Synthetic nutrient medium for growing microorganisms

FIELD: medicine.

SUBSTANCE: synthetic nutrient medium for growing microorganisms contains citric acid, asparagine, potassium phosphate twice substituted, zinc sulfate, magnesium sulfate, iron sulfate, sodium chloride, sodium phosphate twice-substituted, glycine, succinic acid, distilled water and if necessary agar with specified component ratio.

EFFECT: invention makes it possible to increase nutritional properties of synthetic nutrient media.

2 cl, 1 tbl, 3 ex

 

The invention relates to Microbiology and biotechnology, in particular to the design of synthetic environments for cultivation of mycobacteria of different species, Pseudomonas molleo, Pseudomonas aeruginosa Pseudomonas aeruginosa, Staphylococcus, Salmonella, E. coli, Proteus, etc. as well as probiotic microorganisms.

On the origin of the nutrient medium is divided into natural (blood, milk, potato, egg, artificial using the universal source of nitrogen and carbon - peptone obtained by incomplete cleavage of proteins by enzymes (pepsin, trypsin) is different hydrolysates: fish, casein, meat, yeast and synthetic nutrient medium prepared from balanced chemical components (Korotyaev A.I., Babichev S.A. Medical Microbiology, immunology and Virology. SPb., 1998. - P.78-79).

At the same time when producing staphylococcal, komisarkaplan of intoxination, mallein for the purpose of humane and veterinary medicine cultivation of the microorganisms is performed in environments that use mesopatamia-potato broth or hydrolizates meat, fish, casein, characterized by instability of the composition, uneven growth and fibre products.

Known synthetic nutrient medium Kursk Biofactory (p the test environment Engleskog # 1 and # 2) are used for the cultivation of mycobacteria of different species in the manufacture of diagnostic allergens (A.A. Iglewski Experimental rationale and practical aspects of diagnosis and prevention of infectious and allergic diseases of animals. Abstract. dis. ... Prof. wet. Sciences, Lovetension, 1990. - P.8-9).

However, these synthetic environment versatility not possess. And the content of zinc sulfate 0.1 g/l does not always ensure surface cultivation of Mycobacterium tuberculosis. Known environment have also composed of scarce ammonium citrate.

Almost cultivation of Mycobacterium tuberculosis is carried out with the use of synthetic environments to obtain TB allergen - tuberculin PPD instead NKGB. Synthetic environment do not contain dietary fibre meat, casein, peptone, and were first used to obtain PPD-tuberculin (F.Seibert in 1934, Maslennikova in 1939, Ask in 1963)

Known synthetic nutrient medium for cultivation of Mycobacterium tuberculosis (patent No. 2139345, 10.10.1999, author ISI) includes, wt.%: citric acid 6,0±2,0; asparagine 7,5±1,5; ammonium citrate 3,0±1,0; sulfuric acid magnesium 0,75±0,15; iron sulfate 0,075±0,015; zinc sulfate 0,15±0,075; dvuhkamernyi phosphoric acid potassium 7,5±1,5; glycerol - rest.

However, this synthetic environment suitable for growing only Mycobacterium tuberculosis, that is, has no versatility, PR is the suitability for growing number of other pathogenic and probiotic microorganisms in the manufacture of biological products.

The aim of the invention is to increase the nutritional properties of the synthetic environment suitable for stable growth and maximum accumulation of a number of pathogenic and probiotic microorganisms.

This goal is achieved by increasing the content of citric acid prior to 9-11 g/l, zinc sulfate 0.5 g/l, more making succinic acid 3.0 g/l, sodium phosphate 2-substituted 1,5 g/l, glycine 1.0 g/l, NaCl 0.5 g/l, a decrease in the content of expensive asparagine to 2-4 g/l, and the complete withdrawal of scarce ammonium citrate.

Developed synthetic environment contains chemical ingredients in the following proportions, in grams per 1 liter of distilled water, citric acid 9,0-11,0; asparagine - 2,0-4,0; potassium phosphate dvuhkamernyi - 6,0; zinc sulfate is 0.5; magnesium sulfate - 0,7; sulphate of iron - 0,1; sodium chloride and 0.5; sodium phosphate dvuhkamernyi - 1,5; glycine - 1,0; succinic acid - 3.0 and glycerin 40-50 ml.

The reaction medium is set 5-10% ammonia solution to pH 7.0, and 7.1 before autoclaving, and for the cultivation of staphylococci to pH 7.5 and 7.6 with the increase of sodium chloride up to 5-6 g/l

Introduction citric and succinic acids (di-, and tricarboxylic acid cycle Krebs) increases metabolism in microorganisms, improving the solubility of x the chemical ingredients and the formation of ammonium citrate by neutralizing citric acid 5-10% solution of ammonia. The formation of citrate of ammonium as nitrogen compounds needed to build protein molecules of microorganisms, improves metabolism and growth of microbial biomass. Introduction in the nutrient medium composition of glycine in combination with asparagine (amide of aspartic acid) improves the biosynthesis of protein structures of microorganisms, and sodium chloride and sodium phosphate dvuhkamernyi increase the buffering capacity of the environment help to keep the pH during prolonged cultivation of Mycobacterium tuberculosis and Staphylococcus.

The proposed composition of the medium differs from the prototype and analogues qualitative and quantitative ratio of ingredients and provides the accumulation wrung dead Bakassi of Mycobacterium tuberculosis to 130±10.0 g/l after 60 days of cultivation, microbial cells of Salmonella and E. coli after 2-3 days of incubation up to 70±10,0 billion/ml, Staphylococcus after 10-12 days of growing up to 13±1.0 billion/ml, probiotics after 2-3 days of cultivation 70-90 billion/ml Thus, the claimed universal synthetic environment meets the conditions of patentability of "novelty" and "inventive step".

Synthetic nutrient medium, providing consistently high accumulation of microorganisms with subsequent receipt of them tuberculin PPD, TB toxoid, mallein, to bacterioses, komisarkaplan, staphylococcal toxoid vaccines, etc. meets the condition of patentability "industrial applicability".

Synthetic medium for the cultivation of microorganisms was obtained by serial dilution in distilled water ingredients or dissolving them with advanced components are mixed in dry form in the ratio, g/l, citric acid 10.0 g; asparagine 3,0; potassium phosphate dvuhkamernyi 6,0; zinc sulfate to 0.5; magnesium sulfate 0,7; sulphate of iron of 0.1; sodium chloride of 0.5; sodium phosphate dvuhkamernyi 1,5; glycine 1,0; succinic acid 3,0; glycerol 50 ml and subsequent neutralization of the environment 5-10% solution of ammonia and the reduction of the volume of liquid medium by adding distilled water to 1 liter.

For the cultivation of staphylococci in the composition of the synthetic environment, the content of sodium chloride brought to 5 g/l while maintaining the above ratio of all other components, and pH to 7.5 in connection with the physiology of this species of microorganisms.

In the preparation of dense fundamentals of synthetic environments diluted with distilled water in the ratio 1:1 and add 2.5% agar, followed by sterilization at 100°C for 30-60 minutes and packaged in tubes, Petri dishes for sealing and further use for growing microorganism is.

The invention is illustrated by the following examples.

Example 1. Determination of the nutritive value of the synthetic nutrient medium during the cultivation of Mycobacterium tuberculosis.

The studies have used laboratory and industrial strain No. 8 of Mycobacterium tuberculosis and freshly isolated microorganisms.

Seeding of microorganisms was performed using a platinum loop (spiral) on the surface of the liquid synthetic environment. During incubation for 20-30 days there was a full coating film of Mycobacterium tuberculosis entire surface of the medium diameter up to 0.5-0.7 cm, and after 60 days the thickening of the film reached 1.0 cm or more. After autoclaving grown Mycobacterium tuberculosis with the subsequent release of Bakassi through several layers of gauze and drying were weighed.

On average, a constant accumulation of terraria (wrung through several layers of gauze) Bakassi Mycobacterium tuberculosis was 120-130 g/l, and the dry - 12-13 g/l

Example 2. Cultivation of Mycobacterium tuberculosis on solid agar medium.

In the synthetic environment of the claimed composition, as well as when breeding it with distilled water 1:1 was added 2.5% agar, sterilized at 100°C for 30-60 minutes, were Packed up in tubes and grew Mycobacterium tuberculosis. The growth of M. tuberculosis in PR the tags with agar occurs 7-10 days after seeding of microorganisms, as is the case with the stated concentration of the synthetic nutrient medium, and when it is diluted with distilled water 1:1. Thus, in the preparation of dense medium with the addition of agar prepared with liquid synthetic medium is diluted with distilled water in a 1:1 ratio.

Example 3. Test the effectiveness of the growth of staphylococci, Salmonella, and Escherichia coli (E. coli) on the universal synthetic environment and mineral-based in the stated concentration and dilution with distilled water 1:1 with 2.5% agar.

The concentration of staphylococci after 12±2,0 day cultivation in the two-liter beautylab with medium volume, equal to 1.0 liter, is 12±1.0 billion/ml of microbial cells and Salmonella and Escherichia coli after 2-3 days incubation - 70±10,0 billion/ml at continuous sprouting colonies of microorganisms agar surface tubes on a mineral basis. Cultivation of microorganisms on solid agar medium liquid mineral Foundation pre-diluted by half with distilled water. Lower concentrations in combination with 2.5% agar does not affect the growth of the above-mentioned microorganisms.

Examples confirmed the suitability of the nutrient medium in the proposed options for the cultivation not only of M. tuberculosis and other pathogenic microor the mechanisms are: Salmonella, Escherichia coli, Pseudomonas aeruginosa, Proteus, and probiotic microorganisms, i.e. its versatility. For the cultivation of staphylococci in the composition of the medium increase the content of sodium chloride up to 5-6 g/l and pH to 7.5 and 7.6 in accordance with the physiological requirements of these microorganisms.

Results accumulation of bacterial mass cultivation of microorganisms on the claimed synthetic nutrient medium is given in the table.

Table
The accumulation of bacterial mass cultivation of microorganisms on synthetic nutrient medium.
№ p/pName of microorganismsGrowing periodAccumulation (concentration) of microorganisms
1.Mycobacterium tuberculosis (bovine species)30 days60 g of autoclaved and pressed Bakassi
60 days120 g of autoclaved and pressed Bakassi
2.Staphylococci (C is latesty) 7 days7-8 billion/ml
12 days12±1.0 billion/ml
3.Salmonella2-3 days70±10 billion/ml
4.E. coli2-3 days70±10 billion/ml
5.Pseudomonas aeruginosa2-3 daysThick lisaadamsy
weight
6.Bacillus subtilis - Bacillus2-3 daysThick lisaadamsy
subtil isweight
7.Lactobacillus2-3 daysThick lisaadamsy weight

1. Synthetic nutrient medium for exp is farming microorganisms, including citric acid, asparagine, potassium phosphate dvuhkamernyi, zinc sulfate, magnesium sulfate, iron sulfate, glycerin, characterized in that it further comprises succinic acid, glycine, sodium phosphate dvuhkamernyi, sodium chloride and distilled water in the following ratio of components, g/l:

citric acid9,0-11,0
asparagine2,0-4,0
phosphate potassium dvuhkamernyi6,0
sulfate zinc0,5
sulfate magnesium0,7
iron sulfate0,1
sodium chloride0,5
phosphate sodium dvuhkamernyi1,5
glycine1,0
succinic acid3,0
glycerin40-50 ml
distilleria the water output rest

at pH 7.0, and 7.1.

2. Synthetic nutrient medium according to claim 1, characterized in that the dilution with distilled water 1:1 further comprises 2.5% agar.



 

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