Vibrio aquamarinus strain, method of determining sample toxicity using same and testing culture for determining sample toxicity

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology and can be used for biotesting of toxicity of environmental media. The invention discloses a Vibrio aquamarinus bacterial strain, a method of determining toxicity of samples using said strain and use of the strain as a testing culture for determining chemical toxicity of samples. The method comprises measuring luminescence of the Vibrio aquamarinus VKPM V-11245 strain in the presence of at least one chemical toxicant of a sample, said toxicant being ZnSO4 or CuSO4 or K2Cr2O7 or oil or diesel fuel or bilge water or phenol or a heavy metal. Luminescence of said strain is measured without a chemical toxicant and the measured luminescence levels are compared. Change in luminescence indicates toxicity of the analysed sample.

EFFECT: invention improves reliability of detecting toxic substances in the environment.

9 cl, 4 tbl, 4 ex

 

The technical field to which the invention relates.

The invention relates to biotechnology and strains of bacteria for biological testing of the toxicity of environmental objects and can be used when conducting ecological toxicity studies, monitoring of aquatic ecosystems.

The level of technology

A known strain of bacteria Ph. phosphoreum (Cohn) Ford, which is recommended for biotesting of water on the territory of Ukraine (I. Y. Malygin, A. M. Katsev. Luminous bacteria of the Black and Azov seas, KND 211.1.4.060-97) (1, 2). A known strain testing is used in dried form and you want to restore its active form. In addition, the sensitivity of this strain to toxic substances is not high enough, in particular a known strain allows to determine the presence of toxic substances in concentrations exceeding MAC.

Known bacterial test "Ecolum" developed in Russia (Moscow) (3). Biosensor "Ecolum" is a lyophilized culture of genetically engineered strains of luminescent bacteria contained in the inert gas in special glass bottles. The culture obtained through genetic engineering introduction of lux-operon in specially selected strain of E. coli. Produced according to TU 6-09-20-236-93. (3) is Used to determine toxin the STI water (4), soil (5), chemical compounds, polymers, materials, products (6).

The disadvantage of this strain is not sufficiently high sensitivity to toxic substances, in particular to their presence in concentrations at the MPC level and below. The test uses a freeze-dried culture, which is biologically inactive form and to move to an active physiological state, it takes time to go through several cell divisions. The disadvantage should be considered and the high cost of production of the biosensor due to the use of costly lyophilization. In addition, the drug biosensor comes only when the initial purchase of the device ecological control "Biotone-10M.

Known strains of Vibrio fischeri VKPM B-9580 and Vibrio fischeri VKPM B-9579 isolated from waters of the Black sea, which can be used to test the toxicity of the environment (7) (8).

As a prototype of a selected strain of Vibrio fischeri In PMBC-9579 (8). Biotesting is based on determining the change in the intensity of bioluminescence strain Vibrio fischeri VKPM B-9579 when exposed to toxic substances present in the analyzed sample, compared to control.

The sensitivity of this strain to toxic substances is not high enough, in particular a known strain n is sufficiently reliably detects the presence of toxic substances in concentrations at the MPC level and below.

Disclosure of inventions

The purpose of this invention to provide a new, accessible and affordable strain of bacteria, characterized by high sensitivity of bioluminescence to the action of toxic substances of the various chemical nature.

To achieve the objectives proposed bacterial strain Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054), isolated from the Black sea water collected in the area of Abrau-Durso.

One of the embodiments of the present invention is a strain of bacteria Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) to determine the toxicity of the samples.

In another embodiment the present invention relates to a method of determining the toxicity of the samples, including the measurement of luminescence of the bacterial strain Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) in the presence of at least one toxicant samples, the measurement of the luminescence of the specified strain in the absence of toxicant and comparing the measured levels of luminescence.

In another embodiment of the present invention by the above method to determine the toxicity of environmental samples.

In yet another embodiment of the present invention toxicant is a ZnSO4, CuSO4or K2Cr2O7. Also toxicant can be a oil products, polychlorinated biphenyl, or heavy metal.

Also present from the Britania in one of the embodiments is the use of the bacterial strain Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) as a culture test to determine the toxicity of the samples.

The implementation of the invention

To highlight the strain of bioluminescent bacteria from the seawater used method of membrane filters. Bacteria were concentrated from the analyzed water on a membrane filter and then grew them on selective media for fluorescent bacteria. The allocation of bioluminescent bacteria was performed, analyzing visually the presence of bioluminescence. Upon detection of luminescence was isolated pure culture of bacteria by standard methods.

The determination was carried out according to results of the analysis of sequence variable regions of 16S rDNA. The results of the analysis of the tested strain of the microorganism belongs to the genus Vibrio (homology of 98%).

New bioluminescent strain was characterized by phenotypic and genotypic. Identification of the strain was carried out on the basis of a study of its cultural-morphological and physiological-biochemical characteristics in accordance with the description given by the determinant of bacteria Bergey (9).

For the final installation of the type strain is made identification of genetic characteristics in the FSUE "Gosniigenetika", PMBC. The determination was carried out according to results of the analysis of sequence variable regions of 16S rDNA.

Phylogenetic analysis based on comparison of 16S rRNA sequences showed that this strain belongs to novodonesenom species of this genus Vibrio. Based on all these data, the strain was classified as a new kind, called Vibrio aquamarinus VKPM B-11245 {Vibrio aquamarinus DSM 26054).

This strain is characterized by the following cultural-morphological characters: cells with a diameter of about 1-1 .5 μm and a length of 2-3 µm, straight, slightly curved rods, motile due to a single polar flagellum. The strain grows on many natural and synthetic environments. On TCBS (Thiosulfate Citrate Bile Salts Sucrose Agar) colony green, 3-5 mm in diameter. On LB medium with the addition of 1.7% NaCl grows in the form of luminescent, translucent colonies rounded, with smooth edge, yellowish color. For growth requires the presence in the environment from 0.5 to 5% (optimum 1-4%) NaCl (w/v), but not at 8 or 10%; and the temperature 10-35°C (optimum 20-25°C).

Physiological and biochemical characteristics: optional gone anaerobic. Gram-negative, oxidizability, catalogoplantillas. Education indole positive. Hydrogen sulfide is not formed. Gelatin is not hydrolyses. Starch hydrolyses. Reaction Voges-Proskauer (education acetylaminophenol) is negative.

Positive for the following enzymatic activities: Ala-Phe-Pro-arylamidase, β-glucosidase, L-Polyarylamide, amylase, nitroreductase.

Negative on the next enzymatic activity: L-pyrrolidinedione, D-cellobiose, β-galactosidase, the-N-acetylglucosaminidase, glutamyltransferase pNA, γ-glutamyltransferase, β-xyloside, β-aluminosilicate pNA, lipase, palatinose, tyrosinaemia, urease, α-glucosidase, β-N-acetylgalactosaminidase, α-galactosidase, phosphatase, glycinamide, ornithindecarboxilase, isindeterminate, β-glucuronidase, Glu-Gly-Arg-arylamidase, phenylalanine deaminase, arhinencephaly, gelatinase.

Catabolize carbohydrates such as D-glucose, D-maltose. Do not utilize D-cellobiose, arabinose, D-mannose, rhamnose, D-tagatose, D-trehalose, sucrose, raffinose, lactose. Doesn't remove the alcohols of adonitol, L-Arabic, D-mannitol, D-sorbitol, Inositol, dulcet, as well as the following salts: malonate, 3-keto-gluconate, L-lactate, succinate, L-malate, citrate (sodium).

Not resistant to O/129 (2,4-diamino-6,7-diisopropylaniline) vibrostation agent.

Strain has no toxic and pathogenic properties. Emits light, visually recorded in the darkness. The luminescence spectrum of Vibrio aquamarinus falls on the "blue" area. The spectrum maximum bioluminescence is 478 nm.

Stored strain on nutrient agar with 3% NaCl, pH 7,2-7,4.

The method, conditions and medium composition for long-term storage of strain:

- storing bacteria in the refrigerator freezer-refrigerator at -18°C in nutrient broth with 1.7% NaCl containing as cryoprotectant sterile is glycerin;

- storage of bacteria at low temperature refrigerator at a temperature of -60°C in nutrient broth with 3.0% NaCl containing as cryoprotectant sterile glycerol;

- freezing the cells in liquid nitrogen in the nutrient broth with 1.7% NaCl containing as cryoprotectant sterile glycerol;

- in a hermetically sealed ampoule in a freeze-dried state.

The reproduction of culture by subculture on nutrient agar with 3.0% NaCl.

The practical application of this strain is due to its physiological and biochemical characteristics. Strain designed for biotesting toxicity, as characterized by a high sensitivity of bioluminescence to the action of a wide range of toxicants.

Example 1

Implementation of the use of the bacterial strain Vibrio aquamarinus VKPM B-11245 as a culture test to determine the toxicity of environmental factors chemical origin was held at the toxicants ZnSO4, CuSO4, KiCr2O7. sodium dodecyl sulfate (LTOs) and phenol.

The strain of Vibrio aquamarinus VKPM B-11245 were grown on LB-agar with 3.0% NaCl at a temperature of 25°C during the night.

Then preparing a suspension of overnight culture of luminous bacteria. For this night culture was diluted using a densitometer DEN-1 ("BioSan") to turbidity 1 unit Mac-Farland (concentration of 3·108CL is current/ml). Then was diluted suspension in 100 times. The suspension of bacteria was prepared so that the final concentration of NaCl was 3%; glucose - 0,01% in 0.001 M Tris-HCl buffer (pH 7.4).

Aliquots of culture in 180 µl was transferred into the wells, some of them served as a control was added 20 μl of distilled water). In other wells were made in 20 μl solutions of toxicants.

Measurement of luminescence was performed with a microplate luminometer LM-01T (Immunotech). The measurement was carried out for 35 minutes, with an interval of 5 min at 20-25°C.

The results of measurement of the intensity of bioluminescence was presented in arbitrary units of fluorescence (his first-ever competition).

Assessment of the toxicity of the samples was carried out according to the relative difference in intensity of bioluminescence control and experimental samples. For comparative evaluation of the sensitivity of the used characteristic EC (effective concentration) is the concentration of substance that causes 50% reduction in bioluminescence bacterial suspension.

Data on the sensitivity of strains of luminous bacteria (average EU50all investigated toxicants are presented in table 1. The table also shows the sensitivity to the same toxicants lux-strain Vibrio jischeri VKPM B-9579.

Comparison of the sensitivity of bioluminescence strains investigated toxic in which the substances showed what strain of Vibrio aquamarinus VKPM B-11245 more sensitive than Vibrio fischeri VKPM B-9579 (average EU50).

Table 1
EU 50, mg/l
StrainZnSO4CuSO4LTOsK2Cr2O7Phenol
Vibrio aquamarinus VKPM B-112450,30,4101-10225-275
Vibrio fischeri2-31-1,515010-50125-150
VKPM B-9579

The EC50for ZnSO4·7H2O the strain Vibrio aquamarinus VKPM B-11245 is 0.3 mg/l (concentration of zinc is 0,068 mg/l) and the concentration of EU50for CuSO4·3H2O - 0.4 mg/l (respectively, the end of the acidity of copper is is 0.102 mg/l).

The EC50the strain Vibrio aquamarinus VKPM B-11245 for bichromate of potassium is in the range of concentrations from 1 to 10 mg/L.

For phenol value EC50for strain Vibrio aquamarinus VKPM B-11245 was 225-275 mg

Compared to the biosensor strain of Vibrio fischeri In PMBC-9579 (the lower limit of its sensitivity) strain Vibrio aquamarinus VKPM B-11245 more sensitive to ZnSO4(7 times), CuSO4(2.5 times), LTO (15 times). The bichromate of potash strain Vibrio aquamarinus VKPM B-11245 more sensitive (10 times). To the phenol Vibrio aquamarinus a little less sensitive.

High sensitivity of Vibrio aquamarinus VKPM B-11245 shows the viability of using this strain to determine the toxicity of aquatic environments.

Example 2

Of great interest is the reaction of luminous bacteria at low concentrations of contaminants comparable to MAC, and below that allows you to use this strain as the test culture when determining low concentrations of pollutants.

Measurements were carried out, the sensitivity of the bioluminescence of the isolated strains to the toxic action of chemical factors origin at a very low concentration. We used the following toxicants: ZnSO4, CuSO4, K2Cr2O7.

Toxicity testing was performed as described in example 1.

Table 2 presents data on the sensitivity b is luminescence Vibrio aquamarinus VKPM B-11245 to the above toxicants in the concentration range from 0.00001 mg/l to 1.0 mg/L.

As can be seen from table 2, low concentrations of toxicants cause Vibrio aquamarinus VKPM B-11245 stable induction of bioluminescence.

Copper at a concentration of 0.001-0.005 mg/l (MPC copper - 0.005 mg/l for marine water) increases the intensity of luminescence within 35-32%.

Zinc at a concentration of 0.001-0.005 mg/l (MPC zinc - 0.05 mg/l for marine water) increases the intensity of bioluminescence strain within 19-31%.

The bichromate of potash (TLV 0.05 mg/l), at a concentration of 0.001-0.005 mg/l increases the intensity of bioluminescence strain about 38-35%.

It should be noted that a significant induction of luminescence strain Vibrio aquamarinus VKPM B-11245 was observed at the level of the MPC toxicants (10) for copper and potassium bichromate, and below the exposure limits for copper, zinc and potassium bichromate.

According to data given in the patent (8) for Vibrio fischeri VKPM B-9579 have been reported following indicators:

Copper at a concentration of 0.001-0.005 mg/l increased the luminescence within 36-42%. Zinc at a concentration of 0.001-0.01 mg/l increased the intensity of bioluminescence strain within 18-26%. The bichromate of potassium in a concentration of 0.001-0.01 mg/l increased the intensity of bioluminescence strain of about 30%.

Vibrio aquamarinus VKPM B-11245, compared to the biosensor strain of Vibrio jischeri VKPM B-9579 more sensitive to the concentrations of toxicants at MPC and lower - value induction glow strain often achieves greater in the guises and is recorded at lower concentrations of the substances.

The obtained data indicates high sensitivity strain Vibrio aquamarinus VKPM B-11245 and the prospects of its use for determining the toxicity of the components of the aquatic environment.

Example 3

Experiments were carried out on the use of the bacterial strain Vibrio aquamarinus VKPM B-11245 as a culture test on the toxicity of petroleum products.

Were tested oil, diesel fuel and podslanevye (bilge water from the engine room of the vessel in concentrations of from 0.00001 mg/l to 5.0 g/L.

Toxicity testing was performed as described in example 1.

Data on the sensitivity of bioluminescence strain Vibrio aquamarinus VKPM B-11245 all investigated products are presented in table 3.

Oil, starting with concentrations below the exposure limits oil (0.05 mg/l) inhibited luminescence of Vibrio aquamarinus VKPM B-11245. At a concentration of 0.05 mg/l (MPC) reduced the intensity of the glow reached 13%.

Bilge water and diesel fuel also inhibited the glow of this strain, since concentrations below the level of the MPC. At MPC (0.05 mg/l) inhibition of luminescence was 36% and 20%, respectively.

The EC50for oil, diesel fuel and bilge water was 100 mg/L.

The EC50for oil, diesel fuel and bilge water to strain Vibrio fischeri VKPM B-9579, apparently, is more than 1000 mg/L.

With Auntie the sensitivity of the strain Vibrio aquamarinus VKPM B-11245 and Vibrio jlscheri VKPM B-9579 (8) indicates a higher sensitivity of the strain Vibrio aquamarinus VKPM B-11245 and the prospects of its use for determining toxicity components of the marine environment of the waters.

Table 2
The name of the toxicantControlThe concentration of toxicant, mg/l
0,000010,00010,0010,0050,010,050,11
The intensity of the bioluminescence of Vibrio aquamarinus VKPM B-11245, his first-ever competition
Copper349359394473461356,5221,5188,5a 38.5
Zinc349351361417456398309294,597
Ksub> 2Cr7O7349365,5451481473460397346,5181,5
The intensity of the bioluminescence of Vibrio fischeri In PMBC-9579, his first-ever competition
Copper1,622,152,222,3*2,22,081,831,690,803
Zinc0,4440,4950,5190,5260,5400,5590,5000,5200,385
K2Cr2O70,4300,5120,5450,5620,5750,5570,553 0,5440,552

Table 3
Name of productConcentration of oil products, mg/l
Control0,010,050,10,515105010010005000
The intensity of bioluminescence strain Vibrio aquamarinus, yec
oil661617,5577546,5542551,5503486,5498,5361,5284,5138
diesel fuel661438 421416407400399,396382,5350,5336,5197,5
bilge water661538,5531531498of 451.5426,5446,5432,5343,5309214
The intensity of bioluminescence strain Vibrio fischeri VKPM B-9579, yec
oil0,300,490,620,640,560,560,36
diesel fuel0,340,52 0,520,630,550,540,36
bilge water0,310,450,460,480,530,540,30

Example 4.

To determine the toxicity of natural samples using bacterial strain Vibrio aquamarinus VKPM B-11245 samples were collected sediment marine water bodies.

The sample with high content of heavy metals and polychlorinated biphenyls was selected in an area with high anthropogenic load on the Black sea in the port, Tuapse.

Another ground for biotesting was selected in a relatively clean area of the Black sea in the area C. Arkhipo-Osipovka.

Toxicity testing was performed as described in example 1.

Table 4 presents the results of biological testing of the toxicity of the average of the soil samples collected in Uslon the clean area of the sea and harbour area. As control was used 3.0% NaCl solution.

Table 4
Area sampling of sedimentsThe intensity of the bioluminescence of Vibrio aquamarinus VKPM B-11245, his first-ever competition
controlexperiencedeviation from control, %
Water district C. Arkhipo-Osipovka102381220,6
Port, Tuapse179885152,7

From table 3 it follows that the intensity of the bioluminescence of an aqueous extract of bottom sediments taken in a relatively clean area of the Black sea, above the control level by 20.6%, while the intensity of illumination of the sample extract soil from the seaport of - 52.7%. The obtained data demonstrate the sensitivity of bioluminescence strain Vibrio aquamarinus VKPM B-11245 to the action of toxicants present in bottom sediments of water bodies.

Thus, testing the toxicity of one of the main components of environment - sediment, prodemonstrirovavshih use of the proposed strain Vibrio aquamarinus VKPM B-11245 as the test culture.

Sources of information

1. I. Y. Malygin, A. M. Katsev. Luminous bacteria of the Black and Azov seas. Ecology of the sea. 2003. Vol.64.

2. KND 211.1.4.060-97. Viznachennya toxicness water to the bacteria Photobacterium phosphoreum (Cohn) Ford. - 21.05.97.

3. THE 6-09-20-236-93.

4. Mr No. 11-1/133-09. Methodology for rapid determination of water toxicity using luminescent bacterial test "Ellum".

5. Mr No. 11-1/134-09. Determination of total toxic intensity of bioluminescence of bacteria.

6. Mr No. 11-1/131-09. Determination of the toxicity of chemical compounds, polymers, materials and products using luminescent bacterial test.

7. Patent RU 2346035, IPC C12N 1/20, C12R 1/01.

8. Patent RU 2342434, IPC C12Q 1/02, C12R 1/63 (prototype).

9. The determinant of bacteria Burgi. So 1. Ed. j.Holt, N. Krige, P. Snita, S. Williams. - M.: Mir, 1997. - 432 S.; Brief determinant of bacteria Berga. Edited by j.Holt. - M.: Mir, 1980. - 496 S.

10. Standards of water quality fisheries values, including the standards of maximum permissible concentrations of harmful substances in the waters of the water bodies of fishery significance." - Appr. By order of the Russian Federal fisheries Agency on 18.01.2010. No. 20. - 214 S.

1. Bacterial strain Vibrio aquamarinus VKPM B-11245 (Vibrio aquamarinus DSM 26054) to determine the chemical toxicity of the samples.

2. The method of determining chemical toxicity tests, including the measurement of fluorescent lamps is ncii strain under item 1 in the presence of at least one chemical toxicant samples measurement of luminescence strain under item 1 in the absence of this toxicant and comparing the measured levels of luminescence, and the measurement of luminescence indicates the toxicity of the sample.

3. The method according to p. 2, which determine the toxicity of environmental samples.

4. The method according to p. 2 or 3, in which the toxicant is a ZnSO4, CuSO4or K2Cr2O7.

5. The method according to p. 2 or 3, in which the toxicant is a petroleum product.

6. The method according to p. 5, in which the oil is a crude oil, diesel fuel or bilge water.

7. The method according to p. 2 or 3, in which the toxicant is a phenol.

8. The method according to p. 2 or p. 3, in which the toxicant is a heavy metal.

9. The application of strain under item 1 as the test culture to determine the chemical toxicity of the samples.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: method comprises treatment of potato tubers before placement for storage with biological product containing biomass Bacillus amyloliquefaciens RNCIM B-11008 with a titre of vegetative cells and spores of 1.24÷1.30×1010 CFU/ml and humates. At that the content of components of biological product is respectively 99 vol. % and 1.0 vol. %. The biological product is taken in a rate of application of 1.0 l/ton of potato tubers at a rate of application of working fluid of 10.0 l/ton.

EFFECT: inhibition of development of fungal and bacterial diseases of potato tubers during storage.

6 tbl, 5 ex

FIELD: biotechnology.

SUBSTANCE: strain Paenibacillus sp. IB-1 deposited in the Russian Collection of Microorganisms under the registration number VKM B-2823D. Strain Paenibacillus sp. VKM B-2823D has exopolysaccharide-producing ability and can be used, in particular in the petroleum refining industry.

EFFECT: invention enables to increase the yield of exopolysaccharide and expand the range of exopolysaccharides having good rheology and high solubility in highly mineralised formation waters.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology. The method aims at preventing mycotoxicosis in horses. It provides adsorbing the preparation 'Sakhabactisubtil' representing a suspension of the bacterial strain Bacillus subtilis TNP-3-DEP and Bacillus subtilis TNP-5-DEP in equal proportions, adsorbed on oats, and feeding the animals with oats once a day at 10ml of 1kg of oats for 10 days.

EFFECT: invention provides normalising the intestinal microbiocenosis and increasing the animal's body weight.

1 tbl

FIELD: biotechnology.

SUBSTANCE: carrier material for biomass for filtration of oil-contaminated waste waters is proposed. The carrier comprises a filtering material with immobilised cells of oil-oxidising microorganism Rhodotorula sp. VKM Y-2993D with the titer of cells - 106 CFU/cm3. The filtering material is used as basalt fibre STBFconst preliminary modified with cationic starch - oxyamil OPV-1.

EFFECT: proposed carrier material has a high retention capacity of suspended particles and oil products and is used for filling the filters for cleaning oil-contaminated waste waters.

1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: proposed strain Pseudomonas migulae is deposited in the Russian National Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms under the number of VKM B-2761D. The strain is isolated from samples of bottom soil contaminated with oil of freshwater lake of Usinsky district of the Komi Republic in the zone of chronic oil pollution.

EFFECT: strain has high oil-oxidising activity against naphthenic hydrocarbons, is capable of reducing the concentration of hydrocarbons dissolved in water and accelerates the destruction of oil in soils and bottom sediments of water reservoirs.

3 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: inventions relate to a strain of the fungus Penicillium verruculosum B10 EGII and a method of production of the fodder complex enzyme preparation. The presented strain is a producer of endo-1.3/1.4-β-glucanase, cellulase, β-glucosidase and xylanase. It is obtained on the basis of a strain Penicillium verruculosum RNCM F-3764D, by transforming with its plasmids pSTA10 and pPrCBHI-EGII. In the plasmid pPrCBHI-EGII the nucleotide sequence of the structural gene of endo-1.3/1.4-β-glucanase of Penicillium verruculosum is aligned with nucleotide sequences of the promoter region, the signal peptide and the terminator of gene cbhI of cellobiohydrolase I Penicillium verruculosum. The method of production of the preparation containing endo-1.3/1.4-β-glucanase, cellulase, β-glucosidase and xylanase comprises cultivation of the strain of fungus Penicillium verruculosum B10 EGII on the optimised nutrient medium in the fermenter and filtration, ultrafiltration and lyophilisation of the culture liquid.

EFFECT: inventions can be used to produce fodder complex enzyme preparation enriched with endoglucanase.

2 cl, 1 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to microbiology and biotechnology. What is presented is the strain Enterococcus mundtii GKPM (State Collection of Industrial Microorganisms) - Obolensk V-7424 producing a peptide substance having the antilisterial activity.

EFFECT: antimicrobial peptide substance Enterococcus mundtii GKPM - Obolensk V-7424 possesses the activity about 6400 - 12800 antigen units/ml on the test strain Listeria monocytogenes 776 and can be used for the decontamination of bioproducts, as well as materials infected by bacterial pathogens.

2 dwg, 4 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: method comprises inoculation of the test strain on the lawn of the culture, applying a drop of diagnostic phage preparation in centre of the cup, holding at the temperature of 37°C for 20-24 hours and confirmation of its belonging to the microorganisms of the species V. parahaemolyticus in the presence of lysis. At that the diagnostic phage preparation is prepared by mixing phages FC-44 and FC-46, prepared based on the strain Vibrio parahaemolyticus KM-97, in the ratio of 1:1 in the titre n·108 PFU/ml.

EFFECT: present invention enables to accelerate the identification of strains and can be used in the laboratory diagnostics of acute intestinal infections caused by this microorganism.

2 cl, 1 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: bacterial strain Rhodococcus rhodochrous is deposited in the Russian Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms RAS under the registration number RCM Ac-2018 D. The destruction with this strain within 7 days of crude oil is up 100%, oil sludge - up to 98%, black oil - up to 96%.

EFFECT: strain has high destructive activity against petroleum hydrocarbons included in oil sludge, as well as against crude oil and black oil.

1 dwg, 1 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: strain Gordona sp. is deposited in the Russian National Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms RAS under the registration number RNCM Ac-2044 D. The destruction with this strain on the 7th day of crude oil is up to 100%, oil sludge - up to 99%, black oil - up to 97%.

EFFECT: strain has high destructive activity against petroleum hydrocarbons included in oil sludge, as well as against crude oil and black oil.

1 dwg, 1 tbl, 3 ex

FIELD: agriculture.

SUBSTANCE: invention relates to the field of quality control of disinfection. The method comprises disinfection of premises, selection of test objects which are used as a suspension Bacillus subtilis of the strain ATCC PTA-6737 (PB6) sorbed on the carrier - the filter paper with an adhesive surface part. The test objects are placed in the premises of disinfection and added to the nutrient medium. The tubes are heated to 60-80°C and cultured at 37°C for 12-24 hours followed recording the result by recording the growth of bacteria after 12 hours. Clouding of the nutrient medium to form a mucous film is indicative of inadequate quality of disinfection, absence of clouding and formation of a film after 24 hours is indicative of the satisfactory disinfection.

EFFECT: invention enables to simplify the quality control of disinfection of premises.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and microbiology and represents a method for identifying a cluster of antigens coding staphylococcal proteins that are exotoxins. The present method is implemented by performing a single multiple-primer polymerase chain reaction in two reaction mixtures, first of which contains primers to genes coding such staphylococcal proteins, as thermonuclease, beta-glucosidase in the species S.aureus, S.epidermidis, S.haemolyticus, S.lugdunensis, S.saprophyticus, and the second one - to set1, set2, set3, set4, set5 genes coding the exotoxins. That is followed by a comparative analysis of amplified gene fragments prepared in two sample aliquots and a positive control reference according to the identification table. The present invention also discloses a test system for implementing the above method, which contains DNA recovery components, PCR components, and result analysis components. The PCR components contain a 10-merous buffer solution, pH 8.4, deionised sterile water, one positive control reference, Taq polymerase, mixture of four dNTPs, mixture of primers No.1 containing epi-F, epi-R, aur-F, aur-R, hae-F, hae-R, lug-F, lug-R, sap-F, sap-R in a ratio of 1:1:1:1:1:1:1:1:1:1, and a mixture of primers No. 2 containing set1-F, set1-R, set2-F, set2-R, set3-F, set3-R, set4-F, set4-R, set5-F, set5-R in a ratio of 1:1:1:1:1:1:1:1:1:1.

EFFECT: invention enables recovering the cluster of genes coding the staphylococcal proteins, a molecular weight of which makes 25 to 35 kD consisting of almost 200 amino acid residues and having a tertiary structure high-homologous with some staphylococcus superantigens (enterotoxins, TSST-1 toxins) and with pyrogenous streptococcal exotoxin C.

2 cl, 3 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: claimed solutions deal with a method of generating a starter culture, the starter culture and a fermentation method with an application of the said starter culture. The claimed method of generating the starter culture includes impact on a parent bacterial strain, which contains, at least, a part of the locus CRISPR, with a bacteriophage to obtain a mixture of bacteria, which contains a bacteriophage-resistant variant strain, containing the modified locus CRISPR, which contains, at least, one additional spacer in the said modified locus CRISPR; independent impact on the same parent bacterial strain with the same bacteriophage to obtain the mixture of bacteria, which contains other bacteriophage-resistant variant strain, containing the modified locus CRISPR, which contains, at least, one additional spacer in the said modified locus CRISPR, different from the additional spacer in the first bacteriophage-resistant variant strain, selection of the said bacteriophage-resistant variant strains from the mixtures of bacteria and their separation.

EFFECT: claimed inventions make it possible to obtain bacteriophage-resistant cultures and can be applied in the food industry in manufacturing fermented products.

29 cl, 23 dwg, 20 tbl, 23 ex

FIELD: biotechnology.

SUBSTANCE: two suspensions are prepared. Clinical polyantibiotic-resistant strains Escherichia coli are added to an isotonic solution of NaCl to achieve the concentration of 30-40 thousand CFU/ml. Copper nanoparticles are added to the solution of NaCl to achieve the concentration of 0.01-0.05 mg/ml. The suspension of ethylenediaminetetraacetic acid - EDTA is prepared by its dilution in distilled water at the rate of 0.1-0.2:1, respectively. NaOH is added to the prepared suspension to obtain the solution of EDTA with pH=7.6-8. The prepared suspensions are connected with the solution of EDTA in the following ratios by wt %: suspension of copper nanoparticles - 70-85, suspension of microorganisms - 10-20, EDTA - 5.10. It is incubated in the shaker at 100-150 rev/min and a temperature of 36-38°C for 40-60 minutes. The resulting biomass is inoculated on the solid nutrient medium with the volume of 20-25 ml in the amount of 0.1-0.12 ml. It is incubated in the thermostat at a temperature of 36-38°C for 18-24 hours. The sensitivity of E.coli strains to antibiotics is determined.

EFFECT: invention enables to increase the sensitivity of the said bacterial strains to antibiotics gentamicin and ampicillin.

2 tbl, 2 cl, 1 ex

FIELD: biotechnology.

SUBSTANCE: granule of biocatalyst with the cells contained in it of one or more species of Rhodococcus is placed in a well of a 96-well plate, a colorant iodine-nitro-tetrazolium (INT) is added. After the appearance of specific purple staining formazan the optical density of granule is measured using a tablet photometer. In the case of a stained substrate or preliminary presence of the immobilised biocatalyst in the soil the extraction of formazan is carried out with 96% ethanol and the optical density of the extract is measured. The amount of viable Rhodococcus in the granule of the gel is determined using the calibration dependency diagram of the optical density on the number of living cells in suspension as determined by traditional method of plating on solid growth medium. Then, from the granule stained by the colorant of the biocatalyst or from the granule after extraction of the colorant the DNA is isolated and PCR is performed using sets of species-specific primers. The specific position of the immobilised Rhodococcus is determined.

EFFECT: invention enables to reduce the time differentiation of Rhodococcus and to improve the accuracy of the method.

2 cl, 3 tbl, 4 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The method comprises collecting soil samples, adding metsulfuron-methyl (MSM) to the sample in a given amount, followed by incubation and determining specific metabolic activity of the saprophytic soil complex by multisubstrate testing and counting the number of colony-forming units on a diluted agarised medium. The level of the detoxification activity is estimated based on the relative increase in specific metabolic activity of the saprophytic microbial community of the soil when MSM in comparison with characteristics of the soil without adding MSM (Kd factor).

EFFECT: improved method.

4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. Claimed is a method of growing colonies of microbial cells on a surface of a porous plate. The method includes supply of a nutrient solution from bottom to top through the porous plate into zones of growth of colonies of the microbial cells on its upper surface, supply of a suspension of the microbial cells onto the upper surface of the porous plate, creation of controlled conditions for the colony growth, performing observation of the colony growth, separation of the grown colonies of the microbial cells from the zones of growth and their transfer into external means of identification. The nutrient solution is supplied into the zones of growth of the colonies of the microbial cells by creation of a pressure difference between the hole input and output. Holes are made in the plate from an anode aluminium oxide orthogonally to its large plane and are topologically coded. The said zones of growth are formed in them in the form of porous membranes. The porous membranes are located at the same level as the upper surface of the plate or with formation of a hollow and do not pass the microbial cells. After supply of the nutritional solution, the suspension of the microbial cells of a specified concentration is supplied onto the upper surface of the plate until their homogenous distribution is achieved. Between the zones of growth on the surface of the plate a film, preventing attachment of the microbial cells, is formed. Separation of the grown microcolonies from the zones of growth is performed by hydroblow. A hydroblow is directed from the side of the input of cylindrical holes of the plate and spreads along them and farther through the pores of the porous membranes with force, which does not destroy the microcolonies but is sufficient for their separation from the growth zones. Also claimed is a device for growing the colonies of the microbial cells by the claimed method.

EFFECT: providing conditions of automation of processes of the nutrient solution supply and processes of separation and transfer of the grown colonies, possibility of integration into miniature portable devices, and application in laboratories on a chip and provision of the device portability.

6 cl, 14 dwg, 4 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and can be used in monitoring environmental-microbiological investigation of the quality of sea water to determine the amount of oil-oxidising microorganisms. The method involves preparing a mineral medium - bases containing NH4NO3, K2HPO4, KH2PO4, MgSO4, CaCl2, FeCl2, a concentrated solution, agar and distilled water in a given ratio, followed by addition of an oil product in a given amount, said product being bunker oil. Seeding sea water on the surface of the culture medium and incubating the seed for 3-4 hours enables to detect colonies of oil-oxidising bacteria.

EFFECT: invention increases precision of the method when detecting oil and hydrocarbon oxidising bacteria when carrying out environmental monitoring.

2 tbl, 3 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: method of toxicity assessment of products from polymer and textile materials is proposed. The method comprises the use of biosensor based on oxygen electrode, immobilisation of whole cells of bacteria E.coli K-12 on the surface of the oxygen electrode. The immobilisation is carried out using a semipermeable membrane. After immobilisation the respiratory activity of microorganisms is measured in the presence of the sample and standard samples of positive and negative control. Then the toxicity index is calculated and the sample toxicity is evaluated based on the value of the toxicity index.

EFFECT: simplifying assessment of toxicity and improvement of reliability of the results of the sanitary-epidemiological expertise.

2 dwg, 1 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: invention is a method of determining the nonspecific resistance of pathogenic microorganisms to antibiotics and the fact of the presence of bacterial biofilms on the basis of measurement of catalytic activity of phosphodiesterases cleaving the cyclic diguanosine monophosphate, with a threshold sensitivity of 50 pg/ml, comprising: 1) isolating the target-phosphodiesterase from lysed bacterial cells; 2) binding of phosphodiesterase with biotin-conjugated antibodies specific for non-catalytic domains of phosphodiesterase; 3) affinity purification of complexes formed by target-phosphodiesterase and biotin-conjugated antibody using paramagnetic particles containing neutravidin or its analogs that bind biotin; 4) interacting of the complexes of phosphodiesterase/biotin-conjugated antibody, immobilised on paramagnetic particles with complexes containing a-di-GMP in the form of G-quadruplex systems with intercalate dye, which is accompanied by decrease in the intensity while destruction of complexes of intercalate dye with c-di-GMP; 5) measurement of decrease of fluorescence upon hydrolysis with c-di-GMP and destruction of complex of c-di-GMP with intercalate dye, followed by quantitative estimation of the phosphodiesterase activity based on calibration curves made using known amounts of the recombinant enzyme of phosphodiesterase identical to the test target; 6) identification of increased level of phosphodiesterase activity detected by the test antibiotic-resistant bacterial strains capable of biofilm formation, as compared with the level of phosphodiesterase activity that can be detected for the control strains of bacteria of the same species not having the antibiotic resistance and the ability to form biofilms.

EFFECT: method enables to determine the nonspecific resistance of pathogenic microorganisms to antibiotics and to establish the fact of the presence of bacterial biofilms.

4 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: strain Rhodococcus erythropolis is proposed, deposited in the Russian National Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms under the registration number VKM Ac-2611D. The strain is isolated from the bottom sediments of chronically polluted freshwater lakes in the Usinsk region of the Komi Republic in 2007. The destruction efficiency of oil is 97.76%.

EFFECT: strain exhibits oil-oxidising activity to naphthenic, aromatic and polyaromatic oil fractions in a wide temperature range.

1 dwg, 2 tbl, 2 ex

Up!