RU2412240C1 - Culture medium for growing legionella - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: culture medium contains light pig enzymatic hydrolysate with amine nitrogen content of 0.10-0.14 mg %, 1-substituted potassium phosphate, 2-substituted 3-hydrate potassium phosphate, activated carbon, L- cysteine hydrochloride, microorganism embryo growth stimulant, microbiological agar and distilled water.

EFFECT: invention accelerates growth of legionella.

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Description

The invention relates to biotechnology, namely to obtain nutrient media for growing legionella.

Known nutrient medium for growing Legionella, the basis of which is meat water up to 1000.0 ml; peptone enzymatic 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-SP6. - 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: yeast extract 10.0 g; activated carbon 2.0 g; ACES buffer (amino-2-hydroxyethyl-aminoethane sulfonic acid) 10.0 g; a-ketoglutorate 1.0 g; L-cysteine 0.4 g; iron pyrophosphate 0.25 g; agar agar 15.0 g; distilled water up to 1 liter; pH 6.9 ± 0.2. (M.S. Polyak; V.I. Sukharevich; M.E. Sukharevich. Nutrient media for medical and sanitary microbiology. St. Petersburg: ELBI-St. Petersburg. - 2008. - P.268-270).

The disadvantage of this environment is the high cost of a drug not produced in the Russian Federation.

The aim of the present invention is the construction of a high-quality nutrient medium of animal origin, which provides the growth properties of legionella.

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

pig lung enzymatic hydrolyzate with an amine nitrogen content of 0.10-0.14 mg% 260,0 1-substituted potassium phosphate 0.9 2-substituted potassium phosphate 3-water 2.2 Activated carbon 2.0 L-cysteine hydrochloride 0.4 embryonic growth stimulator microorganisms 2.5 microbiological agar 8.5 distilled water to 1 liter

Pig lung TU-8-5344-113607. The biological value of a pig’s lung belonging to category II offal consists in an average of 13.6% protein, minerals of the lung, consisting of sodium, potassium, calcium, phosphorus and high iron; their total amount reaches 1%, 0.5-1.4% of ash. The lung also contains vitamins, trace elements, extractive substances The following amino acids are formed from protein substances in a pig’s lung during enzymatic hydrolysis: lysine, methionine, tryptophan, cystine, threonine, serine, phenylalanine, proline, alanine, glycine, valine, leucine, tyrosine, histidine ( I.V. Petrukhin. Feed and feed additives. Moscow. Rosagropromizdat. 1989. S. - 104-108).

The nutrient medium as a growth promoter contains an embryonic growth stimulator of microorganisms ("Method for producing an embryonic growth stimulator of microorganisms" RF Patent No. 2283347 - Publish. 09/10/2006. - Bull. No. 25).

A method of obtaining an embryonic growth stimulator of microorganisms is as follows: they select fresh, uninfected chicken eggs, then they are incubated for 9 days. Moreover, on the 5th, 6th, 7th day, chicken eggs are irradiated for 30 seconds at a modulation frequency of 9 Hz and a radiation power of 9 mW, then the chicken eggs are cooled for 6-7 days at a temperature of 2-4 ° C, homogenization, centrifugation at 15,000 r / min, then the resulting mass is filtered and subjected to tindialization at a temperature of 60-65 ° C for 5-6 days, packaged and lyophilized.

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: Textbook for medical students / Edited by A.A. Vorobyov. - 2nd ed., rev. and add. - M .: OOO "Medical Information Agency", 2008. - S.-400).

The inclusion of 0.01 M potassium phosphate buffer in the medium (potassium phosphate buffer composition: 1-substituted potassium phosphate; 2-substituted 3-aqueous potassium phosphate) is justified by the widespread use of phosphates in the preparation of culture media, since 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 culture 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-St. Petersburg. - 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, due to their destructive effect.

The combined use of these components provides pure crops after 48 hours

Unlike the prototype, the proposed environment is economical, profitable and provides optimal conditions for the reproduction of legionella in 2 days.

Preparation of enzymatic hydrolyzate

The pig’s lung in the amount of 1 kg is cut into pieces 2 × 2 cm in size, poured into an enameled pan, poured with tap water in an amount of 1.5 l, boiled for 10 minutes, cooled to 46 ° C. The broth is poured into a 3 liter glass bottle. The cooled pieces of the lung are passed through a meat grinder, then placed in a container with a decoction. The enzyme pancreatin in an amount of 9.5 g / l (or the pancreas of cattle, twice passed through a meat grinder, in an amount of 150 g / kg) is placed in a balloon with minced lung and a decoction of it. The pH was adjusted to 8.2 ± 0.1 with potassium hydroxide according to phenolphthalein. Chloroform is added as a preservative in an amount of 1% to the volume of liquid. The cylinder is closed with a cotton-gauze stopper, mixed thoroughly and placed in a heat chamber at a temperature of 37 ± 1 ° C. The contents of the balloon are stirred during the day, every 20 ± 1 minutes for 5 minutes, and the next day after 1.5 ± 0.2 hours for 5 minutes. 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 0.5% -0.6% (after 5 days). After that, heating and stirring are stopped, the hydrolyzate is defended. 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 ° С.

An enzymatic medium on the basis of light pigs for growing legionella is prepared in the following way. Take the enzymatic hydrolyzate from the lungs of a pig diluted with distilled water to an amine nitrogen reading of 0.12% 260.0 ml; potassium phosphate 1-substituted 0.9; potassium phosphate 2-substituted 3-aqueous 2.2; activated carbon 2.0; microbiological agar 8.5; distilled water up to 1 liter. The mixture is brought to a boil and boiled until the agar is completely melted for 2 min, the pH is adjusted with a solution of hydrochloric acid (1: 1) to 6.8 ± 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. within 15 minutes After melting in a boiling water bath, 0.4 g of L-cysteine hydrochloride and 2.5 ml of embryonic growth stimulator of microorganisms are added to the agar cooled to 56 ° C, then it is poured into sterile Petri dishes. Dry for 30 minutes and use for sowing.

The effectiveness of the obtained nutrient medium was evaluated in accordance with the guidelines “Control of diagnostic nutrient media according to biological indicators for plague, cholera, anthrax, tularemia, brucellosis, legioellosis”, (Moscow, 2006), using Legionella pneumophilla ATCC as test cultures 33155 Bloomington, Legionella pneumophilla ATCC 33152 Philadelphia.

The test cultures were grown on solid agar plates with a pH of 6.8 at 37 ° C for 24 hours. From the daily cultures, 1 billion bw was prepared. test strains equal to 10 units according to the optical turbidity standard OSO GISK im. L.A.Tarasovicha then serial tenfold dilutions in saline in a volume of 4.5 ml was adjusted to a content of 1 ml 10 ⁷¹⁰⁰ MK From these dilutions of culture suspensions, 0.1 ml were 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 light pigs diluted with distilled water to an amine nitrogen reading of 0.100% 240.0 ml; potassium phosphate 1-substituted 0.7; potassium phosphate 2-substituted-3-aqueous 2.0; activated carbon 1.0; L-cysteine hydrochloride 0.2; embryonic microorganism growth stimulator 0.5; microbiological agar 6.5; distilled water up to 1 liter. With this ratio of ingredients, the cumulative effect was less than 50% of flat-convex, bluish-grayish colonies with a diameter of 1, -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.

Example 2. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from light pigs diluted with distilled water to an amine nitrogen reading of 0.12% - 260.0 ml; potassium phosphate 1-substituted 0.9; potassium phosphate 2-substituted - 3-aqueous 2.2; activated carbon 2.0; L-cysteine hydrochloride 0.4; embryonic growth stimulator of microorganisms 2.5; microbiological agar 8.5; distilled water up to 1 liter. With this ratio of ingredients, the cumulative effect exceeds 50% of plano-convex, bluish-grayish colonies with a diameter of 1, -2.0 mm after 48 h, while on the SEL control medium growth was observed only after 72 h, no growth was observed on the Hottinger agar.

Example 3. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from light pigs diluted with distilled water to an amine nitrogen reading of 0.14% - 280.0 ml; potassium phosphate 1-substituted 1,1; potassium phosphate 2-substituted - 3-aqueous 2,4; activated carbon 3.0; L-cysteine hydrochloride 0.6; embryonic microorganism growth stimulator 4.0; microbiological agar 10.5; distilled water up to 1 liter. With this ratio of ingredients, the cumulative effect was observed in less than 50% of plano-convex, bluish-grayish colonies with a diameter of 1, -2.0 mm after 48 h, while on the SEL control medium growth was observed only after 72 h, no growth was observed on the Hottinger agar.

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

The proposed environment is of high quality, based on an enzymatic hydrolyzate of the lung of a pig, and as a stimulator, an embryonic stimulator of the growth of microorganisms.

Claims (1)

Nutrient medium for cultivation of legionella containing a nutrient base, activated carbon, microbiological agar and distilled water, characterized in that it additionally contains L-cysteine hydrochloride, embryonic growth stimulator of microorganisms, potassium phosphate 1-substituted, potassium phosphate 2-substituted - 3- water, and as a nutrient base is an enzymatic hydrolyzate of a light pig with an amine nitrogen content of 0.10-0.14 mg% in the following ratio of ingredients, g / l:
Pig lung enzymatic hydrolyzate with an amine nitrogen content of 0.10-0.14 mg% 240.0-280.0 Potassium phosphate 1-substituted 0.7-1.1 Potassium phosphate 2-substituted 3-water 2.0-2.4 Activated carbon 1.0-3.0 L-cysteine hydrochloride 0.2-0.6 Fetal Growth Stimulator microorganisms 0.5-4.0 Microbiological agar 6.5-10.5 Distilled water up to 1 l