Method for quick growth, detection and identification or counting of microcolonies of microorganisms at early stage. RU patent 2505607.

FIELD: biotechnologies.

SUBSTANCE: invention pertains to the method for quick growth, detection and identification or counting of microcolonies of microorganisms at early stage. The described method includes the following stages: obtaining the container with medium with porous element located above or under the top surface of the medium, note that the medium has nutritious substances for quick growth of microcolonies of microorganisms and porous element has pores from 1000 to 107 Da; pouring the liquid sample without serial dilution into container to the area above porous element; capturing the microorganisms in porous element or at the medium above porous element; incubation of container for the time sufficient for quick growth of microcolony at an early stage; transfer of porous element and any medium above it from container to the secondary medium for evaluation, detection and identification of microorganisms; and microcolonies research relatively the growth, detection, identification or counting of microorganisms. The said method for growth, detection and identification or counting of microorganisms takes not more than approximately six hours.

EFFECT: invention allows more quickly, efficiently and less expensively indentifying the total number of revivable microcolonies of microorganisms, their identifying and distinguishing.

7 cl, 10 dwg

LINKS TO RELATED APPLICATIONS

[0001] This application claims priority to the application of the U.S. Ser. No.60/896,321 called “Detection and Identification of Microorganisms on Transparent Permeable Membranes” which is quoted here in full.

BACKGROUND OF THE INVENTION

1. Use of the invention

[0002] the Present invention relates mainly to the field of biology, in particular to the rapid determination, identification and counting microorganisms.

2. Description of previous achievements

[0003] Modern microbiological analysis is based on two main areas: 1) analysis without prior undergrowth, and 2) analysis after the preliminary undergrowth. The first direction includes a group of immunological methods [including , radioimmunoassay, immune-enzyme analysis (IFA) for a single cell]; group of methods based on DNA/RNA analysis using Polymerase Chain Reaction (CPA) and the group of methods for Flow Cytometry [determination of one cell after the label fluorescent antibodies or substrates]. Artificial substrates also used for the analysis of cells in microscopy. However, some of these methods (CPA immunological methods) is not able to define the living cells. Flow Cytometry in combination with artificial (mainly ) substrates capable of identifying living cells, but is very expensive and require highly qualified specialists ' work and concentrated samples. Despite this, the cheap and effective method of determination of living cells in various medical, biotech, food, agricultural, pharmaceutical, natural and military samples is still needed for human needs. However, frequently used tests with the use of cells in Microbiology [chromatography fatty acids, immune-enzyme analysis (ELISA), mass spectrometry, Fourier transforms infrared spectroscopy and immuno-testing], all require preliminary undergrowth of colonies of microorganisms, which represents at least once to long cultivation of primary cells before the definition and identification may be applicable.

[0004] Despite these highly technical capabilities of normal growth in a Petri dish is still the most common method for determination of micro-organisms present in the sample. A typical analysis requires several 10-fold dilutions of the sample followed by the application of one milliliters diluted sample evenly over the surface nutrient agar. Number 10-fold dilutions usually happens in sequence from one to twelve so as to encourage the cultivation of the Petri dish and find the most suitable for the counting of colonies. This might take from a few up to twelve Petri dishes. In practice, suitable breeding is considered to be such, when the number of colonies counted not exceed 250, which are recommended by the FDA. Cups incubated 24-48 hours for bacteria and 72-120 hours for fungi in order to achieve a specified number. This is a relatively long time required for the formation of the colonies visible eye. However, if the sample is relates to an important time or the patient is in critical condition when the time is important - a long incubation and serial cultivation can be a disadvantage with a potentially life-threatening consequences.

[0005] Colony, appearing on a dense medium are calculated to determine and total microbial growth or transferred and analysed in accordance with the normal microbial methods, mass spectrometry, Fourier transforming infrared spectroscopy, chromatography, or CPA.

[0006] Removing suspicious of the colony and the subsequent analysis of this colony, lengthy and cumbersome traditional methods or complex and expensive high-tech tools and techniques led to the invention of CHROMagar™ nutrient media containing special substances, substrates, and antibiotics, which allow growth with simultaneous specific colouring colonies. The CHROMagar™ Candida, CHROMagar™ O157, CHROMagar™ Salmonella, CHROMagar™ Staph. aureus and some other currently used for identification colonies pink, green, or blue. Substances that trigger color, accumulate in the bodies cells, which causes problems for their growth. Therefore, colonies are unusually small and very often require long incubation. Only colony of normal size can be detected by color as the color quite pale, and a small light absorption useless microscopy. This means that micro-colonies (early stage of forming colonies) may not be detected using CHROMagar™. Only certain important species and a number of other species can grow on CHROMagar™. Therefore CHROMagar™ useless full (the Total Number of Living Organisms) detection and counting, which is most often used in Microbiology.

[0007] overall, The growth in a Petri dish is the usual microbiological operation used in medical, industrial, biotechnology, research and pharmaceutical laboratories. Worldwide, hundreds of millions of analyses performed by this method each year. Thus, there is a huge need for more effective, affordable, accurate and fast analysis for the detection, identification and calculation of microbes.

SHORT DESCRIPTION OF THE INVENTION

[0008] In the present description is given one or more incarnations of the device, method and material that surpass the shortcomings of the earlier technologies. In particular, this description does this by using a device for the detection, identification, or counting of microorganisms, which includes container with the medium in order to provide nutrients to the growth of microorganisms and porous element, or porous membrane makes contact with the medium. Himself porous element allows nutrients to pass through him, to support growth and for the colonies in normal size and for (collectively, the "colony"). And porous element can be moved from the container in other places for subsequent processing of the indicators and for further visual analysis.

[0009] As the colonies can be migrated using porous element, growth and colouring with the indicator for cells and can be separated for optimal results: growth stage for rapid growth of the cells without harmful indicators inhibiting growth, and the stage of the indicator for painting and identification, which allows be detected quickly. In addition, if the cells are analyzed in the early stages of , in accordance with the present invention, then the serial dilutions much less necessary. Thus, the present invention surpasses the limitations of the conventional method, which requires the colony normal size and prolonged incubation for example, when you use CHROMagar, after serial dilutions prevent germination colonies on each other. With a small number of dilutions or without dilutions, in accordance with the present invention, the results are faster and lab resources are conserved. In particular, the porous element can be moved together with colonies or on the porous element or on a nutrient medium above the other porous element for the next steps and a wide range of treatments including biochemical indicators or manual or automatic visual determination, calculation or analysis image (analysis according to the form), which is clearly visible in normal light microscope. To to inoculate a sample of the culture media or on the porous element, porous element has a property that allows the liquid part of the sample to be missed down the container. Further, to ensure the growth of cells deposited from the sample, porous element has a feature allowing nutrients nutrient medium to communicate through this feature with the cells. Then, to allow the cells to be processed by the indicator, porous element has a feature that allow biochemical indicators, such as the various types of paints or communicate through this property with cells, while supporting cells and micro-colonies in the integrity of the porous membrane, without breaking down and no blurring the micro-colonies. Finally, to allow the cells and be , porous element is transparent.

[0010] There are two possible locations for the porous element on a nutrient medium. In the first variant, porous element located at the top of the medium and is not covered by any subsequent layers of protection. To make the transportation of cells, porous membrane in this incarnation has porosity more than the smallest cell, to keep the cells on the porous element. So, when the porous element is transferred from a nutrient medium for later analysis, cells and colonies carried along with him. This first incarnation useful for fluorescence analysis, as porous element has its own background fluorescence. In the second incarnation of the porous element is mounted on top of the nutrient layer and then covered with additional environment. In this incarnation cells and micro-colonies remain on top of the additional environment and thus porous element may have pores larger than the smallest cells as cells and micro-colonies are held on the upper surface of the additional environment, from the top of the porous element. Therefore, when the porous element is removed from the container, it brings additional environment on its surface along with located on this additional environment. The second incarnation useful for painting , but not fluorescence analysis as a breeding ground itself has a high level of background fluorescence.

[0012] In General, the present invention to provide a much faster, more efficient, less expensive and more reliable device and means for: the discovery of the total number of viable or microorganisms (Total Living Organisms) through the intensely colored ; differentiation of the same type from another clearly visible , which are for species or groups of species; detection and identification of of antibiotic-resistant microorganisms; identification or radio-labeled antibodies; and detection, differentiation, and / or identification of light absorption or by fluorescent methods. These and other objects and advantages of the existing invention will become apparent to specialist after reading the description of the main embodiments, which are also illustrated with various patterns.

A BRIEF DESCRIPTION TO PICTURES

[0013] the Pictures shown here are part of this description. Drawings illustrate embodiment of the invention and, together with the description, are the explanation of the principle of the invention. Should be understood that the drawings referred to in this description, shown regardless of the scale, if it is not especially noted.

[0014] Figure 1 - functional block diagram illustrating the spatial and functional relations incarnations porosity and structural element and the growth medium for the analysis of sample cells in the solution in accordance with one of the embodiments of the present invention.

[0015] Fig.2 container agar environment with multiple filtering elements on it in accordance with one of the embodiments of the present invention.

[0016] Fig.2 cutaway view of several incarnations of the environment and the filter element, showing the spatial relationships according to one embodiment of the present .

[0017] Figure 3 illustrates the elements and stages determine cells and protection and portable porous element in accordance with one of the embodiments of the present invention.

[0018] figure 4 - the annotated image culture in the container with agar environment with several of porous elements together with secondary environment for application in accordance with one of the embodiments of the present invention.

[0019] Fig.5-5H are several different species of the genus Bacillus identified through the porous element and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0020] 6 - species of the genus Listeria, identified through a porous element and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0021] Fig.7 - species of the genus Staphylococcus identified through the porous element and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0022] Fig.8 - species of the genus Escherichia identified through porous element and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0023] Fig.9 - species of the genus Pseudomonas, identified through a porous element and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0024] 10 - chart the definition and identification of cells and in accordance with one of the embodiments of the present invention.

A DETAILED DESCRIPTION OF THE INVENTION

[0025] Description made in the details of the preferred embodiments of the invention. Examples of the preferred embodiment illustrated with accompanying pictures. While the invention will be described in relation to the preferred embodiments, it is clear that they are not meant to restrict the invention these incarnations. Rather the invention is intended to expand the alternatives, modification and equivalents that may be included in the spirit and opportunities of the invention, as defined in accordance with the attached statements. Additionally, in this detailed description of the invention, numerous specific details formulated to ensure a full understanding of the present invention. However, it is obvious for a professional, that the present invention can be implemented without these specific details. In other cases, the known methods, procedures, components, and microbiological details are not described in detail, so as not to obscure aspects of the present invention.

Terms and explanations

[0026] Environment for growth. Environment for growth is a breeding ground used for the growth of in the context of the present invention. This may be a common environment for bacterial growth, such as Soy Agar, environment for mold and yeast - Saburo Dextrose Agar or " environment for growth of the individual group of microorganisms, such as Agar, used for the growth of species of the genus Pseudomonas, or mcconkey Agar for the identification of Gram-negative enteric pathogens. Growth environment is the basis for the porous element installed on its surface.

[0027] micro-colonies. is a small colony, appearing after 3-6 hours of incubation. The typical size of 10-100 micrometers. They are colourless and are invisible in normal light microscope. Even such as painted in yellow micro-colonies Staphylococcus aureus, are not normally visible in the optical microscope. Therefore, usually, micro-colonies should be painted to become visible. Most of microbial species produce micro-colonies specific form during the early stages of growth (3-6 hours). Quite often, the differences in their form is so obvious that offer a good opportunity to distinguish different types of just form the colony (see Fig.5-5H, and 6-9). Micro-colonies consist of chains of cells, not mechanically connected with each other. Therefore, growth environment and secondary environment should not contain any free liquid, otherwise cell micro-colonies . All nutritional liquid and dissolved materials related or other polymer gel.

[0028] Membrane. Membrane or porous element is an important material for growth and colouring to make them visible and explore their specific form. Requirements to the membrane of the following: it should be transparent to transmit light, when used with a microscope; must be permeable to water and nutrients and indicator substances in the secondary environment in order to feed the microorganisms in the period of formation of micro-colonies and accordingly to paint these micro-colonies; must allow indicators pass through and react with cells ; must be impermeable to cells (except with a layer of a porous membrane environment added on top) so that the cells can grow only from one side of the membrane; must be resistant to external cellular enzymes to hold the membrane is intact and maintain ; must withstand autoclaving, because the membrane must be sterile before use; should be to freely pass water and most of the substances through itself - hydrophobic membranes highly resistant to this kind of ; should be and colourless as background fluorescence intolerant when fluorescent analysis methods, and the painting can mask the color ; must not contain substances prevent or inhibit microbial growth. In addition, the membrane must not visible particles or fibers, as is the membrane to filter since particles and fibers hamper the free growth and are able to change the time of growth and form (except when a layer of protection on top of a porous membrane). Experiments showed that the best polymer for these purposes is regenerated cellulose and its derivatives such as cellophane, and dialysis membrane. Other polymers having the same properties can be used also. Many other types of materials for membranes can be used too: it organic and inorganic materials with natural or artificially pores. Membranes can be transparent to the light transmission for the best visual observation or semi-transparent or white or color, although the latter provide the least desirable quality monitoring.

[0029] Secondary environment. Secondary environment is a gel, polymer or BioNanoChannel™ plate, employees of the carrier for the indicator substance or labeled antibodies. Its basic function is to be the bearer of an indicator substance that is dissolved in a liquid (water or buffer solution), or antibodies and to carry them through the membrane to the by simple diffusion. Such gels as purified Agar (Agarose), Gelatin, and other appropriate polysaccharides with long polymer molecules can be used to link liquids and substances in semi-solid form. The simple use of liquid solutions as indicators should typically be avoided (with the obvious exception for BioNanoChannel plate), because the actual liquid solution quickly penetrates through the membrane and forms large areas where the micro-colonies break up, lose their shape and are joined together again, forming a large lump of cells. Labelled antibodies, however, relatively large molecules (IgG have a size of approximately 150,000 daltons) and can't get out of polymer gel structure freely. In this case BioNanoChannel plate can be used instead of the mentioned gels. Each plate is made up of millions of small nano-channels with a diameter of 10 micrometers or less. So, labelled antibodies are thousands of times smaller than nano-channels and can move freely within the fluid in nano-channels with one side of the plate. When the liquid in a nano-channel, it is associated with strong forces and can not move freely through the membrane and destroy micro-colonies, as it can make a free liquid.

[0030] Indicators. Within the present invention, indicators are substances liable specific or not specific tag micro-colonies. This can be and\or substances or mixtures thereof, and labelled antibody. and substrates produce or fluorescent substances as a result of reaction with specific or non-specific enzymes or groups of enzymes. Small molecules of these substrates are free to leave gel, pass through the membrane and to react with the enzymes of living cells. Antibodies, labelled or radioisotopes and built-in BioNanoChannel plate, may freely leave the nano-channels and pass through the membrane, if the dimensions of the membrane 10^6 Dalton or more. Not yet reaction labelled antibodies are removed by the membrane transfer to another BioNanoChannel plate one or more times.

[0031] Identification, differentiation and identification. The term «definition» is generally understood as the ability to identify and measure any micro-colonies, regardless of their taxonomic facilities. Differentiation refers to the ability of the method used to differentiate two or more species among themselves on the membrane by their shape, color or wavelength\color fluorescence. «Identification» means the identification of the genus and species of the micro-colonies. Definition of needs only chromogenic or substrates. Differentiation is based on the differences in the shape or color of . Identification requires the use of antibodies, preferably monoclonal.

A detailed description of the drawings

[0032] Relative to the Figure 1, functional block diagram 10 illustrates the spatial and functional relations of the two incarnations of porosity and structural element and the element of the growth medium for evaluation of the sample cells in solution according to one embodiment of the present application. The first incarnation uses porosity and structure item 14A, which is installed on top of the surface of 17 the growth medium 16 with the liquid portion of The 18th solution sample 12, passing the filter element 14A down the growth medium 16 in the direction of the bottom of the container containing the growth environment 16, while the portion of cells 20 In the solution of the sample 12 is held or blocked from passing porous element 14A through the pore size of the membrane, which is smaller than the expected cells. Nutrients 22 from a nutrient medium 16 pass through the membrane 14 to 20 cells In the heatsink on top of the porous element 14A.

[0033] In contrast to the first incarnation, the second incarnation of Figure 1 uses an additional layer of the growth medium 13 above to, or located on the porosity or structural element 14A. In this case the liquid portion 18A solution sample 12 passes through both an additional layer growth medium 13 and porous element 14A through growth environment 16, but the portion of cells 20A of the liquid solution 12 is captured or is blocked from passing through an additional layer of the growth medium 13 by the fact that liquid can penetrate into the environment and the cells cannot. As a result of the membrane in the second incarnation 14A not required porosity less than the size of the cells, which should be deducted. Preferred porous element 14A in the second incarnation of the main function to provide structural support to the additional layer of the growth medium 13 and cages with to transfer 24. The second function of the porous element 14A in the second incarnation in the penetration of nutrients 22 of the growth medium 16 to cells and located on additional layer of nutrient medium 13, who may himself have nutrients for the same purpose. Due to this, for the filter element selection of material and the size of the pores can be wider in order to meet the structural requirements and absorption of nutrients.

[0034] an Important function of membrane 14A for all incarnations is to move or remove, function 24 , withheld or painted cells located or on the porous element 14A, or on an additional layer of the growth medium 13 located behind those for later processing withheld or blocked cells at the stage of indicator 31 on the modified porosity\structural element 14 In the way of interaction with the indicator 23.

Possibility of the transfer of living cells and with the maintenance of their integrity allows the growth and indicator stages for cells and be optimally separated: growth stage 21 for rapid cell growth without toxic indicators and indicator stage 31 for special stains and identification, which small size to be defined quickly. Moreover, the cells are determined at an early stage during the initial size , because they can effectively be determined by the present invention, and serial dilution much less necessary. Thus, the present invention overcomes the previous methods, which essentially require the traditional size of the colonies and the long incubation as for example with CHROMagar, after serial dilutions prevent overlap of the colonies.

[0036] Porous element 204 can be porous element allowing the liquid sample and nutrients to pass through them. Porous element can be membrane-permeable membrane, the dialysis membrane or any type of existing elements, including cellulose, plastic materials with holes etc. Specific type of porous element 204 selected depending on how the additional layer of protection is located on it. Porous element can be transparent, and permeable for nutrient and indicator substances, but not for microorganisms. In another embodiment porous element is permeable membrane with one or more of the following characteristics: be , hydrophily, the lack of fluorescence, transparency, resistance to cellular enzymes, availability of regular or irregular pores within: 1000 to 10^7 Dalton (Yes). Material porous element can be selected from the group consisting of: regenerated cellulose, cellophane, , dialysis membranes, or other material with suitable size of the pores and meets the requirements referred to. It is known that the dialysis membrane highly permeable substances\molecules of a certain size, such as nutrients for the growth of microorganisms. The pores of dialysis membranes can be in the range from 1000 up to several hundred thousand Yes (Dalton). A large pore size allows many substances to pass through the membrane. However polymers with long molecules such as polymer galactose agar, used in microbiological dense media cannot pass through membrane. Experiments show that microorganisms can grow on the surface of the dialysis membrane put on the surface of dense nutrient medium due to the passage of nutrients through the membrane to the growing cells. Dialysis membrane on the surface of dense nutrient medium, or integrated into the layer agar allows to transfer the colony and micro-colonies, appearing on their surface to another surface filled indicator substances.

[0037] In another incarnation of the porous element can be a wide variety of filters required for the structural and liquid functions, not for the deposition of cells or blocking feature, which instead is made with an additional layer of protection on top of the filter element, as is provided solely first embodiment Figure 1.

[0038] Regarding Fig.2, section 200 for the various incarnations of the environment and the porous element shows the spatial relationships in accordance with one of the embodiments of the present invention. Section A-A shows the porous element 204A, just installed on top of a dense nutrient medium 206A, allowing easy transfer of the dense nutrient medium 206, but possibly reduces from this application cells of the specimen to the upper surface of the porous element. In contrast, an alternative embodiment in the context of A-A shows the middle 210 on the top layer of dense nutrient medium with a depth sufficient for the porous element 204B from zero to nominal value 208, for example, 0.1-5 mm below the surface of dense nutrient medium, by making a shallow well in which the sample may be and securely distributed under the controlled surface area. Impression 210 can be formed hot element with the form of depressions by fusion of nutrient pollution around the time of laying in the form of depressions, which is removed after the hardening of the environment. Section shows the incarnation, where the first portion of dense nutrient medium is deposited, then hardens with the installation of the porous element 204C and with the subsequent formation of a secondary layer of nutrient medium 206c, which covers the porous element at a depth of layer 212, making spike 214 between 206 In the first and second layer of protection With the invisible. The present invention is well suited to a wide range of methods and procedures for installing the porous element on or in the nutrient medium.

[0039] Figure 3 illustrates the components and steps to set to determine the cell 300 by defining protection and portable porous element in accordance with one of the embodiments of the present invention. Porous element 204D-1, after the transfer of container 202 contains growth environment 321, is transferred to the secondary environment 302 with an indicator substance 331, which changes the cells and micro-colonies on the porous element 204D-2. In one of the incarnations two or more indicator steps are used to satisfy the procedure on determining the sought cells and , for example, the second indicator uses environment 303, which moves the indicator substance to potentially change the cells and micro-colonies on the porous element 204D-3. Secondary environment is selected from the group consisting of agarose, , gelatin or gel, capable to be the bearer with the possibility of release molecules indicator. In another embodiment, the test system has a concentration of the gel in the range of 0.1%-10% in water solutions, buffer, of sodium chloride or liquid medium.

The indicator for may be the addition of a Chromogen substrate, substrate or fluorescently or radio-labeled antibodies. For example, secondary fluid may be pure agar, filled with by methyl (MTT), which stains micro-colonies in the dark purple color. Unpainted micro-colonies practically invisible in the microscope. Many other substances can be used for the detection and differentiation of microorganisms, including those that are toxic to cells or limit growth. Many fast and simple methods for determination and identification can be adapted for use with this method.

[0040] After the indicator of material is used, porous element 204D-5 respectively painted 322 and 322 In can be investigated and calculated, for example using a light microscope or automatic system of identification, recognition and counting images zoom. Selective net 324 given the seal or geometrically shaped by heating or engraving, promoting reliable count ratio of the area among the .

[0041] In the alternative analysis, the colony and micro-colonies can be identified with the help of antibodies labeled with fluorescent dyes. This type of analysis can be performed in one of the incarnations using BioNanoChannel ("BNC") device for determination of the 330 as a carrier of antibodies. In this case, the antibodies are placed in a solution of the liquid inside the nano-channels, on which is placed the porous element with the colonies. The upper surface of the BNC is the support of the porous element 204D-4 to maintain the integrity and cohesion of existing colonies, simultaneously allowing large antibody molecules move in microchannels and communicate with any colonies on the porous element 204D-4 through the top of the open channel. Porous membrane with significantly large pores, such as 10^6 Yes or more or suitable for certain types of antibodies to be transferred to the colony. The colony (antigen) will react with the antibodies, are in contact with her. Excess labeled antibodies can be removed by transfer porous item one or more times to another BNC with a solvent or buffer without fluorescent molecules. In contrast, large molecules antibodies can move in agar or gel because of their large size will prevent this. Moreover, the installation of porous item directly on the open container with liquid medium with antibodies will not maintain the integrity of the colonies if the membrane becomes impregnated, flooding or blurred liquid, dissolving, and blurring the colony. Thus, the use of a portable porous membranes in accordance with the present invention, in combination with the ANS plate, providing support for porous membrane, and delivery mechanism labeled antibodies provides an effective solution, overcoming the limitations of known methods.

[0042] GNH is a plate containing a lot of cylindrical and parallel micro channels open with one or both ends, and with a micro-channels with a diameter of approximately 1 to 30 micrometers with a length of about 100 to 1000 micrometers and the number of micro channels per square centimeter 100000-1000000, long (diameter\length=1\10-1\100). A more detailed description of BioNanoChannel devices and methods is given in the Application USA 11\109,857, Sergey Gazenko, entitled: "Device For Rapid Detection And Identification Of Single Microorganisms Without Preliminary Growth", where the application is given in full.

[0043] Figure 4 shows the annotated image 400 culture in the container with agar environment that has multiple porous elements together with a separate secondary fluid for painting, in accordance with one of the embodiments of the present invention. Petri dish, container 202A with a nutrient medium with multiple porous elements 204E and culture - they demonstrate the successful growth in the stage of growth 421 together with the transparency of the porous element and ease of transfer of the porous element in the container 302 in the stage of indicator 431 with secondary indicator environment in him, where the indicator triggers a darkening of the , hardly determined without the use of the indicator in the container with only one medium 202A.

[0044] Regarding Fig.5-5H; showing several different species of the genus Bacillus identified by using porous element, and a separate secondary environment in according to one of the embodiments of the present invention.

[0045] About 6 shows several different species of the genus Listeria, which are identified using porous element, and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0046] Regarding Fig.7 shows species of the genus Staphyllococcus identified by using porous element, and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0047] Regarding Fig.8 shows several species of the genus Escherichia, identifiable using porous element and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0048] Regarding the Fig.9 shows species of the genus Pseudomonas, identifiable using porous element and a separate secondary environment in accordance with one of the embodiments of the present invention.

[0049] About 10 graph 1000 the definition and identification of colonies, including micro-colonies and ordinary colony, is shown in accordance with one of the embodiments of the present invention. For the initial stage 1002 required container with the medium and porous element mounted on or under the surface of the medium, as shown in Fig.2. In the subsequent stage 1004 poured or liquid sample in the top of the container porous element. The sample can be distributed over the entire surface of the Petri dish or focused only on the porous element, for example, as shown on the section A-A', where a porous element 204B installed in the middle 210, size distribution of the sample porous element 204 Century later in compliance with step 1006, microorganisms seized or blocked on the porous element or on an additional layer of protection, the top of the porous element, the latter is shown on the section B-B Fig.2. Step 1008 is for the purpose of incubation for development , although much less time than the typical known methods of incubation for the development of colonies of normal size, though the latter is another embodiment of the present invention. In a subsequent step, 1010, porous element of any environment over the top of it is transferred from the container with the medium to another environment with the indicator to determine the detection or / identification of microorganisms. In step 1012 determined the need in using additional steps indicator. If the need exists, as often happens, step 1010 repeats itself, but in most cases with a new suitable indicator. In addition, step-1014 provides the final step in the research of the form or colour colonies for the detection, identification, or the computation of the relevant .

[0050] Detection of the total number of viable microorganisms () - one of the most necessary laboratory procedures in the world. It shows the presence and level of microbial contamination in food, biotechnology, environmental, and other industries. Normal is, making a few (3-12) 10-fold dilutions of the sample in the buffer of liquid nutrient mediums, solution or 0.9% NaCl and then distributing one milliliters each dissolution on the surface of the ordinary Petri dishes filled with the appropriate nutrient agar. Cups incubated for 24-48 hours before when 32-37°C. After this Cup is removed from the incubator and colonies are counted. This procedure requires 3-12 tubes , 3-12 Petri dishes and a long incubation. The result appears only after 24-48 hours. This simple procedure is repeated hundreds of millions of times each year worldwide. Method in accordance with the present invention described here is much more faster (3-6 hours), does not require a 10-fold dilutions and requires only 1-2 cups with membranes. Several sterilized (121 o C, 15 min) dialysis membranes (pore size of 12,000-18,000 Yeah) round in shape and mounted on the surface of the hardened nutrient agar in a Petri dish. Other membranes with other pore sizes or plastic membrane can be used also. The surface of the Czech membrane is covered next hot (80-90°C) nutrient medium for 1-2 seconds excess environment not hardened in 1-2 seconds poured. This means that all the membrane will be covered with a thin (0.1-0.2 mm) layer of the medium. The upper layer has a free exchange with bottom layer in the sense that water and nutrients, in addition to the polymer Agarose, can be exchanged between the layers.

[0051] Procedure in accordance with the present invention of the following: one sample transported and distributed on Petri dishes with established membranes (Figure 2). Liquid sample absorbed into the environment, Cup incubated living cells that are present, begin to growth and formation of . show that all the studied bacteria form a micro-colonies within 5 hours at 35-37°C. These micro-colonies consist of from several tens to several hundreds of unstained and almost invisible cells. To paint them, membrane separated from their cups, using sterile forceps, and transferred to the secondary environment. In accordance with the standard method of secondary environment is 1% Agarose in 0.15 phosphate buffer with pH 7.5, and with substrate 3-(4,5--2)-3,5- by methyl at a concentration of 0.7 mg/ml This, slightly yellow, substrate can be transformed into a dark purple after reaction with living cells. This substance is able to change the color of all known aerobic and anaerobic bacteria and yeast, because reacts to the last stage of the electron-conductive circuit microorganisms.

In other words, it does not block the beginning or the middle part of the respiratory chain, which can lead to early death of the cells. So, painted product is collected in a large concentration on the surface of the cell (location dehydrogenases in a cage), giving a dark purple or black bodies cells. The time required for staining usually within 10-20 minutes at 30 to 40 degrees C. This is enough time for the release of molecules of substrate and to allow them to penetrate through the membrane and a thin layer of dense nutrient medium by diffusion and react with . After the reaction, a Cup with a secondary environment and painted transferred under the light microscope and micro-colonies are counted. The size and shape of may be additional features for differentiation of species (Fig.5.1-5.8).

[0052] This method requires only 5-6 hours and much less material and manipulation than the traditional method. It is important to note that this example does not need many 10-fold dilutions, and only 1-2 cultivation in case of extremely high concentrations. This convenience is possible due to the relatively small size of the : the concentration of cells in a sample can be millions in a milliliter, but all micro-colonies will grow separately without overlap, because the area occupied by one colony of thousands of times smaller than the area occupied by the normal colony.

Fluorescent embodiment

[0053] Membrane used in this method are made of regenerated cellulose, which is not a fluorescent and transparent material. Nevertheless, the nutrient medium contains substances. This restricts the use of a thin layer of nutrient medium on top of the porous element or membrane, as described in one of the incarnations. Recommended membrane in this case, this is a 100,000 Yes dialysis membrane.

The relatively large size of the pores in the membrane contributes to satisfactory growth without even a thin layer of protection on top. Additional moisture is also useful for this incarnation, because it creates more favorable conditions (microclimate) to form colonies.

After 5-6 hour growth membrane is transferred to the secondary environment that is filled with one or more substrates. To determine used freshly cooked mixture 4- phosphate and 4- acetate at a concentration of 0.1 mg / ml in acetone. This mixture is poured on the surface of the secondary environment (1% Agarose in distilled water) for a few minutes before the membrane with is hoisted to the top. Incubation takes 10-15 minutes at 35-40 Celsius C. All micro-colonies receive blue fluorescence, it is visible in the fluorescent microscope, because mixture of substrates used responds with a large number of phosphatases and esterases present in all living cells.

Transparent and membrane allows the use of inexpensive fluorescent microscope with UV excitation (e.g., wavelength 350-380 nm) light from below upward, however dear microscopes can be used also.

incarnation; the identification of

[0054] Identification of microorganisms using antibodies, labelled (FITC, , rhodamine, and others), may be carried out through a porous element or membrane without a layer of dense nutrient medium at the top. Immunoglobulins are used as antibodies, are very large protein molecular complexes. For example, often used G (IgG) has a feet of approximately 150,000 Yes. Such a large complex should pass through the membrane and react (antigen-antibody interaction) with special surface antigens cells. Therefore, pore size must be greater than, for example 10^6 Yes or more.

[0055] To execute a method, a sample containing living cells of different types, including target species is distributed on the surface of the membrane. The liquid is absorbed in growth environment and a Cup of incubated for 5-6 hours at 35-37°C in highly humid conditions so as to create a micro-colonies. After this membrane with on the surface is transferred on BioNanoChannel plate filled with a solution containing antibody complementary to the target (for example IgG-FITC). Molecules labeled antibodies float freely inside , each having a diameter of 10 micrometers and length up to 500 microns. Approximately 2.5 million cover BioNanoChannel plate having a diameter of 25 mm Thus, the solution with antibodies «captured» in a robust set of tiny glass channels. This is important to prevent the formation of wet areas with excess fluid, which can blur the micro-colonies and mix the target cells with the cells of other species. Use BioNanoChannel plate is also important because it allows a large IgG molecules move freely, while Agarose used in other incarnations (for small molecule size), is not able to release IgG from its polymeric structure.

[0057] the Definition, identification and/or recognition of forms of micro-colonies can be carried out by the operator using a visual method or the determinant of the images. Different types of identifiers image are currently in use on the basis of CCD cameras and computer programs recognition and connected with a microscope and a computer in order to fix the image and analyze it.

[0058] Identifying can be improved with the use of micromanipulators, which remove a colony of the currently selected form or fluorescence with subsequent identification by PCR or other suitable method. Collection of colonies can be conducted in different ways. However, identification of micro-colonies by PCR does not necessarily mean a specific collection necessarily the colony because PCR techniques allow the detection of target DNA of the target organism against the background of the DNA of other species.

[0059] Membranes with different pore sizes and other skills useful for Virology, especially in cases when the cell cultures are used for growth and virus definitions the holes in the cellular layer.

The quality of decontamination

[0060] Decontamination of the internal condition of buildings after epidemics, the presence of a sick killer diseases or terrorist attacks or military biological attack is the all important problems of Microbiology.

The presence and number of surviving cells after decontamination defines the future of areas. This analysis should be completed in a very short period of time, especially if the areas are important public areas (airports, state military premises or other places where there is a high attendance or social relevance. Only living/surviving cells must be defined in these circumstances.

Moreover, only the real growth of cells known as the most reliable method for determining of living organisms. Normal growth takes 24-48 hours or more for the education of the colonies. The present invention allows the same result for 5-6 hours, a lot cheaper and requires a lot less work, that is, a larger number of samples can be also analyzed. For example, if the areas were contaminated with spores of Bacillus anthracis and decontamination killed vegetative cells and spores, then only the spores of Bacillus can survive. The spores of Clostridium, Actinomycetes and fungi do not grow on the environment used for the growth of B. anthracis for a number of reasons. Thus, only one species of the genus Bacillus, will grow in this environment is B. anthracis. Soy Agar (TSA) can be useful in this case. Fluid samples from different parts of the building are placed on the TSA's membrane and a thin layer of TSA top. After 5 hours of incubation at 37 C, the membrane with possible transferred to a Cup with a 3-(4,5--2)-3,5- bromide (1% of net Agarose in PBS pH 7.5). After 10-20 minutes intensely colored micro-colonies specific forms can be distinguished from other Bacillus species (for example, Fig.5.4 shows like the look of micro-colonies of Bacillus subtilis globigii, known as the model for research Bac. anthracis). These micro-colonies can be captured and analyzed rapid PCR (1-1 .5 hours) to confirm B. anthracis survival after decontamination. Thus, the resolution on the further use of space can be obtained through 6-8 hours instead of 24 to 48 hours.

Determination of antibiotic-resistant microorganisms

[0061] Definition of antibiotic-resistant microorganisms in human samples or hospital acquired infections (Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella oxitoca and others), a fast-growing medical problem. Life patient strongly depends on the speed of diagnosis strain. The growth and formation of the colony in the presence of the antibiotic is the only reliable method is widely used in the diagnosis of antibiotic-resistant strains. Currently it takes 24 hours to detect the growth/colony on a Cup with a nutrient medium with the antibiotic (CHROMagar, MRSA and other). The acceleration of this analysis up to 6-8 hours is able to save thousands of lives every year. As an example, the main method for rapid detection of Staphylococcus aureus following: a sample of blood or the environment), probably containing S. aureus is distributed on a Cup with (MRSA Wednesday) with the inserted discs. After 5-6 hours membrane transferred to the Cup with and substrate. After 10-15 minutes of all micro-colonies dark-violet and visible. Micro-colonies specific form (Fig.7) are defined as antibiotic-resistant S. aureus.

Fast determination of E.coli

[0062] the Presence of E. coli food or environmental samples known as a reliable characteristic of fecal contamination. Only living cells are important for counting; PCR and immunological methods are ineffective in this case. The ability to form a colony is the most reliable characteristic of living E.coli. For growth and determination of E. coli is used mcconkey agar for Gram-negative and -positive microorganisms. In addition to the rapid growth of E. coli can be used CIRCLEGROW > environment. Can also be used LB agar. All of these environments are suitable for the growth of E. coli and certain other microbes present in the samples. Porous element or membrane embedded in the surface of the medium should not have a thin layer of the medium on the surface and must have an appropriate pore size, for example 50,000-100,000 Yes. E.coli generates visible micro-colonies after 4-6 hours of incubation at 37 C (Fig.8). After incubation, the membrane carrier E.coli and other types of micro-colonies is transferred to the BioNanoChannel plate filled with 4--beta-D- (0.1 mg/ml in 35% ethanol). After 5-10 minutes of incubation micro-colonies E.coli acquire a bright blue fluorescence under a fluorescent microscope and UV excitation 350-380 nm. Other micro-colonies remain colorless.

Alternative Incarnation

[0063] This description applies to a wide variety of applications and is not limited to a special type of analysis, environment, filter, diaphragm, or microbe described in this invention. In the review of the applications described the present invention leads method and device overcome the limitations of the current approach, such as multiple serial cultivation, duration and materials consumption and poor results.

[0064] for Example, the present invention can be adapted to the virus definitions growing thin layer of prokaryotic or eukaryotic cells and producing transparent zone on the surface in the places of the propagation of viruses. In this case, will be painted in intact cells, but not infecting virus small zone of transparency or plaques, where the cells and virus and do not have living cells for painting.

[0065] Previous descriptions of specific embodiments of the present invention were presented to the illustrations and the descriptions. They are not intended to be exhaustive or to limit the invention of certain forms. Naturally, many modifications and changes are possible in the light of the above inventions. The incarnation were selected and described to best explain the principles of the invention and its practical significance, and to let the others, qualified specialists is better to use the invention and of its various incarnations in various modifications for practical application. It is assumed that the essence of the invention defined statements, attached here, and their equivalents.

1. Way of fast growing, detection and identification or counting microorganisms an early stage, including the stage of obtaining the container with the environment with a porous element located at the top or below the top surface of the environment and the environment has nutrients for rapid growth microorganisms, and with porous element has pores from 1000 to 10 7 Yes; infusion liquid sample is not subjected to serial dilutions in a container in the area above the porous element; capture of microorganisms in the porous element or the environment above the porous element; incubation of the container to allow for the rapid growth of micro-colonies early stage; the transfer of porous element and any environment above it from the container into the secondary environment for assessment, detection and identification microorganisms; and research in growth, detection, identification or counting of microorganisms, and such way of growing of detection and identification or counting of microorganisms takes no more than about six hours.

2. The method according to claim 1, where porous element represents a permeable membrane, selected from the group consisting of polymers, such as regenerated cellulose, cellophane or and dialysis membrane.

3. The method according to claim 1, wherein the study carried out for identification of microorganisms, using monoclonal antibodies.

4. The method according to claim 1, wherein the stage of studies carried out manually or with the automatic system for detection and recognition of images.

5. The method according to claim 1, further comprising the stage of detection of the total number of viable (TVO).

6. The method according to claim 1, wherein the secondary environment contains a chemical indicator, facilitating the identification of microorganisms on the porous element.

7. The method according to claim 1, wherein the porous element catches or or filtered samples.