Bacteriological Analysis

BACTERIOLOGICAL ANALYSIS, microbiological analysis, serves for the detection of various kinds of microorganisms and determination of their species in the material under examination (in soil, water, various products, secretions of animals, etc.); it consists in the study of the biology of the microbe (its morphological, biological, and immunological properties), and for pathogenic microbes also in determining their ability to cause in experimental animals specific pathological processes. The study of the morphology of microbes is performed with the aid of a microscope, for which the material under examination is subjected to preliminary treatment in order to make the microbes clearly visible among the unorganized decay in which they usually are found. This is achieved by staining preparations from the material under examination with various natural or artificial dyes. Since dead microbes stain better, the preparations are previously subjected to so-called fixation, i.e., treatment with various physical and chemical agents that quickly kill the protoplasm—heat, alcohol, etc. Sometimes the material is microscoped without preliminary treatment when, during the course of analysis, it is required to study living microbes under the microscope (observation of their motility, their relationship to O, etc.). In such cases, a preparation is made in the form of a so-called hanging drop. Observation is conducted either in the usual way or with the dark-field illumination of the microscope. When examining for the presence of Protozoa, the entire analysis is limited to one microscopic examination, which is considered sufficient, since the methods for cultivating protozoa are very complex and have not yet entered into general practice. In bacteriology, the analysis does not stop at the study of the form of the microbe. The extraordinary variability of bacteria under the slightest changes in the environment is primarily reflected in their form; the study of only one form of microbe would lead to the grossest errors in determining their species. Therefore, for judgment about the species of microbe, it is necessary to know, in addition to morphology, all its biological peculiarities, its biochemical and immunological properties, and for pathogenic ones—the result of the animal experiment; only the combination of all these data makes it possible more or less accurately to identify this or that microbe, to determine its species and type. This is what all bacteriological technique serves for. Cultivation of microbes on artificial nutrient media for the study of their biochemical properties is the principal moment of B. a. At present time, such cultivation still encounters a number of difficulties. Many microbes still cannot be obtained in artificial cultures. The obstacle is a number of conditions consisting in the extraordinary diversity of the microbial world, in the sharply expressed specificity of the biochemical functions of microbes and the resulting variability. Our ignorance of many of these conditions does not make it possible to select such nutrient media that would always be suitable. Nevertheless, it is precisely the cultivation of the microbes accessible to us that is the basic method of their B. a. At present time, B. a. begins with obtaining the individual microbes under study in pure cultures, i.e., in cultures consisting of one species of microbe, without admixture of any other. This is achieved by distributing the material under study on the surface of solid nutrient media. This distribution aims to separate the microbes from each other. After a certain period of time, most often after a day, at those places of the substrate where microbial cells have appeared, accumulations of microbes form, so-called colonies of the grown microbe. These colonies in many microbial species have a very characteristic form, which is used for the first orientation in the composition of the microbial flora. Individual colonies are extracted and transferred to suitable nutrient media for further cultivation. It is assumed that each colony grows from one cell; but this is not always the case, and recently the method of obtaining colonies from a known single microbial cell is increasingly entering into practice. Such separation of the microbe is performed under the control of the eye in the microscope with the aid of an instrument called a micromanipulator. Since the material under examination contains an enormous quantity of various microbes, while it is necessary to isolate one specific one from them, in such cases special nutrient media are used. In these either chemical substances are contained that interact with each other under the influence of the growth of microbes, which is detected by some easily accessible manifestation, for example, change in color, precipitation or dissolution of sediment (Endo, Drigalski media, media with chalk, with blood), or these media deliberately favor the growth of the sought microbe and suppress the growth of accompanying microbes, for example, enrichment media for typhoid, diphtheria. Only after such preliminary treatment are pure cultures obtained. In those cases when the sought microbe is present in the original material in very small quantity and is difficult to cultivate directly, resort is made to the experiment on animals (tbc). The material is injected into the animal, and after characteristic pathological changes for this microbe have developed, a pure culture is isolated. Having obtained a pure culture, they proceed to the study of the biochemical peculiarities of this microbe. Since the composition of nutrient media is very complex, for the study of biochemistry to the media are added various, chemically simpler substances, the changes of which under the influence of the growth of microbes can easily be detected by adding some indicator—litmus, neutralrot, etc. For this purpose serve various carbohydrates, mono-, di-, polysaccharides and polyhydric alcohols, as well as gelatin and blood (erythrocytes). On the basis of the change of this or that of these substances, conclusions are drawn about the biochemical peculiarities of this microbe. Immense significance in the determination of the species of microbe has the immunological method, based on the ability of the animal organism to react extremely specifically to the parenteral introduction of foreign proteins by the formation and accumulation in the blood of various so-called antibodies. The blood serum of such animals serves as the reagent. The presence of these antibodies in it can easily be detected by a number of simple reactions. Some of these reactions have become widespread and are obligatory in the identification of microbial species. These reactions—agglutination, precipitation, and the complement deviation reaction. For non-pathogenic microbes, B. a. can be ended at this point. For pathogenic microbes, however, the experiment on animals is still obligatory, especially when discovering a new species. The ideal for the determination of any pathogenic microbe is to obtain in a suitable animal the pathological changes caused by this microbe under natural conditions. However, our knowledge of the conditions necessary for such an experiment is far from complete, which is why experiments are often unsuccessful. Then, the patho-anatomical data obtained as a result of the experiment are not always reliable, since the apparently healthy state of the animal says nothing about infections and other diseases it has undergone; the changes found may be the result of previously undergone diseases. All these considerations make it necessary to treat very cautiously the results obtained in the experiment and never to be satisfied with one successful experiment. Still, in those cases where the reaction of the experimental animal to this microbe is strictly defined and constant (as, for example, the relationship of the guinea pig to diphtheria or tbc, or of the rabbit to smallpox and rabies), there the experiment on animals is obligatory. B. a. without such an experiment is not sufficiently convincing. In some cases, with a successful experiment, it is necessary to isolate in pure culture that microbe which served for the experiment, and to identify it with the original. This is necessary because the introduction of foreign material into the animal organism often awakens dormant pathological processes there and causes the generalization of a limited suffering, increasing the activity of the microbes present there. When isolating a pure culture, it must therefore be kept in mind that one may isolate not the original microbe, but a microbe that happened to be in the organism. In addition, the blood of many apparently healthy animals contains various microbes, which can also be isolated and may mislead the researcher. The general scheme of the course of B. a. can be presented in this way: 1) preparation of preparations from the material under examination; fixation of them; 2) microscopic examination a) in the uncolored state, b) in the colored state; 3) seeding on agar plates for the isolation of pure culture; if necessary, after preliminary seeding on selective or enrichment media; 4) biochemical analysis; 5) immunity reactions; 6) experiment on animals. The purpose of B. a., besides determining the species of bacterium, is also to establish its place in the systematics of microbes. However, the systematics of microbes is not yet sufficiently developed, or rather, there exist several systematics, very incomplete, built on various bases. All this makes the accuracy of the analysis difficult.

In 1917, American microbiologists created a special commission, which developed a systematics of bacteria, based, mainly, on their immuno-biological characteristics and properties detectable in cultures. This commission verified the characteristics of all known microbes in the above-mentioned sense, and the results of its work have been published. All works now published in the English language already adhere to the nomenclature adopted in this manual.