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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.

To

CECELIA DAVEBSO SALLEthis book is affectionately dedicated

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,