Method for determining microorganism sensitivity to antimicrobial substances. RU patent 2505813.
| Method for determining nuclear lens density / 2505812 Patient's venous blood serum is analysed for vascular endothelial growth factor (VEGF). If the derived values in the patients suffering type 1 diabetes mellitus is 141-187 pg/ml, degree 1 and very low nuclear lens density are diagnosed; the value of 188-234 pg/ml enables diagnosing degree 2 and low nuclear lens density; and the value of 235-280 pg/ml shows degree 3 and moderate nuclear lens density. In the patients suffering type 2 diabetes mellitus, the VEGF value of 281-330 pg/ml provides diagnosing degree 2 and low nuclear lens density; the derived value of 331-382 pg/ml shows degree 3 and moderate nuclear lens density; if the value is 383-432 pg/ml, degree 4 and high nuclear lens density are diagnosed; the respective value of 433-478 pg/ml provides diagnosing degree 5 and very high nuclear lens density. In the patients diagnosed with no diabetes mellitus, the VEGF value of 290-336 pg/ml ensures diagnosing degree 2 and low nuclear lens density; the value of 337-379 pg/ml provides degree 3 and moderate nuclear lens density; if the value is 380-426 pg/ml, degree 4 and high nuclear lens density are diagnosed; the derived value of 427-465 pg/ml provides diagnosing degree 5 and very high nuclear lens density. |
 Method for cellular analysis of probe by means of virtual analytical plate / 2504777Probe (2) is processed for cellular analysis. The above processing is intended for marking of pathologic cells among sound cells of a probe. Then, several pictures of probe (6) located on analytical plate (6) are taken to obtain a variety of pictures, each of which displays one zone of analytical plate (6). The above pictures located in a row form composite picture (20) of the sample. Besides, an automatic scrolling of all the taken pictures of sample (2) is performed on a display device at the pre-set scrolling speed. The above provided speed is such that an investigator can analyse a picture and determine whether the displayed sample zone contains potentially pathological cells or not; scrolling stops automatically at detection of an anomaly that can bear evidence of availability of a pathological cell. |
 Diagnostic technique for ischemic nephropathy in newborns / 2504776Diagnosing of ischemic nephropathy in the newborns is ensured by diagnostic testing for 1-3 days of life according to anamnestic and biochemical findings, involving the determination of foetal age, sodium ion concentration, urine microalbumin, urine carbonic anhydrase to creatinine relation and blood free bilirubin concentration; that is followed by the calculation of a probability (P) of ischemic nephropathy by formula. If the value P is more than 50%, ischemic nephropathy is diagnosed in the newborns. |
 Method for assessing inducing action of cytomegalovirus infection on hemoglobin oxygenation in third trimester of gestation with underlying hemoglobin and 2,3-bisphosphoglycerate measurement in peripheral red blood cells / 2504775To assess a threatening hypoxia formation in the third trimester of gestation, peripheral blood is analysed for a cytomegalovirus antibody titre, peripheral blood 2,3-diphosphoglycerate, haemoglobun by cytophotometry, and peripheral blood oxyhaemoglobin. If observing an increase of the cytomegalovirus antibody titre to 1:1600, 2,3-diphosphoglycerate to 7.2 mcmole/ml, a decrease of hemoglobin to 72.25 standard units, a decrease of oxyhaemoglobin to 96.0%, the threatening hypoxia is expected. |
| Method for prediction of infectious inflammation of postoperative wound in external fixation of long bones / 2504774 Blood count is analysed, and a Calph-Caliph leucocytic intoxication index is calculated on the 3rd post-fracture day. If the derived value is 1.8 and more, the following external fixation is expected to be accompanied by the development of an infectious inflammation of the wound. |
| Method of predicting unfavourable course of locally spread forms of human papilloma virus-associated cervical cancer / 2504326 Invention relates to field of medicine, namely to oncogynecology. Immunological parameters are determined: absolute number of lymphocytes (lim_ab), TNFα (TNFα-k) and IL-10 (IL-IL10_k) in peripheral blood, as well as TNFα (TNFα-c) and IL-4 (IL-4_c) in cervical mucus. On the basis of obtained data parameter F(x) is calculated by formula: F(x): P(x)=9.265+1,749x(lim_ab)+0.013×(IL-4_c)+0.005×(IL-10_k)-0.410×(TNFα-k)-0.206×(TNPα-c). If value F(x) is higher than 0.555, unfavourable course of tumour process is predicted, in case if it is lower than 0.555, favourable course is predicted. |
 Method for assessing conditions of proliferative cervical diseases / 2503960Method is staged: 1) cervical canal scrapes and biopsy materials of the uterine cervix are used to recover DNA of human papilloma virus; 2) virus genotype is determined; 3) patients with HPV 16 positive CIN are sampled; 4) real-time PCR is used to determine the number of virus DNA copies and a degree of its integration with a host gene (a physical status); 5) a threshold level of the viral load is stated taking into account the physical status of the virus (6.5 lg HPV DNA copies 16 per one cell); 6) the patients are classified according to the determined threshold viral load and physical status of the virus (the episomal and integrated form); 7) the poorly predicted patients having the high viral load (≥6.5 lg DNA copies per one cell) are detected in the episomal form of the virus, while the low load (<6.5 lg DNA copies per one cell) is shown by the integrated form of the virus. |
| Method for prediction of individual risk of bronchial asthma in regions with high and low incidence of helminth infections / 2503959 Presence or absence of opisthorchiasis is stated; and polymorphous versions of genes and a possibility of referring an individual to a group with a low or high risk of bronchial asthma are found. If observing one of any risk combinations in accordance with the SOCS5-IFNG-opisthorchiasis model in the patient, the high risk of bronchial asthma is diagnosed. If observing one of any protective combinations in accordance with the IL4-TBX21-SOCS5 model in the patient, the low risk of bronchial asthma is diagnosed. |
 Method of individual quantitative assessment of ischemic heart disease development / 2503405Invention relates to cardiology, pathophysiology, biochemistry and pharmacology. To assess development of ischemic heart disease value of discriminant function is calculated by a set of parameters: age, height, weight, Quetelet index, Broca's index, abdominal obesity, triglycerides, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, atherogenicity index, arterial pressure, glucose, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, α-amilase, total protein, albumin, uric acid, urea, creatinine, creatine kinase, alkaline phosphatase, total bilirubin, direct bilirubin, ADP-induced platelet aggregation, collagen-induced platelet aggregation, platelet number, mean platelet volume, number of red blood cells, hematocrit, hemoglobin, mean corpuscular volume, RDW-CV, average content of hemoglobin in blood, number of white blood cells, percent content of neutrophil granulocytes, percent content of eosinophils, percent content of basophils, percent content of lymphocytes, percent content of monocytes, mean content of hemoglobin on red blood cell. Conclusion about initial stage of IHD development, 50% risk of disease or 100% development of IHD is made on the basis of obtained value. |
 Method for assessing disturbance of transamination in plasmodiotrophoblast in third trimester of pregnancy in aggravated herpes viral infection by histochemical method of pyridoxalphosphate dehydrogenase activity in placental plasmodiotrophoblast / 2503007Histochemical method is used to assess pyridoxalphosphate dehydrogenase activity. The disturbance of transamination in placental plasmodiotrophoblast is shown by herpes virus antibody titre 1:12800 and decreased pyridoxalphosphate dehydrogenase activity below 12.5±0.37 standard units that is presented in the form of an increase of transaminase to 0.58±0.002 mmole/l in placental homogenate. |
 Application of indole-3-yl glyoxylamides for suppression of chlamydia infection / 2493259Invention represents new application of indole-3-yl glyoxylamides as specific inhibitors of protease CPAF for suppression of the chlamydia infection. |
 Method to determine bactericide properties of blood serum / 2489489Substance of the invention is quantitative assessment of impact of human or animal blood serum at a laboratory strain Bacillus subtilis VKPM B-10548, efficiently expressing luxAB-genes of gram-negative marine microorganism Photobacterium leiognathi. At the same time extent of this strain luminescence suppression is proportionate to the level of bactericide effect developing in its respect. Total calculation of bactericide properties of blood serum determined by availability of beta-lysine in it, is made according to the formula: B P B S ( B L ) = 100 − ( I exp . 0,5 × I c o n t r . 0 ) ( I exp . 0 × I c o n t r . 0,5 ) × 100. |
 Quality assessment method of biocorrosion damages of thin-walled sealed enclosures of aluminium-magnesium alloys in operation of spacecrafts, and suspension of spore materials for its implementation / 2486250Method of qualitative assessment of biocorrosion damages of thin-walled sealed enclosures of aluminium-magnesium alloys in operation of spacecrafts and the suspension of spore materials fungi of implementation of the said method is proposed. The test and control samples of aluminium and magnesium alloys are prepared. The prepared samples are dried, sterilised. Fungal cultures - strains of microorganisms Paecilomyces variotii Bainier of All-Russian collection of microorganisms F-4039D, Ulocladium botrytis Preuss of All-Russian collection of microorganisms F-4032D, Penicillium chrysogenum Thorn of All-Russian collection of microorganisms F-4034D, Aspergillus sydowii (Bainier et Sartory) Thorn et Church of All-Russian collection of microorganisms F-4037D, Cladosporium sphaerospermum Penz. of All-Russian collection of microorganisms F-4041D are inoculated for growing spores into tubes with a sloped agar medium of Czapek-Dox. The tubes are thermostated at a temperature of (29+2)°C for 14-28 days until appearance of mature spores. Spore materials suspensions of individual cultures of the said fungi are prepared, which are then mixed in equal proportions. And the concentration of spores of each fungi species in the suspension should be in the range of 1-2 mln/cm3. The resulting suspension is applied to the sterilised test samples. Then they are dried and placed as one sample in each Petri dish on the surface of agar medium of Czapek-Dox. The control samples not treated by the spore material are also placed on the surface of the agar medium of Czapek-Dox in the Petri dishes. The Petri dishes with the test and control samples are placed in different desiccators at the bottom of which water is poured to maintain the humidity more than 90%. The desiccators are closed and incubated in the thermostat at a temperature of (29±2)°C. The exposition of test and control samples is carried out for 40, 120 and 180 days. Then the samples are removed from the Petri dishes, and the corrosion products and mycelium are removed from the surface of the samples washing them in running water. The samples are soaked in 70% ethyl alcohol for 30 minutes, then they are washed with detergent and dried. Then, using a scanning electron microscope a qualitative assessment of biocorrosion damages is carried out. According to the change in the appearance of the surface of the samples the initial stages of biocorrosion and its type is evaluated, as well as the distribution of corrosion damage on the surface of the samples, and the dependence of the biocorrosion process on time is determined. |
| Method for evaluating antibacterial action of ozonised physiologic saline / 2425888 Method provides application of a broth agent culture on a plate. These plates are kept for 48 h and triply washed with distilled water to produce biofilms formed by bacteria. A part of the biofilms are processed with ozonised physiologic saline, the other part - by non-ozonised physiologic saline for 10 minutes. They are washed, painted by 1% aqueous solution of gentian violet for 45 minutes, washed and eluted with alcohol and examined for the eluate optical density with using a spectrophotometer. The derived results are matched, and if observing the presence of significant difference p<0.05, antibacterial action of ozonized physiologic saline is stated. |
 Binding domain for bacterial atp-synthase / 2418001Invention describes mutations of the amino acid sequence of the protein of atpE mycobacteria (M. tuberculosis and M. smegmatis), which determine their resistance to DARQ J. Obtained are coding mutant forms of atpE nucleic acids, vectors containing said forms and host cells expressing mutant proteins. |
 Method of determining bacteriostatic, bactericidal and stimulating effect of antibiotic on microorganism / 2410437Method of determining bacteriostatic, bactericidal and stimulating effect of an antibiotic on a microorganism involves preparation of cultures of the analysed antibiotic in a culture medium and detecting growth of microorganisms. Determination can be carried out on 50 test microorganisms, simultaneously deposited on the surface of a solid culture medium with different concentrations of antibiotics. After incubation of inoculations, the diametre of grown colonies of the analysed cultures is measured and compared to that of control inoculations and the effect of the antibiotic is defined as bactericidal, bacteriostatic or stimulating if there is significant increase or reduction the diametre of colonies compared to the control diametre. |
| Method of predicting destabilisation of microbiocenosis of mammal organism / 2377311 Invention relates to medicine, ecology and pharmacology and can be used for determining influence of internal organism media or environmental factors on microbiocenosis of mammal organism biotopes. Liquid sample of internal organism medium or external environment in amount 1-10 vol % is added to culture of microorganisms association in liquid cultivation medium, containing bacteria of genera Bifidobacterium, Lactobacterium, Enterococcus and Escherichia coli, taken with ratio 1011:1010:1010:1010 CFU/l. Then culture of microorganisms association is mixed with liquid sample and obtained mixture is incubated at 37°C during 24 hours. After that ratio of components of microorganisms association is evaluated and prediction is made: absence of destabilisation of microbiocenosis of mammal organism under the impact of internal organism medium or external environment, correspondingly, in absence of not less than 100 times reduction of concentration of one of microorganisms association components. Destabilisation of microbiocenosis of mammal organism under the impact of internal organism medium or external environment, correspondingly, is observed in case of not less than 100 times reduction of one of components of microorganisms association. |
| Method of determination of bacteriostatic properties of antibodies to virulence factors burkholderia pseudomallei / 2337360 Invention is referred to the area of medical microbiology and immunology and concerns selection of highly active monoclonal antibodies (MCA), interacting with the epitopes exhibited on a surface of microbic cells of the melioidosis originator. The method of determination of bacteriostatic properties of antibodies to the biologically active components of Burkholderia pseudomallei cell wall consists in the following: bacteriemic cells preliminary incubated, at various expositions with investigated monoclonal antibodies, and also with MCA in the presence of a complement, plate on a dense nutrient medium, incubate seeding in a thermostat at 37°C and make the account of results of experiences by quantity of evolved colonies in 1-2 days. Estimate protective MCA potential proceeding from indicators of suppression of bacteria growth. |
| Evaluation of antibacterial activity in vitro of patient blood in treatment of infective endocarditis / 2293327 Claimed method includes introducing of blood plasma into culture containing St.aureus and E.coli in amount of 0.03 ml, wherein said plasma was sampled by 2 h after administration of antibiotic to patient. Then on the base of growth suppression zone sensibility of infective process provocative to antibiotic is determined. |
| Method for evaluation of specific activity of immune preparations / 2291425 Invention relates to a method for testing different immune preparations, for example, immunoglobulins. Method involves carrying out the combined culturing test-strain of microorganism with the corresponding immune preparation under standard conditions of the human embryo monolayer of cultured cells. Results are estimated by degree of the adhesion decrease value of test-strain of microorganism as compared with control value. Invention provides elevating rate in evaluation of an anti-infectious activity of immune preparations based on approaching conditions of evaluation to macroorganism conditions. |
 Agent for enhancing sensitivity of microorganisms to antibacterial preparations / 2246541Invention relates to agents used for enhancing sensitivity of microorganisms to antibacterial preparations. Invention involves applying hydrocarbonate chloride-sodium drinking mineral water "Obukhovskaya" with the total mineralization 1.8-2.2 g/l as an agent for enhancing sensitivity of microorganisms to antibacterial preparations that is used early for treatment of atopic dermatitis and as an agent for treatment of iodine-deficient states. It has been found by method using disks that mineral water "Obukhovskaya" provides enhancing sensitivity of microorganisms to such antibacterial preparations as antibiotics and specific preparations against yeast-like fungi. |
FIELD: medicine.
SUBSTANCE: determining the microorganisms sensitivity to antimicrobial substances is ensured by sampling a biological material, incubating the microorganisms found in the biological material on a nutrient medium, introducing the analysed antimicrobial substance into the nutrient medium, and analyzing the prospected result. The microorganisms are cultured using a dense rich nutrient medium. The analysed antimicrobial substance is introduced into the nutrient medium before culturing the microorganisms in the concentration close to the maximum attainable one in the biological material sampling point has started. The microorganism sensitivity to the antimicrobial substances are assessed after the evident microorganism growth is observed in the control inoculation. The nutrient medium can contain vitamins, and/or amino acids, and/or food additives.
EFFECT: using the method enables determining the microorganism sensitivity to the antimicrobial substance more accurately.
12 cl, 6 ex
The invention relates to the means for determining the sensitivity of different microorganisms, including bacteria and fungi that cause disease in humans, animals, plants, and that cause spoilage of food and industrial products, to antimicrobial substances antibiotics and antiseptics, and can be used, mainly, in medicine and in veterinary medicine, agriculture and industry.
One of the most serious problems of modern medicine, veterinary science, plant growing and industry is of vital activity of different microorganisms, and first of all the bacteria and fungi that can lead to disease and food contamination. The fight against bacteria and fungi remains insufficiently effective because of the rapid variability of microorganisms, leading to the emergence of resistant forms, inability to cultivate a significant number of microbes and the lack of effective methods of selection of an antimicrobial agent. In this regard, in the first 3-4 days germicides applied empirically. In medicine and veterinary use broad-spectrum antibiotics, which are limited in number due to which quickly propagated resistance to it, which leads to complications and rarely, death in medicine and veterinary medicine cause irreversible damage and destruction of food and industrial products.
In this regard, fast and accurate evaluation of the sensitivity of microorganisms to antimicrobial drugs - antibiotics and antiseptics remains actual and unsolved problem.
There is a method sensitivity determination to antimicrobial agents, including preliminary allocation of pure culture of a microorganism and identification.
However, the allocation of pure cultures of microorganisms results in a number of negative consequences, first of all, loss of time (48-96 hours). Also, if you have two or more microorganisms some of them are not allocated in the form of pure culture. It should be mentioned that in General are not so-called «until cultivated» (not-yet-is cultivated) microbes, which according to various sources can be up to 90-95%.
Difficulties in working with these microbes are primarily associated with the absence of methods of selection, cultivation and identification. The latter is defined organisation of the life of bacteria in the composition of microbial communities - biofilms, which defines the increasing interdependence of microorganisms. These conditions can't be recreated in the laboratory, see K. Lewis, Epstein S.S. Persisters, biofilms, and the problem of culturability. in incultivated microorganisms. In Series: Microbiology Monographs. Steinbuchel A. (ed.). Berlin / Heidelberg: Springer; 2009. 181-194. Epstein S.S. General model of microbial uncultivability in uncultivated microorganisms. In Series: Microbiology Monographs. Steinbuchel A. (ed.). Berlin / Heidelberg: Springer; 2009, c.c.131-150.
There is a method in which the antimicrobial substances in a certain way inflicted on the strip, see Isenberg H.D. Essential Procedures for clinical Microbiology, ASM Press, 1998, USA, c.c.235-240. Method of serial dilution includes sowing of pure culture on a special fluid Muller Hinton, which also added to antimicrobial agents (antibiotics) in different concentrations, see Isenberg H.D. Essential Procedures for clinical Microbiology, ASM Press, 1998, USA, c.c.216-223. There are ways of semi-automatic determination of antibiotic sensitivity at which the detection is carried out on special equipment (for example, panel Vitec - 2 BioMerie). However, in the basis of the method is also pre-select a pure culture of the alleged agent with subsequent exposure holes in the panel. Genetic methods can identify some of the studied antibiotic resistance genes have identified bacteria or right in the pathological material. This method is relatively fast, but is currently not able to fully assess the susceptibility to antimicrobial agents. It is connected with significant part of micro-organisms and, as a consequence, the lack of data on their genome and genes . The second reason is that even in well-known and cultivated bacteria resistant to one drug can be encoded in many different genes, many of which are still not understood.
There is a method of determining the sensitivity of microorganisms to antimicrobial substance, including the fence of biological material and the allocation of pure culture of the microorganism. To highlight the pure culture of biological material is seeded on nutrient medium, which create favourable conditions for growth of a specific organism that is suspected of causing disease or damage to the products and materials. If the organism grows, carry out its identification. After that determine its sensitiveness to antimicrobial substance, why spend sowing dedicated pure culture of a microorganism culture media Muller Hinton or similar is not a rich environment that does not contain an antimicrobial substance, overlaying the surface sowed microorganisms nutrient medium of paper disks impregnated with various antimicrobial substances, incubation of microorganisms on a nutrient medium in the presence of the studied antimicrobial compounds with subsequent evaluation of the results, see Isenberg H.D. Essential Procedures for clinical Microbiology, ASM Press, 1998, USA, 1998, 205-215.
There is a method of determining the sensitivity of microorganisms to antimicrobial substance (antibiotic), including fence biological material, cultivation contained in the micro-organisms in nutrient medium, does not contain an antimicrobial substance, introduction into the environment of the studied antimicrobial substances and subsequent measurement result; biological material cultivate sugar broth for at least two hours, then enter the standard disk drives with the test antibiotics and additionally incubated for at least two hours, the result is evaluated according to the degree of turbidity content in the sample, considering that less than turbidity, the higher the sensitivity of microorganisms to antibacterial substances, EN 2262533 C2.
Disadvantages of this method, taken as a prototype of the invention consist in the following:
- very low accuracy of the determination of the results, because a decrease in turbidity content in the sample can be associated with inhibition of microbial growth, are not those pathogens whose sensitivity to antimicrobial substance is investigated;
- finding a biological material in the nutrient medium does not contain an antimicrobial substance for a long time (more than two hours) by the selective growth of some microorganisms lead to changes in the overall composition of microorganisms compared to the original material, that sharply distorts the results of the research;
- introduction of antimicrobial substance into the nutrient medium on standard drives makes sense only in conditions of a dedicated pure culture, because only pure cultures of norms values zones inhibit the growth of microorganisms that can assess their sensitivity and resistance to the antimicrobial substance. In this way, because the pure culture of the microorganism is not allocated, cannot be taken into account neither the special characteristics of the microorganism, the concentration of antimicrobial substance, the maximum possible in the place of allocation of biological material;
- rich liquid nutrient medium does not provide the growth of the greater part of microorganisms.
The present invention supposed to solution of the task of improving the accuracy of determining the sensitivity of microorganisms to antimicrobial substance.
According to the invention of the method of determining the sensitivity of microorganisms to antimicrobial substances, which includes the fence biological material, incubation contained in the micro-organisms in nutrient medium, introduction into the medium investigated antimicrobial substance with the subsequent estimation result for cultivation of microorganisms use dense rich medium investigated an antimicrobial substance is introduced into the nutrient medium until the beginning of the cultivation of microorganisms in concentrations close to the maximum achievable in the place of collection of biological material to evaluate the sensitivity of microorganisms to antimicrobial substance is carried out after the appearance of the visible growth microorganisms in the control sowing; as dense nutrient rich environment for the incubation of bacteria use environment agar or environment Colombia agar, and for incubation mushrooms use environment Saburo; to a nutrient medium which may be added serum human or animal and/or red blood cells; to the culture medium can be added vitamins, and/or amino acids and/or food additives; nutrient medium may additionally contain micro-organisms, contributing by co-cultivation growth of the test organism; microorganisms, contributing in a joint cultivation of growth of the test organism, can be pre-transferred genes of resistance to different antimicrobial agents; in a nutrient medium for incubation of mushrooms can be added anti-bacterial preparations; in a nutrient medium for the incubation of bacteria can be added antifungals; incubation of microorganisms can carry out in aerobic conditions; incubation of microorganisms can carry out in anaerobic conditions; incubation can carry out in the presence of one or more antimicrobial substances; nutrient medium may be placed in the Cup Petri; nutrient medium can place on the Board of plastic or paper; the Board can be made for the deepening of the medium.
The Complainant has not identified any technical solution identical to the declared that allows to make the conclusion about conformity of the invention to the condition of «Novelty».
The claimed invention, unlike the original and other well-known analogues does not imply a determination of the sensitivity of certain microorganisms, typical for this disease in humans, animals, plants or damage and spoilage of products and materials.
The new principle underlying the claimed process is the fact that you try to raise the microorganisms contained in material, and then determine what kind of an antimicrobial substance most effectively suppresses the combination of these microorganisms, without having to actually or allegedly to identify the data microorganisms, as is the case in the known methods.
That is, to solve the problems of the struggle with the source of diseases and damage to the products and materials do not need to know that this source represents, it is important to know what tool enables you to eliminate or reduce the harmful effects of this source.
Implementation of the characteristics of the invention provides the above fundamentally new property subject matter of the invention and the technical result consists in the considerable increase of the accuracy of the survey results.
Dense rich breeding ground enables to raise the maximum number of different microorganisms contained in the sample, it is important that the antimicrobial is administered in a nutrient medium to how the cultivation of microorganisms. It prevents changes in the overall composition of microorganisms in the process of cultivation compared to the original material. It is important that the antimicrobial agent is injected at a concentration close to the maximum achievable in the place of collection of biological material and not in any concentration, as is the case with the introduction of antimicrobial substance on the standard disks. The assessment of the sensitivity of micro-organisms is determined not by the degree of turbidity content in a sample which may be due to inhibition of microbial non-pathogenic (harmful), and the results of the appearance of the visible growth of microorganisms in the control sowing.
Significantly new in the proposed method is the fact that the addition of the nutrient medium of some micro-organisms can contribute by co-cultivation with pathogens growth even those microorganisms in the sample, which otherwise are «».
For greater severity of the effect microorganisms, contributing by co-cultivation growth of the test organism, transfer of resistance genes to different antimicrobial agents.
The above new properties of the declared method of cause, in the opinion of the applicant, its conformity to the requirement of patentability of «Inventive step».
Implementation of the method is illustrated by the examples below.
Example 1. Express susceptibility to azithromycin bacteria, while sowing material, obtained from a patient with an infection of the urinary system.
Collected biological material - urine of the patient cystitis.
Nourishing environment: dense, rich - agar enriched horse serum and erythrocytes of human and sheep. On Wednesday previously added azithromycin in the most adequate in the urine concentration of 4 mcg/ml
Material sown in the amount of 0,2 ml in Petri dishes. In control used a similar environment and cultivation conditions. Sowing were incubated at a temperature of 37 deg C.
The result of the study. In the control after 4-6 hours growth at a temperature of 37 C, registered education lawn.
Thus, on a dense rich medium in the control sowing registered the growth of a large number of different unrelated bacteria, which prevented the growth in the presence of azithromycin which can be successfully used for treatment of the patient. You did not have to identification of specific pathogens and allocation of pure culture. The answer to assign the antibiotic was received after 4 hours.
Example 2. Express susceptibility to ampicillin bacteria, while sowing material, obtained from animal (dog) with wound infection.
The method was carried out as described in example 1.
Study material: discharge from the wound.
Nutrient medium: agar Colombia, enriched with horse serum, with sheep red blood cells, vitamins and amino acids. On Wednesday added in the highest practicable at the intake concentration of 1 mg/ml
Material from the wounds were sown swab on Wednesday in a Petri dish. In the control have been using the same environment and conditions of cultivation without ampicillin.
Sowing were incubated at a temperature of 37 deg C.
The result of the study. In the control after 4-6 hours growth at a temperature of 37 C, registered education lawn. On the Cup with ampicillin growth of bacteria is not registered.
Thus, on a dense rich environments registered the growth of a large number of different unrelated bacteria that were not given the growth in the presence of furniture, which can be successfully used for the treatment of the animal. When this did not require identification of specific pathogens and allocation of pure culture. The answer to assign the antibiotic was received after 4 hours.
Example 3. The choice of antiseptics for the destruction of household mold, received from an infected plaster.
Study material: pieces of damaged plaster with signs of mold growth. Prepared suspended plaster (0.5 g) in 1.0 ml izotoniceski solution of sodium chloride.
Nutrient medium: Saburo agar enriched with minerals, is located three Petri dish. On Wednesday pre-added products: the first Cup of antiseptic , derivative , solvent-water at a final concentration of 0,5% (can be achieved without changing the properties of a plaster and similar materials - color, smell and mechanical properties); the second Cup - antiseptics: complex copolymers guanidine. Solvent water (0,5%); third Cup control without antiseptics.
Material sown bacteriological loop.
Crops cultivated at 30 degrees C. Records of the results produced through 4, 6, 8, 12, 20, 24 and 48 hours after planting.
Results. After 6 hours of culturing the growth registered in the control Cup and the Cup, where agar contained a drug . In cups with an growth was absent. Mold has proved resilient to the drug and sensitive to . When this did not require identification of specific pathogens and allocation of pure culture. Reply to select an antiseptic was received after 8 hours.
Example 4. The choice of an antibiotic in the treatment of diseases of the respiratory system.
Material for research - patient's sputum.
Nutrient medium: Б agar enriched horse serum and erythrocytes rights. On Wednesday added Haemophilus influenzae bacteria that cause diseases of the respiratory system and contribute to the growth of another pathogen - Streptococcus pneumoniae, very poorly cultivated in normal conditions. Wednesday was also added to a maximum concentration of levofloxacin attainable in sputum - 10 mcg/ml.
sputum patient lit 5 times in izotonicescom solution of sodium chloride, planted by 0.2 ml/Cup-spatula and incubated at a temperature of 37 deg C. Records of the results produced through 4, 6, 12, 20 and 24 hours after planting.
Results. In the control after 4 hours at the temperature of 37 C, registered education lawn. In the medium with the addition of levofloxacin: 4, 6, 12, 20 and 24 hour - a lack of growth. According to the microscopy: 24 hours Roth smears prepared from bacteria grown in the control sample - over 12 morphotypes of microorganisms; environment adding levofloxacin - no growth.
Thus, the proposed technology allows to fast for 4 hours choose antibiotic which will inhibit the growth of all potential agents of this pathology, including practically not cultivated in normal conditions. This does not require the identification of a particular pathogen and the allocation of pure culture.
Example 5. Express determination of sensitivity to a mixture of the antibiotic gentamicin (acts predominantly on aerobic bacteria) and metronidazole (active against anaerobic bacteria and ), while sowing material obtained from a patient with periodontal disease.
Study material: freshly harvested secretions of the gingival pocket.
Nutrient medium: for cultivation in aerobic conditions used the environment Colombia agar enriched with sheep red blood cells and a horse serum. To identify anaerobic microorganisms used agar containing RAM erythrocytes and serum. On Wednesday were added 2 antibiotics: gentamicin (active against a wide range of aerobes) and metronidazole selectively acting on the anaerobic bacteria. Environment in the cups №1 and №1A contains gentamicin; environment in the cups №2 and №2A contains metronidazole; environment in the cups №3 and №3A contains gentamicin, metronidazole; in the cups №4 and №4 is only nutrient medium (control).
Detachable gingival pocket lit 5 times isotonic raster sodium chloride, planted 0.05 ml/Cup (sowing trowel). Crops for aerobes (cups 1-4) in the normal thermostat and anaerobes (Cup 1a-4A) in thermostat in anaerobic conditions at 37 C 48-72 hours. To create anaerobic conditions used packages. Records of the results produced through 4, 8, 12, 20, 24 and 48 hours after planting (anaerobic bacteria are characterized by long growth).
Results. In the control of aerobic (4 hours) and anaerobic (8 hours) the conditions of growth at a temperature of 37 C, registered education lawn. In aerobic conditions in the experimental Cup №3 contains (gentamicin, metronidazole) growth was absent throughout the observation. In anaerobic conditions in 8 hours in the experimental Cup # 3A (contains gentamicin, metronidazole) growth was absent. Microbial growth registered in the cups №№1, 1a, 2 and 2A. The obtained data testify to the effectiveness of a mixture of powder and metronidazole and the presence of stable clones using drugs separately.
Thus, the proposed method unlike the prototype allows to evaluate the sensitivity to a mixture of antimicrobials.
Example 6. Express determination of sensitivity fungi, plant disease (mildew) antifungal medications.
Study material: wash izotoniceski solution of sodium chloride from the affected areas of the leaf, showing signs of growth of fungi.
Results. After 8 hours of cultivation registered the growth in the control hole and the hole where agar contained medication nystatin. In the coated well with growth was absent. Mold has proved resilient to the drug nystatin. This does not require the identification of a particular pathogen and the allocation of pure culture. The answer for the choice of antifungal drug was obtained after 8 hours.
1. Method of determining the sensitivity of microorganisms to antimicrobial substances, including fence biological material, incubation contained in the micro-organisms in nutrient environment, introduction into the medium investigated antimicrobial substance with the subsequent estimation of the, notable for cultivation of microorganisms use dense rich medium, an antimicrobial substance is injected into the medium before the beginning of the cultivation of microorganisms in concentrations close to the maximum achievable in the place of collection of biological material, as well as exercise control sowing of microorganisms in the nutrient medium does not contain an antimicrobial substance, with an assessment of the sensitivity of microorganisms to antimicrobial substance exercise after the appearance of the visible growth of microorganisms in the control sowing.
2. The method according to claim 1, characterized in that as dense nutrient rich environment for the incubation of bacteria use environment agar or environment Colombia agar, and for incubation mushrooms use environment Saburo.
3. The method of claim 2, characterized in that the nutrition medium add the serum of human or animal and/or red blood cells.
4. The method of claim 2, characterized in that the nutrition medium contains vitamins and/or amino acids and/or supplements.
5. The method according to claim 1, characterized in that the nutrient medium additionally contains micro-organisms, contributing by co-cultivation growth of the test organism.
6. The method according to claim 1, characterized in that the nutrition medium for incubation mushrooms add anti-bacterial preparations.
7. The method according to claim 1, characterized in that the nutrition medium for the incubation of bacteria add antifungal drugs.
8. The method according to claim 1, characterized in that the incubation of microorganisms carried out in aerobic conditions.
9. The method according to claim 1, characterized in that the incubation of microorganisms carried out in anaerobic conditions.
10. The method according to claim 1, characterized in that the incubation carried out in the presence of one or more antimicrobial compounds.
11. The method according to claim 1, characterized in that the nutrition medium is placed in a Petri dish.
12. The method according to claim 1, characterized in that the nutrition medium is placed on a tablet of paper or plastic.