RU2460768C1 - Solid nutrient medium for legionella cultivation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: nutrient medium contains enzyme yolk hydrolyzate containing 0.10-0.14% of amine nitrogen, potassium monophosphate, 3-aqueous disubstituted potassium phosphate, activated carbon, L-cysteine hydrochloride, microbiological agar and distilled water in the preset proportions.

EFFECT: invention allows reducing time for legionella cultivation.

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Description

The invention relates to biotechnology, namely, to obtain culture 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 10.0 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, p. 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 pig lung 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 growth stimulator of microorganisms 2.5; microbiological agar 8.5; distilled water up to 1 liter. (RF patent No. 2412240. Publ. 02.20.2011, Bull. No. 5.)

The disadvantage of this environment is the relatively high cost.

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 chicken yolk, 0.01 M potassium phosphate buffer (potassium phosphate buffer composition: potassium phosphate 1-substituted; potassium phosphate 2-substituted 3-aqueous), and Activated carbon; L-cysteine hydrochloride; microbiological agar, distilled water in the following ratio of ingredients, g / l:

Enzymatic hydrolyzed egg yolk (with an amine nitrogen content of 0.10-0.14%) 132.0-192.0 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 0.25-0.55 Microbiological agar 7.5-11.5 Distilled water up to 1 liter

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 / 100 g: ash 1.7%; potassium 129; calcium 129; magnesium 15; sulfur 170; chlorine 146.8; phosphorus 542; sodium 51, trace elements in micrograms: 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 which - 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: 93% protein (protein); fat 94%. (M.F. Nesterin, I.M. Skurikhin. Chemical composition of food products. Moscow. "Food Industry". 1979. 247 p.)

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 of 0.01 M potassium phosphate buffer (the 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, as these are the only inorganic compounds that have a buffering effect in the physiologically important pH range, they are low toxic. In addition, HPO ₄ is a component of nucleic acids, phospholipids and nucleotides (Guidelines for the manufacture and use of culture media and solutions for microbiological purposes, the cultivation of cells and viruses. - M., 1989).

When preparing the nutrient medium, only distilled water is used without the use of solutions containing sodium ions, due to their destructive effect, they provide pure cultures of continuous growth after 24 hours with sowing of 100 mk.

Unlike the prototype, the proposed environment provides optimal conditions for the reproduction of legionella in 24 hours

Preparation of Enzymatic Hydrolyzate of Chicken Egg Yolk

30 yolks of chicken eggs, which is 550 ml, are poured into a 3-liter glass bottle, filled with drinking water heated to 42 + 1 ° C to 1.5 l, made alkaline with a 20% potassium hydroxide solution to pH 8.2 in an amount 13 ml phenolphthalein, then commercial enzymes are added: trypsin in an amount of 31.0 g and pancreatin 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 mixed after 20 ± 1 min for 5 minutes, and 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, due to a sharp drop in pH to 5 units. 30 ml of 20% potassium hydroxide was added to a pH of 8.2 by 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.

A nutrient medium on the enzymatic basis of the yolk of a chicken egg for growing Legionella is prepared in the following way. Take an enzymatic hydrolyzate from a chicken egg yolk diluted with distilled water to an amine nitrogen reading of 0.12% 162.0 ml, other ingredients in the ratio, g / l: potassium phosphate 1-substituted 0.9; potassium phosphate 2-substituted 3-aqueous 2.2; activated carbon 2.0; microbiological agar 9.5; distilled water - up 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 dilution of hydrochloric acid (1: 1) in an amount of 1.0 ml to a pH of 6.8 ± 0.1. 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. 0.4 g of L-cysteine hydrochloride sterilized by autoclaving at 112 ° C for 30 min is added to agar melted in a water bath and cooled to 56 ° C, then poured into sterile Petri dishes. Dry for 30 minutes and use for sowing.

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

The effectiveness of the obtained culture medium was evaluated in accordance with the guidelines “Control of diagnostic culture media for biological indicators for plague, cholera, anthrax, tularemia, brucellosis, legionellosis” (Moscow, 2007), using Legionella pneumophilla ATCC as test cultures 33155 Bloomington, Legionella pneumophilla ATCC 33152 Philadelphia.

The test cultures were grown on plates of solid agar of a control environment of SEL pH 6.8 at a temperature of 36 ° C for 24 hours. From daily cultures, 1 billion mt suspension was prepared in sterile 0.01 M potassium phosphate buffer. test strains equal to 10 units according to the optical turbidity standard OSO 42-28-85P (pH 6.8) GISK them. L.A. Tarasevich, then serial ten-fold dilutions in potassium phosphate buffer in a volume of 4.5 ml were adjusted to a content of 1000 ml in 1 ml (10 ^-6 ). From the dilution of culture suspensions, 0.1 ml was sown in 3 Petri dishes of the spilled dried test agar and on control media: SEL and Hottinger agar (negative control - there is no growth on it at any observation time). Crops were incubated at 36 ± 1 ° C for 5 days. After 24-120 hours, the results were taken into account by counting the grown colonies.

SEL control medium (Nutrient medium for cultivation and isolation of the causative agent of Legionellosis selective) - composition, g / l: nutrient base of animal origin - liquid spleen fermentolizate of cattle per dry matter - 7.0; activated carbon OU-A 4.0; microbiological agar 17.0; potassium phosphate monosubstituted 1.6; potassium phosphate disubstituted three-water 3.4; pH 7.1. (Producer FGUZ Growth NIIPCH Rospotrebnadzor. 344002, Rostov-on-Don, M. Gorky St., 117.)

Hottinger control agar. Industrial regulation No. 1707-05 for the production of nutrient agar for the cultivation of microorganisms, ready for use (Hottinger agar). Registration certificate No. ФСР 2009/05571 dated August 20, 2009. The validity period is unlimited.

Example 1. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from chicken yolk diluted with distilled water to an amine nitrogen reading of 0.10%, 132.0 ml; potassium phosphate 1-substituted 0.7; potassium phosphate 2-substituted 3-aqueous 2.0; activated carbon 1.0; L-cysteine hydrochloride 0.25; microbiological agar 7.5; distilled water - up to 1 liter. With this ratio of ingredients, more than 50% of flat-convex, bluish-grayish colonies with a diameter of 1.0 mm grew after 24 hours for both strains taken in the experiment, whereas on the SEL control medium growth was observed only after 72 hours in an amount of less than 50% of the colonies , and there was no growth on the Hottinger agar.

Example 2. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from chicken yolk diluted with distilled water to an amine nitrogen reading of 0.12% 162.0 ml; potassium phosphate 1-substituted 0.9; potassium phosphate 2-substituted 3-aqueous 2.2; activated carbon 2.0; L-cysteine hydrochloride 0.4; microbiological agar 9.5; distilled water - up to 1 liter. With this ratio of ingredients, a continuous growth in the form of bluish-gray plaque was observed for both test strains taken in experiment, whereas on the SEL control medium growth was observed only after 72 h in the amount of more than 50% of colonies with a diameter of 1.2 mm from sowing, and no growth on the Hottinger agar.

Example 3. Test strains were grown on a nutrient medium containing, g / l: enzymatic hydrolyzate from chicken yolk diluted with distilled water to an amine nitrogen reading of 0.14% 192.0 ml; potassium phosphate 1-substituted 1,1; potassium phosphate 2-substituted 3-aqueous 2.4; activated carbon 3.0; L-cysteine hydrochloride 0.55; microbiological agar 11.5; distilled water - up to 1 liter. With this ratio of ingredients, more than 50% of flat-convex, bluish-grayish colonies with a diameter of 1.0 mm grew after 24 hours for both test strains taken in the experiment, while growth on the SEL control medium was observed only after 72 hours in an amount of less than 50 % of the colonies, and on the Hottinger agar there was no growth.

The results obtained allowed us to determine the optimal version of the nutrient medium based on the enzymatic hydrolyzate of chicken yolk (example 2).

Claims (1)

Dense nutrient medium for growing Legionella, including a nutrient base, potassium phosphate 1-substituted, potassium phosphate 2-substituted 3-water, activated carbon, L-cysteine hydrochloride, microbiological agar and distilled water, characterized in that it contains enzymatic as a nutrient base hydrolyzed egg yolk hydrolyzate in the following ratio of ingredients, g / l:
enzymatic hydrolyzate of chicken yolk (with an amine nitrogen content of 0.10-0.14%) 132.0-192.0 1-substituted potassium phosphate 0.7-1.1 2-substituted potassium phosphate 3-water 2.0-2.4 Activated carbon 1.0-3.0 L-cysteine hydrochloride 0.25-0.55 microbiological agar 7.5-11.5 distilled water up to 1 l