Method to prepare live preparations of microscopic fungi of coccidioides genus for light microscopy

FIELD: biotechnologies.

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

 

The invention relates to medicine and biotechnology, in particular to methods of preparation of living preparations of microscopic fungi of the genus Coccidioides for light microscopy, can be used to identify, establish the specifics of the structure and development of cells in different physiological States.

In the medical mycological practice in identifying the causative agents of mycotic nature is most frequently used methods aimed at studying the features of the morphology of the structures that vegetative propagation of micromycetes. This concept of diagnosis was named morphological or phenotypic, when to determine the unit type is the determination of the morphological characteristics of the studied strain and comparative analysis by establishing differences with closely related species. This methodological approach is implemented in the cultivation of microscopic fungi in conditions conducive to a culture with a distinctive morphology. The literature describes methods of preparation of live unstained microorganisms for study by light microscopy.

For determination of cell shape and motility of microorganisms examined in the drugs "crushed" or "hanging" drop. Drugs mushrooms for research, as the rule is, prepared from cultures grown on solid agar nutrient media. To do this on a glass slide put a drop of water or isotonic saline (0.15 M) solution of sodium chloride. Bacteriological loop is suspended a small amount of material taken from the colony suspected of cultural characteristics on the colony of the pathogen. Cover glass mounted on a rib at the edge of the drop and slow down, squeezing the air between the objective and the cover glass. Also use the method of preparation of microbial suspensions in vitro, after which a drop of the suspension is applied on the glass. In the case of the preparation of the drug "hanging" drop suspension is applied on the glass with a recess (hole) ["Manual of medical Microbiology" in 3 books edited by A.S. Labinsky, E.V. Valinol. - Moscow: BINOM-2008 - CN p.160-161]. A common shortcoming of such drugs is that they do not allow to evaluate the dynamics of culture. In addition, for their preparation is needed in all cases the preparation of the suspension of the studied culture, accompanied by mechanical impact on the culture, which inevitably leads to the destruction of characteristic structures of the fungus, which is an integral part of its morphological identification.

There is a method of study of living unstained microorganisms in which microcamera with agar nutrient media (agar chambers) ["Medical Microbiology" Ed. Weaponammo, Ochrosia. - Moscow: GEOTAR Medicine. - 1998. - p.114]. The described method of preparation of agar chambers of agar or gelatin-agar plates. A clean glass slides twice (or more times) is dipped in molten agar nutrient medium of the corresponding composition, which is located in the Petri dish. From the bottom of the glass agar nutrient medium wipe with a damp cloth. Using a binocular magnifying glass, the culture of make selected on the upper side of the glass pane with an extruded from a Pasteur pipette, glass fiber or glass rods. The tip of the fiber was soaked in an aqueous solution of peptone, lightly touch them to the colony, transferred adhering microorganisms in a drop of an aqueous solution of peptone on the fresh surface of the agar nutrient medium and distribute a drop on the surface. Cut agar environment around your area, put a cover glass and the edges pour the wax. [Methods of General bacteriology" in 3 volumes edited by F. Gerhardt and other Moscow: Mir. - 1983. 1. - p.46-47]. The disadvantages of this method include high possibility of contamination of the preparation by other organisms, and contamination of personnel when working with pathogenic agents, as in the formation of agarwood camera, namely when closing it on the smooth glass, perhaps squeezing liquid culture outside of the glass cover. In addition, the described method allows to prepare the camera, containing a thin layer of agar nutrient medium, which limits the use of the method when working with micromycetes different slow growth rate.

As a prototype of the selected preparation method of living preparations of fungi NM according to the method of pidoplichko. For this sterile glass slide calcined over the flame of the burner, quickly put the hot loop a small drop of molten agar nutrient medium and allow it to cool slightly. Immediately the burner needle put pre-grown on solid nutrient medium culture, trying to get to the center of the drop. Cover glass, as well as subject, calcined above the burner, a little away from the flames so that it is slightly cooled, and placed on a drop of nutrient agar medium seeded with a culture on a glass slide. For the formation of agarwood camera cover glass gently press down until until agar nutrient medium will not be distributed evenly tapering to a thin layer, and placed under an angle of 10-15° to the subject glass. Prevent drying and contamination of the drug coating glass, except for a raised hand poured paraffin. GE is prepared, the preparation is put into a Petri dish so that to the surface of glass slides on which the drug was directed upwards, and incubated in a humid chamber. ["Methods of experimental Mycology" Ed. by V.I. Bilai. - Kiev: Naukova Dumka. - 1982. - s]. A significant disadvantage of the prototype is seeding culture in negoziroma number that does not allow the growth of the fungus in the form of isolated colonies. In addition, the supply of nutrients in a thin layer of agar camera is limited, which makes it impossible for long-term cultivation of slow-growing fungi, for example, for 3 weeks in the case study mycelial forms of fungi of the genus Coccidioides, and some other pathogenic micromycetes. The disadvantages of the prototype is also the fact that the method involves a subjective assessment of the temperature of the agar nutrient medium in which you are sowing culture. In that case, if the environment is cooled insufficiently studied culture may die, or, on the contrary, when excessive cooling medium prematurely freezes on a slide that prevents preparation.

The task of the invention is to develop a method of preparation of living preparations of microscopic fungi of the genus Coccidioides for examination by light microscopy, which allows to simplify the preparation procedures and to expand on the region of their use.

The technical result is to simplify the method, obtaining predictable results, as well as the opportunity to study in the dynamics of the micro-morphology of fungal cultures.

The technical result is achieved in the method of preparation of living preparations of microscopic fungi of the genus Coccidioides for light microscopy, which consists in pre-grown culture on solid nutrient medium, the seed culture of suspended arthroconidia 104CFU/ml, in the amount of 0.5 ml 4.5 ml of melted and cooled to 45°C agar medium, which is stirred and poured into a Petri dish for education seeded agar plates medium, from which cut out the block for the formation of agarwood camera, and incubated received the product in a humid chamber at 28°C for 3 weeks.

Technical solution allows to predict the result at the expense of seeding culture with a known concentration of CFU (CFU - colony forming unit) and get its growth in the form of isolated colonies. Excludes subjective assessment of the temperature of the agar nutrient medium, in which is the seed culture as the set temperature environment is provided by the device, this simplifies the method and guarantees the suitable drugs. Agar Luggage in the drug has izvestneishim, contains a sufficient amount of nutrients, which eliminates the step of filling the chamber with paraffin wax to prevent drying. In addition, it becomes possible to grow a culture in preparation for a long time and to investigate the dynamics of the development of slow-growing fungi from the stage of germination of spores to the stage of formation of the organs of vegetative propagation. Depending on the purpose of the study is available preparation of a large number of drugs on the same sample.

Example 1 (optimal). Culture microscopic fungus, Coccidioides immitis C-5 grown on agar nutrient medium Saburo at 28°C for 3 weeks. Then the culture is washed off of 0.15 M sodium chloride, pH 6.8 and filtered through a fabric filter. From the filtrate using a sample turbidity gisk named after. Tarasevich (CCA) prepare standard suspension containing 104CFU/ml (CFU - colony forming unit) for Coccidioides immitis C-5. The suspension in the amount of 0.5 ml inoculated into a test tube with 4.5 ml of molten and cooled in a water bath to 45°C agar nutrient medium Saburo containing 2% agar-agar, mixed and poured into a sterile Petri dish. Square Petri dishes 10 cm in diameter makes 78.5 cm2. The thickness of the agar plates when applying 5 ml (cm3medium in a Petri dish - 0,064 mm After hardening seeded plate agaropectin environment is cut into blocks of size 1×1 cm, are in compliance with the rules of asepsis is placed on a glass slide and covered with a cover glass for the formation of agarwood camera. The number of CFU in agar block with an area of 1 cm2is 64 units. Finished products are placed in Petri dishes and incubated in a thermostat at a temperature of 28°C for 3 weeks.

Research drugs is conducted daily using light microscopy with a dry lens when magnification ×100, ×400, which allows to obtain data on the dynamics of cultural development. The size of the cells is determined using an ocular micrometer screw MOU-1-15xin a linear increase in βcf.=46,375.

After 24 h cultivation, arthrospira acquire a rounded shape. There are distinct tortuous growth tubes. After 2 days in growth tubes are formed septa (2-3), are viewed by the kernel. Occasionally observed the formation of secondary branches originating from the primary growth tubes. After 3 day begins education cept in the secondary branches. For 4-6 days formed the branches of the mycelium of the following orders of magnitude (figure 1). On the 7th day of cultivation begins the differentiation of cells of the mycelium. Clearly highlighted cells migrating in arthrospira. In their cytoplasm visible seal gray-blue color, reminiscent of the cell nucleus. Cells migrating in arthrospira, displaced who are stated by the cell-separators, having slightly smaller. To 14th days begin to form arthrospores typical organs of vegetative propagation of Coccidioides immitis (figure 2). Arthroconidia have a rectangular shape. For strains of Coccidioides immitis C-5 sizes arthroconidia are 9,0±0,34×3,6±0,64 mm. After 3 weeks of cultivation in the drug are observed filaments of mycelium in the form of "chains", educated Mature arthroconidia interspersed blank-delimited (figure 3).

Example 2 (optimal). Culture microscopic fungus, Coccidioides posadasii 442 grown on agar nutrient medium Saburo at 28°C for 3 weeks. Then the culture is washed off of 0.15 M sodium chloride, pH 6.8 and filtered through a fabric filter. From the filtrate using a sample turbidity gisk named after. Tarasevich prepare a suspension of arthroconidia 104CFU/ml Suspension in the amount of 0.5 ml inoculated into a test tube with 4.5 ml of molten and cooled in a water bath to 45°C agar nutrient medium Saburo containing 2% agar-agar, which is stirred and poured into a sterile Petri dish. Square Petri dishes 10 cm in diameter makes 78.5 cm2. The thickness of the agar plates when applying 5 ml (cm3medium in a Petri dish - 0,064 mm After hardening seeded plate agar nutrient medium is cut into blocks of size 1×1 cm are in compliance with the rules of asepsis placed on PR is Mednoe glass and covered with a cover glass for the formation of agarwood camera. The number of CFU in agar block with an area of 1 cm2is 64 units. Finished products are placed in Petri dishes and incubated in a thermostat at a temperature of 28°C for 3 weeks.

Research drugs is conducted daily using light microscopy with a dry lens when magnification ×100, ×400, which allows to obtain data on the dynamics of cultural development. The size of the cells is determined using an ocular micrometer screw MOU-1-15xin a linear increase in βcf.=46,375.

After 24 h cultivation, arthrospira acquire a rounded shape. There are distinct tortuous growth tubes. After 2 days in growth tubes are formed septa (2-3), are viewed by the kernel. Occasionally observed the formation of secondary branches originating from the primary growth tubes. After 3 day begins education cept in the secondary branches. For 4-6 days formed the branches of the mycelium of the following orders of magnitude (figure 4). On the 7th day of cultivation begins the differentiation of cells of the mycelium. Clearly highlighted cells migrating in arthrospira. In their cytoplasm visible seal gray-blue color, reminiscent of the cell nucleus. Cells migrating in arthrospira, interspersed cells separators having slightly smaller. To 14th days begin to form arthrospores typical bodies in getting breeding Coccidioides posadasii (figure 5). Arthroconidia have a rectangular shape. For strains of Coccidioides posadasii 442 sizes arthroconidia are 12,0±0,71×3,6±0.29 micrometers. After 3 weeks of cultivation in the drug are observed filaments of mycelium in the form of "chains", educated Mature arthroconidia interspersed blank-delimited (6).

The method of preparation of living preparations of microscopic fungi of the genus Coccidioides for light microscopy, which consists in pre-grown culture on solid nutrient medium, the seed culture of suspended arthroconidia 104CFU/ml, in the amount of 0.5 ml 4.5 ml of melted and cooled to 45°C agar medium, which is stirred and poured into a Petri dish for education seeded agar plates medium, from which cut out the block for the formation of agarwood camera, and incubated received the product in a humid chamber at 28°C for 3 weeks.



 

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