Method of shewanella bacteria recovery and identification

FIELD: medicine.

SUBSTANCE: method provides test inoculation of a nutrient medium containing pancreatic fish flour hydolyzate, fermentative meat peptone, NaCl, Tween-80, CaCl2, sodium thiosulphate (Na2S2O3 × 5H2O), ferrous ammonium sulphate ((NH4)2SO4 × FeSO4 × 6H2O), sorbite, bromthymol blue, irgasan (DP-300), rifampicin, NaOH, agar and distilled water in the preset proportions. Pancreatic fish flour hydolyzate, peptone, NaCl, agar are dissolved with heating, sterilised at 121°C for 20 min and thereafter added in a hot medium of the other components specified above. The inoculations are incubated on the nutrient medium in aerobic conditions at temperature 37°C and/or 28°C for 24-48 h and assessed by the presence of black-centre green or dark grey colonies surrounded by a cloudy precipitate zone in the nutrient medium.

EFFECT: invention allows simplifying and providing higher specificity of Shewanella bacteria recovery and identification.

2 ex

 

The invention relates to the field of Microbiology. Can be used in bacteriological studies on the isolation and identification of bacteria of the genus Shewanella and production of culture media for these studies.

There is a method of separating and identifying bacteria of the genus Shewanella directly from the source material on marine 2216 agar (Difco Lab.) or nutrient agar (Oxoid) without preconcentration. Colonies of bacteria Shewanella often recognized by their white or pink (salmon color) color (Bowman J.P. Genus XIII. Shewanella Mac Donell and Colwell 1986,355 (Effective publication: Mac Donell and Colwell 1985,180), in Bergey''s Manual of Systematic Bacteriology, 2nded., Garriti G.M., D.J. Brenner, N.R. Krieg, and J.T. Staley, Eds., New York: Springer, 2005, Vol.2, part B, pp.480-491).

Also known isolation and identification of bacteria of the genus Shewanella traditional nutrient media, including agar, mcconkey, where they grow well, forming after 18-24 h incubation yellow-brown colonies with a diameter of 1-2 mm Further isolates from yellow-brown colonies identify generic tests (O/f test with glucose in the absence of fermentation, the test oxidase test for production of hydrogen sulfide in the environment Kligler). Then use the tests to identify species S.putrefaciens, S. algae. Both hydrolized gelatin, have nitroreductase, ornithindecarboxilase; unlike S.putrefaciens S.algae has beta hemolysis on blood and the Arab Republic of Egypt with the blood of sheep, grows at 42°C in a nutrient medium with 6% NaCl. Automated identification systems do not distinguish between types S.putrefaciens, S.algae, as in a software system interfaces 20E, 20 NE,I D 32GN, Vitek database include only view S.putrefaciens (..Holt, .Gahrn-Hansen, Vvyp Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics. Clin. Environ. Infect. 2005; Vol.11, No. 5: p.347-352).

There is a method of separating and identifying bacteria Shewanella by accumulation under aerobic conditions in a liquid medium containing 1% peptone, 1% sodium chloride and 0.1% bile salts (Difco). After subsequent reseeding at DHL Agar (deoxycholate-hydrogen sulfide-lactose agar) and incubation under aerobic conditions selected colonies of non-fermentative, producing hydrogen sulfide bacteria. Isolates finally identified as Shewanella and various kinds (S.putrefaciens, S. algae) additional taxonomic tests: determination of oxidase enzyme, fermentation and oxidation of carbohydrates on/f environment, lipase, gelatinase, urease, argininosuccinate, disintegrability, interdiscursivity, production of hydrogen sulfide and trimethylamine, growth on SS-arape citrate and environment Simmons, growth on nutrient agar with 1.5% sodium chloride at 37°C and 42°C for 7 days and 14 days at 4°C; tolerance to 6% of sodium chloride at 30°C for 48 h; hemolysis on blood agar with sheep blood at 30°C for 48 h (.Nozue, .Hayashi, Y.Hashimoto, .Ezaki, .Hamasaki, .Ohwada, Y.Terawaki. Isolation andCharacterization of Shewanella alga from Human Clinical Specimens and Emendation of the Discription of S.alga Simidu et aL, 1990, 335. Intern. J.System. Bacteriology, Oct. 1992, Vol.42, No 4, p.628-634). Note by the way. The composition and application used in the method of the nutrient medium DHL Agar acc. to Sakasaki (Merck Microbiology Manual 12thEdition, 2005, p.262-263). Composition, g/l: peptone from casein 10.0 g; peptone from meat 10,0; extract of meat 3,0; lactose 10.0 g; sucrose 10.0 g; L-cysteine chloride 0,2; sodium citrate 1.0; sodium deoxycholate 1,5; sodium thiosulfate 2,0; ammonium iron (III) citrate; neutral red 0,03; agar 15,0; distilled water to 1 l; pH 7,2±0,2. Preparation protection: all components are suspended in water, dissolved by heating (not to be sterilized in a steam sterilizer), poured into Petri dishes. Crops incubated aerobically at 35°C for 24-48 h Colony fermenting lactose or sucrose bacteria have a pink or red colour; the colonies of their non-fermentative bacteria are colorless; colonies of bacteria that produce hydrogen sulfide, acquire black color. View colonies of Salmonella - colorless with black centre; Citrobacter - colorless. sometimes with a black centre; Proteus - a colorless, surrounded by dark brown area, the center has a slight black color; Escherichia coli - red; Shewanella - colorless with black center.

The prototype of the proposed method, we elected the method according to H.Nozue, T.Hayashi et al using a nutrient medium DHL Agar, since in both methods isolation and identification of bacteria Shewanella held on nutritionally dense the environments on the basis of the production of hydrogen sulfide.

The disadvantages of the prototype method and analogues are:

- lack of specificity of the identification of colonies of bacteria Shewanella on the environment the initial seeding due to the possible presence of colonies of other genera of bacteria with the same differential features (presence in the environment DHL Agar colorless colonies with a black centre at producing hydrogen sulfide bacteria Salmonella, Citrobacter, Proteus; availability on nutrient agar (Oxoid) colonies with pink colouring bacteria Pseudomonas stutzeri, Alcaligenes faecalis, Roseomonas spp.; the presence in the environment McConkie yellow-brown colonies of bacteria Pseudomonas spp., Roseomonas spp.);

- the complexity of research on the identification of bacteria of the genus Shewanella due to the need to use additional tests to study isolated colonies (definition oxidase, fermentation and oxidation of glucose O/f test, production of hydrogen sulfide).

The aim of the invention is to increase the specificity and the facilitation of research on the isolation and identification of bacteria of the genus Shewanella.

In accordance with the invention this objective is achieved in that the material is seeded on solid nutrient medium containing as an inhibitor of foreign microflora irgasan (0.14 to 0.2 g/l) and rifampicin (0,0005-0,001 g/l); sodium thiosulfate (0.35 g/l), salt Mora, sorbitol, bronchiology blue, tween-80, calcium chloride, sodium chloride, sodium hydroxide etc the following contents of the ingredients, g/l: pancreatic hydrolysate of fish meal to 12.0; peptone is an enzymatic meat - 12,0; NaCl - 6,0; tween - 80 - 5,0; CaCl2- 0,2; Na2S2O3·5H20 - 0,35; (NH4)2SO4·FeSO4·6H2O - 0,25; sorbitol - 13,0; bronchiology blue - 0,08; irgasan (DP-300) - 0,14-0,2; rifampicin - 0,0005-0,001; NaOH - 0.8; agar - 12, 0mm; distilled water to 1 l; pH 7,2±0,2; hydrolyzed fish meal, peptone, sodium chloride, agar dissolve when heated, sterilized for 20 min at 121°C, then in a hot environment, add the remaining ingredients, pour into sterile Petri dishes; crops incubated at 37°C and/or 28°C in aerobic conditions for 24 to 48 h; assign the selected bacteria to the genus Shewanella by the presence of green colonies with black centre or dark gray, surrounded by a zone of turbid precipitate in the medium.

Distinguishing the essential feature of the method is the use in a nutrient medium as an inhibitor of foreign microflora irgasan (0.14 to 0.2 g/l) and rifampicin (0,0005-0,001 g/l). Distinctive significant feature was not used in the prototype and the like, and is not known from the prior art. We were first installed on a dedicated strains high level of resistance of bacteria of the genus Shewanella (S.algae, S.putrefaciens) to irgasan: 128-256 mg/l Is allowed to use irgasan (DP-300) in a nutrient medium declare str is both within the range of such high concentrations (0.14 to 0.2 g/l), they are not used in a known selective media with irgasan, for example in a nutrient medium for isolation of Yersinia CIN-Agar (Hi-Media) content irgasan 0.004 g/l; in a nutrient medium for isolation of Pseudomonas Pseudomonas Isolation Agar (Hi-Media, Difco) 0.025 g/l irgasan. The concentration of rifampicin in the medium determined by us experimentally only in the range of 0.0005-0.001 g/l rifampicin does not inhibit the growth of shevenell and products of their sulfide with effective suppression of growth of gram-positive microorganisms. A nourishing environment of the proposed method has a high analytical sensitivity: 1-2 CFU/ml-1bacteria Shewanella (S.algae, S.putrefaciens).

Distinguishing the essential feature of the method directly determines the ultimate goal of improving the specificity of the isolation and identification of bacteria of the genus Shewanella. In the study of 80 strains of 30 species of 24 genera of bacteria, it was found that irgasan and rifampicin as part of the environment of the proposed method completely inhibit the growth of bacteria of the genus Salmonella (S.enterica serovar Enteritidis, S.enterica serovar Typhimurium) and the genus Proteus (P.vulgaris, P.mirabilis, P.penneri), producing hydrogen sulfide, but does not inhibit the growth of bacteria of the genus Citrobacter (complex .freundii, producing hydrogen sulfide). Thus, sharply reduced the number of bacteria colonies which need to be distinguished from colonies of shevenell on the basis of education the project of hydrogen sulfide. Moreover, these inhibitors completely inhibit the growth of bacteria of the genera Escherichia, Shigella, Enterobacter, Klebsiella, Hafnia, Staphylococcus, Bacillus (B.subtilis),but does not inhibit the growth of bacteria of the genera Serratia, Morganella, Providencia (P.rettgeri), Aeromonas, Vibrio, Pseudomonas, Stenotrophomonas, Acinetobacter, Achromobacter, Enterococcus.

The second distinguishing the essential feature of the method is the use of a complex of sodium thiosulfate (0.35 g/l), salt Mora, sorbitol for the detection of production of hydrogen sulfide by bacteria Shewanella and suppressing the formation of hydrogen sulfide by bacteria Citrobacter. This distinctive significant feature was not used in counterparts and only one component (sodium thiosulfate) is used in the prototype. Distinctive significant characteristic not obvious from the prior art. We experimentally found that only significantly reduced the concentration of sodium thiosulfate solution (0.35 g/l), in contrast to the concentrations in the prototype (2.0 g/l), provides in the presence of salt Mora and sorbitol complete suppression of the formation of hydrogen sulfide by bacteria Citrobacter with abundant formation of hydrogen sulphide in the colonies of bacteria Shewanella. Distinctive significant feature provides immediate goal - improving the specificity of the isolation and identification of bacteria shewanella as nutrient environment of the proposed method all the colonies with black coloration (producing Serovo the location) will contain only bacteria shewanella. Bronchiology blue and sodium hydroxide in a nutrient medium to provide an optimal pH environment and additionally differentiate bacteria Citrobacter (yellow color) and shewanella (green color).

The third distinctive essential feature of the method is the use of a complex of tween-80 and calcium chloride for detection of lipase bacteria. This feature was not used in the prototype and the like, but is known from the prior art. It directly provides the goal of increasing the specificity of the selection and identification of shevenell as it will differentiate colonies of shevenell having lipase (zone turbid precipitate around the colonies), from colonies of bacteria Citrobacter without lipase (no zone of precipitate).

The combination of these distinctive essential features determines simplify the isolation and identification of bacteria of the genus Shewanella, as the identification of colonies Shewanella on the basis of the formation of hydrogen sulfide, as well as by the presence of lipase and lack of fermentation of sorbitol reliably provides direct identification of these bacteria on a nutrient medium of the proposed method, which eliminates the need to place additional taxonomic tests generic identification.

Significant features of the process are some of the components of the nutrient medium, which provide a feeder of the diversified needs of the bacteria Shewanella, g/l: pancreatic hydrolysate of fish meal 12,0; peptone is an enzymatic meat 12,0; sodium chloride 6,0; agar 12,0. These components correspond to the composition of commercial nutrient medium "nutrient agar for cultivation of microorganisms dry (RM-agar), which is convenient for making the environment of the proposed method. Growing crops should be at 37°C and/or 28°C, as some species of bacteria of the genus Shewanella (S.baltica) do not grow at 37°C.

Examples confirming the possibility of implementing the method.

Example 1. Study material - bleed and have exudate from the chest cavity of the patient P. sow loops on the nutrient environment of the proposed method in a Petri dish, the composition and preparation of which is described in note; incubated crops at 37°C for 24 h under aerobic conditions and take into account the result: the presence in the medium of colonies with a diameter of 2-3 mm green color with a black center surrounded by a zone of turbid precipitate in the medium indicates their belonging to the genus Shewanella, accompanying the colony diameter of 3 mm yellow without black, surrounded by a zone of turbid precipitate, belong to a different genus of bacteria. Control and identification of selected isolates of additional tests species identification showed that bacteria isolated from green colonies with a black center surrounded by a zone of precipitation include the mind Shewanella algae; bacteria from yellow colonies without black, surrounded by a zone of precipitate, belong to the species Serratia marcescens.

Note to example 1. The method of preparation of the nutrient medium of the proposed method. In 1 l of distilled water contribute, g/l: pancreatic hydrolysate of fish meal to 12.0; peptone is an enzymatic meat - 12,0; NaCl - 6,0; agar - 12, 0mm; dissolve when heated, sterilized in a steam sterilizer at 121°C for 20 min; then in a hot environment add tween-80 (sterile) 5 ml; CaCl2- 0,2 (2 ml sterile 10% solution of CaCl2); sodium thiosulfate - 0,35; salt Mora - 0,25; sorbitol 13,0; bronchiology blue - 0,08 (5 ml of a 1.6% aqueous solution); irgasan (DP-300) to 0.14 (7 ml, 2% solution in 1 N NaOH solution); rifampicin - 0,0005 (0.5 ml 0.1% solution in dimethyl sulfoxide); NaOH - 0,8 (20 ml of 1 N NaOH solution); pH 7,2±0,2; pour into sterile Petri dishes. The environment has a green color, transparent, suitable for use within 14 days when stored between +4°C to +8°C. the Control of the nutrient medium is the presence of colonies with a diameter of 2-3 mm green color with a black center surrounded by a zone of turbid precipitate when using a control strain Shewanella algae 68 according to the method of example 1.

Example 2. The investigated material is water from the river Neva 0.1 ml of rubbing with a spatula on a nutrient medium of the proposed method in Petri dishes containing irgasan 0.2 g/l) and rifampicin 0.001 g/l (the rest of the comp is in the method of preparing the environment for the comment to example 1). Incubated crops at 28°C for 48 h, after which take into account the result of the presence in the medium of the two colonies with a diameter of 3-4 mm green with a wide black area in the center, surrounded by a muddy area of the precipitate in the medium indicates their belonging to the genus Shewanella; there was also a colony of the other genera of bacteria - yellow 3 mm in diameter without precipitation zone; 2 mm diameter blue color with an area of precipitation; 3 mm diameter blue with a broad area of precipitation. Control identification of the selected isolates showed that bacteria from one green colonies with black center and area precipitation are in the form of Shewanella putrefaciens; bacteria from the same colony are in the form of Shewanella baltica; bacteria from colonies yellow diameter, 3 mm without precipitation zone are Citrobacter freundii; bacteria from blue colonies with a diameter of 2 mm with an area of precipitation - Stenotrophomonas maltophilia; bacteria from blue colonies with a diameter of 3 mm with a broad area of precipitation - Aeromonas caviae.

Thus, when all of the limiting concentrations of the main components of the nutrient medium of the proposed method (irgasan, rifampicin), variations of the temperature regime (37°C and 28°C) and exposure time (24-48 h) obtained clear results of the detection and identification of bacteria of the genus Shewanella.

When studying the nutritional environment of the proposed method is of sevov 30 strains of bacteria, producing hydrogen sulfide (5 strains of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Proteus vulgaris, Proteus mirabilis, Citrobacter freuindii, Citrobacter braakii) and 48 26 strains of other bacterial species (except Shewanella)above, did not reveal a single false-positive result of their identification as Shewanella, that is, the inventive method has a high analytical specificity. A nourishing environment of the proposed method has a high analytical sensitivity for bacteria Shewanella - 1-2 CFU/ml-1.

The inventive method for the isolation and identification of bacteria of the genus Shewanella simplifies this study, as in the nutrient environment of the proposed method achieved a direct ancestral identification of Shewanella-type colonies without additional research.

Isolation and identification of bacteria of the genus Shewanella, involving the planting of the studied material on a nutrient medium containing sources of nitrogen, carbon, sulfur, carbohydrates, inhibitors extraneous microflora, pH indicators and the formation of hydrogen sulfide, agar and distilled water; incubation crops at 37°C and/or 28°C in aerobic conditions for 24 to 48 h; identify selected bacteria to the genus Shewanella by the presence of green colonies with black centre or dark gray, surrounded by a zone of turbid precipitate in the medium, characterized in that the culture medium contains as an inhibitor of foreign microflora irgasan (0.14 to 0.2 g/l) and rifampicin (0,0005-0,001 g/l); sodium thiosulfate (0.35 g/l); salt Mora, sorbitol, bronchiology blue, tween-80, calcium chloride, sodium chloride, sodium hydroxide in the following ingredients, g/l:

pancreatic hydrolysate of fish mealto 12.0
peptone is an enzymatic meatto 12.0
NaCl6,0
tween-805,0
CaCl20,2
Na2S2O3·5H2O0,35
(NH4)2SO4·FeSO4·6H2O0,25
sorbitol13,0
bronchiology blue0,08
irgasan (DP-300)0,14-0,2
rifampicin0,0005-0,001
NaOH0,8
agarto 12.0
distilled water

pH 7,2±0,2; hydrolyzed fish meal, peptone, sodium chloride and agar dissolve when heated, sterilized for 20 min at 121°C, then in a hot environment, add the remaining ingredients.



 

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1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method involves treating a water-based medium containing sulphate-reducing bacteria SRB in industrial aqueous systems of chemical production and oil refining. Inhibition of production of biogenic sulphide with SRB takes place as a result of synergetic action of a biocide component in a first concentration and a metabolic inhibitor in a second concentration. The biocide immediately destroys the first portion of SRB. The biocide component is selected from a group comprising aldehydes, amine-type compounds, halogenated compounds, sulphur compounds, salts of quaternary phosphonium and/or combinations thereof. The metabolic inhibitor inhibits growth of a second portion of SRB without its direct destruction. The metabolic inhibitor component is selected from a group comprising nitrite, molybdate, tungstate, selenate, anthraquinone and/or combinations thereof. Contact between the SRB and the biocide and metabolic inhibitor can take place continuously, intermittently or simultaneously.

EFFECT: method ensures efficient inhibition of production of biogenic sulphide with SRB during combined use of components in considerably lower concentrations than if the biocide or metabolic inhibitor was used separately.

25 cl, 2 dwg, 1 ex

FIELD: agriculture.

SUBSTANCE: method to produce a probiotic preparation based on sporogenous strains Bac.subtilis and Bac.licheniformis includes their cultivation, mixing of bacterial cells biomasses with protector of microbial cells and subsequent sterile dehydration. For this purpose the method applies the strain Bac.subtilis VKM V-2287 D and the strain Bac.licheniformis VKM V-2414D. Strain biomasses are mixed at the ratio of 1:1. A protector of microbial cells is lactose in amount that provides for cell titre in the finished product after drying of at least 1X1011 CFU/g.

EFFECT: method makes it possible to produce a pluripotential preparation, which may be used to prevent gastrointestinal diseases in farm animals and birds, prevention of stress actions, correction of microflora in intestine in case of digestion processes disturbance.

6 tbl, 2 ex

FIELD: agriculture.

SUBSTANCE: method to produce a probiotic preparation based on sporogenous strains Bac.subtilis and Bac.licheniformis includes their cultivation, mixing of bacterial cells biomasses with protector of microbial cells and subsequent sterile dehydration. At the same time the method uses the strain Bac. subtilis VKPM V-10172 and the strain licheniformis VPKM V-10135. Biomasses of strains are mixed at the ratio of 1:1. A protector of microbial cells is a gelatine-saccharose protective medium in amount that provides for cell titre in the finished product after drying of at least 2X1012 CFU/g.

EFFECT: method makes it possible to produce a preparation with high efficiency of probiotic component, which ensures higher digestibility of fodders and increases weight gain of farm animals and birds.

3 tbl, 1 ex

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention 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.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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