Possibilities of transmission electron microscopy in assessing the quality of bacterial cells and microbial population.

Determination of changes in the morphostructure of microorganisms under the influence of antibiotics, disinfectants, biocins, physical and chemical agents is carried out using the method of visualization of ultrathin sections in a transmission electron microscope. The aim is to develop methodological techniques for express preparation and visualization of the ultrastructure of bacteria in a transmission electron microscope to detect structural damage to cells and assess the quality of the microbial population. A technique of accelerated sample preparation for visualization of the fine structure of bacteria and diagnosis of structural damage to cells in a transmission electron microscope has been developed. The use of this method will make it possible to more effectively investigate the mechanisms of inactivation of microorganisms at different stages of contact with antibiotics, disinfectants, biocines, including various stressful, damaging and lethal physical and chemical effects.

Peculiarities of the ultrastructure of mixed biofilms of the causing agent of diphtheria and conditionally pathogenic microorganisms isolated from the human respiratory tract.

In the post-epidemic period, the circulation of the causative agent of diphtheria in the population is maintained due to the carrier of bacteria. Entering an organism with a high level of antitoxic immunity, the pathogen enters into intermicrobial interactions with representatives of the opportunistic microflora inhabiting the respiratory tract and forms a biofilm. MATERIALS AND METHODS: Modeling of the biofilm formation process was carried out using the strains C.diphtheriae gravis tox+№. 665, C.pseudodiphtheriticum, S.aureus. Biofilm samples were placed on the stage of a scanning electron microscope and gold-sputtered in an EicoIB-3 ioncoater vacuum deposition unit (Eico, Japan) at an ion current of 6-8 mA. The samples obtained were examined in a JEOL 6510LB scanning electronmicroscope. («JEOL¼ company, Japan) at an accelerating voltage of 30 kV. RESULTS: Electron microscopic examination of samples of biofilms C. diphtheriae gravis tox+ № 665 and opportunistic microorganisms shows groups of 2-7 young bacterial cells packed into a single microcapsule. Much more voluminous accumulations of bacterial cells (more than 10-12) are typical for biofilm samples represented by C. diphtheriae gravis tox+№ 665 and S. aureus cells. On the surface of the biofilm, young bacterial cells with an intact structure are located at various stages of active division. The conglomerates of bacterial cells, covered with a common intermicrobial matrix, adhere tightly to each other and form a multilayer biofilm. CONCLUSION: Features of the ultrastructure of biofilms containing strains of C. diphtheriae and opportunistic bacteria, especially antibiotic-resistant bacteria inhabiting the respiratory tract, can contribute to long-term persistence of the pathogen of diphtheria in the body. They not only significantly complicate the access of antibacterial drugs, but also interfere with the isolation of C.diphtheriae during bacteriological research.

[Features of biofilm morphology, formed by Corynebacterium diphtheriae gravis tox+ strains].

AIM: Study the structure of homogenous microbial communities of Corynebacterium diphtheriae gravis tox+ strains during formation of biofilms in vitro. MATERIALS AND METHODS: Object of study--typical and biofilm cultures of C. diphtheriae gravis tox+ museum and circulating strains. Intensity of biofilm formation was evaluated by OD on microplate reader at 540 nm wave length studying 120 and 720 hour cultures. S-450 (Hitachi, Japan) scanning electron microscope was used. RESULTS: The peak of exopolysaccharide matrix (EPS) formation, that is formed in the process of biofilm formation, by museum strain takes place at earlier terms of cultivation (120 hours) than circulating (720 hours). An inverse correlation was established during analysis of bacterial cells of museum and circulating strains of C. diphtheriae during biofilm formation between them and intensity of EPS formation. At maximum EPS content, that took place at various terms of cultivation of the 2 studied strains of diphtheria causative agent, a reduction of corynebacteria cells was observed. CONCLUSION: Bacterial biofilms of museum and circulating strains of C. diphtheriae and patterns of dynamics of EPS reflect, probably, adaptive abilities of the causative agent, that determine its competitiveness in the fight for adhesion sites, resistance to factors of natural immunity and as a result--prolonged persistence in the organism of bacterial carriers.

[Pancreatic tissue heterotopy in the stomach of a patient with complications due to peptic ulcerative disease].

In the literature there are reports on rare pancreatic tissue heterotopy that promotes the complicated course of duodenal or gastric ulcerative disease. In this connection, the authors have decided to share their observation of a 41-year-old female patient admitted to a hospital for perforated ulcer and ulcerous hemorrhage. Another ulcerative stenosis and ulcer penetration into the pancreas were found at surgery. Partial gastrectomy was carried out due to failure to suture the perforative opening. A gross biopsy specimen from the pyloric portion of the stomach displayed heterotopic parts in the pancreas with and without excretory ducts.

[Disinfectants and main sanitary and preventive measures for protection of ventilation and air-conditioning systems from Legionella contamination].

The study was devoted to selection and assessment of disinfecting preparations for prevention of contamination by Legionella. Using system of criteria for quality assessment of disinfectants, seven newdomestic ones belonging to quaternary ammonium compounds class or to oxygen-containing preparations and designed for disinfecting of air-conditioning and ventilation systems were selected. Antibacterial and disinfecting activities of working solutions of disinfectants were tested in laboratory on the test-surfaces and test-objects of premises' air-conditioning and ventilation systems contaminated with Legionella. High antimicrobial and disinfecting activity of new preparations "Dezactiv-M", "ExtraDez", "Emital-Garant", "Aquasept Plus", "Samarovka", "Freesept", and "Ecobreeze Oxy" during their exposure on objects and materials contaminated with Legionella was shown. Main sanitary and preventive measures for defending of air-conditioning and ventilation systems from contamination by Legionella species were presented.

[Influence of cultivation time and composition of growth medium on formation of capsule, quantity and immunological properties of Pasteurella multocida capsular antigen].

Results of study of capsule formation and immunologic properties of capsular antigen of attenuated strain LA-25 of Pasteurella multocida grown on liquid medium from non-food materials and Hottinger's broth depending on cultivation time are presented.

[Specific features of minute structure of Mycobacteria cultured in murine macrophages]. / Osobennosti tonkoi struktury mikobakterii tuberkuleza, kul'tivirovannykh v makrofagakh myshi.

In experimentally infected murine peritoneal macrophages and murine macrophage-like cells J-774 with different pathogen strains of tuberculos'is, Mycobacterium tuberculosis (MBT) underwent significant morphofunctional changes. In phagocytosis, several live and mycobacteria conventionally referred by the authors to as morphotype I cells come from the environment to the macrophage. Of them, single young and intact mycobacteria are able to multiply and form at 2-3 generations morphotype II microcolonies from 3-9 mycobacteria or more in the phasolysosomes within the first 24 hours after infection. Having taken the form of small-sized cocci and coccoovals having a closely packed cytoplasm, morphotype II cells can be long present intact in the phagocytes. By losing the cellular wall under the action of lytic phagolysosomal enzymes, single mycobacteria turned into L-form or morphotype III MBT. During damage and lysis in the macrophages, single mycobacteria can preserve a part of an intact cytoplasm and genome as ultraminor forms of mycobacteria or morpho-type IV MBT.

[Physicochemical and antimicrobial properties of chlorine-containing compositions ciaref and DP-1]. / Fiziko-khimicheskie i antimikrobnye svoistva khlorsoderzhashchikh kompozitsii tsiaref i DP-1.

The paper presents the results of the physicochemical properties and corrosive activity of the new chlorine-containing compositions Ciaref and DP-1. Various microbiological, biochemical, and biophysical studies were conducted to examine different aspects of the mechanism of action of Ciaref and DP-1 on vegetative (Francisella tularensis) and sporal (Bacillus turingiensis) microorganisms. Suspension, test-surface irrigation, and aerosol techniques were employed under close industrial conditions to study the disinfecting properties of the agents in bacterial and sporal contamination.

[Features of the production of CryIIIA delta-endotoxin from Bacillus thuringiensis in a recombinant strain of Pseudomonas putida under control of Pm and xylS regulatory elements]. / Osobennosti produktsii CryIIIA delta-éndotoksina Bacillus thuringiensis v rekombinantnom shtamme Pseudomonas putida pod kontrolem reguliatornykh élementov Pm i xylS.

The addition of 3-methyl benzoate to the culture of the recombinant strain Pseudomonas putida IPM-36 (bearing the cryIIIA gene of B. thuringiensis subsp, tenebrionis under the control of the Pm promoter and the regulator gene xylS) slowed down the growth rate of the recombinant strain and increased, under non-selective conditions, the number of plasmid-free cells. Intense synthesis of the Coleoptera-specific delta-endotoxin encoded by the cryIIIA gene began 6-8 h after the addition of the inducer 3-methyl benzoate, no matter whether it was added in the early or late logarithmic phase. Maximal production of endotoxin (0.5-0.6 g/l) was observed when the inducer was added in the early logarithmic phase (3 h of growth). Overproduction of delta-endotoxin impaired cell division, so that filamentous cells became predominant in the culture. delta-Endotoxin accumulated in overproducing cells as irregular crystalloid inclusions.

[Morphology, ultrastructure and populational features of bacteria francisella]. / Morfologiia, ul'trastruktura i populiatsionnye osobennosti bakterií roda Francisella.

The morphology, ultrastructure of cells and the structure of microbial populations of various bacteria of the Francisella genus were estimated by electron microscopy. The strain 503 has been found to produce a bacterial population that is most homogeneous in shape and size. It contains microbes of only round and avoid forms, 0.5-0.6 micron in size. In addition of oval and round microbes there are ellipsoid and rod-shaped ones in the strains 15/3M, A. Cole 120, 117, etc. The largest tularemia microbes are typical of the strain Schu. The bacteria of all strains are covered by a capsule-like coat with well-defined borders. A thick capsule (0.12-0.35 micron) is specific for virulent strains whereas a thin capsular coat (0.06-0.12 micron) is encountered in vaccinal and avirulent microbes. The cells of the strain 503 were also shown to have the thickest envelope. All tularemia microbes have an asymmetric structure in the outer and cytoplasmic membranes due to the location of the bulk of intramembrane particles on their inner hydrophobic surfaces. Some F. tularensis microbes are able to produce keel-like protrusions on the outer membrane. The microbial nucleotide occupies 55-65% of the cytoplasmic volume and forms about 20-30 DNA-membrane contacts. Under unfavourable conditions, the microbes are capable of producing cell envelop protrusions and involutional cells, 0.1-0.3 micron in size.

[Biochemical, antigenic and protective properties of the outer membrane of tularemia pathogens]. / Izuchenie biokhimicheskikh, antigennykh i protektivnykh svoistv vneshnei membrany vozbuditelia tuliaremii.

The outer membranes of Francisella tularensis were studied. The membranes were identified morphologically, immunologically and biochemically. They contained 12-20% of protein, 15-30% of carbohydrates, up to 40% of lipids. The main integral proteins of the outer membranes were the 47, 43, 17 and 12 kD proteins. The main protein 63 kD was not integral. The lipopolysaccharides isolated from the outer membranes and acetone-dried cells did not possess the protective properties in experimental tularemia. The preparations of outer membranes possessed the protective properties for mice infected with the virulent strain 503. Chitosan amplified the protective properties of outer membranes.

[An electron microscopic study of the intercellular contact of a vaccinal strain of Francisella tularensis with gram-negative and gram-positive bacteria]. / Elektronno-mikroskopicheskoe izuchenie mezhkletochnogo kontakta vaktsinnogo shtamma Francisella tularensis s gramotritsatel'nymi i grampolozhitel'nymi bakteriiami.

The use of transmission electron microscopy (the negative contrast and ultrathin section techniques) has made it possible to show that F. tularensis vaccine strain is capable, under normal conditions and in mixtures with other gram-negative and gram-positive bacteria, of forming cell aggregations with close contacts between cells, this contact being probably irreversible. The ultrastructure of bacteria taking part in the formation of intercellular contacts remains intact.

[The heterogeneity of collection strains of Yersinia pseudotuberculosis and their molecular biological properties]. / Geterogennost' muzeinykh shtammov Yersinia pseudotuberculosis i ikh molekuliarno-biologicheskie svoistva.

The capacity of Y. pseudotuberculosis strains for disassociation with the appearance of S- and P-forms has been studied. Strains 852 and 9547 show high stability in S-forms, their conversion into R-forms occurring at 40-42 degrees C. Strain 6953 shows pronounced polymorphism and instability of its associations at different growth temperatures. Strain 9532 exists in S- and R-forms which retain their stability during numerous subculturings at different growth temperatures and prolonged storage. This strain has plasmids of 130, 72.2, 5.7 kb. All plasmids are retained in S- and R-forms, i. e. the dissociation of the strain is not accompanied by the loss of plasmids. The conversion of the strain from the S-form into the R-form leads to changes in the structure of lipopolysaccharide and the composition of low-molecular (less than 23 kD) proteins in the outer and inner membranes. In tests on guinea pigs the LD50 of the R-form of the strain is tenfold greater than that of its S-form. The dissociants of strain 9532 are transformed by plasmid DNA with equal efficiency and equally inherit them without selective pressure.

[Comparative study of the morphology and sensitivity to damaging factors of capsular and acapsular variants of Escherichia coli CA 189]. / Sravnitel'noe isuchenie morfologii i chuvstvitel'nosti k povrezhdaiushchim faktoram kapsul'nogo i beskapsul'nogo variantov Escherichia coli CA 189.

E. coli strain SA 189 has been isolated from a calf with E. coli infection. The cultivation of this strain on a solid culture medium has resulted in its dissociation into two variants due to the loss of the capsule by the cells of the initial culture. Both variants of E. coli strain SA 189 (capsular and acapsular) are equally sensitive to antibiotics. The capsular variant has a narrower range of sensitivity of colicins and shows greater resistance to the bactericidal action of normal blood serum than the acapsular one. Bacteria belonging to these two variants differ perceptibly in the kinetics of disturbances in the barrier properties of their membrane apparatus under the action of cetyltrimethylammonium bromide, a cation detergent. The conditions permitting the differentiation of the capsular and acapsular forms of E. coli strain SA 189 by the kinetics of the damaging action of this detergent have been experimentally selected.

[Electron microscopic study of the action of submicroscopic levorin structures on Candida guillermondii]. / Elektronno-mikroskopicheskoe izuchenie deistviia submikroskopiches-kikh struktur levorina na Candida guilliermondii.

In the course of preparation of aqueous solutions of the polyene antibiotic levorin, the latter is recovered in the solid phase forming granular submicroscopic structures. If the cells of Candida guilliermondii are treated with submicroscopic granular structures (SMGS) of levorin, the structures are adsorbed on the surface of the yeast cell walls. Some visible changes occur in the ultrastructure of the yeast cells incubated with SMGS of levorin for 5 min: the inner layer of the cell wall becomes loose, the periplasmic space appears, the cytoplasmic membrane becomes thicker, the mitochondria swell, and fragmentation of the mitochondrial cristae takes place. Dense round alien bodies 20--40 mn in size can be discerned in the periplasmic space of such cells. If the yeast cells are treated with the levorin structures for a longer period of time (15--60 min), the cell ultrastructure is entirely disorganized.

[Fine structure of the outer sheath of the aerial mycelium in Actinomyces levoris]. / Tonkoe stroenie poverkhnostnogo chekhla vozdushnogo mitseliia Actinomyces levoris.

The outer sheath of the aerial mycelium of Actinomyces levoris 64 bears groove-like and granular submicroscopic structures. They are very susceptible to a short-term treatment of the aerial mycelium with aqueous acetone which causes their disintegration. Under specified conditions, submicroscopic structures, viz. granules and threads, assemble from the acetone extract of the aerial mycelium of Actinomyces levoris. These structures possess the anti-yeast activity. The granular structures reorganized from the acetone extract of the aerial mycelium of Actinomyces levoris somewhat resemble in morphology the structures seen in the outer sheath of the aerial mycelium of this actinomycete. The results obtained are discussed within the framework of the hypothesis on a possible participation of polyene antibiotics in the formation of the outer sheath of the aerial mycelium of actinomycetes which produce these antibiotics.