, smoke,
chew gum, chew tobacco, or apply cosmetics when in lab. Never taste
anything in lab. Do not put anything in lab into your mouth. Avoid touch-
ing your face, chewing on pens, and other similar behaviors while in lab.
Always wear shoes in lab.
Unless you are told otherwise by your instructor, assume that all chemicals and
solutions in lab are poisonous, and act accordingly. Never pipette by mouth.
Always use a mechanical pipetting device (e.g., a suction bulb) to pipette solu-
tions. Clean up all spills immediately, and report all spills to your instructor.
Wear safety goggles when working with chemicals. Carefully read the labels
on bottles and know the chemical you are dealing with. Do not use chemicals
from an unlabeled container, and do not return excess chemicals back to their
container. Report all spills to your instructor immediately.
Unless your instructor tells you to do otherwise, do not pour any solutions
down the drain. Dispose of all materials as per instructions from your
instructor.
If you have long hair, tie it back. Don’t wear dangling jewelry. If you are
using open flames, roll up loose sleeves. Wear contact lenses at your own
risk; contacts hold substances against the eye and make it difficult to wash
your eyes thoroughly.
Treat living organisms with care and respect.
Your instructor will tell you the locations of lab safety equipment, including
fire extinguishers, fire blanket, eyewash stations, and emergency showers.
Familiarize yourself with the location and operation of this equipment.
If anything is splashed into your eyes, wash your eyes thoroughly and
immediately. Tell your lab instructor what happened.
Notify your instructor of any allergies to latex, chemicals, stings, or other
substances.
If you break any glassware, do not pick up the pieces of broken glass with
your hands. Instead, use a broom and dustpan to gather the broken glass.
Ask your instructor how to dispose of the glass.
Unless told by your instructor to do otherwise, work only during regular,
assigned hours when the instructor is present. Do not conduct any unau-
thorized experiments; for example, do not mix any chemicals without your
instructor’s approval.
Do not leave any experiments unattended unless you are authorized by your
instructor to do so. If you leave your work area, slide your chair under the lab
table. Keep walkways and desktops clean and clear by putting books, back-
packs, and so on along the edge of the room, in the hall, in a locker, or in an
adjacent room. Keep your work area as clean and uncluttered as possible.
Don’t touch or put anything on the surface of hotplates unless told to do
so. Many types of hotplates have no visible sign that they are hot. Assume
they are hot.
Know how to use the equipment in lab. Most of the equipment is expen-
sive; you may be required to pay all or part of its replacement cost. Keep
water and solutions away from equipment and electrical outlets. Report
malfunctioning equipment to your instructor. Leave equipment in the same
place and condition that you found it. If you have any questions about or
problems with equipment, contact your instructor.
Know what to do and whom to contact if there is an emergency. Know the
fastest way to get out of the lab. Immediately report all injuries—no matter
how minor—to your instructor. Seek medical attention immediately if needed.
If any injury appears to be life-threatening, call 911 immediately.
At the end of each lab, clean your work area, wash your hands thoroughly
with soap, slide your chair under the lab table, and return all equipment
and supplies to their original locations. Do not remove any chemicals or
equipment from the lab.

xiv W–3
 Name _________________________________________

Lab Section _________________________________________

Your lab instructor may require that you submit this page at the end of today’s lab.

1. In the space below, write an analysis of the data shown in figure 1.

After completing table 1, read and sign this statement:

2. I have read and I understand and agree to abide by the laboratory safety rules described in this exercise and discussed
by my instructor. I know the locations of the safety equipment and materials. If I violate any of the laboratory safety
rules, my instructor will lower my grade and/or remove me from the lab.

____________________________________________
Signature

____________________________________________
Name (printed)

____________________________________________
Date

W–4 xv
This page intentionally left blank
 EXER CISE

Scientific Method
The Process of Science 1
Learning Objectives
By the end of this exercise you should be able to:
1. Define science and understand the logic and sequence of the scientific method.
2. Develop productive observations, questions, and hypotheses about the natural world.
3. Calculate the range, mean, and standard deviation for a set of replicate measurements.
4. Design and conduct a controlled experiment to test a null hypothesis.
5. Understand the difference and connection between a hypothesis and a scientific theory.

Please visit connect.mheducation.com to review online resources tailored to this lab.

T he word science brings to mind different things to dif-

ferent students. To some students, science is a textbook.
To others, it’s a microscope, a dissected frog, or a course that
you take. In fact, science is none of those things. Some defini-
tions are more useful than others, but for biological research
a good definition of science is the orderly process of posing
and answering questions about the natural world through
repeated and unbiased experiments and observations. This
definition emphasizes that science is a process rather than a
book, course, or list of facts. Science is not a “thing.” It’s a
way of thinking about and doing things—a powerful way of
learning and knowing about the natural world (fig. 1.1). Sci-
ence has helped us understand phenomena such as climate
change and the coronavirus 2019 (COVID-19) pandemic,
produce antibiotics and other drugs, and improve our lives in
many, many ways.
Our definition also emphasizes that people do science
by asking questions and then doing experiments to answer
those questions. Questions and curiosity are part of human
nature, and science is a human activity. Like any human
task, it takes practice to do science effectively.
Finally, our definition emphasizes that science is a
tool for learning about the natural world. It is ineffective
for moral choices, ethical dilemmas, and untestable ideas.
For example, the scientific method cannot tell us if pollution
is good or bad. It can tell us the environmental consequences
of pollution, but whether these consequences are “good” or
“bad” is a judgment that we make based on our values or
goals, not on science. Although this is an important limi-
tation of the scientific method, science remains one of the
most powerful ways of understanding our world.

Question 1 ​ ​
What practices besides science are used among world
­cultures to learn about the natural world?

The questioning and testing inherent in science sys-

Morsa Images/DigitalVision/Getty Images
Figure 1.1 Science is a process of learning about the natural world.
Doing experiments that involve gathering repeated and unbiased measure-
ments (data) is at the heart of testing hypotheses and answering questions.
tematically sift through natural variation to find underlying
patterns. The natural world includes much variation, and
learning biology would be relatively easy if simple obser-
vations accurately revealed patterns of the natural world.
But they usually don’t—nature is too complicated to rely
solely on simple observation. We can certainly learn much

1–1 Scientific Method 1

about our environment just by looking around us, but casual
observations are often biased and misleading because nature
varies from time to time and from organism to organism.
Biologists need a structured and repeatable process for test-
ing their ideas about the variation in nature. Science is that
process.
Question 2 ​ ​
What factors might be responsible for variation in measure-
ments of traits such as the heights of 10-year-old pine trees
or the kidney filtration rates of 10 replicate lab-mice?
The process of science deals with variation primar-
ily through an organized sequence of steps that maintains
as much objectivity and repeatability as possible. Although
these loosely organized steps, sometimes called the
scientific method, vary from situation to situation, they are
remarkably effective for research and problem solving. The
typical steps in the process of science are:
∙∙ Make insightful observations
∙∙ Pose and clarify testable questions
∙∙ Formulate hypotheses
∙∙ Do experiments to gather data
∙∙ Quantify the data
∙∙ Test the hypotheses
∙∙ Refine hypotheses and retest
∙∙ Answer the questions and make conclusions
DEVELOPMENT OF OBSERVATIONS,
QUESTIONS, AND HYPOTHESES
Make Insightful Observations
Good scientists make insightful observations. But that’s not
as easy as it seems. Consider these two observations:
Observation 1: There are fewer elk in Yellowstone
National Park than there used to be.
Observation 2: The density of elk in Yellowstone
National Park has declined during the
consecutive dry years since the reintro-
duction of the native wolf population.
Which of these two observations is stronger and more
­useful? Both of them may be true, but the second one is
much more insightful because it provides a context to the
observation that the elk population is declining. It also sug-
gests a relevant factor—that is, the reintroduction of the
wolf population—as a productive topic for investigation.
It also suggests a relationship between density of the elk
population and the variation in the local environment.
2 EXERCISE 1
Procedure 1.1 Make insightful observations
1.Consider the following two observations.
Observation 1: Fungi often grow on leftover food.
Observation 2: Fungi such as mold and yeast grow more
on leftover bread than on leftover meat.
Which of the above observations is more useful for
­further investigation? Why?


SAFETY FIRST Before coming to lab, you were asked
to read this exercise so you would know what to do
and be aware of safety issues. Briefly list the safety
issues associated with today’s procedures. If you have
questions about these issues, contact your laboratory
assistant before starting work.
Record the more insightful of the two observations on
Worksheet 1 on page 9.
2. Consider this observation: Pillbugs (sometimes called
roly-poly bugs) often find food and shelter where
fungi are decomposing leaf litter (fig. 1.2).
For this example we are interested in whether
­pillbugs are attracted to leaves or to fungi (including
yeasts) growing on the leaves’ surfaces.
Observation 1: Pillbugs often hide under things.
Propose a more productive observation.
Observation 2:
Record Observation 2 on Worksheet 2 on page 10.
You may revise this later.
Pose and Clarify Testable Questions
Productive observations inspire questions. Humans think in
terms of questions rather than abstract hypotheses or numbers.
But phrasing a good question takes practice and experience,
and the first questions that capture our attention are usually
general. For example, “Which nutrients can yeast most readily
metabolize?” is a general question that expands the observa-
tion posed in procedure 1.1. This question is broadly appli-
cable and is the type of question that we ultimately want to
understand. Enter this as the General Question in Worksheet 1.
Broad questions are important, but their generality
often makes them somewhat vague. The best questions for
the process of science are specific enough to answer clearly.
Therefore, scientists usually refine and subdivide broad
questions into more specific ones. For example, a more spe-
cific question is “What classes of biological molecules are
most readily absorbed and metabolized by yeast?” Enter this
as Specific Question 1 in Worksheet 1.
1–2

©BiologyImaging.com
Figure 1.2 Pillbugs are excellent experimental organisms to test
hypotheses about microenvironments, such as those under logs and
within leaf litter. Pillbugs are readily available and easily cultured in
the lab (10×).
A further clarification might be “Does yeast absorb
and metabolize carbohydrates better than it absorbs and
metabolizes proteins?” This is a good, specific question
because it clearly refers to organisms, processes, and vari-
ables that are likely involved. It also suggests a path for
investigation—that is, it suggests an experiment. Enter this
as Specific Question 2 in Worksheet 1.
Question 3 ​ ​
Consider the questions “What color is your roommate’s
car?” and “How many legs do cats have?” To answer these
questions, would you use the scientific method, or would
you rely on observation? Why?
Procedure 1.2 Posing and refining questions
1. Examine the following two questions.
Question 1: Do songbird populations respond to the
weather?
Question 2: Do songbirds sing more often in warm
weather than in cold weather?
Which of those questions is more useful for further
investigation? Why?
2. Examine the following general question, and record it
in Worksheet 2.
General Question: What influences the distribution of
pillbugs?
Propose a specific question that refers to the food of
pillbugs as a variable, and record it here and in
Worksheet 2. Know that you may revise this later.
1–3
Specific Question 1
Propose a more specific question that refers to pillbugs
eating leaves, as opposed to pillbugs eating fungi grow-
ing on leaves. Record this question here and in Work-
sheet 2. Know that you may revise this later.
Specific Question 2
Formulate Hypotheses
Well-organized experiments to answer questions require that
questions be restated as testable hypotheses. A hypothesis is
a statement that clearly states the relationship between bio-
logical variables. A good hypothesis identifies the organism
or process being investigated, identifies the variables being
recorded, and implies how the variables will be compared.
A hypothesis is a statement rather than a question, and an
analysis of your experimental data will ultimately determine
whether you accept or reject your hypothesis. Remember
that even though a hypothesis can be falsified, it can never
be proved true.
Accepting or rejecting a hypothesis, with no middle
ground, may seem like a rather coarse way to deal with ques-
tions about subtle and varying natural processes. But using
controlled experiments to either accept or reject a hypothesis
is effective. The heart of science is gathering and analyzing
experimental data that lead to rejecting or accepting hypoth-
eses relevant to the questions we want to answer.
In this exercise, you are going to do science as you
investigate yeast nutrition and then experiment with food
choice by pillbugs. As yeast ferments its food, CO2 is pro-
duced as a by-product. Therefore, we can measure the growth
of yeast by measuring the production of CO2 (fig. 1.3).
A hypothesis related to our question about the growth
of yeast might be:
H0: CO2 production by yeast fed sugar is not signifi-
cantly different from the CO2 production by yeast
fed protein.
A related alternative hypothesis can be similarly stated:
Ha: Yeast produces more CO2 when fed sugar than
when fed protein.
Figure 1.3 These tubes of
yeast are fermenting nutrients
provided in solution. The CO2
produced by the yeast accumu-
lates at the top of the test tubes
and indicates that yeast’s rate of
metabolism. From left to right,
the tubes include a control with
no added nutrients, a tube with
low nutrients, and a tube with
high nutrients.
©BiologyImaging.com
Scientific Method 3
The first hypothesis (H0) is a null hypothesis because
it states that there is no difference. This is the most com-
mon way to state a clear and testable hypothesis. (Your
instructor may elaborate on why researchers state and test
null hypotheses more effectively than alternative hypoth-
eses.) Researchers usually find it more useful to associate
statistical probabilities with null hypotheses rather than
with alternative hypotheses. Enter the null hypothesis into
Worksheet 1.
A well-written null hypothesis is useful because it is
testable. In our experiment, the null hypothesis (1) speci-
fies yeast as the organism, population, or group that we
want to learn about; (2) identifies CO2 production as the
variable being measured; and (3) leads directly to an experi-
ment to evaluate variables and compare means of replicated
measurements.
Procedure 1.3 Formulating hypotheses
1. Examine the following two hypotheses:
Hypothesis 1: Songbirds sing more when the weather
is warm.
Hypothesis 2: The number of bird songs heard per hour
during daylight temperatures above
80°F (27°C) is not significantly different
from the number heard per hour at tem-
peratures below 80°F (27°C).
Which of these hypotheses is more useful for further
investigation? Why?
Which of these hypotheses is a null hypothesis? Why?
2. Examine the following hypothesis.
Hypothesis 1: Pillbugs prefer leaves coated with a
thin layer of yeast.
Propose a more effective null hypothesis. Be sure
that it is a null hypothesis, that it is testable, and
that it includes the parameter you will control in an
experiment.
Hypothesis 2 (H0):
Enter your null hypothesis in Worksheet 2.
EXPERIMENTATION AND DATA ANALYSIS:
YEAST NUTRITION
Gather Experimental Data
To test our hypothesis about yeast growth, we must design
a controlled and repeatable experiment. The experiment
suggested by our specific question and hypothesis involves
offering sugar such as glucose to one population of yeast,
4 EXERCISE 1
offering protein to another population of yeast, and then
measuring their respective growth rates. Fortunately, yeast
grows readily in test tubes. As yeast grows in a closed,
anaerobic container, it produces CO2 in proportion to how
readily it uses the available food. CO2 production is easily
measured by determining the volume of CO2 that accumu-
lates at the top of an inverted test tube (fig. 1.3).
Experiments provide data that determine if a hypothesis
should be accepted or rejected. A well-designed experiment
links a biological response to different levels of the vari-
able being investigated. In this case, the biological response
is CO2 production, which indicates growth. The levels of
the variable are sugar and protein. These levels are called
treatments, and in our experiment they include glucose,
protein, and a control. For this experiment the treatment (i.e.,
independent) variable being tested is the type of food mol-
ecule (i.e., protein, sugar), and the response (i.e., dependent)
variable is the CO2 production that indicates yeast growth.
An experiment that compensates for natural variation
must be well designed. It should (1) include replications,
(2) test only one treatment variable, and (3) include controls.
Replications are repeated measures of each treatment under
the same conditions. Replications effectively deal with natu-
rally occurring variation. Usually the more replications, the
better. Your first experiment today will include replicate test
tubes of yeast, each being treated the same. Good design
tests only one treatment variable at a time.
Good experimental design also requires controls to
verify that the biological response we measure is a function
of the variable being investigated and nothing else. Controls
are standards for comparison. They are replicates with all of
the conditions of an experimental treatment except the treat-
ment variable. For example, if the treatment is glucose dis-
solved in water, then a control has only water (i.e., it lacks
only glucose, the treatment variable). This verifies that the
response is to glucose and not to the solvent. Controls vali-
date that our results are due only to the treatment variable.
Procedure 1.4 An experiment to determine the
effects of food type on yeast growth
1. Label 12 test tubes as C1–C4, G1–G4, and P1–P4.
See Worksheet 1.
2. To test tubes C1–C4 add 5 mL of water. These are
control replicates.
3. To test tubes G1–G4 add 5 mL of 5% glucose solu-
tion. These are replicates of the glucose treatment.
4. To test tubes P1–P4 add 5 mL of 5% protein solution.
These are replicates of the protein treatment.
5. Swirl the suspension of yeast until the yeast is distrib-
uted uniformly in the liquid. Then completely fill the
remaining volume in each tube with the yeast suspen-
sion that is provided.
6. For each tube, slide an inverted, flat-bottomed test tube
down over the yeast-filled tube. Hold the yeast-filled tube
1–4
 firmly against the inside bottom of the cover tube and
invert the assembly. Your instructor will demonstrate
how to slip this slightly larger empty tube over the top of
each yeast tube and invert the assembly. If done properly,
no bubble of air will be trapped at the top of the tube of
yeast after inversion.
7. Place the tubes in a rack and incubate them at 50°C.
8. Measure the height (mm) of the bubble of accumu-
lated CO2 after 10, 20, 40, and 60 minutes. Record
your results in Worksheet 1 and graph them here:
Height of CO2
Bubble (mm)
10 20 40 60
Time (min)
9. When you are finished, clean your work area and
dispose of the contents of each tube as instructed by
your lab instructor.
Test Your Predictions by Analyzing
the Experimental Data
Analysis begins with summarizing the raw data for biologi-
cal responses to each treatment. The first calculation is the
mean (x–), which is the average of a set of numbers (e.g.,
measurements) for replicates of each treatment and the con-
trol. That is, the mean is a single number that represents the
central tendency of the response variable. Later the mean of
each treatment will be compared to determine if the treat-
ments had different effects.
The second step in data analysis is to calculate varia-
tion within each set of replicates. The simplest measure
of variation is the range, which is the highest and low-
est values in a set of replicates. A wide range indicates
much variation in the data. The standard deviation (SD),
another informative measure of variation, summarizes
variation just as the range does, but the standard deviation
is less affected by extreme values. Refer to the box “Varia-
tion in Replicate Measures” to learn how to calculate the
standard deviation.
Question 4
Even the seemingly simple question “How tall are mature
males of the human species?” can be difficult to answer.
How would you best express the answer?
1–5
Procedure 1.5 Quantify and summarize the data
1. Examine your raw data in Worksheet 1.
2. Calculate the mean of the response variable (CO2
production) for the four control replicates. To calcu-
late the means for the four replicates, sum the four
values and divide by four. Record the mean for the
control replicates in Worksheet 1.
3. The CO2 production for each glucose and protein
replicate must be adjusted with the control mean.
This ensures that the final data reflect the effects of
only the treatment variable and not the solvent. Sub-
tract the control mean from the CO2 production of
each glucose replicate and each protein replicate, and
record the results in Worksheet 1.
4. Record in Worksheet 1 the range of adjusted CO2
production for the four replicates of the glucose treat-
ment and of the protein treatment.
5. Calculate the mean CO2 production for the four
adjusted glucose treatment replicates. Record the
mean in Worksheet 1.
6. Calculate the mean CO2 production for the four
adjusted protein treatment replicates. Record the
mean in Worksheet 1.
7. Refer to “Variation in Replicate Measures,” and cal-
culate the standard deviation for the four adjusted
glucose treatment values and for the four adjusted
protein treatment values. Record the two standard
deviations in Worksheet 1.
Test the Hypotheses
Our hypothesis about yeast growth is tested by comparing the
mean CO2 production by yeast fed glucose to the mean CO2
production by yeast fed protein. However, only determining
if one mean is higher than the other is not an adequate test
because natural variation will always make the two means at
least slightly different, even if the two treatments have the
same effect on yeast growth. Therefore, the means and the
variation about the means must be compared to determine if
the means are not just different but significantly different.
To be significantly different, the differences between means
must be due to the treatment and not just due to natural vari-
ation. If the difference is significant, then the null hypothesis
is rejected. If the difference is not significant, then the null
hypothesis is accepted. Testing for significant differences is
usually done with statistical methods.
Statistical methods calculate the probability that the
means are significantly different. But these complex calcu-
lations are beyond the scope of this exercise. We will use a
simpler method to test for a significant difference between
the means of our two treatments. We will declare that two
means are significantly different if the means plus or minus
1/2 of the standard deviation do not overlap.
Scientific Method 5
 Variation in Replicate Measures

– = the sample mean

Natural variation occurs in all processes of biology. This varia- x
tion will inevitably produce different results in replicated treat- xi = measurement of an individual sample
ments. One of the most useful measures of variation of values N

about the mean is standard deviation. It’s easy to calculate: The summation sign ( Σ ) means to add up all the squared
i=1
calculate the mean, calculate the deviation of each sample from deviations from the first one (i = 1) to the last one (i = N).
the mean, square each deviation, and then sum the deviations. The sum of squared deviations (10) divided by the num-
This summation is the sum of squared deviations. For example, ber of samples minus one (4 − 1 = 3) produces a value of
data for CO2 production by yeast in four replicate test tubes 10/3 = 3.3 mm2 (the units are millimeters squared). This is
might be 22, 19, 18, and 21 mm. The mean is 20 mm. the variance:
sum of squared deviations
CO2 Production (mm) Mean Deviation Deviation2 Variance =
N−1
22 20 2 4 The square root of the variance, 1.8 cm, equals the standard
19 20 −1 1 deviation
18 20 −2 4 SD = √Variance = √3.3 = 1.8
21 20 1 1
The standard deviation is often reported with the mean in state-
Sum of squared deviations = 10 ments such as, “The mean CO2 production was 20 ± 1.8 mm.”
The standard deviation helps us understand the spread or
The summary equation for the sum of squared deviations is
variation among replicated treatments. For example, if the
N
– 2 standard deviation is zero, all of the numbers in the set are
Sum of squared deviations = Σ (x
i=1
i
− x)
the same. A larger standard deviation implies that individual
where numbers are farther from the mean.
N = total number of samples

For example, consider these two means and their stan- Answer the Questions
dard deviations (SD):
The results of testing the hypotheses are informative, but
Meana = 10 SD = 5 Meanb = 20 SD = 10 it still takes a biologist with good logic to translate these
Meana − (½)SD = 7.5 Meanb − (½)SD = 15 results into the answers of our specific and general ques-
Meana + (½)SD = 12.5 Meanb + (½)SD = 25 tions. If your specific questions were well stated, then
answering them based on the results of your experiment and
Are Meana and Meanb significantly different according to our hypothesis testing should be straightforward.
test for significance? Yes they are, because 7.5 ↔ 12.5 does
not overlap 15 ↔ 25.

Procedure 1.7 Answering the questions: yeast

nutrition
Procedure 1.6 Testing hypotheses
1. Examine the results of hypothesis testing presented in
1. Consider your null hypothesis and the data presented
Worksheet 1.
in Worksheet 1.
2. Specific Question 2 was “Does yeast absorb and
2. Calculate the glucose mean − (½)SD and the glucose
metabolize carbohydrates better than it absorbs
mean + (½)SD. Record them in Worksheet 1.
and metabolizes proteins?” Enter your answer in
3. Calculate the protein mean − (½)SD and the protein Worksheet 1.
mean + (½)SD. Record them in Worksheet 1.
3. Does your experiment adequately answer this ques-
4. Do the half standard deviations surrounding the tion? Why or why not?
means of the two treatments overlap? Record your
answer in Worksheet 1.
5. Are the means for the two treatments significantly
different? Record your answer in Worksheet 1. 4. Specific Question 1 was “What classes of biological
6. Is your null hypothesis accepted? Or rejected? Record molecules are most readily absorbed and metabolized
your answer in Worksheet 1. by yeast?” Enter your best response in Worksheet 1.

6 EXERCISE 1 1–6
5. Does your experiment adequately answer Specific
Question 1? Why or why not?
6. The General Question was “Which nutrients can yeast
most readily metabolize?” After testing the hypoth-
eses, are you now prepared to answer this general
question? Why or why not?
EXPERIMENTATION AND DATA ANALYSIS:
FOOD PREFERENCE BY ​PILLBUGS
In the previous procedures you developed and recorded
observations, questions, and hypotheses concerning food
preference by pillbugs. Pillbugs may be attracted to dead
leaves as food, or they may be attracted to fungi growing on
the leaves as food. Leaves dipped in a yeast suspension can
simulate fungi growing on leaves. Use the following proce-
dures as a guide to the science of experimentation and data
analysis to test the hypothesis you recorded in Worksheet 2.
4. Calculate the range and standard deviation for your
treatments, and record them in Worksheet 2.
5. Test your hypothesis. Determine if the null hypoth-
esis should be accepted or rejected. Record the results
in Worksheet 2.
6. Answer the Specific Question 2, Specific Question 1,
and General Question posed in Worksheet 2.
Procedure 1.9 Answering the questions: food
preference by pillbugs
1. Examine the results of your hypothesis testing pre-
sented in Worksheet 2.
2. Enter your answer to Specific Question 2 in
Worksheet 2. Does your experiment adequately
answer this question? Why or why not?
3. Enter your best response to Specific Question 1
in Worksheet 2. Does your experiment adequately
answer this question? Why or why not?

Procedure 1.8 Design an experiment to test 4. After testing the hypotheses, are you now prepared to
food preference by pillbugs answer your General Question “What influences the
distribution of pillbugs?” Why or why not?
1. Design an experiment to test your hypothesis in
Worksheet 2 about food preference by pillbugs. To do
this, specify:
Experimental setup
Question 5
What are some examples of biological theories?
Treatment 1 to be tested

Treatment 2 to be tested

Control treatment Scientific Theories

Response variable Throughout this course you will make many predictions and
observations about biology. When you account for a group
of these observations with a generalized explanation, you
Treatment variable have proposed a scientific theory.
In science, as opposed to common usage, a theory is a
well-substantiated explanation of some aspect of the natural
Number of replicates world that usually incorporates many confirmed observa-
tional and experimental facts. A scientific theory makes pre-
Means to be compared dictions consistent with what we see. It is not a guess; on the
contrary, a scientific theory is widely accepted within the
scientific community—for example, the germ theory claims
2. Conduct your experiment and record the data in
that certain infectious diseases are caused by microorgan-
Worksheet 2.
isms. Scientific theories do not become facts; scientific the-
3. Analyze your data. Record the control means and ories explain facts.
adjusted treatment-means in Worksheet 2.

1–7 Scientific Method 7

 INQUIRY-BASED LEARNING
How do changes in temperature affect the production of CO2 by yeast?
Observation: Fermentation of nutrients by yeast produces
CO2, and the production rate of this CO2 can be used to mea-
sure growth of the yeast. In this lab you’ve already investi-
gated how the production of CO2 is affected by different
nutrients (i.e., sugar, protein). Here you’ll investigate another
variable: temperature.
Question: How is the production of CO2 by yeast affected by
temperature?
a. Establish a working lab group and obtain Inquiry-Based
Learning Worksheet 1 from your instructor.
b. Discuss with your group well-defined questions rele-
vant to the preceding observation and question. Choose
and record your group’s best question for investigation.
c. Translate your question into a testable hypothesis.
Record this hypothesis.
d. Outline on Worksheet 1 your experimental design and sup-
plies needed to test your hypothesis. Ask your instructor
to review your proposed investigation.
e. Conduct your procedures, record your data, answer
your question, and make relevant comments.
f. Discuss with your instructor any revisions to your questions,
hypothesis, or procedures. Repeat your work as needed.

Questions for Further Study and Inquiry

1. Consider the traits of science. Newspaper articles often refer to a discovery as “scientific” or claim that something has
been proved “scientifically.” What is meant by this description?

2. Experimental results in science are usually reviewed by other scientists before they are published. Why is this done?

3. Have all of our discoveries and understandings about the natural world been the result of testing hypotheses and
applying the scientific method? How so?

4. Suppose that you hear that two means are significantly different. What does this mean? Can means be different but
not significantly different? Explain your answer.

5. Why do scientists refrain from saying, "These results prove that . . ."?

6. How can science be used to address “big” issues such as climate change and COVID-19?

7. Some people dismiss evolution by natural selection as being “only a theory.” Biologists often respond that yes,
evolution is a scientific theory. What does this mean?

8. A hallmark of a scientific theory is that it is falsifiable. What does this mean, and why is it important?

9. Why is there no role for superstition in science?

8 EXERCISE 1 1–8
 Worksheet 1 The Process of Science: Nutrient Use by Yeast

OBSERVATION

QUESTIONS
General Question:

Specific Question 1:

Specific Question 2:

HYPOTHESIS H0:

EXPERIMENTAL DATA: Nutrient Use by Yeast

Treatments Treatments Minus Control x–

Control Glucose Protein Glucose CO2 Protein CO2

CO2 CO2 CO2 Production Production
Production Production Production Adjusted for Adjusted for
Replicate (mm) Replicate (mm) Replicate (mm) the Control –x the Control –x

C1 ______ G1 ______ P1 ______ ______ ______

C2 ______ G2 ______ P2 ______ ______ ______
C3 ______ G3 ______ P3 ______ ______ ______
C4 ______ G4 ______ P4 ______ ______ ______

Control x– = ______ Protein x– = ______

Glucose x– = ______ Protein range = ______ − ______
Glucose range = ______ − ______ Protein SD = ______
Glucose SD = ______

TEST HYPOTHESIS
Glucose x– − (½)SD = Protein x– − (½)SD =

Glucose x– + (½)SD = Protein x– + (½)SD =

Do the half standard deviations surrounding the means of the two treatments overlap? Yes No

Are the means for the two treatments significantly different? Yes No

Is the null hypothesis accepted? or rejected?

ANSWER QUESTIONS
Answer to Specific Question 2

Answer to Specific Question 1

Answer to General Question

1–9 Scientific Method 9

 Worksheet 2 The Process of Science: Food Preference by Pillbugs

OBSERVATION

QUESTIONS
General Question:

Specific Question 1:

Specific Question 2:

HYPOTHESIS H0:

EXPERIMENTAL DATA: Food Preference by Pillbugs

Treatments Treatments Minus Control x–

Treatment 1 Treatment 2
Adjusted for Adjusted for
Replicate Control Replicate Treatment 1 Replicate Treatment 2 the Control –x the Control –x

1 1 1
2 2 2
3 3 3
4 4 4

Control x– = Treatment 2 x– =
Treatment 1 x– = Treatment 2 range = −
Treatment 1 range = − Treatment 2 SD =
Treatment 1 SD =

TEST HYPOTHESIS
Treatment 1 x– − (½)SD = Treatment 2 x– − (½)SD =

Treatment 1 x– + (½)SD = Treatment 2 x– + (½)SD =

Do the half standard deviations surrounding the means of the two treatments overlap? Yes No

Are the means for the two treatments significantly different? Yes No

Is the null hypothesis accepted? or rejected?

ANSWER QUESTIONS
Answer to Specific Question 2

Answer to Specific Question 1

Answer to General Question

10 EXERCISE 1 1–10
 EXER CISE

Measurements in Biology
The Metric System and Data Analysis 2
Learning Objectives
By the end of this exercise you should be able to:
1. Understand the difference between accuracy and precision in measurements.
2. Identify the metric units used to measure length, volume, mass, and temperature.
3. Measure length, volume, mass, and temperature in metric units.
4. Convert one metric unit to another (e.g., grams to kilograms).
5. Use measures of volume and mass to calculate density.
6. Practice the use of simple statistical calculations such as mean, median, range, and standard ­deviation.
7. Analyze sample data using statistical tools.

Please visit connect.mheducation.com to review online resources tailored to this lab.

E very day we’re bombarded with numbers and measure-

ments. They come at us from all directions, including while
we’re at the supermarket, gas station, golf course, and pharmacy,
To help you check your answers, consider an analogy
involving shooting arrows at a bull’s-eye target (fig. 2.1). In
this analogy, each arrow (represented by a dot on the tar-
as well as while we’re in our classrooms and kitchens. Virtually get) would represent a measurement. Accuracy would be the
every package that we touch is described by a measurement. High accuracy, High precision,
Scientists use a standard method to collect data as well low precision low accuracy
as use mathematics to analyze measurements. We must mea-
sure things before we can objectively describe what we are
observing, before we can experiment with biological pro-
cesses, and before we can predict how organisms respond,
adjust to, and modify their world. Once we have made our
measurements, we can analyze our data and look for varia-
tion and the sources of that variation. Then we can infer the
causes and effects of the biological processes that interest us.

ACCURACY AND PRECISION (a) (b)

Low precision, High accuracy,
Scientists strive to make accurate, precise measurements. The low accuracy high precision
accuracy of a group of measurements refers to how closely
the measured values agree with the true or correct value. In
contrast, the precision of a group of measurements refers to
how closely the measurements agree with each other. That
is, precision is the degree to which the measurements pro-
duce the same results, regardless of their accuracy. Scientists
strive to make measurements that are accurate and precise.
Question 1
a. Can measurements be accurate but not precise? Explain.
(c) (d)

Figure 2.1 Precision and accuracy. Measurements can be

b. Can measurements be precise but not accurate? Explain. (a) accurate but not precise, (b) precise but not accurate, (c) neither
precise nor accurate, or (d) both precise and accurate.

2–1 Measurements in Biology 11

closeness of the arrows to the center of the target; arrows The following conversions will help give you a sense
closest to the bullseye would be most accurate. Precision of how some common English units are related to their met-
would be the size of the cluster of arrows, regardless of how ric equivalents:
close they are to the center of the target. 1 inch = 2.5 centimeters
1 foot = 30 centimeters
THE METRIC SYSTEM
1 yard = 0.9 meter
Scientists throughout the world use the metric system to make 1 mile = 1.6 kilometers
measurements. The metric system is also used in everyday 1 fluid ounce = 30 milliliters
life virtually everywhere except the United States. With few 1 pint = 0.47 liter
exceptions (e.g., liter bottles of soda), most measurements in
1 quart = 0.95 liter
the United States use the antiquated ­English system of pounds,
inches, feet, and so on. Check with your instructor about bring- 1 gallon = 3.8 liters
ing to class common grocery store items with volumes and 1 cup = 0.24 liter
weights in metric units or examining those items on display. To learn more about these conversions, see Appendix II.
The most modern form of the metric system is called This exercise will introduce you to making metric
the International System of Units (abbreviated SI). Metric measurements of length, mass, volume, and temperature.
measurement is used worldwide in science to improve com- During this lab, you should spend your time making mea-
munication in the scientific community. Scientists make surements, not reading background information. Therefore,
all of their measurements in the metric system; they do not before lab, read this exercise carefully to familiarize your-
routinely convert from one system to another. When scien- self with the basic units of the metric system.
tists have mixed metric units with English units, the results Metric units commonly used in biology include:
have often been confusing, and have sometimes been disas-
trous. For example, in 1999, the $125-million Mars Climate meter (m)—the basic unit of length
Orbiter was approaching Mars to study the planet’s climate. liter (L)—the basic unit of volume
Lockheed Martin Astronautics, which built the spacecraft, kilogram (kg)—the basic unit of mass
gave NASA critical flight information in English units, degrees Celsius (°C)—the basic unit of temperature
but software aboard the orbiter expected the data in metric
units. As a result, the orbiter was sent into, rather than safely Unlike the English system with which you are already familiar,
above, the Mars atmosphere, where it disintegrated. the metric system is based on units of ten. This simplifies con-
versions from one metric unit to another (e.g., from kilometers
to meters). This base-ten system is similar to our monetary sys-
Hints for Using the Metric System
tem, in which 10 cents equal a dime, 10 dimes equal a d­ ollar,
and so forth. Units of 10 in the metric system are indicated by
1. Use decimals, not fractions (e.g., 2.5 m, not 21/2 m).
Latin and Greek prefixes placed before the base units:
2. Express measurements in units requiring only a few
decimal places. For example, 0.3 m is more easily
Prefix
manipulated and understood than 300000000 nm.
(Latin) Division of Metric Unit
3. When measuring pure water, the metric system offers
an easy and common conversion from volume mea- deci (d) 0.1 10−1 = tenth
sured in liters to volume measured in cubic meters to centi (c) 0.01 10−2 = hundredth
mass measured in grams: 1 mL = 1 cm3 = 1 g. milli (m) 0.001 10−3 = thousandth
4. The metric system uses symbols rather than abbre- micro (µ) 0.000001 10−6 = millionth
viations. Therefore, do not place a period after metric
nano (n) 0.000000001 10−9 = billionth
symbols (e.g., 1 g, not 1 g.). Use a period after a
­symbol only at the end of a sentence. pico (p) 0.000000000001 10−12 = trillionth
5. Do not mix units or symbols (e.g., 9.2 m, not 9 m 200 mm). Prefix
6. Metric symbols are always singular (e.g., 10 km, not (Greek) Multiple of Metric Unit
10 kms). deka (da) 10 101 = ten
7. Except for degrees Celsius, always leave a space hecto (h) 100 102 = hundred
­between a number and a metric symbol (e.g., 20 mm,
kilo (k) 1000 103 = thousand
not 20mm; 10°C, not 10° C).
8. U
 se a zero before a decimal point when the number mega (M) 1000000 106 = million
is less than one (e.g., 0.42 m, not .42 m). giga (G) 1000000000 109 = billion
tera (T) 1000000000000 1012 = trillion

12 EXERCISE 2 2–2
Thus, multiply by
Procedure 2.1 Make metric measurements of
0.01 to convert centimeters to meters length and area
0.001 to convert millimeters to meters Most biologists measure lengths with metric rulers or
1000 to convert kilometers to meters metersticks.
0.1 to convert millimeters to centimeters 1. Examine intervals marked on the metric rulers and
For example, there are 10 millimeters per centimeter. There- metersticks available in the lab.
fore, to convert 62 centimeters to millimeters, 2. Make the following measurements. Be sure to include
10 mm units for each measurement.
62 cm × = 620 mm
cm Length of this page
In these conversion equations, the units being converted from Width of this page
(in this case, centimeters) cancel out, leaving you with the Area of this page
desired units (in this case, millimeters). Also note that when (Area = Length × Width)
units are converted to smaller units, the number associated
with the new units will increase, and vice versa. For exam- Your height
ple, 620 meters = 0.620 kilometer = 620,000 millimeters = Thickness of this manual
62,000 centimeters. Height of a 200-mL beaker
Question 2 Height of your chair
a. Make the following metric conversions: Length of your cell phone
1 meter = centimeters = millimeters
92.4 millimeters = meters = centimeters
Question 3
82 centimeters = meters = millimeters
What are some potential sources of error in your
3.1 kilograms = grams = milligrams
measurements?
281 milliliters = liters = deciliters

b. The spikes on a COVID-19 viral particle are about 11 nm

long. How long, in micrometers, are these spikes?

Length and Area

The meter (m) is the basic unit of length. Units of area are Volume
squared units (i.e., two-dimensional) of length.
Volume is the space occupied by an object. Units of volume
1 m = 100 cm = 1000 mm = 0.001 km = 1 × 10−3 km are cubed (i.e., three-dimensional) units of length. The liter
1 km = 1000 m = 103 m (L) is the basic unit of volume.
1 cm = 0.01 m = 10−2 m = 10 mm 1 L = 1000 cm3 = 1000 mL
470 m = 0.470 km 1 L = 0.1 m × 0.1 m × 0.1 m
1 cm2 = 100 mm2 (i.e., 10 mm × 10 mm = 100 mm2) 1 cm3 = 0.000001 m3

To help you appreciate the size of each of these units, here To help you appreciate the size of each of these units,
are the lengths and areas of some familiar objects: here are the volumes of some familiar objects:
Length Chicken egg 60 mL
Housefly 0.5 cm Coke can 355 mL
Diameter of penny 1.9 cm One breath of air 500 cm3
Diameter of baseball 7.4 cm
Scientists often measure volumes with pipets and graduated
Soda can 12.2 cm
cylinders. Pipets are used to measure small volumes, typi-
Toyota Camry 4.7 m
cally 25 mL or less. Liquid is drawn into a pipet using a bulb
Mt. Everest 8848 m
or pipet pump (fig. 2.2). Never pipet by mouth.
Area Graduated cylinders are used to measure larger vol-
Credit card 46 cm2 umes. To appreciate how to make a measurement accurately,
Total skin area of adult human male 1.8 m2 pour 40–50 mL of water into a 100-mL graduated cylinder,
Ping-pong table 4.18 m2 and observe the interface between the water and air. This
Surface area of human lungs 80 m2 interface, called the meniscus, is curved because of surface
Football field (goal line to goal line) 4459 m2 tension and the adhesion of water to the sides of the cylinder.
Central Park (New York City) 3.4 km2 When measuring the liquid in a cylinder such as a graduated

2–3 Measurements in Biology 13

 cylinder, always position your eyes level with the meniscus
and read the volume at the lowest level (fig. 2.3).

Procedure 2.2 Make metric measurements

of volume
1. Biologists often use graduated cylinders to measure
volumes. Locate the graduated cylinders available in
the lab to make the following measurements. Deter-
mine what measurements the markings on the gradu-
ated cylinder represent. Be sure to include units for
each measurement.
2. Measure the volume (in mL) needed to fill a cup
(provided in the lab).
3. Measure the volume (in L) needed to fill a gallon.

Procedure 2.3 Measure the volume of a solid

object by water displacement
1. Obtain a 100-mL graduated cylinder, a thumb-sized
rock, and a glass marble.
2. Fill the graduated cylinder with 70 mL of water.
 ©BiologyImaging.com
3. Gently submerge the rock in the graduated cylinder.
Figure 2.2 A pipet is used to extract and dispense volumes of liq- Notice that the volume of the contents rises.
uid. A suction device (shown in green on the left) draws fluid into the
pipet, and graduated markings on the pipet allow precise measurement 4. Carefully observe the meniscus of the fluid and
of a fluid’s volume. Never use your mouth to suck fluid into a pipet. record its volume.
5. Calculate and record the volume of the rock by sub-
tracting the original volume (70 mL) from the new
volume.
Rock volume
6. Repeat steps 2–5 to measure and record the volume
of the marble.
Marble volume

Biologists use pipets to measure and transfer small volumes

of liquid from one container to another. The following pro-
cedure will help you appreciate the usefulness of pipets.
improper
position

meniscus proper Procedure 2.4 Learn to use a pipet

reading 20 mL position
1. Add approximately 100 mL of water to a 100-mL beaker.
2. Use a 5-mL pipet with a bulb or another filling d­ evice
improper provided by your instructor to remove some water
position from the beaker.
3. Fill the pipet to the zero mark.
4. To read the liquid level correctly, your eye must be
Figure 2.3 When measuring the volume of liquid in a graduated directly in line with the bottom of the meniscus.
cylinder, always position your eye at the bottom of the meniscus. The 5. Release the liquid into another container.
correct volume is 20 mL.

14 EXERCISE 2 2–4
 a c d

Calibration
(tare) Button

Power Switch

©BiologyImaging.com ©BiologyImaging.com
b
Figure 2.4 Biologists use balances to measure mass. (A) The parts of a triple-beam balance include the (a) zero-adjustment knob, (b) measuring
pan, (c) movable masses on horizontal beams, and (d) balance marks. (B) A top-loading balance has a measuring pan, a power switch, and a zero cali-
bration (“tare”) button.

Question 4 marked with graduations: the closest beam has 0.1-g gradu-
What volume of liquid did you measure? ations, the middle beam has 100-g graduations, and the
­farthest beam has 10-g graduations.

Procedure 2.5 Make metric measurements of mass

Mass 1. If you’re using a triple-beam balance: Before mak-
The kilogram (kg) is the basic unit of mass. A kilogram is 1 ing any ­measurements, clean the weighing pan and
approximately equal to the mass of 1000 cubic centimeters move all of the suspended weights to the far left. The
(cm3) of water at 4°C. Similarly, balance marks should line up to indicate zero grams;
if they do not, turn the adjustment knob until they
1 kg = 1000 g = 103 g 1 mg = 0.001 g = 10−3 g do. Measure the mass of an object by placing it in
Note that the kilogram is the only base-unit of SI that the center of the weighing pan and moving the sus-
includes a prefix (“kilo,” symbol “k”) as part of its name pended masses until the beams balance. The mass of
(see Appendix II). Here are the approximate masses of some the object is the sum of the masses indicated by the
familiar objects: weights on the three beams.
2. If you’re using an electronic balance: Turn on the bal-
Housefly 12 mg Human heart 300 g
ance and let it warm up for 5 minutes. Wait until the
Hummingbird 1.6 g Basketball 0.62 kg
display reads 0.0 g; if the display does not read 0.0 g,
Ping-pong ball 2.45 g $1 bill 1g
press the “tare” button to reset the display to 0.0 g. If you
Quarter 6.25 g Penny 2.5 g
are weighing an object such as a coin or pencil, place the
9V battery 40 g
object on the measuring pan. After the display has stabi-
Biologists usually measure mass with a top-loading bal- lized, read and record the object’s mass.
ance or a triple-beam balance (fig. 2.4). Locate the triple- 3. If you are weighing a liquid, powder, or similar
beam balances or top-loading electronic balances in the lab. specimen, place an empty beaker (in which you will
Triple-beam balances get their names from their three hori- place the liquid) or a piece of weighing paper (on
zontal beams. Suspended from each of the three beams are which you will place the powder) on the balance’s
movable masses. Each of the three beams of the balance is measuring pan. After the display has stabilized,
press the “tare” button to reset the display to 0.0 g.
Place the liquid in the beaker (or the powder on the
1 Remember that mass is not necessarily synonymous with weight. Mass mea-
sures an object’s potential to interact with gravity, whereas weight is the force weighing paper). After the display has stabilized,
exerted by gravity on an object. Thus, a weightless object in outer space has the read and record the mass.
same mass as it has on earth.

2–5 Measurements in Biology 15

4. Measure the masses of the following items. Be sure Temperature
to include units for each measurement. Temperature is the measure of the kinetic energy of
Penny ­molecules—that is, the amount of heat in a system. Biolo-
Paper clip gists measure temperature with a thermometer calibrated in
degrees Celsius (°C). The Celsius scale is based on water
Pencil freezing at 0°C and boiling at 100°C. You can interconvert
Rock (used in procedure 2.3) °C and degrees Fahrenheit (°F) by using the formula 5(°F) =
100-mL beaker (empty) 9(°C) + 160. Here are some typical temperatures:
100-mL beaker containing 50 mL of water −20°C temperature in a freezer
−18°C mixture of ice and salt
0°C water freezes
Question 5 4°C temperature in a refrigerator
a. Density is mass per unit volume. Use data that you’ve 22°C room temperature
gathered to determine the density of water at room 30.6°C butter melts
temperature. 37°C human body temperature
40°C a hot summer day
Density of water = (mass/volume) = 50°C hottest day on record in Phoenix, AZ
71°C flash pasteurization of milk
b. What is the density of the wooden pencil? Does it 75°C hot coffee
float? Why? 100°C water boils
260°C broiler temperature

Procedure 2.6 Make metric measurements

of temperature
c. What is the density of the rock? Does it sink? Why? 1. Obtain a thermometer in the lab. Handle the ther-
mometer with care. If it breaks, notify your instructor
immediately.

Significant Figures

Let’s suppose that you’re measuring the length of a bone, as
shown in figure 2.5. How would you record this length—as 8 cm?
8.3 cm? 8.33 cm? 8.33333 cm? To answer this question, you
need to know something about significant figures.
Significant figures are the number of figures required to
record a measurement so that only the last digit in the number
is in doubt. For example, if the ruler you’re using is calibrated
only in centimeters and you find that the object you’re measur-
ing is between 8 and 9 cm long (fig. 2.5), then you should esti-
mate your measurement only to a tenth of a centimeter. That is,
a measurement of 8.3 cm is acceptable, but 8.33 is not because
it implies a precision that did not exist in the equipment you
used to make the measurement. If, however, your ruler was
calibrated in millimeters, then 8.33 cm would be acceptable.
Remember this: When recording measurements, include all of
the digits you are sure of plus an estimate to the nearest one-
tenth of the next smaller digit.
Here are some other guidelines for using the correct
number of significant figures in your measurements:
∙∙ When adding or subtracting measurements, the answer
should have no more precision than the measurement
having the least number of significant figures. For exam-
ple, suppose the air temperature in an incubator drops from
8.663°C to 8.2°C. This is a difference of 8.663°C – 8.2°C
= 0.5°C, not 0.463°C. If the second temperature reading
had been 8.200°C, then the correct answer would have been
0.463°C.
∙∙ When converting measurements from one set of units
to another, do not introduce precision that is not present
in the first number. For example, 8.3 cm = 83 mm, not
83.0 mm.
∙∙ When manipulating two measurements simultaneously, the
precision of the final measurement should not exceed that
of the least number of significant figures. For example, the
calculation for the mass of 17.2 mL of water is 17.2 mL ×
0.997821 g mL–1 = 17.2 g, not 17.162521 g.
6 7 8 9
cm
Figure 2.5 How long is this
bone? 8 cm? 8.3 cm? 8.33 cm?

16 EXERCISE 2 2–6
 is impossible, so you must choose apples that represent all
Rounding Numbers
of the other apples—that is, you must be working with a
representative sample. A statistical analysis of those sam-
Do not change the value of the last significant digit if that
ple ­apples reduces the sample values to a few characteristic
digit is followed by a number that is less than 5. For exam-
­measurements (e.g., mean mass). As you increase the size
ple, if two significant figures are required, 6.449 rounds to
of the sample, these characteristic measurements provide an
6.4, 66.449 rounds to 66, 66.641 rounds to 67, and 6.591
ever-improving estimation of what is “typical.”
rounds to 6.6. Here is how an original measurement of
There are a variety of software programs that perform
49.5149 rounds to various numbers of significant figures:
statistical analyses of data; all you have to do is enter your data
Five significant figures: 49.515
into a spreadsheet, select the data that you want to analyze,
Four significant figures: 49.51 and perform the analysis. Although these software packages
Three significant figures: 49.5 save time and can increase accuracy, you still need to under-
Two significant figures: 50 stand a few of the basic variables that you’ll use to understand
your numerical data. We’ll start with the mean and median:
One significant figure: 50
Statisticians disagree on what to do when the number follow- The mean is the arithmetic average of a group of measurements.
ing the last significant figure is exactly 5, as in 89.5 (and, in Chance errors in measurements tend to cancel themselves
this case, the precision is limited to two significant figures). when means are calculated for relatively large samples;
Some round the measurement to the higher number, while a value that is too high because of random error is often
others claim that doing so introduces bias into the data. You balanced by a value that is too low for the same reason.
can decide which approach to take, but be consistent. The median is, after arranging the measurements from the
smallest to the largest, the middle value that divides
the set of measurements into two subsets of equal
2. Determine the range of the temperatures that can be size. If there are an even number of measurements, the
measured with your thermometer by examining the median is the mean of the two middle values. In biol-
scale imprinted along the barrel of the thermometer. ogy, the mean is usually preferred to the median when
reporting descriptive statistics.
3. Measure the following temperatures:
Room temperature °C
Cold tap water °C The median is less sensitive to extreme values than is
the mean. To appreciate this, consider a sample consisting of
Hot tap water °C
14 leaves having the following lengths (all in mm):
Inside refrigerator °C
80 69 62 74 69 51 45 40 9 64 65 64 61 67

The mean length is 58.6 mm. However, none of the leaves

UNDERSTANDING NUMERICAL DATA are that length, and most of the leaves are longer than 60 mm.
In biology, the mean is usually preferred to the median when
Statistics offer a way to organize, summarize, and describe reporting descriptive statistics.
data—the data are usually samples of information from a Consider these sets of data:
much larger population of values. Statistics and statistical
1 3 5 7 9 – the mean and median are both 5
tests allow us to analyze the sample and draw inferences
about the entire population. Consequently, the use of statistics 1 3 5 7 14 – the median is 5, but the mean is 6
enables us to make decisions even though we have incomplete 1 3 5 7 34 – the median is 5, but the mean is 10
data about a population. Although this may seem unscientific,
we do it all the time; for example, we diagnose diseases with Question 6
a drop of blood. Decisions are based on statistics when it is a. Does the mean always describe the “typical” measure-
impossible or unrealistic to analyze an entire population. ment? Why or why not?
Let’s say that you want to know the mass of a typi-
cal apple in your orchard. To obtain this information, you
could analyze one apple, but how would you know that
you’d picked a “typical” sample? After all, the batch from b. What information about a sample does a mean not provide?
which you chose the apple may contain many others, each a
little different. You’d get a better estimate of “typical” if you
increased your sample size to a few hundred apples, or even
Determine the median by arranging the measurements in
to 10,000. Or, better yet, to 1,000,000.
numerical order:
The only way to be certain of your conclusions would
be to accurately measure all the apples in your orchard. This 9 40 45 51 61 63 64 64 65 67 69 69 73 80

2–7 Measurements in Biology 17

The median is between the seventh and eighth measurements: b. Could two samples have the same range but different
64 mm. In this sample, the mean differs from the median. means? Explain.

Question 7
a. What is responsible for this difference between the
mean and median?
The standard deviation indicates how measurements
vary about the mean. The standard deviation is easy to cal-
culate. Begin by calculating the mean, measuring the devia-
tion of each sample from the mean, squaring each deviation,
b. How would the median change if the 9-mm-long leaf
and then summing the deviations. This summation results
was not in the sample?
in the sum of squared deviations. For example, consider
a group of shrimp that are 22, 19, 18, and 21 cm long. The
mean length of these shrimp is 20 cm.
c. How would the mean change if the 9-mm-long leaf was
not in the sample? Sample
Value Mean Deviation (Deviation)2
22 20 2 4
19 20 −1 1
d. Consider these samples:
21 20 1 1
Sample 1: 25 35 32 28
18 20 −2 4
Sample 2: 15 75 10 20
What is the mean for Sample 1? Sum of Squared Deviations = 10

What is the mean for Sample 2? The summary equation for the sum of squared deviations is:
N
In most of the exercises in this manual, you’ll have time to Sum of squared deviations = Σ (x i
− x)2
make only one or two measurements of a biological struc- i=1

ture or phenomenon. In these instances, a mean may be the where

only descriptor of the sample. However, if your class com- N = total number of samples
bines its data so that there are many measurements, you’ll x = the sample mean
need to know how to do a couple of other calculations so
xi = measurement of an individual sample
that you understand the variation within your sample.

Variability
As you can see, the samples in Question 7d are different, but
their means are the same. Thus, the mean does not reveal all
there is to know about these samples. To understand how
these samples are different, you need other statistics: the
range and standard deviation.
The range is the difference between the extreme
­mea­surements (i.e., smallest and largest) of the sample. In
Sample 1, the range is 35 − 25 = 10; in Sample 2 the range is
75 − 10 = 65. The range provides a sense of the variation of the
sample, but the range can be artificially inflated by one or two
extreme values. Notice the extreme values in the sample of leaf
measurements previously discussed. Moreover, ranges do not
tell us anything about the measurements between the extremes.
Question 8
a. Could two samples have the same mean but different
ranges? Explain.
N
This formula is simple. The summation sign ( Σ ) means to add
i=1
up all the squared deviations from the first one (i = 1) to the
last one (i = N). The sum of squared deviations (10) divided by
the number of samples minus one (4 − 1 = 3) produces a value
of 10/3 = 3.3 cm2 (note that the units are centimeters squared).
This is the variance:
sum of squared deviations
Variance =
N−1
The square root of the variance, 1.8 cm, equals the standard
deviation (SD):
SD = √Variance = √3.3 = 1.8
The standard deviation is usually reported with the mean
in statements such as, “The mean length of the shrimp was
20 ± 1.8 cm.”
The standard deviation helps us understand the
spread or variation of a sample. For many distributions
of measurements, the mean ± 1 SD includes 68% of the

18 EXERCISE 2 2–8
measurements, whereas the mean ± 2 SD includes 95% of
Range
the measurements.
All classmates to
Male classmates to
Female classmates to
Procedure 2.7 Gather and analyze data Standard deviation
statistically All classmates ±
1. Use a meterstick or tape measure to measure your Male classmates ±
height in centimeters. Record your height here:
Female classmates ±
cm
2. Record your height and gender (male or female) on
the board in the lab. If there is sufficient time, obtain a newspaper that adver-
3. After all of your classmates have reported their tises cars, groceries, or other common commodities. Choose
heights, calculate the following: one example (e.g., new cars) and determine its average price
(e.g., determine the average price of a new car).
Size of sample
All classmates Question 9
Male classmates a. What does your calculation tell you?
Female classmates
Mean height
All classmates b. What are the limitations of your sample?
Male classmates
Female classmates
Median height Your instructor may ask you to do other statistical tests,
All classmates such as Student’s t, chi-square, and analysis of variance
Male classmates (ANOVA). The type of test you’ll do will depend on the
amount and type of data you analyze, as well as the hypoth-
Female classmates
eses you are trying to test.

INQUIRY-BASED LEARNING
How much do the areas and shapes of leaves vary?
Observation: Leaves, which are the primary photosynthetic
organ of most plants, are adapted for absorbing light. This
involves exposing large surface areas to the environment.
Question: How do the surface area and shape of leaves vary on
different parts of plants?
a. Establish a working lab group and obtain Inquiry-Based
Learning Worksheet 2 from your instructor.
b. Discuss with your group well-defined questions relevant
to the preceding observation and question. If leaves are
not available from outdoor plants (e.g., during winter),
use the plants provided by your instructor that were grown
indoors. Choose and record your group’s best question
for investigation.
c. Translate your question into a testable hypothesis and
record it.
d. Outline on Worksheet 2 your experimental design
and supplies needed to test your hypothesis. Ask your
instructor to review your proposed investigation.
e. Conduct your procedures, record your data, answer
your question, and make relevant comments.
f. Discuss with your instructor any revisions to your
questions, hypothesis, or procedures. Repeat your work
as needed.

2–9 Measurements in Biology 19

Questions for Further Study and Inquiry

1. What are the advantages and disadvantages of using the metric system of measurements?

2. Why is it important for all scientists to use a standard system of measures rather than the system that may be most
popular in their home country or region?

3. Do you lose or gain information when you use statistics to reduce a population to a few characteristic numbers?
Explain your answer.

4. Suppose that you made repeated measurements of your height. If you used good technique, would you expect the range
to be large or small? Explain your answer.

5. Suppose that a biologist states that the average height of undergraduate students at your university is 205 cm plus or
minus a standard deviation of 17 cm. What does this mean?

6. What does a small standard deviation signify? What does a large standard deviation signify?

7. Is it possible to make a perfectly precise measurement? Explain.

8. When in our everyday lives do we not want precise measurements?

9. Consider these measurements of the diameter (in nm) of viral particles (“virons”) of COVID-19: 0.12, 0.14, 0.10, 0.12,
0.11, 0.13, 0.14, 0.13, 0.08, 0.13, 0.13. What is the mean? What is the range? What is the median?

20 EXERCISE 2 2–10
 EXER CISE

The Microscope
Basic Skills of Light Microscopy 3
Learning Objectives
By the end of this exercise you should be able to:
1. Identify and explain the functions of the primary parts of a compound microscope and dissecting (stereoscopic)
microscope.
2. Carry and focus a microscope ­properly.
3. Use a compound microscope and dissecting microscope to examine biological specimens.
4. Prepare a wet mount, determine the magnification and size of the field of view, and determine the depth of field.

Please visit connect.mheducation.com to review online resources tailored to this lab.

M any organisms and biological structures are too small

to be seen with the unaided eye (fig. 3.1). Biologists
often use a light microscope to observe such specimens. A
light microscope is a coordinated system of lenses arranged
to produce an enlarged, focusable image of a specimen. A light
microscope magnifies a specimen, meaning that it increases
its apparent size. Magnification with a light microscope is usu-
ally accompanied by improved resolution, which is the abil-
ity to distinguish two points as separate points. Thus, the better
the resolution, the sharper or crisper the image appears. The
resolving power of the unaided eye is approximately 0.1 mm
(1 in = 25.4 mm), meaning that our eyes can distinguish two
points that are 0.1 mm apart. A light microscope, used properly,
can improve resolution as much as 1000-fold (i.e., to 0.1 µm).
The ability to discern detail also depends on contrast,
which is the difference between the lightest and darkest parts
of an image. Therefore, many specimens examined with a
light microscope are stained with artificial dyes that increase
contrast and make the specimen more visible.
The invention of the light microscope was profoundly
important to biology because it was used to formulate the
cell theory and study biological structure at the cellular
level. Light microscopy has revealed a vast new world to the
human eye and mind (fig. 3.2). Today, the light microscope
is the most fundamental tool of many biologists.
THE COMPOUND LIGHT MICROSCOPE
Study and learn the parts of the typical compound light
microscope shown in figure 3.3. A light microscope has two,
sometimes three, systems: an illuminating system, an imag-
ing system, and possibly a viewing and recording system.
Illuminating System
The illuminating system, which concentrates light on the
specimen, usually consists of a light source, condenser lens,
and iris diaphragm. The light source is a lightbulb located
Caring for Your Microscope
Microscopes are powerful tools for understanding biology.
However, they’re also expensive and fragile and require spe-
cial care. When you use your microscope, always do the fol-
lowing to ensure optimal performance and care:
∙∙ Always carry your microscope upright with both
hands—one hand under the base and the other around
the microscope’s arm (fig. 3.3).
∙∙ Always begin by cleaning the ocular and objective lenses
with lens paper.
∙∙ Always start your examinations with the low-power
objective in place.
∙∙ If you shift to the high-power objective, rotate the objective
into place carefully. Never force the objective lens into
place. If the objective lens contacts the slide, stop and
restart your examination with the low-power objective lens.
∙∙ After shifting to the high-power objective, always use
only the fine adjustment to focus the image.
∙∙ When you’ve completed your work with the microscope,
clean the lenses with lens paper, wrap the electrical cord
securely around the microscope’s arm, and return your
microscope to its storage area.

3–1 The Microscope 21

 20 mm
20 μm
20 nm
Figure 3.1 The size of cells and their contents. This diagram shows the size of human skin cells, organelles, and molecules. In general, the
diameter of a human skin cell is about 20 micrometers (µm), of a mitochondrion is 2 µm, of a ribosome is 20 nanometers (nm), of a protein
molecule is 2 nm, and of an atom is 0.2 nm.
at the base of the microscope. The light source illuminates
the specimen by passing light through a thin, almost trans-
parent part of the specimen. The condenser lens, located
imme­diately below the specimen, focuses light from the
light source onto the specimen. Just below the condenser is
the condenser iris diaphragm, a knurled ring or lever that
can be opened and closed to regulate the amount of light
reaching the specimen. When the condenser iris diaphragm
is open, the image will be bright; when closed, the image
will be dim.
Imaging System
The imaging system improves resolution and magnifies the
image. It consists of the objective and ocular (eyepiece)
lenses and a body tube. The objectives are three or four
lenses mounted on a revolving nosepiece. Each objective is
a series of several lenses that magnify the image, improve
22 EXERCISE 3
2 mm 0.2 mm
2 μm 0.2 μm
2 nm 0.2 nm
resolution, and correct aberrations in the image. The most
common configuration for student microscopes includes
four objectives: low magnification (4×), medium magnifi-
cation (10×), high magnification (40×), and oil immersion
(100×). Using the oil immersion objective requires special
instructions, as explained in Exercise 24 to study bacteria.
To avoid damaging your microscope, do not use the oil
immersion objective during this exercise.
The magnifying power of each objective is etched
on the side of the lens (e.g., 4×). The ocular is the lens
that you look through. Microscopes with one ocular are
monocular microscopes, and those with two are binocular
microscopes. Oculars usually magnify the image 10 times.
The body tube is a metal casing through which light
passes to the oculars. In microscopes with bent body-tubes
and inclined oculars, the body tube contains mirrors and a
prism that redirect light to the oculars. The stage secures
the glass slide on which the specimen is mounted.
3–2
 Heritage Image Partnership Ltd/Alamy Stock Photo

Figure 3.2 “Egad, I thought it was tea, but I see I’ve been drinking a blooming micro-zoo!” says this horrified, proper 19th-century
London woman when she used a microscope to examine her tea. People were shocked to learn that there is an active, living world too small
for us to see.

Oculars

Body tube

Arm

Nosepiece

Objectives
Slide holder
to adjust Stage
position Stage clip

Condenser

Condenser iris diaphragm

Coarse focus
adjustment Condenser adjustment

Fine focus
adjustment Light source

Base

©BiologyImaging.com
Figure 3.3 Major parts of a compound light microscope.

3–3 The Microscope 23


A Summary of How to Use
a Compound Light Microscope
1. Place the specimen on the microscope’s stage.
2. Rotate the low-power objective into place. Center the
specimen below the objective.
3. Look through the oculars while using the coarse adjust-
ment to focus on the specimen. Center the area of the
specimen that you want to examine.
4. Slowly rotate the high-power objective into place. Look
through the oculars while you use the fine adjustment to
focus on the specimen.
5. If you “lose” your specimen when you switch from low
power to high power, retrace the previous steps, paying
special attention to placing the specimen in the center of
the field of view.
Viewing and Recording System
The viewing and recording system, if present, converts radi-
ation to a viewable and/or permanent image. The viewing
and recording system usually consists of a camera or video
screen. Most student microscopes do not have viewing and
recording systems.
USING A COMPOUND MICROSCOPE
Although the maximum magnification of light microscopes
has not increased significantly during the last century, the
construction and design of light microscopes have improved
the resolution of newer models. For example, built-in light
sources have replaced adjustable mirrors in the illuminating
system, and lenses are made of better glass than they were
in the past.
Your lab instructor will review with you the parts of
the microscopes (and their functions) you will use in the
lab (fig. 3.3). After familiarizing yourself with the parts of
a microscope, you’re now ready for some hands-on experi-
ence with the instrument. The videos at the website associ-
ated with this manual (connect.mheducation.com) will be
especially useful for helping you understand how to properly
use your microscope.
Procedure 3.1 Use a compound microscope
1. Remove the microscope from its cabinet and carry
it upright with one hand grasping the arm and your
other hand supporting the microscope below its
base. Place your microscope on the table in front
of you.
24 EXERCISE 3
Do not use paper towels or Kimwipes to clean the
lenses of your microscope; they can scratch the
lenses. Clean the lenses only with lens paper.
2. Plug in the microscope and turn on the light source.
3. If it isn’t already in position, rotate the nosepiece
until the lowest-power (4×) objective is in line with
the light source. (The 4× objective is often called
the “scanning objective” because it enables users to
scan large areas of a specimen.) You’ll feel the objec-
tive click into place when it is positioned properly.
Always begin examining slides with the lowest-power
objective.
4. Locate the coarse adjustment knob on the side of the
microscope. Depending on the type of microscope
that you’re using, the coarse adjustment knob moves
either the nosepiece (with its objectives) or the stage
to focus the lenses on the specimen. Only a partial
turn of the coarse adjustment knob moves the stage
or nosepiece a relatively large distance. The coarse
adjustment should only be used when you’re viewing
a specimen with the 4× or 10× objective lens.
5. If your microscope is binocular, adjust the distance
between the oculars to match the distance between
your pupils. If your microscope is monocular, keep
both eyes open when using the microscope; this will
reduce the strain on your eyes. After a little practice
you will ignore the image received by the eye not
looking through the ocular.
6. Adjust the light intensity for comfort and visual quality.
Focus a specimen by using the following steps:
a. Place a microscope slide of newsprint of the
letter e on the horizontal stage so that the e is
directly below the lowest-power objective lens
and is right side up. It should be centered over
the hole in the stage.
b. Rotate the coarse adjustment knob to move the
objective within 1 cm of the stage (1 cm = 0.4 in).
c. Look through the oculars with both eyes open.
d. Rotate the coarse adjustment knob (i.e., raising
the objective lens or lowering the stage) until
the e comes into focus. If you don’t see an
image, the e is probably off center. Be sure that
the e is directly below the objective lens and that
you can see a spot of light surrounding the e.
e. Focus up and down to achieve the crispest image.
f. Adjust the condenser iris diaphragm so that the
brightness of the transmitted light provides the
best view.
g. Observe the letter, then rotate the nosepiece to
align the 10× objective to finish your observation.
Do not use the oil immersion objective.
3–4
Question 1
notice that the image remains near focus and that the
a. As you view the letter e, how is it oriented? Upside
field-of-view has gotten smaller. Most light microscopes
down or right side up?
are parfocal, meaning that the image will remain nearly
focused after the 40× objective lens is moved into place.
Most light microscopes are also parcentered, meaning
that the image will remain centered in the field of view
b. How does the image move when the slide is moved to
after the 40× objective lens is in place.
the right or left? Toward you or away from you?
5. You may need to readjust the iris diaphragm because
the high-magnification objective allows less light to
pass through to the ocular.
c. What happens to the brightness of the view when you 6. To fine-focus the image, locate the fine adjustment
go from 4× to 10×? knob on the side of the microscope. Turning this knob
changes the specimen-to-objective distance slightly
and therefore makes it easy to fine-focus the image.
Use only the fine adjustment when using the
40× (or higher) objective.
Magnification
Never use the coarse adjustment knob to focus
an image on high power.
Procedure 3.2 Determine magnification
1. Estimate the magnification of the e by looking at the
magnified image on lowest magnification (4×), and
then at the e without using the microscope. Question 2
2. Examine each objective and record the magnifica- a. How many times is the image of the e magnified when
tions of the objectives and oculars of your microscope viewed through the high-power objective?
in table 3.1.
3. Calculate and record in table 3.1 the total magnifica-
tion for each objective following this formula:
MagTot = MagObj × MagOcu
b. If you didn’t already know what you were looking at,
could you determine at this magnification that you
where
were looking at a letter e? How?
MagTot = total magnification of the image
MagObj = magnification of the objective lens
MagOcu = magnification of the ocular lens

For example, if you’re viewing the specimen with a 4× Determine the Size of the Field of View
objective lens and a 10× ocular, the total magnifica-
tion of the image is 4 × 10 = 40×. That is, the specimen
The field of view is the area that you can see through the
appears 40 times larger than it is.
ocular and objective (fig. 3.4). Knowing the size of the field
of view is important because you can use it to estimate the
4. Slowly rotate the high-power (i.e., 40×) objective into
size of an object you are examining. The field of view can
place. Be sure that the objective does not touch the slide!
be measured with ruled micrometers (fig. 3.5). An ocu-
If the objective does not rotate into place without touch-
lar micrometer is a small glass disk with thin lines num-
ing the slide, do not force it; ask your lab instructor to
bered and etched in a row. It was put into an ocular on your
help you. After the 40× objective is in place, you should
microscope so that the lines superimpose on the image and

Table 3.1
Total Magnifications and Areas of Field of View (FOV) for Three Objective Lenses
Objective Objective Ocular Total FOV FOV Measurement (mm)
Power Magnification × Magnification = Magnification Diameter (mm) Area (mm2) for 1 Ocular Space

4× × =
10× × =
40× × =

3–5 The Microscope 25

 allow you to measure the specimen. Before you can use the
micrometer you must determine for each magnification the
apparent distance between the lines on the ocular micro­­meter.
This means that you must calibrate the ocular ­micrometer by
comparing its lines to those lines on a standard ruler called a
stage ­micrometer. A stage micrometer is a glass slide hav-
ing ­precisely spaced lines etched at known intervals.

Procedure 3.3 Use a stage micrometer to

calibrate the ocular micrometer, and determine
the size of the field of view
1. Rotate the ocular until the lines of the ocular microm-
eter parallel those of the stage micrometer (fig. 3.5).
2. Align lines at the left edges (0 lines) of the two
micrometers by moving the stage micrometer (fig. 3.5).
3. Count how many spaces on the stage micrometer fit
precisely in a given number of spaces on the ocular
©BiologyImaging.com micrometer. Record the values here:
Figure 3.4 The circular, illuminated field of view of a compound y ocular spaces = x stage spaces
light microscope. Shown here is the letter e from newsprint that is mag-
nified 40 times (i.e., 40×). y=

x=

View of
ocular
micrometer

View of
stage
micrometer

View of both micrometers

aligned at their 0 lines

©BiologyImaging.com

Figure 3.5 Stage and ocular micrometers. A stage micrometer is used to calibrate a microscope with its ocular micrometer to measure the
size of specimens.

26 EXERCISE 3 3–6
 The smallest space on a stage micrometer =
0.01 mm, so
y ocular spaces (mm) = x stage spaces × 0.01
1 ocular space (mm) = (x/y) × 0.01
4. Calculate the distance in millimeters between lines of
the ocular micrometer. For example, if the length of
10 spaces on the ocular micrometer equals the length
of seven spaces on the stage micrometer, then
y = 10
6. The ruler cannot be used to measure the diameters of
the field of view at medium and high magnifications
because the markings are too far apart. Therefore,
these diameters must be calculated using the follow-
ing formula:
FOVlow × Maglow = FOVhigh × Maghigh
where
FOVlow = d
 iameter of the field of view of the
low-power objective

x=7 Maglow = m
 agnification of the low-power
objective (Be consistent and use
10 ocular spaces (mm) = 7 stage spaces × 0.01 mm the magnification of the objective,
1 ocular space (mm) = (7 × 0.01 mm)/10 not total magnification.)

1 ocular space (mm) = 0.007 mm FOVhigh = d

 iameter of the field of view of the
high-power objective
1 ocular space = 7 µm
Maghigh = m
 agnification of the high-power
Therefore, if a specimen spans eight spaces on your objective
ocular micrometer with that objective in place, that
specimen is 56 µm long. For example, if 3.0 mm is the diameter of the field
of view for a 4× low-power objective, then what is
5. Calibrate the ocular micrometer for each objective
the diameter of the field of view of the 40× high-
on your microscope. Record in table 3.1 the diameter
power objective?
of the field of view (FOV) for each objective. Also
record for each objective lens in table 3.1 the mea- 3.0 mm × 4 = FOVhigh × 40
surement (mm) for 1 ocular space. You can use this 0.30 mm = FOVhigh
information in future labs as you measure the sizes of 7. Calculate and record in table 3.1 the diameters of the
organisms and their parts. field of view for the 10× and 40× magnifications.
6. Calculate the radius, which is half the diameter. 8. Calculate and record in table 3.1 the circular area of
7. Use this information to determine the area of the cir- the field of view for the three magnifications by using
cular field of view with the following formula: the following formula.
Area of circle = π × radius2
Area of circle = π × radius2
(π = 3.14)
(π = 3.14)
8. Record your calculated field of view areas in table 3.1.

Alternate Procedure 3.3 Use a transparent Question 3

ruler to determine the size of the field of view
a. Which provides the largest field of view, the 10× or
1. Obtain a clear plastic ruler with a metric scale. 40× objective?
2. Place the ruler on the stage and under the stage
clips of your microscope. If your microscope has a
mechanical stage, ask your instructor how to place
b. How much more area can you see with the 4× objective
the ruler to avoid damage. Carefully rotate the nose-
than with the 40× objective?
piece to the objective of lowest magnification.
3. Slowly focus with the coarse adjustment, and then
with the fine adjustment, until the metric markings on
the ruler are clear. c. Why is it more difficult to locate an object starting
4. Align the ruler to measure the diameter of the circu- with the high-power objective than with the low-power
lar field of view. The space between each line on the objective?
ruler should represent a 1-mm interval.
5. Record in table 3.1 the diameter of this low-
magnification field of view. Also calculate the radius, d. Which objective should you use to initially locate the
which is half the diameter. specimen? Why?

3–7 The Microscope 27

 2. Focus up and down and try to determine the order
of the threads from top to bottom. The order of the
Plane threads will not be the same on all slides.
1. 3. Re-examine the threads using the high-power
objective lens.

1.
Question 4
2. a. Are all three colored threads in focus at low power?
2.

3.
b. Can all three threads be in focus at the same time
using the high-power objective?
3.

Specimen Microscope image

on slide at different levels
of focus
(a) c. Which objective, high or low power, provides the
greatest depth of field?

Preparing a Wet Mount

of a Biological Specimen

Procedure 3.5 Prepare a wet mount of a

©BiologyImaging.com
(b)
Figure 3.6 How focusing at different planes of a specimen would
produce three different images. (a) Focusing up and down when you view
a specimen can help you to understand its three-dimensional structure. (b)
A thin depth of field is apparent in this 100× image of cells of Closterium,
a green alga. The upper and lower layers of cells are out of focus, while
the midlayer of cells is within the thin depth of field and is clearly focused.
Determine the Depth of Field
Depth of field is the thickness of the object in sharp focus
(fig. 3.6). Depth of field varies with different objectives and
magnifications.
Procedure 3.4 Determine the depth of the field
of view
1. Using the low-power objective, examine a prepared
slide of three colored threads mounted on top of
each other.
biological specimen
1. Place a drop of water containing algal cells from a
culture labeled “Algae” on a clean microscope slide.
2. Place the edge of a clean coverslip at an edge of the
drop at a 45° angle; then slowly lower the coverslip
onto the drop so that no air bubbles are trapped
(fig. 3.7). (Your instructor will demonstrate this
technique.) The coverslip holds the specimen in
place and prevents the lens of an objective from
contacting the water and the specimen. This fresh
preparation is called a wet mount and can be viewed
with your microscope.
3. Experiment with various intensities of illumination.
To do this, rotate the 4× objective into place and
adjust the condenser iris diaphragm to produce the
least illumination. Observe the image; note its clarity,
contrast, and color. Repeat these observations with at
least four different levels of illumination. The fourth
level should have the diaphragm completely open.
4. Repeat step 3 for the 10× and 40× objectives.
Question 5
a. Is the image always best with the highest illumination?

28 EXERCISE 3 3–8
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— Ah, quasi — ella ripetè cupamente. — Ci son dunque esempi di
navigli perduti senza che rimanga alcuna traccia di loro, senza che si
salvi un uomo dell’equipaggio, senza che una tavola galleggiante sul
mare dia un indizio della catastrofe?
Non potei negarle che di questi casi ce ne fossero, ma erano così
rari, così eccezionali da non doverci fermare il pensiero.
— Perchè vede, Ceriani — ella ripigliò — non so come sopporterei
l’annunzio positivo d’una sciagura; so che l’incertezza mi
ucciderebbe.... Ma, in nome del cielo — seguitò la povera signora
attorcigliando nervosamente il fazzoletto alle dita — si è poi fatto
tutto quello che si doveva per chiarire questo mistero?... Mio marito
lo afferma; io non lo credo.
Le enumerai le lettere, i telegrammi che si erano spediti; l’assicurai
che si sarebbe tornato a scrivere, a telegrafare.
Ella si strinse nelle spalle. — Scrivere? Telegrafare?... Ah se fossi un
uomo!
Qualcuno s’avvicinava, ed ella mi lasciò con queste parole,
slanciandomi uno sguardo metà di preghiera, metà di rimprovero.
Che pretendeva mai la signora Agnese? Ch’io andassi alla ricerca
del King Arthur come Stanley era andato alla ricerca di
Livingstone?... Ohimè, l’impresa dello Stanley era stata una follia
sublime; la mia non sarebbe stata che una follia ridicola.
Nè la signora Agnese me ne riparlò nei giorni seguenti. La sua
agitazione febbrile aveva ceduto il posto a una calma apparente che
ci impensieriva ancora di più. Ella stava per lunghe ore sdraiata sulla
sua poltrona nel salottino giapponese, senza un libro, senza un
lavoro, immersa in un cupo silenzio. A colazione, a pranzo, toccava
appena il cibo, pronunziava appena qualche monosillabo, si faceva
una legge di non menzionar mai nè il King Arthur, nè il capitano
Atkinson, nè la piccola Ofelia. Solo una volta ella scattò dalla
seggiola quando il dottor Gandolfi le suggerì un viaggetto di un
mese. — Quest’anno non mi muovo da Venezia — ella risposo in
tuono secco, reciso.
Passavano i giorni, passavano le settimane. Eravamo venuti a
sapere d’un tifone che aveva infuriato nei mari della China fra il 25 e
il 28 di giugno ed era penetrato nei nostri animi il convincimento che
in quella occasione appunto il King Arthur si fosse perduto con tutto
l’equipaggio. Ma mentre si conoscevano i nomi d’altri legni ch’erano
scampati miracolosamente al pericolo, e sbattuti, malconci avevano
dovuto ripararsi in qualcheduno di quei porti, del King Arthur
nessuno poteva dir nulla. Nessuno lo aveva visto dopo la sua
partenza da Hiogo.
Anche i danni materiali d’un simile stato di cose erano gravissimi. Le
rimesse fatte a Londra per rimborsare il nostro banchiere
importavano circa ottocentomila lire, somma della quale c’era forza
rimaner scoperti finchè fosse spirato il termine necessario per
acquistare il diritto d’abbandono verso le compagnie assicuratrici, e
non c’è casa di commercio, per potente che sia, a cui non dia
degl’impicci l’immobilizzare un capitale di quasi un milione.
Inoltre tutti i vantaggi sperati da un’iniziativa che doveva riaffermare
la superiorità della nostra ditta andavano in fumo per esser raccolti in
gran parte dai nostri rivali, i Gelardi, che avevano commesso a
Hiogo un carico di riso dopo di noi e che lo aspettavano entro
l’ottobre col vapore inglese The Iron Duke. Noi l’odiavamo questa
Iron Duke che seguiva la via tenuta dal King Arthur, che
probabilmente sarebbe passato sul punto ove il King Arthur era stato
inghiottito dalle onde. Non credo che nessuno di noi gli augurasse
un disastro, ma è certo che a sentirlo nominare ci si rimescolava il
sangue. E lo si sentiva nominare così spesso. I sensali, che in attesa
del King Arthur avevano imbastito degli affari con noi, adesso, con la
compunzione di chi fa una visita di condoglianza, venivano a
sciogliersi da ogni impegno e a dirci della dolorosa necessità in cui si
trovavano di trattare coi Gelardi per l’acquisto della merce di
prossimo arrivo con l’Iron Duke. E poi gli stessi Gelardi, alquanto
vanitosi per loro natura, stimavano opportuno di comunicare ai
giornali cittadini le varie tappe del loro bastimento. Era partito il tal
giorno da Hiogo; aveva nel tal altro toccato Point-de-Galle; era
passato per Aden, era a Suez.... Il comandante dell’Iron Duke non
faceva economia di telegrammi.
Finalmente, ai primi di novembre, una mattina, il bastimento entrò in
porto e andò ad ancorarsi alla Giudecca, proprio dove, in aprile, era
ancorato il King Arthur. Ed io procurai nella giornata medesima di
vedere il capitano per chiedergli se gli fosse venuta all’orecchio
nessuna voce circa al vapore che due mesi prima di lui aveva
lasciato il Giappone alla volta di Venezia. Ma egli non ne sapeva più
di quello che ne sapevamo noi.
Quando tornai in banco dopo questa mia pratica vana, il principale
mi disse: — Mia moglie ha ragione. L’incertezza è il peggiore dei
mali, e una speranza voluta conservare a ogni costo è una fonte
perenne d’inquietudine.... Ma che speranza? — egli corresse con un
gesto d’impazienza. — Noi non ne abbiamo più; noi non dubitiamo
che il King Arthur sia perduto.... Ci manca però la forza di
rassegnarvisi finchè non abbiamo in mano un documento, una
prova.... Ah, questa prova, questa prova chi ce la darà?
Mi guardò in un modo singolare e soggiunse: — Senta, Ceriani. Il
viaggio d’esplorazione che l’Agnese parve consigliarle tempo
addietro è, anche a’ miei occhi, una cosa assurda. Nondimeno
qualche passo si potrebbe fare. Una corsa in Inghilterra per
esempio, tanto da vedere gli armatori, da consultarsi con persone
esperimentate, da recarsi agli uffici del Lloyd ove ci son notizie di
tutto il mondo?... Andrei io, se non avessi scrupolo di piantar quella
disgraziata.... Lei, Ceriani, lei ch’è giovine, ch’è libero, avrebbe
difficoltà di partire per Londra al più presto, domani sera, per
esempio?...
Sollevai alcuni dubbi sull’utilità di questa gita, ma difficoltà ad
abbandonar Venezia per un quindici o venti giorni non ne avevo
affatto. In fondo, lo confesso, l’offerta mi tornava gradita, perchè
ormai il King Arthur pesava sul banco come un incubo. Ora,
quest’incubo io l’avrei subìto anche durante le mie peregrinazioni
che avevano per iscopo preciso di far nuove indagini sulla sorte del
naviglio: ma mi sorrideva l’idea di cambiar aria, di sostituire una
ricerca attiva (fosse pure infruttuosa) a una preoccupazione inerte e
opprimente.

VI.

Partii dunque, munito d’una quantità di commendatizie, partii senza

congedarmi dalla signora Agnese ch’era indisposta, e nel cui animo,
del resto, era inutile far sorgere aspettazioni che non si sarebbero
adempiute.
Che dirò del mio viaggio? Fui a Londra, fui a Glascow, fui a
Liverpool; parlai con gli armatori del King Arthur, mi rivolsi a quel
meraviglioso centro d’informazioni ch’è il Lloyd, conobbi il
comandante d’un vapore ch’era stato investito dal tifone dal 25 al 28
giugno, e non per questo riuscii a dissipare l’oscurità che avvolgeva
il nostro vascello fantasma. M’accorsi bensì che del King Arthur si
discorreva universalmente come di chi sia morto e sepolto da un
pezzo. O, per esser più esatto, m’accorsi che si cominciava a
discorrerne meno, come d’un fatto ormai vecchio.
Gli armatori, ricchi a milioni, proprietari d’una dozzina di piroscafi
sparsi per tutti gli Oceani, erano più che addolorati, inaspriti contro il
capitano Atkinson. Lo accusavano d’imprudenza; già un’altra volta,
parecchi anni addietro, egli aveva, per la sua temerità, tratto a
perdizione un naviglio. Ma allora almeno s’era salvata la gente.
Questa circostanza che noi ignoravamo non era però ignorata dal
Lloyd. Mi mostrarono colà il Captain’s Register, specie di dizionario
biografico dei capitani mercantili inglesi, ove nella forma più succinta
possibile si contengono importanti notizie relative a ciascuno di loro.
E sotto il nome del capitano Giorgio Atkinson, dopo la data e il luogo
della nascita, dopo altre indicazioni varie, si leggevano queste brevi
parole: Lady Hamilton, st. 1863-65 — lost on the 10th May 1865, off
the Isle of Majorca; ciò che significava che il capitano Atkinson avea
dal 1801 al 1865 comandato il vapore Lady Hamilton, e che questo
vapore s’era perduto in vicinanza dell’isola di Majorca il 10 maggio
1865.
— Una disgrazia può succedere al più provetto, — notò la mia guida
chiudendo il volume accusatore; — è però sempre una cattiva
raccomandazione.
Del resto, anche pel Lloyd, il King Arthur era un legno a cui si poteva
recitare il de profundis. Quei preposti, con molta cortesia, mi
lasciarono vedere la corrispondenza, quasi esclusivamente
telegrafica, scambiata coi loro agenti del Giappone, della China,
dell’India su questo argomento. Con parole diverse si arrivava
sempre alla identica conclusione: testimonianze oculari non ce
n’erano, avanzi del bastimento non se ne trovavano, ma il King
Arthur doveva esser naufragato tra il 25 e il 28 giugno. La miglior
prova era la mancanza di qualunque notizia da pressochè cinque
mesi. Nello stato presente delle comunicazioni postali e telegrafiche,
in una navigazione per mari frequentatissimi, ciò non si spiegava che
con un disastro.
E poichè io stentavo a capacitarmi di questa scomparsa assoluta
d’un bastimento, fui condotto in un’altra camera e invitato a dare
un’occhiata alla lista dei missing vessels, cioè dei vascelli mancanti,
su cui pesava lo stesso mistero che sul King Arthur, ma di cui non si
poteva mettere dubbio che fossero stati inghiottiti dal mare con tutti i
loro attrezzi, con tutti i loro uomini. La lista non si riferiva che a pochi
anni, eppure era così lunga. Vi figuravano legni grandi o piccoli, a
vapore ed a vela, col loro nome, col nome del loro capitano, con
l’indicazione del porto dal quale erano partiti e di quello al quale
erano diretti, con la data dell’ultime notizie.... Poi, più nulla.
— E il King Arthur? — chiesi all’impiegato che mi accompagnava.
— Non c’è ancora, — egli mi rispose. — Lo registreremo presto....
Bisogna che passi un certo numero di mesi.
Io non sapevo staccar lo sguardo da quella pagina. Pensavo a tante
tragedie di cui l’Oceano chiude il secreto, a tante esistenze troncate,
a tanti gemiti, a tante imprecazioni, a tante preghiere soffocate dal
fragore del vento e dei flutti. E pensavo alla piccola Ofelia. La
rivedevo nella sala del palazzo Prosperi, ilare e vispa, co’ suoi riccioli
biondi che le svolazzavano sulla fronte; la rivedevo tra il cane Tom e
la signora Agnese. Povera, povera bambina! S’era accorta
dell’imminente catastrofe? O aveva cambiato il sonno con la
morte?... Ma più ancora che ai naufraghi pensavo a quelli che
aspettano.... Madri che l’inutile attesa precipita nella decrepitezza e
nell’imbecillità, spose che avvizziscono nella forzata vedovanza e
che dopo aver pianto tutte le loro lacrime dimandano invano la
libertà di aprir il cuore a nuovi affetti, di farsi una nuova famiglia....
L’impiegato del Lloyd indovinò in parte ciò che mi si agitava nella
mente e disse: — Tristi cose. Ma che sono i legni mancanti, che
sono i legni perduti in confronto di quelli che corrono i mari e
tengono alta la bandiera britannica? La lista dei missing vessels, il
loss-book, sono come il nostro necrologio, e qual’è la famiglia ove
non muoia qualcuno? Che famiglia numerosa sia la nostra lo si rileva
dal nostro registro nautico, il libro dei vivi. Erano l’anno scorso più di
dodici mila navigli.... nè il registro comprende tutti i legni della marina
mercantile inglese.... Sicuro, di quando in quando ci capita un
dispaccio annunziante un disastro, ma novanta volte su cento i
telegrammi che riceviamo a ogni ora del giorno ci parlano di
bastimenti arrivati, di bastimenti partiti, di bastimenti apparsi in
qualche punto remoto del globo e che mandano un saluto alla patria
lontana.... Eh, non ci resta mica tempo d’indugiarci troppo a pianger
sui morti.
Orgoglioso della grandezza della sua patria, della grandezza
dell’istituzione alla quale egli apparteneva, il mio interlocutore, uomo
dall’aria positiva quanto mai, diventava poeta. E io subivo, mio
malgrado, il fascino della sua eloquenza e nelle linee maestose del
quadro ch’egli mi tracciava vedevo ridursi alle proporzioni d’un
dramma domestico il pietoso episodio del King Arthur. Ripetevo a
me stesso quella frase crudele: Non ci resta tempo d’indugiarci
troppo a pianger sui morti — e mi pareva che, pronunziata in quella
sala ove fa capo il commercio marittimo del mondo, ella perdesse
alquanto della sua brutalità. Non ceder mai nè all’accidia, nè allo
scoraggiamento, nè al sentimentalismo, ecco il segreto della forza e
della potenza.
Comunque sia, io ero già al termine della mia missione senz’aver
raggiunto il fine sperato. Indizi in quantità, certezza morale fin che si
vuole; ma prove materiali, palpabili, nessuna. Da Venezia Prosperi
mi scriveva lasciando in mia facoltà di spingermi magari all’India, alla
China, al Giappone se credevo al risultato pratico di questo
viaggio.... Io però non ci credevo, nè ci credeva alcuno di quelli a cui
ne parlai. — Dove andreste? — mi si diceva. — È un’ipotesi
ragionevole che il King Arthur si sia perduto nel tifone dal 25 al 28
giugno. Ma è sempre un’ipotesi. E in ogni modo, pur riuscendo a
precisare il raggio di quel tifone, come scoprirete il punto ove il
bastimento si è sommerso? Che esercito di palombari prenderete
con voi? E vi par possibile ch’essi discendano a una profondità di
migliaia e migliaia di metri?
Quest’era vero, ma io obbiettai che forse qualche uomo
dell’equipaggio s’era salvato, rifugiato in un’isola, in una spiaggia
deserta, che forse si poteva trovarlo....
I miei ascoltatori sorrisero. — Non è più il tempo di Robinson
Crusoè.
Un po’ perchè queste riflessioni non facevano che crescere la mia
sfiducia, un po’ perchè m’impregnavo anch’io della maschia filosofia
anglo-sassone che ci stimola a guardare dinanzi e non dietro a noi,
io abbandonai l’impresa e feci ritorno in Italia. Era inutile ostinarsi, io
dissi al mio principale, era inutile sprecar l’energia, l’ingegno, il
danaro in ricerche fantastiche. Dovevamo mettere il nostro cuore in
pace, dimenticare il King Arthur, lavorar con lena raddoppiata,
mantenere alla nostra casa il posto che le spettava pe’ suoi capitali,
per la sua riputazione, per l’abilità riconosciuta di chi la dirigeva. Nel
fervore del discorso mi sfuggì un’allusione alla frase udita negli uffici
del Lloyd circa alla necessità di non indugiarsi troppo a piangere i
morti.
Il principale m’interruppe. — Non si tratta di morti, caro Ceriani....
almeno per me.... Per me si tratta d’una persona viva che amo e che
avrei voluto render felice a costo del mio sangue, mentre invece un
fatale concorso di circostanze volge ad effetto contrario tutti i miei
sforzi.... Ah è facile dire: quella persona è un’esaltata, ingigantisce i
suoi dolori, non apprezza convenientemente i beni che possede, va
in traccia dello strano, del singolare.... È facile rimproverarle il suo
cieco trasporto per una bambina appena conosciuta, la sua
mancanza di rassegnazione ai decreti della Provvidenza. È facile
infine citarle tante donne che hanno la saviezza di contentarsi di ciò
che hanno, di non accasciarsi sotto il peso di sventure molto più
grandi di quella che l’ha colpita.... Ragioni belle, buone, sacrosante,
ma che non concludono nulla.... Le cose non sono quelle che
dovrebbero essere, ma quelle che sono.... Il fatto si è che le
condizioni di mia moglie sono tali da destar le più legittime
apprensioni.... Quel pensiero assiduo che la tormenta, logora la sua
salute e pur troppo ha un’azione funesta anche sulla sua
intelligenza. La vedrà, Ceriani, la vedrà.... Non è già che ella parli di
continuo del King Arthur o dell’Ofelia; sarebbe meglio che ne
parlasse.... ma si capisce che la sua mente è sempre lì, e la sua
fissazione di voler passar quasi l’intera giornata in quello ch’ella
chiama il suo salottino giapponese basta a dimostrarlo.... Via, siamo
giusti, data una natura nervosa come quella dell’Agnese, non poteva
accader di peggio.... Se lo sciagurato King Arthur si fosse perduto
come si perdono centinaia di navi non saremmo a questo punto.... È
per ciò ch’io insistevo per aver una prova.... Non la si è potuta avere,
pazienza.... Non ne ha colpa nessuno.... Intendo benissimo che
sarebbe una follia il girare il mondo in cerca di un bastimento.... Ma
bisogna convenire ch’è un destino iniquo. E vi sarà della gente che
c’invidia perchè siamo ricchi!
Il signor Roberto non esagerava accennando alle tristi condizioni di
sua moglie. La vidi nella giornata e mi fece una pena immensa.
Aveva dato un crollo in poche settimane. Era ridotta a pelle ed ossa,
aveva gli occhi infossati e più d’un filo bianco si mesceva a’ suoi bei
capelli biondi.... E quello sguardo, quello sguardo!
Mi accolse con una cortesia fredda, ben diversa dall’espansione
ch’ella m’aveva dimostrata negli ultimi tempi. Circa al mio viaggio, mi
disse soltanto: — Non ha saputo nulla.... Già era da immaginarselo.
Lieto ch’ell’avesse rotto il ghiaccio, mi credetti incoraggiato a riferirle
le indagini che avevo fatte, i nuovi indizi che avevo raccolti, i discorsi
che avevo sentiti, la dolorosa convinzione che avevo acquistata
dell’inutilità di ulteriori pratiche per accertare un avvenimento su cui
pur troppo non v’era più dubbio. M’aspettavo ch’ella protestasse
contro le mie parole, che, in un modo o nell’altro, ella sfogasse il suo
dolore.... Niente di tutto ciò.... Teneva la testa chinata sul petto, le
mani incrociate sulle ginocchia, non dava segno di approvazione o
dissenso.
Nell’uscire dalla stanza il signor Roberto sospirò: — È uno strazio.
Così non può durare....
Tale era anche la mia opinione. Ma c’ingannavamo tutti e due. Anzi,
in quanto a salute, la signora Agnese migliorò, riprese un po’ di
polpa e di colore. Non migliorò invece il suo stato morale, non ci fu
verso di scuotere il letargo nel quale ell’era piombata. Ella non
trovava qualche lampo d’energia che per respinger qualunque
proposta suo marito le facesse di viaggi o di distrazioni d’altra
natura.
Compiuto l’anno, gli assicuratori pagarono il risarcimento che ci
spettava, e nella pagina del nostro registro mercanzie intestata al
Riso giapponese col “King Arthur„ noi potemmo inscrivere nella
colonna dell’avere ch’era ancora in bianco la cifra rotonda di un
milione di lire, chiudendo con un utile ragguardevole questo conto,
nonostante le spese impreviste, nonostante la perdita degl’interessi.
Furono pagate contemporaneamente anche le 25 mila lire del
salottino giapponese ch’era stato assicurato a parte.
— Sia ringraziato il cielo, — esclamò quel giorno il ragioniere della
ditta. — Quelle due partite aperte gettavano un’ombra sinistra
sull’intera azienda.... Adesso che ci si è messa su una bella pietra
sepolcrale, si potrà respirar meglio e muoversi con più libertà.
Ebbene, quel giorno stesso il signor Roberto mi esternava per la
prima volta la sua intenzione di liquidare la casa. Non aveva più
amore al lavoro, non aveva più ambizione, non aveva più elasticità di
fibra e di spirito. Sentiva di non esser l’uomo d’un tempo,
d’infastidirsi a ogni contrarietà, di smarrirsi a ogni dubbiezza; quei
lunghi mesi d’ambascia l’avevano spossato, affranto. E poi con che
sugo avrebbe seguitato a logorare il cervello nelle intricate
combinazioni del commercio moderno?... Non aveva figliuoli e non
isperava d’averne; sua moglie non abbisognava di maggiori
ricchezze per vegetar come faceva; le occorrevano soltanto delle
cure sollecite, attente, e queste cure toccava a lui di prestargliele.
Con la coscienza delicata dei buoni egli si caricava di colpe
immaginarie. — Dovevo entrar subito nell’idea dell’Agnese, — egli
ripeteva, — e far sì che il capitano ci lasciasse la bambina
addirittura, chè già con un po’ d’insistenza la si sarebbe spuntata....
Oppure dovevo tagliar corto, dichiarar che non volevo l’Ofelia in casa
nè prima nè dopo, impedire a quella funesta tenerezza di nascere, di
crescere. Il mezzo termine adottato fu la cagion vera di tante
disgrazie.
Ohimè, la vera cagione era da cercarsi nella mente non equilibrata
della signora Agnese, ma questo il signor Roberto non intendeva
ammetterlo. Fermo nel tenersi responsabile di tutto, egli diceva che il
far l’infermiere era per lui, oltre che un debito d’affetto, una giusta
espiazione.
E persisteva nel proposito di ritirarsi dai traffici. Solo studiava il modo
di provvedere all’avvenire de’ suoi commessi, di volgere a loro
vantaggio il credito e le relazioni della sua ditta.
Di qui l’accomandita della quale io sono il gerente e che mi permise
di conservare intimità di rapporti con l’ottimo uomo. Però tra noi non
si discorre d’affari che quando io gli presento il bilancio, e anche
allora se ne discorre poco perchè egli ha in me una fiducia che credo
di non avere demeritata. Anzi talvolta egli mi rimprovera
scherzosamente di aumentar troppo il suo patrimonio.
Per lo più egli mi parla di sua moglie ch’egli ama con l’antico
trasporto e ch’è sempre nel medesimo stato, sospesa tra la sanità e
la malattia, tra la saviezza e la demenza. Quand’io vado a visitarla, e
ci vado ogni tre o quattro settimane, ella mi riconosce, mi porge la
mano, mi ringrazia d’essermi ricordato di lei, mette insieme poche
frasi insignificanti, e quindi ricade in un silenzio penoso. Ma se mi
alzo per accommiatarmi si scuote, e non manca di dire: — Torni: Già
mi trova al solito posto, nel mio salottino giapponese.
E nel salottino che conserva per ironia questo nome si vedono
ancora le fotografie dell’Ofelia e del King Arthur. Ma i mille ninnoli,
ma le lacche colorate, ma gli specchi dipinti, ma le mensole, i vasi
che dovevano adornare il salottino giapponese, dove sono? Dov’è il
King Arthur, dov’è la gentile Ofelia, dov’è il capitano Atkinson, dov’è
il cane Tom? Su quali alghe riposano, quanto mare li copre, chi
saprà mai nulla di loro?

· · · · · · · · · · · · · · · ·
 NELL’ANDARE AL BALLO.

Il servo picchiò leggermente all’uscio, e disse con qualche

esitazione: — Signora....
Sola nel suo salottino, la signora Stella Marioli ripassava della
musica al pianoforte. Un resto di legna si consumava, scoppiettando,
nel caminetto; sopra una tavola sparsa di libri e giornali ardeva un
lume a Carvel.
— Che c’è? — chiese la signora, voltandosi sulla sedia.
— C’è una visita.
— A quest’ora? Lo sapete bene che di sera non ricevo.
— Lo so.... Ma il cavalier Gualberti mi ha ordinato di annunziarlo
ugualmente.
— È Gualberti? — soggiunse la signora Marioli come parlando tra
sè. — E pensò che per due volte consecutive egli era venuto di
giorno senza trovarla in casa. A ogni modo.... — Basta.... Che entri,
— ella disse a voce alta. — E portate da fare il tè.
Spense le due candele del pianoforte e andò a sedere accanto alla
tavola.
Era una donna sulla trentina, non bellissima ma piacente, vedova da
più di tre anni, senza figliuoli; chè il suo primo e unico bimbo l’era
morto in fasce. Viveva con la sua mamma ch’era vedova anch’essa;
ma i due quartierini erano perfettamente disobbligati con porta e
ingresso a parte. Madre e figliuola avevano spiriti indipendenti e
gusti alquanto diversi. Era poi questo spirito indipendente che
rendeva la signora Stella aliena dal rimaritarsi, quantunque, pur che
avesse voluto, non le sarebbero mancate le occasioni. Ma era ben
provvista di beni di fortuna, era in grado di saper difendersi dalle
insidie; che furia doveva avere di rimettere il collo sotto il giogo?
— In verità, Gualberti.... — ella cominciò rivolgendosi all’inatteso
visitatore con aria di mite rimprovero. Ma, al vederlo in abito nero e
cravatta bianca, soggiunse scherzosamente: — Meno male che non
sono che una stazione intermedia.... Via, sedete pure.... Già una
rondine non fa primavera.
Da un pezzo la signora Marioli dava del voi a Gualberti.
— Oh, signora Stella, — egli disse, — non sia così cattiva. Ho
commesso un’indiscretezza, lo so, ma passavo di qui e non ho
potuto resistere alla tentazione. Di giorno non mi riesce mai....
— Mi dispiace che abbiate fatto due volte la strada per nulla. Ero
fuori.... Di venerdì però....
— Il suo giorno di ricevimento?... Non me ne discorra neanche....
Abborro i giorni di ricevimento in generale e il suo in particolare.
— Perchè il mio più degli altri?
— Perchè un salotto pieno di visite è tanto più odioso quanto più è
simpatica la padrona di casa.
— Devo ringraziarvi del madrigale?
— Non è un madrigale; è l’espressione schietta del mio pensiero. E
poi, non lo nego, divento un selvaggio.
— Voi? Non si direbbe. — E la signora Stella diede un’occhiata alla
toilette inappuntabile di Gualberti. Quindi esclamò picchiandosi la
fronte:
— Adesso capisco.... Andate al ballo della contessa Vetturi....
— Sono uscito di casa con questa intenzione, ma....
— Non c’è ma che tenga. Se credete che la Vetturi vi passerebbe
buona la vostra diserzione, v’ingannate a partito.... E fulminerebbe
me se potesse supporre che ne fossi la causa.... Oh non voglio
correre di questi pericoli..... Sono terribili le collere della Vetturi.
— Lei le affronta impavida.... perchè sarà invitata anche lei....
— Oh, il mio è un invito platonico.... Sanno bene che non vado a
feste.... Ma voi....
— Sicchè mi licenzia.... Mi spedisce dalla Vetturi a veder accendere i
lumi.
La signora Stella guardò l’orologio.
— Infatti è presto; sono le nove e tre quarti. Vi concedo di rimaner
fino alle dieci e mezzo, il tempo da prendere una tazza di tè che vi
preparo subito.... Da qui a casa Vetturi ci saranno quindici o venti
minuti di strada; arriverete alle undici; proprio l’ora giusta.
— E dire che rinunzierei tanto volentieri ad andarvi.
— Caro amico, quando non si vuole andare a una festa, non si
comincia col mettersi in frac paré.
— È inesorabile.... Bisogna perdonare alle contraddizioni umane....
Mi son vestito macchinalmente, per forza d’inerzia; adesso domando
a me stesso che cosa vado a fare dalla Vetturi.
— Oh bella, quello che ci faranno gli altri. Ballerete.
— Se non ballo più.
— Vedrete a ballare.
— Non mi piacciono che i balli d’adolescenti.
— Carino, carino. Bals d’enfants addirittura.
— Anche i bals d’enfants. Ma, scherzi a parte, il ballo, se non vuol
essere una cosa ridicola agli occhi di chi vi assiste, deve apparir lo
sfogo ingenuo di una vitalità esuberante. E tale è appunto pei
giovani che hanno un bisogno irresistibile di muoversi, di saltare, di
volare, sarei per dire. Più tardi, dopo vent’anni per le femmine, dopo
venticinque per i maschi, esso non è che un libertinaggio o una
pagliacciata.
— Nientemeno! — proruppe la signora Marioli. — Sicchè voi,
consentaneo ai vostri principî, avete cessato di ballare a
venticinqu’anni?... Io ritenevo....
— Che avessi ballato anche dopo? Lo ammetto. Ma questo non
prova nulla....
— Sarà. Gradirei sapere a ogni modo se ballando nella vostra età
matura facevate atto di libertino, o di...
La signora Stella non finì la frase. Gualberti la finì lui.
— O di pagliaccio? Ecco, siccome l’andar in giro come una trottola
mi pareva supremamente grottesco, crederei piuttosto....
E si fermò lì.
— Intendo, — disse la signora. — La vostra era una colpa di
libertinaggio.... E chi sa che giudizi pronunciavate in cuor vostro sul
conto di quelle povere diavole che accettavano il vostro invito per
una polka o per un valzer?... Buon per me che non abbiamo mai
ballato insieme.
— Io non mi ricordo di averla mai vista ballare, — replicò Gualberti.
— È vero. Anche quando viveva mio marito ballavo pochissimo....
Non in omaggio alla vostra teoria, ma perchè vado soggetta alle
vertigini.... E se ci fosse una signora Gualberti, levatemi una
curiosità.... è il vizio di noi donne l’esser curiose.... come vi
regolereste con la signora Gualberti?
— Eh, chi può dire quel che farei? Se l’amassi, sarei debole, cederei
probabilmente a’ suoi desideri. Ma non nego che dovrei mandar giù
di gran bocconi amari a vederla palleggiata dalle braccia dell’uno a
quelle dell’altro. Senza contare ciò che vien dopo.... Ogni imbecille
che ha ballato con madama si crede in obbligo di portar la mattina
seguente i suoi biglietti da visita, in duplo come le quietanze, che il
marito babbeo è costretto a ricambiare, dando in questo modo la
facoltà a uno stuolo di cretini di venire in casa a corteggiargli la
moglie.
La signora Stella si mise a ridere.
— Che moralista diventate invecchiando, e che marito geloso
sareste!
— Geloso?... Secondo.... Se amassi mia moglie.... Sia sincera,
signora Stella, crede possibile amore senza gelosia?
— No, — ella rispose dopo averci pensato su un momento. — Pur di
non eccedere.
— D’accordo. È come il sale nelle vivande. Non si può farne senza,
ma non si deve abusarne.
— Parlate per aforismi stasera.... Ma torniamo a bomba.... Il vostro
programma coniugale è tuttora oscuro.
— Ha voglia di divertirsi alle mie spalle, lei, — esclamò il cavalier
Gualberti. — Che programma posso avere? È sicuro però che non
adotterei il sistema di coloro i quali nel gran numero di galanti lasciati
ronzare intorno alla moglie vedono una specie di salvaguardia contro
maggiori pericoli.... Tanto più ch’io ho certe opinioni tutte mie.... È la
sera che la faccio maravigliare coi miei paradossi.... Se gliene
dicessi un altro?
Durante questo tempo la signora Stella era stata sempre in piedi
affaccendata intorno alla teiera; adesso il tè era fatto ed ella ne
mescè una tazza al Gualberti, dicendogli:
— Mettete voi a vostro piacere lo zucchero, la panna e il rhum, e
spifferate pure il vostro ultimo paradosso.... Perchè è l’ultimo; sono
già le dieci e mezzo.
— Non baderà poi al minuto. Il mio paradosso è questo. La colpa
che perdono meno alla donna è la civetteria.
— Misericordia! — gridò la padrona di casa alzando le mani al cielo.
— A dir queste cose v’inimicherete l’intera corporazione.... S’è già
passato in giudicato che siamo civette tutte quante?
Gualberti fece un moto vivace di protesta.
— Nemmen per sogno.... Ne conosco una per esempio....
La signora Marioli gli accennò con la mano di non continuare.
— Son io quella, s’intende.... Gualberti, stasera siete in vena
d’originalità; non naufragate in un bicchier d’acqua.
— Le giuro che....
— Tiriamo via.... E invece di perdervi in cerimonie, spiegatevi
meglio.... Per voi dunque la civetteria è un peccato mortale.
— Sarò ingiusto, sarò eccessivo.... Ma già me ne rifaccio con un
eccesso d’indulgenza per altri peccati.
— Oh!... Per esempio?
— Io compatisco la sensualità, compatisco e spesso rispetto la
passione, anche illegittima.
— E non compatite la civetteria?
— No. La sensualità ha qualche cosa d’irresponsabile, è una
malattia del sangue, come la passione è una malattia, una nobile
malattia, del cuore. Sensualità e passione sono necessariamente
sincere; la civetteria non è che un artifizio; è un pervertimento
dell’ingegno rivolto a miseri fini, è un gioco crudele che alla lunga
spegne nell’animo di chi lo fa ogni lampo di gentilezza.... La donna
può esercitarvisi per anni senza restar presa nei lacci che tende; non
importa, ella è mille volte più corrotta, mille volte più condannabile di
quella che ha ceduto all’amore, qualunque sia quest’amore....
— Onde alla signora Gualberti, se ci fosse, — ripigliò in tuono
scherzevole la signora Stella — voi perdonereste un amante, due
amanti....
— Come corre! Veda.... Bisogna distinguere. Quando una donna
maritata ha un amante, il marito è un offeso, e chi è offeso non
guarda tanto pel sottile; non considera il fatto in sè stesso, ma il
danno, la vergogna, il ridicolo che ne deriva a lui.... È probabile ch’io
sarei come gli altri, è possibile che scaccerei dal mio fianco la donna
colpevole.... a’ miei occhi indubbiamente colpevole.... ma non credo
che da una catastrofe di questo genere mi sentirei umiliato come
dall’aver una moglie che tenesse a bada una dozzina di bellimbusti e
girasse tutta la sua vita intorno all’adulterio senza cadervi mai.
— Avete finito?
— Ho finito.
— Ebbene, quantunque siano le dieci e tre quarti, voglio dire anch’io
due parole.... Figuratevi se non protesto in nome del mio sesso
contro le vostre esagerazioni.... Una seconda tazza di tè?
— Sì, grazie, — rispose Gualberti, il quale non domandava di meglio
che di esser trattenuto.
— Nella vostra filippica ci può anche essere un fondo di vero —
soggiunse la signora Stella mentre gli porgeva la tazza colma, —
ma, santo Iddio, le cose che vi dispiacciono le vedete con una lente
d’ingrandimento che vi muta una zanzara in un elefante. Non amo
neppur io la civetta di professione, ma un po’ di civetteria non è poi
quel delitto abbominevole che voi credete. È la nostra arma, la
nostra difesa, la nostra vendetta contro voi altri.
— Una vendetta?
— Appunto, e non è difficile a intenderlo. Con voi uomini una donna
che non sia nè vecchia nè brutta (e già le brutte e le vecchie le
lasciate in disparte) non ha che tre vie da tenere. O consente a
sacrificarvi la sua riputazione, o vi mette alla porta ch’è quello che
meritereste spessissimo, o si prende argutamente gioco di voi.... Ora
capirete che sacrificarvi la propria riputazione è novantanove volte
su cento una follìa, e che il darvi lo sfratto ci condannerebbe
all’isolamento. Non resta quindi che il terzo partito. Con che fronte
venite ad accusarci di finzione, d’artifizio? Siete schietti, siete sinceri
voi altri? Che cosa sono le vostre frasi sdolcinate, le vostre
dichiarazioni patetiche? Sono la bandiera con cui tentate far passare
di contrabbando un vostro desiderio, un vostro capriccio. Voi
c’insultate, noi vi canzoniamo. Ve lo ripeto, io non amo le civette in
genere, però quando sento che una civetta ha corbellato un libertino
biasimo forse la donna, ma in quanto all’uomo dico: Bene gli sta.
— Ah, signora Stella, — replicò Gualberti deponendo la chicchera
sulla tavola. — si capisce che c’è un grande spirito di solidarietà fra
le donne se le migliori prendono con tanto fuoco le parti delle
peggiori!... Cercar scuse alle civette, lei che non ha ombra di
civetteria?
— Ne siete sicuro? E, in ogni caso, credete di farmi un elogio? Se
fosse un difetto? Se per lo meno fosse una disgrazia?
— Come mai?
Ella soggiunse con un sorriso triste:
— Eh caro Gualberti, è quello che dicevo poco fa.... Non volersi
compromettere con uno, non voler prendersi gioco di molti, è il vero
modo di restar sole.
Le parve di essersi lasciata sfuggire qualche parola di troppo e si
alzò bruscamente dalla seggiola.
— Basta così, ormai.... Voi mi avete sciorinato le vostre massime; io
ho rintuzzato la vostra baldanza maschile.... vi ritenete sempre
impeccabili voi uomini.... adesso non mi resta altro che darvi la
felicissima notte e augurarvi buon divertimento dalla Vetturi.
Gualberti s’era alzato anche lui, e stava forse per accommiatarsi
definitivamente, quando ad un tratto abbassò gli occhi sullo sparato
della camicia e mise un piccolo grido.
— Che cos’è accaduto?
— È accaduto.... — rispose alquanto confuso Gualberti — che dalla
Vetturi non ci posso andar più.
— E perchè?
— Perchè m’accorgo d’essermi fatta una macchia di tè sulla camicia.
La signora Stella non potè a meno di sorridere.
— Un altro giorno vi legherete la salvietta al collo.... Per fortuna
avete ancora tempo di passar a casa vostra a mutarvi.
— Questo poi no. Far toilette una volta, transeat, ma due? Non ho
una vocazione così pronunciata per la società. Sia compiacente,
signora Stella, e giacchè sono qui mi permetta di restarci ancora un
pochino. È tanto più gustoso il discorrer con una donna d’ingegno e
di cuore che l’andare a una festa a sentir le solite melensaggini.
Una nuvola si calò sulla fronte della signora Marioli; un sospetto lo
balenò nell’animo. Le venne il dubbio che quella macchia non fosse
accidentale, che Gualberti avesse lasciato cadere apposta una
goccia di tè sulla camicia per avere un pretesto di prolungar la sua
visita. E il pensiero di questo mezzuccio puerile l’offese, e
l’insistenza per rimaner da lei a quell’ora inusata l’afflisse. Ecco,
anche Gualberti del quale ella pregiava infinitamente lo spirito ed il
carattere assumeva dei modi che a lei non potevano convenire.... Le
sarebbe toccato metterlo a posto, forse non riceverlo più come non
aveva ricevuto più tanti altri.... Era un gran dolore.... O forse l’aveva
ella stessa trattato con soverchia familiarità?... Dio buono, che sia
necessario di star sempre in sussiego, di adombrarsi d’ogni atto,
d’ogni parola?... Che ogni minima deferenza debba bastare perchè
un uomo manchi di rispetto?...
— Oh Gualberti — ella ripigliò; e l’intonazione della sua voce
rivelava il suo animo commosso — non mi fate pentire di avervi
perdonata la licenza che vi siete presa.... Perchè io non vi avevo
mai, mai invitato a venire di sera.... Nè voi, nè altri, s’intende.... O
scendo da mia madre, o vado da qualche amica, o rimango sola....
Non è poi la fine del mondo il rimaner sola.... Lo so — ella proseguì,
quasi volendo spiegar a sè stessa la propria condotta — tanti
avevano insistito perchè io ricevessi una sera per settimana.... A che
pro?... Avrei forse potuto ricever soltanto quelli che desideravo?...
No certo, mi sarebbe convenuto subir prima di tutti quelli che
avevano fatto la proposta, i seccatori, gl’importuni, i balordi, quelli
che stimerebbero fallire a un debito d’onore se non facessero la
corte a una donna che non è un mostro, e che non ha nessuno che
la difenda.... Ebbene, no, non era affare per me.... avete ragione,
non sono abbastanza civetta.... D’altra parte, sfido io, una volta detto
di non ricever la sera, come posso fare eccezioni?... Avete avuto
Another random document with
no related content on Scribd:
 "I know," Dorning laughed. "He does drive like a comet. But—er—I'm a
regular snail at the wheel. If Rodrigo doesn't mind——"

"He doesn't," Rodrigo cut in rather sullenly.

John turned eagerly toward her, and she said gayly, "Very well, Mr.
Dorning, and you may drive just as slowly and carefully as you know how."

"Fine," returned John. "We'll take a turn in the park on the way. It's a
wonderful afternoon." He hurried to open the door for her.

"Good afternoon, Count Torriani, the tea was delicious," she said
suavely, dark, ironic eyes upon his grave face. He glanced at the undisturbed
tea things upon the little taboret, shrugged his shoulders, and bent over her
hand. Vexed as he was with her, he could not kiss her hand without feeling a
little emotion within him.

He watched her disappear into the hall. To John Dorning, following her,
he called suddenly, "John, you'll need your hat, won't you?" John
shamefacedly returned for it. Rodrigo handed it to him with a smile.

Rodrigo sank down into a comfortable chair, lighted a cigarette, and

thoughtfully poured himself a cup of the neglected tea.

It was an hour later that John returned, flushed by the wind and
something that had nothing to do with the elements. Rodrigo was still in the
chair, trying to read.

"You didn't mind my running off with Miss Van Zile?" John asked, with
a strange indication in his voice that he didn't care whether his friend minded
or not. He was excited, eager to confide.

"Not at all," returned Rodrigo, "but John——"

John had lighted a cigarette and was walking around the room. "She's
wonderful, isn't she, Rodrigo?" he said suddenly. "A very remarkable and
very beautiful girl. She's never been to New York before, she says. She's
frightened with the city, but eager to see the sights. I've made several
engagements with her to show them to her."
 Rodrigo was silent.

John enthused on. "Rodrigo, if I fell in love, it would be with that kind of
a girl—frank, unspoiled, sweet and lovely. She has something Eastern
women utterly lack. They are all so sophisticated and blasé. You could never
imagine such a woman marrying me for my money, for instance."

Rodrigo wondered if he was a coward. He ought to warn John that he

was playing with dynamite, that this girl was everything that his friend
thought she was not. But John was so utterly absorbed in her. And he,
Rodrigo, had promised Henry Dorning to show his son something of worldly
women. Here was John's opportunity to secure an education. Probably with
no serious results. Elise must be playing with Dorning, and it couldn't last.
She could have no serious intentions toward John. He was exactly the
opposite of the type of man that interested her. Rodrigo, with no sense of
self-flattery, even suspected shrewdly that she had played up to John with
the object of making John's room-mate jealous.

And so, he decided, for the time being, that he would keep silent.

CHAPTER XI

Rodrigo attended a private auction of Flemish art the next morning and
did not reach the office until noon-time. Having glanced through his mail, he
thrust his head into John's office to tell him of the purchases he had made.
He was quite well pleased with himself and was looking forward to
Dorning's commendation on his bargains. Mary Drake was alone in the
office.

"Good-morning, Mary," called Rodrigo. "Has John gone to lunch

already?"

He saw with a little uneasiness that something of the usual warmth with
which she greeted him had fled from her eyes and voice. "Yes, he is lunching
with a Miss Van Zile at the Plaza."

Rodrigo frowned. His high spirits were somewhat quenched. He entered

and walked over toward Mary and sat down. He looked at her a moment,
hesitated, then said abruptly, "Mary, if your best girl friend was attracted to a
chap who you knew was no good, what would you do about it?"

She regarded him seriously and said rather pertly, "I would make very
sure first that my opinion of the man's unworthiness was correct."

"And if you had made sure—then what?"

She gave a little helpless gesture. She was so serious that he was on the
point of asking her what was troubling her. "How can you make sure?" she
asked gravely. And went on, "I used to think that first impressions of people
were instinctively the right ones. That everything after that just had the
effect of clouding things, of leading to wrong judgments. Recently I changed
my mind. I decided that what a person has been in the past has nothing to do
with the present. I thought people could change, could find themselves, and
become new men—or women. Now—I don't know."

He tried to take her delicate, white hand, but it eluded his. "Mary," he
asked softly, "are you thinking of me when you say these things about—first
opinions?"

He took her troubled silence for an affirmation.

"Has someone been talking to you since about me?" he queried

intuitively.

Mary, who was never one for groping about in the dark, replied, "A girl
by the name of Sophie Binner was in this morning. She asked for you. When
she found you weren't here, she grew quite loud and troublesome, and Mr.
Madison referred her to John. I couldn't help but hear some of the
conversation between them, though I left when I discovered its private
nature."

"Sophie Binner?" he repeated, screwing his forehead into a frown. "But I

haven't seen her for several months. She is an actress I knew in England—
and, for a short time, here. But she has been on the road with her company,
and I haven't even written to her."

"You must have written to her some time or other."

"Why, what do you mean, Mary?" He had never seen the usually calm
and capable Mary agitated so. It agitated him in turn. Sophie was not above
making trouble, he knew, especially after the unfriendly manner of their last
parting.

"I don't want you to question me any further, Rodrigo," said Mary
nervously. "I have told you quite all I know. You will have to get the rest
from John. Probably he won't mention it to you. He hates trouble of all kinds
—particularly sordid troubles—and he will be anxious to shield you. And I
think you shouldn't allow yourself to be shielded, in this case."

"Certainly not. I'll ask him what happened at once."

But Rodrigo did not have the opportunity to broach the subject of Sophie
to his partner during the remainder of the day. John did not return from his
luncheon engagement until after three, when he hurried in breezily, a
carnation in his buttonhole and a flush upon his face that caused the
employees out in the gallery to look significantly at each other and smile
approvingly. The head of the concern had never looked so happy. John
closeted himself at once with a couple of art buyers who acted in the
capacity of scouts for Dorning and Son. By the time Rodrigo judged
Dorning was free and went in search of him, John had again disappeared,
this time, Mary said, to dress for dinner.

Rodrigo found John in their apartment, arrayed in his evening clothes,

administering the final touches to his necktie. The Italian told himself a little
ironically that Elise Van Zile had reversed the social order of the day in their
lodgings—now it was John who was donning festive attire almost every
evening and setting out upon social expeditions, and Rodrigo who was left
home to settle in a chair with a book. Formerly it had been the reverse.
Rodrigo remarked banteringly about this.
 "But I have such a wonderful reason for deserting you," John cried.
"How she ever happened to decide to like me, when you were available,
Rodrigo, I don't know. She is such a beautiful creature—she could have the
pick of all the men in the world. And she's just as sweet as she is beautiful.
You don't think that I deliberately went out to oust you from her affections,
do you, Rodrigo?" John spoke so earnestly that Rodrigo gave a short laugh
of reassurance. But there was a note of anxious pity in it also. Poor old John.

"I understand that you saw another friend of mine to-day, also," Rodrigo
said, lighting a cigarette and flicking the match into the open grate.

John dropped his thin fingers from his tie and replied quietly. "Did Mary
tell you? I asked her not to."

"She evidently thought it better that I should know, and I think she is
right, as usual. What did Sophie Binner want of me—and you?"

John walked over to his friend and put his hands upon Rodrigo's
shoulders. He suggested, "Please don't ask me any more about her, Rodrigo.
You'll never see or hear from her again. Why not let it go at that?"

Rodrigo replied impatiently, "I'm not a baby, John, I know more about
women like Sophie than you do. What was she up to?"

John shrugged his shoulders and decided to make a clean breast. "She
looked like the devil—thin and badly dressed. She said her show had failed,
left the whole company stranded out in Pocatello, Idaho. Christy and the
company manager skipped and went back to England. Sophie pawned her
jewels and clothes and just scraped together enough money to get her to
New York. So she came to you for help."

Rodrigo relaxed with relief. "Fair enough," he admitted. "I'll stake her to
a trip home. Why didn't you tell her to go away and come back again when I
was there?"

John hesitated. "She insisted upon some money at once. She had—some
letters from you. I read a couple of them, and they were really pretty serious
stuff, Rodrigo. You were never a calm letter-writer. And writing letters to a
certain type of woman is very had business in this country. There are always
shyster lawyers around ready to pounce upon them and turn them into
money. And she said—well, that you were in her apartment the night her
show opened. She mentioned a colored elevator man whom she could
summon as a witness, if necessary. But, damn it, I don't believe you were,
Rodrigo." John looked at his friend anxiously.

"I was just there for a minute, and it was perfectly harmless," Rodrigo
said at once. "It didn't mean a thing and she probably played it up merely to
give me a black eye with you. As a matter of fact, I recall that the elevator
boy did ride us up and wasn't there when I came down the stairs later. I had a
fearful row with her and she's probably out for revenge. But what's Sophie's
game anyway—blackmail? She can't get away with it."

John replied, "She threatened to sue you for breach of promise to marry
her, said you had jilted her in London once before. She wanted five thousand
dollars to call it off. I knew she didn't have a case, but I thought it was just as
well to keep her quiet. So I gave her two thousand dollars. Then I stopped in
at the apartment house address she gave me and for a fifty dollar bill
persuaded the colored elevator boy that you had never been there."

Rodrigo shook his head and smiled. Was there ever a friend like this
innocent-wise John Dorning?

"You're a prince, John," Rodrigo said sincerely. "But you shouldn't have
done it. You should have let me face the music." He turned almost fiercely
and paced the floor a moment. Returning, he faced John and cried, "I don't
know why you have such a sublime faith in me, John. God knows I've given
you no reason for it. I was in trouble when you first met me. And that wasn't
the first time, as you must have known. And yet you accepted me as a friend
and you gave me a start that's resulted in the happiest time of my life. Now,
damn it, I throw you down again. I guess I'm just bad."

John laid his hand on the Italian's shoulder. "No, I won't have you
condemning yourself. You've been strictly business since you've been over
here, I know. This Binner affair is a carry-over from the past. Your letters
didn't mean anything, even though they sounded pretty intimate. And that
episode in her apartment was just a peculiar combination of circumstances, I
can see that."
 "Oh, don't make me out a saint, John," Rodrigo cried impatiently. "If
those crooks in the hall hadn't jolted it out of my head—oh, well, what's the
use. Once a weakling, always a weakling."

"Not at all," John retorted. "I'll admit there's one kink in your character I
don't understand. I don't see why a chap who is as unselfish, straightforward
and worldly wise as you are, can—well, make a fool out of himself with a
certain type of woman. It's uncanny."

"It's in my blood. I'll never be able to be absolutely sure of myself,"

Rodrigo flung out hopelessly. His hands were nervously fingering the table
against which he was leaning. He was thinking neither of Sophie nor of
Rodrigo. He was seeing the white, disappointed face of Mary Drake, and he
knew now what had been troubling her. It did not occur to him to be thrilled
that she should care enough about him to be troubled. He was afraid, afraid
for his love and his happiness, because he was his own worst enemy. His
nervous, groping fingers closed upon a marble figurine, an exquisite carving
of a hooded cobra, head raised and ready to strike at a tiger. The tiger, about
to spring, had paused and stood, eyes upon the snake, as if fascinated. It was
among his art treasures that he had brought from Italy.

"You'll be sure of yourself," John was saying, "when the right girl comes
along." He smiled, and Rodrigo realized with a pang that John was thinking
of his right girl, Elise Van Zile.

"What chance will I ever have with the right sort of girl when the wrong
sort may come along first?" And Rodrigo too was thinking of Elise. He
suddenly realized that his fingers were digging into something hard until
they hurt. He looked down at the figurine, and lifted it.

"Here I am!" he cried. "I'm this tiger! I never told you why I brought this
figurine with me, why I've always cherished it, have I? Well, one reason is
because my father gave it to me when I was a boy as the memento of a very
exciting afternoon. It happened in India when I was about fourteen years old.
We were riding on an elephant, and we could see over a high wall into a sort
of a lane that led to an enclosure where a chap who used to make a business
of capturing wild animals for museums and circuses kept his stock. He let
the beasts roam around in there, and my father would take me to the other
side of the wall to see them.

"Well, on this afternoon, a big, silky tiger came walking down the lane.
Suddenly, when he was just about opposite us, he stopped short—like this
statue—his head down. He stared at something. We followed his shining
eyes. A cobra had slipped out of the box in which the chap kept his snakes.
The tiger stared as if paralyzed, fascinated, a yard from the snake's head. A
cobra! That's the wrong kind of a girl—a cobra. Mind you, this tiger could
have killed the thing with one blow of his paw. He could have killed a lion,
or scattered a regiment. Yet he stood there, his eyes held by the eyes of the
cobra. All at once he tossed his head up and took a step backward—and the
cobra struck."

"Struck! Did he kill the tiger?"

"I don't know. I felt sick. My father saw how white I was, and we left at
once. Several months later he saw this figurine in a shop in Calcutta and
bought it. He gave it to me."

John looked at him and said slowly, "Perhaps a cobra can't really kill
anything as big and strong as a tiger."

"It can make it bad for him, though. I can remember Dad cursing that he
didn't have a gun with him. A gun! That's you, John. When I've been
walking lately, I've usually had you along, and I've been pretty safe from
cobras."

"It's safer not to go walking at all."

"Well, even a tiger has to have some diversion," Rodrigo tried to lighten
up the serious turn the conversation had taken. As John walked over to the
mirror and resumed his adjusting of his cravat, Rodrigo said suddenly, "And
guns too, John—sometimes guns don't act as they should, very good guns,
too. And cobras raise the dickens with them too."

But John had hardly heard him, much less gotten the meaning of his
friend's cryptic speech. And Rodrigo was instantly glad. John was so
infatuated with Elise that mere words would never undeceive him. It must be
something stronger than words. Likewise, Rodrigo must make very sure that
Elise Van Zile was what he had described to John as the cobra type of
woman.

After John left, Rodrigo sat down and tried to interest himself in a large,
profusely illustrated volume on interior decoration. But he was in no mood
to concentrate upon the hopelessly conventional illustrations and the dry,
prosaic text. He flung the book down at length, and, lighting his pipe,
walked nervously about the apartment. He was thinking of John and Elise
Van Zile, and of himself. His feeling toward the sudden infatuation of his
friend for Mrs. Palmer's niece and Elise's sudden interest in John contained
not one atom of jealousy. Had she been the girl John thought she was,
Rodrigo would have been delighted and would have rendered the match
every assistance.

But Elise, Rodrigo kept telling himself, was the girl he thought she was.
This business to-day of Sophie Binner, this tale of the cobra he had related to
John, this whole raking up of his past had had a depressing effect upon him.
The world looked awry that evening.

He confessed, after fifteen minutes of aimless walking about, that he was

perhaps seeing things through a glass darkly. But of this much he was quite
certain: Elise Van Zile was clever. Though John Dorning was not the type of
man who appealed to her, she might decide to marry him for his money.
Married or single, she would always be selfish, unscrupulous. She wanted a
rich, safe husband.

If the husband were John Dorning, this would bring tragedy.

Having arrived at this conclusion, Rodrigo tried to denounce the whole

thing as nonsense and, catching up his hat, departed from the apartment in
search of something to eat, though he wasn't in the least hungry. He wanted
to get out, get away from himself, get where there were people, noise,
laughter.

He walked over to Broadway and deliberately chose a cheap restaurant

where race-track touts, vaudeville and burlesque actors and actresses,
theatrical agents and motion picture press agents absorb indigestible food.
But the gum-chewing waitresses and clattery crockery failed to divert him.
He hardly touched his food. Rising, he paid what the muscular waitress had
punched on his ticket, and walked back to the apartment through the surging
tide of the Broadway theatre traffic.
 Back in his living-room, he settled down with his book again. But he
could not read. He fell to brooding again. And out of his brooding came
finally a mad plan to save John Dorning. As well have the game as the name,
Rodrigo laughed ironically. He had done so many foolish things for his own
pleasure. God might now forgive this last one if it were done unselfishly, to
save a saint. For John Dorning was almost a saint to Rodrigo. Their
friendship was a thing almost sacred. But it was better to kill even this
sacred thing, Rodrigo reasoned solemnly, than to hurt John Dorning.

So the following afternoon he called Elise Van Zile on the telephone

from his office and, putting into his voice all the mellow intimacy that he
knew so well how to convey, he said, "I have missed you so much, Elise.
John interrupted our last little chat just when it was becoming so interesting,
and took you away. I'd so much more to tell you. We have such a great deal
in common, as you were good enough to say. I'm wondering when I may see
you again."

There was silence for a moment, and then her smooth tones came over
the wire, "Why, certainly. Aunt Helen and I will be delighted to see you any
time."

He lowered his accents. "Not, Auntie—you, you alone. You said you
would like to come again to our apartment. And this time I will promise we
won't be interrupted. Not even by John. I want so badly to see you—Elise.
Won't you come?"

After a long pause, her voice came noncommittedly, "When?"

"On Saturday afternoon at three?"

Another long pause, and then she said faintly, "I shall be there."

Rodrigo hung up the receiver and took a long, deep breath. Then he
walked into John's office and, taking advantage of Mary's temporary
absence, said, "John, I want you to promise me something."

"What is it, old man? And why the terrifically serious look on your
face?"
 Rodrigo forced a smile. "I want you to stay away from the apartment
until three-thirty next Saturday afternoon," he said. "At that time I want you
to meet me there, and probably I'll have something very interesting to show
you?"

"But my birthday isn't until next month, Rodrigo?" John bantered. "Did
you go out and buy that Gainsborough original I fancied so much—or
what?"

"Please don't ask any questions, John. And believe that I'm deadly
serious. Three-thirty. Will you be there?"

"Why, of course—if you say so."

During the rest of the week, Rodrigo was like a man who has had the
date of his electrocution set. He could not work, eat, nor sleep. John
remarked about it. Mary Drake regarded him anxiously from behind his
back.

At noon the following Saturday, Rodrigo heard John leaving his office
and hastened to stop him. He had not reminded John of his engagement of
the afternoon, but now he said,

"You haven't forgotten about coming to the apartment at three-thirty,

have you, John!"

"Oh, I'll be there. You're so darned mysterious about it that you've

aroused my curiosity."

Rodrigo felt a grim satisfaction. He did not purpose to have his

electrocution bungled by the absence of the man who was to turn on the
electricity.

Having with some awkwardness and difficulty disposed of Mrs. Brink,

the housekeeper, who showed a disposition to dawdle at her work so that she
might gossip with him, Rodrigo, at three o'clock that afternoon, was trying
desperately to interest himself in a newspaper. He was arrayed in a purple
silken dressing-gown. Soft cushions were piled invitingly upon the divan.
The shades had been drawn discreetly, so that the room was in a semi-
shadow. Whisky and soda stood upon a slim taboret.

He waited impatiently for fifteen minutes. Then his nerves tingled as he

heard the elevator door outside roll open and someone stepped out into the
corridor. An instant later the apartment bell chimed. Rodrigo gravely arose.
His face broke into an excellent imitation of a smile of hearty welcome. He
opened the door. A freckle-faced, gawky messenger boy grinned on the
threshold, handed him two telegrams and pointed with a chewed stump of a
pencil where to sign in the book.

Rodrigo, mystified and disappointed, broke the envelope of one of the

telegrams. His face turned pale and his chin quivered, like a man suddenly
attacked with a chill, as he read:

Congratulate me. Elise and I married at Greenwich five

minutes ago. I am the happiest man in the world.

JOHN.

He walked falteringly over to the deep armchair and sat down before he
had the courage to open the other yellow container.

Sorry I had to miss our engagement. Just as well perhaps.

Forgive me for influencing John to break his date.

ELISE VAN ZILE.

For the first time in his life, Rodrigo cursed a lady. But mingled with his
resentment against her was a frank tribute to her cleverness. For he hadn't a
doubt in the world now but that Elise had seen through his stratagem and had
taken this decisive step to outwit him.
 CHAPTER XII

One glorious morning, three weeks later, when the June sunshine bathed
Fifth Avenue in a benevolent light and the staff of Dorning and Son edged
over as near the doors and windows as possible and made lugubrious
remarks about their luck at being shut up from the paradise outdoors, the
door of Rodrigo's office was flung open and John Dorning burst in.

"Rodrigo!" he cried, and stood there near the door smiling happily and
blushing furiously, looking wonderfully well and boyish.

Rodrigo sprang up at once and congratulated him heartily.

"I'm the happiest man in the world," John repeated the words of the
fateful telegram, and, Rodrigo admitted, he looked it. His face was bronzed
and suffused with health, the result of many hours upon the golf links and in
the lake adjoining the elaborate Adirondack "lodge" where the Dornings had
been spending their honeymoon. A feeling of relief for the moment and
optimism for the future swept through Rodrigo. Perhaps, after all, he had
misjudged Elise. Though, he told himself, it is a very rare marriage that does
not at least survive the honeymoon.

"Sit right down and tell me how the elopement all happened," invited
Rodrigo gayly, "you old scoundrel."

"Well, to begin at the beginning," said John exuberantly, "I had an

engagement with Elise in the evening on that Saturday you wanted me to
come to the apartment in the middle of afternoon, do you remember?
Around noon-time Elise telephoned me and said she was sorry but she would
have to cancel the evening engagement. She had to go to some charity
committee meeting or other with her aunt. I insisted upon seeing her, and she
finally agreed that we would have luncheon together and go for a short ride
in my car. I told her of my mystery date with you, and we enjoyed a good
laugh about it, old man, though, of course, she insisted upon my keeping it.
And I assure you I had every intention in the world of doing so. But we got
out on the Post Road, and it was such a wonderful afternoon—well, anyway,
I guess I made love to her, and then miraculously she said she would marry
me. I said 'When?' and she replied, 'Oh, it would be so romantic to do it at
once.' She was set against a fussy wedding of any kind. Didn't even want to
let my dad or sister know. I agreed, of course, being darned lucky to get her
any way at all. So we stopped at Stamford. Afterward I telephoned Dad and
sent you a telegram, and we started on our honeymoon."

"Great stuff!" Rodrigo enthused. "John, for a lad who has always fought
shy of the ladies, you certainly put it over in whirlwind style. What are you
going to do now?"

John hitched his chair nearer, beaming with high spirits. "My luck has
kept right on rolling in, Rodrigo. I happened to meet a chap from home at
the place we were staying. He mentioned that Ned Fernald was putting his
new place on the market. It seems Ned isn't so well off as he's supposed to
be, and building the place and outfitting it has strapped him so completely
that now he's anxious to sell. It's a peach of a big house, with lots of ground,
in the Millbank section, a new development. I'm going to get in touch with
Ned, and Elise and I have agreed that if we can arrive at the proper price,
we'll buy it."

Rodrigo averred that it sounded excellent. "But where is the blushing

bride?" he added.

"She's on her way to Greenwich. I just said good-bye to her and her aunt
at Grand Central. She's going to stop with Dad and Alice in Greenwich until
we get a place of our own."

"She's never met your folks, has she?" asked Rodrigo. He wondered
what Henry Dorning would think of his daughter-in-law, whether his
experienced old eyes would penetrate to things in her that his infatuated son
had never dreamed of.

"I'm sure they'll love her as much as I do," John enthused. "They can't
help it. She's the greatest ever. Dad knows Mrs. Palmer, Elise's aunt, very
well, so I got her to go along up."
 Two hours later, he came back into Rodrigo's office to announce that he
was leaving to subway down-town and seek out Edward Fernald, who was a
minor partner in a brokerage house on Nassau Street. John confided further
that he was, as yet, quite unable to settle down to the workaday problems of
Dorning and Son. He was still walking upon air.

"You'll have to put up with my incompetence for a while, till I get used
to the idea of being married to the world's greatest wife," he pleaded
smilingly with Rodrigo.

"Take your time," soothed the latter. "I'll be indulgent. We don't have a
marriage in the firm every day."

"I wish some nice girl like Elise would capture you," John offered
seriously.

Rodrigo laughed. "Oh, that's what all you newlyweds preach to us happy
old bachelors."

Nevertheless he dropped in to see what Mary thought of the returned and

changed Dorning, after John had left.

"Mrs. Dorning is very lucky," said Mary. "John is the sort who will
devote his whole life to making his wife happy."

She said it so positively that she put him a trifle on the defensive. "Any
normal husband would do that, wouldn't he?" he asked a little challengingly.

She was silent a moment, and then she said, evidently out of a troubled
mind and into her typewriter, "Some men aren't equipped to be normal
husbands."

He looked at her gravely, his eyes full of love for her. Some day soon he
was going to have it out with Mary, he told himself. He would have to.
Things couldn't go on with them as they had been. He had called upon her
many times now out of office hours, met her mother, taken Mary to the
theatre, to art exhibitions, and to concerts and the opera. Always he had
avoided making love to her, because he was desperately afraid of losing her
through having his intentions misunderstood. He had wanted, on many
occasions, to sweep her into his arms, to cover her face with kisses, to claim
her for his own, but he was afraid. He could not risk kissing Mary until he
was very sure she loved him. Before the Sophie Binner blackmailing
episode, he had been optimistic about Mary's feelings toward him. But
during the last few months the issue had been cast again into doubt.

Frequently he told himself almost bitterly that if Mary loved him she
would be willing to forget utterly anything that had happened to him in the
past. But this, in his more rational moments, he knew was asking too much.
She was not the sort of girl who rushes blindly into love. Her whole
character and training were influences in the opposite direction. Love must
come upon her gradually. She must be very sure. Americanized though he
was by this time, the very fact that Rodrigo was a man of another country
from her own, with other ideals and up-bringing, made the process of falling
in love with him for this serious-minded American girl groping and slow.
But, once he had won her, he knew that she would be his forever, utterly,
without question or regret. That was Mary Drake's way too.

Two weeks later John Dorning announced that he had bought the Fernald
house, and he eagerly discussed with Rodrigo furnishing the place according
to their high artistic standards. The Italian, on one pretext or another,
declined several invitations to go to Greenwich and look over the Fernald
property and the married Elise. John was insistent that Rodrigo rush up and
congratulate Elise in person, and then just try and deny that John was the
luckiest fellow ever born. Elise had been asking for Rodrigo, John said, had
urged John to invite him up. Rodrigo smiled benevolently, and declined. He
did not, for the time being, wish to face this clever, attractive, and
triumphant young lady.

But, at last, when the John Dornings had actually moved into the Fernald
house and the rare old furniture and objets d'art, which Rodrigo had helped
to select, were installed to the young householder's liking, Rodrigo could no
longer decline the invitation to spend a weekend with them without
offending his friend.

Elise met them at the Greenwich station in a trim new little sedan.
Rodrigo congratulated her heartily, and she gave him very pretty thanks. She
was looking exceptionally alluring, lending an exotic distinction even to the
tweedy sport clothes she was wearing.

"I am especially grateful to you, Rodrigo—I suppose I may call you that
now," she added, "because you were instrumental in bringing John and me
together." Rodrigo glanced at her a little sharply, wondering if there was a
double meaning in this. But her smile was serene, though those enigmatic
eyes were just a little narrower than normal.

"It is glorious out here. I love it," she tossed over her shoulder to him, as
he sat, unusually quiet in the tonneau of the moving car beside his bag and
golf sticks. And as she swept the car into the newly made driveway of their
artistic home of field-stone and stucco, "Aren't we lucky to get this place? It
is the first home of my own that I have ever had. I love every stone in it."

Rodrigo admitted to himself that she was giving an excellent imitation of

a very happy young bride.

John showed him through the house later, and Rodrigo was very sincere
in his praise of their dwelling and its broad, attractive surroundings. The
close-cropped lawn sloped down gradually to a small lake, surrounded by
willow trees, a body of fresh water that eventually found its way into the
neighboring sound. John explained that there was a concrete dam below,
with a private bathing beach of white sand and crystal-clear water. Millbank
was a new development, very much restricted and exclusive, with a fine
nine-hole golf course just across the lake. When Rodrigo cast pleased eyes
upon the links, John recalled that Warren Pritchard, on learning of Rodrigo's
coming, had immediately spoken for the guest's company on Sunday
morning at the Greenwich Country Club.

"I believe Ben Bryon and Lon Sisson are anxious for a revenge match on
account of the beating you and Warren gave them the last time," John
explained, indicating by his tone of voice that he didn't consider the
engagement so pressing as Warren evidently did, and that he would have
preferred to retain Rodrigo's company himself.

"That will be fine," Rodrigo enthused. "That is, if you haven't other plans
for me, John?" John shook his head in the negative.
 He motored to Stamford that evening with his host and hostess and
attended the first night of a polite comedy, destined for its New York
premiere the following week. The play was not particularly interesting, and
Rodrigo paid more attention to the audience than to the stage. It was a mixed
crowd of typical small-towners, well dressed and highly sun-tanned people
from adjacent Long Island Sound resorts, and professionals from Broadway
who were either interested in the production or the players. He recognized
the producer of the piece, a jolly, corpulent individual whom he had met at
the Coffee House Club. They ran into each other in the outside lobby
between the first and second act, and the theatrical admitted blithely that he
had a "flop" and was debating whether to dismiss the company at once and
forfeit his deposit on the lease of the Broadway theatre or chance a
performance in New York.

To Rodrigo, walking down the aisle as the orchestra was playing the
unmelodious prelude to the second act, came the realization anew that Elise
was quite the most striking-looking woman he had ever known. Her creamy
white shoulders billowing up from her black evening dress, her raven hair
sleeked tightly against her skull, her dark eyes either feeling or feigning
vivacious interest as she inclined her head to listen to John's animated
conversation, she was easily the most beautiful person in front or behind the
footlights. He sensed the strong magnetism of her presence as he took the
seat on the other side of her, and she said smilingly to him, "I was telling
John how bad this play is, but he seems only to have noticed that the settings
are in atrocious taste."

"He's right," Rodrigo acknowledged, and, thinking this was rather curt,
added, "And so are you."

"You find the audience more interesting?" she asked shrewdly.

"Yes, part of it," he said quickly, without thinking, and then cursed
himself for betraying that she exerted some of her old spell over him. A
sudden enigmatic smile crinkled her eyes and mouth as she gazed full at him
an instant, then turned abruptly to John.

He played golf with John's brother-in-law and his two companions the
next morning and had the satisfaction of being largely responsible for
another victory for Pritchard and himself. The latter was as tickled as if he
had captured a championship. "Come again next week-end, Rodrigo, and
we'll give these birds a real ride," he proclaimed loudly for the defeated ones'
benefit. But Rodrigo would not promise.

In the afternoon he pleaded pressure of work and an unbreakable dinner

engagement as an excuse for leaving. John protested loudly, but his guest
was adamant. At about five o'clock, they drove him to the station. Elise took
the seat beside him in the tonneau and, just before they reached the station,
she asked, "When are we to see you again? I was in town two or three times
last week. Twice I telephoned John for lunch, and he was too busy or out or
something. The next time, I warn you, I am going to invite you to give me
luncheon, Rodrigo, and you mustn't refuse me." And as if to assure him that
her intentions were innocent, she repeated the same thing to John in a louder
voice. He laughed back and said, "Of course. I want you two to be great
friends."

"Isn't she the most wonderful wife in the world?" John whispered to him
as he grasped the step-rods of the train.

"Yes, she is a wonderful woman," Rodrigo replied sincerely, and looked

over John's head to return her languid wave of good-bye.

Going back in the train, he thought of her and John, and of their chances
for happiness. He recalled the conversation Warren Pritchard had hesitantly
started on the way to the golf links that morning, and then dropped.

"I say, Rodrigo," Warren had begun, after fumbling around obviously for
an opening, "I know it may sound caddish of me, and I shouldn't be talking
this way, but what really do you know of this lady whom my brother-in-law
has married?"

"Oh, I only know her slightly," Rodrigo had replied offhandedly. "She
comes of an excellent San Francisco family, I believe, connected with the
Palmers—your father-in-law knows the Palmers well."

"I wasn't thinking of her family. But will she make old John happy?"

"Why not?"