Cornell Mniversity Litvary BOUGHT WITH THE INCOME FROM THE SAGE ENDOWMENT FUND THE GIFT OF Henrg W. Sage 1891 B25 149.4 sire |i] 1357 iii BACTERIA IN RELATION TO COUNTRY LIFE THE MACMILLAN COMPANY NEW YORK + BOSTON - CHICAGO ATLANTA * SAN FRANCISCO MACMILLAN & CO., Limirep LONDON + BOMBAY « CALCUTTA MELBOURNE THE MACMILLAN CO, OF CANADA, Lt», TORONTO ANTONI VAN LEEUWENHOEK Leeuwenhoek (pronounced la-ven-hook), a Dutch naturalist, 1632-1723, is generally cited as the first to discover bacteria. See page 2 BACTERIA IN RELATION TO COUNTRY LIFE BY JACOB G. LIPMAN, A.M., Pu.D. SOIL CHEMIST AND BACTERIOLOGIST FOR THE NEW JERSEY AGRICULTURAL EXPERIMENT STATION AND ASSOCIATE PROFESSOR OF AGRICULTURE IN RUTGERS COLLEGE THIRD HDITION dew Bork THE MACMILLAN COMPANY 1911 All rights reserved Copyriant. 1908 By THE MACMILLAN COMPANY Set up and electrotyped. Published. September, 1908 Reprinted July, 1909, January, 1911 Mount Pleasant press J. Horace McFarland Company Harrisburg, Pa. a PREFACE Lire in the country, like that in the city, has been rendered less simple by inventions and scientific in- vestigations. A whole series of new problems have arisen within a single generation and have called forth more or less successiui attempts at their solution. Among these new problems we may properly include that of the bacteria, those minute living things that float in the air that we breathe, that exist for our weal or woe in the water that we drink, that perform a mighty work in the soil and thereby make it possible for generations of plants and of animals to come and go. The extreme smallness of the bacteria renders them invisible to the naked eye, and makes it difficult for the layman to think of them as definite living beings entrusted with an important task in the continuing of life. The deepening current of human existence now forces us to study the bacteria and other microérgan- isms. In so far as they are dangerous to our health and happiness we must learn to defend ourselves ; we must learn to destroy them or to render them harm- less. In so far as they are beneficial, we must learn to (vii) viii Preface control them and to make their activities widely useful to human society. The present volume is an attempt to treat, in a simple way, of the bacteria as they concern life in the country. It is an attempt to discuss the char- acter of the bacteria in air, water, sewage, manure, soil, and food products. Techriical terms and expres- sions have been eliminated.as far as practicable, and it is hoped that the general reader may find the book an aid in the understanding of the bacteriological problems as they affect the daily tasks on the farm. The author wishes to express his indebtedness to Messrs. Percy E. Brown and Herbert Seidman for their assistance in the preparation of the index; and to Dr. Edward B. Voorhees for the permission to use a number of the photographs that appear in this volume. JACOB G. LIPMAN. New Jersey Experiment Stations, New Brunswick, N. J. June 22, 1908. CONTENTS PART I Pages Structure anp GrowTH or Bacteria ... 1-45 CHAPTER I Tue Rist oF BacTERIOLOGY. .. 1... 2.2.2 eee 1-12 Contagion—Discovery of bacteria—Spontaneous genera- tion—Spontaneous generation disproved—The physiology of bacteria—Bacteria as a cause of disease—The study of bacteria—Anthrax bacillus—New methods of study— Bacteriology and agriculture. CHAPTER II Tue Form anp Structure or Bacteria. ..... .. . 18-25 Irregular forms—Shape—Size—Motility and organs of motion—Zodgloea—Rate of increase—Conditions that retard bacterial growth—Spores—Spores as a means of preservation—Spore formation— Vitality of spores— Classes of bacteria —Sterilization and pasteurization— Intermittent sterilization. CHAPTER III Tne Cuemistry or THE BacTERIAL CELL 2 fee we « 26-29 The cell-wall and its contents—Content of the proto- plasm—The absorption of food. ‘ (ix) x Contents CHAPTER IV Pages Tue Foop RequireMeNts or BacTeRIA....... . - 30-35 The sources of carbon—The compounds of carbon in bacteria—The source of nitrogen—Enzymes—Pigments —Phosphorescence. CHAPTER V Conpirions AFFECTING THE GROWTH OF BACTERIA . . 36-44 Temperature—Thermophile bacteria—Effect of cold— Attenuated cultures— Moisture content of the culture medium — Relation to oxygen— Action of sunlight — Electric light—Influence of electric currents—Influence of the concentration of the culture medium—The re- action of the culture medium—Importance of reaction of the soil. PART II Bacteria In AIR AND WATER. ..... 45-103 CHAPTER VI BacTERIA IN THE ATMOSPHERE ...... we ee 2 45-55 Bacteria on dust particles—Determination of the bac- teria in the air—Pasteur’s method of determination— Value of Pasteur’s method—Quantitative methods. Influence of locality — Bacteria in city air— Bacteria in the air of the country. Influence of season climate, and altitude—Bacteria in the air at different seasons of the year—The influence of climate—The influence of altitude—Bacteria and respiration. CHAPTER VII Tue Revation or Water to HeatrH AND Disease. . . 56-60 Ancient water-supplies—Relation of drinking-water and disease discovered—Epidemics of cholera and typhoid fever—Other sources of typhoid infection—Other dis- eases arising from use of impure water. Contents xi CHAPTER VIII CONTAMINATION OF STREAMS AND LAKES ......2. 6 61-76 Soils contain bacteria—The character of the bacteria in water—The number of bacteria in water—Causes affect- ing the increase or decrease of bacteria in water—The supply of food—Temperature—Sunlight—Animalcules injurious to bacteria—Sedimentation—Dilution—The contamination of drinking-water by sewage. CHAPTER IX PURIFICATION OF RIVER AND LAKE SUPPLIES . . . « 77-89 The quality of organic matter—Alkaline water— River-water and surface drainage—Self-purification of rivers—The storing and filtration of river-water— Sand-filtration—Other methods of water purification— The alum method—The Clark process—Purification by finely divided solids—The Woolf method—Purification by means of ozone—Filters for domestic purposes— The waters of lakes and ponds. CHAPTER X Bacteria IN WELLS, SprinGs, TANKS ANDIcE ...... 90-98 Bacteria and well-water—Deep wells and springs— Driven wells—Artesian wells—Bacteria in the water.of cisterns and tanks. Bacteria in ice. CHAPTER XI Tue SANrTARY EXAMINATION OF WATER-SUPPLIES . . .99-102 xii Contents PART III Pages BacTerIA AND SewacGe.... . .103-134 CHAPTER XII THE Proptem or SrewaGe-DIsPosaL » ee. » 108-111 The cess-pool—The new method of the nineteenth century —Bacteria in sewage—Growth of the problem—lInland communities and sewage-disposal—The source, com- position and quantity of sewage. CHAPTER XIII BactTeR1AL PURIFICATION OF SEWAGE . . . . . «112-124 Sewage-farms—Sewage tanks and filter beds—Progress in sewage-purification—Intermittent sewage-filtration —Separation of bacterial activities—Temperature and bacterial activities—Hydrolysis—Treatment of effluent —Temperature and filter efficiency—Inoculation—Kinds of bacteria in filter beds and septic tanks— Loss of nitrogen — Bacterial efficiency in sewage-purification — Factory wastes and bacterial efficiency— The working capacity of bacterial filters. CHAPTER XIV SEWAGE-IRRIGATION abd » 4. .125-134 Economic value of sewage-irrigation—Sanitary value of sewage-irrigation—Kinds of irrigation—Broad irriga- tion—Intermittent and mixed irrigation—The crops grown on sewage-farms—Preliminary treatment of sewage for sewage-farms—Objections to sewage-farming —Sanitary efficiency of sewage-purification. Contents xiii PART IV Pages Bacteria IN RELATION To Sow Fertiviry — .135-302 CHAPTER XV NUMBER AND DISTRIBUTION OF BACTERIA IN THE SoIL _ .135-143 Number of bacteria in soil—Distribution of bacteria in the soil. CHAPTER XVI THE RELATIONS oF Bacteria AND Humus . 144-154 The quantity of humus as affecting number of bacteria— Quality of humus as affecting number of bacteria. Bac- teria and the decomposition of soil-humus. CHAPTER XVII BacTERIA AND THE TRANSFORMATION OF SOIL-NITROGEN .155-1§7 The source of nitrogen in the soil—Proportion of nitro- gen in the soil—Nitrogen compounds in the soil-humus— Loss of nitrogen in the soil—Availability of nitrogen— Conditions affecting availability of nitrogen—Soil bac- teria— Ammonification— Ammonifying bacteria— En- zymes—Mutual relations of bacteria—Denitrifying bac- teria—Influence of bacteria on one another—Ammonify- ing power of soils. CHAPTER XVIII NITRIFICATION : . 168-182 True character of nitrification—Pure culture of nitrify- ing bacteria—Importance of nitrification—Conditions affecting nitrification—Loss of nitrates in the soil— Sources of nitrates—Early use of nitrates—Conditions influencing the formation of nitrates—Availability of nitrogenous materials—Influence of crop on availability. Xiv Contents CHAPTER XIX DENITRIFICATION : . . .183-189 Early idea of denitrification—The cause of denitrification —Value of modern discoveries—Modern conclusions con- cerning denitrification—The denitrifying bacteria. Pages CHAPTER XX Tuer Increase or Sort-NiTROGEN : . 190-196 The ammonia theory—The nitrate theory—The bacteria theory. CHAPTER XXI THe Non-Sympiotic Nirrocen-Fixinc Bactrertra’. .197-205 Aérobic and anaérobic bacteria—The aérobie nitrogen- fixing bacteria— Agricultural importance of the two classes of bacteria—Other nitrogen-fixing forms. CHAPTER XXII SympBioTic FrxaTIon oF NITROGEN . . ‘ . .206-220 The value of legumes—The limitations of legumes— Legumes and fertilizers—The cause of the soil-enriching qualities of legumes—Liebig’s theory—Other theories— The solution of the problem—Nodules on the roots of legumes—The nature of the nodules—The bacteria of . legumes—The relations between legumes and the nodule bacteria. CHAPTER XXIII So1t-INocuLATION 78 . . 221-236 Pure cultures—Nitragin—Agar as a food for cultures— Difficulty in using pure cultures—Artificial cultures— Soil-inoculation in the United States—Alinit. Contents XV CHAPTER XXIV Pages GREEN-MaNnvuRING » ee es 6287-264 Green-manures and humus in the soil—Leguminous green-manures and nitrogen in the soil—Green- manures on sandy soils—The cowpea, soybean, sand vetch, crimson clover and velvet bean—Green-manures on loams and clay soils—Leguminous green-manures and the succeeding crops. CHAPTER XXV FALLOWING ek i=. ch . 265-274 Fallow crops—Fallow and cropped soils—Moisture and fallows—Aération and fallows—Fallowing and plant- food—Bare fallows and nitrogen in the soil. CHAPTER XXVI Sow Bacreria In RELATION TO MinerRats IN THE Sor, . .275-302 Soil bacteria in relation to lime and magnesia—The causes of the migration of lime and magnesia—The liming of soils—The iosses of lime—The importance of lime—Bacterial activities and lime. Soil bacteria in re- lation to phosphates and other compounds of phosphorus —Bacterial activities and phosphorus—Bacterial activi- ties and phosphate fertilizers—Ground bone—The effect of using sulfuric acid—Thomas slag—Other sources of phosphoric acid. Activities of soil bacteria in relation to potash—Potash and the weathering process—Lime as an indirect source of potash—Humus in relation to soil- potash—Organic acids and the decomposition of rock fragments—Potash salts and bacteria. Bacteria in rela- tion to sulfur—Sulfuretted hydrogen—Sulfate of lime— The work of bacteria—Sulfur and bacterial development. Bacteria in relation to iron—Iron rust and bacteria—The importance of iron bacteria — Iron in the soil and decay bacteria—Influence of iron on bacteria. xvi Contents PART V Pages BacTeria IN BARNYARD MaNnuRE. . . .303-356 CHAPTER XXVII Manure: Its ComposITION AND LossEs : .3803-317 Bacterial change in manure—Mechanical constitution and bacterial change—Chemical composition and bac- terial change. Losses from farmyard manure—Value of manures—Losses of elements caused by digestion— Other causes for losses—Importance of proper storing— The loss of nitrogen—Aérobic and anaérobic decompo- sition—Bacterial activities and money losses. o CHAPTER XXVIII Tue BacTeRIA IN MANURE—AMMONIFICATION. ‘ . 318-325 Changes in manure-bacteria—Three stages of change— Bacteria and conditions affecting decomposition. Am- monification—Hippuric acid—Uric acid—The formation of ammonia in liquid excreta—Loss of ammonium car- bonate—Urease—Other ammonia-forming bacteria— Importance of ammonification. CHAPTER XXIX DENITRIFICATION IN MANURES . 326-336 Nitrates in manure—Denitrifying Seat Buea ments in denitrification—The danger of denitrification. Losses of elementary nitrogen—The problem of the loss of nitrogen—Recent information on the problem. Soluble nitrogenous substances made insoluble—The change of available into unavailable nitrogen—Conditions affect- ing the change. Conclusion. CHAPTER XXX NIrRiIFICATION IN MANURES .337-341 Nitrification in manures—Effect of liquid—The compost heap—The loss of humus-forming material. Contents XVil CHAPTER XXXI Pages CELLULOSE FERMENTATION ‘ : . 842-347 Gases in the manure pile. Litter and the development of bacteria in manure. CHAPTER XXXII THE CONSERVATION OF MANURIAL CONSTITUENTS . . . .348-356 Chemical methods—Gypsum—Burned lime, shell-lime, and limestone—Superphosphate—Kainit—Sulfuric acid —Value of chemical methods. Mechanical methods— Effect of moisture—The temperature of the manure— Preventing the loss of ammonia. PART VI BAcTERIA IN MILK AND RELATED Propucts — .357—430 CHAPTER XXXIII Mixx as A Foop . . .857-359 Milk a medium for bacterial development CHAPTER XXXIV Source oF Bacteria IN MILK . 3860-368 Bacteria in the udder—Bacteria in the air—Bacteria on the cow’s body—The milker—The ee ane machines—Milk-strainer. CHAPTER XXXV Kinps or Bacteria In Mik .869-377 Species of bacteria in milk—Lactic-acid bacteria—Sweet curdling—Harmless bacteria—Milk faults—Blue milk— Red milk—Bitter milk—Ropy milk. CHAPTER XXXVI Mitk BEVERAGES : . .3878-381 Kefir—Kumiss—Matzoon. XVill Contents CHAPTER XXXVII Pages Tue Keeping QUALITY oF MILK te . 382-396 The so-called germicidal power—Temperature. Means for improving the keeping quality of milk—Pressure— Centrifugal force—Sterilization by heat—Pasteuriza- tion—Treatment with chemicals. Problems of trans- portation and distribution. CHAPTER XXXVIII Disease Bacteria 1n MILK . . . .397-401 Tuberculosis—Typhoid—Diphtheria and Scarlet fever. CHAPTER XXXIX BacTeRIA IN CREAM AND CREAM-RIPENING ... . .402-410 Starters. CHAPTER XL Bacteria IN BUTTER . . . -411-415 The changes occurring in butter—N rnabere and kinds— Disease bacteria in butter—Butter faults. CHAPTER XLI BacTeRIa IN CHEESE ‘ : .416-430 The ripening process—Enzymes and bacteria in the ripening of cheese—Soft cheeses—Hard cheeses— Cheese faults. PART VII BactTEria IN RELATION TO PRESERVATION OF Foop .431-446 CHAPTER XLII BacTERIA IN RELATION TO CANNING ye .431-438 The principles of canning—Development of the industry —Losses through imperfect canning—Temperatures Contents xix Pages required for sterilization—Canned meat—Canned milk —The use of preservatives. CHAPTER XLIIT OTHER MEANS OF PRESERVING Foop Propucts.—PicKLING 439-446 Low temperatures—Drying—Salting, pickling and smoking—Pickled fish—Sauerkraut—Dill-pickles. PART VIII BacTERIA AND FERMENTATION... .447-472 CHAPTER XLIV BacTeria IN Breap-MAKING ......... 447-448 Bread faults. CHAPTER XLV BacTERIA IN THE SuGAR INDUSTRY . 449-450 Streptococcus mesenteroides—Clostridium gelatinosum. CHAPTER XLVI BAcTERIA IN THE PREPARATION OF HAY AND OTHER FoppDERS i eg .451--455 Brown hay—Corn silage. CHAPTER XLVII BacTERIA IN MIscELLANEOUS AGRICULTURAL INDUSTRIES 456-457 The retting of flax and hemp—The preparation of natto—Bacteria and agricultural products. xx Contents CHAPTER XLVIII Pages BacTeriaL Diskases oF FERMENTED Liquors . 458-462 The “turning” of wine and beer—Ropiness in wine-- Sarcina sickness—Loss of color in wine—Mannitic fer- mentation. CHAPTER XLIX VINEGAR-MAKING . . » 2. .463-472 History of the art—Modern knowledge—The “mother of vinegar”—Acetic-acid bacteria—Methods of using acetic ferments—Pure cultures in vinegar-making— The storing of vinegar. ' INDEX AND GLOSSARY .. 0... 0606 25 5 2 473-486 BACTERIA IN RELATION TO COUNTRY LIFE PART I STRUCTURE AND GROWTH OF BACTERIA CHAPTER I THE RISE OF BACTERIOLOGY Bacrerta are minute living things lying in the border- land between plants and animals. Their existence was undreamed of until times comparatively recent, yet their appearance on the earth antedates that of man. Plant and animal remains now turned to stone and dating back to early geological ages have been found to contain the petrified cells of bacteria, some forms of which closely resemble those of today. We know that an almost endless number of plants and animals now extinct have run their course on this earth in passing from lower forms to higher, in adapting themselves to a new environment. We do not know of the cycles of change through which the bacteria have passed, nor do we know of the birth and the passing of forms long ago vanished. We know merely that in the world of today their name is legion, that they differ not only in form and size, but also in the chemical changes that they produce. The rivers, the sea, and the earth all A (1) 2 Bacteria in Relation to Country Life have their specific bacterial inhabitants. There are bacteria that cause abnormal conditions or “disease”’ in plants or animals; there are others that are harm- less; there are still others that are known to be dis- tinctly beneficial and indispensable to the growth of higher organisms. The existence of bacteria was not revealed until the perfecting of the compound microscope, toward the end of the seventeenth century. Yet, though un- known as such, they made themselves manifest many generations previous, by some of their activities. Decay,’ putrefaction and fermentation were familiar phenomena at the dawn of written history. Contagion—The diseases of man and of domestic animals are of very ancient origin. Great epidemics that devastated towns, cities, and entire kingdoms were more common once than they are now. ‘Man learned long ago that disease may be spread from person to person, and that personal contact increases the degree of infection. So certain was this knowledge that attempts were not lacking even in biblical times, and perhaps earlier in old Babylonia, to establish isolation camps for diseased persons, particularly in the case of lepers. It will not be disputed, therefore, that not only were the phenomena resulting from bacterial activities known thousands of years ago, but that a certain suspicion was entertained in the case of human disease that the outbreak was due, in most cases, perhaps to some form of living contagion. Discovery of bacteria.—Leeuwenhoek in Holland who, with his more perfectly constructed lenses, first beheld Leeuwenhoek 3 bacteria in 1675, designated them as ‘‘animalcules.”’ He recognized differences in their appearance and size as well as in their mode of motion, and noted their presence in sea-water and well-water, as well as in various substances of animal origin. These observa- tions, subsequently supplemented by others, gave rise to much speculation and heated discussion concerning the relations of the animalcules to animal diseases. Much interest was attached to the opinion already entertained by some that disease is due to a living contagium. A still greater interest, however, was at- tached, at this time, to the bacteria in connection with the question of spontaneous generation. Spontaneous generation.—The belief in spontaneous generation is of ancient origin. Some of the philosophers of the Middle Ages were firm in this belief. Refer- ence may be found in their writings to the spontaneous generation of mice, worms, maggots, and other forms of animal life. With the advance of knowledge, how- ever, the correctness of their facts and conclusions was questioned, and their statements were, in time, discredited and refuted. The discovery of bacteria seemed to bring new sup- port to the theory of spontaneous generation. Need- ham announced in 1749 that he had observed the development of bacteria in beef-broth which had been boiled and kept, after that, in a well-stoppered flask.. He reasoned that, since no living beings could with- stand boiling, the appearance of the animalcules in the sterile broth must have been due to spontaneous generation. This apparent demonstration of sponta- 4 Bacteria in Relation to Country Lije weous generation was not, however, implicitly accepted by all. Bonnet pointed out the possible existence of organisms, or their modifications, capable of withstand- ing boiling temperatures, and even suggested that Needham’s flasks may have been improperly protected against the entrance of bacteria from without. Spontaneous generation disproved.—In 1765 it was demonstrated by Spallanzani that the development of microérganisms in Needham’s broth resulted from im- perfect sterilization. By boiling the broth in the flask for a long time, both the container and the contents were thoroughly sterilized and no bacterial develop- ment occurred. But, not content with this-proof, the believers in spontaneous generation now maintained that the liquid and the air above it had been so changed by heating as to preclude the formation of microérgan- isms. It was not difficult, of course, to demonstrate that the liquid itself had not lost the power of under- going putrefaction. It was merely necessary to open the flask to the air in order to induce the appearance and development of bacteria in the liquid. The com- position of the atmosphere was still unknown at that time, and no direct proof could, therefore, be adduced in this connection. The matter thus rested, undecided, for several decades, when Schultze brought forward the proof in 1836 that it is not necessary to heat the air in order to deprive it of the microérganisms suspended in it. He proved that this could also be done by passing the air through strong ‘acid or alkaline solutions. Schwann secured similar results in the following year by forcing Spontaneous Generation 5 the air through molten metal; and Schréder and Dusck simplified the matter still further, in 1854, by demon- strating that the air could be deprived of its micro- organisms by being passed through a cotton plug. Hoffmann, and, likewise, Pasteur, went a step further, and proved that even the cotton filter was unnecessary for making the air incapable of setting up putrefaction in sterile meat broth. By drawing out the neck of the flask into a long, capillary tube, and by bending the latter, the dust particles in the air, and, among them, the bacteria, were made to settle out before they reached the liquid, and no putrefaction occurred in the latter. Another proof of the falsity of the idea of sponta- neous generation was given by Tyndall, when he demon- strated that perishable articles did not spoil on the tops of high mountains, due to the fact that the air in those altitudes contained no germs. The belief in spontaneous generation was gradually shown to be untenable. Certain proof was supplied that no putrefaction and decay take place in the absence of bacteria. The occasional failure to secure complete sterilization by boiling was accounted for by Kohn’s discovery in 1875, of spores,—certain rest- ing stages in the growth of bacteria, more resistant to heat than the vegetative cells, and capable of with- standing boiling temperatures for some time. The physiology oj bacteria.—Pasteur’s epoch-making investigations on fermentation shed a broader light on the activities of microdrganisms. His work plainly indicated that various kinds of bacteria possess specific functions and differ in the chemical changes 6 Bacteria in Relation to Country Life which they produce. This work may, therefore, be regarded as the starting point for much fruitful research, the foundation of an extensive knowledge on the physi- ology of bacteria, that is, the chemical changes involved in their life-processes. Bacteria were to be distinguished, henceforth, not by their appearance alone, but by the chemical transformations of which they are capable. They were to be regarded as chemical agents of wide significance, builders and destroyers in vegetable and animal substances, in organic and inorganic materials, in the presence or absence of air. Bacteria as a cause of disease—The study of bac- teria, and of other microdrganisms, as agents of decay, putrefaction and fermentation, gained in interest with the recognition that bacteria may also be the specific cause of disease. As far back as 1762, the belief was expressed by Plenciz, a Vienna physician, that disease is the result of infection by animalcules; and, more important still, that every disease has its particular germ. The views of Plenciz met with no acceptance, and were soon forgotten amid the clashing opinions on spontaneous generation. Towards the middle of the nineteenth century, the question of a living contagium in animal diseases again attracted wide attention. Bassi’s demonstration, in 1837, that a certain disease of the silkworm is due to a specific germ, was the first of its kind, and carried much weight. The question was placed on a broader basis by Henle’s teachings on bacteria. He not only thought that infectious diseases are caused by bacteria, but he outlined the methods of inquiry and pointed out that Bacteria and Disease 7 no organism may be designated as the specific cause of any given disease unless it can be proved to be invariably present in every case, and can be isolated from the infectious materials. Henle’s clear conception of bacteria as agents of infection was evidently too far advanced for his day. At any rate, his views failed to gain general recognition for a long time. Pasteur’s researches prepared the ground for such recognition. A further step in advance was made by. Lemaire when he showed that carbolice acid, poisonous to animals and capable of suspending putrefaction, could also stop pus-formation in wounds. Lister carried the work forward and developed his method of anti- septic surgery, a method through which medical science has achieved such splendid results. Lister’s announce- ment of 1868 stimulated inquiry and brought to light important facts. The investigations of Klebs during the Franco-Prus- sian War traced the entrance and the development of bacteria in wounds and their passing into the circulatory system. Klebs and other investigators also noted the constant presence of bacteria in diphtheritic infections. An apparent relation was likewise found between bacteria and other infectious diseases. For all that, much was sur- mise and speculation rather than. certainty. The bac- teriological methods for the isolation and identification of bacteria had not yet been developed, and no direct proof could be furnished for the facts observed. The study of bacteria.—The systematic study of bac- teria was furthered by the work of Schréter, published in 1872. In his studies of pigment-producing bacteria, 8 Bacteria in Relation to Country Life he found that the organisms could be grown on solid substances, among them boiled potatoes. The bacterial masses which appeared on the potatoes could be em- ployed for infection of new portions of sterile potato, whereby the distinct colors could be maintained. For this reason, Schroter was inclined to think that he was dealing with definite kinds of bacteria. The nucleus was thus created for the opinion that, among bacteria, as among more highly organized or- ganisms, there exist definite species fairly constant in their structure and in their physiological activities. This opinion was given expression by Ferdinand Kohn, whose investigations and writings may be justly marked as the beginning of a new period in modern bacteriology. We owe to him not only the foundation of systematic bacteriology, but the stimulus and the methods for more advanced research. On this foundation, the genius of his pupil, Robert Koch, built a noble structure and established the science of bacteriology. Anthrax bacillus—In 1876, Koch demonstrated clearly and convincingly that anthrax in cattle is due to a specific germ, and thus confirmed a fact already indicated by the observations of others. He isolated the anthrax bacillus in pure culture, studied it under the microscope, and showed that he could produce an- thrax in other animals by inoculation from such cultures. New methods of study.—Soon after this, Koch devel- oped new methods for the study of bacteria, and for the preparation of pure cultures. By the employment of solid media, he demonstrated the comparative ease with which a number of different bacteria in the same Pure Cultures 9 liquid may be separated from one another. He inocu- lated liquefied portions of sterile gelatine with slight quantities of material containing the bacteria which he wished to study, distributed the latter uniformly by shaking the liquid gelatine, and spread it out on a plate where it was solidified by cooling. The germs fixed in isolated spots in the sheet of solid gelatine could multiply only in those particular spots until their numbers became so great as to form a little heap or colony visible to the naked eye. These colonies, each the offspring of a single cell, could furnish only one species of bacteria. When transferred with a sterile platinum needle to some liquid suitable for their de- velopment, the organisms furnished a so-called pure culture, that is, a growth of only one kind of bacteria, uncontaminated by other organisms. When doubt still existed as to the purity of the culture, new plates were prepared from the purified material and the work re- peated several times until, at last, the growth could safely be regarded as pure. , In the course of time, new culture media were de- vised for organisms that would not grow on gelatine,— culture media that made possible the isolation of soil and water bacteria, bacteria of milk and of other food products, and bacteria causing disease in plants. The use of aniline dyes, proposed by Wygert and adopted by Koch, made possible a’ differentiation of the cell structure of the organisms, while the inoculation of mixtures containing disease bacteria into experimental animals, like rabbits or guinea pigs, offered a new means for the-identification and purification of disease germs. 10 Bacteria in Relation to Country Life Diseases like tuberculosis, diphtheria, typhoid, cholera, pleuro-pneumonia and leprosy were proved to be due to specific organisms isolated and studied in pure culture. The rapid advance of bacteriology within the last quarter of a century has carried investigations beyond the isolation of pure cultures of various bacteria. Chem- istry has become a strong ally of bacteriology, and has Fig. 1. Soil-bacteriological investigations at the New Jersey Experiment Station. enabled the latter to recognize the chemical transfor- mations effected by the microdrganisms. Medical bacteriology saw a wonderful development in the study of toxins, the poisonous substances resulting from bacterial activities, and anti-toxins, capable of neutral- izing these poisons. This, in turn, has been the starting point for far-reaching investigations concerning the invasion of the animal system by bacteria, and the protective machinery of the living body. We have learned much concerning the powers of Bacteria and Agriculture 11 resistance possessed by animals, of natural and acquired immunity to disease, and of the methods whereby im- munity may be secured. The mortality from some of the most dreaded diseases has been reduced to an as- tonishing degree, and, with the aid of sanitation, some of them have become almost unknown. The achieve- ments of medical bacteriology, too vast to be reviewed here in detail, are only a promise of the still greater Fig. 2. Cylinders used for chemical and bacteriological investigations of soils. achievements to come, and a vindication of the views set forth by the pioneers in the study,—Kohn, Pasteur, Lister and Koch. Bacteriology and agriculture.—In agriculture, the de- velopment of bacteriology has given us a new insight into the nature of soil fertility. We have learned to regard the soil as a culture medium with its almost endless number of species and varieties of bacteria, specialized to do important work in the transformation of soil, nitrogen, carbon, hydrogen, sulfur; in the trans- 12 Bacteria in Relation to Country Lije ra] 3 oa Fig. 3. Chemical and bacteriological investigations of New Jersey soils. formation, also, of compounds containing lime, magnesia, phosphoric acid, and potash. We have learned to reckon with these organisms in our methods of soil-improve- ment, and have made some progress towards success- ful systems of soil-inoculation. The bacteria concerned in industrial processes have received a not inconsiderable share of attention, and have fully repaid it. The canning industries, the brew- ing industries, the manufacture of wine, cider, and vinegar, the fermentation of tobacco, the retting of flax, the tanning of leather, the pickling of vegetable substances, and of fish, and, above all, the treatment of milk and its products, have been benefited by the study of bacterial friends and foes. We find, thus, that bacteriology, resting on the foundations laid in the latter half of the last century, touches human existence at many points and lights the way for new conquests. CHAPTER II THE FORM AND STRUCTURE OF BACTERIA Tue bacteria that may be observed under the micro- scope in a drop of stagnant water, or of decomposing beef-broth, may be grouped under three main types as regards form. They are, the spherical, the cylindrical, and the spiral forms. These types have been conveniently described as resembling billiard balls, lead pencils, and cork-screws. The organisms occur singly or in aggre- gations of two or more. . 1 dag eeetuneetere YF Fig. 2 Spherical bacteria,—1. Planosarcina urew; X 2,580. {Beverin ck} Streptococcus. (Weigmann.) “3. Coccus lactis viscost. (Gruber.) 4. ee aurantiaca. (Pammel.) 5. Micrococcus Sornthalii; 900. Ca narnate: ) (13) 14 Bacteria in Relation to Country Lije The spherical forms (Fig. 4) differ from the others in multiplying in two or even three planes; hence they may be observed, not only in chains of two or more, but in square or cubical packages, the latter appearing like AY, 2 oN Z : is : 6 ae NE ECE CD ‘Fig. 5. Rod-shaped bacteria. —1. Bacterium panis; X 1,000. (Fubrmann.) 2. Bacillus oxalaticus; X 1,300. (Kuntze.) 3. Bacillus solanisaprus; X. 1,500 (Harrison.) 4. Bacillus casei; X 1,400. (Freudenreich.) 5. Bacillus codes; 1,000. (Nadson.) 6. ‘Pseudomonas trifolii; about X 1,500. (uss) 7. “Pseudomonas dermatogenes; X 1,000. (Fubrmann.) 8 Bacil- lus cerevisie; X 1,000. (Fuhrmann.) 9. Clostridium Pastorianum. (Winogradski.) small bales composed of balls. It has been aptly said that bacteria may be considered immortal, since the same cell may go on multiplying indefinitely. After division, two organisms do not always separate, but Forms of Bacteria 15 sometimes remain attached to each other, giving rise, in time, to long chains of bacteria. Rod-shaped and spiral-shaped organisms (Figs. 5, 6) increase only in one direction, and multiply by elongating and separating into two parts. The spherical species, on the other hand, are of the same diameter in length and breadth, and increase in numbers by dividing in either direction, lengthwise or crosswise, thus giving rise to square and cubical packets. , Irregular forms.—The three main types are not always well defined. The rod-shaped bacteria may be- come so short as to assume a spherical appearance. The spiral forms, also, may become shortened to such an extent as to disguise their true nature. Again, the rods may become thickened in the middle and seem to the eye like boat-shaped masses, designated as clos- tridia; or, they may become thickened at one end and assume the shape of drum-sticks or clubs. Under cer- tain conditions, the organisms may become quite irregu- lar in outline, giving rise to the so-called involution forms. A familiar example of irregular forms may be found in the growth of the organisms in the root nodules of legumes. The organism that penetrates the hair-root of the plant and gives rise to the formation of tubercles, is small and cylindrical in shape. Its offspring, on the other hand, are much larger and different in ap- pearance, for they may not only look like irregular rods, but may also appear pear-shaped, club-shaped, or x- or y-shaped. Shape.—Grouped as to shape, the spherical forms are designated as cocci; the rod forms as bacilli, or bacteria. Fig. 6. Spiral bacteria.—1, 2, and 3. Spirochtee apis; X 2,000. (Maassen.) 4. Spirillum_undula; X_1,300. (Flugge.) 5. Spirillum cholere Asiatice; X 3,000. (Hewlett.) 6. Spirillum volutans.; X1,300. (Cohn.) 7. Thio- spirilum Winogradskii; X 300. (Omelianski.) Size and Motility 17 the spiral forms as spirilla. Besides these, there are also certain groups of bacteria that occur as chains, or aggregations of individuals, encased in sheaths, from which they may escape and go through the process of multiplication. These organisms are well represented by certain classes of the so-called iron- bacteria. Size.-—There are great differences in the size of bac- teria of the same species. Still greater differences in size, however, exist between those of different species. The spherical bacteria have an average diameter of about zsdo0 Of an inch, but those with a diameter of soo00 Of an inch are not uncommon. The rod-shaped bacteria and spirilla attain, in some instances, a much greater size,—up to z,/59 of an inch in length—although such dimensions are very exceptional. The anthrax bacillus, a fairly large organism, is about zso00 of an inch wide, and gséoo to zséoo Of an inch long. There undoubtedly exist bacteria so small as to be practically invisible with the highest magnification. We know, at any rate, that some of them pass through unglazed porcelain filters whose pores are so small as to prevent the passage of ordinary bacteria. Motility and organs of motion.—The bacterial body consists of a cell-wall surrounding the protoplasm. Protoplasm is the living substance of the organism. It may present a homogeneous appearance under the microscope, or it may be granular in structure. The cell-wall and the protoplasm within it are not of the same composition. Attached to the cell-wall are the bacterial organs of motion known as flagella (Fig. 7). B Fig. 7. Flagella—1. Bacillus esterificans; about X 2,000. (Huss.) 2. Bactllus alvei; about X_ 2,000. (Maassen.) 3. Pseudomonas trifolii; about X 2,000. (Huss.) 4. Bacillus Brandenburgiensis; X 1,300. (Maassen.) 5. Bacillus robustus; X 2,800. (Blau.) 6. Pseudomonas cerevisiae; X 1,500. (Fuhrmann.) 7. Pseudomonas dermatogenes; X 1,500. (Fuhrmann ) 8. Urobacillus miqueli; X 3,500. (Beyerinck.) Flagella. Zodglea 19 These may be short or long; at times they are much longer than the body of the organism. Different species show very marked variations in the number of flagella and their arrangement on the body. There are a number of important organisms, among them the nodule bacteria of legumes, that possess only a single flagellum at one end. Such organ- isms are, therefore, designated as pseudomonas, while those with one flagellum at each end are classed as microspira. The spirilla frequently possess a bundle of flagella at each end, while the rod-shaped bacilli ‘may have them attached at any place on the body. The bacteria capable of moving about in a liquid are called motile; the others, non-motile. The latter include species which have, in some instances, been demonstrated to possess flagella. The form of the organ- ism has, also, some relation to motility, for it has been found that among the spherical bacteria, motility is comparatively rare; whereas, among the rod-shaped species, this property is common to many. Zodglea.—In some instances, the cell-wall of the bacteria is surrounded by a zone of a gum-like substance, which may encourage the aggregation of cells into irregular, masses, called zodglea. The formation of | zoégloea may occur in solid, as well as in liquid, media. Membrane formation on the surface of liquid media is characteristic of species that require the unhindered access of air for their development. Rate of increase——Bacteria multiply by splitting into halves. This process may be completed in one- half hour; at times, even more rapidly. Under less 20 Bacteria in Relation to Country Life favorable conditions it may be extended over a much longer period, determined by the food-supply and the character of the organisms. With division occurring every half-hour, a single individual could become, in one day, the ancestor of 280,000,000,000,000 bacteria. Under actual conditions, multiplication never proceeds at such a rapid; uniform rate, for many of the cells die, and the food in the medium is exhausted. Conditions that retard bacterial growth.—The multi- plication of the organisms involves, not only a reduction in the food-supply, but, also, an accumulation of sub- stances injurious to the bacteria. These injurious sub- stances may be formed within the bodies of the bacteria and excreted into the surrounding medium, or they may develop outside of the bacterial bodies. In either case, the growth of the bacteria is retarded more and more as the quantity of these injurious materials is increased and finally comes to a standstill, even though all the food in the medium is not yet entirely consumed. We find an analogy in the injurious effect on plants and animals of their own excretions, as, for instance, in the case of the uncomfortable feeling caused by the crowding of many people into a small room. Under such circumstances, the gaseous exhalations from the lungs and skin may become sufficiently great in amount to produce distress, and, in extreme cases, severe injury and death. A similar accumulation of excreta in their immediate environment reacts unfavorably on bacteria. Their life-processes become more sluggish, and not only is growth retarded, or entirely stopped, but many of the organisms perish and disintegrate. o> Fig. 8. Spores, spore formation, and spore lgermination.—1. Bacillus esteri- ficans; about X 1,900. (Huss.) 2. Bacillus esterificans; about X 1,800. (Huss.) 3. Bacillus tumescens. (Meyer.) 4. Clostridium Pastorianum. (Wino- ete 5. Clostridium Pastorianum. (Winogradski.) 6. Clostridium ‘astorianum. (Winogradski.) 7. Bacillus oxalaticus; X 1,200. (Kuntze.) 8. Bacillus esterificans; about X 1,800. (Huss.) 9. Bacillus esterificans; about X 1,800. (Huss.) 10. Bacillus methanicus; X 1,100. (Omelianski). 11. (a,b and c) Bacillus cylindricus. (d) Bacillus robustus; X 2,800. (Blau.) 12. Bacillus_robustus; X_2,900. (Blau.) 13. Bacillus esterificans; about X 1,500. (Huss.) 14. Bacillus Ellenbachensis. (Kolkwitz.) 15. Bacilli from Armenian Matzoon. (Weigmann, Gruber, Huss.) ae 22 Bacteria in Relation to Country Life Other unfavorable conditions, such as partial ex- haustion of the food-supply, too great concentration of the culture solution, and abnormal temperatures, may contribute to check excessive multiplication. Spores—As a defense against adverse conditions, the bacteria have the power to produce cells known as spores (Fig. 8). These are much more resistant to destructive agents than are the ordinary cells. In form- ing spores, bacteria, at first homogeneous, gradually become granular. The granules thus formed coalesce into a single glistening mass, which surrounds itself with a cell-wall that is tough and not readily penetrated. The newly formed spore may remain in the empty cell- wall of the parent organism, or may pass out into the surrounding medium. The number of such spores in- creases with the age of the culture. Spores a means of preservation.—Spores may be called “bacterial eggs.”” They are, however, a means’ of preservation rather than a means of multiplication. The entire organism is transformed into a single spore. Exceptions have been noted, in the case of one or two species, in which a single organism produces two spores— a fact that does not detract, however, from the truth of the general statement. Spore formation.—Different species show variations in the method of spore formation. With some of them, the cells are transformed into boat-shaped or spindle- shaped masses, containing the spore at one end or in the middle. With others, the cylindrical shape of the parent cell is retained on the spore located in the middle . or at one end. With still others, there may be a thicken- Resistance of Spores 23 ing at one end, giving rise to club-shaped or drum-stick- like cells, with the spores located at ‘the extremity of the thickened part. Vitality of spores.—The resistance of spores to ad- verse conditions is very great. They are not destroyed by: drying, as are most of the ordinary bacterial cells, and may retain their vitality while in a dried state, for along time. Well-authenticated instances are on record when bacterial spores germinated years after they had become dried out. When brought into liquids offering suitable conditions, the spores begin to swell, and, finally, burst open in one spot, giving rise to a new cell, which then elongates, and multiplies in the normal manner. Different species are not alike in their spore germination. Aside from their resistance to drying, bacterial spores can withstand other injurious con- ditions, such as high temperatures, and the action of destructive chemical agents. They are not destroyed ‘by boiling, hence, complete sterilization cannot be effected simply by immersing the contaminated articles in boiling water. Classes of bacteria.—Spore formation has been em- ployed as a means of differentiation, and bacteria are, therefore, classified as spore-jorming and non-spore- forming. A number of the well-known disease-producing, or pathogenic organisms, such as of typhoid, tuberculosis, diphtheria, and cholera, do not produce spores in arti- ficial cultures. This cannot, however, be accepted as absolute proof that they produce no spores under any circumstances whatever. Sterilization and pasteurization—The formation or 24 Bacteria in Relation to Country Life non-formation of spores is utilized in our methods of sterilization in the laboratory, the dairy, and the can- ning industries. A distinction is drawn between pasteuriza- - tion, in which the tempera- ture is raised sufficiently high to kill all of the bacterial cells except the spores, and steriliza- tion, in which the temperature is considerably higher,—enough to insure the destruction of all the bacterial cells. In the case of milk, pasteurization is ad- equate for the complete elimi- nation of the disease germs enumerated above. Intermittent sterilization. — The method of intermittent, sterilization is employed in bacteriological laboratories. It consists in heating the material to be sterilized for one-half hour at a time on three succes- sive days, at the temperature of boiling water. The first heating destroys'all of the cells except the spores. The material is then allowed to cool, the spores ger- minate, and the new vegetative cells thus formed are destroyed by the second boiling on the following day. If any of the organisms escape the second heating, they are destroyed on the third day. Intermittent sterilization is not, however, always effective in practice. Fig. 9. Autoclave. Temperature Apparatus 25 When possible, more certain methods are employed, among them sterilization by superheated steam at increased pressure (Fig. 9) or sterilization by dry heat (Fig. 10) at a greatly increased temperature. Many incubators and other devices are now employed for the cultivation of bacteria, in order that they may be studied under proper conditions of temperature and control. Fig. 9 represents a sterilizing apparatus and not a culture oven. Fig. 10. Hot-air sterilizer, in which articles containing bacteria may be heated until all germs are destroyed. CHAPTER III THE CHEMISTRY OF THE BACTERIAL CELL THERE are certain chemical elements essential to the existence of bacteria. Being composed of protein, carbohydrates, fats and waxes, the bacteria must have the elements that enter into the building of these sub- stances. Hence, carbon, nitrogen, hydrogen, oxygen and sulfur are indispensable for their development. Aside from these, they require, like the higher plants, lime, magnesia, phosphoric acid, potash and, perhaps, iron. The proportion of some of these constituents taken up by. bacteria may be so slight as to preclude their recognition, even by the most refined chemical tests. Moreover, the proportions taken up are affected both by the species of bacteria and by the composition of the culture medium. Certain classes of bacteria—among them the species found in drinking-water—can develop and multiply on quantities of nitrogen compounds so minute as to be altogether insufficient for hundreds of other species. Similarly, certain groups of soil bacteria are known to require much larger quantities of lime and phosphoric acid than are required by other groups. This fact is of great significance in the struggle for existence among the bacterial inhabitants of the soil. (26) Cell-Wall of Bacteria 27 The cell-wall and its contents.—It is not known exactly what substance or substances enter into the composition of the cell-wall. In some cases, cellulose seems to be the main constituent. In the majority of cases, however, cellulose does not seem to be present, but, rather, a Fig. 11. Microscope and accessories for bacteriological work. horny, chitinous matter, the elements of which are as yet unknown. Inclosed within the cell-wall is the protoplasm, a semi-fluid protein substance which is the seat of the chemical changes produced within the cell. The living force in the protoplasm provides for the building up and the tearing apart of chemical compounds, for the pro- cesses of assimilation and decomposition. Content of the protoplasm.—The protoplasm of some species is characterized by its content, glycogen, a kind of sugar—found also in the human liver. Glycogen is 28 Bacteria in Relation to Country Life present in a considerable number of species, among them the nodule bacteria of the legumes. It is appar- ently stored up by the organisms as a reserve food- material. Another carbohydrate, designated as granu- lose, has been found to occur in a well-defined group of organisms. Fats and wax-like substances may also constitute a more or less considerable portion of the bacterial cells. TF Fig. 12. Microscope and accessories for bacteriological work. In the germ of tuberculosis, the cells have been found to contain 26.5 per cent of fat and wax in their dry sub- stance. The nitrogen-fixing organisms of the azoto- bacter group frequently store up very considerable quantities of fat in their cells. Some species are observed to do this more than others. The so-called sulfur bacteria, found in some sulfur springs and in the sea, deposit within their bodies little Content oj the Cell 29 granules of sulfur which they later burn up for the sake of the energy that can be thus derived. The protoplasm also contains various amounts of organic and inorganic salts in solution, derived from the surrounding medium, and compounds formed from these by the bacterial protoplasm. The absorption of food—The substances dissolved in the culture medium pass, without difficulty, through the cell-wall, and the latter is, therefore, said to be permeable to the dissolved substances. On the other hand, the protoplasm is penetrated to only a slight ex- tent, and is, therefore, said to be impermeable. These physical properties are of vital significance, not only in the nutrition of the organisms, but also in their resistance to the entrance of injurious substances. CHAPTER IV THE FOOD REQUIREMENTS OF BACTERIA THE tissues of higher plants contain a large propor- tion of carbon, as may be readily ascertained by charring the substances. Rye straw contains more than half of its weight of fiber, of which 40 per cent or more is carbon. It has been estimated that an acre of beech forest assi- milates nearly a ton of carbon annually. All of this carbon is obtained from the invisible gas, carbon dioxid, which constitutes from .03 to .04 per cent of the total volume of dry air. By means of the green coloring matter, chlorophyll, contained in their leaves, the higher plants decompose the carbon dioxid, and cause its carbon to enter into various combinations, such as starches, sugars, fats, gums and proteins. Lower plants, among them the microscopic alge, are enabled by virtue of their chloro- phyl, or of other coloring matters allied to chlorophyl, to decompose the carbon dioxid of the air, and to build out of the carbon various and numerous complicated organic substances. The decomposition of the carbon dioxid, by means of chlorophyl, can be effected only in the presence of sunlight, for this furnishes the energy for the breaking up of the carbon dioxid molecules. In this way, simple (30) Appropriation of Carbon 31 mineral salts and water derived from the soil, and carbon derived from the air, are changed into the numerous and almost endless varieties of substances found in the vegetable kingdom. The sources of carbon.—The bacteria classed among plants with very few exceptions produce no chloro- phyl, and cannot, therefore, invoke the aid of sunlight for the decomposition of the carbon dioxid of the atmos- phere. They must depend for their carbon on compounds other than carbon dioxid, and they find it in numerous combinations of vegetable and animal origin. We see, thus, that the great majority of bacteria differ from green plants in their inability to decompose the carbon dioxid of the atmosphere. There are, however, a few impor- tant exceptions to this rule. ve ptions s rule The study of the nitrifying bacteria, the organisms which produce nitrates, has demonstrated that they are able to use carbon dioxid as their only source of carbon. Other organisms, recently discovered, can apparently utilize carbon monoxid (coal gas) in a similar manner, while still others may use methane, or marsh gas, for their growth. After making due allowance for these exceptions, we still find it to be true that bacteria differ from green plants in relation to their carbon food. While sugar is undoubtedly an acceptable source of carbon for many species of bacteria, it is not the only source. Preference is frequently given to other carbon compounds. In fact, there are bacteria that are actually hindered in their development, or entirely suppressed, by the presence of sugar in the culture medium. For instance, the germ of cholera, and that of typhoid, are 32 Bacteria in Relation to Country Life injured or suppressed by comparatively slight amounts of sugar, and the failure of putrefying organisms to develop in milk under ordinary conditions is ascribed directly or indirectly to the influence of the milk-sugar. The range of carbon compounds used by bacteria as a source of food is very large. Grape-sugar, cane-sugar, milk-sugar, malt-sugar, and mannite, a compound closely related to the sugars, are readily used by many organisms, as are also such compounds as starch, dex- trin and cellulose, capable of being changed into sugar. The compounds of carbon in bacteria.—The energy stored up in these substances is employed for the manu- facture of the compounds found in the bacterial body. In other words, the bacteria burn up the sugars and allied materials in a manner analogous to the burning up of the food in the animal body. The carbon compounds enumerated consist of three chemical elements—carbon, hydrogen, and oxygen. There are, however, still other classes of carbon com- pounds seized upon with even greater avidity by im- portant groups of bacteria. The compounds in question are the proteins—composed of carbon, nitrogen, hydro- gen, oxygen, and sulfur,—and substances derived from the proteins and composed of carbon, nitrogen, hydro- gen and oxygen. The numerous species of decay- and putrefaction-bacteria are especially favored in their ‘development by protein compounds and grow rapidly in meat and meat extracts, egg-albumin, and other materials of animal or vegetable origin, rich in protein. For this reason, beef-broth, supplemented by mineral salts, is used almost universally in bacteriological labora- x Carbon and Nitrogen 33 tories. Pathogenic bacteria, in particular, seem to re- quire protein compounds as their source of carbon, and some species will not develop without it. Generally speaking, therefore, different species of bacteria are not alike in their preference for one or another source of carbon. Some species will develop in solutions of cane-sugar fully as well as in solutions of grape-sugar, while others will grow in solutions of grape-sugar, but not in solutions of cane-sugar. Analo- gous relations may be observed in the case of other sugars. Again, there are species, like certain kinds of denitrifying bacteria, which will develop perfectly in culture solutions containing salts of citric acid as the only source of carbon, while other organisms will not grow in such solutions at all. Similarly, there are bac-~ teria that will utilize pure cellulose, when the vast majority of microérganisms will utterly fail to develop. These examples will suffice to show that there are deep- seated differences in the chemical machinery of the bacterial cells and in the methods by which the carbon compounds are transformed and assimilated. The source of nitrogen.—Relations like those just described exist also in the assimilation of nitrogen food by bacteria. Just asin the case of field crops, some plants prefer ammonia and others nitrate, as a source of nitrogen, so athong bacteria, different species show analogous preferences. Certain species reject both am- monia and nitrate nitrogen, and demand for their growth some organic nitrogenous compound (prefer- ably protein), or substances derived from protein and known as amino-compounds. The so-called nitrogen- Cc 34 Bacteria in Relation to Country Life fixing bacteria can readily utilize the nitrogen gas of the air, which is inaccessible to the other species. Enzymes.—Valuable knowledge of the mechanism of decomposition by bacteria is supplied by the in- vestigations on the enzymes, other- wise known as unorganized fer- ments. Enzymes are chemical substances of complex constitution, produced by living organisms, both plants and animals. In the human body certain enzymes are found, capable of transforming starch into sugar; others capable of breaking up pro- tegrating fats, and pre- paring them for assimi- lation. The enzymes in the saliva, stomach, liver, and pancreas, are secreted by the animal body for a specific pur- pose in aiding the digestion and assimila- tion of food. A familar instance of enzyme ‘action is that of pepsin, the function of which is to change protein in- to simpler substances. Fig. 13. Apparatus for filtering cultures. Enzymes 35 Plants and bacteria also possess their enzymes. With the aid of these they are able to carry on their life-processes. The different enzymes produced by bacteria are numerous. Produced within the bacterial cell, they may pass outward through the cell-wall and attack the substances in the culture medium, which serve as food for the organisms. Thus, in the case of the cellulose ferments, the inert and insoluble fiber is changed into sugar by the enzyme. The sugar, being soluble, gradually passes into the protoplasm and is used by the latter for the building of other substances. Pigments.—Intimately connected with the life pro- cesses of bacteria is the ability of some species to produce pigments. The coloring matters produced by different species include golden yellow, orange, red, blue, pink, violet, green, brown, and black substances. Blue or red milk, and even bluish or yellow pus in wounds may be produced by bacteria. The production of coloring matter is affected by the food and the culture conditions. Temperature, likewise, plays an important réle in this relation. Phosphorescence.—The ability to produce phosphor- escence is a property held by certain species of bacteria in common with some members of the animal kingdom. These bacteria, designated as photobacteria, are, so far as is known, all inhabitants of the sea. The phosphor- escence of sea-water, a phenomenon much commented upon, is due largely, though not entirely, to bacteria. It is these organisms, also, that produce phosphorescence in decaying fish and meat. CHAPTER V CONDITIONS AFFECTING THE GROWTH OF BACTERIA Tue conditions that favorably or unfavorably: affect the growth of bacteria, include temperature, moisture, aération, light, pressure, and the presence or absence of injurious substances. The first of these (temperature) is an important influence, since -the living protoplasm can perform its functions only within a comparatively narrow range of heat and cold. Just as a few degrees of temperature may hasten or retard the germination of seeds, or the development of fruit-buds, so a similar variation in temperature may increase or diminish the intensity of bacterial development. There are well-defined limits below or above which no bacterial growth whatsoever will occur, yet within these limits there are considerable variations among the different species. Important differences as to suitable growing temperatures occur between the soil and water bacteria, on the one hand, and most of the disease bacteria in warm-blooded animals, on the other. Thermophile bacteria.—There is a third class of bac- teria, known as thermophile that are remarkable for the high-temperature limits within which they will grow. Thermophile bacteria have been isolated from the soil and manure heaps, with the result that it has (36) Effect of Temperatures 37 been learned that, under the direct rays of the sun, the temperature in such places may reach, for short periods at least, the optimum or best for these bacteria. All of the thermophile bacteria form spores. They can, therefore, resist unfavorable conditions for their devel- opment by remaining dormant for more or less consid- erable periods. Effect of cold—Temperatures below the minimum suspend the bacterial activities, but are not very effec- tive in destroying the organisms. Disease bacteria, frozen in ice, are as active as ever after the temperature is raised. It has been demonstrated experimentally that extremely low temperatures injure the bacteria only after prolonged exposure. Even immersion in liquid air does not always succeed in destroying them. Temperatures beyond the maximum lead, on the contrary, to a rapid destruction of the organisms. The non-spore-forming bacteria perish when subjected to a temperature of 130° to 140° Fahr., for ten minutes, and in less time when subjected to still higher temper- atures. Spore-forming bacteria are much more resistant to heat because of the inert character of their spores. They will withstand dry heat well above 250° Fahr., and, while not as resistant to moist heat, will sometimes withstand boiling for an hour or more. Attenuated cultures—When bacteria, or their spores, are subjected to heat insufficient to destroy them en- tirely, yet considerably above their maximum, they may become weakened to a very marked degree. The injury to the organisms may appear then in their less vigorous growth, or in their impaired power to produce 38 Bacteria in Relation to Country Lafe characteristic chemical substances, such as pigments, enzymes, and toxins. Heating within certain limits may, therefore, serve as a means for the production of so-called attenuated or weakened cultures. These may also be produced by other methods, particularly by the limited action on the organisms of germicides, as corrosive sublimate, carbolic acid, and chloroform. Moisture content of the culture medium.—Bacteria growing in solid substances will discontinue their growth when the proportion of moisture in the medium reaches a certain minimum. Studies on the decay of humus in the soil have shown that the decomposition processes are practically at a standstill with 2 to 3 per cent of moisture; that with 4 to 5 per cent they are more active; and that they finally reach a maximum beyond 25 to 30 per cent of moisture. In vegetable and animal sub- stances, the minimum amount of moisture required for the development of bacteria is much higher, scarcely any growth occurring when the moisture content is less than 25 per cent. Relation to oxygen.—Bacteria show widely different relations in their behavior towards the oxygen of the air. Some species will not develop at all when air is excluded or its supply limited beyond a certain point. These organisms are designated as aérobes. Many of the most common soil and water bacteria are strict aérobes, or obligate aérobes (to use a term in vogue among bacteriologists), and they include decay bacteria, nitrifying bacteria, and nitrogen-fixing bacteria. There are, on the contrary, numerous other species that require the entire exclusion of air for their proper soroeds ay} JO UONTUZO0e1 8y} pue ApNys [NJ *pasoduios ere A9y} YoIyA JO jews epem eq Avui aj}e[d ey} uO suieedde -e180 I1eq} ‘asimeyy ‘pus ‘yueudoyeAep palopulyun s1ey} MOT]® 0} Ysnoue | solUO[Od oy} JO JequINU 9y} ‘UIVEIES Jo AyueNnd UsAIs @ OFUL UOT}E[NIOU! 103 ‘;eueyeur J9q}0 Aue Jo JO ‘[los jo saijiyuenb guisveloep Aljenpels Sule, Ag = ‘UOTNIIpP eAlsve00Ns JO FoIyZe O47 suLMogs “SL ‘yesB UO BLIa}OBq [IOS jo SolUO|OD “FT 40 Bacteria in Relation to Country Life development. They are designated as anaérobes, or obligate anaérobes. The obligate anaérobes in the soil include, among others, the cellulose ferments, the nitro- gen-fixing butyric ferments, and the lock-jaw bacillus. Organisms that will develop preferably with an unhindered access of air, but also will grow when it is excluded, are designated as facultative anaérobes. In other words, they are both aérobes and facultative anaérobes. A number of denitrifying bacteria are prominent members of this class. It has been shown that they will not destroy the nitrate when freely sup- plied with oxygen, but that they will derive the latter from the nitrate when atmospheric oxygen is excluded. Still other organisms are designated as facultative aérobes, that is, they will grow, preferably, when oxy- gen is excluded, but will exist also when it is present. Aérobes and anaérobes are found together in water, soil, and elsewhere in nature, and there is no doubt that the first of these facilitate the development of the others by using up the oxygen in the medium. Such relations evidently exist between aérobic and anaérobic water and soil bacteria and account for the abundant presence of anaérobic nitrogen-fixing bacteria even in open and well-ventilated soils. Action of sunlight.—Direct sunshine exerts a more or less destructive action on living cells. The destructive action of direct sunlight is readily observable also in the case of bacteria, sunshine being regarded as one ‘of the most potent forces in nature in the destruction of pathogenic and non-pathogenic germs. A liquid or solid culture of bacteria exposed in thin layers to Action of Light. Electricity 41 the direct action of the sun’s rays may become sterile within a few hours. Hence, in the bacteriological labora- tory, the cultures are kept either in the dark, or in subdued and diffused light. Z In the soil and in sheets of water, also,. strong sun- light exerts some destructive action. Soil exposed in thin layers loses some of its nitrifying power, and very shallow sheets of water in tropical countries become poor in bacteria probably on account of the prolonged exposure to intense sunlight. Direct sunlight affects only the very uppermost layers of soil and water, and its germicidal effect is not, under such conditions, as great as might be expected. Electric light—Very strong electric light has been observed to possess germicidal properties.’ It is asserted, for instance, that cultures of Bacterium prodigiosum, exposed to the ultra-violet rays of the arc light, were found to be sterile at the end of twelve minutes. Influence of electric currents—Experiments on the influence of electric currents on the activities of bac- teria have not revealed any marked injury to the organisms. Experiments with both induction and galvanic currents in the soil showed no injury either to the decay or nitrifying bacteria. Similar results were obtained with some of the pathogenic organisms. Weak electric currents in solutions cause motile bacteria to gather about the negative pole. When the current is reversed, and the positive pole made negative, the organisms change their position and flock toward the new negative pole. Pressure.—A more or less injurious action on bacteria . 42 Bacteria in Relation to Country Lije is exerted by high pressure. It has been found that a gradually increasing pressure up to 2,904 atmospheres fails to destroy many of the bacterial species. When, however, the pressure is raised and lowered successively a number of times, the bacteria are considerably weak- ened, as shown by less active motility, an impairment or loss of the ability to multiply, and impairment or loss of ability to produce typical reactions. Some of the species prove to be more susceptible than others. Influence of the concentration of the culture medium.— Large amounts of soluble salts in the culture solution lead to the injury or destruction of the bacteria. Many substances, when present in small proportion, may serve as food to bacteria, or may be harmless to them, while in greater concentration they may act as poisons. Apart from such substances as alcohol, carbolic acid, or salicylic acid, which are recognized as germicides, there are many neutral salts that are commonly used as nutrients in the preparation of culture media, and yet they are poisonous to the bacteria when present in greater concentration. Common salt and saltpeter (potassium nitrate), both being used as preservatives in the pickling of meats and of other organic materials, may serve as examples of such substances. Because of greater resistance to the injurious action of salt, some species are enabled to develop in herring brine and similar substances, investi- gations having demonstrated the presence of a rich bacterial flora in such brines containing as much as 20 per cent of salt. Among other substances used for the preservation of Influence of the Culture Medium 43 food products is sugar. As is well known, this is em- ployed in the manufacture of condensed milk, as well as in the making of preserves. It seems that the concen- tration of the material to which large amounts of sugar are added precludes the development of the bacteria. Still another instance of the depressing effect of excessive concentration may be found in the relation of the nitrifying bacteria to large amounts of soluble organic matter. The development of these bacteria may be entirely suspended by quantities of soluble organic matter not in the least injurious to other bacteria. Thus the growth of nitrifying bacteria in manure heaps does not begin until the litter and animal excreta have been largely decomposed by the great hosts of decay bacteria and until the soluble organic materials have been changed into insoluble modifications. The reaction of the culture mediwm.—The reaction of the culture medium, that is, the amount of free acid or base present in it, is of the utmost importance for bac- terial growth. The same condition, or acidity, of a culture medium may be remedied by the addition of a base, that is, of a substance which has properties oppo- site to those of an acid. Lime, soda, or potash may be given as examples of bases. All of these may be employed for the neutralization of acidity. A culture medium which is neither acid nor basic (alkaline) is said to be neutral, and is best adapted to the growth of many species. There are numerous or- ganisms that prefer a faintly acid medium, among them some of the common inhabitants of milk. There are others that prefer a slightly alkaline medium. In 44 Bacteria in Relation to Country Life a few instances, bacteria will develop vigorously in distinctly acid media. This is particularly true of the acetic-acid bacteria. On the whole, however, acid media are not acceptable to the bacteria, and, in the struggle for existence, they are replaced in such media by molds better adapted for growth in acid substances. Importance of reaction of the soil—The reaction of the soil is of vital importance in crop production, be- cause it determines what species shall predominate. It determines, also, the rate of decomposition of the soil-humus and the rate of nitrification, or, in other words, it determines the rate at which available nitro- gen is supplied to the crop. The application of lime frequently stimulates plant growth because of the correction of acid conditions, and the favorable influence on decay and nitrifying bacteria. A similar favorable effect may be produced by liming on other desirable bacteria, among them the nodule organisms of clover and other legumes. It should not be supposed that excessive alkalinity is harmless. A large number of experiments have been recorded which show that excessive applications of caustic lime injures the decay and nitrifying bacteria in the soil. This conclusion is further reinforced by the fact that carbon- ate of lime, which is not so caustic as the burned lime, has been found to be less injurious to the soil bacteria. ag PART II BacteERIA IN AIR AND WATER CHAPTER VI BACTERIA IN THE ATMOSPHERE THE air that surrounds our earth is never at a stand- still. It is kept in motion by its waves and storms, and is affected by season, climate, and proximity to human habitation. Mingled with the gases and vapor of which it is composed, there is a quantity, at times very great, of dust particles, derived from the soil, the water, and the streets of cities. These dust particles of varying degree of fineness are borne hither and thither by the changing winds, and, as they pass from place to place, carry with them their minute bacterial passengers. The richness of the air in dust particles is easily per- ceived as we watch the rays of the sun pass into a dark room through a hole in the window curtain. The light reflected from the countless number of these particles shows them to be suspended and restless. The wind, as it sweeps over the bare fields and roadsides, raises clouds of dust, particles of soil and of vegetable and animal materials, and carries them away on a more or less dis- tant journey. The fine spray from the ocean, as it dashes against the shore, and the spray from river and lake, (45) 46 Bacteria in Relation to Country Lije raised by the churning action of innumerable vessels; the busy life of the city, with its hurrying feet, its rattling wheels, and its never-resting chimneys, all show that there is ample cause for the existence of the many dust particles in the air. Bacteria on dust particles.—The bacteria of the soil, water and city streets, are carried along with the dust. A single particle of dust, or the few salt crystals from a drop of water, may carry one or more bacteria. This may be easily demonstrated by exposing sterile plates of gelatin to the air for brief periods of time. The bacteria on the dust particles, as they fall on the nutrient gelatin, begin to grow and form colonies. It frequently happens that a single spot may show a mixed growth of two or even three species, an indication that there were, at least, that number of bacteria on a single dust particle. The dust-laden air currents may, therefore, become the means for conveying bacteria from place to place, sometimes over great distances. Various disease germs including those of typhoid, pneumonia, and tuberculosis, may thus be carried away and become a source of in- fection. Similarly, the nodule bacteria of legumes may be transported from field to field in the soil-dust, may affect soil-inoculation, and may thus lead gradually to the establishment of new legume species in places where they were previously unknown. This may, in part, help to explain the appearance of some of the nodules in newly established alfalfa fields. This assump- tion seems to be supported by the fact that in pot-cul- tures with legumes (particularly peas) nodules frequently Number and Kinds in the Air 47 appear on the young plants notwithstanding the most thorough sterilization of the seed and soil. Number of bacteria in the air—The number of bacteria in the atmosphere, while constantly aug- mented from the various sources enumerated, does not become very large in the open air. This circumstance is readily accounted for by the inroads made upon the bacteria by sunshine and drying. Already dry when raised from the fields and the streets, the bacteria suffer further from the direct light of the sun and succumb to it sooner or later, unless they exist in the spore state. Pasteur’s method of determination.—Pasteur’s early investigations concerning the bacterial content of air demonstrated that the number of organisms in the atmos- phere is not very large and that it is influenced by season, climate, altitude and human activities. Pasteur pre- pared large flasks partly filled with culture solution, drew the neck out to a fine tube, and boiled the contents until all of the air was expelled by the steam. The neck of the flask was then sealed with the aid of a blast lamp, and, on cooling the flask, a partial vacuum was created in it by the condensation of the steam. Such sealed flasks could be taken to any place the atmosphere of which it was desired to examine. On breaking off the neck of the flask, the latter was at once filled with air. The neck was then sealed again. The bacteria, yeasts, and molds present in the air drawn into the flask, soon settled into the liquid and developed there, giving rise to the characteristic turbidity. When the contents of the flask remained clear, the conclusion was inevitable that there were either no microérganisms in that par- 48 Bacteria in Relation to Country Life ticular sample of air, or, at least, none that would grow under those special conditions. Value of Pasteur’s method.—This method of Pasteur merely showed whether there were any microérganisms at allin a given volume of air. It did not show how many there were in it, since the resulting growth could have been due to one or to several organisms. Other methods Fig. 15. Liquefied agar ready for inoculation and pouring into sterile Petri dishes. have been employed, therefore, to show, not only the mere presence, but, also, the number and kind of bac- teria in a given quantity of air. Quantitative methods —The methods employed for this purpose consist in passing measured quantities of the air to be examined through vessels or tubes, whose walls are lined with nutrient gelatin, or through small quantities of solid materials, or of liquid. In the former case, the microdrganisms of the air gradually fall on Germs in the Air of Different Places 49 the nutrient gelatin and, developing there, produce colonies whose number can be counted, and the char- acter of whose organisms can be further investigated. In the second method, the liquid is mixed with nutrient gelatin, the latter spread out on sterile plates of gelatin, and the colonies that gradually appear are counted and studied in a similar manner. Investigations of this nature, with the overwhelming evidence they bring as to the occurrence and the distribution of bacteria, and of other microérganisms in the atmosphere, soon swept away the last vestige of belief in the theory of spontaneous generation. INFLUENCE OF LOCALITY It was observed by Pasteur that the proportion of flasks that showed growth was variable and strongly influenced by the locality whence the air was taken. The air from the city contained more germs than the air from the country, while the air from mountains was poor in bacteria. Bacteria in city air.—The more exact and extensive researches of Miquel have confirmed the observations made by Pasteur. Miquel found in several examinations an average of 3,480 bacteria per cubic meter in the air of the Paris streets, an average of 7,420 in the laboratory air, an average of 36,000 in the air of old houses, and an average of 79,000 in the air of one of the Paris hospitals. These figures are important in showing how human activities lead to an increase of the bacteria in the air. The number of germs in human habitations is undoubt- D 50 Bacteria in Relation to Country Life edly affected not only by conditions more favorable for their development, but, also, by the degree of ven- tilation. Buildings whose air is renewed more frequently, and whose windows allow a free access of sunshine, con- tain fewer germs than similar buildings that are less favorably situated. The dust of the city streets is very rich in bacteria. It is natural to expect, therefore, that the air overlying the streets will also be well stocked with them. An examination of the air over the London streets showed it to contain approximately from 300,000 to 500,000 dust particles per cubic centimeter, while the corresponding determination of the bacteria showed that there was only one germ to every 38,300,000 dust particles. As stated by Fischer, an examination of the street dust in the city of Freiburg showed it to contain from 5 to 17 different species of bacteria included in from 24,000 to -2,000,000 germs per gram. This examination brought out the interesting fact that the sprinkling of streets increases to a very marked extent the number of bacteria in the dust. The sprinkled streets contained a minimum of 1,450,000 germs, and a maximum of 2,896,000 per gram of dust, while the unsprinkled streets contained a minimum of 24,000 and a maximum of 48,000 germs. There is a difference, not only in the number of germs in the’sprinkled and unsprinkled dust, but, also, in their resistance to the destructive effect of drying. The less resistant forms perished in the unsprinkled dust in four days, while, in the sprinkled dust, they survived for fourteen days. Fischer observes, with much justice, that the sprinkling Country Air 51 of streets, even though it increases the number of germs in the road dust, is ngne the less valuable from the hygienic standpoint. He evidently refers to the reduc- tion in the amount of dust in the air caused by sprinkling, whereby the number of: pathogenic germs carried by the wind is very materially lessened. The great value of sprinkling is that it keeps the germs in the road dust from being blown about by the wind. Bacteria in the air of the cowntry—Comparing the number of bacteria in the air of the city and country, we find, according to the examinations of Miquel, very considerable differences. The average for his analyses showed 300 bacteria per cubic meter of air taken outside of the city of Paris, and 5,445 bacteria per cubic meter of air taken within the city. Similar results were secured elsewhere, thus confirming the conclusions reached by Miquel. In the country, the number of bacteria in the air is affected, among other things, by the presence or absence of large forests. It has been demonstrated that the latter may act as filters and hold back a portion of the bacteria brought to them by the wind. This seems to account for the fact that the air within forests is poorer in bacteria than the air on their outskirts. Like the air of the forests, the air over the sea is also poor in bacteria. The greatest number of bacteria was found in sea-air taken in the vicinity of land. With the increasing distance from the coast, the number of bac- teria diminished until there was less than one germ per cubic meter of air. Even poorer in germs than sea-air was that from the polar regions, where but few could be detected by repeated examinations. 52 Bacteria in Relation to Country Life INFLUENCE OF SEASON, CLIMATE, AND ALTITUDE The investigations of Miquel, extending over a period of years, show conclusively that seasonal influences are an important factor in the increase or decrease of bac- teria in the air. There is an evident increase from winter to spring and from spring to summer, and a decrease from summer to autumn. Yet, while the temperature and other seasonal influences can be easily traced in these averages, there are temporary disturbances that threaten to obscure the general results. For instance, the number of bacteria diminishes after a rain, and in- creases rapidly as the soil begins to dry at the surface. When, however, the drying period continues for ten or fifteen days, the number of bacteria decreases again. The causes for these variations are not far to seek. The number of bacteria in the air decreases in periods of rainfall because of their being carried down by the rain. Similarly, in periods of dry weather, the wind carries away some of the finer particles of the surface soil; among them, the bacteria that have multiplied rapidly under favorable moisture conditions. Prolonged drying, on the contrary, and the accompanying germicidal action of direct sunlight, involves the partial destruction of the bacteria floating in the air, as well as of those in the dust mulch at the very surface of the soil; and for these reasons. the dust-laden atmosphere of droughts may not be rich in germs. The seasonable influences are also noticeable in the atmosphere of densely populated districts, although the number of organisms found here is much greater than Seasonal Bacteria in Air of Dwellings 53 that found in the open air. The same increase from win- ter to summer, and decrease from summer to autumn are present,—an evidence that the relative influence of heat, moisture and sunshine is the same in the city as it is in the country. In human dwellings, also, the bacteria in the air are readily affected by seasonal changes. In this instance, however, the differences do not run parallel to those obtained in relation to the open air. Examinations of the air in Paris hospitals demonstrated that there was a steady decrease in the number of bacteria from March to August, and a steady increase from August to Novem- ber. These apparent discrepancies find a ready ex- planation in the fact that the open windows during the warm months allowed a more thorough ventilation of the hospital rooms, and, therefore, a more rapid removal of the organisms suspended in the air. An important point in this connection is the large number of bacteria found in the air of hospitals. It is safe to assume that a considerable portion of the organisms suspended there were pathogenic, and that they were a source of infection to the patients and attendants. The influence of climate.—It is a self-evident truth that the number of bacteria in the atmosphere of any region bears a direct relation to the climate of that region. In warm tropical countries with an abundance of rain- fall, the multiplication of bacteria in the soil, water, and all vegetable and animal matter proceeds with great rapidity. Under such conditions, the particles of dust raised from the earth’s surface carry an abundant num- ber of bacteria and enrich the atmosphere to a very 54 Bacteria in Relation to Country Life marked extent. On the other hand, the frequent and copious rainfall tends to remove the bacteria from the air, while the direct tropical sunshine largely contributes to their destruction. There are, thus, two distinct and opposite tendencies, one favoring the accumulation of bacteria in the atmos- phere, the other hastening their destruction. In dry, tropical countries, the number of bacteria in the air must, of necessity, be limited, a fact also true of dry, cold countries. In countries with temperate or cold climates, the number of bacteria present in the air in the winter is small, because of the retarded or suspended development of the organisms. This is true, also, because of the covering of ice and snow which prevents the re- moval of the bacteria from the soil and the surface of lakes and streams into the atmosphere. The influence of altitude——The, air over mountain peaks contains scarcely any bacteria at all. Their num- ber diminishes as the distance from the level of the sea is increased. Evidently, the lower temperature prevail- ing in the mountains retards bacterial development, even when conditions are otherwise favorable. More- over, bare rock is an unsatisfactory place for bacterial development. Again, the distance from the floor of the valleys is frequently too great to allow the transpor- tation of any large number of their bacteria to the air of the higher peaks. Bacteria and respiration The number of bacteria in the air over the ocean, over high mountains, in polar regions, or in countries of scant rainfall, is relatively very small. Conditions there prevent both the addition Seasonal Bacteria in Air of Dwellings 55 of large numbers to the atmosphere, as well as the sur- vival of the bacteria already there. On the contrary, the air of city streets and of human dwellings is par- ticularly rich in microérganisms owing to conditions favoring both the addition of large numbers to the atmosphere and their survival there. These facts are of very considerable moment from the standpoint of hygiene and sanitation. We know that, notwithstanding the large number of bacteria -in the atmosphere, the air expired from the human lungs is practically germ-free. This means that the microér- ganisms are retained in the nose, mouth, and throat, and that many of them are carried with the dust parti- cles into the lungs. Enormous numbers of bacteria are thus retained, and it is obvious that, everything being equal, the danger from infection is greatest when the number of bacteria in the air is greatest. Persons lead- ing an indoor existence, and those living in large cities, inhale more bacteria and are more exposed to infection than people living in the country. It does not follow, at the same time, that the danger of contracting a disease, say tuberculosis, is greater in the summer than it is in the winter, simply because there are more germs abroad. After all, most of the bacteria in the air are harmless, and it is very likely that the actual number of the germs of tuberculosis and pneumonia in the winter air is greater than that in the summer air. CHAPTER VII THE RELATION OF WATER TO HEALTH AND DISEASE Tus human body contains nearly 60 per cent of water. Some of the individual organs, as the kidneys, heart, liver and pancreas, contain a much larger propor- tion, and all are dependent on a sufficient supply of it for the proper performance of their functions. The human race in its very infancy sought out the springs and streams that yielded a generous supply of cool, refreshing water; it made its home within reach of them; it followed the water-courses to the sea, loath to lose sight of them; and, as it grew in wisdom, it learned to find in them food as well as drink. Long before the dawn of written history, men knew of the life-giving qualities of water, and of its often deadly effects. Ex- perience taught them to preserve it against periods of scanty rainfall, to guard it against pollution, to measure it out with a careful hand to the thirsting crops. Ancient water-supplies.— In selecting their source of drinking-water, the ancient builders of reservoirs and aqueducts were guided by considerations of quality and salubrity. It was known to them and to others before them that there is some relation between drink- ing-water and health and disease. Certain springs were peculiarly noted for their reputed curative proper- (56) Drinking-Water and Disease 57 ties, while other waters were known to be poisonous to man and beast. These observations were, however, for the most part, purely local in character, and could not, in the nature of things, lead to broad views on the general relation of water to health. No general concep- tion could arise then as to the significance of color, hardness, taste, and turbidity, nor as to the more deep- seated distinctions revealed to us by modern research. Individuals, as Hippocrates, who lived four hundred years before the beginning of our era, pointed out the dangers of pollution, and even advised boiling and filter- ing contaminated drinking-water; yet such views met with no general acceptance. In the centuries following, if any relation was observed between the character of the drinking-water and the great epidemics of cholera. and typhoid, which broke out in Europe from time to time, the popular views and the methods of sanitation, such as they were, were not sensibly affected thereby. In the early centuries of the present era, and through the period of the Dark and the Middle Ages, there pre- vailed a vague belief that the outbreak of disease had some connection with the use of water from wells and springs, and many a_ reputed apes ; witch lost her life for the alleged : : poisoning of drinking-water. woe x Relation of drinking-water and disease discovered.—Slowly the re- ~ lation of drinking-water to dis- {~~ Zz ease began to be better under- Meee stood. In the early part of the water Vubria, chor nineteenth century, repeated ref- fhreang j0O (Alter 58 Bacteria in Relation to Country Life erences were made to drinking-water as the cause of malaria and diarrhea. Towards the middle of the cen- tury the accumulation of recorded facts prepared the way for the belief expressed in 1855 by Doctor Michel in France, that there is a seeming relation between the character of drinking-water and the prevalence of typhoid fever. The bacillus of typhoid isolated by Eberth in 1880, and studied in greater detail by Gafky in 1884, is now generally regarded as the specific cause of the disease (Fig. 16). The organism has been repeatedly isolated from the stools of typhoid patients, and a number of in- stances are on record when the bacillus was directly isolated from polluted water. The germ of cholera was isolated by Koch in 1884 from the stools of cholera patients, and from the intestinal contents of persons who died from the disease (Fig. 16). He also found this germ in water from a tank in India. Both of these organisms have been shown to invade the human system through the medium of drinking-water, and the latter has, there- fore, become the object of more thorough care and inspection in all civilized communities. Epidemics of cholera and typhoid fever—The study of the history of epidemics of cholera and typhoid is both interesting and instructive, as marking the growth of our knowledge of these diseases from the standpoint of sanitation. Europe and America have had no serious outbreak of cholera for many years, but epidemics of typhoid are, unfortunately, still too frequent. The re- lation between typhoid fever and drinking-water may be readily traced in the epidemics that have occurred Typhoid Fever 59 within the past twenty-five years. The evidence as to the relation of typhoid fever and drinking-water is further strengthened by a mass of statistics showing a marked decrease of typhoid since the introduction of better water-supplies in the cities. There is an enormous amount of data that might be added to the above as further proof of the intimate connection existing between drinking-water and typhoid epidemics, yet enough has already been said to bring out the importance of this relation. Other sources of typhoid injection.—It should not be supposed that drinking-water is the only source of in- fection, or even the only important source. Milk has been repeatedly shown to be a very serious source of infection, and uncooked vegetables, fruit, and oysters have undoubtedly served as carriers of the disease. There is scarcely a doubt, likewise, that flies have often been responsible for the spread of the disease, particu- larly in army camps. This will explain why the greater care of the water-supplies is not always followed by a corresponding decrease in the number of typhoid fever cases, as is demonstrated, for instance, by the experience of Washington, D. C., and of Youngstown, Ohio. Other diseases arising from use of impure water.— Apart from cholera and typhoid, the use of polluted or ’ otherwise impure water may induce serious intestinal disturbances as a result of the activities of other bacteria. Outbreaks of dysentery of serious proportion are not uncommon in camps. The better care of drinking-water has been responsible for a very marked decrease in the ravages of dysentery 60 Bacteria in Relation to Country Life in temperate climates. It is estimated that the mor- tality from dysentery in England towards the end of the last century was but a fraction of a per cent of that at the middle of the century. In the United States, the death rate from dysentery was 6.32 per cent of the total mortality in 1850; 2.65 per cent in 1860; 1.60 per cent in 1870; and less than 1.5 per cent in 1880. To sum up, therefore, drinking-water is a factor of great moment in the spread of certain diseases and may become the carrier of the germs of cholera, typhoid, and certain forms of dysentery. Its purification and pro- tection from pollution have necessarily become important features of modern sanitation. CHAPTER VIII CONTAMINATION OF STREAMS AND LAKES Bacteria find their way into drinking-water from numerous sources. The dust particles floating in the air, and the bacteria attached to them, are washed down by the rain. The winds that pass over the land carry away with them not inconsiderable numbers of bacteria, and deposit a part of them in the water over which they pass. The surface washings and drainage waters that reach the rivers and lakes contribute to their water a goodly number of bacteria. Soils contain bacteria.—All soils contain large num- bers of bacteria, usually several hundred thousands per gram. In the case of fertile lands, the number may reach several millions per gram of soil. Hence, the rainwater that drains off the surface of the fields, or percolates downward and escapes as subsoil drainage, comes in contact with enormous numbers of bacteria and removes many of them to the water-courses. Anything, there- fore, that tends to increase the number of bacteria in the soil will also tend, indirectly, to increase their num- bers in the neighboring streams. The applications of heavy dressings of animal manure, the plowing under of green crops, the turning under of sod, or the appli- cation of lime and fertilizer, stimulate bacterial develop- (61) 62 Bacteria in Relation to Country Life ment in the soil and, therefore, encourage larger additions of bacteria to the surface waters of the region. Another fruitful source of bacteria for drinking-water is the sewage which finds its way into it. Owing to its origin, sewage is extremely rich in bacteria, frequently containing tens of millions of them in every cubic centi- meter. In view of the enormous volume of sewage poured into some streams, the number of bacteria added to the water is frequently beyond computation (page 73). All of these additions, made more or less constantly, or at irregular intervals determined by the amount of rainfall, provide an abundant supply of bacteria to drinking-water. Various species are thus introduced into the water, some of them capable of adapting them- selves to the new conditions and of multiplying even to a greater or slighter extent. The character of the bacteria in water—The various classes of bacteria in water exist there in different pro- portions. As living organisms they are affected by con- ditions favorable or unfavorable to their survival. The kinds that are best adapted to their environment will emerge victorious from the struggle for existence. Hence, there are groups of bacteria that may be regarded as normal inhabitants of water. These are encountered in large numbers in all drinking-waters. There are others that are brought to the streams in great masses from time to time, but, owing to the com- petition of other bacteria, or to insufficient or improper food, they die out very rapidly. They do not multiply, and so disappear after a longer or shorter period. Such are the numerous species of soil and sewage bacteria, Normal and Not Normal Bacteria of Water 63 which, though frequently found in drinking-water, must be regarded as strangers there. They are not normal inhabitants of water. This class of bacteria may again be divided into two groups. The first includes all the harmless kinds that could produce no illness, even if swallowed in very large numbers. The second is com- posed of disease bacteria proper, such as cholera, typhoid, and dysentery. Since many of the bacteria are not nor- mal inhabitants of water, and since they tend to die out when introduced there, it follows that their presence in large numbers is a certain sign of recent pollution. The number of bacteria in water.—The number of bacteria in water is affected by its composition, by the amount and frequency of rainfall and the resulting drainage, by the contamination with sewage, by the season and climate, the amount and intensity of sun- shine, the depth of the water, the velocity of the cur- rent, and still other factors. Because of the natural variations thus introduced, the number of bacteria in fairly pure river-water may range from a few hundreds, or even less, per cubic centimeter to several thousands. The differences in the bacterial content of water between the winter and summer months are largely attributed to the greater amount of surface drainage in the fall and winter, whereby enormous numbers of bacteria are carried to the streams from the forests, fields, and city streets. In the summer months, on the other hand, much of the lighter rainfall is taken up by the growing vegetation, and a larger portion of it is evaporated from the soil, thus decreasing the amount of surface drainage. The streams must then depend 64 Bacteria in Relation to Country Lije for their main supply on springs, which, as will be shown later, are comparatively poor in bacteria. The determination of the number of bacteria in water may be of distinct advantage in two directions. It has been demonstrated that surface waters are influenced by the character of the soil over which they flow and by the drainage that they receive, thus giving rise to marked variations between one stream and another, even when no actual pollution exists. Yet, in the same stream, and within the limits of seasonal variation, the character and number of bacteria are fairly constant. Hence, any sudden and abnormal increase in the num- ber of organisms in any particular stream should be regarded with suspicion, and as possibly, though not necessarily, due to pollution with sewage. The counting of bacteria in water is also of value in measuring the efficiency of filters whose purpose it is to remove the organisms. It is therefore customary to count the number of bacteria in the unfiltered, as well as in'the filtered water, and a measure is secured thereby of the proportion retained in the filter. Still, even here, mere numbers are not considered a sufficient proof of the efficiency of filtration, and attempts are made to determine the proportion of certain kinds of bacteria that are retained during the process. Causes affecting the increase or decrease of bacteria in water.—The familiar appearance of certain grasses in the meadow, of certain weeds in the fields, and of cer- tain trees in the forest, is largely the outcome of natural selection. The fact, for instance, that sorrel appears in quantity in acid soils is attributed to its adaptation Why Bacteria Increase or Decrease 65 to conditions under which other plants do not thrive. In the struggle for existence, therefore, the sorrel is enabled to crowd out the others when they are not already crowded out by their own inability to grow. Similarly, the addition of lime or gypsum to the soil is followed by the appearance of white clover. This is an indication that conditions have been created favoring the vigorous development of the clover and favoring the suppression by it of other plants not so well adapted to the changed character of the soil. Something of the same nature occurs in water. Just as soils of different origin and composition have their own kinds of weeds, and their own proportions of the different weeds, so waters of different origin and com- position have, though in a more limited sense, their own kinds of bacteria, and their own proportions of the different kinds. Just as there are certain grasses and other plants widely distributed where they are not interfered with by man, so there are certain kinds of bacteria almost universally distributed in our lakes and rivers. They include the normal inhabitants of drinking- water, as well as others which are not, strictly speaking, water bacteria, but are so universally distributed in the soil or in the intestinal tracts of birds, mammals and insects as to gain constant access to surface water. There is, however, an important difference between the normal water bacteria and the others just mentioned. The former can readily multiply in ordinary drinking- water, while the latter are strangers there and perish sooner or later. It thus becomes interesting to inquire why some E 66 Bacteria in Relation to Country Life groups of bacteria find a congenial medium for their development in water, and why others are crowded out and disappear. The factors that influence this are the supply of food, the temperature and sunlight, and the presence of certain products more or less in- jurious to some of the species. The supply of jood.—The first of these factors is of great significance particularly as regards the proportion of organic matter in the water. It should be remembered that, with very few exceptions, bacteria cannot subsist on simple mineral salts. They must have for their de- velopment organic matter elaborated by other organ- isms— plants or animals. There is, however, a wide range of adaptation as to the minimum amounts of organic matter required for the growth of different species. Some organisms will actually multiply in distilled water containing mere traces of organic matter, others will not grow unless there be present large quantities of ‘nitrogenous organic matter. Quite naturally, streams flowing over sandy strata, poor in organic matter, and receiving no additions of sewage, will offer but little food to the latter class of organisms, while streams polluted with sewage and receiving the drainage from fertile soils will offer more favorable conditions for their growth. The organic matter added to surface waters from the drainage area does not long remain unchanged. It tends to disappear, owing to the very class of bacteria that uses it as food. The disappearance of the organic matter necessarily involves, also, the disappearance of the bacteria which prey upon it. Hence, the process of self-purification that occurs in The Food Supply for Water Bacteria 67 streams, lakes, and reservoirs, and the process of puri- fication that occurs in filter beds are both connected with the destruction or removal of the organic matter. On the other hand, the processes of purification that depend upon the destruction of the bacteria themselves, without the accompanying destruction of the organic matter, are wholly inadequate. For instance, the bac- teria in water could be killed by boiling, or by the addi- tion of chloroform, yet after the water becomes cool again, or after the chloroform evaporates, other bacteria may gain access to the liquid, and, with the help of the organic matter which the heat or the chloroform had not destroyed, they may multiply as rapidly as ever. The organic matter may be compared, in this case, to a cask of gun-powder and the bacteria to a spark. While the gun-powder is protected from the spark there is no danger of explosion, yet great care must be exercised to keep the two apart. Perfect safety can be assured only by the removal or the destruction of the gun- powder. The increase of bacteria in drinking-water, due to the additions of organic matter, does not necessarily involve also the increase of typhoid germs that may find entrance to it. On the contrary, a large number of investigations seem to indicate that the typhoid germs and allied species’ survive in water but a short time. Furthermore, it actually appears that larger amounts of organic matter in the water and the resulting active multiplication of certain bacteria inhibit the growth of typhoid germs to a greater extent than when the amounts of organic matter are smaller. At the same 68 Bacteria in Relation to Country Lafe time, the presence of readily decomposable organic matter may prove undesirable because it favors the growth of other bacteria probably responsible for less serious intestinal disturbances. The presence of larger amounts of organic matter is also undesirable because its decomposition gives rise to the formation of nitrates and of other inorganic matter which serves as food for microscopic plants known as alge. This is especially true of lakes and sluggish streams in which the rapid increase of the alge may result in the formation of a green scum on the surface. Under such conditions, the water loses its clear sparkling appearance and acquires a fishy taste, or other undesirable odors and flavors. Since alga, like the green plants, will grow only in the presence of sun- light, their development may be suppressed by keeping the water in the dark. This applies particularly to the storing of drinking-water in reservoirs where the alge are more likely to develop. In a number of European cities, covered reservoirs have been used successfully for years, and the growth of alge has been thus elimi- nated, even in waters rich in mineral salts. Temperature.—The temperature of the water is, undoubtedly, an important factor in the increase and decrease of its bacteria. In so far as the introduction of organisms from outside sources is concerned, it has already been shown that surface waters contain fewer bacteria in the summer than in the winter, at least in moderately temperate climates; and that the greater number of organisms in the winter is due to the greater amount of surface drainage. It may be assumed that Temperature in Relation to Their Development 69 with an equal amount of surface drainage in the summer, the bacterial additions to the surface waters will be much greater than those in the winter. In the water itself, on the other hand, the develop- ment of the bacteria is more intense in the summer than it is in the winter. This involves a more rapid destruction of the organic matter and the corresponding disappearance of the bacteria introduced from out- side sources. In the winter, on the other hand, the destruction of the organic matter is practically at a standstill. Then -the outside bacteria do not suffer so much from the competition of the normal water bacteria, for both are quiescent, and the reduction in the total number of bacteria is not favored to such an extent. This view is encouraged by a number of experiments that show that typhoid germs disappear from unsteril- ized water more quickly in summer than in winter. It is to be further noted in this connection that the normal water bacteria, as well as the great bulk of soil bacteria, develop easily at 68° to 72° Fahr. The bacterial inhabitants of the intestinal tract of warm-blooded animals, however, develop best at blood temperature. It is but natural to expect, for this reason, that these intestinal bacteria will find themselves at a disadvantage when introduced into drinking-water in sewage or in ‘other materials. These organisms actually perish within a comparatively short time, owing largely to the com- petition of the other bacteria. This competition, reacting unfavorably on typhoid. germs and other intestinal bacteria, may be regarded from two different stand- points. 70 Bacteria in Relation to Country Life In the first place, it may be considered that the destruction of the intestinal bacteria is accomplished. by the formation by the other bacteria of substances injurious to them. In other words, the growth of the water and soil bacteria may lead to the formation of substances toxic (poisonous) to the intestinal bacteria. That the excreta or by-products actually formed by bacteria or plants may actually prove poisonous to themselves or to other organisms when accumulated to a large extent, can be well illustrated. An instance of this is the necessity of frequently supplying fresh water to plants grown in water-culture, even when an abundance of available plant-food exists. The other standpoint leads us to assume that the intestinal bacteria are not destroyed by the poisonous excreta of the others, but by actual contact. There is some evidence to how that in case of the typhoid germs, at least, the latter mode of action may serve as the de- structive agents. But whether we regard the disappear- ance of the intestinal bacteria in drinking-water from one or the other standpoint, it remains certain that the activities of the other organisms contribute largely to their rapid disappearance. Numerous experiments carried out with cultures: of the typhoid germ prove conclusively that the organisms survive for a longer time in sterilized than in unsterilized | water. It was shown, for instance, that typhoid germs introduced into unsterilized water from Lake Michigan, survived for only seven or eight days, whereas, in the same water, previously sterilized, they remained alive for twenty-five days, In other experiments, it was Influence of Sunlight 71 demonstrated that a number of soil bacteria and also of water bacteria injuriously affected the typhoid germs, and that such injurious effects were not observed when the typhoid germs were alone. Furthermore, the in- jurious action was found to be dependent to a great extent on the temperature to which the bacteria were exposed. Sunlight.—This is another factor which undoubtedly plays some part in the increase or decrease of bacteria in water. Reference has been made to the germicidal action of direct sunlight, and it has been pointed out that in the deeper layers of water such action can be of only small moment. It is still uncertain to what extent direct sunlight actually contributes to the de- crease of bacteria in water. There can be scarcely any doubt, however, that during the long hours of bright sunshine in the summer months, and, particularly in shallow bodies of water, the germicidal action must be of .considerable significance. Furthermore, some con- sideration must also be given to the indirect action of sunlight in the development of the alge. The latter withdraw in their growth considerable quantities of nitrates and other food material from the water, and thereby render the conditions less favorable for the development of the bacteria. Animalcules injurious to bacteria.—The number of bacteria in water is likewise affected by certain animal- cules that use them as food. It is interesting to watch these little organisms which, though giants.in size as compared with the bacteria, are still so small as to require magnification by a powerful microscope lens 72 Bacteria in Relation to Country Life before they can be seen. When properly magnified, they may be observed rapidly moving about in the drop of water and stirring up currents in it by means of which the bacteria are drawn into the mouth of these animal- cules. Their transparent bodies show the bacteria within them in a moré or less advanced state of de- composition. The development of these animalcules, designated as protozoa, is affected, of course, by the prevailing temperature, and it is quite apparent, there- fore, that in the summer months their consumption of bacteria is a factor to be reckoned with, whereas, in the cold months of the winter their activities are practically suspended. Sedimentation.—The solid particles suspended in water tend'to sink to the bottom when the velocity of the current is reduced. As the stream becomes more sluggish, the diameter of the particles still in suspension becomes smaller and smaller until only the finest frag- ments of silt, clay and organic matter remain floating in the water. When the water is at a standstill, a large proportion of even these finest particles fall to the bot- tom. This gradual settling out of the solid matter in suspension is known as sedimentation, a process which plays an important réle in reducing the number of bac- teria in rivers, lakes and reservoirs. -The décréase in the number of bacteria is encouraged by sédinientation in a twofold manner. In the first place, the settling: out of the suspended matter, which includes, also, a large portion of the organic substances, reduces the quantity of food available to the bacteria and their growth is thereby hindered. In the second Sewage Contamination 73 place, the sedimentation -process carries down large numbers of bacteria which have become entangled in the organic matter. The advantages of sedimentation in reducing the number of bacteria in water have long been recognized in municipal sanitation. Many towns and cities employ either settling basins alone, or settling basins in connec- tion with filter beds for the purification of their drinking- water. The efficacy of sedimentation in removing the bacteria from water has been demonstrated by careful investigation. Dilution.—The influence of sedimentation and of the other factors in reducing the number of bacteria in water, is reinforced by that of the dilution. The water of any river or lake containing large numbers of bacteria, shows a relative decrease of the latter when pure water is added to it from any source. Under actual conditions, the purer water of tributaries frequently serves to reduce, to a very considerable extent, the relative number of bacteria in large rivers. The contamination of drinking-water by sewage.— The vast number of bacteria in sewage, averaging, at times, more than 10,000,000 per cubic centimeter, is frequently a direct source of increase in the number of bacteria in drinking-water. Sewage-polluted streams and other bodies of water show a high bacterial content for the double reason that they are supplied with both the bacteria and the organic matter serving as food for the latter. However, the mere increase of bacteria in water used for drinking purposes cannot be accepted as an infallible proof of sewage contamination, for it has 74 Bacteria in Relation to Country Life already been pointed out that enormous numbers of harmless bacteria may be derived from the soil. To prove that a water-supply has actually been con- taminated by sewage, it is necessary to resort to methods other than the mere counting of the bacteria in it. Chemical methods, bacteriological methods, or both, may be employed for the purpose. The chemical methods are based on the detection of certain substances con- tained in a considerable proportion in sewage. Unfor- tunately, the chemical methods prove insufficiently delicate when the sewage is strongly diluted by the water to which it is added. The bacteriological methods used at present, with considerable success, are more delicate, and are based on the detection of certain kinds of bacteria never absent from sewage, but not regarded as normal inhabitants of pure water. The species of bacteria most widely employed as an indicator of sewage pollution is called Bacillus coli communis, or the colon bacillus, a normal inhabitant of the intestinal tract of man and of domestic animals. Frequent examinations of sewage in this country and in Europe have shown that the colon bacillus is always present in numbers ranging from a few thousand to fifty thousand per cubic centimeter. In occasional instances, this number is greatly exceeded. These results are not surprising in view of the nature and origin of sewage. It has been shown, also, that the colon bacillus can develop and increase in numbers in sterilized sewage, an indication that the sewage itself is adapted for the development of this organism. Under actual conditions, however, the sewage is not sterile, but is inhabitated The Colon Bacillus 75 by hosts of other bacteria whose presence is inimical to the survival of the colon bacillus. The examination of soils of different origin has demonstrated that in those not recently manured the colon bacillus is present in slight numbers or not at all. On the other hand, greater numbers were found in soils that had recently received applications of horse-manure, but the tendency for their gradual decrease was observed here also. Soils which were experimentally inoculated with this organism, were not found, on the whole, to be favorable to its development. It is not, apparently, able to withstand the competition of bacteria normal to cultivated soils, and, while in exceptional cases, it may even increase to a slight extent, its gradual decrease in numbers is inevitable. Water bacteriologists, therefore, agree, for the most part, that the presence of large num- bers of the colon bacillus is a certain indication of recent pollution with sewage. Taking the foregoing facts in their entirety, we find, therefore, that the colon bacillus is present in enor- mous numbers in the excreta of man and of domestic animals; that it is present in large numbers in sewage because this contains human and animal wastes; that it is not present in ordinary soils, except occasionally, or in slight numbers. We are thus forced to the conclu- sion that our surface waters, used for drinking purposes, do not receive large numbers of the colon bacillus from the soils with which they come in contact. The presence of these organisms in surface waters must indicate’ either recent sewage contamination, or the possible multiplication of the colon bacillus in the water itself. 76 Bacteria in Relation to Country Life The presence of large numbers of the colon bacillus becomes thus a danger signal, an indication that it may have been accompanied by the typhoid bacillus. Hence, both the chemical and bacteriological methods employed in the examination of drinking-water aim to detect pollution and its extent, if possible. The best results are secured when the two are combined and a measure thereby secured of the probable addition of sewage materials, on the one hand, and, on the other, of sewage bacteria, some of which may be carriers of disease. Of late years the bacteriological methods have been the subject of much careful study and give promise of becoming more widely applicable in the sanitary examination of drinking-water. CHAPTER IX PURIFICATION OF RIVER AND LAKE SUPPLIES THE composition of river-water is influenced by the geological nature of the region through which it flows, and by the surface drainage it receives. Streams pass- ing through limestone regions carry in solution con- siderable quantities of lime. Their waters are, there- fore, called hard waters. Those that pass through heavily wooded regions and through swamps and meadows acquire large quantities of organic matter, often to such an extent as to give their water a distinct color. Streams that.pass over strata rich in iron may, similarly, be noted for their high content of iron salts, while others may be distinguished for their high content of various salts, as is true of rivers that constantly receive the seepage of alkali soils. The differences in the composition of river-water are not without important influences on the bacteria inhabiting them. It is well known, for instance, that the proportion of lime in the soil affects intimately the character of its bacterial flora. It would be but reason- able to expect analogous relations in surface waters. It still remains to be determined to what extent the varying proportion of lime in water affects the character of its bacteria. (77) 78 Bacteria in Relation to Country Life The quality of organic matter.—It has already been noted that the amount of organic matter in river-water is of paramount importance from the bacteriological standpoint. Something should also be said concerning the quality of the organic matter. There are streams whose waters are brown or almost black on account of the peaty or other vegetable matters dissolved in them. Many such streams furnish a clear, wholesome drinking- water in spite of the high content of organic matter. This is true of some rivers along the Atlantic seaboard. It seems that the organic matter derived from swamps does not decompose as readily as the organic matter derived from sewage. In fact, the organic substances in such waters may exert, to a certain extent, pronounced antiseptic properties, like those of the peat itself. .The development of bacteria in such water must necessarily differ from that in water deriving its organic matter from sewage. The value of the two from the sanitary standpoint must, likewise, be different. However, experts do not entirely agree as to the wholesomeness of peaty water for drinking purposes. Instances are cited when such water has been used for years without ill effects. Instances are likewise cited when the general use of peaty water has led to a markedly increased mortality from intestinal diseases. Alkali water.—But little is known of the bacteriology of waters charged with alkali salts. Such waters are used for drinking purposes when the content of alkali is not too high. Apart from their somewhat laxative effects these waters do not appear to be detrimental to health. Selj- Purification 79 River-water ,and surface drainage.—The differe-ces in the compositon of river-water, as well as their in- fluence on the water bacteria, may be further modified by the quantity and quality of the surface drainage. When the surface drainage includes notable additions of sewage, the influences on the bacterial flora. of the stream may be quite far-reaching. The modifying effect of sewage may be due to its quality as well as to its quantity. For example, the sewage of European cities is more concentrated than that of American cities be- cause of the larger volume of water used by the latter. It is to be expected that equivalent quantities of the two will not have the same effect on any given stream. Moreover, the ratio between the volume of sewage added to a stream, and the volume and velocity of the latter, is of direct significance. A slight quantity of sewage added to a large river would not contaminate the water to such an extent as the addition of a large volume of sewage to a small stream. Self-purification of rivers.—The activities of the water and sewage bacteria rapidly exhaust the store of readily decomposable organic material. The less readily avail- able residues do not furnish the large quantities of food required by vast numbers of bacteria, and the less resistant species are rapidly eliminated in the struggle for existence. The gradual disappearance from the water of the organic substances, as well as of the bacteria introduced with the sewage into flowing streams is designated as self-purification. Differences of opinion still exist as to the extent to which self-purification may be depended upon for 80 Bacteria in Relation to Country Lije the protection of the public health. The older views on the subject are represented in the statement of Pettenkoffer on the one hand, and in that of the sixth report of the Rivers’ Pollution Commissioners of Great Britain, on the other. Pettenkoffer believed nature could be trusted to purify polluted river-waters, provided the amount of sewage added does not exceed 7's of the volume of the stream, and that the velocity of the latter is fully as great as that of the stream of sewage. The Commissioners held, in their report, that nature can not be relied upon to do the work properly, even when the sewage is greatly diluted by pure river-water, and that ‘it is impossible to say how far such water must flow before the sewage matter becomes thoroughly oxidized.”” Their inference was that no river in England was long enough to assure the complete destruction of sewage by oxidation. Modern views steer the middle course between these opposing opinions. We have strong proof that the pro- cess of self-purification is quite variable, even in the same stream, and that the destruction of the polluting substances may be rapid or slow according to.conditions. In the water there is (1) decomposition of the organic matter by bacteria, (2) a removal of a portion of it by sedimentation, (8) and the destruction of disease germs by other bacteria, by sunlight, and by animalcules and other creatures feeding upon them. Gradually, but surely, the putrescible matter largely disappears from the water, and, with it, great hosts of injurious and harmless bacteria. The organic matter carried down by sedimentation is, in its turn, attacked by the classes Purification of Streams 81 of bacteria capable of growing in the absence of air, and is destroyed. The bubbles of gas which rise to the sur- face testify to this. The decomposition of the organic matter in the water and in the mud at the bottom of rivers, lakes and ponds, as well as the destruction of the disease germs introduced with the sewage cannot, however, be depended upon to purify the water so as to make it absolutely safe. The typhoid germs may be destroyed in a few hours or in a few days in sewage-polluted waters, as numerous in- vestigations have clearly demonstrated; yet, under other conditions, they may survive for weeks, or even months, and there is no assurance that twenty, thirty, or even fifty miles of flow would be sufficient for their removal under all circumstances. There is a general agreement on this point among sanitarians and for this reason the practice of pumping sewage-polluted water directly in the city mains is strongly discouraged. The storing and filtration of river-water.— When brought to rest in large reservoirs, river-water is subject to the sedimentation process as it is in rivers, lakes or ponds. The suspended particles in such stored waters settle out gradually. Not only is there a subsidence of the suspended particles, but, also, a partial decomposition of the organic matter in solution, and a very marked general reduction in the number of bacteria. The reservoirs for unfiltered water, or settling basins, as they are often called, appear thus to be a very im: portant means for the purification of drinking-water. The settling basins of the city of Washington, which, before the installation of the filtration plant, were the F 82 Bacteria in Relation to Country Life only means for the bacterial purification of the Potomac water used in the city, showed a bacterial efficiency of 70 to 90 per cent. The city of Baltimore still depends upon the settling basins and the prolonged storage of the drinking-water for the elimination of the typhoid germs from its water. : Notwithstanding the marked decrease of the bacteria in stored water, it is generally conceded that settling basins alone should not be depended upon for the pro- tection of the community against typhoid fever. A small proportion of the disease germs might survive, and, probably, do survive, the sedimentation process. It has become necessary, therefore, to supplement the latter with more efficient means for the elimination of typhoid and other intestinal germs. As a result of this conviction, many modern water-purification plants include in their equipment both settling reservoirs and filter beds, and, likewise, additional reservoirs for the storing of filtered water. Sand-filtration.— After remaining in the settling reser- voirs for a greater or shorter length of time, the water is passed through the filters in order to destroy most of the remaining organic matter, including most of the sewage bacteria. It should not be supposed, however, that the action of the filters is purely mechanical. .On the contrary, they owe their efficiency to the bacteria that establish themselves in the upper layers of the bed and act there as very vigorous scavengers. For this reason, the sand filters do not attain their highest effi- ciency until some days after they are put in operation. It is found then that the grains of sand which compose Sand Filter Beds 83 the upper layer of the filter bed have become covered with a gelatinous film, which has been found to be the seat of the bacterial scavengers. The nitrifying bacteria are found to be quite promi- nent in the filter beds, and it is their function to change the ammonia formed in the processes of decay, into the soluble nitrates. Since the nitrifying bacteria are dis- tinctly aérobic, requiring a plentiful supply of air for their development, the filter beds must be so constructed as to allow the air ready admission. For this reason, layers of coarse materials are placed at the bottom, and on top of that other layers of successively greater fine- ness until the uppermost layer is reached. This consists of sand whose grains are fairly uniform in size. It is recommended that the thickness of the sand layer be about 24 inches and, in any case, not less than 12 inches. Because of the gradually increasing thickness of the gelatinous film around the grains of sand, the filters are apt to become clogged in the course of time. To obviate this difficulty, the surface portion of the sand is pared off from time to time, and the removed material thoroughly cleaned and purified. It has been found in practice that the best results are secured when the filters are worked intermittently ,— that is, when they are given periods of rest in order to allow a more thorough aération of the bed. It has been demonstrated, likewise, that the efficiency of the filters depends, to a great extent, upon the rate at which the water is made to pass through them. Other methods of water purification The bacteria present in drinking-water may be removed or destroyed 84 Bacteria in Relation to Country Lije by methods other than sedimentation and sand-filtration. Some of these involve the removal of the bacteria by chemical substances added to the water, although the process by which this is effected may be, in itself, largely mechanical. The alum method.—One of the methods in question is based on the addition to the water of alum at the rate of 4 to } grain per gallon. In the presence of the car- bonate in the water, the alum is decomposed with the formation of a jelly-like mass, which gradually subsides and carries down with it the suspended parti- cles, including the bacteria. It appears, moreover, that not only are the bacteria carried down by the alum, but that the latter exercises a deleterious effect on their subsequent redistribution in the upper layers of the liquid. The alum possesses the further advantage of combining with the organic substances dissolved in the water and of decolorizing it. After being’ clarified by the precipitated alum, the water is made to pass through layers of sand. Thus it receives the benefit of careful filtration. When the water is soft and deficient in carbonates for the decom- position of the alum, the filtering material is made up of a mixture of sand and crushed marble. In the prac- cal application of the method care is taken, of course, not to add an excess of alum lest a portion of it be carried over into the purified water. It is asserted that water purification involving the use of alum is both efficient and economical, particularly on account of the saving of space that results from its use. The Clark process.—There are other methods of Clark Process of Purification 85 water purification based either on the formation of precipitates which subsequently carry down the sus- pended matter, or on the direct action of the subsiding particles in carrying down the bacteria. One of these, known as the Clark process, involves the use of lime- water and is intended primarily for the softening of hard waters. The lime-water added reacts with the dissolved bicarbonate of lime. The precipitate thus formed gradually subsides, carrying with it, as in the case of alum, the particles in suspension. The efficiency of this method was investigated and it was found that the subsidence of the precipitated matter in open tanks was so rapid as to render the water fit for distribution in three hours. The bacteriological examination of samples of softened and unsoftened water gave the following results: Bacteria in one cc. Unsoftened water 2.20.5 -se.c01se.0 0 seek ves lee sees 322 Water after softening and two days’ subsidence drawn from the main service pipes ..........-.------ 4 Reduction in the number of bacteria, 99 per cent. Purification by finely divided solids.—Still other methods of water purification that have been proposed are based on the direct removal of the bacteria by agi- tating the water with finely divided solids. Spongy iron, animal and vegetable charcoal, coke, ground limestone, chalk, infusorial earth, and the like, have shown a more or less high efficiency in this respect. Spongy iron, charcoal and coke are particularly adapted for this purpose because of their structure. 86 Bacteria in Relation to Country Lije The Woolf method.—Considerable success has attended the use of methods that are distinctly chemical in their action, but involve the employment of electricity. One of these, known as the Woolf method, is based on the decomposition of weak salt solutions by a current from the dynamo, whereby the compound sodium hypochlorite is formed. Sodium hypochlorite has a marked germicidal effect, and the addition of the elec- trolyzed solution to the water causes the destruction of the bacteria in the latter. Because of the quantities of material required to effect the sterilization of water, this method can hardly be regarded as economically practicable. Purification by means of ozone-—There is more prac- tical significance in the purification of water by means of ozone. When electrical discharges occur in the air, a portion of its oxygen is converted into ozone. This phenomenon is utilized in the purification of water. Electric sparks are passed through dry air, and after the latter is ozonized it is made to bubble through the water to be purified. The method is reported to be efficient with water containing moderate quantities of organic matter. The bacteria are readily destroyed, tests with Bacillus coli having demonstrated their entire elimination in a number of instances. The organic matter itself is partly oxidized, and the ammonia converted into nitrates. With water rich in organic matter, the ozonization method does not appear to be as satisfactory as could be desired. Filters for domestic purposes.—There are various types of small filters for domestic use. It is well known Filters 87 that most bacteria are held back by filters made of unglazed porcelain. Some species exist, however, that are so small as to pass the minute pores of these filters. For practical purposes, nevertheless, unglazed porcelain may be regarded as bacteria-tight, and water which passes through it as sterile. This fact has led to the use of such filters for the purification of water used in the home. With many people no doubt seems to exist as to their permanent efficiency. Unfortunately, how- ever, there are conditions under which such filters are not bacteria-tight. To be sure, the organisms can not pass through them directly, yet it has been demonstrated quite forcibly that they can grow through them. Any of these filters, kept moist for some time, and accumulating organic matter on the inside and in their pores, will, finally, become pervious to the bacteria in them. Typhoid germs, as well as other microérganisms, may thus find their way into the supposedly pure water and cause disease. To render the porcelain filters abso- lutely safe it is necessary to burn them out from time to time, so that all of the organic matter contained in them may be destroyed. Unglazed porcelain filters that can be thus renovated with but slight danger of breaking are now being made. Other filters for domestic use, made of charcoal or blocks of sandstone, possess the same defects noted in the porcelain filters, and should not be depended upon for the purification of drinking-water for any consider- able length of tine. They should be thoroughly cleaned and boiled at least once a week if their efficiency is to be assured. 88 Bacteria in Relation to Country Life The waters of lakes and ponds.—These are subject to the same phenomena of purification already noted in the preceding pages in connection with the water of rivers. In lakes, as in rivers, the action of dilution, sunlight, sedimentation, and the decomposition of the organic food bring about the decrease in the numbers of bacteria. In some directions, however, there are important differences, if not in kind, at least in degree. The influence of sedimentation is more strikingly ap- parent in lakes than it is in rivers, for the velocity of the current, so important a factor in the latter, plays but a subordinate part in them. The water of small lakes and ponds is more liable to be affected than that of larger lakes by the growth of alge, and by the drainage from adjacent land. Another important influence that is of more-consequence in this connection is that of sunlight and circulation. The slighter depth of the water allows a more thorough aération of the bottom layers, and the more frequent interchange between the surface and bottom water un- doubtedly exerts a direct effect on the bacteria. The development of plant life in shallow lakes and ponds is, at times, so great as to modify, to a marked degree, the numbers and kinds of bacteria, and the gen- eral character of the water becomes such as to produce diarrhea in most persons who drink it. In smaller ponds, animals frequently come to modify the bacteriological character of the water to their own injury. Comparisons made between ponds to which animals had free access and other similar ponds from which they were excluded showed very marked differences in the bacteriological Bacteria and. Health 89 composition. The fact is thus emphasized that the animal excreta which find their way into ponds may become a bacteriological factor of importance from the standpoint of the health of the animals. Intestinal disturbances among young animals, as- suming, at times, the proportions of an epidemic, may undoubtedly have their origin in the polluted water of ponds and brooks. CHAPTER X BACTERIA IN WELLS, SPRINGS, TANKS AND ICE Witu the introduction of public water-supplies in towns and cities, the use of water from domestic wells has become more restricted. The result of the change is apparent in the gradually dimishing mortality from typhoid fever, as is illustrated, for instance, by the reduction of the typhoid death rate from 34 per 10,000 to 1.1 in the city of Vienna within three years after the installation of a municipal water-supply of good quality. The change in the typhoid fever death rate in Massa- chusetts is fully as striking. BACTERIA AND WELL-WATER Extended observation in many places justifies the belief in the connection existing between the use of water from shallow wells and an increased death rate from typhoid fever. It is a matter of common knowledge that domestic wells are frequently located in places where pollution from various sources is not excluded. It is not, however, generally realized how large a pro- portion of such wells may receive polluted materials without manifesting it in the taste or appearance of the water. The brightness and sparkle of well-water (90) Danger from Wells 91 is largely due to the carbon dioxid contained in it, the latter being derived from the decomposition of organic matter. Soils rich in organic substances, particularly those contaminated with sewage, produce more carbon dioxid than those poor in organic matter, and the brightness of well-water is, therefore, far from being a guarantee of its purity. Soil may be regarded as a good filter. It holds back undissolved organic materials including bacteria, and, to a marked extent, also materials in solution. The water of springs and deep wells is, at times, almost germ-free, largely because of the filtering power of the soil. With that much granted, however, it still remains true that the soil cannot be entirely relied upon to pre- vent the passage of typhoid or other disease germs from cess-pools and privy-vaults into shallow wells located a short distance from them. This fact has long since forced itself upon the attention of municipal au- thorities, and has led to the establishment of regulations calling for a minimum distance of 20 to 50 feet between wells and cess-pools or privy-vaults. Unfortunately, the distance of 25 feet, frequently allowed by the munici- pal regulations, is a very uncertain security against the entrance of disease bacteria into wells. The soil is not a solid mass of material, but an aggre- gation of particles of various sizes, separated by pores, or spaces filled with air. The rain that falls on the soil makes its way downward past the solid particles until it reaches a point where the pores are filled with water instead of air. The depth in the subsoil beyond which the ground is thus saturated with moisture is known 92 Bacteria in Relation to Country Life as the water-table. Wells are made by digging down some feet into this saturated zone. It frequently happens that the cess-pool situated at a distance of 25 feet or less from the well is made to discharge its contents into the latter. The liquid from the cess-pool, which is not as deep as the well, gradually passes downward until the water-table is reached. The air in the ground above the water-table offers more or less resistance to the downward passage of the sewage, and the latter will flow more readily towards the points where the resistance is least. The air in the well, subject to expansion and contraction with changes in temperature, causes the well to become the center of least resistance, and encourages the inflow of water from the surrounding soil. The removal of water from the well also stimulates further additions from the adjacent portions of the saturated zone. When cracks exist in the ground, the passage of water into the well from the surrounding soil becomes even more rapid, and, under such conditions, wells have been known to become pdlluted from sources several hundred feet distant. Should the cess-pool receive additions of disease germs from the house, the well-water nearby is then exposed to serious infection,— the more so since, in sewage-polluted soil, the normal soil bacteria are crowded back by the sewage bacteria, and the disease germs may survive for a longer time under such conditions. It is scarcely necessary to emphasize the evident significance of these observations. They help us to un- derstand why it is so difficult to eradicate the disease, Wells 93 and prove convincingly that shallow wells situated near cess-pools and privy-vaults should be regarded with suspicion. Past freedom from disease germs can never serve as a guarantee of future safety. It has been shown, time and again, that such germs may ultimately find their way into the well, and outbreaks of typhoid may occur, not only on account of the direct use of the water, but, also, on account of the use of such water in the washing of milk cans and other dairy utensils. It happens that milk is a good culture medium for typhoid bacilli. A few drops of well-water containing a single typhoid germ may be sufficient for the production of vast num- bers. Some severe typhoid epidemics have been traced directly to the consumption of infected milk. Deep wells and springs—The thorough filtration of the ground-water which finds its way into deep wells makes it almost germ-free. Numerous examinations of such waters have shown them to contain usually less than 50 bacteria per cubic centimeter. At times the number does not exceed 4 or 5 per cc., and it is very seldom that as many as 100 or 150 per cc. are found. This does not mean that deep-well-water is incapable of supporting a vigorous growth of bacteria. On the contrary, samples of deep-well-water, when allowed to stand for a few days, frequently show an enormous multiplication of their bacteria, while similar samples of surface water containing a much larger number of organisms, show a development decidedly more feeble. The difference is probably due to the fact that the organisms in the surface water suffer from com- petition among themselves, and, likewise, from the 94 Bacteria in Relation to Country Lije attack of animalcules which devour them in great numbers; hence the decrease. Deep wells, improperly protected from surface wash- ing, may also contain considerable numbers of bacteria. It has been observed that the first portions of the water pumped out of wells contain large numbers of bacteria, whereas the portions brought up later contain compara- tively few. The phenomenon in question is explained by the rapid multiplication of the bacteria in the slimy layer covering the walls of the well, and their washing down into the surface water. Hence, when the water in the well had been left undisturbed for some hours, the accumulation of the bacteria in the surface layer may be quite considerable. Later on their numbers will tend to decrease again, owing to the action of sedimen- tation. The influence of sedimentation in this direction is made evident in the comparison of the bacteria in the undisturbed and the disturbed water. When the bottom layers of the well-water are thus disturbed, the number of bacteria per cc. may increase to a very striking extent. Springs.—The bacterial nature of spring-water does not differ much from that of deep-well-water. When contamination from the surroundings is excluded, it is not unusual to find the samples germ-free, or contain- ing a few organisms per cubic centimeter. Driven wells.— Wells of this class have the advantage over open wells in that they are better protected against pollution from surface drainage. Under certain condi- tions, however, surface washings may pass downward along the tube until the ground-water is reached. Wells 95 Moreover, they do not exclude infiltration of sewage liquids below the water-table, and, when the distance from cess-pools, stables, or privy-vaults is slight, pol- lution may occur in a manner noted in connection with open wells. When pollution is excluded, the water from driven wells contains but few bacteria, and, from deeper wells, it may be practically germ-free. Artesian wells.—The water of artesian wells is de- rived from sources more or less distant, and makes its way between two impervious strata towards lower levels. When the upper stratum is punctured, the water is driven to the surface with a force proportional to the elevation of the region whence it is derived. A porous layer of sand or gravel, situated between two layers of rock or clay, and placed in a vertical position, may thus collect large quantities of rain- or snow-water and con- vey it hundreds of miles to the plain below. Owing, however, to the efficient filtering action of the water- bearing stratum, the comparatively small number of microérganisms derived from the rain or snow are not carried far, and artesian waters are, therefore, generally very poor in bacteria. BACTERIA IN THE WATER OF CISTERNS AND TANKS In regions where the ground-water is difficult to reach, or where it contains a large proportion of unde- sirable substances, like bicarbonate of lime, or other salts, the collection and use of rain-water is generally resorted to. Its freedom from lime makes it highly desirable for laundry purposes. When carefully collected 96 Bacteria in Relation to Country Life and carefully kept, it makes, also, a good and healthful drinking-water. Cistern-water is collected by allowing the rain that falls on the roof to run into receptacles made of wood, stone, concrete, or metal. This method of collection exposes the water to more or less contamination, for, apart from the dust particles and bacteria in the air, carried down by the falling rain, the roof itself supplies varying quantities of dust, droppings of birds, insects, and other objectionable materials. Small animals, birds, and insects may also find their way to the cistern itself, thus adding still further to the amount of con- tamination. When we remember that the particles of dust borne by the winds may contain disease germs still capable of development, for example, those of ty- phoid, tuberculosis, and diphtheria, we realize at once that cistern-water may become a carrier of disease. To make matters worse, the cisterns are frequently located in the ground near sources of pollution, and are not always impervious to infiltration from adjacent soil. Cisterns made of wood, brick, or stone, may thus be- come polluted from nearby cess-pools and privy-vaults. This is not a mere assumption, but a fact repeatedly demonstrated by actual examination. Cases of typhoid, sometimes several in one family, have been traced to the use of cistern-water. Numerous instances of other intestinal disturbances are also on record. A large pro- portion of the cisterns examined have been found to contain the colon bacillus and, likewise, considerable numbers of other bacteria. This condition is not at all surprising in view of the very infrequent cleaning of ER ffect of Freezing 97 such cisterns, and the large amount of filth that may have accumulated in them. BACTERIA IN ICE Freezing temperatures destroy a large proportion of the bacteria in the water. They cannot, however, be depended upon to destroy all of the bacteria, and thus render the water sterile. This has been repeatedly demonstrated by various investigators. Careful studies have been made in this connection with the typhoid bacillus, the cholera germ, the anthrax bacillus, and a number of non-pathogenic organisms. It has been proved that ice may become a source of infection and disease. The process of freezing is often, in itself, insufficient for the complete destruction of all the bacteria present in the water. Under certain conditions, the longevity of the typhoid germs and other bacteria may be considerably reduced. This is particularly so when the process of freezing is not continuous, but consists of alternate periods of freez- ing and thawing. Such intermittent freezing may lead to the destruc- tion of the typhoid, cholera, and other organisms within a few days; whereas, spore-producing species, like the anthrax bacillus, are not thus destroyed, owing to the great resistance of the spores. Everything considered, then, ice made from polluted water must be regarded with suspicion. Artificial ice, on the other hand, when made from distilled water, is almost free from bacteria, and may be used safely in the household. Artificial ice made from river-, well-, or spring-water contains a G 98 Bacteria in Relation to Country Life variable number of bacteria, depending on the purity of the water employed. Bacteriological examinations of thick cakes of natural ice have shown that, as the freezing proceeds from the top downward, the number of bacteria included in the ice diminishes. The greatest proportion of bacteria has been found to occur in the snow-ice, although, on the whole, there seems to be no uniform distribution of the bacteria in any one layer. Their number may vary from less than one hundred to several thousand . per cubic centimeter. As the ice melts, the number of bac- teria in the ice-water begins to increase, attaining at times, very considerable proportions. One instance is reported in which a piece of ice was melted and im- mediately examined. The number of organisms per cubic centimeter was 1,020, whereas, eleven days later the ice-water was found to contain 220,000 bacteria. The partial destruction of the bacteria by freezing, and their subsequent multiplication in the ice-water, may have a direct bearing on the typhoid question, since it has been observed that not all of the bacteria are affected to the same extent by freezing. It is quite possible that the disease germs may survive in much greater proportion than the harmless bacteria, and may subsequently multiply as the ice melts. Typhoid germs do not appear to suffer from the competition of other bacteria at lower temperatures so much as they suffer from it at higher temperatures. Savage states that ‘At the temperature of the ice-chest, the typhoid germ may grow in the by-products of other germs, which, at higher temperatures, are quickly fatal to it.” CHAPTER XI THE SANITARY EXAMINATION OF WATER-SUPPLIES On account of the important interests at stake, sanitary examinations of water-supplies should be thor- oughgoing and complete as far as it is practicable. It is not sufficient to determine whether pollution of drinking- water has actually occurred: the inspection should extend to possible future pollutions and the value of sources of supply judged accordingly. Streams that once supplied pure water are now grossly polluted by towns and cities that have grown up within recent years. The expanding limits of cities and the development of industries are calling for greater and greater quantities of pure water and are disposing of a constantly growing volume of waste. The problem is thus complicated in both directions. Its solution must be found ultimately in accordance with the statement made by Mason that ‘a land should be looked upon as watered by its smaller lakes, its springs, and its brooks, and sewered by its great, especially its navigable, rivers. Its water-sources should be protected by law with exceeding care, and no river or stream should be added to its list of drains except after proper consider- ation by the State Board of Health, followed by legis- lative permission.’ Larger cities are carefully guarding the area from which their drinking-water is drawn. In some instances, (99) 100 Bacteria in Relation to Country Life the entire water-shed is fenced in and men ‘and animals are excluded. In other instances, a careful record is kept of the dwellings on the water-shed, and the cases of typhoid occurring there are isolated as far as possible. In still other instances, such inspections are either not made at all, or made in a very incompetent, or perfunc- tory manner, to the ultimate detriment of the com- munity. It is scarcely necessary, therefore, to empha- size here the importance of the topographical inspection of the area whence a city’s water-supply is derived. It is necessary to determine whether sources of pollution exist on the water-shed, and the extent to which the drinking-water may become contaminated from such sources. This much established, the aid of chemical and bacteriological methods may be invoked for the measure of the pollution ‘that has already taken place, and, to some extent also, for the measure of pollution that may take place. The chemical examination seeks to discover in the water certain substances derived from human wastes. For instance, sewage contains a much larger proportion of common salt than does pure water, since salt is always used in the kitchen. If the salt content of the drinking- water is found to be greater than that of water known to be pure and derived from the same locality, it may be safely assumed that pollution has occured. Great care must be exercised, however, in the interpretation of the results, for the amount of salt present in the surface- and well-water of different regions is variable. In some localities, deposits of rock-salt exist, and their well- waters contain, therefore, considerable quantities of Testing Water 101 salt, enough to condemn the water as polluted had they been derived from other localities. It is evident, therefore, that, in judging the purity of a sample of water by its salt-content, we must be informed previously as to the normal salt-content in the waters of that locality. Similarly, in the case of free ammonia, albuminoid ammonia, and nitrates— substances which serve as indicators of pollution— the source of the water must be known to allow an intelligent interpretation of the analytical results. Common salt, and other substances readily detected in water, may also be employed as an indicator of possible pollution. If it is desired to know, for instance, whether the contents of a certain well make their way into a stream, or whether the contents of a certain cess-pool make their way into'a well nearby, it is merely necessary to add larger quantities of salt to the well or to the cess-pool in question, and to examine, subse- quently, the supposedly polluted water for an increase in its salt-content. When the pollution is slight, the chemical methods may fail to show it. For example, a typhoid fever patient may be located within a few hundred yards of a stream used as a source of drinking-water, and some of the infected wastes may find their way into the stream. The quantity of polluting material being slight, and the amount of dilution great, the chemical methods would prove to be not delicate enough for the detection of this pollution. A count of the total number of bacteria growing on ordinary media would probably prove un- satisfactory, because of the natural variations in the 102 Bacteria in Relation to Country Lije bacterial content of the water. On the other hand, the determination of the number of Bacillus coli and, per- haps, also of other characteristic intestinal bacteria, would probably show definitely the extent of pollution. It will be seen, therefore, that topographical, chemical, and bacteriological examinations may each serve to throw some light on the problem. The topographical examination must show whether danger of pollution exists, the chemical and bacteriological examinations must show whether pollution has taken place. That thoroughgoing inspections of the water-supplies are a good investment of time and money is evidenced by the data on the cost of typhoid epidemics, as collected by Mason. According to him, the cost of the typhoid epidemic at Plymouth, Pennsylvania, was: Loss of wages for those who recovered........ $30,020 80 Care ‘of ‘the: sick. ssiss vewas pacieec a were oecleiss x 67,100 17 Yearly earnings of those who died ........... 18,419 52 The epidemic started on account of the improper protection of the water-supplies from the wastes of a single typhoid fever patient; and the figures just cited are eloquent as a condemnation of municipal negligence. PART III BactTERIA AND SEWAGE CHAPTER XII THE PROBLEM OF SEWAGE- DISPOSAL EaR.y in his history man learned to know that the committal to earth of his waste products was quite effective in rendering them harmless and inoffensive. As long as he retained his nomadic existence, and kept moving from place to place, the problem of refuse- disposal was not a vital one. The time came, however, when he established himself more or less permanently in camps and villages. It then became necessary for him to protect himself against being poisoned by his own excreta. Trenches and pits were therefore located outside of the habitations to receive the waste and offal capable of undergoing putrefaction. But the tent and the hut were finally replaced by the more permanent home. Human life itself became more complex. Man’s mind developed and his wants, straightway, became more numerous. He began to cook his food, to wear garments, - to wash them, and to use considerable quantities of water for purposes other than drinking. The shallow trench being no ‘longer suf- (103) 104 Bacteria in Relation to Country Life ficient for the reception of the solid and liquid refuse, larger and deeper pits had to be dug—such was the origin of the cess-pool. Later on, it was found necessary to build a roof over the cess-pool to keep out the rain, and to provide an opening in the roof for the escape of the gases generated in the putrefying masses. Still later, it was found desirable to line the pits with brick and stone, in order to prevent, to some extent, the dif- fusion of the offensive liquid into adjoining soil, and as a protection against the pollution of shallow wells. Sub- sequent improvements in the construction of cess-pools made them less unsightly architecturally and more easily accessible from the house. The cess-pool—The contents of the cess-pools and privies had to be removed from time to time by scaveng- ing. In the pail system, as well as in the dry-earth- closet system, it was aimed to render the human excreta less offensive by covering them up with ashes, lime, or dry earth, and to remove them from time to time. In the dry-earth system, particularly, the organic matter was rapidly destroyed by bacteria and the same earth could be used over and over again. The waste materials removed by scavenging were either buried in trenches or used as fertilizer on tilled land. Generally speaking, therefore, these methods of disposal depended on bac- terial action. It was the bacteria in the soil and in the water that effected the rapid and’ satisfactory decom- position of the waste products. From the agricultural standpoint, much may be said in favor of these methods, since they proyided for the return to the earth of the plant-food that had been Cess- Pools 105 taken from it. They also prevented the squandering of much national wealth. The impoverishment of large tracts of once fertile soil in Europe and America may be contrasted with the undiminished fertility of the culti- vated lands in China and Japan, where great care is taken to return the waste products from towns and villages to the soil. The rapid growth of cities in the nineteenth century, the increasing density of population, and the constantly growing volume of human waste, created conditions which, in the course of time, became a menace to health. The numerous cess-pools made the soil in cities black and soggy with fetid, undecomposed wastes; the shallow wells became polluted by surface washing and infiltration, and the death-rate from typhoid and other intestinal diseases became abnormally high. The old methods of sewage-disposal were no longer adequate for the changed needs of modern life. Sewerage systems, based on the dilution and removal of the human wastes in water, came into existence; the privy and cess-pool were, to a great extent, abolished in the larger cities. The sanitary conditions at once showed a marked improvement. The new method of the nineteenth century.—It was not long, however, before the new method of sewage-disposal gave rise to serious misgivings. The rivers into which the constantly growing volume of sewerage was being poured became grossly polluted and frequently offensive to sight and smell. Fish could not live in such water, and complaints came from towns and cities farther down on the streams that their water-supplies werc being poisoned. These conditions finally forced munici- 106 Bacteria in Relation to Country Life pal and state authorities to pass regulations for the treatment and purification of sewage previous to its discharge into rivers or lakes. The early legislation on sewage-purification was antecedent to the development of modern bacteriology. The Rivers Pollution Act, which made it compulsory for some communities to take steps toward the purifi- cation of their sewage, was passed in England in 1865. It was but natural at that time to seek in chemical methods a means for the proper purification of sewage, and we find, accordingly, a number of such methods suggested, or actually employed for the purpose. A patent for the chemical purification of sewage by the lime process had been taken out in 1846 by Higgs. Other patents were taken out by Wickstead in 1851 and in 1854, and a company was organized at Leicester for the manufacture of fertilizer out of sewage. In 1852 a patent was taken out for the treatment of sewage by means of alumina and charcoal. Similar patents were granted in the period from 1853 to 1860. All of these processes are based on the more or less thorough re- moval of the organic matter in the sewage, thus making it. non-putrescible. The salts of alumina or of iron react with the substances present in the sewage and form flocculent masses, which gradually settle out to the bottom, dragging down the bacteria entangled in them. The chemical methods of sewage-purification proved satisfactory up to a certain point. They freed the sewage from the suspended matter and reduced the number of bacteria in this way, producing effluents in some cases, which did not putrefy as readily as the Purification of Sewage 107 original sewage. They were incapable, however, of freeing the sewage from most of the putrescible matter, for the reason that about one-third of the organic nitro- gen and about one-half of the carbonaceous matter in the sewage are contained in the suspended solids. The rest is held in solution. The dissolved substances in the clear effluents were still liable to create serious pollution of surface water. The disease bacteria, also, that were not carried down by the chemicals, still re- tained their virulence. Moreover, the machinery, chemi- cals, and labor required for the chemical treatment of sewage, involved considerable expense, and the sludge removed from the sewage did not prove as valuable for manurial purposes as was anticipated. In some instances, companies that had been organized for the recovery of fertilizer substances from sewage were compelled to dispose of their plants after sustaining large monetary losses. More recently, bacteriological methods for the puri- fication of sewage have come into more general use, and have already demonstrated their efficiency when prop- erly applied. Reference has been made to the self- purification of streams, a process dependent upon the activities of microscopic organisms. Similarly, in sewage, the vast number of bacteria attack and destroy both the suspended and dissolved organic matter, and render the liquid non-putrescible and inoffensive. However, this process is gradual and, under natural conditions, gives rise to foul odors in its early stages. The artificial methods in the bacteriological purifi- cation of sewage aim to intensify the activities of cer- 108 Bacteria in Relation to Country Life tain groups of bacteria by which the organic matter is rapidly destroyed without giving rise to undue offense. The purified sewage is thus comparatively free from putrescible substances and contains comparatively few of the intestinal bacteria whose presence in drinking- ji nas ” “fy “N oe Lf oy. f 7 Ps e Fig. 17. Sewage bacteria.—1. Bacterium mesentericus; X 2,000. (Rideal.) 2. Bac- terium subtilissimus; X 2,000. (Rideal.) 3. Bacterium mem- braneus fatulus; X 2,000. (Rideal.) 4. Bacteriwm fusi- formis; . X 2,000. (Rideal.) 5. Bacterium entiritidis sporo- genes; X 2,000. (Hewlett.) 6. Bacterium coli communis; X 2,000. (Hewlett.) 7. Pro- teus vulgaris; X 2,000, (Ro- della.) water is objectionable. The bacteriological methods of sewage- purification promise to be more economical and more efficient from the sani- tary standpoint than the older chemical methods. Bacteria in sewage.—Sew- age may become a menace to public health for the two- fold reason that it contains organic substances and dis- ease germs. The disposal of sewage with the least danger to public safety becomes, therefore, a problem of great moment. By sterilizing sew- age, by means of heat or anti- septics, all the bacteria con- tained in it can be destroyed, and, by keeping it sterile, its decomposition can be pre- vented. The fact cannot be ignored, however, that steri- lized sewage, when discharged into any body of water, will Sewage- Disposal 109 immediately begin to decompose, because of the pres- ence in the air and water of certain bacteria that find the organic matter in the sewage proper food for their development. Sewage can be made entirely harmless only by the destruction of its organic matter, as well as by the destruction of the disease germs contained in it. Since the destruction of the organic matter is almost exclu- sively a bacteriological process, whether taking place naturally in the self-purification of streams, or more or less artificially in sewage-purification plants and sewage- farms, it is evident that the matter, to be properly under- stood, should be regarded from the bacteriological rather than the chemical standpoint. Growth of the problem.—The problem of sewage- disposal, like that of water-supply, has grown in magni- tude within the last few decades. The gathering of vast numbers of people on comparatively small areas, and the diversified interests of the city with its human and manufacturing wastes, are rendering the proper solution of this problem more and more difficult. Like a dread specter, threatening disease and destruction, it has disturbed the peace of inland cities. Notwith- standing the progress of modern sanitation, it still continues to be the subject of careful inquiry. Even communities like that of Greater New York, favored in their situation on the ocean coast, are not entirely free from the care and anxiety of rendering their sewage harmless to human health and comfort. Inland communities and sewage-disposal.—Away from the coast, the problem of sewage-disposal is rendered 110 Bacteria in Relation to Country Life more or Jess complicated and difficult by the fact thai the rivers and lakes used ‘as a source of water-supply by some communities receive the sewage of other com- munities. Progressive legislation in this respect in some states has contributed much towards a rational solution of the problem.. The pollution of rivers by sewage is prohibited in some states, exception being made only in the case of rivers already much polluted before enter- ing the boundaries of the state. It is unnecessary to add proof of the growing realiza- tion of the importance of proper sewage-disposal, and of the awakened public interest in the matter, as ex- pressed in recent legislation. The work is, nevertheless, still in its very beginning. The source, composition and quantity of sewage.—City sewage consists of human and animal excreta, refuse from the kitchen and laundry, various manufacturing wastes, and the dust and dirt of streets and roads, dis- tributed in a greater or slighter quantity of water. Some cities provide a separate system for storm water, thus facilitating the purification of the sewage, when this is found necessary. For instance, in the city of Paris, where the sewage is utilized for irrigation purposes, a large portion of the water used for flushing the streets finds its way back to the Seine without previous puri- fication. The composition of sewage is variable. Variations in sewage of the same town occur at different periods in the day. The sewage that is produced in the forenoon and afternoon shows the influence of human activities in the greater volume and in the greater amount of Composition of Sewage 111 substances in suspension and in solution. At night, the volume of sewage is diminished and its composition like- wise modified. Different sewers in the same town may, likewise, show well-marked differences in the character of their sewage as affected by the density of population and its mode of life. The quantity of sewage produced in any town or city is affected by the water- supply, as well as by the nature and extent of their industries. From the sanitary standpoint, the composition of sewage is to be considered both chemically and bac- teriologically. The solid matter held in suspension and solution is potential food for bacteria and other micro- organisms. It may undergo putrefaction and become offensive to sight and smell and may lead to the destruc- tion of large quantities of fish in inland waters. From the bacteriological standpoint, it may act as a carrier of disease germs, and may be the cause of serious out- breaks of disease, even when diluted with a large volume of bacteriologically pure water. The chemical examination of sewage serves to indi- cate the extent of possible pollution when it is added to a given volume of surface water. It serves still another purpose when employed in connection with the various methods of sewage-purification. It becomes a means, then, for the gauging of the efficiency of these methods. The analyses performed at the different stages of every process show clearly to what extent the objectionable “materials are removed or rendered harmless. This ‘procedure finds an analogy in the bacteriological ex- aminations in which the numbers and kinds of certain groups of bacteria are determined. CHAPTER XIII BACTERIAL PURIFICATION OF SEWAGE Tue decomposition of manure and humus in the soil, the destruction of organic matter in surface waters, and its gradual disappearance in the cess-pool, filter- beds, or septic tanks, are all brought about by the same forces—the vital activities of bacteria. The uni- versal distribution of the microérganisms in the air, dust, water and soil, assures a speedy inoculation of all materials capable of undergoing decay. Over and above these sources of inoculation, sewage contains enor- mous numbers of bacteria derived from human excreta and kitchen wastes. It is no wonder, therefore, that sewage becomes foul so rapidly. Sewage-farms.—The purifying power of soil, due to its bacteria, was recognized in early times. Hence, when the purification of sewage was absolutely necessary for a number of cities, land treatment was one of the first means considered for such purification. Sewage-farms thus came into existence after the middle of the last century. Some of these have been successfully managed to this day. In many towns, however, there were no suitable areas of land available for this purpose and sewage-irrigation was entirely impracticable. Sewage tanks and filter beds.—It was discovered that (112) Sewage Tanks 113 large volumes of sewage can be exposed to bacterial activities in specially constructed tanks and filter beds, and the organic matter destroyed in a comparatively short time. This discovery, the outcome of widely scat- tered observations, has finally led to the evolution of the modern plants for the bacteriological purification of sewage. In September, 1881, a patent for ‘‘The Automatic and Odorless Scavenger’? was granted to Mouras in France. The American patent granted to Mouras is dated November 28, 1882. The patents were preceded by twenty years of practical experience, which left no doubt as to the remarkable efficiency of the process in destroying organic matter. The construction of the “Scavenger’’ was very simple. It consisted of an air- tight, hermetically sealed tank, supplied with a feed- pipe to receive evacuations, kitchen wastes, and the like, and an outlet in the upper part of the tank for the discharge of the sewage. Both of the pipes dipped under the surface of the liquid in the tank, which was completely filled with water before being placed in ser- vice. When anything was discharged into the feed-pipe, an equal volume of liquid was expelled from the tank. The liquid expelled contained disintergrated and largely decomposed material. From the inventor’s description of the process, it is evident that the organic matter introduced into the tank was destroyed by anaérobic bacteria. So rapid was the process of decomposition that the excreta was dissolved in eighteen days, while resistant substances, like paper, disappeared in a com- paratively ‘short time, with the formation of products H 114 Bacteria in Relation to Country Life largely gaseous in character. The Mouras “ Automatic Scavenger’? may thus be regarded as the predecessor of the modern “septic tank,” an important feature of all efficient sewage-purification plants. In 1895, Cameron, then City Surveyor of Exeter, England, introduced his so-called “septic tank” as an efficient medium for the treatment of sewage. The septic tank constructed by him consisted of a cemented pit, arched over, covered with sod, and provided with a vent for the gases generated from the decomposing materials. The crude sewage was introduced five feet below the surface of the liquid, so as to disturb it as little as possi- ble, and made to pass through the tank so slowly as to occupy about twenty-four hours in the process. In the absence of air and light, the anaérobic organisms soon attained an intense destructive activity. A leathery scum was observed to have formed on the surface, thus excluding the air still more effectively, while from the interior of the liquid, bubbles of gas arose in large vol- ume and the solid matter was constantly undergoing liquefaction. The sediment accumulated very slowly, being scarcely sufficient in amount to require removal at the end of a year. Subsequent experiments, conducted elsewhere, demonstrated that it is not even necessary to cover the septic tank, for the leathery scum at the surface is evidently adequate for the exclusion of air. On the whole, however, the covered tank is preferable to the open tank, as furnishing more uniform conditions for the undisturbed action of the bacteria. Progress in sewage-purification—In the period during which the septic tank was gradually evolved from the Septic Tanks 115 ‘ Mouras “ Automatic Scavenger,” bacteriological purifi- cation of sewage saw notable progress in other directions. The fact that sewage may be partly purified by filtra- tion through sand or other finely divided material, was a matter of common observation. Yet it was believed in the third quarter of the last century that the purifying effect of such filtration was entirely mechanical and due to the retention of the suspended matter by the filter. Chemical analyses soon showed, however, that the filtration process led to marked changes in the compo- sition of the sewage. The opinion was expressed that the finely divided material of the filter facilitated the union of atmospheric oxygen with the constituents of the sewage. At the beginning of the seventies, the study of micro- organisms had made sufficient progress, and the work of Pasteur had made an impression deep enough, to raise the question in the minds of some as to the relation of bacteria to the purification of water and sewage. In 1877, the French chemists, Schlésing and Mintz, demonstrated that nitrification is a biological process, thus confirming the opinion expressed some years previously by Alexander Miller in Germany. Other investigators demonstrated that the putrefaction of nitrogenous materials is brought about by microdégan- isms. The ground was thus prepared for a better under- standing of the changes that take place in sewage. Intermittent sewage - filtration—Practical experience with the land treatment of sewage demonstrated that the greater purifying effect, as well as the best plant growth, was secured on rather porous soils properly 116 Bacteria in Relation to Country Lije underdrained. On heavy, ill-drained soils, the sewage became foul and was purified but little. Moreover, even the lands adapted to sewage-farming could not be forced beyond a certain point in purifying the sewage applied. A proper supply of air was apparently neces- sary for the rapid and thorough purification of the liquid wastes, and the most satisfactory results were obtained when the land was worked intermittently, that is, allowed periods of rest and aération. With the discovery of the biological nature of nitrification, and the further demonstration by Schlésing and Mintz in France, and Warrington in England, that the nitrifying bacteria need a plentiful supply of air for their development, new light was thrown on the efficacy of intermittent filtration. The system of intermittent filtration is based pre- eminently on the action of aérobic bacteria. The Mouras scavenger and the septic tank depend for their efficiency on the work of anaérobic organisms. The earlier instal- lations of bacterial filters relied largely on the activities of the former class of organisms. The most satisfactory results were secured when the sewage had been previously subjected to screening and chemical precipitation. When this preliminary treatment was left out the filters showed a marked reduction in capacity, due to the deposition of solid materials around the grains of the filter. In other words, .the total pore-space between the solid particles of the filter was reduced on account of the accumulation of materials which the aérobic bacteria were evidently unable to destroy. On further examina- tion, these substances were found to consist of woody Aérobic and Anaérobic Agencies 117 fiber, like straw, chaff, paper and fragments of the wooden pavements. Here, then, is an important differ- ence between aérobic and anaérobic action. In the case of the latter, organic matter is almost completely de- stroyed in the septic tank with the formation of a very slight amount of sediment. In view of these facts, a sewage-purification plant that provides for the activities of both classes of bac- teria will allow more complete decomposition of the organic matter than can be accomplished by either the septic tank or the aérobic filter alone. Furthermore, the septic tank will dispense with the preliminary chemical treatment for the removal of the suspended matter, since the latter not only settles out in the tank, but is also liquefied by the anaérobic bacteria. Separation of bacterial activities—Thus the gradual development of sewage-purification methods, as based on bacterial activities, may be noted. The clearer un- derstanding of the chemical reactions caused by the various microérganisms and of the part played by at- mospheric oxygen in these reactions, made possible the differentiation of the aérobic and anaérobic changes. Following this differentiation came the septic tank, reserved for the activities of anaérobic germs alone, and the aérobic filters, reserved almost entirely for the aérobic bacteria. While in the contact beds the two processes were somewhat mixed, the conditions were so adjusted as to favor one or the other set of changes. In more recent installations, greater heed has been paid to the separation of the anaérobic and aérobic 118 Bacteria in Relation to Country Life activities, and the septic tank has been accorded a prominent réle in the purification of sewage. Temperature and bacterial activities—The low tem- perature of the winter months seriously retards bacterial activities. The rising temperatures of spring and summer stimulate these activities to such an extent as to permit the organisms to regain the lost ground. Some classes of bacteria are more susceptible than others to cold weather, yet all are retarded in their growth, as is proved by the diminished efficiency of the bacteriological pro- cesses in very cold weather. Warm countries offer favorable conditions for con- tinuous and intense bacteriological development, and permit the completion of the biological changes in a shorter period. Due consideration should always be given to the paramount influence of temperature in the biological purification of sewage (page 120). Hydrolysis.—The anaérobic changes that take place in the septic tank involve the breaking down of the complex nitrogenous substances known as proteins, or albuminoids. This transformation, which may be accom- plished by a large variety of anaérobic ferments, is known as hydrolysis. The nitrogen of the protein bodies is changed to a large extent into ammonia and other nitrogenous substances somewhat more complicated than the latter, yet simple in composition as compared with the proteins themselves. The non-nitrogenous substances, including the starches, sugars, and cellulose, are also decomposed and transformed, for the most part, into gaseous products. On account of the absence of atmospheric oxygen in the septic tank, these gases are The Effluent 119 still capable of comb’ning with oxygen. In other words, they are cotnbustible. Hence, the gases generated in the septic tank and consisting of nitrogen, carbon dioxid, marsh gas and hydrogen, are, in a number of places, usec for illumination purposes after the previous removal of the inert carbon dioxid. Treatment of the effluent—When the sewage is very strong, the effluent from the septic tank may contain a high proportion of ammonia and other decomposition products, and the concentration may be great enough to retard, for a time, the development of aérobic bacteria. With proper aération, however, and with dilution in extreme cases, the effluent may be subjected to further rapid change in the contact beds, or filters. Contact beds, when worked on the intermittent plan, do not exclude the activities of aérobic bacteria. The alternate filling, emptying and resting interfere, however, with the best development of either aérobic or anaérobic organisms. In the filters proper, on the contrary, ade- quate underdraining and intermittent working permit a better aération and higher aérobic efficiency. The latter is further enhanced by sprinkling the effluent over the entire surface of the filter. By reducing the amount of liquid passed through the filter, so as to admit a large volume of air, or by forcing air through the filter by some artificial means, the oxidation pro- cesses may be made quite intense. The nitrifying bac- teria become more aggressive under such conditions and change the ammonia in the septic-tank effluent into nitrites and nitrates in an incredibly short space of time. The destruction of the organic matter in the 120 Bacteria in Relation to Country Lije sewage is then almost complete and the effluent from the filter beds is non-putrescible, and not ‘injurious to fish. Temperature and filter efficiency—The efficiency of the aérobic filters, like that of the septic tank is affected by changes in temperature. The aérobic decay bacteria, and the nitrifying bacteria in the filters, multiply more and more slowly, and cause less and less chemical change as the, temperature falls. Still, their activity is not entirely suspended, even after a coating of ice has formed on the surface. It may not, however, be vigorous enough to produce the desired transformation. In order to remedy this retarded bacterial action in the winter months, it has been proposed to raise the temperature of the beds by artificial means. In some of the filters designed for this purpose, the filter beds are provided with steam-pipes. However, considerable expense is involved in such treatment—expense not always justified by the circumstances. It is well known that, among the aérobic as well as among the anaérobic bacteria, there are races that can grow at lower temperatures than others. This leads to a natural selection and the establishment of certain combinations of species under any given set of conditions. It follows, therefore, that the breaking down of the animal and vegetable materials in the septic tank is not necessarily accomplished by the same species in the different localities, or even in two different septic tanks. Inoculation.— Aside from the temperature, the estab- lishment of definite kinds of bacteria in the tank orin the beds is influenced by the composition and concentration Kinds of Germs in Beds 121 of the sewage and the kind of germs native to the water and soil. It thus comes about that the best results from septic tanks, or contact and filter beds, are not obtained until after a more or less prolonged period of preparation. In order to facilitate the process, septic tanks have been inoculated with sewage from old tanks in active opera- tion, and gratifying results have been secured thereby. Kinds of bacteria in filter beds and septic tanks.—While there is thus a natural variation in the kinds and pro- portions of bacteria occurring in different sewage- purification plants, and an accompanying variation in the amounts and composition of the products formed by them, it still remains true that certain definite groups of bacteria may be found in all septic tanks and filter beds. The septic tanks all contain rod-shaped, spore- forming cellulose ferments that can destroy woody tissue with the formation of the combustible gases, hydrogen and marsh gas. They also contain several kinds of rod- shaped putrefaction-bacteria, and a small proportion of spherical organisms. The contact and filter beds con- tain, among others, a number of species of small rod- shaped bacteria forming no spores and developing preferably in the presence of atmospheric oxygen. They contain, also, the nitrous and nitric ferments whose function it is to change ammonia into nitrites and ni- trates, as will be described more fully in the discussion of soil bacteria. Moreover, the bacterial beds likewise contain denitrifying bacteria—organisms that have the power to destroy the nitrates already formed. In the filter beds, a thorough aération and comparatively small proportion of ammonia may favor the activities of the 122 Bacteria in Relation to Country Life nitrifying bacteria. In the same filter beds, under different conditions, a less thorough aération and a larger supply of soluble organic matter, may encourage the growth of the denitrifying bacteria and the destruc- tion of the nitrates already formed. Loss of nitrogen.—The extent of denitrification, that is, the extent of destruction to which the nitrates are subjected, is naturally variable. The same may be said of the bacterial processes in the septic tank and beds, which involve losses of gaseous nitrogen in the course of putrefaction and decay occurring there. Economically, the differences in question are of some moment, especially when the effluents are used for irrigation purposes. It seems, therefore, that with the better understanding of the bacteriological changes in sewage-purification, means will be found to avoid unnecessary losses of nitrogen without detracting from the efficiency of the process. Nitrogen-fixing bacteria have also been found in ‘the bacteria-beds, and actual gains of combined nitrogen observed. Information on this point, however, is very meager. Bacterial efficiency in sewage -purification—The ca- pacity of bacteria-beds for the purification of sewage depends on the numbers and kinds of bacteria present there, as well as on the vigor of the organisms. But the bacterial work accomplished in the filter beds is by no means determined by the numbers alone. It may readily happen that 1,000,000 bacteria in one filter bed will perform as much work as 2,000,000 or 3,000,000 of the same kind in another bed. The difference is in vigor, or, to use a more exact term, in physiological efficiency. Physiological Efficiency 123 Climatic conditions and the methods of construction of filter beds are, therefore, important in so far as they affect the numbers and physiological efficiency of the bacteria. It will be readily perceived, then, that in tropical and sub-tropical countries, the work of bacterial decom- position is intensified by the more rapid multiplication of the organisms. Furthermore, conditions favorable for the rapid multiplication of bacteria are also favorable for the development of a high degree of physiological efficiency. On the other hand, cold weather retards the multiplication of the organisms and also reacts unfavor- ably on their physiological efficiency. As an illustration of this are the observations made in Massachusetts in the year following the very severe winter of 1903-1904. When cold weather set in, the filters showed a marked falling off in their efficiency and did not fully recover during the following year. Factory wastes and bacterial efficiency.—The numbers and physiological efficiency of the organisms may be affected by factors other than temperature and aération. In industrial centers where the factory wastes are added to the sewage, a favorable effect may be exerted on the sewage bacteria in occasional cases. More frequently, however, such factory wastes are decidedly injurious, and may seriously interfere with the proper working of the bacterial filters. The use of acids in many industrial processes leads to the production of acid sewage. When the volume of such sewage forms a considerable proportion of the total, the bacteria which are very sensitive to even a 124 Bacteria in Relation to Country Life slight excess of acid in their medium, may be injured or entirely destroyed. Injury may also be caused by other substances not necessarily acid. Thus, the ammoniacal spent liquors from gas works and coke ovens, when forming 1 per cent of the total volume of sewage, ap- parently do not prevent or retard the purification pro- cesses in the bacterial beds. When, however, the spent liquors form as much as 3 per cent of the entire volume of sewage, the injury to the bacteria is at once apparent. The working capacity of bacterial filters—There are natural variations in the working capacity of filters of different construction and location. . Apart from the thorough digestion in the septic tank and the skilful construction of the filters, the high efficiency in some cases may be ascribed to the favorable temperatures that prevail throughout the year. CHAPTER XIV SEWAGE-IRRIGATION THE application of sewage to the land may be prompted by economic or sanitary considerations, or by both. In countries of slight rainfall, sewage possesses a certain value entirely apart from the plant-food it may contain, since it may be advantageously employed for the sake of its water alone. The arid and semi-arid lands of the West yield profitable returns from sewage- irrigation for this, if for no other reason. Even in regions of more abundant rainfall, the application of sewage to light, sandy soils with small capacity for retaining water, is very beneficial. Soils of this type need large and frequent applications of water for the production of maximum crops, even when plant-food is abundant. On the sandy soils of southern New Jersey, irrigation has been found to increase the yields notwithstanding the forty-five to fifty inches of annual rainfall. Economic value of sewage-irrigation.—Sewage pos- sesses a still further interest, economically, on account of the plant-food constituents contained in it. The nitrogen, phosphoric acid, potash, lime and other plant- food removed by the crops from the soil are carried in part to the city to be discharged ultimately into the sewers and thence to the sea. The land is thus gradually (125) 126 Bacteria in Relation to Country Life deprived of its fertility, and loses, in time, its power to produce profitable harvests when none of the fertility is restored. It has been estimated by Crookes that England alone wastes in the sewage and drainage of her cities, nitrogen to the value of $80,000,000 a year. It has been computed, also, that the conversion of 90 per cent of the nitrogen in the sewage into nitrates, and their utilization, would add $70,000,000 annually to the wealth of England. We see, thus, that the value of combined nitrogen and of other plant-food drained away from the cities, towns and villages, must be truly enormous. Hilgard states that nearly 5,000,000,000 tons of mineral matter in solution are annually removed by the rivers from the earth’s surface, and that the amount of sediment simi- larly carried away is much greater. It is safe to assume that the drainage from human habitations forms an appreciable portion of the substances thus constantly added to the sea. Knowledge of these facts has naturally encouraged attempts to utilize the manurial ingredients of sewage for crop-production. Efforts have not been wanting to encourage the utilization of the sludge obtained by treating sewage with lime, alumina, or salts of iron. How- ever, the fertilizers manufactured by these processes did not meet with favor among farmers, and the cost of their preparation rendered profitable production diffi- cult. From time to time, enthusiasts still appear who would, in one way or another, extract the valuable fertilizer constituents from sewage. But, while the extraction of the manurial constituents Manurial Value of Sewage 127 of sewage by chemical means has been found unprofitable on account of the slight concentration of the liquid their utilization for crop growth is occasionally found profitable on sewage-farms. Profitable sewage-farming is, however, exceptional. By far the greatest number of sewage- farms do not yield a profit. Especially is this the case when the initial outlay for land and equipment is in- cluded in the charges. The failure of sewage-farms to return a profit is not difficult to understand if we remem- ber that the land near large cities is very costly, that large areas are required for sewage treatment, and that the range of crops grown is frequently limited. More- over, the proportion of manurial constituents in sewage is, after all, so slight as to make the application of very large quantities necessary in order that an adequate supply of plant-food may be furnished to the soil. At a generous estimate, English sewage may be allowed a value of three or four cents per ton; while American sewage cannot be valued at much more than a cent per ton on the basis of its manurial ingredients. “‘ As Profes- sor Anderson suggested long ago,” says Storer, ‘‘it would be about as reasonable to expect the farmers to manure their land with the smoke of cities as with sewage; for, as every one knows, enormous quantities of ammonia must be lost in the aggregate from cities where domestic fires are fed with soft coal. But precisely as it is with the smoke, so it is with sewage; that is to say, the fluid is so very dilute that it cannot be put to use.” Sanitary value of sewage-irrigation.—While sewage- irrigation has little to recommend it from the economic’ 128 Bacteria in Relation to Country Life standpoint, as indicated by the facts just noted, more may be said in its favor from the sanitary standpoint. The soil readily retains a large portion of the materials suspended or dissolved in the sewage, and, under favor- able conditions, purifies it to such an extent as to make the effluent resemble good drinking-water. The sub- stances retained in the soil are rapidly decomposed by the bacteria and rendered harmless, while the plant-food contained in them is made available. The purifying power of the soil was well known, of course, in ancient times, and utilized to a large extent in protecting the health of man. With the growth of cities in the last century, and the establishment of sewage systems, the large streams became the receptacles of much sewage until the resulting serious pollution called forth protests and led to remedial legislation. Many communities were thus compelled by law to purify their sewage before discharging it into surface waters. They turned to land treatment as a convenient method for their purpose. Kinds of irrigation—The application of sewage to the land is designated as broad irrigation when the liquid is distributed over a large area in order to promote the growth of some cultivated crop. It is designated as intermittent irrigation when much larger quantities are applied, at frequent.intervals, to open, well-under- drained soil. In this case, the land may be seeded and cultivated, or left uncropped. When the volume of sewage is large, and the area of soil available for broad irrigation limited, the two methods may be combined, giving rise to the mized system of irrigation. Soils and Sewage 129 The early experience with sewage-irrigation soon taught that soils differ strikingly in their ability to effect purification. It was also noted in those days that the purifying power of peat and heavy clay soils was rather limited. We readily see now why this should have been so, for the acid character of the peat retards the growth of decay bacteria, and, more particularly, of nitrifying bacteria. The retarding action in the heavy clay soils is due, on the other hand, to their compact nature and improper aération, which is a serious hindrance to the aérobic nitrifying ferments. The best results in the purification of sewage were obtained with light sandy soils underlaid by a porous gravelly subsoil, and pro- vided with a sufficient amount of lime. Additions of the latter to the land were found to intensify its purifying power. Broad irrigation.—The amount of sewage that may be successfully treated by broad irrigation is limited by the area available. Land to which too much sewage is applied, ceases to purify it. It becomes wet and foul; the aérobic bacteria, whose function it is to accomplish much of the purification, are crowded out, and the sewage runs off unpurified. Matters are made worse even in suitable soils, by the deposition at the surface of various organic materials. These form a felt-like layer and tend to keep out the air. This circumstance necessitates the occasional stirring of the surface soil, as well as the limiting of the amounts applied per given area within a limited time. It is estimated that one acre of land should not receive the sewage from more than one hundred persons. This is, of course, only an arbitrary measure, I ® 130 Bacteria in Relation to Country Lije for the character of the land and of the sewage, as well as the climatic conditions, necessarily play here a pre- dominating part. The effectiveness of broad irrigation in sewage- disposal is further affected. by the constant supply of sewage, irrespective of the season or the needs of the crops. It should be remembered that in the summer there is a rapid evaporation of moisture, not only from the soil, but also a transpiration of water from the foliage of growing plants. The pumping action of the latter is extremely important in quickly disposing of excessive moisture, since it is estimated that about three hundred tons of water must be transpired through the foliage in the production of one ton of dry matter. Moreover, the summer temperatures stimulate the ac- tivities of the soil bacteria and make possible thereby a rapid decomposition of the organic matter. In the winter months, on the other hand, the evaporation of water directly from the soil is greatly reduced, the removal of moisture by transpiration entirely dis- continued, and the decomposition of the organic matter markedly retarded. It frequently happens, thus, that offensive conditions are created in the vicinity of sewage- irrigated farms in the fall and winter months, even when no cause for complaint exists in the summer. Intermittent and mixed irrigation.—In intermittent irrigation, the business of crop-production becomes of secondary moment. The purpose sought here is to secure the greatest efficiency for any given soil area in the purification of sewage. Porosity, aération, and a vigorous bacterial flora are the main desiderata here. The land Kinds of Crops Under Sewage 131 is prepared carefully by thorough underdrainage, and the influences injurious to intense bacterial activity are eliminated as far as possible. The treatment of sewage by intermittent irrigation is, therefore, much akin to its treatment in bacterial filters. Satisfactory results from sewage-irrigation are se- cured when broad and intermittent irrigation are com- bined. A portion of the land, properly prepared and underdrained, is employed for intermittent irrigation when the sewage cannot be used to advantage on the growing crops. In this manner, the land is not made to receive more sewage than it can readily purify. More- over, the entire process becomes, in a way, intermittent, since the application of sewage is not too frequent, nor too severe to permit adequate aération. The crops grown on sewage-jarms.—These must be capable of transpiring large quantities of water, and must otherwise be adapted to the soil conditions. Italian rye grass has been grown extensively on the sewage- farms in England and Scotland. It grows very rapidly, crowds out weeds, and yields several heavy cuttings in one season. It requires reseeding every three years, although usually it is succeeded at the end of that time by other crops, like mangolds or cabbages. On some of the irrigated meadows the rye grass has been replaced ‘by a mixture of native grasses which likewise produce heavy yields of dry matter. Alfalfa has also been grown successfully on sewage-irrigated lands near Paris and in our western states. Like the Italian rye grass, it trans- pires enormous quantities of water. On the whole, how- ever, leguminous crops are not adapted for sewage-farms. 132 Bacteria in Relation to Country Life Preliminary treatment of sewage for sewage-jarms.— The clogging of the surface soil by the suspended matter in the sewage has led, in some places, to its preliminary treatment with chemicals. The suspended solids are precipitated by means of lime, or salts of alumina and iron, and the resulting deposit (sludge) is removed. The clear liquid still contains a considerable amount of organic matter in solution. When allowed to stand, it undergoes decomposition and gives rise to the same offensive conditions created by the untreated sewage. When applied to the land, however, it decomposes more readily, does not clog the soil to such an extent, and is evidently more suited than untreated sewage to promote the activities of the nitrifying bacteria. This is par- ticularly true of the sewage that has been clarified by lime, for the latter promotes the desirable changes. The advantages of preliminary treatment, aside from those already mentioned, include the greater capacity of any given area for sewage-purification. For instance, the official regulations in England required one acre of gravelly loam soil for every one hundred persons when the sewage was untreated, but allowed one acre for every four hundred persons when prelimi- nary treatment was carried out. The favorable influence of the preliminary treatment of the decomposition of the sewage in the soil is offset by the accumulation of large quantities of sludge that results. When left to ‘itself, the sludge does not dry rapidly, but undergoes putrefaction and creates a nuisance. It must be disposed of in one way or another, thus involving considerable expense to the community. Healthfulness of Sewage- Farms 133 Objections to sewage-farming.—Objections have been raised against sewage-farming on account of the possible dangers to public health arising therefrom. It has been urged that the dust particles and tiny drops of moisture carried away by the wind from the sewage-irrigated land may contain disease germs that may be thus brought to the city. It has been urged likewise that the disease germs may be carried away from the irrigated land by flies and other insects that frequent it. It has been asserted, furthermore, that there is great danger in using the vegetables and other products raised on sewage- farms, because the germs in the sewage readily cling to the leaves, stems and roots of the plants. The cows pasturing on the meadows, or consuming the grass and root crops from the irrigated land aré liable to come in contact with the disease bacteria. Actual experience in sewage-irrigated districts has failed, however, to confirm these fears. The very considerable number of gardeners on the sewage-farms near Paris and Berlin show no greater amount of disease than the people in the city. The grass from the sewage- irrigated meadows near. Edinburgh have been used for many years in large dairies as well as by owners of single cows, yet there is no record of any outbreaks of sickness that could be directly attributed to the consumption of such crops. Individual cases of sickness may, how- ever, have thus arisen in the past. If any serious danger at all exists in this direction, it is the danger that a portion of the sewage will escape unpurified into wells or surface water used for drinking purposes, a danger which sewage-irrigation shares with other methods. 134 Bacteria in Relation to Country Life Sanitary efficiency of sewage-purification.—The various methods of biological treatment of sewage are, to a great extent, effective, in that they destroy the organic matter and produce a non-putrescible effluent. Purified effluents of this character do not seem to affect injuriously the potable qualities of the surface waters to which they are added. Instances are even recorded in which they actually improved the water into which they were discharged. The water that drains away from the sewage-irrigated lands near Paris is clear and sparkling. It is used for drinking and fish thrive in it. When sewage-purification is considered as a means for the destruction of the disease germs contained in it, there is no certainty of its absolute reliability. In this case it is not so much the number as the kind of bacteria that survive the purification process, that is of impor- tance. Much work has been done in the study of this problem, but the results are somewhat conflicting. It has been shown that the growth of the sewage bacteria is inimical to the survival of typhoid germs. Cultures of the latter, introduced into unsterilized sewage, tended to dis- appear rapidly, and only an occasional individual survived. In order to eliminate any possible contamination of drinking-water by the disease germs, it has been pro- posed to treat the effluents with what Rideal calls Jinishers, that is, substances that will effect the steriliza- tion when added to the purified sewage at the rate of a few grains per gallon. Ozone could probably be used for the same purpose, as recent experiments indicate. It remains to be demonstrated whether large volumes of effluents can be thus treated effectively and economically. PART IV BaActTERIA IN RELATION TO Sor FERTILITY CHAPTER XV 5 NUMBER AND DISTRIBUTION OF BACTERIA IN THE SOIL THERE are species of bacteria so common in cultivated soils as to constitute a definite bacterial flora. This flora may vary with climatic conditions, the composition of the soil, and the methods of tillage and cropping. How- ever, it shows fairly constant characteristics. By agi- tating a small quantity of fresh soil with some sterile water, a turbid liquid, in which the bacteria remain in suspension for a considerable length of time, is obtained. When placed under the microscope, a drop of this liquid will be found to contain not merely a large number of microérganisms, but, also, numerous species, as indicated by differences in shape and size. Rod-shaped, spherical, spiral and boat-shaped forms may be distinguished among them without great difficulty. The rod-shaped organisms will be present in by far the greatest pro- portion. As in the case of water and sewage, some of the organisms are endowed with the power of motion and others are devoid of it. There will also be spore- (135) 136 Bacteria in Relation to Country Life forming and non-spore-forming organisms, and aérobic and anaérobic bacteria. But whatever interest is attached to the size and shape of the soil bacteria as viewed under the micro- . Fig. 18. Colonies of soil bacteria. scope, it must be remembered that there is reserved for them a certain task, upon the proper performance of which depends the well-being of more highly organized creatures. They are the connecting link between the Numberless Soil Organisms 137 world of the living and the world of the dead. They are the great scavengers intrusted with restoring to circu- lation the carbon, nitrogen, hydrogen, sulfur, and other elements held fast in the dead bodies of plants and animals. Without them, dead bodies would accumulate, and the kingdom of the living would be replaced by the kingdom of the dead. And yet the soil bacteria are not mere destroyers, for there are among them species that do constructive work, also indispensable. Number of bacteria in soil—The number of bacteria in arable soils is large, ranging from several hundreds of thousands to several millions per gram of soil (about vg of an ounce). In dry, sandy soils, very poor in humus, their numbers may be low, scarcely more than a few thousands per gram. In rich loam soils, they may reach the enormous total of fifteen to twenty millions per gram. In soils polluted with sewage, the number of bacteria may, at times, exceed one hundred millions per gram. There is a very intimate relation between the mois- ture content of the soil and the number of its bacterial inhabitants. Periods of rainfall are followed by a very marked increase in the number of soil bacteria. Periods of drought are-inimical to their development. The temperature of the soil has also a very direct relation to the number of its bacteria. Like higher plants, they need a certain degree of warmth for the manifestation of their vital activities. They cannot grow in the frozen earth, and they must wait for moisture and warmth to wake them up and to stir them into activity. With the coming of spring and of the longer hours of sunshine, the water moves more freely through the 138 Bacteria in Relation to Country Life soil; the rock particles give up some of their constituents which serve as nourishment for the soil bacteria, as well as for the higher plants. The microdrganisms, under such conditions, grow in numbers and vigor. Fig. 19. Colonies of soil bacteria. It is for this reason that in the warm summer months the moist soil is in slighter need of additional fertiliza- tion. The chemical and bacterial activities are then intense enough to provide for an adequate supply of Bacteria in Different Soils 139 food for the growing crop. On the other hand, the colder months of early spring do not provide the best con- ditions for the rapid transformation of plant-food im the soil. There is a certain relation between the character of the soil and the numbers and kinds of bacteria growing in it. Sandy and sandy loam soils allow the air to pene- trate them rather freely. Thus the development of the so-called aérobic species, that is, the kinds of bacteria that will not grow when the supply of air is excluded or limited, is favored. ‘Heavy clay soils and clay loams dc not permit the air to circulate freely in them. For this reason the-aérobic bacteria do not find in them the best conditions for their development. However, the anaéro- bic species, that is, the kinds that grow by preference when the supply of air is cut off or limited, are favored in their growth. It happens thus that the proportion of the aérobic and anaérobic species is not the same in the two classes of soil. The altitude of the field is of some importance in determining the numbers and kinds of its bacteria. The distance above sea-level affects the pressure and, therefore, the circulation of the air in the soil. The bacteria in the soil may also be affected to some extent by the character of the prevailing winds and the expos- ure of the land. Mountain slopes and hill-sides turned to the south offer different conditions for the growth of soil bacteria than those offered by northern exposures. There exists unquestionably an important relation between the crop on the soil and the numbers and kinds of bacteria within it. For one thing, the leafy crops that 140 Bacteria in Relation to Country Life shade the soil create conditions as to moisture, tem- perature and light that are different from those created by cereal crops. The differences do not, by any means, stop there. The crops take plant-food and moisture from ee Fig. 20. Colonies of soil bacteria. the soil and give back to it some of their substance, something that passes out of the roots and into the soil. Our knowledge of the amount and nature of the substan- stances thus given up to the soil by the plants is meager. It is not known as yet to what extent these secretions Bacteria and Crop Rotation 141 influence the numbers and kinds of bacteria in the soil. There is reason to believe, however, that a decided in- fluence is thus exerted by the growing crops. These affect the growth of soil bacteria in still another way. It is a well-known fact that different crops do not take out of the store of available constituents in the soil the same amounts and proportions of plant-food. For this reason, they affect the composition of the soil to an unequal extent and unequally change the numbers and character of the soil bacteria. The effects of differ- ent systems of cropping are clearly distinguishable both in the size and quality of the harvests and in the endur- ance of soil fertility. The pernicious effects of the con- tinuous growing of cereals were noted generations ago, and led gradually to the introduction of rotation systems. It seems that the evil effects of continuous grain-growing are due in part to the one-sided and wasteful changes in the soil-humus caused by bacteria. On the other hand, a succession of different crops, including members of the legume family, creates conditions favoring an economical transformation of the soil-humus. It will thus be seen that there is a direct relation between the crops on the soil and the bacteria in the soil. The number of soil bacteria varies likewise with the methods of tillage. All of the mechanical operations, such as plowing, harrowing, disking, hoeing, and rolling, which affect the evaporation from the soil, the penetra- tion of air, or the supply of moisture from the subsoil by capillary action, affect the rate of increase of the soil bacteria. The numbers of bacteria in the soil are readily affected by the application of manures and fertilizers as 142 Bacteria in Relation to Country Life well as by the turning under of green-manures. When any of these manurial substances are introduced into the soil, there are changes produced in its content of soluble salts, and, likewise, modifications in its moisture and aération conditions. An additional factor is introduced in the case of barnyard manure, since the latter is a material rich in bacteria. An application of several tons of manure per acre introduces into the soil many millions of bacteria, and not only adds thus to the num- bers already present there, but also influences the rate of subsequent increase. The kinds and numbers of bacteria in the soil bear also a certain relation to the processes of irrigation and drainage, to subsoiling, to the once prevalent practice of paring, burning, sanding, or claying, and, more par- ticularly, to the important processes of marling and liming. The application of lime, or of lime marl, exerts a far-reaching effect on the numbers and species rela- tionsh'ps of the soil bacteria. The effect of such appli- cation may be observed for years in the size and quality of the crops grown. Distribution of bacteria in the soil.—After the middle of the last century, when the universal presence of bac- ter a began to be more widely recognized, bacteriologists turned their attention to the soil as a breeding-place for various microérganisms. Miquel in France showed in 1879 that the soil, at a depth of several inches below the surface, may contain very large numbers of bacteria. Several German investigators who studied the subject in the eighties of the last century confirmed Miquel’s results. It was demonstrated by them that the greatest Bacteria at Different Depths 143 numbers of bacteria are present, not immediately at the surface, but at a slight distance below it. It was also shown that the number diminishes rapidly as the dis- tance from the surface is increased. The smaller number of bacteria at the surface, as com- pared with that four or five inches below, is due, largely, to less favorable conditions in regard to moisture and organic matter. Beyond the zone of root-development, the decrease of humus naturally involves a rapid de- cline in the number of bacteria. Moreover, the air is not renewed as readily at greater depths from the sur- face, and the aérobic organisms find conditions there unfavorable for survival. There is a tendency, of course, for the bacteria to be carried to the deeper soil layers by the rain-water percolating downward. Yet this tendency is checked by the filtering action of the soil, the organ- isms being held back in the fine pores of the latter. It is evident, likewise, that in the more open sandy soils the bacteria are not filtered out as rapidly, and are, therefore, scattered through a greater depth. CHAPTER XVI THE RELATIONS OF BACTERIA AND HUMUS Humus is defined as ‘decaying organic matter in the soil.”” It is the seat of all the important bacteriologi- cal activities for the very reason that it furnishes food and energy to the microérganisms. The bacteria are unlike the green plants in that they do not depend for their food on the roots, stubble and other remains of plants and animals. These organic remains are capable of furnishing nourishment and energy to the bacteria. In other words, they possess potential energy. It follows, therefore, that, everything else being equal, the greater the amount of humus in the soil, the greater the num- ber of its bacteria. The quantity of humus as affecting number of bacteria.— Besides furnishing food to the bacteria, the soil-humus also favors their development by providing better moisture and temperature conditions. Because of its great water-holding power, the humus enables the soil to retain greater quantities of moisture. The latter, in turn, provides for a more un:form temperature. It has been demonstrated by experiment that soils well pro- vided with humus do not become warm, or cool as quickly as do similar soils poor in humus. The application of barnyard manure multiplies the (144) Kinds of Humus and Bacteria 145 soil bacteria since it not only adds directly to their number, but also furnishes food for their further devel- opment. On the other hand, green-manures and organic fertilizers, like cottonseed meal, castor- pomace, or tankage, do not add as great ~% numbers of bacteria to the soil, and are chiefly valuable for the food they furnish to | Can, those already in the soil. Arid and semi-arid soils contain smaller -— proportions of humus than those found in humid soils. It would seem, therefore, 4 that aside from the moisture conditions in ™#:,21, Bact; the former, the two classes of soils must becllloa ‘ce show very considerable bacteriological dif- Sour 3'2608 ferences. ees Quality of humus as affecting number of bacteria.— The influence of the quality of humus on the soil bacteria is also important. The so-called mild humus, or mull, of arable soils, or of woodland, is different in its compo- sition from the raw humus of heaths, meadows, and swamps. It influences in an entirely different way the numbers and character of the bacteria. The differences observed are due largely to the origin and mode of formation of the two classes of humus substances. The mild humus is formed under conditions admitting the free access of air and through the activities largely of aérobic organisms. It is either neutral or alkaline in reaction. The raw humus is formed through the process of putrefaction rather than that of decay. It is acid in reaction, and is not a suitable medium for the develop- ment of most bacteria. It has been shown that peat J 146 Bacteria in Relation to-Country Life lands contain a relatively slight number of bacteria before they are reclaimed and placed under cultivation. When drained and limed, the number of bacteria soon increases from a few thousands to many millions per. gram of soi. BACTERIA AND THE DECOMPOSITION OF SOIL-HUMUS The dark-colored humus substances in the soil, the extensive deposits of peat in certain localities, and the beds of bituminous and anthracite coal have a common origin. They are all derived largely from atmospheric air. The atmosphere that surrounds our earth is a mixture of transparent gases, two of them, nitrogen and oxygen, being present in large proportions. A third, carbon dioxid, is present only in a small proportion,—three of four parts in ten thousand parts of dry air. It is believed that at an earlier period in the history of our earth the atmosphere contained a larger proportion of carbon dioxid. Owing to the latter, and to an abundance of moisture, the plants of that period grew more luxu- riously and accumulated an enormous amount of vege- table matter which was transformed, in the course of many centuries, into bituminous and anthracite coal. The growth of plants in our own day, while not so luxuriant as that in the carboniferous era, still takes place in accordance with the same laws. The colorless gas, carbon dioxid, is decomposed by green plants into its constituent parts, carbon and oxygen. The carbon is utilized by the plants for the building of their tissues. Restoration in Nature 147 It thus comes to pass that plants lock up in their body- substance enormous amounts of carbon combined with other elements, chiefly oxygen, hydrogen, and, to a smaller extent, nitrogen. When the plants die and mingle with the soil, their carbon becomes a part of the latter as soil-humus. Hence, soil-humus, the decay- ing remains of plants, owes its existence to the carbon and nitrogen of the air, as well as to the hydrogen and oxygen derived from the water-vapor of the atmosphere. But if the vegetation of forests, meadows, and prairies continued indefinitely to draw upon the com- paratively small amount of carbon dioxid in the air for their carbon, the time would come when the atmosphere would have but little left. The surface of the earth would become covered with vast accumulations of fallen trees and tangled herbs, and, in time, plant and animal life would cease because of the exhaustion of the carbon dioxid in the atmosphere. Fortunately, however, there is provision in nature for the restoration of this carbon dioxid. In the burning of wood and of coal, the carbon of these materials is changed again into carbon dioxid; in the respiration of animals the carbon of their food is changed to carbon dioxid, and, more important still, in all the processes of decay and putrefaction carbon dioxid is formed. Indeed, life could not persist without decay. The dead tissues must be- resolved into simple substances that new life may arise and find expression in an almost endless variety of material forms. The vastly important processes of decay and putre- faction are biological in character. They would not take 148 Bacteria in Relation to Country Life place in the absence of bacteria and of other microérgan- isms. The bacteria are thus the great scavengers of the living world, supplementing the work of the green plants. These are the builders of organic materials, the bacteria are the tireless destroyers. Under their attack the roots and the stubble of (fh a ew - WV 3 g cultivated crops, 2 ~4 the leaves and twigs 1 of forest trees, or j f e the bulk of barn- f yard and green-ma- * 6 \ , ures are changed ( J \ fa > slowly into dark- / colored humus sub- Fig. 22. Cellulose ferments, causing the breaking stances. Without down of woody tissue.—1, 2,and 3. Hydrogen . bacillus; x 2,000. (Omelianski.) 4, 5 and bacteria and other 6. Methane bacillus; X 2,000. (Omelianski.) microorganisms such changes would not take place at all, or else very slowly. It has been demonstrated time and again that soils sterilized by heat, or treated with antiseptic substances, like chloroform, carbon bisulfid, or carbolic acid, either cease to give off carbon dioxid, or yield only minute quantities of it. Similar soils, not sterilized, continue to form large amounts of this gas. Furthermore, soils or quantities of manure that have been sterilized begin to give off carbon dioxid in large quantity soon after they are inoculated either with pure cultures of certain bacteria, or with mixtures of several species. It has thus been established that the decomposition of vege- Decomposition of Woody Cell- Tissue 149 table and animal substances in the soil, and the return of the carbon dioxid to the air, are accomplished by bacteria. To them has been assigned the important task of main- taining the proper circulation of carbon in the living world, and, from the very beginning of organized life, they have unceasingly worked at their task. The rate at which the soil-humus decays is of vital concern to the farmer. Under certain conditions it Fig. 23. Strips of filter paper (woody fiber), showing gradual decomposition by cellulose ferments. (Omelianski.) vanishes from the soil quite rapidly, notwithstanding the utmost efforts of the farmer to enrich his soil. Large applications of animal manures and the turning under of green crops seem to leave no lasting effect. The soil remains light in color, and capable only of retaining a small fraction of the rainfall. Under other conditions, the soil does not seem to digest its humus properly. It becomes sour to an increasing extent, and its crops languish. The same manures that rapidly disappear from one soil accumulate in another. The. different results thus noted should be attributed to the micro- organisms of the soil. 150 Bacteria in Relation to Country Life The soils that allow a rapid disappearance of their’ humus are usually sandy or sandy loams, warm and well drained. The air circulates freely in them down to a considerable depth; the soil bacteria, particularly the aérobic species, develop in great numbers, and the carbon of the humus is returned to the air as carbon dioxid. Decay, under such conditions, is really slow burning, and the organic matter is, in time, reduced to a little heap of ash, as if it had been destroyed -by fire. The term ‘“‘eremakausis,’’ which means slow burning, has been applied to this process. It has been demonstrated by analyses of air contained in the soil that its oxygen is used up rapidly in this bacterial burning of the humus. Such soil-air has been found to contain as much as 9 per cent of carbon dioxid and only about 11 per cent of oxygen, instead of the 20.90 per cent of oxygen and .03 to .04 per cent of carbon dioxid found, on the average, in the air above the soil. The average of nineteen analyses of soil-air, made by von Fodor, showed 2.54 per cent of carbon dioxid and 18.33 per cent of oxygen; whereas, the air above the soil contained .04 per cent of carbon dioxid and 20.96 per cent of oxygen. , All this goes to show that not only is the carbon in the humus changed to carbon dioxid, but that this is accomplished at the expense of the oxygen in the soil- air. When the oxygen is thus used up rapidly, the de- composition of the humus is checked somewhat, and it is conceivable that soil-air deprived of all, or nearly all, of its oxygen will no longer supply the proper condi- tions for decay. It happens, however, that in the open Soil Conditions and Humus Decomposition 151 sandy soils the interchange of gases between soil and overlying air is rapid, the supply of oxygen is renewed without great difficulty, and the processes of decay go on uninterrupted. In fine-grained, compact soils, the conditions are different. These soils do not part as readily with the rain that falls upon them; their power of lifting water from the subsoil is greater, and a larger portion of their air-space is therefore occupied by water. Furthermore, because of their compactness, the air does not enter them or circulate in them as freely. Hence, the decay. processes in such soils are not so intense. In extreme cases, the humus accumulates more rapidly than it is destroyed. This is true of water-logged soils, of swamps and meadows, and also of upland moor and heath soils. In the presence of excessive moisture the aérobic bacteria are almost entirely suppressed, and the anaéro- bic species become prominent. The decomposition pro- cess then partakes of the nature of putrefaction, which does not involve as far-reaching destruction of the organic matter. The air being argely or entirely ex- cluded, the carbon of the humus is changed only partly to carbon dioxid, and that at the expense of the oxygen derived from decomposing vegetable matter. Another portion of the carbon passes off as marsh-gas, while.a third portion remains in the soil in the form of sour compounds—so-called humic acids. The humic acids and other substances formed with them are more or less antiseptic in character. Peat,-for instance, is a material that is resistant to decay even after it is re- moved from the swamp. The sour humus of upland 152 Bacteria in Relation to Country Lije moors and heaths is due not so much to absence of air as to absence of moisture. The place of the bacteria is taken, in this case, by various molds and fungi which, in their development, give rise to the formation of acid compounds. When the soil-moisture is neither excessive nor de- ficient, fine-grained soils, like those of coarser texture, allow the change of their carbon to carbon dioxid, but at a less rapid rate. For this reason, the disappearance of humus from such soils is comparatively slow, even when the land is frequently tilled. The effects of barn- yard manure, or of green-manure, may be observed years after the influence of similar treatment is no longer apparent on sandy soils. Taking it altogether, then, clay soils, or clay loams do not part with their humus as rapidly as do sandy soils or sandy loams. When they are very compact, the normal rate of decom- position is too slow for profitable crop-production, and’ resort is then had to drainage, liming, or manuring,— operations which serve, among other things, to admit more air into the soil. The soil bacteria are then per- mitted to develop more rapidly, and to cause the de- sired changes. In most soils under cultivation, the exhaustion of humus is rapid. To offset this loss, the soil is seeded down to grass from time to time, and receives, also, applications of barnyard manure. The undisturbed sod diminishes the aération of the soil and reduces the rapid oxidation of the humus. The latter is thus allowed to increase in quantity. Barnyard manure furnishes humus-forming material and serves to replenish the Manure and the Humus Supply 153 depleted stores. The roots and stubble added every year are a further addition to the soil-humus. Hence, the losses and gains bear a certain relation to one another. The crop residues and small or moderate applications of manure on heavy soils may be sufficient to maintain them in a satisfactory physical condition for many years. The supply of commercial fertilizers alone may prove adequate under such conditions for profitable crop-pro- duction. On the other hand, the crop residues and small applications of manure on sandy soils are not adequate in replenishing their humus. Humus-forming material must be applied in large amounts if the soil is to remain fertile. Hence, the practice of market-gardeners on such soils, of buying manures, or of raising green-manuring crops. It is because of the more rapid work of soil bacteria in the well-aérated, light soils that the latter have shown themselves capable of profitable cultivation under systems of green-manuring. They digest the organic matter rapidly and can, therefore, in a com- paratively short time, unlock the insoluble plant-food contained in it, and place it at the disposal of the grow- ing crop. As the fresh roots and stubble, turned under from the green crop, or the animal manures applied, are attacked by the soil bacteria, they begin to change rapidly at first, and more and more slowly as time goes on. From practical experience and from vegetation experiments it is known that these materials usually benefit the crop most in the first year after their application. In the second growing season the benefit is less pronounced, 154 Bacteria in Relation to Country Lije and in the third, still slighter. This diminishing effect from the application of the manures is due partly to the reduction of their plant-food by the first crop and partly to the gradually decreasing availability of the remaining portion. It seems that the bacteria first attack the substances they can destroy most readily. After these are used up, they cannot grow very fast on account of the greater difficulty in securing their food from the residues. In the course of time, only the most resistant portions of the soil-humus remain, portions that change so slowly as to prohibit normal crop growth. Chemical analysis shows that the carbon disappears from the humus at a more rapid rate than does the nitrogen. Hence, in old humus, a smaller proportion of carbon is found, and a larger proportion of nitrogen. In regions of scant rainfall, the proportion of humus in the soil is much smaller than that in humid regions. But the humus of arid sois is richer in nitrogen to such an extent as to be able to supply a sufficient amount of it to the growing -crops in the irrigated districts. CHAPTER XVII BACTERIA AND THE TRANSFORMATION OF SOIL-NITROGEN NITROGEN is one of the essential elements. There can be no life without it. It makes up nearly four-fifths of the gaseous envelope surrounding the earth, there being about thirty-five thousand tons of it over every acre of ground. Like the air above the ground, the soil atmosphere contains almost four-fifths nitrogen. This nitrogen, however, cannot ordinarily serve as food for plants. It must be combined with other elements in order to do so. The source of nitrogen in the soil—The crops growing on the land must depend for their supply of nitrogen on the humus, for this contains practically all of the com- bined nitrogen in the soil. There is but one exception to this,—the plants of the legume family. These possess the power of utilizing free nitrogen for their growth. But even they are deprived of this power in the absence of certain bacteria. Proportion of nitrogen in the soil—Cultivated soils contain, on the average, 0.1 to 0.2 per cent of nitrogen in the surface portion. Taking the average weight of an acre of ground to a depth of nine inches to be about 3,000,000 pounds, we find 3,000 to 6,000 pounds of combined (155) 156 Bacteria in Relation to Country Life nitrogen per acre to that depth. Smaller, but still con- siderable, quantities of nitrogen are found in the subsoil. It has been estimated that in some of our rich prairie soils there are present in every acre of ground 25,000 pounds of nitrogen to a depth of three or four feet. All this nitrogen exists in the humus in the decaying tissues of plants that once grew in the soil. The interesting question arises as to the origin of all this nitrogen. We know that the phosphorus and potash, as well as all the other mineral ingredients of plant-food, are derived from the rocks of which the soil is made. We know, also, that the rocks of the earth’s surface do not, as a rule, contain any compounds of nitrogen. We must conclude, therefore, that all of the combined nitrogen in our soils is derived from the nitro- gen in the air. Soil bacteria have played, throughout many ages, a predominating réle in the accumulation of humus-nitrogen. The activities of other soil bacteria, that concern the various changes that the humus- nitrogen undergoes, are varied and numerous. \ Nitrogen compounds in.the soil-humus.—The nitro- gen compounds in the soil-humus are complex. They are akin to the protein substances of food, and, as such, cannot be utilized by the crops. They must first be broken down and changed into simple substances,— ammonia, nitrites and nitrates. The breaking down of the nitrogen compounds in the humus is accomplished by bacteria and fungi in the soil. For them is reserved the task of providing building-material for the tissues of higher plants. These destructive activities of the soil bacteria mean so much to cultivated crops that it be- Loss and Availability of Nitrogen 157 comes proper to inquire whether the transformations due to them may not, at times, be wasteful. Loss of nitrogen in the soil_—Many facts recorded in agricultural science prove that under certain conditions the nitrogen removed by crops forms but a small por- tion of the total quantity lost from the soil. Investi- gations show that in the continuous cultivation of wheat the changes in the humus may be so wasteful as to con- stitute a most serious drain on the nitrogen resources of the soil. The losses thus occasioned bear a certain relation to the physical character of the soil, to its water- holding power, to its chemical composition, to the methods of tillage to which it is subjected, and to the crops grown upon it. The bacterial digestion of humus proceeds quite rapidly in the open sandy soils. The de- composition processes there may be so rapid as to pre- clude the accumulation in them of any considerable quantities of humus. Under such conditions, the organic nitrogen applied is changed more or less wastefully. Availability of nitrogen.—In assigning a certain degree of availability to nitrogenous substances from one source or another, the significant part played by the soil and its bacteria in the transformation of such sub- stances is frequently overlooked. Barnyard manure, green-manure, or tankage, that show a high rate of availability in one soil may prove much less available in another. For instance, out of every one hundred pounds of nitrogen applied in barnyard manure, the crops of one soil may recover thirty-five pounds in the first year, ten pounds in the second year, and five pounds in the third year. The same crops may recover from the 158 Bacteria in Relation to Country Life same manure in a different soil twenty pounds in the first year, five pounds in the second year, and two or three pounds in the third year. Evidently, the trans- formation would be more economical in the first instance; and the differences noted might be attributed to a greater proportionate loss from the soil, or the change of a greater proportion of the manure nitrogen to very inert com- binations. The losses responsible for a low rate of availability may be twofold. They may be due to the setting free of gaseous nitrogen in the course of decomposition, or they may be due to the leaching out of the soluble nitrates. In the first instance, there are conditions ex- tremely favorable to rapid decay, that is, a rapid union of the carbon and hydrogen of the humus with atmos- pheric oxygen. The humic nitrogen is not allowed to retain its hold on the hydrogen, as it would do when the processes of decay are more gradual; hence, it is forced to return to the atmosphere in the gaseous state. Rapid decomposition of this character can only occur in very open soils in which the aérobic bacteria may develop unhindered in the presence of sufficient moisture. The losses from the leaching of nitrates will depend again upon the amount of rainfall and the character of the crop. Excessive precipitation may wash the nitrates into the subsoil beyond the reach of the roots, and thus diminish the proportion of nitrogen available to the crop. Some crops possessing a deeper root system will forage more thoroughly in the soil, and will prevent large losses of nitrate. It is thus evident that the economical utilization of Varying Vigor in Bacteria 159 humus- or manure-nitrogen is affected by the character of the soil, by climatic conditions, and by the crop. The character of the soil bacteria is also important. The soil bacteria accomplish their work because of their vast numbers, but the amount of work accom- plished is not necessarily proportionate to their num- bers. The soil bacteriologists have come to realize, more and more, that bacteria, like higher plants, show differences in vigor and in their ability to survive the competition of other species. A cultivated field aban- doned to itself is soon overrun by weeds; a certain number of the latter become more prominent than others, and a characteristic flora is, in time, established. The nature of this flora is determined by soil and climate, and likewise by the adaptation of the predominant species to soil and climatic conditions. Something of the same nature holds good in regard to the bacteria of our soils. The natural methods of selection bring about not only a predominance of certain species under given conditions of soil and climate, but also endow these species with varying degrees of vigor which may find expression in the rate of increase or in the ability to form characteristic products. For instance, there are certain species of bacteria capable of fixing atmospheric nitrogen. Two strains of these bacteria may be apparently alike in every particular, one multi- plying as rapidly as the other. The amount of nitrogen fixed by these two may show considerable differences. We find analogous conditions when, by breeding or selection. a strain of wheat, corn or potatoes is developed that yields larger amounts of plant substance. In con- 160 Bacteria in Relation to Country Life sidering the work of soil bacteria, therefore, not only numbers must be considered, but, also, physiological efficiency. ; Conditions affecting availability of nitrogen.—In the decomposition of soil-humus and in the economy of its nitrogen transformation, it has been seen that soil and climatic conditions may modify the numbers, physiological efficiency and species relationship of the soil organisms. Certain conditions may not only favor the predominant development of certain species, but also a decided increase in the physiological efficiency of the latter. There is reason to believe that in the breaking down of the complex nitrogenous substances in the humus some species may occasion slighter losses than others. For this reason, the wasteful change of humus- nitrogen may be due to certain species rather than to others. Some of them are capable of causing intense oxidation processes; that is, extremely rapid decay, while others have but a feeble power in this direction. Hence, the greater losses of free nitrogen under certain conditions already noted. It seems highly important and desirable, in view of the facts just stated, that our knowledge of the various conditions of decay in the soil and of the manner in which the bacteriological transformation of humus- nitrogen is affected by soil, manuring, cultivation, and crop-rotations, be increased. Such increased knowledge would enable us to provide for a better conservation of soil-nitrogen, and would add greatly to the economy of crop-production. Bacteria as chemical agents.—The soil bacteria are Bacteria as Competitors with Crops 161 not destroyers only. To some extent, they are also builders, for any change caused by them.in the. soil- humus, whether it be ammonification, nitrification, or denitrification, involves necessarily their multiplication. Since, however, the bacterial bodies are complex in their structure, they add to the soil considerable quan- tities of highly organized materials derived from the simple decomposition products of humus. Soil bacteria, like higher plants, utilize for their growth the soluble mineral salts in the soil, particularly the soluble phos- phates and sulfates. They also make use of the am- monia, and, particularly, the nitrates derived from the humus for the building of their bodies. Indeed, certain bacterial species, which can rapidly transform large quantities of nitrate nitrogen into organic combinations, have been isolated from the soil. It may thus happen that the soil bacteria actually compete with the crop for the available nitrogen, and conditions probably exist in which the crop-yields are considerably reduced on account of the transformation of the soluble nitrogen compounds in the soil into the insoluble portions of the bacterial bodies. Soil bacteria are, then, the indispensable agents in the breaking down of the soil-humus, and in the supplying of simple com- pounds of nitrogen to higher plants. They may per- form this work economically or wastefully, depending upon soil and climatic conditions whereby their physio- logical efficiency becomes greater or less. By proper methods of cultivation, crop-rotation, or manuring, the bacteriological efficiency of these organisms can be controlled to advantage. The withdrawal of soluble K 162 Bacteria in Relation to Country Life plant-food from the soil-moisture by certain species to the disadvantage of the crop is an important factor in cultivation of the soil. Ammonification—Plants take up most of their nitrogen in the form of nitrates, which are simple sub- stances when compared with the humus from which they are drawn. According to our present knowledge, the change of humus-nitrogen into nitrate cannot be accomplished by a single kind of bacteria. There are at least three well-defined steps in this process of change, each accomplished by a different species. The first step in the transformation involves the production of am- monia, the second, the change of ammonia to nitrite, the third, the change of nitrite to nitrate. The last named is the final product of these combined activities which may serve as a splendid illustration of the speciali- zation in the work of soil bacteria. In the course of many ages there has been established this division of labor by the adaption of certain bacteria to only one kind of work. The microérganisms that can change protein nitrogen to ammonia cannot transform the latter to nitrite; nor can the nitrite bacteria change nitrite to nitrate. The formation of ammonia as the first step in the decomposition of protein substance is designated as “ammonification.” Ammonifying bacteria.—The bacteria capable of producing ammonia out of protein compounds are called ‘‘ammonifying bacteria.”’” There are many kinds of ammonifying bacteria. Some of them are aérobic others are anaérobic. Some can produce large amounts of ammonia in a given time, others but a slight amount ~ Ammonia Bacteria 163 of it. There are differences in the chemical processes by means of which the different species produce am- monia. The protein substances from which it is derived are complex and insoluble, and the bacteria cannot use them as food in this insoluble form. The bacteria are like higher plants in this respect, and can only draw their food from the dissolved materials in the medium in which they live. Fig. 24. Ammonifying bacteria—1. Bacterium mycoides; X 3,000. (Nadson.) 2. Baclerium mycoides; involution forms; X 3,000. (Nadson.) 3. Bacterium tumescens. (Myer.) 4. Proteus vulgaris; X 3,000. (Nadson.) 5. Proteus vulgaris; involution forms; X 3,000. (Nadson.) 164 Bacteria in Relation to Country Life Enzymes.—In order to hasten the solution of the protein, many bacteria produce chemical ferments called ‘enzymes.’ The latter are like the pepsin, or trypsin, produced for the same purpose in the animal system. The bacteria, like animals, must digest their food before they can assimilate it. However, animals digest their food after it is eaten, whereas the bacteria digest their food before it is eaten. In other words, the animals have special organs in which the processes of digestion are conducted, while the bacteria, as single cells, have no special digestive organs; hence, the soluble enzymes produced by them pass out through the cell- wall and cause a chemical change in the protein sub- stances that happen to be present in the material in which the bacteria are developing. The bacteria possessing the ability to produce en- zymes are known as ‘“‘peptonizing”’ organisms. Peptone is one of the products formed in the breaking down of protein by means of enzymes. It is soluble and much more simple in composition than protein, and is readily changed still further with the production of ammonia. Since the bacteria differ not only as to the amount, but, also, as to the quality of the enzymes produced by them, they must, necessarily, show marked differences in their ability to decompose protein substances. Mutual relations of bacteria.—The decomposition of: protein substances and the production of ammonia should not be regarded as the result of the independent activities of several kinds of bacteria. Living in the same soil with many other species, each kind is influenced by its neighbors. The struggle for existence develops a Mutual Relations of Different Species 165 combination of species designated the ‘‘bacterial flora.” adapted in each case to any particular set of soil and climatic conditions. In the com- petition for food, the various species in this combination hold one another in check and influ- ence, thereby, the degree and kind of the chemical changes produced. For in- stance, any one NY kind of anaérobic se bacteria living by it- if self will not develop \ under conditions \ where air is freely ™ admitted, because presence of free oxy- gen. When, however, aérobic species are living with them, they begin to grow and multiply as if no oxy- gen were present. This phenomenon is they cannot stand the N \ explained by the fact y 3 that the aérobic or- ganisms use up the 1 i 25. A if bacteria.—1. Pro- oxygen in the im- Fis. camer ee ee teus vulgaris; X 2 (Rodella.) 2. mediate vicinity of Bacillus megatherium; xX 2,600 i ie nel e800. chin their anaérobic neigh- pa ey ae i aerg pee . Bacillus 166 Bacteria in Relation to Country Life bors, and the latter are enabled thereby to grow normally. Denitrifying bacteria.—There are in the soil certain bacteria capable of breaking down nitrates and of returning their nitrogen to the air in the gaseous state. These organisms are called ‘‘denitrifying’”’ bacteria. There are other organisms in the soil which possess this power only in a limited degree. Some of them can reduce nitrates to nitrites, or to ammonia; others can reduce nitrites to nitrogen gas. But when two of these species are living together the destruction of the nitrates may be complete, since each performs part of the work the other cannot do. The combined work of the two or more species, which may be referred to as “associative action,” is seen, also, in the ammonification of humus- nitrogen. There are species that are incapable of pro- ducing ammonia in the soil, even in slight amounts, yet are capable of developing a marked ammonifying power in the presence of other organisms, or of influenc- ing favorably the ammonifying action of their neigh- bors. ’ Influence of bacteria on one another.—There is still much to be learned concerning the influence of the dif- ferent kinds of bacteria on one another. We are still ignorant of the manner in which the associative action is modified by climate and the mechanical and chemical constitution of the soil. In the case of ammonification, we know that the transformation of the humus-nitrogen may be rapid or slow, that it may be accompanied by large or slight losses of nitrogen in the gaseous state. We know, also, something of the conditions that hasten The Nitrifying Agencies 167 or retard such losses. We know next to nothing of the bacterial relationships under the changing conditions. Ammonifying power of soils —Attempts are being made now in soil-bacteriological laboratories to measure the ammonifying power of different soils under varying conditions of tillage and fertilization. Attention is being given, likewise, to the separation of certain species from their neighbors in different soils. To use the ex- pression employed in the bacteriological laboratories, pure cultures of the same species are made from different soils, and compared as to their vigor in the production of ammonia. That this work is of considerable impor- tance is evident from the fact that ammonification is an essential step in the transformation of soil-nitrogen. Soils that have but a feeble ammonifying power will not allow a healthy growth of crops, irrespective of the vigor of any of the other important soil bacteria. Proper and profitable plant development is dependent on the rapid and economical transformation of the humus-nitrogen; also, on the rapid and abundant supply of ammonia to the bacteria that change it into nitrites and nitrates. Nitrification —The processes involved in the change of nitrogen compounds are at least three (page 161): Ammonification, nitrification, and dentrification. Am- monification we have just discussed. The other two processes are so important that we shall consider each of them in a special chapter (XVIII, XIX). CHAPTER XVIII NITRIFICATION THE term nitrification is used to designate the change of humus-nitrogen, or of any nitrogen in vegetable and animal substances, into a nitrate. It is also used, at times, to designate the change of ammonia, or of nitrites, into nitrates. The first definition is broader and includes the work not only of the nitrifying bacteria proper, but, also, that of the ammonifying bacteria. A nitrate (NO3) contains one more atom of oxygen than does a nitrite (NOs). Until within a generation ago it was not known that nitrification is a bacterial process. The transformation of organic nitrogen into nitrate was regarded as a purely chemical reaction and was extensively studied from that standpoint. Important economic issues were in- volved in such studies. The matter was of lively con- cern, not only to agriculture, but, also, to military organizations. Since gunpowder is composed largely of saltpeter (nitrate of potassa, or potash), some pro- cess whereby this could be obtained was eagerly sought. Numerous methods were discovered and employed with various degrees of efficiency. The fact that saltpeter has its origin in organic sub- stances was suspected by at least a few chemists early in the eighteenth century. It was stated in 1717 that (168) Nitrification Due to Bacteria 169 this salt is present in vegetable and animal materials in a disguised state, and is set free when the substances decay. Notwithstanding this view, there were many chemists a hundred years later who believed that nitrates are formed by the oxidation of the gaseous nitrogen of the air in the soil, or in the compost heap. The studies conducted by Kuhlmann between 1825 and 1838 indicated that nitrates are not formed in the soil out of nitrogen gas, but out of ammonia. The ex- periments carried on by Boussingault between 1858 and 1871 showed that nitrates in the soil are formed at the expense of the humus-nitrogen and not out of nitrogen gas. It was thus demonstrated that nitrifi- cation in the soil, or in compost heaps, depends on the decomposition of the organic substances containing nitrogen and the formation of ammonia. Nevertheless, it was still believed that the process of nitrification was purely chemical in its nature and that the superior nitrifying powers of some soils were due to their content of certain compounds of lime, iron, and the like. True character of nitrification.—After the brilliant investigations of Pasteur, in the sixties of the last cen- tury, on the nature of fermentation, the ground was prepared for the understanding of the true character of nitrification. Indeed, in the early seventies, the belief ‘was expressed by at least one investigator that nitri- fication is of bacteriological origin. The proof for this assumption was furnished in 1877 by two French investigators, Schlésing and Mintz. In passing diluted sewage through glass tubes filled with soil, they found that after some days the liquid that had passed through 170 Bacteria in Relation to Country Life the soil had lost its ammonia, and had gained an equiva- lent quantity of nitrate. On treating the soil with chloro- form vapors, nitrification was discontinued and the ammonia in the sewage passed through unchanged. The process of nitrification was reéstablished after the soil had received fresh soil in which nitrification was active. Similarly, the nitrification of of ammonia in the soil was stopped by 4A o boiling. This showed that the change of es ammonia to nitrate was of bacterial oe %:. origin, since the process could be sus- Fie. 26. are pended by antiseptics and by boiling, bacteria-—1. and reéstablished by the addition of Nitrous fer- ments; 2,000. very minute quantities of fresh soil. Tents: x 2,000; These experiments were amply con- Stutzer.) firmed by other investigations. Pure culture of nitrifying bacteria.— With the bacterial nature of nitrification thus demonstrated, attempts were soon made to secure pure cultures of the nitrify- ing organisms. For a number of years such attempts were not crowned with success. To be sure, various investigators claimed to have detected the ability to produce nitrates in one or another of the soil bacteria studied by them. More careful examination showed, however, that, in all probability, they did not work with pure cultures. To Warrington belongs the credit of having isolated, after many years of study, organ- isms capable of changing ammonia to nitrites. He ob- served other organisms also capable of changing nitrites to nitrates. Warrington’s investigations indicated that the oxidation of ammonia occurs in two distinct stages. Isolation of Nitrogen Bacteria 171 The first reaction involves the change of ammonia to nitrite, the second change of a nitrite to a nitrate. The Russian bacteriologist, Winogradsky, not only confirmed Warrington’s observations, but, by a series of highly ingenious experiments, made clear the causes of the failure of other bacteriologists. He showed, in 1890 and 1891, that the nitrify- ing-organisms do not develop in the culture solutions and on the gela- tine plates em- ployed for the isolation of vari- ous soil bacteria. He was, therefore, led to employ cul- ture media con- Fig. 27. Nitrite bacteria, showing the aitieianl taining only min- form of individual cells. eral salts. The gelatin was replaced by the mineral silica jelly, on which colonies of the nitrifying bacteria developed without great difficulty. The organisms capable of changing ammonia to a nitrite were found by him in two varieties, one from the Old World, which he named Nitrosomonas, the other from the New World and named Nitrosococcus. Both of these belong to the group of nitrous ferments and are spherical in shape. The so-called nitric ferments, those capable of changing nitrites to nitrates, were designated by him as Nitrobacter. The latter are small, 172 Bacteria in Relation to Country Life rod-shaped organisms. The demonstration was thus furnished that the oxidation of ammonia to nitrites is performed by two distinct groups of organisms found in all soils. They seem to work in unison, since the nitrites are changed into nitrates almost as fast as they are formed. Under abnormal conditions favoring the growth of the nitrous ferment, but not of the nitric ferment, there may be an accumulation of nitrites. Recently it was announced by Kaserer that he had isolated an organism capable of changing ammonia directly into nitrate. This discovery, if substantiated by other investigators, promises important revelations concern- ing the transformation of nitrogenous organic materials in the soil. Importance of nitrification —The vast practical sig- nificance of nitrification processes is apparent from the fact that most of the nitrogen used by crops is taken up in the nitrate form. While there is much evi- dence at hand to show that many plants are capable of utilizing am- monia as readily as nitrate nitrogen, yet, because of the very rapid con- version of ammonia into nitrate, the Fig. 28. Nitrite bacteria, /atter is almost the exclusive source Foor tne ope, oof nitrogen. The rapidity with prepared azar. which ammonia salts are changed in the soil to nitrates is attested by the experience at Rothamsted in England. It was the practice there to apply the nitrogen on certain plots in the form of am- monium sulfate in the fall. It was soon noticed, how- Soil Conditions Affecting Nitrification 173 ever, that, notwithstanding the lateness of the season, the ammonia was rapidly converted into nitrate as shown by the increased content of the latter in the drainage- water from those plots. In fact, the application of the ammonia salts was practically equivalert to the appli- cation of nitrate, and, in order to ‘guard against the loss of nitrogen, the fall applications of ammonia salts were discontinued. Loss of nitrates in the soil.—The soil-nitrates all dissolve in water, diffuse themselves readily in the soil- moisture, and, when not taken up by the crops, are liable to be washed into the drains. This property of nitrates accounts for their presence in comparatively slight amounts in the soil. In certain rainless regions of the earth there are large accumulations of nitrate. This is particularly true of the extensive deposits of nitrate of soda in some provinces in Chile, South America. This salt, known also as Chile saltpeter, occurs in crystal form throughout a considerable thickness of solidified earth, and is secured from the latter by the crushing and leaching of the earthy material, and subsequent crystallization of the nitrate from these leachings. Sources of the nitrate deposits—Various theories have been proposed to account for these deposits. One of these theories, accepted by many, assumes that, in the gradual rising of the coast of western South Amer- ica, a portion of the sea was cut off, forming a great inland bay whose waters could be replenished by the ocean only at high tides. Immense quantities of sea- weed developed in this shallow bay, and, on their decay, were changed by the nitrifying bacteria into nitrate. 174 Bacteria in Relation to Country Life Owing to the warm climate, the decay of the organic matter must have proceeded very rapidly, while the absence of rain prevented the nitrate formed from being leached out and carried away in the- drainage of the country. The natives of Peru and Chile have known the agricultural value of the nitrate for a long time, and have probably used it to stimulate plant growth. The first ship-load of nitrate of soda was taken to England in 1827. Another was taken there in 1830, but there was scarcely any demand for it on account of the high price. As its value in agriculture and in the chemical industries was recognized, the demand for it increased rapidly until in 1903 it showed a consumption -of 1,429,150 tons, including the 264,000 tons brought to the United States. Considerable quantities of nitrates, particularly nitrate of potash, are also derived from other sources. After the rainy season, portions of the plains of the Ganges river in East India, become covered with a deposit of white crystals consisting largely of nitrate of potash. Evidently, the abundant moisture and favorable temperature encourage the vigorous development of the nitrifying bacteria, and a rapid production of the nitrates of lime, magnesia and potash. As the moisture brought up by capillary action from the subsoil is evaporated at the surface, the nitrates it contains are left behind in crystalline form. The nitrates of lime and mag- nesia have, however, a great affinity for water-vapor, which they absorb from the air, and do not, therefore, crystallize out to the same extent as the nitrate of potash. Commercial Nitrates 175 Early use of nitrates—The natives of India recog- nized long ago the stimulating action of nitrate in plant growth. It is stated that the natives of certain districts in Bengal, “particularly a caste called Quirees (hereditary gardeners), who cultivate the best lands and produce the best crops, are in the habit of irrigating their fields with water from wells so strongly impreg- nated with saltpeter and other salts as to be brackish. They consider onions, turnips, and peas to be most benefited by this irrigation.” Saltpeter is also believed to have been used as a manure by the peasants of Mantua, and is known to have been employed by Digby in Eng- land in the reign of Charles I. It was likewise recom- mended as a top-dressing by Evelyn in the reign of Charles II. Early in the nineteenth century, the use of saltpeter was slight, but not uncommon, in England, and a few decades later the application of Chile saltpeter was resorted to by many farmers to supplement the natural production of nitrates in the soil. Conditions influencing the formation of nitrates.—The rapidity with which nitrates are formed from the soil- humus is determined largely by soil and climatic con- ditions. The nitrifying bacteria must have sufficient moisture and a favorable temperature for their develop- ment. They must have the proper supply of humus as the source of nitrogen; they will not develop in the absence of lime or magnesia, which serve, in the average soil, to neutralize the nitric acid formed by the bacteria. The presence of lime and magnesia, or of other basic substances, as they are called, is of extreme importance. They combine with the nitric acid produced by the bac- 176 Bacteria in Relation to Country Life teria to form nitrates of lime, magnesia and, potash— substances not injurious to the bacteria even when present in the soil in considerable quantities. In the absence of basic substances, the nitric acid produced by the nitrifying ferments accumulates and reacts injuriously on the latter. Small amounts of nitric acid are sufficient to retard seriously the nitrification processes in the soil. The supply of air also exerts a direct influence on these processes. Nitrates are formed most rapidly in sandy loam soils well supplied with humus. On the other hand, heavy clay soils are too compact and fine-grained to allow satisfactory aération; hence they allow a gradual nitrification of their humus-nitrogen. In fairly open calcareous soils, the nitrification processes may proceed with great intensity, whereas, in coarse sandy soils they may be quite irregular on account of the rapid depletion of the soil-moisture. The process of nitrification in cultivated lands is in- fluenced by the chemical and mechanical composition of the soil as well as by the prevailing climatic conditions. We are indebted for much of our knowledge on the subject to the careful researches at Rothamsted, Eng- land. Many interesting facts contributed by these in- vestigators show that, among other things, the pro- duction of nitrates takes place almost exclusively in the surface soil. It was found at Rothamsted that drain gages situated at a depth of 40 inches and 60 inches respectively, yielded no more nitrates than did the drain gages situated at a depth of 20 inches. It is not difficult to account for this fact, since the nitrifying Physical Conditions Affecting Nitrification 177 bacteria will develop only where there is a supply of air and of nitrogenous material capable of being con- verted into nitrate. It seems, therefore, that in the more open sandy loams, or sandy soils, where the air circulates more freely at greater depths, the development of the nitri- fying bacteria is favored somewhat in the deeper layers of the soil. In regard to the material capable of nitri- fication, it is true that in the soil this consists of roots, stubble, and other plant residues accumulated mostly in the surface soil. For this reason, the nitrifying bacteria will grow almost exclusively in the place where these accumulations are concentrated, that is, in the surface soil. Indeed, it has been shown, experimentally, that samples of soil from depths greater than three feet usually fail to cause nitrification in nitrifiable materials. Soil bacteriologists are now endeavoring to compare the nitrifying power of different soils under identical conditions, and to study the various influences that encourage or discourage the bacteriological activities in the soil. The fact has been recognized for some time that not only are the nitrogenous materials nitrified at a different rate in different soils, but that the order of nitrification may be different. For instance, it has been observed that sulfate of ammonia is usually changed to nitrate more quickly than dried blood or cottonseed meal. Nevertheless, soils are occasionally met with in which the reverse seems to be true. According to the views held at present, the organic nitrogen in dried blood or cottonseed meal must be changed first to am- L 178 Bacteria in Relation to Country Life monia by ammonifying bacteria before it can be util- ized by the nitrous and nitric ferments. The nitrogen of ammonium sulfate can be attacked directly. It fol- lows from these facts that there must exist a certain relation between the ammonifying and nitrifying bac- teria in the soil, of which we know very little. The determination of the nitrates, at Rothamsted, in the drainage wastes from soils that had been kept fallow, showed an average annual removal of 40.2 pounds of nitrate nitrogen per acre. The least amount of nitrate nitrogen in the drainage waste was found in the spring, the greatest amount in July. In the soil which had borne crops, and received applications of nitrogenous materials, the production of nitrate bore a direct relation to the amount and character of the substances applied. The least amount of nitrates was found in the soil receiving annual applications of barn- yard manure at the rate of fourteen tons per acre. The soils receiving applications of nitrate of soda and of ammonium salts showed a higher content of nitrate than the corresponding unmanured soil. The differences may be due to two causes. In the first place, the soils receiving nitrogenous materials contain more nitrate because these substances contribute directly some nitrogen to the soil. The nitrogen thus added may be in the nitrate form, as in the case of the applications of nitrate of soda, or it may be in a form capable of more or less rapid conversion into nitrate, e.g., sulfate of ammonia, or barnyard manure. Not all of the nitrate nitrogen thus contributed is removed by the crops or in drainage. In the second place, the Acid Soils and Nitrification 179 crops growing on the manured land are more yigorous and leave greater amounts of root and stubble residue in the soil, hence, also, more nitrifiable material. It has been demonstrated that the nitrogen of organic matter is not all capable of nitrification to an equal extent. A portion will change to nitrate quite readily, the following successive portions less and less readily, until, finally, a stage is reached when the remaining nitrogen nitrifies with extreme difficulty. The great resistance to the activities of nitrifying bacteria is characteristic of the humus in exhausted soils. On the other hand, the nitrogen of ammonia salts, of liquid manure, or of such compounds as dried blood, nitrifies very rapidly. It has already been stated that the application of ammonia salts in the fall is wasteful because of its ready conversion into nitrate, even in the late fall. In fact, nitrification seems to go on until the soil is almost frozen. When a crop is occupying the land, the nitrate as it is formed is taken up by the plants and but little allowed to escape into the drains. When’ the land is kept bare, the nitrates formed are washed into the deeper layers of the soil and may be carried off by the drainage. It is for this reason partly that the continuous growing of wheat is a wasteful procedure. The land is kept bare at a time of the year when the nitrification processes in the soil are most active, resulting in the loss of very considerable quantities of nitrates. In- vestigations have shown that in the continuous growing of wheat there may be four to six pounds of nitrogen lost from the soil to every pound removed in the crop. 180 Bacteria in Relation to Country Life Acid soils—There is an intimate relation between the amount of lime and magnesia and the nitrifying power of the soil. It should be remembered that the activity of the organisms leads to the formation of acids; that is, of sour substances, and that in the absence of lime the acids accumulate and react injuriously on the bac- teria. The deleterious effects of acid substances may be. seen readily on light soils, naturally poor in lime, and upon which applications of sulfate of ammonia are made from time to time. A comparatively large amount of lime is required to neutralize the acids formed in the trans- formation of ammonia salts to nitrate. Because, also, of the severe drain on the lime resources of the soil, the acid conditions become more pronounced. In soils in which large amounts of nitrogen are being nitrified there is a continuous production of the nitrates of lime and magnesia, which are either taken up by the crops or removed in the drainage. It is well known, also, that soils yielding large amounts of nitrate, that is, those rich in humus, lose their lime in still other ways. One in par- ‘ticular is on account of the formation of bicarbonate of lime, which is soluble in the soil-water. It becomes necessary, therefore, on practically all but limestone soils, to apply lime from time to time, lest the soil become sour and the nitrification processes feeble. Difference in organisms.—When favorable conditions for nitrification exist, there comes to be established in time a vigorous combination of nitrifying organisms, capable of accomplishing much work in a short time. This circumstance accounts for the marked differences in the nitrifying power of bacteria from different soils. Availability in Various Substances 181 The increased vigor, due to long-continued development under favorable conditions, may, in a measure, become fixed in the bacteria. There are facts, at least, that point strongly in that direction. The significance of this circumstance will be discussed under soil-inoculation. Availability of nitrogenous materials.—The relation must play an important part in determining the relative availability of the various nitrogenous materials em- ployed for manuring purposes. Agricultural chemists and practical farmers know that substances like dried blood, meat meal and ground fish yield their nitrogen rather rapidly to the growing crop, while other substances like leather-meal, wool and peat are a very unsatis- factory source of nitrogen to plants. The first of these are designated as available, while the last are called unavailable, or difficultly available. Experience and investigation have arranged such nitrogenous substances in the order of their availability in somewhat the fol- lowing fashion: Nitrates. cisse gas eae eee eee eee 100 Ammonium sulfate ..........-...----22--0-55 90 Dried blood, horn meal, green clover.......---- 70 Fine bone meal, ground fish, meat meal ....... 60 Manin 3 eet: canine o cigieed be ses Sarena ds A ee 45 Wool waste.......---.- seb iSdebahdad Rcpenedi ad wa REST 30 Ground leather................--..-2---+++5- 20 This table brings out the existing relations in a general way. It should not be forgotten, however, that the absolute and relative availabilities are modified by soil and climatic conditions. The differences in availa- - bility as brought out in the table are determined largely 182 Bacteria in Relation to Country Life by bacteriological activities. Some of these substances are more resistant to the attacks of the organisms and are converted into ammonia and nitrate slowly, while others are changed rapidly. It may also be that in the process of ammonification and nitrification, gaseous nitrogen is set free, and that these losses are different with different substances. The available substances, that is, those that nitrify rapidly, are more like nitrate in their action than are the slowly available substances. Influence of the crop on availability—It has been demonstrated in the case of some plants that the crop may favor or retard nitrification, not only by with- drawing greater or slighter amounts of moisture from the soil, but, also, by modifying, however slightly, the chemical nature of the latter. It was thus shown, in the case of wild mustard, that the nitrifying action of the soil had been diminished, the effect showing also in the following season. An interesting field of inquiry is thus opened to us. Studies in this direction will undoubtedly prove of great value, and will help us to understand, perhaps, the beneficial effect of crop-rotations in so far as they concern the soil bacteria. CHAPTER XIX DENITRIFICATION DENITRIFICATION is the reverse of nitrification. The latter process has been defined as the gradual changing of the nitrogen of vegetable and animal substances (organic nitrogen) into nitrates. It is therefore, an oxidation process, involving the addition of oxygen to the nitrogen through the activities of the nitrifying bacteria. Denitrification is a reducing process whereby the nitrate is made to part with some or all of its oxygen, and is changed to a nitrite, to ammonia, or to nitrogen gas. A distinction should be drawn, therefore, between the complete destruction of nitrates with the formation of nitrogen gas, and the partial decomposition in which a nitrite or- ammonia is formed. The first instance, which: represents denitrification proper, is of much greater importance from the economic standpoint, since the nitrogen, once returned to the. air, is lost to the soil and crops. On the other hand, the reduction to nitrite, or ammonia, does not remove the nitrogen from the soil. It is still there, though in a.changed.form,. and may again be oxidized -to nitrate.. - Early idea of denitrification. —The earlier obiervers: who noted the reduction of nitrates ascribed.it to -re-.. actions purely chemical. In the sixtics of the last cen- (183) 184 Bacteria in Relation to Country Lije tury, a number of investigators were “Already familiar with the fact that such reductions of nitrate are likely to take place in the presence of organic matter. They were not a little puzzled at the apparently contradictory properties of soils. The fact that the reduction of nitrates in the soil is more likely to occur when the latter contains an ex- cess of moisture, or an excess of organic matter, was recognized by these men. They did not know, however, that microscopic organisms in the soil are intimately connected with the reduction processes. The German scientist, Schénbein, suggested in 1868 that the reduc- tion of nitrates might be due to fungi and bacteria. A few years later his views were confirmed by the ob- servations of others, particularly as to the reduction of nitrates in sewage and in drinking-water. The cause of denitrification.—These views found strong support in the studies of the French investigators, Gayon and Dupetit. They actually observed, at the beginning of the eighties, cultures of a bacillus, or “ferment,” as they called it, that was found capable of reducing nitrates with the production of nitrogen gas. In 1886, they described two denitrifying ferments that. they had isolated and studied under varying con- ditions. Cultures of a denitrifying bacillus were also prepared by Giltay and Aberson in Holland. The latter found that when grown in meat broth or other solutions containing nitrate, this organism is capable of reducing nitrate with the transformation of almost all of its nitrogen into gas. It was thus demonstrated that soil contains bacteria that cause reduction of nitrates, _ Significance of Denitrification 185 ‘Value of the modern discoveries.—No practical signifi- cance was attached to these discoveries until 1895, when the statement of Wagner, in Germany, that the use of barnyard manure leads frequently to serious losses’ of nitrogen from the soil recalled the attention of chemists and bacteriologists to the subject. It was held by Wagner that the application of manure and nitrate is a very wasteful practice, since the denitri- fying bacteria, present in large numbers in the manure, cause the destruction of the nitrate in the fertilizer. Furthermore, they reduce the nitrates formed from the soil-humus by the nitrifying bacteria. Wagner’s state- ment naturally aroused much comment and stimu- lated extensive investigations in Germany, England, France, the United States, and elsewhere. Modern conclusions concerning denitrification.—The conclusions as they were reached after five or six years of study may be summarized briefly as follows: It was demonstrated that under certain conditions the applications of large quantities of manure may really decrease the yields, in some cases to a very serious extent. The injury thus occasioned may be due to the direct action of the organic materials in the manure on the plants, or it may be bacteriological in character. As to the former source of injury, it should be remembered that excessive amounts of soluble organic materials like those in liquid manure may, of themselves, prove injurious to the plants. In ordinary field practice the amounts of manure applied are never large enough to cause such injury. In market-gardening, or in green- house work, in both of which applications of as much as 186 Bacteria in Relation to Country Life fifty tons of manure per acre are at times made, there is some probability of injury thus resulting. When such injury does occur, it cannot, however, be designated as denitrification. , The depression of the crop-yields arising out of bac- teriological causes may be due to the suppression of nitrification in the soil, to the transformation of the nitrate into organic nitrogen by certain classes of bac- teria, or to the actual reduction of the nitrate by soil or manure bacteria. It is well known that nitrifying bacteria are quite susceptible to the presence of soluble organic substances. This may be easily demonstrated by the addition of a solution of sugar to a soil in which nitrification is taking place. When large amounts of manure are added to the soil, the soluble materials ‘in it will discourage nitrification. The striking influence of soluble organic materials on nitrification in the soil is particularly apparent when sewage-irrigation is practiced. Effect not permanent.—The depressing effect on nitrification is not permanent. After a longer or shorter interval, the nitrifying bacteria become active again, and . the supply of nitrates proceeds normally. The length of the period during which nitrification is suspended, or is very feeble, is determined by the amount of organic matter applied; hence very large applications of manure will suppress nitrification for a longer period than smaller applications. During this time the conditions for crop growth are not favorable. When ordinary applications of manure are made, that is, in amounts not exceeding twenty or even thirty tons per acre, the interference with Nitrogen-Consuming Bacteria 187 nitrification is scarcely appreciable. When such inter- ference does occur, on account of excessive applications of manure, the injury to crop growth cannot be ascribed to denitrification. Bacteria that consume nitrates.—Crop growth may also be affected unfavorably by certain classes of soil bacteria that use up the nitrate for their own growth. This cir- cumstance will be less confusing if we remember that bacteria are plants competing, at times, with the higher plants for their food in the soil. When conditions peculiarly favor the development of these nitrate- consuming bacteria, the higher plants are rapidly de- prived of a part of their food, for the bacteria change the nitrate into organic nitrogenous materials that do not again become available until after the bacteria are decayed and their bodies nitrified. We are still in ignorance as to the soil and climatic conditions that will favor the rapid increase of these bacteria in the soil, nor do we know to what extent they are economi- cally important. Even assuming that they do, at times, interfere with plant growth, their activities. cannot be designated as denitrification. The denitrifying bacteria.—Finally, the injurious action from excessive applications of animal manures, or of other organic materials, may be really ascribed to denitrification. Animal manures, particularly those mixed with straw, contain vast numbers of denitri- fying bacteria. When a quantity of such manure ~ is placed in a solution of nitrate, and the latter kept in 9 warm place, the nitrate is rapidly destroyed. Within two or three days, the surface of the liquid will be found 188 Bacteria in Relation to Country Life covered with a fine foam produced by the small bubbles of nitrogen gas passing out of the liquid. A chemical examination will show that the nitrate has all disap- peared. No denitrification occurs when both the manure and solution are sterilized. Applications of manure carry with them to the soil millions of denitrifying bacteria. It has been shown that the soil itself contains several species of denitrifying © organisms. In the presence of large quantities of manure or of other organic matter, therefore, these bacteria find favorable conditions for the destruction of the nitrates applied with the manure, or formed from the soil- humus. Large quantities of organic matter are essen- tial for the rapid growth of the denitrifying bacteria. When these are absent, the microérganisms fail to find enough food and energy for the destruction of the ni- trates. It is for this reason that, under ordinary soil conditions, denitrification does not play a significant part in the nitrogen-feeding of crops. It has been demonstrated that annual applications of cow manure at the rate of sixteen tons per acre, together with quantities of nitrate equivalent to 320 pounds per acre, fail to cause any appreciable loss of nitrogen that can be directly. attributed to denitrifi- cation. The destruction of nitrates by denitrifying or- ganisms does not occur, therefore, in arable soils under ordinary conditions of farm practice. These losses may take place, however, in greenhouse or market-garden soils on account of the excessive amounts of manure used, and because of the activities of the denitrifying bacteria in the manure and soil. These bacteria need The Denitrifying Bacteria 189 large amounts of organic food for their development, and are, therefore, most injurious when developing in the presence of large quantities of fresh vegetable or animal matter. Hence. denitrification may also occur at times when abundant green-manuring crops are turned under. It is not advisable, therefore, to top- dress the soil with nitrate of soda shortly after large applications of manure or of other organic material. After the organic matter has partly undergone decay, the denitrifying bacteria no longer find in the residues an acceptable source of food for their rapid develop- ment. For this reason, well-composted manure is not liable to cause denitrification in the soil, even when applied in large amounts. The same applies also to vigorous crops of green-manures which cease to be a source of danger after they have undergone partial decay. Denitrification is favored by the exclusion of air. The denitrifying bacteria, it seems, require a certain amount of oxygen for their growth. When it is absent they take it out of the nitrates. This explains the re- duction of nitrates in the deeper layers of the soil, or in water-logged surface soil. It accounts also for the greater tendency to denitrification in heavy, compact soils as compared with the more open, sandy loams. Drainage, liming, and thorough tillage greatly lessen the danger from denitrification by improving the cir- culation of air in the soil. Even when air is excluded, the presence of nitrate and of easily decomposable organic matter is a prerequisite for the rapid growth of the denitrifying bacteria. Soils poor in humus, therefore. are not liable to cause the reduction of nitrate. CHAPTER XX THE INCREASE OF SOIL-NITROGEN SCIENTIFIC agriculture has been called on within the last one hundred years to solve an almost endless number of problems. Some of these have been solved without difficulty, others only after they have taxed the efforts of many men of science. Many are yet to be solved. Of all the problems that have been at last ‘made clear, none has created so much discussion or stimulated so much research as that concerning the source of nitrogen to plants. Since it became known, toward the end of the eigh- teenth century, that we are moving about at the bottom of a great ocean composed mainly of two gases, nitro- gen and oxygen, the question as to the part played by the former in the growth of plants has been before scien- tists. Chemists realized in the early years of the last century that the rocks of the earth’s crust do not ordi- narily contain compounds of nitrogen. Not many years later, it became known that very productive soils contain five to ten thousand pounds of nitrogen per acre to a depth of one foot, that many soils contain much greater amounts, and that all this vast quantity of combined nitrogen in the earth’s surface has been derived in some way from the gaseous nitrogen of the air. (190) The Appropriation of Nitrogen fromthe Air 191 As they became more skilled in analytical methods, the chemists could not remain blind to the fact that there is a constant drain on the nitrogen resources of the soil. They knew that much nitrogen is removed in the crops. They have learned that, in the decomposition of humus, some nitrogen returns to the air in the gaseous state, that another part is changed to soluble com- binations and finds its way to the rivers and the sea. Here then is a puzzling problem. Bare, nitrogenless rock falls into fragments under the attack of the sun, the winds and the rain. It crumbles more and more and living things come to find lodgment in it. The weathered part becomes deeper with the passing years, and its vegetation grows in splendor. The rock loses its identity and becomes soil, the receptacle of enor- mous amounts of humus and nitrogen. In what manner does the free nitrogen gas floating in the atmosphere pass into the plant body, and, on the death of the latter, into the soil-humus? What means are there in nature’s laboratory for replacing the constant losses of soil- nitrogen? It seemed simple enough, at first, to assume that the great aérial ocean furnished nitrogen directly to all green plants. Such an assumption agreed well with ancient traditions. The experience of many generations had forced into the consciousness of the farmer the belief that certain crops possessed soil-enriching quali- ties. The scientific men of a hundred years ago were inclined to ascribe these to the utilization of atmos- pheric nitrogen. Further investigations demonstrated apparently that plants cannot make use of nitrogen 192 Bacteria in Relation to Country Life gas for their growth. It seemed that the nitrogen must be combined with other elements to form either ammonia or nitrate before it could be employed by plants. The ammonia theory.—Toward the middle of the nineteenth century the opinion came to prevail that ammonia is the important source of nitrogen to plants. Liebig, in Germany, known as the father of agricultural chemistry, demonstrated that ammonia is a constant constituent of the atmosphere. He maintained that the natural supply of ammonia is usually sufficient for the growth of crops. According to him, the exhaustion of soils should be ascribed to their decreased content of mineral ingredients rather than to decrease in nitrogen. When careful study of the composition of the atmos- phere, conducted in France, England, and Germany, had proved that the amount of ammonia brought down to the earth by rain and snow scarcely exceeded a few pounds per acre annually, Liebig maintained that plants are capable of directly absorbing ammonia by means of their leaves. He pointed out that the beneficial effects of nitrogenous manures are most apparent in the case of cereal crops with a comparatively short vegetation period, and least’ apparent in the case of leafy crops with a long vegetation period. Experience had taught the farmer, he said, that it was useless to apply nitrogenous materials to clover. No benefit at all; or only slight benefit, was likely to result therefrom. The long vegetation period of crops like clover allowed the gradual utilization of the ammonia in the air, and no artificial supply was necessary. On the other hand, The Ammonia Theory 193 crops with a short vegetation period had a limited power for accumulating ammonia from the air, and gratefully responded to applications of nitrogenous materials. So great was the weight of Liebig’s authority that his views were widely accepted in spite of many facts, rapidly growing in number, that seemed to be contrary to these views. Lawes and Giibert, of England, denied Liebig’s claims on the strength of their experiments at Rothamsted. They soon became involved in a contro- versy with the great German chemist. It was not always free from bitterness. The investigations stimulated by this controversy proved that ammonia does not directly feed the crops, that the amounts brought down in rain or snow are but slight, and that the atmosphere contains but minute quantities of it. The adherents of the am- monia theory sought to strengthen their position by pointing to experiments that, apparently, proved the extensive formation of ammonia in nature. They asserted that either in the burning of organic materials contain- ing no nitrogen, or in the decay of such substances in the soil, ammonia was always formed. The German chemist, Schoenbein, thought he had demonstrated that nitrate of ammonia is formed in the simple evaporation of water in shallow vessels. Further investigation, under more rigid experimental conditions, disproved these statements. The experi- ments of Boussingault in France, and of Lawes, Gilbert and Pugh in England, conducted in the fifties of the last century, seemed to have established with certainty that gaseous nitrogen cannot be used by plants. There remained, therefore, but one logical explanation of the M * 194 Bacteria in Relation to Country Life supply of nitrogen to plants. All of the nitrogen of crops, so it appeared, was taken up by them from the soil either in the form of ammonia, or of nitrates. The nitrate theory.—It thus came to be believed, in the sixties and seventies, that the soil and the soil alone could feed the plants with nitrogen. But, assuming that all of this nitrogen food was derived from the humus by the formation from the latter of ammonia and of nitrates, how could the maintenance of the nitrogen store in the soil be explained? The harvests remove annually twenty to forty pounds of nitrogen,—in the case of crops like clover, as much as one hundred pounds. At times, double that quantity is withdrawn. The drainage waters, moreover, frequently carry away from the land as much nitrogen as is removed by the crops. Surely, with the atmospheric nitrogen not available, the origin and maintenance of the supplies of combined nitrogen in the soil seemed shrouded in mystery. Thus, after the middle of the last century, men came to believe more and more that the evident ability of the soil to restore its lost nitrogen was connected in some way with the formation of nitrates. This belief was strengthened by the experiments of Boussingault, . who showed that nitrates were readily utilized by plants, that the growth of the latter was in proportion to the nitrate supplied, and that humus-nitrogen in its un- changed state offered no nitrogen food to plants. The conviction grew, therefore, that soils possess the ability not only to form ammonia and nitrates, by the decay of their humus, but, also, to produce nitrate directly by the condensation of the nitrogen and oxygen Discovery of Bacterial Relation 195 present in the soil-air. It was assumed, therefore, that the production of nitrate in the soil out of the gases, nitrogen and oxygen, was affected by certain compounds of iron, by ozone, and by sulfate of lime. It was assumed, also, that the electrical discharges in the atmosphere not only caused the formation of small quantities of nitric acid which was washed into the soil by rain, but that electrical discharges led also to the formation of nitric acid in the soil itself. The bacteria theory.—The proof furnished in 1877 by Schléesing and Mintz that nitrification is a bacterio- logical process and that it does not take place in sterile soil, disproved the claim that nitrogen and oxygen are directly condensed in the soil to nitric acid. The earlier experiments were recalled that indicated the formation of nitrates only when nitrogenous organic materials were supplied, and the non-formation of nitrates in soils devoid of humus. It was remembered that the nitric acid brought down in the rain scarcely exceeded three or four pounds per acre, annually, and the wonder grew again as to the manner in which the supply of com- bined nitrogen in the world is maintained. The existing uncertainty was dispelled in 1886 with the announcement of the German investigator, Hell- riegel, that certain plants are capable of using for their development the nitrogen gas of the air, but that they are enabled to do so only with the aid of bacteria which live in their roots. The power of thus gathering atmos-_ pheric nitrogen was confined, with few exceptions, to the plants of the legume family. A few years later, the Russian bacteriologist, Winogradsky, furnished ‘the 196 Bacteria in Relation to Country Life proof that there is in the soil and living outside of plants still another class of bacteria that are capable of utiliz- ing nitrogen gas for their growth. Thus it was learned that the supply of combined nitrogen to the plant world would soon fail but for the activities of bacteria which, living either within the roots or in the soil by themselves, provide for the continuance of life on this earth. They are different from the other classes of bacteria already considered in that they are concerned with the addition of nitrogen compounds to the resources of the plant and animal world, and are known, therefore, as nitrogen-fixing, or nitrogen-gathering bacteria. The others can only change the form of combination, and are concerned only with the transformation of soil-nitrogen, a process that fre- quently involves considerable losses of nitrogen. The nitrogen-transforming bacteria we have already discussed. We will now turn our attention to the very important nitrogen-fixing kinds. The nitrogen-fixing bacteria may be divided broadly into two classes, non-symbiotic and symbiotic. The former live in the soil itself and develop there even when no crop is growing upon it. The latter may also grow in bare soils, but attain a pronounced power of gathering atmospheric nitrogen only after they had in- vaded the roots of some leguminous crop and had at- tained a certain development there. The term sym- biotic is derived from the word symbiosis, which means living together. It is applied to describe a condition in which two organisms, rather different in character, live together to the advantage of both. CHAPTER XXI THE NON-SYMBIOTIC NITROGEN-FIXING BACTERIA THE non-symbiotic nitrogen-fixing bacteria thus far known may be divided into two classes: (1) Anaérobic ferments, first described by Winogradsky in 1893; (2) aérobic bacteria described by Beyerinck in 1901. Anaérobic bacteria.—The anaérobic ferment described by Winogradsky and named by him Clostridium Pastori- anum (Fig. 29), is a rod-shaped organism, developing © in the absence of air (anaérobic), and producing spores and boat-shaped masses (clostridia). Pure cultures of this organism, when grown in solutions of sugar and the necessary mineral salts, but containing no nitrogen compounds, develop rapidly and assimilate the free nitrogen of the air. This may be readily demonstrated by analyzing the cultures at the end of ten days or two weeks, when considerable quantities of combined nitrogen will be found in the liquid. While Clostridium Pastorianum is a distinctly anaérobic organism, it is yet capable of developing under conditions allowing a more or less ready access of air, provided other bacteria are present. Under these circumstances, the accom- panying bacteria use up the oxygen in the solution, and thus make it possible for the anaérobic Clostridium Pastorianum to develop properly. (197) 198 Bacteria in Relation to Country Lije Winogradsky showed also that the amount of nitro- gen fixed bears a certain relation to the amount of sugar supplied. In other words, the bacteria employed used the sugar not only as food, but also as fuel, the energy of which was utilized partly for making the free nitrogen of the air to combine with other elements. Winogradsky demonstrated, likewise, that the fixation of nitrogen is discouraged when nitrogenous substances are present in the culture medium. Thus, when salts of ammonia, Fig. 29. Non-symbiotic nitrogen-fixing bacteria.—Rods, diostridia, and spores of Clostridium Pastorianum. (Winogradski.) were purposely added, the fixation of free nitrogen de- creased in proportion to the combined nitrogen sup- plied. A point was finally reached when there was practically no fixation of nitrogen by the bacteria. This fact is of great interest in teaching us that the nitrogen- gathering bacteria, like leguminous -plants, prefer to employ the nitrogen compounds already at hand, and turn to the atmosphere only when combined nitrogen is not to be had. Clostridium Pastorianum has been isolated also by other investigators. It seems to be widely distributed in cultivated soils, although we are still in ignorance of the The Anaérobic Form 199 actual work performed by these organisms in the field. We do not know how they are affected by various modes of soil treatment, tillage, and crop-rotation. We know that they are influenced in their growth by the mechanical composition of the soil, by the amount of humus present, and by the proportion of moisture. While found in both open and compact soils, their growth is favored by the less thorough aération of the latter. As to their distribution in the different soil- layers, it will probably be found that they occur at greater depths in sandy loams than in heavy clays or clay loams. The aérobic nitrogen-fixing bacteria.—These organisms, described by Beyerinck in 1901, consisted of two species, Azotobacter chrodcoccum and Azotobacter agilis (Fig. 30). Three additional species, making five in all, were de- scribed by the writér in 1903 and 1904. They were all large bacilli, quite characteristic in their appearance, and widely distributed in arable soils. Being distinctly aérobic in character, they develop only when air is freely admitted. Their power to fix atmospheric nitrogen is more pronounced than that of Clostridium Pastori- anum, and the highest yield of combined nitrogen thus far recorded has been given by Azotobacter Vinelandti (Fig. 31), isolated from a New Jersey soil. From the standpoint of crop-production, this cir- cumstance is of great significance, since the fixation of nitrogen is accomplished at the expense of the humus. The latter furnishes the food and energy to the bacteria and they use it up in their growth. For the fixation of nitrogen a smaller quantity of humus suffices for the azotobacter species. Azotobacter Vinelandii can fix, 200 Bacteria in Relation to Country Lije with a given quantity of humus, two or three times as much nitrogen as can be fixed by Clostridium Pas- torianum. The differences in the nitrogen-fixing power of the anaérobic and aérobic bacteria are due to the differences in their mode of action. The chemical pro- cesses which they employ are not the same, hence the results cannot be expected to be the same. However, the power of fixing atmospheric nitrogen is quite vari- able in either class of or- ganisms. It is modified essentially by soil and climatic conditions, and may be increased by favor- able soil conditions, or de- creased by unfavorable conditions. . The quantity of lime in Fig. 30. Non-symbiotic nitrogen-fix- the soil bears a striking re- ing bacteria.—1. Azotobacter agilis; A X 2,000. 2. Azotobacter chrodcoc- lation to the development aaa ania ‘21500: of azotobacter. Being more (Beyerinck.) * s i susceptible to acid condi- tions than Clostridium Pastorianum they will not develop in soils that are more or less sour. They grow best, there- fore, in soils containing an abundance of lime. It has been found that they can be most readily secured by inoculating culture solutions of the proper composition with small pieces of undecomposed lime carbonate found in the soil. Investigations carried on in Germany and in Sweden show the importance of lime for the development of these organisms. The unlimed soils in these experiments failed, for the most part, to yield a The -Azotobacters 201 growth of azotobacter even when the land was in good condition. It is not at all impossible that the persistent fertility of limestone soils is due in part to their vigorous flora of azotobacter species and the consequent steady gains of nitrogen through the activities of these organisms. Much interesting light is still to be thrown on the relation of azotobacter to other soil bacteria. There is good reason to be- lieve that this re- lation is important, not only from the standpoint of nitrogen-fixation (addition) but also from the _ stand- point of nitrogen- transformation. Beyerinck thought at first that azoto- bacter have not, by themselves, the power of fixing at- Fig. 31. Azotobacter Vinelandit: X 1,000.—One of the non-symbiotic nitrogen-fixing bacteria. mospheric nitro- gen, and that they become endowed with this power only in the presence of certain other soil bacteria. Sub- sequent investigations by others demonstrated the fallacy of his views, and the writer showed experiment- ally why Beyerinck came to hold such views. But, while azotobacter can undoubtedly fix atmos- pheric nitrogen when in pure culture, its power of nitro- gen-fixation is enhanced by other species themselves 202 Bacteria in Relation to Country Life not nitrogen-fixing organisms. The writer demonstrated that the increased power thus lent to the nitrogen- fixing bacteria may be very considerable, and that the several azotobacter species show differences in this respect. For in- stance, it was found by the writer that 2,000. (itinter- 1 } nt erger. . ‘ubercle acillus; lished that milk may be x 000, {itewect) 4. Typhoid . acillus; ; i ewlett. 3 come the carrier of the Typhoid bacillus; x 2,000. Hin: germs of these diseases. terberger.) The rules of sanitation require that the utmost care be exercised in assuring the exclusion of these germs from milk. Persons coming in contact with diphtheria or scarlet fever patients, should not be allowed to enter the dairy, nor should convalescents from these diseases be permitted to become a source of infection. Other disease bacteria may likewise be distributed in milk. Z CHAPTER XXXIX BACTERIA IN CREAM, AND CREAM-RIPENING THE numbers -and kinds of bacteria found in cream are determined to a great extent by the method of its production. Cream secured by separating sweet milk will differ bacteriologically from cream prepared by the shallow or deep-setting systems, at low or at compara- tively high temperatures. Separator cream made from fresh milk, with a low bacterial content, will contain’ frequently but a few thousands of bacteria per cubic centimeter. For instance, one of the cream samples at the milk and cream contest at Cleveland, in 1907, contained only 1,100 bacteria per cubic centimeter, while three others contained from 20,700 to 29,200 per cubic centimeter. In two samples, on the other hand, the numbers were 392,000 and 402,500 per cubic centimeter, respectively. These differences were un- doubtedly due to the care observed in the production of the milk and its subsequent treatment. It is generally true that, everything else being equal, milk of good keeping quality will furnish cream of good keeping quality. In the production of good cream, therefore, the same precautions as to cleanliness and temperature should be observed as were already noted in the dis- cussion of milk. (402) Sweet-Cream Butter 403 But, aside from the use of sweet cream for direct consumption or for the making of ice-cream, we must consider it in its important relation as the raw material for the manufacture of butter. The vast significance of the latter as an article of diet will be readily realized from the fact that the product of more than one-half of all the cows in the United States (there were 18,000,000 of them in 1900) is turned over to the butter-maker. The important material interests involved in this agri- cultural industry have stimulated extensive inquiry into the economy of butter-making and have thus brought to light a long array of interesting facts. The dairy bacteriologist has played a prominent part in discover- ing and explaining these facts and the butter-maker owes to him much of his ability to produce a uniform and high-grade product. Sweet-cream butter is made from cream immediately or soon after it is separated. In some of the European countries the demand for such butter is very large. In the United States the demand for it is almost wholly confined to the cities with large foreign. populations. The success in the making of sweet-cream butter de- pends essentially on the checking of bacterial growth in the cream. As far as possible, the latter must re~ main unchanged in flavor and composition; hence, in its preparation, the milk is quickly removed from the barn, passed through the centrifuge, and the cream at once cooled to a low temperature. The opera- tion of churning is hastened as far as possible, and is performed so as to exclude bacterial contamina- tion. 404 Bacteria in Relation to Country Life But, even when the most scrupulous care is observed in excluding dirt and bacteria from the milk and cream, the keeping quality of sweet-cream butter is poor at best, and the butter must be, therefore, consumed within a short time after its production. Some grades of sweet-cream butter are made out of pasteurized cream and possess, then, fairly good keeping quality, besides being freed, incidentally, from any disease bacteria that may have been present in the milk or cream. Ripened cream .butter—By far the greatest propor- tion of butter made in the United States, and, for that matter, in foreign countries, is prepared from cream that has undergone bacterial change. The ripening process involves changes that affect the yield of butter from any given quantity of cream, the keeping quality of the butter produced, as well as the flavor of the latter. It is well known that ripened cream produced by the gravity system will yield a larger quantity of butter than the same amount of unripened cream. In the case of separator cream, the difference is not so apparent. The greater yield of butter from ripened cream is accounted for by the assumption that the fat globules in milk are separated by or perhaps coated with protein substances, that in the course of ripening the peptonizing bacteria attack these substances, and gradually decom- pose them. This change is hastened by the lactic acid formed by the lactic-acid bacteria. The digestion of the protein substances by the bacteria becomes, therefore, an aid to the rapid coalescing of the fat globules in the subsequent churning. That the bacteria must necessarily Ripened Cream 405 play an important part in the ripening process is evi- denced by their presence in such truly enormous num- bers. According to Conn, normally ripened cream con- tains 100,000,000 to 1,500,000,000 of bacteria per cubic centimeter, the average number being about 500,000,000. The organisms grow rapidly at the temperature of ripen- ing cream and produce the changes that subsequently modify the flavor and probably also the keeping quality of the butter. In cream, as in milk, the various kinds of bacteria must compete for their food. Only those best adapted to their environment finally emerge victorious from the struggle. We have already seen that in milk the lactic- acid bacteria, largely on account of the lactic-acid pro- duced by them, usually gain the upper hand and pre- vent the growth of the other species. We have seen, also, that there is more or less associative action in the bac- terial decomposition of milk, for the lactic-acid germs are evidently stimulated in their development by the changes caused by some of the other species. Similar conditions prevail in cream. While it is still sweet, the peptonizing bacteria grow freely, decompose the protein substances and prepare the ground for the lactic-acid germs. These, in their turn, become more and more prominent, and, finally, crowd out the other species. As a result of the activities of the two groups of organisms, we have an accumulation of protein de- composition products on the one hand, and of lactic acid on the other. These substances impart distinct tastes and flavors to the cream and to the butter made from the cream. 406 Bacteria in Relation to Country Life We see, therefore, that the ripening of cream is equivalent to the accumulation in it of certain products. Under properly controlled conditions, the quantity and quality of these products are satisfactory to the butter- maker, for they impart to his butter the desired proper- ties. Experience has taught him, at the same time, that it is not safe to depend on the spontaneous bacterial changes in the ripening cream for the production of high-grade butter. The method is not only slow, but uncertain. Occasionally the. lactic-acid bacteria do not gain the ascendancy in the ripening cream. Other species become prominent and produce substances that impart to the butter objectionable flavors, or injuriously affect its keeping quality. The butter-maker must attempt, therefore, to control the ripening process by adding to the cream large quantities of desirable bac- teria, such additions being known as “‘starters.”’ Starters.—If a quantity of cream sours normally and produces butter of superior quality, it must contain bacteria capable of bringing about this result. A starter is a quantity of inoculating material and is valuable only when it contains the proper kinds of bacteria, . that is, bacteria capable, not merely of producing cer- tain chemical changes, but, also, of growing with suf- ficient rapidity to overcome the other organisms present in the cream. A pure culture of some germ capable of carrying forward the ripening process in a satisfactory manner may be added to the cream. This is actually done on a more or less extensive scale in the dairy districts of northern Europe, notably in Denmark. A limited use The Starter 407 of pure cultures is made also in the United States and Canada. The method itself is fairly simple. A laboratory culture of some lactic-acid germ is added to a small quantity of sterilized or pasteurized milk, or to skimmed milk, and is allowed to grow at a suitable temperature for a day or two. This milk, with its bacteria multi- plied to vast numbers, is added to a still larger quantity of pasteurized milk or cream. After a similar period of increase, the latter is ready to be added to the mass of cream that is to be ripened. The starter is thus gradu- ally built up, and added to the sweet cream in the pro- portion of about one to ten. The building-up process is almost indispensable. It is depended on to furnish num- bers of bacteria great enough to permit the suppression of the organisms in the cream, as well as to allow the ripening process to run to completion rapidly. The direct addition of such laboratory cultures to the body of cream has, for these reasons, proved unsatisfactory in most instances. Starters may be built up in a similar manner out of samples of milk, skim-milk, or cream, that have been allowed to sour spontaneously. They are known as natural starters, in contradistinction to the pure-culture starters just described. A natural starter may frequently happen to be practically a pure culture of some lactic- acid bacterium, since such organisms readily become prominent in milk or cream. In order to secure a natural starter, the butter-maker obtains, with every precaution as to cleanliness, a quantity of milk from a healthy cow. This milk is set aside, partly skimmed, warmed, ‘and 408 Bacteria in Relation to Country Life allowed to turn sour naturally. Separator skim-milk may be used instead of whole milk or partly skimmed milk. The following steps are much like those noted in connection with the building up of pure-culture starters. It is not necessary to build up a new starter for each churning, for a quantity of ripened cream or of butter- milk reserved from the preceding lot may be used for the preparation of the starter. From time to time, however, the building up of a new starter becomes essential. Pure-culture starters possess certain advantages which render their use acceptable to the butter-maker. Experience soon teaches him how to control his ripening process within narrow time limits, and he can, there- fore, carry on his work with a precision not attainable in the use of natural starters. He is more certain of uniformity in his product, and finds himself, therefore, able to place a standard article on the market. The natural starters, on the other hand, cannot always be relied on. In spite of the care observed in their preparation, they are apt, at times, to yield an unsatis- factory product, both as to flavor and keeping quality. With that much admitted, however, the skilled butter- maker who uses natural starters believes that he can, with their aid, produce a better-flavored product than he could with the use of pure-culture starters. This is not difficult to understand, since no single species has yet been discovered that is capable of producing all of the substances found in properly ripened and highly flavored cream. According to Grotenfelt, Jensen char- acterizes the ideal lactic-acid bacterium as possessing Natural Starters 409 the following properties: (1) ‘That it will sour the cream rather strongly in a comparativly short time, so that it can compete with other bacteria present; (2) that it will thrive at a relatively low temperature (60° to 72° Fahr.); (3) that it will coagulate the cream and milk to a uniform homogenous mixture, and give it a slightly sour taste and odor; (4) that it will produce an agreeable aromatic taste and flavor.’ Since no single organism thus far known is capable of meeting all these requirements, the cream ripened by means of pure cultures must necessarily be inferior to good cream ripened under the best conditions by natural starters. It should be remembered, however, that the average sample of cream ripened under the average conditions by means of natural starters will probably be inferior to the same cream ripened under average conditions by means of pure-culture starters. The more general introduction of the separator adds, in some respects, to the superiority of the pure-culture starters, since the proportionate amount of cream brought by farmers to the creameries, is increasing, while that of whole milk is decreasing. By separating the milk on the place, the farmer not only simplifies the problem of transportation, but he also has the skim-milk at his immediate disposal, and, moreover, reduces the danger of infecting his cattle and his pigs with tubercle bacilli, brought from the creamery in skimmed milk. Advantages and disadvantages.—These advantages to the farmer involve certain disadvantages to the creamery manager; for the cream is accumulated at the farm and is delivered in a condition frequently far 410 Bacteria in Relation .to Country Life from satisfactory. Under the older system of delivering whole milk, the farmer found procrastination less prac- ticable. This state of affairs is not only unjust to the farmer whose dairy is clean, and whose cream is of good quality, but, also, complicates the work of the butter- maker and, at times, reduces him to despair. He cannot make good butter out of inferior cream, however care- ful he may be in the preparation and use of his starters. When confronted by this situation, his chances for turn- ing out a fairly uniform product are improved by the use of pure-culture starters and the pasteurization of his cream. The best results from pure-culture starters are to be expected, theoretically, when the latter are used with pasteurized cream. The bacteria present in the fresh cream are either destroyed or weakened by pasteuriza- tion, and the organisms supplied in the starter are per- mitted to develop unhindered. As already noted, a uniform product is thus assured. When two or three different species are furnished in the culture, the flavor, as well as the keeping quality of the butter are, on the whole, satisfactory. In some localities in Europe, and particularly in Denmark, the pasteurization of cream and its ripening by means of culture starters is the common practice. In this country, a more strongly flavored butter is pre- ferred, and unpasteurized cream is found, therefore, to yield a more satisfactory product. Either artificial or nat- ural starters are employed, and are added in proportions sufficiently large (usually about 10 per cent) to assure a preponderance of the desirable lactic-acid bacteria. CHAPTER XL BACTERIA IN BUTTER BurTer, as it grows older, usually deteriorates in quality. It loses its flavor and begins to undergo decom- position. This proceeds slowly at low temperatures, but very noticeably at higher temperatures. Ultimately it assumes the rancid character of old butter. The changes thus occurring are partly chemical and partly bacteriological. The loss of flavor is accounted for readily enough by the escape of the small amounts of the volatile compounds present in fresh butter. On the other hand, the develop- ment of rancidity, accompanied by the accumulation of acid substances, is not so simple. There is scarcely a doubt that bacteria are intimately concerned with the development of rancidity. For one thing, butter kept at low temperatures not only retains its good qualities, but, also, for some time after it is removed from cold storage. Moreover, butter made from cream delivered sweet at the creamery, has a better keeping quality than butter made from cream that was sour. The differences are not apparent while the butter is in cold storage, but subsequently become noticeable. It has been likewise demonstrated experimentally that butter made from pasteurized cream does not become (411) 412 Bacteria in Relation to Country Life rancid so readily as butter made from unpasteurized cream. In fact, butter made from sterilized cream does not seem to become rancid at all as long as it remains sterile. In preventing butter from becoming rancid, antiseptics are as effective as sterilization by heat. Butter made from pasteurized or sterilized cream may be made to become rancid by the addition to it of a small quantity of old butter. Furthermore, butter contain- ing a large proportion of casein or milk-sugar becomes rancid more readily than butter poor in these substances. When air and light are excluded, the butter does not deteriorate so rapidly. In hermetically sealed cans, it keeps best when the latter are full, that is, when the air is excluded. Numbers and kinds.—Bacteriological examinations of fresh butter show large numbers of organisms, fre- quently many millions per gram. The number is inti- mately affected by the source and character of the cream. Cream from the milk of clean cows and in a clean dairy- house will yield butter with a much smaller number of bacteria than filthy cream. Clean cream contains, for the most part, only lactic-acid bacteria that rapidly decrease in numbers in the butter, whereas filthy cream contains other species that do not decrease as rapidly, and, in some instances, actually multiply in the butter. A study of the various microérganisms found in butter will show that different species are prominent in the same sample at different times. Fresh butter neces- sarily contains lactic-acid bacteria in predominating numbers. There are usually present, also, Bacillus fluorescens liquejaciens, derived from the wash-water, Kinds in Butter 413 Oidium lactis and other molds, and various yeasts. In stored butter, the lactic-acid species decrease rapidly, while Bacillus fluorescens liquefaciens may increase for atime. Oidium lactis is always prominent, but is finally suppressed by another common butter mold, Clados- porium butyri. Certain yeasts also persist in stored butter, and become so prominent at times as to constitute nearly the entire flora. There is still a difference of opin- ion as to the part played by these various organisms in the development of rancidity in butter. It has been found that samples of butter prepared under aseptic conditions become rancid when inoculated with a culture of Bacillus fluorescens liquefaciens, or of Bacterium prodigi- osum. On the other hand, the mold, Oidiwm lactis, while capable of developing strong acidity in butter and of decomposing the butter-fats, does not cause rancidity when present alone. Neither does Cladosporium butyri; yet, when the two are growing together, rancidity is produced. We note thus associative action in the pro- duction of rancidity by two prominent butter molds, and, in general, it may be said that bacteria, molds and yeasts contribute to the development of rancidity and to other changes that occur in butter. Disease bacteria in butter.—The spread of infectious diseases by means of butter has been made the subject of considerable discussion. The wide prevalence of bovine tuberculosis, and the frequent presence of tuber- cle bacilli in milk, are the cause of the presence of the bacilli of tuberculosis in butter. An examination of forty samples of market butter in Posen revealed the presence of tubercle bacilli in 22 per cent of these sam- 414 Bacteria in Relation to Country Life ples. In another instance, an examination of one hun- dred samples of butter selected in Stuttgart showed that living tubercle bacilli were present in nearly one out of every ten samples. There is, therefore, more or less danger of transmission of tuberculosis by means of butter, and the pasteurization of cream in butter- making, whenever practicable, must be regarded as a commendable practice from the standpoint of public hygiene. In the case of typhoid and diphtheria, on the other hand, the danger of transmission in butter, if it exists at all, can be only slight. The germs of these diseases do not seem to occur in market butter. Typhoid bacilli, purposely added, disappeared gradually and could not be detected at the end of ten days. Tubercle bacilli also gradually disappear in stored but- ter, although they are hardier than the typhoid germs and may persist for weeks, perhaps for months. Butter jfaults.—Occasionally butter acquires unde- sirable characteristics, quite different from the ordinary development of rancidity. The objectionable changes known as butter faults or butter diseases may affect the appearance and taste of the butter, giving rise to mottled, putrid, bitter or tallowy butter. Such faults have been traced to microérganisms that may be derived from the cream or from other sources. Samples of inferior cream produced in a filthy environment often contain species of bacteria that later develop in the butter and destroy its value as a food product. The pasteurization of such cream undoubtedly improves the keeping qual- ity of the butter made from it. Not infrequently, butter faults have their origin not Butter Faults 415 _in the cream supplied by the patron but in the water used at the creamery. Certain bacteria present in wells, brooks, or springs, are introduced into the butter with the wash-water, and bring about undesirable changes. Even creameries with an established reputation for excellent butter are now and then confronted by the sudden deterioration of their product on account of microorganisms causing butter faults. In such cases, normal conditions are reéstablished only after the most thorough cleaning of the premises, and, when this is insufficient, by the boiling of the water used in the creamery. CHAPTER XLI BACTERIA IN CHEESE FresH cheese curd consists of rather tough, elastic material, which, according to popular belief, is not easily digestible. Ripened cheese is soft and waxy, in some varieties nearly semi-liquid, and rich in water-soluble nitrogenous nutrients. The transformation of the fresh curd into ripened cheese may be accomplished in four or five weeks in the soft varieties. It may require several. months for its completion in the hard varieties. The ripening process.—The three important constitu- ents of the curd—the fat, milk-sugar and casein—are affected by this process of transformation and undergo more or less deep-seated changes. The fat, while fre- quently modified to a very slight extent, contributes none the less to the pungent taste and smell of the ripened cheese. The milk-sugar is changed into lactic acid, which, in its turn, plays an important réle in the reaction that subsequently occurs in the ripening mass. But, more important than these are the changes that occur in the casein, or paracasein, as the chemists call the rennet coagulated mass. The proteid in the insoluble curd (Calcium para- casein) is rapidly modified so as to become soluble in a 5 per cent salt solution at 50° to 55° C. (122° to 131° Fahr.). (416) Curd Protein 417 After this reaction is completed, the curd proteid is again changed into an insoluble form. The latter is in turn gradually transformed in the ripening process into water-soluble compounds. This last series of changes, which are intimately associated with the final digesti- bility and flavor of the cheese, involve the gradual breaking down of the proteid substances, and the formation of the so-called amides (amido compounds). It has been found that a properly ripened Cheddar or Swiss cheese may contain about a third of its total nitrogen in the amid form, and that 3 to 5 per cent may be present as ammonia. It is to these various amides and the ammonia that the cheese flavors are largely due, at least, in the case of the hard cheeses. The well- known influence of temperature on the rate of ripening and the flavor may be explained by the differences in the proportionate amounts of the various amides formed. We see, thus, that the ripening of cheese is not unlike the putrefaction of other protein bodies. However, some of the characteristic putrefaction products are absent from normally ripened cheese. It is only in ex- ceptional cases that such putrefaction products appear. They may give rise, then, to ptomaine poisoning. While the changes leading to the formation of poisonous sub- stances in cheese have not yet been made clear, it is assumed that these compounds are most likely to appear in over-ripe or improperly ripened cheese. Enzymes and bacteria in the ripening of cheese.— When modern methods of investigation were first brought to bear on the problem of ¢cheese-ripening, it was believed that bacteria and other microérganisms were the sole AA 418 Bacteria in Relation to Country Life cause of the changes observed. It was thought that the organisms themselves or the enzymes produced by them, accomplished the digestion of the curd. There was a strong difference of opinion, however, as to the kinds of bacteria responsible for the important transforma- tions. Some bacteriologists asserted that the digestion was performed by peptonizing bacteria, while others maintained that it was effected by the lactic-acid bac- teria. In order to decide 2 \ the point at issue, various wy v7 organisms occurring in \ \ cheese were isolated in pure : culture and were employed for the ripening of sterile 4 ( 4“ curd. Large numbers of i. such experimental cheeses “as were prepared and studied, Fig . 68. Lactic acid ferments in : : cheese -ripening.— 1. Bacterium and it was found that in wets $506.3. Bocilus covert, most instances they failed X 3,000. 4. Bacillus casei A; é X 3,000. (Freudenreich and to ripen normally. To be shen sure, Freudenreich, in Switz- erland, was able to secure a partial ripening of cheese made out of pasteurized milk and inoculated with pure cultures of lactic-acid bacteria, yet the results were not fully satisfactory. Moreover, cheese made from boiled milk and similarly inoculated failed to show any ap- preciable ripening. Other investigators were believed to have secured more or less satisfactory ripening, with the aid of peptonizing bacteria, but their results were, in no case, above criticism. Meanwhile, the investigations of Babcock and Rus- Enzymes in Cheese-ripening 419 sel, and the studies conducted independently by Jensen, in Europe, demonstrated that there is another impor- tant factor in cheese-ripening, that has hitherto been overlooked. Babcock and Russell, and, likewise, Jen- sen, showed that the rennet employed in cheese-making contains pepsin. The latter, as is well known, is capable of causing the digestion of protein substances. By using increased quantities of rennet in the preparation of cheese, the experimenters found that there was a corresponding increase in the amounts of total soluble nitrogen, and of albumoses and peptones formed in the course of six months. Furthermore, they found that still another enzyme, galactase, plays a prominent réle in cheese-ripening. This enzyme has its origin apparently in the cow’s body, for it is secreted with the milk. It resembles some of the bacterial enzymes in its action on protein substances and in the end products formed. Unquestionably, the two enzymes are prominent in cheese-ripening and are instrumental in the breaking down of the complex protein substances into more simple compounds. Additional investigations carried out in Europe, and, more particularly, at the New York Experiment Sta- tion, make it practically certain that the pepsin and galactase are by themselves insufficient for the complete ripening of cheese. In the investigations at the New York station, a lot of Cheddar cheese was divided into two portions, one of which was allowed to ripen under normal conditions, while the other was treated with chloroform so as to exclude the action of microérganisms but not of the two enzymes. When examined at dif- 420 Bacteria in Relation to Country Life ferent intervals for a period of nine months, the normal. cheese showed a fairly small variation in the content of albumoses and peptones and a gradual and constant increase of amides and‘of ammonia. On the other hand, the chloroform cheese showed a much larger accumu- lation of albumoses and peptones, a much smaller ac- cumulation of amides and no accumulation at all of ammonia. It would seem, thus, that the pepsin and galactase are incapable of carrying the ripening process to completion and that the normal changes in cheese are dependent on still another factor or factors. Bacteriological studies of cheese by different investi- gators, at different times, agree in showing that the bacteria multiply rapidly in freshly prepared cheese. In the hard varieties, the increase may continue for a month or for a few days, depending largely on the tem- perature at which the cheese is kept. The organisms consist almost entirely of the lactic acid speciesy for the other bacteria, initially present, do not seem to be able to maintain their ground and are rapidly crowded out. In experiments in which large numbers of pep- tonizing bacteria were purposely introduced, their disappearance was fully as marked. Evidently, then, organisms, other than the lactic-acid species, do not play, numerically, a significant part in the ripening of cheese. Since the lactic-acid bacteria constitute nearly the entire flora of fresh cheese, and since, furthermore, normal ripening of hard cheese does not occur when they are excluded, the thought naturally suggests itself that these organisms are intimately concerned with the ripen- Bacteria and the Ripening Process 421 ing process. Indeed, numerous attempts have not been wanting to prove that they are thus directly concerned. However, the adherents of this theory have a serious difficulty to overcome in proving that the lactic-acid bacteria are capable of causing the digestion of protein substances and the formation of amides and of ammonia. In some instances, it was undoubtedly demonstrated Fig. 64. Bacteria in cheese-ripening.—1 and 2. Sections through Emmentaler (Swiss) cheese, showing enclosed bacteria. 3. Section through Gorgonzola cheese. (Rodella.) that there are species of lactic-acid bacteria that pro- duce proteolytic, that is, protein-digesting, enzymes. Most investigators, however, have been unable to dis- cover the production of such enzymes by the lactic-acid bacteria. The question as to the direct influence of these organisms in the ripening of cheese is, therefore, still an open one. On the other hand, it is generally agreed that the lactic-acid bacteria are indirectly of great moment for the ripening process. Their rapid increase in the fresh ~ 422 Bacteria in Relation to Country Life cheese and the production of lactic acid by them ex- cludes the predominance of other species. Moreover, it would seem that the lactic acid thus produced unites with the casein (paracasein), forming thereby a com- pound, which, there is reason to think, behaves differ- ently towards the galactase and pepsin than does the casein (paracasein) itself. In some of the experiments on cheese-ripening the curd was prepared from pasteurized or sterilized milk in which the galactase and pepsin had been destroyed by heating. The enzymes being thus excluded, the subsequent inoculation with lactic- acid bacteria did not reéstablish normal conditions. In other experiments, on the contrary, the bacteria, but not the galactase and pepsin, were excluded, lactic acid was not formed, chemical reaction between the casein (paracasein) and lactic acid did not occur, and enzyme action was unquestionably modified thereby. It will.be seen, therefore, that the question is still far from being settled. The indirect importance of the lac- tic-acid bacteria in cheese-ripening having been estab- lished, it remains for future experiments either to con- firm the belief as to their direct action, held by many, or to prove it untenable. Sojt cheeses——The soft cheeses include Roquefort, Camembert, Limburger, Stilton, Brie, Gorgonzola, Backstein, Gammelost, Port de Salut, and others with reputations more or less local. They differ from the hard cheeses both in their method of preparation and their appearance, taste and flavor. They are not sub- jected to pressure, the whey is not as completely removed, and they contain, in consequence, more moisture. Air Ripening of Soft Cheeses 423 and moisture being present in greater amounts, they allow a more rapid development of microérganisms, and ripen more rapidly than the hard cheeses. The bacteria and molds concerned are not the same in the different varieties of cheese, some containing character- istic species of molds, others of bacteria. The soft cheeses have their origin in Europe. They have been manufactured in some localities for many generations. The different conditions of moisture and temperature in the different localities, and the differ- ences in the composition of the milk itself, have led. to the establishment of combinations of microérganisms that may be characteristic of one locality but not of another. For this reason, brands of the same variety of cheese from different localities may show a very marked divergence in taste and flavor. The soft cheeses do not keep so well as the hard cheeses, and are not so adapted for export trade. Because of the high prices commanded by the best grades of soft cheeses, and, likewise, because of the grow- ing demand for some of the brands in the United States, attempts are being made here to develop the soft cheese industry. Considerable progress has been made in this direction by commercial concerns employing imported cheese-makers, and much has been contributed by the investigations at the Storrs (Connecticut) Experiment Station. The work of microérganisms in the ripening of some of the soft cheeses has been made clearer by the ex- periments at the Storrs station on the manufacture of Camembert cheese. The milk is heated to about 85° 424 Bacteria in Relation to Country Life Fahr., and a pure-culture starter of lactic-acid bacteria is added. After the desired degree of acidity is developed by the bacteria, the casein is coagulated: by means of rennet, the curd is cut, stirred and dipped into forms. It is then allowed to drain, without artificial pressure, for four or five hours, and is inoculated with spores of the proper mold. After being salted, the cheese is ready to go through the ripening process, which requires about four weeks for its completion, and which involves the gradual change of the hard curd into a soft, waxy substance. The softening begins near the surface and slowly spreads towards the center of the mass. ‘‘ When in prime condition, the cheese is soft enough to spread upon bread or crackers, but not soft enough to run. An over-ripened cheese, however, becomes still softer, until, in time, the whole interior of the cheese below the rind is converted into a nearly liquid consistency, which will run out of the cheese readily, if the rind is broken. On the other hand, an under-ripened cheese will show more or less of the sour curd in the center, which has not been affected by the softening agents. The cheeses purchased in the market are, very frequently, in one of these two con- ditions, either over-ripe or under-ripe.”’ The lactic-acid bacteria of the milk and the starter soon become predominant in the cheese, almost entirely excluding the other species. In some of the experiments, the lactic-acid bacteria reached a maximum number of about 900,000,000 per cubic centimeter in two days, constituting then practically a pure culture. This number was maintained throughout most of the ripening Ripening of Soft Cheeses 425 period, but declined towards the end. Peptonizing bac- teria were found in considerable numbers only occasion- ally, such cheeses manifesting a somewhat abnormal ripening, but yielding fairly satisfactory final results. The sour curd formed by the lactic-acid bacteria is a favorable medium for the development of molds. Two of these appear to play a predominant réle in the ripen- ing of Camembert cheese, namely, Penicillium camem- berti and Oidium lactis. The molds gradually reduce the acidity of the curd until its reaction is markedly modified. Enzymes are produced which gradually diffuse towards the center of the cheese, the curd is digested and becomes, to a marked extent, soluble in water, and the transfor- mation thus proceeds until the ripening process is com- plete. Experiments with pure cultures of the molds showed definitely that they are capable of causing these digestive changes when not associated with bacteria. A number of chéeses that were prepared by means of a pure-culture starter and inoculated with Penicilliwm camemberti ripened properly, but showed no distinct flavor. But when Oidium lactis was also introduced in the cheese, there was developed the characteristic flavor. ‘Bacteria or other molds,” says Thom, ‘in many cases modify the flavor of Camembert cheese, but do not seem to produce it independently of the mold. There thus arise characteristic secondary flavors which are associated with the output of certain factories and which command special markets. These varieties are usually more highly flavored than what we have re- garded as typical. The essential relation of the Camem- 426 Bacteria in Relation to Country Lije bert Penicillium and Oidium lactis to the production of Camembert cheese is, therefore, well established.” The ripening of Camembert cheese is, therefore, due to lactic-acid bacteria and molds, the former bringing about the initial changes, the latter modifying the reaction, digesting the curd and developing the flavors. Lactic-acid bacteria, with Penicillium camemberti alone, were sufficient for the ripening and the proper texture but were not adequate for the production of the typical ‘flavor. The latter appeared only when Oidium lactis was also present. As to actual conditions in the manu- facture of Camembert cheese, it still remains to be de- termined whether the miscellaneous bacteria that appear in large numbers in the later stages of ripening are of decided significance. Similarly, in the case of other soft cheeses, molds are almost invariably present. In Roquefort cheese, made out of goat’s or sheep’s milk, lactic-acid bacteria and a characteristic Penicillium, different from Penicillium camemberti, seem to be the. principal organisms concerned. In Stilton and Gorgon- zola, made out of cow’s milk, a green Penicillium re- sembling that of Roquefort is prominent; and the same may be said of Hungarian Brinse, prepared from sheep’s milk. The differences in flavor which these cheeses exhibit must be sought, therefore, in differences of qual- ity and treatment of the materials employed, as well as in the varying predominance of the several groups of microérganisms more or less common to them all. It may be added that the American Brie and Isigny cheeses examined by Thom, contained no trace of Peni- cillium camemberti, but always bore a growth of Oidium Ripening of Hard Cheeses 427 lactis. He concludes that there is in this case associative action between Oidiwm lactis and various species of bacteria. The function of these organisms, as well as of the yeasts that are frequently present, on the ripening of American Brie and Isigny is still a matter of uncer- tainty. Hard cheeses—The growth of molds is excluded in hard cheeses by the strong salt content and the hard- ness and compactness of the rind. Hence, these cheeses, prominently represented by the Swiss Emmenthaler, the English and American Cheddar, and the Dutch Edam, in so far as they are at all affected by micro- organisms, must depend for their ripening, apart from the enzymes pepsin and galactase, on the action of bac- teria. At high temperatures, the numbers of bacteria in hard cheeses evidently decrease more rapidly than they do at lower temperatures, a difference coincident with the rate of ripening. The cheeses kept at the higher temperatures not only ripen more rapidly, and develop a stronger flavor, but soon become over-ripe. It seems, furthermore, that the accumulation of amides and of ammonia goes on for some time after the bacteria had become greatly reduced in numbers. This fact indi- cates, therefore, that if the latter stages of ripening are due to bacteria, they must be carried out by means of enzymes secreted by the organisms. It follows, likewise, that at the higher temperatures the bacterial enzymes are produced early in the ripening process in larger amounts, and are, therefore, enabled to accomplish their work more quickly than it is ac- 428 Bacteria in Relation to Country Life complished at the lower temperatures. Practical studies on the ripening of American Cheddar cheese at different temperatures have shown conclusively that at the lower temperatures the ripening is more uniform, and the keeping quality of the cheese much better. In fact, ripening was shown to be experimentally practicable at temperatures but little above freezing. Commercially, Fig. 65. Section through inflated and Te collapsed Emmenthaler cheese. (1/5 natural size. Freudenreich.) however, this extension of the period of ripening has its limitations. Cheese faults—Abnormal ripening and the conse- quent production of cheese that is not marketable, occasion, at times, large monetary losses to the cheese- maker. Among such faults, the most common is that which leads to the formation of gassy curd. In this case, large quantities of gas are generated by certain gas-producing bacteria and the curd becomes filled with cavities of various shapes and sizes. The surface of the cheese bulges out, and, in extreme cases, the rind is split open. Other abnormal characteristics, particularly Cheese Faults 429 as to taste, accompany this phenomenon and deduct from the value of the cheese. The trouble is due most frequently to one or two lactic-acid species, notably B. lactis aérogenes, and can be partly overcome by the use of larger amounts of a good lactic-acid starter. Other faults that, as a rule, are less troublesome than the preceding, involve the development of objectionable Fig. 66. Section through an inflated Emmenthaler cheese. (Freudenreich.) tastes and odors. In soft cheeses considerable loss is now and then occasioned by yeasts which develop on the surface. The cheese becomes slimy, adheres to the board, and is liable to lose part of its surface containing the mold, thereby interfering with the proper ripening. In still other instances, the soft cheese may be invaded by putrefaction bacteria which change it into an offen- sive slimy mass. More or less loss to the cheese-maker is likewise occasioned by discolorations of the hard cheeses, that is, the formation of black, red, blue, or rusty spots. These are due to molds or bacteria. In all these instances, the remedy must be sought in thor- 430 Bacteria in Relation to Country Life ough cleanliness. When objectionable bacteria, molds, or yeasts, have invaded the premises, it may become necessary, therefore, to resort to sterilization of the utensils and disinfection of the rooms. Again, certain faults occurring in the output of the cheese factory may not be due to microérganisms, but to faulty manipu- lation. An instance of this is found in the manufacture of soft cheeses, when some of the products become too dry on account of the lack of sufficient moisture in the atmosphere of the drying room. PART VIL BacTERIA IN RELATION TO PRESERVATION oF Foop ; CHAPTER XLII BACTERIA IN RELATION TO CANNING THE perishable products of the farm have but a brief existence. The succulent fruits and vegetables decay and vanish; milk turns sour; butter becomes rancid; wine, juices and cider are changed into acid liquids; and meat and eggs undergo putrefaction. Decay and putre- faction, as natural phenomena, were forced upon the attention of man as he emerged from savagery. When he realized that food-preservation and his own well- being were so intimately related, he endeavored to de- vise means for arresting the course of dissolution. The passing centuries taught him that moisture and warmth furnish suitable conditions for the rapid decay of vege- table and animal materials. They taught him that, for a short time at least, boiling will arrest the decom- position of food. They taught him that the addition of substances like salt or saltpeter, or the natural pro- cesses of souring, may be utilized in the preservation of food products. (431) 432° Bacteria in Relation to Country Life With this knowledge came its application. There came the drying or salting of fish and meat; the storing of grain in cool, dry chambers; the pickling of vegetables in tight receptacles. The seasons of plenty were made to contribute to seasons of scarcity, and these contri- butions were made effective largely by the natural re- sistance to decay of dry grains and grasses. Modern research has made clear to us the nature of decay, putrefaction and fermentation, and has helped us to account for the instability of vegetable and ani- mal substances. Knowing, as we do, of the universal presence of microérganisms, and of the conditions suitable for their growth and survival, we have gradually perfected the means for excluding them from our food products. We have thus developed important agricul- tural industries. The principles of canning.—The successful canning of fruits, vegetables, meat and fish depends upon the de- struction of all of the bacteria present in these materials, and the sealing of the cans so that no new invasion by bacteria or other microédrganisms can occur. This is readily accomplished by heating the cans and their contents to a temperature that kills not only the bac- teria themselves, but, also, the most resistant spores. The. complete sealing of the cans previous to heating is inadvisable, for the steam pressure inside of the can might lead to the formation of leaks that would en- courage subsequent infection. Hence, a small hole is punched in the cover, through which the air and steam escape during the heating process. After the steriliza- tion, the hole is closed with a drop of solder and, if the History of Canning 433 work is effectively performed, the contents of the can will remain unchanged for an indefinite period. Development of the canning industry.—An applica- tion of the principle involved in canning was suggested as early as 1782 by the Swedish chemist, Scheele, when he advised the exposure of vinegar to the temperature of boiling water, in order to assure its conservation. Some time after this, the Parisian confectioner, Appert, demonstrated that meats, vegetables and other perish- able products enclosed in sealed vessels could be pre- served by placing them for a short time in boiling water. The practice of canning was thus established prior to the development of modern bacteriology, for Appert’s book, recounting the results of his experience, first ap- peared in 1810. Gradually, the methods of canning were improved; the open tanks, in which the cans were heated to 212° Fahr., gave way to closed vessels, in which steam under pressure made possible the use of temperatures ranging up to 250° Fahr. The higher temperatures are convenient, not merely because they are more effective in destroying the bacteria, but also because they shorten the period of heating. The constant improvement of the mechanical ap- pliances and the growing demand for canned. goods have both contributed to the wonderful development of the canning industries. Tomatoes, corn, peas, beans, asparagus, okra, succotash, squash, pumpkins, and other products of the garden and orchard are now placed before the consumer in a palatable and succulent state at a time of the year when, a generation ago, they were merely the subject of pleasant reminiscence. BB 434 Bacteria in Relation to Country Life The canning industries have been responsible for the growth of skill and intelligence in many farming com- munities, and have, thereby, contributed much toward the development of American agriculture. They have benefited both the producer and the consumer and have indirectly affected the growth and prosperity of other important industries, notably the manufacture of tin- cans, of glassware, and of fertilizers. Losses through imperfect canning.—Occasionally, the elimination of the bacteria from the canned goods is not complete. Spores of resistant species survive the heating process, develop later, and spoil the contents of the package. Such -spoiled packages frequently burst on account of the accumulation of gas in the cans, or merely bulge outward. They are designated as ‘‘swells’’ at the canneries, and their contents, on opening, are found to be decomposed and offensive to the smell. In other instances, the imperfectly sterilized cans do not swell, but the bacteria within them cause the souring of the material. The losses thus occasioned to the canneries are, at times, considerable. Entire shipments are now and then rendered worthless. This involves not only a direct monetary loss, but, also, that of reputation, since the swelling does not become apparent, at times, until after the goods are in the hands of the jobber or of the ‘retailer. Temperatures required jor sterilization—Some fruits or vegetables require higher temperatures than others. For instance, pie-plant will keep with less heating than is required for asparagus, a difference ascribed to the acid in pie-plant. Again, the heat does not pass so readily Sterilizing the Canned Product 435 through a given weight of one vegetable as it does through that of another. With a temperature of 236° Fahr., the center of a two-pound can of peas has been known to attain the maximum in ten minutes, whereas, in the case of corn, this temperature was not attained at the center until after the can had been exposed to 250° Fahr. for forty minutes. Hence, the sterilization of canned corn requires higher temperatures and longer exposure than are required for the sterilization of canned peas. “The bacteria capable of destroying canned goods are not only of different species, but, what is of more im- portance to the canners, the spores of different species are capable of withstanding different amounts of heat- ing. As a result of this, canners who have been processing successfully at a low temperature for a number of sea- sons suddenly find themselves in trouble when a more resistant species gets into the cans.”’ In an outbreak of swelling, two-pound cans of peas were processed at 230° Fahr. for thirty minutes, and the swelling of the cans was noticed in the stock-room in twenty days. The peas in most of the swelled cans emitted a disagreeable odor, “the bodies of the peas were mushy and the skins inflated with gas, like miniature balloons. The liquor was darkened and of a greenish tinge, due to the small particles of the ruptured peas.’’ Large numbers of bac- teria were present in the spoiled cans, while similar unspoiled cans proved to be sterile. The organism that caused this trouble was an anaéro- bic, rod-shaped, spore-forming bacillus, that produced the characteristic swelling when inoculated into cans of sterile peas. Such cans, when kept at blood-heat, 436 Bacteria in Relation to Country Life swelled within twenty-four hours, and, in some instances, the internal pressure was great enough to burst the can. In order to determine its resistance to heat, a number of inoculated cans were heated at 230° for varying lengths of time, sealed in the usual manner, and kept under observation. Nearly all of the cans heated for twenty or twenty-five minutes swelled within ten days. Of the cans heated for thirty minutes only 16 per cent swelled in the same length of time, while none of the cans heated for thirty-five or forty minutes showed any evidence of swelling. ; Further tests with a temperature of 240° Fabhr. demonstrated that at least thirty minutes were neces- sary for the efficient sterilization of the cans, although even with this treatment, a very few showed signs of swelling. An examination of the spoiled cans showed that those that had been heated for ten minutes contained a mixture of several organisms, while those heated for a longer time contained only the characteristic resistant species. A less resistant form that caused the souring of the peas without the production of marked quantities of gas was also found. This form, when inoculated into sterile peas, produced the characteristic souring of the liquid, which assumed a milky appearance. We are taught, thus, that there are organisms in canned goods capable of withstanding temperatures of 230° or 240° Fahr. for varying lengths of time, and that there may be others not yet studied whose resistance to heat is even greater. These organisms constitute, at times, a serious problem for the canner, since he cannot afford Sterilizing by Heat 437 to ruin his goods by excessive heating. A circular letter, sent out to canners in New York State, brought replies from twenty-nine, of whom eleven were of the opinion that a temperature of 240° Fahr. is liable to injure the quality of the peas. The others had observed no bad effects. We must conclude, however, that ex- posures of more than thirty minutes at 240°, or shorter exposures at higher temperatures, are apt to diminish the commercial value of canned vegetables. 4 Ve a z) dy > ee in meat ee and allied products.—1. Bacterium insulosum. ai . . Bacillus carniphilus. 3. Bacterium ruses 4. Bacillus carniphilus. z Bacillus micans. 6. Booitine glaciformis. Bacillus carnis. 8. Bacillus intermittans. 9. Bacillus levis. 10. Bacillus " Zaleuianpieus. 11. Bacillus levis. 12. Bacillus vegetus. (All after Wilhelmy; X 2, 000.) 438 Bacteria in Relation to Country Life Canned meat.—Canned sardines, salmon, or meat, are similarly liable to be spoiled by bacteria in imper- fectly sterilized goods. In the case of canned fish, the organisms develop, at times, in the liquid rather than on the fish itself. Various objectionable tastes and odors are developed, rendering the product unfit for consump- tion. Among the organisms found in spoiled meat or fish, spore-forming species as well as members of the coli group may be noted. Canned milk.—In the case of canned milk, commonly known as condensed milk, complete sterilization of the material is not necessary. The milk is pasteurized and concentrated in vacuum pans after the addition of about 12 per cent of cane-sugar. Condensed milk contains, therefore, about 25 per cent of moisture and 50 per cent of sugar, the remainder being composed of fat, protein and ash. This comparatively small amount of moisture and high content of sugar make conditions unfavorable for the growth of those bacteria still present in the sealed cans. The use of preservatives—Since the enactment of the National Pure Food law, the canneries are no longer permitted to use antiseptics or preservatives in their canned goods without making a direct statement to this effect. They must largely depend, therefore, on heat alone for the proper conservation of their products. They must exercise greater care as to cleanliness, in order that fewer bacteria be present in the cans before heat- ing. They must also determine carefully the safe limit of heating, so that efficiency of sterilization be combined with the greatest economy of time and fuel, CHAPTER XLIII OTHER MEANS OF PRESERVING FOOD PRODUCTS.— PICKLING Foop preservation in the canning industries is made practicable and efficient by the destruction of the micro- organisms by heat and their subsequent exclusion from the sealed can. On a smaller scale, the housewife makes use of the same principle in preserving fruits, berries, and vegetables. She may also eliminate bacterial growth by the addition of larger quantities of sugar. There are, however, still other important methods for the preserva- tion of perishable foods from destruction by bacteria. Low temperatures.—Reference should be made to the extensive employment of low temperatures, particu- larly for the preservation of meat and dairy products. At times, such materials are kept and transported in a frozen state. This is true of meats exported from Aus- tralia and of fish exported from Scandinavia. The effi- ciency of the method is determined, in this case, not by the destruction of the bacteria, but by the more or less complete checking of their activities. Drying.—Another method, more or less efficient, for the preservation of food products, is that of drying. When the moisture content of food products is reduced below 25 to 30 per cent, the bacteria no longer find con- (439) 440 Bacteria in Relation to Country Life ditions favorable for their development. Hence, by drying vegetable and animal substances, we can pre- vent the multiplication of bacteria in them. The pre- servation by cold and by drying ceases to be effective when, on the one hand, the temperature rises above 50° Fahr., as was already noted in the discussion of milk, or when, on the other hand, enough moisture gains access to the dry material to allow bacterial growth. Salting, pickling and smoking.—Still another method of food preservation is based on the salting or pickling of animal and vegetable substances. It is well known that meat or fish, immersed in very strong brine, is more or less effectively protected from bacterial decom- position. Similarly, meat of healthy animals may be preserved by smoking, as is actually done on a large scale at the meat-packing establishments. All these methods are effective and desirable in so far as they permit the exclusion of the destructive bac- teria, or, better still, if they allow the preservation of foods with the retention of the desirable tastes and flavors. Hence, canning, refrigeration, drying, salting and smoking each have a distinct value. From the bac- terial standpoint, however, they aim only at the ex- clusion of microdrganisms, or the suppression of their activities. In some of the pickling industries, more than mere suppression of bacterial growth is aimed at. The value of certain fish products and of dill-pickles and sauerkraut is directly dependent upon certain bacterial changes, which impart to the materials distinct tastes and flavors. Pickled fish.—Bacteriological and chemical studies Pickled Fish 441 of herring brine have shown the latter to contain large numbers of bacteria. The maximum number is attained in fresh herring brine, the organisms ranging from several hundred thousands to a million or more per cubic centimeter. With the increasing age of the brine, this number gradually diminishes to a few thousands or a few hundreds per cubic centimeter. Not all of the bacteria disappear, even in several years, for brines five years old have been found to contain several hundreds of living bacteria per cubic centimeter. It appears, therefore, that there is at first, a rapid increase of bacteria in the fresh brine, and, subsequently, a gradual dying off of the organisms. The bacteria are undoubtedly retarded in their activities by the large proportion of salt in the brine, yet it is certain that they are not entirely suspended, and that the changes pro- duced by them affect the taste and flavor of the fish im- mersed in the brine. Some of the bacteria commonly occurring in herring brine have been isolated and studied in pure culture, and it has been observed that they can still grow in media containing as much as 20 per cent of salt. It is interesting to note in this connection that, notwithstanding the large numbers of bacteria in- the. brine, the fish itself is not penetrated by the organisms. It is only. when the saturated brine is diluted with water that the bacteria begin to multiply again rapidly and, with sufficient dilution, may. attack the fish and induce various putrefactive changes. Sauerkraut.—In the preparation of sauerkraut, the bacterial activities serve a twofold purpose. In the first place, the bacteria and other microdrganisms 442 Bacteria in Relation to Country Life produce certain changes that impart the characteristic taste and flavor to the material. In the second place, the substances thus formed by them inhibit the growth of the common putrefactive germs. Owing to the bac- terial activities in question, the preparation of sauerkraut has grown into an industry of considerable magnitude. On the continent of Europe, there are single establish- lishments whose yearly output amounts to thousands of tons, while in the United States the production of sauerkraut is undoubtedly of growing importance among the minor agricultural industries. Briefly stated, the process of sauerkraut-making is as follows: The cabbage as it arrives at the factory is washed, the outer, green leaves and stem are removed, and the residue washed and shredded. The shredded cabbage is packed tightly into casks, salted and weighted to facilitate the expression of the juice. In this manner, considerably more than half of the 90 per cent of juice present in fresh cabbage is squeezed out. The signifi- cance of this will be appreciated from the fact that the bacterial changes take place in the juice and not in the particles of the shredded cabbage. The tight packing and compression of the latter drives out the air, and the living cells of the cabbage die the more rapidly and part with the soluble substances contained in them, a pro- cess that is further hastened by the salt that is added. The changes that occur after the cabbage is packed into the casks may be classified as follows: (1) Juice- formation; (2) the evolution of gas; (3) souring; (4) membrane-formation on the surface. The changes do not stop here, however, for, if the sauerkraut is kept Jong Sauerkrout 443 enough, the acid gradually disappears again, and finally ‘the entire mass undergoes decomposition. As the juice passes out of the shredded material, the volume of the latter diminishes until it occupies two- thirds of the former space, and, at times, only one-half or even less. In the juice itself, which contains sugar and other carbohydrates, proteins, amides, organic acids, and the like, bacteria and yeasts multiply rapidly. The bacteria that belong to the lactic-acid species, prominent among them being Bacterium lactis acidi (B. Giintheri), change the sugar into lactic acid, while the yeasts change & it into alcohol and carbon Oo dioxid. The latter accumu- \ gy lates in considerable quantities 0 and leads to the character- © % istic foam-formationinfreshly Fig. 68. Microdrganisms of sauer- pickled cabbage. kraut. (Wehmer.) The bacteria and yeasts apparently present, in this case, another instance of associative action, for both seem to derive a benefit from their partnership. With the increasing amount of bacterial change, the lactic acid accumulates in the juice until it reaches a maximum of about 1 per cent and imparts the familiar sour taste to the material. Under commercial conditions, suffi- cient souring may occur to permit the placing of the sauerkraut on the market within two weeks. In most instances, however, it is not marketed in such a short time. The manufacturer knows that there is but little danger of his goods spoiling under the prevailing con- ditions, namely, the acidity of the juice and the 444 Bacteria in Relation to Country Life comparatively low temperatures of the late fall and winter. In the course of time, a membrane or skin is formed on the surface of the sauerkraut. It is found to consist largely of Oidium lactis, and of certain yeasts. These organisms begin to consume the lactic acid formed by the bacteria, finally reducing its amount to such an extent as to permit the growth of decay and putrefaction bacteria and the decomposition of the sauerkraut. It has been observed that at a certain point in this change the juice may be alkaline, while the sauerkraut itself is still acid. Higher temperatures encourage membrane- formation and the destruction of the acid, hence, the sauerkraut may be kept in condition for a longer time when stored in cold cellars. Under the best of conditions, however, the acid will gradually disappear and the sauer- kraut will then spoil. It should be noted at the same time that properly prepared and stored sauerkraut will keep for a year, and can undoubtedly be made to keep longer when the growth of Oidiwm lactis is more or less com- pletely suppressed. The fermentation in the different casks of the same factory is remarkably uniform. Bacterium lactis acidi and Bacillus coli communis appear with great regu- larity in the early stages of the process, as do certain yeasts. Similarly, in the later stages, Oidiwm lactis always becomes prominent. It would seem, therefore, that the organisms present on the cabbages, as well as the com- position of the juice and the prevailing temperatures in the pickling establishments, are, on the whole, fairly constant. Dill Cucumber Pickles 445 Dill-pickles.—The art of preparing dill-pickles was probably unknown in western Europe until late in the Middle Ages. It was introduced there by the Slavs, with whom dill-pickles have long been a favorite food and condiment. The art itself is based on the same princi- ples as that of sauerkraut-making. The sugars and other soluble organic substances in the cucumbers are made to pass out into the liquid in which they are immersed. The juice thus formed undergoes spontaneous fermen- tation through the activities of lactic-acid ferments, principally Bacterium lactis acidi and Bacillus coli communis. In its turn, the lactic acid produced serves to keep out the various putrefactive bacteria that would otherwise destroy the pickles. As in the case of sauerkraut, the acid ultimately decreases in quantity through the activities of Oidiwm lactis and of other molds and yeasts. The conditions finally become favor- able for the growth of the decay bacteria and the pickles are attacked and decomposed. In the case of sauerkraut, the juice in which the microérganisms multiply is pressed out of the cabbage itself, in the preparation of dill-pickles, it is supplied by the addition of water. After the washed cucumbers are placed in the cask, they are covered with clean water (in some cases with boiled water). Aside from the small quantity of salt that is added there are placed in the casks various other substances like dill, laurel leaves, oak leaves or paprica, the one used depending largely upon local preferences. It is important to add enough water to cover the cucumbers entirely and to exclude the air from the 446 Bacteria in Relation to Country Life souring mass. It is also important that the formation of lactic acid by the bacteria take place rapidly, since, the more rapid the accumulation of acid at the beginning of the process, the better the keeping quality of the pickles. Hence, various expedients are employed or have been suggested for hastening the initial souring, such as the maintaining of higher temperatures in the cellar for a day or two; the pricking of the pickles with needles to facilitate the penetration of the juice and the more rapid diffusion outward of the sugar; the addition of sugar that will be transformed by the bacteria into acid; the addition directly of small quantities of lactic acid; or the addition of pure cultures of lactic acid ferments. There is no doubt that some of these expedients may be employed to advantage in assuring a more uniform product and in improving its keeping quality.. This is especially true of the addition of large numbers of vigorously growing lactic-acid ferments. Lactic-acid starters can be prepared in this case by allowing pure cultures of Bacterium lactis acidi to develop in pasteur- ized milk which is to be added subsequently to the pickle casks. On the whole, a better understanding of the micro- érganic processes underlying the pickling of cucumbers, and, similarly, the pickling of tomatoes, apples, melons, and the like, as is practiced on the continent of Europe, will allow a greater uniformity and palatability of product. Among the Slav peoples of eastern Europe, the lactic fermentation of vegetable substances is also extensively employed in the preparation of borsch from beets. PART VIII BactTERIA AND FERMENTATION CHAPTER XLIV BACTERIA IN BREAD-MAKING In the making of ordinary bread, bacteria play a subordinate part. The work of fermentation is accom- plished by yeasts which change the sugar derived from the starch into alcohol and carbon dioxid. On the other hand, in the making of bread from sour dough, bacteria are associated with the yeasts and produce the flavors characteristic of such bread. Large quantities of sour- dough bread are made in certain parts of Europe, where rye flour is used almost exclusively in the baking of bread. It has been found that the bacteria of the lactic- acid group are prominently represented in sour dough. Among the acids formed by them, acetic and lactic acid are present in large proportion, particularly the latter, which is not driven off in the process of baking and im- parts a sour taste to the bread. Bread faults—Apart from the réle played by bacteria in the making of bread, as just noted, microérganisms bear, under certain conditions, a direct relation to what may be termed its keeping quality. It has been observed (447) 448 Bacteria in Relation to Country Life that, in the summer months especially, the interior of bread loaves may become soft and slimy. When the decomposition is sufficiently advanced, the crust sinks, the bread gives off a rather sharp, unpleasant odor, the crumb being then sufficiently viscous to allow its being @ drawn out into long threads. Such viscosity in bread is more ee Ds | A apt to occur when the dough Rn @ has not been previously soured. / One or two bacteria to which the formation of viscous bread \ Soe ore ie is usually due have been iso- We tetgy or sumy dough es lated, and it has been demon- 1. Bacterium panis; X : a 3,000. (Fuhrmann.) 2. strated that viscosity may be Bacillus mesentericus pc nis é 3 viscost; X 3,000. (Em- developed in sound bread by in- merling.) 7 < . oculation with these organisms. The occurrence of this bread fault is favored by moist, warm weather, and is most common in damp, dark bakeries. The corresponding organisms are rarely ab- sent from commercial flours, but may be prevented from becoming prominent and injurious to the baker, by storing the fresh bread in cold closets, or by adding to the dough small quantities of lactic acid or of sour skim-milk. The latter makes the bread sufficiently sour to prevent the growth of the organisms producing viscosity. CHAPTER XLV BACTERIA IN THE SUGAR INDUSTRY THE proportion of crystallizable sugar secured from the juices of sugar-beets, sugar-cane, sorghum and other plants used for the manufacture of cane-sugar, is often reduced by the activities of certain microérganisms. Sugar refiners realized many years ago that*the gelat- inous masses that appear in the juice are a hindrance to its profitable utilization. They knew, also, that these gelatinous masses increase in amount, and that they may, in aggravated cases, convert the entire juice into a gelatinous mucus. The true nature of this substance was not recognized, however, until 1875, when the opinion was expressed by Jubert that it is due to a “ferment,’”’—that it is, in other words, of bacterial origin. A few years later, it was shown that the gelatinous substances consist of swollen and gelatinized cell-walls of bacteria, and the organism was named Ascococcus mesenterioides, renamed Leuconostoc mesenterioides, and, finally, Streptococcus mesenterioides. Streptococcus mesenteroides.—This organism does not always enclose itself in a gelatinous sheath. On media like potato, meat-broth, gelatin and milk-gelatin, it is an ordinary spherical bacterium arranged in chains of ec (449) 450 Bacteria in Relation to Country Lije two or more. However, when growing in media contain- ing cane-sugar or grape-sugar, it produces the character- istic gelatinous mucus. The gelatinous envelope serves as a protection to the organism against unfavorable conditions. It enables it to withstand drying for a long time and makes it, resistant to heat. For this reason, it survives in the heated juice, when other bacteria are destroyed, and retains its ability to cause mischief. The injury caused by Streptococcus mesenteroides is twofold. It destroys a certain amount of sugar by converting it into mucus, and it produces an enzyme, invertin, which changes cane-sugar into grape-sugar, increasing, thereby, the proportion of molasses. It probably finds its way into the sugar refinery with the raw materials employed, for it may be assumed to occur in the soil. Clostridium gelatinosum.—Another organism that is frequently a troublesome pest in the sugar industry is Clostridium gelatinosum. This organism forms resistant spores and is not destroyed when the sugar is heated to from 122° to 158° Fahr. It destroys the sugar with the production of butyric acid and of other substances. Similarly, among the large variety of other bacteria found in the raw juice and derived from the air, water, and soil, there are species that produce organic acids and, likewise, large amounts of gaseous products. It is not at all surprising, of course, that bacteria are numerous in the raw juice, for the latter offers an abundant supply of food to most species. The manufacturer of sugar must, therefore, be constantly on his guard against the in- vasion of the raw juice by microérganisms. CHAPTER XLVI BACTERIA IN THE PREPARATION OF HAY AND OTHER FODDERS THE preservation of animal foods on the farm is made effective by the removal of water from plant sub- stances until the amount of moisture left in them is no longer sufficient for the growth of bacteria. Hay, straw and corn fodder are thus preserved for the future needs of the animals by drying. However, even with these substances, the water may not be removed rapidly enough to check bacterial decompositions. Freshly cut grass gathered in heaps shows a rise in temperature that may be high enough at times to cause serious deterioration in its value as an animal food. As the temperature of the grass in the interior of the heap gradually rises, the number of bacteria increases until the heat is too great for all except the most resis- tant forms. Later, even these are destroyed and the material becomes practically sterile. The question at once arises whether the organisms have anything to do with the heating of the grass or of other vegetable materials placed in heaps. Bacteriologists are not yet fully agreed as to the part played by microérganisms in this process. Some even go so far as to assert that the elevation of temperature may be accomplished without (451) 452 Bacteria in Relation to Country Life microérganisms, and that the phenomenon is due to the activities of the cells of the dying grass, or of the enzymes contained in them. On the other hand, it has been shown that unwashed, moist cotton, when stored in large heaps, undergoes a rise in temperature. The latter rose to 155° Fahr. within twenty-four to thirty hours and after a few days gradu- ally declined. No elevation of temperature occurred in sterilized cotton, but this was induced by moistening the sterilized material with a small quantity of water pressed out of moist, unsterilized cotton. The process is analogous here to that occurring in the heating of hay, and the experiments just referred to would indi- cate that it is of bacterial origin. More recently, ex- periments have been made on the heating of hay in specially constructed vessels that would allow a thor- ough sterilization of the material, and its subsequent inoculation when necessary. As in the case of cotton, no heating occurred in the sterilized samples, yet these manifested a rise in temperature soon after they were moistened with a little infusion from unsterilized hay, from soil and the like. A large number of species have been isolated and studied as to their ability to cause the heating of hay. Sterile material inoculated with such pure cultures failed, in most instances, to undergo the characteristic rise in temperature. In one instance, positive results were secured from inoculation with a certain species of mold. Resistant organisms belonging to the group of thermophile bacteria have also been found in large num- bers in hay heated to 122° to 140° Fahr. It is not at Heating of Hay 453 all unlikely, therefore, that these organisms, capable of growing at comparatively high temperatures, play a direct réle in the changes that occur at elevated tem- peratures in large masses of vegetable materials. The process of heating, as it occurs in moist hay, may, therefore, be described as follows: The soluble substances that pass out of the moist material serve as food for a host of bacteria. The chemical changes thus occasioned, and perhaps also the respiration of the vegetable cells that are still living, lead to temperature elevation. The heat accumulated in the mass of hay destroys the less resistant forms, and, as the temperature rises higher and higher, even the most resistant species are killed off. Hay infusions, inoculated with particles of hay so heated, at times remain sterile, a fact that tends to prove that the process may amount to self-sterilization. Brown hay.—Bacterial activities, in so far as they are at all concerned in the temperature changes that occur in the curing of hay, are utilized in some localities for the preparation of so-called brown hay. In this in- stance, the wilted grass is arranged in large, well-com- pacted piles, and is protected from the rain. The de- velopment of heat becomes manifest at the end of two or three days, when the mass begins to steam. It retains its high temperature for from eight to fourteen days, as may be readily shown by placing a thermometer in a metal pipe driven into the heated material. After the steaming subsides, the hay is allowed to remain undisturbed for several weeks longer. It is then of a pale to a dark brown color, rather firm and dry, and somewhat aromatic. As chemical analyses have 454 Bacteria in Re'ation to Country Life shown, marked losses of dry matter occur in the process of making brown hay. The losses fall most heavily on the carbohydrates and on the amides and soluble pro- tein compounds. The making of brown hay, cannot therefore, be regarded as economical, and the practice is justifiable only in locations where rains are very frequent and interfere with the ordinary process of curing. Under such conditions, the farmer finds the preparation of brown hay a guarantee against spon- taneous combustion. Corn silage.—This substance is one of great economic importance in the United States. Vast quantities of corn are annually stored away in silos and form, later, a palatable and nutritious food for dairy cattle. The process of ensiling corn involves the cutting of the immature plants into small pieces which are then carried into square or circular structures known as silos. The material thus placed in the silos is compacted to exclude the air and is allowed to undergo fermentation. A rise of temperature occurs, large quantities of gas are evolved, and acids are produced which impart a characteristic taste to the material. Careful experiments indicate that for the first two or three days the cells of the corn plants still have life in them. Their vital activities induce chemical changes that are evidently of moment in the making of silage. A part of the starch and sugar is thus transformed into gaseous products, some of the soluble substances are transformed into insoluble modifications, and slight losses of amides, of protein and of fat undoubtedly occur. The changes, in so far as they are of a purely chemical Silage 455 nature, may be attributed to the dying cells themselves or to the enzymes produced within them. It is well known that large numbers of bacteria are present on the corn plants and that they are carried with the latter into the silo. What part do these bacteria play in the process? Do they contribute to the digesti- bility, the taste, or the flavor of the silage? or are they of no significance in silage-making? The data thus far available are not sufficient for a definite answer to these questions. In the investigations conducted at the Wis- consin station, it was demonstrated that the changes that take place in the ordinary silo may occur also when the corn is kept in an atmosphere of ether or chloroform. The product secured in the presence of these antiseptics was much like normal silage except that it contained a smaller proportion of acid. It would seem then that silage may be produced even when the bacteria and other microérganisms are excluded, and that the changes occurring in the silo are independent of bacterial activities. However, it still remains to be demonstrated whether, under practical conditions of silage-making, the activities of the large numbers of microérganisms present are really of no significance in determining the digestibility, taste and aroma of the resulting product. CHAPTER XLVII BACTERIA IN MISCELLANEOUS AGRICULTURAL INDUSTRIES Tue fibers of flax and hemp which serve as raw ma- terial in the textile industries are held together in the plant by means of certain pectin compounds. These must be removed in order to render the fiber suitable for spinning. The retting of flax and hemp.—The removal of the pectin substances is accomplished in practice by means of bacteria, which dissolve all of the organic compounds except the resistant fibers. The organisms in question have been isolated in pure cultures and proved capable of accomplishing the work unaided by other bacteria. Since the germs concerned in the retting of flax and ‘hemp are anaérobic, they do their work when these materials are covered with water, as is done in the actual operations of retting. The preparation of. natto.—Natto is a vegetable cheese made in Japan by fermenting boiled soybeans. The fermenting mass is kept in a warm place for one or two days, at the end of which time it has become filled with vast numbers of bacteria. The material is then found to contain a large proportion of a mucilaginous, viscous substance, which is highly esteemed by the Japanese, (456) Natto, and Other Products 457 The bacterial flora of natto consists at first largely of bacilli, but subsequently spherical forms become promi- nent. : Two rod-shaped organisms, isolated by Sawamura, were found to change boiled soybeans. into a product similar to natto. One of these produced the character- istics taste and aroma, but did not develop a strong viscosity in the beans. The other organism was found to possess a more pronounced ability to form mucilaginous materials, but did not develop as desirable a taste and aroma. The changes produced by these organisms in the preparation of natto were shown to be due to en- zymes secreted by them. Bacteria and agricultural products.—Bacteria are also concerned more or less intimately in the preparation of other products of more or less remote agricultural origin. Among these may be included tanned hides and leather, and the various grades of fermented tobacco. In case of the latter, the exact significance of the bac- terial activities has not yet been definitely established. CHAPTER XLVIII BACTERIAL DISEASES OF FERMENTED LIQUORS THE transformation of sugar into alcohol is a phenom- enon that was well known to the ancients. From time immemorial, advantage has been taken of this knowledge for the preparation of a great many fermented liquors. The organisms chiefly concerned in the alcoholic fer- mentation of sugar are yeasts, and not bacteria, although the ability to produce alcohol is more or less common, also, to bacteria and molds. It is beyond the scope of the present work, therefore, to treat of alcoholic fer- mentations, even though they be of considerable interest to agriculture in their relation to grapes, grain, potatoes, and other raw materials of the fermentation industries. The “turning” of wine and beer.—There are some changes, so-called diseases, to which wine, beer, cider, and other alcoholic beverages, are subject. The manu- facture of vinegar, like these, is due to, or dependent upon, bacterial activities. The turning of wine and beer may be occasioned by acetic ferments, or by lactic fer- ments. The lactic-taint appears most frequently in wine that is still young. The wine becomes turbid, acquires an irritating taste or flavor, and may subsequently be changed to a brown or black liquid. A slimy precipi- tate is formed and gradually accumulates at the bottom (458) Turning of Wine and Beer 459 of the container. Wine thus turned always contains considerable quantities of lactic acid produced by cer- tain lactic-acid ferments. The disease is most liable to occur in wine made from musts that are ‘not, in all respects, normal, particularly from those whose acidity is below the average. The turning of beer is characterized by a gradually increasing turbidity and the development of unpleasant tastes and odors. A deposit is also formed in the course of time. An organism isolated from samples of turned beer and named Saccharobacillus pasteurianus has been found to a cause the disease when inoculated ! into sound beer. The alcohol in | the beer does not prevent the de- velopment of the organism except l ~ * when present in amounts greater ) 2 than 7 per cent. It seems, also, 19. 70, Lacticacid bac teria, used for the that it is affected by the composi- souring of distillery tion of the wort, since a larger pro- $5,000; (b). X 2,700 portion of hop extract either pre- (mmerling.) vents or retards its growth in the beer. The brewers guard against the turning of their product by careful refrigeration and resort to pasteurization when the beer is intended for export to warm countries. For certain varieties of beer, the lactic fermentation in beer wort is encouraged, as is done, for instance, in the brewing of “‘ Weissbier”’ (white beer). Ropiness in wine.—Another disease that leads to the development of ropiness in wine was investigated by Pasteur in 1861. He and other investigators after him 460 Bacteria in Relation to Country Lije found bacteria in ropy wine, to which they attributed the disease, and showed that sound wine may be made to become ropy by the addition of some diseased wine. It is not yet known whether this malady is caused by a single organism, or whether there are several species capable of producing the same result. Wine affected by this disease becomes turbid and gradu- ally more viscid until, like ropy milk, it can be drawn out into threads. The phenomenon is attributed to the transformation of the sugar into mucilaginous substances by the bacteria. Besides this bacterial mucilage or gum, the ropy wine is found to contain a white, sweetish sub- stance called mannite. Wines that contain more than 10 per cent of alcohol are not subject to this disease. Beer wort and beer are similarly subject to ropiness. A number of organisms that may cause such ropi- ness have been isolated; some of them capable of growing only in unhopped wort, others developing in hopped worts and in beers. Worts rich in nitrogenous matter are most subject to this disease, while a high degree of acidity is inimical to the bacteria. The same or similar organisms cause ropiness in cider. Sarcina sickness.—There is still another malady of beer, “sarcina sickness,” caused by spherical bacteria. Additions of hops to the beer, or additions of salicylic acid in small quantities, may be employed for the ‘sup- pression of this disease. Loss of color in wine.—Among the common diseases of wine, of bacterial origin, may be included that which causes the loss of color. The color of red wine is changed to brown. The alcohol is changed into acetic acid, and Diseases of Swine 461 the entire liquid finally undergoes putrefactive decom- position. The disease is more frequent in southern than in northern countries and often involves extensive pe- cuniary losses. The bacterial nature of the disease is proved, not only by the presence of large numbers of bacteria in diseased wine, but, also, by direct inoculation of sound wine with a minute quantity of diseased wine. The presence of large amounts of protein favors the development of the disease, while the presence of any but minute quantities of acid retards it. It has been recommended, therefore, that citric acid be added to young wine as a preventive against the loss of color. Mannitic fermentation—This disease of wine is familiar to wine-growers. It is most prevalent in warm countries, largely because the bacteria causing the dis- ease will develop best at higher temperatures. The dis- ease is characterized by the conversion of the sugar into mannite. Still another common disease, the bittering of wine, has also been traced to bacteria. This disease, which usually affects only red wines, is manifested by a reduction of acidity, the loss of color, and the deposi- tion of a sediment on the bottom and walls of the con- tainer. Objectionable tastes and odors are likewise developed in the wine and finally render it worthless. The diseases of fermented liquors already enumerated by no means exhaust the list. There are others that are similarly responsible for serious injury to stored wine. Climatic conditions, locality, and the methods of manu- facture employed, are factors of importance in influen- cing the character and prevalence of such maladies. Much bacteriological and chemical work has already 462 Bacteria in Relation to Country Life been performed in the attempt to determine the nature ‘and origin of these diseases. Much still remains to be done for their complete elucidation. All of them are of interest to agriculture in that they bear a certain re- lation to the value of vineyards and their crops. CHAPTER XLIX VIN EGAR-MAKING Tue transformation of sugar into alcohol by yeasts and other microérganisms opens the way for still other changes. Apart from the various bacteriological diseases of alcoholic beverages already noted, the alcohol itself is the raw material for the preparation of acetic acid. It is well known that when wine, beer, or cider are left to themselves, they are likely to turn sour in the course of time; that is, they are likely to change into vinegar. Vinegar is, therefore, a sour liquid containing variable quantities of acetic acid made out of alcohol. History of the art.—Old as is the art of vinegar-mak- ing, the true nature of the processes involved was not even suspected in the early days of the last century. In 1837, Kiitzing expressed the belief that the change of alcohol into vinegar is accomplished by living organ- isms. His statement was not accepted, however, as the true explanation of the phenomenon. The chemists, led by Liebig, carried the day, and it came to be believed for a time that the formation of acetic acid is a purely chemical process. This claim was supported by the discovery made by Davy in 1821, that when alcohol is poured on platinum- black the latter becomes very hot and transforms the (463) 464 Bacteria in Relation to Country Life alcohol into acetic acid. Similarly, the spontaneous souring of wine and beer was thought to be accomplished: by the condensation of oxygen in the pores of the mem- brane formed on the surface of the liquid. The mem- brane itself was regarded by the chemists as a lifeless mass of albuminous matter. This purely chemical explanation of acetic fermenta- tion fell to the ground, however, when Pasteur, in 1864, upheld the contention of Kiitzing in 1837. Pasteur demonstrated that the ‘mother of vinegar,” the mem- brane on the surface of souring alcoholic liquids, was composed of minute living cells, to whose activities the formation of vinegar was due. He was not willing to admit, however, that the bacteria were directly con- cerned with the change of alcohol into acetic acid, but thought that they helped merely to condense the oxy- gen in the membrane and facilitated thus the oxidation of the alcohol. Subsequent investigations showed that the micro- organisms must play a direct part in the production of acetic acid. It was found that the transformation was accomplished best at certain temperatures. When the latter was too low, acetic acid was formed very slowly or not at all. When it exceeded 104° Fahr., the formation of acetic acid stopped. Liquids containing more than 14 per cent of alcohol did not undergo acetic fermenta- tion. On the other hand, the oxidation of alcohol by platinum-black was favored by high temperatures, and concentrated alcohol was changed as readily as was dilute alcohol. A further step in advance was made when the acetic ferments were isolated in pure culture History of Vinegar-Making 465 and were shown to have the ability to change alcohol into acetic acid. When the organisms were kept out, this change did not take place. Modern knowledge —Within the last fifteen or twenty years, our knowledge concerning acetic-acid fermenta- tion has been enriched by many interesting facts. New species have been gradually added to the two acetic ferments, Mycoderma aceti and Mycoderma Pastorianum, described by Hansen in 1878, until the list includes now at least fifteen distinct organisms. The different organ- isms show characteristic variations as to the temper- ee é eA atures at which they will Lee grow best; as to develop- veg 2 te ment in liquids of different “ys . ‘ HPre re concentration and compo- oe PO ae oye o sition, and as to the amount ‘ Fig. 71. Acetic-acid bacteria.—1 and of vinegar produced in a 2. Bacteria from sour beer; s . X 1,600. (Emmerling.) 3. Bac- given time. Henneberg terium agelosum; X 2,000, (Em- merling. found, for instance, that Bacterium vini acetati showed a moderate growth at 97° Fahr., while Bacterium cxylinioides and Bacterium orleanense developed to a very slight extent at this temperature. Similarly, some of the species grew best at 77° Fahr., and others at 86° Fahr. It may be concluded, therefore, that the different temperatures prevailing in different vinegar factories will, among other conditions, help to determine which of of the acetic ferments shall predominate. In the same way, it has been found that the various species will be unequally affected by the proportion of alcohol in the DD 466 Bacteria in Relation to Country Life liquid, although they may become accustomed slowly to gradually increasing concentrations until an alcohol content of 10 per cent is reached. Even then, however, the growth is markedly retarded. Thus, it was found by Henneberg, that in the case of Bacterium xylinoides, growth occurred in five days with 0.2 per cent of alcohol; in thirty-two days, with 3 to 5 per cent of alcohol; and in forty-five days with 6 to 8 per cent of alcohol. The differ- ent species are unequally affected, likewise, by the con- centration of acetic acid. According to Henneberg, the greatest amount of acid that would still allow bacterial development under certain laboratory conditions was 9.3 per cent for Bacterium xylinoides; 9 per cent for Bac- terium orleanense; 8 per cent for Bacterium vini acetati, and 10.9 per cent for Bacterium Schiizenbachi. The ‘mother of vinegar.”—An examination of the “mother of vinegar,” or ‘‘mycoderma,” as it was called by Persoon as early as 1822, will show it to consist of small, usually cylindrical cells imbedded in a mucilagi- nous substance. The latter causes the entire mass to form a continuous skin or membrane of variable thick- ness. The organisms imbedded in the membrane may occur singly, in twos, or in chains, and may also undergo more or less striking modifications in shape and size, as the temperature falls below or rises above certain limits. At temperatures below 60° Fahr., the cells become long and exhibit pear-shaped swellings, while at 104° Fahr. they not only undergo variations in shape, but may attain an extraordinary length, occasionally one hundred times that of normal cells. As the temperature is again lowered to approximate Mother of Vinegar 467 the optimum, the long chains break up into the short rods. Apart from temperature, the age of the inocu- lating material and the composition of the liquid are of considerableinfluence in the formation of the extremely long or of the thickened cells. The proportion of acid in the liquid is of importance here, for with increasing acidity the degeneration forms become prominent even at optimum temperatures, and a point is finally reached when most of the cells die off on account of the unfavor- able conditions. Acetic-acid bacteria.—These bacteria are distinguished for their ability to oxidize not only alcohol, but a whole series of other compounds. The products formed de- pend entirely on the nature of the original substance. For instance, oxalic acid is made by them out of grape- sugar, cane-sugar, milk-sugar, and the like, the differ- ent species showing marked variations as to preference for certain compounds. Other compounds made by acetic-acid ferments include glycerine, mannite, butyric acid, gluconic acid, levulose, and various other substances. Moreover, the acetic acid itself made by the ferments out of grain alcohol may be burned up further to car- bon dioxid and water. This accounts for the fact that in sour wine, cider, and similar compounds, the acid accumulates up to a certain point and then begins to decrease in amount until, in some extreme cases, all of it has been used. Under such conditions, the ordinary decay bacteria are no longer kept out by the acid and the liquid undergoes putrefaction. The disappearance of the acetic acid in vinegar is favored by its dilution, for, having changed 468 Bacteria in Relation to Country Life the alcohol into acetic acid, the bacteria proceed to oxi- dize it further. On the other hand, when the vinegar is quite strong, the bacteria and certain yeasts no longer find conditions suitable for their growth, and the acid is not burned up to water and carbon dioxid. Methods of using acetic ferments.—In the commercial . preparation of vinegar, the acetic ferments may be util- ized either according to the Orleans method, or to the so-called ‘‘ quick vinegar’ (German, Schnellessig) method. In the first of these, named after the locality in France where it has long been employed, wine is allowed to turn sour in barrels. The latter have a capacity of about fifty-five gallons, and are provided with two holes near the top, one for the introduction of the wine and the removal of the vinegar, the other for the proper supply of air. The casks used in the process are thoroughly scalded with hot water, and then with hot vinegar. When thus made ready for use, they receive about twenty-two gallons of good, strong vinegar and about one-half gallon of wine. At the end of eight days, a further quantity of wine (somewhat more than one-half gallon) is added, and in eight days more a somewhat larger amount; the addi- tions being continued at similar intervals until the cask contains forty to forty-four gallons. The vinegar is then drawn off to leave about twenty-two gallons in the cask, and about two and one-fifth gallons of fresh wine added. After this, two and one-half gallons of vinegar are re- moved every week and are replaced by the same quantity of wine. When once properly started, the casks may be suitable for a continuous use of six to eight years, at Vinegar Methods 469 the end of which time they require emptying and clean- ing. In the “quick vinegar” method, the change of alcohol into acetic acid is accomplished in vats filled with beech shavings. Grain alcohol diluted with the vinegar is allowed to trickle slowly through the shavings which, on account of the enormous surface exposed, present extremely favorable conditions for the development of the air-loving acetic ferments. We see, therefore, that while in the Orleans method the bacteria multiply at the surface of the liquid, in the “quick vinegar” method they multiply throughout the body of the shavings. When once thoroughly established on the surface of the shavings, the ferrnents of the ‘quick vinegar’’ method work with intense rapidity and accomplish the transformation of large quantities of alcohol in a comparatively short time. Under actual conditions in the factory, various species may come to predominate, among them yeasts and bacteria which oxidize the acetic acid to water and carbon dioxids. The uncertainty as to the prevailing species is well illustrated by some investigations of Henneberg. In his studies of the acetic ferments in two vinegar factories in Berlin, where the Orleans method was employed, he found in casks standing close to one another Bac- terium xylinum, Bacterium cylinoides, and Bacterium vini acetati. In the other factory, the differences were even more striking. Now, since the different species differ not only in the amount of acetic acid produced by them in a given time, but, also, in the quality, that is, the aroma, taste, and appearance of the product, it is 470 Bacteria in Relation to Country Life not to be wondered that a uniform product is so hard to secure. Moreover, the manufacture of vinegar in accordance with the old methods not only makes it difficult to secure a uniform and high-grade product, but is also very wasteful of alcohol. In many instances, the liquid becomes infested with certain yeasts that burn up the acetic acid, and also with vinegar eels which, besides destroying large numbers of bacteria, also detract from the appearance of the vinegar. Pure cultures in vinegar-making.—In order to make vinegar manufacture more certain as to results, it has been proposed that pure cultures be employed. Vinegar eels and injurious yeasts and bacteria would be thus eliminated, and the product would be uniform in char- acter. The species that give the largest yields and pro- duce the best-flavored vinegar could be selected, and the vinegar industry thus placed on a more firm basis. Years ago an attempt was made to render vinegar- making more certain in its results by the employment of a method somewhat analogous to that of natural starters used in cream-ripening. Quantities of wine were allowed to sour in small vessels, and the skin formed on the sur- face was carefully lifted off and placed on the surface of larger quantities of wine which was tg be soured. Of course, care was taken to employ only such skin for inoculation as was, to all appearances, healthy and free from ‘vinegar eels.’ In practice, this method failed to yield satisfactory results, for the simple reason that the spontaneous skin formation in the small vessels was, at times, due to one kind of organisms, and at other times to entirely different organisms, Storing of Vinegar 471 Apart from the possible elimination of the vinegar eels, all of the old uncertainty as to results accompanied this method, and led to its abandonment. On the other hand, the use of pure cultures proper promises to gain favor with vinegar-makers. The difficulties involved are not at all insurmountable, and, as laboratory ex- periments have shown, pasteurization at comparatively low temperatures may be employed to assure the estab- lishment of the desirable ferments. The storing of vinegar—The vinegar itself, like souring wine or cider, is liable to deteriorate on standing. Stored vinegar, as is well known, may lose its flavor, or may become turbid or slimy. ‘The same undesirable organisms that become prominent in the process of vinegar-making may become prominent also in the manufactured vinegar, especially in the home-made product. Practical experience teaches us that the de- terioration of stored vinegar is most marked when the proportion of acetic acid in the liquid is least. Careful investigations have demonstrated that the vinegar eel multiplies only when the proportion of acid falls below 6 per cent. When more than this quantity is present, the growth of vinegar eels is suppressed. Similarly, the acid-consuming yeasts are not allowed to multiply when the proportion of acid exceeds 3 per cent; frequently their growth stops even when the quan- tity of acid is only 2 per cent of the entire volume. On the contrary, the acetic-acid bacteria are much more resistant to large accumulations of acid, some of them continuing to grow when the acid content reaches 11 per cent. Losses of acetic acid may occur under these 472 Bacteria in Relation to Country Life conditions on account of the oxidizing activities of the bacteria. . It has been suggested, therefore, that the pasteuri- zation of vinegar will prevent the loss of acid by the destruction of the non-spore-forming acetic ferments. , Since spore-forming species are excluded by the acid, the heating of the vinegar need not exceed a compara- tively low temperature, say 120° Fahr. The presence of the acid hastens the destruction of the bacteria in the heating process, hence, in stronger vinegar, the organisms will be killed more quickly than in dilute. vinegar. Moreover, the low temperature of pasteuriza- tion will not cause the escape of the volatile substances that impart the aroma to vinegar, nor of the acid itself. The introduction of pure cultures in the manufactur- ing of vinegar promises to place the entire industry on a more certain and economical basis, while the resort to pasteurization for both the raw material and the finished vinegar will prove a boog for the manufacturer, farmer and dealer. The farmer, especially, should re- member that the method of making cider vinegar, as employed by him at present, is too uncertain as to the quality of the product secured, and too wasteful of the ‘alcohol. -There is no reason why pasteurization and inoculation with pure cultures of desirable acetic fer- ments should not be accessible also for him. INDEX AND GLOSSARY Aberson, 184. Acetic acid, 465. Acetic-acid bacteria, 44, 466, 467, 468 Acid media, 44. Acids, organic, 292. Adametz, 376. Aérobic bacteria (bacteria requiring an abundant supply of air), 38, 40, 117, 139, 309, 312, 345. Air, bacteria in, 45-54. Albumoses (compounds formed in the decomposition of proteids), 420. Alfalfa, 131, 230, 231. Alge (minute green plants), 30, 88, 203. Alinit (a commercial culture of bac- teria at one time sold in Germany, and supposedly capable of fixing at- mospheric nitrogen), 235. + Alkali salts, 78. Alkalinity, 44. Altitude, effect of, on soil bacteria, 139. Alum method, 84. American Brie, 427. Amide (Amides, or Amido-compounds substances formed in the decompo- sition of proteids and more simple in composition than albumoses), 417, 420. Amino-compounds, 33. Ammonia, 192, 193, 321, 331, 333; salts of, 172. Ammonification, 161,.162, 320, 324. Ammonifying bacteria (bacteria capa- ble of decomposing nitrogenous sub- stances of animal or vegetable ori- gin, with the formation of ammonia) 162. Ammonium carbonate, 322-324. Ammonium nitrate, 193. Anaérobic bacteria, 40. Aniline dyes, 9. Animalcules, (a name formerly em- ployed to designate all very small living organisms), 71. Animal excreta, tubercle bacilli in, 399. , Anthrax (a bacterial disease usually fatal to cattle and sheep and due to a specific germ, Bacillus anthracis), 8. Antitoxins (substances that can coun- teract the effect of toxins, or poi- sons, produced by bacteria), 10. Appert, 433. Artesian wells, 95. Ascococcus mesenteroides, 449. Attenuated (a term employed to designate cultures of bacteria that have been weakened by unfavor- able conditions of growth), 37, 38. Atwater, 210. Automatic scavenger, 113. Available substances, 182. _ Azotobacter (a group of aérobic bac- teria possessing a very pronounced power of fixing atmospheric nitro- gen), 200-204, 235, 272, 289, 302. Babcock, 419. Bacilli (rod-shaped bacteria possess- ing the power of motion), 15. Bacillus aérogenes, 370, 384, 429; caset E, 373, 374; coli communis 74, 370, 444, 445; cyanogenus, 375; Ellenbachensts, 235; fluorescens lique- (473) 474 faciens, 412, 413; lactis acidi, 372, 373, 381, 384, 443-445; lactis ery- throgenes, 375; lactis viscosus, 376; mycoides, 295; radicicola, 213, 226, 227; subtilis, 319. Backstein cheese, 422. Bacteria (rod-shaped organisms not capable of moving about in the cul- ture solutions. In a broader sense, all microscopical organisms of a certain character) 17; aérobic, 38; ammonifying, 162; and disease, 6; and respiration, 55; carbon com- pounds in, 32; carbon source of, 33; conditions affecting growth of, 36; eylindrical, 13; denitrifying, 166; digestion of food by, 164; discovery of, 2; effect of cold on, 37, — of con- centration of medium on, 42, — of electricity on, 41, — of germicides on, 42, — of preservatives on, 42, — of pressure on, 41, 42, — of reaction of medium on, 43, — of sugar on, 43, — of sunlight on, 40, 41; food requirements of, 30; form and struc- ture of, 13; in air currents, 46; in air, determination of, 47, — in- fluence of altitude on, 55, — in- fluence of climate on, 53, — in- fluence of dry weather on, 52, — in- fluence of season on, 52, — num- bers and kinds of, 48, — of cities, 49, — country, 51, — of Paris hos- pitals, 53, — of polar regions, 51; in atmosphere, 45; in bread, 447; in butter, 411-415, — numbers and kinds of, 412; in canning industries, 431-438; in cheese, 416-430; in cisterns and tanks, 95, 96; in cream, 402-410; in dust, 50; in fermented liquors, 458-461; in filter beds, 121; in hay, 451-453; in herring brine, 441; in ice, 97, 98; in legume nod- ules, 212, 214; in manure, 303-306, 309, 312, 318-345; in mountain air, 54; in peat, 146; in pickles, 445; m yivers and Jakes, 77; in sauer- Index and Glossary kraut, 442-444; in sea air, 51; in sewage, 103, 108; in silage, 454, 455; in soil, 275, 276, — decomposition of humus by, 146, — distribution of, 142, — effect of altitude, lime, manure and tillage on, 189-142, — numbers of at surface, 143, — physiological efficiency of, 160, — relation of, to humus, 144, — trans- formation of nitrogen by, 155, — variations in vigor of, 159; in the sugar industry, 449, 450; in vinegar- making, 463-472; in water, 61-96, — character of, 62, — competition among, 69, — increase and decrease of, 64; influence of, on one another, 166; iron, 299, 302; lactic acid, 370-373, 390, 405, 406, 412, 418, 420-424, 459; nitrate-consuming, 187; nitrifying, 170, 171, 177, 181; nitrogen-fixing, 196-206; nitrogen, source of, 33; nitrogen-transform- ing, 196; numbers and kinds of in butter, 412; numbers of in soil, 137; peptonizing, 164, 418, 425; relation of nitrifying to ammonifying, 181; spherical, 13; sulfate-reducing, 297; sulfur, 294-298; thermophile, 36; tubercle, 217-219, — forms of, 215; tuberculosis, in butter, 413, — in manure, 398, — in milk, 397. Bacterial activities, influence of lime on, 28, — of phosphate on, 282, 284. Bacterial cell, chemistry of, 26. Bacterial flora (the various species of bacteria characteristic of the soil or other natural media), 165. Bacteroids (that is, bacteria-like, small bodies found in the nodules on the roots of leguminous plants), 213. Bacteriology and agriculture, 11. Bacterium mazun, 381; orleanense, 465; prodigiosum, 375, 376, 413; Schutzenbachi, 466; vini acetati, 465, 469; xylinoides, 465, 466, 469; aylinum, 469. Bare fallows, 266, 270. Index and Glossary Barns, bacteria in atmosphere of, 361. Bassi, 6. Beal, 309. Berthelot, 211. Beyerinck, 197, 199, 202, 213. Boeckhout, 372. Bone, phosphoric acid in, 285. Bonnet, 4. Boussingault, 169, 193, 209. Bread, faults, 447; sour dough, 447; viscous, 448. Brie cheese, 422, 426, 427. Broad irrigation, 128-130. Brown hay, 453. Brunchorst, 213. Butter, bacteria in, 411-415; changes occurring in, 411; disease bacteria in, 413; faults, 414, 415; lactic-acid bacteria in, 412; numbers and kinds of bacteria in, 412; rancidity of, 412, 413. Cameron, 114. Camembert cheese, 422. Canning industry, bacteria in, 436; development of, 433; principles of, 432; temperatures required in, 434, 435; use of preservatives in, 438. Cans, swelling of, 434, 436. Carbon, amount of in plants, 147; bisulfide, 272, 329; compounds of, in bacteria, 32; dioxid, 30, 147, 148, 388; disappearance of, from humus, 154; monoxid, 31; restoration of, to air, 147; sources of, to bacteria, 31, 33. Caron, 235, 272. Cell, bacterial, chemistry of, 26; wall, 27. Cellulose, 27; digestibility of, 342; fermentation, 342; fermentation of, in manures, 342; ferments, 342-345. Cess-pools, 105; as sources of pollution, 92; origin of, 104. Cheddar cheese, 427, 428. Cheese, bacteria in, 416-430; lactic- acid bacteria in, 418; multiplication 475 of bacteria in, 420; peptonizing bac- teria in, 418; ptomaine poisons in, 417. Cheese-ripening, 416, 417; effect of chloroform on, 420; enzymes in, 419, 421, 422. Cheeses, hard, 428-430; soft, 423-427; lactic-acid bacteria in, 424; mclds, presence of, in ripening, 425; pep- tonizing bacteria in, 425; ripening of, 424. Chemical methods of sewage-disposal, 107. Chili saltpeter, source of, i74. Chloroform, 272. Chlorophyl (a green coloring matter found in the leaves or stems of plants, which is indispensable for the assimilation of their food under normal conditions), 30. Cholera, 58. Cisterns, 96. City air, bacteria in, 51. Cladosporium butyricus, 413. Cladothrix dichotoma, 301. Clark process, 84; efficiency ‘of, 85. Climate, influence of, on bacteria, 53. Clostridia (boat-shaped cells of cer- tain spore-forming bacteria), 15. Clostridium gelatinosum, 450; pastori- anum, 197-203. Clover, failure of, 206. Coal, anthracite and bituminous, 146. Cocci (spherical bacteria), 15. Cold, effect of, on bacteria, 37. Colon bacillus in soils, 75. Colonies (little heaps of bacteria of one kind, visible to the naked eye), 9. Color, loss of, in wine, 460. Compost heap, nitrate formation in, 339. Conditions affecting bacterial growth, 20, 36. Contact beds (pits filled with coke- breeze, burnt clay ballast, and simi Jar substances; sewage is here ‘ 476 periodically admitted and with- drawn), 117. Contagion, 2. Corn, 383; silage, 454, 455. Cotton cultures, 233; commercial preparation of, 234; defects of, 234; tests of, 234. Cotton, moist, heating of, 452. Country air, bacteria in, 51. Cowpeas, 245; and vetch, 252; wide usefulness of, 247. Cream, bacteria in, 402-410; lactic- acid, bacteria in, 406; pasteurized, use of, with pure cultures, 410; ripened, number of bacteria in, 405; ripening, 402, 404; separating of, on farms, 409. Crenothriz: Kuhniana, 301. Crimson clover, 245; amount of nitro- gen in, 251; nitrogen-gathering, power of, 250; value of, on sandy soils, 249. Crops, fallow, 237; green-manuring, 237; relation of, to soil bacteria, 141; rotations of, 239, 241. Culture medium (any substance or combination of substances that offers suitable conditions for the growth of bacteria), concentration of, 42; moisture in, 38; new methods for preparation of, 43; reaction of, 43. Culture (a growth of any species of bacteria on suitable mete liquid, 234; pure, 9. Dark Ages, condition of agriculture in, 240. a2 Deep wells, bacteria in, 93. Dehérain, 343. Denitrification (the decomposition of nitrates by bacteria with the evo- lution, usually, of nitrogen gas), 161, 183-185, 188, 189, 326-229. Denitrifying bacteria, 166, 187, 188, B27. Diarrhoea, 58, Index and Glossary Diaspora caucasica, 379. Digby, 176. Digestion, losses of elements in, 307. Dill-pickles, 445, 446. Disease bacteria in milk, 397; butter, 413. Diseases of fermented liquors, 458. Domestic filters, 87. Drying of food products, 439.. Dupetit, 184. Dust, bacteria in, 50. Dust particles in air, 45, 46, 50. Dysentery, 59, 60. Eberth, 58. Economy of fallowing, 270. Edam cheese, 427. Effluent (purified, or partly. purified, sewage, running out of filter beds, contact beds, etc.), treatment, 119. Electricity, influence of, on bacteria, 41. Elementary nitrogen, losses of, from mauure, 331. Emmenthaler cheese, 427. Emmerling, 381. Enzymes (chemical compounds elab- orated by animals, plants and bac- teria, or other microérganisms, and indispensable to them for the proper digestion and assimilation of food), 34, 35, 164, 419, 421, 422. Eremakausis (the decay of vegetable or animal substances with a plenti- ful supply of air), 150. Ether, 272. European agriculture, revival in, 241. Evelyn, 176. Facultative aérobes (bacteria prefer- ring to develop in the absence of air, but capable, also, of growing when air is admitted), 40; anaérobes (bacteria preferring to develop in the presence of air, but capable, also, of growing when air is ex- cluded.) 40, Index and Glossary Fallow crops, 237, 266. Fallowing (the cultivation of land which is allowed to remain bare for an entire growing season), defini- tion of, 265; economy of, 270; in ancient times, 265; objections to, on sandy soils, 270; wastefulness of, 271. Fallow soils, 267, 268; bacteria in, 273. Fallows and aération, 269; bare, 266, 270, 272; characteristics of, 267; conservation of moisture in, 269; elimination of, 267. Farms, abandoned, 271; sewage, 131. Farmyard manure, losses from, 306. Fats, 28. Fermented liquors, bacteria in, 458. Ferments (substances or organisms capable of causing fermentation, that is, chemical change in organic materials, which is rather intense in character), nitric, 171. Filter beds (masses of coke, broken stone, clinkers, sand, and the like, arranged in layers of varying de- gree of fineness and employed for the filtration of water or sewage), 82, 83. Filters, domestic, 86, 87, 121, 122; efficiency and temperature, 120; working capacity of, 124. Filtration of sewage, 116; of water, 81, 82, 83. Finishers (chemicals that may be used to purify still further the filtered sewage), 134. Fish, 440. Fisher, 50. Flagella (long, thread-like appendages on the bodies of bacteria employed as organs of locomotion), 17. Flax, bacteria in the preparation of, 456. ; Floats (finely ground raw phosphate rock), 284, 285. Flora, bacterial, 165. Food, absorption of, by bacteria, 164; 477 digestion of, by bacteria, 164; products, pickling of, 439; require- ments of bacteria, 30. Foods, exclusion of preservatives from, 440; preservation of, and bacteria, 431. Fore-milk, bacteria in, 360. Form and structure of bacteria, 13. Formaldehyde, use of, in milk, 393. Forcing effects of green-manures, 263. Forests, influence of, on bacteria in air, 51. Fraser, 362. Freudenreich, 318, 418. Fruit, keeping quality of, and green- manures, 261. Galactase (an enzyme found in cow's milk, and of considerable impor- ance in the ripening of cheese), 419, 422. Gammelost, 422. Gayon, 184. Gelatine, use of, in plate methods, 9 Germicidal power of milk, 382, 383. Germicides (chemical substances de- structive to bacteria even when used in comparatively small amounts), 42. * Gilbert, 193, 209, 211. Giltay, 184. Glycogen (a kind of sugar found in the liver, and, also, in some species of bacteria), 27. Goodwin, 310. Gorgonzola, 422, 426. Granluose (a starch-like substance found in some species of bacteria). 28. Green-manuring, 237; practice of, 242. Green-manures, 237; accumulation of nitrogen by, 261; decomposition of in the soil, 255; depth of covering of, 259; draught on _ soil-moisture, 244; effect of on keeping quality of fruits, 261; — on soil bacteria, 244, — succeeding crops, 247, 259; 478 forcing effect of, 263; for loam and clay soils, 253; functions of, on light soils, 254; legumes suitable for heavy soils, 259; leguminous, value of, origin of, 239; nitrogen content of, 245; on sandy soils, 241, 243; relation to soil-humus, 237; smaller returns from on heavy soils, 259; value of, for soil-improvement 242. Grotenfelt, 362, 367, 408. Gypsum (sulfate of lime), 295, 297, 348. Hall, 312. Haubner, 342. Hauser, 465. Hay, bacteria in, 451; brown, bac- teriological activities in, 453; freshly cut, rise of temperature in, 451; heating of, 452, 453; preparation of, 451; relation of bacteria to, 452. Health and disease, relation of water to, 56. Hellriegel, 195, 210, 212, 213, 221. Hemp, bacteria in the preparation of, 456. Henneberg, 307, 465, 466, 469. Herring briné, bacteria in, 441. Henle, 6. ‘ Higgs, 106. Hilgard, 279. Hiltner, 227, 229, 268, 272. Hippocrates, 57. Hippuric acid (a nitrogenous sub- stance found in considerable pro- portion in the urine of herbivorous animals), 321. Hoffmann, 5. Hospitals, bacteria in the air of, 53. Humic acids (sour substances found in humus), 151. Humus, decomposition of by bacteria, 146; disappearance of carbon from, 154; exhaustion of in cultivated soils, 152; formation of in moor and heath soils, 152; in sandy soils, 153; nitrogen compounds in, 156; phos- Index and Glossary phoric acid in, 283, 284; proportion of in acid soils, 145; rate of decay of, 149; raw, 145; relation of crop-ro- tations to, 239, — bacteria to, 144, — green-manuring to, 238, — potash to, 291, — to water-holding power of soil, 144. Hydrolysis (chemical change prcm- inent among others in the decom- position of proteids by bacteria, 118. Ice, bacteria in, 97, 98. Ice, use of, in the transportation of milk, 395. Immunity (ability to resist infection), 1l. Impermeable, 29. Inoculation (the introduction of definite species of bacteria, or other microérganisms, into animals, plants, or any media presumably suitable for their development), on various soils, 224; soil, cotton cul- tures, 233, 234, — history of, in U.S., 230, — liquid cultures, 234, — Moore’s method, 232, — with alinit, 235,— with pure cultures, 226,— method of securing, 226; soil 222, — defects of, 225, — diseases intro- duced by, 225, — lessons taught by, 225. Intermittent filtration (that is, not continuous filtration), 116. Intermittent sterilization, 24. Involution forms (bacterial cells of irregular or abnormal shape, and size, usually due to unfavorable conditions of growth), 15. Iron bacteria, 301; as geological agents, 302; importance of, 301; influence of, on azotobacter, 302; potential energy in, 300; produc- tion of nitrates by, 195; relation of bacteria to, 299, 300; relation of decay bacteria to, 302; rust, 300; soluble compounds of, 301; sul- fate of, 299. Index and Glossary Irrigation, broad, mittent, 128, mixed, 128, 130. Isigny cheese, 426. inter- 128; 128-130; 130; kinds, Jensen, 408, 419. Jubert, 449. Kainit, 350. Kalantarjanz, 381. Kaserer, 172, 333. Keeping quality of milk, 382-385, 387, 388. Kefir (cow's milk, fermented by means of Kefir grains, which are composed of a mixture of certain yeasts and bacteria), 378, 379; grains, 378. Khiin, 204. King, 268. Klebs, 7. Koch, 8, 9, 58. Kohn, 8, 9. Kumiss, (originally prepared by fer- menting mare’s milk by means of a@ mixture of yeasts and bacteria), 379, 380. Kuhlmann, 169. Katzing, 463. Lactic-acid bacteria (organisms capa- ble of transforming milk-sugar, or other sugars, into lactic acid), 370- 373, 390, 405, 406, 412, 418, 420- 422, 424, 459. Lactic acid, formation of, pickles, 445, 446. Lakes and ponds, circulation of water in, 88; pollution of, 89; sedimenta- tion in, 88. Lakes, bacteria in, 77. Lawes, 193, 209. Leéuwenhoek, 2. Legumes. as green-manures, 239-259; cause of soil-enriching qualities of, 207; entrance by nodule bacteria, 217; limitations of, 206; nodules, bacteria in, 212; tubercles, arrange- in dill- 479 ment, of on roots, 214; value of, 206. Lemaire, 7. Liebig, 192, 208, 209, 460. Limburger, 422. Lime, bicarbonate, 277; causes of migration of, 277; effect of, on soil bacteria, 142; importance of, 279; influence of, on azotobacter, 201, 280; — on bacterial activities, 280; in manure, conservation of, 349; losses of, from soil, 278, 279; phos- phate, 281, 286; relation of bac- teria to, 276,—of potash to, 291,— to nitrifying power of soil, 179; re- moval of, to the sea, 277; required for nitrification, 179; restoration of, by bacterial activities, 280; solubility of, 277; sulfate, 196, 295. Limestone, decomposition of, 276; soils, 201. Liming of soils, 278. Liquid cultures, 234. Liquid manure, ammoniacal fermen- tation of, 322. Lister, 7, 11. Litter, relation of bacteria to, 346; resistance to decay, 305. Lockjaw bacillus, 40. Losses from farmyard manure, 306. Loss of color in wine, 460. Maercker, 311, 335. Magnesia, relation of, 276, 277. Malaria, 58. Malpighi, 212. Mannitic ( fermentation of wine unde- sirable changes in wine, caused by bacteria and accompanied by the formation of a white crystalline substance, mannite), 461. Manure, aérobic bacteria in, 309; ammonification in, 320, 324; bac- terial changes in, 303, 304; barn- yard, bacteria in, 303; cellulose fer- mentation in, 342-344; changes of to bacteria, 480 bacterial species in, 319; character of bacteria in, 319; compacting of, 309; composition of, 304, 305; con- ditions affecting bacterial decom- position of, 320; conservation of, 349-356; cost of organic matter in, 316; danger of denitrification from, 329; decomposition of, 313; de- nitrification in, 326, 327; denitri- fying bacteria, carried by, 188; denitrifying, power of, 328, 329; depressing effect of, on nitrifica- tion, 186; farmyard, losses from, 306, 308, 309; formation of ammon- ium carbonate in, 322; formation of gases in, 344; growth of bacteria in, 306; importance of proper stor- ing of, 308; increase of insoluble nitrogen in, 334, 335; influence of, on soil bacteria, 141; liquid, am- moniacal fermentation of, 322, — formation of ammonia in, 321, — separate collection of, 312; losses of ammonium carbonate from, 323, — of elementary nitrogen from, 330-332, — of nitrogen from, 310, 311, 312, — of organic matter from, 309; measurement of temperature of, 355; mechanicai constitution of, 304; nitrates in, 326; nitrification in, 337, 338, 340; numbers of bacteria in, 318; organic matter in, 307; tem- perature of, 314; value of, 307; — of lost portions from, 315,—of or- ganic matter in, 316. Marshall, 373. Marsh gas, 151, 343, 344. Matzoon (fermented milk, originally prepared in Armenia by means of a mixture of certain yeasts and bacteria), 381, 382. Meat, canned, 438. Mechanical methods for conservation of manure, 30, 356. Membrane-formation, 19. Methane (or marsh gas, an inflamma- ble gas occurring in swamps, petrol- Index and Glossary eum wells, volcanoes and cval mines), 31, 344. Michel, 58. Milk, as a food, 357; beverages, 378; bitter, 376; blue, 374; canned, 438; cooling of, importance of, 384; eurdling of, 370; diphtheria and scarlet fever in, 401; dirt in, 362, 363; disease bacteria in, 397; elimi- nation of bacteria from, 386, 387; faults, 374; germicidal power of, 382, 383; keeping quality of, 382- 384, 387, 388; kinds of bacteria in, 369; machine-drawn, bacteria in, 365, 366; numbers of bacteria in, 366, 382, 395; pails, 361, 365, 387; pasteurization of, 389, 391; pre- servatives, 392; progress in produc- tion of, 358; red, 375; ropy, 376, 377 suitability for bacterial growth, 358; source of bacteria in, 360; spontane- ous souring of, 373; sterilization of, by heat, 388, 389; strainers, 367, 368; transportation and distribu- tion of, 394, 395; treatment of, with chemicals, 392; tuberculosis, bacteria in, 397; typhoid bacilli in, 400; typhoid infection of, 59; utensils, 385. Miquel, 49, 51, 52. Mixed irrigation, 128, 130. Moisture in fallow soils, 268. Molds in Camembert cheese, Roquefort cheese, 426. Moore’s method, 232. Moors, formation of humus in, 152. Mother of vinegar (a heavy membrane formed on the surface of alcoholic liquids which are being changed into vinegar—acetic fermentation), 464, 466. Motile bacteria (bacteria that pos- sess the power of motion), 19. Motility, 17. Mountain air, bacteria in, 5, 54. Mouras, Automatic Scavenger, 113. Miiller, 115. 425; Index and Glossary 481 Muntz, 115, 116, 169, 195. Mycoderma aceti, 465; pastorianum, 465. Natto (a vegetable cheese made in Japan out of soy beans), bacteria in, 456, 457. Natural starters (impure cultures of lactic-acid bacteria secured by al- lowing milk to turn sour spontan- eously), 407, Needham, 2. Nitragin, cause of failure of, 228; failure of, 228; further studies with, 228; gratifying returns from, 229; improvement of, 228; new, 235; preparation of, 227; recent ex- perience with, 229. Nitrate (or nitrite, a salt resulting from the union of nitric acid with a base. Nitric acid has three equiva- lents of oxygen, HNO3. Nitrous acid has only two equivalents of nitrogen, HNOo, and the union of this with a base produces a nitrite. Nitrate of soda is NaNO3,—Na standing for sodium, N for nitro- gen, O for oxygen. Nitrite of soda is NaNOg.), accumulation of, in manures, 340; formation of, 176, 178, — in compost heaps, 339, — in electrical discharges, 195; in gunpowder-making, 168; leaching of, from soil, 158, 194; loss of, in soils, 174; of ammonia, 193; of potash, 175; of soda, consumption of, in 1903, 175; production of, by iron, ozone, and sulfate of lime, 195, — by organic matter, 184; relation of, to plant growth, 175; removal of, in drainage, 177; utili- zation of, by plants, 194. Nitric ferments, 171. Nitrifying bacteria, 170, 181; culture media for,-171; development of, in deeper soil layers, 177; in sand filters, 83. EE Nitrification (the gradual change of nitrogenous yegetable or animal substances into nitrate), 160; bac- teriological nature of, 195; charac- ter of, 169; conditions, affected by, 178; definition of, 168; depressing effect on, of manures, 186; effect of crop on, 182; importance of, 172; in different soils, 173; in manure, 337, 338, 340; lime required for, 179; study of, at Rothamsted, 176. Nitrites, 171. ° Nitrobacter, 171. Nitrogen, accumulation of, in the soil, 261; addition of, to soil, by azotobacter, 204; amount of, in atmosphere, 155, — in crimson clover, 251; and bare fallows, 272; available and unavailable, 335, 336; conditions affecting availability of, 160; content of, in productive soil, 191; elementary, losses of from manure, 330-332; fixation, 199, 205, 209, 211; fixing bacteria (or- ganisms capable of causing the nitrogen gas of the air to combine with other elements. This property enables them to enrich the soil in nitrogenous substances), 196, 199, 200, 202, — in filter beds, 122; gas, utilization of, by plants, 195; hunger, 217; increase of, in manure, 334; insoluble, 334; in soil-humus, 156; in subsoil, 156; loss of, in con- tinuous growing of wheat, 179; loss of, in-soil, 157; losses of, in filter beds, 122; proportion of, -in soil, 155; soil, 190, — bacteriologi- cal efficiency in transformation of, 161; soil, increase of, 190; source of, for bacteria, 35; source of, in soil, 155; theory of source of, to plants, 208; transformation of, by bacteria, 155; transforming bacteria, 196, 197; utilization of, by bacteria, 190. Nitrogenous materials, availability of, 181, 482 Nitrosococcus, 171. Nitrosomonas, 171. Nobbe, 227, 233. Nodules (little swellings that are formed on the roots of legumes through the agency of a certain species of bacteria), depth of for- mation, 259; nature of, 213; on roots of legumes, 210, 212, 217. Non-motile bacteria, 19. Oidium lactis, 413, 425-427, 444. Omelianski, 344. ' Organic acids (sour substances con- taining the element carbon. Car- bonic acid is usually not included among these.), effect of, on rock decomposition, 292. Organic matter, destruction of, in water, 67, 81; oxidation of, in ani- mal body, 307; production of ni- trates by, 184; quality of, in water, 78; relation of, to denitrification, 189. Orleans method, 469. Oxidation of ammonia, 331, 334. Oxygen, relation of bacteria to, 38. Ozone (a modification of oxygen, found in minute quantities in the atmosphere, and produced aarti- ficially by chemical or electrical methods), 86, 195. Pasteur, 5, 169, 390, 459. Pasteurization (the heating of mater- ials, usually to 140° to 165° Fahr., a temperature sufficient to kill the bacteria themselves, but not their resistant spores), 23, 24, 389-391, 472. Pathogenic bacteria (capable of caus- ing disease), 23. Peat, number of bacteria in, 146. Penicillium camembertt, 425, 426. Pepsin, 34, 422. Peptones (substances akin to al- 11, 49, 115, Index and Glossary bumoses, derived from the decom- position of proteids), 420. Peptonizing bacteria (organisms pro- ducing enzymes that lead to the breaking down of proteids and the formation of albumoses, peptone, amides and ammonia), 418, 425. Permeable, 29. Persoon, 466. Pfeiffer, 332, 350. Phosphate, fertilizers and bacterial activities, 284; lime, 281, 286. Phosphates, influence of, on humus, 282; relation of, to soil bacteria, 281; soil, relation of, to carbon dioxid, 282; soluble, production of, by bacteria, 282. Phosphorescence, 35. Phosphoric acid, action of, 289; availability of, in bone, 286; availa- bility of, in Thomas slag, 287; in bone, 285; in humus, 283; in manure 289; relation of, to soil bacteria, 289; solution of, in soil, 281. Phosphorus, in humus, 284; inorganic, and organic, 282; relation of, to soil bacteria, 282. Photobacteria (bacteria that cause phosphorescence in the media in which they are growing), 35. Physiology of bacteria (the chemical changes effected by bacteria in their life processes), 5. Pickles, 445, 446. Pigments, 35. Plant-food, loss of in drainage, 126. Plant-food, removal of, to the sea, 276. Plate methods (the isolation of bac- teria from colonies on gelatin, agar, and similar materials that can be spread out and solidified by cooling on glass plates, or shallow glass dishes), 9. Plenciz, 6. Popov, 342. Port de Salut, 422, Index and Glossary Potash, carbonate of, 253; character of, in soil, 290; influence of carbon dioxid on, 290; relation of humus to, 291; relation of lime to, 291; re- lation to soil bacteria, 292, 293; relation to weathering, 290; salts, influence of, on soil bacteria, 292. Prazmowski, 213. Preservatives, 42, 438. Pressure, influence of, on bacteria, 41, 42. Proteins (complex organic substances containing about 16 per cent of nitrogen), 32. Protoplasm, (the active substance of living cells in which the chemical reactions necessary for organized life take place), 17, 27, 29. Protozoa (minute, one-celled animals), 72 Pseudomonas, 19. Ptomaines, 417. Pugh, 193. Pure cultures (the growth of any sin- gle species of microérganisms in suitable media), in vinegar-making, 472. Quick vinegar method, 468, 469. Rancidity of butter, 412, 413. Reaction of soils, 44. Rennet, 419. Resistance, 11. Respiration and bacteria, 54, 55. Ripening of cheese, 416, 417, 419-422; cream, 402, 404. Rivers and sewage, 105; bacteria in, 77; Pollution Act, 106; self-purifi- cation, of 79. Rod-shaped forms, 15. Ropy wine, 460. Roquefort cheese, 422, 426. Rotation of crops, 239, 241. Rothamsted soils, azotobacter in, 204. Russel, 310, 419. 483 Rye grass, 131. Rye straw, 30. Saccharobacillus pastorianus, 459. Saccharomyces pastorianus, 381. Salting of foods, 440. Saltpeter, origin of, 168. Sand filters, 82, 83. Sand vetch, 251. Sarcina, sickness of wine, 460. Sauerkraut, bacteria in, 441, 4438; preparation of, 442; presence of lactic acid in, 443. Scarlet fever and milk, 401. Schloésing, 115, 116, 168, 195. Schneidewind, 355. Schoenbein, 184, 193. Schroeder and Dush, 5. Schroeter, 7. Schubert, 206. Schultz, 242, 259, 261, 288. Schultze, 4. Schwann, 6. Sea-air, bacteria in, 51. Sedimentation (the gradual settling out of solids suspended in water), 72, 73, 88. Self-purification (the natural tendency for the decomposition and disap- pearance of organic substances in streams or other bodies of water), of streams, 66, 79. Septic tank, 114, 117, 118, 122. Septic tanks (covered or uncovered pits in which a large portion of the organic matter in sewage is decom- posed by anaérobic bacteria), in- oculation of, 121; kinds of bacteria in, 121. Settling basins (tanks, pits, or de- pressions in the ground, in which suspended solids in sewage or water gradually settle out), 81. Severin, 306. : Sewage, and rivers, 105; bacteria in, 103, 108; bacterial purification of, 112, 122; clogging of soil with, 129; 484 composition of, 110; decomposi- tion, aérobic and anaérobic action in, 117; disposal, bacteriological methods of, 107, — chemical methods of, 106, 107, importance of, 110, — problem of, 103, 109; examination of, 111; factory wastes in, 123; farming, objections to, 133; farms, 112, 127, 131; intermittent filtration of, 115; irrigation, value of, 125, 127; preliminary treatment of, 132; purification, relation of temperature to, 118; purification, sanitary efficiency of, 134; source of, 110; spent liquors in, 124; steri- lization of, 108; value of, per ton, 127. Shape of bacteria, 15. Silage corn, 454. Size of bacteria, 17. Sludge (the material that settles out of sewage, or is precipitated out by means of chemicals), 126, 132. Smoking of foods, 440. Soft cheeses, 422; lactic-acid bacteria in, 424; peptonizing bacteria in, 425; production of, in the U. S., 423. Soil, aérobic species in, 139; air, com- position of, 150; anaérobic species in, 139; bacteria, effect of lime on, 142, — influence of tillage on, 141, — numbers of, at surface, 143, — physiological efficiency of, 160, — relation of crops to, 1389, — varia- tions in vigor of, 159; clogging of, by sewage, 129; colon bacillus in, 75; distribution of nitrogen in, 199; fertility and bacteria, 135; formation of nitrates in, 178; gases, 151; humus, decomposition of, by bacteria, 146, — nitrogen com- pounds in, 156; improvement, 242; inoculation, 221-226, 230, 236; leaching of nitrates from, 158, 174; loss of nitrogen in, 157; nitrifying power of, 179, 180; nitrogen, in- Index and Glossary crease of, 190; numbers of bacteria in, 137; proportion of nitrogen in, 155; removal of nitrogen from, 191; source of nitrogen in, 155. Soils, ammonifying power of, 167; arid, proportion of humus in, 145; cultivated, exhaustion ‘of humus in, 152; fallow, 267, 273; greenhcuse and market-garden, denitrification in, 188; liming of, 278; sandy, green- manures in, 241, 243, — humus in, 153, — value of crimson clover on, 249; sterilized, carbon dioxid in, 148. Souring of canned vegetables, 436. Soybeans, 230, 245. Spherical bacteria, 13, 14. Spiral bacteria, 13, 14. ; Spirilla (spiral-shaped bacteria), 17. Spontaneous generation (the forma- tion of living organisms out of dead matter), 3, 4. Spore-formation, 22. Spore-forming bacteria, 23. Spores (certain cells, formed in some species of bacteria, and capable of withstanding adverse conditions much better than the bacteria themselves), 22; discovery of, 5; vitality of, 23. Starters, building up of, 407. Starters (pure or impure cultures of lactic-acid bacteria added in con- siderable proportion to cream that is to be ripened), definition of, 406; natural, 407, 409; preparation of, 407, 408; pure culture, 407-410. Sterilization (the complete destmuc- tion of bacteria and other micro- organisms in any material), 23, 24; by superheated steam, 25; of milk, 388, 389; under increased pressure, 25, Stilton cheese, 422, 426. Stocking, 365. Stored water, bacteria in, 82. Storer, 127. Index and Glossary : Stormer, 268, 272. ; Straw, denitrifying bacteria in, 187. Streptococcus mesenteroides, 449, 450. Strippings, bacteria in, 360. Stutzer, 313, 350. Subsoil, nitrogen in, 156. Sugar, 32, 43. Sulfate of iron, 299; -reducing bac- teria, 297. Sulfates, influence of soil bacteria, 299. Sulfur, 28, 293; bacteria, 294, 298; relation of, to bacterial develop- ment, 298; transformation of, by bacteria, 295. Sulfuretted hydrogen (a gaseous sub- stance composed of the elements, hydrogen and sulfur), 293, 297; production by Proteus vulgaris, 295. Sulfuric acid, in manure conservation, 349. Sunlight, influence of, on bacteria, 41, 71. Superphosphates (fertilizers made out of ground phosphate, rock, or bone, by treatment with sulfuric acid), 287; in manure conservation, 349. Swelling of canned vegetables, 436. Sweet-cream butter, 403. Sweet clover, 231. Symbiosis (the living together of two distinct organisms with benefit to both, as, for instance, in the case of legumes and their nodule bac- teria), 197. Symbiotie nitrogen-fixing bacteria, 2C6. Tanks, 95. Tanning industry, bacteria in, 457. Temperature, relation of bacteria to, 36. Thaer, 266. Thermophile bacteria (organisms de- veloping only at comparatively high temperatures), 36. 485 Thom, 425, 426. Thomas slag (a by-product in the re- fining of iron ore, containing phos- phorus. The slag, when finely ground, is a valuable source of phos- phoric acid), 287, 288. Tillage, influence of, on soil bacteria, 141; relation to denitrification, 189. Toxins (poisons produced by bacteria, or other living organisms), 10. Transportation of milk, 394, 395. Tubercle bacteria (the organisms in the nodules on the roots of legumi- nous plants), forms of, 215; return of, to the soil, 215. © Tubercle-formation, 216. Tubercles, arrangement of, on roots, 214. Tuberculosis bacteria in animal ex- creta, 398; in butter, 413; in milk, 397, 399. Tull, 206, 207, 266, 339. Typhoid, 58; bacteria in butter, 414, — in ice, 97, — in milk, 400, — in sterilized. and unsterilized water, 70; epidemics, cost of, 102; infec- tion and milk, 59. suppression of, Udder, bacteria in, 360. Urease (an enzyme produced by cer- tain bacteria, and capable of trans- forming the substance urea into ammonium carbonate), 323, 324. Uric acid (a nitrogenous compound found in the urine of animals), 321. Uro-bacilli, 322. Uro-bacteria (organisms in the decomposition of urine), 324. Utensils as a source of bacteria in milk, 364. prominent 322 Vegetables, use of, in canning indus- tries, 433. Vetch, 251, 252. Vinegar, eels, 470; formation, chemi- 486 Index and Glossary cal explanation of, 464; making, 463, 470, 472; pasteurization of, 472; storing of, 471. de Vries, 372. Virulence (the pronounced power of bacteria to produce severe attacks of disease. The term is also em- ployed in the case of tubercle bac- teria to denote a pronounced power of tubercle-formation.), increase of, 218, 219; influence of nitration, 219. Voélcker, 311. Wagner, 185, 312, 321, 329, 330, 332, 335. Warrington, 116, 170. Water, alkali salts in, 78; alkaline, 78; as a source of disease, 57; bacteria, competition among, 69; bacteria, in winter and summer, 69; charac- ter of bacteria in, 62, 65; cisterns, 95, 96; composition of, 77; colon bacillus in, 76; destruction of or- ganic matter in, 67, 81; dilution of, 73; increase or decrease of bacteria in, 64; in lakes and ponds, 88; num- ber of bacteria in, 68; peaty, 78; proportion of organic matter in, 66; purification of, 84, 85, — by ozone, 86, — by the Woolf method, 86; quality of organic matter in, 78; relation of, to health and disease, 56; self-purification of, 80; sewage bacteria in, 61; sewage contamina- tion of, 73, 74; shed, inspection of, 100; soil bacteria, in 61; storing of, 68, 81; supplies, ancient, 56, — bacteriological examination of, 102, — chemical examination of, 100, — sanitary examination of, 99; table, 92; well-, bacteria in, 90, — bright- ness of, 91, — number of bacteria in, 94, — pollution of, 91, — ty- phoid mortality from, 90. Waxes, 28. Wells, artesian, 95; deep, 93; driven, 94. Wickstead, 106. Wilfarth, 211, 212. Wine, mannitic fermentation of, 461; ropiness in, 459, 460; turning of, 458. Winogradsky, 171, 195, 197, 198. Wollny, 268. Woolf method, 86. Woronin, 212. Wutrich, 318. Wyeert, 9. Yeasts, 444; in vinegar, 470. CYCLOPEDIA OF AMERICAN AGRICULTURE Edited by L. H. BAILEY Of Cornell University, Editor of “Cyclopedia of American Horticulture,” Author of “Plant Breeding,” “‘Principles of Agriculture,” etc. WITH 100 FULL-PAGE PLATES AND MORE THAN 2,000 ILLUS- TRATIONS IN THE TEXT—FOUR VOLUMES—THE SET- CLOTH, $20 NET—HALF-MOROCCO, $32 NET—CARRIAGE EXTRA Volume I—Farms The Agricultural Regions—The Projecting of a Farm—The Soi! Environment—The Atmosphere Environment. Volume II-—Crops The Plant and Its Relations—The Manufacture of Crop Products~ North American Field Crops. Volume IIIJ—Animals The Animal and Its Relations—The Manufacture of Animal Prod- uets— North American Farm Animals. 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