Who's who among the microbes

Preface

The following sketches grew out of a series of radio talks on communicable diseases and their microbes. The increasing demand for information suggested that peo¬ ple were deeply interested in these invisible, omnipresent enemies and friends. The thought occurred to The Cen¬ tury Co. that the information given in these talks might be more useful in a permanent shape. We were consulted with the result that the subject matter was elaborated to form this small volume.

The question of the relationship of microbes to man s welfare is so vitally important and many sided that any effort made to show some of its sides seems worth while.

We have endeavored to described simply and ac¬ curately the most important facts known that help us determine how and why some microbes are harmful to man, others harmless and still others helpful. We also tell how man can use available knowledge to protect himself against harmful ones and utilize more fully the

activities of the useful. <

We wish to express our great appreciation of the

kindly aid of Anna I. and Harriet von Sholly m reading the manuscript and of Amelia R. Wilson m its pre¬ liminary critical typing.

The Authors.

Contents

OHAPTEK

I. Early Discoveries .

Menace of the unknown — Invisible forces — Fighting in the dark — Glimmerings of dawn — Revelations of first lenses — Whence? — How ? — Why ?

Chapter I

Menace of the unknown — Invisible forces — Fighting in the dark — Glimmerings of light — Revelations of the first lenses — Whence ? — How ? — Why ?

The world of microbes, that world of minutest living beings, had existed, invisible and unknown to man, throughout the ages. Only comparatively recently have its ever-present myriads been forced upon our atten¬ tion. As late as the middle of the nineteenth century disease and death brought about by germs remained a mystery — due, believed the many, to malignant spirits. Only with the development of the microscope was it possible for man to be certain of the presence of such tiny creatures and to begin intelligently his battle to study and control them.

It is true that even as early as 40 b.c. an occasional real thinker surmised that minute organisms which the eye cannot see enter the body and cause disease.1 And a few investigators of pre-microscope days went so far as to say that certain diseases were probably caused by a contagmm-vivmm — a living virus or poison. But the majority of people in those days still thought that the mystery of disease must be referred to gods or devils either directly or through magic or

1 Osier, “The Evolution of Modern Medicine.” New Haven; Yale University Press, 1923.

witchery. And to what lengths their obsession led them for many years we all know not only from the customs of ignorant savages but from the tragic history of more intelligent people. Witchcraft in Salem and else¬ where may be recalled as one of the many horrible situ¬ ations resulting from the ignorance of fanatics.

This belief that disease was due to some malign in¬ fluence that used an agency often belonging to the in¬ visible world was true in a sense other than these be¬ wildered people realized. The idea that disease was sometimes sent as a punishment or corrective by the gods went along with other early crude imaginings. Little wonder that people prayed and made sacrifices to their gods to avert the pestilences that descended upon them and the dread diseases that stayed with them they knew not why except it might be for their sins known or unknown. And the conviction that the gods in their wise providence sent or allowed these terrors as punishment became firmly established. Men exercised their ingenuity in devising ways of propi¬ tiating the angry gods. In very early times feasts and dances were given to appease divine wrath. Later, fasts with sackcloth and ashes were resorted to in endeavors to obtain the favor of the offended deity.

In this day of our knowledge of the prevention of infectious diseases we can scarcely realize the state of mind of people who constantly faced the menace of the unknown in the fear of being attacked by such terrible epidemic scourges as the black death (bubonic plague), yellow fever, cholera, smallpox, and the endemic or ever-present diseases of tuberculosis, syphilis and ma¬ laria, without knowing anything about their cause, prevention and cure. And in addition they saw their crops destroyed by blights and their flocks swept away by still other mysterious diseases.

Defoe’s famous “Plague Year” (1665) gives a mi¬ nute vivid picture of the fearful ravages from plague in those times and of the horrible way the authorities, in their ignorance, handled the victims. One in every six or seven persons died, and the dead, and sometimes the dying, were thrown together into large pits. It is true they were covered with quicklime, which is a good germicide. Now with our knowledge of the nature of plague and its spread we have demonstrated one of the most clear-cut relationships between cause and pre¬ vention, and have shown the satisfactory results of pre¬ ventive treatment based upon this knowledge in the areas where it is possible to carry out these measures. Thus in the United States there is now only an occa¬ sional sporadic case started by infection brought in from outside, though earlier a small epidemic of plague developed even here before we had learned how com¬ paratively easy it is to prevent its spread.

Copeman 2 dramatically describes the early history of the terrors from smallpox, perhaps the most dreaded of all the formerly uncontrolled scourges, because it had become endemic, always present. Not only did it frequently cause death, but, what might be worse, it often destroyed the beauty, the physical personality even, of the survivors, “turning the babe into a change¬ ling at which the mother shuddered, and making the eyes and cheeks of the betrothed maiden objects of horror to the lover.” 3

* Macaulay’s “History of England.” Account of the year 1694.

These shocking conditions were due chiefly to the fact that people knew nothing about the activities of these microscopic or ultra-microscopic organisms in disease and health.

The truth only commenced to be discovered, and then slowly, when the microscope began to reveal its won¬ ders. Even when the stupendous discovery was made that minute forms of life too small to be seen with the naked eye were swarming in myriads throughout na¬ ture, in air, water, soil, observers did not at first connect these tiny creatures with the cause of definite diseases. For they were primarily found by van Leeuwenhoek, the first of de Kruif’s magnetic microbe hunters, in quite harmless vehicles, such as rain-water, the top layers of the soil and in healthy human excretions.

When these minute germs were first seen swimming around in fluids as fish do, their discoverers thought that they had found some new little animals — animal¬ cules; so they searched for their heads, eyes, tails and other parts similar to those belonging to the larger known animals. When they could not find these struc¬ tures, the hunters thought it was due to the fact that their lenses did not magnify enough, and many were the attempts to make better lenses.

Now we know that the majority of the forms they first saw were the very simplest of living creatures, for the most part with no clearly discernible structure ; that is, they were motile bacteria belonging to the veg¬ etable kingdom. Only a small number were animals, and even these were single-celled with simply a few differentiated parts called organelles.

While it was exciting enough to watch through the lenses these little creatures girate around with all sorts of fantastic motions, the thoughts as to what they might mean, where they came from, how they live were far more alluring to the few who knew about this great discovery and busied themselves with attempts to find out all about these mysterious forms of life.

First the searchers asked themselves, “Where did these minute living forms come from?” “How were they created?” The majority thought that the answer to these questions was that these creatures were continu¬ ally being generated spontaneously under certain con¬ ditions of heat, moisture and food; and many were the grotesque ideas these observers had of just how this generation took place.

The few most thoughtful investigators believed that these lowly organisms grew from preexisting like or¬ ganisms and that their beginnings lay, as did that of the beginning of all life, in the dim unknown past, or that they were created “in the beginning by God.”

The many who believed that all germs continued spontaneously to generate were superficial observers, who thought in ruts. The occasional ones like Redi (1688) or Spallanzani (1769), who believed the oppo¬ site, were not only close observers but showed that they were right by actually doing the things they claimed could be done, in such a logical way and so fully con¬ trolled against error that nearly everybody with at least average intelligence who became acquainted with their work, even some of the most doubtful, were finally convinced that under known conditions germs did not spontaneously generate. The story of their convincing demonstrations has often been told, but it cannot be too often repeated, because it is such a good illustration of wrong deductions from insufficient checks or controls against error, and right conclusions from sufficient evidence properly controlled.

The sponsors of spontaneous generation said that if one boiled broth made from vegetable or animal matters the germs that might be present would certainly be killed, and yet if such broth was kept from the air by corking the bottles containing it, in a few days it would be swarming with germs. Spallanzani decided that these broths had not been cooked long enough to kill the most resistant germs or that it had not been properly corked. So he made a large quantity of broth and put some into each of many glass flasks. Then after closing some of the flasks by fusing with heat, and others by corking with imperfectly fitted porous corks, he kept some in boiling water for a few minutes and others for at least one hour. After removing the flasks from the water he kept them under observation for some time. At length he saw that in all flasks that had been in boiling water for only a short time the broth had become turbid, due to the growth of germs, and that of those that had been in the boiling water for about an hour the fused flasks remained free from germs, while a number of the im¬ perfectly corked ones showed them.

His opponents answered him by claiming that the boiling for so long a time without access to any air had destroyed the power of the broth to generate germs spontaneously.

Spallanzani soon showed that this was not true by the simple process of exposing some of the boiled broth in the fused flasks to the air, when the broth soon be¬ came filled with growing germs. He said that germs must produce seeds that are very resistant to heat. He thus concluded before they were seen that these tiny creatures could develop still more minute parts like seeds which would contain the germ of the individual. Pasteur, much later, showed that the reason why some fluids required so much boiling was because of the presence of these very resistant seeds or spores which had just been demonstrated by Perty (1852).

Pasteur’s clear-cut experiments showed that no life occurred in any fluids, however rich the mixture, if such fluids were boiled sufficiently and kept protected from unfiltered air or other source of contamination. These investigations of his, his telling demonstrations and the heated arguments of his opponents lasted over four years. They are vividly told in Yallery-Radot’s 4 interesting “Life of Pasteur.”

Finally, after more than a century of controversy, nearly every one agreed that these minute creatures did not suddenly come into life under any of the con¬ ditions observed.

One of the early practical results of this study on spontaneous generation — one of far-reaching impor¬ tance — was the introduction by Appert in 1804 of the preserving of foods by sterilizing them in cans.

With this greatly contested point of spontaneous generation apparently settled, there was more time to study other questions concerning these little creatures ; but with all their endeavors, students did not get very far in learning how the germs lived and what they mean, because nobody had yet learned how to separate them easily one from the other so that each kind might be grown by itself in pure cultures. That came much later. In the meantime, Ehrenberg (1838) and others

4 Translated by Mrs. R. L. Devonshire. New York: McClure, Phillips & Co., 1902.

gave rough classifications of these minute forms based upon physical appearance or morphology alone.

During all this time there slowly multiplied the num¬ ber of observers who claimed that some of these germs were probably the cause of certain diseases, and, as usual, one or two were far ahead of others in their powers to observe and deduce. First came Boyle just about the time of Kircher’s and Leeuwenhoek’s dis¬ coveries of germs (1659-1675), who said that infec¬ tious diseases were probably due to fermentation caused by animalcules. Then Lancisi (1718) inferred that malaria was due to an animalcule. In 1753 Linnaeus emphatically stated that all disease was due to fermen¬ tation and putrefaction from an unknown living cause which he called chaos. And Plenciz (1762), a Viennese physician, published a more clear-cut statement. He maintained that all infectious diseases must be caused by micro-organisms, and each disease probably has its own kind of germ. He gave as his reasons for this belief the mode of infection, the unlimited development among large numbers of individuals with the gradual spread over wide areas, the incubation, course and resulting immunity.

It had already been shown by the Chinese and the Hindus that the inoculation of a susceptible person with smallpox virus taken from a person suffering from smallpox (which method was introduced into England by Lady Montague in 1721) usually reproduced the disease in a light form and caused immunity. Then in 1796 Jenner made his world-famous demonstrations with cowpox virus. He was led to make his first trials of it because of the oft-reiterated opinion of dairy people that milkmaids, after developing cowpox con¬ tracted while milking, did not get smallpox after ex¬ posure to it. He showed that when inoculated into the skin of human beings, this cowpox virus produced a localized pustule resembling those of smallpox, but not spreading, and that, marvelous to relate, after this healed, the individual was subsequently protected from infection with smallpox virus. Thus he proved that the dairy people had been right in their observation.

That was indeed a wonderful discovery. Of course, there were detractors and still are. That some parts of the world are free from smallpox to-day because of wide-spread and thorough vaccinations these detractors will not accept. They claim there would be no smallpox if there were no vaccination. They have only to watch reports of outbreaks of smallpox and study the cases minutely to become convinced of the efficacy of thor¬ ough vaccination. These carpers should learn, since they apparently don’t know, that each individual re¬ sponds to vaccination in a different degree, so that in some the immunity lasts only one year, in others longer and in many for life ; while there are even a very few that do not make immune antibodies, so there are ex¬ ceptional cases that appear to receive no benefit from vaccination.

The next virus that was shown to produce a distinct disease was the rabies virus (1813). The saliva of rabid animals was shown to have the power of producing rabies when inoculated into well animals. It had been observed much earlier that man became “mad” after the bite of a rabid animal. Much later, Pasteur did his monumental work on the nature of the rabies virus and showed that a vaccine could be made that would keep the vast majority of those bitten from developing rabies.

Up to the middle of the last century certain medical people continued to assert that the living nature of contagion was too absurd an idea to waste time in re¬ futing, but enthusiasts on the other side studied more than ever to find evidence pointing to the truth of the idea that germs are the cause of infectious diseases.

Henle in 1840 enunciated practically all of the points quoted later as Koch’s postulates. He said that if a given germ causes a given disease, it should always be found in that disease and in no other, and the tissues containing it should produce the disease when inocu¬ lated into other animals. When, finally, Pollender in 1849 actually demonstrated bacterial rods in the blood of animals dying of anthrax, and Davaine (1850) proved that only blood that contained these rods pro¬ duced the disease when inoculated into healthy sheep, many investigators were ready to go to the limit in believing that all diseases are caused by germs and that all germs are dangerous.

Later discoveries proved, of course, that such was an extreme view. It is interesting to note as we scan the history of medicine the fluctuating curve of empha¬ sis placed by different writers upon the importance of microbes to man. First germs were ignored or con¬ sidered merely a curiosity. We still have ignorers. Then when dangerous germs were demonstrated, all germs were damned ; some have been praised and others damned irregularly since then.

Even to-day two popular books illustrate the fact that we still have with us those who emphasize the extremes of both sides. In one, entitled “The Conquest of Disease,” 5 the statement is emphatically made that all germs are harmful to humans. On the other hand, the book “Civilization and the Microbe” 6 states very clearly and convincingly that the great majority of these minute forms are extremely useful directly or in¬ directly to civilized man. Many instances are given of their direct as well as indirect use to man. To believe that all are dangerous is the depth of pessimism; on the other hand, to believe that all may become useful or rendered innocuous within an appreciable time is the height of optimism. Both appear to be unwarranted by the known facts.

Notwithstanding their dread importance as disease producers and their helpful importance in breaking down complex dead materials into simple compounds for plant use and so enable life to continue, microbes are still ignored by the majority of people. Many people don’t want to be bothered with the thought of all of the difficult problems concerning the relationship ot these minute forms to our welfare. But to those people in whom the quality of curiosity, of wondering over the mysteries of our beginnings and our endings, is well developed, the fact that we know so little about germs is all the more reason for being interested in learning more about them. And when these eager searchers think of the many minute forms that may still be undiscovered because there is no way yet known to demonstrate them, and that while some of these germs may be harmful, some may be useful, and some

“David Masters, “The Conquest of Disease.” New York: Dodd, Mead & Co., 1925.

“Arthur I. Kendall, “Civilization and the Microbe.” Houghton, Mifflin Company, 1923.

of the many harmless ones ‘may be made useful, then is their eagerness to learn about all these forms greatly enhanced.

Fortunately for our welfare, there have always been a few enthusiasts who have continued to study these elusive protista, or first forms of life, and so we have gradually come to know many of their characteristics.

An important characteristic to which we have al¬ ready alluded is the formation of resistant spores or seeds by some varieties. Perty, in 1852, was the first to observe them. Others also saw them. Then Pasteur, as we have said, showed that the presence of spores ex¬ plained why we still have a growth of germs after they have been submitted to a boiling temperature for a short time.

A big discovery made by Perkins (1856) helped ma¬ terially in the study of these forms. Perkins discovered accidentally the value of aniline in dyes. This led to the brilliant work of Weigert (1871) and of Ehrlich (1887) on the use of these dyes in staining micro¬ organisms, and later still to the work of Churchill and others in the use of the dyes in differential cultural methods and in treatment of certain infectious diseases.

Then the discovery of how to get pure cultures of these germs was of inestimable importance in studying them. The first method devised by Lister (1858), Pas¬ teur and others was a dilution method. This method required great patience and an almost innumerable number of dilutions in order to catch a single organ¬ ism. It is still used to obtain pure cultures of certain protozoa, but it is now seldom employed for that pur¬ pose in studying bacteria. In 1871 Klebs used hen’s eggs as a culture medium. At the same time Koch developed a jelly medium in which he obtained isolated colony growth. Then came Hesse (1881) with her agar-agar medium, which Koch later substituted so successfully for the gelatine medium.

Just at this time, when it was demonstrated that the suppuration of wounds was due to bacteria, the great English surgeon Lister, incited by Pasteur’s work, introduced the use of antiseptics in his surgical opera¬ tions. His work was epoch-making in its saving of life. Thus was stopped the formation of pus, even laudable pus (really praiseworthy pus it was considered then), as the small amount was called that was wrongly thought necessary for the quick healing of wounds. While later it was learned that the use of chemical antiseptics in surgery could be largely replaced by heat sterilization of apparatus and by the surgical cleaning of the hands of the operator and by the wearing of a mask, and so on, still, Lister’s work was an immense advance over old methods either of letting “nature take its course” or of pouring hot oil on the wounds or using other barbarous procedures. It is true that long before Lister’s time Oliver Wendell Holmes, in America (1843), maintained that puerperal fever (childbed fever), that former bane of childbirth, was due to a virus that might be carried by attendants, physicians as well as nurses, from patient to patient. And while he was ridiculed by his colleagues as an immature raver about a condition that they declared was plainly due to “Providence,” he maintained his contention. He was soon brilliantly corroborated by Semmelweiss in Aus¬ tria (1847), who showed that if examiners in a lying-in ward first washed their hands in a chloride of lime solution, puerperal fever practically stopped in that ward. Of course, we know now that the disinfecting of hands, while perhaps the chief means of preventing puerperal fever and a wonderful advance over the old custom of merely washing the hands, as was ordinarily done, even though coming from the dissecting room to the wards, does not wholly prevent the danger from the carriers, which we will tell about later.

This skeptical reaction of many of the medical pro¬ fession to the brave and logical presentations of Holmes and Semmelweiss illustrates again how difficult it seems to be for the majority to break away from custom. Most of the doctors strongly opposed the communicable na¬ ture theory of disease, and it was not until Lister’s time that it was generally accepted.

From the discoveries in rapid succession of the spe¬ cific microbes of many of the infectious diseases, and the demonstration by Pasteur that other vaccines than that against smallpox might prevent each its own dis¬ ease, may be dated the beginning of the fight against dangerous microbes which constitutes a large part of modern preventive medicine. Facts in regard to these later discoveries will be given in the following chapters.

Chapter II

Through the microscope — By the use of stains — By the use of

culture media.

Our chief aid in becoming acquainted with microbes must be, of course, the microscope. The revelations through the first lenses spoken of in our previous chap¬ ter gave only a glimpse of the subvisible world that we know to-day. Through the series of lenses in the com¬ pound microscope of to-day the lower limits of vision are extended about twentyfold over those early lenses. That is, we may see germs magnified as much as 2000 diameters, which allows us to distinguish some indi¬ vidual characteristics of all but a few of even these minute beings.

This wonder, the modern microscope, has been gradually evolved from the imperfectly ground low magnification lenses of Jansen in 1590, Kircher in 1659 and Leeuwenhoek in 1675. While Leeuwenhoek’s lenses were an improvement over the others, their power to magnify was not much greater and they were still full of imperfections that often caused the objects studied to appear like distorted and indefinite dots and lines. One has only to visit the Army Medical Museum in Washington and view their wonderful and fantastic collection of old microscopes, one of Jansen’s time and a number dating before the time of van Leeuwenhoek, to realize what improvements have been made. With the development much later of the Huyghenian com¬ pensation eyepieces, the immersion lens, the Abbe sub¬ stage condenser with its iris blender, and the series of finely ground compensation lenses of special glass in the objectives and eyepieces, our microscopes of to-day allow us to perceive more or less clear-cut pictures of all of the known microbes.

A bifocal or binocular microscope adds greatly to the comfort of the observer. Then there is the dark field microscope that was invented to help study very minute particles and so-called ultra-microscopic forms. These little points of matter catch the oblique rays of light and reflect them to the eye, thus standing out on an otherwise dark field.

In order to understand fully the structure and work¬ ings of our microscopes, a special book on the subject should be consulted.1

We may study individual germs through the micro¬ scope in either the living or dead condition. Since through our high-power lenses these minute organisms when living appear for the most part only as tiny bits of translucent jelly-like substances containing one or more retractile granules, they must be killed, fixed to a glass slide and stained in certain ways in order to learn more details of their structural differences and of their individual reactions to special stains.

Living, the microbes are examined in what is called a hanging drop of a fluid menstrum, or they may be planted on a hanging mass of a transparent solid medium. The hanging drop or mass is placed on the

1 One of the good ones is Gage’s “The Microscope.” Ithaca, N. Y.: The Comstock Publishing Company, 1925.

center of a little square piece of thin glass (cover glass) and then inverted over a glass slide at the place where there is a hollow of about a half inch diameter. The surface of the glass around the hollow is smeared with a little vaseline or other inert oil. This allows the cover glass to stick after being pressed down and pre¬ vents the drying of the drop. In this way we can study the individual form of the microbes, also their motion, size, shape, growth, division, spore formation, vital staining, and their reactions to specific serums, such as the Widal reaction (the agglutination test) and other so-called serologic tests which we will explain later.

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Fig. 1

Hollow slide with cover-glass for studying a hanging drop of

GERMS THROUGH THE MICROSCOPE.

To examine the organisms dead they must be spread in an even film on a glass slide, dried, fixed usually by heat or alcohol and finally stained. In tissues the organ¬ isms must be examined stained in sections.

The microscope and stains alone are not enough for complete identification of most micro-organisms. We must use cultural methods as well in order to get pure cultures of the germs and bring out the mass growth characteristics of these pure cultures. In order to ob¬ tain pure cultures we must get colony growths from a single individual. We obtain these colony growths by one of two methods. In the first we make high dilutions of the menstrum containing the germ and mix each in a transparent jelly medium while it is in a fluid state; then we pour the mixture into a Petri dish which is made of clear glass, allow it to solidify and let it stand at the right temperature for growth. In the other method we spread the menstrum thinly over the surface of a solid transparent medium in a Petri dish. Each germ then multiplies many thousand times until there is a little mass growth or colony, usually becoming large enough to be seen with the naked eye. The colonies of many varieties of bacteria present so characteristic an appearance on certain culture media that they are of aid in the identification of the organism. From each of these colonies a pure culture of the organism may be obtained by “fishing” or touching a portion of a single colony with a sterile platinum needle and transferring it to a plate or tube of a fresh sterile medium. In many cases this fishing has to be done under a low-power lens of the microscope, in order to be sure we have touched with our fishing needle no other colonies invisible to the unaided eye (see Frontispiece). The hand of the “fisher” must be steady and his eye true to be able to touch the right colony and only that one.