Part I, continued

The Principles of Classification of Microorganisms

   The classification of microorganisms is veryunsatisfactory. There is no common principle of classification in microbiology. Theclassification of bacteria and actinomycetes is especially inadequate. This can beexplained by the peculiarity of those organisms, the simplicity of their structureand growth and lack of external properties for differentiation. An immense numberof 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 classificationof bacteria and actinomycetes lies in the absence of factual material on phylogenyof individual species, groups and subgroups. The natural classification should reflectthe stages of evolutionary development. The principle of phylogenetic structure ofclassification requires a thorough knowledge of the organisms and species relationship.

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

   The great shortcoming of every classificationis 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 ofnew forms, species, varieties, etc.

   Classification also becomes complicated by thefact 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 accidentalforeign organisms. The natural classification should embrace only the former organismswhich determine real affinity, while the foreign convergent forms should be excluded.

   Microbiological literature contains numerousdata on the morphological similarity of entirely different organisms. For example,a large group of nonsporeforming yeasts, comprising the family Torulaceae, in realityrepresents a mixture of different species and not only species but also genera, andgenerally speaking in a mixture of different unrelated groups. The bacteria of thegenus Micrococcus are characterized by their spherical shape. Into this grouporganisms which in fact belong to coccoid bacteria are included and also not infrequentlyspecimens of actinomycetes are included in the genus Mycococcus. To the rodlikebacteria of genus Bacterium very diverse microbial forms are referred. Bacteriafrom the genus Bacterium are frequently linked to Mycobacterium onlybecause the former sometimes possess lateral branches which are characteristic ofthe genus Mycobacterium. Thereby the specificity of this property is not accountedfor. Properties which are natural and fixed in one genus may be abnormal and unnaturalin others.

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

   The lack of knowledge of the life cycle of thisor another microbe frequently misleads the investigator. For example for this reasonmycobacteria 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 propertyis of systematic value. The problems of identification and classification shouldbe distinguished from each other although in some cases they are interwoven and overlap.Fixed properties which are transmitted by heredity, which characterize the natureof the species, and which are manifest under determined growth condittons, are takeninto consideration for classification. 

The Species Concept in Microorganisms

   The concept of species is the most difficultproblem 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 branchof biology.

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

   Often authors consider bacterial species accordingto 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 theknowledge of the organism. It was a long way from the Linnean idea of a species asa fixed and not changing entity to Darwin's teaching and present ideas of the ever-developingspecies. In the determination of species, Linnaeus stressed two basic properties--theconstancy of properties and their sharp demarcation in various species. Later onKorzhinskii (1892) introduced a geographical principle into the concept of specieswhich was more thoroughly elaborated by Wetstein (1898). According to this principle,the species morphology is correlated with the place of its habitation. Approximatelyat the same time, a historical moment entered the concept of species; this was especiallypronounced in the works of Semenov-Tyan'Shanskii (1910), who pointed out that thesum of architectonic properties is a result of interaction of a complex of physicogeographicalfactors which took place in a bygone geological period (according to Krasil'nikov,1938a).

   The establishment of phylogenetic relationshipswithin the species led to the separation of the main and the subordinate taxonomicentities--varieties, forms, etc. The species was divided into main and secondaryforms; binomial nomenclature became tri-and quadrinomial nomenclature.

   With the development of genetics the conceptof 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"--asimple means of classification of lower organisms. "Linneon"--the complexof "jardanons"--according to the Russian concept, the inner species varietyof forms does not exceed the limits of qualitative unity of the species.]

   The experimental verification is a new stagein the development of the determination of species. Of the works in this directionthe most important ones are those of Touresson, (1922-1925) which showed the dependenceof the external form of plants on the ecological conditions of habitation. He introducedthe 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 Linneanspecies are definite systems of forms, and not an accidental motley of races. Komarovin his work "Flora of the Kamtchatka Peninnsula" (1920) gives the followingdefinition of species: species is a morphological system multiplied by geographicaldefiniteness (Komarov, 1940).

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

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

   As can be seen from the aforesaid, the conceptof species is considered differently. Every specialist has his own narrow idea aboutspecies, moreover, the factual material accumulated by others to not always takeninto 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 toelaborate it according to their specific needs, we point out that those concernedwith classification should adhere to the main outlines and taxonomic rules, establishedby international congresses and by conferences of specialists. Besides, there aregeneral biological features which characterize species of higher and lower organismsalike.

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

   The distinguishing feature of microorganismsis their lack of multitude of morphological characteristics. Therefore, determinationof 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 employedfor the determination of microbial species. No data are available in microbiology,which could show some definite regularity in the distribution of separate microbialspecies in nature. Even less data exist on the fossil microbial forms. The scantdata on the presence of microbial cells in microsections of coal, shales and otherdeposits gave no grounds for establishment of even large taxonomic entities. Methodsemployed in cytology and genetics are not suitable in that case. Bacteria and actinomycetesdo 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 ofhigher organisms. The sexual process in those organisms is either lacking or, whenpresent, is a unique one and cannot serve as a means for the establishment of a geneticrelationship of the organisms.

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

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

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

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

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

   Such profound and comprehensive studies enablethe 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 organismswhich were formerly thought to represent a homogenous species. Organisms which areassumed to belong to one species prove to be a complex group consisting of severalor many species.

   There are comparatively few species which areutilized 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 isclear that the usual methods employed for the determination of bacterial speciesare not uniform. There are many more species in nature than we are able to determineby the relatively primitive methods at our disposal. The heterogeneity of the knownspecies of bacteria and actinomycetes is becoming more and more apparent with themore thorough study to which they are subjected.

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

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

   The comparative studies of pure cultures of microbesenable one to study the species not only by itself but also with regard to the entirepopulation during their variations and deviations which take place in the processof variability.

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

   Physical properties are not infrequently employedfor 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 otherproperties 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 inorganicnutrients may be properties of a species only in a restricted sense.

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

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

   Microbiological literature contains data on thephylogenetic affinity of some specimens of mycobacteria (Imshenetskii, 1940). Osterleand Stahl (1929) and then Rautenstein (1946) obtained variants of Bac. mycoideswhich did not differ from Bac. effusus, Bac. olfactortus, Bac. nanus, Bac.vulgatus, Bac. cereus, Bac. brevis, Bac. panis. and Bac. mesentericus. A multitudeof 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, andBac. mesentericus panis described in the literature represent one specieswith two varieties. Gibson (1949) brings a voluminous experimental material concerningthe scope of the species Bac. subtilis. He identified this species with thefollowing 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 strainof Az. chroococcum yielded colorless, capsuleless variants with small atypicalcells which grew on protein media and did not fix nitrogen. Such variants were alsoisolated from the soil.

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

   Even more variants are encountered which differfrom the original cultures in their physiological and biochemical properties. Inthe majority of cases such variants are not distinguished in laboratory practiceand remain unrecognized.

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

   Employing this method we succeeded (Krasil'nikov1947b) in establishing the affinity between certain species of nonsporeforming bacteriaof the genus Bacterium and Pseudomonas and then between the sporeformingbacteria and other groups. During the course of the dissociation of two differentcultures of the genus Pseudomonas similar variants (Figure 39) were obtainedwhich 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 yieldidentical variants as was mentioned above. All this indicates the similarity of hereditaryand species properties of organisms.

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

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

   The serological method of diagnosis of pathogenicbacteria is widely used in medical bacteriological practice. This method is a verysensitive one and enables the detection of small differences between strains whichare formed under the conditions of variability of these or other cultures. The sensitivityis determined by the chemical nature of the antigens and the capacity of the methodto distinguish differences between the various molecules, predominantly protein molecules,which cannot be differentiated by chemical methods. With the aid of antigens thesmallest differences in the protein composition can be detected and consequentlysubtle culture variations can be diagnosed. Nonetheless, this method is not beingused in the classification of microbes to the extent that could be expected. Thoughit is an excellent and sensitive method for the differentiation of related strainsIt is at the same time unsuitable in the majority of cases for the classificationand differentiation of species. Attempts to divide the root-nodule bacteria by serologicalmethods yielded contradictory results which did not agree with the classificationdone by conventional methods. Entirely different species are linked to one taxonomicgroup 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 wasmade to classify strains of Azotobacter, Radiobacter, bacteria of the genusPseudomonas, Bacterium, Clostridium, streptococci, and others (Mayr and Harting,1948; Fred et al., 1932; Frances-Shattock, 1955).

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

   Concerning the lactic bacteria of the genus Lactobacteriumthere was no success recently in classifying them by serological means. Quite recentlySharpe succeeded in obtaining precipitin, with the aid of which these bacteria areas if differentiated into types, which can be correspondingly determined by physiological-morphologicalmethods. (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 elaboratedfor 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 bythis method are of a specific character and do not always correspond to the groupsdetermined by other methods. Experience shows that the determination of species byserological methods should be carried out with precaution and the results must becarefully analyzed. It is only of secondary importance and complements other methodsof microbiological analysis.

   Attempts are made to identify bacterial speciesby means of phages. This method is based on the capacity of phages to lyse definedbacterial species. Among phages there are strains with strictly specific speciesproperties attacking only one bacterial species. There are also group-specific phageswhich attack bacteria belonging to different species or even genera. It is assumedthat the former phages are suitable for species differentiation and the latter forgroup differentiation.

   Data exist on species differentiation by meansof phages of bacteria of the coli group, staphylococci, nonsporeforming and sporeformingsoil bacteria, and others. Katznelson and Sutton (1951) pointed out that phytopathogentcbacteria (in various substrates) can be detected by means of phages. They detectedPs. phaseolicola in seeds of kidney beans without isolating them in pure cultures.The seeds were washed with water, the washings were inoculated with the specificphage 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, accordingto the authors, can multiply only in the cells of the afore-mentioned species.

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

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

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

   These properties are of a narrow specific characterand can he employed only for a restricted group of microbes. It may happen that whendifferent representatives of microorganisms are studied from the point of view offormation of specific toxic and nontoxic metabolites this principle will find wideapplication in classification of microorganisms.

   According to our data, specificity of antagonismconstitutes an essential species indicator. We have shown that microbes grown togetherin 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 importancein species determination. Each species of an antagonist synthesizes its specificantibiotic and sometimes two and three antibiotics simultaneously. As a rule, thecharacteristic property of antibiotics to the fact that they do not suppress thegrowth of the producing organism and other organsms belonging to the same species.The action of the antibiotic and, consequently, of the organism which produces theantibiotic is directed toward the organism's competitors. This specificity is strictlyconstant for the antagonists and can serve as a means for species determinaion. Onthe basis of this principle, we have elaborated a method of species differentiationof actinomycetes and sporeforming antagonistic bacteria (Krasil'nikov, 1951c, Krasil'nikov,Korenyako, Nikitina, Skryabin, 1951, Afrikyan, 1951a). Employing this method we wereable to establish a multitude of species among certain groups of organisms whichwere classified as one species. Similar data were obtained by Teillon (1953) andsome other investigators (see below).

   From the afore-mentioned it may be concludedthat species is a really existing taxonomic entity, consisting of different individuals,strains or cultures (in the laboratory), forms and varieties. They all possess oneproperty which can be manifested in different forms. This property characterizesthe 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. Individualsand varieties may differ from each other but the species properties common for themremain unchanged.

   Consequently, species as a link in the evolutionarychain 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 relationto one another. The relations within the species differ from the relations betweenorganisms of different species. This is clearly shown in examples of microbial antagonism.

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

   For example, the actinomycetes which producestreptomycin and comprise one species Act. streptomycini are widely distributedin nature; individual strains live in different geographical zones, in differentsoils, in the north and south, east and west, in the silts of rivers and lakes, andon 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 samefundamental characteristics.

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

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

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

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

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

Subdivision of Microorganisms into Main Groups

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

   This series is divided into two groups:

   1) Schizophyceae--lower organisms, possessingchlorophyll, phycocyanin or phycoerythrin;

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

   We divide them into four classes:

Class I--Actinomycetes

Class II--Bacteriae

Class III--Myxobacteriae

Class IV--Spirochaetae



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

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

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

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

   Sporangia containing spores are formed on thethreads 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 between0.5-1 and even 5-7 and more. The coils in some cases are densely packed, they frequentlyhave 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, lessfrequently they are rodlike, and cylindrical forms with sharp ends (Figure 41). Sporesof many actinomycetes have different forms--spherical, oval, and rodlike. In youngsporangia 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 intoseparate cells which part and become mature spores (Figure 42b). In the second case,the protoplasm of the sporangium becomes fragmented into separate sectors or lumpswithout formation of septa. The lumps become rounded at the ends, assume rodlike,oval or spherical form, become covered with their own membrane and transform intomature spores. Afterward the membrane of the thread is destroyed and the spores arereleased (Figure 42a).


Figure 42. Spore formation by actinomycetes:

a) fragmentation; b) segmentation.

   Upon submerged growth in liquid media with constantshaking (on shakers) many actinomycetes have a fragmented mycelium resembling thatof 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 mistakenfor 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 growthof the culture. Cells which are formed upon fragmentation of the vegetative myceliumunder the condition of submerged growth are ordinary vegetative forms like thoseof mucor fungi.

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

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

   The physiological property of actinomycetes istheir capacity to utilize many organic substrates even such which are not utilizedby other bacteria and fungi. In natural conditions on plant residues, actinomycetesbegin to grow after all the readily assimilated substances have been decomposed andassimilated and the fungi and bacteria have stopped growing. Actinomycetes grow abundantlyon 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 becovered 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 northand in the tropics, on barren rocks and in fertile chernozems, on mountaintops andin valleys. They live in water, silts, in the soil, on various plant and animal residues.

   Upon growth in artificial nutrient media, actinomycetesform a number of peculiar substances. They have a characteristic smell somewhat resemblingthe odor of earth. Some species have a smell of camphor, fruits, etc. The chemicalnature of those odors is not known.

   Among the metabolic products of actinomycetesdifferent biotic, antibiotic and toxic substances are found. Of the biotic substancesthe 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 actinomycetesis widespread. Approximately 50-70 % of isolated actinomycetes produce antibiotics.There are species which form substances toxic for plants and species which form substancesstimulating the growth of plants.

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

   Genus Proactinomyces. Proactinomycetesresemble actinomycetes by their structure. They form a well-developed mycelium inthe early stage of growth on ordinary nutrient media. This mycelium is similar tothat of actinomycetes, and soon becomes segmented into rode and cocci. The segmentationof 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 usuallybare and devoid of serial mycelium, rough, wrinkled, seldom smooth, and are lesscompact 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 relatedto actinomycetes have colonies covered with weak coating of an aerial mycelium withstraight sporangia. Spores in that case are usually rodlike. The characteristic propertyof proactinomycetes is the disintegration of the mycelium during their growth intorods of different length and into cocci.

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

   Genus Mycobacterium. This genus consistsof rodlike organisms,, resembling bacteria. Their cells are of irregular form (Figure45), immobile and grampositive; they do not form genuine spores, but in some speciesthe 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 thisis sharply pronounced. Cells possess 1-2 branches in the form of lateral appendices.In some species branching is rarely encountered. After inoculation the rodlike cellsdisintegrate into cocci relatively rapidly (after 1-2 days and sometimes after only10-15 hours). In this stage of growth mycobacteria may be mistaken for micrococci.Gray and Thornton (1928) described bacteria closely resembling mycobacteria; theywere curved, branched, gram-positive but they possessed flagella and were mobile.These bacteria were called Mycoplana. Similar bacterial forms were describedby 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. Inordinary nutrient media they are of pasty or slimy consistency, the colonies areconvex, more seldom flat, and sometimes assume gluey droplike form. The color ofthe colonies may be red, orange, pink, yellow, blue or brown. There are many colorlessforms.

   In the course of variation, mycobacteria canform variants of the structure of proactinomycetes. Mycobacteria do not possess sharplypronounced biochemical or physiological properties. This group is similar to bacteriaand 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 substancesnot utilized by ordinary bacteria. Among mycobacteria, oligonitrophilic forms areencountered, which grow well on nitrogen-free medium; they apparently fix atmosphericnitrogen.

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

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

   Mycobacteria live in soils of different geographicalzones and their population there is abundant. Their numbers depend on the state ofthe soil and on external conditions. Mycobacteria similar to actinomycetes, growwell in soils of low humidity. Owing to this fact they are frequently the prevailingorganisms in arid regions.

   Genus Mycococcus. In their external appearancethe mycococci resemble micrococci, or, more strictly, mycobacteria in their lastphase 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 areangular, pearlike, irregularly spherical (Figure 46). They multiply by fission, buddingand constriction. Before fissions cells elongate slightly and assume a rodlike form.Mycococci are gram-positive, immobile, and nonsporeforming. In the course of variationof mycococci, variants are obtained of the mycobacterial type, which provides thegrounds for considering them genetically related to the latter.


Figure 46. Mycococci. Mycoc. ruber

   Physiologically and biochemically they do notdiffer from mycobacteria. In soil they are rarely encountered.

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


Figure 47. Micromonospora globosa:

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

   The colonies of micromonospora are compact. mergewith 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 ordinarymedia, the majority of species prefer a temperature of 40-45° C or higher. Theyare rarely encountered in soil.

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


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

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

   Cocci--family Coccaceae. The characteristic propertyof this group of bacteria is the spherical shape of their cells. Their shape is regularand 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 differentplanes. The cells are nonmottle, and with the exception of one species (Sarcinaureae) 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 otheror united in aggregate pairs, arranged by fours into platelets of packet-shaped aggregates.According to this property, cocci are subdivided into groups: 1) micrococci--genusMicrococcus, 2) diplococci--genus Diplococcus, 3) streptococci--genusStreptococcus and 4) sarcina--genus Sarcina.

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

   In the genus Diplococcus the cells appearin pairs. Sometimes they resemble pairs of beans flattened laterally along the longaxis.

   The genus Streptococus is characterizedby chain formation. Cell division takes place in one plane, perpendicular to thelong axis. The length of chains varies.

   In the genus Sarcina the cells are combinedin packets in regular cubical forms. The number of cells in each packet varies from8 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 inone 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, formationprocesses is very restricted, if it takes place at all. In soil these organisms arerarely 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 sometimesto rodlike bacteria.

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

Asporogenous Bacteria

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

   Genus Bacillus. Bacteria allocated tothis genus have both motile and nonmotile rodlike cells of 2-10 x O.7-1 µ. Theflagella are usually peritrichous. Their plasma is homogeneous, sometimes containinggranules of reserve food stuff. Granuloma is absent. They multiply by division. Manyspecies form long chains--threads. Upon spore formation the cells do not swell orswell slightly. The spores are terminal and central or located in any part of thecell. Most of thorn are aerobes, but anerobes are also encountered among them. Physiologicallyand biochemically this bacterial group is very divers. Among them there are manyspecies with sharply pronounced proteolytic activity: they do compose proteins withthe formation of ammonia, H2Sand other odorous products of the putrefactive process.

   There are bacteria which vigorously hydrolyzestarch, sugars, alcohols, organic acids, and many other organic compounds. Amongthe sporeforming bacteria of the genus Bacillus there are autotrophs, chemotrophsand organisms which oxidize hydrogen, ammonia, methane, and other compounds. Organismsare described which are capable of fixing molecular nitrogen. Bacteria which synthesizevarious active organic compounds--biotics, antibiotics, toxins, and others belongto this group.

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

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

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

   They swell before spore formation, assuming alemonlike, 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 cellscontain granuloma which stains with I + KI in dark-blue almost black color. The sporesare not fundamentally different from those of the genus Bacillus, in somespecies they are somewhat larger.

   Bacteria of this genus are anerobes. They vigorouslyhydrolyze starch and pectinic substances and ferment sugars and other carbohydrateswith the formation of butyric, proptonic and other acids. Some of them are characterizedby acetone--butyrtc fermentation.

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

   The cultures of the genus Clostridiumare widely distributed in soils, predominantly in those which contain humus and arerich in organic substances.

Sporeforming Bacteria

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

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

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

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

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

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

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

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

   Genus Azotobacter. This genus representsa group of asporogenous bacteria with peritrichous flagella, capable of fixing molecularatmospheric nitrogen. This group will be described in detail in the chapter on nitrogenfixation. Here we shall only mention that cells of Azotobacter differ fromthose of other soil bacteria by their markedly larger size. Many cultures are rodlikein the initial stages of growth (Figure 2b, c), and afterward become spherical. Thecells are often united in packetlike complexes, and are coated with a slimy, compactcapsule.

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

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

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

   Presently, the genus Azotobacter is representedby few specie --Az. chroococcum, Az. azile, Az. vinelandii, and others. Thereare reasons to assume that there is a much greater number of species and varietiesin nature.

Bacteria with polar flagella.

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

   Genus Peoudomonas. Apart from bacteriaof the genus Bacterium this group of organisms to the most widely distributedin soils. There are millions, tens and hundreds of millions and often billions per1 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. Atthe end of the cell there is one or a few flagella (Figure 1, a-e). The spores arenot formed inside the cells. They multiply like other bacteria by division.

   The appearance of the culture of this group doesnot differ from that of other groups of asporogenous bacteria. In nutrient mediathey produce colonies which are smooth, shining, colorless or rarely pigmented. Manyspecies form a fluorescent green-yellow pigment which diffuses into the medium.

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

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

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

   The role of Pseudomonas in soil-formationprocesses was not studied in detail but judging from its activity, it should be assumedto be considerable.

   Genus Rhizobium. This genus includes asporogenousbacteria 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 thebacteria of the genus Pseudomonas in structure. The cells are rodlike, sometimescurved like mycobacteria (3-5 x 0.7 µ) and motile. In nodules and in certainartificial media they are deformed and possess bacteroid forms--swollen, irregularlyspherical, 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 cellsin general do not survive.

   The cultures of root-nodule bacteria resemblethose 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 plantorganic substances (soy bean extracts and tissues of soybean plants). Some speciesdo not grow in media with animal proteins (meat-peptone agar, meat-peptone broth,and others).

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

   The root-nodule bacteria are specific in thateach culture forms nodules in the roots of strictly specific plants or groups ofrelated species of plants. The division of these bacteria into species to made accordingto the plant host.

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

   Genus Azotomonas. Asporogenous bacteria3-7 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 biochemicallythey do not differ from other nonsporeforming bacteria. They grow well on many usualnutrient media, both protein and nonprotein, and on organic and synthetic media witha mineral source of nitrogen.

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

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

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

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

   This group is subdivided into five genera.

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

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

   Genus Thiospira. Organisms of this genusare colorless autotrophs which ozidize H2S. Inside their cells, drops of sulfur are accumulated, which are also oxidizedand 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. Theyare 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 constitute a special group of organisms.Their size to measured in millimicrons (m µ) usually 50-100 m µ. Smallerphages are also encountered.

   Phage particles have an oval or spherical shapeeither with a short tail or without it. They multiply only inside the microbial hostand then only in a young vegetative living cell. In dead and old cells they do notreproduce.

   The reproduction process is as follows: phagesbecome attached to the cell surface with their tails and enter it through the cellmembrane; after they have penetrated the cell, pronounced structural and chemicalchanges of the protoplast take place. Desoxyribonucleic acid is formed in large amountsand all its mass becomes fragmented into separate granules or prophages. Subsequently.the cell membrane is destroyed and prophages are liberated in the form of maturephages (Figure 48). The entire process takes 15-40 minutes. In some bacteria thisprocess 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 cellvaries from 50-1,000, most often 100-200, and varies according to growth conditions,stage and bacterial species.

   The characteristic property of phages is theirability to lyse bacterial cells. Phages possess defined specificity, attacking microbesof one or several species. Some phages attack only few strains of one species. Thereare 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 yeastsand fungi, however these data have not yet been confirmed.

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

   Different phages are endowed with different resistanceto the action of environmental factors--temperature, salt solutions, etc. Some phagesare 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 radiationthan bacteria. They may be preserved for long periods of time in a dry state. Theyare easily inactivated by certain substances such as citric acid, phenanthrene, andothers.

   Phages differ from each other in their lyticactivity. Some of them lyse calls quickly and completely; others weakly and not tocompletion. 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 "negativecolonies", in other words the form of lysis plaques. Their resistance to thisor other external factors can also change (Rautenstein, 1955).

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

   The clover exhaustion of the soil, accordingto the authors, is indeed conditioned by much a phenomenon. Phages exert a greateffect on the variability of bacteria and actinomycetes and favor formation of newforms and variants. In other words, phages constitute one of the powerful factorsof species formation.

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