Detection method of microfungi coccidioides posadasii 36 s and coccidioides immitis c-5
SUBSTANCE: detection method of microfungi Coccidioides posadasii 36 S and Coccidioides immitis C-5 in vitro involves pre-growth of culture in mycelial phase, preparation of a suspension corresponding to 5 units of activity of a standard opacity sample, possibility of spherules formation and detection of spherules filled with endospores. Culture in mycelial phase is grown during 3 days. Possibility of spherules formation is provided by infection of the one-day culture of cells of murine splenocytes, which is obtained on RPMI-1640, and further cultivation during 5 days at the temperature of 37°C, with content of CO2 in atmosphere of 5%. In order to detect spherules in the form of round double-outline formations filled with endospores, a sample is taken, deactivated with formalin and investigated by means of a light microscopy method.
EFFECT: invention allows simplifying the method and reducing the investigation period.
The invention relates to medicine, for laboratory diagnosis of causative agents of coccidioidomycosis.
AIDS (disease Wernicke-Posada) - non-communicable systemic mycosis, which is manifested as a primary pulmonary infection, or as a progressive granulomatous lesions of the skin, joints, bones, internal organs and brain membranes. The causative agents of coccidioidomycosis, Coccidioides spp. - until recently, were represented by single species .immitis. In-depth study of the genome of fungi is possible to divide the strains into two groups - California and ncalifornia. In accordance with these data, since 2004, fungi of the genus Coccidioides are divided into two types: .immitis and .posadasii. However, to date the morphological differences among the cultures of different species is not installed.
The problem of identification of phytopathogenic fungi (microscopic fungi of importance in the development of pathological conditions, highly relevant, as it determines the further tactics of treatment and selection of effective drugs. In mycological practice, the most frequently used methods, aimed at the study of macro - and micro-morphology of the cultures. This concept of diagnosis was named morphological or phenotypic, when the determination unit type is defined is the morphological characteristics of the studied strain and comparative analysis by establishing differences with closely related species. This methodological approach is implemented by microscopic fungi cultivation under conditions that allow to obtain a culture of micromycetes with a characteristic morphology.
The causative agents of coccidioidomycosis refer to dimorphic fungi, characterized by the ability to exist in two different physiological phases and respectively in two morphological forms. Saprophyte phase represented by the filamentous form in which the causative agents of coccidioidomycosis live in the environment, and grow on nutrient media used for culturing, microscopic fungi. Strains of Coccidioides spp. within 3-5 days of incubation at 28°C on the surface of the dense nutrient agar form a grayish mold colonies. Microscopic study of the cultures found in filamentous form, revealed hyphae, consisting of a barrel-shaped of arthropod, alternating with smaller sterile dividing cells. Along with the development and maturation of the mycelium is its disintegration into smaller fragments and detached arthroconidia (4-6 µm in length) with the "scraps" cell separators in the form of antennae located at the corners. However, the formation of arthroconidia characterized not only by Coccidioides spp., but some yeast-like fungi and dermatophytes, which has a similar structure: Auxarthron spp., Oidiodendron spp. Gymnoascus spp., followed the flax morphology of filamentous forms of Coccidioides spp, is not strictly specific.
Inhalation of arthroconidia Coccidioides spp. in the tissues of the lung is the development of tissue or parasitic phase. It is represented by spherule - rounded formations from 10 to 200 microns in diameter, filled with endospore (from single to several hundred). Endospores are oval or irregular in shape. Double wall Sterol up to 2 microns in diameter. The final diagnosis of coccidioidomycosis is the identification of the morphological elements of microscopic fungus, characteristic of the parasitic phase, namely Sterol. Note that the immature spherule, i.e. undifferentiated education may not be taken into account in the absence of typical signs, such as a double shell, and the presence of endospore.
Cultivation of fungi in tissue (parasitic) phase in nutrient medium, i.e. iv vitro is difficult. There is a method of cultivation 3-4 monthly cultures of Coccidioides spp. in the medium RPMI 1640 containing 10% inactivated serum, 2,835 mg Na2PO4×7H2O per liter, of 0.8 mg N-Tamol, 40 units of penicillin and 40 mg of streptomycin per milliliter, in an atmosphere of 5% CO2at 35°C. After 48 h after initial inoculation of fungal suspension is filtered and re-seeded in fresh portion of the nutrient medium of the same composition. Full spherule formed within 8 days it is mandatory re-seeding culture in a fresh portion of the nutrient medium every 48 h of cultivation (Alan F. Petkus, Linda L. Baum, Robert B. Ellis, Malvin Stern and David L. Danley "Pure Spherules of Coccidioides immitis in continuous culture", Journal of Clinical Microbaiology, Aug., 1985, p.165-167). The presented method is time-consuming, costly and very time-consuming, making impractical their utilization for detection of microscopic fungi of the genus Coccidioides spp.
The closest analogue is the way to detect the causative agents of coccidioidomycosis in vivo. It provides for the infection of white mice particles 2-week cultures of Coccidioides spp., and observing them within 7 days. After that, kill mice, extract the liver from three animals and add 10 ml of 10%KOH solution, heated in a boiling water bath for 15 min and after dissolution bioprobes material sterilized with formalin. Then decontaminated suspension in a 1.5 ml centrifuged at 5000 rpm, the supernatant removed, and the precipitate prepare smears for light microscopy without staining and detection of Sterol judge the presence of the causative agent of coccidioidomycosis. (T.A. Grishkin, B.C. forest, E.V. Timofeeva, V.A. Spiridonov "Method for detection of the causative agent of coccidioidomycosis". RF patent №2265844, C1 G01N 33/48, 2004). A significant disadvantage of this method is that its implementation required odimo work with infected bioprobe animals as well as a longer research time for comparison with the proposed method.
The task of the invention is to devise simple and effective way of detecting microscopic fungi of the genus Coccidioides spp. in vitro.
The technical result is a reduction in terms of research and simplification of the method, due to the fact that the study does not require multiple transfers, performed in vitro without the use of biotronik animals.
The technical result is achieved by conducting a preliminary crop in the mycelial phase, the preparation of the suspension corresponding to 5 units at a time standard sample turbidity conversion in tissue culture phase and detection Sterol in the form of a rounded double-loop formations filled with endospores. The method differs in that the culture is grown for 3 days and cooked it in suspension infect the daily cell culture murine splenocytes, cultured for 5 days at 37°C, with content in the atmosphere of 5% CO2. then select a sample volume of 0.1 ml, sterilized with formalin and examined by light microscopy in drug "crushed" drop at magnification ×600.
Example 1 (optimal):
Strains of Coccidioides posadasii 36 S and Coccidioides immitis C-5 grown on the environment Saburo at 28°C for 3 days. Then the cult of the s wash 0.15 M solution of sodium chloride, pH 6.8, prepare a suspension, corresponding to the 5 UNITS of the industry standard sample turbidity gisk named after. Lytvyn (CCA) and 0.5 ml of suspension make tablets with cell cultures of splenocytes.
Cell culture murine splenocytes in the form of a monolayer prepare for the day before infection with fungal cultures. As a donor splenocytes using inbred BALB/c mice. Allocate the spleen in compliance with the rules of asepsis, homogenize it in a serum-free medium RPMI-1640 with glass cage, then twice washed cell suspension by centrifugation 1000 rpm for 10 min, resuspended sediment in 5-10 ml of the same medium. Counting splenocytes hold the camera Goryaeva, evaluate their concentration and viability using a 0.4% solution Trypanosoma blue. Seeding of splenocytes to produce tablets based 5,0-8,0×104CL/cm2. Cultivation of murine splenocytes carried out in medium RPMI-1640, pH 7.2 in the presence of 2 mM sodium bicarbonate, 20 mM Hepes, 10% ETS (Biolot"), without the addition of antibiotics, in CO2incubator (Hereus) at 37 atmosphere containing 5% CO2within 24 hours, and then infect obtained in the form of a monolayer culture of murine splenocytes by suspensions of the fungi and additionally cultivated under the same conditions for 5 days. Samples decontaminated by adding formalin at 10% of otoyama samples within 24 h, prepare native drugs "crushed" drop and examined by light microscopy (magnification ×600). The result is that in all fields of view observed spherule different degrees of maturity, the young form having a size of 5-10 microns, to Mature, the size of 60-80 microns, with a pronounced two-shell and formed by endospore.
The detection of microscopic fungi of the genus Coccidioides posadasii 36 S and Coccidioides immitis C-5 in vitro, including preliminary cultivation of rice in the mycelial phase, the preparation of the suspension corresponding to 5 units at a time standard sample turbidity, providing the possibility of formation of Sterol and detection Sterol in the form of a rounded double-loop formations filled with endospore, characterized in that the culture in the mycelial phase is grown within 3 days, moreover, providing the possibility of formation of Sterol carried out by infecting the daily cell culture murine splenocytes obtained in the medium RPMI-1640, and subsequent culturing for 5 days at 37°C content in the atmosphere of 5% CO2and to detect Sterol in the form of a rounded double-loop formations filled with endospore selected sample, sterilized with formalin and examined the sample by light microscopy.
SUBSTANCE: method involves preparation of bacterial suspension, separation of the obtained biomass of bacteria by centrifugation, dilution of the obtained biomass in physiological solution, preparation of monolayer of CaCo-2 cells, introduction of bacterial culture, cultivation of cells, flushing with physical solution, removal of monolayer with bacterial cells and counting of the amount of bacterial cells related to 1000 cells of CaCo-2; with that, bacteria refer to highly-adhesive ones if the number of bonded cells is 1010 to 3000, to average-adhesive ones of 210 to 1000, and to low-adhesive ones of 0 to 200.
EFFECT: improvement of the method.
SUBSTANCE: preparation containing biodestructor of oil contamination represents centrate of cultural liquid of microbial mass of consortium of oil-oxidising microorganisms immobilised on peat carrier. Consortium composition includes the following: Rhodococcus eqvi SRI MCC B-1115 bacteria strain, Rhodotorula glutinis SRI MCC Y-1113 yeast and Rhodotorula glutinis ARSRI MCC Y-1114 yeast strain in the ratio of 1:1:1 respectively, which have been grown jointly.
EFFECT: invention allows improving quality of soil cleaning from oil and oil products under conditions with reduced temperatures.
SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.
EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.
4 cl, 4 dwg, 3 ex
SUBSTANCE: 1% sterile solution of glucose is prepared on the basis of a physiological solution, which is used as a nutrient medium. An absorber by Zaytsev is connected to an aspirator of "Briz-1" grade, into the flask of which 10 ml of the prepared 1% glucose solution is placed. The device is placed into the investigated room, and the aspirator is switched on for 15 min. Microorganisms that are in air go through the glucose solution and are retained in it. The solution is placed into a test tube and thermostatted at 37°C for 2 hours. Solution electroconductivity is measured with the help of a sensor KDS-1038. The number of microorganisms in air is determined according to the curve of empirical dependence of solution electroconductivity on the number of microbes, which is built in accordance with the produced values.
EFFECT: invention makes it possible to reduce time for determination of microorganism number in the air of the working area down to 2 hours and 20 min.
SUBSTANCE: method to protect yeast Saccharomyces cerevisiae against oxidising stress as a result of exposure to hydrogen peroxide includes growing of yeast culture under standard conditions till the end of the logarithmic or the start of the stationary phase of growth, incubation with a protective agent, exposure to hydrogen peroxide with subsequent determination of the number of survived cells. The protective agent is represented by soy inhibitors of proteases in concentration of 0.1-2.0 g/l. The time of incubation with the protective agent makes 5-6 hr, concentration of hydrogen peroxide is 700-750 mM. The invention provides for high extent of yeast cells production with no depressant action.
EFFECT: share of survived yeast cells during method realisation increases in comparison with the reference one 1,2-5,4 times.
SUBSTANCE: composition for production of organosilicon sol-gel matrix for immobilisation of microorganisms in biosensor analysers is provided. The composition consists of 20% polyethyleneglycol solution in phosphate buffer solution, Tetraethoxysilane, and 0.2 mol/dm3 catalyst solution NaF, an additionally introduced hydrophobic additive - methyltriethoxysilane. The components are taken at a volume ratio of PEG:TES:MTES: NaF 4:(18-3.4) (2-16.6):1.
EFFECT: reducing the toxic effect of the matrix on the biomaterial and increase in its mechanical strength.
1 tbl, 1 ex
SUBSTANCE: present invention relates to microbiology and a method of detecting and counting viable Legionella pneumophila microorganisms in a sample. The described method involves: (1) contacting said microorganisms of said sample with at least one reducing compound which contains glutamate and pyruvate, and a culture medium which is a buffered charcoal yeast (BCYE) or GVPC agar culture medium, (2) incubating the product of step (1), and (3) detecting and determining the number of viable microorganisms. The reducing compound used directly or indirectly affects metabolism, reducing oxidative stress of the microorganism by reducing formation and/or breaking down reactive forms of oxygen.
EFFECT: disclosed method enables to accurately count Legionella pneumophila microorganisms in a stressed condition.
7 cl, 5 dwg, 5 ex
SUBSTANCE: method of qualitative assessment of biocorrosion damages of thin-walled sealed enclosures of aluminium-magnesium alloys in operation of spacecrafts and the suspension of spore materials fungi of implementation of the said method is proposed. The test and control samples of aluminium and magnesium alloys are prepared. The prepared samples are dried, sterilised. Fungal cultures - strains of microorganisms Paecilomyces variotii Bainier of All-Russian collection of microorganisms F-4039D, Ulocladium botrytis Preuss of All-Russian collection of microorganisms F-4032D, Penicillium chrysogenum Thorn of All-Russian collection of microorganisms F-4034D, Aspergillus sydowii (Bainier et Sartory) Thorn et Church of All-Russian collection of microorganisms F-4037D, Cladosporium sphaerospermum Penz. of All-Russian collection of microorganisms F-4041D are inoculated for growing spores into tubes with a sloped agar medium of Czapek-Dox. The tubes are thermostated at a temperature of (29+2)°C for 14-28 days until appearance of mature spores. Spore materials suspensions of individual cultures of the said fungi are prepared, which are then mixed in equal proportions. And the concentration of spores of each fungi species in the suspension should be in the range of 1-2 mln/cm3. The resulting suspension is applied to the sterilised test samples. Then they are dried and placed as one sample in each Petri dish on the surface of agar medium of Czapek-Dox. The control samples not treated by the spore material are also placed on the surface of the agar medium of Czapek-Dox in the Petri dishes. The Petri dishes with the test and control samples are placed in different desiccators at the bottom of which water is poured to maintain the humidity more than 90%. The desiccators are closed and incubated in the thermostat at a temperature of (29±2)°C. The exposition of test and control samples is carried out for 40, 120 and 180 days. Then the samples are removed from the Petri dishes, and the corrosion products and mycelium are removed from the surface of the samples washing them in running water. The samples are soaked in 70% ethyl alcohol for 30 minutes, then they are washed with detergent and dried. Then, using a scanning electron microscope a qualitative assessment of biocorrosion damages is carried out. According to the change in the appearance of the surface of the samples the initial stages of biocorrosion and its type is evaluated, as well as the distribution of corrosion damage on the surface of the samples, and the dependence of the biocorrosion process on time is determined.
EFFECT: inventions enable to carry out the qualitative assessment of the initial stages of biocorrosion damages of thin-walled sealed enclosures of aluminium-magnesium alloys with the thickness of more than 2 mm.
2 cl, 9 tbl
SUBSTANCE: invention relates to means of controlling quality of organic and inorganic products, and can be used to evaluate safety of food and fodder products, natural water and waste water, ground, soil, determining the maximum allowable concentration of contaminants, as well as the impact of human activities on the environment, including oil extraction and refining products. The apparatus consists of a computer with a software system and a biodetector. The biodetector has a housing 1 inside of which there is a displacement controller for a board 2 with containers 3 for test objects, a light source - light-emitting diodes 4, an optical system with a television camera 5 and a lens 6 mounted on a support 7. The apparatus is provided with a light-proof casing 8 for closing the top of the board with containers for test objects, the inner surface of which is coated with white paint. The optical system with a television camera 5 and a lens 6 is mounted on the support 7 by a turning mechanism 9 in form of a ball head. Inside the housing 1 there is a stepper motor 10 for rotating the board 2 with containers 3.
EFFECT: invention increases measurement accuracy while reducing the measuring time, simplifying the design and reducing the size of the apparatus.
2 cl, 2 dwg
SUBSTANCE: device comprises a through cell equipped with holes, where at least one hole represents an inlet hole for intake of fluid medium from the specified technological flow, and at least one hole is an outlet hole for discharge of fluid medium from the specified through cell. To one of specified holes an RK probe is attached, possibly, an OVP probe, a cleaning accessory. The first pipeline is connected to the inlet hole. Possibly, the second pipeline is connected to the outlet hole. A valve is connected to the specified through cell. With the help of the specified devices and methods they measure volume microbiological activity and surface microbiological activity in a process flow of water by means of measurement of dissolved oxygen concentration.
EFFECT: method improvement.
45 cl, 10 dwg, 2 tbl, 4 ex
SUBSTANCE: production method of keratinase provides for directed adaptation of strain Penicillium citrinum PC-54-91"ВИЛАР" by three-time subcultivation on Czapek agar medium containing 2% of hair keratin as a carbon source. Cultivation under deep conditions on Czapek medium containing 10% of hair keratin and 0.5% of saccharose during 6 days. Separation of biomass from culture fluid with further extraction of target product by two-stage chromatographic cleaning involving gel filtration on TSK-Gel TOYOPEARL HW-40 and affine chromatography on protein A to sepharose CL-4B with further lyophilisation.
EFFECT: invention allows increasing ferment yield, obtaining keratinase preparation decomposing the hair α-keratin, and improving cleaning degree of ferment preparation.
2 tbl, 5 ex
SUBSTANCE: proposed method provides for preparation of inoculating mycelium of basidiomycete, which has been chosen from the following groups: Flammulina velutipes (Curtis) Singer and/or Hericium erinaceus (Bull.) Pers. Preparation of culture medium containing pulverised cold-pressed sunflower cake, soya flour, potassium dihydrogen phosphate, magnesium sulphate and water in the specified ratios. Inoculation of the obtained culture medium with the inoculating mycelium of basidiomycete, cultivation of basidiomycete with further separation of mycelium biomass and extraction of an antitumour agent from the biomass.
EFFECT: invention allows improving an environmental situation due to utilisation of production wastes of food industry.
3 cl, 1 tbl, 4 ex
SUBSTANCE: strain Aspergillus oryzae RCAM01135 is a producer of proteolytic and amylolytic ferments, which has ability to produce proteolytic and amylolytic ferments. It was deposited at GNU VNIISKHM with the following registration number: RCAM01135. It can be used at production of different food products (fermented spices, additives, and drinks).
EFFECT: invention allows increasing the growth speed and intensity of spore formation.
4 tbl, 2 ex
SUBSTANCE: strain of unicellular algae Dunaliella salina IPPAS D-295-producer of biologically active substances having antioxidant activity was deposited in the culture collection of microalgae of the Institute of Plant Physiology n.a. K.A.Timiryazev RAS (IPP RAS (IPPAS)) under registration number IPPAS D-295 and can be used to produce biologically active substances having antioxidant activity and antagonistic activity against opportunistic pathogenic bacteria.
EFFECT: invention enables to increases the yield of antioxidant substances.
3 tbl, 2 ex
SUBSTANCE: alternative methods are proposed to produce a yeast extract to give taste of "kokumi" to food products, containing peptide γ-Glu-X or γ-Glu-X-Gly. One version of proposed methods includes growing of yeast in a nutrient medium containing peptide selected from the group that consists of γ-Glu-X, γ-Glu-X-Gly and X-Gly, and preparation of the yeast extract from the produced cells. Another version includes interaction of γ-glutamiltransferase on the yeast extract containing X or X-Gly, produced from yeast grown in the nutrient medium, to which amino acid X or peptide X-Gly is added. X is an amino acid or its derivative, different from Cys and its derivatives. The yeast extract is described to give "kokumi" taste to food products and produced by the specified methods, containing peptide selected from the group that consists of γ-Glu-X and γ-Glu-X-Gly, in the amount of 0.005% or more of dry weight of the yeast extract, differing by the fact that X is amino acid or its derivative, different from Cys and its derivatives.
EFFECT: invention makes it possible to produce a yeast extract with improved properties.
24 cl, 8 dwg, 14 tbl, 13 ex
SUBSTANCE: method provides for growing of a culture of microscopic fungi on a dense nutrient medium. A standard suspension containing 104CFU/ml is prepared from the grown culture of microscopic fungi. The suspension in the amount of 0.5 ml is seeded into 4.5 ml of molten agar nutrient medium cooled down to 45°C. Mixed, poured into a Petri cup to produce a seeded plate of the agar nutrient medium. The seeded agar plate is cut into blocks with the specified size, which are placed onto the microscope slide and covered with the cover slop with formation by the agar chamber - production of the preparation. The produced preparation is incubated in the moist chamber at 28°C for 3 weeks.
EFFECT: invention makes it possible to simplify methodology of preparations making and to expand area of application of the produced preparations.
3 dwg, 2 ex
FIELD: food industry.
SUBSTANCE: additive is produced from wheat offal fermented with mould fungi. The additive agents are at least one ferment and a mixture of nutritional ingredients for yeast: ergosterol, N-acetylglucosamine, vitamins, nucleic acids and amino acids. The said additive is also used for activation of alcohol fermentation or pre-fermentation intended for preparation of yeast under aerobic conditions.
EFFECT: reduction of the simultaneous saccharification-fermentation stage time in the process of ethanol production, increase of yeast growth and efficiency.
21 cl, 2 dwg, 4 tbl, 4 ex
SUBSTANCE: composition contains an agent designed for biocontrol, for reduction of level of pollution of food and fodder with aflatoxin, a binding agent, an agent having osmoprotector and adhesive properties, a carrier and a source of nutrient elements for an agent, designed for biocontrol.
EFFECT: reduced level of food and fodder contamination with aflatoxin in raw materials.
29 cl, 4 tbl, 6 ex
FIELD: food industry.
SUBSTANCE: invention relates to a food product with extended storage life and to usage of probiotic spores and/or resting cells as a probiotic ingredient in the said product. The product is chosen from the group containing milk products, fruit juices and fruit beverages. The probiotic spores are chosen from the group containing bacterial, yeast and fungous spores. The resting cells are chosen from bacterial, fungous and/or yeast and/or their mixtures. The produced is stored under uncooled conditions during a long period of time by way of probiotic bacteria spores inclusion.
EFFECT: probiotic organisms contained in fruit juices and beverages remain viable during a longer period of time in comparison with live probiotics.
23 cl, 9 ex
SUBSTANCE: strain of fungus Trichoderma harzianum Rifai VKPM F-180 is used as a producent of an inhibitor of a stimulant of bacterial burn of fruit cultures.
EFFECT: invention makes it possible to reduce losses of decorative and fruit cultures caused by bacteria Erwinia amylovora.
FIELD: biotechnology, in particular reagent for structural protein hydrolysis.
SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.
EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.
2 dwg, 12 ex