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

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

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.

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.

Fig. 2.—The first drawings of bacteria by Leeuwenhoek. The dotted line C–D indicates the movement of the organism.

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.

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.

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.

Fig. 6.—Pasteur’s flask.

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.

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.

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.

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:

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.

Fig. 8.—A thread of blue-green algæ.

Fig. 9.—A thread of small blue-green algæ.

Fig. 10.—A thread of bacteria. Compare with [Figs. 8] and [9].

Fig. 11.—A chain of spherical blue-green algæ.

Fig. 12.—A chain of spherical bacteria.

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.

Fig. 14.—Spherical bacteria. Several pairs are shown.

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]

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.

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.

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.

Fig. 19.—Bacteria seen within capsules.

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.

Fig. 21.—A bacterium showing a single flagellum at the end—monotrichic.

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.

Fig. 23.—A bacterium showing flagella at each end—amphitrichic.

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]).

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.

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.

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.

Fig. 28.—Spores in the middle of the rod without enlargement of the rod. The lighter areas in the rods are spores.

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.

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.

Fig. 31.—Spores in the end of the rod with enlargement of the rod, A, A, A, A.

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”:

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.

Fig. 33.—Cocci.

Fig. 34.—Bacilli.

Fig. 35.—Short bacilli.

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.

Fig. 37.—Spirilla.

Fig. 38.—Vibrio forms of spirilla. Compare with [Fig. 36].

Fig. 39.—Spirochetes.

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.

Fig. 41.—Streptospirillum grouping.

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.

Fig. 43.—Streptobacillus

grouping.

Fig. 44.—Typical diplococcus grouping. Note that the individual cocci are flattened on the apposing sides.

Fig. 45.—Long streptococcus grouping.

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.

Fig. 47.—Staphylococcus grouping. The large flat masses are staphylococcus grouping. Diplococcus grouping, tetrads and short streptococci are also evident.

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.

• diplococcus—in 2’s.
• streptoccus—in chains.
• tetracoccus, tetrads—in 4’s.
• staphylococcus—irregular
• flat masses.
• sarcina—regular, solid packets, multiples of 8.

• diplobacillus—in 2’s.
• streptobacillus—in chains.

• diplospirillum—in 2’s, little used.
• 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.

Fig. 49.—Illustrates the genus Streptococcus. Typical chains, no staphylococcus grouping, no sarcina grouping, no flagella.

Fig. 50.—Illustrates the genus Micrococcus. Diplococcus, tetrads short chains and staphylococcus; no sarcina, no flagella.

Fig. 51.—Illustrates the genus Sarcina. Sarcina grouping, no flagella.

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:

Fig. 53.—Illustrates the genus Pseudomonas. A bacillus with flagella at the end only.

Fig. 54.—Illustrates the genus Microspira. It is (though the photograph does not prove it) a short spiral with one flagellum at the end.

Fig. 55.—Illustrates the genus Spirillum. Spiral bacteria with more than three, in this case four, flagella at the end.

Fig. 56.—Illustrates the genus Spirochæta

.

Fig. 57.—Illustrates the genus Chlamydothrix. Fine threads with a delicate sheath.

Fig. 58.—Illustrates the genus Crenothrix. The thickness of the cell walls is due to deposits of iron hydroxide. (After Lafar.)

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.

• Genus 1. Streptococcus Billroth: division in one plane only ([Fig. 49]).
• Genus 2. Micrococcus (Hallier) Cohn: division in two planes only ([Fig. 50]).
• Genus 3. Sarcina Goodsir: division in three planes only ([Fig. 51]).

• Genus 4. Planococcus Migula: division in two planes only.
• Genus 5. Planosarcina Migula: division in three planes only.

• Genus 1. Bacterium (Ehrenberg) Migula: no flagella; non-motile.
• Genus 2. Bacillus (Cohn) Migula: flagella peritrichic ([Fig. 52]).
• Genus 3. Pseudomonas Migula: flagella at the end: monotrichic, lophotrichic, amphitrichic ([Fig. 53]).

• Genus 1. Spirosoma Migula: non flagellated; non-motile.
• Genus 2. Microspira (Schrœter) Migula: flagella one to three at the end ([Fig. 54]).
• Genus 3. Spirillum (Ehrenberg) Migula: flagella more than three at the end ([Fig. 55]).

• Genus 4. Spirochæta Ehrenberg: motile; no flagella ([Fig. 56]).

• Genus 1. Chlamydothrix Migula ([Fig. 57]).
• Genus 2. Crenothrix Colin ([Fig. 58]).
• Genus 3. Pragmidiothrix Engler.
• Genus 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.
• Genus 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.