RU2841394C1 - Method for producing liquid culture medium for culturing and accumulating microbial cells of leptospirosis agent - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a method for preparing a liquid culture medium for culturing and accumulating microbial cells of a leptospirosis agent involving mixing dry weights of: 12-aqueous disubstituted sodium phosphate – 1.00 g, monosubstituted potassium phosphate – 0.30 g, sodium chloride – 1.00 g, ammonium chloride – 0.25 g, thiamine – 0.005 g in a container and adding up to 1 l of distilled water, mixture is brought to the boiling point, boiled until all the ingredients are dissolved, allowed to cool, pH is corrected to 7.4 and dispensed into graduated flasks, canted and sterilized at temperature of 115 °C for 15 minutes, cooled to temperature 56 °C, normal inactivated rabbit serum is added in a sterile manner at rate of 40 ml/l and normal horse blood serum for cultivating mycoplasmas on nutrient media, liquid sterile, at rate of 20 ml/l, and sterilely dispensed from flasks by bacteriological pipettes into bacteriological test tubes.

EFFECT: invention enables preparing the easy-to-prepare liquid culture medium for culturing and accumulating microbial cells of the leptospirosis agent with increasing the number of formed microbial cells with preserving their typical morphology and active mobility in strain passage up to 14 days (follow-up period).

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Description

The invention relates to microbiology and biotechnology, namely, to the production of nutrient media that create optimal conditions for cultivating the causative agent of leptospirosis.

To accumulate the bacterial mass of leptospira, the VGNKI medium is used, which is prepared as follows: the required amount of Zerensen buffer mixture (pH 7.2) is added to a bottle with a siphon and sterilized in an autoclave at 0.8 atm for 30 min, 5-8% inactivated rabbit or ram serum is added to the cooled mixture until the protein concentration in the medium is 0.22-0.25% and vitamins: B ₁₂ - to a concentration of 1 mg / ml and B ₁ - to a concentration of 0.25 ml / l of 6% solution.

Zerensen's mixture: solution A: 11.867 g of sodium dibasic phosphate are dissolved in 1 liter of distilled water; solution B: 9.067 g of potassium monobasic phosphate are dissolved in 1 liter of distilled water. To 72 ml of solution A add 28 ml of solution B and bring the volume to 1000 ml with distilled water.

The medium is filtered through sterilizing Seitz filter plates and poured into sterile 5-10 ml test tubes or bottles. To test for sterility, the medium is kept at 37°C for 3 days and at room temperature for up to 10 days [1].

The disadvantages of this environment are the complexity and time required to manufacture it.

The closest to the proposed invention is Terskikh's medium, consisting of Zerensen's phosphate mixture and rabbit serum.

The Zerensen mixture is filtered twice through a two-layer paper filter, poured into 5 ml test tubes, sterilized in an autoclave at 120°C for 20 min, and inactivated rabbit serum is sterilely added to each test tube at a rate of 1-2 drops per 1 ml of medium. The test tubes with the medium are heated twice in a water bath at 56°C for 1 hour [1].

The disadvantages of this medium are the complexity of production and the reduction of growth properties during long passages of cultures.

The aim of the invention is to develop a simple-to-manufacture liquid nutrient medium for cultivating and accumulating microbial cells of the leptospirosis pathogen with improved growth properties.

The essence of the proposed invention is that a commercial preparation - normal horse blood serum for cultivating mycoplasmas in liquid sterile nutrient media - is used as the main source of nitrogen nutrition in the preparation of a liquid nutrient medium for cultivating and accumulating microbial cells of the leptospirosis pathogen together with freshly taken rabbit serum, inactivated by heating in a water bath for 30 minutes at a temperature of 56°C.

The technical result is achieved in that the liquid nutrient medium for cultivating and accumulating microbial cells of the leptospirosis pathogen, in addition to the main source of nutrition - serums, contains ammonium chloride and thiamine, which increase the accumulative properties of the medium as additional growth factors; phosphates with a buffering effect - 12-aqueous dibasic sodium phosphate and monobasic potassium phosphate, as well as sodium chloride and distilled water, with the following ratio of ingredients, g / l:

12-hydrate sodium dihydrogen phosphate 1.00 Potassium monophosphate 0.30 Sodium chloride 1.00 Ammonium chloride 0.25 Thiamine 0.005 Rabbit serum normal inactivated 40.0 ml Horse blood serum is normal for cultivation of mycoplasmas on nutrient media liquid sterile 20.0 ml Distilled water up to 1 l

Phosphates, sodium diphosphate dodecyl phosphate and potassium diphosphate are widely used in the preparation of nutrient media, which is justified by their buffering action in the physiologically important pH range and low toxicity [2].

Sodium chloride is necessary to maintain the isotonicity of the medium. Dissolved in the nutrient medium and in a state of electrolytic decomposition, ionizable mineral compounds are simultaneously catalysts for various chemical processes occurring in the microbial cell. Sodium ions (Na ⁺ ) participate in the transport of substances through cell membranes and in the regulation of protein synthesis [3].

Freshly taken normal inactivated rabbit serum is a source of nitrogen, albumins and globulins [4] in a liquid nutrient medium for culturing and accumulating microbial cells of the causative agent of leptospirosis.

Blood for obtaining serum was taken from rabbits weighing 1.5-3 kg. The animals were fixed on their backs, the hair was cut off at the site of palpation of the cardiac impulse, the surface was treated with alcohol and iodine. Blood was taken by inserting a syringe needle into the heart, in an amount of 30-40 ml and poured into sterile test tubes or bottles. The container with blood was placed in a thermostat at 37 ° C for 30-40 minutes. The resulting clot was outlined with a sterile metal needle and placed in the refrigerator for 1-2 days.

The settled serum was poured into sterile 100-200 ml vials and inactivated at a temperature of 56-58°C for 30 min. The temperature was strictly monitored, since overheated serum is unsuitable for the preparation of nutrient media. The inactivated serum was stored at a temperature of 2-4°C [1].

Horse blood serum normal for cultivating mycoplasmas on nutrient media, liquid sterile - a commercial preparation, used to intensify growth and accelerate the process of accumulation of leptospira, while maintaining their active mobility.

Ammonium chloride increases the accumulative properties of nutrient media, being an additional growth factor; participates in the regulation of osmotic pressure inside cells and the direction of biochemical reactions; has a beneficial effect on the colloidal properties of living protoplasm. It is known from literary sources that leptospires can utilize inorganic ammonium salts as the main source of nitrogen [1].

Thiamine (vitamin B ₁ ) is a growth factor that is required by the causative agents of leptospirosis [1]. Many bacteria lack the ability to synthesize all the organic compounds necessary for growth and depend on the presence of certain growth factors in the environment. The need for vitamins is a common property of all microorganisms.

Distilled water, in accordance with sanitary and hygienic requirements, is a transparent, colorless, odorless liquid containing: ammonium ions, nitrate ions, zinc - no more than 0.2 mg / dm ³ ; sulfate ions and chloride ions - no more than 0.5 mg / dm ³ ; aluminum, iron, lead - no more than 0.05 mg / dm ³ ; calcium - no more than 0.8 mg / dm ³ ; copper - no more than 0.02 mg / dm ³ ; having a specific conductivity at a temperature of 20 ° C of no more than 4.3⋅10 ^-4 S / m and pH - from 5.0 to 7.0 [5].

Water makes up 80-90% of the cellular mass of microorganisms and plays a vital role in their physiological functions, is part of the structural elements of the cell, serves as a medium for biochemical reactions, a source of oxygen in metabolic processes, and is directly involved in metabolic reactions and hydrolysis reactions.

Preparing the dishes

The dishes intended for culturing leptospires were disinfected and washed separately from other dishes. The test tubes used were processed using a special method, which included the following steps:

- immersion for 24 hours in a 1-2% solution of hydrochloric acid in a glass or enamel container;

- rinsing with running tap water (12 - 15 times);

- treatment with brushes in a hot soapy solution (1 bar of laundry soap per 10 liters of water);

- thorough rinsing with running tap water (12-15 times);

- immersion in distilled water for 24 hours;

- drying and sterilization together with cotton plugs.

New rubber hoses and plugs were washed with running tap water, boiled for 30-40 minutes in a 2% solution of baking soda, washed with tap water and boiled again in distilled water for 30-45 minutes, after which they were dried [1].

Preparation of nutrient medium

To prepare the liquid nutrient medium, the following dry samples were weighed on scales: 12-hydrate dibasic sodium phosphate - 1.00 g; monobasic potassium phosphate - 0.30 g; sodium chloride - 1.00 g; ammonium chloride - 0.25 g, thiamine - 0.005 g. The samples were poured into an enamel container and distilled water was added to 1 liter. On a gas stove, the mixture was brought to a boil, boiled until all the ingredients were dissolved, allowed to cool, the pH was adjusted to 7.4 and poured into 333 ml graduated bottles with a volume of 450 ml, crimped and sterilized at a temperature of 115 ° C for 15 minutes.

The medium cooled to a temperature of 56°C was poured into bacteriological tubes of high purity using sterile bacteriological pipettes with a capacity of 5 ml, after first adding in a sterile manner over a torch normal inactivated rabbit serum - 13.3 ml per bottle (at the rate of 40 ml/l) and normal horse blood serum for cultivating mycoplasmas on nutrient media, liquid sterile - 6.7 ml per bottle (at the rate of 20 ml/l). In the examples given, the added amount of serums was varied in increments of 10 ml/l.

The sterility of the medium was controlled by keeping it at a temperature of 37°C for 2 days and at room temperature (20-25°C) for up to 7 days; the medium should remain transparent.

Direct microscopy technique

Leptospires do not perceive color well, so all observations were carried out with live pathogens in the dark field of view of the microscope. For this purpose, a microscope with a dark field condenser was used. To identify leptospires by microscopy, "crushed drop" preparations were prepared: 5 μl of test tubes with the studied media after thermostatting were applied with a loop onto a slide no more than 1.0-1.1 mm thick and covered with a cover glass 0.2 mm thick. Due to the significant size of the microbe, dry systems were used for the study: a ×40 objective and an ×10 eyepiece.

The growth properties of the proposed liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen were assessed in accordance with the guidelines MU 3.1.1128-02 Epidemiology, diagnostics and prevention of human diseases caused by leptospirosis [6] and taking into account the requirements of SanPiN 3.3686-21 Sanitary and epidemiological requirements for the prevention of infectious diseases [7], using diagnostic virulent strains of Leptospira interrogans P.O.5621 (serogroup Pomona); strain M20 (serogroup Icterohaemorrhagiae); strain Perepelitsin (serogroup Tarassovi); strain Ballico (serogroup Australis); strain Akiyami A (serogroup Autumnalis).

The Terskikh nutrient medium served as the comparison medium. 0.5 ml of suspensions from the original test tubes with stored cultures were inoculated into each test tube with the declared and control media and cultivated at a temperature of 29±1°C. The growth of leptospirosis pathogens in the proposed and comparison nutrient media was observed as a barely noticeable turbidity. To detect the growth of leptospirosis pathogens from the 5th to the 14th day of cultivation, “crushed drop” preparations were prepared from the test tubes and examined in a dark field microscope by viewing 10 fields of vision at a magnification of ×400. Mobility, the number of cells in the field of vision, as well as a characteristic morphological feature of leptospira - a hook-shaped end of the microbial body - were taken into account.

When leptospires were detected in the test tubes after thermostatting, their contents were transferred by 0.5 ml into test tubes with 5 ml of fresh liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen in each for further preservation of the cultures.

A table is presented with data on the number of leptospira cells observed in the “crushed drop” preparations from the comparison medium and the proposed nutrient medium.

In the “crushed drop” preparations from the comparison medium cultures after 5 days of culturing the diagnostic strains of L. interrogans, an average of 63±11.8 leptospira cells of typical morphology were observed: from 48±2.6 m.c. (in the Ballico strain, serogroup Australis) to 79±4.0 m.c. (in the Akiyami A strain, serogroup Autumnalis).

When re-examined 14 days after sowing, a slight decrease in the number of microbial cells in the field of view was observed for all five strains.

Example 1. The test strains were grown on a liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen at an optimal pH of 7.4±0.2, containing, g/l: 12-aqueous dibasic sodium phosphate - 1.00; monobasic potassium phosphate - 0.30; sodium chloride - 1.00; ammonium chloride - 0.25, thiamine - 0.005. In an enamel container, adding distilled water to 1 l, boiled for 1-2 minutes until the ingredients were completely dissolved, allowed to cool and adjusted to pH 7.4. Poured 333 ml into graduated 450 ml bottles, crimped and sterilized at a temperature of 115 ° C for 15 min. The medium cooled to 56°C was poured into high-purity bacteriological test tubes using sterile bacteriological pipettes with a capacity of 5 ml, after first adding in a sterile manner over a torch normal inactivated rabbit serum - 10.0 ml per bottle (at the rate of 30 ml/l) and normal horse blood serum for cultivating mycoplasmas on nutrient media, liquid sterile - 3.3 ml per bottle (at the rate of 10 ml/l).

With this ratio of ingredients in the liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen in the “crushed drop” preparations from the cultures kept in a thermostat for 5 days at a temperature of 29±1°C, an average of 76±5.2 typical, actively motile leptospira cells were observed in the dark field of view at a magnification of ×400: from 68±3.4 m.c. of the strain P.O.5621 (serogroup Pomona) to 93±3.6 m.c. of the strain Akiyami A (serogroup Autumnalis). When re-examining 14 days after culture, the picture did not change significantly.

Example 2. The test strains were grown on a liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen at an optimal pH of 7.4±0.2, containing, g/l: 12-aqueous dibasic sodium phosphate - 1.00; monobasic potassium phosphate - 0.30; sodium chloride - 1.00; ammonium chloride - 0.25, thiamine - 0.005. In an enamel container, adding distilled water to 1 l, boiled for 1-2 minutes until the ingredients were completely dissolved, allowed to cool and adjusted to pH 7.4. Poured 333 ml into graduated 450 ml bottles, crimped and sterilized at a temperature of 115 ° C for 15 min. The medium cooled to 56°C was poured into high-purity bacteriological test tubes using sterile bacteriological pipettes with a capacity of 5 ml, after first adding in a sterile manner over a torch normal inactivated rabbit serum - 13.3 ml per bottle (at a rate of 40 ml/l) and normal horse blood serum for cultivating mycoplasmas on nutrient media, liquid sterile - 6.7 ml per bottle (at a rate of 20 ml/l).

With this ratio of ingredients in the liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen in the “crushed drop” preparations from the crops kept in a thermostat for 5 days at a temperature of 29±1°C, an average of 119±3.8 typical, actively motile leptospira cells were observed in the dark field of view at a magnification of ×400: from 107±2.5 m.c. of the M20 strain (Icterohaemorrhagiae serogroup) to 126±3.6 m.c. of the Akiyami A strain (Autumnalis serogroup). When viewed after 14 days, the number of typical and motile cells remained the same.

Example 3. The test strains were grown on a liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen at an optimal pH of 7.4±0.2, containing, g/l: 12-aqueous dibasic sodium phosphate - 1.00; monobasic potassium phosphate - 0.30; sodium chloride - 1.00; ammonium chloride - 0.25, thiamine - 0.005. In an enamel container, adding distilled water to 1 l, boiled for 1-2 minutes until the ingredients were completely dissolved, allowed to cool and adjusted to pH 7.4. Poured 333 ml into graduated 450 ml bottles, crimped and sterilized at 115°C for 15 min. The medium cooled to 56°C was poured into high-purity bacteriological test tubes using sterile bacteriological pipettes with a capacity of 5 ml, after first adding in a sterile manner over a torch normal inactivated rabbit serum - 16.7 ml per bottle (at a rate of 50 ml/l), and normal horse blood serum for cultivating mycoplasmas on nutrient media, liquid sterile - 10.0 ml per bottle (at a rate of 30 ml/l).

With this ratio of ingredients in the liquid nutrient medium for cultivating and accumulating microbial cells of the leptospirosis pathogen in “crushed drop” preparations from crops kept in a thermostat for 5 days at a temperature of 29±1°C, an average of 251±13.9 leptospira cells were observed in the dark field of vision at a magnification of ×400: from 221±3.2 m.k. strain Ballico (serogroup Australis) to 281±3.8 m.c. strain Akiyami A (serogroup Autumnalis), however, due to spontaneous agglutination, cell counting of all tested strains was difficult. In all test tubes, when viewed after 14 days of cultivation, a decrease in the number of leptospira was observed, their number, on average, was 134±2.7 m.c.: from 130±3.0 m.c. strain Ballico (serogroup Australis) to 142±8.7 m.c. strain P.O.5621 (serogroup Pomona).

Thus, the declared liquid nutrient medium for cultivating and accumulating microbial cells of the leptospirosis pathogen (example 2) is optimal in terms of the number of leptospira cells obtained with typical morphology and active motility, observed in “crushed drop” preparations in the dark field of view of a microscope at a magnification of ×400.

The nutrient medium is high-quality, easy to manufacture, when cultivating leptospirosis pathogens, full-fledged microbial cells are formed on the 5th day, and no decrease in growth is observed up to the 14th day (observation period).

List of references

1. Laboratory diagnostics of dangerous infectious diseases. Practical guide / Edited by Academician of the Russian Academy of Medical Sciences G.G.

Onishchenko and Academician V.V. Kutyrev. 2nd edition, revised and supplemented. - Moscow: ZAO Shiko, 2013. - P. 468-498.

2. Methodological recommendations for the preparation and use of nutrient media and solutions for microbiological purposes, cell and virus cultivation. - M., 1986.

3. Medzhidov M.M. Handbook of microbiological nutrient media. Moscow, "Medicine", 2003. - P. 18.

4. Peretrukhina A.T., Blinova E.I. Bacterial and viral preparations: a textbook for students of higher educational institutions studying in the specialty: 020209.65 - "Microbiology" and areas of training 020400.62 - "Biology" (bachelor's degree), profile "Microbiology"; 020400.68 - "Biology" (master's degree), profile "Microbiology" / A.T. Peretrukhina, E.I. Blinova; Federal Agency for Fisheries, FGBOU HPE "Murm State Technical University". - Moscow: Publishing House of the Academy of Natural Sciences, 2011. - 311 p.

5. GOST R 58144-2018. Distilled water. Technical conditions.

6. MU 3.1.1128-02 Epidemiology, diagnostics and prevention of human diseases with leptospirosis. - Ministry of Health of Russia, - Moscow. - 2002.

7. Sanitary rules and regulations SanPiN 3.3686-21 Sanitary and epidemiological requirements for the prevention of infectious diseases. - Moscow, Ministry of Health of the Russian Federation. - 2021.

Claims (1)

A method for obtaining a liquid nutrient medium for culturing and accumulating microbial cells of the leptospirosis pathogen, including mixing dry samples of: 12-aqueous sodium dibasic phosphate - 1.00 g, monobasic potassium phosphate - 0.30 g, sodium chloride - 1.00 g, ammonium chloride - 0.25 g, thiamine - 0.005 g in a container and adding up to 1 l of distilled water, the mixture is brought to a boil, boiled until all ingredients are dissolved, allowed to cool, the pH is adjusted to 7.4 and poured into graduated vials, sealed and sterilized at a temperature of 115 ° C for 15 minutes, cooled to a temperature of 56 ° C, normal inactivated rabbit serum is sterilely added at a rate of 40 ml / l and normal horse blood serum for cultivating mycoplasmas on nutrient media, liquid sterile at a rate of 20 ml/l, and sterilely poured from vials with bacteriological pipettes into bacteriological test tubes.