RU2841400C1 - Method of producing liquid enriched nutrient medium for culturing leptospira - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a method for preparing a liquid enriched nutrient medium for leptospira cultivation involving mixing the following components: sodium chloride – 0.5 g; dry enzymatic peptone – 1.0 g; thiamine – 0.005 g in a container, adding 12.5 ml of Tween-80 to the mixture and adding the ready working solution of the phosphate-buffer mixture in distilled water to 1.0 l, boiling until all the ingredients are completely dissolved, the pH of medium is 7.4±0.2, and dispensed into graduated flasks, followed by sealing and sterilization at temperature 115 °C for 15 minutes, cooling down to temperature 56 °C, then in each bottle with a sterile bacteriological pipette, normal horse serum for bacteriological nutrient media liquid is added in amount of 10 ml/l and the medium is poured into sterile bacteriological test tubes.

EFFECT: invention enables to develop a liquid enriched culture medium for culturing leptospira with high growth properties with respect to leptospirosis agents and a simple manufacturing technique.

1 cl, 1 tbl, 3 ex

Description

The invention relates to microbiology and biotechnology, namely, to the production of liquid nutrient media for cultivating leptospires.

Tween-albumin medium is used for bacteriological examination for leptospirosis and for accumulation of leptospira bacterial mass. The medium is prepared in stages. Basic solutions are obtained by dissolving the following ingredients (each in 100.0 ml of distilled water): NH ₄ Cl - 25.0 g, ZnSO ₄ ⋅7H ₂ O - 0.4 g, MgCl ₂ ⋅6H ₂ O and СаС1 ₂ ⋅2H ₂ O - 1.5 g each, FeSO ₄ ⋅7H ₂ O - 0.5 g, sodium pyruvate - 10.0 g, glycerol - 10.0 g, Tween-80 - 10.0 g, thiamine - 0.5 g and cyanocobalamin - 0.02 g. The FeSO ₄ solution should be freshly prepared and transparent. Preparation of albumin supplement: Dissolve 10.0 g of bovine serum albumin (fraction V) in 50.0 ml of distilled water, stir with a magnetic stirrer, and add the following solutions: CaCl ₂ or MgCl ₂ -1.0 ml; FeSO ₄ -10.0 ml; cyanocobalamin - 1.0 ml; Tween-80 - 12.5 ml. Adjust pH to 7.4 and add distilled water to a final volume of 100.0 ml. Sterilize the albumin supplement by filtration. Prepare the base medium: Add 1 g of Na ₂ HPO ₄ (dehydrated) to 996 ml of distilled water; 0.3 g of K ₂ HPO ₄ (dehydrated) and 1.0 g of NaCl. Then add the following solutions: 1.0 ml of NH ₄ Cl; thiamine - 1.0 ml; sodium pyruvate - 1.0 ml and glycerol - 1.0 ml. The pH of the medium base is adjusted to 7.4 and then sterilized by autoclaving at 120°C for 20 min. Preparation of liquid twin-albumin medium: 1 volume of albumin supplement is mixed with 9 volumes of the medium base while maintaining asepsis.

The disadvantages of this environment are its multi-component nature, duration and complexity of production.

The closest to the proposed invention is the Ferworth-Wolf nutrient medium modified by S.I. Tarasov. Composition of the medium: distilled water - 900 ml, sodium chloride - 0.5 g, peptone (Difco or Witte) - 1 g, stock solution of phosphate buffer with pH 7.2 - 100 ml. The mixture in the flask is autoclaved at 120 ° C for 30 minutes. The next day, it is filtered twice through a two-layer paper filter, poured into test tubes of 5 ml and autoclaved again at 120 ° C for 30 minutes. To the test tubes with a transparent medium, 0.5 ml of inactivated (at 56 ° C for 30 minutes in a water bath) rabbit serum are added. The medium is heated twice in a water bath at a temperature of 56-58 ° C for 30 minutes. To check for sterility, test tubes with the medium are kept in a thermostat at a temperature of 37°C for 3-5 days.

The disadvantages of this medium include the lengthy, multi-stage production process, as well as rapid depletion, which leads to the weakening and death of diagnostic strains that require long-term preservation.

The aim of the invention is to develop a liquid enriched nutrient medium for cultivating leptospira with high growth properties in relation to leptospirosis pathogens and a simple manufacturing method.

The essence of the proposed invention is that enrichment of the nutrient medium composition with polysorbate Tween-80 and thiamine has a favorable combined effect on the growth rate, quantity and stability of leptospira cells formed on it, while replacing rabbit serum inactivated at a temperature of 56°C for 30 minutes in a water bath with a ready-made commercial preparation - normal horse serum for bacteriological nutrient media liquid allows to simplify and shorten the process of preparing the medium by eliminating the stage of its double heating in a water bath after adding the serum. In addition, there is no need for repeated autoclaving at 120°C for 30 minutes after pouring the medium into test tubes, which also simplifies the method of preparing the proposed medium.

The technical result is achieved by the fact that the liquid nutrient medium includes: normal horse serum for liquid bacteriological nutrient media - as the main source of nitrogen; dry enzymatic peptone and thiamine - as additional nutrient factors; polysorbate Tween-80 - as a source of fatty acids, to intensify the growth of leptospira; a working solution containing sodium and potassium phosphates - to ensure buffering of the medium; sodium chloride - to maintain the osmotic pressure level in the medium, distilled water - as a solvent, with the following ratio of ingredients, per 1 l:

Twin-80 12.5 ml Horse serum normal for bacteriological nutrient media liquid 10.0 ml Dry enzymatic peptone 1.0 g Thiamine 0,005 Sodium chloride 0.5 g Working solution of phosphate buffer mixture in distilled water up to 1 l

Obtaining a working solution of phosphate buffer mixture. Prepare two initial 1/15-M stock solutions: dissolve 11.867 g of sodium dibasic phosphate in 1 L of distilled water; dissolve 9.067 g of potassium monobasic phosphate in 1 L of distilled water. Store the solutions at 2-4°C for up to 1 month. To obtain a working buffer solution (pH 7.2), mix 72 ml of a 1/15-molar solution of sodium dibasic phosphate (alkaline solution) and 28 ml of a 1/15-M solution of potassium monobasic phosphate (acidic solution). Add 900 ml of distilled water to the resulting 100 ml of phosphate buffer. If the pH deviates towards the acidic or alkaline side, add a solution of sodium dibasic phosphate or potassium monobasic phosphate, respectively. The base is filtered twice through a two-layer paper filter, poured into vials and sterilized in an autoclave at 120°C for 20 minutes. After autoclaving, vials with a cloudy or sedimented solution are discarded. The shelf life of the working solution is 1 month at a temperature of 2-4°C [1].

Tween-80 is an oxyethylated monoester of fatty acid anhydrohexavites with the chemical formula C ₆₄ H ₂₆ O ₁₂₄ . It is a transparent, oily, slightly viscous liquid from yellow, bright amber, to brown in color with a slight characteristic odor, soluble in water and vegetable and animal oils. Adding small amounts of Tween-80 to essential and fatty oils gives them the ability to easily dissolve in water, while preserving all the beneficial properties of the oils in the mixture. It also dissolves well in isopropyl and ethyl alcohol, benzene. It does not dissolve in mineral oils. It is obtained chemically from sorbitol and olive oil fatty acids. It is used for bacteriological research, used as a component for enriching nutrient media, to maintain the viability of microorganisms, and to neutralize disinfectants in sanitary samples [2].

Dry enzymatic peptone for bacteriological purposes (GOST 13805-76), or pancreatic digest, is obtained from pancreatic enzymes. Peptone is produced mainly in a dry state, in the form of a yellow-beige powder. Peptone is characterized by high nutritional value for microorganisms [3].

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.

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 [4].

Horse serum normal for bacteriological nutrient media liquid - a commercial preparation, is the main source of nitrogen in the liquid nutrient medium, ensures the formation of full-fledged, actively mobile microbial cells of leptospira.

Preparing the dishes

The dishes intended for culturing leptospires must be subjected to special treatment, disinfected and washed separately from other dishes. In this work, we used test tubes treated according to a special method [1], 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.

Preparation of nutrient medium

To prepare a liquid enriched nutrient medium for culturing leptospira, sodium chloride - 0.5 g; dry enzymatic peptone - 1.0 g; thiamine - 0.005 g were weighed on a scale. The samples were poured into an enamel container, 12.5 ml of Tween-80 was added and the ready working solution of phosphate-buffer mixture in distilled water was added to 1.0 l. Boil for 1-2 minutes until all ingredients are completely dissolved. The pH of the medium was measured, it should be within 7.4 ± 0.2, and 333 ml were poured into graduated 450 ml bottles, then sealed and sterilized at a temperature of 115 ° C for 15 minutes. Cool to 56°C, then add 3.3 ml of normal horse serum for bacteriological nutrient media liquid (at a rate of 10 ml/l) to each vial over the torch of an alcohol lamp using a sterile bacteriological pipette, and pour 5 ml of the medium into sterile bacteriological tubes of high purity using sterile bacteriological pipettes. The amount of polysorbate Tween-80 and normal horse serum for bacteriological nutrient media liquid, added at a rate of 1 l of medium, in the given examples from the first to the third was increased by 2.5 ml for Tween-80 and by 5 ml for the serum, starting with doses of 10 ml and 5 ml, respectively.

Evaluation of growth properties of the proposed liquid enriched nutrient medium for the cultivation of leptospira

To control sterility, the medium was kept 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.

The growth properties were assessed in accordance with the guidelines MU 3.1.1128-02 Epidemiology, diagnostics and prevention of human diseases with leptospirosis [5] and taking into account the requirements of SanPiN 3.3686-21 Sanitary and epidemiological requirements for the prevention of infectious diseases [6], using cultures of 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), attenuated by multiple passaging on the Verworth-Wolff nutrient medium modified by SI. Tarasov. The same medium was used in experiments to compare the growth properties.

The cultures on both media were made in parallel, 0.5 ml per test tube from the original test tubes with stored cultures, after which they were placed in a thermostat at a temperature of 29±1°C. The growth of leptospires in the proposed and comparison nutrient media was observed as a barely noticeable turbidity. Since these pathogens poorly perceive color, bacterioscopic studies with leptospires are carried out in a dark field of a microscope, preparing a “crushed drop” preparation.

To detect the growth of leptospira from the 5th to the 14th day of culturing the strains, we prepared “crushed drop” preparations by applying 5 μl drops from test tubes with a loop onto slides no more than 1.0-1.1 mm thick and covering with a 0.2 mm thick cover glass. Live pathogens were observed using a microscope with a dark field condenser and, due to the relatively large size of the microbe, dry systems: a ×40 objective and an ×10 eyepiece. The preparations were examined by viewing 10 fields of view at a magnification of ×400. The presence of thin, flexible, mobile silvery threads with hooked ends, the activity of the microbes, and their number in the field of view were taken into account.

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±0.6 leptospira cells of typical morphology were observed: from 62±1.7 m.c. (in the Perepelitsin strain (serogroup Tarassovi) to 65±2.3 m.c. (in the Akiyami A strain, serogroup Autumnalis).

At 14 days after sowing, a repeat examination showed a slight decrease in the number of microbial cells in the field of view for all tested strains.

Example 1. The test strains were grown on a liquid enriched nutrient medium for culturing leptospira at an optimal pH of 7.4±0.2, containing, g/l: sodium chloride - 0.5 g; dry enzymatic peptone - 1.0 g; thiamine - 0.005 g; polysorbate Tween-80 - 10.0 ml; normal horse serum for liquid bacteriological nutrient media - 5 ml, ready-made working solution of phosphate-buffer mixture in distilled water - up to 1.0 l. Dry samples were placed in an enamel container, Tween-80 and a working solution of phosphate-buffer mixture in distilled water were added. Boil for 1-2 minutes until all ingredients are completely dissolved. The pH was measured, it was confirmed that it was within the range of 7.4±0.2, and the medium was poured into graduated 450-ml flasks (333 ml), then sealed and sterilized at 115°C for 15 min. Cooled to 56°C, 1.67 ml of normal horse serum for liquid bacteriological nutrient media was added to each flask over an alcohol lamp torch using a sterile bacteriological pipette, and 5 ml of the medium was poured into sterile high-purity bacteriological test tubes using sterile bacteriological pipettes. The medium was maintained at 37°C for 2 days and at room temperature (20-25°C) for 7 days to control sterility, after which the cultures were inoculated at 0.5 ml per test tube and cultivated at 29±1°C.

With this ratio of ingredients in the liquid enriched nutrient medium for culturing leptospira 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 41±0.4 typical, actively motile leptospira cells were observed in the dark field of view at a magnification of ×400: from 41±1.7 m.c. of the Ballico strain (Australis serogroup) to 43±1.0 m.c. of the P.O.5621 strain (Pomona serogroup). When re-examined 14 days after culture, the number of microbial cells and their morphology did not change significantly.

Example 2. The test strains were grown on a liquid enriched nutrient medium for culturing leptospira at an optimal pH of 7.4±0.2, containing, g/l: sodium chloride - 0.5 g; dry enzymatic peptone - 1.0 g; thiamine - 0.005 g; polysorbate Tween-80 - 12.5 ml; normal horse serum for bacteriological nutrient media, liquid - 10 ml; ready-made working solution of phosphate-buffer mixture in distilled water - up to 1.0 l. Dry samples were placed in an enamel container, Tween-80 and a working solution of phosphate-buffer mixture in distilled water were added. Boil for 1-2 minutes until all ingredients are completely dissolved. The pH was measured, it was confirmed that it was within the range of 7.4±0.2, and the medium was poured into graduated 450-ml flasks (333 ml), then sealed and sterilized at 115°C for 15 min. Cooled to 56°C, 3.3 ml of normal horse serum for liquid bacteriological nutrient media was added to each flask over an alcohol lamp torch using a sterile bacteriological pipette, and 5 ml of the medium was poured into sterile high-purity bacteriological test tubes using sterile bacteriological pipettes. The medium was maintained at 37°C for 2 days and at room temperature (20-25°C) for 7 days to control sterility, after which the cultures were inoculated at 0.5 ml per test tube and cultivated at 29±1°C.

With this ratio of ingredients in the liquid enriched nutrient medium for culturing leptospira 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 121±4.0 actively motile leptospira cells with typical morphological features were observed in the dark field of vision at a magnification of ×400: from 111±3.3 m.c. of the M20 strain (Icterohaemorrhagiae serogroup) to 130±4.1 m.c. of the Akiyami A strain (Autumnalis serogroup). On the 14th day, the number of typical and motile cells remained the same.

Example 3. The test strains were grown on a liquid enriched nutrient medium for culturing leptospira at an optimal pH of 7.4±0.2, containing, g/l: sodium chloride - 0.5 g; dry enzymatic peptone - 1.0 g; thiamine - 0.005 g; polysorbate Tween-80 - 15.0 ml; normal horse serum for bacteriological nutrient media, liquid - 15 ml; ready-made working solution of phosphate-buffer mixture in distilled water - up to 1.0 l. Dry samples were placed in an enamel container, Tween-80 and a working solution of phosphate-buffer mixture in distilled water were added. Boil for 1-2 minutes until all ingredients are completely dissolved. The pH was measured, it was confirmed that it was within the range of 7.4±0.2, and the medium was poured into 333 ml graduated 450 ml flasks, then sealed and sterilized at 115°C for 15 min. Cooled to 56°C, 5.0 ml of normal horse serum for liquid bacteriological nutrient media was added to each flask over an alcohol lamp torch using a sterile bacteriological pipette, and 5 ml of the medium was poured into sterile high-purity bacteriological test tubes using sterile bacteriological pipettes. The medium was maintained at 37°C for 2 days and at room temperature (20-25°C) for 7 days to control sterility, after which the cultures were inoculated at 0.5 ml per test tube and cultivated at 29±1°C.

With this ratio of ingredients in the liquid enriched nutrient medium for culturing leptospira in “crushed drop” preparations from crops kept in a thermostat for 5 days at a temperature of 29±1°C, an average of 161±1.6 leptospira cells were observed in the dark field of vision at a magnification of ×400: from 156±1.1 m.c. of the M20 strain (serogroup Icterohaemorrhagiae) to 164±3.7 m.c. of the Akiyami A strain (serogroup Autumnalis), however, cell counting was difficult due to spontaneous agglutination observed in all the strains tested. When examining after 14 days of cultivation, a slight decrease in the number of leptospires was observed in all test tubes: their number, on average, was 154±1.2 m.c.: from 151±1.1 m.c. of the M20 strain (serogroup Icterohaemorrhagiae) to 157±1.8 m.c. of the Perepelitsin strain (serogroup Tarassovi).

Thus, the declared enriched liquid nutrient medium for cultivating leptospira, prepared according to the method described in Example 2, is optimal in terms of the number of leptospira cells obtained, possessing typical morphological characteristics and active motility, observed in “crushed drop” preparations in the dark field of view of a microscope at a magnification of ×400.

The proposed nutrient medium is prepared using a simplified method and has high growth properties, allowing the restoration of the viability of cultures weakened by multiple passages, which is essential for preserving diagnostic strains of leptospirosis pathogens.

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. Partanen, L., Marttinen, N., and Alatossava, T. (2001). Fats and fatty acids as growth factors for Lactobacillus delbrueckii. System. Appl. Microbiol. 24, 500–506. doi: 10.1078/0723-2020-00078.

3. Polyak M.S., Sukharevich V.I., Sukharevich M.E. Nutrient media for medical and sanitary microbiology. - St. Petersburg: ELBI-SPb, 2008. -352 p.

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

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

6. 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 enriched nutrient medium for culturing leptospira, comprising mixing the following components: sodium chloride - 0.5 g; dry enzymatic peptone - 1.0 g; thiamine - 0.005 g in a container, add 12.5 ml of Tween-80 to the mixture and top up with a ready-made working solution of a phosphate-buffered mixture in distilled water to 1.0 l, boil until all ingredients are completely dissolved, the pH of the medium is 7.4 ± 0.2, and pour into graduated vials, after which they are sealed and sterilized at a temperature of 115 ° C for 15 minutes, cooled to a temperature of 56 ° C, then normal horse serum for bacteriological nutrient media, liquid, is added to each vial using a sterile bacteriological pipette at a rate of 10 ml / l and the medium is poured into sterile bacteriological test tubes.