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[Illustration: PLATE I

ANTHONY VON LEEUWENHOEK

Who first saw bacteria]




  THE FUNDAMENTALS
  OF
  BACTERIOLOGY

  BY
  CHARLES BRADFIELD MORREY, B.A., M.D.

  PROFESSOR OF BACTERIOLOGY AND HEAD OF THE DEPARTMENT
  IN THE OHIO STATE UNIVERSITY,
  COLUMBUS, OHIO


  ILLUSTRATED WITH 171 ENGRAVINGS AND 6 PLATES

  _Second Edition, thoroughly Revised_

  [Illustration: Publisher’s logo]

  LEA & FEBIGER
  PHILADELPHIA AND NEW YORK
  1921

  COPYRIGHT
  LEA & FEBIGER
  1921


  TO
  GRACE HAMILTON MORREY
  AMERICAN PIANIST




PREFACE TO SECOND EDITION


The first edition seems to have fulfilled a need for a general
text-book on the subject of bacteriology. The original method of
presentation is preserved. The text-book idea is adhered to, so that
the individual instructor may have full liberty to expand on topics in
which he is especially interested. A number of illustrations have been
added, the text has been improved in many instances by the addition
of further explanatory matter and the most recent general advances in
the Science. Examples are the System of Classification of the Society
of American Bacteriologists, which is used throughout the text, their
Key to the Genera of Bacteria, a discussion of the H-ion concentration
method of standardization, the selective action of anilin dyes, the
mechanism of entrance of pathogenic organisms into the body, a more
detailed explanation of the origin of antibodies, the nature of
antigens and a table of antigens and antibodies.

Professor Vera McCoy Masters has assisted in the revision by aiding
in the preparation of manuscript and the reading of proof and in the
making of the index, for which services the author’s thanks are hereby
expressed.

    C. B. M.
    Columbus, Ohio, 1921.




PREFACE TO FIRST EDITION


An experience of nearly twenty years in the teaching of Bacteriology
has convinced the author that students of this subject need a
comprehensive grasp of the entire field and special training in
fundamental technic before specializing in any particular line of work.
Courses at the University are arranged on this basis. One semester is
devoted to General Bacteriology. During the second semester the student
has a choice of special work in Pathogenic, Dairy, Soil, Water, or
Chemical Bacteriology. A second year may be devoted to advanced work in
any of the above lines, to Immunity and Serum Therapy, or to Pathogenic
Protozoa.

This text-book is intended to cover the first or introductory
semester’s work, and requires two classroom periods per week. Each
student is compelled to take two laboratory periods of three hours per
week along with the class work. The outline of the laboratory work is
given at the end of the text. Results attained seem to justify this
plan. A text-book is but one of many pedagogical mechanisms and is not
intended to be an encyclopedia of the subject.

The author makes no claim to originality of content, since the facts
presented are well known to every bacteriologist, though the method
of presentation is somewhat different from texts in general. During
the preparation of this work he has made a thorough review of the
literature of Bacteriology, covering the standard text-books as well
as works of reference and the leading periodicals dealing with the
subject. Thus the latest information has been incorporated.

No attempt has been made to give detailed references in a work of this
character.

The photomicrographs are original except where otherwise indicated
and are all of a magnification of one thousand diameters where no
statement to the contrary appears. These photographs were made with
a Bausch & Lomb Projection Microscope fitted with a home-made camera
box. Direct current arc light was used and exposures were five to ten
seconds. Photographs of cultures are also original with a few indicated
exceptions. All temperatures are indicated in degrees centigrade.

For use of electrotypes or for prints furnished the author is indebted
to the following: A. P. Barber Creamery Supply Company, Chicago, Ill.;
Bausch & Lomb Optical Company, Rochester, N. Y.; Creamery Package
Manufacturing Company, Chicago, Ill.; Davis Milk Machinery Company,
North Chicago, Ill.; Mr. C. B. Hoover, Superintendent of Sewage
Disposal Plant, Columbus, O.; Mr. C. P. Hoover, Superintendent of Water
Filtration Plant, Columbus, O.; The Hydraulic Press Manufacturing
Company, Mt. Gilead, O.; Loew Manufacturing Company, Cleveland, O.;
Metric Metal Works, Erie, Pa.; Sprague Canning Machine Company,
Chicago, Ill.; U. S. Marine Hospital Service; Wallace and Tiernan
Company, New York City, N. Y.

For the preparation of many cultures and slides, for great assistance
in the reading of proof and in the preparation of the index, Miss Vera
M. McCoy, Instructor in Bacteriology, deserves the author’s thanks.

The author trusts that the book will find a place in College and
University courses in Bacteriology.

    C. B. M.




CONTENTS


  Historical Introduction--Spontaneous Generation--Causation
  of Disease--Putrefaction and Fermentation--Study of
  Forms--Chronological Table                                        17

  CHAPTER I.

  Position of Bacteria--Relationships to
  Algæ--Yeasts--Molds--Protozoa                                     37


  PART I.

  MORPHOLOGY.

  CHAPTER II.

  Cell Structures--Cell Wall--Protoplasm--Plasmolysis
  --Plasmoptysis--Nucleus--Vacuoles--Capsules--Metachromatic
  Granules--Flagella--Spores                                        41

  CHAPTER III.

  Cell Forms--Coccus--Bacillus--Spirillum--Involution Forms         52

  CHAPTER IV.

  Cell Groupings                                                    55

  CHAPTER V.

  Classification--Migula’s--Society of American
  Bacteriologists’--Key to the Latter                               59


  PART II.

  PHYSIOLOGY.

  CHAPTER VI.

  Occurrence--General Conditions for
  Growth--Moisture--Temperature--Light--Oxygen--
  Osmotic Pressure--Electricity--X-rays and Radium
  Emanations--Pressure--Mechanical Vibration                        71

  CHAPTER VII.

  Chemical Environment--Reaction of Medium--Chemical
  Composition                                                       81

  CHAPTER VIII.

  Chemical Environment (Continued)--General Food
  Relationships--Metabolism of Elements                             86

  CHAPTER IX.

  Physiological Activities--Fermentation of
  Carbohydrates--Splitting of Fats                                  93

  CHAPTER X.

  Physiological Activities (Continued)--Putrefaction of
  Proteins--Cycles of Nitrogen, Carbon, Sulphur, Phosphorus        102

  CHAPTER XI.

  Physiological Activities (Continued)--Production of
  Acids, Gases, Esters, Alcohols, Aldehydes, Aromatic
  Compounds--Phosphorescence--Chromogenesis--Reduction--
  Oxidation--Production of Heat--Absorption of Free
  Nitrogen--Nitrogen Nutrition of Green Plants                     110

  CHAPTER XII.

  Physiological Activities (Continued)--Production of
  Enzymes--Discussion on Enzymes--Toxins--Causation of
  Disease                                                          121

  CHAPTER XIII.

  Disinfection--Sterilization--Disinfectants--Physical
  Agents--Pasteurization                                           130

  CHAPTER XIV.

  Disinfection and Sterilization (Continued)--Chemical
  Agents--Anilin Dyes                                              156

  CHAPTER XV.

  Disinfection and Sterilization (Continued)--Choice
  of Agent--Standardization of Disinfectants--Phenol
  Coefficient--Practical Sterilization and Disinfection            164


  PART III.

  THE STUDY OF BACTERIA.

  CHAPTER XVI.

  Culture Media--Broth, Milk, Gelatin, Agar, Potatoes, Blood
  Serum--Standardization of Media--H-ion Concentration
  Method--Synthetic Media                                          171

  CHAPTER XVII.

  Methods of Using Culture Media--Culture Tubes--Plates--
  Anaërobic Cultures--Vignal Tubes--Fermentation Tubes--
  Deep Culture Tubes--Novy Jars--Inoculation of Culture Media      184

  CHAPTER XVIII.

  Isolation of Bacteria in Pure Culture--Dilution
  --Plating--Streaking--Barber Apparatus--Aids in
  Isolation--Heat--Selective Antiseptics--Selective
  Food---Indicators--Animal Inoculation                            194

  CHAPTER XIX.

  Study of the Morphology of Bacteria--Bacteriological
  Microscope--Hanging Drop Slides--Staining--Gram’s
  Method--Spores--Acid-fast Bacilli--Capsules--
  Flagella--Metachromatic Granules                                 200

  CHAPTER XX.

  Study of the Physiology of Bacteria--Temperature
  --Incubators--Thermal Death Point--Oxygen Relationships
  --Study of Physiological Activities--Appearance of Growth
  on Culture Media--Appearance of Molds on Plate Cultures          213

  CHAPTER XXI.

  Animal Inoculation--Material for Bacteriological
  Examination                                                      227


  PART IV.

  GENERAL PATHOGENIC BACTERIOLOGY.

  CHAPTER XXII.

  Introduction--Infection--Acute Infection--Chronic
  Infection--Specific--Non-specific--Koch’s
  Postulates--Virulence--Susceptibility                            231

  CHAPTER XXIII.

  Pathogenic Bacteria Outside the Body--As Saprophytes--As
  Facultative Saprophytes--Latent--Carriers--Universal
  Carriers--Accidental Carriers--Necessary Intermediate Hosts      237

  CHAPTER XXIV.

  Channels of Infection--Skin--Mucosæ--Respiratory Tract
  --Alimentary Tract--Mechanism of Entrance of Organisms
  --Dissemination in the Body--Paths of Elimination--
  Specificity of Location                                          243

  CHAPTER XXV.

  Immunity--Natural--Artificial--Active--Passive--
  Production of Immunity--Vaccine--Antiserum--Practical
  Applications of Immunity Reactions                               250

  CHAPTER XXVI.

  Theories of Immunity--Pasteur--Chauveau--Baumgärtner
  --Metchnikoff--Ehrlich--Principles of Ehrlich’s Theory           256

  CHAPTER XXVII.

  Ehrlich’s Theory (Continued)--Receptors of the First
  Order--Antitoxin--Antienzyme--Preparation of Antitoxins
  --Units                                                          261

  CHAPTER XXVIII.

  Ehrlich’s Theory (Continued)--Receptors of the Second
  Order--Agglutinins--Agglutination Reaction--Precipitins
  --Precipitin Test                                                265

  CHAPTER XXIX.

  Ehrlich’s Theory (Continued)--Receptors of the Third
  Order--Cytolysins--Amboceptor--Complement--Anti-amboceptors
  --Antisnake Venoms--Failure of Cytolytic Serums in
  Practice--Complement-fixation Test                               271

  CHAPTER XXX.

  Phagocytosis--Opsonins--Opsonic Index--Bacterial
  Vaccines--Preparation of--Use of--Lipovaccines
  --Aggressins                                                     280

  CHAPTER XXXI.

  Anaphylaxis--Author’s Theory--Tuberculin Test--Table of
  Antigens and Antibodies--Summary of Immunity as Applied to
  Protection from Disease                                          289




BACTERIOLOGY.




HISTORICAL INTRODUCTION.


Bacteriology as a science is a development of the latter half of the
nineteenth century. It may be said to have begun in the decade between
1870 and 1880, due largely to the wide circulation given to Koch’s
work in proving that _Bacillus anthracis_ is the cause of Anthrax in
1876, in devising new culture methods and in demonstrating that wound
infections are due to microörganisms, 1878. Associated with this
work were the great improvements in the microscope by Abbé and the
introduction of anilin dyes for staining bacteria by Weigert. These
results attracted workers throughout the world to the “new science.”
Nevertheless, this work of Koch’s was preceded by numerous observations
and experiments which led up to it. Certainly the most important
discoveries immediately responsible were those of Pasteur. He must be
considered as the greatest of the pioneer bacteriologists since he
worked in all fields of the subject. Some of the antecedent work was
done in attempting to disprove the old “spontaneous generation” theory
as to the origin of organisms; some in searching for the causes of
disease and some in the study of fermentation and putrefaction.


SPONTANEOUS GENERATION.

Speculation as to the first origin of life is as old as history and
doubtless older. Every people of antiquity had its own legends, as for
example, the account in Genesis. This question never can be definitely
settled, even though living matter should be made in the laboratory.

The doctrine of the “spontaneous origin” of particular animals or
plants from dead material under man’s own observation is a somewhat
different proposition and may be subjected to experimental test. The
old Greek philosophers believed it. Anaximander (B.C. 610-547) taught
that some animals are derived from moisture. Even Aristotle (B.C.
384-322) said that “animals sometimes arise in soil, in plants, or
in other animals,” _i.e._, spontaneously. It can be stated that this
belief was general from his day down through the Dark and Middle Ages
and later. Cardano (A.D. 1501-1576) wrote that water gives rise to
fish and animals and is also the cause of fermentation. Van Helmont
(1578-1644) gives directions for making artificial mice. Kircher
(1602-1680) describes and figures animals _produced under his own eyes_
by water on plant stems.

However, many thinkers of the seventeenth century doubted the truth
of this long-established belief. Francesco Redi (1626-1698) made a
number of experiments which tended to prove that maggots did not
arise spontaneously in meat, as was generally believed, but developed
only when flies had an opportunity to deposit their eggs on the
meat. It seems that by the latter part of this century the idea that
organisms large enough to be seen with the naked eye could originate
spontaneously was generally abandoned by learned men.

The work of Leeuwenhoek served to suspend for a time the subject of
spontaneous generation, only to have it revived more vigorously later
on. He is usually called “The Father of the Microscope,” though the
compound microscope was invented probably by Hans Zansz or his son
Zacharias, of Holland, about 1590. Leeuwenhoek used a simple lens,
but his instruments were so much more powerful that they opened up an
entirely new and unknown world. (Fig. 1.)

Anthony van Leeuwenhoek (1632-1723) was apprenticed to a linen draper
and accumulated a comfortable fortune in this business. He became
interested in the grinding of spectacle lenses, then an important
industry in Delft, Holland, where he lived, and did a great deal of
experimental work in this line, mainly for his own enjoyment. Finally
he succeeded in making a lens so powerful that he could see in water
and various infusions very minute living bodies never before observed.
Leeuwenhoek contributed 112 papers to the Royal Society of Great
Britain, the first in 1673, many of them accompanied by such accurate
descriptions and drawings, for example a paper submitted September
12, 1683, that there is no doubt that he really saw bacteria and was
the first to do so (Fig. 2). Rightly may he be styled “The Father
of Bacteriology,” if not of the microscope. He says in one paper:
“With the greatest astonishment I observed that everywhere through
the material I was examining were distributed _animalcules_ of the
most microscopic dimness which moved themselves about in a remarkably
energetic way.” Thus he considered these living objects to be animals,
from their motion, and this belief held sway for nearly two hundred
years.

[Illustration: FIG. 1.--Leeuwenhoek’s Microscope. A is the simple
bi-convex lens held firmly in place. In front of this is the small
table, B, with the support, C, on the tip of which the object to be
examined was held. This support could be brought nearer to or removed
further away from the lens and held firmly in place by the screw, D.
E is a second screw for raising or lowering the entire table. A concave
mirror that Leeuwenhoek sometimes used to focus more light on the
object under examination, is shown at the right.]

Leeuwenhoek was a pure observer of facts and made no attempt at
speculation, but his discoveries soon started the theorists to
discussing the origin of these minute organisms. Most observers, as
was probably to be expected, believed that they arose spontaneously.
Needham, in 1749, described the development of microörganisms
around grains of barley in water. Bonnet, in 1768, suggested that
probably Needham’s animalcules came from ova in the liquid. The Abbot
Spallanzani, in 1769, called attention to the crudeness of Needham’s
methods and later, in 1776, attempted to disprove spontaneous origin
by heating infusions of organic material in flasks and then _sealing_
them. His critics raised the objections that heating the liquids
destroyed their ability to support life, and that sealing prevented
the access of fresh air which was also necessary. The first objection
was disproved by the accidental cracking of some of the flasks which
thereafter showed an abundant growth. This accident seemed also to
support the second objection, and Spallanzani did not answer it. Though
Spallanzani’s experiments failed to convince his opponents, they led to
important practical results, since François Appert, in 1810, applied
them to the preserving of fruits, meats, etc., and in a sense started
the modern canning industry.

[Illustration: FIG. 2.--The first drawings of bacteria by Leeuwenhoek.
The dotted line _C-D_ indicates the movement of the organism.]

[Illustration: FIG. 3.--Schultze’s experiment. The set of bulbs next to
the face contained KOH and the other set concentrated H₂SO₄. Air was
drawn through at frequent intervals from May until August but no growth
developed in the boiled infusion.]

From Spallanzani to Schultze, there were no further experiments to
prove or disprove spontaneous generation. Schultze, in 1836, attempted
to meet the second objection to Spallanzani’s experiment, _i.e._,
the exclusion of air, by drawing air through his boiled infusions,
first causing it to bubble through concentrated sulphuric acid to
kill the “germs” (Fig. 3.). His flasks fortunately showed no growths,
but his critics claimed that the strong acid changed the properties
of the air so that it would not support life. This experiment of
Schultze’s, though devised for a different purpose, was really the
first _experiment_ in the use of _chemical disinfectants_, though
Thaer (page 31) had used chemicals in a practical way. Schwann, in
1837, modified this experiment, by drawing the air through a tube
heated to destroy the living germs (Fig. 4). His experiments were
successful but the “spontaneous generation” theorists raised the same
objection, _i.e._, the change in the air by heating. This was the first
_experiment_ in which the principle of “_dry heat_” or “_hot air_”
sterilization was used. Similar arguments were brought forward, also
to the use of _cotton plugs_ as filters by Schroeder and Dusch in 1859
(Fig. 5). This was the first use of the principle of _sterilization by
filtration_. It remained for Chevreuil and Pasteur to overcome this
objection in 1861 by the use of flasks with long necks drawn out to a
point and bent over. These permitted a full access of air by diffusion
but kept out living germs, since these cannot fly but are carried
mechanically by air currents or fall of their own weight (Fig. 6.).
Hoffman, the year before (1860), had made similar experiments but these
remained unnoticed. The Pasteur flasks convinced most scientists that
“spontaneous generation” has never been observed by man, though some
few, notably Dr. Charlton Bastian, of England, vigorously supported the
theory from the early seventies until his death in November, 1915.

[Illustration: FIG. 4.--Schwann’s experiment. After boiling, as shown
in the diagram, and cooling, air was drawn into the flask by aspiration
while the coiled tube was kept hot with the flame.]

[Illustration: FIG. 5.--Schroeder and Dusch’s experiment. The
aspirating bottle drew the air through the flask after it had been
filtered by the cotton in the tube.]

[Illustration: FIG. 6.--Pasteur’s flask.]

[Illustration: FIG. 7.--Tyndall’s box. One side is removed to show
the construction. The bent tubes at the top are to permit a free
circulation of air into the interior. The window at the back has one
corresponding in the front (removed). Through these the beam of light
sent through from the lamp at the side was observed. The three tubes
received the infusion and were then boiled in an oil bath. The pipette
was for filling the tubes. (Popular Science Monthly, April, 1877.).]

John Tyndall, in combating Bastian’s views showed that boiled infusions
left open to the air in a closed box through which air circulated
did not show any growth of organisms provided the air was so free of
particles that the path of a ray of light sent through it from side to
side could not be seen (Fig. 7). Or if such sterilized infusions were
exposed to dust-free air, as in the high Alps, the majority showed
no growth, while all infusions in dusty air did show an abundance of
organisms. Tyndall’s experiments confirmed those of Pasteur and his
predecessors and showed that the organisms developed from “germs”
present in the air falling into the liquids and not spontaneously.

While Tyndall’s experiments were of great value as indicated, they
probably were harmful in another way. These “germs in the air” were
considered by bacteriologists as well as laymen to include necessarily
many _disease germs_ and to indicate the very general, if not
universal, presence of these latter _in the air_. This idea led to many
erroneous practices in sanitation and disinfection which even to this
day are not eliminated.


CAUSATION OF DISEASE.

The transmission of disease from person to person was recognized by the
ancients of European and Asiatic countries. Inoculation of smallpox was
practiced in China and India probably several thousand years ago and
was introduced by Lady Mary Wortley Montague into England in 1721, from
Constantinople. These beliefs and practices do not seem to have been
associated with any speculations or theories as to the cause of the
disease.

Apparently the first writer on this subject was Varo, about B.C. 70,
who suggested that fevers in swampy places were due to invisible
organisms. The treatment of wounds during the thirteenth and fourteenth
centuries by hot wine fomentations and by the application of plasters
was based on the theory that the _air_ brought about conditions in the
wounds which led to suppuration. These practices were indeed primitive
antisepsis, yet were not based on a _germ theory_ of the conditions
which were partially prevented. Fracastorius (1484-1553), in a work
published in 1546, elaborated a theory of “disease germs” and “direct
and indirect contagion” very similar to modern views, though based
on no direct pathological knowledge. Nevertheless Kircher (mentioned
already) is usually given undeserved credit for the “contagium vivum”
theory. In 1657 by the use of simple lenses he observed “worms” in
decaying substances, in blood and in the pus from bubonic plague
patients (probably rouleaux of corpuscles in the blood, certainly not
bacteria in any case). Based on these observations and possibly also on
reading the work of Fracastorius, his theory of a “living cause” for
various diseases was published in 1671, but received little support.

The discoveries of Leeuwenhoek which proved the existence of
microscopic organisms soon revived the “contagium vivum” idea of
Kircher. Nicolas Andry in a work published in 1701 upheld this view.
Lancisi in 1718 advanced the idea that “animalcules” were responsible
for malaria, a view not proved until Laveran discovered the malarial
parasite in 1880.[1] Physicians ascribed the plague which visited
Southern France in 1721 to the same cause, and many even went so far as
to attribute all disease to animalcules, which brought the theory into
ridicule. Nevertheless the “contagium vivum” theory survived, and even
Linnaeus in his _Systema Naturæ_ (1753-6) recognized it by placing the
organisms of Leeuwenhoek, the contagia of diseases and the causes of
putrefaction and fermentation in one class called “Chaos.”

Plenciz, a prominent physician and professor in the Vienna Medical
School, published in 1762 a work in which he gave strong arguments for
the “living cause” theory for transmissable diseases. He taught that
the agent is evidently transmitted through the air and that there is
a certain period of incubation pointing to a multiplication within
the body. He also believed that there was a specific agent for each
disease. His writings attracted little attention at the time and the
“contagium vivum” theory seems to have been almost lost sight of for
more than fifty years. Indeed, Oznam, in 1820, said it was no use to
waste time in refuting hypotheses as to the animal nature of contagium.

Isolated observers, were, however, keeping the idea alive, each in
his own locality. In 1787 Wollstein, of Vienna, showed that the pus
from horses with glanders could infect other horses if inoculated
into the skin. Abilgaard, of Copenhagen, made similar experiments at
about the same time. In 1797 Eric Viborg, a pupil of Abilgaard’s,
published experiments in which he showed the infectious nature not
only of the pus but also of the nasal discharges, saliva, urine, etc.,
of glandered horses. Jenner in 1795-98 introduced vaccination as a
method of preventing smallpox. This epoch-making discovery attracted
world wide attention and led to the overcoming of this scourge which
had devastated Europe for centuries, but contributed little or nothing
to the question of the causation of disease. Prevost’s discovery of
the cause of grain rust (_Puccinia graminis_) in 1807 was the _first
instance of an infectious disease of plants_ shown to be _due to a
microscopic plant organism_, though not a bacterium in this case.

Doubtless one reason why the work on glanders and grain rust attracted
little attention among the practitioners of human medicine was owing to
the prevalent belief in man’s complete separation from all lower forms
of life. The evolutionists had not yet paved the way for experimental
medicine.

In 1822 Gaspard showed the poisonous nature of material from infected
wounds by injecting it into animals and causing their death. Tiedemann
(1822), Peacock (1828) described “little bodies” in the muscles of
human cadavers which Hilton (1832) considered to be parasitic in
nature. Paget (1835) showed that these bodies were round worms and
Owen (1835) described them more accurately and gave the name _Trichina
spiralis_ to them. Leidy (1846) found organisms in the muscles of
hogs which he considered to be the same as Owen’s Trichina and paved
the way for the work of Zenker (1860) in showing the pathological
relation between the Trichina of pork and human Trichinosis. Bearing
on the “contagium vivum” theory was the rediscovery of the “itch mite”
(_Sarcoptes scabiei_) by Renucci (1834), an Italian medical student.
This had been declared several hundred years before but had been lost
sight of. Chevreuil and Pasteur, in 1836, showed that putrefaction did
not occur in meat protected from contamination, and suggested that
wound infection probably resulted from entrance of germs from without.
Bassi, investigating a disease of silkworms in Italy, demonstrated
that a certain mold-like fungus (_Botrytis bassiana_) was the cause in
1837. This was the _first instance of a microscopic vegetable organism_
proved to be capable of _causing disease in an animal_.

Boehm, in 1838, observed minute organisms in the stools of cholera
patients and conjectured that they might have a causal connection
with the disease. Dubini of Milan in 1838 discovered the _Ankylostoma
duodenale_ which later was further described by Omodei in 1843 and
shown to be the cause of Egyptian chlorosis by Griesinger (1851).
The fungous nature of favus, a scalp disease, was recognized by
Schönlein in 1839, and the organism was afterward called “_Achorion
schoenleinii_.” Berg, in 1839-41, showed that thrush is likewise due to
a fungus, “_Oidium albicans_.”

These discoveries led Henle, in 1840, to publish a work in which
he maintained that all contagious diseases must be due to living
organisms, and to propound certain postulates (afterward restated
by Koch and now known as “Koch’s postulates” p. 233) which must be
demonstrated before one can be sure that a given organism is the
specific cause of a given disease. The methods then in vogue and the
instruments of that period did not enable Henle to prove his claims,
but he must be given the credit for establishing the “contagium vivum”
theory on a good basis and pointing the way for men better equipped to
prove its soundness in after years.

[Illustration: PLATE II

SIR JOSEPH LISTER]

In 1842-43 Gruby showed that Herpes tonsurans, a form of ringworm, is
due to the fungus _Trichophyton tonsurans_. Klencke, in 1843, produced
generalized tuberculosis in a rabbit by injecting tuberculous material
into a vein in the ear, but did not carry his researches further.
In 1843, Doctor Oliver Wendell Holmes wrote a paper in which he
contended that puerperal fever was contagious. Liebert identified the
_Peronospora infestans_ as the cause of one type of potato rot in 1845.
The skin disease Pityriasis (tinea) versicolor was shown to be due to
the _Microsporon furfur_ by Eichstedt in 1846. In 1847 Semmelweiss of
Vienna recommended disinfection of the hands with chloride of lime by
obstetricians because he believed with Holmes in the transmissibility
of puerperal fever through poisons carried in this way from the
dissecting room but his theories were ridiculed.

[Illustration: PLATE III

ROBERT KOCH]

Pollender, in 1849, and Davaine and Rayer, in 1850, independently
observed small rod-like bodies in the blood of sheep and cattle
which had died of splenic fever (anthrax). That Egyptian chlorosis,
afterward identified with Old World “hookworm disease,” is caused by
the _Ankylostoma duodenale_ was shown by Greisinger in 1851. In the
same year the _Schistosomum hematobium_ was shown to be the cause
of the “Bilharzia disease” by Bilharz. Küchenmeister discovered the
tapeworm, _Tænia solium_, in 1852, Cohn, an infectious disease of
flies due to a parasitic fungus (_Empusa muscæ_) in 1855, and Zenker
showed the connection between trichinosis of pork (“measly pork”)
and human trichinosis (1860) as indicated above. The organisms just
mentioned are, of course, not bacteria, but these discoveries proved
conclusively that _living things of one kind or another, some large,
most of them microscopic, could cause disease in other organisms_ and
stimulated the search for other “living contagiums.” In 1863 Davaine,
already mentioned, showed that anthrax could be transmitted from
animal to animal by inoculation of blood, but only if the blood
contained the minute rods which he believed to be the cause. Davaine
later abandoned this belief because he transmitted the disease with
old blood in which he could find no rods. It is now known that this
was because the bacilli were in the “spore” form which Davaine did not
recognize. He thus missed the definite proof of the bacterial nature
of anthrax because he was not familiar with the life history of the
organism which was worked out by Koch thirteen years later. In 1865
Villemin repeatedly caused tuberculosis in rabbits by subcutaneous
injection of tuberculous material and showed that this disease must be
infectious also. In the same year Lord Lister introduced antiseptic
methods in surgery. He believed that wound infections were due to
microörganisms getting in from the air, the surgeon’s fingers, etc.,
and without proving this, he used carbolic acid to kill these germs
and prevent the infection. His pioneer experiments made modern surgery
possible. In this year also, Pasteur was sent to investigate a disease,
Pebrine, which was destroying the silkworms in Southern France. He
showed the cause to be a protozoan which had been seen previously by
Cornalia and described by Nägeli under the name _Nosema bombycis_ and
devised preventive measures. This was the _first infectious disease_
shown to be _due to a protozoan_. In 1866 Rindfleisch observed small
pin-point-like bodies in the heart muscle of persons who had died
of wound infection. Klebs, in 1870-71, published descriptions and
names of organisms he had found in the material from similar wounds,
though he did not establish their causal relation. Bollinger, in
1872, discovered the spores of anthrax and explained the persistence
of the disease in certain districts as due to the resistant spores.
In 1873 Obermeier observed in the blood of patients suffering from
recurrent fever long, flexible spiral organisms which have been named
_Spirochæta obermeieri_. Lösch ascribed tropical dysentery to an ameba,
named by him _Amœba coli_, in 1875. Finally, Koch, in 1876, isolated
the anthrax bacillus, worked out the life history of the organism
and reproduced the disease by the injection of pure cultures and
recovered the organism from the inoculated animals, thus establishing
beyond reasonable doubt its causal relationship to the disease. This
was the _first instance of a bacterium_ proved to be the cause of a
_disease in animals_. Pasteur, working on the disease at the same time,
confirmed all of Koch’s findings, though his results were published
the next year, 1877. Bollinger determined that the _Actinomyces bovis_
(_Streptothrix bovis_) is the cause of actinomycosis in cattle in
1877. Woronin in the same year discovered a protozoan (_Plasmodiophora
brassicæ_) to be the cause of a disease in cabbage, the _first proved
instance of a unicellular animal causing a disease in a plant_. In 1878
Koch published his researches on wound infection in which he showed
beyond question that microörganisms are the cause of this condition,
though Pasteur in 1837, had suggested the same thing and Lister had
acted on the theory in preventing infection.

These discoveries, especially those of Koch, immediately attracted
world-wide attention and stimulated a host of workers, so that within
the next ten years most of the bacteria which produce disease in
men and animals were isolated and described. It is well to remember
that the first _specific_ disease of man proved to be caused by a
_bacterium_ was _tuberculosis_, by Koch in 1882.

Progress was greatly assisted by the introduction of anilin dyes as
suitable stains for organisms by Weigert in 1877, by Koch’s application
of special technic and gelatin cultures for isolation and study, 1881,
and the great improvements in the microscope by Prof. Abbé, of Jena.

Laveran’s discovery of the malarial parasite in 1880 turned attention
to protozoa as the causes of disease and led to the discovery of the
various piroplasmoses and trypanosomiases in man and the lower animals.

Pasteur’s protective inoculations in chicken cholera and anthrax
directed attention to the possibility of using bacteria or their
products as a specific protective or curative means against particular
diseases. This finally led to the discovery of diphtheria antitoxin by
Behring, and independently by Roux, in 1890, a discovery which opened
up the wide field of immunity which is so persistently cultivated at
the present time.

[Illustration: PLATE IV

LOUIS PASTEUR]

While the causation of disease by bacteria has probably attracted
most attention, especially in the popular mind, it should not be
forgotten that this is but one of the numerous ways in which these
organisms manifest their activities, and in a sense it is one of
their least-important ways, since other kinds are essential in many
industries (dairying, agriculture) and processes (sewage purification)
and are even _indispensable for the very existence of all green plants
and hence of animals, including man himself_.


PUTREFACTION AND FERMENTATION.

The idea that there is a certain resemblance between some infectious
diseases and the processes of putrefaction and fermentation seems to
have originated during the discussion on spontaneous generation and
the “contagium vivum” theory which followed Leeuwenhoek’s discoveries.
Plenciz (1762) appears to have first formulated this belief in writing.
He considered putrefaction to be due to the “animalcules” and said that
it occurred only when there was a coat of organisms on the material
and only when they increased and multiplied. Spallanzani’s experiments
tended to support this view since his infusions did not “spoil” when
boiled and sealed. Appert’s practical application of this idea has been
mentioned.

Thaer, in his _Principles of Rational Agriculture_, published in the
first quarter of the nineteenth century, expressed the belief that the
“blue milk fermentation” was probably due to a kind of fungus that
gets in from the air, and stated that he had prevented it by treating
the milk cellars and vessels, with sulphur fumes or with “oxygenated
hydrochloric acid” (hypochlorous acid).

In 1836 Chevreuil and Pasteur showed that putrefaction did not occur
in meat protected from contamination. In 1837 Caignard-Latour, in
France, and Schwann, in Germany, independently showed that alcoholic
fermentation in beer and wine is due to the growth of a microscopic
plant, the yeast, in the fermenting wort. C. J. Fuchs described the
organism which is commonly called the “blue milk bacillus” in 1841 and
conjectured that the souring of milk was probably bacterial in origin.
It remained for Pasteur to prove this in 1857. During the following
six or seven years Pasteur also proved that acetic acid fermentation,
as in vinegar making, butyric acid fermentation (odor of rancid butter
and old cheese) and the ammoniacal fermentation of urea, so noticeable
around stables, were each due to different species of bacteria. Pasteur
also, during the progress of this work, discovered the class of
organisms which can grow in the absence of free oxygen--the anaërobic
bacteria. There is no question that Pasteur from 1857 on did more to
lay the foundations of the science of bacteriology than any other
one man. Influenced by Pasteur’s work von Hesseling, in 1866, stated
his belief that the process of cheese ripening, like the souring of
milk, was associated with the growth of fungi, and Martin also, in
1867, stated that cheese ripening was a process which was akin to
alcoholic, lactic and butyric fermentations. Kette, in 1869, asserted
the probability of Pasteur’s researches furnishing a scientific basis
for many processes of change in the soil. In 1873 Schlösing and Müntz
showed that nitrification must be due to the action of microörganisms,
though the discovery of the particular ones remained for Winogradsky
in 1889. Thus the belief that fermentation and putrefaction are due to
microörganisms was as well established by the early eighties of the
last century as that similar organisms are the causes of infectious
diseases.


STUDY OF FORMS.

An important part of the scientific knowledge of living organisms is
dependent on a study of their forms and relationships. As has been
stated, Leeuwenhoek considered bacteria to be “animalcules” because
they showed independent movement. But little attention was paid to
the natural history of these animalcules for nearly a hundred years
after Leeuwenhoek. During the last quarter of the eighteenth century,
however, workers busied themselves chiefly with the discovery and
description of new forms. Among these students were Baron Gleichen,
Jablot, Lesser, Reaumur, Hill and others. Müller, of Copenhagen, in
1786 published the first attempt at classification, a most important
step in the study of these organisms. Müller introduced the terms
Monas, Proteus and Vibrio, which are still in use. Ehrenberg, in his
work on _Infusoria_, or the organisms found in infusions, published
in 1838, introduced many generic names in use at present, but still
classed the bacteria with protozoa. Joseph Leidy, the American
naturalist, considered that the “vibrios” of previous writers were
plants and not “animalcules.” He seems to have been the first to have
made this distinction (1849). Perty (1852) recognized the presence of
spores in some of his organisms. Ferdinand Cohn (1854) classed the
bacteria among plants. Nägeli (1857) proposed the name “Schizomycetes”
or “fission fungi,” which is still retained for the entire class of
bacteria. Cohn in the years 1872-1875 established classification on
a modern basis and added greatly to the knowledge of morphology and
natural history of bacteria. He described spore formation and the
development of spores into active bacteria, and showed the close
relationships as well as differences between the bacteria and the lower
algæ. Robert Koch was a pupil of Cohn.

An examination of the accompanying chronological table will show how
the investigations and discoveries in connection with “spontaneous
generation,” the “contagium vivum” theory and putrefaction and
fermentation must have been mutually suggestive:

  1546. Fracastorius, disease germs theory and direct and indirect
  contagion.

  1671. Kircher, “contagium vivum” theory.

  1675. Leeuwenhoek, first saw bacteria, “animalcules.”

  1701. Andry, “animalcules” cause of diseases.

  1718. Lancisi, “animalcules” cause of malaria.

  1749. Needham, described development of organisms in water around
  barley grains.

  1762. Plenciz, arguments for “living cause” theory and that
  “animalcules” cause putrefaction.

  1768. Bonnet, suggested that probably Needham’s organisms came from
  germs in the liquid.

  1776. Spallanzani, boiled and sealed infusions.

  1786. Müller, first classified “animalcules.”

  1787. Wollstein, glanders pus infectious.

  1795-1798. Jenner, vaccination against smallpox.

  1797. Viborg, transmitted glanders repeatedly.

  1807. Prevost, grain rust, _Puccinia graminis_. _The first instance
  of a microscopic plant organism shown to be the cause of a disease in
  a higher plant._

  1810. Appert, directions for “canning.”

  1822. Gaspard, infectiousness of material from wounds.

  1834. Renucci, itch--itch mite (_Sarcoptes scabiei_).

  1835. Paget and Owen, _Trichina spiralis_.

  1836. Schultze, air through acid to kill “germs.”

  1837. Chevreuil and Pasteur, protected meat did not putrefy;
  suggested wound infection due to entrance of germs from without.

  1837. Caignard-Latour, Schwann, alcoholic fermentation--yeast.

  1837. Schwann, air through heated tubes to kill germs.

  1837. Bassi, muscardine of silkworms, _Botrytis bassiana_. _The first
  instance of a microscopic plant organism shown to be the cause of a
  disease in an animal._

  1838. Boehm, cholera, saw organisms in stools (not the cause).

  1838. Dubini discovered _Ankylostoma duodenale_.

  1838. Ehrenberg, study of forms.

  1839. Schönlein, Favus, _Achorion schoenleinii_.

  1839-41. Berg, Thrush, _Oidium albicans_.

  1840. Henle, theory of contagious diseases.

  1841. Fuchs, bacterial cause of blue milk.

  1842-43. Gruby, Herpes tonsurans, _Trichophyton tonsurans_.

  1843. Klencke, inoculations of tuberculous material into rabbit.

  1843. Holmes, puerperal fever contagious.

  1845. Liebert, a potato rot, _Peronospora infestans_.

  1846. Leidy, Joseph (American Naturalist), _Trichina spiralis_ in
  pork.

  1846. Eichstedt, Pityriasis versicolor, _Microsporon furfur_.

  1847. Semmelweiss, recommended disinfection to prevent puerperal
  fever. Not followed.

  1849. Leidy, considered “vibrios” to be plants.

  1849. Pollender, Anthrax, saw rods in blood.

  1850. Davaine and Rayer, Anthrax, saw rods in blood.

  1851. Griesinger, Egyptian chlorosis, _Ankylostoma duodenale_.

  1851. Bilharz, Bilharzia disease, _Schistosomum hematobium_.

  1852. Kückenmeister, tapeworm, _Tænia solium_.

  1852. Perty, saw spores in bacteria.

  1854. Cohn, classed bacteria as plants.

  1855. Cohn, disease of flies, _Empusa muscæ_.

  1857. Nägeli, named bacteria, Schizomycetes.

  1857. Pasteur, lactic, acetic, butyric acid fermentation.

  1860. Zenker, Trichinosis, _Trichinella spiralis_.

  1861. Pasteur, disproof of spontaneous generation.

  1863. Davaine, transmitted anthrax by blood injections.

  1865. Pasteur, Pebrine of silkworms, _Nosema bombycis_. _The first
  instance of a protozoan shown to be the cause of a disease in a
  higher animal._

  1865. Villemin, repeatedly transmitted tuberculosis to rabbits.

  1865. Lister, introduced antisepsis in surgery.

  1860. Rindfleisch, Pyemia, organisms in the pus.

  1866. Von Hesseling, cheese ripening.

  1867. De Martin, cheese ripening akin to alcoholic fermentation.

  1869. Kette, Pasteur’s researches scientific basis for many processes
  in the soil.

  1871. Klebs, Pyemia, organisms in the pus.

  1872. Bollinger, spores in anthrax.

  1872-75. Cohn, definite classification.

  1873. Obermeier, recurrent fever, _Spirochæta obermeieri_.

  1873. Schlösing and Münz, nitrification due to organisms.

  1875. Lösch, amebic dysentery, _Amœba coli_.

  1875-76. Tyndall, germs in the air.

  1876. Robert Koch, anthrax, _Bacillus anthracis_. _The first instance
  of a bacterium shown to be the cause of disease in an animal._

  1877. Bollinger, actinomycosis, _Actinomyces bovis_ (_Streptothrix
  bovis_).

  1877. Weigert, used anilin dyes for staining.

  1877. Woronin, cabbage disease, _Plasmodiophora brassicæ_. _The first
  instance of a protozoan shown to be the cause of a disease in a
  plant._

  1878. Koch, wound infections, bacterial in origin.

  1881. Koch, gelatin plate cultures, Abbé, improvements in the
  microscope.




CHAPTER I.

POSITION--RELATIONSHIPS.


Bacteria are considered to belong to the plant kingdom not because of
any one character they possess, but because they most nearly resemble
organisms which are generally recognized as plants. While it is not
difficult to distinguish between the higher plants and higher animals,
it becomes almost, if not quite, impossible to separate the lowest,
forms of life. It is only by the method of resemblances above mentioned
that a decision is finally reached. It has even been proposed to make a
third class of organisms neither plants nor animals but midway between
in which the bacteria are included, but such a classification has not
as yet been adopted.

In many respects the bacteria are most nearly related to the lowest
_algæ_, since both are unicellular organisms, both reproduce by
transverse division and the forms of the cell are strikingly similar.
The bacteria differ in one important respect, that is, they do not
contain _chlorophyl_, the green coloring matter which enables all
plants possessing it to absorb and break up carbon dioxide in the
light, and hence belong among the fungi. Bacteria average much smaller
than even the smallest algæ.

Bacteria are closely connected with the _fission yeasts_ and the
_yeasts_ and _torulæ_. All are unicellular and without chlorophyl. The
bacteria, as has been stated, reproduce by division but the others
characteristically by budding or gemmation, though the fission yeasts
also by division.

There is a certain resemblance to the _molds_ in their absence
of chlorophyl. But the molds grow as branching threads and also
have special fruiting organs for producing spores as a means of
reproduction, neither of which characteristics is found among the
_true_ bacteria. The higher thread bacteria do show true branching
and rudimentary fruiting bodies (Streptothrix) and appear to be a link
connecting the true bacteria and the molds.

[Illustration: FIG. 8.--A thread of blue-green algæ.]

[Illustration: FIG. 9.--A thread of small blue-green algæ.]

[Illustration: FIG. 10.--A thread of bacteria. Compare with Figs. 8
and 9.]

[Illustration: FIG. 11.--A chain of spherical blue-green algæ.]

[Illustration: FIG. 12.--A chain of spherical bacteria.]

[Illustration: FIG. 13.--A pair of spherical blue-green algæ.]

Further the _chemical composition_ of bacteria is more like that of
other fungous plants than of any of the forms classed as animals.

[Illustration: FIG. 14.--Spherical bacteria. Several pairs are shown.]

[Illustration: FIG. 15.--Yeast cells. Some show typical budding.]

The food of bacteria is always taken up in solution by diffusion
through the outer covering of the cell as it is in all plants. Plant
cells never surround and engulf particles of solid food and digest them
within the cell as many single-celled animals do, and as the leukocytes
and similar ameboid cells in practically all multicelled animals do.[2]

[Illustration: FIG. 16.--A portion of the mycelium of a mold. Note the
large size and the branching.]

One of the most marked differences between animals and plants is with
respect to their energy relationships. Plants are characteristically
storers of energy while animals are liberators of it. Some bacteria
which have the power of swimming in a liquid certainly liberate
relatively large amounts of energy, and in the changes which bacteria
bring about in the material which they use as food considerable heat is
evolved (“heating” of manure, etc.). Nevertheless the evidence is good
that the bacteria as a class store much more of the energy contained
in the substances actually taken into the body cell as food than is
liberated in any form.

Bacteria do show some resemblance to the protozoa, or single-celled
animal forms, in that the individuals of each group consist of one cell
only and some bacteria have the power of independent motion from place
to place in a liquid as most “infusoria” do, but here the resemblance
ceases.

Bacteria are among the smallest of organisms, so small that it requires
the highest powers of the microscope for their successful study, and
the use of a special unit for their measurement. This unit is the
one-thousandth part of a millimeter and is called the micro-millimeter
or micron. Its symbol is the Greek letter _mu_ (µ).

The size varies widely among different kinds but is fairly constant in
the same kind. The smallest described form is said to be only 0.18µ
long by 0.06µ thick and is just visible with the highest power of the
microscope, though it is possible and even probable that there are
forms still smaller which cannot be seen. Some large rare forms may
measure 40µ in length, but the vast majority are from 1µ to 4µ or 5µ
long, and from one-third to one-half as wide.

From the above description a bacterium might be said to be a
_microscopic, unicellular plant, without chlorophyl, which reproduces
by dividing transversely_.




PART I.

MORPHOLOGY




CHAPTER II.

CELL STRUCTURES.


The _essential_ structures which may by appropriate means be
distinguished in the bacterial cell are _cell wall_ and _cell
contents_, technically termed _protoplasm_, cytoplasm. The cell wall is
not so dense, relatively, as that of green plants, but is thicker than
the outer covering of protozoa. It is very similar to the cell wall
of other lower fungi. Diffusion takes place readily through it with
very little selective action on substances absorbed as judged by the
comparative composition of bacteria and their surrounding medium.

=Cytoplasm.=--The cytoplasm according to Bütschli and others is
somewhat different and slightly denser in its outer portion next to the
cell wall. This layer is designated the _ectoplasm_, as distinguished
from the remainder of the cell contents, the _endoplasm_. When bacteria
are suddenly transferred from a given medium into one of decidedly
_greater_ density, there sometimes results a contraction of the
_endoplasm_, due to the rapid diffusion of water. This phenomenon is
designated _plasmolysis_ (Fig. 17), and is similar to what occurs in
the cells of higher plants when subjected to the same treatment. This
is one of the methods which may be used to show the different parts of
the cell just described.

If bacteria are suddenly transferred from a relatively dense medium
to one which is of decidedly _less_ density, it occasionally happens
that water diffuses into the cell and swells up the endoplasm so much
more rapidly than the cell wall that the latter ruptures and some of
the endoplasm exudes in the form of droplets on the surface of the cell
wall. This phenomenon is called _plasmoptysis_. Students will seldom
observe the distinction between cell wall and cell contents, except
that in examining living bacteria the outer portion appears more highly
refractive. This is chiefly due to the presence of a cell wall, but is
not a proof of its existence.

[Illustration: FIG. 17.--Cells of bacteria showing plasmolysis. The
cell substance of three of the cells in the middle of the chain has
shrunk until it appears as a round black mass. The cell wall shows as
the lighter area.]

[Illustration: FIG. 18.--Vacuoles in the bacterial cell. The lighter
areas are vacuoles.]

=Nucleus.=--Douglas and Distaso[3] summarize the various opinions with
regard to the nucleus in bacteria as follows:

1. Those who do not admit, the presence of a nucleus or of anything
equivalent to it. (Fischer, Migula, Massart).

2. Those who consider that the entire bacterial cell is the equivalent
of a nucleus and contains no protoplasm. (Ruzicka).

3. Those who admit the presence of nuclein but say that this is not
morphologically differentiated from the protoplasm as a nucleus.
(Weigert).

4. Those who consider the bacterial protoplasm to consist of a central
endoplasm throughout which the nuclein is diffused and an external
layer of ectoplasm next to the cell wall. (Bütschli, Zettnow).

5. Those who say that the bacterial cell contains a distinct nucleus,
at least in most instances. These authors base their claims on staining
with a Giemsa stain. (Feinberg, Ziemann, Neuvel, Dobell, Douglass and
Distaso).

That nucleoproteins are present in the bacterial cell in relatively
large amounts is well established. Also that there are other proteins
and that the protoplasm is not all nuclein.

Some workers as noted above have been able to demonstrate collections
of nuclein by staining, especially in very young cells. In older cells
this material is in most instances diffused throughout the protoplasm
and can not be so differentiated.

The following statement probably represents the generally accepted view
at the present time:

A nucleus _as such_ is not present in bacterial cells, except in a few
large rare forms and in very young cells. _Nuclein_, the characteristic
chemical substance in nuclei, which when aggregated forms the nucleus,
is scattered throughout the cell contents and thus intimately mingled
with the protoplasm, and cannot be differentiated by staining as in
most cells.

The close association of nuclein and protoplasm may explain the rapid
rate of division of bacteria (Chapter VIII, p. 91).

The chemical composition of the bacterial cell is discussed in Chapter
VII.

In addition to the _essential_ parts just described the bacterial cell
may show some of the following _accidental_ structures: _vacuoles_,
_capsules_, _metachromatic granules_, _flagella_, _spores_.

=Vacuoles.=--_Vacuoles_ appear as clear spaces in the protoplasm when
the organism is examined in the living condition or when stained very
slightly (Fig. 18). During life these are filled with liquid or gaseous
material which is sometimes waste, sometimes reserve food, sometimes
digestive fluids. Students are apt to confuse vacuoles with spores (p.
47). Staining is the surest way to differentiate (Chapter XIX, p. 209).
If vacuoles have any special function, it is an unimportant one.

[Illustration: FIG. 19.--Bacteria seen within capsules.]

[Illustration: FIG. 20.--Metachromatic granules in bacteria. The dark
round spots are the granules. The cells of the bacteria are scarcely
visible.]

=Capsule.=--The _capsule_ is a second covering outside the cell wall
and probably developed from it (Fig. 19). It is usually gelatinous,
so that bacteria which form capsules frequently stick together
when growing in a fluid, so that the whole mass has a jelly-like
consistency. The term _zoöglœa_ was formerly applied to such masses,
but it is a poor term and misleading (zoön = an animal) and should
be dropped. The masses of jelly-like material frequently found on
decaying wood, especially in rainy weather, are in some cases masses
of capsule-forming bacteria, though a part of the jelly is a product
of bacterial activity, a gum-like substance which lies among the
capsulated organisms. When these masses dry out, they become tough
and leathery, but it is not to be presumed that capsules are of this
consistency. On the contrary, they are soft and delicate, though they
certainly serve as an additional protection to the organism, doubtless
more by selective absorption than mechanically. Certain bacteria
which cause disease form capsules in the blood of those animals which
they kill and not in the blood of those in which they have no effect
(_Bacterium anthracis_ in guinea pig’s blood and in rat’s blood). The
presence of capsules around an organism can be proved only by staining
the capsule. Many bacteria when stained in albuminous fluids show a
clear space around them which appears like a capsule. It is due to the
contraction of the fluid away from the organism during drying.

=Metachromatic Granules.=--The term “_metachromatic_” is applied to
granules which in stained preparations take a color different from
the protoplasm as a whole (Fig. 20). They vary widely in chemical
composition. Some of them are glycogen, some fat droplets. Others are
so-called “granulose” closely related to starch but probably not true
starch. Others are probably nuclein. Of many the chemical composition
is unknown. They are called “Babes-Ernst corpuscles” in certain
bacteria (typhoid bacillus). Since they frequently occur in the ends
of cells the term “polar granules” is also applied. Their presence is
of value in the recognition of but few bacteria (“Neisser granules” in
diphtheria).

=Flagellum.=--A _flagellum_ is a very minute thread-like process
growing out from the cell wall, probably filled with a strand of
protoplasm. The vibrations of the flagella move the organism through
the liquid medium. Bacteria which are thus capable of independent
movement are spoken of as “motile bacteria.” The actual rate of
movement is very slight, though in proportion to the size of the
organism it may be considered rapid. Thus Alfred Fischer determined
that some organisms have a speed for short periods of about 40 cm. per
hour. This is equivalent to a man moving more than 200 miles in the
same time.

It is obvious that bacteria which can move about in a liquid have an
advantage in obtaining food, since they do not need to wait for it to
be brought to them. This advantage is probably slight.

[Illustration: FIG. 21.--A bacterium showing a single flagellum at the
end--monotrichic.]

[Illustration: FIG. 22.--A bacterium showing a bundle of four flagella
at the end--lophotrichic.]

An organism may have only one flagellum at the end. It is then said
to be monotrichic (Fig. 21) (μόνος = alone, single; τριχος = hair).
This is most commonly at the front end, so that the bacterium is drawn
through the liquid by its motion. Rarely it is at the rear end. Other
bacteria may possess a bundle of flagella at one end and are called
_lophotrichic_ (Fig. 22) (λοφος = tuft). Sometimes at approaching
division the flagella may be at both ends and are then _amphitrichic_
(Fig. 23) (αμφι = both). It is probable that this condition does not
persist long, but represents the development of flagella at one end
of each of a pair resulting from division of an organism which has
flagella at one end only. In many bacteria the flagella arise from
all parts of the surface of the cell. Such bacteria are _peritrichic_
(Fig. 24) (περι = around). The position and even the number of the
flagella are very constant for each kind and are of decided value in
identification.

[Illustration: FIG. 23.--A bacterium showing flagella at each
end--amphitrichic.]

[Illustration: FIG. 24.--A bacterium showing flagella all
around--peritrichic.]

Flagella are too fine and delicate to be seen on the living organism,
or even on bacteria which have been colored by the ordinary stains.
They are rendered visible only by certain methods which cause a
precipitate on both bacteria and flagella which are thereby made thick
enough to be seen (Chapter XIX, p. 210). The movement of liquid around
a bacterium caused by vibrations of flagella can sometimes be observed
with large forms and the use of “dark-field” illumination.

Flagella are very delicate and easily broken off from the cell body.
Slight changes in the density or reaction of the medium frequently
cause this breaking off, so that preparations made from actively motile
bacteria frequently show no flagella. For this reason and also on
account of their fineness the demonstration of flagella is not easy,
and it is not safe to say that a non-motile bacterium has no flagella
except after very careful study.

The motion of bacteria is characteristic and a little practice in
observing will enable the student to recognize it and distinguish
between motility and “Brownian” or molecular motion. Dead and
non-motile bacteria show the latter. In fact, any finely divided
particles suspended in a liquid which is not too viscous and in which
the particles are not soluble show Brownian motion or “pedesis.” This
latter is a dancing motion of the particle within a very small area
and without change of place, while motile bacteria move from place to
place or even out of the field of the microscope with greater or less
speed. There is a marked difference in the character of the motion of
different kinds of bacteria. Some rotate around the long axis when
moving, others vibrate from side to side.

Among the higher thread bacteria there are some which show motility
without possessing flagella. Just how they move is little understood.

=Spores.=--Under certain conditions some bacterial cells undergo
transformations which result in the formation of so-called _spores_.
If the process is followed under the microscope, the changes observed
are approximately these: A very minute point appears in the protoplasm
which seems to act somewhat like the centrosome of higher cells as a
“center of attraction” so that the protoplasm gradually collects around
it. The spot disappears or is enclosed in the collected protoplasm.
This has evidently become denser as it is more highly refractive than
before. In time all or nearly all of the protoplasm is collected. A new
cell wall is developed around it which is thicker than the cell wall of
the bacterium. This thickened cell wall is called the “spore capsule.”
Gradually the remnants of the former cell contents and the old cell
wall disappear or dissolve and the spore becomes “free” (Fig. 25).

[Illustration: FIG. 25.--The smaller oval bodies in the middle of the
field are free spores.]

If the spore is placed in favorable conditions the protoplasm absorbs
water, swells, the capsule bursts at some point, a cell wall is formed
and the bacterium grows to normal size and divides, that is, it is an
active growing cell again. This process is called “germination” of the
spore. The point at which the spore capsule bursts to permit the new
cell to emerge is characteristic for each kind of bacterium. It may be
at the end when the germination is said to be _polar_ (Fig. 26). It may
be from the middle of one side which gives _equatorial_ germination
(Fig. 27). Rarely it is diagonally from a point between the equator and
the pole, which type may be styled _oblique_ germination. In one or
two instances the entire spore swells up, lengthens and becomes a rod
without any special germination unless this type might be designated
_bi-polar_.

[Illustration: FIG. 26.--Spores showing polar germination. The lighter
part of the two organisms just below A and B is the developing
bacterium. In the original slide the spore was stained red and the
developing bacterium a faint blue.]

[Illustration: FIG. 27.--A spore showing equatorial germination.
The spore in the center of the field shows a rod growing out of it
laterally. In the original slide the spore was stained red and the
developing bacterium blue.]

[Illustration: FIG. 28.--Spores in the middle of the rod without
enlargement of the rod. The lighter areas in the rods are spores.]

[Illustration: FIG. 29.--Spores in the middle of the rod with
enlargement of the rod around them. The lighter areas in the rods are
spores.]

Spores are most commonly oval or elliptical in shape, though sometimes
spherical. A spore may be formed in the middle of the organism without
(Fig. 28) or with (Fig. 29) a change in size of the cell around it.
If the diameter through the cell is increased, then the cell with
the contained spore becomes spindle-shaped. Such a cell is termed a
“_clostridium_.” Sometimes the spore develops in the end of the cell
either without (Fig. 30) or with enlarging it (Fig. 31). In a few
forms the spore is placed at the end of the rod and shows a marked
enlargement. This is spoken of as the “_plectridium_” or more commonly
the “drumstick spore” (Fig. 32). The position and shape of the spore
are constant for each kind of bacteria. In one or two instances only,
two spores have been observed in a single organism.

[Illustration: FIG. 30.--Spores in the end of the rod with no
enlargement of the rod around them. The lighter areas in the rods are
spores.]

[Illustration: FIG. 31.--Spores in the end of the rod with enlargement
of the rod, _A_, _A_, _A_, _A_.]

[Illustration: FIG. 32.--Drumstick spores at the end of the rod.]

The fact that the protoplasm is denser and the spore capsule thicker
(the percentage of water in each is decidedly less than in the growing
cell) gives the spore the property of much greater resistance to all
destructive agencies than the active bacterium has. For example, all
actively growing cells are destroyed by boiling in a very few minutes,
while some spores require several hours’ boiling. The same relation
holds with regard to drying, the action of chemicals, light, etc. That
the coagulation temperature of a protein varies inversely with the
amount of water, it contains, is shown by the following table from
Frost and McCampbell, “General Bacteriology”:

  Egg albumin plus 50 per cent. water coagulates at  56°
   „     „     „   25 per cent.   „       „      „   74-80°
   „     „     „   18 per cent.   „       „      „   88-90°
   „     „     „    6 per cent.   „       „      „  145°
   „     „    dry                 „       „      „  160-170°

This resistance explains why it happens that food materials boiled
and sealed in cans to prevent the entrance of organisms sometimes
spoil. The spores have not been killed by the boiling. It explains
also in part the persistence of some diseases like anthrax and black
leg in pastures for years. From the above description it follows that
the spore is to be considered as _a condensation of the bacterial
protoplasm surrounded by an especially thick cell wall_. _Its function
is the preservation of the organism under adverse conditions._ It
corresponds most closely to the encystment of certain protozoa--the
ameba for example. Possibly the spore represents a very rudimentary
beginning of a reproductive function such as is gradually evolved in
the higher thread bacteria, the fission yeasts, the yeasts, the molds,
etc. Its characteristics are so markedly different, however, that the
function of preservation is certainly the main one.

It must not be supposed that spores are formed under adverse conditions
only, because bacteria showing vigorous growth frequently form spores
rapidly. Special conditions are necessary for their formation just as
they are for the growth and other functions of bacteria (Chapters VI
and VII).




CHAPTER III.

CELL FORMS.


Though there is apparently a wide variation in the shapes of different
bacterial cells, these may all be reduced to _three_ typical _cell
forms_. These are: first and simplest, the round or _spherical_,
typified by a ball and called the _coccus_ form, or _coccus_, plural
cocci[4] (Fig. 33). The coccus may be large, that is, from 1.5µ to 2µ
in diameter. The term _macrococcus_ is sometimes applied to these large
cocci. If the _coccus_ is less than 1µ in diameter, it is sometimes
spoken of as a _micrococcus_; in fact, this term is very commonly
applied to any coccus. When cocci are growing together, many of the
cells do not appear as true spheres but are more or less distorted
from pressure of their neighbors or from failure to grow to full size
after recent division. Most cocci divide into hemispheres and then each
half grows to full size. A few cocci elongate before division and then
appear oval or elliptical.

The second cell form is that of a _cylinder_ or rod typified by a
section of a lead-pencil. The name _bacillus_, plural _bacilli_, is
applied to this type (Fig. 34). The bacillus may be short (Fig. 35),
1µ or less in length, or long, up to 40µ in rare cases. Most bacilli
are from 2µ to 5µ or 6µ long. The ends of the rod are usually rounded,
occasionally square and very rarely pointed. It is evident that a very
short rod with rounded ends approaches a coccus in form and it is not
always easy to differentiate in such cases. Most bacilli are straight,
but some are slightly curved (Fig. 36).

The third cell form is the _spiral_, typified by a section of a
cork-screw and named _spirillum_, plural _spirilla_ (Fig. 37). A very
short spiral consisting of only a portion of a turn is sometimes called
_vibrio_ (Fig. 38). Vibrios when seen under the microscope look like
short curved rods. The distinction between the two can be made only by
examining the organism alive and moving in a liquid. The vibrio shows a
characteristic spiral twisting motion. Very long, flexible spirals are
usually named _spirochetes_ (Fig. 39). The spirochetes are motile but
flagella have not been shown to be present.

[Illustration: FIG. 33.--Cocci.]

[Illustration: FIG. 34.--Bacilli.]

[Illustration: FIG. 35.--Short bacilli.]

[Illustration: FIG. 36.--Curved bacilli. Only the one in the center of
the field is in focus. The others curve out of focus.]

Besides the three typical cell forms bacteria frequently show
very great irregularities in shape. They may be pointed, bulged,
club-shaped or even slightly branched. These peculiar and bizarre
forms practically always occur when some of the necessary conditions
for normal growth, discussed in Chapters VI and VII, are not fulfilled.
They are best regarded as _involution_ or _degeneration_ forms for this
reason (Fig. 40). In a very few cases it is not possible to obtain the
organism without these forms (the diphtheria group). It is probable
that these cell forms are normal in such cases, or else conditions
suitable for the normal growth have not been obtained.

[Illustration: FIG. 37.--Spirilla.]

[Illustration: FIG. 38.--Vibrio forms of spirilla. Compare with Fig.
36.]

[Illustration: FIG. 39.--Spirochetes.]

[Illustration: FIG. 40.--Involution forms. The organisms are tapering
and branched at one end.]




CHAPTER IV.

CELL GROUPINGS.


It has been stated that bacteria reproduce by transverse division, that
is, division across the long axis. Following repeated divisions the new
cells may or may not remain attached. In the latter case the bacteria
occur as separate isolated individuals. In the former, arrangements
characteristic of the particular organism almost invariably result.
These arrangements are best described as _cell groupings_ or _growth
forms_.

[Illustration: FIG. 41.--Streptospirillum grouping.]

[Illustration: FIG. 42.--Diplobacillus grouping.]

In the case of spiral forms it is obvious that there is only one
possible grouping, that is, in chains of two or more individuals
adherent end to end. A chain of two spirilla might be called
a _diplospirillum_ (διπλός = double); of three or more, a
_streptospirillum_ (στρεπτός = necklace, chain) (Fig. 41). These terms
are rarely used, since spirilla do not ordinarily remain attached.
Likewise the bacillus can grow only in chains of two or more, and
the terms _diplobacillus_ (Fig. 42), bacilli in groups of two, and
_streptobacillus_ (Fig. 43), bacilli in chains are frequently used.
Still the terms _thread_, _filament_, or _chain_ are more common for
_streptobacillus_.

[Illustration: FIG. 43.--Streptobacillus grouping.]

[Illustration: FIG. 44.--Typical diplococcus grouping. Note that the
individual cocci are flattened on the apposing sides.]

[Illustration: FIG. 45.--Long streptococcus grouping.]

[Illustration: FIG. 46.--Short streptococcus grouping.]

Since the coccus is spherical, _transverse_ division may occur in any
direction, though in three planes only at right angles to each other.
Division might occur in _one plane only_ as in spirilla and bacilli,
or in _two planes only_ or in _all three planes_. As a matter of fact
these three methods of division are found among the cocci, but only one
method for each particular kind of coccus. As a result there may be a
variety of cell groupings among the cocci. When division occurs in one
plane only, the possible groupings are the same as among the spirilla
or bacilli. The cocci may occur in groups of two--_diplococcus_
grouping (Fig. 44), or in chains--_streptococcus_ grouping (Figs. 45
and 46). When the grouping is in _diplococci_, the individual cocci
most commonly appear as hemispheres with the plane surfaces apposed
(Fig. 44). Sometimes they appear as spheres and occasionally are even
somewhat elongated. The individuals in a streptococcus grouping are
most commonly elongated, either in the same direction as the length of
the chain, or at right angles to it. The latter appearance is probably
due to failure to enlarge completely after division. Streptococci
frequently appear as chains of diplococci, that is, the pair resulting
from the division of a single coccus remain a little closer to each
other than to neighboring cells, as a close inspection of Fig. 45 will
show.

If division occurs in _two planes only_, there may result the above
groupings and several others in addition. The four cocci which result
from a single division may remain together, giving the _tetracoccus_
or _tetrad_ grouping. Very rarely all the cocci divide evenly and the
result is a regular _rectangular flat mass_ of cells, the total number
of which is a multiple of four. The term merismopedia (from a genus of
algæ which grows the same way) is applied to such a grouping. If the
cells within a group after a few divisions do not reproduce so rapidly
(lack of food), as usually happens, the number of cells becomes uneven
or at least not necessarily a multiple of four and the resultant _flat
mass_ has an _irregular_, _uneven outline_. This grouping is termed
_staphylococcus_ (σταφυλος = a bunch of grapes) (Fig. 47). It is the
most common grouping among the cocci.

When division occurs in all three planes, there is in addition to all
the groupings possible to one- and two-plane division a third grouping
in which the cells are in _solid packets_, _multiples of eight_. The
name _sarcina_ is applied to this growth form (Fig. 48). The individual
cells in a sarcina packet never show the typical coccus form so long as
they remain together, but are always flattened on two or more sides.

The above descriptions indicate how the method of division may be
determined. If in examining a preparation the _sarcina_ grouping
appears, that shows _three-plane division_. If there are no sarcina,
but _tetrads_ or _staphylococci_ (rarely merismopedia), then the
division is in _two planes_. If none of the foregoing is observed but
only _diplo-_ or _streptococci_, these indicate _one-plane division_
only. Cocci show their _characteristic_ groupings only when grown in a
liquid medium, and such should always be used before deciding on the
plane of division.

[Illustration: FIG. 47.--Staphylococcus grouping. The large flat masses
are staphylococcus grouping. Diplococcus grouping, tetrads and short
streptococci are also evident.]

[Illustration: FIG. 48.--Sarcina grouping.]

As the above description shows, these terms which are properly
adjectives describing the cell grouping, are quite generally used as
nouns. Thus the terms a diplococcus, a tetrad, a streptococcus, etc.,
are common, meaning a bacterium of the cell form and cell grouping
indicated.

    CELL FORM.             CELL GROUPING.

  coccus--             {diplococcus--in 2’s.
  round or spherical.  {streptoccus--in chains.
                       {tetracoccus, tetrads--in 4’s.
                       {staphylococcus--irregular flat masses.
                       {sarcina--regular, solid packets, multiples of 8.

  bacillus--           {diplobacillus--in 2’s.
  rod-shaped           {streptobacillus--in chains.
  or cylindrical.

  spirillum--          {diplospirillum--in 2’s, little used.
  spiral-shaped.       {streptospirillum--in chains, little used.




CHAPTER V.

CLASSIFICATION.


The arrangement of living organisms in groups according to their
resemblances and the adoption of _fixed names_ is of the greatest
advantage in their scientific study. For animal forms and for the
higher plants this classification is gradually becoming standardized
through the International Congress of Zoölogists and of Botanists
respectively. Unfortunately, the naming of the bacteria has not as
yet been taken up by the latter body, though announced as one of the
subjects for the Congress of 1916 (postponed on account of the war).
Hence there is at present no system which can be regarded as either
fixed or official.

[Illustration: FIG. 49.--Illustrates the genus Streptococcus. Typical
chains, no staphylococcus grouping, no sarcina grouping, no flagella.]

[Illustration: FIG. 50.--Illustrates the genus Micrococcus.
Diplococcus, tetrads short chains and staphylococcus; no sarcina, no
flagella.]

[Illustration: FIG. 51.--Illustrates the genus Sarcina. Sarcina
grouping, no flagella.]

[Illustration: FIG. 52.--Illustrates the genus Bacillus. A bacillus
with peritrichic flagella. (Student preparation.)]

Since Müller’s first classification of “animalcules” in 1786 numerous
attempts have been made to solve the problem. Only those beginning with
Ferdinand Cohn (1872-75) are of any real value. As long as bacteria
are regarded as plants it appears that the logical method is to follow
the well-established botanical principles in any system for naming
them. Botanists depend on morphological features almost entirely in
making their distinctions. The preceding chapters have shown that
the minute plants which are discussed have very few such features.
They are, to recapitulate, _cell wall_, _protoplasm_, _vacuoles_,
_metachromatic granules_, _capsules_, _flagella_, _spores_, _cell
forms_ and _cell groupings_. Most bacteria show not more than three
or four of these features, so that it is impossible by the aid of
morphology alone to distinguish from each other the large number of
different kinds which certainly exist. In the various systems which
are conceded to be the best these characteristics do serve to classify
them down to genera, leaving the “species” to be determined from their
_physiological_ activities. One of these systems was adopted by the
laboratory section of the American Public Health Association and by the
Society of American Bacteriologists and was practically the standard
in this country until superseded by the Society’s own classification.
It is that of the German Bacteriologist Migula and is given below for
comparison. Since practically the entire discussion in this book is
concerned with the first three families the generic characteristics
in these only will be given. The full classification as well as a
thorough discussion of this subject is given in Lafar’s _Handbuch_,
whence the following is adopted:

[Illustration: FIG. 53.--Illustrates the genus Pseudomonas. A bacillus
with flagella at the end only.]

[Illustration: FIG. 54.--Illustrates the genus Microspira. It is
(though the photograph does not prove it) a short spiral with one
flagellum at the end.]

[Illustration: FIG. 55.--Illustrates the genus Spirillum. Spiral
bacteria with more than three, in this case four, flagella at the end.]

[Illustration: FIG. 56.--Illustrates the genus Spirochæta.]

[Illustration: FIG. 57.--Illustrates the genus Chlamydothrix. Fine
threads with a delicate sheath.]

[Illustration: FIG. 58.--Illustrates the genus Crenothrix. The
thickness of the cell walls is due to deposits of iron hydroxide.
(After Lafar.)]

[Illustration: FIG. 59.--Illustrates the genus Beggiatoa. The filament
_A_ is so full of sulphur granules that the individual cells are not
visible. _B_ has fewer sulphur granules. In _C_ the granules are
nearly absent and the separate cells of the filament are seen. (After
Winogradsky, from Lafar.)]


ORDER I. Eubacteria.

Cells without nuclei, free from sulphur granules and from
bacteriopurpurin (p. 112); colorless, or slightly colored.

1. Family: COCCACEÆ (Zopf) Migula, all cocci.

                   {Genus 1. _Streptococcus_ Billroth:
                   {          division in one plane only (Fig. 49).
  Non-flagellated, {  „   2. _Micrococcus_ (Hallier) Cohn:
  Non-motile       {          division in two planes only (Fig. 50).
                   {  „   3. _Sarcina_ Goodsir:
                   {          division in three planes only (Fig. 51).

                   {  „   4. _Planococcus_ Migula:
  Flagellated,     {          division in two planes only.
  motile           {  „   5. _Planosarcina_ Migula:
                   {          division in three planes only.

2. Family: BACTERIACEÆ Migula, all bacilli.

  Genus 1. _Bacterium_ (Ehrenberg) Migula: no flagella; non-motile.
    „   2. _Bacillus_ (Cohn) Migula: flagella peritrichic (Fig. 52).
    „   3. _Pseudomonas_ Migula: flagella at the end:
              monotrichic, lophotrichic, amphitrichic (Fig. 53).

3. Family: SPIRILLACEÆ Migula, all spirilla.

                {Genus 1. _Spirosoma_ Migula:
                {          non flagellated; non-motile.
                {  „   2. _Microspira_ (Schrœter) Migula:
  Cells stiff   {          flagella one to three at the end (Fig. 54).
                {  „   3. _Spirillum_ (Ehrenberg) Migula:
                {          flagella more than three
                {          at the end (Fig. 55).

  Cell flexible {  „   4. _Spirochæta_ Ehrenberg:
                {          motile; no flagella (Fig. 56).

4. Family: CHLAMYDOBACTERIACEÆ.

Cells cylindrical in long threads and surrounded by a sheath.
Reproduction also by gonidia formed from an entire cell.

  Genus 1. _Chlamydothrix_ Migula (Fig. 57).
    „   2. _Crenothrix_ Colin (Fig. 58).
    „   3. _Pragmidiothrix_ Engler.
    „   4. _Spherotilus_ (including Cladothrix).


ORDER II. THIOBACTERIA: SULPHUR BACTERIA.

Cells without a nucleus, but containing sulphur granules, may be
colorless or contain bacteriopurpurin and be colored reddish or violet.

1. Family BEGGIATOACEÆ.

  Genus 1. _Thiothrix_ Winogradsky.

    „   2. _Beggiatoa_ Trevisan. Of interest since it is without a
            sheath, is motile, but without flagella (Fig. 59).

2. Family RHODOBACTERIACEÆ.

This has five subfamilies and twelve genera, most of which are due to
the Russian bacteriologist Winogradsky who did more work than anyone
else with the sulphur bacteria.


THE CLASSIFICATION OF THE SOCIETY OF AMERICAN BACTERIOLOGISTS.

The Committee on Classification of the Society of American
Bacteriologists at the meeting held in December, 1919, submitted its
final report. This report has not been formally adopted as a whole,
but in all probability will be substantially as outlined below. This
outline does not attempt to give the detailed characterizations of the
different groups as defined by the committee, but does show the names
to be applied to the commoner organisms. These organisms are included
in the 4th and 5th orders. Details of the first three orders have not
been worked out. They are listed merely for completeness.

CLASS SCHIZOMYCETES.

Unicellular, chlorophyl-free plants, reproducing by transverse division
(some forms by gonidia also).

ORDERS:

  A. Myxobacteriales--Cells united during vegetative stage into
  a pseudo-plasmodium which passes over into a highly developed
  cyst-producing resting stage.

  B. Thiobacteriales--Sulphur bacteria.

  C. Chlamydobacteriales--Iron bacteria and other sheathed bacteria.

  D. Actinomycetales--Actinomyces, tubercle and diphtheria bacilli.

  E. Eubacteriales--All the other common bacteria.

GENERA OF ORDERS D AND E.

  D. ACTINOMYCETALES--
      FAMILY I. ACTINOMYCETACEÆ Buchanan, 1918.
        Genus 1. _Actinobacillus_, Brampt, 1900.
          Type species, _Actinobacillus lignieresi_ Brampt, 1900.
        Genus 2. _Leptotrichia_ Trevisan, 1879.
          Type species, _Leptotrichia buccalis_ (Robin, 1847) Trevisan.
        Genus 3. _Actinomyces_ Harz, 1877.
          Type species, _Actinomyces bovis_ Harz.
        Genus 4. _Erysipelothrix_ Rosenbach, 1909.
          Type species, _Erysipelothrix rhusiopathiæ_ (Kitt, 1893)
              Rosenbach, swine erysipelas.
      FAMILY II. MYCOBACTERIACEÆ Chester, 1897.
        Genus 1. _Mycobacterium_ Lehmann and Neumann, 1896.
          Type species, _Mycobacterium tuberculosis_ (Koch, 1882) L.
              and N.
        Genus 2. _Corynebacterium_ Lehmann and Neumann, 1896.
          Type species, _Corynebacterium diphtheriæ_ (Loeffler, 1882)
              L. and N.
        Genus 3. _Fusiformis_ Hoelling, 1910.
          Type species, _Fusiformis termitidis_ Hoelling. Vincent’s
              angina.
        Genus 4. _Pfeifferella_ Buchanan, 1918.
          Type species, _Pfeifferella mallei_ (Loeffler, 1896) Buchanan.
              Glanders bacillus.

  E. EUBACTERIALES
      FAMILY I--NITROBACTERIACEÆ--Proto- or autotrophic for N
            or C and sometimes for both (except Acetobacter).
        TRIBE I--NITROBACTEREÆ--autotrophic for C.
          Genus 1. _Hydrogenomonas_ Jensen, 1909.
            Type species, _Hydrogenomonas pantotropha_ (Kaserer, 1906)
                Jensen; oxidizes free H.
          Genus 2. _Methanomonas_ Jensen, 1909.
            Type species, _Methanomonas methanica_ (Söhngen) Jensen;
                oxidizes CH₄.
          Genus 3. _Carboxydomonas_ Jensen, 1909.
            Type species, _Carboxydomonas oligocarbophila_ (Beijerinck
                and Van Delden, 1903) Jensen; oxidizes CO.
          Genus 4. _Acetobacter_ Fuhrman, 1905.
            Type species, _Acetobacter aceti_ (Thompson, 1852) Fuhrman;
                oxidizes alcohol to acetic acid.
          Genus 5. _Nitrosomonas_ Winogradsky, 1892.
            Type species, _Nitrosomonas europoea_ Winogradsky; oxidizes
                ammonia or ammonium salts to nitrous acid,
                hence nitrites.
          Genus 6. _Nitrobacter_ Winogradsky, 1892.
            Type species, _Nitrobacter Winogradskyi_ Committee of 1917;
                oxidizes nitrous acid (nitrites)
                to nitric acid (nitrates).
        TRIBE II--AZOTOBACTEREÆ--prototrophic for N.
          Genus 7. _Azotobacter_ Beijerinck, 1901; large, free-living,
              aerobic N absorbers.
            Type species, _Azotobacter chroococcum_ Beijerinck.
          Genus 8. _Rhizobium_ Frank, 1889.
            Type species, _Rhizobium leguminosarum_ Frank; root tubercle
                bacteria of legumes.
      FAMILY II--PSEUDOMONADACEÆ, Committee of 1917.
        Genus 1. _Pseudomonas_ Migula, 1894.
          Type species, _Pseudomonas violacea_ (Schroeter, 1872) Migula.
      FAMILY III--SPIRILLACEÆ Migula, 1894--all spiral bacteria.
        Genus 1. _Vibrio_ Müller, 1786, emended by E. F. Smith, 1905.
          Type species, _Vibrio choleræ_ (Koch, 1884) Schroeter, 1886.
        Genus 2. _Spirillum_ Ehrenberg, 1830, emended Migula, 1894.
          Type species, _Spirillum undula_ (Müller, 1786) Ehrenberg.
      FAMILY IV--COCCACEÆ Zopf, 1884, emended Migula, 1894--all cocci.
        Tribe I--NEISSEREÆ.
          Genus 1. _Neisseria_ Trevisan, 1885.
            Type species, _Neisseria gonorrhoeae_ Trevisan.
        Tribe II--STREPTOCOCCEÆ Trevisan, 1889.
          Genus 2. _Diplococcus_ Weichselbaum, 1886.
            Type species, _Diplococcus pneumoniae_ Weichselbaum.
          Genus 3. _Leuconostoc_ Van Tieghem, 1878.
            Type species, _Leuconostoc mesenterioides_ (Cienkowski) Van
                Tieghem.
          Genus 4. _Streptococcus_ Rosenbach, 1884; emended Winslow
                and Rogers, 1905.
            Type species, _Streptococcus pyogenes_ Rosenbach.
        Tribe III--MICROCOCCEÆ Trevisan, 1889.
          Genus 5. _Staphylococcus_ Rosenbach, 1884; animal parasites.
            Type species, _Staphylococcus aureus_ Rosenbach.
          Genus 6. _Micrococcus_ Cohn, 1872, emended Winslow and Rogers,
              1905. Facultative parasites or saprophytes.
            Type species, _Micrococcus luteus_ (Schroeter, 1872) Cohn.
          Genus 7. _Sarcina_ Goodsir, 1842, emended Winslow and
                Rogers, 1905.
            Type species, _Sarcina ventriculi_ Goodsir.
          Genus 8. _Rhodococcus_ Zopf, 1891, emended Winslow and
                Rogers, 1905; cocci with red pigment.
            Type species, _Rhodococcus rhodochrous_ Zopf.
      FAMILY V--BACTERIACEÆ Cohn, 1872, emended by Committee of 1917;
                bacilli without spores not above included.
        Tribe I--CHROMOBACTEREÆ Committee of 1919; producing red or
                violet pigment, mainly water forms.
          Genus 1. _Erythrobacillus_ Fortineau, 1905.
            Type species, _Erythrobacillus prodigiosus_
                (Ehrenberg, 1848) Fortineau.
          Genus 2. _Chromobacterium_ Bergonzini, 1881.
            Type species, _Chromobacterium violaceum_ Bergonzini.
        Tribe II--ERWINEÆ Committee 1919; plant pathogens.
          Genus 3. _Erwinia_ Committee 1917.
            Type species, _Erwinia amylovora_ (Burrill, 1883) Committee
                1917.
        Tribe III--ZOPFEÆ Committee of 1919; Gram +, no pigment,
                non-carbohydrate-fermenting.
          Genus 4. _Zopfius_ Wenner and Rettger, 1919.
            Type species, _Zopfius zopfii_ (Kurth) Wenner and Rettger.
        Tribe IV--BACTEREÆ Committee of 1919; Gram -, carbohydrate
                fermenters.
          Genus 5. _Proteus_ Hauser, 1885; liquefy gelatin.
            Type species, _Proteus vulgaris_ Hauser.
          Genus 6. _Bacterium_ Ehrenberg, 1828, emended Jensen, 1909;
                liquefy gelatin rarely.
            Type species, _Bacterium coli_.
        Tribe VI--LACTOBACILLEÆ Committee of 1919; Gram +, high acid,
                thermophils.
          Genus 7. _Lactobacillus_ Beijerinck, 1901.
            Type species, _Lactobacillus caucasicus_ (Kern?) Beijerinck;
                Bulgarian bacillus.
        Tribe VI--PASTEURELLEÆ Committee of 1919; organisms of
                hemorrhagic septicemia.
          Genus 8. _Pasteurella_ Trevisan, 1888.
            Type species, _Pasteurella cholerae-gallinarum_
                (Flügge, 1886); Trevisan.
        Tribe VII--HEMOPHILEÆ Committee of 1917; require hemoglobin for
                growth.
          Genus 9. _Hemophilus_ Committee of 1917.
            Type species, _Hemophilus influenzae_ (Pfeiffer, 1893)
                Committee of 1917.
      FAMILY VI--BACILLACEÆ Fischer, 1895. Spore forming rods.
          Genus 1. _Bacillus_ Cohn, 1872; aerobic, no change of form
                around the spore.
            Type species, _Bacillus subtilis_ Cohn.
          Genus 2. _Clostridium_ Prazmowski, 1880; anaërobic, frequently
                enlarged around spore.
            Type species, _Clostridium butyricum_ Prazmowski.

As compared with Migula’s classification it is to be noted that there
are 38 genera listed by the Committee instead of 13 in the same general
groups.

The following list of _Genera conservanda_ submitted by the Committee
was formally adopted by the Society and these are therefore its
official names for the organisms included in these genera.

  _Acetobacter_ Fuhrman
  _Actinomyces_ Harz
  _Bacillus_ Cohn
  _Bacterium_ Ehrenberg
  _Chromobacterium_ Bergonzini
  _Clostridium_ Prazmowski
  _Erythrobacillus_ Fortineau
  _Leptotrichia_ Trevisan
  _Leuconostoc_ Van Tieghem
  _Micrococcus_ Cohn
  _Rhizobium_ Frank
  _Sarcina_ Goodsir
  _Spirillum_ Ehrenberg
  _Staphylococcus_ Rosenbach
  _Streptococcus_ Rosenbach
  _Vibrio_ Müller

_It is greatly to be desired that the Society’s Classification when
finally completed shall become the standard in the United States at
least._

_Such names as have been adopted by the Society are used throughout
this work._

The Committee also submitted the following artificial key for
determining the genera in the two orders _ACTINOMYCETALES AND
EUBACTERIALES_:

  A--Typically filamentous forms                       _Actinomycetacae_
    B--Mycelium and conidia formed                     _Actinomyces_
    BB--No true mycelium
      C--Cells show branching
        D--Gram negative                               _Actinobacillus_
        DD--Gram positive                              _Erysipelothrix_
      CC--Cells never branch. Gram positive threads later fragmenting
          into rods                                    _Leptotrichia_
  AA--Typically unicellular forms (though chains of cells may occur)
    B--Cells spherical--_COCCACEÆ_
      C--Parasitic forms (except Leuconostoc), cells generally grouped
          in pairs or chains, never in packets, generally active
          fermenters.
        D--Cells in flattened coffee-bean-like pairs, gram -.
                                                       _Neisseria_
        DD--Not as D
          E--Saprophytes in zoögloea masses in sugar solutions.
                                                       _Leuconostoc_
          EE--Not as E. Gram +.
            F--Cells in lanceolate pairs or in chains. Growth on
                media not abundant.
              G--Cells in lanceolate pairs. Inulin generally fermented.
                                                       _Diplococcus_
              GG--Cells in chains. Inulin not generally fermented.
                                                       _Streptococcus_
            FF--Cells in irregular groups. Growth in media fairly
                vigorous. White or orange pigment.
                                                       _Staphylococcus_
      CC--Saprophytic forms. Cells in irregular groups or packets,
          not in chains. Fermentative powers low.
        D--Packets                                     _Sarcina_
        DD--No packets.
          E--Yellow pigment                            _Micrococcus_
          EE--Red pigment                              _Rhodococcus_
    BB--Rods:
      C--Spiral rods
        D--Short, comma-like rods. One to three flagella.
                                                       _Vibrio_
        DD--Long spirals. Five to twenty flagella.     _Spirillum_
      CC--Straight rods.
        D--No endospores.
          E--Rods of irregular shape or showing branched or filamentous
              involution forms.
            F--Cells irregular in shape. Staining unevenly. Animal
              parasites.
              G--Acid fast                             _Mycobacterium_
              GG--Not acid fast.
                H--Cells elongated, fusiform           _Fusiformis_
                HH--Cells not elongated, sometimes branching.
                  I--Gram positive. Slender, sometimes club-shaped.
                                                       _Corynebacterium_
                  II--Gram negative. Rods sometimes form threads.
                      Characteristic honey-like growth on potato.
                                                       _Pfeifferella_
            FF--Cells staining unevenly but with branched or filamentous
                forms at certain stages. Never acid fast.
                Not animal parasites.
              G--Metabolism simple, growth processes involving oxidation
                  of alcohol or fixation of free N (latter in symbiosis
                  with green plants).
                H--Cells minute. Symbiotic in roots of legumes.
                                                       _Rhizobium_
                HH--Oxidizing alcohol. Branching forms common.
                                                       _Acetobacter_
              GG--Not as G. Proteus-like colonies.
                H--Not attacking carbohydrates         _Zopfius_
                HH--Fermenting glucose and sucrose at least.
                                                       _Proteus_
          EE--Regularly formed rods.
            F--Metabolism simple, growth processes involving oxidation
                of C, H, or their simple compounds or the fixation
                of free N.--_NITROBACTERIACEÆ._
              G--Fixing N or oxidizing its simple compounds.
                H--Fixing N, cells large, free in soil _Azotobacter_
                HH--Oxidizing N compounds.
                  I--Oxidizing NH₄ compounds           _Nitrosomonas_
                  II--Oxidizing nitrites               _Nitrobacter_
              GG--Not as G.
                H--Oxidizing free H                    _Hydrogenomonas_
                HH--Oxidizing simple C compounds, not free H.
                  I--Oxidizing CO                      _Carboxydomonas_
                  II--Oxidizing CH₄                    _Methanomonas_
            FF--Not as F.
              G--Flagella usually present, polar--_PSEUDOMONADACEÆ_
                                                       _Pseudomonas_
              GG--Flagella when present peritrichic--_BACTERIACEÆ_
                H--Parasitic forms showing bi-polar staining.
                                                       _Pasteurella_
                HH--Not as H.
                  I--Strict parasites growing only in presence
                     of hemoglobin
                                                       _Hemophilus_
                  II--Not as I.
                    J--Water forms producing red or violet pigment.
                      K--Pigment red                   _Erythrobacillus_
                      KK--Pigment violet               _Chromobacterium_
                    JJ--Not as J.
                      K--Plant pathogens               _Erwinia_
                      KK--Not plant pathogens.
                        L--Gram +, forming large amount of acid
                          from carbohydrates, sometimes CO₂,
                          never H                      _Lactobacillus_
                        LL--Gram -, forming H as well as CO₂ if
                          gas is produced              _Bacterium_
        DD--Endospores present--_BACILLACEÆ_
          E--Aerobes, rods not swollen at sporulation. _Bacillus_
          EE--Anaërobes, rods swollen at sporulation.  _Clostridium_




PART II.

PHYSIOLOGY.




CHAPTER VI.

GENERAL CONDITIONS FOR GROWTH.


OCCURRENCE.

Bacteria are probably the most widely distributed of living organisms.
They are found practically everywhere on the surface of the earth.
Likewise in all surface waters, in streams, lakes and the sea. They
occur in the air immediately above the surface, since they are carried
up mechanically by air currents. They cannot fly of themselves. There
is no reason to believe that any increase in numbers occurs to an
appreciable extent in the air. The upper air, for example, on high
mountains, is nearly free from them. So also is the air over midocean,
and in high latitudes. As a rule, the greater the amount of dust in
the air, the more numerous are the bacteria. Hence they are found more
abundantly in the air in cities and towns than in the open country.
The soil is especially rich in numbers in the upper few feet, but they
diminish rapidly below and almost disappear at depths of about six
feet unless the soil is very porous and open, when they may be carried
farther down. Hence the waters from deep wells and springs are usually
devoid of these organisms. In the sea they occur at all levels and have
been found in bottom ooze dredged from depths of several miles. It is
perhaps needless to add that they are found on the bodies and in the
alimentary tract of human beings and animals; on clothing, utensils;
in dwellings, stables, outhouses, etc. From one-fourth to one-half of
the dry weight of the feces of animals and men is due to the bacteria
present. The urine is practically free from them in health.

While bacteria are thus found nearly everywhere, it is an entirely
mistaken idea to suppose that all are injurious to man. As a matter
of fact, those which are dangerous are relatively few and are for the
most part found only in close association with man. Most bacteria are
harmless and the vast majority are beneficial or even essential to
man’s existence on the earth. These facts must be constantly borne in
mind, and it is hoped that the pages which follow will make them clear.

In order that any organism may thrive there are a number of general
environmental conditions which must be fulfilled. These conditions
vary more or less for each kind of organism. Bacteria are no exception
to this general rule. These conditions may be conveniently considered
under the general heads of _moisture_; _temperature_; _light_; _oxygen
supply_; _osmotic pressure_; _action of electricity_; of _Röntgen_
and _radium rays_; _pressure_; _mechanical vibration_; and _chemical
environment_, including the _reaction of the medium_, _the effect
of injurious chemicals_, and especially the _food requirements of
bacteria_. For each of these conditions there is a _maximum_, meaning
the greatest amount of the given condition which the organism can
withstand, a _minimum_, or the least amount, and an _optimum_ or that
amount which is most favorable for development. Further, there might be
distinguished a maximum for _mere existence_ and a lower maximum for
_development_; also a minimum for _mere existence_ and a higher minimum
for _development_. These maxima, minima, and optima for bacteria have
been determined with exactness for only a very few of the general
conditions and for comparatively few kinds.


MOISTURE.

The _maximum_ moisture is absolutely pure water, and no organism can
thrive in this alone owing to the factor of too low osmotic pressure
and to the further factor of absence of food material. There are many
bacteria which thrive in water containing only traces of mineral salts
and a large class whose natural habitat is surface water. These “water
bacteria” are of great benefit in the purification of streams. They are
as a class harmless to men and animals. Some of the disease-producing
bacteria like _Bacterium typhosum_ (of typhoid fever) and _Vibrio
choleræ_ (of Asiatic cholera) were undoubtedly originally water
bacteria, and it is rather striking that in these diseases conditions
are induced in the intestine (diarrheas) which simulate the original
watery environment. The _minimum_ moisture condition is absolute
dryness, and no organism can even exist, not to say develop, in such
a condition since water is an essential constituent of living matter.
Some bacteria and especially most spores may live when dried in the
air or by artificial means for months and even years, while some are
destroyed in a few hours or days when dried (typhoid, cholera, etc.).
The optimum amount of moisture has not been determined with any great
accuracy and certainly a rather wide range in percentage of water is
permissible with many, though a liquid medium is usually most favorable
for artificial growth. The “water bacteria” have been mentioned. In the
soil a water content of 5 to 15 per cent. seems to be most suitable for
many of the organisms which aid in plant growth. In animals and man the
organisms infecting the intestinal tract prefer a high percentage of
moisture as a rule, especially those causing disease here. Those found
on the surface of the body (pus cocci) need a less amount of water,
while those invading the tissues (tuberculosis, black-leg, etc.) seem
to be intermediate in this respect. In artificial culture media a water
content of less than 30 per cent. inhibits the growth of most bacteria.

As a general rule those bacteria which require the largest percentage
of water are most susceptible to its loss and are most readily killed
by drying. The typhoid and cholera organisms die in a few hours when
dried, while pus cocci and tubercle bacilli live much longer.


TEMPERATURE.

The temperature conditions for bacterial existence and growth have been
determined more accurately than any of the other general conditions.
The maximum for existence must be placed at or near 100° since it is
known that all bacteria including spores may be killed by boiling in
time. Nevertheless, certain forms have been reported as thriving in hot
springs where the water temperature was 93°. This is the highest known
temperature for development. The minimum for existence lies at or near
the absolute zero (-273°) since certain organisms have been subjected
to the temperature produced by the sudden evaporation of liquid
hydrogen (-256° to -265°) and have remained alive. Whether they could
withstand such temperatures indefinitely is not known. The minimum for
development is near the freezing-point of water, since reproduction
by division has been observed in the water from melting sea-ice at
a temperature of -1.5°. Thus bacteria as a class have a range for
existence of about 373° (-273° to +100°) and for development of 94.5°
(-1.5° to +93°) certainly much wider ranges than any other group of
organisms.[5]

The optimum temperature for development varies within rather wide
limits for different organisms. In general it may be stated that the
optimum temperature is approximately that of the natural habitat of
the organism, though there are exceptions. The optimum of the “hot
spring” bacteria just mentioned is apparently that of the springs (93°
in this case). Many soil organisms are known whose optimum is near
70° (a temperature rarely, if ever, attained in the soil), _but only
when grown in air or oxygen_; but is very much lower when grown in the
_absence of oxygen_. Many other soil organisms exhibit very little
difference in rate or amount of growth when grown at temperatures which
may vary as much as 10° or 15°, apparently an adaptation to their
normal environment. The disease-producing organisms show much narrower
limits for growth, especially those which are difficult to cultivate
outside the body. For example, the bacterium of tuberculosis in man
scarcely develops beyond the limits of 2° or 3° from the normal body
temperature of man (37°), while the bacterium of tuberculosis in birds
grows best at 41° to 45°, the normal for birds, and the bacterium of
so-called tuberculosis of cold-blooded animals at 14° to 18°.

Those bacteria whose optimum temperature is above 40° are sometimes
spoken of as the “_thermophil_” bacteria. The fixing of the “thermal
death-point” that is, the minimum temperature at which the bacteria
are killed is a matter of great practical importance in many ways
and numerous determinations of this have been made with a great many
organisms and by different observers. The factors which enter into such
determinations are so many and so varied that unless all the conditions
of the experiment are given together with the time of application,
the mere statements are worthless. It may be stated that all _young,
actively growing_ (non-spore-containing) _disease-producing bacteria,
when exposed in watery liquids and in small quantities are killed at
a temperature of 60° within half an hour_. It is evident, that this
fact has very little practical application, since the conditions stated
are rarely, if ever, fulfilled except in laboratory experiments. (See
Sterilization and Pasteurization, Chapter XIII.)


LIGHT.

Speaking generally, it can be said that light is destructive to
bacteria. Many growing forms are killed in a few hours when properly
exposed to direct sunlight and die out in several days in the diffuse
daylight of a well-lighted room. Even spores are destroyed in a
similar manner, though the exposure must be considerably longer.
Certain bacteria which produce colors may grow in the light, since
the pigments protect them. Some few kinds, like the sulphur bacteria,
which contain a purplish-red pigment that serves them to break up
H₂S, need light for their growth. Since disease-producing bacteria
are all injuriously affected by light, the advantage of well-lighted
habitations both for men and animals is obvious.


OXYGEN SUPPLY.

Oxygen is one of the constituents of protoplasm and is therefore
necessary for all organisms. This does not mean that all organisms
must obtain their supply from _free oxygen_, however, as animals and
plants generally do. This fact is well illustrated by the differences
among bacteria in this respect. Some bacteria _require free oxygen_ for
their growth and are therefore called _aërobic_ bacteria or _aërobes_
(sometimes _strict aërobes_, though the adjective is unnecessary).
Others _cannot grow in the presence of free oxygen_ and are therefore
named _anaërobic bacteria_ or _anaërobes_ (strict is unnecessary).
There are still other kinds which may grow either in the presence of
free oxygen or in its absence, hence the term _facultative anaërobes_
(usually) is applied to them. The distinction between _facultative
aërobe_ and _facultative anaërobe_ might be made. The former means
those which grow best in the absence of free oxygen, though capable of
growing in its presence, while the latter term means those which grow
best in the presence of free oxygen, but are capable of growing in its
absence. The amount of oxygen in the atmosphere in which an organism
grows may be conveniently expressed in terms of the oxygen pressure,
_i.e._, in millimeters of mercury. It is evident that the maximum,
minimum and optimum oxygen pressures for anaërobic bacteria are the
same, namely, 0 mm. Hg. This is true only for natural conditions,
since a number of anaërobic organisms have been gradually accustomed
to increasing amounts of O, so that by this process of training they
finally grew in ordinary air, that is, at an oxygen pressure of about
150 mm. Hg. (Normal air pressure is 760 mm. Hg. and oxygen makes up
one-fifth of the air.) The minimum O pressure for facultative anaërobes
is also 0 mm. Hg. Some experiments have been made to determine the
limits for aërobes, but on a few organisms only, so that no general
conclusions can be drawn from them. To illustrate: _Bacillus subtilis_
(a common “hay bacillus”) will grow at 10 mm. Hg. pressure but not at 5
mm. Hg. It will also grow in compressed oxygen at a pressure of three
atmospheres (2280 mm. Hg.), but not at four atmospheres (3040 mm. Hg.),
though it is not destroyed.

Parodko has determined the oxygen limits for five common organisms as
follows:

                                                        Minimum
                                 Maximum.            Vol.     Mm.
                      In atmospheres.  Mm. Hg.     per cent.  Hg.

  _Bacterium          1.94 to 2.51  1474 to 1908  0.00016 = 0.0012
   fluorescens_
  _Sarcina lutea_     2.51 to 3.18  1908 to 2417  0.00015 = 0.0011
  _Proteus vulgaris_  3.63 to 4.35  2749 to 3306      0        0
  _Bacterium coli_    4.09 to 4.84  3108 to 3478      0        0
  _Erythrobacillus    5.45 to 6.32  3152 to 4800      0        0
   prodigiosus_

These few instances do not disclose any general principles which may
be applied either for the growth or for the distinction of aërobes or
facultative anaërobes.

It has been shown that compressed oxygen will kill some bacteria but
this method of destroying them has little or no practical value. Oxygen
in the form of ozone, O₃, is rapidly destructive to bacteria, and this
fact is applied practically in the purification of water supplies for
certain cities where the ozone is generated by electricity obtained
cheaply from water power. The same is true of oxygen in the “nascent
state” as illustrated by the use of hypochlorites for the same purpose.

It was stated (p. 74) that certain thermophil bacteria in the soil have
an optimum temperature for growth _in the air_ which is much higher
than is ever reached in their natural habitat and that they grow at a
moderate temperature under _anaërobic_ conditions. It has been shown
that if these organisms are grown with aërobes or facultative anaërobes
they thrive at ordinary room temperature. These latter organisms by
using up the oxygen apparently keep the tension low, and this explains
how such organisms grow in the soil.[6]


OSMOTIC PRESSURE.

Like all living cells bacteria are very susceptible to changes in
the density of the surrounding medium. If placed in a medium less
concentrated than their own protoplasm water is absorbed and they
“swell up”; while if placed in a denser medium, water is given off and
they shrink (plasmoptysis or plasmolysis). Should these differences
be marked or the transition be sudden, the cell walls may even burst
and the organisms be destroyed. If the differences are not too great
or if the transition is made gradually, the organisms may not be
destroyed, but will either cease to grow and slowly die out, or will
show very much retarded growth, or will produce abnormal cell forms.
This is illustrated in the laboratory in attempting to grow bacteria on
food material which has dried out. A practical application of osmotic
effects is in the use of a high percentage of sugar in preserving
fruits, etc., and in the salting of meats. Neither the cane-sugar nor
the common salt themselves injure the bacteria chemically, but by the
high concentration prevent their development. In drying material in
order to preserve it there are two factors involved: first, the loss of
water necessary for growth and second, the increased osmotic pressure.

In a medium of greater density diffusion of water is outward from the
cell and this will continue until an equilibrium is established between
cell contents and medium. Food for the organism _must be in solution
and enter the cell by diffusion_. Therefore, growth ceases in a medium
too dense, since water to carry food in solution does not enter the
cell.


ELECTRICITY.

Careful experimenters have shown that the electric current, either
direct or alternating, has no direct destructive effect on bacteria.
In a liquid medium the organisms may be attracted to or repelled
from one or the other pole or may arrange themselves in definite
ways between the poles (galvanotaxis), but are not injured. However,
electricity through the _secondary_ effects produced, may be used to
destroy bacteria. If the passage of the electric current _increases the
temperature_ of the medium sufficiently, the bacteria will be killed,
or if _injurious chemical substances_ are formed (ozone, chlorine,
acids, bases, etc.), the same result will follow (see Ozone, pp. 77 and
157).


RADIATIONS.

Röntgen or _x_-rays and radium emanations when properly applied to
bacteria will destroy them. The practical use of these agents for
the direct destruction of bacteria in diseases of man or animals is
restricted to those cases where they may be applied directly to the
diseased area, since they are just as injurious to the animal cell
as they are to the bacteria, and even more so. Their skilful use as
_stimuli to the body cells_ to enable them to resist and overcome
bacteria and other injurious organisms or cell growths is an entirely
different function and will not be considered here.


PRESSURE.

Hydrostatic pressure up to about 10,000 pounds per square inch is
without appreciable effect on bacteria as has been shown by several
experimenters and also by finding living bacteria in the ooze dredged
from the bottom of the ocean at depths of several miles.

Pressures from 10,000 to 100,000 pounds show variable effects. Some
bacteria are readily killed and others, even non-spore formers,
are only slightly affected. The time factor is important in this
connection. The presence of acids, even CO₂, or organic acids, results
in the destruction of most non-spore formers.


MECHANICAL VIBRATION.

Vibrations transmitted to bacteria in a liquid may be injurious to them
under certain circumstances. Some of the larger forms like _Bacillus
subtilis_ may be completely destroyed by shaking in a rapidly moving
shaking machine in a few hours. Bacteria in liquids placed on portions
of machinery where only a slight trembling is felt, have been found
to be killed after several days. Reinke has shown that the passing of
strong sound waves through bacterial growths markedly inhibits their
development.




CHAPTER VII.

CHEMICAL ENVIRONMENT.


REACTION OF MEDIUM.

Most bacteria are very susceptible to changes in the degree of acidity
or alkalinity of the medium in which they grow. Some kinds prefer a
slightly acid reaction, some a slightly alkaline, and some a neutral
(with reference to litmus as indicator). The organism which is the
commonest cause of the souring of milk thrives so well in the acid
medium it produces that it crowds out practically all other kinds,
though its own growth is eventually stopped by too much acid. Acid
soils are usually low in numbers of bacteria and as a consequence
produce poor crops. The disease-producing bacteria as a class grow best
in a medium which is slightly alkaline.

Accurate determination of limits have been made on but few organisms.
The reaction is a most important factor in growing bacteria on
artificial media (see Making of Media, Chapter XVI).


INJURIOUS CHEMICAL SUBSTANCES.

(SEE DISINFECTION AND DISINFECTANTS, Chapter XIII.)


CHEMICAL COMPOSITION.

The chemical composition is subject to wide variation chiefly for
two reasons: First, the cell wall in most instances seems to exert
only a slight selective action in the absorption of mineral salts
so that their concentration within the cell is very nearly that of
the surrounding medium. Second, the chief organic constitutents vary
remarkably with the kind and amount of food material available--a
rich protein pabulum increases the protein, a plentiful supply of
carbohydrates or of fat results in the storing of more fat, especially
and _vice versa_. These facts must be borne in mind in considering the
chemistry of bacteria.

Of the chemical elements known, only the following seem to be essential
in the structure of bacteria: carbon, hydrogen, oxygen, nitrogen,
sulphur, phosphorus, chlorine, potassium, calcium, magnesium, iron,
manganese. Other elements, as sodium, iodine, silicon, aluminum,
lithium, copper, etc., have been reported by different analysts, but
none of them can be regarded as essential, except possibly in isolated
instances.

These elements exist in the bacterial cell in a great variety of
combinations of which the most abundant is _water_. The amount of water
varies in different species from 75 to 90 per cent. of the total weight
in growing cells, and is less in spores. The amount of _ash_ has been
shown by different observers to vary from less than 2 per cent. to as
much as 30 per cent. of the _dry weight_. The following table compiled
from various sources will give an idea of the relative abundance of the
different elements in the ash.

  S  as SO₃         7.64 per cent. (much more in sulphur bacteria)
  P  as P₂O₅       18.14    „      to 73.94 per cent.
  Cl                2.29    „
  K  as K₂O        11.1     „      to 25.59    „
  Ca as CaO        12.64    „      to 14.0     „
  Mg as MgO         0.7     „      to 11.55    „
  Fe as Fe₂O₃       1.0     „      to 8.15     „   (iron bacteria)
  Mn               traces

As to the form in which the last six elements in the table exist in
the cell, little is known. The sulphur and phosphorus are essential
constituents of various proteins. The high percentage of phosphorus
points to nuclein compounds as its probable source.

The carbon and nitrogen, together with most of the hydrogen and oxygen
not united as water, make up the great variety of organic compounds
which compose the main substances in the bacterial cell.

It has already been stated that the essential structures in the
bacterial cell are cell wall and protoplasm, including the nuclein.
These differ markedly in chemical composition. It is well known that
the cell walls of green plants consist largely of cellulose and closely
related substances.[7] _True cellulose_ has been recognized in but
very few bacteria. (_Sarcina ventriculi_, Migula; _Mycobacterium
tuberculosis_, Hammerschlag, Dreyfuss, Nishimura; _Bacillus
subtilis_, Dreyfuss; _Acetobacter xylinum_, Brown; _Acetobacter
acidi oxalici_, Banning; and a few others.) It is certainly not an
important constituent of the cell wall in many. On the other hand,
_hemicellulose_ and _gum-like_ substances have been identified in
numerous organisms of this class as important constituents of the cell
wall and of the capsule which is probably an outgrowth from the latter.
Practically always associated with these substances are compounds
containing nitrogen. One of these has been certainly identified as
_chitin_ or a closely similar substance. Chitin is the nitrogenous
substance which enters largely into the composition of the hard parts
of insects, spiders and crustaceans. It is an interesting fact to find
this substance characteristic of these animals in bacteria, as well as
other fungi.

Though it is extremely difficult to separate the cell wall of bacteria
from the cell contents, in the light of our present knowledge it can be
stated that the cell walls are composed of a carbohydrate body closely
related to cellulose, though not true cellulose, probably in close
combination with chitin.

Of the organic constituents of the cell contents the most abundant are
various proteins which ordinarily make up about one-half of the dry
weight of the entire cell. The “Mycoproteid” of Nencki, 1879, and other
earlier workers is deserving of little more than historical interest,
since these substances were certainly very impure and probably
consisted of mixtures of several “proteins” in the more recent sense.

From later studies it seems probable that substances resembling
the albumin of higher forms do not occur in bacteria, at least in
appreciable quantities. Globulin has been reported by Hellmich in an
undetermined bacterium, but is certainly not commonly found. The larger
portion of the protein is of a comparatively simple type, in fact,
consists of protamins most of which are in combination with nucleic
acid as nucleoprotamins. Practically all recent workers find a high
percentage of nuclein, both actually isolated and as indicated by the
amounts of purin bases--xanthin, guanin, adenin--obtained, as well as
by the abundance of phosphorus in the ash, already mentioned. Some of
these nucleins have been shown to have poisonous properties.

Closely related to but not identical with the proteins are the enzymes
and toxins which are formed in the cell and exist there as endo-enzymes
or endo-toxins respectively. These substances will be discussed later
under the heading “Physiological Activities of Bacteria” (Chapter XII).

Carbohydrates are not commonly present in the cell contents, though
glycogen has been observed in a few and a substance staining blue
with iodine in one or two others. This latter substance was at first
considered to be starch “granulose,” but is probably more closely
related to glycogen.

Fats seem to be very generally present. The commoner fats--tri-olein,
tri-palmitin, tri-stearin have been found by many analysts. The
“acid-fast bacteria” are particularly rich in fatty substances,
especially the higher wax-like fats. Lecithins (phosphorized fats) and
cholesterins (not fats but alcohols) have been repeatedly observed and
probably occur in all bacteria as products of katabolism.

Organic acids and esters occur as cell constituents but will be
discussed in connection with their more characteristic occurrences
as products of bacterial activity, as will also pigments which may
likewise be intracellular in some instances.

The following analysis of tubercle bacilli, from de Schweinitz and
Dorset, while not intended as typical for all bacteria, still
illustrates the high percentage of protein compounds which undoubtedly
occurs in most, as well as showing the large amount of fatty substance
in a typical “acid-fast” organism:

                {  8.5 per cent. tuberculinic acid
                { 24.5    „      nucleoprotamin    }
  In the dried  { 23.0    „      neucleoprotein    } 55.8 per cent.
    organisms   {  8.3    „      proteinoid        } protein.
                { 26.5    „      fat and wax
                {  9.2    „      ash




CHAPTER VIII.

CHEMICAL ENVIRONMENT (CONTINUED).


GENERAL FOOD RELATIONSHIPS. METABOLISM.

The foregoing brief review of the chemical composition of the bacterial
cell illustrates the variety of compounds which necessarily occurs,
but affords no definite clue as to the source of the elements which
enter into these compounds. These elements come from the material
which the organism uses as food. Under this term are included elements
or compounds which serve as building material, either for new cell
substance or to repair waste, or as sources of energy.

An organism which is capable of making use of an element in the free
state is said to be _prototrophic_ for that particular element.
Thus aërobes and facultative anaërobes are prototrophic for O. The
“root-tubercle bacteria” of leguminous and other plants and certain
free living soil organisms are prototrophic for N.[8]

On the other hand, if the element must be secured from compounds, then
the organism is _metatrophic_ in respect to the element in question.
Should the compound be inorganic, the term _autotrophic_ is applied
to the organism and _heterotrophic_ if the compound is organic. It is
very probable that anaërobes, exclusive of a few nitrogen absorbers,
are metatrophic for all the elements they utilize. With the exception
of the anaërobes it seems that all bacteria are _mixotrophic_, that is,
prototrophic for one or two elements and auto- or heterotrophic for the
others.[9]

Those bacteria whose food consists of dead material are spoken of as
_saprophytes_, while those whose natural habitat, without reference to
their food, is in or on other living organisms are called _parasites_.
The _host_ is the organism in or on which the parasite lives.
Parasites may be of several kinds. Those which neither do injury nor
are of benefit to the host are called _non-pathogenic_ parasites or
_commensals_; many of the bacteria in the intestines of man and other
animals are of this class. Those which do injury to the host are
called _pathogenic_ or disease-producing, as the organisms causing the
transmissible diseases of animals and plants.[10] Finally, we have
those parasites which are of benefit to and receive benefit from the
host. These are called _symbionts_ or _symbiotic parasites_ and the
mutual relationship _symbiosis_. Certain of the intestinal bacteria in
man and especially in herbivorous animals are undoubted _symbionts_, as
are also the “root-tubercle bacteria” already mentioned.

It is evident that all parasites that may be cultivated outside
the body are for the time _saprophytic_, hence the terms _strict_
parasites and _facultative_ parasites, which should require no further
explanation.

The changes which the above-mentioned types of food material undergo
in the various anabolic and katabolic processes _within the cell_ are
as yet but very slightly known. Nevertheless there are a number of
reactions brought about by bacteria acting on various food materials,
partly within _but largely without the cell_ which are usually
described as “physiological activities” or “biochemical reactions.”
Some of these changes are to be ascribed to the utilization of certain
of the elements and compounds in these materials as tissue builders,
some as energy-yielding reactions and still others as giving rise to
substances that are of direct benefit to the organism concerned in its
competition with other organisms.

Though all of the twelve elements already mentioned are essential for
the growth of every bacterium, two of them are of especial importance
for the reason that most of the “physiological activities” to be
described in the next chapters are centered around their acquisition
and utilization. These elements are _carbon_ and _nitrogen_. Some few
of the special activities of certain groups have to do with one or the
other of the remaining nine, as will be shown later. But generally
speaking _when a bacterium under natural conditions secures an adequate
supply of carbon and nitrogen, the other elements are readily available
in sufficient amount_.

Carbon is necessary not only because it is an essential constituent
of protoplasm but because its oxidation is the chief source of the
energy necessary for the internal life of the cell, though nitrogen
and sulphur replace it in this function with a few forms. This
latter use of carbon constitutes what may be called its _respiratory
function_. Bacteria like other organisms in their respiration utilize
oxygen and give off carbon dioxide. The amount of the latter given off
from the cell in this way is very small as compared with that which
is frequently produced as an accompaniment of other reactions (see
Fermentation, next chapter). But there is no doubt of its formation and
it has been determined by a few investigators. On account of this use
of carbon, bacteria require relatively large amounts of this element.
One group of bacteria concerned in the spontaneous heating of coal
seems to be able to use free carbon from this material. Another group
is said to be able to oxidize marsh gas, CH₄, and use this as its
source of carbon. The nitrite, nitrate and sulphur bacteria mentioned
later utilize carbon dioxide and carbonates as their carbon supply,
and one kind has been described which uses carbon monoxide. With these
few exceptions bacteria are dependent on _organic compounds_ for their
carbon and cannot use CO₂ as green plants do.

The oxygen requirement is high partly for the same reason that
carbon is, _i.e._, respiration. Oxygen is one of the constituents of
protoplasm, and combined with hydrogen forms water which makes up such
a large part of the living cell. Anaërobic bacteria are dependent on
so-called “molecular respiration” for their energy. That is, through
a shifting or rearrangement of the atoms in the compounds used as
food the oxidation of carbon is brought about. Enzymes are probably
responsible for this action. Carbon dioxide is produced by anaërobes
as well as by aërobes, and frequently in amounts readily collected. A
carbohydrate is usually though not always essential for the growth of
anaërobes and serves them as the best source of energy.

Nitrogen is the characteristic element of living material. Protoplasm
is a chemical substance in unstable equilibrium and nitrogen is
responsible for this instability. No other of the commoner elements
is brought into combination with such difficulty, nor is so readily
liberated when combined (all commercial explosives are nitrogen
compounds). Bacteria, like other forms of protoplasm, require nitrogen.
More marked peculiarities are shown by bacteria with reference to the
sources from which they derive their nitrogen than for carbon. Some can
even combine the free nitrogen of the air and furnish the only natural
means of any importance for this reaction. Some few forms (the nitrite
and nitrate formers, Chapter XI) obtain their energy from the oxidation
of inorganic nitrogen compounds, ammonia and nitrites respectively, and
not from carbon. These latter bacteria use carbon from carbon dioxide
and carbonates. A great many bacteria can secure their nitrogen from
nitrates but some are restricted to organic nitrogen. Many bacteria
obtain their carbon from the same organic compounds from which their
nitrogen is derived.

Sulphur serves mainly as a constituent of protein compounds in the
protoplasmic structure. In some of the _sulphur_ bacteria it is a
source of energy, since either free sulphur or H₂S is oxidized by them.
Some of these bacteria can obtain their carbon from CO₂ or carbonates,
and their nitrogen from nitrates or ammonium salts.

Whether the _iron_ bacteria, belonging to the genus _Crenothrix_ of the
higher, thread bacteria, use this element or its compounds as sources
of energy is still a disputed question. The evidence is largely in
favor of this view.

Free hydrogen has been shown to be oxidized by some forms which obtain
their energy in this way.

Whether there is a special class of _phosphorus_ bacteria remains to be
discovered. That phosphorus is oxidized during the activity of many
bacteria is undoubted, but whether this represents a source of energy
or is the accidental by-product of other activities is undetermined.

Practically nothing is known about the metabolism of the other elements
as such.

From the preceding brief review of the relation of certain bacteria
to some of the elements in the free state and from the further fact
that there is scarcely a known natural organic compound which cannot
be utilized by some kind of bacterium, it is evident that this class
of organisms has a far wider range of adaptability than any other
class, and this adaptability helps to explain their seemingly universal
distribution.

As to the metabolism _within the cell_, no more is known than is the
case with other cells, nor even as much. The materials used for growth
and as sources of energy are taken into the cell, built up into various
compounds some of which have been enumerated and in part broken down
again. Carbon dioxide and water are formed in the latter process. What
other katabolic products occur it is not easy to determine. Certainly
some of the substances mentioned in the next chapters are such products
but it is not always possible to separate those formed _inside_ the
cell from those formed _outside_. Perhaps most of the latter should be
considered true metabolic products. It would seem that on account of
the simplicity of structure of the bacterial cell and of the compounds
which they may use as food they would serve as excellent objects
for the study of the fundamental problems of cell metabolism. Their
minuteness and the nearly impossible task of separating them completely
from the medium in or on which they are grown makes the solution of
these problems one of great difficulty.

When all of the environmental conditions necessary for the best
development of a given bacterium are fulfilled, it will then develop
to the limit of its capacity. This development is characterized
essentially by its reproduction, which occurs by transverse division.
The rate of this division varies much with the kind even under good
conditions. The most rapid rate so far observed is a division in
eighteen minutes. A great many reproduce every half-hour and this may
be taken as a good average rate. If such division could proceed without
interruption, a little calculation will show that in about sixty-five
hours a mass as large as the earth would be produced.

  Starting with 1 coccus, 1µ in diameter,
    its volume = 0.0000000000005 cc.

       1/2 hour  =                 2
         1 hour  =                 4
         2 hours =                16
         4 hours =               256
         5 hours =              1024 = 10³+
        15 hours =     1,000,000,000 = 10⁹ = 0.5 cc.
        35 hours =     10²¹+ = 500.0 cu.m.
  About 65 hours = 2 × 10⁴²+ = 5 × 10²⁰ cu.m. = a mass as large
                                               as the earth.

Such a rate of increase evidently cannot be kept up long on account of
many limiting factors, chief of which is the food supply.

The foregoing calculation is based on the assumption that the organism
divides in one plane only. If it divides in 2 or 3 planes, the rate is
much faster, as is shown by the following formulæ, which indicate the
theoretical rate of division:

  S = number of bacteria after a given number of divisions.
  a = number at the beginning, and n = number of divisions.
              1 plane division S = 2_ⁿ_a
              2   „      „     S = 2²_ⁿ_a
              3   „      „     S = 2³_ⁿ_a

With two-plane or three-plane division, assuming that each organism
attains full size, as was assumed in the first calculation, the “mass
as large as the earth” would be attained in about thirty-two and
twenty-two hours respectively.

This extraordinary rate of increase explains in large measure why
bacteria are able to bring about such great chemical changes in so
short a time as is seen in the rapid “spoiling” of food materials,
especially liquids. The reactions brought about by bacteria on
substances which are soluble and diffusible are essentially “surface
reactions.” The material diffuses into the cell over its entire surface
with little hindrance. The bacteria are usually distributed throughout
the medium, so that there is very intimate contact in all parts of
the mass which favors rapid chemical action. The following calculation
illustrates this:

  The volume of a coccus 1µ in diameter is 0.5236 × 10⁻¹³ cc.
  The surface of a coccus 1µ in diameter is π × 10⁻⁸ sq. cm.

It is not uncommon to find in milk on the point of souring
1,000,000,000 bacteria per cc.

Assuming these to be cocci of 1µ diameter the volume of these bacteria
in a liter is only 0.05 cc. or in the liter there would be 19999 parts
of milk and only 1 part bacteria. The surface area of these bacteria is
3141.6 sq. cm. With this large surface exposed, it is not strange that
the change from “on the point of souring” to “sour” occurs within an
hour or less.

Although large numbers of bacteria can and do cause great chemical
changes the amount of material actually utilized for maintenance of
the cell is very slight, infinitesimal almost, and yet is fairly
comparable to that required for man, as is illustrated by the following
computations:

E. Kohn has shown that certain water bacteria grew well in water to
which there was added per liter 0.000002 mg. dextrose, 0.00000007
mg. (NH₄)₂SO₄ and 0.0000000007 mg. (NH₄)₂HPO₄. The bacteria numbered
about 1000 per cc. Taking the specific gravity at 1 (a little too
low) the mass of the bacteria in the liter was about 0.001 mg. Hence
the bacteria used 0.002 of their weight of carbohydrate and 0.00007
of ammonium sulphate. A 150-pound (75-kilo) man can live on 375 g.
of sugar (0.005 of his weight) and 52.5 g. of protein (0.0007 of his
weight). From these figures it can be calculated that the man utilizes
about two and a half times as much carbohydrate and about seven times
as much nitrogen as the bacterium, relatively speaking.




CHAPTER IX.

PHYSIOLOGICAL ACTIVITIES.


The physiological activities of motion, reproduction and metabolism
within the cell have been discussed in previous chapters. The
objects in view in the discussion of the “physiological activities”
(sometimes spoken of as “biochemical” activities) of bacteria in
this and subsequent chapters are to familiarize the student to some
extent with the great range of chemical changes brought about by these
minute organisms, to show their usefulness, even their necessity, and
to impress the fact that it is chiefly by a careful study of these
“activities” that individual kinds of bacteria are identified. It
should always be borne in mind that the bacteria, in bringing about
these changes which are so characteristic in many instances, are simply
engaged in their own life struggle, in securing the elements which
they need for growth, in liberating energy for vital processes, or
occasionally in providing conditions which favor their own development
and hinder that of their competitors.


FERMENTATION OF CARBOHYDRATES.

By this is meant the changes which different carbohydrates undergo when
subjected to bacterial action.[11]

These changes are marked chiefly by the production of gas or acid. The
former is called “gaseous fermentation” the latter “acid fermentation.”
The gases commonly produced are carbon dioxide (CO₂) hydrogen and
marsh gas (CH₄). Other gases of the paraffin series may also be formed
as ethane (C₂H₆), acetylene (C₂H₂), etc. CO₂ and H are the ones usually
formed from sugars by the few gas-forming bacteria which produce
disease, though even here some CH₄ is present. The common _Bacterium
coli_ forms all three, though the CH₄ is in smallest quantity.

[Illustration: FIG. 60.--Cylinder to show the formation of gas by
bacteria. The gauge shows 265 pounds. It went beyond 500 pounds.]

[Illustration: FIG. 61.--A burning natural gas well at night. From a
photograph colored.]

In the fermentation of the polysaccharids--starch and especially
cellulose and woody material--large amounts of CH₄ occur, particularly
when the changes are due to anaërobic bacteria. This phenomenon may be
readily observed in sluggish streams, ponds and swamps where vegetable
matter accumulates on the bottom. The bubbles of gas which arise when
the mass is disturbed explode if a lighted match is applied to them.

The author has conducted a number of experiments to demonstrate this
action as follows: Material taken from the bottom of a pond in the
fall after vegetation had died out was packed into a cylinder five
feet long and six inches in diameter, water was added to within about
2 inches of the top. After leaving them open for a few days to permit
all the dissolved oxygen to be used up by the aërobes, the cylinders
were tightly capped and allowed to stand undisturbed. Pressure gauges
reading to 500 lbs. were attached (Fig. 60). At the end of six months
the gauge showed a pressure beyond the limits of the readings on it.
Most of the gas was collected and measured 146 liters. An analysis
of portions collected when about one-half had been allowed to escape
showed the following composition, according to Prof. D. J. Demorest of
the Department of Metallurgy:

  CO₂     18.6 per cent.
  CH₄     76.1    „
   H       1.0    „
   N       4.3    „

In the author’s opinion natural gas and petroleum have been formed in
this way[12] (Figs. 61 and 62).

[Illustration: FIG. 62.--A “flowing” oil well.]

One of the very few practical uses of the gaseous fermentation of
carbohydrates is in making “salt rising” bread. The “rising” of the
material is due not to yeasts but to the formation of gas by certain
bacteria which are present on the corn meal or flour used in the
process (Fig. 63).

[Illustration: FIG. 63.--A loaf of “salt rising” bread. The porous
structure is due to the gas formed by bacilli and not by yeasts.]

Another is in the formation of the “holes” or “eyes” so characteristic
of Swiss and other types of cheese (Fig. 64).

[Illustration: FIG. 64.--Ohio Swiss cheese. The “eyes” are due to gas
formed by bacteria during the ripening of the cheese.]

A great many organic acids are formed during the “acid fermentation”
of carbohydrates by bacteria. Each kind of bacterium, as a rule, forms
several different acids as well as other substances, though usually one
is produced in much larger amounts, and the kind of fermentation is
named from this acid. One of the commonest of these acids is lactic.
The “lactic acid bacteria” form a very large and important group and
are indispensable in many commercial processes. In the making of butter
the cream is first “ripened,” as is the milk from which many kinds of
cheese are made (Fig. 65). The chief feature of this “ripening” is the
formation of lactic acid from the milk-sugar by the action of bacteria.
A similar change occurs in the popular “Bulgarian fermented milk.” The
reaction is usually represented by the equation:

  Milk-sugar.                  Lactic acid.
  C₁₂H₂₂O₁₁ + H₂O + (bacteria) = 4C₃H₆O₃

It is not probable that the change occurs quantitatively as indicated,
because a number of other substances are also formed. Some of these
are acetic and succinic acids and alcohol. Another industrial use of
this acid fermentation is in the preparation of “sauer kraut.” These
bacteria are chiefly anaërobic and grow best in a relatively high salt
concentration. They occur naturally on the cabbage leaves.

[Illustration: FIG. 65.--A cream ripener. In this apparatus cream is
“ripened,” _i.e._, undergoes lactic acid fermentation, preparatory to
making it into butter.]

In the formation of ensilage (Fig. 66) the lactic acid bacteria play
a very important part, as they do also in “sour mash” distilling, and
in many kinds of natural “pickling.” In fact, whenever green vegetable
material “sours” spontaneously, lactic acid bacteria are always present
and account for a large part of the acid. This property of lactic acid
formation is also taken advantage of in the preparation of lactic acid
on a commercial scale in at least one plant in this country.

[Illustration: FIG. 66.--Filling a silo on the University farm.]

Acetic acid is another common product of acid fermentation. However, in
vinegar making the acetic acid is not formed directly from the sugar in
the fruit juice by bacteria. The sugar is first converted into alcohol
by yeasts, then the alcohol is _oxidized_ to acid by the bacteria (Fig.
67). The reaction may be represented as follows:

  Dextrose. Ethyl alcohol.  Acetic acid.
  C₆H₁₂O₆ = 2C₂H₅OH + 2CO₂

  C₂H₅OH + O₂ + (bacteria) = CH₃COOH + H₂O.

Butyric acid is generally produced where fermentation of carbohydrates
occurs under _anaërobic_ conditions. Some of the “strong” odor of
certain kinds of cheese is due to this acid which is formed partly
from the milk-sugar remaining in the cheese. Most of it under these
conditions comes from the proteins of the cheese and especially from
the fat (see page 101).

As has been indicated alcohol is a common accompaniment of most acid
fermentations, as are the esters of acids other than the chief product.
Bacteria are not used in a commercial way to produce alcohol, however,
as the yield is too small. There are some few bacteria in which the
amount of alcohol is prominent enough to call the process an “alcoholic
fermentation” rather than an acid one. In brewing and distilling
industries, _yeasts_ are used to make the alcohol, though molds replace
them in some countries (“sake” and “arrak” from rice).

[Illustration: FIG. 67.--A vinegar ripener. The tank shown opened at
the side is filled with a special type of beech shavings which thus
provide a very large surface. The apple juice which has been previously
fermented with yeast, which converts the sugar into alcohol, is allowed
to trickle through the openings at the top over the shavings. The
acetic acid bacteria on the shavings rapidly oxidize the alcohol to
acetic acid. The vinegar is drawn off below.]

Under ordinary conditions the carbohydrate is never completely
fermented, since the accumulation of the product--acid--stops
the reaction. If the acid is neutralized by the addition of an
alkali--calcium or magnesium carbonate is best--then the sugar
may all be split up. Where such fermentation occurs under natural
conditions, the products are further split up, partly by molds and
partly by acid-destroying bacteria into simpler acids and eventually
to carbon dioxide and water, so that the end-products of the complete
fermentation of carbohydrate material in nature are carbon dioxide,
hydrogen, marsh gas, and water.

In all of these fermentations the bacteria are utilizing the _carbon_
both as building material and for oxidation and the fermentations
are incidental to this use. As a rule, the acid-forming bacteria can
withstand a higher concentration of acid than the other bacteria that
would utilize the same material, and in a short time crowd out their
competitors or inhibit their growth, and thus have better conditions
for their own existence, though finally their growth is also checked by
the acid.


SPLITTING OF FATS.

The _splitting of fats_ into glycerin and the particular acid or
acids involved may be brought about by bacteria. An illustration
is the development of rancidity in butter at times and the
“strong” odor of animal fats on long keeping and of many kinds of
cheese--“limburger”--in this country. Generally speaking, however, fats
are not vigorously attacked, as is illustrated by the difficulties due
to accumulation of fats in certain types of sewage-disposal works. The
chemical change is represented by the equation:

   Fat.                            Glycerin.     Fatty acid.
  C₃H₅(C{_n_}H₂{_n_}₋₁O₂)₃ + 3 H₂O = C₃H₅(OH)₃ + 3 (C{_n_}H₂{_n_}O₂).




CHAPTER X.

PHYSIOLOGICAL ACTIVITIES (CONTINUED).


PUTREFACTION OF PROTEINS.

The word “_putrefaction_” is now restricted to the action of bacteria
on the _complex nitrogen-containing substances_, proteins, and their
immediate derivatives. The process is usually accompanied by the
development of foul odors.

Bacteria make use of proteins chiefly as a source of nitrogen, but also
as a source of carbon and other elements. Proteins contain nitrogen,
carbon, hydrogen, oxygen, sulphur and frequently phosphorus. Some of
the metals--potassium, sodium, calcium, magnesium, iron and manganese
and the non-metal chlorine--are nearly always associated with them more
or less intimately. Since these bodies are the most complex of natural
chemical substances it follows that the breaking up of the molecule to
secure a part of the nitrogen gives rise to a great variety of products.

There are marked differences among bacteria in their ability to
attack this class of compounds. Some can break up the most complex
natural proteins such as albumins, globulins, glyco-, chromo-, and
nucleoproteins, nucleins and albuminoid derivatives like gelatin. The
term _saprogenic_ (σαπρος = rotten) is sometimes applied to bacteria
which have this power. These proteins are large-moleculed and not
diffusible, so that the first splitting up that they undergo must occur
outside the bacterial cell. The products of this first splitting may
diffuse into the cell and be utilized there. The bacteria of this class
attack not only these proteins in the natural state or in solution,
but also in the coagulated state. The coagulum becomes softened and
finally changed into a liquid condition. The process when applied to
the casein of milk is usually called “digestion,” also when coagulated
blood serum is acted on. In the latter case the serum is more commonly
said to be “liquefied” as is the case when gelatin is the substance
changed. Most of these bacteria have also the property of coagulating
or curdling milk in an alkaline medium, and then digesting the curd.
A second class of bacteria has no effect on the complex proteins just
mentioned but readily attacks the products of their first splitting,
_i.e._, the proteoses, peptones, polypeptids and amino-acids. They are
sometimes called _saprophilic_ bacteria.

Other bacteria derive their nitrogen from some of the products of the
first two groups, and still further break down the complex protein
molecule. Under normal conditions these various kinds of bacteria
all occur together and thus mutually assist one another in what is
equivalent to a symbiosis or rather a metabiosis, a “successive
existence,” one set living on the products of the other. The result
is the complete splitting up of the complete protein molecule. A part
of the nitrogen is built up into the bodies of the bacteria which are
using it as food. A part is finally liberated as _free nitrogen_ or as
_ammonia_ after having undergone a series of transformations many of
which are still undetermined.

One class of compounds formed received at one time much attention
because they were supposed to be responsible for a great deal of
illness. These are the “ptomaines,” basic nitrogen compounds of
definite composition--amines--some few of which are poisonous, most of
them not. The basic character of ptomaines may be understood if they be
regarded as made up of one or more molecules of ammonia in which the
hydrogen has been replaced by alkyl or other radicals. Thus ammonia
(NH₃) may be represented as

    /H
   /
  N--H
   \
    \H.

The simplest ptomaine is

    /CH₃
   /
  N--H
   \
    \H,

in which one H is replaced by methyl, methylamine, a gaseous ptomaine.
With two hydrogens replaced by methyl,

    /CH₃
   /
  N--CH₃
   \
    \H,

dimethylamine, also a gas at ordinary temperature, is formed.
Trimethylamine,

    /CH₃
   /
  N--CH₃
   \
    \CH₃,

a liquid, results when three hydrogens are similarly replaced.
All three of these occur in herring brine and are responsible
for the characteristic odor of this material. Putrescin and
cadaverin--tetramethylene--diamine, and pentamethylenediamine
respectively--occur generally in decomposing flesh, hence the names.
They are only slightly poisonous. One of the highly poisonous ptomaines
is neurin C₅H₁₃NO or C₂H₃N(CH₃)₃OH = trimethyl-vinyl ammonium
hydroxide. This is a stronger base than ammonia, liberating it from
its salts. Numerous other ptomaines have been isolated and described.
These bodies were considered for a long time to be the cause of various
kinds of “meat poisoning,” “ice cream poisoning,” “cheese poisoning,”
etc. It is true that they may sometimes cause these conditions, but
they are very much rarer than the laity generally believe. Most of
the “meat poisonings” in America are due, not to ptomaines, but to
infections with certain bacilli of the _Bacterium enteritidis_ group.
Occasionally a case of poisoning by the true toxin (see Chapter XII) of
_Clostridium botulinum_ occurs, and in recent years has become entirely
too common due to insufficient heating of canned goods. _The boiling of
such material will destroy this toxin. The safest rule to follow is not
to eat any canned material that shows any departure from the normal in
flavor, taste or consistency._

As ptomaines result from the putrefaction of proteins, so they are
still further decomposed by bacteria and eventually the nitrogen is
liberated either as free nitrogen or as ammonia.

Another series of products are the so-called aromatic compounds--phenol
(carbolic acid), various cresols, also indol and skatol or methyl indol
(these two are largely responsible for the characteristic odor of human
feces). All of these nitrogen compounds are attacked by bacteria and
the nitrogen is eventually liberated, so far as it is not locked up in
the bodies of the bacteria, as free nitrogen or as ammonia.

The carbon which occurs in proteins accompanies the nitrogen in many of
the above products, but also appears in nitrogen-free organic acids,
aldehydes and alcohols which are all eventually split up, so that the
carbon is changed to carbon dioxide or in the absence of oxygen partly
to marsh gas.

The intermediate changes which the sulphur in proteins undergoes are
not known, but it is liberated as sulphuretted hydrogen (H₂S) or as
various mercaptans (all foul-smelling), or is partially oxidized to
sulphuric acid. Some of the H₂S and the sulphur of the mercaptans
are oxidized by the sulphur bacteria to free sulphur and finally to
sulphuric acid.

Phosphorus is present especially in the nucleoproteins and nucleins.
Just what the intermediate stages are, on whether there are any, so
far as the phosphorus is concerned, in the splitting up of nucleic
acid by bacterial action is not determined. The phosphorus may occur
as phosphoric acid in such decompositions, or when the conditions are
anaërobic, as phosphine (PH₃), which burns spontaneously in the air to
phosphorus pentoxide (P₂O₅), and water.[13]

The hydrogen in proteins appears in the forms above indicated: H₄C,
H₃N, H₃P, H₂S, H₂O and as free H. The oxygen as CO₂ and H₂O.

In the breaking down of the complex protein molecule even by a single
kind of bacterium there is not a perfect descending scale of complexity
as might be supposed from the statement that there result proteoses,
peptones, polypeptids, amino-acids. These substances do result, but at
the time of their formation simpler ones are formed also, even CO₂,
NH₃ and H₂S. It appears that the entire molecule is shattered in such
a way that less complex proteins are formed from the major part, while
a minor portion breaks up completely to the simplest combinations
possible. A more complete knowledge of these decompositions will aid
in the further unravelling of the structure of proteins. The presence
or absence of free oxygen makes a difference in the end-products, as
has been indicated. There are bacteria which oxidize the ammonia to
nitric acid and the H₂S to sulphuric acid. (See Oxidation, Chapter XI.)
Bacteria which directly oxidize phosphorus compounds to phosphoric
acid have not been described. It does not seem that such are necessary
since this is either split off from nucleic acid or results from the
spontaneous oxidation of phosphine when this is formed under anaërobic
conditions.

Not only are proteins decomposed as above outlined, but also their
waste products, that is, the form in which their nitrogen leaves the
animal body. This is largely urea in mammals, with much hippuric acid
in herbivorous animals and uric acid in birds and reptiles. These
substances yield NH₃, CO₂ and H₂O with a variety of organic acids
as intermediate products in some cases. The strong odor of ammonia
in stables and about manure piles is the everyday evidence of this
decomposition.

Where the putrefaction of proteins occurs in the soil with moderate
amounts of moisture and free access of air a large part of the products
is retained in the soil. Thus the ammonia and carbon dioxide in the
presence of water form ammonium carbonate; the nitric, sulphuric and
phosphoric acids unite with some of the metals which are always present
to form salts. Some of the gases do escape and most where the oxygen
supply is least, since they are not oxidized.

The protein-splitting reactions afford valuable tests in aiding in
the recognition of bacteria. In the study of pathogenic bacteria the
coagulation and digestion of milk, the digestion or liquefaction
of blood serum, the liquefaction of gelatin and the production of
indol and H₂S are those usually tested for. In dairy bacteriology
the coagulation of milk and the digestion of the casein are common
phenomena. Most bacteria which liquefy gelatin also digest blood serum
and coagulate and digest milk, though there are exceptions. In soil
bacteriology the whole range of protein changes is of the greatest
importance.

[Illustration:

     +----<----------------------+
     |                           |
     |                           |
     v                           |
  _Nuclein of                    |
  animal cells_                  |
     |                           |
     |                           |
     v                           |
     |                           |
  _Decomposition                 |
  bacteria_                      |
     |     \                     |
     |      \                    ^
     v       \                   |
  _Unknown   _PH₃                |
     P        oxidizes           |
  compounds_  spontaneously      |
     |        to_                |
     |        /                  |
     v       /                   |
  _Phosphoric                    |
    acid_                        |
     |                           |
     |                           |
     v                           |
  _Phosphates                    |
   in the                        |
    soil_                        |
     |                           ^
     |                           |
     v                           |
  _Green                         |
  plants_                        |
     |                           |
     |                           |
     v                           |
  _Nuclein of                    |
   plant                         |
   cells_                        |
     |                           |
     |                           |
     v                           |
  _Animals_----->----------------+

FIG. 68.--Diagram to illustrate the circulation of phosphorus through
the agency of bacteria.]

[Illustration:

  _Fats and
  various C <---------------------------+
  compounds_                            |
      |                                 |
      |                                 |
      v                                 |
  _Decomposition                        |
  bacteria_                             |
      |                                 |
      |                                 |
      v                                 |
  _Various                              |
      C                                 |
  compounds                             |
  eventually                            ^
     to_                                |
      |                                 |
      |                                 |
      v                                 |
  _CO₂    <---------------+             |
    in                    |             |
  the air_<-+             |             |
      |     |             |             |
      |     |       _Plant respiration_ |
      v     |             |             |
  _Green    |             |             |
  plants_---+             |             |
      |                   |             |
      |                   |             |
      v                   |             |
  _Carbohydrates,         |             |
  fats and                |             |
  other C compounds_      |             |
      |                   |             |
      |                   |             |
      v                   |             |
  _Animals_---->_Animal respiration_    |
      |                                 |
      |                                 |
      +---->----------------------------+

FIG. 69.--Diagram to illustrate the circulation of carbon through the
agency of bacteria.]

[Illustration:

                         +---------------<-----------------+
                         |                                 |
                    _Dead animal                           |
                      protein_                             |
                         |                                 ^
                         |                                 |
                         v                                 |
  _Free N taken <-----_Decomposition <-------------+       |
  up by free living    bacteria_ <----------+      |       |
  N absorbers and        |                  |      |       |
  root tubercle          |                  |      |       |
  bacteria_              v                  |      |       |
        |        _NH₃ compounds             |      |       |
        |             in soil_              |      |       |
        |                |                  |      |       |
        |                |                  |      |       |
        |                v                  |      |       |
        |       _Nitrite bacteria_          |      |       |
        |                |                  |      |       |
        |                |                  |      |       |
        |                v                  |      |       |
        |            _Nitrites_             |      |       |
        |                |                  |      |       |
        |                |                  |      |       |
        |                v                  |      |       |
        |       _Nitrate bacteria_          |      |       |
        |                |                  ^      ^       ^
        |                |                  |      |       |
        |                v                  |      |       |
        |       _Nitrates in soil_          |      |       |
        |                |                  |      |       |
        |                |                  |      |       |
        |                v                  |      |       |
        |         _Green plants_            |      |       |
        |                |                  |      |       |
        |                |                  |      |       |
        |                v            _Dead plant  |       |
        +------->_Plant protein_----> protein_     |       |
                         |                         |       ^
                         |                         |       |
                         v               _Animal waste,    |
                     _Animals_---------->urea, etc._       |
                         |                                 |
                         |                                 |
                         +-------->------------------------+

FIG. 70.--Diagram to illustrate the circulation of nitrogen through the
agency of bacteria.]

[Illustration:

      +--------------<---------------+
      |                              |
      |                              |
      v                              |
  _Dead animal                       |
   protein_                          |
      |                              |
      |                              |
      v                              |
  _Decomposition bacteria_           |
      |                 |            |
      |                 |            |
      v                 ^            |
    _H₂S_               |            |
      |                 |            |
      |                 |            |
      v                 |            |
  _Sulphur bacteria_    |            |
      |                 |            |
      |                 |            |
      v                 |            |
  _Free S_              |            ^
      |                 |            |
      |                 |            |
      v                 |            |
  _Sulphur bacteria_    |            |
      |                 |            |
      |                 |            |
      v                 |            |
  _Sulphates            |            |
   in the               |            |
    soil_               |            |
      |                 |            |
      |                 |            |
      v                 |            |
  _Green plants_        ^            |
      |                 |            |
      |                 |            |
      v                 |            |
  _Plant protein_----->_Dead plant   |
      |                 protein_     |
      |                              |
      v                              |
  _Animals_                          |
      |                              |
      |                              |
      +-------------->---------------+

FIG. 71.--Diagram to illustrate the circulation of sulphur through the
agency of bacteria.]

The three physiological activities already discussed explain how
bacteria break down the chief complex, energy-rich substances
--carbohydrates, fats and proteins which constitute the bulk of the
organic material in the bodies of plants and animals, as well as
the waste products of the latter--into energy-free compounds like
carbon dioxide, water, ammonia, nitric, sulphuric and phosphoric
acids--mineralize them, as is frequently said. By so doing the bacteria
act as the great scavengers of nature removing the dead animal
and vegetable matter of all kinds which but for this action would
accumulate to such an extent that all life, both on land and in the
water, must cease. It is further to be noted that not only is all this
dead organic matter removed; but it is converted into forms which are
again available for plant growth. Carbon dioxide forms the source of
the carbon in all green plants, hence in all animals; the sulphates and
phosphates are likewise taken up by green plants and built up again
into protein compounds; the ammonia is not directly available to green
plants to any large extent but is converted by the nitrifying bacteria
(Chapter XI) into nitrates which is the form in which nitrogen is
assimilated by these higher types. Even the free nitrogen of the air
is taken up by several kinds of bacteria, the symbiotic “root-tubercle
bacteria” of leguminous and other plants, and some free-living forms,
and made available. Hence bacteria are indispensable in nature,
especially in keeping up the circulation of nitrogen. They are also of
great service in the circulation of carbon, sulphur and phosphorus.
Though some few kinds cause disease in man and animals, if it were not
for the saprophytic bacteria above outlined, there could be no animals
and higher plants to acquire these diseases.




CHAPTER XI.

PHYSIOLOGICAL ACTIVITIES (CONTINUED).


PRODUCTION OF ACIDS.

The production of organic acids has been sufficiently discussed in
preceding chapters. It should be noted that not only these in great
variety are produced by bacteria but that under certain conditions
mineral acids, such as nitric, sulphuric and phosphoric may be formed
(see Oxidation, p. 114). Acid production is of great value in the
identification of bacteria in dairy and soil work and in connection
with certain types of pathogenic bacteria.


GAS PRODUCTION.

It will be sufficient merely to enumerate collectively the various
gases mentioned in preceding paragraphs and to state that those
commonly observed in the study of pathogenic bacteria are the first
six mentioned. Most of them come in in dairy work either in the study
of bacteria causing milk and cheese “failures” or as affecting the
flavors of butter or cheese. In the study of soil organisms, any or all
of them are liable to be of importance. The gases are: CO₂, H, CH₄, N,
NH₃, H₂S, gaseous mercaptans, gaseous ptomaines, volatile fatty acids,
ethereal salts or esters and others, both of pleasant and of foul odor,
but of unknown composition.


PRODUCTION OF ESTERS.

The production of esters, as mentioned in Chapters IX and X, of various
alcohols and aldehydes are activities which are sometimes of value in
the study of bacteria, but need not be further discussed.


PRODUCTION OF “AROMATIC” COMPOUNDS.

These have been mentioned in discussing the putrefaction of proteins,
as indol, skatol, phenol and various cresols. Of these only the first
is ordinarily tested for in the study of bacteria, though others of the
group become of value in certain special cases.

[Illustration: FIG. 72.--Culture of phosphorescent bacteria in an
Ehrlenmeyer flask photographed by their own light. Time of exposure
twelve hours. (Molisch, from Lafar.)]


PHOSPHORESCENCE OR PHOTOGENESIS.

This is a most interesting phenomenon associated with the growth of
some bacteria. The “fox fire” frequently seen on decaying wood which
is covered with a slimy deposit is most commonly due to bacteria,
though also to other fungi. Phosphorescent bacteria are very common
in sea water, hence they are frequently found on various sea foods,
especially when these are allowed to decompose, such as fish, oysters,
clams, etc. The light is due to the conversion of the energy of
unknown easily oxidizable compounds directly into _visible_ radiant
energy through oxidation without appreciable quantities of heat.
The light produced may be sufficient to tell the time on a watch in
absolute darkness, and also to photograph the growths with their own
light, but only after several hours’ exposure (Fig. 72). None of the
phosphorescent bacteria so far discovered produce disease in the higher
animals or man.


PRODUCTION OF PIGMENT OR CHROMOGENESIS.

One of the most striking results of bacterial activity is this
phenomenon. The particular color which results may be almost any one
throughout the range of the spectrum, though shades of yellow and of
red are of more frequent occurrence.

In the red sulphur bacteria the “bacteriopurpurin” which they contain
appears to serve as a true respiratory pigment in a manner similar to
the chlorophyl in green plants, except that these bacteria oxidize
H₂S in the light as a source of energy instead of splitting up CO₂.
The red pigment produced by certain bacteria has been shown to have
a capacity for combining with O resembling that of hemoglobin, and
some investigators have believed that such bacteria do store O in this
way for use when the supply is diminished. With these few exceptions
the pigments seem to be merely by-products of cell activity which are
colored and have no known function.

The red sulphur bacteria above mentioned and one or two other kinds
retain the pigments formed within the cell. Such bacteria are called
_chromophoric_ as distinguished from the _chromoparic_ bacteria whose
pigment lies outside the cell.

The chemical composition of no bacterial pigment has been determined
up to the present. Some are soluble in water, as shown by the
discoloration of the substances on which they grow. Others are not
soluble in water but are in alcohol, or in some of the fat solvents
as ether, chloroform, benzol, etc. These latter are probably closely
related to the _lipochromes_ or “fat colors” of higher plants and
animals. Attempts have been made to render the production of pigments
a still more reliable means of identification of species of bacteria
through a careful examination of the spectra of their solutions, but
such study has not as yet led to any valuable practical results.

The production of pigment depends on the same general factors which
determine the growth of the organism but does _not necessarily run
parallel_ with these. It is especially influenced by the oxygen
supply (only a very few organisms are known which produce pigment
anaërobically--_Spirillum rubrum_ is one); by the presence of
certain food substances (starch, as in potato, for many bacteria
producing yellow and red colors; certain mineral salts, as
phosphates and sulphates, for others); by the temperature (many
bacteria cease to produce color at all if grown at body temperature,
37°--_Erythrobacillus prodigiosus_--or if grown for a longer time at
temperatures a few degrees higher).


REDUCING ACTIONS.

Reduction of nitrates to nitrites or to ammonia or even to free
nitrogen is brought about by a great many different kinds of bacteria.
In many instances this phenomenon is due to a lack of free oxygen,
which is obtained by the bacteria from these easily reducible salts.
In other cases a portion of the nitrogen is removed to be used as food
material in the building up of new protein in the bacterial cell. This
latter use of the nitrogen of nitrates by bacteria might theoretically
result in considerable loss of “available nitrogen” in the soil as has
actually been shown in a few experiments. The reduction of nitrates as
above mentioned would also diminish this supply, but probably neither
of these results has any very great practical effect on soil fertility.
The building up of protein from these mineral salts by bacteria in the
intestines of herbivorous animals has been suggested by Armsby as a
considerable source of nitrogenous food, and this suggestion appears
possible.

The liberation of nitrogen from nitrates or nitrites, either as free
nitrogen or as ammonia, is spoken of as “dentrification,” though this
term was formerly applied to such liberations, from compounds of
nitrogen generally even from proteins.

Certain bacteria may also reduce sulphates and other sulphur compounds
to H₂S, a phenomenon frequently observed in sewage and likewise of
importance in the soil. It is possible that phosphates may be similarly
reduced.[14] Further and more careful study of the reducing actions of
bacteria is needed.


OXIDATION.

As has been stated in discussing the respiration of bacteria (Chapter
VIII) most of these organisms gain their energy through the oxidation
of carbon in various forms, chiefly organic, so that CO₂ is a product
of the activity of nearly all bacteria. Some few oxidize CO to CO₂,
others CH₄ and other paraffins to CO₂ for this purpose. One class of
bacteria even oxidizes H in small amounts for its energy and uses the
carbon dioxide of the air or traces of organic carbon in the air as a
source of carbon for “building” purposes.

One of the familiar oxidations of organic carbon is that of the acetic
acid bacteria in the making of vinegar. These oxidize the alcohol
which results from the action of yeast to acetic acid according to the
formula CH₃CH₂OH + O₂ = CH₃COOH + H₂O (see Fig. 67).

Of the various phenomena of oxidation due to bacteria, the formation of
nitrites and nitrates has the greatest practical importance, since it
is by this means that the ammonia which results from the decomposition
of animal and vegetable tissue and waste products is again rendered
available to green plants as food in the form of nitrates. Practically
all the nitrates found in nature, sometimes in large quantities, are
formed in this way. There are two distinct kinds of bacteria involved.
One, the nitrous bacteria, oxidizes the ammonia to nitrous acid
which forms nitrites with bases, and the other, the nitric bacteria,
oxidizes the nitrous to nitric acid, giving nitrates with bases. A
striking peculiarity of these two classes of organisms is that they
may live entirely on inorganic food materials, are proto-autotrophic,
prototrophic for oxygen (aërobic) and autotrophic for the other
elements. Their carbon is derived from CO₂ or carbonates. The
importance of such organisms in keeping up the supply of nitrates in
the soil can scarcely be overestimated.

[Illustration: FIG. 73.--Sprinkling filters of the Columbus
sewage-disposal plant--devices which provide a good supply of oxygen
for the bacteria that oxidize the organic matter in the sewage.]

The oxidation of the H₂S, which is formed in the putrefaction
of proteins, to free S by the sulphur bacteria and the further
oxidation of this free S to sulphuric acid, and of the phosphorus, so
characteristic of the nucleins, to phosphoric acid have been referred
to. These activities of bacteria are of great value in the soil.
Doubtless the commercial “phosphate rock” owes its origin to similar
bacterial action in ages past.

The oxidation of H₂S to free S may be an explanation of the origin of
the great deposits of sulphur which are found in Louisiana and along
the Gulf coast. These deposits occur in the same general regions as
natural gas and oil. The sulphur might have been derived from the same
organic material carried down by the Mississippi which yielded the oil
and gas.[15]

A purposeful utilization of the oxidizing power of bacteria is in
“contact beds,” “sprinkling filters” and “aërated sludge tanks” in
sewage disposal works. In these instances the sewage is thoroughly
mixed with air and brought in contact with large amounts of porous
material so as to expose an extensive surface for oxidation (Fig. 73).

[Illustration: FIG. 74.--One of the University hot beds.]


PRODUCTION OF HEAT.

A direct result of the oxidizing action of bacteria is the production
of heat. Under most conditions of bacterial growth this heat is not
appreciable. It may become well marked. The “heating” of manure is one
of the commonest illustrations. The temperature in such cases may reach
70°. The heating of hay and other green materials is due chiefly to
bacterial action. This heating may lead to “spontaneous combustion.”
The high temperatures (60° to 70°) favor the growth of thermophil
bacteria which cause a still further rise. The heat dries out the
material, portions of which are in a state of very fine division due
to the disintegrating action of the organisms. The hot, dry, finely
divided material oxidizes so rapidly on contact with the air that it
ignites.

A practical use of heat production by bacteria is in the making of “hot
beds” for forcing vegetables (Fig. 74).


ABSORPTION OF FREE NITROGEN.

[Illustration: FIG. 75.--Root tubercles on soy bean. × 3/7.]

This is likewise one of the most important practical activities of
certain types of bacteria present in the soil. The ability of plants
of the legume family to enrich the soil has been known and taken
advantage of for centuries, but it is only about thirty years since
it was demonstrated that this property is due to bacteria. These
plants, and several other kinds as well, have on their roots larger or
smaller nodules (Fig. 75) spoken of as “root tubercles” which are at
certain stages filled with bacteria. When conditions are favorable,
these bacteria live in symbiotic relationship with the plant tissues,
receiving carbonaceous and other food material from them and in return
furnishing nitrogenous compounds to the plant. This nitrogenous
material is built up from free nitrogen absorbed from the air by the
bacteria. The utilization of this peculiar property through the proper
cultivation of clover, alfalfa, soy beans and other legumes is one of
the best ways of building up and maintaining soil fertility in so far
as the nitrogen is concerned. The technical name of these bacteria is
_Rhizobium leguminosarum_.

[Illustration: FIG. 76.--Free-living nitrogen absorbing bacteria
“Azotobacter.” Note their large size as compared with other bacteria
shown in this book.]

There are also types of “free-living,” as distinguished from these
symbiotic, bacteria which absorb the free nitrogen of the air and aid
materially in keeping up this supply under natural conditions. One
of the most important of these types is the aërobic “Azotobacter”
(Fig. 76), while another is the anaërobic _Clostridium pasteurianum_.
The nitrogen which is absorbed is built up into the protein material
of the cell body and this latter must in all probability be “worked
over” by various types of decomposition bacteria and by the nitrous
and nitric organisms and be converted into utilizable nitrates just
as other protein material is, as has been discussed in Chapter X. At
any rate there is as yet no definite knowledge of any other method of
transformation. Up to the present no intentional practical utilization
of this valuable property of these free-living forms has been made.

=Nitrogen Nutrition of Green Plants.=--It is the belief of botanists
that green plants obtain their nitrogen chiefly in the form of
nitrates, though ammonium salts may be utilized to some extent by
certain plants at least. Exceptions to this general rule are those
plants provided with root tubercles (and the bog plants and others
which have mycorrhiza?). These plants obtain their nitrogen in the
form of organic compounds made for them by the bacteria growing in
the tubercles. That nitrogen circulates throughout the structure of
plants in organic combination is certain. There does not appear to
be any reason why similar compounds which are soluble and diffusible
(amino-acids?) should not be taken up through the roots of plants and
utilized as such. _It seems to the author that this is very probably
the case._ Arguments in favor of this view are: (1) The nitrogen
nutrition of leguminous and other plants with root nodules. (2) The
close symbiosis between “Azotobacter” and similar nitrogen-absorbing
bacteria and many species of algæ in sea water at least. (3) The
vigorous growth of plants in soils very rich in organic matter, which
inhibits the production of nitrates by the nitrous-nitric bacteria
when grown in culture, and possibly (?) in the soil, so that nitrates
may not account for the vigorous growth. (4) The effect of nitrate
fertilizers is to add an amount of nitrogen to the crop much in excess
of the amount added as nitrate. (5) The most fertile soils contain
the largest numbers of bacteria. The doctrine that nitrates furnish
the only nitrogen to plants was established before the activities of
bacteria in the soil were suspected, and, so far as the author is
aware, has not been supported by experiments under conditions rigidly
controlled as to sterility.

It would seem that one of the chief functions of soil bacteria is to
prepare soluble organic compounds of nitrogen for the use of green
plants and thus to make a “short cut” in the nitrogen cycle (p. 107),
as now believed in, direct from the “decomposition bacteria” to green
plants.

Experiments have been made by different observers in growing seedling
plants of various kinds in water culture with one or in some cases
several of the amino-acids as sources of nitrogen. Most of these
experiments were disappointing. Plant proteins are not so different
from animal proteins, or plant protoplasm (apart from the chlorophyl
portions of plants) from animal protoplasm as to lead one to suppose
that it could be built up from one or two amino-acids any more than
animal protoplasm can. The author is strongly convinced that this
subject should be thoroughly investigated. It will require careful
experimentation and perhaps rather large funds to provide the amounts
of amino-acids that would probably be needed, but might result in a
decided change in our ideas of soil fertility, and especially in the
use of nitrogen fertilizers.




CHAPTER XII.

PHYSIOLOGICAL ACTIVITIES (CONTINUED).


PRODUCTION OF ENZYMES.

Most of the physiological activities of bacteria which have been
discussed are due to the action of these peculiar substances, so that
a knowledge of their properties is essential. This knowledge cannot as
yet be exact because no enzyme has, up to the present, been obtained in
a “pure state,” though it must be admitted that there are no certain
criteria which will enable this “pure state” to be recognized. It was
formerly thought that they were protein in nature, but very “pure” and
active enzymes have been prepared which did not give the characteristic
protein reactions, so this idea must be abandoned. That they are large
moleculed colloidal substances closely related to the proteins in many
respects must still be maintained. There are certain characteristics
which belong to enzymes, though no one of them exclusively. These may
be enumerated as follows:

1. Enzymes are _dead_ organic chemical substances.

_Dead_ is used in the sense of non-living, never having lived, not in
the sense of “ceased to be alive.”

2. They are always produced by _living_ cells:

Sometimes as active enzymes, sometimes as _pro-enzymes_ or _zymogens_
which are converted into enzymes outside the cell by acids, other
inorganic substances or other enzymes.

3. They produce very great chemical changes without themselves being
appreciably affected.

Enzymes will not continue to act indefinitely, but are used up in the
process (combination with products?). The amount of change is so great
in proportion to the amount of enzyme that the above statement is
justified in the relative sense. Thus a milk-curdling enzyme has been
prepared that would precipitate 100,000,000 times its own weight of
caseinogen.

4. Their action is specific in that each enzyme acts on one kind of
chemical substance only, and the products are always the same.

The substance may be combined with a variety of other chemical
substances so that the action appears to be on several, but in reality
it is on a definite group of molecules in each instance. For example,
emulsin attacks several different glucosides but always sets free
dextrose from them.

5. The action is inhibited and eventually stopped, and in some cases
the enzyme is destroyed by an accumulation of the products of the
action. If the products are removed, the action will continue, if the
enzyme is not destroyed. This effect is explained partly because the
enzyme probably combines with some of the products, since it does
not act indefinitely, and partly because of the reversibility of the
reaction.

6. Like many chemical reactions those of enzymes are reversible, that
is, the substance broken up may be reformed by it from the products
produced in many instances. Thus:

  maltose + maltase ⇆ glucose + glucose + maltase.

      fat + lipase ⇆ glycerin + fatty acid + lipase.

7. The presence of certain mineral salts seems to be essential for
their action. These and other substances which are necessary are
sometimes called _co-enzymes_. A salt of calcium is most favorable for
a great many.

8. They may be adsorbed like other colloids by “shaking out” with
finely divided suspensions like charcoal or kaolin, or by other
colloids like aluminum hydroxide or proteins.

9. When properly introduced into the tissues or blood of an animal,
they cause the body cells to form _anti-enzymes_ which will prevent the
action of the enzyme (see Chapter XXVII).

10. Though inert, they show many of the characteristics of living
organisms, that is

(_a_) Each enzyme has an optimum, a maximum and a minimum temperature
for its action.

All chemical reactions have such temperature limits, the distinction
is that for enzymes as for living substance the _range_ is relatively
narrow.

(_b_) High temperatures destroy enzymes. All in water are destroyed
by boiling in time and most at temperatures considerably below the
boiling-point. When dry, many will withstand a higher degree of heat
than 100° before they are destroyed.

(_c_) Temperatures below the minimum stop their action, though they are
not destroyed by cold.

(_d_) Many poisons and chemical disinfectants (Chapter XIV) which kill
living organisms will also stop the action of enzymes, though generally
more of the substance is required, so that it is possible to destroy
the living cells by such means and yet the action of the enzyme will
continue.

(_e_) Most enzymes have an optimum reaction of medium either acid,
alkaline or neutral, depending on the particular enzyme, though some
few seem to act equally well within a considerable range on either side
of the neutral point.

_The final test for an enzyme is the chemical change it brings about in
the specific substance acted on._

The most prominent characteristic of enzymes is that they bring about
very great chemical changes without themselves being appreciably
affected. This property is also shown by many inorganic substances
which are spoken of as “catalytic agents” or “catalyzers” so that
enzymes are sometimes called “organic catalyzers.” The function of
catalytic agents seems to be to hasten the rate of a reaction which
would occur spontaneously, though in a great many cases with extreme
slowness.

Just how enzymes act is not certain and probably will not be until
their composition and constitution are known. Most probably they form a
combination with the substance acted on (_the substrate_) as a result
of which there is a rearrangement of the atoms in such a way that new
compounds are formed, nearly always at least two, and the enzyme is at
the same time set free. It is rather remarkable that chiefly optically
active substances are split up by enzymes and where two modifications
exist it is usually the dextro-rotatory one which is attacked. No
single enzyme attacks both. This probably means that the structure of
the enzyme corresponds to that of the substrate, “fits it as a key fits
a lock,” as Emil Fischer says.

The production of enzymes is by no means restricted to bacteria since
all kinds of living cells that have been investigated have been shown
to produce them and presumably _all_ living cells do. Hence the
number of different kinds of enzymes and of substances acted upon
is practically unlimited. Nevertheless they may be grouped into a
comparatively few classes based on the general character of the change
brought about by them.

I. Class I is the so-called _“splitting” enzymes_ whose action is
for the most part hydrolytic, that is, the substance takes up water
and then splits into compounds that were apparently constituents of
the original molecule. As examples may be mentioned _diastase_, the
enzyme first discovered, which changes starch into a malt-sugar, hence
is more commonly called _amylase_[16] (starch-splitting enzyme);
_invertase_,[16] which splits cane-sugar into dextrose and levulose:
C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ + C₆H₁₂O₆. _Lipase_[16] or a fat-splitting
enzyme, which decomposes fat into glycerin and fatty acid:

  C₃H₅(OC{n}H₂{n}₋₁O)₃ + 3H₂O = C₃H₅(OH)₃ + 3C{n}H₂{n}O₂.
   Fat                        Glycerin   Fatty acid

_Proteases_, which split up proteins into proteoses and peptones.

Other classes of “splitting enzymes” break up the products of complex
protein decomposition, such as proteoses, peptones and amino-acids. A
variety of the “splitting enzymes” is the group of

_“Coagulases” or coagulating enzymes_ as the rennet (lab, chymosin)
which curdles milk; fibrin ferment (thrombin, thrombase) which causes
the coagulation of blood. These apparently act by splitting up a
substance in the fluids mentioned, after which splitting one of the
new products formed combines with other compounds present (usually a
mineral salt, and in the cases mentioned a calcium salt) to form an
insoluble compound, the curd or coagulum.

_Another variety is the “activating” enzymes or “kinases”_ such as the
enterokinase of the intestine. The action here is a splitting of the
_zymogen_ or mother substance or form in which the enzyme is built up
by the cell so as to liberate the active enzyme.

Of a character quite distinct, from the splitting enzymes are

II. The _zymases_. Their action seems to be to cause a “shifting on
rearrangement of the carbon atoms” so that new compounds are formed
which are not assumed to have been constituents of the original
molecule. Most commonly there is a closer combination of the carbon and
oxygen atoms, frequently even the formation of CO₂ so that considerable
energy is thus liberated. Examples are the _zymase_ or _alcoholase_ of
yeast which converts sugar into alcohol and carbon dioxide; C₆H₁₂O₆
= 2C₂H₆O + 2CO₂: also _urease_, which causes the change of urea into
ammonia and carbon dioxide. Another common zymase is the _lactacidase_
in lactic acid fermentation.

III. _Oxidizing enzymes_ also play an important part in many of the
activities of higher plants and animals. Among the bacteria this action
is illustrated by the formation of nitrites, nitrates and sulphates and
the oxidation of alcohol to acetic acid as already described.

IV. _Reducing enzymes_ occur in many of the dentrifying bacteria and
in those which liberate H₂S from sulphates. A very widely distributed
reducing enzyme is “catalase” which decomposes hydrogen peroxide.

As previously stated, most of the physiological activities of
bacteria are due to the enzymes that they produce. It is evident
that for action to occur on substances which do not diffuse into the
bacterial cell--starches, cellulose, complex proteins, gelatin--the
enzymes must _pass out_ of the bacterium and consequently may be
found in the surrounding medium. Substances like sugars, peptones,
alcohol, which are readily diffusible, may be acted on by enzymes
_retained within_ the cell body. In the former case the enzymes are
spoken of as extra-cellular or “_exo-enzymes_,” and in the latter as
intra-cellular or “_endo-enzymes_.” The endo-enzymes and doubtless also
the exo-enzymes may after the death of the cell digest the contents
to a greater or less extent and thus furnish substances that are
not otherwise obtainable. This process of “self-digestion” is known
technically as “_autolysis_.”

A distinction was formerly made between “organized” and “unorganized
ferments.” The former term was applied to the minute living organisms,
bacteria, yeasts, molds, etc., which bring about characteristic
fermentative changes, while the latter term was restricted to enzymes
as just described. Since investigation has shown that the changes
ascribed to the “organized ferments” are really due to their enzymes,
and that enzymes are probably formed by all living cells, the
distinction is scarcely necessary at present.


PRODUCTION OF TOXINS.

The injurious effects of pathogenic bacteria are due in large part to
the action of these substances, which in many respects bear a close
relationship to enzymes. The chemical composition is unknown since
no toxin has been prepared “pure” as yet. It was formerly thought
that they were protein in character, but very pure toxins have been
prepared which failed to show the characteristic protein reactions. It
is well established that they are complex substances, of rather large
molecule and are precipitated by many of the reagents which precipitate
proteins. Toxins will be further discussed in Chapter XXVII. It will
be sufficient at this point to enumerate their chief peculiarities in
order to show their marked resemblance to enzymes.

1. Toxins are _dead_ organic chemical substances.

2. They are always produced by _living_ cells.

3. They are active poisons in _very small quantities_.[17]

4. Their action is specific in that each toxin acts on a particular
kind of cell. The fact that a so-called toxin acts on several
different kinds of cells, possibly indicates a mixture of several
toxins, or action on the _same substance_ in the cells.

5. Toxins are very sensitive to the action of injurious agencies such
as heat, light, etc., and in about the same measure that enzymes are,
though as a rule they are somewhat more sensitive or “labile.”

6. Toxins apparently have maxima, optima, and minima of temperature for
their action, as shown by the destructive effect of heat and by the
fact that a frog injected with tetanus toxin and kept at 20° shows no
indication of poison, but if the temperature is raised to 37°, symptoms
of poisoning are soon apparent. Cold, however, does not destroy a toxin.

7. When properly introduced into the tissues of animals they cause the
body cells to form antitoxins (Chapter XXVII) which are capable of
preventing the action of the toxin in question.

8. _The determining test for a toxin is its action on a living cell._

It is true that enzymes are toxic, as are also various foreign
proteins, when injected into an animal, but in much larger doses than
are toxins.

A marked difference between enzymes and toxins is that the former may
bring about a very great chemical change and still may be recovered
from the mixture of substances acted on and produced, while the toxin
seems to be permanently used up in its toxic action and cannot be
so recovered. _Toxins seem very much like enzymes whose action is
restricted to living cells._

Just as enzymes are probably produced by all kinds of cells and not by
bacteria alone, so toxins are produced by other organisms. Among toxins
which have been carefully studied are _ricin_, the poison of the castor
oil plant (_Ricinus communis_); _abrin_ of the jequirity bean (_Abrus
precatorius_); _robin_ of the common locust (_Robinia pseudacacia_);
poisons of spiders, scorpions, bees, fish, snakes and salamanders.

It has been stated that some enzymes are thrown out from the cell and
others are retained within the cell. The same is true of toxins, hence
we speak of _exo-toxins_ or toxins excreted from, and _endo-toxins_
or toxins retained within the cell. Among the pathogenic bacteria
there are very few which secrete toxins when growing outside the body.
_Clostridium tetani_ or lockjaw bacillus, _Corynebacterium diphtheriæ_
or the diphtheria bacillus, _Clostridium botulinum_ or a bacillus
causing a type of food poisoning, _Pseudomonas pyocyanea_ or the blue
pus bacillus are the most important. Other pathogenic bacteria do not
secrete their toxins under the above conditions, but only give them up
when the cell is disintegrated either within or outside the body. For
the reason that endotoxins are therefore difficult to obtain, their
characteristics have not been much studied. The description of toxins
as above given is intended to apply to the _exo-toxins_ of bacteria,
sometimes spoken of as _true toxins_, and to the vegetable toxins
(phytotoxins) which resemble them.

The snake venoms and probably most of the animal toxins (zoötoxins) are
very different substances. (See Chapter XXIX.)


CAUSATION OF DISEASE.

This subject belongs properly in special pathogenic bacteriology. It
will be sufficient to indicate that bacteria may cause disease in one
or more of the following ways: (_a_) blocking circulatory vessels,
either blood or lymph, directly or indirectly; (_b_) destruction of
tissue; (_c_) production of non-specific poisons (ptomaines, bases,
nitrites, acids, gases, etc.); (_d_) production of specific poisons
(toxins).


ANTIBODY FORMATION.

Bacteria cause the formation of specific “antibodies” when properly
introduced into animals. This must be considered as a physiological
activity since it is by means of substances produced within the
bacterial cell that the body cells of animals are stimulated to form
antibodies. (See Chapters XXVI-XXIX.)


STAINING.

The reaction of bacteria to various stains is dependent on their
physico-chemical structure and hence is a result of physiological
processes, but is best discussed separately (Chapter XIX).


CULTURAL CHARACTERISTICS.

The same is true of the appearance and growth on different culture
media. (Chapter XX.)




CHAPTER XIII.

DISINFECTION--STERILIZATION--DISINFECTANTS.


The discussion of the physiology of bacteria in the preceding chapters
has shown that a number of environmental factors must be properly
correlated in order that a given organism may thrive. Conversely, it
can be stated that any one of these environmental factors may be so
varied that the organism will be more or less injured, may even be
destroyed by such variation. It has been the thorough study of the
above-mentioned relationships which has led to practical methods for
destroying bacteria, for removing them or preventing their growth when
such procedures become necessary.

The process of killing all the living organisms or of removing them
completely is spoken of as _disinfection_ or as _sterilization_,
according to circumstances. Thus the latter term is applied largely in
the laboratory, while the former more generally in practice outside the
laboratory. So also disinfection is most commonly done with chemical
agents and sterilization by physical means, though exceptions are
numerous. The original idea of disinfection was the destruction of
“infective” organisms, that is, organisms producing disease in man or
animals. A wider knowledge of bacteriology has led to the application
of the term to the destruction of other organisms as well. Thus the
cheese-maker “disinfects” his curing rooms to prevent abnormal ripening
of cheese, and the dairy-worker “disinfects” his premises to avoid bad
flavors, abnormal changes in the butter or milk. _Sterilization_ is
more commonly applied to relatively small objects and _disinfection_ to
larger ones. Thus in the laboratory, instruments, glassware, apparatus,
etc., are “sterilized” while desks, walls and floors are “disinfected.”
The surgeon “sterilizes” his instruments, but “disinfects” his
operating table and room. The dairy-workers mentioned above sterilize
their apparatus, pails, milk bottles, etc. Evidently the object of the
two processes is the same, removing or destroying living organisms, the
name to be applied is largely a question of usage and circumstances.
Any agent which is used to destroy microörganisms is called a
“disinfectant.” Material freed from _living_ organisms is “sterile.”

The process of _preventing the growth_ of organisms without reference
to whether they are killed or removed is spoken of as “_antisepsis_,”
and the agent as an _antiseptic_. Hence a mildly applied “disinfectant”
becomes an “antiseptic,” though it does not necessarily follow that
an “antiseptic” may become a disinfectant when used abundantly. Thus
strong sugar solutions prevent the development of many organisms,
though they do not necessarily kill them.

_Asepsis_ is a term which is restricted almost entirely to surgical
operations and implies the taking of such precautions that foreign
organisms are _kept out_ of the field of operation. Such an operation
is an _aseptic_ one, or performed _aseptically_.

A “deodorant or deodorizer” is used to destroy or remove an odor and
does not necessarily have either antiseptic or disinfectant properties.

The agents which are used for the above-described processes may be
conveniently divided into _physical agents_ and _chemical agents_.


PHYSICAL AGENTS.

=1. Drying.=--This is doubtless the oldest method for _preventing the
growth_ of organisms, and the one which is used on the greatest amount
of material at the present time. A very large percentage of commercial
products is preserved and transported intact because the substances
are kept free from moisture. In the laboratory many materials which
are used as food for bacteria (see Chapter XVI) “keep” because they
are dry. Nevertheless, drying should be considered as an _antiseptic_
rather than as a _disinfectant_ process. While it is true that the
_complete_ removal of water would result in the death of all organisms
this necessitates a high temperature, in itself destructive, and does
not occur in practice. Further, though many pathogenic bacteria are
killed by drying, many more, including the spore formers, are not.
Hence drying alone is not a practical method of _disinfecting_.

[Illustration: FIG. 77.--A small laboratory hot-air sterilizer.]

=2. Heat.=--The use of heat in some form is one of the very best
means for destroying bacteria. It may be made use of by combustion,
or burning, as direct exposure to the open flame, as dry heat (hot
air), or as moist heat (boiling water or steam). Very frequently in
veterinary practice, especially in the country, occasionally under
other conditions, the infected material is best burned. This method is
thoroughly effective and frequently the cheapest in the end. Wherever
there are no valid objections it should be used. Exposure to the open
flame is largely a laboratory procedure to sterilize small metallic
instruments and even small pieces of glassware. It is an excellent
procedure in postmortem examinations to burn off the surface of the
body or of an organ when it is desired to obtain bacteria from the
interior free from contamination with surface organisms.

_Dry Heat._--Dry heat is not nearly so effective as moist heat as a
sterilizing agent. The temperature must be higher and continued longer
to accomplish the same result. Thus a dry heat of 150° for thirty
minutes is no more efficient than steam under pressure at 115° for
fifteen minutes. Various forms of hot-air sterilizers are made for
laboratory purposes (Fig. 77). On account of the greater length of
time required for sterilization their use is more and more restricted
to objects which must be used dry, as in blood and serum work, for
example. In practice the use of hot air in disinfecting plants is now
largely restricted to objects which might be injured by steam, as
leather goods, furs, and certain articles of furniture, but even here
chemical agents are more frequently used.

_Moist Heat._--Moist heat may be applied either by boiling in water
or by the use of steam at air pressure, or, for rapid work and on
substances that would not be injured, by steam under pressure.
Boiling is perhaps the best household method for disinfecting all
material which can be so treated. The method is simple, can always
be made use of, and is universally understood. It must be remembered
that all pathogenic organisms, even their spores, are destroyed by a
few minutes’ boiling. The process may be applied to more resistant
organisms, such as are met with in canning vegetables, though the
boiling must be continued for several hours, or what is better,
repeated on several different days. This latter process, known as
“_discontinuous sterilization_,” or “_tyndallization_,” must also be
applied to substances which would be injured or changed in composition
by too long-continued heating, such as gelatin, milk, and certain
sugars. In the laboratory such materials are boiled or subjected to
steaming steam for half an hour on each of three successive days. In
canning vegetables the boiling should be from one to two hours each
day. The principle involved is that the first boiling destroys the
growing cells, but not all spores. Some of the latter germinate by the
next day and are then killed by the second boiling and the remainder
develop and are killed on the third day. Occasionally a fourth boiling
is necessary. It is also true that repeated heating and cooling is more
destructive to bacteria than continuous heating for the same length of
time, but the development of the spores is the more important factor.
Discontinuous heating may also be used at temperatures below the
boiling-point for the sterilization of fluids like blood serum which
would be coagulated by boiling. In this case the material is heated at
55° to 56° for one hour, but on each of seven to ten successive days.
The intermittent heating and cooling is of the same importance as the
development of the spores in this case. (Better results are secured
with such substances by collecting them aseptically in the first place.)

[Illustration: FIG. 78.--The Arnold steam sterilizer for laboratory
use.]

[Illustration: FIG. 79.--Vertical gas-heated laboratory autoclave.]

[Illustration: FIG. 80.--Horizontal gas-heated laboratory autoclave.]

_Steam._--Steam is one of the most commonly employed agents for
sterilization and disinfection. It is used either as “streaming steam”
at air pressure or confined under pressure so that the temperature is
raised. For almost all purposes where boiling is applicable streaming
steam may be substituted. It is just as efficient and frequently
more easily applied. The principle of the numerous forms of “steam
sterilizers” (Fig. 78) is essentially the same. There is a receptacle
for a relatively small quantity of water and means for conducting the
steam generated by boiling this water to the objects to be treated,
which are usually placed immediately above the water. Surgical
instruments may be most conveniently sterilized by boiling or by
steaming in especially constructed instrument sterilizers. If boiled,
the addition of carbonate of soda, about 1 per cent., usually prevents
injury.

[Illustration: FIG. 81.--A battery of two horizontal autoclaves in one
of the author’s student laboratories. Steam is furnished direct from
the University central heating plant.]

_Steam under pressure_ affords a much more rapid and certain method of
destroying organisms. Fifteen to twenty pounds pressure corresponding
to temperatures of 121° to 125° is commonly used. Variations depend on
the bulk and nature of the material. Apparatus for this purpose may
now be obtained from sizes as small as one or two gallons up to huge
structures which will take one or two truckloads of material (Figs.
79-91). The latter type is in common use in canning factories, dairy
plants, hospitals, public institutions, municipal and governmental
disinfecting stations. Very frequently there is an apparatus attached
for producing a vacuum, both to exhaust the air before sterilizing,
so that the steam penetrates much more quickly and thoroughly and for
removing the vapor after sterilizing, thus hastening the drying out of
the material disinfected.

[Illustration: FIG. 82.--A “process kettle” (steam-pressure sterilizer)
used in canning. Diameter, 40 inches; height, 72 inches.]

The smaller types of pressure sterilizers are called “autoclaves”
and have become indispensable in laboratory work. Fifteen pounds
pressure maintained for fifteen minutes is commonly sufficient for a
few small objects. For larger masses much longer time is needed. The
author found that in an autoclave of the type shown in Fig. 81 it
required ten minutes for 500 cc. of water at 15 pounds pressure to
reach a temperature of 100°, starting at room temperatures, 20° to
25°. Autoclaves may be used as simple steam sterilizers by leaving the
escape valves open so that the steam is not confined, hence they have
largely replaced the latter.[18]

[Illustration: FIG. 83.--Horizontal steam chest used in canning.
Height, 32 inches; width, 28 inches; length, 10 feet.]

[Illustration: FIG. 84.--A battery of horizontal rectangular steam
chests in actual use in a canning factory.]

[Illustration: FIG. 85.--A battery of cylindrical process kettles in
actual use in a canning factory.]

A process closely akin to sterilization by heat is _pasteurization_.
This means the heating of material at a temperature and for a time
which will destroy the actively growing bacteria but not the spores.
The methods for doing this vary but are essentially two in principle.
1. The material in small quantities in suitable containers (bottles) is
placed in the apparatus; the temperature is raised to 60° to 65° and
maintained for twenty to thirty minutes and then the whole is cooled
(beer, wine, grape juice, bottled milk) (Figs. 92, 93 and 94).

[Illustration: FIG. 86.--A steam chamber used in government
disinfection work. Size, 4 feet 4 inches × 5 feet 4 inches × 9 feet.]

[Illustration: FIG. 87.--Circular steam chamber used in government
disinfection work, 54 inches in diameter.]

[Illustration: FIG. 88.--Portable steam chamber used in government
disinfection work.]

[Illustration: FIG. 89.--Steam chambers on deck of the U. S. quarantine
station barge “Defender.”]

[Illustration: FIG. 90.--Steam chambers in hold of U. S. quarantine
station barge “Protector.” Disinfected space.]

[Illustration: FIG. 91.--Municipal disinfecting station, Washington,
D. C.]

[Illustration: FIG. 92.--A pasteurizer for milk in bottles.]

[Illustration: FIG. 93.--A pasteurizer for grape juice, cider, etc., in
bottles.]

2. Pasteurizing machines are used and the fluid flows through
continuously. In one type the temperature is raised to 60° and by
“retarders” is kept at this temperature for twenty to thirty minutes
(Figs. 95 to 98). In another type the temperature is raised to as
high as 85° for a few seconds only, “flash process” (Fig. 99), and then
the material is rapidly cooled. It is certain that all pathogenic
microörganisms, except the very few spore formers in that stage, are
killed by proper pasteurization. The process is largely employed in the
fermentation and dairy industries.

[Illustration: FIG. 94.--A pasteurizer for beer in bottles.]

[Illustration: FIG. 95.--A continuous milk pasteurizer.]

[Illustration: FIG. 96.--A pasteurizer for cream to be used in making
ice-cream.]

[Illustration: FIG. 97.--A continuous milk pasteurizer with holder;
capacity 1500 pounds per hour. _A_, pasteurizer--the milk flows in
tubes inside of a jacket of water heated to the proper temperature;
_B_, holder; _C_, water cooler; _D_, brine cooler.]

[Illustration: FIG. 98.--A continuous pasteurizing plant in operation.
Similar to Fig. 97 but larger. Capacity, 12,000 pounds per hour. _A_,
pasteurizer; _B_, seven compartment holder; _C_, _D_, coolers.]

=3. Cold.=--That _cold_ is an excellent _antiseptic_ is illustrated
by the general use of refrigerators and “cold storage.” Numerous
experiments have shown that although many pathogenic organisms of a
given kind are killed by temperatures below freezing, not all of the
same kind are, and many kinds are only slightly affected. Hence cold
cannot be considered a practical means for _disinfection_.

[Illustration: FIG. 99.--A “flash process” pasteurizing outfit, with
holder. _A_, flash pasteurizer; _B_, holder; _C_, cooler.]

=4. Light.=--It has been stated (p. 75) that light is destructive to
bacteria, and the advisability of having well-lighted habitations
for men and animals has been mentioned. The practice of “sunning”
bedclothing, hangings and other large articles which can scarcely be
disinfected in a more convenient way is the usual method of employing
this agent. Drying and the action of the oxygen of the air assist
the process to some extent. Undoubtedly large numbers of pathogenic
organisms are destroyed under natural conditions by the combined
effects of drying, direct sunlight and oxidation, but it should not
be forgotten that a very slight protection will prevent the action of
light (Figs. 100 and 101).

[Illustration: FIG. 100.--Effect of light on bacteria. × 7/10. The
plate was inoculated in the usual way. A letter _H_ of black paper was
pasted on the bottom. The plate was then exposed for four hours to the
sun in January outside the window and then incubated. The black paper
protected the bacteria. Outside of it they were killed except where
they happened to be in large masses. Hence the letter shows distinctly.
(Student preparation.)]

=5. Osmotic Pressure.=--Increase in the concentration of substances
in solution is in practical use as an _antiseptic_ procedure.
Various kinds of “sugar preserves,” salt meats and condensed milk
are illustrations. It must be remembered that a similar increase in
concentration occurs when many substances are dried, and is probably
as valuable in the preservative action as the loss of water. That the
process cannot be depended on to _kill_ even pathogenic organisms is
shown by finding living tubercle bacilli in condensed milk. The placing
of bacteria in water or in salt solution in order to have them die and
disintegrate (greatly aided by vigorous shaking in a shaking machine)
(“autolysis,” p. 126) is a laboratory procedure to obtain cell
constituents. It is not a practical method of disinfection, however.

[Illustration: FIG. 101.--Effect of light on bacteria. × 7/10. This
plate was treated exactly as the plate in Fig. 100, except that the
letter is _L_, and that it was exposed inside the window and wire
screen. The window was plate glass. It is evident that few of the
bacteria were killed, since the letter _L_ is barely outlined. The
exposure was at the same time as the plate in Fig. 100. (Student
preparation.)]

=6. Electricity.=--Electricity, though not in itself injurious to
bacteria, is used as an indirect means for destroying bacteria in a
practical way. This is done by electrical production of some substance
which is destructive to bacteria as in ozone water purification
(Petrograd, Florence, and elsewhere), or the use of ultra-violet rays
for the same purpose (Marseilles, Paris) and for treatment of certain
disease conditions. Electricity might be used as a source of heat for
disinfecting purposes should its cheapness justify it. It has also
been used in the preservation of meats to hasten the _penetration of
the salt_ and thus reduce the time of pickling. Electrolyzed sea water
has been tried as a means of flushing and disinfecting streets, but
it is very doubtful if the added expense is justified by any increased
benefit. A number of electric devices have been put forth for various
sterilizing and disinfecting purposes and doubtless will continue to
be, but everyone should be carefully tested before money is invested in
it.[19]

[Illustration: FIG. 102.--An electric milk purifier (pasteurizer). The
milk flowing from cup to cup completes the circuit when the current is
on. The effect is certainly a heat effect. Sparking occurs at the lips
of the cups.]

[Illustration: FIG. 103.--One of the ten filter beds of the Columbus
water filtration plant with the filtering material removed. Sand is
the filtering material. All of the beds together have a capacity of
30,000,000 gallons daily.]

[Illustration: FIG. 104.--Suction filtration. _A_, Berkefeld filter in
glass cylinder containing the liquid to be filtered; _B_, sterile flask
to receive the filtrate as it is drawn through; _C_, water pump; _D_,
manometer, convenient for detecting leaks as well as showing pressure;
_E_, bottle for reflux water.]

[Illustration: FIG. 105.--Pressure filtration. _A_, cylinder which
contains the filter candle; _B_, cylinder for the liquid to be
filtered; _C_, sterile flask to receive the filtrate; _D_, air pump to
furnish pressure.]

=7. Filtration.=--Filtration is a process for rendering fluids sterile
by passing them through some material which will hold back the
bacteria. It is used on a large scale in the purification of water for
sanitary or manufacturing reasons (Fig. 103). Air is also rendered
“germ free” in some surgical operating rooms, “serum laboratories” and
breweries by filtration. In the laboratory it is a very common method
of sterilizing liquids which would be injured by any other process.
The apparatus consists of a porous cylinder with proper devices for
causing the liquid to pass through either by suction (Fig. 104) where
the pressure will be only one atmosphere (approximately 15 pounds per
square inch), or by the use of compressed air at any desired pressure
(Fig. 105). The two main types of porous cylinders (“filter candles,”
“bougies”) are the Pasteur-Chamberland (Fig. 106) and the Berkefeld.
The former are made of unglazed porcelain of different degrees of
fineness, the latter of diatomaceous earth (Fig. 107) The Mandler
filter of this same material is now manufactured in the United States
and is equal if not superior to the Berkefeld. The designs of complete
apparatus are numerous.

[Illustration: FIG. 106.--Pasteur-Chamberland filter candles about
one-half natural size.]

=8. Burying.=--This is a time-honored method of disposing of infected
material of all kinds and at first thought might not be considered
a means of _disinfection_. As a matter of fact, under favorable
conditions it is an excellent method. The infected material is
removed. Pathogenic organisms tend to die out in the soil owing to an
unfavorable environment as to temperature and food supply, competition
with natural soil organisms for what food there is, and the injurious
effects of the products of these organisms. Care must be taken that
the burial is done in such a way that the _surface_ soil is not
contaminated either directly or by material brought up from below by
digging or burrowing animals, insects, worms, or movement of ground
water to the surface. Also that the underground water supply which is
drawn upon for use by men or animals is not contaminated. Frequently
infected material, carcasses of animals, etc., are treated in some
way so as to aid the natural process of destruction of the organisms
present, especially by the use of certain chemical agents, as quicklime
(see p. 158).

[Illustration: FIG. 107.--Berkefeld filter candles about one-half
natural size.]




CHAPTER XIV.

DISINFECTION AND STERILIZATION (CONTINUED).


CHEMICAL AGENTS.

A very large number of chemical substances might be used for destroying
bacteria or preventing their growth either through direct injurious
action or by the effect of concentration. Those which are practically
useful are relatively few, though this is one of the commonest methods
of disinfecting and the word “disinfectant” is frequently wrongly
restricted to chemical agents.

Chemical agents act on bacteria in a variety of ways. Most commonly
there is direct union of the chemical with the protoplasm of the cell
and consequent injury. Some times the chemical is first precipitated
on the surface of the cell without penetrating at once. If removed
soon enough, the organism is not destroyed. This is true of bichloride
of mercury and formaldehyde. If bacteria treated with these agents in
injurious strength be washed with ammonia or ammonium sulphate, even
after a time which would otherwise result in their failure to grow,
they will develop. Some chemicals change the reaction of the material
in a direction unfavorable to growth, and if the change is enough,
may even kill the bacteria. Some agents remove a chemical substance
necessary to the growth of the organism and hence inhibit it. Such
actions are mainly preventive (antiseptic) and become disinfectant only
after a long time.


ELEMENTS.

=Oxygen.=--Oxygen as it occurs in the air is probably not injurious
to living bacteria but aids them with the exception of the anaërobes.
In the nascent state especially as liberated from ozone (O₃)
hydrogen peroxide (H₂O₂) and hypochlorites (Ca(ClO)₂) it is strongly
bactericidal.

=Chlorine.=--Chlorine is actively disinfectant and is coming into use
for sterilizing water on a large scale in municipal plants (Fig. 108).

[Illustration: FIG. 108.--Apparatus for sterilizing water with liquid
chlorine.]

_Iodine_ finds extended use in aseptic surgical operations and
antiseptic dressings. Bromine, mercury, silver, gold, nickel, zinc
and copper are markedly germicidal in the elemental state but are not
practical.


COMPOUNDS.

=Calcium Oxide.=--Calcium oxide (CaO), _quick lime_, is an excellent
disinfectant for stables, yards, outhouses, etc., where it is used
in the freshly slaked condition as “white wash;” also to disinfect
carcasses to be buried. It is very efficient against the typhoid
bacillus in water, where it is much used to assist in the softening.

=Chloride of Lime.=--Chloride of lime, _bleaching powder_, which
consists of calcium hypochlorite, the active agent, and chloride and
some unchanged quicklime is one of the most useful disinfectants. It
is employed to sterilize water for drinking purposes on a large scale
and to disinfect sewage plant effluents. A 5 per cent. solution is the
proper strength for ordinary disinfection. Only a supply which is fresh
or has been kept in air-tight containers should be used, as it rapidly
loses strength on exposure to the air. The active agent is nascent
oxygen liberated from the decomposition of the hypochlorite.

=Sodium Hypochlorite.=--Sodium hypochlorite prepared by the
electrolysis of common salt has been used to some extent.

=Bichloride of Mercury.=--Bichloride of mercury, _mercuric chloride,
corrosive sublimate_ (HgCl₂), is the strongest of all disinfectants
under proper conditions. It is also extremely poisonous to men and
animals and great care is necessary in its use. It is precipitated by
albuminous substances and attacks metallic objects, hence should not be
used in the presence of these classes of substances.

It is used in a strength of one part HgCl₂ to 1000 of water for general
disinfection. Ammonium chloride or sodium chloride, common salt, in
quantities equal to the bichloride, or citric acid in one-half of the
amount should be added in making large quantities of solution or for
use with albuminous fluids to prevent precipitation of the mercury
(Fig. 109).

None of the other metallic salts are of value as practical
disinfectants aside from their use in surgical practice. In this
latter class come boric acid, silver nitrate, potassium permanganate.
The strong mineral acids and alkalies are, of course, destructive to
bacteria, but their corrosive effect excludes them from practical use,
except that “lye washes” are of value in cleaning floors and rough
wood-work, but even here better _disinfection_ can be done more easily
and safely.

[Illustration: FIG. 109.--Tanks for bichloride of mercury, government
quarantine disinfecting plant.]


ORGANIC COMPOUNDS.

=Carbolic Acid or Phenol.=--Carbolic acid or phenol (C₆H₅ OH) is one
of the commonest agents in this class. It is used mostly in 5 per
cent. solution as a disinfectant and in 0.5 per cent. solution as an
antiseptic. For use in large quantities the crude is much cheaper and,
according to some experimenters, even more active than the pure acid,
owing to the cresols it contains. The crude acid is commonly mixed with
an equal volume of commercial sulphuric acid and the mixture is added
to enough water to make a 5 per cent. dilution, which is stronger than
either of the ingredients alone in 5 per cent. solution.

=Cresols.=--The cresols (C₆H₄CH₃OH, ortho, meta and para), coal-tar
derivatives, as phenol, are apparently more powerful disinfectants.
A great number of preparations containing them have been put on the
market. _Creolin_ is one which is very much used in veterinary practice
and forms a milky fluid with water, while _lysol_ forms a clear frothy
liquid owing to the presence of soap. Both of these appear to be more
active than carbolic acid and are less poisonous and more agreeable to
use. They are used in 2 to 5 per cent. solution.

=Alcohol.=--Ordinary (ethyl) alcohol (C₂H₅OH) is largely used as a
_preservative_, also as a disinfectant for the body surface, hands, and
arms. Experiments show that alcohol of 70 per cent. strength is most
strongly bactericidal and that absolute alcohol is very slightly so.

=Soap.=--Experimenters have obtained many conflicting results with
soaps when tested on different organisms, as is to be expected from
the great variations in this article. Miss Vera McCoy in the author’s
laboratory carried out experiments with nine commercial soaps--Ivory,
Naphtha, Packer’s Tar, Grandpa’s Tar, Balsam Peru, A. D. S. Carbolic,
German Green, Dutch Cleanser, Sapolio--and obtained abundant growth
from spores of _Bacillus anthracis_, from _Bacterium coli_ and from
_Staphylococcus pyogenes aureus_ in all cases even when the organisms
had been exposed twenty-four hours in 5 per cent. solutions. From
these results and from the wide variations reported in the literature
it is clear that _soap solutions alone cannot be depended on_ as
disinfectants. Medicated soaps do not appear to offer any advantages in
this respect. The amount of the disinfectant which goes into solution
when the soap is dissolved is too small to have any effect.

=Formaldehyde.=--Formaldehyde (HCHO) is perhaps the most largely used
chemical disinfectant at the present time. The substance is a gas
but occurs most commonly in commerce as a watery solution containing
approximately 40 per cent. of the gas. This solution is variously known
as formalin, formol, and formaldehyde solution. The first two names
are patented and the substance under these names usually costs more.
It is used in the gaseous form for disinfecting closed spaces of all
kinds to the exclusion of most other means today. A great many types
of formalin generators have been devised. The gas has little power of
penetration and all material to be reached should be exposed as much as
possible. The dry gas is almost ineffective, so that the objects must
be moistened or vapor generated along with the gas. A common method
in use is to avoid expensive generators by pouring the formaldehyde
solution on permanganate of potash crystals placed in a vessel removed
from inflammable objects on account of the heat developed which
occasionally sets the gas on fire. The formalin is used in amounts
varying from 20 to 32 ounces to 8-1/2 to 13 ounces of permanganate to
each 1000 cubic feet of space. This method is expensive since one pint
(16 ounces) of formalin is sufficient for each 1000 cubic feet, and
since the permanganate is an added expense. Dr. Dixon, Commissioner of
Health of Pennsylvania, recommends the following mixture to replace the
permanganate, claiming that it works more rapidly and is less expensive
and just as efficient:

  1. Sodium bichromate, ten ounces.
  2. Saturated solution of formaldehyde, sixteen ounces.
  3. Common sulphuric acid, one and a half ounces.

Two and three are mixed together and when cool are poured on the
bichromate which is placed in an earthenware jar of a volume about ten
times the quantity of fluid used. The quantities given are for each
1000 cubic feet of space.

A very simple method is to cause the formalin, diluted about twice
with water to furnish moisture enough, to drop by means of a regulated
“separator funnel” on a heated iron plate. The dropping should be so
regulated that each drop is vaporized as it falls. The plate must
have raised edges, pan-shaped, to prevent the drops rolling off when
they first strike the plate. Formaldehyde has no corrosive (except on
iron) or bleaching action, and is the most nearly ideal closed space
disinfectant today. In disinfecting stations it is made use of in
closed sterilizers such as were described under steam disinfection
particularly in connection with vacuum apparatus. It is also used
in solution as a preservative and as a disinfectant. The commonest
strength is 2 or 3 per cent. of formalin or 0.8 to 1.2 per cent. of
the formaldehyde gas. As an _antiseptic_ it is efficient in dilutions
as high as 1 to 2000 of the gas. It is very irritant to mucous
membranes of most individuals.

=Anilin Dyes.=--Some of the anilin dyes show remarkable selective
disinfectant and antiseptic action on certain kinds of bacteria with
little effect on others. This has been well shown by Churchman in his
work on Gentian Violet. This dye inhibits the growth of _Gram positive_
organisms up to a dilution of one part in 300,000 while for _Gram
negative_ organisms it is without effect even in saturated solution.
This is nicely shown in the accompanying illustration. This inhibiting
effect of anilin dyes is taken advantage of in several methods of
isolating bacteria (Chapter XVIII).

[Illustration: FIG. 110.--The lower half of the plate is plain agar
medium, the upper half the same medium plus gentian violet to make one
part in 300,000. The Gram positive organism is on the right and the
Gram negative on the left. Streak inoculations were made across both
media.]

In addition to the above-discussed disinfectants a large number of
substances, particularly organic, are used in medicine, surgery,
dentistry, etc., as more or less strong antiseptics, and the list is a
constantly lengthening one.

In the laboratory chloroform, H₂O₂, ether and other volatile or easily
decomposable substances have been used to sterilize liquids which could
not be treated by heat or by filtration. The agent is removed either
by slow evaporation or by exhausting the fluid with an air pump. The
method is not very satisfactory, nor is absolute sterilization easily
accomplished. It is much better to secure such liquids aseptically
where possible.




CHAPTER XV.

DISINFECTION AND STERILIZATION (CONTINUED).


CHOICE OF AGENT.

The choice of the above-described agents depends on the conditions.
Evidently a barn is not to be disinfected in the same way that a
test-tube in the laboratory is sterilized. Among the factors to be
considered in making a choice are the thing to be disinfected or
sterilized, its size and nature, that is, whether it will be injured by
the process proposed, cost of the agent, especially when a large amount
of material is to be treated. Among the conditions which affect the
action of all agents the following should be borne in mind particularly
when testing the disinfecting power of chemical agents:

1. _The kind of bacterium_ to be destroyed, since some are more readily
killed by a given disinfectant than others, even though no spores are
present.

2. _The age of the culture._ Young bacteria less than twenty-four hours
old are usually more readily killed than older ones since the cell wall
is more delicate and more easily penetrated, though old growths may
be weakened by the accumulation of their products and be more easily
destroyed.

3. _Presence of spores_, since they are much more resistant than the
growing cells.

4. Whether the organism is a _“good” or “bad” growth_, _i.e._, whether
it has grown in a favorable environment and hence is vigorous, or under
unfavorable conditions and hence is weak.

5. _The number of bacteria present_, since with chemical agents the
action is one of relative masses.

6. _Nature of the substance in which the bacteria are._ Metallic salts,
especially bichloride of mercury, are precipitated by albuminous
substances and if employed at all must be used in several times the
ordinary strength. Solids require relatively more of a given solution
than liquids.

7. _State of the disinfectant_, whether solid, liquid or gas, and
whether it is ionized or not. Solutions penetrate best and are
therefore more quickly active and more efficient.

8. _The solvent._ Water is the best solvent to use. Strong alcohol (90
per cent. +) diminishes the effect of carbolic acid, formaldehyde and
bichloride of mercury. Oil has a similar effect. The action is probably
to prevent the penetration of the disinfectant.

9. _Strength of solution._ The stronger the solution, the more rapid
and more certain the action, for the same reason as mentioned under 5.
In fact, every disinfectant has a strength below the lethal at which it
stimulates bacterial growth.

10. _Addition of salts._ Common salt favors the action of bichloride
of mercury and also of carbolic acid. Other salts may hinder by
precipitating the disinfectant.

11. _Temperature._ Chemical disinfectants, as a rule, follow the
general law that chemical action increases with the temperature, up to
the point where the heat of itself is sufficient to kill.

12. _Time of action._ It is scarcely necessary to point out that a
certain length of time is necessary for any disinfectant to act. One
may touch a red hot stove and not be burned. All the above-mentioned
conditions are influenced by the time of action.


STANDARDIZATION OF DISINFECTANTS--“PHENOL COEFFICIENT.”

Many attempts have been made to devise standard methods for testing
the relative strengths of disinfectants. The one most widely used in
the United States is the so-called “Hygienic Laboratory” method of
determining the “phenol coefficient” of the given substance and is a
modification of the method originally proposed by Rideal and Walker
in England. In this method as proposed by Anderson and McClintic,
formerly of the above laboratory, the strengths of the dilution of the
disinfectant to be tested which kills a culture of _Bacterium typhosum_
in 2-1/2 minutes is divided by the strength of the dilution of carbolic
acid which does the same; and the dilution which kills in 15 minutes is
likewise divided by the corresponding dilution of carbolic acid. The
two ratios thus obtained are averaged and the result is the “phenol
coefficient.” For example

        Phenol      1:80 killed in 2-1/2 minutes
  Disinfectant “A”  1:375  „    „    „      „
        Phenol      1:110  „    „   15      „
  Disinfectant “A”  1:650  „    „    „      „
                   375 ÷ 80  = 4.69
                   650 ÷ 110 = 5.91
                              -----
                            2)10.60
                              -----
                   Average = 5.30 = “phenol coefficient.”

Standard conditions of temperature, age of culture, medium, reaction,
etc., and of making the dilutions and transfers are insisted on.
Details may be found in the Journal of Infectious Diseases, 1911, 8, p.
1.

This is probably as good a method as any for arriving at the relative
strengths of disinfectants and in the hands of any given worker
concordant results in comparative tests can usually be attained.
Experience has shown that the results obtained by different workers
with the same disinfectant may be decidedly at variance. This is to
be expected from a knowledge of the factors affecting the action of
disinfectants above stated and from the known specific action of
certain disinfectants on certain organisms (compare anilin dyes, p.
162).

It seems that the only sure way to test the action of such a substance
is to try it out in the way it is to be used. It is scarcely wise to
adopt the “phenol coefficient” method as a legal standard method as
some states have done.


PRACTICAL STERILIZATION AND DISINFECTION.

The methods for sterilizing in the laboratory have been discussed and
will be referred to again in the next chapter.

In practical disinfection it is a good plan always to _proceed as
though spores were present_ even if the organism is known. Hence use an
_abundance of the agent_ and _apply it as long as practicable_. Also
it is best to secure the _chemical substances used as such_ and _not
depend on patented mixtures purporting to contain them_. As a rule the
latter are _more expensive_ in _proportion to the results secured_.

_Surgical instruments_ may be sterilized by boiling in water for
fifteen minutes, provided they are clean, as they should be. If dried
blood, pus, mucus, etc., are adherent, which should never be the case,
they should be boiled one-half hour. The addition of sodium carbonate
(0.5 to 1 per cent.) prevents rusting. Surgeons’ sterilizers are to
be had at reasonable prices and are very convenient. Whether the
instruments are boiled or subjected to streaming steam depends on
whether the supporting tray is covered with water or not. The author
finds it a good plan to keep the needles of hypodermic syringes in a
small wire basket in an _oil bath_. The oil may be heated to 150° to
200° and the needles sterilized in a very few minutes. The oil also
prevents rusting.

_Rooms_, _offices_ and all spaces which may be readily made practically
gas-tight are best disinfected by means of formaldehyde by any of the
methods above described (Figs. 111 and 112).

_Stables_ and _Barnyards_ (Mohler): “A preliminary cleaning up of all
litter is advisable together with the scraping of the floor, mangers
and walls of the stable with hoes and the removal of all dust and
filth. All this material should be burned since it probably contains
the infective agent. Heat may be applied to the surfaces, including
barnyard, by means of a ‘cyclone oil burner.’ When such burning is
impracticable, the walls may be disinfected with one of the following:

  1. Whitewash 1 gallon + chloride of lime 6 ounces.

  2. Whitewash 1 gallon + crude carbolic acid 7 ounces.

  3. Whitewash 1 gallon + formalin 4 ounces.

The same may be applied with brushes or, more rapidly, sprayed on with
a pump; the surface soil of the yard and surroundings should be removed
to a depth of 5 or 6 inches, placed in a heap and thoroughly mixed
with quicklime. The fresh surface of soil thus exposed may be sprinkled
with a solution of a chemical disinfectant as above described.

[Illustration: FIG. 111.--Formaldehyde generator used in city work for
room disinfection.]

[Illustration: FIG. 112.--Government formaldehyde generator.]

“Portions of walls and ceiling not readily accessible may be
disinfected by chlorine gas liberated from chloride of lime by crude
carbolic acid. This is accomplished by making a cone of 5 or 6 pounds
of chloride of lime in the top of which a deep crater is made for the
placement of from 1 to 2 pints of crude carbolic acid. The edge of
the crater is thereupon pushed into the fluid, when a lively reaction
follows. Owing to the heat generated, it is advisable to place the
chloride of lime in an iron crucible (pot), and to have nothing
inflammable within a radius of two feet. The number and location of
these cones of chloride of lime depend on the size and structure
of the building to be disinfected. As a rule it may be stated that
chlorine gas liberated from the above sized cone will be sufficient for
disinfecting 5200 cubic feet of air space.”

_Liquid manure_, _leachings_, etc., where collected are thoroughly
disinfected by chloride of lime applied in the proportion of 2 parts to
1000 of fluid.

[Illustration: FIG. 113.--Chamber used in government work for
formaldehyde disinfection. The small cylinder at the side is the
generator.]

_Vehicles_ may be thoroughly washed with 2 per cent. formalin solution,
or if closed space is available, subjected to formaldehyde gas
disinfection, after cushions, hangings, etc., have been removed and
washed with the disinfectant.

_Harness_, _brushes_, _combs_ should be washed with a solution of
formalin, carbolic acid, or creolin as given under these topics.

_Washable articles_ should be boiled, dropped into disinfectant,
solutions as soon as soiled, and then boiled or steamed.

_Unwashable articles_--burn all possible. Use formaldehyde gas method
in a closed receptacle (Fig. 113).

_Stock cars_--the method described for stables is applicable here.

_Animals, large and small_, may have the coat and surface of the body
disinfected by washing with 1 to 1000 bichloride or strong hot soapsuds
to which carbolic acid has been added to make a 5 per cent. solution;
they should then be given a good warm bath.

Frequently time and money are saved by a combination of steam and
formaldehyde disinfection. This is a regular practice in municipal and
quarantine disinfection (Fig. 114).

[Illustration: FIG. 114.--Chamber in actual use at government
quarantine station for disinfecting baggage and dunnage with steam
or formaldehyde or both. The small cylinder at the side is the steam
formaldehyde generator.]

Persons engaged in disinfection work should wear rubber boots, coats
and caps which should be washed in a disinfectant solution and the
change to ordinary clothing made in a special room so that no infective
material will be taken away.




PART III.

THE STUDY OF BACTERIA.




CHAPTER XVI.

CULTURE MEDIA.


The study of bacteria may be taken up for the disciplinary and
pedagogic value of the study of a science; with the idea of extending
the limits of knowledge; or for the purpose of learning their
beneficial or injurious actions with the object of taking advantage of
the former and combating or preventing the latter.

Since bacteria are classed as plants, their successful study implies
their cultivation on a suitable soil. A growth of bacteria is called
a “_culture_” and the “soil” or material on which they are grown is
called a “_culture medium_.” In so far as the culture medium is made up
in the laboratory it is an “artificial culture medium” as distinguished
from a natural medium. A culture consisting of one kind of bacteria
only is spoken of as a “pure culture,” and accurate knowledge of
bacteria depends on obtaining them in “pure culture.” After getting
a “pure culture” the special characteristics of the organism must be
ascertained in order to distinguish it from others. The discussion of
the _morphology_ of bacteria in Chapters II, III, and IV shows that
the morphological structures are too few to separate individual kinds.
They serve at best to enable groups of similarly appearing forms to be
arranged. Hence any further differentiation must be based on a study
of the _physiology_ of the organism as discussed in the chapters on
Physiological Activities of Bacteria.

The thorough study of a bacterium involves, therefore:

1. Its isolation in pure culture.

2. Its study with the microscope to determine morphological features
and staining reactions.

3. Growth on culture media for determining its physiological activities
as well as morphological characteristics of the growths themselves.

4. Animal inoculations in certain instances.

5. Special serum reactions in some cases.

Since isolation in pure culture requires material for growing the
organism, the first subject to be considered is culture media.

A culture medium for a given bacterium should show the following
essentials:

1. It must contain all the elements necessary for the growth of the
organism except those that may be obtained from the surrounding
atmosphere.

2. These elements must be in a form available to the organism.

3. The medium must not be too dry, in order to furnish sufficient
moisture for growth and to prevent too great a concentration of the
different ingredients.

4. The reaction must be adjusted to suit the particular organism dealt
with.

5. There must be no injurious substances present in concentration
sufficient to inhibit the growth of the organism or to kill it.

Ordinarily, more attention must be paid to the sources of the two
elements N and C than to the others, for in general the substances
used to furnish these two and the water contain the other elements in
sufficient amount. For very exact work on the products of bacteria,
_synthetic media_ containing definite amounts of chemicals of known
composition have been prepared, but for most of the work with bacteria
pathogenic to animals such media are not needed.

Culture media may be either _liquid_ or _solid_, or for certain
purposes may be liquid at higher temperatures and solid at lower, as
indicated later. Liquid media are of value for obtaining bacteria for
the study of morphology and cell groupings and for ascertaining many of
the physiological activities of the organisms. Solid media are useful
for studying some few of the physiological activities and especially
for determining characteristic appearances of the isolated growths
of bacteria. These isolated growths of bacteria on solid media are
technically spoken of as “_colonies_,” whether they are microscopic in
size or visible to the unaided eye.

It is clear that the kinds of culture media used for the study of
bacteria may be unlimited but the undergraduate student will need to
use a relatively small number, which will be discussed in this section.

=Meat Broth (Bouillon).=[20]--This itself is used as a medium and as
the basis for the preparation of other solid and liquid media.

Finely ground _lean_ beef is selected because it contains the necessary
food materials. Fat is not desired since it is a poor food for most
bacteria and in the further processes of preparation would be melted
and form an undesirable film on the surface of the medium. The meat is
placed in a suitable container and mixed with about twice its weight of
_cold_ water (not distilled) and allowed to soak overnight or longer.
The cold water extracts from the meat water-soluble proteins, blood,
carbohydrates in the form of dextrose (occasionally some glycogen),
nitrogenous extractives and some of the mineral salts. The fluid is
strained or pressed free from the meat. This “meat juice” should now
be thoroughly boiled, which results in a coagulation of a large part
of the proteins and a precipitation of some of the mineral salts,
particularly phosphates of calcium and magnesium, both of which must be
filtered off and the water loss restored by adding the proper amount of
distilled water. The boiling is done at this point because the medium
must later be heated to sterilize it and it is best to get rid of the
coagulable proteins at once. The proteins thus thrown out deprive
the medium of valuable nitrogenous food material which is replaced
by adding about 1 per cent. by weight of commercial peptone. It is
usual also (though not always necessary) to add about 0.5 per cent. by
weight of common salt which helps to restore the proper concentration
of mineral ingredients lost by the boiling. The chlorine is also an
essential element. The reaction is now determined and adjusted to the
desired end point, “standardized,” as it is called. The medium is
again _thoroughly_ boiled and filtered boiling hot. The adjusting of
the reaction and the boiling ordinarily cause a precipitate to form
which is largely phosphates of the alkaline earths with some protein.
The filtered medium is collected in suitable containers, flasks or
tubes, which are plugged with well-fitting non-absorbent cotton plugs
and sterilized, best in the autoclave for twenty minutes at 15 pounds
pressure, or discontinuously in streaming steam at 100°. If careful
attention is paid to _titration_ and to _sufficient boiling_ where
indicated, the meat broth prepared as above should be clear, only
faintly yellowish in color and show no precipitate on cooling.

The conventional method for standardizing an acid medium is as follows:
Take 5 cc of the medium, add 45 cc of distilled water and 1 cc of
_phenolphthalein_ as indicator. Boil the solution and while still hot
run in from a burette N/20 NaOH solution until a faint pink color
appears. From the number of cc of N/20 NaOH used to “neutralize” the
5 cc of medium it is calculated how many cc of N/1 NaOH are necessary
to give the desired end reaction to the volume of medium which is to
be standardized. The resulting reaction is expressed as % _acid or
alkaline to phenolphthalein_. If it is necessary to add to each 100 cc
of the medium 1 cc of N/1 NaOH to make it neutral to phenolphthalein,
the reaction is called 1% acid: if to each 100 cc of medium there is
added 1 cc of N/1 alkali in addition to the quantity necessary to
neutralize, the reaction is called 1% alkaline.

In order to obtain a pink color when titrating with this indicator
not only must the “free acid” be neutralized by the alkali but also
loosely combined acid and any other substances present which will
combine with the alkali rather than with the indicator so that in many
media _more alkali_ is added than is necessary to neutralize the “free
acid,” _i.e._, the free H ions present.

It is well established that the controlling factor in the growth of
bacteria in so far as “reaction” is concerned is not the _titratable
substances_ present but only the “free acid,” _i.e._, the _number of
free H ions_, consequently it is better to determine the concentration
of H ions and to _standardize to a definite H ion concentration_.
Phenolphthalein as shown above is not a good indicator for this purpose.

The H ions present can be determined accurately in all cases only by
electrolytic methods. The apparatus necessary is usually relatively
expensive and scarcely adapted to the use of large classes of students.
There are a number of indicators each of which will show color changes
within rather narrow ranges of H ion concentration. Standardization by
the use of these indicators, the “colorimetric method,” is recommended
by the Society of American Bacteriologists and is coming into general
use.

The H ion concentration is ordinarily _indicated_ by the conventional
symbol P{H}, _e.g._, the concentration in pure water which is regarded
as neutral is expressed as P{H} 7; of normal HCl, P{H} 0; of normal
NaOH, P{H} 14. The figure after P{H} does not in reality represent the
concentration of H ions in the solution. This, like the concentration
of acids, is expressed on the basis of normality, _i.e._, as compared
with the concentration of a normal solution (1 g. equivalent) of H
ions. Concentration of H ions in pure water is N/10,000,000, _i.e._,
is 1/10,000,000 of the concentration in a normal solution of H ions.
Expressed in other words, it is the concentration in a normal solution
of H ions diluted ten million times. 10,000,000 = 10 to the 7th power
= 10⁷. Hence the figure after the P{H} indicates the _logarithm of
the number of times the solution is diluted_. Therefore this number
_increases with the dilution_, and the larger the figure after the
P{H}, the _less acid the solution is_.

Most saprophytic organisms and many parasitic ones grow within a wide
range of H ion concentration so that titration with phenolphthalein
gives sufficient accuracy for media for such organisms. On the
other hand, many organisms grow within a very narrow range of H ion
concentration, hence accurate standardization to a definite H ion
concentration is necessary. It is also evident that for comparative
work, such standardization is essential because this reaction can be
reproduced in other media and by other workers.[21]

Broth may be prepared from Liebig’s or Armour’s meat extract by adding
5 grams of either, 10 grams peptone and 5 grams NaCl to 1000 cc of
water, boiling to dissolve, then titrating and filtering as above.

The author after much experience finds _meat juice_ preferable to meat
extract for broth and other media for pathogenic bacteria, and has
abandoned the use of meat extracts for these organisms.

=Glycerin Broth.=--Glycerin broth is made by adding 4 to 6 per cent. of
glycerin to the broth just previous to the sterilization. The glycerin
serves as a source of carbon to certain bacteria which will not grow on
the ordinary broth--as _Mycobacterium tuberculosis_.

=Sugar Broths.=--Sugar broths are used for determining the action of
bacteria on these carbohydrates, since this is a valuable means of
differentiating certain forms, especially those from the intestinal
tract. Broth _free from sugar_ must first be made. This is done by
adding to broth prepared as already described, _just previous to final
filtering and sterilization_, a culture of some sugar-destroying
organism (_Bacterium coli_ is ordinarily used), and then allowing the
organism to grow in the raw broth at body temperature for twenty-four
hours. Any carbohydrate in the broth is destroyed by the _Bacterium
coli_. This mixture is then boiled to kill the _Bacterium coli_,
restandardized and then 1 per cent. by weight of required sugar is
added. Dextrose, saccharose and lactose are the most used, though
many others are used for special purposes. After the sugar is added
the medium must be sterilized by _discontinuous heating_ at 100° for
three or four successive days, because long boiling or heating in the
autoclave splits up the di- and polysaccharids into simpler sugars and
may even convert the simple sugars (dextrose) into acid.

Various other _modified broths_ are frequently used for special
purposes but need not be discussed here.

=Dunham’s peptone solution=, frequently used to determine indol
production, is a solution of 1 per cent. of peptone and 0.5 per cent.
of salt in tap water. It does not need to be titrated, but should be
boiled and filtered into tubes or flasks and sterilized.

=Nitrate Broth.=--Nitrate broth for determining nitrate reduction
is 1 per cent. of peptone, 0.2 per cent. of C. P. potassium nitrate
dissolved in distilled water and sterilized.

=Milk.=--Milk is a natural culture medium much used. It should be
fresh and thoroughly skimmed, best by a separator or centrifuge to
get rid of the _fat_. If the milk is not fresh, it should be titrated
as for broth and the reaction adjusted. The milk should be sterilized
discontinuously to avoid splitting up the lactose as well as action on
the casein and calcium phosphate.

_Litmus Milk._--Litmus milk is milk as above to which litmus has been
added as an acid production indicator. The milk should show blue when
the litmus is added or be made to by the addition of normal NaOH
solution. It should be sterilized discontinuously. Frequently on
heating litmus milk the blue color disappears due to a reduction of
the litmus. This blue color will reappear on shaking with air or on
standing several days, due to absorption of O and oxidation of the
reduced litmus, provided the heating has produced no other change in
the milk, as proper heating will not.

=Gelatin Culture Medium.=--Gelatin to the extent of 10 to 15 per
cent. is frequently added to broth and gives a culture medium of
many advantages. It is solid at temperatures up to about 25° and
fluid above this temperature, a property which is of great advantage
in the isolation of bacteria. (See Chapter XVIII.) Further gelatin
is liquefied (that is digested, converted into gelatin proteose and
gelatin peptone, which are soluble in water and do not gelatinize)
by many bacteria and not by others, a valuable diagnostic feature.
The gelatin colonies of many bacteria are very characteristic in
appearance, as is the growth of many on gelatin in culture tubes.

Gelatin medium may be prepared by adding the proper amount of gelatin
(10 to 15 per cent. by weight) broken into small pieces (powdered
gelatin in the same proportion may be used) to broth, gently warming
until the gelatin is dissolved, standardizing as for broth, filtering
and sterilizing. It is usually cleared before filtering by stirring
into the gelatin solution, cooled to below 60°, the white of an egg
for each 1000 cc., and then thoroughly boiling before filtering. The
coagulation of the egg albumen entangles the suspended matter so that
the gelatin filters perfectly clear, though with a slight yellowish
color. The filtering may be done through filter paper if the gelatin
is well boiled and filtered boiling hot, but is more conveniently done
through absorbent cotton, wet with boiling water.

Or, the gelatin may be added to _meat juice before it is boiled_, then
this is heated to about body temperature (not too hot, or the proteins
will be coagulated too soon) until the gelatin is dissolved. Then
the material is standardized and thoroughly boiled and filtered. The
proteins of the meat juice coagulate and thus clear the medium without
the addition of egg white. Commercial gelatin is markedly acid from the
method of manufacture, hence the medium requires careful titration,
even when made from a standardized broth.

Gelatin should be sterilized by discontinuous heating at 100° on three
successive days, because long boiling or heating above 100° tends to
hydrolyze the gelatin into gelatin proteose and peptone and it will
not gelatinize on cooling. It may be heated in the autoclave for
ten to fifteen minutes at 10 pounds’ pressure and sometimes not be
hydrolyzed, but the procedure is uncertain and very resistant spores
may not be killed. The medium should be put into the culture tubes in
which it is to be used as soon as filtered, and sterilized in these,
since, if put into flasks these must be sterilized, and then when
transferred to tubes for use, it must be again sterilized unless great
care is taken to have the tubes plugged and sterilized first, and in
transferring aseptically to these tubes. These repeated heatings are
very apt to decompose the gelatin, so it will not “set” on cooling. The
prepared and sterilized tubes of gelatin should be kept in an ice-box
or cool room, as they will melt in overheated laboratories in summer or
winter.

=Agar Medium.=--Agar agar, usually called agar, is a complex
carbohydrate substance of unknown composition obtained from certain
seaweeds along the coast of Japan and Southeastern Asia. It occurs
in commerce as thin translucent strips or as a powder. It resembles
gelatin only in the property its solutions have of gelatinizing when
cooled. Gelatin is an albuminoid closely related to the proteins,
agar a carbohydrate. Agar is much less soluble in water, 1 or 1.5
per cent. of agar giving a jelly as dense as 10 to 15 per cent. of
gelatin. It dissolves only in water heated to near the boiling-point
(98° to 99°) and only after much longer heating. This hot solution
“jells,” “sets” or gelatinizes at about 38° and remains solid until
again heated to near boiling. Hence bacteria may be grown on agar at
the body temperature (37°) and above, and the agar will remain solid,
while gelatin media are fluid above about 25°. No pathogenic bacteria
and none of the saprophytes liable to be met with in the laboratory are
able to “liquefy” agar.

An agar medium is conveniently prepared from broth by adding 1 or
1.5 per cent. of the finely divided agar to the broth and boiling
until dissolved, standardizing, clearing, filtering, and sterilizing.
The agar must be thoroughly boiled, usually for ten to fifteen
minutes, and the water loss made up by the addition of distilled
water before titration. Agar is practically neutral so that there is
little difference between the titration of the dissolved agar and
the original broth. The agar solution should be kept hot from the
beginning to the end except the cooling down to below 60°, when the
egg white for clearing is added. Though filtration through paper is
possible as with gelatin, if the agar solution is thoroughly boiled
and filtered boiling hot, it is more satisfactory for beginners to
use absorbent cotton wet with boiling water and to pour the hot agar
through the same filter if not clear the first time. The solidified
agar medium is never perfectly clear, but always more or less
opalescent. The agar medium may be sterilized in the autoclave for
fifteen minutes at 15 pounds pressure as the high temperature does not
injure the agar.

=Potato Media.=--Potatoes furnish a natural culture medium which is
very useful for the study of many bacteria. The simplest, and for most
purposes the best, way to use potatoes is in culture tubes as “potato
tube cultures” (No. 8, Fig. 119). These are prepared as follows:
Large tubes are used. Large healthy potatoes are selected. Each end
of the potato is sliced off so as to have parallel surfaces. With a
cork-borer of a size to fit the tubes used, cylinders about one and
one-half inches long are made. Each cylinder is cut diagonally from
base to base. This furnishes two pieces each with a circular base and
an oval, sloping surface. The pieces are then washed clean and dropped
for a minute into boiling water to destroy the oxidizing enzyme on the
surface which would otherwise cause a darkening of the potato. (The
darkening may also be prevented by keeping the freshly cut potatoes
covered with clean water until ready to sterilize.) A bit of cotton
one-fourth to one-half inch in depth is put into each of the test-tubes
to retain moisture and a piece of potato dropped in, circular base
down. The tubes are then plugged with cotton and sterilized in the
autoclave at 15 pounds pressure for not less than twenty-five minutes,
since potatoes usually harbor very resistant spores, and it is not
unusual for a few tubes to spoil even after this thorough heating.

Potatoes are sometimes used in “potato plate cultures.” The term “plate
culture” is a relic of the time when flat glass plates were used for
this and other “plate cultures.” Now glass dishes of the general
form shown in Fig. 115, called “Petri dishes,” or plates are used for
practically all plate culture work. For “potato plates” slices from
potatoes are cut as large and as thick as the relative sizes of potato
and dish permit (Fig. 116). The slices should be thin enough not to
touch the lid and thick enough to be firm.

[Illustration: FIG. 115.--Petri dish with the lid partly raised. × 1/2]

[Illustration: FIG. 116.--A potato plate. × 1/2]

It is a good plan to wrap each dish separately in paper to retain the
lid securely, then sterilize as for potato tubes, and leave plates
wrapped until wanted.

It sometimes happens that the natural acidity of potatoes is too great
for the growth of many organisms. The acidity is sufficiently corrected
by soaking the pieces of potato in a 1 per cent. solution of sodium
carbonate for an hour before they are put into the tubes or plates.

_Glycerinized potato tubes_ are conveniently prepared by covering the
potato in the tube with glycerin broth, sterilizing and pouring off the
excess broth immediately after sterilizing, taking care that the tubes
do not become contaminated which is not very probable if the work is
quickly done while the tubes are still hot.

=Blood Serum Media.=--Blood serum, usually from the larger, domestic
animals on account of convenience in securing it in quantity, is used
in the study of the bacteria causing disease in man and animals. Most
commonly the serum is collected from the clotted blood after it has
well separated (usually about forty-eight hours is required for this).
It is then run into tubes which are plugged with cotton and placed in
an apparatus for coagulating the serum by heat. A copper water bath
with a tightly closed air compartment or the horizontal autoclave (Fig.
81) is sufficient for this purpose, though special forms of apparatus
are to be had. It is important that the temperature be raised slowly so
that the blood gases escape gradually. Three to five hours or longer
should be allowed for the temperature to reach the boiling-point. If
the tubes are heated too rapidly, the serum is filled with bubbles and
badly torn since the gases are driven off suddenly. _Löffler’s serum_
is made by adding one part of dextrose broth to three parts of serum
and then coagulating as above. The solidified serum in either case is
best sterilized discontinuously, though with care the autoclave at 15
pounds pressure may be used for a single sterilization. This is very
apt to cause a greater darkening of the serum and frequently also a
laceration of the solid mass by escaping gases.

Blood serum is also used in the liquid state. For this purpose it is
best to collect it aseptically; or it may be sterilized discontinuously
at a temperature of 55° or 56° on seven to ten consecutive days. Novy
has recently suggested dialyzing the serum to free it from salts and
thus prevent its coagulation when heated. Whether the removal of the
various “extractives” which diffuse out with the salts deprives the
serum of any of its advantageous properties remains to be ascertained.

From the discussion of the physiological activities of bacteria in
Chapters IX-XII it is apparent that a very great variety of culture
media other than those described is necessary for the study of special
types of bacteria, but such media are beyond the scope of the present
work.

The ideal culture media are without a doubt the _synthetic media_, that
is media of definite known chemical composition, so that the various
changes due to the growth of bacteria can be accurately determined
and thus a means of sharply differentiating closely related organisms
be secured. Such media have been prepared and every bacteriologist
believes strongly in their future usefulness when media of wider
application shall have been devised. An example of this type of culture
media is Uschinsky’s synthetic medium, of which the following is one of
the modifications:

  Distilled-water              1000 parts
  Asparagin                       4  „
  Ammonium lactate                6  „
  Disodium phosphate              2  „
  Sodium chloride                 5  „

A criticism of this medium is that the elements K, Ca, Mg, Fe, Mn, and
S which have been shown to be essential are not present if chemically
pure salts are used in the preparation.




CHAPTER XVII.

METHODS OF USING CULTURE MEDIA.


The way in which culture media shall be used depends on the purpose
in view. By far the larger part of bacteriological work is done with
cultures in “bacteriological culture tubes.” Various laboratories have
their own special types but all are more or less after the “Board of
Health” form. They differ from ordinary chemical test-tubes in that
they are usually longer, have no “lip” and have much thicker walls to
prevent breakage and consequent loss of the culture as well as danger
from pathogenic organisms. The author finds two sets of tubes most
serviceable for student use--one size 15 cm. long by 19 mm. outside
diameter (No. 9, Fig. 119), the other 15 cm. long by 13 mm. (Nos. 1 to
7, Fig. 119). Culture tubes are conveniently used in “wire baskets”
circular or square in section and of a size to correspond with the
length and number of tubes used. These baskets are light, do not break,
and if made of good galvanized wire netting do not readily rust (Figs.
117 and 118).

Liquid media such as broth, milk, litmus milk, indol and nitrate broths
are used in the above-mentioned tubes when small quantities only are
to be worked with. The tubes are filled approximately one-third full,
then plugged with _non-absorbent_ cotton and sterilized. _Cotton plugs_
are used so much in bacteriological work because they permit a free
circulation of air and gases and at the same time act as filters to
keep out the bacteria of the air.

Sugar broths or other media in which gas may be produced are used in
fermentation tubes (Smith tubes) of the type shown in Fig. 120 so that
the gas may be collected in the closed arm of the tube, measured (Fig.
121) and tested if desired.

[Illustration: FIG. 117.--Round wire basket.]

[Illustration: FIG. 118.--Square wire basket.]

[Illustration: FIG. 119.--Culture tubes with media in them. × 2/3. _1_
to _7_ are the smaller tubes mentioned in the text; _9_ the larger
tube; _8_ is extra large for potato tubes; _1_, plain broth; _2_, plain
milk; _3_, litmus milk; _4_, gelatin for “stab” or “puncture” culture;
_5_, agar for “stab” or “puncture” culture; _6_, agar for “slope” or
“slant” culture; _7_, blood serum; _8_, potato tube; _9_, agar for
plating. Note the transparency of the broth and gelatin and the slight
opalescence of the agar.]

One method of using gelatin and also agar is as “puncture” or “stab”
cultures. The tubes (the narrower tubes are to be preferred for most
“stab” cultures) are filled one-third full of the medium while it is
still fluid, plugged, sterilized and allowed to cool in the vertical
position. The medium is then “inoculated” with a _straight_ platinum
needle by plunging this into the center of the surface down to the
bottom of the tube (Fig. 119, Nos. 4 and 5).

[Illustration: FIG. 120.--Fermentation tubes. _1_, filled ready for
use; _2_, shows a cloudy growth and the development of gas in the
closed arm.]

Agar and blood serum are frequently used in the form of “slope” or
“slant” cultures. That is, the medium solidifies with the tubes lying
on their sides which gives a long, sloping _surface_ on which the
bacteria are inoculated (Fig. 119, Nos. 6 and 7).

[Illustration: FIG. 121.--Method of estimating percentage of gas in a
fermentation tube by means of the “gasometer”, the reading is 45 per
cent.]

[Illustration: FIG. 122.--A toxin flask showing a large surface
growth.]

Potato tubes are likewise used for “slant” or “slope” cultures (Fig.
119, No. 8). Potatoes as “plate cultures” have been referred to. Agar
and gelatin are very largely used in the form of “plate cultures”
also. For this purpose Petri dishes are first sterilized, then the
melted agar or gelatin poured into them and allowed to “set” while the
plates are kept horizontal. The melted media may be “inoculated” before
they are poured, or a portion of the material to be “plated” may be
placed in the dish, then the melted medium poured in and distributed
over the dish by tilting in various directions, or the medium after
solidifying may be inoculated by “strokes” or “streaks” over its
surface, according to the purpose in view in using the plate. The
larger sized tubes should be used for making plates in order to have
sufficient medium in the plate (No. 9, Fig. 119).

For using large quantities of medium, Florence flasks, Ehrlenmeyer
flasks, special toxin flasks (Fig. 122) or various other devices
(Vaughan and Novy’s “mass cultures,” Figs. 123 and 124) have been
employed.

For growing _anaërobic organisms_ it is evident that some method for
removing and excluding the oxygen of the air must be used. A very great
variety of appliances have been devised for these purposes. Some are
based on the principle of the vacuum, exhausting the air with an air
pump; some on replacing the air with a stream of hydrogen; others on
absorbing the oxygen by chemical means, as with an alkaline solution
of pyrogallic acid, or even by growing a vigorous aërobe in the
same culture or in the same container with the anaërobe, the aërobe
exhausting the oxygen so that the anaërobe then develops, or finally
by excluding the air through the use of deep culture tubes well filled
with the medium, or in the closed arm of fermentation tubes. For many
purposes a combination of two or more of the above methods gives good
results.

In any event the culture medium should have been _freshly sterilized_
just before use, or _should be boiled_ in order to drive out the
dissolved oxygen. For most, anaërobes the presence in the medium of
about 1 per cent. of a carbohydrate, as dextrose, is advisable.

A description of all the various devices is unnecessary in this work,
but the following have answered most of the purposes of general work in
the author’s laboratories.

[Illustration: FIG. 123.--Tank with raised lids. (Vaughan.)]

[Illustration: FIG. 124.--Tank with lids lowered. (Vaughan.)

FIGS. 123 and 124.--Vaughan and Novy’s mass culture apparatus.]

_A._ “_Vignal tubes_” of the style shown (Fig. 125) are made from
glass tubes of about 6 to 8 mm. outside diameter, sealed at the small
end, plugged with cotton above the constriction and sterilized. The
medium, agar or gelatin, which has been previously inoculated with the
anaërobic culture, is then drawn up into the tube, after breaking off
the tip, as far as the constriction. The tube is then sealed in the
flame at the small end and also at the constriction. Since it is full
of the medium and sealed, access of air is prevented. This forms an
excellent means for “isolation” (Chapter XVIII); the tube needs merely
to be cut with a file at the point where colonies appear, then these
may be readily transferred.

[Illustration: FIG. 125.--Vignal tubes. × 1/3 _1_, the sterile tube
ready for inoculation; _2_, fourth dilution tube showing a few isolated
colonies, one near the figure; _3_, third dilution showing colonies
isolated but numerous; _4_, second dilution tube showing colonies still
more numerous; _5_, first dilution tube showing colonies so numerous
and small as to give a cloudy appearance to the tube. In use tube _2_
would be filed in two at the colony and inoculations made from it.]

_B._ “_Fermentation tubes_” form a simple means for growing liquid
cultures of anaërobes, the growth occurring in the closed arm only,
while with facultative anaërobes, growth occurs both in the closed arm
and in the open bulb. A little “paraffin oil” (a clear, heavy petroleum
derivative) may be poured on the fluid in the open bulb as a very
efficient seal, though it is not usually necessary.

_C._ “_Deep culture tubes._”--The medium, agar, gelatin or a liquid is
poured into tubes until they are approximately one-half full, a little
paraffin oil is poured on the surface (not essential always), then the
tubes are plugged and sterilized. Inoculation is made to the bottom
and anaërobes grow well (Fig. 126).

[Illustration: FIG. 126.--Deep tubes showing anaërobic growth. _1_,
shows a few small gas bubbles; _2_, shows the medium broken up by the
excessive development of gas.]

_D._ For slope or plate, or any type of surface cultures the Novy jar
(Fig. 127) is the most practical device. It is good practice to combine
the vacuum method, the hydrogen replacement method and the oxygen
absorption method in using these jars. In operation a solution of 20
per cent. NaOH is poured on the bottom of the jar to a depth of 1 or 2
cm., the cultures are placed on glass supports above the alkali and a
short wide tube of strong pyrogallol is set in on the bottom in such a
way that it may be easily upset and mixed with the alkali when it is
desired to do so. The cover is now clamped in position with all joints
well vaselined. Then the outlet tube is connected with a suction pump
and the air drawn out. Meanwhile the inlet tube has been connected
with a hydrogen generator, and after the jar is exhausted hydrogen is
allowed to flow in, and this process is repeated until one is satisfied
that the air is replaced. The suction exhausts the air from the tubes
or plates so that much less time is required to replace the air with
hydrogen. Finally the stop-cock is closed, and the pyrogallol solution
is gently shaken down and mixed with the alkali so that any remaining
oxygen will be absorbed.

[Illustration: FIG. 127.--Novy jars.]

It must be remembered that facultative anaërobes as well as anaërobes
will grow under any of the above conditions, so that cultures of
organisms so obtained must be further tested aërobically in order to
determine to which group the organisms belong.

Reference has been made above to the “inoculation” of culture media,
which means introducing into the medium used the desired material in
the proper way. For small quantities this is most conveniently done
with platinum “needles,” that is, pieces of platinum wire inserted
into the ends of glass rods. The “straight” needle is a piece of
heavy platinum wire of about 0.022 inch in diameter (Fig. 128). It is
used most frequently to inoculate all forms of _solid media_. The
platinum loop is of lighter wire, 0.018 inch. The loop in the end is
conveniently made by twisting the wire around the lead of an ordinary
lead-pencil. The “loop needle” (Fig. 129) is most used in transferring
liquid media. On account of the high price of platinum, the author
has substituted “nichrome” wire for student use. This is stiffer, not
so easily made into loops and breaks out of the rods more easily.
The latter defect is remedied to some extent, by imbedding the wire
only slightly for about one-fourth of an inch on the side of the end
portion of the rod. The low cost, less than one-twentieth of platinum,
justifies its use.

[Illustration: FIG. 128.--Straight needle.]

[Illustration: FIG. 129.--Straight and loop needles.]

[Illustration: FIG. 130.--Pasteur flask--“ballon pipette.”]

Sterile graduated pipettes varying in capacity from 1 cc graduated in
hundredths, upward, permit the transfer of definite amounts of liquids.
Large quantities are conveniently transferred by means of Pasteur
flasks (Fig. 130). The details of inoculation are best derived from
laboratory practice.




CHAPTER XVIII.

ISOLATION OF BACTERIA IN PURE CULTURE.


As has been stated, the thorough study of a bacterium depends on
first getting it in pure culture. In the early days of bacteriology
supposedly pure cultures were obtained by (1) _dilution in liquid
media_. A series of tubes or flasks containing sterile liquid media was
prepared. Number one was inoculated with the material to be examined
and thoroughly mixed. A small portion of the mixture was transferred
to number two, and mixed; from this to number three, and so on until a
sufficient number were inoculated, the last three or four in the series
receiving the same amounts of a very high dilution of the original
material. If one or two of these latter showed a growth and the others
not, it was assumed that the dilution had been carried so far that only
a single organism was transferred and therefore the culture obtained
was “pure.” The method in this crude form is too uncertain to be of
value today and recourse is had to more exact means. The procedure most
widely used is that of (2) “_plating out_” by means of gelatin or agar
plates. The material to be plated out is diluted by transferring to
three or more tubes of melted gelatin or agar as in the first method
and then all the tubes are poured into Petri dishes and grown under
suitable conditions. By proper mixing in the tubes the bacteria are
well scattered through the medium which holds the individual organisms
separate when it solidifies. On some of the plates a sufficient
dilution will be reached so that the colonies developing from the
bacteria will be so few that they are separate and pure cultures may
be obtained by inoculating from one of these a tube of the appropriate
medium (Figs. 131 to 134). The chief uncertainty with this method is
that occasionally two kinds of bacteria stick together so closely that
even the separate colonies contain both organisms. This is not common,
however. The plate colonies frequently develop from groups of bacteria
which were not separated, but as these are of the same kind the culture
is essentially pure.

[Illustration: FIG. 131.--Dilution plates. × 3/10. _1_, shows the
first dilution, the colonies are so numerous and small that they
are invisible (compare Fig. 132); _2_, shows fewer and hence larger
colonies, but too crowded to isolate (compare Fig. 133); _3_, shows the
colonies larger and well separated, so that it is easy to isolate from
them (compare Fig. 134).]

[Illustration: FIG. 132.--A portion of plate _1_ in Fig. 131 as seen
under the low-power objective. × 100. Very small, closely crowded
colonies.]

Another method which is frequently applicable with material from human
or animal sources is to (3) _rub the material over the surface_ of a
slope tube or of medium solidified in a Petri dish with a sterile heavy
platinum needle, glass rod, or cotton swab. If the bacteria are not
too numerous, pure cultures may frequently be obtained. A modification
of this method is to make a series of (4) _parallel streaks on a
slope tube or plate of medium_ with a needle inserted _but once_ into
the material to be plated. On the first streak most of the bacteria
are rubbed off and a continuous growth results, but usually on the
last of a series only isolated colonies appear, which are presumably
pure. The ideal method for securing pure cultures is to be absolutely
certain that the culture starts from a single organism. This may be
accomplished by means of the (5) _apparatus and pipettes devised by
Professor Barber_ of the University of Kansas (Figs. 135 and 136). With
this instrument a single organism is picked out under the microscope
and isolated in a drop of culture medium and observed until it is
seen to divide, thus proving its viability. Transfers are then made
to the proper media. The method requires much practice to develop the
necessary skill in the making of pipettes, determining the proper
condition of the large cover-glasses used over the isolating box, and
in manipulation, but the results fully compensate.

[Illustration: FIG. 133.--From the thinnest part of plate _2_, Fig. 131
as seen under the low-power objective. × 100. Colonies much larger than
on plate _1_, but still crowded.]

[Illustration: FIG. 134.--The smallest colony on plate _3_, Fig. 131,
as seen under the low-power objective. × 100. Large, single, isolated
colony.]

Professor W. A. Starin of the author’s department, a former student of
Professor Barber, has done some excellent work with this apparatus.

[Illustration: FIG. 135.--Diagram of Barber’s isolation apparatus. _b_,
moist chamber; _ms_, large cover-glass over moist chamber; _p_, small
pipette drawn out to a fine point; _k_, _r_, _g_, pipette holder; _f_,
screw for raising and lowering _k_, _r_, _g_; _s_, screw for lateral
motion of _k_, _r_, _g_; _n_, screw for clamp on pipette which allows
it to be moved in or out; _m_, mechanical stage of microscope; _t_,
rubber tube held in the mouth and used to move the liquid culture
medium in the pipette. (Journal of Infectious Diseases, October 20,
1908, vol. 5, No. 4, p. 381.)]

[Illustration: FIG. 136.--Photograph of microscope with Barber’s
isolation apparatus set up to use.]

A number of procedures may be used to greatly facilitate the above
methods of isolation by taking advantage of the different physiological
properties of different organisms in a mixture such as ability
to form spores, different resistance to antiseptics, special food
requirements, and pathogenic properties. (_a_) If material contains
resistant spores, it may be _heated to temperatures high_ enough to
kill all of the organisms except the spores (80° for half an hour, for
example) and then plated out. Or (_b_) _an antiseptic which restrains
the growth_ of some organisms and not others may be placed in the
culture media (carbolic acid, various anilin dyes, (p. 162), excess
acid, or alkali, ox bile, etc.), when the more resistant organisms grow
on the final plates, the others not. (_c_) _Special food substances_
(various carbohydrates) from which the organism desired forms special
products (acids, aldehydes) that may be shown on the plates by various
indicators, is one of the commonest means. Or media in which certain
organisms thrive and others not, so that the former soon “crowd out”
the latter (unsterilized milk for lactic acid bacteria, inorganic
media in soil bacteriology) may be used. A combination of the general
methods (_b_) and (_c_) is much used in the separation of the organisms
of the “intestinal group” in human practice. (_d_) _The inoculation
of a susceptible animal_ with a mixture suspected to contain a given
pathogenic bacterium frequently results in the development of the
latter in pure culture in the body of an animal, from which it may be
readily recovered. In all of the above methods (except Barber’s) the
first “pure culture” obtained should be “purified” by replating in a
series of dilution plates to make sure that it is pure.




CHAPTER XIX.

STUDY OF INDIVIDUAL BACTERIA--STAINING.


When an organism has been obtained in pure culture by any of the
methods described in the preceding chapter the next step is the study
of its morphology as discussed in Chapters II--IV. This involves the
use of the microscope, and since bacteria are so small, objectives
of higher power than the student has presumably used will be needed.
Doubtless only the two-thirds inch or 16 mm. and the one-sixth inch or
4 mm. objectives are all that have been used in previous microscopic
work, while for examining bacteria a one-twelfth inch or 2 mm. is
necessary. It will have been observed that the higher the power of
the objective the smaller is the front lens or object glass and
consequently the less is the amount of light which enters. With the use
of the one-twelfth inch or 2 mm. objective it is necessary to employ
two devices for increasing the amount of light entering it, with which
the student is probably not familiar. One of these is to place a drop
of cedar oil between the front lens and the object and to immerse the
lens in this oil--hence the term “oil-immersion objective;” the other
is the substage or Abbé condenser. The latter is a system of lenses
placed below the stage and so constructed as to bring parallel rays of
light--daylight--from an area much larger than the face of the front
lens of the objective to a focus on the object to be examined, thus
adding very greatly to the amount of light entering the objective.
Since the condenser brings _parallel_ rays to a focus on the object,
the _flat-mirror_ is always used with the condenser when working with
daylight. With _artificial light close_ to the microscope, the concave
mirror may be used to make the divergent rays more nearly parallel and
thus give better illumination.

The function of immersion oil is to prevent the dispersion of
considerable light that would otherwise occur owing to refraction
as the light passes up through the slide and into the air. The
accompanying diagram will help to make this clearer (Fig. 137). A ray
of light (_A B_) coming through the slide will be refracted in the
direction _B C_ if the medium has a lower refractive index than the
slide, as air has, and hence will not enter the objective _O_. If,
however, there is interposed between the objective and the slide a
medium which has the same refractive index as the slide, as immersion
oil has, then the ray will continue in the same direction (_B D_) at
the point _B_ and hence enter the objective. Evidently the immersion
oil causes much more light to enter the front lens and makes the field
brighter and at the same time prevents considerable refraction and
dispersion of light from the object seen and hence this appears more
distinct and sharply defined. The Abbé condenser and the oil-immersion
objective are practically always used in the microscopic study of
bacteria (Fig. 138).

[Illustration: FIG. 137.--Diagram of use of immersion oil.]

[Illustration: FIG. 138.--Diagram of paths of rays of microscope.]


HANGING DROP SLIDE.

It is sometimes necessary to examine living bacteria and for this
purpose the device known as the “hanging drop slide” is used (Fig.
139). The slide has a slight concave depression ground in the middle
of one face. A ring of vaseline is placed around this depression with
the loop needle. On a clean cover-glass, large enough to fit over the
ring of vaseline, several drops of a broth culture, or of material
from a solid culture suspended in broth or physiological normal salt
solution are placed. The slide is inverted on the cover-glass in such
a way that the ring of vaseline seals the latter to the slide. When
the whole preparation is quickly turned cover side up, the drops are
seen “hanging” to the under side of the cover over the depression in
the slide. In examining such a preparation with the microscope great
care is necessary in order to focus on the bacteria, without breaking
the cover. To see the organisms distinctly the _lower iris diaphragm of
the condenser must be nearly closed_, so that the light coming through
consists mainly of parallel vertical rays, otherwise the transparent
bacteria themselves refract and diffract the light and appear blurred
and indistinct. By studying living bacteria with this device it can
be determined whether they are motile or not. The motility should not
be confounded with the familiar “Brownian movement” of all minute
insoluble inert particles which non-motile living bacteria and
also dead bacteria show. The hanging drop slide is of value in the
measurement of bacteria, since this is properly done on the living
organism. Measurement is done with a calibrated ocular micrometer as in
other kinds of measurement with the microscope with which the student
is presumably familiar. The direct effect of various agents on living
bacteria as light, electricity, heat, etc., in the study of “tropisms”
and “taxes” has been investigated on various modifications of the
above-described hanging drop slide.

[Illustration: FIG. 139.--Hanging drop slide.]

Cell forms and cell groupings may be studied in the same way but
these features are best determined on _stained_ preparations in many
instances.

“Dark field” illumination and the ultramicroscope are of great value in
the study of living bacteria and other minute objects, but apparatus
of this type would scarcely be used by the student in an introductory
course, so that they will not be discussed in the present volume.


STAINING.

The main use of the microscope in bacteriology is in the study of
_stained preparations_ of the organisms. Staining makes bacteria opaque
and hence more easily seen than the transparent unstained forms. Some
methods of staining also show morphological structures which are either
imperfectly recognized in the unstained cell, spores, or are not
visible at all--capsules, metachromatic granules, flagella. Finally
certain bacteria are colored by special methods of staining which do
not affect others, so that under proper conditions these bacteria may
be recognized by staining methods alone--tubercle bacilli in the organs
of animals.

The phenomena of staining are essentially chemical, though sometimes
the chemical union is a very weak one, even resembling an absorption of
the dye rather than true chemical union--most watery stains. In other
cases the chemical compounds formed are decidedly stable and are not
decomposed even by strong mineral acids--staining of tubercle bacilli
and other “acid-fast” organisms. In still other cases the principal
action is a precipitation on the surface of the object stained--methods
for staining flagella.

In many methods of staining in addition to the dyes used other
substances are added to the solution which assist in fixing the dye in
or on the organism stained. Such substances are called _mordants_. The
principal mordants used are alkalies, anilin, carbolic acid, iodine,
metallic salts, tannic acid.

While it is true that some bacteria may be stained by that standard
histological nuclear dye, hematoxylin, it is of little value for this
purpose. Practically all bacteriological stains are solutions of the
_anilin dyes_. These dyes, as is well known, are of nearly every
conceivable color and shade but relatively very few are used in
bacteriological work. The beginning student will rarely use solutions
of other than the three dyes _fuchsin_ (red), _methylene blue_ and
_gentian violet_ for staining bacteria, with occasionally Bismarck
brown, or eosin, or safranin as tissue contrast stains.

The bacteriological dyes are kept “in stock” as saturated solutions in
95 per cent. alcohol which are _never used as stains_, but merely for
convenience in making the various staining solutions.

The approximate percentages of the three common dyes in such solutions
are indicated in the following table adapted from Woods _Chemical and
Microscopical Diagnosis_, Third Edition, 1917, Appendix:

  Fuchsin           3.0%
  Gentian Violet    4.8%
  Methylene Blue    2.0%

The stains made from these dyes which are in most common use are the
following:

  1. Aqueous (watery) gentian violet solution.

  Saturated alcoholic solution of gentian violet      1 part
  Distilled water                                    20 parts
  Mix well and filter.

  2. Anilin gentian violet.

  Saturated alcoholic solution of gentian violet      1 part
  Anilin water (see below)                           10 parts
  Mix well and filter.

  3. Anilin Fuchsin.

  Saturated alcoholic solution of fuchsin             1 part
  Anilin water (see below)                           10 parts
  Mix and filter.

These stains rarely keep longer than ten days in the laboratory (unless
kept in the ice-box) and must be made fresh on the first sign of a
deposit on the glass of the container.

=Anilin Water.=--Anilin water is made by putting 3 or 4 cc of anilin
“oil” in a 120 cc. flask, adding 100 cc of distilled water, shaking
vigorously for a minute or so and filtering through a wet filter, in
other words, a saturated solution of anilin in water.

  4. Löffler’s (methylene) blue.

  Saturated alcoholic solution of methylene blue      3 parts
  Aqueous solution of NaOH (or KOH), 1 to 10,000     10   „
  Mix and filter.

  5. Carbol-fuchsin (Ziehl’s solution).

  Saturated alcoholic solution of fuchsin             1 part
  5 per cent. aqueous solution of carbolic acid      10 parts
  Mix and filter.

  6. Gabbet’s (methylene) blue (solution).

  Dry methylene blue                                  4 parts
  Concentrated H₂SO₄                                 25   „
  Distilled water                                    75   „
  Dissolve the dry dye in the acid and add the solution to the
    distilled water and filter.

[Illustration: FIG. 140.--Author’s staining set. Square bottles are set
in square holes in the block. The capacity of each bottle is 30 cc.]

Staining solutions are conveniently kept in square dropping bottles
inserted in a block as shown in Fig. 140. This form of holder
necessitates the use of _one hand only_ in securing the stain and
dropping it on the preparation.

The actual staining of bacteriological preparations can be learned only
by repeated laboratory practice, yet the following methods have given
such uniform results in class work that it is felt they are not out of
place in a text-book.

=Preparation of the “Film.”=--The author learned to stain bacteria,
on the “cover-glass” but does not recall having used this method in
fifteen years and does not teach it to his students. All staining is
done on the slide. To prepare a film from a solid culture medium the
procedure is as follows:

First, be sure the slide is clean and _free from grease_. This is
accomplished most readily by scouring a few minutes with finely
ground pumice stone and a little water, then washing and drying with
a grease-free cloth, handkerchief, or piece of cheese-cloth. With the
“loop” needle place in the middle of the slide a small loop of water.
This is best done by filling the loop by dipping in water, then tapping
it gently so that all that remains is the water that just fills the
loop level full, and this amount is placed on the slide by touching
the flat side of the loop to the glass. Then the _straight needle_ is
sterilized, dipped into the culture and just touched once into the
small drop of water on the slide. The remainder of the culture on the
straight needle is then burned off and the needle is used to spread the
drop of water containing the bacteria into a thin even film, which will
result, provided the slide is free from grease. This is dried and then
“fixed” by passing three times through the Bunsen flame at intervals of
about one second, passing through slowly for thick slides and a little
more rapidly for thin ones. If the culture is in a liquid medium, the
use of the loop of water is unnecessary; a loop of the fluid from the
surface, middle or bottom as the culture indicates is spread out to a
thin film, dried and fixed.

After the film is fixed the stain desired is dropped on, allowed
to act for the proper time, which will depend on the stain and the
preparation, washed in water, dried thoroughly and examined with the
oil-immersion lens, without a cover. If it is desired to preserve the
preparation it may then be mounted in balsam. This is not necessary, as
they keep just as well, provided the immersion oil is removed. To do
this, fold a piece of filter paper so that at least three thicknesses
result. Lay this on the slide and press firmly several times, when the
surplus oil will be taken up by the paper. Slides not mounted in balsam
are more apt to become dusty than those that are. This is the only
disadvantage.

=Gram’s Method of Staining.=--It has been ascertained that some
bacteria contain a substance, possibly a protein, which forms a
compound with gentian violet and iodine, which compound is insoluble in
alcohol, and other bacteria do not contain this substance. Consequently
when bacteria are stained by Gram’s method (given below), those that
contain this chemical remain colored, while if it is not present the
dye is washed out by the alcohol and the bacteria are colorless and may
be stained by a contrast stain. The bacteria which stain by this method
are said to “take Gram’s” or to be “Gram-positive,” while those that
decolorize are called “Gram-negative.” The method is:

1. Prepare the film as above given.

2. Stain with fresh anilin gentian violet 1 minute.

3. Wash in tap water.

4. Cover with Gram’s solution 1 minute.

5. Wash in tap water.

6. Wash with 95 per cent. alcohol three times or until no more color
comes out.

7. Dry and examine.

Gram’s solution is:

  I           1 part
  KI          2 parts
  H₂O       300   „

This method is excellent for differentiating Gram-positive and
Gram-negative organisms on the same slide. First stain by this
method and after washing with alcohol stain with a counter-stain,
carbol-fuchsin diluted ten to fifteen times with water is excellent.
The Gram-positive bacteria are violet and the Gram-negative are red.

It is also of great value in staining Gram-positive bacteria in
tissues, but the sections should be stained about five minutes in
the anilin gentian violet and be left about two minutes in the Gram’s
solution. Sections are to be counter-stained in Bismarck brown, dilute
eosin or safranin solutions and cleared in oil of bergamot, lavender or
origanum and not in clove oil or carbol-xylol, as these latter dissolve
out the dye from the bacteria.

=Staining of Spores in the Rod.=--Prepare the films as usual. Cover
with carbol-fuchsin, using plenty of stain so that it will not dry on
the slide; heat until vapor arises, not to boiling; cool until the
stain becomes cloudy and heat again until the stain clears, and repeat
once more; wash in tap water and then wash in 1 per cent. H₂SO₄ three
times, dropping on plenty of acid, tilting and running this over the
slide three times and then pour off and use fresh acid and repeat this
once. Wash thoroughly in _distilled_ water, then stain with Löffler’s
blue one to three minutes. Wash, dry and examine. The spores should be
bright red in a blue rod.

This method will give good results if care is taken to secure cultures
of the right age. If the culture is too old the spores will all be free
outside the rods, while if too young they will decolorize with the
acid. For _Bacillus subtilis_ and _Bacillus anthracis_, cultures on
agar slants forty-eight hours in the 37° incubator are just right. For
the spores of _Clostridium tetani_, the culture should be three days
old, but may be as old as a week.

=Staining of “Acid-fast” Bacilli.=--_Mycobacterium tuberculosis,
Mycobacterium of Johne’s disease, “grass” and “butter bacilli,”
Mycobacterium lepræ, Mycobacterium smegmatis._

_Gabbet’s method_:

  1. Prepare the film as usual.

  2. Stain with carbol-fuchsin as given above for spores.

  3. Wash with tap water.

  4. Decolorize and stain at the same time with Gabbet’s blue, two or
  three minutes.

  5. Wash, dry and examine.

The sulphuric acid in Gabbet’s blue removes the carbol-fuchsin from
everything except the “acid-fast” bacteria, which remain red, and the
blue stains the decolorized bacteria and nuclei of any tissue cells
present.

_Ziehl-Neelson method_:

  1, 2, 3, as in Gabbet’s method.

  4. Decolorize with 10 per cent. HCl until washing with water shows
  only a faint pink color left on slide.

  5. Wash thoroughly.

  6. Stain with Löffler’s blue one or two minutes.

  7. Wash, dry and examine.

The results are the same as with Gabbet’s method.

=Staining of Capsules.=--_Räbiger’s Method._--Films of the organism
to show capsules should be _freshly prepared, dried but not fixed_.
Material is usually obtained from milk or blood. A drop of the fluid
is placed on the middle of a slide about one-fourth of the distance
from one end. The narrow edge of another clean slide is placed in this
drop and then drawn lengthwise across the slide with firm pressure.
This gives a _thin layer_ which is necessary if good results are to
be expected. The preparation is covered with a _freshly prepared_
saturated solution of gentian violet in formalin and this allowed
to stain for 30 seconds. Then wash _lightly_, dry and examine. The
organisms appear deeply violet and much larger than with ordinary
stains and capsules are well stained and show well.

_Welch’s Method._--Prepare films as in the above method. Cover with
glacial acetic acid for 10 to 20 seconds. Wash off the acid with
carbol-fuchsin. Wash the stain off with physiological normal salt
solution (0.85 per cent.) until all surplus stain is removed. Dry and
examine. Capsules and bacteria are red.

=Staining of Flagella.=--The rendering of flagella visible is
considered one of the most difficult processes in staining. Experience
of a number of years during which whole classes numbering from one
hundred to three hundred students accomplish this result shows that it
is no more difficult than many other staining processes. The essentials
are: (1) clean slides, (2) young cultures on agar slopes, (3) freshly
prepared mordant and stain which are kept free from precipitate,
(4) gentle heating. The author’s students are furnished only stock
materials and make their own cultures, mordants and stains.

The slides are cleaned with pumice in the usual way. An agar slope
culture of the organism to be stained from six to twenty-four hours
old is selected. A bit of the culture is removed and placed in a
watch-glass of water. The bacteria are allowed to diffuse of themselves
without stirring. After several minutes a loop of this water is removed
and three streaks are made across the slide, one in the middle and one
on each side of this about one-quarter of an inch from it. This gives
well scattered bacteria in one of the three streaks at least and very
little other material on the slide to cause precipitates. The slide is
carefully dried and fixed and then covered with an abundance of the
mordant by filtering through a small filter onto the slide so that the
mordant shows transparent on the slide. The preparation is then gently
warmed and cooled three times, adding mordant if necessary. _Do not
heat to steaming._ After mordanting for about five minutes the excess
is washed off under the tap. It is a good plan to hold the slide level
and allow the water to run into the center of the mordant and flow it
off. Inclining the slide is apt to cause the film on the surface of the
mordant to settle down on the slide and spoil the preparation. After
the mordant is washed off and all traces of it removed with a clean
cloth if necessary the stain is applied and gently heated and cooled
the same way for from three to five minutes. The preparation is then
washed, dried and examined.

The mordant used is a modification of Löffler’s which is somewhat
simpler in preparation since the stock solution of FeCl₃ is more
permanent than FeSO₄ solution.

Mordant sufficient for one student:

  5 per cent. solution of FeCl₃           20.0 cc
  25 per cent. solution of tannic acid    20.0 cc
  Anilin fuchsin                           4.0 cc
  Normal NaOH                              1.5 cc

The solution of FeCl₃ is made up in the cold and must be perfectly
clear. The tannic acid solution must be thoroughly boiled and filtered
until clear. The iron and the acid are carefully mixed, boiled and
filtered clear. The anilin fuchsin must be added slowly with constant
stirring and the mixture boiled and filtered. The NaOH is added in the
same way and this mixture boiled and filtered. The final mordant should
not leave a film on a clean slide when poured on and allowed to run
off. Unless the mordant is in this condition and perfectly clear, it
should not be used, but a new one must be made up. Time and care in the
preparation of the mordant are essential.

The stain to follow this mordant is anilin fuchsin.

=Staining of Metachromatic Granules.=--_Neisser’s Method._ Prepare the
film in the usual way. Stain with Neisser’s stain a few seconds only.
Wash and stain with Bismarck brown a few seconds only.

  _Neisser’s Stain_:

  Sat. alcoholic solution of methylene blue   1.0 part
  Glacial acetic acid                         2.5 parts
  Distilled water                            50.0 parts

  _Bismarck Brown_:

  Bismarck brown (dry dye)                      2 parts
  Distilled Water                            1000 parts

By the use of the hanging drop slide and the methods of staining just
described all the various morphological features of the bacterial cell
may be ascertained.

It is necessary when _cell groupings_ as characteristic of definite
modes of division are to be determined to make slides from a liquid
culture, as broth. Place a drop of the material, preferably from the
bottom of the tube in most instances, from the top in case a pellicle
or scum is formed on the surface, on the slide and allow this to dry
_without spreading it out_, fix, wash gently with water, then stain
lightly with Löffler’s blue. Such slides also show characteristic
_cell forms_ as well. Slides should be made from solid media to show
variations in form and size and involution forms. These latter are
especially apt to occur on potato media.




CHAPTER XX.

STUDY OF THE PHYSIOLOGY OF BACTERIA.


Of the environmental conditions influencing the growth of bacteria the
following are the chief ones ordinarily determined:

_A._ Temperature.--The optimum temperature for growth is usually
about the temperature of the natural environment and ordinarily one
determines merely whether the organism grows at body temperature (37°)
and at room temperature (20°) or not. For exact work the maximum,
minimum and optimum temperature must be ascertained by growing in
“incubators” with varying temperatures.

A bacteriological incubator is an apparatus for growing bacteria at a
constant temperature. This may be any temperature within the limits for
bacterial growth. If temperatures above that of an ordinary room are
desired, some source of artificial heat is needed. Electricity, gas or
oil may be used. A necessary adjunct is some device for maintaining
the temperature constant, a “thermoregulator” or “thermostat.” For
lower temperatures a cooling arrangement must be installed. For the
great part of bacteriological work only two temperatures are used, 20°
so-called “room temperature” (this applies to European “rooms” not to
American) and 37° or body temperature. Incubators for 37° of almost any
size and style desired may be secured from supply houses and need not
be further described. Figs. 141 and 142 illustrate some of the types.

For use with large classes “incubator rooms” are to be preferred. The
author has one such room for 37° work with 200 compartments for student
use which did not cost over $60 to install.

[Illustration: FIG. 141.--Small laboratory incubator, gas heated.]

[Illustration: FIG. 142.--Electric incubator.]

The styles of incubators for lower temperatures, 20° and below, are not
so numerous nor so satisfactory. The author has constructed a device
which answers every purpose for a small class. The diagram, Fig. 143,
explains it.

[Illustration: FIG. 143.--Diagram of fittings for a cold incubator.
_1._ small tank for constant head, about 1 foot in each dimension. _a_,
inflow; _b_, overflow; _c_, lead pipe. _2_, refrigerator. _a′_, ice;
_b′_, flat coil under ice; _c′_, outflow to incubator. _3_, incubator.
_a″_, cold water inflow; _b″_, overflow; thermometer and burner
omitted. The diagram explains the construction. The water cooled to
about 14° with artificial ice by flowing through the lead coil under
the ice, flows into the incubator which may be heated and regulated in
the usual way.]

The thermal death-point is determined by exposing the organisms in
thin tubes of broth at varying temperatures for ten-minute periods and
then plating out to determine growth. The effect of heat may also be
determined by exposing at a given temperature, _e.g._, 60°, for varying
lengths of time and plating out.

_B._ Oxygen relations--whether the organism is aërobic, anaërobic, or
facultative is determined by inoculation in gelatin or agar puncture or
stab cultures and noting whether the most abundant growth is at the
top, the bottom or all along the line of inoculation.

_C._ Reaction of the medium--acid, alkaline or neutral as influencing
the rate and amount of growth.

_D._ The kind of medium on which the organism grows best.

_E._ The effect of injurious chemicals, as various disinfectants, on
the growth.

_F._ Osmotic pressure conditions, though modifying decidedly the growth
of bacteria, are not usually studied as aids in their recognition, nor
are the effects of various forms of energy, such as light, electricity,
_x_-rays, etc.

Among the “Physiological Activities” discussed in Chapters IX-XII those
which, in addition to the staining reactions described, are of most
use in the identification of non-pathogenic bacteria are the first ten
listed below. For pathogenic bacteria the entire thirteen are needed.

1. Liquefaction of gelatin.

2. Digestion of blood serum.

3. Coagulation and digestion of milk.

4. Acid or gaseous fermentation in milk, or both.

5. Acid or gaseous fermentation of various carbohydrates in
carbohydrate broth, or both.

6. Production of indol in “indol solution.”

7. Production of pigments on various media.

8. Reduction of nitrates to nitrites, ammonia, or free nitrogen.

9. Production of enzymes as illustrated in the above activities.

10. Appearance of growth on different culture media.

11. Production of free toxins as determined by injection of animals
with broth cultures filtered free from bacteria.

12. Causation of disease as ascertained by the injection of animals
with the bacteria themselves, and recovery of the organism from the
animals.

13. Formation of specific antibodies as determined by the
proper injection of animals with the organism or its products
and the subsequent testing of the blood serum of the inoculated
animals.

For special kinds of bacteria other activities must be determined
(oxidation, nitrate and nitrite formation, action of sulphur and iron
bacteria, etc.).

The first nine activities are determined by inoculating the different
culture media already described and observing the phenomena indicated,
making chemical tests where necessary.


APPEARANCE OF GROWTH ON DIFFERENT CULTURE MEDIA.

In addition to those changes that are associated with the manifestation
of different physiological activities, many bacteria, show
characteristic appearances on the various culture media which are of
value in their identification.

Too much stress should not be laid on these appearances alone, however,
since slight variations, particularly in solid media due especially to
the age of the medium, may change decidedly the appearance of a colony.
This is true of variations in the amount of moisture on agar plates.
Colonies which are ordinarily round and regular may assume very diverse
shapes, if there chance to be an excess of moisture on the surface.

Also in slope and puncture cultures on the various solid media much
variation results from the amount of material on the inoculation needle
and just how the puncture is made, or the needle drawn over the slope.
These variations are largely prevented by the use of standard media and
by inoculating by standard methods. The Laboratory Committee of the
American Public Health Association has proposed standard methods for
all culture media and tests and for methods of inoculation, and these
have been generally adopted in this country for comparative work.

Likewise the Society of American Bacteriologists has at different times
(1904, 1914, 1917) adopted “descriptive charts” for detailing all the
characteristics of a given organism. A committee is at present working
on a revision of the 1917 chart to be presented at the 1920 meeting.
One of the earlier charts which includes a glossary of descriptive
terms is inserted in this chapter.

Among the cultural appearances the following are of most importance:

[Illustration: FIG. 144.--Broth cultures × 2/3. _1_ uninoculated
transparent broth; _2_, broth cloudy from growth of organisms; _3_,
broth slightly cloudy with a deposit in bottom; _4_, broth slightly
cloudy with a heavy membrane at the surface.]

[Illustration: FIG. 145.--A filiform stab or puncture culture. × 3/5.]

[Illustration: FIG. 146.--A beaded stab or puncture culture. × 1/2.]

[Illustration: FIG. 147.--A villous stab or puncture culture. × 1/2.]

In broth cultures the presence or absence of growth on the surface
and the amount of the same. Whether the broth is rendered cloudy or
remains clear, and whether there is a deposit at the bottom or not
(Fig. 144). An abundant surface growth with little or nothing below
indicates a strict aërobe, while a growth or deposit at bottom and a
clear or nearly clear medium above, an anaërobe. These appearances are
for the first few days only of growth. If the broth is disturbed, or
after the culture stands for several days many surface growths tend to
sink to the bottom. So an actively motile organism causes in general
a cloudiness, especially if the organism is a facultative anaërobe,
which tends to clear up by precipitation after several days when the
organisms lose their motility. Non-motile facultative anaërobes
usually cloud the broth also, but settle out more rapidly than the
motile ones.

In gelatin and agar punctures the oxygen relationship is shown by
surface growth for aërobes, growth near the bottom of the puncture
for anaërobes, and a fairly uniform growth all along the line of
inoculation for facultative anaërobes. In the case of these last
organisms, a preference for more or less oxygen is indicated by the
approach to the aërobic or anaërobic type of growth.

[Illustration: FIG. 148 FIG. 149 FIG. 150 FIG. 151

FIG. 148.--Crateriform liquefaction of gelatin. × 1/2.

FIG. 149.--Funnelform liquefaction of gelatin. × 1/2.

FIG. 150.--Saccate liquefaction of gelatin. × 1/2.

FIG. 151.--Stratiform liquefaction of gelatin. × 1/2.]

Along the line of puncture the commonest types are _filiform_ (Fig.
145), which indicates a uniform growth; _beaded_ (Fig. 146), or small
separate colonies; _villous_ (Fig. 147), delicate lateral outgrowths
which do not branch; _arborescent_, tree-like growths branching
laterally from the line. In agar these branchings are usually short and
stubby, or technically, _papillate_.

[Illustration: FIG. 152.--Filiform slope culture. × 1/2.]

[Illustration: FIG. 153.--Filiform, slightly spreading, slope culture.
× 1/2.]

[Illustration: FIG. 154.--Beaded slope culture. × 1/2.]

Further, in the gelatin puncture the liquefaction which occurs is
frequently characteristic. It may be _crateriform_ (Fig. 148), a
shallow saucer at the surface; or _funnel-shaped_ (Fig. 149); or it
may be of uniform width all along the puncture, _i.e._, _saccate_ (Fig.
150); or it may be _stratiform_, (Fig. 151), _i.e._, the liquefaction
extends to the sides of the tube and proceeds uniformly downward.

[Illustration: FIG. 155 FIG. 156 FIG. 157 FIG. 158

FIG. 155.--Effuse slope culture. × 1/2.

FIG. 156.--Rhizoid slope culture. × 1/2.

FIG. 157.--Rugose slope culture. × 1/2.

FIG. 158.--Verrucose slope culture. × 1/2.]

On agar, potato and blood serum slope tubes the amount of growth, its
form and elevation, the character of the surface, and the consistency
should be carefully noted, and in some few cases the character of the
edge. Figures 152 to 158 show some of the commoner types.

[Illustration: FIG. 159.--Punctiform colonies on a plate. × 1/2.]

[Illustration: FIG. 160.--A rhizoid colony on a plate. Natural size.]

[Illustration: FIG. 161.--Ameboid colonies on a plate. × 1/2.]

[Illustration: FIG. 162.--Large effuse colony on a plate. The edge is
lacerated. Incidentally the colony shows the rate of growth for six
successive days. × 2/3.]

[Illustration: FIG. 163.--Colony with edge entire as seen under the
low-power objective. × 100.]

[Illustration: FIG. 164.--Colony with edge coarsely granular as seen
under the low-power objective. × 100.]

[Illustration: FIG. 165.--Colony with edge curled as seen under the
low-power objective. × 100.]

[Illustration: FIG. 166.--Colony with edge rhizoid as seen under the
low-power objective. × 100.]

[Illustration: FIG. 167.--A small deep rhizoid colony as seen under the
low-power objective. × 100.]

On agar and gelatin plates made so that the colonies are well isolated,
the form of the latter, the rate of their growth, the character of the
edge and of the surface, the elevation and the internal structure
as determined by a low-power lens are often of almost diagnostic
value. Also in the case of the gelatin plates, the character of the
liquefaction is important. Figs. 159 to 167 show some of the commoner
characteristics to be noted.

[Illustration: FIG. 168.--A small mold colony natural size as viewed by
transmitted light.]

[Illustration: FIG. 169.--The same colony as viewed by reflected light.]

[Illustration: FIG. 170.--A portion of the thin edge of the same colony
as seen with the lower-power objective. × 100.]

[Illustration: FIG. 171.--A single fruiting body (sporangium) from the
same colony as seen under the lower-power objective. × 100.]

Colonies of mold frequently appear on plates. These are readily
differentiated from bacterial colonies after a little experience. With
the naked eye usually the fine radiations of the edge of the colony
are apparent. The surface appears duller and by reflected light more
or less “fuzzy.” With the low-power objective the relatively large,
branching threads of the mold (mycelia) show distinctly. Also the large
fruiting bodies (sporangia) are easily distinguished. Figs. 168 to 171
illustrate a common black mold (_Rhizopus nigricans_).




CHAPTER XXI.

ANIMAL INOCULATION.


Animal inoculation has been referred to (1) as a method of assisting
in the preparation of pure cultures of pathogenic organisms; (2) as a
means of testing the poisonous properties of substances produced in
bacterial cultures; (3) in order to test the ability of an organism to
cause a disease; (4) for the production of various antibodies; it may
be added (5) that some bacteria produce in the smaller experimental
animals lesions which do not occur in animals naturally infected, but
which nevertheless are characteristic for the given organism. The best
illustration is the testicular reaction of young male guinea-pigs to
intraperitoneal injections of glanders bacilli. Experimental animals
are also inoculated (6) to test the potency of various bacterial and
other biological products, as toxins, antitoxins, etc.

Guinea-pigs are the most widely used experimental animals because they
are easily kept and are susceptible to so many diseases on artificial
inoculation. Rabbits are used very largely also, as are white mice. For
special purposes white rats, pigeons, goats and swine are necessary.
For commercial products horses (antitoxins) and cattle (smallpox
vaccine) are employed. In the study of many human diseases the higher
monkeys and even the anthropoid apes are necessary, since none of the
lower animals are susceptible.

The commonest method of animal inoculation is undoubtedly the
_subcutaneous_. This is accomplished most readily with the hypodermic
needle. The skin at the point selected (usually in guinea-pigs the
lateral posterior half of the abdominal surface, in mice the back near
the root of the tail) is pinched up to avoid entering the muscles and
the needle quickly inserted. Clipping the hairs and washing with an
antiseptic solution should precede the inoculation as routine practice.
Frequently a small “skin pocket” is all that is needed. The hair is
clipped off, the skin pinched up with small forceps and a slight snip
with sharp scissors is made. The material may be inserted into this
pocket with a heavy platinum needle. _Cutaneous_ inoculation is made
by shaving the skin and rubbing the material onto the shaved surface
or scratching with a scalpel or special scarifier, but without drawing
blood, and then rubbing in the material to be inoculated.

_Intravenous_ injections are made with larger animals. In rabbits the
posterior external auricular is a convenient vein. In larger animals
the external jugular is used.

_Intraperitoneal_, _-thoracic_, _-cardiac_, _-ocular_, _-muscular_
injections, and injections into the parenchyma of internal organs are
accomplished with the hypodermic needle. In the case of the first two,
injury to contained organs should be carefully avoided. Intracardiac
injection, or aspiration of the heart to secure blood, requires
considerable practice to be successful without causing the death of the
animal at once through internal hemorrhage. In _subdural_ injections
into the cranial cavity it is necessary to trephine the skull first,
while such injections into the spinal canal may be accomplished between
the vertebra with needles longer and stronger than the usual hypodermic
needle. Occasionally animals are caused to _inhale_ the organisms, or
are _fed_ cultures mixed with the feed.


SECURING AND TRANSPORTING MATERIAL FROM ANIMALS FOR BACTERIOLOGICAL
EXAMINATION.

If the site of the lesion is readily accessible from the exterior,
material from the _living animal_ should be collected with sterile
instruments and kept in sterile utensils until the necessary tests can
be made. Testing should be done on material as soon after collection
as possible, in all cases, to avoid the effects of “decomposition”
bacteria.

If the blood is to be investigated it may be aspirated from a
peripheral vein with a sterile hypodermic syringe of appropriate size
or allowed to flow through a sterile canula into sterile receptacles.
The site of the puncture should be shaved and disinfected before the
instrument is introduced.

Discharges of whatever kind should likewise be collected in sterile
receptacles and examined as soon as may be.

If internal organs are to be examined it is best to kill a moribund
animal than to wait for death, since after death, and in severe
infections even sometimes before, the tissues are rapidly invaded by
saprophytic bacteria from the alimentary and respiratory tracts which
complicate greatly the isolation of the specific organism. Hence the
search for specific bacteria in carcasses or organs several hours after
death is frequently negative. Animal inoculation with such material is
very often followed by sepsis or septicemia in a few hours, so that the
specific organism has no opportunity to manifest itself.

In securing material for cultures from internal organs it is a good
plan to burn the surface of the organ with a gas or alcohol flame, or
to sear it with a hot instrument to kill surface organisms, then make
the incision or puncture through the burned area and secure material
from the interior of the organ. Such punctures made with a stiff
platinum needle frequently give pure cultures of the organism sought.
Slides may be made from such material and culture media inoculated at
once.

Since a bacteriological diagnosis depends most commonly on growing the
organisms, it is evident that material sent for examination must _never
be treated with an antiseptic or preservative_. If decomposition is to
be feared the only safe procedure is to _pack the material in ice_ and
forward in this way.

_Tuberculous material from the parenchyma_ of internal organs may be
forwarded in a preservative (not _formalin_, since this makes it very
difficult to stain the bacteria) as _in this special_ case a very
positive diagnosis may be made by staining alone. Even here it is
better to _pack in ice_ in order that the diagnosis by staining may be
confirmed by inoculating the living organisms into guinea-pigs.

In the case of material _from a rabid animal_ and many protozoal
diseases the rule against preservatives is not absolute, since staining
is a reliable diagnostic means. Even in these cases it is often
desirable to inoculate animals, hence, as before stated, it is best to
make it a uniform practice to _pack material for examination in ice and
use no preservatives_.




PART IV.

GENERAL PATHOGENIC BACTERIOLOGY.




CHAPTER XXII.

INTRODUCTION.


Pathogenic Bacteriology treats of the unicellular microörganisms which
are responsible for disease conditions, _i.e._, pathological changes
in other organisms. Hence not only are bacteria considered, but also
other low vegetable forms, as yeasts and molds, likewise protozoa in
so far as they may be pathogenic. For this reason the term pathogenic
“Microbiology” has been introduced to include all these organisms. It
is largely for the reason that the methods devised for the study of
bacteria have been applied to the investigation of other microörganisms
that the term “bacteriology” was extended to cover the entire field.
The general discussion in this chapter is intended to include,
therefore, microörganisms of whatever kind pathogenic to animals.

The term pathogenic as applied to an organism must be understood in a
purely _relative_ sense, since there is no single organism that can
cause disease in all of a certain class, but each is limited to a more
or less narrow range. Some form of tuberculosis attacks nearly all
vertebrates, but no other classes of animals and no plants. Lockjaw or
tetanus attacks most mammals, but not any other vertebrates naturally.
Typhoid fever affects human beings; hog cholera, swine, etc. This point
is more fully discussed in Chapter XXIII but can not be too greatly
insisted upon.

    “The greatest enemy to mankind is man.”

Exceptions to this statement do occur and are important and must be
considered in efforts to protect completely human beings from disease
(tuberculosis from cattle, glanders from horses, poisoning from spoiled
canned goods, anthrax from hair, hides, wool, of animals dead of the
disease), but the most common human diseases are derived from other
human beings directly or indirectly.

Diseases which are due to unicellular pathogenic microörganisms are
called _infectious_ diseases, while if such diseases are transmitted
under natural conditions from organism to organism they are spoken of
as _contagious_ diseases. Most infectious diseases are contagious but
not all. Tetanus is a good illustration of a non-contagious infectious
disease. There are very few such diseases.

When a unicellular microörganism gains entrance into the body and
brings about any pathological changes there, the result is an
_infection_. Undoubtedly many pathogenic organisms get into the body
but never manifest their presence by causing disease conditions, hence
do not cause an infection. It is the pathological conditions which
result that constitute the infection, and not the mere _invasion_.

The time that elapses between the entrance of the organism and the
appearance of symptoms is called the _period of incubation_ and varies
greatly in different diseases.

The term _infestation_ is used to denote pathological conditions due to
_multicellular_ parasites. Thus an animal is _infested_ (not infected)
with tapeworms, roundworms, lice, mites, etc. Many of these conditions,
probably all, are contagious, _i.e._, transmissable naturally from
animal to animal. The word _contagious_ has been used in a variety
of ways to mean _communicated by direct contact_, communicated by a
living something (_contagium_) that might be carried to a distance
and finally _communicable_ in any manner, transmissable. The agency
of transmission may be very roundabout--as through a _special tick_
in Texas fever, a _mosquito_ in malaria, etc.,--or by direct personal
contact, as generally in venereal diseases. After all, though exactness
is necessary, it is better to learn all possible about the _means of
transmission of diseases_, than quibble as to the terms to be used.

An infectious disease may be _acute_ or _chronic_. An acute infection
is one which runs for a relatively short time and is “self-limited,”
so-called, _i.e._, the organisms cease to manifest their presence after
a time. In some acute infections the time is very short--German measles
usually runs five or six days. Typhoid fever may continue eight to
ten weeks, sometimes longer, yet it is an acute infectious disease.
It is not so much the time as the fact of _self-limitation_ that
characterizes acute infections.

In chronic infections there is little or no evidence of limitation of
the progress of the disease which may continue for years. Tuberculosis
is usually chronic. Leprosy in man is practically always so. Glanders
in horses is most commonly chronic; in mules and in man it is more apt
to be acute.

Many infections begin acutely and later change to the chronic type.
Syphilis in man is a good illustration.

The differences between acute and chronic infections are partly due
to the nature of the organism, partly to the number of organisms
introduced and the point of their introduction and partly to the
resistance of the animal infected.

An infectious disease is said to be _specific_ when one kind of
organism is responsible for its manifestations--as diphtheria due
to the _Corynebacterium diphtheriæ_, lockjaw due to _Clostridium
tetani_, Texas fever due to the _Piroplasma bigeminum_, etc. It is
_non-specific_ when it may be due to a variety of organisms, as
_enteritis_ (generally), _bronchopneumonia_, _wound infections_.

Henle, as early as 1840, stated certain principles that must be
established before a given organism can be accepted as the cause of a
specific disease. These were afterward restated by Koch, and have come
to be known as “Koch’s postulates.” They may be stated as follows:

1. The given organism must be found in all cases of the disease in
question.

2. No other organism must be found in all cases.

3. The organism must, when obtained in pure culture, reproduce the
disease in susceptible animals.

4. It must be recovered from such animals in pure culture and this
culture likewise reproduce the disease.

These postulates have not been fully met with reference to any disease,
but the principles embodied have been applied as far as possible
in all those infections which we recognize as specific, and whose
causative agent is accepted. In many diseases recognized as infectious
and contagious no organism has been found which is regarded as the
specific cause. In some of these the organism appears to be too small
to be seen with the highest powers of the microscope, hence they are
called “_ultramicroscopic_” organisms. Because these agents pass
through the finest bacterial filters, they are also frequently called
“_filterable_.” The term “_virus_” or “_filterable virus_” is likewise
applied to these “ultramicroscopic” and “filterable” agents.

The term _primary infection_ is sometimes applied to the first
manifestation of a disease, either specific or non-specific, while
_secondary_ refers to later developments. For example, a _secondary_
general infection may follow a _primary_ wound infection, or _primary_
lung tuberculosis be followed by _secondary_ generalized tuberculosis,
or _primary_ typhoid fever by a _secondary_ typhoid pneumonia. The
terms _primary_ and _secondary_ are also used where the body is
invaded by one kind of an organism and later on by another kind; thus
a _primary_ measles may be followed by _secondary_ infection of the
middle ear, or a _primary_ influenza may be followed by a _secondary_
pneumonia, or a _primary_ scarlet fever by a _secondary_ nephritis
(inflammation of the kidney). Where several organisms seem to be
associated simultaneously in causing the condition then the term _mixed
infection_ is used--in severe diphtheria, streptococci are commonly
associated with the _Corynebacterium diphtheriæ_. In many cases of
hog-cholera, mixed infections in the lungs and in the intestines are
common. Wound infections are usually _mixed_. _Auto-infection_ refers
to those conditions in which an organism commonly present in or on the
body in a latent or harmless condition gives rise to an infectious
process. If the _Bacterium coli_ normal to the intestine escapes into
the peritoneal cavity, or passes into the bladder, a severe peritonitis
or cystitis, respectively, is apt to result. “Boils” and “pimples”
are frequently autoinfections. Such infections are also spoken of as
_endogenous_ to distinguish them from those due to the entrance of
organisms from without--_exogenous_ infections. _Relapses_ are usually
instances of autoinfection.

Those types of _secondary infection_ where the infecting agent is
transferred from one disease focus to another or several other points
and sets up the infection there are sometimes called _metastases_. Such
are the transfer of tubercle bacilli from lung to intestine, spleen,
etc., the formation of abscesses in internal organs following a primary
surface abscess, the appearance of glanders nodules throughout various
organs following pulmonary glanders, etc.

The characteristic of a pathogenic microörganism which indicates
its ability to cause disease is called its _virulence_. If slightly
virulent, the effect is slight; if highly virulent, the effect is
severe, and may be fatal.

On the other hand, the characteristic of the host which indicates
its capacity for infection is called _susceptibility_. If slightly
susceptible, infection is slight, if highly susceptible, the infection
is severe.

Evidently the degree of infection is dependent in large measure on
the relation between the _virulence_ of the invading organism and the
_susceptibility_ of the host. High virulence and great susceptibility
mean a severe infection; low virulence and little susceptibility a
slight infection; while high virulence and little susceptibility or
low virulence and great susceptibility might mean a moderate infection
varying in either direction. Other factors influencing the degree of
infection are the number of organisms introduced, the point where they
are introduced and various conditions. These will be discussed in
another connection (Chapter XXV).

The study of pathogenic bacteriology includes the thorough study
of the individual organisms according to the methods already given
(Chapters XVIII-XXI) as an aid to diagnosis and subsequent treatment,
bacteriological or other, in a given disease. Of far greater
importance than the _treatment_, which in most infectious diseases
is not specific, is the _prevention_ and _ultimate eradication_ of
all infectious diseases. To accomplish these objects involves further
a study of the _conditions under which pathogenic organisms exist
outside the body_, _the paths of entrance into and elimination from
the body_ and those _agencies within the body itself_ which make it
_less susceptible to infection or overcome the infective agent after
its introduction_. That condition of the body itself which prevents any
manifestation of a virulent pathogenic organism after it has been once
introduced is spoken of as _immunity_ in the modern sense. Immunity is
thus the opposite of susceptibility and may exist in varying degrees.

That scientists are and have been for some years in possession of
sufficient knowledge to permit of the prevention and eradication
of most, if not all, of our infectious diseases can scarcely be
questioned. The practical application of this knowledge presents
many difficulties, the chief of which is the absence of a public
sufficiently enlightened to permit the expenditure of the necessary
funds. Time and educative effort alone can surmount this difficulty. It
will probably be years yet, but it will certainly be accomplished.




CHAPTER XXIII.

PATHOGENIC BACTERIA OUTSIDE THE BODY.


Pathogenic bacteria may exist outside the body of the host under a
variety of conditions as follows:

  I. In or on inanimate objects or material.
    (_a_) As true saprophytes.
    (_b_) As facultative saprophytes.
    (_c_) Though obligate parasites, they exist in a latent
        state.
  II. In or on other animals, or products from them:
    A. Susceptible to the disease.
      (_a_) Sick themselves.
        (As far as human beings are concerned these are
        mainly:
        1. Other human beings for most diseases.
        2. Rats for plague.
        3. Dogs for rabies.
        4. Horses for glanders.
        5. Cattle, swine, parrots for tuberculosis).
      (_b_) Recovered from illness.
      (_c_) Never sick but “carriers.”
    B. Not susceptible.
      (_d_) Accidental carriers.
      (_e_) Serving as necessary intermediate hosts for certain
        stages of the parasite--this applies to _protozoal_
        diseases only, as yet.


I.

(_a_) The bacilli of tetanus, malignant edema and the organisms of
“gas gangrene” are widely distributed. There is no evidence that their
entrance into the body is at all necessary for the continuation of
their life processes, or that one case of either of these diseases
ever has any connection with any other case; they are true saprophytes.
Manifestly it would be futile to attempt to prevent or eradicate such
diseases by attacking the organism in its natural habitat. _Clostridium
botulinum_, which causes a type of food poisoning in man, does not even
multiply in the body, but the disease symptoms are due to a soluble
toxin which is produced during its growth outside the body.

(_b_) Organisms like the bacterium of anthrax and the bacillus of
black-leg from their local occurrence seem to be distributed from
animals infected, though capable of a saprophytic existence outside the
body for years. These can no more be attacked during their saprophytic
existence than those just mentioned. Doubtless in warm seasons of the
year and in the tropics other organisms pathogenic to animals may live
and multiply in water or in damp soil where conditions are favorable,
just as the cholera organism in India, and occasionally the typhoid
bacillus in temperate climates do.

(_c_) Most pathogenic organisms, however, when they are thrown off
from the bodies of animals, remain quiescent, do not multiply, in fact
always tend to die out from lack of all that is implied in a “favorable
environment,” food, moisture, temperature, light, etc. Disinfection is
sometimes effective in this class of diseases in preventing new cases.


II. A.

(_a_) The most common infectious diseases of animals are transmitted
more or less directly from other animals of the same species. Human
beings get nearly all their diseases from other human beings who are
sick; horses, from other horses; cattle, from other cattle; swine,
from swine, etc. Occasionally transmission from one species to another
occurs. Tuberculosis of swine most frequently results from feeding
them milk of tuberculous cattle or from their eating the droppings of
such cattle. Human beings occasionally contract anthrax from wool,
hair and hides of animals dead of the disease or from postmortems
on such animals; glanders from horses; tuberculosis (in children)
from tuberculous milk; bubonic plague from rats; rabies practically
always from the bites of dogs and other rabid animals, etc. The
mode of limiting this class of diseases is evidently to isolate the
sick, disinfect their discharges and their _immediate_ surroundings,
sterilize such products as must be handled or used, kill lower animals
that are dangerous, and disinfect, bury properly, or destroy their
carcasses.

Classes of the sick that are especially dangerous for the spread of
disease are the mild cases and the undetected cases. These individuals
do not come under observation and hence not under control.

(_b_) This class of carriers offers a difficult problem in the
prevention of infectious diseases since they may continue to give off
the organisms indefinitely and thus infect others. Typhoid carriers
have been known to do so for fifty-five years. Cholera, diphtheria,
meningitis and other carriers are well known in human practice.
Carriers among animals have not been so frequently demonstrated,
but there is every reason for thinking that hog-cholera, distemper,
roup, influenza and other carriers are common. Carriers furnish the
explanation for many of the so-called “spontaneous” outbreaks of
disease among men and animals.

It is the general rule that those who are sick cease to carry the
organisms on recovery and it is the occasional ones who do not that are
the exceptions. In those diseases in which the organism is known it
can be determined by examination of the patient or his discharges how
long he continues to give off the causative agent. In those in which
the cause is unknown (in human beings, the commonest and most easily
transmitted diseases, scarlet fever, measles, German measles, mumps,
chicken-pox, small-pox, influenza), no such check is possible. It is
not known how long such individuals remain carriers. Hence isolation
and quarantine of such convalescents is based partly on experience
and partly on theory. It is highly probable that in the diseases just
mentioned transmission occurs in the _early stages only_, except
in small-pox and chicken-pox where the organism seems to be in the
pustules and transmission by means of material from these is possible,
though only by direct contact with it.

The fact that such individuals are _known to have had the disease_ is a
guide for control. The methods to be used are essentially the same as
for the sick, (_a_), though obviously such human carriers are much more
difficult to deal with since they are well.

(_c_) Another class of carriers is those who have never had the
disease. Such individuals are common and are very dangerous sources
of infection. Many of them have _associated with the sick or with
convalescents_ and these should always be suspected of harboring the
organisms. Their control differs in no way from that of class (_b_).
Unfortunately a history of such association is too often not available.
Modern transportation and modern social habits are largely responsible
for the nearly universal distribution of this type of carrier. Their
detection is probably the largest single problem in the prevention
of infectious diseases. A partial solution would be universal
bacteriological examination. In our present stage of progress this is
impossible and would not detect carriers of diseases of unknown cause.

The various classes of carriers just discussed are in a large part
responsible for the continued presence of the commoner diseases
throughout the country. The difficulties in control have been
mentioned. A complete solution of the problem is not yet obtained. The
army experience of the past few years in the control of infectious
diseases shows what may be done.

There is another class of carriers which might be called the “universal
carrier,” _i.e._, there are certain organisms which seem to be
constantly or almost constantly present in or on the human body. These
are _micrococci_, _streptococci_ and _pneumococci_, all _Gram positive_
organisms. They are ordinarily harmless parasites, but on occasion may
give rise to serious, even fatal, infection. Infected wounds, pimples,
boils, “common colds,” most “sore throats,” bronchitis, pneumonia are
pathological conditions that come in this class. Such infections are
usually autogenous. There is a constant interchange of these organisms
among individuals closely associated, so that all of a group usually
harbor the same type though no one individual can be called _the_
carrier. Whenever, for any reason, the resistance of an individual (see
Chaps. XXV et seq.) is lowered either locally or generally some of
these organisms are liable to gain a foothold and cause infection. It
sometimes happens that a strain of dangerous organisms may be developed
in an individual in this way which is passed around to others with its
virulence increased and thus cause an epidemic. Or, since all of the
group are living under the same conditions the resistance of all or
many of them may be lowered from the same general cause and an epidemic
result from the organism common to all (pneumonia after measles,
scarlet fever and influenza in camps). Protection of the individual is
chiefly a personal question, _i.e._, by keeping up the “normal healthy
tone” in all possible ways: The use of protective vaccines (Chap. XXX)
appears to be advisable in such instances (colds, pneumonia after
measles and influenza, inflammation of throat and middle ear following
scarlet fever and measles). Results obtained in this country during
the recent influenza epidemic have been conflicting but on the whole
appear to show that preventive vaccination against _pneumonia liable to
follow_ should be practiced.

It would seem that among groups of individuals where infection may be
expected the proper procedure would be to prepare autogenous vaccines
(Chapter XXX) from members of the group and vaccinate all with the
object of protecting them.


II. B.

(_d_) In this class come the “accidental carriers” like flies, fleas,
lice, bed-bugs, ticks, and other biting and blood-sucking insects,
vultures, buzzards, foxes, rats, and carrion-eating animals generally;
pet animals in the household, etc. Here the animals are not susceptible
to the given disease but become contaminated with the organisms
and then through defilement of the food or drink or contact with
individuals or with utensils pass the organisms on to the susceptible.
Some biting and blood-sucking insects transmit the organisms through
biting infected and non-infected animals successively. The spirilloses
and trypanosomiases seem to be transmitted in this way, though there
is evidence accumulating which may place these diseases in the next
class. Anthrax is considered in some instances to be transmitted by
flies and by vultures in the southern United States. Transmission
of typhoid, dysentery, cholera and other diseases by flies is well
established in man. Why not hog-cholera from farm to farm by flies,
English sparrows, pigeons feeding, or by turkey buzzards? Though this
would not be easy to prove, it seems reasonable.

Preventing contact of such animals with the discharges or with the
carcasses of those dead of the disease, destruction of insect carriers,
screening and prevention of fly breeding are obvious protective
measures.

(_e_) In this class come certain diseases for which particular
insects are necessary for the parasite in question, so that certain
stages in its life history may be passed therein. The surest means
for eradicating such diseases is the destruction of the insects
concerned. Up to the present no _bacterial_ disease is known in which
this condition exists, unless Rocky Mountain spotted fever and typhus
fever shall prove to be due to bacteria. Such diseases are all due to
protozoa. Among them are Texas fever, due to _Piroplasma bigeminum_
in this country which has been eradicated in entire districts by
destruction of the cattle tick (_Margaropus annulatus_).

Piroplasmoses in South Africa among cattle and horses, and in other
countries are transmitted in similar ways. Probably many of the
diseases due to spirochetes and trypanosomes are likewise transmitted
by _necessary_ insect intermediaries. In human medicine the eradication
of yellow fever from Panama and Cuba is due to successful warfare
against, a certain mosquito (_Stegomyia_). So the freeing of large
areas in different parts of the world from _malaria_ follows the
destruction of the mosquitoes. The prevention of typhus fever and
of trench fever by “delousing” methods is familiar from recent army
experience though for typhus this method has been practiced in Russia
for more than ten years to the author’s personal knowledge. The
campaign against disease in animals and man from insect sources must be
considered as still in its infancy. The full utilization of tropical
lands depends largely on the solution of this problem.




CHAPTER XXIV.

PATHS OF ENTRANCE OF PATHOGENIC ORGANISMS,

OR

CHANNELS OF INFECTION.


_A._ =The Skin.=--If the skin is healthy there is no opportunity for
bacteria to penetrate it. It is protected not only by the stratified
epithelium, but also in various animals, by coats of hair, wool,
feathers, etc. The secretion pressure of the healthy sweat and oil
glands acts as an effective bar even to motile bacteria. Nevertheless a
very slight injury only is sufficient to give normal surface parasites
and other pathogenics, accidentally or purposely brought in contact
with it, an opportunity for more rapid growth and even entrance
for general infection. Certain diseases due to higher fungi are
characteristically “skin diseases” and rarely become general--various
forms of favus, trichophyton infections, etc. A few disease organisms,
tetanus, malignant edema, usually get in through the skin; others,
black-leg, anthrax, quite commonly; and those diseases transmitted
by biting and blood-sucking insects, piroplasmoses, trypanosomiases,
spirilloses, scarcely in any other way. Defective secretion in the skin
glands from other causes, may permit lodgment and growth of bacteria
in them or in the hair follicles. “Pimples” and boils in man and local
abscesses occasionally in animals are illustrations. Sharp-edged and
freely bleeding wounds are less liable to be infected than contusions,
ragged wounds, burns, etc. The flowing blood washes out the wound and
the clotting seals it, while there is less material to be repaired
by the leukocytes and they are free to care for invading organisms
(phagocytosis). Pathogenic organisms, especially pus cocci, frequently
gain lodgment in the _milk glands_ and cause local (mastitis) or
general infection.

_B._ =Mucosæ directly continuous with the skin and lined with
stratified epithelium= are commonly well protected thereby and by the
secretions.

(_a_) The external auditory meatus is rarely the seat even of local
infection. The tympanic cavity is normally sterile, though it may
become infected by extension through the Eustachian tube from the
pharynx (_otitis media_).

(_b_) The conjunctiva is frequently the seat of localized, very rarely
the point of entrance for a generalized infection, except after severe
injury. Those diseases whose path of entrance is generally assumed to
be the respiratory tract (see “Lungs” below) might also be admitted
through the eye. Material containing such organisms might get on the
conjunctiva and be washed down through the lachrymal canal into the
nose. Experiment has shown that bacteria may pass in this way in a
few minutes. In case masks are worn to avoid infection from patients
suffering with these diseases, the eyes should therefore be protected
as well as the nose and mouth.

(_c_) The nasal cavity on account of its anatomical structure retains
pathogenic organisms which give rise to local infections more
frequently than other mucosæ of its character. These may extend from
here to middle ear, neighboring sinuses, or along the lymph spaces of
the olfactory nerve into the cranial cavity (meningitis). Acute coryza
(“colds” in man) is characteristic. Glanders, occasionally, is primary
in the nose, as is probably roup in chickens, leprosy in man. The
meningococcus and the virus of poliomyelitis pass from the nose into
the cranial cavity without local lesions in the former.

(_d_) The mouth cavity is ordinarily protected by its epithelium
and secretions, though the injured mucosa is a common source of
_actinomycosis_ infection, as well as thrush. In foot-and-mouth disease
no visible lesions seem necessary to permit the localization of the
unknown infective agent.

(_e_) The tonsils afford a ready point of entrance for ever-present
_micrococci_ and _streptococci_ whenever occasion offers (follicular
tonsillitis, “quinsy”), and articular rheumatism is not an uncommon
sequel. The diphtheria bacillus characteristically seeks these
structures for its development. Tubercle and anthrax organisms
occasionally enter here.

(_f_) The pharynx is the seat of localized infection as in
_micrococcal_, _streptococcal_ and diphtherial “sore throat” in human
beings, but both it and the esophagus are rarely infected in animals
except as the result of injury.

(_g_) The external genitalia are the usual points of entrance for
the venereal organisms in man (gonococcus, _Treponema pallidum_, and
Ducrey’s bacillus). The bacillus of contagious abortion and probably
the trypanosome of dourine are commonly introduced through these
channels in animals.

_C._ =Lungs.=--The varied types of pneumonia due to many different
organisms (tubercle, glanders, influenza, plague bacilli, pneumococcus,
streptococcus, micrococcus and many others) show how frequently these
organs are the seat of a localized infection, which may or may not be
general. Whether the lungs are the actual point of entrance in these
cases is a question which is much discussed at the present time,
particularly with reference to tuberculosis. The mucous secretion
of the respiratory tract tends to catch incoming bacteria and other
small particles and the ciliary movement along bronchial tubes and
trachea tends to carry such material out. “Foreign body pneumonia”
shows clinically, and many observers have shown experimentally that
microörganisms may reach the alveoli even though the exchange of
air between them and the bronchioles and larger bronchi takes place
ordinarily only by diffusion. The presence of carbon particles in the
walls of the alveoli in older animals and human beings and in those
that breathe dusty air for long periods indicates strongly, though it
does not prove absolutely, that these came in with inspired air. On the
other hand, experiment has shown that tubercle bacilli introduced into
the intestine may appear in the lungs and cause disease there and not
in the intestine. It is probably safe to assume that in those diseases
which are transmitted most readily through close association though
not necessarily actual contact, the commonest path is through the
respiratory tract, which may or may not show lesions (smallpox, scarlet
fever, measles, chicken-pox, whooping-cough, pneumonic plague in man,
lobar and bronchopneumonias and influenza in man and animals, some
cases of glanders and tuberculosis). On the other hand, the fact that
the _Bacterium typhosum_ and _Bacterium coli_ may cause pneumonia when
they evidently have reached the lung from the intestinal tract, and the
experimental evidence of lung tuberculosis above mentioned show that
this route cannot be excluded in inflammations of the lung.

_D._ =Alimentary Tract.=--The alimentary tract affords the ordinary
path of entrance for the causal microbes of many of the diseases of
animals and man, since they are carried into the body most commonly and
most abundantly in the food and drink.

(_a_) The stomach is rarely the seat of local infection, even in
ruminants, except as the result of trauma. The character of the
epithelium in the rumen, reticulum and omasum in ruminants, the
hydrochloric acid in the abomasum and in the stomachs of animals
generally are usually sufficient protection. Occasionally anthrax
“pustules” develop in the gastric mucosa. (The author saw nine such
pustules in a case of anthrax in a man.)

(_b_) The intestines are frequently the seat of localized infections,
as various “choleras” and “dysenteries” in men and many animals,
anthrax, tuberculosis, Johne’s disease. Here doubtless enter the
organisms causing “hemorrhagic septicemias” in many classes of animals,
and numerous others. These various organisms must have passed through
the stomach and the question at once arises, why did the HCl not
destroy them? It must be remembered that the acid is present only
during stomach digestion, and that liquids taken on an “empty stomach”
pass through rapidly and any organisms present are not subjected to
the action of the acid. Also spores generally resist the acid. Other
organisms may pass through the stomach within masses of undigested
food. The fact that digestion is going on in the stomach of ruminants
practically all the time may explain the relative freedom of _adult_
animals of this class from “choleras” and “dysenteries.”


MECHANISM OF ENTRANCE OF ORGANISMS.

In the preceding chapters statements have been made that “bacteria
enter” at various places or they “pass through” different mucous
membranes, skin, etc. Strictly speaking such statements are
incorrect--bacteria do not “enter” or “pass through” of themselves.
It is true that some of the intestinal organisms are motile, but most
of the bacteria which are pathogenic are non-motile. Even the motile
ones can not make their way against fluids secreted or excreted on free
surfaces. Bacteria cannot pass by diffusion through membranes since
they are finite particles and not in solution.

In the case of penetrating wounds bacteria may be carried mechanically
into the tissues, but this is exceptional in most infections. Also
after gaining lodgment they may gradually grow through by destroying
tissue as they grow, but this is a minor factor. Evidently, there
must be some mechanism by which they _are carried_ through. The known
mechanisms for this in the body are ameboid cells, especially the
phagocytes. It is most probable that these are the chief agents in
getting bacteria into the tissues through various free surfaces. The
phagocytes engulf bacteria, carry them into the tissues and either
destroy them, are destroyed by them, or may disgorge or excrete them
free in the tissues or in the blood.


DISSEMINATION OF ORGANISMS.

Dissemination of organisms within the tissues occurs either through
the lymph channels or the bloodvessels or both. If through the lymph
vessels only it is usually much more restricted in extent, or much more
slowly disseminated, while blood dissemination is characterized by the
number of organs involved simultaneously.


PATHS OF ELIMINATION OF PATHOGENIC MICRÖORGANISMS.

I. Directly from the point, of injury. This is true in infected
wounds open to the surface, skin glanders (farcy), black-leg,
surface anthrax, exanthemata in man and animals (scarlet fever (?),
measles (?), smallpox; hog erysipelas, foot-and-mouth disease):
also in case of disease of mucous membranes continuous with the
skin--from nasal discharges (glanders), saliva (foot-and-mouth
disease), material coughed or sneezed out (tuberculosis, influenza,
pneumonias), urethral and vaginal discharges (gonorrhea and syphilis
in man, contagious abortion and dourine in animals), intestinal
discharges (typhoid fever, “choleras,” “dysenteries,” anthrax,
tuberculosis, Johne’s disease). Material from nose, mouth and lungs
may be swallowed and the organisms passed out through the intestines.

II. Indirectly through the secretions and the excretions where the
internal organs are involved. The _saliva_ of rabid animals contains
the ultramicroscopic virus of rabies (the sympathetic ganglia
within the salivary glands, and pancreas also, are affected in this
disease as well as the cells of the central nervous system). The
_gall-bladder_ in man is known to harbor colon and typhoid bacilli,
as that of hog-cholera hogs does the virus of this disease. It may
harbor analogous organisms in other animals, though such knowledge
is scanty. The _kidneys_ have been shown experimentally to excrete
certain organisms introduced into the circulation within a few minutes
(micrococci, colon and typhoid bacilli, anthrax). Typhoid bacilli occur
in the urine of typhoid-fever patients in about 25 per cent. of all
cases and the urine of hogs with hog cholera is highly virulent. Most
observers are of the opinion, however, that under natural conditions
the kidneys do not excrete bacteria unless they themselves are infected.

The _milk_ both of tuberculous cattle and tuberculous women has been
shown to contain tubercle bacilli _even when the mammary glands are not
involved_. Doubtless such bacteria are carried through the walls of the
secreting tubules or of the smaller ducts by phagocytes and are then
set free in the milk.


SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS.

It is readily apparent that certain disease organisms tend to locate
themselves in definite regions and the question arises, Is this due
to any specific relationship between organism and tissue or not?
Diphtheria in man usually attacks the tonsils first, gonorrhea and
syphilis the external genitals, tuberculosis the lung, “choleras” the
small intestine, “dysenteries” the large intestine, influenza the
lungs. In these cases the explanation is probably that the points
attacked are the places where the organism is most commonly carried,
with no specific relationship, since all of these organisms (Asiatic
cholera excepted) also produce lesions in other parts of the body _when
they reach them_. On the other hand, the virus of hydrophobia attacks
nerve cells, leprosy frequently singles out nerves, glanders bacilli
introduced into the abdominal cavity of a young male guinea-pig cause
an inflammation of the testicle, malarial parasites and piroplasms
attack the red blood corpuscles, etc. In fact, most _pathogenic
protozoa_ are specific in their localization either in certain tissue
cells or in the blood or lymph. In these cases there is apparently a
real chemical relationship, as there is also between the _toxins_ of
bacteria and certain tissue cells (tetanus toxin and nerve cells).
Whether “chemotherapy” will ever profit from a knowledge of such
chemical relationships remains to be developed. It appears that a
search for these specific chemical substances with the object of
combining poisons with them so that the organisms might in this way be
destroyed, would be a profitable line of research.




CHAPTER XXV.

IMMUNITY.


Immunity, as has already been stated, implies such a condition of the
body that pathogenic organisms after they have been introduced are
incapable of manifesting themselves, and are unable to cause disease.
The word has come to have a more specific meaning than resistance in
many instances, in other cases the terms are used synonymously. It is
the opposite of susceptibility. The term must be understood always in a
relative sense, since no animal is immune to all pathogenic organisms,
and conceivably not entirely so to anyone, because there is no question
that a sufficient number of bacteria of any kind might be injected
into the circulation to kill an animal, even though it did it purely
mechanically.

Immunity may be considered with reference to a single individual or to
entire divisions of the organic world, with all grades between. Thus
plants are immune to the diseases affecting animals; invertebrates to
vertebrate diseases; cold-blooded animals to those of warm blood; man
is immune to most of the diseases affecting other mammals; the rat to
anthrax, which affects other rodents and most mammals; the well-known
race of Algerian sheep is likewise immune to anthrax while other sheep
are susceptible; the negro appears more resistant to yellow fever than
the white; some few individuals in a herd of hogs always escape an
epizoötic of hog cholera, etc.

Immunity within a given species is modified by a number of
factors--age, state of nutrition, extremes of heat or cold, fatigue,
excesses of any kind, in fact, anything which tends to lower the
“normal healthy tone” of an animal also tends to lower its resistance.
Children appear more susceptible to scarlet fever, measles,
whooping-cough, etc., than adults; young cattle more frequently have
black-leg than older ones (these apparently greater susceptibilities
may be due in part to the fact that most of the older individuals
have had the diseases when young and are immune for this reason).
Animals weakened by hunger or thirst succumb to infection more readily.
Frogs and chickens are immune to tetanus, but if the former be put
in water and warmed up to and kept, at about 37°, and the latter be
chilled for several hours in ice-water, then each may be infected.
Pneumonia frequently follows exposure to cold. The immune rat may
be given anthrax if first he is made to run in a “squirrel cage”
until exhausted. Alcoholics are far less resistant to infection than
temperate individuals. “Worry,” mental anguish, tend to predispose to
infection.

The following outlines summarize the different, classifications of
immunity so far as mammals are concerned for the purposes of discussion.

Immunity.

            {              {1. Inherited through
            {              {   the germ cell or cells.
            {              {                {(_a_) By having the
            {A. Congenital {2. Acquired     {      disease _in utero_.
 I. Natural {              {   _in utero_.  {(_b_) By absorption
            {              {                {      of immune
            {                               {      substances
            {B. Acquired by                 {      from the mother.
            {   having the disease.

 II. Artificial--acquired through human agency by:
               1. Introduction of the organism or its products.
               2. Introduction of the blood serum of an immune animal.

Immunity.

 I. Active--due to the introduction of the organism or due to the
    introduction of the products of the organism.
      A. Naturally by having the disease.
      B. Artificially.
        1. By introducing the organism:
                                    {1. Passage through another animal.
                                    {2. Drying.
          (_a_) Alive and virulent. {3. Growing at a higher temperature.
          (_b_) Alive and virulence {4. Heating the cultures.
                  reduced by        {5. Treating with chemicals.
          (_c_) Dead.               {6. Sensitizing.
                                    {7. Cultivation on artificial media.
        2. By introducing the products of the organism.

 II. Passive--due to the introduction of the blood serum of an actively
     immunized animal.

Immunity present in an animal and not due to human interference is to
be regarded as _natural_ immunity, while if brought about by man’s
effort it is considered _artificial_. Those cases of natural immunity
mentioned above which are common to divisions, classes, orders,
families, species or races of organisms and to those few individuals
where no special cause is discoverable, must be regarded as instances
of true _inheritance_ through the germ cell as other characteristics
are. All other kinds of immunity are _acquired_. Occasionally young are
born with every evidence that they have had a disease _in utero_ and
are thereafter as immune as though the attack had occurred after birth
(“small-pox babies,” “hog-cholera pigs”). Experiment has shown that
immune substances may pass from the blood of the mother to the fetus
_in utero_ and the young be immune for a time after birth (tetanus).
This is of no practical value as yet. It is a familiar fact that with
most infectious diseases recovery from one attack confers a more or
less lasting immunity, though there are marked exceptions.

=Active Immunity.=--By active immunity is meant that which is due
to the actual introduction of the organism, or in some cases of its
products. The term active is used because the body cells of the animal
immunized perform the real work of bringing about the immunity as
will be discussed later. In _passive_ immunity the blood serum of an
actively immunized animal is introduced into a second animal, which
thereupon becomes immune, though its cells are not concerned in the
process. The animal is _passive_, just as a test-tube, in which a
reaction takes place, plays no other part than that of a passive
container for the reagents.

In _active_ immunity the organism may be introduced in what is to
be considered a natural manner, as when an animal becomes infected,
has a disease, without human interference. Or the organism may be
purposely introduced to bring about the immunity. For certain purposes
the introduction of the products of the organism (toxins) is used to
bring about active immunity (preparation of diphtheria and tetanus
antitoxin from the horse). The method of producing active immunity by
the artificial introduction of the organism is called _vaccination_,
and a _vaccine_ must therefore contain the organism. _Vaccines_ for
_bacterial_ diseases are frequently called _bacterins_. The use of
the blood serum of an immunized animal to confer passive immunity on
a second animal is properly called _serum therapy_, and the serum so
used is spoken of as an _antiserum_, though the latter word is also
used to denote any serum containing any kind of an antibody (Chapters
XXVII-XXXI). In a few instances both the organism and an antiserum are
used to cause both active and passive immunity (_serum-simultaneous
method_ in immunizing against hog cholera).

In producing active immunity the organism may be introduced (_a_)
_alive and virulent_, but in very small doses, or in combination with
an immune serum, as just mentioned for hog cholera. The introduction
of the live virulent organism alone is done only experimentally as
yet, as it is obviously too dangerous to do in practice, except under
the strictest control (introduction of a _single tubercle_ bacillus,
followed by gradually increasing numbers--Barber and Webb). More
commonly the organisms are introduced (_b_) alive but with their
_virulence reduced_ (“attenuated”) in one of several ways: (1) By
passing the organism through another animal as is the case with
_smallpox vaccine_ derived from a calf or heifer. This method was
first introduced by Jenner in 1795 and was the first practical means
of preventing disease by _vaccination_. This word was used because
material was derived from a cow--Latin _vacca_. (2) By drying the
organism, as is done in the preparation of the vaccine for the _Pasteur
treatment of rabies_, where the spinal cords of rabbits are dried for
varying lengths of time--one to four days, Russian method, one to three
days, German method, longer in this country. (It is probable that the
passage of the “fixed virus” through the rabbit is as important in this
procedure as the drying, since it is doubtful if the “fixed virus” is
pathogenic for man.) It would be more correct to speak of this as a
_preventive vaccination against rabies_, since the latter is one of the
few diseases which is not amenable to _treatment_. The patient always
dies if the disease develops. (3) The organism may be attenuated by
growing at a temperature above the normal. This is the method used in
preparing _anthrax vaccine_ as done by Pasteur originally. (4) Instead
of growing at a higher temperature the culture may be heated in such
a way that it is not killed but merely weakened. _Black-leg_ vaccines
are made by this method. (5) Chemicals are sometimes added to attenuate
the organisms, as was formerly done in the preparation of black-leg
vaccine by Kruse’s method in Germany. The use of toxin-antitoxin
mixtures in immunizing against diphtheria and in the preparation
of diphtheria antitoxin from horses is an application of the same
principle, though here it is the _product_ of the organism and not the
organism whose action is weakened. (6) Within the past few years the
workers in the Pasteur Institute in Paris have been experimenting with
vaccines prepared by treating living virulent bacteria with antisera
(“sensitizing them”) so that they are no longer capable of causing
the disease when introduced, but do cause the production of an active
immunity. The method has been used with typhoid fever bacilli in man
and seems to be successful. It remains to be tried out further before
its worth is demonstrated (the procedure is more complicated and the
chance for infection apparently much greater than by the use of killed
cultures). The term _sero-bacterins_ is used by manufacturers in this
country to designate such bacterial vaccines. (7) Growing on artificial
culture media reduces the virulence of most organisms after a longer
or shorter time. This method has been tried with many organisms in the
laboratory, but is not now used in practice. The difficulties are that
the attenuation is very uncertain and that the organisms tend to regain
their virulence when introduced into the body.

In producing active immunity against many bacterial diseases the
organisms are introduced (_c_) dead. They are killed by heat or by
chemicals, or by using both methods (Chapter XXX).

When the products of an organism are introduced the resulting immunity
is against the products only and not against the organism. If the
organism itself is introduced there results an immunity against it
and in some cases also against the products, though the latter does
not necessarily follow. Hence the immunity may be _antibacterial_ or
_antitoxic_ or both.

Investigation as to the causes of immunity and the various methods by
which it is produced has not resulted in the discovery of specific
methods of treatment for as many diseases as was hoped for at one
time. Just at present progress in serum therapy appears to be at a
standstill, though vaccines are giving good results in many instances
not believed possible a few years ago. As a consequence workers in all
parts of the world are giving more and more attention to the search for
_specific chemical substances_, which will destroy invading parasites
and not injure the host (_chemotherapy_). Nevertheless, in the study
of immunity very much of value in the treatment and prevention of
disease has been learned. Also much knowledge which is of the greatest
use in other lines has been accumulated. Methods of _diagnosis_ of
great exactness have resulted, applicable in numerous diseases. Ways
of _detecting adulteration_ in foods, particularly foods from animal
sources, and of _differentiating proteins_ of varied origin, as well
as means of establishing _biological relationships_ and differences
among groups of animals through “immunity reactions” of blood serums
have followed from knowledge gained by application of the facts or the
methods of immunity research. Hence the study of “immunity problems”
has come to include much more than merely the study of those factors
which prevent the development of disease in an animal or result in
its spontaneous recovery. A proper understanding of the principles of
immunity necessitates a study of these various features and they will
be considered in the discussion to follow.




CHAPTER XXVI.

THEORIES OF IMMUNITY.


Pasteur and the bacteriologists of his time discovered that bacteria
cease to grow in artificial culture media after a time, because of
the exhaustion of the food material in some cases and because of the
injurious action of their own products in other instances. These facts
were brought forward to explain immunity shortly after bacteria were
shown to be the cause of certain diseases. Theories based on these
observations were called (1) “_Exhaustion Theory_” of _Pasteur_, and
(2) “_Noxious Retention Theory_” of _Chauveau_ respectively. The fact,
soon discovered, that virulent pathogenic bacteria are not uncommonly
present in perfectly healthy animals, and the later discovery that
immunity may be conferred by the injection of dead bacteria have led
to the abandonment of both these older ideas. The (3) “_Unfavorable
Environment_” theory of _Baumgartner_, _i.e._, bacteria do not grow
in the body and produce disease because their surroundings are not
suitable, in a sense covers the whole ground, though it is not true as
to the first part, as was pointed out above, and is of no value as a
working basis, since it offers no explanation as to _what the factors
are_ that constitute the “_unfavorable environment_.” Metchnikoff
brought forward a rational explanation of immunity with his (4)
“_Cellular or Phagocytosis Theory_.” As first propounded it based
immunity on the observed fact that certain white blood corpuscles,
_phagocytes_, engulf and destroy bacteria. Metchnikoff has since
elaborated the original theory to explain facts of later discovery.
Ehrlich soon after published his (5) “_Chemical or Side-chain Theory_”
which seeks to explain immunity on the basis of _chemical substances_
in the body which may in part destroy pathogenic organisms or in
part neutralize their products; or in some instances there may be
an absence of certain chemical substances in the body cells so that
bacteria or their products _cannot unite_ with the cells and hence can
do no damage.

[Illustration: PLATE VI

PAUL EHRLICH]

At the present time it is generally accepted, in this country at
least, that Ehrlich’s theory explains immunity in many diseases as
well as many of the phenomena related to immunity, and in other
diseases the phagocytes, frequently assisted by chemical substances,
are the chief factors. Specific instances are discussed in _Pathogenic
Bacteriologies_ which should be consulted. It is essential that the
student should be familiar with the basic ideas of the chemical
theory, not only from the standpoint of immunity, but also in order to
understand the principles of a number of valuable methods of diagnosis.

The chemical theory rests on three fundamental physiological
principles: (1) the response of cells to stimuli, in this connection
_specific chemical stimuli_, (2) the presence within cells of _specific
chemical groups_ which combine with chemical stimuli and thus enable
them to act on the cell, which groups Ehrlich has named _receptors_,
and (3) the “_over-production_” activity of cells as announced by
Weigert.

1. That cells respond to stimuli is fundamental in physiology. These
stimuli may be of many kinds as mechanical, electrical, light, thermal,
chemical, etc. The body possesses groups of cells specially developed
to _receive_ some of these stimuli--touch cells for mechanical stimuli,
retinal cells for light, temperature nerve endings for thermal,
olfactory and gustatory cells for certain chemical stimuli. _Response_
to chemical stimuli is well illustrated along the digestive tract. That
the chemical stimuli in digestion may be more or less specific is shown
by the observed differences in the enzymes of the pancreatic juice
dependent on the relative amounts of carbohydrates, fats, or proteins
in the food, the specific enzyme in each case being increased in the
juice with the increase of its corresponding foodstuff. The cells of
the body, or certain of them at least, seem to respond in a specific
way when substances are brought into direct contact with them, that is,
without having been subjected to digestion in the alimentary tract,
but injected directly into the blood or lymph stream. Cells may be
affected by stimuli in one of three ways: if the stimulus is too weak,
there is no effect (in reality there is no “stimulus” acting); if the
stimulus is too strong, the cell is injured, or may be destroyed; if
the stimulus is of proper amount then it excites the cell to increased
activity, and in the case of _specific chemical stimuli_ the increased
activity, as mentioned for the pancreas, shows itself in an _increased
production of whatever is called forth by the chemical stimulus_. In
the case of many organic chemicals, the substances produced by the
cells under their direct stimulation are markedly specific for the
particular substance introduced.

2. Since chemical action always implies at least two bodies to react,
Ehrlich assumes that in every cell which is affected by a chemical
stimulus there must therefore be a chemical group to unite with this
stimulus. He further states that there must be as many different
kinds of these groups as there are different kinds of chemicals which
stimulate the cell. Since these groups are present in the body cells to
_take up_ different kinds of chemical substances, Ehrlich calls them
_receptors_. Since these groups must be small as compared with the cell
as a whole, and must be more or less on the surface and unite readily
with chemical substances he further speaks of them as “side-chains”
after the analogy of compounds of the aromatic series especially. The
term _receptors_ is now generally used. As was stated above, the effect
of _specific chemical stimuli_ is to cause the production of _more of
the particular substance_ for which it is specific and in the class
of bodies under discussion, the _particular product is these cell
receptors_ with which the chemical may unite.

3. Weigert first called attention to the practically constant
phenomenon that cells ordinarily respond by doing more of a particular
response than is actually called for by the stimulus, that there is
always an “overproduction” of activity. In the case of chemical stimuli
this means an _increased production of the specific substance_ over and
above the amount actually needed.

The student will better understand this theory if he recalls his
fundamental physiology. Living substance is characterized, among other
things, by irritability which is instability. It is in a constant,
state of unstable equilibrium. Whenever the equilibrium becomes
permanently stable the substance is dead. It is also continually
attempting to restore disturbances in its equilibrium. Whenever a
chemical substance unites with a chemical substance in the cell, a
receptor, the latter is, so far as the cell is concerned, _thrown out
of function_ for that cell. The chemical equilibrium of the latter
is upset. It attempts to restore this and does so by making a _new_
receptor to take the place of the one thrown out of function. If this
process is continued, _i.e._, if the new receptor is similarly “used
up” and others similarly formed are also, then the cell will prepare
a supply of these and even an excess, according to Weigert’s theory.
Whenever a cell accumulates an excess of products the normal result
is that it excretes them from its own substance into the surrounding
lymph, whence they reach the blood stream to be either carried to
the true excretory organs, utilized by other cells or remain for a
longer or shorter time in the blood. Hence the excess of receptors
is _excreted from the cell that forms them_ and they become _free_
in the blood. These free receptors are termed _antibodies_. _They
are receptors_ but instead of being retained in the cell are _free
in solution in the blood_. One function of the free receptor, the
antibody, is _always to unite with the chemical substance which caused
it to be formed_. _It may have additional functions._ The chemical
substance which caused the excess formation of receptors, antibodies,
is termed an _antigen_ for that particular kind of antibody.

To recapitulate, Ehrlich’s theory postulates _specific chemical
stimuli_, which react with _specific chemical substances in the body
cells, named receptors_, and that these _receptors_, according to
Weigert, are _produced in excess_ and hence are excreted from the
cell and become _free receptors_ in the blood and lymph. These _free
receptors_ are the various kinds of _antibodies_, the kind depending
on the nature of the stimulus, antigen, the substance introduced. Any
substance which when introduced into the body causes the formation of
an antibody of any kind whatsoever is called an _antigen_,[23] _i.e._,
anti (body) former.

The foregoing discussion explains Ehrlich’s theory of immunity.
According to this theory the _manner of formation of all antibodies_ is
the same. The _kind of antibody_ and the _manner of its action_ will
differ with the _different kinds of antigens_ used.

The succeeding chapters discuss some of the kinds of antibodies, the
theory of their action and some practical applications. It must be
borne in mind throughout the study of these, as has been stated, that
_every antibody has the property of uniting with its antigen whether it
has any property in addition or not_.

Just what antibodies are chemically has not been determined because
no one has as yet succeeded in isolating them chemically pure. To the
author they appear to be enzymes.

Antigens were considered by Ehrlich to be proteins or to be related to
proteins. Most workers since Ehrlich have held similar views. Dr. Carl
Warden of the University of Michigan has been doing much work in recent
years in which he is attempting to show that the antigens are not
proteins but are fats or fatty acids. Mr. E. E. H. Boyer, in his work
(not yet published) in the author’s laboratory for the degree of Ph.D.,
received in June, 1920, succeeded in producing various antibodies from
_Bacterium coli_ antigens. In these antigens he could detect only fatty
acids or salts of fatty acids. If the work of these men is confirmed,
it will open up a most interesting and extremely important field in
immunity and in preventive medicine. It is not apparent that the nature
of the antigen would affect Ehrlich’s theory of the formation of
antibodies.

The author has no doubt that eventually the formation of antibodies and
the reactions between them and their antigens will be explained on the
basis of physical-chemical laws, but this probably awaits the discovery
of their nature.




CHAPTER XXVII.

RECEPTORS OF THE FIRST ORDER.


ANTITOXINS--ANTIENZYMES.

The general characteristics of toxins have been described (Chapter
XII). It has been stated that they are more or less specific in
their action on cells. In order to affect a cell it is evident that
a toxin must enter into chemical combination with it. This implies
that the toxin molecule possesses a chemical group which can combine
with a receptor of the cell. This group is called the _haptophore_ or
combining group. The toxic or injurious portion of the toxin molecule
is likewise spoken of as the _toxophore_ group. When a toxin is
introduced into the body its _haptophore_ group combines with suitable
_receptors_ in different cells of the body. If not too much of the
toxin is given, instead of injuring, it acts as a chemical stimulus
to the cell in the manner already described. The cell in response
produces more of the specific thing, which in this instance is more
receptors which can combine with the toxin, _i.e._, with its haptophore
group. If the stimulus is kept up, more and more of these receptors
are produced until an excess for the cell accumulates, which excess is
excreted from the individual cell and becomes free in the blood. These
free receptors have, of course, the capacity to combine with toxin
through its haptophore group. When the toxin is combined with these
free receptors, it cannot combine with any other receptors, _e.g._,
those in another cell and hence cannot injure another cell. These free
receptors constitute, in this case, _antitoxin_, so-called because
they can combine with toxin and hence neutralize it. Antitoxins are
specific--that is, an antitoxin which will combine with the toxin of
_Clostridium tetani_ will not combine with that of _Corynebacterium
diphtheriæ_ or of _Clostridium botulinum_, or of any other toxin,
vegetable or animal.

When a toxin is kept in solution for some time or when it is heated
above a certain temperature (different for each toxin) it loses its
poisonous character. It may be shown, however, that it is still capable
of uniting with antitoxin, and preventing the latter from uniting with
a fresh toxin. This confirms the hypothesis that a toxin molecule has
at least two groups: a combining or _haptophore_, and a poisoning or
_toxophore_ group. A toxin which has lost its poisonous property, its
toxophore group, is spoken of as a _toxoid_. The theory of antitoxin
formation is further supported by the fact that the proper introduction
of _toxoid_, the _haptophore_ group, and hence the real stimulus, can
cause the production of _antitoxin_ to a certain extent at least.

The close relationship between toxins and enzymes has already been
pointed out. This is still further illustrated by the fact that when
enzymes are properly introduced into the tissues of an animal there
is formed in the animal an _antienzyme_ specific for the enzyme in
question which can prevent its action. The structure of enzymes,
as composed of a _haptophore_, or uniting, and a _zymophore_ or
_digesting_ (or other activity) group, is similar to that of toxins,
and _enzymoids_ or enzymes which can combine with the substance acted
on but not affect it further, have been demonstrated.

These free cell receptors, antitoxins or antienzymes, which are
produced in the body by the proper introduction of toxins or enzymes,
respectively, have the function of _combining_ with these bodies
_but no other action_. As was pointed out above, this is sufficient
to neutralize the toxin or enzyme and prevent any injurious effect
since they can unite with nothing else. Since these receptors are the
simplest type which has been studied as yet, they are spoken of by
Ehrlich as _receptors of the first order_. Other antibodies which are
likewise free receptors of the first, order and have the function of
combining only have been prepared and will be referred to in their
proper connection. They are mainly of theoretical interest.

Ehrlich did a large part of his work on toxins and antitoxins
with _ricin_, the toxin of the castor-oil bean, _abrin_, from the
jequirity bean, _robin_ from the locust tree, and with the toxins and
antitoxins for diphtheria and tetanus. Antitoxins have been prepared
experimentally for a large number of both animal and vegetable poisons,
including a number for bacterial toxins. The only ones which, as yet,
are of much practical importance are _antivenin_ for snake poison, (not
a true toxin, however, see p. 275), _antipollenin_ (supposed to be
for the toxin of hay fever) and the antitoxins for the true bacterial
toxins of _Corynebacterium diphtheriæ_ and _Clostridium tetani_.

The method of preparing antitoxins is essentially the same in all
cases, though differing in minor details. For commercial purposes
large animals are selected, usually horses, so that the yield of
serum may be large. The animals must, of course, be vigorous, free
from all infectious disease. The first injection given is either a
relatively small amount of a solution of toxin or of a mixture of
toxin and antitoxin. The animal shows more or less reaction, increased
temperature, pulse and respiration and frequently an edema at the
point of injection, unless this is made intravenously. After several
days to a week or more, when the animal has recovered from the first
injection, a second stronger dose is given, usually with less reaction.
Increasingly large doses are given at proper intervals until the animal
may take several hundred times the amount which would have been fatal
if given at first. The process of immunizing a horse for diphtheria or
tetanus toxin usually takes several months. Variations in time and in
yield of antitoxin are individual and not predictable in any given case.

After several injections a few hundred cubic centimeters of blood
are withdrawn from the jugular vein and serum from this is tested
for the amount of antitoxin it contains. When the amount is found
sufficiently large (250 “units” at least for diphtheria per cc.)[24]
then the maximum amount of blood is collected from the jugular with
sterile trocar and cannula. The serum from this blood with the addition
of an antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes
“antidiphtheritic serum” or “antitetanic serum,” etc. All sera which
are put on the market must conform to definite standards of strength
expressed in “units” as determined by the U. S. Hygienic Laboratory.
In reality a “unit” of diphtheria antitoxin in the United States is
an amount equivalent to 1 cc. of a given solution of a _standard_
diphtheria _antitoxin_ which is kept at the above-mentioned laboratory.
This statement, of course, gives no definite idea as to the amount
of antitoxin actually in a “unit.” Specifically stated, a “unit” of
antitoxin contains approximately the amount which would protect a 250
gram guinea-pig from 100 minimum lethal doses of diphtheria toxin, or
protect 100 guinea-pigs weighing 250 grams each from one minimum lethal
dose each. The minimum lethal dose (M. L. D.) of diphtheria toxin is
the least amount that will kill a guinea-pig of the size mentioned
within four days. Since toxins on standing change into toxoids to a
great extent, the amount, of antitoxin in a “unit,” though protecting
against 100 M. L. D., in reality would protect against about 200 M. L.
D. of toxin containing no toxoid.

The official unit for tetanus antitoxin is somewhat different, since it
is standardized against a _standard toxin_ which is likewise kept at
the Hygienic Laboratory. The unit is defined as “ten times the amount
of antitoxin necessary to protect a 350 g. guinea-pig for 96 hours
against the _standard test dose_” of the standard toxin. The standard
test dose is 100 M. L. D. of toxin for a 350 g. guinea-pig. To express
it another way, one could say that a “unit” of tetanus antitoxin
would protect one thousand 350 g. guinea-pigs from 1 M. L. D. each of
standard tetanus toxin.

Various methods have been devised for increasing the amount of
antitoxin in 1 cc. of solution by precipitating out portions of the
blood-serum proteins and at the same time concentrating the antitoxin
in smaller volume. It is not considered necessary in a work of this
character to enter into these details nor to discuss the process of
standardizing antitoxin so that the exact amount of “units” per cc. may
be known.




CHAPTER XXVIII.

RECEPTORS OF THE SECOND ORDER.


AGGLUTININS.

Charrin and Rogers appear to have been the first (1889) to observe the
clumping together of bacteria (_Pseudomonas pyocyanea_) when mixed
with the blood serum of an animal immunized against them. Gruber and
Durham (1896) first used the term “agglutination” in this connection
and called the substance in the blood-serum “agglutinin.” Widal (1896)
showed the importance of the reaction for diagnosis by testing the
blood serum of an infected person against a known culture (typhoid
fever).

It is now a well-known phenomenon that the proper injection of cells
of any kind foreign to a given animal will lead to the accumulation in
the animal’s blood of substances which will cause a clumping together
of the cells used when suspended in a suitable liquid. The cells settle
out of such suspension much more rapidly than they would otherwise
do. This clumping is spoken of as “agglutination” and the substances
produced in the animal are called “agglutinins.” If blood cells are
injected then “hemagglutinins” result: if bacterial cells “bacterial
agglutinins” for the particular organism used as “glanders agglutinin”
for _Pfeifferella mallei_, “abortion agglutinin” for _Bacterium
abortus_, “typhoid agglutinin” for _Bacterium typhosum_, etc.

The phenomenon may be observed either under the microscope or in small
test-tubes, that is, either _microscopically_ or _macroscopically_.

In this case the cells introduced, or more properly, some substances
within the cells, act as stimuli to the body cells of the animal
injected to cause them to produce more of the specific cell receptors
which respond to the stimulus. The substance within the introduced
cell which acts as a stimulus (_antigen_) to the body cells is called
an “_agglutinogen_.” That “agglutinogen” is present in the cell has
been shown by injecting animals experimentally with extracts of cells
(bacterial and other cells) and the blood serum of the animal injected
showed the presence of agglutinin for the given cell. It will be
noticed that the receptors which become the free agglutinins have at
least _two functions_, hence at least _two chemical groups_. They must
combine with the foreign cells and also bring about their clumping
together, their agglutination. Hence it can be stated technically that
an agglutinin possesses a _haptophore group_ and an _agglutinating
group_.

It is probable that the agglutination, the clumping, is a secondary
phenomenon depending on the presence of certain salts and that the
agglutinin acts on its antigen as an enzyme, possibly a “splitting”
enzyme. This is analogous to what occurs in the curdling of milk
by rennet and in the coagulation of blood. This probability is
substantiated by the fact that suspensions of bacteria may be
“agglutinated” by appropriate strengths of various acids.

The formation of agglutinin in the body for different bacteria does
not as yet appear to be of any special significance in protecting the
animal from the organism, since the bacteria are not killed, even
though they are rendered non-motile, if of the class provided with
flagella, and are clumped together. The fact that such bodies are
formed, however, is of decided value in the diagnosis of disease, and
also in the identification of unknown bacteria.

In many bacterial diseases, agglutinins for the particular organism
are present in the blood serum of the affected animal. Consequently
if the blood serum of the animal be mixed with a suspension of the
organism supposed to be the cause of the disease and the latter be
agglutinated, one is justified in considering it the causative agent,
provided certain necessary conditions are fulfilled. In the first place
it must be remembered that the blood of normal animals frequently
contains agglutinins (“normal agglutinins”) for many different
bacteria when mixed with them in full strength. Hence the serum must
always be diluted with physiological salt solution (0.85 per cent.).
Further, closely related bacteria may be agglutinated to some extent by
the same serum. It is evident that if they are closely related, their
protoplasm must contain some substances of the same kind to account for
this relationship. Since some of these substances may be agglutinogens,
their introduction into the animal body will give rise to agglutinins
for the related cells, as well as for the cell introduced. The
agglutinins for the cell introduced “chief agglutinins,” will be
formed in larger quantity, since a given bacterial cell must contain
more of its own agglutinogen than that of any other cell. By _diluting
the blood serum_ from the animal to be tested the agglutinins for the
related organisms (so-called “coagglutinins” or “partial agglutinins”)
will become so much diminished as to show no action, while the
agglutinin for the specific organism is still present in an amount
sufficient to cause its clumping. _Agglutinins are specific for their
particular agglutinogens_, but since a given blood serum may contain
many agglutinins, the _serum’s specificity for a given bacterium_
can be determined only by diluting it until this bacterium alone
is agglutinated. Hence the necessity of diluting the unknown serum
in varying amounts when testing against several known bacteria to
determine for which it is specific, _i.e._, which is the cause of the
disease in the animal.

The agglutinins in the serum may be removed from it by treating it with
a suspension of the cells for which agglutinins are present. If the
“chief” cell is used all the agglutinins will be absorbed. If related
cells are used, only the agglutinins for this particular kind are
removed. These “absorption tests” furnish another means of determining
specificity of serum, or rather of determining the “chief agglutinin”
present.

Just as an unidentified _disease_ in an animal may be determined by
testing its serum as above described against _known_ kinds of bacteria,
so _unknown bacteria_ isolated from an animal, from water, etc., may
be identified by testing them against the _blood sera_ of different
animals, each of which has been properly inoculated with a different
kind of _known bacteria_. If the unknown organism is agglutinated
by the blood of one of the animals in high dilution, and not by the
others, evidently the bacterium is the same as that with which the
animal has been inoculated, or _immunized_, as is usually stated. This
method of identifying cultures of bacteria is of wide application,
but is used practically only in those cases where other methods of
identification are not readily applied, and especially where other
methods are _not sufficient_ as in the “intestinal group” of organisms
in human practice.

The diagnosis of disease in an animal by testing its serum is also a
valuable and much used procedure. This is the method of the “Widal” or
“Gruber-Widal” test for typhoid fever in man and is used in veterinary
practice in testing for glanders, contagious abortion, etc. In some
cases a dilution of the serum of from 20 to 50 times is sufficient for
diagnosis (Malta fever), in most cases, however, 50 times is the lowest
limit. Evidently the greater the dilution, that is, the higher the
“titer,” the more specific is the reaction.


PRECIPITINS.

Since agglutinins act on bacteria, probably through the presence of
substances within the bacterial cell, it is reasonable to expect that
if these substances be dissolved out of the cell, there would be some
reaction between their (colloidal) solution and the same serum. As
a matter of fact Kraus (1897) showed that broth cultures freed from
bacteria by porcelain filters do show a precipitate when mixed with
the serum of an animal immunized against the particular bacterium and
that the reaction is specific under proper conditions of dilution.
It was not long after Kraus’s work until the experiments were tried
of “immunizing” an animal not against a bacterium or its filtered
culture, but against (colloidal) solutions of proteins, such as white
of egg, casein of milk, proteins of meat and of blood serum, vegetable
proteins, etc. It was ascertained that in all these cases the animal’s
serum contains a substance which causes a _precipitate_ with solutions
of the protein used for immunization. The number of such precipitating
serums that have been made experimentally is very large and it appears
that protein from any source when properly introduced into the blood
or tissues of an animal will cause the formation of a precipitating
substance for its solutions. This substance is known, technically as a
“_precipitin_.” The protein used as antigen to stimulate its formation,
or some part of the protein molecule (haptophore group), which acts
as stimulus to the cell is spoken of as a “precipitinogen,” both
terms after the analogy of “agglutinin” and “agglutinogen.” In fact
the specific precipitation and agglutination are strictly analogous
phenomena. Precipitins act on proteins in (colloidal) _solution_ and
cause them to settle out, agglutinins act on substances within cells
which cells are in _suspension_ in a fluid and cause the cells to
settle out. Ehrlich’s theory of the formation of precipitins is similar
to that of agglutinins, and need not be repeated. Substitute the
corresponding words in the theory of formation of agglutinins as above
given and the theory applies.

The precipitin reaction has not found much practical use in
bacteriology largely because the “agglutination test” takes its place
as simpler of performance and just as accurate. The reaction is,
however, generally applicable to filtrates of bacterial cultures and
could be used if needed. The so-called “mallease” reaction in glanders
is an instance.

Precipitins find their greatest usefulness in legal medicine and
in food adulteration work. As was noted above, if animals, rabbits
for example, are immunized with the blood of another animal (human
beings) precipitins are developed which are specific for the injected
blood with proper dilution. This forms an extremely valuable means
of determining the _kind of blood_ present in a given spot shown by
chemical and spectroscopic tests to be blood and has been adopted as
a legal test in countries where such rules of procedure are applied.
Similarly the test has been used to identify the different kinds of
meat in sausage, and different kinds of milk in a mixture. An extract
of the sausage is made and tested against the serum of an animal
previously treated with extract of horse meat, or hog meat, or beef,
etc., the specific precipitate occurring with the specific serum. Such
reactions have been obtained where the protein to be tested was diluted
100,000 times and more. Biological relationships and differences have
been detected by the reaction. Human immune serum shows no reaction
with the blood of any animals except to a slight extent with that of
various monkeys, most with the higher, very slight with the lower Old
World and scarcely any with New World monkeys.

It is a fact of theoretical interest mainly that if agglutinins
and precipitins themselves be injected into an animal they will
act as _antigens_ and cause the formation of _antiagglutinins_ or
_antiprecipitins_, which are therefore receptors of the first order
since they simply combine with these immune bodies to neutralize their
action, have only a combining or haptophore group. Also if agglutinins
or precipitins be heated to the proper temperature they may retain
their combining power but cause no agglutination or precipitation,
_i.e._, they are converted into agglutinoid or precipitinoid
respectively after the analogy of toxin and toxoid.

Precipitins like agglutinins possess at least two groups--a combining
or _haptophore_ group and a _precipitating_ (sometimes called
zymophore) group. Hence they are somewhat more complex than antitoxins
or antienzymes which have a combining group only. For this reason
Ehrlich classes agglutinins and precipitins as _receptors of the second
order_.




CHAPTER XXIX.

RECEPTORS OF THE THIRD ORDER.


CYTOLYSINS.

Before Koch definitely proved bacteria capable of causing disease
several physiologists had noted that the red corpuscles of certain
animals were destroyed by the blood of other animals (Creite, 1869,
Landois, 1875), and Traube and Gescheidel had shown that freshly drawn
blood destroys bacteria (1874). It was not until about ten years
afterward that this action of the blood began to be investigated in
connection with the subject of immunity. Von Fodor (1885) showed that
saprophytic bacteria injected into the blood are rapidly destroyed.
Flügge and his pupils, especially Nuttall in combating Metchnikoff’s
theory of phagocytosis, announced in 1883, studied the action of the
blood on bacteria and showed its destructive effect (1885-57). Nuttall
also showed that the blood lost this power if heated to 56°. Buchner
(1889) gave the name “alexin” (from the Greek “to ward off”) to the
destroying substance and showed that the substance was present in
the _blood serum_ as well as in the whole blood, and that when the
serum lost its power to dissolve, this could be restored by adding
fresh blood. Pfeiffer (1894) showed that the destructive power of the
blood of animals immunized against bacteria (cholera and typhoid) was
markedly specific for the bacteria used. He introduced a mixture of
the blood and the bacteria into the abdominal cavity of the immunized
animal or of a normal one of the same species and noted the rapid
solution of the bacteria by withdrawing portions of the peritoneal
fluid and examining them (“Pfeiffer’s phenomenon”). Belfanti and
Carbone and especially Bordet (1898) showed the specific dissolving
action of the serum of one animal on the blood corpuscles of another
animal with which it had been injected. Since this time the phenomenon
has been observed with a great variety of cells other than red blood
corpuscles and bacteria--leukocytes, spermatozoa, cells from liver,
kidney, brain, epithelia, etc., protozoa, and many vegetable cells.

It is therefore a well-established fact that the proper injection of
an animal with almost any cell foreign to it will lead to the blood of
the animal injected acquiring the power to injure or destroy cells of
the same kind as those introduced. The destroying power of the blood
has been variously called its “cytotoxic” or “cytolytic” power, though
the terms are not strictly synonymous since “cytotoxic” means “cell
poisoning” or “injuring,” while “cytolytic” means “cell dissolving.”
The latter term is the one generally used and there is said to be
present in the blood a specific “cytolysin.” The term is a general one
and a given cytolysin is named from the cell which is dissolved, as a
_bacteriolysin_, a _hemolysin_ (red-corpuscle-lysin), _epitheliolysin_,
_nephrolysin_ (for kidney cells), etc. If the cell is _killed_ but
not _dissolved_ the suffix “cidin” or “toxin” is frequently used as
“bacteriocidin,” “spermotoxin,” “neurotoxin,” etc.

The use of the term “cytolysin” is also not strictly correct, though
convenient, for the process is more complex than if _one substance
only_ were employed. As was stated above, the immune serum loses its
power to dissolve the cell if it is heated to 55° to 56° for half an
hour, it is _inactivated_. But if there be added to the heated or
inactivated serum a small amount of _normal serum_ (which contains only
a very little cytolytic substance, so that it has no dissolving power
when so diluted) the mixture again becomes cytolytic. It is evident
then that in cytolysis there are _two distinct substances_ involved,
one which is _present in all serum, normal or immune_, and the other
_present only in the immune cytolytic_ serum. This may be more apparent
if the facts are arranged in the following form:

  I. Immune serum dissolves cells in high dilution.

  II. Heated immune serum does not dissolve cells.

  III. Normal serum in high dilution does not dissolve cells.

  II. + III., _i.e._, Heated immune serum plus diluted normal serum
  dissolves cells.

Therefore, there is something in heated immune serum necessary for
cell dissolving and something different in diluted normal serum which
is necessary. This latter something is present in unheated immune
serum also, and is destroyed by heat. Experiment has shown that it is
the substance present in all serum both normal and immune that is the
true dissolving body, while the immune substance serves to unite this
body to the cell to be destroyed, _i.e._, to the antigen. Since the
immune body has therefore _two uniting groups_, one for the dissolving
substance and one for the cell to be dissolved, Ehrlich calls it the
“_amboceptor_.” He also uses the word “_complement_” to denote the
dissolving substance, giving the idea that it completes the action of
dissolving after it has been united to the cell by the amboceptor, thus
replacing Buchner’s older term “alexin” for the same dissolving body.


AMBOCEPTORS.

The theory of formation of amboceptors is similar to that for the
formation of the other types of antibodies. The cell introduced
contains some substance, which acts as a chemical stimulus to some of
the body cells provided with proper receptors so that more of these
special receptors are produced, and eventually in excess so that they
become free in the blood and constitute the free amboceptors. It will
be noticed that these free receptors differ from either of the two
kinds already described in that they have _two uniting groups_, one for
the antigen (cell introduced) named _cytophil-haptophore_, the other
for the complement, _complementophil haptophore_. Hence amboceptors
are spoken of as _receptors of the third order_. They have no other
function than that of this double combining power. The action which
results is due to the third body--the complement. It will be readily
seen that complement must possess at least two groups, a combining or
_haptophore group_ which unites with the amboceptor, and an active
group which is usually called the _zymophore_ or _toxophore_ group.
Complements thus resemble either toxins, where the specific cell
(antigen) is injured or killed, or enzymes, in case the cell is
likewise dissolved. This action again shows the close relation between
toxins and enzymes. Complement may lose its active group in the same
way that toxin does and becomes then _complementoid_. Complement is
readily destroyed in blood or serum by heating it to 55° to 56° for
half an hour, and is also destroyed spontaneously when serum stands for
a day or two, less rapidly at low temperature than at room temperature.

Amboceptors appear to be _specific_ in the same sense that agglutinins
are. That is, if a given cell is used to immunize an animal, the
animal’s blood will contain amboceptors for the cell used and also for
others closely related to it. Immunization with spermatozoa or with
epithelial or liver cells gives rise to amboceptors for these cells
and also for red blood corpuscles and other body cells. A typhoid
bactericidal serum has also some dissolving effect on colon bacilli,
etc. Hence a given serum may contain a chief amboceptor and a variety
of “coamboceptors,” or one amboceptor made up of a number of “partial
amboceptors” each specific for its own antigen (“amboceptorogen”).
Amboceptors may combine with other substances than antigen and
complement, as is shown by their union with lecithin and other
“lipoids,” though these substances seem capable of acting as complement
in causing solution of blood corpuscles.


COMPLEMENTS.

As to whether complements are numerous, as Ehrlich claims, or there
is only one complement, according to Buchner and others, need not be
discussed here. In the practical applications given later as means of
diagnosis it is apparent that all the complement or complements are
capable of uniting with at least two kinds of amboceptors.

If complement be injected into an animal it may act as an antigen and
give rise to the formation of _anticomplement_ which may combine with
it and prevent its action and is consequently analogous to antitoxin.
If amboceptors as antigen are injected into an animal there will be
formed by the animal’s cells _antiamboceptors_. As one would expect,
there are two kinds of antiamboceptors, one for each of its combining
groups, since it has been stated that it is always the combining group
of any given antigen that acts as the cell stimulus. Hence we may have
an “anticytophil amboceptor” or an “anticomplementophil amboceptor.”
These antiamboceptors and the anticomplements are analogous to
antitoxin, antiagglutinin, etc., and hence are receptors of the first
order.


ANTISNAKE VENOMS.

A practical use of antiamboceptors is in antisnake venoms. Snake
poisons appear to contain only _amboceptors_ for different cells of the
body. In the most deadly the amboceptor is specific for nerve cells
(cobra), in others for red corpuscles, or for endothelial cells of the
bloodvessels (rattlesnake). The complement is furnished by the blood
of the individual bitten, that is, in a sense the individual poisons
himself, since he furnishes the destroying element. The antisera
contain antiamboceptors which unite with the amboceptor introduced and
prevent it joining to cells and thus binding the complement to the
cells and destroying them. With this exception these antibodies are
chiefly of theoretical interest.


FAILURE OF CYTOLYTIC SERUMS.

The discovery of the possibility of producing a strongly bactericidal
serum in the manner above described aroused the hope that such sera
would prove of great value in passive immunization and serum treatment
of bacterial diseases. Unfortunately such expectations have not been
realized and no serum of this character of much practical importance
has been developed as yet (with the possible exception of Flexner’s
antimeningococcus serum in human practice. What hog cholera serum is
remains to be discovered).

The reasons for the failure of such sera are not entirely clear.
The following are some that have been offered: (1) Amboceptors do
not appear to be present in very large amount so that relatively
large injections of blood are necessary, which is not without risk
in itself. (2) Since the complement is furnished by the blood of the
animal to be treated, there may not be enough of this to unite with a
sufficient quantity of amboceptor to destroy all the bacteria present,
hence the disease is continued by those that escape. (3) Or the
complement may not be of the right kind to unite with the amboceptor
introduced, since this is derived from the blood of a _heterologous_
(“other kind”) species. In hog-cholera serum, if it is bactericidal,
this difficulty is removed by using blood of a _homologous_ (“same
kind”) animal. Hence Ehrlich suggested the use of apes for preparing
bactericidal sera for human beings. The good results which have been
reported in the treatment of human beings with the serum of persons
convalescing from the same disease indicate that this lack of proper
complement for the given amboceptor is probably a chief factor in the
failure of sera from lower animals. (4) The bacteria may be localized
in tissues (lymph glands), within cavities (cranial, peritoneal), in
hollow organs (alimentary tract), etc., so that it is not possible to
get at them with sufficient serum to destroy all. This difficulty is
obviated by injecting directly into the spinal canal when Flexner’s
antimeningococcus serum is used. (5) Even if the bacteria are dissolved
it does not necessarily follow that their _endotoxins_ are destroyed.
These may be merely liberated and add to the danger of the patient,
though this does not appear to be a valid objection. (6) Complement
which is present in such a large excess of amboceptor may just as
well unite with amboceptor which is not united to the bacteria to be
destroyed as with that which is, and hence be actually prevented from
dissolving the bacteria. Therefore it is difficult to standardize the
serum to get a proper amount of amboceptor for the complement present.


COMPLEMENT-FIXATION TEST.

Although little practical use has been made of bactericidal sera,
the discovery of receptors of this class and the peculiar relations
between the antigen, amboceptor and complement have resulted in
developing one of the most delicate and accurate methods for the
diagnosis of disease and for the recognition of small amounts of
specific protein that is in use today.

This method is usually spoken of as the “complement-fixation” or the
“complement-deviation test” (“Wassermann test” in syphilis) and is
applicable in a great variety of microbial diseases, but it is of
practical importance in those diseases only where other methods are
uncertain--syphilis in man, concealed glanders in horses, contagious
abortion in cattle, etc. A better name would be the “Unknown Amboceptor
Test” since it is the amboceptor that is searched for in the test by
making use of its power to “fix” complement.

The principle is the same in all cases. The method depends, as
indicated above, on the ability of complement to combine with at least
two amboceptor-antigen systems, and on the further fact that if the
combination with one amboceptor-antigen system is once formed, it
does not dissociate so as to liberate the complement for union with
the second amboceptor-antigen system. If an animal is infected with a
microörganism and a part of its defensive action consists in destroying
the organisms in its blood or lymph, then it follows from the above
discussion of cytolysins that there will be developed in the blood of
the animal amboceptor specific for the organism in question. If the
presence of this _specific amboceptor_ can be detected, the conclusion
is warranted that the organism for which it is specific is the cause of
the disease. Consequently what is sought in the “complement-fixation
test” is a _specific amboceptor_. In carrying out the test, blood
serum from the suspected animal is collected, heated at 56° for half
an hour to destroy any complement it contains and mixed in definite
proportions with the specific antigen and with complement. The
antigen is an extract of a diseased organ (syphilitic fetal liver,
in syphilis), a suspension of the known bacteria, or an extract of
these bacteria. Complement is usually derived from a guinea-pig,
since the serum of this animal is higher in complement than that
of most animals. The blood of the gray rat contains practically as
much. If the specific amboceptor is present, that is, if the animal
is infected with the suspected disease, the complement will unite
with the antigen-amboceptor system and be “fixed,” that is, be no
longer capable of uniting with any other amboceptor-antigen system. No
chemical or physical means of telling whether this union has occurred
or not, except as given below, has been discovered as yet, though
doubtless will be by physico-chemical tests, nor can the combination
be seen. Hence an “indicator,” as is so frequently used in chemistry,
is put into the mixture of antigen-amboceptor-complement after it has
been allowed to stand in the incubator for one-half to one hour to
permit the union to become complete. The “indicator” used is a mixture
of sheep’s corpuscles and the heated (“inactivated”) blood serum of
a rabbit which has been injected with sheep’s blood corpuscles and
therefore contains a _hemolytic amboceptor specific_ for the corpuscles
which is capable also of uniting with complement. The indicator is
put into the first mixture and the whole is again incubated and
examined. If the mixture is _clear_ and _colorless_ with a _deposit
of red corpuscles_ at the bottom, that would mean that the complement
had been bound to the first complex, since it was not free to unite
with the second sheep’s corpuscles (antigen)--rabbit serum (hemolytic
amboceptor) complex--and destroy the corpuscles. Hence if the
complement is bound in the first instance, the _specific amboceptor_
for the first antigen must have been present in the blood, that is, the
animal was infected with the organism in question. Such a reaction is
called a “positive” test.

On the other hand, if the final solution is _clear_ but of a _red_
color, that would mean that complement must have united with the
corpuscles--hemolytic amboceptor system--and destroyed the corpuscles
in order to cause the _clear red_ solution of hemoglobin. If complement
united with this system it could not have united with the first system,
hence there was no _specific amboceptor_ there to bind it; no specific
amboceptor in the animal’s blood, means no infection. Hence a _red
solution_ is a “negative test.”

The scheme for the test may be outlined as follows:

  Antigen         +  Patient’s Serum, heated  +  Complement
  (specific for      (unknown amboceptor)        (derived from
  the amboceptor                                 guinea pig’s serum)
  sought)

Incubate one-half hour in a water bath or one hour in an incubator.

Then add the indicator which is

  Antigen                 +  Amboceptor
  (red blood corpuscles)     (for corpuscles, serum of
                             a rabbit immunized against
                             the red corpuscles)

Incubate as above.

In practice all the different ingredients must be accurately tested,
standardized and used in exact quantities, and tests must also be run
as controls with a known normal blood of an animal of the same species
as the one examined and with a known positive blood.

  It should be stated that in one variety of the Complement-Fixation
  Test, namely, the “Wassermann Test for Syphilis” in human beings,
  an antigen is used which is not derived from the specific organism
  (_Treponema pallidum_) which causes the disease nor even from
  syphilitic tissue. It has been determined that alcohol will extract
  from certain tissues, _human or animal_, substances which _act
  specifically_ in combining with the syphilitic amboceptor present
  in the blood. Alcoholic extracts of beef heart are most commonly
  used. Details of this test may be learned in the advanced course in
  Immunity and Serum Therapy.

The complement-fixation test might be applied to the determination
of unknown bacteria, using the unknown culture as antigen and trying
it with the sera of different animals immunized against a variety
of organisms, some one of which might prove to furnish _specific
amboceptor_ for the unknown organism and hence indicate what it is. The
test used in this way has not been shown to be a practical necessity
and hence is rarely employed. It has been used, however, to detect
traces of unknown proteins, particularly blood-serum proteins, in
medico-legal cases in exactly the above outlined manner and is very
delicate and accurate.




CHAPTER XXX.

PHAGOCYTOSIS--OPSONINS.


It has been mentioned that Metchnikoff, in a publication in 1883,
attempted to explain immunity on a purely cellular basis. It has
been known since Haeckel’s first observation in 1858 that certain of
the white corpuscles do engulf solid particles that may get into the
body, and among them bacteria. Metchnikoff at first thought that this
engulfing and subsequent intracellular digestion of the microörganisms
were sufficient to protect the body from infection. The later
discoveries (discussed in considering Ehrlich’s theory of immunity)
of substances present in the blood serum and even in the blood plasma
which either destroy the bacteria or neutralize their action have
caused Metchnikoff to modify his theory to a great extent. He admitted
the presence of these substances, though giving them other names,
but ascribed their formation to the phagocytes or to the same organs
which form the leukocytes--lymphoid tissue generally, bone marrow. It
is not within the province of this work to attempt to reconcile these
theories, but it may be well to point out that Ehrlich’s theory is one
of _chemical substances_ and that the _origin_ of these substances is
not an _essential_ part of the theory, so that the two theories, except
in some minor details, are not necessarily mutually exclusive.

[Illustration: PLATE V

ELIE METCHNIKOFF]

Sir A. E. Wright and Douglas, in 1903, showed that even in those
instances where immunity depends on phagocytosis, as it certainly does
in many cases, the phagocytes are more or less inactive unless they are
aided by chemical substances present in the blood. These substances
_act on the bacteria, not on the leukocytes_, and change them in such
a way that they are more readily taken up by the phagocytes. Wright
proposed for these bodies the name _opsonin_, derived from a Greek
word signifying “to prepare a meal for.” Neufeld and Rimpau at about
the same time (1904), in studying immune sera, observed substances of
similar action in these sera and proposed the name _bacteriotropins_,
or bacteriotropic substances. There is scarcely a doubt that the two
names are applied to identical substances and that Wright’s name
_opsonin_ should have preference.

The chemical nature of opsonins is not certainly determined, but they
appear to be a distinct class of antibodies and to possess two groups,
a combining or haptophore and a preparing or opsonic group and hence
are similar to antibodies of Ehrlich’s second order--agglutinins and
precipitins. Wright also showed that opsonins are just as specific as
agglutinins are--that is, a micrococcus opsonin prepares micrococci
only for phagocytosis and not streptococci or any other bacteria.

Wright showed that opsonins for many bacteria are present in normal
serum and that in the serum of an animal which has been immunized
against such bacteria the opsonins are _increased_ in amount. Also
that in a person infected with certain bacteria the opsonins are
either increased or diminished, depending on whether the progress of
the infection is favorable or unfavorable. The _opsonic power_ of a
serum normal or otherwise is determined by mixing an emulsion of fresh
leukocytes in normal saline solution with a suspension of the bacteria
and with the serum to be tested. The leukocytes must first be washed in
several changes of normal salt solution to free them from any adherent
plasma or serum. The mixture is incubated for about fifteen minutes
and then slides are made, stained with a good differential blood
stain, Wright’s or other, and the average number of bacteria taken up
by at least fifty phagocytes taken in order in a field is determined
by counting under the microscope. The number so obtained Wright calls
the _phagocytic index_ of the serum tested. The phagocytic index of a
given serum divided by the phagocytic index of a normal serum gives
the _opsonic index_ of the serum tested. Assuming the normal opsonic
index to be 1, Wright asserts that in healthy individuals the range
should be not more than from 0.8 to 1.2, and that an index below 0.8
may show a great susceptibility for the organism tested, infection with
the given organism if derived from the individual, or improper dosage
in case attempts have been made to immunize by using killed cultures,
vaccines, of the organism.

On the occasion of the author’s visit to Wright’s clinic (1911) he
stated that he used the determination of the _opsonic index_ chiefly as
a _guide to the dosage_ in the use of vaccines.

Most workers outside the Wright school have failed to recognize any
essential value of determinations of the opsonic index in the use
of vaccines. Some of the reasons for this are as follows: The limit
of error in phagocytic counts may be as great as 50 per cent. in
different series of fifty, hence several hundred must be counted, which
adds greatly to the tediousness and time involved; the variation in
apparently healthy individuals is frequently great, hence the “normal”
is too uncertain; finally the opsonic index and the clinical course of
the disease do not by any means run parallel. Undoubtedly the method
has decided value in the hands of an individual who makes opsonic
determinations his chief work, as Wright’s assistants do, but it can
scarcely be maintained at the present time that such determinations
are necessary in vaccine therapy. Nevertheless that opsonins actually
exist and that they play an essential part in phagocytosis, and hence
in immunity, is now generally recognized.


BACTERIAL VACCINES.

Whether determinations of opsonic index are useful or not is largely
a matter of individual opinion, but there is scarcely room to doubt
that Wright has conferred a lasting benefit by his revival of the
use of _dead cultures of bacteria_, _bacterial vaccines_, both for
protective inoculation and for treatment. It is perhaps better to use
the older terms “vaccination” and “vaccine” (though the cow, _vacca_,
is not concerned) than to use Wright’s term “opsonic method” in this
connection, bearing in mind that the idea of a vaccine is that it
contains the _causative organism_ of the infection as indicated on p.
253.

As early as 1880 Touissant proposed the use of dead cultures of
bacteria to produce immunity. But because injections of such cultures
were so frequently followed by abscess formation, doubtless due to
the _high temperatures_ used to kill the bacteria, the method was
abandoned. Further, Pasteur and the French school persistently denied
the possibility of success with such a procedure, and some of them
even maintain this attitude at the present time. The successes of
Wright and the English school which are being repeated so generally
wherever properly attempted, leave no doubt in the unprejudiced of the
very great value of the method and have unquestionably opened a most
promising field both for preventive inoculation and for treatment in
many infectious diseases. That the practice is no more universally
applicable than are immune serums and that it has been and is still
being grossly overexploited is undoubted.

The use of a vaccine is based on two fundamental principles. The first
of these is that the cell introduced must not be in a condition to
cause serious injury to the animal by its multiplication and consequent
elaboration of injurious substances. The second is that, on the other
hand, it must contain antigens in such condition that they will act as
stimuli to the body cells to produce the necessary antibodies, whether
these be opsonins, bactericidal substances, or anti-endotoxins. In the
introduction of living organisms there is always more or less risk
of the organism not being sufficiently attenuated and hence of the
possibility of its producing too severe an infection. In using killed
cultures, great care must be exercised in destroying the organisms,
_so that the antigens are not at the same time rendered inactive_.
Hence in the preparation of bacterial vaccines by Wright’s method the
_temperature and the length of time used to kill the bacteria are most
important factors_. This method is in general to grow the organisms
on an agar medium, rub off the culture and emulsify in sterile normal
salt solution (0.85 per cent. NaCl). The number of bacteria per cc.
is determined by staining a slide made from a small volume of the
emulsion mixed with an equal volume of human blood drawn from the
finger and counting the relative number of bacteria and of red blood
corpuscles. Since the corpuscles are normally 5,000,000 per c.mm.,
a simple calculation gives the number of bacteria. The emulsion of
bacteria is then diluted so that a certain number of millions shall
be contained in each cc., “standardized” as it is called, then heated
to the proper temperature for the necessary time and it is ready for
use. A preservative, as 0.5 per cent. phenol, tricresol, etc., is added
unless the vaccine is to be used up at once. The amounts of culture,
salt solution, etc., vary with the purpose for which the vaccine is to
be used, from one or two agar slant cultures and a few cc. of solution,
when a single animal is to be treated, to bulk agar cultures and liters
of solution as in preparing antityphoid vaccine on a large scale.

Agar surface cultures are used so that there will be as little
admixture of foreign protein as possible (see Anaphylaxis, p. 289 _et
seq._). Normal saline solution is isotonic with the body cells and
hence is employed as the vehicle.

=Lipovaccines.=--The suspension of bacteria in neutral oil was first
used by Le Moignac and Pinoy who gave the name “lipovaccines” (λιπος
= fat) to them. It was claimed that the reaction following injection
of these vaccines was less severe than with saline vaccines in many
instances; also, that the bacteria were much more slowly absorbed. For
these two reasons it was hoped that much larger numbers of bacteria
could be injected at one dose and one injection would suffice instead
of three or more as ordinarily used. The technique of preparation,
standardization and killing of the organisms has not as yet been
sufficiently well established to warrant the general substitution of
lipovaccines for ordinary saline suspensions.

Vaccines are either “_autogenous_” or “_stock_.” An “autogenous”
vaccine is a vaccine that is made from bacteria derived from the
individual or animal which it is desired to vaccinate and contains
not only the particular organism but the particular strain of that
organism which is responsible for the lesion. Stock vaccines are
made up from organisms like the infective agent in a given case but
derived from some other person or animal or from laboratory cultures.
Commercial vaccines are “stock” vaccines and are usually “polyvalent”
or even “mixed.” A “polyvalent” vaccine contains several strains of the
infective agent and a “mixed” contains several different organisms.

Stock vaccines have shown their value when used as preventive
inoculations, notably so in typhoid fever in man, anthrax and black-leg
in cattle. The author is strongly of the opinion, not only from the
extended literature on the subject, but also from his own experience
in animal, and especially in human cases, that stock vaccines are
much inferior and much more uncertain in their action when used in
the _treatment_ of an infection, than are autogenous vaccines. This
applies particularly to those instances in which _pneumococci_,
_streptococci_, _micrococci_, and _colon bacilli_ are the causative
agents but to others as well. The following are some of the reasons for
this opinion: The above organisms are notoriously extremely variable in
their virulence. While there is no necessarily close connection between
virulence and antigenic property, yet since virulence is so variable,
it is rational to assume that antigenic property is also extremely
variable. Individuals vary just as much in susceptibility and hence in
reactive power, and generally speaking, an individual will react better
in the production of antibodies to a stimulus to which he has been more
or less subjected, _i.e._, to organisms derived from his own body.

In the preparation of a vaccine great care must be used in heating so
that the organisms are killed, but the _antigens_ are not destroyed.
Many of the enzymes present in bacteria, especially the proteolytic
ones, are not any more sensitive to heat than are the antigens, hence
are not destroyed entirely. Therefore a vaccine kept in stock for a
long time gradually has some of its antigens destroyed by the uninjured
enzymes present with them, and so loses in potency. Therefore in
treating a given infection it is well to make up a vaccine from the
lesion, use three or four doses and if more are necessary make up a new
vaccine.

If the above statements are borne in mind and vaccines are made and
administered accordingly, the author is well satisfied that much better
results will be secured.

In accordance with the theory on which the use of vaccines is based,
_i.e._, that they stimulate the body cells to produce immunizing
antibodies, it is clear that they are especially suitable in those
infections in which the process is _localized_ and should not be of
much value in _general_ infections. In the latter case the cells of
the body are stimulated to produce antibodies by the circulating
organisms, probably nearly to their limit, hence the introduction of
more of the same organisms, capable of stimulating though dead, is apt
to overtax the cells and do more harm than good. It is not possible to
tell accurately when this limit is reached, but the clinical symptoms
are a guide. If vaccines are used at all in general infections they
should be given in the early stages and in small doses at first with
close watch as to the effect. In localized infections only the cells in
the immediate neighborhood are much stimulated, hence the introduction
of a vaccine calls to their aid cells in the body generally, and much
more of the resulting antibodies are carried to the lesion in question.
Manifestly surgical procedures such as incision, drainage, washing
away of dead and necrotic tissue with normal saline solution, not
necessarily antiseptics, will aid the antibodies in their action and
are to be recommended where indicated.

In the practical application of any remedy the _dosage_ is most
important. Unfortunately there is no accurate method of determining
this with a vaccine. Wright recommended determining the number of
the organisms per cc. as before mentioned, and his method or some
modification of it is still in general use. From what was said with
regard to variation, both in organisms and in individuals, it can
be seen that the number of organisms is at least only a very rough
guide. This is further illustrated by the doses of micrococcus
(staphylococcus) vaccines recommended by different writers, which
vary from 50,000,000 to 2,000,000,000 per cc. The author is decidedly
of the opinion that _there is no way of determining the dosage of
a vaccine in the treatment of any given case except by the result
of the first dose_. Hence it is his practice to make vaccines of a
particular organism of the same approximate strength, and to give a
dose of a measured portion of a cubic centimeter, judging the amount
by what the individual or animal can apparently withstand, without too
violent a reaction. If there is no local or general reaction or if it
is very slight and there is no effect on the lesion, the dose is too
small. If there is a violent local reaction with severe constitutional
symptoms clinically, and the lesion appears worse, the dose is too
large. There should be some local reaction and some general, but not
enough to cause more than a slight disturbance, easy to judge in human
subjects, more difficult in animals. In cases suitable for vaccine
treatment no _serious_ results should follow from a properly prepared
vaccine, though the process of healing may be delayed temporarily.
Wright claimed, and many have substantiated him, that always following
a vaccination there is a period when the resistance of the animal is
diminished. This is called the “negative phase,” and Wright considered
this to last as long as the opsonic index remained low, and when this
latter began to increase the stage of the “positive” or favorable phase
was reached. As has been stated the opsonic index is pretty generally
regarded as of doubtful value, though the existence of a period of
lowered resistance is theoretically probable from the fact that
antibodies already present in the blood will be partially used up in
uniting with the vaccine introduced and that the body cells are called
upon suddenly to do an extra amount of work and it takes them some time
to adapt themselves. This time, the “negative phase,” is much better
determined by the clinical symptoms, general and especially local. It
is good practice to begin with a dose relatively small. The result
of this is an indication of the proper dosage and also prepares the
patient for a larger one. The second dose should follow the first not
sooner than three or four days, and should be five to seven days if the
first reaction is severe. These directions are not very definite, but
clinical experience to date justifies them. It is worth the time and
money to one who wishes to use vaccines to learn from one who has had
experience both in making and administering them, and then to remember
that each patient is an individual case, for the use of vaccines as
well as for any other kind of treatment.


AGGRESSIN.

Opsonins have been shown to be specific substances which act on
bacteria in such a way as to render them more readily taken up by the
leukocytes. By analogy one might expect to find bacteria secreting
specific substances which would tend to counteract the destructive
action of the phagocytes and bactericidal substances. Bail and his
co-workers claim to have demonstrated such substances in exudates in
certain diseases and have given the distinctive name “aggressins” to
them. By injecting an animal with “aggressins,” antiaggressins are
produced which counteract their effects and thus enable the bacteria to
be destroyed. The existence of such specific bodies is not generally
accepted as proved. The prevailing idea is that bacteria protect
themselves in any given case by the various toxic substances that they
produce, and that “aggressins” as a special class of substances are not
formed.




CHAPTER XXXI.

ANAPHYLAXIS.


Dallera, in 1874, and a number of physiologists of that period,
observed peculiar skin eruptions following the transfusion of blood,
that is, the introduction of foreign proteins. In the years subsequent
to the introduction of diphtheria antitoxin (1890) characteristic
“serum rashes” were not infrequently reported, sometimes accompanied by
more or less severe general symptoms and occasionally death--a train of
phenomena to which the name “serum sickness” was later applied, since
it was shown that it was the horse serum (foreign protein) that was
the cause, and not the antitoxin itself. In 1898 Richet and Hericourt
noticed that some of the dogs which they were attempting to immunize
against toxic eel serum not only were not immunized but suffered even
more severely after the second injection. They obtained similar results
with an extract of mussels which contain a toxin. Richet gave the name
“anaphylaxis” (“no protection”) to this phenomenon to distinguish it
from immunity or prophylaxis (protection).

All the above-mentioned observations led to no special investigations
as to their cause. In 1903, Arthus noticed abscess formation,
necrosis and sloughing following several injections of horse serum
in immediately adjacent parts of the skin in rabbits (“Arthus’
phenomenon”). Theobald Smith, in 1904, observed the death of
guinea-pigs following properly spaced injections of horse serum. This
subject was investigated by Otto and by Rosenau and Anderson in this
country and about the same time von Pirquet and Schick were making a
study of serum rashes mentioned above. The publications of these men
led to a widespread study of the subject of injections of foreign
proteins. It is now a well-established fact that the injection into an
animal of a foreign protein--vegetable, animal or bacterial, simple or
complex--followed by a second injection after a proper length of time
leads to a series of symptoms indicating poisoning, which may be so
severe as to cause the death of the animal. Richet’s term “anaphylaxis”
has been applied to the condition of the animal following the first
injection and indicates that it is in a condition of supersensitiveness
for the protein in question. The animal is said to be “sensitized”
for that protein.[25] The sensitization is specific since an animal
injected with white of chicken’s egg reacts to a second injection of
chicken’s egg only and not pigeon’s egg or blood serum or any other
protein. The specific poisonous substance causing the symptoms has
been called “anaphylotoxin” though what it is, is still a matter of
investigation. It is evident that some sort of an antibody results from
the first protein injected and that it is specific for its own antigen.

A period of ten days is usually the minimum time that must elapse
between the first and second injections in guinea-pigs in order that a
reaction may result, though a large primary dose requires much longer.
If the second injection is made within less time no effect follows,
and after three or more injections at intervals of about one week the
animal fails to react at all, it has become “immune” to the protein.
Furthermore, after an animal has been sensitized by one injection and
has reacted to a second, then, if it does not die from the reaction, it
fails to react to subsequent injections. In this latter case it is said
to be “antianaphylactic.”

It must be remembered that proteins do not normally get into the
circulation except by way of the alimentary tract. Here all proteins
that are absorbed are first broken down to their constituent
amino-acids, absorbed as such and these are built up into the proteins
characteristic of the animal’s blood. Hence when protein as such gets
into the blood it is a foreign substance to be disposed of. The blood
contains proteolytic enzymes for certain proteins normally. It is
also true that the body cells possess the property of digesting the
proteins of the blood and building them up again into those which are
characteristic of the cell. Hence it appears rational to assume that
the foreign proteins act as stimuli to certain cells to produce more
of the enzymes necessary to decompose them, so that they may be either
built up into cell structure or eliminated as waste. If in this process
of splitting up of protein a poison were produced, then the phenomena
of “anaphylaxis” could be better understood. As a matter of fact
Vaughan and his co-workers have shown that by artificially splitting
up proteins from many different sources--animal, vegetable, pathogenic
and saprophytic bacteria--a poison _is produced_ which appears to be
the same in all cases and which causes the symptoms characteristic of
anaphylaxis. On the basis of these facts it is seen that anaphylaxis
is simply another variety of immunity. The _specific antibody_ in
this case is an _enzyme_ which decomposes the protein instead of
precipitating it. The enzyme must be specific for the protein since
these differ in constitution. Vaughan even goes so far as to say that
the poison is really the central ring common to all proteins and that
they differ only in the lateral groups or side chains attached to this
central nucleus. The action of the enzyme in this connection would be
to split off the side chains, and since these are the specific parts of
the protein, the enzyme must be specific for each protein. The pepsin
of the gastric juice and the trypsin of the pancreas split the native
proteins only to peptones. As is well known, these when injected in
sufficient quantity give rise to poisonous symptoms, and will also
give rise to anaphylaxis under properly spaced injections. They do not
poison normally because they are split by the intestinal erepsin to
amino-acids and absorbed as such. Whether Vaughan’s theory of protein
structure is the true one or not remains to be demonstrated. It is
not essential to the theory of anaphylaxis above outlined, _i.e._,
a phenomenon due to the action of specific _antibodies_ which are
enzymes. On physiological grounds this appears the most rational of the
few explanations of anaphylaxis that have been offered and was taught
by the author before he had read Vaughan’s theory along the same lines.

On the basis of the author’s theory the phenomena of protein immunity
and antianaphylaxis may be explained in the following way which the
author has not seen presented. The enzymes necessary to decompose the
injected protein are present in certain cells and are formed in larger
amount by those cells to meet the increased demand due to injection
of an excess of protein. They are retained in the cell for a time at
least. If a second dose of protein is given before the enzymes are
excreted from the cells as waste, this is digested within the cells in
the normal manner. If a third dose is given, the cells adapt themselves
to this increased intracellular digestion and it thus becomes normal
to them. Hence the _immunity_ is due to this increased intracellular
digestion.

On the other hand, if the second injection is delayed long enough, then
the _excess_ enzyme, but not all, is excreted from the cells and meets
the second dose of protein in the blood stream and rapidly decomposes
it there, so that more or less intoxication from the split products
results. This uses up _excess_ enzyme, hence subsequent injections are
not digested in the blood stream but within the cells as before. So
that “antianaphylaxis” is dependent on the exhaustion of the excess
enzyme in the blood, and the condition is _fundamentally_ the same as
protein immunity, _i.e._, due to _intracellular_ digestion in each case.

As has been indicated “serum sickness” and sudden death following
serum injections are probably due to a sensitization of the individual
to the proteins of the horse in some unknown way. Probably hay fever
urticarial rashes and idiosyncrasies following the ingestion of certain
foods--strawberries, eggs, oysters, etc., are anaphylactic phenomena.

In medical practice the reaction is used as a means of diagnosis in
certain diseases, such as the tuberculin test in tuberculosis, the
mallein test in glanders. The individual or animal with tuberculosis
becomes sensitized to certain proteins of the tubercle bacillus and
when these proteins in the form of tuberculin are introduced into the
body a reaction results, local or general, according to the method of
introduction. The practical facts in connection with the tuberculin
test are also in harmony with the author’s theory of anaphylaxis
as above outlined. Milder cases of tuberculosis give more vigorous
reactions because the intracellular enzymes are not used up rapidly
enough since the products of the bacillus are secreted slowly in such
cases. Hence excess of enzyme is free in the blood and the injection of
the tuberculin meets it there and a vigorous reaction results. In old,
far-advanced cases, no reaction occurs, because the enzymes are all
used in decomposing the large amount of tuberculous protein constantly
present in the blood. The fact that an animal which has once reacted
fails to do so until several months afterward likewise depends on the
fact that the _excess_ enzyme is used in the reaction and time must
elapse for a further excess to accumulate.

The anaphylactic reaction has been made use of in the identification of
various types of proteins and is of very great value since the reaction
is so delicate, particularly when guinea-pigs are used as test animals.
Wells has detected the 0.000,001 g. of protein by this test. It is
evident that the test is applicable in medico-legal cases and in food
examination and has been so used.


A TABULATION OF ANTIGENS AND ANTIBODIES AS AT PRESENT RECOGNIZED.

                                                                CLASS OF
 ANTIGEN         ANTIBODY         ACTION OF ANTIBODY            RECEPTOR

 Toxin           Antitoxin        Combines with toxin and             I.
                                    hence prevents toxin
                                    from uniting with a
                                    cell and injuring it,
                                    _i.e._, neutralizes toxin.

 Enzyme          Antienzyme       Combines with enzyme                I.
                                    and thus prevents enzyme
                                    from uniting
                                    with anything else and
                                    showing its action, _i.e._,
                                    neutralizes enzyme.

 Solution of     Precipitin       Unites with its antigen            II.
   protein                          and causes its precipitation
                                    from solution.

 Solution of          ?           Causes phenomenon of               (?)
   protein                          anaphylaxis(?)

 Suspension of   Agglutinin       Unites with its antigen            II.
   cells                            causes its clumping together
                                    and settling out
                                    of suspension.

 Suspension of   Opsonin          Unites with its antigen            II.
   cells                            and makes the cells              (?)
                                    more easily taken up
                                    by phagocytes.

 Suspension of   Amboceptor       Unites with its antigen           III.
   cells                            and also with complement
                                    which latter then
                                    dissolves the antigen.

 Precipitin      Antiprecipitin   Neutralizes precipitin.             I.

 Agglutinin      Antiagglutinin   Neutralizes agglutinin.             I.

 Opsonin         Antiopsonin      Neutralizes opsonin.                I.

 Amboceptor      Antiamboceptor   Neutralizes amboceptor.             I.
                   (two kinds)

 Complement      Anticomplement   Neutralizes complement.             I.


SUMMARY OF IMMUNITY AS APPLIED TO PROTECTION FROM DISEASE.

The discussion of “immunity problems” in the preceding chapters serves
to show that protection from disease either as a condition natural to
the animal or as an acquired state is dependent on certain properties
of its body cells or fluids, or both. The actual factors so far as at
present known may be summarized as follows:

1. _Antitoxins_ which neutralize true toxins; shown to exist for very
few diseases.

2. _Cytolytic substances_ which destroy the invading organism: in
reality two substances; amboceptor, which is specific, and complement,
the real dissolving enzyme.

3. _Phagocytosis_ or the destruction of the invading organisms within
the leukocytes.

4. _Opsonins_ which render the bacteria more readily taken up by the
phagocytes.

5. _Enzymes_ other than complement possibly play a part in the
destruction of some pathogenic organisms or their products. This
remains to be more definitely established.

6. It is possible that in natural immunity there might be no receptors
in the body cells to take up the organisms or their products, or the
receptors might be present in certain cells but of a very low chemical
affinity, so that combination does not occur. It is even highly
probable that many substances formed by invading organisms which might
injure specialized cells, such as those of glandular, nervous or muscle
tissue, have a more rapid rate of reaction with, or a stronger affinity
for, lower unspecialized cells, such as connective and lymphoid tissue,
and unite with these so that their effects are not noticed.

The importance of these different, factors varies in different diseases
and need not be considered in this connection.

The question “which of the body cells are engaged in the production
of antibodies” is not uncommonly asked. On physiological grounds it
would not seem reasonable that the highly specialized tissues above
mentioned could take up this work, even though they are the ones which
suffer the greatest injury in disease. Hence it is to be expected that
the lower or unspecialized cells are the source, and it has been shown
that the antibodies are produced by the phagocytes (though not entirely
as Metchnikoff maintained), by lymphoid tissue generally, by the bone
marrow and also by connective-tissue cells, though in varying degrees.

Since immunity depends on the activity of the body cells it is evident
that one of the very best methods for avoiding infectious diseases is
to keep these cells up to their highest state of efficiency, to keep in
“good health.” Hence good health means not only _freedom from disease_
but also _protection against disease_.




LIST OF LABORATORY EXERCISES GIVEN IN CONNECTION WITH THE CLASS WORK
INCLUDED IN THIS TEXT-BOOK.

  Exercise 1. Cleaning glassware.

  Exercise 2. Preparation of broth medium from meat juice.

  Exercise 3. Preparation of gelatin medium from broth.

  Exercise 4. Preparation of agar medium from broth.

  Exercise 5. Potato tubes.

  Exercise 6. Potato plates.

  Exercise 7. Plain milk tubes.

  Exercise 8. Litmus milk tubes.

  Exercise 9. Sugar broth media.

  Exercise 10. Blood-serum tubes.

  Exercise 11. Inoculation of tubes. Action on complex proteins.

  Exercise 12. Production of gas from carbohydrates.

  Exercise 13. Production of indol.

  Exercise 14. Reduction of nitrates.

  Exercise 15. Chromogenesis: Illustrates nicely the variation with
  environment.

  Exercise 16. Enzyme production.

  Exercise 17. Making of plate cultures; isolation in pure culture.

  Exercise 18. Stain making and staining.

  Exercise 19. Cell forms and cell groupings.

  Exercise 20. Hanging drop slides.

  Exercise 21. Staining of spores.

  Exercise 22. Staining of acid-fast bacteria.

  Exercise 23. Staining of capsules.

  Exercise 24. Staining of metachromatic granules.

  Exercise 25. Staining of flagella.

  Exercise 26. Study of individual species.

  Exercise 27. Determination of thermal death-point.

  Exercise 28. Action of disinfectants on bacteria.

  Exercise 29. Action of sunlight on bacteria.




DESCRIPTIVE CHART--SOCIETY OF AMERICAN BACTERIOLOGISTS.

_Prepared by Committee on Methods of Identification of Bacterial
Species.--F. D. Chester, F. P. Gorham, Erwin F. Smith._

_Endorsed by the Society for general use at the Annual Meeting,
December, 1907._


GLOSSARY OF TERMS.

AGAR HANGING BLOCK, a small block of nutrient agar cut from a pour
plate, and placed on a cover-glass, the surface next the glass having
been first touched with a loop from a young fluid culture or with a
dilution from the same. It is examined upside down, the same as a
hanging drop.

AMEBOID, assuming various shapes like an ameba.

AMORPHOUS, without visible differentiation in structure.

ARBORESCENT, a branched, tree-like growth.

BEADED, in stab or stroke, disjointed or semiconfluent colonies along
the lines of inoculation.

BRIEF, a few days, a week.

BRITTLE, growth dry, friable under the platinum needle.

BULLATE, growth rising in convex prominences, like a blistered surface.

BUTYROUS, growth of a butter-like consistency.

CHAINS,
    Short chains, composed of 2 to 8 elements.
    Long chains, composed of more than 8 elements.

CILIATE, having fine, hair-like extensions, like cilia.

CLOUDY, said of fluid cultures which do not contain pseudozoogleæ.

COAGULATION,[22] the separation of casein from whey in milk. This may
take place quickly or slowly, and as the result either of the formation
of an acid or of a lab ferment.

CONTOURED, an irregular, smoothly undulating surface, like that of a
relief map.

CONVEX surface, the segment of a circle, but flattened.

COPROPHYL, dung bacteria.

CORIACEOUS, growth tough, leathery, not yielding to the platinum needle.

CRATERIFORM, round, depressed, due to the liquefaction of the medium.

CRETACEOUS, growth opaque and white, chalky.

CURLED, composed of parallel chains in wavy strands, as in anthrax
colonies.

DIASTASIC ACTION, same as DIASTATIC, conversion of starch into
water-soluble substances by diastase.

ECHINULATE, in agar stroke a growth along line of inoculation, with
toothed or pointed margins; in stab cultures growth beset with pointed
outgrowths.

EFFUSE, growth thin, veily, unusually spreading.

ENTIRE, smooth, having a margin destitute of teeth or notches.

EROSE, border irregularly toothed.

FILAMENTOUS, growth composed of long, irregularly placed or interwoven
filaments.

FILIFORM, in stroke or stab cultures a uniform growth along line of
inoculation.

FIMBRIATE, border fringed with slender processes, larger than filaments.

FLOCCOSE, growth composed of short curved chains, variously oriented.

FLOCCULENT, said of fluids which contain pseudozoogleæ, _i.e._, small
adherent masses of bacteria of various shapes and floating in the
culture fluid.

FLUORESCENT, having one color by transmitted light and another by
reflected light.

GRAM’S STAIN, a method of differential bleaching after gentian violet,
methyl violet, etc. The + mark is to be given only when the bacteria
are deep blue or remain blue after counter-staining with Bismarck brown.

GRUMOSE, clotted.

INFUNDIBULIFORM, form of a funnel or inverted cone.

IRIDESCENT, like mother-of-pearl. The effect of very thin films.

LACERATE, having the margin cut into irregular segments as if torn.

LOBATE, border deeply undulate, producing lobes (see _Undulate_).

LONG, many weeks, or months.

MAXIMUM TEMPERATURE, temperature above which growth does not take place.

MEDIUM, nutrient substance upon which bacteria are grown.

MEMBRANOUS, growth thin, coherent, like a membrane.

MINIMUM TEMPERATURE, temperature below which growth does not take place.

MYCELIOID, colonies having the radiately filamentous appearance of mold
colonies.

NAPIFORM, liquefaction with the form of a turnip.

NITROGEN REQUIREMENTS, the necessary nitrogenous food. This is
determined by adding to _nitrogen-free_ media the nitrogen compound to
be tested.

OPALESCENT, resembling the color of an opal.

OPTIMUM TEMPERATURE, temperature at which growth is most rapid.

PELLICLE, in fluid bacterial growth forming either a continuous or an
interrupted sheet over the fluid.

PEPTONIZED, said of curds dissolved by trypsin.

PERSISTENT, many weeks, or months.

PLUMOSE, a fleecy or feathery growth.

PSEUDOZOOGLEÆ, clumps of bacteria, not dissolving readily in water,
arising from imperfect separation, or more or less fusion of the
components, but not having the degree of compactness and gelatinization
seen in zoogleæ.

PULVINATE, in the form of a cushion, decidedly convex.

PUNCTIFORM, very minute colonies, at the limit of natural vision.

RAPID, developing in twenty-four to forty-eight hours.

RAISED, growth thick, with abrupt or terraced edges.

RHIZOID, growth of an irregular branched or root-like character, as in
_B. mycoides_.

RING, same as RIM, growth at the upper margin of a liquid culture,
adhering more or less closely to the glass.

REPAND, wrinkled.

SACCATE, liquefaction the shape of an elongated sac, tubular,
cylindrical.

SCUM, floating islands of bacteria, an interrupted pellicle or bacteria
membrane.

SLOW, requiring five or six days or more for development.

SHORT, applied to time, a few days, a week.

SPORANGIA, cells containing endospores.

SPREADING, growth extending much beyond the line of inoculation,
_i.e._, several millimetres or more.

STRATIFORM, liquefying to the walls of the tube at the top and then
proceeding downward horizontally.

THERMAL DEATH-POINT, the degree of heat required to kill young fluid
cultures of an organism exposed for ten minutes (in thin-walled
test-tubes of a diameter not exceeding 20 mm.) in the thermal
water-bath. The water must be kept agitated so that the temperature
shall be uniform during the exposure.

TRANSIENT, a few days.

TURBID, cloudy with flocculent particles; cloudy plus flocculence.

UMBONATE, having a button-like, raised centre.

UNDULATE, border wavy, with shallow sinuses.

VERRUCOSE, growth wart-like, with wart-like prominences.

VERMIFORM-CONTOURED, growth like a mass of worms or intestinal coils.

VILLOUS, growth beset with hair-like extensions.

VISCID, growth follows the needle when touched and withdrawn, sediment
on shaking rises as a coherent swirl.

ZOOGLEÆ, firm gelatinous masses of bacteria, one of the most typical
examples of which is the _Streptococcus mesenterioides_ of sugar vats.
(_Leuconostoc mesenterioides_), the bacterial chains being surrounded
by an enormously thickened, firm covering inside of which there may be
one or many groups of the bacteria.


NOTES.

(1) For decimal system of group numbers see Table I. This will be found
useful as a quick method of showing close relationships inside the
genus, but is not a sufficient characterization of any organism.

(2) The morphological characters shall be determined and described
from growths obtained upon at least one solid medium (nutrient agar)
and in at least one liquid medium (nutrient broth). Growths at 37° C.
shall be in general not older than twenty-four to forty-eight hours,
and growths at 20° C. not older than forty-eight to seventy-two hours.
To secure uniformity in cultures, in all cases preliminary cultivation
shall be practised as described in the revised Report of the Committee
on Standard Methods of the Laboratory Section of the American Public
Health Association, 1905.

(3) The observation of cultural and biochemical features shall cover
a period of at least fifteen days and frequently longer, and shall be
made according to the revised Standard Methods above referred to. All
media shall be made according to the same Standard Methods.

(4) Gelatin stab cultures shall be held for six weeks to determine
liquefaction.

(5) Ammonia and indol tests shall be made at end of tenth day, nitrite
tests at end of fifth day.

(6) Titrate with N/20 NaOH, using phenolphthalein as an indicator; make
titrations at same time from blank. The difference gives the amount of
acid produced.

The titration should be done after boiling to drive off any CO₂ present
in the culture.

(7) Generic nomenclature shall begin with the year 1872 (Cohn’s first
important paper).

Species nomenclature shall begin with the year 1880 (Koch’s discovery
of the pour plate method for the separation of organisms).

(8) Chromogenesis shall be recorded in standard color terms.


TABLE I.

A NUMERICAL SYSTEM OF RECORDING THE SALIENT CHARACTERS OF AN ORGANISM.
(GROUP NUMBER.)

  100           Endospores produced
  200           Endospores not produced
   10           Aërobic (strict)
   20           Facultative anaërobic
   30           Anaërobic (strict)
    1           Gelatin liquefied
    2           Gelatin not liquefied
    0.1         Acid and gas from dextrose
    0.2         Acid without gas from dextrose
    0.3         No acid from dextrose
    0.4         No growth with dextrose
    0.01        Acid and gas from lactose
    0.02        Acid without gas from lactose
    0.03        No acid from lactose
    0.04        No growth with lactose
    0.001       Acid and gas from saccharose
    0.002       Acid without gas from saccharose
    0.003       No acid from saccharose
    0.004       No growth with saccharose
    0.0001      Nitrates reduced with evolution of gas
    0.0002      Nitrates not reduced
    0.0003      Nitrates reduced without gas formation
    0.00001     Fluorescent
    0.00002     Violet chromogens
    0.00003     Blue chromogens
    0.00004     Green chromogens
    0.00005     Yellow chromogens
    0.00006     Orange chromogens
    0.00007     Red chromogens
    0.00008     Brown chromogens
    0.00009     Pink chromogens
    0.00000     Non-chromogenics
    0.000001    Diastasic action on potato starch, strong
    0.000002    Diastasic action on potato starch, feeble
    0.000003    Diastasic action on potato starch, absent
    0.0000001   Acid and gas from glycerin
    0.0000002   Acid without gas from glycerin
    0.0000003   No acid from glycerin
    0.0000004   No growth with glycerin

The genus according to the system of Migula is given its proper symbol
which precedes the number thus:(7)

  BACILLUS COLI (Esch.) Mig.        becomes B.     222.111102
  BACILLUS ALCALIGENES Petr.        becomes B.     212.333102
  PSEUDOMONAS CAMPESTRIS (Pam.) Sm. becomes Ps.    211.333151
  BACTERIUM SUICIDA Mig.            becomes Bact.  222.232103

Source............ Date of Isolation.............. Name........
Group No.(1)...............




DETAILED FEATURES.

NOTE--Underscore required terms. Observe notes and glossary of terms on
opposite side of card.


I. MORPHOLOGY(2)

  1. Vegetative Cells, Medium used.............................
  temp....................age.................days

  Form, _round_, _short rods_, _long rods_, _short chains_, _long
  chains_, _filaments_, _commas_, _short spirals_, _long spirals_,
  _clostridium_, _cuneate_, _clavate_, _curved_.

  Limits of Size..........................

  Size of Majority.............................

  Ends, _rounded_, _truncate_, _concave_.

                  {Orientation (grouping)............................
  Agar            {Chains (No. of elements)........................
  Hanging-block   {_Short chains_, _long chains_
                  {Orientation of chains, _parallel_, _irregular_.

  2. Sporangia, medium
  used.....................temp..............age..............days

  Form, _elliptical_, _short rods_, _spindled_, _clavate_, _drumsticks_.

  Limits of Size................

  Size of Majority..............

  Agar            {Orientation (grouping)........
  Hanging-block   {Chains (No. of elements)......
                  {Orientation of chains, _parallel_, _irregular_.

  Location of Endospores, _central_, _polar_.

  3. Endospores.

  Form, _round_, _elliptical_, _elongated_.

  Limits of Size................

  Size of Majority..............

  Wall, _thick_, _thin_.

  Sporangium wall, _adherent_, _not adherent_.

  Germination, _equatorial_, _oblique_, _polar_, _bipolar_, _by
  stretching_.

  4. Flagella, No........Attachment _polar_, _bipolar_,
  _peritrichiate_. How Stained.........

  5. Capsules, present on.............

  6. Zooglea, Pseudozooglea.

  7. Involution Forms, on........in.....days at....° C.

  8. Staining Reactions.

  1:10 watery fuchsin, gentian violet, carbol-fuchsin, Loeffler’s
  alkaline methylene blue.

  Special Stains.
  Gram....................Glycogen...............
  Fat.....................Acid-fast................
  Neisser.................


II. CULTURAL FEATURES(3)

1. Agar Stroke.

  Growth, _invisible_, _scanty_, _moderate_, _abundant_.

  Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_,
  _plumose_, _arborescent_, _rhizoid_.

  Elevation of growth, _flat_, _effuse_, _raised_, _convex_.

  Lustre, _glistening_, _dull_, _cretaceous_.

  Topography, _smooth_, _contoured_, _rugose_, _verrucose_.

  Optical characters, _opaque_, _translucent_, _opalescent_,
  _iridescent_.

  Chromogenesis(3)................

  Odor, _absent_, _decided_, _resembling_............

  Consistency, _slimy_, _butyrous_, _viscid_, _membranous_,
  _coriaceous_, _brittle_.

  Medium _grayed_, _browned_, _reddened_, _blued_, _greened_.

2. Potato.

  Growth _scanty_, _moderate_, _abundant_, _transient_, _persistent_.

  Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_,
  _plumose_, _arborescent_, _rhizoid_.

  Elevation of growth, _flat_, _effuse_, _raised_, _convex_.

  Lustre, _glistening_, _dull_, _cretaceous_.

  Topography, _smooth_, _contoured_, _rugose_, _verrucose_.

  Chromogenesis(3)...........Pigment in water _insoluble_, _soluble_:
  other solvents.....................

  Odor, _absent_, _decided_, _resembling_....................

  Consistency, _slimy_, _butyrous_, _viscid_, _membranous_,
  _coriaceous_, _brittle_.

  Medium, _grayed_, _browned_, _reddened_, _blued_, _greened_.

3. Loeffler’s Blood-serum.

  Stroke _invisible_, _scanty_, _moderate_, _abundant_.

  Form of growth, _filiform_, _echinulate_, _beaded_, _spreading_,
  _plumose_, _arborescent_, _rhizoid_.

  Elevation of growth, _flat_, _effuse_, _raised_, _convex_.

  Lustre, _glistening_, _dull_, _cretaceous_.

  Topography, _smooth_, _contoured_, _rugose_, _verrucose_.

  Chromogenesis(3)..........................

  Medium _grayed_, _browned_, _reddened_, _blued_, _greened_.

  Liquefaction begins in.............d, complete in................d,

4. Agar Stab.

  Growth _uniform_, _best at top_, _best at bottom_: surface growth
  _scanty_, _abundant_: _restricted_, _wide-spread_.

  Line of puncture, _filiform_, _beaded_, _papillate_, _villous_,
  _plumose_, _arborescent_: _liquefaction_.

5. Gelatin Stab.

  Growth uniform, _best at top_, _best at bottom_.

  Line of puncture, _filiform_, _beaded_, _papillate_, _villous_,
  _plumose_, _arborescent_.

  Liquefaction _crateriform_, _napiform_, _infundibuliform_,
  _saccate_, _stratiform_: begins in....................d. complete
  in....................d

  Medium _fluorescent_, _browned_...............

6. Nutrient Broth.

  Surface growth, _ring_, _pellicle_, _flocculent_, _membranous_,
  _none_.

  Clouding _slight_, _moderate_, _strong_: _transient_, _persistent_:
  _none_: _fluid turbid_.

  Odor, _absent_, _decided_, _resembling_..................

  Sediment, _compact_, _flocculent_, _granular_, _flaky_, _viscid on
  agitation_, _abundant_, _scant_.

7. Milk.

  Clearing without coagulation.

  Coagulation _prompt_, _delayed_, _absent_.

  Extrusion of whey begins in............days.

  Coagulum _slowly peptonized_, _rapidly peptonized_.

  Peptonization begins on....d, complete on ....d.

  Reaction, 1d...., 2d...., 4d...., 10d...., 20d....

  Consistency, _slimy_, _viscid_, _unchanged_.

  Medium _browned_, _reddened_, _blued_, _greened_.

  Lab ferment, _present_, _absent_.

8. Litmus Milk.

  _Acid_, _alkaline_, _acid then alkaline_, _no change_.

  _Prompt reduction_, _no reduction_, _partial slow reduction_.

9. Gelatin Colonies.

  Growth _slow_, _rapid_.

  Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_,
  _filamentous_, _rhizoid_.

  Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_,
  _crateriform_ (_liquefying_).

  Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_,
  _fimbriate_, _filamentous_, _floccose_, _curled_.

  Liquefaction, _cup_, _saucer_, _spreading_.

10. Agar Colonies.

  Growth _slow_, _rapid_ (temperature..............)

  Form, _punctiform_, _round_, _irregular_, _ameboid_, _mycelioid_,
  _filamentous_, _rhizoid_.

  Surface _smooth_, _rough_, _concentrically ringed_, _radiate_,
  _striate_.

  Elevation, _flat_, _effuse_, _raised_, _convex_, _pulvinate_,
  _umbonate_.

  Edge, _entire_, _undulate_, _lobate_, _erose_, _lacerate_,
  _fimbriate_, _floccose_, _curled_.

  Internal structure, _amorphous_, _finely_, _coarsely granular_,
  _grumose_, _filamentous_, _floccose_, _curled_.

11. Starch Jelly.

  Growth, _scanty_, _copious_.

  Diastatic action, _absent_, _feeble_, _profound_.

  Medium stained...................

12. Silicate Jelly (Fermi’s Solution).

  Growth _copious_, _scanty_, _absent_.

  Medium stained..................

13. Cohn’s Solution.

  Growth _copious_, _scanty_, _absent_.

  Medium _fluorescent_, _non-fluorescent_.

14. Uschinsky’s Solution.

  Growth _copious_, _scanty_, _absent_.

  Fluid _viscid_, _not viscid_.

15. Sodium Chloride in Bouillon.

  Per cent. inhibiting growth........................

16. Growth in Bouillon over Chloroform, _unrestrained_,
      _feeble_, _absent_.

17. Nitrogen. Obtained from _peptone_, _asparagin_, _glycocoll_,
      _urea_, _ammonia salts_, _nitrogen_.

18. Best media for long-continued growth...................
      .....................................................

19. Quick tests for differential purposes..................
      .....................................................
      .....................................................


III. PHYSICAL AND BIOCHEMICAL FEATURES.

  +----------------------------------+---+---+---+---+---+---+---+---+
  |                                  | D | S | L | M | G | M |   |   |
  |                                  | e | a | a | a | l | a |   |   |
  |                                  | x | c | c | l | y | n |   |   |
  |                                  | t | c | t | t | c | n |   |   |
  | 1. Fermentation-tubes containing | r | h | o | o | e | i |   |   |
  |    peptone-water or              | o | a | s | s | r | t |   |   |
  |    sugar-tree bouillon and       | s | r | e | e | i |   |   |   |
  |                                  | e | o |   |   | n |   |   |   |
  |                                  |   | s |   |   |   |   |   |   |
  |                                  |   | e |   |   |   |   |   |   |
  +----------------------------------+---+---+---+---+---+---+---+---+
  | Gas production, in per cent.     |   |   |   |   |   |   |   |   |
  +----------------------------------+---+---+---+---+---+---+---+---+
  | (H/CO₂)                          |   |   |   |   |   |   |   |   |
  +----------------------------------+---+---+---+---+---+---+---+---+
  | Growth in closed arm             |   |   |   |   |   |   |   |   |
  +----------------------------------+---+---+---+---+---+---+---+---+
  | Amount of acid produced 1d.      |   |   |   |   |   |   |   |   |
  +----------------------------------+---+---+---+---+---+---+---+---+
  | Amount of acid produced 2d.      |   |   |   |   |   |   |   |   |
  +----------------------------------+---+---+---+---+---+---+---+---+
  | Amount of acid produced 3d.      |   |   |   |   |   |   |   |   |
  +----------------------------------+---+---+---+---+---+---+---+---+

  2. Ammonia production, _feeble_, _moderate_, _strong_, _absent_,
  _masked by acids_.

  3. Nitrates in nitrate broth.

  _Reduced_, _not reduced_.

  Presence of nitrites...........ammonia..................

  Presence of nitrates...........free nitrogen............

  4. Indol production, _feeble_, _moderate_, _strong_.

  5. Toleration of Acids, _great_, _medium_, _slight_.

  _Acids tested_..............

  6. Toleration of NaOH, _great_, _medium_, _slight_.

  7. Optimum reaction for growth in bouillon, stated in terms of
  Fuller’s scale..........................

  8. Vitality on culture media, _brief_, _moderate_, _long_.

  9. Temperature relations.

  Thermal death-point (10 minutes’ exposure in nutrient broth when this
  is adapted to growth of organism)............C.

  Optimum temperature for growth......° C.; or best growth at 16° C.,
  20° C., 25° C., 30° C., 37° C., 40° C., 50° C., 60° C.

  Maximum temperature for growth.......... ° C.

  Minimum temperature for growth.......... ° C.

  10. Killed readily by drying: resistant to drying.

  11. Per cent. killed by freezing (salt and crushed ice or liquid
  air)................

  12. Sunlight: Exposure on ice in thinly sown agar plates; one-half
  plate covered (time 15 minutes), _sensitive_, _not sensitive_.

  Per cent. killed................

  13. Acids produced.................

  14. Alkalies produced...............

  15. Alcohols.......................

  16. Ferments, _pepsin_, _trypsin_, _diastase_, _invertase_,
  _pectase_, _cytase_, _tyrosinase_, _oxidase_, _peroxidase_,
  _lipase_, _catalase_, _glucase_, _galactase_, _lab_,
  _etc._........................

  17. Crystals formed:.....

  18. Effect of germicides:

  +-----------+-------------+---+---+---+---+---+
  |           |             | M | T | K | A | r |
  |           |             | i | e | i | m | e |
  |           |             | n | m | l | t | s |
  |           |             | u | p | l | . | t |
  |           |             | t | e | i |   | r |
  |           |             | e | r | n | r | a |
  |           |             | s | a | g | e | i |
  | Substance | Method used |   | t |   | q | n |
  |           |             |   | u | q | u |   |
  |           |             |   | r | u | i | g |
  |           |             |   | e | a | r | r |
  |           |             |   |   | n | e | o |
  |           |             |   |   | t | d | w |
  |           |             |   |   | i |   | t |
  |           |             |   |   | t | t | h |
  |           |             |   |   | y | o |   |
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+
  |           |             |   |   |   |   |   |
  +-----------+-------------+---+---+---+---+---+


IV. PATHOGENICITY.

  1. Pathogenic to Animals.

  _Insects_, _crustaceans_, _fishes_, _reptiles_, _birds_, _mice_,
  _rats_, _guinea-pigs_, _rabbits_, _dogs_, _cats_, _sheep_, _goats_,
  _cattle_, _horses_, _monkeys_, _man_..........................

  2. Pathogenic to Plants:
  .........................................................
  .........................................................
  .........................................................

  3. Toxins, _soluble_, _endotoxins_.

  4. Non-toxin forming.

  5. Immunity bactericidal.

  6. Immunity non-bactericidal.

  7. Loss of virulence on culture-media: _prompt_, _gradual_, _not
  observed in_.....................months.

  +-------------------------------------+
  |   BRIEF CHARACTERIZATION.           |
  |                                     |
  |   Mark + or 0, and when two terms   |
  | occur on a line erase the one which |
  | does not apply unless both apply.   |
  |                                     |
  +--------------------------------+----+
  | M | Diameter over 1µ           |----|
  | O | Chains, filaments          |----|
  | R | Endospores                 |----|
  | P | Capsules                   |----|
  | H | Zooglea, Pseudozooglea     |----|
  | O | Motile                     |----|
  | L | Involution forms           |----|
  | O | Gram’s stain               |----|
  | G |                            |----|
  | Y |                            |----|
  |(2)|                            |----|
  +---+----------------------------+----+
  | C | B   | Cloudy, turbid       |----|
  | U | r   | Ring                 |----|
  | L | o   | Pellicle             |----|
  | T | t   | Sediment             |----|
  | U | h   |                      |----|
  | R +-----+----------------------+----+
  | A | A   | Shining              |----|
  | L | g   | Dull                 |----|
  |   | a   | Wrinkled             |----|
  | F | r   | Chromogenic          |----|
  | E +-----+----------------------+----+
  | A | G   | Round                |----|
  | T | e   | Proteus-like         |----|
  | U | l.  | Rhizoid              |----|
  | R |     | Filamentous          |----|
  | E | P   | Curled               |----|
  | S | l   |                      |----|
  |(3)| a   |                      |----|
  |   | t   |                      |----|
  |   | e   |                      |----|
  |   +-----+----------------------+----+
  |   | G S | Surface growth       |----|
  |   | e t | Needle growth        |----|
  |   | l a |                      |----|
  |   | . b.|                      |----|
  |   +-----+----------------------+----+
  |   | P   | Moderate, absent     |----|
  |   | o   | Abundant             |----|
  |   | t   | Discolored           |----|
  |   | a   | Starch destroyed     |----|
  |   | t   |                      |----|
  |   | o   |                      |----|
  |   +-----+---------------------------+
  |   | Grows at 37° C.            |----|
  |   | Grows in Cohn’s sol.       |----|
  |   | Grows in Uschinsky’s sol.  |----|
  |---+-----+----------------------+----+
  | B | L f | Gelatin(4)           |----|
  | I | i a | Blood-serum          |----|
  | O | q c | Casein               |----|
  | C | u t |                      |----|
  | H | i i |                      |----|
  | E | - o |                      |----|
  | M |   n |                      |----|
  | I +-----+----------------------+----+
  | C | M   | Acid curd            |----|
  | A | i   | Rennet curd          |----|
  | L | l   | Casein peptonized    |----|
  |   | k   |                      |----|
  | F +-----+----------------------+----+
  | E | Indol(3)                   |----|
  | A | Hydrogen sulphide          |----|
  | T | Ammonia(3)                 |----|
  | U | Nitrates reduced(3)        |----|
  | R | Fluorescent                |----|
  | E | Luminous                   |----|
  | S |                            |----|
  +---+----------------------------+----+
  | D | Animal pathogen, epizoon   |----|
  | I | Plant pathogen, epiphyte   |----|
  | S | Soil                       |----|
  | T | Milk                       |----|
  | R | Fresh water                |----|
  | I | Salt water                 |----|
  | B | Sewage                     |----|
  | U | Iron bacterium             |----|
  | T | Sulphur bacterium          |----|
  | I |                            |----|
  | O |                            |----|
  | N |                            |----|
  +---+----------------------------+----+




FOOTNOTES.


[1] Sir H. A. Blake has called attention to the fact that the “mosquito
theory” of malaria is mentioned in a Sanscrit manuscript of about the
6th century A.D.

[2] Myxomycetes excepted, and they are probably to be regarded as
animals--Mycetozoa.

[3] Centralblatt f. Bakteriologie, etc. LXIII. 1 Abt. Orig. 1912, 4,
idem LXVI. 1 Abt. Orig. 1912, 323.

[4] The pronunciation of this word according to English standards
is kok-si; the continental pronunciation is kok-kee; the commonest
American seems to be kok-ki. We prefer the latter since it is easier
and more natural and should like to see it adopted. (Author.)

[5] With the possible exception of blue green algæ which have been
found with bacteria in the above-mentioned hot springs. Seeds of
many plants have been subjected to as low temperatures as those
above-mentioned without apparent injury.

[6] It is popularly supposed that in canning fruit, vegetables, meats,
etc., all the air must be removed, since the organisms which cause
“spoiling” cannot grow in a vacuum. The existence of anaërobic and
facultative anaërobic bacteria shows the fallacy of such beliefs.

[7] “By cellulose is understood a carbohydrate of the general formula
C₆H₁₀O₅ not soluble in water, alcohol, ether, or dilute acids but
soluble in an ammoniacal solution of copper oxide. It gives with iodine
and sulphuric acid a blue color and with iodine zinc chloride a violet
and yields dextrose on hydrolysis.”--H. Fischer.

[8] The sulphur bacteria are partially prototrophic for S; probably the
iron bacteria also for Fe. Some few soil bacteria have been shown to
be capable of utilizing free H, and it seems certain that the bacteria
associated with the spontaneous heating of coal may oxidize free C. So
far as known no elements other than these six are directly available to
bacteria.

[9] Only a few kinds of bacteria so far as known are proto-autotrophic.
The nitrous and nitric organisms of Winogradsky which are so essential
in the soil, and which might have been the first of all organisms so
far as their food is concerned, and some of the sulphur bacteria are
examples.

[10] The term _pathogenic_ is also applied to certain non-parasitic
saprophytic bacteria whose products cause disease conditions, as one
of the organisms causing a type of food poisoning in man (_Clostridium
botulinum_), which also probably causes “forage poisoning” in domestic
animals.

[11] The term “fermentation” was originally used to denote the process
which goes on in fruit juices or grain extracts when alcohol and gas
are formed. Later it was extended to apply to the decomposition of
almost any organic substance. In recent years the attempt has been made
to give a chemical definition to the word by restricting its use to
those changes in which by virtue of a “wandering” or rearrangement of
the carbon atoms “new substances are formed which are not constitutents
of the original molecule.” It may be doubted whether this restriction
is justified or necessary. A definition is at present scarcely possible
except when the qualifying adjective is included as “alcoholic
fermentation,” “ammoniacal fermentation,” “lactic acid fermentation,”
etc.

[12] See “Oil and Gas in Ohio,” Bownocker: Geological Survey of Ohio,
Fourth Series, Bull. I, pp. 313-314.

[13] It is probable that this is the way “Jack o’lanterns” or “Will o’
the wisps” are ignited. Marsh gas is produced as above outlined from
the vegetable and animal matter decomposing in swampy places under
anaërobic conditions and likewise phosphine. These escape into the air
and the “spontaneous combustion” of the phosphine ignites the marsh gas.

[14] Dr. H. H. Green, of Pretoria, South Africa, has isolated from
“cattle dips” a bacterium that _reduces arsenates_ to _arsenites_.

[15] Dr. Green (l. c.) has also isolated an organism which causes some
deterioration of cattle dips by _oxidizing arsenites to arsenates_.

[16] It will be noted that the names of enzymes (except some of
those first discovered) terminate in _ase_ which is usually added to
the _stem of the name of the substance acted on_, though sometimes
to a word which indicates the substance formed by the action, as
_lactacidase_, _alcoholase_.

[17] Tetanus toxin is about 120 times as poisonous as strychnin, both
of which act on the same kind of nerve cells.

[18] In the author’s laboratory in the past ten years all sterilization
except those few objects in blood and serum work which must be dry, has
been done in autoclaves of the type shown in Fig. 81 which are supplied
with steam from the University central heating plant. A very great
saving of time is thus secured.

[19] The author has tested an “electric milk purifier” (Fig. 102)
which was as efficient as a first-class pasteurizer and left the milk
in excellent condition both chemically and as far as “cream line” was
concerned. The cost of operation as compared with steam will depend on
the price of electricity.

[20] The exact laboratory details for preparing various media are
not given in this chapter. It is the object to explain the choice of
different materials and the reasons for the various processes to which
they are subjected.

[21] For a discussion of this method of standardization consult the
following:

  Clark & Lubs--J. Bact., 1917, II, 1-34, 109-136, 191-236.
  Committee Report--Ibid., 1919. IV, 107-132.
  Jones--J. Inf. Dis., 1919, 25, 262-268.
  Fennel & Fisher--Ibid., 444-451.

Additional references will be found in these articles.

[22] Term also applied to the solidification of serum in media: _e.g._,
the Hiss inulin medium for the differentiation of pneumococci (see
diplococcus of pneumonia).

[23] The term “antigen” is also used to designate substances which may
take the place of what are supposed to be the true antigens in certain
diagnostic reactions (Chapter XXIX, Complement Fixation Test for
Syphilis).

[24] If the antitoxin is later concentrated (see last paragraph in
this chapter) a serum containing as little as 175 units per cc. may be
commercially profitable.

[25] Tho term “allergie” was introduced by Von Pirquet to designate the
state of the animal’s being sensitized and “allergic” as the adjective
derived therefrom. It does not seem to the author that there is any
advantage gained by the introduction of these terms.




INDEX


  A

  ABBÉ, 17
    condenser, 200
    microscope, improvements in, 30, 36

  ABILGAARD, 26

  Abrin, 262

  Absorption of free nitrogen, 117
    tests, 267

  Accidental carriers, 241
    structures, 43

  Acetic acid, 99
    bacteria, carbon oxidation, 114
    fermentation, 32

  _Acetobacter acidi oxalici_, 83
    _xylinum_, 83

  _Achorion schœnleinii_, 27, 34

  Acid, acetic, 99
      fermentation, 32
    agglutination, 266
    amino, relation to green plants, 119
    butyric, 99
      fermentation, 32, 99
    carbolic, first used, 29
    disinfectant action of, 159
    fast bacteria, fat content, 84
      staining of, 209
    fermentation, 93
      Bulgarian fermented milk, 98
      ensilage, 98
      industrial uses, 97
      lactic acid, 96
      sauerkraut, 98
    hydrochloric, 246
    production of, 110
    soils, 81

  Acquired immunity, 251, 252

  _Actinomyces bovis_, 30, 36

  Actinomycosis, cause of, 30, 36
    path of entrance of, 244

  Actions, reducing, 113

  Activating enzymes, 125

  Active immunity, definition of, 251, 252
    production of, 252

  Activities of bacteria, importance of, 31
    overproduction, of cells, 258
    physiological, definition of, 87
      in identification, 216

  Acute coryza, 244
    disease, 233

  Adulteration of food, anaphylactic test in, 293
      complement-fixation test in, 279
      immunity reactions in, 255
      precipitin test in, 269

  Aërobes, facultative, 76
    strict, 76

  Aërobic, 76, 215

  Agar, composition of, 179
    gelatinizing temperature, 179
    medium, preparation of, 179
    melting point of, 179
    plating in, 188
    sterilization of, 180

  Agent, chemical, for disinfection, 156-163
    choice of, for disinfection, 164
    physical, for disinfection, 131

  Agglutinating group, 266

  Agglutination, acid, 266
    diagnostic value of, 266
    in identification of bacteria, 266
    macroscopic, 265
    microscopic, 265
    phenomenon, 265

  Agglutinin, 265
    absorption test for, 267
    action of, 266
    anti-, 270
    antigenic action of, 270
    bacterial, 265
    chief, 267
    co-, 267
    function of, 266
    normal, 266
    partial, 267
    relation to precipitins, 269
    specificity of, 267
    theory of formation, 265
    use of, 266

  Agglutinogen, 266

  Agglutinoid, 270

  Aggressins, 288

  Air, bacteria in, 71
    filtration of, 153
    “germ-free,” 153

  Albumin in bacteria, 84

  Alcohol as antiseptic, 160
    as disinfectant, 160

  Alcoholase, 125

  Alcoholic fermentation, 31, 100

  Alexin, 271, 273

  Algæ, relation to bacteria, 37

  Alimentary tract as path of entrance, 246

  Alkalies as disinfectants, 158

  Allergic, 290

  Amboceptor, 273
    anti-, 275
    co-, 274
    in cobra, 275
    formation of, 273
    hemolytic, 278
    partial, 274
    in rattle snake, 275
    specificity of, 274
    theory of formation, 273

  Amboceptorogen, 274

  Amebic dysentery, 29, 35

  Ameboid cells, 247
    colonies, 224

  Amino-acids, relation to green plants, 119

  Ammonia, structural formula, 103

  Ammoniacal fermentation, 32

  _Amœba coli_, 29, 35

  Amphitrichic, 46

  Amylase, 124

  Anaërobes, 76
    cultivation, methods of, 188
      principles underlying, 188
    facultative, 76
    isolation of, 190
    relation to elements, 86
    strict, 76

  Anaërobic, 76, 215
    acid, butyric, 99
    acid fermentation, 98
    bacteria, first discovered, 32
    fermentation of polysaccharides, 95

  Analysis of ash, 82
    chemical, of tubercle bacilli, 85

  Anaphylactic, anti-, 290
    phenomena, 292
    reaction, uses of, 293

  Anaphylatoxin, 290

  Anaphylaxis, 289
    anti-, 292
    antibodies in, 291
    theory of, 290, 291, 292

  ANAXIMANDER, 18

  ANDERSON, 289

  ANDERSON and MCCLINTIC, phenol coefficient, 165

  ANDRY, 25, 33

  Anilin dyes, as antiseptic, 162
      as disinfectants, 162
      introduction of, 30
      as stains, 204
      Weigert, 36
    fuchsin, 205
    gentian violet, 205
    water, 205

  Animal carriers, 239
    inoculation, uses of, 227

  Animalcules, 19, 33

  Animals, disinfection of, 170
    experimental, 227
    food relationships of, 39

  _Ankylostoma duodenale_, discovery of, 27, 34
    Egyptian chlorosis, cause of, 28, 35
    hookworm disease, cause of, 28

  Anthrax, 17, 28, 35
    bacterium a facultative saprophyte, 238
      isolation of, 29
    due to a bacterium, 29
    in human beings, 238
    path of entrance, 243
      intestine, 246
      stomach, 246
    persistence due to spores, 251
    produced by exhaustion, 251
    protective inoculation in, 30
    spores, 29, 35
    transmission by flies, 242
    vaccine, 254

  Anti-agglutinins, 270
    aggressins, 288
    amboceptors, 275
      antisera in snake poisoning, 275
    anaphylactic, 290
    anaphylaxis due to intracellular digestion, 292
      protein immunity compared to, 292
    bacterial immunity, 254, 255
    bodies, 259
      place of production, 295
      tabulation of, 294
    body, action, 260
      chemical composition, 260
      formation of, 128, 260
    complement, 274
    complementophil amboceptor, 275
    cytophil amboceptor, 275
    diphtheritic serum, 263
    enzyme, 122, 262
      function of, 262

  Antigen, 259
    chemical composition of, 260
    in complement-fixation, 277
    syphilitic, 277, 279
    in Wassermann test, 279

  Antigens, fats and fatty acids as, 260
    in preparation of vaccine, 285
    tabulation of, 294

  Antipollenin, 263

  Antiprecipitins, 270

  Antisepsis, 131
    Lister, introduced, 35
    primitive, 25

  Antiseptic, 131
    action of anilin dyes, 162
    carbolic acid as, 159
    cold as, 148

  Antisera in snake poisoning, 275

  Antisnake venoms, 275

  Antitetanic serum, 263

  Antitoxic immunity, 254, 255

  Antitoxin, 261
    collection of, 263
    diphtheria, 30, 252
    preparation of, 263
    standard, 264
    tetanus, 252

  Antitoxins, 261-264
    as factors in immunity, 295
    preservative in, 263
    specific, 261

  Antivenin, 263

  Apes, 227

  Apparatus of Barber, 196

  Appearance of growth on culture media, 217

  APPERT, 20, 31, 34

  Aqueous gentian violet, 205

  Arborescent growth, 221

  ARISTOTLE, 18

  Aromatic compounds, production of, 104, 111

  Arrak, 100

  Arsenate, reduction of, 114

  Arsenite, oxidation of, 115

  ARTHUS, 289
    phenomenon, 289

  Articles, unwashable, disinfection of, 169
    washable, disinfection of, 169

  Artificial immunity, 251, 252

  Ase, termination of name of enzyme, 124

  Asepsis, 131

  Aseptic, 131

  Ash, analysis of, 82

  Asiatic cholera, 27, 34, 73, 238, 239, 246, 248, 249

  Attenuated, 253

  Autoclave, air pressure sterilizer, 138
    pressure sterilizer, 138

  Autogenous vaccines, 284
    in epidemic, 241

  Autoinfection, 234

  Autolysis, 149
    self-digestion, 126

  Autotrophic, 86

  Available nitrogen, loss of, 113

  Azotobacter, 118


  B

  BABES-ERNST corpuscles, 45

  Bacilli, butter, 209
    colon, 248
    grass, 209
    size and shape of, 52
    tubercle, chemical analysis of, 85

  Bacillus, 52, 60, 62
    _anthracis_, 17, 36
      spore staining, 209

  Bacillus of blue milk, 31
    Ducrey’s, 245
    _subtilis_, 77, 83
      spore staining, 209

  Bacteria, absorption of N by, 117
    acid fast, 84, 209
    adaptability, range of, 90
    advantage of motility to, 45
    aids in isolation of, 197
    anaërobic, 32
    cause of disease in animals, 30
      of souring of milk, 32
    cell groupings of, 55
    chains of, 38
    chemical composition of, 39, 81
      elements in, 82
    classed as fungi, 37
      as plants, 33, 35
    definition of, 40
    development of, 90
    distribution of, 71
    energy relationships, 39
    environmental conditions for growth, 72
    first classification of, 34
      drawings of, 20
      seen, 19, 33
    food relationships of, 39
    injurious, 72
    isolation of, 194
    measurement of, 40, 203
    metabolism of, 86
    methods of study of, 171
    morphology of, 41
    motile, 45
    nitric, 114
    nitrous, 114
    nucleus of, 42
    occurrence, 71
    pathogenic, outside the body, 237
    phosphorescent, 111, 112
    position of, 37
    rate of division, 43
      of motion, 45
    relation to algæ, 33, 37
      to elements, 86
      to gas and oil, 95
      to phosphate rock, 115
      to protozoa, 40
      to soil fertility, 120
      to sulphur deposits, 116
      to yeasts and torulæ, 37
    reproduction of, 37, 55
    root tubercle, 86, 87
    size of, 37, 40
    soil, chief function of, 119
    source of N, 102
    speed of, 45
    spiral, 53
    staining of, 204-212
    sulphur, 63
    thermophil, 75, 77
    universal distribution of, 90
    in vinegar-making, 99

  BACTERIACEÆ, 62, 66, 70

  Bacterial agglutinin, 265
    vaccines, 282
      preparation of, 283, 284

  Bacterin, 253

  Bacteriocidin, 272

  Bacteriological culture tubes, 184
    examination, material for, 228
    microscope, 200

  Bacteriology, pathogenic, definition of, 231
    reasons for study of, 217
    as a science, 17, 32

  Bacteriolysin, 272

  Bacteriopurpurin, 62, 63, 112

  Bacteriotropin, 281

  _Bacterium abortus_, agglutinin of, 265
    _coli_ in autoinfection, 234
      gas formation by, 95
      oxygen limits for, 77
      pneumonia through intestinal route, 246
      in preparation of sugar broths, 176
    definition of, 62, 67, 70
    _enteriditis_, cause of food poisoning, 104
    _fluorescens_, oxygen limits, 77
    _typhosum_, 73
      agglutinin, 265
      in phenol coefficient method, 166
      pneumonia through intestinal route, 246

  Ballon pipette, 193

  Balsam, mounting in, 207

  BARBER, 253
    apparatus, 196

  Barnyards, disinfection of, 167

  Baskets, wire, 184

  BASSI, 27
    silkworm disease, 34

  BASTIAN, 24

  BAUMGÄRTNER, 256

  Beaded growth, 221

  Bed-bugs, 241

  Beds, contact, 116
    hot, 117

  Beer, pasteurization of, 141, 144, 145

  _Beggiatoa_, 63

  BEGGIATOACEÆ, 63

  BEHRING, 30

  BELFANTI, 271

  BERG, 27, 34

  Berkefeld filter, 154

  Bichloride of mercury as disinfectant, 158

  BILHARZ, 28, 35

  Bilharzia disease, 28, 35

  Biochemical reactions, definition of, 87

  Biological relationships, immunity reactions, 255, 270

  Bipolar germination of spore, 48

  Bismarck brown, 209, 212

  Black-leg, 51, 73, 238, 243, 248, 251
    vaccine, 254

  Bleaching powder as disinfectant, 158

  Blood, collection of, 228
    cytolytic power of, 272
    detection of, 269
    serum, liquid, sterilization of, 182
      Loeffler’s, 182
      medium, preparation of, 182, 183
      sterilization of, 182
    vessels in dissemination of organisms, 247

  Blue milk, bacterial cause of, 34
      fermentation of, 31, 34

  BOEHM, 27, 34

  Boiling as disinfectant, 133

  Boils, 237, 240, 243

  BOLLINGER, 29, 30, 35, 36

  BONNET, 20, 33

  BORDET, 271

  _Botrytis bassiana_, 27, 34

  Bottles, staining of, 206

  Bougies, 154

  Bouillon, 173

  BOYER, 260

  Bread, salt rising, 95, 97

  Bronchopneumonia, 233, 246

  Broth, appearance of growth in, 218
    extract of, 176
    glycerine, 176
    medium, 173
    nitrate, 177
    sterilization of, 174
    sugar, 176

  Brownian movement, 47, 203

  Brushes, disinfection of, 169

  Bubonic plague, 239

  BUCHNER, 271

  Budding of yeasts, 37

  Bulgarian fermented milk, 98

  Burning as disinfectant, 132

  Burying as disinfectant, 154

  BÜTSCHLI, 41, 43

  Butter, 97
    bacilli, staining of, 209
    rancidity of, 101

  Butyric acid fermentation, 32, 99

  Buzzards, 241


  C

  CABBAGE disease due to protozoa, 36

  Cadaverin, 104

  CAIGNARD-LATOUR, 31, 34

  Calcium hypochlorite as disinfectant, 158
    oxide as disinfectant, 158

  Candles, filter, 153, 154

  Canned goods, food poisoning by, 104
    spoilage of, 51

  Canning, introduced, 21, 34
    principles involved, 133

  Capsule, 44, 45
    of spore, 48
    staining of, 210

  Carbohydrates in bacterial cell, 84
    fermentation of, 93-101

  Carbol-fuchsin, 206

  Carbolic acid as antiseptic, 159
      as disinfectant, 159
      first used, 29

  Carbol-xylol, 209

  Carbon cycle, 107
    dioxide, 108
    function of, in bacteria, 88, 101
    oxidation of, 114
    in proteins, liberation of, 105
    source of, 88
    uses of, 88, 101

  CARBONI, 271

  CARDANO, 18

  Carrier problem, solution of, 240

  Carriers, 239
    accidental, 241
    carrion eating animals as, 241
    control of, 240
    intermediate hosts as, 242
    protective measures against, 242
    universal, 240
    of unknown organisms, 239

  Cars, stock, disinfection of, 170

  Catalase, 125

  Catalytic agents, function of, 123

  Catalyzer, 123

  Cattle, 227

  Causation of disease, 24, 128

  Cell, constituents of, 84
    contents of, 41, 83
    forms of, 58, 59
      staining for, 212
      typical, 52
    groupings, 55, 58, 59
      staining for, 195
    metabolism, 90
    structures of, 41
    wall, 41, 59
      composition of, 83

  Cells, chemical stimuli of, 257
    overproduction activity of, 258
    specific chemical stimuli of, 258

  Cellular theory of immunity, 256, 280

  Cellulose, definition of, 83
    occurrence of, 83

  Chain, 56

  Channels of infection, 243
      alimentary tract, 246
      conjunctive, 244
      external auditory meatus, 244
        genitalia, 245
      intestines, 246
      lungs, 245
      milk glands, 244
      mouth cavity, 244
      mucosæ, 244
      nasal cavity, 244
      pharynx, 245
      skin, 243
      stomach, 246
      tonsils, 245

  Chaos, 25

  Characteristic groupings, 58

  Characteristics of enzymes, 121
    of toxins, 126

  CHARRIN, 265

  Chart, descriptive, 217

  CHAUVEAU, 256

  Cheese, eyes in, 96
    failures, 110
    Limburger, 101
    odor of, 99
    poisoning, 104
    ripening of, 35

  Chemical composition of bacteria, 39, 81, 85
    elements in bacteria, 82
    disinfectants, action of, 156-163
    stimuli, 257-260
    theory, fundamentals of, 256

  Chemotherapy, 249, 255

  CHEVREUIL, 21, 27, 31, 34

  Chicken cholera, 30
    pox, 239, 246

  Chief agglutinin, 267
    cell, 267

  Chitin, 72

  CHLAMYDOBACTERIACEÆ, 63

  _Chlamydothrix_, 63

  Chloride of lime as disinfectant, 158

  Chlorine as disinfectant, 157

  Chloroform as antiseptic, 162
    as disinfectant, 162

  Chlorophyl, 37, 112

  Chlorosis, Egyptian, 27, 35

  Cholera, Asiatic, carriers of, 239
      organisms in, 27, 34
        facultative saprophytes, 238
        path of elimination of, 248
          of entrance of, 246
        relation to moisture, 73
        specific location of, 249
    hog, 242, 248, 252

  Cholesterins as cell constituents, 84

  Chromogenesis, 112

  Chromoparic, 112

  Chromophoric, 112

  Chronic disease, 232

  Chronological table, 33-36

  Chymosin, 124

  Circulation of carbon, 107
    of nitrogen, 107
    of phosphorus, 107
    of sulphur, 108

  Classification, advantage of, 59
    early, 33, 35, 59
    Migula’s, 62-63
    S. A. B., 63-70

  Cleaning of slides, 207

  Clearing of sections, 209

  Closed space disinfection, 161

  _Clostridium_, 49
    _botulinum_, 87, 104, 128, 238, 261
    _pasteurianum_, 118
    _tetani_, 128, 209, 233, 261, 263

  Clothing, disinfection of, 170

  Coagglutinins, 267

  Coagulases, 124

  Coagulating enzymes, 124

  Coagulation temperature of proteins, 51

  Coal, spontaneous heating of, 88

  Coamboceptors, 274

  Cobra, 275

  COCCACEÆ, 62, 66, 68

  Coccus, appearance of, on dividing, 57
    cell form of, 52
      groupings of, 52, 56, 57
    division of, 52

  Coenzymes, 122

  COHN, 28, 33, 35, 59

  Cold as antiseptic, 148
    incubator, 215
    storage, 148

  Colds, due to universal carriers, 240
    path of entrance of, 244
    vaccines in, 241

  Colonies, characteristics of plate, 223-226
    definition of, 173

  Color production, 112

  Colorimetric method of standardization, 175

  Combustion, spontaneous, 116

  Commensal, 87

  Commercial preparation of lactic acid, 99
    products, why keep, 131
    vaccines, 285

  Communicable disease, 232

  Complement, 273
    deviation test, 277
    effect of temperature on, 274
    fixation test, 276-279
    lecithin as, 274
    relation to toxins and enzymes, 273
    source of, 277

  Complementoid, 274

  Complementophil haptophore, 273

  Complements, nature of, 274

  Composition, chemical, 81-85
      related to fungi, 39
      relation to food, 81

  Concentration of antitoxin, 264

  Condenser, 200

  Conditions for growth, general, 72
      maximum, 72
      minimum, 72
      optimum, 72
      spore formation, 51

  Congenital immunity, 251, 252

  Conjunctiva as path of entrance, 244

  Constant temperature apparatus, 213

  Contact beds, 116

  Contagion, direct and indirect, 34

  Contagious abortion, agglutination test, 268
      complement-fixation text, 277
      path of elimination, 248
        of entrance, 245

  Contagium, definition of, 232
    vivum theory, 25, 28, 33

  Contamination of food by carriers, 241

  Continuous pasteurization, 141

  Contrast stains, 205

  Convalescents, control of, 239-240

  CORNALIA, 29

  Corpuscles, Babes-Ernst, 45
    red, in complement-fixation test, 278, 279
      malaria, etc., in, 249

  Corrosive sublimate as disinfectant, 158

  _Corynebacterium diphtheriæ_, 64, 69, 128, 233, 234, 261, 263

  Coryza, acute, 244

  Cotton plugs, 21, 184

  Coughing, 248

  Crateriform liquefaction, 221

  Cream ripening, 97

  CREITE, 271

  _Crenothrix_, 61

  Creolin as disinfectant, 160

  Cresols as disinfectants, 159

  Culture, definition of, 171
    medium, definition of, 171
      essentials of, 172
      inoculation of, 186, 192
      kinds of, 172
      liquid, 172, 173
      methods of using, 184
      optimum moisture for, 73
      plating of, 188
      reaction of, 81, 216
      selective, 198, 199
      solid, 172, 173
      standardization of, 174, 175
      synthetic, 183
      titration of, 174
      use of, 173
    tubes, description of, 184

  Cultures, anaërobic, 188-192
    from internal organs, 229
    mass, 188
    plate, 188
    potato, 186
    puncture, 185
    pure, definition of, 171
      isolation of, 194-199
    slant, 186
    slope, 186
    stab, 185

  Curled edge, 225

  Cutaneous inoculation, 228

  Cycle, carbon, 107
    nitrogen, 107
    phosphorus, 107
    sulphur, 108

  Cystitis, 234

  Cytolysin, 272

  Cytolysins, 271-279

  Cytolytic, 272
    power of blood, 272
    serums, failure of, 275
    substances in immunity, 295

  Cytophil group, 273

  Cytoplasm, 41

  Cytotoxic, 272


  D

  DALLERA, 289

  Dark field illumination, 204

  DAVAINE, 28, 35

  Death-point, thermal, 75
      determination of, 215

  Decomposition, how caused, 108
    importance of, 108
    of urea, 106

  Deep culture tubes, 190-191

  Degeneration forms, 54

  Delousing method in typhus, 242

  DE MARTIN, 35

  Denitrification, 114

  Deodorant, 131

  Descriptive chart, 217

  Diagnosis, agglutination test in, 265-267
    anaphylaxis in, 292
    complement-fixation test in, 277
    immunity reactions in, 255
    material for bacteriological, 228-229
    precipitin test in, 269

  Diastase, 124

  Diffusion of food through cell wall, 41

  Digestion of proteins, 102

  Dilution method of isolation, 194
    plates, 194, 195

  Dimethylamine, structural formula, 103

  Diphtheria antitoxin, 30, 263, 264
    bacilli, granules in, 45
      involution forms, 54
    carriers, 239
    location of, 245, 249
    path of entrance, 245
    toxin, M. L. D., 264

  Diplobacillus, 55

  Diplococcus, 56

  _Diplococcus_, 66, 69

  Diplospirillum, 55

  Discharges, 228
    intestinal, 248
    nasal, 248
    urethral, 248
    vaginal, 248

  Discontinuous sterilization, 133

  Disease, acute, 233
    of animals to man, 232
    Bilharzia, 28, 35
    cabbage, 30, 35
    causation of, 24, 128
    communicable, 232
    contagious, 34, 232
    of flies, 28, 35
    germ, 25, 27, 33
    hookworm, 28, 35
    infectious, 232, 240
    Johne’s, 246, 248
    non-specific, 233
    protozoal, eradication, 242
      transmission, 242
    silkworm, 27, 29, 34, 35
    skin, 243
    specific, 27, 30, 233
    transmission of, 26, 232

  Dishes, Petri, 181

  Disinfectant, 131
    action of anilin dyes, 162
    closed space, 161
    dry heat as, 133
    moist heat as, 132, 133
    standardization of, 165
    steam as, 132, 133

  Disinfectants, chemical, action of, 156-163
      first experiment in, 21
    factors affecting, 164-165

  Disinfection, agents in, 131-163
    by boiling, 132, 133
    by burning, 132
    by burying, 154
    definition of, 130
    first chemical, 34
    hot air, 21, 133
    physical agents, 131-155
    practical, 166-170
    precautions in, 170
    puerperal fever, 28, 34
    steam, 134-138
    surgeon’s hands, 28

  Dissemination of organisms, 247

  DISTASO, 42, 43

  Distilling sour mash, 98

  Division, planes of, 55-58
    rate of, 43, 91

  DOBELL, 43

  DORSET, 84

  Dosage of vaccines, 286

  Dose, minimum lethal, 264
    standard test, 264

  DOUGLAS, 42, 43, 280

  Dourine, 245, 248

  Drumstick spore, 49

  Dry heat, 21, 133

  Drying, 131, 132

  DUBINI, 27, 34

  Ducrey’s bacillus, 245

  Dunham’s peptone, 177

  DURHAM, 265

  Dyes, anilin, as antiseptics, 162
      introduction of, 30
      as stains, 204

  Dysenteries, 242, 246, 248, 249

  Dysentery, amebic, 29, 35
    tropical, 29


  E

  ECTOPLASM, 41

  Edema, malignant, 237, 243

  Edge of colony, 225

  Effuse colony, 224

  Egg sensitization, 292

  EHRENBERG, 33, 34

  EHRLICH, 256, 276

  Ehrlich’s theory, 256-260

  EICHSTEDT, 28, 34

  Electric milk purifier, 152

  Electricity, 79, 150

  Elements in bacteria, 82, 86, 88, 89

  Elimination of organisms, 248

  _Empusa muscæ_, 28, 29, 35

  Emulsin, 122

  Endo-enzymes, 126

  Endogenous infection, 235

  Endoplasm, 41

  Endotoxins, 128, 276

  Energy relationships, 39
    transformations, 86-90

  Ensilage, 98

  Enteritis, 233

  Entire edge, 225

  Entrance of organisms, 243-246, 247

  Environmental conditions, 72, 130, 213

  Enzymes, 84, 121-126
    in anaphylaxis, 291
    in immunity, 295

  Enzymoid, 262

  Epidemics, 241

  Epitheliolysin, 272

  Eosin, 204

  Equatorial spore, 49
      germination of, 48, 49

  Eradication of disease, 236, 242

  Erysipelas, hog, 248

  _Erythrobacillus prodigiosus_, 66, 68, 70, 77, 113

  Essential structures, 41

  Essentials of a culture medium, 172

  Esters, 84, 110

  Ether as disinfectant, 162

  EUBACTERIA, 62

  Exanthemata, 248

  Exhaustion factor in immunity, 251
    theory of immunity 256

  Existence, conditions for, 72

  Exo-enzymes, 126

  Exogenous infection, 235

  Exotoxins, 128

  Experiment, Pasteur’s, 21
    Schroeder and Dusch’s, 22
    Schultze’s, 21
    Schwann’s, 22
    Tyndall’s, 24

  Experimental animals, 227

  External auditory meatus, 244
    genitalia, 245

  Extracellular enzymes, 126

  Extract broth, 176

  Eyes in cheese, 96, 97


  F

  Factors affecting disinfectants, 164, 165
      immunity, 250, 251
    in immunity to disease, 295

  Facultative, 215
    aërobes, 76
    anaërobes, 76, 192
    parasites, 87
    saprophytes, 238

  Failure of cytolytic serums, 275
    of vaccines, 286

  Fat colors, 112
    splitting enzymes, 124

  Father of bacteriology, 19
    of microscope, 19

  Fats as antigens, 260
    occurrence of, 84
    rancidity of, 101
    in sewage disposal, 101
    splitting of, 101

  Favus, 27, 34, 243

  Feces, bacteria in, 72

  Feeding, as inoculating method, 228

  FEINBERG, 43

  Ferment, organized, 126
    unorganized, 126

  Fermentation, 31, 93
    acid, 93, 96
      acetic, 32, 99
      butyric, 32, 99
    alcoholic, 31, 34, 100
    ammoniacal, 32
    bacterial, 32
    blue milk, 31, 34
    of carbohydrates, 93-101
    definition of, 93
    gaseous, 93, 94, 96
    tubes, 184, 190
    yeast, 34, 99

  Fermented milk, Bulgarian, 98

  Fever, due to invisible organisms, 25
    Malta, 268
    recurrent, 29, 35
    Rocky Mountain spotted, 242
    scarlet, 246, 248
    Texas, 232, 233, 242
    typhoid, 232, 248
    typhus, 242
    yellow, 242

  Fibrin ferment, 124

  Filament, 56

  Filiform growth, 221

  Film, fixing of, 207
    preparation of, 207

  Filter, Berkefeld, 154
    candles, 153-154
    Mandler, 154
    Pasteur-Chamberland, 154
    sprinkling, 115, 116

  Filterable virus, 234

  Filtration, 152-154

  First order, receptors of, 261, 262

  FISCHER, 42, 45

  Fixation test, complement, 276

  Fixed virus, 253

  Fixing of film, 207

  Flagella, 45-47
    staining of, 210

  Flash process of pasteurization, 145

  Fleas, 241

  FLEXNER, 276

  Flies, 28, 35, 241

  FLÜGGE, 271

  FODOR, von, 271

  Food adulteration, complement-fixation test in, 279
      immunity reactions in, 255
      precipitin test in, 269

  Food contamination by carriers, 241
    poisoning, 87, 104, 238
    requirement compared with man, 92
    uses of, 86

  Foot-and-mouth disease, 244, 248

  Forage poisoning, 87

  Foreign body pneumonia, 245

  Formaldehyde as disinfectant, 160

  Formalin, 160

  Formol, 160

  Forms, cell, 52-54
    degeneration, 54
    growth, 55
    involution, 54
    study of, 32-34

  Fox fire, 111

  Foxes, 241

  FRACASTORIUS, 25, 33

  Free acid, 175
    receptors, 259
    spores, 48

  Fruiting organs, 37

  FUCHS, 31, 34

  Fuchsin, 205
    anilin, 205
    carbol, 206

  Fungi, bacteria as, 37

  Funnel-shaped liquefaction, 221


  G

  GABBET’S blue, 206
    method of staining, 209

  Gall-bladder, 248

  Galvanotaxis, 79

  Gas formation in cheese, 96, 97
    natural, 95
    production of, 110

  Gaseous fermentation, 93-95

  GASPARD, 26, 34

  Gelatin, advantage of, 178
    composition of, 179
    cultures, first used, 30, 36
    liquefaction of, 103
    medium, 177
    plating of, 188
    standardization of, 178
    sterilization of, 178

  Gemmation, 37

  General conditions for growth, 72
    infections, vaccines in, 286

  Generation, spontaneous, 17-24

  Generic names introduced, 33

  Genitals, 245

  Gentian violet, selective action of, 162
      stain, 205

  Germ, free air, 153
    theory of disease, 25

  German measles, 233

  Germination of spore, 48

  Germs, 33
    in air, 24, 35

  GESCHEIDEL, 271

  Giemsa stain, 43

  Glanders, 26, 233, 238, 244, 248, 249, 268, 277

  Glands, mammary, 248
    salivary, 248

  GLEICHEN, 32

  Globulin in bacteria, 84

  Glycerine broth, 176

  Glycerinized potato, 172

  Glycogen as cell constituent, 84

  Goats, 227

  Gonidia, 63

  Gonococcus, 245

  Gonorrhea, 248, 249

  Good health, 296

  Grain rust, 26, 34

  Gram positive organisms, 162, 208
    negative organisms, 162, 208

  Gram’s method of staining, 208
    solution, 208

  Granular edge, 225

  Granules, metachromatic, 212
    Neisser’s, 45
    polar, 45

  Granulose in bacteria, 84

  Grape juice, pasteurization of, 141

  Grass bacilli, 209

  Green plants, N nutrition of, 118

  GRIESINGER, 27, 28, 35

  Group, agglutinating, 266
    haptophore, 261, 262, 266, 270, 273
    precipitating, 270
    toxophore, 261, 262
    zymophore, 262, 270, 273

  Groupings, cell, 55-58

  Growth, appearance in media, 217

  _Gruber_, 265, 268

  _Gruby_, 28, 34

  Gum-like substance in bacteria, 83


  H

  HAECKEL, 280

  Hanging drop slide, 203

  Haptophore, complementophil, 273
    cytophil, 273
    group, 261, 262, 266, 270, 273

  Harness, disinfection of, 169

  Hay fever, 263, 292

  Health, 296

  Heat as disinfectant, 132-144
    due to oxidation, 112
    production of, 116

  Heated serum, 271, 277, 278, 279

  Heating of manure, 116

  HELLMICH, 84

  HELMONT, VAN, 18

  Hemagglutinin, 265

  Hemicellulose, 83

  Hemolysin, 272

  Hemolytic amboceptor, 278

  Hemorrhagic septicemia, 246

  HENLE, 27, 34, 233

  HERICOURT, 289

  Herpes tonsurans, 28, 34

  HESSELING, von, 32

  Heterologous sera, 276

  Heterotrophic, 86

  HILL, 33

  HILTON, 27

  HOFFMAN, 24

  Hog cholera, 231, 242, 248, 252, 253
    erysipelas, 248

  Holders, 143

  HOLMES, 28, 34

  Homologous sera, 276

  Hookworm disease, 28, 34

  Horses, 227, 263

  Host, 87

  Hot beds, 117

  Hunger in immunity, 251

  Hydrochloric acid, 246

  Hydrogen, function of, 98
    ion concentration standardization, 175, 176
    oxidation of, 114
    peroxide, 162
    sulphide, 115

  Hydrophobia, 249

  Hydrostatic pressure, 79

  Hygienic laboratory, 165

  Hypochlorites, 157, 158


  I

  Ice cream poisoning, 104

  Identification of bacteria, 216, 217
      in blood, 269
      in meat, 269
      in milk, 269

  Immersion oil, 201

  Immunity, 236, 250-296
    acquired, 251, 252
    active, 251, 252-255
    antibacterial, 254, 255
    antitoxic, 254, 255
    artificial, 251, 252
    classification of, 251
    congenital, 251
    factors in, 295
      modifying, 250
    inherited, 251, 252
    natural, 295
    passive, 251, 252, 253
    to protein, 290
    reactions, value, 255
    relative, 250
    summary of, 295
    theories of, 256

  Inactivate, 272

  Incubation period, 26, 232

  Incubator, 213
    cold, 215
    room, 213

  Index, chronological, 31
    opsonic, 281
    phagocytic, 281

  Indicator, 278

  Indol, 104

  Infection, 232
    auto, 234
    channels of, 243
    endogenous, 235
    exogenous, 235
    mixed, 234
    primary, 234
    secondary, 234
    wound, 17, 25, 26, 27, 30, 34, 36, 233, 234, 240, 243, 248

  Infectious diseases, 232
      control of, 240

  Infective organisms, specificity of location, 249

  Infestation, 232

  Infested, 232

  Influenza, 239, 241, 246

  Infusoria, 33

  Inhalation, 228

  Inherited immunity, 251, 252

  Inoculation of animals, 227
      uses of, 227
    of cultures, 186, 188
    definition of, 192
    methods of, 227
    needles, 192

  Inoculations, first protective, 30
    of smallpox, 24

  Insects, 241, 242

  Instruments, sterilization, 136, 167

  Intracardiac, 228

  Intracellular enzyme, 166

  Invasion, 232

  Invertase, 124

  Involution forms, 53, 212

  Iodine, 157

  Iron bacteria, 86
    function of, 89

  Irregular forms, 53

  Isolation of anaërobes, 190
    of pure cultures, aids in, 197-199
      methods, 194-196

  Itch mite, 27, 34


  J

  JABLOT, 32

  Jack-o-lantern, 105

  Jar, Novy, 192

  JENNER, 26, 34, 253

  Johne’s disease, 248


  K

  KETTE, 32, 35

  Kidneys, 248

  Kinase, 125

  KIRCHER, 18, 25, 33

  KLEBS, 29, 35

  KLENCKE, 28, 34

  KOCH, 17, 27, 29, 30, 33, 36

  Koch’s postulates, 233

  KRAUS, 268

  KRUSE, 254

  KÜCHENMEISTER, 28, 35


  L

  LAB, 124

  Lachrymal canal, 244

  Lactacidase, 125

  Lactic acid bacteria, 97
    fermentation, 96-99

  LANCISI, 25, 33

  LANDOIS, 271

  LATOUR, 31, 34

  LAVERAN, 25, 30

  Lecithin as antigen, 279
    as cell constituent, 84
    as complement, 274

  LEEUWENHOEK, 19, 32, 33

  Legumes, 118

  LEIDY, 27, 33, 34, 35

  LE MOIGNAC, 284

  Leprosy, 233, 244, 249

  LESSER, 32

  Lethal dose, 264

  Leukocytes, washing of, 281

  Lice as carriers, 241

  LIEBERT, 28, 34

  Light, action on bacteria, 75
    as disinfectant, 148
    production of, 111

  LINNÆUS, 25

  Lipase, 124

  Lipochromes, 113

  Lipoids as antigen, 274

  Lipovaccines, 284

  Liquefaction of gelatin, 221
    of protein, 103

  Liquid blood serum, 182
    manure, disinfection of, 169
    media, 172

  Liquids, sterilization of, 153

  LISTER, 29, 30, 35

  Litmus milk, 177

  Living bacteria, examination of, 201
    cause theory, 28, 33

  Localized infections, vaccines in, 286

  Location of organisms, specificity of, 249

  Lockjaw, 231, 233

  Loeffler’s blood serum, 182
    blue, 206

  Loop needles, 193

  Lophotrichic, 46

  LÖSCH, 29, 35

  Lungs, 245, 249

  Lye washes as disinfectants, 159

  Lymph channels in dissemination, 247

  Lysol as disinfectant, 160


  M

  MCCLINTOCK, 165

  MCCOY, 160

  Macrococcus, 52

  Macroscopic agglutination, 265

  Malaria, 25, 30, 32, 242

  Malarial parasite, 30, 249

  Malignant edema, 237, 243

  Mallease reaction, 269

  Mallein test, 292

  Malta fever, 268

  Mammary glands, 248

  Mandler filter, 154

  Manure, liquid, disinfection of, 169
      heating of, 40

  _Margaropus annulatus_, 242

  MARTIN, 32

  Mass cultures, 188

  MASSART, 42

  Maximum conditions, 72, 73, 74, 76

  Measles, 246, 248, 250
    German, 233, 239

  Measly pork, 28

  Measurement of bacteria, 203
      special unit of, 40

  Meat broth, 173
    identification of, 269
    juice, 173
    poisoning, 104

  Mechanical vibration, 80

  Medico-legal examination, 269, 279, 293

  Medium. _See_ Culture medium

  Meningitis, 239, 244

  Meningococcus, 244

  Mercuric chloride, 158

  Merismopedia, 57

  Metabiosis, 103

  Metabolism, 86-91

  Metachromatic granules, 44, 45, 59, 212

  Metastases, 235

  Metatrophic, 86

  METCHNIKOFF, 256, 280

  Methods of inoculation of animals, 227
      of cultures, 186-188
    of obtaining pure cultures, 194

  Methylamine, 103

  Methylene blue, 205, 206

  Mice, white, 227

  Microbiology, 231

  Micrococcus, 52, 60, 62, 66, 68, 69, 245

  Micrometer, 203

  Micromillimeter, 40

  Micron, 40

  Microörganisms, 32

  Microscope, improvements in, 30, 36
    invention, 19
    Leeuwenhoek’s, 19
    use of, 200

  Microspira, 61, 63

  _Microsporon furfur_, 28, 34

  Middle ear, 241

  Migula’s classification, 62

  Milk, blue, 31, 34
    Bulgarian, 98
    digestion of, 102
    flavors in, 110
    glands, 244
    identification of, 269
    litmus, 177
    pasteurization of, 141, 144-147
    as path of elimination, 248
    preparation of, 177
    purifier, electric, 152
    souring of, 32
    sterilization of, 177
    tuberculous, 248

  Minimum conditions, 72, 73, 74, 77
    lethal dose, 264

  Mirror, use of, 200

  Mixed infection, 234
    vaccine, 285

  Mixotrophic, 86

  M. L. D., 264

  MOHLER, 167

  Moist heat, 133

  Moisture, 73

  Mold colonies, 226

  Molds in alcoholic fermentation, 100
    in relation to bacteria, 37, 39

  Molecular respiration, 88, 89

  Monas, 33

  Monkeys, 227

  Monotrichic, 45

  MONTAGUE, 24

  Mordants, 204, 211

  Morphology, 41-58
    in identification, 171, 212

  Mosquitoes and malaria, 25, 242

  Motile bacteria, 45

  Motion of bacteria, 47
    Brownian, 47, 203

  Mounting in balsam, 207

  Mouth cavity, 244

  Mu, 40

  Mucosæ as channels of infection, 244

  MÜLLER, 33, 34, 59

  Mumps, 239

  Municipal disinfection, 170

  MÜNTZ, 32, 35

  Muscardine, 34

  Mycelia, 39, 226

  _Mycobacteriaceæ_; 64

  _Mycobacterium_, 64, 69
    of Johne’s disease, 209
    _lepræ_, 209
    _smegmatis_, 209
    _tuberculosis_, 83, 176, 209

  Mycoproteid, 83

  Mycorrhiza, 119

  Myxomycetes, 38


  N

  NÄGELI, 29, 35

  Nasal cavity, 244
    discharges, 248

  Natural gas, 95
    immunity, 251, 252, 296

  NEEDHAM, 20, 33

  Needles, inoculation, 192

  Negative complement-fixation test, 278
    phase, 287

  Neisser’s granules, 45
    stain, 212

  NENCKI, 83

  Nephrolysin, 272

  NEUFELD, 281

  Neurin, 104

  Neurotoxin, 272

  NEUVEL, 43

  Nichrome wire, 193

  Nitrate broth, 177

  Nitrates in soil, 115

  Nitric bacteria, 114

  Nitrification, 32, 35

  Nitrite, oxidation of, 114

  Nitrogen, absorption of, 117
    in bacterial cell, 89
    circulation, 109
    cycle, 107
    fertilizers, 120
    liberation, 104
    nutrition of green plants, 118
    use of, 103

  Nitrous bacteria, 114

  Non-pathogenic, 87

  Non-specific disease, 233

  Normal agglutinins, 266
    serum, 272

  _Nosema bombycis_, 29, 35

  NOVY, 183
    jar, 192

  Noxious retention theory, 255

  Nuclein, 42, 43

  Nucleoprotein, 43

  Nucleus, 42, 43

  Nutrition of green plants, 118

  NUTTAL, 271


  O

  OBERMEIER, 29, 35

  Objective, oil immersion, 200, 201

  Oblique germination of spore, 48

  Occurrence of bacteria, 71

  Official classification, 59

  _Oidium albicans_, 27, 34

  Oil bath, 167
    essential for clearing, 209
    immersion objective, 200, 201
    relation of bacteria to, 116

  OMODEI, 27

  Opsonic index, 281, 282, 287
      method, 282
      power, 287

  Opsonin, 281

  Opsonins, 281, 282, 295

  Optimum conditions, 72, 73, 74

  Order, receptors of first, 261-264
      of second, 265-270, 281
      of third, 271-279

  Organic acids, 84, 110
    catalyzers, 123

  Organisms, dissemination of, in body, 247
    filterable, 234
    pathogenic, elimination of, 248
      entrance of, 243-246, 247
    specific relation to tissue, 249
    ultramicroscopic, 234

  Organized ferments, 126

  Osmotic pressure, 78, 149, 216

  Otitis media, 244

  OTTO, 289

  Overproduction theory, 257, 258

  OWEN, 27, 34

  Oxidation, 114, 115

  Oxidizing enzymes, 125

  Oxygen, compressed, 77
    as disinfectant, 156
    function of, 88
    nascent, 77
    relationships, 215, 220
    requirement, 88
    source, 76, 77

  Oyster sensitization, 292

  OZNAM, 26

  Ozone, 77, 150, 157


  P

  PANCREAS, 248

  Papillate, 221

  PAGET, 27, 34

  Paraffin oil, 190

  Parasite, 87
    facultative, 87
    strict, 87

  PARODKO, 77

  Partial agglutinin, 267
    amboceptor, 274

  Passive immunity, 251, 252

  PASTEUR, 17, 21, 29, 30, 31, 32, 35, 253, 256, 283
    flask, 21, 23, 24, 193
    treatment of rabies, 253

  Pasteur-Chamberland filter, 154

  Pasteurization, 139-147
    continuous, 141
    flash process, 145

  Pathogenic, 87
    bacteria, definition of, 231
      outside the body, 237
    bacteriology, scope of, 235
    organisms, destroyed by boiling, 133
      elimination of, 248
      entrance of, 243-247

  Paths of elimination, 248
    of entrance, 243-247

  PEACOCK, 26

  Pebrine, 29, 35

  Pedesis, 47

  Peptone solution, Dunham’s, 177

  Period of incubation, 26, 232

  Peritonitis, 234

  Peritrichic, 46

  _Peronospora infestans_, 28, 34

  PERTY, 33, 35

  Pet animals, 241

  Petri dishes, 181, 188

  Petroleum, 95

  PFEIFFER, 271

  Pfeiffer’s phenomenon, 271

  _Pfeifferella mallei_, 65, 69, 265

  Phagocytes, 247

  Phagocytic index, 281

  Phagocytosis, 243, 280-288, 295
    theory, 256

  Pharynx, 245

  Phase, negative, 287
    positive, 287

  Phenol coefficient, 165, 166
    as disinfectant, 159
    production of, 104, 111

  Phenolphthalein, 174

  Phenomenon, anaphylactic, 292
    Arthus’, 289
    Pfeiffer’s, 271

  Phosphate reduction, 114
    rock, 115

  Phosphorescence, 111

  Phosphorus cycle, 108
    in proteins, 105
    uses of, 89

  Photogenesis, 111

  Physical agents for disinfection, 131-155

  Physiological activities, 93-129
    definition of, 87
    in identification, 216

  Physiology of bacteria, 71-171

  Phytotoxins, 127, 128

  Pickling, 98

  Pigeons, 227

  Pigments, 84, 112, 113

  Pimples, 234, 240, 243

  PINOY, 284

  Pipettes for inoculation, 193

  _Piroplasma bigeminum_, 233, 242, 249

  Piroplasmoses, 242, 249

  PIRQUET, von, 289

  Pityriasis versicolor, 28, 34

  Plague, 246

  Planes of division, 56, 57

  _Planococcus_, 62

  _Planosarcina_, 62

  Plants and animals, 39

  _Plasmodiophora brassicæ_, 30, 36

  Plasmolysis, 41, 42, 78

  Plasmoptysis, 42, 78

  Plate colonies, study of, 224-226
    cultures, 180, 188, 191

  Plates, dilution, 194, 195
    gelatin first used, 30, 36

  Platinum needles, 193

  Plectridium, 49

  PLENCIZ, 26, 31

  Plugs, cotton, 21, 184

  Pneumococcus, 240, 245

  Pneumonia, 240, 245, 246, 248
    vaccination against, 241

  Poisoning, cheese, 104
    food, 87, 104, 238
    ice cream, 104
    meat, 104

  Polar germination, 48, 49
    granules, 45

  Poliomyelitis, 244

  POLLENDER, 28, 35

  Polysaccharides, fermentation of, 95

  Polyvalent vaccine, 285

  Pork, measly, 28

  Position of bacteria, 37
    of flagella, 45, 46
    of spore, 49

  Positive phase, 287
    test, 278

  Postulates, Koch’s, 233

  Potato, acidity of, 182
    glycerinized, 182
    media, 180-182
    rot, 28, 34

  Power, opsonic, 287

  Practical sterilization and disinfection, 166-170

  _Pragmidiothrix_, 63

  Precipitinogen, 269

  Precipitinoid, 270

  Precipitins, 268-270
    anti-, 270

  Preparation of antitoxin, 263
    of bacterial vaccines, 283, 284
    of film, 207

  Preservation of slides, 207, 208

  Preservative, alcohol as, 160
    in vaccine, 284

  Pressure, hydrostatic, 79
    osmotic, 78, 149
    oxygen, 76, 77
    steam, 136
    sterilization, 136-139

  Prevention of disease, 235, 236, 253, 255, 283

  Preventive vaccination, colds, 241
      pneumonia, 241
      rabies, 253
      smallpox, 26, 34, 253
    vaccines, autogenous, 285
      stock, 285

  PREVOST, 26

  Primary infection, 234

  Process kettle, 137

  Pro-enzyme, 121

  Prophylaxis, 289

  Protamine in bacteria, 84

  Protease, 124

  Protective inoculation, first, 30

  Protein in bacteria, 84
    coagulation temperature, 51
    composition of, 102
    decomposition of, 105
    differentiation of, 255
    foreign, 289
    identification of, 293
    immunity, 290
    putrefaction of, 102-109
    split products of, 291
    splitting of, 106
    structure of, 291
    synthesis of, 113

  _Proteus vulgaris_, 67, 70, 77

  Protoautotrophic, 115

  Protoplasm, 41, 59

  Prototrophic, 86

  Protozoa, cause of disease, 30
    cell wall in, 41
    in intermediate hosts, 242
    relation to bacteria, 40
    specificity of localization, 249

  Protozoal diseases, transmission of, 242

  PSEUDOMONADACEÆ, 65, 70

  _Pseudomonas pyocyanea_, 62, 65, 70, 128, 265

  Ptomaines, 103, 104

  _Puccinia graminis_, 26, 34

  Puerperal fever, 28, 34

  Punctiform colonies, 223

  Puncture cultures, 185

  Pure culture, 171, 194-199

  Purification of streams, 73
    of water, 150

  Purin bases in bacteria, 84

  Pus cocci, 73
    infectious, 26
    organisms in, 35

  Putrefaction, 27, 31, 33
    definition of, 102
    of proteins, 102-109
      end products of, 103
      in soil, 106

  Putrescin, 104


  Q

  QUARANTINE, 239
    disinfection, 170

  Quicklime as a disinfectant, 155, 158

  Quinsy, 245


  R

  RABBITS, 227

  Rabies, bacteriological examination in, 229
    Pasteur treatment of, 253
    path of elimination in, 248
    transmission of, 239
    specificity of localization in, 249

  Räbiger’s method of staining, 210

  Radiations, 79

  Radium, 79

  Rancidity of butter, 101

  Rashes, serum, 289
    urticarial, 292

  Rate of division, 43, 91
    of movement, 45

  Rats, 227, 241

  RAYER, 28, 35

  Reaction of medium, 81, 174, 175, 216

  Reactions, biochemical, 87
    immunity, 255, 269, 279, 292, 293
    surface, 91

  REAUMUR, 33

  Receptors, 257, 258, 259, 261-280
    as factors in immunity, 295
    of first order, 262
    free, 259, 261, 262
    of second order, 265
    tabulation of, 294
    of third order, 273

  Recurrent fever, 29, 35

  Red corpuscles, 249, 278, 279

  REDI, 19

  Reducing actions, 112, 113
    enzymes, 125

  Refrigeration as antiseptic, 148

  REINKE, 80

  Relapses, 235

  Relationships of bacteria, 37-40
    biological, 255, 270

  Rennet, 124

  RENUCCI, 27, 34

  Reproduction, 37, 63, 90

  Resistance to disease, 241, 250
    of spores, 50

  Respiratory function, 88
    tract, 246

  Retarders, 143

  Rheumatism, 245

  _Rhizobium leguminosarum_, 65, 68, 69, 118

  Rhizoid colonies, 222, 223

  RHIZOPUS NIGRICANS, 226

  RHODOBACTERIACEÆ, 63

  _Rhodococcus_, 66, 69

  RICHET, 289

  Ricin, 262

  RIDEAL, 165

  Rideal-Walker method, 165

  RIMPAU, 281

  RINDFLEISCH, 29, 35

  Ringworm, 28

  Ripening of cheese, 32
    of cream, 97

  ROBIN, 262

  Rock, phosphate, 115

  Rocky Mountain spotted fever, 242

  ROGERS, 265

  Röntgen rays, 79

  Room temperature, 213

  Rooms, disinfection of, 167
    incubator, 213

  Root tubercle bacteria, 86, 87, 108
    tubercles, 117

  ROSENAU, 289

  Rot, potato, 28, 34

  Round worm, 232

  Roup, 244

  ROUX, 30

  Rubbing as inoculation, 195

  Rust, grain, 26, 34

  RUZICKA, 42


  S

  SACCATE liquefaction, 222

  Safranin, 205

  Saliva, 248

  Salivary glands, 248

  Sake, 100

  Salt-rising bread, 95

  Saprogenic, 102

  Saprophilic, 103

  Saprophyte, 87, 238

  _Sarcina_, 57, 58, 60, 66, 68, 69
    _lutea_, 77
    _ventriculi_, 83

  _Sarcoptes scabiei_, 27, 34

  Sauerkraut, 98

  Scarlet fever, 246, 248, 250

  Scavengers, bacteria as, 108

  SCHICK, 289

  _Schistosomum hematobium_, 28, 35

  SCHLÖSING, 32, 35

  SCHÖNLEIN, 27, 34

  SCHROEDER and DUSCH, 21

  SCHULTZE, 21, 34

  SCHWANN, 21, 31, 34

  Sea, bacteria in, 71, 111

  Sealing air-tight, 20

  Secondary infection, 234

  Sections, staining of, 209

  Selective media, 198, 199

  Self-limited, 233

  SEMMELWEISS, 28, 35

  Sensitization, 290

  Sensitized animal, 290
    bacteria, 254
    vaccine, 254

  Septicemias, hemorrhagic, 246

  Sero-bacterins, 254

  Serum, antidiphtheritic, 263
    antitetanic, 263
    heated, 271, 277, 279
    rashes, 289
    sickness, 289, 292
    simultaneous method, 253
    therapy, 253

  Serums, cytolytic, failure of, 275

  Sewage disposal, 101, 116
    sulphate, reduction in, 114

  Shape of spore, 48

  Sickness, serum, 289, 292

  Side-chain theory, 256, 258

  Silkworm disease, 27, 29, 34, 35

  Size of bacteria, 37, 40

  Skatol, 104

  Skin, channel of infection, 243
    diseases, 243
    glanders, 248
    lesions, 228
    pocket, 227

  Slant cultures, 186

  Slide, cleaning of, 207
    hanging drop, 203
    staining on, 207

  Slope cultures, 186

  Sludge tanks, 116

  Small intestine, 249

  Smallpox, 24, 26, 34, 239, 246, 248
    babies, 252
    vaccines, 253

  SMITH, 289
    tubes, 184

  Snake poisons, 263, 275
    venoms, 128

  Sneezing, 248

  Soap, 160
    medicated, 160

  Society of American Bacteriologists, classification, 63
    descriptive chart, 217
    key, 68

  Sodium hypochlorite, 158

  Soil, acid, 81
    bacteria, 119
    bacteriology, 35
    enrichment, 117
    fertility, 120
    organisms, 74

  Solid media, 172, 173

  Solution, Gram’s, 208
    stock, 205

  Sore throat, 240, 241

  Sound, 80

  Sour mash, 98

  Source of complement, 277

  Souring, 98

  SPALLANZANI, 20, 31, 34

  Species determination, 59, 60

  Specific amboceptor, 274, 278, 279
    antibody, 291
    chemical stimuli, 257, 258, 259
    disease, 27, 30, 233

  Specificity of agglutinins, 267
    of amboceptor, 274
    of location, 249
    of opsonins, 281

  Spermotoxin, 272

  Spherical form, 52

  _Spherotilus_, 63

  _Spirillaceæ_, 63, 65

  Spirilloses, 241, 242

  _Spirillum_, 53, 54, 55, 61, 63, 66, 68, 69
    _rubrum_, 113

  _Spirochæta_, 61
    _obermeieri_, 29, 35

  Spirochetes, 53, 242

  _Spirosoma_, 63

  Splenic fever, 28

  Split products of proteins, 291

  Splitting enzymes, 124
    of fats, 101

  Spoilage of canned goods, 51, 78

  Spoiling of food, 91

  Spontaneous combustion, 105, 116
    generation, 17-24, 33, 34
    outbreaks of disease, 239

  Sporangia, 226

  Spore, 47-51
    anthrax, 29, 35
    capsule, 48
    germination, 48

  Spores, cause spoiling of canned goods, 51
    destroyed by boiling, 133
    first recognized, 33, 35
    light on, 75
    in pasteurization, 146
    resistance of, 50, 51
    staining of, 209
    two in bacterium, 50

  Sprinkling filters, 116

  Stab cultures, 185

  Stables, disinfection of, 167

  Stain, anilin fuchsin, 205
      gentian violet, 205
    aqueous gentian violet, 205
    Bismarck brown, 212
    carbol fuchsin, 206
    contrast, 205
    Gabbet’s blue, 206
    Loeffler’s blue, 206
    Neisser’s, 212

  Staining, 204-212
    acid-fast bacteria, 209
    bottles, 206
    capsules, 210
    cell forms, 212
      groupings, 212
    flagella, 210
    Gabbet’s method, 209
    Gram’s method, 208
    metachromatic granules, 212
    Neisser’s method, 212
    Räbiger’s method, 210
    reasons for, 204
    sections, 209
    spores, 209
    Welch’s method, 210
    Ziehl-Neelson, 210

  Standard antitoxin, 264
    methods, 217
    test dose, 264
    toxin, 264

  Standardization, colorimetric method, 175
    of culture media, 174
    of disinfectants, 165
    H-ion method, 175
    of vaccines, 284

  Staphylococcus, 57, 58

  _Staphylococcus_, 66, 68, 69

  STARIN, 196

  Steam at air pressure, 134
    sterilizers, 135
    streaming, 135
    under pressure, 136

  _Stegomyia_, 242

  Sterile, 131

  Sterilization, 130
    in canning, 133
    discontinuous, 133
    by filtration, 21, 152
    first experiment by boiling (moist heat), 20
      by chemicals, 21
      by dry heat (hot air), 21
      by filtration, 21

  Sterilizers, pressure, 137
    steam, 135

  Stimuli, chemical, 257, 258, 259

  Stock cars, 170
    solutions, 205
    vaccines, 285

  Stomach, 246

  Straight needles, 192

  Stratiform liquefaction, 222

  Strawberry poisoning, 292

  Streak methods of isolation, 196
    plates, 188

  Streptobacillus, 53, 56

  Streptococcus, 56, 60, 245

  _Streptococcus_, 60, 62, 66, 68, 69

  Streptospirillum, 55

  Streptothrix, 38

  _Streptothrix bovis_, 30, 36

  Strict aërobe, 76
    anaërobe, 76
    parasite, 87

  Structures, accidental, 43
    cell, 41
    essential, 41

  Subcutaneous inoculation, 227

  Subdural inoculation, 228

  Substrate, 123

  Successive existence, 103

  Sugar broth, 176, 177

  Sulphate reduction, 114

  Sulphur bacteria, 63, 86, 115
    deposits, 116
    function of, 89
    in proteins, 105

  Summary in immunity, 295
    Ehrlich’s theory, 259

  Sunning, 148

  Surface reactions, 91, 92

  Surgical instruments, 167

  Susceptibility, 235

  Swine, 227

  Symbionts, 87, 103

  Symbiosis, 87

  Synthetic media, 172, 183

  Syphilitic antigen, 277, 279

  Syphilis, 233, 245, 248, 249
    Wassermann test, 277, 279


  T

  TABULATION of antigens and antibodies, 294

  _Tænia solium_, 28, 35

  Tapeworm, 28, 35, 232

  Taxes, 203

  Temperature conditions, 74
    effect on growth, 213
    factor in immunity, 251
    room, 213

  Test, complement deviation, 277
      fixation, 276, 279
    dose, 264
    for enzymes, 123
    Gruber-Widal, 268
    mallein, 292
    negative, 278
    positive, 278
    for toxins, 127
    tuberculin, 292
    Wassermann, 277, 279
    Widal, 268

  Testicle, 249

  Tetanus, 231, 238, 243, 249, 251, 252
    antitoxin, 252
    toxin, 126

  Tetracoccus, 57

  Tetrad, 57

  Texas fever, 232, 233, 242

  THAER, 31

  Theories of immunity, 256

  Theory, anaphylaxis (author’s), 290-292
    cellular, 256
    chemical, 256
    contagious disease, 34
    contagium vivum, 25, 28, 33
    Ehrlich’s, 256-260
    exhaustion, 256
    germ, 25
    living cause, 33
    mosquito, 25
    noxious retention, 256
    overproduction, 257, 258
    phagocytosis, 256
    side-chain, 256
    spontaneous generation, 17
    unfavorable environment, 256

  Thermal death point, 75, 215

  Thermophil bacteria, 75, 77

  Thermoregulator, 213

  Thermostat, 213

  THIOBACTERIA, 63

  _Thiothrix_, 63

  Thread, 56

  Thrombin, 124

  Thrush, 27, 34, 244

  Ticks, 241

  TIEDEMANN, 26

  Tinea, 28

  Tissue contrast stains, 205

  Titer, 268

  Titration, 174

  Tonsil, 245, 249

  Tonsillitis, 245

  TOUISSANT, 283

  Toxin, diphtheria, 264
    effect of temperature, 262
    final test for, 127
    in food poisoning, 104
    molecule, 261, 262
    standard, 264
    tetanus, 264

  Toxin-antitoxin method, 254

  Toxins and enzymes compared, 127
    as cell constituents, 84
    production of, 126-128
    of other organisms, 127
    specific localization, 249
    true, 128

  Toxoid, 262

  Toxophore group, 261, 262, 273

  Tract, alimentary, 246

  Transmission, accidental carriers in, 241
    agency of, 232
    of contagious diseases, 232
    of disease, 26, 28, 35, 239
    of glanders, 26, 34
    of protozoal diseases, 242
    of tuberculosis, 28, 29, 34, 35, 238

  Transverse division, 54, 56

  TRAUBE, 271

  _Treponema pallidum_, 245

  Trichina, 27

  _Trichina spiralis_, 27, 34, 35

  Trichinosis, 28, 35

  Trichophyton, 243

  _Trichophyton tonsurans_, 28, 34

  Trimethylamine, 104

  Tropical dysentery, 29
    lands, 242

  Tropisms, 203

  True toxins, 128

  Trypanosomes, 242

  Trypanosomiases, 241, 243

  Tubercle bacteria, 85, 209

  Tuberculin reaction, 292, 293

  Tuberculosis, 73, 233, 238, 245, 246, 248, 249
    due to bacteria, 30
    produced experimentally, 28, 34
    proved infectious, 29, 35

  Tuberculous milk, 248

  Tubes, culture, 184
    deep, 190
    fermentation, 184, 190
    Smith, 184
    Vignal, 189

  Two spores in a bacterium, 50

  TYNDALL, 24

  Tyndallization, 133

  Tyndall’s box, 23, 24, 35

  Typhoid bacilli, 73, 238
    bacillus, 45
    carriers, 239
    fever, 231, 233, 248, 265, 268
    transmission by flies, 242
    vaccine, 254

  Typhus, 242

  Typical cell forms, 52


  U

  ULTRAMICROSCOPE, 204

  Ultramicroscopic organisms, 234

  Ultraviolet rays, 150

  Unfavorable environment theory, 256

  Unit of antitoxin, 264
    of measurement, 40

  Universal carrier, 240

  Unorganized ferment, 126

  Unwashable articles, 169

  Urea, 106

  Urease, 125

  Urethral discharges, 248

  Urine, 72

  Urticarial rashes, 292


  V

  VACCINATION in chicken cholera, 30
    negative phase in, 287
    in pneumonia, 241
    in smallpox, 26, 34, 253

  Vaccine, 253
    age of, 285
    anthrax, 254
    antigens for, 285
    autogenous, 285
    black-leg, 254
    derivation of, 253
    mixed, 285
    polyvalent, 285
    preservative in, 284
    sensitized, 254
    smallpox, 253

  Vaccines, bacterial, 283
    in colds, 241
    dosage of, 286
    in epidemics, 241
    failure of, 285-286
    in infections, 286
    preparation of, 283
    standardization of, 284
    stock, 284
    theory of, 286
    use of, 283

  Vacuoles, 42, 43, 44, 59

  Vaginal discharges, 248

  VARO, 25

  VAUGHAN, 291

  Vaughan and Novy’s mass cultures, 188

  Vegetable toxins, 127, 128

  Vegetables, forcing of, 117

  Vehicles, disinfection of, 169

  Venoms, antisnake, 275

  VIBORG, 26, 34

  Vibration, mechanical, 80

  Vibrio, 33, 35, 53, 65, 68, 69
    _choleræ_, 66, 73

  Vignal tubes, 189

  VILLEMIN, 29, 35

  Villous growth, 219, 221

  Vinegar, 99, 114

  Virulence, 235

  Virus, 234

  Vultures, 241


  W

  WALKER, 165

  Wall, cell, 41
      composition of, 82, 83

  WARDEN, 260

  Washable articles, disinfection of, 169

  Washing leukocytes, 281

  Wassermann test, 277

  Water, bacteria in, 73
    filtration of, 153
    purification of, 77, 150
    sterilization of, 157

  WEBB, 253

  WEIGERT, 17, 30, 36, 42, 257, 258

  Welch’s method of staining, 210

  Whooping cough, 246, 250

  WIDAL, 265
    test, 268

  Will o’ the wisp, 105

  Wine, pasteurization of, 141

  WINOGRADSKY, 32, 63, 86

  Wire baskets, 184
    nichrome, 193

  WOLLSTEIN, 26, 34

  WORONIN, 30, 36

  Wound infections, 17, 25, 26, 27, 30, 34, 36, 233, 234, 240, 243, 248

  WRIGHT, 280


  X

  X-RAYS, 79

  _Xylinum, acetobacter_, 83


  Y

  YEAST, fermentation, 31, 34, 99, 100, 114
    relation to bacteria, 37
    reproduction of, 37, 39

  Yellow fever, 242


  Z

  ZANZ, 18

  ZENKER, 27, 28, 35

  ZETTNOW, 43

  ZIEMANN, 43

  Ziehl-Neelson method of staining, 210

  Ziehl’s solution, 206

  Zoögloea, 44

  Zoötoxins, 128

  Zymase, 125

  Zymogens, 121, 125

  Zymophore group, 273




Transcriber’s Notes.

Punctuation has been standardised and simple typographical errors have
been repaired. Hyphenation, quotation mark usage, and obsolete/variant
spelling (including variant spellings of proper nouns) have been
preserved as printed.

In the original book, the page numbering goes xiii, blank, unnumbered,
18. This is a printer’s error: no pages are missing.

The descriptive chart insert has been moved from between pages 216 and
217 to the end of the book.

The following changes have also been made:

  Page 26: ‘this scourge which had devastated’
  for ‘this scourge which had devasted’

  Page 30: ‘to be the cause of a disease in cabbage,’
  [added comma]

  Page 32: ‘alcoholic, lactic and butyric’
  for ‘alcoholic, lactic and butryic’

  Page 32: ‘however, workers busied themselves’
  for ‘however, workers, busied themselves’ [deleted extra comma]

  Page 56: ‘Fig. 43.--Streptobacillus’
  for ‘Fig. 43.--Steptobacillus’

  Page 57: ‘from a genus of algæ’
  for ‘from a genus of algae’

  Page 58: ‘staphylococcus--irregular’
  for ‘staphylococcus--irrgular’

  Page 59: ‘so that it is impossible’
  for ‘so that is is impossible’

  Page 62: ‘Illustrates the genus Spirochæta’
  for ‘Illustrates the genus Spirochaeta’

  Page 63: ‘since it is without a sheath’
  for ‘since it is without a a sheath’

  Page 64: ‘Corynebacterium diphtheriæ’
  for ‘Corynebacterium diphtheriae’

  Page 67: ‘Prazmowski, 1880; anaërobic’
  for ‘Prazmowski, 1880; anaerobic’

  Page 70: ‘growth processes involving oxidation’
  for ‘growth processes involving oxidadation’

  Page 70: ‘EE--Anaërobes, rods swollen at sporulation’
  for ‘EE--Anaerobes, rods swollen at sporulation’

  Page 73: ‘percentage of water is permissible’
  for ‘percentage of water is permissable’

  Page 95: ‘Material taken from the bottom’
  for ‘Material taken from the botton’

  Page 102: ‘large-moleculed and not diffusible’
  for ‘large-moleculed and not diffusable’

  Page 104: ‘various kinds of “meat poisoning,”’
  for ‘various kinds of “meat posisoning,”’

  Page 106: ‘formed under anaërobic conditions’
  for ‘formed under anaerobic conditions’

  Page 110: ‘volatile fatty acids, ethereal’
  for ‘volatile fatty acids, etheral’

  Page 127: ‘but in much larger doses’
  for ‘but in much large doses’

  Page 131: ‘“antiseptic” may become a disinfectant’
  for ‘“antiseptic” may become a disfectant’

  Page 141: ‘quarantine station barge’
  for ‘quaratine station barge’

  Page 147: ‘A continuous milk pasteurizer.’
  for ‘A continuous milk pastuerizer.’

  Page 163: ‘especially when a large amount of material’
  for ‘expecially when a large amount of material’

  Page 179: ‘these must be sterilized’
  for ‘these must be steriliized’

  Page 191: ‘Deep tubes showing anaërobic’
  for ‘Deep tubes showing anaerobic’

  Page 193: ‘less than one-twentieth of platinum’
  for ‘less than one-twentieth of platimum’

  Page 207: ‘grease-free cloth, handkerchief’
  for ‘grease-free cloth, handerchief’

  Page 210: ‘Stain with Löffler’s blue’
  for ‘Stain with Löffller’s blue’

  Page 211: ‘slide to cause precipitates’
  for ‘slide to cause preciptates’

  Page 213: ‘grows at body temperature (37°)’
  [added closing parenthesis]

  Page 217: ‘working on a revision’
  for ‘working on a revission’

  Page 220: ‘inoculation for facultative anaërobes’
  for ‘inoculation for facultative anërobes’

  Page 231: ‘the unicellular microörganisms’
  for ‘the unicellular micro-organisms’ [split across line]

  Page 232: ‘unicellular pathogenic microörganisms’
  for ‘unicellular pathogenic micro-organisms’ [split across line]

  Page 233: ‘the fact of self-limitation’
  for ‘the fact of self-limitaion’

  Page 242: ‘the cattle tick (_Margaropus annulatus_).’
  for ‘the cattle tick (_Margaropus annulatus_.)’

  Page 244: ‘B. Mucosæ directly continuous’
  for ‘f. Mucosæ directly continuous’

  Page 245: ‘localized infection as in micrococcal, streptococcal’
  for ‘localized infection as in micrococcal, strepococcal’

  Page 248: ‘ELIMINATION OF PATHOGENIC MICROÖRGANISMS.’
  for ‘ELIMINATION OF PATHOGENIC MICRO-ORGANISMS.’ [split across line]

  Page 254: ‘sometimes added to attenuate’
  for ‘sometimes added to attentuate’

  Page 256: ‘Metchnikoff has since elaborated’
  for ‘Metchinkoff has since elaborated’

  Page 266: ‘This is analogous to what’
  for ‘This is analagous to what’

  Page 280: ‘other names, but ascribed’
  for ‘other names, but asscribed’

  Chart: ‘(10 minutes’ exposure in nutrient broth when this is adapted
      to growth of organism)’
  for ‘(10) minutes’ exposure in nutrient broth when this is adapted
      to growth of organism)’

  Page 299: ‘Allergic, 290’
  [index entry was printed twice]

  Page 308: ‘Foreign body pneumonia’
  for ‘Foreignbody pneumonia’

  Page 309: ‘oxidation of’
  for ‘ox dation of’

  Page 311: ‘Microspira’
  for ‘Miscospira’

  Page 311: ‘Microsporon’
  for ‘Miscrosporon’

  Page 314: ‘Plasmodiophora brassicæ’
  for ‘Plasmodiophora bassicæ’

  Page 317: ‘Starin, 196’
  [index entry was printed between Standardization and Staphylococcus]

  Page 318: ‘Thermostat’
  for ‘Thermostadt’