Method for microorganism adhesive potential rapid test |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
IPC classes for russian patent Method for microorganism adhesive potential rapid test (RU 2537128):
 Method of determining substituted 2-methoxyhydroxybenzene in biological material / 2537125
Invention relates to biology and toxicological chemistry and can be used in practice of sanitary and epidemiological stations, chemical-toxicological, forensic and veterinary laboratories. A biological material, containing substituted 2-methoxyhydroxybenzene, is two times (each time for 30 minutes) infused with ethylacetate with mixing, separate extracts are separated from solid particles of the biological material, combined, ethylacetate is evaporated in an air flow at 18-22°C, residue is repeatedly processed with acetone, acetone extracts are separated, combined, dehydrated, evaporated in an air flow at 18-22°C, and then in a nitrogen flow until a solvent is completely removed, residue is dissolved in hexane, extracted with a buffer solution with pH 12-13, a water-alkaline extraction is separated, acidified to pH 2-3, saturated with sodium sulphate, extracted with diethyl ether, the ether extract is separated, dehydrated, evaporated in an air flow at 18-22°C, and then - in a nitrogen flow until the solvent is completely removed, residue is dissolved in a mixture of solvents hexane-dioxane-propanol-2, taken in a ratio of 20:5:1 by volume, chromatographed in macrocolumn with silicagel KSS No 3 80/120 mcm with the application of a mobile phase hexane-dioxane-propanol-2 in a ratio of 20:5:1 by volume, eluate fractions, containing the analysed substance, are combined, the eluent is evaporated first in an air flow at a temperature of 18-22°C, and then in a nitrogen flow until the solvent is completely removed, residue is dissolved in dichloromethane, processed for 20 minutes with N-tert-butyl-dimethylsilyl-N-methyltrifluoroacetamide under conditions of heating at a temperature of 60°C with carrying out determination by a chromatography-mass spectrometry method with the application of a capillary column 25 m long with an internal diameter of 0.2 mm with an immobile phase (5%-phenyl)-methylpolysiloxane, with the application of a mass-selective detector, working in an electron impact mode, an initial temperature of the column thermostat constitutes 70°C, the said temperature is kept for 3 minutes, further the temperature is programmed from 70°C to 290°C at a rate of 20°C per minute, the final column temperature is kept for 10 minutes, the injector temperature constitutes 250°C, the quadrupole temperature is 150°C, the temperature of a ion source is 230°C, the temperature of the detector interface is 300°C, intensity of a signal, conditioned by charged particles, formed in the bombardment of the analysed substance, leaving the capillary column and getting into the ion source, with a ionising beam of electrons with the energy of 70 eV, is registered, the mass-spectrum by the complete ion flow is registered and an amount of substituted 2-methoxyhydroxybenzene is calculated by the area of a chromatographic peak of its trimethylsilyl derivative.
 Method of determining n-(4-nitro-2-phenoxyphenyl)-methanesulphonamide in biological material / 2537121
Invention relates to biology and toxicological chemistry and can be used in chemical-toxicology, expert forensic and clinical laboratories. The method includes: crushing a biological object containing N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide, settling twice for 45 minutes with portions of an organic isolating agent which is methyl acetate, combining the obtained extracts, evaporating the solvent from the resultant extract, treating the residue with acetone, separating the acetone extract, evaporating the solvent from resultant extract, dissolving the residue in diethyl ether, extracting the ether solution with a buffer solution at pH 9-10, acidifying the aqueous alkaline extract with 24% hydrochloric acid to pH 2-3, saturating the obtained solution with sodium bromide, extracting with ethyl acetate, evaporating the obtained extract in an air current at 20-22°C until a dry residue is obtained, dissolving the residue in a mixture of hexane and acetone taken in volume ratio of 8:2, performing chromatography on a macrocolumn with silica gel L 40/100 mcm using a hexane-acetone mobile phase in volume ratio of 8:2, combining eluate fractions containing the analysed substance, evaporating the eluent in an air current at 20-22°C until complete removal of the solvent, dissolving the residue in methanol and performing determination via a combined physical-chemical method in the form of chromatography-mass spectrometry, using a DB-5 MS EVIDEX capillary column with a mobile phase which is 5% phenyl-95% methylpolysiloxane, using a mass-selective detector operating in electron impact mode, the initial thermostat temperature of the column is 70°C, maintaining said temperature for 3 minutes, further raising the temperature from 70°C to 290°C at a rate of 20°C per minute, maintaining the final temperature of the column for 16 minutes, the temperature of the injector is 250°C, the temperature of the quadrupole is 150°C, the temperature of the detector interface is 300°C, detecting strength of the signal resulting from charged particles formed when bombarding the analysed substance coming from the capillary column and falling into an ion source with an ionising electron beam with energy of 70 eV, recording the mass spectrum on the full ion current, while calculating the amount of N-(4-nitro-2-phenoxyphenyl)-methanesulphonamide from the area of the chromatographic peak.
 Method of obtaining standard quality sample of saliva facia for crystallography / 2536950
Invention relates to medicine, namely to clinical, laboratory diagnostics, microbiological methods of research, and is aimed at standardisation of saliva analysis by method of wedge dehydration/crystallography. Claimed is method of obtaining standard, quality sample of mixed saliva facia for crystallography with application of portable laboratory device with inbuilt levels on axes X and Y, legs, regulated by height and isolating cover. From 20 to 40 microscope slides are simultaneously placed on the surface of portable laboratory device after obtaining smooth horizontal without inclination angle surface. After that, one sample of mixed saliva in amount 0.02 ml, preliminarily centrifuged for 20 min at 3000 rev/min, is applied on each microscope slide by means of micropipette. As a result a round 1.0 mm high drop of mixed saliva with diameter from 4.0 to 5.0 mm, corresponding to standard parameters, is obtained. Then, standard in dimensions drop on microscope slide placed on the surface of portable laboratory device, adjusted by levels, is dried at room temperature +18…+25°C under isolating cover for 5 hours, with further performance of microscopy of standard quality sample of saliva facia.
 Method for measuring maximum allowable blood concentration of heavy metals in children after integrated exposure / 2536268
Invention aims at asserting the maximum allowable blood concentrations (MAC) of heavy metals in the children living in the dirty environment as shown by health risk criteria after the chronic integrated exposure. An environmentally neglected zone is selected; a representative sampling of the children for the examination is drawn that is a basic group with using biological, social and hygienic criteria; the same criteria are used to draw a representative sampling of the children to a reference group living in the environmentally friendly zone. In the territory of the above zones, the chronic exposure of the analysed heavy metal is qualitatively assessed by establishing its average daily concentration in the ambient environment; the derived value is used to calculate a total average daily doses of a heavy metal supplied from various sources into a child's body averaged over the annual exposure for the children of both groups. Blood is sampled from the children every three months for one year to determine the content of the analysed heavy metal and also to measure the biochemical values of blood plasma and serum characterizing body responses presented by actual or potential health problems that are response markers. That is followed by calculating the average blood concentration of the analysed heavy metal and comparing it to the reference for the same heavy metal with using a Student two-sample test, thereby stating whether the children were sampled from the main and reference groups adequately. A mathematical modelling procedure is used to establish a relation between the exposure that is the total average daily doses of the analysed metal, and the exposure marker that is the average blood metal concentration. A sliding window technology is used to assert the response markers selected. The maximum allowable concentration of the exposure marker and respective marker is determined by a technique based on ratio analysis.
Instant diagnostic technique for acute intestinal infections / 2536243
Invention represents an instant diagnostic technique for acute intestinal infections (AIIs), involving detecting indication markers of the AII aetiology with the use of laboratory immunology tests, differing by the fact that the AII aetiology is stated in children of an early age category, preferentially in the newborn children; that is accompanied by measuring the concentration of cytokine, interleukin IL-10 in coprofiltrate and diagnosing chronic placental insufficiency (CPI); a probability (P) of the bacterial AII aetiology is calculated; the value P of more than 50% testifies to the bacterial AII aetiology, while the value P being less than 50% shows the absence of the bacterial AII aetiology, and enables considering the diagnostics second stage to be necessary, which implies measuring the concentration of cytokine, interleukin IL-4 in coprofiltrate; the time of latching the newborn child to the breast is established with considering the type of feeding; that is combined with calculating a probability (P) of the viral or viral-bacterial AII aetiology, with the value P of more than 50% testifying to the viral AII aetiology, while the value being less than 50% makes it possible to state the viral-bacterial AII aetiology.
 Molecular diagnostic technique for genetic risk of complicated urogenital chlamydial infection causing reproductive dysfunction in individual / 2535724
Polymerase chain reaction method is used to recognise polymorphous variants of IL6 and TGFb1 genes. Recognising the homozygous genotype CC in -174 position of IL6 gene in males and females, as well as the heterozygous genotype GC in -915 position of TGFb1 gene in females enables predicting the high risk of the complicated clinical course of the urogenital Chlamydial infection.
Diagnostic technique for undifferentiated connective tissue dysplasia in females with personal history of miscarriage / 2535071
Invention concerns diagnosing undifferentiated connective tissue dysplasia (UCTD) in females with a personal history of miscarriage. The technique involves determining the fibrinolytic activity in an endometrial biopsy sample. If the value is less than 23.55 mm2, undifferentiated connective tissue dysplasia is diagnosed.
 Oral diagnostic instrument / 2534519
Instrument comprises an oral sample collection vessel, a detector able to detect a marker in this sample, an indicator actuated by a detector signal. The above vessel is detachably connected to an oral cavity instrument. The vessel comprises a sample collection element, a sample storage container, and a passage connecting the collection element and the container to supply the sample to the container by capillary action. The indicator is integrated into the container. The declared instrument is used to diagnose oral diseases by collecting the oral sample, detecting one or more markers in this sample and indicating the presence of one of the disease markers.
 Prenosological diagnostic technique for sportsmen health / 2534403
Technique involves the clinical-laboratory examination of a sportsman who completed heavy physical activity 12-16 hours ago. The examination extent is determined taking into account the organs and systems most vulnerable to the physical activity while deriving the prognostically significant criteria of the morphofunctional body state. The examination involves measuring and analyzing the biochemical, haematological, immunological and functional values, as well as vitamin-mineral saturation. And if the above values are stably unchanged, reliably different from the norm, nonspecific changes of the sportsman's organs and systems are diagnosed.
 Method of predicting efficiency of treatment of patients with high grade non-hodgkin lymphoma / 2533816
Efficiency of treatment of patients with high grade non-Hodgkin malignant lymphoma is determined by the likelihood of achieving remission, and 5-year total and relapse-free survival. The method comprises the study of polymorphism G13494A 6th intron of gene TP53 of the patient. In case of revealing in the patient of homozygous genotype G/G in a given locus the low efficiency of treatment is predicted, namely, low likelihood of 5-year survival of the patient and low likelihood of absence of relapse. In the case of revealing in the patient of the genotype A/A or G/A in a given locus, the high efficiency of treatment is predicted, namely the high likelihood of remission and 5-year survival of patient.
Vibrio aquamarinus strain, method of determining sample toxicity using same and testing culture for determining sample toxicity / 2534819
Group of inventions relates to biotechnology and can be used for biotesting of toxicity of environmental media. The invention discloses a Vibrio aquamarinus bacterial strain, a method of determining toxicity of samples using said strain and use of the strain as a testing culture for determining chemical toxicity of samples. The method comprises measuring luminescence of the Vibrio aquamarinus VKPM V-11245 strain in the presence of at least one chemical toxicant of a sample, said toxicant being ZnSO4 or CuSO4 or K2Cr2O7 or oil or diesel fuel or bilge water or phenol or a heavy metal. Luminescence of said strain is measured without a chemical toxicant and the measured luminescence levels are compared. Change in luminescence indicates toxicity of the analysed sample.
Method of quality control of disinfection of poultry premises / 2534356
Invention relates to the field of quality control of disinfection. The method comprises disinfection of premises, selection of test objects which are used as a suspension Bacillus subtilis of the strain ATCC PTA-6737 (PB6) sorbed on the carrier - the filter paper with an adhesive surface part. The test objects are placed in the premises of disinfection and added to the nutrient medium. The tubes are heated to 60-80°C and cultured at 37°C for 12-24 hours followed recording the result by recording the growth of bacteria after 12 hours. Clouding of the nutrient medium to form a mucous film is indicative of inadequate quality of disinfection, absence of clouding and formation of a film after 24 hours is indicative of the satisfactory disinfection.
Method for identifying staphylococcal exotoxin genes with high level of structural homology with superantigens by multiplex polymerase chain reaction and test system for implementing it / 2532225
Invention refers to biotechnology and microbiology and represents a method for identifying a cluster of antigens coding staphylococcal proteins that are exotoxins. The present method is implemented by performing a single multiple-primer polymerase chain reaction in two reaction mixtures, first of which contains primers to genes coding such staphylococcal proteins, as thermonuclease, beta-glucosidase in the species S.aureus, S.epidermidis, S.haemolyticus, S.lugdunensis, S.saprophyticus, and the second one - to set1, set2, set3, set4, set5 genes coding the exotoxins. That is followed by a comparative analysis of amplified gene fragments prepared in two sample aliquots and a positive control reference according to the identification table. The present invention also discloses a test system for implementing the above method, which contains DNA recovery components, PCR components, and result analysis components. The PCR components contain a 10-merous buffer solution, pH 8.4, deionised sterile water, one positive control reference, Taq polymerase, mixture of four dNTPs, mixture of primers No.1 containing epi-F, epi-R, aur-F, aur-R, hae-F, hae-R, lug-F, lug-R, sap-F, sap-R in a ratio of 1:1:1:1:1:1:1:1:1:1, and a mixture of primers No. 2 containing set1-F, set1-R, set2-F, set2-R, set3-F, set3-R, set4-F, set4-R, set5-F, set5-R in a ratio of 1:1:1:1:1:1:1:1:1:1.
 Method of generating starter culture, starter culture and fermentation method with its application / 2531343
Claimed solutions deal with a method of generating a starter culture, the starter culture and a fermentation method with an application of the said starter culture. The claimed method of generating the starter culture includes impact on a parent bacterial strain, which contains, at least, a part of the locus CRISPR, with a bacteriophage to obtain a mixture of bacteria, which contains a bacteriophage-resistant variant strain, containing the modified locus CRISPR, which contains, at least, one additional spacer in the said modified locus CRISPR; independent impact on the same parent bacterial strain with the same bacteriophage to obtain the mixture of bacteria, which contains other bacteriophage-resistant variant strain, containing the modified locus CRISPR, which contains, at least, one additional spacer in the said modified locus CRISPR, different from the additional spacer in the first bacteriophage-resistant variant strain, selection of the said bacteriophage-resistant variant strains from the mixtures of bacteria and their separation.
Method of increasing sensitivity of microorganisms to antimicrobial agents / 2529367
Two suspensions are prepared. Clinical polyantibiotic-resistant strains Escherichia coli are added to an isotonic solution of NaCl to achieve the concentration of 30-40 thousand CFU/ml. Copper nanoparticles are added to the solution of NaCl to achieve the concentration of 0.01-0.05 mg/ml. The suspension of ethylenediaminetetraacetic acid - EDTA is prepared by its dilution in distilled water at the rate of 0.1-0.2:1, respectively. NaOH is added to the prepared suspension to obtain the solution of EDTA with pH=7.6-8. The prepared suspensions are connected with the solution of EDTA in the following ratios by wt %: suspension of copper nanoparticles - 70-85, suspension of microorganisms - 10-20, EDTA - 5.10. It is incubated in the shaker at 100-150 rev/min and a temperature of 36-38°C for 40-60 minutes. The resulting biomass is inoculated on the solid nutrient medium with the volume of 20-25 ml in the amount of 0.1-0.12 ml. It is incubated in the thermostat at a temperature of 36-38°C for 18-24 hours. The sensitivity of E.coli strains to antibiotics is determined.
 Method of specific differentiation of viable rhodococcus immobilised in gel carrier / 2525934
Granule of biocatalyst with the cells contained in it of one or more species of Rhodococcus is placed in a well of a 96-well plate, a colorant iodine-nitro-tetrazolium (INT) is added. After the appearance of specific purple staining formazan the optical density of granule is measured using a tablet photometer. In the case of a stained substrate or preliminary presence of the immobilised biocatalyst in the soil the extraction of formazan is carried out with 96% ethanol and the optical density of the extract is measured. The amount of viable Rhodococcus in the granule of the gel is determined using the calibration dependency diagram of the optical density on the number of living cells in suspension as determined by traditional method of plating on solid growth medium. Then, from the granule stained by the colorant of the biocatalyst or from the granule after extraction of the colorant the DNA is isolated and PCR is performed using sets of species-specific primers. The specific position of the immobilised Rhodococcus is determined.
Method of evaluating detoxification activity of black soils in farming ecosystems / 2525677
Invention relates to biotechnology. The method comprises collecting soil samples, adding metsulfuron-methyl (MSM) to the sample in a given amount, followed by incubation and determining specific metabolic activity of the saprophytic soil complex by multisubstrate testing and counting the number of colony-forming units on a diluted agarised medium. The level of the detoxification activity is estimated based on the relative increase in specific metabolic activity of the saprophytic microbial community of the soil when MSM in comparison with characteristics of the soil without adding MSM (Kd factor).
 Method of growing colonies of microbial cells and device for its realisation / 2522005
Group of inventions relates to biotechnology. Claimed is a method of growing colonies of microbial cells on a surface of a porous plate. The method includes supply of a nutrient solution from bottom to top through the porous plate into zones of growth of colonies of the microbial cells on its upper surface, supply of a suspension of the microbial cells onto the upper surface of the porous plate, creation of controlled conditions for the colony growth, performing observation of the colony growth, separation of the grown colonies of the microbial cells from the zones of growth and their transfer into external means of identification. The nutrient solution is supplied into the zones of growth of the colonies of the microbial cells by creation of a pressure difference between the hole input and output. Holes are made in the plate from an anode aluminium oxide orthogonally to its large plane and are topologically coded. The said zones of growth are formed in them in the form of porous membranes. The porous membranes are located at the same level as the upper surface of the plate or with formation of a hollow and do not pass the microbial cells. After supply of the nutritional solution, the suspension of the microbial cells of a specified concentration is supplied onto the upper surface of the plate until their homogenous distribution is achieved. Between the zones of growth on the surface of the plate a film, preventing attachment of the microbial cells, is formed. Separation of the grown microcolonies from the zones of growth is performed by hydroblow. A hydroblow is directed from the side of the input of cylindrical holes of the plate and spreads along them and farther through the pores of the porous membranes with force, which does not destroy the microcolonies but is sufficient for their separation from the growth zones. Also claimed is a device for growing the colonies of the microbial cells by the claimed method.
 Method of counting oil-oxidising bacteria in sea water / 2520084
Invention relates to microbiology and can be used in monitoring environmental-microbiological investigation of the quality of sea water to determine the amount of oil-oxidising microorganisms. The method involves preparing a mineral medium - bases containing NH4NO3, K2HPO4, KH2PO4, MgSO4, CaCl2, FeCl2, a concentrated solution, agar and distilled water in a given ratio, followed by addition of an oil product in a given amount, said product being bunker oil. Seeding sea water on the surface of the culture medium and incubating the seed for 3-4 hours enables to detect colonies of oil-oxidising bacteria.
 Method of toxicity assessment of products from polymer and textile materials / 2518306
Method of toxicity assessment of products from polymer and textile materials is proposed. The method comprises the use of biosensor based on oxygen electrode, immobilisation of whole cells of bacteria E.coli K-12 on the surface of the oxygen electrode. The immobilisation is carried out using a semipermeable membrane. After immobilisation the respiratory activity of microorganisms is measured in the presence of the sample and standard samples of positive and negative control. Then the toxicity index is calculated and the sample toxicity is evaluated based on the value of the toxicity index.
 Method for biotesting water and aqueous extract samples / 2245367
Invention is designated for biotesting water and aqueous extract samples. Daphnia are immersed firstly in solution or water for culturing to be tested and then daphnia are transferred into lightproof chamber with outlet hole for culturing and this chamber is immersed into water. Time passing out daphnia from the testing chamber to light is measured and toxicity of analyzed solution is estimated by difference time in daphnia passing out. Method allows carrying out the rapid control of water and aqueous extracts toxicity in laboratory and field conditions.
|
FIELD: medicine. SUBSTANCE: method involves the preliminary sorption of a specific ligand in polystyrene tray wells; the above ligand is specified in: haemoglobin, myoglobin, collagen, fibrinogen, fibronectin, immunoglobulin A; that is followed by adding a microorganism cell suspension into the tray wells with the sorbed ligand, incubating the cell suspension in the tray wells for 15 minutes, sampling a suspension aliquot from the well and adding it to wells of another or the same tray containing 0.5% sodium chloride or 0.2 M sodium phosphate; the bacterial cell adhesion is assessed by measuring a decrease of the optical density of the prepared diluted suspensions at wave length 600 nm as compared to the well references free from ligands. EFFECT: invention is characterised by a high test rate of the microorganism adhesion to ligands of various nature, high reproducibility, using a minimum amount of the microorganism biomass, with no need for hazardous chemicals to be used. 7 tbl, 7 ex
The invention relates to Microbiology and can be used in medical bacteriology and medical Mycology to quantify the adhesive properties of conditionally pathogenic microorganisms isolated from human and environmental objects, to characterize their virulent properties in vitro the level of adhesion to ligands of different nature. There are different approaches to quantify the level of adhesion of cells of microorganisms (bacteria and yeast-like fungi) to ligands of different nature. There is a method of quantitative determination of cell adhesion of microorganisms to collagen and fibronectin, based on the specific staining of adhered cells dye crystal violet, however, such method requires deficitii cells with glutaraldehyde, the use of additional procedures for cell disruption using the detergent Triton X-100 [1]. A simpler approach is to use membranes that enclose the appropriate ligand (e.g., hemoglobin) [2]. In this case, the cells of the microorganisms associated with membrane carrier ligands, using specific adhesins, located on the surface of their cell walls. Quantitative adhesion level in this case is determined by counting the number of glue is OK microorganisms, adhered on the membrane with ligands, or by reducing the optical density of a suspension of cells of the microorganism after interaction with the membrane. However, this approach requires waste approach in the manufacture of membranes using specific components of nitrocellulose, organic solvents. In practice requires the use of relatively large amounts of membranes (to achieve a large area of the working surface of several square centimeters) and large volume (several milliliters) of a suspension of microorganisms, which is not always possible to use this method with a small number of biomaterial, for example in the presence of one small colonies of microorganisms growing on solid (agar) nutrient medium. The closest to our invention include a method for determining the adhesion of microorganisms to the ligands adsorbed on the polystyrene surface of a 96-well plate [3]. This approach allows to avoid the use of different, including unsafe substances, as well as to bring the work to the use of micro volumes (about 100 µl), do not require a large amount of source material. However, the described invention has a number of disadvantages. First, to quantify the adhesion of cells of microorganisms are offered either the adding in of the wells (containing the adhered cells) liquid nutrient medium, either selection aliquots of a suspension of microorganisms from wells in the liquid (or culture on solid nutrient medium followed by incubation and growth assessment (counting the resulting colonies) culture of microorganisms, which allows to obtain results only after a significant amount of time (from 6 to 48 h depending on the incubation conditions and the type of microorganism). In addition, in this case, do not take into account the possibility of differences in growth rate among different strains of the same species of microorganism that can artificially increase or dilute the real difference in adhesion potential of specific strains. Secondly, the use of quantitative assessment of adhered cells of microorganisms for determining the activity of bacterial enzymes in adhered cells is practically unusable due to the variation of the activity (number) enzymes in different strains of the same species of microorganism [4] and, consequently, the lack of absolute correlation between the level of enzyme activity and the level of adhesion potential. This approach can also increase or dilute the real difference in adhesion potential of the compared strains. In this regard, the objective of the claimed invention is to develop a simple way to quickly, less h is m for 1 hour, using the minimum amount of biomass of microorganisms in microvolumes without the use of chemical reagents to quantify the level of adhesion of cells of microorganisms (bacteria and yeast-like fungi) to ligands of different nature. This object is achieved through the development of a detection method, which involves a preliminary sorbirovaniya in the wells of polystyrene tablet specific ligand, is added to wells of a suspension of cells of microorganisms, a short incubation of the cell suspension in the wells, the subsequent selection of an aliquot of the suspension from the wells and add it to the wells of another or of the same tablet, containing an aqueous solution of salt (for example, 0.5% sodium chloride or 0.2 M of sodium phosphate), followed by assessment of the level of adhesion of bacterial cells by determining the decrease of optical density obtained with the diluted suspension at a wavelength of 600 nm. On the basis of data obtained from optical density to determine the index of adhesion. The adhesion index (in percent) is calculated by the formula: IA=100-100×(OP600experience/OP600control) where OP600experience the value of optical density of the suspension at 600 nm, selected from the wells containing ligand; OP600control - the value of optical density is spencie at 600 nm, selected from the wells not containing ligand. The higher the index of adhesion, the higher the level of adhesion of the microorganism to the selected ligand. The technical result of the claimed invention is to develop a rapid method for determining the level of adhesion of microorganisms to ligands of different nature, with good reproducibility, a minimum quantity of biomass of microorganisms, using the minimum amount of biomass of microorganisms in microvolumes, there is no need to use dangerous chemicals. Example 1. The determination of the level of adhesion of Staphylococcus aureus to the hemoglobin. In wells pre-Sorb ligand - human hemoglobin. For this hemoglobin is dissolved in 0.2 M sodium phosphate buffer solution with pH 7.4 rate of 20 μg/ml of the Prepared solution in a volume of 50 μl contribute to the wells, incubated for 24 h at 4°C. the wells washed three times with 0.2 M sodium phosphate buffer solution, pH 7.4, containing 0.05% tween-20, filling 100 ál in each well, then the tablet drained by shaking out the remaining liquid. The thus prepared tablets can be stored for years at 4°C in dry conditions. In the finished wells made in 200 μl of a suspension of Staphylococcus aureus in 0.2 M sodium-fo is Putnam buffer solution at pH 7.4 with an optical density at 600 nm in the range from 0,500 to 0,600. Incubare for 15 minutes, then extracted with 100 μl of the suspension of the pit and move it to another well containing 100 μl of 0.2 M sodium phosphate buffer solution with pH 7.4, stir the diluted suspension and then measure the optical density of the obtained diluted suspensions at 600 nm. The control options are wells that do not contain hemoglobin. The results are presented in table 1. Example 2. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use the myoglobin. The results are presented in table 2. Example 3. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use collagen. The results are presented in table 3. Example 4. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use fibronectin. The results are presented in table 4. Example 5. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use the fibrinogen. The results are presented in table 5. Example 6. Determination of the level is desii strains of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, using immunoglobulin A (IgA). The results are presented in table 6. Example 7. Determination of the adhesion level is carried out analogously to example 1, but as the microorganism used yeast-like fungus Candida albicans. The results are presented in table 7. 0,269
LITERATURE 1. Pynnonen M., Stephenson, R. E., Schwartz K., M. Hemandez, B. R. Boles / Hemoglobin promotes Staphylococcus aureus nasal colonization // PLoS Pathogens, 2011. 2. Lisovskaya S. A. / the dissertation on competition of a scientific degree of candidate of biological Sciences "a New approach to the assessment of the pathogenic potential of clinical strains of Candida albicans" // Kazan, 2008. 25 C. 3. Tyurin, Y. A., pasahow R. S., Mustafin, I. / the Method to determine the adhesion of Staphylococcus spp.to hemoprotein the m // Patent RU No. 2393229. 4. Bayazitova L. T., Tyurin, Y. A., Kulikov S. N., Dolbin D. A., pasahow R. S. / study of the antibacterial activity of the drug, "Skin cap" against Staphylococcus aureus when local therapy of atopic dermatitis // Practical Medicine, 2009, №3 (35), S. 89-91. The method for determining the adhesive potential of microorganisms, characterized in that in the wells of polystyrene tablet absorb specific ligand selected from the range: hemoglobin, myoglobin, collagen, fibrinogen, fibronectin, immunoglobulin And add in wells with sorbed ligand cell suspension of the microorganism, spend incubation of the cell suspension in the wells for 15 minutes, take an aliquot of the suspension from the wells and add it to the wells of another or the same tablet containing an aqueous salt solution of 0.5% sodium chloride or 0.2 M of sodium phosphate and assess the level of adhesion of bacterial cells by determining the decrease of optical density obtained with the diluted suspension at a wavelength of 600 nm when compared with the control variant holes, not containing ligand.
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||