RU2728379C1 - Nutrient medium dense for brucella cultivation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is represented by a dense nutrient medium for Brucella cultivation, including mixing dry fermentative peptone, sodium chloride, microbiological agar, mixed hydrolyzate of embryo and extraembryonic birds tissues – stimulator C with distilled water, heating to boiling, boiling till complete agar melting, filtration, pH adjustment with hydrochloric acid solution (1:1) to pH 7.2±0.1, pouring finished filtrate into flasks and hermetic sealing, sterilization in autoclave.

EFFECT: use of the given medium enables to grow a sufficient number of colonies of the brucellosis agent early in 24–48 hours.

1 cl, 3 ex

Description

The invention relates to biotechnology and microbiology, namely, to the development of nutrient media that create optimal conditions for the cultivation of the brucellosis microbe.

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

The disadvantage of this medium is the lack of nutritional value for brucella.

Closest to the claimed invention is a method of obtaining a nutrient medium-substitute for "Albimi" -agar for the cultivation of brucella, containing, per 1 liter: 20.0 g of dry peptone, 20 ml of yeast water, 5 g of chemically pure sodium chloride and 20 g of agar agar, pH 7.3. These ingredients are dissolved in an autoclave under flowing steam and filtered through a cotton-gauze filter (pre-moistened with warm water and well wrung out). Then add 1 g of glucose and 0.1 g of sodium bisulfite, adjust the pH to 7.2-7.3. It is poured into the necessary dishes and sterilized for 40 minutes under flowing steam, and then at 110 ° C for 20 minutes. Yeast water: 1 kg of bread yeast and 1 liter of distilled water are boiled until dissolved. Then filtered through a web. It should be stored under chloroform in the dark for no more than two weeks (Epidemiological surveillance and laboratory diagnosis of brucellosis. Guidelines MU 3.1.7. 3402-16. Official edition. Ministry of Health of Russia, Moscow. 2017, p. 58).

The disadvantage of the nutrient medium obtained by this method is the slow growth of the pathogen.

The aim of the invention is to develop a method for producing a dense nutrient medium for the cultivation of brucella, providing the growth of pathogen colonies after 24 and 48 hours, which is essential for the diagnosis of infection and the production of biological products.

The achieved technical result consists in the fact that the production of a stimulator C is introduced into the technology of manufacturing a nutrient medium, which is a mixed hydrolyzate of embryonic and extraembryonic tissues of birds subjected to special treatment. As a nutrient base and a source of nitrogen, the medium contains enzymatic dry peptone, and also includes: sodium chloride, microbiological agar and distilled water.

The essence of the invention lies in the use of stimulant C, which, in the composition of the nutrient medium for the cultivation of the pathogen of brucellosis, provides a significant reduction in the growth time of Brucella colonies formed in the early stages - after 24 and 48 hours, on plates of the nutrient medium containing the following ingredients:

peptone dry enzymatic 20.0 g; sodium chloride 5.0 g; microbiological agar 9.0 g; stimulator C (mixed hydrolyzate of embryonic and extraembryonic tissues of birds) 35.0 ml; distilled water up to 1 l

Embryonic and extraembryonic tissues of birds are used as a raw material for obtaining stimulator C. After multi-stage pretreatment of raw materials by first acidic, then enzymatic hydrolysis, a mixed hydrolyzate is obtained (Rzhepakovsky IV, Timchenko LD, Areshidze DA, Avanesyan SS, Budkevich EV, Piskov SI, Mannino S., Lodygin AD, Kovalev DA, Kochergin SG Antioxidant activity of chicken embryo tissues powder obtained by different methods of hydrolysis / Journal of Hygienic Engineering and Design (Macedonia, electronic.). - 2019. - Vol. 27. - pp. 127-133. E-ISSN: 1857-8489 http: // www .jhed.mk / categories / view / 478/457.

Characteristics of the resulting hydrolyzate: sterile, transparent brown liquid, without preservatives; dry matter content (35.0 ± 3.0) g / l, hydrolyzate pH 7.0-7.5, reaction with sulfosalicylic acid for proteins - negative, total nitrogen (0.37 ± 0.03)%, amine nitrogen (0.11 ± 0.02)%, degree of hydrolysis (30.8 ± 3.0)%.

The scheme for obtaining a mixed hydrolyzate (stimulant C):

- preliminary processing of embryonic and extraembryonic tissues of birds during incubation of 9-10 day old chicken embryos to stimulate metabolic processes;

- keeping in a refrigerator for 7 days;

- tissue homogenization;

- nanostructuring of the mass in a high-pressure homogenizer;

- freezing and lyophilization of the resulting mass;

- fractionation of the obtained protein-containing powder into proteins, peptides, amino acids, carbohydrates, nucleic acids, mineral compounds, etc .;

- acid hydrolysis;

- enzymatic hydrolysis;

- substance purification;

- autoclaving.

Sodium chloride - chemically pure grade, GOST 4233-77, batch 24. Produced in Barnaul. Date of manufacture 06.2017 Expiry date 3 years. Bacteria need a minimum amount of salt. Dissolved in a nutrient medium and being in a state of electrolytic decay, ionizable mineral compounds are simultaneously catalysts for various chemical processes occurring in the microbial cell. Sodium chloride is necessary to maintain the isotonicity of the medium and the redox potential.

Dry enzymatic peptone for bacteriological purposes - GOST 13805-76. Producer LLC NPO Port-Petrovsk. Date of manufacture 05.2018 Expiry date 05.2021. Peptone is a mixture of various protein breakdown particles that do not coagulate when heated (peptones, peptides, amino acids, etc.). Enzymatic peptone contains 16 amino acids, mg%: aspartic acid 0.40 ± 0.03; threonine 0.30 ± 0.01; series 0.40 ± 0.03; glutamic acid 0.50 ± 0.03; glycine 0.50 ± 0.01; alanine 1.0 ± 0.02; valine 0.80 ± 0.02; methionine 0.40 ± 0.02; isoleucine 0.70 ± 0.01; leucine 2.40 ± 0.06; tyrosine 0.40 ± 0.01; phenylalanine 1.30 ± 0.01; lysine 1.30 ± 0.05; histidine 0.20 ± 0.02; arginine 1.70 ± 0.09; tryptophan 0.06 ± 0.01 (Shepelin A.P., Dyatlov I.A. Nutrient media for enterobacteria. Obolensk 2017, p. 43; p. 232).

Microbiological agar - bacteriological agar (European type). Manufacturer: "Pronadisa" Spain. Lot LF 15130184. Date of manufacture: October 2017 Expiry date: until 10.2022 Powder of light creamy grayish color, odor without foreign matter, characteristic of jelly with a mass fraction of dry agar 0.85%. The melting point of jelly with a mass fraction of dry agar of 0.85% is not lower than 80 ° C, the gelation temperature is 30-37 ° C, the strength of the jelly is not less than 350 ± 40 g. The main feature of agar is its gelling ability at 90 ° C.

Distilled water - GOST 6709-72, meets all sanitary and hygienic requirements. Appearance - transparent liquid, chlorides, no mg / dm, nitrates, no mg / dm. Specific electrical conductivity at 20 ° C, S / m - 0.38, pH - 6.5. Water makes up 80-90% of the cell mass of microorganisms and plays an important role in their physiological functions, and is also part of the structural elements of the cell, serves as a medium for biochemical reactions, a source of oxygen in metabolic processes, directly participates in metabolic reactions, for example, in reactions hydrolysis.

Obtaining a mixed hydrolyzate from embryonic and extraembryonic tissues of birds (stimulant C).

Lipids are removed from the sublimate of embryonic and extraembryonic tissues of 9-10-day-old chicken embryos (Incubator for eggs ILB-0.5, Russia and Lyophilic dryer LS-500, Prointech, Russia) with petroleum ether (Rotary evaporator RV 10 Basic V, IKA, Germany ) with subsequent drying of the defatted protein-containing residue (Lyophilic dryer LS-500, Prointech). Take 500 ml of distilled water (distiller, Liston, Russia), add to 20 g of protein-containing powder, mix and place in a shaker-thermostat ES 20/60 (Biosan, Latvia) for incubation at 50 ° C for 30 min and with shaking 100 vol. ./min. Then 35% HC1 is added to the solution to its concentration of 0.5% and kept at 50 ° C for 60 min and with shaking at 100 rpm in an ES 20/60 shaker thermostat (Biosan, Latvia). Then the resulting mass is autoclaved at 125 ° C for 60 min in a steam sterilizer SPVA-75-1NN (Trans-signal, Russia). The autoclavate cooled to 37 ° C is neutralized with 1 M NaOH to pH 7.0-7.5, trypsin is added 25 μg / ml, kept with stirring for 120 min at 37 ° C in an ES 20/60 shaker thermostat (Biosan, Latvia) at shaking 100 rpm. Fermentation is stopped by heating to 90 ° C for 10 min on a thermoban LAB-TZh-TB-01/26 (Medlab, Russia). The resulting hydrolyzate is centrifuged (SL40R refrigerated centrifuge (Thermo fisher scientific, USA) at 4000-5000 g for 120 minutes at 4 ° C. The liquid obtained after centrifugation is successively filtered using a Vivaflow 50 filtration system (Sartorius, France) using PES cassettes (pore sizes 0.2 μm, 30 kDa and 10 kDa) and autoclaved at 120 ° C for 10 min in a steam sterilizer SPVA-75-1NN (Trans-signal, Russia).

Preparation of a dense nutrient medium

To prepare a dense nutrient medium, dry enzymatic peptone is weighed on the scales - 20.0 g, sodium chloride - 5.0 g, microbiological agar - 9.0 g, a mixed hydrolyzate of embryonic and extraembryonic tissues of birds (stimulant C) - 35.0 ml (based on the dry residue (based on the dry matter content of 35.2 g / l). All ingredients are introduced into an enamel pan, distilled water is poured into the pan up to 1 l. Then everything is heated to a boil, boiled for two minutes until complete melting the agar, filter through a cotton-gauze filter, adjust the pH with a solution of hydrochloric acid (1: 1) to pH 7.2 ± 0.1.The finished light yellow filtrate is poured into graduated 450 ml vials of 250.0 ml, which closed with cotton-gauze plugs and Kraft paper, hermetically fixed with rubber rings, sterilized in an autoclave at 121 ° C for 20 minutes.

The prepared solid nutrient medium is cooled to (53 ± 3) ° С, poured into Petri dishes, 35-40 ml each. Used for the cultivation of brucella.

The growth properties of the resulting medium are assessed in accordance with the methodological instructions MU 3.3.2.2124-06 "Control of diagnostic nutrient media by biological indicators for causative agents of plague, cholera, anthrax, tularemia, brucellosis, legionellosis", Moscow, 2007, using test strains Brucella abortus 19 BA and B. melitensis Rev I. The test cultures of the test strains are grown on the surface of a slant agar, pH 7.2 ± 0.1, at a temperature of 37 ° C for 48 hours. From two-day cultures of test strains of Brucella, washed away from the surface of the slanting agar with 0.9% sodium chloride solution, suspensions are prepared with a concentration of 10 units according to the optical turbidity standard of bacterial suspensions OSO 42-28-85-2018 FSBI Scientific Center for the Expertise of Medicinal Products of the Ministry of Health of Russia, equivalent to 1.7 × 10 ⁹ brucella in 1 ml. The resulting microbial suspensions of the test strains are first diluted to a concentration of 1 × 10 ⁹ mc / ml, then, by serial tenfold dilutions, up to 1 × 10 ³ mc / ml. In the process of dilution, the suspension is transferred to the next tube using a sterile 1 ml pipette, changing the pipette for each dilution. 0.1 ml of each culture of B. abortus 19 BA and B. melitensis Rev I from dilutions 10-6 (100 m.c.) are sown on 3 Petri dishes with the test medium and evenly distributed by rocking over the entire surface. Crops are incubated at a temperature of (37 ± 1) ° C.

Accounting for results. Evaluation of the growth properties of the nutrient medium dense for the cultivation of brucella is carried out by counting the number of grown colonies of test strains B. abortus 19 BA and B. melitensis Rev I after 24, 48 and 72 hours. Albimi agar is used as a control.

Example 1. Cultures of test strains B. abortus 19 BA and B. melitensis REV I were grown on a dense nutrient medium for the cultivation of brucella containing the following ingredients: dry enzymatic peptone - 10.0 g, sodium chloride - 4.0 g, microbiological agar - 8.0 g, mixed hydrolyzate of embryonic and extraembryonic tissues of birds (stimulator C) - 30.0 ml, distilled water up to 1 liter.

With this ratio of ingredients in the test medium during cultivation on it of the test strains B. abortus 19 BA and B. melitensis REV I from crops of 100 m.k. colonies began to form, a visible growth of colonies appeared after 24 hours, and after 48 hours the number of colonies with an average diameter of 1.3 mm for both strains averaged 87 and 126, respectively, while on the control Albimi agar, growth at 100 m.k. averaged 71 and 99 colonies, respectively, with an average diameter of 1.3 mm after 72 hours.

Example 2. Cultures of test strains B. abortus 19 BA and B. melitensis REV I were grown on a dense nutrient medium for the cultivation of brucella containing the following ingredients: dry enzymatic peptone - 20.0 g; sodium chloride - 5.0 g; microbiological agar - 9.0 g; mixed hydrolyzate of embryonic and extraembryonic tissues of birds (stimulator C) - 35.0 ml, distilled water up to 1 liter.

With this ratio of ingredients in the test medium during cultivation on it of the test strains B. abortus 19 BA and B. melitensis REV I from crops of 100 m.k. colonies began to form after 24 hours, and after 48 hours the number of colonies with an average diameter of 1.5 mm for both strains averaged 115 and 157, respectively, while on the control Albimi agar, the growth at seeding 100 m.k. averaged 71 and 99 colonies, respectively, with an average diameter of 1.3 mm after 72 hours.

Example 3. Cultures of test strains B. abortus 19 BA and B. melitensis REV I were grown on a dense nutrient medium for the cultivation of brucella containing the following ingredients: dry enzymatic peptone - 30.0 g; sodium chloride - 6.0 g; microbiological agar - 10.0 g; mixed hydrolyzate of embryonic and extraembryonic tissues of birds (stimulator C) - 40.0 ml, distilled water up to 1 liter.

With this ratio of ingredients in the test medium during cultivation on it of the test strains B. abortus 19 BA and B. melitensis REV I from crops of 100 m.k. colonies began to form after 24 hours, and after 48 hours the number of colonies with an average diameter of 1.3 mm for both strains averaged 75 and 116, respectively, while on control Albimi agar, growth at 100 m.k. averaged 71 and 99 colonies, respectively, with an average diameter of 1.3 mm after 72 hours.

Thus, the claimed method of obtaining a dense nutrient medium for cultivating brucella using a mixed hydrolyzate of embryonic and extraembryonic tissues of birds (stimulator C) (example No. 2) is optimal in terms of the number of selected ingredients, which allows growing a sufficient number of colonies as early as possible - after 24 -48 hrs

Claims (2)

Dense nutrient medium for the cultivation of brucella, including mixing of dry enzymatic peptone, sodium chloride, microbiological agar, mixed hydrolyzate of embryonic and extraembryonic tissues of birds - stimulator C with distilled water, heating to a boil, boiling until the agar is completely melted, filtering, adjusting the pH with a solution of hydrochloric acid (1: 1) to pH 7.2 ± 0.1, pouring the finished filtrate into vials and hermetically sealing, sterilizing in an autoclave with the following ratio of starting ingredients: