Method of phagocyte activation test

FIELD: medicine.

SUBSTANCE: method provides phagocyte recovery from a cell mixture. The recovered cells are mixed with medium 199 in 18 bottles, and the cells are attached to glass at room temperature for 60 minutes. In 9 bottles, a culture medium containing medium 199, L-glutamine and mixed human serum heated for 30 min at 52°C is added to the cells in the preset amounts to produce cell samples. A culture medium of said composition containing 0.01% of zymosan particles is added to another 9 bottles with the cells to produce the cell samples. The prepared cell samples are divided into three portions. One one-third potion of zymosan and zymosan-free samples are frozen. The second portion of the zymosan and zymosan-free cell samples are cultivated at temperature 37°C for 4 to 6 h to be frozen, and the third portion of the zymosan and zymosan-free samples are cultivated at temperature 37°C for 18 to 24 h to be frozen respectively. All 18 bottles are exposed to simultaneous multiple freezing to -10°C and thawing at room temperature to complete recovery of intracellular lysozyme with determination of amount by a micromethod and calculation of synthesised lysozyme and a phagocyte activation value (PAV) by formula: PAV=Z Lsynth - Z-free Lsynth, mcg/ml, where Z Lsynth is a difference of lysozyme amount in the cultivated zymosan cells and lysozyme amount in the uncultivated zymosan cells, mcg/ml; Z-free Lsynth is a difference of lysozyme amount in the cultivated zymosan-free cells and lysozyme amount in the uncultivated zymosan-free cells, mcg/ml; the phagocyte activation value for long-term activation mechanism assessment is calculated by said formula by results of cell cultivation for 4 to 6 hours, and the phagocyte activation value for long-term activation mechanism assessment is calculated by said formula by results of cell cultivation for 18 to 24 hours.

EFFECT: invention allows more precise phagocyte activation assessment.

4 tbl, 2 ex

 

The invention relates to the field of biology, medicine, namely cell biology, immunology, and can be used to estimate the initial state faguoqitirute cells (neutrophils, monocytes, macrophages) in the ability to activate in normal and pathological conditions.

Known methods for determining the activation faguoqitirute cells (phagocytes) to change the metabolic shift in the cells in the absorption phagocyting object involving enzymatic and non-enzymatic systems - lysosomal cationic proteins (Kozhemyakin L.A. and other Evaluation of the immune status of the organism in hospitals SA Navy. Methodological manual / Loukoumades, Amorous, Vgoranov, Wahawisal, Iggnorance. - Leningrad. - 1987. - 62 S.; Khaitov R.M P.M., Pinegin B.V. Assessment of the main stages of the phagocytic process: current approaches and research perspectives // Immunology. - 1995. No. 4. - P.3-9). Widely used are test recovery narasinga of tetrazole (NBT-test) and lysosome-cation test based on the study, respectively oxybiotic and kislorodozavisimogo activation mechanism faguoqitirute cells (neutrophils, monocytes, macrophages).

Known methods involve the steps of allocating faguoqitirute cells, their interaction with phagocytophilum objects (zymosan, bacteria), differential histochem the ical coloring, the accounting result and the calculation of indicators of activation.

Lysosome-cation test (LKT)characterizing the degree of activity kislorodozawisimam microbicide systems phagocyte is in the manufacture of a blood smear, bone marrow, imprint of inflamed tissue fat-free sterile slide, drying in the air. Smears, fingerprints fixed in a mixture of formalin with ethyl alcohol (immersed drug for 15-20 seconds in a mixture of 1 part formalin and 19 parts of 96% alcohol). Stained for 20 min in buffered ethanol solution of fast green with pH 8.1-8.2. Stained smears immediately rinsed with distilled water, Domracheva for 15-20 sec 0.25% aqueous solution of Azur, and washed with distilled water, dried, take into account the results of microscopy. While looking at 100 granulocytes (neutrophils). The intracellular content of cationic proteins in the blood is measured semi-quantitatively by the average cytochemical coefficient (CCS), calculated by the formula:

,

where a-e - a number of identical cells with a certain degree of okrashivaemoy cytoplasm solid green, and the numbers indicate the severity and the intensity of staining;

0 - no staining of cytoplasmic and lysosomal granules;

0.5 to the presence in the cytoplasm of single who's colored granules;

1 - half of the cytoplasm is filled with light-colored granules;

1,5 - cytoplasm uniformly filled with granules, painted in light green color (meet single dark green granules);

2 - cytoplasm contains 1/3 dark green pellets;

3 is contained in 2/3 cytoplasm.

In normal adult human CCS is equal to 1,5-1,7, children - about 1.9.

In lysosome-cation test semiquantitatively assessed the status of microbicide system phagocyte defining kislorodozawisimae change their activity in lysosomal cationic proteins under the influence of microorganisms (bacteria).

As the prototype was taken closest way of learning activation faguoqitirute cells - nst-test proposed Meiksin and Animalscom in 1979, the advanced and widely implemented in practical health (Kozhemyakin L.A. and other Evaluation of the immune status of the organism in hospitals SA Navy. Methodological manual / Loukoumades, Amorous, Vgoranov, Wahawisal, Iggnorance. - Leningrad. - 1987. - 62 C.). It is based on the evaluation of activation on respiratory or respiratory burst by increasing consumption O2in the reaction catalyzed by nikotinamidadenindinukleotida ·H-oxidase (NADP·H-oxidase).

For its implementation in two agglutination test tubes contribute to 0.05 ml to perarnau blood, then one of them added to 0.025 ml of isotonic phosphate buffer, in other 0,025 ml suspension zymosan. After that the tubes make a 0,025 ml of 0.15% solution of HCT (solution narasinga of tetrazole in 0.1 M isotonic phosphate buffer with a pH of 7.2). The contents of the tube gently mixed and incubated in a water bath at 37°C for 30 min, stirring every 10 minutes After incubation the contents of the tubes are mixed, put one drop on thoroughly cleaned and skimmed mixture Nikiforova slides, make smears and dried in air. Ready smears fixed with methanol for 10 min, dried, stained with 2% aqueous solution of methyl green for 20 min, washed, dried and mikroskopiruyut. Each brushstroke count 100 neutrophils, among which the percentage of cells containing deposits of deformazione (NBT-positive neutrophils). Next, calculate the index of the activation of neutrophils (IAN) according to the formula:

,

where a is the number of cells that do not contain deformographic deposits or contain them in the form of dust a few inclusions;

In the number of cells, in which the area of sediments deformazione does not exceed 1/3 of the surface area of the core;

With the number of cells in which these deposits occupy 1/3 to the total value of the square of the kernel;

D - the number of CL is current with deformative sediments, area exceeding the size of the kernel.

The magnitude IAN ranges from 0.1 to 0.15 in basal conditions and 0.5 to 1.5 during stimulation of neutrophils.

Known methods involve the determination of the activation faguoqitirute cells to change metabolic shift in the cells in the absorption phagocyting object with the participation of the enzyme systems (nikotinamidadenindinukleotida·H-oxidase - NADP·H-oxidase) and non-enzymatic - lysosomal cationic proteins based on the study, respectively oxybiotic (NBT-test) and kislorodozavisimogo (lysosome-cation test) mechanism of activation faguoqitirute cells (neutrophils, monocytes, macrophages).

Known methods allow us to estimate only the urgent mechanism of activation faguoqitirute cells while absorbing object. Estimated activation mainly of neutrophils due to the fact that monocytes and macrophages, this mechanism is 3 times less pronounced (Roos D., Reiss, M., Balm, A.I. EA. A metabolic comparison between human blood monocytes and neutrophils. - In: Macrophages and lymphocytes. New-York, 1980, p.29-36). Both methods are very time-consuming, are semi-quantitative, are used to study urgent mechanism of activation mainly of neutrophils, one of the populations faguoqitirute cells and limited monocytes and macrophages.

The task of the invention is to develop an effective method of quantifying the evaluation of the activation faguoqitirute cells and to extend the possibility of evaluation by examining not only urgent, but long-term activation of both neutrophils - a population of phagocytes and monocytes (macrophages blood) in healthy and sick people.

Technical result - increase the accuracy of the estimated activation faguoqitirute cells by using as a criterion the activation quantitative evaluation of cellular synthesis of lysozyme in time and when you activate cells simhasanam, as well as expanding the capabilities of the method of estimating the activation faguoqitirute cells by studying the immediate and long-term mechanism of activation of both neutrophils and monocytes (macrophages blood).

This technical result is achieved in that in the method of determining activation faguoqitirute cells, including the selection of cells, the interaction with simhasanam, assessing the activation of the enzymatic activity of cells as a criterion activation faguoqitirute cells using the increase of synthesis of cell lysozyme; where to selected cells, neutrophils or monocytes add medium 199 with 0.5% L-glutamine and 2.5% mixed human serum, heated for 30 min at 52°C, containing 0.01% of the particles zymosan (Z) and not containing particles of zymosan is equal to the number of cell samples, then all of the above cell sample is divided into three equal parts, one third part of them with simhasanam and without zymosan freeze, two droguerie part of the cell samples equally with simhasanam and without zymosan cultivated at 37°C, moreover, one third of the sample within 4 to 6 hours and freeze, and another 18 to 24 hours, then all cell samples sequentially and repeatedly (three-to fourfold) freeze at temperatures up to -10°C and thawed at room temperature for a full selection of intracellular lysozyme, determines the amount of lysozyme (L) micromethod, then expect synthesized lysozyme (L Sint.) and carry out the calculation of the indicator activation of phagocytes (PAF) by the formula:

PAF=L Z - L without Z,

where L with Z (L synthesized when exposed to zymosan (Z)) is the difference in the number of lysozyme in cultured cells with particles zymosan and the amount of lysozyme in rekultivirovannyh cells with particles zymosan determined after 3-4 times of freezing and thawing, in µg/ml;

L without Z (L without zymosan) - the difference in the number of lysozyme in cultured cells without particles zymosan and the amount of lysozyme in rekultivirovannyh cells without particles zymosan determined after 3-4 times of freezing and thawing, in µg/ml;

when this indicator activation of phagocytes to assess urgent mechanism of activation calculated from the formula according to the results of culturing the cells for 4 to 6 hours, and the activation of phagocytes to assess long-term mechanism of an asset is the AI calculated from the formula according to the results of culturing cells within 18 to 24 hours.

These culturing conditions do not violate the cell viability, which was verified experimentally.

The proposed method unlike the prototype has the following advantages:

1. The use of quantitative evaluation of the difference in the cellular synthesis of lysozyme (L), which is a constitutive enzyme to the lysosomes faguoqitirute cells - neutrophils, monocytes and macrophages before and after 4-6 hours (urgent mechanism) and 18-24 hours (long-term mechanism) cultivation faguoqitirute blood cells in culture medium (medium 199 with 0.5% L-glutamine and 2.5% mixed human serum, heated for 30 min at 52°C) without particles zymosan and 0.01% of the particles zymosan (Z)

2. As the activation criterion is proposed to use the lysosomal enzyme lysozyme: estimated increase in its intracellular amounts in faguoqitirute cells due to the ongoing strengthening of synthesis when activated under the influence of particles zymosan (Sharonov AS Phagocytes, complementary mechanism, membrane. - Vladivostok (in Russian) Dalnauka. - 2007. - 127 S.). Lysozyme is a marker enzyme of lysosomes and lysosomal membranes along with acid phosphatase and cathepsin D (AA Pokrovsky, tutelian V.A. complementary mechanism. - M.: Nauka, 1976. - 382 C.). Lysozyme most actively synthesized faguoqitirute cells and is part of both primary and secondary l is the AIA (Freidlin I.S. The system of mononuclear phagocytes. - M.: Medicine, 1984. - 272 S.; Roit A. fundamentals of immunology. - M.: Mir, 1991. - 327 C.). Lysozyme has a wide range of regulatory and effector actions, which is an important manifestation of activated phagocytes (Sharonov AS Phagocytes, complementary mechanism, membrane. - Vladivostok (in Russian) Dalnauka. - 2007. - 127 S.).

3. To calculate the concentration of lysozyme expressed in µg/ml and calculation of PAF in µg/ml, the authors have developed a table-a program allowing a computer evaluation of the result, which also increases the accuracy of the method and makes it easy and affordable to use.

4. Use as faguoqitirute cells as neutrophils and monocytes (macrophages blood), which are the main cells producing lysozyme, and determining the growth of cellular synthesis of lysozyme through 4-6 and 18-24 hours for a quantitative assessment of immediate and long-term activation mechanism faguoqitirute cells under the influence of particles simhasana that extends the capabilities of the method.

The method is as follows.

Example 1. The allocation of a blood mononuclear cells (lymphocytes and monocytes) produce in the density gradient of ficoll-urografin on A.Boyum (1968) in the description Antheridia (Chiradeep A.N. Quantitative and functional assessment of T and b systems of immunity in humans // General questions pathology. Imaginaci and technology. - M.: VINITI, 1976. - V.4. - p.124-160) with the additional introduction of gradient solution with EDTA-Na to 0.18%.

The selected ring mononuclear blood sucked off by pipette from the bottom layer of blood plasma and part of the gradient of the solution is transferred into a clean tube and medium 199 concentration of EDTA-Na bring to 0,056% by mixing 1 part of the obtained cell suspension and 2.2 parts of medium 199. 18 2 ml flat-bottomed vials contribute to 0.25 ml of the diluted cell suspension (concentration of 2×106cells/ml) and produce the attachment of cells to the glass for 60 min at room temperature, after which no adherent cells (lymphocytes) remove the 2-fold laundering medium 199. Nine vials with attached faguoqitirute cells add culture medium, namely the medium 199 with 0.5% L-glutamine and 2.5% mixed human serum, heated for 30 min at 52°C, and in nine bottles of 0.01% suspension of particles zymosan in culture medium. Six bottles (three bottles with cells in culture medium and culture medium with particles zymosan) immediately freeze in the freezer for later evaluation of the initial content of intracellular lysozyme. Six vials of cells in culture medium and culture medium with particles zymosan cultivated at 37°C for 4 to 6 hours, followed samorost the fifth and six vials of cells in culture medium and culture medium with 0.01% of the particles zymosan - within 18 to 24 hours at t=37°C, and then frozen. All eighteen (18) bottles are subjected to simultaneous three-to fourfold freezing to -10°C and thawing at room temperature for cell disruption and lysosomes to the complete isolation of cell lysozyme.

All vials to determine the number of lysozyme by micromethods (Matakena NS, Sharonov A.S., Kovalev BM micro methods in immunology. - Vladivostok (in Russian), FENU. - 1987. - 182 C.): used indicator culture of Micrococcus lysodeicticus to obtain in advance which makes seeding in stubble MPA.

The obtained microbial culture wash with joints 2 ml of phosphate buffer using a glass rod, bring the suspension to a homogeneous state. Prepare a working concentration of 5×107MIC./Jr.

To 8.0 ml of phosphate buffer in a glass Cup, add a portion of the agarose 0,1, Suspension of agarose is melted on the tile, lead three times before boiling. The molten agarose is cooled to 45°C and mixed with 2.0 ml of the suspension culture test. The mixture is stirred and poured into the chamber, consisting of two plates and a U-shaped strip between them, fastened with clothespins. After solidification of the gel top plate sliding movement is removed and the gasket are removed. In the gel gently gel-punch make holes at a distance of 1.0 cm from each other. In the wells by capillary is whether microspace make 0.003 ml (the volume of the wells) of the analyzed samples with lysozyme. New or washed capillary is used to make subsequent samples. At the same time on the same plate make a standard solution of crystalline lysozyme, taken in the concentration 0,76; 1,53; 3,06; 6,125; 12.5 µg/ml Plate filled with holes incubated in thermostat in a humid chamber for 2-3 hours, after which the plate is soaked in a 5% solution of sodium chloride at 15-18 hours to stop the reaction, soak in tap water for 2 hours. Measure the diameter of the rings lysis test culture immediately after drying of the gel. On the basis of the results obtained with the standard solution of lysozyme via a calibration curve produced translation of the diameter of the rings lysis at a concentration of lysozyme expressed in µg/ml.

The difference between the amount of lysozyme after cultivation and before cultivation is the number of synthesized lysozyme (L) and shows the synthetic activity faguoqitirute cells. The difference between the synthetic lysozyme faguoqitirute cells cultured with particles zymosan and without particles zymosan, is evaluated as an indicator of activation of phagocytes (PAF):

PAF=L Z - L without Z μg/ml where:

L Z - difference of the amount of lysozyme in cultured cells with particles zymosan and the amount of lysozyme in rekultivirovannyh cells with particles is mi zymosan, determined after 3-4 times of freezing and thawing, in µg/ml;

L without Z - difference of the amount of lysozyme in cultured cells without particles zymosan and the amount of lysozyme in rekultivirovannyh cells without particles zymosan determined after 3-4 times of freezing and thawing, in µg/ml.

PAF to assess urgent mechanism of activation is calculated according to the formula according to the results of culturing the cells for 4 to 6 hours.

PAF to assess long-term mechanism of activation is calculated according to the formula according to the results of culturing cells within 18 to 24 hours. Using the developed by the authors table of the program is a computer translation of the diameters of the rings lysis at a concentration of lysozyme expressed in μg/ml and the calculation of PAF in µg/ml, which increases the accuracy of the method and makes it easy and affordable to use.

Example 2. Neutrophils secrete described method (Frick,, Preißner z Allocation of human granulocytes // Immunological methods. Ed. Hrimaly. - M.: Mir, 1979. - s-332) from a mixture of cells (erythrocytes, neutrophils), who gradient ficoll-urografin when selecting blood mononuclear cells (lymphocytes and monocytes), destruction of red blood cells five times the volume of 0.87% solution of ammonium chloride for 10 min at room temperature and laundering from zrusenych erythrocytes saline solution. The selected cells are transferred into a clean tube and mixed with medium 199, bringing the cell concentration up to 6×106cells/ml of the Subsequent stages of the research carried out analogously to example 1.

The result of determining the activity of faguoqitirute cells without activation simhasanam and after activation is shown in table 1.

Table 1
The cell typeThe concentration of intracellular lysozyme before and after cultivation with particles zymosan and without particles during different time
Cultivation without particles zymosanCultivation with 0.01% of the particles zymosan
0 hours4-6 hours18-24 hours0 hours4-6 hours18-24 hours
Neutrophils1,31,51,71,31,92,9
Monocytes1,92,51,92,8a 3.9

The calculated data of the synthesized intracellular lysozyme when you activate the particles zymosan in the process of cultivation and without activation by particles zymosan presented in table 2.

Table 2
The cell typeThe number of synthesized lysozyme (L) in mcg/ml
Cultivation without particles zymosanCultivation with 0.01% of the particles zymosan
4-6 hours18-24 hours4-6 hours18-24 hours
Neutrophils0,20,40,61,6
Monocytes0,30,60,92,0

For the time mode when assessing activation faguoqitirute cells - neutrophils and monocytes adopted a period of 4-6 hours on the I assessment term activation mechanism and 18-24 hours long term, allowing to evaluate the combined term and long-term outcome of activation.

The difference between the number of synthesized lysozyme in activated and unactivated faguoqitirute cells is an indicator of activation of phagocytes (PAF). PAF urgent activation of neutrophils is 0.4 μg/ml, monocytes - 0.6 ág/ml In the long-term activation of the total PAF neutrophils based on the data presented in table 2 is 1.2 µg/ml PAF monocytes to 1.4 mcg/ml

Monitoring time spent on the execution of 10 and 100 research proposed and known method of setting NBT-test in accordance with the steps of the methods is the selection of cells, the interaction with activating the object, estimating the change in the activity faguoqitirute cells with the calculation of activation, indicating that increased productivity in accordance with the proposed method, shown in table 3.

Table 3
The research methodTime to perform 10 and 100 studies in hours
Total costContinuous costs
10 person-sample10 person-sample10 person-sample10 person-sample
Nst-test9 hours54 hours7 hours52 hours
The proposed method24 hours24-25 hours4 hours5-6 hours

The result of the study, activation faguoqitirute cells known (index of activation of neutrophils - IAN) and the proposed method (in terms of activation of phagocytes - PAF) in acute and chronic metroendometrit indicates the possibilities of different ways that can be seen from table 4.

Table 4
The cell typeIAN and PAF faguoqitirute cells in acute and chronic metroendometrit
Acute metroendometritChronic metroendometrit
IAN PAFIANPAF
Neutrophils0,7±0,11,8±0,50,08±0,010,75±0,2
Monocytes-2,0±0,7-0,7±0,18

In healthy people IAN is 0.1-0.15 usled PAF neutrophils is 1.2±0.3 ág/ml PAF monocytes - 1,4±0.25 microgram/ml

As can be seen, first, the orientation changes IAN and PAF depending on the course of inflammatory diseases of the match. Secondly, the study of the activation faguoqitirute cells proposed method involves the study of the activation process not only neutrophils and monocytes.

The invention can be used in research to study the mechanism of activation faguoqitirute cells is an important stage of phagocytosis and immune response, the search and selection of immunotropic drugs and in medical practice for the diagnosis of disorders of phagocytic immune system diseases, including infertility of unknown origin, forecast trends, evaluate the effectiveness of treatment.

The method of determining activation faguoqitirute cells, including : the separation of cells, interaction with simhasanam, assessing the activation of the enzymatic activity of the cells, characterized in that as a criterion activation faguoqitirute cells using the increase of synthesis of cell lysozyme, where to selected cells add medium 199 with 0.5% L-glutamine and 2.5% mixed human serum, heated for 30 min at 52°C, containing 0.01% of the particles zymosan (Z) and not containing particles of zymosan when they are equal to the ratio of the number of cell samples; further one third of the samples with simhasanam and without him freeze, and the other two equal parts of cell samples and simhasanam without it cultivated at 37°C, and one part of the samples within 4 to 6 h, and frozen, the other from 18 to 24 h, after that, all cell samples repeatedly and consistently freeze at temperatures up to -10°C and thawed at room temperature until complete selection of intracellular lysozyme (L), then determine the number of lysozyme by micromethods, calculate the quantity of synthesized lysozyme (L) and the indicator of the activation of phagocytes (PAF) by the formula:
PAF=L Z - L without Z in µg/ml, where
L Z - difference of the amount of lysozyme in cultured cells with particles zymosan and the amount of lysozyme in rekultivirovannyh cells with particles zymosan in µg/ml;
L without Z - difference of the number of lizol is and in cultured cells without particles zymosan and the amount of lysozyme in rekultivirovannyh cells without particles zymosan in μg/ml; when this indicator activation of phagocytes to assess urgent mechanism of activation calculated from the formula according to the results of culturing cells within 4 to 6 h, and the activation of phagocytes to assess long-term mechanism of activation calculated from the formula according to the results of culturing cells within 18 to 24 hours



 

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3 ex

FIELD: physics.

SUBSTANCE: method for bioindication of water bodies involves collecting samples of planktons inhabiting in a water body, determining the contamination level by analysing said samples and assessing the analysis results. The contamination level is determined via phylogenetic analysis of ribosomal RNA genes (18S rRNA) of planktons in the sample. Phylogenetic trees built from the conservative 18S rRNA gene are determined and evolutionary relationships of the analysed object with other saprobionts are identified. Analysis results are assessed as follows: at high (over 85%) value of bootstrap support of clusters containing the analysed planktons and resistant saprobionts, the following conclusions are made: resistant indicator organisms xeno- or oligosaprobic (or exclusively xenosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in a safe ecological state and there is no threat of negative anthropogenic action, if resistant indicator organisms oligo- and mesosaprobic (or exclusively oligosaprobic) of the water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unstable (transition from safe to unsafe state) ecological state, is under insignificant anthropologic load, is capable of self-recovery and does not need additional environmental protection measures, if resistant indicator organisms meso- and polysaprobic (or exclusively mesosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unsafe state and is under considerable anthropologic load, natural capability of self-recovery is insufficient and the water body needs environmental protection measures, if resistant indicator organisms of polysaprobic water bodies and the analysed plankton merge into one cluster, it is concluded that there is a local ecological disaster and there is need for urgent recovery measures.

EFFECT: high reliability of the biomonitoring result for use without territorial limit, independent of the geographical location of the investigated water body.

3 ex

FIELD: medicine.

SUBSTANCE: immunoglobulin work solution migration time specific to each series and manufacturer is set; specific immunoglobulin is used as a reference mark (control). Further, an analysed sample suspected for the presence of sporous bacteria is prepared for an antigen-antibody reaction by adding the specific immunoglobulin work solution in a phosphate buffer to the sample. In reaction, a complex of bacterium in the sporous form and specific immunoglobulin is produced. It is followed with capillary electrophoresis, UV detection, complex migration time test in the capillary electrophoresis system to be compared with the migration time in the capillary electrophoresis system of the specific immunoglobulin (control). If the complex migration time exceeds the specific immunoglobulin migration time by 50 seconds, the presence of bacteria in the sporous form is detected in the sample. Also, the analysis conditions and parametres are described.

EFFECT: invention allows the high-reliable specific identification of bacteria in the sporous form.

6 cl, 2 dwg, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to methods of determining degree of sensitivity of bacteria to damaging effect of bacteriolytic enzymes. The method involves growing bacteria on dense culture medium for 20-24 hours at temperature 37 °C. The obtained culture of bacteria is washed with 0.9% NaCl solution and centrifuged. After centrifuging, the bacteria cells are resuspended in a buffer whose pH is optimum for the enzyme with subsequent measurement of optical density of the suspension at wavelength 540 nm, setting the density on the level of 0.5 units and determination of the absolute quantity of bacteria in 1 ml of the suspension. 0.2 ml lysozyme with concentration of 10 mcg/ml or lysostaphin in concentration of 2.5 mcg/ml is added to 2.8 ml of the obtained suspension. The suspension of cells and enzyme solution are mixed. Optical density is measured every 10 second for 600 seconds from the moment the enzyme solution is added, with subsequent evaluation of bacteria sensitivity on the section with maximum rate of reaction for its characteristic with number of bacteria cells lysed in unit volume (ml) per unit time (sec).

EFFECT: invention enables to carry out sound correction of a system of treatments with a positive clinical result.

1 dwg,1 ex

FIELD: medicine.

SUBSTANCE: method includes incubation of microorganism E. coli in soil suspension, containing investigated substance, taken at the level of its limit permissible concentration (experiment) and without it (control) with further comparison of lipase activity of bacteria from control and experimental samples and in case of its reduction compared to control one, this concentration of substance is considered to be dangerous.

EFFECT: method makes it possible to increase sensitivity, accuracy, reduction of time required for sanitary-hygiene normalising of hazardous substances in soil with simultaneous simplicity and affordability of this method for wide practical use.

1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: tested culture of B. pseudomallei or B. Mallei in dose 104 mc is inoculated on Muller Hinton medium, disc, usually applied for treatment of acute forms of melioidosis (ceftazidime, meropenem, ciprofloxacin, doxycycline, rifampicillin, chloramphenicol, co-trimoxazole) are added and cultivated at 37°C for 24 hours. Simultaneously the same concentrations of tested cultures are inoculates on the same medium with addition of 10% of blood of laboratory animals - golden hamsters, discs are applied and cultivated at 37°C in atmosphere, containing 5% CO2, for 24 hours, after which results are counted. As index of antibacterial medication activity serves preservation of activity in conditions close to in vivo, at the level of bacteria growth indexes in standard conditions, zone of growth retardation essentially exceeding index of bacteria sensitivity to said antibacterial medication. Simultaneously determined is comparative efficiency of medications in experiments on treatment of experimental animals.

EFFECT: invention allows to determine which antibacterial medication is efficient for treatment of diseases caused by pathogenic burkholderias more accurately.

2 cl, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: cell material containing marrow stem cells in a liquid culture medium is placed over a prepared agar medium containing marrow cells producing homing factors. After incubation of the prepared two-layer culture, inadherent cells are transferred to a semi-viscous culture medium and incubated in a mode required for colony-formation. Then by recording the difference of stem cell count in the cell material in the reference, and after placing on the agar medium containing homing factors, stem cells migrated by stem cell homing factor are counted.

EFFECT: invention allows simplifying the method for determination of production of stem cell homing factors due to the use of the agar medium as a semipermeable membrane and the introduction of colony-forming ability change of the cell material as an evaluation criterion of production of stem cell homing factors.

1 tbl, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to a method of determining death rate of microbes. The method enables determination of effectiveness of antimicrobial agents by determining death rate of microbes. The method involves determination of the death rate of microbes by calculating the logarithm of ratios of time values at which the microbes die, where the said logarithm is standardised on a time interval and the said time values are spaced apart by the said interval.

EFFECT: invention cuts on time, increases accuracy and simplifies determination of the death rate of microbes.

2 cl, 2 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: luminescent biocatalyst contains immobilised cells of luminescent bacteria which are included in a polyvinyl alcohol based cryogenic gel at given ratio of components.

EFFECT: prolonged possible use of the biocatalyst, simplification of its composition and production technology.

1 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: study of biomaterials from primary and secondary lesions for identifying a infectious agent is conducted, as well as immunoglobulins IgG and kappa and lambda of free light chains (FLC) and for values of lambda FLC in spinal fluid ≥0.01 mcg/ml and kappa FLC ≥0.5 mcg/ml, in additional the antibodies to beta-2-glycoprotein-I, to endothelium, to complement factor Clq and cryoglobulins as cryocrit and rheumatoid factor activity are defined. If values of antibodies to beta-2-glycoprotein-I ≥5 U/ml and/or to endothelium ≥1:10 and/or to complement factor Clq ≥10 U/ml and/or to cryoglobulins as cryocrit >0% and/or rheumatoid factor activity ≥1:20 and when detecting chlamydia and/or pathogenic forms of bacteroids in cerebrospinal fluid and/or blood and/or in scrapings from nasal and/or conjunctiva vascular lesions of CNS is detected.

EFFECT: development of methods for determining vascular lesions of CNS associated with chlamydial infection.

2 tbl

FIELD: medicine.

SUBSTANCE: invention relates to composition, which contains Arthrospira maxima subjected to physiological stress, for application as biocide and/or therapeutic medication. Invention also relates to method of prevention or treatment of infection or contamination of subject with organism, where method includes stage of introduction of efficient amount of composition, containing Arthrospira maxima subjected to physiological stress, to subject.

EFFECT: increase of efficiency of biocidal compositions and therapeutic preparations.

30 cl, 34 dwg, 23 tbl, 13 ex

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