(east a ena Reser : erat i ON thks en ate ath Eby vi rateHS rhe pbc a, ri 7 fis y i PO Hae RE EAE EL Eh eae fat Secaguuu ia scenes EASUELPLL APA nk) bc, : Read oie erent elie Pate cnet ve bv oc sk Viele et Py thihe if , fl Seal tens ts Fairer trea oe eI Are eae CS Nahas ora AHS ELOORt Es Fa NeTCU NAH PAM AA AA Aenea 5 Kea a PTT EP ER rt i atteeate cretatai| ne te ; He GOURD UE AERA? & : ' i AAAs Cts vy of age eae Weare ie i . Neth ae Cee ata ia Adair at POU athe Wy a erie ry rene tu TL Tote eal piceishe tee clon Py reer hy? las ee cnt ae tole LEA AMA HAG die vate (4 iA4 yy CERO AGH Ot eee lettres A EAs Og) Fa wt eA Peer purer ee Phe MAMA A AL BT i tel lly ity. Peron et ry dy ee Dry Mey Pay = ALBERT R. MANN LIBRARY AT CORNELL UNIVERSITY una Cornell University The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www. archive.org/details/cu31924054708544 Rack and Pinion for coarse adjustment Micrometer 7 VMs, 7 _ Screw for s LY) fine adjustment 3 . : “SS des, Stage " Abbe Condenser Iris Diaphragm Pinion for oblique light Plane and ’ Concave Mirror. LABORATORY WORK IN BACTERIOLOGY, BY FREDERICK G. NOVY, Sc.D., M.D., JUNIOR PROFESSOR OF HYGIENE AND PHYSIOLOGICAL CHEMISTRY, UNIVERSITY OF MICHIGAN. ‘ SECOND EDITION, REVISED AND ENLARGED, WITH FRONTISPIECE AND SEVENTY-—SIX ILLUSTRATIONS. ANN ARBOR: GEORGE WAHR, PUBLISHER. 1899. Copyright, 1899, by F. G. Novy. PREFACE. A thorough course of laboratory instruction in bacter- iology is absolutely essential to the proper education of the medical student of the present day. The practical knowl- edge thus acquired in the methods of handling bacteria, in the precautions necessary to the prevention of personal infection, and in the methods for the recognition and for the destruction of disease-producing organisms is funda- mental and invaluable. Such information is directly useful as a means of diagnosis; it is necessary to the successful performance of antiseptic operations and is indispensable to the proper execution and understanding of the common hygienic measures for the prevention of communicable diseases. It is therefore evident that the course in bacteriology should not be inferior, either in length or in the character of the instruction, to any other laboratory course offered in the medical curriculum. The student should be taught to work, not merely with a few harmless bacteria, but espe- cially with all of the common pathogenic organisms. The exclusion of the latter organisms from a laboratory course on the plea of danger is an admission of weakness in instruction or in supervision. The danger ina laboratory is avoidable, and is not to be compared with that encountered 4 PREFACE. in practice in connection with cases of actual disease. Accidents may occur, it is true, but they are extremely rare as compared with the numerous instances of infection _ incurred in post-mortems or in surgical operations. Bacteriology, as an educational measure of the first importance, belongs in the first, or at the latest, in the second year of a medical course. The student is thus enabled to make use of his knowledge in connection with his clinical studies. The spirit of scientific investigation, and not mere book reading, must be fostered in the student from the outstart, since it is this that leads to progress in medicine and serves to distinguish the true physician from those bound down through blind faith, commercialism or ignorance. This edition is thoroughly revised and greatly enlarged. It should be noted that it is a text-book of laboratory work for students. The arrangement of the subject matter is different from that usually met with in text-books, for the reason that it conforms as much as possible to the actual work as carried on, day by day, in the Hygienic Laboratory of the University of Michigan. During the past ten years three laboratory courses in bacteriology have been given annually. Each course covers a period of twelve weeks of daily work, to which the entire afternoon is devoted. Inas- much as this laboratory work is required of all medical students, the number of students’which annually take the course at times exceeds two hundred. Since it is a beginner’s guide and not a manual, it. obviously is undesirable to introduce the numerous modifi- PREFACE, 5 cations that can be found of almost every known procedure. The methods that are described are those which have stood the severe test of the practical instruction indicated above. Many of the methods, as well as some of the apparatus described, have originated in this laboratory. Illustrations of the various bacteria and of their cultural characteristics have been expressly omitted, inasmuch as the student is expected to sketch from observation the form of each organism and its. peculiarities of growth in the colony, and in tube culture. Blank pages are provided for this purpose and for such additional notes as may be desirable. More space has not been given to the consideration of the individual pathogenic bacteria, and of questions of immunity, for the reason that these and allied subjects are treated of in a general course of lectures which is wholly independent from the laboratory work. These lectures on general bacteriology are given daily and extend throughout a semester. From the standpoint of a teacher it is desir- able that the two courses be kept distinct, and for that reason the treatment of the general subject should be pub- lished separately, if at all. An exception is made in the case of the first five chapters, which deal with the general properties of bacteria, and which, as such, are necessary for reading and reference in connection with the laboratory work. The last two chapters are devoted to special methods which will be of value to advanced students. Several of the characteristic ingenious methods of the Pasteur school are given at length. Here, as elsewhere in the book, the 6 PREFACE, attempt is made to supply the fullest details to the student in order to insure satisfactory results. A complete index will serve to render the text easily accessible. It is hardly necessary to state that the various journals and the standard works on bacteriology have been freely drawn upon in the preparation and revision of these pages It has been deemed desirable to introduce illustrations especially of such apparatus as is actually employed Thanks are due to the Bausch & Lomb Optical Co., of Rochester, N. Y., also to Himer & Amend, and Stransky & Co., of New York, for kindly supplying several of the cuts. F. G. NOVY. ANN ARBOR, MicuH., April 15, 1899. CONTENTS. CHAPTER I. Page. Form and Classification of Bacteria Definition of bacteria.—Microscopic plants and animals, distinction.——-Bacteria as plants.——Bacteria, fission fungi or schizomycetes; moulds, thread fungi or hyphomycetes; yeasts, pudding fungi or blastomycetes.— Absence of a natural classi- fication.— Bacteria classified according to form; micrococcus, bacillus, spirillum.——Modifications; bacterium, vibrio, spiro- chzte.——Division into species. Influence of environment on form and size.——Involution forms.——Vdriations due to meth- ods of examination.——Plasmolytic changes. —-Pleomorphism. — Constancy of species. Attenuation. ——Origin of new spe- cies. CHAPTER II. Size and Structure of the Bacterial Cell Bacteria as unicellular organisms.——Called the smallest of living beings.——Existence of still more minute life.—— Micromillimeter or micron.——Size of bacteria. The cell-wall, composition, demonstration.—Plasmolysis. —- Softening of the outer layer, the capsule.——Zooglea. The contents of the cell, composition. —Existence of a nucleus.—A ppearance of contents; granulations, polar bodies, color, absence of chlorophyll. Granulose reaction. Motility——Molecular or Brownian movement.——Real motion, flagella or whips.—-Number and arrangement.—— Giant whips. CHAPTER III. The Life-history of Bacteria Rapidity of multiplication.—Cell division: ‘dip noetim threads; diplococcus, streptococcus, tetrad, sarcine, staphylo- coccus; vibrio, spirillum. 15 24 41 8 CONTENTS. Spores.—Vegetating and reproductive forms.——Endos- pore, arthrospore.——Sporulation.——Position of the spore; median, terminal, intermediate.—Resultant cell forms; clos- tridium, “‘drum-sticks.”——Cause of spore formation.— Aspor- ogenic bacteria.—Germination.—-Spore structure.——Re- sistance of spores.—Spontaneous generation. é CHAPTER Iv. The Environment of Bacteria Conditions of growth——Necessity of moisture.——Chem- ical composition of the cell.— Sources of carbon, nitrogen, hydrogen, oxygen, and other elements.—Reaction of the medium.——Distribution of bacteria in nature, where absent. Classification according to habitat.——Saprophytic and parasitic bacteria. Obligative and facultative forms.—— Classification according to oxygen requirements.— Aerobic and anaerobic bacteria.—Obligative and facultative forms. ——Microbic association. Temperature, minimum, maximum and optimum, —Ther- mophilic bacteria Effect of cold and heat on vitality.—— Action of light, high pressure, electricity, —-Chemotaxis. CHAPTER V. The Chemistry of Bacteria : : . ; ‘ The number and kind of products vary with each species. — Influence of environment.— Accumulation of waste-pro- ducts.——Synthetic and analytic, primary and secondary pro- ducts. Bacterial proteins, tox-albumins.-—Venoms, plant albu- moses as abrin and ricin.——Toxins; synthetic products, elabor- ated within the cell, not bases or proteins.—Ferments, organized and unorganized. ——Enzymes, their classification, —— Ptomains.— Alkalis.— Acids.——Alcohols. — Gases.——. Classification of bacteria according to function. Fermentations.——Their cause, the nature of the chemical changes induced.—Diverse fermentations, alcoholic; acetic acid, vinegar, summer complaint; lactic acid, dental caries, stomach and intestinal disorders, souring of milk, koumiss, cheese; butyric acid, sauerkraut, ensilage, cheese, retting; viscous or slimy fermentations.——Fermentations of fats, flavor of butter.—Hydrogen sulphide and ammoniacal fermentations of urine.— Nitrification in water and soil, saltpeter.——Deni- trification.— Indigo reduction.——Putrefaction, bacteria as scavéngers perpetuate life. 58 79 CONTENTS. Pigment production.—Phosphorescence.——Heat produc- tion.—Toxicogenic and pathogenic bacteria. CHAPTER VI. The Microscope. Staining ‘ ‘ ‘ Simple and compound microscopes.—— Spherical and chro- matic aberration.— Achromatic and apochromatic objectives. —Under-correction.—Magnifying power, equivalent focal distance.——Defining power.——Resolving and penetrating power.— Numerical aperture.—Designation of objectives. Huyghenian eye-piece.——-Compensating eye-pieces.— Designation of oculars. Abbe condenser, iris diaphragm: —-Structural and colored images.—Rules for use of condenser, to secure illumination. The stand, graduation of draw-tube.——Coarse and fine adjustment.—Nose-piece.—Care of microscope. Measurement of an object.——Stage and ocular microme- ters:——Micromillimeter or micron.—-Micrometer value of an objective. Slides and cover-glasses.——Cleaning of the latter.— Cover-glass forceps for bacteriological work. Examination of living bacteria.——The hanging-drop.—— Laboratory work. : Staining of bacteria.— Acid and basic anilin dyes.— Stock solutions.——Dilute stains.—~—Simple staining.——Labor- atory work. The Hanging Drop.—Simple CHAPTER VII. Gelatin and Potato Media.—Cultivation of Bacteria -Preparation of the meat extract.—Alkalization.—— Cleaning and plugging of tubes. —Dry and moist heat steriliza- tion.——Fractional sterilization at 100°, at 60°.——Steam steril- izer.——The autoclave. Preparation of potato media.-—Dilution and mass cul- tures.——The pure culture.—Precautions in work.—Labora- tory work. Gelatin plate cultivation.——Advantages over potato and liquid media. —-Glass plates: _—-Platinum wires. —Dilution in gelatin.——Pouring on plates.——Plating apparatus.——Moist chambers.— Apparatus for constant low temperature.— Laboratory work. Modified gelatin plate method.—Petri dishes.——Esmarch roll-tubes.— Laboratory work. 128 152 10 CONTENTS. Modified potato cultures———Esmarch dishes.——Potato tubes. Preparation of Roux tubes.——Laboratory work. Examination of colonies, macroscopic and microscopic.— Transplantation of colonies——Stab cultures.——The plan of study.— Laboratory work. 1 CHAPTER VIII. The Non-Pathogenic Bacteria . Bacillus prodigiosus.—B. Indicus.——B. Faber of Kiel. ——B. rubidus.—B. violaceus.——B. fluorescens putidus.— B. phosphorescens. —Orange sarcine.——Yellow sarcine. —B. subtilis —B. mesentericus vulgatus.—B. megaterium.——B. ramosus.——Proteus ‘vulgaris.——Bacterium Zopfii.m—Spiril- lum rubrum.——B. acidi lactici.——B. butyricus. ——B. cyano- genus. CHAPTER IX. cd Bouillon, Agar, Milk and Modified Media.—The Incubator and Accessories ; F Advantages of bouillon, agar and milk.——Preparation of beuillon.—Preparation of agar.——Sterilization of milk.—— Modified media: peptonless agar, glycerin agar; glucose, lactose, litmus and serum media.— Laboratory work. The incubator.—— Thermo-regulators. —— Gas-pressure regulator.— Micro-burners.—— Koch’s safety lamp.—— Ther- mometers. CHAPTER X. Relation of Bacteria to Disease.—Methods of Infec- tion and Examination Infectious diseases.—Infection and intoxication.—— Rules of Koch.—Generation.—Attenuation.——Bacterial, fungous and protozoal diseases. Unknown causes, Methods of infection.—Cutaneous application.-—-Subcu- taneous injection.——Animal holders. —Intravenous injection. ——Intraperitoneal.— Intrapleural.——Anterior chamber of the eye.—Lymphatics.—Intracranial.— Infection along respiratory tract.——Alimentary tract. — Observation of infected animals. ——Keeping of record,—— Food and drink. Post-mortem examination.— Sterilizing case for instru- ments.—Preparation of the animal.—Examination of the sub-cutis.——Peritoneal and pleural cavities—Removal of 193 232: 253. CONTENTS. portions of organs.—Drawing of heart blood.t™——Cover-glass preparations of tissues and organs, of blood.——Staining of the streak preparations.——Precautionary measures. ' Laboratory work with anthrax animal.——Gelatin plates. Preparation of agar plates.——Impression preparation of col- onies.——Hanging-drop examination of blood.—Simple stains of streak preparations.—Gram’s method.——Anilin-water gentian violet.——Spore staining.—cCarbolic futhsin.— Phagocytes.—Summary. a CHAPTER XI. The Pathogenic Bacteria Bacillus anthracis——B. anthracis symptomatici.m—B. cedematis maligni.im—B. cedematis maligni No. II.——B. tetani. -——Culture of anaerobic bacteria.— Staining of flagella.— Giant whips.——B. lepre.——B. tuberculosis.——B. mallei.—— B. diphtheriz.—-M. pneumoniz croupose.——B. pneumoniz. ——B. rhinoscleromatis.— Vibrio cholerze Asiaticaze.— Vibrio Deneke.——-Vibrio Finkler-Prior.—Vibrio Metschnikovi.—— B. coli communis.—B. typhosus.——B. icteroides.——B. pestis pubonice.——B. influenze.——B. pyocyaneus.——Streptococcus pyogenes.——Staphylococcus pyogenes aureus.—Micrococcus gonorrhee.—-M. tetragenus.——Spirillum Obermeieri.——B., cholere gallinarum.——B. cholere suis.— B. rhusiopathiz suis. ——B. muriepticus. CHAPTER XII. Yeasts, Moulds and Streptotrices : Yeasts, size and multiplication.—Saccharomyces. — Torula or wild yeasts. Pathogenic forms. Moulds. ——~ Mycelium. -— Hyphe. —— Fruit-organs. —— Characteristics of the mucor, aspergillus, penicillium, oidium, —Relation to fermentation.———Pathogenic forms.—Skin diseases. : Streptotrices.—Relation to moulds and to bacteria. Laboratory work.—Preparation of bread flasks.——Exam- ination of moulds. Saccharomyces cerevisie.——S. glutinis——Oidium lactis. —Monilia candida.—-Mucor corymbifer.——M. rhizopodi- formis.— Aspergillus niger.——A. flavescens.— A. fumigatus. —Penicillium glaucum.——Achorion Schénleinii.m—Strep- tothrix actinomyces.——S. Madurz.——S. farcinica. 1k 295. 385. 12 CONTENTS. CHAPTER XIII. Examination of Water, Soil and Air . . ‘ ‘ Water asa carrier of disease.——Limitations of a chemi- cal analysis.—Recognition of typhoid bacilli—Cholera vibrios.—+Method of analysis.——Counting of colonies, direct and by aid of microscope.—Number and kind of species.—— Detection of pathogenic bacteria’ by animal inoculation.— Bacterial.contents of snow, ice, rain-water, lakes, rivers, wells and sea-water. The bacteria of the soil. Their action._—Pathogenic forms.— Effect of rain and snow.——Filtering action of the soil._M ethod of analysis. The source of air bacteria.._—- Effect of moist surfaces.—— Purification of air by sedimentation.——By rain and snow.— Expired air— Number of organisms in the air.——Country and city air.—Pathogenic bacteria.— Methods of analysis.— ‘Laboratory work. CHAPTER XIV. Special Methods of Work Making of Pasteur pipettes. = Baie of ined from animals, from man.—Oxalate blood and plasma.——Prepara- tion of blood-serum,——Sterilization of serum by filtration.— Fractional sterilization at 58°, at 75°, at 100°,——Solidification of serum.— Modified serum media.—Filtration of bacterial liquids. —Tuberculin.——Diphtheria toxin.——The minimum fatal dose.—Testing of antitoxin———Immunization against diphtheria.— Antitoxic and anti-infectious serum.— Active and passive immunity.—— Pfeiffer’s reaction.—— Elsner’s medium.—Stoddart’s medium.——Hiss’ tube medium.—Usch- insky’s medium.—Preparation and use of collodium sacs.—— Inoculation for rabies. CHAPTER XV. Special Methods of Work, Continued Serum agglutination. Poisonous foods. = Pizinvation of litmus.—The tubing of media, The sealing and keeping of cultures.—Thermal death point.——Moist and dry heat.—— Testing of disinfectants.—-Testing of antiseptics— Room disinfection. Hardening, imbedding and cutting of sections. -——The staining of sections.——Simple, and Gram’s method. Staining of anthrax, tubercle leprosy and typhoid bacilli, and actinomyces in sections. List of apparatus and chemicals . ; i Index : 422 456 505 547 549 LIST OF ILLUSTRATIONS. Frontispiece showing component parts of a microscope. PAGE.. Fig. 1.—Micrococcus, bacillus, spirillum...... .. SEG Rad Moamese was 17 2.—Bacterium, vibrio, spirochete..............:e eee eee eee 18. 3.—Involution forms: 22.666 cs cece Sede eee eee eet eed wens 21 4.—Plasmolytic changes......... wee Meee ee ees4d Pe Gaang awe 27 5.—Capsulated bacteria.............. 0 cece cece et tee tena ees 29: 6.—Motile organs or whips on bacteria.............. Fat aenre 36- Te=Gilant= Wipes caisignn vadvrnseacked ve hacieon aka eitoee ahepbetiances 39 8.—Cell division........ espa asp Bea ceo sete mek Woe sapas Seu rolbe seater 42 9.—Bacilli, single, in pairs and in threads.................. 43. 10.—Division forms of micrococci........... cave N ase ienstmaaichncns 44 11.—Spirillum forms. i:scys aasieiies seas see susGavedioeeens 46. 42.—Sporvlation vac: ia oees ssewiiaweasenee deed yone ye ves adiecs 49. 13.—Position of spores and resultant forms.................. 50- 14.—Spore germination........... 0... cece cece cece ee eee eee 54 15.—Virtual image, simple microscope (Carpenter).......... 123 . 16.—Real image (Carpenter)...............e sees eee e eee 124. 17.—Principle of the compound microscope (Carpenter),.... 125. 18,—Arrangement of lenses in an objective. Angle of aper- ture (Carpenter})............. bik ae ue awa aaway gene Aiea 130: 19.—Structure and action of the Abbe condenser.... ....... 133 20.—Cover-glass forceps (F. G. N.)... 0... c eee ce nee e eee eens 141 21.—The ‘‘hanging-drop”........ 6.0 e sce e eee eee eens eee ee ee 143. 22.—Water-bath with hot iron plate (F. G. N.).............. 150 23.—Enamelled jar for making media.................. 0... 153. 24.—Burettes for titrating media (F. G. N.)................. 155 25.—Dry-heat sterilizer... 0.0.06. cee cece cece eee cece 160- 26.—Steam sterilizer (F. G. N.)........ 0.660 eee cece eee eee 164 DT —Avitoclaversesceis ye secas preset 4 5ehosciseey aus woke 165. 28.—Apparatus used in cultivation...............-.......06. 172. 29.—Inoculation of a single tube........-.-... see ee eee eee 173 30.—Inoculation from tube to tube when diluting........... 174 31.—Ice apparatus for cooling plates............-.......505. 176. 32.—Water apparatus for cooling plates (F. G. N.).......... 177 33.—Constant low temperature apparatus (F. G. N.)......... 179: 34.—Potato tubes, ordinary and Roux form. ................ 184. 35.--Apparatus for filtering agar (F.G. N.). ..............5. 237 36.—The incubator or thermostat......-. ...............0.. 244. 14 ‘ LIST OF ILLUSTRATIONS. q 37,—Thermo-regulator (F. G. Nu)... ce eee eee eee eee oe 38.—Murrill’s gas pressure regulator...... Sosa eaaielGumecen ie 89.—Koch’s safety burner..............+00 ig beashangus duces’ sess 40.— Adjustable syringe........... eee e eee eee cree eens 41.—Syringe and holder, water-bath and radial burner,..... 42,—Injection apparatus............c0eeee ee eee ee pane nantes 43.—Sterile conical test-glass.........6. 62 se eee eect cere ees 44,—Voges’ cylindrical holder..............c cece eeee een eee! 45.—Latapie’s animal holder............ 0.000 ese ceeee eens 46.—Rat cage and forceps............. cee eee pee cae w ote La 47.—Vaughan’s cage for rabbits, etc......-..-..- see eee eee 48.--Instrument sterilizing case and searing iron........... 49.—The Roux spatula.and Nuttall needle................+-5 50.— Simple bottle for anaerobes (F. G. N.).......-..seeee ees 51.—Bottle for tube culture of anaerobes (F. G. N.)........5 52,—Apparatus for plate cultivation of anaerobes (F. G. N.) 53.— Apparatus for plate cultivation of anaerobes (F. G. N.) 54.—Tube culture of tubercle bacillus on potato (F. G. N.).. 55.—Yeast cells with spores (Hansen)..................4. 56,—Fruit-organs of moulds (Lehmann).........-......0. 008. 57.—Wolffhiigel’s counter................ Soar Guaed Ae eta Sanat 58.—Lafar’s and Jeffer’s counters ...........--.-2 02. eee eee 59.—Esmarch’s roll-tube counter..............00e eee eee si 60.—Apparatus for the examination of air................4. 61.—Drawn-out tube pipettes of Pasteur....... .........05- 62.—Sealing cultures in capillaries.................-00. sees 63.—Pipette used in drawing blood........ .....-.. eee wees ‘64,—Roux water-bath for serum sterilization................ 65.—Koch’s serum sterilizer.........- ese c cent eee e eee eee ees 66.—Apparatus for filtering bacterial. liquids (F. G. N.)..... 67.—Connections for filtering apparatus (F. G. N.).. oaks 68—Berkefeld filter; globe receiving flask (F. G. N.).. ntesyesiew 2 69.—Roux flask for surface cultures...... ip Mises eas camiy Sen 70.—Rolling of collodium sacs............. cee e eee eee eee T1:—CON Odi SACK. cwacdecascedsan cuvrweeneuerascdheewceys 72,.—Tubing of media (F. G.N.)..... ccc eee eee ee cee cere een 73.—Keeping of cultures (F. G. N.)o. cee ccc eee cence eee 74,—Filter for bacterial suspensions,............ 02.0000 eee 15.—Determination of the thermal death-point (F. G. N.)... 16.—Formaldehyde. apparatus for room disinfection (F. G.N) CHAPTER I. FORM AND CLASSIFICATION OF BACTERIA. . Bacteria may be characterized as unicellular, micro- scopic plants. Their vegetable nature has been established only within comparatively recent years. From the time of Leeuwenhoek, who in 1683 discovered the first representa- tive of this group, until 1854 these microscopic organisms were spoken of as animalcule or minute forms of animal life. Indeed, it was not until about 1875 that the true relationship of bacteria to plants was definitely settled, largely through the labors of Cohn, Naegeli, DeBary and other botanists. : When studying the exceedingly minute forms of life observed under the microscope, one recognizes that it is not always possible to indicate the dividing line between the animal and vegetable kingdoms. There is no one charac- teristic which will serve -for this purpose. From the very nature of things it must be expected that the lowest forms of animal and plant life will approach one another. The striking differences which are seen between the higher forms of animal and plant life gradually disappear as the comparisons are carried down the scale to the lowest forms. The latter merge into one another indicating a common origin in the remotest past. Under these conditions it is evidently impossible to characterize certain forms of life as plants or as animals. Inasmuch as bacteria belong to the lowest and simplest forms of life, it cannot be expected that they will show any marked differentiation into plants. They are classified, however, among plants because of their evident relation. 16 BACTERIOLOGY. ship to certain well recognized types of plant life. In their characteristics of growth, multiplication and reproduction they resemble the group of algzw more than any other group of living beings, and it is this general relationship, rather than any one peculiarity, which has led to their being placed in the vegetable kingdom. As will be indicated later, bacteria multiply by division or fission, and for this reason they are spoken of as fission- fungi or schizomycetes. The word germ or microbe is often employed to designate bacteria. It should be understood, however, that these two terms have a broader significance and include all microscopic life, whether animal or plant. Bacteria therefore constitute a definite group of germs or microbes. In addition to their relationship to alge, the bacteria resemble in many respects the moulds or fungi, and the yeasts. These two groups will be discussed more in detail in a subsequent chapter. It will be sufficient to indicate in this connection the more marked distinctions between the moulds, yeasts and bacteria. ‘The former, as is well known, appear in velvety or cotton-like spreading growths which even the unaided eye can often resolve into a net- work of threads. The moulds are therefore spoken of as thread fungi or hyphomycetes. Growth results not by division, as in the case of bacteria, but by the lengthening of a cell or thread, that is to say by continuous end-growth. Moreover, the moulds are relatively higher forms of plant- life inasmuch as many species of this group possess special fruit-organs. The threads which make up the mass of a mould are much thicker than bacterial cells. The yeasts are uni-cellular plants which differ from bacteria in size, method of multiplication and in other respects. The yeast cell may be considered as a giant in comparison with the minute bacterial cell. Moreover, yeasts multiply, not by fission, but by a process known FORM AND CLASSIFICATION OF BACTERIA. 17 as budding. They are designated by the term bdlastomy- cetes. A great many elaborate attempts have been made at classifying bacteria. Owing to their extremely minute size it is, as a rule, impossible to follow out the life-history of each individual species. The characteristic development of the fruit-organs in higher plants affords a basis for a natural classification whereby the various species are grouped into genera and families. Such a classification is natural, because it brings together the various individuals which possess the same structure and development. Inas- much as bacteria are unicellular it follows that they do not possess definite fruit-organs, and owing to their size very little indeed can be said of their structure. The various classifications proposed are based upon characteristics such as form, size, manner of division, presence of spores, motion, number and arrangement of whips, etc. It is evi- dent, therefore, that all such systems of classification are more or less artificial. For practical purposes it is sufficient to divide bacteria according to their external form into three groups. These are: Micrococci, or spherical bacteria; Bacilli, or rod-like bacteria; Spirilla, or screw-shaped bacteria. Fic. 1. a@—Micrococcus; 4—Bacillus; c—Spirillum. In a few instances special names are applied to certain forms of one or another of these three primary types. Thus, the term Uacterium is occasionally applied to a very short bacillus. It has the same significance as the word cocco-bacillus, which indicates that the organism may at 2 18 BACTERIOLOGY. times be almost spherical, at other times rod-shaped. Again, the term vibrio is applied to certain bacteria which may form spirals, but which commonly grow in segments of a spiral. They may therefore be considered as bent, twisted rods which appear under the microscope as comma- like forms. The union of two of these ‘‘comma bacilli” gives an elongated S form, and when more of these elements unite thus they give rise to a spirillum. Long, slender, flexible spirals are frequently designated as spirochetes. Additional terms will be met with when describing the various characteristics of growth of bacteria. i @ } rol Fic. 2 a—Bacterium; 4—Vibrio; c—Spirochaete. The above classification is based upon the external forms of bacteria, and is therefore artificial in character. A similar classification applied to the higher forms of life would lead to gross error. Thus, the worm, eel and snake, viewed at a distance, show the same general appear- ance, and yet they are wholly different, unrelated types of life. A close inspection at once reveals striking differences. In the case of bacteria, owing to the extremely small size and the simplicity of the bacterial cell, it is not possible to establish structural differences, and for that reason the form classification, imperfect as it may be, is of necessity adopted. The various species of micrococci will show differences in their size, that is in their diameter, but otherwise they closely resemble one another. Occasionally, when micro- cocci grow in pairs they may show flattened, apposed sur- faces, as if two biscuits were brought together. In other in- stances they may be elongated or lance-shaped (see fig. 10 a). In the rod-shaped bacteria or bacilli greater differences will FORM AND CLASSIFICATION OF BACTERIA. 19 be observed in the form and size of various species. Thus, one species may be very short and thick, while another may be considerably longer and narrower, or longer and thicker. Again, in one species of bacillus the ends may be square, while in others they may be rounded or ellipsoidal. Among the spirilla marked variations in form or size will be observed. So much so that it is very doubtful at times whether certain species really belong to the group of bac- teria. While some species of bacteria show marked differences in form and size, it must not be supposed that this is always the case. On the contrary many undoubtedly distinct species of bacteria may have the same form and size, so that, as far as the microscope is concerned, they cannot be distinguished one from another. The division of micro- cocci, bacilli and spirilla into species is based, as a rule, not upon the mere microscopical appearance, but rather upon the sum total of the properties they possess. The characteristics of growth on various artificial media, the behavior to staining reagents, and the effect on the living animal must all be considered when an attempt is made to to distinguish one species from another. For this reason it is not possible, except in very few instances, to identify species among bacteria by mere microscopic examination. In the majority of cases the identification of species neces- sitates a careful, long and tedious study of all the proper- ties possessed by that organism. The higher forms of plant and animal life possess, as a rule, a constant form and size. Marked changes in the environment, such as temperature, and altitude, are neces- sary to produce type variations. Bacteria, however, are extremely liable to undergo alterations in form, size and other characteristics. Owing to their simple nature they are readily influenced, in some way or another, by the slightest change in their environment. A variation of a few degrees in temperature, a trifling difference in 20 BACTERIOLOGY. reaction or chemical composition of the soil, may profoundly affect the form and size of bacteria. It is evident, therefore, that a given species of bacteria does not possess a constant form or size. Under certain conditions of environment it may be a large, thick bacillus, while at other times it may appear almost like a coccus. By the typical form of a given species is meant that form which is met with when the best conditions of temperature, soil, oxygen supply, etc., are provided. The slightest variation from these optimum conditions will, as indicated above, cause a deviation from the typical form. Variations in form and size may be considered as arising from natural causes, the environment; and from artificial causes due to methods of manipulation. When a perfectly pure specimen is examined under the microscope more or less marked differences in the size of the various cells will be observed. Such differences must be expected among living, actively growing forms. The small, young cells will always be present beside the large, old cells. Again, most of the bacterial cells in a given specimen may be single, but, now and then, some will be found forming threads, or filaments, many times longer than the single cell. These thread-like forms are not con- taminations due to the presence of a different species, as might at first thought be supposed. : Another variation may be expected when the adult cell develops a spore or seed. Very characteristic forms result from the presence of a spore within the bacterial cell. These will be given special attention in a subsequent chapter. The composition of the medium on which the bacteria grow will exert a marked influence upon their form charac- teristics. The same organism planted on solid media, such as coagulated blood serum, agar and potato, will show dif- ferences inform. The addition of small amounts of glycerin- FORM AND CLASSIFICATION OF BACTERIA, 21 glucose, or an acid, or alkaline reaction likewise may affect the appearance of the cell. A comparison of the growths on solid and on liquid media will show peculiarities and modifications in the form and size of a given species. Thus, single cells or at most short threads may predominate on solid media, whereas in liquid media very long threads or filamentous growths may be found. The temperature exerts a profound effect upon the form characteristics of bacteria. At low temperature the growth is slower, and hence the individual cell may attain an unusual size, whereas at a higher temperature multipli- cation results so rapidly that the cells are considerably smaller. : Under unfavorable conditions of soil or temperature certain bacteria will show remarkable variations from the normal type. What is ordinarily a perfect rod becomes distorted out of all semblance to the original form. Club- shaped, spindle-shaped, dumb-bell-like forms are produced. Sometimes they become twisted into irregular, spherical bodies. These peculiar, deformed cells are considered as degenerations. They are commonly designated as involu- tion forms: The organism is struggling for existence under adverse conditions which, if they persist, will eventually cause its destruction. Transplantation to a favorable medium will promptly restore the typical form. AO 208 Fic. 3. Involution forms. The alterations mentioned above are the result of environment. Whatever may be the variation, it is to be considered always as temporary, inasmuch as the typical 22 ; BACTERIOLOGY. form can be reproduced whenever the organism is trans- planted and grows under its most favorable conditions of soil and temperature. The methods of examination may apparently influence the form of a given species. This is very often seen in stained preparations. Asa result of heavy staining, such as is resorted to in order to demonstrate flagella, the organism appears much larger and thicker than when stained in the ordinary way. The successive deposition of the dye, layer on layer, causes an apparent increase in the size of the cell. Again, in the method of double-staining tubercle and leprosy bacilli, the bacteria, after being heavily stained, are partly decolored with alcohol. Depending upon the extent of the decoloration, they may appear as relatively thick or as extremely thin rods. As a rule the bacteria present in tissues, when stained, appear smaller and narrower than in the fresh material. This variation is in part due to the contraction of the pro- toplasm or contents of the cell by the alcohol employed to harden the tissue. It is also due to partial decoloration which is unavoidable when the bacteria are to be differen- tiated from the tissue in which they lie imbedded. Asa result of the action of various chemical substances the protoplasm of the bacterial cell may be contracted or drawn up into irregular masses or granules. When such forms are stained they may appear like minute micrococci, although the original form was that of a typical rod. This action on protoplasm is designated as plasmolysis. EXxpos- ure of bacteria to iodine is likely to result in plasmolytic changes. Similar alterations are met with as the result of overheating specimens in the process of preparation (Fig. 4). From what has been said above with reference to variation in the form of a given species, it is evident that pleomorphism is very common among bacteria. Certain species are more prone to undergo modifications of this FORM AND CLASSIFICATION OF BACTERIA. 23 kind than others. Variations in the form and sizé of the individuals of a given species must be expected. The same form will be maintained only when all the conditions of environment are constant. In view of the marked alterations in the form of bac- teria, it has been supposed that there is a corresponding variability of species. Under certain conditions of environ- ment a given species may cease to produce the pigment which under normal conditions it would elaborate. Again, similar unfavorable conditions may cause a disease-pro- ducing germ to lose its property of growing in the living body. In other words, not only the mere form and size, but also the physiological activities of the cell may be altered. This weakening of the functional activity of a bacterial cell is known as attenuation. The attenuated or weakened germ constitutes a variety of the original type, but it does not constitute a new species. On reversing the conditions of environment, that is rendering these more favorable, the attenuated form can be brought back to the full possession of its original properties. Undoubtedly, species have originated in the past as a result of growth under altered conditions, and new species may even now be in process of formation. But under ordinary -conditions of observation this change of one- species into another does not and cannot take place. The hay bacillus cannot be transformed into the anthrax bacil- lus, nor can the germ of pneumonia be converted into that of consumption. They may both be greatly modified, giving rise to varieties, but so long as these exist they still represent the original species. The typical form and the typical species can always be reproduced by restoring the most favorable conditions of growth. CHAPTER II. SIZE AND STRUCTURE OF THE BACTERIAL CELL. Bacteria have been described as uni-cellular micro- organisms. The fact that they consist of a single cell indicates at once their microscopic character. The micro- scope reveals a world of animate beings which differ enormously in size. Some of these organisms may be large while others are exceedingly small. The bacteria are usually spoken of as the smallest of living beings, the ‘infinitely small.” Until very recently no form of life was known smaller than these. The discovery of the microbe of pleuro-pneumonia has revealed the existence of organ- isms considerably smaller than the bacteria. This new organism is so small that the very highest magnification of the microscope still leaves its form uncertain. The size of microscopic objects is usually expressed in micro-millimeters. A micro-millimeter or micron may be defined as the thousandth part of a millimeter. Inas- much as a millimeter corresponds approximately to vs of an inch, it follows that one micro-millimeter repre- sents stv of an inch. It is customary to speak of a micro-millimeter as a micron and to designate it by the Greek letter ». The largest micrococcus known has a diameter of 2 or rtoo Of an inch. There are some micrococci that have a diameter only one-tenth as large as this. The common pus-producing micrococci have a diameter of about 0.8 » or stévs Of an inch. Inasmuch as the diameter of a red blood cell is about 8 », or about #0 of an inch, it is evident that ten of these cocci placed in a row would correspond to the SIZE AND STRUCTURE OF THE BACTERIAL CELL. 25 diameter of a blood cell. The average micrococcus has a diameter of about 1 », or zste0 of an inch. The width of the average bacillus is about lw. The length will vary usually from 2 to4,. The thickest bacilli, like the B. crassus, are said to have a width of 44. Usually several bacilli must be placed end to end to correspond to the diameter of the red blood corpuscle. From the dimensions given it is evident that an enor- mous number of bacteria may be present in a relatively small volume. One milligram of the pure cells of the golden pus-producing micrococcus will contain about 2,000,000,000 individuals. One grain of this material would therefore contain 128,000,000,000 cells. In view of the extremely minute size of bacteria it is evident that they are very close to the limit of micros- copic vision. It is manifestly impossible to make out much of any structure in such small organisms. The ordinary plant or animal cell can be readily shown to consist of a cell-wall, a protoplasm and a nucleus. It may there- fore be expected that the bacterial cell will likewise consist of a cell-wall, containing the protoplasm and the nucleus. It has been supposed in the past that the bacterial cell ‘possessed a cell-wall which did not stain readily and which was composed of cellulose or woody fibre. There can be no question regarding the existence of an outer membrane ‘or envelope, but its chemical composition is undetermined. ‘Certain sarcines and a few bacilli have been said to give ‘cellulose reactions, but these observations have not been confirmed by subsequent investigators. Some of the reac- tions employed in the detection of cellulose cannot be considered as characteristic. Thus, the production of a reducing substance on treatment with an-acid may be due to cellulose, but it may also be due to the presence of a glyco-proteid. 26 BACTERIOLOGY. There is reason to believe that the cell-wall proper consists chiefiy of protein substances. The cell-wall may be considered as a layer of hardened or condensed proto- plasm. It is stained by anilin dyes and is not digested by proteolytic ferments such as pepsin and trypsin. It appar- ently responds to Millon’s reagent, but is insoluble in Schweitzer’s reagent, which, as is known, dissolves cellu- lose. It is quite possible that certain species, especially when growing on special media, acquire some cellulose-like substance. In other species dextrin-like products seem to be elaborated. Again, granulose, or a chitinous substance may be present at times in the cell-wall. The existence of a cell-wall is indicated by various characteristics exhibited by bacteria. Thus, the constant form of a spirillum or of rods can only be accounted for by the presence of an outer hard envelope. Occasionally the contents of a cell may die out and disappear, in which case the empty shell remains and is easily distinguished from the normal cells, which stain perfectly. The presence of a cell-wall is especially clearly demon- strated in certain cases. Thus, the contents of the cell contain, in addition to the protoplasm, more or less of a watery cell fluid. Asaresult of surface tension this may gather in minute droplets between the semi-solid proto- plasm. Occasionally the protoplasm will show marked adhesion to the wall, and as a result the fluid is prevented from forming globules and causes a separation of the con- tents of the cell into discs. A disc of protoplasm will alternate with a disc of the cell fluid. On staining such an organism it will exhibit transverse bands. The cell fluid holds in solution various mineral salts which exert a certain osmotic or internal pressure which is counteracted by the protoplasm and especially by the firm outer cell-wall. The normal internal pressure of the cell contents depends upon the composition of the fluid sur- rounding the cell. When the cell is placed in water which SIZE AND STRUCTURE OF THE BACTERIAL CELL. 2T contains relatively less osmotic substances than the cell itself, there is a tendency for the cell fluid and the dissolved substances to pass out into the surrounding water. This is prevented by the outer layer of protoplasm, and hence the osmotic or internal pressure. When, however, the cell is placed in a liquid which is richer in osmotic substances, a 2.5 per cent. saltpeter or a 1 per cent. salt solution, the osmotic pressure of the outside liquid overcomes that of the cell fluid. A current is thus established into the cell. The protoplasm as a result retracts from the cell wall, and this retraction of protoplasm is known as plasmolysis. aoe (6 me a & | Fic. 4. Plasmolytic changes, after A. Fischer. @—Cholera vibrio; é—Typhoid bacillus; c—Spirillum undula, In the above case the salts in the outer liquid, owing to the relatively impermeable wall, do not easily penetrate into the cell. If they did the internal pressure of the cell fluid would soon rise above that of the surrounding liquid, and as a result the protoplasm would expand and refill the cell. This actually does occur when a saltpeter solution of double the strength given is employed. When the plasmo- lyzed cell is placed in pure water the protoplasm returns. promptly to its original position. In the case of the micrococcus the protoplasm contracts. to a small globular mass, while in the bacillus two round bodies form, one at each end. As a result the cell-wall between the polar bodies is rendered visible. These plas- molytic changes occur in the living cell. As will be seen later, the flagella or motile organs seem to arise from the outer layer of the cell-wall, in which case this membrane is functionally active and is not a passive structure, as in the case of the cellulose;wall of the ordinary plant cell. 28 BACTERIOLOGY, The presence of a cell-wall is also demonstrated by the action of iodine on the cells. An organism treated with iodine solution and then fixed and stained will show the cell membrane more or less distinctly. The protoplasm has contracted from the wall and a number of colorless globules or vacuoles will be seen, due to the cell fluid that has been Squeezed out. Moreover one or more heavily stained roundish bodies, or ‘‘chromatin granules,” can also be observed. There is abundant evidence, therefore, which shows that a definite membrane is present in all the stages of the development of the bacterial cell. Moreover, plasmo- lytic experiments indicate that the protoplasm is not firmly united to this wall. The fact that strong solutions of salts do penetrate the interior of the cell indicates that the cell- wall is permeable. This of course is necessarily so, since the nourishing material present in the surrounding fluid must pass through this wall in order to reach the proto- plasm. Similarly the waste products of the cell must pass outward through a permeable cell-wall. The bacterial membrane or cell-wall is usually very thin and colorless, and for this reason cannot be ordinarily seen. As indicated above, it probably consists of a protein and not of cellulose. Under special conditions the outer layer of the cell-wall takes up water and gelatinizes. In this case the cell becomes surrounded by a broad, colorless zone which does not'stain. This softened, expanded cell- wall is known as the capsule. Owing to its soft, slimy character it causes the cells to stick together, thus giving rise to masses of cells which are, as it were, cemented together. Such a mass, when touched with a wire, can be drawn out into long, slimy threads. This massed condition of certain bacteria is designated as a zooglea. Well marked capsule formation is met with in only comparatively few bacteria. In the majority of these organisms it is either very feebly developed or entirely SIZE AND STRUCTURE OF THE BACTERIAL CELL. 29 absent. Capsulated forms are met with most often when staining the bacteria that may be present in the fluids of the animal body. Such forms are therefore met with oc- casionally in saliva, sputum and in blood. Certain species, however, may give rise to pronounced capsules, when grown on artificial media (Fig. 5 a). Gee ree Cooder a ec cnie Iiellite iueinted ome Mieat oe. capsules formed on glucose agar; 4é—Micrococcus tetragenus with cap- Gislocacous of pueamonia wilt cavailer ak ve thie § ted Bloodaell chow. ing false capsule, due to shrinkage in drying. The chemical composition of the soil influences the’ formation of the capsule. Thus, the leuconostoc, when grown on sugar media, develops remarkably large capsules, whereas in the absence of sugar this gelatinization of the cell-wall does not take place. Itis evident that the capsule is not a degenerative product, but a normal reaction induced by certain constituents of the medium. While the presence of capsules gives rise to slimy growths, it must not be inferred that they are the invariable cause of a slimy consistence. Bacteria may actually secrete a mucin-like sticky substance, and as a result the liquid, will be slimy although no capsules will be found surround- ing the individual cells. Instances of slimy milk, beer, wine, etc., from causes of this kind are not uncommon. The detection of the presence.of a capsule is not always. an easy matter. Frequently, as a result of manipulation capsule-like forms may be met with. This is invariably the case when the bacteria are present in an albuminous. 30 BACTERIOLOGY. fluid like blood. The organic matter dries out on the cover- glass first, whereas the bacterial cell dries later. In the process of drying it naturally shrinks somewhat in size, and hence a clear zone results between the dried organic matter and the retracted dried cell. On staining the speci- men a colorless zone will surround each cell and may there- fore be easily mistaken for a capsule (Fig. 5 ¢c). The capsule, unlike the real membrane, does not stain easily with anilin dyes. In certain organisms related to the bacteria, as the beggiatoa, a hardening rather than softening of the mem- brane takes place. This gives rise to a sheath or tube which surrounds the individual elements and may be consid- ered as analogous to the cell-wall of higher plants. Such sheaths, however, arenot met with among the true bacteria. The contents of the bacterial cell. The relatively hard and more or less impenetrable cell-wall encloses the soft proto- ‘plasm which is the living portion of the cell. In it, there- fore, are carried on the chemical and physical changes necessary to life. It contains, like all protoplasm, a rela- tively large amount of water. The solid constituents of the protoplasm are chiefiy nitrogenous, that is to say protein, in character. Moreover, fatty substances are always pres- ent, and in some species, as the tubercle bacillus, they may make up a large percentage (30-40 per cent.) of the total solids. There is reason to believe that at times a carbohy- drate, like granulose, is present. The inorganic constitu- ents, or ash, constitute about 10 per cent. of the dried cells. The composition of the bacterial cell will vary accord- ing to the soil on which it grows. Moreover, it is undoubt- edly influenced by the age of the individual cell. In view of the different products elaborated by various species of bacteria, it is evident that their chemical composition must necessarily vary. SIZE AND STRUCTURE OF THE BACTERIAL CELL. 31 The higher animal and plant cell always contains a nucleus within the protoplasm. The nucleus unquestion- ably plays a most important part in the division of the cell. So much so that it has been considered essential to repro- ductive changes. Inasmuch as bacteria possess the power of multiplication, it might be expected that they would contain, like higher forms, a well defined nuclear body. The direct examination of a bacterial cell fails to reveal the presence of any form analogous toa nucleus. Usually, the cell appears perfectly homogeneous, and the closest examination will not bring out a structure. The question of the existence of nuclei in bacteria has been the subject of numerous extensive investigations which have led to very different conclusions. In fact, it may be said that the presence of a nucleus is not demonstrated. There are some who consider bacteria as wholly devoid of nuclei. The minute granules which are not infrequently present, especially in older cells, are believed by some to be the first indication of the formation of a nucleus. Some have endeavored to show the existence of a cen- tral body which, while it is not a nucleus in the sense that this term is usually employed, nevertheless possesses certain properties of a nucleus, and may therefore be considered as a rudimentary form of that body. On the whole the opinion prevails that bacteria consist essentially of nuclear matter surrounded by a very thin protoplasmic layer and by the cell-wall. It is well known that in embryonic cells the nucleus almost fills the cell. This view is especially strengthened by the behavior of bacteria to anilindyes. The bacterial cells stain readily and intensely, like the nuclei of higher cells. This is assumed to be due to the presence of similar chemical sub- stances such as nucleins. The presence of a nuclein com- pound in the bacterial cell is indicated by the fact that nuclein bases and even a protamin-like body have been isolated. 32 BACTERIOLOGY. As stated above, the cellular contents of most bacteria. appear perfectly homogenous. Certain species, however, show the presence of various sized granules. These are especially present in old cultures and seem to have a protein composition. They may be related to the chromatin of higher cells and, as pointed out above, they are believed by some to represent the earliest form in the development of a. nuclear body. These granules may be large or small, very numerous or apparently absent. It is quite possible that these are due to condensation of the protoplasm as a result. of plasmolytic changes. Similar bodies appear in the cell contents invariably previous to spore formation, and these have been termed sporogenic granules. In the process of drying, fixing and staining bacteria, artificial changes not infrequently occur which may be con- sidered as evidence of a structure which in reality does not. exist. Forinstance, a bacterial suspension is allowed to dry onacover-glass. As the water evaporates the concentration of the salts present is increased, and as a result marked plas- molytic changes mayresult. Thecell may theu show marked granules or polar bodies and vacuoles. The socalled spores of the tubercle bacillus may be due to changes of this kind. Certain bacteria, such as the typhoid and chicken cholera group, on feeble staining show well stained ends. separated by an almost colorless zone. This is spoken of as the bi-polar stain, and is supposed to be due to the pres- ence of a vacuole or cell fluid in the middle of the cell. The protoplasm is consequently pushed to each side and, owing to its dense condition, readily takes up the stain. This may represent the normal conditions of the cell but, on the other hand, it may be due to the plasmolytic changes mentioned above. These polar bodies are certainly not spores, as has been at one time supposed. The contents of the bacterial cell are, as a rule, per- fectly colorless. This is true even though the organism SIZE AND STRUCTURE OF THE BACTERIAL CELL. 33 produces a brilliant pigment. In this case the pigment, or rather its antecedent, is made within the cell and is then excreted into the surrounding medium. A few bacteria show a faint red coloration when exam- ined under the microscope. This is due to the presence of a red pigment known as bacterio-purpurin. This substance, in its microchemical reactions, appears to be identical with a similar coloring matter present in certain protozoa. It is believed to exercise a role similar to that of chlorophyll. A green chlorophyll-like pigment has been observed to be present in a small number of bacteria. The absence of chlorophyll from the majority indicates that these organ- isms, unlike the higher plants, cannot assimilate carbonic acid. This, however, does not necessarily follow, since certain nitrifying bacteria are capable of assimilating car- bonic acid even in the absence of light. A number of bacteria have been met with in the mouth which show a yellowish or brownish tint. The exact nature of the pigment in either of these cases is unknown. On contact with iodine, protoplasm in general takes on a light yellow color. A number of bacteria, however, give with iodine a blue or dark violet color. Inasmuch as this reaction is similar to that with boiled starch or granulose, it is spoken of as the granulose reaction. Such bacteria would seem to contain, therefore, a carbohydrate similar to soluble starch. This substance may be present only in scattered granules, or these may accumulate so that the entire cell is deeply stained. In some bacteria the substance is not present in the cell until just previous to spore forma- tion. Bacteria giving this reaction are especially found in the mouth. , Motility of bacteria. When bacteria are examined in the living condition they will, as a rule, show motion. The movements observed may be apparent or they may. be real.. In the latter case the organism is in active motion, travel- 3 34 BACTERIOLOGY. ing from one place to another. The impulse that creates this real, active motion comes from within the cell, in other words from the living protoplasm, On the other hand, many bacteria will show an apparent motion. The — cells move to and fro, trembling as it were, but do not actually change their relative position. The impulse in this case comes from without the cell, which itself remains passive. The apparent motility of bacteria is known as Brownian, or molecular, or physical motion. All exceedingly minute objects, when suspended in the air or in a fluid, will show this peculiar swaying or pendulum movement. The mole- cules composing the air‘or fluid are in constant motion, and consequently strike, again and again, the minute objects that may be in their path. This molecular bombardment of an otherwise motionless bacterial cell causes it to sway to and fro. It may at times be very difficult to distinguish between Brownian and real motion. In that case it is necessary to resort to certain experiments. Brownian motion is obviously manifested by dead as well as by living cells. The organism may be destroyed by heating at 60° for one hour, or it may be treated with a germicidal substance such as carbolic acid, or mercuric chloride. If the dead cell exhibits the same motion as the living cell, it is clearly due to purely physical causes. Again, by growing the organism at a constant low temperature of 15° or less it will in many cases become larger than usual, and consequently will respond less readily to the impact of molecules. This pro- cedure is especially useful in doubtful cases. Finally, asa rule, it is possible to distinguish between the two kinds of motion by demonstrating the presence or absence of the characteristic organs of motion. If these are present there can be no doubt of the true motility of the organism. On the other hand, failure to find these motile organs does not prove that they are absent, inasmuch as in some undoubt- SIZE AND STRUCTURE OF THE BACTERIAL CELL. 35 edly motile bacteria it is very difficult to demonstrate their presence. Bacteria exhibit real motion only in liquid or on moist media, and then only when in the actively growing con- dition. When in the seed or spore form they do not possess motion; neither do they possess active motion when floating about in the air as fine dust-like particles. Real motion is observed in most of the spiral-shaped bacteria. The bacilli are, as a rule, motile. The micro- cocci are,.on the other hand, usually non-motile. Two or three micrococci are known to possess motion. Two motile sarcines have also been studied. The motion exhibited by bacteria will vary with the different species, and probably depends upon the number and arrangement of the organs of locomotion. Some rods show a slow, forward, wabbling movement. Others glide rapidly and steadily forward. In some the forward motion is accompanied by a rotation of the cell around its long axis, while in others a ‘‘somersault” movement is to be seen. Frequently the actively motile cell will suddenly reverse its motion and travel backward. It may come toa sudden stop, and then as suddenly, again begin to move. The motility of a culture depends largely upon its age, upon the composition of the soil, and upon. the temperature. In the case of anaerobic bacteria the presence of oxygen soon inhibits motion. The higher the temperature, as a rule, the more marked will be the movement. Thus, certain bacteria scarcely show real motion at the ordinary room temperature because of the presence of a slimy secretion. When placed, however, at the temperature of the body the motion becomes well marked. Active motion is carried on by means of certain organs known as flagella or whips. The whips are very delicate, long, thin threads of protoplasmic substance. They are analogous to similar appendages on motile infusoria, on plants and on ciliated epithelium. Owing to their extreme 36 BACTERIOLOGY. delicacy they can only very rarely be seen in the living condition. Moreover, they do not color with anilin dyes in the same way as the mass of the bacterial cell, and for that reason they are not visible in the ordinary stained prepara- tion. In order to demonstrate the presence of flagella or whips it is necessary to resort to a very special method of staining. They then appear as slender, wavy filaments. It is the lashing of these whips that propels the organism through the liquid. fil avtipts ¢ Maciine with aiftoss while, @ “Winter Single aperlllum with Enel Ab whips; * Spirillum after division with whips at each end. The flagella will vary considerably in size in different species, and even at times in. individuals of the same spe- cies. Their width is usually less than s of the width of of the cell. Their length will usually be two or three times the length of the cell, but it is not uncommon to find some bacteria with whips that are ten or twenty times as long as the cell. The flagella project from the outer border of the organ- ism. They are supposed by some to be directly continuous with the protoplasm within. the cell. In that case the protoplasmic threads are supposed to pass through minute openings in the cell-wall. Plasmolytic experiments, how- ever, do not indicate a protoplasmic continuity. On treatment with saline solutions, as indicated above, the protoplasm of the living cell withdraws completely from the cell-wall and gathers in one or two round masses.. If SIZE AND STRUCTURE OF THE BACTERIAL CELL. 37 the flagella are merely projecting threads of protoplasm it might be expected that in plasmolysis they would be with- drawn within the cell and that motion would cease. This, however, is not. the case. The plasmolyzed living cell continues to move the same as in the beginning. The flagella, therefore, are not directly connected with the inner protoplasm of the cell. They are given off by the cell-wall and chemically they would seem to be identical with the outer, softened layer of this structure. As indicated above, the cell-wall is essentially protein matter and, unlike the cellulose wall of higher plant cells, it takes an active part in the life of the cell. The cell-wall receives its nourishment from the protoplasm and is itself, therefore, a living structure. The filaments or whips given off by the outer layer of this wall are also protein in nature and are also living. The flagella are unquestionably the organs of motion. By inducing movements in the liquid they renew continually the food supply. Moreover, it is possible that the protoplasmic whip.is a means of absorb- ing nourishment for the cell. In the latter case it might be expected that flagella would also be present on non-motile bacteria. Flagella, however, are never found on strictly non-motile organisms. Pseudo-flagella, due to a mucous- like secretion, are sometimes met with in such cases. The arrangement of flagella on a given species is fairly constant, but will vary with different species. In the vibrio of Asiatic cholera, and in the bacillus of green pus, there is usually only one whip present and that is attached to one end. At times, the cholera vibrio may have two, three or four whips at one pole, and again it may have none. In old cultures it may have a whip at each end (Fig. 6 d). The spiral forms, as a rule, have a bunch of whips at one end. When each end is equipped with a bunch of whips it is because there are really two cells present. The whips on the spiral forms are not as flexible and wavy as those on the bacilli. They appear rather stiff and are 38 BACTERIOLOGY. slightly curved, like an eye-lash. They can therefore be designated as cilia rather than as flagella (Fig. 6 ¢, f). In many bacilli the flagella are very numerous, and are diffuse, or distributed all over the surface of the cell. In such cases a perfect fringe of delicate wavy lines can be seen surrounding the organism. The Proteus vulgaris, typhoid bacillus and the anaerobic bacteria are especially well provided with flagella (Fig. 6 c). Flagella are especially abundant on fresh young growths, about one day old. They disappear in old cul- tures by being torn off. Usually, they also disappear just before spore formation. This, however, is not the case with anaerobic bacteria. The formation of whips depends upon the composition of the soil. Thus, cultures of the cholera and typhoid bacilli which have been grown on the ordinary artificial media by the author for more than ten years show scarcely any motion. It has been proposed to employ the fact of the number and arrangement of whips as a means of identification of species, but for reasons indi- cated above this is not feasible. In 1890 Léffler observed in stained preparations and in a living culture of the bacillus of symptomatic anthrax enormous spindle-shaped spiral bodies, which he believed to be formed like a braid of hair, by the twisting together of a large number of the ordinary whips. Three years later the author met with these same ‘‘giant-whips ” while study- ing a new anaerobic bacillus, and was also able to repeat- edly demonstrate their presence in three other anaerobic bacteria. In the same year Sakharoff met with these pecu- liar forms in gelatin cultures of the B. Asiaticus. Recently, A. Fischer has described giant-whips in several species, and Sames has found the same forms in cultures of a motile sarcine. The author, during the past year, has studied the development of these enormous spirals in cultures of the typhoid, coli, psittacosis, and icteroides bacilli. There is SIZE AND STRUCTURE OF THE BACTERIAL CELL. 39 reason, therefore, to believe that all motile bacteria give rise to these so-called giant-whips. It is possible that the spirals observed in the intestinal contents of cholera, and those present in hospital gangrene, are not distinct organ- isms, but rather altered flagella. The author has found giant-whips in the bodies of animals (Fig 7 a)’. Their size can be inferred from the fact that they can be seen in unstained specimens. The larger forms can be easily seen with a No. 3 objective. The author has repeat- edly found giant-whips that were 70 » in length. In one instance the length was 132 », or rts of an inch. y iy Fic. 7._ Giant whips. a@ and 4 from photographs of Bacillus oedematis maligni No. II. a@—Colorless spiral in streak preparation from peritoneum of a guinea-pig— Sherrie Cie tea spindle-shaped spiral, compared with ordinary whips; c— The giant-whips are invariably motionless and usually are spindle-shaped. In this case the borders are wavy and corresponding diagonal bands will be seen, resembling the twisted appearance of a rope. Sometimes a spindle seems to divide lengthwise, so that it appears as if two spindles diverged from acommon point. The thick spindle is not the only form in which the giant-whip is met with. It may be a slender, wavy, very long spiral, without any enlarge- ment or thickening. Such spirals may extend through the entire field. These thread-shaped giant-whips may be single, but they may also be bunched, proceeding, as it were, from acommon point. The giant-whips are especially abundant in the water of condensation which is present in 1 Zeitschrift fir Hygiene, 17, plates 1 and 2. 40 BACTERIOLOGY. a tube of freshly inclined agar. The method for their detection will be given in Chapter XI. As mentioned above, Léffier, the discoverer of these strange forms, considered them to be woven masses of the ordinary whips, and this view has been quite generally accepted. It is doubtful, however, that this explana- tion of their origin is correct. If they result from a twist- ing process some motion should at times be observable, but such is not the case. The author has observed beauti- ful small spindles in cultures only eight hours old, but at no time could motion be observed. Moreover, the spindles, especially in the earliest stage, might be expected to be surrounded by the bacteria which have had their whips entangled. Usually, however, the whips stand out sharp by themselves. Furthermore, the braiding process does not satisfactorily explain the formation of the perfectly even and very thin spirals which frequently attain a length of 100 # or more. It has been supposed by some that the ordinary flagella, which, as pointed out, may be considered as living protoplasmic matter, when torn loose from the cell, may continue to move about for a short time, and in this way lead to the formation of giant-whips. There is rea- son to believe that the motile organs on certain flagellates, or animal organisms, are endowed with contractility and, for a short time at least, may live and move about after separation from the cell proper. It is possible, however, that these forms are unusually developed flagella, either as a result of involution changes, or because of a softening of the whip substance, corresponding to that observed in capsule formation. CHAPTER IIL THE LIFE HISTORY OF BACTERIA. Growth and multiplication is a characteristic of living organisms. As a rule the plant or animal cell, when it reaches the fully developed, adult stage, divides and thus gives rise to two newcells. The young bacterial cell, like- wise, grows, attains its full size, and then multiplies by division or fission. Bacteria are, for this reason, designated as schizomycetes or fission-fungi. The multiplication, or actual increase in number, of bacteria always results by the process of division whereby one cell forms two, and only two, newcells. There are instances, as will presently be seen, where apparently one cell gives rise to four or to eight cells, but in all such cases the division is consecutive and not direct. That is to say, the cell does not divide directly into fourths or eighths, but does form two halves which, subsequently dividing, yield four cells, and the next division yields eight cells. Cell-division among uni-cellular plants and animals is completed ina very short length of time. This, perhaps, is especially true of the bacteria. Given a suitable soil, the rapidity of growth and multiplication of bacteria will depend upon the temperature. The nearer the temperature approaches the freezing point the slower will be the rate of multiplication. On the other hand a temperature of 80° to 87° gives. the most rapid growth. Under such conditions the average bacterial cell will probably divide in less than ahalf anhour. This rate of multiplication cannot be main- tained for any length of time, owing to the exhaustion of the soil and above all to the accumulation of waste products 42 BACTERIOLOGY. which retard and eventually stop growth. Assuming the continuance of favorable conditions, a single cell, dividing in 80 minutes, would be represented at the end of 12 hours by more than sixteen million descendants. In 24 hours the number would rise to more than two hundred and eighty billions. : The above figures will serve to impress the fact that bacteria multiply with extreme rapidity. Moreover, they will help to understand what marked changes may result in a comparatively short period of time when certain bacteria develop in milk, meats or in the living animal body. Small as bacteria are, owing to their rapid multiplication, they may give rise to poisonous substances in sufficient amount to cause in a few hours profound poisoning, or even death. K soursinsal Gout Wow utlh YO GC) GZ CO’ (at eae a—Bacillus, showing deflection of outer The process of. division can best be observed in large bacilli. Owing to the absence of a definite nucleus, and of any cell structure, the change that takes place during multiplication is very simple. When the bacillus has attained the fully developed stage, a slight transverse constriction appears at the middle of the rod. The ring- like process of the cell-wall gradually extends toward the center of the cell until eventually the protoplasm becomes divided into two halves. Usually the first indication that cell division has taken place is the appearance of the clear, delicate transverse line. The division of bacilli and of spirals is always transverse, never longitudinal. The wall that divides the cell into two is to be consid- ered as an ingrowth of the cell membrane. Inasmuch as in THE LIFE HISTORY OF BACTERIA. 43. stained preparations this dividing line remains colorless, it is evident that it largely consists of the same material as the outer layer of the cell membrane, namely, the softened, gelatinized mantle which, unlike the membrane proper, does not stain with anilin dyes. Obviously the entire cell- wall, inner as well as outer membrane, is depressed at the zone of constriction (Fig. 8 a). When the ingrowing wall reaches the center the inner membrane coalesces and divides, leaving the space between the two new end walls filled with the gelatinized outer membrane. Continuous filaments showing no apparent division into cells are some- times met with. In such cases division may have occurred, but is not visible, owing to the absence of the outer mem- brane. 4 7 oe - : 3 o-oo oe Fic. 9. Division forms of bacilli. a@—single; 4—in pairs; c—in threads. As soon as the cell has completely divided, the two new cells may at once tear apart and lead a separate exist- ence. Many bacilli are therefore characterized as growing singly. In some species there is a tendency for the two cells to remain attached owing to the firm union of the two cells, which are held together by the gelatinous connecting zone. The bacillus is then spoken of as growing in pairs— diplo-bacillus. In other species the cells are likely to remain attached even after repeated division. The individual rods remain attached, end to end, by the undivided outer layer of the cell-wall, and thus give rise to long filaments which are commonly designated as threads. A thread may be long or short; that is, it may consist of four or five, or fifty to one 44 BACTERIOLOGY. hundred or more cells. A thread is always cOmposed of rod-shaped bacteria, bacilli, and may be compared to a row of bricks in a wall. Micrococci multiply in like manner by division. The Spherical organism is usually supposed to elongate some- what just previous to fission (Fig. 8 b). The transverse constriction and dividing line then appear as in the case of a bacillus. According to some the micrococcus does not elongate first, but divides directly into two halves. It may increase in size previous to division, but its form remains spherical. On the other hand the bacillus always grows in length, and it would seem as if this fact could be utilized in distinguishing between a true micrococcus and a very short rod. 8 8 © dictiened-appoeedl auntace Geoneegseust, lameolaly tore Canedsioaiae Eaeta: . coccus: Fecera are euaeien veins atetrad; a@—Sarcine form resulting from The two half-spheres which result from the division of micrococcus may retain this form and remain attached by the undivided cell-wall. In-this case the two cells are spoken of as being biscuit-shaped and in pairs. The germ of gonorrhea presents this characteristic appearance. When micrococci grow in pairs, as in this instance, they ate designated as diplococci (Fig. 10 a). On the other hand, as soon as the division is complete, the two new cells may gradually round out and assume a spherical form. They may tear apart and grow singly; or, the two cells may remain attached by the narrow zone of undivided cell-wall, forming thus a diplococcus. If each of these two attached cells now divides in the same direction as the original one, a row of four spherical organisms will result.