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   Various representatives of microorganisms inhabit the soil--bacteria, actinomycetes, fungi, algae, protozoa and more highly organized animals. There are also various ultramicrobes--phages and others. Our knowledge of the structure and development of soil microorganisms is obtained by observation of their growth on artificial nutrient media, but little is known about the form in which they inhabit the soil and what their dimensions, structure, growth, and multiplication are. Does our knowledge, obtained by investigation of growth of microorganisms on artificial media, reflect the state in which they occur in soil? Many microbes are known only because they occur in soil. From general observations of bacterial growth on nutrient media we know that the structure and form of their cells is relatively simple and monotypic. Three types of cell structure are recognized--spherical. rod-shaped and spiral form. According to this, bacteria are divided into groups: cocci, bacilli, and spirilla. In every group there are subgroups.

   The cell structure of these organisms is monotypic. As in higher organisms the bacterial cells possess a cell wall and a protoplast. The internal structure of the protoplast and cell wall differ from the structure of higher forms.

   The cells of bacteria, fungi, actinomycetes, and yeasts, like other organisms of plant origin have a cell wall. Very few bacteria (myxobacteria) do not possess one. It forms the external skeleton of bacteria, actinomycetes and fungi, and determines the cell shape of these organisms. Cells having a cell wall do not change their shape while moving; they are rigid. The cell wall itself has some elasticity, due to its physical and chemical structure.

   There are data showing that bacterial cell walls have a complex structure differing in various representatives. In some gram-negative bacteria (Shigella and others), the cell wall is composed of polysaccharides, proteins and residual phospholipids. Immunological properties of intact cells, separation of the "O" antigen and its components from them, show that a polysaccharide is the basic substance of the bacterial cell surface. The protein component of the "O" antigen as well as the phospholipid part of the cell wall is evidently located deeper.

   In gram-positive bacteria the inner layer of the cell wall, close to the plasma membrane (the upper layer of plasma or cytolemma), is composed of protein molecules which contain a considerable number of basic amino acids and sulfhydryl groups. In this layer there are compounds which condition the gram-positive staining. The layer in question is covered on the outside with a second layer composed of magnesium ribonucleate, which is in the gram-positive complex.

   On the outside of this second layer desoxyribonucleoproteins are located. With their aid specific polysaccharides are produced by the smooth (S) bacteria forms on their surfaces. This three-component layer of nucleoproteins represents the outer system of various enzymes and coenzymes. On the one hand the first phases of metabolism, synthesis and resynthesis of molecules from elements entering from the environment take place here, on the other hand, the decomposition of complex molecules leaving the organism also takes place here. In smooth forms of bacteria (S-forms) on the surface, the outer layer of desoxyribonucleic proteins produces specific polysaccharides which enter into the composition of the capsules. This synthesis is carried out by an enzyme, containing magnesium. In the absence of magnesium in the medium, cells appear, as a result of magnesium starvation, that do not contain the gram-positive substance and do not synthesize the capsular polysaccharide. In such cases the cells are gram-negative. having properties of rough forms and grow as R-forms (Stacey, 1949, Peshkov, 1955).

   With the aid of electron microscopy it was revealed that the surface of the bacterial cell wall often has a fibrous structure. Separate submicroscopic fibers of a surface mucous substance, mucomucin, extend beyond the cell wall, in the form of slender filaments sometimes imitating flagella.

   In this way we imagine the structure of the cell wall as a complex multi-layer formation, covering the living substance of the cell and having a sufficiently loose structure to let various compounds through and to secure an exchange of substances of the cell protoplasm with the environment.

   The mechanical strength of the bacterial cell wall is relatively great. This is proved by the fact that upon disruption of the cells, the cell walls are often completely preserved. One may achieve a complete destruction of the cell, fully preserving the cell walls. For instance by shaking the bacterial cells with microscopically small glass beads the cells are fractionated, yet the cell walls remain intact. The cell wall is disrupted in one spot and the plasma leaks out through the opening. Upon rupture, the broken edges of the cell walls may be seen. This destruction with rupture of cell walls is also observed after ultrasonic and other mechanical action.

   The cell walls of bacteria as well as those of higher plants are formed by the membrane of the periplast or cytolemma.

   As was mentioned above, this bacterial surface excretes a mucous substance, composing the mucous capsule. When this substance is formed in large amounts, the capsule becomes thick, well outlined under the microscope, and the whole culture assumes a mucous character. In cases of scanty excretion of the mucous substance, the capsule around the cell wall is very small and not often visible by ordinary microscopy.

   Mucous capsules of various bacteria differ in their chemical composition. Thus, for instance, the capsule of the acetic bacteria--Acetobacter xylinum consists of pure cellulose, the micelles of which were successfully revealed and photographed by electron microscopy, (Van Iterson, 1949). In the butyric acid bacteria Clostridium pasterianum, the mucous capsule consists of hemicellulose and may be stained blue with iodine (according to Peshkov, 1955).

   The mucous capsular substance may be mechanically or chemically separated from the cell. It may be dissolved In aqueous, alkaline, or buffer solutions. From acid solutions it may be precipitated by alcohol. Upon acid hydrolyasis and subsequent chemical treatment, one succeeds in differentiating two groups of compounds in the capsular mucous substance. One group does not contain nitrogenous substances or contains them in negligible quantities. The substances of the other are composed essentially of nitrogenous compounds Knaysi et al., 1950).

   The capsular substance of the first group in some root-nodule bacteria decomposes during hydrolysis with the formation of glucose, in pneumonia bacteria--with formation of galactose. Beside these two sugars, in the mucous capsular substance fructose and arabinose, or a mixture of these substances were found. The capsular substance of the second group of compounds found in the sporeforming bacteria, Bacillus anthracis and others, contains 7.4 - 8.0% nitrogen and is considered as a glucoprotein of the pseudomucin group (Kramer, 1921). A large amount of nitrogen was found in the mucous capsule of Rhizobium leguminosarum and in some lactic acid bacteria. In the capsules of bacteria, amino acids, ribonucleic and desoxyribonucletc acids are found. (Catlin, 1956, Smith et al., 1957).

   The study of the capsules of many bacteria and primarily of pneumococci, is of special interest due to the presence in them of substances which condition virulence and the gram-positive staining. The chemical composition of the capsule determines the specific serological and antigenic reactions. The capsular antigen is connected with the phenomenon of gram-staining. An autolyzed culture of pneumococci, having lost the ability to stain gram-positively, becomes unable to provoke the formation of specific precipitins for the capsular polysaccharide when injected, subcutaneously, into a rabbit. Upon autolysis, 4-10% of the dry weight of the cells is obtained and this substance consists mainly of ribonucleoproteins and ribonucleic acid Dubos, 1948).

   The ability of bacterial cells in the animal body to provoke the formation of precipitins for the capsular polyeaccharides is inseparably connected with the integrity of the gram-positive complex. This property in attributed to smooth forms (S). The rough forms (R) having completely lost the ability to form capsules, do not stain gram-positively.

   Some investigators (Bisset, 1950) consider the cell wall a dead skeleton, a product of secretion of the cell protoplast. The data cited prove the contrary. The cell wall represents an organ of a living organism able, not only to carry out the function of a skeleton, but also to perform a series of purely biochemical processes of great importance in the metabolism of the cell, as well as in the exchange of substances between the inner and outer medium, between the organism and the substrate. The cell wall, together with the protoplast, comprises an entity--the bacterial cell.

   The cell membrane plays a great role in the multiplication of the cell. It forms two transverse, protoplasmic, parallelly located threads, with a true system between them. This system appears as a result of secretion of transverse membranes and in fact consists of two thin septa which part and perform the division.

   On reproduction of cells by formation of a septum, the cell membrane at the site of division is constricted inside the protoplast to complete closure or a small area in the form of a canal is left. In both cases the membrane forms the cell wall simultaneously with constriction inside the protoplast.

   The transverse membrane in some bacteria in strongly thickened at the ends of the cell. Since it stains strongly with basic dyes, due to its basophilic character, in such cases it takes the form of caps or even polar bodies, assumed by some investigators to be nuclear elements (see Imshenetskii, 1950, Peshkov, 1955).

Figure 1. Polar flagella in bacteria: A. Monotrichous

a) Pseudomonas fluorescens (1:3,000); b) Pseudomonas malvacearum (Azerbaijan strain, 1:9,000) ; c) Pseudomonas malvacearum (Fergana strain. 1:9,000), d) Rhizobium trifolii (1:3,000); e) Rhizobium meliloti (1:3,000); f) Vibrio metchnikovii (1:18,000, after Iterson, 1949).

Figure 1. (continuation) B. Lophotrichous:

Spirillum serpens (after Iterson, 1949, 1:18,000).

   Flagella in bacteria were revealed for the first time by Ehrenberg (1838); later, they were studied by many investigators in various bacterial species--sporiferic and nonsporiferic, spiral forms, some cocci and others.

   According to the location of flagella, bacteria are divided into: monotrichous forms--those which have a single polar flagellum; amphitrichous forms--a single flagellum at both ends of the cell; lophotrichous forms--with tufts of flagella at the ends of the cell (Figure 1A, B); peritrichous forms--with flagella distributed over the whole cell surface (Figure 2). In monotypes one may frequently observe the flagellum located not on the end of the cell but at the side and sometimes in the middle of the cell. Such an anomaly appears in root-nodule bacteria, vibrio and others. The cause of this has not been clarified: it is not known if this is an anomaly caused by a pathological development or is an accidental event in the development and formation of the flagellum an a result of internal disturbances in the protoplasm or cell membrane.

   According to our observations, the lateral flagella are formed by a dislocation of the polar flagella during the growth of the cell. The latter grow in length with the end attached at the point of growth. Sometimes the site of attachment of the flagellum is moved by some event from the side of the growth point and, as the cell lengthens, it moves farther away from the end (Krasil'nikov. 1932, 1935).

   Flagella are formed by the protoplast, they are organically connected with the membrane and obtain impulses for movement from it. Contemporary methods of investigation elucidated that at the base of flagella there are granules located directly under the membrane. These granules are similar to the basal bodies of cilia in protozoa.

Figure 2. Bacteria with peritrichous flagella:

a) Bact. proteus (1:17,900, after Iterson, 1949); b) Azotobacter chroococcum (Moscow strain); c) Azotobacter chroococcum (Central Asia strain, Vakhsha Valley, 1:8,000).

 

   Loeffler (1889) established that flagella have a spiral-like form. They often stick together into tufts and locks which may be seen by ordinary microscopy without having been specially stained. As shown by recent studies, the separate flagella are of a more complex structure than was earlier assumed. In spiral bacteria the flagellum consists of many very thin elements, visible only under electron microscopy. There are 17-20 in one flagellum. A similar flagellum structure was described in protozoa.

   The structure of flagella is different in sporeforming bacteria. According to observations of Roberts and Franchini (1950), the cells of Bac. cereus have flagella, consisting of a spirally-twisted axial thread and an outer layer. The length of the spiral twist equals 80 Å. The spiral consists of two very thin inter-woven threads. These spirals appear to be the motion apparatus of the flagellum.

   The length of flagella differ in various bacteria. In some species the flagella are long, sometimes being more than a hundred times the length of the cell. In other species they are short, distributed on the surface of the cell in the form of bristles. The width of flagella is several Å or millimicrons, the length attains several tens of microns.

   The development of flagella proceeds consecutively. At first, in young, developing cells they are very thin and short. Then, as the cell grows, they become longer and larger. The growth of flagella begins from the periplast, directly from the basal or kinetoblast body (Erikson, 1949).

   With the aid of flagella the bacterial cells move actively in the liquid medium. The movement of bacteria is a progressive motion. The cells move rapidly or slowly forward, backward or sideways. More often than not, the movement is uneven, sometimes rapid, sometimes slow with sudden halts. The cells move by jumping.

   There are indications in the literature that in some bacterial species flagella occur, but the cells themselves are nonmotile. Some authors assume that in such bacteria the flagella are paralyzed.

   The movement may be stopped artificially by placing the cells in strong solutions of salts or sugars, by lowering the pH of the medium, by strong illumination by increasing or lowering temperature and by other means. Frequently, the flagella movement is stopped as a result of abundant slime formation. If they are grown on unsuitable media, bacteria may not display any motility. Often motility may only be seen in young cultures. as for instance observed in the hay bacillus Bac. subtilis, When the cells form long filaments, they become nonmotile. This in observed in Bac. mycoides, Bac. megatherium, Bac. mesentericus, Bac. cereus and other bacteria. Migula (1892) observed the formation of generations in Bac. subtilis, which, although they possessed flagella, remained nonmotile. The same was noted in Micrococcus agilis, Sarcina mobilis and other bacteria.

   On the basis of the data cited, some investigators are inclined to assume that there are no bacteria in nature without flagella (Meyer, 1912). Some investigators tried to prove this assumption experimentally. Thus, for instance. Kobblmüller (1934, 1937) observed motility in lactic-acid streptococci considered to be motionless; Clark and Carr (1951) found that mycobacteria and corynebacteria--Mycob. phlei, Mycob. fimi, are motile. They claimed that they observed flagella in these organisms through electron microscopy. These data are as yet not confirmed and it to doubtful whether they are correct. Mycobacteria according to their nature are related not to bacteria but to the group of actinomycetes. One must assume, that the above investigators dealt with root-nodule bacteria or with Mycoplana. or they observed mixed (impure) cultures. Perhaps artifacts which were formed upon the treatment of the external cell-wall layer with reagents were taken to be flagella as Ptjper (1931, 1949) observed.

   In many works Ptjper tried to show that bacteria do not possess flagella at all. and what in considered to be flagella are artifacts in the form of thin threads obtained an treatment, at the expense of the mucus of the cell capsule.

   The theory of Ptjper was not confirmed by other investigators. Bolsches (1948. 1949) disproved Ptjper's statements experimentally. Weiball's a data (1948, 1949) on the protein nature of flagella and the works of Fleming et al., (1950), of Van Iterson (1949) and many others are in variance with Ptjper's a observations. The structure of the flagella, their subsequent development, and the connection with the membrane and basal bodies, an was mentioned above, all speak against the theory of Ptjper.

   There are data in the literature, with the aid of which some investigators try, to prove that, in general, bacterial motility is caused not by flagella but by another mechanism, In particular, Ptjper explains the motility of cells by an outflow of mucus, an it taken place in myxobacteria and blue algae. The motility of microbial cells without flagella has for a long time attracted the attention of investigators. An yet, there is no full and clear idea on the mechanism of motility in these microorganisms. It in assumed to be essentially similar to that of diatoms and bluegreen algae, i.e. , of the reactive type. Myxobacteria evidently move by contraction of the whole cell peroplast. Wavelike contractions, accompanied by a longitudinal shrinkage and stretching of the protoplast. brings the cell to a sliding movement (Peshkov, 1955). On moving, jets of mucus are excreted. The cell moves according to the principle of recoil i. e , in the direction opposite to the direction of ejection of the mucus.

   The chemical composition of flagella differs from that of the cell wall. The basic chemical component of flagella is protein; polysaccharides are not present.

   On the basis of data of physicochemical analysis and study of X-ray spectrum. an individual flagellum is considered to be a gigantic muscle macromolecule capable of rhythmic movements. However, cystine, characteristic of muscle protein--myosin, was not detected in flagella. The protein nature of flagella structure is also indicated by immunological reactions. These reactions also reveal differences between the protein of flagella and that of the cell protoplasm.

   Flagella protein is the basis of the H-antigen which provokes formation of specific antibodies in the animal body. The presence of these antibodies in serum causes the agglutination of the flagella of bacterial cells, and differs from O agglutination, during which the agglutination of the cells themselves takes place.

   The bacterial cell contains plasma, which, with its inclusions is called the protoplast; in the immediate vicinity of the cell wall it is covered with a condensed plasma layer which is called the membrane. The plasma, as in all other cells, consists of a living substance of a very complex structure--proteins. polysaccharides, fats and other compounds.

   The structure of bacterial plasma is also very complex and of rather great diversity, depending upon the bacterial species, age and the conditions of growth.

   The plasma of young cells is optically homogeneous. there are no inclusions, no fat or volutin; they are without vacuoles. As the culture grows older, small granules of a different nature, and vacuoles appear in the plasma. In old cultures the plasma of the cells becomes fine-grained, strongly vacuolized with a larger or smaller content of various granules and bodies--volutin, chromatin; fat droplets and other inclusions stained with various dyes appear in it.

   The detailed structure of bacterial protoplasm has not been investigated. It in known that protoplasm has great viscosity which varies greatly in various species. The lowest viscosity of the protoplasm, according to data of Gostev (1951), exceeds that of water approximately 3-4 times; in the majority of cases it exceeds the viscosity of water 800-8, 000 times. The viscosity of plasma depends directly upon the condition of the culture, its age, nutritional conditions, etc. As in other organisms, the viscosity of bacterial plasma changes strongly in response to external factors (temperature, mechanical damage), under the effect of radiant energy, chemical reagents and other factors.

   The internal osmotic pressure of bacterial cells equals on the average 3-6 atmospheres. In some bacterial species it amounts to considerable values--300 atmospheres and more (Mishustin, 1947). The isoelectrIc point of bacterial protoplasm in the majority of species to of the range of pH = 3.0-4.0. The point of acid agglutination of bacteria also lies within these limits of pH. In smooth variants the point of acid agglutination lies a somewhat lower (pH from 3.0 to 4.0) than in rough forms (pH from 4.0 to 4.5). The specific gravity of bacterial plasma is 1.055.

   The chemical composition of bacterial protoplasm is hardly known. More detailed investigations of the chemical composition have only begun in recent years. General data on the total chemical composition of bacterial plasma are known. For instance. the plasma of higher organisms consists essentially of protein substances, ribonucleotides, lipides, polysaccharides, fats and water. The latter constitutes 90-95% on the average.

   Data of chemical analysis of the bacterial cell. obtained by Peshkov (1955), Belozerskii (1941) and others, show that the quantitative composition of the mentioned substances in the plasma changes, depending on the age of the cell. For instance, in a 5-hour-old culture of B. coli, the content of nucleic acids in the cell plasma is greater than that in cells of a 40-hour-old culture (respectively 22, 30 and 9.66%); on the contrary, the general amount of proteins increases with the age of the culture: in 5 hours--57.0 %, in 40 hours-70.4 % of the dry weight. The same relationships are also found in Shigella. The basophilic character also changes simultaneously with the change in content of these substances, and with it the stainability of the plasma. In young 5-hour-old cultures, the plasma absorbs aniline dyes better than that of 40-hour-old cultures. In young cells, just beginning to develop, the ribonucleic acids are firmly bound to the proteins. With the aging of the culture this tie becomes weaker (Belozerskii, 1941).

   Detailed data on the chemical composition of the bacterial plasma are given in the books of Gubarev (1952), Gostev (1951), Kuzin (1946), Model (1952) and others.

   Different microorganisms have different nuclear structures. In protozoa as well as in fungi and yeasts there is a fully formed distinct nucleus with a characteristic internal structure and development. attributed to that of higher organisms. In blue-green algae it is represented by a primitive structure in the central part of the cell called the "central body".

   The "central body" occupies the greatest part of the cell. It consists of a thin reticulum with distinct granules of chromatin distributed in the loops. This part stains well with basic dyes. Located on the periphery, close to the cell wall, is a thin layer of protoplasm. There are often granules of cyanophycin. Before the cell divides. the "central body" splits into two portions by the aid of a transverse septum (Figure 3).

Figure 3. Central body in blue-green algae:

1--oscillatoria; 2--Nostoc; 3--Oscillatoria;
A--central body; a--chromatin net.

   In fungi and yeast the nuclear aparatus was thoroughly studied. It does not differ essentially from that of higher plants. As in the latter, the nucleus of yeasts and fungi have a vesicular structure and contain nucleoli; when dividing, chromosomes are formed in the nucleus with characteristic structural figures according to the phases of development, the division of the nucleus proceeds mitotically. Detailed investigations of the nucleus in yeast and fungi were performed by Guilliermond (1920). Until his investigations, the yeast cells were regarded as being without a nucleus; the distinct bodies found inside the cells were considered as nucleus-like formations, but not as true nuclei (Kursanov. 1940; Nadson, 1935; Guilliermond, 1941). The conceptions of the nucleus of bacteria and actinomycetes are not as clear.

   For a long time bacteria were regarded as organisms without nuclei. At the end of the nineteenth century, Büchili (1880) suggested that bacteria, like all other organisms, possess a nucleus. Ten years later, after a very careful study of the protoplast, he came to the conclusion that bacteria do in fact possess a nucleus. but not like that of higher organisms. According to his data, the nucleus in bacteria constitutes the central part of the protoplast and is constructed like the nuclei of blue-green algae. As in the latter, the central body or the prototype of the nucleus is surrounded by a thin layer of protoplasm directly adjacent to the cell wall.

   This opinion was shared by many other investigators of that time--Weigert (1887), Tsetnov (1891), Frenzel (1892), Ruzhichka (1909), Mitrofanov (1093). Shevyakov (1893), and others (see Peshkov, 1955).

   Fisher (1902) developed another point of view on the question of the existence of a nucleus in bacteria. According to his data, there is no nucleus in bacteria, or more precisely, the whole protoplast of the bacterial cell constitutes the nucleus. The author subjected the cells to microscopic analysis, but did not find any inclusions which resembled a nucleus in the plasma. According to his data, the separate bodies and granules mentioned did not have anything in common with it. Only some granules like the chromatin, stained with aniline dyes.

   This opinion was also shared by Migula (1892). Like Fisher. he found only small granules which upon staining were similar to the nuclear substance. According to these authors, the nucleus or nuclear substance in bacteria exists in a diffused or fragmented state.

   In developing the theory of the diffused nucleus, Hertwig (1902), proceeded from this analogy, with the formation of the so-called chromidia upon decomposition of the nucleus in some protozoa (Heliozoa). According to his opinion, bacteria do not possess an individual nucleus, but a nuclear substance, which is in the form of tiny granules or threads, and is distributed as a net (chromidial net) in the protoplast throughout the cell. The cbromidial net may occupy the whole cell or a greater part of it. In the latter case the plasma is located on the periphery. Schaudin (1902), conducted extensive investigations on the nuclear apparatus in the gigantic sporeforming bacillus--Bac. bütchlii, which he found; he confirmed what has been stated above. This microbe is 80 µ long and 3.5 u wide and is very motile; it is peritrichous. The protoplast of the cells consists of two parts: a peripheral portion, in the form of a light rim, and a central portion. According to the author's observations, the first represents a thin layer of plasma, the second--the central body, or a primitive nucleus. It has a vesicular structure, staining well with basic dyes, and small granules--the chromidia--are embedded in it. Prior to spore formation the granules of the central body move to the center of the cell and form a thread along the longer axis of the cell. This zigzag-like chromatin thread extends from one end of the cell to the other, stains strongly, and strongly refracts light. After some time the granules of the thread begin to move to the poles where they assemble and form one large body of round or oval form. The spore is later formed from this body. A central body was noted by Schaudin In the sporeforming bacillus--Bac. Sporonema.

   The theory of diffused nucleus was also elaborated by Guilliermond, Swellengrebel (1909) and other investigators. At present this is accepted by many physiologists (see Imshenetskii, 1940). Swellengrebel described the chromatin thread, formed from chromidia in the sporeforming bacillus Bac. maximus buccalis. In other bacteria he found nuclear threads which were formed either from round nuclear bodies or from chromidia.

   Guilliermond, an outstanding specialist in cytology, did much work investigating the protoplast of protist organisms--algae. fungi, yeasts and bacteria. In bacteria he did not find nuclei. but found a chromatin substance which was present in the protoplasm in a soluble or fragmented state. During spore formation Guilliermond observed precipitation of chromatin in the form of separate granules.

   Contrary to the opinions on the diffuse structure of the nucleus just cited , there are in the literature statements as well-founded, on the presence of a well-defined, fully formed nucleus in bacteria.

   This point of view was expressed for the first time by Meyer (1897). According to him, bacteria possess a true nucleus similar to that of higher organisms. He based his conclusions on data from analysis of fungal organisms in which the nuclei are very well defined and manifest themselves clearly. The author assumed that fungi and bacteria are organisms phylogenetically close to each other. If nuclei occur in fungi they should occur also in bacteria. As an object of his investigations, Meyer chose the large sporeforming bacillus Bac. asterosporus. He established the presence of defined bodies, which he regarded an nuclei inside this bacillus. He counted from 3 to 4 such bodies of a diameter of 0.3 µ in the cell.

   The followers of these opinions extensively developed the theory of a defined nucleus in bacteria. At present this theory is the most popular one.

   Data recently obtained by electron microscopy are of interest. By introducing some improvements, it was possible to disclose and differentiate very small separate cell structures.

   It is known that the most characteristic substances in the composition of the nucleus are nucleic acids, namely thymonucleic or desoxyribonucleic acid in the nucleus, and ribonucleic acid or plasma acid in the protoplasm.

   The penetrability, i. e., transparency to a beam of electrons, of the nuclear or chromatin substance which consists essentially of thymonucleic acid, differs from that of plasma nucleic substances (ribonucleic acid). Owing to this it is possible to differentiate and to discern nuclear from nonnuclear elements.

   In the study of the nucleus and nuclear substance of bacterial cells great importance is attached to chemical methods. By chemical reactions one succeeds in revealing the separate components of the nucleus, and in establishing their chemical nature. Among the chemical reactions, the Feulgen reaction is noteworthy. It is based on the hydrolysis of thymonucleic acid by hydrochloric acid. During hydrolysis guanine and adenine, as well as the polysaccharide fraction, are released. The latter, when treated with fuchsin sulfate acquires a bright pink-violet color. This stain is characteristic of aldehydes, and, consequently, shows that the polysaccharide part of thymonucleic acid consists of aldehydes.

   Feulgen (1926) applied this reaction in order to reveal thymonucloic acid in protozoa and obtained a positive result. He did not find this substance in bacteria and yeasts, on which he based the erroneous conclusion that in bacteria the essential nuclear substance was absent.

   Subsequent investigations of many other authors showed that this conclusion was at fault.

   By this method thymonucleic acid was found in bacteria of various groups and species, in actinomycetes, yeasts, fungi and other representatives of microorganisms (Imshenetskii, 1940; Dubos, 1948; Guillermond, 1941 and others).

   The studies showed that the distribution of thymonucleic acid in bacterial cells as established by the Feulgen method fully corresponds to the localization of structural formations revealed by microscopic methods. Depending on the bacterial species, their age and growth conditions, thymonucleic acid in distributed either diffusely or is concentrated in the form of small bodies and masses of various dimensions and configurations.

   A valuable microchemical method for the establishment of the composition of nuclear substance is the enzymatic treatment. Pepsin with hydrochloric acid (stomach juice) dissolves bacterial cells and proteins of various composition, but not the chromatin substance. Neither does it dissolve nucleic acids. Peshkov (1955) applied ferments of nuclease to discern nuclear substances in basophilic bodies of bacteria.

   A macrochemical method is also applied for the investigation of the nuclear substance. Belozerskii (1944, 1945) extracted nucleoproteins from the bacterial mass with alkali and separated them into two fractions. nuclear and protoplasmatic. The first contains a typical thymonucleic or desoxyribonucleic acid. The second - only the plasma or ribonucleic acid.

   Recently another very sensitive method of recognizing nuclear substances was suggested--the method of spectroscopy. It is based on the ability of the chromatin substance or nucleoli and other nuclear structures to absorb certain parts of the spectrum of ultraviolet light. By this method it has been successfully    shown that the nucleoli, and, in general, the nuclear substance of bacteria, has exactly the same absorption spectrum (in the region of 2,600Å) as that characteristic of the chromatin of higher organisms (Peshkov, 1955).

   As a result of the application of all these methods in the cytological analysis of the microbial cell, one may obtain quite convincing date on the presence of a nucleus in bacteria. At present one can definitely say that bacteria possess a nuclear substance, not differing in its composition from the nuclear substance of higher plants. However, in contradistinction to the latter, the nuclear substance of chromatin in bacteria in not always distributed in the form of defined bodies or organelles. It occurs in various forms depending on the species, age of cells, and growth conditions of the culture. The chromatin may occur in bacterial cells in a diffuse state or in the form of tiny bodies and granules distributed throughout the protoplast of the cell. At given stages of development the nuclear substance is present in bacterial cells in the form of defined structural formations, having dimensions, forms and patterns characteristic of the true nuclei of other organisms--fungi, yeasts and others.

   Such distinct structural formations of chromatin, in contradistinction to true nuclei are called bacterial nuclei or nucleoids. The most characteristic and convincing proof that these formations are cell organelles and, not some other inclusion, is their ability to reproduce themselves during the process of the multiplication of the cells. This property is known to be characteristic of every cell organelle and primarily of the nucleus. This distinguishes it from superficially similar nonliving inclusions as for instance reserve food substances, various metabolites, etc.

 

Figure 4. Division of nucleoli in bacteria (after Robinow, 1942):

Bac. cereus; b) Bac. mycoides; c) Bac. mesentericus; d) Bact. proteus.

 

   As shown by many recent investigations, nucleoids multiply by simple division or constriction, in other words, by amitosis. The round body extends and becomes rodlike; a furrow appears in the center, with the aid of which the splitting into two bodies occurs (Figure 4). Sometimes one may observe a splintering of the nucleoids, i. e. , division of the chromatin mass into several pieces simultaneously (Figure 5). The nuclear substance of the nucleoids may reproduce by budding. A protrusion appears on the surface of the body which gradually increases in size and after reaching certain dimensions is pinched off from the parent body. From one body several daughter nucleoids may be pinched off.

 

Figure 5. Splintering of nucleoids in bacteria:

a) Bac. megatherium (De Lamater, 1951); b) Bac. proteus (after Stempen and Hutchinson, 1951); c) Microc. cryophilus (De Lamater, 1951).

   Budding and splintering of the chromatin mass of nucleoids is usually observed in distended, so-called involution, cells. Such forms of reproduction of nucleoids are very often accompanied by formation of special spores, reproductive or regenerative bodies, inside the cell. These formations were observed by us in Azotobacter, root-nodule bacteria and actinomycetes (Krasil'nikov, 1932 d, 1954 e).

   Following the division of nucleoids there is cell division. In bacteria a plural cell reproduction is often observed, multiplication by fragmentation when the cell divides simultaneously into several daughter cells. Such division is observed in micrococci, mycrobacteria, sporeforming bacteria--Bac. megatherium, Bac. mesentericus and others, in Azotobacter and in certain filamentous bacteria. In these cases in every daughter cell one small chromatin body or nucleoid may be observed (Peshkov, 1955; Robinow, 1942; Knaysi, 1950; Bisset, 1950; Pickarski, 1937, and others).

   Nucleoids are always formed during spore formation. The fragmented chromatin in the form of tiny granules concentrates in small bodies in a part of the cell, more often in the part where the spore is formed. These bodies become rounded; a thin rim of plasma appears and then the cell wall is formed. A ripe spore is obtained. In all species of sporeforming bacteria, mycobacteria, actinomycetes, and, one should assume, in all other microorganisms which produce spores internally or have other reproductive bodies, there are always nucleoids in the mature spore.

   The internal structure of the "bacterial nucleus" is not apparent; the whole body represents a homogeneous mass, strongly stained with basic dyes. Inside such bodies neither chromosomes nor nuclear grains are found.

   Some investigators try to show the presence of defined structures inside the "bacterial nucleus", they describe chromosomes and various bodies, and are of the opinion that these bodies are like the chromidial net of true nuclei. By making comparisons with nuclei of higher organisms, mitosis with its various phases was described in bacteria. For instance. De Lamater (1951-1952) notes in Bac. Megatherium, a prophase, metaphase, anaphase, telophase, then formation of a typical spindle with centrioles. He observed this picture of nuclear division in micrococci, Micr. cryophilus and in Bac. coli. The author indicates that various bacterial species contain a different number of chromosomes in the nucleus. Analagous data on structure and development of the nucleus in bacteria are presented by Lindegren (1950) and some other investigators.

   The material cited by these investigators is not convincing. The structural changes of the chromatin accumulation bodies described by them, are of a quite diverse character. Chromatin or chromatin-like substances in bacteria quite often assume various and rather indefinite configurations and dimensions. These changes are without any regularity and connection with the process of cell division. Among the various formations of chromatin accumulation one may always find fortuitous figures somehow reminding one of the forms of one or another phase of nuclear division.

   Bisset (1953) could not confirm the data of De Lamater. He considers his classification of the structures observed inside the cell during the process of division as erroneous. What De Lamater assumed to be centrioles, proved in fact to be rudiments of transverse septa. Neither could Bisset find mitochondria in mycobacteria as did De Lamater.

   The nuclear substance in the form of nucleoids occurs in the cells for a short time, usually only in the period of their early development. As the culture ages the chromatin substance in the calls increases noticeably. It often occupies a considerable part of the cell, almost filling it completely. In such cases the chromatin substance, as a rule, has indefinite patterns and configurations. Large masses of chromatin stain densely with nuclear dyes, giving the Feulgen reaction, and acquiring a loose vesicular structure, often the whole mass is broken into separate parts and small lumps or separate small pieces. Some authors consider these enormous accumulations as one large nucleus, and consider its fragmentation as cell division (Peshkov, 1955, Bisset, 1950, Robinow, 1951, Pickarski, 1937 and others). However, it is quite impossible to agree with this. As a rule, the enormous chromatin aggregations are observed during the abnormal development of a culture, when the cells are in a state of involution. They are always observed during unfavorable growth conditions, under the effect of increased temperature, irradiation with ultraviolet, radium X-rays, etc. An active and rapid formation of chromatin substance occurs under the influence of phages on bacteria and actinomycetes. According to observations of Gerchik (1945). a penetration of the phage tail into the cell body suffices to provoke in it, all the indicated changes. After several minutes of contact between the protoplast of bacteria or actinomycetes with the phage, large lumps of chromatin or chromatin-like substance are formed. The cells swell and assume unusual forms and dimensions.

   As a rule the accumulation of chromatin substance in cells is accompanied by a decrease in their viability.

   Formation of such a large amount of nuclear substance can hardly be regarded as a normal function of development and cell multiplication. It must be assumed that this process reflects an abnormal development and disturbance of certain biochemical reactions of metabolism. The chromatin accumulations in the form of various masses represent a result of an unnatural metabolism and in no case, a phase in the development of the nucleus. In these small masses distinct rudiments of germs, the so-called regenerative bodies, may be formed.

   Nucleoids have been described in bacteria of the coli group - Bac. coli, Bac. typhi, Bac. dysenteriae, Bact. proteus and other species.

   We have observed nucleoids in actinomycetes, as a rule in young individuals. They are particularly noticeable when the organisms are cultivated in a liquid synthetic medium. In the threads of the mycelium separate bodies which are located far away from each other are revealed. One may often see two nucleoids closely located or even fused together. We regard this phenomenon as the process of division of these formations. In old cultures the threads do not possess definite nucleoids, instead granules, or irregular small blocks and large aggregations of chromatin are found. They are dispersed in a disorderly manner over the entire mycelium or in separate threads only. These chromatin aggregations are not organelles of normal cell development, but represent aggregations resulting from a degenerative change of the whole or part of the protoplast, namely that connected with the nucleoproteins.

   It was shown experimentally that with the disorderly accumulation of chromatin masses in the cells, biochemical processes noticeably change as well as the nature of exchange and formation of metabolic products. This may easily be seen particularly in actinomycetes during their process of formation of antibiotic substances. The indicated changes in chromatin structures of the actinomycete mycelium are quite constant and regular during culture development and are often used in antibiotic practice as an indication of the current state of the process. It must be assumed that chromatin may possibly be one of the potent factors of metabolism regulation. Unfortunately we know very little of the relationship between the biochemical processes and the formation and aggregation of chromatin substance in the cell.

   There are bacteria in which the nuclear substance is distributed in the form of a central body in the cells as in blue-green algae. Such nuclei have been described by us in Pontothrix longissima, in Oscillospira guilliermondii, and in Anabaeniolum sp. These bacteria consist of threadlike individuals of various lengths. Every individual or thread is divided by transverse septa in a series of short cells, whose length usually does not exceed the width and is more often less than the latter. The internal structure of such cells reminds one of the structure of the protoplasts of blue-green algae. A great part of the cell is occupied by the central body. On the periphery the plasma is distributed in the form of a thin layer. The central body stains well with aniline dyes and is easily found by ordinary magnification of the microscope owing to its dimensions (Figure 6).

 

Figure 6. Central body in A--Oscillospira guilliermondii, B--Anabaeniolum langeroni and in C--Pontothrix longissima

a--central body; b-nucleoids; c-prospore; d--division of central bodies.

   Such a distribution of chromatin has also been described in Caryophanon by Sall and Mudd (1955).

   In many bacteria so-called polar bodies or small grains at the ends of the cells are noted. Like chromatin, they stain densely with stains. The role of these bodies is not exactly known. Some authors regard them as nuclear structures, some others as volutin. There are indications that polar bodies represent a specific enlargement of the cell wall. Recently many investigators are inclined to regard these enlargements as points of growth of cells, directing the process of elongation of the individuals.

   Thus, one may conclude that the nuclear apparatus in bacteria and actinomycetes is of a peculiar nature. It differs strongly from the nuclear apparatus of fungi, yeasts, protozoa and higher plants or animals.

   The nucleus in bacteria and actinomycetes is primitive. It does not have a constant, established, structural formation. It occurs either in a diffuse dissolved state, or in the form of small grains and bodies which are dispersed all over the protoplast of the cell, or in the form of an organized intracellular organoid-nucleoid. In the latter case, such a body, according to its external appearance, form or, dimension, and patterns, calls to mind the true nucleus of fungi or yeasts. However, in contraditinction to the latter, the nucleoids of bacteria are structurally undifferentiated, the characteristic formations of true nuclei as for instance nucleoli, chromosomes or other structures, are not found in them. The most convincing proof of the nuclear nature of nucleoids is their ability to reproduce during the process of cell division.

   It is a characteristic property of nucleoids that they are often reformed from diffusely dissolved or dispersed chromatin during spore formation in sporiferous bacteria, mycobacteria and actinomycetes; they are also organized inside the vegetative cells at certain stages of their growth.

   It should be noted that some forms of manifestation of the bacterial nucleus are attributed also to the nuclear apparatus of higher organisms.

   Unusual methods of cell multiplication and division of the nucleus have been noted long ago in the cytology of the plant cell. Under certain conditions the nuclei of cells of some tissues begin to multiply in a way not peculiar to ordinary cells of higher organisms; this multiplication is neither by complex development and formation of various phases, nor by mitosis, but by a simple division or splitting of the nuclear mass, or amitosis. In amitosis the nuclei multiply by division, constriction, fragmentation and budding. Amitosis, as shown by recent investigations, is widespread among the representatives of the vegetable kingdom. It in found in cells of animal organisms (Polyakov, 1949; Usov, 1924; Ellenhorn, 1951; Glushchenko et al. 1953 and others).

   Amitotic division of the nucleus is found in the cells of callus, cicatrization, regeneration and in tissues formed anew. In these cases the cells undergo a series of cytochemical and morphological changes. In the nucleus processes of direct division or fragmentation go on and several daughter nuclei are formed consecutively; sometimes the nucleus splits simultaneously into several daughter nuclei. The latter separate and are distributed in various parts of the plasma, where they become the centers of formation of daughter cells. According to Glushchenko et al, (1953) the budding of nuclei was established in the cells of cicatricating tissue of the potato fiber. Such nuclei undergo deformations, swell. acquire various patterns, and oarlike or budlike protrusions appear on their surface. The latter gradually become rounded, pinch off from the maternal nucleus and transform into separate daughter nuclei.

   Ellenhorn and Zhironkin (1953) showed that in certain cases, upon the development of the primordial root, its cells possess no nuclei at all. These cells, lacking nuclei, multiply by fragmentation. In the subsequent development of the rootlet the cells lacking nuclei form primitively organized nuclei or "protokaryons". The "protokaryon" multiplies by simple division or binary fission. Mitosis is absent. Only afterward when the rootlet develops sufficiently do the primitive nuclei begin to multiply mitotically in their cells.

   Cells without nuclei were found by Glushchenko in tissues of lentils of black currants, where they form rootlets, and later in cells of cicatricating Neder's tissue. These calls multiply by fragmentation in the early state of rootlet formation without any indication of the presence of a nucleus. Also, according to the experiments of Ellenhorn, after some time primitive nuclei are formed anew in the cells. They multiply by simple division, binary fission. In these nuclei--"protokaryons", neither nucleoli nor chromosomes are present.

   In these instances (there are many in the literature) some similarity of nuclear formation and structure in plants and bacteria or actinomycetes has been shown. As in the latter. the nuclei of plant cells may be formed anew from chromatin substance diffusely distributed in the protoplast. Such a way of formation and development of primitive nuclei in plants evidently reflects the picture of the early stage of evolution and formation of cells.

   During the elaboration of methods for differentiating bacterial cells in tissues of animal organisms, Christian Gram (1884) and his collaborators suggested a special method of staining. By this method the division of all bacteria into two groups, gram-positive and gram-negative bacteria. has been established.

   This staining method afterward underwent some changes and improvements, but in principle remained the same. The technique of staining is an follows: the bacterial cells are stained with a slightly alkaline basic stain such as crystal-violet; then they are treated with mordant iodine in the form of potassium iodide, or with picric acid. The stained preparations are washed with water and neutral alcohol or acetone. By this treatment the stain in removed in some bacterial species and retained in others. The former are called gram-negative, the latter, gram-positive bacteria.

   The fact that different bacteria retain the stain in a different manner is important, not only from the point of view of the problem of staining, but also in its broader significance, since it indicates a chemical difference in the cells of these microorganisms. The gram stain is an indication of the biological or hereditary properties and state, in other words, of the nature of the organism. These properties should be used, not only as a diagnostic, but also as a systematic indication in bacterial taxonomy.

   In clinical laboratory practice, this indication is widely used and yields good results. However, it is not taken into account in classification or only considered to a small degree since it in undoubtedly underestimated here.

   The ability to absorb and to retain the stain according to Gram is characteristic of many organisms of the bacterial class of almost all actinomycetes and mycobacteria, fungi and yeasts. The majority of the cells of higher plants and animals are gram-negative. However, separate inclusions in the cells of these organisms, in particular the nucleus, the nuclear hyalin and the nuclear substance, etc, are gram-positive. Viral proteins, bacteriophages and actinophages are gram-positive.

   Many other bacterial properties are also related to gram staining. Thus, for instance, gram-positive bacteria are more resistant to the lysing effect of alkalies and the proteolytic effect of enzymes--trypsin, pepsin, pancreatic juice; they are more sensitive to many inhibitors--to antibiotics, aniline, phenol, ethanol, toluene, benzene, xylene, chloroform, ether, iodine, to basic dyes and other substances. Concentrated in the cells of these bacteria are such amino acids as arginine, glutamic acid, histidine, lysine and tyrosine. Upon staining with methylene-blue and eosin, they become more sensitive to light, etc, (see Bartholomew and Mittwer, 1952).

   The property of gram staining may change to a considerable degree even in the same species, depending on the age of the culture, the nutrient medium and other external conditions. For instance, on unfavorable media, bacteria often lose the ability to stain gram-positively or they stain weakly. The hay bacillus--Bac. subtilis becomes gram-negative when grown in a medium with immune serum (Simonini, 1914). Some authors observed gram- positiveness in Bac. coli, grown in a liquid protein medium with a high content of glucose and the salts MgSO₄, NaCl. The sporeforming bacillus--Bac. cerus, after having been in distilled water or tap water, stains gram-positively rather weakly, and many cells become completely gram-negative. How water acts is unknown. Whether the gram-positive substance in used as a nutrient during starvation, or if this substance is dissolved as a result of autolytic processes which proceed quite intensely under these conditions was not elucidated.

   Knaysi et al. (1950) established that small doses of benzimidazole added to the Dubos medium transforms the tubercle bacillus--Mycob. tuberculosis, avium type - from a gram-positive, acid fast form into a gram-negative, nonacid fast form. The authors assume that benzimidazole inhibits the synthesis of ribonucleic acid--the essential ingredient in the composition of the gram-positive substance.

   As seen from the previously-mentioned data, the medium is of great importance in connection with the gram stainability of cells. A medium not sufficiently well chosen may mislead the investigator in his differentiation of bacterial species. Therefore, in all doubtful cases of uncertain staining, the use of several nutrient media in recommended. The gram stainability is also greatly affected by the age of the culture. As a rule, young cells stain more strongly than old ones. A 24-hour culture is more gram-positive than a two-to three-day culture and a five-to six-day-old culture even more so. In some cases old cultures are more gram-positive than young cultures. In the sporeforming bacteria--Bac. mesentericus, Bac. subtilis and other species; the gram-positive substance occurs in cells in the sporulation phase and even after the spore had already been formed, i.e. , in the residual plasma (epiplasma). This substance is not decolorized on treatment for 10 minutes with 95% ethanol.

   The better gram stainability of young cells is evidently caused by the basophily of the protoplast and its ability to strongly absorb stains in general.

   The stainability of cells depends to a considerable degree upon their individual features. Two cells of the same age placed side by side often stain with a different intensity. The degree of the stainability changes depending upon the duration of the decolorization, the fixation method and, in general, upon the preparation of the cells and reagents. Thick smears take longer to decolorize than thin ones.

   Gram stainability is connected with the species characteristics of organisms. Certain species, for example gonococci and certain mycobacteria easily change their staining characteristics when grown under different conditions, while others preserve those characteristics in a more or less stable fashion. Consequently, the gram staining depends not only on the staining technique but also on the species characteristics of the culture and the properties of the cell substance. It is obvious, that when gram stainability is being determined one should adhere to certain standards in the methods of making the preparations, as well an in the following procedures--fixation, staining. decolorization and others.

   The essence of gram stainability in spite of numerous investigations performed, has not yet been elucidated, Various opinions and theories have been expressed. They can all be reduced to three basic ideas: a chemical theory, an isoelectric theory, and that of cell permeability.

   The chemical theory of the gram-positive staining of bacteria in based on the particular composition of the cell plasma, According to this theory, there are in the cells of gram-positive bacteria particular substances which retain the stain and do not release it, or release it with difficulty upon washing. Some authors relate the gram stain to the presence of fatty acids of lecithin or lipoproteins in the plasma. These substances strongly combine with the stain and iodine and do not decolorize on treatment with ethanol, Schumacher (1920 isolated fatty acids from the cells of yeasts; afer this treatment the cells lost the ability to stain gram-positively. However, the same cells regained their gram stainability after treatment with fatty acids. According to the works of Peterson (1955) and of other authors, the role played by unsaturated fatty acids in gram stainability was refuted In connection with the fact that gram-negative bacteria do not contain fewer of these acids than gram-positive bacteria,

   Recently gram stainability has been attributed to a particular nucleoprotein gram-positive substance, ribonucloic acid, more precisely, to magnesium ribonucleate. It was established that by hydrolysis or treatment with bile salts one can deprive the cells of the gram-positive substance and transform them into gram-negative (Denesen, 1948; Stacey et al., 1940; Stacey, 1949). Bartholomew and Umbrait (1944) removed the magnesium ribonucleate by crystalline ribonuclease and transformed the cells into gram-negative ones. The artificially removed magnesium ribonucleate may be returned to the cells.

   Although this theory sounded convincing. it proved to be groundless. It was revealed that when the gram-positive substance is separated from the cells, it does not transform naturally gram-negative bacteria. In the latter there is no less ribornucleic acid and magnesium ribonucleate. These substances isolated from bacteria of the coli group do not transform gram-positive cells (after previous ,separation of the gram-positive substance) for instance Clostridium cells--Clostridium welchii (Jones, Mugglestone and Stacey, 1950).

   Henry, Stacey and others came to the conclusion that the gram stain depends upon the ratio of nucleoproteins to ribonucloic and desoxyribonucleic acids. In streptococci and in Clostridium--Clostridium welchii this ratio is 8: 1. and, in gram-negative bacteria--1: 3 (Stacey, 1949; Henry, Stacey, 1943). Other authors did not confirm the regularity of these relationships.

   Mitchell and Moyle (1950) attribute greatest importance to a particular substance, "XP", of unknown nature and containing phosphorus which is combined with the ribonucleate of the gram-positive substance. According to their data. the substance "XP" is always found in the cells of gram-positive bacteria. However, subsequent studies showed that this substance also occurs in large quantities in gram-negative bacteria and in some of them--Bac. coli, Bac. aerogenes, Nelsseria catarrhalis--even more is found than in the gram-positive bacillus--Bac. subtilis and in brewer's yeasts. Shugar and Baranowska (1954) came to the conclusion that a decisive role in the gram stain belongs not to, ribonucloic acid but to the protein of the cells.

   'The theory of the isoelectric point, suggested by Stearn and Stearn (1926-1931) is based on the fact that the isoelectric state of the plasma in gram-positive bacteria differ from that in gram-negative bacteria. The authors determine this state of the plasma by the call's ability to stain or to adsorb acid and basic dyes, at various values of pH. If one determines the pH of the cell protoplast at a stage when it absorbs basic and acidic dyes to the same degree, then it may be noted that In gram-positive bacteria this state occurs at pH 2 and in gram-negative at pH 5. Bacteria whose gram stainability is not completely clear occupy an intermediate position (according to Dubos, 1948).

   It should be noted that the establishment of the isoelectric point is carried out in the cell treated with iodine and, if it conditions the result of the gram stain, it does so in nonliving cells. The applied fixation method by iodine causes oxidation of plasma. Oxidizing agents are also such substances as bromine, picric acid, potassium bichromate, tri-nitrobenzene, etc. These substances oxidize some components of the protoplasts, render it more acid and shift the isoelectric zone. As shown by Stearn and Stearn (1931), the displacement occurs in all bacteria. but is expressed more strongly in gram-positive bacteria than in gram-negative ones. Due to this fact, the difference in the degree of acidity in given groups of bacteria increases considerably under the action of iodine and other mordants. This fact, if true, only shows that the biochemical condition of the plasma of gram-positive bacteria differs from that of gram-negative bacteria. In other words, the living substance in the former and the latter organisms is different.

   The given theory of gram stain of bacteria has not been sufficiently confirmed by a series of experiments. First, some investigators did not confirm that iodine as an oxidizing agent may be replaced by other reagents in the same treatment of cells. Second, if the theory is true, then iodine should also produce this effect before staining, but this does not occur (Bartholomew and Mittwer, 1952). Further, if the theory in correct, then decomposed cells or the content of decomposed cells should be stained by this staining procedure as well an the plasma of whole cells, however, in fact, this is not observed; the protoplast of the disrupted cells is not gram-stainable.

   At the basis of the permeability theory is the different ability of the cell wall and cell membrane to be permeated by stains and mordants. A suggestion was put forward that, in the cell, insoluble precipitates of the stain and iodine are formed, which are not washed out by ethanol. It in known that iodine does indeed form a colloidal complex with methylviolet which in not soluble in water. In favor of this theory are some other facts; for instance, that decomposed cells are gram-negative. It is sufficient to destroy the integrity of the cell wall by some method (grinding with sand, autolysis, lysis, etc) to make the cell lose its ability to stain gram-positively. Benians (1920) divides bacteria according to their stainability into three groups.

   The first group comprises bacteria whose cell wall allows stains and iodine to permeate but the molecules of iodine and stain combined inside the cell are retained. As a result. the whole protoplast remains stained after washing with ethanol. The second group of bacteria possess cell walls which do not allow stains to permeate and. due to this, are easily decolorized upon gram staining. In the third group of bacteria, the cell wall allows the stain and iodine to permeate, but the complex formed inside the cell is freely released upon decolorization.

   Stearn and Stearn refute Benians' point of view on the basis that iodine and the stain do not form such large particles that they cannot penetrate into the cell. Besides this, the addition product of iodine with the stain dissociates in ethanol. Consequently, on washing the preparation, the obtained complex should also be dissolved and go out through the cell wall.

   It was suggested that the permeability, for iodine itself, of the cell walls of gram-positive and gram-negative bacteria is different. For instance, in order to prevent the exit of the stain from the cell of gram-negative bacteria a strong concentration of 0. 01% or less of iodine in methanol is sufficient (Mitchell, Bartholomew. Kallman, 1950).

   Summing up, it may be stated that the essence of the gram stain has not been solved until now. No single hypothesis or theory explains the diversity of cell stainability in various species and bacterial groups.

   It must be assumed that the ability of gram staining is determined by the property of the whole protoplast and cell wall together and not by any one part of the cell. The degree of stainability or stability of the combination of the plasma with the stain depends upon external growth conditions of the culture, its age and other causes.