[Show abstract][Hide abstract] ABSTRACT: We isolated 50 strains of free-living ultrasmall bacteria with a cell volume that varies from 0.02 to 1.3 μm3 from a range of natural biotopes, namely permafrost soils, oil slime, soils, lake silt, thermal swamp moss, and the skin integuments of the clawed frog, Xenopus laevis. Of them, 23 isolates, characterized by a cell size of less than 0.1 μm3 and a genome size from 1.5 to 2.4 Mb, were subsumed to ultramicrobacteria belonging to different philogenetic groups (Alphaproteobacteria, Bacteroidetes, Actinobacteria) and genera (Kaistia, Chryseobacterium, Microbacterium, Leucobacter, Leifsonia, and Agrococcus) of the Bacteria domain. They are free-living mesophilic heterotrophic aerobic bacteria. The representatives of Kaistia and Chryseobacterium genera were capable of facultative parasitism on other species of chemo-organotrophic bacteria and cyanobacteria. The ultramicrobacteria differed in their morpholgy, cell ultrastructural organization, and physiological and biochemical features. According to the fine structure of their cell walls, the isolates were subdivided into two groups, namely Gram-positive and Gram-negative forms.
Prikladnaia biokhimiia i mikrobiologiia 03/2015; 51(2):151-160. DOI:10.7868/S055510991502021X
[Show abstract][Hide abstract] ABSTRACT: The number of spores formed in a single cell of Anaerobacter polyendosporus PS-1(T) is significantly influenced by the composition of nutrient media. Depending on carbohydrate concentration in synthetic medium, the number of spores may vary from one or two to as many as five to seven. Investigation of spore formation process by fluorescence and electron microscopy revealed that on media with 0.5-1.0% glucose or galactose most of vegetative cells remained rod-shaped after cessation of cell division in the culture. The nucleoids of these cells were localized at cell poles close to the polar site of the cytoplasmic membrane. Fore-spores were formed at one or both of these poles. A satellite nucleoid (operator) was observed close to each forespore. In the variant with bipolar organization of mother cells, only one or two spores per cell were formed. In the second variant of culture development, when the cells were grown at low galactose concentrations (0.1-0.3%), most of vegetative cells increased in volume and became oval or spherical after cessation of cell division in the culture. Epifluorescence microscopy with nucleic acid-specific fluorochromes (DAPI and acridine orange) revealed the presence of multiple (six to nine) nucleoids in these cells. The nucleoids were located at the cell periphery in close contact with the cytoplasmic membrane. These nucleoids became the centers (poles) for forespore formation. Thus, in the early stationary phase transversion from bipolar to multipolar cells occurred. Cessation of cell division combined with continuing replication of the nucleoids resulted in formation on multinuclear cells. The multiplicity of nucleoides and multipolarity of these cells were prerequisites determining endogenous polysporogenesis, occurring as synchronous formation of three to seven twin spores in many of the oval and spherical cells.
[Show abstract][Hide abstract] ABSTRACT: Ultramicrobacteria (UMB) are species of the domain Bacteria characterized by very small sizes of
proliferating cells (less than 0.1 μm3 in volume) and small genomes (3.2 to 0.58 Mb). Some authors use the
term nanobacteria as a synonym of UMB. Several tens of UMB species have been isolated from various nat�
ural habitats: sea water, soil, silt, Greenland ice sheet, permafrost soils, and intestines of humans and insects.
Under laboratory conditions, they are cultivated on different nutrient media. In the second prokaryotic
domain, the Archaea, ultrasmall forms (ultramicroarchaea) have also been described, including nanoarchaea
(members of the genus Nanoarchaeum) with a cell volume of less than 0.1 μm3. The term nanobacteria is used
in the literature also to denote ultrasmall bacterium�like particles occurring in rocks, sands, soils, deep sub�
surface layers, meteorites, and clinical samples. The systematic position and the capacity for self�reproduc�
tion of these particles are still unclear. The cultured UMB forms are characterized by highly diverse morphol�
ogy, ultrastructural organization, physiology, biochemistry, and ecology. UMB form three groups according
to the type of cell wall structure and the reaction to Gram staining: (1) gram�negative, (2) gram�positive, and
(3) cell wall�lacking. Their cells divide by constriction, septation, or budding. The unique processes per�
formed by UMB are dehalorespiration and obligate or facultative epibiotic parasitism. The UMB that syn�
thesize organic compounds in ocean waters with the involvement of proteorhodopsin play a great role in the
biosphere. UMB have been found in seven large phylogenetic groups of prokaryotes, where their closest rel�
atives are organisms with larger cells typical of bacteria, which is evidence of the polyphyletic origin of the
currently known UMB species and the reductive mode of their evolution.
[Show abstract][Hide abstract] ABSTRACT: Two strains, NF4 and NF5, of a yellow-colored gram-negative bacterium were isolated from sediments of Lake Baikal and from
old oil sludge of the Nizhnekamsk oil-processing plant. The cells of the strains are ultrasmall coccoids or short rods, measuring
0.2–0.4 × 0.2–0.5 μm; the average cell volume ranges from 0.004 to 0.04 μm3. A considerable proportion (30–60%) of cells have nanometer dimensions (180–300 nm in diameter and 0.004–0.02 μm3 in volume). The new isolates are thus among the smallest representatives of presently known free-living ultramicrobacteria.
The two studied isolates are gram-negative nonmotile cells possessing a pronounced outer membrane. The cells do not have flagella
and are not capable of gliding motility. They divide by constriction, budding, and multiple septation. The multiplicity of
reproduction mechanisms results in a high degree of cell polymorphism. The isolates are chemoorganotrophic, aerobic, psychrotolerant,
oxidase- and catalase-positive. Their characteristic trait is the absence of extracellular hydrolytic enzymes, such as proteases,
lipases, pectinases, and cellulases. Menaquinone MK-6 is the main respiratory quinone; the flexirubin pigment was not detected.
The G + C contents of the DNA of strains NF4 and NF5 are 40.8 and 40.5 mol %, respectively. The DNA-DNA hybridization level
of strains NF4 and NF5 was close to 100%. Analysis of the 16S rRNA gene sequences and the fatty acid compositions showed that
the isolates are most closely related to certain representatives of the genus Chryseobacterium (C. solincola, C. antarcticum, and C. jeonii). However, the differences in the 16S rRNA gene sequences, as well as in the phenotypic properties, such as formation of
ultrasmall cells, the absence of extracellular hydrolases, oligotrophy, and the capacity for epibiosis on bacterial cells,
suggest that the studied strains belong to a new species of the genus Chryseobacterium. The capacity for epibiosis, i.e., the ability to exist in a tightly adhered state on the surfaces of host Bacillus subtilis cells, is a peculiar trait of the studied isolates. It is assumed that adhesion of the cells of strains NF4 and NF5 (members
of the phylum Bacteroidetes) occurs via by the same unique mechanism as the mechanism that we previously described for representatives of Alphaproteobacteria (Kaistia sp., NF1, and NF3), which use polysaccharide chains equipped with sticky granules as trapping and constricting cords.
[Show abstract][Hide abstract] ABSTRACT: The morphology of microbial cells was studied, and the biomass of microorganisms was estimated in the modern steppe soils
and paleosols buried under kurgans in the Lower Volga region with the methods of electron microscopy. The shape and ultrastructure
of the cells in the modern soils and paleosols were similar, though their average volumes differed (0.37 and 0.28 μm3, respectively). The portion of cells with a volume above 1 μm3 in the surface soils and paleosols reached 10.9 and 9.2%, respectively, and the portion of cells with a volume less than
0.01 μm3 in the surface soils was 10% lower than that in the buried paleosols. It was found that the cells of the microorganisms have
an external organomineral layer, which increases the cell volume by 4.9 times, and this fact was taken into account in the
calculation of the microbial biomass. In the chestnut and light chestnut paleosols, the latter comprised 1500 and 230 μg of
C/g soil, respectively.
[Show abstract][Hide abstract] ABSTRACT: The non-spore-forming gram-positive bacterium Mycobacterium smegmatis mc2 155, related to M. tuberculosis, was revealed to be capable of forming different types of dormant forms (DFs) during the life cycle of its cultures. The
relationship between the intraspecies diversity of DFs and the cultivation conditions of the mycobacterium was established.
The DFs possessed the following common properties: (i) maintenance of viability for a long period of time (5 months), (ii)
resistance to deleterious factors such as heat treatment, and (iii) morphological and ultrastructural peculiarities that distinguish
DFs from vegetative cells. The diversity of M. smegmatis DFs manifested itself in differences in terms of structural organization, conditions required for growth renewal, and capacity
to produce antibiotic-resistant variants upon germination on selective media. Well-differentiated cystlike dormant cells (CDCs)
were formed in the cultures grown in synthetic SR1 medium with fivefold-decreased nitrogen content. The structural organization
of CDCs differed from that of other DF types mainly in the presence of club-shaped cells, thickened lamellar cell walls, coarse
cytoplasm texture, and large electron-transparent triacylglyceride inclusion bodies. It was possible to use mycobacterial
CDCs as a source of PCR-competent DNA. CDC populations were heterogeneous in cell buoyant density, and the individual fractions,
which we isolated, were found to differ in thermal stability and the ability to revert to growth under standard conditions.
Coccoid DFs, which retained their colony-forming capacity for a long time but were less heat-resistant than the CDCs, were
formed by mycobacteria grown in standard Sauton’s medium with initial pH value decreased to 6.2. Poorly differentiated DFs
resulted from growing mycobacterial cultures in Sauton’s medium with a fivefold-decreased phosphorus content. Upon germination
of various DF types, the variants resistant to kanamycin (200 μg/ml) and tetracycline (20 μg/ml) were obtained. CDC suspensions
incubated for 5 months demonstrated the highest percentage (1.5%) of antibiotic-resistant clones. The data obtained on the
DF diversity of M. smegmatis, a fast-growing relative of M. tuberculosis, contribute to our understanding of the flexibility of the survival strategy of this bacterium in nature and in the host
Key wordsmycobacteria-dormancy-diverse morphological types of dormant forms-cystlike dormant cells-persistence-stress resistance
[Show abstract][Hide abstract] ABSTRACT: The colony-forming ability of long (3–9 months) incubated cystlike resting cells (CRC) of the nonspore-forming gram-positive
bacteria Micrococcus luteus and Arthrobacter globiformis was studied in this work. The preservation of the CRC proliferative potential as assayed by plating on standard LB agar was
shown to depend on the conditions of the formation of the dormant cells. In aged post-stationary cultures of micrococci and
arthrobacters grown under carbon and phosphorus limitation the number of colony-forming units (CFU/ml) of CRC decreased in
the course of 3–9 month incubation to the level of 106–107 CFU/ml. However, M. luteus CRC obtained under carbon and nitrogen limitation and A. globiformis CRC obtained under nitrogen limitation and starvation completely lost their ability to form colonies on standard solid medium
after 4–6 months of incubation and turned into a ‘non-culturable’ (non-platable) state. In this case, the ratio of live cells
in the population of M. luteus and A. globiformis ‘non-culturable’ CRCs (determined by the Live/Dead staining test) was 10–44% of the total cell number. To study the possible
preservation of proliferative potential in non-platable CRCs, various methods of their reactivation were applied. Although
preincubation of CRC suspensions in a buffer solution of 0.1 M K2HPO4 (pH 7.4) or in the presence of lysozyme (1 or 10 μg/ml) resulted in increased numbers of live cells (determined by the Live/Dead
test) or in disruption of the cell conglomerates, it did not increase considerably the CFU titer on LB medium. Variations
in the medium composition, such as addition of sodium pyruvate as an antioxidant or dilution of the medium, promoted the formation
of macrocolonies by a small portion of nonplateable CRC of M. luteus (50−80 CFU/ml), whereas the number of the cells capable of microcolony formation (mCFU) was 1.8–6.8 × 105 mCFU/ml, exceeding the CFU titers by four orders of magnitude. The application of semisolid agar and the most probable number
(MPN) method was the most efficient for determination of the mCFU titer, and an almost complete reversion of ‘non-culturable’
micrococcal CRCs to microcolony formation was observed (up to 2.3 × 107 mCFU/ml). The usefulness of diluted complete media for the restoration of the colony-forming ability of the dormant forms
was confirmed in experiments with ‘nonculturable’ CRCs of A. globiformis. The development of special procedures and methods for determining actively proliferating cells not detected by ordinary
methods is of great importance for advanced monitoring studies.
[Show abstract][Hide abstract] ABSTRACT: Differences in generation of dormant forms (DF) were revealed between two strains of non-sporeforming gram-negative bacteria
Azospirillum brasilense, Sp7 (non-endophytic) and Sp245 (endophytic strain). In post-stationary ageing bacterial cultures grown in a synthetic medium
with a fivefold decreased initial nitrogen content, strain Sp7 formed two types of cyst-like resting cells (CRC). Strain Sp245
did not form such types of DF under the same conditions. CRC of the first type were formed in strain Sp245 only under phosphorus
deficiency (C > P). The endophytic strain was also shown to form structurally differentiated cells under complete starvation,
i.e. at a transfer of early stationary cultures, grown in the media with C > N unbalance, to saline solution (pH 7.2). These
DF had a complex structure similar to that of azotobacter cysts. The CRC, which are generated by both azospirilla strains
and belong to distinct morphological types, possessed the following major features: absence of division; specific ultrastructural
organization; long-term maintenance of viability (for 4 months and more); higher heat resistance (50–60°C, 10 min) as compared
with vegetative cells, i.e. the important criteria for dormant prokaryotic forms. However, CRC of non-endophytic strain Sp7
had higher heat resistance (50, 55, 60°C). The viability maintenance and the portion of heat-resistant cells depended on the
conditions of maturation and storage of CRC populations. Long-term storage (for 4 months and more) of azospirilla DF populations
at −20°C was optimal for maintenance of their colony-forming ability (57% of the CFU number in stationary cultures), whereas
the largest percentage of heat-resistant cells was in CRC suspensions incubated in a spent culture medium (but not in saline
solution) at room temperature. The data on the intraspecies diversity of azospirilla DF demonstrate the relation between certain
type DF formation to the type of interaction (non-endophytic or endophytic) with the plant partner and provide more insight
into the adaptation mechanisms that ensure the survival of gram-negative non-spore-forming bacteria in nature.
[Show abstract][Hide abstract] ABSTRACT: The phenotypic characteristics of the species Sulfobacillus thermotolerans Kr1T, as dependent on the cultivation conditions, are described in detail. High growth rates (0.22–0.30 h−1) and high oxidative activity were recorded under optimum mixotrophic conditions at 40 °C on medium with inorganic (Fe(II),
S0, or pyrite-arsenopyrite concentrate) and organic (glucose and/or yeast extract) substrates. In cells grown under optimum
conditions on medium with iron, hemes a, b, and, most probably, c were present, indicating the presence of the corresponding cytochromes. Peculiar extended structures in the form of cylindrical
cords, never observed previously, were revealed; a mucous matrix, likely of polysaccharide nature, occurred around the cells.
In the cells of sulfobacilli grown litho-, organo-, and mixotrophically at 40 °C, the enzymes of the three main pathways of
carbon utilization and some enzymes of the TCA cycle were revealed. The enzyme activity was maximum under mixotrophic growth
conditions. The growth rate in the regions of limiting temperatures (55 °C and 12–14 °C) decreased two-and tenfold, respectively;
no activity of 6-phosphogluconate dehydrogenase, one of the key enzymes of the oxidative pentose phosphate pathway, could
be revealed; and a decrease in the activity of almost all enzymes of glucose metabolism and of the TCA cycle was observed.
The rate of 14CO2 fixation by cells under auto-, mixo-, and heterotrophic conditions constituted 31.8, 23.3, and 10.3 nmol/(h mg protein),
respectively. The activities of RuBP carboxylase (it peaked during lithotrophic growth) and of carboxylases of heterotrophic
carbon dioxide fixation were recorded. The physiological and biochemical peculiarities of the thermotolerant bacillus are
compared versus moderately thermophilic sulfobacilli.
[Show abstract][Hide abstract] ABSTRACT: Cystlike resting cells (CRC) of non-spore-forming gram-negative bacteria of the genus Pseudomonas, P. aurantiaca and P. fluorescens, were obtained and characterized for the first time; their physiological and morphological diversity was demonstrated. The
following properties were common for all the revealed types of CRC as dormant forms: (1) long-term (up to 6 months or longer)
maintenance of viability in the absence of culture growth and cell respiration; (2) absence of an experimentally detectable
level of metabolism; (3) higher resistance to damage and autolysis under the action of provoking factors than in metabolically
active vegetative cells; and (4) specific features of ultrastructural organization absent in vegetative cells: thickened and
lamellar envelopes, clumpy structure of the cytoplasm, and condensed DNA in nucleoid. The differences in various types of
CRC concern the thickness and lamellar structure of cell envelopes, as well as the presence and thickness of the capsular
layer. In particular, forms ultrastructurally similar to typical bacterial cysts were revealed in pseudomonad populations
growing on soil agar. Physiological diversity was revealed in different levels of viability preservation and thermal resistance
in various types of CRC and depended on the conditions of their formation. The optimal conditions and procedures for obtaining
P. aurantiaca and P. fluorescens CRC that retain the ability to form colonies on standard nutrient media are as follows: (1) a twofold decrease of nitrogen
content in the growth medium; (2) an increased level of anabiosis autoinducer (C12-AHB, 10−4 M) in stationary cultures; (3) transfer of the cells from stationary cultures to a starvation medium with silica; (4) cultivation
in soil extract; and (5) development of cultures on soil agar. The CRC from the cultures grown in soil extract or starvation
medium with silica proved to be resistant to heat treatment (60°C, 5 min). In the CRC formed in nitrogen-limited media, the
degree of heat resistance increased at longer incubation (1.5 to 6 months). CRCs on soil agar surface were resistant to desiccation.
The ultrastructure of the morphologically varied types of P. aurantiaca CRC formed under simulated natural conditions is described for the first time. The data on the intraspecies diversity of
pseudomonad dormant forms contribute to the concept of plasticity of the life style and adaptive reactions that ensure survival
of these bacteria in unfavorable environmental conditions.
[Show abstract][Hide abstract] ABSTRACT: Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gram-negative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated, but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.
[Show abstract][Hide abstract] ABSTRACT: The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1-3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29-43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of < or =0.3 microm and volume of < or =0.014 microm3) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18-0.20 x 0.20-0.30 microm in size, forming spherical aggregates (microcolonies) of dividing cells in situ. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.
[Show abstract][Hide abstract] ABSTRACT: Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development
cycle, the strains formed small coccoid cells of 400–800 nm and ultrasmall cells of 200–300 nm. Phylogenetically, the strains
NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that
the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated.
The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm.
The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB; these include
the following: (a) piles of rod-like subunits, ca. 30 Å in diameter and 150–250 Å in length; (b) long bunches (up to 300–400
Å) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200–300 Å in diameter) localized
in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial
(CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on
the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB cytoplasm.
UMB cells have been shown to reproduce by budding, with buds (up to 2–3) located directly on the mother cell, without formation
of intermediate hyphae.
[Show abstract][Hide abstract] ABSTRACT: The changes in the state of Bacillus subtilis spores that occur during germination were analyzed using dynamic phase microscopy (DPM). DPM is based on monitoring and analyzing
the interference image of a specimen in a coherent laser beam. The optical path difference (the phase thickness of the specimen,
PT) depends on the geometrical height of the specimen and its refractive index. We demonstrated that the maximum PT value
is a convenient criterion of the physiological state of the organism involved: PT is ≥ 80 nm, ∼40–50 nm, and ≤ 20 in dormant,
developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40°C results
in a reversible twofold decrease (from 80 to 40 nm) in their PT under conditions that do not promote the development of the
bacteria; this decrease is irreversible under growth-promoting conditions; (ii) the PT values of germinating spores oscillate
with a considerable fluctuation amplitude (up to 7 nm), in contrast to the limited fluctuation amplitude (within 1 nm) in
dormant spores; (iii) activated spores were heterogenous with respect to the PT pattern: a majority of the spores exhibited
a usual spatial profile (with a maximum thickness in the center), whereas a minor fraction of them were characterized by an
erythrocyte-like profile with a concave center; this implies that the central zone of the spore was more rapidly hydrated
(with a decrease in refractive index) than the peripheral zone.
[Show abstract][Hide abstract] ABSTRACT: The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.
[Show abstract][Hide abstract] ABSTRACT: The changes in the state of Bacillus subtilis spores that occur during germination were analyzed using dynamic phase microscopy (DPM). DPM is based on monitoring and analyzing the interference image of a specimen in a coherent laser beam. The optical path difference (the phase thickness of the specimen, PT) depends on the geometrical height of the specimen and its refractive index. We demonstrated that the maximum PT value is a convenient criterion of the physiological state of the organism involved: PT is > or = 80 nm, 40-50 nm, and < or = 0 in dormant, developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40 degrees C results in a reversible twofold decrease (from 80 to 40 nm) in their PT under conditions that do not promote the development of the bacteria; this decrease is irreversible under growth-promoting conditions; (ii) the PT values of germinating spores oscillate with a considerable fluctuation amplitude (up to 7 nm), in contrast to the limited fluctuation amplitude (within 1 nm) in dormant spores; (iii) activated spores were heterogenous with respect to the PT pattern: a majority of the spores exhibited a usual spatial profile (with a maximum thickness in the center), whereas a minor fraction of them were characterized by an erythrocyte-like profile with a concave center; this implies that the central zone of the spore was more rapidly hydrated (with a decrease in refractive index) than the peripheral zone.
[Show abstract][Hide abstract] ABSTRACT: One of the challenging tasks in monitoring studies is to estimate heterogeneity of microbial populations by the physiological state and potential viability of individual cells, especially with regard of their ability to withstand various environmental assaults. Previously, we described some approaches based on electron microscopy methods to discriminate vegetative, dormant, and dead cells in both aged microbial cultures and environmental samples, including permafrost. We propose to extend the arsenal of microscopy methods for monitoring studies by a new non-invasive and informative method – dynamic phase microscopy (DPM). The substantial advantage of DPM is that it gives quantitative (digitized) data of undestroyed (living) microscopic objects, exemplified in our work by Bacillus licheniformis spores. Using DPM made it possible to record interference images of objects (spores) and to produce picture of their “phase thickness” (PT) that is the optical path difference in nm. Thus, it was demonstrated the remarkable difference in the PT of spores at different physiological states: dormant, germinating, and heat-killed spores had PT values of 80, 40–50, and 20 nm, respectively. The other found criterion to distinguish between spores was the PT fluctuations. In contrast to dormant and killed spores, the PT of germinating spores oscillated with amplitude of up to 7 nm, with typical frequencies of 1.3 and 3.4 Hz. A combination of the recorded PT values and PT fluctuations gave a key to detect viable and dead cells. Under the conditions that did not support germination (the lack of nutrients), we were able to follow the response of a single dormant spore and a spore population to heating from 25 °C to 70 °C. Thus, a very small temperature change (from 40 °C to 42 °C) under conditions non-favorable for germination, caused a drastic decrease in the spores’ PT; the second drop in the PT values was observed during heating from 60 °C to 70 °C. These changes were reversible: after cessation of heating, PT values became similar to dormant spores. So, DPM allowed us to track the first, reversible stage of activation, when a spore maintains the attributes of the dormant state. Under the conditions that favor germination (in the presence of nutrients), irreversible changes in the PT and spore diameter, d, were detectable in a single germinating spore and spore population. In addition, DPM allowed an easy estimation of the heterogeneity of spore populations. It is a great advantage of DPM that it makes possible to reveal the ability of spores to respond to various stimuli with or without further germination and outgrowth – the salient feature of a living cell. DPM may have a high potential in general microbiology and astrobiology, enabling to: (1) estimate the heterogeneity of spore populations either under standard conditions and subjected to solar radiation and simulated extraterrestrial factors; (2) to track a response of spores to changing conditions at the early germination stage, even if they do not enter further outgrowth; (3) to develop some approaches for monitoring studies and appraisal of the physiological state of dormant cells in situ, in samples of dry soils, permafrost, etc. regarded as models for searching life beyond the Earth.
Advances in Space Research 01/2007; 40(11-40):1678-1685. DOI:10.1016/j.asr.2007.03.090 · 1.35 Impact Factor