Method for sampling for detecting microorganism, method for detecting microorganism and set for detecting microorganism

FIELD: medicine.

SUBSTANCE: according to the following stages, flow cytometry is used to detect living cells, damaged cells, VNC cells and dead cells of a microorganism in a tested sample: a) the stage of processing the tested sample with enzyme chosen from lipolytic enzymes and protease with activity to destruct the cells differing from those of the microorganism, colloid protein particles or lipids found in the analysed sample; b) the stage of processing the tested sample with topoisomerase inhibitor and/or DNA-gyrase inhibitor; e) the stage of processing the tested sample been processed at the stages a) and b) with a kernel-dyeing agent, and d) the stage of detecting microorganisms in the tested sample processed by the kernel-dyeing agent with using flow cytometry.

EFFECT: convenient and fast detecting of live microorganisms and identification of the damaged and dead cells in foodstuff and clinical samples.

19 dwg, 8 tbl, 8 ex

 

The technical field to which the invention relates

The present invention relates to a method of detecting a microorganism contained in food or clinical samples, the method of obtaining the sample used for the method, and kit for detecting a microorganism. More specifically the present invention relates to a method and kit for detecting a microorganism, which enables you to evenly distinguish live cells, injured cells and dead cells of the microorganism contained in food and clinical samples.

The level of technology

Method of cultivation on cups traditionally used to measure the total number of live bacteria in food products, clinical samples or the environment. However, the method of cultivation on cups requires to get the results in about two days time. Moreover, bacteriological test, culture-based, when using the commonly used environment it is difficult to detect damaged bacteria in the environment, bacteria, damaged artificial influence (the first may refer to alive, but not cultivated (VNC) cells, and the latter may refer to damaged cells, especially in the narrow sense of the word), etc. and it was desirable to develop a fast and n is unreliable method for counting living bacteria.

Flow cytometry (FCM) is a method of transmission of the sample flow in the flow cell at a constant flow rate passing through the laser beam, and measuring the light scattering cells or other particulate or fluorescence emitted by the cells or other particles. Because it allows to detect microorganisms at the level of a single cell, in recent years, this method is used for detection of microorganisms not only in the field of molecular biology and cell biology, but also for detection of microorganisms in the environment, dairy products, drinks, clinical samples etc (for example, patent documents 1 and 2, non-patent documents 1 to 5).

However, the apparatus for FCM (flow cytometry)used for the purposes of this method, are extremely expensive and have large sizes, and they also require skill to use them. Moreover, for these devices to date are requiring improvements or solve problems related to economy, safety, simplicity, reliability, and validity of the distinction of live cells and dead cells of microorganisms for the valid applications in the food industry, where there is a wide variety of bacterial contaminants, as a direct relationship is established by bacteria, damaged by bacteria and dead bacteria.

For example, patent document 1 describes a method of detecting the total content of bacteria in a liquid sample using chelating ion, protease, surfactants and bacteriologically-specific fluorescent dye. Chelating ion, a typical example of which is EDTA, must be used in a concentration of from 1 to 5 mm, and if the concentration exceeds this level, then the cell wall and cell membrane of live bacteria, the cell walls are not damaged, can be destroyed. The preferred concentration of chelating ion used in the method of patent document 1, is from about 6 to 17 mm, and, on this basis, there is a problem, which is that undergo lysis as dead bacteria, and live bacteria. Moreover, the detection limit of this method is approximately 104CFU/ml, and, on this basis, if the number of live bacteria as low as 103CFU/ml or less) in a liquid sample, provided that only one living bacterium exists in a liquid sample, the bacteria must proliferate to levels above the detection limit. Accordingly, this method cannot certainly be considered as a quick method.

Patent document 2 on Easyway method of processing samples of body fluids by the protease, lipase and nuclease, lysis of leukocytes, platelets and red blood cells using staining the ethidium bromide buffer including sodium borate, EDTA, formaldehyde and a non-ionic surfactant (Triton X-100 etc), for staining the ethidium bromide only bacteria, detection and quantification of bacteria on the basis of fluorescence microscopy, flow cytometry or the like, However, it is assumed that the leukocytes and platelets do not undergo lysis, remaining in the body fluid sample even after treatment with protease, lipase and nuclease, and adsorbed live bacteria with the formation of complexes, and that are painted as live bacteria and dead bacteria, and thus, is difficult to determine whether the bacterium is alive or dead. Moreover, although patent document 2 describes that the method is a method of detecting bacteria in such a low density as 10 cells/ml (sample) over time about 2 hours or less, often 45 minutes or less, patent document actually describes an example in which the detection was not possible, while in the liquid sample does not have at least 104CFU/ml or more bacteria, and thus the method is not suitable for detecting small quantities of microorganisms, such as the cow's milk.

Non-patent document 1 describes how to use the properties SYTO63, consisting in the fact that it penetrates through the cell wall and cell membrane of living cells and dead cells, and property, TO-PRO3, consisting in the fact that it penetrates through the cell wall and cell membrane of dead cells, in an attempt detection of live bacteria and dead bacteria on the basis of flow cytometry. Additionally, the document describes an example in which living cells and dead cells suspended in sterilized water and were trying detection of living cells and dead cells in a specified environment. However, dead cells consisted of cells that are boiled for 15 minutes, and their cell wall and cell membrane were significantly damaged compared to dead cells in real food. Accordingly, this method is a method suitable only for dead bacteria in a limited range of food products, such as thermally processed food, while not rated conditions for ultra-high-temperature pasteurization, used for cow's milk, etc. and modern products, and destroys bacteria without denaturation of proteins in food products.

Non-patent document 2 describes a method that allows the protease To deistvovat what I have in UHT (pasteurized at ultra high temperature) milk from the cow to digest micellar casein, with the removal of lipids by centrifugation at cooling for the detection of bacteria in cow's milk and measure it the total number of bacteria (including live bacteria and dead bacteria), and how to add 0,1% Triton X-100 as nonionic surfactants for raw milk in addition to the above proteinase K for detection of bacteria in raw milk and measurements of the total bacterial number (the number of live bacteria and dead bacteria). However, in the methods of non-patent document 2, even if provided the protease to act on UHT cow's milk, micellar casein is not fully digested and there are a large number of not fully digested products with size comparable to the size of bacteria. If the fluorescent nuclear dye, such as SYTO BC or SYTO9, acts on such products, there is a strong non-specific absorption, impairing their distinction from live bacteria. Moreover, there is a problem, consisting in the fact that the cell membrane of somatic cells, such as bovine leukocytes and epithelial cells of the mammary gland, considered as part of the components, which contaminate the milk, are damaged only to a minor extent, and if they are subjected to staining with SYTO BC, SYTO9 or propedy iodide in the form in which they are present, then p is opici iodide does not penetrate into them, and as a result of chromosomal DNA emits green fluorescence, which complicates the distinction between somatic cells and live bacteria.

Non-patent document 3 describes a method similar to the method of patent document 1, with the exception that they did not use the processing of the protease as a way of measuring the number of living bacteria of lactic acid bacteria in yogurt or sourdough, yogurt, and described as a method using nonionic surfactants in combination with a chelating agent. As a distinctive characteristic of the invention are described that way you can destroy somatic cells, acting as contaminants, and to effectively separate globules of fat. However, the samples subjected to the above processing, contain many contaminants originating from milk, and the limit of detection of live lactic acid bacteria, is reduced to so small content of about 105CFU/ml for yogurt or starter yogurt because of the presence of contaminants. Accordingly, the method requires a particularly sensitive determination of conditions for the destruction exclusively somatic cells and the lack of damage to cell walls and cell membranes of live bacteria by regulating the concentration of nonionic surfactants and gelatinous what about the agent. Thus, the method is not suitable as a convenient and highly sensitive method of detection for distinguishing live bacteria and dead bacteria.

Although ethidium monoacid (EMA, 8-azide-3-amine-6-phenyl-5-ethylphenethylamine chloride) in General known as having anti-cancer effect, it is a poison against topoisomerase II (type II topoisomerase), existing in mammalian cells (for example, non-patent document 5). EMA randomly intercalary in the chromosomal DNA, and then only intercalated EMA turns into nitren under the action of irradiation of visible light and is associated with chromosomal DNA through covalent binding. For example, under the action of topoisomerase cancer cells regulate the degree of helicity of DNA chains or unwind the DNA chain, in order to carry out the replication of DNA chains and gene expression (transcription DNA), and the untwisting is achieved by splitting the relevant sections of chromosomal DNA and re-ligating the products of cleavage. In this case, as for the functions of the EMA, re-ligation of DNA topoisomerase II is inhibited as a result of covalent joining nitrene obtained from the EMA, at the time of re-ligation, and this results in the increasing fragmentation of DNA. EMA, not intercalated in the DNA chain and the existing free-f is RME, turns under the action of visible light hydroxylamine, but the hydroxylamine did not inhibit the activity of topoisomerase II.

As compounds inhibiting this activity of topoisomerase II, in addition to the ethidium monoacid mentioned above, a well-known amsacrine, doxorubicin, ellipticine, etopside, mitoxantrone, saintaubin etc. as compounds inhibiting the activity of topoisomerase I, which has activity similar to topoisomerase II, known camptothecin, topotecan, etc. (for example, non-patent document 6). Additionally, in the field of bacteria as compounds inhibiting the activity of bacterial DNA gyrase, with activity similar to the above-mentioned enzymes, known ciprofloxacin, ofloxacin, enoxacin, pefloxacin, fleroxacin, norfloxacin, nalidixic acid, oxolinic acid, pyramidula acid and so on (for example, non-patent document 7).

To date, however, nothing was reported about the use of these poisons topoisomerase I poisons topoisomerase II poisons bacterial DNA gyrase for pre-treatment of samples, such as food and clinical samples containing microorganisms, methods of study to distinguish between living cells and dead cells of microorganisms in order to implement a fast and highly sensitive detection.

p> As another method of detection of live bacteria was proposed automatic system for easy and quick detection of respiratory activity and esterase activity (patent document 3). However, detection using this method is limited to cases in which it may be accurately measured respiratory activity and esterna activity of the studied bacteria.

As a state, other than live bacteria, there are damaged cells, VNC (living, but non-cultivated cells and dead cells. Describes how to determine their flow cytometry using cFDA (diacetate carboxyfluorescein), which emits green fluorescence in the presence of esterase and propedy iodide (PI) (non-patent document 8). However, this method is also a way by which you can easily distinguish live cells, injured cells and dead cells, only if the damage to cell walls in the damaged cells expressed significantly.

Thus, if the damaged cells are damaged cells with a low degree of damage caused by prolonged time pasteurization at low temperature (LTLT) or short-time pasteurization at high temperature (HTST), or damaged cells from the bottom of the second degree of damage, caused by exposure to the environment, living cells and damaged cells cannot be distinguished using this method.

Patent document 1: international patent application unexamined publication in Japanese No. 9-510105.

Patent document 2: Japanese patent publication (Kokoku) No. 6-55157.

Patent document 3: Japanese published patent No. 2002-281998.

Non-patent document 1: Bokin Bobai, vol. 31, No.7, 2003, pp. 357-363.

Non-patent document 2: Applied and Environmental Microbiology, vol. 66, No. 3, 2000, pp. 1228-1232.

Non-patent document 3: Applied and Environmental Microbiology, vol. 68, No. 6, 2002, pp. 2934-2942.

Non-patent document 4: Applied and Environmental Microbiology, vol. 60, No. 12, 1994, pp. 4255-4262.

Non-patent document 5: Biochemistry, vol. 36, No. 50, 1997, pp. 15884-15891.

Non-patent document 6: The Journal of Biological Chemistry, vol. 270, No. 37, 1995, pp. 21429-21432.

Non-patent document 7: The New England Journal of Medicine, vol. 324, No. 6, 1991, pp. 384-394.

Non-patent document 8: Applied and Environmental Microbiology, vol. 68, 2002, pp. 5209-5216.

Description of the invention

Problems solved by the present invention

One of the purposes of the present invention is to provide a method of detecting a microorganism, which enables easy and fast detection of living microorganisms in food and clinical samples using economically advantageous flow cytometry, and can be used in wybron the x inspection of food businesses or in the clinical field, and the method of obtaining the sample used in the following way. Moreover, another objective of the present invention is to provide a set, enabling the detection of live cells, injured cells and dead cells.

Part of the solution

From the point of view of the above prior art, the applicants of the present invention thoroughly researched convenient way to test the truth or accuracy and application to the detection of living cells of the microorganism contained in food and clinical samples, particularly for distinguishing live cells, injured cells and dead cells. As a result, applicants have found that even if the number of microorganisms contained in the sample, infinitely small, it is possible to distinguish between live cells and dead cells with a high degree of sensitivity, by identifying various contaminants, including dead cells, contained in food and clinical samples by pre-treatment of the food product or the clinical specimen lipase, protease, ethidium monosiga, as a DNA intercalating agent, etc. in order to effectively remove contaminants at the stage of preliminary processing of the sample, the fluorescent staining pattern and exposure measurement when using the FR is knogo cytometer. Thus, the applicants have carried out the present invention.

Thus, the present invention provides a method of obtaining a measured sample for detecting live cells of a microorganism in a test sample using flow cytometry, which includes the following stages:

a) processing stage of the test sample with an enzyme having the activity from the point of view of destruction of cells different from the cells of the microorganism, colloidal particles, proteins or lipids present in the sample, and

b) the processing stage of the test specimen poison topoisomerase and/or poison DNA gyrase.

The present invention also provides a method for detecting live cells of a microorganism in a test sample using flow cytometry, which includes the following stages:

a) processing stage of the test sample with an enzyme having the activity from the point of view of destruction of cells different from the cells of the microorganism, colloidal particles, proteins or lipids present in the sample,

b) the processing stage of the test specimen poison topoisomerase and/or poison called DNA gyrase,

(C) processing stage of the test specimen processed in the stages (a) and (b) the agent, coloring kernel, and

d) stage detection of microorganisms in the test sample, obrabotan the m-agent, dye core flow cytometry.

In a preferred embodiment, the method for obtaining the measured sample for detecting live cells of a microorganism in a test sample using flow cytometry and method for detecting live cells of a microorganism in a test sample using flow cytometry, stage b) is carried out after stage a).

In preferred embodiments of the above methods, the test sample is a sample of milk, milk product, food product, obtained by using milk or milk product as a starting material, blood sample, urine sample, a sample of cerebral spinal fluid sample sinovialnoj fluid and a sample of pleural fluid.

In preferred embodiments, the implementation of the above-mentioned methods, the microorganism is a bacterium.

In preferred embodiments, the implementation of the above-mentioned methods, the enzyme is selected from lipolytic enzymes and proteases.

In preferred embodiments, the implementation of the above-mentioned methods, the poison of topoisomerase choose from amsacrine, camptothecin, doxorubicin, ellipticine, etoposide, mitoxantrone, syntopia, topotecan and CF-115953.

In preferred embodiments, implementation of the latter is above the poison DNA gyrase is selected from ciprofloxacin, ofloxacin, enoxacin, pefloksatsina, fleroxacin, norfloxacin, nalidixic acid, oksolinovoj acid and pyrimidinones acid.

In preferred embodiments, the implementation of the above-mentioned methods, the poison of topoisomerase represents the ethidium monoacid, and the method includes a step of exposure of the test specimen, to which add the ethidium monoacid, the irradiation of visible light.

In preferred embodiments, the implementation of the above methods, the methods further include stage C) processing the test specimen processed in stages a) and b), agent, coloring kernel.

In preferred embodiments, the implementation of the above-mentioned methods, the agent, coloring kernel includes a first coloring agent that can penetrate through the cell walls of living cells and dead cells, and the second coloring agent, which is more easily penetrate cell walls of dead cells compared with cell walls of living cells, compared with the first coloring agent.

In preferred embodiments, the implementation of the above-mentioned methods, the agent, coloring kernel is propedy iodide and SYTO9.

The present invention also provides a kit for obtaining a measured sample for detecting live cells of a microorganism in the test sample and the use of flow cytometry, which includes the following elements: an enzyme selected from the lipolytic enzymes and proteases, poison topoisomerase and/or poison DNA gyrase and agents, coloring kernel.

In a preferred embodiment, the above-mentioned set of poison topoisomerase choose from amsacrine, camptothecin, doxorubicin, ellipticine, etoposide, mitoxantrone, syntopia, topotecan and CF-115953.

In a preferred embodiment, the above-mentioned set of poison DNA gyrase is selected from ciprofloxacin, ofloxacin, enoxacin, pefloksatsina, fleroxacin, norfloxacin, nalidixic acid, oksolinovoj acid and pyrimidinones acid.

In a preferred embodiment, the above-mentioned set of poison topoisomerase represents the ethidium monoacid.

Effective action inventions

The present invention provides a convenient and quick way to distinguish live cells, injured cells and dead cells in food and clinical samples using flow cytometry. Methods and kit of the present invention can be applied in disaster inspections, and it is advantageous from an economic point of view.

Brief description of drawings

Fig. 1. Graphs showing the results of FCM measurements after SYTO9/PI staining LP-treated group suspensionsEscherichia coli(live the bacteria and damaged bacteria) and LP-treated group suspensions Staphylococcus epidermidis(live bacteria and damaged bacteria)and untreated groups suspensionsEscherichia coli(live bacteria and damaged bacteria) and untreated groups suspensionsStaphylococcus epidermidis(live bacteria and damaged bacteria).

Fig. 2. Graphs showing the results of FCM measurements after SYTO9/PI staining LP-treated UHT homogenized milk inoculated withEscherichia coli(live bacteria), and LP-treated UHT homogenized milk not inoculated with bacteria.

Fig. 3. Graphs showing the results of FCM measurements after SYTO9/PI staining LP-treated LTLT dehomogenization milk inoculated withEscherichia coli(live bacteria and damaged bacteria), LP-treated LTLT dehomogenization milk inoculated withStaphylococcus epidermidis(live bacteria and damaged bacteria), and LP-treated LTLT dehomogenization milk not inoculated with bacteria.

Fig. 4. Graphs showing the results of FCM measurements after SYTO9/PI staining for LP-treated and EMA-treated suspensionsEscherichia coli(live bacteria and damaged bacteria) and suspensionsStaphylococcus epidermidis(live bacteria and damaged bacteria).

Fig. 5. Graphs showing the results of FCM measurements after SYTO9/PI staining for LP-treated and EMA-treated UHT is gomogenizirovannogo milk, inoculatedEscherichia coli(live bacteria), and UHT homogenized milk inoculated withStaphylococcus epidermidis(live bacteria), and UHT homogenized milk not inoculated with bacteria.

Fig. 6. Graphs showing the results of FCM measurements after SYTO9/PI staining for LP-treated and EMA-treated dehomogenization milk inoculated withEscherichia coli(live bacteria and damaged bacteria).

Fig. 7. Graphs showing the results of FCM measurements after SYTO9/PI staining for LP-treated and EMA-treated LTLT dehomogenization milk inoculated withStaphylococcus epidermidis(live bacteria and damaged bacteria).

Fig. 8. Graphs showing the results of FCM measurements after SYTO9/PI staining for UHT homogenized milk inoculated withEscherichia coli(live bacteria) andStaphylococcus epidermidis(live bacteria) after LP-processing and processing a) amsacrine, b) ellipticine,) camptothecin or d) ciprofloxacin.

Fig. 9. Graphs showing the relationship between the time of immersion in boiling water microtube containing physiological salt solution, and the temperature of the liquid in microprobing.

Fig. 10. Pictures of electrophoresis of chromosomal DNAEscherichia coli, Klebsiella, CitrobacterandSalmonella(live bacteria and damaged bacteria), extracted and cleaned up the x to (N) or after (E) EMA-processing.

Fig. 11. Pictures of electrophoresis of chromosomal DNAEscherichia coli(damaged bacteria and dead bacteria), extracted and purified to (N) or after (E) EMA-processing.

Fig. 12. Pictures of electrophoresis of chromosomal DNAStaphylococcus epidermidis(live bacteria, damaged, bacteria and dead bacteria), extracted and purified to (N) or after (E) EMA-processing.

Fig. 13. Graphs showing the results of FCM measurements for live bacteria, dead bacteria and dead bacteriaEscherichia colibefore and after EMA-processing.

Fig. 14. Graphs showing the results of FCM measurements for live bacteria, dead bacteria and dead bacteriaStaphylococcus epidermidisbefore and after EMA-processing.

Fig. 15. Graphs showing the results of FCM measurements for live bacteriaMycobacterium tuberculosisand dead bacteria and dead bacteriaMycobacterium tuberculosistreated with isonicotinic acid hydrazide and rifampicin before and after EMA-processing.

Fig. 16. Graphs showing the results of FCM measurements for live bacteria,and dead bacteria and dead bacteriaListeriatreated with ampicillin and gentamicin before and after EMA-processing.

Fig. 17. Graphs showing classifications of live bacteria, dead bacteria and dead bacteriaEscherichia coli, Staphylococcus epidermidis, Mycobacterium tuberculosi andListeriaaccording to the ATP method and their distinction according to the method of the present invention.

Fig. 18. Graphs showing classifications of live bacteria, dead bacteria and dead bacteriaEscherichia coli, Staphylococcus epidermidis, Mycobacterium tuberculosisandListeriaaccording asteracea method and their distinction according to the method of the present invention.

Fig. 19. Graphs showing the results of FCM measurements forListeriain human blood before and after EMA-processing

The best option of carrying out the invention

Next, here will be explained in detail preferred embodiments of the invention. However, the present invention is not limited to the following preferred options for implementation and can be freely modified within the scope of the present invention.

The method of obtaining the measured sample for flow cytometry (hereinafter also abbreviated as "FCM") in accordance with the present invention is a method for obtaining a measured sample for detecting live cells of a microorganism in a test sample using flow cytometry and is a method including the following stages:

a) processing stage of the test sample with an enzyme having the activity from the point of view of cell disruption, Otley is audacia from the cells of the microorganism, colloidal particles, proteins or lipids present in the sample, and

b) the processing stage of the test specimen poison topoisomerase and/or poison DNA gyrase.

Method of detecting live cells of a microorganism in the test sample is a method of detecting living cells when using the sample obtained above to obtain a measured sample for FCM, and additionally includes the following stages in addition to the above stages a) and b):

(C) processing stage of the test specimen processed in the stages (a) and (b) the agent, coloring kernel, and

d) stage detection of microorganisms in the test sample treated with the agent, dye core flow cytometry.

In this specification "test sample" refers to the object for which the detected present it live cells of microorganism, and it is not restrictive to the extent until the microorganism can be detected FCM.

Examples include milk, dairy products and food products obtained when using milk or milk product as a starting material, blood samples, urine samples, samples of cerebral spinal fluid samples sinovialnoj fluid and samples of pleural fluid, etc. are particularly preferred Molok is, dairy products, food products, obtained using milk or milk product as the starting material. In the present invention, the test sample can be any of the above products and biological samples by themselves and can be a sample obtained by dilution or concentration of any of the above products and biological samples, or by exposure to any of the above products or biological samples prior to processing different from the processing according to the method of the present invention. Examples of the pretreatment include thermal processing, filtering, processing, antibiotic etc.

"Microorganism" is detected when using the method of the present invention, and it is not restrictive to the extent until the microorganism can be detected FCM, and poison topoisomerase poison DNA gyrase or ethidium monoacid different effects on live cells, injured cells and dead cells of the microorganism. Preferred examples include bacteria, hyphomycetes, yeast, etc. Bacteria include gram-positive bacteria and gram-negative bacteria. Examples of gram-positive bacteria include bacteriaStaphylococcussuch asStaphylococcus pidermidis, bacteriaStreptococcus,bacteriaListeria,bacteriaBacillusbacteriaMycobacteriumetc. are Examples of gram-negative bacteria include bacteriaEscherichiasuch asEscherichia coliintestinal bacteria, a typical representative of which are bacteriaEnterobacter,bacteriaSalmonellabacteriaVibrio,bacteriaPseudomonasetc.

In the present invention "live bacteria (living cell)" refers to a cell that can proliferate in its cultivation, in General, preferred culture conditions, and can proliferate in a state such that the cell exerts metabolic cellular activity (status live and cultivated in favorable terms, and cells substantially no damage to the cell wall. As examples of the above metabolic cell activity can be specified ATP activity, esterna activity, etc.

"Damaged bacteria" (damaged cell or alive, but not cultured cell) is a cell in a state in which it is almost impossible to proliferate even under cultivation under optimal culture conditions, because the cell is damaged due to artificial stress or environmental stress, and shows metabolic activity at a low level compared with a living cell, but is a significant level compared with a dead cell is damaged or alive, but not cultured [VNC]). Although VNC cells and damaged cells can be discerned in the narrow sense based on the type of exposure that causes damage, VNC cells and damaged cells in the narrow sense can be combined under the name of the damaged cells of the present invention compared with live cells or dead cells.

Detection of bacteria, showing the condition of damaged cells using heat treatment in mild conditions or the introduction of antibiotics, attracts attention especially in the field of sanitary inspection of food products and clinical tests, and the present invention provides a method for detecting a microorganism, which allows us to distinguish all States of the cells, including not only the detection of living cells, but the distinction between living cells and dead cells and the distinction of live cells and damaged cells.

"Dead cell" is a cell in a state in which it cannot proliferate, does not show metabolic activity (dead state), even if it is cultivated under optimal culture conditions. Moreover, it is in a state in which although the structure of the cell wall is supported, but the cell wall of substantially damaged, and the agent, coloring the core region the surrounding low permeability, such as propedy iodide, can pass through the cell wall.

Unit number of cells live cells, injured cells and dead cells is generally the number of cells (cells/ml the Number of living cells can be approximated by the number of colonies (CFU/ml (colony forming units/ml))that are formed during the cultivation of cells under optimal conditions in a suitable medium in Petri dishes. The standard sample of dead cells can be obtained by exposure of a suspension of living cells to heat treatment such as heat treatment in boiling water. In this case, the number of dead cells in this sample can be approximately described as CFU/ml suspension of living cells before heat treatment. Although the time of heat treatment in boiling water to get the dead cells varies depending on the type of microorganisms, dead cells, bacteria, described in the examples can be obtained, for example, heat treatment for about 12 minutes. The damaged cells can be obtained by heat treatment in boiling water for a shorter time compared to the time used to get the dead cells. For example, the damaged cells of the bacteria described in the examples can be obtained by heat treatment for about 50 seconds. In this case, the number of damaged cells may be about is isano as CFU/ml suspension of living cells before heat treatment. Additionally, the standard sample of the damaged cells can also be obtained by treatment with an antibiotic. In this case, the number of damaged cells can be roughly estimated by the number of colonies (CFU/ml)that are formed during the cultivation of cells under optimal conditions in a suitable medium for Petri dishes, by removal of the antibiotic after treatment of a suspension of living cells with the antibiotic, measurement of the transmittance of visible light (wavelength: 600 nm), which is an optical turbidity, and comparison of optical turbidity with the optical turbidity of the suspension of living cells, for which you know the density of living cells.

"Colloidal particles" proteins are colloidal particles contained in the test sample comprising proteins as components, and nonspecific painted by the agent, coloring kernel, and example of which includes micellar casein.

Hereinafter, the method of the present invention will be explained for each stage.

(1) stage a)

In this phase, the test sample is treated with an enzyme having the activity from the point of view of destruction of cells different from the cells of the microorganism, colloidal particles, proteins or lipids present in the sample.

In General indicates that, in order to detect bacteria FCM, bacteria clean the Dimo be passing through the detector in the amount of at least about 100 CFU. However, if the living cells in the tested sample, such as milk, detects using FCM, a large number of contaminants, such as somatic cells, globules of fat and micellar casein, not only the bacteria that are in the target area (bacterial target region) FSC (forward scattered light) - SSC (side scattered light), and therefore, the bacteria may not be detected, even if the detector is about 100 CFU of bacteria. Based on the foregoing, it is preferable to remove or reduce the number of cells different from the cells of microorganisms, colloidal particles, proteins, lipids, etc. existing in the test sample, by treatment with an enzyme.

In that case, if the test sample is a milk, milk product or a food product obtained when using milk or milk product as a starting material, examples of cells that differ from the cells of the microorganisms present in the test sample include bovine leukocytes, epithelial cells of the mammary gland, etc. moreover, if the test sample is a biological sample such as a blood sample, a urine sample, a sample of cerebral spinal fluid sample sinovialnoj fluid and a sample of pleural fluid, examples of the cells include erythrocytes, leukocytes (granulate the s, neutrophils, basophils, monocytes, lymphocytes etc), platelets, etc.

As mentioned above, the enzyme is not limiting to the extent until the enzyme can destroy the above contaminants and does not damage the living cells of the microorganism, which is selected as the detected object, and examples of enzymes include lipolytic enzymes and proteases. Although the enzyme can be used independently of one type of enzyme or may be used in combination of two or more types of enzymes, it is preferable to use both the lipolytic enzyme and the protease.

Examples of lipolytic enzymes include lipases, proteases, etc. and examples of the protease include proteinase K, pronase etc.

Although the processing conditions, these enzymes is not specifically limited, they may be suitable determined, for example, lipases can be specified conditions final concentration of from 10 to 50 units/ml, temperature from 25 to 37°C. and the processing time of 30 minutes or more, and proteinase K can be specified conditions final concentration of from 10 to 50 units/ml, temperature from 25 to 37°C. and the processing time of 30 minutes or more.

The processing of a lipolytic enzyme and the protease is preferably carried out in a manner: (i) processing a lipolytic enzyme and (ii) treatment with protease, or processing can be carried out od vremennym add them. Although these enzymes can be left in the test sample after the treatment, it is preferable to separate the enzymes from the cells by centrifugation or the like

(2) stage b)

In this phase, the test sample is treated with poison topoisomerase and/or poison DNA gyrase. Stage b) is preferably carried out after stage a).

Venom and poison topoisomerase DNA gyrase used in the present invention, are poisons that do not inhibit the activity topoisomerase and DNA gyrase, respectively, in the DNA cleavage, but inhibit the re-ligation of DNA or improve the return rate of DNA cleavage. Venom and poison topoisomerase DNA gyrase preferably are poisons that are associated with the chromosomal DNA of the microorganism using covalent joining that intercalary in chromosomal DNA and bind to chromosomal DNA through covalent joining under irradiation of visible light, poisons that are easy intercalary in the chromosomal DNA, or poisons, which form a complex or topoisomerase DNA gyrase.

Although it is preferable to simultaneously use poison and poison topoisomerase DNA gyrase, can also be used any of them.

Venom and poison topoisomerase DNA gyrase preferably are poisons that have different effects on living cells, damages the basic cells, dead cells, somatic cells, such as bovine leukocytes, leukocytes and platelets, etc. and, more specifically, poisons, which exhibit higher permeability of the cell walls of damaged cells and dead cells and cell membranes of somatic cells, such as bovine leukocytes, leukocytes and platelets, etc. compared to the permeability of cell walls of living cells.

Examples of poison topoisomerase include amsacrine, camptothecin, doxorubicin, ellipticine, etoposide, mitoxantrone, saintaubin, topotecan, WED-115953, etc. Can be used independently of one type poison topoisomerase or a combination of two or more types of poison topoisomerase.

Examples of poison DNA gyrase include ciprofloxacin, ofloxacin, enoxacin, pefloxacin, fleroxacin, norfloxacin, nalidixic acid, oxolinic acid, pyrimidinone acid, etc. May be used independently of one type of poison called DNA gyrase, or a combination of two or more types of poison DNA gyrase.

Handling of poison topoisomerase or poison DNA gyrase can be defined in an appropriate way. For example, conditions that make it possible to easily distinguish living cells from damaged cells and dead cells can be determined by adding poison or poison topoisomerase DNA gyrase at various concentrations to the suspensions of living cells, damaged the s cells or dead cells of the microorganism, representing object detection, keeping them within different periods of time, then collecting the cells by centrifugation, etc., staining of the cells with the agent, coloring kernel, and analysis of cells using FCM. Moreover, the conditions that allow for easy distinction of live cells of microorganism, representing the object detection from somatic cells, such as bovine leukocytes, platelets, etc. can be defined by adding poison topoisomerase at various concentrations to the suspensions of living cells and the above mentioned different cells (living cells containing damaged cells), keeping them within a predefined time, then the collection of living cells and the above mentioned different cells by centrifugation, etc., staining of the cells with the agent, coloring kernel, and analysis of cells using FCM. Examples of such conditions include, specifically, for amsacrine final concentration of from 1 to 100 µg/ml, the temperature from 25 to 37°C. and the processing time is from 5 minutes to 48 hours, for ellipticine final concentration of from 0.05 to 5 μg/ml, the temperature from 25 to 37°C. and the processing time from 10 minutes to 48 hours, for camptothecin final concentration of from 1 to 100 µg/ml, the temperature from 25 to 37°C. and the processing time from 10 minutes to 48 hours, for the ciprofloxacin konechno the concentration from 0.4 to 40 μg/ml, the temperature from 25 to 37°C. and the processing time from 10 minutes to 48 hours, for etoposide final concentration of from 1 to 100 µg/ml, the temperature from 25 to 37°C. and the processing time is from 5 minutes to 48 hours, for mitoxantrone final concentration of from 0.1 to 10 μg/ml, the temperature from 25 to 37°C. and the processing time from 10 minutes to 48 hours. After the test sample is treated under predetermined conditions, processing preferably is stopped by removal, thinning and/or separation by centrifugation, etc.

Above venom and poison topoisomerase DNA gyrase rather penetrate the cell wall of damaged cells and dead cells compared with cell walls of living cells. Thus, it is believed that if the processing time is within the above range, the poisons largely do not penetrate through the cell walls of living cells, but they penetrate into the damaged cells, dead cells and live somatic cells, including damaged cells, because they have only a cell membrane and do not have cell walls. It is established that the venom or poison topoisomerase DNA gyrase penetrates into somatic cells, injured cells and dead cells, then randomly associated with chromosomal DNA through covalent joining, intercalary in DNA or forms a complex with topo is samarati and additionally inhibits the re-ligation of DNA topoisomerase II and topoisomerase I in somatic cells or topoisomerase IV, or topoisomerases I, III, or DNA Girasol in damaged cells, or increase the return rate of DNA cleavage, resulting in fragmentation of chromosomal DNA.

I believe that if the chromosomal DNA damaged cells preferably fragmented compared with the chromosomal DNA of living cells when coloring agent, coloring cores that penetrate cell walls of live cells, injured cells and dead cells, such as SYTO9, the intensity of staining of damaged cells is reduced compared to the intensity of the staining of living cells, and as a result, it becomes possible to distinguish between living cells and damaged cells for detecting when using FCM.

In another preferred embodiment of the present invention poison topoisomerase represents the ethidium monoacid, and the method includes a step of exposure of the test specimen, to which add the ethidium monoacid, the irradiation of visible light. The ethidium monoacid (EMA) more easily penetrate cell walls of damaged cells compared with cell walls of living cells of microorganisms. On this basis, it is believed that the EMA substantially does not penetrate the cell walls of living cells, but EMA penetrates the cell walls of damaged cells and across cell membranes of living somatic cells including damaged cells, since the cell membrane of somatic cells do not represent a cell wall. In the case when the leukocytes and platelets in the blood are living cells, EMA more easily penetrates the cell membrane of cells in sterilized water or hypotonic salt solution. EMA enters into somatic cells and damaged cells and chaotic intercalary in the chromosomal DNA, and then only intercalated EMA turns into nitren under the action of irradiation of visible light and is associated with chromosomal DNA through covalent attachment. Installed that, then it inhibits the re-ligation of DNA topoisomerase II in somatic cells, topoisomerase IV in damaged cells or DNA Girasol, resulting in fragmentation of chromosomal DNA.

The machining conditions EMA can be determined appropriately. For example, conditions that make it possible to easily distinguish living cells from damaged cells can be determined by adding the EMA at various concentrations to the suspensions of living cells and damaged cells of the microorganism, representing the object detection, keeping them within different periods of time, then the irradiation by visible light, collecting the cells by centrifugation, etc. in accordance with the requirement staining glue is OK agent, coloring kernel, and analysis of cells using FCM.

Preferred conditions for the irradiation of visible light can also be determined as appropriate, by conducting this experiment, as described above, using different exposure time. Specifically, the processing EMA preferably carried out at a final concentration of from 0.5 to 100 μg/ml, at a temperature of from 4 to 10°C for a time from 5 minutes to 48 hours. Moreover, processing EMA preferably carried out while shielding light. As visible light is preferred visible light containing components from 500 to 700 nm. Specific examples of the conditions for irradiation of visible light include the irradiation of visible light from 100 to 750 W for 5 minutes to 2 hours at a distance of from 10 to 50 cm to the test specimen. The irradiation of visible light is preferably carried out at a low temperature, for example, when the sample cooling with ice.

(3) stage C)

In this phase, the test sample treated in stages a) and b), process agent, coloring kernel. Agent, coloring kernel contains at least a first coloring agent that can penetrate through the cell walls of live cells, injured cells and dead cells. The expression "a coloring agent can penetrate through the cell walls of living cells, damaged the cells and dead cells" refers simultaneously to that case, when the permeability of the agent in respect of the cell walls of live cells, injured cells and dead cells is substantially identical, and to the case where even if the permeability is different, then the differences in permeability of the agent in respect of cell walls of living cells and dead cells are smaller compared with the second coloring agent, described below. Specific examples of the first coloring agent include, for example, SYTO9.

Moreover, agent, coloring kernel, preferably contains a second coloring agent, which increasingly permeates through the cell walls of dead cells compared with cell walls of living cells and damaged cells compared with the first agent. In other words, the first coloring agent is a coloring agent that can stain live cells and dead cells in a variety of colors. Examples of the second coloring agent include propedy iodide (PI). If these agents, coloring kernel, penetrate into the cells, they intercalary in the chromosomal DNA, and are excited by the irradiation of laser beams emitting fluorescence. For example, if SYTO9 and PI intercalated in DNA irradiated with a laser beam with λ488 nm, they become excited and SYTO9 and PI emit respective what about the green fluorescence with a Central wavelength λ518 nm and red fluorescence with a Central wavelength λ617 nm.

When coloring a coloring agent, if there is a clear distinction between living cells and dead cells, and somatic cells other than the microorganism present in the sample, living cells can be distinguished to some extent by using a similar coloring agent. However, even a coloring agent that can penetrate into physically damaged dead cells may not penetrate poorly damaged cells and, thus, has a slight difference in staining compared with live cells. In this case, it is difficult to clearly distinguish between living cells and damaged cells exclusively in the second coloring agent.

However, the chromosomal DNA of damaged cells become fragmented when the processing at the above stage (b) and, thus, become less painted coloring agent, and thus, it becomes possible to distinguish living cells from damaged, etc. by colouring the first coloring agent. Moreover, when using a second coloring agent, it becomes possible two-dimensional detection of cells using FCM. Accordingly, in the present invention, although the living cells can be detected solely by the staining of the first coloring agent, use is their second coloring agent makes it possible to distinguish living cells and dead cells to improve the accuracy of detection, and thus, it is preferable to use as the first coloring agent and the second coloring agent.

The staining of the first coloring agent and the second coloring agent may be carried out simultaneous use of agents and separate use.

Terms of coloring agents for coloring are not in a special way limited, and you can use conditions, typically used for staining of chromosomal DNA of microorganisms. Specifically, it is preferable to add a coloring agent to the cell suspension at a final concentration of from 4.0 to 6.0 μm in the case of SYTO9 or from 25.0 to 35.0 μm in the case of PI and to carry out the reaction at from 15 to 25°C for 10 to 20 minutes.

(4) stage (d)

In this stage, the microorganism in the test sample treated with the agent, coloring kernel, in the above stage (C) detects when using FCM. Principles of detection of microorganism using FCM are as set forth below.

If particles such as microorganisms, are in line and sequentially irradiated by the beam of an argon laser (λ488 nm), the light scatters in a small angle range from 1.5 to 19° with respect to the axis of the laser beam. This scattered light is called the direct scattered light (FSC), and the degree of scattering is largely proportionate is and particle size. Light, simultaneously dissipated at an angle of approximately 90° with respect to the laser beam, called the side scattered light (SSC), and it is believed that it reflects the complexity of the internal structure of the particles, including the structure of DNA. Thus, if the FSC-SSC schedule get in the form of an x-y graph, the horizontal axis represents the particle size, and the vertical axis represents the complexity of the internal structure of the particles.

If bacteria are painted above the first agent, coloring kernel, and the second agent, coloring kernel, and irradiated by a laser beam, the point on the graph for individual cells are in a certain specific area of FSC-SSC graphics. By environment a specific area with four sides (the target region) when using software FCM analysis (e.g., Cell Quest Ver. 3.1, Becton Dickinson, Sydney, Australia), selectively choosing only particles of bacterial field and paying attention to only the selected particles can be obtained graphics point of living cells and dead cells (refer to Fig. 1-8).

FCM measurement is preferably carried out under the following conditions. They are the preferred application of 15 mW argon laser beam (wavelength λ=488 nm) for excitation light and the direct measurement of scattered light (FSC <15°), side scattered light (> 15°) and three types of fluorescent signals from FL1 3, respectively. For the measurement of fluorescent signals from FL1 to 3, preferably using a filter with a bandwidth of from 515 to 545 nm, particularly 530 nm for green fluorescence (FL1), preferably using a filter with a bandwidth from 564 to 606 nm, in particular 585 nm, yellow-orange fluorescence (FL2), preferably using long-wave filter with a bandwidth from 655 nm to 800 nm, in particular at 670 nm for red fluorescence (FL3).

Moreover, the measurement is preferably carried out in the following settings detectors FSC: E02, SSC: 376, FL1: 709, FL2: 736 and FL3: 811 (all presented using a logarithmic gain), setting to % compensation FL1-FL2: 0,0, FL2-FL1: 0,0, FL2-FL3: 0.0 and FL3-FL2: 0,0, signal FSC 150% threshold (cutoff value), the download speed suspension with a low flow rate for FCM test: 12 ál/min, the number of cells trapped in the target area on the FSC-SSC plot (number of particles), 5000000 and measurement time 30 seconds.

For example, under the preferred conditions of living cells and damaged cells are on schedule in SYTO9 positive and PI negative (SYTO9(+)·PI(-)), and dead cells mainly fall on the chart in the SYTO9 positive and PI positive (SYTO9(+)·PI(+)) on SYTO9/PI chart. The border of the negative and positive values for SYTO9 and the border of the negative and positive values for PI presents the fluorescent intensity of 10 3in the case of SYTO9 and the fluorescence intensity of 2×102in the case of PI. Install the target area preferably represent for FSC from 102up to 2×103and SSC from 10 to 2×102.

Analysis using FCM can be performed using commercially available FCM devices. Conditions for FCM are not in a special way limited, and conditions commonly used for detection and separation of microorganisms, are such that the present invention could be applied with regard to bacteria.

In the present invention the detection of living cells" includes both the determination of the presence or absence of living cells in the tested sample, and determining the number of living cells in the tested sample. Moreover, the detection of living cells" includes distinguishing living cells and damaged and/or dead cells and determining the presence or absence of each of the live cells, injured cells and dead cells. The number of living cells is not limited by an absolute amount and can be a relative amount relative to the amount in the control sample.

<3> Sets according to the present invention

The first set of the present invention is a kit for obtaining the first sample for FCM measurements, including erment, selected from lipolytic enzymes and proteases as element represents enzyme poison topoisomerase and/or poison DNA gyrase and agent, coloring kernel.

The second set of the present invention is a kit for obtaining the above second sample for FCM measurement, comprising an enzyme selected from the lipolytic enzymes and proteases as element represents enzyme ethidium monoacid and agent, coloring kernel.

The above sets the enzyme is selected from lipolytic enzymes and proteases, poison topoisomerase and/or poison DNA gyrase and agent, coloring kernel are the same as indicated above for the methods of the present invention.

The kits of the present invention in addition to the above elements may also include a diluent, a statement describing the method of the present invention, etc.

EXAMPLES

Hereafter the present invention will be more specifically explained by reference to the following examples. However, the present invention is not limited to the following examples.

The tested samples

In the following examples and control examples, the experiments were conducted using the test samples obtained by exposure of the samples to the following types of processing when usingEscherichia coli/i> DH5α (hereinafter referred to here denoted as "Escherichia coliandStaphylococcus epidermidisstrain KD (hereinafter referred to here denoted as "Staphylococcus epidermidis").

(1) Living cells and damaged cells in suspension in physiological saline were detected using flow cytometer (labeled "raw force").

(2) a Suspension of living cells and damaged cells in physiological saline solution was treated with lipase and proteinase K, and then cells were detected using flow cytometer (labeled "LP-treated group). The results of the above samples (1) and (2) shown in the control example 1.

(3) the Living cells and damaged cells suspended in cow's milk is subjected to pasteurization at ultra high temperature (130°C, 2 seconds, referred to as "UHT"), and cow's milk is subjected to pasteurization at low temperature for a long time (63°C, 30 minutes, referred to as "LTLT"), and the suspension was treated with lipase and proteinase K. Cell suspensions were detected using flow cytometer (marked as "M-LP-treated group). Moreover, cells in cow's milk treated with lipase and proteinase K were detected using flow cytometer. The results are shown in the test examples 2 and 3.

(4) Specified to enter the sample (2) was further subjected to processing by ethidium monosiga (EMA), and cells in the sample were detected using flow cytometer (marked as "LPE-treated group). The results shown in example 1.

(5) the above sample (3) was further subjected to processing EMA, and cells in the sample were detected using flow cytometer (marked as "M-LPE-treated group). The results are shown in examples 2 and 3.

(6) the Sample was obtained in a similar manner to the above (5) to ensure that the EMA treatment was replaced by treatment with a poison topoisomerase or treatment poison DNA gyrase (marked as "M-LPD-treated group). The results shown in example 4.

Control example 1

Detection of living cells and damaged cellsEscherichia coliandStaphylococcus epidermidissuspended in physiological saline (LP-processed or unprocessed), when using a flow cytometer.

(1) Obtaining samples

The suspensions of living cells and damaged cells

1-1-1. SuspensionEscherichia coli

Escherichia coliDH5α was inoculable in a nutrient medium L, and cultured at 10°C for 12 hours, as a stationary culture, and then the culture was subjected to centrifugation at cooling at 4°C and 3000×g for 10 minutes to collect cells. The collected cells are suspended in physiological saline (Otsuka Phamaceutical, a similar solution was used in subsequent descriptions) and the suspension is again centrifuged to collect cells. A similar procedure was repeated again for washing cells.

The washed cells suspended in physiological saline and the suspension is appropriately diluted, and 0.1 ml of the suspension was placed on agar medium L and cultivated for measuring the number of living cells. The above cell suspension was brought to a final concentration of 4×106CFU/ml to obtain a suspension of living cells.

A suspension of living cells in a volume of 1 ml was placed in microprobing volume of 2 ml and was immersed in boiling water at 100°C for 50 seconds to obtain a suspension of damaged cells. Separately received another suspension of damaged cells and volume of 0.1 ml was placed on L agar medium, to confirm that the cells do not form colonies.

1-1-2. SuspensionStaphylococcus epidermidis

Staphylococcus epidermidisstrain KD was inoculable in the medium L and cultured at 37°C for 18 hours, as a stationary culture, and then the culture was subjected to centrifugation at cooling at 4°C and 3000×g for 10 minutes to collect cells. The collected cells are suspended in physiological saline (Otsuka Pharmaceutical) and the suspension is again centrifuged to collect cells. A similar procedure was repeated again to wash the glue is OK. The washed cells suspended in physiological saline and the suspension is appropriately diluted, and 0.1 ml of the suspension was placed on agar medium L and cells were cultured for measuring the number of living cells. The above cell suspension was brought to a final concentration of 4×107CFU/ml to obtain a suspension of living cells.

A suspension of living cells in a volume of 1 ml was placed in microprobing volume of 2 ml and microprobing was immersed in boiling water at 100°C for 50 seconds to obtain a suspension of damaged cells. Separately received another suspension of damaged cells and volume of 0.1 ml was placed on agar medium L, to confirm that the cells do not form colonies.

1-2. Treatment of suspensions of living cells and damaged cells lipase and proteinase K.

Each of the suspensionsEscherichia coli(live cells and injured cells) and suspensionsStaphylococcus epidermidis(live cells and injured cells)obtained in the above 1-1-1 and 1-1-2, in the amount of 1 ml was placed in microprobing volume of 2 ml of the suspension was added 100 μl of lipase (E.C. 3.1.1.3, Sigma), increased physiological saline solution to 189 units/ml, and the suspension is incubated at 37°C for 30 minutes for treatment with lipase.

Each cell suspension subjected to processing by the lipase solution was added 25 μl 1250 units/ml proteinase K (E.C. 3.4.21.64, Siga) and the mixture is incubated at 37°C for 30 minutes to effect the treatment with proteinase K.

After treatment with lipase and treatment with proteinase K (hereafter referred to as "LP processing) cells were collected by centrifugation at cooling at 4°C and 14000×g for 10 minutes and the collected cells are suspended in physiological saline to obtain the LP-treated group suspensionEscherichia coli(live cells and injured cells) and LP-treated group suspensionStaphylococcus epidermidis(live cells and injured cells), respectively.

Additionally, in the above method was added 100 μl of physiological saline instead of the lipase and 20 μl of physiological saline instead of proteinase K and were treated in the same way to obtain the raw group suspensionEscherichia coli(live cells and injured cells) and untreated group suspensionStaphylococcus epidermidis(live cells and injured cells), respectively.

(2) Method FCM test

Each LP-treated group suspensionEscherichia coli(live cells and injured cells), LP-treated group suspensionStaphylococcus epidermidis(live cells and injured cells), untreated group suspensionEscherichia coli(live cells and injured cells) and untreated group suspensionStaphylococcus epidermidis(live cells and injured cells) in a volume of 300 μl was added to 0.9 ál SYTO9/PI fluorescent acrasia the corresponding agent (LIVE/DEAD BacLight TMBacterial Viability kit, Molecular Probes, SYTO9/PI=1/1 mixture) and the reaction was carried out at room temperature for 15 minutes under light shielding for each sample. For these samples measured using FCM instrumentation FACS Calibur (Becton Dickinson).

FCM measurements were carried out as follows. As the exciting light used 15 mW beam of an argon laser (wavelength λ=488 nm) and used the product from Becton Dickinson as a protective solution for downloading the sample fluid FCM. Additionally, we measured direct the scattered light (FSC <15°), side scattered light (> 15°) and three types of fluorescent signals from FL1 to 3, respectively. For the measurement of fluorescent signals from FL1 to 3 used a filter with a bandwidth of 530 nm (from 515 to 545 nm) for green fluorescence (FL1)was used filter with a bandwidth of 585 nm (from 564 to 606 nm) to yellow-orange fluorescence (FL2) and used long-wave filter with a bandwidth of 670 nm (655 nm to 800 nm) for red fluorescence (FL3).

For measurements used for the installation of detectors FSC: E02, SSC: 376, FL1: 709, FL2: 736 and FL3: 811 (all presented using a logarithmic gain), setting to % compensation FL1-FL2: 0,0, FL2-FL1: 0,0, FL2-FL3: 0.0 and FL3-FL2: 0,0, signal FSC 150% threshold (cutoff value), the download speed of the suspension on the FCM I test installed with 12 μl/min, the number of cells trapped in the target area on the FSC-SSC plot (number of particles), was established at 5000000, and the measurement time was 30 seconds.

(3) the results of the test

The results of this test are shown in Fig. 1.

SYTO9 demonstrates the high permeability of the cell wall and penetrate cell walls of living cells and dead cells, whereas PI exhibits low permeability of cell walls and penetrates through the cell wall physically damaged dead cells. Hence, initially, the results for living cells, should fall on the chart in the area of SYTO9(+)·PI(-), the results for dead cells should get on the chart in the area of SYTO9(+)·PI(+) on the graph SYTO9/PI. In fact, when comparing the results for the untreated group suspensionEscherichia coli(live cells and injured cells) and untreated group suspensionStaphylococcus epidermidis(live cells and injured cells), PI is not penetrated not only in living cells, but also in the damaged cells and living cells and damaged cells could clearly be distinguished. Therefore appreciated that the degree of damage to the cell walls of bacteria after immersion in boiling water at 100°C for 50 seconds, which is the similar conditions with pasteurized at ultra-high temperature (industrial cow's milk), is not significant. Moreover, OBS who was a minor difference in the fluorescence intensity of staining of SYTO9 between living cells and damaged cells and assessed, this difference was due to the fact that the chromosomal DNA of damaged cells was partially destroyed when heated, and thus, the efficiency of intercalation SYTO9 in the chromosomal DNA is reduced.

Even if it was carried out LP treatment, living cells and damaged cells cannot be easily seen in the case, in particular,Escherichia coli. If you compare the results of the untreated groups suspensionsEscherichia coli(living cells) and LP-treated groups suspensionsEscherichia coli(living cells), you can see that some of the points that hit the SYTO9(+)·PI(-), moved into the area SYTO9(-)·PI(-) on the graph of the livingEscherichia coliand efficient detection of living cells decreases, since it was a LP processing. This phenomenon is even more pronounced in the case ofStaphylococcus epidermidis. However, if the living cells and damaged cells were detected separately from cow's milk using FCM, contaminants, i.e. somatic cells, such as bovine leukocytes and epithelial cells in the breast, micellar casein and lipids must be removed, and thus, it is considered that the LP processing inevitable.

Control example 2

Detection of living cellsEscherichia coli(LP-treated), suspended in gomogenizirovannom milk subjected to pasteurization at the ultra-high is the temperature value, when using the flow cytometer

(1) Obtaining samples

To 1 ml of 1.1×107CFU/ml suspensionEscherichia coli(living cells)obtained in the same manner as in reference example 1, was added 9 ml of a commercially available homogenized cow's milk (subjected to pasteurization at ultra high temperature (130°C, 2 seconds), here also called "UHT homogenized milk) for 10-fold dilution of the suspension, and the diluted suspension was sequentially diluted in the same way with getting UHT homogenized milk inoculated from 1.1×102to 1.1×106CFU/mlEscherichia coli(living cells). It was shown that the above mentioned UHT homogenized milk does not form colonies during incubation at 37°C for 48 hours and at 25°C for 72 hours when using agar medium.

Then, UHT homogenized milk, in various degrees of dilution inoculatedEscherichia coli(living cells), and UHT homogenized milk not inoculated withEscherichia coliwere subjected to the same processing procedure lipase and proteinase K (LP processing)as in the control example 1. The procedure is that each of the samples of UHT homogenized milk inoculated withEscherichia coli(living cells), and UHT homogenized milk not inoculated withEscherichia colivoyame 1 ml was placed in a 2 ml microprobing To the sample was added 100 μl of lipase (E.C. 3.1.1.3, Sigma), increased physiological saline solution to 189 units/ml, and the suspension is incubated at 37°C for 30 minutes for treatment with lipase.

To each sample subjected to processing by the lipase solution was added to 20 ál 1250 units/ml proteinase K (E.C. 3.4.21.64, Sigma) and the mixture is incubated at 37°C for 30 minutes to effect the treatment with proteinase K.

After treatment with lipase and treatment with proteinase K to each sample was added 880 μl of physiological saline solution and the sample was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. The lipid layer, existing in the upper layer were completely removed the tampon and the aqueous layer existing in the middle part, was also removed. The remaining lower layer was added 2 ml of physiological salt solution, and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes for washing the collected precipitate after centrifugation and to the precipitate after centrifugation was added 300 μl of physiological saline.

(2) Method of test

To each sample LP-treated UHT homogenized milk inoculated withEscherichia coli(living cells), and LP-treated UHT homogenized milk, prepared above, and translated into the volume of 300 μl was added to 0.9 ál SYTO9/PI fluore the interest of coloring reagent and the reaction was left to proceed at room temperature for 15 minutes under light shielding for each sample. For these samples measured using FCM instrumentation FACS Calibur (Becton Dickinson). The measurement conditions were the same as in test example 1, except that the measurement time was 5 minutes.

(3) the results of the test

The results of this test are shown in Fig. 2.

Although the number of data points in the SYTO9(+)·PI(-) region varies depending on inoculated concentration ofEscherichia coli(living cells), in this area there are many points on the graph, even if the LP-treated UHT homogenized milk not inoculated withEscherichia coli(living cells), and opposed toEscherichia coli(live cells) was difficult. It was assumed that these contaminants consist of somatic cells and cells originally present in cow's milk and damaged by heat. It was assumed that, however, because the cell membrane of somatic cells and the cell walls of the cells originally present in the milk, largely unaffected by the sterilized at ultra high temperature, SYTO9 penetrates into the cells, but PI is not going to penetrate. As a comparison, the results obtained by subtracting the number of data points in the SYTO9(+)·PI(-) region for the LP-treated UHT homogenized milk not inoculated withEscherichia coli(living cells, each of the number of data points in the same region for the LP-treated UHT homogenized milk inoculated withEscherichia coli(living cells), shown in table 1. According to the results, it is considered that the detection limit (Escherichia coli(living cells) is 1.1×104CFU/ml under the conditions of this test.

Table 1
The detection results liveEscherichia coliin the LP-treated UHT gomogenizirovannom milk
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)
00
of 1.1×1020
of 1.1×1030
of 1.1×104311
of 1.1×1052755
of 1.1×106128457

Reference example 3

Detection of living cells and damaged cellsEscherichia coliandStaphylococcus epidermidis(LP-treated), suspended in dehomogenization milk subjected to DL the more time pasteurization at low temperature (LTLT), when using the flow cytometer

(1) Obtaining samples

To 1 ml each of the 1.5×107CFU/ml suspensionEscherichia coli(live cells and injured cells)obtained in the same manner as in reference example 1, was added 9 ml of a commercially available are not subjected to homogenization of cow's milk (dehomogenization cow's milk subjected to long-time pasteurization at low temperature (63°C, 30 minutes), here also called "LTLT dehomogenization milk) for 10-fold dilution of the suspensions, and the diluted suspension was sequentially diluted in the same way with getting LTLT dehomogenization milk inoculated with 1.5×102up to 1.5×106CFU/mlEscherichia coli(live cells and injured cells). It was shown that the above mentioned LTLT dehomogenization milk does not form colonies during incubation at 37°C for 48 hours and at 25°C for 72 hours when using agar medium.

In addition to 1 ml each of 1.8×108CFU/ml suspensionStaphylococcus epidermidis(live cells and injured cells)obtained in the same manner as in reference example 1, was added 9 ml not subjected to homogenization LTLT cow's milk for 10-fold dilution of the suspensions, and the diluted suspension was sequentially diluted in the same way with the item is the receiving LTLT dehomogenization milk, inoculated from 1.8×102to 1.8×107CFU/mlStaphylococcus epidermidis(live cells and injured cells).

Then LTLT dehomogenization milk inoculated withEscherichia coli(live cells and injured cells) with different degree of dilution, LTLT dehomogenization milk inoculated withStaphylococcus epidermidis(live cells and injured cells) with different degree of dilution, and LTLT dehomogenization milk not inoculated with bacteria were subjected to the same treatment with lipase and proteinase K (LP processing)as in the control example 1. The procedure is that each of the samples LTLT dehomogenization milk inoculated withEscherichia coli(live cells and injured cells), LTLT dehomogenization milk withStaphylococcus epidermidis(live cells and injured cells) and LTLT dehomogenization milk not inoculated with bacteria in a volume of 1 ml was placed in microprobing volume of 2 ml To the sample was added 100 μl of lipase (E.C. 3.1.1.3, Sigma), increased physiological saline solution to 189 units/ml and the sample incubated at 37°C for 30 minutes for treatment with lipase.

To each sample subjected to processing by the lipase solution was added to 20 ál 1250 units/ml proteinase K (E.C. 3.4.21.64, Sigma) and the sample incubated at 37°C for 30 minutes to effect the treatment with proteinase K.

the donkey treatment with lipase and treatment with proteinase K to each sample was added 880 μl of physiological saline solution and the sample was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. The lipid layer, existing in the upper layer were completely removed the tampon and the aqueous layer existing in the middle part, was also removed. The remaining lower layer was added 2 ml of physiological salt solution, and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes for washing the collected precipitate after centrifugation and to the precipitate after centrifugation was added 300 μl of physiological saline.

(2) Method of test

To each sample LP-treated LTLT dehomogenization milk inoculated withEscherichia coli(live cells and injured cells), LP-treated LTLT dehomogenization milk inoculated withStaphylococcus epidermidis(live cells and injured cells), and LP-treated LTLT dehomogenization milk not inoculated with bacteria prepared above and translated into the volume of 300 μl was added to 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was left to proceed at room temperature for 15 minutes under light shielding for each sample. For these samples measured using FCM instrumentation FACS Calibur (Becton Dickinson). The measurement conditions were the same as in test example 1, except that the measurement time was 5 minutes.

(3) the results of the test

the results of this test are shown in Fig. 3.

In the SYTO9(+)·PI(-) region, there are many points on the graph for LP-treated LTLT dehomogenization milk not inoculated with bacteria, and they degrade the limits of detection ofEscherichia coli(living cells) andStaphylococcus epidermidis(living cells). The point on the chart forEscherichia coli(damaged cells) andStaphylococcus epidermidis(damaged cells) were hidden by the points on the graph arising from the LP-treated LTLT dehomogenization milk not inoculated with bacteria.

Because LTLT dehomogenization milk initially contained the damaged cells and dead cells in a concentration of from 103up to 105CFU/ml, the likelihood is high that the points on the graph of damaged cells, again inoculated in test case 3, it will be masked points initially contained cells. Additionally, from the point of view of the location of the points on the graph for LP-treated suspensionsEscherichia coliandStaphylococcus epidermidis(damaged cells), which can be seen from the results of control example 1 shown in Fig. 1, it is assumed that with high probability the point on the chart again inoculated damaged cells will be hidden by the points on the graph arising from the LP-treated LTLT dehomogenization milk.

The number of data points in the graph area SYTO9(+)·PI(-), udovletvoryayushchaya, intensity of ≥7×103shown in table 2 for the LP-treated LTLT

dehomogenization milk inoculated withEscherichia coli(living cells), and LP-treated LTLT dehomogenization milk. It is believed that under the conditions of this test, the limit of detection of the method LP-treatment forEscherichia coli(living cells) in the LP-treated LTLT dehomogenization milk is 1.5×105CFU/ml

Table 2
The detection results liveEscherichia coliin the LP-treated LTLT dehomogenization milk for the LP-processing
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)1)
09
of 1.5×1028
of 1.5×10310
of 1.5×1049
of 1.5×10518
of 1.5×106111
1)It has been estimated number is about the points on the graph in the graph area SYTO9(+)·PI(-), satisfying the condition that the calculated intensity SYTO9 ≥7×103.

Additionally, the number of data points in the graph area SYTO9(+)·PI(-), satisfying the condition that the intensity of ≥7×103shown in table 3 for LP-treated LTLT dehomogenization milk inoculated withStaphylococcus epidermidis(living cells), and LP-treated LTLT dehomogenization milk. It is believed that under the conditions of this test, the limit of detection of the method LP-treatment forStaphylococcus epidermidis(living cells) in the LP-treated LTLT dehomogenization milk is 1.8×106CFU/ml

Table 3
The detection results liveStaphylococcus epidermidisin the LP-treated LTLT dehomogenization milk for the LP-processing
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)1)
09
1,8×1028
1,8×10310
1,8×1048
57
1,8×10647
1,8×107296
1)It was estimated the number of data points in the graph area SYTO9(+)·PI(-), satisfying the condition that the calculated intensity SYTO9 ≥7×103.

Example 1

Detection in processed by ethidium monosiga (EMA) suspensionEscherichia coliand suspensionsStaphylococcus epidermidis(LP-treated) using the flow cytometer

(1) Obtaining samples

Each of the suspensionsEscherichia coli(live cells and injured cells, 4×106CFU/ml) and suspensionsStaphylococcus epidermidis(live cells and injured cells, 4×107CFU/ml)obtained in the same manner as in reference example 1 in a volume of 1 ml was placed in a microtube with a volume of 2 ml and then subjected to treatment with lipase and proteinase K treatment (LP processing) in the same manner as in test example 1, and then subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. After I removed the upper water layer, the cells of the lower layer (sludge) was added 1 ml of physiological saline for suspension cells.

The ethidium monoacid (hereafter also oboznacheniyalari "EMA", Sigma, catalog number: E2028) was dissolved in sterilized water at a concentration of 1000 µg/ml and filtered through a 0,45 µm filter. Specified aqueous solution VOLUME of 10 µl was added to the above LP-treated cell suspension and the suspension was left at 4°C for 30 minutes under light shielding. Then the suspension was placed on ice and irradiated with visible light from the lamp 500 W (FLOOD PRF, 100 V, 500 W, Iwasaki Electric Co., Ltd.), located at a distance of 20 cm from the suspension for 10 minutes. This procedure of adding a solution of EMA and irradiation with visible light is also referred to as EMA processing. Then, to the suspension was added to 990 μl of physiological saline and the suspension was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. After the top water layer was removed, to the precipitate after centrifugation of the lower layer was added 300 μl of physiological saline, and then to the mixture was added to 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was conducted for 15 minutes at room temperature under light shielding for each sample.

(2) Method of test

FCM measurements were carried out for each of the samples obtained above (measurement time: 30 seconds) in the same manner as in test example 1.

(3) the results of the test

The results of this test are shown in the IG. 4.

As shown in Fig. 1, referenced above, living cells and damaged cellsEscherichia colicannot be clearly distinguished, if only LT-processing. However, as is clear from the results of this test, if after LP treatment is done EMA processing, it becomes possible to clearly distinguish between living cells and damaged cellsEscherichia coli. Additionally, the same results were also obtained forStaphylococcus epidermidis. It is assumed that the result was due to the fact that EMA has low permeability to cell walls, do not penetrate cell walls of living cells, but penetrates through the cell wall damaged cells, in which the damage is not too much.

EMA, penetrating into the damaged cells, randomly intercalary in the chromosomal DNA in cells damaged bacteria, then turns into nitren under the action of irradiation of visible light and is associated with chromosomal DNA through covalent binding. Because the activity of DNA gyrase or topoisomerase is stored in the cells after a short time pasteurization, there untwisting the strands of DNA for transcription of genes during metabolism and, thus, there is cleavage and re-ligation of chromosomal DNA. At the same time re-ligation of DNA under bastiaansen above enzymes inhibited as a result of covalent joining nitrene, obtained from EMA, and this results in the increasing fragmentation of chromosomal DNA. If enable SYTO9 to act on damaged cells containing fragmented chromosomal DNA, the intensity of its fluorescence is significantly reduced compared to the intensity observed before the EMA treatment. It is believed that this is due to the fact that the DNA fragmentation reduces the efficiency of intercalation SYTO9 in DNA. However, since the EMA cannot penetrate the cell walls of living cells, chromosomal DNA of living cells the effect is not provided. Thus, it is believed that even if conduct EMA treatment, living cells do not show changes in the intensity of fluorescence due to SYTO9.

Example 2

Detection of living cellsEscherichia coliandStaphylococcus epidermidis(LP-treated), suspended in UHT gomogenizirovannom milk, processed by ethidium monosiga (EMA), when using the flow cytometer

(1) Obtaining samples

To 1 ml of 6×107CFU/ml suspensionEscherichia coli(living cells)obtained in the same manner as in reference example 1, was added 9 ml of UHT homogenized milk, used in the control example 2, for a 10-fold dilution of the suspension, and the diluted suspension was sequentially diluted in the same way with getting the UHT homogenizing the data of milk, inoculated from 6×101up to 6×105CFU/mlEscherichia coli(living cells).

Additionally, 1 ml of 1.9×108CFU/ml suspensionStaphylococcus epidermidis(living cells)obtained in the same manner as in reference example 1, was added 9 ml of UHT homogenized milk for 10-fold dilution of the suspension, and the diluted suspension was sequentially diluted in the same way with getting UHT homogenized milk inoculated from 1.9×102to 1.9×107CFU/mlStaphylococcus epidermidis(living cells). Separately also made UHT homogenized milk not inoculated with bacteria.

Each of the samples of UHT homogenized milk inoculated withEscherichia coli(living cells), UHT homogenized milk inoculated withStaphylococcus epidermidis(living cells), and UHT homogenized milk not inoculated with bacteria in a volume of 1 ml was placed in microprobing volume of 2 ml and subjected to the same processing procedure lipase and proteinase K (LP processing)as in the control example 1. To the suspension was added 880 μl of physiological saline and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. The upper lipid layer was removed with a swab and the aqueous layer existing in the middle part, was also removed. Then added 1 ml of physiological saline is Astara to the cells of the lower layer (sludge) for suspension cells. Each LP-treated cell suspension and UHT gomogenizirovannom milk not inoculated with bacteria, was added 10 μl of 1000 µg/ml aqueous solution of EMA used in example 1, the mixture was left at 4°C for 5 minutes under light shielding, and was irradiated with visible light 500 watt ice from the lamp (FLOOD PRF, 100 V, 500 W, Iwasaki Electric Co., Ltd) for 5 minutes (EMA processing). Then the sample was added to 990 μl of physiological saline solution and the sample was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. Then the upper water layer was removed, was added 300 μl of physiological saline to the precipitate after centrifugation of the lower layer, then to the mixture was added to 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was carried out at room temperature for 15 minutes under light shielding for each sample.

(2) Method of test

FCM measurements were carried out for each of the samples obtained above (measuring time: 5 minutes) in the same manner as in test example 1.

(3) the results of the test

The results of this test are shown in Fig. 5.

As shown in reference example 2 above, the results shown in Fig. 2, when liveEscherichia coliwere detected in the UHT gomogenizirovannom milk with LT-processing, a large number of points on the graph is for contaminating somatic cells and damaged cells were present in the field of SYTO9(+)·PI(-), this area corresponds to the presence ofEscherichia coli(living cells), as a result, the detection limit was reached 1.1×104CFU/ml

However, as clearly seen from the results of this test, SYTO9/PI schedule for LP-treated and EMA-treated UHT homogenized milk not inoculated withEscherichia coli, SYTO9 intensity decreases from 1×101up to 5×101and the results were difficult to plot in the field of SYTO9(+)·PI(-), showing the presence of living cells.

Moreover, the number of data points in the field of SYTO9(+)·PI(-) changes depending on the inoculated concentration ofEscherichia coli(living cells). The number of data points in this area for each inoculated concentration shown in table 4. It is believed that under the conditions of this test, the limit of detection forEscherichia coli(living cells) in UHT gomogenizirovannom milk subjected LP processing and EMA processing (LPE processing)is 6.0×102CFU/ml

Table 4
The detection results liveEscherichia coliin UHT gomogenizirovannom milk for the LPE-processing
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)
05
6,0×1013
6,0×10272
6,0×103157
6,0×104376
6,0×1052316

Additionally conducted the above LP processing and EMA processing in a similar manner except that the used sample volume for the above LP processing and EMA treatment was changed from 1 ml to 10 ml (the concentration of lipase, proteinase K and EMA were the same) and the measurements were carried out using the flow cytometer. The measurement results of the number of data points in the SYTO9(+)·PI(-) region are shown in table 5. It is believed that under the conditions of this test, the limit of detection ofEscherichia coli(living cells) in UHT gomogenizirovannom milk is 6.0×101CFU/ml compared with the detection limit obtained using the LP processing specified in the control example 2 to 1.1×104CFU/ml, the limit of detection forEscherichia coli(live cells) was improved to such a low concentration as approximately 1/(1,8×102).

Table 5
The detection results liveEscherichia coliin UHT gomogenizirovannom milk for the LPE-processing (reaction scale 10 ml)
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)
01
6,0×10114
6,0×102113
6,0×103506
6,0×1044360
6,0×10545541

The number of data points in the SYTO9(+)·PI(-) region, calculated forStaphylococcus epidermidis,shown in table 6. According to the results, the detection limit forStaphylococcus epidermidis(living cells) in UHT gomogenizirovannom milk was 1.9×104CFU/ml Causes a higher detection limit compared toEscherichia coliconsider an extremely strong trendStaphylococcus epidermidisto be adsorbed on the lipid layer is formed in the middle of the LP treatment, and a significant decrease in the number of data points for LP-treatedStaphylococcus epidermidis(living cells) in the sphere of the SYTO9(+)·PI(-), serving as the field of living cells, in comparison with the data points in the field of SYTO9(+)·PI(-)observed rawStaphylococcus epidermidis(living cells), which are responsible for the majority of cells.

Table 6
The detection results liveStaphylococcus epidermidisin UHT gomogenizirovannom milk for the LPE-processing
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)
028
of 1.9×10224
of 1.9×10317
of 1.9×10495
of 1.9×105149
of 1.9×106584
of 1.9×1075263

Example 3

Detection of living cells and damaged cellsEscherichia coliandStaphylococcus epidermidis(LP-treated), suspended in LTLT dehomogenization milk, processed by ethidium monosiga (EMA),when using the flow cytometer

(1) Obtaining samples

To each 1 ml of 1.5×107CFU/ml suspensionEscherichia coli(live cells and injured cells)obtained in the same manner as in reference example 1, was added 9 ml of LTLT dehomogenization milk used in control example 2, for a 10-fold dilution of the suspension, and the diluted suspension was sequentially diluted in the same way with getting LTLT dehomogenization milk inoculated with 1.5×102up to 1.5×106CFU/mlEscherichia coli(live cells and injured cells).

In addition to each of 1 ml of 1.8×108CFU/ml suspensionStaphylococcus epidermidis(live cells and injured cells)obtained in the same manner as in reference example 1, was added 9 ml of LTLT dehomogenization milk used in control example 2, for a 10-fold dilution of each suspension, and the diluted suspension was sequentially diluted in the same way with getting LTLT dehomogenization milk inoculated with 1.8×102to 1.8×107CFU/mlStaphylococcus epidermidis(live cells and injured cells). Separately also made LTLT dehomogenization milk not inoculated with bacteria.

Each of the samples LTLT dehomogenization milk inoculated withEscherichia coli(living cells), LTLT dehomogenization milk, a monk of the reach Staphylococcus epidermidis(living cells), and LTLT dehomogenization milk not inoculated with bacteria were subjected to the same treatment with lipase and proteinase K (LP processing), and EMA treatment, in the same manner as the processing in example 2. Then the sample was added to 990 μl of physiological saline and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. After the upper lipid layer was removed, was added 300 μl of physiological saline to the precipitate after centrifugation of the lower layer, then to the mixture was added to 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was carried out at room temperature for 15 minutes under light shielding for each sample.

(2) Method of test

FCM measurements were carried out for each of the samples obtained above (measuring time: 5 minutes) in the same manner as in test example 1.

(3) the results of the test

The results of this test are shown in Fig. 6 and 7. As shown in the above reference example 3, according to the results shown in Fig. 3, when liveEscherichia coliandStaphylococcus epidermidiswere detected in the LP-treated LTLT dehomogenization milk, a large number of data points for contaminating somatic cells and damaged cells were present in the field of SYTO9(+)·PI(-), this region is the industry responds to the presence of Escherichia coli(living cells) andStaphylococcus epidermidis(living cells), and the limit of detection ofEscherichia coliamounted to 1.5×105CFU/ml, and limit of detection ofStaphylococcus epidermidis1.8×106CFU/ml, both of which represented a high level.

However, as clearly seen from the results of this test, SYTO9/PI schedule for LP-treated and EMA-treated (LPE-treated) LTLT dehomogenization milk not inoculated with bacteria, SYTO9 intensity decreases to the level of 5×102and the results were difficult to plot in the field of SYTO9(+)·PI(-), showing the presence of living cells.

Moreover, the number of data points in the field of SYTO9(+)·PI(-) changes depending on the inoculated concentration ofEscherichia coli(living cells). The number of data points in this area for each inoculated concentration shown in table 7. It is believed that under the conditions of this test, the limit of detection forEscherichia coli(living cells) in LTLT dehomogenization milk subjected LP processing and EMA processing (LPE processing), 1.5×104CFU/ml. moreover, LTLT dehomogenization milk inoculated withEscherichia coli(damaged cells) at a density of 1.5×106CFU/ml, was subjected LPE process and investigated the number of data points in the field of SYTO9(+)·PI(-), indicating the presence of W is o cells. As a result, the point on the graph is almost not present in this area. On this basis, LPE treatment makes possible a clear separation of living cells and damaged cellsEscherichia coli.

Table 7
The detection results liveEscherichia coliin LPE-treated LTLT dehomogenization milk
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)
01
of 1.5×10220
of 1.5×10320
of 1.5×10431
of 1.5×105349
of 1.5×1062095

The measurement results of the number of data points in the field of SYTO9(+)·PI(-) forStaphylococcus epidermidisshown in table 8. According to the results, detection limitStaphylococcus epidermidis(living cells) in UHT gomogenizirovannom milk was 1.8×105CFU/ml is Considered that the reasons for the higher detection limit compared toEschericia coli are similar to the reasons specified in example 2.

Table 8
The detection results liveStaphylococcus epidermidisin LPE-treated LTLT dehomogenization milk
The number of inoculated bacteria (CFU/ml)The value of FCM (actual measured value)
01
1,8×1022
1,8×1032
1,8×1041
1,8×10513
1,8×10685
1,8×107530

Example 4

Investigation of the effect of various poisons and poisons topoisomerase DNA gyrase

Conducted research detection using the flow cytometerEscherichia coli(living cells) andStaphylococcus epidermidis(living cells)suspended in a commercially available cow's milk, when using the compounds belonging to other poisons topoisomerase (amsacrine, ellipti is in, camptothecin) and compounds related to poisons DNA gyrase (ciprofloxacin), whose activity is similar to activity ethidium monoacid used in examples 1 and 2.

(1) Obtaining samples

To 1 ml of 7.5×107CFU/ml suspensionEscherichia coli(living cells), obtained similarly as in reference example 1, was added 9 ml of UHT homogenized milk for 10-fold dilution of the suspension and, thus, received UHT homogenized milk inoculated with 7.5×106CFU/mlEscherichia coli(living cells).

In addition to 1 ml of 2.0×108CFU/ml suspensionStaphylococcus epidermidis(living cells), obtained similarly as in reference example 1, was added 9 ml of UHT homogenized milk for 10-fold dilution of the suspension and, thus, received UHT homogenized milk inoculated with a 2.0×107CFU/mlStaphylococcus epidermidis(living cells). Separately also made UHT homogenized milk not inoculated with bacteria.

Each of the samples of UHT homogenized milk inoculated withEscherichia coli(living cells), UHT homogenized milk inoculated withStaphylococcus epidermidis(living cells), and UHT homogenized milk not inoculated with bacteria in a volume of 1 ml was placed in microprobing volume of 2 ml and subjected to treatment with lipase and proteinase K (L treatment) in the same way, as in the control example 1. To the sample was added 880 μl of physiological saline solution and the sample was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. The lipid layer, existing in the upper layer was removed with a swab and the aqueous layer existing in the middle part, was also removed. Then to the cells of the lower layer (draft) was added 1 ml of physiological saline to obtain each LP-treated suspension.

Each LP-treated suspension was subjected to treatment with a solution (a) amsacrine, b) ellipticine,) camptothecin and d) ciprofloxacin and to the suspension was added to 0.9 ál SYTO9/PI fluorescent dye reagent to obtain each sample for FCM measurements. Specific procedures are described below with (a) through (d).

a) Processing amsacrine

Each LP-treated suspension was added 10 μl of solution amsacrine (Sigma, catalog number: A), dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml, and the mixture was left at 37°C for 10 minutes. Then to the mixture was added to sterilized water to obtain total volume of 2 ml and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. After the water layer is the upper layer was removed, was added 300 μl of physiological saline to the precipitate after centrifugation of the lower layer, then to the mixture was added is of 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was carried out at room temperature for 15 minutes under light shielding for a sample.

b) Processing ellipticine

Each LP-treated suspension was added 5 μl of a solution of ellipticine (Sigma, catalog number: E), dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml, and the mixture was left at 37°C for 30 minutes. Then to the mixture was added to sterilized water to obtain total volume of 2 ml and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. After the water layer is the upper layer was removed, was added 300 μl of physiological saline to the precipitate after centrifugation of the lower layer, then to the mixture was added to 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was carried out at room temperature for 15 minutes under light shielding for a sample.

(C) Processing camptothecin

Each LP-treated suspension was added 10 μl of a solution of camptothecin (Sigma, catalog number: C), dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml, and the mixture was left at 37°C for 30 minutes. Then to the mixture was added to sterilized water to obtain total volume of 2 ml and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. After the water layer is the upper layer was removed, was added 300 μl of physiological saline to the precipitate after centrifugation of the lower layer, then to the mixture is added to 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was carried out at room temperature for 15 minutes under light shielding for a sample.

d) Treatment with ciprofloxacin

Each LP-treated suspension was added 8 μl of a solution of ciprofloxacin (Fluka, catalogue number: 17850), dissolved in dimethyl sulfoxide (DMSO) at a concentration of 0.5 mg/ml, and the mixture was left at 37°C for 30 minutes. Then to the mixture was added to sterilized water to obtain total volume of 2 ml and the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes. After the water layer is the upper layer was removed, was added 300 μl of physiological saline to the precipitate after centrifugation of the lower layer, then to the mixture was added to 0.9 ál SYTO9/PI fluorescence staining reagent and the reaction was carried out at room temperature for 15 minutes under light shielding for a sample.

(2) Method of test

FCM measurements were carried out for each of the samples obtained above (measuring time: 5 minutes) in the same manner as in test example 1.

(3) the results of the test

The results of this test are shown in Fig. 8.

For samples subjected only LP processing, the area on the graphEscherichia coli(living cells) and area contaminants (somatic cells, damaged cells, products of incomplete decomposition michelango casein caused LP processing), originating from UHT homogenized milk, was located next to one another, and h is partially overlap (intensity SYTO9 LP-treated UHT homogenized milk, not inoculated with bacteria, contaminants may significantly exceed 103depending on the type, manufacturer, etc. of cow's milk, and in this case, the area on the graph of living cells and contaminants significantly overlap with one another) and, thus,Escherichia coli(living cells) cannot be explicitly recognized. However, when after LP treatment were treated with a solution a) amsacrine, b) ellipticine,) camptothecin or (d) of ciprofloxacin, the area on the graphEscherichia coli(living cells) and contaminants are easily separated, and as a result facilitate the detection ofEscherichia coli(living cells). Moreover, similar results were also observed forStaphylococcus epidermidis.

With a high probability of amsacrine and camptothecin at the concentrations used in example 3, and within the response time specified above (10 minutes and 30 minutes respectively), did not penetrate through the cell wallEscherichia coliandStaphylococcus epidermidis(living cells), whereas they penetrated through the cell membrane of somatic cells, which were transformed into dead cells under the action of instant pasteurization, and cell wall damaged cells. It is believed that two types of the above agents, penetrating into somatic cells and damaged cells, randomly associated with chromosomal DNA by kovalentnosvyazannoi with inhibition of the re-ligation of DNA topoisomerase II and topoisomerase I in somatic cells or topoisomerase IV, topoisomerase I or topoisomerase III in damaged cells or DNA Girasol, and thus, the chromosomal DNA is fragmented, leading to a decrease in the fluorescence intensity of SYTO9. On the other hand, these agents do not affect the chromosomal DNA of living cells, and thus, the intensity of SYTO9 fluorescence in living cells is not reduced. On this basis, the area on the graph were clearly separated.

With high probability ellipticine and ciprofloxacin at the concentrations used in example 3, and within the response time specified above (30 minutes), also did not penetrate through the cell walls of living cells, whereas they penetrated through the cell membrane of somatic cells and cell walls of damaged cells, similarly to the above. Although it is believed that ellipticine uses the action of topoisomerase II in somatic cells and DNA gyrase in damaged cells or topoisomerase IV, it did not inhibit the re-ligation of DNA under the action of these enzymes, but promotiom cleavage of DNA chains under the action of these enzymes, leading to fragmentation of chromosomal DNA. On the contrary, it is believed that ciprofloxacin with high probability inhibits re-ligation of DNA chains under the action of DNA gyrase in damaged cells and also inhibits re Legerova the e-chain DNA under the action of topoisomerase II in somatic cells.

Example 5

Detection of live cells, injured cells and dead cells on the basis of electrophoretic spectrum of chromosomal DNA obtained after EMA processing four types of gram-negative bacteria (bacteriaEscherichia coliDH5α,Klebsiella, CitrobacterandSalmonella) and gram-positive bacteria (Staphylococcus epidermidisused as test materials

(1) Obtaining samples

1-1) the suspensions of four types of gram-negative bacteria (live cells, injured cells and dead cells)

Escherichia coliDH5α,Klebsiella oxytocaJCM 1665 (hereafter also referred to as the "Klebsiella"), Citrobacter koseriJCM 1658 (hereafter also referred to as the "Citrobacter"andSalmonella enteridisIID 604 (hereafter also referred to as the "Salmonella") were cultured at 37°C in a nutrient medium BHI and 40 ml of each medium in which the cells were in the last stage of the logarithmic growth phase were subjected to centrifugation at cooling at 4°C and 8000×g for 15 minutes. The supernatant was removed, and then to the residue was added 40 ml of physiological salt solution, the mixture was stirred and subjected to centrifugation at cooling under the same conditions. The supernatant was removed and to the residue was added 10 ml of physiological saline to obtain a suspension of living cells. The number of live CL is current in these suspensions of living cells was: Escherichia coli: 3,2×108CFU/ml,Klebsiella: 4,8×108CFU/ml, Citrobacter: 6,7×107CFU/ml andSalmonella: 1,9×108CFU/ml + 1 ml of each suspension of living cells was placed in microprobing 1.5 ml, microprobing was immersed in boiling water for 50 seconds and then rapidly cooled by immersion in ice water to obtain a suspension of damaged cells. In addition, forEscherichia coliseparately received a suspension of dead cells from 1 ml of a suspension of living cells by immersing in boiling water for 12 minutes and rapid cooling in ice water. Confirmed that as the suspension of damaged cells and dead cells do not form colonies on agar medium L.

1-2) suspensions of gram-positive bacteria (live cells, injured cells and dead cells)

Staphylococcus epidermidis(Staphylococcus epidermidisstrain KD) were cultured at 37°C in a nutrient medium BHI and 40 ml of medium in which cells were in the last stage of the logarithmic growth phase were subjected to centrifugation at cooling at 4°C and 8000×g for 15 minutes. The supernatant was removed, and then to the residue was added 40 ml of physiological salt solution, the mixture is sufficiently stirred and subjected to centrifugation at cooling under the same conditions. The supernatant was removed and to the residue was added 10 ml of physiological SALEWA what about the solution to obtain a suspension of living cells. The number of living cells in this suspension of living cells was 1.9×108CFU/ml. Additionally, 1 ml of a suspension of living cells was placed in microprobing 1.5 ml, microprobing was immersed in boiling water for 50 seconds and then rapidly cooled by immersion in ice water to obtain a suspension of damaged cells. In addition, we have obtained a suspension of dead cells from 1 ml of a suspension of living cells by immersing in boiling water for 12 minutes and rapid cooling in ice water. Confirmed that as the suspension of damaged cells and dead cells do not form colonies on agar medium L.

In order to obtain the time dependence of immersion in boiling water the temperature of the liquid in advance, 1 ml of physiological saline was placed in microprobing 1.5 ml at room temperature and completely sealed by a cover, the cover has a little hole and in the hole was placed temperature sensor type thermocouple (TX10, Yokogawa M & C). Then microprobing substantially fully placed in boiling water and measure the water temperature with time.

(2) test Time

2-1) Stage of processing by ethidium monosiga and irradiation with visible light

In volume 1 ml of each suspension gram-negative bacteria (live cells, injured cells and dead cells) and graphological the first bacteria (living cells, damaged cells and dead cells) was subjected EMA processing in the same manner as in example 3. After adding the EMA solution to the suspensions, suspensions of gram-negative bacteria were left at 4°C for 30 minutes under light shielding, and suspensions of gram-positive bacteria were left at 4°C for 5 minutes under light shielding, while they were not subjected to irradiation with visible light.

Separately, 1 ml of each suspension gram-negative bacteria (live cells, injured cells and dead cells) and gram-positive bacteria (live cells, injured cells and dead cells) instead of EMA was added 10 μl of sterilized water and then subjected to the same procedure used for the above EMA processing (EMA-raw).

2-2) the stage of DNA extraction

Each microprobing containing live cells, injured cells and dead cells of gram-negative bacteria and gram-positive bacteria (EMA-raw and processed)were subjected to centrifugation at cooling at 4°C. and 15,000×g for 10 minutes and remove the supernatant. Each microprobe was added 1 ml of physiological salt solution, the mixture was sufficiently stirred, the mixture was moved into microprobing 2 ml and was subjected to centrifugation at cooling at 4°C. and 15,000×g for 10 minutes. The supernatant is idcast was removed to obtain a precipitate of cells after centrifugation.

In the case of gram-positive bacteria DNA was extracted as follows. To every draught of cells after centrifugation was added to 0.5 mm etilenditiodiuksusnoi acid (EDTA) was added 20 μl of a solution of chromopeptide in advance received a 5 mg/ml of 10 mm aqueous NaCl solution (Wako Pure Chemical Industries, catalog number: 014-09661), and the mixture was left at 50°C for 30 minutes. Then to the mixture was added 0.5 ml of 10 mm Tris-HCl (pH 8.0), was added 20 μl 1250 units/ml proteinase K (Sigma, catalog number: Y.S. 3.4.21.64), was added 400 μl of SDS solution, prepared at a concentration of 10% (wt./about.) in sterilized water, and the reaction was left over night at 50°C.

Each of the treated suspension was placed in two microtube volume of 2 ml in each half of the volume, to the suspension was added 0.5 ml of saturated phenol and the mixture was gently stirred for 15 minutes. Then to the mixture was added 0.5 ml of chloroform and the mixture was gently stirred for 5 minutes. The mixture was subjected to centrifugation at cooling at 4°C and 6000×g for 10 minutes, the aqueous layer of the upper layer was transferred into a new tube with a volume of 2 ml, to the mixture was added 70 μl of sodium acetate 3M (pH 5,2) and to 1.21 ml of cold ethanol of 99.5% and the mixture is gently stirred. The mixture was subjected to centrifugation at cooling at 4°C. and 15,000×g for 10 minutes, the supernatant was removed and then the residue is washed and 0.4 ml of 70% cold ethanol (the above procedure is also referred to as extrace phenol/chloroform"). To the precipitate after centrifugation was added 0.5 ml of TE buffer (10 mm Tris-HCl, 1 mm etilenditiodiuksusnoi acid (EDTA)·2Na) and the mixture was left overnight at 4°C to dissolve the DNA.

Pre-obtained solution of 5 μl of ribonuclease (Sigma, catalog number: E.C. 3.1.27.5) with a concentration of 10 mg/ml in sterilized water was added to the above DNA solution and the mixture is incubated at 37°C for 1 hour. To the mixture was added 0.25 ml of a mixture of phenol/chloroform (1/1) and the mixture was gently stirred for 10 minutes, to the mixture was further added 0.25 ml of chloroform and the mixture was gently stirred for 5 minutes. The mixture was subjected to centrifugation at cooling at 4°C and 6000×g for 10 minutes, the aqueous layer of the upper layer was transferred into a new microprobing volume of 2 ml, to the mixture was added 50 μl of 3 M aqueous solution of sodium acetate and 1 ml of 99.5% cold ethanol and the mixture is gently stirred. The mixture was subjected to centrifugation at cooling at 4°C. and 15,000×g for 10 minutes, the supernatant was removed, then the residue was washed with 0.4 ml of 70% cold ethanol and the precipitate after centrifugation were dried (the above procedure is also designated as "processing by ribonuclease"). To the dried precipitate after centrifugation was added 125 μl of TE buffer and the mixture was left overnight at 4°C to dissolve the DNA. The concentration of purified DNA solution was measured on the Snov absorption at 260 nm (UV), OD260(50 ág DNA/ml was considered as OD=1, length cuvette: 1 cm) and the purity of the purified DNA was evaluated on the basis OD260/OD280.

For gram-negative bacteria DNA was extracted by using the next method. By the above draught of cells after centrifugation was added 0.5 ml of 10 mm Tris-HCl (pH 8.0), was added 10 μl 1250 units/ml proteinase K (Sigma, catalog number: Y.S. 3.4.21.64) was added in 200 µl SDS solution, prepared at a concentration of 10% (wt./about.) in sterilized water, and the reaction was left over night at 50°C. Then carried out the extraction of DNA in a similar manner with the DNA extraction of gram-positive bacteria.

2-3) Electrophoresis of extracted DNA in agarose gel

Got to 0.8% agarose gel from Seakem GTG agarose (FMC, catalogue number: 50070) and TAE buffer (4,84 g/l Tris, 1,142 ml of acetic acid, 0,149 g/l etilenditiodiuksusnoi acid (EDTA)·2Na), and λ-EcoT14I hydrolyzate (Takara Shuzo, code: 3401) and 100 bp (base pairs) DNA Ladder (Takara Shuzo, code: A) were used as markers. For each of the gram-negative bacteria and gram-positive bacteria EMA-raw suspension of living cells, EMA-treated suspension of living cells, EMA-raw suspension of damaged cells and EMA-treated suspension of damaged cells were placed in wells in the specified order in the amount of about 1 μg and subjected to electrophor the memory at 100 C. After the gel has migrated approximately 90% bromophenol blue (BPB), electrophoresis was completed. Separately forEscherichia coliandStaphylococcus epidermidiswas also subjected to a similar procedure electrophoresis EMA-raw suspension of dead cells and EMA-treated suspension of dead cells.

The gel, which was performed electrophoresis, was immersed in an aqueous solution of 1 µg/ml ethidium bromide for 20 minutes and twice washed with Milli-Q water, and then the degree of cleavage of chromosomal DNA was observed using UV transilluminator (254 nm).

(3) the results of the test

The relationship between the time of immersion in boiling water and the temperature of the liquid is shown in Fig. 9. It was confirmed that at least heat treatment by immersion in boiling water for 50 seconds corresponds to the processing pasteurization is somewhat more powerful than the pasteurization within a short time (HTST pasteurization, from 72 to 75°C, from 15 to 16 seconds). Thus, it was confirmed that the above heat treatment equivalent to a heat treatment such as sterilization from the viewpoint of suppressing the denaturation of food, i.e. low-temperature pasteurization in a long time (LTLT pasteurization) and pasteurized at ultra high temperature (UHT pasteurization). Thus, the damaged cells, obtained the ri method, described in (1) Obtaining samples specified above represent the biochemical and enzymatic equivalents of dead cells in food succumbing to suppress the denaturation of food ingredients, and physical damage are also equivalent. For dead cells, obtained using the method described in (1) Obtaining samples specified above, since the liquid temperature was maintained at 100°C for 10 minutes after the temperature reached 100°C., and the suspension was also heated for two minutes before the temperature reached 100°C, according to the dependence shown in Fig. 9, most of the enzymes in dead cells was inactivated, and damage to the cell walls was so significant, that part of the chromosomal DNA released from the cells.

Further, the results distinguish between living cells and damaged cells for each gram-negative bacteria is shown in Figure 10, the results distinguish between damaged cells and dead cellsEscherichia colifigure 11 and the results distinguish live cells, injured cells and dead cells to gram-positive bacteriaStaphylococcus epidermidisshown in Fig. In Fig. 10 was marked by extremely long strip (LL band)corresponding chromosomal DNA, localized slightly below the brand is and λ-EcoT14I of the hydrolyzate 19329 bp, and it was accepted that the presence of this band was identified as (+) and the absence of this band was identified as (-). The results for the four types of gram-negative bacteria from the point of view of this band represented, in order EMA-raw and EMA-treated samples (+)·(+) painting for living cells, and (+)·(-) the picture for damaged cells. In addition, the picture for damaged cells and dead cellsEscherichia coli, shown in Fig. 11, was a (+)·(-) picture and (-)·(-) picture, respectively. Thus, the EMA has made possible the simultaneous distinction of living cells, damaged and dead cells. In Fig. 12 similarly shows that the distinction of live cells, injured cells and dead cells is also possible for gram-positive bacteria, such asStaphylococcus epidermidis.

The EMA applies in particular to topoisomerase II in tumor cells having a high rate of mitosis, as the poison of topoisomerase II in mammalian cells, and the action of the enzyme, which consists in splitting the DNA and re-legirovanii, inhibits re-ligation of DNA. Thus, the EMA is expected as an anti-cancer agent, repeatedly cleave chromosomal DNA of the cancer cells to kill cancer cells. Originally EMA showed low permeability of cell membranes and, in application of the x in the field of bacteriology, it was estimated solely as a so-called DNA-crosslinking agent, which cannot penetrate the cell wall of live bacteria, but can penetrate the cell wall of dead bacteria to bind chromosomal DNA of dead bacteria, as described in the international patent application unexamined publication application Japan No. 2003-530118. Also in this example was observed to chromosomal DNA of living cells, left for 30 minutes, specifically split under the action of the EMA, as shown in Fig. 10, and the chromosomal DNA of damaged cells left at the same time, there was a significant split under the action of the EMA. Thus, it was hypothesized that the EMA substantially does not penetrate the cell walls of living cells, but penetrates through the greater part of the cell walls of damaged cells. What should particularly be noted is that, despite the fact that EMA initially inhibits the action of topoisomerase II cells of the mammal from the point of view of re-ligating and, thus, causes multiple cleavage of chromosomal DNA, resulting in cell death of the mammal, the EMA does not affect the living cells in the present example, suggesting that the EMA inhibits the activity of bacterial DNA gyrase and/or topoisomerase IV activity that save is applied in cells and causes multiple cleavage of chromosomal DNA damaged cells.

Example 6

Simultaneous discernment EMA-treated live cells, injured cells and dead cellsEscherichia coliandStaphylococcus epidermidiswhen using FCM

(1) Obtaining samples

1-1) suspensionsEscherichia coli(live cells, injured cells and dead cells)

According to the method of example 5, (1) Obtaining samples, 1-1)got live cells, injured cells and dead cellsEscherichia coli(number of living cells: 4×106CFU/ml, the number of damaged cells: 4×106CFU/ml, the number of dead cells: 4×106CFU/ml).

1-2) suspensionsStaphylococcus epidermidis(live cells, injured cells and dead cells)

According to the method of example 5, (1) Obtaining samples, 1-2)got live cells, injured cells and dead cellsStaphylococcus epidermidis(number of living cells: 4×107CFU/ml, the number of damaged cells: 4×107CFU/ml, the number of dead cells: 4×107CFU/ml).

(2) Method of test

2-1) Processing by ethidium monosiga and stage irradiation of visible light

To each sample 1 ml of the above suspension of live cells, injured cells and dead cellsEscherichia coliadded 10 μl of an aqueous solution EMA 1000 µg/ml and the suspension was left at 4°C for 30 minutes under light shielding. Then the suspension was placed on ice and irradiated with visible light of 500 W lamp (FLOOD PRF, 100 V, 500 W, Iasaki Electric Co., Ltd), located at a distance of 20 cm from the suspension for 10 minutes. Live cells, injured cells and dead cellsStaphylococcus epidermidiswere subjected to the same treatment.

2-2) Staining of nuclei and FCM measurements

Each of the above suspensions treated with EMA were subjected to centrifugation at cooling at 4°C. and 15,000×g for 15 minutes. After removal of the supernatant liquid to the residue was added 1 ml of physiological salt solution, the mixture is sufficiently stirred and subjected to centrifugation at cooling under the same conditions. The supernatant was removed and added to the residue, 1 ml of physiological saline. To the mixture was added 3 μl of SYTO9/PI fluorescence staining reagent (LIVE/DEAD BacLightTMBacterial Viability kit, Molecular Probes, SYTO9/PI = 1/1 mixture) and the reaction was carried out at room temperature for 15 minutes under light shielding for each sample suspension. For these samples measured using FCM instrumentation FACS Calibur (Becton Dickinson). The measurement conditions were the same as conditions in the control example 1.

(3) the results of the test

The test results using FCM for live cells, injured cells and dead cellsEscherichia colibefore and after EMA processing shown in Fig. 13 and the same results forStaphylococcus epidermidisshown in f is, 14.

The boundary intensity SYTO9 staining is defined as the intensity of the 103results that exceed this value, represented as SYTO9(+), and the results are below specified values are represented as SYTO9(-). The boundary value of the intensity of staining PI is defined as the intensity of 2×102results that exceed this value, represented as PI(+), and the results are below specified values are represented as PI(-). Living cellsEscherichia colibefore EMA treatment was mainly distributed in the SYTO9(+)·PI(-) region, the damaged cells mainly distributed in the SYTO9(+)·PI(-) region, and the dead cells are mainly distributed in the SYTO9(+)·PI(+) region. Thus, for cells not treated with EMA, living cells and damaged cells cannot be distinguished, but the damaged cells and dead cells can be distinguished even in the case when the cells not treated with EMA. Moreover, after the EMA processing the distribution of living cells has not changed, i.e. they are distributed in the SYTO9(+)·PI(-) region, but the intensity of SYTO9 staining for damaged cells was significantly decreased, shifting their main distribution in the SYTO9(-)·PI(-) region, and thus, living cells and damaged cells could be easily distinguished. Without processing the simultaneous distinction of live cells, injured cells and dead cells was impossible.however, after EMA processing, although the main distribution of dead cells moved from the SYTO9(+)·PI(+) region in the SYTO9(-)·PI(+) region, the area in which live cells, injured cells and dead cells in the main distribution do not overlap, and thus, it is possible to distinguish between cells.

In the case of damaged cells retained enzymatic activity of damaged cells and there is also metabolic activity. Thus, EMA penetrate cell walls of damaged cells and knits chromosomal DNA damaged cells with inhibition of re-ligating the bacterial DNA gyrase in damaged cells. The result is stored in such a condition that repeatedly cleaved chromosomal DNA and the chromosomal DNA is significantly fragmented. This in particular leads to a decrease in the number of intercalated SYTO9, and, thus, decrease the intensity of the staining. In the case of dead cells metabolic functions cease, and genes are not transcribed. However, it is assumed that, because of bacterial DNA gyrase and bacterial topoisomerase IV are resistant to heat, part of their activity is maintained, and thus, the above enzymes operate by themselves. Thus, it is estimated that, if the EMA treatment, the intensity of the staining is Denmark SYTO9 is significantly reduced.

Example 7

Simultaneous distinction of live cells, injured cells and dead cellsMycobacterium tuberculosis(Mycobacterium tuberculosisH37RA, hereafter also referred to as"Mycobacterium tuberculosis"andListeria monocytogenes(Listeria monocytogenesJCM 2873, hereafter also referred to as the "Listeria"when using FCM

(1) Obtaining samples

1-1) suspensionsMycobacterium tuberculosis(live cells, injured cells and dead cells)

Mycobacterium tuberculosisput on oblique agar medium Ogawa and cultivated at 37°C for three weeks (in the environment of 20% oxygen and 5% carbon dioxide). Then the cells were inoculable in a liquid environment Saucon containing 0.05% Tween 80, and cultivated at 37°C for 3 weeks under these aerobic conditions. Cultivated environment serially diluted with physiological saline containing 0.05 Tween 80, and were cultured on Petri dishes with agar medium Middelbrook 7H10 to confirm that the number of living cells was 7.7×108CFU/ml of Cultured medium in a volume of 2 ml was inoculable in 200 ml of their liquid environment Saucon containing 0.05% Tween 80 was added 200 μl of a solution of rifampicin 150 mg/ml (dissolved in sterilized water) to the culture (final concentration: 148,5 µg/ml)was further added 100 μl of the hydrazide of isonicotinic acid 10 mg/ml (dissolved in territans water) to the medium (final concentration: 5 μg/ml) and cells were cultured at 37°C for 3 months under these aerobic conditions. After culturing for 1 month the number of living cellsMycobacterium tuberculosismeasured on N environment, and it was confirmed that the cells do not form colonies.

Cultured medium after cultivation for 3 months in the amount of 200 ml was subjected to centrifugation at cooling at 4°C and 8000×g for 10 minutes. The supernatant was completely removed, and then to the residue was added 200 ml of physiological salt solution, the mixture was stirred and subjected to centrifugation at cooling under the same conditions as described above, and the supernatant was completely removed. The washing procedure is additionally carried out again, and it was confirmed that the supernatant does not show the brown color is caused by rifampicin. To the precipitate after centrifugation, obtained by centrifugation at cooling, was added 20 ml of physiological saline containing 0.005% Tween 80, to obtain a suspension of damaged cellsMycobacterium tuberculosis.

The number of bacterial cells in the above suspension of damaged cellsMycobacterium tuberculosiswas measured using the following method. How is that 1 ml of the above cultured environment (living cells) of 7.7×108CFU/mlMycobacterium tuberculosiswere extracted and subjected to centrifugation under cooling at 4°C. and 15,000 rpm during the 10 minutes. After the supernatant was removed, the residue was added 1 ml of physiological saline containing 0.05% Tween 80, and the mixture was stirred and subjected to centrifugation at cooling under the same conditions as described above, and the supernatant was completely removed. To the precipitate after centrifugation was added 1 ml of physiological saline containing 0.05% Tween 80, (suspension of living cellsMycobacterium tuberculosisof 5.2×108CFU/ml). To obtain a dilute suspension, the suspension was further diluted 10, 100, 1000 and 10000 times with physiological saline containing 0.05% Tween 80, and each suspension was measured by absorption of visible light of 600 nm, OD600Nm. On the schedule was delayed density of living cellsMycobacterium tuberculosisand the OD value to obtain the calibration curve and the concentration of damaged cells was calculated based on the OD values above suspension damaged cellsMycobacterium tuberculosis(the number of damaged cells in suspension damaged cellsMycobacterium tuberculosis:6×107CFU/ml).

Once again obtained a suspension of living cellsMycobacterium tuberculosiswas diluted with physiological saline containing 0.05% Tween 80, so that he had the same OD value, and that the suspension of damaged cells.

Additionally, the suspension of living cellsMycobacterium tuberculosisthe same raft the spine, that and the suspension of damaged cells, immersed in boiling water for 12 minutes to obtain a suspension of dead cellsMycobacterium tuberculosis.Since it is impossible to transform cellsMycobacterium tuberculosisin dead cells without damaging the cells, or no confidence in the transformation of cellsMycobacterium tuberculosisin dead cells without damaging the cells by long-term use of anti-TB agent, from the viewpoint of the mechanism of action of anti-TB agent, dead cells was obtained using thermal processing.

1-2) suspensionsListeria(live cells, injured cells and dead cells)

Listeriawas inoculable in a nutrient medium L, and cultured at 30°C for 48 hours (3×108CFU/ml). Cultivated environment in a volume of 3 ml was inoculable in 300 ml of culture medium L, was added 1.5 ml of ampicillin 100 mg/ml (dissolved in sterilized water) and 600 μl of a solution of gentamicin 100 mg/ml (dissolved in sterilized water) to the cultivated medium (final concentration: 500 μg/ml and 200 μg/ml, respectively) and the cells were cultivated at 30°C for 3 weeks. After cultivation was confirmed that the cells do not form colonies on agar medium L. Cultivated environment in a volume of about 200 ml was subjected to centrifugation at cooling at 4°C and 8000×g for 15 the minutes and completely remove the supernatant. To the precipitate after centrifugation was added 300 ml of physiological salt solution, the mixture was stirred and then subjected to centrifugation at cooling under the same conditions, the supernatant was completely removed and to the precipitate after centrifugation was added 3 ml of physiological saline to obtain a suspension of damaged cellsListeria. When using substantially the same procedure as that for measuring the number of damaged cellsMycobacterium tuberculosismeasured the number of damaged cells in suspension damaged cellsListeria(2×108CFU/ml).

Separately received a suspension of living cellsListeriaaccording to the method of example 6, (1) Obtaining samples 1-1), and was diluted with physiological saline to a density that is the OD of the suspension would be the same as the OD value of the suspension damaged cellsListeria.

Additionally, the suspension of dead cellsListeriawas obtained by immersion of a suspension of living cellsListeriathe same density as the suspension of damaged cells in boiling water for 12 minutes.

(2) Method of test

2-1) Stage of processing by ethidium monosiga and irradiation with visible light

To each sample in a volume of 1 ml of the above suspension of live cells, injured cells and dead cellsMycobacterium tuberculosisand suspensions of living cells, damages the different cells and dead cells Listeriaadded 30 μl volume aqueous solution of EMA 1000 µg/ml forMycobacterium tuberculosis(final concentration: about 30 μg/ml) or 10 µl forListeria(final concentration: 10 μg/ml) and each suspension was left at 4°C for 2.5 hours toMycobacterium tuberculosisor at 4°C for 5 minutes forListeriawhile shielding light. Then the suspension was placed on ice and irradiated with visible light of 500 W lamp (FLOOD PRF, 100 V, 500 W, Iwasaki Electric Co., Ltd), located at a distance of 20 cm from the suspension for 5 minutes.

2-2) Staining of nuclei and FCM measurements

Each of the above suspensions treated with EMA were subjected to centrifugation at cooling at 4°C. and 15,000×g for 15 minutes. After removal of the supernatant liquid to the residue was added 1 ml of physiological saline containing 0.05% Tween 80, forMycobacterium tuberculosisor 1 ml of physiological saline for Listeria, and the mixture is sufficiently stirred and subjected to centrifugation at cooling under the same conditions. The supernatant was removed and added to the residue, 1 ml of liquid medium of Sauton containing 0.05% Tween 80, forMycobacterium tuberculosisor 1 ml of physiological saline forListeria. ForMycobacterium tuberculosiscells were cultured at 37°C for 24 hours, then the culture was subjected to centrifugation at cooling at 4°C. and 15,000×g for 15 minutes, nagoshi is Chou liquid was removed and then the residue was added 1 ml of physiological saline, containing 0.05% Tween 80.

For each treated mixture was added 3 μl of SYTO9/PI fluorescence staining reagent (LIVE/DEAD BacLightTMBacterial Viability kit, Molecular Probes, SYTO9/PI=1/1 mixture) and the reaction was carried out at room temperature for 15 minutes under light shielding for each sample suspension. For these samples measured using FCM instrumentation FACS Calibur (Becton Dickinson). The measurement conditions were the same as conditions in the control example 1.

(3) the results of the test

The test results using FCM for living cells, damaged cells treated with isonicotinic acid hydrazide and rifampicin, and dead cellsMycobacterium tuberculosisbefore and after treatment EMA shown in Fig. 15, and the results for living cells,damaged cells treated with ampicillin and gentamicin, and dead cellsListeriabefore and after treatment EMA shown in Fig. 16.

As in example 6, (3) the results of the test, the boundary value of the intensity of SYTO9 staining is defined as the intensity of the 103results that exceed this value, represented as SYTO9(+), and the results are below specified values are represented as SYTO9(-). The boundary value of the intensity of staining PI is defined as the intensity of 2×102results that exceed this value, represented as PI(+) and the results below the specified values are represented as PI(-). From the results shown in Fig. 15, it is clear that EMA processing makes it possible to distinguish living cellsMycobacterium tuberculosisand damaged cellsMycobacterium tuberculosistreated with isonicotinic acid hydrazide and rifampicin. As for dead cells, recognizing them from living cells and damaged cells have already been implemented on the basis of SYTO9/PI to add the EMA. Additionally, although due to the volume of the processing region, which was mainly distributed dead cells, is shifted from the SYTO9(+)·PI(+) region to region SYTO9(±)·PI(±), the region does not overlap with areas in which mainly distributed in living cells and damaged cells treated with EMA. Thus, it becomes possible to distinguish live cells, injured cells and dead cells. The intensity of SYTO9 damaged cellsMycobacterium tuberculosisunder the action of the EMA treatment significantly shifted to the left, and the region is shifted under the action of EMA processing from the SYTO9(+)·PI(-) to SYTO9(-)·PI(-). Rifampicin binds to the β-subunit of bacterial DNA-dependent RNA polymerase, inhibition of binding of DNA polymerase to promoter DNA. In the result, prevents the initiation of transcription. Because it has no effect on RNA polymerase, already associated with the promoter, already initiated by the transcription reaction is not inhibited. Thus, even the EU and the cells by adding rifampicin become damaged cells, who lost the ability to form colonies retained the activity of different enzymes produced before adding rifampicin to the living cellsMycobacterium tuberculosissuch as bacterial DNA gyrase and bacterial topoisomerase IV, and the walls of the cells are also saved from the point of view of the mechanism of action of rifampicin. Despite the fact that the isonicotinic acid hydrazide inhibits the synthesis of microway acid in cell walls, it substantially no effect on the already formed cell wall or, thus, does not lead to the destruction of cells during cell division. By adding the EMA in this state, is suppressed by re-ligation of DNA gyrase and/or bacterial topoisomerase IV, as a result, the process of splitting will be observed throughout the chromosomal DNA and, thus, DNA is fragmented. Believe that obviously reduces the efficiency of intercalation SYTO9, and thus, the intensity of staining of damaged cells is significantly shifted to the left. In the case of dead cells, the intensity of SYTO9 staining is significantly reduced according to the same mechanism of action as for dead cells mentioned in example 6. Additionally, live cells, injured cells and dead cellsListeriaalso showed the same reaction as demonstratedMycobacteium tuberculosis .

Reference example 4

Receiving and distinguishing live cells, injured cells and dead cells using traditional methods

Receipt of samples and the methods for testing

1-1) Applications in sanitary food inspection

Received suspensions of living cells in physiological salineEscherichia coli(from 2.6×103to 2.6×108CFU/ml) andStaphylococcus epidermidis(6.7×102to 6.7×107CFU/ml) (A), and immersed in boiling water for 50 seconds and rapidly cooled (In) or immersed in boiling water for 12 minutes and quickly cooled (With). In these suspensions (A), (b) and (C) live cells, injured cells and dead cells were classified with the same density of cells using ATP-method and measurement esterase activity. Additionally evaluated how different States of the bacteria, defined as live cells, injured cells or dead cells according to the method of the present invention examples 5 and 6 were correlated with the classification of these cells defined using ATP-method (KIKKOMAN reagent kit for the measurement of ATP (ATP measurement reagent kit), Lucifer 250 Plus and KIKKOMAN set of reagents to inhibit ATP (ATP eliminating reagent kit), Lucifer ATP Eliminating Reagent, KIKKOMAN CORP.) and astaranga method (Applied and Environmental Microbiology, 2002, 68: 5209-5216).

1-2) used in clinical test

Were obtained suspension of the LM is s cells Mycobacterium tuberculosisin physiological saline containing 0.05% Tween (D1, from 5.3×103to 5.3×108CFU/ml), and the suspension of living cellsListeriain physiological salt solution (E1, from 3.1×104to 3.1×109CFU/ml). If you are using the suspensions of living cells receivedMycobacterium tuberculosistreated with isonicotinic acid hydrazide (INH, final concentration: 5 μg/ml) and rifampicin (REF, 150 μg/ml) for 3 months (D2),Mycobacterium tuberculosistreated with streptomycin (SM, 300 μg/ml) and kanamycin (KM, 300 μg/ml) for 3 months (D3),Mycobacterium tuberculosisprocessed REF (150 µg/ml) and SM (300 µg/ml) for 3 months (D4) andListeria monocytogenestreated with gentamicin (200 mg/ml) and ampicillin (500 µg/ml) for 3 weeks (E2). D1 and E1 were immersed in boiling water for 12 minutes to obtain the dead cells. Cells in all samples above were classified as live cells, injured cells and dead cells at the same density of cells using ATP-method and measurement esterase activity and evaluate how different States of the bacteria, defined as live cells, injured cells or dead cells according to the method of the present invention of example 6 was correlated with the classification of these cells defined using ATP-method and astaranga method.

(2) the results of the t of the one hundred and discussion

The test results shown in Fig. 17 and 18. The test results show that when comparing features distinguish the method of the present invention and ATP method, the results coincide with one another forMycobacterium tuberculosisandListeriawhileEscherichia coliandStaphylococcus epidermidiscells that are defined when using the ATP method, as being in a state of dead cells were classified as damaged cells and dead cells by the method of the present invention. Thus it is considered that the fine distinction of damaged cells and dead cells can be carried out using the method of the present invention with high sensitivity.

Additionally, in the event estratega way esterna activity of living cellsEscherichia coliwas below the detection limit, and esterna cell activityEscherichia coliimmersed in boiling water at 100°C for 12 minutes and scientific way, defined as dead cells was higher than the detection limit. Thus, it is considered that the measure was problematic. When comparing the abilities of discernment of the method according to the present invention and astaranga way for other bacteria, it was found that the results forListeriabasically coincided with one another, whereas cellsStaphylococcus epidermidisandMycobacterium tuberculsis in the state, defined as the dead cells on the basis of estratega way, were classified as two types of the condition, the damaged cells and dead cells, according to the method of the present invention. Thus, it is believed that the method according to the present invention is more suitable for accurate separation of damaged cells and dead cells.

Example 8

Detection of living cells in clinical samples

(1) preparation of samples, and test

Heparinised blood (group a) was collected from healthy human specimens, and blood was diluted 10-fold suspension of living cellsListeria1,8×106CFU/ml Suspension additionally serially diluted blood to get blood, inoculated from 1.8×103up to 8×107CFU/mlListeria monocytogenes(living cells).

Selected each blood sample, not inoculatedListeria(living cells), and blood samples inoculated withListeria(living cells), in a volume of 1 ml of each blood sample was added 10 μl of an aqueous solution EMA 1000 µg/ml and the mixture was maintained at 4°C for 5 minutes under light shielding. Then, each sample was irradiated with visible light of 500 W for 5 minutes on ice. To each sample was added 750 μl of physiological saline, 200 μl of a solution of lipase 189 units/ml and 10 μl of a solution deoxyribonuclease 1000 units/ml and the sample on which he had laid down at 30°C for 30 minutes. Then the sample was added to 40 ál of proteinase K 1250 units/ml and the sample was maintained at 30°C for 30 minutes. The total amount of the above EMA-treated blood was carefully applied to the 2-ml ficoll-Pak, previously placed in a polypropylene tube with a volume of 15 ml, and subjected to centrifugation at 100×g for 5 minutes. The supernatant was extracted in a volume of 1 ml were subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes and the supernatant was removed. To the residue was added 200 μl of physiological saline and 0.6 µl agent, coloring kernel (SYTO9/PI=1/1), and the mixture was left at room temperature for 15 minutes under light shielding. Then the mixture was subjected to centrifugation at cooling at 4°C and 14000×g for 10 minutes, the supernatant was removed, and then to the residue was added 1 ml of physiological salt solution, and the mixture was subjected to centrifugation at cooling under the same conditions and to the residue was added 200 μl of physiological saline to obtain a sample for FCM measurements.

To obtain a sample for FCM measurements are also performed in parallel procedure (denoted as "LNP-FP method"), similar to the above method (referred to as "EMA-LNP-FP method"), except that not conducted EMA processing.

(2) the results of the test

The result of the test is shown in Fig. 19. In the way EMA-LNP-FP and the way LNP-FP the number of data points that meet the SYTO9(+) (intensity 2×103or above) and PI(-), were proportional to the concentration of inoculatedListeria(living cells), and the detection limit (Listeria monocytogenes(living cells) in the blood was considered to be equal to 1.8×104CFU/ml. Additionally, because the point of the contaminants on the graph corresponding to the SYTO9(-)·PI(-), did not move to the left when the EMA treatment, it was believed that they did not constitute menagerie cells, such as monocytes and leukocytes or lymphocytes, called granulocytes. Because the white cells do not have cell walls, but only the cell membrane, EMA enters them, even if cells are not damaged. Additionally, as the penetrating EMA inhibits re-ligation of chromosomal DNA in cells leukocytes by splitting and re-legirovanii chromosomal DNA under the action of topoisomerase II, chromosomal DNA is repeatedly split, and this state is maintained. Thus, the efficiency of intercalation SYTO9 in the chromosomal DNA is greatly reduced and, as a result, the point on the graph is significantly shifted to the left. However, in this experience the mentioned phenomenon was not observed.

Additionally, because the red blood cells that do not contain chromosomal DNA, including blood cells had the highest density, not what to read, they were containerbase supernatant even after centrifugation at low speed when using ficoll pack, and in fact they did not give points contaminants on the chart if using SYTO9/PI, similar to the point of this example. Moreover, because the sample was treated with lipase, nuclease and proteinase K, lipids and components remaining in the blood in small quantities, also cannot be considered as elements of the contaminants.

From the above, these contaminants are considered as fragments of the splitting of the cell membrane formed during phagocytosis bacteriaListeriapart of red blood cells or hemolysis part of red blood cells. In the present example, living cells can be somewhat detected, even if not conducted EMA treatment. However, the size and complexity of the internal structure managernew cells, such as monocytes and lymphocytes, close to the size and complexity of the internal structure of bacteria, and they can demonstrate the intensity of SYTO9 and PI-like bacteria (living cells). Thus, when the contamination they can be mistakenly identified as living cells. Thus, from the point of view of accuracy more preferably EMA treatment. Additionally, because in addition to living cells, for example, in the blood of patients with sepsis with disabilities f is NCLI liver, there are many damaged cells caused by the action of the antibiotics from the point of view of this aspect is required EMA processing.

1. The method of obtaining the measured sample for detecting live cells of a microorganism in a test sample using flow cytometry, which includes the following stages:
a) processing stage of the test sample with an enzyme selected from lipolytic enzymes and proteases with activity on the destruction of cells different from the cells of the microorganism, colloidal particles, proteins or lipids present in the sample,
b) the processing stage of the test specimen by a topoisomerase inhibitor and/or an inhibitor of DNA gyrase, and
c) processing stage of the test specimen processed in stages a) and b), agent, coloring kernel.

2. The method according to claim 1, wherein the test sample consists of either: milk, milk product, food product, obtained by using milk or milk product as the source material, blood sample, urine sample, the sample of cerebral spinal fluid sample sinovialnoj fluid and a sample of pleural fluid.

3. The method according to claim 1, wherein the microorganism is a bacterium.

4. The method according to claim 1, wherein the topoisomerase inhibitor selected from amsacrine, camptothecin, doxorubicin, is ellipticine, etoposide, mitoxantrone, syntopia, topotecan and CF-115953.

5. The method according to claim 1, wherein the inhibitor of DNA gyrase is selected from ciprofloxacin, ofloxacin, enoxacin, pefloksatsina, fleroxacin, norfloxacin, nalidixic acid, oksolinovoj acid and pyrimidinones acid.

6. The method according to claim 1, wherein the topoisomerase inhibitor is an ethidium monoacid, and the method includes a step of exposure of the test specimen, to which add the ethidium monoacid, the irradiation of visible light.

7. The method according to claim 1, in which the agent, coloring kernel includes a first coloring agent that can penetrate through the cell walls of live cells, injured cells and dead cells, and the second coloring agent, which is more easily penetrate cell walls of dead cells compared with cell walls of living cells and damaged cells, compared with the first coloring agent.

8. The method according to claim 7, in which the agent, coloring kernel consists of propedy iodide and SYTO9.

9. Method of detecting live cells of a microorganism in a test sample using flow cytometry, which includes the following stages:
a) processing stage of the test sample with an enzyme selected from lipolytic enzymes and proteases with activity from the point of view of destruction of cells, different from the cells of the microorganism, colloidal particles, proteins or lipids present in the sample,
b) the processing stage of the test specimen by a topoisomerase inhibitor and/or an inhibitor of DNA gyrase,
c) processing stage of the test specimen processed in stages a) and b), agent, coloring kernel, and
d) stage detection of microorganisms in the test sample treated with the agent, dye core flow cytometry.

10. The method according to claim 9, in which the test sample consists of any milk, milk product, food product, obtained by using milk or milk product as the source material, blood sample, urine sample, the sample of cerebral spinal fluid sample sinovialnoj fluid and a sample of pleural fluid.

11. The method according to claim 9, in which the microorganism is a bacterium.

12. The method according to claim 9, in which the topoisomerase inhibitor selected from amsacrine, camptothecin, doxorubicin, ellipticine, etoposide, mitoxantrone, syntopia, topotecan and CF-115953.

13. The method according to claim 9, in which the inhibitor of DNA gyrase is selected from ciprofloxacin, ofloxacin, enoxacin, pefloksatsina, fleroxacin, norfloxacin, nalidixic acid, oksolinovoj acid and pyrimidinones acid.

14. The method according to claim 9, in which the topoisomerase inhibitor is an ethidium monoazo is, and the method includes a step of exposure of the test specimen, to which add the ethidium monoacid, the irradiation of visible light.

15. The method according to claim 9, in which the agent, coloring kernel includes a first coloring agent that can penetrate through the cell walls of live cells, injured cells and dead cells, and the second coloring agent, which is more easily penetrate cell walls of dead cells compared with cell walls of living cells and damaged cells, compared with the first coloring agent.

16. The method according to item 15, in which the agent, coloring kernel consists of propedy iodide and SYTO9.

17. Set to obtain a measured sample for detection of live cells, injured cells and dead cells of the microorganism in the test sample using flow cytometry, which includes the following elements:
the enzyme is selected from the lipolytic enzymes and proteases, a topoisomerase inhibitor and/or an inhibitor of DNA gyrase and agents, coloring kernel.

18. Set on 17, which is a topoisomerase inhibitor selected from amsacrine, camptothecin, doxorubicin, ellipticine, etoposide, mitoxantrone, syntopia, topotecan and CF-115953.

19. Set on 17, in which the inhibitor of DNA gyrase is selected from ciprofloxacin, ofloxacin, enoxacin, pefloksatsina, fleroxacin, nor is loxacin, nalidixic acid, oksolinovoj acid and pyrimidinones acid.

20. Set on 17, in which the topoisomerase inhibitor is an ethidium monoacid.



 

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