Nutrient medium for growing legionella. RU patent 2510828.

IPC classes for russian patent Nutrient medium for growing legionella. RU patent 2510828. (RU 2510828):

C12R1/00 - INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C-C12Q; OR C12S, RELATING TO MICRO-ORGANISMS

C12N1/20 - Bacteria; Culture media therefor

Another patents in same IPC classes:

Nutritive medium for extraction of lactobacteria / 2510416
Nutritive medium includes lactoserum, yeast autolysate, acetic acid 70%, agar and cabbage brew at the specified component ratio.

Method for biosorption purification of water from heavy metal ions using saccharomyces cerevisiae yeast / 2509734
Invention relates to biotechnology and can be used in biological treatment of waste water from electroplating plants from heavy metal salts. The method involves adding yeast biomass to waste water, said biomass being in form of brewery wastes containing a combination of yeasts of different strains of Saccharomyces cerevisiae with viability of 90-95% in a given amount. The biomass is mixed with the waste water to obtain a suspension. The obtained suspension is held for 8 hours at temperature of 10°C-29°C and solution pH of 5.5-8.0, followed by recycling spent yeast containing heavy metals by treating with lime Ca(OH)2, with the ratio of yeast biomass to lime of 1:5-8, to obtain a mixture. The obtained mixture is subjected to wet treatment at temperature of 90°C for 1 hour, followed by isolation of the obtained mixture, which contains heavy metals, in concrete paste.

Diagnostics method of sensitivity of mycobacterium tuberculosis strains to injection antituberculous preparations of reserve row (aminoglycosides and capreomycin) / 2509158
Invention presents a diagnostics method of sensitivity of M. tuberculosis (MBT) to injection antituberculous preparations of a reserve row. The method involves a DNA extraction stage, amplification of the investigated DNA sections by means of a polymerase chain reaction method and analysis of conformational polymorphism of single-chain fragments (SSCP). Gene section rrs is amplified in 50 mcl of a reaction mixture with addition of 5 mcl of the specimen. Promoter gene section eis is amplified in 30 mcl of the reaction mixture containing direct 5'CGGAGCCGTCGGGGTATGC and reverse 5'GCCGCGGCCAGTAGGAACA primers and 3 mcl of the specimen as per the amplification programme: 1-st stage - 95°- 4 min; 2-nd stage - 95° - 20 sec, 59° - 30 sec, 72° - 20 sec (30 cycles); 3-rd stage: 72° - 4 min; 10° - storage. Separation of amplification products in the ratio of 4 mcl of the specimen and 6 mcl of a denaturing dye is performed by electrophoresis in 8% polyacrylamide gel with 5% glycerin at voltage of 400 Volts during 5 hours at 8°C. Gel painting is performed by means of caustic silver.

Association of strains of bacteria-oil decomposers, and remediation method of oil-contaminated objects / 2509150
Invention proposes an association of strains of bacteria-oil decomposers, which have been extracted from oil-contaminated soil, Acinetobacter species B-1037, Pseudomonas species B-989, Bacillus species B-1040, deposited at The State Research Centre of Virology and Biotechnology VECTOR. Besides, at least 30% bacteria of each strain is contained in the association. Remediation of oil-contaminated soils includes water suspension of lyophilic dried biomass of the strain association based on 109 cells per square metre. Strains of the association can utilise a wide range of oil components at the temperature of 10-15°C.

Bacillus subtilis subsp subtilis strain having apparent antagonism in relation to escherichia coli, salmonella typhi, staphylococcus aureus, listeria monocytogenes and resistance to streptomycin and tetracycline / 2509149
Bacillus subtilis subsp.subtilis BKM B-2711D strain has apparent antagonism in relation to Escherichia coli, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes, and resistance to streptomycin and tetracycline antibiotics. It is deposited in the All-Russia Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms Named after G.K. Skryabin of the Russian Academy of Science (IBFM RAN) and has the following registration number: BKM B-2711D, and can be used at production of probiotic bacterial preparations that can be used in veterinary medicine.

Bacillus amyloliquefaciens bkm b-2714d strain having apparent antagonism in relation to salmonella typhi, staphylococcus aureus, listeria monocytogenes and resistance to tetracycline and trimethoprim / 2509148
Bacillus amyloliquefaciens BKM B-2714D strain has apparent antagonism in relation to Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes, and resistance to tetracycline and trimethoprim antibiotics. It is deposited in the All-Russia Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms Named after G.K. Skryabin of the Russian Academy of Science (IBFM RAN) and has the following registration number: BKM B-2714D, and can be used at production of probiotic bacterial preparations that can be used in veterinary medicine.

Dry chromogenic feed medium for detection of coliform bacteria and e.coli (versions) / 2508400
Invention 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.

Strain of chlorella vulgaris microalgae for obtaining lipids as raw material for production of motor fuel / 2508398
Invention propose a strain of Chlorella vulgaris IPPAS C-616 microalgae for obtaining lipids as raw material for production of motor fuel.

N-glycan from c jejuni presented in salmonella enterica and its derivatives / 2507253
Invention refers to genetically modified bacteria Salmonella enterica, which contain at least one operon pgI from Campylobacter jejuni or its functional derivative and refers to presentation of at least one N-glycan from Campylobacter jejuni or derivative of this N-glycan on their cell surface. In bacteria one or several genes for biosynthesis of bacillozamine are inactivated by mutation and/or partial or complete deletion of genes pgID, E, F, G. Invention provides method for obtainment of genetically modified bacteria Salmonella enterica. Invention is directed to application of modified bacteria in pharmacological compositions of medical and veterinary purpose and methods for treatment and/or prevention of infections Campylobacter and, not obligatory, Salmonella.

Method of obtaining preparation based on vaccine strain of plague microbe / 2510825
Invention relates to field of biotechnology and deals with method of obtaining preparation based on vaccine strain of plague microbe. Claimed invention includes preparing inoculation native culture of plague microbe, concentration of microbe suspension, preparing vaccine suspension and obtaining dry form of preparation, with process of preparing inoculation culture including cultivation of microbes in liquid nutritional medium in flasks for 48 h at temperature 26…28°C and contibuous aeration with not less than 10 l min-1. with passaged stabilised starting culture, obtained as a result of three successive passages through organism of guinea pigs and mixed with glycerol-lactose-polyglucinum liquid in ratio 2:1; for preparation of vaccine suspension used is optimised in component composition protective drying medium, lyophilisation being carried out with observance of the specified regimen.

Nutritive medium for extraction of lactobacteria / 2510416
Nutritive medium includes lactoserum, yeast autolysate, acetic acid 70%, agar and cabbage brew at the specified component ratio.

Method for obtaining spore material of bacteria of clostridium type / 2509151
Method for obtaining spore material of bacteria of Clostridium type provides for production of inoculum of bacteria in a full synthetic growth medium, seeding of inoculum and cultivation under the corresponding conditions in growth medium including potato, glucose, ammonium sulphate and chalk. During the main fermentation process for 18-28 parts of bacterial culture growth depending on time of occurrence in one field of view of at least 80-100 thickened forms of cells, n-butanol in the amount of 0.16-0.81 wt % is added to growth medium. Number of formed spores is 1.08-2.86·108 in one millilitre of the medium.

Method for obtaining actinobacillus pleuropneumonia apxi or apxiii toxins in liquid culture media supplemented by air enriched with carbon dioxide / 2507267
Invention relates to method for obtaining of RTX-toxins Apxl or ApxIII by culturing of Actinobacillus pleuropneumoniae bacteria in liquid culture media. Characterised method consists in the following: during exponential growth phase of bacteria and production of RTX-toxins air passes through the medium, carbon dioxide content in air is above normal atmospheric level and is up to 10 % vol.

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 / 2243779
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.

FIELD: biotechnology.

SUBSTANCE: invention is production of the nutrient medium, which creates optimal conditions for growing legionella, comprising: enzymatic hydrolyzate of pig lung, enzymatic hydrolyzate of chicken egg yolk, potassium monophosphate, trihydrate disubstituted potassium phosphate, L-cysteine hydrochloride, activated carbon, microbiological agar and distilled water at a predetermined ratio of ingredients.

EFFECT: invention enables to produce the high-quality, easy-to-prepare nutrient medium, to reduce the time of growing legionella.

The invention refers to biotechnology, namely to receive the nutrient media for cultivation of Legionella.

Known nutrient medium for cultivation of Legionella, which is based on the meat water to 1000,0 ml; fermentative peptone - 10,0 g, sodium chloride - 5,0 g; blood - 10 g; agar-agar - 15,0-20,0 g, pH 7,3±0,2. Wednesday autoclave 30 minutes at a temperature of 121 C (Mosk; Diotalevi; Measureswhich. The nutrient medium for medical and sanitary Microbiology. SPb.: ALBI-SPb.-2008.-P.64-65.-C.-269).

The disadvantage of this environment is the lack of effectiveness.

Closest to the proposed culture medium is the medium containing, in g/l: enzymatic hydrolysate easy pig - 260,0; potassium phosphate 1-substituted - 0,9; potassium phosphate 2-substituted 3-water - 2,2; activated carbon - 2,0; L-cysteine hydrochloride - 0,4; embryonic growth of microorganisms - 2,5; agar microbiological - 8,5; distilled water up to 1 L. (RF Patent №2412240. Publ. 20.02.2011, bul. №5).

The disadvantage of this environment is a multistage and the difficulty of obtaining embryonic growth of microorganisms.

The aim of the present invention is to construct high quality and simple to prepare nutrient medium of animal origin, which provides growth properties Legionella in 36 hours

This goal is achieved by the fact that nutrient medium as nutrition contains enzymatic hydrolysate easy pigs; 0.01 M potassium phosphate buffer (composition potassium phosphate buffer: potassium phosphate 1-substituted; potassium phosphate 2-substituted 3-water), and also a coal active, L-cysteine hydrochloride; distilled water, agar microbiological with the addition Wednesday as growth stimulator enzymatic hydrolysate yolk eggs in the following ratio of ingredients (g/l:

Enzymatic hydrolysate easy pigs

with the amine nitrogen content of 0.10 to 0.14%

240,0-280,0

Enzymatic hydrolysate yolk eggs

3,0-7,0

Potassium phosphate 1-substituted

0,7-1,1

Potassium phosphate 2-substituted

3-water 2,0-2,4

Coal active

1,0-3,0

L-cysteine hydrochloride

0,2-0,6

Agar microbiological

6,5-10,5

Distilled water

up to 1 l

Easy pigs TU-8-5344-113607. The biological value of the lung pig-related by-products category II, is to have a average of 13.6% protein, minerals (salts of sodium, potassium, calcium, phosphorus) and high content of iron; the overall amount reaches 1%, 0,5-1,4% ash. Light also contains vitamins, microelements, extractive substances. From the proteins in the light pig enzymatic hydrolysis formed following amino acids: lysine, methionine, tryptophan, cystine, threonine, serine, phenylalanine, Proline, alanine, glycine, valine, leucine, tyrosine, histidine (Iwithin. Fodder and fodder additives. Moscow. Rosagropromizdat. 1989. C.-104-108).

The biological value of the yolk of the egg is on average 50% of water, protein 16,2%, amino acids, macro-elements in mg per 100 g of potassium - 129; calcium - 129; magnesium - 15; sulfur - 170; chlorine - 146,8; phosphorus - 542; sodium - 51, microelements in micrograms per 100 g: iron - 6700; iodine - 23; cobalt - 23; manganese - 37; copper - 139; of molybdenum - 11,8; chromium - 7,8; zinc - 3105. Vitamin content mg kg: vitamin a - 0,35; carotene - 0,06; vitamin B 1 - 1,6; vitamin D - 4,70; vitamin E - 2,0; Pantothenic acid - 1,3; vitamin a 5 - 3,0; 6 - 0,14; Niacin - 0,19; Riboflavin - 0,44; folacin - 7,0; choline - 251,7. The conversion factor of essential amino acids in g/kg is 6558, of which: valine - 937; isoleucine - 907; leucine - 1381; lysine - 1156; methionine - 415; threonine - 830; tryptophan - 236 and phenylalanine - 696. The conversion factor of essential amino acids is 9331, including: alanine - 854; arginine - 1156; aspartic acid - 1339; histidine - 383; glycine -514; glutamic acid - 2051; Proline - 695; serine - 1365; tyrosine - 699; cystine - 275. Microelements mg per kg: zinc - 14,0; manganese - 0,8; copper - 0,8; cobalt - 0,07. Organic substances: proteins - 93% (protein); fat - 94% (Mfreeman, Imiquimod. The chemical composition of food. Moscow. "Food industry". 1979. 247).

L-cysteine hydrochloride - amino acid (Panreac 15 V/ Party Lot 0000054464), which accelerates the growth of Legionella.

Hydrochloric acid GOST 3118-77 (analytical grade, CHP) is used to measure the pH. Mass fraction of the basic substance, % 35-38.

Agar microbiological - agar bacteriological (European type). Manufacturer: "Pronadisa" Spain. The party LF 15110173. Production date: April 2012

Coal active wood powder, brand OU-A. Fine-dispersed powder of black color. Without foreign inclusions, corresponds to GOST 4453-74. Absorbs in itself waste products of metabolism of Legionella.

Legionella are facultative intracellular parasites, and so grow in the yolk sack chick embryos, in the culture of cells of animals and humans (lung fibroblasts). (Medical Microbiology, Virology and immunology: Textbook for medical students /edited by BEA. - 2-e Izd., Corr. and extra - M OOO "Medical informational Agency", 2008. - S.-400).

The inclusion of environment potassium phosphate buffer 0.01 M (composition potassium phosphate buffer: potassium phosphate 1 substituted; potassium phosphate 2-substituted 3-water) justified the extensive use of phosphates in the preparation of nutrient mediums, because it is the only inorganic compounds with a buffer action in the physiologically important pH range, they have low toxicity (Methodical recommendations for the production and use of nutrient media and solutions for microbiological purposes, the cultivation of cells and viruses. -M, 1989). (Mosk; Diotalevi; Measureswhich. The nutrient medium for medical and sanitary Microbiology. SPb.: ALBI-SPb.-2008. - Pp.37-38).

When preparing the nutrient use only distilled water without the use solutions containing sodium ions, because of their destructive actions.

Unlike the prototype combined application of these components provides obtaining pure cultures after 36 PM

Preparation of enzymatic hydrolysate easy pigs.

Easy pigs in quantity of 1 kg cut into pieces 2x2 cm, pouring in enamelled pot, pour water tap in the amount of 1.5 l, cook for 10 minutes, cooled to a 46o C. the Broth is poured in 3 l glass bottle. Cold bits of light passed through a meat grinder, then placed in the cylinder with the broth. Enzyme Pancreatin in the amount of 9.5 g/l (or the pancreas of cattle, twice, missed through Mincer in the amount of 150 g/kg) is placed into the balloon stuffing easy and broth. Bring the pH to 8.2±0,1 using potassium hydroxide to phenolphthalein. As preservative add chloroform in the amount of 1% by volume of liquid. The container closed cotton-gauze tube, carefully mix and put in heat-chamber at a temperature of 37±1 degree C. The contents of the container during the day mix, every 20 of + / -1 min for 5 min, and in the next day after 1.5±0.2 h in 5 minutes Dynamics of the process of hydrolysis control by the amine nitrogen. About the end of hydrolysis judged upon the achievement of amine nitrogen 0,5%and-0.6% (after 5 days). After this the heating and stirring stop, hydrolyzed defend. Settled top layer of the hydrolysate decanted with a rubber hose and filtered through a cloth filter. The leachate lead in glass bottle with capacity of 3 years, for preservation add chloroform in the amount of 1% by volume of the contents of a container, cover with a rubber stopper and transported in a cold chamber, where it is stored at a temperature of from +2 to +8 degrees C.

Preparation of enzymatic hydrolysate yolk chicken eggs

The yolks of eggs in the amount of 30 pieces, that is 550 ml, pour in 3-liter glass bottle, pour warmed up to 42+1 degree of drinking water to 1.5 l, alkalinized 20% solution of potassium hydroxide in the amount of 13 ml to a pH of 8.2 on phenolphthalein, then add commercial enzymes: trypsin in number - 31.0 g and Pancreatin - 10,0 g as preservative add chloroform in the amount of 1% by volume of liquid. The container closed cotton-gauze tube, carefully mix and put in heat-chamber at a temperature of 42 degrees C. The contents of the container for the first night mix in 20 of + / -1 min, 5 min, in the next - in 1,5-2,0 h for 5 minutes every day is the measurement of pH and slew amino nitrogen. Two days due to a sharp drop in pH to 5% add 30 ml of 20% potassium hydroxide to pH of 8.2 to phenolphthalein. The dynamics of the process of hydrolysis control by the amine nitrogen. About the end of hydrolysis judged upon the achievement of amine nitrogen 0,7%-0,8%. After this the heating and stirring stop, hydrolyzed assert within 2 days. Settled top layer of the hydrolysate decanted with a rubber hose and filtered through a cloth filter. The leachate lead in glass bottle with capacity of 3 years, for preservation add chloroform in the amount of 1% by volume of the contents of a container, cover with a rubber stopper and transported in a cold chamber, where it is stored at a temperature of from +2 to +8 degrees C.

Nutrient medium on enzymatic the basis of light pigs for breeding Legionella is prepared in the following way. Take enzymatic hydrolysate of light pigs, diluted with distilled water to the testimony of amine nitrogen 0,12% - 260,0 ml; enzymatic hydrolysate yolk eggs - 5,0 g/l; potassium phosphate 1-substituted - 0.9 g/l; potassium phosphate 2-substituted 3-water - 2.2 g/l; coal active - 2.0 g/l; agar microbiological is 8.5 g/l; distilled water up to 1 L. The mixture to the boil and boil until complete melt the agar for 2 min, then add 0.4 g L-cysteine hydrochloride, dissolved in 10 ml of distilled water, pH correct solution of hydrochloric acid (1:1) to 6.9±in the amount of 0,5 2,2 ml Cooked medium poured into graduated bottles of 200±10.0 ml, which is hermetically sealed cotton-gauze plugs and Kraft paper. Sterilized at 1 ATM for 20 minutes After melting in a boiling water bath and cooled down to 56 degrees With agar poured into a sterile Petri dishes. Dry for 30 minutes and are used for planting.

The subjects of the cultures were grown on the plates of thick agar pH 6.8 at a temperature of 37 C for 24 hours Of daily subsistence crops cooked 1 billion suspension M.K. test strains of 10 units on the optical turbidity standard of CCA gisk named after. Laurukovich, then tenfold serial dilutions in the physiological solution of 4.5 ml brought to content in 1 ml 100 M.K. of these dilutions of sediment crops were sown on - 0.1 ml of 3 Petri dishes spilled dried agar. The crops were incubated at 37 C for 5 days at the daily view. Through 24-120 h took into account the results by calculations of grown colonies.

Example 1. Test strains were grown on a nutrient medium, containing (g/l: enzymatic hydrolysate of light pigs, diluted with distilled water to indications amino nitrogen 0,100% - 240,0 ml; enzymatic hydrolysate yolk eggs - 3.0 ml; potassium phosphate 1-substituted - 0,7; potassium phosphate 2-substituted 3-water - 2,0; angle active - 1,0; L-cysteine hydrochloride - 0,2; agar microbiological - 6,5; distilled water up to 1 L. At this ratio, of ingredients cumulative effect was less than 50%, after 36 hours of grown colonies from sowing test strains in the amount of 2 (PLANO-convex, bluish-grayish with a diameter of 1.0-1.1 mm), then as to the control environment SAL growth was observed only in 72 hours on agar of Hottinger growth was observed.

Example 2. Test strains were grown on a nutrient medium, containing (g/l: enzymatic hydrolysate of light pigs, diluted with distilled water to the testimony of amine nitrogen 0,12% - 2600 ml; enzymatic hydrolysate yolk of eggs 5.0 ml; potassium phosphate 1-substituted - 0,9; potassium phosphate 2-substituted 3-water - 2,2; angle active - 2,0; L-cysteine hydrochloride - 0,4; agar microbiological - 8,5; distilled water up to 1 L. At this rate ingredients cumulative effect exceeds 50%of grown colonies from sowing test strains of 7 (PLANO-convex, bluish-grayish colonies in diameter, 1.2-1.5 mm) after 36 hours, whereas in the control environment SAL growth was observed only in 72 hours on agar of Hottinger growth was observed.

Example 3. Test strains were grown on a nutrient medium, containing (g/l: enzymatic hydrolysate of light pigs, diluted with distilled water to the testimony of amine nitrogen 0,14%-280,0 ml; enzymatic hydrolysate yolk of eggs to 7.0 ml; potassium phosphate 1-substituted - 1,1; potassium phosphate 2-substituted 3-water - 2,4; coal active - 3,0; L-cysteine hydrochloride - 0,6; agar microbiological - 10,5; distilled water up to 1 L. At this ratio, of ingredients cumulative effect was observed less than 50%, of grown colonies from sowing test strains in the amount of 3 (PLANO-convex, bluish-grayish with a diameter of 1.0-1.1 mm) after 36 hours, whereas in the control environment SAL growth was observed only in 72 hours on agar of Hottinger growth was observed.

The obtained results have allowed to determine the optimal variant of nutrient medium on enzymatic the basis of light pigs (example 2).

The proposed environment quality, easy to prepare, based on enzymatic hydrolysate easy pigs, and as a stimulator of growth of Legionella - enzymatic hydrolysate yolk of the egg. Profitability is 29%.

The nutrient medium for cultivation of Legionella, containing, in g/l: