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a University UbrarqCall No. < ' $ $ t Q I" Accesion No.
Title
Thia book should ba returned on or befoVe/the date last marked below
FUNDAMENTAL PRINCIPLES
of
BACTERIOLOGY
This book is produced in juil compliance
with the government's regulations for con-
serving paper and other essential materials.
Fundamental Principles
of
Bacteriology
BY
A. J. SALLE, B.S., M.S., PH.D.Associate Professor of Bacteriology
University of California
Los Angeles
SECOND EDITION
SIXTH IMPRESSION
McGRAW-HILL BOOK COMPANY, INC.
NEW YORK AND LONDON1943
FUNDAMENTAL PRINCIPLES OF BACTERIOLOGY
COPYRIGHT, 1939, 1943, BY THE
McGRAw-HiLL BOOK COMPANY, INC.
PRINTED IN THE UNITED STATES OF AMERICA
All rights reserved. This book, or
parts thereof, may not be reproduced
in any form without permission of
the publishers.
THE MAPLE PRESS COMPANY, YORK, PA.
PREFACE TO THE SECOND EDITION
The advancements that have taken place in all phases of bacteriologysince this book was first published have made it necessary to prepare a
second edition. In order to bring the material up to date, the book has
been thoroughly revised and entirely rewritten.
To mention the chapters that have been revised would mean the
inclusion of the entire book. However, the chapters that show the most
significant changes are the following: The Morphology of Bacteria; The
Microscope, including a brief discussion of the newer results and possi-
bilities with the electron microscope; The Effect of Environment uponBacteria; The Nutrition of Bacteria, including a discussion of the various
growth factors or vitamins required by organisms; The Enzymes of
Bacteria; the Respiration of Bacteria; The Fermentation of Carbo-
hydrates and Related Compounds; Associations of Bacteria, im-biil'mo
the newer work on bacterial antagonisms; Differentiation and Classifica-
tion of Bacteria; Bacteriology of Air; Bacteriology of Soil; Bacterial andVirus Diseases of Plants; and Specific Infections.
The first edition contained a considerable amount of chemistrybecause it is believed that no student can intelligently understand or
pursue research in bacteriology without first having had a sound knowl-
edge of at least inorganic and organic chemistry. This feature of the
first edition has been retained and perhaps emphasized to a greater
extent in the present revision.
Considerably more textbook material has been incorporated in this
edition. Because of this fact it was considered advisable to separate the
laboratory procedures from the text material; otherwise, the book would
have been too bulky. The experimental procedures have been incor-
porated into a laboratory manual to accompany the textbook. The
separation of the laboratory material from the textbook will answer the
objection of those instructors who prefer to use their own manuals.
The author has attempted to give credit for all illustrations and text
material used in the book. Any omissions or errors are entirely uninten-
tional. He wishes to thank his wife and Mitchell Korzenovsky and
Harvey C. Upham for their aid in reading and checking the proof, andalso those who have offered valuable suggestions during the preparationof this manuscript.
Los ANGELES, CALIF.,A. J. oALLE.
December, 1942.
vii
PREFACE TO THE FIRST EDITION
"This is frankly a plea for the return of the dignity and importance of the
preface. Too often the writing of a preface has become a chore, a necessary
evil prescribed by custom. But like many practices which have become common
through familiarity, the original purpose of the preface has perhaps been obscured
by time and by the careless reading habits of the average reader who wishes to
get to the meat of the book as quickly as possible. The preface is a vital part
of the book and for good reasons. . . .
"
"All of us come to a book loaded with prejudices. We are not as impartial
as we think we are. Mention a topic or theme and we can be sure to express a
certain point of view right or wrong. It is the function of the preface to
modify these prejudices by suggesting what presumably are new points of view.
Thus, the preface is an exercise in persuasion. It must break down 'reader
resistance7
;it must put the reader in the proper frame of mind to approach the
reading of the book. If the preface is written with this idea in mind, the reader
will come to the book proper already favorably disposed toward the author.
If the author is inclined to evade actualities, he must then be prepared for reader
apathy and perhaps neglect"
JOHN R. WILBUR.
This book has been written for those who are beginning the study of
bacteriology and especially for those who plan to specialize in the subject.
It is concerned chiefly -with a discussion of the important principles
and facts of bacteriology which a student should acquire in order to
realize to the fullest extent the more advanced work on the subject.
The book is, as its name implies, a textbook on fundamentals. Theauthor has tried at all times to keep this thought in mind in the prepara-tion of the manuscript. The usual textbooks either are too elementaryor do not contain sufficient fundamental material to give the beginningstudent a solid foundation on which to build for more specialized work on
the subject. The author has tried to give explanations of all phenomenadescribed in the book insofar as it is possible to do so, a point which has
been greatly neglected in most texts. The book is profusely illustrated
with chemical formulas because it is believed that no student can intelli-
gently understand bacteriology without first having had at least inorganicand organic chemistry. This statement applies especially to the chap-ters on Biological Stains, Disinfection and Disinfectants, Enzymes of
Bacteria, The Respiration of Bacteria, Protein Decomposition, Industrial
Fermentations, The Bacteriology of Water, and The Bacteriology of Soil.
X PREFACE TO THE FIRST EDITION
The book differs in one important respect from practically all texts on
fundamental bacteriology in that it is written as a combination textbook
and laboratory manual. The experimental portion is not added as
an appendix but is woven into the body of the manuscript under the
appropriate chapters. The textbook material goes hand in hand with
systematically arranged laboratory procedures. It is believed that
bacteriology cannot properly be understood or appreciated unless studied
in conjunction with experimental laboratory work. The incorporation
of laboratory exercises into the body of the book permits the reader better
to understand the textbook material, and the addition of text to the
laboratory portion aids the student better to understand the experimental
procedures.
Sufficient experimental material has been included to meet the
fundamental requirements of briiinniii^ students in the bacteriology
major and of students in tho various divisions of agriculture, forestry,
home economics, sanitary engineering, physical education, hygiene, public
health, etc. The number of experiments should prove ample for a one-
semester course. The author has purposely included a large numberin order that the instructor may make a selection if desired.
The names of the organisms used are those recommended by the
Committee on Classification of the Society of American Bacteriologists.
Although the system does not satisfy everyone, it comes nearer to being a
standard classification than any that has been used before and it is
now in general use in this country.
The author has attempted to indicate in the text the sources of -the
material and illustrations used. He wishes to thank all who have offered
Mijiut^lion- and have been of assistance in the preparation of the manu-
script. He is especially indebted to his wife and to I, L. Shephmeister
for their aid in reading and checking the proof. The author alone accepts
responsibility for any defects that may be inherent in the plan and scopeof the book and for errors that may have escaped detection.
A. J. SALLB.
BERKELEY, CALIFORNIA,
December, 1938.
CONTENTSPAGE
PREFACE TO THE SECOND EDITION vii
PREFACE TO THE FIRST EDITION ix
CHAPTER I
INTRODUCTION
CHAPTER II
THE MICROSCOPE
CHAPTER III
BIOLOGICAL STAINS 26
CHAPTER IV
MORPHOLOGY OF BACTERIA 45
CHAPTER VYEASTS 68
CHAPTER VI
MOLDS 88
CHAPTER VII
TECHNIQUE OF PURE CULTURES 114
CHAPTER VIII
EFFECT OF ENVIRONMENT UPON BACTERIA 132
CHAPTER IX
STERILIZATION 163
CHAPTER XDISINFECTION AND DISINFECTANTS 179
CHAPTER XI
NUTRITION OF BACTERIA 208
CHAPTER XII
ENZYMES OF BACTERIA 238
xi
xii CONTENTS
CHAPTER XIII PAGE
RESPIRATION OF BACTERIA 270
CHAPTER XIV
DECOMPOSITION AND PUTREFACTION OF PROTEINS 307
CHAPTER XVFERMENTATION OF CARBOHYDRATKS AND RELATED COMPOUNDS. . . 327
CHAPTER XVI
DIFFERENTIATION AND CLASSIFICATION OF BACTERIA 358
CHAPTER XVII
DISSOCIATION OF BACTERIA 377
CHAPTER XVIII
ASSOCIATIONS OF BACTERIA 392
CHAPTER XIXBACTERIOLOGY OF AIR 401
CHAPTER XXBACTERIOLOGY OF WATER 415
CHAPTER XXI
BACTERIOLOGY OF MILK AND MILK PRODUCTS 438
CHAPTER XXIIBACTERIOLOGY OF FOOD 466
(HIAFTER XXIII
BACTERIOLOGY OF SOIL 483
CHAPTER XXIVINFECTION AND IMMUNITY 525
CHAPTER XXVBACTERIAL AND VIRUS DISEASES OF PLANTS 546
CHAPTER XXVISPECIFIC INFECTIONS 563
CHAPTER XXVIITHE HISTORY OF BACTERIOLOGY 598
INDEX 619
FUNDAMENTAL PRINCIPLESOF BACTERIOLOGY
CHAPTER I
INTRODUCTION
Bacteriology is the science that deals with the study of the organismsknown as bacteria ^ingiilnr. bacterium). M: M,-
1 !;.: . in its broadest
meaning is the science that deals with the study of all mirmoruniiNm^.
such as bacteria, yeasts, molds, and algae. The word germ is probably
synonymous with bacterium. Although this book will include a dis-
cussion of microorganisms in general, the major portion of the material
will be devoted to the study of bacterial organisms.
Man, who is forever classifying things, has placed living organismsinto either the plant or the animal kingdom. Most living organisms
possess the characteristics of one kingdom or the other and may be sharply
differentiated. However, bacterial microorganisms display the char-
acteristics of both plants and animals and, for this reason, it is not possible
to place them in one group or the other.
Haeckel (1884) believed that considerable confusion could be avoided
if the bacteria were placed in a new kingdom which he named the Protista.
He grouped into this kingdom all microorganisms such as yeasts, molds,
bacteria, protozoa, algae, which were placed with difficulty into the twoolder kingdoms. His suggestion did not gain wide acceptance and
Haeckel finally abandoned the idea. After all, it makes little difference
whether bacteria are plants or animals as long as their fundamental
characteristics have been studied and are understood.
CHARACTERISTICS OF PLANTS AND ANIMALS
Bacteria are among the simplest forms of life known and hence showcharacteristics of both plants and animals. For the sake of convenience
they have been grouped under the plant kingdom. According to Tanner
(1937) some of the characters used to distinguish plants from animals
are given in Table 1.
1
2 FUNDAMENTAL PRINCIPLES OF BACTERIOLOGY
TABLE 1*
Plants Animals
Store energy Liberate energy
Cell walls composed of cellulose (carbo- Cell walls composed of nitrogenous corn-
hydrate) pounds and carbohydrate
Root hairs and stomata absorb H 2 and Possess an alimentary canal in which
gases. No digestion digestion takes place
Take CO 2 and H 2O from the atmosphere Cannot utilize CO 2 and H 2O from the
and nitrogen salts from the soil to build atmosphere. Use organic compounds
up their proteins and carbohydrates. and liberate C(>2. Chemoanalytic
Oxygen liberated. ChemosyntheticVacuoles well-developed Vacuoles absent or not well developed
Nucleoproteins contain a pentose Nucleoproteins contain a desoxypentoseDo not have sensory organs or nervous Have sensory organs and nervous system
system* Reprinted by permission from "Bacteriology A Textbook of Microorganisms," by Tanner,
published by John Wiley & Sons, Inc., New York, 1937.
A condensed classification of the plant kingdom is as follows:
CLASSIFICATION OF PLANTS
Phylum I. Thallophyta. The thallophytes or thallus plants do not have roots, stems,or leaves.
Subphylum 1. Algae. The algae possess the green coloring matter chlorophyll
and are capable of manufacturing their own food from water and carbon
dioxide of the atmosphere in the presence of sunlight.
Class I. Diatomaceaej the diatom ks.
Class II. Cyanophyceae, tho blue-green algae.
Class III. Chlorophyceae, the green algae.
Class IV. Phaeophyceae, the brown algae.
Class V. Rhodophyceae, the red algae.
Subphylum 2. Fungi. The fungi, with the possible exception of a few species,
i do riot contain chlorophyll and are unable to synthesize their own food from
water and carbon dioxide of the atmosphere. They must have their food
materials in a preformed condition. In this group are placed the bacteria,
molds, and yeasts.
Class I. Schizomyceies, the bacteria.
Class II. Saccharomycetes, the yeasts."
Class III. Phycomycetes, the alga-like fungi.
Class IV. Ascomycetcs, the sac fungi.-'
Class V. BasidiomyceteSj the basidia fungi.
Phylum II. Bryophyta. The bryophytes are the mosses.
Class I. Hepaticae, the liverworts.
Class II. Muscineae, the mosses.
Phylum III. Pteridophyta. The pteridophytes are the fern plants.
Class I. FilicaleSj the true ferns.
Class II. Equisetales, the horsetails.
Class III. Lycopodiales, the club mosses.
Phylum IV. Spermatophyta. The spermatophytes includes the seed plants.
Subphylum 1. Gymnospermae, the cone-bearing plants, pines, hemlocks, etc.
Subphylum 2. Angiospermae, the flowering plants.
Class I. Monocotyledons, endogenous plants.
Class II. Dicotyledons, exogenous plants. .
!
INTRODUCTION 3
The characteristics of the classes of the subphylum Fungi, which
includes the bacteria, yeasts, and molds, are as follows:
Subphylum 2. Fungi. The thallus plants have neither roots, stems, nor leaves.
Class I. Schizomycetes. All the organisms are single-celled, with a possible few
exceptions, contain no chlorophyll, and multiply normally by a process of
transverse or binary fission. The cells may be spherical, cylindrical, comma-
shaped, spiral, or filamentous and are often united into chains or into flat
or cubical aggregates.
Class II. Saccharomycetes. The saccharomycetes include the yeasts. Theyare generally easily distinguishable from the bacteria in being larger and in
having well-defined nucleuses. Yeasts multiply by budding, spore formation,
fission, and by copulation, but usually by the process of budding.Class Til. Phycornycetes. The phycornycetes are filamentous alga-like fungi
which do not form cross walls (nonseptate). Sexual spores are producedwhich are known as zygospores.
Class IV. Ascomycetes. The ascomycetes produce spores within sacs known as
/ asci (singular, ascus). The spores are known as ascospores.
Class V. Basidiornycetes. The basidiomycetes reproduce by the formation of
basidia. The mycelium is septate. Asexual spores and chlamydospores are
also formed.
Class VI. Fungi Imperfecti. The fungi in this group are separated from the
other fungi in not having well-defined fruiting bodies. The fungi that cannot
be classified with the Phycornycetes, Ascomycetes, or Basidiomycetes are
placed in this group. Some of the genera of the Fungi Imperfecti are Oidium,
ilidj Endomyces, Torula, Mycoderma, etc.
DISTRIBUTION OF BACTERIA
Bacteria are widely distributed in nature, being found nearly every-
where. They are found in the soil, air, water, foods, in decaying organic
matter of all kinds, on the body surface, within the intestinal tract of
man and animals, etc. The numbers vary from one place to another,
depending upon the environmental conditioas.
Some bacteria are more commonly distributed in certain places than
others. The common occurrence of a species in a certain environment is
spoken of as the natural flora of that particular environment. Changesin the environmental conditions produce changes in the bacterial flora.
Soil^The numbers %and kinds of organisms present in soils depend
upon the kind of soil, quantity of plant and animal debris (humus),
acidity or alkalinity, depth, moisture content, and treatment. Thenumbers decrease with depth, owing to lack of oxygen and food materials.
A rich garden soil contaias many more organisms than a poor uncul-
tivated soil. The great majority of soil organisms are found in the
surface layers.
Air. Bacteria are found in the atmosphere, being carried there byair currents. Organisms do not grow and multiply in air because condi-
tions are not favorable. There is no such thing as a normal atmospheric
4 FUNDAMENTAL PRINCIPLES OF BACTERIOLOGY
flora. The numbers and kinds depend upon location, amount of moisture,
dust particles, wind currents, and the presence of toxic gases. The air
over the ocean far removed from shore is practically free from micro-
organisms. The same holds true for air over high mountains. The air
of city and country differ as to the numbers and kinds of species present.
Dusty rooms usually show considerably more organisms than do rooms
kept free from dust. Bacteria are usually found adhering to particles
of dust. This means that the more particles suspended in air the greater
will be the extent of bacterial contamination. Spores of yeasts, molds,and bacteria are commonly found in air owing to the fact that these
bodies are more resistant to the ultraviolet rays of the sun than are the
vegetative cells producing them. These bodies are a frequent cause of
air contaminations in bacteriological laboratories and, because of their
great resistance to heat, require high temperatures to destroy them.
Water. Most waters contain large numbers of bacterial organisms.
TteT^numbers vary considerably, depending upon the source of the
water, e.g., from deep or shallow wells, springs, rivers, lakes, ponds,
streams, etc. Water polluted with sewage may contain thousands or
even millions of organisms per cubic centimeter. Under some conditions
disease organisms may also be present. Some bacterial species are
constantly present and constitute the natural flora of that water. Usu-
ally fewer bacterial species occur in sea water than in the soil. The
absence of a high bacterial population in sea water is probably due to its
poor qualities as a culture medium.
Foods. Foodstuffs are rarely free from living organisms. Some of
the organisms are of benefit in producing desirable fermentations, such as
occur in the oxidation of alcohol to acetic acid or vinegar, the lactic
fermentation of cabbage to sauerkraut, etc. Frequently undesirable
organisms are found in foods and bring about abnormal changes. Some-
times foods are the cause of certain types of intoxications and disease
processes due to the presence of pnlliogonic o'^.-iiii-ii!*.
Normal udders of cows are probably never free from bacteria, which
means that freshly drawn milk is not sterile. The first milk to be drawn
always contains more organisms than milk drawn at the close of the
milking operation owing to the fact that the bacteria are washed awayfrom the udders early in the process. However, most of the organismsfound in milk are chiefly those which gain entrance during the operationsof milking, handling, and storing. Unless the milk is stored at a low
temperature immediately after collection, these organisms are capableof producing undesirable changes, making the milk unfit for human
consumption.
Bodjk^The outer surface or skin of the body always contains micro-
organisms. The same applies to the alimentary tract and respiratory
INTRODUCTION 5
passages of man and animals. The skin, intestinal contents, and the
respiratory passages contain a normal bacterial flora. These organismsare for the most part harmless. Occasionally some species penetratethe broken skin and intestinal wall, resulting in the establishment of a
disease process. Usually the organisms are destroyed by the defensev
mechanisms of the host. It has been said that as much as one-fourth
of the dry weight of the intestinal contents of man is composed of bacterial
cells.
Escherichia cdi is always found in the large intestine of man. There
arTTofHer organisms present but in an adult on a mixed diet this organism
predominates. The organism E. coli, then, is largely responsible for the
natural flora of the large intestine. Changes in the environmental
conditions produce changes in the bacterial flora. If the diet of an adult
is changed from a high-protein to a high-carbohydrate diet the E. coli
organisms will be gradually reduced in numbers only to be replaced bya much larger organism known as Lactobacillus acidophilus. If this
particular diet is maintained L. acidophilus will now become the pre-
dominating organism of the large intestine.
FUNCTIONS OF BACTERIA
Those who are not familiar with the activities of bacteria usually
believe that the vast majority of them are harmful; that their chief func-
tion in this world is to gain entrance to the body and produce various
kinds of diseases. This statement is entirely erroneous. The great
majority of the bacteria are not only harmless but absolutely necessaryfor the existence of living things. Life could not exist in the completeabsence of bacteria. They are necessary for the disposal of human and
animal carcasses,. The remains of plant crops, plant stubble, leaves,
etc., are converted into soluble compounds by the soil organisms and
made available to the new plants. Some species are capable of taking
nitrogen from the air and converting it into compounds that are utilized
by the plants. In the absence of fertilizers such as animal manures,
nitrates, and ammonium salts, there would be no nitrogen in the soil
were it not for the activities of these organisms. Sulfur and phosphorus,two necessary elements for plant growth, are also converted into soluble
inorganic compounds and absorbed by plant roots.
Fertile soils may always be distinguished from poor soils in containing
greater numbers of viable organisms. If the soil is rich in plant remains,
contains sufficient moisture, and shows the right temperature and hydro-
gen-ion concentration (reaction), many organisms will be present to
attack the plant and animal residues, converting the insoluble and
indiffusible constituents into soluble, diffusible compounds utilizable bythe plants.
6 FUNDAMENTAL PRINCIPLES OF BACTERIOLOGY
Bacteria are necessary for the disposal of .sewage.^. They convert
the insoluble proteins, fats, carbohydrates (cellulose) into soluble odorless
compounds, which may be disposed of in an inoffensive manner.
The souring of milk is the result of bacterial action. This is the
first step in the preparation of butter and various types of cheeses. The
ripening of cheese is brought about by the action of bacteria and molds,
which are responsible for the odors and flavors imparted to cheeses.
These are only a few examples of the part played by the associated .
activities of organisms in nature. Many other useful purposes will be
discussed under the various chapters in this book.
References
BUCHANAN, E. D., and R. E. BUCHANAN: "Bacteriology," New York, The Macmillan
Company, 1938.
COULTER, M. C.: "The Story of the Plant Kingdom," Chicago, University of Chicago
Press, 1935.
HAECKEL, ERNST: "The History of Creation," Vol. II, New York, D. Appleton-
Century Company, Inc., 1884.
TANNER, F. W.: "Bacteriology," New York, John Wiley & Sons, Inc., 1937.
CHAPTER II
THE MICROSCOPE
The compound microscope may be defined as an optical instrument
consisting of a combination of lenses for making enlarged or magnified
images of minute objects. The term is compounded from the two Greek
words, jufcp6s, micro, small, and 0-/co7rei*>, scope, to view.
Bacteria are so small that they cannot be seen with the naked eye.
They must be greatly magnified before they can be clearly seen and
studied. The use of a microscope is, therefore, absolutely indispensable
to the bacteriologist and to the biologist in general.
The student should first understand the principles involved in order
that the microscope may be employed to the greatest advantage. As
Sir A. E. Wright (1907) stated,
Every one who has to use the microscope must decide for himself the ques-
tion as to whether he will do so in accordance with a system of rule of thumb, or
whether he will seek to supersede this by a system of reasoned action based upona study of his instrument and a consideration of the scientific principles of micro-
scopical technique.
GENERAL PRINCIPLES OF OPTICS
A simple microscope, or a single microscope, consists merely of a single
lens or magnifying glass held in a frame, usually adjustable, and often
provided with a stand for conveniently holding the object to be viewed
and a mirror for reflecting the light. A compound microscope differs
from a simple microscope in that it consists of two sets of lenses, one
known as an objective and the other as an eyepiece, commonly^ mountedin a holder known as a body tube (Fig. 1). Accurate focusing is attained
Try a gpecial screw" appliance known as a fine adjustment. Compoundmicroscopes give much greater magnifications than simple microscopesand are necessary for viewing and examining such minute objects as
bacteria.
The path of light through a microscope is illustrated in Fig. 2. The
light, in passing through the condenser, object in Plane I, and objective
lens would form a real and inverted image in Plane II if the ocular or
eyepiece were removed. In the presence of the ocular F the rays are
intercepted, forming the image in Plane III. The real image is then
examined with the eye lens. E of the ocular acting AS a single magnifier
7
8 FUNDAMENTAL PRINCIPLES OF BACTERIOLOGY
and forming a virtual image in Plane IV. The distance between the
virtual image (Plane IV) and the eyepoint is known as the projection
distance. The object is magnified first by the objective lens and second
Eyepiece
Coarse Adjustment Button
Body Tube
Revolving Nosepiece
Inclination
Joint
Stage
AbbeCondenser
Base
FIG. 1. Compound microscope and its parts. (Courtesy of Bauach and Lomb Optical
Company.)
by the ocular or eyepiece. With a tube length 6f 160 mnu (most micro-
scope manufacturers have adopted 160 mm. as the standard tube length),
the total magnification of the microscope is equal to the magnifying
THE MICROSCOPE 9
power of the objective lens multiplied by the magnifying power of the
ocular.
MECHANICAL TUBELENGTH (160mm)
EYEPOINT
REAR INTERMEDIATE
PLANE H
IMAGE OF OBJECT
OF OBJECTIVE
PLANE I
/
/
REAR FOCAL PLANE, /
/
/
/
]U.
FIG. 2. Path of light through a microscope. (From Photomicrography', courtesy of EastmanKodak Company.)
The above magnifications are obtained on a ground glass placed 10 in.
from the ocular of the microscope. After the microscope has been set at
the proper tube length, the total magnification may be computed by
10 FUNDAMENTAL PRINCIPLES OF BACTERIOLOGY
Red
multiplying the magnifying power of the objective by that of the eyepiece
and by one-tenth of the distance from the eyepiece to the ground glass
measured in inches. For example, if the ground glass is placed 10 in.
from the eyepiece of the microscope the total magnification will be as
given on the ocular and objective. If the ground glass is placed 20 in.
from the eyepiece the miiuMific.-iiioM will be twice as great. If placed
5 in. from the eyepiece the magnification will be one-half as great. To
take a specific example:
Magnification of objective 97XMagnification of ocular 10XDistance of ground glass from ocular 7 in.
Total magnification 97 X 10 X 0.7(0.1 X 7) = 679X
It may be seen that almost any degree of m.'iuiiirir.'ithm could be
obtained by using oculars of different
miigmfying powers or by varyingthe length of the draw tube. Even
though the magnifying powers of the
microscope could be greatly increased
in this manner, the amount of detail
that can be seen is not improved since
this is strictly limited by the structure
of light.
Structure of Light. It is generally
agreed that light is transmitted from
luminous bodies to the eye and other
objects by the undulating or vibra-
tional movement of the ether. This
is known as the undulatory or wave
theory of light. Light waves travel
at the rate of about 186,300 miles
per second and the vibrations are transverse to the direction,' of the
propagation of the wave motion.
When a beam of white light is passed through a prism, a spectrum is
obtained in which several colors form a series from deep red through
orange, yellow, green, blue, and indigo to deepest violet. It is knownthat the wave lengths of the various colors are different, that red shows
the longest and violet the shortest waves of the visible spectrum.
The length of a light wave is the distance from the crest of one wave
to the crest of the next (Fig. 3) . The unit of measurement is the angstromunit (A.) which is equal to 1/10,000,000^ mm. or to ^approximately
17350^000^000 "in. The'Visible* spectrum, together with the corresponding
wave" lengths of the light rays in angstrom units, may be represented
Green
BlueFio. 3. Wave lengths of light of dif-
ferent colors.
THE MICROSCOPE 11
as shown in Fig. 4. Visible light waves, ranging in length from 4000 to
7000 A., may be roughly divided into three portions: blue violet, from
4000 to 5000 A.; green, from 5000 to 6000 A.; red, from 6000 to 7000 A.
4000 5000 6000 7000
FIG. 4. Light rays of the visible spectrum and their corresponding wave lengths in ang-strom units.
OBJECTIVES
The objective is the most important lens on a microscope because its
properties may make or mar the final image. The chief functions of the
objective lens are (1) to gather the light rays coming from any point of the
object, (2) to unite the light in a point of the image, and (3) to magnifythe image.
Numerical Aperture. The resolving power of an objective may be
defined as its ability to separate distinctly two small elements in the
structure of an object, which are a short distance apart. The measure
for the resolving powers of an objective is the numerical aperture (N.A.).
The larger the numerical aperture the greater the resolving power of the
objective and the finer the detail it can reveal.
Since the limit of detail or resolving power of an objective is fixed
by the structure of light, objects smaller than the smallest wave length of
visible light cannot be seen. In order to see such minute objects it would
be necessary to use rays of shorter wave length. Invisible rays, such as
ultraviolet light, are shorter than visible rays but, since they cannot be
used for visual observation (photography only), their usefulness is
limited.
The image of an object formed by the passage of light through a
microscope will not be a point but, in consequence of the diffraction of
the light at the diaphragm, will take the form of a bright disk surrounded
by concentric dark and light rings (Fig. 5). The brightness of the central
disk will be greatest in the center, diminishing rapidly toward the edge.
The image cone of light composed of a bright disk surrounded by con-
centric dark and light rings is spoken of as the antipoint. If two inde-
pendent points in the object are equidistant from the microscope lens,
each will produce a disk image with its surrounding series of concentric
dark and light rings. The disks will be clearly visible if completely
separated but, if the images overlap, they will merge into a single bright
area the central portion of which appears quite uniform. The two disks
will not, therefore, be seen as separate images. It is not definitely known
12 FUNDAMENTAL PRINCIPLES OF BACTERIOLOGY
just how close the centers of the images can be and still allow them to be
seen as separate antipoints.
The minimum distance between the images of two distinct object
points decreases as the angle of light AOC (Fig. 2), coming from the object
0, increases. The angle formed by the extreme rays is known as the
aperture of the objective. The ability of the objective lens system to
(d)
FIG. 5. Resolving power of an objective, (a) The rays from the object at O form an
image at /. (b) Distribution of light in the image at /. The bright disk, dd, is surrounded
by concentric dark and light rings, (c) Two independent points in the object O and O',
form two images at / and /'. (d\ The two independent object points O and O' are so close
together that their images overlap at / and /' and merge into a single bright area, the cen-
tral portion of which appears quite uniform. (From Sir Herbert Jackson and H. Moore,Microscope, courtesy of the Encyclopaedia Britannica, Inc.)
form distinct images of two separate object points is proportional to the
trigonometric sine of the angle. The latter, then, is a measure of the
resolving power of the objective. Actually, however, the sine of angle
AOB is used, which is just one-half of angle AOC. This is usually
referred to as sin u. Since the sine of an angle may be defined as the
ratio of the side opposite the angle in a right-angled triangle to the
hypotenuse then,