Zur Untersuchung von pathogenen Organismen

(On the investigation of pathogenic organisms)

by Robert Koch, published 1881, here Google-translated to English. The original article is available at https://archive.org/details/b21303095/page/1/mode/1up.

Introduction.

Hygiene has thus far been able to derive relatively little benefit from the advances in our knowledge of pathogenic organisms. This is due to the fact that the vast majority of questions relevant to hygiene regarding pathogenic microorganisms can only be resolved using reliable methods for separating the different types of these organisms. For example, hygiene is not merely concerned with determining whether fungi, bacteria, and other lower organisms are present in a particular soil or drinking water, but specifically whether disease-causing organisms are among them.

Furthermore, once the presence of a notoriously harmful or even suspected organism has been established, the next step is to study it in all its aspects, separate from other organisms that would interfere with and confuse observation. This includes investigating its living conditions, its developmental history, and everything that promotes or hinders its growth. This knowledge, however, can only be gained through continuous cultures of individual species, so-called pure cultures, for which there are currently no universally applicable and reliable methods.

I have made considerable efforts to fill this gap and have finally arrived at results that are certainly capable of and in need of further improvement, but which, even in their present form, have proven quite effective in the investigations into infectious diseases and disinfection carried out at the Public Health Office. Partly to make these highly versatile methods accessible to a wider audience, and partly to facilitate understanding of the work carried out using these methods, which will be published in these journals, they will be described below.

Description of the task.

In general, the study of lower organisms for the purposes of public health will have to consider the following points. First and foremost, it must be determined whether the organisms in question are pathogenic at all, i.e., capable of causing disease. This is followed by proof of their infectivity, i.e., transmissibility to other, hitherto healthy individuals, either those of the same species as the first spontaneously or artificially infected individual, or those belonging to other species. Furthermore, the manner in which the pathogenic organisms enter the animal body, their behavior outside the body in the air, water, and soil must be observed, and finally, the influence exerted on them by inhibitory and destructive substances must be determined.

Their presence in the body is of interest to hygiene only insofar as it provides information about the nature of the infection, for example, localization of pathogenic organisms in the intestine, transmission into the bloodstream, formation of persistent conditions within the body. Frequently, as long as the pathogens characteristic of a particular infectious disease, such as cholera, plague, etc., are not yet known, or when the general aim is to recognize the harmful properties of air, water, and soil and to assess the disinfectant value of certain substances, it will be possible to consider the occurrence and behavior of the bacteria and fungi most closely related to pathogenic organisms based on experience, and from this to infer, with a greater or lesser degree of probability, the behavior of the presumed but not yet known pathogens.

Determination of Pathogenic Properties.

Let us now turn to the individual tasks, beginning with the determination of the pathogenic properties and infectiousness of microorganisms.

Some researchers still maintain that bacteria are present in the blood and tissues of a healthy body; however, this assertion is not based on direct microscopic detection, but rather partly on theoretical assumptions and partly on experiments concerning the putrefaction of healthy organ parts isolated under antiseptic conditions. However, very significant objections can be raised against these experiments, the discussion of which would go beyond the scope of this text. It is certain that, using the microscope and with the aid of examination methods that can reliably detect even isolated bacteria in animal organs, it has not yet been possible to prove their presence in the blood and tissues of healthy individuals to such an extent that no doubt can arise about their presence there during life.

Therefore, as soon as bacteria—and the same applies equally to other microorganisms—are found inside organs, whether in the blood or lymph vessels or in the tissue itself, in configurations that can only occur in a living body, or when the unmistakable influence of the microorganisms on the tissue affected by their invasion is evident, e.g., necrosis of cells located in a certain area, accumulation of round cells in the vicinity, penetration of the foreign organisms into the cells, etc., then such microorganisms must be considered pathogenic, or at the very least, they must appear suspicious and warrant further investigation and clarification of the findings.

Determining the pathogenicity of microorganisms found on the surface of the body and on its mucous membranes is more difficult. Only the massive occurrence and the differences in form between the presumably pathogenic organisms and those known to be harmless and usually parasitic in or on the body can be decisive here. Unfortunately, too little attention has been paid to these harmless parasites so far; a deficiency that is particularly noticeable with regard to intestinal disorders and necessitates a certain degree of caution regarding all information about pathogenic bacteria in the intestines until all doubt has been eliminated that there is a confusion with habitual inhabitants of the intestines, which multiply under exceptional but favorable conditions and become noticeable through their large numbers.

However, the time is certainly not far off when the harmless microorganisms parasitizing the healthy body will be so well known that they can be immediately identified as such when it comes to distinguishing them from pathogenic entities, and the newly emerging pathogenic organisms can be reliably excluded from their number.

Detection of pathogenic microorganisms.

When it comes to identifying the pathogenic organisms suspected in the diseased body, primarily bacteria, ordinary microscopic examination, performed without special preparation or techniques, encounters considerable, sometimes even insurmountable, obstacles. While some pathogenic bacteria are distinguished by their size, characteristic shape, and motility, making them difficult to overlook, others have such a simple form and are so small that, when mixed with the similarly shaped breakdown products of tissue cells, they cannot be distinguished from them.

Fortunately, however, bacteria possess a property that allows us to overcome all these difficulties: their remarkable ability to absorb certain dyes, especially aniline dyes. However, the fluids in which the bacteria are located—blood, mucus, tissue fluids, etc.—form precipitates when directly mixed with aniline dyes. These precipitates are also colored and can either mimic bacteria through their granular or thread-like structure, or obscure the bacteria present due to their voluminous mass. Further preparations are therefore necessary to make the bacteria clearly visible using the dye solutions.

Furthermore, when examining the stained specimens under a microscope, a very special device for illuminating the preparation is required if the advantages of the staining method are to be fully realized.

Several years ago, I described in detail the most suitable methods for detecting bacteria when they are present in liquids or animal tissues, and published them partly in a paper appearing in F. Cohn’s “Beiträgen zur Biologie der Pflanzen, Band 2, Heft 3”, and partly in a treatise on wound infection diseases. Regarding the details of these methods, I must, in order to avoid going into too much detail, refer the reader to the aforementioned publications; here, I will only address those points that have been improved since then or that have been subject to misunderstandings and therefore require clarification.

Microorganisms in liquids.

The method of identifying bacteria in liquids, e.g., in blood, pus, or tissue fluid, using dyes consists of spreading the liquid in the thinnest possible layer on a coverslip, allowing it to dry, and then exposing it to the dye solution. If the liquids containing bacteria are protein-free or contain very little protein, the staining is almost always successful. However, as soon as they contain more protein, the layer does not adhere firmly enough, even for a considerable time after drying, to prevent it from being largely softened, torn, and even partially washed off the coverslip by the dye solution. Furthermore, the protein does not become insoluble upon drying; it largely dissolves into the dye solution and forms precipitates with the dye, which adhere firmly to the coverslip, obscuring everything and rendering it unrecognizable.

This problem can be almost entirely avoided if, instead of the aqueous solutions of fuchsin, methyl violet, etc., which are almost exclusively used for staining, aniline brown dissolved in glycerol is employed. In the aforementioned work on bacterial examination, glycerol brown is strongly recommended on page 407, and all the photograms accompanying that article, which depict bacteria in blood or tissue fluids, were prepared from preparations stained with glycerol brown.

Nevertheless, some have repeatedly attempted to stain blood preparations in aqueous solutions, and incomprehensibly, the inevitable failures have been blamed on the method itself. Even recently, M. Wolff * asserted that a reliable diagnosis of bacterial infection cannot be made using the method I have described. He struggled in vain to get rid of the "grains and spheres" in his blood preparations stained with aqueous solutions. That something can also be achieved with the existing method in skilled hands is demonstrated, besides many other successful investigations carried out using the same method, by the work of Ogston, who in numerous cases detected various types of bacteria in fluids taken from human bodies, and whose experiences led him to the following statement: "It is impossible to confound the (microorganisms) with such granular bodies as those alluded to by Wolff (The British Medical Journal 1881, March 12)."

*) Virchow’s Archiv für pathologische Anatomie, Bd. LXXXI Hft. 2 u. 3.

It was desirable to improve the staining of bacteria in protein-containing liquids so that even inexperienced users would achieve reliable results. The simplest way to achieve this was to convert the protein present in the layer adhering to the coverslip into an insoluble form. Even when storing the prepared coverslips, one can observe that after a few days, sometimes only after weeks, the layer has become firmer, adheres better to the coverslip, and forms less precipitate when the dye is added. Better results and faster insolubility of the dried layer can be achieved by placing the coverslips in solutions that coagulate albumin, such as solutions of chromic acid, chromic acid salts, alum, or tannin.

The beneficial effect that alcohol has on bacteria-containing tissues during hardening, particularly with regard to the proteins they contain, ultimately led me to also harden the protein layer on the coverslip with alcohol, which indeed had the desired effect. After the preparations had been in absolute alcohol for some time, the layer became completely insoluble and stained uniformly and excellently. No granules or other interfering precipitates impaired the diagnosis of micrococci or other bacterial species present in blood, pus, etc.

Only one thing is uncertain with this alcohol hardening method: determining the exact time the preparations must remain in the alcohol. Sometimes a few days are sufficient, but sometimes the required degree of insolubility of the protein layer is only achieved after several weeks. It is therefore advisable to prepare a sufficient number of coverslips and to remove one from the alcohol from time to time to test its staining.

Very often, however, in investigations of infectious diseases, it is desirable to be immediately informed about the presence of bacteria in the organs of the animal body, in order to be able to assess, for example, the success of an inoculation and the need for further inoculation, or similar situations, directly during the necropsy. In such cases, one would naturally not be able to wait for the success of alcohol hardening. It was necessary, if the method was to be effective in every respect, to also develop a solution for this.

When Ehrlich's* investigations became known, the excellent results he had obtained on heated blood preparations for distinguishing the differently granulated blood cells naturally prompted the study of the effect of heat on bacterial preparations. Ehrlich exposed the coverslips, coated with the dried blood layer, to high temperatures (120° to 130°C) for one to several hours. Such intense heat exposure renders the blood layer completely solid and insoluble, but, as the experiments showed, the bacteria lose their ability to absorb dye.

*) Verhandlungen der physiologischen Gesellschaft zu Berlin, 1878/79 No. 20. Zeitschrift für klinische Medicin, Bd. I Hft. 3.

For our purposes, however, it was sufficient to apply the heat only long enough to render the proteins insoluble, and this can be achieved in a much shorter time. If the coverslips are exposed to a temperature of 120° to 130°C for just a few minutes, the layer is already so solid that it no longer forms precipitates with the dye solutions and stains very well. The exact duration of the required heat exposure cannot be specified. Sometimes the preparation is sufficiently heated after only 2 minutes, sometimes only after 5 to 10 minutes.

It should also be noted that some bacteria, e.g., anthrax bacilli, appear somewhat altered when heated first and then stained; they look thinner and more delicate than when stained with glycerol brown, and they do not exhibit the characteristic structure of anthrax bacilli as clearly. I would like to take this opportunity to point out that slight differences, similar to those just discussed, can be observed primarily in the lateral diameter of the bacteria when the preparations are made in different ways or stained with different dyes. Therefore, only preparations made using a completely identical procedure can be used for comparison.

If, for example, one wants to most clearly demonstrate the characteristic structure of anthrax bacilli, which allows for an infallible diagnosis, then, as already mentioned, staining with glycerol brown is the most effective method. This particular form of the anthrax bacilli also becomes apparent to a greater or lesser extent with other stains, but not reliably enough to establish a diagnosis. And if, with another preparation method, this characteristic of the anthrax bacilli is not clearly visible, then one is certainly not yet justified in asserting, as Zürn did, that there can be no question of the bacilli being segmented.

Despite the aforementioned shortcomings, the heating method is a significant enhancement of the methods used to examine bacteria. It is used continuously in the work on infectious diseases at the public health office and has become virtually indispensable. In every necropsy of an animal that has succumbed to an infectious disease, blood, tissue fluid from the inoculation site, lungs, spleen, and, if necessary, from other organs are immediately examined in the manner described. The further course of the experiment is then determined by the findings, which are naturally only preliminary and are supplemented by careful subsequent examination of the organs preserved in alcohol.

Regarding the choice of dyes, we owe to Ehrlich* the introduction of a new, highly recommended aniline dye, methylene blue, which is particularly suitable for staining heated preparations. In difficult and doubtful cases, however, it is advisable to try other aniline dyes as well, since some bacteria behave quite peculiarly with respect to their staining properties, a point to which I will return later.

*) Zeitschrift für klinische Medicin, Bd. 2 S. 710.

Wherever possible, some preparations should be stained with brown dyes to enable the urgently needed photographic imaging of the bacteria. The eye is, of course, far more pleased by the extraordinarily strong and saturated tones of the red and blue aniline dyes than by the brown stains, which usually appear somewhat dull. But so far, it has not been possible to obtain good photographs of blue or red-colored bacteria preserved in Canada balsam, while bacteria colored brown or yellow pose no difficulties whatsoever for photographic imaging.

Preparations whose layer has been rendered insoluble by alcohol treatment or heat in the manner described and stained with a suitably chosen dye solution should be free of granular precipitates, dye particles, and the like; they contain only the formed elements originally present in the liquid spread on the coverslip, and if the staining is neither too weak nor too strong, only these latter elements should appear stained, while the dried residue of the liquid or the dried plasma is barely indicated by a faint tint. Therefore, only the cells and their products, whether naturally or artificially produced, can give rise to confusion with microorganisms.

Regarding the aforementioned artificial products, anyone who has conducted some examinations of blood, pus, tissue fluid, etc., will soon discover that the thinner the fluid under investigation, the less the shape of the cells it contains is altered when spread on the coverslip. In blood, for example, white blood cells, with few exceptions, retain their round shape and thus appear after drying as circles containing the diverse, often ribbon-like nucleus.

However, if the fluid is thick and viscous, which is especially true of the tissue fluid of organs, such as the spleen or lungs, then it is usually impossible to spread it into a thin layer without the cellular elements being more or less distorted, even completely torn and shattered. This results in comet-like figures, with the remaining nucleus forming the head and the spread-out, often elongated, remaining nuclear material forming the tail. The interpretation of these often fantastically shaped structures is self-evident. A few adjacent fields of view reveal all the transitional forms, from the thread-like figures located at the edges of the smeared mass, where it was thinnest and the smearing needle most severely compressed and crushed the cells, to the unchanged, i.e., undamaged, cell nuclei in the thicker areas of the preparation. One would therefore assume that these distorted cell nuclei, which at first glance are clearly recognizable as such, could not be mistaken for microorganisms, and yet this has been the case.

Fokker* believed that in his investigations into anthrax he had discovered two types of the disease. In one, the familiar bacilli are present, while in the other, they are entirely absent or only present sporadically. Instead, he found long threads which, he claimed, were connected to lymph cells and resembled spermatozoa, with the lymph cell forming the head and the thread the tail. Fokker considered these structures, which he termed fungal threads, to be actual fungi transmitted through inoculation. These fungi, absorbed by the lymph cells, grew within them, elongated them, and pierced them at one end.

*) Centralblatt für die medicinischen Wissenschaften, 1880 No. 44, 1881 No. 2. Weekblad van hed Nederlandsch Tydschrift voor Geneeskunde, 1881 No. 4.

Finally, Fokker found the same structures in the normal spleen. But even this did not enlighten him about the true nature of his supposed fungal threads; he consoled himself with the fact that fungi are among the ordinary components of the body. An illustration he included in one of his publications leaves no doubt that Fokker's fungal hyphae are smeared cell nuclei.

A more forgivable mistake would be the confusion of micrococci with the granules of granulated cells, especially those Ehrlich called mast cells. The granules of some of these cells appear to be very loosely connected; the cells easily disintegrate when spread on the coverslip, their granules are scattered, and to the untrained eye, they can give the impression of individual micrococci arranged in groups. Particularly large and regularly developed cells of this type are found in the blood, and especially in the spleen and lungs, of white rats, and less frequently in white mice. I recall having seen preparations from these organs in which the intensely stained granules of the crushed cells were scattered over such a wide area that this sight would undoubtedly have elicited a cry of joy from an enthusiastic micrococci hunter.

However, the preparations came from healthy animals, and upon closer examination, some intact cells were still found in which the granules surrounded a faintly stained nucleus, thus revealing themselves as components of granulated cells. These granules can almost always be distinguished from micrococci by their unequal size and often also by the peculiar color they exhibit. In any case, caution is advised regarding such findings, and if doubt remains, a comparison should be made with corresponding specimens taken from normal animals, as well as with sections of hardened tissue that show the suspicious granule clusters in context and in their natural location. I have yet to encounter a situation where it has not been possible to make a definitive determination between micrococci on the one hand and the components of granulated cells on the other.

However, in order to gain the necessary experience in this field, anyone involved in experimental investigations of infectious diseases is strongly advised to familiarize themselves with the results of Ehrlich's work on granular cells.

I would also like to emphasize the importance of Ehrlich's examination method for another reason related to infectious diseases. Ehrlich provided proof that among the cellular elements of the blood, which were generally considered equivalent, differences can be detected with the aid of dyes—that is to say, with the aid of chemical reactions—differences that must lead to the assumption that these differences are related to the origin and physiological significance of the cells.

But what does the differentiating staining of blood cells have to do with infectious diseases? Simply that in one or more groups of infectious diseases, the pathogens might possibly occur in a form similar to white blood cells, e.g., amoeba-like, and that in this case, it would be of the greatest value to possess reliable distinguishing characteristics, such as those undoubtedly provided by Ehrlich's staining method. It is certainly a one-sided, albeit currently widely accepted, opinion that all as yet unknown infectious agents must be bacteria. Why should other microorganisms not be equally capable of leading a parasitic life in the animal body? I do not wish to claim that these would be exclusively amoeba-like entities. Other members of the protist kingdom are also suspect. The idea that amoeba-like structures could play a role as parasites is only suggested so readily because there is a very striking example from the plant world. This concerns a peculiar disease of the cabbage plant, which remained a puzzle to botanists for a long time until Voronin**) found the solution. He demonstrated that an organism, probably belonging to the simplest forms of myxomycetes, penetrates the root of the cabbage plant in the form of a colorless, fine-grained droplet of plasma. Upon reaching a parenchyma cell of the root, the parasite, which Woronin termed Plasmodiophora brassicae, mixes with the cell's cytoplasm and is initially indistinguishable from the cell contents. Only later does its presence become noticeable through characteristic changes in the cell. (Cf. Table XIV, Photos 83, 84.)

**) Pringsheim’s Jahrbücher für wissenschaftliche Botanik, 11. Bd. 187 S.

The further, highly interesting developmental course of Plasmodiophora is not of interest to us here, but rather the initial phase of its presence in the infected root. For, assuming that colorless, extremely small clumps of cytoplasm similarly found their way into the fluid mass of the animal's body and multiplied there, would the process be very different than in the root of the cabbage plant, where it is impossible to differentiate the parasite from the cell's cytoplasm? Certainly, such plasma clumps in the blood would be mistaken for fragments or breakdown products of white blood cells if it were not possible to differentiate them using more refined staining methods. Voronin's study of plasmodiophores had already prompted similar considerations. He suspects “that the appearance and development of many pathological growths and swellings occurring on the animal body are caused by small myxamoebae that penetrate the living organism, develop into plasmodia, and cause significant irritation, etc.”

Eidam*), who confirms Voronin's statements, also expresses this view and considers it possible that in some infectious diseases with as yet unexplained etiologies, where bacteria have been sought in vain, parasites might appear that initially do not differ from the body's tissue elements and would therefore behave similarly to Plasmodiophora.

*) Der Landwirth. Allgemeine landwirtschaftliche Zeitung, 1880 No. 97.

The example of Plasmodiophora was discussed in some detail because it serves as a stark reminder that when searching for living pathogens, one should not only hunt for bacteria, as is currently the norm, but should also focus attention on other formed elements of the blood or the infected organ.

Microorganisms in animal tissues.

The examination of fluids described above is immediately followed by that of the animal organs themselves, which is intended to provide information about the storage and distribution of pathogenic organisms in the tissues, their relationships to neighboring cells, etc.

These are mostly objects of the smallest dimensions, which can only be seen in very thin sections of the tissue under investigation, and therefore the microtome is the most advantageous tool for preparing these sections. Regarding the further treatment of the sections with the dye solutions, dehydration, lightening, and preservation in Canada balsam, as well as the use of the Abbe light-gathering apparatus, I must refer to the detailed description I gave in my work on wound infections. I have little to add to it.

First, I would like to reiterate that the examination should not be limited exclusively to bacteria, but should also consider other microorganisms that may be present. This must be taken into account during preparation, especially during the hardening of the tissue samples. To date, alcohol hardening has consistently proven to be the most suitable hardening method for bacterial preparations; However, whether this is also true for all microparasites seems at least doubtful, and it is certainly advisable in some cases to use other hardening agents, e.g. chromic acid, osmic acid.

Then, another experience seems worth mentioning, which I gained during my attempts to stain pathogenic bacteria with various dyes, particularly the brown dyes I will discuss shortly. This is the often quite different behavior of individual bacterial species towards certain dyes. Initially, it seemed as if all bacterial species behaved the same in this respect, but this is not the case; just as bacterial species differ from one another in many other particular properties, so too do they differ in their staining power.

To cite one of the most striking examples of this kind, relapsing fever spirochetes stain intensely with fuchsin, methyl violet, gentian violet, etc., in the blood layer dried on the coverslip, while it is not possible to stain them with the same dyes in sections using nuclear staining. In my experiments, however, brown aniline dyes are effective, though unfortunately not very strongly, so that locating spirochetes in sections is not among the easiest tasks. Since most considered it impossible to detect spirochetes in hardened specimens, I have included two photographs here as proof of their stainability. These photographs depict sections from the brain of a monkey that had been inoculated with relapsing fever spirochetes and subsequently killed on the brink of death. (Table IV, Photos 23, 24.)

Leprosy bacilli behave in almost the opposite way. They can only be stained when very fresh on a coverslip; even a short time after drying, they no longer accept the dye. However, when hardened in alcohol, they stain excellently with fuchsin, gentian violet, etc., for a long time, at least several years, but very poorly with aniline brown.

Almost all micrococci stain intensely with blue, red, and brown aniline dyes when wet. But differences emerge again among the bacilli. Some accept all aniline dyes strongly, others, e.g., the short typhus bacilli first described by Eberth, to a lesser degree, although not as weakly as Eberth's description might suggest. (See Table IX, Photos 49–53.)

These differences in the staining properties of bacteria deserve attention insofar as they partly provide evidence for the chemical diversity of bacterial species, but also call for a cautious evaluation of negative results, since, based on current experience, it does not seem impossible that one or another bacterial species does not accept any of the dyes currently in common use.

A small trick in staining may be mentioned here, as it can be quite helpful under certain circumstances. By significantly heating the dye solution, the time required for the staining process can be considerably shortened, and at the same time, a stronger color can be achieved. It even appears that some pathogenic bacteria can only be stained sufficiently intensely using this method. However, the temperature should not exceed approximately 40 to 50°C during heating, otherwise tissue sections rich in connective tissue will begin to shrink.

Photographic Images of Microorganisms.

Of paramount importance for the study of microorganisms is their photographic representation. If anywhere a purely objective approach, free from all prejudice, is necessary, it is in this field. But precisely the opposite has occurred so far, and there are probably no places with more subjectively colored views and, consequently, more disagreements than in the study of pathogenic microorganisms.

No one will deny that the difference in the understanding of the relationships of one and the same subject almost always stems from the fact that this subject appeared to the first researcher in a different light than to the second. One should remember that we are dealing exclusively with microscopic objects, and that in microscopy, two observers cannot simultaneously view the same object and communicate about it. Rather, each observer sees the object in question one after the other, and, as every microscopist knows, even the slightest adjustment of the micrometer screw causes objects as small as bacteria to either disappear entirely from the field of view or appear with completely different outlines and shadows. Nevertheless, communication about the observed object is more readily possible if it is observed with one and the same instrument, that is, with the same illumination, the same lens system, and the same magnification.

However, if the many conditions under which the microscopic image is formed are different, communication becomes much more difficult. For example, if one observer uses a narrow diaphragm while another uses a wide one, one uses a weak eyepiece while the other uses a strong one, and so on, or if the preparation and staining of the object are already different, or if it is furthermore immersed in liquids of varying refractive indices, how can it be surprising that one microscopist claims to have seen an object quite differently—perhaps thicker or thinner, more or less shiny—or if he possibly cannot find it at all and therefore denies its existence? And how, in such cases, can the error of observation, whether it lies on one side or the other, be proven among the many possibilities mentioned above? Was it the preparation or the handling of the microscope that led the observers to different results regarding the same object? Deciding this will almost never succeed without other means of testing; each of the disputing parties naturally remains in their own opinion, and medical science does not know whom to believe.

For these shortcomings, which have countless times manifested themselves in microscopy to the greatest detriment of science, there is only one remedy: photography, which must act as a mediator, a balancing force, and an instructive guide. The photographic image of a microscopic object is sometimes more important than the object itself. For if I hand someone a microscopic specimen with the intention of examining specific parts of it, such as bacteria-laden lymphatic vessels, I have no guarantee that the correct location will actually be found and, if so, that the correct settings, illumination, and so forth will be chosen. Photography, on the other hand, reproduces the microscopic image once and for all, without the slightest possibility of deception, precisely in the settings, magnification, and illumination in which it was captured. Nothing is easier than agreeing on what a photograph represents, because any number of observers can simultaneously examine the image previously accessible only to one individual; one can point at the object in question, measure it with a compass, directly compare it with other photographs of the same or other objects, in short, do everything that can serve to reach an understanding about the disputed subject.

Another, perhaps even more significant, benefit of photography lies in the rigorous scrutiny it forces upon the microscopist regarding their own observations. Drawings of microscopic objects are almost never true to life; they are always more beautiful than the original, with sharper lines and stronger shadows. And what can a sharper line or a darker shadow in a suitable place not do to give the image a completely different meaning? The selection of the specimen is also irrelevant for the drawing; for even from a poor and unsubstantiated specimen, a correct and seemingly conclusive drawing can be produced.

This is, of course, impossible with photographic imaging. Here, the shadow of the specimen itself is captured as the image, and the microscopic object draws itself; it is therefore not at all possible to exert any corrective influence on the individual parts of the image. Therefore, nothing remains but to produce specimens that not only meet one's own standards but are also capable of withstanding comprehensive criticism regarding their evidentiary value.

Anyone who publishes drawings of their microscopic specimens can hardly expect criticism, since the drawing is inevitably made according to the author's subjective perception. However, anyone who publishes a photogram relinquishes any subjective influence on the depiction of their specimen; they essentially present the specimen itself to their audience and allow the latter to participate directly in their observation. This awareness of having to openly expose the specimen, reproduced in a photographic image, to the scientific world for criticism compels the microscopist to repeatedly account for the accuracy of their observation and not to make the results of their investigation public until they are absolutely certain of their findings. The widespread use of photography in microscopic work would certainly have prevented a large number of immature publications.

A few examples may illustrate the value of photography, especially for research in the field of infectious diseases.

Lewis* devoted considerable attention to the study of bacteria and, among other things, examined the relapsing fever spirochetes during the relapsing fever epidemic in India. He concluded that the relapsing fever spirochetes found in India were thicker than the European ones he assessed based on my photographs in Cohn's articles. Lewis is known as a meticulous observer, and his findings deserve full consideration. Science would thus have had to grapple with these two different relapsing fever spirochetes, the Indian and the European, and would also have had to reckon with the possibility suggested by Lewis: that, due to this difference in the spirochetes, the disease they caused would also be different.

*) The microscopic organisms found in the blood of man and animals. Calcutta 1879.

Fortunately, however, Lewis simultaneously published photographs of his Indian spirochetes, and the alleged difference is immediately cleared up. In Lewis's photographs, one can see both the blood cells and the spirochetes surrounded by the lines of interference, an unmistakable sign that an illumination aperture too small in relation to the intensity of the light was used during the examination and presumably also during the crucial observation. Every microscopist knows that the narrower the illumination aperture, the darker and broader the contours of the objects appear, and that if the light is also very intense, e.g., sunlight, as was probably the case with Lewis, the dark and broad edges of the object are immediately surrounded by the color fringes caused by interference.

Furthermore, every microscopist familiar with modern examination methods knows that stained bacteria are not illuminated with narrow apertures, but on the contrary, with the widest possible apertures, and in some cases, the aperture is omitted entirely, and diffuse light is used, in order to fully exploit the color effect and to see perfectly sharp, clear outlines. My photographs were also taken using diffuse illumination, and one will not notice the slightest trace of interference lines on them. Lewis thus measured the true diameter of the spirochetes on my photographs, and simultaneously the broad interference fringe on his. Had he only published a drawing, on which interference lines are not known to be shown, then the error might never have been cleared up.**

**) In addition, I would like to mention that I had the opportunity to photograph genuine Indian relapsing fever spirochetes, which I received from Dr. Caster in Bombay, in my own way (Table IV, Photo 21), and there they appear to be completely identical to the European ones.

Clarity also came to me only through photography in another respect. When I found descriptions of plasma cells, plasma spheres, swarming micrococci, etc., in Letzerich's publications, I simply couldn't imagine what Letzerich actually meant or what he had seen until the photographic illustrations accompanying his work on morphological differences in schistomycetes were published.*** A glance at these photographs immediately reveals that the plasma cells and spheres are quite ordinary, developing micrococcal colonies embedded in isinglass (a gelatinous substance). They remain in a closed mass for a longer period than if they were in a liquid. Eventually, the gelatinous substance liquefies, and the cluster of micrococci disperses (swarms).

***) Archiv für experimentelle Pathologie und Pharmakologie, Band 12 Heft 5.

I have had the opportunity to comment on Zürn's photographs before. They suffer from almost every flaw that can occur in photomicrography, for they lack any sharpness, are mostly not even properly aligned, have very pronounced interference lines, and, most importantly, are partly retouched. Nevertheless, these imperfect photographs are still infinitely more valuable to me than the most beautiful drawing.

Zürn's photographs demonstrate at first glance what to make of his statements about anthrax, anthrax-like diseases, and the bacilli involved. Although Zürn claims that anthrax bacilli have no characteristic shape, even in the blurred images of anthrax bacilli in Zürn's photographs, the actual anthrax bacilli can be immediately and unmistakably distinguished from other bacilli. Anyone who takes the trouble to compare my photographs (Cohn’s Beiträge, Vol. 2, Issue 3, Plate XVI, No. 5 and 6) with Zürn's will immediately recognize the anthrax bacilli in Zürn's Figure 4 on Plate II and the putrefactive bacilli depicted in my photograph No. 6 in Zürn's Figures 2 and 4 on Plate I, which are also supposed to be anthrax bacilli. In describing this latter photograph, I already pointed out the danger of confusing these similar, yet, as can be seen here again, clearly distinguishable bacilli. The Zürn case proves that this warning was justified; I can therefore only repeat it and, moreover, refer the reader to the subsequent works on anthrax and septicemia for further details on this subject.

The necessity of photography for illustrating publications about infectious diseases will be explained using an example.

Semmer** enriched science with numerous reports on pathogenic bacteria that he claimed to have found in rabies, distemper, septicemia, rinderpest, glanders, and typhus. But what are we to make of Semmer's claims when we consider the illustrations of his pathogenic bacteria that accompany the aforementioned treatise? I certainly don't want to claim that Semmer had no bacteria at all before him, although his figures could just as easily represent anything other than bacteria. But what kind of bacteria they were, and whether they can truly be considered pathogenic, seems at least doubtful to me, especially when one compares the bacteria of rabies with those of rabies in cattle, and the anthrax bacteria, which look almost like dental spirochetes, with the bacteria of distemper and typhus.

**) Virchow's Archiv, Bd. 70, S. 371.

In light of what I have said, and considering what I have already reported about Fokker's investigations and Semmer's illustrations, to which I could add much more, I am sure no one will hold it against me if I remain highly skeptical of any bacterial drawing that I cannot verify for accuracy on a specimen. I cannot urge all those working in this field strongly enough to support their discoveries with photographic images as evidence. This is not to say that photography should replace all drawing; it never can or will, and drawing will remain irreplaceable in many cases. But where photography is applicable—and experience has shown that it is almost without exception for microorganisms—it must be fully utilized in the interest of the subject.

Anyone who is unwilling or unable to undertake the somewhat more difficult task of photographing sections should prepare simple coverslip slides of the microorganisms they are studying, which are easily obtained from blood as well as from the tissue fluid of any organ, and photograph them themselves or have them photographed. It is essential, however, to keep in mind that the photograph should reproduce the object as it appears under ordinary microscopy. Therefore, the illumination must correspond precisely to that which is otherwise considered most advantageous for observing the object in question. If the pattern on diatom shells is best seen in direct sunlight and under oblique illumination, then it should also be best photographed under the same lighting conditions. However, no one examines stained bacteria in direct sunlight, so they should not be photographed in such conditions.

To obtain good photographs of stained objects, three conditions must be met. The preparation must be impregnated as intensively as possible with a dye that does not transmit blue light, thus having the same effect on the light-sensitive layer as a black color that absorbs all light. These are primarily yellow and brown dyes. The correct choice of dye can be immediately assessed by viewing the stained preparation in monochromatic blue light, for example, in the light passed through a copper ammonia solution. In this case, the cell nuclei, bacteria, etc., should appear more or less intensely black against a blue background.

Strong magnifications can only be achieved with the help of sunlight, but for the reasons already explained, sunlight projected directly onto the object to be photographed is not advantageous for our eyes and must therefore be diffused by one or more frosted panes.

The third requirement is a condenser or lighting apparatus designed so that the scattered sunlight illuminates the object brightly from all directions in the widest possible cone of light, thus obscuring the structural image. Essentially, these are the same conditions used to achieve the best possible optical image, and only someone with no understanding of photography might suspect special tricks employed to create the illusion of more photographs than actually exist.

But even in the further processing of the negatives and the production of the prints, one should never forget that the photographic image is not merely an illustration, but primarily a piece of evidence, a document, so to speak, whose credibility must not be subject to even the slightest doubt. Therefore, any touch-up of the negative or the print, however insignificant, would rob it of its entire value. It's actually so obvious that one hardly needs to say a word about it. But because retouched micro-photographs have nevertheless been published, it was necessary to address this point and to protest most vehemently once and for all against the exploitation of retouched negatives.

The great importance I attach to microphotography led me to make the best possible use of it for my investigations. After successfully photographing bacteria that had dried onto coverslips, I also aimed to photograph bacteria still embedded in tissues, i.e., in sectioned preparations. Since achieving good brown staining of nuclei and bacteria is not easy, my initial goal was to photograph blue- and red-stained preparations using dry plates and appropriately colored lenses.

However, after many unsuccessful attempts, this plan had to be abandoned, and I had to resort to the other method, already proven effective with coverslip preparations, of staining the objects brown. In some cases, this yields excellent results; in others, it leaves much to be desired compared to blue or red staining and requires further improvement. However, in order to give an idea of what can currently be achieved in this field, I will publish a number of examples from my collection of negatives following this work, which should be of interest not only as photographs, but also because of the subject they depict.

Transmissibility of pathogenic microorganisms.

The methods discussed so far demonstrate the presence of microorganisms in the animal body. If the examination reveals that the parasites are present in large quantities, or that they have caused irritation, necrosis, etc., in the affected tissues, then their pathogenic nature is established.

However, our primary interest lies in the question of whether the microorganisms identified as pathogenic are also infectious, transmissible from one body to another. The two terms pathogenic and infectious must not be confused. One can easily imagine organisms capable of migrating into the animal body and causing illness—that is, pathogenic—but lacking the ability to directly spread from one body to another and infect it. Assuming that intermittent fever is a bacterial disease, which still requires further proof, it would provide an excellent example of the existence of a pathogenic but non-infectious microorganism.

The properties of pathogenic and infectious are therefore not synonymous, and if a parasite is identified as pathogenic, it must also be experimentally determined whether it is transmissible or not. The method used for this purpose, if it is to be successful, must closely resemble naturally occurring conditions—a principle that was disregarded in the early days of experimental investigations of infectious diseases and is often still disregarded today. Attempts have been made, in the most primitive way, to transmit diseases previously observed only in humans to dogs, cats, rabbits, guinea pigs, and similar animals. Gradually, however, experience taught us that the choice of animal species for infection experiments is by no means irrelevant, and that, moreover, the method of transmission has the greatest influence on the success of the experiment. To discuss all these factors in detail would go too far here, and only the most essential aspects can be briefly highlighted to characterize the principles according to which our work was carried out.

Regarding the selection of experimental animals, it is advisable to first use animals of the same species as those from which the infectious material originates. Only if this is not feasible should related species be used. In the case of human infectious diseases, then it is likewise necessary to use the animals most closely related to humans, namely monkeys, as the striking example of relapsing fever demonstrates, which, so far, can be transferred to no other animal species but monkeys, and to them with ease and certainty.

However, the experiment must not stop at transferring the infectious agent to individuals of the same or related species: the reaction of as many different animal species as possible to the infectious agent must then be examined. In doing so, one will encounter quite peculiar variations in the mode of action of the parasitic organisms, which are important for the study of the infectious disease in question. There are animal species that react most promptly and without exception to the infectious agent introduced into them; others, however, remain more or less immune.

Differences also exist with regard to the spread within the animal's body; for the same bacteria that immediately cause a fatal systemic disease in one animal species can induce a localized, non-fatal infection in another. Highly instructive observations can be made in such experiments regarding the extraordinary sensitivity of pathogenic bacteria to the medium on which they can thrive or which they reject. Within the same class of animals, e.g., rodents, infection succeeds in some but not in others. On a previous occasion, I was able to point to a very striking example of this kind: the ease with which house mice are infected with the small bacilli of mouse septicemia, while it was not possible to kill a field mouse with the same parasite. This sounds quite paradoxical, and yet this fact has been established through numerous experiments and later extended to other cases through many similar observations. To highlight just a few, mice are so susceptible to anthrax infection that they can be used as a completely reliable test for the efficacy of anthrax bacilli. Rats, on the other hand, are more or less immune to anthrax. Rabbit septicemia kills rabbits and mice with absolute certainty, leaves guinea pigs and rats unaffected, but can also be very easily transmitted to sparrows and pigeons.

Also very remarkable in this respect is the different behavior of animals of the same genus but of different ages, which has been observed several times, particularly in the case of anthrax infections, and has been mentioned by various authors. Very young rabbits are apparently quite easily infected with anthrax, while older ones are hardly infected at all. Rats behave similarly with anthrax. The same is true for mouse septicemia, which, when administered to very young rabbits, causes a systemic infection, just as in mice, and kills the animals, while in older animals it only produces a local effect.

A more detailed discussion of these highly interesting relationships will be given in the relevant works. At this point, I only wanted to mention them to show how important the correct selection of test animals is. This applies especially to the immunity experiments currently being pushed so much to the forefront, and, based on what has been said, it is probably only necessary to hint at the errors that can arise when, in such experiments, young and old animals are mixed together without further consideration and subjected to infection tests against which the older animals might already be immune.

The particular prevalence of pathogenic bacteria for certain animal species is reminiscent of the similar behavior of parasites in general, which often restrict themselves in the most idiosyncratic ways to a single type of plant or animal as their host. For higher parasites, these are such well-known facts that they are almost taken for granted. Therefore, it would never occur to anyone, for example, to conduct breeding experiments with tapeworms in water, simply because tapeworm relatives live in water.

But isn't it almost the same undertaking when, as one still hears and reads daily, breeding experiments with the most sensitive microparasites are carried out quite stereotypically in Cohn's or Pasteur's nutrient solutions? It is not enough to advise all those who wish to conduct cultivation experiments with pathogenic organisms to consider these conditions.

This is not enough. The manner in which the infectious agent is transmitted deserves no less attention.

The most frequently practiced procedure is vaccination. We are accustomed to understanding vaccination as a very small, superficial injury to the epidermis followed by the application of the vaccine, and accordingly, it is no longer considered a true vaccination if the injury penetrates the epidermis and extends into the subcutaneous tissue. In modern times, however, the term vaccination no longer seems to be so narrowly defined; now, all sorts of things are called vaccination, and French experimenters, in particular, have been prolific in subsuming under the term "vaccination" the most diverse types of subcutaneous, intravenous, and other methods of transmission.

Such a broadening of the term would be meaningless if it were not simultaneously a confusion of terms, as in this case; for these different methods of transmitting infectious agents are by no means identical in their effect. As the example of the bacilli of malignant edema (the so-called Vibrions septiciques), discussed in another work, demonstrates, vaccination can, under certain circumstances, have a completely different effect than subcutaneous injection, even when using the same material. Far too little attention is usually paid to the quantity of the infectious agent administered. Only when very small quantities are used can the disruptive side effect of dissolved, chemically active substances, which could cause intoxication instead of the intended infection, be avoided. However, there are also pathogenic bacteria that must be administered in larger quantities to produce effects. Therefore, it is all the more important to employ a wide variety of methods successively in transmission experiments, and never to omit the precise description of the mode of infection—whether simple vaccination, subcutaneous injection, transplantation, etc.—when describing the experiment.

Whenever vaccination is mentioned in the following works, it always refers to actual vaccination. Whenever any other method is used, it is described in such a way as to leave no doubt about the mode of transmission. I have a few brief remarks to make about some infection methods.

Actual vaccination is hardly feasible in mice; at most, such a minimal injury can be achieved on the ear that it is equivalent to a purely cutaneous injury. Any reasonably deep incision in the skin penetrates the subcutaneous tissue and should actually be called a subcutaneous application. It is by no means a simple vaccination if, for example, it is necessary to examine soil for infectious agents, one creates a pocket-shaped skin wound and introduces the infectious material into it. Infection via subcutaneous insertion of [unintellible] and similar methods, to which I will return on another occasion, belong to the same category.

Naturally, all instruments used in the infection experiments must undergo reliable disinfection, which, in my experience, can only be achieved in this case by prolonged heating to 150°C and above. It is often reported that disinfection was carried out with alcohol, carbolic acid, and the like. However, the unreliability of these substances will be evident from the disinfection experiments with anthrax spores, which will be described later. Therefore, disinfection by high temperatures remains the only option. For some instruments, such as knives, needles, etc., this presents no difficulties; they are simply heated to a high temperature.

However, disinfecting syringes used for subcutaneous injection is somewhat more complicated. Ordinary syringes, even those made of metal and glass, become completely unusable after several hours at 150°C, and lower temperatures are by no means sufficient for reliable disinfection. I cannot deny that many experiments have failed due to this obstacle, and that many inexplicable results of subcutaneous injections can be attributed to insufficient disinfection of the syringes.

Therefore, to counter any such objection, specially designed syringes were used for our infection experiments. On these syringes, the metal barrel is connected to the glass cylinder by a screw thread ground into the glass, and this connection is sealed by a perforated cork plug, which is replaced as needed. The plunger is wrapped with thread and cotton wool until it seals completely. Before each use, the syringe is heated in a drying box at 150°C for one or more hours, and then the plunger is moistened with distilled water sterilized in a pressure boiler. With these measures in place, any transfer of the infectious agent from one experiment to another via the syringe is entirely impossible.

For all cases where the local effect of the infectious agent is to be observed, inoculation in the ear, on the cornea, and in transplantation into the anterior chamber of the eye are particularly advantageous. A specific description of the procedures commonly used in these cases seems unnecessary, as they are already almost universally established.

Artificial infection by inhalation has been attempted several times, but unfortunately, a completely safe method has not yet been found. With inhalation through tracheal fistulas, infection of the tracheal wall remained a possibility; with inhalation through the mouth or nose, simultaneous ingestion of the infectious agent was a risk; and with Büchner's method of dusting the entire animal, infection of any minor injury to the body could not be ruled out. It would be highly desirable if a reliable method for this route of infection were discovered very soon.

In all infectious disease experiments, it should be an indispensable condition that one does not limit oneself to a single experiment and never neglects the necessary control experiments. How often do we still encounter reports that some suspicious substance or liquid was injected or subcutaneously into an animal, that the animal became ill, perhaps even died, and it is then taken for granted that the death resulted from the inoculation and the infectious disease in question? And yet it is obvious that a single such experiment proves virtually nothing.

First, it must be demonstrated that the single success was not apparent or accidental, and that the inoculation causes illness or death in the animals every time, or at least in such a large number of cases, that any possibility of chance is ruled out. Then, however—and I must emphasize this point because it has very often been overlooked—it must under all circumstances be proven that the substance in question is indeed a genuine infectious agent. The fact that a substance exhibits a pathogenic effect when administered subcutaneously or intravenously, or introduced into the abdominal cavity or otherwise into the body, does not in the slightest prove its infectious properties. Even unorganized, soluble substances can have similar effects. Only when transmission from one individual to another succeeds via such quantities of the vaccine that its reproduction and multiplication within the infected body are demonstrated, only then can this substance be considered infectious.

It therefore follows that anyone who wants to prove that they experimented with an infectious agent cannot possibly leave it at a single experiment, but must carry out a more or less long series of continuous transmissions, from one experimental animal to the second, from this to the third, etc., if they do not want to expose themselves to the justified objection that they were not dealing with an infectious disease at all, but with an intoxication disease.

Pure culture.

After establishing the presence of pathogenic microorganisms in the patient's body, their reproductive capacity within the body, and their transmissibility to other individuals, the most important task—and the one of greatest interest to hygiene—remains: investigating their living conditions. As already emphasized in the introduction to this work, this task can only be accomplished with the aid of pure culture, and therefore it is no exaggeration to say that pure culture is the focus of all investigations into infectious diseases.

Because the importance of pure culture has long been understood, all those who research in the field of infectious diseases have made every conceivable effort to perfect the methods of pure culture. The results of recent and very recent studies, however, prove most evidently that progress beyond the initial, weak attempts has been minimal. At best, we have learned to eliminate the most egregious errors, and even these are not always successful.

The essence of the pure culture method, as it is currently practiced, can be summarized approximately as follows:

A suitable, sterilized nutrient solution is placed in a disinfected container, which is sealed "fungus-tight" with disinfected cotton wool. This solution is then "inoculated" with the substance containing the microorganisms to be cultured. Once bacterial growth has occurred, the inoculation can be transferred from the first container, using disinfected instruments, to a second, similarly prepared container, and so on. In short, it is almost the same process as the transmission of an infectious disease from one organism to another.

Of course, certain precautions are taken during this process. Several prerequisites had to be met, namely, firstly, that the culture vessel was indeed disinfected. However, the debate between Pasteur and Bastian regarding spontaneous generation, and the famous question posed by Pasteur to Bastian—“Do you flame your glassware before using them?”*, which Bastian had to answer in the negative—demonstrates how unnecessary this disinfection process has sometimes been perceived to be.

*) Bulletin de l' Academie de med., 1879 p. 1230.

Secondly, that the disinfected cotton wool actually seals against fungi, which, according to Nägeli's** investigations, cannot be assumed to be true in all cases.

**) Ueber die Bewegungen kleinster Körperchen, 1879.

Thirdly, the nutrient solution must be both suitable and sterilized. I have already discussed what constitutes a suitable nutrient solution and why it is not always easy to obtain. Here, we will assume that a suitable nutrient solution has been found and only needs to be sterilized. Anyone who has had the opportunity to work with hay infusion, meat extract, or malt extract solutions as nutrient solutions knows the difficulties and risks associated with this task. Small quantities of such and similar nutrient solutions can be sterilized fairly reliably in suitable apparatus, but how difficult it is to render larger quantities free of viable bacterial germs can be seen from several series of experiments described in the paper on disinfection with steam.

Fourthly, the inoculation substance must contain no microorganisms other than those intended for pure cultivation. If even a very slight contamination of the inoculation substance exists with a more rapidly multiplying type of organism than those to be cultivated in pure form, the intended purification can never succeed, as Büchner aptly demonstrated. Therefore, in order to obtain pure starting material for his experiments with anthrax bacilli, Büchner employed a peculiar method. He infected the nutrient solutions with anthrax substance diluted to such an extent that, according to approximate calculations, only one bacillus entered the culture vessel, and then concluded from the characteristic macroscopic appearance of the developing culture that the purification had been successful.

However, I will later demonstrate that there are bacilli which develop macroscopically in nutrient solutions in exactly the same way as anthrax bacilli, and which, if accidentally mixed with the latter, would be indistinguishable using Buchner's method. This method would be entirely impossible to apply to bacilli which do not exhibit any characteristic forms in the nutrient solution, but perhaps only cause simple turbidity, like so many other bacilli. The difficulty of obtaining completely pure material for inoculation therefore remains for the vast majority of cases and cannot be eliminated at all for these cases using the currently customary methods of pure culture.

Fifthly, that no germs from foreign organisms enter the culture fluid during the initial inoculation and likewise during each subsequent inoculation; a danger against which the experimenter, even if he only briefly lifts the protective cotton plug, can never be certain of preserving his pure cultures. Even if the first, second, and third inoculations are successful, the probability of contamination of the culture increases with the number of subsequent inoculations. To counteract this eventuality as much as possible, one usually continues the pure culture simultaneously in several samples and inoculates only from the one that, as visual inspection or microscopic examination shows, has remained pure.

Unfortunately, however, one cannot rely on this either. For, as I have already stated above, the uncertainty of macroscopic differentiation of such cultures is, and microscopic examination can only ever provide information as to whether the droplet taken as a sample and placed between the slide and coverslip is free of foreign admixtures. Even if isolated other microorganisms are already present in this droplet, how can one reliably identify them? The very first signs of contamination cannot be unequivocally detected macroscopically or microscopically. And if, by chance, further inoculation is carried out from such a supposedly pure but in reality already impure culture, and the invading organisms are superior to the cultured ones in their developmental capacity, then the further pure culture is irretrievably lost. In the next generation, the microscope will leave little doubt about the contamination, but this realization comes too late because it is impossible to get rid of the now massively present uninvited guests.

To gain a reasonable degree of certainty when carrying out a longer series of pure cultures in nutrient solutions, in my experience there is only one means of verification, which I have also used in my earlier experiments and especially in the investigations on the development of anthrax bacilli. This consists of limiting the quantity of culture fluid to such a small amount that it can be completely examined under a microscope and checked for purity. This is done by placing a drop of nutrient solution on a number of glass cells formed from a hollow slide and coverslip. The liquid is placed on the underside of the coverslip and must be spread into a fairly thin layer so that it can be completely viewed under the microscope even at the magnification required for bacterial examination. The culture is then placed at the edge of the nutrient solution, and its development and purity are monitored periodically under a microscope. If the preparation of the glass cells and the inoculation are carried out skillfully and at a moderate pace, one can be confident that at least half, and usually more, of the cultures will remain pure and suitable for further propagation.

Unfortunately, this method, which has proven effective for cultures of easily identifiable anthrax bacilli, also fails when very small and less characteristic microorganisms are to be cultured in purity. Further drawbacks of this method are that the organisms being cultivated can only be provided with a limited supply of air, and that the gaseous decomposition products, which inhibit the further development of the culture, accumulate in the confined space. Therefore, this method is only applicable in isolated cases.

All in all, the situation with pure cultures is rather bleak, and no one who has undertaken the cultivation of microorganisms in the previously customary manner, and who has not absolutely avoided all the sources of error I have indicated (which, in my opinion, is entirely impossible), can complain if the results of their experimental research are not considered to have been obtained by exact means under the current conditions and are therefore not recognized by science as conclusive evidence. This probably applies most strongly to the work, carried out with a commendable but simultaneously blind zeal, that is now emerging en masse from the Pasteurian school and is achieving incredible results with pure cultures of organisms of rabies, smallpox, pneumonia, etc.

As has been repeatedly emphasized, pure culture is absolutely indispensable for the further development of the study of pathogenic organisms and everything connected with them, and some way must be found to obtain an easy-to-use and precise method for it. On the path now being pursued, there seems to me no prospect of sufficient improvement. Attempts have been made to carry out inoculations and booster vaccinations under the protection of an antiseptic spray. It is not impossible that this might destroy some airborne bacteria that are still capable of development; however, how little the usual antiseptic agents are effective against spores can be seen from my disinfection experiments with anthrax bacilli and other bacteria, which I will mention later. These experiments have established that a spray containing a solution of carbolic acid, salicylic acid, potassium permanganate, etc., has absolutely no effect on bacterial spores upon brief contact, and that, therefore, despite the spray, an entire category of airborne germs can contaminate culture media at any time.

Klebs* describes an improvement consisting of a fungus-proof plug in the upper part of the culture vessel, formed from calcined asbestos. Before taking samples for examination or further cultivation, the asbestos layer is reheated, destroying any germs that may have accumulated on it in the meantime. The plug is then pierced with a calcined needle, and the sample is taken from the culture vessel using a calcined glass tube. I can only see this as a further complication of the already very cumbersome procedure of purification, without it providing any corresponding protection against contamination during further injection. Even if only a very small opening is made in the plug, the risk of germ ingress is only reduced, but never completely eliminated. Furthermore, when transferring the sample from one container to another, it must always pass through the air, either directly or through the glass tube, and can pick up contaminants along the way.

*) Archiv für experimentelle Pathologie und Pharmakologie, Bd. XIII, S. 432.

It is quite obvious that all efforts in this direction are futile. Therefore, I have completely abandoned the principle of pure culture I have followed until now and embarked on a new path. A simple observation, which anyone can easily repeat, led me to this point.

If you halve a cooked potato and leave it cut-side up in the air for a few hours, then place it in a humid room, for example under a moistened glass bell jar to prevent it from drying out, you will notice, depending on the temperature of the room, on the following, second, or third day, that various very small droplets form on the potato, almost all of which seem to be different from one another. Some of these droplets are whitish, porcelain-like in color; others are yellowish, brown, light gray, or reddish; some look like flattened water droplets; others are hemispherical; and still others are wart-like. But all of them enlarge to a greater or lesser extent, and mycelium of mold spores appears among them. Finally, the individual droplets merge, and soon the potato begins to rot.

If these droplets are now examined microscopically while still isolated, ideally after being spread on a coverslip, heated, and stained, it turns out that each one consists of a specific type of microorganism. For example, one droplet contains large micrococci, another very small ones, a third micrococci arranged in chains, while other colonies, especially the flat, membrane-like ones, are formed by bacilli of varying sizes and arrangements. Some consist of yeast cells, and occasionally, the mycelium of a mold sprouting from a spore is found.

The origin of all these different organisms will not be in doubt for long if another potato, peeled with a hot knife to remove the bacilli spores that always adhere to the peel along with the soil and are not killed by the brief boiling, is kept away from the air in a disinfected beaker with a cotton wool plug and observed. No droplets form on the potato treated in this way, no organisms settle on it, and it remains unchanged until it gradually dries out after several weeks. Therefore, the germs that developed into the small, droplet-like colonies on the first potato could only have settled from the air, and one often finds, indeed, a clearly recognizable dust particle or fiber in the center of the small colony, which served as a carrier for the germs, be they dried, still viable bacteria, yeast cells, or spores.

However, to avoid any misunderstanding, I must add that on the unpeeled potato, individual colonies develop from the edges, arising from germs that are located on the peel, specifically in the soil adhering to it.

What conclusions can be drawn from this observation of the colonies growing on the potato? Most striking is the fact that, with a few exceptions—where presumably two different germs were so close together that the resulting colonies were bound to merge, or where the same speck of dust was laden with different germs and these began to develop simultaneously—each droplet or colony is a pure culture and remains so until, during further growth, it collides with its neighbor and the individuals of one colony mix with those of another.

If, instead of the potato, the same surface area of a nutrient solution had been exposed to the air, then germs from the air would undoubtedly have settled on its surface, and indeed approximately the same number and the same types as on the potato. The development of these germs would have proceeded in a completely different manner than previously described. The motile bacteria would have rapidly dispersed in the liquid, mingling with the initially somewhat cohesive, floating colonies of immotile bacteria. Their lively movements would have also disturbed and scattered these colonies far and wide. Some organisms would drift at the bottom of the liquid, others in the upper layers. Some that found a spot on the potato where they could multiply undisturbed would be suffocated by the more vigorously growing organisms in the nutrient solution as soon as they began to grow, and would never develop at all.

In short, from the very beginning, a microscopic examination of the entire liquid would reveal a chaotic mixture of forms and shapes, never even remotely resembling a pure culture. But what is the fundamental difference between the nutrient medium that the potato offers the microorganisms and that provided by the nutrient solution? The difference lies solely in the fact that one is solid and prevents the different species, even if they are mobile, from being mixed together, while in the other liquid nutrient substrate, there is no question of the species remaining separate at all.

It was therefore logical to further exploit the advantages offered by a stable substrate for pure cultures. Individual colonies, previously described as arising on cooked potatoes, were spread as widely as possible onto other freshly cut potatoes and placed in the humid room. Soon, as early as the following day or the day after, a plentiful development of the inoculated microorganisms occurred, and they retained precisely the same characteristic properties as the original droplet. If this droplet was yellow and consisted of small micrococci, then an extensive yellow layer, consisting of exactly the same small micrococci, now appeared on the infected potato. Other micrococci, the various types of bacilli, yeast, fungi, etc., behaved in exactly the same way. All of these colonies, initially very small, were maintained quite quickly from a few propagations on further potatoes in plentiful quantities and in perfectly pure cultures.

A very careful sealing off of the air from these cultures was not at all necessary, for even if here and there germs from other organisms found their way onto the potatoes, they could only develop locally and spread slowly, but were never able to endanger the entire culture; moreover, they were immediately identifiable as foreign organisms among the cultured organisms by the different appearance of their colonies, so that all these accidental contaminations of the culture could easily be avoided during further inoculation. Only if one waited too long did such a proliferation of foreign organisms occur that the culture was endangered. But experience soon taught one to observe the right time for further propagation in order to always maintain completely pure cultures.

Thus, the possibility of producing entirely flawless pure cultures in a most simple way was given. At least with all those organisms for which cooked potatoes are a suitable nutrient medium, and their number is not insignificant. As already mentioned, numerous different micrococci and bacilli grow abundantly on potatoes, and it was natural to also transplant other known and practically interesting bacteria onto this medium. Hay bacteria were therefore introduced onto cooked potatoes, and very vigorous cultures were obtained, which formed a whitish, creamy coating on the cut surface of the potato and were easily distinguishable from other bacilli colonies that spontaneously arise on the potato, particularly from the bacilli that most frequently appear as a small wet spot at the edge of the potato and soon transform into a veil-like, folded membrane, producing a tenacious, stringy mucus.

After this experiment proved successful, another was conducted with anthrax bacilli, and this too was highly successful, as I will discuss in more detail elsewhere. However, with other bacteria that had proven pathogenic in animal experiments, all culture trials on potatoes were unsuccessful.

But the principle had been found, and it was simply a matter of giving it a form suitable for all cases. It would be pointless to describe all the experiments that were conducted to find a firm nutrient medium similar to a cooked potato, but suitable for all microorganisms, including pathogenic ones. I will now present the final result of these experiments, which in its current form already provides a perfectly adequate method for pure cultures in the vast majority of cases and, with further refinement over time, will undoubtedly satisfy all requirements.

Having realized that it is virtually impossible to construct a universal nutrient solution equally suitable for all microorganisms, I limited myself to converting already known and other new, proven nutrient solutions from a liquid to a solid, rigid form. The most suitable means of achieving this is the addition of gelatin to the nutrient solution. Isinglass and other gelatinizing substances are far less suitable.

The mixture of nutrient solution and gelatin, which for brevity I will call nutrient gelatin, is prepared as follows: The gelatin is allowed to swell in distilled water and then dissolved by heating. The nutrient solution is also prepared separately, and both liquids are given such a concentration that, after mixing them in a specific ratio, the desired final content of gelatin and nutrients is achieved. In my experiments, I found that the most suitable gelatin concentration for nutritional gelatin is 2.5 to 3 percent. Therefore, if the gelatin solution and the nutrient liquid are to be mixed in equal parts, then to achieve a gelatin concentration of 2.5 percent, the gelatin solution must be prepared with 5 percent gelatin. Similarly, the nutrient solution would need to be doubled in concentration; for example, to prepare nutritional gelatin with 1 percent meat extract, a 2 percent aqueous meat extract solution would be used. Incidentally, the gelatin can also be allowed to swell and dissolve directly in the nutrient liquid.

Gelatin is usually weakly acidic, and therefore it is necessary, at least when nutrient gelatin is to be used for cultivating bacteria, to neutralize it with either potassium carbonate, sodium carbonate, or sodium phosphate. The neutralized nutrient gelatin is then boiled again and, because precipitates form either during this process or even earlier during mixing and neutralization, and because the gelatin is often contaminated, it is filtered.

Meanwhile, a container sealed with cotton wool is disinfected for an extended period by heating to 150°C. The nutrient gelatin is then poured into this container, sealed with the cotton wool plug, and boiled again. The boiling process only needs to be very brief, as it is intended only to render harmless the easily killed microorganisms present in the nutrient gelatin. The spores present would only be destroyed by prolonged boiling, which is not feasible here because it reduces the gelatin's ability to gelatinize. For the same reason, sterilizing the nutritional gelatin with steam at higher temperatures is also ineffective. [Note: Brock, in his translation of part of Koch’s text, comments that “Nutrient gelatin can be steam-sterilized if higher concentrations of gelatin are used.”] Therefore, the manipulations performed so far have not yet definitively sterilized the nutritional gelatin; however, this is not a problem. If the nutrient solution were liquid, any remaining viable spores would develop into bacteria, multiply rapidly, and spread throughout the entire liquid, only revealing their presence on the second or third day through cloudiness. At that point, however, the liquid would be beyond saving, as its original composition would already be altered, and it might even be contaminated with countless newly formed spores.

However, the situation is quite different with nutritional gelatin. Here, the enormous advantage that the solid consistency of the nutrient substrate offers for assessing its bacterial content becomes apparent. The next day, or a little later, one will notice a few or even numerous small, opaque, whitish dots, scattered fairly evenly throughout the previously clear, solidified gelatin. If the gelatin were left to its own devices, these dots would soon enlarge into small spheres, which would steadily increase in size, gradually enclosing a space that, as becomes apparent when the container is moved, is filled with liquefied gelatin, and finally, by merging, transform the entire nutritional gelatin into a cloudy liquid. The small colonies growing from the whitish dots consist of bacilli, which can easily be verified by microscopic examination.

However, if one is aware of this and wants to sterilize the gelatin for culture purposes, then one naturally doesn't wait until the bacteria have reached a considerable size, but kills them by boiling the gelatin as soon as they are visible to the naked eye. This, as already mentioned, is a significant advantage of nutritional gelatin: the very first stages of bacterial development cannot be overlooked within it. For the bacteria emerging from a germ must remain concentrated in one spot for a long time, thus becoming noticeable to the eye even when their numbers are still relatively small, and so early that they have not yet formed spores and have not yet significantly altered the nutrient solution itself.

A further advantage of nutrient gelatin over liquid nutrients is that one can, to a certain extent, deduce the quantity of remaining germs, considered impurities, from the number of colonies that have formed. Furthermore, the manner in which the germs entered the vessel containing the nutrient gelatin is readily apparent; for all germs that remained capable of development after the gelatin was boiled distribute themselves fairly evenly throughout the liquid mass and later appear as colonies embedded within the solidified gelatin mass.

In contrast, everything that comes into contact with the nutrient gelatin after it has solidified—for example, germ-laden fibers falling from incompletely disinfected cotton wool, dust particles passing through the poorly sealed cotton plug, or germs adhering to the upper part of the container, particularly its neck—must settle on the surface of the gelatin and can only grow into colonies there. Thus, one is always able to monitor the condition of the nutrient material for the pure cultures and immediately recognize and correct any errors that may have crept in during preparation. The value of this constant oversight of any errors, and how it quickly leads to practice and great confidence in handling the procedure, hardly needs further explanation.

One soon learns whether the nutrient gelatin prepared with a particular nutrient solution is easy or difficult to sterilize. With some solutions, such as alkaline urine or Pasteur's nutrient solution in the form of nutrient gelatin, sterilization is easy, usually achieved with a single boiling. With others, such as meat extract or hay infusion gelatin, it is more time-consuming; therefore, it is boiled once a day for several days. This is because one must not assume that all spores germinate at the same time. [This method of sterilization (a short boiling once a day, repeated over consecutive days) is called “Tyndallization” and was first described by John Tyndall in a text dating from 1877. Brock includes a part of that text in “Milestones in Microbiology”.]

Sometimes, isolated colonies can still appear for days after the last boiling. These colonies, as their position within the gelatin proves, were present from the beginning and did not enter later. However, as mentioned, even if this were the case, they would be detected early enough during the frequent inspection of the nutrient gelatin, which is essential during the first week, and rendered harmless by boiling again.

Regarding the further processing and application of the nutrient gelatin to pure cultures, it is particularly practical to fill the nutrient gelatin into a number of test tubes sealed with cotton wool and thoroughly disinfected by heat, along with the cotton wool. This allows for the immediate availability of a suitable quantity of the nutrient gelatin without having to liquefy the entire quantity each time and thereby risk contamination by opening the container. Since this suitable quantity, as will soon be seen, is only a small amount, approximately 10 to 15 cubic centimeters, no more is added to each individual test tube.

Because the pure cultures were so convenient and safe to prepare with potatoes, I preferred to give the nutrient gelatin a similar form. It can be poured into shallow watch glasses, small glass dishes, or the like, but in my experience, the most practical way to handle the cultures, especially for microscopic examination, was to spread the nutrient gelatin in the form of a long, wide droplet onto microscope slides. This is done with a previously disinfected pipette, and the slides themselves are also thoroughly cleaned before use and exposed to a temperature of 150°C for an extended period. The gelatin drop is made to a thickness of about two millimeters. After a few minutes, the gelatin sets, and the slides are placed on small glass trays wide enough to hold two or three slides side by side. Finally, several such trays, about four to six, are stacked on top of each other and placed in a constantly humid environment. For this purpose, I use glass dishes covered with flat bell jars and lined inside with moistened blotting paper. In such a environment, the gelatin drops are preserved from drying out for two to three weeks.

The inoculation of the organisms to be cultivated is then carried out by using a flamed needle or a flamed platinum wire to pick up the smallest possible amount of the liquid or substance containing them and then applying it in several, about three to six, transverse lines to the gelatin. The needle is handled in approximately the same way as the implanter is used when inoculating with sections. It is also good to keep the incision lines as shallow as when inoculating. The term "inoculation" would therefore be quite fitting for this manipulation. The inoculation is carried out in the same way on several slides, so that twelve to fifteen individual cultures are initiated without any effort or significant loss of time; for each inoculation incision represents a self-contained culture, completely independent in its development from the others. Actually, the number is even greater, because the individual sections of a incision can be examined separately and used for further cultivation.

No further protection is needed for the cultures against the ever-present dangers of contamination beyond the glass bell jar, which is not even completely airtight. It is inevitable that foreign organisms will enter the cultures during inoculation of the nutrient gelatin, when the bell jar is opened, and during microscopic examination of the cultures; however, these organisms can only develop at the point on the gelatin onto which they fall. Only occasionally does one of the inoculation points itself, or its immediate vicinity, become the site of foreign colonies.

But it is hardly conceivable that all the cultures would become so infested with germs within a short time that they would be unusable for further cultivation, and this does not actually occur, especially if the bell jars are not opened too frequently. Within a few days, the pure cultures have grown sufficiently to reach their maximum development and can be inoculated further. Especially when, as is the case with some bacteria, the gelatin liquefies during rapid growth, and furthermore when spore formation has already occurred, then allowing the culture to remain for any length of time is pointless and it must be transferred as soon as possible.

If individual cultures are to be protected from contamination for extended periods, they must, of course, be kept under cotton wool. However, even in this case, nutrient gelatin proves to be a reliable substrate because, with just a little practice, the purity of the colonies resulting from the inoculation can be determined with considerable certainty based on their shape and other characteristic features, even without the otherwise indispensable microscopic examination. Contaminants located outside the inoculation site can also be immediately identified as such.

At low temperatures, the development of cultures proceeds very slowly; some organisms even require a specific temperature to thrive. Gelatin cultures grow most abundantly at 20 to 25°C, and so far I have not encountered an organism that could not grow at this temperature, assuming it is even amenable to artificial cultivation.

However, should it become necessary to use temperatures above 30°C, at which the gelatin becomes liquid, then one would have to forgo gelatin altogether or utilize its properties to the extent that inoculation is carried out into the gelatin at a lower temperature, protected by a cotton wool seal. Only when no foreign colonies have appeared in the rigid gelatin after approximately 24 hours at 25°C, thus ensuring the greatest probability of a successful, contaminant-free inoculation, should the cultures then be brought up to incubation temperature.

A very important task in carrying out pure cultures, the difficulty of solving which, as I have explained previously, has caused most previous pure culture attempts to fail—namely, obtaining absolutely pure material for the initial inoculation—can be very easily accomplished with the aid of nutrient gelatin. If, for example, blood from a septicemic animal is to be used for cultures and the septicemia bacteria contained therein are to be cultivated in pure form, then there is absolutely no need for extraordinary preparations with antiseptic sprays, heated capillary tubes, etc., which ultimately all fail. It is perfectly sufficient, while avoiding any major contamination, which certainly presents no difficulties, to take, for example, some blood from the freshly opened heart or from any blood vessel using a heated needle and inoculate it onto the nutrient gelatin in a considerable number of strokes. Isolated fungal mycelia then grow in some sections, as well as some clusters of micrococci, whose germs can hardly be completely excluded during an animal dissection. In addition, there is a smaller or larger number, depending on the content of septicemia bacteria, of completely pure colonies of septicemia bacteria, immediately recognizable at low magnification by their peculiar matte sheen and extremely fine granulation. Among these are enough from which further cultivation can be easily initiated, and, if necessary, with the aid of a dissecting microscope. (Compare Tab. XII, Photo 70.) In this case, the foreign admixtures were in the minority, and therefore a larger quantity of pure colonies of the bacteria intended for further cultivation could be expected from the outset.

However, even if this ratio were to be reversed, or if only a very few of the bacteria to be sought were present in the mixture, the experiment would still succeed, albeit not as easily, but just as reliably. It would then only be necessary to inoculate the bacterial mixture in a highly diluted form and in numerous inoculations. Inoculating the mixture into still-liquid gelatin is also very advantageous under such circumstances, in order to distribute the various microbes over a larger area. Alternatively, one can thoroughly mix the liquefied nutrient gelatin with the smallest possible amount of the substance, then pour it onto microscope slides and select the relevant colonies from among the resulting ones using a microscope.

I have previously emphasized that different nutrient substrates are required for different microorganisms. To recall just one of the most obvious examples, bacteria and fungi cannot be cultivated to advantage on the same medium, because, generally speaking, the former thrive better on acidic substrates, while the latter prefer neutral or slightly alkaline ones. It is therefore necessary to work with as many different nutrient gelatins as possible, tailored to the requirements of the various groups of microorganisms and even individual species within the same group.

In our investigations, we used various nutrient gelatins, each offering greater advantages in different cases. Particularly noteworthy are: hay infusion gelatin, which provides an excellent nutrient material for some types of bacilli; wheat infusion gelatin; gelatin prepared with aqueous humor; gelatin with meat extract and peptone; another gelatin with meat infusion and peptone is especially suitable for some pathogenic bacteria; however, the best nutrient material for pathogenic bacteria is undoubtedly a nutrient gelatin made from blood serum and gelatin, about which I will make a few remarks due to its indispensability.

Sterilization of this nutrient gelatin cannot be achieved by boiling, as this would cause the proteins in the serum to coagulate; therefore, care must be taken from the outset to keep the blood serum as free as possible from impurities. Fresh blood is collected directly into a clean glass container and left to stand undisturbed until solid coagulation has occurred. The clot is then carefully loosened at the top and placed, covered, in a cool place for one to two days until a sufficient quantity of clear, slightly colored serum has separated. This serum is then drawn up with a heated pipette and mixed in equal parts with liquefied, previously well-sterilized gelatin (5%) into disinfected test tubes, which are immediately sealed with disinfected cotton wool.

To further sterilize the serum gelatin, the test tubes are placed several times, initially once a day, later at longer intervals, in a water bath at 52°C for half an hour to an hour. Using this method, I have always succeeded in obtaining completely sterilized blood serum gelatin.

For fungal cultures, nutrient gelatins with plum or horse manure decoction were used, which also provide an extremely favorable growing medium for them.

In addition to the advantages already discussed, cultures grown with nutrient gelatin offer the significant benefit that they can be examined under a microscope at any time without being damaged or having their development disrupted in any way. Admittedly, only low magnifications can be used, but these are perfectly sufficient for monitoring the cultures and selecting suitable areas for further cultivation. The slides containing the nutrient gelatin can easily be placed under a microscope and examined, for example, with a Hartnack System 4 and Ocular 3 or a Zeis System A.A., along with a high-powered eyepiece and a narrow aperture on the illumination apparatus. Very often, even at these magnifications, the individual bacteria within the colonies can be discerned, at least at the edges; larger bacilli, sarcina, and yeast are immediately and clearly identifiable as such within the individual colonies.

If higher magnifications are required in doubtful cases, one or more inoculation lines must be sacrificed and covered with a coverslip; in this way, even immersion systems can be used for the direct examination of the colonies. Usually, however, this is not necessary. If one observes a larger number of spontaneously settled and inoculated bacterial, fungal, etc., colonies on nutrient gelatin under a microscope at low magnification, one very quickly becomes convinced that each individual species possesses quite characteristic and easily recognizable properties in the form, shape, color, and growth of its colonies on nutrient gelatin.

This phenomenon is not remarkable when one remembers that similar conditions are repeated everywhere in the vast field of natural observation, namely wherever there is an accumulation of individuals of the same species. Even if the distance is so great that the individual is no longer clearly recognizable as such, or even if the size of the individual members is so small that they are no longer perceptible to the naked eye, one can still deduce with more or less certainty from the characteristics of the entire group, the heap, the swarm, the colony, the specific species to which it belongs; for most of the characteristics of the swarm are ultimately nothing more than the sum of the characteristics of the individual members.

Let us take color as an example. With a single animal or a single plant, the color may no longer be clearly recognizable due to the distance or the small size of the object, but as soon as a larger number of individuals of the same species are located close together, the color effect of all of them adds up, and an effect is created that is clearly recognizable to the observing eye. The same applies to movement. A single small object, barely perceptible to the eye or even invisible to it altogether, e.g., a bird, is not easily discernible. For example, a bird flying in the distance is either not perceived at all or perceived so indistinctly that it is impossible to form a judgment about its nature. The situation changes immediately when a flock of birds moves at the same distance. Not only does the larger number immediately catch the eye, but a trained eye can also recognize, from the shape of the flock and its overall movements, the species to which the individual birds comprising it belong. In the same way, other characteristics of the flock as a whole can be traced back to those of its constituent parts.

The situation is exactly the same in our case with the swarms and colonies formed by microorganisms, except that here, the naked eye is usually unable to adequately discern the characteristics of the swarm and must resort to a massive magnification, namely a microscope. With the aid of a microscope, however, the distinctive characteristics of the individual colonies—color, size, shape, etc.—can be perceived so clearly that it is easy to distinguish colonies belonging to different species.

For example, anthrax bacilli and hay bacilli in gelatin cultures are not at all confusable. The anthrax bacilli are never motile and always form clumps consisting of long, wavy, and curly threads, often twisted around each other. Hay bacillus, on the other hand, only develop into longer filaments in very young colonies. As soon as they develop further and, as is regularly the case, liquefy the gelatin, they are seen only as lively, mobile rods filling the interior of the colony and, at its periphery, burrowing into the still-solid gelatin in perfectly regular masses directed perpendicularly to the periphery, so that the colony appears as if surrounded by a radiating halo.

This creates such a characteristic image, so different from that of the anthrax colony, that one can immediately recognize both types of bacillus among all other microorganisms by the described characteristics. Other bacillus exhibit yet other forms; the mobile ones mostly form wreath-like figures similar to those of the hay bacillus, but differing from it in the shape and width of the radiating halo. Still other bacillus form colonies that resemble a far-reaching, intricately intertwined root system. In the disinfection experiments, we obtained one of the Bacillus strains that withstood heat the longest. It is rather plump and forms flat colonies on the gelatin, in which the individual bacilli are arranged close together in a mosaic pattern, thus forming no pseudo-filaments and showing no movement.

However, this is far from the complete range of different bacillus forms; it would be too lengthy to list all the species I have observed so far, and how many more might there be? Even more numerous are the various forms of micrococcal colonies, ranging from simple, colorless, spherical structures to fine- to coarse-grained masses, as well as brownish, reddish, yellow, white, etc., spirally coiled or leaf-like lobed, spreading masses. The clusters of Sarcina are easily recognizable and also occur in several different sizes. Yeast species behave in a very similar way. The fungi can be easily distinguished from one another by the fruiting bodies that develop so abundantly on the gelatin. (See Tab. IX, Photo 54.)

When bacterial colonies are located within the gelatin, their special characteristics are not as apparent as when they can develop completely unhindered and in contact with the air on the surface of the gelatin. It is therefore advisable to compare only the colonies located on the surface and to inoculate those located deeper within, whose origin is uncertain, onto the surface and allow them to develop fully there.

Some examples of gelatin cultures can be found among the photograms, to whose description I refer at the end of this work.

I have conducted numerous and often lengthy series of pure cultures with pathogenic and non-pathogenic microorganisms on cooked potatoes and nutrient gelatin, and not once have I encountered any organisms that exhibited any discernible changes in their properties. They all retained their external characteristics as well as their physiological properties, insofar as these could be ascertained, in exactly the same way from the beginning to the end of the observation, no matter how often they were examined or maintained in pure cultures for months.

Even when the nutrient substrate was temporarily changed, or when the intervals between propagation were made as long as possible in one series and as short as possible in another, or when spore formation was always awaited in one series while inoculation continued before spore formation in another, none of this had any influence whatsoever on the properties of the cultivated organisms.

Naturally, impurities of various kinds occurred. But if, for example, among fifteen inoculated strips of nutrient gelatin containing anthrax bacilli, twelve develop completely pure, in two brown micrococci clusters have formed alongside the anthrax bacilli, and in one, but only at one point along the long strip, hay bacilli have settled, and if, in addition, at individual points on the nutrient gelatin distant from the inoculated strips, some further micrococci clusters, several hay bacilli colonies, and fungal mycelia have developed, then surely no one would claim that in that one inoculated strip, and only at one point therein, the anthrax bacilli had immediately transformed into fully veritable hay bacilli without any further transitional forms. Such a claim would lead to the consequence that, because all other inoculation lines are under exactly the same conditions, one would have to conclude that in the two lines contaminated with micrococci, the anthrax bacilli transformed directly into micrococci, and that the micrococcal, hay bacillus, and fungal colonies that arose freely and away from the inoculation lines must have arisen by equivalent generation. It will probably be most difficult to accept this last consequence, and one will say that the freely formed colonies originate from airborne germs that fell onto the gelatin. However, nothing then precludes the assumption that, quite by chance, micrococcal germs settled on two of the inoculation lines and a hay bacillus spore settled on one spot of the other inoculation line. This example is by no means taken from entirely unusual circumstances, but rather, precisely as just described, and in a similar manner, the admixture of foreign organisms occurs in pure cultures.

Therefore, the objection that could be raised against the constancy of species in my pure-culture method—namely, that only the best and purest inoculation lines are always selected for further inoculation and that, consequently, a transformation of the species into another cannot occur—is not valid. In my method, only those species already recognizable by their broader characteristics are kept separate from the purebred species, which are separated by a wide gap. If very gradual transitions from one species to another were to occur, they would no longer be clearly perceptible, as they can only be minimal. Undoubtedly, these minimally altered organisms would also be further inoculated, and ultimately, without being able to prevent it, the morphologically altered species would be obtained.

My method would also not present the slightest obstacle to the transformation into a physiologically different variety, since the selection for further breeding is based on morphological, not physiological, criteria. But, I repeat, I have not observed any morphological or physiological changes in the species during my experiments.

In botany and zoology, it is a generally accepted principle to accurately describe, name, and provisionally register all previously unknown living organisms as distinct species. It has occasionally happened, however, that individual species initially considered independent have later turned out to be forms belonging to the evolutionary cycle of an already known species. But far more frequently, closer examination and the application of more refined methods and better instruments have necessitated the division of a species previously considered a single entity into several.

Remarkably, this established and universally valid principle—to distinguish all new forms that differ significantly in their characteristics until their relatedness is irrefutably proven—has been frequently disregarded in the field of microorganisms, particularly in the field of bacteria. From the very beginning of bacterial research to the present day, from Hallier to Nägeli and Büchner, we encounter attempts to lump together bacteria that, undeniably, differ greatly in their characteristics, and to create a single species, or at most a few.

If it should ever truly be possible to transform bacterial species from one known form to another through conversion or selective breeding, then it will certainly still be time to group these proven related forms into a single species. To date, this proof has not been provided, and there is not the slightest reason to deviate from the principles of general natural science in the study of bacteria.

Even if initially a few too many species were assumed, this would not harm science. However, if the usefulness and necessity of researching the various forms of bacteria and making them accessible to science is dismissed from the outset, then all further research and progress in this field will be thwarted, and this is certainly to the greatest detriment of the development of this young and promising discipline.

Truth and knowledge will undoubtedly prevail here as well, just as in other fields of knowledge, and overturn all untenable hypotheses. But as so often happens, true progress, which only moves along the path of arduous and slow-moving research, can be pushed into the background for a time by promising theories that seem to solve even the most difficult problems with ease, and even if no lasting harm comes to science as a result, the wrong direction can cause great harm by gaining influence for a time in some of the most important areas of healthcare and by having its teachings put into practice.

It therefore seems to me quite unobjectionable, and not only that, but the only correct thing to do, to allow a very careful separation of all microorganisms and especially bacteria encountered in our investigations, and with regard to the latter to adhere very strictly to the principle that all those bacteria which, on the same nutrient medium and under otherwise identical conditions, retain unchanged their properties by which they differ from one another through several cultivations or so-called generations, are to be regarded as different, whether one calls them species, varieties, forms, or however else one wishes.

Before I conclude this chapter on pure culture, I want to address an objection that I am certain I will face. It will be argued that my pure culture method is nothing new and that cultivating bacteria on potatoes and in gelatin is nothing new. That is indeed true. It has long been known that some bacteria grow quite well on cooked potatoes, and bacteria have also been cultivated in gelatin and isinglass jelly, but the advantages afforded by a solid culture medium were not recognized, because the isinglass and gelatin were used in such small quantities in the nutrient solution that they could not gelatinize, could not become a solid culture medium, or even if enough isinglass was present in the nutrient solution to solidify, the culture was started with impure material and, moreover, the cultures were kept at incubation temperatures, at which the jelly had to become liquid again. And how little the cultivations previously established on potatoes have to do with real pure cultures is shown by Wernich's investigations on Micrococcus prodigiosus, regarding which I refer to the work of Gaffky (cf. this publication), in which they are discussed in detail.

The distinctive feature of my method is that it uses a solid, preferably transparent, nutrient medium; that the nutrient substrates are varied as much as possible and chosen appropriately for the organisms to be cultivated; that all precautionary measures to protect against subsequent contamination are unnecessary; that further cultivation is carried out in a larger number of individual cultures, of which only the purest remain for further cultivation; and that, finally, continuous monitoring of the quality of the cultures is performed using a microscope. Thus, my method differs in almost every one of these points from the previously customary methods and especially from the earlier potato and isinglass cultures mentioned above.

It was very obvious to utilize the excellent properties of nutritional gelatins for other relevant investigations, especially wherever it is important to learn about the quantity and types of microorganisms present, e.g., in the air, water, soil, on transport objects, foodstuffs, etc.

Air examination.

How easily air transfers its components to nutrient gelatin, and with what ease and certainty the number and type of viable organisms that have settled on the nutrient gelatin can be determined, so to speak, will be evident to anyone who has conducted or observed even a few gelatin cultures. To obtain comparable figures, it would only be necessary to supply arbitrarily large quantities of air to a nutrient gelatin of a specific surface area, such that the latter would transfer all the microbes it contains to the former.

As simple as it initially seemed to fulfill this condition, its execution proved quite difficult. An initial attempt was made to filter air through disinfected cotton wool using an aspirator and to transfer the cotton plug, laden with airborne dust, into liquid nutrient gelatin, distribute it within, and protect it from further penetration of airborne germs by a completely airtight or merely dustproof seal. This experiment was successful insofar as the bacterial and fungal colonies developed well; however, being partially obscured within the gelatin and by the fibers of the cotton plug, they did not provide nearly the clear and well-defined picture that colonies formed from spontaneously deposited germs on the surface of nutrient gelatin. Therefore, this method was temporarily abandoned. However, although it does not seem particularly suitable for general air analysis, it could be used in certain cases, for example, when very specific quantities of air need to be examined within a short period.

Then, following the procedure of other known air testing methods, the air drawn in by the aspirator was passed against a drop of glycerin or a glass plate coated with glycerin gelatin. The glycerin droplet or the glycerin gelatin was mixed with enough nutrient gelatin so that, as preliminary tests had shown, the amount of added glycerin would not have any adverse effect on the nutrient gelatin. Other experiments conducted at the same time on the effect of glycerin on microorganisms, however, showed that glycerin has no adverse effect on spores of bacilli and fungi, nor on yeast, but kills many freshly dried and still viable bacteria after a fairly short time. Therefore, one obtains almost exclusively fungal, yeast, and bacillus colonies in the nutrient gelatin.

This method does not provide accurate information about the concentration of viable organisms in the air, because a number of them are destroyed before they can be exposed to conditions in which they could develop. Furthermore, I got the impression that the strong airflow required here carries away many dust particles and germs from the glycerin or glycerin gelatin and prevents them from adhering to it. Equal quantities of air, filtered through cotton wool at the same time and in the same place, would allow far more fungal mycelia and bacillus colonies to develop, quite apart from the micrococci that might be destroyed by the glycerin.

Attempts were also made to direct the airflow directly against the gelatin. When this was done using a narrow tube, the gelatin dried out on its surface opposite the air intake, and naturally, no dust particles could adhere to it. However, even with the widest possible opening, control tests showed that the air released only a few germs onto the gelatin surface, and this device could not be used either.

The limited success I had with the more or less rapidly moving airflow led me to the idea of allowing the air components to settle from a layer of air that was either barely or not at all moving. To a certain extent, if the still air layer was given a sufficiently high height, it was possible to expect equal volumes of air to deposit their solid components onto the nutrient gelatin within the same timeframe. However, it was necessary to arrange the vessel containing the nutrient gelatin in such a way that, similar to the previously described slide cultures, the surface of the gelatin could be brought directly under the microscope without damaging the colonies on it.

Thus, a very simple apparatus was created, but, it seems to me, also very easy to use and sufficient for ordinary investigations. It is set up as follows. At the bottom of a cylindrical glass vessel (5 cm in diameter and 18 cm high) is a shallow glass dish, 1 cm high (excluding the thickness of the base) and 5.5 cm in diameter, intended to hold the nutrient gelatin. To facilitate the removal of this glass dish from the cylindrical vessel for filling with the gelatin and for microscopic examination of the cultures, a narrow, right-angled metal strip is used. The glass dish rests on the short leg of this strip, which is positioned transversely inside the cylindrical vessel, and can be easily moved up and down using it. For continuous air examinations, a sufficient number of such vessels, at least twenty, are required. The cylindrical glass vessel, into which the thoroughly cleaned glass dish and the metal strip are inserted, is sealed with a large, firm cotton plug and exposed to a temperature of 150°C for one to two hours. After cooling, and with the cotton plug vented as briefly as possible, the glass dish is lowered to the edge of the cylindrical vessel using the metal strip. The cylindrical vessel is lifted and filled with 0.5 cm of sterilized nutrient gelatin, then lowered again and immediately sealed with the cotton plug. Even if individual germs from the air in the work area should happen to enter the gelatin during this process, they will sink to the bottom and, unlike the germs that later settle on the solidified surface, will not develop on the gelatin but within it.

Once the gelatin has solidified, the apparatus can be used immediately. At the location where the air is to be tested, the cotton plug is removed and stored in a way that prevents contamination; the simplest method is to place it in a second, disinfected cylindrical vessel kept as a spare. The vessel containing the nutrient gelatin is then left open for a specific number of hours, e.g., 5, 10, 12, or 24 hours. It is then sealed again with its cotton plug to prevent further contamination and are kept at a temperature of 20 to 25°C until the colonies have fully developed.

After just 24 to 30 hours, the first small colonies appear on the gelatin in the form of droplets or small, circular spots. By the second day, development has usually progressed to the point where microscopic examination and counting of individual colonies with the aid of a magnifying glass are possible. This should not be done later, otherwise the colonies will become too large and partially merge.

The properties of the gelatin are essential for the vigorous growth of such various microbes as those carried in the air, because it should simultaneously provide a favorable nutrient medium for molds and yeasts, as well as for bacteria. In an experiment conducted simultaneously with a number of different nutrient gelatins, it seemed to me that a gelatin prepared with wheat infusion would be best suited for air analysis, because on this the various categories of microorganisms developed most uniformly.

However, if time and manpower are available, I would consider it expedient to expose as many different nutrient substrates as possible to air simultaneously in separate glasses, e.g., cooked potato, plum infusion gelatin, blood serum gelatin, wheat infusion gelatin. If the sole purpose is the detection of pathogenic organisms, e.g., when examining air in hospital rooms, then meat infusion-peptone gelatin and especially blood serum gelatin should be used.

Over the course of last winter, I conducted fairly regular air tests using wheat infusion gelatin for several weeks to convince myself of the method's usefulness and to gain some insight into the presence of viable microorganisms in the air. The air testing methods used so far, such as filtering through cotton wool, aspiration against a drop of glycerin, etc., provide fairly accurate information about the quantity of dust-like particles in the air; even larger microorganisms, like fungal spores, can be counted.

However, no testing method has yet been able to determine the number of viable microorganisms in the air. To a certain extent, my method accomplishes this quite reliably. It is impossible to say, however, how much air the germs deposited on the nutrient gelatin were contained in, but on the whole, in the individual experiments, a fairly constant quantity of air, even when the apparatus is set up outdoors in more or less moving air, will always allow its dust-like particles to fall onto the gelatin, because the glass cylinder is so tall that the air in its lower part can always be assumed to be still.

In the aforementioned experiments, which, as mentioned, are only intended as preliminary tests, it turned out that there were far fewer bacterial germs but more mold spores in my workrooms than in the open air, which was examined in front of a window facing the garden of the veterinary school. In a collection room, which was rarely entered at the time the jars of nutrient gelatin were set up, considerably fewer bacteria and fungi were found than in the workrooms. A few isolated fungal mycelia and a handful of bacterial colonies had developed in a glass jar that had been left open for three days in a cupboard with a loose door. In contrast, almost as many fungi and bacteria had grown on gelatin placed next to the containers of the experimental animals as on gelatin exposed to the open air. However, even in winter, the open air contained so many viable microorganisms that after 24 hours of continuous opening of the jars, often well over a hundred individual colonies had formed on the gelatin, and the latter appeared densely covered with droplets and small spots once development had begun. Even after opening the jars for only 12 hours, the number of colonies still fluctuated between forty and eighty, which was still too many for a quick overview and for further examination of the individual colonies. Therefore, it seems most practical to me to open the jars for only 4 to 6 hours, and perhaps for an even shorter time in very polluted air.

A proper investigation of the organisms obtained from the air would have required cultivating them in pure cultures and testing their pathogenic and other properties. I lacked the time for this at the time, and I reserve the right to conduct further investigations in this direction at a later date.

Soil Examination.

Examining soil samples for their content of viable microbes is much simpler. No special preparations are required. The sample is spread in streaks, ensuring that the individual particles are not too densely packed, onto slides coated with nutrient gelatin. If the subsequent ingress of airborne microbes is to be completely ruled out, a similar or identical device to that used for air examination would be necessary. Incidentally, it is hardly possible to confuse microbes sown with soil with those that fall onto it later, because the colonies that develop from the former always originate from the sand grains and clods of earth.

A nutrient gelatin made from wheat infusion or meat infusion with added peptone is particularly suitable for these examinations as well. A small number of soil samples that I have been able to test for their microorganism content, which yielded fairly consistent results, suggest that the upper soil layers are exceptionally rich in bacterial germs. Remarkably, these are predominantly bacilli. In freshly extracted soil, micrococci are also found, but almost always in the minority. In soil samples taken from heavily contaminated sites, such as areas saturated with liquid manure, the micrococci outnumbered the bacilli, and molds were also present; however, this is only a local occurrence. Bacilli, on the other hand, appear to be consistently present in large quantities in the upper cultivated layers of inhabited areas and wherever horticulture and agriculture are practiced. They were found in soil from the veterinary school garden in Berlin, as well as in the soil of a disused burial ground and in soil samples from gardens and fields located far from densely populated areas.

If the soil samples are allowed to dry for several weeks, even the few micrococci in the cultures disappear, leaving only the bacilli, and in the same abundance as before drying. Since it is known that microorganisms that have not transitioned into dormant forms do not remain viable for long in a dried state, this phenomenon suggests that while the micrococci perished upon drying, the bacilli must have been present in dormant forms, i.e., as spores, in the soil. This assumption is further supported by the fact that, as repeatedly demonstrated in heat disinfection experiments, the bacilli germs in the soil withstood high temperatures, which only spores can survive.

It is highly probable to me that, because only spores and no or very few bacilli are found in the soil, these spores did not originate in the place where they were found, but rather entered the soil with agricultural waste, manure, and products of decay and decomposition. They may also have been carried far away by airborne dust from traffic areas where they could have formed, deposited on the soil, and mixed with its upper layers. The soil primarily contained the bacilli and hay bacilli mentioned earlier, which form root-like colonies on nutrient gelatin. In addition, approximately six to eight other well-characterized bacilli species were found in varying numbers.

I was able to observe a very striking fact, though based on only a few investigations, so I cannot yet claim its general validity. It appeared that the abundance of microorganisms in the soil decreases very rapidly with depth, and that at a depth of barely one meter, undisturbed soil is almost devoid of bacteria. Even in the heart of Berlin, in soil samples taken from freshly excavated building sites, I found no bacilli at a depth of one meter and only very isolated colonies of very small micrococci after sowing them on nutrient gelatin. In one instance, the soil came from a new building erected directly next to the Panke River on Philippstrasse, taken from a depth of two meters, at the level of the Panke water and barely two meters away from it, and this sample also proved to be exceptionally poor in microorganisms.

My investigations, however, were conducted only in winter, which should be taken into account. Conditions might be different in summer. However, if, according to the now universally accepted assumption, a vibrant microbial life exists in groundwater and the adjacent soil layers, even if only in summer, then the dormant forms of these organisms should remain there and, just as they are easily detectable in the upper layers, should also be found in the lower layers, even in winter. Since this is not the case, it seems questionable to me whether many microorganisms exist in the deeper soil layers at all.

Water Examination.

Examining water using nutrient gelatin also presents no difficulties. A specific quantity of the water to be tested is mixed with a corresponding amount of liquefied nutrient gelatin. The container is then immediately sealed with disinfected cotton wool, and the colonies developing inside the gelatin are allowed to grow large enough to be easily visible under a microscope and for samples to be taken for further cultivation. To accommodate this latter requirement, it is advisable to prepare the mixture in a shallow container and allow it to solidify, ensuring that the individual colonies are as spread out as possible and easily accessible with a dissecting needle. It is also advantageous to use a clear and colorless nutrient gelatin, such as wheat infusion gelatin, because in this examination, it is unavoidable that the colonies develop within the gelatin.

Regarding the amount of water to be added, in some water samples I obtained only very isolated bacterial colonies with 1 cubic centimeter of water to 10 cubic centimeters of nutrient gelatin; in others the microorganisms, including in particular colonies formed from very short rods, i.e. the actual bacteria, and many mold mycelia, were so numerous that they were no longer manageable and the water had to be diluted 10 to 20 times with sterilized water in order to obtain usable cultures.

Dust Analysis.

Dust is a very interesting object of study for gelatin cultures. Initially, I believed I could obtain from it a map of all possible airborne microorganisms, but I soon realized that reality did not meet my expectations. While a fair number of fungal mycelia and some bacilli develop from freshly settled dust, relatively few micrococci are present compared to the numerous germs that directly transfer from the air to the gelatin. Old dust, on the other hand, which accumulates inside furniture or in remote corners, allows almost exclusively fungal mycelia and a considerable number of bacilli to develop on nutrient gelatin. Thus, this again confirms that the vast majority of airborne germs die off quite quickly in the dried state, and that only the dormant forms of fungi and bacilli, especially the latter, remain viable and gradually accumulate.

Examination of various objects.

It only requires a brief mention that, in the same or a similar way as air, water, soil, and dust, a wide variety of other objects can also be tested for their content of viable microorganisms. The field of work opened up by this new examination method is so vast that it would be highly desirable for it to be undertaken by a large number of people. In addition to regular and thorough general air, water, and soil examinations, with particular attention to groundwater and rainwater, in many different locations and at different times, it would be necessary to carry out special investigations of the air in inhabited and uninhabited rooms, classrooms, hospital rooms, mortuaries, and workplaces, especially those that are overcrowded or where easily decomposable and putrefactive substances are processed, etc.

Furthermore, masonry, wooden walls, wallpaper, clothing, all kinds of consumer goods, money, and especially foodstuffs, etc., would also need to be examined, for example milk, sausage (in view of the recently more frequent cases of sausage poisoning), in short everything that can serve as a habitat or carrier of pathogenic microorganisms, should be examined for its content of the latter.

Description of the photograms.

I must first draw attention to a few points to consider when viewing and evaluating photograms.

While observing an object, the microscopist almost constantly adjusts the micrometer screw, sometimes moving the tube closer to, sometimes away from, the point of focus. This rapidly provides not only a visual impression of the object in question and everything lying in the same plane, but also allows him to immediately orient himself to what lies directly above and below it. Photography cannot offer this advantage, as a photographic image can only ever reproduce a single, very thin layer of the specimen. What lies precisely in this plane appears with sharp outlines; everything else, depending on its distance from the focused plane, appears indistinct, and further away it appears blurred. Objects furthest from the fixed point along the microscope axis produce only a shadow in the image. The greater the magnification, the more pronounced this becomes.

At first glance, therefore, the photographic image of specimens of a certain thickness, i.e., of all tissue sections, appears somewhat strange. It contains many objects with indistinct outlines, and shadows whose origin is not apparent. These are the nuclei, bacterial clusters, etc., that are not located in the focused plane and should be disregarded. Instead, focus on the areas of the photograph that were in sharp focus. Often, only a small group of bacteria could be brought into focus at a single plane; however, these few individuals are perfectly sufficient to indicate the size relationships, groupings, etc. To obtain clear images of the arrangement of bacterial colonies inside organs, lower magnifications, ideally 100x, are suitable.

On this occasion, I would like to reiterate that cell nuclei, like bacteria, do not appear the same when diffusely stained on a coverslip or treated with nuclear staining. This is particularly striking in anthrax bacilli, which present a significantly different appearance when nuclear stained in tissue sections compared to when stained on a coverslip. Therefore, comparisons in the photograms can only be made with bacteria that have been prepared and stained in the same manner.

Incidentally, to facilitate the necessary comparative observations of the pathogenic bacteria, I have chosen almost uniform magnifications. The overview images were taken at 100x magnification, and the highly magnified images at 700x. As numerous experiments have shown me, even higher magnifications offer no benefit, since, as is well known, the performance limit of our best systems is reached approximately at the indicated magnification.

All the photograms were taken with Seibert's objective systems; the weakest enlargements with the 1-inch photographic objective, the 100x magnifications with the 1/4-inch photographic objective, and the 700x magnifications with the immersion system VII.

During the preparation for photogravure, which consisted of removing the negative varnish and transferring the negative to gelatin film, some negatives were damaged, and a few lost some of their original sharpness. I have deliberately avoided correcting these and other flaws, which are never entirely avoidable in the photographic process and are corrected by professional photographers through retouching. Not the slightest corrective or otherwise altering intervention has been made to any of the images; they are completely free of any kind of retouching and reproduce the unadulterated and perfectly true-to-life image of the objects. I must therefore ask the viewer to overlook the streaks, spots, etc., found here and there, particularly at the corners and edges of the images, which are, incidentally, always easily recognizable as not belonging to the actual image, or at least to consider them as proof of the purely objective nature of the images.

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