Marine Biological Laboratory Library [ Woods Hole, Mass. I I [[ I i I I The Macmillan Co. ^ New Yoric City (jj Ij II D Presented, by Microbes and You THE MACMILLAN COMPANY NEW YORK . CHICAGO DALLAS • ATLANTA • SAN FRANCISCO THE MACMILLAN COMPANY OF CANADA. LIMITED TORONTO '1/- 7/ Microbes and You STANLEY E. WEDBERG, Ph.D. ASSOCIATE PROFESSOR OF BACTERIOLOGY UNIVERSITY OF CONNECTICUT STORRS, CONNECTICUT NEW YORK THE MACMILLAN COMPANY Copyright, 1954, by The MacmiUan Company ALL RIGHTS RESERVED— NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM WITHOUT PER- MISSION IN WRITING FROM THE PUBLISHER, EXCEPT BY A REVIEWER WHO WISHES TO QUOTE BRIEF PAS- SAGES IN CONNECTION WITH A REVIEW WRITTEN FOR INCLUSION IN MAGAZINE OR NEWSPAPER. Printed in the United States of America First Printing TO Carol, Karen, and Robin Preface Microbes and You has been written as a text for an introductory, terminal, survey course in microbiology for students with little or no background in science. The book should fill a need in schools— both large universities and small liberal arts colleges— where students are required to complete at least one course in a biological science as part of their general education. This text is not intended primarily for those planning to pursue microbiology as their major field of interest, nor for those individ- uals preparing for a career in medicine. Because Microbes and You is designed for use in a cultural course in science, it is written in a style which should help to hold the interest of the reader. Practical everyday applications of microbiology are incorporated into the text, and the instructor can supplement the reading material with lectures and laboratory exer- cises slanted toward his particular course objectives. This text is more concise than other similar presentations, and students are likely to read it more thoroughly than longer textbooks. Stanley E. Wedberg Storrs, Connecticut vtv Acknowledgments It is through the courtesy of a number of individuals, corpora- tions, and publishers that many of the illustrations are included in this book. Specific acknowledgment is found in the legends accompanying these figures. The editors of the Journal of Bac- teriology, the Journal of Botany, the Journal of Experimental Medicine, and the Journal of Infectious Diseases have been very cooperative in allowing material to be reproduced from original articles in their publications. Some of the prints are from the collection of the Committee for Visual Education in Microbiology of the Society of American Bacteriologists. Copyright owners, including Appleton-Century-Crofts, Inc., Harper and Brothers, D. C. Heath and Company, McGraw-Hill Book Company, Inc., C. V. Mosby Company, W. B. Saunders Company, D. Van Nostrand Company, Inc., and John Wiley and Sons, Inc., have given permission for the use of designated material from books published by them. Their courtesy is appreciated. I am indebted to my colleague, Dr. Robert C. Cleverdon, for his critical reading of the manuscript and for his invaluable sug- gestions for improvement of the text. My thanks are extended to Mrs. Gordon R. Hanks who typed and retyped the manuscript. And in conclusion, I acknowledge sincere gratitude to Dr. Walter L. Kulp, Dr. Milton J. Foter, Mr. Vinton E. White, Miss Christie J. Mason, Dr. Leo F. Rettger, Dr. George Valley, Dr. George H. Smith, and Dr. Philip B. Cowles— my microbiology teachers— for the foundation they have given me in this science. viii Contents 1. Introduction 1 2. Highlights in the History of Microbiology 6 3. Bacteria Are Classified as Plants 51 4. Microbes Must Eat 57 5. Microbial Structures and Staining Reactions 84 6. Cultivation and Identification of Bacteria 116 7. Bacterial Multiplication 137 8. Effects of Physical Forces on Bacteria 154 9. Effects of Bacterial Growth on the Environment 174 10. The Effect of Chemicals on Microorganisms 195 11. Polluted Water Can Kill You 219 12. Biological Sewage Disposal 238 13. The Air We Breathe 251 14. The Soil and They That Dwell Therein 265 15. Food Poisoning and Food Infection 277 16. Disease Transmission and Man's Resistance 294 17. Pathogenic Bacteria 328 18. Arthropods and Disease Transmission 348 19. The Fungi-Molds 358 20. The Fungi- Yeasts 369 21. The Rickettsiae 378 22. Viruses 392 23. Blood Grouping 408 References /\nl^ ^^^ Index ^^07 ( 425 tx CHAPTER 1 Introduction We are privileged to live in an age when, as the result of the hard labors and the genius of men, a new world is opened up for our studv— a fascinatinor, vibrant, sub-visible world hidden from the eyes of human beings since the beginning of time. Since man first viewed these interesting microorganisms, or germs, only about two hundred years ago, much has been sacrificed, including the very lives of some of the microbe hunters who have attempted to ferret out the mysteries of these minute forms of life. It might be well at this point to distinguish between bacteria and microorganisms. The former word includes only those single- celled, non-chlorophyll-containing plants which multiply by binary fission, or equal division. It may be a revelation to you to dis- cover that bacteria are not "bugs," but plants. The word micro- organism implies a much broader field and includes many different microscopic forms of both plant and animal life, including molds, yeasts, bacteria, protozoa, and even rickettsiae and viruses. Each of these organisms will be considered separately in later chapters. This book will deal principally with the bacteria, but because the technics for studying the other microorganisms are similar to those employed with bacteria, and because these other microscopic forms are oftentimes found intimately associated with bacteria, we shall not overlook them by any means. These organisms face many of the same problems confronting 1 2 Microbes and You you and me— obtaining food, digestion, excretion, respiration, repro- duction, etc. But in contrast to man, who possesses specialized organs for these important functions, the lowly bacteria must carry out all of these activities within the confines of a single cell, a cell so minute that 25,000 of them standing side by side would hardly occupy an inch! Microbes cannot walk, so they are unable to crawl up over our shoes to get at us, but some do have the power of locomotion in liquids. These have been endowed with special hair-like projec- tions, all part of the single cell, called flagella. By a whipping motion these flagella are able to propel the bacteria through liquids. The typhoid bacterium has been clocked at nearlv 65 millimeters (about 2.5 inches) an hour! The swimming speed of a given organism is influenced by such factors as temperature, available food, nature of the suspending medium, age of the organism, and undoubtedly by other motivating forces. Some bacteria lack flagella, yet they make progress through liquids by a twisting, corkscrew-like motion. It should be made clear that many bacteria lack the power of independent motion; they just sit around and wait to be pushed. There is little that is static in a science as young as micro- biology. It is a dynamic phase of biology which has many prac- tical, everyday applications. As a science, bacteriology is hardly more than one hundred years old. Some persons date this branch of science to 1857 when Pasteur first demonstrated that micro- organisms could sour milk. Sir William Osier (1849-1919) said that this publication of Pasteur's, together with his demonstration that the transformation of sugar into alcohol and carbon dioxide was a phenomenon of life, set the date for a new era in medicine. This killed the notions that ma^ic and air caused disease in some mysterious way, and it dragged the enemy into the open. All modern medical and surgical technics employed to prevent and to combat disease are based upon this germ concept. Too often, however, the word germ or bacteria conjures up in the mind of the layman but one thought— disease. Disease mav be defined as an abnormal condition of anv part or organ of the body or of the Introduction 3 mind. It is possible to name a number of diseases not caused by bacteria or other microbes, and with a httle further probing one could undoubtedly think of many uses to which mankind has pressed microbes for useful ends. Yet if a psychologist uttered the word "bacteria" in an association test where the test subject is re- quested to give the first word that comes to his mind, the response would undoubtedly be "disease," "sickness," or some similar con- notation more times than not. Just how did this young branch of science originate? To state with finality that a given day of a given year launched microbiology is an impossibility. It is probably easier to pinpoint the beginning of the atomic age to the first successful application of the cyclotron or to that early morning hour on July 16, 1945, when the first man- made atomic bomb was set off in the desert near Alamo^ordo, New Mexico. Nevertheless, it must be admitted that a great deal of both pure and applied research preceded the first atomic blast. The same statement can be made with respect to microbiology. A number of modern inventions are the product of the accumulation of vast storehouses of smaller, minor discoveries which, when tied together, provided background material for the development of the finished product. The fortunate accidents which have been capitalized upon by clever observers should not be ignored. At the same time that mention is made of the harmful effects produced by these organisms, the tremendous good that they accomplish for mankind should not be overlooked. The statement has been made that were it not for microbes, you and I would not be here. That is a rather strong assertion, yet there is plenty of evidence to add weight to such a contention. Without the chemical activity of organisms in their never-ending quest for food, no trees, plants, or animals would be consumed after their death unless they were burned or destroyed by some means other than biological activity. The vast accumulation of ancestors, plant and animal, would soon leave little room for the living. The chemical elements borrowed for a while by living things in the past would not be available for present generations, and in time life could very well grind to a creaking^ halt. Bacteria and other microorganisms 4 Microbes and You depend upon other living things for their food and for their very existence. Man's diet has been broadened as the result of micro- bial activity in the manufacture of such things as cheeses, sauer- kraut, pickles, beer, and leavened bread, to mention but a few. Once it had been established that bacteria were not "animal- cules," as Antony van Leeuwenhoek (1632-1723) designated them in the seventeenth century, this new branch of plant science appropriately found its way into the field of botany, where it still remains as an ugly-duckling in some institutions. Botanists do not employ the same technics as those used by microbiologists, so it was only natural that the new science should eventually break away from botany and stand on its own tsvo feet. It seems unfortunate that so many bacteria in the distant past became unhappy with their saprophvtic existence and decided to become pathogens, invading the tissues of plants and of animals. But this characteristic of microorganisms has presented one of the great challenges to microbiologists in the search for new ways to lengthen the span of man's life to the Biblical three score years and ten. We have come astonishingly close to this goal in recent years as the following statements will indicate. Actuarial figures released by a large life insurance company demonstrate in a clear fashion the influence of scientific progress on the life span. The expectation of life at birth is five years greater today than it was a decade ago, and double that existing between 1879-1889, the earliest period for which experience tables are available. In round numbers, since 1911 we have gained some- thing over twenty-one years, and since 1879 the average human life has been prolonged by thirty-five years! This is primarily a victory for preventive medicine, with a heavv assist in recent years from antibiotics. It is also a victory for nutritionists, who in the past two decades have told us more about what is good for us to eat than we ever knew before. An average human life two thousand years ago was about twenty-five years. In 1900 this figure had climbed to forty-nine and today we can look forward to better than sixty-eight years of life, females, on the average, living longer than males. Introduction 5 These changes in hfe expectancy will have, and already are having, a tremendous impact on problems connected with care of the aged. The social security program must carefully weigh these factors. We now have two chances out of three that an eighteen-year old will live to the retirement age of sixtv-five. A forty-five-year-old man has seventy chances in one hundred of reaching sLxty-five. Projecting our tables to 1980, about one person in ten will be sixty-five years of age or older, as compared with one person in twenty-five back in 1900. Some insurance companies have already adjusted premiums downward on straight life in- surance policies as a reflection of this trend toward long life. We have not reached the end of possible means for increasing the span of human life, and before too many decades slip by, it seems fair to assume that the three score years and ten figure will be readily surpassed. CHAPTER 2 Highlights in the History of Microbiology THE DEVELOPMENT OF THE MICROSCOPE THEORIES CONCERNING SPONTANEOUS GENERATION THEORIES CONCERNING FERMENTATION THEORIES CONCERNING DISEASE THE DEVELOPMENT OF THE MICROSCOPE Studying the history of some subjects represents a necessary evil to an undue number of individuals, but without some historical foundation, modern students of science would miss a great deal of the tingle that accompanied great and small discoveries. In microbiology it is possible to look back with considerable pride at the fascinating way in which small pieces of an intricate puzzle have fallen into place to bring the picture to its present stature. Do you enjoy reading stimulating tales of adventure, such as those conjured up by Robert Louis Stevenson and others? The events described in these best sellers sound like commonplace happenings when they are compared with the adventures of the pioneers in microbiology. These scientists did not discover a mere island or a simple continent, they opened up an entirely new world! The discovery of microbes, some of which kill and maim, but others Highlights in the History of Microbiology 7 of which work for the good of mankind, is a bright chapter in the history of biology. Long before the advent of television, the radio, the motor car, the refrigerator, and many other conveniences that enrich our lives to the point where we consider them as necessities, microbiological history was slowly being written. In 100 B.C. we find records of a Roman named Marcus Varro (116-27 B.C.) who speculated: "Certain minute invisible animals develop which, carried by the air, may enter the body through mouth or nose and cause serious ailments." How do you suppose Varro would react were he to peer through some of our modern instruments and see these microscopic living forms? The eyes of what we term a normal individual cannot see objects smaller than about 30 microns (about Yiooo oi an inch) in diameter, but by grinding lenses in certain ways we have created greater near-sightedness, if you will, which allows us to view objects much smaller than 30 microns. The origin of the first ground lenses is lost in history, although reference to magnifying glasses can be found in the writings of the ancient Greeks and Romans. As early as 1267 a Franciscan monk, Roger Bacon ( 1214- 1294), clarified some of the principles of optics, and he is usually credited with being the founder of the science of optics. Bacon suggested, probably for the first time, that these lenses could be fashioned into spectacles for persons with poor eyesight. A report from Florence dated 1299 states: "I find myself so pressed by age that I can neither read nor write without those glasses they call spectacles, lately invented to the great advantage of poor old men when their eyesight grows weak." While reading the latest Book of the Month offerings did not take much of a person's time back in the thirteenth or fourteenth century, what a joy it must have been for the aged in the twilight of their lives to again see the world about them. We have good reason to assume that the crudeness of these early spectacles, while affording temporary help to failing eyes, might have done a great deal of harm over a period of time to persons suffering from certain eye disorders. Carefully compounded prescriptions for glasses were not available for centuries after these first spectacles were marketed. 8 Microbes and You By accident, or by logic, it was discovered that if a single ground lens could make things look larger, an object could be magnified still further by using two or more lenses set up in a definite relationship to each other. This compounding of lenses is usually credited to a spectacle maker in Holland in the year 1590. Considerable disagreement exists as to the exact name of this individual and as to the spelling of his name. Many books call him Janssen; other histories say his name was Zacharius (miscalled Jansen), the son of John, the spectacle maker. Other books refer to these persons as Hans Jansen and his son Zacharius, with the latter person being the discoverer of the principle of the telescope- two lenses in a tube. The earliest compound microscope was pro- vided with a concave ocular and convex objective. Practical uses of compound lenses were not put to serious use in biological science until the middle of the seventeenth century by Robert Hooke (1635-1723), Antony van Leeuwenhoek (1635^1703), Marcello Malpighi (1628-1694), and others of whom more shall be written later. One of the earliest reports on the existence of microorganisms can be found in the writings of an Austrian Jesuit priest, Athanasius Kircher (1601-1680), who reported on the cause of plague as seen in blood of infected individuals. Since his microscopes were ex- tremely crude affairs and his lenses had magnifying limits of only about 32 diameters, it is quite doubtful that Kircher actually saw the organisms we attribute today as the etiologv of this scourge. Kircher was trained in physics, medicine, mathematics, and music, and in 1658 he published a treatise on medical microscopy. The name of Galilei Galileo (1564-1642) should not be passed over without at least a mention of his work on lenses. Some historians go so far as to credit this man with the discoveiy of the compound microscope, but because Galileo failed to leave complete records of his work and his findings, other individuals who had left such reports were credited with manv discoveries which might originally have stemmed from the brain of Galileo. It is of interest to note that the word "microscope" was coined in 1625 by Giovanni Faber. Hooke is credited with the discovery of what we know Highlights in the History of Microbiologtj 9 today as cells, and his name is included as a milestone in cytology. He published his "Micrographia" in 1665 in which his compound microscope was described and pictured. Another pioneer micro- scopist was Malpighi, who viewed, probably for the first time, circulation of blood in capillaries. Among his interests are in- cluded studies of animal and vegetable materials, and his re- Fig. 1. Antony van Leeuwenhoek (1632-1723) -The "Father of Microbiology." (Courtesy of the Lambert Pharmacal Company, Division of the Lambert Company, St. Louis, Missouri.) searches are recorded in papers submitted to the Royal Society of London. One of the most interesting of the microbe hunters was Antony van Leeuwenhoek, who was born in Delft, Holland, the son of well-to-do tradespeople. This person's early years are not well documented, but when his father died, Antony's mother wanted him to become a government official, a member of a respected profession. When he was sixteen years old he left school and 10 Microbes and You became an apprentice in a dry goods store in Amsterdam until he reached the age of twenty-one. For the next twenty years Leeuwen- hoek ran his private dry goods estabHshment, but Httle is recorded of this period in his hfe. It is beheved, however, that he was twice X Fig. 2. One of Leeuwenhoek's microscopes. {Courtesy of the American Optical Company, Instrument Division, Buffalo, New York.) married and had children, but most of the children died. Some- where during this period he was appointed janitor of the Cit}^ Hall of Delft, and sandwiched in between his official duties he began his passionate lens-grinding activities which must have Highlights in the History of Microbiology 11 drained off most, if not all, of his spare time. He is reported to have constructed 247 complete microscopes with magnifying powers of from 40 to 270 diameters. These might more accurately be described as simple lenses rather than microscopes. Some 419 individual lenses are credited to his patient grinding activities during his lifetime. Leeuwenhoek's neighbors did not look kindly on the strange things to which Antony devoted his energies. But Leeuwenhoek, the only man in Holland who knew how to grind pieces of clear rock crystal in such a way as to magnify objects too small to be seen with the naked eye, actually felt sorry for his neighbors and once made the statement: "We must forgive them, seeing that they know no better." Science has not always been a respectable profession, and much laboratory work was conducted behind closed doors. After all, had not Galileo been imprisoned because he dared to sugsiest that the earth moved around the sun? Burning at the stake was the price paid by some who ventured to cut up a human body in an attempt to discover what made it function. Learned men of this interesting era spoke Latin, but poor Leeuwenhoek could only read the Dutch Bible. He was a religious man and referred to God as the Maker of the Great All. Because of his relative ignorance, Antony was not fettered with a great deal of nonsense subscribed to bv the so-called learned professions. He built up his storehouse of knowledge by employing the scientific method, unbiased by the printed word which was so often based upon fallacy and not upon fact. A half-dozen observa- tions of a given reaction were not sufficient for Leeuwenhoek be- fore he put his findings in writing. Each experiment had to be repeated hundreds of times to eliminate any chance misconception of what had taken place. However, once he felt that he could record his observations, strong-willed Leeuwenhoek could be swayed by no one. Many writings describe the man as being strongly opinionated; perhaps he had every justification for being so sure of himself. Who among these Latin speaking scholars had soiled his hands by working tediously in poorly lighted labora- tories to squeeze out one scientific fact from the secrets to be 12 Microbes and You unfolded to those who pursue the truth? They could read the works of others written in fancy Latin, but they were not scientists in any sense of the word, according to Leeuwenhoek. Just as it is true today, this devoted scholar had little or no time for his family. From all appearances Leeuwenhoek richly deserves the title often given to him— "Father of Microbiology," although many persons today reserve that honor for Louis Pasteur who lived nearly two centuries later. If a father is one who gives origin, then we must admit that Pasteur, as great as he was, must be relegated to the rank of stepfather since he came after Leeuwenhoek. The majority of persons today give the honor to Leeuwenhoek, and apparently the Society of American Bacteriologists has leanings in that same direction since this group prints a small picture of Leeuwenhoek on the cover of its monthly publication, the Journal of Bacteriology, and has done so since the Society was founded in 1899. The careful records compiled by this man and sent in great volume to the Royal Societ)^ of London are sufficient evidence that this microbe hunter was much more patient and much more con- servative than many scientists are willing to be before publishing results of their experimentations. We recognize from his draw- ings many organisms which we associate with certain parts of our bodies, such as scrapings from the teeth. The Royal Society to which Antony sent his observations had a most humble origin. A band of individuals, curious about the surrounding world and strong-willed enough to overcome opposition, risked public ridicule and even death to eke out the truths of science. They joined a sort of secret fraternity and did not come out in the open until the reign of Charles II when they emerged as the Royal Society of London and gained respectability. The membership boasted such names as the founder of the science of chemistry, Robert Boyle, Samuel Pepys, Isaac Newton, Christopher Wren, and others of equal stature in the scientific world. While most of Leeuwenhoek's countrymen scoffed at his ex- Highlights in the History of Microbiology 13 periments and his boasts of the "beasties" he saw under his lenses, one man, Regnier de Graaf (1641-1673), became truly curious and eventually was accorded the rare privilege of peeping through some of these lenses. To say that the observer was agog would phrase the reaction in mild terms. Having been appointed a correspond- ing member of the Royal Society for his interesting observations on the subject of the human ovary, de Graaf implored the Society to request a written account from Leeuwenhoek of his unbelievable discoveries. As suspicious and jealous as Antony was, he finally consented to the Society's invitation, and in his humble, unpolished way he wrote a letter entitled "A Specimen of Some Observations Made by the Microscope Contrived by Mr. Leeuwenhoek Con- cerning Mould upon the Skin, Flesh, etc.; the Sting of a Bee, etc." Quite a title. It is perfectly true that Leeuwenhoek had not mastered the fine art of writing, but what a contribution this man made to biological science! When the Royal Society, in its re- served manner, asked that this original letter be followed by others, they did not have long to wait. Records show that during the next fifty years hundreds of such communications reached the Society from Leeuwenhoek's laj3oratory in Holland. While he tended to ramble in his writings and loved to discuss topics not always pertinent to the subject at hand, each letter he wrote did contain some gem, or gems, of a scientific nature. Bacteria were first described by him in a letter written on October 9, 1676, to Henry Oldenburg, secretary of the Royal Society. And in a letter written in 1783 he sketched the three principal shapes of bacteria we accept today: the rods (tube-like), the spheres (circles), and the spirals (snake-like). Insatiable curiosity led this man to examine a wide variety of objects under his lenses, including stagnant water, rain water, scrapings of his teeth and the teeth of perfect strangers when he thought their brown stains might reveal something his own white teeth might not harbor, bodies of a wide variety of insects, the intestinal contents of frogs, horses, and humans, spermatozoa of man and lower animals, human skin, whale fibers, hairs of sheep. 14 Microbes and You beaver, and elk, wood from many types of trees, etc. He was amazed to discover that the sperms of an ox and of a mouse were much ahke in size. It was not until he examined drops of water, however, that Leeuwenhoek began to see amazing creatures— a thousand times smaller than the limits for the human eye. Since the beginning of time these sub-visible creatures had wreaked their havoc, had killed the innocent child as well as the adult rascal; had played important roles in the essential process of decay and putrefaction, in soil fertility, and in fermentations resulting in the production of wine and other beverages. "They stop, they stand still as 'twere upon a point, and then turn themselves round with that swiftness, as we see a top turn round, the circumference they make being no bigger than that of a fine grain of sand," he wrote. It is always a pleasant experience to observe the reaction of students peering through a microscope and viewing for the first time the nervous activity of the strange, new world in a drop of stagnant water. Students are informed that there are such things in existence, but how would a person react were he living back in the seventeenth century looking at this same drop of stagnant water and seeing sub-visible families that no other person had ever seen before? Leeuwenhoek was jubilant! As we examine these early records of his observations we find them startlingly accurate. He once wrote that one of his "beasties" was one-thousand times smaller than the eye of a large louse. We have since learned that the eyes of all adult lice are no smaller nor larger than the eyes of sister and brother lice. His findings in stagnant water drove him mercilessly on in his observations, and he naturally wondered whether these "animal- cules" arrived on earth in rain water. Samples of rain water were carefully collected in clean containers, and examination of drops of this fluid revealed no organisms. However, after dust and lint had fallen into his container and sufficient time had elapsed, he was able to show that life abounded in his stored liquid. While trying to find out what made pepper bite his tongue, he cut the condiment into pieces for easier microscopic examination. Highlights in the History of Microbiology 15 but when these pieces were still too large to be conveniently placed under his lenses, he decided to soak the pepper in water to facilitate cutting into smaller units. Lo and behold, four days later the pepper water was teeming with life. This might be considered as the first bacteriological medium devised for growing organisms in the laboratory. With his calculating mind he reported to the Royal Society that a single drop of his pepper water contained more than 2,700,000 of these little animals— more than the total population of his native country! This was, after all, a rather startling revelation, and scoffers were in the majority. A few in- dividuals, however, remembered how accurate his previous ob- servations had been, and they tried to get Leeuwenhoek to reveal his technic of manufacturing microscopes. He was a jealous man and refused the request, but he finally did condescend to sub- mit his calculations to show how he had arrived at his conclusions relative to the pepper water populations. People might look at some of his instruments, but touch them— never. In his famed letter of October 9, 1676, to the Royal Society he wrote: "My method for seeing the very smallest animalcules and minute eels, I do not impart to others; nor how to see very many animalcules at one time. That I keep for myself alone." Barnett Cohen in 1937 offered the following explanation of Leeuwenhoek's success which others at the time could not seem to duplicate. "One can augment the effectiveness of a simple lens by suitably utilizing the inherent optical properties of the spherical drop of fluid containing the objects under observation. The advantages of a water-immersion objective are too well known to require comment, but the added advantage of what amounts to the super-position of a relatively thick meniscus lens (of water) may be worthy of mention. There is apparently no way to prove that Leeuwenhoek did actually employ either of the simple devices set forth above; but certainly, their production was well within the facilities and competence of that clever manipulator." It is recorded that the Royal Society instructed two of its members— Robert Hooke and Nehemiah Grew— to build the best. 16 Microbes and You most modern instrument they could devise. It was a memorable day for science on November 15, 1677, when this microscope was presented to the Society and all could see for themselves that the poor Dutch lens grinder from Delft was not fabricating;; his find- ings. This instrument did not, however, measure up to the ones that Leeuwenhoek had perfected. An invitation for Leeuwenhoek to become a Fellow of the Society was soon on its way, and he accepted the high honor and promised to serve faithfully during the rest of his life. He never went back on his word. But he never sent them a single microscope. In fact, he possessed one instrument that no one was allowed to even look at— not even members of his immediate family. In the tail of a small fish he saw capillary blood vessels through which the blood passes from the arteries to the veins. This com- pleted Harvey's discovery of the theory of blood circulation. As Antony watched the blood cells passing through capillaries, prac- tically in single file, a bright idea occurred to him which eventually resulted in probably one of the earliest cures for a hangover. He wrote that after a night of drinking he awoke in the morning feeling sluggish, because his blood thickened, he postulated. Several cups of black coffee in the morning, taken as hot as possible until sweat broke out on his face, made him feel better. If this treatment did not cure his sluggishness by opening up the capillaries or by the thinning of his blood, he felt sure that no prescription by an apothecary could cure the condition either. He chanced to examine some of his teeth scrapings after such a hot coffee episode, and he found that the small organisms scraped from his front teeth no longer exhibited their frisky movements. His back teeth, however, where the coffee had not come in contact at such a high temperature, still showed active organisms when scrapings were examined under his lenses. Selective heating in flasks revealed the truth of his suspicions that the organisms could be killed by heat. Of all the microbiologists none was so accurate, none so com- pletely honest, and none had such common sense as Leeuwenhoek, Highlights in the History of Microbiology 17 Fixed Draw Tube Substage Adjustment Knob Fig. 3. Parts of the compound microscope. {Courtesy of the Amer- ican Optical Company, Instrument Division, Buffalo, New York.) but when he died in 1723 at the age of ninety-one, this field of science went into a dormant stage for almost 150 years. Com- petition stirs activity. Leeuwenhoek was not in competition with anyone except himself. Had he cooperated with others, micro- biology might have used his work as a springboard, instead of waiting until Louis Pasteur (1822-1895), Robert Koch (1843- 1910), and others gave it the necessary impetus during the Golden 18 Microbes and You Age of Microbiology, commonly designated as the period between 1850 and 1900. As we increase the magnification of our lenses with a light-type instrument, we must have a greater concentration of light if we are to see our objects clearly. An important contribution in this di- rection was the introduction of the immersion lens which provides a homogeneous refraction system for the light as it passes from Fig. 4. Ferdinand Cohn (1828-1898). (By permission from In- troduction to the Bacteria, by C. E. Clifton. Copyriglit, 1950. Mc- Graw-Hill Book Company, Inc.) below up through the lenses. In other words, by placing a drop of oil on top of the preparation on the slide to be examined, if the oil has the same index of refraction as the lenses of the microscope, the light coming from below will not be lost after hitting the object, but will continue through the oil and will be reflected through the objective lens of the instrument. Along with this improvement, the perfection of the substage condenser by Ernst Abbe (1840-1905) about 1870 made possible a more brilliant illumination of the microscopic field. With the use of improved microscopes observers were better Highlights in the Hist cry of Microbiology 19 able to study in more detail the finer characteristics of bacterial morphology which spurred schemes for attempting to classify these newly discovered living forms. A German botanist, Ferdinand Cohn (1828-1898), worked out beUveen 1872-1876 the first scheme for classifying bacteria as plants rather than as animals. Can we Fig. 5. Table model electron microscope. (Courtesy of Radio Cor poration of America, RCA Victor Division, Camden, New Jersey.) say that modern bacteriology began with this piece of work? At this point the useful magnification of instruments was quite similar to those employed today. A clear view of an object is not possible when the object is smaller than half the wavelength of the light being used to illuminate the field. This sets the limitations of 20 Microbes and You light-type microscopes and explains why we can't just keep setting up stronger and greater numbers of lenses to raise magnifying powers of the instruments. When we employ light of shorter wavelength, such as ultra-violet light, as our source of illumina- I ■,w Fig. 6. Universal type electron microscope. (Courtesy of Radio Corporation of America, RCA Victor Division, Camden, New Jersey.) tion, we can increase the magnifying power about two or three times that of ordinary light instruments. However, even with ultra-violet light we are unable to photograph these particles which we now call viruses. Highlights in the History of Microbiology 21 Research continued in an effort to devise some means of in- creasing useful magnifications without sacrificing resolving power (the ability to detect small objects that are close together), and this resulted in the development of the electron microscope in which electrons replace light waves. The wavelength of an electric beam is about 1/100,000 that of light, and the Radio Corporation of America developed an electron scope with mag- nification as high as 100,000 diameters in one of the earliest attempts to employ this new principle. Recent improvements have pushed these limits up to 200,000 diameters, whereas the practical limits of optical scopes is 2500-3000 diameters. Certain magnetic and electric fields act on an electric beam in the same manner that a lens acts on a light beam. High velocity electrons and electro- magnetic or electrostatic "lenses" serve in place of the condenser, objective, and ocular of optical instruments. The image produced is viewed on fluorescent screens or is registered on a photographic plate. THEORIES CONCERNING SPONTANEOUS GENERATION Having developed the tools necessary to see these microscopic plants we know as bacteria, a second major problem confronting biologists was the question of the origin of life. Could living things arise spontaneously from dead matter ( abiogenesis ) , or do all living things have to have parents (biogenesis)? While at- tempting to thrash out the solution, many discoveries were made which were incidental to the main objective, but which contributed greatly to the over-all growth of the science of microbiology. It is just as true today as it was centuries ago that certain persons greatly influence the thinking and the beliefs of their time. Almost all so-called scientists from the time of Aristotle (384-322 B.C.) to the middle of the nineteenth century, believed that animals could be generated from non-living matter. The ancient teachings of Aristotle were accepted, unfortunately, and because of the man's stature, progress along certain lines was materially retarded. According to this Greek naturalist, living things were the result of passive matter and active form, the latter 22 Microbes and You representing the soul. Only after the soul enters the matter, does life originate. He said in 354 B.C., "Animals sometimes arise in soil, in plants or in other animals." Facts are established by repeated, confirmed observations. But as stated in the introduction of this book, scientific facts remain only until something proves them to be otherwise. The develop- Fig. 7. Francesco Redi (1626-1697). (From Elementary Bac- teriology, /. E. Greaves and E. O. Greaves, 5th. ed. Copyright 1946, W. B. Saunders Company, Philadelphia.) ment of newer knowledge may completely nullify previously accepted facts. Some unusual recipes for creating living forms are inserted here for your edification. They were based upon repeated observations and were accepted at the time. Publius Vergil (70-19 B.C.) in the Georgics suggested this technic for producing swarms of in- sects: "First, a space of ground of small dimensions is chosen; this they cover with the tiling of a narrow roof with confining walls, and add four openings with a slanting light turned toward the four points of the compass. Then a bullock, just arching his horns upon his forehead of two years old, is sought out; whilst he struggles fiercely, they close up both nostrils and his mouth; and Highlights in the History of Microbiology 23 when they have beaten him to death, his battered carcass is macerated within the hide which remains unbroken. Then they leave him in the pent-up chamber, and lay under his sides frag- ments of boughs, thyme, and fresh cassia. This is done when first the zephyrs stir the waves, before the meadows blush new colors, before the twittering swallow suspends her nest upon the rafters. Meanwhile, the animal juices, warmed in the softened bones, ferment; and living things of wonderful aspect, first devoid of feet, and in a little while buzzing with wings, swarm together, and more and more take the thin air, till they burst away like a shower poured down from the summer clouds; or like an arrow from the impelling string, when the swift Parthians first began to fight." This formula sounds like the hallucinations of an alcoholic, but the "facts" were undoubtedly believed to be true, at least by Vergil. An interesting comment by Homer indicates that he knew the origin of flies. He put these words into the mouth of Achilles: "But I greatly dread that flies may enter into the mighty son of Menoities through the wounds made by the bronze weapons, and beget worms in him and defile his corpse." The proof of Homer's words was not forthcoming until 1668 when Francesco Redi ( 1626- 1697), poet and physician of Arezzo, showed that if meat was properly covered with gauze, no maggots would develop on the meat, and only when the egg-laying flies gained access to the meat, were mas^gots able to arise. Theophrastus Paracelsus (1493-1541), a Swiss medical phi- losopher, offered his formula for the creation of human beings ( homunculi ) . "Place certain substances in a bottle, stopper it, and bury it in a dung heap. Everv day certain incantations must be uttered over the submerged bottle. In time, a small being will appear in the bottle." However, he did admit that he was never successful in keeping the homunculus alive after taking it from the bottle. His instructions, I'm sure vou will agree, were rather vague, and he never was able to demonstrate publicly his spon- taneous generation. Should you desire to produce mice, Jean Baptiste van Helmont (1577-1644), a physician and alchemist, offered this: "Place a 24 Microbes and You dirty shirt in a vessel containing wheat, and after twenty-one days' storage in a dark place, to allow fermentation to be completed, the vapors of the seeds and the germinating principle in human sweat contained in the dirty shirt will generate live mice." An English naturalist says of the views of a doubter of abiogenesis: "So may we doubt whether, in cheese and timber, worms are generated, or if beetles and wasps in cow dung, or if butterflies, locusts, shell- w^yf^^^^ Fig. 8. Theophrastus Paracelsus (1493-1541). {From Elementary Bacteriology, /• E. Greaves and E. O. Greaves, 5th. ed. CopyrigJit 1946, W. B. Saunders Company, PJiiJadelpJiia.) fish, snails, eels, and such life procreated of putrefied matter which is to receive the forms of that creature to ^^4lich it is bv formative power disposed. To question this is to question reason, sense and experience. If he doubts this, let him go to Egypt, and there he will find the fields swarming with mice begot of the mud of Nylus, to the great calamity of the inhabitants." These examples are enough to give you an idea of how fantastic concepts can become when a limited bit of knowledge is available. In the light of our present information vv^e can partially explain how many of these recipes for creating life resulted in the evolution Highlights in the History of Microbiology 25 of animals under the conditions set forth by these early workers. Other formulae are probably pure fabrications. After Leeuwenhoek and others enlightened the world with the discovery of bacteria, theories concerning the origin of these small (microscopic) forms of life were soon forthcoming, just as there had been explanations for the creation of large ( macroscopic ) visible forms of life. John T. Needham (1713-1781), a Roman Catholic priest, firmly believed that a "productive" or a "vegeta- tive" force was responsible for the creation of living things. This was in opposition to Georges Buff on (1708-1788), the naturalist, who felt that all life possessed certain chemical constituents in common. After death, he postulated, these constituents were released and remained very active until they could locate and combine with other similar particles and form a new microscopic organism. Needham was one of the first research workers to con- duct scientific laboratory experiments in support of abiogenesis, and the Royal Society was convinced by his proof of the theory, which resulted in his election as a Fellow of the Society. Not to be outdone, the Academy of Science in Paris made him an As- sociate Member of their organization. These honors irked Lazzaro Spallanzani (1729-1799) who re- peated the work of Needham and arrived at the opposite con- clusion—life does not arise spontaneously. Needham had boiled meat juice or vegetable infusions in corked flasks, and upon standing he found that life had been generated in these containers. Spal- lanzani (the maker of the doll in Offenbach's opera. Tales of Hoff- man) boiled his infusions for a longer period and then sealed the openings of his flasks in a flame. None of these revealed spoilage. To quote Spallanzani: "I used hermetically sealed vessels. I kept them for one hour in boiling water, and after opening and ex- amining their contents, after a reasonable interval I found not the slightest trace of animalcules, though I had examined the infusion from nineteen different vessels." This might be considered to be the first laboratory proof that abiogenesis was not founded upon fact. He criticized Needham for using such porous material as cork which allowed the entrance of microorsjanisms into his boiled 26 Microbes and You infusions, especially during the cooling stage when negative pressure within the flasks tended to suck contaminated air into the vessels. Spallanzani, exhibiting a truly scientific approach to the prob- lem, tried to beat his own theory disproving spontaneous genera- tion. His critics claimed that in boilino; his infusions for such a long time, he had devitalized the substrate and organisms could not grow. Spallanzani took some seeds which he had found to be Fig. 9. Lazzaro Spallanzani (1729-1799). {From Fundamentals of Bacteriology, M. Frobisher, 4th. ed. Copyright 1949, W. B. Sound ers Company, Fhiladelphia.) good food for microbes, and he roasted these seeds until they were black— certainly devitalizing them, if such was the case. When he added water to this charred medium, he found that access to air soon provided the necessary germs which readily multiplied in his seed infusion. This proof was finally accepted by most persons, and he was proclaimed all over Europe, but a few persisted in their denunciation of the man because they felt that sealing the flasks had removed a vital force in air necessary for microbial growth. To combat this criticism Franz Schultze (1836) passed air through his infusions after forcing the air through caustic potash Highlights in the History of Microbiology 27 and sulfuric acid to filter out any suspended organisms. He aspirated his flasks daily for three months, and at the end of that period no flasks exhibited the slightest suggestion of microbial growth. Theodore Schwann ( 1837 ) passed the air through heated tubes before it was allowed to come in contact with his boiled infusions, and he showed that growth was absent. Some biological historians credit Schwann as founder of the science of disinfection. It was only natural that opponents should accuse these two workers of chemically devitalizing the air by the chemical treatment and by the heating technics they employed. One of the greatest con- tributions to microbiology resulted from this argument when Schroeder and von Dusch (1853-1854) suggested the use of wool stoppers in flasks to allow ready access of air without devitalizing it in any way. These wool plugs are capable of mechanically screening out the tiny microbes, and the bacteria-free atmosphere can provide the necessary vital factors for growth of any organisms present in the infusions. As long as these plugs are kept dry, they are effective, but wet stoppers allow migration of organisms through an otherwise effective filter. Today laboratories through- out the world employ non-absorbent cotton as a standard pro- cedure for stoppering test tubes and flasks to be used in the cultivation of microorganisms. To add weight to Spallanzani's contention that microbes must come from other microbes, he took a flask of broth containing: actively growing bacteria, and he diluted this culture until only a few bacteria were present in each drop of infusion. By placing these drops under his microscope, he was able to observe that simple method of reproduction which we now call binary fission— equal splitting. These cells became longer, pinched in the center, and finally separated into two organisms. He was probably the first person to observe this process, but his keen observations were for the most part lost in the many controversies relative to spon- taneous generation. The pressure being exerted on science for more and more proof of commonly accepted theories spurred microbiological progress, just as the pressure of war results in rapid expansion of practically all scientific knowledge. 28 Microbes and You Useful applications of Spallanzani's discovery were made by Nicholas Appert (1750-1841), a French confectioner, in 1810 when the French government offered 20,000 francs to the first person who could perfect a method for preserving food. He founded the modern canning industry when he demonstrated that food placed in sealed containers and boiled for suitable time periods could be stored indefinitely without spoilage. Appert did not try to explain how this technic worked, but Joseph Gay-Lussac (1778-1850) stated that air was necessary before fermentation and spoilage could occur. Since Appert's containers were sealed when hot, a vacuum was created, and both Joseph Priestley (1733-1804) and Antoine Lavoissier (1743-1794) had previously contended that oxygen, a constituent of air, was essential for life. In spite of mounting evidence that microbes must have parents, Felix-Archimede Pouchet (1800-1872), a famous naturalist and member of the French Academy of Sciences, lead concerted attacks in support of the theorv of abiogenesis. The Academy had made an offer in 1860 to anyone who could, by scientific methods, pro- vide indisputable proof one way or the other on the question of spontaneous generation. When Pouchet gave a paper before the Academy presenting his views, many influential scientists rallied to his support and Louis Pasteur was compelled to make public his opposite point of view. Pasteur insisted that air contained the necessary spark that reproduced cells. Microbes ride through the air on dust particles, and he insisted that a dust-free atmosphere harbored no microbes. We know today, however, that droplets expelled from the nose and throat of man and lower animals can also contain bacteria, even in the absence of dust particles. An English physicist, John Tyndall (1820-1893), added support to Pasteur's claims when he demonstrated that an open vessel con- taining a fermentable infusion would remain sterile when placed in a dust-free, or optically empty atmosphere within a chamber. By passing a beam of light through his chamber, Tyndall was able to see whether motes were dancing in the light beam. If no motes were evident to the eye, he found that there was no growth of organisms in his infusions. Highlights in the History of Microbiology 29 Pasteur felt that since bacteria cannot walk, they must hav.:? vehicles to carry them. Dust particles provide this necessary transportation. Pouchet's rebuttal is contained in this quotation: "How could orerms contained in the air be numerous enough to develop in every organic infusion? Such a crowd of them would produce a thick mist as dense as iron." Undoubtedly the first air analysis experiments were those per- formed by Pasteur as he added new evidence in support of his dust-borne theory of microbial dissemination. He noted that when he broke the tip from his flasks sealed with their infusion contents while hot, air rushing into the vessels confirmed the existence of a vacuum. This observation gave Pasteur an idea which he was to employ later in analyzing difterent air samples. If dust carried microbes, then the streets of Paris on a windv day were certainly a good source of organisms. So he prepared a number of such sealed flasks and opened them in the streets of the French Capitol. Everv flask revealed microbial activity upon incubation. Similar flasks opened in the relatively calm, dustless atmosphere of his cellar showed some infusions positive for growth, while still others remained sterfle. Even fewer flasks were positive when the ves- sels were opened on the Jura Mountains at an altitude of 2500 feet, and only one flask in 20 showed growth when the vacuum of the flasks was broken in the clear air on the Mer de Glace at a height of 6000 feet. Pouchet performed similar experiments, but every one of his containers opened at the 9000 foot level of the Pyrenees showed growth of microorganisms. Pouchet became so incensed when Pasteur continued to report results supporting the dust theory, that Pouchet challenged Pasteur to a duel, which never materialized. Wouldn't present day science leap ahead if every time research workers disagreed the issue had to be settled with drawn swords? It would be a boon to the sword manufacturers, but science would undoubtedly lose many brilliant scholars whose adeptness with the sword was limited! How can these opposite results obtained by these two workers be explained? The one difference in their research was the me- dium employed for growing the organisms. Pasteur used sugar. 30 Microbes and You yeasts and water, a relatively easy medium to sterilize. Pouchet, unfortunately, had chosen a hay infusion as his substrate, and we know that such material abounds with spores, those resistant bodies so difficult to kill by mere boiling for the usual time periods. Pouchet's experiments did help eventually to prove that spores are more resistant to heating than are vegetative cells, but he did not have spontaneous generation. These tough endospores help to explain the many irregularities obtained by the pioneers as they fumbled and groped with ideas and technics in their search for proof of one theory or another. The great resistance of these spores to heat was not conclusively demonstrated until 1877 when Ferdinand Cohn described them in the so-called hay bacillus, Bacillus suhtilis. Cohn also showed that spores could be made to germinate into vegetative cells which were not as resistant to boiling and to treatment with chemicals as were the spores. In fact, our present methods for sterilizing materials in bacteriological laboratories and in hospitals are based upon the time, and the temperature, necessary to inactivate these resistant stages in the growth of some, but not of all, bacteria. Although Pasteur was essentially a chemist, he had a flair for microscopy, and his ability to see through the clouds of miscon- ceptions to the clear air of reality led him to some of the greatest discoveries in biology up to that time. To overcome all criticism about devitalizing the air by heating it, by passing it through strong chemicals, or by screening it through such an innocuous substance as wool or cotton plugs, Pasteur placed a fermentable substrate in a flask and boiled this medium for a sufficiently long time to insure destruction of all living^ thino[s in this infusion. He then heated the neck of the flask in a flame and drew the glass out into an S-shaped curved capillary tube which he left open to the outside air. Since microbes cannot ambulate, he postulated that it would be impossible for contamination of his infusion to occur unless he tilted the flasks and allowed some of the sterile liquid to come in contact with the tip of the capillary tube containing dust particles. Others had amply demonstrated that boiling in- fusions did not devitalize them, and since ready access to vital Highlights in the History of Microbiology 31 substance in the atmosphere was not impaired, microbes incUned to generate spontaneously had every opportunity to do so in his open flasks. We know that no growth took place until Pasteur either broke off the neck of his flasks or until he tilted the contents Fig. 10. Louis Pasteur (1822-1895). {Courtesy of the Centra! Scientific Company, Chicago, Illinois.) into the dust-laden tip. This was conclusive proof that spontane- ous generation was a myth, and the arguments of his opponents had been nullified once and for all. Some of these flasks are still on display at the Pasteur Institute in Paris, France, and they re- main bacteria-free to this day. Pasteur was hailed as a hero and 32 Microbes and You the French Academy awarded him in 1862 the prize they had offered in 1860 to the first person who could prove or disprove abioijenesis. One of the highHghts of Pasteur's hfe occurred on April 7, 1864, when he deUvered his now-famous address at the Sorbonne in Paris in defense of his disproof of spontaneous generation: "There is no condition known today in which you can affirm that micro- scopic beings come into the world without germs, without parents like themselves. They who allege it have been the sport of illu- sions, of ill-made experiments, vitiated by errors which they have not been able to perceive, and have not known how to avoid." In another passage he described how he watched his flasks, pleading for them to g^ive him a sign of life, and could not . . . "for I have kept from them, and am still keeping from them, that one thing which is above the power of man to make; I have kept from them the germs which float in the air; I have kept from them life." He postulated his germ theory when he stated so concisely that life is the crerm and the o;erm is life. THEORIES CONCERNING FERMENTATION Another overwhelming problem needing clarification and sound proof before microbiology could become a firmly established sci- ence was the riddle of fermentation. What initiates this change in fruit juices and other sugar-containing substances, and what main- tains the reaction? Is fermentation the same as decay? Once bacteria had been discovered many persons pondered over the question revolving around whether bacteria were the cause or the direct result of fermentation. We can find Biblical references relative to the transmissibility of ferments, including the "little leaven that leaveneth the whole loaf." Since earliest times man has employed the process of fer- mentation for making bread rise, for souring milk, and for making alcoholic beverages without knowing how it all came about. Suc- cesses and failures could never be explained. However, until the nineteenth century was well along, we had little, if any, concrete evidence on the matter. Cag^niard-Latour in 1836, Theodore Highlights in the History of Microbiology 33 Schwann (1810-1882) in 1837, and Friedrich Kiitzing (1807- 1893) in 1837 independently reported that yeasts play a role in the process o£ fermentation. Witnessing budding in veast water, Charles Cagniard-Latour ( 1777-1859 ) referred to these objects as livino; substances. Ferments, as far as he was concerned, were composed of cells susceptible to reproduction by a sort of budding process, and these living objects were capable of acting upon sugars through "some effect of their vegetation." Schwann des- cribed yeasts as vegetative germs. These adherents of a biological theory to explain fermentation were scorned by most scientists of their day because the biological explanation was in direct con- tradiction to the physico-chemical theory of that renowned German organic chemist, Justus von Liebig (1803-1873). Von Liebig held sway from 1840-1860, and few individuals dared to question his decisions. When he announced that microbes were not the cause of fermentation, his faithful followers went along with the idea and helped to perpetuate the untruth. Molecules are in a constant state of motion— are chemically unstable— according to von Liebig, and when small amounts of decomposing stuff are mixed with fresh fermentable material, a chain-like reaction is initiated and continues until the fermentation is complete. Fermentation was believed to be a natural physical decomposition of large molecules with bacteria and other organisms capitalizing on this more readily available food supply for their metabolism. This supported the concepts of Georg Stahl (1660-1734) expressed in 1697 when he suggested that the process of fermentation was the result of the shattering of molecules by forces either from within or from without. Just what set off the reaction was never made quite clear by any of the proponents of the mechanistic theory. In 1869 when Berzelius, the renowned Swedish chemist of Upsala, supported the mechan- istic approach with his explanation that it was due to contact of catalytic forces, things looked dark for the opponents. The genius of Louis Pasteur arrived on the scene in time to set up one of the more famous controversies in microbiology: Pasteur's biological vs. von Liebig's mechanistic theory of fermentation. Believing that the proof of such knotty problems lay in the experi- 34 Microbes and You mental method, Pasteur went to work in his laboratory. He did not sit down and try to explain reactions until he had enough careful experimentation to back up his contentions. He v/as a Professor at the University of Lille, France, right in the heart of the wine and beer industries, and just at this time France was experiencing serious wine spoilage which no one seemed able to control. Pasteur was commissioned to undertake a study in the hope that a "cure" might be found for the "disease" of the wine. In his in- vestigations he found that each type of microbe will produce a predictable ferment. Pasteur's first paper on fermentation appeared in 1857. He described a grayish color in sugars undergoing fer- mentation, and his microscopic observations revealed the presence of small globules or short rods which, when transferred to fresh sugar solutions, perpetuated the process. Heating solutions so inoculated resulted in no fermentation. He stressed that spoilage of wine could be directly attributed to the action of certain microbes which produced undesirable end products and "diseased" the wines. By selectivelv heating the fresh juice after it was bottled, such diseases could be prevented. This prescribed heating has since been given the designation of pasteurization. In further studies on fermentation, Pasteur reported that among other end products in the reaction v/as amyl alcohol. If von Liebig's theory were true, then amyl alcohol should be a constituent of sugar merelv waiting release when the larger molecules shat- tered. This discovery made such a theory untenable since amyl alcohol is too difterent from sugar in its structure. Lavoisier and Gay-Lussac had reported that the weight of carbon dioxide gas and alcohol formed in sugar fermentation was practically equal to the weight of the sugar. By a series of clever experiments, Pasteur was able to give uncontestable proof that fermentation was a biological process, initiated and perpetuated bv living substances. Moritz Traube (1826-1894) in 1858 was apparently the first person to suggest the existence of enzymes, those remarkable digestive juices so essential to all life. The work of Edward Buchner (1860-1917) in 1897 conn med the enzyme theory when Highlights in the History of Microbiology 35 he was able to demonstrate the presence of zymase, the ferment which attacks gkicose. A temporary wave of excitement arose when Buchner demonstrated that a yeast-free extract could still cause fermentation— apparent evidence in favor of von Liebig's original non-biological concept. But it had to be admitted by even the most ardent supporters of the mechanistic theory that the enzvmes had originallv come from a living cell; not any old juice could start the reaction on its w^ay. Durins: the course of his feiTnentation studies, Pasteur dis- ci ^ covered anaerobes, those interesting microorganisms which grow in the absence of free atmospheric oxygen, in contrast to the aerobes which can develop only when free atmospheric oxygen is available. Fermentation is life without air, according to Pasteur. The first step involves growth of the organisms, and air is the source of assimilated oxygen. Alcohol production in this stage of fermen- tation is insicrnificant. During; the second stao;e the veast is com- pelled to act upon the sugar in the absence of atmospheric oxygen, and the essential gas is abstracted from the sugar. Pasteur's final conclusions were that (1) ferments are living organisms, (2) each ferment is produced by a special organism, and (3) ferments are not formed spontaneouslv. Liebig and his proponents believed fermentation to be a function of death, but Pasteur proved it to be a function of life. If wine could be diseased, the next logical step was to assume that human beings and other living things could also be afflicted as the result of specific microbial activity. It is of interest to note that Robert Boyle (1627-1691) in 1663 had stated that until the nature of fermxCntation v/as clearly understood, we could ha dly expect a logical explanation of disease. As a direct result of the Pasteur-von Liebig controversy, the solution of many other prob- lems, including the disposal of wastes, the purification of water, etc., were given a decided impetus, and progress was recorded. Lord Joseph Lister (1827-1912), a surgeon of Glasgow, Scotland, was so impressed with Pasteur's conclusions with respect to fer- mentation that he decided to apply these same biological principles 36 Microbes and You to the fermentation of wounds which too often were the aftermath of surgery. Could he kill the fermenting agents before they could destroy flesh? An Institute was erected in Paris in honor of Pasteur, and the building was dedicated in 1888. Pasteur was its first director, and he remained there until his death on September 28, 1895. He was succeeded by Emile Duclaux (1840-1904) who made the Institute Fig. 11. Joseph Lister (1827-1912). (Courtesy of Kelly and Rite, Microbiology, Appleton-Century-Crofts, Inc., and Ethicon Suture Lab- oratories, Inc., New Brunswick, New Jersey.) a great research center for scholars from all over the world. A scientific journal was begun by Duclaux in 1887, before the Institute actually was dedicated, and this famous journal is still devoted to the publication of articles relative to microbiology and related fields. THEORIES CONCERNING DISEASE Another vital concept that had to be crystallized before micro- biology could emerge on a firm foundation was the etiology ( cause ) of disease. Without reviewing the minute history of this phase of our science, and eliminating the evil spirits as the cause of man's Highlights in the History of Microbiology 37 miseries, we can note that the ancient Greeks suggested worms, too small to be seen, as the explanation of disease. Marcus Varro, in about 100 B.C., expressed the idea that invisible animals are carried through the air and enter the body by way of the nose and mouth. The Italian physician, Hieronymus Fracastorius ( 1483- 1553 ) , in 1546 postulated the theory of contagion, but since he had not actually seen the inciting agents, his thoughts represented pure Fig. 12. Robert Koch (1843-1910). {By permission from Intro- duction to the Bacteria, by C. E. Cliftoti. Copyright, 1950. McGraw- Hill Book Company, Inc.) speculation. Some two hundred years later (1762) an Austrian physician, Marcus von Plenciz (1705-1786), put forth a new con- cept that not only were diseases caused by microscopic organisms, but each disease was caused by a specific germ capable of being transmitted to other individuals via the air. Glossing over these early suggestions, we come to the year 1840 when Jacob Henle (1809-1885), a German pathologist, laid down the principles for our present germ theory of disease which led directly to the fundamental work of Robert Koch (1843-1910). 38 Microbes and You Koch emphasized the importance of proving concepts by employ- ing the scientific approach of actual laboratory experimentation. Before an organism could be said to be the cause of a specific disease, the agent had to fulfill the following postulates which Koch formulated in 1882: 1. The suspected organism must be found in every case of the disease. 2. The organism must be isolated in pure culture from every case of the disease. 3. These isolated pure cultures, when introduced into susceptible animals, must be capable of reproducing the original disease in its typical clinical form. 4. The same organism must be re-isolated from the injected test animal. If the postulates were fulfilled, Koch was willing to admit that the cause of that particular disease had been proven. We should point out here, however, that Koch's postulates cannot be applied in some cases. When you try to select a susceptible animal in postulate three, you find that human beings are the only animals you can use in some diseases and that is not always practical. It can be said, however, that accidental laboratory infections have permitted proof of the etiology of some diseases, such as t)^phoid fever, which man alone seems to contract. The strictly parasitic nature of such microorganisms as the leprosy organisms does not allow their isolation on ordinary laboratory media, and that sets up a block in the necessary cycle of proof. There are some diseases in which the clinical symptoms are the result of multiple infections, and trving to prove that a single organism is the cause of the disease may lead to confusing results. The common cold, for example, probably is initiated by a virus, or bv viruses, but the misery of a cold seems to be associated with the activitv of secondary invaders, usually bacteria, which are opportunists. In 1863 Casimer Joseph Davaine ( 1812-1882 ) reported that the blood of animals infected with anthrax contained rod-shaped organisms which could be transferred bv blood from infected Highlights in the History of Microbiology 39 animals to the blood of healthv animals and could cause the same disease. Pasteur reported similar findings with respect to silk worm disease caused bv protozoan parasites. Robert Koch con- firmed the findings of Davaine and expanded materially on the subject as we shall see later in the chapter. Because Koch is considered high on the list of famous micro- biologists, second probably only to Pasteur, his accomplishments deserve more detailed evaluation than many other workers men- tioned in this chapter on the highlights of microbiology. He was a rural practitioner in Wollstein, Germany, and at the time that news of Pasteur's work reached him, he was employed at an in- sane asvlum in Hamburg. The potential possibilities in this new biological field intrigued him, and his tendency to let his medical practice fall by the wayside became more pronounced with the passage of time. His first microscope was a present from his wife on his twenty-eighth birthday. In this dvnamic period during the latter half of the nineteenth century, Koch became increasingly alarmed at the tremendous economic loss being incurred as the result of anthrax infection in domestic animals. Those who could least afford to lose their animals— in some cases the family's sole support— seemed to be taking the brunt of this unconquerable disease. Perfectly healthy- looking sheep would die during the relatively short space of a single night, and postmortem examination would reveal the tell- tale black blood so characteristic of anthrax. The farmer, or members of his family, might contract horrible-looking boils, and in some instances they would contract the pneumonic type of the disease from breathing in the infectious agent, with painful death culminating their losing battle with respiration. Koch laboriously examined untold numbers of blood specimens drawn from healthy sheep and he compared these samples with the blackened blood of the stift^ carcasses of the infected animals. Without exception he found that the blackened blood contained rod-like sticks among the remaining undissolved blood-cells, but these rods were never found in blood from healthy sheep. Since those rods exhibited no locomotion, Koch was hesitant about calling 40 Microbes and You them microbes. We know today that typical anthrax bacteria are non-motile, but movement was accepted as an important criterion of microbes by many workers in those days. Being a man of modest means and unable to equip his labora- tory with large numbers of animals for experimentation, Koch was r - * .- , * *"* * > A' H Fig. 13. Koch's photographs of anthrax bacilli. {From Fundamen- tals of Bacteriology, M. Frobisher, 2nd. ed. Copyright 1942, W. B, Saunders Company, Philadelphia.) obliged to conduct much of his early research under field condi- tions. This added complications but it also added weight to his final conclusions. Mice were available and were relatively cheap to maintain, but he wasn't even sure that they were susceptible to this disease. He proposed to inject some of the infected blood containing these rod-shaped "things" into mice, but with no con- Highlights in the History of Microbiology 41 venient syringe handy for the injection, he was obUged to im- provise, an important necessity for those individuals who do ex- tensive research. If these rods were really microbes, he hoped that the mice would contract the disease and show active multiplication of the organisms within the body. His improvised syringe turned out to be splinters of wood, sharpened to a point, washed thoroughly, and heated in a drying oven to destroy any other microbes the wood might harbor. He made a small slit with a knife at the base of the mouse's tail and inserted the sharpened splinter which had been dipped into the blood of a recently expired sheep that had been suffering from anthrax. After a night's sleep, which we can presume was probably not too restful as he pondered over his problems, he returned to his laboratory and examined the injected mice. They were all dead, and at autopsy he found the internal organs and the blood to be harboring these rod forms. Whereas he had injected only a few microbes, there were millions of them to be seen in the dead mice, an indication that active multiplication had occurred. Most persons would have accepted this as conclusive proof that the rods were the etiological agent in the disease. Koch, being a cautious research worker, repeated his experiment over and over again, transferring the blood of successive dead mice to new healthy mice using his splinter technic. The results always came out the same. At this juncture Koch hit upon an idea which was to develop into a simple yet vital technic in microbiology. He wanted to cultivate his suspected organisms in such a manner that he could watch actual multiplication occurring before his very eyes. If he could see this step in their development, he felt certain that he was on safe ground if he announced that anthrax was caused bv a microbe which grew and multiplied within the susceptible animals. For some reason known only to Koch, he decided to place some liver from an infected mouse into some fluid drained from the eye of a recently slaughtered ox. We know today that such body fluids are normally free of bacteria, and are an excellent culture medium for growing organisms away from living hosts. 42 Microbes and You For hours he watched intently to see whether these rods under his microscope changed their size or their shape, but nothing happened. Then suddenly he was able to observe that the rods were becoming longer, and these elongated forms eventually broke in half and formed two short rods. These short rods in turn lengthened and subdivided by splitting in the very center, until within a few more hours he had many times the number of organ- isms that he started out with in his ox-eye fluid medium. This was trulv an astounding discovery. Here was an organism probably one-billionth the size of a sheep. Bv gaining access to the internal organs of a sheep, the microbe could set up housekeeping, settle down, and have a family within a relatively few minutes, and before a single day had passed, untold numbers of offspring could point with pride to their parent who came over on a blade of grass. The multiplication of rabbits is put to shame when compared with the ability of bacteria to reproduce several times during a single hour! When these overwhelming hordes of microorganisms had parasitized enough cells of the sheep, the afflicted animal could no longer overcome their deadlv eftect, and the sheep succumbed. After growing these bacteria away from the originally infected sheep for generation after generation, would the far-removed off- spring be able to establish themselves once more in the species of animal from whence they originated in his experiments? This was most important for Koch to know, and he promptly set about to find the answer. He still had some of the mice he had killed with blood-soaked splinters dipped in the juices of previously sacrificed mice. It had been weeks since these offspring of the original anthrax bacteria had been near sheep. If he could inject some infected mouse blood back into sheep and have the larcrer animal contract the disease, he felt that he could say with assurance that these microbes were the cause of anthrax. As he hoped, the sheep died with typical clinical symptoms of the disease. Not wanting to shout from the housetops that he had solved the problem, Koch very cautiously sent out feelers by casually telling some of his intimate friends and colleagues what he had done. The)^ naturallv raised questions which he could not always answer to their satis- Highlights in the History of Microbiology 43 faction. One of these embarrassingly perplexing problems was that of the "curse" of some fields in which grazing sheep died like flies long after any infected sheep had succumbed on the premises. If bacteria were spread from one person to another or from one animal to another, how did Koch propose to explain the infectivity of these grazing lands after the broiling sun had baked the area for months and the freezing winds of winter had subjected the land to such rigorous treatment? At the moment Koch had no answer, but he proposed to find the explanation if one was to be had. It was a puzzling situation indeed, for had he not seen these same organisms shrivel up and disintegrate on his glass slides when they were allowed to dry? Certainly that would indicate that the bacteria were dead and no longer able to generate their own kind. Fortunate accidents have resulted in many great discoveries, and fate decided to step in at this juncture and give the answer to a man who was clever enough to capitalize on an accident. One of his ox-eye fluid cultures happened to be left out on his laboratory table for a twenty-four-hour period. Normally such preparations would have been discarded in some chemical fluid, but Koch de- cided to take just one more look at this dried specimen under his microscope before discarding the slide. Lo and behold, his thread-like germs had been transformed into a string of glistening bead-like structures. Upon closer examination he discovered that these beads were within faintly outlined rod-like objects, slightly suggestive of his original organisms. At the time he felt that some contaminating bacteria had found their way into his carefully pre- pared slides, so he didn't think too much about it. Nevertheless, he did, again quite by chance rather than by design, keep his slide for several more months before he came across it once more and decided to confirm his original observation of the beads. Things had not changed in the interval, the beads were there glistening as before. He added some fresh ox-eye fluid to the dried slide and watched under his lenses to see what these supposedly contaminat- ing forms would turn out to be. Within a few hours he really was jolted when before his eyes he watched the individual beads dis- 44 Microbes and You appear and turn into the original rod-shaped microbes so char- acteristic of the ones in the anthrax-infected animals. Like a flash, the truth of the situation occurred to him. Here was the answer to the "cursed" pasture land; these beads were spores capable of withstanding drying for extended periods of time, and the rigors of winter could not kill them. Those pasture lands were con- taminated with resistant forms of these bacteria, and when sheep grazed on these fields, they ingested the spores and became in- fected. Now he was ready in 1876 to announce to the world the cause of anthrax. With self-assurance for the first time in many long, arduous months, Koch determined to journey to Breslau to visit an old friend who had encouraged him durino; his earlv ventures in re- search. This friend was Professor Ferdinand Cohn, the first person to work out a scheme for classifying bacteria in the plant kingdom. He carried along some of his deadly anthrax organisms and some of his mice on this particular trip, since he proposed to show Professor Cohn the completeness of his findings with respect to the deadly disease. Cohn invited anvbodv who was anvbody in the scientific world to attend a lecture bv Koch in which he was to present his research findings. Few had ever heard of this man who had been working quietly without the aid of fancy laboratory equipment, but many came to the presentation more out of curiosity than faith in what Koch might have to say. Because Koch was not an outstanding orator, he used the demonstration technic of teaching in place of the lecture method, and he put on a show that amazed the most learned scientists in the audience. He gave his theories and then proved his beliefs with actual animal experimentation. The renowned Professor Julius Cohnheim ( 1839- 1884), who first demonstrated that pus was composed largely of white blood cells, and who was without doubt the leading authority on disease in Europe, was most impressed with the clarity and conclusiveness of Koch's presentation. Koch had converted a disciple! Because of Cohnheim's influence, Koch was compelled to turn away scholars who came in droves to study under his tutelage. When he announced to members of his class, including Highlights in the History of Microbiology 45 Paul Ehrlich (1854-1915), what he had heard and seen relative to the etiology of anthrax, Koch found himself with still other followers who all wanted to join in these investigations of microbes. This was the year 1876, and Louis Pasteur had made a rather sweeping statement just seven years previously to the effect that man held the power to wipe parasitic maladies from the face of the earth. To say that this pronouncement was scoffed at is to put it mildlv. Koch was now leadino; the fight in that direction, and while we must agree that we still have a long way to go before Pasteur's statement can be fulfilled, the strides we have made would astonish Pasteur, were he to rise today from his tomb in the basement of the Pasteur Institute in Paris. Koch's reputation continued to grow, and even though the years immediatelv following his announcement of his anthrax findings were not prosperous ones for this great discoverer, he emerged in 1880 with an appointment by the German government to the position of Extra-ordinary Associate of the Imperial Health Office, with a fine, well-equipped laboratory and with assistants to help him in his research activities. People clamored to be allowed to study under him in Germany, and the list of his pupils is a "Who's Who in Microbiology." Laboratory procedures were in a very chaotic state in 1860, and Koch felt that order had to emerge from this chaos if microbiology ever expected to amount to very much. If each disease is caused by a single species of microbes, Koch realized that he would have to devise some technic for separating organisms from other organisms. There are few places in nature where a pure culture of an organism can be found. Bacteria are usually mixed with all forms of microscopic and macroscopic life in the keen battles for survival. Fate once again came to Koch's rescue and he was smart enough to capitalize on the chance observation. It seems that a sliced, cooked potato had been left on one of the tables in his laboratory. He happened to observe several colored spots on this potato and his curiousity got the better of him. What are those colored spots? Streaking a little of the pigmented material on a glass slide in a drop of water, Koch was thrilled to find that each spot was composed of a pure culture of 46 Microbes and You microbes. Nature had provided the answer to the problem of culturing microbes as separate species. If a single micro- organism carried through dust in the air, as Pasteur had shown, landed on a cooked potato, it was capable of multiplying into a visible growth called a colony. All members of a colony represent the progeny of the original organism and hence we have a pure culture. If he could just work out a few of the details, he had the answer to what might have become a perplexing problem. Finally, with the assistance of two military doctors, Friedrich Loeffler (1852-1915) and Georg Gaffky (1850-1918), he announced that by streaking mixtures of microbes over the surface of a fresh boiled potato free of other organisms, colonies of the bacteria contained in the mixtures could be made to develop on the potato. When these colonies were well-enough isolated, they could be picked from the potato with the assurance that all microbes from such a colonv were alike. This was revolutionary! Before too many organisms had been cultivated, however, Koch began to appreciate that the food requirements of bacteria varied tremendously, some being a great deal more exacting about the diet set before them. Joseph Schroeter (1835-1895) first separated chromogens from each other in 1872 bv growing them on such solid substances as potatoes, coagulated egg white, starch paste, and meat, but he ran into difficultv^ when he tried to cultivate non- pigmented organisms. Koch came along and knew what he wanted— a solid, transparent, sterile medium. Gelatin, as a solidi- fying agent, seemed to best fulfill these requirements, and since mycologists had been using this material for thirty years, Koch adopted it in 1881, thus revolutionizing bacteriological technic. As is so often true with new ideas, time modifies their original seem- ingly wonderful characteristics. Gelatin has two major limitations. Some bacteria can utilize the substance as a source of food, thus turning it into a liquid. If one is seeking Ivtic ferments, gelatin serves a useful purpose, but the enzyme also makes the medium useless from the standpoint of trying to isolate pure cultures on a solid medium. A second serious defect of grelatin is that it is a Highlights in the History of Microbiology 47 liquid at body temperature, that vital warmth needed to cultivate many pathogenic organisms. At this point let us introduce a man who had worked in Koch's laboratory-Dr. Walther Hesse ( 1846-1911) -and his wife, Fanny Eilshemius (1850-1934), his faithful technician. He had labored Fig. 14. Frau Fanny Eilshemius and Dr. Walther Hesse. {Courtesy of Morris C. Leikind. From the Journal of Bacteriology, 19S9, 37, 487.) on the bacteriology of the atmosphere using gelatin as a solidifying agent for his many bouillon concoctions necessary to cultivate the organisms in the air. His maddening failures when the gelatin was attacked by the organisms drove Hesse to seek new solidif>dng agents. Mrs. Hesse made an epic suggestion for which Koch is often given more credit than is probablv due him. She had been using agar-agar (derived from the Malayan agal-agal, which 48 Microbes and You means very gelatmous) as a solidifying agent in her own jelly and jam recipes at home. She picked up this technic from her mother, who in turn received it from some Dutch friends who had lived in Java. In the East Indies agar-agar had been employed for generations as a thickening agent for both soups and jellies. Why not try agar-agar in place of gelatin, she suggested to her husband. An historic moment in microbiology was reached by this simple, yet necessary, substitution. An occasional contribution of an un- known individual can make discoveries of lasting value. Oliver Wendell Holmes (1809-1894) stated that medicine learned "from a Jesuit how to cure agues, from a friar how to cut for the stone, from a soldier how to treat gout, from a sailor how to keep oflF scurvy, from a postmaster how to sound the eustachian tube, from a dairymaid how to prevent smallpox, and from an old market woman how to catch the itch-insect." To this imposing list we can add a housewife who helped her husband solve the perplexing problem of pure culture isolation technics. Without delay this new substance derived from Japanese seaweed ( Gelidium corneum ) was reported to Koch, probably in the latter half of 1881. Koch adopted it and in his famous publication of 1882 in which he announced his preliminary investigation on the tubercle organism (Mijcohacterium tuberculosis), he made reference to agar-agar in one brief sentence. Fanny Hesse, who had been born in 1850 in New Jersey in the present locality of Jersey Cits^, died in 1934, with few bacteriologists realizing that the credit often ascribed to Koch originated with her suggestion to her research-minded husband. When our source of agar was cut off during World War II, the United States was obliged to seek a new supply. After some intensive searching, beds of the species GeUdeum cartUagineum were discovered off the coast of California, and much of our war- time agar came from this source. By perfecting new technics and by making use of this new- found agar base for his culture media, Koch began a long series of fruitful discoveries, complementing much of the work of his French colleague in microbiology, Louis Pasteur. Koch proved Highlights in the History of Microbiology 49 the etiology of cholera epidemics after his studies in Calcutta, India, where the disease was endemic. The Emperor of Germany bestowed the Order of the Crown, with star, on Robert Koch for his brilliant discoveries. His laboratories in Berlin became the focal point for training laboratory technicians. The germ theory of disease was now firmly established, and Koch, together with his ever-widening circle of trained personnel, began a chain reaction of discoveries. Once we knew the cause of disease, isolated the germ, and found ways to destroy it, we were able to cut out a link in the chain of the progress of disease, giving us the greatest control in the spread of diseases of man in recorded history. What has been accomplished has already been reviewed in the intro- ductory chapter under the discussion of life expectancy since the turn of the twentieth century. Pasteur had not been idle while Koch and his disciples were busy perfecting new technics, and Pasteur's armouncements also began to stir the imagination of men. Lord Lister carried out his first aseptic surgery with the help of a fine mist of carbolic acid playing on the field of operation, after soaking his instruments in the same solution to destroy these, germs that Pasteur and Tyndall had so conclusively demonstrated were present in the air. This is the foundation of modern surgical procedures, based today upon a combination of asepsis and disinfection. We can divide this history of microbiology into three periods. People do not always agree with the limiting dates of these periods, but in general we can say that until the year 1850 most micro- biology was purely speculative in nature. From 1850 until about 1900 the important fundamental discoveries were made, preparing for the so-called modern era from 1900 to the present. Too often students of this science feel that everything has been discovered; nothing remains to be done. This may be the tendency of some individuals in the twilight of their lives, but any real student of bacteriology can hardly read a single printed page in a research journal, without realizing that much remains to be done. If one thinks in terms of bacterial diseases, we will have to agree that many of the former scourges of mankind have been fairly-well 50 Microbes and You checked. But the field of Virology is a young, very young, out- growth of our science of microbiology, and it hides many secrets. This chapter was purposely titled "Highlights in the History of Microbiology." Included in the list of persons mentioned were individuals whom Kitchens and Leikind so aptly described as the "bead strincrers" in contrast to the "bead collectors." Each bead o represents a scientific fact. But isolated facts, to be reallv useful, must be strung together by those rare individuals who have the insight to string these beads into a useful necklace. Bead collectors are by far more numerous, but this does not detract from their usefulness in science. Other famous bead stringers have plaved vital roles in the science of microbiology, but we shall weave their contributions into subsequent chapters as we discuss specific phases and applications of their discoveries. CHAPTER 3 Bacteria Are Classified as Plants DIFFERENCE BETWEEN PLANTS AND ANIMALS CLASSIFICATION OF BACTERIA DIFFERENCES BETWEEN PLANTS AND ANIMALS Before the perfection of ground lenses opened up a new vista for scientists to explore, it was a relatively simple matter for an individual to distinguish a plant from an animal. After all, an animal was an animal, and it was a mighty peculiar person who could not tell a plant from an animal! The microscope, however, made the distinction more complex. Since bacteria have some characteristics of each kingdom, the question of where to place them in a systematic scheme was a good topic for brisk debate during the infancv of this new branch of science. As evidence continued to accumulate, it became increasingly clear that more plant traits were being exhibited by bacteria than were the animal char- acteristics. Biologists today generally agree that we should con- sider these microorganisms as plants. However, the decision is not a unanimous one; a few die-hards are still a bit hesitant in making the concession. It is only a natural reaction for beginning students in bac- teriology to evidence surprise that bacteria are plants, especially after they view motility of microorganisms under the microscope for the first time. Who ever saw a plant that could swim under 52 Microbes and You its own power? We can answer that inquiry by stating that loco- motion, in itself, is a poor criterion for judging whether a living thing should be classified as a plant or as an animal. There are other more important differences which have been accepted by persons who devote their lives to this field of study we call Taxonomij— the science of classification. While bacteria do not exhibit all of the characteristics generally ascribed to plants, they do have more plant features than animal characteristics. The presence of a firm, thick, demonstrable cell wall, the ability to combine simple substances for their own use ( CHEMOSYNTHESis ) , and the capacity to utilize only relatively simple compounds taken in solution (holophytic nutrition), are strong arguments favoring the classification of bacteria as plants. The dividing line between the plant and the animal kingdoms might be said to pass through the bacteria. CLASSIFICATION OF BACTERIA It has been convenient for scientists to place living things into various groups, or classifications, in order to point out their relation to other living; thino;s, and to demonstrate how much more specialized some organisms are than others. The "big words" emploved by scientists are often frightening to the novice, but don't think for one moment that these same words are not also disturbing to workers who have been engaged in the field for a good many years. Not all bacteriologists agree with the names given to microorganisms by taxonomists. The very fact that taxonomy is not static, that changes are continually being suggested and adopted by those individuals most concerned, indicates that even the leaders in the field cannot always agree. Compromises must be made if we are to have any kind of a workable system for classifying microbes. This agreement becomes important not only for American biologists, but for scientists engaged in labora- tories throughout the world. An International Microbiological Congress meets every few years in an attempt to thrash out knotty biological problems, and the questions relative to taxonomy have a habit of finding their way near the top of the agenda. Without Bacteria are Classified as Plants 53 such international understanding, science would soon turn into a series of closed cells, each country adhering to its own, oftentimes narrow, opinions. The concept of One World is also important in biology. Classification supplies valuable information to a person trained in the field. It is often uninteresting to many students who are taking a terminal survey course in microbiology, and it is not the intention of this book to perpetuate this natural reluctance to learn new names. But a brief discussion of this topic will help to em- phasize the relative position that bacteria occupy in the plant kingdom. Since some plants closely resemble others, and are quite distinct from still other plants, we can group them together under ap- propriate headings. The following is the generally accepted simplified breakdown, or table of organization, for bacteria : KINGDOM: Plant PHYLUM: Thallophyta (exhibit no roots, no stems, and no leaves.) CLASS: Schizomijcetes (microscopic, unicellular, chlorophyll-free plants that reproduce asexually by fission, and exist either as rods, spheres, or spirals). ORDER: I. Euhacteriales (The true bacteria, including most of the organisms discussed in an elementary course of this type.) II. Actinomycetales (Elongated cells with a definite tendency to branching.) III. Chlamydobacteriales (Filamentous, colorless, alga- like bacteria which may or may not be en- sheathed.) IV. Myxobacteriales (Slime bacteria, exhibiting group movement as a unit; crawling, creeping motion away from the center of the colony.) V. Spirochaetales (Slender, flexuous cell body in the form of a spiral with at least one complete turn —from 6 to 500 microns in length.) Each order, in turn, is subdivided into families; the families contain genera, and the genera include various species. It should be made clear that sharp lines of demarcation do not always exist between these man-made groupings. S4 Microbes and You It seems important to review the thinking that preceded this final classification whereby bacteria are catalogued under the Thollopliijta. You may recall that Antony van Leeuwenhoek, the first person to leave written descriptions of bacteria, referred to these organisms as "animalcules," since their active motility sug- gested small, darting animals. Carl von Linne (1707-1778), the Swedish botanist who is better known as Linnaeus, couldn't decide where to place bacteria in his Sijstema Naturae, in which he listed all plants and animals recognized up to that time. However, he finallv did call bacteria animals in the class Vermes and the order Chaos, where they remained until more could be learned about them. The first organized attempt to bring order out of this chaos was in 1774 with the work of Otto F. Miiller (1730-1784), a Dutch naturalist, who placed bacteria among the ciliated protozoa. He included a genus he called Vibrio, a term still in common usage. Felix Dujardin (1801-1860), not knowing whether bacteria were plants or animals, reached a happy solution by naming them Zoophvtes, which means animal-plants. Christian Ehrenberg (1795-1879) published his grouping of organisms in 1839, and he includes four sjenera familiar to modern bacteriologists: Bacterium. SpiriUum, Spirochaeta, and Vibrio. As early as 1857 Karl Nageli ( 1817-1891 ) introduced the word Schizomifcetes (fission fungi) and this is the class under which we find bacteria in modern classification schemes. This helped to set the pattern for workers interested in supporting the idea that bacteria belong to the plant kingdom. A great lift was given to this school of thought when Ferdinand Cohn in 1872 published the first systematic classification of bacteria. He pointed out that group- ing these organisms into genera and into species was not only possible, but that it was logical. It was not, however, until Gual- terio Migula at the turn of the twentieth century classified bacteria on the basis of morphology (size, shape, and structure)— especially motility and arrangement of the flagella— that wide acceptance of any scheme was encouraged. The technical difficulties involved in trying to stain flagella soon became onlv too apparent, and Orla- Jensen in 1909 expanded the base for criteria emploved in taxonomy Bacteria are Classified as Plants 55 hv including biochemical characteristics along with morphology. C.-E. A. Winslow (1877-) had made the same suggestion in 1908 when he classified the Coccaceae. Other schemes were advocated from time to time, but the general acceptance o£ any classification was still lacking. Through the initiative of a volunteer committee consisting of A. C. Abbott, Professor of Hygiene and Bacteriology at the Uni- versit)^ of Pennsylvania, H. W. Conn, Professor of Biology at Wesleyan University in Middletown, Connecticut, and E. O. Jordan, Assistant Professor of Bacteriology at the University of Chicago, a national organization called the Society of American Bacteriologists (abbreviated S.A.B.) was founded in 1899 at New Haven, Connecticut. This group naturally became interested in the problem of bacterial taxonomy, and at its annual meeting held at Urbana, Illinois, in 1915, a committee was appointed to review the problem of classification and to report back to a later annual gathering of the S.A.B.^ A progress report was submitted in 1916 and the final recommendations were published in 1920. While this report was not adopted as official by the S.A.B. , it did serve as a valuable framework for future deliberations. Other taxonomists pursued the problem further with the result that bacteria were finally classified into groups on the basis of all ^ Some of you may be wondering how one goes about becoming a member of a national scientific society. The S.A.B. is not Hke an exclusive college fraternity. Members are not picked after a period of "rushing," criticism, and selection. To quote Article III, Section 2a of the S.A.B. Constitution: "Any person interested in the objects of the SOCIETY shall be eligible for election as a member." And to quote Article II: "The objects of the SOCIETY shall be to promote scientific knowledge of bacteriology and related subjects through discussions, reports and publications, to stimulate scientific investigations and their applications, to plan, organize and ad- minister projects for the advancement of knowledge in this field, and to im- prove professional quafifications." If you care to join nearly five thousand individuals who are presently listed on the membership rolls of the S.A.B., you merely have a member nominate you on a prescribed formal application blank, have another member second the nomination, send in your annual dues, and after the National Council approves the nomination (which is practically an automatic matter), you are a member of an outstanding national scientific society! Anyone who plans to work in the field of microbiology should be encouraged to join the S.A.B. and to help promote its important program. 56 Microbes and You considerations— morphology, cultural characteristics, habitat, bio- chemical reactions, etc. Through the leadership o£ Robert E. Buchanan and David H. Bergey, among others, the 1923 publica- tion of the Manual of Determinative Bacteriology came into being. Modern terminology calls this Bergey s Manual. The sixth edition of this bacteriologists' bible was published in 1948, and this volume lists 1,630 species of bacteria with descriptions of each organism. Bergey s Manual is widely accepted today as the standard reference work in the field. Just as it is customary for human beings, at least in large areas of the civilized world, to have a first and a last name, scientists employ a two-name system, called the Binomial System of Nomen- clature, for plant and animal designations. Linnaeus introduced this system to science, but evidence tends to show that he was not its originator, contrary to popular opinion. Latin, or Latinized, names are used in biology. Man, for ex- ample, is called Homo sapiens. Homo is the generic name, while the species name is sapiens. When writing such scientific names we underscore them, or in print we italicize the names. Sometimes we find organisms with three names, not to be outdone by many humans, such as Thiohacillus thiooxidans Beijerinck. The dis- coverer of the organism, in this instance Beijerinck, is occasionally honored in this way. Notice too that the generic name is always started with a capital letter, while the species name is written with a small letter. Names of individuals tacked on the end are always capitalized. These details may seem very exacting to be- ginners in science, but orderliness is important. Most of the bacteria studied in an elementary course in micro- biology fall into the order Eubacteriales— the so-called true bacteria. But when disease-producing (pathogenic) organisms are con- sidered, interesting^ members of some of the other orders under the class Schizomycetes will be considered. They will include the Actinomycetales under which is found the tuberculosis organism {Mycobacterium tuberctdosis), and the order Spirochaetales in- cludes the syphilis spirochaete, Treponema pallidum, to mention just tv.'o. CHAPTER 4 Microbes Must Eat PREREQUISITES FOR A GOOD MICROBIOLOGICAL MEDIUM Proper moisture content Readily available food materials Correct pH Sterility Desired physical properties GELATIN VS. AGAR CLASSIFICATION OF MEDIA PREPARATION OF STANDARD NUTRIENT AGAR It may be difBciilt for you to visualize a living entity the size of a microbe sitting down to an abundant repast, but like every other living thing the microscopic organism must ingest food to provide energy and to allow metabolic processes to take place. Being single cells, bacteria have not been endowed with specialized organs, such as a mouth, a stomach, or intestines. But an in- genious process has been evolved whereby a single-celled organism, whether it be a plant or an animal, can absorb dissolved nutrients directly through the cell wall membrane— holophytic feeding. The outer shell is fastidious in that it selects what shall go into the cell and what shall be secreted or excreted. Such food intake Js called diffusion and the exacting nature of the cell wall depends 57 58 Microbes and You upon the permeability of its membrane. The passage of fluids across a membrane is termed osmosis. We all know individuals who are particular about their diets; they refuse to eat this or that because they just don't like it. Microbes also exhibit this type of rejection, in some cases to a marked degree. While differences in the permeability of the cell walls partially explains this selectivity, we find some foods that can be absorbed only to be rejected by a cell that doesn't thrive on them. Some organisms have had their own way for such a long time that thev are no longer able to metabolize certain foods. Since a number of organisms are unable to swim around in their quest for food, the nutrients must be provided nearbv at the right time, and in an acceptable concentration, otherwise the cells may starve to death. Such extreme dependence upon a narrowh^ defined diet sometimes works to the disadvantage of pathogenic bacteria. When more than a bare minimum of food is available the organ- ism gets larger, just as man puts on weight, but the microbe goes us one step better. When it reaches a predetermined size, it splits in two by a process called binary fission ( equal division ) . Under optimum conditions the dividing process occurs once in about twenty to thirty minutes, with notable exceptions including Mi/co- bacterium tuberculosis whose generation time is much greater. But this splitting cannot go on indefinitely, because natural checks and balances come into play to keep other forms of life from being forced from the face of the earth bv growth of overwhelminsj numbers of microbes. Some persons keep cows, others keep pigs or sheep, and the breed of scientists we call a microbiologist, tends microbes. In each case the nutritional needs of the living things being cared for must receive careful attention by the keepers. While minute amounts of food are required by a single bacterial cell, the masses of organ- isms capable of being generated in only a few hours put increasing demands upon the food supply. In general, we are not interested in prolonging the life span of microbes the way we are striving to increase man's life expectancy. However, in the process of Microbes Must Eat 59 training microbiologists or in research investigations, increased numbers and increased longevity of bacteria may be important. We cannot do too effective a job by studying only a few of these microscopic forms, but masses of actively growing cultures can provide valuable information relative to the physiology and the ultimate identity of a given species. In addition to preparing a diet which is most favorable for as manv species of bacteria as possible, we can also blend chemi- cals with our nutrient materials in such a manner that selectivity of growth can be accomplished. Often, particularly in clinical laboratories when we are attempting to isolate the etiological agent in a given disease, it is highly desirable to inhibit the growth of abundant organisms which we know are normally not pathogenic, and to encourage the cultivation of our suspected pathogens. Such a medium emplovs the principle of bacteriostasis for its selectivitv. No culture medium has yet been devised which will allow all known bacteria to grow under one set of conditions. To meet this problem, microbiologists have been forced to concoct hundreds, even thousands, of combinations of substances to fulfill growth requirements. There are a few basic media to provide general uniformity in running such standardized tests as milk and water analyses, otherwise no two laboratories would be able to compare a given sample with reproducible results. A great boon to uniformity has been the manufacture by biological supply houses of dehydrated media, and standardized procedures play an im- portant part in modern laboratory technics. PREREQUISITES FOR A GOOD MICROBIOLOGICAL MEDIUM Any material employed to grow bacteria is termed a culture MEDIUM, and the resultant growth is termed a culture. A satis- factory microbiological medium should fulfill the following pre- requisites. It must: 1. Have the proper moisture content. 2. Contain readily available food materials. 3. Have the correct acid-base balance, called pH. 60 Microbes and You 4. Be sterile— in a bacteriological sense. 5. Provide desired physical properties (clarity, liquid, solid, etc.). Since modern microbiology is founded upon our abilits' to isolate pure strains of specific species of organisms through the use of culture media, it seems wise at this point to elaborate on these five prerequisites of a good microbiological medium. PREREQUISITE I: PROPER MOISTURE CONTENT When we analyze bacterial cells, we find that their moistiue content approaches 80%, with a range approximatelv 15% either side of this figure. The technic generally used in the determination of water content of cells is to dry them at 100-110° C. in the air, or in a vacuum oven at a lower temperature, and to observe their weight loss. Slimy capsular layers surrounding some organisms tend to raise their relative moisture level. In compounding media we try to provide a high water content, usually 75 to 95%, to ful- fill this growth requirement of microorganisms. Bacteria are more closely related to aquatic plants than to terrestrial plants; they thrive best when the organisms are sur- rounded by moisture containing readily available food substances. An abundance of water is just as important for bacterial growth as is the presence of available food, since moisture serves as a vehicle for the food and provides transportation for the egress of waste products built up within the cell during metabolism. Water is the most universal solvent. Its specific heat aids in the ab- sorption of heat liberated during metabolism of the cells, and equally important is the conductivity of water which facilitates the dissipation of heat generated by living organisms. The concentration of food in solution directlv affects the speed of flow and the direction of flow of water with respect to the suspended cells. Too high a concentration of food draws water from the organism and tends to shrink the cell, while too low a concentration of nutrients induces the entrance of excessive water through the cell wall with resulting swelling and eventual harm to the organism. Microbes Must Eat 61 PREREQUISITE II: READILY AVAILABLE FOOD MATERIALS Organisms vary tremendously in their nutritional requirements, varying from simple inorganic salts up to and including living tissues of species of plants or animals. But in general most common bacteria are not too exacting, with the result that many types of food substances can be utilized for food. Minute amounts of accessory growth substances, or vitamins, are required by some organisms, and to that end some species manufacture their own vitamins. This brings in an interesting sidelight in nutritional studies. If an organism requires a given vitamin and this vitamin is not provided by the organism's own chemosynthetic activity, the vitamin content of foods can be determined by attempting to grow these vitamin-dependent microbes on this food. This is called a VITAMIN ASSAY determination, employing biological rather than strictly chemical test tube technics in the analysis. Experience has demonstrated the value and the accuracv of this biolo^^ical method. Organisms can be classified on the basis of their nutritional needs into two major groupings, the autotrophs and the hetero- TROPHS. The former bacteria use simple elements or simple com- binations of inorganic material ( iron, manganese, etc. ) as their principal source of food, and carbon dioxide is utilized as a carbon source. Heterotrophs, in general, are unable to assimilate carbon from carbon dioxide and their energy is primarily derived through the chemical breakdown (analysis) of more complex food ma- terials (nitrites, nitrates, etc.), especially organic compounds. An organic compound is a carbon-containing compound derived from plants or from animals. Heterotrophs can be further subdivided into SAPROPHYTES which thrive on dead orsjanic matter, and into PARASITES which depend upon living cells for their survival. There are degrees of parasitism. The term facultative parasites is used to designate those organisms capable of thriving on either living or on inanimate materials, such as common laboratory media. Strict parasitism is not nearly as common as is facultative para- sitism. It is generallv conceded that heterotrophs probably evolved S2 Microbes and You from the autotrophs, although this conjecture provides the sub- ject for hvely discussions in seminar sessions. It should be em- phasized that while it is convenient to speak of various nutritional groupings of organisms, clear-cut differences do not always exist. There are plenty of "in between" foiTns to complicate these neat cataloguing schemes. If bacteria were compelled to depend upon nature to supply the exact chemical substances required for their existence, survival of these microscopic forms might probably be even more difficult than is actuallv true under existing conditions. Whenever the food particles ( molecules ) are too large for heterotrophs to absorb directly, the cells must secrete digestive juices, called enzymes, to attack these large molecules as the initial step in making the food parcels small enough to pass through the membrane of the organ- ism via the fluid menstrum. Enzymes may be defined in simple language as organic catalysts^ produced by living cells. A catalyst may be considered to be an agent which accelerates a chemical reaction without itself being consumed in the reaction. A little bit of catalyst goes a long way, and it has something which "sparks" a reaction. The orderliness of science demands categorizing and classifica- tion. Enzymes are no exceptions. We can divide these digestive ferments into two major groupings, the extracellular enzymes and the intracellular enzymes. When an organism is obliged in its search for food to secrete enzymes to attack large molecules surrounding the cell, these juices are referred to as extracellular enzymes. While it is true that they are formed within the living cell, such exo-enzymes are able to leave the confines of the cell and ^ If you would like to demonstrate a catalytic reaction as a parlor trick, take a piece of lump sugar and try to make it burn with the flame from a match. It will not ignite. Now dip the sugar into some cigarette ash and the sugar will burn with a faint blue flame when a lighted match is touched to the catalyst in the cigarette ash on the lump of sugar. No doubt a good chemist using micro-technics could analyze the end products of such combustion, and the catalyst in the original ash would be found to be intact. To prevent damage during a demonstration, it is recommended that the experimenter carry out the ignition over a plate or xessel to catch any hot dripping sugar. Microbes Must Eat 63 to carry out their assigned functions extracellularly. Once the food has been broken down to a size permitting passage through the semi-permeable covering of the organism, then the absorbed food is further attacked within the cell by the endo-enzymes, which convert the food into an available form. A rebuilding process mav also be employed by intracellular enzvmes to convert the food into chemical configurations required in the metabolism of the specific microbe. Enzymology is a complex science, and a book of this nature is not intended to do much more than introduce the beginning student to general concepts needed to familiarize him with the underlying technics employed by single-celled, mouthless, micro- scopic organisms when they are faced with the problem of obtain- ing food "too tough to chew" without some preliminary breakdown. The action of enzymes may be influenced by many of the same forces affecting other chemical reactions. Temperature, moisture, acid balance (pH), presence of chemical poisons, and other factors have their effect on enzymes. Enzymes are highly specific for given substrates. Enzyme "A" only works on substrate "A," and substrate "B" can only be attacked by enzyme "B." Since phys- iological reactions determine to a great degree where organisms are placed in classification schemes, it becomes apparent that there is a close relationship between enzymes which initiate physiological reactions and taxonomy. Higher plants have the advantage over bacteria in that the former possess chloroplujU, that green pigment so essential to higher plant life. Chlorophyll is another example of a catalyst, where the cholorphyll is activated by sunlight to allow water and carbon dioxide gas to combine into plant substance in a process called photosynthesis ( putting together in the presence of light). Much of the food taken in by bacteria must be drawn upon for respiration. When free gaseous atmospheric oxygen is involved in respiration, we speak of this as aerobic respiration, in con- trast to ANAEROBIC RESPIRATION which iuvolvcs the breakdown or re-arrangement of molecules in a food substrate as a means of 64 Microbes and You obtaining oxygen. All living cells must have oxygen, but the origin of this oxygen varies with the type of respiration employed by the cell. Some organisms have adjusted their lives in such a way that they can respire either aerobically or anaerobically. Such bacteria are termed facultative. Some persons go so far as to subdivide the facultative microbes into the facultative aerobes, which grow either aerobically or anaerobically but prefer aerobic respiration, and facultative anaerobes which prefer anaerobic to aerobic respiration. When bacterial cells are subjected to chemical analysis, a number of elements can be detected. Sometimes an element may be fortuitous, just entering the cell wall for the ride at the same time that essential elements are being absorbed. Still other elements, even in minute traces, are vital to survival and multiplica- tion of the organism. The five pillar elements— carbon, hydrogen, oxygen, phosphorous, and nitrogen— must be supplied in an avail- able form to all living cells. In addition to these five, however, trace amounts of other elements must be included in good bacteriological media. The names of the essential elements for cellular growth are conveniently remembered in the expression: C HOPKINS CAFE MG, an abbreviation for Carbon (C), Hydro- cren (H), Oxvgen (O), Phosphorous (P), Potassium (K), Iodine (I), Nitrogen (N), Sulfur (S), Calcium (CA), Iron (FE), and Magnesium (MG). Energy is required to blend these elements into useful combination for cell substance, and this energy ma\' be derived in one of two general ways. Bv directly absorbing energy foods, such as available sugars, and breaking down these sub- stances through the activity of enzymes, energy can be released for cell use. Or the organism may liberate energy through oxida- tion, a process tied up with respiration in all living cells. Fulfilling the prerequisite of providing readih' available food in a microbiological medium is a great deal more involved than the simple statement might lead you to believe, and unless the provider understands these ramifications of nutrition, healthy, actively grow- ing; crops cf microoro;anisms misiht not materialize. Microbes Must Eat 65 PREREQUISITE III: THE CORRECT ACID-BASE BALANCE- THE ;;H If all of the other requirements for a good microbial medium are supphed, but the acidity or the alkahnity is not properly con- trolled, poor growth of organisms, or no growth at all, may result The usual ingredients incorporated into common media are acid in character, and we must neutralize at least part of this acidity by adding alkaline substances called bases. Most organisms prefer a medium that is close to neutrality, but as might be expected, some species require marked deviations from neutrality before thev find optimum growth conditions. All organisms have a maximum, an optimum, and a minimum chemical reaction with respect to growth of that species. We learn in chemistry that it is the concentration of dissociated or ionized hydrogen (H) or hydroxyl (OH) that determines the effective acidity or alkalinity of a solution. The theory of dis- sociation of electrolytes was formulated in 1887 bv the Swedish chemist, Svante Arrhenius (1859-1927), and measurement of the acid-base reaction of a medium in microbiology is determined by the HYDROGEN ION CONCENTRATION, abbreviated pH. Equal con- centrations of so-called weak acids and strong acids show marked o diflferences in true acidity, or sourness. This difference depends upon the ability of the acid to ionize, or dissociate. Strong acids dissociate into relatively large numbers of hydrogen ions, while weak acids do not ionize to such a degree. Pure water dissociates very slightly into equal numbers of hydrogen ( H ) ions and hydroxyl ( OH ) ions, and hence such water is neutral in reaction. At 22°C. the hydrogen ion concentration of pure water is 1 X 10"^ gram ions per liter. Hydrogen ion potential is expressed as the logarithm of the reciprocal of the hydrogen ion concentration, and this expression was given the symbol pH by S. P. L. Sorensen in 1909. In other words, pH can be determined bv the formula pH = Loe — - 66 Microbes and You Exact determinations have revealed that one hter of pure water (H2O) contains 0.000,000,000,000,01 (10-^^) gram of hydrogen and hydroxyl ions. The hydrogen and the hydroxyl ions are always equal in pure water. It follows, therefore, that the hydrogen ions are found in a concentration of 0.000,000,1 gram per liter. The logarithm of this fraction (0.000,000,1 gram) is minus 7. For con- venience we express this as a positive number and call it pH 7.0. Numbers less than 7.0 on the pH scale, therefore, represent greater acidities. Figures above pH 7.0 indicate lesser hvdrogen ion concentrations, but greater hydroxyl ion concentrations, or alkalinities. The normality of the hydrogen ions times the normality of hydroxyl ions is a constant number, called the dis- sociation constant. This number must always be approximately 10"^^, so if we know the concentration of hydrogen ions, we can determine the hydroxyl ion concentration by subtraction. Each whole number on the pH scale represents a ten-fold difference from the number above or below it. A pH of 6.0 would therefore have a hydrogen ion concentration ten times that of pH 7.0. We can express these facts about pH in tabular form. Table ] L HYDROGEN ION CONCENTRATION AND pH GRAMS OF HYDROGEN IONS NUMBER OF TIMES ACIDITY OR ALKA- PER LITER PB. LINITY EXCEEDS THAT OF PURE WATER 1.0 (10-0) 0.0 10 ,000,000 0.1 (10-1) 1.0 1 ,000,000 0.01 (10-2) 2.0 100,000 Acid 0.001 (10-3) 3.0 10,000 0.000,1 (10-^) 4.0 1,000 0.000,01 (10-s) 5.0 100 0.000,001 (10-«) 6.0 10 , , 0.000,000,1 (10--) 7.0 0 Neutral (pure H2O) 0.000,000,01 (10-«) 8.0 10 ' 0.000,000,001 (10-9) 9.0 100 0.000,000,000 ,1 (10-i«) 10.0 1,000 0.000,000,000 01 (10-11) 11.0 10,000 0.000,000,000 001 (10-12) 12.0 100,000 Alkaline 0.000,000,000 000,1 (10-13) 13.0 1 ,000,000 0.000,000,000 000,01 (10-14) 14.0 10 ,000,000 ., , Microbes Must Eat 67 Several rather simple technics have been devised for measuring pH in the laboratory. One of these methods, and the more accurate of the two we shall mention, depends upon electrometric devices, called potentiometers. The other technic, and the more commonly employed method, depends upon indicator dye solutions, usually weak organic acids or bases, which change color as the pH is altered. When attempting to determine the pH of colored solutions, or of solutions containing high concentrations of proteins which mav absorb dye from the indicator, the electrometric devices must be used, since the color changes of pH indicators may be masked under these conditions. However, for most pH determinations, the colorometric technics lend themselves well for use in bacteriological laboratories, in spite of the sacrifice of some accuracy. The series of color standards can readily be checked electrometrically before labeling the tubes with the determined pH. The effective range (the useful range) of many indicators commonly employed in pH determinations covers 1.6 points on the pH scale, but again we find exceptions to the rule, some cover- ing a greater spread on the scale. On page 68 is a partial list of some of the satisfactory indicator dyes, with their color changes and their pH ranges. Notice in Table 2 that thymol blue has both an acid range (1.2-2.8) and an alkaline range (8.0-9.6). This characteristic is true of a few pH indicators. The question may arise as to which indicator one should choose, if several dye ranges overlap on the pH scale. It depends upon the particular use to which the dye is to be put, but when at- tempting to adjust the pH of a medium, choose an indicator whose mid-point is close to the final desired pH of your medium. The degree of color change at the mid-point with each minor alteration in pH is more pronounced than are the color changes at the low end or at the high end of the indicator's effective range. One of the characteristics of many microorganisms is their ability to attack sugars or proteins present in a medium, and the resulting concentrations of acids or alkalies may be sufficient to kill the very cells that produced them. When attempting to harvest Microbes and You Table 2 INDICATORS AND THEIR pR RANGES EFFECTIVE pH COLOR ON LOWER COLOR ON UPPER NAME OF INDICATOR RANGE SIDE SIDE Thymol blue 1.2-2.8 Red Yellow Bromphenol blue 3.0-4.6 Yellow Blue Congo red 3.0-5.0 Blue Red Methyl orange 3.1-4.4 Orange red Yellow Broni cresol green 3.8-5.4 Yellow Blue Methyl red 4.4-6.0 Red Yellow Chlorophenol red 4.8-6.4 Yellow Red Litmus 4.5-8.3 Red Blue Brom cresol purple 5.2-6.8 Yellow Purple Brom thymol blue 6.0-7.6 Yellow Blue Phenol red 6.8-8.4 Yellow Red Cresol red 7.2-8.8 Yellow Red Thymol blue 8.0-9.6 Yellow Blue Cresolphthalein 8.2-9.8 Colorless Red Phenolphthalein 8.3-10.0 Colorless Red Alizarine yellow 10.0-12.0 Colorless Yellow LaMotte sulfo orange 11.0-12.6 Pale yellow Deep orange a maximum crop of organisms we must minimize pH changes in our medium by incorporating substances called buffers. Buffers may be defined as substances, which by their presence in solutions, increase the amount of acid or alkali that must be added to cause material change in pH of the solution. The word is derived from the German word Puffer (plug or bung), and the most efficient buffers are mixtures of weak acids or weak bases, in combination with their salts and certain other amphoteric substances. Ampho- teric substances are able to dissociate so that under one set of conditions they yield hydrogen ions, and under another set of conditions hydroxyl ions are liberated. Sometimes both types of ions are released simultaneously. Alkaline dissociation pre- dominates in an acid medium, and acidic dissociation of ampho- teric substances can be expected when the medium is alkaline. The hydrogen ion concentration at which this type of dissociation is at a minimum is called the isoelectric point of the amphoteric substances. Buffers do not stop pH changes, they merely retard Microbes Must Eat 69 rapid changes upon additions of sjnall amounts of acids or alkalies. To help visualize degrees of sourness as they correlate with pH, the following list of common substances is presented: Table 3 THE ;;H OF COMMON MATERIALS pa Hydrochloric acid 1.0 Human gastric contents 2.0 Ginger ale 3.0 Wines 3.0 Sour pickles 3.2 Tomatoes 4.2 Beans 5.5 Human saliva 7.0 Human blood plasma 7.4 Sea water 8.2 It has been mentioned that each organism has its own pH range which limits growth of that species, and Table 4 lists a few such organisms. Table 4 THE pH LIMITS FOR MICROBIAL GROWTH L Molds 1.4-9.0 2. Yeasts 2.5-9.0 3. Most bacteria 5.0-9.0 A. Escherichia coli, the common organism found in the intestines of all warm-blooded animals 4.8-10.0 B. Clostridium tetani, the etiological agent in lockjaw 5.5-8.3 C. Salmonella typhosa, the typhoid organism 5.6-8.5 PREREQUISITE IV: STERILITY Physical Methods Moist Heat Sterilization. Sterility means one thing to the layman and something quite different to a microbiologist. So often we hear people say that they have sterilized the baby's bottle by boiling it for ten minutes. In some cases this treatment may very well sterilize the bottle, but because of spores, those resistant bodies so useful in helping some organisms withstand periods of unfavorable environment, we cannot always be sure that mere 70 Microbes and You boiling kills all microorganisms. It is not meant to imply that boiling a baby's bottle for ten minutes is not a safe procedure. Quite to the contrary. This common practice is based upon sound scientific principles. Pathogenic bacteria are not normally able to withstand such heat treatment, and the spore-formers which might survive are not likely to cause any upset in the infant who drinks milk stored under proper refrigeration in such a boiled bottle. To a microbiologist sterility means the killing, or the removal, of all living cells, whether they be plant or animal, microbe or whale! The living protoplasm of bacterial cells is composed of protein distributed in a well balanced state known as a colloid. Matter is said to be in a coLLomAL state when it is dispersed permanently and so finely that the individual particles cannot be seen with the ordinary microscope, even though the particles may be larger than molecules. Anything that we can do to tip this colloidal protoplasm out of balance is going to adversely affect the organism, and a severe unbalance will result in the eventual death of the cell. We can coagulate cell protein in much the same way that we con- geal an egg by heating it, and such disruption of the colloidal balance is enhanced in the presence of water, as will be evident from the figures in Table 5. Table 5 RELATIONSHIP OF MOISTURE CONTENT TO COAGULATION TEMPERATURE OF EGG ALBUMIN IN THIRTY MINUTES Egg albumin +50% water coagulates at 56° C. Egg albumin +25% water coagulates at 74-80° C. Egg albumin + 18% water coagulates at 80-90° C. Egg albumin + 6% water coagulates at 145° C. Egg albumin + NO water coagulates at 160-170° C. Boiling. Under certain conditions, in the absence of resistant spores, boiling will free liquids of living cells, but as previously mentioned, since one cannot be sure when spores are present, boiling should not be relied upon to insure bacteriological sterility. An interesting and important sidelight is the influence of elevation upon the boiling point. At sea level water boils in an open con- Microbes Must Eat 71 tainer at 100° C. (212° F. ), but as the elevation increases above sea level, the boiling point decreases. On the top of Pikes Peak in the Rocky Mountains of Colorado where the elevation reaches 14,109 feet above sea level, water boils in an open vessel at about 87° C. (187° F.). Persons residing in a locality that is appreciably Fig. 15. Ai-nold Sterilizer. (Manufactured by Wilmot Castle Com- pany. Will Corporation catalog 25101.) above sea level should remember that boiling occurs at such low temperatures that cooking periods often must be double that at sea level where boiling takes place at a hotter temperature. It is not the mere bubbling of water that determines effectiveness of heating, it is the temperature at which this boiling occurs. 72 Microbes and You Intermittent Heating (Arnold sterilization). This technic is advocated in the sterihzation of theraio-labile substances ( broken down by heat) which can withstand a temperature of 60 to 65° C, the point where most vegetative protein coagulates. The underly- ing principle of intermittent heating is to destroy the vegetative ( non-spore-containing ) cells present in the material to be sterilized, incubate the product at a temperature which will encourage germination of spores, heat once more to destroy the new crop of vegetative cells, re-incubate, etc., until the third heating has dis- posed of the last germinated spores. This method does have certain advantages, but few microbiological laboratories employ the technic extensively. Autoclaving. One of the most efficient and reliable methods of sterilizing liquids is the use of steam under pressure. The instru- ments employed in laboratories for accomplishing this are called AUTOCLAVES, and they resemble home pressure cookers. Water boils at 100° C. at atmospheric pressures of 760 millimeters of mercury, but if we build up the pressure in a sealed container, water can be kept from boiling until the temperature goes well above 100° C. Table 6 reviews the relationship of steam under pressure at sea level to temperatures attained. Table 6 CORRELATION OF STEAM PRESSURE WITH TEMPERATURE TEMPERATURE POUNDS OF STEAM PRESSURE DEGREES C. DEGREES F. 0 100.0 212.0 5 109 . 0 228.2 10 115.5 239.9 15 121.5 250.7 20 126.5 259.6 The usual rule of thumb for operating an autoclave is to subject the liquids being sterilized to 15 pounds of live steam pressure for at least 15 minutes. But as the volume of material to be sterilized increases, sufficient time must be added to allow the Microbes Must Eat 73 entire bulk to be in contact with 121° C. for at least the minimum 15-minute time period. In general, a liter flask of broth should be kept for 30 minutes under 15 pounds steam pressure, while lesser volumes require shorter sterilization times. Remember, it is not the pressure that kills the organisms, it is the temperature created Flow Rcgulato Atmosphere Sofe»y Valve Chamber Gouge Ex'housf Valve Chamber Droin ShufOft Valve Fig. 16. Steam sterilizer (autoclave). (Courtesy of the American Sterilizer Compamj, Erie, Pennsylvania.) by raising steam pressure to 15 pounds above normal atmospheric pressure. To insure that the material in the autoclave is being subjected to live steam under pressure, not merely to a mixture of cold air and steam, rely more upon the reading of the temperature gauge than upon the pressure indicator. After securely fastening the door of the sterilizer, drive out the trapped cold air by allowing 74 Microbes and You steam to enter the chamber and to escape through the drain before closing all outlets. As soon as the temperature approaches 121° C, the pressure gauge, if it is functioning properly, should register close to 15 pounds. At the conclusion of the sterilization period, do not release the pressure quicklij or the liquids in the autoclave will "blow their tops" and spill the carefully prepared media. For best results, merely shut off the steam inlet valve and allow the pressure to decrease gradually. If the autoclave has no serious leaks in the gaskets or valves, this cooling-off period should reduce the pressure to zero in from 10 to 20 minutes after closing the steam valve. Tubes or flasks of liquids being autoclaved are commonly stoppered with plugs of non-absorbent cotton to allow ready access to live steam. Under no circumstances should absorbent cotton be substituted, since once the cotton becomes soaked with moisture, it loses its capacity to retard the entrance of contaminating organisms into the sterile fluids. The importance of the introduction by Schroder and von Dusch of cotton plugs to microbiology cannot be overemphasized. Dry Heat Sterilization. Indirect Heat. Most of the glass- ware, such as pipettes and petri dishes, used in microbiological laboratories must be dry after sterilization, and this precludes the wet autoclave as a means of sterilizing much of this equipment. However, a common baking oven, heated with gas, kerosene, or electricity, serves as a useful instrument for dry heat sterilization. In addition to glassware, all oils and greases should be subjected to drv oven treatment to sterilize them. A number of hospitals still mistakenly "sterilize" such things as mineral oil and vaseline gauze in the autoclave, forgetting the old adage that oil and water do not mix. Onlv the outer exposed portion of such greasy sub- stances which come in intimate contact with the live steam are being subjected to moist heat at 121° C. The rest of the material is practically water-free, and 121° C. dry heat is not sufficient to sterilize in the usual 15-minute contact period. The chart in Table 5 indicates that protein in the absence of moisture does not coagu- late until the temperature approaches 160° C. Therefore, when Microbes Must Eat 75 employing oven heat in sterilization, the temperature is raised to 170° C. (a 10-degree temperature margin) and is maintained for at least one hour, with two hours' contact being more common in many laboratories. Open Flame Sterilization. In transferring growths from one container to another, the usual practice is to handle these organ- isms by means of sterile wires or wires fashioned into loops. The most convenient method of sterilizing these needles and loops is to plunge them into the direct flame of a bunsen burner or an alcohol lamp. Incineration insures sterility. Surgical instruments cannot, however, be subjected to such rough treatment. Filtration as a Means of Sterilization. Some liquids cannot be sterilized by heat because the elevated temperature will coagu- late the material or will adversely affect the chemical structure of the compound. Various types of filters have been developed to overcome the objections to heat treatment, and today we employ filters to sterilize blood sera, to separate exotoxins from their parent cells, to separate viruses from bacteria and other organisms, to aid in the isolation of enzymes, and to free thermo-labile liquids from contaminating organisms. It is not the mere physical removal of organisms by the minute pores in the filters that accomplishes sterilization; adsorption phenomena associated with electrical charges are also instrumental in this filtering process. Liquids may be drawn through filters at a rate faster than gravity would normally allow by applying positive pressure as the liquid is introduced into the filter, but a more common practice is to provide negative pressure (suction) to the receiving flask. Too much positive or negative pressure, however, will create an undesirable differential which may nullify the surface action of the filters by drawing the bacteria away from the sides of the pore spaces where thev have been trapped. A differential of 150 to 200 millimeters of mercury is usually recommended to speed up filtration without sacrificing dependability. We shall mention but four of the commonlv- employed filters. Those readers interested in more complete discussions of the subject are referred to advanced textbooks in the field. 76 Microbes and You Chamberland Candles. These are composed of mixtures of silicon and kaolin, and are unglazed porcelain filters shaped like a hollow candle open at one end. The candles can be sterilized in the autoclave, attached to suction flasks, and the liquid to be freed of organisms can be introduced into the open end of the candle Fig. 17. Some of the common filters used by bacteriologists. (A) Seitz filter designed to operate with external pressure; (B) Small Seitz filter, suction model; (C) and (D) Berkefeld filter candles mounted in glass mantles. (From Textbook of Bacteriology, E. O. Jordan, and W. Burroivs, 14th ed. Copyright 1945, W. B. Saunders Company, Philadelphia.) and allowed to pass through the filter with the aid of negative pressure applied to the side arm of the receiving flask. Bacteria normally carry a negative charge. Chamberland candles possess a positive charge. Great care must be exercised to insure that the candles are not cracked. A minute crevice in the filter can allow organisms to squeeze into the filtrate. Microbes Must Eat 77 Berkefeld Filters. These filters of varying porosities are com- posed of diatomaceous earth which is obtained from the siUca-hke skeletons of marine or fresh-water algae. These are coarse Berke- feld V (German viel), medium N (German normal), and fine Berkefeld W (German wenig) filters, but the effectiveness of any of these devices may be materially impaired unless the filters are kept scrupulously clean. The V filter may allow some of the smaller bacteria to pass through, but it serves a useful purpose as the first step in filtering masses of organisms from fluids when centrifugation is not convenient. The N filters usually remove 100% of the suspended bacteria, and the W filter, having a fine porosity, can usually be relied upon to free fluids of all cells. It is a wise precaution, however, to run sterility checks on all filtrates whenever freedom from microbes is essential. If the pores become coated with grease, the adsorptive power of the pore walls may be neutralized, and organisms may slip through the filter. Stuart Mudd has calculated that Chamberland and Berkefeld filters that are "tight" to bacteria have mean pore space sizes in the range of 3 to 4 microns. Cleaning of filters can sometimes be accomplished by merely passing a continuous stream of clear water through them for several hours. Heating in ovens is usually discouraged because the ex- treme temperature changes may cause the filter to crack, often inside where the eye cannot readily detect the fissure. A suggested cleaning technic involves passing a five-tenths per cent solution of potassium permanganate through the filter, followed by a 5% sodium bisulfite solution before allowing streams of clear water to wash out o all traces of these cleansing agents. Seitz Pads. Discussion of these filters can be dismissed by saying that they are made by compressing shredded asbestos into pads which can be inserted into special chambers, sterilized in the autoclave, attached to side-arm flasks, and used to filter liquids with the aid of negative pressure to speed up the process. Sintered Glass Filters. Germany led in the production of these devices made from Jena glass, but American pyrex glass is now being manufactured into excellent sintered glass filters. This spe- 78 Microbes and You cial glass is ground up very fine, placed in molds, and heated just to the point where the glass particles adhere to each other, leaving pore spaces through which liquids may pass. The fineness of the filters is directly correlated with the size of the glass particles Fig. 18. Sintered glass filter. Corning, New York.) (Courtesy of Corning Glass Works, employed in their manufacture. Cleaning such sintered glass is accomplished by passing sulfuric acid containing 1% sodium nitrate through the filter. Bichromate cleaning fluid should be avoided since it is adsorbed on the glass and may adversely affect future filtrates. Microbes Must Eat 79 In brief, we autoclave all liquids except those which are thermo- labile. Such fluids are usually filtered or sterilized by inter- mittent selective heating over a period of days. All glassware that must be dry after sterilization and all oils and greases should be oven-sterilized. Glassware that need not be dry before use may be autoclaved, which is a shorter process than the oven treatment. Chemical Methods of Sterilization Chemical sterilization of bacteriological media is impractical. Any chemical used to free a medium of organisms will nullify the usefulness of that substrate for the subsequent cultivation of cells. Selected chemicals are being employed for the sterilization of surgical instruments which cannot undergo the rigors of heat treat- ment, but the choice of chemical and the length of the contact period are prime considerations. In a later chapter the use of chemicals as antiseptics and as disinfectants will be discussed, pointing out the limitations as well at the strong points in favor of representative chemical agents. PREREQUISITE V: DESIRED PHYSICAL PROPERTIES Every medium blended in the laboratory has a definite use, and the physical properties of that medium should help to fulfill specific requirements. In studying motility of organisms the culture should be grown in a liquid medium which encourages flagella to develop. Organisms picked from a colony growing on a solid medium cannot be expected to exhibit active motility, if they show locomotion at all. In nutritional studies where development of turbidity is to be the criterion of growth, it is imperative that the culture medium be as clear as possible before inoculation with the test organisms. If characteristics of colonies are to be studied, our microbes must be cultivated on a medium containing some solidifying agent. In brief, a liquid medium, a clear medium, or a solid medium is pro- vided depending upon the ultimate use to which the substrate is to be put. The effect of such factors as surface tension, concentration of ingredients, etc., will be discussed in a later chapter. 80 Microbes and You GELATIN VS. AGAR Of all the solidifying agents employed in microbiology, agar is by far the most common. Robert Koch introduced gelatin as one of the first substances, but because of undesirable characteristics of this solidifying agent, it was replaced by agar. A brief com- parison of gelatin and agar is presented in Table 7. Table 7 CHARACTERISTICS OF GELATIN AND AGAR 1. Food value. 2. Chemical composition. 3. Melting point. 4. Solidifying point. 5. Other properties. GELATIN May be used by some bacteria. An incomplete protein. About 25° C. About 23° C. Forms no water of con- densation. Clear in appearance. AGAR No food value. A hemicellulose. About 90° C. About 40° C. Produces water of con- densation. Clear when melted, but slightly opalescent upon solidification. Agar is a complex sugar, a POLYSACCHAmDE, that for all practical purposes is not attacked by organisms in their quest for food, but gelatin is not very resistant to attack by microbial enzymes. If gelatin is being employed as the solidifying agent in the attempted isolation of bacterial colonies, and if the organisms are capable of liquefying the gelatin through enzymatic activity, much of the value of the gelatin has been sacrificed. It should be emphasized, however, that nutrient gelatin serves as a useful medium in the study of physiological reactions as part of the procedure in the identification of organisms, since some organisms can attack gelatin while others cannot. In the isolation, study, and identification of organisms derived from warm-blooded animals, it is oftentimes imperative that the cultures be incubated at body temperature or above. Since gelatin becomes liquid without anv physiological activity when the temper- ature approaches 25° C, it is obvious that this solidifying agent Microbes Must Eat 81 cannot be used in such a medium. Some bacteria, includinsf those which Hve and thrive in such locations as hot springs, cannot be cultivated unless the optimum temperature for the growth is pro- vided. This may mean incubation of the cultures at 50° C. or higher. With agar as a base, the medium can be relied upon to remain solid even at these elevated temperatures, since agar normally does not melt until the temperature approaches 90° C. Gelatin has a narrow differential between the melting point and the solidifying point, about 2 degrees, while agar has a 50-degree spread, which is an advantage under some circumstances. When incubating cultures in special plates called fetri dishes, such plates must be inverted if agar is the solidifying agent. The water is not bound the way it is in gelatin media, and the heat of the incubator allows moisture to escape from agar media and to condense on the lid of the petri dish. When the drops of con- densed moisture become large enough, they can drop onto the agar surface and link up the growths from well-isolated colonies, nullifying the otherwise effective isolation procedure. Gelatin does not produce water of condensation, and hence it does not require incubation in an inverted position. In fact, gelatin plates should be kept upright in the incubator because of the liquefying ability of some organisms. The slight opalescence imparted to solidified agar does not interfere with its usefulness in distinguishing col- onies of organisms growing either in or on the medium, while the clarity of gelatin is completelv overshadowed by the serious dis- advantages previouslv discussed. CLASSIFICATION OF MEDIA We can conveniently arrange the types of media under four major headings: I. Natural Media— Substances occurring in nature. 1. Milk. 2 Eo-cTs 3. Blood and other body fluids and tissues. 4. Extracts of plant and animal tissues. 82 Microbes and You II. Derived Media— Comprised of known substances but the exact chemical composition of which is not known. 1. Nutrient broth. 2. Nutrient agar. 3. Nutrient gelatin. III. Synthetic Media— The exact chemical composition is known. IV. Special Media— Combinations of the other three types of media. PREPARATION OF STANDARD NUTRIENT AGAR There are literally hundreds of different combinations of in- gredients comprising the workable list of media employed by micro- biologists. Trying to remember the formula for very many media is generally a waste of a person's time. But it doesn't seem too much to ask students, even in an elementary course, to remember the constituents of the most commonly used medium, Standard Nutrient Agar. During a single semester each student may use a hundred or more tubes of media. In many bacteriology courses one of the early laboratory exercises is devoted to having students prepare a batch of standard agar. Such an exercise should ac- complish two objectives. First, the very fact that the operation involves "doing" will help the student to remember the constituents and the technic for blending them much better than merely hearing the steps presented in a lecture. But even more important, per- haps, the student should be impressed with the amount of time, effort, and expense involved in the preparation of even one tube of culture medium. STANDARD NUTRIENT AGAR GRAMS /LITER Bacteriological peptone (0.5%) 5.0 Beef extract (0.3%) 3.0 Agar (1.5%)* 15.0 Distilled water Up to 1 liter (1000 ml.) Final pH of medium- -6 8 to 7.0 *A firmer medium can be obtained by adding a higher concentration of agar -usually up to two per cent. Microbes Must Eat 83 The peptone and the beef extract are heated in the distilled water just sufficiently to dissolve them. After determining the pH of the broth, either electrometrically or colorimetrically, 10% sodium hydroxide (NaOH) is added to raise the pH to the proper level (about neutrality), before the carefully-weighed agar is intro- duced. The percentage of agar added to the broth varies from lVo% to 27c, depending upon the hardness desired in the finished medium. Several minutes of a rolling boil (avoid burning or boil- ing over) will be required to dissolve the agar. The hot medium is transferred to flasks or to tubes, depending upon the ultimate use of the medium, and each container is plugged with non-ab- sorbent cotton. After autoclaving^ to sterilize the nutrient asjar, the tubes or flasks are kept in the refrigerator until readv for use. If the medium is to be used within a few days, storage at room temperature is satisfactory, but refrigeration will materially reduce dehydration during prolonged storage. The refinements of blending the ingredients, adjusting the pH, plugging the tubes, and autoclaving the finished product are covered more fully in laboratory manuals, and hence will not be discussed in detail here. From these observations on the preparation of a suitable medium, it should become evident that feeding microbes is not a simple matter of throwing them a bone, the way one might satisfy the desires of a hungry canine blessed with sharp incisors. Run- ning a restaurant for microorganisms is a scientific endeavor based upon table service rather than cafeteria style of mass feeding. In devising a menu for microbes, the microbiologist attempts to pro- vide an adequate supply of available food, served in the same stvle to which the organisms are accustomed in nature where thev are eking out their own livelihood in a highly competitive environment. CHAPTER 5 Microbial Structures and Staining Reactions REPRODUCTION OF BACTERIA SHAPE THE METRIC SYSTEM ARRANGEMENT SIZE WEIGHT COLLOIDAL NATURE OF PROTOPLASM INDIVIDUAL STRUCTURES OF BACTERIA STAINING OF MICROBES To think in terms of living things as minute as bacteria is more difficult for some individuals than it is for others, and becoming familiar with the concept of a world of microscopic plants and animals requires orientation. A few students never make this transition and their laboratory technic is a direct reflection of this maladjustment. When the instructions call for inoculating a medium with a few bacteria, the idea of transferring a barely visible amount of microbial growth cannot be fathomed by persons whose world is entirely populated with humans, horses, elephants, the neighbor's dog, etc. REPRODUCTION OF BACTERIA Higher plants and animals possess specialized organs for carry- ing on the process of reproduction, just as they are endowed with 84 Microbial Structures and Staining Reactions 85 structures particularly adapted for breathing, digestion, and excre- tion. Bacteria, however, must metabolize and reproduce within the confines of a single microscopic cell. It appears that reproduc- tion in living things as small as bacteria cannot be too involved, and when compared with multicellular organisms, bacteria do em- ploy a relatively simple reproductive process. At a given signal, best comprehended by bacteria, the single cells split in half across their short axis ( at right angles to the long axis ) , in a process called BINARY FISSION. It is this simple equal division which gives bacteria their class designation of Schizomycetes (schizo, split; mijket, fungus). Strictly speaking, growth means enlargement or increase in cell substance, but in bacteriology the terms "growth" and "re- production" are sometimes loosely used as synonyms. Just prior to cell division the protoplasm appears to gather at opposite ends of the organism. A cell wall puts in its appearance across the middle of the cell, with or without a visible constriction, and separation into two smaller cells takes place. Each offspring tlius retains a part of the parent cell in this asexual tvpe of repro- duction. Recent evidence, particularly by geneticists, leads us to believe that sexuality in bacteria has been demonstrated. This is not meant to imply that "female" and "male" bacteria exist in the same form that we think of females and males in higher planes of life. But it has been shown that when different strains of a given species of bacteria are grown together in a test tube (in vitro), selected isolations from these mixtures can be demonstrated to have nutritional requirements different from those exhibited by either of the two original strains. Some tvpe of conjugation has ap- parently occurred, and this type of combination stronglv suggests sexual reproduction in contrast to simple binary fission. When a bacterial cell divides, the two small offspring waste little time growing to a predetermined maximum size. These two new cells are then ready to divide, and under ideal conditions binary fission can take place on an average of once every 20 or 30 minutes for many species. Generation times longer than this are not un- ccmmon, however, among some of the slower-growing bacteria. Fortunately for mankind this multiph'cation of microorganisms does S6 Microbes and You not continue indefinitely at this alarming rate. A few examples of what could conceivably occur should microbial growth go un- hampered at this optimum rate might be of general interest. A sphere that normally grows in a chain formation (called a strepto- coccus ) in forty-eight hours would extend over a mile and a quarter in length, according to one calculation. When you consider that the progeny of a single microbe mav amount to over 300,000,000,000 in just 24 hours, Lohnis and Fred have figured that in thirty-six hours the bulk of organisms would fill 200 trucks of five ton capacity each. After a full week at this rate of multiplication the microbial volume would exceed that of the world itself! These are rather revealing theoretical numbers, but whv are they prevented from becoming reality? Unless food is available in the immediate vicinity of the microbes, multiplication of the organisms is prevented. This food shortage when coupled with the accumulation of waste products by the growing bacteria and the natural antagonisms existing between microorganisms in their fight for survival explains in large measure the inability of organisms to multiply indefinitely at the optimum rate of speed. THE METRIC SYSTEM Because of the minuteness of bacteria, the use of decimals to express fractions of an inch the\' represent would be too cumber- some, so we speak of their size in terms of microns ( designated by the Greek lower case letter mu, abbreviated /x). It is to be hoped that some day the Congress of the United States will take productive steps to replace the antiquated system of weights and measures we adhere to so tenaciously with the more logical metric system. It seems odd indeed, to a scientist, that a progressive country like ours has allowed itself to be saddled with such an outmoded system, but it has become so firmly entrenched in our way of life that get- ting in step with a great segment of the rest of the world's popula- tion will not be an easy matter. From time to time feeble attempts have been made to initiate steps to have America convert to the metric system, but progress on a national scale can be reported as nil. Education is admittedly a slow process. Scientific publica^ Microbial Structures and Staining Reactions 87 tions, however, have led the way by expressing weights and mea- sures in that metric language understandable to fellow scientists throughout the world. Since this is the first contact some students will have with the formal field of science, they would do well to gain a mental picture of the metric equivalents of some of our weights and measures. A review for other individuals might be profitable. A cubic centi- meter (cc), also commonly referred to as a milliliter (ml), should come to mean about twenty small drops, just as a liter (1000 ml) should be visualized as a volume slightly greater than a quart. An object described as being ten millimeters (mm) long should be thought of as about two-fifths of an inch in length. Beginners in science are cautioned not to confuse millimeters (mm) with milliliters (ml). The former is a linear measurement (length), while the latter term refers to a volumetric deteraiination. It is unnecessary to outline the complete metric svstem in a book of this type, but the mention of a few of the more commonly em- ployed terms seems justified. Prefix Mecming deci- tenth centi- hundredth milli- thousandth micro- millionth kilo- thousand mega- million Length: One United States yard is ''' ^' *^ % 9 ;{ 7 meter. Or to express this in another way, 1 meter equals 39.37 inches. 1 meter =10 decimeters 1 decimeter =10 centimeters 1 centimeter = 10 millimeters 1 millimeter = 1000 microns 1 micron = approximately 1/25,000 of an inch. In measuring light waves we employ the Angstrom unit, which is 1/10,000 of a micron, or 1/10,000,000 of a millimeter. Ordinary light-type microscopes cannot distinguish objects smaller than 88 Microbes and You about 0.1 micron in size. As an object for comparison, a human red blood cell measures 7.5 microns in diameter. Area: 1 square meter =100 square decimeters 1 square decimeter =100 square centimeters 1 square centimeter = 100 square millimeters 1 square millimeter = 1,000,000 square microns Capacity: One liter is defined as the volume of pure water at 4° C. (point of maximum density) and 760 millimeters pressure which weighs one kilogram ( 1000 grams ) . 1 liter = 1000 milliliters or 1000 cubic centimeters 1 liter = 33.8 fluid ounces (32 ounces in one quart) 1 cubic centimeter = 1000 cubic millimeters Mass: 1 gram =10 decigrams 1 decigram =10 centigrams 1 centigram = 10 milligrams 1 gram = 1000 milligrams SIZE While the exact size of bacteria does vary from one species to another, in general these organisms lie within the range of 1 to 5 microns. Variations in size within this group of microorganisms, when they do occur, tend to be reflected in the length rather than in the width of the bacteria. One of the smallest microbes classified as a bacterium is found in the nasopharynx of certain persons in the early stages of influenza, and it has the scientific name Dialister pneumonsintes (from the Greek pneumon, lung, and sintor, murderer or devastator). This tiny organism measures only 0.15 fx by 0.3 jx, and it is pathogenic for rabbits and guinea pigs. On the other end of the scale may be cited Bacillus hutschlii, originally isolated from the cockroach, Periplaneta orientalis. This bacillus measures 3.0 /x to 6.0 fx in width and up to 80 ix in length. Most cocci fall within the range Microbial Structures and Staining Reactions 89 of 0.5 fA, to l.O iJL in diameter, while most spirals are larger than either the rods or the cocci. Some spirals have been reported to be as long as 500 fx, but the general length is under 16 /x. The syphilis germ, Treponema pallidum, measures 0.3 /x in width and from 6 to 14 fx in length. The spirochete associated with trench mouth, BorreJia vincentii, is very similar in size to the syphilis organism, but the nature of the spiral twists in the two organisms readily differentiates them. SHAPE Although variations in morphology (size, shape, and structure) of bacteria may be induced by altering the environment in which the organisms are growing, in general only three principal shapes of bacteria exist: spheres, called cocci, (singular coccus); rods, called bacilli ( singular bacillus ) ; and twisted rods, called spirilla ( singular spirillum). It might be well to briefly mention the Latin endings used to designate singular or plural nouns in scientific terminology. Many Latin nouns ending in -us form the plural by converting the word ending to -i (as bacillus to bacilli). Many words ending in -a become plural by changing to -ae (such as sarcina to sarcinae). Other nouns ending in -um form the plural by changing the ending to -a. Occasionally persons prefer the word mediums for the more correct plural media, and these liberties with Latin may be adopted in time and become part of the accepted terminology. ARRANGEMENT After binary fission has taken place, many bacteria separate from their twins and go about their carefree way, but with some species the tendency to remain attached after division has occurred results in an arrangement of the cells which is a characteristic aiding in the identification of the species. To best determine this typical arrangement, cultures should be cultivated in liquid media for from 18 to 24 hours prior to being examined in a so-called hanging drop preparation under the high dry objective of the compound micro- scope. Thus a living culture can be studied and typical arrange- ment for the species can be determined. 90 Microbes and You Since multiplication of true bacteria occurs at right angles to the long axis (although there is evidence that at least some spirilla multiply by longitudinal division), it should become apparent that if complete separation of cells does not materialize after fission, the possible arrangement of the cells will depend upon the original shape of the organism. Since cocci possess no long axis, division can take place at any angle with the result that the following arrangements can and do exist: SPHERES Coccus — Individual spheres (Coccus comes from the Greek kokkos, meaning berry.) Diplococcus This is formed by simple division of a sphere into two cocci which remain attached. Such an arrangement is relatively common and includes such organisms as the pneumococcus (Diplococcus pneumoniae), the gonococcus {Neisseria gonorrheae), and the meningococcus (Neisseria intracellularis) . While all three of these examples happen to be pathogens, it is not the intention to imply that all diplococci are disease-producers. Streptococcus Continued division of a diplococcus in the same plane forms a chain of spheres resembling a strand of beads. The organism (Streptococcus lactis) which plays such an important role in the souring of milk and dairy products is an example of this beaded arrangement. Streptococcus pyogenes, which is the etiological agent in a number of human and possibly animal infections, is another example of chain formation. Tetrads When a diplococcus divides in such a manner as to form a group of four spheres in the same plane, rather than in a straight line, we Microbial Structures and Staining Reactions 91 call the resulting formation a tetrad. Micrococcus tetragemis is a common skin contaminant. Sarcina If a tetrad divides to form four spheres back to back with four other spheres, this packet of eight cocci is a sarcina. An organism commonly found floating on dust particles in the atmosphere is Sarcina Jiitea. The specific name lutea is derived from the yellow color this organism produces when grown on a solid medium under aerobic conditions. Staphylococcus Haphazard multiplication in all planes resulting in masses of cocci in grape-like clusters gives us an arrangement called a staphylococcus. Representative members of this group include Micrococcus pyogenes variety alhus (formerly called Staphylo- coccus albus), a pus-producing inhabitant of normal skin, and its golden-colored cousin Micrococcus pyogenes var. aureus, the common cause of pimples, boils, carbuncles, osteomyelitis (a bone disease), and blood poisoning. Both the albus and the aureus varieties of this organism may be implicated in cases of food poison- ing, but the golden variety is more often the causative agent. Many workers in the field of microbiology, probably from force of habit, prefer the term Staphylococcus food poisoning to Micro- coccus food poisoning. The typical arrangement of mature cultures of bacteria is characteristic of the species and aids in the identification process. While it is true that a single sphere placed in a liquid medium must first pass through the diplococcus stage before it can become a tetrad, a streptococcus, a sarcina, or a staphylococcus, if that single sphere was destined to be a staphylococcus, it will eventually mature and have that arrangement. If a coccus is destined to be a diplococcus, it will stop at the diplo stage. However, a diplo- coccus may temporarily appear as a tetrad just at the time that it is dividing into another diplococcus, but the characteristic arrange- 92 Microbes and You ment of the spheres in pairs will eventually predominate in the mature culture. RODS In contrast to the cocci, rod-shaped bacteria can only divide one way— at right angles to the long axis, and this limits the number of possible arrangements such rods can have. Diplobacillus If the single rod divides and the two newly formed rods adhere to one another, the result is a diplobacillus. Streptobacillus Should the diplobacillus in turn divide and the four rods form a chain, we speak of this arrangement as a streptobacillus. SPIRALS Spirals do not ordinarily remain in close proximity after the signal for them to divide has been relayed to the proper trigger mechanism. It is relatively uncommon, therefore, to see spirals in any arrangement other than singles and occasionally pairs. The looseness or the tightness of the twists in a spiral are also of some diagnostic significance. The tight cork-screw appearance of a living syphilis germ (Treponema pallidum) as it spins its way through the fluid obtained from a suspected lesion being examined under the darkfield microscope is so characteristic that a trained observer can confirm a clinical diagnosis of this terrible disease. In trench mouth the appearance of long, loose spirals found in association with fusiform bacteria (rods pointed at each end) in a stained smear made from material swabbed from the gum line of the teeth is a laboratory confirmation of a clinical diagnosis of this troublesome affliction. THE WEIGHT OF BACTERIA A curious individual might be tempted to speculate as to the weight of a single bacterium. To dismiss the inquiry by stating that they don't weigh very much is avoiding the issue. People Microbial Structures and Staining Reactions 93 have faced this problem and have arrived at definite answers con- firmed by other workers applying similar or different technics. It is true that when you compare a mouse to an elephant, the mouse really isn't very big. But to an inquisitive bacterial cell, a mouse is a monstrous thing, while a virus undoubtedly looks extremely tiny to this same bacterial cell. Everything is relative. Bearing in mind that weight depends upon size and density, it has been calculated that it would take five billion bacteria to weigh one milligram. To express this in another way, a single organism weighs about 0.000,000,000,000,2 gram. A moderate sized drop of milk can easily harbor a number of bacteria ex- ceeding the entire human population of New York City, and there would still be plenty of room to spare in that drop of milk. A farmer, when informed that the milk he had shipped to the creamery a few days previously had a bacterial count exceeding one million, struck his forehead in amazement and remarked, "I wonder how I ever managed to get the lid on the can!" His surprise would have been still greater had he been told that every twenty drops of milk in that forty quart can contained over a million bacteria. It is true that bacteria don't weigh much, nor do they occupy much space, if we are thinking purely in terms of macroscopic rather than of microscopic objects. COLLOIDAL NATURE OF PROTOPLASM Protoplasm (Greek protos, meaning first, and plasma, meaning formed substance) is the basis of all life, and it is still a biological mystery. Even though scientists have compounded materials ap- parently identical with protoplasm, the synthetic material lacks the spark of life. Hugo von Mohl in 1846 originated the word protoplasm to describe that colorless or gray, translucent, semi- fluid, colloidal substance found in all cells. As our knowledge of cells increased, other words were introduced to describe definite structures within the protoplasm. The term nucleoplasm (some- times called karyoplasm) was applied to the material within the nucleus, and cytoplasai for the material surrounding the nucleus. Protoplasm has certain characteristics in general, but specific 94 Microbes and You cells have their own chemical configuration. Protoplasm has been defined as a system of chemical compounds held together in a colloidal suspension and containing carbon, hydrogen, oxygen and nitrogen, among other elements, in the form of proteins, carbo- hydrates and fats. The English physicist, Thomas Graham, intro- duced the word colloid in 1861 to describe substances that will not diffuse through a membrane. Those substances which can diffuse are called crystalloids. The minute colloid particles are suspended in a liquid, and there is a constant interplay and exchange of various atoms, molecules, electrical forces, physical and chemical stresses and attractions— all in a delicately balanced state of equi- librium. Any abnormal alteration of any of these forces will affect the functioning of the cell and may result in its death. Microbiologists employ this knowledge in their attempts to create as favorable conditions for growth as possible, and also in their attempts to destroy microbes by chemical and physical forces when destruction is desirable. The properties of protoplasm are those which distinguish living from dead substance: movement, irritability, reproduction, metab- olism, and death. The latter term, death, is the final distinguish- ing characteristic of protoplasm, in a given stage, but to define death is not easy. Just to say that it is cessation of life is hardly satisfactory, because a definition of life is equally difficult. We do know that the colloidal structure of protoplasm is markedly altered when death steps in, but other than that, our knowledge is fragmentary. INDIVIDUAL STRUCTURES OF RACTERIA CELL WALL AND CELL MEMBRANE The cell walls of plants are more rigid than the walls normally found on animal cells. This membrane varies in its permeability between organisms, and the permeability within a given species can be materially altered by application of chemical or physical forces. Most evidence points to the conclusion that the wall functions principally as a protective device for the underlying structures and gives the cell its shape. If it were not fairly rigid. Microbial Structures and Staining Reactions 95 bacteria would all tend to assume a spherical shape. When it is said that the wall is firm, it should also be pointed out that it is somewhat elastic. Ordinary staining usually fails to distinguish the wall from the rest of the cell unless the organism has been grown in a concentrated solution which causes the cytoplasm to shrink away from the outer wall, leaving a ghost-like shell. Schematic drawing of a hypothetical bacterial cell. Cellulose, hemicellulose, or mucin (nitrogen-containing com- pounds) are the common constituents of bacterial cell walls. The cell wall proper is probably not as discriminatory as is the underlying cell membrane, which limits the cytoplasm of the organism. Because of its selectivity we refer to this membrane as being semi-permeable in nature. Theories have been proposed to explain this selectivity, and these explanations vary from a simple mechanical sieve theory to complex physico-chemical re- 96 Microbes and You actions. There is little doubt that a number of forces are active in the process, and to try to pinpoint a single explanation is prob- ably not feasible. NUCLEUS A typical cell contains a nucleus, usually rather spherical in shape, enclosed within a thin membrane, and surrounded by ma- terial called CYTOPLASM. But bacteria, just to be different, do not usually possess a well-defined nucleus such as we observe in most other cells. Among other things nuclear material consists of CHROMATIN, a substaucc believed to be vital to all living cells. The name chromatin is derived from the strong affinity it has for certain coal-tar dyes, and the nucleus of cells stains more deeply than other parts of the protoplasm. Some investigators go so far as to claim that the primitive nature of bacteria makes a well-defined nucleus unnecessary, but others hold the opposite view, and they claim that a bacterial cell is extremely complex. They feel that it has to be, in order to cope with all of the life processes within the borders of a single cell. The nucleus is the heart of the cell— the control panel of a complex mechanism. A nucleus is usually considered to be the determiner of heredi- tary characteristics, and to some persons the nucleus is a single chromosome which must undergo division before new cells can be formed. If each offspring is to resemble the parent cell, there must be some mechanism for the transmission of these hereditary characteristics, usually a function of chromatin material. When we subject bacteria to nuclear dyes, the entire cell be- comes stained, suggesting that the nuclear material may be diffuse. There is some evidence to support the concept that at certain stages in the growth of bacteria the nuclear material may undergo a local concentration. Common beliefs relative to the bacterial nucleus include the following: 1. The entire bacterial cell is composed of nuclear material, with little or no cytoplasm. Microbial Structures and Staining Reactions 97 2. The entire cell is composed of cytoplasm with no nucleus. 3. There is a definite nucleus in bacterial cells but the usual stain- ing technics do not reveal a nucleus except when the cells are grown under specific conditions, and stains are made at a precise stage in their growth. 4. Chromatin granules are spread out in the cytoplasm and are abundant enough to give the impression that the entire cell is composed of chromatin material. This latter theory of a diffuse nucleus has considerable support in the bacteriology profession. Blue-green algae have such a nuclear structure, but because these bodies are larger than in bacteria, they are more readily observed under the microscope. Electron microscope studies have revealed distinct nuclear-like material in some bacterial species and undifferentiated nuclear matter in still other cells. By adhering to the structure definition of a nucleus, it must be admitted that bacteria probably have no nucleus. But if function is considered, all cells must have a regulatory body, and bacteria are probably no exception. From the functional point of view, the structure of the nucleus is of relatively little importance. CAPSULES AND SHEATHS Although it is not possible to demonstrate capsules on all bacteria, it may be a safe assumption that all bacteria possess a slime layer, sometimes of a thickness not detectable by the usual technics employed for their demonstration. There is evidence that capsules serve as a protective material for some organisms, by slowing down or preventing penetration of chemicals and body juices. Most capsular material is carbohydrate or carbohydrate- like in nature, but lipoid material predominates in a few selected species. The capsule may be a mere thickening of the outer membrane or more probably a secretion or excretion which deposits itself around the cell. Capsule formation can be enhanced by animal passage of certain organisms or by growing them in high carbohydrate media. Microbes and You W Fig. 20. Electrun micrograph of Diplocucciis pneumoniae. Type I. The capsules are swollen as a result of exposure to Type I antiserum. (From Mudd, S., Heinmets, F., and Anderson, T. F. The Journal of Experimental Medicine, 194S, 78, 327-332.) Capsules are of more than just passing interest to bacteriologists. They have played an important role in medicine particularly with respect to pneumococcus pneumonia. Before the advent of the sulfa drugs and the more recent antibiotics, it was necessary for a physician to know which of the many ( seventy-five or more ) types of pneumonia a patient was suffering from before antiserum could be administered. Type III pneumococcus infections were par- Microbial Structures and Staining Reactions 99 ticularly difficult to treat because their capsule is so much thicker than we find in other types of pneumococci. Treatment with the antibiotics is not dependent upon the specific type of capsular material; all pneumococci react about the same to these newer drut^s. Now that pneumococcus typing by a qualified technician Fig. 21. Ropy milk caused by the growth of Alcaligenes viscosiis. (From Microbiology, W. B. Sarles, W. C. Frazier, J. B. Wilson, and S. D. Knight. Copyright 1951, Harper and Brothers, New York.) is unnecessary, the time saved before specific treatment can be initiated can mean the difference in the outcome of the disease. Another interesting sidelight on capsules is their role in pro- ducing slimy, or ropy, milk. Some organisms (Alcaligenes viscosus ) commonly found in swampy areas, among other locations, are endowed with greatly enlarged capsules, and if these bacteria become established in a milk processing plant, they can cause the milk to become stringy. The relatively high resistance of these en- capsulated forms to the normal heat and chemical treatments em- 100 Microbes and You ployed by milk-processing plants makes them difficult to eliminate, and by the time the situation has been cleared up, many customers have found new sources of supply for their milk. Ropy bread will result when the flour from which it is made becomes contaminated with encapsulated organisms. Ropiness is due to a breakdown of starch by the organisms and the synthesis of gums from the resulting carbohydrates. Should these forms of bacteria become established in a sugar-refining plant, they are capable of interfering with normal crystallization of the sugar, a costly affair for a large plant. Constant vigilance and strict sani- tary measures must be the watch-word in food-processing plants. Many pathogenic bacteria lose their virulence when they are stripped of their slime layer, but Bacillus anthracis, the etiological agent in anthrax, becomes encapsulated after it gets into a susceptible animal, and it may eventuallv destrov the host. It is also possible for sheep to ingest spores of the anthrax bacilli, and these spores can germinate and become encapsulated in vivo with similar fatal results for the host. Certain bacteria, particularly the iron bacteria, are capable of secreting or excreting a substance called a sheath, which becomes quite firm in contrast to the slimy nature of representative capsules. Iron compounds may be deposited in these sheaths, and when their volume builds up, these bacteria are capable of completelv occlud- ing water pipes. The importance of transfusions with whole blood or with blood plasma in saving human life has been amply demonstrated during World War II and in the Korean conflict, as well as in civilian hospitals. With the source of supply of these vital fluids relatively limited, research has been undertaken to provide needed sub- stitutes. A substance called dextran has been found to be such a possibility. While dextran cannot be considered to be a complete substitute for either whole blood or plasma, its use as a so-called "extender" is proving valuable in restoring the balance so essential in the blood stream in cases of shock. Dextran is produced by the action of certain species of the bacterial genus Leuconostoc. These organisms are gram positive Microbial Structures and Staining Reactions 101 spheres occurring in pairs and in chains, and when they are cultivated in sucrose sokitions, the chains are surrounded by a thick, gelatinous, colorless membrane consisting essentially of dextran. The dextran is removed bv selective chemical action and is purified for use in human transfusions. GRANULES Ordinary staining technics commonly employed with bacteria reveal at times deeplv stained bodies called metachromatic granules within the cells. Definite agreement as to the origin and the func- tion of these bodies is still not available. Some workers feel that they are particles of reserve food material. Others call them waste products since the granules in some bacteria do not appear until the twilight of the microbe's life, and granules tend to disappear when active microbial multiplication is encouraged by subculturing in a new medium. Chemical analysis reveals these particles to be fat, carbohydrate, or complex nitrogenous compounds. Pronounced development of granules is a characteristic feature aiding in the identification of the etiological agent in diphtheria {Cori/nebac- terium diphtlieriae). The arrangement of granules at the ends (poles) in the plague organism (Pasteurella pestis) is in contrast to the bands and scattered dots seen in the diphtheria organisms. Sulfur and iron granules may be seen in some of the higher bacteria. SPORES When bacteria are gradually subjected to increasingly unfavor- able conditions, including lack of water, depletion of available food, and marked temperature deviations from the optimum, many organisms will die. Certain rod-shaped species classified under the family Bacillaceae are able to develop structures called endospores (spore within a cell), which can withstand relatively undesirable environmental conditions. On the other hand, few cocci or spirals exhibit spores. Bacteria lacking these structures are termed vege- tative CELLS. Bacterial spores might be considered to represent a resting stage similar, perhaps, to the hibernation of some higher animals and the encysted stage present among protozoa. Since 102 Microbes and You but one spore is formed by a single cell, there is reproduction without multiplication. This is in contrast to mold spores which are reproductive bodies, many of which are formed by a single mold plant. Frequent transplantation under ideal growth conditions may prevent spore-formation, and some strains so cultivated may lose their ability to sporulate even when conditions become unfavorable for the cell. The cause of sporulation is unknown, but it is not necessarily a response to marked unfavorable conditions, since some bacteria sporulate early in their lifetime when conditions are apparently still favorable for microbial growth and multiplication. After the danger has passed, the "possum-like" microbes emerge from their dormancy, or resting stage, and revert to their original vegetative state. The first visible change when spores are placed in a suitable medium is an enlargement of the spore, probably due to the absorption of water. After losing its refractive nature, the spore elongates. Some shed their "skin" as they germinate, while others appear to absorb the spore material. There is some evidence that the "shedding" type of spore can withstand more unfavorable environmental conditions than can the "absorbing" type. A given species of organism will exhibit a constant size, shape, and location of the spore (terminal, suhterminal, or central) within the cell, and in some cases this structure aids in the tentative identi- fication of an organism. Should the swelling occur in the center of the rod, it gives a spindle-shaped appearance, and if the bulge is located terminally, the cell takes on a drumstick shape. Not all spores are equally resistant to chemical and physical forces, but the most resistant spores are the basis for the time and temperature relationships employed in sterilization technics. Were there no spores, we could drastically revise our sterilizing tem- peratures downward. The common soil organism Bacillus stib- tilis has been found to withstand 100° C. dry heat for three hours, and Clostridium hotulinum, the causative a2;ent in a highly fatal type of food poisoning to be discussed in a later chapter, may withstand four or five hours of boiling. The spores of some Microbial Structures and Staining Reactions 103 THERMOPHILIC ( heat-loving ) bacteria are much more resistant to heat, whereas vegetative bacteria are killed by an exposure to a temperature of 65-80° C. moist heat for only a few minutes. Fortunately, few spore-formers are capable of causing disease in man. But the most important ones are probably Clostridium tetani, the cause of lockjaw, Clostridium perfringens, the etiological agent of gas gangrene, the previously mentioned Clostridium hotulinum, and Bacillus anthracis, the biological agent in anthrax. FLAGELLA Rapid locomotion by bacteria is not uncommon, and this move- ment is brought about principally through the influence of hair- like projections termed flagella ("little whips"). All spirals are motile, probably half of the rods are motile, and practically none of the cocci exhibit independent movement. The position and the number of flagella will vary between species, but constancy of arrangement of flagella is an aid in the identification of species. Since bacteria are single-celled organisms, we must consider the flagella to be a continuation of the cell with a direct connection to the underlying cytoplasm. The electron microscope substantiates this claim. Electronographs reveal many flagella to be much longer than the cells from which thev arise, so it is not too sur- prising to find free flagella floating around in a medium, since flagella are delicate and can be broken off by even gentle agitation. Motility is a valuable asset to bacteria, just as it is important to man and to other animals. The chances of survival are greatly enhanced when an organism has an opportunity to go out and look for food if the supply begins to run low. Being trapped in an unfavorable locality without means of escape can be as critical for a bacterium as it is for a helpless bedridden patient in a locked room that has caught fire when no one is around to offer assistance. One of the topics of the day in active bacteriological circles is this entire question of flagella. Do bacteria have them, or are they artifacts which appear as a result of locomotion? Are flagella the cause or the effect of motility? Until definitely proven othersvise, 104 Microbes and You we will continue to speak of flagella as the cause of bacterial locomotion. If we consider the time in which bacteria can cover a distance equal to their own length, their speed becomes jet-like in character. Fig. 22. Electronograph of a Proteus species prepared by a shadow- ing technic, showing flagella. (Courtesy of C. F. Rohinow and J. Hillier.) It has been calculated that a car traveling at comparable speed for its mass as some of our more active microbes would have to zo over 1000 miles an hour! What induces a microbe to changje direction is still not known, but a car attempting to alter its course Microbial Structures and Staining Reactions 105 to at the same relative speed would turn over. Perhaps bacteria do execute a "barrel roll" as they change direction. After all, which side is up for a bacterium? When objects are very small they are bombarded by molecular forces external to the cell, and the particles undergo a vibrating, trembling-like motion called Brownian movement. This phe- nomenon was reported in 1827 by Robert Brown, a botanist. As the particle size diminishes, Brownian movement increases. To distinguish this reaction from true movement caused by forces from within the cell, more than mere vibration must be detected. True motility means making progress through liquids— actually getting somewhere rather than merely vibrating in one spot. Motion and motility, in this sense, are not synonymous. The sluggish move- ment of some bacteria makes the distinction between the two quite difficult unless the culture is in the active stage of growth (less than twenty-four hours old), and is examined at the optimum temperature under the high dry objective of the microscope in a preparation called a hanging drop. Drifting with the tide is often mistaken for motility; bacteria must buck the tide and swim upstream, as it were, before we can call the motion true motility. The natural slow movement of some microorganisms makes distinction between motility and Brownian movement difficult, but speed of the bacteria may be enhanced by examining hanging drop preparations on a warm microscope stage with the culture in the active phase of growth. Not all rods are flagellated, but rods ex- hibiting motility have all been shown to possess flagella. Some of the spiral forms of bacteria appear to move more by a twisting of their elongated cells than by flagella, although this point is still debatable. Because of their narrow width (about 0.03 ^ in diameter), flagella are not visible when the usual staining technics are em- ployed. The extreme fragility of these structures is another factor complicating their observation. Spirals tend to have flagella located at the ends of the cells but not on the sides. With straight rods, however, we find singles, tufts, and dispersed flagella arranged around the cell's perimeter. 106 Microbes and You The following terminology has been commonlv accepted to describe arrangement of flagella: Monotrichic a single flagellum at one end. Lophotrichic a tuft of two or more flagella at one end. Amphitrichic one or more flagella at each end. Peritrichic flagella around the entire perimeter. When flagella are lacking, the organism is said to be atrichic. It has been suggested by some workers that amphitrichic flagella- tion as such does not exist. What is being viewed is two mono- trichic or two lophotrichic organisms about to divide but not as yet completely separated into individual organisms. Others classify motile bacteria into but two categories: Those having only lateral flagella, and those possessing only terminal organs of locomotion. Any finer breakdown, to these scientists, seems unjustified. Movement of flagella is not mere thrashing about. An apparent rotating motion propels them through fluids. Periodic contractions run around the flagella from one end to another, giving the appearance of a rotary motion. These rhythmic contractions are capable of forcing organisms through liquids at a rate exceeding 100 microns per second, although 25 to 30 microns per second is more common. The cholera organism has sped through a measured course at a registered speed of 8 inches per hour, and this speed can apparently be maintained for considerable time. The angle formed by the flagella with the cell body determines the direction of movement. These protoplasmic threads, as they are referred to by some investigators, have a constant arrangement for each species on which flagella are found, and this can usually be relied upon in the identification of organisms. We can slow down the speed of organisms by growing them in a semi-solid medium which is more viscous than nutrient broth. STAINING OF MICROBES Bacteria stain well with basic (in the sense of pH) dyes be- cause of the dye affinity of the nucleic acids contained in the Microbiol Structures and Staining Reactions 107 oro;anisms. These stains belong to the group of anihne (coal-tar) dves and include crystal violet, basic fuchsin, safranin, methylene blue, cosine, etc. After flooding a properly prepared smear on a glass slide with the particular dye and allowing a contact of from a few seconds to a minute, enough dye will be taken up by the cell contents to make the organism readily visible under the microscope. Stained slides are usually best examined under the oil immersion objective, which allows magnifications of one thousand or more diameters. Different stains and combinations of stains have been devised for specific purposes. A brief discussion of a few of these will follow. An important prerequisite for any staining operation is to have a clean slide on which the smear is to be prepared. Greasy slides do not allow even distribution of the test material, and much of the bacterial film may wash off during the staining processes. Be- ginners in bacteriology have a tendency to prepare films much too crowded with organisms for satisfactory examination of individual cells— the ultimate goal of any bacterial staining process. With a little practice the novice soon learns that a little bacterial culture will go a long way. Once the organisms have been introduced into the drop of liquid on the slide, the film should be spread out to provide thick as well as thin areas of material to be stained. Drying is usually best accomplished by allowing the smears to air-dry, but if more rapid drying is desired, no more heat should be applied to the slide than would be comfortable for your own fingers. It it is too hot for your hand, it is certainly too hot for the bacteria in the wet smear where abundant moisture intensifies the heating action. Bacteria can tolerate higher temperatures of dry heat than they can with- stand moist heat. It's not the heat, it's the humidity! Over-heat- ing wet smears is undesirable, otherwise staining mav not be characteristic. Some persons advocate a quick passage of the dried film through an open flame to "fix" the smear more firmlv to the slide. The value of this technic is debatable. Stains may be classified into two major groups: (1) General Stains in which basic dyes are usually employed to make bacteria 108 Microbes and You more readily observable, and (2) Differential Stains in which technics are used to divide bacteria into groups or to bring out some specific structure of organisms. The idea of staining microbes was first introduced into micro- biology by Carl von Weigert in 1871 when he first stained bacteria with carmine and later aniline dves. Dozens of dyes have come into common use since those early experiments of Weigert. A dye is a colored organic compound which has the ability to combine with certain substances and to impart color to them. It is possible to en- hance staining ability by adding intensifiers. For example, if a basic dye solution is made more alkaline, bacteria tend to stain more intensely. The introduction of surface tension depressants (wetting agents) may allow more intimate contact of dye with protoplasm. By applying heat and by adding carbolic acid (phenol) it is possible to intensify the dye-protoplasm union. Staining is a chemical or a physical union between the dye and components of the cell. If it is a chemical reaction, a new com- pound is foiTned, and simple washing in water does not liberate the bound dye. But if it is mere physical union, it is easier, as a rule, to decolorize such organisms. Many staining reactions are undoubtedly a combination of physical and chemical unions. NEGATIVE STAINS Negative stains, including India ink, nigrosine, and Congo red, do not have an affinity for bacterial protoplasm. Negative staining is also known as relief staining, since the background material retains the dye while the bacteria stand out in relief as unstained areas. To prevent bacterial growth in these dyes during storage— a common occurrence and a troublesome one— the addition of 0.5% formalin is advocated. India ink is a fine suspension of carbon particles in an aqueous gelatinous medium, but because it lacks uniformity this dye is not as popular as some of the others, particularly nigrosine which is a colloidal suspension. When Congo red is the dye being em- ployed, some persons recommend that the completed preparation be dipped in acid-alcohol (1% hydrochloric acid in ethyl alcohol) Microbial Structures and Staining Reactions 109 for a few seconds. This will convert the Congo red to a blue color, which appears to be easier on the eyes for some individuals. Sharp boundaries of organisms are characteristic when negative dyes are used, and in studving sizes and shapes of bacteria, particularly the spiral forms, this is a decided advantage. Students should be cautioned not to confuse negative staining with a gram negative reaction; they bear no relationship to each other. GRAM STAIN The gram stain is by far the most important differential stain employed in bacteriology. The technic was introduced in 1884 by Christian Gram, a Danish scientist, after a chance observation that tissues stained by his method could be made to release the dye but the bacteria embedded in the tissue retained their color. Further experimentation revealed that organisms could be divided into GRAM POSITIVE bactcria (those bacteria retaining the dye) and GRAM NEGATIVE bactcria ( those organisms giving up the dye ) when the slide was placed in alcohol. Gram's original method was to treat the smear with crystal violet followed by exposure to a dilute solution of potassium iodide. In order to distinguish the gram negative bacteria which have lost their color after the alcohol treatment, a counterstain of safranin or of carbol fuchsin is em- ployed. Thus, a mixture of gram positive and gram negative organisms after having been subjected to this differential staining procedure will reveal purple cells (the gram positive bacteria) and pink cells ( the gram negative organisms ) . The binding effect of the iodine with the crystal violet is less pronounced with the protoplasm comprising the gram negative organisms, and subsequent decolorization in alcohol is more easily accomplished. It should be pointed out, however, that excessive exposure to alcohol will also decolorize gram positive bacteria. The contact periods in each solution varv with the preference of individual laboratories, and procedures designated to acquaint students with the gram stain will outline specific methods. There are degrees of gram positiveness and gram negativeness. To minimize the number of stained preparations that are difficult no Microbes and You to call one reaction or the other, it is important that young, actively growing cultures be employed as source material. After twenty-four hours of growth on laboratory media many gram positive bacteria tend to lose their dye-holding capacity, and they will yield a gram negative or a gram variable reaction. Table 8 SOxME ORGANISMS THAT EXHIBIT A GRAM NEGATIVE STAINING REACTION NAME OF ORGANISM FUNCTION, SOURCE OR HABITAT Aerobacter aerogenes Azotohacter chroococcum Brucella abortus Escherichia coli Hemophilus pertussis Neisseria intracellularis Proteus vulgaris Salmonella typhosa Found in the soil, on plants and grains. Fixes atmospheric nitrogen non-symbiotically in the soil. The cause of contagious abortion in cattle and undulant fever in man. Inhabitant of the intestines of warm-blooded animals. The cause of whooping cough. The cause of epidemic meningitis. Found in certain infections and in putrefying material. The cause of typhoid fever. Table 9 SOME ORGANISMS THAT EXHIBIT A GRAM POSITIVE STAINING REACTION NAME OF ORGANISM FUNCTION, SOURCE OR HABITAT Bacillus anthracis Bacillus cereus Corynebacterium diphtheriae Clostridium tetani Diplococcus pneumoniae Sarcina lutea Streptococcus lactis The cause of anthrax. Found in soil and dust. The cause of diphtheria. The cause of tetanus, or lock-jaw. The cause of lobar pneumonia. Found in air, soil, water, and on skin surfaces. Found in milk and milk products. Plants may be the natural habitat. When attempting to identify a bacterial culture, the gram stain is one of the first tests conducted on the organisms. If the stain reveals a gram positive cell, the investigator can immediately Microbial Structures and Staining Reactions 111 dismiss the hundreds of gram negative organisms described in Bergeys Manual of Determinative Bacteriology. Identification of organisms is an ehmination procedure, and the gram stain represents a very important early step in this process. Without discussing complex chemical and physical theories proposed to explain this staining difference of bacteria, a few broad concepts will be mentioned. Some persons believe that there is a difference in the permeability ( intactness ) of the cell walls, with gram negative cells being more permeable than the gram positive bacteria, both for the entrance and for the egress of the crystal violet dye. The chemical composition of the surface of the cells is another theory put forth to explain the difference in gram stain- ing reactions between cells. Gram positive bacteria contain a chemical compound called magnesium ribonucleate at or near the cell surface. When these organisms are stripped of this chemical, they become negative in their staining reactions. If the magnesium ribonucleate is "replated" on these stripped cells, they revert to their gram positive status. Attempts to convert true gram negative cells by this plating technic had met with failure until recent studies indicated that if a viscous ribonucleate is employed, it ;s possible to change gram negative bacteria into gram positive organisms. That the gram stain depends upon surface phenomena is fairly well agreed, but the exact mechanism of the reaction is still in doubt. CAPSULE STAIN It is sometimes difficult to demonstrate capsules on bacteria, and some failures undoubtedly are due to the ease with which some slime layers can slip off the cells during the staining process. In fact, slime can be demonstrated in some liquid cultures, but the bacteria in that culture may fail to show capsules around the cell wall. A generally accepted technic for staining capsules employs India ink, nigrosine, or Congo red (so-called negative stains) as background material against which the unstained organisms stand out. By counter-staining with a basic dye like crystal violet, the bacterial cell will take up the dye while the capsule stains only 112 Microbes and You faintly, if at all. This type of staining can be enhanced if the organisms are grown or suspended in blood serum or in milk. Many workers in the field are reluctant to accept the relatively wide halo seen around such cells as representing the true size of the capsule. Some shrinking occurs in the staining process and capsules are exaggerated. By employing mordants and by ex- amining undried preparations, more reliable results should be obtained. A rather widely accepted technic for demonstrating capsules is the Hiss Stain. This method recommends serum or ascitic (body) fluid as background material in the preparation of the smear. After allowing the slide to dry rather slowly at room tem- perature, the smear is flooded with a crystal violet solution and heated just enough to make steam visible. By washing off the excess dye with a solution of copper sulfate (use no water any- where in the process), draining away the excess fluid and blotting gentlv until the slide is dry, the capsule will be seen as a faint purplish halo about the more deeply stained underlying organism when the oil immersion objective is employed. ACID-FAST STAIN As the name suggests, this stain remains fast even in the presence of mineral acid. There are limits to this fastness, how- ever, and just as timing is important in the various steps of the gram stain, we must understand the limits of exposure of acid-fast bacteria to acid. A relatively small group of organisms, principally members of the genus Mycobacterium, possess chemical or physical properties which make the cells difficult to stain, and once stained they are difficult to de-stain by ordinary methods. Robert Koch's difficulty in finding the causative agent in "consumption" ( tuberculosis ) may be traced to this peculiarity in staining of the organisms. The fatty-waxy nature of the capsule surrounding these organisms has been the usual explanation of why the tuberculosis organism is difficult to penetrate with dves. Once this tough barrier has been breached, the reverse process of removing the dye is equally Microbial Structures and Staining Reactions 113 difficult. By applying heated dye, such as carbol fuchsin (com- posed of basic fuchsin dye, alcohol, and phenol), the permeability of the capsule is increased, possibly due to the softening effect of the heat on the capsule. A common procedure employed in staining acid-fast material is that method proposed by Ziehl-Neel- sen in which hot carbol fuchsin is applied to the dried smear for about 5 minutes. Decolorization is carried out using a mixture of ethyl alcohol and 2 or 3% hydrochloric acid. This is called acid alcohol. The length of exposure to this decolorizer depends upon the thickness of the preparation, but in general a few seconds are sufficient to remove the color from everything except the acid-fast organisms. The decolorization is stopped by plunging the slide into water, and the preparation is then counterstained in methylene blue or some similar dye. The blue background facilitates locating the pink acid-fast organisms. Paul Ehrlich noted this acid-fast phenomenon in 1882 when he was studying the tuberculosis organisms. Recent evidence points to mycolic acid, a constituent of the waxy material, as the cause of acid-fastness. When mycolic acid is in combination with complex sugars (polysaccharides), it is even more acid-fast than when it is tested alone. However, just to make the problem more interesting, some workers have shown that not all acid-fast bacteria possess mycolic acid. Undoubtedly, more than one explanation of acid-fastness probably exists, and the full story is yet to be revealed. In recent years acid-fast bacteria have been stained with a dyestuff called aurmnine, which possesses properties of fluorescence. When such stained organisms are examined with ultaviolet light, using a yellow filter to block out the blue light, the field is dark but the auramine-stained organisms stand out as luminous yellow bodies. This technic has value in the diagnosis of tuberculosis. Persons suffering from leprosy harbor acid-fast bacteria within their diseased tissues, and these organisms resemble the tubercu- losis microbes. Mi/cobacteriwn leprae is the name given to the leprosy acid-fast cells. To date these organisms have not been isolated and grown on artificial media, but the fact that they are 114 Microbes and You seen in diseased tissue of lepers leads us to believe that they are the etiology of the disease. Saprophytic acid-fast bacteria are troublesome and can lead to false conclusions unless the clinician understands the problem. An organism known as Mijcohacterium smegmatis is a normal saprophytic inhabitant of the prepuce of males and the external labia of women. If urine specimens are collected without taking proper precautions, such as the use of a catheter which permits the collection of urine directly from the bladder or from the kidneys, acid-fast bacteria found in urine specimens might lead to a false diagnosis of tuberculosis of the genito-urinary tract. GRANULE STAINS Specialized stains have been developed to detect more easily granules in bacteria. Loeffler's alkaline methylene blue encourages the irregular staining so characteristic of t)'pical diphtheria organ- isms. Ponder's stain and Gohar's technic have also come into popular use for similar studies. The ingredients of all these stains and the technics for their use may be found in bacteriology laboratory manuals. SPORE STAINS Bacterial spores are usually refractile to the common technics employed for staining bacteria, but once the spores have stained, they retain the dye longer than the vegetative protoplasm. Mala- chite green is a very satisfactory dye to be forced into spores with the aid of heat. A counterstain of safranin will yield a pink vegeta- tive cell in which the green-stained spore can be observed. Carbol fuchsin is another satisfactory dye for primary staining with methylene blue as a counterstain. Many combinations have been suggested for spore-staining, and a number of these methods are quite satisfactory. The low permeabilit)^ of the spore walls to dyestuffs is the most common explanation of spore resistance to staining and to subsequent decolorization. Without the use of heat, a staining time of from 2 to 4 hours is not uncommon. In ordinary staining without the application of heat the spore stands out as a clear refractile bodv within the cell. At times it is Microbial Structures and Staining Reactions 115 difficult to demonstrate the actual spore, but the resistance of a culture to 80° C. for 10 minutes suggests the presence of spore- forming organisms or the presence of cells that are unusually resistant to heat. FLAGELLA STAINS Before attempting to demonstrate flagella by staining reactions, the culture must be grown under conditions optimum for the en- couragement of the formation of these structures. One theory, which in practice has been shown to possess considerable merit, is to make the organism go hunting for food in a large tube of distilled water after the bacteria have been grown in the conden- sation water of an agar slant and serially transferred each twenty- four hour period for several days. By dashing about in the dis- tilled water in search of food, development of flagella is theoret- ically encouraged. Some persons, on the other hand, believe it is the slow growth or static condition of the cultures which permits development of flagella to take place. Flagella apparently become thicker and longer with age and are more readily stained than when they are in the active stages of their development. By treat- ing the dry film on the slide with a mordant (a complex colloidal solution), the diameter of the flagella is built up to within micro- scope range by the packing-on of mordant. Subsequent staining with either methylene blue or carbol fuchsin will usually reveal the thread-like flagella, particularly at the edge of the stained smear. Strict attention to details is of the utmost importance in this delicate staining procedure, and scrupulously clean slides free of scratches are essential. The presence of even minute amounts of organic matter interferes with good staining, because the debris may react with the mordant and absorb some of the dye. The discussion of staining and staining technics in this chapter has been brief. Definite procedural details vary so greatly from one laboratory to another that it seems wise to leave the fine de- tails of specific staining to the individual instructors as part of their lecture or laboratory discussions. CHAPTER 6 Cultivation and Identification of Bacteria INTRODUCTION PURE CULTURE ISOLATION TECHNICS Broth dilution Agar dilution Streak plates Selective and enrichment media Micromanipulators for single cell isolation Selective heating Use of laboratory animals IDENTIFICATION OF PURE CULTURES Morphology Cultural characteristics Physiology Serology INTRODUCTION Because microbiology is an entirely new field to most persons en- rolled in a survey course of this type, the student must be intro- duced to a new language. With a little effort, a student can readily expand his vocabulary by several hundred scientific words, and if he has studied Latin or has been exposed to some Greek, many terms used in microbiology will be familiar to him. An agreement as to the meaning of common terms seems ap- propriate at the outset of this chapter. When bacteria are trans- ferred from one medium to another, the material being transferred 116 Cultivation and Identification of Bacteria 117 is called the inoculum and the resultant growth, whether it be in a liquid or in a solid medium, is termed a culture, or subculture. If the transfer is made to a solid medium, the visible growth which appears after a suitable incubation period is a colony. If but one species of organism is involved in this cultivation technic, it is said to be a PURE cultl^re. A single kind of organism may be isolated Fig. 23. Colonies developing on nutrient agar exposed for ten minutes to the air in a classroom. {By permission frotn Introduction to the Bacteria by C. E. Clifton. Copyright, 1950. McGraw-Hill Book Company, Inc.) from mixtures of organisms by a purification process to be discussed in this chapter. Attempts to study the metabolism of a single bacterial cell are impractical, in fact, probably impossible. But by cultivating masses of organisms, as long as they arise from a single cell or from a group of like organisms, the results of a study can be relied upon, and reproducible reactions are possible. It therefore be- comes important to grow volumes of bacteria by employing the prerequisites of a good microbiological medium discussed pre- 118 Microbes and You viously in Chapter 4. To be assured of a good harvest a farmer not only has to plant good seed in fertile ground, he must also rely upon Providence to furnish the necessary warmth, sunshine, and moisture to allow these seeds to germinate and to develop into mature plants. Once the plants begin to grow, the care-taker must control inevitable pests— the insects and the diseases to which these growing plants are susceptible. A bacteriologist in cultivating microbes is able to control these factors of heat, moisture, and contaminants. If he knows the food requirements of his proposed crop, and if he understands the ele- mentary principles of aseptic technic (not allowing undesirable organisms to get into his cultures), the unwanted "weeds" can be eliminated, and he can be assured of harvesting a good crop of pure culture. Controlled cultivation is within the reach of all laboratory workers who follow the fundamental rules of the game. No reliable physiological determinations can be made on mixed cultvires of bacteria. The purity of the microbial culture is just as important, if not more so, than it is in growing grain crops. In the latter case, should an undesirable seed find its wav into the batch, the growing plant can readily be pulled up and discarded prior to the harvest. But in the bacteriology laboratory, once the contami- nant gains entrance into a culture, especiallv liquids, it may be necessary to plant a new crop, sometimes repeatedly, until the cul- ture has been purified. Bacteria are ubiquitous, and if technicians will keep that thought constantly in mind, proper technics for handling and cul- tivating pure bacterial masses are more likely to be employed. Careful attention to what may seem like exacting details will pay dividends in the long run. Not onlv will contamination be mini- mized, but the worker may save himself the painful experience of contracting a laboratoiy infection when he handles pathogenic organisms. PURE CULTURE ISOLATION TECHNICS There are very few places where pure cultures of organisms exist in nature. As long as bacteria are found practically every- Cultivation and Identification of Bacteria 119 where, it must be expected that they will occur as mixed cultures. A number of technics designed to separate bacteria from their neighbors are available, but before much can be done in the way of identifying these organisms, it is necessary that the culture be pure. BROTH DILUTION This method represents one of the earliest attempts to secure pure cultures of organisms. The first in a series of broth tubes is inoculated with the material containing the bacteria, and after thorough mixing, a small quantity ( a loopf ul or a drop ) from tube 4^1 is transferred to tube #2. After mixing the contents of tube #2, a transfer is carried from #2 to tube #3, etc., in series. A decreasing number of organisms is carried over into each succeed- ing tube of broth, and if this dilution procedure is carried along through a sufficient number of transfers, the point is eventually reached where the inoculum consists of only one or a very few cells. The species predominating in the original material logically would be found in pure culture in the last tube of the series show- ing visible growth after a suitable incubation period. A little contemplation should make it obvious that this broth dilution technic has several serious drawbacks. First of all, one can never be sure that the last dilution tube will always contain a pure culture. If two species of organisms are found in about equal numbers in the original test material, they might both be carried over into the last dilution tube and yield a mixed culture. But a serious disadvantage of the method is that it denies the opportunity for isolating those species which happen to be in the minority in the original microbial mixture. AGAR DILUTION Bv incorporating a solidifying agent in the broth to be em- ployed in the serial dilutions, it is possible to anchor the organisms in the solid medium. If the melted agar is cooled to between 45-50° C. before inoculation and is poured into a culture dish be- fore the medium thickens, the poured agar will solidify when the temperature approaches 40° C, and the organisms are trapped Fig. 24. (A) One of the accepted technics for holding test tubes and cotton stoppers during transfer of cultuies. (B) A close-up of the same technic. 120 Cultivation and Identification of Bacteria 121 in the solidified agar. Each separate cell develops in the medium producing a visible growth, called a colony, and these usually represent pure cultures which arise from either a single cell or from a group of like cells. If the dilutions are carried out in series, the developing colonies will be far enough apart to facilitate their being picked from the agar with the aid of a sterile wire or loop. By subculturing these isolations to tubes of sterile broth or to solid media, many different isolations are possible from different colonies on a single culture plate, and they will represent those organisms found in low numbers as well as the predominating species in a given mixed culture. This solid medium technic is a decided improvement over the serial broth procedure, but it still has certain undesirable features which can readily be overcome. One of the principal disadvantages of the method is the dif- ference in the appearance of colonies of the same species when the organisms develop at different oxygen tensions. Colonies growing on the surface of agar plates and having full access to atmospheric concentrations of oxygen are usually larger than subsurface, im- bedded colonies. In the discussion of chromogenesis in the pre- vious chapter it was emphasized that only in the presence of an abundant oxygen supply can pigmentation by chromogenic organ- isms be assured. Upon examination of an agar dilution plate made of such organisms, it would appear on the basis of differences in pigmentation that more than one species of organism was present in the plate. By inhibiting chromogenesis and reducing colony size of sub-surface colonies, one is faced with serious diagnostic disadvantages. Plates over-crowded with colonies is another un- desirable feature of this culturing technic. STREAK PLATES It is possible to overcome the above criticisms by a simple ex- pedient. Instead of mixing the bacteria with the liquefiable-solid medium before transferring it into a petri dish, the agar can be poured into the dish first, allowed to harden, and then the culture can be smeared or streaked on the hardened surface of the medium. 122 Microbes and You It may seem incredible to a novice that it is possible to take a loopful of culture containing hundreds or thousands of bacteria, and by simply streaking that loop back and forth in an orderly manner over the surface of nutrient agar in a 65 square centimeter area of a petri dish, one can deposit in certain areas on that agar sur- face single, well-isolated bacteria which are capable of growing into Fig. 25. When making streak platings, the lid of the petri dish should be held in a position that will protect the surface of the agar in the plate from outside contamination. distinct colonies. There are probably almost as many modifications of technics for streaking plates as there are teaching institutions, but most methods will produce satisfactory results if the prescribed directions are carefully followed. Rather than outline any one technic to the exclusion of others, it seems best to leave that spe- cific detail to the individual instructor who undoubtedly has a method he has previously found to be satisfactory. The streak plate is the most universally accepted procedure for obtaining isolations from mixed cultures, and if a reasonable Cultivation and Identification of Bacteria 123 amount of inoculum is used, isolated colonies representing even some of the minorities in mixed cultures can be examined for dif- ferences in their size, shape, elevation, and pigmentation— the com- mon criteria for identifying different species on a streak plate. At times, however, it becomes necessary to employ enrichment (selec- tive diet) technics to encourage the growth of certain organisms found in small numbers in a given mixed culture. Final identifica- tion of these isolated species involves a systematic elimination procedure to be discussed later in this chapter. SELECTIVE AND ENRICHMENT MEDIA The studies of Churchman and others have been instrumental in the development of selective media for the isolation of specific bacteria from mixtures. Certain dyes have been found to exert a growth-inhibitory effect upon gram positive bacteria as a group, while other dyes act similarly toward gram negative organisms. In general, the gram positive species are more susceptible to dye action than is true for gram negative bacteria, and diflFerences within a given staining group also exist. By incorporating one or more dyes into a medium, the task of isolating selected bacteria is sim- plified. This principle has decided value in clinical laboratory work, particularly in the detection of intestinal (enteric) patho- gens, including typhoid and dysentery organisms. Such dyes as malachite green, brilliant green, gentian violet, and others, can be added in low concentration to various media for retarding growth of selected organisms. Escherichia coli is one of the more numerous gram negative species found in feces of warm-blooded animals, and because of its aggressive characteristics, it might readily overgrow the enteric pathogens one is trying to isolate by cultivation of feces organisms in a clinical laboratory. Selective media provide a valuable means of retarding the growth of Escherichia coli. Another gram negative species, Proteus vulgaris, has the peculiar characteristic, especially on primary isolation, of spreading over the entire surface of a cul- ture plate, and this may cover up the growth of other organisms being sought in clinical material. By adding chloral hydrate to 124 Microbes and You the medium, the spreading of Proteus can be prevented and dis- crete colonies may be more readily examined for some of the cul- tural characteristics mentioned previously. Bacteria, like humans, vary in their food demands. A person who has been on a diet of filet mignon finds it difficult to switch to a steady diet of pork and beans. Some bacteria are also very fastidious about what they will eat, and if they are deprived of their high living, they would prefer not to put up a struggle to change their mode of life, and in other instances they may be pre- vented from doing so. The "vampire-like" Hemophilus (blood- loving) group of bacteria, including the organism which causes whooping cough (Hemophilus pertussis), is incapable of growing unless it has access to blood and blood derivatives. The more parasitic an organism becomes, the more exacting it may be in its food demands. When viruses are discussed, you will discover that not only must viruses have cells upon which to grow, but they must have living cells, and sometimes even particular living cells from designated tissues. Such extreme dependency limits chances for survival of many parasitic organisms when the environment is altered. MICROMANIPULATORS FOR SINGLE CELL ISOLATION At times, particularly in research investigations involving the genetics of bacteria, it becomes desirable to isolate single bacterial cells to insure the purity of a given strain of organism under in- vestigation. This type of isolation is not practical on a routine basis, but its mention should be included in a discussion of technics employed for obtaining pure cultures of bacteria. By placing a series of small drops of diluted liquid culture on a slide and examining them with the aid of a microscope to find a drop containing only a few bacteria (or only one), it is possible with the aid of a micromanipulator to isolate an organism from its fellow microbes in that fluid drop. Without going into the minute details as to the operation of this microscope attachment, the tech- nic involves drawing isolated organisms into a fine capillary pi- pette and transferring the trapped cells into a suitable broth Cultivation and Identification of Bacteria 125 medium. Not all such isolated organisms survive, but if growth occurs in the broth, the offspring can all be traced back to a single cell, and studies relative to nutrition and genetics will have more significance when they are based upon cultures originating from known single cells. SELECTIVE HEATING It has been pointed out that spores resist higher temperatures for longer periods of time than do vegetable cells. To separate a spore-former from a mixed culture, the culture is subjected to varying degrees of heating, and the surviving spores can then be separated. Not all spores are equally resistant to heat, so heating of parts of the suspension at different temperatures may be neces- sary to separate mixtures of spore-formers. USE OF LABORATORY ANIMALS Some animals are known to be extremely susceptible to the ac- tion of specific organisms. The mouse, for example, can be in- jected with a mixture of many organisms, but if in that mixture is found a virulent pneumococcus, it is quite possible for the natural destructive forces of the mouse to dispose of all of the injected bacteria except the pneumococcus. This organism and its progeny mav eventually destroy the mouse, oftentimes in less than 24 hours. Other organisms, however, are also capable of being pathogenic for mice. An examination of the peritoneal cavity of the mouse immediately after death will frequently reveal a pure culture of the pneumococcus. The guinea pig serves as a similar filter for separating the tuberculosis microbes from materials such as sputum. This purification process may take from 4 to 6 weeks, however, because of the slow metabolism of the tuberculosis organisms. Autopsy examination of the lungs, liver, spleen, and other organs will generally reveal pure cultures of packed organisms in tubercles —visible growths of the pathogenic agent. When Koch's postulates were discussed it was made clear that unless a susceptible animal is used, it is not possible to prove the etiology of all diseases by his postulates. This is true for some 126 Microbes and You viruses, and the number of animals that can be used for such studies is more hmited. Mice are susceptible to the action of some viruses, rhesus monkeys to others, and ferrets to still others, but many animals are completely refractory to some viruses. IDENTIFICATION OF PURE CULTURES After primary isolation procedures have assured that the cul- tures so obtained are pure (only one species), the next step is to subject the pure culture to an orderly sequence of morphological, cultural, and physiological tests. Trying to determine the accepted name of an organism involves an elimination procedure, each test narrowing down the possibili- ties. Bergeijs Manual of Determinative Bacteriology is the stand- ard reference work used in the final identification of bacteria. Since the number of organisms studied in a course of this nature is usually not very extensive, many institutions incorporate into their laboratory manuals simplified reference charts to which stu- dents may refer, rather than have the beginning students labori- ously thumb through bergey's manual. By employing a chart similar to the one which follows, all pertinent information for the organisms under investigation can be tabulated in an orderly fashion for handy reference. It seems appropriate to discuss in some detail the theory and the significance of some of these tests commonly used in describing the growth and activities of micro- organisms. This should aid the student to understand better the why and the wherefore of what might appear to be hocus-pocus practiced in bacteriology laboratories. MORPHOLOGY Gram Stain A carefully prepared gram stain will immediately place the or- ganism in question into one of two major groups— the gram posi- tive bacteria or the gram negative bacteria. This is the first im- portant step in the elimination process. The value of a reliable gram stain cannot be over-emphasized. Much time and effort can be needlessly wasted by trying to identify an organism that doesn't Cultivation and Identification of Bacteria 127 DESCRIPTIVE CHART FOR BACTERIA I. Morphology VEGETATIVE CELLS: Gram staining reaction: Form: spheres, short rods, long rods, filaments, spirals. Size : Sketch : Motility: present, absent. Arrangement: singles, pairs, chains, tetrads, clusters, cubical packets. Spores: present, absent. Location: central, terminal, subterminal. Sporangia: swollen, not swollen. II. Cultural Characteristics COLONY: Medium Age: Form: punctiform, circular, rhizoid, irregular. Surface: smooth, rough, dry, moist, dull, glistening. Elevation : flat, raised. Edge: entire, wavy, filamentous. Growth: slow, moderate, rapid. SLANT: Medium: Age: Form: thread-like, beaded, root-like, spreading. Consistency: butyrous, viscid, brittle. Medium: grayed, browned, greened, unchanged. Optical characters: translucent, opaque, irridescent. Color: water soluble, water insoluble. NUTRIENT BROTH: Age: Surface growth: ring, pellicle, none. Clouding: slight, moderate, heavy, none. Amount of sediment: none, scanty, abundant. Type of Sediment : flaky, granular, vis- cid on agitation. EN DO OR EOS IN METHYLENE BLUE AGAR: Growth: present, absent. Color: present, absent. Metallic sheen: present, absent. III. Physiology FERMENT A TIONS: Glucose : acid, gas, negative. Age: Lactose: acid, gas, negative. Age: BROM C RE SOL PURPLE MILK: Reaction: acid, neutral, or alkaline. Age : Curds: acid, rennet, none. Proteolysis: none, slight, moderate, complete. Age : METHYLENE BLUE MILK: Reduction: none, slight, moderate, complete. Age : GELATIN LIQUEFACTION: None, moderate, complete. Slow, moderate, rapid. INDOLE TEST: positive, negative. METHYL RED TEST: positive, negative. VOGES-PROSKAUER TEST: posi- tive, negative. CITRATE TEST: positive, negative. UREA TEST: positive, negative. IV. Additional Data Name of organism concluded from the above reactions: Student: Dates of study: 128 Microbes and You exist, and this is exactly what might occur if false conclusions are drawn from improperly prepared gram stains. A microscopic ex- amination, usually with the oil immersion objectiye, will reyeal the form and the size of an organism, in addition to its gram reaction. By underlining or circling the applicable terms that appear on the descriptive chart, a quick glance will point out to the observer the outstanding characteristics of the organism. Hanging Drop By examining a hanging drop of a young (less than twent)^- four hours) broth culture under the high dry objective of the microscope, motility and typical arrangement of the species can be determined. Spores Spores fail to stain during ordinary gram staining, but by the application of heat with dyes such as malachite green or carbol fuchsin, spores can be stained and their size and location can be ascertained after suitably counter-staining the preparation with a contrast dye such as safranin or methylene blue which colors the vegetative cells and the non-spore components of spore-bearing bacteria. CULTURAL CHARACTERISTICS To a trained observer the cultural characteristics can supply valuable clues as to the possible genus, and sometimes even as to the species of the test organism. It is unwise, however, to allow these criteria to be the only studies made, since closely related, yet distinct, species may have similar cultural characteristics. Streak Plate Because a streak plate allows all colonies to develop on the surface of the medium, such considerations as form, surface ap- pearance, elevation, edge of the colony, and the speed of microbial growth can be compared. Again, these characteristics in them- selves are not conclusive evidence as to the genus or species name C'ultivation and Identification of Bacteria 129 of an organism, but these data do add weight to the other factors considered in the identification process. Slant Cultures Media containing a soHdifying agent such as agar can be dis- pensed in tubes while the media are still hot and in a liquid state. By placing these tubes at an angle during the solidifying process, the material will harden into what is termed a slant. Such a preparation provides more surface area for inoculation. Care must be exercised to avoid wetting the cotton stoppers with the medium during the slanting operation, otherwise the natural filtering ability of the cotton will be lost and contamination of the tube's contents might well be expected. In addition to furnishing information as to the form, con- sistency, and optical characters of the bacterial growth, the agar slant also provides information relative to the color-producing capacity ( chromogenesis ) of the species. Some media are better than others for stimulating pigmentation of cultures; meat infusion agar is one such medium. If the color produced by organisms is water soluble, the pigment will diffuse throughout the agar. Most bacterial pigments, however, are of the water-insoluble type, and fail to leave the cell, at least not in detectable amounts. Nutrient Broth A young broth culture, in addition to providing information about motility and natural arrangement of organisms, also displays cultural characteristics, such as surface growth, clouding of the medium, and sediment formation. A sudden jarring of the tube will suspend the sediment, and the type of sediment can be de- termined and recorded under the appropriate heading on the de- scriptive chart. Selective Media This has been discussed earlier in this chapter and needs little more elaboration than to point out that organisms which do grow 130 Microbes and You on these media may exhibit characteristic cultural appearances, some of which are of diagnostic significance to a trained eye. PHYSIOLOGY In simple terms, physiology involves the enzyme systems pos- sessed by bacteria and the effects these enzymes have on the sub- strates employed for the cultivation of these organisms. The re- actions to be discussed represent only a few of the fundamental considerations in the identification of bacteria. Fermentation In advanced courses in microbiology it is not uncommon to study the fermentation of a dozen or more substances. Two sugars you can expect to find in all such lists are glucose (also known as dextrose) and lactose (milk sugar). Glucose is a simple sugar— a monosaccharide, while lactose is more complex— a disaccharide. Different species of bacteria attack some sugars and not others, and the type of physiological reaction produced is also variable. The fermentation process may produce, among other things, vari- ous acids, and to detect the presence of acid it is customary to in- corporate a pH indicator dye in the broth to which the sugar has been added. The indicator chosen varies from one laboratory to another depending upon individual preferences. Brom thymol blue, the same indicator employed in the pH adjustment of stand- ard nutrient agar, is commonly used in fermentation studies. Andrade's indicator is another. As the growing organisms attack the sugar in the broth, the acid produced depresses the pH level to the point where color changes are brought about in the dye indicators. In addition to acids formed, various gases, notably carbon dioxide and hydrogen, may be evolved. A gas trap is included in these fermentation tubes by placing a small test tube, or vial, in an inverted position within the tube containing the sugar broth. Gas being liberated during the breakdown of the sugar rises in the Cultivation and Identification of Bacteria 131 medium and some of it is caught in the gas trap. Displacement of the Hquid in the inner inverted tube is evidence of gas formation. Five visible changes may take place in tubes of sugar broth under- going microbial action. If the bacteria do not possess the enzymes required to attack specific sugars, growth in the broth will occur, as evidenced by clouding of the medium, but no color change will take place in the medium and no gas will be liberated to be trap- ped in the inverted vial. Acid production without gas formation is a second possibility, and acid together with gas evolution rep- resents the third visible change in the sugar broth. Alkali pro- duction is a fourth possible change, and alkali production coupled with