RU2522005C2 - Method of growing colonies of microbial cells and device for its realisation - Google Patents

Abstract

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

Description

The invention relates to analytical instrumentation and can be used in the study of liquid biological and medical samples. They are most effectively used in express microbiological diagnostics, including determining the sensitivity of bacteria to antibiotics, as part of miniature analytical devices - laboratories on a chip for portable microbiological control systems.

Portable microbiological control systems with laboratories on a chip can work in laboratory and field conditions, and in the corresponding telemetry systems. The system should be easily reconfigurable to work with various clinical material and to search for pathogens of various groups of infections: respiratory, gastrointestinal, urogenital, nosocomial infections, assessment of bacterial contamination of the external environment.

The analysis of microbiological samples includes the isolation of microbial cells, the accumulation of biomass and the identification of strains of microbial cells, the determination of their number, as well as the determination of their sensitivity to antibiotics. This is necessary for the diagnosis of infectious bacterial diseases and the correct appointment of antibacterial therapy. This problem is especially relevant in connection with an increase in the incidence of infectious bacterial diseases and an increase in the number of strains of pathogenic bacteria resistant to many antibiotics. In 2010, the number of infectious and parasitic diseases in Russia reached 30069567 cases (according to Rospotrebnadzor: / asset_publisher / N2qH / content / 2). Traditional microbiological research is time-consuming and, more significantly, lengthy (3-7 days). It is carried out only in specialized institutions and, as a rule, for inpatients. The creation of miniature mobile devices for the rapid identification of pathogens and determining their sensitivity to antibiotics will make it possible to choose the right tactics for treating infectious diseases, increase its effectiveness, decentralize microbiological analysis and make it accessible to the entire population, including remote areas. The low cost and availability of the analysis will allow it to improve laboratory diagnostics for infectious diseases and improve the quality of treatment.

The longest (limiting) stage of conducting cultural microbiological analysis (the most reliable diagnostic method for infectious diseases) is the accumulation of microorganisms on nutrient media. It is this stage that needs improvement to increase the speed of analysis. The duration of this stage is determined by the need for the accumulation of colonies visible with the eye with obvious visual characteristics. After the initial incubation and accumulation, the material is manually selected for further studies, namely, identification, isolation and accumulation of a pure pathogen culture, and sensitivity to antibiotics. Known automatic and semi-automatic instrumental methods of microbiological analysis, implemented using stationary devices, however, they are also lengthy and require primary accumulation of cells. Comparative data on the technical and operational characteristics of the device using the inventive device and industrial analogues are presented in table 1.

The semi-automatic method of microbiological analysis is known (Table 1, column 2), which uses the inoculation of biological samples in Petri dishes with nutrient medium, incubation of samples in order to accumulate colonies of microbial cells, isolation of individual colonies and the study of their sensitivity to antibiotics according to the Kirby method Baer. In this method, many operations are carried out manually, which significantly reduces the performance of the method. An incubator of high cost and dimensions is used, which makes the method fundamentally unsuitable for working in remote places. A large amount of consumables is required. The method is not suitable for automation and cannot fulfill the technical task set in this invention.

The well-known automatic microbiological analyzer "VITEK ^® 2 compact 60" (Table 1, column 3), developed by bioMerieux SA (France), automatically identifies the type of pathogenic microbial cells and determines their sensitivity to antibiotics on separate microbiological cards for each of these types analyzes. For work, preliminary accumulation and isolation of a strain of pathogenic bacteria is required. According to the manufacturer, the analysis time is up to one day, and taking into account the preliminary accumulation of pathogenic microbial cells, two or more days. The device is stationary and not suitable for work in remote places of assistance, including in clinics. Its high cost determines the need for placement in large centralized microbiological laboratories. The device does not automate the procedure of preliminary accumulation and sorting of microbial cells with the isolation of strains of pathogenic or diagnostically significant microbial cells. Thus, this well-known industrial device cannot achieve the technical objectives set in this invention.

Advances in microelectronics technology, microsystem technology, and the principles of miniaturization in instrumental analysis have opened up the possibility of developing a new generation of technical tools - microanalytical systems, also called µ-TAS, lab-on-a-chip (lab on a chip), microfluidic systems, etc. (Manz A., Graber N., Widmer N.M. // Sensors and Actuators. 1990. B.1. P.244-248; Zimina T.M. Petersburg Journal of Electronics. 2000. No. 3-4. 79). Representing miniature devices made using planar and hybrid technologies, microanalytical systems are designed for various types of chemical analyzes and biomedical tests. Such systems can improve productivity in the implementation of biomedical and environmental analysis, the study of living organisms. The development of microanalytical systems allows us to solve the problem of reducing the cost, material and energy intensity of products, to increase the productivity of analysis. These improvements are possible due to the formation of such devices with the help of micro- and nanotechnologies, which allow to implement precision geometry, providing the possibility of geometric complementarity of components at the molecular level, increase the speed of analysis without additional costs, control mass transfer and automatic micro-scale control of the stages of the analytical process in integrated functional modules and subsystems. The transfer of biological objects in such systems can be carried out using flow analysis techniques, using the principles of microfluidics. Improving the methods of traditional microbiological analysis can be carried out precisely using the capabilities of micro- and nanoelectronics technologies.

A known method of growing cell cultures, in which the cells are grown in a growth reservoir containing a nutrient medium connected to a reservoir for a nutrient medium with the formation of a tangential flow in the growth reservoir using flow control and an external pump, the membrane is also located tangentially in the form of hollow fibers or frames with a filter providing a sterile barrier for cells, a node for selecting grown cells with the possibility of its opening and closing, a system for ensuring mass transfer of cultures with possible the ability to change the direction of the medium flow using a four-way valve, a filtration system in the form of a "stack of plates" and hollow fibers, for separating solid impurities, a means of replacing the nutrient medium, a means of concentrating cells, by removing excess fluid (US 6022742, C12M 1 / 36,2000 )

This method does not provide for the controlled growth of individual microcolonies attached to growth zones in order to reduce the growth time of identified microcolonies of cells, and their controlled transfer to external devices for identification and sorting of colonies is not possible.

A known method of growing biological cells, implemented in a liquid medium on a platform in microwells, connected by microchannels, through which the mediums that provide the growth of colonies. Microwells and channels are formed in the form of a profile, which, after coating with a cover plate, forms microchambers and capillaries. The cover plate has openings for access to the cameras. The cameras and channels are formed radially on a disk that can rotate to move the cameras through the aperture of the optical recording device for analyzing colonies without disturbing their structure in the cameras (US 6,632,656 B1, C12M 3/06, C12Q 1 / 20,2003).

This method does not provide for the possibility of separation and transfer of microcolonies for identification to external devices in order to isolate bacterial colonies and their subsequent accumulation to study sensitivity to antibacterial agents.

Known methods of growing colonies, carried out using miniature cells for cell growth, formed by applying films of polymers and / or oxides and fluorides of metals with a thickness of 0.01-0.2 mm and, then, forming by partial removal of the films of sections of a given geometry, and with surface properties that control 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 these methods, it is not possible to automatically detach and transfer the colonies to the identification and sorting unit in external devices in order to isolate colonies of microbial cells and test their sensitivity to antibiotics.

Closer to the claimed method is a method of cultivation and analysis of individual cell cultures, in which, after preliminary cleaning of the sample containing a variety of cells, the cells are placed in growth cells (or platforms) to conduct their simultaneous growth under specified conditions (humidity, temperature, composition of the liquid medium ) to obtain individual microcolonies. The method is carried out using growth cells, for example, formed using microtechnologies, of a very small size, so as to ensure the growth of individual cells in a size comparable to the size of the growth zone. In this method, the cell suspension is placed in the growth zones after separation of the suspension into separate volumes small enough to further ensure the growth of a homogeneous culture in each volume, they are seeded and incubated in separate microwells having a cylindrical shape, not separated by less than 1.8 mm, and the surface between them is covered with a water-repellent layer and is common to all "microcultures" or individual microcolonies, and during the incubation, the necessary pits come to the cultures substances through membranes with a pore size of 0.1 × 10 ^-7 ÷ 4 × 1 ^-6 with a thickness of 0.2 × 10 ^-7 ÷ 10 × 10 ^-6 (EP 1425384 (A2), C12M 1/00, 1/20 , 1/26, 1/34; C12N 1/00, 1/02; 2002).

However, in this method, the problem of the rapid separation of individual microcolonies and their transfer to external identification and sorting devices was not solved. The method is complex and not suitable for rapid analysis, and the corresponding device cannot be used in a portable device due to the complex design and low strength of the dispensers, it does not provide for the possibility of isolation and identification of pathogenic strains, with the aim of their subsequent accumulation and study of sensitivity to antibacterial drugs.

The closest in technical essence to the claimed method - the prototype is the method presented in the article Ingham, C. J., Sprenkels, A., Bomer, J., Molenaar, D., van den Berg, A., van Hylckama Vlieg, JET ., de Vos, WM “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 of anodic alumina, on the surface of which special zones are formed for the growth of microorganisms, limited by the walls of the polymer network formed by the lamination method followed by photolithography and plasma etching.

When implementing such a method of growing colonies of microbial cells, a nutrient solution is supplied by seeping through the pores of a porous anodic alumina plate conjugated with an agar layer through capillary forces. Next, microbial cells are manually fed, distributing them among the growth zones. Incubated under anaerobic conditions at 37 ° C, or other specified conditions, observing the growth of colonies to the required size using a microscope. After reaching a sufficient size, colonies of microbial cells are manually disconnected from the growth zones using a sharp sterile toothpick, and they are also manually transferred to external means of identification.

The main disadvantages of the prototype method is the use of manual labor when feeding cells into the growth zone, as well as when disconnecting the grown colonies and transferring them to identification tools and the inability to automate the process.

The objective of the proposed method is to develop a method for growing colonies of microbial cells, which allows to obtain a technical result, which consists in creating conditions for automation of the process of growing, separation and transfer of microcolonies, which makes it possible to use the method in laboratories on a chip.

The essence of the proposed method lies in the fact that in the method of growing colonies of microbial cells, the nutrient solution is supplied from the bottom up through the holes in the plate and the porous membranes located in them, into the growth zones of the colonies of microbial cells formed on porous membranes; the supply of microbial cells is carried out on the upper surface of the plate until they are evenly distributed in the growth zones; creation of conditions for the growth of microorganisms in the form of colonies is ensured under conditions of monitoring environmental parameters (temperature, humidity, oxygen partial pressure, etc.); colony growth is monitored; the grown microcolonies of microbial cells are separated from the growth zones by water hammer and transfer them to external means of identification in the stream.

Significant differences of the proposed method are the commensurability of the growth zones and microcolonies, which implies the isolation of a purely culture of microorganisms, the ability to control the supply of nutrient medium, the ability to control colony growth in situ, the possibility of creating shock conditions using shock pressure at the inlet of the holes in the porous plate to separate the colonies from the surface of growth zones on a porous membrane located in the hole, with the aim of their automatic movement without mechanical damage to the microcolonies in external devices for their further research, as well as the possibility of implementing the method in the laboratory on a chip. The supply of the nutrient solution is carried out due to the pressure drop when the solution is supplied from the bottom to the top, which allows you to control the process of supplying the solution to the growth zones, as well as automate it (for example, pumping). Submission of the suspension using automated means (pump) allows you to evenly distribute it on the surface. The application of a hydrophobic film to the surface of the plate with holes prevents the attachment of microorganisms to it, which will be delayed by growth zones with an increased concentration of nutrients supplied from below. Thanks to the regulated supply of nutrient solution to the growth zones, colony growth is ensured. Upon reaching, by dividing, a size of the order of N = 100-1000 cells in the colony, the microcolonies are separated from the growth zones for transfer to external means of identification. Separation of microcolonies in the claimed method is carried out using a water hammer. Creating a water hammer regime allows you to automate the process of separation of colonies for their subsequent transfer.

A portable device for growing cell colonies is known, which contains a cassette with a container for cell growth, a container for growth medium, a container for draining waste, a unit for collecting grown colonies, connected to a single device that is not connected with the external environment. The device can interface with various external devices, for example, a unit for mixing cells, a processor, a thermostat, a mass transfer unit, filters for regulating the composition of the medium, and a unit for selecting and concentrating cells. The mass transfer unit in the device provides a medium supply, a change in the direction of the tangential growth surface of the stream to optimize cell growth and increase the yield of culture, as well as a sterile growth medium and removal of inhibitory substances (US 6096532, C12M 3 / 02.1997).

This device provides the ability to automate and optimize the process of cell growth and collection, but it is complex, cumbersome and does not provide for the ability to automatically move individual microcolonies of cells to external devices to identify them and further determine their sensitivity to antibiotics, i.e. does not complete the task. The described device is not intended to be integrated into the laboratory on a chip.

A device is known in which a semipermeable membrane is used as a substrate for the growth of cell strains, through the pores of which a nutrient solution enters. For example, a device for growing cell and bacterial cultures, which is placed in a Petri dish containing a liquid or nutrient solution, which is a ring or a frame on which a porous lattice coated with a porous membrane is fixed, so that its lower surface is in full contact with the liquid and cells, bacteria or other organisms that are fed from the pores of the membrane (GR 1003266 (B2), C12M 1/12, 3/06, 1997) are placed on the upper plane of the membrane. A device for analyzing cells is also known in which microwells are made using a photoresist film with a thickness of 15-60 μm forming the walls of the wells (RU 2298798, G01N 33/546).

These devices do not have the automation potential and the ability to select microcolonies, i.e. cannot be used to obtain in a short time a pure culture of microorganisms.

Known devices for growing cell colonies that contain a mass transfer site, filters for regulating the composition of the medium and a site for the selection and concentration of cells. The mass transfer unit in such devices provides a medium to optimize cell growth and increase the yield of culture, as well as a sterile growth medium and removal of inhibitory 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 these devices, providing the ability to automate and optimize the process of cell growth, are complex, cumbersome and do not provide for the ability to automatically move individual microcolonies of cells to external devices to identify and determine the sensitivity of the resulting cultures to antibiotics.

Closest to the claimed is the device presented in the article Ingham, C. J., Sprenkels, A., Bomer, J., Molenaar, D., van den Berg, A., van Hylckama Vlieg, JET., De Vos, WM “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 anodic alumina, on the surface of which a network of polymer septa is formed, forming zones of colony growth with a characteristic size of 7 to 200 microns. The porous plate is located on the agar layer with the nutrient solution in the Petri dish to achieve full liquid contact of the device with the surface of the agar to wet the bottom surface of the porous plate.

The main disadvantages of the device of the prototype is that it does not provide the ability to automate the process of cultivation and cell growth, when working with it, manual labor is used when feeding cells into the growth zones, as well as when disconnecting grown colonies and transferring them to identification and control using a microscope . The device analyzes at low speed, does not have portability, does not provide the ability to integrate into miniature portable devices and use in laboratories on a chip

The objective of the invention is to develop a device for implementing a method of growing colonies of microbial cells, which allows to obtain a technical result, which consists in providing conditions for the automation of the processes of feeding the nutrient solution and the processes of separation and transfer of grown colonies, the possibility of integration into miniature portable devices, and use in laboratories on a chip and device portability.

The essence of the claimed device lies in the fact that in the device for growing colonies of microbial cells containing a porous plate of anodic alumina with zones of growth of colonies of microbial cells formed on its upper surface, the lower surface of which is coupled to a means of supplying a nutrient solution, a porous plate of anode oxide aluminum is located in the housing with the formation of the upper and lower capacities of the housing, which are equipped with an inlet and outlet for liquid media, and the specified porous plate from the anode of aluminum oxide has cylindrical holes formed orthogonally to its large plate plane and topologically encoded, with porous membranes with pores not passing microbial cells located in each hole, the upper surface of the casing is capable of passing gases, but not permeable to moisture and particles external environment, as well as with the ability to monitor the growth of colonies. Growth zones can take the form of either a platform, with the location of the porous membrane flush with the surface of the porous plate of anodic alumina, or wells, with the location of the porous membrane below the surface of the plate. The surface of the porous plate of anodic alumina between the growth zones can be coated with a layer of material that prevents the attachment of cells to it, for example, metal, polymer, hydrophobic material. Porous membranes can be made, for example, from anodic alumina, ceramics, polymer, metal. The lid of the upper tank can be equipped with a central window, with an optical glass fixed in it to monitor the growth of colonies. The lid of the upper tank can be equipped with two windows closed with semipermeable membranes, which serve to pass gases, but which are not permeable to moisture and environmental particles.

The formation of growth zones in the holes of the porous plate by placing porous membranes in them and the presence of a coating between the growth zones allows the colonies to be concentrated and quickly grown without special means of limiting the growth zones (polymer network in the prototype). The porous membranes in the holes act as barriers that retain cells in the growth zones after suspension is fed before the colonies grow. The holes in the plate are made in the form of cylinders with smooth walls of the orthogonally large plane of the porous plate and are topologically encoded, which makes it possible to improve the process of manipulating colonies and observing the progress of their growth. Topological coding of the holes ensures their predetermined location in the porous plate. This makes it easier to observe through the camera. The cylindrical shape of the holes and their orthogonal arrangement provides a reduction in energy costs for separation of colonies during water hammer. Water hammer is carried out according to the principle of "hydraulic ram", i.e. has a stage of acquiring a sufficient linear velocity in the part of the device that serves as an accelerating chamber by the fluid flow, and a step of abruptly closing the valve with the formation of a shock wave front moving in the opposite direction to the initial fluid flow. The holes functionally play the role of nozzles, which determine the flow rate of its uniformity. The use of nanotechnology allows you to adjust the size of the nozzle and the configuration of the porous membrane supporting colonies of microorganisms. The pore size of the porous membrane that does not allow microbial cells to pass through ensures the sterility of the lower container, which leads to a one-time use of the device.

The invention is illustrated by the following graphic materials and tables:

Figure 1. Device design.

Figure 2. Section A of the 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 of microbial cells.

Figure 4. Photographs of a fragment of a porous plate of anodic alumina and a porous membrane.

a - with holes and porous membranes;

b - with colonies on porous membranes.

in - a porous membrane.

Figure 5-8. Phase 1-Phase 7 device operation.

Fig.9. Timing diagram of the device.

Figure 10. Section of a plate with a hole and a porous membrane:

a - with a microcolony attached to the growth zone;

b - with a microcolony in the initial stage of movement of the microcolony into external devices after separation from the growth zone.

11. SEM photograph of an anodic alumina porous membrane:

and - top view, the average pore size of 105 nm, the porosity of φ = 0.1;

b - chip;

c - top view, average pore size 198 nm, porosity φ = 0.71;

g - chipped.

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

a - section along the axis of the pores;

b - top view.

Fig.13. A model of a fragment of a porous plate with holes and porous membranes in them for hydraulic calculation.

Fig.14. Species of Staphylococcus aureus Colony 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 microbial cells (S.Aureus) in suspension and the size of the growth zones of the porous plate on the growth efficiency of the colonies.

Table 3. Effect of suspected S.Aureus clinical patient dilution on the percentage of grown colonies.

Table 4. The dependence of the number of grown (N ₀ ) and separated (N _d ) from the growth zones of the colonies on the flow rate (ν) in the accelerating chamber of the device for plates with 400 growth zones and different diameters (D) of the holes for the suspension containing S.aureus 1 · 10 ⁷ CFUs ¹ .

The device consists of (Fig. 1) a casing 1, separated by a partition 2 with the formation of two containers 3 and 4. The container body 3 has an opening 5 for supplying a nutrient solution, and a check valve 6 connected with it, designed to protect the pump connected to this hole, from the return flow of the nutrient medium. The container body 4 has an opening 7 for supplying bacteria, equipped with a valve 8. The upper surface of the housing 1, which is the cover of the container 4, is equipped with two windows 9 closed by semipermeable membranes 10 for access of oxygen or another gaseous medium, as well as a central window 11, s an optical glass 12 fixed therein to observe the growth of microbial cell colonies. The observation can be carried out using a video camera, the lens 13 of which is located above the window 11 with the optical glass 12. In the tank 4, a second hole 14 is made for the output of grown colonies, in which the valve 15 is installed. In the tank 3, a second hole 16 is made in which the valve 17 is installed .

The partition 2 has a Central hole 18, in which is fixed a plate 19 of porous alumina, thickness H. The plate 19 has holes 20 (figure 2), diameter D, in each of which are porous membranes 21, thickness h. Cylindrical holes 20 are formed orthogonally to the large plane of the plate and are topologically encoded and can be chemically modified. Porous membranes 21 located in the openings 20 have pores 22.

Perhaps a different arrangement of the porous membranes 21 in the holes 20 of the plate, for example, figure 2 shows the location below the surface level of the plate 19 with the formation in each hole of the hole 23, depth a .

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

An example implementation of the design of the device based on thick-film technologies is shown in figure 3, where the device is a sandwich structure. On the basis of 1-a, made of a polymer plate, a layer 1-b is placed, containing a container 3 and its openings 5 (b) and 16 (b), which are the input and output for the nutrient solution, made by laser ablation, on the layer 1-b, a layer 2 is placed, containing a hole 18, the radius of which corresponds to the radius of the porous plate 19 made of anodic aluminum oxide, and holes 5 and 16, conjugated with holes 5 (b) and 16 (b), which are the input and output for the nutrient solution, respectively. A layer 1 (c) is placed on the layer 2 with the plate 19, containing an opening 4 forming a container 4 (Fig. 1) with channels connected thereto, connecting the opening 4 with openings 5 (c) and 16 (c), for supplying a suspension of microorganisms and output of the grown colonies to external devices, respectively. Layer 1 (d) is located on layer 1 (c), containing holes 5 (d) and 16 (d), conjugated with holes 5, 5 (b, c) and 16, 16 (b, c), respectively, forming a channel for feeding the nutrient solution into the tank 3 and its output, holes 7 (d) and 14 (d), paired with holes 7 (c) b 14 (c), respectively, designed to supply a suspension of microbial cells in the tank 4 and the conclusion of the colonies external devices, as well as an opening with an optical window 11, 12. On a layer 1 (d) are valves 6 and 17, interfaced with holes 5 (d) and 14 (d), designed to control the flow of nutrient solution a, and valves 8 and 15, coupled with holes 7 (d) and 14 (d), designed to regulate the flow of a suspension of microbial cells and the withdrawal of grown colonies. A porous plate 19 with a thickness of 80 μm is made of anodic aluminum oxide with the formation of cylindrical holes and porous membranes located in them, by the method of reverse photolithography and anodization. Layer 2 of the device with a thickness of 80 μm is made of lavsan film by laser processing. Layers 1 (b) and 1 (c) are made of a polyvinyl chloride film 400 microns thick by laser processing. Layers 1 (a) and 1 (d) are made of extruded acrylic film 1 mm thick. Holes with a diameter of 1 mm are machined. Valves 6 and 17 are made using precision casting, and valves 8 and 15 are made by machining polycarbonate. The sealing of the device was carried out by compression with bolts in a specially made frame.

The porous plate 19 has topologically coded holes 20 (FIG. 4-a) in which the porous membranes 21 are located (FIG. 4-b). The diameter range 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. Colonies 24 grow on porous membranes 21 of anodic alumina located in holes 20 (FIG. 4-c). Porous membranes 21 formed from anodic alumina (Fig. 4-c) are characterized by porosity values φ = S _meme / S _pore = 0.1 ÷ 0.7, where S _meme is the membrane area, S _pore is the pore area on the surface membranes.

The operation of the device is as follows and has phases:

Phase 1, see FIG. 5-a.

- filling the container 3 and through the openings of the plate of porous anodic alumina 19 of the container 4 with a liquid medium, for example, a nutrient solution with the displacement of air from the containers and pores. The pump H1 creates a flow of the nutrient medium with a volume velocity of Q ₁ , the check valve 6 and valve 15 are open, valves 8 and 17 are closed;

Phase 2, see FIG. 5-b.

- filling the tank 4 with a suspension of microbial cells, valves 8 and 17 are open, check valve 6 and valve 15 are closed. The H2 pump delivers a flow of a suspension of microbial cells into the vessel 4 with a space velocity Q ₂ ;

Phase 3, see FIG. 6-a.

- flushing the cavity 3, the check valve 6 and the valve 17 are open, the valves 8 and 15 are closed, the pump H1 feeds the flow of the washing solution into the tank 3 with a high volume flow rate Q ₃ ;

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 into the tank 3 with a low space velocity Q ₄ , providing recharge of growth zones in the tank 4;

Phase 5, see FIG. 7-a.

- creating conditions for the occurrence of water hammer, the non-return valve 6 is open, the valves 15 and 17 are open, the valve 8 is closed, the pump H1 creates a flow of the nutrient medium in the tank 3 with a high space velocity Q ₅ ;

Phase 6, see FIGS. 7-6.

- the creation of a water hammer, the check valve 6, the valves 8 and 17 are closed, the valve 15 is open, the shock wave, which creates additional pressure R _beats , passing through the nutrient medium in the tank 3 and through the holes and pores in the plate, tears the grown colonies from the growth zones.

Phase 7, see Fig. 8

- transfer of the grown colonies of microbial cells to external devices, the check valve 6 and valves 8 and 17 are closed, the pumps H1 and H2 create a flow of nutrient medium with a volume velocity Q _6, which ensures the transfer of grown colonies of microbial cells to external devices through valve 15.

The timing diagram shown in Fig. 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 created by the pumps H1 and H2, the state sensor (camera), the dependence of the pressure P in the tank 3 in the mode of water hammer on time. On the abscissa axis, time intervals corresponding to the phases of operation of the device are highlighted, Phase 1-Phase 7. The following notation is used in the diagram (Fig. 9): Q ₁ is the space velocity created by an external pump H1, which provides a flow of nutrient medium for washing tanks 3 and 4 , and transfer of the grown colonies of microbial cells. Q ₂ - space velocity created by an external pump H2, providing a flow of suspension of microorganisms. Q ₃ - high volumetric flow rate created by the pump H1, providing a flushing chamber 3 and water hammer. Q ₄ - low space velocity created by the pump H1, providing wetting of the growth zones. D _VK - the sensor (video camera, see figure 1), which provides a signal to start the operation of the pump H1 in the mode of hydroblow (PG) to separate the grown colonies. P is the pressure in the tank 3.

A method of growing colonies of microbial cells is as follows. To fill the device with a nutrient medium during Phase 1 (Fig. 5-a), a nutrient solution with a volumetric flow rate Q _{1 is} supplied to the tank 3 using, for example, an external pump H1. The pump H1 provides a pressure sufficient for the flow of the nutrient medium through the holes 20 of the plate 19 and the pores 22 of the membrane 21, wetting and degassing of the tanks 3 and 4. During Phase 2 (Fig. 5-b), a suspension of microbial cells of a certain concentration is supplied to the tank for 4 s volumetric speed Q ₂ , for example, using an external pump H2, a microsyringe or an automatic pipette. The suspension is fed until it is evenly distributed over the surface of the plate 19 (if it is necessary to remove the air bubble, semipermeable membranes 10 (Fig. 1) or a valve 15, which is temporarily opened, are used. The distribution of the nutrient solution is monitored through a window 11 with an optical glass 12 fixed in it , over which the video camera lens is located 13. Then, before the growth begins in Phase 3 (Fig. 6-a), the composition of the nutrient medium in the tank 3 is brought to a standard state by washing with the pump H1 at a speed of Q ₃ . baseline 4 (Fig. 6-b) colony growth, during which the necessary conditions for the division of microorganisms are provided: nutrition, temperature, humidity (by placing the device in an incubator or by installing mode sensors and ensuring maintenance of parameters). Necessary conditions for the growth of aerobic cells provides the presence of windows 9 with semipermeable membranes 10, which are permeable to gases, but not permeable to moisture and environmental particles. The nutrient solution enters from the cavity 3 through the openings 20 in the plate 19 and the pores 21 using an external pump H1, providing a volumetric speed. Observation of the growth of colonies is conducted through the window 11 with an optical glass 12 fixed therein, over which the lens 13 of the video camera is located. After reaching a sufficient size (of the order of N = 100 ÷ 1,000 cells in a colony) which is fixed either by using the camcorder D _cr, acting as sensor, to supply an appropriate control signal or the time determined experimentally colony microbial cells detached from the zones hydraulic impact growth . For this, in Phase 5, the external pump H1 is transferred to the high space velocity Q ₅ mode (Fig. 7-a). After the set volumetric flow rate Q _{5 is} established in the vessel 3, valves 17 and 6 are sharply closed for a time t (Phase 6). In the container 3, the valve 17 increases the shock pressure and a shock wave occurs, directed from the valve 17 (Fig.7-b). The shock wave propagates into the openings 20 and pores 22, providing a pressure jump without significant fluid movement, as a result of which the colonies are disconnected from the growth zones.

Due to the passage in the growth zone of the shock wave, which has increased pressure, the grown microcolonies break away from the growth zones (removed from the sites or pushed out of the holes). Then the flow with a space velocity Q ₆ transfers the colonies to external devices, for example, identification (Fig. 8).

Microbial cell colonies grown during Phase 4 are fixed in the growth zones, in the wells (Fig. 10-a), since the remaining surface of the plate 19 is covered with a layer of material that prevents the cells from attaching to it. During phase 6, colonies 24 are disconnected from the growth zones, as shown in FIG. 10-b.

To clarify the operation parameters of the device, it is necessary to evaluate the following technical characteristics:

1) The characteristic energy and binding strength of the bacteria (and, accordingly, the colony) with the surface of the growth zone.

2) The pressure P _beats required to detach the bacteria (valid for colonies).

3) The parameters of water hammer (speed in the booster chamber, closing speed of the valve 17), providing pressure P _beats .

4) The parameters of the flow, providing the transfer of colonies to external devices.

Consider the above technical specifications 1-4 of the device.

1) Consider a porous membrane of anodic alumina (Fig. 11), on the surface of which there is a microbial cell in the form of a disk with a diameter of D _mcl = 10 ^-6 m (suppose that the cell takes the form of a disk on the surface). From Fig. 11 it can be seen that the pores of the porous membrane are located quite regularly (Figs. 11a, b), are characterized by a narrow size distribution and, depending on the anodizing conditions, may have pores of a given average diameter, for example, d _cf = 105 nm (FIG. 10-a) or d _cp = 208 nm (FIG. 10-c). Moreover, it can be seen from FIGS. 11 b, d that the pores are characterized by a high aspect ratio and a shape close to cylindrical. Therefore, to assess the adhesion force of microbial cells to the surface of the membrane, we use a model in the form of a plate with cylindrical pores, as shown in Fig. 12-a, -b. Then, if the contact area S _pole bacteria on a solid flat surface is:

S _{pole m.kl} = πD ^2/4 = 3.14 (10 ^-6 m) ^2/4 = 7.8 10 ^-13 m ^2,

and the cross-sectional area of the pores of the porous membrane S of the _pore (Fig.12-b) diameter d = 2 · 10 ^-7 m:

_Pores S = πd ^2/4 = 3.14 ⁽² · 10 ^-7 m) ^2/4 = 3.14 × 10 ^-14 m ^2,

which, in turn, for the four pores that the typical bacterium overlaps (Fig. 12-a, -b), is the area S:

S = 4 · 3.14 · 10 ^-14 m ² = 1.26 · 10 ^-13 m ²

then the contact area of the bacteria on the porous surface will be:

S _{cont / pore} = 7.8 10 ^-13 m ² -1.26 · 10 ^-13 m ² = 6.54 · 10 ^-13 m ² .

To assess the adhesion force of the bacteria to the surface of the growth zones (Fig. 10, Fig. 12), we assume that the average bond density is three bonds per 1 · 10 ^-18 m ² . Then the number of bonds N _sv to the entire contact area of the bacteria will be: N _sv = 3 · (6.54 · 10 ^-13 / 10 ^-18 ) = 2 · 10 ⁶ bonds.

The maximum energy of a single van der Waals bond (V-d-V) type (which prevail under the conditions of the method) according to the literature (Kaplan IG Introduction to the theory of intermolecular interactions. - M .: Nauka. mathematical literature, 1982. 312 p.) is, E _V-d-V (max) = 1.36 · 10 ^-23 J (Table 2), and the total energy of V-d-B bonds for the entire bacterium will be E = N _sv E _B-d-B = (1.36 · 10 ^-23 ) · (2 · 10 ⁶ ) = 3 · 10 ^-17 J.

Then the binding force of the entire bacterium F ₁ (taking the van der Waals interaction as the main one) can be estimated as:

F ₁ = E / R _W-d-B = 3 · 10 ^-17 / 0.6 · 10 ^-9 = 5 · 10 ^-8 H.

Here, R _B-d-B = 0.6 · 10 ^-9 m is the characteristic length of the V-d-B connection.

2) The pressure required to separate the bacteria from the growth zone can be obtained by dividing F ₁ by the area S from Example 1, we obtain P _spr = F ₁ / S = 5 · 10 _-8 H / 1,26 · 10 ^-13 m ² = 3.9 · 10 ^-5 Pa.

In the general case, for a bacterium (colony) of arbitrary shape, the following criterion for determining the pressure required to disconnect them from growth zones can be derived:

$$R_{\mathrm{about}tR}\ge \frac{\left(\mathrm{one}-\varphi \right)}{\varphi }{n}_{\mathrm{AT}-d-\mathrm{AT}}{F}_{\mathrm{at}-d-\mathrm{at}}\left(\mathrm{one}\right)$$

Here φ = 0.6 is the value of porosity of the membrane sections containing pores, defined as the ratio of the cross-sectional area of cylindrical pores to the membrane area.

Since the pressure necessary for separation of bacteria (at the maximum estimate) is quite high - 3.9 atm., It is advisable to use a hydraulic shock or pressure jump caused by a rapid change in speed to separate the colonies (Fig. 10-b) from the growth zones fluid flow for a very short period of time (Kalitsun V.I., Drozdov E.V., Komarov A.S., Chizhik K.I. Fundamentals of hydraulics and aerodynamics, Stroyizdat, 2002). The use of water hammer will make it possible, when using miniature low-pressure pumps, most suitable for portable diagnostic equipment, to achieve, at short intervals, sufficiently high values of the shock pressure necessary to carry out the claimed function of the device — separation and movement of colonies.

A number of studies performed a numerical study of the propagation of a shock wave in a thin capillary. For example, in the work of M.S. Ivanov, G.V.Shoyev, E.A. Bondar, D.V. Khotyanovsky, A.N. Kudryavtsev "Modeling the propagation of a shock wave in a microchannel, taking into account the effects of viscosity" (International Conference . "Modern problems of applied mathematics and mechanics: theory, experiment and practice", Novosibirsk, Russia, May 30 - June 4, 2011 90 / fulltext / 47499/47500 / Ivanov_full_paper.pdf). conducted numerical modeling of the processes of entry and propagation of a shock wave into a thin capillary for a gaseous medium taking into account viscosity using, among other things, the near-continuum approach. For the latter case, in a numerical experiment, an insignificant attenuation of the shock wave was obtained as it moves in the capillary. Thus, it can be assumed that the shock wave formed during a sharp overlap of the valve 17, entering the holes 20 and pores 21, propagates to the exit, gradually damping. In this article, an experiment is conducted for gas at Re = 400. Using the principle of similarity with respect to the Reynolds number, we find that in order to achieve conditions similar to a numerical experiment for gas, in the proposed device, the fluid flow in the tank 3, which acts as an accelerating chamber, should acquire a velocity of the order of 4 m · s ^-1 , which corresponds to the range of permissible velocities functioning of the device. Then the use of data obtained in the above article is legal. In this case, it can be assumed that during propagation in the openings 20 and pores 22 (Fig. 13), the shock wave gradually attenuates, losing at most 30% of the energy. Thus, taking into account the attenuation inlet hole must develop a pressure of at least 5.57 atm to obtain at the outlet of the capillary pressure R≥R _Neg.

3) Pressure increase during direct hydraulic shock, i.e. such that the shock phase T is shorter than the closing time (τ) of the valve 17, is ideally determined for the rigid walls of the chamber and low compressibility of the liquid by the formula 2:

$$R_{\mathrm{at}d}=\rho \left({\nu }_0-{\nu }_{\mathrm{one}}\right)c\left(2\right)$$

where R _beats is the pressure increase in Pa, ρ is the fluid density in kg / m ³ , ν ₀ and ν ₁ are the average fluid flow rates in the channel before and after closing valve 17 (Fig.6-a, -b) in m s ^-1 , c is the velocity of the shock wave in the liquid in m · s ^-1 .

To estimate the value of ν ₀ necessary to obtain a pressure of P _beats ≥Р _otr = 5.57 · 10 ^-5 Pa, providing separation of bacteria, we will consider the speed of sound in the device’s capacity to be equal to the speed of sound in an unlimited aqueous medium with _water = 1.348 · 10 ³ ms ^-1 , and the velocity ν ₁ = 0.

Let us evaluate the nature of the hydroblow, i.e. the ratio of the phase of the shock wave and the valve closing time. The characteristic size of the booster chamber l in the inventive device will be taken equal to 2 mm, then the impact phase T will be 2.5 · 10 ^-5 s. If the closing time (τ) of valve 17, τ <2.5 · 10 ^-5 , then direct water hammer is carried out, calculated by the formula (1). Then, substituting in the formula (1) as P _{beats the} corresponding value, we obtain:

ν ₀ = P _beats / (ρc _water ) = 5.57 · 10 ⁵ / (10 ³ · 1.348 · 10 ³ ) = 4.13 · 10 ^-1 m · s ^-1

If τ> 2.5 · 10 ^-5 , then the hydroblow is called indirect and the formula is applied

$$R_{\mathrm{at}d}=\frac{2\rho {\nu }_{0}l}{\tau }\left(3\right)$$

Then, at τ = 10 ⁻³ s, l = 2 · 10 ⁻² m, we obtain ν ₀ = P _beats τ / (2ρl) = 3.9 · 10 ⁵ · 10 ^-3 / (2 · 10 ³ · 2 · 10 ^-2 ) = 10 m · s ^-1 , and at τ = 10 ^-4 s, ν ₀ = 1 m · s ^-1 .

In the General case, from expressions 1 and 3 for indirect water hammer can be obtained by the formula:

$${\mathrm{<figure-callout\; id="0"\; label="\nu "\; filenames="00000021.png"\; state="\{\{state\}\}">\nu </figure-callout>}}_0\ge \frac{\left(\mathrm{one}-\varphi \right)}{\varphi }{n}_{\mathrm{AT}-d-\mathrm{AT}}{F}_{\mathrm{AT}-d-\mathrm{<figure-callout\; id="2"\; label="AT\; \tau "\; filenames="00000015.png,00000017.png"\; state="\{\{state\}\}">AT\; </figure-callout>}}\frac{\tau }{}\frac{}{2\rho l}\left(\mathrm{four}\right)$$

Thus, for use in laboratories on a chip, where the volumetric flow of working fluids is limited by the miniature size of the pumps and the hydraulic resistance of small diameter pipes (capillaries), practical considerations require the use of water hammer (both direct and non-direct). In any case, the implementation of such a water hammer will require the use of high-speed locking micro-valves, such as described in the well-known patent (US 20110073788, F16K 31/12, F16K 31/02, 2011).

For a more accurate calculation of the critical velocity ν _0, it is necessary to take into account the dependence of the velocity with the propagation of the shock wave in the liquid on the size and characteristics of the materials of the accelerating chamber.

When the valve is instantly actuated, the critical velocity ν ₀ can be found using formulas 1 and 2. From the condition P _beats ≥Р _otr with ν ₁ = 0, we obtain

$$\rho {\nu }_{0}c\ge \frac{\left(\mathrm{one}-\varphi \right)}{\varphi }{n}_{\mathrm{AT}-d-\mathrm{AT}}{F}_{\mathrm{AT}-d-\mathrm{AT}}\left(5\right)$$

$${\mathrm{<figure-callout\; id="0"\; label="\nu "\; filenames="00000021.png"\; state="\{\{state\}\}">\nu </figure-callout>}}_0\ge \frac{\left(\mathrm{one}-\varphi \right)}{\varphi }\frac{\mathrm{one}}{\rho c}{n}_{\mathrm{AT}-d-\mathrm{AT}}{F}_{\mathrm{AT}-d-\mathrm{AT}},\left(6\right)$$

Where:

$$c=\frac{\mathrm{one}}{\sqrt{\rho \left(\frac{\mathrm{one}}{E_{\mathrm{at}\mathrm{about}d\mathrm{but}}}+\frac{a_{\mathrm{at}n\mathrm{at}tR}}{b{E}_{\mathrm{to}\mathrm{about}RP\mathrm{at}\mathrm{from}}}\right)}}\left(7\right)$$

Then for the speed in the booster chamber ν ₀ we get:

$${\mathrm{<figure-callout\; id="0"\; label="\nu "\; filenames="00000021.png"\; state="\{\{state\}\}">\nu </figure-callout>}}_0\ge \frac{\left(\mathrm{one}-\varphi \right)}{\varphi }\frac{\mathrm{one}}{\rho c}{n}_{\mathrm{AT}-d-\mathrm{AT}}{F}_{\mathrm{AT}-d-\mathrm{AT}}\sqrt{\frac{\left(\frac{\mathrm{one}}{E_{\mathrm{at}\mathrm{about}d\mathrm{but}}}+\frac{a_{\mathrm{at}n\mathrm{at}tR}}{b{E}_{\mathrm{to}\mathrm{about}RP\mathrm{at}\mathrm{from}}}\right)}{\rho }}\left(8\right)$$

where: a _int - diameter of the booster chamber - 1 mm;

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

E _water is the modulus of elasticity of water 2 · 10 ⁹ n · m ^-2 ;

E _casing - the modulus of elasticity of the casing material (for example, for anodic alumina - 0.6 · 10 ¹¹ n · m ^-2 , for aluminum - 0.71 · 10 ¹¹ n · m ^-2 , for polymer - 0.032 · 10 ¹¹ n M ^-2 ).

4) Let us estimate the volumetric flow rates passing through the porous membrane at a constant pressure drop ΔP = 10 ⁵ Pa created by the pump H1 in Phase 1 and Phase 7. For example, in Phase 7 the flow Q ₆ will be determined by the number (N), radii (r) and pore length (h) (FIG. 13), since they will create the greatest hydrodynamic resistance of the passing fluid. The calculation of the space velocity Q in one pore can be done using the Poiseuille formula (2) (L.G. Loytsyansky. Mechanics of liquid and gas. - L., 1950. - 676 p.):

$$Q=\frac{\pi r^{\mathrm{four}}}{8\eta h}\Delta P,\left(9\right)$$

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

$$Q=\frac{3.14{\left({10}^{-7}\right)}^{\mathrm{four}}}{8\cdot {10}^{-3}{10}^{-5}}{10}^5\cong \mathrm{four}\cdot {10}^{-16}{\mathrm{<figure-callout\; id="3"\; label="m"\; filenames="00000015.png,00000017.png"\; state="\{\{state\}\}">m</figure-callout>}}^3\cdot {\mathrm{from}}^{-\mathrm{one}}$$

Let us estimate the total number of pores of porous membranes throughout the porous plate. To estimate the density of pores on the surface (ns), one can use the following expression:

n _S = φ / (π · r ² ) = 0.6 / (3.14 · (10 ^-7 ) ² ) = 1.91 · 10 ¹³ m ^-2 .

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

S = N ₁ · π · R ² = 1.6 · 10 ³ · 3.14 · (5 · 10 ^-6 ) ² = 1.26 · 10 ^-7 m ² , where N ₁ = 1.6 · 10 ³ - the number of holes on the plate, R = 5 · 10 ^-6 m is the radius of the hole.

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

N = n _S · S = 2.4 · 10 ⁶ .

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

Q ₆ = N · Q = 2.4 · 10 ⁶ · 4 · 10 ^-16 ≅ 10 ^-9 m ³ · s ^-1 .

We calculate the linear velocity (ν _n ) of microbial cell transfer in Phase 7 under the influence of flow Q ₆ . To do this, it is necessary to evaluate the linear flow rate in the tank 4 and the associated communications with external devices (Fig. 8), with a volume flow of Q ₆ . When the size of the booster chamber is 10 ^-4 m × 10 ^-3 m (cross-sectional area s = 10 ^-7 m ² ), the average linear flow velocity ν _{n is} determined by the expression:

ν _n = Q / s = 3 · 10 ^-9 / 10 ^-7 = 3 · 10 ^-2 m · s ^-1

This value of speed Q _{6 is} sufficient to maintain the colonies in the flow and to compensate for sedimentation on the porous plate, as well as to transfer the colonies to an external device. For additional flow control to external devices, an H2 pump in Q ₂ mode can also be used.

EXAMPLE 1

A suspension of a strain of microbial cells was prepared at a concentration of 10 ⁷ CFU / ml ^-1 (CFU - colonies of the forming units) and was successively diluted to concentrations of 1 · 10 ² , 1 · 10 ³ 1 · 10 ⁴ 1 · 10 ⁵ 1 · 10 ⁶ CFU / ml ^-1 , to obtain six samples of a suspension of S.aureus. To fill the device with a liquid medium (Phase 1, Fig. 5-a, physiological solution was supplied to vessel 3 with a volumetric flow rate Q ₁ = 10 μl · s ^-1 using a syringe pump H1. Thus, wetting and degassing of containers 3 and 4 were provided Then, a suspension of microbial cells of each of the samples with a volume of 20 μl was fed into the container 4 using a microsyringe with a bulk velocity Q ₂ = 2 μl · s ^-1 (Phase 2, Fig.5-b), until it is evenly distributed over the surface of the porous plate 19 The distribution of the suspension was monitored through window 11 using an external video camera. it before growth composition of the nutrient medium in the vessel 3 resulted in a standard state by washing H1 via pump at the rate Q = ₃ 10 l · s ^-1 (Phase 3). Then the incubation phase was started and the growth of microbial colonies. The nutrient solution fed via pump H1 with a space velocity of Q ₁ = 0.16 pl · s ^-1 and set the temperature regime for growth to 37 ° C using an external thermostat (Phase 4). A porous plate containing 400 growth zones with a diameter of 20 μm and a pore diameter of 200 nm was used. and porosity φ = 0.6. After incubation for 3 hours (Phase 4), a porous plate with holes was examined using a microscope. Microscopic examination showed that, depending on the concentration of the initial suspension, grown colonies of S. Aureus are observed in part of the growth zones (from 0.5% to 60%), as in Fig. 14. We studied the effect of the initial concentration of S.aureus on the proportion of growth zones in which the colonies grew, observing the results of incubation using a microscope. The observation results are given in table 2, column 7.

The experiment was repeated with plates having 400 growth zones with a hole size of 2 μm, 7 μm and 10 μm, an average pore diameter of 200 nm and a porosity of φ = 0.6. The results are shown in table 2, columns 4, 5, 6.

Then, the grown colonies of microorganisms were disconnected and transferred to external devices for plates with openings of 7 μm, 10 μm and 20 μm. The transfer was carried out hydraulically using water hammer. To carry out water hammer, the external pump H1 was switched to the high volumetric velocity mode Q ₅ = 0.8 · 10 ^-6 m · s ^-1 (Phase 5), which corresponded to a linear speed of 1 m · s ^-1 (cross-sectional area of the booster chamber 3 in FIG. .3 is 0.8-10 ^-6 m ² ), then the valves 6 and 17 (Fig. 3) were blocked for a time t in accordance with Phase 6 of the operation of the device. As a result, a shock wave appeared in the vessel 3, propagating in the direction of the growth plate 19. Then, the valve 8 was opened and the pump H1 was put into Q ₆ = 1 · 10 ⁹ m ³ · s ^-1 mode and was observed using a microscope in channel 14 of the device and carried out their counting. The data are shown 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 shown in table 3 columns 5 and 7, respectively.

From table 3 it is seen that at speeds below 1 m · s ^-1 there is a significant decrease in the proportion of 14 colonies transferred to the channel. When the speed is exceeded 5 m · s ^-1 , a destruction of the porous plate with holes is observed.

EXAMPLE 2

Sowing was carried out in the proposed device (see Example 1) of the microorganisms of the patient's sample with suspected damage to the nasopharynx of S.aureus. A patient’s nasopharyngeal mucus smear was prepared, and a suspension of the microorganism population was prepared by washing the mucus sample from a cotton swab into a 1 ml physiological solution and sedimenting the fibers and particles by sedimentation for 5 min. Suspension samples of various concentrations were prepared by ten-fold dilution, obtaining dilutions of 10, 100, and 1000 times. Transferred 20 μl of the suspension from each sample to the device as in example 1. Used a plate containing 400 holes, with a diameter of 10 μm. After carrying out phases 1-3 of the incubation process under conditions as in example 1, the result was examined using a microscope. The total filling of the growth zones of the device was 10–40%, and according to the data of a visual expert assessment, four types of colonies were observed: S. Aureus, Neisseria meningitidis, S. pneumoniae, Enteroccocus. The results are shown in table 4.

Then, the grown colonies of microorganisms were disconnected and transferred to external devices (channel 14) for plates with openings of 7 μm, 10 μm and 20 μm. The transfer was carried out hydraulically using water hammer. To carry out water hammer, the external pump H1 was transferred to the high volumetric velocity mode Q ₅ = 0.8 · 10 ^-6 m ₃ · s ^-1 (Phase 5), which corresponded to a linear speed of 1 ms ^-1 (cross-sectional area of the booster chamber 3 on figure 3 is 0.8 · 10 ^-6 m ² ), then the valves 6 and 17 were closed for a time t (figure 3) in accordance with Phase 6 of the operation of the device. As a result, a shock wave appeared in the vessel 3, propagating in the direction of the growth plate 19. Then, the valve 8 was opened and the pump Н1 was put into Q ₆ = 1 · 10 ^-9 m · s ^-1 mode and was observed using a microscope in the channel 14 of the device and carried out their counting. The data are shown in table 3, column 3.

Table 1 Technical and operational characteristics of the device using the inventive device and industrial analogues Technical solution
Product Feature Kirby Bauer Way Automatic device VITEK ^® 2 compact 60 (Biomerieur, France) Laboratory on a chip in which the proposed method and device can be used one 2 3 four Instrument composition Petri dishes with nutrient medium, sets of discs impregnated with antibiotics, microbiological incubator with cooling KV-23 (Binder, Germany), biological microscope. Reader, disposable microbial cell analysis cards, personal computer. Portable automatic reading device, one-time laboratories on a chip, containing modules: sample preparation, incubation and growth of bacteria, identification, determination of viability, microcomputer on board, navigation, communication module. terms of Use Specialized Laboratory (GOST R ISO / IEC 17025-2000) Specialized Laboratory (GOST R ISO / IEC 17025-2000) Accreditation in accordance with GOST R ISO / IEC 17025-2000, autonomous conditions is not required. Appointment The study of the sensitivity of microorganisms to antibiotics. Identification of bacteria and yeast. Determination of the sensitivity of microorganisms to antibiotics. Sample processing, creating conditions for the growth of microorganisms, differentiating and accounting for the number of microorganisms, determining the sensitivity of microorganisms to antibiotic drugs. Method for reading identification information Absent Colorimetry Pattern recognition by three or more signs.

one 2 3 four Method for reading antibiotic sensitivity information A manual way to measure the clearance around discs impregnated with antibiotics using a ruler. Turbidimetry Microturbidimetry, the position of the border in the microchannel: video camera, pattern recognition. Overall dimensions, mm 433 × 516 × 618 590 × 715 × 6725 <150 × 200 × 200 Weight, kg 44 75 <3 Power, W 340 1025 thirty Number of modules 3 2 (to one computer) one Automation level Low Tall Tall Productivity: samples per day 40 60 twenty Analysis time 1 hour 48-72 26-34 6-8 Identification Not produced Up to view Up to view

one 2 3 four Antibiotic count 6 (per petri dish) twenty twenty Reliability,% 95 90 91 Changeover flexibility Yes No Yes Operating costs for 1 sample,
rub. 1000 1000 350 Price, million rubles 0.175 (import into the Russian Federation) 6.0 (import into 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 growth efficiency of the colonies The concentration of microbial cells of S.aureus in suspension, CFU ml ^-1 The number of microbial cells of S.aureus in the sample The size of the growth zones of the porous plate, microns 2 7 10 twenty one 2 3 four 5 6 7 The share of grown colonies in the growth zones,% 1 · 10 ² 2 0 0 0.25 0.5 1 · 10 ³ twenty 0.25 1,2 1,5 4,5 1 · 10 ⁴ 2 · 10 ² 0.5 four 6.25 12.5 1 · 10 ⁵ 2 · 10 ³ 2,5 10 25 35 1 · 10 ⁶ 2 · 10 ⁴ fifteen twenty 32 45 1 · 10 ⁷ 2 · 10 ⁵ twenty 25 45 60

Table 3 The dependence of the number of grown (N ₀ ) and separated (N _d ) from the growth zones of the colonies on the flow rate (ν) in the accelerating chamber of the device for plates with 400 growth zones and different diameters (D) of the holes for the suspension containing S.aureus 1 · 10 ⁷ CFU ml ^-1 . The flow velocity ν m · s ^-1 The number of grown N ₀ and moved to channel 14 (Fig.3-b) (N _d ) colonies d = 20 μm d = 10 μm d = 1 μm N ₀ N _d N ₀ N _d N ₀ N _d one 2 3 four 5 6 one 0.01 220 10 178 10 97 10 0.1 235 fifty 179 one hundred 105 19 one 250 150 189 160 104 25 2 245 210 185 183 99 70 3 256 225 190 180 one hundred 80 four 239 230 176 175 101 99 5 241 240 180 195 95 94

Table 4

The effect of dilution of a suspected S.Aureus clinical patient sample on the proportion of colonies N ₀ grown in the growth zones and the proportion of 14 colonies N _d transferred to the channel Clinical Dilution Type of microbial cells S.aureus,% Neisseria meningitidis,% S. pneumoniae,% Enteroccocus,% N ₀ N _d N ₀ N _d N ₀ N _d N ₀ N _d one 2 3 four 5 6 7 8 9 1:10 ⁶ 0 0 0 0 0 0 0 0 1:10 ⁵ 0 0 0 0 0 0 0 0 1:10 ⁴ 0 0 0 0 0 0 0 0 1:10 ³ 3 3 0 0 0 0 one one 1:10 ² 8 8 0 0 2 2 3 3 1:10 fifty 48 2 2 10 9 fifteen fifteen

Claims (6)

1. A method of growing colonies of microbial cells on the surface of a porous plate, including supplying a nutrient solution from the bottom up through a porous plate to the growth zones of colonies of microbial cells formed on its upper surface, feeding a suspension of microbial cells to the upper surface of the porous plate, creating controlled conditions for the growth of colonies, observing the growth of the colonies, colonies grown detach the microbial cells from the growth zone and transfer them into the external identification means, characterized in that the pit The solid solution is fed into the growth zones of microbial cell colonies by creating a pressure differential between the inlet and outlet of the holes made in the plate of anodic alumina orthogonally to its large plane and topologically encoded, in which these growth zones are formed in the form of porous membranes, while porous membranes, placed either flush with the upper surface of the plate, or with the formation of a hole, do not let microbial cells pass, then a suspension of microbial cells of a given concentration is fed to the upper surface the plate to their uniform distribution, while a film is formed on the surface of the plate between the growth zones that prevents the attachment of microbial cells, and the growing microcolonies are disconnected from the growth zones by water hammer directed from the inlet side of the cylindrical holes of the plate and propagating along them further through the pores of the porous membranes with a force that does not destroy the microcolony, but sufficient to separate them from the growth zones, while the pressure in the zone of hydroshock

where:

- the porosity of the porous membrane
n is the number of bonds of the microbial cell and the membrane surface per unit surface area;
F is the average strength of a single van der Waals-type bond between the surface of the microbial cell and the surface of the membrane. 2. The device for growing colonies of microbial cells by the method according to claim 1, containing a porous plate of anodic alumina with zones of growth of colonies of microbial cells formed on its upper surface, characterized in that the porous plate of anodic alumina is located in the housing with the formation of the upper and lower containers of the body, which are equipped with an inlet and outlet with valves for liquid media, while the lower tank is equipped with an inlet and outlet for the nutrient medium of microbial cells, and its inlet is designed to connect I am with an external pump, the upper tank is equipped with an entrance for a suspension of microbial cells and an outlet for grown microcolonies, and the upper surface of the body is made permeable to gases, but impermeable to moisture and environmental particles, the lower tank is designed to form a water hammer, with a force sufficient to detach colonies from growth zones but not destructive colonies, while the porous plate of anodic alumina has cylindrical holes formed orthogonally to the large plane of the plate and topologically porous membranes with pores that do not allow microbial cells forming growth zones are located, and porous membranes are either flush with the top surface of the plate or with the formation of a hole, while a film is formed on the surface of the plate between the growth zones that prevents attachment microbial cells. 3. A device for growing colonies of microbial cells according to claim 2, characterized in that the film is made of metal, polymer or hydrophobic material that prevents the attachment of microbial cells to the surface of the plate. 4. A device for growing colonies of microbial cells according to claim 2, characterized in that the porous membranes are made of anodic aluminum oxide, ceramic, polymer or metal. 5. A device for growing colonies of microbial cells according to claim 2, characterized in that the lid of the upper container is equipped with a central window, with an optical glass fixed in it for monitoring the growth of colonies. 6. The device for growing colonies of microbial cells according to claim 2, characterized in that the lid of the upper tank is equipped with windows closed with semipermeable membranes, which serve to pass gases, but which are not permeable to moisture and environmental particles.

Images