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Part I, continued

The Principles of Classification of Microorganisms

   The classification of microorganisms is very unsatisfactory. There is no common principle of classification in microbiology. The classification of bacteria and actinomycetes is especially inadequate. This can be explained by the peculiarity of those organisms, the simplicity of their structure and growth and lack of external properties for differentiation. An immense number of species is not fully described and is not classified in an appropriate way. Besides, the classification is not an objective one, there are different directions in it, which often do not agree with each other and are often even contradictory.

   The main reason for the inadequacy of classification of bacteria and actinomycetes lies in the absence of factual material on phylogeny of individual species, groups and subgroups. The natural classification should reflect the stages of evolutionary development. The principle of phylogenetic structure of classification requires a thorough knowledge of the organisms and species relationship.

   Unfortunately this principle is absent in the modern classifications. The classification of bacteria is made according to separate, frequently accidental properties. One and the same homogenous group is frequently divided into separate taxonomic entities often without sufficient grounds.

   The great shortcoming of every classification is the fact that it is built in only one plane, a descending or an ascending one, while in reality each taxonomic entity should represent a center of formation of new forms, species, varieties, etc.

   Classification also becomes complicated by the fact that phylogenetically foreign organisms are concentrated around these centers, the morphology of which is close to that of the main specimens of the studied group. Each group of bacteria, actinomycetes or fungi comprises main and convergent forms. All these organisms may appear homogenous according to their appearance and physiology, while phylogenetically some of them comprise a uniform group and others are accidental foreign organisms. The natural classification should embrace only the former organisms which determine real affinity, while the foreign convergent forms should be excluded.

   Microbiological literature contains numerous data on the morphological similarity of entirely different organisms. For example, a large group of nonsporeforming yeasts, comprising the family Torulaceae, in reality represents a mixture of different species and not only species but also genera, and generally speaking in a mixture of different unrelated groups. The bacteria of the genus Micrococcus are characterized by their spherical shape. Into this group organisms which in fact belong to coccoid bacteria are included and also not infrequently specimens of actinomycetes are included in the genus Mycococcus. To the rodlike bacteria of genus Bacterium very diverse microbial forms are referred. Bacteria from the genus Bacterium are frequently linked to Mycobacterium only because the former sometimes possess lateral branches which are characteristic of the genus Mycobacterium. Thereby the specificity of this property is not accounted for. Properties which are natural and fixed in one genus may be abnormal and unnatural in others.

   The shortcomings of the bacteriological classification have their origin in our scant knowledge of the life of the organisms. In order to be able to speak of the phylogenetic relations between the organisms, it is not sufficient to know and study one randomly chosen stage of the life cycle of the microbe. A thorough knowledge of its growth, development, structure, reproduction, life cycle, polymorphism, variability, etc, is needed. In order to obtain much knowledge, the organism in question should be studied not only in laboratory conditions but also in natural surroundings.

   The lack of knowledge of the life cycle of this or another microbe frequently misleads the investigator. For example for this reason mycobacteria are considered by some authors as micrococci or as rodlike bacteria.

   Each property, biological, physiological or morphological, may be employed for the identification of the organism, but not each diagnostic property is of systematic value. The problems of identification and classification should be distinguished from each other although in some cases they are interwoven and overlap. Fixed properties which are transmitted by heredity, which characterize the nature of the species, and which are manifest under determined growth condittons, are taken into consideration for classification. 

The Species Concept in Microorganisms

   The concept of species is the most difficult problem in classification and is of the utmost importance in taxonomy of microorganisms. In microbiology this basic taxonomic entity was less studied than in any other branch of biology.

   Different authors, according to their specialization, much as mycologists, bacteriologists, phytopathologists, medical bacteriologists, and applied microbiologists consider microbial species differently. Each branch of microbiology chooses different properties for the determination of species. In yeasts, for example, certain indicators are taken as a basis for species determination (sugar fermentation of Saccharomyces, Zygosaccharomyces and others) in bactoria, other and moreover, different properties are employed in different groups; actinomycetes have still other properties which are employed for this purpose.

   Often authors consider bacterial species according to their own views and whims, which do not agree with the factual material.

   The more thorough the knowledge of the organism, the more strict the term species becomes. It progresses with the development of the knowledge of the organism. It was a long way from the Linnean idea of a species as a fixed and not changing entity to Darwin's teaching and present ideas of the ever-developing species. In the determination of species, Linnaeus stressed two basic properties--the constancy of properties and their sharp demarcation in various species. Later on Korzhinskii (1892) introduced a geographical principle into the concept of species which was more thoroughly elaborated by Wetstein (1898). According to this principle, the species morphology is correlated with the place of its habitation. Approximately at the same time, a historical moment entered the concept of species; this was especially pronounced in the works of Semenov-Tyan'Shanskii (1910), who pointed out that the sum of architectonic properties is a result of interaction of a complex of physicogeographical factors which took place in a bygone geological period (according to Krasil'nikov, 1938a).

   The establishment of phylogenetic relationships within the species led to the separation of the main and the subordinate taxonomic entities--varieties, forms, etc. The species was divided into main and secondary forms; binomial nomenclature became tri-and quadrinomial nomenclature.

   With the development of genetics the concept of species widened according to the ideas of variability and heredity of organisms. New terms were introduced for the determination of species subdivision, such as "biotype", "pure line", "jardanon", "linneon", etc. ["Jardanon"--a simple means of classification of lower organisms. "Linneon"--the complex of "jardanons"--according to the Russian concept, the inner species variety of forms does not exceed the limits of qualitative unity of the species.]

   The experimental verification is a new stage in the development of the determination of species. Of the works in this direction the most important ones are those of Touresson, (1922-1925) which showed the dependence of the external form of plants on the ecological conditions of habitation. He introduced the term "ecotype". Species is now being looked upon as a definite system. This is most sharply pronounced in works of Vavilov (1932) who showed that Linnean species are definite systems of forms, and not an accidental motley of races. Komarov in his work "Flora of the Kamtchatka Peninnsula" (1920) gives the following definition of species: species is a morphological system multiplied by geographical definiteness (Komarov, 1940).

   Lysenko (1952) considers species as separate links of a single chain of the organic living world. Species, he writes, is a unique qualitative state of the living matter. The basic characteristics of animal, plant and microbial species are defined intraspecies relationshps between the individuals. These intraspecies relationships are qualitatively different from those between the individuals of different species.

   This definition is based upon the biological properties of organisms and their specific properties, the knowledge of which should be the subject of a detailed study.

   As can be seen from the aforesaid, the concept of species is considered differently. Every specialist has his own narrow idea about species, moreover, the factual material accumulated by others to not always taken into account. In spite of the diversity of concepts and radical biological differences, the principles of classification of different groups of organisms should be the same. Admitting the right of the different specialists to probe into the problem and to elaborate it according to their specific needs, we point out that those concerned with classification should adhere to the main outlines and taxonomic rules, established by international congresses and by conferences of specialists. Besides, there are general biological features which characterize species of higher and lower organisms alike.

   Concerning the concept of species in microorganisms, certain specific properties which preclude the possibility of deploying all the principles which are being applied in the classification of higher organisms, should be mentioned.

   The distinguishing feature of microorganisms is their lack of multitude of morphological characteristics. Therefore, determination of species on the basis of morphology in the overwhelming majority of cases is impossible. Morphology can be used only for the determination of higher taxonomic entities--genus, family and higher.

   The geographical principle or more strictly speaking, the ecological-spatial principle, and the historical principle can not be employed for the determination of microbial species. No data are available in microbiology, which could show some definite regularity in the distribution of separate microbial species in nature. Even less data exist on the fossil microbial forms. The scant data on the presence of microbial cells in microsections of coal, shales and other deposits gave no grounds for establishment of even large taxonomic entities. Methods employed in cytology and genetics are not suitable in that case. Bacteria and actinomycetes do not possess a genuine, well-defined and isolated nucleus (only nucleoids are present), they do not possess plastids or any other organelles, characteristic of cells of higher organisms. The sexual process in those organisms is either lacking or, when present, is a unique one and cannot serve as a means for the establishment of a genetic relationship of the organisms.

   The biochemical method of species determination unfortunately is not systematically elaborated. Separate attempts to divide species according to their biochemical properties are as yet of no importance.

   Biochemical properties can only be employed in individual cases for determination of species. Products utilized by the microorganisms and decompositIon of some organic and inorganic compounds can serve as a basis for such determination. In other cases organisms are determined by metabolic products of a specific nature, for instance by their capacity to form antibiotics, toxins, slimes and other features.

   The mode and sequence of the decomposition of organic compounds such as carbohydrates, with the formation of intermediate compounds is of great interest for classification of organisms.

   Unfortunately, this direction is not sufficiently elaborated for the purpose of classification (Shaposhnikov, 1944; Knight, 1955). It should be noted that species of practical importance are the most thoroughly studied ones. In such cases species is determined by the leading biochemical or biological property. Antagonists of actinomycetes as well as of other microbes can be divided into species according to the antibiotic they produce. In higher plants species are not infrequently established according to their economic importance, or according to some property utilized by men in everyday life or industry.

   Organisms of any importance in economy were more thoroughly studied and naturally are among the best known organisms. Many biochemical, physiological and biological properties of such organisms have been studied, and methods for their determination are well elaborated.

   Such profound and comprehensive studies enable the revelation of the nature of the species and its relationship to other species. Often, owing to such a thorough study, the heterogeneous quality is revealed in organisms which were formerly thought to represent a homogenous species. Organisms which are assumed to belong to one species prove to be a complex group consisting of several or many species.

   There are comparatively few species which are utilized by men in everyday life and in industry. The majority of organisms, however, were studied only to an extent which seemed necessary for general purposes. It is clear that the usual methods employed for the determination of bacterial species are not uniform. There are many more species in nature than we are able to determine by the relatively primitive methods at our disposal. The heterogeneity of the known species of bacteria and actinomycetes is becoming more and more apparent with the more thorough study to which they are subjected.

   What are the accepted methods for the determination of species of bacteria, actinomycetes, and microorganisms in general?

   The ability to grow organisms in artificial nutrient media in pure cultures and to follow their growth directly under the microscope under different growth conditions is of great advantage. The microbes proliferate at a high rate and, due to this, many problems connected with their polymorphism and variability during their life cycle can be solved in shorter time than when working with higher organisms. This is especially important in determining the variability or stability of taxonomic properties over a large number of generations.

   The comparative studies of pure cultures of microbes enable one to study the species not only by itself but also with regard to the entire population during their variations and deviations which take place in the process of variability.

   In spite of what was said before, that the morphological principle is in many cases unsuitable for the determination of species of bacteria and actinomycetes, it should not be neglected. In some cases it is a trustworthy method for species determination. Such organisms as Azotobacter--Az. chroococcum, Bac. megatherium, threadlike bacteria, many sulfur bacteria, iron bacteria and other species can be determined and identified by their external cultural and morphological properties.

   Physical properties are not infrequently employed for the determination of species. This method is trustworthy in many cases. However, it is of limited importance and may lead to errors if employed without taking other properties into account. The afore-mentioned method requires further elaboration. Such indexes as microbial behavior toward oxygen, temperature, pH of the medium, fermentative capacity of the organism, its requirements of organic and inorganic nutrients may be properties of a species only in a restricted sense.

   The phylogenetic principle can be employed in microbiology with success. The afore-mentioned biological properties of microbes enable one to determine their genetic relationship experimentally. The method of experimental variability reveals not only heterogeneity of cells in the culture and their polymorphism in the life cycle but also the frequency of the formation of variants and, according to the latter, the affinity to the naturally existing forms and species of microorganisms.

   Employing this method, we were able to establish the affinity between specimens of actinomycetes. The transformation of mycococci into mycobacteria, mycobacteria into proactinomycetes, and the latter into actinomycetes was experimentally proven. Data were obtained on the affinity of lactic bacteria, propionic bacteria, certain micrococci and pseudobacteria to a group of organisms close to actinomycetes (Krasil'nikov, 1938a, 1947b, 1949c, 1955b).

   Microbiological literature contains data on the phylogenetic affinity of some specimens of mycobacteria (Imshenetskii, 1940). Osterle and Stahl (1929) and then Rautenstein (1946) obtained variants of Bac. mycoides which did not differ from Bac. effusus, Bac. olfactortus, Bac. nanus, Bac. vulgatus, Bac. cereus, Bac. brevis, Bac. panis. and Bac. mesentericus. A multitude of variants of Bac. mycoides was also obtained by us (Figure 38) (1947b). Medvins'ka (1946) experimentally established that the sporeforming bacteria Bac. mesentericus fuscus, Bac. mesentericus niger, Bac. mesentericus vulgatus, and Bac. mesentericus panis described in the literature represent one species with two varieties. Gibson (1949) brings a voluminous experimental material concerning the scope of the species Bac. subtilis. He identified this species with the following bacteria: Bac. aerrimus, Bac. globigii, Bac. leptosporus, Bac. levaniformans, Bac. panis, Bac. niger, Bac. viscogenes, Bac. vulgatus and some other varieties.

 

Figure 38. Variants of the culture Bac. mycoides obtained experimentally:

(+) and (-) variants: according to the utilization of nutrients vertical rows; according to colony structure--horizontal rows.

   Nakhimovskaya (1948) obtained variants of Ps. aurantiaca which did not differ from Ps. fluorescens. The typical strain of Az. chroococcum yielded colorless, capsuleless variants with small atypical cells which grew on protein media and did not fix nitrogen. Such variants were also isolated from the soil.

   Widely known are the variants R, S, M, G and others in various specimens of bacteria. They markedly differ from the original cultures and often appear to be like independent species which exist in nature.

   Even more variants are encountered which differ from the original cultures in their physiological and biochemical properties. In the majority of cases such variants are not distinguished in laboratory practice and remain unrecognized.

   With the aid of the experimental variability method we have shown, the affintty between the individual species of root-nodule bacteria and also the phylogenetic closeness of the latter to certain root-nodule bacteria of the genus Pseudomonas. It was established that certain asporogenous species of Pseudomonas can be transformed into root-nodule bacteria under certain conditions (Krasil'nikov, 1954b, 1955b).

   Employing this method we succeeded (Krasil'nikov 1947b) in establishing the affinity between certain species of nonsporeforming bacteria of the genus Bacterium and Pseudomonas and then between the sporeforming bacteria and other groups. During the course of the dissociation of two different cultures of the genus Pseudomonas similar variants (Figure 39) were obtained which were identical morphologically, physiologically and biochemically.

 

Figure 39. The scheme of formation of monotype variants by different cultures of Pseudomonas fluorescens

(strains A and B): fermentation of: G-glucose, L-lactose; MC--milk coagulated; MP--mtlk peptonized; N03--reduction of nitrates; F--fluorescence; FM--fluorencence on meat-peptone agar or meat-peptone broth; FCh--fluorescence on Chapek medium; + reaction positive, - reaction negative (according to Krasil'nikov, 1947).

   Species of sporeforming bacteria such as Bac. mesentericus, Bac. subtilis, Bac. cereus, and Bac. licheniformis yield identical variants as was mentioned above. All this indicates the similarity of hereditary and species properties of organisms.

   The method of experimental variability also reveals the phylogenetic relations between yeasts. Filippov (1932) established the affinity between various specimens of nonsporeforming yeasts by this method. Cultures of the genus Torulopsis which were exposed to X-ray irradiation produced a number of stable variants which should be included in the genera Torulopsis and Mycotorula (Filippov 1932) according to their morphological and physiological properties, if the rules of present classification were applied.

   The yeast fungus, Sporobolomyces, yielded through the method of dissociation many (more than twenty) variants some of which were identical with cultures of Torula. Torulopsis, and Mycotorula; others had well-developed mycelia which did not differ from the mycelia of mycelial fungi. We obtained these variants by relaxing their heredity with subsequent adaptation. These data show that expertmental variability is a valuable method for the study of a species and its relationship to other species, and therefore should be employed in the classification of microorganisms in general and of bacteria and actinomycetes in particular.

   The serological method of diagnosis of pathogenic bacteria is widely used in medical bacteriological practice. This method is a very sensitive one and enables the detection of small differences between strains which are formed under the conditions of variability of these or other cultures. The sensitivity is determined by the chemical nature of the antigens and the capacity of the method to distinguish differences between the various molecules, predominantly protein molecules, which cannot be differentiated by chemical methods. With the aid of antigens the smallest differences in the protein composition can be detected and consequently subtle culture variations can be diagnosed. Nonetheless, this method is not being used in the classification of microbes to the extent that could be expected. Though it is an excellent and sensitive method for the differentiation of related strains It is at the same time unsuitable in the majority of cases for the classification and differentiation of species. Attempts to divide the root-nodule bacteria by serological methods yielded contradictory results which did not agree with the classification done by conventional methods. Entirely different species are linked to one taxonomic group and vice versa (Zipfel, 1911; Simon, 1914; Stevens, 1923; Fred et al., 1932; lzrail'skii, et al., 1933). Similar results were also, obtained when an attempt was made to classify strains of Azotobacter, Radiobacter, bacteria of the genus Pseudomonas, Bacterium, Clostridium, streptococci, and others (Mayr and Harting, 1948; Fred et al., 1932; Frances-Shattock, 1955).

   It is apparent that some points of the method are not sufficiently elaborated. Experiments showed that not all antigens of microorganisms and higher organisms could be detected (Lamana, 1940; Davis, 1951). These authors made an attempt to classify species of sporeforming bacteria of the genus Bacillus. They could not detect specific antigens in vegetative cells; neither the somatic antigen (0) nor the flagella antigen (H). More encouraging results are obtained with spore antigens.

   Concerning the lactic bacteria of the genus Lactobacterium there was no success recently in classifying them by serological means. Quite recently Sharpe succeeded in obtaining precipitin, with the aid of which these bacteria are as if differentiated into types, which can be correspondingly determined by physiological-morphological methods. (Sharpe, 1955, Briggs, 1953.)

   Fourteen year studies of Mez and his collaborators (1922, 1926, 1936) on plant classification by serological methods were unsuccessful.

   The method of serological diagnosis is well elaborated for colic bacteria--Bact. coli, Bact. typhi, Bact. paratyphi , Bact. dysenteriae, and others. This method is successfully used for the classification of streptococcii, pneumococci, staphylococci, and especially of viruses (Bawden, 1955; Holmes, 1955; Andrews, 1955; Ryzhkov, 1952). It should be pointed out that groups established by this method are of a specific character and do not always correspond to the groups determined by other methods. Experience shows that the determination of species by serological methods should be carried out with precaution and the results must be carefully analyzed. It is only of secondary importance and complements other methods of microbiological analysis.

   Attempts are made to identify bacterial species by means of phages. This method is based on the capacity of phages to lyse defined bacterial species. Among phages there are strains with strictly specific species properties attacking only one bacterial species. There are also group-specific phages which attack bacteria belonging to different species or even genera. It is assumed that the former phages are suitable for species differentiation and the latter for group differentiation.

   Data exist on species differentiation by means of phages of bacteria of the coli group, staphylococci, nonsporeforming and sporeforming soil bacteria, and others. Katznelson and Sutton (1951) pointed out that phytopathogentc bacteria (in various substrates) can be detected by means of phages. They detected Ps. phaseolicola in seeds of kidney beans without isolating them in pure cultures. The seeds were washed with water, the washings were inoculated with the specific phage of Ps. phaseolicola and after some time the titer of the phage was determined. By this means the degree of phage multiplication was determined. The phage, according to the authors, can multiply only in the cells of the afore-mentioned species.

   Smith, Gordon and Clark (1952) have shown that sporeforming bacteria can be differentiated by the phage method. Data are available on the differentiation of species and groups of the coli bacteria--Bact. coli, Bact. typhi, Bact. paratyphi, Bact. dysenteriae. Stocker (1955) and others think that all these organisms represent one closely related bacterial group. To this group, according to him, Bact. pestis and Bact. pseudotuberculosis also belong. As can be seen from this, the bacteriophages and actinophages are very sensitive, but are far from specific to the degree required in classification. The phage method as well as the serological method can be employed in separate cases and then only as auxiliary tests.

   Parasitological and toxicological methods of species classification are employed in phytopathology. Certain parasites are very specific and parasitize only defined plant species, therefore this method can be employed for the differentiation of the parasite by its host.

   It is known also that certain parasites form toxins, for example the diphtheria bacillus, the causative agent of gas gangrene, Cl. botulinum and others (Oakley, 1955).

   These properties are of a narrow specific character and can he employed only for a restricted group of microbes. It may happen that when different representatives of microorganisms are studied from the point of view of formation of specific toxic and nontoxic metabolites this principle will find wide application in classification of microorganisms.

   According to our data, specificity of antagonism constitutes an essential species indicator. We have shown that microbes grown together in mixtures often exhibit antagonism. One culture supresses the growth of the other. This process can be caused by specific and nonspecific substances.

   Specific substances as antibiotics are of importance in species determination. Each species of an antagonist synthesizes its specific antibiotic and sometimes two and three antibiotics simultaneously. As a rule, the characteristic property of antibiotics to the fact that they do not suppress the growth of the producing organism and other organsms belonging to the same species. The action of the antibiotic and, consequently, of the organism which produces the antibiotic is directed toward the organism's competitors. This specificity is strictly constant for the antagonists and can serve as a means for species determinaion. On the basis of this principle, we have elaborated a method of species differentiation of actinomycetes and sporeforming antagonistic bacteria (Krasil'nikov, 1951c, Krasil'nikov, Korenyako, Nikitina, Skryabin, 1951, Afrikyan, 1951a). Employing this method we were able to establish a multitude of species among certain groups of organisms which were classified as one species. Similar data were obtained by Teillon (1953) and some other investigators (see below).

   From the afore-mentioned it may be concluded that species is a really existing taxonomic entity, consisting of different individuals, strains or cultures (in the laboratory), forms and varieties. They all possess one property which can be manifested in different forms. This property characterizes the species an a whole, and as long as it Is preserved, the species remains unchanged. Individuals and varieties are only forms in which the species exists. Individuals and varieties may differ from each other but the species properties common for them remain unchanged.

   Consequently, species as a link in the evolutionary chain of development represents, in a certain sense, a closed entity of living organisms. It is closed because all the individuals of the species have a definite relation to one another. The relations within the species differ from the relations between organisms of different species. This is clearly shown in examples of microbial antagonism.

   Proximity of species is manifested in the requirements for certain life conditions, nutrition, light, temperature and other factors. All organisms of the same species, no matter how different they may be, require the same basic factors for existence, which may be different for other species. The organisms of one and the same species always assimilate the same medium regardless of the geographical zone in which the individual organisms or cultures may grow.

   For example, the actinomycetes which produce streptomycin and comprise one species Act. streptomycini are widely distributed in nature; individual strains live in different geographical zones, in different soils, in the north and south, east and west, in the silts of rivers and lakes, and on different plant residues. Their ecological conditions of habitation are very different. Nonetheless all strains of this species are of the same heredity and of the same fundamental characteristics.

   Species is an isolated link in the evolution of organisms consisting of qualitatively homogenous forms, which require the same basic life conditions, have the same origin and are characterized by definite morphological and physiological properties, and also by the degree of variability which is transmitted by heredity.

   The scope of a species is determined by the extent of the variability of the organism in question, by its morphological-genetic capacity. The species manifests itself in varieties, forms and individuals (cells) which represent species variations and polymorphism under defined conditions of existence.

   Species is an ever-changing and developing system of closely related organisms. Its beginnings can be seen in individual changes of cells. These changes widen and become fixed by natural selection in the course of adaptation to the environment and dominating growth.

   Experiments showed that microbes are not monomorphous either under the conditions of growth in artificial nutrient media or in nature. In natural conditions microbial cells are subjected to stronger and more diverse interactions; it is natural to expect more frequent and diverse formation of variants under these conditions. This is confirmed by microbiological analyses of natural substrates. In the soil, as it will be shown later, species exist in very diverse forms, morphological and functional. Diversity of strains, forms and varieties of each species under natural conditions are determined by the properties of the latter and hereditary properties of the organism.

   In classification systems species are grouped into larger taxonomic entities--genera, genera into families, etc.

Subdivision of Microorganisms into Main Groups

   In our manual for determination of species all microorganisms except protozoa are included in one series of primitive organisms of the plant kingdom--Protophyta.

   This series is divided into two groups:

   1) Schizophyceae--lower organisms, possessing chlorophyll, phycocyanin or phycoerythrin;

   2) Schizomyceae--chlorophyll-less organisms. Here belong all bacteria, fungi and actinomycetes described in the literature. The organisms of this group are very diverse in their structure, growth, biological essence and phylogeny. They are divided into classes; each of them has its genealogy and it should be assumed, its own unique roots of origin. In other words, organisms of this group are of polyphytic origin.

   We divide them into four classes:

Class I--Actinomycetes

Class II--Bacteriae

Class III--Myxobacteriae

Class IV--Spirochaetae

 

Actinomycetes

   The class of actinomycetes is a homogenous, well-studied group of microbes. According to our data, the group of actinomycetes included various subgroups: actinomycetes, micromonospora Waksmania, proactinomycetes, mycobacteria, and mycococci; the affinity between them was established by us experimentally. Beside the above-listed organisms, the group of actinomycetes comprises to a greater or lesser extent specimens of the so-called pseudobacteria, lactic and propionic bacteria and some others (Krasil'nikov, 1938 a).

   Genus Actinomyces. Actinomycetes are higher organisms than bacteria, according to their structure and growth. As it is known they form a well-developed mycelium. The mycelial threads are thin, 0.5- 1.0 µ in diameter, without septa. Branching is similar to that of fungi, from the main branch side branches of the first, second, third order, etc originate. The mycelial threads are thinner than in fungi, more fragile, are easily broken and destroyed, forming shreds and splinters.

   Actinomycetes grow on dense nutrient media in the form of compact, dense, gristlelike leathery colonies. The latter have a smooth, granular, rough or plicated surface. The colonies grow into the medium with their threads, and have a flat or convex form. They are of such density that platinum wire is unsuitable for removing part of the colony, for this purpose special loops have to be used.

   Actinomycetes possess a substrate and aerial mycelium. The former really represents the entire colony. The aerial mycelium consists of hyphae which originate in the threads of the substrated mycelium. It may be abundant and cover the entire colony with a fluffy, velvety or floury coating. It may also be weakly developed in the form of separate bundles of threads located on the surface of the colony.

   Sporangia containing spores are formed on the threads of the aerial mycellum. The structure of sporangia varies in different species. Some actinomycetes have a spiral or more precisely spindle like sporangia, others, straight or undulant. The number of coils in the spiral sporangia can fluctuate between 0.5-1 and even 5-7 and more. The coils in some cases are densely packed, they frequently have the form of spheres, in others they are stretched (Figure 40A).

 

Figure 40. The structure of sporangia of actinomycetes:

A) spiral sporangia with coils of different character; B) nonspiral sporangia of actinomycetes.

   Nonspiral sporangia also differ from each other. In some species they are very short, straight, in the form of bristles, in others-long. straight or undulant but not spiral (Figure 40B).

   Spores are often of oval, spherical forms, less frequently they are rodlike, and cylindrical forms with sharp ends (Figure 41). Spores of many actinomycetes have different forms--spherical, oval, and rodlike. In young sporangia spores are frequently rodlike, in mature sporangia they are oval and spherical. The rodlike spores gradually become round until they assume spherical form.

 

Figure 41. The form of spores of actinomycetes:

a) spherical; b) elongated, c) cylindrical with cut-off ends.

   The spores are formed by segmentation or fragmentation. In the first case, the sporeforming branch is fragmentated by transverse septa into separate cells which part and become mature spores (Figure 42b). In the second case, the protoplasm of the sporangium becomes fragmented into separate sectors or lumps without formation of septa. The lumps become rounded at the ends, assume rodlike, oval or spherical form, become covered with their own membrane and transform into mature spores. Afterward the membrane of the thread is destroyed and the spores are released (Figure 42a).

 

Figure 42. Spore formation by actinomycetes:

a) fragmentation; b) segmentation.

   Upon submerged growth in liquid media with constant shaking (on shakers) many actinomycetes have a fragmented mycelium resembling that of proactinomycetes. The threads form septa and disintegrate into rods and cocci. Frequently the disintegration of the mycelium proceeds by fragmentation (Figure 43). Cells thus formed resemble the spores described above. These cells are usually mistaken for spores, which of course is erroneous.

 

Figure 43. Fragmentation of mycelial threads of actinomycetes upon submerged growth in shakers. The threads of Act. streptomycini disintegrate into rodlike elements

   The formation of spores ends the cycle of growth of the culture. Cells which are formed upon fragmentation of the vegetative mycelium under the condition of submerged growth are ordinary vegetative forms like those of mucor fungi.

   As it is known, the mycelial threads of mucor fungi are devoid of septa and upon growth in submerged cultures transform into yeast cells called mucor yeasts through fragmentation. These cells differ from the spores formed in the sporangium of the fungus.

   Many actinomycetes form various pigments on nutrient media--blue, violet, red, orange, yellow, sky-blue, green and others. The pigments are either diffuse or not. There are cultures whose pigment diffuses in the medium leaving the colony colorless.

   The physiological property of actinomycetes is their capacity to utilize many organic substrates even such which are not utilized by other bacteria and fungi. In natural conditions on plant residues, actinomycetes begin to grow after all the readily assimilated substances have been decomposed and assimilated and the fungi and bacteria have stopped growing. Actinomycetes grow abundantly on semi-rotten residues. When a lump of peat or humus is inoculated with actinomycetes, it will soon be penetrated by the threads of the latter, and its surface will be covered with a white coat of the aerial mycelium.

   Due to the fact that they are not fastidious, they are widespread in nature, they can be detected everywhere--in the extreme north and in the tropics, on barren rocks and in fertile chernozems, on mountaintops and in valleys. They live in water, silts, in the soil, on various plant and animal residues.

   Upon growth in artificial nutrient media, actinomycetes form a number of peculiar substances. They have a characteristic smell somewhat resembling the odor of earth. Some species have a smell of camphor, fruits, etc. The chemical nature of those odors is not known.

   Among the metabolic products of actinomycetes different biotic, antibiotic and toxic substances are found. Of the biotic substances the following were found: vitamins Bl, B2, B6, B12, biotin, folic acids, auxins, pantothenic acid, nicotinic acid and others, in addition they form some amino acids which serve as complementary nutrients.

   The formation of antibiotics by actinomycetes is widespread. Approximately 50-70 % of isolated actinomycetes produce antibiotics. There are species which form substances toxic for plants and species which form substances stimulating the growth of plants.

   Actinomycetes not infrequently form dark-brown substances in substrates which by their chemical composition resemble the humic acids of soil (Flaig, 1952; Küster, 1952, and others).

   Genus Proactinomyces. Proactinomycetes resemble actinomycetes by their structure. They form a well-developed mycelium in the early stage of growth on ordinary nutrient media. This mycelium is similar to that of actinomycetes, and soon becomes segmented into rode and cocci. The segmentation of threads is accomplished through transverse septa (Figure 44).

 

Figure 44. Proactinomyces ruber. 48 hour culture on must agar

a, b, c--disintegration of mycelium into rods and cocci.

   Colonies of the typical specimens are usually bare and devoid of serial mycelium, rough, wrinkled, seldom smooth, and are less compact than the colonies of actinomycetes; sometimes they are of a doughy consistency. Hyphae near the base of the colony often grow into the agar. Some species more related to actinomycetes have colonies covered with weak coating of an aerial mycelium with straight sporangia. Spores in that case are usually rodlike. The characteristic property of proactinomycetes is the disintegration of the mycelium during their growth into rods of different length and into cocci.

   There are pigmented and colorless species among the proactinomycetes. The pigments are often red or orange, more rarely yellow or brown; only the colonies are colored while the surrounding medium remains colorless. Physiologically, proactinomycetes do not differ markedly from actinomycetes. The nutritional requirements and fermentative capacity is similar in both genera. Few antagonists can be found among them. In soil proactinomycetes are rarely encountered.

   Genus Mycobacterium. This genus consists of rodlike organisms,, resembling bacteria. Their cells are of irregular form (Figure 45), immobile and grampositive; they do not form genuine spores, but in some species the cell plasma becomes fragmented into 3-5 parts, in the same manner as in actinomycetes. The characteristic property of mycobacteria is their branching. In many species this is sharply pronounced. Cells possess 1-2 branches in the form of lateral appendices. In some species branching is rarely encountered. After inoculation the rodlike cells disintegrate into cocci relatively rapidly (after 1-2 days and sometimes after only 10-15 hours). In this stage of growth mycobacteria may be mistaken for micrococci. Gray and Thornton (1928) described bacteria closely resembling mycobacteria; they were curved, branched, gram-positive but they possessed flagella and were mobile. These bacteria were called Mycoplana. Similar bacterial forms were described by Köhler (1955), who considered them mycobacteria, which is erroneous.

 

Figure 45. Mycobacteria. Different types of structure:

a--Mycob. hyalinum; b--Mycob. rubrum; c--Mycob. cyaneum; d--Mycob. bifiidum; d--Mycob. citreum; f--Mycob. filiforme. Magnified about 600 times.

   Their colonies resemble those of bacteria. In ordinary nutrient media they are of pasty or slimy consistency, the colonies are convex, more seldom flat, and sometimes assume gluey droplike form. The color of the colonies may be red, orange, pink, yellow, blue or brown. There are many colorless forms.

   In the course of variation, mycobacteria can form variants of the structure of proactinomycetes. Mycobacteria do not possess sharply pronounced biochemical or physiological properties. This group is similar to bacteria and consists of representatives with different physiological and biochemical properties. Some species decompose proteins, carbohydrates, sugars, organic acids and alcohols. Others decompose hydrocarbons, products of oil, paraffin, tars and other substances not utilized by ordinary bacteria. Among mycobacteria, oligonitrophilic forms are encountered, which grow well on nitrogen-free medium; they apparently fix atmospheric nitrogen.

   Mycobacteria do not possess antagonistic properties. They were not found to produce antibiotics.

   Among mycobacteria some phytopathogenic forms are described, such as Mycob. michiganense and others (see Krasil'nikov, 1949 c). The well-known causative agents of tuberculosis and diphtheria belong to mycobacteria.

   Mycobacteria live in soils of different geographical zones and their population there is abundant. Their numbers depend on the state of the soil and on external conditions. Mycobacteria similar to actinomycetes, grow well in soils of low humidity. Owing to this fact they are frequently the prevailing organisms in arid regions.

   Genus Mycococcus. In their external appearance the mycococci resemble micrococci, or, more strictly, mycobacteria in their last phase of growth. The cells of mycococci are coccoid of 0.5-1.0 µ in diameter. They differ from micrococci by the irregular shapes of their cells. The latter are angular, pearlike, irregularly spherical (Figure 46). They multiply by fission, budding and constriction. Before fissions cells elongate slightly and assume a rodlike form. Mycococci are gram-positive, immobile, and nonsporeforming. In the course of variation of mycococci, variants are obtained of the mycobacterial type, which provides the grounds for considering them genetically related to the latter.

   

Figure 46. Mycococci. Mycoc. ruber

   Physiologically and biochemically they do not differ from mycobacteria. In soil they are rarely encountered.

   Genus Micromonospora. Organisms belonging to this genus resemble actinomycetes in their external appearance. They produce a well-developed mycelium in the form of thin branching threads. Their formation of spores differs from that of actinomycetes. The spores (conidia) are formed on short branches or directly on mycelial threads. The spores are single (Figure 47), of spherical or oval form (they rarely have an elongated form). Their size is similar to that of actinomycetes--about 1 µ.

 

Figure 47. Micromonospora globosa:

a--branches with sporangia; b--spores.

   The colonies of micromonospora are compact. merge with agar, and lack the aerial mycelium; upon spore formation a weak coating appears. consisting of short aerial branches--sporangia. The colonies are red-brown, yellow-brown, and of other colors. The pigment does not diffuse into the medium.

   The organisms grow slowly and poorly in ordinary media, the majority of species prefer a temperature of 40-45° C or higher. They are rarely encountered in soil.

   Their physiological and biochemical properties do not differ from those of actinomycetes. Some species form antibiotics.

Bacteria

   Microorganisms designated as bacteria are diverse in their structure, nature, growth, and biological properties.

   According to external appearance, bacterial calls are divided into three types which are distinctly different from each other: coccoid, rodlike, and spiral forms. According to this they are divided into groups: cocci or Coccaceae, rods--Bacteriaceae and spirals--Spirillaceae. Each group is further subdivided.

   Cocci--family Coccaceae. The characteristic property of this group of bacteria is the spherical shape of their cells. Their shape is regular and they possess a well-defined membrane. The diameter of the cells is 0.2-1 µ, more frequently 0.6-0.7 µ. They divide by fission, which occurs in different planes. The cells are nonmottle, and with the exception of one species (Sarcina ureae) do not form flagella or spores. The majority of cocci are gram-positive, only gonococci are gram-negative.

   The cells are either separated from each other or united in aggregate pairs, arranged by fours into platelets of packet-shaped aggregates. According to this property, cocci are subdivided into groups: 1) micrococci--genus Micrococcus, 2) diplococci--genus Diplococcus, 3) streptococci--genus Streptococcus and 4) sarcina--genus Sarcina.

   In the genus Micrococcus the cells are individual and do not form complexes. Only during their division do the cells remain in pairs for some time. Sometimes the cells are glued mechanically together in small formless clusters and easily separate into individual cells.

   In the genus Diplococcus the cells appear in pairs. Sometimes they resemble pairs of beans flattened laterally along the long axis.

   The genus Streptococus is characterized by chain formation. Cell division takes place in one plane, perpendicular to the long axis. The length of chains varies.

   In the genus Sarcina the cells are combined in packets in regular cubical forms. The number of cells in each packet varies from 8 to 54 and more. Cell division takes place in three reciprocally perpendicular planes. In some species the calls divide in two planes; then they form plates located in one plane. Such cell location is conditioned by growth conditions of the sarcina. Some investigators are inclined to consider them as a separate species--Pediococcus; however, there are not sufficient grounds for doing so.

   The participation of Coccaceae in the soil, formation processes is very restricted, if it takes place at all. In soil these organisms are rarely encountered, but if so, only in small numbers.

   In laboratory practice during analysis of soils, coccoid forms of mycobacteria, proactinomycetes and mycococci are mistaken for micrococci. On Kohlodny's growth plates the coccoid forms often belong to actinomycetes and sometimes to rodlike bacteria.

   Rodlike bacteria--family Bacteriaceae. The rodlike bacteria are the most varied and widespread group in the soil. According to the structure and development of the cells, rodlike bacteria subdivide into two large subgroups: sporeforming and nonsporeforming.

Asporogenous Bacteria

   Sporeforming bacteria have a rodlike form and are gram-positive. The cells are 2-10 x 0.5-1.2 µ in size. They form spores according to the character of spore formation, the shape of the calls, and the cytochemical composition. The sporeforming bacteria are divided into the genus Bacillus and the genus Clostridium.

   Genus Bacillus. Bacteria allocated to this genus have both motile and nonmotile rodlike cells of 2-10 x O.7-1 µ. The flagella are usually peritrichous. Their plasma is homogeneous, sometimes containing granules of reserve food stuff. Granuloma is absent. They multiply by division. Many species form long chains--threads. Upon spore formation the cells do not swell or swell slightly. The spores are terminal and central or located in any part of the cell. Most of thorn are aerobes, but anerobes are also encountered among them. Physiologically and biochemically this bacterial group is very divers. Among them there are many species with sharply pronounced proteolytic activity: they do compose proteins with the formation of ammonia, H2S and other odorous products of the putrefactive process.

   There are bacteria which vigorously hydrolyze starch, sugars, alcohols, organic acids, and many other organic compounds. Among the sporeforming bacteria of the genus Bacillus there are autotrophs, chemotrophs and organisms which oxidize hydrogen, ammonia, methane, and other compounds. Organisms are described which are capable of fixing molecular nitrogen. Bacteria which synthesize various active organic compounds--biotics, antibiotics, toxins, and others belong to this group.

   Among the sporeforming bacteria--Bacillus, antagonists are frequently encountered. The latter are second to actinomycetes with respect to distribution in nature.

   The sporeforming bacteria are widely distributed in soils, in numbers which reach tens and hundreds of thousands and even millions.

   Genus Clostridium. Cells of this genus of sporeforming bacteria differ from those, of the genus Bacillus in their structure and physiological properties.

   They swell before spore formation, assuming a lemonlike, or club shape. They are of a quite large in size (7-15 x 1.5-2 µ). The cells in young cutures are somewhat smaller (5-10 x 0.8-1 µ). The cells contain granuloma which stains with I + KI in dark-blue almost black color. The spores are not fundamentally different from those of the genus Bacillus, in some species they are somewhat larger.

   Bacteria of this genus are anerobes. They vigorously hydrolyze starch and pectinic substances and ferment sugars and other carbohydrates with the formation of butyric, proptonic and other acids. Some of them are characterized by acetone--butyrtc fermentation.

   Many species fix molecular nitrogen. The best known of them is Clostridium pastourianum described by Vinogradskii.

   The cultures of the genus Clostridium are widely distributed in soils, predominantly in those which contain humus and are rich in organic substances.

Sporeforming Bacteria

   Asporogenous bacteria are the most diverse and most widely distributed soil organisms. Their diversity to manifest in their size, form, growth, cultural pecularities and especially in their physiological and biochemical properties.

   The overwhelming majority of asporogenous bacteria do not differ from each other with respect to their external appearance--structure, size and type of colonies. They cannot be differentiated by ordinary laboratory methods according to their physiological properties. Nonetheless, the group of asporogenous bacteria comprises biologically different species, which can be established only upon thorough study of a few of them.

   Asporogenous bacteria an a rule, are gram-negative and motile, although their mobility to not always apparent. The motion in due to flagella. According to the location of the flagella they can be divided into peritrichous (genus Bacterium) and lophotrichous (genus Pseudomonas). Among the peritrichous soil bacteria there to a great number of diverse groups. We shall describe here only the most important and widely distributed genera Bacterium and Azotobacter.

   Genus Bacterium. The bacterial cells of this genus are rodlike, their size is 1.5-10 x 0.5-1 µ, more often 3-7 µ in length and 0.5-7 µ in diameter. They are gram-negative and motile. The flagella are located along the whole periphery of the cell, peritrichously. The number of the flagella vary; sometimes they are very numerous. They do not form spores. Their motility and the presence of flagella is not always apparent. In some species motility and the presence of flagella are apparent only under strictly specific conditions. The cells are flexible, their external form does not change. In many organisms the form and the size of the cells varies with the age.

   Cultural, physiological and biochemical properties of the different representative of this genus are diverse. There are species which decompose organic and inorganic compounds and are capable of decomposing and synthesizing, oxidizing and reducing many substances.

   There are among them pigmented and nonpigmented forms, which decompose various plant and animal residues and cause putrefaction of proteins with the formation of NH3, H2S, and other compounds.

   This is the most numerous and varied group among the soil bacteria. Their numbers in 1g of the soil approach millions and billions.

   The species diversity of the genus Bacterium is very great, but the number of species described to relatively small. This can be explained by the fact that the external features of these bacteria, as is the case in other bacteria, are very weakly expressed.

   Genus Azotobacter. This genus represents a group of asporogenous bacteria with peritrichous flagella, capable of fixing molecular atmospheric nitrogen. This group will be described in detail in the chapter on nitrogen fixation. Here we shall only mention that cells of Azotobacter differ from those of other soil bacteria by their markedly larger size. Many cultures are rodlike in the initial stages of growth (Figure 2b, c), and afterward become spherical. The cells are often united in packetlike complexes, and are coated with a slimy, compact capsule.

   The cultures grow well on synthetic nitrogen-free media (Ashby medium, Beijerink medium, and others). The majority of cultures and species do not grow on protein media nor, in general on media containing organic substances (meat-peptone agar, soybean agar, must agar).

   Azotobacter to not found in all soils; the degree of its distribution to not uniform.

   Great importance in soil fertility to ascribed to this group of bacteria.

   Presently, the genus Azotobacter is represented by few specie --Az. chroococcum, Az. azile, Az. vinelandii, and others. There are reasons to assume that there is a much greater number of species and varieties in nature.

Bacteria with polar flagella.

   Asporogenous bacteria with polar flagella, mono-and lophotrichous, are divided, according to their physiological and biochemical properties, into the following genera: Pseudomonas, Rhizobium, Acetomonas, Azotomonas, and others.

   Genus Peoudomonas. Apart from bacteria of the genus Bacterium this group of organisms to the most widely distributed in soils. There are millions, tens and hundreds of millions and often billions per 1 g of soil, depending on the properties of the soil and climatic conditions.

   Cells of this group are rodlike and 2-5 x 0. 6-0.7 µ in size. Larger as well as smaller forms are rarely encountered. At the end of the cell there is one or a few flagella (Figure 1, a-e). The spores are not formed inside the cells. They multiply like other bacteria by division.

   The appearance of the culture of this group does not differ from that of other groups of asporogenous bacteria. In nutrient media they produce colonies which are smooth, shining, colorless or rarely pigmented. Many species form a fluorescent green-yellow pigment which diffuses into the medium.

   Bacteria of this genus are very diverse in their physiological and biochemical properties. There are representatives among them which are similar to those of other bacterial groups, which decompose various organic and inorganic, protein and nonprotein compounds. Many species decompose organic compounds with the formation of final and intermediate decomposition compounds. There are species which fix molecular nitrogen, the so-called oligontrophils. This group includes autotrophs, heterotrophs, pathogens, saprophytes, etc.

   Abundant growth of Pseudomonas is seen in the rhizosphere of vegetative plants as well as on rotting animal and plant residues.

   Species with antagonistic properties are encountered among the representatives of Pseudomonas and Bacterium. Some species form antibiotics, others form toxins. But the majority of species of the genus Pseudomonas form biotic substances --vitamins (B1, B2, and others), auxins, various amino acids. etc.

   The role of Pseudomonas in soil-formation processes was not studied in detail but judging from its activity, it should be assumed to be considerable.

   Genus Rhizobium. This genus includes asporogenous bacteria with polar flagella which form nodules on the roots of leguminous plants. For this reason they are called root-nodule bacteria. They do not differ from the bacteria of the genus Pseudomonas in structure. The cells are rodlike, sometimes curved like mycobacteria (3-5 x 0.7 µ) and motile. In nodules and in certain artificial media they are deformed and possess bacteroid forms--swollen, irregularly spherical, retortlike, amoeboid, etc. Sometimes they possess lateral protrusions. However. the latter do not appear during the course of branching, as they do in mycobacteria, but to a result of degenerative process. Cells with protrusions and bacteroid cells in general do not survive.

   The cultures of root-nodule bacteria resemble those of ordinary asporogenous bacteria. Their colonies are colorless, smooth, slimy, convex or flat. They grow well in many synthetic media and also in media with plant organic substances (soy bean extracts and tissues of soybean plants). Some species do not grow in media with animal proteins (meat-peptone agar, meat-peptone broth, and others).

   Physiologically and biochemically the root-nodule bacteria do not differ from other bacteria. Their only specific property in the capacity to form nodules on the roots of legumes. The cells penetrate the cells of root tissue and develop there symbiotically, securing the fixation of nitrogen.

   The root-nodule bacteria are specific in that each culture forms nodules in the roots of strictly specific plants or groups of related species of plants. The division of these bacteria into species to made according to the plant host.

   The root-nodule bacteria are widely distributed in soils. They can be found everywhere, where the corresponding legumes are found and often even when such plants are absent. The number of these bacteria in soils varies in relation to the conditions and properties of the soil itself.

   Genus Azotomonas. Asporogenous bacteria 3-7 x 0.8 µ in size, and having polar flagella are included in the genus Azotomonas. They are motile, and multiply by division. Morphologically, culturally and biochemically they do not differ from other nonsporeforming bacteria. They grow well on many usual nutrient media, both protein and nonprotein, and on organic and synthetic media with a mineral source of nitrogen.

   The characteristic property of these bacteria to their ability to fix molecular nitrogen. Some species form a yellow-green fluorescent pigment, which diffuses into the substratum. They resemble Az. Vinelandii. They are rarely encountered in soil.

   Among the asporogenous bacteria, apart from the above-mentioned, there are many organisms grouped in separate genera which possess special and unique physiological functions as for example the sulfur bacteria, iron bacteria, nitrifiers, denitrifiers, etc. Different threadlike and other bacteria also belong here (see Krasil'nikov, 1949a; Bergey, 1948).

   Spiral bacteria--family Spirillaceae. Bacteria of this family have spiral and corkscrew-shaped cells, which can easily be distinguished by their appearance. Some of them are long with a large number of coils, thin or thick, others are short with 1-2 coils. There are also very small forms with half a turn.

   The bacteria are motile, with polar flagella, mono- and lophotrichous (Figure 1 a-f). The cells multiply by fission and constriction. They live predominantly in water but are frequently encountered in soils. Many of them participate in the sulfur cycle.

   This group is subdivided into five genera.

   Genus Vibrio or vibrions. The cells are small 1.5-3 µ in length and 0.5-0.7 µ in diameter. They are bent in the form of a comma, and are very motile due to their polar flagella. They are widely distributed In water reservoirs and in soil. Among them there are pathogenic species. Of the saprophytic forms, many reduce sulfates. They probably are of great importance in the soil.

   Genus Spirillum (spirilla). The cells are large, of various length from 3 µ to 70 µ and more, and 1-2 µ in diameter. They possess polar flagella and move actively, multiply by division and constriction, do not form pigments, and live in water. They are rarely encountered in soil.

   Genus Thiospira. Organisms of this genus are colorless autotrophs which ozidize H2S. Inside their cells, drops of sulfur are accumulated, which are also oxidized and serve as energy source. They live in water and are rare in soil.

   Genus Thiospirillum and genus Rhodospirillum. These genera differ from the preceding one due to their red-purple pigment. They are autotrophs, and synthesize organic compounds, some at the expense of light energy (genus Rhodospirillum) and others at the expense of light and chemical energy (genus Thiospirillium). They live in water reservoirs.

Phages

   Phages constitute a special group of organisms. Their size to measured in millimicrons (m µ) usually 50-100 m µ. Smaller phages are also encountered.

   Phage particles have an oval or spherical shape either with a short tail or without it. They multiply only inside the microbial host and then only in a young vegetative living cell. In dead and old cells they do not reproduce.

   The reproduction process is as follows: phages become attached to the cell surface with their tails and enter it through the cell membrane; after they have penetrated the cell, pronounced structural and chemical changes of the protoplast take place. Desoxyribonucleic acid is formed in large amounts and all its mass becomes fragmented into separate granules or prophages. Subsequently. the cell membrane is destroyed and prophages are liberated in the form of mature phages (Figure 48). The entire process takes 15-40 minutes. In some bacteria this process takes five or more hours.

 

Figure 48. Phage particles, released after cell destruction. Phage of mycobacteria Mycobacterium sp. (according to Battagini, 1953)

   The number of phage particles formed in one cell varies from 50-1,000, most often 100-200, and varies according to growth conditions, stage and bacterial species.

   The characteristic property of phages is their ability to lyse bacterial cells. Phages possess defined specificity, attacking microbes of one or several species. Some phages attack only few strains of one species. There are phages which attack only bacteria--bacteriophages, and phages which attack actinomycetes-actinophages. Phages attacking other groups of microorganisms--fungi, yeasts, algae, etc, are unknown. There are descriptions of individual cases where phage particles were found in yeasts and fungi, however these data have not yet been confirmed.

   The chemical composition of phages resembles that of animal viruses. They contain protein, lipides and carbohydrates, as well as deoxyribonucleic acid in large quantity. All phages possess antigenic properties, ie., when they are introduced into the body of animals, they are capable of causing the production of antibodies with specific properties. Because of this, specific antiphage sera are available.

   Different phages are endowed with different resistance to the action of environmental factors--temperature, salt solutions, etc. Some phages are inactivated by a relatively low concentration of salts or low temperature, others, on the contrary, survive 75° C for one hour. Phages are more resistant to radiation than bacteria. They may be preserved for long periods of time in a dry state. They are easily inactivated by certain substances such as citric acid, phenanthrene, and others.

   Phages differ from each other in their lytic activity. Some of them lyse calls quickly and completely; others weakly and not to completion. They may be differentiated according to the character of lysis of colonies. Some produce lytic zones with sharply defined borders, others with diffuse borders.

   Phages are relatively easily subjected to variation. They change their specificity, and the character of the formation of "negative colonies", in other words the form of lysis plaques. Their resistance to this or other external factors can also change (Rautenstein, 1955).

   Phages, as shown by studies, are widely distributed in nature. They can be found in water, animal and plant residues. They are found in large quantities in soil. The works of Rautenstein and Khavina (1954) and of others have shown that actinophages live in the soil in considerable numbers. It can be assumed, according to some data, that their role in the soil to considerable, Demolon and Dunes (1933, 1939) for instance assume that phages of root-nodule bacteria inactivate the latter.

   The clover exhaustion of the soil, according to the authors, is indeed conditioned by much a phenomenon. Phages exert a great effect on the variability of bacteria and actinomycetes and favor formation of new forms and variants. In other words, phages constitute one of the powerful factors of species formation.

   There are various points of view on the problem of the nature of phages which can be reduced to two essential ones. According to one-phages are ultramicroscopic organisms or microbial viruses (viruses of bacteria and actinomycetes) (Zilber, 1953; Ryzhkov, 1952; Sukhov, 1951, 1955). According to the other theory, phages are biocatalysts, i.e., active substances with biocatalytic or enzymatic properties capable of reproduction under certain conditions (Kriss, 1944). Both points of view present elements which cannot be neglected.



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