Test-system for differentiating species and biotypes of bacteria of genus yersinia. RU patent 2518297.
 Nutrient medium for extraction of bacteria of shigella kind from water objects / 2517750Nutrient medium is proposed for extraction of Shigella kind bacteria from water objects. The nutrient medium contains the following components: 0.1% alcohol solution of bromcresol green - 9.0 ml; 0.2% alcohol solution of methyl red - 3.0 ml; fermentative peptone - 0.5 g; extract of fodder yeast - 4.5 g; potassium dihydrophosphate - 8.7 g; caustic soda - 1.4 g; sodium chloride - 5.0 g; distilled water - up to 1000 ml; medium pH is 6.70-6.85. |
 Method of differentiating bacillus anthracis from other closely related species of genus bacillus based on determining differences in structure of chromosomal genes / 2514663Method includes sample preparation, DNA isolation, PCR statement. At that in carrying out PCR, the oligonucleotide primers are used, which are complementary to sequences of the chromosomal genes fliC and hom2, having the following sequences: fliC-F: 5'-TGGAGCAGTAACAATTGG-3', fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3', hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3', hom2-R: 5'-CACCAATTTCGTCTTTTACA-3', followed by electrophoretic analysis of the amplification products, when the formation of the amplification product is 153 bps in size it is indicative of belonging of the strain under study to the species B.anthracis, formation of the amplification product with size of 550 bps is indicative of belonging of the strain under study to the other species of the genus Bacillus. |
 Differential diagnostic nutrient medium for identification of yersinia bacterium / 2511436Invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions. |
| Method for preparing microgravimetric immunosensor / 2510830 Invention refers to biotechnology and microbiology. What is presented is a method for preparing a microgravimetric immunosensor. A surface of a quartz crystal resonator is activated first by plasma spray coating with polyethylene imine of molecular weight less than 10000 Da for 10 s in a vacuum unit at UHF field power 30 Wt. Thereafter, immunoglobulins from a solution with the protein concentration of 0.125 mg/ml are immobilised on the activated surface of the quartz crystal resonator. That is followed by washing in distilled water and drying in an air flow. |
 Method for quantitative assessment of bactericidal activity of disinfectants / 2510610Invention refers to microbiology and biotechnology. Analysed bacterial strains are inoculated on a dense nutrient medium. Paper disks impregnated with a disinfectant are applied. They are incubated in a temperature chamber until the bacteria start growing. The bacterial growth inhibition areas are measured. A quantity of the grown colonies is counted to construct a dependence diagram of the bacterial growth inhibition area and the quantity of the grown colonies after the disinfectant reaction. The diagram and Shughart inspection sheet are used to assess the disinfectant activity on specific types of the microorganisms. The disinfectants with mean measurements of the bacterial growth inhibition area are above an upper control limit of the Shughart inspection sheet are considered to be high bactericidal activity agents. The disinfectants with mean measurements of the bacterial growth inhibition area are below a lower control limit of the Shughart inspection sheet are considered to be low bactericidal activity agents. The disinfectants with mean measurements of the bacterial growth inhibition area between the control limits of the Shughart inspection sheet are considered to be mean bactericidal activity agents in relation to all analysed agents. |
 Method of species identification of l.casei/paracasei, l.fermentum, l.plantarum and l.rhamnosus / 2508406Invention refers to a method of species identification of L.casei/paracasei, L.fermentum, L.plantarum, L.rhamnosus lactobacilli. The proposed method involves performance of a PNR reaction with species-specific primers; besides, primers are built, which are specific to the first gene of operon FIFO ATP of synthase (a gene of subunit a) and a gene of uracylphosphoribosyltransferase, which precedes it, for L.casei/paracasei and L.rhamnosus and a gene of uracyltransport protein for L.plantarum and L. fermentum. |
 Dry chromogenic feed medium for detection of coliform bacteria and e.coli (versions) / 2508400Invention can be used for detection of coliform bacteria and E.coli in specimens of food products and water at performance of bacteriological tests. Feed medium includes a nitrogen source represented by meat peptone or pancreatic hydrolysate of fish flour, sodium chloride, dibasic sodium phosphate, potassium monophosphate, sodium pyruvate, L-tryptophane, sodium dodecyl sulphate, 6-chloro-3-indolyl-β-D-galactopyranoside (Salmon - GAL), 5-bromine-4-chloro-3-indolyl-β-D-glucoronide-(X-GLUC), isopropyl- β-D1-tiogalactopyranoside (IPTG) and microbiological agar in the specified ratio. |
| Dry differential diagnostics feed medium for detection and consideration of e.coli and coliform bacteria / 2508399 Invention can be used for detection and considering of E.coli and coliform bacteria in water, food products, clinic material, etc. Feed medium contains pancreatic hydrolysate of fish flour dried with Tergitol 7 on the bases of 0.1 g of Tergitol 7 per 6 g of dry pancreatic hydrolysate of fish flour, yeast extract, 1-water D (+) lactose, bromthymol blue, sodium dodecyl sulphate, 2,3,5-triphenyltetrazolium chloride, sodium carbonate and microbiological agar in the specified ratio. |
| Method of identifying vibrio bacteria / 2506313 Invention relates to microbiology and biotechnology. Material to be investigated - pure culture of rod-like, gram-negative, glucose-fermenting, oxidase-positive or oxidase-negaive bacteria - is collected first. The investigated daily bacterial culture is seeded on the surface of nonselective nutrient agar (GRM-agar) with 1% sodium chloride. A paper disc is then placed seeded surface, said disc containing vibriostatic substance niclosamide (2,5-dichloro-4-nitrosalicylanilide) in amount of 10 mcg or 16 mcg per disc. The seeded material is incubated in aerobic conditions at 35°C for 24 hours. Vibrio bacteria are indicated a zone of inhibited bacterial growth around the disc. |
 Method for quick growth, detection and identification or counting of microcolonies of microorganisms at early stage / 2505607Invention pertains to the method for quick growth, detection and identification or counting of microcolonies of microorganisms at early stage. The described method includes the following stages: obtaining the container with medium with porous element located above or under the top surface of the medium, note that the medium has nutritious substances for quick growth of microcolonies of microorganisms and porous element has pores from 1000 to 107 Da; pouring the liquid sample without serial dilution into container to the area above porous element; capturing the microorganisms in porous element or at the medium above porous element; incubation of container for the time sufficient for quick growth of microcolony at an early stage; transfer of porous element and any medium above it from container to the secondary medium for evaluation, detection and identification of microorganisms; and microcolonies research relatively the growth, detection, identification or counting of microorganisms. The said method for growth, detection and identification or counting of microorganisms takes not more than approximately six hours. |
 Anti-obesity preparation, anti-obesity food product or drink, preparation improving tolerance to glucose and food product or drink for improving tolerance to glucose / 2518293Group of inventions relates to field of biotechnology. Strain Bifidobacterium breve MCC 1274 FERM BP-11175 demonstrates low coefficient of conversion of linoleic acid into conjugated linoleic acid. Strain demonstrates coefficient of conversion of linoleic acid into conjugated linoleic acid not higher than 10%. To reduce or prevent obesity or to improve tolerance to glucose said strain is introduced in efficient quantity to subject requiring it. Also claimed are food product and drink, which contain preparation based on said strain. |
| Nutrient medium for submerged cultivation of tularemia microbe / 2518282 Nutrient medium for submerged cultivation of tularemia microbe contains as a base, that provides the content of amino nitrogen in the medium of not less than 0.32%, dry enzymatic hydrolyzate of fibrin obtained from waste of whey-vaccine production, and the components - glucose and calcium pantothenate at a predetermined ratio. |
| Nutrient medium for extraction of bacteria of shigella kind from water objects / 2517750 Nutrient medium is proposed for extraction of Shigella kind bacteria from water objects. The nutrient medium contains the following components: 0.1% alcohol solution of bromcresol green - 9.0 ml; 0.2% alcohol solution of methyl red - 3.0 ml; fermentative peptone - 0.5 g; extract of fodder yeast - 4.5 g; potassium dihydrophosphate - 8.7 g; caustic soda - 1.4 g; sodium chloride - 5.0 g; distilled water - up to 1000 ml; medium pH is 6.70-6.85. |
 Method to prepare medicated product from live strains and microorganisms of lactobacilli and bifidobacteria "lb-complex plus" / 2517734Method provides for cultivation at 37±1°C of strains of lactobacilli and bifidobacteria in the medium and packing of liquid product with account of daily dose necessary for patients. The medium contains components in the following quantities: caseine hydrolysate dissolved by distilled water, 0.33-0.4 g/l, sodium chloride 5 g/l, fructose 10 g/l, peptone 2 g/l, agar-agar 0.75 g/l for lactobacilli, for bifidobacteria - 1.0 g/l, ascorbic acid 0.25 g/l, distilled water 0.67-0.6 g/l. Strains-producers are Lactobacillus plantarum 8 RA-3, Lactobacillus fermentum 39, Lactobacillus fermentum 90 TC-4, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Bifidobacterium bifidum 1. For lactobacilli and bifidobacteria they prepare accordingly media with different content of agar-agar, amine nitrogen in caseine hydrolysate 160-170 and 180-200 mg% and medium pH 7.8-8.0 and 8.5-8.6. Starting from the first generation the bifidobacteria and lactobacilli are cultivated for 24±1 hours, two strains together, one separately. The produced biomass is mixed with the fresh nutrient medium at the ratio of 1:1000, and strains of lactobacilli are cultivated 24±1 hours, strains of bifidobacteria - 48±1 hours. Upon completion of cultivation of biomass of strains they are mixed at the ratio of 2:1:2:1. |
 Nourishing composition, including probiotics and improving sleep pattern / 2517616Invention relates to application of probiotic bacterial strain for production of a probiotic composition for reducing sleep disorders and/or improvement of sleep quality in people and animals. As a bacterial strain used is Lactobacillus reuteri DSM 17938 or Bifidobacterium longum NCC 3001 (ATCC BAA-999). |
| Strain of fungus stagonospora cirsii davis having herbicidal activity against canada thistle / 2515899 Strain of the fungus Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 has herbicidal activity against canada thistle and species closely related to it - S. Cirsii. It was deposited in the State Collection of Microorganisms of All-Union Research Institute of Plant Protection under the collection number of Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 and can be used for biological control of canada thistle. |
 Medication (versions), composition (versions) and application of medication (versions) for reduction of halitosis / 2515113Invention relates to versions of medication for reduction of halitosis, versions of compositions based on said medications and versions of application of said medications. Device for reduction of halitosis represents strains of microorganisms Lactobacillus acidophilus, selected from group Lactobacillus acidophilus DSM 19825, Lactobacillus acidophilus DSM 19826 and Lactobacillus acidophilus DSM 19827. Version of said medication represents culture supernatant of said strains. Also claimed are compositions, which contain said microorganisms, and their application for reduction of halitosis. |
| Method of culturing bifidobacteria in milk / 2515048 Invention relates to biotechnology and can be used in the dairy industry. Pasteurised milk is cooled to a given temperature and MnSO4, ZnSO4, KJ, CuSO4, FeSO4 and selexen are then added in a given ratio, followed by addition of bifidobacteria. The mixture is stirred and soured until a clot of given titratable acidity is obtained. |
| Method of obtaining toxin actinobacillus pleuropneumoniae apxi, applying culture medium, containing calcium-borogluconate complex / 2514667 Claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex. |
| Hafnia alvei bacteria strain, capable of producing thermolabile lt-enterotoxin / 2514656 Invention relates to biotechnology and can be used to produce thermolabile enterotoxin (LT-enterotoxin) and Hafnia alvei anatoxin when producing a vaccine. The strain is deposited in the State Collection of Pathogenic Microorganisms of FBSI Scientific Centre for Evaluation of Medical Products of the Ministry of Public Health and Social Development of Russia under number 294. |
 Strain of lactobacilli lactobacillus paracasei cncm i-2116 (ncc 2461) eliciting ability to prevent intestine colonization with pathogenic microorganisms causing diarrhea, supernatant of its culture and oral agent for prophylaxis and/or treatment of diarrhea-associated disorders / 2243779Invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea. |
FIELD: chemistry.
SUBSTANCE: invention relates to field of microbiology and deals with test-system for differentiation of species and biotypes of bacteria of genus Yersinia. Claimed invention consists of a set of nutritional media, which contain substrates and reagents for determination of presence in microorganism of lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, urease, acetoin production, indole production, meliobiose fermentation, rhamnose fermentation, saccharose fermentation, sorbite fermentation, lipase, xylose fermentation, maltose fermentation, α-methyl-D-glucopyranoside fermentation, salicin fermentation, sorbose fermentation and raffinose fermentation.
EFFECT: claimed invention makes it possible to increase specific activity in differentiation of bacteria of genus Yersinia due to extension of the range of determinable species and biotypes.
3 tbl, 5 ex
The invention relates to Microbiology and may be used in bacteriological research for vnutrirodovoe and intraspecific differentiation of bacteria of the genus Yersinia, and to determine biotype of some kinds of Yersinia.
The genus Yersinia includes a wide group of gram-negative anaerobic micro-organisms, belonging to the family Enterobacteriaceae. Among the 17 members of the genus Yersinia, only attributed to three pathogenic for humans and animals - the plague pathogen Y.pestis, pseudotuberculosis pathogen Y.pseudotuberculosis and separate representatives Y.enterocolitica, which cause intestinal yersiniose. The incidence of intestinal yersiniosis registered in Russia almost everywhere, but there is uneven distribution of infection in separate territories.
Type Y. enterocolitica is widely distributed in nature. Representatives of the species are found in the gastrointestinal tract (GIT) of various animals, water, soil, can contaminate raw vegetables, milk and other products stored in the refrigerator [Bottone E.J. Yersinia enterocolitica: overview and epidemiologic correlates / J.Microb. Infect. - 1999. - №1. - P.323-333]. In accordance with the results of biochemical reactions known six biotypes Y.enterocolitica: 1A, 1B, 2, 3, 4, 5. Representatives of biotypes 1B, 2-5 are pathogenic and contain chromosomal and plasmid genes determining adhesiveness, invasiveness, thermostable toxins and other virulent properties microbe [Thoerner P., Bin Kingombe S, Bogli-Stuber K., et al. PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution / J. Appl. Env. Microb. - 2003. - Vol.69, №3. - P.1810-1816]. Representatives of biotype 1A classified as non-pathogenic as not having a chromosomal and plasmid genes virulence [Grant So, Bennet-Wood V., Robbins-Brown R. Identification of virulence-associated characteristics in clinical isolates of Yersinia enterocolitica lacking classical virulence markers / Infection and Immunity. - 1998. - Vol 66. - №3. - P.1113-1120]. However, in recent years there is evidence of the presence of this biotype pathogenic variants that can cause gastrointestinal diseases accompanied by diarrhea [Tennant S.M., Grant T.N., Robins-Browne R. Pathogenicity of Yersinia enterocolitica biotype 1A / FEMS Immun. Med. Microb. - 2003. - №38. - P.127-137].
Other representatives of the genus classified as non-pathogenic, but can be agents of opportunistic infections. Until recently on biochemical properties they are divided into eight types: Y.aldovae, Y.bercovieri, Y.frederiksenii, Y.intermedia, Y.kristensenii, Y.mollaretii, Y.rohdei, Y.ruckeri. The development of molecular-genetic methods allowed in recent years (2006-2011) to Supplement the genus Yersinia six representatives - Y.aleksiciae [Spraguet L.D., Neubauer N. Yersinia aleksiciae sp.nov. / Int. J. of Syst. and Evol. Microb., 2005. - Vol.55. - P.831-835], Y. similis [Spraguet L.D., Scholz H.C., Amman s, et al. Yersinia similis sp.nov. / Int. J. of Syst. and Evol. Microb., 2008. - Vol.58. - P.952-958], Y.massiliensis [Merhej V., Adekambi So, Pargnier I., et al. Yersinia massiliensis sp.nov., isolated from fresh water / Int. J. Syst. Evol. Environ, 2008. - Vol.58. - P.779-784], Y. nurmii [Murros-Kontiainen A., Fredriksson-Ahomaa M., Korkeala h, et al. Yersinia nurmii sp.nov. / Int. J. Syst. Evol. Environ., 2011. - Vol.61. - 2368-2372], Y.pekkanenii [Murros-Kontiainen A., P. Johansson, Niskanen So, et al. Y.pekkanenii sp.nov. / Int. J. Syst. Evol. Environ., 2011. - Vol.61. - P.2363-2367], Y. entomophaga [Hurst M.R.H., Becher S.A. Young S.D., et al. Yersinia entomophaga sp.nov., isolated from the New Zealand grass grub Costelytra zealandica I Int. J. Syst. Evol. Environ, 2011 - Vol.61. - P.844-849]and type Y. intermedia - two biotypes (at the moment there are 10 biotypes Y.intermedia) [Martin L., Leclercq A., Savin C, and E. Carniel Characterization of atypical isolates of Yersinia intermedia and definition of two new biotypes / J. Clin. Environ., 2009. - Vol.47, no. 8. - P.2376-2380]. The diversity of the genus and the heterogeneity of individual species complicates the identification of Yersinia. Existing commercial test-systems on the biochemical composition of the tests focused on the identification of the more common pathogenic representatives of the family Enterobacteriaceae genera such as Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella, Shigella. In clinical microbiologists using such test systems, there are often problems with identification and differentiation of some kinds of Yersinia, especially Y.enterocolitica biotype 1A, Y.intermedia, Y.frederiksenii, Y.bercovieri, Y.mollaretii, due to the insufficient number of required tests [Hallanvuo S., Peltola J., Heiskkanen So, Siitonen A. Simlified phenotypic scheme evaluated by 16S rRNA sequencing for differentiation between Yersinia enterocolitica and Y.enterocolitica-like species / J.Clin. Environ., 2006. - Vol.44, no. 3. - P.1077-1080]. In General, there is no public system vnutrirodovoe differentiation of bacteria of the genus Yersinia. All this leads to errors and incorrect results, especially in the study of environmental swabs from vegetables and other food products - the main sources of representatives Y.enterocolitica biotype 1A and closely related species.
Known methods of generic and specific phenotypic identification of bacteria of the genus Yersinia and differentiation of representatives Y.enterocolitica on biotypes, including the aggregate of many biochemical signs - fermentation some carbohydrates, utilization of certain amino acids and the formation of metabolites.
The determinant of bacteria Burgi [Determinant of bacteria Burgi, Ed. by J.. Howth [and others], 9th edition, Vol. 1. - M: Mir, 1997 - 432 S., Il.] phenotypic identification of Yersinia produced by 56 characteristics (table 5.2 str-229; 5.40, 5.41, 5.42, 5.43 str-258), including 22 test for differentiation of 11 species (mobility, products indole, products acetone in the reaction of Fogasa-Proskauer; disposal citrate; the presence of urease, lizindekarboksilazy, Intergeneratsiya, gelatinase, pyrazinamidase, b-keloids, t-glutamyl transferase; the formation of acid from cellobiose, melibiose, α-methyl-D-glucoside, raffinose, L-ramnose D-sorbitol, sucrose, matinata, L-fucose, L-sorbose) and 7 tests to determine biotypes Y.enterocolitica (production of indole; deoxyribonuclease, the presence of a lipase, nitrate reduction, education acid from sucrose, trehalose, D-xylose). The disadvantages of species differentiation determinant of bacteria Burgi is inaccuracy differentiation between species Y.frederiksenii, Y.intermedia, Y.enterocolitica 1 biotype, as well as the lack of differentiation biotype 1 Y.enterocolitica on biotype 1A, the representatives of which are non-pathogenic, and biotype 1B, the representatives of which are pathogenic. Interspecific differentiation Y.frederiksenii and Y.intermedia complicated by the heterogeneity of representatives Y.intermedia, the number of biotypes which has 10.
In manual of clinical Microbiology [Wagner A. Yersinia. Manual of Clinical Microbiology, editor Patrick R. Murray, 9 th edition, vol. 1. - Washington, DC: ASM Press, 2007. - P.688-697] proposed the identification of 11 species of Yersinia 14 phenotypic (mobility, presence of urease production acetone in the reaction of Fogasa-Proskauer, Intergeneratsiya, utilization citrate, products indole, fermentation of carbohydrates: ramnose, sucrose, cellobiose, corboz, sorbitol, melibiose, raffinose, fucose) and differentiation biotypes Y.enterocolitica nine biochemical properties (presence of lipase, hydrolysis of esculin, products indole, nitrate reduction test for Tnkase, pyrazinamidase, fermentation of salicin, xylose, trehalose). Differentiation over the specified collection phenotypic characteristics is similar to the previous method disadvantages due to lack of necessary tests and more complete data of the biochemical properties of Yersinia.
In MU 3.1.1.2438-09 [Epidemiological surveillance and prevention of pseudotuberculosis and intestinal yersiniose. MU 3.1.1.2438-09. M: the Federal state educational establishment "NERC's Roszdrav", 2009] presents biochemical properties 10 species of Yersinia (view Y.pestis not included in the table) as determinant Burgi. Accordingly, this table, as well as differentiation of species and biotopes of Yersinia the determinant Burgi, has a number of drawbacks and does not allow to fully differentiate certain bacteria of the genus Yersinia.
In the basis of implementation of existing commercial test-systems is the aggregate of the phenotypic characteristics of bacteria that are listed in the keys Burgi and other generally accepted guidelines.
Test-system for biochemical identification and differentiation enterobacteria "DS-DIF-ENTERO-24" (NGO Diagnostic Systems, N. Novgorod) is designed for phenotypic identification of microorganisms of the family Enterobacteriaceae within 24 hours in 24 biochemical characteristics. The kit DC-DIF-ENTERO-24" includes: tablets polystyrene 96-well coated folding to the bottom of wells substrate-the indicator of nutrient media, stable polyvinyl alcohol; solution sterile physiological preparation suspension cultures of microorganisms; vaseline oil sterile to create anaerobic conditions biochemical reactions; reagent to test indole" (3,5% solution paradimethylaminobenzaldehyde); reagent to test phenylalanine" (10% solution of iron chloride (III) of uranyl); reagent 1 for the test "AMK" (12% solution alpha-naphthol), reagent 2 for the test "AMK" (40% solution of potassium hydroxide). The test system allows to determine the following biochemical properties of enterobacteria: recycling citrate, malonate sodium, Inositol, sorbitol, mannitol, adonia, dulcita, glucose, lactose, sucrose, arabinose, maltose, ramnose, trehalose, hydrolysis of esculin; education indole, hydrogen sulphide, acetylaminophenol (reaction of Fogasa-Proskauer); presence of urease, beta-galactosidase, decarboxylase ornithine and lysine, desaminase phenylalanine, dihydrolase arginine. Test-system identifies these types of Yersinia as Y.enterocolitica, Y.frederiksenii, Y.intermedia, Y.kristensenii, Y.pseudotuberculosis, but does not identify other types of Yersinia set biotypes Y.enterocolitica and Y.intermedia and to differentiate between members of the species Y.frederiksenii and some biotypes Y.intermedia.
The test-system "ENTEROtest" (and its modification "ENTEROtest") (Embalagem, Czech Republic). Set "ENTEROtest" contains: microtiter plates (each for 4 identification of strains); frame plate with cover for incubation; plastic bags for incubation; forms for registration of the results. Also necessary reagents supplied separately: the reagent test INDOLE, reagent to test PHENYLALANINE, reagent to test ACETON, paraffin oil sterile. The test system is used for identification of enterobacteria to 24 features and includes a panel of 24 biochemical substrates. The test-system "ENTEROtest" includes 24 test to determine such biochemical properties as education indole, the formation of hydrogen sulphide, lizindekarboksilazy, Intergeneratsiya, urease, egindykol, utilization citrate, utilization of malonate, the presence phenylalkylamines, beta-galactosidase, hydrolysis of esculin, education acetone (reaction of Fogasa-Proskauer), fermentation, Inositol, adonitol, cellobiose, sucrose, trehalose, mannitol, sorbitol, ramnose, melibiose, raffinose, dulcita, glucose. The test system "ENTEROtest N" consists of 24 tests, including 5 tests that distinguish it from "ENTEROtest": instead of the definition of indole production, availability phenylalkylamines, education acetone, fermentation ramnose and glucose entered tests to determine beta-glucuronidase, b-celoxica, fermentation of salicin, lactose, Arteta. It may be supplemented produced separately tests to education indole and acetone that are necessary for differentiation of Yersinia. Using the test-system "ENTEROtest" ("ENTEROtest") it is possible to identify these types of Yersinia as Y.enterocolitica, Y.frederiksenii, Y.intermedia, Y.kristensenii, Y.pestis, Y.pseudotuberculosis, but it cannot be used to identify other Yersinia species, to differentiate between Y.frederiksenii, some biotypes Y.intermedia and Y.enterocolitica biotype 1A, and set the biotypes Y.enterocolitica and Y.intermedia.
Microdiscectomy for biochemical identification of enterobacteria ("MTS-M-12") produced by NPO "Nutrient medium" (Makhachkala). Set "MTS-M-12" is a container made of neutral transparent polystyrene with cells on the bottom of which are substrate-the indicator of nutrient medium, stable polyvinyl alcohol. The test system allows to determine the following biochemical properties of enterobacteria: recycling citrate, malonate sodium, glucose, lactose, mannitol, maltose, education indole, hydrogen sulfide, urease (3-galactosidase, lysine decarboxylase, desaminase phenylalanine. This test-system allows to identify the genus Yersinia, but offer 12 alone is not enough to determine the species of Yersinia and the more biotypes.
For the prototype selected the test system "AP1 20E" (bioMerieux, France). Set "AP1 20E" is intended for the identification of bacteria of the family Enterobacteriaceae and other undemanding to nutrients gram-negative bacilli in 20 signs. The kit AP1 20E": strips API 20E, containers for incubation, forms for accounting of results, instruction. The necessary reagents and materials, not included in the set: mineral oil; saline API NaCl 0,85%; the reagent to strip API 20E TDA to determine triptofangidroksilazy; the reagent to strip API 20E JAMES to determine indoleamine; the reagent to strip API 20E 1 VP VP and 2 to determine products acetone. Strip API 20E consists of 20 microwells containing dehydrogenated substrate for determination of beta-galactosidase, argininosuccinate, lizindekarboksilazy, Intergeneratsiya, utilization of citrates, production of hydrogen sulphide, urease, triptofangidroksilazy, production of indole, production acetone in the reaction of Fogasa-Proskauer, gelatinase, fermentation D-glucose, D-mannitol, Inositol, D-sorbitol, L-ramnose, D-sucrose, D-melibiose, amygdalin, L-arabinose. With the help of test-system it is possible to identify these types of Yersinia as Y.enterocolitica, Y.frederiksenii, Y.intermedia, Y.kristensenii, Y.pestis, Y.pseudotuberculosis, but this test-system is able to identify any of the other Yersinia species, and to differentiate between species Y.frederiksenii and Y.intermedia and install the biotypes Y.enterocolitica and Y.intermedia.
The prior art, it follows that none of the existing commercial test-systems does not allow full definition of species and biotopes bacteria of the genus Yersinia. Some types of Yersinia often not included in the range defined test systems of microorganisms. Species such as Y.aldovae, Y.bercovieri, Y.mollaretii, Y.rohdei and Y.aleksiciae often defined as Y.enterocolitica due to the absence of analogues as some tests for identification and data on biochemical properties of these species. In turn, representatives Y.frederiksenii, Y.intermedia differentiate wrong or not differentiate at all. Often the errors of determination of view, especially when you need to differentiate representatives biotype 1A Y.enterocolitica which are widely distributed in the environment and often are allocated from the swabs from vegetables and other food products.
nutrient medium for determining lizindekarboksilazy - 0,15
nutrient medium for determining intergeneratsiys - 0,15
nutrient medium for determining triptofangidroksilazy - 0,15
reagent for the determination triptofangidroksilazy - 0,050 0,025...
nutrient medium for determining urease - 0,15
nutrient medium for defining products acetone - 0,15
reagent for the determination of products acetone - 0,050 0,025...
nutrient medium for defining products indole - 0,15
reagent for the determination of products indole - 0,050 0,025...
nutrient medium for determining fermentation melibiose - 0,15
nutrient medium for determining fermentation ramnose - 0,15
nutrient medium for determining the fermentation of sucrose - 0,15
nutrient medium for determining the fermentation of sorbitol - 0,15
nutrient medium for definitions - 5,00
nutrient medium for determining xylose fermentation - 0,15
nutrient medium for determining fermentation maltose - 0,15
nutrient medium for determining fermentation
α-methyl-D-glucopyranoside - 0,15
nutrient medium for determining the fermentation of salicin - 0,15
nutrient medium for determining fermentation corboz - 0,15
nutrient medium for determining fermentation raffinose - 0,15.
Biochemical properties of bacteria of the genus Yersinia in respect of substrates that are used to define the attributes shown in table 1.
The selection and study of pure culture of the plague pathogen Y.pestis can only be held specialized laboratories anti-plague institutions of Rospotrebnadzor in strict compliance with the regime of work with microorganisms of I-II groups of pathogenicity. Why let biochemical properties of Y.pestis only for comparison with other representatives of the genus Yersinia.
Determination of the presence of the enzyme urease, carrying out the hydrolysis of urea. This characteristic is essential for almost all kind of Yersinia. The urease have such species as Y.aldovae, Y.aleksiciae, Y.bercovieri, Y.enterocolitica, Y.frederiksenii, Y.intermedia, Y.kristensenii, Y.massiliensis, Y.mollaretii, Y.pseudotuberculosis, Y.rohdei and Y.similis. He have the ability to hydrolyze urea Y.pestis and Y.ruckeri. Some representatives Y.mollaretii can be reasonation, in this case, they can be differentiated from Y.ruckeri (and Y.pestis) the results of the fermentation of sucrose.
Sucrose oxidized with formation of acid representatives Y.bercovieri, Y.enterocolitica, Y.frederiksenii, Y.intermedia, Y.massiliensis, Y.mollaretii, Y.rohdei, not fermented representatives Y.aldovae, Y.aleksiciae, Y.kristensenii, Y.pseudotuberculosis, Y.ruckeri, Y.similis, Y.pestis.
Determination of the presence of the enzyme intergeneratsiys. The test allows for differentiation between species Y.kristensenii and Y.pseudotuberculosis.
Determination of the presence of the enzyme lizindekarboksilazy. Generally not used for the differentiation of species of Yersinia. However, only this test allows to differentiate Y.kristensenii from Y.aleksiciae. Lizindekarboksilaza also defined in Y.massiliensis and some representatives Y.ruckeri.
Determination of the presence of the enzyme triptofangidroksilazy. All of Yersinia except representatives Y.massiliensis, this enzyme is missing.
Fermentation maltose with the formation of acid is observed in all of Yersinia, in addition to representatives Y.aldovae and Y.rohdei, differentiation between which, in turn, can be performed at fermentation of sucrose (above).
An important role in species differentiation within the genus Yersinia plays such a biochemical property bacteria, as the ability of education acetylaminophenol (acetone) in the reaction of Fogasa-Proskauer. If (37 of + / -1)OC With all the Yersinia aceton not formed. However, when (26 of + 2)OC With observed results are different for different species. So, the ability to form aceton have representatives species Y.aldovae, Y.enterocolitica, Y.frederiksenii, Y.intermedia; not form aceton Y.bercovieri, Y.kristensenii, Y.mollaretii, Y.pestis, Y.pseudotuberculosis, Y.rohdei. This test is important to differentiate Y.mollaretii and Y.bercovieri from Y.enterocolitica. Between Y.mollaretii and Y.bercovieri can be divided in determining the fermentation L-sorbose.
The ability of decomposition ramnose with the formation of acid is used to differentiate between Y.frederiksenii from Y.enterocolitica, unable to ferment rhamnose. Rhamnose also disposed of representatives of the different biotypes Y.intermedia and Y.aldovae, Y.pseudotuberculosis and Y.similis.
The absence of representatives of all biotypes Y.enterocolitica ability to ferment with the formation of acid rhamnose, raffinose and melibiose, they differentiate from Y.intermedia. View Y.intermedia biochemically heterogeneous and includes, according to the reference center of the Paris Institut Pasteur [Martin L., Leclercq A., Savin, and E. Carniel Characterization of atypical isolates of Yersinia intermedia and definition of two new biotypes / J.Clin. Environ., 2009. - Vol.47, no. 8. - P.2376-2380], 10 biotypes. Although unlike biotypes Y.enterocolitica biotypes Y.intermedia not have important clinical significance, high heterogeneity biochemical properties of this type requires intraspecific differentiation for a correct determination of other types of Yersinia in the study. Division by biotypes produced by the ability to ferment melibiose, rhamnose, α-methyl-D-glucopiranozid (alpha-methylglucose), raffinose and ability to dispose of sodium citrate. Distribution by biotypes are reflected in table 1. It is seen that the representatives of biotypes 1-7 Y.intermedia fermented to melibiose unlike Y.frederiksenii. The definition of the ability to ferment α-methyl-D-glucopiranozid is necessary to differentiate "ramnopiranozil" strains Y.frederiksenii from representatives Y.intermedia 8-10 biotypes.
View Y.enterocolitica on biochemical properties are divided into six biotypes [Bottone E.J. Yersinia enterocolitica: overview and epidemiologic correlates / J.Microb. Infect, 1999. - №1. - P.323-333]. The establishment of biotype has important epidemical significance. Thus, representatives of biotype 1A are considered non-pathogenic, while Y.enterocolitica biotypes 1B and 2-5 have a set of virulence genes and can cause disease in humans and animals. Distribution by biotypes are reflected in table 1. Intraspecific differentiation should be performed using the following tests. Determining the ability of the fermentation of salicin, which have representatives biotype 1A. Determination of the presence of the enzyme lipase, which have representatives biotype 1A and 1B, but absent in biotypes 2-5. This test allows to differentiate pathogenic representatives of American biotype 1B from other pathogenic Y.enterocolitica. Determining the ability of indoleamine allows to differentiate between the 2-nd and 3-th biotypes Y.enterocolitica, indole capable of forming representatives biotypes 1A, 1B and 2, while not forming indole. To differentiate between the 3rd and 4th biotypes Y.enterocolitica allows the definition of xylose fermentation. Representatives of the 3rd biotype unlike the 4th able to ferment xylose with the formation of acid. The ability to fermentation of sorbitol can be differentiated 4th biotype from 5th. Sorbitol fermented representatives of the 4th biotype, while strains Y.enterocolitica 5th biotype this ability does not possess the rare representatives of this biotype may not have interdiscursivity and not to ferment sucrose.
The invention is realized as follows.
All biochemical reactions carried out by standard methods. Analyzed daily agar culture of Yersinia sow on one loop (diameter 2 mm) in environments containing various substrates.
Carbohydrate substrates. The test is based on the ability of the microorganism tested to dispose of carbohydrate, introduced in quantitative ratio of 1% in liquid nutrient medium contains peptone (1%), sodium chloride (0,5%), bronchiology blue or phenol red (0,002%), with the formation of acid, determined to change the color of the indicator environment [Manual of medical Microbiology. General and sanitary Microbiology. The Book I /Coll. Authors // ed Labinsk A.S., Volnoy Mrs x - M: Publishing house BINOM, 2008. - 1080 S.: Il.]. Fermentation of carbohydrates accompanied by changes in blue (indicator bromothymol blue) were found or red (indicator phenol red) colors environment to yellow.
Urea. Microorganisms having the enzyme urease, able to hydrolyze urea, forming in the form of end-products ammonia and carbon dioxide, thus changing the pH of the environment in the alkaline side. Change is visualized with the help of the indicator. Environment to determine urease contains 100 ml 0,067 M phosphate buffet, pH 7.0 (40 ml of potassium dihydrophosphate in concentration 9,078 g/l, 60 ml dowolnego sodium hydrogen phosphate concentration 11,876 g/l); 2.5 g of sodium chloride; 2.5 ml of 0.4% caustic solution phenol red; 10 ml of 50% urea; finite volume brought to 500 ml of distilled water [Methodical recommendations for microbiological diagnosis of wound infections in the medical centers of the army and Navy / Dobrynin V. M. [and other] // decl. Beg. The main military medical Department of the Ministry of defense of the Russian Federation. - SPb.: Savor, 1999. - with 75]. Reaction into account through one, two and 24 h of incubation of the studied culture (26 of + 2)OC S.
The amino acids lysine and ornithine. Tests aimed at the detection of enzymes can catalyze the decarboxylation of amino acid with the formation of alkaline products, which can be seen on the change of indicator environment. The composition of the medium: peptone (0.5%), glucose (0,1%), bronchiology blue (0,003%), L-amino acid (lysine, ornithine) (1%), distilled water; pH (6,5±5) [Manual of medical Microbiology. Private medical Microbiology and etiological diagnosis of infection. Book II /Coll. Authors // ed Labinsk A.S., Kostjukova N.N., Ivanova S.M. - M: Publishing house BINOM, 2010. - 1152 S.: Il.]. The decomposition of the substrate yellowish-green environment changes to blue.
Tryptophan. The test is based on the ability of microorganisms with the growth in nutrient medium, containing L-tryptophan (alpha-amino-3-intelliprofile acid), to produce specific desaminase in respect of L-tryptophan that put him oxidative deamination with the formation of indole-3-pyruvic acid, which can be detected by qualitative reaction with the addition of iron chloride (III). Liquid environment to determine desaminase contains peptone enzymatic dry (1%), sodium chloride (0,5%), L-tryptophan (0,5%), distilled water. To determine in an environment of indole-3-pyruvate use reagent containing 50 ml of 10% aqueous solution of iron chloride (III) (FeCl 3 ) and 50 ml of 10% solution of hydrochloric acid [Methodical recommendations for microbiological diagnosis of wound infections in the medical centers of the army and Navy / Dobrynin V. M. [and other] // decl. Beg. The main military medical Department of the Ministry of defense of the Russian Federation. - SPb.: Savor, 1999. - with 75]. The presence triptofangidroksilazy detected by the appearance of dark-brown or red-brown colouring of the environment in 1-2 minutes after making 1-2 drops of reagent with ferric chloride.
Education indole. The test is based on the fact that in the process of propagation in a medium containing tryptophan, microorganisms form the enzyme tryptophanase that splits heterocyclic amino acid L-tryptophan forming indol and other substances. Environment for indoleamine besplodna, contains L-tryptophan (0,25%) (or source), sodium chloride (0,5%), potassium dihydrophosphate (0,25%), distilled water; pH (7,3 of + / -1) [Manual of medical Microbiology. General and sanitary Microbiology. The Book I /Coll. Authors // ed Labinsk A.S., Volnoy Mrs x - M: Publishing house BINOM, 2008. - 1080 S.: Il.]. The content of indole define the addition of benzoic aldehyde (reagent Kovacs or Ehrlich), the reaction which leads to the formation of compounds pink or red. For preparation of reagent Kovacs 5 g of parametermetadata dissolved in 75 ml of amyl alcohol, then add 25 ml of concentrated hydrochloric acid [Methodical recommendations for microbiological diagnosis of wound infections in the medical centers of the army and Navy / Dobrynin V. M. [and other] // decl. Beg. The main military medical Department of the Ministry of defense of the Russian Federation. - SPb.: Savor, 1999. - with 75]. The presence of indole in the environment after incubation judged by the appearance of red rings on the surface of the environment in 1-2 minutes after making 1-2 drops of reagent Kovacs.
Education acetone. Anaerobic fermentation of glucose goes with the transformation of pyruvic acid metabolite in aceton (acetylenedicarbonic). Environment for the reaction of Fogasa-Proskauer contains peptone (0,5%), potassium hydrogen phosphate (0,5%), glucose (0,5%), distilled water; pH (6,9 of + / -1). After incubation at (26 of + 2)OC education acetone identified on the surroundings due to oxidation acetone in the presence of alpha-naphthol (add 1-3 drops 6% alcohol solution alpha-naphthol) in alkaline medium (add 1-3 drops 40%aqueous solution of potassium hydroxide) to diacetyl, which forms complexes cherry-red color, reacting with nitrogen bases peptone [Manual of medical Microbiology. General and sanitary Microbiology. The Book I /Coll. Authors // ed Labinsk A.S., Volnoy Mrs x - M: Publishing house BINOM, 2008. - 1080 S.: Il.].
Environment with appropriate substrates can fit in a standard bacteriological test tubes with traffic in the volume of 0.5-1.0 ml, and in the holes of sterile 96-well polymer tablet in the amount of 100-200 ml (mikroebene technology studies). Mikroebene technology research biochemical properties of bacteria most cost-effective, simple, suitable for automation and standardization research. Can be used as a round bottom and flat-bottomed polystyrene plates. The first is convenient for seeding the studied organism in the environment. In turn, flat-bottomed tablets are issued in the form of folding on the individual strips. Strippersindia tablets enable research from one culture. To create anaerobic conditions and to prevent volatile metabolites, such as ammonia, which can alter the pH of the environment in the neighbouring pits, after inoculation of culture on Wednesday in well put 2-3 drops sterile mineral oil. Before incubation tablet (or strips) stick with film or take in a thick plastic bag to prevent contamination and dry environment.
Tween 80 (ether oleic acid) is used as substrate to determine whether an organism of the enzyme lipase. To identify lipase microorganisms cultivated environments libasterisk substrates, hydrolytic cleavage of which is accompanied visually determined by changing the appearance of the environment. Used agar medium with the addition of 0.01% odnofaznogo of calcium chloride and 1% tween 80 [common Methods of bacteriology: Per. from English. Ed. Ferhard [and others], in three volumes, V.3. - M: Mir, 1984. - 264 S.: Il.]. As agar bases used agar of Hottinger. Incubation on the environment is made within 24-48 hours (26 of + 2)OC C. Positive result is the appearance of the misty halo around the colonies, consisting of crystals of calcium soap. Environment for determination of lipase is dense and in the preparation of bottled in Petri dishes.
Important conditions of use of the invention are also age bacteria cultures, temperature, and time. The optimum mode of study - incubation daily agar at (26 of + 2)OC for 24 hours. This is due to the psychrophilic properties of representatives of the genus Yersinia where the metabolism of some substances may vary for (37 of + / -1)C and (26 of + 2)OC S.
A distinctive feature of the test system is the integrated use of in one set of various substrates in a nutrient medium, allowing to define 17 biochemical properties for full differentiation of species and biotopes bacteria of the genus Yersinia.
The specific activity of the test-system was installed in the study, 27 reference strains of Yersinia (26 obtained from the collection of the reference center on Yersinia the Paris Institut Pasteur, one culture Y.enterocolitica 174 km - from the State collection of pathogenic bacteria "Microbe" FKUS antiplague research Institute "Microbe" Rospotrebnadzor): two strains Y.pseudotuberculosis; 14 strains Y.enterocolitica, including four strains of biotype 1B, four strains of biotype 1A, two strains of biotype 2, one strain of biotype 3, two strains of biotype 4 and one strain of biotype 5; three strains Y.frederiksenii; three strain various biotypes Y.intermedia; three strains Y.kristensenii; two strains Y.bercovieri. The results are presented in table 2.
Specificity of the test system was evaluated in the identification of some representatives of the family Enterobacteriaceae. It was investigated eight cultures: the two cultures of Escherichia coli, one culture Klebsiella oxytoca, one culture Klebsiella pneumoniae, one culture Proteus mirabilis, one culture Enterobacter amnigenes, one culture Shigella sp., one culture Salmonella enteritidis. Generic and species of the studied crops was determined using commercial test-systems API 20E (BioMerieux, France), ENTEROtest N (Erba Lachema, Czech Republic), DC-the WPPT-Entero-24 (Diagnostic Systems, N. Novgorod). None of the investigated eight crops was not identified as Yersinia test-system for vnutrirodovoe and intraspecific differentiation of bacteria of the genus Yersinia.
The result from the use of the invention is increasing the efficiency vnutrirodovoe and intraspecific differentiation of bacteria of the genus Yersinia through the use of the proposed tests, the zoom range is defined types and biotypes, raising the standard of research by eliminating subjective choice of different tests and methods of their preparation.
Provide examples of the use of the test systems. The material culture of bacteria isolated from swabs with vegetables and of the material from the person and animals and identified as Yersinia sp. For the establishment of the species and biotype used a test system for vnutrirodovoe and intraspecific differentiation of bacteria of the genus Yersinia. In the wells of sterile polystyrene tablet previously been spilled 16 liquid media with substrates 0.15 ml Daily agar culture placed a noose (diameter 2 mm) to each well with substrate-indicator environment. In wells with urea and amino acids made sterile mineral oil. On a dense medium in a Petri dish containing tween 80, culture sowed stroke. After incubation at (26 of + 2)OC for 24 hours in one hole with tryptophan (Wednesday to determine indoleamine) made 1-2 drops of reagent Kovacs, in well with the environment to determine the education acetone made serially 1 drop of 40% aqueous solution of potassium hydroxide and 1 drop of 6% alcohol solution alpha-naphthol. After incubation at (26 of + 2)OC for 30 minutes in another hole with tryptophan (Wednesday to determine triptofangidroksilazy) made 1-2 drops of reagent with ferric chloride (III), and produced records reactions. Comparison and used the prototype - commercial test-system API 20E (BioMerieux, France), which was the highest percentage of correct identification of reference strains of Yersinia of various kinds.
Example 1.
From the laboratory of practical health care was obtained culture №1574 isolated from mouse-like rodent (Bank vole), and on phenotypic identification was attributed to the genus Yersinia. In the course of research with use of the developed test systems this culture is brought to mind Y.frederiksenii. Namely, it was determined the presence of urease, intergeneratsiys; education indole, acetone; fermentation of sucrose, sorbitol, xylose, salicin, corboz, ramnose, maltose. The rest of the tests yielded negative result. Also biochemical properties of the culture studied commercial test kit API 20E (BioMerieux, France). Such tests as the detection of b-galactosidase, Intergeneratsiya, urease production indole production acetone, fermentation D-glucose, D-mannitol, D-sorbitol, Inositol, L-ramnose, D-sucrose, amygdalin, L-arabinose were positive, the other negative. According to the results of determination of biochemical properties of culture identified a view of Yersinia. According to the test-the system API 20E culture related either to mind Y.frederiksenii or mind Y.intermedia. Set just been possible with use of the developed test systems. The results are presented in table 3.
Example 2.
From the laboratory of practical health care was obtained culture №663 allocated from the material from the person, and on phenotypic identification was attributed to the genus Yersinia mind Y.enterocolitica. In the course of research with use of the developed test systems, was confirmed view, and revealed biotype of this strain. Culture had urease, interdiscursivity; has produced aceton in the reaction of Fogasa-Proskauer; was fermentive sucrose, sorbitol, sorbose, maltose. The rest of the tests have given a negative result, namely, culture is not formed indole; had lsendcardstatus, triptofangidroksilazy, lipase; not fermentive xylose, salicin, melibiose, α-methyl-D-glucoside, rhamnose, raffinose. In the study of biochemical properties of commercial test kit API 20E (BioMerieux, France), revealed the presence of b-galactosidase, Intergeneratsiya, urease; products acetone; ability to ferment D-glucose, D-mannit, D-sorbite, Inositol, sucrose, amygdalin, L-arabinose. The other test is negative. According to the test-the system API 20E was defined only kind of investigated strains Y.enterocolitica. The results are presented in table 3.
Example 4.
From the laboratory of practical health care was obtained culture №1303 allocated from flushing with potatoes, and on phenotypic identification was attributed to the genus Yersinia mind Y.mollaretii. In the course of research with use of the developed test systems this culture is brought to mind Y.enterocolitica and biotype 1A. By the results of biochemical tests culture had urease, interdiscursivity, lipase, had no lsendcardstatus, triptofangidroksilazy; was produced indole, aceton; was fermentive sucrose, maltose, sorbitol, sorbose, xylose and salicin; not fermentive melibiose, rhamnose, raffinose and II-methyl-D-glucoside. Also biochemical properties of the culture studied commercial test kit API 20E (BioMerieux, France), which has identified the presence of b-galactosidase, Intergeneratsiya, urease; products of indole and acetone; fermentation D-glucose, D-mannitol, D-sorbitol, sucrose, Inositol, amygdalin, L-arabinose; there was no fermentation ramnose and melibiose; culture had no lsendcardstatus, triptofangidroksilazy, argininosuccinate, gelatinase, has not produced hydrogen sulfide, lacked the capacity to recycle sodium citrate. According to the results of determination of biochemical properties of culture with the API 20E, established the form Y.enterocolitica, but biotype is not defined. The results are presented in table 3.
Example 5.
From the laboratory of practical health care was obtained culture №1482 allocated from flushing with potatoes and on phenotypic identification referred to the genus Yersinia mind Y.frederiksenii. In the course of research with use of the developed test systems were defined urease, intergeneratsiys; education indole, acetone; fermentation of sucrose, sorbitol, xylose, salicin, corboz, maltose, melibiose, ramnose, α-methyl-D-glucoside, raffinose. The rest of the tests yielded negative result. According to the results of determination of biochemical properties of the studied strains belonged to mind Y.intermedia. Also biochemical properties of the culture studied commercial test kit API 20E (BioMerieux, France). Revealed: the presence of b-galactosidase, Intergeneratsiya, urease; products of indole, acetone; fermentation D-glucose, D-mannitol, D-sorbitol, L-ramnose, D-sucrose, amygdalin, L-arabinose. The other test is negative. However, according obtained using the test system API 20E results culture related either to mind Y.frederiksenii or mind Y.intermedia. Set accurately you using the developed test systems. The results are presented in table 3.
Thus, the application of the developed test systems allows to raise specific activity during differentiation of bacteria of the genus Yersinia by expanding the range defined types and biotypes of Yersinia.
Test-system for differentiation of species and biotopes bacteria of the genus Yersinia, consisting of a set of nutrient media containing substances and reagents to determine whether an organism of lizindekarboksilazy, Intergeneratsiya, triptofangidroksilazy, urease production acetone, production of indole, fermentation melibiose, fermentation ramnose, fermentation of sucrose, fermentation sorbitol, characterized in that it additionally contains environment with substrates to determine whether an organism lipase, xylose fermentation, fermentation maltose, fermentation α-methyl-D-glucopyranoside, fermentation of salicin, fermentation corboz, fermentation raffinose the next quantitative content of components, test-systems for one definition, ml: nutrient medium for determining lizindekarboksilazy - 0,15 nutrient medium for determining intergeneratsiys - 0,15 nutrient medium for determining triptofangidroksilazy - 0,15 reagent for the determination triptofangidroksilazy - 0,050 0,025...nutrient medium for determining urease - 0,15 nutrient medium for defining products acetone - 0,15 reagent for the determination of products acetone - 0,050 0,025...nutrient medium for definition of products of indole - 0,15 reagent for the determination of products indole - 0,050 0,025...nutrient medium for determining fermentation melibiose - 0,15 nutrient medium for determining fermentation ramnose - 0,15 nutrient medium for determining the fermentation of sucrose - 0,15 nutrient medium for determining the fermentation of sorbitol - 0,15 nutrient medium for determination of lipase - 5,00 nutrient medium for determining xylose fermentation - 0,15 nutrient medium for determining fermentation maltose - 0,15 nutrient medium for determining fermentation α-methyl-D-glucopyranoside - 0,15 nutrient environment to determine the fermentation of salicin - 0,15 nutrient medium for determining fermentation corboz - 0,15 nutrient medium for determining fermentation raffinose - 0,15.