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Part I, continued:

Growth and Multiplication of Microorganisms

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

   It is quite natural that the character and regularitiesof growth of separate cells and whole associations of the same microbial speciessharply differ.

Growth of Cells and Development of Cultures

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

   In sporeforming bacteria spore formation occursat 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 characterizedby an increase in its volume. The separate cells increase in length; the growth occursat 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 colorwith aniline dyes. This substance is often regarded as chromatin or metachromatin.The studies of Bielig et. al., (1949), Bergersen (1953) and Bisset (1953) provedthat 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 increasesthere. Because of its properties, this part of the cell may be regarded as a growthpoint. Such distal cell growth occurs in actinomycetes, protoactinomycetes and mycobacteria.The elongation of the separate hyphae at the growth point in easily noticed uponobservation of a hanging drop. With the growth of the mycelium the length increasesjust 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 (Figure7).

   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) thegrowth point may occur at various areas on the periphery of the cell. Respectivelytheir division takes place in various planes, in sarcina the growth is orientatedin three perpendicular directions, in streptococci in one direction, and in micrococci--inall directions.

   A multilateral increase of the volume of cellsoccurs during the growth of the culture in yeast, which proliferate by budding. Asknown many yeasts multiply by budding. On the surface of the cell a small knob isformed--the bud. The dimensions of the bud increase, its growth proceeds uniformlyin all directions, or unequally. In the former case spherical cells are obtainedand 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: al. 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 cellsof microorganisms start to multiply when they reach certain dimensions. At this stageof life various cytochemical transformations occur inside the cell, which lead tothe maturation of the individual. The state of its nuclear apparatus changes, anda redistribution of chromatin and other structural substances occur. These processeshave been little studied in bacteria, but one must assume that they are no less complicatedthan in higher organisms.

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

   The dimensions of cells are in general constantand characteristic for separate microbial groups or even their different species.Under unfavorable growth conditions or under the action of special agents as wellas 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 correspondencebetween the growth of the cells and proliferation. The cells continue to grow, whereasthe multiplication slows down or stops completely. Strongly elongated individualsappear 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--andassumed 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 noticeablechange in width. Elongated, threadlike cells are obtained. In all cases of such anabnormal growth the internal regulation of reproduction is disturbed, the systemmay be artificially evoked by means of external factors.

   Upon development of cultures on artificial nutrientmedia, definite and well-expressed. regularly proceeding phases are observed. Theyfollow consecutively.

   Four phases are observed: the lag phase, phaseof logarithmic growth, phase of stationary growth, and phase of reverse development.In the first phase--the lag phase--the cells do not proliferate. After inoculationof 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 showthat in this phase an intense preparatory activity of cells proceeds. The cells increasein volume, become elongated and enlarge; the plasma acquires a more strongly expressedbasophily, and is optically homogeneous, without granular inclusions. The cells remainin 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 thecells 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 newtransfer the process of adaptation of the cells to the fresh medium proceeds veryslowly; 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 shorterthan upon transfer of old, (5-7 days) cultures. The number of surviving cells isgreater 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 adaptthemselves. Some remain in a resting state without any apparent development, andfinally die. Separate cells grow after a great delay, while the neighboring individualsmay already have produced several generations. A delayed development is observedin weak cells.

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

   In microbes the cells are polymorphous. Theydiffer from each other in many respects, for example, in viability. Some of themreveal a high degree of adaptability, growth and proliferation, in others the lifefunctions 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 Ashbymedium approximately 10-30% of the cells grow; upon transfer of a young 24-hour culture70-90% grow (observations were carried out in a hanging drop).

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

   The phase of logarithmic growth is characterizedby a rapid growth and proliferation of cells. Their size increases, and they assumethe usual dimensions characteristic of the respective species. When they reach thelimiting size, the cells multiply. The daughter cells formed grow to their limitingsize and soon also begin to multiply. The plasma of the cells in this period of growthis less basophilic and less homogeneous, small granules and various other structuralformations appear in it, but in a very small quantity, without changing the homogeneityof 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 thenumber of cells increases exponentially, if the logarithms of the survivors are plottedon the ordinate and the time on the abscissa (Figure 8). Such an increase of thecell number in the culture lasts until a definite value is reached, then the intensityof growth decreases, the proliferation slows down and the culture passes into thethird phase--the stationary phase. In this phase the number of living cells doesnot increase, the formation of new cells equalling the number of dying ones. Theculture reaches its limiting age. The plasma loses the basophily, becomes granularand 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 ofthe culture or phase of accelerated death. Its main feature is that the number ofdying cells proves to be greater than the newly formed ones. The multiplication ofcells in this period slows down. The culture becomes old, senile and dies.

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

   Beside normal cells, various deviating formsare found in varying quantities. As a rule, in old cultures there are many stronglyfractionated germ formations almost invisible by optical microscopy and of a sizesmaller than the resolving power. In these cultures one may also find many largespecimens, often reaching enormous dimensions, ten times greater than those of normalones.

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

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

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

Table 1

Intensity of cell metabolism depending upon the phase of development of a Bac. coli culture. Observations in a peptone-glucose medium (Hungington and Winslow, 1937)

Time,
hours

Release of
CO
2

Volume of cells
µ
3

Rate of multiplication (number of cells formed during one hour)

0

--

0.41

--

1

78

0.77

0.13

2

86

1.03

1.04

3

79

0.93

1.41

4

59

0.91

0.64

5

31

0.81

0.42

6

24

0.84

0.04

7

19

0.89

0.18

23

--

0.76

--

25

7

0.75

0.05

   Note. Calculation of CO2 in mg x 10-11 per1 µ3 viable cells duringone hour.

   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 lessresistant to higher temperature and salt concentrations, to various chemicals, stains,antibiotics, etc, (Peahkov, 1955).

 

Proliferation of Bacteria

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

   Mostly they multiply by division. This processproceeds in the following manner. According to recent data, the division of the cellis preceded by a differentiation of the protoplast. The amount of chromatin or nucleoproteinsincreases in it. Various inclusions of reserve food substances and others are formed.Consecutively an intracellular separation of the protoplast into two daughter unitsfollows. 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 conditionsa 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 analmost invisible constriction is formed, it deepens gradually separating the cellinto two halves. Sometimes the constriction is not complete and leaves a small bridgein the form of a copulation canal between the formed daughter halves. Finally thisbridge 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 Azotobacterduring growth on mustagar, In mycobacterium, in purple sulfur bacteria, Chromatiumand other bacteria.

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

 

Figure 12. Multiplication of bacterial cells by constriction:

Bac. cereus (after Johnson, 1944).

   Sometimes under unfavorable conditions, smallparticles in the form of cocci, detach themselves from the end of the cell. Theysplit 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, andothers (Kudrayvtsev, 1932; Krasil'nikov. 1932 and others). They may often be seenin filamentous bacteria - Pontothrix and others Krasil'nikov, 1932a, 1945B),

   One and the same organism may proliferate bydivision, 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 normalgrowth conditions. It is found in old cultures or in cultures subjected to the actionof unfavorable factors. It is expressed in the following way. On the surface of thecell, on any part of it, a tiny body appears, which gradually increases, reachesdefinite dimensions and acquires a contour in the form of a more or less distinctmembrane 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 doesone succeed in following their further development. A great many of these buds becomeswollen and after some time burst. In single cases one may observe buds growing intonormal rod-like cells, while still affixed to the maternal cell. Such a formationof 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 andother specimens of Actinomycetales, budding as a form of multiplication is oftenobserved, the process of budding hereby proceeds exactly as that in the yeastlikefungi. At first a small protrusion appears on the surface of the cell wall, thenit enlarges. reaches a definite size and then either splits off from the maternalcell and continues to grow and develop,or it grows without separation from the initialcell (Figure 14). In Actinomycetales, the process of budding should be regarded asthe normal way of proliferation. The buds formed in them possess a completely normaldense protoplasm which strongly refracts light and stains intensely. A small granuleof 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 proceedby the formation of special germ cells, the so-called regenerative bodies insidethe cells. These bodies are usually formed during the degenerative process, in oldcultures or under unfavorable growth conditions of the culture. The cells undergodeformation, the plasma changes noticeably and granules of chromatin and other structuralbodies appear. In such degenerative protoplasma, separate particles or granules ofchromatin become centers of formation of very small germ cells. Around the chromatingranule a zone of plasma concentration is formed, on the surface of which there isa thin, hardly visible membrane. A tiny germ cell is obtained, which is usually scarcelynoticeable in the protoplasm. Refraction of light of this germ cell hardly differsfrom that of the cell protoplasm. The diameter of the whole germ does not exceed0. 5µ, more often 0.1-0. 2µ (Figure 15). When the cells undergo autolysistheir cell wall disintegrates, the germ cells are released, and under favorable conditionsthey 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 commonwith endogenous spores in sporeforming bacteria. They are closer to the regenerativeforms occurring an budding described above. Their characteristic feature is a lowviability. 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 bodiesinside the cells, then the fragmentation of the cells into tiny germ elements andthe branching off of small particles from the extremity of the cell--all these methodsof reproduction take place in particular pathological states in organisms under unfavorablegrowth conditions. Evidently, these forms of reproduction constitute a biologicaladaptation of the species. The probability is not excluded. that in many bacterialspecies they are the most frequent under natural conditions. In the soil the cellsof bacteria and actinomycetes occur in other forms and states, and, consequently,the ways of reproduction may sharply differ from those observed by us in artificiallaboratory media.

   Under conditions of the normal growth and developmentof microbes a definite and quite constant relationship between the growth of cellsand their proliferation is observed. Multiplication of cells begins after the latterreach a certain size. Growth and multiplication of cells occur at a definite ratewhich differs in various species.

   If growth and multiplication is suppressed forsome reason, various kinds of formation disturbances are observed. Under the influenceof environmental factors the process of multiplication of microbes may be suppressedor stopped, while the growth function is preserved or only slightly suppressed. Theopposite may also occur--the growth is stopped, but the process of multiplicationproceeds normally or only slowly.

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

   In those cases where the growth function is sloweddown 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, mycobacteriaand 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); Al) the same--microphotography; B) Bac. mycoides; C) Mycob. rubrum; arrows show consecutive transformations of cells.

   The viability of such forms decreases or vanishescompletely at a determined stage of this process during both a strong increase anda strong decrease in size. The cells of decreased. size stop growing on nutrientlaboratory media. Particularly the viability of ultramicroscopic elements decreasedwhen they were passed through fine filters.

   On studying the process of bacterial proliferationa biologically important question arises: are the daughter cells formed equivalent?It is usually assumed that during proliferation bacterial cells divide into two equalparts; on division the maternal cell is transformed into two identical "sister"cells. On this basis some investigators altogether negate the development of bacterialcells. The latter grow and increase in length, but do not develop; qualitativelythey do not change. According to these opinions the newly formed daughter cells donot 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 divisionof the cell, the daughter cells which are formed are identical in nature with theinitial cell before the division. In other words bacterial cells are invariable.

   This opinion is not correct. It has been formedunder the influence of observations which are primarily of a morphological nature.Modern improved methods of investigation show that in the cells, formation processesproceed de novo and that the protoplast of the cell is not equivalent in all itsparts. The daughter cells formed are not identical in their physiological and biochemicalproperties.

   It was assumed earlier that the viability ofthe "sister" cells is the same, but it was recently clearly establishedthat this is not always the came. Developing cultures of bacteria, even in the mostactive growth phase (logarithmic growth phase), under the most favorable conditionsof nutrition and respiration, contain a considerable number of dead cells. Thesecells die a natural death as a result of exhaustion after a consecutive series ofmultiplications (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 cellsin yeast organisms, budding--Saccharomyces cerevisiae and proliferating Schizosaccharomycesoctosporus. In the maternal cell, upon senescence, the process of bud formationis slowed down, the plasma becomes more granular, and fat inclusions appear. Soonafter, such a cell stops budding and perishes. If at the onset, the maternal cellwas similar to the daughter cells, it now becomes very different, not only physiologically,but also cytochemically. The latter difference is not always well expressed. Sometimesthe dying maternal cell cannot be distinguished externally from the young daughtercells. Only cessation of growth and multiplication indicates its death.

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

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

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

   In bacteria as in Saccharomycetes, thegrowth of the protoplast proceeds from one end of the cell. Their content undergoesdifferentiation 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. Oftenone cell ceases to grow and multiply while the other divides intensely. The two cellsformed as a result of the division are, in fact, not "sisters": one ofthem in the maternal cell and the other is a daughter cell. Bisset (1950, 1951) showedthat the young growing part of the bacterial cell does not possess flagella. Thelatter are formed later, when the transverse septum appears and the daughter cellsplits 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 protoplasmof the bacterial cell varies in quality during the growth process. There is in thecell, an older and a younger part. The latter is principally connected with the formationof daughter cells. Malek (1955) showed that formation of the daughter cell proceedsin the maternal cell. Before starting division, the cell increases in mass of livingsubstance and length and also undergoes qualitative changes. A definite cycle ofdevelopment takes place--formation of a qualitatively different portion of protoplasmoccurs 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 microorganismsbegin to form spores. This process proceeds in various microbial groups in a differentmanner. Spares may be formed exogeneously as for instance, in actinomycetes, andendogeneously--in bacteria (sporeformers). Both the manner in which they are formedand the properties of spores in various specimens vary.

   In sporeforming bacteria endogenous spores areformed in the following way. As was indicated above, at first a chromatin substancein the form of a distinct body appears inside mature cells. This body is regardedas a rudimental spore. Around it, the protoplasm concentrates into a large roundformation, the prospore. The prospore soon becomes dense, decreases in volume, thecontour 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, Inthis way, the prospore matures and is transformed into a spore.

   Upon formation of the spore the chromatin bodydisappears; it is, not found, in the prospore but in the mature spore it appearsonce 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 stainingproperties. The plasma which is concentrated around the chromatin body, stains moreintensely than normal protoplasm; the prospore stains most densely of all. The latteris also well seen without staining. Due to its great refraction of light under themicroscope, the content of the prospore appears to be glistening. Mature spores losethe ability to stain. Only after treatment with a weak solution of hydrochloric acidat 60°C does the plasma of the spore take on stain. The stained spores are decolorizedby acid more slowly than vegetative cells. The method of differentiation and recognitionof spores is based on this fact. The inability of the mature spore to stain by usualmethods is ascribed to the relative lack of permeability of the membrane. However,analysis shows that isolated membranes stain well, but the protoplasm of the sporeitself does not stain. Consequently, the plasma of the cell undergoes essential changesduring the process of spore formation. The plasma of mature spores has physicochemicalproperties which differ from those of the plasma of vegetative cells, although thecomposition 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 groupsare not destroyed upon heating.

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

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

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

   The factors which condition spore formation arenot 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 thanin a deficient medium. Neither aeration, nor temperature nor other factors constituteby themselves a direct cause of spore formation; they only create conditions affectingthe given process.

   The process of spore formation in bacteria issubject to the same rules, as those which are noted for spore formation or offspringproduction 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 biologicaladaptation of bacteria for preserving the species.

   We assume that the biological essence of sporeformation consists not solely in the preservation of the species but it has perhapsanother 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 unfavorableconditions. According to our observations, it does not occur in bacteria of the temperatezone 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 manyantibiotics and some chemicals.

   Neither does spore formation occur under manynatural 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 timewhen spores have not yet been formed, the latter do not appear. We did not succeedin obtaining spores in soil (podsol), from Bac. mycoides, Bac. mesentericusand Bac. megatherium.

   Under conditions of the Far North. on the islandsof the Northern Arctic Ocean, (islands of Franz-Josef, Severnaya Zemlya and others)sporeforming bacteria even lose the ability to form spores. In our investigationsand those of Sushkina and Ryzhkova (1955) the majority of these bacteria neitherform spores in artificial nutrient media nor in the soil itself. We tested many mediaand grew cultures under various conditions; but the majority of them did not formspores. Only some, under particular growth conditions at an increased temperature(36-38°C). started spore formation. but they were not many. In some organismsthe process is incomplete, only prospores being formed, i.e. , not fully mature spores,without membranes. Spores, as shown by daily observations, are not particularly resistantto unfavorable environmental factors. They often die with the same rapidity as vegetativecells. For instance, we observed their death simultaneously with that of vegetativecells under the action of some chemical antiseptics. They are not always resistantto high temperature. There are species whose spores die, like the vegetative cells,at a temperature of 80-100°C. Frequently, nonsporeforming bacteria occur, whichare as resistant or even more so, to unfavorable factors, than species of sporeformingbacteria.

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

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

   Some authors (Sorokin, 1890; Gibson, 1935; Starkey.1938; Rubenchik. 1953) indicate that the described formation of endogenous sporesin 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 ofspirilla whose external form resembles spores in sporeforming bacteria. For theirformation the whole protoplast or a considerable part of it in used. Sometimes twoor three such sporelike bodies are formed simultaneously in the cell. We observedthe formation of similar bodies in Spirillum voluntans and other bacteria(Krasil'nikov, 1949 B), by placing them in an artificial synthetic medium or in adrop of the same water as that from which they were obtained. The same could be observedin a hanging drop of this medium. The motility of the spirillum slows down aftersome time the protoplast begins fragmentation into separate parts which become roundand assume the forms and sizes of large spores. The number of such sporelike bodiesvaries 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 formationsare not like the true spores of sporeforming bacteria. They represent fragments ofprotoplasts, divided into parts as a result of these or other causes. Their formationrather resembles fragmentation of the filaments of mycelium in actinomycetes, mycobacteriaand in some filamentous and other bacteria.

   In actinomycetes, as will be shown further, sporeformation proceeds in two ways--endogenously and exogenously. Spores or reproductiveelements are formed on special branches of the aerial mycelium. and inside vegetativefilaments of the substrate mycelium. They are formed by segmentation or fragmentation.

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

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




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