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Part I

FUNDAMENTALS OF THE STRUCTURE AND DEVELOPMENT OF MICROORGANISMS,AND THEIR CLASSIFICATION

   Various representatives of microorganisms inhabit the soil--bacteria,actinomycetes, fungi, algae, protozoa and more highly organized animals. There arealso various ultramicrobes--phages and others. Our knowledge of the structure anddevelopment of soil microorganisms is obtained by observation of their growth onartificial nutrient media, but little is known about the form in which they inhabitthe soil and what their dimensions, structure, growth, and multiplication are. Doesour knowledge, obtained by investigation of growth of microorganisms on artificialmedia, reflect the state in which they occur in soil? Many microbes are known onlybecause they occur in soil. From general observations of bacterial growth on nutrientmedia we know that the structure and form of their cells is relatively simple andmonotypic. Three types of cell structure are recognized--spherical. rod-shaped andspiral 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 higherorganisms the bacterial cells possess a cell wall and a protoplast. The internalstructure of the protoplast and cell wall differ from the structure of higher forms.

 

STRUCTURE OF BACTERIAL CELLS

Cell Wall

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

   There are data showing that bacterial cell walls have a complexstructure differing in various representatives. In some gram-negative bacteria (Shigellaand others), the cell wall is composed of polysaccharides, proteins and residualphospholipids. Immunological properties of intact cells, separation of the "O"antigen and its components from them, show that a polysaccharide is the basic substanceof the bacterial cell surface. The protein component of the "O" antigenas 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 composedof protein molecules which contain a considerable number of basic amino acids andsulfhydryl groups. In this layer there are compounds which condition the gram-positivestaining. The layer in question is covered on the outside with a second layer composedof magnesium ribonucleate, which is in the gram-positive complex.

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

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

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

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

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

   As was mentioned above, this bacterial surface excretes a mucoussubstance, composing the mucous capsule. When this substance is formed in large amounts,the capsule becomes thick, well outlined under the microscope, and the whole cultureassumes 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 ordinarymicroscopy.

   Mucous capsules of various bacteria differ in their chemicalcomposition. Thus, for instance, the capsule of the acetic bacteria--Acetobacterxylinum consists of pure cellulose, the micelles of which were successfully revealedand photographed by electron microscopy, (Van Iterson, 1949). In the butyric acidbacteria Clostridium pasterianum, the mucous capsule consists of hemicelluloseand may be stained blue with iodine (according to Peshkov, 1955).

   The mucous capsular substance may be mechanically or chemicallyseparated 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 andsubsequent chemical treatment, one succeeds in differentiating two groups of compoundsin the capsular mucous substance. One group does not contain nitrogenous substancesor contains them in negligible quantities. The substances of the other are composedessentially of nitrogenous compounds Knaysi et al., 1950).

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

   The study of the capsules of many bacteria and primarily ofpneumococci, is of special interest due to the presence in them of substances whichcondition virulence and the gram-positive staining. The chemical composition of thecapsule determines the specific serological and antigenic reactions. The capsularantigen is connected with the phenomenon of gram-staining. An autolyzed culture ofpneumococci, having lost the ability to stain gram-positively, becomes unable toprovoke the formation of specific precipitins for the capsular polysaccharide wheninjected, subcutaneously, into a rabbit. Upon autolysis, 4-10% of the dry weightof the cells is obtained and this substance consists mainly of ribonucleoproteinsand ribonucleic acid Dubos, 1948).

   The ability of bacterial cells in the animal body to provokethe formation of precipitins for the capsular polyeaccharides is inseparably connectedwith the integrity of the gram-positive complex. This property in attributed to smoothforms (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 deadskeleton, a product of secretion of the cell protoplast. The data cited prove thecontrary. The cell wall represents an organ of a living organism able, not only tocarry out the function of a skeleton, but also to perform a series of purely biochemicalprocesses of great importance in the metabolism of the cell, as well as in the exchangeof 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 ofthe cell. It forms two transverse, protoplasmic, parallelly located threads, witha true system between them. This system appears as a result of secretion of transversemembranes and in fact consists of two thin septa which part and perform the division.

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

   The transverse membrane in some bacteria in strongly thickenedat the ends of the cell. Since it stains strongly with basic dyes, due to its basophiliccharacter, in such cases it takes the form of caps or even polar bodies, assumedby 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

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

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

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

   Flagella are formed by the protoplast, they are organicallyconnected with the membrane and obtain impulses for movement from it. Contemporarymethods of investigation elucidated that at the base of flagella there are granuleslocated directly under the membrane. These granules are similar to the basal bodiesof 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-likeform. They often stick together into tufts and locks which may be seen by ordinarymicroscopy without having been specially stained. As shown by recent studies, theseparate flagella are of a more complex structure than was earlier assumed. In spiralbacteria the flagellum consists of many very thin elements, visible only under electronmicroscopy. There are 17-20 in one flagellum. A similar flagellum structure was describedin protozoa.

   The structure of flagella is different in sporeforming bacteria.According to observations of Roberts and Franchini (1950), the cells of Bac. cereushave 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 verythin inter-woven threads. These spirals appear to be the motion apparatus of theflagellum.

   The length of flagella differ in various bacteria. In some speciesthe flagella are long, sometimes being more than a hundred times the length of thecell. In other species they are short, distributed on the surface of the cell inthe form of bristles. The width of flagella is several Å or millimicrons, thelength 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 inthe liquid medium. The movement of bacteria is a progressive motion. The cells moverapidly or slowly forward, backward or sideways. More often than not, the movementis uneven, sometimes rapid, sometimes slow with sudden halts. The cells move by jumping.

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

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

   On the basis of the data cited, some investigators are inclinedto assume that there are no bacteria in nature without flagella (Meyer, 1912). Someinvestigators tried to prove this assumption experimentally. Thus, for instance.Kobblmüller (1934, 1937) observed motility in lactic-acid streptococci consideredto be motionless; Clark and Carr (1951) found that mycobacteria and corynebacteria--Mycob.phlei, Mycob. fimi, are motile. They claimed that they observed flagella in theseorganisms through electron microscopy. These data are as yet not confirmed and itto doubtful whether they are correct. Mycobacteria according to their nature arerelated not to bacteria but to the group of actinomycetes. One must assume, thatthe above investigators dealt with root-nodule bacteria or with Mycoplana. or theyobserved mixed (impure) cultures. Perhaps artifacts which were formed upon the treatmentof 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 possessflagella at all. and what in considered to be flagella are artifacts in the formof 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 withthe membrane and basal bodies, an was mentioned above, all speak against the theoryof Ptjper.

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

   The chemical composition of flagella differs from that of thecell wall. The basic chemical component of flagella is protein; polysaccharides arenot present.

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

   Flagella protein is the basis of the H-antigen which provokesformation of specific antibodies in the animal body. The presence of these antibodiesin serum causes the agglutination of the flagella of bacterial cells, and differsfrom O agglutination, during which the agglutination of the cells themselves takesplace.

 

Protoplast

   The bacterial cell contains plasma, which, with its inclusionsis called the protoplast; in the immediate vicinity of the cell wall it is coveredwith a condensed plasma layer which is called the membrane. The plasma, as in allother 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 ofrather great diversity, depending upon the bacterial species, age and the conditionsof growth.

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

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

   The internal osmotic pressure of bacterial cells equals on theaverage 3-6 atmospheres. In some bacterial species it amounts to considerable values--300atmospheres and more (Mishustin, 1947). The isoelectrIc point of bacterial protoplasmin the majority of species to of the range of pH = 3.0-4.0. The point of acid agglutinationof bacteria also lies within these limits of pH. In smooth variants the point ofacid 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 recentyears. 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 constitutes90-95% on the average.

   Data of chemical analysis of the bacterial cell. obtained byPeshkov (1955), Belozerskii (1941) and others, show that the quantitative compositionof 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 acidsin the cell plasma is greater than that in cells of a 40-hour-old culture (respectively22, 30 and 9.66%); on the contrary, the general amount of proteins increases withthe 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 characteralso changes simultaneously with the change in content of these substances, and withit the stainability of the plasma. In young 5-hour-old cultures, the plasma absorbsaniline dyes better than that of 40-hour-old cultures. In young cells, just beginningto develop, the ribonucleic acids are firmly bound to the proteins. With the agingof the culture this tie becomes weaker (Belozerskii, 1941).

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

 

Nucleus and Nucleoli

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

   The "central body" occupies the greatest part of thecell. It consists of a thin reticulum with distinct granules of chromatin distributedin the loops. This part stains well with basic dyes. Located on the periphery, closeto 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 bythe 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, thenucleus of yeasts and fungi have a vesicular structure and contain nucleoli; whendividing, chromosomes are formed in the nucleus with characteristic structural figuresaccording 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 withouta nucleus; the distinct bodies found inside the cells were considered as nucleus-likeformations, 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 withoutnuclei. At the end of the nineteenth century, Büchili (1880) suggested thatbacteria, like all other organisms, possess a nucleus. Ten years later, after a verycareful study of the protoplast, he came to the conclusion that bacteria do in factpossess 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 constructedlike the nuclei of blue-green algae. As in the latter, the central body or the prototypeof the nucleus is surrounded by a thin layer of protoplasm directly adjacent to thecell wall.

   This opinion was shared by many other investigators of thattime--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 questionof the existence of a nucleus in bacteria. According to his data, there is no nucleusin bacteria, or more precisely, the whole protoplast of the bacterial cell constitutesthe nucleus. The author subjected the cells to microscopic analysis, but did notfind 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 ina 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 decompositionof the nucleus in some protozoa (Heliozoa). According to his opinion, bacteriado not possess an individual nucleus, but a nuclear substance, which is in the formof tiny granules or threads, and is distributed as a net (chromidial net) in theprotoplast throughout the cell. The cbromidial net may occupy the whole cell or agreater 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 giganticsporeforming bacillus--Bac. bütchlii, which he found; he confirmed whathas been stated above. This microbe is 80 µ long and 3.5 u wideand is very motile; it is peritrichous. The protoplast of the cells consists of twoparts: a peripheral portion, in the form of a light rim, and a central portion. Accordingto the author's observations, the first represents a thin layer of plasma, the second--thecentral body, or a primitive nucleus. It has a vesicular structure, staining wellwith basic dyes, and small granules--the chromidia--are embedded in it. Prior tospore formation the granules of the central body move to the center of the cell andform a thread along the longer axis of the cell. This zigzag-like chromatin threadextends from one end of the cell to the other, stains strongly, and strongly refractslight. After some time the granules of the thread begin to move to the poles wherethey assemble and form one large body of round or oval form. The spore is later formedfrom 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 manyphysiologists (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 nuclearbodies or from chromidia.

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

   Contrary to the opinions on the diffuse structure of the nucleusjust cited , there are in the literature statements as well-founded, on the presenceof 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 higherorganisms. He based his conclusions on data from analysis of fungal organisms inwhich the nuclei are very well defined and manifest themselves clearly. The authorassumed 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 hisinvestigations, Meyer chose the large sporeforming bacillus Bac. asterosporus.He established the presence of defined bodies, which he regarded an nuclei insidethis bacillus. He counted from 3 to 4 such bodies of a diameter of 0.3 µin the cell.

   The followers of these opinions extensively developed the theoryof 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 verysmall separate cell structures.

   It is known that the most characteristic substances in the compositionof the nucleus are nucleic acids, namely thymonucleic or desoxyribonucleic acid inthe 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 thymonucleicacid, differs from that of plasma nucleic substances (ribonucleic acid). Owing tothis it is possible to differentiate and to discern nuclear from nonnuclear elements.

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

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

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

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

   The studies showed that the distribution of thymonucleic acidin bacterial cells as established by the Feulgen method fully corresponds to thelocalization of structural formations revealed by microscopic methods. Dependingon the bacterial species, their age and growth conditions, thymonucleic acid in distributedeither diffusely or is concentrated in the form of small bodies and masses of variousdimensions and configurations.

   A valuable microchemical method for the establishment of thecomposition of nuclear substance is the enzymatic treatment. Pepsin with hydrochloricacid (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 bodiesof bacteria.

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

   Recently another very sensitive method of recognizing nuclearsubstances was suggested--the method of spectroscopy. It is based on the abilityof the chromatin substance or nucleoli and other nuclear structures to absorb certainparts of the spectrum of ultraviolet light. By this method it has been successfully   shown that the nucleoli, and, in general, the nuclear substanceof 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 cytologicalanalysis of the microbial cell, one may obtain quite convincing date on the presenceof a nucleus in bacteria. At present one can definitely say that bacteria possessa nuclear substance, not differing in its composition from the nuclear substanceof higher plants. However, in contradistinction to the latter, the nuclear substanceof chromatin in bacteria in not always distributed in the form of defined bodiesor 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 cellsin a diffuse state or in the form of tiny bodies and granules distributed throughoutthe protoplast of the cell. At given stages of development the nuclear substanceis present in bacterial cells in the form of defined structural formations, havingdimensions, forms and patterns characteristic of the true nuclei of other organisms--fungi,yeasts and others.

   Such distinct structural formations of chromatin, in contradistinctionto true nuclei are called bacterial nuclei or nucleoids. The most characteristicand convincing proof that these formations are cell organelles and, not some otherinclusion, is their ability to reproduce themselves during the process of the multiplicationof the cells. This property is known to be characteristic of every cell organelleand primarily of the nucleus. This distinguishes it from superficially similar nonlivinginclusions 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 bysimple division or constriction, in other words, by amitosis. The round body extendsand becomes rodlike; a furrow appears in the center, with the aid of which the splittinginto two bodies occurs (Figure 4). Sometimes one may observe a splintering of thenucleoids, i. e. , division of the chromatin mass into several pieces simultaneously(Figure 5). The nuclear substance of the nucleoids may reproduce by budding. A protrusionappears on the surface of the body which gradually increases in size and after reachingcertain dimensions is pinched off from the parent body. From one body several daughternucleoids 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 isusually observed in distended, so-called involution, cells. Such forms of reproductionof nucleoids are very often accompanied by formation of special spores, reproductiveor regenerative bodies, inside the cell. These formations were observed by us inAzotobacter, 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 fragmentationwhen the cell divides simultaneously into several daughter cells. Such division isobserved in micrococci, mycrobacteria, sporeforming bacteria--Bac. megatherium,Bac. mesentericus and others, in Azotobacter and in certain filamentousbacteria. In these cases in every daughter cell one small chromatin body or nucleoidmay be observed (Peshkov, 1955; Robinow, 1942; Knaysi, 1950; Bisset, 1950; Pickarski,1937, and others).

   Nucleoids are always formed during spore formation. The fragmentedchromatin in the form of tiny granules concentrates in small bodies in a part ofthe 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 shouldassume, in all other microorganisms which produce spores internally or have otherreproductive 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 withbasic dyes. Inside such bodies neither chromosomes nor nuclear grains are found.

   Some investigators try to show the presence of defined structuresinside 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 phaseswas described in bacteria. For instance. De Lamater (1951-1952) notes in Bac.Megatherium, a prophase, metaphase, anaphase, telophase, then formation of atypical spindle with centrioles. He observed this picture of nuclear division inmicrococci, Micr. cryophilus and in Bac. coli. The author indicatesthat various bacterial species contain a different number of chromosomes in the nucleus.Analagous data on structure and development of the nucleus in bacteria are presentedby 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, areof a quite diverse character. Chromatin or chromatin-like substances in bacteriaquite often assume various and rather indefinite configurations and dimensions. Thesechanges are without any regularity and connection with the process of cell division.Among the various formations of chromatin accumulation one may always find fortuitousfigures 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 considershis classification of the structures observed inside the cell during the processof division as erroneous. What De Lamater assumed to be centrioles, proved in factto be rudiments of transverse septa. Neither could Bisset find mitochondria in mycobacteriaas did De Lamater.

   The nuclear substance in the form of nucleoids occurs in thecells for a short time, usually only in the period of their early development. Asthe culture ages the chromatin substance in the calls increases noticeably. It oftenoccupies a considerable part of the cell, almost filling it completely. In such casesthe chromatin substance, as a rule, has indefinite patterns and configurations. Largemasses of chromatin stain densely with nuclear dyes, giving the Feulgen reaction,and acquiring a loose vesicular structure, often the whole mass is broken into separateparts and small lumps or separate small pieces. Some authors consider these enormousaccumulations 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 aggregationsare observed during the abnormal development of a culture, when the cells are ina 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 influenceof 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, allthe indicated changes. After several minutes of contact between the protoplast ofbacteria or actinomycetes with the phage, large lumps of chromatin or chromatin-likesubstance are formed. The cells swell and assume unusual forms and dimensions.

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

   Formation of such a large amount of nuclear substance can hardlybe regarded as a normal function of development and cell multiplication. It mustbe assumed that this process reflects an abnormal development and disturbance ofcertain biochemical reactions of metabolism. The chromatin accumulations in the formof various masses represent a result of an unnatural metabolism and in no case, aphase in the development of the nucleus. In these small masses distinct rudimentsof 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 youngindividuals. They are particularly noticeable when the organisms are cultivated ina liquid synthetic medium. In the threads of the mycelium separate bodies which arelocated far away from each other are revealed. One may often see two nucleoids closelylocated or even fused together. We regard this phenomenon as the process of divisionof these formations. In old cultures the threads do not possess definite nucleoids,instead granules, or irregular small blocks and large aggregations of chromatin arefound. They are dispersed in a disorderly manner over the entire mycelium or in separatethreads only. These chromatin aggregations are not organelles of normal cell development,but represent aggregations resulting from a degenerative change of the whole or partof the protoplast, namely that connected with the nucleoproteins.

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

   There are bacteria in which the nuclear substance is distributedin the form of a central body in the cells as in blue-green algae. Such nuclei havebeen described by us in Pontothrix longissima, in Oscillospira guilliermondii,and in Anabaeniolum sp. These bacteria consist of threadlike individualsof various lengths. Every individual or thread is divided by transverse septa ina series of short cells, whose length usually does not exceed the width and is moreoften less than the latter. The internal structure of such cells reminds one of thestructure of the protoplasts of blue-green algae. A great part of the cell is occupiedby the central body. On the periphery the plasma is distributed in the form of athin layer. The central body stains well with aniline dyes and is easily found byordinary 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 inCaryophanon by Sall and Mudd (1955).

   In many bacteria so-called polar bodies or small grains at theends of the cells are noted. Like chromatin, they stain densely with stains. Therole 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 specificenlargement of the cell wall. Recently many investigators are inclined to regardthese enlargements as points of growth of cells, directing the process of elongationof the individuals.

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

   The nucleus in bacteria and actinomycetes is primitive. It doesnot have a constant, established, structural formation. It occurs either in a diffusedissolved state, or in the form of small grains and bodies which are dispersed allover 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 contraditinctionto the latter, the nucleoids of bacteria are structurally undifferentiated, the characteristicformations 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 nucleoidsis their ability to reproduce during the process of cell division.

   It is a characteristic property of nucleoids that they are oftenreformed from diffusely dissolved or dispersed chromatin during spore formation insporiferous bacteria, mycobacteria and actinomycetes; they are also organized insidethe vegetative cells at certain stages of their growth.

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

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

   Amitotic division of the nucleus is found in the cells of callus,cicatrization, regeneration and in tissues formed anew. In these cases the cellsundergo a series of cytochemical and morphological changes. In the nucleus processesof direct division or fragmentation go on and several daughter nuclei are formedconsecutively; sometimes the nucleus splits simultaneously into several daughternuclei. The latter separate and are distributed in various parts of the plasma, wherethey become the centers of formation of daughter cells. According to Glushchenkoet al, (1953) the budding of nuclei was established in the cells of cicatricatingtissue of the potato fiber. Such nuclei undergo deformations, swell. acquire variouspatterns, and oarlike or budlike protrusions appear on their surface. The lattergradually become rounded, pinch off from the maternal nucleus and transform intoseparate 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 developmentof the rootlet the cells lacking nuclei form primitively organized nuclei or "protokaryons".The "protokaryon" multiplies by simple division or binary fission. Mitosisis absent. Only afterward when the rootlet develops sufficiently do the primitivenuclei begin to multiply mitotically in their cells.

   Cells without nuclei were found by Glushchenko in tissues oflentils of black currants, where they form rootlets, and later in cells of cicatricatingNeder's tissue. These calls multiply by fragmentation in the early state of rootletformation without any indication of the presence of a nucleus. Also, according tothe experiments of Ellenhorn, after some time primitive nuclei are formed anew inthe 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 similarityof nuclear formation and structure in plants and bacteria or actinomycetes has beenshown. As in the latter. the nuclei of plant cells may be formed anew from chromatinsubstance diffusely distributed in the protoplast. Such a way of formation and developmentof primitive nuclei in plants evidently reflects the picture of the early stage ofevolution and formation of cells.

 

Gram-staining of Bacteria

   During the elaboration of methods for differentiating bacterialcells in tissues of animal organisms, Christian Gram (1884) and his collaboratorssuggested a special method of staining. By this method the division of all bacteriainto 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: thebacterial 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 withpicric acid. The stained preparations are washed with water and neutral alcohol oracetone. By this treatment the stain in removed in some bacterial species and retainedin others. The former are called gram-negative, the latter, gram-positive bacteria.

   The fact that different bacteria retain the stain in a differentmanner 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 inthe cells of these microorganisms. The gram stain is an indication of the biologicalor 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 systematicindication in bacterial taxonomy.

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

   The ability to absorb and to retain the stain according to Gramis characteristic of many organisms of the bacterial class of almost all actinomycetesand mycobacteria, fungi and yeasts. The majority of the cells of higher plants andanimals are gram-negative. However, separate inclusions in the cells of these organisms,in particular the nucleus, the nuclear hyalin and the nuclear substance, etc, aregram-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 effectof 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, glutamicacid, 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 degreeeven in the same species, depending on the age of the culture, the nutrient mediumand other external conditions. For instance, on unfavorable media, bacteria oftenlose 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 liquidprotein medium with a high content of glucose and the salts MgSO4,NaCl. The sporeforming bacillus--Bac. cerus, after having been in distilledwater or tap water, stains gram-positively rather weakly, and many cells become completelygram-negative. How water acts is unknown. Whether the gram-positive substance inused as a nutrient during starvation, or if this substance is dissolved as a resultof autolytic processes which proceed quite intensely under these conditions was notelucidated.

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

   As seen from the previously-mentioned data, the medium is ofgreat importance in connection with the gram stainability of cells. A medium notsufficiently well chosen may mislead the investigator in his differentiation of bacterialspecies. Therefore, in all doubtful cases of uncertain staining, the use of severalnutrient media in recommended. The gram stainability is also greatly affected bythe 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-tosix-day-old culture even more so. In some cases old cultures are more gram-positivethan young cultures. In the sporeforming bacteria--Bac. mesentericus, Bac. subtilisand other species; the gram-positive substance occurs in cells in the sporulationphase 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 causedby the basophily of the protoplast and its ability to strongly absorb stains in general.

   The stainability of cells depends to a considerable degree upontheir individual features. Two cells of the same age placed side by side often stainwith a different intensity. The degree of the stainability changes depending uponthe duration of the decolorization, the fixation method and, in general, upon thepreparation of the cells and reagents. Thick smears take longer to decolorize thanthin ones.

   Gram stainability is connected with the species characteristicsof organisms. Certain species, for example gonococci and certain mycobacteria easilychange their staining characteristics when grown under different conditions, whileothers 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 speciescharacteristics 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 standardsin 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 investigationsperformed, 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 bacteriain 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 retainthe stain and do not release it, or release it with difficulty upon washing. Someauthors relate the gram stain to the presence of fatty acids of lecithin or lipoproteinsin the plasma. These substances strongly combine with the stain and iodine and donot decolorize on treatment with ethanol, Schumacher (1920 isolated fatty acids fromthe 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 fattyacids. According to the works of Peterson (1955) and of other authors, the role playedby unsaturated fatty acids in gram stainability was refuted In connection with thefact that gram-negative bacteria do not contain fewer of these acids than gram-positivebacteria,

   Recently gram stainability has been attributed to a particularnucleoprotein gram-positive substance, ribonucloic acid, more precisely, to magnesiumribonucleate. It was established that by hydrolysis or treatment with bile saltsone can deprive the cells of the gram-positive substance and transform them intogram-negative (Denesen, 1948; Stacey et al., 1940; Stacey, 1949). Bartholomew andUmbrait (1944) removed the magnesium ribonucleate by crystalline ribonuclease andtransformed the cells into gram-negative ones. The artificially removed magnesiumribonucleate 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 noless ribornucleic acid and magnesium ribonucleate. These substances isolated frombacteria of the coli group do not transform gram-positive cells (after previous ,separationof the gram-positive substance) for instance Clostridium cells--Clostridiumwelchii (Jones, Mugglestone and Stacey, 1950).

   Henry, Stacey and others came to the conclusion that the gramstain depends upon the ratio of nucleoproteins to ribonucloic and desoxyribonucleicacids. In streptococci and in Clostridium--Clostridium welchii this ratiois 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 aparticular substance, "XP", of unknown nature and containing phosphoruswhich is combined with the ribonucleate of the gram-positive substance. Accordingto their data. the substance "XP" is always found in the cells of gram-positivebacteria. However, subsequent studies showed that this substance also occurs in largequantities 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 conclusionthat a decisive role in the gram stain belongs not to, ribonucloic acid but to theprotein of the cells.

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

   It should be noted that the establishment of the isoelectricpoint is carried out in the cell treated with iodine and, if it conditions the resultof the gram stain, it does so in nonliving cells. The applied fixation method byiodine causes oxidation of plasma. Oxidizing agents are also such substances as bromine,picric acid, potassium bichromate, tri-nitrobenzene, etc. These substances oxidizesome components of the protoplasts, render it more acid and shift the isoelectriczone. 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 bacteriaincreases considerably under the action of iodine and other mordants. This fact,if true, only shows that the biochemical condition of the plasma of gram-positivebacteria differs from that of gram-negative bacteria. In other words, the livingsubstance in the former and the latter organisms is different.

   The given theory of gram stain of bacteria has not been sufficientlyconfirmed by a series of experiments. First, some investigators did not confirm thatiodine as an oxidizing agent may be replaced by other reagents in the same treatmentof cells. Second, if the theory is true, then iodine should also produce this effectbefore staining, but this does not occur (Bartholomew and Mittwer, 1952). Further,if the theory in correct, then decomposed cells or the content of decomposed cellsshould 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 isnot gram-stainable.

   At the basis of the permeability theory is the different abilityof the cell wall and cell membrane to be permeated by stains and mordants. A suggestionwas put forward that, in the cell, insoluble precipitates of the stain and iodineare formed, which are not washed out by ethanol. It in known that iodine does indeedform a colloidal complex with methylviolet which in not soluble in water. In favorof 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 (grindingwith 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 stainsand iodine to permeate but the molecules of iodine and stain combined inside thecell are retained. As a result. the whole protoplast remains stained after washingwith ethanol. The second group of bacteria possess cell walls which do not allowstains to permeate and. due to this, are easily decolorized upon gram staining. Inthe 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 basisthat iodine and the stain do not form such large particles that they cannot penetrateinto the cell. Besides this, the addition product of iodine with the stain dissociatesin ethanol. Consequently, on washing the preparation, the obtained complex shouldalso be dissolved and go out through the cell wall.

   It was suggested that the permeability, for iodine itself, ofthe 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 bacteriaa 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 stainhas not been solved until now. No single hypothesis or theory explains the diversityof cell stainability in various species and bacterial groups.

   It must be assumed that the ability of gram staining is determinedby the property of the whole protoplast and cell wall together and not by any onepart of the cell. The degree of stainability or stability of the combination of theplasma with the stain depends upon external growth conditions of the culture, itsage and other causes.



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