RU2399660C2 - Nutrient medium for bacteria cultivation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: nutrient medium contains salmon milt broth, sodium chloride, yeastrel, glucose, microbiological agar and distilled water.

EFFECT: extended range of nutrient mediums and simplified method for preparing a nutrient medium.

2 cl, 3 tbl, 4 ex

Description

The invention relates to general and medical microbiology and can be used both in research and in practice for the cultivation of a wide range of microorganisms (bacteria of the genus Listeria, genus Yersinia - Y. pseudotuberculosis, Y. enterocolitica, Escherihia coli, Shigella flexneri, Salmonella typhimureum, Staphylococcus aureus, etc.).

Currently, there is a sufficient number of dense nutrient media for the cultivation of microorganisms. As a rule, the nutrient medium consists of a protein and carbohydrate base, as well as additional components that enhance the growth of microorganisms: vitamins, amino acids, mineral components.

The nutritional properties of a dense nutrient medium mainly depend on the composition of the protein base. For their preparation, as a rule, animal proteins are used - meat (mainly beef), fish, meat and bone meal, casein, high-grade meat substitutes - placenta, domestic animal blood, yeast, vegetable proteins (soybeans, peas, barley) .

Nutrient media are known for the cultivation of microorganisms, including conditionally pathogenic bacteria of the genus Listeria, in the composition of which both broths and various hydrolysates are used as a protein base:

1) a nutrient medium for the cultivation of bacteria of the genus Listeria, consisting of a protein component, which is used meat-peptone broth, sodium chloride and water (French patent No. 2671516, CL C12Q 1/04);

2) a nutrient medium for the cultivation of bacteria of the genus Listeria, consisting of a protein component, which is used as an enzymatic blood hydrolyzate, sodium chloride, glucose, microbiological agar, meat water (RF patent No. 2118363, IPC 6 C12N 1/04).

The disadvantage of these nutrient media is an insufficient amino acid composition, which leads to an uneven growth of microorganisms on them, including bacteria belonging to the genus Listeria.

Closest to the claimed solution are:

1) Timing-agar (Nutrient agar for the cultivation of microorganisms, №), consisting of a protein base, which is used as (g / l): pancreatic hydrolyzate of fish meal (24.0), pancreatic hydrolyzate of casein ( 10.0), sodium chloride (4.0), agar (12.0 ± 2.0), water - the rest.

2) Timing medium No. 1 (Nutrient medium for the cultivation and counting of a wide range of aerobic bacteria, including bacteria of the genus Listeria, which require more efficient reproduction of glucose, consisting of a protein base, which is used (g / l): pancreatic fish meal hydrolyzate (15.0), casein pancreatic hydrolyzate (10.0), sodium chloride (3.5), yeast extract (2.0), glucose (1.0), agar (10.0 ± 2.0 ), water the rest (Catalog "Bacteriological environments for sanitary and clinical microbiology, biotechnology and drug control resources, Obolensk, 1999. 4th edition, 56 pp.).

The disadvantage of these culture media is their multicomponent nature. As the protein base, protein hydrolysates are used, the preparation of which is rather laborious, in addition, these nutrient media have an insufficient amino acid composition, which leads to an uneven growth of microorganisms on them, including bacteria belonging to the genus Listeria.

The problem solved by the invention is the creation of a universal nutrient medium for a wide range of bacteria, including bacteria of the genus Listeria, by maintaining a constant amino acid composition in the nutrient medium, expanding the raw material base for the preparation of the protein base of the nutrient medium, reducing its cost by using fish waste production, availability in practical laboratories.

The problem is solved as follows.

A nutrient medium for the cultivation of bacteria containing a protein base, microbiological agar, sodium chloride, distilled water, according to the invention additionally contains yeast extract, and as a protein base, a decoction of salmon milk in the following ratio, g / l:

- decoction of salmon milk - 6.0

- microbiological agar 10.0 ± 2.0

- sodium chloride - 5.0

- yeast extract - 4.0-10.0

- distilled water - the rest

- pH of the medium is 7.2-7.4,

while culturing bacteria of the genus Listeria, the nutrient medium additionally contains glucose in an amount of 1 g / l.

The preparation of a nutrient medium for the cultivation of a wide range of bacteria, both conditionally pathogenic and pathogenic, based on a decoction of salmon milk is carried out as follows.

First, prepare a decoction of salmon milk. To 500 g of pre-crushed salmon milk is added tap water in a ratio of 1: 2 and boiled for 1 hour. The resulting broth is filtered, sterilized with a pressure of 1 atm for 30 minutes.

The broth can be prepared for future use, stored in a household refrigerator at a temperature of + 4-8 ° C for 30 days or more or dried (“dry” broth), and then used as necessary.

A “dry” decoction of fish milk can be stored for a long time in a household refrigerator (+ 4-8 ° C) or at room temperature, it can be transported to other regions, while its quality is preserved.

To prepare the claimed nutrient medium, take 4.0-10.0 g of yeast extract, 5.0 g of sodium chloride, 10.0-12.0 g of microbiological agar, 6.0 g of “dry” broth from salmon milk or 600, 0-750.0 ml of liquid broth from salmon milk and add up to 1 liter of distilled water. The mixture is stirred, the pH of the medium is adjusted to 7.2-7.4 using a well-known method using 20% NaOH. The resulting culture medium is sterilized at 1 atm for 30 minutes.

Ready sterile medium, poured into Petri dishes, has a yellowish color, transparent or slightly opalescent.

The use in the proposed nutrient medium as a protein base, a decoction of salmon milk, leads to the growth of microorganisms not inferior to the prototype due to the content of a sufficient amount of amino acids and microelements in it. Fish milk is an affordable, inexpensive source of raw materials. They contain nucleoproteins, consisting of nucleic acids and simple proteins - protamines and histones. Protamines and histones are built from amino acids, among which arginine, lysine and histidine predominate; protamines include tyrosine, tryptophan, serine, valine and proline, and histones include serine, valine, alanine, phenylalanine and tyrosine. In the milk of a number of fish, there are such water-soluble vitamins: ₁₂ in amounts from 7 to 50 μg%; B ₁ (thiamine) - 50-185 mcg%; In ₂ (riboflavin) - 40-600 mcg%; In _s (folic acid) - 125-1300 mcg%; RR (nicotinic acid) - 1.1-5.7 mg%, etc. (V.P. Zaitsev, I.V. Kizevetter, L.L. Lagunov and others. "Technology of fish products. M: Publishing house "Food Industry", 1965, p. 73-74). The chemical composition of chum milk of the 6th stage of maturity is reflected in table No. 1 (Yu.I. Kasyanenko, T. N. Pivnenko. “Comparative physicochemical characteristics of low molecular weight deoxyribonucleic acid (DNA) from marine hydrobionts” - “Izvestiya TINRO”, 1999, vol. 125, p. 152-158).

Table 1 The chemical composition of milk of chum salmon of the 6th stage of maturity,% The name of the substance. Content Deoxyribonucleic Acid (DNA) 12.0 Ribonucleic Acid (RNA) 0.3 Acid soluble products 0.1 Protein 8.7 Water 78.5 Other 0.4

The specified technical result in the implementation of the invention is achieved due to the fact that the nutrient medium for culturing a different spectrum of bacteria according to the invention contains:

1) as a protein base and mineral substances - a decoction (liquid or dry) from salmon milk,

2) as a source of B vitamins - yeast extract,

3) to ensure isotonicity of the medium - sodium chloride.

For the cultivation of bacteria belonging to the genus Listeria, an additional 1.0 g of glucose is used as a carbohydrate base.

Preparation of a dense nutrient medium based on a dry broth of salmon milk is carried out as follows.

To 4.0-10.0 g of yeast extract, 1.0 g of glucose, 5.0 g of sodium chloride, 10.0 ± 2.0 g of microbiological agar, add 6.0 g of “dry” or 600.0-750, 0 ml of liquid broth from salmon milk and add up to 1 liter of distilled water. The mixture is stirred, the pH of the medium is adjusted to 7.2-7.4 using a well-known method using 20% NaOH. The resulting culture medium was sterilized at 0.5 atm for 20 minutes.

Ready sterile medium, poured into Petri dishes, has a yellowish color, transparent or slightly opalescent.

The mass of "dry" broth or liquid from salmon milk was determined experimentally based on the required amount of amino acids in the nutrient medium. Moreover, its qualitative and quantitative composition should be considered essential features of the proposed dense nutrient medium, as the values of the content of components in the claimed nutrient medium are optimal and determine the relative balance of the cost of the medium with its growth characteristics. Going beyond the declared values of the components leads either to an overexpenditure of raw materials, or to a decrease in its growth properties.

To determine the growth properties of the claimed medium, bacterial test strains were used from the collection of the Research Institute of Epidemiology and Microbiology SB RAMS (Vladivostok): 1) bacteria of the genus Listeria — L. monocytogenes NCTC 10527 (4b serovariant); L. ivanovii NCTC 11846; L. innocua SLCC 3379; L. seeligeri SLCC 5921; L. welschimeri; L. grayi 17; 2) bacteria of the genus Yersinia - Y. pseudotuberculosis 2781; Y. enterocolitica 164; 3) Escherihia coli; 4) Staphylococcus aureus; 4) Shigella flexneri 73.

To determine the sensitivity and growth rate of the claimed nutrient medium, the daily cultures of test strains grown on mowed agar were washed off with physiological NaCl solution and adjusted to the GISK optical standard. Tarasovich, corresponding to a dilution of 10 units. turbidity. 10-fold dilutions were prepared from the initial standard suspension of each test strain by sequential transfer of at least 0.5 ml of culture in 8 test tubes with 4.5 ml of physiological saline and from dilutions of 10 ³ CFU / ml and 10 ² CFU / ml, 0 1 ml of suspension of each test strain was made in 3 cups with experimental medium. A suspension of culture was distributed with a spatula over the surface of the agar. Cups with crops were placed for 24-48 hours in a thermostat at 37 ° C. Analysis was carried out daily.

At the same time, the biological properties of cultures grown on plates with the claimed medium were determined (determination of the form, growth pattern of colonies, their diameter, crop morphology, motility, biochemical properties, hemolytic and lecithinase activity, determination of catalase) with the study of at least 3 colonies from each cup (according to Guidelines for the control of nutrient media for biological indicators. Moscow, 1980).

As control media, GRM-AHAR (Nutrient agar for the cultivation of microorganisms, Patent No. 2089609) and Medium No. 1 Timing medium (Nutrient medium for the cultivation and counting of a wide range of aerobic bacteria, Obolensk) were used.

The results on the number of grown colonies of test strains on the claimed medium are presented in tables 2, 3.

As can be seen from the data in Tables 2, 3, the claimed dense nutrient medium, prepared on the basis of a liquid or “dry” decoction of salmon milk, was not inferior to the control in the number of colonies grown on a cup after 24 ± 2 hours of incubation of crops, and also had a high sensitivity (10 ^-7 ), because it allowed to detect the growth of colonies during sowing of single microbial cells. We studied the biological properties of the grown colonies of test strains on the claimed medium (different bases) after their cultivation on it for 24 and 48 hours, which did not differ from the controls.

Table 2 shows that the claimed nutrient medium is universal and bacteria can grow on it as pathogenic Escherihia coli; Staphylococcus aureus; Shigella flexneri 73, as well as opportunistic Yersinia - Y. pseudotuberculosis 2781; Y. enterocolitica 164.

The study of the effectiveness indicator of the claimed nutrient medium for the cultivation of listeria was carried out on an expanded set of strains belonging to different types of Listeria. As can be seen from the data in table 3, the number of listeria grown on the claimed medium was on average 100 m.k. / ml (for species L.monocytogenes, Livanovii, L.innocua). Some species of listeria grew better (L.monocytogenes, L.ivanovii, L.innocua), others worse (L.welschimeri, L..grayi, L.seeligeri). On the claimed nutrient medium from salmon milk (MLR), the listeria was formed transparent with a pearlescent shade, round with even edges of a colony with a diameter of 2-4 mm.

When determining the morphology of listeria in smears, typical small gram-positive bacilli or ovoid cells were observed, identical with the control test strain of listeria.

As can be seen from table 2, the growth properties of the test strains of bacteria in the claimed nutrient medium, prepared on the basis of both liquid and "dry" decoction of fish milk, are identical.

The claimed technical solution meets the criterion of "inventive step" in view of the fact that for the preparation of a dense nutrient medium for the cultivation of bacteria, a liquid or "dry" broth made from salmon milk is used as a protein base.

A significant difference of the proposed technical solution is:

- the possibility of using the proposed environment for the cultivation of a wide range of bacteria, both opportunistic and pathogenic;

- Replacement of an expensive nutrient medium with a cheap, easily accessible nutrient medium prepared on the basis of a decoction of salmon milk.

The result is:

- cheapening of the nutrient medium;

- expansion of the raw material base for its preparation.

The claimed technical solution meets the criterion of "industrial applicability", as the medium is easy to manufacture and it can be prepared both in any microbiological laboratory and on an industrial scale.

Examples of specific production of a nutrient medium.

Example 1

For the cultivation of bacteria of the genus Yersinia take a nutrient medium of the following composition, g / l:

"Dry" decoction of fish milk - 6.0

microbiological agar - 10.0

yeast extract - 5.0

sodium chloride - 5.0

distilled water - up to 1 l

pH 7.2-7.4

The mixture is stirred. The resulting nutrient medium is poured into containers, the well-known method using pH 20% NaOH in each container of the nutrient medium, the pH is adjusted to 7.2-7.4, and then sterilized at 1 ATM for 30 minutes.

Ready sterile medium, poured into Petri dishes, has a yellowish color, transparent or slightly opalescent.

The cultivation of bacteria of the genus Yersinia was carried out according to the above method. The results are shown in table 2.

Example 2

For the cultivation of bacteria of the genus Listeria take a nutrient medium of the following composition, g / l:

“Dry” broth from salmon milk - 6.0

microbiological agar - 11.0

yeast extract - 5.0

glucose - 1,0

sodium chloride - 5.0

distilled water up to 1 l

pH 7.2-7.4

The mixture is stirred. The resulting nutrient medium is poured into containers, the well-known method using pH 20% NaOH in each container of the nutrient medium, the pH is adjusted to 7.2-7.4, and then sterilized at 0.5 ATM for 20 minutes.

Ready sterile medium, poured into Petri dishes, has a yellowish color, transparent or slightly opalescent.

Cultivation of bacteria belonging to the genus Listeria was carried out according to the above method. The results are shown in table 3.

Example 3

The nutrient medium is prepared analogously to example 1, however, the medium was prepared on the basis of a liquid decoction of fish milk, g / l:

Microbiological agar - 12.0

sodium chloride - 5.0

glucose - 1,0

Yeast Extract - 10.0

Liquid decoction of salmon milk - 600.0

Distilled water - the rest

pH 7.2-7.3

Cultivation of bacteria belonging to the genus Listeria was carried out according to the above method. The results are shown in table 3.

Example 4

The nutrient medium is prepared analogously to example 1, however, the medium was prepared on the basis of a liquid decoction of fish milk, g / l:

Microbiological agar - 10.0 NaCl - 5.0

Glucose - 1.0

Yeast Extract - 7.0

Liquid decoction of salmon milk - 750.0

Distilled water - the rest

pH 7.2-7.3

The cultivation of bacteria belonging to the genus Escherihia coli was carried out according to the above method. The results are shown in table 2.

The nutrient medium is developed on the basis of cheap domestic raw materials, easy to manufacture, much cheaper than the prototype environment. For its preparation, you can use substandard milk of fish 5-6 degrees of maturity, i.e. flowing that cannot be used for cooking foods. Moreover, its growth properties are not inferior to control environments, and the preparation technology is much simpler, which allows to expand the raw material base and reduce the cost of the nutrient medium for the cultivation of bacteria.

Claims (2)

1. Nutrient medium for the cultivation of bacteria, containing a protein base, microbiological agar, sodium chloride, distilled water, characterized in that the nutrient medium additionally contains yeast extract, and as a protein base, a decoction of salmon milk in the following ratio, g / l:
salmon milk broth 6.0 microbiological agar 10.0 ± 2.0 sodium chloride 5,0 yeast extract 4.0-10.0 distilled water rest pH of the medium 7.2-7.4. 2. Nutrient medium according to claim 1, characterized in that the nutrient medium additionally contains glucose in an amount of 1.0 g / L.