Method of growing colonies of microbial cells and device for its realisation

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. Claimed is a method of growing colonies of microbial cells on a surface of a porous plate. The method includes supply of a nutrient solution from bottom to top through the porous plate into zones of growth of colonies of the microbial cells on its upper surface, supply of a suspension of the microbial cells onto the upper surface of the porous plate, creation of controlled conditions for the colony growth, performing observation of the colony growth, separation of the grown colonies of the microbial cells from the zones of growth and their transfer into external means of identification. The nutrient solution is supplied into the zones of growth of the colonies of the microbial cells by creation of a pressure difference between the hole input and output. Holes are made in the plate from an anode aluminium oxide orthogonally to its large plane and are topologically coded. The said zones of growth are formed in them in the form of porous membranes. The porous membranes are located at the same level as the upper surface of the plate or with formation of a hollow and do not pass the microbial cells. After supply of the nutritional solution, the suspension of the microbial cells of a specified concentration is supplied onto the upper surface of the plate until their homogenous distribution is achieved. Between the zones of growth on the surface of the plate a film, preventing attachment of the microbial cells, is formed. Separation of the grown microcolonies from the zones of growth is performed by hydroblow. A hydroblow is directed from the side of the input of cylindrical holes of the plate and spreads along them and farther through the pores of the porous membranes with force, which does not destroy the microcolonies but is sufficient for their separation from the growth zones. Also claimed is a device for growing the colonies of the microbial cells by the claimed method.

EFFECT: providing conditions of automation of processes of the nutrient solution supply and processes of separation and transfer of the grown colonies, possibility of integration into miniature portable devices, and application in laboratories on a chip and provision of the device portability.

6 cl, 14 dwg, 4 tbl, 2 ex

 

The invention relates to analytical instrumentation and can be used in the study of liquid biological and medical samples. Most effectively used for microbiological Express-diagnostics, including the determination of sensitivity of bacteria to antibiotics, consisting of miniature analytical devices laboratories on a chip for portable systems for microbiological control.

Portable microbiological control laboratories on a chip can work in laboratory and field conditions, and associated telemetry systems. The system should be easily to work with different clinical material and search agents of different groups of infections: respiratory, gastrointestinal tract, urogenital tract, nosocomial infections, evaluation of bacterial contamination of the external environment.

Analysis of microbiological samples includes the isolation of microbial cells, the accumulation of biomass and identification of strains of microbial cells, determining their number, and determination of their sensitivity to antibiotics. It is necessary for the diagnosis of infectious bacterial diseases and proper antibiotic therapy. This issue is particularly relevant in connection with the growing incidence of detail is klonnie bacterial diseases and the increasing number of resistant to many antibiotics, strains of pathogenic bacteria. In 2010, the number of infectious and parasitic diseases in Russia has reached 30069567 cases (according to Rospotrebnadzor: /asset_publisher/N2qH/content/2). Traditional microbiological research is time-consuming and, more importantly, long-term (3-7 days). It is only in specialized institutions and, as a rule, for inpatients. Creating a miniature mobile devices for rapid identification of pathogens and determine their sensitivity to antibiotics will correctly choose the tactics of treatment of infectious diseases, to increase its efficiency, to decentralize microbiological analysis and make it available for the whole population, including remote areas. Low cost and availability analysis will use it to improve laboratory diagnostics in infectious diseases and improve the quality of treatment.

The longest (limiting) the stage of carrying out microbiological culture analysis (the most reliable method for the diagnosis of infectious diseases is the accumulation of microorganisms on nutrient media. This stage requires improvements to increase the speed of analysis. The duration of this stage is determined by the necessity of accumulation of visible eye colonies with distinct visual characteristics of the mi. After primary incubation and accumulation of material manually selected for further research, namely, identification, allocation and accumulation of a pure culture of the pathogen, setting the sensitivity to antibiotics. Known automated and semi-automated instrumental methods of microbiological analysis, implemented using stationary devices, but they are also long lasting and require primary accumulation of cells. Comparative data of technical and operational characteristics of the device using the inventive device and industrial analogues shown in table 1.

Known semi-automatic method for microbiological analysis (table 1, column 2), which use the seeding of biological samples in Petri dishes with culture medium, incubation of the samples with the purpose of accumulation of colonies of microbial cells, isolation of individual colonies and the study of their sensitivity to antibiotics using the Kirby-Baer. In this method, many operations are carried out manually, which significantly reduces the performance of the method. Used incubator high cost and dimensions, which makes this method not suitable for operation in remote locations. Requires a large number of consumables. The method is not suitable for automation and can't do-the th task delivered in this invention.

Known automated microbiological analyzer VITEK®2 compact 60" (table 1, column 3), developed by bioMerieux SA (France), performs automatic identification of species of pathogenic microbial cells and determination of their sensitivity to antibiotics on a separate microbiological cards for each of these types of analyses. For reservation accumulation and release of strain of pathogenic bacteria. The analysis according to the manufacturer, up to one day, and taking into account the accumulation of pathogenic microbial cells two or more days. The device is stationary and is not adapted to work in remote areas of assistance, including in clinics. Its high cost determines the need for accommodation in large centralized microbiological laboratories. The device is not automated procedure for preliminary collection and sorting of microbial cells with the release of pathogenic strains or diagnostically significant microbial cells. Thus, this known industrial unit is unable to achieve the technical objectives of this invention.

Achievements of microelectronics, Microsystem technology and principles of miniaturization in instrumental analysis revealed who is agnosti development of a new generation of hardware - microanalytical systems, also known as: µ-TAS, lab-on-a-chip (lab-on-chip), microfluidic systems, and others (Manz, A., Graber, N., Widmer N.M. // Sensors and Actuators. 1990. B.1. P.244-248; Zimin T.M. // St. Petersburg journal of electronics. 2000. No. 3-4. S). Representing a miniature device, manufactured using planar and hybrid technologies, microanalytical systems are designed for various chemical analyses and biomedical testing. Such systems can provide improved performance when carrying out biomedical and environmental analysis, the study of living organisms. Development of microanalytical systems allows to solve the problem of reducing the cost, material and energy intensity of products, increase productivity analysis. These improvements possible due to the formation of such devices using micro - and nanotechnology, enabling precise geometry, providing the opportunity geometric complementarity of the components at a molecular level, increasing the speed of analysis without additional costs, management of mass transfer and automatic control at the microscale stages of the analytical process in integrated functional modules and subsystems. The transfer of biological objects in such systems can be implemented using the techniques PR is an accurate analysis, using the principles of microfluidics. Improvement of traditional methods of microbiological analysis can be performed using technologies of micro - and nanoelectronics.

A known method of growing cell cultures in which the cells are grown in the growing reservoir containing nutrient medium, which is connected with a reservoir for the nutrient medium with the formation of tangential flow in the growing tank by means of the control flow and the external pump, membrane, also located tangentially, in the form of hollow fibers or a frame with a filter that provides a sterile barrier for cells, the site selection of cells grown with the possibility of opening and closing system ensure mass transfer of cultures with the possibility of changing the direction of flow of the medium through four-way valve, filter system in the form of stacked plates and hollow fibers, for the separation of solid impurities, the solution to replace the nutrient medium, a means of concentrating the cells by removing excess liquid (US 6022742, C12M 1/36,2000).

This method does not allow for controlled growth of individual microcolonies attached to the areas of growth in order to reduce the time of identifiable growth of microcolonies of cells, and not possible a controlled transfer to an external device and is entifically and sorting colonies.

There is a method of cultivation of biological cells, implemented in a liquid medium on the platform in the microwells, connected by microchannels, which are environment conducive to the growth of the colonies. The microwells and channels are formed in the form of a profile, after which the top cover plate forms microcamera and capillaries. The cover plate has openings to access the camera. The chambers and channels are formed radially on the drive that has the possibility of rotation to move the camera through the aperture of the optical recording device for analysis of colonies without disturbing their structure in the cells (US 6632656 B1, C12M 3/06, C12Q 1/20,2003).

This method does not allow for the separation and transfer of microcolonies to identify the external device, to isolate bacterial colonies and their subsequent accumulation to study the sensitivity to antibacterial agents.

Known methods of growing colonies, carried out with the help of tiny cells for growth of the cells formed using thin film deposition of polymers and/or oxides and fluorides of metals with a thickness of 0.01 to 0.2 mm and, then, forming by partial removal of the film plots the given geometry, and surface properties that provide control over cell growth (JP 1141588, C12M 3/00, 1989; JP 7075547, C08F 120/28,20/26, C12M 1/00, 3/00, 5/07, 5/0783, 1993).

In the shown how there is no automatic tear-off and transfer of colonies in site identification and sorting in external devices with the aim of identifying colonies of microbial cells and testing their sensitivity to antibiotics.

Closer to the claimed method is a method of cultivation and analysis of individual cell cultures, in which, after pre-treatment of samples containing a variety of cells, cells are placed in a growth cell (or site) to conduct simultaneous growth under specified conditions (humidity, temperature, composition of the liquid medium) to obtain the individual microcolonies. The method is carried out using the growth of cells, for example, formed using microfabrication, very small size, so as to ensure the growth of individual cells, with a size comparable to the size of the growth zone. In this method, the cell suspension is placed in a growth zone after separation of the suspension into separate volumes, small enough in order to ensure the growth of a homogeneous culture in each volume, spend their seeding and incubation in a separate microwells having a cylindrical shape, separated from each other by a distance not less than 1.8 mm, and the surface between them is covered with a water-repellent layer and is common to all "microculture or individual microcolonies, and during incubation the cultures supplied with the necessary nutrients through membranes with pore size of 0.1×10-7÷4×1-6mi thickness of 0.2×10-7÷10×10-6(EP 1425384 (A2), C12M 1/00, 120, 1/26, 1/34; C12N 1/00, 1/02; 2002).

However, this method does not solved the problem of rapid separation of individual microcolonies and their transfer to an external device identification and sorting. The method is complicated and is not suitable for rapid analysis, and the appropriate device cannot be used in a portable device because of the complex structure and low strength of the dispensers, it does not allow for the isolation and identification of pathogenic strains, with a view to their subsequent accumulation and study of sensitivity to antibiotics.

The closest to the technical nature of the claimed method of the prototype is the method presented in the article Ingham, .J., Sprenkels, A., Bomer, J., Molenaar, D., van den Berg, A., van Hylckama Vlieg, J.E.., de Vos, W.M. "The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms" 04/46/18217 .full.

The method is carried out on a porous plate made of anodic aluminum oxide on the surface of which is formed of a special zone for the growth of microorganisms, limited by the walls of the polymeric grid formed by a method of laminating with the subsequent photolithography and plasma etching.

When implementing such a method of growing colonies of microbial cells nutrient solution served by leakage due to capillary forces through the pores of the plates of porous anodic oxide is of luminia, paired with a layer of agar. Then manually served microbial cells, distributing them across the growth areas. Incubate under anaerobic conditions at 37°C, or other specified conditions, observing the growth of the colonies to the required size using a microscope. After reaching a sufficient size, colonies of microbial cells detach from the growth zones manually using sharp sterile toothpick, and also manually perform their transfer to an external means of identification.

The main disadvantages of the prototype of this method is the use of manual labor when filing cells in the growth zone, and also when removing the grown colonies and their transfer means of identification and the inability to automate the process.

The objective of the proposed method is to develop a method of growing colonies of microbial cells, allowing to obtain the technical result consists in creating the conditions to allow the automation of the process of cultivation, separation and transfer of microcolonies, which provides the possibility of using the method in the laboratories on a chip.

The essence of the proposed method lies in the fact that in the method of growing colonies of microbial cells supply the nutrient solution is bottom-up through the holes in the plate and placed them in the porous membrane, in the zone of growth of colonies of microbial cells, is formed on the porous membrane; submission of microbial cells is carried out on the upper surface of the plate to their uniform distribution in the growth areas; ensures the creation of conditions for growth of microorganisms in the form colonies in the monitoring of environmental parameters (temperature, humidity, partial pressure of oxygen, and others); observing the growth of the colonies; is the gap of the grown microbial microcolonies of cells from growth zones by surge and transfer them to the external means of identification in the stream.

Significant differences of the proposed method are co measurement of growth zones and microcolonies, which involves the allocation of the right pure culture of microorganisms, the ability to control supply of the nutrient medium, the ability to control the growth of colonies in situ, the possibility of creating conditions of water hammer with the use of shock pressure at the inlet of the holes in the porous plate for the separation of the colonies from the surface of the growth zones on a porous membrane located in the hole so that they automatically move without mechanical damage of microcolonies in the external device for further research, and the ability of the method in the laboratory on a chip. The flow of the nutrient solution at the expense of pressure drop when the flow of solution from the bottom to the top that allows you to control the submission process p is the target for growth zones, and can be automated (e.g., pumping). The flow of suspension through any automated means (pump) allows you to evenly distribute it over the surface. Applying a hydrophobic film on the surface of the plate with the holes prevents the attachment of the microorganisms that will be delayed growth zones with high concentration of nutrients supplied from below. Thanks to the adjustable supply of the nutrient solution in a growth zone provided by the growth of colonies. When reached by dividing the size of the order N=100-1000 cells in the colony, micro-colonies are separated from the growth zones for the transfer of external means of identification. Separation of microcolonies in the inventive method is carried out using a hydraulic impact. Create mode surge allows you to automate the process of separation of the colonies for their subsequent transfer.

Known portable device for growing colonies of cells that contains the cartridge, with the capacity for cell growth, the container for the growth environment, the container for draining the waste site to gather grown colonies United into a single device that is not associated with the external environment. The device can be interfaced with a variety of external devices, for example, the node for mixing cells, a processor, a thermostat, a site of mass transfer, the filters for the regulated what I medium composition and site selection and concentration cells. Site of mass transfer in the device allows flow environment, the change of direction tangential growth surface flow to optimize cell growth and enhance the yield of the culture, and the supply of sterile growth medium and removal of inhibiting substances (US 6096532, C12M 3/02,1997).

This device provides the ability to automate and optimize the process of growth and harvesting of cells, but is complex, cumbersome and does not provide the ability to automatically move the individual microcolonies of cells in the external device to identify and further define their sensitivity to antibiotics, i.e. does not perform the task. The described device is not intended for integration into lab-on-chip.

It is known a device in which as a substrate for growth of strains of cells uses a semi-permeable membrane through pores which is supplied nutrient solution. For example, a device for growing cell cultures and bacteria that is placed in a Petri dish containing liquid or nutrient solution, representing a ring or frame, which is fixed to the porous lattice covered with a porous membrane, so that its bottom surface is in full contact with the liquid, and on the upper surface of the membrane are placed cells, bacteria or other organizations who isms, fed from the pores of the membrane (GR 1003266 (B2), C12M 1/12, 3/06, 1997). It is also known a device for analysis of cells in which the microwells fabricated using photoresistive film thickness of 15-60 μm, forming the walls of the holes (EN 2298798, G01N 33/546).

These devices do not have the potential of automation and the possibility of selection of microcolonies, i.e. cannot be used to obtain in a short period of pure cultures of microorganisms.

The known device for growing colonies of cells that contain the node mass transfer, filters for environmental control and site selection and concentration cells. Site of mass transfer in these devices supply the environment for optimizing cell growth and enhance the yield of the culture, and the supply of sterile growth medium and removal of inhibiting substances (US 6022742, C12M 1/36, 1988; US 4885087, B01D 13/00, C12N 5/00, 1986; US 6127141, C12P 1/00, 1999; US 5240854, C12M 3/00, 1991; US 6096532, C12M 3/02, 1997; US 7320889 B2, C12M 1/08, 2004). All of these devices, providing the ability to automate and optimize the process of cell growth are complex, cumbersome and do not provide the ability to automatically move individual microcolonies of cells in the external device to identify and determine the sensitivity of the obtained cultures to antibiotics.

Closest to the claimed device is presented in the article Ingham, .J., Sprenkels, A., Bomer, J., Molenaar, D., van den Berg, A., van Hylckama Vlieg, J.E.., de Vos, W.M. "The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms)) .

The device consists of a porous plate of the anodic aluminum oxide on the surface of which is formed a grid of polymer walls, forming a zone of growth of colonies with a characteristic size of from 7 to 200 μm. The porous plate is located on the layer of agar with a nutrient solution in a Petri dish with the achievement of full liquid contact device with the surface of the agar for wetting the lower surface of the porous plate.

The main drawbacks of the prototype is that it does not provide the possibility of automating the process of cultivation and growth of cells, it is all manual labor when filing cells in the growth zone, and also when removing the grown colonies and their transfer means of identification and control using a microscope. The device analyzes at a low speed, does not have the portability, does not provide the possibility of integration into miniature portable devices and use in laboratories on a chip

The task of the claimed invention to provide an apparatus for carrying out the method of growing colonies of microbial cells, allowing to obtain the technical result consists in providing the author is matinale processes feeding the nutrient solution and processes branch, and transfer of grown colonies, the possibility of integration into miniature portable devices, and use in laboratories on a chip and software portability.

The essence of the proposed device is that the device for growing colonies of microbial cells containing a porous plate made of anodic aluminum oxide formed with its upper surface areas of growth colonies of microbial cells, the lower surface of which is associated with means for supplying nutrient solution, a porous plate made of anodic aluminum oxide is the case with the formation of the top and bottom of the tank corps, which is equipped with inlet and outlet for liquid media, and the said porous plate made of anodic aluminum oxide has openings of cylindrical shape, formed orthogonal her big plane of the plate and topologically encoded, with each hole are a porous membrane with pores, preventing microbial cells, the upper surface of the housing is arranged to pass gas, but not permeable to moisture and particles in the external environment, and monitor the growth of the colonies. The growth zone can be either site, at the location of the porous membrane is flush with the surface of the porous plate of the anodic aluminum oxide, or moons and - at the location of the porous membrane below the surface of the plate. The surface of the porous plate of the anodic aluminum oxide between growth zones may have a coating layer of material that prevents the attachment of cells, for example, metal, polymer, hydrophobic material. The porous membrane can be produced, for example, anodic aluminum oxide, ceramic, polymer, metal. Cover the top of the tank may be provided with a Central window, with fixed therein an optical glass for monitoring the growth of the colonies. Cover the top of the tank may be provided with two Windows, closed semi-permeable membranes, serving for the transmission of gases, but which is not permeable to moisture and particles in the external environment.

The formation of growth zones in the holes of the porous plate by placing a porous membrane in them and the availability of coverage between growth zones allows concentrated and rapidly growing colonies, without special means of limiting growth zones (polymer network in the prototype). The porous membrane in the holes play the role of barriers that delay cells in the growth zones after the filing of the suspension before the growth of the colonies. The holes in the plate is made in the form of cylinders with smooth walls orthogonal to the big plane porous plate and topologically encoded, which allows improvements is painted a process of manipulating colonies and monitor their growth. Topological encoding of holes provides them to a given location in the porous plate. This allows you to simplify the process monitoring using cameras. Cylindrical holes and their orthogonal arrangement provides a reduction in energy costs for the separation of the colonies when the hammer. Hammer is based on the principle of "hydracarina", i.e. the stage of acquisition of fluid flow sufficient linear velocity of the device, which serves as the upper chamber, and the stage of the abrupt closing of the valve with the formation of a shock front moving in opposite primary fluid flow direction. Holes functional role of the nozzles, which determine the flow velocity of its homogeneity. The application of nanotechnology allows you to adjust the nozzle size and configuration of the porous membrane, supporting colonies of microorganisms. The pore size of the porous membrane, impermeable to microbial cells, ensures the sterility of the bottom of the vessel, which leads to a single use device.

The invention explain the following graphics and tables:

Figure 1. The design of the device.

Figure 2. The cross-section of A hole in the plate with a porous membrane.

Figure 3. An example of a device made in the form of a sandwich structure for growing colonies Mick is one of the cells.

Figure 4. Pictures of a fragment of a porous plate of the anodic aluminum oxide and porous membrane.

a - holes and porous membranes;

b - with colonies on porous membranes.

in the porous membrane.

Figure 5-8. Phase 1 to Phase 7 of the device.

Fig.9. Timing chart of operation of the device.

Figure 10. The cross-section of a plate with a hole and a porous membrane:

and with microcolonies attached to the growth zone;

b - microcolonies in the initial stage of movement of the micro-colonies in the external device after separation from the growth zone.

11. SEM picture of the porous membrane from the anodic aluminum oxide:

a - top view, the average pore size of 105 nm, a porosity φ=0,1;

b - split;

in view of above, the average pore size of 198 nm, a porosity φ=0,71;

Mr. spall.

Pig Model of a fragment of a porous membrane with the cell attached to its surface:

a - section along the axis of the pores.

b - top view.

Fig. Model fragment porous plate with holes and porous membranes for hydraulic calculation.

Fig. View colonies of Staphylococcus aureus after 2 hours of growth on a porous plate, containing about 800 colony forming units (CFU).

Table 1. Technical and operational characteristics of the device using the inventive device and industrial analogues.

Table 2. The effect of the concentration of micro is different cells (S.Aureus) in suspension and the size of the growth zones of the porous plate on the efficiency of growth of the colonies.

Table 3. The effect of dilution of a clinical sample of a patient with suspected S.Aureus share of grown colonies.

Table 4. The dependence of the number of grown up (N0) and separated (Ndfrom the areas of growth colonies of flow velocity (ν) in the upper chamber of the device for plates with 400 growth zones and different diameter (D) holes for suspension with a content of S.aureus 1·107Meml1.

The device consists of (1) of the housing 1 divided by a partition 2 with the formation of two containers 3 and 4. The body of the vessel 3 has an opening 5 that is designed to supply the nutrient solution, and mated with him the check valve 6, which is designed to protect the pump, attached to this hole, from the back-flow of the nutrient medium. The body of the vessel 4 has an opening 7 that is designed to feed bacteria, provided with a valve 8. The upper surface of the housing 1, which is the lid of the container 4 containing two Windows 9, closed by a semi-permeable membrane 10 for oxygen or other gaseous environment, as well as the Central window, 11, with fixed therein an optical glass 12 to monitor the growth of colonies of microbial cells. The observation can be carried out using a video camera, a lens 13 which is fitted over the window 11 with optical glass 12. In the vessel 4 made the second hole 14 to exit the grown colonies, in which is installed a valve 15. In the vessel 3 made the second hole 16, which is equipped with a valve 17.

The partition 2 has a Central opening 18, which is fixed to the plate 19 of the porous aluminum oxide, the thickness h of the Plate 19 has openings 20 (2), of diameter D, each of which are porous membrane 21, the thickness h. The hole 20 has a cylindrical shape formed orthogonal to a large plane of the plate and are topologically encoded and can be chemically modified. The porous membrane 21, located in the holes 20, have pores 22.

Maybe a different location porous membrane 21 in the holes 20 of the plate, for example, figure 2 shows the location below the surface of the plate 19 with the formation of each hole of the hole 23, the depth ofa.

The surface of the plate 19 between the outputs of the holes is covered with a layer 24 of a material that prevents the attachment of cells.

An example implementation of the device design based on thick-film technology is shown in figure 3, where the device is a sandwich structure. On the base 1, made from polymer plates, placed the layer 1 b containing a capacitor 3 and associated holes 5(b) and 16(b), which is the entrance and exit to the nutrient solution, made by laser ablation, layer 1 b is placed a layer 2 containing hole 18, R is dius which corresponds to the radius of the porous plate 19, made of anodic aluminum oxide, and the holes 5 and 16 associated with the holes 5(b) and 16(b), which is the entrance and exit to the nutrient solution, respectively. On layer 2 with the plate 19 is placed a layer 1(c)containing 4 hole forming capacity 4 (figure 1) with paired him with channels connecting hole 4 hole 5(c) and 16(c), for the filing of a suspension of microorganisms and output of grown colonies in the external device, respectively. On layer 1(c) is a layer 1(d)containing holes 5(d) and 16(d), paired with holes 5, 5(b), (C) and 16, 16(b), (C), respectively, forming a channel for feeding the nutrient solution in the container 3 and its output, the holes 7(d) and 14(d), paired with holes 7(c) 14 b(c), respectively, for submitting suspension of microbial cells into the vessel 4 and the conclusion of the colonies in external device, and a hole with an optical window 11, 12. On layer 1(d) are the valves 6 and 17 associated with the holes 5(d) and 14(d), intended to regulate the flow of the nutrient solution, and the valves 8 and 15, associated with the holes 7(d) and 14(d), intended to regulate the flow of a suspension of microbial cells and output of grown colonies. The porous plate 19, a thickness of 80 μm made of anodic aluminum oxide with the formation of cylindrical holes and located in these porous membranes, the method of the inverse is autolithography and anodizing. The layer 2 device thickness of 80 μm made of Mylar film by laser processing. Layers 1(b) and 1(c) is made of PVC film thickness of 400 μm by laser processing. Layers 1(a) and 1(d) made of extruded acrylic film with a thickness of 1 mm diameter Holes 1 mm is made by machining. The valves 6 and 17 are made using precision casting and valves 8 and 15 manufactured using machining polycarbonate. The sealing device carried by the compression bolts in a specially made frame.

The porous plate 19 has a topologically encoded holes 20 (figure 4-a), which are porous membrane 21 (figure 4-b). The diameters of the holes in the porous plate is from 2 μm to 20 μm. Figure 4-a shows a porous plate with a hole diameter of 6 μm. Colony 24 grow on the porous membrane 21 of the anodic aluminum oxide located in the holes 20 (figure 4-b). The porous membrane 21, formed of anodic aluminum oxide (figure 4), are characterized by values of porosity φ=Smeme/Sthen=0,1÷0,7, where Smeme- size of the membrane, Sthen- the area of the pores on the membrane surface.

The operation of the device is as follows and has a phase:

Phase 1, see figure 5.

- lling 3 and through the holes of the plates of porous radnog the aluminum oxide 19 capacity 4 liquid medium, for example, a nutrient solution with the exclusion of air from the tanks and then. Pump N1 creates a flow of a nutrient medium with a bulk velocity Q1, the check valve 6 and the valve 15 are opened, the valves 8 and 17 are closed.

Phase 2, see figure 5-b.

- lling 4 a suspension of microbial cells, the valves 8 and 17 are open, the check valve 6 and the valve 15 closed. Pump H2 feeds into the tank 4 flow of suspensions of microbial cells with a bulk velocity Q2;

Phase 3, see Fig.6.

- rinse the cavity 3, the check valve 6 and the valve 17 are opened, the valves 8 and 15 are closed, the pump H1 sends a stream of wash solution into the container 3 with a high volumetric flow rate of Q3;

Phase 4, see Fig.6-b.

the growth of colonies of microbial cells, the check valve 6 is open and the valves 8, 15, 17 are closed, the pump H1 creates a flow of nutrient medium in a container 3 with a low volumetric rate of Q4ensuring sustenance of growth zones in the vessel 4;

Phase 5, see Fig.7.

- creation of conditions for the occurrence of water hammer, the check valve 6 is open, the valves 15 and 17 are open, the valve 8 is closed, the pump H1 creates the flow of the nutrient medium in the vessel 3 with high volumetric rate of Q5;

Phase 6, see Fig.7-6.

- creation of wet running, the check valve 6, valve 8 and 17 are closed, the valve 15 is open, the shock wave, creating additional pressure Pbeatspassing through the medium in the vessel 3 is through the holes and pores in the plate, separates the grown colonies of the growth zones.

Phase 7, cm Fig

- transfer of grown colonies of microbial cells in the external device, the check valve 6 and the valve 8 and 17 are closed, the pumps H1 and H2 generate a flow of nutrient medium with a bulk velocity Q6,providing transfer of grown colonies of microbial cells in the external device through the valve 15.

The timing diagram in figure 9 illustrates the operation of the device, namely the state of the valves (closed - "0" or open- "1") 6, 8, 15 and 17, the dependence of the volumetric flow rates generated by the pumps H1 and H2, the state of the sensor (camera), the dependence of the pressure P in the vessel 3 mode hammer from time to time. On the x-axis selected time intervals, corresponding to the phases of operation of the device, Phase 1 to Phase 7. In the diagram (Fig.9), the following notation: Q1- volumetric rate that is generated by an external pump H1, ensuring the flow of the nutrient medium for flushing tanks 3 and 4, and transfer of grown colonies of microbial cells. Q2- volumetric rate that is generated by an external pump H2, ensuring the flow of suspensions of microorganisms. Q3- high volumetric rate of flow created by the pump H1, providing the washing chamber 3 and the hammer. Q4- low space velocity created by the pump H1, ensuring wetting of the growth zones. Din the sensor (camera, see figure 1), providing a signal to start operation of the pump H1 mode surge (SU) for the separation of the grown colonies. P is the pressure in the vessel 3.

The method of growing colonies of microbial cells is as follows. To fill the device nutrient medium during Phase 1 (figure 5-a) into the tank 3 serves nutrient solution with a volumetric flow rate of Q1using, for example, the external pump H1. Pump N1 provides sufficient pressure for the flow of the nutrient medium through the holes 20 in the plate 19 and the pores 22 of the membrane 21, wetting and degassing tanks 3 and 4. During Phase 2 (figure 5-b) serves a suspension of microbial cells a certain concentration into the container 4 with the volumetric rate of Q2for example, using an external pump H2, microspace or automatic pipettes. The suspension serves up her uniform distribution on the surface of the plate 19 (if necessary, remove the air bubble using a semi-permeable membrane 10 (1) or valve 15, which is temporarily open. The distribution of the nutrient solution are monitoring through the window 11, with fixed therein an optical glass 12 on which is located the lens 13 of the video camera. Then before growth in Phase 3 (6-a) the composition of the nutrient medium in the vessel 3 lead standard condition by washing with a pump N1 is ri speed Q 3. Begins Phase 4 (6-b) growth of the colonies, during which provided the necessary conditions for dividing microorganisms: power, temperature, humidity, (by placing the device in the incubator or by installing sensors regimes and to ensure the maintenance of parameters). The necessary conditions for the growth of aerobic cells provides Windows 9 with a semi-permeable membrane 10, which is permeable to gases but impervious to moisture and particles in the external environment. Nutrient solution is fed from the cavity 3 through the holes 20 in the plate 19 and the pores 21 using an external pump N1, providing volumetric rate. Monitoring the growth of the colonies lead through the window 11 with fixed therein an optical glass 12 on which is located the lens 13 of the video camera. After reaching a sufficient size (N=100÷1000 cells in the colony), which is fixed either by using the camera DVKthe role of the sensor, with the submission of the respective control signal, or a time determined experimentally, colonies of microbial cells detach from the growth zones to water hammer. To do this, in Phase 5 external pump N1 is switched to the high flow rate of Q5(Fig.7-a). After establishing in the vessel 3 of a given volume flow rate Q5abruptly closed at time t valves 17 and 6 (Phase 6). In the vessel 3 at clap the 17 increasing shock pressure and shock wave occurs, directed from the valve 17 (Fig.7-b). The shock wave propagates into the holes 20 and pores 22, providing a pressure surge without substantial movement of the fluid, resulting colonies are detached from growth zones.

Thanks to the passage in the growth zone of the shock wave, high pressure micro-colonies grown detached from growth zones (removed from the sites or are pushed out of the holes). Then the volumetric flow rate Q6transfers colonies in the external device, for example, identification (Fig).

Having grown up during Phase 4 colonies of microbial cells are fixed in the growth zones in the wells (figure 10-a), as the remaining surface of the plate 19 is covered with a layer of material that prevents the attachment of cells. During the implementation phase 6 colony 24 are detached from growth zones, as shown in figure 10-b.

To clarify the operation of the device, you must evaluate the following specifications:

1) is Characterized by energy and power connection bacteria (and, respectively, of the colony) with surface areas of growth.

2) the Value of the pressure Pbeatsrequired for the separation of bacteria (true for colonies).

3) Parameters of water hammer (the speed in the accelerating chamber, closing speed valve 17), providing a pressure Pbeats.

4) flow Settings, providing transfer of colonies in the external device is STV.

Consider the above specifications 1-4 operation.

1) Consider the porous membrane from the anodic aluminum oxide (11), which on the surface is a microbial cell in the form of a disk with a diameter of DMCL=10-6m (assume that the cell takes the form of a disk on the surface). From 11 shows that the pores of the porous membrane are quite regularly (11-a, -b), are characterized by a narrow size distribution and, depending on the anodizing conditions, may have pores of a given average diameter of, for example, dcf=105 nm (figure 10-a) or dcp=208 nm (figure 10). Moreover, 11-b, -g shows that the pores are characterized by a high aspect ratio and shape close to cylindrical. Therefore, to evaluate the strength of adhesion of microbial cells to the surface of the membrane using the model in the form of a plate with cylindrical pores, as shown in Fig-and-B. Then, if the contact area Spinbacteria on a solid flat surface is:

Spin=πDMCL2/4=3,14(10-6m)2/4=7,8 10-13m2,

and the cross-sectional area of the pores of the porous membrane Spores(Fig-b) diameter d=2·10-7m:

Spores=πd2/4=3,14(2·10-7m)2/4=3,14·10-14m2,

that, in turn, for four long that peregrinae is a typical bacterium (Fig, -b)is the area of S:

S=4·3,14·10-14m2=1,26·10-13m2,

the area of contact of the bacteria on porous surfaces will be:

Spin/long=7,8 10-13m2-1,26·10-13m2equals 6.54·10-13m2.

To evaluate the strength of the adhesion of the bacteria to the surface of the growth zones (figure 10, Fig) assume that the average density of links is three connection 1·10-18m2. Then the number of links NSton the whole contact area bacteria will be: NSt=3·(6,54·10-13/10-18)=2·106links.

The maximum energy of a single van der Valsava (d-In) type (which predominate in terms of the implementation of the method) according to literature data (Kaplan I.G. introduction to theory of intermolecular interactions. - M.: Nauka. The main edition of physico-mathematical literature, 1982. 312 C.) is, EIn-d-In(max)=1,36·10-23J. (table 2), and the total energy In-d-In ties on all the bacteria will be E=NStEIn-d-In=(1,36·10-23)·(2·106)=3·10-17J.

Then the strength of connections all bacteria F1(taking the van der Waals interaction for the major) can be estimated as:

F1=E/RIn-d-In=3·10-17/0,6·10-9=5·10-8H.

Here RIn-d-In=0,6·10-9m characteristic length In-d-In connection.

2) the pressure required for the separation of the bacteria from the growth zone, the can is about to receive, dividing F1square S from example 1, we obtain RRef=F1/S=5·10-8H/1,26·10-13m2=3,9·10-5PA.

In the General case for bacteria (colonies) of arbitrary shape can derive the following criterion for determining the amount of pressure needed to detach from the growth areas:

Pabouttp(1-φ)φnIn-d-InFin-d-in(1)

Here φ=0,6 - porosity value plots of the membrane containing pores defined as the ratio of the sectional area of cylindrical pores to the area of the membrane.

Because the pressure required for the separation of bacteria (at maximum rating) has a quite high value - 3.9 ATM., it is advisable to use for the separation of the Colo is s (figure 10-b) from growth zones hydraulic shock, or the pressure surge caused by a rapid change of velocity of fluid flow in a very short period of time (Calico VI, thrushes E.V., Komarov A.S., Chizhik SURDS Fundamentals of hydraulics and aerodynamics, "Stroyizdat", 2002). The use of water hammer will allow the use of miniature pumps low pressure, the most suitable for portable diagnostic equipment, to achieve in a short period of time sufficiently high values of the shock pressure required for carrying out the functions of the device - isolation and moving colonies.

A number of studies conducted numerical study of the propagation of shock waves in a thin capillary. For example, in the work Mcevenue, Gview, Ehara, Dwhatever, Angurbala "Modeling the propagation of shock waves in the microchannel, with consideration of the effects of viscosity" (proc. proc. "Modern problems of applied mathematics and mechanics: theory, experiment and practice", Novosibirsk, Russia, 30 may - 4 June 2011 90/fulltext/47499/47500/Ivanov_full_paper.pdf). conducted numerical simulation of the processes of entry and propagation of shock waves in a thin capillary gas environment, taking into account the viscosity using, including okololiteraturnogo approach. For the last case, in the numerical experiments obtained a slight attenuation of the shock wave as magic cube MOV is I in the capillary. Thus, it can be assumed that the shock wave generated by a sudden closing of the valve 17, entering into the holes 20 and pores 21 extends to the exit, gradually fading. In this article, the experiment is conducted for gas at Re=400. Using the principle of similarity by the Reynolds number, get that to achieve conditions similar numerical experiment for gas, in the proposed device the fluid flow in the vessel 3, performs the function of the accelerating chamber must purchase a speed of 4 m·s-1that corresponds to the range of speeds of operation of the device. Then using the data obtained in the above article is true. In this case, we can assume that the distribution in the holes 20 and then 22 (Fig) shock wave is fading, losing at most 30% of energy. Thus, taking into account attenuation, at the entrance to the hole it is necessary to develop a pressure of not less than 5,57 ATM to obtain at the outlet of the capillary pressure P≥RRef.

3) Increase of pressure in direct hydraulic impact, i.e. that at which the phase of the kick T shorter closure time (τ) of the valve 17, is determined in the ideal case for the hard walls of the chamber and the low compressibility of the liquid by the formula 2:

Pyd=ρ( ν0-ν1)c(2)

where Rbeats- increase pressure in PA, ρ is the liquid density in kg/m3, ν0and ν1- the average velocity of fluid flow in the channel before and after closing of the valve 17 (6-a, -b) m·s-1and c is the speed of propagation of the shock wave in the fluid in m·s-1.

To estimate the ν0necessary to obtain the pressure Pbeats≥RRef=5,57·10-5PA, providing separation of bacteria, we assume the speed of sound in the capacity of the device is equal to the speed of sound in unlimited aquatic environment cwater=1,348·103MS-1and the speed ν1=0.

Evaluate the nature of the hammer, i.e. the ratio of the phase of the shock wave and the time of closure of the valve is. The characteristic size of the accelerating chamber l in the inventive device is assumed to be 2 mm, then the phase of the kick T 2.5·10-5c. If the closing time (τ) of the valve 17, τ<a 2.5·10-5then is a direct hammer, calculated according to the formula (1). Then, substituting in the formula (1) as Pbeatsthe relevant value, we obtain:

ν0=Pbeats/(ρcwater)=5,57·105/(103·1,348·103)=4,13·10-1m·s-1

If τ>2,5·10-5then the hammer is called indirect and applies the formula

Pyd=2ρν0lt(3)

Then, p and τ=10 -3c, l=2·10-2m, we get ν0=Pbeatsτ/(2ρl)=3,9·105·10-3/(2·103·2·10-2)=10 m·s-1and when τ=10-4s, ν0=1 m·s-1.

In the General case of the expressions 1 and 3 for indirect hammer you can get the formula:

ν0(1-φ)φnIn-d-InFIn-d-Int2ρl(4)

Thus, for use in laboratories on a chip, where the magnitude of the volumetric feed fluid is limited by the small size pumps and hydraulic resistance of small-diameter tubes (capillaries), practical considerations require the use of hydraulic impact (both direct and not direct). In any case, R is the realization of such a surge would require the application of high-speed locking mikrolimano, like described in the patent (US 20110073788, F16K 31/12, F16K 31/02, 2011).

For more accurate calculation of the critical velocity ν0it is necessary to consider the dependence of the speed of propagation of the shock wave in the fluid from the dimensions and material characteristics of the accelerating chamber.

At the instant the valve critical speed ν0can be found using formulas 1 and 2. From the condition Rbeats≥RRefwhen ν1=0, we

ρν0c(1-φ)φnIn-d-InFIn-d-In(5)

ν0(1-φ)φ1ρcnIn-d-InF In-d-In,(6)

where:

c=1ρ(1Einaboutdand+ainnytpbEKaboutppywith a)(7)

Then for speed in the accelerating chamber ν0received:

ν0 (1-φ)φ1ρcnIn-d-InFIn-d-In(1Einaboutdand+ainnytpbEKaboutppywith a)ρ(8)

where:aintthe diameter of upper chamber is 1 mm;

b - thickness of the walls of the upper chamber (the thickness of the thin wall - plate with holes is 100 μm, the other wall thick - 2÷5 mm);

Ewater- the modulus of elasticity of water 2·109n·m-2;

Ecase- the modulus of elasticity of the material of the body (for example, anodic aluminum oxide - 0,6·1011n·m-2for aluminum - 0,71·1011nm-2for polymer - 0,032·1011n·m-2).

4) Estimate the volumetric flow rate passing through the porous membrane at a constant pressure differential ΔP=10 5PA generated by the pump H1 in Phase 1 and Phase 7. For example, in Phase 7, the flow Q6will be determined by the number (N), radius (r) and length (h) then (Fig), because they will create the greatest hydrodynamic resistance of the flowing fluid. The calculation of the flow rate Q at the same time can be produced using the formula (2) Poiseuille flow (Lgini. Fluid and gas mechanics. - Leningrad, 1950. - 676 S.):

Q=πr48ηhΔP,(9)

where η is the dynamic viscosity of the fluid. For water at temperature T=20°C, η=10-3PA·S. At ΔP=105PA, r=10-7m, h=10-5m Substituting these values into formula (9), we obtain:

Q=3,14(10-7 )4810-310-5105410-16m3with a-1

Estimate the total number of pores of porous membranes throughout the porous plate. For the evaluation of pore density on the surface (ns), you can use the following expression:

nS=φ/(π·r2)=0,6/(3,14·(10-7)2)=1,91·1013m-2.

The surface area covered with pores, can be estimated by the formula

S=N1·π·R2=1.6 x 103·3,14·(5·10-6)2=1,26·10-7m2where N1=1.6 x 103the number of holes on the plate, R=5·10-6m is the radius of the hole.

Then the total number of pores of porous membranes throughout the porous plate can be calculated by the formula

N=nS·S=2.4 x 106.

Full flow through the pores of porous membranes throughout the porous plate will be equal to:

Q6=N·Q=2.4 x 106·4·10-16≅10-9m3·with-1.

Calculate the linear speed (νn) transfer of microbial cells in Phase 7 under the action of flow Q6. For this purpose it is necessary to estimate linear / min net is ü flow in the vessel 4 and related communications with external devices (Fig), at volumetric flow Q6. When the size of the upper chamber 10-4m×10-3m (cross-sectional area s=10-7m2), the average linear flow velocity νndetermined by the expression:

νn=Q/s=3·10-9/10-7=3·10-2m·s-1

The velocity of Q6sufficient to maintain colonies in the stream and compensation sedimentation on a porous plate and to transfer the colonies to the external device. For additional regulation of the flow in the external device may also be used to pump H2 in the regime Q2.

EXAMPLE 1

Preparing a suspension of a strain of microbial cells at a concentration of 107CoML-1(CFU - colony forming units) and serially diluted to concentrations of 1·102, 1·1031·1041·1051·106CoML-1pick six samples of the suspension S.aureus. To fill the device with liquid environment (Phase 1, figure 5-capacity 3 filed saline solution with a volumetric flow rate of Q1=10 ál·-1using a syringe pump H1 . Thus, provided the wetting and degassing tanks 3 and 4. Then the suspension of microbial cells of each of the samples with a volume of 20 μl was applied to the tank 4 through microspace with a bulk velocity Q2=2 ál·-1(Phase 2, figure 5-b)to its uniform distribution over the spine of the porous plate 19. The distribution of the suspension was observed in the window 11 with the external camera. Then before growth, the composition of the nutrient medium in the vessel 3 made in the standard condition by washing with a pump H1 at a speed of Q3=10 ál·-1(Phase 3). Then began the phase of the incubation and growth of colonies of microbial cells. Nutrient solution was applied using a pump H1 with a bulk velocity Q1=0,16 PL·-1and set temperature for growth is 37°C using an external thermostat (Phase 4). Used porous plate containing 400 growth zones with a diameter of 20 μm, a pore diameter of 200 nm and a porosity φ=0,6. After incubation for 3 hours (Phase 4) investigated porous plate with holes with a microscope. Microscopic examination showed that, depending on the concentration of the initial suspension, part of the growth zones (from 0.5% to 60%) are observed grown colonies of S.Aureus as Fig. Investigated the influence of the initial concentration S.aureus per share growth zones, which were growing colonies, watching the results of the incubation using a microscope. The results of the observations are given in table 2, column 7.

The experience was repeated with plates with 400 growth areas with a hole size of 2 μm, 7 μm and 10 μm, an average pore diameter of 200 nm and a porosity φ=0,6. The results are shown in table 2, columns 4, 5, 6.

Then there was a disconnect and transfer of grown colonies of microorganisms in the external device for plates with holes 7 μm, 10 μm and 20 μm. The transfer was carried out hydraulically by using a hammer. For the implementation of water hammer external pump H1 resulted in a high volume rate of Q5=0,8·10-6m·s-1(Phase 5), which corresponded to a linear speed of 1 m·s-1(the cross-sectional area of the accelerating chamber 3 figure 3 is 0.8 to 10-6m2), then blocked at time t valves 6 and 17 (figure 3) in accordance with the Phase 6 of the device. Resulting in the vessel 3 there was a shock wave, propagating in the direction of the growth plate 19. Then the valve 8 is opened and the pump H1 translated in the regime Q6=1·109m3·with-1and observed with a microscope in the channel 14 of the device and have them count. The data are given in table 3, column 3.

Next, the experiment was repeated for plates with holes with a diameter of 7 μm and 10 μm. The data are given in table 3, columns 5 and 7, respectively.

From table 3 it can be seen that at speeds below 1 m·s-1there has been a significant decline in the share moved in the channel 14 of the colonies. When exceeding a speed of 5 m·s-1there is destruction of the porous plate with holes.

EXAMPLE 2

Were sown in the proposed device (like 1) microorganisms samples of a patient with suspected lesions of the nasal S.aureus. Made a swab of the nasopharynx of the patient and by flushing the sample of mucus with a cotton swab in saline in a volume of 1 ml and deposition of fibers and particles by sedimentation for 5 min was preparing a suspension of the microbial population. Prepared samples of suspensions of different concentrations by step tenfold dilution after dilution with 10, 100 and 1000 times. Moved 20 µl of the suspension from each sample into the device as in example 1. Used the plate containing 400 holes, with a diameter of 10 μm. After conducting phases 1-3 of incubation under the conditions as in example 1, was investigated with the aid of a microscope. The total filling of the growth zones of the device was 10-40%, and according to visual expert evaluation was observed colonies of four types: S.Aureus, Neisseria meningitidis, S.pneumoniae, Enteroccocus. The results are shown in table 4.

Then there was a disconnect and transfer of grown colonies of microorganisms in the external device (channel 14) for plates with holes 7 μm, 10 μm and 20 μm. The transfer was carried out hydraulically by using a hammer. For the implementation of water hammer external pump H1 resulted in a high volume rate of Q5=0,8·10-6m3·with-1(Phase 5), which corresponded to a linear speed of 1 MS-1(the cross-sectional area of the accelerating chamber 3 figure 3 is 0.8·10-6 m 2), then blocked at time t valves 6 and 17 (figure 3) in accordance with the Phase 6 of the device. Resulting in the vessel 3 there was a shock wave, propagating in the direction of the growth plate 19. Then the valve 8 is opened and the pump N1 translated in the regime Q6=1·10-9m·s-1and observed with a microscope in the channel 14 of the device and have them count. The data are given in table 3, column 3.

Table 1
Technical and operational characteristics of the device using the inventive device and industrial analogues
Technical solution
Characteristic of the device
Method Kirby-BauerAutomatic device VITEK®2 compact 60 (Biomerieur, France)Lab on a chip, which can be used by the proposed method and device
1234
The composition of the devicePetri dishes with nutrient medium, the sets of discs impregnated with antibiotics, microbiological incubator with cooling KW (Binder, Germany), biological microscope.The read-out device, disposable card for analysis of microbial cells, a personal computer.Portable automatic reading device, disposable lab on a chip containing the following modules: preparation, incubation and growth of bacteria, identification, determination of viability, the microcomputer on-Board module navigation links.
Conditions of useSpecialized laboratory (GOST R ISO/IEC 17025-2000)Specialized laboratory (GOST R ISO/IEC 17025-2000)No accreditation is required by GOST R ISO/IEC 17025-2000, Autonomous conditions.
PurposeStudy of the sensitivity of microorganisms to antibiotics.Identification of bacteria and yeast. Determination of the sensitivity of microorganisms to antibiotics.Sample processing, creation of conditions for microbial growth, differentiation and counting of microorganisms, determination of the sensitivity of microorganisms to antibiotic drugs.
The method of reading information on identification NoColorimetricThe recognition of three or more attributes.

1234
The method of reading information on sensitivity to antibioticsThe manual method of measuring the clearance around the discs impregnated with antibiotics with a ruler.TurbidimetryMicrodensitometry, position in the microchannel: camera, image recognition.
Overall dimensions, mm433×516×618590×715×6725<150×200×200
Weight, kg4475<3
Power, W340102530
The number of modules32 (a single computer)1
Level automates the tion LowHighHigh
Performance: samples per day406020
Time 1 analysis, h48-7226-346-8
IdentificationNot be performedToTo

1234
The number of antibiotics6 (per Petri dish)2020
Accuracy, %959091
Flexibility flexibilityYesNoYes
Operating costs for 1 sample
RUB
10001000 350
Price, million rubles0,175 (import in the Russian Federation)6,0 (import in the Russian Federation)0,03.

Table 2
The effect of the concentration of microbial cells (S.Aureus) in suspension and the size of the growth zones of the porous plate on the efficiency of growth of colonies
The concentration of microbial cells of S.aureus in suspension CFU ml-1The number of microbial cells of S.aureus in the sampleThe size of the growth zones of the porous plate, microns
271020
1234567
The share of grown colonies in areas of growth, %1·1022000,250,5
1·103200,251,21,54,5
1·1042·1020,546,2512,5
1·1052·1032,5102535
1·1062·10415203245
1·1072·10520254560

160
Table 3
The dependence of the number of grown up (N0) and separated (Ndfrom the areas of growth colonies of flow velocity (ν) in the upper chamber of the device for plates with 400 growth zones and different diameter (D) of the holes for the suspension with a content of S.aureus 1·10 7CFU ml-1.
The flow velocity ν m·s-1The number of grown N0and moved into the channel 14 (Fig 3-b) (Nd) colonies
d=20 mmd=10 µmd=1 µm
N0NdN0NdN0Nd
1234561
0,0122010178109710
0,12355017910010519
125015018910425
22452101851839970
325622519018010080
423923017617510199
52412401801959594

Table 4

0
The effect of dilution of a clinical sample of a patient with suspected S.Aureus per share has grown in the growth zones of colonies N0and the share moved to channel 14 colonies Nd
Dilution of the clinical sampleView of microbial cells
S.aureus, %Neisseria meningitidis, %S.pneumoniae, %Enteroccocus, %
N0NdN0NdN0NdN0Nd
123456789
1:10600000000
1:10500000000
1:1040000000
1:10333000011
1:10288002233
1:105048221091515

1. The method of growing colonies of microbial cells on the surface of the porous plate, including the supply of the nutrient solution from the bottom up through the porous plate in the zone of growth of colonies of microbial cells, formed on its upper surface, the flow of suspensions of microbial cells on the upper surface of the porous the second plate, controlled environment growth of the colonies, for the monitoring of the growth of the colonies, removing the grown colonies of microbial cells from growth zones and transfer them to the external identification means, characterized in that the,nutrient solution serves in the zone of growth of colonies of microbial cells by creating a pressure differential between the entrance and exit holes made in the plate of the anodic aluminum oxide orthogonal her big plane and topologically encoded, which formed the said growth zone in the form of porous membranes, with a porous membrane placed either flush with the top surface of the plate, or with the formation of the hole, do not miss the microbial cells, continue to serve the suspension of microbial cells of a given concentration at the top surface of the plate to a uniform distribution on the surface of the plate between growth zones formed film, preventing the attachment of microbial cells, and disconnecting growing microcolonies from growth zones is carried out by the hammer, aimed on the input side of the cylindrical holes of the plate and extending along them and then through the pores of the porous membrane with a force, not a destructive micro-colonies, but sufficient for their isolation from the growth zones in the area of water hammer pressure
where:
- the value of the porosity of the porous membrane
n- the number of relationships of microbial cells and the membrane surface per unit surface area;
F- the average power of a single communication van der Valsava type between the surface of microbial cells and the surface of the membrane.

2. Device for growing colonies of microbial cells by the method according to p. 1, containing a porous plate made of anodic aluminum oxide formed with its upper surface areas of growth colonies of microbial cells, wherein the porous plate made of anodic aluminum oxide is the case with the formation of the top and bottom of the tank corps, which are equipped with input and output valves for liquids, the bottom tank is provided with entrance and exit to the nutrient medium of microbial cells, and its input is for connection with an external pump, the top of the tank is provided with an inlet for the suspension of microbial cells and access to growing microcolonies, and the upper surface of the case is made permeable to gases but impermeable to moisture and particles in the external environment, the lower container is intended for formation of the hammer with enough force for the separation of the colonies from growth zones, but do not destroy the colony, with a porous plate made of anodic aluminum oxide has a hole in cilindrica the some form formed orthogonal to a large plane of the plate and topologically encoded, and each hole is located a porous membrane with pores, preventing microbial cells, forming a zone of growth, and the porous membrane are either flush with the top surface of the plate, or with the formation of holes on the surface of the plate between growth zones formed film, preventing the attachment of microbial cells.

3. Device for growing colonies of microbial cells according to claim 2, characterized in that the foil is made of metal, polymer or hydrophobic material that prevents the attachment of microbial cells to the surface of the plate.

4. Device for growing colonies of microbial cells according to claim 2, characterized in that the porous membrane is made of anodic aluminum oxide, ceramic, polymer or metal.

5. Device for growing colonies of microbial cells according to claim 2, characterized in that the upper cover of the container is equipped with a Central window, with fixed therein an optical glass for monitoring the growth of the colonies.

6. Device for growing colonies of microbial cells according to claim 2, characterized in that the upper cover of the container is equipped with Windows closed semi-permeable membranes, serving for the transmission of gases, but which is not permeable to moisture and particles is the current environment.



 

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2 dwg, 1 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: invention is a method of determining the nonspecific resistance of pathogenic microorganisms to antibiotics and the fact of the presence of bacterial biofilms on the basis of measurement of catalytic activity of phosphodiesterases cleaving the cyclic diguanosine monophosphate, with a threshold sensitivity of 50 pg/ml, comprising: 1) isolating the target-phosphodiesterase from lysed bacterial cells; 2) binding of phosphodiesterase with biotin-conjugated antibodies specific for non-catalytic domains of phosphodiesterase; 3) affinity purification of complexes formed by target-phosphodiesterase and biotin-conjugated antibody using paramagnetic particles containing neutravidin or its analogs that bind biotin; 4) interacting of the complexes of phosphodiesterase/biotin-conjugated antibody, immobilised on paramagnetic particles with complexes containing a-di-GMP in the form of G-quadruplex systems with intercalate dye, which is accompanied by decrease in the intensity while destruction of complexes of intercalate dye with c-di-GMP; 5) measurement of decrease of fluorescence upon hydrolysis with c-di-GMP and destruction of complex of c-di-GMP with intercalate dye, followed by quantitative estimation of the phosphodiesterase activity based on calibration curves made using known amounts of the recombinant enzyme of phosphodiesterase identical to the test target; 6) identification of increased level of phosphodiesterase activity detected by the test antibiotic-resistant bacterial strains capable of biofilm formation, as compared with the level of phosphodiesterase activity that can be detected for the control strains of bacteria of the same species not having the antibiotic resistance and the ability to form biofilms.

EFFECT: method enables to determine the nonspecific resistance of pathogenic microorganisms to antibiotics and to establish the fact of the presence of bacterial biofilms.

4 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to medical microbiology and a method of determining activation of plasminogen with bacteria. The method involves adding protamine sulphate to a prepared supernatant fluid, incubating the obtained mixture, depositing cells by centrifuging, incubating the supernatant fluid with the protamine sulphate, depositing protein and detecting activation of plasminogen with bacteria from the amount of split arginine, content of which is determined by Sakaguchi method from the red colour of the sample.

EFFECT: invention enables to detect activation of plasminogen with bacteria in vitro using protamine.

4 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is container for isolation and identification of microorganism. Container includes upper part, which has wide internal diameter, and lower part, which has capillary tube, middle conic part, connecting said upper and lower parts, and optic window on the bottom and/or on one or more than one wall of container. Optic window is less than 0.1 inch (2.54 mm) thick and is transparent for wavelength of near-infrared, visible and/or ultraviolet light spectrum. Window contains quartz, quartz glass, sapphire, acrylic resin, methacrylate, cyclic olefin copolymer, cycloolefin polymer or any their combination. Capillary tube has internal diameter from 0.01 inch (0.03 mm) to 0.04 inch (1.02 mm).

EFFECT: container ensures isolation of microorganisms, which are free of interfering materials and compatible with fast identification technologies, from hemoculture and other complex samples.

8 cl, 12 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to microbiology and biotechnology. Analysed bacterial strains are inoculated on a dense nutrient medium. Paper disks impregnated with a disinfectant are applied. They are incubated in a temperature chamber until the bacteria start growing. The bacterial growth inhibition areas are measured. A quantity of the grown colonies is counted to construct a dependence diagram of the bacterial growth inhibition area and the quantity of the grown colonies after the disinfectant reaction. The diagram and Shughart inspection sheet are used to assess the disinfectant activity on specific types of the microorganisms. The disinfectants with mean measurements of the bacterial growth inhibition area are above an upper control limit of the Shughart inspection sheet are considered to be high bactericidal activity agents. The disinfectants with mean measurements of the bacterial growth inhibition area are below a lower control limit of the Shughart inspection sheet are considered to be low bactericidal activity agents. The disinfectants with mean measurements of the bacterial growth inhibition area between the control limits of the Shughart inspection sheet are considered to be mean bactericidal activity agents in relation to all analysed agents.

EFFECT: method enables assessing the bactericidal activity of the disinfectants.

3 dwg, 3 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used in biological treatment of waste water from electroplating plants from heavy metal salts. The method involves adding yeast biomass to waste water, said biomass being in form of brewery wastes containing a combination of yeasts of different strains of Saccharomyces cerevisiae with viability of 90-95% in a given amount. The biomass is mixed with the waste water to obtain a suspension. The obtained suspension is held for 8 hours at temperature of 10°C-29°C and solution pH of 5.5-8.0, followed by recycling spent yeast containing heavy metals by treating with lime Ca(OH)2, with the ratio of yeast biomass to lime of 1:5-8, to obtain a mixture. The obtained mixture is subjected to wet treatment at temperature of 90°C for 1 hour, followed by isolation of the obtained mixture, which contains heavy metals, in concrete paste.

EFFECT: invention increases efficiency of purifying waste water from heavy metal ions.

3 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to molecular biology. Disclosed is a method of detecting frameshift and nonsense mutations in the BRCA1 gene, which involves construction of recombinant plasmids where the amplified gene fragment is located in a single translation frame with the alkaline phosphatase gene of E.coli (phoA). A plasmid vector pPhoA-frame, which contains a DNA sequence which encodes alkaline phosphatase of E.coli was constructed. A DNA fragment containing restriction endonuclease recognition sites BgIII, StuI, Apal, SacII and intended for cloning BRCA1 gene fragments (polylinker) was inserted into said DNA sequence. The amplified BRCA1 gene fragment is inserted into the plasmid vector pPhoA-frame in a single translation frame with phoA. The occurrence of mutations which violate reading frame integrity in the investigated gene fragment is evaluated visually from the absence of colour of E.coli. cell colonies transformed by the obtained recombinant plasmid on an indicator dish containing a substrate for alkaline phosphatase. The disclosed method enables to detect only mutations that are significant for development of pathology since it avoids detection of polymorphous versions which do not lead to stop codons and most cases have not significant effect on protein function. The method enables to detect any, including unknown, mutations which violate frame integrity.

EFFECT: method can be used to detect frameshift and nonsense mutations that are responsible for the development of a range of cancerous diseases in human genes.

2 cl, 3 dwg, 2 ex

FIELD: biotechnologies.

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.

1 ex

FIELD: biotechnologies.

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.

FIELD: biotechnology.

SUBSTANCE: strain of bacteria Exguobacterium mexicanum RNCIM V-11011 is grown, and the suspension is made from it, which is applied in the cryomorphic soil and water environment. It is exposed under the specified parameters from 7 to 60 days and the quantitative content of oil and petroleum products in the test soil and water environment is determined.

EFFECT: invention enables to reduce the time of denaturation of oil and petroleum products and to reduce the concentration of oil and petroleum products in the soil and water environment.

3 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed is a method of purifying an aqueous solution containing a nickel salt from nickel ions. The method involves contacting an aqueous solution containing 20 mg Ni2+/l with 0.2 g suspension of a homogenised culture per 1 l solution. The suspension has the following composition of dominant types of cyanobacteria: Phormidium ambiguum (Jom.), Phormidium boryanum (Kutz.), Leptolyngbya foveolarum (Rabenhor stex Gom), Plectonema boryanum (Gom.f.boryanum). Contacting is carried out for 1-3 hours.

EFFECT: invention enables to purify an aqueous solution from divalent nickel ions to concentration close to or lower than the maximum allowable concentration of 0,1 mg/l.

4 ex

FIELD: biotechnology.

SUBSTANCE: strain Moraxella bovoculi has antigenic, virulent properties and immunogenic activity. It is deposited under the number "SH-Ch6 N-DEP" in the All-Russian State Collection of strains of microorganisms used in veterinary medicine and animal husbandry FSBI "Russian national centre of quality and standardisation of drugs for animals and feed". The strain can be used in manufacture of diagnostic preparations and vaccines against infectious keratoconjunctivitis of cattle.

EFFECT: invention enables to produce diagnostic preparations and vaccines against infectious keratoconjunctivitis of cattle.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to biotechnology. The strain Bifidobacterium lactis CNCM I-3446 is applicable in preparing a probiotic composition for stimulating development of initial bifidogenic intestinal microbiota in the children delivered by caesarean section. The strain may be used in preparing the probiotic composition for reducing a risk of further development of allergy, as well as for preventing or treating diarrhoea in these children.

EFFECT: group of inventions provides stimulating the intestinal colonisation in the children delivered by caesarean section both with the above strain, and with the other species.

78 cl, 6 dwg, 4 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: bacterial strain Rhodococcus aetherivorans ofRussian classification of microorganisms BKM Ac-2610D is proposed. The bacterial strain is different in growth on the common organic-mineral medium at high nitrile hydrase activity, reaching 332 U/mg at 20°C or 521 U/mg at 25°C. The nitrile hydrase of the bacterial strain Rhodococcus aetherivorans of Russian classification of microorganisms BKM Ac-2610D is thermostable. Also the method of culturing this strain is provided. The cells of the strain are inoculated on the slant meat-and-peptone agar and grown for 24-48 hours. Then the biomass is washed with sterile saline solution with pH 7.0-7.4. The resulting suspension is inoculated to the first container with the nutrient medium. The process is carried out for 24-48 hours at 28-30°C, with the circumferential stirring at a speed of 140-160 rev/min until obtaining the optical density of the suspension of 2-16 units at 540 nm and the magnitude of the optical layer of 5 mm. The resulting suspension is inoculated to the second container, which volume is 10-100 times greater than that of the first container. The value of optical density in the second container is adjusted to 0.1-0.3. The strain is cultured for 48-120 hours at 26-31°C, the aeration of 0.5-1.0 the air volume/medium volume per minute until the value of optical density reaches 36-40 and pH 7.5-7.8. The resulting biomass is separated. Also the method of production of acrylamide is provided. Hydration of acrylonitrile is carried out with the acrylonitrile concentration not exceeding 0.5%. The hydration is carried out using the biomass of bacterial strain Rhodococcus aetherivorans of Russian classification of microorganisms BKM Ac-2610D based on the order of 400-500 g of dry weight of strain per 1 ton of the final product - acrylamide with the concentration of 45-49%. The inventions enable to obtain cells of Rhodococcus aetherivorans of Russianclassification of microorganisms BKM Ac-2610D of 10-18 g/l with the nitrile hydrase enzyme activity of 250-332 U/mg.

EFFECT: improvement of strain quality.

5 cl, 6 ex, 1 tbl

FIELD: biotechnologies.

SUBSTANCE: pharmaceutical composition includes bacteriophages obtained by cultivation on nutritional medium containing glucose, sodium chloride, twice-substituted sodium phosphate, liquid autolysed yeast and clean water in the specified ratio, and dried and having a filler without lyophilisation, in the form of pills with a gastral-resistant coating.

EFFECT: invention allows increasing safety of pharmaceutical composition.

7 ex

FIELD: biotechnologies.

SUBSTANCE: bacteria strain Salmonella enteritidis var. Issatschenko 32/3 deposited in the Departmental Collection of Beneficial Microorganisms for Agricultural Purpose of All-Russia Research Institute for Agricultural Microbiology (GNU VNIISHM Rosselkhozakademii) with registration No. RCAM 00149 has expressed pathogenic properties against mouse-like rodents. Efficiency of bioattractant obtained based on Salmonella enteritidis var. Issatschenko 32/3 bacteria strain and grain in grain and vegetable warehouses and green-houses was more than 90% in relation to sewer rat and house mouse.

EFFECT: improving efficiency.

1 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes Aeromonas bestiarum bacteria strain - producer of alkaline ribonuclease having antiviral activity and deposited in a collection of bacteria, bacteriophages and fungi of the Federal Budgetary Scientific Institution "The State Scientific Centre of Virology and Biotechnology "Vector" with registration No. B-1270. Strain has high production rate of alkaline ribonuclease - 921.8 U/ml and activity against A/H5N1 bird and A/Aichi/2/68 (H3N2) human being flu viruses.

EFFECT: improving strain properties.

2 dwg, 7 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to a preparation for babies for reduction or prevention of inflammation in a baby. The preparation for babies includes a source of protein, providing from 1 to 5 g of protein per 100 kkal of the preparation, a source of fat or lipids, providing from 3 to 7 g of fat or lipids per 100 kkal of the preparation, a source of carbohydrates, providing from 8 to 12 g of carbohydrates per 100 kkal of the preparation, a source of long-chain polyunsaturated fatty acids, including docosahexaenoic acid. The preparation also includes from 1×104 CFU to 1×1010 CFU of Bifidobacterium longum AH1206 NCIMB 41382 per a gram of the preparation. Also claimed are probiotic baby food and a method of reduction or prevention of inflammation in the baby or child with application of the said food.

EFFECT: invention ensures induction of anti-inflammatory response, makes it possible to reduce secretion of anti-inflammatory cytokines.

14 cl, 19 dwg, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and can be used in monitoring environmental-microbiological investigation of the quality of sea water to determine the amount of oil-oxidising microorganisms. The method involves preparing a mineral medium - bases containing NH4NO3, K2HPO4, KH2PO4, MgSO4, CaCl2, FeCl2, a concentrated solution, agar and distilled water in a given ratio, followed by addition of an oil product in a given amount, said product being bunker oil. Seeding sea water on the surface of the culture medium and incubating the seed for 3-4 hours enables to detect colonies of oil-oxidising bacteria.

EFFECT: invention increases precision of the method when detecting oil and hydrocarbon oxidising bacteria when carrying out environmental monitoring.

2 tbl, 3 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed is device for obtaining nanoparticles by reduction of metals from initial salts in presence of cultivated cells of microorganisms. Device includes control computer (1), connected with it electronic block of regulation and control (2) of all functional units and blocks of fermenter (3), pH-stabilising block (4) with pH sensor (5) and hoses for supply of titering solutions by pumps (6, 7), block (8) for regulation of redox-potential of culture mixture, provided with redox sensor (9), independently controlled pumps (10, 11) for introduction of initial solutions of metal salts, reducing agents and growth factors into fermenter (3), block (12) for regulation of dissolved oxygen level with sensor pO2 (13), pump (14) for supply of growth substrate, block (15) for measurement of optic culture density with application of optic fibre sensor (16), block (17) for measurement of spectral characteristics of culture mixture with application of optic fibre sensor (18), isolated with impermeable for cells membrane with pore size 100-250 nm, block (19) for thermoregulation of fermenter (3), equipped with temperature sensor (20), block (21) for regulation of culture mixture mixing, which brings into motion blade mixer (22), block (23) for regulation of culture mixture illumination in case of cultivating phototrophic microorganisms and control of spectral parameters of submersible diode lamp (24), block (25) for ultrafiltration of sampled culture mixture with sterilising membrane with pore size 100-250 nm with possibility of output of only nanoparticle suspension from fermenter, condenser of output moisture (26), preventing loss of culture mixture.

EFFECT: invention contributes to extension of arsenal of technological methods of obtaining nanoparticles of metals and makes it possible to achieve controllability of modes of nanoparticle formation.

2 dwg, 3 ex

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