HOME      AG LIBRARY       KRASILNIKOV TABLE OF CONTENTS

Part I, continued:

   Microorganisms, like all other living creatures, grow, develop, multiply, undergo changes, age and die. They have their own cycle of development. One must differentiate between the growth and development of separate individuals or microbial cells and the growth and development of their cultures. The latter consist of an enormous number of separate individuals and represent large populations or associations of various individuals.

   It is quite natural that the character and regularities of growth of separate cells and whole associations of the same microbial species sharply differ.

   The growth and development of separate bacterial individuals are very simple in their external manifestations. When they are observed under laboratory conditions of growth on nutrient media, the following may be noted. The cells elongate, reach definite dimensions and start division. The daughter cells repeat the same cycle and this continues until the nutrient substances become exhausted, the medium and environmental conditions change.

   In sporeforming bacteria spore formation occurs at a definite phase of development. Upon inoculation of a fresh nutrient substrate, the spores grow into rodlike individuals, which begin the cycle anew.

   The growth process of the microbial cell is characterized by an increase in its volume. The separate cells increase in length; the growth occurs at the ends. It was established that in bacteria there are accumulations (polar bodies), at the ends of the cell, which are of a particular substance, and stain a dark color with aniline dyes. This substance is often regarded as chromatin or metachromatin. The studies of Bielig et. al., (1949), Bergersen (1953) and Bisset (1953) proved that these bodies are related to the growth of the cell. According to their observations, intense biochemical processes take place in this area and the body of the cell increases there. Because of its properties, this part of the cell may be regarded as a growth point. Such distal cell growth occurs in actinomycetes, protoactinomycetes and mycobacteria. The elongation of the separate hyphae at the growth point in easily noticed upon observation of a hanging drop. With the growth of the mycelium the length increases just at the distal part, from the youngest apical branch to the end of the hyphae; the distance between consecutive branches of the same sprig remains unchanged (Figure 7).

   We observed growth at the end of cells in Azotobacter, in purple sulfur bacteria (Krasil'nilkov, 1932, 1935) and in some other bacteria. Streshinskii (1955, 1956) observed such growth of cells in sporeforming bacteria--Bac. subtilis and Bac. megatherium.

   In spherical bacteria (family Coccaceae) the growth point may occur at various areas on the periphery of the cell. Respectively their division takes place in various planes, in sarcina the growth is orientated in three perpendicular directions, in streptococci in one direction, and in micrococci--in all directions.

   A multilateral increase of the volume of cells occurs during the growth of the culture in yeast, which proliferate by budding. As known many yeasts multiply by budding. On the surface of the cell a small knob is formed--the bud. The dimensions of the bud increase, its growth proceeds uniformly in all directions, or unequally. In the former case spherical cells are obtained and in the latter-oval or oblong ones.

Figure 7. Growth of microbial cells. Body increase at the distal part of the cell, at the "growth point"

A. Actinomyces streptomycini: a, b, c--cell increase from the site of sprig formation; B. Azotobacter chroococcum growth of the distal part of the cell: a^l. a² and a³--increase of the cell at the "growth point" from the site of attachment of the septum.

    Under conditions of normal growth the cells of microorganisms start to multiply when they reach certain dimensions. At this stage of life various cytochemical transformations occur inside the cell, which lead to the maturation of the individual. The state of its nuclear apparatus changes, and a redistribution of chromatin and other structural substances occur. These processes have been little studied in bacteria, but one must assume that they are no less complicated than in higher organisms.

   The cells under conditions of normal growth are of definite dimensions. For many species or groups of microbes this value is quite constant. For instance in representatives of the Pseudomonas bacteria the dimensions of the cell are on the average 3-5 x 0.7 µ, while the representatives of sporeforming bacteria of the group Bac. subtilis and Bac. mesentericus consist of larger cells--5-7 x 1.0µ. In representatives of the Clostridium bacteria the cells are of still larger dimensions 5-10 x 1.5µ. The representatives of Azotobacter, etc, have quite large cells.

   The dimensions of cells are in general constant and characteristic for separate microbial groups or even their different species. Under unfavorable growth conditions or under the action of special agents as well as upon the aging of the culture the cells acquire different and variable sizes. As a rule they increase greatly in volume, assume various forms and patterns.

   In these cases there is a lack of correspondence between the growth of the cells and proliferation. The cells continue to grow, whereas the multiplication slows down or stops completely. Strongly elongated individuals appear with a specific internal structure of the protoplast and nuclear substance. Some investigators observed in such cells formation of many nuclei or their analogues--nucleoids--and assumed them to be polynuclear cells (Peshkov, 1955, De Lamater and others, 1955). Under unfavorable growth conditions the cell may grow only in length without a noticeable change in width. Elongated, threadlike cells are obtained. In all cases of such an abnormal growth the internal regulation of reproduction is disturbed, the system may be artificially evoked by means of external factors.

   Upon development of cultures on artificial nutrient media, definite and well-expressed. regularly proceeding phases are observed. They follow consecutively.

   Four phases are observed: the lag phase, phase of logarithmic growth, phase of stationary growth, and phase of reverse development. In the first phase--the lag phase--the cells do not proliferate. After inoculation of a culture of bacteria, yeasts, or other microbes on a fresh nutrient substrate, the cells act for some time as if they are at rest, their number does not increase. However. one should not regard this period as a resting state. Investigations show that in this phase an intense preparatory activity of cells proceeds. The cells increase in volume, become elongated and enlarge; the plasma acquires a more strongly expressed basophily, and is optically homogeneous, without granular inclusions. The cells remain in this phase from 2 to 10 hours and more, depending upon the microbial species, the composition of medium and the environmental conditions. During this period the cells undergo reorganization, they adapt themselves to the new conditions of life. For an old culture the fresh nutrient substrate becomes a new medium. Upon each new transfer the process of adaptation of the cells to the fresh medium proceeds very slowly; the older the transferred culture, the slower the process. When young cells, for instance those 5-6 hours old are transferred, the lag phase is considerably shorter than upon transfer of old, (5-7 days) cultures. The number of surviving cells is greater in the former case than in the latter.

   Upon the inoculation of bacteria. actinomycetes, yeasts or other microorganisms in a fresh nutrient medium, not all the cells adapt themselves. Some remain in a resting state without any apparent development, and finally die. Separate cells grow after a great delay, while the neighboring individuals may already have produced several generations. A delayed development is observed in weak cells.

   They evidently lack the growth factors, whose ability to be formed is lost. When all those substances are produced in a sufficient amount by the neighboring, normal cells, then the weak and the abnormal cells develop.

   In microbes the cells are polymorphous. They differ from each other in many respects, for example, in viability. Some of them reveal a high degree of adaptability, growth and proliferation, in others the life functions are weak. As the culture becomes old the number of weakened cells increases. In Azotobacter, when an old 7-10 day culture is transferred to a fresh Ashby medium approximately 10-30% of the cells grow; upon transfer of a young 24-hour culture 70-90% grow (observations were carried out in a hanging drop).

   The number of growing cells of an old culture may be increased by selection of suitable conditions, or by a change of the composition of the medium. When a little of the filtrate of the old Azotobacter or yeast culture and often of other microbes, is added to the medium, the percentage of growing cells increases.

   The phase of logarithmic growth is characterized by a rapid growth and proliferation of cells. Their size increases, and they assume the usual dimensions characteristic of the respective species. When they reach the limiting size, the cells multiply. The daughter cells formed grow to their limiting size and soon also begin to multiply. The plasma of the cells in this period of growth is less basophilic and less homogeneous, small granules and various other structural formations appear in it, but in a very small quantity, without changing the homogeneity of the protoplast.

Figure 8. Typical growth curve of bacterial culture; 1g² of growth as a function of time (after Stephenson, 1951)

    In the phase of logarithmic growth the number of cells increases exponentially, if the logarithms of the survivors are plotted on the ordinate and the time on the abscissa (Figure 8). Such an increase of the cell number in the culture lasts until a definite value is reached, then the intensity of growth decreases, the proliferation slows down and the culture passes into the third phase--the stationary phase. In this phase the number of living cells does not increase, the formation of new cells equalling the number of dying ones. The culture reaches its limiting age. The plasma loses the basophily, becomes granular and has various inclusions: chromatin, metachromatin, fat droplets and other substances. The growth curve in this period is parallel to the abscissa.

   The fourth phase is a reversed development of the culture or phase of accelerated death. Its main feature is that the number of dying cells proves to be greater than the newly formed ones. The multiplication of cells in this period slows down. The culture becomes old, senile and dies.

   In this phase of senescence and degeneration the cells undergo considerable cytomorphological changes. While in the second phase the cells assumed their usual form and size for the given species, and the plasma showed a somewhat weaker basophily in comparison with the first phase, in the third phase the cells become more polymorphous. In this period of development the cultures reach maximal variation in size and form, as well as in the internal structure of the protoplast; they also differ biochemically.

   Beside normal cells, various deviating forms are found in varying quantities. As a rule, in old cultures there are many strongly fractionated germ formations almost invisible by optical microscopy and of a size smaller than the resolving power. In these cultures one may also find many large specimens, often reaching enormous dimensions, ten times greater than those of normal ones.

   These swollen cells differ from normal ones by unusually diverse forms. The deformed cells also differ in internal structure, structure of protoplast, in the quantity and formation of chromatin and other granular formations.

   The physiology and fermentative properties of cells in the lag phase differ from those in the logarithmic phase of growth; cells of a later growth period differ from those of the two preceding phases (Ierusalimskii, 1949).

   Enlarged cells of the lag phase with a more basophilic plasma, strongly absorb stains. A shift of the isoelectric point of the protoplasm to the acid side may be noted in them. In young cells passing into the second phase or already being in this phase, the metabolism is much more strongly expressed than in the preceding and in the following phases. The oxygen uptake, the release of carbonic acid, and the heat formation increase: upon the decomposition of proteins, the release of ammonia and other decomposition products is increased. Intensification of the process is caused not by the increase of the cell mass or total volume of individual cells but by the condition and properties of the living substance of the individual cells. This may be seen from the data in Table 1.

   The rate of metabolism (from CO₂) per unity of living substance in the lag phase is considerably higher.

   According to the statements of some authors, cells of the lag phase are more sensitive to environmental factors. They are less resistant to higher temperature and salt concentrations, to various chemicals, stains, antibiotics, etc, (Peahkov, 1955).

   Bacterial cells proliferate in various ways--by division, constriction and sometimes by budding and fragmentation.

   Mostly they multiply by division. This process proceeds in the following manner. According to recent data, the division of the cell is preceded by a differentiation of the protoplast. The amount of chromatin or nucleoproteins increases in it. Various inclusions of reserve food substances and others are formed. Consecutively an intracellular separation of the protoplast into two daughter units follows. Each forms its own membrane by which they are separated from one another. The membrane of each daughter protoplast forms a transverse septum on its exterior. Afterward, division of the cell into two daughter cells occurs (Figure 9).

Figure 9. Division of cells by means of transverse septa:

a) Bac. anthracis; b and c) Bac. megatherium (after Robinow from Dubos, 1948),

 

Figure 10. Fragmentation of bacterial cells:

a) Bac. megatherium (after Robinow from Dubos, 1948); b) Bac. megatherium (after Kudryavtsev, 1932); c) Act. globisporus (after Krasil'nikov, 1938).

   In separate cases under unfavorable growth conditions a simultaneous division of the cell into several daughter cells is observed in bacteria. The cell splits or undergoes fragmentation to three, four, five and more small individuals (Figure 10). Such splitting is observed in sporeforming bacteria--Bac. megatherium (Kudryavtsev, 1932), in Bac. mesentericus, Bac. mycoides in lactic bacteria (Krasil'nikov, 1954 B), in filamentous bacteria--Pontothrix longissima (Krasil'nikov, 1932a) and in many others (see Dubos, 1948; Bisset, 1950; Robinow, 1942, 1951; Malek, 1955 and others). During the process of splitting, septa are formed in various directions--transverse. longitudinal and oblique, like those formed in micrococci.

   Constriction is less often observed than division. It may, however, be seen quite often in various specimens of bacteria and mycobacteria. This process proceeds in the following manner. In the central part of the cell an almost invisible constriction is formed, it deepens gradually separating the cell into two halves. Sometimes the constriction is not complete and leaves a small bridge in the form of a copulation canal between the formed daughter halves. Finally this bridge breaks and the daughter cells part (Figure 11).

Figure 11. Multiplication of bacterial cells by constriction:

a--Azotobacter chroococcum (the author's observations); b--Rhizobium trifolii (the author's observations).

   Such a multiplication is observed in Azotobacter during growth on mustagar, In mycobacterium, in purple sulfur bacteria, Chromatium and other bacteria.

   Often a mixed type of proliferation is noted in bacteria, division is combined with constriction. At first a small annular depression appears--a constriction--then a transverse septum is formed and the cell divides. The process begins with constriction and ends with division (Figure 12). A similar type of proliferation is observed in Azotobacter, in various specimens of sporeforming and other bacteria.

Figure 12. Multiplication of bacterial cells by constriction:

Bac. cereus (after Johnson, 1944).

   Sometimes under unfavorable conditions, small particles in the form of cocci, detach themselves from the end of the cell. They split off from the maternal cell, and under favorable conditions, grow into new organisms. Such coccuslike germs, formed by splitting off, are found in many bacterial species, and in mycobacteria: in Bac. mycoides, Bac. megatherium, Bac. cereus, and others (Kudrayvtsev, 1932; Krasil'nikov. 1932 and others). They may often be seen in filamentous bacteria - Pontothrix and others Krasil'nikov, 1932a, 1945 B),

   One and the same organism may proliferate by division, constriction and some times by fragmentation (splitting into small fragments) depending upon the growth conditions of the culture.

   Budding in bacteria is not observed under normal growth conditions. It is found in old cultures or in cultures subjected to the action of unfavorable factors. It is expressed in the following way. On the surface of the cell, on any part of it, a tiny body appears, which gradually increases, reaches definite dimensions and acquires a contour in the form of a more or less distinct membrane or cell wall. Such a body has the form of a budlike cell (Figure 13).

Figure 13. Formation of budlike bodies in bacteria:

a) Azotobacter chroococcum Krasil'nikov, 1931); b) Bac. megatherium (after Kudryavtsev, 1932); c) Bact. vulgaris (after Peshkov, 1955); d) Bac. mesentericus (after Robinow from Dubos, 1948); e) Bact. proteus (after Stempen and Hutchinson, 1951).

   As a rule under conditions of the hanging drop. where the formation of buds proceeds, they do not develop. Only in rare cases does one succeed in following their further development. A great many of these buds become swollen and after some time burst. In single cases one may observe buds growing into normal rod-like cells, while still affixed to the maternal cell. Such a formation of reproductive forms was observed by us in Azotobacter, in root-nodule bacteria, in some sporeforming, and nonsporeforming bacteria, Acetobacter and others (Krasil'nikov. 1954 B).

   In mycobacteria, mycococci, actinomycetes and other specimens of Actinomycetales, budding as a form of multiplication is often observed, the process of budding hereby proceeds exactly as that in the yeastlike fungi. At first a small protrusion appears on the surface of the cell wall, then it enlarges. reaches a definite size and then either splits off from the maternal cell and continues to grow and develop,or it grows without separation from the initial cell (Figure 14). In Actinomycetales, the process of budding should be regarded as the normal way of proliferation. The buds formed in them possess a completely normal dense protoplasm which strongly refracts light and stains intensely. A small granule of chromatin may be differentiated inside the bud (Krasil'nikov, 1938 b).

Figure 14. Budding in Actinomycetales

A--Act. candidus: a--filaments of mycelium with short rodlike sprigs--buds, b--germination of rodlike appendages. B--Proact ruber: a--chain of rodlike cells, b--cells with buds, c--process of bud formation. C--Mycobac. nigrum, D--Mycoc. ruber: a--formation of the bud on the surface and further development b, c, d and e. a₁-- e₁--the same. The arrows show the sequence of development.

   The reproduction of bacterial cells may proceed by the formation of special germ cells, the so-called regenerative bodies inside the cells. These bodies are usually formed during the degenerative process, in old cultures or under unfavorable growth conditions of the culture. The cells undergo deformation, the plasma changes noticeably and granules of chromatin and other structural bodies appear. In such degenerative protoplasma, separate particles or granules of chromatin become centers of formation of very small germ cells. Around the chromatin granule a zone of plasma concentration is formed, on the surface of which there is a thin, hardly visible membrane. A tiny germ cell is obtained, which is usually scarcely noticeable in the protoplasm. Refraction of light of this germ cell hardly differs from that of the cell protoplasm. The diameter of the whole germ does not exceed 0. 5µ, more often 0.1-0. 2µ (Figure 15). When the cells undergo autolysis their cell wall disintegrates, the germ cells are released, and under favorable conditions they may grow and produce a normal generation (Krasil'nikov, 1954B).

Figure 15. Formation of regenerative bodies inside swollen and dying cells of Azotobacter

   Such regenerative bodies have nothing in common with endogenous spores in sporeforming bacteria. They are closer to the regenerative forms occurring an budding described above. Their characteristic feature is a low viability. Under laboratory condition they do not, as a rule. grow, or very seldom, there by producing only several generations.

   The formation of buds, and regenerative bodies inside the cells, then the fragmentation of the cells into tiny germ elements and the branching off of small particles from the extremity of the cell--all these methods of reproduction take place in particular pathological states in organisms under unfavorable growth conditions. Evidently, these forms of reproduction constitute a biological adaptation of the species. The probability is not excluded. that in many bacterial species they are the most frequent under natural conditions. In the soil the cells of bacteria and actinomycetes occur in other forms and states, and, consequently, the ways of reproduction may sharply differ from those observed by us in artificial laboratory media.

   Under conditions of the normal growth and development of microbes a definite and quite constant relationship between the growth of cells and their proliferation is observed. Multiplication of cells begins after the latter reach a certain size. Growth and multiplication of cells occur at a definite rate which differs in various species.

   If growth and multiplication is suppressed for some reason, various kinds of formation disturbances are observed. Under the influence of environmental factors the process of multiplication of microbes may be suppressed or stopped, while the growth function is preserved or only slightly suppressed. The opposite may also occur--the growth is stopped, but the process of multiplication proceeds normally or only slowly.

   In the former case, when the function of multiplication is suppressed, the cells increase, reach enormous sizes, undergo deformation and are transformed into so-called involution forms (see below). Such forms occur in old cultures which are influenced by their own metabolic products. They can be obtained by the action of penicillin, lithium chloride and other substances.

   In those cases where the growth function is slowed down or suppressed and the process of proliferation continues, small cells are produced. Smaller cells are formed by each division until ultramicroscopic cells are formed (Figure 16). This decreasing cell size is observed in many bacteria, mycobacteria and actinomycetes.

Figure 16. Decrease of bacterial cell size in the process of consecutive divisions during slowed growth

A) Azotobacter chroococcum; inside the capsula consecutive division of cells leads to formation of small elements, like camocytes in algae (a - f); A_l) the same--microphotography; B) Bac. mycoides; C) Mycob. rubrum; arrows show consecutive transformations of cells.

   The viability of such forms decreases or vanishes completely at a determined stage of this process during both a strong increase and a strong decrease in size. The cells of decreased. size stop growing on nutrient laboratory media. Particularly the viability of ultramicroscopic elements decreased when they were passed through fine filters.

   On studying the process of bacterial proliferation a biologically important question arises: are the daughter cells formed equivalent? It is usually assumed that during proliferation bacterial cells divide into two equal parts; on division the maternal cell is transformed into two identical "sister" cells. On this basis some investigators altogether negate the development of bacterial cells. The latter grow and increase in length, but do not develop; qualitatively they do not change. According to these opinions the newly formed daughter cells do not differ in their properties from the initial maternal cell. They are only shorter. Consequently, an ontogenetic development does not occur in bacteria.

   If it is indeed so, then after each division of the cell, the daughter cells which are formed are identical in nature with the initial cell before the division. In other words bacterial cells are invariable.

   This opinion is not correct. It has been formed under the influence of observations which are primarily of a morphological nature. Modern improved methods of investigation show that in the cells, formation processes proceed de novo and that the protoplast of the cell is not equivalent in all its parts. The daughter cells formed are not identical in their physiological and biochemical properties.

   It was assumed earlier that the viability of the "sister" cells is the same, but it was recently clearly established that this is not always the came. Developing cultures of bacteria, even in the most active growth phase (logarithmic growth phase), under the most favorable conditions of nutrition and respiration, contain a considerable number of dead cells. These cells die a natural death as a result of exhaustion after a consecutive series of multiplications (Malek, 1954, 1955; Streshinskii, 1955, 1956).

Figure 17. A. Aging and death of cells in yeasts Schizosaccharomyces octosporus: a--young viable cells; b--dying and dead old maternal cells B. Enlargement of cells in yeasts:

a--Saccharomyces cerevisiae; growth and multiplication of cells in all directions; b--Saccharomycodes ludwigie; growth of cells in one direction along the long axis; c cells multiply by budding; the formed buds branch off by transverse septa; c--Schizosaccharomyces octosporus: polar growth from one end (a₁); multiplication by division.

   We observe senescence and the death of cells in yeast organisms, budding--Saccharomyces cerevisiae and proliferating Schizosaccharomyces octosporus. In the maternal cell, upon senescence, the process of bud formation is slowed down, the plasma becomes more granular, and fat inclusions appear. Soon after, such a cell stops budding and perishes. If at the onset, the maternal cell was similar to the daughter cells, it now becomes very different, not only physiologically, but also cytochemically. The latter difference is not always well expressed. Sometimes the dying maternal cell cannot be distinguished externally from the young daughter cells. Only cessation of growth and multiplication indicates its death.

   A similar aging of cells was noted in proliferating yeasts Schizosaccharomyces octosporus. This organism multiplies like bacteria. Its cells on reaching a definite form a transverse septum and separate. The daughter cells prove to be externally identical, as in bacteria it is hard to see any difference whatsoever between "sister" cells, when the culture is still young. However, after a prolonged series of division one of the daughter cells becomes weaker, lags in growth, and its division slows down or ceases altogether. Soon its protoplast noticeably also changes, the plasma becomes coarse-grained with a fatty degeneration; the permeability of the cell wall to, stains, increases considerably. After a successive division, one may often see that one of the cell a die immediately, and the other continues to develop and to multiply normally. In two daughter cells which are still attached to each other, one is often dead and the other alive. This is well seen upon cytochemical analysis (Figure 17A).

   In budding yeasts--Saccharomyces the formation of daughter cells proceeds at various places on the periphery of the cell. The growth of the protoplast in them is evidently homogeneous. The buds in the early growth phase stand out sharply in form and size as well as in the state of the plasma. When they reach maturity. they are hardly distinguishable from the not yet aged maternal cell.

   In some yeasts (Saccharomycodes) growth of the protoplast proceeds in one direction, along the longer axis of the cell. Cells of these organisms multiply by budding in the following manner. At the end of the cell a bud is formed, which grows and reaches a definite size, afterward a transverse septum is formed at the site of the constriction and branching off from the maternal cell occurs (Figure 17B). This type of multiplication is intermediary between budding and typical division. Upon division of yeast organisms (Schizosaccharomyces) growth of the protoplast proceeds at one end at the point of growth. In this case constriction does not occur. The cells divide into two equal parts by means of a transverse septum.

   In bacteria as in Saccharomycetes, the growth of the protoplast proceeds from one end of the cell. Their content undergoes differentiation as growth and at the same time, different cells are formed on division. One of them has an older type of structure, and is less viable than the other. Often one cell ceases to grow and multiply while the other divides intensely. The two cells formed as a result of the division are, in fact, not "sisters": one of them in the maternal cell and the other is a daughter cell. Bisset (1950, 1951) showed that the young growing part of the bacterial cell does not possess flagella. The latter are formed later, when the transverse septum appears and the daughter cell splits off (Figure 18).

Figure 18. Enlargement of cells in bacteria (diagram after Bisset, 1950):

1-5 polar growth of cell at the "growing point": a--external cell wall of maternal cell; b--cytolemma; c--"growing point"; d--external cell wall in "growing point"; e--cell wall of daughter cell; f--flagella in state of formation.

   As seen from the afore-mentioned, the protoplasm of the bacterial cell varies in quality during the growth process. There is in the cell, an older and a younger part. The latter is principally connected with the formation of daughter cells. Malek (1955) showed that formation of the daughter cell proceeds in the maternal cell. Before starting division, the cell increases in mass of living substance and length and also undergoes qualitative changes. A definite cycle of development takes place--formation of a qualitatively different portion of protoplasm occurs in it. This is indispensable for the formation of the daughter cell. Consequently, in bacterial cells an ontogenetic development takes place.

   At a definite stage of development, many microorganisms begin to form spores. This process proceeds in various microbial groups in a different manner. Spares may be formed exogeneously as for instance, in actinomycetes, and endogeneously--in bacteria (sporeformers). Both the manner in which they are formed and the properties of spores in various specimens vary.

   In sporeforming bacteria endogenous spores are formed in the following way. As was indicated above, at first a chromatin substance in the form of a distinct body appears inside mature cells. This body is regarded as a rudimental spore. Around it, the protoplasm concentrates into a large round formation, the prospore. The prospore soon becomes dense, decreases in volume, the contour becomes clearer ,and a thin membrane, the intima, appears on its surface. This membrane is in turn covered by an external, thicker membrane, the enzyme, In this way, the prospore matures and is transformed into a spore.

   Upon formation of the spore the chromatin body disappears; it is, not found, in the prospore but in the mature spore it appears once more in the form of a definitely formed, nucleus-like formation (Figure 19).

Figure 19. Spore formation in bacteria Bacillus sp.:

a--vegetative mature cell with chromatin and metachromatin granules inside it, b--chromatin aggregated into a separate body--nucleoid, around which plasma is concentrated; c--formation of prospore, nucleoid is absent; d--maturation of prospore, plasma becomes dense, chromatin appears in the form of nucleoid; e--mature spore, chromatin in the form of nucleoid, membrane is clearly visible.

   Upon spore formation the plasma changes its staining properties. The plasma which is concentrated around the chromatin body, stains more intensely than normal protoplasm; the prospore stains most densely of all. The latter is also well seen without staining. Due to its great refraction of light under the microscope, the content of the prospore appears to be glistening. Mature spores lose the ability to stain. Only after treatment with a weak solution of hydrochloric acid at 60°C does the plasma of the spore take on stain. The stained spores are decolorized by acid more slowly than vegetative cells. The method of differentiation and recognition of spores is based on this fact. The inability of the mature spore to stain by usual methods is ascribed to the relative lack of permeability of the membrane. However, analysis shows that isolated membranes stain well, but the protoplasm of the spore itself does not stain. Consequently, the plasma of the cell undergoes essential changes during the process of spore formation. The plasma of mature spores has physicochemical properties which differ from those of the plasma of vegetative cells, although the composition of their ash is the same.

   Free enzymes are not found in mature spores. It is assumed that they are present in a bound form, such that its active groups are not destroyed upon heating.

   Spores, as is known, are resistant to heating. The mechanisms and causes for this thermostability of spores are not known. Some authors ascribe it to a lowered water content of the plasma, but this was not confirmed by investigations; it was found that the amount of water in the plasma of the spore and vegetative cells is equivalent.

   Recently, the resistance of spores has been related to an increased concentration of calcium. Attempts have been made to explain the thermoresistance of spores by their content of lipides and other factors. However, neither hypothesis was confirmed experimentally.

   Formation of spores in bacteria proceeds under various conditions. It is observed in both deficient and enriched media. In the former, spores appear earlier.

   The factors which condition spore formation are not clear. There is no basis for explaining spore formation by a deficiency of nutrition. In a rich medium the total quantity of spores in the culture is always greater than in a deficient medium. Neither aeration, nor temperature nor other factors constitute by themselves a direct cause of spore formation; they only create conditions affecting the given process.

   The process of spore formation in bacteria is subject to the same rules, as those which are noted for spore formation or offspring production in other groups of microorganisms--actinomycetes, fungi, yeasts. and others. Nutritional deficiency is an accelerating factor under conditions of spore formation. The resistance of the spores to unfavorable life conditions is regarded as a biological adaptation of bacteria for preserving the species.

   We assume that the biological essence of spore formation consists not solely in the preservation of the species but it has perhaps another biologically essential purpose

   It is not always conducive to species preservation. In the majority of the soil bacteria, an a rule, it does not occur under many unfavorable conditions. According to our observations, it does not occur in bacteria of the temperate zone at increased temperatures (36-38°C), or at a low temperature 3-5°C). We did not succeed in obtaining spores in nutrient media in the presence of many antibiotics and some chemicals.

   Neither does spore formation occur under many natural conditions. Cells with spores are seldom found during microscopic soil analysis. If a young culture of sporeforming bacteria is introduced into the soil, at a time when spores have not yet been formed, the latter do not appear. We did not succeed in obtaining spores in soil (podsol), from Bac. mycoides, Bac. mesentericus and Bac. megatherium.

   Under conditions of the Far North. on the islands of the Northern Arctic Ocean, (islands of Franz-Josef, Severnaya Zemlya and others) sporeforming bacteria even lose the ability to form spores. In our investigations and those of Sushkina and Ryzhkova (1955) the majority of these bacteria neither form spores in artificial nutrient media nor in the soil itself. We tested many media and grew cultures under various conditions; but the majority of them did not form spores. Only some, under particular growth conditions at an increased temperature (36-38°C). started spore formation. but they were not many. In some organisms the process is incomplete, only prospores being formed, i.e. , not fully mature spores, without membranes. Spores, as shown by daily observations, are not particularly resistant to unfavorable environmental factors. They often die with the same rapidity as vegetative cells. For instance, we observed their death simultaneously with that of vegetative cells under the action of some chemical antiseptics. They are not always resistant to high temperature. There are species whose spores die, like the vegetative cells, at a temperature of 80-100°C. Frequently, nonsporeforming bacteria occur, which are as resistant or even more so, to unfavorable factors, than species of sporeforming bacteria.

   We assume that spore formation in bacteria is a biological form of renewal of the organism a means of increasing the viability of the cells and, consequently, of the whole species. The spore may be regarded as a zygote cell, formed after fusion of various parts of the protoplast, as it takes place upon autogamous copulation.

   The bacterial cells reach a definite size, and after a series of consecutive divisions begin spore formation. which is accompanied by a complex picture of microscopic transformations in the protoplast, leading to the fusion of separate chromatin elements and other parts into a compact body--the center of spore formation (see sexual process).

   Some authors (Sorokin, 1890; Gibson, 1935; Starkey. 1938; Rubenchik. 1953) indicate that the described formation of endogenous spores in characteristic, not only of sporeforming bacteria, but also of spirilla and vibriones. According to their observations, a large oval body is formed inside the cells of spirilla whose external form resembles spores in sporeforming bacteria. For their formation the whole protoplast or a considerable part of it in used. Sometimes two or three such sporelike bodies are formed simultaneously in the cell. We observed the formation of similar bodies in Spirillum voluntans and other bacteria (Krasil'nikov, 1949 B), by placing them in an artificial synthetic medium or in a drop of the same water as that from which they were obtained. The same could be observed in a hanging drop of this medium. The motility of the spirillum slows down after some time the protoplast begins fragmentation into separate parts which become round and assume the forms and sizes of large spores. The number of such sporelike bodies varies from one to four or more (Figure 20.)

Figure 20. Formation of sporelike bodies (fragmentation spores) in Spirillum volutans (after Krasil'nikov, 1949)
a, b, c, d--consecutive stages of spore formation.

   According to our observations. these formations are not like the true spores of sporeforming bacteria. They represent fragments of protoplasts, divided into parts as a result of these or other causes. Their formation rather resembles fragmentation of the filaments of mycelium in actinomycetes, mycobacteria and in some filamentous and other bacteria.

   In actinomycetes, as will be shown further, spore formation proceeds in two ways--endogenously and exogenously. Spores or reproductive elements are formed on special branches of the aerial mycelium. and inside vegetative filaments of the substrate mycelium. They are formed by segmentation or fragmentation.

   Spore formation by fragmentation is observed in some species of mycobacteria and lactic acid bacteria (Krasil'nikov, 1938a, 1952d). As in actinomycetes, the protoplast of the cell splits into separate parts, which become round bodies--spores. The number of such spores in the cell varies from 2 to 6, or more. The quantitative regularity so characteristic of yeasts and fungi is absent.

   In all cases of multiple spore formation in actinomycetes, mycobacteria or lactic acid bacteria a concentration of chromatin substance as a rudiment or center for their formation is noted.

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