LIBRARY OF THE DENTAL DEPARTMENT, UNIVERSITY OF CALIFORNIA. This book must be returned within four days. Fine, five cents each day for further detention. PHARMACEUTICAL BACTERIOLOGY SCHNEIDER PHARMACEUTICAL BACTERIOLOGY WITH SPECIAL REFERENCE TO DISINFECTION AND STERILIZATION JIM BY ALBERT SCHNEIDER, M. D., Ph. D. . = (Columbia University) PROFESSOR OF PHARMACOGNOSY, HISTOLOGY, AND BACTERIOLOGY, CALIFORNIA COLLEGE OF PHARMACY; PHARMACOGNOSIST, u. s. DEPARTMENT OF AGRICULTURE. WITH 86 ILLUSTRATIONS PHILADELPHIA P. BLAKISTON'S SON & CO. 1012 WALNUT STREET 1912 COPYRIGHT, 1912, BY P. BLAKISTON'S SON & Co. Printed by The Maple Press York, Pa. PREFACE. The recent growth and development of the professional side of pharmacy has made new text-books necessary. The present volume is the product of such progress. The illustrations have been selected with a view to a fuller explanation of the text. The descriptions of the illustrations have been made unusually complete. This is to make it possible for the student to ascertain the use of every article illustrated without the necessity of searching for additional information in the text itself. Some of the illustrations are from original drawings, others are from electros supplied by the Bausch & Lomb Optical Company and the Cutter Biological Laboratory of Berkeley, California. Still others are taken from recent works on bacteriology, notably Williams' "Manual of Bacteriology." Attempts have been made to adhere strictly to the subject from the standpoint of the pharmacist, with only enough treatment of general bacteriology to make clear the collateral relationships, especially as it pertains to medical and commercial or industrial bacteriology. While this volume is primarily intended for students in colleges of pharmacy, it is hoped it will also be found useful by practising pharmacists. SAN FRANCISCO. CONTENTS. PAGE. CHAPTER I. — GENERAL INTRODUCTION. i Introduction of bacteriology into colleges of pharmacy. Relationship of pharmaceutical and medical bacteriology. Reasons why pharmacists should study bacteriology. CHAPTER II.— HISTORICAL 5 Introduction. From Hippocrates (300 B. C.) to Leeuwenhoek (1656), the earliest ideas regarding infections, epidemics and spontaneous generation. From Leeuwenhoek (1656) to Schwann (1837), the discovery of micro-organisms and the earliest observations regarding their activities. From Schwann (1837) to Pasteur (1862), the earlier investigations pertaining to the relationship of micro- organisms to fermentation and to disease. From Pasteur (1862) to Behring (1890), period of remarkable activity in bacteriological pathology, listerism, antiseptic surgery, etc. From Behring (1890) to Wright (1907), discovery of serum therapy, bacterial vaccines and development of utilitarian bacteriology. CHAPTER III. — GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA .... 21 Classification of microbes. General morphology. General physiology. CHAPTER IV. — RANGE AND DISTRIBUTION OF BACTERIA 33 Bacteria of earth, air and water. Bacteria found in animals, in plants, on non- living objects, etc. Altitudinal range. Latitudinal range. CHAPTER V.— BACTERIOLOGICAL TECHNIC 36 Cleaning glassware. Plugging containers with cotton. Filling test-tubes with culture media. Preparation of culture media. Sterilization of culture media. Neutralization of culture media. Making bacterial cultures. Making bacterial counts. Preparing bacterial stains. Examining bacteria. CHAPTER VI.— BACTERIA IN THE INDUSTRIES 93 The function of bacteria in agriculture. Bacteria in milk and in the dairying industry. Bacterial pest exterminators. Bacteria in the tanning industry. Rotting bacteria. Cider making. CHAPTER VII. — IMMUNITY, BACTERIAL ACTIVITIES AND BACTERIAL PRODUCTS 114 Immunity, natural and acquired. Race, age and sex immunity. Anaphylaxis. Phagocytosis. Ehrlich's side-chain theory. Toxins and antitoxins. Agglutin- ins. Precipitins. Lysins. Opsonins and the opsonic index. CHAPTER VIII. — THE MANUFACTURE AND USE OF SERA AND VACCINES . . . 125 Antidiphtheric serum. Other sera. Bacterial vaccines (bacterins). Concen- trated diphtheria antitoxin. Manufacture of bacterial vaccines. Tuberculins. Small-pox vaccine. Rabies vaccine. vii Vlll CONTENTS. PAG* CHAPTER IX.— YEASTS AND MOULDS 142 Yeast organisms. Moulds. Organisms active in fermented drinks, as beer, wine and sake. Yeast cakes. CHAPTER X.— PROTOZOA IN DISEASE ' 156 Rhizopoda. Flagellata. Sporozoa. CHAPTER XI. — DISINFECTANTS AND DISINFECTION. FOOD PRESERVATIVES. INSECTICIDES 159 Principles of sterilization and disinfection. Pasteurization. Standardization of disinfectants. Methods of sterilization. Disinfectants and their use. Disin- fection of rooms and buildings. Disinfection of sewage. Sterilization of water supplies. Food preservatives, their use and abuse. Insecticides and other pest exterminators. CHAPTER XII.— STERILIZATION AND DISINFECTION IN THE PHARMACY .... 190 Sterilization of containers, stoppers, etc. Surgical supplies. Dusting powders. Salves and pastes. Gargles, washes, etc. Chemicals and galenicals. Solutions for hypodermic use. Ampuls. CHAPTER XIII.— COMMUNICABLE DISEASES WITH SUGGESTIONS ON PREVENTIVE MEDICINE 202 Causes of disease. Tuberculosis. Typhoid fever. Pneumonia. Small-pox. Malaria. Diphtheria. Cancer. Plague. Asiatic cholera. Yellow fever. Pel- lagra. Syphilis. Gonorrhea. Tabulation of diseases. CHAPTER XIV. — A BACTERIOLOGICAL AND MICROSCOPICAL LABORATORY FOR THE PHARMACIST 225 Position of laboratory. Size of laboratory. Furnishings. Equipment. Appa- ratus. Reference library. Outline of microscopical and bacteriological work. GENERAL INDEX 231 PHARMACEUTICAL BACTERIOLOGY. CHAPTER I. GENERAL INTRODUCTION. In introducing the first of a new series of text-books, certain explanations are necessary or at least desirable, which, after the subject is well established, become superfluous. Comparatively speaking, the science of bacteriology is not new, but its introduction into pharmacy is of very recent date. Medical bacteriology forms the very framework of medical practice. It has brought about our modern antiseptic surgery which has been the means of saving countless lives. It has led to the still more recent discoveries in serum therapy and the opsonic theory of disease. About 1896 a few of the colleges of pharmacy in the United States gave optional courses of instruction in bacteriology. At the present time nearly all of the leading colleges of pharmacy give instruction in bacteriology and in many of these institutions the courses are compulsory, forming a part of the prescribed curriculum, represented by lectures and laboratory work. In some universities the students of pharmacy receive their bacteriological instruction in the department of medicine or perhaps dentistry. However, pharmaceutical bacteriology and medical bacteriology are quite distinct. Medical students study this subject from the standpoint of pathology and disease, matters which concern the pharmacist but little. Students of pharmacy do not have the time necessary to devote themselves extensively to special laboratory methods and technic, nor is it advisable that they should receive extensive laboratory instruction in pathology. Pharmaceutical bacteriology must be suitably adapted to the practice of pharmacy. The pharmacist should have a fair knowledge of general bacteriology, in order that he may realize what important relationships bacteria bear to human activities in general, to medical practice more especially, and in order that he may comprehend quite fully the significance of these minute organ- isms in pharmaceutical practice. He should know what pharmaceutical prep- arations and what medicinal substances are likely to be attacked by bacteria, and what changes they are capable of producing in such substances. He 2 PHARMACEUTICAL BACTERIOLOGY. should have some knowledge of the effects that bacterially deteriorated sub- stances may have when introduced into the human organism. He should be qualified to sterilize pharmaceuticals as is now required in the pharmacopoe- ias of several foreign countries as Austria, Italy, and Belgium. He should know something of the comparative value of the numerous disinfectants and antiseptics used and found upon the market and should know how to stand- ardize these agents according to recent bacteriological methods. The pharmacist should know that bacteria, yeasts, and related organisms develop very promptly and profusely in all aromatic waters; in carelessly manipulated boiled and distilled water; in dilute solutions of all acids and alkalies; in dilute alcohol and alcoholic liquids; tinctures; infusions; extracts, solid and liquid; decoctions; in dilute salt solutions; in plant juices; mucilages; emul- sions; elixirs; wines; in syrups of all kinds; in carelessly manipulated vege- table drugs, crude and powdered; in drugs from the animal kingdom, as ox-gall, lard, oils, fats, pepsin, etc. He should have a clear comprehension of antiseptics as germ destroyers, and should know how to prepare and use them. He should have a general knowledge of alimentary and systemic phagocytosis; of leucocytosis in inflammatory processes, in pus formation, necrosis, etc. He should comprehend immunity, natural and acquired; he should know about opsonins and the opsonic index. He should have a general knowledge of bacterial enzymes; of toxins, ptomaines, leucomaines; of antitoxins; of bacterial vaccines. He should have a special knowledge of the source, manufacture, and use of antitoxins and toxins, modified toxins, vaccine virus, and related products used in medical practice. He should have a general knowledge of the causation of the more common bacterial and protozoic diseases. He should have special instruction in the disinfection of public and private dwellings, and should be able to cooperate with the physician in stamping out threatened epidemics and in carrying out public prophylactic and hygienic measures. To attain these ends a knowledge of bacteriology, specialized to suit the needs of the pharmacist, is absolutely essential. It is not the so-called practical side of bacteriology, represented by dollars and cents, which should interest the pharmacist in this science, but rather the broader view of his profession which will enable him to perform his duties more intelligently and more efficiently. The man whose actions are altogether prompted and directed by the dollar sign has no place in pharmacy or in medicine. He should turn to some non-professional enterprise. As yet there are no text-books or other works devoted especially to phar- maceutical bacteriology. Text-books on bacteriology for use in universities, medical colleges, and technical schools are not suitable for use in colleges of pharmacy. Some of these books are excellent collateral reading for phar- macists, but most of them are of such a highly specialized nature that they GENERAL INTRODUCTION. 3 would no doubt do more harm than good should the average pharmacist attempt to use them as a practical guide in the performance of his duties. Bacteriology must not be made discouragingly difficult to the pharmacist, in order that the best results may be attained. Wherever possible the college instruction in pharmaceutical _ bacteri- ology should be supplemented by visits to biological laboratories for the manufacture of sera and bacterial vaccines, to board of health laboratories, quarantine stations, garbage reduction works, etc. Students should also be assigned special reading. Journals and special treatises on bacteriology and on public sanitation should be consulted. The reports on bacteriological and related subjects issued from time to time by the United States Public Health and Marine Hospital Service are of special interest. The following references are given for the benefit of those students who may desire further information regarding the earlier conceptions of phar- maceutical bacteriology. It will be found that the opinions advanced by the authors cited differ considerably. 1. Bacteriology for Pharmacists. Pharm. Journ. Trans,, 23 (III), 565, 865; 24 (III), 101, 1893. Largely a description of the apparatus employed in bacteriological work, giving special attention to the value and use of the compound microscope in such work. 2. H. P. Campbell. Bacteria Dangerous to Medicines. Am. Journ. Pharm., 72, 113-118, 1890. 3. R. G. Eccles. Pharmaceutical Bacteriology. Proc. A. Ph. A., 42, 225-230, 1894. A very interesting paper on the theoretical possibilities of pharmaceutical bacteriology. 4. J. L. Hatch. Bacteriology. Pharm. Journ. Trans., 22 (III), 271, 289> 33°> l89J- A series of lectures delivered before the alumni association of the Phila- delphia College of Pharmacy, devoting the major attention to the morph- ology, physiology, and classification of bacteria. 5. R. T. Hewlett. Bacteriology in its Practical Aspects. Pharm. Journ. Trans., 25 (III), 819-820, 893-894, 1895. A general retrospect of bacteriology as a possible source of financial gain to the pharmacist. 6. Smith Ely Jeliffe. Moulds and Bacteria. Druggists Circular, 94-95, 1897. A description of some of the more common moulds and bacteria found medicinal solutions. Good illustrations. 7. E. Klein. Bacteria, Their Nature and Function. Pharm. Journ. Trans., 23 (III), 15, 35, 1893. 4 PHARMACEUTICAL BACTERIOLOGY. 8. W. H. Lymans. Bacteriological Culture Apparatus. Pharm. Journ. Trans., 1893. (National Druggist, 173, 1893.) 9. Albert Schneider. Pharmaceutical Bacteriology. Proc. A. Ph. A., 48, 186-189, 1894. 10. Specialism in Pharmacy, begotten by Progress in Bacteriology. Pharm. Journ. Trans., 25 (III), 625, 1895. Points out the necessity of a suitable preparatory training; the importance of a knowledge of the use of antiseptics. 11. R. Warrington. The Chemistry of Bacteria. Pharm. Journ. Trans., 23 (III), 402, 1893. (Pharm. Era., 104, 1894.) CHAPTER II. HISTORICAL. It must be evident that the science of bacteriology had its inception with the discovery of the compound microscope. For some time the progress in bacteriological investigation continued parallel with the progress in the mechanical perfection of the microscope and with the advance in microscop- ical technic. Gradually, however, the chemical and physiological inve^ti- gations pertaining to bacteria gained in importance and significance. Our knowledge of the morphology of bacteria as revealed through the compound microscope has been practically stationary for two decades, but not so our knowledge of bacterial products and bacterial action. The methods of bacteriological technology have been gradually perfected, and the prog- ress along this line has kept pace with the chemical and physiological investigations. Although, as indicated, the science of bacteriology is of comparatively recent origin, yet we must not lose sight of the fact that many of the ideas underlying this science, as now comprehended, were advanced in remote an- tiquity. For this reason it is desirable to set forth these earlier concepts in a historical review. Most of the writers on general bacteriology, who make reference to the history of the subject, almost invariably mention the older ideas regarding spontaneous generation as being the forerunners of the mod- ern ideas of bacteriology. It is, however, the ancient theories and beliefs pertaining to the cause of decay, disease, and epidemics which are even more directly associated with the first more important discoveries pertaining to modem bacteriological pathology. For the purposes of simplification, condensation, and greater clearness the historical review is divided into periods or epochs. It is not possible, in the following brief outline, to cite all investigations of importance. Only a few of the epoch-making specialists are mentioned. Period I. From Hippocrates (300 B. C.) to Leeuwenhoek (1656). (The earliest ideas regarding epidemics and spontaneous generation.) From the earliest times the more scholarly writers mentioned certain noxious gaseous, and odoriferous substances or erfluvias as being the cause of epidemics. These effluvias were supposed to emanate from the soil, from 5 0 PHARMACEUTICAL BACTERIOLOGY. the air, from water, stagnant pools, marshes, from decaying and putrescent substances, from crowded habitations, army camps, etc. The common people throughout the world and throughout all ages have held the belief that pestilence and disease was the manifestation of divine or supernatural influence, the judgment of an angry deity, a punishment inflicted on mankinti for their sins and iniquities, beliefs which are occasionally asserted even at the present time. Changes of season, climatic conditions, arid the influence of heavenly bodies were also considered as causative of diseases of an epi- demic nature. /"'Animals, such as rats, mice, and insects, have long been recognized as possible carriers of disease. An English investigator has recently discovered some very excellent sanitary rules in the Vedas of the Hindus. The follow- ing is a translation from Book VI, verse 50, of the Atharva-Veda. "Destroy the rat, the mole, the boring beetle; cut off their heads, O asvins. "Bind fast their mouths; let them not eat our barley; so guard ye twain our growing corn from danger. " Hearken to me, lord of the female borer, lord of the female grub ! Ye rough-toothed vermin. "Whate'er ye be, dwelling in woods, and piercing, we crush and mangle all those piercing insects." By " piercing insects" no doubt mosquitos are meant. If the injunctions were literally obeyed, plague, malaria, and certain protozoic diseases would be abolished from India. f- Hippocrates (460-377 B. C.), the father of medicine, considered seasons and winds as the cause of pestilence, particularly the long continued south- erly winds (for Greece), and a warm, humid, clouded atmosphere. Galen (130-220 A. D.) held similar beliefs. He declared that diseases arose from a putridity of the air or from atmospheric and weather conditions. Mar- cellinus (359 A. D.), a warrior as well as philosopher and historian, declared that the decomposing bodies left on the battlefield were the cause of " pesti- lential distempers," also caused by extremes in weather, by marsh effluvias, violent heat, and a vitiated atmosphere. Aetiusj^ fifth century), an eminent physician, declared that epidemics or common diseases were caused by bad food, bad water, immoderate grief, hunger, excesses, particularly abundance following extreme want, lack of exercise, excessive humidity, and putrid sub- stances. Alpinus, a Venetian physician of the sixteenth century, explained how the cause of plagues and epidemics may be carried by persons or in cargoes. He pointed out that a given ' disease from one country is more malignant than the same disease from another country. During the dark and middle ages various ecclesiastical and lay writers ascribed epidemics and pestilence to a variety of causes — the wrath of God, to demons or evil spirits, comets, meteors, earthquakes, volcanic eruptions, cyclones, eclipses of the sun, terrific storms, wars, famines, great fires, etcl Even as late as 1799 HISTORICAL. 7 no less an authority than Noah Webster makes the following declaration: "All the great plagues which have afflicted mankind have been accompanied with violent agitations of the elements. The phenomenon most generally and closely connected with pestilence is an earthquake. From all the facts which I can find in history, I question whether an instance of any considerable plague, in any country, can be mentioned which has not been immediately preceded by, or accompanied with, convulsions of the earth. If any excep- tions have occurred, they have escaped my researches. It does not happen that every place where pestilence prevails is shaken; but during the progress of the disease which I denominate pestilence, and which runs, in certain periods, over large portions of the globe, some parts of the earth, and especi- ally those which abound most with subterranean fire, are violently agitated." Were Noah Webster alive, he would certainly cite the recent plague on the Pacific Coast as bearing out his assertions. On April 18, 1906, the coast region about San Francisco was certainly "violently agitated," and this phe- nomenon was followed by the plague (black pest, bubonic plague). But what were the actual facts? The plague had, in all probability, existed in a sporadic form in "Chinatown," in San Francisco, and in other places on the Pacific coast for many years. In 1903 several authentic cases came to notice and were reported. The reasons why the disease had not previously gained a stronger foothold in San Francisco are several. Chinatown is more or less isolated (socially, at least) from the rest of the city, and the poorer, more filthy class of the Chinese do not as a rule mingle with the white population. The disease is an Oriental filth disease. After the earthquake and fire of April 18-22, 1906, the Chinese of all classes, the plague-infected rats and fleas of the Chinese quarters, became thoroughly intermingled with the rest of the stricken population, and as a result there were established several new foci of plague infection, which accounted for the increase in plague cases in 1907, a condition which was soon under control, thanks to the strenuous efforts of the federal government, the board of health, and various citizens' organizations Several writers of remote times, as well as occasional writers of the dark and middle ages, held the opinion that the cause of disease, the disease- producing effluvias, might be carried long distances by air currents, in ships, or by caravans, and that the poison may enter the system via the air pas- sages, through the skin, or through the digestive tract. Hodges, an English- . man, who wrote a treatise on the London plague of 1665, declared that some essential alteration in the air is necessary to the propagation of this disease. That is, the " nitro-aerial " principle, which causes or invigorates plant and animal life, is supposed to become vitiated. The corrupting principle is a "subtle aura or vapor" which is "extricated from the bowels of the earth." This plague-causing poison was said to 8 PHARMACEUTICAL BACTERIOLOGY. affect trees and other plants, fishes and other animals, as well as man. Dr. Mead declared that epidemics were caused by (i) diseased persons, (2) goods imported from infected places, and (3) a vitiated or poisoned state of the air, notions which may be considered as the direct forerunners of the germ theory of disease. Let us now go back and consider the ancient ideas regarding spontane- ous generation. Anaximander, of Miletus, who lived during the forty- third Olympiad (610 B. C.), believed that many animals developed de novo, from moisture and water acted upon by^sun and warmth. The extremist, Empedodes of Agrigentum (450 B. C.), declared that all living things upon the earth were capable of originating spontaneously. Aristotle (384 B. C.) taught that some plants and animals originated spontaneously. Ovid, some FIG. i. — From the Arcana Natures of A. van Leeuwenhoek. The first published illustration of bacteria. These bacilli of the mouth cavity were seen with the aid of simple lenses only, a, b, bacilli; c, a spirillum; e, perhaps chain forms of bacilli; d. illustrating the characteristic motion of certain bacilli (n to m). three centuries later, gives instructions how to create bees spontaneously in the carcasses of horses. To within recent times the belief that certain animals could originate spontaneously, that is, without a pre-existing parent, was quite general, and differed only in grotesqueness. Cardan as late as 1542 declared that water created fishes, and that many fermentative proc- esses created animals. Van Helmont gives instructions how to produce mice artificially. Kircher boldly declared that he had seen certain animals develop spontaneously before his eyes. Paracelsus gives instructions how to make homunculi. The instructions are quite simple. Certain substances are placed in a bottle, the bottle is well stoppered and buried in a manure heap. Every day certain incantations must be pronounced over the bottle in the manure heap. In time, Paracelsus declared, a small living human being (homunculus) will appear in the bottle. Paracelsus, however, naively HISTORICAL. 9 admits that he has never succeeded in inducing the homunculus to continue alive after being taken from the bottle. Gradually these grotesque and extreme opinions regarding spontaneous generation were abandoned, and it was declared that only the lower plants and animals, such as seaweeds, algae, lichens, lice, mites, maggots, etc., could develop spontaneously. -In fact, we can find fairly intelligent individuals to-day who firmly believe that certain animals, as lice, mites, etc., can originate without a parent, and that the hair from the tail or mane of a horse will change into a worm or snake if placed in a bottle of water and exposed to light and warmth. From the earliest records we learn that the value of disinfectants in pre- venting the spread of infectious diseases (epidemics and plagues) was known. Ovid states that the shepherds of his time used burning sulphur for bleach- ing wool and to free it from infectious diseases. In times of plagues, big fires were made to stay the ravages of pestilential diseases. The Mosaic law is replete with instructions regarding cleanliness as a means of pre- venting disease. Wine was highly valued as a dressing for wounds, having the effect of preventing or checking pus formation. ) Period II. From Leeuwenhoek (1656) to Schwann (1837). (Discovery of micro-organisms and the early investigations regarding their activities.) As early as 1646 Kircher suggested that certain diseases might be due toj£ery_jnjnutfi organisms which were supposed to originate spontaneously FIG. 2. — From Arcana Naturae. Cell structure of cork. Cell-lumen is shaded and cell-walls are shown light. under certain conditions. Anton van Leeuwenhoek is very justly called the father of microscopy, and to him must undeniably be given the credit of first having discovered and actually figured microbes and other micro- organisms. His Arcana Nature was published in 1656 in four volumes. It is a most interesting work, and contains many good illustrations showing 10 PHARMACEUTICAL BACTERIOLOGY. microbes of the mouth cavity, infusoria of stagnant water and cellular structure of vegetable tissues. He observed the motion of bacteria and in- fusoria, made measurements, illustrated capillary circulation in the web of the frog's foot, etc. He was closely followed by Robert Hooke, who published his Micrographia in 1658. The discoveries of Leeuwenhoek and Hooke were certainly epoch-making. A new world of minute organisms was made known, the question of spontaneous generation received a new turn, and the way to the discovery of the causes of disease and fermentation was paved. In 1660 Leeuwenhoek discovered yeast cells. From 1660 to 1760 the microscope was actively employed by a few investigators, and addi- tions were slowly made to the list of micro-organisms. Audry (1701) desig- nated microbes worms. Muller, of Copenhagen (1786), grouped them under two divisions, monas and vibrio. In 1743 Henry Baker, of England, published his work, "The Microscope Made Easy," from which it would appear that very little progress had been made since the time of Leeuwen- hoek (1656). As early as 1686 Franceso Redi doubted that maggots were generated de novo in putrid meats. He noticed that the presence of the maggots was preceded by swarms of flies which, he concluded, had something to do with the development of the maggots. He found that meat from which the flies were excluded by means of paper or a very fine mesh wire screen, simply decayed without any development of maggots. The paper cover and the fine screen kept the eggs of the flies from being deposited on the meat, and the meat was not infested by maggots, which, as Redi rightly conjectured, developed from the eggs of the fly-like imago. This very simple but reli- able experiment did much to create doubt as regards the correctness of the theory of spontaneous generation and other related beliefs. Spallanzani (1777) was among the first to demonstrate experimentally that boiling and hermetically enclosing fermentable liquids prevented fer- mentation. Ehrenberg (1828) discovered microscopic organisms in dust and in water, and in 1833 he classified all known bacteria under four orders, bacterium, vibrio, spirillum, and spirocheta. Cagniard-Latour and von Schwann (1836) discovered the vegetable nature of yeast, and in 1837 Schwann decleared that yeast was the direct cause of fermentative changes resulting in the liberation of alcohol and CO2, and that the causes of decay were to be found in the atmosphere. Berzelius (1827) declared that the yeast cells were the direct cause of fermentation. F. Schulze (1836) prevented decay in liquids containing certain organic substances by first heating or boiling them and excluding the air by means of a layer of oil or by closing the container with cotton and supplying it with air which had been ster- ilized by passing through sulphuric acid. Braconnot (1831) advanced the theory that yeast cells had the power of holding, and condensing within the HISTORICAL. II cell-substance, the oxygen of the air and conducting it to the substances undergoing fermentation, resulting in the splitting up of sugar into alcohol and carbonic acid gas. The question of spontaneous generation was again discussed with renewed energy. The belief that larger animals could originate de novo was quite generally abandoned, but it was very persistently argued that micro-organisms, maggots and a few other very small animals could thus develop. Bastian was perhaps the leader in the arguments in favor of spontaneous generation, opposed by Schwann, Pasteur, and others. Schroeder and von Dusch dem- onstrated that decay could be prevented by boiling and supplying air that had been filtered through cotton. Pasteur (1862) used bent tubes to supply air to the previously sterilized (by heating) substance, as shown in Fig. 3. FIG. 3. — Flask, containing an organic substance, a, hermetically closed by means of a stopper, b. The bent tube is open at e, admitting air. Dust and microbes lodge at the bends d and c. The microbes in the air passing through the tube are deposited (by gravity) in the lower bends of the tube. Those favoring the theory of spontaneous generation nevertheless continued their arguments. It was pointed out that changes of decay took place in eggs, in internal tissues and organs of the dead as well as in the living, etc., where, it was supposed, microbes could not possibly have access. However, further convincing experiments gradually silenced all opposition. Bastian and a few followers took practi- cally their last stand in 1875, an^ smce tnat time no scientist of repute has ever argued in favor of spontaneous generation, though the question of the primal origin of living things remains unanswered. Vaccination as a protection against virulent small-pox was practised early in the eighteenth century in Turkey and other Oriental countries, and was introduced into Europe via England through the influence of Lady Mary Wortley Montagu. A. von Humboldt states that the Mexicans practised vaccination at a very early period. This early vaccination mate- 12 PHARMACEUTICAL BACTERIOLOGY. rial was obtained from a pustule of a small-pox patient, and not from the cow, as at present. The immunity against subsequent attacks was established, but the disease transmitted through this older method of vaccination was severe and often fatal; besides, the general vaccination was a source of spreading the disease. In 1840 this form of vaccination was prohibited in England by act of Parliament. In 1768 Jenner's attention was attracted to the value of vaccination, and after a series of patient researches he perfected the method of vaccination by means of the virus obtained from a cow which had been inoculated with small-pox (vaccinia). Jenner established the first public institution for vaccination in 1799, and in the following year the practice was introduced into France, Germany, and the United States. Vaccination writh vaccinia material is now universal in all civilized countries and in countries under civilized control, and as a result small-pox in an epidemic form does not occur in these countries, and the disease has become less and less virulent, so that it is no longer the dreaded scourge that it was two centuries ago. In spite of the beneficent influence of vaccination, there are individuals who oppose this simple, harmless operation with all the energy that ignorance is capable of. Civilized countries are beginning to raise the long-enforced small-pox quarantine as a wholly unnecessary infliction, because vaccination makes the spreading of small-pox impossible. France has raised the quarantine, and so have several other countries, examples which will no doubt soon be followed generally. In conclusion, it is of interest to note that the primary cause of small-pox is unknown even to this day. No organism has thus far been isolated from diseased tissues to which small-pox manifestations could be ascribed. Period III. r From Schwann (1837) to Pasteur (1862). (Investigations per- taining to the relationship of micro-organisms to fermentation and disease.) The discoveries of the cause of fermentation, of decay, and of wound infection are closely associated. Many centuries ago Varro expressed it as his opinion that certain minute animals, breeding in marshy places, got into the system through mouth and nostrils and caused the disease and decay of tissues. Theodoric (1260) taught that wound infection came from the air. To prevent such infection he applied wine, which is known to be somewhat antiseptic. John Colbach (1704) described a "new and secret method of treating wounds by which healing took place without inflammation or suppuration." From earliest time up to as late as 1860, it was quite generally taught HISTORICAL. 13 that all normal healing of wounds and cuts must be preceded by pus-for- mation. A "laudable pus" was recognized, the presence of which was looked upon as a hopeful sign and indicated that repair was proceeding favorably. If the laudable pus which was of a whitish creamy consistency changed to a watery consistency, it was considered an unfavorable sign. After Schwann and others had demonstrated that fermentation was due to the presence of yeast cells, and it was proven conclusively that decay was caused by bacteria, the relationship of bacteria to disease began to receive consideration. Rayner and Devaine (1850) found bacterial rods in animals suffering from splenic fever. As early as 1840 Henle, who is by some con- sidered the father of modern bacteriology, made some very valuable deduc- tions regarding the relationship of micro-organisms to disease. He recog- nized a "contagium" (the active cause of the disease associated with micro- organisms), which was supposed to be air-like and yet at the same time fixed. It was supposed to retain its activity for years in the dry state. An unweighable and unmeasurable quantity of this substance may cause an extensive epidemic. Air currents can carry the contagium great distances and cause epidemics in widely separated areas. Bassi (1835) declared that a fungus was the cause of the muscardine disease of silkworms. Pollender (1855) reported that bacteria caused anthrax, verified by Devaine in 1863. Hallier, an enthusiast but not reliable as an investigator, declared that scarlet fever, measles, typhus, and cholera were caused by bacteria. His deductions were, however, not based upon scientific research and proof. Rindfleisch (1866) and Waldeyer (1868) gave considerable attention to wound infection, which, they declared, was due to microbic invasion. In i86o_Pasteur demonstrated the microbic cause of the silkworm disease which interfered very seriously with the silk industry in France. Pasteur and Klebs demonstrated experimentally that bacteria could be grown in artificial culture media, and Robert Koch proved that the pathogenic mi- crobes actually secreted the disease-causing substance. This was demon- strated by transferring an infinitely small quantity of the germ material from a diseased organ to a suitable culture medium and making sub-cultures, until the last culture must contain less than the trillionth part of the original substance. Nevertheless, inoculations from the last culture developed the disease with full energy. This experiment was made to meet the assertions that the cause of the disease did not reside in the bacterium, and that the bacterium, if present in the disease, was merely incidental to and not causa- tive of the disorder. A heated controversy continued for some time. Such authorities as Liebig, Na'geli, Bastian, Cohrij Billroth, Hiller, Schroeder, Hoppe-Seyler, Kiihne, Tiegel, Sanderson, Nencki, Serval, and Paschutin declared that micro-organisms were not the cause of decay, fermentation, and disease; 14 PHARMACEUTICAL BACTERIOLOGY. that these changes were due to chemical substances. However, such men as Pasteur, Koch, Panum, Klebs, and others forged link after link in the chain of evidence connecting the causative relationship of bacteria to disease. Period IV. From Pasteur (1862) to Behring (1890). (Period of remarkable activity in pathological bacteriology.) It would be impossible in a brief review to cite all of the important investigations of this period. Pasteur, Koch, and others had already given the subject of bacteriological technic considerable attention. The most suitable culture media, laboratory apparatus, stains, etc., were determined. The compound microscope had now reached a high degree of perfection, and the oil-immersion lenses made the closer study of the morphology of bacteria possible. As might be expected, the importance of germicides irr^urgery received first attention. The "laudable pus-" formation ideas were at^andoned. It became the surgeon's duty to induce " primary union" or healing by " first intention," that is, healing without any pus formation whatever. This demanded that the surfaces of the incision be brought in close contact, and that all bacterial infection be prevented by the use of antiseptic dressings, antiseptic solutions in the form of irrigations and sprayings, etc. Sir Joseph Lister, of Scotland (1875), brought the use df disinfectants in surgery to a high degree of perfection, and modern antiseptic surgery is often designated "Listerism." The modern proprietary antiseptic "listerine" is named after this eminent surgeon. The chief antiseptic of Lister and his followers was carbolic acid, which was used for free wound irrigation and general disinfection. He operated in a spray of carbolic acid solution. As late as 1890 there was to be found an occasional lecturer in a college of medicine who held out against the germ theory, and not a small number of the eminent opponents mentioned in the previous period carried their mistaken notions with them to the grave. The name of Robert Koch will stand throughout the ages as the leader in modern bacteriological science. Early in life he was convinced of the cor- rectness of the germ theory of disease, but his first contributions to bacterio- logical'science awakened a storm of opposition. Billroth, of Vienna, and others persisted in declaring that microbes were not causative of pus-forma- tion or of the development of disease; but that microbes might be accident- ally present, due to the action of a " phlogistic zymoid" which developed in the animal organism. In 1882 the French government sent a medical commission to India to determine if possible the cause of Asiatic cholera, but the commission re- HISTORICAL. 15 turned with a negative report as far as a bacterial cause of the disease was concerned. In 1883 the German government sent a similar commission, headed by Robert Koch, and the report of this commission was that Asiatic cholera was caused by a bacillus, the famous comma bacillus of Koch. The work of Koch in connection with the study of cholera seemed to act as a wonderful stimulus, and other eminent investigators made important dis- coveries within the year or two following. Klebs and Loffler discovered the diphtheria bacillus in 1884. Fraenkel, Weichselbaum, and Friedlander discovered the pneumococcus in 1884. Nicolaier and Kitasato discovered the tetanus bacillus in 1884. Loffler and Schiitz discovered the glanders bacillus in 1882, and the bacillus of hog erysipelas (Rothlauf) in 1885. Pasteur in 1881 made his first experiments in reproducing rabies in susceptible animals by inoculation with material obtained from the spinal cord, medulla oblongata, and lobes of the brain o£ animals dead from rabies. In 1884 he reported his experiments pertaining to the modification of the virulence of rabies by successive inoculations into susceptible animals. His use of this modified rabies virus as a means of preventing a severe and fatal course of the disease in those bitten by animals suffering from hydro- phobia, is familiar to all. Thousands of cases have been treated successfully at Pasteur institutes established throughout the larger cities of the civilized world. The above are only a few of the important investigations of this period. The causative relationship of microbes to certain diseases was undeniably established. The voices of opposition were silenced. This period is especially nntahlp fpr the dpv^npmppt nf flntisppfir surgery. As a result, operations were no longer dreaded as in former times. Fatal infections following operations now became rare. Thousands of lives are saved. To remove or destroy the pus germs in open wounds or to pre- vent the access of germs to wounds, cuts, and abrasions, has become a simple matter, a simple mechanical application of suitable antiseptics. The progress of purely medical bacteriology was not so marked. Al- though it was proven that certain diseases were due to bacteria, there were no satisfactory means of destroying them in the system. Internal antiseptics were tried, but without satisfactory results, as a rule. However, preventive medicine based on a bacteriological knowledge gave good results. Period V. From Behring (1890) to Wright (1907). (Discovery of serum therapy, bacterial vaccines, and development of utilitarian bac- teriology.) The subject of immunity from disease received early attention. Age 1 6 PHARMACEUTICAL BACTERIOLOGY. immunity, race immunity, animal immunity, individual immunity, artificial immunity, natural immunity, acquired immunity, etc., attracted attention and received careful consideration. Metchnikoff (1884) explained im- munity on the supposition that certain white corpuscles (leucocytes, phago- cytes) of the blood devoured the microbes which entered the system. These white blood corpuscles are the guardians of health. They attack and feed upon any disease germs which may enter the body, either via the digestive tract, the respiratory tract, or via the circulatory system. If the leucocytes are deficient in number, or if the microbes are excessive in number, disease will develop. This theory had numerous followers, as well as opponents. It is now generally accepted as correct, borne out by observation and by experimental evidence. The next important discovery was that blood serum had bactericidal prop- erties in a varying degree, and that in- addition to this there was something in the blood which had a tendency to neutralize or destroy the action of the poisons or toxins formed by pathogenic .microbes. No one particular bacteriologist can be said to have made these discoveries. We can only name a few of the leading investigators who worked along these lines, leading to the discovery of the relationship of immunity and antitoxins — Behring, Brieger, Buchner, Calmette, Chamberland, Ehrlich, Emmerich, Fliigge, Frankel, Hiieppe, Jetter, Kitasato, Klemperer, LofBer, Rankin, Roux, Wassermann, and others. These eminent authorities have demonstrated the possibility of developing or aiding the antitoxic or immunizing power of the blood or of the body cells by introducing sera obtained from the blood of animals in which the antitoxic power is naturally high or is made so as the result of special treatment. Numerous sera (containing antitoxins and toxins) were tried; the one which first proved entirely satisfactory was the diphtheria antitoxin of Behring, which is now in universal use. O.thers are used more or less successfully, and some are still in the experimental stage. In 1890 Koch reported on a "lymph" to be used in the treatment of tuberculosis. This lymph was a glycerin extract of the toxin of the bacillus of tuberculosis, and was to be used in the treatment of this dread disease, but the hopes of Koch were not realized, as the remedy proved a failure, and it soon fell into disuse, to be again taken up very recently. In 1907 Wright made known his discovery of opsonins. According to this authority, there exist in the blood certain substances which have the power of acting on the invading bacteria in such a manner as to render them more liable to be attacked and assimilated by the white blood-corpuscles or leucocytes. There are possibly as many opsonins as there are microbes capable of being di- gested by the leucocytes. The microbe-devouring power of the leucocytes can be increased by the use of bacterial vaccines, which consist of suspensions HISTORICAL. 17 of microbes. Very minute quantities are injected into the system, and the resulting reaction increases the power referred to. Toxins of bacterial origin received the attention of investigators, and antibodies (antitoxins) were extensively discussed as to their possible relation- ship to health and disease. Enzymes, in their relationship to life processes in plants and in animals, were investigated. It is now supposed that soil toxins of plant origin, as well as those of bacterial origin, influence plant growth. Glandular preparations (ductless glands) have been care- fully tested, and several of these are in use. As the result of Wright's discovery of the use of bacterial vaccines in increasing the opsonic index, the tuberculin (lymph) of Koch was again tried in the treatment of tuberculosis, apparently with some success. It was found that there were many bacteria other than those which caused disease in animals and plants. Some were found to be decidedly beneficial. Bacterial cultures were employed in butter-making (ripening of cream), in cheese-making, in tanning, in paper-making, siloing, etc. Some bacteria are employed to exterminate certain pest animals. A microbic chintz bug exterminator was tried in 1895-97, but it proved a failure. Microbic rat and mice exterminators (azoa, ratite, mouratus, etc.) are now being tested, and they appear to be quite successful, at least in certain localities and under certain conditions. A microbic rabbit exterminator has been tried in Australia. In 1879 Dr. Frank, of Berlin, began his investigations of the leguminous root nodule microbes. In 1893 the writer attempted to utilize these microbes in increasing the yield of certain gramineous crops. In 1896 Nobbe and Hiltner, of Germany, introduced a patented microbic fertilizer for legumin- ous plants. In 1907 a California soil microbe was isolated which appears to be especially active in promoting the growth of sugar beets. This experi- ment led to the supposition that perhaps every species of plant has its' pecu- liar bacterial flora, symbiotically (mutually beneficent) associated with the root system, mutually essential to active development. The importance of soil bacteria in setting free plant foods has been demonstrated by numerous investigators of Europe and of the United States. Yeast and mould organ- isms are practically utilised in the manufacture of beer, sake, and other food and drink products. outline of the history of bacteriology may be The above condensed summed up as follows: 1. Ancient conceptions of disease and of spontaneous generation, dating back to 500 years B.C. 2. Discovery of micro-organisms about 1660 by Leeuwenhoek, followed by the work of Robert Hooke and a few others. 3. Discovery of bacteria in air, dust, and decaying substances, and the 2 1 8 PHARMACEUTICAL BACTERIOLOGY. causal relationship of microbes to decay, and of the yeast organisms to fermentation. 4. Disproving the theory of spontaneous generation, by Schwann and others, about 1840. 5. Discovery of the bacterial origin of certain diseases — 1862 to 1880. 6. Introduction of small-pox vaccination into England by Jenner in 1796. 7. Development of antiseptic surgery or Listerism — 1875. 8. Period of great activity in pathological bacteriology — 1880 to 1890. 9. Discovery of the causes of immunity to disease, antitoxin of diphtheria and other antitoxins, serum therapy, etc. — 1886 to 1894. 10. Introduction of the use of certain bacteria in commerce and agri- culture. 11. Discovery of opsonins and the use of bacterial vaccines. Reintro- duction of Koch's lymph in the treatment of tuberculosis. Useful Works of Reference to Bacteriology and Related Topics. The following references are selected for collateral reading. A few of these works are rare, and can be found only in some of the leading libraries. A reading of these and other related works will serve as a supplement to this text-book. It is not intended to imply that all of the works cited should be procured. Others besides those mentioned may be consulted as oppor- tunity presents itself. Some of them can be obtained from public libraries; others may be ordered through the local book dealer, and a few may be borrowed from professional friends. HENRY BAKER. The Microscope Made Easy. London. 1743. Like the work of R. Hoke, this is of great historical interest, and is quite rare. Much of it is a copy of the work of Leeuwenhoek. B. M. BOLTON (H. U. Williams). A Manual of Bacteriology. P. Blakiston's Son & Co., Philadelphia. 1910. A most excellent work for medical students, also of value to students of pharmacy. H. W. CONN. Agricultural Bacteriology. This is a most excellent little work treating of bacteria in water, in the soil, in farm products, in the dairying industry, and in plants and domestic animals. It is well written in a simple, clear style. H. W. CONN. Bacteria, Yeasts and Moulds in the Home. Ginn & Co. 1903. This is \ VAN LEEUWENHOEK. Arcana Naturae. Four volumes. London. 1656. This is by far the most important historical work on the use of the microscope. In Latin. Some very good illustrations. Very rare; found in a few libraries only. K. C. MKX. Alikroskopische Wasser Analyse. Berlin. 1898. 20 PHARMACEUTICAL BACTERIOLOGY. An excellent German work treating of the bacteriological investigation of drinking water and sewage waters. GEO. NEWMAN. Bacteria. G. Putnam's Sons, New York. 1899. Treats of bacteria in industrial processes, bacteria in public health, in nature, in soil, etc. A very valuable work, excellent for general reading. T. M. PRUDDEN. The Story of Bacteria. G. P. Putnam's Sons, New York. 1889. Very interesting reading on general bacteriology and on the relationship of bacteria to health and disease. M. J. ROSENAU. The Bacteriological Impurities of Vaccine Virus. U. S. Public Health and Marine Hospital Service. Hygienic Lab. Bui., No. 12. 1903. Of special interest to pharmacists. It should be borne in mind, however, that since the publication of this report the methods of vaccine manufacture have been modified somewhat, and the figures and results given may no longer apply. M. J. ROSENAU. An Investigation of a Pathogenic Microbe of Rats and Mice (B. typhi- murium Danysz.). Washington, D. C. 1903. This treatise is also of special interest to pharmacists, as the microbe referred to is the active ingredient of several patented rat and mouse exterminators sold under proprietary names as Azoa (Parke, Davis & Co.), Rattite, Mouratus (Pasteur Co.), etc. These exterminators are still under investigation, testing, etc., and the findings in the above report should not be considered final or conclusive. W. G. SAVAGE. The Bacteriological Examination of Water Supplies. Philadelphia. 1906. A valuable treatise. Contains a citation of the more valuable literature on the subject. An excellent laboratory guide for the specialist. DR. C. STICH. Bacteriologie und Sterilization im Apothekerbetrieb. Berlin. 1904. In German only. Contains many valuable suggestions but too incomplete and too much lacking in detail for the student. E. R. STITT. Practical Bacteriology, Blood Work and Animal Parasitology. P. Blakis- ton's Son & Co., Philadelphia. 1909. Primarily for medical students, especially those interested in the parasitology of the tropics. Complete on methods. Full details regarding blood work and use of hemacy- tometer. JOHN TYNDALL. Floating Matter in the Air. London. 1881. A very interesting popular work on the micro-organisms of the air and their relationship to fermentation and putrefaction. For general information. NOAH WEBSTER. A Brief History of Epidemics and Pestilential Diseases. Two volumes. Hartford. 1799. Of great historical ^interest, though entirely antiquated and of no scientific value. CHAPTER III. GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. Bacterium (plural, bacteria) is a misleading term, though firmly estab- lished in general usage. It means "a small rod," the name being applied because it was believed that these minute organisms were mostly, if not all, rod-shaped. This is not the case, as will be explained later. Further- more, the term is used in a generic sense, and again applied to the group of organisms as a whole. This causes confusion. Therefore, the generic term Bacterium is now abandoned and the term Bacillus is used to include all of the micro-organisms which are rod-shaped although generic sub-divisions are being made of this now very large group. The term "microbes" (micros, small, and bios, life) or micro-organisms would be far more suitable than the term " bacteria," as applied to the entire group of organisms included in the subject of bacteriology. Microbiology is no doubt more correctly descriptive than bacteriology, but the latter term is so firmly established in general usage that it would be unwise to urge a change at the present time. Whereas the general morphology of microbes is apparently quite simple, the physiology and chemistry is extremely complex, and as yet not fully understood. The morphological simplicity is no doubt only apparent, and not real. Perhaps, with the greater perfection of the compound microscope, we may discover marked structural differences which thus far have escaped our notice. i. Classification Of Microbes. Microbes are the smallest of the known living organisms. It is wholly impossible to see the single individual, even the largest, with the naked eye. The rod-shaped microbes (bacilli) range from 0.5^ to 10/1 in length. Some are so minute as to pass through the pores of the finest elay filters (microbes of foot and mouth disease). To study them a good compound micro- scope is absolutely necessary, though, as stated in the historical review (Period II), Leeuwenhoek and others observed the larger forms under the simple microscope. The systematic position of microbes has from time to time received much attention. The great majority of biologists now unhesitatingly class them as plants, belonging to the group fungi. It cannot be denied, however, that their origin (phylogeny) is still shrouded in mystery. Some suggest that they are derived from degenerate algal forms, in common with most of the fungi, while others declare that they in all probability originated as microbes. A 21 22 PHARMACEUTICAL BACTERIOLOGY. few of the philosophical biologists, as Ernst Haeckel, place them in a separate group, the Monera, which is supposed to form the connecting link between plants and animals. Without entering into lengthy discussion, we shall, in conformity with the opinion of the majority, class them as plants, belonging to the lowest of the group fungi (the fungi includes rust, smuts, cup fungi, moulds, spot fungi, toad-stools, etc.), namely, the Schizomycetes or fission fungi, so called because they multiply by fission or division. They are related to the yeasts, though somewhat lower in the scale of evolution. They are single-celled, each cell forming a complete living unit, though the several units may be variously arranged into chains or clusters or groups known as zoogolea. The scientific grouping of microbes is as yet very unsatisfactory because so little is known of their ultimate morphology, their physiology and chemis- try. Some have attempted to classify them as to form, others as to occur- rence, as to action, etc. Thus, we have: a. Micrococci or Coccaceae. — Globular or non-elongated microbes. b. Bacilli or Bacteriaceae. — Cells more or less elongated. Rod-shaped microbes. c. Spirillae or Spirillaceae. — Cells elongated and more or less spirally twisted. Or, we may have: a. Bacteria of earth. b. Bacteria of air. c. Bacteria of water. Or, again: a. Chromogenic. b. Zymogenic. c. Pathogenic, etc. These artificial groupings could be extended indefinitely, but such sys- tems of classification would be as unsatisfactory as they are unscientific. The best system makes use of all of the known facts of bacteriology. Several such systems have been proposed from time to time, but the new discoveries along bacteriological lines make it necessary to change them in the course of two or three years. Migula, Fischer, Eisenberg and others have proposed general systems, and a host of investigators have submitted more limited group sys- tems. The following classification will serve to convey some idea as to the structural characteristics of the more important groups: BACTERIA OR MICROBES. (Schizomycetes or Fission Fungi.) I. Family COCCACEAE. — Micrococci. Cells globular or not elongated. Division in two or three directions of space. Spore formation rare. GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. 23 1 . Micrococcus. — Cells spherical or biscuit-shaped. Division in one direction of space. With or without flagellae. A large genus, represented by numerous species, pathogenic and non-pathogenic, chromogenic, zymo- genic, etc. 2. Streptococcus. — Generic limitation not clearly denned. Often merely chain forms of above, resulting from cohesion of cells dividing in one direction of space. 3. Sarcina. — Division in three directions of space. Cells often in fours (Tetracoccus) — as for example, the sarcina of the stomach. With or without flagellae. II. Family BACTERIACE.E. — Bacilli. Cells more or less elongated, cylindrical, straight ; some are somewhat curved or irregular in outline. With or without flagellae. Endospore formation. Transverse septation. 1. Bacillus. — Variable in size and length of cell. Numerous flagellae. Endospore formation common. A very large group, to which belong many of the most important microbes. Includes the old genus Bacterium. 2. Pseudomonas. — Said to have only polar flagellae. Doubtful genus, by many relegated to the group bacillus. III. Family SPIRILLACE.E. — Spirillae. Cells elongated and spirally twisted. Transverse septation. Body fixed, with polar flagellae. 1. Spirillum. — Numerous polar flagellae. Large group. 2. Microspira. — Few polar flagellae. A group Spirosoma is said to be without flagellae. IV. Family SPIROCHETACE^:. — Spirocheta. Long, single-celled, flex- ible, spirally twisted threads without flagellae. One genus — Spirocheta. (Some authorities place these organisms in the animal kingdom with the Protozoa.) V. Family MYCOBACTERIACE.E. — Filamentous organisms, perhaps form- ing a connecting link between bacteria proper and the lower filamentous fungi. Cells filamentous but not enclosed in a sheath. To this family belong the groups Mycobacterium and Actinomyces (ray fungus). No flag- ellae have been observed. Mostly transverse septation. Gonidial (spore) formation has been observed. VI. Family CHLAMYDOBACTERIACE^E. — Resembling above family, but the cell filaments are enclosed in a sheath. The following not very clearly defined groups are recognized: Cladothrix, Crenothrix, Phragmidiothrix, and Thiothrix. VII. Family BEGGIATOACE.E. — Beggiatoa. Family characters not clearly defined. Motile, though no flagellae have been observed. Beggiatoa is the most important genus. The uncertainty in the systematic grouping of microbes need not cause worry, as even the leading specialists do not give the matter any con- 24 PHARMACEUTICAL BACTERIOLOGY. siderable attention, for the simple reason that their time is taken up by matters of far greater importance, namely, the determination of the role which the microbe plays in the life economy. Should the student ever be placed in position to justify him in attempting to identify a given microbe, he will find an extensive literature which will aid him in his efforts. Undoubtedly in time there will be a fairly simple, scientific, and complete system of classi- fication of all known bacteria. As yet such a system does not exist. 2. General Morphology of Microbes. As already stated, the morphology of microbes is simple. They consist of a single cell composed of cell-wall and cell-contents. The cell-wall con- FIG. 4. — Illustrating the general morphology of microbes, a, showing general structure of a bacillus, endospore formation, and development of new bacillus from a spore; b, showing manner of transverse septation; c, arrangement of flagellae, single unipolar, single bipolar; and multiple, polar and general; d, cocci; e, flagellae of cocci; / spirillum with single polar cilia. sists of cellulose, and is very thin; stains readily with the various bacterial stains. The chief cell-contents is the cytoplasmic or protoplasmic living base commonly designated as the nucleoplasm, which is of a granular nature, GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. FIG. 5. — Illustrating the general morphology of Coccaceae. a, b, micrococci (a) differing in size, showing chain formation or streptococci (b);c, diplococcus; d, diplococcus;e, tetra- coccus;/, gelatinized tetracoccus; g, gelatinized diplococcus. FIG. 6. — General morphology of Bacteriaceae. a, b, c, d, bacilli differing in size and orm; c, shows curved bacilli like those of Asiatic cholera; e, hay bacillus (B. subtilis); /, Y-shaped or branched bacilli, as of clover root nodules; g, drum-stick (Trommelsch lager) bacilli, as of tetanus — form due to the enlarged endospores. 26 PHARMACEUTICAL BACTERIOLOGY. and by some is supposed to be a nucleus in a divided state. A nucleus proper does not exist, or, rather, has not been demonstrated. The cyto- plasm, as a rule, stains quite readily.. Distributed through the cytoplasm may be found various substances, elaborated by cytoplasmic activity. Polar granules (metachromes or Babes-Ernest granules) have been observed. Sulphur, fat, pigment, chlorophyll, etc., may be found. The cell-walls of many species undergo a gelatinous change. This change may affect the outer layers only, or it may involve the entire thickness of the wall, forming the gelatinous substances noticeable in bacterial cultures and in other substances (stringy cultures, stringy milk, etc.) . This gelatin- FIG. 7.— General morphology of the Spirillaceae. a, S-shaped or single spiral; b, double spiral; c, multiple spirals; d, slender threads; a and b have fixed bodies, motion being caused by flagellae; c and d, bodies flexible, motion not due to flagellae. ous substance also causes the individual organisms to cling to each other, thus causing the formation of the peculiar zooglea masses in natural as well as in artificial culture media. The cilia or flagellae are very delicate threads, supposed to extend from the cell-plasm, through the cell-wall, into the surrounding medium. The delicate threads are probably cytoplasmic in nature, and by their rapid vibratory motion enable the microbe to move about within liquid media. Some microbes are apparently without flagellae, nor is it definitely deter- mined that all motile microbes have flagellae. Some authorities are inclined GENERAL- MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. 2J to the belief that perhaps nearly all, if not all, micrococci and bacilli have active motion under certain conditions. This makes it clear that the attempt to group microbes into motile and non-motile must result in failure. The attempt to make generic distinctions based upon the absence or presence of few or many flagellae, upon the existence of polar or non-polar flagellae, etc., is also unsatisfactory. Special staining methods are necessary to demon- strate the presence or absence of flagellae. Some investigators declare that it is almost impossible to demonstrate them ocularly. That they do exist is fully demonstrated, but it is not demonstrated to any degree of satisfaction that it is practicable to make finely drawn numerical and structural distinc- tions in the flagellae of the different species of microbes. The rate of motion of bacteria has been measured. The cholera bacillus moves at the rate of 18 cm. per hour. The typhoid bacillus is slower moving a distance of 4 mm. in one hour. The rate of motion in one and the same < m Fig. 8. — Illustrating polymorphism or pleomorphism. Involution forms of the bacillus of Asiatic cholera. (Williams.) species is, however, variable, being comparatively rapid at one time under certain conditions of food supply, warmth, etc., and at other times com- paratively slow. When the microbe approaches the end of the life cycle, or when the con- ditions for growth and septation are no longer good, spore formation is apt to take place. This spore formation is of two kinds, endospore formation and arthrospore formation. The former predominates, and occurs largely in the group bacilli, though it is also noticeable among the micrococci and the spirillae. Endospores are usually spherical, though they may be slightly elongated, and usually occur near one end of the cell, and usually there is only one in each cell. Generally the diameter of the spore is equal to or somewhat less than the diameter of the cell-lumen. Sometimes, however, the diameter of the spore exceeds that of the cell-lumen, causing a charac- teristic bulging, as in the tetanus bacillus (drum-stick bacillus, Trommel- hldger Bacillus). The spore is formed from the cytoplasm, and differs 28 PHARMACEUTICAL BACTERIOLOGY. from it in its higher refractive index and its peculiar resistance to the action of stains. As soon as spore formation is complete, the rest of the cytoplasm dies, the cell- wall disintegrates, and the spore is thus set free. Spores have a remarkable resisting power to high temperatures and other unfavorable conditions. In a dry atmosphere they may lie dormant for a long time, even several years. Boiling from one to two hours does not kill some of them (spores of hay bacillus) . As soon as the spores are placed in suitable media (adequate warmth, moisture, and food supply) they develop into new individuals, which continue to septate until spore formation again takes place. ^Vt ff i FIG. 9. — Illustrating polymorphism or pleomorphism. a to d, inclusive, represent different forms of the same organism — the Diphtheria bacillus. (See also Figs. 46-50 inclusive.) Arthrospore formation is less common, and occurs mostly among the micrococci. The entire cell is converted into a spore, which becomes some- what enlarged and encapsuled, in which state it is enabled to tide over cer- tain conditions unfavorable to normal growth and septation. Arthrospore formation is not well understood as yet. It may also be that some of the phenomena described as arthrospore formations are in reality endospore formations. The classification given above, into families and genera, and Figs. 2 to 10, inclusive, will serve to give a fairly good idea of the general structural char- acteristics of microbes. 3. General Physiology of Microbes. Microbes, in common with living things generally, spring from pre- existing parents, take in and assimilate food, grow and multiply, and finally die. The rate of growth and of multiplication (septation or division) varies GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. 29 somewhat, depending on temperature, moisture, and food supply. The average life of one individual (from septation to septation) is perhaps thirty minutes. Under favorable condition the period is much shortened. This life period of the individual cell must not be confounded with the life cycle of the individuals resulting from a single cell or parent. It is known that under uniform conditions of temperature, moisture, food supply, and the environ- ment generally, the progenations from a single parent cell show an increasing rate of septation, a stationary period, followed by a gradual decline, ending in total cessation of all septation, and in death. These life cycles have not CO a '%. FIG. 10. — Illustrating zooglea formation, a, bacillar aggregates resulting from cohesion; b, aggregates resulting from cohesion of bacilli with gelatinized cell- walls; cy streptococcus formation resulting from the septation of a coccus form; d, cohering cocci forms; e, bacilli united end to end (resulting from septation), enclosed in a gelatinous coat; /, bacillar thread enclosed in gelatin; g, mycobacterial form; h, irregular cell forms, as My coder ma aceti. yet been carefully determined; in fact, they are but little understood. It is highly probable that the cycles of existence play a very important part in the course and development of diseases of bacterial origin. Whereas the period from one septation to another septation is very short, the life cycle referred to is often quite long, perhaps months and, under certain conditions, lasting for years. The period of the life cycle can be modified artificially by food supply, chemicals, etc. 30 PHARMACEUTICAL BACTERIOLOGY. Investigators have succeeded in prolonging the life cycle of Paramedum. Normally P. caudatum dies out in about 175 generations; but by applying alcohol (1-5000 to 1-10,000) the cycle has been increased to 860 generations. Very dilute solutions of strychnine gave similar results. If the life cycle or vital impulse of these simple organisms can be prolonged it is probable that similar effects can be produced in higher organisms. Numerous investi- gators have from time to time sought after agents which might inhibit the senile changes in cells and circulatory system (arteriosclerosis) but thus far without conclusive results. It is, however, highly probable that within a comparatively short time means may be found to prolong the life of the higher animals from 10 to 20 per cent, and even more. Microbes feed upon organic substances generally. Those which feed upon dead organic substances are said to be saprophytic; those feeding upon living substances are said to be parasitic. If they can live on dead organic substances only, they are obligatively saprophytic; if they can feed on both dead and living organic substances, they are facultatively saprophytic, or, vice versa, facultatively parasitic. The great majority of microbic parasites are facultatively so, as is evidenced by the fact that they can be grown in artificial culture media. Many of the microbic saprophytes will develop on living substances under certain conditions, thus showing that they are facul- tatively parasitic. It is no doubt true that no. known microbic parasite actually feeds upon the living substances of the various hosts, since the cyto- plasm is in all instances dead before it is taken up and assimilated by the microbe. It would therefore be more correct to say that parasitic microbes are biologically associated with living organisms, while the saprophytes are biologically associated with dead organic substances, and that they all feed upon and assimilate dead organic substances. In certain mutualistic symbioses (as in the root nodules of the Leguminosse) the biological rela- tionship of microbe and host plant is very intimate, but there is no actual interchange of living material. All microbes require moisture and warmth (comparatively speaking) for their development, although they are enabled to withstand greater ex- tremes of heat and cold than other organisms. The temperature of liquid air (about — 270° F.) does not kill them at once, and the spores may be boiled for some time without destroying their germinating power. Cold (freezing temperature) promptly checks growth and septation, and so does dryness and excessive warmth, although life may not be destroyed. The majority of microbes develop most actively at a temperature of 25° C., a few species develop more actively at a lower temperature (20° C.), and a few others at a higher temperature (38° C.). Those which develop at a temperature rang- ing from o° C. to 30° C. are said to be cold loving (psychrophile), from 10° to 45° C., mesophile, from 40° to 70° C., thermophile. Thermophile species are GENERAL MORPHOLOGY AND PHYSIOLOGY OF BACTERIA. 31 found in decaying vegetable matters, whereas psychrophile species are found in cold water and cold soils. Bacterial life processes result in the formation of many substances, some of which are of the greatest importance. It is impossible to estimate prop- erly the enormous tasks performed by these minute organisms, nor shall we at this time make any attempt to set forth the great good and the apparent great harm done by them. We need only state that without rotting microbes soil formation would be impossible, and without soil, higher plant and animal life, as we now know them, would be impossible. Without plant food digesting microbes crop growing would be impossible. The saltpeter depos- its in South America and the iron deposits of the Mesabi range of Minnesota are said to be the result of bacterial action. We make extensive practical use of microbes in medical practice, in the dairying industry, etc. We will mention ohly a few substances of undoubted microbic origin. Ptomaines and toxalbumins are well-known poisons elaborated by sapro- phytic microbes which feed on meats and other organic substances, causing the familiar putrefactive changes. Pathogenic microbes elaborate toxins to which are due the manifestations of the disease. Acetic acid, lactic acid, and butyric acid are elaborated by Bacillus aceticus, B. acidi lactici, and B. butyricus, respectively. Some species liberate odoriferous substances, others gases, coloring substances, phosphorescence, etc. The phosphor- escence observed on the ocean is supposed to be due to bacteria (Bacillus phosphorescent indicus). Phosphorescent bacteria occur in dead fish and in meat. Old cultures in animal nutrient media and in the presence of sodium salts are phosphorescent in the dark, sufficiently so, to have sug- gested making bacterial lamps and signal lights: It has been suggested that certain diseases, of which the causes are at present unknown (as yellow fever, measles, whooping cough), may be due to organisms so small as to be invisible (ultra micro-organisms). It is known that the virus of yellow fever will pass through the most compact clay or por- celain filter. Attempts have been made to demonstrate the presence of ultra micro-organisms by special photomicrographic methods, aided by special illuminating devices (the ultra microscope of Siedentopf and Szigmondy) but without success. Furthermore, no one has succeeded in culturing such theoretically surmised organisms in artificial media, which would certainly render them visible en masse. It may, however, be possible that some ultra-organisms are obligative parasites hence will not develop in artificial media. The biological (symbiotic) relationship of different species of bacteria to each other and to their host are, in many instances at least, not well under- stood. For example, it is not clear what biological relationship the different species of bacteria in a mixed infection bear to each other. In the case of the 32 PHARMACEUTICAL BACTERIOLOGY. root nodule organisms of the Leguminosae it is known that there is a mutu- ally beneficial (mutualistic symbiosis, mutualism) relationship between mi- crobe and host but it is not obligatively so, since the symbionts can exist independently of each other. In most diseases due to microbic invasion there is one species of bacterium which acts as the primary cause. It is known that tuberculosis, especially the pneumonic form, usually shows a mixed infec- tion, and it is probable that the associated organisms as bacteria and higher fungi act as predisposing causes, preparing the tissues so as to yield more readily to the invasion of the primary cause, the Bacillus tuberculosis. Such an association may be designated compound symbiosis, in which the relation- ship of the invading organisms (secondary and primary) is mutualistic and the relationship of these to the host is antagonistic. It is known that certain microbic diseases predispose to other microbic invasions, thus we may say that these organisms are mutualistically disposed toward each other. Since it is possible to cultivate most disease germs in and upon artificial culture media (hence dead organic substances) it is evident that they are only facultatively parasitic. In many instances the biological association of bacteria and higher plants and animals is loosely mutualistic, as the bacteria upon roots and rootlets of all plants and the bacteria lining the intestinal tract of animals. The hay bacillus (Bacillus subtilis) is a constant associate with the Graminege and serves an important function, assimilating or binding for the use of the host plant, the free nitrogen of the air. Certain soil organisms (Bacillus megatherium, B. ellenbachiensis,B.mesentericus,B. pyocyaneus, B. prodigiosus, the Azoto- bacter group, Clostridium pastorianum, certain moulds as Aspergillus niger and Penicillium glaucum) are capable of assimilating the free nitrogen of the air thus enriching the soil for the benefit of higher plants. CHAPTER IV. RANGE AND DISTRIBUTION OF MICROBES. Microbes are omnipresent over the surface of the earth. In number and in bulk they exceed all other organisms (plants and animals) put together. They form a large precentage of the bulk of the soil. They occur in the air, in water, in snow, in hail, in raindrops, in and upon plants, in and upon animals. All substances with which we come in contact are likely to hold microbes. Our clothing teems with them. They are in the air we breath, in the food we eat, and in the liquids we drink. The floating dust particles of the air carry microbes; the particles of organic matter in water harbor microbes; they are found on wood, on cloth, on paper, on metal, glass, and rock surfaces, in fact on all exposed surfaces. The hands, the hair, the entire body surface of man and of the lower animals contain or hold mi- crobes. They line all mucous membranes. The mouth cavity is a veritable bacteriological laboratory. The entire intestinal tract teems with millions upon millions of these minute beings. Each animal and each plant has a microbic flora peculiar to itself. Each portion of the plant or animal, again, has distinctive bacterial groups. The microbic flora of the intestinal tract of the dog is different from that of the pig, or cat, or fowl, or man. Certain species predominate in the mouth cavity, others in the stomach, still others in the small intestine, in large intestine, etc. Microbes are found on the highest mountain peaks and in the deepest valleys. It is, however, true that the higher atmospheric strata contain fewer microbes than the lower strata. The deeper layers of soil contain fewer microbes than the upper. The atmosphere of the country contains fewer microbes than that of the cities and towns. Since sunlight and absence of moisture are natural enemies of microbes, we may expect to find microbes more abundant in dark, damp, and moist places and areas. Mi- crobes are always more abundant in cellars, basements, dark hall-ways, and alleys than they are in attics, sunlit living rooms, and along broad boule- vards and highways. Good drinking water, whether from hydrant, spring, or well, contains only a comparatively few microbes, from fifty to one hundred per c.c., or even less. Stagnant, foul water teems with microbes, besides other organisms, such as protozoa. So-called pure milk contains comparatively more microbes than pure water. The average good milk contains as many as 30,000 microbes 3 33 34 PHARMACEUTICAL BACTERIOLOGY. per c.c. Filthy milk may contain millions of microbes per c.c. From 100,000 to 3,000,000 microbes per c.c. is not uncommon in some milk which careless dairymen declare to be "good." Soups, broths, etc., boiled squash, potatoes, meats, and cooked organic substances generally, if allowed to stand for a day or two, contain many living microbes. In the course of two or three days, if the weather is warm, these substances teem with microbes and are rendered wholly unfit for food because of the predominating rotting microbes which develop the highly poisonous ptomaines. Microbes do not live and multiply in aseptic and antiseptic substances, such as strong solutions of acids, of alkalies, of salts, etc. Used and dirty cups, drinking vessels, milk bottles, dishes, cooking utensils, knives, spoons and forks, hold numerous microbes. The public drinking cup has been the source of numerous disease infections. Disease is carried by the tools of the careless dentist and by the clothing, the apparatus and the clinical thermom- eter of the indifferent and careless physician. The hand-shaking and kiss- ing habits spread disease. These facts are generally known and indicate the wide dissemination of the different kinds of microbes. From the foregoing it becomes clear that microbes are present almost everywhere, and that it is impossible to escape them. It is the aim of the science of bacteriology to distinguish between good and bad microbes, between those which are desirable and those which are undesirable, between useful and harmful microbes. It is not the aim of the science of bacteriology to destroy them all, or to devise ways and means to escape from all of them. In fact, we owe our very existence to these very minute organisms, as has already been explained. Under certain conditions bacteria multiply very rapidly. Such sub- stances as meat, milk, and organic foods of all kinds, if exposed to moisture, warmth and removed from sunlight, soon swarm with microbes. Certain non-pathogenic microbes, as the root nodule bacteria (of the Leguminosse) , multiply very rapidly within the tissue cells. Others multiply upon the exterior of roots and of root hairs, where they no doubt serve a useful pur- pose to the plant. In bacterial diseases of plants and animals the microbes multiply very rapidly and form large aggregates, as a rule. To pathological conditions accompanied by extensive and general bacterial or microbic inva- sion, we apply the term bacteremia. In some diseases the microbic invasion remains localized and yet there are pronounced general or systemic effects, due to the absorption, into the system, of the toxins liberated by the microbes. To such conditions we apply the term toxemia. Toxemia may, however, also occur in bacteremia. Microbes do not multiply in the air itself, rather upon the organic dust particles present, provided warmth and moisture are adequate. Since microbes multiply rapidly, perhaps one septation in from twenty to RANGE AND DISTRIBUTION OF MICROBES. 35 thirty minutes, it is evident that the rate of numerical increase, under favor- able conditions, is very great. Allowing thirty minutes for each septation, there would be a colony of 2,097,152 microbes in ten hours, developed from a single cell, or about 75,000,000,000,000 cells in twenty-four hours. However, under natural conditions septation never proceeds in such uniform ratio. All manner of checks to septation come into play sooner or later which may finally bring about complete cessation of septation and sporulation. CHAPTER V. BACTERIOLOGICAL TECHNIC. As may readily be supposed, the minuteness and wide distribution of microbes call for special methods of study and examination. Even the largest forms are far below the ken of unaided vision. Their general dis- semination through organic substances calls for special methods for the separation and isolation of individuals or of single bacterial cells. The difficulties of technic are further increased by the resistance of spores to various agents and substances which are readily fatal to higher organisms. The methods of examination are also greatly complicated by the marked polymorphism of many species. Bacteriological technic comprises the use of glassware, compound microscope, and other apparatus, a thorough knowledge of sterilization and disinfection, the preparation and use of culture media, the making of micro- FIG. ii. — a, Nest of beakers and reagent bottles. The smaller and medium size beakers are more desirable for bacteriological work. The reagent bottles are for Canada balsam, stains, clearing fluid, etc. bic cultures, and the study of cultures. Methods vary greatly. The follow- ing represents a brief summary of general methods which are noted for simplicity and which have proven very satisfactory after years of testing. i. Cleaning the Glassware. All glassware, such as test-tubes, flasks, beakers, Petri dishes, pipettes, shells, bottles, etc., which is to be used in bacteriological work must be clean; that is, free from all extraneous organic as well as inorganic matter. To accomplish this, it is necessary to use an abundance of pure water, hot as well as cold, aided by sand, paper shreds, brushes, towels, alcohol, acids, soap, sodic and potassic hydroxides, and whatever else may be necessary. Boil, wash, rinse, and wipe within and without repeatedly until it looks, and 36 BACTERIOLOGICAL TECHNIC. 37 is, absolutely clean. The following solution will be found useful as a cleans- ing agent for old as well as new glassware: Potassium Bichromate, 6 parts. Sulphuric Acid, 30 parts. Water, 40 parts. Of course, the sulphuric acid must be added little by little with constant stirring, in order to avoid excessive heat development. Soak the glassware FIG. 12. — Wire baskets for holding test-tubes. Cylindrical form and square form. Each basket holds about fifty test-tubes. The wire is galvanized to prevent rusting. The round wire baskets should be used. in this solution for some time, several hours or more, and rinse, wash, drain, and wipe thoroughly afterward. The sole object to be attained is cleanliness in the true sense of the word. The glassware must be clean bacteriologically and chemically; that is, it must be free from mi- crobes and chemical substances. 2. Plugging Containers with Cotton. After the thorough cleansing above outlined, the test-tubes and flasks are plugged with a good quality of non-absorbent commercial cotton. The dry cotton plug forms a most efficient germ filter. All microbes are caught and held in the meshes of the cotton, and yet the air is permitted to pass through into the tube or flask. Open a roll of cotton, find the free end, and lay it out on the work table. Take the test-tube in the left hand; remove a goodly tuft of cotton with right hand, using thumb and first and second fingers. Place this over the mouth of the tube or flask, and push it down to a distance of 1/2 to 3/4 inch glass rod rounded (by heat) at the ends. The Fig. 13. — Wire basket filled with test-tubes plug- ged with cotton. A little cotton should be placed in the bottom of the bas- ket to lessen the danger of breaking the test-tubes. (Williams.) by means of a solid rod must not be too 38 PHARMACEUTICAL BACTERIOLOGY. thick, as it will then not permit enough cotton to enter the opening n or yet too thin, as it will then be forced through the cotton. The plug must not be too tight, as that would interfere with subsequent manipulations nor, yet too loose, for obvious reasons. Enough cotton should project above the opening to permit of ready grasping between the fingers in the later operations. Plugging may also be done with fingers alone, but this is tedious and non- professional. A far better method is to use a pair of fairly large blunt- FIG. 14.— A hot air sterilizer. These sterilizers are double-walled, on stand, with per- forations at top for thermometers. Ordinary baking ovens which can be secured from hardware dealers will serve the purpose. pointed pincers. Remove the cotton from the roll by means of the pincers and insert it into the test-tube with the pincers. Whatever method is used, remove the amount of cotton required to plug one tube or flask at one time. Do not attempt to plug with several small pieces. If an excess of cotton projects above the opening, pluck it away with the fingers; do not cut it away with scissors. Plug the tubes as uniformly as possible. 3. Filling Test-tubes with Culture Media. The rule is to pour the culture media hot, although this is not absolutely essential. For example, if the media are liquid in the cool or cold state, as bouillon, serum, milk, etc., they may be poured cold. A good rule is to pour a desired amount of the media just as soon as they are prepared, whether they are still hot or merely warm or cold. Of course, gelatin and agar media must be poured hot or must be liquefied before they can be poured. BACTERIOLOGICAL TECHNIC. 39 Fill a small to medium-sized beaker about two-thirds full of the culture medium. Grasp a plugged tube near the upper end, holding it between thumb and first two fingers of the left hand. Remove the cotton plug by means of the first and second, second and third, or third and fourth fingers of the right hand, grasping the free portion of the plug with the back of the fingers toward the cotton. Holding the tube slightly inclined on a level with the mouth, take beaker with medium in right hand (at the same time holding the cotton plug as described), see that the beak rests lightly upon and projects slightly over the edge of the tube, and pour, at the same time shifting the eyes to the lower end of the tube to watch the filling process. Fill tubes one-third full. Set down the beaker and replace the cotton plug. Place the filled tubes in special wicker baskets, with a little cotton at the bottom to prevent breaking. Some practice is necessary in order to pour so that none of the liquid comes in contact with the upper third of the tube. This must be avoided, in order to prevent the cotton plug from sticking. Tubes may also be filled from funnel with rubber hose, stop-cock, and glass nib attachment. Occasionally it is desirable to place exact amounts of culture media in the tubes, in which case a graduate, a burette, a pipette, or other convenient measuring device may be used. FIG. 15. — Diagrammatic sectional view of Arnold steam sterilizer illustrating the principle of steam formation, circulation and condensation. 4. Sterilization of Culture Media. All culture media in tubes as above set forth, and the portions remaining after the desired number of tubes are filled, must be considered as being contaminated with living microbes and their spores. These microbes and spores are killed by the sterilizing process. For all ordinary purposes the PHARMACEUTICAL BACTERIOLOGY. discontinuous or fractional method answers the purpose admirably. Place the test-tubes, flasks, and other cotton-plugged containers with culture media, in a steam sterilizer (Arnold steam sterilizer, either board of health or cylin- drical form; or kitchen vegetable cooker or steamer). The test-tubes are placed in wire baskets (rectangular or cylindrical) . These several containers with culture media are exposed to live steam for about thirty minutes, where- upon the flame is turned out, and if convenient the containers are allowed to remain in the sterilizer. Caution must be observed to guard against con- densed steam running into the several containers. The better way is to remove the containers and place them in an incubator kept at a temperature of 20° C. In twenty-four hours, or thereabouts, steam is again applied for thirty minutes. This is re- peated a third time on the second day after the first sterilization. The first sterilization presumably kills most of the vegetative cells. During the first interval of twenty-four hours most of the spores present develop into vege- tative cells, which are killed at the second sterilization. Should any survive the second steaming, they are sure to be killed during the third sterilization. During this time the cotton plugs have not been removed. The media thus fractionally or discontinuously sterilized are now ready for use in making microbic cultures, or they may be set aside for an indef- inite period of time. It is, of course, evident that in the above process of sterilization the temperature does FIG. 16.— Autoclave for using not exceed ioo° C., and it may be less in cer- steam under pressure for pur- poses of sterilization. tain portions of the sterilizer, steamer, or cooker, say, 95° to 97° C. In large or well- equipped bacteriological laboratories certain kinds of sterilizations are done by steam under pressure. The apparatus used for this purpose is known as autoclave. It consists of a strong steam cylinder with a screwed-down top safety valve, steam gauge, and thermometer. The articles (media, etc.) to be sterilized are placed inside, the top is securely fastened down, steam is generated until the thermometer registers, say, 120° C. The temperature is kept up to that degree for about five to ten minutes, which is sufficient to destroy all life, including spores. For ordinary purposes the autoclave is not essential. In fact, its use is rather limited. Blood serum, gelatin media, and all media containing carbohydrates, undergo certain chemical changes BACTERIOLOGICAL TECHNIC. 41 when the temperature is raised above 100° C, or even if kept at 100° C. for a long time or for a short time, if oft repeated. The autoclave is convenient for sterilizing discarded cultures, test-tubes, and glassware generally, and such media as beef broth and agar. In many instances it is desirable to sterilize at a temperature lower than 100° C. Albumen and blood serum, for instance, will coagulate at that temperature. Again, it is desired to kill the microbes without destroying the toxins which they form, as in the manufacture of bacterial vaccines. In the sterilization (pasteurization) of milk, a lower temperature is employed. In the sterilization of these and other substances the temperature ranges from 50° C. to 85° C. The discontinuous method is employed, differing from the method already described in that the period of exposure is much prolonged, about one hour. The number of daily exposures ranges from one to six. For example, milk exposed to a temperature of 60° to 70° C. for one hour is considered sufficiently sterilized, whereas blood serum is subjected to hourly exposures of a temperature of 60° C. for six successive days before it is pronounced completely sterilized. 5. Preparation of Culture Media. The pharmacist should give especial attention to the preparation of bacterial culture media, as in this he may be of service to the physician. The busy general practitioner who is not eqipped with a suitable bacterio- logical laboratory, or who does not have time to prepare culture media, would no doubt consider it a very decided advantage should the pharmacist offer to assist him. This will be more fully set forth in the last chapter. In brief, it may be stated that microbes feed upon the same substances that we feed upon. In the presence of adequate warmth and moisture they attack all organic substances. This being the case, it may readily be as- sumed that there are many substances or media which can be used as food for bacteria. Such is the case, and the number of media which have been used is legion. Almost any organic substance may be used, provided it is not aseptic or antiseptic in its properties. Culture media are liquid or solid, simple or compound. In the case of liquid or liquefiable solid media, the following physical properties are de- sired, in so far as it is possible to attain them: a. Culture media should be perfectly clear. There should be no sedi- ment, no opacity or flocculent suspension, and no floating matter. In the case of broths, extracts generally, gelatin media, and blood serum, these require- ments are easily attained. Perfectly clear agar is difficult to obtain. Milk is normally opaque. b. Media should be neutral or very slightly alkaline to litmus, which is PHARMACEUTICAL BACTERIOLOGY. equivalent to a slightly acid reaction to phenolphthalein, at a temperature of about 20° C. Most microbes develop best in media of such reaction. c. They must be free from living microbes and their spores, and from FIG. 17. — Arnold Steam Sterilizer. Boston Board of Health Form. This sterilizer is square, and constructed with a side-door all in accordance with the recommendation of the Boston Board of Health. Its large size makes it well suited to the requirements of Board of Health laboratories, and it has been found to be very serviceable and convenient. It is made of copper throughout, following the same principles as employed in the construc- tion of the other sterilizers. FIG. 18. — Arnold Steam and Hot-Air Sterilizer for Surgical Instruments. This sterilizer is a combination and portable sterilizer, so designed that instruments may be both sterilized and then dried by hot air, if desired. About 100° C. can be attained with the hot air by simply turning the valve shown in the illustration, which turns the steam as it escapes from the chamber into the base. other organism. This requirement is attained by sterilization as already described. Culture media contaminated with living organisms are not usable in bacteriological work. BACTERIOLOGICAL TECHNIC. 43 The essential requirements given under a, b, and c are obtained by nitra- tion, neutralization, and sterilization, as will be more fully explained. Non- liquefiable solid media, as potato, bread, squash, etc., must be clean, free from living microbes and other organisms, and there should be a compara- tively smooth exposed inoculating surface. These requirements are attained by washing and otherwise cleansing, disinfecting, rinsing, and heat sterili- zation (dry heat, steam or hot- water bath). The following are the more important media: A. Nutrient Bouillon. — Beef Extract (Armour's, Liebig's, etc.), 3 gm. Peptone, 10 gm. Salt, 5 gm. Distilled Water, 1000 c.c. Mix ingredients and boil for a few minutes. Filter through filter paper. This bouillon may be modified by adding glycerin (6 per cent.) and sugars, as dextrose, saccharose, or lactose (i per cent.). B. Loeffler's Blood Serum. — Very largely used in making diagnostic diphtheria bacillus cultures. In many cities this medium, with sterilized cotton swabs, in sterilized test-tubes, is furnished free to physicians by the board of health. In cities and towns where this is not done, the pharmacist should be prepared to fur- nish the materials to the physicians. The medium con- sists of Bouillon with i per cent. Glucose, Blood Serum, i part. 3 parts. FIG. 19. — Culture tube and swab tube used by physicians in the diagnosis of diph- theria. The swab tube should be long enough to have the entire length of swab inside, not projecting as shown in the fig- ure. (Williams.} The bouillon is prepared as above described, with i per cent, of glucose added. The blood serum can be obtained from calf, sheep, ox, or cow, through the butcher or at the abattoir. Collect the blood in a clean, sterile jar or flask, closed with cotton plug. Place on ice for twenty-four to forty-eight hours, during which time coagulation has taken place; the serum may then be siphoned off. The proper sterilization of Loeffler's serum re- quires care. After the bouillon and serum are mixed, pour into test-tubes and coagulate in a Koch-serum coagulator at a temperature of 80° C. Any form of sterilizer may, however, be used. The essentials are that the tern: perature should be raised very gradually and must be kept below the boil- ing-point, and the tubes should be slanted at a degree which will bring the medium close to the cotton plug, making what are commonly called tube slants. After the medium is coagulated in the tubes it is sterilized frac- 44 PHARMACEUTICAL BACTERIOLOGY. tionally on three successive days (one hour each day) at a temperature of 80° C. These tube slants are now ready for the physician. To prevent evaporation of the medium in the test-tubes, cover the cotton plug and upper end of tube with tin foil fastened with thread, and dip into melted paraffin several times. Tubes thus sealed can be kept for a year or more without any considerable shrinking of the medium. Dip the tin foil in a 1:2000 corrosive sublimate solution before capping on tubes. A simpler way is to use rubber caps which are especially made to fit over the end of the test-tube and the cotton plug. These rubber caps must be sterilized before applying them, for which purpose the 1-2000 corrosive sublimate solution will be found satisfactory. Rubber stoppers may also be used but they are more expensive and inferior to the rubber cap or the tin foil with coat of paraffin. C. Liquid Blood Serum. — Obtained as for Loeffler's serum. Sterilize fractionally at a temperature of from 56° to 58° C. for one hour on each of six days. The serum will be liquid and clear. D. Milk. — Secure fresh milk directly from cow, or, if in cities, demand certified milk. Keep on ice, in a covered jar, for twenty-four hours. Siphon off the middle portion, rejecting cream and sediment. Sterilize like Loeffler's blood serum. Litmus milk is prepared by adding i per cent, of azolitmin before sterilizing. This indicator will show whether or not acids are formed by the microbes which may be cultivated in the milk. Only pure milk will answer the purpose. Milk to which preservatives (formaldehyd, salicylic acid, borax, boric acid) have been added must not be used. E. Peptone Solution. — The medium is employed to test for the develop- ment of indol by certain bacteria. It consists of Peptone, 10 gm. Salt, 5 gm. Distilled Water, 1000 c.c. Boil, filter, and sterilize as for bouillon. The bacteriological indoF test is of great importance in medical practice, and the chances are that physi- cians will require this medium. However, sugar-free beef broth is also used for this test; in fact, it is generally preferred. Beef contains a small amount of muscle sugar, which must first be removed. F. Sugar-free Bouillon. — Grind the fat-free beef through a meat grinder; add water, and inoculate at once with a pure culture of Bacillus coli communis, and allow to incubate for twelve to fifteen hours at 38° C., then boil, filter, add peptone and salt, and prepare like bouillon; or, inoculate nutrient bouillon with the colon bacillus and prepare as above. However, before using the medium it should be tested for indol, as it has been proved that B. coli communis may form indol in beef extract. The indol test in bacterial cultures is made by adding two drops of concentrated sulphuric acid and one BACTERIOLOGICAL TECHNIC. 45 drop of a o.oi per cent, sodium nitrite solution to a four-day peptone-broth culture. If a pink color appears at the end of one-half hour it indicates the presence of indol. G. Beef Broth. — This medium is now not as extensively used as formerly. It is more difficult to prepare, and shows no advantages over the bouillon already described. Ground or Chopped Lean Beef, 500 gm. Peptone, 10 gm. Salt, 5 gm. Distilled Water. 1000 c.c. Add the water to the minced meat, shake frequently, and keep on ice for twenty-four hours, then strain forcibly through cloth, or press out in a hand press. Add the salt to the liquid, boil, make up to 1000 c.c., and add the peptone. Neu- tralize, filter, and sterilize. It will be ap- parent that the cold water meat infusion contains merely the meat salts, meat sugar, and acids, and a certain proportion of the albumens. The albumens are coagulated and removed in the filtering process, so that nothing remains of the meat but the salts, acids, and the trace of muscle sugar. Nearly the whole of the meat proper is wasted. It FIG. 20. FIG. 21. FIG. 20. — Test-tube cultures, a, Stab culture. This tube is closed with a rubber stopper to prevent drying of medium ; b, streak or smear culture on slant, tube closed with rubber cap. (Williams?) FIG. 21. — The ordinary rice cooker. A most valuable apparatus in preparing culture media and for sterilizing test-tubes and other objects. is apparent, therefore, that the meat extract bouillon answers all the pur- poses of the beef broth. H. Gelatin Medium. — Beef Extract, 3 gm. Gelatin, 100 gm. Salt, 5 gm. Peptone, 10 gm. Distilled Water, 1000 c.c 46 PHARMACEUTICAL BACTERIOLOGY. Mix ingredients in a rice cooker and boil for one-half hour, stirring fre- quently; neutralize and filter. This forms a very efficient culture medium for most bacteria, and is clear and remains solid at ordinary temperatures. It must be borne in mind, however, that frequent or prolonged heating tends to liquefy gelatin permanently. FIG. 22. — This is a copper double-walled incubator covered with non-conducting material and provided with a water gauge, tubulations for thermometer and thermostat, a ventilating strip, enclosed base and inner glass door. The incubating chamber is 24 cm. high, 30 cm. wide and 24 cm. deep. I. A gar Medium. — Agar is a seaweed found on the Japanese coast. It forms an important article of diet among the Japanese and Chinese. The medium consists of Beef Extract, Agar, Salt, Peptone, DistiUed Water, 3 gm. J5 gm- 5 gm. 10 gm. 1000 c.c. Prepare like the gelatin medium. Agar is difficult to filter, and the medium is never quite clear. The agar medium liquefies at a higher tem- perature than gelatin, and does not tend to remain liquid, no matter how often or how long it may be heated. BACTERIOLOGICAL TECHNIC. 47 J. A gar-gelatin Medium. — This has the advantage of both media, and is now much used in general bacteriological work. Agar, Gelatin, Salt, Peptone, Distilled Water, 8 gm. 40 gm. 5 gm. 10 gm. IOOO C.C. Mix, boil in rice cooker, stir; neutralize, filter, and sterilize as for other media. The above includes the more important culture media used in bacteriolog- ical work. Others can be prepared as occasion requires. It is not neces- FIG. 23. FIG. 24. FIG. 23. — MurrilPs Gas Pressure Regulator. This apparatus in its most improved form is to be used in connection with a thermostat for the maintenance of a constant emperature. The use of this regulator relieves the thermostat of the necessity of caring for the wide viriation which is apt to occur in the gas pressure, and with it the temperature may be held constant to within 0.1° C. FIG. 24. — Reichert thermo-regulator or thermostat used with incubator and other apparatus requiring a uniform degree of temperature. May be used in conjunction with the gas pressure regulator. sary to make up the full amounts indicated if it is evident that smaller quantities will suffice. The student should prepare all of the media in small amounts (one-quarter the quantities given) several times, in order to get the necessary experience and practice. 6. General Directions for the Preparation of Culture Media. Book information alone is not sufficient. Experience must be added. 48 PHARMACEUTICAL BACTERIOLOGY. Also, brief, concise explanations are far more valuable than lengthy descrip' tions of unessential details. Those possessed of good judgment do not re- quire lengthy explanations, and lengthy explanations would certainly be wasted on those who lack good judgment. This does not imply, however, that it is unnecessary to adhere strictly to established methods. The novice must follow closely the methods formulated by those who have devoted many years to some one particular mode of procedure, as it is wholly unlikely that he can improve upon them. Furthermore, when a physician calls for Loeffler's blood serum, for example, he wishes to be assured that the medium has been prepared according to the standard method. Any sub- stitution or deviation, no matter how slight, may bring about wholly neg- ative or erroneous results and conclusions. With this in mind the follow- ing suggestions are added: A. Selection of Ingredients. — Great care must be observed in the selection of the ingredients used in the preparation of culture media. Meats used must be from healthy animals, and there must be absolute certainty that no preservative has been added. Buy the meat personally from the nearest reliable butcher who keeps fresh meats only. Remove as much of the fat as possible. The so-called round steak of beef is usually employed. Use only the best gelatin; the so-called best French gelatin is usually employed, although much of the " French gelatin" comes from Berlin, Chicago, Omaha, or other places equally remote from France. Do not attempt to use old friable gelatin. The milk requirements have already been referred to. The milk must be fresh, placed on ice at once, and sterilized within twenty-four hours after it is taken from the cow. If the milk is obtained from an unknown dealer, test it for the presence of added water, preservatives, and other foreign matter. Agar does not deteriorate readily, and may be kept in good condition for a long time. Other highly gelatinous seaweeds may be used, although this is not permissible in the preparation of any of the standard culture media. Serum, egg albumen, peptone, various indicators, etc., must be pure. Too much caution cannot be observed in this regard. Secure the blood for serum personally wherever possible, from healthy animals. Use egg albu- men from fresh eggs, not from cold-storage eggs. Peptone and other chem- icals should be secured from reliable dealers. B. Suggestions on the Preparation of Culture Media. — First of all, some experience is necessary before a neat article can be prepared. Do not expect to prepare a medium which meets all of the requirements the very first time. In preparing gelatin media, remember that these are injured by excess- ive heating, and in preparing agar media, remember that they are very difficult BACTERIOLOGICAL TECHNIC. 49 to filter. Both must be filtered hot, using hot-water funnels; or the ordinary filtering device can be used by keeping the unfiltered portion hot and pouring into the funnel from time to time. Cover funnel with filter paper to keep out dust, and keep in the heat as much as possible. In so far as possible filter all media through filter paper (one thickness, properly folded), but it is practi- cally impossible (for reasons of time) to pass agar through filter paper. This medium is usually filtered through cotton upon which a neatly folded and perforated sheet of filter paper has been placed. Puncture the filter paper several times with a small knife blade. Filtering through cotton is quick, but the media are much less clear than when filtered through filter paper. The filtering process may also be hastened by means of pressure (suction) ; connect funnel with aspirator bottle and pump, but see to it that the connec- tions with the hydrant are properly made and that the flow is properly reg- ulated, in order to guard against any back pressure, which may cause the receiver to fill with hydrant water. This accident is best avoided by interpolating a flask or bottle. Agar may also be clarified by pre- cipitation. Pour the hot agar into an ordinary percolator used by phar- macists. The dirt particles and other impurities will gradually settle to the bottom. When cool, take out the solid medium and cut away the lower portion cantaining the sediment. C. Neutralization of Culture Media. — As already stated, most bacteria grow best in neutral or very slightly alkaline (to litmus) media, and since most media are quite decidedly acid in reaction, it is desirable to alkalinize. This is done by means of normal sodium hydroxide solution. In order to understand the method of procedure clearly, it is necessary to make certain explanations. A normal (N/i) solution of any substance contains as many grams per liter of the substance as there are units in its molecular weight, if the substance contains one atom of replaceable hydrogen. If it contains two atoms of replaceable hydrogen, the number of grams used equals the molecular weight divided by two, and so on. According to this, a normal solution of sodium hydroxide contains 40 gm. of sodium hydroxide in a liter. Exact normal solutions are, however, not prepared by weight. Crystallized oxalic acid is used as the basis for making normal solutions. This acid has a molecular weight (including a molecule of water of crystallization) of 126, and, since it is dibasic, 63 gm. per liter are taken. Any normal acid solution will exactly neutralize an equal volume of normal alkaline solution. To make a normal sodium hydroxide solution, add about 41 gm. of pure caustic soda to one liter of distilled water. Determine the amount of this solution required to just neutralize i c.c. of normal oxalic acid solution. This volume contains the quantity of sodium hydroxide which should be present in i c.c. of normal solution, and from this we may 4 50 PHARMACEUTICAL BACTERIOLOGY. calculate the volume of distilled water to be added in order that i c.c. of sodium hydroxide solution will neutralize i c.c. of normal oxalic acid solution. Having a normal solution of sodium hydroxide, it is now possible to prepare a normal solution of hydrochloric acid, etc. A tenth- (N/io), twentieth- (N/2o), fiftieth- (N/5o) normal solution is a normal solution diluted ten, twenty, and fifty times. An acid reaction is indicated by + , and an alkaline by — . The degree of acidity of any culture medium in preparation may be indicated by the amount of normal sodium hydroxide solution required to render it neutral to phenolphthalein. Neutralization by titration is done as follows: Place 5 c.c. of the medium to be neutralized in a dish, add 45 c.c. of distilled water, stir, and bring to a boil. Add i c.c. of phenolphthalein solution (0.5 per cent, of phenolphthalein in 50 per cent, alcohol). Add enough of twentieth-normal sodium hydroxide solution (in a burette), with constant stirring, to give a faint but distinct pink color. Read the amount of twentieth- normal sodium hydroxide necessary to neutralize the 5 c.c. of medium, and from this calculate the amount of normal sodium hydroxide solution neces- sary to neutralize the entire quantity of culture medium. Now boil the medium, and again titrate, when it will be found that there is a slight acid reaction. A third titration is rarely necessary. Another method is to take 10 c.c. of the culture medium, add a few drops of the phenolphthalein solution. From a burette add, drop by drop, with constant stirring, a normal sodium hydroxide solution (0.4 per cent.) until a faint pink color appears, which indicates the beginning of the alkaline reac- tion. Repeat this with two more samples. Note the amount of sodium hydroxide solution required in each case, and take the average and calcu- late the amount required for the entire quantity of medium. If, for example, the average was i c.c. for each 10 c.c. of medium, then 1000 c.c. of bouillon would require 100 c.c. of the sodium hydroxide solution; a concentrated solution being used, in order to avoid the dilution of the medium with the water of the caustic-soda solution. Flocculency of the medium usually indicates excessive alkalinity. The old, crude, rough-and-ready method is to add, from a beaker, drop by drop, a tenth-normal sodium hydroxide solution, with constant stir- ring, until red litmus paper just begins to turn blue. In practice it is found that when a culture medium is neutral or slightly alkaline to litmus it is still acid to phenolphthalein. In fact, it is claimed that most bacteria develop best in a medium having a reaction indicated by + 1 or +0.5; that is, it is sufficiently acid to phenolphthalein to require i per cent, or 0.5 per cent, of normal sodium hydroxide solution to render it neutral to phenolphthalein. D. Suggestions on the Preparation of Culture Media for Physicians. — First of all, the pharmacist must have the necessary laboratory equipment and BACTERIOLOGICAL TECHNIC. 51 necessary skill and experience to prepare culture media. He should explain to a few representative physicians that he is ready to prepare such media as the busy physician may require. The physicians will in all probability indicate what media are likely to be needed in the course of their practice. Allow yourself to be guided by these several suggestions and prepare the media accordingly. Make sure that the culture media are clear. There must be no sediment and no flocculency. Not infrequently the medium fails to become sufficiently clear, even though every precaution has been taken. In such cases clari- fication may be tried, rather than to discard it. Add the white of an egg, thor- oughly beaten, to a liter of the medium in the liquid state and at a tempera- ture below the coagulating point for albumen, mix thoroughly; boil for ten minutes, and filter. The coagulating albumen takes up the impurities which remain upon the filter with the albumen, while the medium comes through perfectly clear. Media which have become infected with bacteria as the result of inadequate setrilization should be discarded. Do not attempt to clarify them. They may become clear, but they are nevertheless objectionable because of the substances which the bacteria may have liberated and which might interfere with the development of the , FIG. 25.— Koch safety burner. Should the bacteria to be grown in it subsequently. flame be blown out, an Most of the tubes with solid media (Loefifler's automatic devise shuts . 1111°" the 8as- serum, gelatin, agar, and gelatme-agar) should be slants. The slanting surface offers certain advantages in making diag- nostic bacterial cultures. The usual, non-slanting tubes, for deep stab cultures, should, however, also be held in readiness. Keep all tubes in suitable containers, in a dry, cool, clean place. To guard against infection by mould and other organisms, it is well to cap all tubes with the rubber caps or the tin foil dipped in corrosive sublimate and parafhn, as already suggested. In case of liquid media, the rubber stoppers or the rubber caps are much preferred, or the hot paraffin may be painted over the tin foil and upper end of tube by means of a small brush. Apply two or three coats. Thus protected, there is no danger of outside infection. The chances are that the physician who calls for tube culture media will also require the use of an incubator. This the pharmacist should have in readiness. The usual copper double-walled water-jacket incubator, with thermo-regulator, kept at a temperature of about 25° C., will serve the purpose. The swab to be supplied with each tube of slanted Loeffler's serum consists of a piece of wire or of pine wood four inches long, around the 52 PHARMACEUTICAL BACTERIOLOGY. lower end of which a pledget of absorbent cotton has been wound and firmly tied by means of thread. This is placed in a test-tube, which is then plugged with cotton and sterilized in the dry sterilizer (one hour at a tempera- ture of 140° C.) The physician wipes the cotton end of the swab over the suspected throat area, and then lightly rubs it over the surface of the serum FIG. 26. — Hot water funnel with stand and ring gas burner. FIG. 27. — Hot water funnel with stand. tube 'slant. The swab is returned to the tube, the cotton plug is restored and then returned to the board of health to be destroyed in stove or furnace fire, or destroyed by the attending physician in case there is no board of health to recieve it. FIG. 28. — Glass rods with platinum wire, straight and loop, for inoculating culture tubes. Petri plates, etc.— (Williams.) 7. Making Bacterial Cultures. This branch of the science of bacteriology is of comparatively little importance to the pharmacist. While it is desirable to know what bacterial cultures are and how to make some of them, it is wholly unlikely that the pharmacist will be called upon to do extensive work along this line. This is BACTERIOLOGICAL TECHNIC. 53 the work of those who make bacteriology a specialty. Such bacterial cultures as are likely to come to the notice of pharmacists will most generally be prepared by physicians, health officers, and other specialists in bacteri- ology. The pharmaceutical bacteriologist may be called upon to make bacterial examinations of drinking water, of milk, of ice cream, and other food materials; of syrups, liquors, aquae, tinctures, fluidextracts, infusions, etc., and he should, if possessed of some skill and adequate laboratory facilities, be able to do so. The prime object in growing bacteria in artificial culture media is to make possible their further more careful and more extended study. The FIG. 29. — Cover-glass pincers. a and b are self-clamping but the pressure is often enough to break thin covers. study of bacteria in their natural or normal surroundings is all-important, but is not complete without the artificial culturing. As a rule, bacteria are biologically associated with other organisms, and it is unusual to find pure cultures in nature or in natural media. An open sore may contain several or many species and varieties of bacteria, in addi- tion to the pus germs. The intestinal tract of the typhoid patient contains bacteria other than the comma bacillus of Koch. The tubercular bron- chials always show a mixed infection. The diphtheric membrane con- tains some foreign germs, etc. Some infections, particularly those of internal tissues or organs, as lymphatic glands for example, may present practically pure cultures. However, no matter how mixed an infection 54 PHARMACEUTICAL BACTERIOLOGY. may be, there is always a predominating type present, or, to state it more correctly, it is the unusual development of the predominating type which determines the diagnostic characteristics of the infection. It must also be borne in mind that bacteria behave differently when taken out of their natural environment and placed in artificial culture media. It does not at all follow that, in the case of a mixed infection, the predominat- ing and diagnostic microbe will remain the predominating type when said mixed infection is transferred to some artificial culture medium. In fact, the predominating microbe may develop very slowly or with great difficulty, if at all, in the artificial culture media; whereas one or more of the associated microbes may thrive remarkably well, soon entirely over- shadowing the former. These and other conditions occasion some of the great difficulties encountered in determining the primary causes of some microbic and protozoic diseases and infections. A. Test-tube Cultures. — Inoculate several test-tubes, containing nutrient gelatin or agar gelatin, with any FIG. 30. Fig. 30. — Cotton plugged tube with a potato slant resting on a bit of glass rod to keep the potato out of the water in the bottom of the tube. (Williams.} FIG. 31. — Manner of holding tubes when making subcultures. The cotton plugs, removed from the two tubes, should be held in hand holding the platinum rod, as explained in the text. (Williams.} material which is known to be bacterially infected. This is done by touching the infected material with the tip of a heat-sterilized (by holding in flame of Bunsen burner until red hot) platinum needle (pre- pared by fusing a platinum wire, 11/2 inches long, into the end of a glass rod, six to seven inches long), then removing the cotton plug from the test-tube, and pushing the needle, carrying the microbes, into the culture medium down to the very bottom of the tube. Replace the cotton plug at once, pass the needle into the flame of the Bunsen burner until red hot, to sterilize it, and lay aside for the next tube inoculation. This is known as a deep stab tube inoculation. In this manner inoculate some five or six tubes. BACTERIOLOGICAL TECHNIC. 55 Also make streak inoculation in tube slants by simply passing the infected platinum needle over the middle of the tube slant surface, from lower end toward the top, observing the instructions regarding the cotton plug and needle sterilization, with each tube inoculation. Number the tubes seri- ally, and in a special notebook make entry of all desirable data pertaining to each inoculation, making such entries under each tube number. Place tubes vertically in a suitable holder, as tumbler, beaker, wire basket, etc., and set aside in incubator or in some container to which you alone have access. In warm weather the first bacterial growths may appear at the end of thirty-six hours. In cold or cool weather nothing may appear for two, three, and even four to five days. Note the nature of the bacterial growth in a deep stab inoculation and in the streak inoculation, as to FIG. 32. — Making an Esmarch roll-tube culture. A lump of ice is placed in a dish and the inoculated tube is placed horizontally in a groove in the ice and revolved until the medium is well set. The groove may be made with test-tube full of hot water. (Williams.} a. Growth — scanty, moderate, abundant, slow, rapid. b. Form of growth — outline clearly defined, spreading, rugose, beaded, etc. c. As to surface — flat, raised, concave, convex. d. Color — translucent, glistening, waxy, transparent, opaque, light, chalky white, grayish-white, dark red, green, blue, yellow, lemon color, purple, etc. e. Odor — comparative description. f. Consistency — viscid, slimy, stringy, membranous, friable or brittle, dry, watery, etc. g. Changes in medium — gelatin liquefied, gelatin not liquefied; colored, as grayed, browned, reddened, blued, etc. In case indicators are used, the possible color changes should be noted. 50 PHARMACEUTICAL BACTERIOLOGY. h. Deep stab culture — where is growth most active? If at bottom, it indicates anaerobic tendencies. If limited to top of medium, it indicates de- cidely aerobic tendencies. (Most bacteria are decidedly aerobic; that is, they require oxygen to thrive.) The test-tube cultures do not necessarily represent pure cultures, and the student cannot know whether the growths in the test-tubes represent the predominating bacterial flora in the substance from which the inoculations were made. The chief object in making the above cultures is to enable the student to get practice in this preliminary work, particularly as to making the cultural observations above indicated. FIG. 33. — Kitasato filter for filtering hypodermic solutions, culture media, sera, water, etc. The material to be filtered is placed in the globose container and forced through the clay (infusorial earth) tube (Berkefeld filter bougie) by connecting the receiver with a vacuum pump. All parts of the filter must, of course, be sterilized by heat before and after using. (Williams.} The student should now make transfers (subcultures) from the first tube cultures into second tubes, and note whether or not the characteristics originally noted are continued or repeated. If the transfer cultures are the same as the originals, it is an indication that the first cultures were pure (representing one species or variety), which is generally the case, though it must be borne in mind that one and the same species of microbe may undergo considerable change in extended culturing, as indicated in the changed culture characters. In fact, some of the changes are so extreme as to confuse even the most expert bacteriologists. BACTERIOLOGICAL TECHNIC. 57 B. Isolating Bacteria by the Plate Method. — In order to separate or isolate the several species and varieties of bacteria in any contaminated substance, it is only necessary to dilute the inoculating material sufficiently. For this purpose there is necessary, sterilized Petri dishes containing heat-sterilized gelatin or other solid media through which the bacteria from the contami- nated substance are disseminated in numbers so small that the colonies from each and every microbe present may be visible to the naked eye (or aided by a simple lens). This is done as follows: FIG. 34. — Streak culture on agar in a Petri dish. (Delafteld and Prudden.) To obtain isolation cultures of air bacteria it is only necessary to expose the Petri dish (with a layer of gelatin or agar-gelatin medium, sterilized) for about two minutes, immediately closing the dish and setting it aside to await developments. Making isolation cultures from contaminated solids or liquids is not quite so simple. Proceed as follows: Liquefy the gelatin in four or five test-tubes and keep them at a temperature of not more than 30° C., 58 PHARMACEUTICAL BACTERIOLOGY. just high enough to keep the contents liquid; set them in a beaker filled with warm water (30° C.) until needed. Number the tubes from i to 5. Dip a platinum loop (bend the end of a straight needle into a small loop) into the infected liquid, as bouillon, milk, water, tea, syrup, tincture, fluid- extract, etc., etc., and pass one loopful into tube No. i (sterilize loop and return to its proper place). Rotate tube (replugged with the cotton and held vertically) rapidly between the hands for twenty seconds, to mix contents. By means of the platinum loop take two loopfuls (one loopful may serve) from tube No. i (which you have just inoculated and rotated) and pass them into tube No. 2. Plug both tubes, set aside tube No. i, and rapidly rotate tube No. 2. Take two loopfuls from tube No. 2 and transfer to tube No. 3, and proceed as before. Now pour contents of tube No. i into a sterile FIG. 35. — Appearance of colonies on gelatin in a Petri dish. Differences in size of colonies may indicate different species. Differences in color also indicating different species, cannot be shown in the figure. (Williams.) Petri dish, also numbered i; contents of tube 2 into Petri dish 2; and tube 3 into Petri dish 3. Wait until the media in the Petri dishes are solidified, and then set aside at the room temperature to await developments. In the course of two or three days it will perhaps be found that very many minute specks are visible in dish No. i, some one hundred or more may appear in dish No. 2, and perhaps not more than ten or twenty in dish No. 3. Observe carefully the several growths in dishes 2 and 3. Each visible growth in- dicates the development from a single microbe. Are the several growths all alike, or do they differ? Differences in color and in outline of growths indicate different species of bacteria. The several different kinds of bacteria BACTERIOLOGICAL TECHNIC. 59 may now be transferred to test-tubes by means of the straight platinum needle or the loop, and the observations may thus be extended. Transfers can be made to different kinds of media, as agar, gelatin, agar-gelatin, beef broth, milk, prepared potato, etc. C. Making Bacterial Counts. — In order to determine the number of bac- teria in any given substance the same procedure as was just described is fol- lowed, with the difference that a definite amount of the thoroughly mixed contaminated substance is added to a definite amount of culture medium in the test-tubes in which the dilution mixtures are made. For example, we will suppose that it is desired to determine the number of bacteria (per c.c.) in milk: Thoroughly mix the sample of milk by shaking it in the container. Take o.i, 0.2, 0.5, or i c.c. of the milk (by means of a sterilized graduated pipette) and add it to 9 c.c. of the liquefied culture medium in tube No. i; i c.c. of tube No. i to tube No, 2, also with 9 c.c. of medium; i c.c. of tube No. 2 to tube No. 3 (with 9 c.c. of medium), following the other directions as already given. Plate out as already explained, and watch developments. In Petri dish No. i the number of bacterial growths (colonies) will no doubt FIG. 36. — Petri dish. These dishes are among the essentials in the bacteriological laboratory. (Williams.) be so great as to make counting impossible. Petri dish No. 2 may contain 360 colonies, and dish No. 3 may contain not more than 40. An average is obtained by repeating the test (using the same milk sample) a number of times. In the above milk sample the average may be 42,000 microbes per c.c. If the bacterial content is high, it is necessary to extend the attenuation four and even five times. If it is desired to determine the number of bacteria per gram of dry soil, it- will be necessary to carefully weigh a small quantity (i gm., more or less) of average soil, triturate the entire sample with say, 100 c.c. of sterile distilled water, and from this make the dilution cultures as above described, using i c.c. or less of the soil triturate. To compute the number of bacteria per gram of dry soil, it will now be necessary to determine the moisture percentage in a sample of soil taken from the same place as the sample which was used in making the triturate. The solution is simple. We will suppose the tritu- rate sample weighed 0.856 gm. and the number of bacteria found was 6o PHARMACEUTICAL BACTERIOLOGY. 3,000,000; and the percentage of moisture was 10. From these data it would be found that i gm. of dry soil will contain 3,855,011 microbes. The above is sufficient to make clear how one might proceed to deter- mine the number of microbes in and upon old pills, tablets, powders; on one ivory vaccine tip, in one glycerinated vaccine tube, in i c.c. of bacterial vaccine, antitoxin, syrup, tincture, fluidextract, camphor water, distilled water, sewage, drinking water, etc. Naturally, great caution and care must be observed to avoid errors and faulty conclusions. In fact, no one should attempt such work in actual practice until after considerable preliminary laboratory experience. It is not practicable nor is it necessary to give fuller information regarding bacterial cultures. We have not touched upon the various methods for determining whether or not the microbes under investigation are essentially FIG. 37. — Graduated fermentation tube. These tubes are required for gas determination with colon bacillus and other gas-forming micro-organisms. aerobic or essentially anaerobic; the manner of determining the thermal death- point; relationship of rate of growth to temperature, etc. We have said nothing of the use of indicators added to culture media, as litmus, rosolic acid, and phenolphthalein, nor have we explained the special use of special culture media in determining the nature and identity of bacteria. These and many other details we must omit, merely stating that, should it become desirable to make such investigations, the necessary information must be secured elsewhere, as in some standard laboratory guide in bacteriological technic. The following outline of special methods will serve as a guide in making bacteriological examinations of soils, air, pharmaceuticals, liquids, etc. D. Cultur ing Soil Bacteria. — Soil is a mixture of dead and decayed organic matter, sand and living organisms and their spores. Near the surface the soil contains large numbers of bacteria, from 10,000 to 10,000,000 per gram, BACTERIOLOGICAL TECHNIC. 6 1 and more. In fact the fertility of the soil is practically proportional to the number of bacteria present. Most species of soil bacteria are harmless to man though the bacilli of tetanus (lockjaw), of typhoid fever, of malignant edema, of anthrax, and of pus formation may be present. The tetanus germ is quite common in garden soils and the anthrax germ is apt to occur in cattle pens, pastures and other places frequented by cattle. Other soil bacteria are decidedly useful as will be more fully explained elsewhere. Some soil bacteria (the nitrifiers) do not grow on the usual media while others thrive exceedingly well in such media. Anaerobic forms must be cultured in the absence of air or oxygen. The root nodule bacteria of the leguminosae can be grown readily on gelatin or agar. The tubercles or nodules must be thoroughly cleansed and repeatedly washed in boiled distilled water, then rinsed for ten seconds in a i-iooo corrosive sublimate solution, and finally thoroughly rinsed (three minutes) in boiled distilled water. Crush several of the sterilized nodules in a sterile watch crystal, by means of a sterile glass rod and from this make the dilution plate cultures and set aside at room temperature. Colonies of small motile bacteria (Rhizobium mutabile) will appear in about four days. To test the soil bacterially, select thoroughly mixed samples and plate out as already suggested, using every precaution to prevent the introduction of extraneous germs. Cultures can also be made from internal plant tissues by following, in general, the directions given under root nodule bacteria,' excepting that after the washing and rinsing, the root, instead of being crushed, is cut or broken across and the inoculation material is taken from the inner tissue by means of a platinum needle or scalpel. E. Bacteria of the Air. — Air currents carry the germ-laden dust and dirt particles. The number and kind of air bacteria depends upon environment, climatic conditions, moisture, sunlight, etc. The air currents are the main factors in germ dissemination. Spores and dry (though not dead) bacilli may be carried many miles. Air microbes are derived from the soil surface and from the objects surrounded by the air. Bacteria are exhaled with the breath and are carried and distributed from and by animals, plants and clothing. The air carries pus germs, tubercle bacilli, anthrax bacilli and their spores, besides other pathogenic microorganisms, including also yeast cells and the spores of higher fungi. Air microbes may be studied by exposing a Petri dish containing steril- ized agar or gelatin, for two minutes or longer. The number of colonies that will appear will depend upon the locality, season, air moisture, etc. To determine the number of microbes in a given volume of air the Sedgwick- Tucker aerobioscope is used, though similarly constructed apparatus may be made by any fairly skillful student. The aerobioscope consists of a glass cylinder as shown in the illustration. The open ends are plugged with cotton. 62 PHARMACEUTICAL BACTERIOLOGY. Granulated sugar is loosely packed into the narrow end and all is then sterilized in a hot-air sterilizer (not over 120° C.). Pass a given quantity of air through the aerobioscope by attaching an aspirator bottle to the narrow end and allowing a given volume of water to run out of the bottle. The volume of air drawn through equals the volume of water run from the bottle. Of course the cotton plug is removed from the larger end of tube while the water is running. The bacilli and spores are caught in the sugar, while the air passes through. Replace cotton plug and shake the sugar into the larger end of tube. Remove cotton plug again and pour in about 10 to 15 c.c. of liquefied (40° C., not hot) gelatin. Roll the tube held horizontally. The gelatin dissolves the sugar and mixes with it. Roll on ice to hasten the hardening of the gelatin. Set aside in incubator, at room temperature (20° C., about). The number of colonies which appear indicates approx- FIG. 38. — Aerobioscope after Sedgwick-Tucker, plugged with cotton. The larger end in which the cultureing is done is ruled to facilitate the counting of colonies. imately the number of microbes in the volume of air aspirated. Let us suppose that the number of colonies was 125, the volume of air aspirated 10 liters, from which we would get 1250 bacteria per cubic meter of air. F. Bacteria of Liquid Substances. — The bacteria of water, milk, tinctures, fluidextracts, aquae, aerated waters, mineral waters, distilled water, broth, and liquids generally, can be studied quantitatively in a comparatively simple manner. By means of a sterile i c.c. graduated pipette, run o.i c.c. to 0.5 c.c. of the liquid into the center of a sterilized petri dish, pour upon this enough (about 10 c.c.) melted (sterile) agar or gelatin and mix by tilting the dish slightly from side to side. Set aside for the medium to harden and incubate at the room temperature, or at 25° C., if quicker results are desired. This method is satisfactory if the number of bacilli present is comparatively small. If very abundant, dilutions must be made in the manner already described. The following general suggestions should be observed in making bacterio- logical determinations of liquids: a. Containers for samples (other than the original containers) must be sterile and closed with sterile corks or cotton plugs. If the samples are to be carried any distance they should be packed in ice. In no case is it wise to keep a sample longer than forty-eight hours before culturing it. If the sample is to be examined within two or three hours after collecting it, placing on ice is not absolutely necessary. BACTERIOLOGICAL TECHNIC. 63 b. Every sample should be thoroughly mixed before making cultures. Shake well, about twenty times. This is very important. c. All glassware, pipettes, etc., must be thoroughly sterilized by washing, use of disinfectants, rinsing, wiping, hot air and steam sterilization, etr. d. Incubate at room temperature, as a rule. Colonies will begin to appear in forty-eight hours. The maximum development will be in three or four days, in most instances, provided the temperature does not fall below 20° C. e. As a rule the presence of abundant gelatin-liquefying organisms may be looked upon with suspicion. Certain sewage organisms liquefy gelatin very actively. f. The colon bacillus and some sewage cocci give pink colonies with lactose litmus agar medium. The cocci colonies are a deeper vermilion than the colon colonies'. Sewage-contaminated water will show many pink colonies. g. Certified milk (just delivered) should not show more than from 1000 to 10,000 colonies per c.c. h. Wholesome uncertified milk should not show more than from 30,000 to 50,000 colonies per c.c. The number of colonies permissible varies in different states and in different localities in the same state. The number of colonies may range from 25,000 to 1,000,000 per c.c., and even more, and yet the milk may be pronounced wholesome. No pink colonies should be present. No pus cells should be present (centrifugalized sediment). i. Good drinking water should not show more than 50 to 100 colonies per c.c. and there should be no pink colonies, only a few liquefying colonies (i-io) and most of the colonies should develop best at 20° C. If 50 per cent, of the colonies develop best at 30° to 38° C. this indicates probable sewage contamination or contamination with intestinal bacteria. This differential temperature test is considered of importance in the bacterial examination of drinking waters. Normal water gives a proportion of i colony of high temperature organisms to from 25 to 50 colonies of low temperature organisms. In sewage contaminated water the proportion is i to 4 and even less. j. Thus far there are no standards for the bacteriological testing of phar- •maceuticals. Tinctures and fluidextracts should show only few colonies per c.c., not over 30 to 60. Sera should show none. Well prepared and prop- erly ripened small-pox vaccine should show only a few colonies per point or per glycerinated tube. Aquae often show abundant colonies, from 10,000 to 10,000,000 per c.c. and more. k. The colon bacillus should not be present in drinking water, in milk or in pharmaceuticals. If present, it indicates sewage or other objectionable contamination. The colon bacillus is motile in young broth cultures, forms 64 PHARMACEUTICAL BACTERIOLOGY. no spores, is gas- (dextrose broth cultures in fermentation tube) and indol- forming, reduces nitrates to nitrites, does not liquefy gelatin and is not stained by Gram's method. 1. Syrups of all kinds, unless very carefully prepared and carefully kept to prevent fermentation, are apt to show numerous bacteria, yeasts and moulds. Any syrup showing signs of yeast fermentation (gas bubbles, vinous odor) or mouldiness, is not fit for use and should be rejected. The attempt to render it usable by boiling is unsatisfactory, furthermore the changes produced by the organisms are always objectionable and cannot be rectified by heating or by other methods of sterilization. m. Recent investigations have shown that many of the marketed (bottled) mineral waters contain numerous bacteria, from 10,000 to 300,000,000 and more per c.c. In some cases colon bacilli have been found. These find- ings prove that in many instances the methods of bottling must be careless or otherwise unsatisfactory since sewage contamination is not reasonably possible under proper sanitary conditions. Undoubtedly the contamination is in some instances due to reused and inadequately cleaned and sterilized containers and in other instances to impure and inadequately sterilized mineral water. A popular opinion prevails that the chemicals in the min- eral waters are sufficiently germicidal to destroy bacteria but this is not the case. Bacteria may develop actively in a great variety of solutions of high concentration provided such solutions are chemically balanced. Loeb, Osterhaut and others have shown, for example, that ocean water is chemically balanced, thus being suitable to maintain life in a great variety of organisms. G. Bacteria in Canned Fruits. — The work recently demanded by the pure food laws (federal and state) has shown that such food substances as canned fruits of all kinds, including jams, jellies, preserves, catsups, tomato pastes, etc., are frequently highly contaminated with yeast cells, moulds and their spores, and other higher fungi, and bacteria. It is, however, evident that the food products named may be quite free from such contamination as may be seen from the examination of canned food products prepared by the careful housewife. That manufacturers may approximate the home condition is demonstrated by the fact that factory products are found on the market, which are quite free from contamination. Since wholesome ripe fruit contains yeast cells, bacteria and mould in very small numbers only, and since most of these organisms are removed in the various steps of the processing, as washing, peeling, steaming, etc., it is evident that the finished factory product should, like the home-made product, contain these organisms in negligibly small numbers only, provided, of course, that wholesome fruit is used. However, most of the factory samples thus far examined have shown numerous dead yeast cells, mould spores, mould hyphae, and bacteria, indicating the use of fruit, fruit pulp, fruit BACTERIOLOGICAL TECHNIC. 65 juices, fruit refuse, etc., which was decomposed or undergoing fermentation or decomposition prior to or at the time of manufacture. The organisms named prevail in varying amounts in different products. Yeast-organisms are apt to predominate in jellies, fruit juices and fruit pulp; bacteria in catsups and pastes; and moulds in certain fruits as strawberries, blackberries and raspberries. The presence of numerous dead yeast cells (1,000,000 to 50,000,000 per c.c.) is evidence that the material was undergoing alcoholic fermentation just prior to or at the time of manufacture. Tomato pastes have been found on the market showing over 400,000,000 bacteria per c.c. besides numerous yeast cells and considerable mould. The bacterial content of catsups is apt to run high, from 10,000,000, to 50,000,000 and more per c.c. Not m Areas I sa. mm.. Cu.bic Contents So*,SW:> O.Z.C.mm^. O.ooSC.nim.. FIG. 39. — Counting apparatus for mould, yeast cells and spores. From the measuring values marked on the slide it is easy to determine the number of mould hyphal clusters, yeast cells and spores per c.c. of the substance under examination. Used with No. 2 ocular and No. 3 and No. 5 objectives. The rulings are as follows: There are 75 square millimeters in the entire area, of three squares of 25 square millimeters each. The one-millimeter areas are to be used in determining the quantity of mould, dirt, sand and other impurities present. The one- millimeter areas indicated black in the figure are marked off into 1/25 (0.04) square millimeters. These smallest areas are used in making spore and yeast cell counts. The depth is o. 2 millimeter. We will suppose that a given fruit sample, as strawberry jam, shows 30 yeast cells in the smallest area (0.2 mm. Xo.o4 mm. =0.008 cm.), then i c.c. of the substance would contain 3,750,000 yeast cells. including the vinegar bacteria, which are introduced into catsups and pastes, such high bacterial content is generally due to bacterial development during or after manufacture. The presence of mould organisms and their spores (other than penicillium) indicates the use of mould-infested fruit. Pen- icillium, which is entirely saprophytic in habit, may develop after manu- facture, particularly on the surface of inadequately sterilized fruit products in containers not entirely filled. "Swelling" of cans containing fruit products is generally due to yeast development though it may also be due to bacterial activity, and indicates inadequate sterilization of either the container or of the fruit or both. Exam- ination will show the presence of living yeast cells, or bacteria, perhaps air bubbles, and the characteristic vinous odor of yeast may be noted. 5 66 PHARMACEUTICAL BACTERIOLOGY. Based upon such conditions as can be made to prevail in carefully oper- ated factories, the following may be given as the limits of the number of organisms permissible in the fruit products under discussion. a. Yeast cells, either living or dead, not to exceed 1,000,000 per c.c. b. Mould spores not to exceed 50,000 per c.c. c. Hyphal clusters and hyphal fragments not to exceed 10,000 per c.c.; or not over 50 per cent, of separate and distinct fields of view under the compound microscope should show hyphal clusters or hyphal fragments. d.% Bacteria (either living or dead but not including vinegar bacteria in products to which vinegar is added) not to exceed 5,000,000 per c.c. The, above figures apply only to fruit products supposedly made from comparatively fresh fruits and fresh fruit juices. The yeast, bacterial and spore counts are made with a Thoma-Zeiss hemacytometer (Turck ruling) using a No. 5 (1/5 in.) objective with No. 2 (i in.) ocular. H. Quantitative and Qualitative Bacteriological Testing. — The following will serve as a general outline of bacteriological analyses which may be made in food and drug laboratories. The substances which require such bacterio- logical examination include catsups, tomato pastes, vinegars, water supplies, mineral waters, milk, ice creams, any and all substances which are suspected to be sewage contaminated, etc., etc. The sequence of processes here given bear a progressive relationship. Whether process II is carried out will depend upon the findings under I and whether III shall be undertaken will depend upon the findings under II. The essential facts to be ascertained are whether or not there is possible sewage contamination as indicated by the presence of the colon bacillus, sewage streptococci and possibly the typhoid bacillus. The typhoid agglutinating tests are apt to prove unsatisfactory. In most instances this test will be unnecessary as the presence of the colon bacillus is evidence that the food, drug or drink is contaminated with sewage and is hence unfit for human use. I. Direct Count. — For this purpose the Thoma-Zeiss hemacytometer with Turck ruling1 is used (No. 2 ocular with 1/5 in. objective) which can be secured from any bacteriological supply house. The instructions for using it can be obtained from the dealer, though the measuring values indicated on the hemacytometer are sufficient to indicate the manner of making the counts. The rulings generally used for bacterial countings are 1/25 sq. mm. X i/io mm. deep, making an area of 1/250 cu. mm., or reduced to decimal fractions, 0.04 sq. mm. X o.i mm. deep = 0.004 cu. mm. We will suppose that the average of 20 counts shows 5 bacilli, then i cu. mm. would contain 1,250 bacilli or 1,250,000 in i c.c. The direct count is, in many instances, very unsatisfactory for several 1 To render the ruled lines visible rub a very soft pencil over the ruled area. BACTERIOLOGICAL TECHNIC. 67 reasons. Particles other than micro-organisms may be mistaken for bacilli or cocci and, furthermore, it cannot be known for a certainty that the organ- isms are dead or alive. If they are present in great abundance (10,000,000 to 100,000,000 and more per c.c.), ordinary smear preparations maybe stained, using methyl blue or fuchsin. Dead bacilli, that is those which have been dead for some time, do not take the stain well, due" to the fact that the cell- plasm is disintegrated. Tomato pastes, anchovy pastes, catsups, some mineral waters and similar preparations, may contain bacteria in such numbers that dilutions are desirable or necessary to make counting possible. A dilution of one in ten will, as a rule, be sufficient. Weigh or measure one part (i gm or r c.c.) of the substance, add it to nine parts filtered distilled water and mix thoroughly by shaking. If the direct count shows bacilli in great numbers or if for any reason sewage contamination is suspected, and also to determine the number of living bacilli and spores suspected, proceed as follows: II. Plate Culture Counts. — Make one set of plate cultures, using lactose litmus agar,1 and incubate at 20° C. Make a second set of plate cultures, also upon lactose litmus agar, and incubate at 38° C. The usual dilution methods are followed when necessary, using preferably o.i c.c. quantities for the plates. This temperature differential test is considered of great impor- tance. Colon bacilli and other micro-organisms, whose natural habitat is the intestinal canal, will develop actively at the higher temperature (38° C.), whereas the usual air, soil and water bacteria develop best at the lower tem- perature (20° C.). If the high temperature colonies approximate the low temperature colonies, sewage contamination may be suspected. If in addi- tion many of the high temperature lactose litmus agar colonies show pink or light vermilion, the sewage contamination is practically proven. The colon bacillus, as well as sewage streptococci, give pink colonies, the latter being the brighter, more vermilion in coloration, due to the formation of acid (in the fermenting lactose) . Examine the pink colonies under the microscope. The colon microbe is rod-shaped, rather thick, non-sporing. and shows motility in recent broth cultures, whereas the streptococci are smaller and are not rod- shaped. High temperature colonies as compared with low temperature col- onies should not exceed 1:100 or 1:25. If the proportion is 1:4 or less, sewage contamination is very likely. After 36 hours the pink colonies may turn blue, due to the development of ammonia and amines. Naturally the high temperature colonies must be studied within twenty- four to thirty hours whereas the low temperature cultures require much more time, two to four days. 1 Add i per cent, of lactose to the usual agar medium and enough tincture of litmus- to give it a lilac tinge. 68 PHARMACEUTICAL BACTERIOLOGY. If the temperature and color differential tests indicate sewage contamina- tion, then the following additional tests should be carried out. III. Indol Reaction and Gas Formula. — The indol reaction has already been explained. The gas formula is determined as follows: To sets of four grad- uated fermentation tubes containing glucose bouillon and lactose bouillon, add o.i, 0.2, 0.5, and 10 c.c. of the suspected liquid. If gas formation is observed the presence of colon bacilli may be suspected. If the o.i c.c. tubes show gas formation then the presence of colon bacilli may be assumed. Fill the bulb of a tube, showing gas formation, with a 2-per cent, solution of sodic hydrate, hold thumb tightly over the opening and mix contents by tilting back and forth carefully. The portion of gas absorbed is CO2 whereas the unabsorbed portion is supposedly hydrogen. The colon bacillus shows a gas formation of 1/3 hydrogen. Of course the total volume of gas is recorded before the sodic hvdrate is added. FIG. 40. — Colon bacillus. This microbe is quite large, in the comparative sense, and is morphologically typical of the group bacillus. The flagellae are few in number and comparatively long. The gas formula with a positive indol reaction is practically conclusive as far as the presence of the colon bacillus is concerned. Add to this the other tests and we have presumptive evidence of sewage contamination, and any article of food or drink showing such contamination is unfit for human consumption. The colon bacillus, ,the bacilli of the hog cholera group and others have the power of reducing neutral red; producing a greenish-yellow fluorescence. For this reaction use glucose bouillon to which has been added i per cent, of a 0.5 per cent, solution of neutral red. In examining milk, the pus cell and leucocyte count is considered important; centrifugalize 10 c.c. of milk for thirty minutes, pour off supernatant milk and mix residue with BACTERIOLOGICAL TECHNIC. 69 0.5 c.c. normal salt solution and make counts of pus cells and leucocytes per c.c. from the amount (0.5 c.c.). Abundant pus cells and leucocytes indi- cate abscess or other pathological condition of milk ducts or glands. This test is, however, of little significance excepting in the hands of authorities on diseases of cows. It is stated that as many as 100,000 leucocytes per c.c. may occur in apparently healthy animals. Gelatin-liquefying organisms may be looked upon with suspicion when found in milk, water and other liquid-food substances intended for human consumption, as has already been explained. It should be borne in mind that the colon bacillus is one of a group of some fifteen or more species and varieties of closely related micro-organisms which resemble each other in the following particulars: 1. Do not form spores. 2. Do not liquefy gelatin. 3. Produce acid in milk and cause milk coagulation. 4. Produce acid and gas in glucose and lactose media. 5. Produce acid and gas in bile-salt-glucose broth. 6. Grow well in temperatures ranging from 38° to 42° C. In differentiating the colon bacillus, remember that this organism is rod-shaped (2 to 3/1 long by 0.5 to o.6/* wide), is motile, produces indol, gives rise to pink colonies on lactose (or glucose) litmus agar and reduces neutral red glucose (or lactose) agar with a greenish-yellow fluorescence. It should also be remembered that sewage is a highly complex substance and contains micro-organisms in great variety and in great abundance. Among the organisms present are species of Spirillum, Vibrio, Proteus and Beggiatoa in addition to the bacilli and streptococci already mentioned. The typhoid bacillus does not thrive well in sewage. The number of bac- teria present in crude or ordinary sewage (domestic, city, hospital, mixed, etc.) ranges from 1,000,000 to 100,000,000 and more per c.c. The work of these organisms is to break down and render soluble and assimilable (for plants) the organic matter composing the sewage, thus assisting the work of rotting bacteria generally. The following is a tabulation of the bacteriological testing that should be made of foods (including pastes, catsups, milk, ice creams, water supplies, mineral waters, alcoholic beverages, etc.) that may show an excess of bacterial growth or which may be sewage contaminated: BACTERIOLOGICAL EXAMINATION. I. Direct Count. 1. Bacilli per c.c 2. Cocci, per c.c . 70 PHARMACEUTICAL BACTERIOLOGY. II. Plate and Tube Cultures. (Lactose-litmus-agar.) 1. Temperature differential test. a. (20° C.) Colonies per c.c b. (38° C.) Colonies per c.c 2. Color differential test. a. Pink and yellow colonies per c.c c. Not pink or yellow colonies per c.c. . 3. Colorless gelatin liquefying colonies per c.c. 4. Neutral red reduction, + or — . 5. Indol reaction, + or — . 6. Gram stain behavior, + or — . 7. Gas (hydrogen) formula. III. Agglutinating tests for Typhoid Germs. * 8. Staining Bacteria. Staining consists of the infiltration of the cell-substance with solutions of various coloring materials obtained for the most part from the group of coal- tar derivatives known as the aniline dyes. As is generally known, different cells and different portions of one and the same cell react differently with the various dyes used. This peculiar behavior brings out contrasts in appearances which aid very materially in determining the morphological characters. The prime object, therefore, in using stains is to aid in the study of cell morphology. Different bacteria react differently with the several stains used. Some species take certain stains very readily, while they are quite indifferent to other stains. The vegetative cell stains much more readily than do the spores. In fact, spores are stained with great difficulty; however, after they are once thoroughly stained they hold the color persistently. The dyes which may be used in bacteriologic work are of many kinds, differing as to color and as to staining powers with different cells, cell-con- tents, and cell-parts. They are usually classified as acid or basic. Eosin, acid fuchsin, and picric acid are acid stains, and are said to be diffuse in their effects, having no special affinity for any special cell structure. Fuch- sin, methylene blue, and gentian violet are basic, and appear to have special attraction for bacteria and for plasmic and nuclear substances of cells gener- ally, for which reasons they are most generally employed as bacterial stains. Fuchsin is, in fact, about the only efficient stain for endospores, while gentian violet and methylene blue are excellent stains for the bacterial cell- wall. It is known that certain substances possess the property of preparing the bacterial cells in such a way as to induce them to take up the dye more BACTERIOLOGICAL TECHNIC. 7 1 readily, thus intensifying the stain, as aniline oil and carbolic acid. Such substances are called mordants, and may be used separately or added directly to the stain itself. Certain liquids or solutions remove the stain from the bacterial cell more or less readily, as water and alcohol, but more particularly solutions of acids. Such substances are quite generally employed for removing any excess of stain from the bacterial cell or from the matrix in which the bacteria are fixed or embedded. Acidulated (with HC1) alcohol is most commonly employed. Ordinarily, rinsing in a small stream of water is sufficient. Some bacteria resist the decolorizing process with acids more strongly than others, and are said to be acid fast or acid proof, as, for example, the bacilli of leprosy and of tuberculosis, while the great majority of species give up the stain very readily. It is a fact that one and the same species of microbe reacts variably with one and the same stain, depending upon a variety of causes. Moderate heat hastens and intensifies the staining. For ordinary purposes a single stain only is used, but sometimes struc- tural differences are more clearly shown by what is known as double or con- trast staining. Take, for example, a spore-bearing microbe, as that of anthrax. The spores may be stained by means of carbol fuchsin; the entire cell, excepting the spore, can be completely, decolorized in acidulated alcohol, and then methylene blue or gentian violet applied as the contrast stain. We then have a blue cell-wall with a red spore. However, the beginner is apt to be disappointed in his attempts at double staining; in fact, even the most skilled bacteriological technologists are apt to meet with small success, and generally rest satisfied with the use of the single stain. The pharmacist will have comparatively little to do as far as the actual staining of bacteria is concerned. He should, however, be able to prepare the more important stains, mordants, and other solutions which may be required by the city or health board bacteriologist or the physician, and we shall therefore give the more commonly employed preparations. A. Stock Solutions. — Make saturated solutions of the basic dyes (fuch- sin, gentian violet, and methylene blue) in 95 per cent, alcohol. Keep these in glass-stoppered bottles in a cool, dark place, ready for use in preparing the stains. The stock solutions should in all intances be filtered before using. Secure the dyes from reliable dealers and in small quantities. Do not make up large quantities of stock solutions or stains proper, as they gradually deteriorate, particularly if exposed to light. B. Mordants. — The principal substances used are aniline, carbolic acid, tannic acid, glacial acetic acid, ferrous sulphate, sodium hydroxide solution, chromic acid, and a few others. Those in general use are the two first named. The others have a more limited use in special cases. 72 PHARMACEUTICAL BACTERIOLOGY. i. Aniline Water. Aniline, 2 c.c. Distilled Water, 98 c.c. Shake frequently, and finally filter several times through filter paper. It should be perfectly clear. This preparation deteriorates rapidly. Make up small amounts and keep in a dark place. It becomes .worthless, even when observing all precautions, in a few weeks. 2. Carbolic Acid- Solution. Carbolic Acid, 20 c.c. Distilled Water, 100 c.c. Filter. This mordant is rarely used by itself. C. Stains. — We give here the more important stains, approximately in the order of preferred use. i. Loeffler's Methylene Blue. Stock Solution (saturated) Methylene Blue, 30 c.c. i : 10,000 Sol. KHO in Dist. Water, 100 c.c. Mix, shake, filter. This stain is much used as a general bacterial stain and in the examination of blood, pus, etc. 2. Aniline Gentian-Violet. Aniline Water, 75 c.c. Stock Solution Gentian-Violet, 25 c.c. Mix, shake, filter. This is an excellent bacterial stain. 3. Carbol-Fuchsin. Stock Solution of Basic Fuchsin, 10 c.c. 5 per cent. Sol. Carbolic Acid, 100 c.c. Mix, shake, filter. This is one of the most useful stains with the so-called acid-proof microbes. It is also a spore stain, and is the most commonly em- ployed stain used in contrast or double staining. It is a comparatively slow stain, but is permanent. 4. Gram's Stain. Gram's stain is used for diagnostic purposes, and is perhaps the best known stain in the entire field of bacteriological technic. Its value depends upon the fact that certain microbes, when stained and afterward treated BACTERIOLOGICAL TECHNIC. 73 with a solution of iodine and washed in alcohol, give up the stain. Such microbes are known as Gram-negative, whereas those which do not give up the stain are said to be Gram-positive. The method of using this stain is somewhat complicated, requires care, and, with a beginner, often yields disappointing results. Keeping in mind the following will minimize the disappointments: a. Long-continued (one year or more) subcultures frequently lose the Gram-stain behavior. b. Old cultures, that is, those which have been growing in the same medium for several days or more, as a rule do not stain characteristically. With such cultures the results are often neither negative nor positive, just enough to be confusing and perplexing. c. The solutions used must be fresh. The gentian-aniline solution, as well as the iodine solution, deteriorates quite rapidly. d. Do not overstain, and do not decolorize too long. Stop decolorizing as soon as no more violet color comes away. In the Gram method two solutions are used, namely: 1. Aniline gentian-violet, and 2. Gram's iodine solution. Iodine, i gm. Potassium Iodide, 2 gm. Distilled Water, 300 c.c. The method, briefly outlined, is as follows: a. Spread the bacteria evenly and thinly over the cover-glass (the usual smear preparation). Stain with the aniline gentian-violet for from two to five minutes. Warming will hasten and intensify the staining. Wrash in water to remove excess of stain. b. Drop on the iodine solution and allow it to act for about one minute or until the preparation assumes a coffee-brown color. It may be desirable to apply the iodine a second time. c. Wash off the excess of iodine in water and then decolorize by dropping on 95 per cent, alcohol. Tip the slide and allow alcohol to run over the preparation; continue until the violet color ceases to stream away. d. Finally rinse in water and examine in water. If desired, dry and mount permanently in Canada balsam or some other suitable mounting medium. e. A contrast stain, such as eosin, fuchsin, safranin, or Bismarck brown, may be used, following (c). Keeping in mind the difficulties already referred to in using the Gram method, and the additional possible source of error due to the fact that one and the same microbe will stain but feebly at one time and very intensely 74 PHARMACEUTICAL BACTERIOLOGY. at another time, we now name the principal organisms which are Gram- positive or Gram-negative. Bacteria and other Organisms Stained by the Gram Method. Staphylococcus pyogenes aureus. Staphylococcus pyogenes albus. Streptococcus pyogenes. Micrococcus tetragenus. Micrococcus lanceolatus. Bacillus diphtheriae. Bacillus tuberculosis. Bacillus 6f anthrax. Bacillus of tetanus. Bacillus of leprosy. Bacillus aerogenes capsulatus. Oidium albicans. Actinomyces (of actinomycosis). Bacteria not Stained by the Gram Method Diplococcus of meningitis (intracellular) . Diplococcus of gonorrhea. Micrococcus melitensis. Bacillus of chancroids (Ducrey's). Bacillus of dysentery (Shiga's). Bacillus of typhoid fever. Bacillus of bubonic plague. Bacillus of influenza. Bacillus coli communis. Bacillus pyocyaneus. Bacillus of Friedlander. Bacillus proteus. Bacillus pyocyaneus. Bacillus mallei (glanders). Spirillum of Asiatic cholera. Spirillum of relapsing fever. Bacillus of pneumonia. 5. Pappenheim's Stain. Sat. Aqueous Sol. Methyl Green, 50 c.c. Sat. Aqueous Sol. Pyronine, 15 c.c. Mix and filter. This is much used for staining bacteria in pus and other pathological secretions. The bacteria are stained a bright red, while the cell nuclei are blue to purple. 6. Smith's Stain. Stock Sol. Basic Fuchsin, 10 c.c. Methyl Alcohol, Formaldehyde, each 10 c.c. Distilled Water, to make 100 c.c. BACTERIOLOGICAL TECHNIC. 75 Mix and filter. Let stand for twenty-four hours before using. Renew in three weeks. This stain is much used to distinguish between bacteria and nuclear substances. Allow the stain to act for from two ta tea. minutes. 7. Flagella Staining. Care is necessary in staining flagellae. Numerous methods have been recommended, but Pitfield's method, as modified by Muir, is perhaps the best and at the same time comparatively simple. The following solutions are required: a. Mordant. Tannic Acid (10 per cent. Aq. Sol.), . 10 c.c. Sat. Aq. Sol. Mercuric Chlor., 5 c.c. Sat. Aq. Sol. Alum, 5 c.c. Carbol-Fuchsin, 5 c.c. Mix, shake, filter or centrifuge. This solution does not keep longer than one week. b. Stain. Sat. Aq. Sol. Alum, 10 c.c. Stock Sol. Gentian-Violet, 2 c.c. Mix, filter. Carbol-fuchsin may be used instead of gentian-violet. This stain will not keep longer than a few days. The method is as follows: 1. Drop on mordant. Leave for one minute, with gentle heat. 2. Rinse in water for two minutes. 3. Dry carefully at slight warmth. 4. Stain for one minute with gentle heat. 5. Wash, dry, and mount in Canada balsam. In making the cover-glass preparation, take a loopful from a young aqueous subculture of some motile bacillus and touch it on the carefully cleaned cover and allow the drop to spread by rotating and tilting the cover. Do not use the loop more than is necessary. Flagellae are very delicate and easily destroy* d. Dry very carefully, and do not pass through flame more than three times. 8. Spore Staining. As already stated, spores (endospores) of microbes stain with great diffi- culty, for which reason a contrast is effected negatively; that is, the rest of the cell is quickly stained, leaving the unstained, highly refractive spore to appear like a bit of glass within the colored frame. This is in many ways the most satisfactory way of demonstrating the presence of spores. The spores may, 76 PHARMACEUTICAL BACTERIOLOGY. however, be stained by the usual acid-fast or acid-proof methods, care being observed in decolorizing. Stain with hot carbol-fuchsin for a few minutes, wash, and decolorize quickly with 3 per cent, hydrochloric acid in 95 per cent, alcohol, and then use a contrast stain, as gentian-violet or methylene blue. The red spores will then appear in the violet or blue frame. 9. Capsule Staining. The gelatinous capsule of microbes is also stained with great difficulty, and requires special methods and experience to yield anything like satisfactory results. The methods of Welch and Hiss are quite satisfactory. The cap- sule is, however, generally visible without any staining because of the light contrast that naturally exists. Certain substances, as glacial acetic acid (Welch method), cause the capsule to enlarge and take up the stain more readily. Certain staining methods bring out the capsule of certain mi- crobes, as, for example, the Gram method as applied to pneumonia sputum. The Muir method is perhaps the best for capsule staining. It is as follows: 1. Stain in carbol-fuchsin for one-half minute, with gentle heat. 2. Wash lightly in alcohol (95 per cent.). 3. Wash well in water. 4. Flood with mordant of Sat. Aq. Sol. Mercuric Chlor., 2 c.c. Tannic Acid (20 per cent. Aq. Sol.), 2 c.c. Sat. Aq. Sol. Potassium Alum, 5 c.c. 5. Wash in water. 6. Wash in 95 per cent, alcohol, one minute. 7. Wash in water. 8. Stain with methylene blue for one-half minute. 9. Dehydrate in alcohol. 10. Clear in xylene, and mount in Canada balsam. There are numerous other special stains and special staining methods, which need not be mentioned here. Should the pharmacist be called upon to prepare any of these, he will find full particulars in any standard work on medical bacteriology. 9. Studying Bacteria. The complete study of any one species of microbe with a view to deter- mining its identity is a long and tedious process. It involves a study of the organism in its natural element and in artificial culture media, and its behavior in animal inoculation tests, etc. Special apparatus, experimental animals (as rats, mice, guinea-pigs, dogs, etc.), and technical experience and BACTERIOLOGICAL TECHNIC. 77 skill are necessary. Just what kind of observations are involved in such study is indicated in the complete method as outlined by the Society of American Bacteriologists (Jan., 1908), which is hereby submitted for the Benefit of those who may wish to acquaint themselves with such details. The glossary of terms should be carefully considered first of all. The decimal system for indicating group relationships of microbes (Table I) is most unique and is very convenient for active workers. Those interested will find the desired explanations of the methods and reagents mentioned, in any of the larger works on medical bacteriology and in bacteriological technology. It is not at all likely that the pharmacist will ever have occasion to make use of the special methods cited. He should nevertheless acquaint himself with them sufficiently to comprehend their application in the study of pathogenic bacteria. Our bacteria nomenclature is in some confusion, and unless the methods of naming bacteria are corrected, the confusion is certain to become much greater. The trouble lies in the failure to define group or generic delimita- tions. The present generic terms, "bacillus" and "micrococcus," include too many species. We have a confusing and almost incomprehensible array of synonyms, of which those applied to Rhizobium mutabile may serve as an example. The different names that have been given to this organism may be arranged as follows: Pasteuracese, Laurent. Bacteria, Woronin, 1866. Bakteroiden, Brunchorst and Frank, 1885. Microsymbiont, Atkinson, 1893. Spores or gemmules, Ward and Ericksson. Bacillus radicicola, Beyerinck, 1888. Cladochytrium leguminosarum, Vuellemin. Phytomyxa leguminosarum, Schroeter. Schinzia leguminosarum, Woronin. Rhizobium leguminosarum, Frank, 1890. Rhizobium Frankii, Schneider, 1892. Rhizobium mutabile, Schneider, 1902. Pseudomonas radicicola, Moore, 1905. The above synonomy is also interesting because it indicates a most remark- able difference of opinion regarding the nature and identity of this root- nodule organism. Further, as the result of the wholly inadequate group delimitations we have such name-monstrosities as Granulobacillus saccharo- butyricus mobilis nonliquifaciens , and Micrococcus acidi paralactici liquifaciens Halensi. Reform in nomenclature is very desirable, and it must come through a careful definition of generic groups based on physiological charac- ters, rather than upon largely morphological characters, as is done now. 78 PHARMACEUTICAL BACTERIOLOGY. It is advised that the pharmacist refrain from experimenting with patho- genic organisms, excepting in so far as he may act in cooperation with the physician. When experimenting with pathogenic organisms the greatest caution is necessary to guard against autoinoculation and the spreading of disease. It should be made a rule to treat every microbe studied as though it were virulently pathogenic, capable of spreading an epidemic. Never expose a colony (plate culture, tube culture, etc.) in such a way as to permit the escape of the organisms into the air. Pour a disinfecting solution (5 per cent, carbolic acid) into cultures that are to be discontinued and then boil container and all, for thirty minutes, before washing and cleaning the glassware. Never -forget to sterilize the platinum needle before and after making an inoculation or a culture transfer. DESCRIPTIVE CHART— SOCIETY OF AMERICAN BACTERIOLOGISTS. 1 GLOSSARY OF TERMS. Agar Hanging Block, a small block of nutrient agar cut from a poured plate, and placed on a cover-glass, the surface next the glass having been first touched with a loop from a young fluid culture or with a dilution from the same. It is examined upside down, the same as a hanging rock. Ameboid, assuming various shapes like an ameba. Amorphous, without visible differentiation in structure. Arborescent, a branched, tree-like growth. Beaded, in stab or stroke, disjointed or semi-confluent colonies along the line of inoculation. Brief, a few days, a week. Brittle, growth dry, friable under the platinum needle. . Bullate, growth rising in convex prominences, like a blistered surface. Butyrous, growth of a butter-like consistency. Chains, short chains, composed of 2 to 8 elements. Long chains, composed of more than 8 elements. Ciliate, having fine hair-like extensions, like cilia. Cloudy, said of fluid cultures which do not contain pseudozooglea. Coagulation, the separation of casein from whey in milk. This may take place quickly or slowly, and as the result either of the formation of an acid or of a lab ferment. Contoured, an irregular, smoothly, undulating surface, like that of a relief map. Convex, surface the segment of a circle, but flattened. Coprophyl, dung bacteria. Coriaceous, growth tough, leathery, not yielding to the platinum needle. Crater if orm, round, depressed, due to the liquefaction of the medium. Cretaceous, growth opaque and white, chalky. Curled, composed of parallel chains in wavy strands, as in anthrax colonies. Diastasic Action, same as Diastatic, conversion of starch into water-soluble substances by diastase. Prepared by F. D. Chester, F. P. Gorham, Erwin F. Smith, Committee on Methods of Identification of Bacterial Species. Endorsed by the Society for general use at the annual meeting, January, 1908. . BACTERIOLOGICAL TECHNIC. 79 Echinulate, in agar stroke a growth along the line of inoculation, with toothed or pointed margins; in stab cultures growth beset with pointed outgrowths. Effuse, growth thin, veily, unusually spreading. Entire, smooth, having a margin destitute of teeth or notches. Erose, border irregularly toothed. Filamentous, growth composed of long, irregularly placed or interwoven filaments. Filiform, in stroke or stab cultures a uniform growth along line of inoculation. Fimbriate, border fringed with slender processes, larger than filaments. Floccose, growth composed of short curved chains, variously oriented. Flocculent, said of fluids which contain pseudozooglea, i.e., small adherent masses of bacteria of various shapes and floating in the culture fluid. Fluorescent, having one color by transmitted light and another by reflected light. Gram's Stain, a method of differential bleaching after gentian- violet, methyl- violet, etc. The + mark is to be given only when the bacteria are deep blue or remain blue after counterstaining with Bismarck brown. Grumose, clotted. Infundibuliform, form of a funnel or inverted cone. Iridescent, like mother-of-pearl. The effect of very thin films. Lacerate, having the margin cut into irregular segments as if torn. Lobate, border deeply undulate, producing lobes (see Undulate). Long, many weeks or months. Maximum Temperature, temperature above which growth does not take place. Medium, several weeks. Membranous, growth thin, coherent, like a membrane. Minimum Temperature, temperature below which growth does not take place. Mycelioid, colonies having the radiately filamentous appearance of mould colonies. Napiform, liquefaction with the form of a turnip. Nitrogen Requirements, the necessary nitrogenous food. This is determined by adding to nitrogen-free media the nitrogen compound to be tested. Opalescent, resembling the color of an opal. Optimum Temperature, temperature at which growth is most rapid. Pellicle, in fluid bacterial growth either forming a continuous or an interrupted sheet over the fluid. Peptonized, said of curds dissolved by trypsin. Persistent, many weeks, or months. Pseudozooglece, clumps of bacteria, not dissolving readily in water, arising from imperfect separation, or more or less fusion of the components, but not having the degree of compactness and gelatinization seen in zooglea. Pulvinate, in the form of a cushion, decidedly convex. Punctiform, very minute colonies, at the limit of natural vision. Rapid, developing in twenty-four to forty-eight hours. Raised, growth thick, with abrupt or terraced edges. Repand, wrinkled. Rhizoid, growth of an irregular branched or root-like character, as in B. mycoides. Ring, same as Rim, growth at the upper margin of a liquid culture, adhering more or less closely to the glass. Saccate, liquefaction the shape of an elongated sac, tubular, cylindrical. Scum, floating islands of bacteria, an interrupted pellicle or bacterial membrane. Slow, requiring five or six days or more for development. Short, applied to time, a few days, a week. 80 PHARMACEUTICAL BACTERIOLOGY. Sporangia, cells containing endospores. Spreading, growth extending much beyond the line of inoculation, i.e., several millimeters or more. Stratiform, liquefying to the walls of the tube at the top and then proceeding downward horizontally. Thermal Death-point, the degree of heat required to kill young fluid cultures of an organism exposed for ten minutes (in thin-walled test-tubes of a diameter not exceeding 20 mm.) in the thermal water-bath. The water must be kept agitated so that the temperature shall be uniform during the exposure. Transient, a few days. Turbid, cloudy with flocculent particles; cloudy flocculence. Umbonate, having a button-like, raised center. Undulate, border wavy with shallow sinuses. Verrucose, growth wart-like, with wart-like prominences. Vermiform-contoured, growth like a mass of worms, or intestinal coils. Villous, growth beset with hair-like extensions. Viscid, growth follows the needle when touched and withdrawn, sediment on shaking rises as a coherent swirl. Zooglea, firm gelatinous masses of bacteria, one of the most typical examples of which is the Streptococcus mesenteroides of sugar vats (Leuconostoc mesenterioides} , the bacterial chains being surrounded by an enormously thickened firm covering inside of which there may be one or many groups of the bacteria. NOTES. (r) For decimal system of group numbers see Table i. This will be found useful as a quick method of showing close relationships inside the genus, but is not a sufficient characterization of any organism. (2) The morphological characters shall be determined and described from growths obtained upon at least one solid medium (nutrient agar) and in at least one liquid medium (nutrient broth). Growths at 37° C. shall be in general not older than twenty-four to forty-eight hours, and growths at 20° C. not older than forty-eight to seventy-two hours . To secure uniformity in cultures, in all cases preliminary cultivation shall be practised as described in the revised Report of the Committee on Standard Methods of the Laboratory Section of the American Public Health Association, 1905. (3) The observation of cultural and bio-chemical features shall cover a period of at least fifteen days and frequently longer, and shall be made according to the revised Stand- ard Methods above referred to. All media shall be made according to the same Standard Methods. (4) Gelatin stab cultures shall be held for six weeks to determine liquefaction. (5) Ammonia and indol tests shall be made at end of tenth day, nitrate tests at end of fifth day. N (6) Titrate with NaOH, using phenolphthalein as an indicator: make titrations at 20 same time from blank. The difference gives the amount of acid produced. The titrations should be done after boiling to drive off any CO2 present in the culture (7) Generic nomenclature shall begin with the year 1872 (Cohen's first important paper). Species nomenclature shall begin with the year 1880 (Koch's discovery of the poured plate method for the separation of organisms). (8) Chromogenesis shall be recorded in standard color terms. BACTERIOLOGICAL TECHNIC. 8 1 TABLE i. A NUMERICAL SYSTEM OF RECORDING THE SALIEXT CHARACTERS OF AN ORGANISM (GROUP NUMBER). 100 Endospores produced. 200 Endospores not produced. 10 Aerobic (Strict). 20 Facultative anaerobic. 30 Anaerobic (Strict). i Gelatin liquefied. 2 Gelatin not liquefied. o.i Acid and gas from dextrose. o . 2 Acid without gas from dextrose. 0.3 No acid from dextrose. 0.4 No growth with dextrose. .01 Acid and gas from lactose. .02 Acid without gas from lactose. .03 No acid from lactose. .04 No growth with lactose. .001 Acid and gas from saccharose. .002 Acid without gas from saccharose. .003 No acid from saccharose. .004 No growths with saccharose. .0001 Nitrates reduced with evolution of gas. .0002. Nitrates not reduced. . 0003 Nitrates reduced without gas formation. .0000 r Fluorescent. .00002 Violet chromogens. .00003 Blue chromogens. .00004 Green chromogens. . 00005 •••••••• Yellow chromogens. . 00006 Orange chromogens. . 00007 Red chromogens. .00008 Brown chromogens. . 00009 Pink chromogens. . ooooo Non-chromogenic. .000001 Distasic action on potato starch, strong. .000002 Distasic action on potato starch, feeble. .000003 Distasic action on potato starch, absent. .0000001 Acid and gas from glycerin. .0000002 Acid without gas from glycerin. . 0000003 No acid from glycerin. . 0000004 No growth with glycerin. The genus according to the system of Migula is given its proper symbol which precedes the number thus: (7) BACILLUS COLI (Esch.) Mig. becomes B. 222.111102 BACILLUS ALCALIGENES Petr. becomes B. 212.333102 PSEUDOMONAS CAMPESTRis (Pam.) Sm. be- comes Ps. 211 .333151 BACTERIUM SUICIDA Mig. becomes Bact. 222.232103 6 82 PHARMACEUTICAL BACTERIOLOGY. DETAILED FEATURES. NOTE. — Underscore required terms. Observe notes and glossary of terms. I. MORPHOLOGY (2) i. Vegetative Cells, Medium used temp age days Form, round, short rods, long rods, short chains, long chains, filaments, commas, short spirals, long spirals, clostridium, cuneate, clavate, curved. Limits of Size Size of Majority Ends, rounded, truncate, concave. ( Orientation (grouping) , Agar J Chains (No. of elements) Hanging-block 1 Short chains, long chains. ^ Orientation of chains, parallel, irregular. 2. Sporangia, medium used temp age days Form, elliptical, short rods, spindled, clavate, drum-sticks. Limits of Size Size of Majority A f Orientation (grouping) Hanging-block Chains -*N iHtN HM «!-* *?} ro H M . 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A great variety of substances are recommended for the extermination of ants, as borax, camphor, balsam of Peru, spraying with benzine, etc. In lawns and in the open generally (in ant hills) they are most quickly destroyed by means of carbon bisulphide. This kills the ants as well as the larvae. The exterminators for pests of all sorts is legion and those especially interested must consult some standard work on formulas such, as the Scien- tific American Cylcopedia of Formulas (Hopkins) . CHAPTER XII. STERILIZATION AND DISINFECTION IN THE PHARMACY. It is only within very recent years that sterilization in the pharmacy has received any serious attention. Certain pharmacopeias, notably those of Austria and Beligum, give specific directions regarding the sterilization of certain medicamenta, particularly those intended for hypodermic use. The German, English, Italian, Swiss and other pharmacopeias give direc- tions regarding certain sterilizing processes which may be applied to a few articles. Fischer, Stich, Deniges, Mario, Schoofs and other European investigators have given the subject much attention and have perfected many of the details of procedure. Some of the non-official methods of sterilization are of very doubtful practicability. Particularly the methods recommended for the sterilization of pharmaceutical solutions by means of the ultra-violet rays and by means of chemical disinfectants. Lesure sums up the use of the ultra-violet rays as follows: "A series of experiments shows that, at present, the ultra-violet rays can scarcely be regarded as a practical means of sterilizing pharmaceu- tical solutions, such as hypodermic injections. It is not yet possible to sterilize liquids in small closed glass vessels, since the glass absorbs the rays of shortest wave length, which are precisely those of most active sterilizing power. Possibly on a large scale solutions could be sterilized in bulk and then filled, in vacuo, into sterilized small receivers. The rays might be useful for substances which are decomposed by treatment in the autoclave. Some substances are, however, so readily decomposed by ultra-violet rays, that their solutions can never be sterilized therewith. Such are solutions of quinine salts, of mercuric iodide, of atoxyl, of eserine, of apomorphine and some glucosides, as for example gentiopicrin. Opaque solutions and suspensions of solids cannot be thus sterilized. The permeability of the different solutions to the rays also varies very greatly. Apart from the question of decomposition, it is found that, in the case of gentiopicrin, completely sterile solutions were not obtained even after an exposure of half an hour; on the other hand ancubin solutions were completely sterilized in thirty seconds." The decomposition changes due to the ultra-violet rays are not clearly understood. The indications are that there are no very marked chemical changes in such substances as cocaine and pilocarpin hydrochloride after three hours' exposure. Arbutin shows a change in a few 190 STERILIZATION AND DISINFECTION IN THE PHARMACY. IQI minutes. There is so much uncertainty as to the results that the method cannot as yet be recommended for practical use. The addition of disinfectants to medicines for purposes of sterilization has recently received some attention. The use of formaldehyde, ether, chloroform and alcohol, have been recommended, each having its special use in practice. The general criticisms made regarding the use of the ultra- violet rays also apply here. Currie recommends a formalin method as follows: applicable to infusions of calumba, gentian, quassia and senega "The infusions of calumba and quassia are simply evaporated to one-eighth of their bulk, filtered, and 4 minims of the ordinary 40 per cent, solution of formaldehyde added to each fluid ounce of the concentrated infusion. On dispensing, the requisite amount is put in a shallow basin and brought sharply to the boil, thus dissipating the formaldehyde. The infusion is then diluted to the normal strength with sterilized distilled water. Infusion of gentian is made from gentian root alone, and concentrated. To this is added essence of lemon (i in 10), and the official tincture of orange in the proportion of 2 fluid drams of the former and i fluid ounce of the latter to each pint of the infusion. There is also added 4 minims of 40 per cent, solu- tion of formaldehyde to each fluid ounce of infusion. Infusion of senega is concentrated by evaporation and to prevent precipitation, 5 grains of potassium bicarbonate are added to each fluid ounce of the concentrated solution, and 4 minims of 40 per cent, solution of formaldehyde. In case of both gentian and senega infusion, the formaldehyde is dissipated at the time of dispensing, in the manner already described. The advantages of this process are ease of manipulation, cheapness, and the certainty of the antiseptic condition of the infusion while being kept in stock and until dis- pensed. The quantity of formaldehyde remaining in the diluted infusion is infinitesimal, and may be ignored for all practical purposes." It is known that weak solutions of hypodermic and intravenous solutions, unless sterilized, will show numerous bacteria upon standing for a time. One per cent, solutions of pilocarpin, atropin, cocaine, morphine, and fluid- extract of ergot have been found to contain millions of bacteria per c.c. However, loper cent, iodoform glycerin, camphorated oil (i in 10), solutions of apomorphin (0.2 in 20), quinine (i in 10), antipyrin (5 in 10), cocaine (10 per cent.) are usually quite free from bacteria. In a general way the bacterial content of medicinal solutions decreases directly with the degree of concentration. Pus microbes die at once in ether and in a saturated solution of quinine, whereas they remain active in a 10 per cent, solution of cocaine. A 2 per cent, solution of morphine kills pus microbes in twenty-four hours, while pure glycerin kills them only after an exposure of six to eight days. A perfectly safe rule for the pharmacist is to consider all medicamenta which he handles and which he may be called upon to dispense, as being IQ2 PHARMACEUTICAL BACTERIOLOGY. possibly contaminated and to sterilize and disinfect all articles which in his judgment as a qualified pharmacist may require such treatment, in so far as it is practically possible. The retail pharmacist must not place too much confidence in the assertions of comparatively little known manufacturers and wholesale houses, regarding the sterile conditions of the articles which they may supply. The medicines found in a drug store and dispensed by the pharmacist may be grouped as follows: A. Medicines which do not Generally Require Sterilization. a. For internal administration per mouth. They may be contaminated or may become contaminated on standing for a time. Such medicines should be rejected. Do not attempt to render them usable by sterilization. b. Mouth washes and gargles. c. Enemas. Enemas for young children and such enemas as are to be applied to inflamed or otherwise pathologic conditions of the intestinal mucous membrane, should be sterilized. d. Medicamenta which are to be applied to the intact skin, or to the scalp. B. Medicines Which Require Sterilization. a. Those intended for intravenous and hypodermic use. Not only must these be absolutely sterile but they must be in perfect solution, before using. b. Those to be applied to cuts, bruises, abrasions, wounds, ulcers, sores, and to the broken skin generally. c. Those to be applied to inflamed mucous membranes, as enemas, douches, etc. d. Solutions for the irrigation of the bladder. e. Eye medicines, as washes and other solutions, intended for direct application to the eye. i. Methods of Sterilization. The following methods of sterilization are applicable in the pharmacy and should be consistently practised : A. Sterilization of Containers. — The glassware and other containers used in the pharmacy should be cleaned and sterilized as follows: a. Bottles and Glassware Generally. — Wash and rinse in warm water to remove dust, dirt, sand, straw, etc., then wash and rinse in hot water with 2 to 5 per cent, sodic hydrate. Neutralize the sodic hydrate by washing and rinsing in 2 to 5 per cent, hydrochloric acid. Finally wash and rinse in hot sterile water and allow to drain. Wipe dry and plug lightly with STERILIZATION AND DISINFECTION IN THE PHARMACY. 193 cotton. Place the plugged bottles in a hot-air sterilizer and heat for one hour at 120° C. to 130° C. Keep these cleaned, sterilized, and cotton-plugged bottles in clean container in a dry clean store-room, until wanted foroise. b. Porcelain and Similar Containers. — May be cleaned and sterilized like glassware. Plugging with cotton is as a rule inadmissible. c. Large Flasks, Jugs, Etc. — Large containers are as a rule difficult to sterilize and for this very reason are often subject to special neglect. Proceed much as for bottles, observing greater caution as to changes in temperature. Large bottles, carboys and similar containers cannot be sterilized by means of boiling hot water as they are very apt to crack. They may be sterilized by means of carbolic acid (5 per cent.), lysol (1.5 per cent.) or formaldehyde (4 per cent.), then thoroughly rinsed in sterile water, allowed to drain, plugged with cotton, carefully heated in hot-air sterilizer for one hour or more at 115° to 120° C. Cool gradually. d. Tin Containers. — Wash and rinse thoroughly in water; boil for thirty minutes, drain and dry and sterilize in dry-air sterilizer for one hour at 100° C. B. Sterilization of Apparatus and Tools. — It is of the highest importance that mortar and pestle, spatulas, percolators, pill and suppository machines, mixing plates, etc., etc., should be clean and sterile. This means a liberal use of hot water, green or soft soap, and clean towels. The sink, the floor of the dispensing room, the tables, chairs, desks, in fact everything in and about the dispensing room should be scrupulously clean. C. Sterilization of Corks and Other Stoppers for Containers. — It would be energy wasted to clean and sterilize the containers if the stoppers are not also clean and sterile. Sterilize corks by washing in hot 60 to 75 per cent, alcohol, drain and heat in hot-air sterilizer for one hour at 130° C. Keep these corks in sterilized wide-mouthed ground-glass capped bottles. Take out corks as wanted by means of a sterile pair of pincers, not by means of fingers. Other stoppers, as of glass, of wood, of rubber, must also be cleaned and sterilized. Rubber caps, rubber stoppers, and other rubber goods may be sterilized by boiling in water for thirty minutes. D. Sterilization of Surgical Supplies. — a. Bandaging materials, cotton, absorbent gauze, etc., may be sterilized by wrapping in cheese cloth or filter paper, first placing a grain of fuchsin or other aniline dye in the center of the package (wrapped in paper or cloth), and sterilizing in steam for one hour. The dye particle is introduced as a test object to ascertain if the steam has penetrated the entire package. If it has penetrated the entire package it will be indicated by a spreading of the color. Afterward, dry for one hour at 100° C. in the hot-air sterilizer. For this purpose the form of Arnold steam sterilizer shown in Fig. 18 will be found very useful. b. Sewing materials, such as needles, forceps, catgut, etc., require careful sterilization before using. All metal instruments and appliances, including IQ4 PHARMACEUTICAL BACTERIOLOGY. silver wire, can be sterilized in 5 per cent, carbolic acid if necessary or they may be boiled for 30 to 50 minutes. Wipe perfectly dry with sterile towels and place in hot-air sterilizer for one hour at 100° C. In order to keep them in sterile condition for immediate use they must be kept wrapped in sterilized cloth or cotton. c. Catgut requires thorough sterilization as not infrequently spores of disease germs (as anthrax) are present. The so-called cumol (cumene) method of catgut sterilization is quite generally adopted in the hospitals of Germany and of other European countries. Wind the catgut in the usual ring form, dry in hot-air sterilizer for two hours at 70° C., place rings in a vessel (beaker, etc.) with cumol on sand-bath and heat to 155° C. or 165° C. (the boiling-point of cumol), turn off the gas and allow to remain in the hot cumol for one hour. The cumol dish should be covered with a fine mesh wire screen to guard against catching fire. Take the catgut rings out of the cumol by means of sterile pincers and place in benzine for three hours, then allow the benzine to evaporate in sterile Petri dishes. d. Silver catgut is preferably sterilized in i per cent, silver citrate (itrol) or i per cent, silver lactate (actol), allowing it to remain for six hours, which destroys even the anthrax spores. Next expose the catgut to light (in sterile dishes) for a day or two, then wind or fasten on glass and preserve in 95 per cent, alcohol with 10 per cent, glycerin. Actol and itrol ionize silver far less actively than silver nitrate, hence their preference. e. Catheters, drainage tubing and other rubber materials are sterilized by boiling in water with 5 per cent, sodic hydrate. Rubber goods will not stand prolonged and frequent boiling. Do not sterilize metal ware with rubber goods. e. Sterilization of Medicines. — As a rule, medicines which are prepared under aseptic surroundings and conditions do not require sterilization. However, the ideal conditions rarely exist and subsequent sterilizations become desirable and even necessary. Tooth powders, dusting powders and similar substances may be ster- ilized at a dry temperature of 70° C., for three to four hours. Salves and pastes are difficult to sterilize. Low temperatures (from 60° C. to 70° C.) for several hours may be employed. Solutions for subcutaneous injection, for wound irrigation, for bladder irrigation, solutions of boric acid, of tannic acid, aquae, normal salt solu- tions and all weaker solutions of chemicals, intended for washes and irriga- tion in surgery, should be sterilized by boiling for five minutes. Strong solutions of chemicals (as acids, alkalies, etc.) do not require sterilization as they are themselves strongly germicidal. Alkaloidal and glucosidal solutions, and solutions of alkaloidal salts, tinctures and fluidextracts, should be carefully filtered and sterilized in STERILIZATION AND DISINFECTION IN THE PHARMACY. 195 sealed containers at a temperature of 60° C., one hour each day for six days. Concentrated alkaloidal solutions may be similarly sterilized. It is not advised to employ a higher temperature for these substances inasmuch as the decomposition changes, if any, which may take place at 100° C. are not clearly understood. To be on the safe side, the lower temperature (60° C.) should be employed. In the case of solutions or emulsions for hypodermic use, prepared with oil, the oil is first to be treated with alcohol (95 per cent.) to remove the oleic acid. Oily solutions of calomel, yellow oxide of mercury, lecithin, and of camphor are to be prepared with sterile materials, then placed in a boiling- water-bath for ten minutes or in an air-bath at 100° C. An interesting requirement is exacted by the Italian Pharmacopeia as regards the glass- of the containers for hypodermic injections: Ten to twelve ampuls or five or six bottles are filled with a clear solution of i per cent, mercuric chloride, then sealed. They are then left in an autoclave at 112° C. for half an hour, at the expiration of which time no brownish turbidity should be perceptible. Some of the points pertaining to the sterilization of alkaloidal, glucosidal and other substances which are quite readily decomposed or altered by light and heat, will be treated under ampuls. 2. Preparation of Ampuls. Ampuls (Lat. ampulla ;Fr. ampoule; — a flask) are small glass containers filled with medicinal substances usually in solution. These have come into great prominence within recent years, due to the methods of sterilization now required and practised in well regulated pharmacies. Ampuls are really nothing more than very small flasks, the size being suited to single doses of the medicine, as a rule. They were introduced into France about thirty years ago by Limousin and have now come into general use in France, Italy, Spain, Holland and England. It is only recently that they have come into use in the United States. C. A. Mayo was among the first Amer- ican writers to publish the first more complete information regarding their origin, manufacture and use. (See Proc. A. Ph. A., vol. 57, 1909.) They are generally adopted by the navies and armies of all civilized countries, because of the advantage which they offer for the preservation, storage and transportation of all manner of medicines, particularly those which require sterilization and which are generally wanted for immediate administration. From the standpoint of the physician they are wonderfully convenient and are great time savers. Ampuls may have any desired capacity, from i c.c. up to 100 c.c., and more, but the more usual capacities are i c.c., 2 c.c., 5 c.c., and 10 c.c. They are made of alkali-free glass, white or colored (amber) . Those supplied by ig6 PHARMACEUTICAL BACTERIOLOGY. French, German and Italian makers are of different forms, as flask-like, bulb-like, spindling, globose, etc. The following are some of the reasons why ampuls have come into use: a. Most of the liquid medicamenta and those which are to be dissolved before using have little or no antiseptic power and under the usual conditions 0 FIG. 75. — Making ampuls, a, Piece of glass rod to make two ampuls; b, rod a heated in the middle and nearly drawn apart; c, d, two half ampuls filled; e,f, the completed ampuls. readily become highly contaminated with different organisms. The use of such contaminated medicines has led to serious infections. b. The necessity of direct administration of medicinal solutions, by hypo- dermic, intramuscular and intravenous injection, is due to the desirability of getting prompt therapeutic effects. c. The direct (hypodermic, intramuscular and intravenous) adminis- STERILIZATION AND DISINFECTION IN THE PHARMACY. 197 tration of medicamenta is very frequently necessary because administration per mouth is impossible or undesirable. As a rule the pharmacist will purchase ampuls, ready for immediate use by the physician, from some reliable wholesale manufacturing house. In certain districts and under certain conditions this may not always be possible, in which case the pharmacist must prepare the ampuls. The pharmacist should be prepared to make all ampuls which may be desired by the physi- cians in his community. The following suggestions can be carried out readily: A. Glass Tubing. — Ampuls can readily be made from ordinary alkali- free glass tubing, selecting rods of a diameter to make ampuls of i c.c., 2 c.c., 5 c.c., and 10 c.c. capacity. This tubing can be secured from any chemical or pharmaceutical supply house. Select rods which are quite free from bubbles and of fairly uniform diameter and thickness. B. Breaking the Tubing into Suitable Lengths. — Break the tubing in lengths of from five to six inches, by filing a scratch with a small file and breaking, with the hands protected by gloves to avoid injury by small bits of glass. C. Sterilizing and Neutralizing the Glass Tubing. — Place the lengths of glass rods into water with 5 per cent, of soda and boil for thirty minutes. Neutralize in 5 per cent, hydrochloric acid, rinse thoroughly and again boil in distilled water. Let drain until dry. May be dried in hot-air ster- ilizer at 140° C. D. Making the Half Ampul. — Take one glass tube and heat the middle part in a bunsen burner with rotation until red hot and soft, and pull apart with a fairly quick strong pull. Break off the thin hairlike ends and hold the tips in the flame to seal them securely. A smalt bead should form as shown in Fig. 75, c, d, e, f. A little practice with a steady hand is neces- sary to do this neatly. The half ampuls (one end open, the other sealed as explained) are now laid aside in a sterile box or other container, until ready to be filled. Or the two ends of the ampul can be reduced to a capillary tube as follows. Heat the glass tubing in the blow-pipe flame, beginning at one end, until soft and draw out a short distance with a firm pull. Heat at a point about i to 3 inches from the narrowing portion of the glass tube and repeat as before. Repeat this until there are a series of tubes of normal diameter with capillary connections. Breaking these apart with the aid of a file, gives empty ampuls open at the two capillary ends. E. Filling the Half Ampuls. — This can be done by means of a burette, a pipette or a medicine dropper. The burette has many advantages. Many ampuls can be filled from one burette, the exact amounts can easily be measured. The pipette is far less convenient than- the burette and is more easily contaminated. A well graduated medicine dropper is very con- 198 PHARMACEUTICAL BACTERIOLOGY. venient, but all things considered the burette is recommended. The points to be kept in mind are. a. The finished ampul should not be more than three-quarters full. The length (of untapered portion of tube) of a neat looking ampul is about three or four times the diameter of the tubing used. b. In filling, introduce at least 10 per cent, more than the actual dose required, that is, the i c.c. tube should contain i.io c.c.; the 5 c.c. tube should contain 5.50 c.c. of the medicinal substance, etc. This is to make sure that the physician may get a full i c.c., 5 c.c., etc., dose after allowing for unavoid- able loss (portion clinging to inside of ampul, remaining in narrowed ends, etc.). c. In filling do not allow any of the liquid to come in contact with the upper end (open end) of the tube as that would interfere with sealing. There are many different methods for filling ampuls which may be classed under three heads; filling by gravity, by pressure, and by vacuum; the latter two being but modifications of the same principle involved. There are on the market (France, Holland, Germany) several devices made ex- pressly for filling and sealing ampuls. F. Sealing the Filled Half Ampuls. — This is done by means of suitable side-flame blow-pipe burner, pinching together and drawing out the soft end of the glass by means of pincers and sealing in same manner as the other end. Do not upend the ampul until it is cool, to avoid cracking the glass. G. Sterilizing the Ampuls. — The hypodermic and other solutions usually put up in ampuls can be divided into three classes or groups according to the degree of heat which may or must be used in sterilizing, namely, those which cannot withstand a temperature above 60° C., those which can be sterilized at 100° C., and those which may be sterilized in an autoclave at 120° C. Inasmuch as the autoclave is rarely usable and also because the ordinary steam temperature (100° C.) will meet all of the requirements of the autoclave, the latter piece of apparatus may be left out of consideration by the practising pharmacist. To bring about a complete sterilization of the ampuls, the discontinued or fractional method should in all cases be carried out. Place the ampuls in a container (beaker, tumbler, etc.) with water to which enough methyl blue or fuchsin has been added to give it a very marked color and sterilize as follows: If a temperature of 60° C. is to be used, apply this temperature (in incubator with Reichert thermo regulator) for one hour each day for four to eight days. Some manufacturers recommend a period of ten days. If the 100° C. is to be used, apply this temperature (in an ordinary Arnold steam sterilizer) for from 20 to 30 minutes once each day for three days. Should the autoclave be used, an exposure for a period of 20 minutes at 120° C. is sufficient to kill all organisms, including spores. STERILIZATION AND DISINFECTION IN THE PHARMACY. 199 It is of vital importance in preparing liquids for hypodermic and intra- venous injection to have absolutely perfect solutions. There must be no insoluble particles as these might cause serious harm. After the solutions are made they should be forced through a Berkefeld or Pasteur- Chamber- land filter. All operations should be done under aseptic conditions, using only chemically pure materials and boiled distilled water. If the contents of the ampuls become cloudy after sterilization or if the inside of the glass tubes show opacities something is wrong and such ampuls should be rejected. Also reject all " leaks," indicated by the aniline color which will appear on the inside of the tube. The finished ampuls are now ready for use. The physician simply breaks off one end of the ampul, inserts the hypodermic needle (sterilized), upends the ampul and aspirates the contents of the ampul into the syringe by simply drawing down the piston. A second method is to remove the piston from the syringe tube, break off one end of the ampul, insert this end into the open end of the piston tube, break off the other end of the ampul, whereupon the contents will flow into the piston tube; afterward replace the piston rod. In this latter method great care must be observed so as not to get small particles of broken glass into the hypodermic syringe. Use white glass for making ampuls. Those filled with solutions which are affected by light may be kept in an amber-colored bottle or other con- tainer which is impervious to light. The following substances are commonly put up in ampuls. Many others can be so put up. Each ampul should contain enough material for one dose or for one application, as the case may be. In the columns to the right are given the sterilization temperatures; the preferred or only usable temperatures being given in degrees, the permissible method being indi- cated by "Yes" and the inadmissible method being indicated by "No." In case of doubt it is always advisable to use the lower temperature (60° C., hourly for from four to eight days) . Sterilizing Temperatures Name of Article Incubator Steam Steam (auto- 60° C. 100° C. clave) 120° C. No No No No No No No No Adrenalin Yes 100° C. Alkaloidal salts generally Alkaloids generally 60° C. 60° C. Yes? Yes? Antitoxins 60° C. No Argyrol 60° C. Yes? Arsacetin. Yes 100° C. Arsenate of iron Yes 100° C. Arsenic Yes 100° C. 2OO PHARMACEUTICAL BACTERIOLOGY. Name of Article Sterilizing Temperatures Incubator 60° C. Steam 100° C. Steam (auto- clave) I20°C. Atoxyl 60° C. 60° C. 60° C. 60° C. Yes 60° C. Yes Yes Yes 60° C. 60° C. 60° C. 60° C. 60° C. Yes 60° C. 60° C. Yes Yes 60° C. 60° C. Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 60° C. 60° C. Yes 60° C. 60° C. Yes Yes Yes 60° C. 60° C. 60° C. 60° C. No Yes? No No 100° C. No 100° C. 100° C. 100° C. No No No No No 100° C. No No 100° C. 100° C. No No 100° C. 100° C. 100° C. 100° C. 100° C. 100° C. 100° C. 100° C. 100° C. 100° C. No No 100° C. No No 100° C. 100° C. 100° C. No No No No No No No No No No No Yes Yes? No No No No No No No No No? No No No No No Yes Yes No No No Yes Yes No No No No No No No No No No No No No Atropin .... Bacterins Cacodylates Caffeine . Caffeine benzoate Calomel cream Camphorated oil Chemicals in solution Cocaine Duboisine Ergot . . . . . Eserine sulphate Eucaine Gelatin Glucosides Glycerophosphates Grey oil Gums Hyoscine Iron cacodylate Mercury benzoate Mercury cacodylate Mercury salicylate Mercury sozo-iodolate Mercurial salts generally. ... Morphine Mucilaginous substances Normal salt solution Oils Paraffins Quinine .... Physostigmine Salvarsan Scopalamine Sera Sodium cacodylate Stovaine Strophantin Strychnine Toxins ... Tryosin Vaccines. . STERILIZATION AND DISINFECTION IN THE PHARMACY. 2OI Empty ampuls of German and French make can be secured from dealers in glassware and chemical supplies, likewise the appliances for filling and sealing. These ready-made empty and filled, ampuls vary in form as already indicated. Those with a flat bottom and which will remain stand- ing when placed on a flat surface are preferred by some physicians. The ready-made empty ampuls (still sealed) may be sterilized by boiling for fifteen minutes in a 5 per cent, solution of phenol, rinsing thoroughly in boiling hot sterile water, draining and drying. With the aid of a small sharp file, break off the tips of the ampuls to be filled. Place them in dis- tilled water, bring to a boil, take vessel from the fire for a few moments, pour cold distilled water upon the empty floating ampuls, a partial vacuum is produced in the interior of the ampuls and they quickly fill with water. Now boil for thirty minutes. When water is sufficiently cool take out the ampuls, shake out the water and dry in the hot-air sterilizer at 100° C. They are then ready to be filled, sealed and finally sterilized in the manner already described. An ordinary sterilized hypodermic syringe will be found very satisfactory for filling the ampuls. The suggestions regarding the amount of material to be placed in the ampul, sealing, sterilization, use of the aniline solution, etc., already given, also apply here. CHAPTER XIII. COMMUNICABLE DISEASES WITH SUGGESTIONS ON PRE- VENTIVE MEDICINE. The pharmacist should be prepared to assist the physician and the health authorities in the enforcement of the sanitary rules and regulations. To this end he should be informed as regards the source of the more im- portant contagious and infectious diseases and the causes of epidemics and the means available to prevent or to combat such conditions. This does not mean that the pharmacist must have a full knowledge of the pathology FIG. 76. — Bacillus botulinus. This bacillus causes botulism, a form of meat poisoning. There are numerous cases of poisoning resulting from eating infected meats. It should be kept in mind, however, that meat may not be decomposed and may be without bacilli and yet ptomaines may be present. Therefore absence of bacilli and of bad odor does not prove that the meat is wholesome. Meat from animals recently killed, which has been well cared for and which is without bad odor and shows no bacilli, is in all probability whole- some. Ham, canned meats, cold storage meats, etc., may have taken up toxins from contaminated meats, thus being made unfit for consumption even though no bacteria are found. and therapeutics of disease. He should have at least a general knowledge of the causes of disease in order that he may assist in applying the means for preventing disease. It is not within the province of the pharmacist to cure disease, but he should be a potent factor in preventive medicine. A contagious disease is one which is readily communicable, from one person or animal to another, either through direct contact or very close proximity. An infectious disease is communicable through a considerable 202 COMMUNICABLE DISEASES. 203 interval of space. Itch, for example, is contagious, but not in the least infectious, whereas whooping-cough is infectious, but not contagious. Some diseases are both contagious and infectious, as small-pox and diph- theria. Malaria and yellow fever are infectious, but not in the least con- tagious. However, the distinctions between infectious and contagious are often not very clear. It would be better to discontinue these terms and say that certain diseases are communicable from man to man or from animals to man. When a disease picks its victims rather promiscuously, in a cir- cumscribed area, with none of the usual characteristics of a contagion or infection, we usually apply the term epidemic. For example, cerebro- spinal meningitis and pneumonia may be epidemical. Diphtheria is often FIG. 77. — Bacillus anthracis. This bacillus is spore-forming and causes the cattle disease known as anthrax. This disease is especially common among sheep and cattle and may be transmitted to man, especially those working with the wool, hides and meat of infected animals. The two chief forms of anthrax in man are malignant pustule and woolsorter's disease. The dried spores of this bacillus will live for years and will withstand the boiling temperature for hours. Vaccinating animals against anthrax is commonly practised now. Anthrax is frequently confused with glanders, an equine disease caused by the Bacillus mallei, a, Non-spore-bearing bacilli; b, chains of cells; c, spore-bearing bacilli. Cell-walls and plasmic contents are stained, the spores are unstained. epidemic in a community^ and as above stated, it is likewise infectious and contagious. The term epidemic is, however, also applied to any communi- cable disease which has become general in a given community. A more or less common or spreading disease which is limited to and recurs in a given district or country is said to be endemic in that district or country. En- demics are usually due to climatic conditions which encourage certain microbic and other disease-producing invasions. The causes of disease are of two kinds, primary or inciting and secondary or predisposing. The primary cause of a disease is that factor or influence which must invariably be active before the disease can possibly develop. For example, the primary cause of diphtheria is the diphtheria bacillus; the predisposing causes are exposure to wet and cold, impoverished condition 204 PHARMACEUTICAL BACTERIOLOGY. of body, etc. No matter how numerous or how active the predisposing causes may be, the disease cannot develop until the primary cause acts. There are numerous abnormal or pathological states or conditions without recognizable primary causes, as gout, rheumatism and the senile changes in the body: and again there are certain diseases which evidently have primary causes, as whooping-cough, small-pox and yellow fever, but in which said primary causes are not yet discovered. The following tabulation outlines the primary and secondary causes of disease: Communicable diseases. Primary causes (inciting). Bacteria, as in typhoid and Asiatic cholera. Protozoa, as in malaria. Parasitic higher animals, as tape-worm and itch. Fungi, as in ring-worm and pellagra. Undetermined, as in whooping-cough and small-pox. Secondary causes (predisposing). Heredity. . Age. Sex. Environment. Race. Family. Individual (ontogenetic). (Phylogenetic). Habits. . . Infancy. Childhood. Adolescence. Adult. Old age. Climate. Altitude. Seasons. Unsuitable food. Unsuitable clothing. Poisons. Occupation. Injuries. Alcoholic. Tobacco. Drugs. Coffee and tea. Gourmandage. In a general way it may be stated that any cause, factor or influence, which tends to lower the vitality, predisposes to disease. Individuals with a well-balanced physical and mental development are less liable to disease, and when attacked are more apt to recover, than those individuals who have a poor physical development. Undue abstinence is as harmful as over-indulgence. The ascetic is as pathologic as the gouty gourmand. COMMUNICABLE DISEASES. 205 Irrational diet, drink and food fads, sooner or later leave their pernicious effects upon the system and predispose to certain diseases. Overeating is as objectionable as starvation. Lack of adequate physical exercise has its evil effects as does also over-exertion. Trained or professional athletes / / IS ^ >> FIG. 78. FIG. 79. FIG. 78. — Bacillus mallei, the cause of glanders in horses. This disease can be trans- mitted to man where it causes symptoms of a suppurative infection of the lymphatic glands. Mallein, which is used in testing horses for glanders, consists of the nitrate (Berkefeld filter) of dead cultures (glycerin bouillon) of the bacillus. A positive malleiu reaction consists in a rise in temperature and local swelling. The dose is i c.c. FIG. 79. — Bacillus tetani, an anaerobic spore-bearing bacillus, the cause of tetanus or lockjaw. This bacillus is found in soils and may infect abrasions, cuts and wounds. Treatment with tetanic antitoxin is successful if begun before the symptoms develop. The best time to administer the antitoxin is at the time the injury is received. FIG. 80. — A spore-bearing bacillus stained with methyl blue leaving the spores unstained. Fortunately most of the bacilli pathogenic to man do not bear spores. are not long lived, many are hopelessly afflicted with enlarged and weakened heart and arteries (aneurism). Pernicious habits of all kinds indicate weakness and further develop the weakness, which in turn predisposes to certain diseases and render the individual less resistant to the ravages of disease. A good ancestry and inheritance, good wholesome food, comfort- 206 PHARMACEUTICAL BACTERIOLOGY. able clothing, the right sort of exercise for body and mind, the simple life rather than the strenuous life, avoiding bad habits of all kinds, abundant fresh air, etc., all tend toward longevity. To argue that we should go un- clothed is as absurd and unreasonable as to teach that sheep should be shaved. To adhere to a wholly vegetable diet is irrational simply because we are organically adapted to a mixed diet. An excessive meat diet is also very pernicious. Occupation is a potent factor in predisposing to disease, and in lon- gevity. The following table adapted from a report by Ogle will serve to make this clear. The high mortality rate -among street-hawkers is due to several causes chief of which are low-living, exposure to inclement weather, and the greater exposure, in the squalid districts of large cities, to the primary causes of disease. The low mortality rate among clergymen is due to a comparatively simple though comfortable mode of living; while in the case of the farmer and gardener, the out-of-door life is the favorable influence. The list represents ages ranging from twenty-five to sixty years, therefore adults. Occupation Comparative Mortality Clergymen, priests and ministers 100 Gardeners 108 Fanners 114 Carpenters 147 Lawyers 152 Coal miners 160 Bakers 172 Builders, masons, bricklayers 174 Blacksmiths 175 Commercial clerks 179 Tailors 189 Cotton manufacturers 196 Medical men 202 Stone, slate quarries 202 Book-binders 210 Butchers 211 Glass workers 214 Plumbers, painters, glaziers 216 Cutler, scissors makers 229 Brewers 245 Innkeeper, liquor dealers 274 File makers 300 Earthenware workers 314 Street hawkers 338 Inn, hotel service 396 The following are the more important communicable diseases with suggestions on prevention. The information is given for the sole purpose COMMUNICABLE DISEASES. 207 to better qualify the pharmacist to cooperate with the health officers in safeguarding the public health. A. Tuberculosis. — Commonly known as consumption and the " white plague." A universal disease, essentially infectious, especially peculiar to crowded habitations and to lack of pure fresh air. The primary cause is the Bacillus tuberculosis (bacillus of Koch), a non-spore-bearing microbe, which is somewhat more resisting to disinfectants and other destructive agencies than most other pathogenic bacteria. The chief predisposing causes are living in crowded habitations; inherited low vitality, especially weak lungs; and exposure to inclement weather. The disease may be general (general tubercular infection) or it may be localized in any one or in several organs or tissues. Commonly localized in the lungs (phthisis, con- sumption) and in lymph glands. Lupus and many so-called scrofulous conditions are tuberculosis of the skin; the disease often attacks bones and f -. IN FIG. 81. — Bacillus tuberculosis. Although this organism does not form spores it is quite resistant to the action of germicides. The bacillus causing the bovine type of tuber- culosis differs slightly in several characteristics from the bacillus of human tuberculosis. joints (hip-joint disease of children). It attacks young and old and may occur in all walks of life. The disease enters via the air passages and per mouth with food and drink, or through cuts, bruises, wounds and abrasions. It is contracted by inhalation through close association with consumptives, and the bovine form or type of tuberculosis is acquired from the milk of tubercular cows. Bovine tuberculosis is especially liable to affect the lymph glands and the joints. The disease sometimes runs a quick course (quick consumption), but more generally it makes an insidious start and runs a chronic course. Many people have limited local infections which are only discovered at an autopsy. There are many spontaneous recoveries from tuberculosis. Since it is very important to begin early treatment, the physician resorts to several tests for the purpose of determining the possible existence of masked or incipient forms of the disease. These tests are as follows and all depend 2O8 PHARMACEUTICAL BACTERIOLOGY. upon the reactions produced by tuberculins when introduced into the system: a. The Calmette or Ophthalmo Test. — Old tuberculin, precipitated by alcohol is used. The precipitate is dried and made into a i per cent, solu- tion in sterilized distilled water or sterile physiologic salt solution. This substance is put up in sterile capillary pipettes, ready for use. A drop of the solution is placed in one eye, using the other eye as a control. Any abnormality in the eye is regarded as a contraindication. If tuberculosis exists in the system it is indicated by an inflammation in the eye tested. Also known as the Wolff-Eisner test or reaction. It may be necessary to repeat the test several times before satisfactory results are obtained. b. The von Pirquet or Cutaneous Test. — A 25~per cent, solution of tuber- culin (O. T.) is applied to the skin with scarification, as in vaccination. The skin is first cleansed with alcohol and control scarifications are made near the test area. This test is also known as the "skin reaction." It is not very reliable. The inflammatory reaction may be simulated by other substances in persons that are known to be entirely free from tuberculosis. c. The Moro, Percutaneous or Ointment Test. — Fifty per cent, tuberculin (O. T.) in lanolin is rubbed into the skin, without scarification. The prep- aration is put up in collapsible tubes, one tube containing enough material for several tests. If tuberculosis exists, small reddened vesicles appear at the point of inunction, usually on the second day. d. The Thermal Test. — A solution of tuberculin (O. T.), put up in 8 c.c. bottles, representing one milligram per c.c. (i-iooo) is injected hypo- dermically. If tuberculosis is present there is a rise in temperature, usually within ten to twenty-four hours after injection. e. The Detre Differential Test. — This test is intended to differentiate between tuberculosis of human origin and that of bovine origin. Three tuberculins are required. Tuberculin O. T., tuberculin B. F., made from tubercle bacilli of human origin and tuberculin B. F., made from tubercle bacilli of bovine origin. Three small skin areas are scarified. Into one tuberculin O. T. is rubbed, into the second humanized tuberculin, and into the third bovinized tuberculin. The resulting reactions indicate whether tuberculosis is of human or cf bovine origin. We cannot go into the details of the reactions. They are not always reliable, neither the positive nor the negative reactions. In the advanced stages of tuberculosis and in moribund cases, the reaction is usually negative. . Indeed, in such cases the test is unnecessary as the existence of the disease is evident without special tests. Tuberculosis is not as infectious as is generally supposed. Those who are in good condition physically may live for years with those afflicted with the disease without becoming infected. Yet, tubercular patients should be COMMUNICABLE DISEASES. 209 isolated from well people as much as possible. The sputum is the principal source of infection, also other secretions; and the breath as in sneezing, laugh- ing and coughing. Plenty of fresh pure dry air should be supplied to patients, large airy sleeping rooms and easily digested wholesome food is essential. Consumptives should not marry, should not kiss healthy individuals, espe- cially children. Expectorated material should be disinfected at once. Treat- ment should be begun early. The propaganda favoring well constructed, well ventilated, comfortably warmed homes and less close segregation in cities and a general improvement in sanitation will do much toward eradi- cating tuberculosis. Tenement houses and large or small crowded houses .of all kinds should not be tolerated for moral as well as for sanitary reasons. Above all, see to it that the milk used is free from tubercular infection. B. Typhoid Fever. — This is a filth disease. If the environment were made clean and sanitary, typhoid fever could not exist. The primary cause is the non-sporulating Bacillus typhosis which is found in filthy water, in milk and in food materials. Slops, sewage, wash water, etc., poured on the soil may seep into the well water and finally enter the system in drinking. The bacillus develops readily in the intestinal tract where the reaction is alkaline. It is quite susceptible to the action of weak acids and is easily killed by boiling and by disinfectants. Typhoid is a widely disseminated dangerous infectious as well as contagious disease. In large cities the mortality rate from this disease is directly proportional to the filthiness of the drinking-water supply. In country districts epidemics are very fre- quently due to contaminated well-water (contaminated from kitchen refuse, barns, cow-sheds, etc.). Epidemics often follow in the wake of the dairy- man, who supplies cow's milk in cans washed with or which contain milk, adulterated with polluted water. Typhoid fever is carried in vegetables from truck gardens where human and other excrement are used for fertilizing purposes. The Chinese truck gardeners are particularly culpable in this regard. Again, the vegetables are irrigated with stagnant and sewage- polluted water. House flies are carriers of typhoid. The mortality rate in typhoid is high and the disease runs its course in about five weeks. There are some mild cases, the so-called walking or ambulatory cases. All of the excreta from the patient should be disin- fected, using corrosive sublimate solution (i-iooo), copperas solution (10 to 20 per cent.), blue vitriol solution (5 to 15 per cent.), milk of lime (for stools), etc. All bed linen,' clothing, etc., used by the patient should be disinfected in 5-per cent, carbolic acid before washing. Everything used by the patient should be sterilized, disinfected and kept away from the rest of the family. Those who nurse typhoid patients must be extremely care- ful not to carry the infection to others. Pillows, mattresses and other large articles used by the patient should be steam sterilized, or if that cannot be 14 210 PHARMACEUTICAL BACTERIOLOGY. done conveniently, they should be destroyed by burning. In simple words, everything about the patient must be scrupulously sterilized in order to avoid spreading the infection. A national department of health should see to it that the water supply of large cities is free from sewage contamination. Our streams, lakes and reservoirs supplying drinking water require careful guarding against typhoid infection. There should be a compulsory regulation regarding the position and depth of wells in farm yards and as regards the position of the well relative to barns, cow sheds, privy vaults, etc. Typhoid ^ 0 ^ - fever will continue its ravages as long as filth ^ ^v ^ ^ contamination of water supplies and food sup- \ £* &^> 0 # plies is permitted. «=» ^ ^ f\ The Gruber-Widal test for typhoid is an 4=9 ^ *=> ^ agglutination phenomenon. The agglutinating ^ ff 0 ^ ^ f) power of the blood of a typhoid patient is usually ^ 0 /) 0<=3 ==> noticeable as early as the fifth day of the dis- <^ ^ *^>v^ ease- Preventive inoculation with typhoid bac- ^ ^ j) terin has been used with considerable success, particularly in the British and German armies, Fig. 82. — Bacillus pneu- , • ., , , . monia of Friedlander, also and IS now