RU2300560C2 - Method for preparing nutrient medium for culturing yersinia enterocolitica - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves preparing beef-extract agar, its cooling to temperature 45-50°C and addition of cattle brain extract to agar. Extract is prepared by extraction of milled cattle brain in tap water for 2 h at temperature 37°C with addition of 0.5% sodium chloride solution. Invention provides abundant growth of culture Yersinia and allows reducing culturing time. Invention can be used in culturing microorganisms, accumulation of microorganism biomass and preparing biopreparations.

EFFECT: improved preparing method.

5 tbl, 4 ex

Description

The invention relates to veterinary microbiology and can be used for the cultivation of microorganisms, the accumulation of biomass of microorganisms, as well as the preparation of biological products (antigens for agglutination reactions, indirect hemagglutination).

It is known to cultivate pure cultures of microorganisms on ordinary nutrient media, in particular, on meat peptone agar (MPA) (Rozanov NI, Handbook of microbiological technology. Moscow, 1957, p. 46). This method is based on adding 10 g peptone and 5 g table salt in 1 liter of meat water. The components of the nutrient medium are heated to boiling for 30 seconds, the pH is adjusted to 7.2-7.4, 30 g of agar agar are added, heated to complete dissolution and the pH is checked, filtered through a cotton-gauze filter, then the medium is sterilized at 120 ° C for 15-20 minutes. However, the known method has significant disadvantages: when growing pure cultures of microorganisms of the genus Yersinia on the surface of MPA, there is a sparse growth of Y. enterocolitica and prolonged cultivation (for 48-72 hours).

Isolation of microorganisms and the production of bacterial preparations depend on the full composition of the applied nutrient media: this primarily concerns the content of plastic material in the nutrient medium for building the body of microbial cells, as well as energy sources.

The objective of the invention is to increase the efficiency of the cultivation of microorganisms of the genus Yersinia, the method is carried out by preparing meat peptone agar, sterilizing and cooling it, and it is cooled to 45-50 ° C, cattle brain extract is added to it in an amount of 5-10 wt.%, Obtained by extracting the crushed cattle brain in tap water for two hours at 37 ° C, adding a 0.5% sodium chloride solution to it and sterilizing it in an autoclave for 15-20 mi ut at 121 ° C, the resulting culture medium is agitated.

The proposed method allows to reduce the time of cultivation of microorganisms in 2-2.5 times and provides abundant growth of Yersinia culture.

Cattle brain extract stimulates the growth of bacteria due to the content of various substances: proteins, carbohydrates, lipids, mineral and extractive substances.

The chemical composition of the nervous tissue is very complex. The gray and white matter of the brain contains water - 74.0-82.7; proteins - 33.0-51.0; total nitrogen 2.06-3.19 and lipids 25.0-40.0 (Chechetkin A.V. et al. Animal Biochemistry, M., 1982, p. 493).

The gray matter of the brain contains proteins neuroglobulins and neurostromin, which contain up to 0.5% phosphorus. About 20-45% of all organic substances of the nuclei of gray and white matter account for nucleic acids. The nervous tissue contains glycogen (100-150 mg%), glucose 150 mg%, pentoses, glycolipids, which consist of glucose, galactose, sphingosine, higher fatty acids and neuraminic acid residues. The composition of the lipid substances of the brain differs from their composition in other organs and tissues by a large number of phospholipids, cholesterol and cholesterides, cerebrosides and, in a small amount, glycerides. Mineral substances of the nervous tissue are distributed in different parts of the nervous system evenly. In the mass of the brain there are K, Na, Ca, Mg, Cu, Fe, Al, Zn, Mn, Co, P, Cl, I, S. Nitrogen and nitrogen-free low-molecular substances are contained in the nervous tissue. Of the nitrogenous extractives, there are creatine, phosphocreatine, ATP and ADP, free amino acids, choline, acetylcholine, serotonin, aminobutyric acid, uric acid, glutamine, histamine, asparagine and others. Nitrous extractives include glucose, inositol, lactate - and hexose phosphates.

Table 1
Comparative indicators of the mineral composition of skeletal muscle tissue and the brain The content of chemical elements, mg% to fresh weight Skeletal muscle Brain Na 65-150 150-220 TO 250-400 290-340 Sa 4-9 8-10 Mg 2.2-2.8 14-17 Cl 60-70 108-222 R eighteen 7 S 6 10

Thus, the foregoing indicates that the brain of cattle contains more diverse mineral, extractive substances, proteins, carbohydrates and lipids than skeletal muscle.

Description of experience

Cultures of Y. enterocolitica serovariants 0: 3 and 0: 9 were grown in Petri dishes on the surface of nutrient agar containing 10% cattle brain extract, incubated aerobically at + 28 ° C.

Nutrient agar was prepared according to the instructions indicated on the bottle (production date 06.2002. Lot No. 103. Yaroslavl region, Uglich, OOO BioKompas-S. TU 10-02-02-789-176-94).

Brain extract was prepared according to the following method: a fresh, cleaned shell of a cattle brain weighing 200 g was diced and passed through a nozzle (hole diameter 5 mm) of a household meat grinder, mixed with a double volume of tap water. In order to extract for two hours, the resulting slurry was incubated at + 37 ° C.

The extract was filtered through a cotton-gauze filter into a clean bottle and a 0.5% sodium chloride solution up to 400 ml (pH 7.0-7.2) was added as a buffer. After that, a bottle with brain extract was placed in an autoclave at 121 ° C for 15-20 minutes.

The components of the experimental nutrient medium were combined aseptically in a sterile box: 100 ml of nutrient agar was cooled to 45-50 ° C, after which 10 ml of brain extract was added with a sterile pipette.

Suspensions of 0: 3 and 0: 9 serovariants Y. enterocolitica were sown on the experimental medium at a dilution of 10 ⁵ with an initial concentration of 1 billion bw, prepared in a 0.5-0.55% sodium chloride solution. A suspension of Yersinia in a volume of 0.4 ml was applied to the surface of the agar plate and triturated with a spatula.

The growth results were taken into account after incubation of crops at a temperature of 28 ° С for 24 h, comparing the formed yersinia colonies in experimental and control Petri dishes (MPA without brain extract).

Biochemical properties of cultures of Y. enterocolitica serovariants 0: 3 and 0: 9, grown on experimental and control media, were compared using a set of biochemical tests (Enterobacteria. A guide for doctors, edited by Pokrovsky V.I. Moscow, 1985, p. 226 )

Example 1. The selection of the optimal temperature MPA

MPA was prepared according to the instructions, after which a brain extract was introduced into the medium at various temperature conditions.

It was experimentally established that at temperatures below 45-50 ° С MPA acquires a dense texture and does not mix with the extract, and at 50-55 ° С and 55-60 ° С MPA retains a liquid consistency and gray-white flakes form when the extract is introduced into the medium due to coagulation of proteins.

Therefore, we believe that the optimal MPA temperature for mixing with brain extract is 45-50 ° C.

Example 2. The ratio of the components of the nutrient medium

The study of the growth pattern of Yersinia on a nutrient medium was carried out at the following ratios of the extract to MPA: 1/100, 5/100, 10/100, 15/100, 20/100 and 0/100 (control).

table 2
Selection of the amount of brain extract in MPA The content of the brain extract,% Nutrient components Morphological characteristics of the colonies of Y. Enterocolitica MPA, ml Brain extract, ml one one hundred one small (0.3-0.5 mm), gray-white, opaque, with smooth edges and a shiny, smooth surface 5 one hundred 5 juicy, convex, opaque, gray-white colonies, with a diameter of 2-6 mm, with smooth edges and a shiny, smooth surface 10 one hundred 10 juicy, convex, opaque, gray-white colonies, with a diameter of 2-6 mm, with smooth edges and a shiny, smooth surface fifteen one hundred fifteen juicy, convex, opaque, gray-white colonies, with a diameter of 2-6 mm, with smooth edges and a shiny, smooth surface twenty one hundred twenty juicy, convex, opaque, gray-white colonies, with a diameter of 2-6 mm, with smooth edges and a shiny, smooth surface 0 (control) one hundred - small (0.3-0.5 mm), gray-white, opaque, with smooth edges and a shiny, smooth surface

From table 2 it is seen that when making 1 ml (1 wt.%) Of the extract in 100 ml of MPA, the rate and nature of the growth of microorganisms did not differ significantly from the control. So, after 48 hours, small (0.3-0.5 mm), gray-white, opaque colonies of rounded shape with a shiny, smooth surface and even edges were formed on the surface of the MPA with Yersinia extract.

When 100 ml of MPA was added to 5-10 ml (5-10 wt.%) Of the extract, abundant growth of yersinia was observed after 20-24 hours of incubation. Yersinia formed juicy, convex, opaque, gray-white colonies, with a diameter of 2 to 6 mm, with smooth edges and a shiny, smooth surface. Subsequently, with an increase in the content of the extract in MPA to 20 ml (20 wt.%), The rate and nature of the growth of Yersinia did not differ from the indicators of the previous version (Table 2).

Thus, the optimal content of the brain extract in MPA is 5-10 wt.% Or 5-10 ml in 100 ml of MPA.

Example 3. Exposure and temperature extraction of the brain

Extraction of the crushed cattle brain was carried out at a temperature of 25, 37, and 45 ° C in a thermostat for 1, 2, and 4 hours. Then, the filtered extract was introduced into MPA, after which the number of yersinia colonies formed after 20-24 hours of incubation in a thermostat at 28 ° C on the surface of a known (control) and proposed (with brain extract) nutrient medium was compared (Table 3).

Table 3
The number of yersinia colonies on the experimental medium under various conditions The temperature of extraction, ° C Exposition, hour one 2 four 25 98 119 127 37 135 Solid growth Solid growth 45 141 123 117

In control Petri dishes (without extract) 84 colonies of Y. enterocolitica were formed.

It was experimentally established that the number of yersinia colonies formed on the surface of the nutrient medium with brain extract obtained by extraction for 1-4 hours at 25 ° C did not differ significantly from the control and amounted to 98, 119 and 127 colonies, respectively.

135 colonies formed on the surface of the experimental medium with a brain extract obtained by extraction at 37 ° C for one hour, and continuous extraction of Y. enterocolitica was observed upon extraction for 2-4 hours.

Subsequently, with an increase in the extraction temperature to 45 ° C for 1, 2, and 4 hours, the number of Yersinia colonies formed on the surface of the nutrient medium with brain extract decreased to 141, 123, and 117, respectively.

Thus, the optimal mode for obtaining the extract of the brain of cattle is an exposure of 2 hours at 37 ° C.

Example 4. Comparison of the cultural and biochemical properties of Yersinia with a known and proposed method

On the surface of MPA with brain extract after 18 h of incubation, the formation of single colonies was noted, after 20-24 h, Yersinia cultures formed juicy, convex, opaque, gray-white colonies with smooth edges and a shiny, smooth surface, up to 6 mm in diameter. In this case, colonies of Yersinia serovariant 0: 9 were larger (4-6 mm) than colonies of Yersinia serovariant 0: 3 (2-4 mm). After 36 h of cultivation, the edges of the yersinia colonies were combined, forming a continuous growth on the surface of the experimental medium (Tables 4, 5).

Table 4
Morphological characteristics of colonies and biochemical properties of Y. Enterocolitica serovariants 0: 3 and 0: 9 Prototype (cultivation of Yersinia on nutrient agar) The proposed method (cultivation of Yersinia on nutrient agar with brain extract) Y. enterocolitica Y. enterocolitica 0: 3 0: 9 0: 3 0: 9 Colony morphology small (0.3-0.5 mm), gray-white, opaque, with smooth edges and a shiny, smooth surface small (0.3-0.5 mm), gray-white, opaque, with smooth edges and a shiny, smooth surface juicy, convex, opaque, gray-white colonies, 2-4 mm in diameter, with smooth edges and a shiny, smooth surface juicy, convex, opaque, gray-white colonies, with a diameter of 4-6 mm, with smooth edges and a shiny, smooth surface Biochemical tests Glucose + + + + Sucrose + + + + Lactose - - - - Sorbitol + + + + Galactose + + + + Maltose + + + + Ramnose - - - - Raffinose - - - - D-arabinose - - - - Gelatin - - - - Mannitol + + + + Cytochrome oxidase - - - - Nitrate reductase + + + + Catalase + + + + Mobility + + + + Phenylalanine desaminase - - - - Argenine dehydrolase - - - - Lysine decarboxylase - - - - Indole - - - - Hydrogen sulfide - - - - Urease + + + + Simmons Citrate - - - - Sodium Acetate - + - + Voges-Proskauer reaction + + + + Methyl Red Reaction + + + +

In the control (MPA without brain extract), the formation of small (0.3-0.5 mm), gray-white, opaque colonies of Y. enterocolitica with smooth edges and a shiny, smooth surface was observed after 48 hours (Table 5).

In Gram stained smears prepared from Yersinia colonies grown on experimental and control media, Yersinia enterocolitica represented small gram-negative bacilli with rounded ends.

When studying the motility of Y. enterocolitica, the isolated cultures were seeded in semi-liquid agar and cultured at 28 ° C for 48 hours. The turbidity of the medium in test tubes was evidence of the mobility of Yersinia. Yersinia cultures fermented glucose, sucrose, sorbitol, galactose, maltose, mannitol already in the first day; possessed enzymes nitrate reductase, catalase and did not cleave D-arabinose, ramnose, raffinose, lactose, did not thin the gelatin; did not possess enzymes oxidase, arginine dehydrolase, phenylalanine deaminase, tryptophandesaminase, lysine decarboxylase; did not produce hydrogen sulfide. Urea was fermented for 24 hours at 28 ° C. Y. enterocolitica did not utilize sodium citrate, and the sodium acetate microorganisms of the 0: 9 serovariant were used. The Voges-Proskauer reaction with methyl red at 28 ° C is positive.

It was experimentally established that yersinia grown on MPA and MPA with brain extract do not differ in biochemical properties, which allows us to recommend the proposed environment for the accumulation of Yersinia biomass.

Table 5 Comparative table of the cultivation dates of Y. enterocolitica Cultivation time, hour Way Known (MPA), number of colonies Offered (MPA with brain extract), the number of colonies 12 - - 16 - - eighteen - 7 twenty - 145 24 - 254 36 6 Solid growth 48 99 Solid growth

The data in table 5 prove that the number of colonies of yersinia on the proposed nutrient medium increases, and the period of appearance of colonies Y. enterocolitica is reduced by 2-2.5 times

Thus, the proposed nutrient medium for the accumulation of biomass of microorganisms allows you to:

1) to grow microorganisms;

2) receive abundant growth of cultures of microorganisms;

3) isolate pure cultures of microorganisms by inoculation on MPA with the addition of 5-10 wt.% Extract of the brain of cattle;

4) use the resulting biomass of microorganisms for the preparation of biological products;

5) to preserve freshly allocated and industrial crops for a long time;

6) reduce the cultivation time by 2-2.5 times.

Claims (1)

A method of obtaining a culture medium for the cultivation of Yersinia enterocolitica, involving the preparation of meat peptone agar, its sterilization and cooling, characterized in that the meat peptone agar is cooled to 45-50 ° C., cattle brain extract is added to it in an amount of 5-10 wt.% obtained by extracting the crushed cattle brain in tap water for two hours at 37 ° C by adding a 0.5% sodium chloride solution to it and sterilizing it in an autoclave for 15-20 minutes at 121 ° C, the resulting nutrient medium is shaken.