RU2528101C2 - Nutrient medium for legionella culture - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry, namely to a nutrient medium for a legionella culture. The nutrient medium for the legionella culture which contains: fermentative hydrolysate of soya beans, potassium dihydrogen phosphate, disubstituted potassium phosphate trihydrate, L-cysteine hydrochloride, activated charcoal, microbiological agar and fermentative hydrolysate of the chicken yellow yolk, distilled water taken in certain relations.

EFFECT: above nutrient medium enables reducing the length of legionella growth.

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Description

The invention relates to biotechnology, namely to obtain nutrient media for the cultivation of Legionella.

Known nutrient medium for growing Legionella, the basis of which is meat water up to 1000.0 ml; enzymatic peptone - 10.0 g; sodium chloride - 5.0 g; blood - 10 g; agar-agar - 15.0-20.0 g; pH 7.3 ± 0.2. The medium is autoclaved for 30 min at a temperature of 121 ° C (M.S. Polyak; V.I. Sukharevich; M.E. Sukharevich. Nutrient media for medical and sanitary microbiology. St. Petersburg: ELBI-St. Petersburg. - 2008. - P.64 -65; S. - 269).

The disadvantage of this environment is the lack of effectiveness. Closest to the proposed nutrient medium is a medium containing, g / l: enzymatic hydrolyzate of a lung of a pig - 260.0; potassium phosphate 1 - substituted - 0.9; potassium phosphate 2 - substituted 3 - aqueous - 2.2; activated carbon - 2.0; L-cysteine hydrochloride - 0.4; embryonic microorganism growth stimulator - 2.5; microbiological agar - 8.5; distilled water up to 1 liter (RF Patent No. 2412240. Publish. February 20, 2011 Bull. No. 5).

The disadvantage of this environment is the multistage and complexity of obtaining an embryonic growth stimulator of microorganisms.

The aim of the present invention is the construction of high-quality and easy to prepare nutrient medium of plant origin, which provides the growth properties of legionella after 48 hours

This goal is achieved in that the nutrient medium as a nutrient base contains an enzymatic hydrolyzate of soybeans; 0.01 M potassium phosphate buffer (composition of potassium phosphate buffer: potassium phosphate 1 - substituted; potassium phosphate 2 - substituted - 3 - aqueous), as well as active carbon, L-cysteine hydrochloride; distilled water, microbiological agar with the addition of chicken egg yolk enzymatic hydrolyzate to the medium as a growth promoter in the following ratio of ingredients, g / l:

Bean Soybean Enzymatic Hydrolyzate 60.0-140.0 Enzymatic hydrolyzed egg yolk 10.0-30.0 Potassium phosphate 1 - substituted 0.7-1.1 Potassium Phosphate 2 - Substituted 3 - water 1.2-3.2 Active charcoal 1.0-3.0 L-cysteine hydrochloride 0.2-0.6 Microbiological agar 6.5-10.5 Distilled water up to 1 l

Soy is used as a raw material, the fruits of which - beans contain an average of 40% protein, in addition, soy is rich in trace elements and vitamins. As is known (Y.P. Yupta, 1987) soy proteins are characterized by an increased content of glutamic and aspartic acids, which supply macroergic bonds. Soy proteins also contain a large number of such essential amino acids as lysine, leucine, arginine. There is also information about the ability of leucine, as branched chain amino acids, to initiate protein synthesis (E.I. Chazov, H.S. Morgan, / USA / 1989).

Preparation of Enzymatic Hydrolyzate of Chicken Egg Yolk

The biological value of the yolk of a chicken egg is the presence of an average of 50% water, protein 16.2%, amino acids, macronutrients in mg per 100 g: potassium - 129; calcium - 129; magnesium - 15; sulfur - 170; chlorine - 146.8; phosphorus - 542; sodium - 51, trace elements in micrograms per 100 g: iron - 6700; iodine - 23; cobalt - 23; Manganese - 37; copper - 139; molybdenum - 11.8; chromium - 7.8; zinc - 3105. The content of vitamins mg in 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 B ₅ - 3.0; B 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 them: valine - 937; isoleucine - 907; leucine - 1381; lysine - 1156; methionine - 415; threonine - 830; tryptophan - 236 and phenylalanine - 696. The conversion factor for non-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. Micronutrients mg in kg: zinc - 14.0; Manganese - 0.8; copper - 0.8; cobalt - 0.07. Organic matter: protein - 93% (protein); fat - 94%. (M.F. Nesterin, I.M. Skurikhin. Chemical composition of food products. Moscow. "Food Industry". 1979. 247 p.).

L-cysteine hydrochloride is an amino acid (Panreac 15 B512 / Lot Lot 0000054464) that accelerates the growth of Legionella.

Hydrochloric acid GOST - 3118-77 (bhd, hch) is used to establish pH. Mass fraction of the main substance,% 35-38.

Microbiological agar - bacteriological agar (European type). Producer: "Pronadisa" Spain. Lot LF 15110173. Date of manufacture: April 2012

Powdered active charcoal, OU-A brand. Fine powder, black. Without foreign matter, complies with GOST 4453-74. Absorb on itself the legionella metabolic waste products.

Legionella are facultative intracellular parasites; therefore, they grow in the yolk sac of chicken embryos, in the culture of animal and human cells (lung fibroblasts). (Medical Microbiology, Virology, and Immunology: A Textbook for Students of Medical Universities / Edited by A.A. Vorobyov. - 2nd ed., Rev. And add. - M.: Medical Information Agency LLC, 2008. - P. - 400).

The inclusion in the medium of potassium phosphate buffer 0.01 M (composition of potassium phosphate buffer: potassium phosphate 1 substituted; potassium phosphate 2 - substituted - 3 - aqueous) is justified by the widespread use of phosphates in the preparation of nutrient media, because these are the only inorganic compounds that have a buffering effect in a physiologically important pH range, they are low toxic (Guidelines for the manufacture and use of nutrient media and solutions for microbiological purposes, the cultivation of cells and viruses - M., 1989). (M.S. Polyak; V.I. Sukharevich; M.E. Sukharevich. Nutrient media for medical and sanitary microbiology. St. Petersburg: ELBI-SP6. - 2008. - P.37-38).

When preparing a nutrient medium, it is allowed to use only distilled water without the use of solutions containing sodium ions, in view of their destructive effect.

Unlike the prototype, the combined use of these components provides pure crops for 2 days.

Preparation of enzymatic hydrolyzate of soybeans.

The nutrient-based nutrient medium of soybeans for the cultivation of Legionella is prepared in the following way. Take an enzymatic hydrolyzate from soybeans diluted with distilled water to an amine nitrogen reading of 0.12% - 110.0 ml; enzymatic hydrolyzate of egg yolk - 20.0 ml of potassium phosphate 1 - substituted - 0.9; potassium phosphate 2 - substituted 3 - aqueous - 2.2; activated charcoal - 2.0; microbiological agar - 0.9; distilled water to - 1 liter. The mixture is brought to a boil and boiled until the agar is completely melted for 2 minutes, the pH is adjusted with a solution of hydrochloric acid (1: 1) to 6.9 ± 0.1 in an amount of 5 ml. The prepared nutrient medium is poured into graduated bottles of 200 ± 0.5 ml, which are hermetically sealed with cotton gauze plugs and Kraft paper. Sterilized at 1 atm for 15 minutes. After melting in a boiling water bath, a sterile solution of L-cysteine hydrochloride in an amount of 0.4 g in 10 ml of distilled water is added to the agar cooled to 56 ° C, then it is poured into sterile Petri dishes. Dry for 30 minutes and use for sowing.

Preparation of enzymatic hydrolyzate of chicken yolk.

30 yolks of chicken eggs, which is 550 ml, are poured into a 3-liter glass bottle, poured with drinking water heated to 42 ± 1 ° C to 1.5 l, made alkaline with a 20% potassium hydroxide solution in an amount of 13 ml to pH 8 , 2 for phenolphthalein, then commercial enzymes are added: trypsin in an amount of 31.0 g and pancreatin in an amount of 10.0 g. Chloroform in an amount of 1% by volume of liquid is added as a preservative. The cylinder is closed with a cotton-gauze plug, mixed thoroughly and placed in a heat chamber at a temperature of 42 ° C. The contents of the balloon during the first day are stirred after 20 ± 1 min for 5 minutes, and in the next - after 1.5-2.0 hours for 5 minutes. Daily measurements are made of the pH of the medium and the growth of amine nitrogen. After two days, in view of a sharp drop in pH to 5 units, add - 30 ml of 20% potassium hydroxide to a pH of 8.2 for phenolphthalein. The dynamics of the hydrolysis process is controlled by determining amine nitrogen. The end of hydrolysis is judged by the achievement of amino nitrogen of 0.7% -0.8%. After this, the heating and stirring are stopped, the hydrolyzate is defended for 2 days. The settled upper layer of the hydrolyzate is decanted with a rubber hose and filtered through a linen filter. The filtrate is collected in a glass container with a capacity of 3 l, chloroform is added in an amount of 1% to the volume of the contents of the container for preservation, closed with a rubber stopper and transported to a cold chamber where it is stored at a temperature of +2 to + 8 ° C.

The effectiveness of the obtained culture medium was evaluated in accordance with the guidelines “Monitoring of diagnostic culture media by biological indicators for plague, cholera, anthrax, tularemia, brucellosis, legionellosis” (Moscow, 2006) using Legionella pneumophila ATCC 33155 Bloomington as test cultures Legionella pneumophila ATCC 33152 Philadelphia.

The test cultures were grown on solid agar plates, pH 6.8, at 37 ° C for 24 hours. From daily cultures, 1 billion m.c. test strains equal to 10 units according to the optical turbidity standard OSO GISK im. L.A. Tarasovich, then serial ten-fold dilutions in physiological saline in a volume of 4.5 ml were brought to a content of 1 ml 100 mk From the dilution of the suspension of cultures, 0.1 ml was sown in 3 Petri dishes of spilled dried agar. Crops were incubated at 37 ° C for 5 days with daily viewing. After 24-120 hours, the results were taken into account by counting the grown colonies.

Example 1. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from soybeans, diluted with distilled water to an amine nitrogen reading of 0.100% - 60.0 ml; enzymatic hydrolyzate of egg yolk - 10.0 ml; potassium phosphate 1 - substituted - 0.7; potassium phosphate 2 - substituted 3 - water - 1.2; active angle - 1.0; L-cysteine hydrochloride 0.2; microbiological agar - 7.0; distilled water to - 1 liter. With this ratio of ingredients, the cumulative effect was less than 50% of flat-convex, grayish-yellowish colonies with a diameter of 1.0-2.0 mm after 48 hours, while on the SEL control medium growth was observed only after 72 hours, on the Hottinger growth agar not observed.

Example 2. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from soybeans diluted with distilled water to an amine nitrogen reading of 0.120% - 110.0 ml;

enzymatic hydrolyzate of egg yolk - 20.0 ml; potassium phosphate 1 - substituted - 0.9; potassium phosphate 2 - substituted 3 - aqueous - 2.0; activated charcoal - 2.0; L-cysteine hydrochloride - 0.4; microbiological agar - 9.0; distilled water to - 1 liter. With this ratio of ingredients, the cumulative effect exceeds 50% of plano-convex, yellowish-gray colonies with a diameter of 1.0-2.0 mm after 48 hours, while on the SEL control medium growth was observed only after 72 hours, on the Hottinger agar no growth was observed

Example 3. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from soya beans, diluted with distilled water to an amine nitrogen reading of 0.140% - 140.0 ml; enzymatic hydrolyzate of chicken yolk - 30.0 ml; potassium phosphate 1 - substituted - 1.1; potassium phosphate 2 - substituted 3 - aqueous - 3.2; activated charcoal - 3.0; L-cysteine hydrochloride - 0.6; microbiological agar - 11.0; distilled water to - 1 liter. With this ratio of ingredients, the cumulative effect was observed in less than 50% of plano-convex, yellowish-gray colonies with a diameter of 1.0-2.0 mm after 48 hours, whereas on the SEL control medium growth was observed only after 72 hours, on the Hottinger agar there was no growth observed.

The results obtained allowed us to determine the optimal variant of the nutrient medium on an enzymatic basis of soybeans (example 2).

The proposed environment of plant origin provides the growth of legionella. Profitability of production is 27.3%.

Claims (1)

Culture medium for the cultivation of Legionella, containing, g / l:
Enzymatic Soya Bean Hydrolyzate 60.0-140.0 Enzymatic hydrolyzed egg yolk 10.0-30.0 Potassium phosphate 1 - substituted 0.7-1.1 Potassium Phosphate 2 - Substituted 3 - water 1.2-3.2 Active charcoal 1.0-3.0 L-cysteine hydrochloride 0.2-0.6 Microbiological agar 7.0-11.0 Distilled water up to 1 l