Nutrient medium for growing filamentous fungi-dermatophytes from clinical material
SUBSTANCE: nutrient medium for growing filamentous fungi-dermatophytes from clinical material comprises glucose, bacteriological agar, meat peptone, casein hydrolyzate, yeast extract, sodium chloride, sodium carbonate, L-cysteine, thioglycolic acid and distilled water in a predetermined ratio of components.
EFFECT: invention enables to reduce time of growing filamentous fungi-dermatophytes from clinical material.
1 tbl, 2 dwg, 4 ex
The invention relates to medical Microbiology and can be used for the selection of clinical material is the most important pathogens - pathogenic fungi-dermatomitsetov, agents of fungal diseases of humans and animals.
The essence of the invention is to design an efficient dense semi-synthetic nutrient medium to obtain primary cultures of fungi-dermatomitsetov from clinical material, taking into account their needs batteries and physiological characteristics.
For isolation and identification of fungi currently in clinical Microbiology, they use solid and liquid nutrient media Saburo, approved by the USSR Ministry of health order No. 535 of April 22, 1985, "On the unification of microbiologic methods used in clinical diagnostic laboratories medical institutions". It relates to semi-synthetic environments, which, along with the substances of uncertain composition includes compounds of known chemical nature. Wednesday Saburo: peptone - 10 g, glucose or maltose) - 40 g, agar - 18... 20 g, distilled water 1 l, pH 5,6 5,8..., broth prepared similarly, but without agar .
Today, however, the percentage of positive results of culture experiments is of dermatomitsetov on the environment Saburo if positive direct microscopy and undoubted clinical picture remains low. Reportedly, A. Sergeev, to highlight the culture of the pathogen in 1994-1996 he succeeded in 36% of cases , which cannot be considered optimal for laboratory diagnosis. There are foreign corporate environment for Trichophyton (Mycosel, Mycobiotic) with similar indices in Visavuori mushrooms from primary clinical material. This is probably related to a number of features of this kind of fungi. First, the relatively slow growth - unlike most mizelialnah fungi, the growth of Trichophyton begins 5-7 days after sowing (fungi on day 2), and secondly, higher nutrient needs of Trichophyton. Most species dermatomitsetov to start growth requires the presence in the medium of a complete set of vitamins and proteins of animal origin.
The purpose of the claimed invention is to develop a dense semi-synthetic nutrient medium to obtain primary cultures of fungi-dermatomitsetov from clinical material meeting the established performance criteria, which is a necessary prerequisite for reliable results of any clinical, microbiological, and providing accurate and objective results.
The technical result of the invention is to obtain a dense semi-synthetic nutrient medium is La obtain primary cultures of fungi-dermatomitsetov from clinical material.
The implementation of the invention is achieved by preparing a nutrient medium containing 1 liter of distilled water: glucose - 30,0 g bacteriological agar - 20,0 g, peptone meat - 5.0 g, casein hydrolysate - 2.5 g yeast extract, 0.8 g sodium chloride 0.4 g sodium carbonate is 0.16 g, L-cystine - 0.12 g, acid thioglycolate - 0.08 g, pH of the medium is 6.5.
Components are successively dissolved in water, make agar and heated to melt the agar. If necessary, the medium is filtered. Then add the glucose, mix thoroughly and set a pH of 6.5. Then poured into a sterile vessel and sterilized for 60 minutes at 120°C.
Example 1. The method of obtaining the composition. In 1 l of distilled water was dissolved sample: meat peptone - 5.0 g, casein hydrolysate - 2.5 g yeast extract, 0.8 g sodium chloride 0.4 g sodium carbonate is 0.16 g, L-cystine - 0.12 g, acid thioglycolate to 0.08, Stirred the resulting solution. To the dissolved components were added to the agar bacteriological - 20,0 g and heated to melt the agar. Filtered if necessary (in case of presence of insoluble particles). Then add the glucose - 30,0 g and thoroughly mixed. Established a pH of 6.5.
The prepared medium was poured into sterile dishes and sterilized for 60 minutes at 120°C.
Freshly prepared medium was poured into Petri dishes in an amount not IU the 20 ml and the same day produced planting material.
The difference between the proposed composition of the medium is that to improve the effectiveness of seeding dermatomitsetov on Wednesday added L-cystine and sodium carbonate to neutralize free radicals and reduce the redox potential of the environment and thioglycolic acid that helps to release elements of the fungus from keratin masses due to the destruction of disulfide bonds in the keratin of pathogenic material (skin, scales, nails, hair), which provides the contact elements of the fungus with nutrients environment and a more rapid onset of growth dermatomitsetov.
For a better understanding of the invention examples of effectiveness of the proposed environment.
Study of the effectiveness of the proposed environment, in contrast to the environment Saburo, was performed by the method of comparative analysis of fungal growth-dermatomitsetov as in model experiments and clinical material. In model experiments took into account the speed and pattern of growth of the colony and manifestations of morphological characteristics of fungi dermatomitsetov. The study was conducted for 21 days with 3 species of fungi (Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum). These types of fungi are the most frequently isolated from patients with causative agents of fungal infections. The strains were obtained from the collection model and pathogenic cultures mycological laboratory Kazanskogo the scientific research Institute of epidemiology and Microbiology of the CPS. Were used as Museum strains and isolated from patients with a diagnosis of mycosis.
Example 2. Nourishing environment conducive to accelerating the growth of fungi dermatomitsetov, allows you to achieve greater efficiency in identifying dermatomitsetov, since the rate of growth dermatomitsetov is an important factor in the efficiency of the environment due to frequent contamination of clinical material growing mold fungi. It is known that mushrooms dermatomitsety grow in 2, and sometimes 4 times slower fungi . For typical signs dermatomitsetov - branching, sporulation, pigmentation currently recommended to maintain the incubation period crops up to 1 month .
To study the effect of medium composition on the linear growth of colonies Museum of test strains of Trichophyton rubrum and Epidermophyton floccosum were sown into the center of a dense nutrient medium small one AMF inoculum density. The colony diameter was measured in two mutually perpendicular directions in two or three repeats every 24 hours . The sowing of the test strains on our proposed environment showed an increase in the rate of growth in 1,5-2 times in comparison with the crops on the environment Saburo (figure 1, 2). However, given that these species of fungi on the surface of some limiting growth in dense environments form a widely rasprostranen the Yu, but thin filmy colony, and with the growth of dense optimal environment colony of the fungus can reach the same diameter, but can be high, fluffy, have a richly developed aerial mycelium, in addition to the growth rate of colonies was determined the density of hyphae colony . The density of hyphae was determined by their total length, representing the total length of the main hyphae and branches of all orders in the same field of view under a microscope at magnification 20x20. The density was determined in different parts of a growing colony. On the sixth day the length of hyphae (H) of Trichophyton rubrum on the environment Saburo and the environment amounted to 648 and 817 μm, respectively. The length of the hyphae Epidermophyton floccosum 547 μm on the environment Saburo and 651 μm on the environment.
Example 3. Getting the nutrient medium on which the fungus would give a typical colony and more differentiated fruiting bodies, is an important task to identify dermatomitsetov. Analysis of differential diagnostic features of strains of fungi Trichophyton rubrum, Trichophyton mentagrophytes, grown on the environment and the environment comparison Saburo, showed the effectiveness of the proposed environment. In ten cases out of ten strains of Trichophyton rubrum was isolated in an environment of bright pigment rich red color, which is one of the main diagnostic characters of this species (table 1). While strains, wirawan the e on the environment Saburo, allocated in the environment pigment pale pink color, and in 3 cases the pigment was absent.
Microscopic picture of the different strains of Trichophyton mentagrophytes grown on medium Saburo, differed from those grown in the proposed environment. Seven out of ten strains were not fertile hyphae, microconidii was absent.
Example 4. Evaluation of the effectiveness of the proposed environment on clinical material obtained from patients with limited ringworm-onychomycosis. Criteria for inclusion in the study: patients with onychomycosis confirmed by the positive results of mycological examination (microscopy of the nail plates in 20% KOH or fluorescent microscopy with calcofluor white).
Large pieces of nails cut into several pieces under sterile conditions. Then an equal amount of material at the same time planted on the studied environment. On the seventh day after inoculation of the 25 samples, sown on Wednesday, Saburo, growth dermatomitsetov was detected in 44% (11 samples). Culture the culture on offer Wednesday revealed mushrooms dermatomitsety in 18 samples, which accounted for 72%. Thus the contamination of mold fungi is not hindered identification dermatomitsetov.
Thus, we offer nutritional environment allows you to complete the task to get rapid growth dermatomitsetov, vladusic characteristic features (pigmentation, the sporulation), allowing their detection and identification in primary culture.
1. Order of the Ministry of health of the USSR No. 535 of April 22, 1985, "On the unification of microbiologic methods used in clinical diagnostic laboratories medical institutions".
2. Semenov S.M. Laboratory environment for actinomycetes and fungi. The Handbook. - M.: Agropromizdat, 1990. - 240 S.
3. Sergeev, A., Sergeev, Y.V. Fungal infections. A guide for physicians. 2 ed. - M.: Publishing house of the BINOMIAL, 2008. - 480 S.
4. Sergeev, A. Fungal infections of the nails. 2 ed. - M.: national Academy of Mycology, 2007. - 164 S.
5. Methods experimental Mycology. Reference (Dudka I.A., Vaser S. p., Elanskaya I.A. and others). - Kiev.: Naukova Dumka, 1982. - 550 C.
6. Medical Mycology: management / V.A. Andreev, A.V. Zachinaeva, A. Moskalev, V.B. have been Konstantin; Ed. by V.B. have been Sboychakov. - M.: GEOTAR-Media, 2008. - 208 S.
|Formation of differential diagnostic elements in fungi dermatomitsetov under cultivation in the investigated environments.|
|Strains||Growth of strains is of fungal on the environment Saburo, day||Growth of the strains on the environment, the day|
|T. rubrum||P||P||M And|
|T. rubrum||P||P||M And|
|T. rubrum||P||P||M And|
|T. mentagrophytes||P, C||P||C|
|T. mentagrophytes||P||P, C|
|T. mentagrophytes||P, C||P, C|
|T. mentagrophytes||P, C|
|T. mentagrophytes||P||P||C, M|
|P - education pigment M - microconidii And - arthroconidia, C - crimps or knots.|
Nutrient medium for you is asiania filamentous fungi-dermatomitsetov from clinical material, containing in one liter of distilled water: glucose - 30,0 g bacteriological agar - 20,0 g, peptone meat - 5.0 g, casein hydrolysate - 2.5 g yeast extract, 0.8 g sodium chloride 0.4 g sodium carbonate is 0.16 g, L-cystine - 0.12 g, acid thioglycolate - 0.08 g, pH of the medium is 6.5.
SUBSTANCE: invention relates to biotechnology. The method involves treating the sensitive layer of a biosensor with solution of a common fraction of protease of hepatopancreas of king crab in a buffer comprising tris-HCl 50 mM, CaCl2 3 mM, NaCl 100 mM, pH 8.0 at solution temperature of 35-40°C. The process is carried out in multiple successive steps with a solution of a solution of a common fraction of protease of hepatopancreas of king crab in said solvent at temperature of 35-40°C followed by exposure. The sensitive layer of the biosensor is successively washed with dodecyl sulphate dissolved in the same buffer in concentration of 0.1% and then with water at each step. The treatment process is carried out three times.
EFFECT: invention reduces the duration of the process.
SUBSTANCE: method of obtaining an antiviral preparation is carried out by preparation of an inoculation mycelium of basidiomycete Enokitake Flammulina velutipes (Curtis) Singer, preparation of a liquid nutritional medium, which contains water, vegetable oil and molasses as a carbon source, as a nitrogen source - corn flour, as mineral salts - potassium dihydrophosphate and magnesium sulphate, its sterilisation, inoculation of the sterile nutritional medium with the prepared inoculation mycelium, cultivation of basidiomycete in it in an aerobic conditions; the obtained submerged culture is divided into basidiomycete biomass and a culture liquid, from which a clot is separated by addition to the latter of ethyl alcohol, which is pressed, dried and crushed with obtaining the antiviral preparation under specified conditions.
EFFECT: method makes it possible to simplify technological process, increase the output of the antiviral preparation, possessing higher activity.
2 cl, 1 tbl, 4 ex
SUBSTANCE: strain of fungus Aspergillus oryzae Amy T-52-3-21 produces maltogenic α-amylase, and it is deposited in the All-Russian Collection of Microorganisms Institute of Biochemistry and Physiology of Microorganisms n.a. GK Scriabin RAS under the number F-4476D. The strain is made on the basis of strain Aspergillus oryzae of All-Russian Collection of Microorganisms F-3927D using the genetic engineering methods. Activity of α-amylase at 120 h of growth of the strain is 600-640 units/ml.
EFFECT: higher level of activity of maltogenic α-amylase.
1 tbl, 2 ex
SUBSTANCE: invention relates to biotechnology. Claimed is strain of Fusarium sambucinum, deposited in VKPM collection under number F-1161. Claimed strain is producent of protein food biomass.
EFFECT: invention makes it possible to accumulate biomass with high protein content with higher quantity of valuable unsaturated fatty acids, complete in composition.
2 tbl, 3 ex
SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining food fungal biomass with high protein content. Multicycle deep cultivation of Fusarium sambucinum All-Russian collection of industrial microorganisms F-1161 on liquid nutritional medium, containing sources of carbon, nitrogen, mineral salts, separation and drying of wet fungus biomass are carried out. Cultivation is performed at pH from 3.5 to 7.0 under conditions of air aeration from 0.5 to 2.0 l/l/min. Temperature mode in each cycle of fermentation is supported from the beginning of the cycle to the point of switch at the level from 26 to 30°C, and further to the end of the cycle at the level from 22 to 25°C. Point of switch is determined by accumulation of biomass to concentration from 45 to 60% from maximally achievable in fermentation apparatus, or point of switch is determined by concentration of dissolved oxygen by its reduction to the value from 20 to 40% of saturation ( calculated per atmospheric air pressure).
EFFECT: invention makes it possible to obtain the largest accumulation of biomass with high protein content with specified quantity of nucleic acids.
SUBSTANCE: mutant strain is obtained by impact on Glarea lozoyensis ATCC 20957 strain by nitrosoguanidine and it is deposited in CGMCC with number CGMCC 2933.
EFFECT: fungi strain has stable genetic and productive properties, produces low quantity of impurities during fermentation and is acceptable for commercial production of antibiotic.
6 cl, 2 dwg, 2 tbl, 3 ex
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.
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
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
FIELD: microbiological industry.
SUBSTANCE: invention relates to nutrient media used for culturing producers of carotene. Invention proposes nutrient medium containing barley flour, soybean meal, potassium dihydrogen phosphate, sunflower oil, vitamin B1, β-ionone and tap water being components are taken in the following ratio, wt.-%: barley flour, 3.0-4.0; soybean meal, 4.0-4.7; sunflower oil, 3.8-4.0; potassium dihydrogen phosphate, 0.04-0.05; vitamin B1, 0.0002-0.0005; β-ionone, 0.098-0.099, and tap water, the balance. The proposed nutrient medium is low-priced and enhances the biosynthetic ability of producer of carotene.
EFFECT: valuable properties of nutrient medium.
3 tbl, 3 ex
FIELD: biotechnology, microbiology.
SUBSTANCE: invention relates to producing ethyl alcohol or fodder for monogastral animals. The polyenzyme product with glucoamylase, proteolytic and xylanase activities is prepared by fermentation of wheat bran using microorganism Aspergillus niger. Indicated glucoamylase, proteolytic and xylanase activities have the following minimal values: glucoamylase activity - at least 100 U/g of dry matter; proteolytic activity - at least 100 U/g of dry matter; xylanase activity - at least 100 U/g of dry matter under condition that glucoamylase activity has to be at least 750 U/g of dry matter and/or xylanase activity has to be at least 300 U/g of dry matter. Invention provides enhancing the soluble nitrogen content in wort after saccharification, reducing viscosity and effectiveness in using.
EFFECT: improved preparing method, valuable properties of hydrolyzed bran.
14 cl, 10 tbl, 7 ex
FIELD: biotechnology, microbiology.
SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.
EFFECT: improved preparing method.
15 cl, 4 ex
FIELD: agriculture, fruit storage.
SUBSTANCE: the present innovation deals with technology to treat fruit before their laying for storage. For the purpose to decrease the loss from microbial spoiling and increase the terms of storage fruit essential oils and epiphytic substances of cuticular layer of plant raw material should be applied onto the surface from mycella obtained after extracting Saprolegnia parasitica micromycete biomass with nonpolar extragent in supracritical state.
EFFECT: higher efficiency of protection.
FIELD: food industry, confectionary industry.
SUBSTANCE: the suggested concentrate should be prepared due to pressing the juice under aseptic conditions out of sugar beet prepared, deodorated and sterilized with carbon dioxide in supracritical state in the field of ultrasound fluctuations, cultivating upon residues of mycellial fungi of Trichoderma and Aspergillus species of citric acid fermentation, separating culture liquid, its mixing with juice and introducing liquid ammonia into the blend along with supracritical CO2-extract out of Mortierella nigrescens micromycete biomass further extracted according to the preset technique to obtain a solid residue treated with liquid ammonia followed by concentrating the blend, treatment of concentrate with liquid carbon dioxide, mixing Mortierella nigrescens micromycete biomass with a treated solid residue and heating the mixture up to 60 C, not less. The innovation enables to obtain a concentrate of improved structure-forming properties and increased thermal stability.
EFFECT: higher efficiency of manufacturing.
FIELD: food industry, confectionary industry.
SUBSTANCE: the suggested concentrate should be prepared due to pressing the juice out of sugar beet prepared, deodorated with carbon dioxide in supracritical state, extracting residues at mixing water and liquid hydrogen chloride at extractional volume, mixing juice and extract and introducing liquid ammonia into the blend along with supracritical CO2-extract out of Mortierella zychae micromycete biomass further extracted according to the preset technique to obtain a solid residue treated with liquid ammonia followed by concentrating the blend, treatment of concentrate with liquid carbon dioxide, mixing Mortierella zychae micromycete biomass with a treated solid residue and heating the mixture up to 60 C, not less. The innovation enables to obtain a concentrate of improved structure-forming properties and increased thermal stability.
EFFECT: higher efficiency of manufacturing.
FIELD: microbiology, biotechnology.
SUBSTANCE: invention proposes the strain Mucor racemosus № 195 for preparing immunogenic preparations used against mucorosis in agricultural animals. This strain elicits the high spore-forming capacity, absence of pathogenicity and it produces polysaccharides eliciting immunogenic properties.
EFFECT: valuable properties of strain.
2 tbl, 2 ex
FIELD: biotechnology and agriculture, in particular fungi production.
SUBSTANCE: claimed method includes preparation of mycelium biomass on nutrient medium in presence of growth stimulator (e.g., Azospirillum bacterium suspension) and seeding of mycelium biomass on cereal nutrient medium. Mycelium biomass is prepared by deep cultivation; and as nutrient medium potato-wheal medium is used.
EFFECT: accelerated method for production of edible fungal seed mycelium.
FIELD: food industry.
SUBSTANCE: prepared sugar beet should be deodorated and sterilized with supercritical carbon dioxide in the field of ultrasound fluctuations, juice pressing should be carried out under aseptic conditions followed by cultivation upon residues of mycellar fungi of Trichoderma and Aspergillus species of acetic acid fermentation. Then comes separation of culture liquid, its blending with juice, addition of ammonia and supercritical CO2-extract into the blend out of Mortierella reticulata micromycete biomass extracted then according to the preset technique to obtain solid residue treated with liquid ammonia followed by concentrating the blend, treating the concentrate with liquid carbon dioxide, mixing with solid residue-treated Mortierella reticulata micromycete biomass and heating the mixture up to 60 C, not less. The innovation provides improved structure-forming capacity and increased thermal stability of gelling concentrate.
EFFECT: higher efficiency.